Escherichia coli

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 to cancer development . Here, we report the characterization of isoforms of human beta-pol mRNA in different cells by reverse transcription polymerase chain reaction (RT-PCR) . DNA sequence analysis of RT-PCR products revealed eight alternative splicing mRNA isoforms in the brain cancer cell line, SK-N-MC . These various isoforms were consistent with alternative splicing of four exons (II, IV, V, and VI) and with a 105-nucleotide insertion (exon alpha) between exons VI and VII . We also found an isoform with a 19-nucleotide sequence inserted into the exon IV and V junction, which resulted from usage of a different 3' splice site . Seven of the isoforms resulted in truncated open reading frame (ORF); five corresponded to deduced peptide of amino acids 1-20 of beta-pol and two corresponded to amino acids 1-60 of beta-pol . Only one of the right mRNA isoforms, that with the exon alpha insertion, was in-frame with the entire wild-type ORF resulting in a deduced protein of 370 residues, compared with the wild-type protein of 335 residues and 39 kD . This longer ORF was shown to be capable of encoding a beta-pol protein, larger than wild-type beta-pol, that cross-reacted with beta-pol antibody and exhibited beta-pol enzymatic activity . The mRNA isoform with the exon alpha insertion was not tumor specific because it as detected in low abundance in all cells tested, except the colon cell line CCD18 Co where the isoform was absent . The genomic location of exon alpha is in intron VI, 990 bp upstream of exon VII and flanked by consensus splice sites . Thus, this 105-bp genomic sequence is a beta-pol exon present in a low-abundance beta-pol mRNA isoform capable of encoding an approximately 42-kD beta-pol.

Int Arch Allergy Immunol, 1996 Aug, 110(4), 348 - 53
Cloning and expression of a murine Fab fragment recognizing a defined linear epitope of Chironomus thummi thummi major allergen Chi t 1-9; Kipp B et al.; We have cloned and expressed in bacteria the genes coding for the Fab fragment of the monoclonal antibody (MoAb) M3 . M3 is a murine IgG1 antibody reactive with the major allergen Chi t 1-9 of Chironomus thummi thummi . The major allergen Chi t 1-9 is known to be an aggressive inhalant allergen and causes type-I allergy . The immunoglobulin (Ig) fragment genes were cloned as a synthetic dicistronic operon . In this operon each gene is preceded by a bacterial signal sequence to direct the recombinant protein to the periplasmic space of the bacteria . The cloned genes were expressed in Escherichia coli using the strong T7-RNA-polymerase-based system . Sequence analysis revealed that the light and heavy chains of MoAb M3 belong to the V kappa II and V kappa IIC group of the Ig family, respectively . Genes of the V kappa II and V kappa IIC group are known to be used in response to haptens . The apparent affinity constants of the parent antibody M3 and the recombinant Fab fragment are nearly equivalent.

Carcinogenesis, 1996 Aug, 17(8), 1729 - 33
Recombinant rat and hamster N-acetyltransferases-1 and -2: relative rates of N-acetylation of arylamines and N,O-acyltransfer with arylhydroxamic acids; Jones RF et al.; Genes for the 290 amino acid, 33-34 kDa cytosolic acetyltransferases (NAT1* and NAT2*) from rat and hamster were cloned and expressed in Escherichia coli . Active clones were selected by a simple visual test for their ability to decolorize 4-aminoazobenzene in bacterial medium by acetylation . These recombinant acetyltransferases were analyzed for: (i) N-acetyltransferase, which was assayed by the rate of acetyl coenzyme A-dependent N-acetylation of 2-aminofluorene (2-AF) or 4-aminoazobenzene (AAB); (ii) arylhydroxamic acid acyltransferase, assayed by N,O-acyltransfer with N-hydroxy-N-acetyl-2-aminofluorene . Both NAT2s showed first order increases in N-acetylation rates with increasing 2-AF or AAB concentrations between 5 and 100 microM, with apparent K(m) values of 22-32 and 62-138 microM respectively . Although under the same conditions the N-acetylation rates for the two NAT1s declined by > 50%, below 5 microM 2-AF or AAB, the NAT rate data fit Michaelis-Menten kinetics, and the apparent K(m) values were 0.2-0.9 microM . For N,O-acyltransferase, the apparent K(m) values of the NAT1s were approximately 6 microM, while the K(m) values of the NAT2s were approximately 20- to 70-fold higher . SDS-PAGE/Western blot analysis of the recombinant acetyltransferases gave apparent relative molecular weights (MWr) of approximately 31 kDa for both NAT1s and rat NAT2 and approximately 29 kDa for hamster NAT2 . Comparable MWr values were observed for native hamster liver NAT1 and NAT2 and for rat NAT1 under the same conditions . Although we did not detect NAT2-like activity in rat liver cytosol previously, the present data show that the rat NAT2* gene does code for a functional acetyltransferase, with properties similar to those of hamster liver NAT2 . The data also indicate that at low substrate concentrations, NAT1 would apparently play the predominant role in vivo in N-acetylation and N,O-acyltransfer of aromatic amine derivatives, including their metabolic activation to DNA-reactive agents.

Carcinogenesis, 1996 Aug, 17(8), 1609 - 14
Contribution of ogt-encoded alkyltransferase to resistance to chloroethylnitrosoureas in nucleotide excision repair-deficient Escherichia coli; Abril N et al.; We investigated the relative contribution of the two Escherichia coli DNA alkyltransferases (ATases) to the increased sensitivity of ATase-deficient bacteria to the mutagenic and lethal effects of chloroethylnitrosoureas (CNU) . The ogtencoded protein was the principal determinant in resistance to the mutagenic effects of CNU in E.coli . Thus, only when the ogt gene was inactivated was sensitivity to mutagenesis greatly increased; the contribution of inactivation of the ada gene was relatively minor . Furthermore, induction of the adaptive response provided essentially no protection against CNU mutagenesis in either an ogt+ or ogt- background . Finally, overexpression of the ogt gene into ogt- ada- double mutants provided the greatest protection against CNU; introduction of the full-length or truncated ada gene was protective, but to a much lesser extent . Mammalian ATases were not as protective against mutation induction by CNU as Ogt, even though they were apparently expressed at higher level . In order of effectiveness the ATases ranked Ogt > human > truncated Ada = Ada > rat . This order was not observed in the protection against killing by 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, where truncated Ada = human > Ogt > rat = Ada . Higher mutation frequency and toxicity were observed in uvr- mutants, suggesting that one or more of the potentially mutagenic and/or toxic lesions are also substrates for the excision repair proteins.

Microbiology, 1996 Aug, 142 ( Pt 8), 2175 - 80
Rapid invasion by colicinogenic Escherichia coli with novel immunity functions; Tan Y et al.; Bacteriocins have been suggested to play an important role in the invasion dynamics of bacteria . Recently, the 'diversifying selection' hypothesis has been proposed, which addresses the origin and diversification of one group of bacteriocins, the colicins of Escherichia coli . According to this hypothesis, novel colicin gene clusters arise from mutations generating expanded immunity functions . Positive selection, favouring these novel immunities, then rapidly drives strains carrying the evolved colicin gene clusters to fixation in the local population . To test this fixation step driven by selection, invasion experiments were carried out by introducing novel colicinogenic strains into established colicinogenic populations . In all cases, invasion by strains expressing novel immunity functions occurred rapidly, even when initial frequencies of the invader were quite low . These invasions were attributed primarily to colicin killing effect . Other factors, such as growth rate, level of colicin production and stationary-phase survival rate, were shown to play very minor roles in the invasion process . These results provide direct evidence for the hypothesis of diversifying selection acting on colicin gene clusters and shed light on the ecological role of colicins.

Microbiology, 1996 Aug, 142 ( Pt 8), 2057 - 69
Translational control of puf operon expression in Rhodobacter sphaeroides 2.4.1; Gong L et al.; The puf operon of Rhodobacter sphaeroides 2.4.1 encode the beta- and alpha-polypeptides of the B875 complex, the L and M polypeptides of the reaction centre and the pufX gene product . A previous report from the authors' laboratory indicated the potential existence of a 20-codon open reading frame (orfK, now designated pufK) located immediately upstream of the pufB structural gene . It is now demonstrated that pufK is translated in vivo and that the specific levels or nature of the rare codons within pufK affect the expression of pufK . Using a series of pufK-specific mutations, both in trans as lacZ translational fusions and incorporated into the genome in single copy, evidence has been obtained that translation initiation through pufK may be essential to translation of pufB . Further, the abundance, quality and distribution of rare codons within pufK may serve to 'gate' the entry of ribosomes at pufB . The data also suggest that translation of pufB is uncoupled from that of pufA, with the latter capable of being produced in excess of the former . It is also revealed that the secondary structure at the 5' end of the large and small puf transcripts may play a role in mRNA stability and that stability of the small puf transcript is independent of translation.

Microbiology, 1996 Aug, 142 ( Pt 8), 1997 - 2004
The modE gene product mediates molybdenum-dependent expression of genes for the high-affinity molybdate transporter and modG in Azotobacter vinelandii; Mouncey NJ et al.; The Azotobacter vinelandii mod locus, which is involved in high-affinity molybdate transport and the early event in Mo metabolism, consists of two divergently transcribed operons, modG and modEABC . modA, modB and modC encode the components of the high-affinity molybdate transporter, and modG encodes a Mo-binding protein . High concentrations of Mo repressed transcription of both operons . The modEABC operon was also repressed by tungstate and to a lesser extent by vanadate . modE, the first gene in the modEABC operon, controlled the Mo-dependent transcription of both operons . It was not involved in the metal regulation of alternative nitrogenase gene expression . Although a modE mutant constitutively expressed genes encoding the molybdate transporter, it had a reduced rate of Mo accumulation.

Nucleic Acids Res, 1996 Aug 1, 24(15), 3118 - 9
A simple assay to determine the functionality of Cre or FLP recombination targets in genomic manipulation constructs; Buchholz F et al.; We report the construction of two Escherichia coli strains (294-Cre and 294-FLP) which express either Cre- or FLP-recombinase . Plasmids containing authentic recognition targets for either recombinase (loxPs or FRTs) are recombined when propagated in the appropriate strain . 294-Cre and 294-FLP thus provide a simple test for the recombination competence of constructs that are designed for use in Cre- or FLP-mediated genomic manipulations.

Nucleic Acids Res, 1996 Aug 1, 24(15), 3065 - 70
RNase E processing of essential cell division genes mRNA in Escherichia coli; Cam K et al.; The ratio of the FtsZ to FtsA proteins determines the correct initiation of cell division in Escherichia coli . The genes for these proteins are contiguous on the chromosome . Although both genes are transcribed from common promoters, the presence of ftsZ-specific promoters, along with differences in the efficiency of translation of their respective mRNAs, contribute to the increased relative expression of ftsZ . We report here that the polycistronic ftsA-ftsZ transcripts are cleaved by RNase E and that this cleavage affects the decay of ftsA and ftsZ mRNA . As a consequence of the cleavage, RNase E also contributes to the differential expression of the two genes.

Nucleic Acids Res, 1996 Aug 1, 24(15), 2894 - 9
Codon-reading specificity of an unmodified form of Escherichia coli tRNA1Ser in cell-free protein synthesis; Takai K et al.; Unmodified tRNA molecules are useful for many purposes in cell-free protein biosynthesis, but there is little information about how the lack of tRNA post-transcriptional modifications affects the coding specificity for synonymous codons . In the present study, we prepared an unmodified form of Escherichia coli tRNA1Ser, which originally has the cmo5UGA anticodon (cmo5U = uridine 5-oxyacetic acid) and recognizes the UCU, UCA and UCG codons . The codon specificity of the unmodified tRNA was tested in a cell-free protein synthesis directed by designed mRNAs under competition conditions with the parent tRNA1Ser . It was found that the unmodified tRNA with the UGA anti-codon recognizes the UCA codon nearly as efficiently as the modified tRNA . The unmodified tRNA recognized the UCU codon with low, but detectable efficiency, whereas no recognition of the UCC and UCG codons was detected . Therefore, the absence of modifications makes this tRNA more specific to the UCA codon by remarkably reducing the efficiencies of wobble reading of other synonymous codons, without a significant decrease in the UCA reading efficiency.

J Trauma, 1996 Aug, 41(2), 222 - 8; discussion 228-30
Induction of cytokine-induced neutrophil chemoattractant (CINC) mRNA in the lungs of septic rats; Lukaszewicz GC et al.; OBJECTIVE: To study cytokine-induced neutrophil chemoattractant (CINC) mRNA induction in lungs of normal, neutropenic, and adrenalectomized rats after intraperitoneal Escherichia coli lipopolysaccharide (LPS) administration and in cultured rat pulmonary cell lines after exposure to mediators of the septic response . MATERIALS AND METHODS: Northern blotting was used to assay relative CINC mRNA levels and a colorimetric myeloperoxidase assay was used as a measure of neutrophil infiltration . RESULTS: After a single dose of LPS, rapid induction of CINC mRNA coincided with neutrophil infiltration into lungs, a response that lasted approximately 12 to 24 hours . Multiple LPS treatments resulted in a similar CINC response, but a more prolonged myeloperoxidase response . CINC mRNA induction in lungs was heightened 30% in adrenalectomized animals and 400% in neutropenic ones . LPS and cytokines induced CINC mRNA in cultured endothelial and epithelial cells . CONCLUSIONS: Induction of CINC mRNA expression in pulmonary endothelial and/or epithelial cells by systemic LPS or cytokines may play a role in mediating neutrophil infiltration into lungs during sepsis . Markedly increased CINC induction in the lungs of neutropenic animals suggests that neutrophils may act to inhibit expression of this chemoattractant via a negative feedback mechanism.

J Gen Virol, 1996 Aug, 77 ( Pt 8), 1975 - 83
Immunological characterization of rice tungro spherical virus coat proteins and differentiation of isolates from the Philippines and India; Druka A et al.; Rice tungro spherical virus (RTSV) has an RNA genome of more than 12 kb with various features which classify it as a plant picornavirus . The capsid comprises three coat protein (CP) species, CP1, CP2 and CP3, with predicted molecular masses of 22.5, 22.0 and 33 kDa, respectively, which are cleaved from a polyprotein . In order to obtain information on the properties of these proteins, each was expressed in E . coli, purified as a fusion to the maltose-binding protein and used for raising a polyclonal antiserum . CP1, CP2 and CP3 with the expected molecular masses were detected specifically in virus preparations . CP3 is probably the major antigenic determinant on the surface of RTSV particles, as was shown by ELISA, Western blotting and immunogold electron microscopy using antisera obtained against whole virus particles and to each CP separately . In some cases, especially in crude extracts, CP3 antiserum detected several other proteins (40-42 kDa), which could be products of CP3 post-translational modification . No serological differences were detected between the three CPs from isolates from the Philippines, Thailand, Malaysia and India . The CP3-related 40-42 kDa proteins of the Indian RTSV isolate have a slightly higher electrophoretic mobility (42-44 kDa) and a different response to cellulolytic enzyme preparations, which allows them to be differentiated from south-east Asian isolates.

J Gen Virol, 1996 Aug, 77 ( Pt 8), 1893 - 9
Functional analysis of C-terminal deletion mutants of Epstein-Barr virus thymidine kinase; Hsu TY et al.; Thymidine kinase (TK) activity was detected following expression of the TK gene of Epstein-Barr virus (EBV) using the pET expression plasmid and E . coli BL21 (DE3)pLysS . To study the amino acid residues required at the C terminus of the EBV TK protein for enzymatic activity, a series of C-terminal deletion mutants was generated by direct truncation, linker insertion or PCR mutagenesis to create stop codons at particular sites . Deletion of nine residues from the C terminus caused a 35% reduction in TK activity, while a ten-residue deletion completely abolished the activity . A single point mutation at residue Cys570, corresponding to Cys336 of herpes simplex virus TK, did not alter the TK activity . Single amino acid changes within the last seven to ten residues also did not affect activity . The results indicate that maintenance of the conformation of the C terminus is important for enzyme activity.

J Gen Virol, 1996 Aug, 77 ( Pt 8), 1865 - 74
Characterization of the genes, including that encoding the viral proteinase, contained in BamHI restriction fragment 9 of the pseudorabies virus genome; Camacho A et al.; We describe the nucleotide sequence, transcription pattern and open reading frames (ORFs) located on BamHI restriction fragment 9 (0.406-0.435 map units) in the unique long segment of the pseudorabies virus (PRV) genome . The fragment contains three nested genes with a common 3' end . The 5' ends of the corresponding 0.9, 1.7 and 3.3 kb mRNAs have been mapped . Fragment BamHI-9 contains three complete ORFs, ORF1, ORF2 and ORF2.5 . ORF1, which is within the 3.3 kb transcript, encodes a protein with an apparent molecular mass of 60 kDa which is homologous to the product of the herpes simplex virus type 1 UL25 gene . The 1.7 kb mRNA contains ORF2, whose product is homologous to the herpesvirus proteinases, while the 0.9 kb transcript contains ORF2.5, which probably encodes the assembly protein precursor . ORF2 was identified as the PRV proteinase gene following expression in E . coli, using the product of ORF2.5 as the substrate protein.

J Gen Virol, 1996 Aug, 77 ( Pt 8), 1847 - 51
Immediate early protein IE63 of herpes simplex virus type 1 binds RNA directly; Ingram A et al.; The herpes simplex virus type 1 (HSV-1) immediate early protein IE63 acts post-transcriptionally to affect RNA 3'-processing and splicing . Functional domains such as the RGG box and zinc-finger motifs potentially provide the protein with RNA binding capacity . Here, IE63 protein expressed in E . coli, purified by affinity chromatography and used in RNA binding assays, demonstrated similar binding to RNA substrates containing poly(A) sites from different temporal classes of HSV-1 genes, RNA containing splice site recognition sequences and RNA containing no recognized processing motifs . Competition binding assays showed that IE63 binding could be competed out, suggesting that IE63 binds RNA weakly . HSV-1 infection results in an increase or stabilization in vitro of protein binding to poly(A) site-containing RNAs; IE63 is required for this effect . RNA binding assays combining purified IE63 with protein from mock-infected and HSV-1 infected nuclear extracts demonstrated no effect on protein-RNA binding patterns.

J Gen Virol, 1996 Aug, 77 ( Pt 8), 1821 - 4
Activation of the protease from human adenovirus type 2 is accompanied by a conformational change that is dependent on cysteine-104; Jones SJ et al.; Adenovirus codes for a protease the activity of which can be regulated in vitro by an 11 residue peptide (GVQSLKRRRCF) derived from another viral protein, pVI . Three cysteine residues, one in the activating peptide and two in the protease (C104 and C122), play a central role in both activation and catalysis . Expression of protease mutants in insect cells has shown that C104 is not essential for proteolytic activity . GVQSLKRRRCF also caused a concentration-dependent increase in tryptophan fluorescence of protease expressed in Escherichia coli that paralleled the increase in proteolytic activity, indicating that activation was accompanied by a conformational change . Tryptophan fluorescence of C104S was not increased by the addition of GVQSLKRRRCF, nor was the fluorescence of wild-type protease increased by the addition of the peptide analogues where cysteine is replaced by aspartic acid or serine, suggesting that C104 is involved in activation and C122 in catalysis.

Biochem J, 1996 Aug 1, 317 ( Pt 3), 945 - 54
Insulin-responsive tissues contain the core complex protein SNAP-25 (synaptosomal-associated protein 25) A and B isoforms in addition to syntaxin 4 and synaptobrevins 1 and 2; Jagadish MN et al.; SNAP-25 (synaptosomal-associated protein 25), syntaxin and synaptobrevin are the three SNARE {soluble NSF attachment protein receptor (where NSF = N-ethylmaleimide-sensitive fusion protein)} proteins that form the core complex involved in synaptic vesicle docking and subsequent fusion with the target membrane . The present study is aimed at understanding the mechanisms of fusion of vesicles carrying glucose transporter proteins with the plasma membrane in human insulin-responsive tissues . It describes the isolation and characterization of cDNA molecules encoding SNAP-25 A and B isoforms, syntaxin 4 and synaptobrevins (also known as vehicle-associated membrane proteins) from two major human insulin-responsive tissues, skeletal muscle and fat . The DNA and deduced amino acid sequences of SNAP-25 revealed perfect identity with the previously reported human neural SNAP-25 A and B isoforms . Our results indicate the presence of both isoforms both in insulin-responsive tissues and in in vitro cultured 3T3-L1 cells, but suggest a differential pattern of gene expression: isoform A is the major species in adipose tissue, and isoform B is the major species in skeletal muscle . The presence of SNAP-25 protein in 3T3-L1 cells was demonstrated by immunofluorescence microscopy using an anti-SNAP-25 monoclonal antibody . Immunoprecipitation experiments using the same monoclonal antibody also revealed the presence of SNAP-25 protein in plasma membrane fractions from rat epididymal fat pads . The syntaxin 4-encoding region from skeletal muscle contains five nucleotide differences from the previously reported placental cDNA sequence, two of which result in amino acid changes: Asp-174 to Glu and Val-269 to Ala . The synaptobrevin 1 cDNA from skeletal muscle contains two nucleotide differences when compared with the corresponding clone from neural tissues, one of which is silent and the other resulting in the amino acid change Thr-102 to Ala . The cDNA sequence of the protein from fat is identical with that of human synaptobrevin 1 from neural tissues . Furthermore, we have confirmed the presence of syntaxin 4 in fat and of synaptobrevin 2 in skeletal muscle by PCR amplification and Southern hybridization analysis . Using the yeast two-hybrid system, an interaction was observed between the full-length cytoplasmic domains of syntaxin 4 and synaptobrevin 2, a vesicle membrane SNARE previously shown by others to be associated with vesicles carrying the GLUT4 glucose transporter protein, but no interaction was seen with synaptobrevin 1 . Flow cytometry of low-density microsomes isolated from fat cells was used to demonstrate the binding of syntaxin 4 to a subset of vesicles carrying GLUT4 protein; whereas SNAP-25 on its own bound poorly to these vesicles, the syntaxin 4-SNAP-25 complex gave a strong interaction.

Biochem J, 1996 Aug 1, 317 ( Pt 3), 891 - 9
Purification of a rat neurotensin receptor expressed in Escherichia coli; Tucker J et al.; A truncated rat neurotensin receptor (NTR), expressed in Escherichia coli with the maltose-binding protein fused to its N-terminus and the 13 amino acid Bio tag fused to its C-terminus, was purified to apparent homogeneity in two steps by use of the monomeric avidin system followed by a novel neurotensin column . This purification protocol was developed by engineering a variety of affinity tags on to the C-terminus of NTR . Surprisingly, expression levels varied considerably depending on the C-terminal tag used . Functional expression of NTR was highest (800 receptors/cell) when thioredoxin was placed between the receptor C-terminus and the tag, indicating a stabilizing effect of the thioredoxin moiety . Several affinity chromatography methods were tested for purification . NTR with the in vivo-biotinylated Bio tag was purified with the highest efficiency compared with NTR with the Strep tag or a hexa-histidine tail . Co-expression of biotin ligase improved considerably the in vivo biotinylation of the Bio tag and, therefore, the overall purification yield . Proteolysis of the NTR fusion protein was prevented by removing a protease-sensitive site discovered at the N-terminus of NTR . The ligand binding properties of the purified receptor were similar to those of the membrane-bound protein and the native receptor . The scale-up of this purification scheme, to provide sufficient protein for biophysical studies, is in progress.

J Bacteriol, 1996 Aug, 178(16), 5053 - 6
Identification of a second RcsA protein, a positive regulator of colanic acid capsular polysaccharide genes, in Escherichia coli; Dierksen KP et al.; A second form of RcsA, a positive activator of the capsular polysaccharide genes (cps), has been identified in Escherichia coli . Ferguson plot analysis suggests that the two RcsA proteins differ by size rather than by charge . Both RcsA proteins are expressed from a single rcsA gene . Detection of both RcsA proteins in delta lon cells is RcsB dependent.

J Bacteriol, 1996 Aug, 178(16), 5045 - 8
Escherichia coli DNA repair genes radA and sms are the same gene; Song Y et al.; Escherichia coli strains carrying radA100 or sms mutations were identical in their sensitivities to either methyl methanesulfonate or UV radiation treatment and in their plasmid complementation patterns for UV radiation survival . DNA sequencing analysis of the radA mutant and radA+ strains and comparison of their sequences with the published sms gene sequence showed the radA mutant to differ only by a G-to-A transition mutation, which is predicted to change a cysteine in a zinc-finger motif to tyrosine . The sms gene is concluded to be identical to the previously described radA gene.

J Bacteriol, 1996 Aug, 178(16), 5042 - 4
Single-stranded DNA-binding protein enhances the stability of CTG triplet repeats in Escherichia coli; Rosche WA et al.; The stability of CTG triplet repeats was analyzed in Escherichia coli to identify processes responsible for their genetic instability . Using a biochemical assay for stability, we show that the absence of single-stranded-DNA-binding protein leads to an increase in the frequency of large deletions within the triplet repeats.

J Bacteriol, 1996 Aug, 178(16), 4919 - 25
The role of the 5'-end untranslated region of the mRNA for CspA, the major cold-shock protein of Escherichia coli, in cold-shock adaptation; Jiang W et al.; During cellular adaptation to low temperature, Escherichia coli transiently synthesizes the major cold-shock protein CspA . It was found that adaptation to cold shock is blocked when the 143-base sequence of the 5' untranslated region (5' UTR) of the cspA mRNA is overproduced . The overproduction of this UTR at 15 degrees C caused the synthesis of not only CspA but also other cold-shock proteins such as CspB and CsdA to be no longer transient but rather prolonged . In addition, inhibition of both the synthesis of cellular proteins other than cold-shock proteins and cell growth was observed . Interestingly, when CspA was also overproduced together with the 5' UTR, normal cold-shock adaptive response was resumed without a prolonged lag period of cell growth . This indicates that the 5' UTR of the cspA mRNA and its gene product CspA play a critical role in the regulation of the expression of cold-shock genes and cold-shock adaptation . An 11-base common sequence (cold box) was found in the 5' UTRs of cspA, cspB, and csdA mRNAs . Indeed, the 25-base sequence within the 5' UTR of the cspA mRNA containing the cold-box sequence was able to prolong CspA production at 15 degrees C . We propose that a putative repressor binds to the cold-box sequence of the cold-shock mRNAs during the adaptive process and this binding in turn blocks the transcription of the cold-shock genes or destabilizes their mRNAs . CspA appears to promote either directly or indirectly the repressor function.

J Bacteriol, 1996 Aug, 178(16), 4909 - 18
Porin activity of the native and recombinant outer membrane protein Oms28 of Borrelia burgdorferi; Skare JT et al.; The outer membrane-spanning (Oms) proteins of Borrelia burgdorferi have been visualized by freeze-fracture analysis but, until recently, not further characterized . We developed a method for the isolation of B . burgdorferi outer membrane vesicles and described porin activities with single-channel conductances of 0.6 and 12.6 nS in 1 M KCI . By using both nondenaturing isoelectric focusing gel electrophoresis and fast-performance liquid chromatography separation after detergent solubilization, we found that the 0.6-nS porin activity resided in a 28-kDa protein, designated Oms28 . The oms28 gene was cloned, and its nucleotide sequence was determined . The deduced amino acid sequence of Oms28 predicted a 257-amino-acid precursor protein with a putative 24-amino-acid leader peptidase I signal sequence . Processed Oms28 yielded a mature protein with a predicted molecular mass of 25,363 Da . When overproduced in Escherichia coli, the Oms28 porin fractionated in part to the outer membrane . Sodium dodecyl sulfate-polyacrylamide gel-purified recombinant Oms28 from E . coli retained functional activity as demonstrated by an average single-channel conductance of 1.1 nS in the planar lipid bilayer assay . These findings confirmed that Oms28 is a B . burgdorferi porin, the first to be described . As such, it is potential relevance to the pathogenesis of Lyme borreliosis and to the physiology of the spirochete.

J Bacteriol, 1996 Aug, 178(16), 4870 - 6
Replication of a plasmid lacking the normal site for initiation of one strand; Becker EC et al.; The origin of replication of the plasmid R1162 contains an initiation site for the synthesis of each DNA strand . When one of these sites (oriL) is deleted, synthesis on the corresponding strand is no longer initiated efficiently in vitro by the R1162-encoded replication proteins, and the plasmid is no longer stably maintained in the cell . However, in vivo the two strands of the plasmid duplex molecule are active at a similar level as templates for DNA synthesis, and newly synthesized copies of each strand are incorporated into daughter molecules at a similar rate . No secondary, strong initiation sites on the delta oriL strand were detected in the region of the origin . The delta oriL plasmid induces the SOS response, and this is important for plasmid maintenance even in a recombination-proficient strain . Our results indicate that an SOS-induced host system can maintain an R1162 derivative lacking one of its initiation sites.

