Escherichia coli

Protein Expr Purif, 1996 Dec, 8(4), 463 - 8
Purification of porcine phospholamban expressed in Escherichia coli; Yao Q et al.; Phospholamban (PLB) is a small hydrophobic protein that regulates contractility in the heart . This membrane protein expressed in bacterial cells is resistant to purification by conventional strategies that have been used to isolate expressed soluble proteins . Therefore, in order to obtain both wild-type and mutant PLB proteins, we have amplified the PLB gene by the polymerase chain reaction from genomic DNA of porcine heart and inserted it into the pGEX-2T plasmid expression vector . In this vector, the gene product fused to glutathione S-transferase has been expressed in JM109 Escherichia coli cells . The expressed fusion protein was found associated predominantly with insoluble cellular constituents . However, most of the fusion protein was readily extracted with SDS . PLB was subsequently purified by a simple procedure consisting of isolation of the fusion protein by preparative SDS-gel electrophoresis, followed by a second electrophoretic separation of PLB after its cleavage from the fusion protein by thrombin . This isolation method yields 3-4 mg of PLB per liter of cells, in a form which is capable of functional interaction with the Ca-ATPase in reconstituted proteoliposomes.

Protein Expr Purif, 1996 Dec, 8(4), 430 - 8
Isolation, overexpression, and biochemical characterization of the two isoforms of glutamic acid decarboxylase from Escherichia coli; De Biase D et al.; Escherichia coli glutamic acid decarboxylase is a pyridoxal phosphate-dependent enzyme that catalyzes the alpha-decarboxylation of glutamate to yield 4-amino-butyrate and CO2 . The E . coli chromosome contains two genes encoding for this enzyme, gadA and gadB, which map at distinct loci . Their protein products differ in only five amino acid residues, four of which are located in the N-terminal region (Smith et al., 1992, J . Bacteriol . 174, 5820-5826) . Herein, we report the sequences of the two gad genes, including their regulatory regions . Both genes were separately cloned into the vector pQE60, for overexpression under the control of the lac promoter . In this way, we have succeded in separately expression large quantities of each pure isoform . The two isoforms were characterized biochemically and all evidence, including that from analysis of the complex pre-steady-state kinetic behavior of the enzymes, indicates that the functional properties of the two isoenzymes are identical.

Protein Expr Purif, 1996 Dec, 8(4), 423 - 9
Two phenol sulfotransferase species from one cDNA: nature of the differences; Yang YS et al.; A phenol sulfotransferase from rat liver (EC 2.8.2.9), expressed in Escherichia coli from a single cDNA, was purified as two separable but catalytically active proteins . The proteins appeared to be identical to each other and to the natural liver sulfotransferase by comparison of their amino acid constitution, amino-terminal end group, and interaction with a polyclonal antibody raised against the liver enzyme . Each of the recombinant forms, alpha and beta, catalyzed the sulfuryl group transfer from 4-nitrophenylsulfate to an acceptor phenol, a reaction in which 3'-phospho-adenosine 5'-phosphate (PAP) is a necessary intermediate . Only form beta, however, catalyzes the physiological transfer of a sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the free phenol . Evidence is presented that sulfotransferase alpha, but not beta, has 1 mol of PAP tightly bound per enzyme dimer . The ability to utilize PAPS as a sulfate donor could be altered: form alpha could be treated and purified as form beta to acquire the ability to use PAPS, whereas form beta was treated by extended incubation with PAP, lost its ability to use PAPS, and was purified as form alpha.

Protein Expr Purif, 1996 Dec, 8(4), 409 - 15
Cloning of cDNA encoding rat spermatidal protein TP2 and expression in Escherichia coli; Meetei AR et al.; Rat spermatidal protein TP2 is a basic nuclear protein containing two atoms of zinc bound per molecule . We report here cloning of complementary DNA encoding rat TP2 by the RT-PCR method . The nucleotide sequence of cloned TP2 cDNA differs at a few positions from the sequence already reported in the literature . We have cloned rat TP2 cDNA into the expression vector pTrc 99A . Upon induction with 1 mM IPTG, there was a low level of expression of TP2 which could be recovered in the soluble form . Recombinant TP2 was purified from the soluble form . Recombinant TP2 was purified from the soluble extract of E . coli using nickel-agarose and heparin-agarose chromatography and was shown to be identical to native rat TP2 as revealed by immunoblotting with anti-rat TP2 antibodies and radioactive 65Zn-blotting.

Genomics, 1996 Dec 1, 38(2), 238 - 42
Molecular cloning and characterization of the mouse Kin17 gene coding for a Zn-finger protein that preferentially recognizes bent DNA; Tissier A et al.; We report the isolation of the mouse Kin17 gene, located on chromosome 2, coding a nuclear Zn-finger protein that has a 39-residue region homologous to Escherichia coli RecA protein and that is recognized by anti-RecA antibodies . Kin17 protein preferentially binds to curved DNA in vitro and in vivo, suggesting a role in illegitimate recombination and in regulation of gene expression . We have shown that the Kin17 gene is about 8 kb in length and displays three exons and two introns . The 5' flanking region lacks a canonical TATAA box but presents several putative regulatory domains . A major transcription initiation site is located 322 nucleotides upstream of the translation start site . The 1.7-kb transcript of the Kin17 gene is weakly and ubiquitously expressed in murine tissues and cell lines as determined by Northern analysis . The cross-hybridization of Kin17 cDNA with the genomic DNA of other species in Southern analysis indicates the conservation of the gene among mammals and suggests that the Kin17 gene plays a conserved role in DNA metabolism.

Gen Comp Endocrinol, 1996 Dec, 104(3), 330 - 6
Cloning and expression of somatolactin, a pituitary hormone related to growth hormone and prolactin from gilthead seabream, Sparus aurata; Astola A et al.; A pituitary hormone, somatolactin (SL), belonging to the GH/PRL family, is produced in the intermediate lobe of the teleost pituitary . The function of this protein is uncertain . Clones coding for SL were isolated and sequenced from a gilthead seabream pituitary cDNA expression library . The nucleotide sequence of the larger cDNA isolated was 1.5 kb containing a 0.8-kb 3'-untranslated region and two potential polyadenylation signals (AATAAA) . The mature polypeptide is composed of 207 amino acids, and a signal peptide of 24 residues was also found in the SL precursor . A potential N-glycosylation site Asn-Lys-Thr was identified in gilthead seabream SL . A comparison of the SL amino acid sequences of several fishes indicated that seven cysteine residues are characteristically present in all the SLs so far isolated . Six of those residues are present in homologous positions in SL and GH Sparus aurata proteins . SL and GH from S . aurata showed a 43% homology at the nucleotide level and 22% identity at the amino acid level . Expression of recombinant SL (rSL) in Escherichia coli and isolation from inclusion bodies led to a monomeric form of SL identical in electrophoretic mobility to one of the two forms of the native SL secreted from gilthead seabream pituitaries cultured in vitro . Further, a native glycosylated modified SL secreted in vitro as shown by N-glycosidase treatment was identified . Specific anti-SL antibodies that discriminate well against gilthead sea-bream GH and PRL in immunoblotting were also raised against rSL.

Anal Biochem, 1996 Dec 1, 243(1), 176 - 80
Construction of biotinylated firefly luciferases using biotin acceptor peptides; Tatsumi H et al.; A cDNA for a thermostable mutant of Luciola lateralis (a Japanese firefly) luciferase was fused with either a gene for an artificial biotin acceptor peptide No . 84 {P . J . Schatz (1993) Bio/Technology 11, 1138-1143} or a gene for the carboxyl-terminal 87 residues of Escherichia coli biotin carboxyl carrier protein . The fused genes, when introduced into separate E . coli, directed the expression of luciferases that were able to bind with streptavidin . We purified them and showed that their specific activities and thermal stabilities remained unchanged . We also found that more than 95% of each fusion protein was biotinylated, suggesting that the biotin holoenzyme synthetase in the host cells worked efficiently . Using the biotinylated luciferases, we developed a highly sensitive bioluminescent enzyme immunoassay system.