J Bacteriol, 1996 Aug, 178(16), 4847 - 53
A study of mycobacterial transcriptional apparatus: identification of novel features in promoter elements; Bashyam MD et al.; Our earlier studies on transcriptional signals of mycobacteria had revealed that (i) strong promoters occur less frequently in the slowly growing pathogen Mycobacterium tuberculosis H37Rv than in the fast-growing saprophyte M . smegmatis and (ii) mycobacterial promoters function poorly in Escherichia coli . We now present evidence that RNA polymerases of M . smegmatis, M . tuberculosis, and M . bovis BCG recognize promoter elements with comparable efficiencies . Analysis of these randomly isolated mycobacterial promoters by DNA sequencing, primer extension, and deletion experiments revealed that their -10 regions are highly similar to those of E . coli promoters, in contrast to their -35 regions, which can tolerate a greater variety of sequences, owing presumably to the presence of multiple sigma factors with different or overlapping specificities for -35 regions, as reported earlier for the Streptomyces promoters . A comparison of the -10 and -35 binding domains of MysA, HrdB, and RpoD (the principal sigma factors of M . smegmatis, Streptomyces aureofaciens, and E . coli, respectively) showed that all three sigma factors have nearly identical -10 binding domains . However, the -35 binding domains of the principal mycobacterial and streptomycete sigma factors, although nearly identical to each other, are vastly different from the corresponding region of the sigma factor of E . coli . Thus, the transcriptional signals of mycobacteria have features in common with Streptomyces promoters but differ from those of E . coli because of major differences in the -35 regions of the promoters and the corresponding binding domain in the sigma factor.

J Bacteriol, 1996 Aug, 178(16), 4830 - 8
Modulation of NifA activity by PII in Azospirillum brasilense: evidence for a regulatory role of the NifA N-terminal domain; Arsene F et al.; Azospirillum brasilense NifA, which is synthesized under all physiological conditions, exists in an active or inactive from depending on the availability of ammonia . The activity also depends on the presence of PII, as NifA is inactive in a glnB mutant . To investigate further the mechanism that regulates NifA activity, several deletions of the nifA coding sequence covering the amino-terminal domain of NifA were constructed . The ability of these truncated NifA proteins to activate the nifH promoter in the absence or presence of ammonia was assayed in A . brasilense wild-type and mutant strains . Our results suggest that the N-terminal domain is not essential for NifA activity . This domain plays an inhibitory role which prevents NifA activity in the presence of ammonia . The truncated proteins were also able to restore nif gene expression to a glnB mutant, suggesting that PII is required to activate NifA by preventing the inhibitory effect of its N-terminal domain under conditions of nitrogen fixation . Low levels of nitrogenase activity in the presence of ammonia were also observed when the truncated gene was introduced into a strain devoid of the ADP-ribosylation control of nitrogenase . We propose a model for the regulation of NifA activity in A . brasilense.

Invest Ophthalmol Vis Sci, 1996 Aug, 37(9), 1914 - 20
Gene transfer with liposomes to the intraocular tissues by different routes of administration; Masuda I et al.; PURPOSE . To determine whether a reporter gene carried by liposomes can be introduced into the ocular tissues in vivo by different routes of administration . METHODS . Three different kinds of liposomes carrying plasmid DNA with beta-galactosidase gene were applied topically to the eye or were injected into the anterior chamber, subretinal space, and vitreous of adult Wistar rats . Gene expression was detected by enzymatic color reaction using X-gal as a substrate in enucleated eyes 1 day, 1 week, and 1 month after topical application or injection . RESULTS . Topical application could transfer the gene to retinal ganglion cells . Injection into the anterior chamber delivered the gene to the basal layer of the corneal epithelium, ciliary epithelium, stroma of the ciliary body and iris, and retinal ganglion cells . Injection into the vitreous or subretinal space resulted in the expression of the gene in the ciliary epithelium, stroma of the ciliary body and iris, retinal ganglion cells, and retinal pigment epithelial cells . CONCLUSIONS . Efficient and stable transfer of the functional gene could be achieved by liposomes in the cornea, iris, ciliary body, and retina of rats . Liposomes appear to be a promising vehicle for delivering therapeutic genes in vivo to mammalian intraocular tissues.

Infect Immun, 1996 Aug, 64(8), 3416 - 8
Adhesion of K88ab to guinea pig erythrocytes: effect on membrane enzyme activities; Caloca MJ et al.; Nontoxigenic Escherichia coli strains bearing K88 fimbriae have been associated with diarrhea in piglets . We have used guinea pig erythrocytes as a model of the host cell to study the cellular alterations after adherence of purified K88ab fimbriae . Although Mg2+-dependent ATPase was inhibited (up to 61%), Na/K ATPase was not . Metabolic enzymes were not significantly affected.

Infect Immun, 1996 Aug, 64(8), 3294 - 300
Translocation of Shiga toxin across polarized intestinal cells in tissue culture; Acheson DW et al.; Escherichia coli strains producing Shiga toxins (Stx) 1 and 2 colonize the lower gastrointestinal tract in humans and are associated with gastrointestinal and systemic diseases . Stx are detectable in the feces of infected patients, and it is likely that toxin passes from the intestinal tract lumen to underlying tissues . The objective of this study was to develop an in vitro model to study the passage of Stx across intact, polarized cell monolayers . Translocation of biologically active Stx was examined in four cell lines grown on polycarbonate filters . Stx1 translocated across intestinal cell monolayers (CaCo2A and T84 cells) in an energy-requiring and saturable manner, while the monolayers maintained a high level of electrical resistance . Stx1 had no effect on electrical resistance or inulin movement across these cell lines for at least 24 h . Induction of specific Stx receptors with sodium butyrate reduced the proportion of toxin translocated across CaCo2A monolayers but had no major effect on the movement of horseradish peroxidase or {3H}inulin . We have shown that biologically active Stx1 is capable of moving across intact polarized intestinal epithelial cells without apparent cellular disruption, probably via a transcellular pathway . The data also suggest that the presence of Stx receptors on the apical surface of intestinal epithelial cells may offer some protection against the absorption of luminal Stx1.

Infect Immun, 1996 Aug, 64(8), 3236 - 43
Roles for tumor necrosis factor alpha and nitric oxide in resistance of rat alveolar macrophages to Legionella pneumophila; Skerrett SJ et al.; Legionella pneumophila is an intracellular parasite of alveolar macrophages, and recovery from legionellosis is associated with activation of alveolar macrophages to resist intracellular bacterial replication . Gamma interferon (IFN-gamma) is known to activate alveolar macrophages to suppress L . pneumophila, but the role of macrophage-derived cytokines in modulating alveolar macrophage resistance is unknown . To test the hypothesis that macrophage-derived mediators contribute to the resistance of alveolar macrophages to L . pneumophila, we incubated adherent rat alveolar macrophages with Escherichia coli lipopolysaccharide (LPS), recombinant tumor necrosis factor alpha (TNF-alpha), recombinant IFN-gamma, neutralizing anti-TNF-alpha, and/or N(G)-monomethyl-L-arginine (L-NMMA) for 6 h before challenge with L . pneumophila . Monolayers were sonically disrupted and quantitatively cultured on successive days . We also measured bioactive TNF-alpha release by infected macrophages in the presence or absence of IFN-gamma . We found that pretreatment of alveolar macrophages with LPS or, to a lesser degree, TNF-alpha, significantly inhibited intracellular replication of L . pneumophila . Both LPS and TNF-alpha acted synergistically with IFN-gamma at less than the maximally activating concentration to suppress L . pneumophila growth . The independent and coactivating effects of LPS were blocked by anti-TNF-alpha . Killing of L . pneumophila by IFN-gamma at the maximally activating concentration was inhibited by anti-TNF-alpha . The synergistic effects of TNF-alpha . or LPS in combination with IFN-gamma were inhibited by L-NMMA . Infected alveolar macrophages secreted TNF-alpha in proportion to the bacterial inoculum, and secretion of TNF-alpha was potentiated by cocultivation with IFN-gamma . These data indicate that secretion of TNF-alpha is an important autocrine defense mechanism of alveolar macrophages, serving to potentiate the activating effects of IFN-gamma through costimulation of nitric oxide synthesis.

Infect Immun, 1996 Aug, 64(8), 3218 - 23
Pathogenicity island evaluation in Escherichia coli K1 by crossing with laboratory strain K-12; Bloch CA et al.; In bacterial pathogens, strain-specific chromosomal segments often contain genes encoding strain-specific traits, and because these genes often appear to be dedicated to pathogenic interactions with eucaryotic hosts, the segments containing them may be considered so-called pathogenicity islands (G . Blum, M . Ott, A . Lischewski, A . Ritter, H . Imrich, H . Tschape, and J . Hacker, Infect . Immun . 62:606-614, 1994) . We evaluated the contribution to pathogenesis of a recently identified strain-specific chromosomal segment from an Escherichia coli K1 mammalian-newborn sepsis strain: transfer of E . coli K-12 DNA sequences near 64 min, by P1 transduction, into K1 strain RS218 resulted in an RS218-K-12 chimera that (i) contained a shortened NotIotl restriction fragment (relative to wild-type RS218) encompassing the 64-min region; (ii) lacked invasiveness in newborn rats; and (iii) grew in vitro, in both rich and minimal laboratory media, indistinguishably from strain RS218 . In addition, genomic DNA from the chimera failed to hybridize with sequences of the K1 capsule genes from strain RS218, suggesting that the chromosomal segment near 64 min which was lost contained these sequences and indeed contained K1-specific virulence genes . Transfer of K-12 sequences resulting in deletion of E . coli pathogen-specific chromosomal segments may afford a general method of detecting genes encoding virulence and/or other distinguishing traits.

Infect Immun, 1996 Aug, 64(8), 3048 - 54
Localization of a yeast-phase-specific gene product to the cell wall in Histoplasma capsulatum; Weaver CH et al.; A yeast-phase-specific gene, yps-3, has been identified in the virulent Histoplasma capsulatum strain, G217B . Although DNA sequencing of the genomic yps-3 gene from G217B failed to detect homologies with known proteins, the 5' end of a yps-3 cDNA contained a consensus signal sequence . A 519-bp fragment of the cDNA containing the translational stop codon was linker modified and inserted into the bacterial expression vector, pATH 1 . Escherichia coli extracts containing the pATH 1 vector alone expressed a major 34-kDa TrpE polypeptide following induction with indoleacrylic acid, while the pATH 1/yps-3 construct produced a predominant 54-kDa TrpE/yps-3 fusion protein . Polyclonal rabbit sera directed against G217B reacted exclusively with the 54-kDa fusion protein in Western blots (immunoblots); serum samples from three patients with acute pulmonary or disseminated histoplasmosis were also positive . To localize the yps-3 protein within G217B, a monoclonal antibody (MAb 7.1) which recognized the yps-3 portion of the fusion protein was generated . A 17.4-kDa protein was detected with MAb 7.1 in Western blots prepared from cell wall fractions of G217B; cytoplasmic fractions were unreactive . No yps-3 antigen was detected in either fraction of the Downs strain, which fails to express the yps-3 gene . MAb 7.1 also detected a 17.4-kDa antigen in ethanol-precipitated culture supernatants derived from G217B . These findings localize the yps-3 gene product to the cell wall and culture supernatants, where the protein may influence the phase transition or the maintenance of the yeast state.

Infect Immun, 1996 Aug, 64(8), 3038 - 47
Novel insights into the genetics, biochemistry, and immunocytochemistry of the 30-kilodalton major extracellular protein of Mycobacterium tuberculosis; Harth G et al.; The 30/32-kDa complex of major secretory proteins are among the most important and intensively studied proteins of Mycobacterium tuberculosis . The proteins have been demonstrated to be immunoprotective and to play a central role in the physiology of the mycobacterium . In this study, we present a series of novel insights into this key protein complex arising out of a combination of genetic, biochemical, and immunocytochemical analyses . Our genetic analyses (i) indicate that the genes are arranged as separate transcription units, (ii) demonstrate that the mature 30-kDa protein of M . tuberculosis differs from the corresponding 30-kDa proteins of two strains of Mycobacterium bovis BCG by only 1 and 5 amino acids, (iii) suggest that expression of the proteins is regulated at the transcriptional level, and (iv) map the transcriptional start site of the 30-kDa protein gene . Our biochemical analyses provide evidence that (i) the 30-kDa protein and the two 32-kDa proteins (i.e., 32A and 32B) are secreted at a ratio of approximately 3:2:1, respectively, (ii) the proteins exist as monomers, (iii) the proteins are not posttranslationally modified by the addition of carbohydrates and lipids, (iv) the 30-kDa and 32A proteins contain one disulfide bridge, and (v) high-level expression and leader peptide processing are achievable in Escherichia coli . Our immunocytochemical analyses demonstrate that the 30/32-kDa complex is expressed in human monocytes and that the proteins are localized to the phagosomal space and the mycobacterial cell wall . These analyses fill important gaps in our knowledge of this critical protein complex of M . tuberculosis and, at the same time, raise new and fundamental questions regarding regulatory mechanisms that control coordinate expression of the proteins at a fixed ratio.

Infect Immun, 1996 Aug, 64(8), 2917 - 22
Epidermal growth factor-binding protein in mycobacterium avium and mycobacterium tuberculosis: a possible role in the mechanism of infection; Bermudez LE et al.; Epidermal growth factor (EGF) is a potent mitogen for a variety of eukaryotic cells . EGF is found in a number of tissues and is prevalent in necrotic tissues and granulomata . The biological effect of EGF on mammalian cells is initiated by the binding to a specific receptor . Both Mycobacterium avium and Mycobacterium tuberculosis cause lung infections and localized or disseminated disease in both patients without AIDS and those with AIDS . Histopathologic studies show necrosis in the lung, liver, and splenic tissues of patients with disseminated mycobacterial infection . In the course of experiments to examine the effect of growth factors on macrophages, it was observed that M . avium and M . tuberculosis but not Mycobacterium smegmatis cultured in the presence of 5, 50, or 500 ng of EGF per ml grew significantly faster than mycobacteria cultured in the absence of EGF . 125I-EGF was found to bind to M . avium and M . tuberculosis, and the binding was competitively inhibited by unlabeled EGF . A receptor for EGF was identified on mycobacteria . Incubation of mycobacteria with EGF prior to infection of macrophage monolayers resulted in faster bacterial growth within macrophages compared with that of mycobacteria not incubated with EGF . EGF-binding protein was cloned and expressed in Escherichia coli, and subsequently the protein was purified and the N-terminal amino acids were sequenced . These results suggest that EGF is a growth factor for pathogenic mycobacteria in granulomatous tissues and within macrophages and might enhance growth rates of both intracellular and extracellular mycobacteria in the site of infection.

Ann Surg, 1996 Aug, 224(2), 213 - 8
Endotoxic shock after long-term resuscitation of hemorrhage/reperfusion injury decreased splanchnic blood flow and eicosanoid release; Myers SI et al.; OBJECTIVE: The authors examine the hypothesis that hemorrhage/reperfusion injury predisposes the splanchnic bed to decreased prostacyclin (PGl2) release and blood flow after subsequent endotoxin challenge . SUMMARY BACKGROUND DATA: Prostacyclin is a potent vasodilator that has been demonstrated to be an important regulator of splanchnic blood flow . Previous studies have demonstrated that during resuscitation from severe hemorrhage, there is a marked reduction in intestinal PGl2 levels, which is associated with reduced splanchnic perfusion . METHODS: Anesthetized Sprague-Dawley rats underwent hemorrhage to a mean arterial pressure of 30 mmHg for 30 minutes followed by the reinfusion of shed blood . Then the animals were maintained on total parenteral nutrition (TPN) for 10 days, after which time they received 20 mg/kg Escherichia coli endotoxin intraperitoneally . Aortic and superior mesenteric artery (SMA) blood flow was monitored with a Doppler flow probe . The splanchnic bed was excised and perfused in vitro for measurement of venous effluent eicosanoid concentrations . Controls consisted of animals that received TPN and endotoxin but did not undergo hemorrhage and resuscitation (sham) . RESULTS: Total parenteral nutrition support of sham animals followed by endotoxin challenge did not alter splanchnic eicosanoid release or blood flow . Hemorrhage/reperfusion animals supported by long-term TPN and challenged with endotoxin demonstrated a threefold decrease in splanchnic prostacyclin metabolite (6-keto-PGF1 alpha) release and a 50% decrease in SMA blood flow . CONCLUSIONS: Hemorrhage/reperfusion injury predisposes the splanchnic bed from rats sustained with long-term TPN to decreased release of PGl2 and SMA blood flow when challenged with endotoxin as a second injury.

J Histochem Cytochem, 1996 Aug, 44(8), 919 - 27
Immunohistochemical localization of mouse Clara cell 10-KD protein using antibodies raised against the recombinant protein; Ray MK et al.; To investigate the developmental regulation of the mouse Clara cell 10-KD protein (mCC10), we raised an antibody against the recombinant mCC10 protein . The coding region for the mature mCC10 protein was placed in frame with the glutathione-S-transferase gene in the pGEX2-T bacterial expression vector . The GST-mCC10 fusion protein was expressed in E . coli DH5 alpha cells . The fusion protein was purified and eluted using glutathione-Sepharose beads . The GST-mCC10 fusion protein was injected into rabbits to raise antibodies . The rabbit anti-mCC10 antibody was tested by immunoblot analysis using both purified protein as well as extracts of lung, liver, and uterus . The antibodies produced were used in immunohistochemistry and immunoelectron microscopy to detect the cellular localization of this protein in the above organs . This anti-mCC10 antibody will be useful for future investigation of the developmental biology of the lung.

Plant Physiol, 1996 Aug, 111(4), 1067 - 75
The cyanobacterial thioredoxin gene is required for both photoautotrophic and heterotrophic growth; Navarro F et al.; The gene encoding thioredoxin in the facultative heterotrophic cyanobacterium Synechocytis sp . PCC 6803 (trxA) has been cloned by heterologous hybridization using the corresponding gene trxM from the cyanobacterium Anacystis nidulans as a probe . The deduced amino acid sequence of trxA predicts a protein of relative molecular weight of 11,750 and has strong identity with its cyanobacterial counterparts and other m-type thioredoxins of photo-synthetic eukaryotes . The trxA gene has been expressed Escherichia coli as a functional protein of 12 kD and has been shown by western blot analysis to be the same size as in Synechocystis . The trxA gene is transcribed in Synechocystis as a single transcript of 450 nucleotides and accumulates to the same level under photosynthetic and heterotrophic growth conditions . The trxA gene was inactivated with a kanamycin-resistant cassette, and total segregation of the disrupted trxA gene was obtained only when the trxM gene from A . nidulans (E.D.G . Muller, B.B . Buchanan {1989} J Biol Chem 264: 4008-4014) was simultaneously expressed in Synechocytis . Our results suggest an essential role of thioredoxin not only when cells grow photosynthetically but also under heterotrophic conditions.

Biotechnol Appl Biochem, 1996 Aug, 24 ( Pt 1), 79 - 82
Production, purification and characterization of an anti-(carcinoembryonic antigen) recombinant single-chain Fv antibody fragment; Perez L et al.; Specific targeting of radioactive agents to tumour cells has been the main objective of the in vivo use of monoclonal antibodies and their fragments . In particular, specific antibodies to carcinoembryonic antigen (CEA)-expressing tumours can be used either for diagnosis or therapy, if targeting could be improved . The expression of antibody fragments in bacteria allows the preparation of engineered molecules with antigen-binding properties and a better penetration into the tumour . A specific anti-CEA single-chain Fv fragment was produced in bacteria and purified . Its binding activity has been demonstrated in ELISA, immunocytochemistry, immunohistochemistry, fluorescence-activated cell sorting and the kinetic parameters determined by the plasmon surface resonance.

Acta Chem Scand, 1996 Aug, 50(8), 688 - 96
Expression of de novo designed alpha-helical bundles; Betz SF et al.; The successful design of proteins requires careful consideration of the multiplicity of forces that stabilize their three-dimensional structures including hydrophobic interactions, hydrogen-bonding, electrostatics and weakly polar interactions . Early attempts to design proteins relied too heavily on hydrophobic interactions to provide stability, resulting in structures with dynamic properties . Addition of more specific interactions to these initial designs gives rise to proteins with more native-like properties . This manuscript describes the design of native-like three- and four-helix bundles, and their cloning and expression of these proteins.

J Bacteriol, 1996 Aug, 178(15), 4704 - 9
Regulated expression of a repressor protein: FadR activates iclR; Gui L et al.; The control of the glyoxylate bypass operon (aceBAK) of Escherichia coli is mediated by two regulatory proteins, IclMR and FadR . IclMR is a repressor protein which has previously been shown to bind to a site which overlaps the aceBAK promoter . FAR is a repressor/activator protein which participates in control of the genes of fatty acid metabolism . A sequence just upstream of the iclR promoter bears a striking resemblance to FadR binding sites found in the fatty acid metabolic genes . The in vitro binding specificity of FadR, determined by oligonucleotide selection, was in good agreement with the sequences of these sites . The ability of FadR to bind to the site associated with iclR was demonstrated by gel shift and DNase I footprint analyses . Disruption of FadR or inactivation of the FadR binding site of iclR decreased the expression of an iclR::lacZ operon fusion, indicating that FadR activates the expression of iclR . It has been reported that disruption of fadR increases the expression of aceBAK . We observed a similar increase when we inactivated the FadR binding site of an iclR+ allele . This result suggests that FadR regulates aceBAK indirectly by altering the expression of IclR.

J Bacteriol, 1996 Aug, 178(15), 4620 - 7
Defect in general priming conferred by linker region mutants of Escherichia coli dnaB; Stordal L et al.; The dnaB gene of Escherichia coli encodes a bifunctional primase accessory protein/helicase necessary for chromosomal replication . Monomers of DnaB comprise two trypsin-resistant domains connected by a 45-amino-acid linker . To investigate the role of the linker in the structure and function of DnaB, we have purified and characterized three DnaB mutant proteins having single amino acid substitutions in the linker . We find that the mutant proteins retain the two-domain structure and assemble into hexamers that may be less stable than hexamers formed by wild-type DnaB . These mutant hexamers have hydrodynamic properties slightly different from those of the wild type, suggestive of a more open structure . The mutant proteins had reduced or absent ability to stimulate primase and also exhibited slight alterations in ATPase activity compared with the wild type . We conclude that the linker region promotes primase-DnaB interaction, but this effect may be indirect . We propose a model involving repositioning of N-terminal domains to explain the properties of the mutant proteins.

J Bacteriol, 1996 Aug, 178(15), 4530 - 9
Temperature-dependent growth kinetics of Escherichia coli ML 30 in glucose-limited continuous culture; Kovarova K et al.; Detailed comparison of growth kinetics at temperatures below and above the optimal temperature was carried out with Escherichia coli ML 30 (DSM 1329) in continuous culture . The culture was grown with glucose as the sole limiting source of carbon and energy (100 mg liter(-1) in feed medium), and the resulting steady-state concentrations of glucose were measured as a function of the dilution rate at 17.4, 28.4, 37, and 40 degrees C . The experimental data could not be described by the conventional Monod equation over the entire temperature range, but an extended form of the Monod model {mu = mu(max) x (s - s(min))/(Ks + s - s(min))}, which predicts a finite substrate concentration at 0 growth rate (s(min)), provided a good fit . The two parameters mu(max) and s(min) were temperature dependent, whereas, surprisingly, fitting the model to the experimental data yielded virtually identical Ks values (approximately 33 microg liter(-1)) at all temperatures . A model that describes steady-state glucose concentrations as a function of temperature at constant growth rates is presented . In similar experiments with mixtures of glucose and galactose (1:1 mixture), the two sugars were utilized simultaneously at all temperatures examined, and their steady-state concentrations were reduced compared with to growth with either glucose or galactose alone . The results of laboratory-scale kinetic experiments are discussed with respect to the concentrations observed in natural environments.

J Bacteriol, 1996 Aug, 178(15), 4515 - 21
O2 as the regulatory signal for FNR-dependent gene regulation in Escherichia coli; Becker S et al.; With an oxystat, changes in the pattern of expression of FNR-dependent genes from Escherichia coli were studied as a function of the O2 tension (pO2) in the medium . Expression of all four tested genes was decreased by increasing O2 . However, the pO2 values that gave rise to half-maximal repression (pO(0.5)) were dependent on the particular promoter and varied between 1 and 5 millibars (1 bar = 10(5) Pa) . The pO(0.5) value for the ArcA-regulated succinate dehydrogenase genes was in the same range (pO(0.5) = 4.6 millibars) . At these pO2 values, the cytoplasm can be calculated to be well supplied with O2 by diffusion . Therefore, intracellular O2 could provide the signal to FNR, suggesting that there is no need for a signal transfer chain . Genetic inactivation of the enzymes and coenzymes of aerobic respiration had no or limited effects on the pO(0.5) of FNR-regulated genes . Thus, neither the components of aerobic respiration nor their redox state are the primary sites for O2 sensing, supporting the significance of intracellular O2 . Non-redox-active, structural O2 analogs like CO, CN-, and N3-, could not mimic the effect of O2 on FNR-regulated genes under anaerobic conditions and did not decrease the inhibitory effect of O2 under aerobic conditions.

J Bacteriol, 1996 Aug, 178(15), 4429 - 37
The integrons In0, In2, and In5 are defective transposon derivatives; Brown HJ et al.; The class 1 integrons In0, In2, and In5, found in different locations in pVS1, Tn21, and pSCH884, have closely related structures . All three integrons contain an insertion sequence, IS1326, that is a new member of the IS21 family . IS1326 has caused deletions of adjacent 3'-conserved segment and transposition module sequences, and all three integrons retain a complete copy of only one of four genes required for transposition of related transposons and are thus defective transposon derivatives . In2 contains an additional insertion sequence, IS1353, located within IS1326 . IS1353 is a member of the IS3 family and appears to have been acquired after the integron was inserted into an ancestral mercury resistance transposon to create the ancestor of Tn21 and several other transposons that are close relatives of Tn21.

J Bacteriol, 1996 Aug, 178(15), 4400 - 11
The genetic requirements for UmuDC-mediated cold sensitivity are distinct from those for SOS mutagenesis; Opperman T et al.; The umuDC operon of Escherichia coli, a member of the SOS regulon, is required for SOS mutagenesis . Following the posttranslational processing of UmuD to UmuD' by RecA-mediated cleavage, UmuD' acts in concert with UmuC, RecA, and DNA polymerase III to facilitate the process of translesion synthesis, which results in the introduction of mutations . Constitutive expression of the umuDC operon causes an inhibition of growth at 30 degrees C (cold sensitivity) . The umuDC-dependent physiological phenomenon manifested as cold-sensitive growth is shown to differ from SOS mutagenesis in two respects . Intact UmuD, the form inactive in SOS mutagenesis, confers a significantly higher degree of cold sensitivity in combination with UmUC than does UmuD' . In addition, umuDC-mediated cold sensitivity, unlike SOS mutagenesis, does not require recA function . Since the RecA protein mediates the autodigestion of UnmD to UmuD', this finding supports the conclusion that intact UmuD is capable of conferring cold sensitivity in the presence of UmuC . The degree of inhibition of growth at 30 degrees C correlates with the levels of UmuD and UmuC, which are the only two SOS-regulated proteins required to observe cold sensitivity . Analysis of the cellular morphology of strains that exhibit cold sensitivity for growth led to the finding that constitutive expression of the umuDC operon causes a novel form of sulA- and sfiC-independent filamentation at 30 degrees C . This filamentation is observed in a strain constitutively expressing the single, chromosomal copy of umuDC and can be suppressed by overexpression of the ftsQAZ operon.

J Bacteriol, 1996 Aug, 178(15), 4392 - 9
Genes encoding the pKM101 conjugal mating pore are negatively regulated by the plasmid-encoded KorA and KorB proteins; More MI et al.; The IncN plasmid pKM101 contains a group of 11 genes thought to be required for the synthesis of its conjugal pilus and mating pore . Within this region are two genes, kilA and kilB, either of which is conditionally lethal to the cell . kilA was previously shown to be allelic with traL, and we now show that kilB is allelic with traE . In the same region, genetic studies previously defined two loci, korA and korB (kor for kill override), which together prevent lethality mediated by kilA and kilB . We now identify the genes that encode KorA and KorB functions . To determine whether KorA and KorB proteins influence tra gene transcription, we constructed beta-galactosidase fusions to three promoters in this region and measured their expression in the presence of KorA, KorB, and both proteins . KorA and KorB together repressed transcription of all three promoters, while neither protein alone affected transcription . We identified all three transcriptional start sites by primer extension analysis . Two putative binding sites for these proteins, designated kor boxes, contain 26 identical nucleotides in a 29-nucleotide region . The electrophoretic mobilities (of DNA fragments containing kor boxes were retarded by cell extracts containing both KorA and KorB but were not retarded by extracts containing just KorA or just KorB . DNase I footprinting analysis of one of these promoters demonstrates that KorA and/or KorB binds to a region containing a kor box.

J Bacteriol, 1996 Aug, 178(15), 4344 - 66
Global analysis of proteins synthesized during phosphorus restriction in Escherichia coli; VanBogelen RA et al.; The pattern of proteins synthesized in Escherichia coli during steady-state growth in media with ample inorganic phosphate (Pi), upon limitation for Pi (without an alternative phosphorous compound), and during steady-state growth in media containing phosphonate (PHN) as the sole P source was examined by two-dimensional gel electrophoresis . Of 816 proteins monitored in these experiments, all those with differential synthesis rates greater than 2.0 or less than 0.5 upon phosphate limitation (P limitation) or during growth on PHN compared with their rates in the cultures with Pi were classified as belonging to the PL or PHN stimulon, respectively . The PL stimulon included 413 proteins, 208 showing induced synthesis and 205 showing repressed synthesis . The PHN stimulon was smaller: it included 257 proteins; 227 showed induced synthesis and 30 showed repressed synthesis . The overlap of the two stimulons included 137 proteins: most (118) were ones showing induced synthesis . The promoter regions of genes for several of the proteins with induced or repressed synthesis contained sequences which resembled the consensus sequence for PhoB binding . The aggregate mass of proteins responding to P limitation or growth on PHN was 30 to 40% of the cells' total mass . By comparing the proteins responding to P limitation with those responding to growth on PHN, one can speculate which proteins are likely involved in adapting cells to new P sources or in preparing cells to survive stationary phase.

J Bacteriol, 1996 Aug, 178(15), 4335 - 43
Probing the structure, function, and interactions of the Escherichia coli H-NS and StpA proteins by using dominant negative derivatives; Williams RM et al.; Twelve different dominant negative mutants of the Escherichia coli nucleoid-associated protein, H-NS, have been selected and characterized in vivo . The mutants are all severely defective in promoter repression activity in a strain lacking H-NS, and they all disrupt the repression normally exerted by H-NS at two of its target promoters . From the locations of the alterations in these mutants, which result in both large truncations and amino acid substitutions, we propose that H-NAS contains at least two distinct domains . The in vitro protein-protein cross-linking data presented in this report indicate that the proposed N-terminal domain of H-NS has a role in H-NS multimerization . StpA is a protein with known structural and functional homologies to H-NS . We have analyzed the extent of these homologies by constructing and studying StpA mutants predicted to be dominant negative . Our data indicate that the substitutions and deletions found in dominant negative H-NS have similar effects in the context of StpA . We conclude that the domain organizations and functions in StpA and H-NS are closely related . Furthermore, dominant negative H-NS can disrupt the activity of native StpA, and reciprocally, dominant negative StpA can disrupt the activity of native H-NS . We demonstrate that the N-terminal domain of H-NS can be chemically cross-linked to both full-length H-NS and StpA . We account for these observations by proposing that H-NS and StpA have the ability to form hybrid species.

Surgery, 1996 Aug, 120(2), 360 - 6
Diethylmaleate attenuates endotoxin-induced lung injury; Nathens AB et al.; BACKGROUND: Alterations in the cellular redox state play a critical role in cell signaling and cell activation, suggesting that administration of sulfhydryl-reactive agents may have important modulatory effects on the inflammatory response . We postulated that intracellular thiol depletion may attenuate the pulmonary inflammatory response after intratracheal administration of endotoxin (LPS) and that this attenuation would supersede the reduction in antioxidant defenses associated with depletion of the endogenous antioxidant, glutathione . METHODS: Sprague Dawley rats were administered diethylmaleate (6 mmol/kg intraperitoneally), a rapidly acting glutathione-depleting agent, followed by intratracheal administration of LPS . Lung injury was assessed by measuring the transpulmonary flux of 125-I albumin and expressed as a permeability index . RESULTS: Administration of diethylmaleate reduced lung glutathione levels from 1310 +/- 114 to 185 +/- 48 nmol/gm . This was associated with a reduction in the permeability index after LPS treatment (LPS, 0.22 +/- 0.03 versus LPS + diethylmaleate, 0.03 +/- 0.01) . Bronchoalveolar lavage fluid polymorphonuclear neutrophil counts were markedly reduced in animals pretreated with diethylmaleate (LPS, 90.5 +/- 24 x 10(6) versus LPS + diethylmaleate, 1.9 +/- 0.4 x 10(6)) . Peripheral blood polymorphonuclear neutrophils isolated from animals treated with diethylmaleate had equivalent chemotactic responses to n-formyl-methionyl-leucyl-phenylalanine and normal up-regulation of CD11b as determined by flow cytometry . Levels of bronchoalveolar lavage fluid tumor necrosis factor-alpha were unaffected . CONCLUSIONS: Diethylmaleate attenuates LPS-induced lung injury through a reduction in lung polymorphonuclear neutrophil sequestration . Normal peripheral blood neutrophil chemotactic responses and CD11b expression suggest that thiol depletion might mediate this effect through inhibition of endothelial cell adhesion molecule activity.

J Nucl Med, 1996 Aug, 37(8), 1392 - 7
Detecting infection and inflammation with technetium-99m-labeled Stealth liposomes; Oyen WJ et al.; The performance of 99mTc Stealth liposomes was investigated in various rat models . METHODS: Preformed polyethyleneglycol-containing liposomes with encapsulated reduced glutathione, were radiolabeled using the lipophilic 99mTc-HMPAO . The labeled liposomes were intravenously administered to rats with focal S . aureus or E . coli infection, or turpentine-induced inflammation . For comparison, Tc-99m-nanocolloid- and 99mTc-labeled nonspecific IgG were tested . In rats with Pneumocystis carinii pneumonia (PCP), Tc-99m-liposomes were directly compared to In-111 labeled nonspecific IgG . RESULTS: Technetium-99m-liposomes accumulated in the infectious and inflammatory muscle foci over 24 hr (0.59% injected dose per gram tissue (%ID/g) for S . aureus; 1.18 %ID/g for turpentine) . Abscess-to-muscle ratios increased to values as high as 24.0, 41.7 and 44.5 for the respective models at 24 hr postinjection . Technetium-99m-liposomes visualized the foci as early as 1 hr postinjection . Technetium-99m-IgG visualized S . aureus infection, but abscess-to-muscle ratios and abscess uptake at the later time points were significantly lower . Technetium-99m-nanocolloid failed to visualize any of the muscle foci . In PCP however, 99mTc-liposomes did not show preferential localization in the infection . The control agent 111In-IgG showed a significant, two-fold increase in lung uptake . CONCLUSION: Technetium-99m-Stealth liposomes preferentially accumulated in abscesses, leading to very high target-to-nontarget ratios . This property appears to be related to a process based on uptake of long-circulating particles . In a specific type of infection, i.c . PCP, 99mTc-liposomes did not accumulate in diseased lung tissue, thus mimicking the in vivo behavior of labeled leukocytes.

Crit Care Med, 1996 Aug, 24(8), 1373 - 80
Alterations of myocardial and vascular adrenergic receptor-mediated responses in Escherichia coli-induced septic shock in the rat; Boillot A et al.; OBJECTIVES: To investigate responsiveness to exogenous catecholamines in rat bacteremic shock by studying both myocardial and vascular functional parameters; to determine in the same study the relationship of these parameters with other relevant biological parameters of the adrenergic pathway, such as myocardial beta-adrenergic receptors and cyclic adenosine monophosphate (cAMP); and to indirectly approach the roles of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide . DESIGN: Experimental, comparative study . SETTING: Laboratory in a university hospital . SUBJECTS: Male Sprague-Dawley rats, weighing 270 to 320 g . INTERVENTIONS: Intravenous injection of live Escherichia coli DH5 alpha (2 x 10(10) organisms/kg) or saline (0.6 mL) and comparison of the two groups . MEASUREMENTS AND MAIN RESULTS: Mean arterial pressure and heart rate (HR) were recorded, and circulating TNF-alpha concentrations were measured, during the first 3 hrs after E . coli administration . Myocardial and vascular functional parameters were obtained, respectively, from Langendorff-perfused hearts and isolated aortic rings . Adrenergic biochemical parameters (catecholamines, density and affinity of beta-receptors, and isoproterenol-stimulated myocardial cAMP) were determined 3 hrs after E . coli injection . Mean arterial pressure decreased within 5 to 60 mins after bacteria injection and returned to basal levels in the last 2 hrs; HR was unchanged . Serum TNF-alpha concentrations peaked at 120 mins (7333 +/- 672 pg/mL) and were still increased at 3 hrs . Plasma concentrations of epinephrine and norepinephrine were significantly (p < .05) increased . Baseline values for differential left ventricular pressure and coronary flow were significantly (p < .0001, p < .001, respectively) reduced; HR remained unchanged . Isoproterenol induced a similar increase in differential left ventricular pressure and in HR . There was no decrease in the functional myocardial response to adrenergic stimulation . beta-adrenergic receptors were similar in density and in affinity in the two groups . Isoproterenol-stimulated myocardial cAMP was significantly (p < .01) reduced compared with the control group . In aortic rings, bacteria administration significantly (p < .01) shifted the dose-response curve to norepinephrine to the right, both in the presence and absence of endothelium . NG-monomethyl-L-arginine significantly increased the contractions to attain the control level: p < .001 in presence of endothelium; p < .05 in absence of endothelium . CONCLUSIONS: In ex vivo experiments, 3 hrs after E . coli injection, vascular responsiveness was sharply decreased . This impaired response was improved by inhibition of nitric oxide . The heart, nevertheless, was still able to modulate its inotropic and chronotropic response to isoproterenol, even though an impaired beta-adrenergic-receptor stimulation of cAMP was already present.

Crit Care Med, 1996 Aug, 24(8), 1345 - 51
Endotoxic shock alters distribution of blood flow within the intestinal wall; Revelly JP et al.; OBJECTIVE: To investigate whether a redistribution of blood flow from the mucosa to the muscular layer of the intestinal wall contributes to the observed increased arterial-mucosal Pco2 gradient and the decreased mucosal tonometric pH during endotoxic shock . DESIGN: A prospective, controlled, animal study . SETTING: Animal laboratory in a university medical center . SUBJECTS: Ten domestic pigs . INTERVENTIONS: Pigs were anesthetized with ketamine and pentobarbital, mechanically ventilated, hemodynamically monitored, and then challenged with Escherichia coli endotoxin (10 micrograms/ kg i.v.) . MEASUREMENTS AND MAIN RESULTS: Cardiac output, mesenteric artery blood flow, and systemic, pulmonary, and portal pressures were measured . Intestinal mucosa tonometric Pco2 and pH were determined with saline-filled balloon tonometers . The tissue blood flow to the mucosa and the muscular layer were independently measured with colored microspheres, using the arterial reference sample method . Thus, total intestinal blood flow was evaluated with respect to its transmural (mucosa vs . muscularis) and geographical (proximal jejunum, mid-small intestine, and terminal ileum) distribution . Endotoxin administration with fluid resuscitation induced a distributive shock with a decrease in intestinal mucosa tonometric pH . Under endotoxemic conditions, the mucosal flow increased in each geographical area, with the increase being larger proximally in the jejunum than distally in the ileum . The mucosal tonometric pH was found to correlate inversely with mucosal blood flow . The increase in blood flow to the mucosa was balanced by a decrease in blood flow to the muscularis, with total mesenteric flow remaining unchanged . CONCLUSIONS: Mucosal hypoperfusion does not account for the acidotic mucosal tonometric pH in endotoxic shock . The results suggest either a primary cytotoxic effect or an enhanced counter-current-mediated hypoxic insult in the apical villus . The decrease in blood flow to the muscularis may contribute to loss of intestinal wall peristaltic activity and structural wall integrity.

Arthritis Rheum, 1996 Aug, 39(8), 1308 - 12
Novel autoantibodies directed against the common tertiary configuration of transfer RNA in a patient with interstitial lung disease; Matsumura M et al.; OBJECTIVE . To identify and characterize a novel autoantibody, anti-WS, that binds total transfer RNA (tRNA) . METHODS . Serum from patient WS, who had polyarthritis, Sjogren's syndrome, Raynaud's phenomenon, and interstitial pulmonary fibrosis, was used in this study . Characteristics of anti-WS and antibody-reactive determinants of tRNA were investigated by 32P immunoprecipitation using HeLa cell RNA and deletion mutants of tRNA transcribed in vitro . RESULTS . WS serum produced nucleolar and cytoplasmic staining on indirect immunofluorescence . 32P immunoprecipitation assays demonstrated that this serum immunoprecipitated total tRNAs and 5.8S and 5S ribosomal RNAs from 32P-labeled HeLa cell extract . When deproteinized RNA was used as antigen source, total tRNAs were still precipitated by WS serum . An immunoprecipitation study, using various deletion mutants of Escherichia coli tRNA, demonstrated that both D and T psi C loops were needed for antibody binding . Substitution of nucleotide 18G with 18A of E coli tRNA(Trp), which is essential in the formation of the tertiary "L" shape of tRNA, inhibited binding by anti-WS antibodies . CONCLUSION . Anti-WS antibodies are novel autoantibodies directed against tRNAs . The antibody binding site is the common L-shaped tertiary structure conformed by the D loop and T psi C loop of tRNA, suggesting that the antibodies are induced by a conserved sequence among all species . Furthermore, these antibodies could be a marker for a newly recognized subset of connective tissue disease.

EMBO J, 1996 Aug 1, 15(15), 3993 - 4000
The small RNA, DsrA, is essential for the low temperature expression of RpoS during exponential growth in Escherichia coli; Sledjeski DD et al.; dsrA encodes a small, untranslated RNA . When over-expressed, DsrA antagonizes the H-NS-mediated silencing of numerous promoters . Cells devoid of DsrA grow normally and show little change in the expression of a number of H-NS-silenced genes . Expression of a transcriptional fusion of lacZ to dsrB, the gene next to dsrA, is significantly lower in cells carrying mutations in dsrA . All expression of beta-galactosidase from the dsrB::lacZ fusion is also dependent on the stationary phase sigma factor, RpoS . DsrA RNA was found to regulate dsrB::lacZ indirectly, by modulating RpoS synthesis . Levels of RpoS protein are substantially lower in a dsrA mutant, both in stationary and exponential phase cells . Mutations in dsrA decrease the expression of an RpoS::LacZ translational fusion, but not a transcriptional fusion, suggesting that DsrA is acting after transcription initiation . While RpoS expression is very low in exponential phase at temperatures of 30 degrees C and above, at 20 degrees C there is substantial synthesis of RpoS during exponential growth, all dependent on DsrA RNA . dsrA expression is also increased at low temperatures . These results suggest a new role for RpoS during exponential growth at low temperatures, mediated by DsrA.

EMBO J, 1996 Aug 1, 15(15), 3912 - 22
A novel role for the budding yeast RAD9 checkpoint gene in DNA damage-dependent transcription; Aboussekhra A et al.; Cells respond to DNA damage by arresting cell cycle progression and activating several DNA repair mechanisms . These responses allow damaged DNA to be repaired efficiently, thus ensuring the maintenance of genetic integrity . In the budding yeast, Saccharomyces cerevisiae, DNA damage leads both to activation of checkpoints at the G1, S and G2 phases of the cell cycle and to a transcriptional response . The G1 and G2 checkpoints have been shown previously to be under the control of the RAD9 gene . We show here that RAD9 is also required for the transcriptional response to DNA damage . Northern blot analysis demonstrated that RAD9 controls the DNA damage-specific induction of a large 'regulon' of repair, replication and recombination genes . This induction is cell-cycle independent as it was observed in asynchronous cultures and cells blocked in G1 or G2/M . RAD9-dependent induction was also observed from isolated damage responsive promoter elements in a lacZ reporter-based plasmid assay . RAD9 cells deficient in the transcriptional response were more sensitive to DNA damage than wild-type cells, even after functional substitution of checkpoints, suggesting that this activation may have an important role in DNA repair . Our findings parallel observations with the Escherichia coli SOS system and suggest the existence of an analogous eukaryotic network coordinating the cellular responses to DNA damage.

Biochim Biophys Acta, 1996 Jul 31, 1308(1), 88 - 92
Naturally occurring splicing variants of the hMSH2 gene containing nonsense codons identify possible mRNA instability motifs within the gene coding region; Marshall B et al.; We have identified certain unusually spliced cDNA species following PCR amplification of peripheral blood lymphocyte (PBL) mRNA from the hMSH2 gene . A naturally occurring transcript containing a nonsense codon due to the skipping of 5 exons was amplified from PBLs of several healthy individuals . A feature of this and another unusual splicing product was the presence of sequence motifs which bore significant similarity to mRNA instability determinants in the region immediately downstream of the stop codon . In particular, the rare tetranucleotide GAUG, previously identified in yeast as being of critical importance to the rapid degradation of nonsense-containing mRNAs was situated 23 base pairs downstream of the stop codon . Furthermore the region downstream of the stop codon was A:U rich and contained 2 copies of the AUUUA motif . As other forms of alternative splicing would not result in the same juxtaposition of stop codons and instability motifs, we suggest that the stop codons may have been deliberately introduced by the splicing process for their proximity to these destabilising motifs, and that splicing may play a role in channeling mRNAs into degradative pathways . These results are consistent with the hypothesis that nuclear factors may scan pre-mRNAs prior to splicing.

Biochim Biophys Acta, 1996 Jul 31, 1308(1), 12 - 4
Molecular cloning and expression of the gene encoding Chromatium vinosum 2{4Fe-4S} ferredoxin; Moulis JM; The gene encoding Chromatium vinosum 2{4Fe-4S} ferredoxin has been cloned and expressed in Escherichia coli . It is flanked by a gene starting with the rare codon UUG and homologous to E . coli kdtB . Chromatium vinosum ferredoxin may represent a new sub-class of iron-sulfur proteins widely distributed among diverse, including non-photosynthetic, bacteria.

Curr Genet, 1996 Jul 31, 30(2), 159 - 65
Identification of a new antifungal target site through a dual biochemical and molecular-genetics approach; Gustafson G et al.; The target site of the antifungal compound LY214352 {8-chloro-4-(2-chloro-4-fluorophenoxy) quinoline} has been identified through a dual biochemical and molecular-genetics approach . In the molecular-genetics approach, a cosmid library was prepared from an Aspergillus nidulans mutant that was resistant to LY214352 because of a dominant mutation in a single gene . A single cosmid (6A6-6) that could transform an LY214352-sensitive strain of A . nidulans to LY214352-resistance was isolated from the library by sib-selection . Restriction fragments from cosmid 6A6-6 containing the functional resistance gene were identified by transformation, and sequenced . The LY214352-resistance gene coded for a protein of 520 amino acids that had a 34% identity and a 57% similarity in a 333 amino-acid overlap to E . coli dihydroorotate dehydrogenase (DHO-DH) . The results of a series of biochemical mechanism-of-action studies initiated simultaneously with molecular-genetic experiments also suggested that DHO-DH was the target of LY214352 . Assays measuring the inhibition of DHO-DH activity by LY214352 in a wild-type strain (I50=40 ng/ml) and a highly resistant mutant (I50>100 microgram/ml) conclusively demonstrated that DHO-DH is the target site of LY214352 in A . nidulans . Several mutations in the DHO-DH (pyrE) gene that resulted in resistance to LY214352 were identified.

Biochemistry, 1996 Jul 30, 35(30), 9907 - 16
The transient kinetics of Escherichia coli chorismate synthase: substrate consumption, product formation, phosphate dissociation, and characterization of a flavin intermediate; Bornemann S et al.; Chorismate synthase is the seventh enzyme of the shikimate pathway and catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate . The reaction involves the 1,4-elimination of phosphate and the C-(6proR) hydrogen of the substrate with unusual anti stereochemistry and requires a reduced flavin cofactor . This paper describes the kinetics of the formation and decay of a flavin intermediate, EPSP consumption, chorismate and phosphate formation, and phosphate dissociation during single and multiple turnover experiments, determined using rapid reaction techniques . The kinetics of phosphate dissociation using the substrate analogues (6R)-{6-2H}EPSP and (6S)-6-fluoro-EPSP have also been determined . The observations are consistent with a nonconcerted chorismate synthase reaction . The flavin intermediate is not simply associated with the conversion of substrate to product because it forms before the substrate is consumed . The transient spectral changes must be associated primarily with events such as protonation of the reduced flavin, a charge transfer complex between reduced flavin and an aromatic amino acid, or a conformational change in the protein . This does not rule out the direct role of flavin in catalysis.

Biochemistry, 1996 Jul 30, 35(30), 9864 - 72
A fluorescence anisotropy study of DNA binding by HPV-11 E2C protein: a hierarchy of E2-binding sites; Alexander KA et al.; Association of the human papillomavirus (HPV) E2 protein with its palindromic DNA-binding site is a necessary step for transcriptional trans-activation . To study the interaction between DNA and E2, the carboxyl-terminal domain of HPV-11 E2 protein (E2C) was expressed in Escherichia coli and purified to homogeneity . The binding affinity of the recombinant E2C protein for a single palindromic DNA recognition site was determined using a 5'-fluorescein-labeled 24 base pair oligonucleotide . Competitive titrations between the fluorescein-labeled oligonucleotide and an unlabeled oligonucleotide of identical sequence yielded a native affinity of 4.5 x 10(-9)M . Sequences from the seven E2-binding sites within the HPV-11 genome were titrated to establish a hierarchy of binding site affinities . All high-affinity E2-binding sites are located within or near the HPV-11 LCR . E2-binding sites distant from the LCR appear to have low affinity for E2 . When the location and affinity of each E2-binding site are plotted in relation to a transcription map of HPV-11, it is apparent that the major RNA transcripts produced reflect the high-affinity E2-binding sites within the HPV LCR . To assess the E2C-binding contribution of specific base pairs within the oligonucleotide palindrome, additional double-stranded oligonucleotides were prepared in which the central nonpalindromic sequences were varied . While simple strand transposition of the A4.T4 center had a minimal effect upon the E2C-oligonucleotide binding affinity, replacement with TATA.ATAT or CGCG.GCGC centers substantially decreased the affinity of E2C for its binding site . Alteration of the canonical portions of the E2-binding palindrome reduced the DNA-protein binding affinity dramatically.

Biochemistry, 1996 Jul 30, 35(30), 9840 - 9
Steady-state and pre-steady-state kinetic analysis of dNTP insertion opposite 8-oxo-7,8-dihydroguanine by Escherichia coli polymerases I exo- and II exo-; Lowe LG et al.; Escherichia coli polymerases (pol) I exo-(KF-) and pol II exo- (pol II-) were used as model enzymes with a DNA primer/template complex (12/16-mer) to examine the kinetics of incorporation of dCTP and dATP at the site of an 8-oxo-7,8-dihydroguanine (8-oxoGua) residue; compared to guanine (Gua) . In steady-state assays (with DNA in excess) the rate of incorporation (kcat) was dCTP > dATP and the K(m),dATP < K(m),dCTP during incorporation opposite 8-oxoGua with both polymerases . Pre-steady-state kinetic curves (rapid-quench analysis) for the addition of C opposite 8-oxoGua or Gua by KF- and pol II- were all biphasic, with a rapid initial single-turnover burst followed by a slower multiple turnover rate, while addition of A opposite 8-oxoGua did not display burst kinetics with either enzyme . Reduced rates of incorporation of the dCTP alpha S and dATP alpha S phosphorothioate analogs suggest that the rates of incorporation of A and C opposite 8-oxoGua are limited during polymerization by the rate of phosphodiester bond formation . Neither polymerase appears to discriminate between adducted and nonadducted DNA substrate for binding . Kinetic assays performed with varying dCTP concentrations indicate that dCTP has a higher K(d) and lower k(p) (polymerization rate) for incorporation opposite 8-oxoGua compared to Gua . Furthermore, the dATP binding affinities with KF- and pol II- were approximately 10- and approximately 3-fold lower, respectively, than that of dCTP as determined in competition assays with mixtures of dCTP and dATP . Microscopic rate constants were estimated by mathematical analysis of dNTP concentration dependence curves . Both polymerases preferentially extended the A:8-oxoGua pair while extension of the C:8-oxoGua pair was greatly impaired . Based on these findings, the fidelity of KF- and pol II- during replication of 8-oxoGua depends on contributions from nucleotide binding, the rate of phosphodiester bond formation, and the ease of base pair extension.

Biochemistry, 1996 Jul 30, 35(30), 9797 - 806
Complementation of coq3 mutant yeast by mitochondrial targeting of the Escherichia coli UbiG polypeptide: evidence that UbiG catalyzes both O-methylation steps in ubiquinone biosynthesis; Hsu AY et al.; Ubiquinone functions in the mitochondrial electron transport chain . Recent evidence suggests that the reduced form of ubiquinone (ubiquinol) may also function as a lipid soluble antioxidant . The biosynthesis of ubiquinone requires two O-methylation steps . In eukaryotes, the first O-methylation step is carried out by the Coq3 polypeptide, which catalyzes the transfer of a methyl group from S-adenosylmethionine to 3,4-dihydroxy-5-polyprenylbenzoate . In Escherichia coli, 2-polyprenyl-6-hydroxyphenol is the predicted substrate; however, the corresponding O-methyltransferase has not been identified . The second O-methylation step in E . coli, the conversion of demethylubiquinone to ubiquinone, is carried out by the UbiG methyltransferase, which is 40% identical in amino acid sequence with the yeast Coq3 methyltransferase . On the basis of the chemical similarity of the first and last methyl-acceptor substrates and the high degree of amino acid sequence identity between Coq3p and UbiG, the ability of UbiG to catalyze both O-methylation steps was investigated . The current study shows that the ubiG gene is able to restore respiration in the yeast coq3 mutant, provided ubiG is modified to contain a mitochondrial leader sequence . The mitochondrial targeting of O-methyltransferase activity is an essential feature of the ability to restore respiration and hence ubiquinone biosynthesis in vivo . In vitro import assays show the mitochondrial leader sequence present on Coq3p functions to direct mitochondrial import of Coq3p in vitro and that processing to the mature form requires a membrane potential . In vitro methyltransferase assays with E . coli cell lysates and synthetically prepared farnesylated-substrate analogs indicate that UbiG methylates both the derivative of the eukaryotic intermediate, 3,4-dihydroxy-5-farnesylbenzoate, as well as that of the E . coli intermediate, 2-farnesyl-6-hydroxyphenol . The data presented indicate that the yeast Coq3 polypeptide is located in the mitochondria and that E . coli UbiG catalyzes both O-methylation steps in E . coli.

Biochemistry, 1996 Jul 30, 35(30), 9700 - 9
The plasma and cytoplasmic forms of human gelsolin differ in disulfide structure; Wen D et al.; Gelsolin is a widely distributed actin binding protein that regulates actin filament length . It exists in both an intracellular and an extracellular form that is derived from a single gene by alternative splicing . Both forms contain the six homologous domains that are responsible for function . Little is known regarding differences between the forms . We have used a combination of cysteine-specific modification with 4-vinylpyridine, HPLC peptide mapping methods, and mass spectrometry to analyze the disulfide structures of human plasma and cytoplasmic gelsolin . Of the five Cys residues in the human gelsolin sequence, all were present in the free thiol form in human cytoplasmic gelsolin, while only three of them were free thiols in the human plasma form . Cys residues 188 and 201 in domain 2 of plasma gelsolin were disulfide linked . Recombinant human plasma gelsolin that had been expressed intracellularly in Escherichia coli and as a secreted protein from Cos green monkey cells was also investigated . The E . coli product lacked the disulfide but could be converted to the plasma-like structure with mild oxidation while the mammalian product formed the correct disulfide prior to isolation . Structural differences were also detected by limited proteolysis with plasmin . The differences in proteolytic susceptibility were also due to perturbations in domain 2 . These studies demonstrate that the intracellular and extracellular gelsolins are structurally distinct and suggest that at least some of the preparations of recombinant gelsolin that are being used to study structure/function may be improperly folded . The experiments also demonstrate a general method for the location of disulfide bonds in proteins.

Biochemistry, 1996 Jul 30, 35(30), 9661 - 6
Backbone dynamics of the C-terminal domain of Escherichia coli topoisomerase I in the absence and presence of single-stranded DNA; Yu L et al.; The backbone dynamics of the C-terminal DNA-binding domain of Escherichia coli topoisomerase I has been characterized in the absence and presence of single-stranded DNA by NMR spectroscopy . 15N spin-lattice relaxation times (T1), spin-spin relaxation times (T2), and heteronuclear NOEs were determined for the uniformly 15N-labeled protein . These data were analyzed by using the model-free formalism to derive the model-free parameters (S2, tau e, and R(ex)) for each backbone N-H bond vector and the overall molecular rotational correlation time (tau m)., The molecular rotational correlation time tau m was determined to be 7.49 +/- 0.36 ns for the free and 12.7 +/- 1.07 ns for the complexed protein . Several residues were found to be much more mobile than the average, including 11 residues at the N-terminus, 2 residues at the C-terminus, and residues 25 and 31-35 which are located in a region of the protein that binds to DNA . The binding of ssDNA to the free protein causes a slight increase in the order parameters (S2) for a small number of residues and a slight decrease in the order parameters (S2) for the majority of the residues . In particular, upon binding to ssDNA, the mobility of the first alpha-helix and the two beta-sheets was slightly increased, and the mobility of a few specific residues in the loops/turns was restricted . These results differ from the previous studies on the backbone dynamics of molecular complexes in which reduced mobilities were typically observed upon ligand binding.