Anal Biochem, 1996 Dec 1, 243(1), 150 - 3
A nonradioactive assay for ribosome-inactivating proteins; Langer M et al.; A sensitive nonradioactive method to determine the activity of ribosome-inactivating proteins (RIPs) based on a combined transcription/translation in vitro assay was established . Using this assay we investigated the RIP activities of the heterodimeric toxic plant lectins ricin and mistletoe lectin I (ML-I) . The enzymatic activities of the holoproteins were compared to that of the RIP-active chain of ML-I (ML-I A-chain) and recombinant ML-I A-chain expressed in Escherichia coli . The IC50 values determined for the plant toxins showed that the translation-inactivating activity of ML-I (39.8 pM) is about four times higher than that of ricin (176.0 pM) . The plant-derived ML-I A-chain is more toxic (3.4 pM) in the cell-free translation system than the respective holoprotein . The recombinant ML-I A-chain was found to be about three times less active (IC50 10.6 pM) than the A-chain from plant . The in vitro assay described here is a convenient method for the fast determination of RIP activity with a 1000-fold lower detection limit than that of commonly used RIP assays.

Appl Environ Microbiol, 1996 Dec, 62(12), 4621 - 6
Retention of enteropathogenicity by viable but nonculturable Escherichia coli exposed to seawater and sunlight; Pommepuy M et al.; The effect of natural sunlight on culturability and persistence of pathogenicity of Escherichia coli was examined in the field, i.e., in the Morlaix Estuary, France, using an enterotoxigenic strain of Escherichia coli H10407 . Results showed that E . coli responds to the estuarine diurnal solar cycle by entering the viable but nonculturable state upon exposure to sunlight . That is, direct counts of viable cells remained stable without significant change, but E . coli cells remained fully culturable only when exposed to seawater in control chambers in the dark, i.e., without solar irradiation . The effect of sunlight on the pathogenicity of E . coli H10407 was studied, using both the rabbit intestinal loop assay and ganglioside-enzyme-linked immunosorbent assay (GM1-ELISA), a sensitive procedure for testing for production of enterotoxin . Results of the GM1-ELISA demonstrated that strains of E . coli, after exposure to sunlight and entering the viable but nonculturable state, as well as culturable E . coli, retained pathogenicity, i.e., produced enterotoxin . The GM1-ELISA is concluded to be more sensitive than the rabbit intestinal loop assay for analysis of enterotoxin in natural water samples.

Hum Gene Ther, 1996 Dec 1, 7(18), 2235 - 45
Killing Epstein-Barr virus-positive B lymphocytes by gene therapy: comparing the efficacy of cytosine deaminase and herpes simplex virus thymidine kinase; Rogers RP et al.; Epstein-Barr virus (EBV)-positive lymphomas are frequent among immunosuppressed patients . We have examined the feasibility of killing EBV-immortalized B lymphocytes by gene transfer involving the use of "suicide" genes whose expression in target cells renders them susceptible to killing by a prodrug . We examined two gene/prodrug pairs: the Escherichia coli cytosine deaminase (CD) gene with the prodrug 5-fluorocytosine (5-FC), and the herpes simplex virus thymidine kinase (HSV-TK) gene with the prodrug ganciclovir . Retroviral vectors and drug selection were used to obtain CD or HSV-TK expression in cells . Both the CD/5-FC and the HSV-TK/ganciclovir combinations yielded substantial killing of EBV-immortalized B lymphocytes in vitro, although the CD/5-FC regimen had a significantly greater therapeutic margin than the HSV-TK/ganciclovir combination . The CD/5-FC pair, but not the HSV-TK/ganciclovir pair, was shown to have a "bystander killing effect" in vitro . When only 30% of the cells expressed the suicide gene, scid mouse tumors regressed in both the CD/5-FC regimen and the HSV-TK/ganciclovir regimen, documenting an in vivo bystander effect with both regimens . However, a greater percentage of tumors completely regressed with the CD/5-FC regimen . Overall, the sum of our data indicates that the CD/5-FC combination is the more promising regimen for treatment of EBV-associated lymphomas in vivo.