Biochemistry, 1996 Jul 30, 35(30), 9637 - 46
NMR analysis of site-specific ligand binding in oligomeric proteins . Dynamic studies on the interaction of riboflavin synthase with trifluoromethyl-substituted intermediates; Scheuring J et al.; The binding of small ligands to symmetrical oligomeric proteins may lead to a number of different partially ligated intermediates but should finally yield a symmetrical fully ligated enzyme/ligand complex . In the case of the trimeric protein, riboflavin synthase, some ligands form an unexpected protein/ligand complex, even in the presence of a large excess of ligand . Three different bound forms were observed by 19F NMR spectroscopy, and Scatchard-type analysis suggested binding sites of similar affinities . NOESY analysis of the kinetic network revealed that the three bound states exchange with free ligand, but not with each other, thus suggesting that the trimeric enzyme could be asymmetrical . This information permits appropriate precautions to be taken during X-ray structure analysis of riboflavin synthase, which is in progress . Quantitative analysis of the NOESY spectra yielded different rate constants for the different binding sites . For comparison, the monomeric lumazine protein was investigated as an example of a case with simple two-site exchange . For such systems, all kinetic parameters including kon and the dissociation constant can be determined from the NOESY spectrum . The data show that NMR spectroscopy can produce qualitative and quantitative information in cases of nonequivalent binding sites in oligomeric proteins if isolated NMR signals of the different forms can be observed . The technique is not limited to 19F as reporter nucleus.

Mol Gen Genet, 1996 Jul 26, 251(6), 699 - 706
Localization of nusA-suppressing amino acid substitutions in the conserved regions of the beta' subunit of Escherichia coli RNA polymerase; Ito K et al.; Escherichia coli RNA polymerase is composed of four different subunits, alpha (present in two copies), beta, beta' and sigma . Among these, the beta' polypeptide shares nine conserved regions with the largest subunits of eukaryotic RNA polymerases, but its role is poorly understood . We isolated novel mutations in a plasmid-borne copy of rpoC, which encodes beta', as dominant suppressors of two temperature-sensitive nusA alleles . All 20 suppressors of nusA11 (single missense mutation) isolated had either of two specific substitutions: Lys for Glu-402 (rpoC10) and Thr for Ala-904 (rpoC111) in the beta' subunit . In vivo and in vitro transcription assays revealed that the rpoC10 allele of beta' participates in Rho-dependent transcription termination . On the other hand, of 20 suppressors of nusA134 (deletion of C-terminal one-third) scattered at 18 distinct sites, 16 were assigned to one of six conserved regions C-I . These results suggested that the conserved domains of the beta' subunit of E . coli RNA polymerase are involved in transcript termination or interaction with termination factor(s).

Mol Gen Genet, 1996 Jul 26, 251(6), 657 - 64
Preferential replication-dependent mutagenesis in the lagging DNA strand in Escherichia coli; Iwaki T et al.; The mutation frequencies attributable to -1 frameshift or one-base substitution in the structural genes coding for resistance to chloramphenicol (Cm) and tetracycline (Tc) were followed over several cycles of DNA replication, and found to differ several-fold, depending on the orientation of the gene on the plasmid with respect to the direction of (unidirectional ColE1-type) replication . The mutation frequency was higher when the reporter gene was present in the plasmid in the same orientation as the direction of the origin, i.e., when the transcription template is the lagging daughter strand, than when the gene was inserted in the opposite orientation . This significant difference in reversion frequencies of genes with different polarities was demonstrated only for a brief period of cell growth (several cycles of replication) after induction of the dnaQ49 mutator, but was not observed when an increased number of replication cycles, was permitted, most probably due to fixation of the mutation into both strands . The mutated intermediate DNA which possesses a misaligned basepair in the Cm gene was demonstrated to be replicated into two progeny DNA molecules; one is the chloramphenicol-resistant (CmR) DNA synthesized from the template strand having the mutation and the other is the CmS DNA from the template strand without mutation . Our results suggest that replication-dependent mutagenesis may occur preferentially in the lagging strand.

Mol Gen Genet, 1996 Jul 26, 251(6), 628 - 34
Interactions among the bHLH domains of the proteins encoded by the Enhancer of split and achaete-scute gene complexes of Drosophila; Gigliani F et al.; The Enhancer of split and achaete-scute gene complexes {E(spl)-C and AS-C} encode helix-loop-helix proteins required for neurogenesis in Drosophila . Using a heterologous bacterial system, we show that (i) the bHLH domains of the proteins encoded by the two gene complexes differ in their ability to form homo- and/or heterodimers; (ii) the bHLH domains of the E(spl)-C proteins m5, m7 and m8 interact with the bHLH domains of the Ac and Sc proteins . These bHLH domains form an interaction network which may represent the molecular mechanism whereby the competent state of the proneural cells is maintained until the terminal determination to neuroblast occurs . Also, the pattern of interactions of the bHLH domains of certain proteins encoded by the two gene complexes may explain their functional redundancy.

Cell, 1996 Jul 26, 86(2), 321 - 9
Structural basis for the excision repair of alkylation-damaged DNA; Labahn J et al.; Base-excision DNA repair proteins that target alkylation damage act on a variety of seemingly dissimilar adducts, yet fail to recognize other closely related lesions . The 1.8 A crystal structure of the monofunctional DNA glycosylase AlkA (E . coli 3-methyladenine-DNA glycosylase II) reveals a large hydrophobic cleft unusually rich in aromatic residues . An Asp residue projecting into this cleft is essential for catalysis, and it governs binding specificity for mechanism-based inhibitors . We propose that AlkA recognizes electron-deficient methylated bases through pi-donor/acceptor interactions involving the electron-rich aromatic cleft . Remarkably, AlkA is similar in fold and active site location to the bifunctional glycosylase/lyase endonuclease III, suggesting the two may employ fundamentally related mechanisms for base excision.

Cell, 1996 Jul 26, 86(2), 311 - 9
Three-dimensional structure of a DNA repair enzyme, 3-methyladenine DNA glycosylase II, from Escherichia coli; Yamagata Y et al.; The three-dimensional structure of Escherichia coli 3-methyladenine DNA glycosylase II, which removes numerous alkylated bases from DNA, was solved at 2.3 A resolution . The enzyme consists of three domains: one alpha + beta fold domain with a similarity to one-half of the eukaryotic TATA box-binding protein, and two all alpha-helical domains similar to those of Escherichia coli endonuclease III with combined N-glycosylase/abasic lyase activity . Mutagenesis and model-building studies suggest that the active site is located in a cleft between the two helical domains and that the enzyme flips the target base out of the DNA duplex into the active-site cleft . The structure of the active site implies broad substrate specificity and simple N-glycosylase activity.

Cell, 1996 Jul 26, 86(2), 233 - 42
Alpha L beta 2 integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1, a cytoplasmic regulatory molecule; Kolanus W et al.; The avidity of integrin adhesion receptors for extracellular ligands is subject to dynamic regulation by intracellular programs that have yet to be elucidated . We describe here a protein, cytohesin-1, which specifically interacts with the intracellular portion of the integrin beta 2 chain (CD18) . The molecule shows homology to the yeast SEC7 gene product and bears a pleckstrin homology (PH) domain . Overexpression of either the full-length cytohesin-1 or the SEC7 domain induces beta 2 integrin-dependent binding of Jurkat cells to ICAM-1, whereas expression of the isolated cytohesin-1 PH domain inhibits T cell receptor-stimulated adhesion . Similar inhibition is not exhibited by PH domains taken from other proteins, showing that the interaction is specific and that individual PH domains are capable of discriminating between alternative targets.

Biochem Pharmacol, 1996 Jul 26, 52(2), 281 - 8
Structural studies of a human pi class glutathione S-transferase . Photoaffinity labeling of the active site and target size analysis; Whalen R et al.; The glutathione S-transferases (GSTs; EC 2.5.1.18) are a family of dimeric proteins that catalyze reactions between glutathione (GSH) and various electrophiles . A partial cDNA for human GST pi was obtained and the open reading frame completed . The completed cDNA was cloned, and GST pi protein was expressed in bacteria . Cloned enzyme was purified and had the same kinetic constants, molecular mass, pI value, and N-terminal sequence as placental GST pi except that some of the polypeptides had N-terminal methionines . A radiolabeled azido derivative of GSH, S-(p-azidophenacyl)-{3H}glutathione, was used to photoaffinity-label the active site of the cloned enzyme . Labeled enzyme did not bind to a GSH-agarose affinity column . Labeling was prevented in the presence of S-hexylglutathione, and noncovalently-bound azido affinity label was a competitive inhibitor towards 1-chloro-2,4-dinitrobenzene and GSH . These results suggest that the azido label was binding at the active site of the enzyme . Photoaffinity-labeled enzyme was trypsinized, and two labeled peptides were purified and sequenced . One peptide corresponded to residues 183-188, whereas the other corresponded to residues 183-186 . These residues appear to form part of the hydrophobic (H-site) binding region of human GST pi that has not been shown previously . Cloned enzyme was subjected to radiation inactivation to assess the importance of subunit interactions in the maintenance of catalytic activity . The target size of enzymatic activity (23 kDa) was not significantly different from that of the protein monomer (24 kDa) . Therefore, each subunit of human GST pi appears to be capable of independent catalytic activity.

J Biol Chem, 1996 Jul 26, 271(30), 17927 - 31
COOH-terminal extended recombinant amphiregulin with bioactivity comparable with naturally derived growth factor; Thompson SA et al.; The mature secreted form of the epidermal growth factor (EGF) receptor ligand amphiregulin (AR) is reported to be an 84-amino acid residue polypeptide, which is generated by proteolytic processing of a 252-amino acid precursor . This form of recombinant AR (rAR84) and two forms with COOH-terminal extensions corresponding to sequences from the AR precursor (rAR87 and rAR92) were expressed at high levels in Escherichia coli, oxidized to the correct disulfide arrangement, and purified to homogeneity . rAR84 competed poorly for binding of radiolabeled EGF to the EGF receptor and had little ability to stimulate growth of Balb/c/3T3 cells . In striking contrast, rAR87 and rAR92 possessed 42- and 20-fold greater receptor binding activity and 55- and 14-fold greater bioactivity, respectively . Furthermore, addition of the COOH-terminal four amino acids from transforming growth factor alpha to the COOH terminus of rAR84 improved the activity of rAR84 by 100- and 1000-fold, respectively, in these assays . rAR87 was found to have approximately 32% of the specific activity of natural AR from MCF-7 cells when compared in two different bioassays . These findings strongly suggest that the 84-amino acid sequence is not the correct structure of the naturally occurring secreted form of AR and that natural AR contains additional amino acid residues at the COOH-terminal end.

J Biol Chem, 1996 Jul 26, 271(30), 18277 - 84
Mutational analysis of the ligand binding site of the inositol 1,4,5-trisphosphate receptor; Yoshikawa F et al.; To define the structural determinants for inositol 1,4, 5-trisphosphate (IP3) binding of the type 1 inositol 1,4, 5-trisphosphate receptor (IP3R1), we developed a means of expressing the N-terminal 734 amino acids of IP3R1 (T734), which contain the IP3 binding region, in Escherichia coli . The T734 protein expressed in E . coli exhibited a similar binding specificity and affinity for IP3 as the native IP3R from mouse cerebellum . Deletion mutagenesis, in which T734 was serially deleted from the N terminus up to residue 215, markedly reduced IP3 binding activity . However, when deleted a little more toward the C terminus (to residues 220, 223, and 225), the binding activity was retrieved . Further N-terminal deletions over the first 228 amino acids completely abolished it again . C-terminal deletions up to residue 579 did not affect the binding activity, whereas those up to residue 568 completely abolished it . In addition, the expressed 356-amino acid polypeptide (residues 224-579) exhibited specific binding activity . Taken together, residues 226-578 were sufficient and close enough to the minimum region for the specific IP3 binding, and thus formed an IP3 binding "core." Site-directed mutagenesis was performed on 41 basic Arg and Lys residues within the N-terminal 650 amino acids of T734 . We showed that single amino acid substitutions for 10 residues, which were widely distributed within the binding core and conserved among all members of the IP3R family, significantly reduced the binding activity . Among them, three (Arg-265, Lys-508, and Arg-511) were critical for the specific binding, and Arg-568 was implicated in the binding specificity for various inositol phosphates . We suggest that some of these 10 residues form a basic pocket that interacts with the negatively charged phosphate groups of IP3.

J Biol Chem, 1996 Jul 26, 271(30), 17986 - 9
Conformational changes in the Escherichia coli ATP synthase (ECF1F0) monitored by nucleotide-dependent differences in the reactivity of Cys-87 of the gamma subunit in the mutant betaGlu-381 --> Ala; Feng Z et al.; Cys-87, one of two intrinsic cysteines of the gamma subunit of the Escherichia coli ATP synthase (ECF1F0), is in a short segment of this subunit that binds to the bottom domain of a beta subunit close to a glutamate (Glu-381) . Cys-87 was unreactive to maleimides under all conditions in wild-type ECF1 and ECF1F0 but became reactive when Glu-381 of beta was replaced by a cysteine or alanine . The reactivity of Cys-87 with maleimides was nucleotide-dependent, occurring with ATP or ADP + EDTA in catalytic sites, in the presence of AMP.PNP + Mg2+ but not with ADP + Mg2+ bound, whether Pi was present or not, and not when nucleotide binding sites were empty . Binding of N-ethylmaleimide had no effect, whereas 7-diethyl-amino-3-(4'-maleimidylphenyl)-4-methylcoumarin increased the ATPase activity of ECF1 more than 2-fold by reaction with Cys-87 . In ECF1F0, these reagents inhibited activity . The nucleotide dependence of the reaction of Cys-87 of the gamma subunit depended on the presence of the epsilon subunit . In epsilon subunit-free ECF1, maleimides reacted with Cys-87 under all nucleotide conditions, including when catalytic sites were empty . These results are discussed in terms of nucleotide-dependent movements of the gamma subunit during functioning of the F1F0-type ATPase.

J Biol Chem, 1996 Jul 26, 271(30), 17881 - 9
Maltose-binding protein containing an interdomain disulfide bridge confers a dominant-negative phenotype for transport and chemotaxis; Zhang Y et al.; Bacterial substrate-binding proteins exist in an equilibrium among four forms: open/substrate-free, open/substrate-bound, closed/substrate-free, and closed/substrate-bound . Ligands stabilize the closed conformation, whereas the open conformation predominates in the substrate-free species . In its closed form, the NH2-terminal and COOH-terminal domains of maltose-binding protein (MBP) are proposed to be aligned to allow residues in both domains to interact simultaneously with complementary sites on the MalF and MalG proteins of the maltodextrin uptake system or with the Tar chemotactic signal transducer . However, the initial interaction might occur with an open/substrate-bound form of the binding protein, which would then close in contact with MalFG or Tar . Ligand would help stabilize this complex . We introduced cysteines (G69C and S337C) by site-directed mutagenesis into each domain of MBP and found that they formed an interdomain disulfide cross-link that should hold the protein in a closed conformation . This mutant MBP confers a dominant-negative phenotype for growth on maltose, for maltose transport, and for maltose chemotaxis . The growth and transport defects are partially reversed when the cells are exposed to the reducing agent dithiothreitol . We conclude that the cross-linked form of MBP competes with wild-type MBP in vivo for interaction with MalFG and Tar.

J Biol Chem, 1996 Jul 26, 271(30), 17932 - 6
Modulation of the thermosensing profile of the Escherichia coli aspartate receptor tar by covalent modification of its methyl-accepting sites; Nara T et al.; The Escherichia coli aspartate receptor Tar is involved in the thermotactic response . We have studied how its thermosensing function is affected by the modification of the four methyl-accepting residues (Gln295, Glu302, Gln309, and Glu491), which play essential roles in adaptation . We found that the primary translational product of tar mediates a chemoresponse, but not a thermoresponse, and that Tar comes to function as a thermoreceptor, once Gln295 or Gln309 is deamidated . This is the first identification of a thermosensing-specific mutant form, suggesting that the methylation sites of Tar constitute at least a part of the region required for thermoreception, signaling, or both . We have also investigated the inverted thermoresponse mediated by Tar in the presence of aspartate . We found that, whereas the deamidated-and-unmethylated form functions as a warm receptor, eliciting a smooth-swimming signal upon increase of temperature, the heavily methylated form functions as a cold receptor, eliciting a smooth-swimming signal upon decrease of temperature . Thus, it is suggested that Tar exists in at least three distinct states, each of which allows it to function as a warm, cold, or null thermoreceptor, depending on the modification patterns of its methylation sites.

J Biol Chem, 1996 Jul 26, 271(30), 17845 - 51
Thermal stability of hexameric and tetrameric nucleoside diphosphate kinases . Effect of subunit interaction; Giartosio A et al.; The eukaryotic nucleoside diphosphate (NDP) kinases are hexamers, while the bacterial NDP kinases are tetramers made of small, single domain subunits . These enzymes represent an ideal model for studying the effect of subunit interaction on protein stability . The thermostability of NDP kinases of each class was studied by differential scanning calorimetry and biochemical methods . The hexameric NDP kinase from Dictyostelium discoideum displays one single, irreversible differential scanning calorimetry peak (Tm 62 degrees C) over a broad protein concentration, indicating a single step denaturation . The thermal stability of the protein was increased by ADP . The P105G substitution, which affects a loop implicated in subunit contacts, yields a protein that reversibly dissociates to folded monomers at 38 degrees C before the irreversible denaturation occurs (Tm 47 degrees C) . ADP delays the dissociation, but does not change the Tm . These data indicate a "coupling" of the quaternary structure with the tertiary structure in the wild-type, but not in the mutated protein . We describe the x-ray structure of the P105G mutant at 2.2-A resolution . It is very similar to that of the wild-type protein . Therefore, a minimal change in the structure leads to a dramatic change of protein thermostability . The NDP kinase from Escherichia coli behaves like the P105G mutant of the Dictyostelium NDP kinase . The detailed study of their thermostability is important, since biological effects of thermolabile NDP kinases have been described in several organisms.

Biochem Biophys Res Commun, 1996 Jul 25, 224(3), 638 - 44
Functional analysis of the human guanylin gene promoter; Pardigol A et al.; Guanylin (GCAP-I, guanylate cyclase activating peptide I) and uroguanylin (GCAP-II, guanylate cyclase activating peptide II) are regulatory peptides involved in the regulation of the intestinal chloride / water balance . They share significant structural homology to the E . coli enterotoxin STa, which binds to the particulate guanylyl cyclase C causing diarrhea in mammals . In this study we report the functional analysis of the guanylin / GCAP-I gene promoter region . By means of the luciferase reporter gene assay, we demonstrate a strong promoter activity in T84 cells . Especially the first 160 bp of the 5'-flanking region of the gene seem to be essential for gene induction . Our findings are the basis for further identification of important regulatory elements of the corresponding gene.

Biochim Biophys Acta, 1996 Jul 25, 1282(2), 240 - 8
Alteration of Na(+)-coupled transport in site-directed mutants of the melibiose carrier of Escherichia coli; Franco PJ et al.; Asn-58 of the Escherichia coli melibiose carrier was replaced by Ala, Leu, Ser, and Gln . Trp-54 was replaced by Leu and a double mutant Leu-54/Ala-58 was constructed using site-directed mutagenesis . Cation/sugar cotransport and sugar-induced cation uptake were studied for each mutant . The change of Asn-58 to Ala results in a nearly complete loss of Na(+)-stimulated galactoside transport as well as sugar-stimulated Na+ uptake . Substitutions of Leu, Gln, and Ser for Asn-58 were also defective in Na(+)-stimulated sugar transport . The Trp-54 to Leu mutant shows moderate sugar accumulation with cation selectivity similar to wild-type . The double mutant Leu-54/Ala-58 shows elevated H(+)-melibiose cotransport as well as reduced Na(+)-stimulated melibiose cotransport . These results suggest that Asn-58 is important for Na+ recognition.

Nature, 1996 Jul 25, 382(6589), 319 - 24
A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A; Yang XJ et al.; The adenoviral oncoprotein E1A induces progression through the cell cycle by binding to the products of the p300/CBP and retinoblastoma gene families . A new cellular p300/CBP-associated factor (P/CAF) having intrinsic histone acetylase activity has been identified that competes with E1A . Exogenous expression of P/CAF in HeLa cells inhibits cell-cycle progression and counteracts the mitogenic activity of E1A . E1A disturbs the normal cellular interaction between p300/CBP and its associated histone acetylase.

Biochemistry, 1996 Jul 23, 35(29), 9625 - 30
Histidine-450 is the catalytic residue of L-3-hydroxyacyl coenzyme A dehydrogenase associated with the large alpha-subunit of the multienzyme complex of fatty acid oxidation from Escherichia coli; He XY et al.; Multienzyme complexes of fatty acid oxidation from Escherichia coli with Gln or Ala substituting for His450 or with Ala in place of Gly322 in the large alpha-subunit have been purified and characterized . The alpha/Gly322-->Ala mutation did not significantly affect the catalytic efficiencies (kcat/k(m)) of different component enzymes except for a 6.1-fold decrease in the kcat/k(m) of L-3-hydroxyacyl-CoA dehydrogenase and a 10-fold increase in the k(m) for NADH . This observation confirms the prediction {Yang, X.-Y . H., Schulz, H., Elzinga, M., & Yang, S.-Y . (1991) Biochemistry 30, 6788-6795} that the E . coli dehydrogenase has an NAD-binding site at its amino-terminal domain and structurally resembles the pig heart dehydrogenase . The pH dependence of the kcat/k(m) of the E . coli dehydrogenase suggested the catalytic involvement of an amino acid residue with a pKa of 6, which is presumably a histidine residue as proposed previously on the basis of chemical modifications . Since His450 of the E . coli multifunctional protein is the only histidine conserved in all known L-3-hydroxyacyl-CoA dehydrogenases, and since its counterpart in pig heart enzyme appeared to be close to the 3-keto group of the fatty acyl moiety of the substrate, His450 was replaced by either Gln or Ala . The catalytic properties of 3-ketoacyl-CoA thiolase, enoyl-CoA hydratase, and delta 3-cis-delta 2-trans-enoyl-CoA isomerase of the alpha/His450-->Gln mutant complex were virtually unchanged except for a small decrease in the kcat values of the latter two enzymes . In contrast, the dehydrogenase of this mutant complex was almost inactive due to a greater than 3000-fold decrease in its kcat and a 6-fold increase in the k(m) for NADH . The alpha/His450-->Ala mutant complex showed similar catalytic behaviors . Taken together, several lines of evidence lead to the conclusion that His450 is the catalytic residue of L-3-hydroxyacyl-CoA dehydrogenase of the E . coli multifunctional fatty acid oxidation protein.

Biochemistry, 1996 Jul 23, 35(29), 9610 - 6
Evidence for the interaction of avian 3-hydroxy-3-methylglutaryl-CoA synthase histidine 264 with acetoacetyl-CoA; Misra I et al.; Previous work on HMG-CoA synthase has implied the presence of a reactive active site histidine, prompting our examination of the possible function of invariant histidine residues by site-directed mutagenesis . Mutations encoding H197N, H264N/A, and H436N HMG-CoA synthases were constructed, and the mutant enzymes were overexpressed in Escherichia coli BL21(DE3) . Kinetic characterization of the isolated synthase variants indicates that, while H197N and H436N enzymes behave similarly to wild-type synthase, H264N and H264A synthases exhibit significant differences . Although the k(m) for acetyl-CoA is not substantially altered, H264N/A synthases catalyze production of HMG-CoA at a diminished (approximately 25-fold slower) rate . In contrast, H264N/A synthases can efficiently catalyze the acetyl-CoA hydrolysis partial reaction exhibiting a k(m) for acetyl-CoA that, again, approximates the value obtained with the wild-type enzyme . These mutants also retain the ability to form significant levels of the acetyl-S-enzyme reaction intermediate . The functional catalysis of partial reactions argues that the H264 mutant proteins retain substantial structural integrity . In this context, it appears significant that the H264N/A synthases exhibit a approximately 100-fold increase in the k(m) for acetoacetyl-CoA . In order to test whether the two orders of magnitude effect may be largely attributed to a decreased affinity of acetoacetyl-CoA for these enzymes and, more specifically, whether H264 interacts with the carbonyl oxygen of acetoacetyl-CoA's thioester, turnover of S-(3-oxobutyl)-CoA, a thioether analog of acetoacetyl-CoA, was investigated . This alternative substrate, in which a methylene group replaces the thioester carbonyl, is utilized by wild-type synthase with an apparent Vmax that is approximately 100-fold lower and an apparent k(m) that is 25-fold higher than the values obtained using the physiological substrate, acetoacetyl-CoA . H264A synthase also catalyzes the turnover of S-(3-oxobutyl)-CoA; the diminution in rate supported by the alternative substrate is comparable in magnitude to the effect observed for wild-type enzyme . In contrast, H264A exhibits comparable apparent k(m) values for S-(3-oxobutyl)-CoA and acetoacetyl-CoA . Thus, unlike wild-type synthase, there is no penalty in terms of efficiency of H264A saturation when the alternative thioether substrate replaces the physiological substrate . These data suggest that the imidazole of H264 in avian enzyme may play a role in anchoring the second substrate, acetoacetyl-CoA, by interacting with the carbonyl oxygen of the thioester functionality.

Biochemistry, 1996 Jul 23, 35(29), 9594 - 602
Sequence preference of 7,12-dimethylbenz{a}anthracene-syn-diol epoxide-DNA binding in the mouse H-ras gene detected by UvrABC nucleases; Chen JX et al.; We have found that 7,12-dimethylbenz{a}anthracene-syn-diol epoxide (syn-DMBADE)-modified DNA fragments are sensitive to UvrABC incision . The incisions occur mainly seven bases 5' and four bases 3' of a syn-DMBADE-modified adenine or guanine residue . The kinetics of UvrABC incision at different sequences in a DNA fragment are the same, and the extent of UvrABC incision is proportional to the syn-DMBADE concentration . On the basis of these results, we have concluded that UvrABC incision on syn-DMBADE-DNA adducts is independent of DNA sequence and is quantitative . Using the UvrABC incision method, we have analyzed the syn-DMBADE-DNA binding spectrum in several defined DNA fragments, including the first two exons of the mouse H-ras gene . We have found that both guanine and adenine residues in codons 12, 13, and 61 of the H-ras gene are strong syn-DMBADE binding sites . These results suggest that the initial binding of DMBADE may greatly contribute to the frequency of H-ras mutations . Results from dinucleotide binding analysis indicate that the 5'-nearest neighbor displays a greater effect on syn-DMBADE-DNA binding than the 3'-nearest neighbor.

Biochemistry, 1996 Jul 23, 35(29), 9505 - 12
Three-dimensional solution structure and 13C nuclear magnetic resonance assignments of the colicin E9 immunity protein Im9; Osborne MJ et al.; The 86-amino acid colicin E9 immunity protein (Im9), which inhibits the DNase activity of colicin E9, has been overexpressed in Escherichia coli and isotopically enriched with 15N and 13C . Using the 3D CBCANH and CBCA(CO)NH experiments, we have almost completely assigned the backbone 13C resonances and extended previously reported 15N/1H backbone assignments {Osborne et al . (1994), Biochemistry 33, 12347-12355} . Side chain assignments for almost all residues were made using the 3D 13C HCCH-TOCSY experiment allied to previous 1H assignments . Sixty solution structures of Im9 were determined using the DIANA program on the basis of 1210 distance restraints and 56 dihedral angle restraints . The 30 lowest-energy structures were then subjected to a slow-cooling simulated annealing protocol using XPLOR and the 21 lowest-energy structures, satisfying the geometric restraints chosen for further analysis . The Im9 structure is well-defined except for the termini and two solvent-exposed loops between residues 28-32 and 57-64 . The average RMSD about the average structure of residues 4-84 was 0.94 A for all heavy atoms and 0.53 A for backbone C alpha, C = O, and N atoms . The Im9 fold is novel and can be considered a distorted antiparallel four-helix bundle, in which the third helix is rather short, being terminated close to its N-terminal end by a proline at its C-terminus . The structure fits in well with available kinetic and biochemical data concerning the interaction between Im9 and its target DNase . Important residues of Im9 that govern specificity are located on the molecular surface in a region rich in negatively charged groups, consistent with the proposed electrostatically steered association {Wallis et al . (1995a), Biochemistry 34, 13743-13750}.

Biochemistry, 1996 Jul 23, 35(29), 9488 - 95
Human ferredoxin: overproduction in Escherichia coli, reconstitution in vitro, and spectroscopic studies of iron-sulfur cluster ligand cysteine-to-serine mutants; Xia B et al.; Human ferredoxin, the human equivalent of bovine adrenodoxin, is a small iron-sulfur protein with one {2Fe-2S} cluster . It functions, as do other vertebrate ferredoxins, to transfer electrons during the processes of steroid hormone synthesis . A DNA fragment encoding the mature form of human ferredoxin was cloned into an expression vector under control of the T7 RNA polymerase/promoter system . The protein was overproduced in Escherichia coli, and the {2Fe-2S} cluster was incorporated into the protein by in vitro reconstitution . The overall yield was approximately 30 mg of purified, reconstituted ferredoxin per liter of culture . Four of the five cysteines in human ferredoxin are coordinated to the iron-sulfur cluster . First, the non-ligand cysteine (cysteine-95) was mutated to alanine, and then double mutants were created in which each of the other four cysteines (at positions 46, 52, 55, and 92) were mutated individually to serine . The wild-type ferredoxin and each of the five mutant proteins were studied by UV-visible spectroscopy and electron paramagnetic resonance spectroscopy . The EPR gav values of all five mutants were very similar to that of wild-type human ferredoxin . In the reduced state, three of the cysteine-to-serine mutants exhibited axial EPR spectra similar to that of wild-type, but one of the double mutants (C52S/C95A) exhibited a rhombic EPR spectrum . The UV-visible spectroscopic properties of the wild-type and the C95A mutant ferredoxins were identical, but those of the other cysteine-to-serine mutant proteins of human ferredoxin were quite different from those of the wild-type protein and each other . These results, along with those from cysteine-to-serine mutations in other ferredoxins, provide the basis for a more comprehensive theoretical and practical understanding of the features important to the ligation of {2Fe-2S} clusters, although they do not yet permit determination of which two cysteines ligate Fe(II) and which ligate Fe(III) in the reduced protein.

Biochemistry, 1996 Jul 23, 35(29), 9475 - 87
Tumor suppressor p16INK4A: structural characterization of wild-type and mutant proteins by NMR and circular dichroism; Tevelev A et al.; The tumor suppressor p16INK4A with eight N-terminal amino acids deleted (p16/delta 1-8) was expressed in Escherichia coli without any fusion artifacts and purified . The integrity of p16/delta 1-8 was confirmed by mass spectrometry, and its activity was demonstrated by in vitro cdk4 inhibition assay . Various physical methods were used to characterize the molecular and structural properties of p16/delta 1-8 . The protein was found to oligomerize in vitro, as demonstrated by gel electrophoresis, mass spectrometry, and NMR . Various approaches, including changes of concentration and pH, additions of salts, detergents, and various organic solvents, and construction of a C-terminal deletion mutant and a cysteine mutant were used to try to reduce the extent of oligomerization . Only decreasing the protein concentration was found to reduce oligomerization . The affinity between p16 molecules in vivo was demonstrated by the yeast two-hybrid system . The protein was found to be very unstable on the basis of urea- and guanidinium chloride-induced denaturation studies monitored by NMR and CD, respectively . Despite these unfavorable properties, total NMR assignments were accomplished with uniform 13C and 15N isotope labeling . All multidimensional NMR experiments were performed at a very low concentration of 0.2 mM . The secondary structure was then determined from the NMR data . The results of NMR and CD studies indicate that the protein is highly alpha-helical, and the ankyrin repeat sequences show helix-turn-helix structures . This is the first structural information obtained for the important motif of ankyrin repeats . Overall, p16/delta 1-8 appears to be conformationally flexible . In order to understand the structural basis of the functional changes for some mutants existing in tumor cells, several missense mutants of p16/delta 1-8 were constructed . Four of them were expressed at high levels and purified . The molecular and structural properties of these mutants were analyzed by CD and NMR and compared with the corresponding properties of wild-type p16/delta 1-8 . The results suggest that the functional changes in P114L and G101W are likely to be related to global conformational changes . In addition, we have demonstrated that the tendency of aggregation increases significantly by a single D84H mutation.

Biochemistry, 1996 Jul 23, 35(29), 9460 - 8
V92A mutation altered the folding propensity of chicken apocytochrome c and its interaction with phospholipids; Tong JC et al.; Chicken apocytochrome c has been shown to possess a much stronger tendency to fold spontaneously in aqueous solution than the equivalent enzyme from other species . In the present work, the amino acid that determines its folding ability was elucidated by site-directed mutagenesis . Wild-type chicken apocytochrome c and three mutants V92A, S103A, and V92A/S103A were expressed in Escherichia coli . The wild-type apoprotein and S103A exhibited the same folding property during dialysis renaturation processes as that chemically prepared from chicken cytochrome c, while those containing V92A mutation did not . Quantitative studies by 2,2,2-trifluoroethanol (TFE) and sodium perchlorate (NaClO4) titration demonstrated that the V92A mutation decreased the helix content that could be induced and confirmed that valine 92 is the major determinant of the folding propensity of chicken apocytochrome c . Furthermore, CD spectra, turbidity measurements, and a translocation assay on a model membrane system showed that the V92A mutation also drastically altered the conformation of apocytochrome c after being incorporated into lipid bilayer and decreased the aggregation of phospholipid vesicles after association of the apoprotein, thus rendering the molecule more competent for translocation across the membrane . Our results showed that a single amino acid substitution could radically alter the folding propensity of an unfolded polypeptide chain and thus influence the conformation following its insertion into phospholipid bilayer.

Biochemistry, 1996 Jul 23, 35(29), 9415 - 23
ptl-1, a Caenorhabditis elegans gene whose products are homologous to the tau microtubule-associated proteins; McDermott JB et al.; The tau microtubule-associated proteins are axonal proteins that have been implicated in axonal outgrowth, microtubule spacing, and microtubule bundling . Moreover, tau is the major structural component of the paired helical filaments present in the brains of Alzheimer's disease patients . The Caenorhabditis elegans Genome Sequencing Consortium identified a genomic sequence with homology to the repeat region of tau . PCR, Northern analyses, and cDNA sequencing were used here to identify transcripts containing the tau homology region . The gene that encodes these transcripts was named ptl-1 for protein with tau-like repeats . The ptl-1 transcript, like mammalian tau transcripts, is alternatively spliced to produce messages that encode proteins with variable numbers of repeats . The predicted ptl-1 products have strong sequence homology to tau over the repeat region and are similar to tau in several other important respects including size, amino acid content, charge distribution, predicted secondary structure, hydrophobicity, and flexibility . Both proteins contain several potential glycosylation sites and numerous phosphorylation sites . Bacterially expressed PTL-1 bound to microtubules in vitro . These results show that tau-like proteins evolved early and suggests that they may be present in many different phyla . C . elegans is a powerful system amenable to genetic, molecular, and cellular analysis in which to study the functions of this important class of proteins.

Biochemistry, 1996 Jul 23, 35(29), 9385 - 91
Mercaptide formed between the residue Cys70 and Hg2+ or Co2+ behaves as a functional positively charged side chain operative in the Arg70-->Cys mutant of the metal-tetracycline/H+ antiporter of Escherichia coli; Someya Y et al.; The bacterial tetracycline/H+ antiporter (TetA) mediates active efflux of a chelation complex of tetracycline with a divalent cation such as Mg2+, Co2+, or Mn2+ {Yamaguchi, A., Udagawa, T., & Sawai, T . (1990a) J . Biol . Chem . 265, 4809-4813} . The positive charge of Arg70 in the antiporter is important for the transport function {Yamaguchi, A., Someya, Y., & Sawai, T . (1992c) J . Biol . Chem . 267, 19155-19162} . Out of six site-directed mutants of Arg70, only the Lys70 mutant retained moderate transport activity, whereas the Ser70, Ala70, Trp70, Leu70, and Asp70 mutants had no or extremely low transport activity . In this study, we constructed the Cys70 mutant and found that the Cys70 mutant showed, unexpectedly, a significant activity comparable to that of the Lys70 mutant in the presence of Co2+ ions, whereas it showed very low activity as well as the Ala70 mutant in the presence of Mg2+ or Mn2+ ions . Hg2+, which is known to be a cysteine specific modifier but has no ability to form a complex with tetracycline, caused a dramatic increase in the Vmax value of Co(2+)-dependent tetracycline transport mediated by the Cys70 mutant without affecting the k(m) value, whereas activities of the wild-type and the Lys70 and Ala70 mutants were not affected by Hg2+, Hg2+ alone without Co2+ could not support the transport activity at all, because Hg2+ does not form a chelation complex with tetracycline . These observations suggest that a mercaptide formed between the SH group of Cys70 and Hg2+ or Co2+ works as a positively charged side chain like that of Arg or Lys . When the SH group of the Cys70 mutant was masked with modification by sulfhydryl reagents, the residual activity was no longer affected by Hg2+ . Inversely, when the Cys70 mutant was preincubated with Hg2+, it was protected from the inactivation by sulfhydryl reagents . These observations also confirm the mercaptide formation between the Cys70 and a divalent cation as a functional side chain.

Biochemistry, 1996 Jul 23, 35(29), 9335 - 48
Backbone dynamics, amide hydrogen exchange, and resonance assignments of the DNA methylphosphotriester repair domain of Escherichia coli Ada using NMR; Habazettl J et al.; The 10kDa amino-terminal fragment of Escherichia coli Ada protein (N-Ada10) repairs methyl phosphotriesters in DNA and possesses a tightly bound zinc ion . The complete resonance assignments of this protein domain have been obtained using multidimensional homonuclear and heteronuclear NMR experiments . The assignments served to study the internal mobility of this protein domain via 15N relaxation experiments . This involved the measurement of longitudinal and transverse 15N relaxation rates, as well as the amide proton solvent exchange rates . Relaxation rates in the rotating frame, R1 rho, of 15N nuclei were measured at different spin-lock field strengths, leading to the detection of two slow conformational exchange processes at Gly-25 and Gln-73 . For the latter, which is next to the active site of this protein domain, the characteristic time of this process was found to be around 60 microseconds . The other relaxation experiments unveiled some regions of fast internal motions, faster than the overall correlation time . These motions were found in the N- and C- terminal tails, in segment 33-35 which forms the turn between beta-strands S1 and S2, and residues 47-52 located in a long loop preceding strand S3 . The latter loop belongs to the potential DNA binding surface of N-Ada10 . While the structure from residue 18 to residue 26 appears not well defined in the calculated structure, the relaxation experiments do not indicate higher mobility for this region . Residues at the N-terminal portion, including the first helix, the sequentially adjacent loop, and part of the second helix, exhibit internal motions close to the time scale of the overall rotational correlation time . This appears to be related to the fact that the first helix has no hydrogen bonds or salt bridges to the rest of the protein and is stabilized only by the involvement of some of its side chains in a hydrophobic core consisting of the side chains of two phenylalanines, a tryptophan, a leucine, and a valine . The four cysteines which bind the zinc show motions on different time scales ranging from microseconds to picoseconds . Thus the motions in the immediate region around the bound zinc of the DNA methyl phosphotriester repair domain are of relatively small amplitude but take place over a wide time range . On the other hand, high mobility is found in the turn connecting S1 and S2 and in the loop preceding S3, regions of the potential DNA binding surface.

Proc Natl Acad Sci U S A, 1996 Jul 23, 93(15), 7882 - 7
Pore-forming toxins trigger shedding of receptors for interleukin 6 and lipopolysaccharide; Walev I et al.; Cleavage of membrane-associated proteins with the release of biologically active macromolecules is an emerging theme in biology . However, little is known about the nature and regulation of the involved proteases or about the physiological inducers of the shedding process . We here report that rapid and massive shedding of the interleukin 6 receptor (IL-6R) and the lipopolysaccharide receptor (CD14) occurs from primary and transfected cells attacked by two prototypes of pore-forming bacterial toxins, streptolysin O and Escherichia coli hemolysin . Shedding is not induced by an streptolysin O toxin mutant which retains cell binding capacity but lacks pore-forming activity . The toxin-dependent cleavage site of the IL-6R was mapped to a position close to, but distinct from, that observed after stimulation with phorbol myristate acetate . Soluble IL-6R that was shed from toxin-treated cells bound its ligand and induced an IL-6-specific signal in cells that primarily lacked the IL-6R . Transsignaling by soluble IL-6R and soluble CD14 is known to dramatically broaden the spectrum of host cells for IL-6 and lipopolysaccharide, and is thus an important mechanism underlying their systemic inflammatory effects . Our findings uncover a novel mechanism that can help to explain the long-range detrimental action of pore-forming toxins in the host organism.

Proc Natl Acad Sci U S A, 1996 Jul 23, 93(15), 7805 - 10
Cellular strategies for accommodating replication-hindering adducts in DNA: control by the SOS response in Escherichia coli; Koffel-Schwartz N et al.; The replication of double-stranded plasmids containing a single adduct was analyzed in vivo by means of a sequence heterology that marks the two DNA strands . The single adduct was located within the sequence heterology, making it possible to distinguish trans-lesion synthesis (TLS) events from damage avoidance events in which replication did not proceed through the lesion . When the SOS system of the host bacteria is not induced, the C8-guanine adduct formed by the carcinogen N-2-acetylaminofluorene (AAF) yields less than 1% of TLS events, showing that replication does not readily proceed through the lesion . In contrast, the deacetylated adduct N-(deoxyguanosin-8-yl)-2-aminofluorene yields approximately 70% of TLS events under both SOS-induced and uninduced conditions . These results for TLS in vivo are in good agreement with the observation that AAF blocks DNA replication in vitro, whereas aminofluorene does so only weakly . Induction of the SOS response causes an increase in TLS events through the AAF adduct (approximately 13%) . The increase in TLS is accompanied by a proportional increase in the frequency of AAF-induced frameshift mutations . However, the polymerase frameshift error rate per TLS event was essentially constant throughout the SOS response . In an SOS-induced delta umuD/C strain, both US events and mutagenesis are totally abolished even though there is no decrease in plasmid survival . Error-free replication evidently proceeds efficiently by means of the damage avoidance pathway . We conclude that SOS mutagenesis results from increased TLS rather than from an increased frameshift error rate of the polymerase.

Proc Natl Acad Sci U S A, 1996 Jul 23, 93(15), 7767 - 71
Replication infidelity during a single cycle of Ty1 retrotransposition; Gabriel A et al.; Retroviruses undergo a high frequency of genetic alterations during the process of copying their RNA genomes . However, little is known about the replication fidelity of other elements that transpose via reverse transcription of an RNA intermediate . The complete sequence of 29 independently integrated copies of the yeast retrotransposon Ty1 (173,043 nt) was determined, and the mutation rate during a single cycle of replication was calculated . The observed base substitution rate of 2.5 x 10(-5) bp per replication cycle suggests that this intracellular element can mutate as rapidly as retroviruses . The pattern and distribution of errors in the Ty1 genome is nonrandom and provides clues to potential in vivo molecular mechanisms of reverse transcriptase-mediated error generation, including heterogeneous RNase H cleavage of Ty1 RNA, addition of terminal nontemplated bases, and transient dislocation and realignment of primer-templates . Overall, analysis of errors generated during Ty1 replication underscores the utility of a genetically tractable model system for the study of reverse transcriptase fidelity.

Proc Natl Acad Sci U S A, 1996 Jul 23, 93(15), 7655 - 60
Isolation and characterization of mutant cell lines defective in transforming growth factor beta signaling; Hocevar BA et al.; To isolate and characterize effector molecules of the transforming growth factor beta (TGFbeta) signaling pathway we have used a genetic approach involving the generation of stable recessive mutants, defective in their TGFbeta signaling, which can subsequently be functionally complemented to clone the affected genes . We have generated a cell line derived from a hypoxanthine-guanine phosphoribosyltransferase negative (HPRT-) HT1080 clone that contains the selectable marker Escherichia coli guanine phosphoribosyltransferase (gpt) linked to a TGFbeta-responsive promoter . This cell line proliferates or dies in the appropriate selection medium in response to TGFbeta . We have isolated three distinct TGFbeta-unresponsive mutants following chemical mutagenesis . Somatic cell hybrids between pairs of individual TGFbeta-unresponsive clones reveal that each is in a distinct complementation group . Each mutant clone retains all three TGFbeta receptors yet fails to induce a TGFbeta-inducible luciferase reporter construct or TGFbeta-mediated plasminogen activator inhibitor-1 (PAI-1) expression . Two of the three have an attenuated TGFbeta-induced fibronectin response, whereas in the other mutant the fibronectin response is intact . These TGFbeta-unresponsive cells should allow selection and identification of signaling molecules through functional complementation.

Proc Natl Acad Sci U S A, 1996 Jul 23, 93(15), 7618 - 22
Sequence-specific recognition of cytosine C5 and adenine N6 DNA methyltransferases requires different deformations of DNA; Garcia RA et al.; DNA methyltransferases modify specific cytosines and adenines within 2-6 bp recognition sequences . We used scanning force microscopy and gel shift analysis to show that M.HhaI, a cytosine C-5 DNA methyltransferase, causes only a 2 degree bend upon binding its recognition site . Our results are consistent with prior crystallographic analysis showing that the enzyme stabilizes an extrahelical base while leaving the DNA duplex otherwise unperturbed . In contrast, similar analysis of M.EcoRI, an adenine N6 DNA methyltransferase, shows an average bend angle of approximately 52 degrees . This distortion of DNA conformation by M.EcoRI is shown to be important for sequence-specific binding.

Proc Natl Acad Sci U S A, 1996 Jul 23, 93(15), 7464 - 9
In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains; Fernandes-Alnemri T et al.; Emerging evidence suggests that an amplifiable protease cascade consisting of multiple aspartate specific cysteine proteases (ASCPs) is responsible for the apoptotic changes observed in mammalian cells undergoing programmed cell death . Here we describe the cloning of two novel ASCPs from human Jurkat T-lymphocytes . Like other ASCPs, the new proteases, named Mch4 and Mch5, are derived from single chain proenzymes . However, their putative active sites contain a QACQG pentapeptide instead of the QACRG present in ail known ASCPs . Also, their N termini contain FADD-like death effector domains, suggesting possible interaction with FADD . Expression of Mch4 in Escherichia coli produced an active protease that, like other ASCPs, was potently inhibited (Kj = 14 nM) by the tetrapeptide aldehyde DEVD-CHO . Interestingly, both Mch4 and the serine protease granzyme B cleave recombinant proCPP32 and proMch3 at a conserved IXXD-S sequence to produce the large and small subunits of the active proteases . Granzyme B also cleaves proMch4 at a homologous IXXD-A processing sequence to produce mature Mch4 . These observations suggest that CPP32 and Mch3 are targets of mature Mch4 protease in apoptotic cells . The presence of the FADD-like domains in Mch4 and Mch5 suggests a role for these proteases in the Fas-apoptotic pathway . In addition, these proteases could participate in the granzyme B apoptotic pathways.

FEBS Lett, 1996 Jul 22, 390(2), 211 - 6
A covalently bound catalytic intermediate in Escherichia coli asparaginase: crystal structure of a Thr-89-Val mutant; Palm GJ et al.; Escherichia coli asparaginase II catalyzes the hydrolysis of L-asparagine to L-aspartate via a threonine-bound acyl-enzyme intermediate . A nearly inactive mutant in which one of the active site threonines, Thr-89, was replaced by valine was constructed, expressed, and crystallized . Its structure, solved at 2.2 A resolution, shows high overall similarity to the wild-type enzyme, but an aspartyl moiety is covalently bound to Thr-12, resembling a reaction intermediate . Kinetic analysis confirms the deacylation deficiency, which is also explained on a structural basis . The previously identified oxyanion hole is described in more detail.

FEBS Lett, 1996 Jul 22, 390(2), 207 - 10
Identification and characterisation of a homologue of p64 in rat tissues; Howell S et al.; Previous work has suggested that the gene encoding p64, a component of a bovine kidney intracellular chloride channel, may be a member of a gene family . We have raised a polyclonal antibody to an E . coli fusion protein which has sequence similarity to p64 . Immunoblotting detected a protein in rat brain, kidney, liver and lung . In rat brain, the protein was enriched in cerebellar microsomal membranes . Western blot analyses of denaturing and blue native polyacrylamide gels indicated that the protein is a single non-disulphide-linked polypeptide chain with an apparent M(r) of 43 kDa that contributes to a native protein complex with an apparent M(r) of 130 kDa.

FEBS Lett, 1996 Jul 22, 390(2), 179 - 82
4-O-phosphoryl-L-threonine, a substrate of the pdxC(serC) gene product involved in vitamin B6 biosynthesis; Drewke C et al.; The Escherichia coli pdxC(serC) gene codes for a transaminase (EC 2.6.1.52) . The gene is involved in both pyridoxine (vitamin B6) and serine biosynthesis and was overexpressed as a MalE/PdxC(SerC) fusion protein . The fusion protein was purified by affinity chromatography on an amylose resin and hydrolyzed in the presence of protease factor Xa . Both the fusion protein and the PdxC(SerC) protein were characterized (K(M) value, turnover number, optimum pH) . Both enzymes used 4-O-phosphoryl-L-threonine rather than 4-hydroxy-L-threonine as a substrate indicating that the phosphorylated rather than the non-phosphorylated amino acid is involved in pyridoxine biosynthesis . Pyridoxal phosphate was shown to be the cofactor for both enzymes and therefore seems to be involved in its own biosynthesis.

FEBS Lett, 1996 Jul 22, 390(2), 142 - 4
N-(2-ferrocene-ethyl)maleimide: a new electroactive sulphydryl-specific reagent for cysteine-containing peptides and proteins; Di Gleria K et al.; We report the synthesis and application of a specific electroactive label, N-(2-ferrocene-ethyl)maleimide, which provides new redox properties to organic compounds and proteins possessing sulphydryl groups . Its reaction conditions with the cysteine-containing peptide, glutathione, and a terminal monooxygenase enzyme, cytochrome P450cam are presented . The labelled peptide and enzyme acquired reversible electrochemical properties due to the attached ferrocene moiety.

J Theor Biol, 1996 Jul 21, 181(2), 111 - 24
The relation between codon usage, base correlation and gene expression level in Escherichia coli and yeast; Li H et al.; Based on the investigation of the relation between gene expression and the usage of synonymous codons, a method of classifying and predicting the gene expression level is proposed which is called the Self-consistent Information Clustering (SCIC) . Using the modified Codon Adaption Index (CAI) values, we have accomplished the linear regression analysis on the relation between base composition, base correlation and gene expression level in Escherichia coli and yeast . The assumption of Expression-Enhancing-Network Site (EENS) is proposed, the existence of which can be demonstrated by the linear equations between gene expression and base correlations in a codon, in adjacent codons and in non-adjacent codons . The modes of base correlation of E . coli and yeast which are important to gene expression have been found and listed in this paper.

J Mol Biol, 1996 Jul 19, 260(3), 347 - 58
The mechanism of early transcription termination by Rho026; Washburn RS et al.; We previously found that nusD-type mutations in Escherichia coli transcription termination factor Rho enhance in vitro transcription termination at four points within the lambdacro gene . Here we show that the early termination points are part of one Rho-dependent termination site, tRE, with properties like those of previously characterized Rho-dependent sites lamda tR1 and trpt' . The early termination points are all RNA polymerase pause sites, and by deletion analysis and oligonucleotide blocking experiments, a common 5' Rho entry site for the early termination points (rutE) is identified . We show that both Rho026 and Rho+ can use rutE as an entry point for termination, but that Rho026 is more efficient in releasing the nascent RNA at tRE . The RNA-dependent ATPase activities of wild-type and mutant Rhos are similar, as are their abilities to bind free RNA and to use (rC)10 oligomers for ATPase activation . We therefore suggest that Rho-RNA polymerase interactions that define the site of RNA 3' end formation are altered in NusD Rho mutants . NusD Rho mutants are less dependent on, but still responsive to, the transcription termination factor NusG . However, addition of NusG to in vitro termination assays allows Rho+ to terminate more efficiently at tRE . These results suggest that NusG aids in the 3' end formation process . The decreased dependence on NusG for termination by the mutant Rhos in vitro provides an explanation for poorer lambda growth in rho(nusD) cells by interference with lamdaN-mediated antitermination at Rho-dependent sites.

Mol Gen Genet, 1996 Jul 19, 251(5), 591 - 8
Regulation of the ribA gene encoding GTP cyclohydrolase II by the soxRS locus in Escherichia coli; Koh YS et al.; We isolated a promoter that is inducible by paraquat, a superoxide-generating agent, from Escherichia coli using the promoter-probe plasmid pRS415 . Sequence analysis revealed that the promoter derives from the ribA gene encoding GTP cyclohydrolase II, which is the first enzyme in the biosynthetic pathway of riboflavin . We fused the lacZ gene with the ribA promoter to monitor the expression of the gene in the single-copy state . LacZ expression from the ribA promoter was induced about eight-fold by 200 microM paraquat . Other known superoxide generators, menadione and plumbagin, also induced the expression of beta-galactosidase in the fusion strain . On the other hand, no significant induction was observed following treatment with hydrogen peroxide, ethanol or heat shock . Induction of beta-galactosidase was significantly reduced by the introduction of a delta sox-8::cat or soxS3::Tn10 mutation into the fusion strain, indicating that the ribA gene is a member of the soxRS regulon . The transcriptional start site was determined by primer extension analysis and putative binding sites for SoxS in both orientations were identified . GTP cyclohydrolase II activity in soluble extracts of E . coli increased more than three-fold on treatment with paraquat . This increase was dependent on the soxRS locus, and reflects the increase in transcript levels . However, flavin pools did not change significantly . A possible role for ribA induction during superoxide stress is discussed.

Mol Gen Genet, 1996 Jul 19, 251(5), 573 - 9
Induction of the Escherichia coli UVM response by oxidative stress; Wang G et al.; UVM (ultraviolet modulation of mutagenesis) is a recently described recA-independent, inducible mutagenic phenomenon in which prior UV irradiation of Escherichia coli cells strongly enhances mutation fixation at a site-specific 3-N4-ethenocytosine (epsilon C) lesion borne on a transfected single-stranded M13 DNA vector . Subsequent studies demonstrated that UVM is also induced by alkylating agents, and is distinct from both the SOS response and the adaptive response to alkylation damage . Because of the increasing significance being attributed to oxidative DNA damage, it is interesting to ask whether this class of DNA damage can also induce UVM . By transfecting M13 vector DNA bearing a site-specific epsilon C lesion into cells pretreated with inducing agents, we show here that the oxidative agent H2O2 is a potent inducer of UVM, and that the induction of UVM by H2O2 does not require oxyR-regulated gene expression . UVM induction by H2O2 appears to be mediated by DNA damage, as indicated by the observation of a concomitant reduction in cellular toxicity and UVM response in OxyRc cells . Available evidence suggests that UVM represents a generalized cellular response to a broad range of chemical and physical genotoxicants, and that DNA damage constitutes the most likely signal for its induction.

J Biol Chem, 1996 Jul 19, 271(29), 16987 - 90
Overexpression of catalytic subunit p110alpha of phosphatidylinositol 3-kinase increases glucose transport activity with translocation of glucose transporters in 3T3-L1 adipocytes; Katagiri H et al.; To elucidate the mechanisms of phosphatidylinositol (PI) 3-kinase involvement in insulin-stimulated glucose transport activity, the epitope-tagged p110alpha subunit of PI 3-kinase was overexpressed in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system . Overexpression of p110alpha was confirmed by immunoblot using anti-tagged epitope antibody . p110alpha overexpression induced a 2.5-fold increase in PI 3-kinase activity associated with its regulatory subunits in the basal state, an increase exceeding that of the maximally insulin-stimulated control cells, while PI 3-kinase activity associated with phosphotyrosyl protein was only modestly elevated . Overexpression of p110alpha induced an approximately 14-fold increase in the basal glucose transport rate, which was also greater than that observed in the stimulated control . No apparent difference was observed in the cellular expression level of either GLUT1 or GLUT4 proteins between control and p110alpha-overexpressing 3T3-L1 adipocytes . Subcellular fractionation revealed translocation of glucose transporters from intracellular to plasma membranes in basal p110alpha-overexpressing cells . The translocation of GLUT4 protein to the plasma membrane was further confirmed using a membrane sheet assay . These findings indicate that an increment in PI 3-kinase activity induced by overexpression of p110alpha of PI 3-kinase stimulates glucose transport activity with translocation of glucose transporters, i.e., mimics the effect of insulin.

J Biol Chem, 1996 Jul 19, 271(29), 17199 - 204
Phosphorylation of the MADS-Box transcription factor MEF2C enhances its DNA binding activity; Molkentin JD et al.; Members of the myocyte enhancer factor-2 (MEF2) family of transcription factors activate muscle gene expression by binding an A/T-rich DNA sequence in the control regions of muscle-specific genes . There are four MEF2 factors in vertebrates, MEF2A-D, which share homology in an amino-terminal MADS domain and an adjacent region known as the MEF2 domain, that together mediate DNA binding and dimerization . We show that serine 59 located between the MADS and MEF2 domains of MEF2C is phosphorylated in vivo and can be phosphorylated in vitro by casein kinase-II (CKII) . Phosphorylation of this site enhanced the DNA binding and transcriptional activity of MEF2C by increasing its DNA binding activity 5-fold . In vivo 32P labeling experiments showed that serine 59 is the only phosphorylation site in the MADS and MEF2 domains . Mutagenesis of this serine to an aspartic acid resulted in an increase in DNA binding and transcriptional activity of MEF2C comparable to that observed when this site was phosphorylated, suggesting that phosphorylation augments DNA binding activity by introducing negative charge . This phosphorylation site, which corresponds to a CKII recognition site, is conserved in all known MEF2 factors in organisms ranging from flies to humans, consistent with its importance for the functions of MEF2C.