Hum Gene Ther, 1996 Dec 1, 7(18), 2217 - 24
Rapid production of interleukin-2-secreting tumor cells by herpes simplex virus-mediated gene transfer: implications for autologous vaccine production; Tung C et al.; Production of autologous tumor vaccines would be facilitated by the development of a rapid and efficient method for the transfer of genes into freshly isolated cells . To evaluate the potential of replication defective herpes simplex viral (HSV) amplicon vectors as gene transfer vehicles for tumor vaccine generation, a vector that expresses the human interleukin-2 (IL-2) gene (HSV-IL2) and one that expresses Escherichia coli beta-galactosidase (HSVlac) were tested in hepatoma cells of both murine and human origin . Gene transfer into murine hepatoma cells (HEPA 1-6) was both rapid and highly efficient: greater than 50% of cells expressed beta-Gal when infected at a multiplicity of infection (m.o.i.) of 1 with an exposure period of 20 min . Moreover, gene transfer was as efficient in tumor cells after irradiation with 10,000 rads as in nonirradiated tumor cells . Irradiated HEPA 1-6 cells infected with HSV-IL2 for 20 min secreted IL-2 at a rate of 1,200 +/- 160 ng/10(6) cells per day . C57B1/6J mice immunized with irradiated, HSV-IL-2-transduced tumor cells produced in this way demonstrated specific tumor immunity by in vitro splenocyte tumoricidal activity and by in vivo protection against tumor challenge . Human hepatobiliary tumor specimens harvested at the time of operation, irradiated, and infected with HSV-IL-2 also produced nanogram quantities of IL-2/10(6) cells per 24 hr . These results indicate that the HSV amplicon vector is a good candidate vehicle for gene transfer in the production of autologous tumor vaccines . By allowing rapid gene transfer to freshly harvested tumor specimens, these vectors bypass the requirement for cell culture and make feasible reinfusion of genetically modified and irradiated autologous cells within hours of tumor harvest.

Free Radic Res, 1996 Dec, 25(6), 541 - 6
Determination of manganese superoxide dismutase activity by direct spectrophotometry; Bolann BJ et al.; A method to determine Mn-superoxide dismutase activity by measuring directly the rate of decay of O2- in a spectrophotometer, is described . Decay of O2- generated by KO2 at pH 9.5, was monitored as the fall in absorbance (A250nm-A360nm) . Mn-superoxide dismutase was determined as the activity of cyanide-resistant superoxide dismutase, calculated from the rate of O2- dismutation . Mn-superoxide dismutase could be determined in the presence of a 700 times higher Cu,Zn-superoxide dismutase activity . The alkaline pH did not cause analytical problems . The assay was used to measure both Mn- and Cu,Zn-superoxide dismutase activity in mitochondrial preparations . The assay had a detection limit of 2.8 ng/ml when Mn-superoxide dismutase from E . coli was used, and the between-day CV was 5.8% . The assay is an alternative to indirect methods for detecting superoxide dismutase activity.

Chem Res Toxicol, 1996 Dec, 9(8), 1375 - 81
Mechanistic studies of the inhibition of MutT dGTPase by the carcinogenic metal Ni(II); Porter DW et al.; Promutagenic 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) levels are increased in DNA of animals exposed to carcinogenic metals, such as Ni(II) . Besides being generated directly in genomic DNA, 8-oxo-dG may be incorporated there from 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP), a product of oxidative damage to the nucleotide pool . The Escherichia coli dGTPase MutT, and analogous dGTPases in rats and humans, have been suggested as a defense against such incorporation because they hydrolyze 8-oxo-dGTP to 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-monophosphate (8-oxo-dGMP) . MutT and its mammalian counterparts are Mg(II)-dependent enzymes . Ni(II), in turn, is known to interact antagonistically with Mg(II) in biological systems . Thus, we hypothesized that Ni(II) might inhibit the activity of MutT . As an initial examination of this hypothesis, we conducted enzyme kinetic studies of MutT to determine the effect of Ni(II) on MutT activity and the mechanisms involved . As found, Ni(II) inhibited MutT in a concentration-dependent manner when either dGTP or 8-oxo-dGTP was the nucleotide substrate . Ni(II) was determined to be an uncompetitive inhibitor of MutT with respect to Mg(II) when dGTP was the substrate, with apparent Ki of 1.2 mM Ni(II), and a noncompetitive inhibitor with respect to Mg(II) when 8-oxo-dGTP was the substrate, with apparent Ki of 0.9 mM Ni(II) . Hence, the two metal cations did not compete with each other for binding at the MutT active site . This makes it difficult to predict Ni(II) effects on 8-oxo-dGTPases of other species . However, based upon the amino acid sequences of human and rat MutT-like dGTPases, their capacity for Ni(II) binding should be greater than that of MutT . Whether this could lead to stronger inhibition of those enzymes by Ni(II), or not, remains to be investigated.