J Biol Chem, 1996 Jul 19, 271(29), 17485 - 90
Characterization of Trypanosoma brucei gamma-glutamylcysteine synthetase, an essential enzyme in the biosynthesis of trypanothione (diglutathionylspermidine); Lueder DV et al.; The parasitic protozoan Trypanosoma brucei maintains redox balance by synthesizing a conjugate of glutathione and spermidine termed trypanothione . The first committed step in the biosynthesis of glutathione, and thereby trypanothione, is catalyzed by the enzyme gamma-glutamylcysteine synthetase (gammaGCS) . We have cloned and sequenced the 2037-base pair gene coding for the catalytic subunit of T . brucei gammaGCS . T . brucei gammaGCS appears to be encoded by a single copy gene . A transcript of about 2.3 kilobases was observed in procyclic trypomastigotes . The deduced amino acid sequence of 679 amino acids shares 45, 41, and 36% sequence identity with mammalian, Caenorhabditis elegans, and yeast gammaGCS, respectively . The T . brucei gammaGCS gene was expressed in E . coli; the purified 77.4-kDa enzyme catalyzes the ligation of L-Glu to L-Cys with a kcat of 10 s-1, confirming that the gene encodes the functional catalytic subunit of gammaGCS . The apparent Km values measured for the three natural substrates L-Glu, L-Cys, and ATP are 0.24, 0.69, and 0.07 mM, respectively . Unlike the mammalian enzyme, L-alpha-aminobutyrate (apparent Km = 10 mM) is a poor substitute for L-Cys in the T . brucei gammaGCS-catalyzed reaction . T . brucei gammaGCS is feedback-inhibited by glutathione (apparent KI = 1.1 mM), and it is inactivated by cystamine and buthionine sulfoximine . The kinetic properties of recombinant T . brucei gammaGCS suggest that the substrate binding pocket and the mechanism of enzyme regulation differ from the mammalian enzyme, providing evidence that T . brucei gammaGCS could be a selective chemotherapeutic target for the treatment of trypanosomiasis.

J Biol Chem, 1996 Jul 19, 271(29), 17439 - 44
Characterization of a potential catalytic residue, Asp-133, in the high affinity ATP-binding site of Escherichia coli SecA, translocation ATPase; Sato K et al.; The high affinity ATP-binding site of SecA is located in its amino-terminal domain possessing amino acid sequences, the Walker A (GXXXXGKT) and B (ZZZZD) motifs, that are characteristic of a major class of nucleotide-binding sites (Walker, J . E., Saraste, M., Runswick, M . J., and Gay, N . J . (1982) EMBO J . 1, 945-951) . Recently, we proposed that proteins possessing a typical set of Walker A and B motifs contain a conserved Glu or Asp between the two motifs . This Glu or Asp acts as a "catalytic residue" that activates a water molecule for an in-line attack on the gamma-phosphate of ATP (Amano, T., Yoshida, M., Matsuo, Y., and Nishikawa, K.(1995) FEBS Lett . 359, 1-5) . In the present study, the aspartate residue at position 133 in Escherichia coli SecA, which could be the "catalytic residue," was mutated to an asparagine . The mutant SecA (SecA D133N) protein was expressed in E . coli CK4706, encoding a duplication of the secA gene, and purified to homogeneity . The in vitro protein translocation activity and membrane vesicle stimulated ATPase activity of SecA D133N were drastically reduced . Proteolytic studies indicated that the conformational changes of the mutant SecA occurring on interaction with ATP, presecretory proteins, phospholipids, and membrane vesicles, were similar to those of wild-type SecA . The mutant SecA allowed the signal peptide cleavage of proOmpA during translocation, indicating that the mutant retains the ability to bind ATP to perform the initial step of the translocation reaction . These data indicate that the carboxyl group of Asp-133 plays a role as a catalytic carboxylate, which activates a water molecule to attack gamma-phosphate of ATP, and the mutant lacking this residue cannot perform the total translocation but can still perform the initial step of the protein translocation.

J Biol Chem, 1996 Jul 19, 271(29), 17035 - 40
Ordered and sequential binding of DnaA protein to oriC, the chromosomal origin of Escherichia coli; Margulies C et al.; DnaA protein of Escherichia coli acts in initiation of chromosomal DNA replication by binding specific sequences, termed DnaA boxes in the chromosomal origin, oriC . On binding, it induces a localized unwinding to create a structure recognized by other replication proteins that act subsequently in the initiation process . In this report, we examined the binding of DnaA protein to each of the DnaA boxes in oriC . By gel mobility shift assays, DnaA protein formed at least six discrete complexes . ATP or ADP included in the reaction mixture prior to electrophoresis was required . Chemical cleavage of isolated complexes with 1,10-phenanthroline-copper revealed that DnaA protein binds in an ordered manner to the DnaA boxes in oriC . Preferential binding to one DnaA box (R4) was confirmed by demonstration that a DNA fragment containing it was bound with greater affinity than another DnaA box sequence (R1) . In vitro replication activity correlated with a complex formed at a ratio of 30 DnaA monomers/oriC in which all DnaA boxes are occupied . The last site bound is DnaA box R3 . This event may be critical in promoting initiation from oriC as it correlates with in vivo observations that binding of DnaA protein to box R3 occurs at the time of initiation of chromosomal replication, whereas other DnaA boxes are bound by DnaA protein throughout the cell cycle (Cassler, M . R., Grimwade, J . E., and Leonard, A . C.(1995) EMBO J . 14, 5833-5841).

J Biol Chem, 1996 Jul 19, 271(29), 17013 - 20
Assembly of the human neutrophil NADPH oxidase involves binding of p67phox and flavocytochrome b to a common functional domain in p47phox; De Leo FR et al.; The human neutrophil NADPH oxidase is a multi-component complex composed of membrane-bound and cytosolic proteins . During activation, cytosolic proteins p47(phox), p67(phox), Rac2, and possibly p40(phox) translocate to the plasma membrane and associate with flavocytochrome b to form the active superoxide-generating system . To further investigate the role of p67(phox) in this complex assembly process, experiments were performed to identify possible regions of interaction between p67(phox) and other NADPH oxidase proteins . Using random sequence peptide phage-display library analysis of p67(phox), we identified a novel region in p47(phox) encompassing residues 323-332 and a previously identified SH3 binding domain encompassing p47(phox) residues 361-370 as p67(phox) binding sites . Synthetic peptides mimicking p47(phox) residues 323-332 inhibited the p47(phox)-p67(phox) binding interaction in an affinity binding assay; however, peptides mimicking flanking regions were inactive . Surprisingly, this same region of p47(phox) was found previously to represent a site of binding interaction for flavocytochrome b (DeLeo, F . R., Nauseef, W . M., Jesaitis, A . J., Burritt, J . B., Clark, R . A., and Quinn, M . T.(1995) J . Biol . Chem . 270, 26246-26251), and this observation was confirmed in the present report using two different in vitro assays that were not evaluated previously . Using affinity binding assays, we also found that p67(phox) and flavocytochrome b competed for binding to p47(phox)after activation, suggesting that prior to full NADPH oxidase assembly the 323-332 region of p47(phox) is associated with p67(phox) and at some point in the activation process is transferred to flavocytochrome b . Thus, taken together our data demonstrate that both p67(phox) and flavocytochrome b utilize a common binding site in p47(phox), presumably at distinct stages during the activation process, and this p47(phox) region plays a key role in regulating NADPH oxidase assembly.

J Biol Chem, 1996 Jul 19, 271(29), 17234 - 40
Distal switch II region of Ras2p is required for interaction with guanine nucleotide exchange factor; Crechet JB et al.; The interaction of Saccharomyces cerevisiae Ras2p with the catalytic domain of the GDP/GTP exchange factors (GEFs) mouse CDC25(Mm), yeast Cdc25p, and Sdc25p was analyzed by introducing the substitution R80D/N81D into Ras2p S24N, a mutant that is shown to interfere with the Ras2p wild type (wt)-GEF interaction by forming a stable complex . The triple mutant, like Ras2p R80D/N81D, did not interfere with the action of GEF on Ras2p wt (or H-Ras p21) and was unable to form a stable complex with GEF . The GEF stimulation of the nucleotide dissociation of the triple mutant was virtually abolished and strongly decreased with the double mutant . The affinity of Ras2p S24N/R80D/N81D for GDP and GTP was decreased 3 and 4 orders of magnitude, respectively, like that of Ras2p S24N, whereas the double mutant behaved as Ras2p wt . Like Ras2p S24N and unlike Ras2p R80D/N81D, the GTP-bound triple mutant did not activate adenylyl cyclase . Thus, the triple mutant and Ras2p S24N have opposite properties toward the binding to GEF but similarly modified behaviors toward GDP, GTP, and adenylyl cyclase . This work emphasizes the determinant role of the distal switch II region of Ras2p for the interaction with GEF and the different structural background of the interaction with adenylyl cyclase.

J Biol Chem, 1996 Jul 19, 271(29), 17469 - 75
Molecular dissection of a protein SopB essential for Escherichia coli F plasmid partition; Hanai R et al.; Biochemical and genetic experiments were carried out to deduce the structural and functional domains of SopB protein involved in the equipartition of F plasmid . The protein is dimeric . Proteolytic and chemical footprinting studies support earlier genetic analyses that the binding of SopB to specific sites within the F plasmid sopC locus involves mainly the C-terminal region . In vivo, the expression of a high level of SopB protein is known to repress sopC-linked genes . This silencing activity is shown to be unaffected by the deletion of 35 N-terminal residues, but abolished when 71 or more were removed from the N terminus . An excess of SopB protein does not extend its in vitro binding outside sopC, implicating participation of a host factor(s) in SopB-mediated gene silencing . A data base search identified a number of SopB homologues, including both chromosomally encoded bacterial proteins and phage- and plasmid-encoded proteins known to be involved in partition . Sequence homology is limited to the N-terminal half, suggesting that the N-terminal regions of these proteins are conserved to interact with a conserved cellular structure(s), whereas the C-terminal regions have diverged to bind different nucleotide sequences.

J Biol Chem, 1996 Jul 19, 271(29), 17124 - 31
Cellular mechanisms for human procollagenase-3 (MMP-13) activation . Evidence that MT1-MMP (MMP-14) and gelatinase a (MMP-2) are able to generate active enzyme; Knauper V et al.; Gelatinase A and membrane-type metalloproteinase (MT1-MMP) were able to process human procollagenase-3 (Mr 60,000) to the fully active enzyme (Tyr85 N terminus; Mr 48,000) . MT1-MMP activated procollagenase-3 via a Mr 56,000 intermediate (Ile36 N terminus) to 48,000 which was the result of the cleavage of the Glu84-Tyr85 peptide bond . We have established that the activation rate of procollagenase-3 by MT1-MMP was enhanced in the presence of progelatinase A, thereby demonstrating a unique new activation cascade consisting of three members of the matrix metalloproteinase family . In addition, procollagenase-3 can be activated by plasmin, which cleaved the Lys38-Glu39 and Arg76-Cys77 peptide bonds in the propeptide domain . Autoproteolysis then resulted in the release of the rest of the propeptide domain generating Tyr85 N-terminal active collagenase-3 . However, plasmin cleaved the C-terminal domain of collagenase-3 which results in the loss of its collagenolytic activity . Concanavalin A-stimulated fibroblasts expressing MT1-MMP and fibroblast-derived plasma membranes were able to process human procollagenase-3 via a Mr 56,000 intermediate form to the final Mr 48,000 active enzyme which, by analogy with progelatinase A activation, may represent a model system for in vivo activation . Inhibition experiments using tissue inhibitor of metalloproteinases, plasminogen activator inhibitor-2, or aprotinin demonstrated that activation in the cellular model system was due to MT1-MMP/gelatinase A and excluded the participation of serine proteinases such as plasmin during procollagenase-3 activation . We have established that progelatinase A can considerably potentiate the activation rate of procollagenase-3 by crude plasma membrane preparations from concanavalin A-stimulated fibroblasts, thus confirming our results using purified progelatinase A and MT1-MMP . This new activation cascade may be significant in human breast cancer pathology, where all three enzymes have been implicated as playing important roles.

J Biotechnol, 1996 Jul 18, 48(1-2), 97 - 105
High-level expression and simple purification of recombinant human insulin-like growth factor I; Kim SO et al.; Human insulin-like growth factor I (IGF-I) was expressed in Escherichia coli as a truncated beta-galactosidase-IGF-I fusion protein . The Lac Z" gene was truncated by removal of a 490 bp fragment which encoded 163 N-terminal residues of beta-galactosidase and was connected to the IGF-I cDNA by a linker encoding hydroxylamine cleavage recognition sequence . By truncating Lac Z" gene, the overall yield and purification procedures of IGF-I from fusion protein have been improved . The fusion protein was produced in the form of insoluble inclusion bodies with isopropyl-1-thio-beta-D-galactoside (IPTG) induction . After cleavage of the fusion protein with hydroxylamine, the released IGF-I was purified to homogeneity by a cation exchange chromatography, refolding and reverse-phase high performance liquid chromatography (rp-HPLC) . The purified IGF-I was found to be indistinguishable from the native IGF-I by N-terminal amino acid sequence, SDS-polyacrylamide gel electrophoresis, and rp-HPLC and by biological activities such as thymidine uptake, protein synthesis and receptor binding . These results suggest that the expression and simple purification of recombinant human IGF-I described in this paper may be useful for large scale production of IGF-I.

J Biotechnol, 1996 Jul 18, 48(1-2), 1 - 8
Cooperativity of stabilized mRNA and enhanced translation activity in the cell-free system; Kitaoka Y et al.; Detailed analysis of cell-free translation, coupled transcription-translation in static conditions and a continuous flow system based on E . coli S30 extracts was performed . Degradation of template mRNA was the predominant trigger to terminate the protein synthesis . In a coupled system, mRNA was preserved by repeated transcription whereas the starvation of nucleotide triphosphates led to the termination of protein synthesis in less than 1 h . In the CFCF system, NTP was held at the level of initial concentration and therefore did not arrest the translation for 15 h . The accurate coupling of transcriptional rate and translational rate was also crucial to enhance the efficiency of protein synthesis.

Nature, 1996 Jul 18, 382(6588), 278 - 81
Structure of Taq ploymerase with DNA at the polymerase active site; Eom SH et al.; The DNA polymerase from Thermus aquaticus (Taq polymerase) is homologous to Escherichia coli DNA polymerase I (Pol I) and likewise has domains responsible for DNA polymerase and 5' nuclease activities . The structures to the polymerase domains of Taq polymerase and of the Klenow fragment (KF) of Pol I are almost identical, whereas the structure of a vestigial editing 3'-5' exonuclease domain of Taq polymerase that lies between the other two domains is dramatically altered, resulting in the absence of this activity in the thermostable enzyme . The structures have been solved for editing complexes between KF and single-stranded DNA and for duplex DNA with a 3' overhanging single strand, but not for a complex containing duplex DNA at the polymerase active-site . Here we present the co-crystal structure of Taq polymerase with a blunt-ended duplex DNA bound to the polymerase active-site cleft; the DNA neither bends nor goes through the large polymerase cleft, and the structural form of the bound DNA is between the B and A forms . A wide minor groove allows access to protein side chains that hydrogen-bond to the N3 of purines and the O2 of pyrimidines at the blunt-end terminus . Part of the DNA bound to the polymerase site shares a common binding site with DNA bound to the exonuclease site, but they are translated relative to each other by several angstroms along their helix axes.

Oncogene, 1996 Jul 18, 13(2), 381 - 9
Formation of Shc/Grb2- and Crk adaptor complexes containing tyrosine phosphorylated Cbl upon stimulation of the B-cell antigen receptor; Smit L et al.; B-cell antigen receptor (BCR) stimulation induces tyrosine phosphorylation of the Shc adaptor protein and its association with Grb2 . The Shc/Grb2 complex may be involved in Ras activation, since Grb2 interacts with the guanine nucleotide exchange factor Sos . We reveal here an additional complexity of the BCR-induced Shc/Grb2 complex: it contains tyrosine phosphorylated proteins of 130, 110 and 75 kDa . The 130 kDa molecule inducibly associates with Shc, while the 75 kDa protein interacts with the carboxy-terminal SH3 domain of Grb2 . The 110 kDa molecule is defined as Cbl, the product of the c-cbl oncogene, which is strongly phosphorylated on tyrosine upon BCR stimulation . Cbl constitutively interacts with the SH3 domains of Grb2, with a preference for the amino-terminal domain, and is in this way recruited to Shc upon BCR stimulation . Immunodepletion studies showed that Grb2-associated Cbl can be phosphorylated by BCR-induced tyrosine kinases independent of a Shc/Grb2 interaction . This indicates that the BCR can also couple to a Grb2 complex without the involvement of Shc . Cbl not only interacts with Grb2, but also with the adaptor protein Crk . In contrast to its constitutive interaction with Grb2, tyrosine-phosphorylated Cbl only associates with Crk after BCR stimulation . In summary, we observe that the BCR activates Shc/Grb2-, Grb2- and Crk adaptor complexes of distinct composition, which may allow selective coupling to different signal transduction cascades . Cbl participates in all three adaptor complexes and is tyrosine phosphorylated upon BCR stimulation, pointing to a central role for this molecule in the regulation of antigen receptor-induced B cell responses.

Biochim Biophys Acta, 1996 Jul 18, 1295(2), 165 - 73
Ligation of the iron in the heme-heme oxygenase complex: X-ray absorption, electronic absorption and magnetic circular dichroism studies; Hawkins BK et al.; Heme oxygenase (HO) catalyzes the first steps in the breakdown of heme to biliverdin and carbon monoxide . It is a membrane-bound protein that has been shown to exist in two isoforms, HO-1 and HO-2 . Recently, a soluble, truncated form of rat HO-1 (rHO) lacking the 23 amino-acid membrane anchor has been expressed in E . coli . Extended X-ray absorption fine structure (EXAFS) data on ferric rHO and its fluoride derivative support assignment of the axial iron ligands as oxygen and/or nitrogen donors having distances similar to ferric myoglobin . The electronic absorption and magnetic circular dichroism (MCD) spectra of the ferric and ferrous protoheme complexes of rHO as well as various ligand adducts are very similar to the corresponding spectra of myoglobin . The present study is the first investigation of the heme-heme oxygenase complex with EXAFS and MCD spectroscopy and establishes that the proximal ligand to the heme in rHO is histidine . Furthermore, the close similarity between the electronic absorption and MCD spectra of ferric rHO and myoglobin over the pH range 6 to 10 is consistent with distal heme ligation of ferric rHO as a water molecule or hydroxide ion, depending on pH . Taken together and in conjunction with the results of earlier studies, EXAFS, electronic absorption, and MCD spectroscopy solidly establish that the ligands to the heme in rHO are identical to those in myoglobin, namely, histidine/H2O at low pH and histidine/OH at high pH.

Biochim Biophys Acta, 1996 Jul 18, 1295(2), 119 - 24
A cDNA clone from Arabidopsis thaliana encoding plastidic ferredoxin:sulfite reductase; Bruhl A et al.; A cDNA with an open reading frame of 1929 bp (termed sir) was isolated from a lambda ZapII library of Arabidopsis thaliana leaf tissue . The polypeptide sequence deduced from the cDNA is homologous to the ferredoxin-dependent sulfite reductase (EC 1.8.7.1) from Synechococcus PCC7942 and distantly related to the hemoprotein subunit of Escherichia coli NADPH-dependent sulfite reductase (EC 1.8.1.2) . A molecular mass of 71.98 kDa can be predicted for a ferredoxin sulfite reductase from A . thaliana . The polypeptide consists of 642 amino acids including a transit peptide of 66 residues (6.72 kDa) that is assumed to direct the protein into the plastid . For expression and enzymatic characterization of a putative A . thaliana ferredoxin sulfite reductase, the DNA of the transit peptide was deleted by a PCR method . The truncated cDNA clone was expressed as his-tag fusion protein . The modified gene product was enzymatically inactive but specific cross-reaction with polyclonal antibodies against ferredoxin sulfite reductase from Synechococcus is seen as confirmation of its identity as higher plant ferredoxin sulfite reductase.

Biochim Biophys Acta, 1996 Jul 18, 1275(1-2), 96 - 100
ATP hydrolysis by membrane-bound Escherichia coli F0F1 causes rotation of the gamma subunit relative to the beta subunits; Zhou Y et al.; We recently demonstrated that the gamma subunit in soluble F1-ATPase from Escherichia coli rotates relative to surrounding beta subunits during catalytic turnover (Duncan et al . (1995) Proc . Natl . Acad . Sci . USA 92, 10964-10968) . Here, we extend our studies to the more physiologically relevant membrane-bound F0F1 complex . It is shown that beta D380C-F1, containing a beta-gamma intersubunit disulfide bond, can bind to F1-depleted membranes and can restore coupled membrane activities upon reduction of the disulfide . Using a dissociation/reconstitution approach with crosslinked beta D380C-F1, beta subunits containing an N-terminal Flag epitope (beta flag) were incorporated into the two non-crosslinked beta positions and the hybrid F1 was reconstituted with membrane-bound F0 . Following reduction and ATP hydrolysis, reoxidation resulted in a significant amount of crosslinking of beta flag to the gamma subunit . This demonstrates that gamma rotates within F1 during catalytic turnover by membrane-bound F0-F1 . Furthermore, the rotation of gamma is functionally coupled to F0, since preincubation with DCCD to modify F0 blocked rotation.

J Immunol Methods, 1996 Jul 17, 194(1), 13 - 25
Implications for the assay and biological activity of interleukin-4 . Results of a WHO international collaborative study; Mire-Sluis AR et al.; Five ampouled preparations of interleukin-4 (IL-4) have been evaluated by 36 laboratories in 14 countries for their suitability to serve as an international standard for this material in a joint international collaborative study for interleukin-3 (IL-3) and IL-4 . The preparations were assayed in a wide range of in vitro bioassays and immunoassays . It is clear from the study that different recombinant preparations of IL-4 can have very different biological specific activities, including those from the same source (i.e., E . coli) . In addition, immunoassay estimates of IL-4 levels did not correlate with those of bioassays, illustrating the fact that immunoassays do not necessarily measure biologically active cytokine . It is of interest that the estimates provided by the different bioassays were less variable than those produced by the immunoassays, suggesting that bioassays can be as accurate, if not more so, than immunoassays . The large reduction in the variability of estimates with the inclusion of a single reference preparation clearly illustrates the need for a single standard to assay IL-4 . On the basis of the results reported here, with the agreement of the participants of the study and with the authorisation of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) the preparation of IL-4 (88/656) was established as the international standard for interleukin-4 with an assigned unitage of 1000 IU/ampoule.

J Immunol Methods, 1996 Jul 17, 194(1), 1 - 12
Implications for the assay and biological properties of interleukin-3 . Results of a WHO international collaborative study; Mire-Sluis AR et al.; Five preparations of interleukin-3 (IL-3) have been evaluated by 28 laboratories in 12 countries for their suitability to serve as an international standard for this material in a joint international collaborative study for IL-3 and interleukin-4 (IL-4) . The preparations were assayed in a wide range of in vitro bioassays and immunoassays . It is clear from the biological assays contributed to this study that different recombinant preparations of IL-3 can have very different biological specific activities, including those from the same source (i.e., E . coli) . Biological assays of IL-3 were significantly more consistent in their estimates of levels of IL-3 than the immunoassays, suggesting an unusual pattern of epitope recognition amongst the antibodies included in the immunoassays . This study also illustrates the point that the level of cytokine measured by immunoassay does not necessarily reflect the biological potency of the cytokine . On the basis of results reported here, with the agreement of the participants of the study and with the authorisation of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) the preparation of IL-3 (91/510) was established as the international standard for interleukin-3 with an assigned unitage of 1700 IU/ampoule.

Biochim Biophys Acta, 1996 Jul 17, 1307(3), 325 - 30
Studies on electrotransfer of DNA into Escherichia coli: effect of molecular form of DNA; Kimoto H et al.; Effects of several molecular forms of DNA were examined on voltage-pulse-mediated transfection or transformation . Among circular DNAs, the single-stranded microvirid DNA was less infective than the double-stranded replicative form, whereas transfectivity of the relaxed or nicked molecule was nearly equal to or slightly lower than that of the supercoiled DNA . The linearized double-stranded DNA derived from phage or plasmid electrotransfects Escherichia coli, albeit at a reduced efficiency . Alkaline denaturation of the linearized DNA resulted in complete loss of the infectivity . Relationship between terminal structure of the linearized DNA and efficiency of the transfection was investigated . Host recombination function did not significantly affect the infectivity of the linearized DNA.

Biochim Biophys Acta, 1996 Jul 17, 1307(3), 309 - 17
Cloning and characterization of cDNAs encoding the epsilon-subunit of eukaryotic initiation factor-2B from rabbit and human; Asuru AI et al.; A rabbit reticulocyte lysate cDNA library was screened with a polyclonal antiserum directed against eukaryotic initiation factor eIF-2B (eIF-2B) . A 2508 base pair cDNA (pA1) was isolated and determined to encode the epsilon-subunit of eIF-2B based on the immunoreactivity of the fusion protein expressed from the cDNA in Escherichia coli and the presence of two peptide sequences obtained from two V8 fragments of purified nonrecombinant eIF-2B epsilon in the deduced amino acid sequence of the cDNA . The open reading frame of the cDNA began with the third nucleotide of the cDNA with the first AUG codon at nucleotide 522 . Mutational analysis of pA1 indicated that the cDNA did not code for full-length eIF-2B epsilon . Seven missing codons of the reading-frame and the 71 nucleotide 5' non-coding region of the eIF-2B epsilon mRNA were obtained by 5' RACE . A human eIF-2B epsilon cDNA fragment, which corresponded to a similar 2.3 kb fragment generated by digestion of the rabbit pA1 cDNA with EcoRI, was isolated from a human histiocytic lymphoma (U-937) cell cDNA library constructed in lambda gt10 . The nucleotide and amino acid sequences were highly conserved between the rabbit and human cDNAs, showing approx . 90% sequence identity within the open reading frame . Northern and Western blot analyses of reticulocyte lysate and other rabbit tissue extracts indicated that the eIF-2B epsilon polypeptide has a similar apparent molecular weight in all tissues examined, and is coded for by a single approximately 2.8 kilobase mRNA species which is ubiquitously expressed.

Biochim Biophys Acta, 1996 Jul 17, 1307(3), 259 - 62
A duplicated sequence in sugarbeet mitochondrial transcripts is differentially edited: analysis of orfB and its derivative orf324 mRNAs; Kubo T et al.; RNA editing of the duplicated sequences was investigated in the transcripts of orfB and orf324 genes from sugarbeet mitochondria . The orf324 shares the first 59 bp of the reading frame and 321 bp upstream sequence with orfB . Two cytidine-to-uridine editing sites were found in orfB, but the corresponding cytidine residues remained unchanged in the transcripts of orf324 . In the vicinity of the non-edited cytidine residues within the shared sequence element of orf324 were identified three point mutations that may abolish orf324 editing . Our results also suggest that selection of editing sites depends on primary sequence.

Biochemistry, 1996 Jul 16, 35(28), 9150 - 7
Motional dynamics of a buried tryptophan reveals the presence of partially structured forms during denaturation of barstar; Swaminathan R et al.; A double mutant of the single-domain protein barstar having a single tryptophan (W53) was made by mutating the remaining two tryptophans (W38 and W44) into phenylalanines . W53 is buried in the core of barstar . Time-resolved fluorescence of the mutant barstar (W38FW44F) showed that W53 has a single fluorescence lifetime in the native (N) state and has three lifetimes in the molten globule-like low-pH (A) form . Quenching of fluorescence by either KI or acrylamide showed that W53 is solvent inaccessible in the N-state and fairly accessible in the A-form . The denaturation of W38FW44F by guanidine hydrochloride (GdnHCI) was monitored by several probes: near-UV and far-UV circular dichroism (CD), fluorescence intensity, and steady-state and time-resolved fluorescence anisotropy . While the unfolding transitions observed through CD and fluorescence intensity coincided with each other (midpoint approximately 1.8 M GdnHCI), the transition observed through the steady-state fluorescence anisotropy was markedly different from others . Initially, the anisotropy increased with the increase in the concentration of GdnHCI and decreased subsequently . The midpoint of this titration was 2.2 M GdnHCI . Picosecond time-resolved fluorescence anisotropy showed that W38FW44F has a single rotational correlation time of 4.1 ns in the native (N) state and 1.5 as in the unfoled (U) state (6 M GdnHCI) . These could be explained as being due to the absence of motional freedom of W53 in the N-state and the presence of rotational freedom in the U-state . In the intermediate concentration region (1.8-3.0 M GdnHCI), the anisotropy decays showed at least two coorrelation times, approximately 1 and 6-12 ns . These two correlation times are ascribed to partially structured forms leading to hindered rotation of W53 . Thus, the usefulness of time-resolved fluorescence anisotropy in detecting partially folded structures is demonstrated.

Biochemistry, 1996 Jul 16, 35(28), 9128 - 32
Structural determinants of the uridine-preferring specificity of RNase PL3; Vicentini AM et al.; RNase PL3 is a structurally highly conserved, pyrimidine-specific RNase, which strongly prefers to cleave at the 3'-side of uridine . Here, question of which residues are involved in determining substrate specificity is addressed . The difference in the rate of cleavage of UpA and CpA was found to result from a 375-fold larger kcat for the former substrate, whereas the values of Km were essentially the same . The pyrimidine specificity of this class of RNases is thought to result from hydrogen bonds between the base and a threonine residue in the B1 subsite . Mutation of this residue (Thr-44) in RNase PL3 resulted in strongly reduced activity with UpA and poly(U) . However, the activity with CpA and poly(C) had increased . Comparison with the effect of the same mutation in RNase A {delCardayre, S . B., & Raines, R . T . (1994) Biochemistry 33, 6031-6037} and angiogenin {Curran et al . (1993) Biochemistry 32, 2307-2313} showed that the function of this threonine in substrate recognition is different in three RNase subfamilies . Previous studies have shown that the 36-42 region contains one or more residues that are involved in substrate recognition {Vicentini et al . (1994) Protein Sci . 3, 459-466} . Site-directed mutagenesis of amino acids in this region identified Phe-42 as the only single residue that affected the cytidine/uridine specificity ratio . The mutation F42V resulted in a 10-fold increase in kcat and a 1.9-fold decrease in Km for CpA . The properties of the double mutant F42V/T44A suggested that a suboptimal binding of cytidine is caused by Phe-42, partially through an effect on Thr-44.

Biochemistry, 1996 Jul 16, 35(28), 9069 - 75
Development of a sensitive peptide-based immunoassay: application to detection of the Jun and Fos oncoproteins; Heuer KH et al.; c-Jun and c-Fos belong to the bZIP class of transcriptional activator proteins, many of which have been implicated in the neoplastic transformation of cells . We are interested in engineering dominant-negative leucine zipper (LZ) peptides as a means of sequestering these proteins in vivo in order to suppress their transcriptional regulatory activity . Toward this end, we have developed a novel immunoassay for measuring the dimerization affinities of dimeric Jun and Fos complexes . This peptide-based ELISA relies on the fact that Jun and Fos preferentially form heterodimers via their leucine zipper domains . Recombinant Jun leucine zipper peptides (either native JunLZ or a V36 --> E point mutant) were labeled with biotin and specifically bound through a leucine zipper interaction to a FosLZ-glutathione S-transferase fusion protein adsorbed onto the wells of an ELISA tray . Jun:Fos complexes were subsequently detected using a recently developed streptavidin-based amplification system known as enzyme complex amplification {Wilson, M . R., & Easterbrook-Smith, S.B . (1993) Anal . Biochem . 209, 183-187} . This ELISA system can detect subnanomolar concentrations of Jun and Fos, thus allowing determination of the dissociation constants for complex formation . The dissociation constant for formation of the native JunLZ:FosLZ heterodimer at 37 degrees C was determined to be 0.99 +/- 0.30 nM, while that for JunLZ(V36E):FosLZ heterodimer was 0.90 +/- 0.13 microM . These results demonstrate that the novel peptide-based ELISA described herein is simple and sensitive and can be used to rapidly screen for potential dominant-negative leucine zipper peptides.

Biochemistry, 1996 Jul 16, 35(28), 9034 - 41
Identification of residues critical to the activity of human granulocyte colony-stimulating factor; Reidhaar-Olson JF et al.; Alanine scanning mutagenesis of human granulocyte colony-stimulating factor (G-CSF) was used to identify residues critical for the cell-proliferative activity of the protein . Fifty-eight residues, most of them on the protein surface, were independently mutated to alanine . Most of the variants retained full biological activity; however, 15 mutants were significantly impaired in their ability to stimulate bone marrow cell proliferation in vitro . Four of these variants contain mutations at buried residues and two have substitutions at side chains involved in intramolecular hydrogen bonds . The remaining nine down mutations identify two regions on the surface of the molecule important for biological activity . Consistent with these observations, measurements of binding to NFS-60 cells indicate that the residues most important for receptor binding are Lys40 and Phe144 in site 1 and Glu19 in site 2 . In addition to these residues, Val48 and Leu49 in site 1 and Leu15, Asp112, and Leu124 in site 2 are also important for biological activity . These results suggest the presence of two binding sites on the cytokine surface required for dimerization of the G-CSF receptor.

Biochemistry, 1996 Jul 16, 35(28), 9024 - 33
Involvement of arginine 143 in nucleotide substrate binding at the active site of adenylosuccinate synthetase from Escherichia coli; Moe OA et al.; Adenylosuccinate synthetase from Escherichia coli is inactivated in a biphasic reaction by guanosine 5'-O-{S-(4-bromo-2,3-dioxobutyl)thio}phosphate (GMPSBDB) at pH 7.1 and 25 degrees C . Reaction of the enzyme with {8-3H}GMPSBDB results in the incorporation of 2 mol of the reagent/mol of subunit; in the presence of active site ligands the incorporation is reduced to 1 mol of reagent/mol of subunit . GMPSBDB reacts with Cys-291 in the initial rapid reaction which is accompanied by loss of 50% of the enzymatic activity; this reaction is not affected by the presence of active site ligands . In the slower reaction, GMPSBDB inactivates the enzyme by reacting with Arg-143 . The inactivation kinetics of the slower phase are consistent with the formation of an enzyme--GMPSBDB complex having a Kd of 42 microM . Active site nucleotides, either adenylosuccinate or IMP + GTP, prevent both slower phase inactivation and labeling of Arg-143 . Replacement of Arg-143 with a Leu by site-directed mutagenesis does not change the catalytic constant or the K(m) for aspartate but does significantly impair nucleotide binding: the Michaelis constants for IMP and GTP increase by 60-fold and 10-fold, respectively, in the R143L mutant . The crystal structure of the E . coli enzyme {Poland, B.W., Silva, M.M., Serra, M.A., Cho, Y., Kim, K . H., Harris, E.M.S., & Honzatko, R.B . (1993) J . Biol . Chem . 268, 25334--25342} shows that Arg-143 from one subunit projects into the putative active site of the other subunit . These results indicate that both subunits of dimeric adenylosuccinate synthetase contribute to each active site and that Arg-143 plays an important role in nucleotide binding.

Biochem Biophys Res Commun, 1996 Jul 16, 224(2), 474 - 8
Cytochrome f encoded by the chloroplast genome is imported into thylakoids via the SecA-dependent pathway; Nohara T et al.; In vitro import of the precursor of tobacco cytochrome f, which is is encoded by the chloroplast genome, into isolated pea thylakoids was analyzed . Upon incubation with thylakoids and a stromal fraction, the precursor of cytochrome f was efficiently imported into thylakoids . The imported cytochrome f was tightly integrated into the thylakoid membrane . Insertion of cytochrome f into the thylakoid membrane was blocked by nigericin, sodium azide, and antibodies against pea chloroplast SecA . These results suggest that cytochrome f utilizes the bacterial-type SecA-dependent pathway to be imported into thylakoids.

Biochem Biophys Res Commun, 1996 Jul 16, 224(2), 351 - 7
Chemostat selection of an Escherichia coli mutant containing permease with enhanced lactose affinity; Tsen SD et al.; Chemostats supplied with limited lactose were used to ask whether it was possible to generate and isolate any mutant of Escherichia coli lactose permease which allowed cells to grow faster . The permease and beta-galactosidase activities of the chemostat culture initially rose together to reach a plateau . After 30 days, the former underwent a second increase alone . From this culture, a faster-growing mutant was isolated . Its permease gene was cloned, sequenced, and found to have a single base pair changed . Thymine at position 199 was changed to guanine, resulting in serine 67 being substituted by alanine . Cells bearing this mutant in the plasmid could grow faster than parents in 10 microM lactose . The Km of the mutant permease toward lactose was 1.4 mM, about half of the wild-type value . Thus, a mutant with higher affinity for substrate could be selected from the chemostat.

Clin Chim Acta, 1996 Jul 15, 251(1), 41 - 52
Enzymatic diagnosis of holocarboxylase synthetase deficiency using apo-carboxyl carrier protein as a substrate; Suzuki Y et al.; We developed a simple and sensitive method for assessing holocarboxylase synthetase (HCS) activity that is based on measuring incorporation of {3H}biotin into apo-carboxyl carrier protein, a subunit of acetyl-CoA carboxylase from E . coli . Kinetic analysis of HCS from normal fibroblasts showed that the Km for biotin was 260 +/- 94 nmol/l (mean +/- S.D.; n = 5) . In contrast, the Km values of HCS from two cell lines derived from patients with HCS deficiency were 7200 and 3700, clearly distinguishable from the control value . The sensitivity of this assay was so high that we were able to characterize a mutant enzyme whose activity had not been previously detected . Our method is useful for enzymatic diagnosis of HCS deficiency and characterization of HCS.

Nucleic Acids Res, 1996 Jul 15, 24(14), 2790 - 2
Cloning and characterization of Sse9I DNA-methyltransferase recognizing 5'-AATT-3'; Gonchar DA et al.; The gene from Sporosarcina species 9D encoding Sse9I DNA-methyltransferase (M.Sse9I) was cloned and expressed in Escherichia coli . The recombinant plasmid pMSse-1 contains the M.Sse9I gene 1086 bp in length, corresponding to a protein of 362 amino acid residues . M.Sse9I recognizes the tetranucleotide sequence 5'-AATT-3' and modifies the second adenine within the recognition sequence . The amino acid sequence of M.Sse9I was compared with those of other methylases . According to mutual positions of four conservative domains the new enzyme belongs to a subgroup of D12 class . This subgroup includes Sse9I, CviAII, NlaIII and N-terminal domains of LlaI, FokI and StsI DNA-methyltransferases.

Nucleic Acids Res, 1996 Jul 15, 24(14), 2706 - 11
In vitro and in vivo function of the C-terminus of Escherichia coli single-stranded DNA binding protein; Curth U et al.; We constructed several deletion mutants of Escherichia coli single-stranded DNA binding protein (EcoSSB) lacking different parts of the C-terminal region . This region of EcoSSB is composed of two parts: a glycine and proline-rich sequence of approximately 60 amino acids followed by an acidic region of the last 10 amino acids which is highly conserved among the bacterial SSB proteins . The single-stranded DNA binding protein of human mitochondria (HsmtSSB) lacks a region homologous to the C-terminal third of EcoSSB . Therefore, we also investigated a chimeric protein consisting of the complete sequence of the human mitochondrial single-stranded DNA binding protein (HsmtSSB) and the C-terminal third of EcoSSB . Fluorescence titrations and DNA-melting curves showed that the C-terminal third of EcoSSB is not essential for DNA-binding in vitro . The affinity for single-stranded DNA and RNA is even increased by the removal of the last 10 amino acids . Consequently, the nucleic acid binding affinity of HsmtSSB is reduced by the addition of the C-terminus of EcoSSB . All mutant proteins lacking the last 10 amino acids are unable to substitute wild-type EcoSSB in vivo . Thus, while the nucleic acid binding properties do not depend on an intact C-terminus, this region is essential for in vivo function . Although the DNA binding properties of HsmtSSB and EcoSSB are quite similar, HsmtSSB does not function in E.coli . This failure cannot be overcome by fusing the C-terminal third of EcoSSB to HsmtSSB . Thus differences in the N-terminal parts of both proteins must be responsible for this incompatibility . None of the mutants was defective in tetramerization . However, mixed tetramers could only be formed by proteins containing the same N-terminal part . This reflects structural differences between the N-terminal parts of HsmtSSB and EcoSSB . These results indicate that the region of the last 10 amino acids, which is highly conserved among bacterial SSB proteins, is involved in essential protein-protein interactions in the E.coli cell.

Nucleic Acids Res, 1996 Jul 15, 24(14), 2701 - 5
The influence of base identity and base pairing on the function of the alpha-sarcin loop of 23S rRNA; O'Connor M et al.; The alpha-sarcin loop of large subunit rRNAs is one of the sites of interaction of elongation factors with the ribosome, and the target of the cytotoxins alpha-sarcin and ricin . Using a genetic selection for increased frameshifting in a reporter gene, we have isolated a C --> U mutation at position 2666 in the alpha-sarcin loop . In the NMR-derived structure of the loop, bases equivalent to 2666 and 2654 are paired via a non-canonical base pairing interaction . Each of the three base substitutions at C2666 and A2654 was constructed by site-directed mutagenesis of a plasmid borne copy of the rrnB operon of Escherichia coli . Only the C2666 --> U and A2654 --> G mutations that resulted in the formation of canonical A-U and C-G base pairs respectively, increased the levels of stop codon readthrough and frameshifting . The effects of different base pair combinations at positions 2666 and 2654 on ribosome function were then tested by constructing and analyzing all possible base combinations at these sites . All A --> G base substitution mutations at position 2654 and C --> U substitutions at position 2666 increased the levels of translational errors . However, these effects were greatest when G2654 and U2666 had the potential to engage in standard Watson-Crick base pairing interactions . These data indicate that base identity as well as base pairing interactions are important for the function of this essential component of the large subunit rRNA.

Nucleic Acids Res, 1996 Jul 15, 24(14), 2690 - 6
Requirements for cleavage by a modified RNase P of a small model substrate; Liu F et al.; M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, has been covalently linked at its 3' terminus to oligonucleotides (guide sequences) that guide the enzyme to target RNAs through hybridization with the target sequences . These constructs (M1GS RNAs) have been used to determine some minimal features of model substrates . As few as 3 bp on the 3' side of the site of cleavage in a substrate complex and 1 nt on the 5' side are required for cleavage to occur . The cytosines in the 3' terminal CCA sequence of the model substrates are important for cleavage efficiency but not cleavage site selection . A purine (base-paired or not) at the 3' side of the cleavage site is important both for cleavage site selection and efficiency . M1GS RNAs provide both a simple system for characterization of the reaction governed by M1 RNA and a tool for gene therapy.

Nucleic Acids Res, 1996 Jul 15, 24(14), 2673 - 8
Is the in-frame termination signal of the Escherichia coli release factor-2 frameshift site weakened by a particularly poor context?
Major LL, Poole ES, Dalphin ME, Mannering SA, Tate WP.
The synthesis of release factor-2 (RF-2) in bacteria is regulated by a high efficiency +1 frameshifting event at an in-frame UGA stop codon . The stop codon does not specify the termination of synthesis efficiently because of several upstream stimulators for frameshifting . This study focusses on whether the particular context of the stop codon within the frameshift site of the Escherichia coli RF-2 mRNA contributes to the poor efficiency of termination . The context of UGA in this recoding site is rare at natural termination sites in E.coli genes . We have evaluated how the three nucleotides downstream from the stop codon (+4, +5 and +6 positions) in the native UGACUA sequence affect the competitiveness of the termination codon against the frameshifting event . Changing the C in the +4 position and, separately, the A in the +6 position significantly increase the termination signal strength at the frameshift site, whereas the nucleotide in the +5 position had little influence . The efficiency of particular termination signals as a function of the +4 or +6 nucleotides correlates with how often they occur at natural termination sites in E.coli; strong signals occur more frequently and weak signals are less common.

Nucleic Acids Res, 1996 Jul 15, 24(14), 2666 - 72
Structure of a U.U pair within a conserved ribosomal RNA hairpin; Wang YX et al.; A conserved hairpin corresponding to nt 1057-1081 of large subunit rRNA (Escherichia coli numbering) is part of a domain targeted by antibiotics and ribosomal protein L11 . The stem of the hairpin contains a U.U juxtaposition, found as either U.U or U.C in virtually all rRNA sequences . This hairpin has been synthesized and most of the aromatic and sugar protons were assigned by two-dimensional proton NMR . Distances and sugar puckers deduced from the NMR data were combined with restrained molecular dynamics calculations to deduce structural features of the hairpin . The two U residues are stacked in the helix, form one NH3-O4 hydrogen bond and require an extended backbone conformation (trans alpha and gamma) at one of the U nucleotides . The hairpin loop, UAGAAGC closed by a U-A pair, is the same size as tRNA anticodon loops, but not as well ordered.

Nucleic Acids Res, 1996 Jul 15, 24(14), 2627 - 31
Hypermutagenic PCR involving all four transitions and a sizeable proportion of transversions; Vartanian JP et al.; Very complex mutant libraries of the dihydrofolate reductase (DHFR) gene encoded by the Escherichia coli plasmid R67 were created using hypermutagenic PCR with biased deoxynucleotide triphosphate (dNTP) concentrations . Exploiting the particular stability of the G:T mismatch, the DHFR gene could be enriched in A+T by employing biased deoxypyrimidine triphosphate concentrations, i.e . {dTTP} > {dCTP} . A sizeable fraction of hypermutants were functional . A combination of {dTTP} > {dCTP} and {dGTP} > {dATP} biases generated mutations at unexpectedly low frequencies . This could be overcome by the addition of Mn2+ cations . Overall mutation frequencies of 10% per amplification (range 4-18% per clone) could be attained . All four transitions and a smaller number of transversions were produced throughout the gene . PCR mutagenesis could be so extensive as to inactivate all amplified versions of the gene.

J Clin Invest, 1996 Jul 15, 98(2), 285 - 9
Defective cystathionine beta-synthase regulation by S-adenosylmethionine in a partially pyridoxine responsive homocystinuria patient; Kluijtmans LA et al.; We determined the molecular basis of cystathionine beta-synthase (CBS) deficiency in a partially pyridoxine-responsive homocystinuria patient . Direct sequencing of the entire CBS cDNA revealed the presence of a homozygous G1330A transition . This mutation causes an amino acid change from aspartic acid to asparagine (D444N) in the regulatory domain of the protein and abolishes a TaqI restriction site at DNA level . Despite the homozygous mutation, CBS activities in extracts of cultured fibroblasts of this patient were not in the homozygous but in the heterozygous range . Furthermore, we observed no stimulation of CBS activity by S-adenosylmethionine, contrary to a threefold stimulation in control fibroblast extract . The mutation was introduced in an E . coli expression system and CBS activities were measured after addition of different S-adenosylmethionine concentrations (0-200 microM) . Again, we observed a defective stimulation of CBS activity by S-adenosylmethionine in the mutated construct, whereas the normal construct showed a threefold stimulation in activity . These data suggest that this D444N mutation interferes in S-adenosylmethionine regulation of CBS . Furthermore, it indicates the importance of S-adenosylmethionine regulation of the transsulfuration pathway in homocysteine homeostasis in humans.

FEBS Lett, 1996 Jul 15, 390(1), 85 - 90
Characterization of an Arabidopsis thaliana cDNA encoding an S-adenosylmethionine-sensitive threonine synthase . Threonine synthase from higher plants; Curien G et al.; An Arabidopsis thaliana cDNA encoding an S-adenosylmethionine-sensitive threonine synthase (EC 4.2.99.2) has been isolated by functional complementation of an Escherichia coli mutant devoid of threonine synthase activity . Threonine synthase from A . thaliana was shown to be synthesized with a transit peptide . The recombinant protein is activated by S-adenosylmethionine in the same range as the plant threonine synthase and evidence is presented for an involvement of the N-terminal part of the mature enzyme in the sensitivity to S-adenosylmethionine.

FEBS Lett, 1996 Jul 15, 390(1), 69 - 72
GTPase properties of the interferon-induced human guanylate-binding protein 2; Neun R et al.; Guanylate-binding proteins (GBPs) were originally described as proteins that are strongly induced by interferons and are capable of binding to agarose-immobilized guanine nucleotides . hGBP1, the first of two members of this protein family in humans, was recently shown to represent a novel type of GTPase that hydrolyzes GTP predominantly to GMP . We now report that purified recombinant hGBP2 also hydrolyzes GTP very efficiently, although GDP rather than GMP was the major reaction product . The biochemical parameters of this reaction were as follows: Km = 313 microM, turnover number = 22 min-1 . Both hGBP1 and hGBP2 failed to hydrolyze GDP, however, GDP was an effective inhibitor of the hGBP2- but not the hGBP1-catalyzed GTP hydrolysis reaction . Thus, hGBP1 and hGBP2 have similar biochemical properties, but show pronounced differences in product specificity.

FEBS Lett, 1996 Jul 15, 390(1), 53 - 8
The construction and characterization of an effective transpositional system based on IS30; Farkas T et al.; We constructed an in vivo system to detect transpositional rearrangements induced by the insertion sequence IS30 . The transposase protein expressed from the transposase producer plasmids catalyzed rearrangements on different target sequences presented in trans . High yields, up to 83%, of transpositional frequencies were observed . The frequency of rearrangements correlated with the amount of transposase protein produced and the attractivity of the target sequences . Alteration in the frequency of transposition was observed in the recA- E . coli strains JM109 and TG2 . Remarkable structural and functional analogy was found with site-specific recombination systems.

FEBS Lett, 1996 Jul 15, 390(1), 39 - 43
Cloning and expression of human mitochondrial deoxyguanosine kinase cDNA; Wang L et al.; Mammalian mitochondrial deoxyguanosine kinase (dGK) is responsible for phosphorylation of purine deoxyribonucleosides in the mitochondrial matrix . Using a RT-PCR-generated probe, based on amino acid sequence information from proteolytic fragments of purified bovine dGK, we have cloned a cDNA from a human brain cDNA library that encodes a 30 kDa protein . The deduced amino acid sequence of this protein included the sequence of all six peptides isolated and sequenced from purified dGK . Expression and purification of recombinant protein from induced Escherichia coli extracts revealed that it catalyses efficient phosphorylation of dGuo, arabinosyl guanine, dAdo, 2-chloro-2'-deoxyadenosine and dIno similar to purified dGK . Northern blot analysis demonstrated one dominant positive mRNA of 1.35 kb and it was found in several tissues at similar levels . The coding sequence of dGK showed 46% identity to the coding sequence of cytosolic deoxycytidine kinase, and conserved sequence motifs among the known deoxynucleoside kinase were identified.

FEBS Lett, 1996 Jul 15, 390(1), 34 - 8
Transmembrane topology of Escherichia coli H(+)-ATPase (ATP synthase) subunit a; Yamada H et al.; Escherichia coli H(+)-ATPase subunit a is a hydrophobic F0 subunit . To investigate the topology of the subunit in the membrane, we prepared site-specific polyclonal antibodies against amino-terminal (Ser-3 to Leu-16), middle loop (Lys-167 to Gln-181), and carboxyl-terminal (Thr-259 to His-271) peptide segments . Enzyme-linked immunosorbent assay revealed that these antibodies specifically reacted with subunit a of inside-out membrane vesicles, but not with that of right-side-out spheroplasts . Full reactivity appeared when spheroplasts were disrupted with Triton X-100 (0.5%) or by sonication . These results suggest that at least parts of the three peptide segments of subunit a face the cytoplasm . Based on these observations, we propose a novel transmembrane topology of subunit a.

FEBS Lett, 1996 Jul 15, 390(1), 113 - 8
Identification of an Arabidopsis thaliana cDNA encoding a HSP70-related protein belonging to the HSP110/SSE1 subfamily; Storozhenko S et al.; Heat-shock protein 70 (HSP70)-related proteins are classified in two main subfamilies: the DnaK subfamily and the HSP110/SSE1 subfamily . We have characterized the first plant member of the HSP110/SSE1 subfamily, HSP91 . At least two, tightly linked genes encoding HSP91 are present per haploid Arabidopsis genome . HSP91 is constitutively expressed in non-stressed Arabidopsis plants and is transiently induced by heat shock.

FEBS Lett, 1996 Jul 15, 390(1), 109 - 12
Characterization of recombinant human HBP/CAP37/azurocidin, a pleiotropic mediator of inflammation-enhancing LPS-induced cytokine release from monocytes; Rasmussen PB et al.; Neutrophil-derived heparin-binding protein (HBP) is a strong chemoattractant for monocytes . We report here for the first time the expression of recombinant HBP . A baculovirus containing the human HBP cDNA mediated in insect cells the secretion of a 7-residue N-terminally extended HBP form (pro-HBP) . Deletion of the pro-peptide-encoding cDNA sequence resulted in correctly processed HBP at the N-terminus . Electrospray mass spectrum analysis of recombinant HBP yielded a molecular weight of 27.237 +/- 3 amu . Consistent with this mass is a HBP form of 225 amino acids (mature part +3 amino acid C-terminal extension) . The biological activity of recombinant HBP was confirmed by its chemotactic action towards monocytes . Furthermore, we have shown that recombinant HBP stimulates in a dose-dependent manner the lipopolysaccharide (LPS)-induced cytokine release from human monocytes.

FEBS Lett, 1996 Jul 15, 390(1), 10 - 4
A possible role for CYP27 as a major renal mitochondrial 25-hydroxyvitamin D3 1 alpha-hydroxylase; Araya Z et al.; A mitochondrial cytochrome P450 fraction catalyzing 1 alpha- and 27-hydroxylation but not 24-hydroxylation of 25-hydroxyvitamin D3 was purified from pig kidney . The ratio between the 1 alpha- and 27-hydroxylase activities was the same in all purification steps including a side fraction . Attempts to separate the 1 alpha- and 27-hydroxylase activities were unsuccessful . A monoclonal antibody directed against purified pig liver CYP27 recognized a protein of the same apparent M(r) and immunoprecipitated both the 1 alpha- and 27-hydroxylase activities towards 25-hydroxyvitamin D3 in the purified kidney enzyme fraction as well as in a solubilized, crude cytochrome P450 extract considered to represent the major part of the 25-hydroxyvitamin D3 hydroxylases in kidney mitochondria . Taken together, the results from the purification and the experiments with CYP27 antibody, substrate inhibition, and recombinant expressed human liver CYP27 strongly indicate that CYP27 is able to catalyze 1 alpha-hydroxylation but not 24-hydroxylation of 25-hydroxyvitamin D3 in kidney . In conclusion, the results provide evidence for a role for CYP27 as a major renal mitochondrial 25-hydroxyvitamin D3 1 alpha-hydroxylase.

Eur J Biochem, 1996 Jul 15, 239(2), 532 - 8
Structural analysis of the O-antigenic polysaccharide from the enteropathogenic Escherichia coli O125; Kjellberg A et al.; The structure of the polysaccharide part of the lipopolysaccharide obtained from the enteropathogenic Escherichia coli O125 has been investigated . Methylation analysis, 1H-NMR spectroscopy and 13C-NMR spectroscopy revealed that the polysaccharide is composed of repeating hexasaccharide units . Smith degradation of the native O-polysaccharide resulted in a polysaccharide with four sugar residues in the repeating unit . Information on the sequence of the native O-polysaccharide and the Smith-degraded product was obtained by two-dimensional techniques, namely heteronuclear-multiple-bond-connectivity and NOESY experiments . The structure of the repeating unit of the O-polysaccharide of E . coli strain O125, which has two adjacent branch-point residues, is {sequence: see text}.

Eur J Biochem, 1996 Jul 15, 239(2), 427 - 33
L-aspartate oxidase from Escherichia coli . II . Interaction with C4 dicarboxylic acids and identification of a novel L-aspartate: fumarate oxidoreductase activity; Tedeschi G et al.; L-Aspartate oxidase is a monomeric flavoprotein that catalyzes the first step in the de novo biosynthetic pathway for pyridine nucleotide formation under both aerobic and anaerobic conditions . In spite of the physiological importance of this biosynthesis in particular in facultative aerobic organisms, such as Escherichia coli, little is known about the electron acceptor of reduced L-aspartate oxidase in the absence of oxygen . In this report, evidence is presented which suggests that in vitro fumarate can play such a role . L-Aspartate oxidase binds succinate and fumarate with Kd values of 0.24 mM and 0.22 mM, respectively . A competitive behaviour was observed for these two dicarboxylic acids towards iminoaspartate and sulfite ions . Photoreduction experiments suggest that fumarate and succinate bind at or close to the active site of the molecule . A new fumarate reductase activity of L-aspartate oxidase is reported using benzylviologen or L-aspartate as reductants and fumarate as oxidant . Steady-state kinetics for the oxidase and the fumarate reductase activity of L-aspartate oxidase were obtained using either fumarate or oxygen as electron acceptor and L-aspartate as electron donor . Finally, succinate was identified as the product of the L-aspartate:fumarate oxidoreductase activity using radiolabeled fumarate under anaerobic conditions . The results suggest that fumarate can be a valuable alternative to oxygen as a substrate for L-aspartate oxidase.