Chem Res Toxicol, 1996 Dec, 9(8), 1350 - 4
Using UvrABC nuclease to detect 7,12-dimethylbenz{a}anthracene anti-diol epoxide-DNA binding specificity in the mouse H-ras gene; Chen JX et al.; DNA fragments modified with chemically synthesized 7,12-dimethylbenz{a}anthracene anti-diol epoxide (anti-DMBADE) are sensitive to UvrABC nuclease incision . The incisions occur mainly 7 bases 5' and 4 bases 3' of an anti-DMBADE-modified adenine or guanine residue, and the kinetics of incision at different sequences in a DNA fragment are the same . These results indicate that UvrABC incision on anti-DMBADE-DNA adducts is independent of DNA sequences and is quantitative, the same as on syn-DMBADE-DNA adducts . This method was used to analyze the anti-DMBADE-DNA binding spectrum in the exon 2 region of the mouse H-ras gene, and it was found that anti-DMBADE binds to the two adenine residues at codon 61 of the H-ras gene with an average affinity . Previously, we have demonstrated that syn-DMBADE binds strongly to the adenines at codon 61 of H-ras; these results together suggest that the oncogenic mutation in H-ras may be induced by anti- and syn-DMBADE-DNA adducts.

Chem Res Toxicol, 1996 Dec, 9(8), 1278 - 84
Synthesis, miscoding specificity, and thermodynamic stability of oligodeoxynucleotide containing 8-methyl-2'-deoxyguanosine; Kohda K et al.; 8-Methyl-2'-deoxyguanosine (8-MedG) was synthesized by reacting dG under the methyl radical generating system and incorporated into oligodeoxynucleotides using phosphoramidite techniques . The site-specifically modified oligodeoxynucleotide containing a single 8-MedG was then used as a template for primer extension reactions catalyzed by the 3' --> 5' exonuclease-free (exo-) Klenow fragment of Escherichia Coli DNA polymerase I and mammalian DNA polymerase alpha . Primer extension catalyzed by the exo- Klenow fragment readily passed the 8-MedG lesion in the template while that catalyzed by pol alpha was retarded opposite the lesion . The fully extended products formed during DNA synthesis were analyzed to quantify the miscoding specificities of 8-MedG . Both DNA polymerases incorporated primarily dCMP, the correct base opposite the lesion, along with small amounts of incorporation of dGMP and dAMP . In addition, two-base deletion was observed only when the exo- Klenow fragment was used . The thermodynamic stability of 8-MedG in the duplex was also studied . The duplex containing 8-MedG:dG was more thermally and thermodynamically stable than that of dG:dG . The duplex containing 8-MedG:dA was more thermodynamically stable than that of dG:dA . We conclude that 8-MedG is a miscoding lesion and capable of generating G --> C and G --> T transversions and deletion in cells.

Arch Biochem Biophys, 1996 Dec 1, 336(1), 151 - 6
Human endothelial nitric oxide synthase: expression in Escherichia coli, coexpression with calmodulin, and characterization; Rodriguez-Crespo I et al.; Human endothelial nitric oxide synthase (eNOS) has been cloned and expressed in Escherichia coli . The spectroscopic properties and specific activity (100-130 nmol x min(-1) x mg(-1) at 37 degrees C) of the recombinant protein are similar to those of the bovine enzyme . FPLC and low-temperature SDS-PAGE indicate that the protein is mostly dimeric in both the absence and presence of tetrahydrobiopterin . Human eNOS thus has a higher tendency to dimerize than the bovine enzyme . A chloramphenicol-resistant, trc promoter-based plasmid has been constructed that allows coexpression of human calmodulin (CaM) . Coexpression of CaM increases more than threefold the amount of expressed eNOS, stabilizes the recombinant protein, and significantly augments its specific activity (to 140-170 nmol x min(-1) x mg(-1) at 37 degrees C) . The cytochrome c reduction activity is also improved by CaM coexpression . These increases in activity are not achieved by the addition of CaM to eNOS expressed in the absence of CaM . Gel filtration studies suggest that CaM coexpression produces a more elongated eNOS structure and alters the NADPH binding domain . CaM coexpression has been shown previously to be required for successful expression of the inducible NOS isoform, but this is the first demonstration that CaM coexpression improves the expression of a constitutive isoform.

Arch Biochem Biophys, 1996 Dec 1, 336(1), 105 - 12
Cloning and expression of a cDNA encoding uridine kinase from mouse brain; Ropp PA et al.; Uridine kinase is the rate-limiting enzyme in the pyrimidine salvage pathway of all mammalian cells . A cDNA for uridine kinase from mouse brain has been isolated, sequenced, and characterized . This is the first report of a complete nucleotide sequence for mammalian uridine kinase . The isolated cDNA is only 95% complete, missing the first 17 codons . The correct 5'-terminus sequence was obtained from high-stringency screening of a mouse liver genomic DNA library . The translated cDNA sequence encodes a protein of 277 amino acids (Mr 31,068) . A truncated form of the cDNA was expressed in Escherichia coli . The expressed protein displayed uridine kinase activity and readily formed a tetramer, the most active form of the wild-type enzyme . Analysis of the amino acid sequence identified the three ATP-binding site consensus motifs . The predicted secondary structure for uridine kinase and the sequence comparison with three kinases having known crystal structures are consistent with uridine kinase having an alpha/beta core structure of the nucleotide-binding fold found in many kinases . We have also isolated and cloned a nonfunctional, processed pseudogene from mouse genomic DNA . This pseudogene sequence is 94% identical with the coding DNA.

Pediatr Res, 1996 Dec, 40(6), 822 - 6
Endotoxin induces biphasic alterations in small intestinal myoelectric activity in fasted newborn piglets; Li JX et al.; Previous studies have shown that i.v . endotoxin infusion causes gastrointestinal dysfunction and intestinal injury in piglets . The aim of this study was to investigate the effects of endotoxin on intestinal myoelectric activity in newborn swine and to correlate this with gastrointestinal and hemodynamic events . Three pairs of electrodes were implanted in the jejunal wall of piglets, and after recovery, intestinal myoelectric activity was continuously recorded in the conscious, fasted condition . The intestinal myoelectric activity on the control day showed regular, repeating migrating myoelectric complex (MMC) cycles, each of which was composed of the classic phases I, II, and III . Mean cycle duration was 67.0 +/- 18.7 min (+/- SD), and phase III comprised 9.1 +/- 2.2% of each cycle . On the next day, infusion of 30 micrograms/kg endotoxin caused an initial, prolonged quiescent period and delayed the appearance of the first postendotoxin phase III complex . After the quiescent period, there was a period of irregular spiking activity followed by several shortened MMC cycles (47.9 +/- 22.7 min, p < 0.01 versus control) with a prolongation of the percentage of time spent in phase III (15.4 +/- 11.3%, p < 0.01) . Endotoxin thus produced biphasic alterations in intestinal myoelectric activity characterized by an initial quiescence followed by increased gastrointestinal smooth muscle activity . Animals developed diarrhea, hypotension, and tachycardia about 1 h after endotoxin infusion in temporal association with increased spiking activity and MMC cycling . These studies are the first to show this biphasic response to endotoxin.

Infect Immun, 1996 Dec, 64(12), 5434 - 8
Mutations in the A subunit affect yield, stability, and protease sensitivity of nontoxic derivatives of heat-labile enterotoxin; Magagnoli C et al.; Heat-labile toxin (LT) is a protein related to cholera toxin, produced by enterotoxigenic Escherichia coli strains, that is organized as an AB5 complex . A number of nontoxic derivatives of LT, useful for new or improved vaccines against diarrheal diseases or as mucosal adjuvants, have been constructed by site-directed mutagenesis . Here we have studied the biochemical properties of the nontoxic mutants LT-K7 (Arg-7-->Lys), LT-D53 (Val-53-->Asp), LT-K63 (Ser-63-->Lys), LT-K97 (Val-97-->Lys), LT-K104 (Tyr-104-->Lys), LT-K114 (Ser-114-->Lys), and LT-K7/K97 (Arg-7-->Lys and Val-97-->Lys) . We have found that mutations in the A subunit may have profound effects on the ability to form the AB5 structure and on the stability and trypsin sensitivity of the purified proteins . Unstable mutants, during long-term storage at 4 degrees C, showed a decrease in the amount of the assembled protein in solution and a parallel appearance of soluble monomeric B subunit . This finding suggests that the stability of the B pentamer is influenced by the A subunit which is associated with it . Among the seven nontoxic mutants tested, LT-K63 was found to be efficient in AB5 production, extremely stable during storage, resistant to proteolytic attack, and very immunogenic . In conclusion, LT-K63 is a good candidate for the development of antidiarrheal vaccines and mucosal adjuvants.