Eur J Biochem, 1996 Jul 15, 239(2), 418 - 26
L-aspartate oxidase from Escherichia coli . I . Characterization of coenzyme binding and product inhibition; Mortarino M et al.; This paper reports the biochemical characterization of the flavoprotein L-aspartate oxidase from Escherichia coli . Modification of a previously published procedure allowed overexpression of the holoenzyme in an unproteolysed form . L-Aspartate oxidase is a monomer of 60 kDa containing 1 mol of noncovalently bound FAD/mol protein . A polarographic and two spectrophotometric coupled assays have been set up to monitor the enzymatic activity continuously . L-Aspartate oxidase was subjected to product inhibition since iminoaspartate, which results from the oxidation of L-aspartate, binds to the enzyme with a dissociation constant (Kd) equal to 1.4 microM . The enzyme binds FAD by a simple second-order process with Kd 0.67 microM . Site-directed mutagenesis of the residues E43, G44, S45, F47 and Y48 located in the putative binding site of the isoallossazinic portion of FAD reduces the affinity for the coenzyme.

Eur J Biochem, 1996 Jul 15, 239(2), 410 - 7
Protein engineering studies of dichloromethane dehalogenase/glutathione S-transferase from Methylophilus sp . strain DM11 . Ser12 but not Tyr6 is required for enzyme activity; Vuilleumier S et al.; The structural gene for dichloromethane dehalogenase/glutathione S-transferase (GST, EC 2.5.1.18) from Methylophilus sp . strain DM11 was subcloned into a multicopy plasmid under the control of the T7 polymerase promoter, allowing expression in Escherichia coli and easy purification of the enzyme in good yield . Several point mutations leading to amino acid changes at residues Tyr6, His8 and Ser12 of the protein were introduced in this gene . Mutations at Tyr6, the N-terminal tyrosine known to be essential for enzymatic activity in glutathione S-transferases of the alpha, mu, and pi classes, had little effect on the activity of dichloromethane dehalogenase . The same applied for mutations at residue His8, which from multiple alignments of GST sequences may also correspond to the conserved N-terminal tyrosine residue of GST enzymes . The higher turnover rate of the wild-type enzyme with dibromomethane compared with dichloromethane was lost in mutants with amino acid replacements at residue His8, but retained in mutant proteins at Tyr6 . Mutations at Ser12 led to mutants with drastically reduced enzymatic activity, pinpointing this residue as an essential determinant of catalytic efficiency.

Eur J Biochem, 1996 Jul 15, 239(2), 397 - 402
An essential lysine in the substrate-binding site of ornithine carbamoyltransferase; Valentini G et al.; Treatment of ornithine carbamoyltransferase from dolphin Stenella with pyridoxal phosphate, followed by reduction with NaBH4 resulted in complete loss of enzyme activity . The phosphate alone or the substrate analogue 2-aminovaleric acid moderately decreased the extent of inactivation, while carbamoyl phosphate plus 2-aminovaleric acid provided complete protection from inactivation . The partially inactivated enzyme showed K(m) values for substrates equivalent to those of native enzyme and lowered Kcat values . Two lysyl residues were substantially modified in the absence of ligands but only one of them was responsible for the inactivation of catalytic activity . Modification of a single subunit was sufficient to completely abolish the catalytic activity of the trimeric enzyme . The lysine involved has been identified as lysine 56 on the known primary structure of homologous human liver enzyme.

Eur J Biochem, 1996 Jul 15, 239(2), 272 - 80
Clavin, a type-1 ribosome-inactivating protein from Aspergillus clavatus IFO 8605 . cDNA isolation, heterologous expression, biochemical and biological characterization of the recombinant protein; Parente D et al.; We describe the cloning and expression of a new cDNA from the filamentous fungus Aspergillus clavatus IFO 8605 . This cDNA contains an open reading frame (ORF) that predicts a putative ribonuclease precursor with high similarity to the alpha-sarcin family of ribosome-inactivating proteins (RIPs) . The cDNA encoding the mature protein was expressed in Escherichia coli, and the recombinant protein, a 17-kDa polypeptide designated clavin was purified and characterized . Clavin shows typical type-1 RIP properties: specific cleavage of ribosomal and synthetic RNA and inhibition of protein synthesis in cell-free and cellular systems . When selectively targeted to a tumour cell antigen by coupling to a monoclonal antibody (mAb) clavin was able to inhibit protein synthesis at nanomolar concentration . Pharmacokinetics analysis in mice indicated an elimination half-life (t1/2 beta) of 7.4 h with no particular accumulation in major organs . Liver toxicity was very limited and transient while no alteration of kidney function was observed . Clavin induced a late and very low antibody response in mice . The in vitro and in vivo biological characteristics of clavin, together with its availability in large amounts, suggest the usefulness of this toxin in the production of toxic chemical conjugates.

Circulation, 1996 Jul 15, 94(2), 197 - 206
Recombinant staphylokinase variants with altered immunoreactivity . I: Construction and characterization; Collen D et al.; BACKGROUND: Recombinant staphylokinase offers promise for thrombolytic therapy in acute myocardial infarction, but it is immunogenic . Although reduced immunogenicity of heterologous proteinaceous drugs by protein engineering has not previously been reported, an attempt was made to achieve this in staphylokinase by site-specific mutagenesis . METHODS AND RESULTS: Biospecific interaction analysis of a panel of 17 murine monoclonal antibodies against recombinant staphylokinase (SakSTAR variant) identified three nonoverlapping immunodominant epitopes, two of which could be eliminated by substitution mutagenesis of clusters of two or three charged amino acids with alanine . Circulating anti-staphylokinase antibodies elceted in patients by treatment with SakSTAR were incompletely (< 90%) absorbed by these mutants . Therefore, the combination variants K35A,E38A,K74A,E75A,R77A (SakSTAR.M38) and K74A,E75A,R77A,E80A,D82A (SakSTAR.M89) were constructed, expressed in Escherichia coli, highly purified by ion-exchange and hydrophobic interaction chromatography, and characterized . These variants had specific activities that were approximately half that of SakSTAR, and they combined the reduced reactivity with the panels of monoclonal antibodies of their parent molecules . Absorption of circulating antibodies elicited in patients by treatment with SakSTAR was incomplete in 13 of 16 patients (median values, 68% and 65% with SakSTAR.M38 and SakSTAR.M89, respectively) . CONCLUSIONS: SakSTAR contains three immunodominant epitopes, two of which were eliminated by site-directed mutagenesis, yielding combination mutants with relatively maintained specific activities that were not recognized by a significant fraction of the antibodies elicited in patients by treatment with wildtype SakSTAR . These mutants appear to be suitable for more detailed investigation of their thrombolytic and antigenic properties.

EMBO J, 1996 Jul 15, 15(14), 3524 - 8
Porins of Escherichia coli: unidirectional gating by pressure; Le Dain AC et al.; OmpC and PhoE porins of Escherichia coli were examined by the patch-clamp technique following reconstitution in liposomes, and were observed primarily in the open (conducting) state . With application of negative voltage and positive hydrostatic pressure, OmpC exhibited marked gating towards a more closed state whereas PhoE remained largely unaffected by pressure application . Hybrid chimeric OmpC-PhoE proteins showed an increased tendency for pressure-dependent gating as the OmpC proportion in the chimeric molecule increased . In addition, several PhoE mutants with amino acid substitutions and insertions in either the L3 or L4 loop of the monomer exhibited pressure sensitivity comparable with the wild-type OmpC porin . Our data support the structural plasticity model of porins and are consistent with the 'charge-screening-unscreening' hypothesis that describes how these proteins may exist in distinct conformations.

Virology, 1996 Jul 15, 221(2), 351 - 4
SFV topoisomerase: sequence specificity in a genetically mapped interval; Palaniyar N et al.; Poxviral DNA topoisomerases are sequence-specific enzymes whose activities are thought to influence such diverse processes as transcription, DNA replication, and genetic recombination . To obtain further insights into the relatedness of these enzymes, and their influence on virus-mediated recombination, we have determined the target-specificity and other catalytic properties of the Shope fibroma virus (SFV) topoisomerase . SFV topoisomerase was expressed in Escherichia coli and purified as a glutathione S-transferase (GST) or (his)6-tagged fusion protein . The recombinant Leporipox-virus (SFV) enzyme displayed catalytic properties very similar to vaccinia topoisomerase . In particular SFV topoisomerase recognizes the same pentanucleotide motif {5'-(C/T)CCTT-3'} and promotes the same DNA relaxation, strand transfer, and strand cleavage reactions catalyzed by the Orthopoxviral (vaccinia) enzyme . The SFV enzyme can also efficiently cleave DNA 3' of the variant site 5'-CCCTG-3' in certain sequence contexts . These studies identified several sites where SFV topoisomerases interact with a recombinational substrate and permitted a comparison of recombination frequencies across intervals which did, or did not, span these sites . We failed to detect any effect of topoisomerase-recognition sites on recombination frequencies, except for a small (< 2-fold) stimulation seen when the substrates encoded a nearby poxviral promoter . This and other work shows that poxviral topoisomerases from several genera share common target specificities, but other enzymatic systems probably catalyze the high-frequency recombination seen in poxvirus-infected cells.

Virology, 1996 Jul 15, 221(2), 335 - 45
Proteolytic activity of purified avian sarcoma and leukemia virus NC-PR protein expressed in Escherichia coli; Sellos-Moura M et al.; Processing of the internal structural and enzymatic proteins of retroviruses occurs during or shortly after budding and is accomplished by the viral protease (PR), which belongs to the large family of aspartic proteases . It is not known how the activity of PR is regulated so that proteolysis occurs at this time . Cellular aspartic proteases are synthesized as zymogens with short N-terminal extensions that are proteolytically removed to generate the free active enzyme . In the avian sarcoma and leukosis viruses (ASLV), PR is expressed as the carboxy-terminal domain of the Gag polyprotein, which thus has a structure analogous to such a zymogen . We have investigated the enzymatic properties of ASLV PR when it is part of a longer protein, NC-PR, serving as a model for Gag . This protein represents about one-third of Gag and consists of the nucleocapsid (NC) domain fused to the N-terminus of PR . NC-PR and derivatives of NC-PR were expressed in bacterial cells and purified . In short-term assays, these fusion proteins lacked measurable protease activity toward an exogenous substrate prepared by in vitro translation . In contrast to PR, which is a homodimer, NC-PR migrated as a monomer both by glycerol gradient sedimentation and by gel filtration chromatography . Thus the NC domain appears to inhibit enzymatic activity by altering the dimerization potential of the PR domains . However, upon long incubations NC-PR was found to cleave itself to generate free and fully active PR, implying that dimerization was not prevented entirely . On the basis of these results, we hypothesize that the Gag protein in vivo is also incompletely active as a protease, because upstream portions of Gag interfere with proper interaction of the PR domains . The eventual dimerization, perhaps triggered by other events, then could lead to a cascade whereby PR is proteolytically freed from Gag and thereby gains enzymatic activity.

Arch Biochem Biophys, 1996 Jul 15, 331(2), 170 - 6
A role for threonine 302 in the mechanism-based inactivation of P450 2B4 by 2-ethynylnaphthalene; Roberts ES et al.; 2-Ethynylnaphthalene (2EN) is a mechanism-based inactivator of P450 2B4 that covalently modifies an amino acid in the peptide Glu273-Met314 with a 2-naphthylacetyl group {Roberts et al . (1994) Biochemistry 33, 3766-3771} . Truncated 2B4 lacking amino acids 2-27, 2B4 (Delta2-27), was expressed in Escherichia coli, purified, and found to catalyze the oxidation of 2EN to 2-naphthylacetic acid (2NA) . The metabolism of 2EN resulted in the inactivation and covalent modification of the protein moiety as we have previously reported with P450 2B4 purified from the livers of phenobarbital-induced rabbits . The rate constants of inactivation of the O-deethylation activity of 7-ethoxy-4-trifluoromethylcoumarin (EFC) were 0.15 +/- 0.01 and 0.20 +/- 0.05 min-1 for the protein purified from rabbit liver and 2B4 (Delta2-27), respectively . A protein in which threonine 302 was replaced with alanine, P450 2B4 (Delta2-27, T302A), was inactivated by 2EN with a much slower rate constant (0.05 +/- 0.01 min-1) and formed 1.8-fold more 2NA as compared to P450 2B4 (Delta2-27) over a 10-min incubation . When the formation of 2NA was supported by cumene hydroperoxide, 2B4 (Delta2-27, T302A) formed 30% less product than 2B4 (Delta2-27) over a 5-min incubation . After incubation with {3H}2EN and NADPH, P450 2B4 (Delta2-27) had significant radioactivity associated with the P450 in an NADPH-dependent manner when the incubation mixture was analyzed by SDS-PAGE followed by autoradiography and 10-fold more radioactivity associated with the P450 as compared to P450 2B4 (Delta2-27, T302A) when analyzed by reverse-phase HPLC . Thus, threonine 302 is not required by 2B4 for oxidation of 2EN or EFC but appears to play an important role in the inactivation of P450 2B4 by 2EN and the covalent labeling of the P450 protein by 2EN.

Anal Biochem, 1996 Jul 15, 239(1), 25 - 34
Monitoring cleavage of fusion proteins by matrix-assisted laser desorption ionization/mass spectrometry: recombinant HIV-1IIIB p26; Parker CE et al.; Matrix-associated laser desorption ionization/mass spectrometry (MALDI/MS) has been used to examine whole bacteria for the presence of a recombinant HIV p26 fusion protein . MALDI/MS, combined with affinity-purification techniques, is also shown to be very useful in monitoring the enzymatic cleavage of both affinity-bound fusion protein and fusion protein in solution . The combination of mass resolution, sensitivity, and speed of analysis makes MALDI/MS an attractive alternative to SDS-PAGE.

J Mol Biol, 1996 Jul 12, 260(2), 251 - 60
The motile major sperm protein (MSP) from Ascaris suum is a symmetric dimer in solution; Haaf A et al.; The major sperm protein (MSP) of Ascaris suum mediates amoeboid motility by forming an extensive intermeshed system of cytoskeletal filaments analogous to that formed by actin in many amoeboid cells . We have used a combination of biochemical and NMR methods to show that, in contrast to actin, MSP exist in solution as a symmetrical dimer . This result has important implications for the mechanism of both MSP filament assembly and the recognition of different MSP isoforms in vivo.

J Mol Biol, 1996 Jul 12, 260(2), 236 - 50
NMR structure of the J-domain and the Gly/Phe-rich region of the Escherichia coli DnaJ chaperone; Pellecchia M et al.; The recombinant N-terminal 107-amino acid polypeptide fragment 2-108 of the DnaJ molecular chaperone of Escherichia coli, which contains the J-domain (residues 2 to 76) and the Gly/Phe-rich region (residues 77 to 108), was uniformly labeled with nitrogen-15 and carbon-13 . The complete NMR solution structure of the J-domain was determined with the program DIANA on the basis of 682 nuclear Overhauser enhancement (NOE) upper distance limits and 180 dihedral angle constraints . It contains three well-defined helices comprising residues 6 to 10, 18 to 32 and 41 to 57, and a fourth helix, consisting of residues 61 to 68, which is well defined as a regular secondary structure but for which the location relative to the remainder of the molecule is not precisely determined . The helices II and III form an antiparallel helical coiled-coil . Helix I is approximately parallel to the plane defined by the helices II and III and runs from the carboxy-terminal end of the helix III to the center of helix II . Helix IV is positioned near the carboxy-terminal end of helix III and is on the same side of the coiled coil as helix I, but it is oriented approximately perpendicular to the plane of the helices II and III . This novel alpha-protein topology leads to formation of a hydrophobic core involving side-chains of all four helices . A strong correlation is seen between the extent of sequence-conservation of hydrophobic residues in the family of J-domain homologues, and the structural organization of the hydrophobic core in these proteins . The residues which have key roles for the specificity of the interaction of DnaJ-like proteins with their corresponding Hsp70 counterparts are located on the outer surfaces of the helices II and III, and in the loop connecting these two helices . Measurements of backbone amide proton exchange rates, 15N spin relaxation times and heteronuclear 15N inverted question mark1H inverted question mark NOEs provided additional insights into local conformational equilibria and internal rate processes in DnaJ(2-108) . In the Gly/Phe-rich region, which is poorly ordered in the NMR solution structure and does not form a globular core, the polypeptide segment 90 to 103 differs from the segments 77 to 89 and 104 to 108 by reduced local flexibility . Considering that this same segment shows sequence conservation with corresponding segments in the Gly/Phe-rich regions of other DnaJ-like proteins, its reduced flexibility may be directly linked to the formation of the ternary DnaJ-DnaK-polypeptide complex.

J Mol Biol, 1996 Jul 12, 260(2), 224 - 35
Nuclear magnetic resonance solution structure of the human Hsp40 (HDJ-1) J-domain; Qian YQ et al.; The J-domain is a highly conserved domain found in all members of the DnaJ family of molecular chaperones . The three-dimensional structure of a recombinant, uniformly 15N-labeled 77-residue polypeptide containing the complete J-domain from human Hsp40 (HDJ-1) has been determined by nuclear magnetic resonance (NMR) spectroscopy in solution . On the basis of 876 upper distance constraints derived from nuclear Overhauser effects (NOE) and 173 dihedral angle constraints, a group of 20 conformers representing the solution structure of the HDJ-1 J-domain was computed with the program DIANA and energy-minimized with the program OPAL . The average of the pairwise root-mean-square deviations of the individual NMR conformers relative to the mean coordinates for the backbone atoms N, C2 and C' of residues 4 to 54 and 4 to to 66 is 0.88 and 0.99 A respectively . The molecular architecture includes four helices composed of residues 5 to 9, 15 to 28, 40 to 54 and 60 to 66 . A turn composed of residues 10 to 14 links helices I and II, and a loop composed of residues 29 to 39 containing a highly conserved tripeptide HPD (residues 31 to 33) connects the antiparallel helices II and III . The tertiary fold formed by helix I-turn-helix II-loop-helix III forms a closed structural core; the less defined helix IV stands away from the core of the domain . The side-chains of the tripeptide HPD extend out from the core of the structure in the opposite direction from helix IV . The structure supports the hypothesis that the highly conserved tripeptide could play a key role in the interaction of Hsp40 with the molecular chaperone, Hsp70.

J Biol Chem, 1996 Jul 12, 271(28), 16773 - 83
Cloning, characterization, and properties of seven triplet repeat DNA sequences; Ohshima K et al.; Several neuromuscular and neurodegenerative diseases are caused by genetically unstable triplet repeat sequences (CTG.CAG, CGG.CCG, or AAG.CTT) in or near the responsible genes . We implemented novel cloning strategies with chemically synthesized oligonucleotides to clone seven of the triplet repeat sequences (GTA.TAC, GAT.ATC, GTT.AAC, CAC.GTG, AGG.CCT, TCG.CGA, and AAG.CTT), and the adjoining paper (Ohshima, K., Kang, S., Larson, J . E., and Wells, R . D.(1996) J . Biol . Chem . 271, 16784-16791) describes studies on TTA.TAA . This approach in conjunction with in vivo expansion studies in Escherichia coli enabled the preparation of at least 81 plasmids containing the repeat sequences with lengths of approximately 16 up to 158 triplets in both orientations with varying extents of polymorphisms . The inserts were characterized by DNA sequencing as well as DNA polymerase pausings, two-dimensional agarose gel electrophoresis, and chemical probe analyses to evaluate the capacity to adopt negative supercoil induced non-B DNA conformations . AAG.CTT and AGG.CCT form intramolecular triplexes, and the other five repeat sequences do not form any previously characterized non-B structures . However, long tracts of TCG.CGA showed strong inhibition of DNA synthesis at specific loci in the repeats as seen in the cases of CTG.CAG and CGG.CCG (Kang, S., Ohshima, K., Shimizu, M., Amirhaeri, S., and Wells, R . D.(1995) J . Biol . Chem . 270, 27014-27021) . This work along with other studies (Wells, R . D.(1996) J . Biol . Chem . 271, 2875-2878) on CTG.CAG, CGG.CCG, and TTA.TAA makes available long inserts of all 10 triplet repeat sequences for a variety of physical, molecular biological, genetic, and medical investigations . A model to explain the reduction in mRNA abundance in Friedreich's ataxia based on intermolecular triplex formation is proposed.

J Biol Chem, 1996 Jul 12, 271(28), 16734 - 40
Cloning, characterization, and modeling of mouse and human guanylate kinases; Brady WA et al.; Guanylate kinase catalyzes the phosphorylation of either GMP to GDP or dGMP to dGDP and is an essential enzyme in nucleotide metabolism pathways . Despite its involvement in antiviral drug activation in humans and in mouse model systems and as a target for chemotherapy, the human and mouse primary structures have never been elucidated . Full-length cDNA clones encoding enzymatically active guanylate kinase were isolated from mouse B-cell lymphoma and human peripheral blood lymphocyte cDNA libraries . Multiple tissue Northern blots demonstrated an mRNA species of approximately 1 kilobase for both mice and humans in all tissue types examined . The mouse cDNA is predicted to encode a 198-amino acid protein with a molecular mass of 21,904 daltons . The human cDNA is predicted to encode a 197-amino acid protein with a molecular mass of 21,696 daltons . These proteins share 88% sequence identity with each other and 52-54% identity with the yeast guanylate kinase . Molecular modeling using the yeast diffraction coordinates indicates a high degree of conservation within the active site and maintenance of the overall structural integrity, despite a lack of similarity along the periphery of the enzyme.

J Biol Chem, 1996 Jul 12, 271(28), 16967 - 74
Purification and characterization of a soluble form of mammalian adenylyl cyclase; Dessauer CW et al.; An engineered, soluble form of mammalian adenylyl cyclase has been expressed in Escherichia coli and purified by three chromatographic steps . The enzyme utilizes one molecule of ATP to synthesize one molecule of cyclic AMP and pyrophosphate at a maximal specific activity of 12.8 micromol/min/mg, corresponding to a turnover number of 720 min-1 . Although devoid of membrane spans, the enzyme displays all of the regulatory properties that are common to mammalian adenylyl cyclases . It is activated synergistically by Gsalpha and forskolin and is inhibited by adenosine (P-site) analogs with kinetic patterns that are identical to those displayed by the native enzymes . The purified enzyme is also inhibited directly by the G protein betagamma subunit complex . After adenovirus-mediated expression in adenylyl cyclase-deficient HC-1 cells, the enzyme can be stimulated synergistically by Gs-coupled receptors and forskolin.

J Biol Chem, 1996 Jul 12, 271(28), 16662 - 7
Expression of the chlI, chlD, and chlH genes from the Cyanobacterium synechocystis PCC6803 in Escherichia coli and demonstration that the three cognate proteins are required for magnesium-protoporphyrin chelatase activity; Jensen PE et al.; Magnesium-protoporphyrin chelatase catalyzes the first step unique to chlorophyll synthesis: the insertion of Mg2+ into protoporphyrin IX . Genes from Synechocystis sp . PCC6803 with homology to the bchI and bchD genes of Rhodobacter sp . were cloned using degenerate oligonucleotides . The function of these genes, putatively encoding subunits of magnesium chelatase, was established by overexpression in Escherichia coli, including the overexpression of Synechocystis chlH, previously cloned as a homolog of the Rhodobacter bchH gene . The combined cell-free extracts were able to catalyze the insertion of Mg2+ into protoporphyrin IX in an ATP-dependent manner and only when the products of all three genes were present . The ChlH, ChlI, and ChlD gene products are therefore assigned to the magnesium chelatase step in chlorophyll a biosynthesis in Synechocystis PCC6803 . The primary structure of the Synechocystis ChlD protein reveals some interesting features; the N-terminal half of the protein shows 40-41% identity to Rhodobacter BchI and Synechocystis ChlI, whereas the C-terminal half displays 33% identity to Rhodobacter BchD . This suggests a functional as well as an evolutionary relationship between the "I" and "D" genes.

J Biol Chem, 1996 Jul 12, 271(28), 16656 - 61
Is the NAD(P)H:flavin oxidoreductase from Escherichia coli a member of the ferredoxin-NADP+ reductase family? . Evidence for the catalytic role of serine 49 residue; Niviere V et al.; The NAD(P)H:flavin oxidoreductase from Escherichia coli, Fre, is a monomer of 26.1 kDa which catalyzes the reduction of free flavins by NADPH or NADH . The flavin reductase Fre is the prototype of a new class of flavin reductases able to transfer electrons with no prosthetic group . It has been suggested that the flavin reductase could belong to the ferredoxin-NADP+ reductase (FNR) family, on the basis of limited sequence homologies . A sequence, conserved within the ferredoxin-NADP+ reductase family and present in the flavin reductase, is important for recognition of the isoalloxazine ring . Within this sequence, we have mutated serine 49 of the flavin reductase into alanine or threonine . kcat value of the S49A mutant was 35-fold lower than kcat of the wild-type enzyme . Determination of real Kd values for NADPH and lumichrome, a flavin analog, showed that recognition of the flavin is strongly affected by the S49A mutation, whereas affinity for the nicotinamide cofactor is only weakly modified . This suggests that serine 49 is involved in the binding of the isoalloxazine ring . Moreover, the Kd value for 5-deazariboflavin, in which the N-5 position of the isoalloxazine ring has been changed to a carbon atom, is not affected by the serine 49 to alanine mutation . This is consistent with the concept that the N-5 position is the main site for serine 49-flavin interaction . In the ferredoxin-NADP+ reductase family, the equivalent serine residue, which has been shown to be essential for activity, is hydrogen-bonded to the N-5 of the FAD cofactor . Taken together, these data provide the first experimental support to the hypothesis that the flavin reductase Fre may belong to the ferredoxin-NADP+ reductase family.

Science, 1996 Jul 12, 273(5272), 211 - 7
Transcription processivity: protein-DNA interactions holding together the elongation complex; Nudler E et al.; The elongation of RNA chains during transcription occurs in a ternary complex containing RNA polymerase (RNAP), DNA template, and nascent RNA . It is shown here that elongating RNAP from Escherichia coli can switch DNA templates by means of end-to-end transposition without loss of the transcript . After the switch, transcription continues on the new template . With the use of defined short DNA fragments as switching templates, RNAP-DNA interactions were dissected into two spatially distinct components, each contributing to the stability of the elongating complex . The front (F) interaction occurs ahead of the growing end of RNA . This interaction is non-ionic and requires 7 to 9 base pairs of intact DNA duplex . The rear (R) interaction is ionic and requires approximately six nucleotides of the template DNA strand behind the active site and one nucleotide ahead of it . The nontemplate strand is not involved . With the use of protein-DNA crosslinking, the F interaction was mapped to the conserved zinc finger motif in the NH2-terminus of the beta' subunit and the R interaction, to the COOH-terminal catalytic domain of the beta subunit . Mutational disruption of the zinc finger selectively destroyed the F interaction and produced a salt-sensitive ternary complex with diminished processivity . A model of the ternary complex is proposed here that suggests that trilateral contacts in the active center maintain the nonprocessive complex, whereas a front-end domain including the zinc finger ensures processivity.

Mutat Res, 1996 Jul 10, 369(1-2), 87 - 96
Effect of eugenol on the mutagenicity of benzo{a}pyrene and the formation of benzo{a}pyrene-DNA adducts in the lambda-lacZ-transgenic mouse; Rompelberg CJ et al.; To study the possible reduction by eugenol of the mutagenicity and genotoxicity of benzo{a}pyrene (B{a}P) in vivo, the lambda-lacZ-transgenic mouse strain 40.6 (Muta Mouse) was used . Male mice were fed a diet containing 0.4% (w/w) eugenol or a control diet for 58 days . On day 10, half of the mice received an i.p . dose of 100 mg/kg b.w . B{a}P . The lacZ mutants were recovered by packaging of DNA isolated from liver into lambda phage, and expressed in E . coli C lacZ-recA-galE- bacteria . In both control mice and mice fed the eugenol diet, B{a}P treatment resulted in a similar, significant increase in lacZ mutant frequency . Eugenol was not mutagenic by itself . By 32P-postlabelling analysis of the liver DNA using an analysis method with chromatographic conditions for B{a}P-DNA adducts, no effect of eugenol on the formation of B{a}P-DNA adducts in the lambda-lacZ-transgenic mouse was found . By 32P-postlabelling analysis using an alkenylbenzene solvent system the amount of B{a}P-DNA adducts was lower in mice fed the eugenol diet than in mice fed the control diet but the decrease was not statistically significant . However, one spot indicative of an eugenol-associated DNA adduct was detected . The present data provide no evidence for antimutagenic or antigenotoxic potential of eugenol in vivo . Furthermore, they suggest genotoxicity in vivo of eugenol per se.

Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7381 - 6
High affinity type I interleukin 1 receptor antagonists discovered by screening recombinant peptide libraries; Yanofsky SD et al.; Two families of peptides that specifically bind the extracellular domain of the human type I interleukin I (IL-1) receptor were identified from recombinant peptide display libraries . Peptides from one of these families blocked binding of IL-lalpha to the type I IL-1 receptor with IC50 values of 45-140 microM . Affinity-selective screening of variants of these peptides produced ligands of much higher affinity (IC50 approximately 2 nM) . These peptides block IL-1-driven responses in human and monkey cells; they do not bind the human type II IL-1 receptor or the murine type I IL-1 receptor . This is the first example (that we know of) of a high affinity peptide that binds to a cytokine receptor and acts as a cytokine antagonist.

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