Infect Immun, 1996 Dec, 64(12), 5413 - 6
Mutants of the Escherichia coli heat-labile enterotoxin with reduced ADP-ribosylation activity or no activity retain the immunogenic properties of the native holotoxin; de Haan L et al.; The Escherichia coli heat-labile enterotoxin (LT) is a potent inducer of mucosal immune responses . In a previous study (L . DeHaan, W . R . Verweij, M . Holtrop, E . Agsteribbe, and J . Wilschut, Vaccine 14:620-626, 1996), we have shown that efficient induction of an LTB-specific mucosal immune response by LT requires the presence of the LTA chain, suggesting a possible role of the enzymatic activity of LTA in the induction of these responses . In the present study, we generated LT mutants with altered ADP-ribosylation activities and evaluated their immunogenicity upon intranasal administration to mice . The results demonstrate that the mucosal immunogenicity of LT is not dependent on its ADP-ribosylation activity.

Infect Immun, 1996 Dec, 64(12), 5390 - 4
Influence of cloned tRNA genes from a uropathogenic Escherichia coli strain on adherence to primary human renal tubular epithelial cells and nephropathogenicity in rats; Susa M et al.; The uropathogenic Escherichia coli strain 536 (O6:K15:H31) possesses pathogenicity islands which are incorporated into two tRNA genes, the selC and the leuX gene . The leuX gene influences the expression of different putative virulence factors . We demonstrate an effect of the leuX-specific tRNA on adherence and uropathogenicity.

Infect Immun, 1996 Dec, 64(12), 5290 - 4
Biphasic, organ-specific, and strain-specific accumulation of platelets induced in mice by a lipopolysaccharide from Escherichia coli and its possible involvement in shock; Shibazaki M et al.; Platelets contain a large amount of 5-hydroxytryptamine (5HT, serotonin) . Intravenous injection into BALB/c mice of a Boivin's preparation of lipopolysaccharide (LPS) from Escherichia coli induced rapid 5HT accumulation in the lung (within 5 min) and slow 5HT accumulation in the liver (2 to 5 h later) . The rapid response required high doses of LPS (more than 0.1 mg/kg) . On the basis of 5HT measurements, 70% or more of the platelets which disappeared from the blood appeared to have accumulated rapidly in the lung, and a large number of platelets were found there by electron microscopy . A shock, which was manifested by crawling, convulsion, or prostration, followed shortly after the rapid accumulation of 5HT in the lung . On the other hand, the slow accumulation of 5HT in the liver could be induced by much lower doses of LPS (1 microg/kg or less), even when given by intraperitoneal injection . This 5HT accumulation appears to be a reflection of platelet accumulation in the liver (Y . Endo and M . Nakamura, Br . J . Pharmacol . 105:613-619, 1992) . The combination of a low dose of LPS with D-galactosamine amplified the hepatic accumulation of 5HT, and the mice developed a severe hepatic congestion resulting in death . The rapid response was not induced at all in C3H/HeN mice . These results and comparison with other LPS preparations indicate that some component(s) of LPS from E . coli induces a biphasic, organ-specific and strain-specific accumulation of platelets, and it is proposed that this effect is involved in the development of shock.

Infect Immun, 1996 Dec, 64(12), 5171 - 7
A 35-kilodalton protein is a major target of the human immune response to Mycobacterium leprae; Triccas JA et al.; The control of leprosy will be facilitated by the identification of major Mycobacterium leprae-specific antigens which mirror the immune response to the organism across the leprosy spectrum . We have investigated the host response to a 35-kDa protein of M . leprae . Recombinant 35-kDa protein purified from Mycobacterium smegmatis resembled the native antigen in the formation of multimeric complexes and binding by monoclonal antibodies and sera from leprosy patients . These properties were not shared by two forms of 35-kDa protein purified from Escherichia coli . The M . smegmatis-derived 35-kDa protein stimulated a gamma interferon-secreting T-cell proliferative response in the majority of paucibacillary leprosy patients and healthy contacts of leprosy patients tested . Cellular responses to the protein in patients with multibacillary leprosy were weak or absent, consistent with hyporesponsiveness to M . leprae characteristic of this form of the disease . Almost all leprosy patients and contacts recognized the 35-kDa protein by either a T-cell proliferative or an immunoglobulin G antibody response, whereas few tuberculosis patients recognized the antigen . This specificity was confirmed in guinea pigs, with the 35-kDa protein eliciting strong delayed-type hypersensitivity in M . leprae-sensitized animals but not in those sensitized with Mycobacterium tuberculosis or Mycobacterium bovis BCG . Therefore, the M . leprae 35-kDa protein appears to be a major and relatively specific target of the human immune response to M . leprae and is a potential component of a diagnostic test to detect exposure to leprosy or a vaccine to combat the disease.

Infect Immun, 1996 Dec, 64(12), 5144 - 50
Identification of ligand recognition sites in heat-stable enterotoxin receptor, membrane-associated guanylyl cyclase C by site-directed mutational analysis; Wada A et al.; Guanylyl cyclase C (STaR), a receptor protein for heat-stable enterotoxin (STa) elaborated by Escherichia coli, is associated with and spans the plasma membrane of mammalian intestinal cells . The extracellular domain functions in the binding of STa and the association of each domain to an oligomeric form . Two amino acid residues, Arg-136 and Asp-347, were identified as the residues binding to STa in the extracellular domain of pig STaR by site-directed mutagenesis and analysis of expression on 293T cells . Replacement of these residues by other amino acid residues resulted in the loss of binding of pig STaR to STa, and as a result, STa-induced guanylyl cyclase activity was eliminated . Furthermore, mutation in a region (from Asp-347 to Val-401) which is close to the transmembrane domain caused a significant reduction in both STa-binding activity and guanylyl cyclase catalytic activity . These results suggest that the region adjacent to the transmembrane domain plays an important role in facilitating a favorable conformation of STaR for STa binding.

Infect Immun, 1996 Dec, 64(12), 4967 - 75
Cloning and expression of the complement fixation antigen-chitinase of Coccidioides immitis; Zimmermann CR et al.; A chitinase had been isolated from the culture filtrates of Coccidioides immitis endosporulating spherules and from hyphae and shown to be the coccidioidal complement fixation (CF) and immunodiffusion-CF antigen . In the present study, we made use of our previously determined amino-terminal (N-terminal) sequence of the CF-chitinase to design degenerate oligonucleotide primers and to amplify and sequence a PCR product that coded for the N-terminal portion of the CF-chitinase . The PCR product was used as a hybridization probe to screen a developing spherule-(lambda)ZAP cDNA library, and three hybridizing clones were selected . These clones were converted into their pBluescript expression plasmid form in Escherichia coli and induced to express their recombinant proteins . Lysate from only one clone, pCTS 4-2A, yielded an enzymatically functional CF-chitinase and a line of identity with control immunodiffusion-CF-positive antigen . The pCTS 4-2A insert was sequenced and found to contain a deduced open reading frame coding for a 427-amino-acid polypeptide with an approximate molecular weight of 47 kDa . When purified by a chitin adsorption-desorption method, the recombinant protein exhibited virtually identical characteristics to those of the original C . immitis CF-chitinase . Nondenaturing gels of the pCTS 4-2A E . coli lysates and the purified C . immitis and recombinant CF-chitinase revealed proteins that had chitinase activity and similar relative electrophoretic mobilities . The appearance and relative levels of hybridizing RNA from the developing spherules-endospores (SEs) and hyphae correlated with the appearance or presence and level of CF-chitinase enzyme activity found in SEs culture filtrate and in cellular extracts of developing SE and hyphae . Thus, a functional recombinant CF-chitinase antigen was produced in E . coli and was used in serological diagnostic applications . These results also suggest a functional role for this chitinase in SE development and maturation.

Nat Genet, 1996 Dec, 14(4), 430 - 40
Identification of a RING protein that can interact in vivo with the BRCA1 gene product; Wu LC et al.; The hereditary breast and ovarian cancer gene, BRCA1, encodes a large polypeptide that contains the cysteine-rich RING motif, a zinc-binding domain found in a variety of regulatory proteins . Here we describe a novel protein that interacts in vivo with the N-terminal region of BRCA1 . This BRCA1-associated RING domain (BARD1) protein contains an N-terminal RING motif, three tandem ankyrin repeats, and a C-terminal sequence with significant homology to the phylogenetically conserved BRCT domains that lie near the C terminus of BRCA1 . The BARD1/BRCA1 interaction is disrupted by BRCA1 missense mutations that segregate with breast cancer susceptibility, indicating that BARD1 may be involved in mediating tumour suppression by BRCA1.

J Immunol, 1996 Dec 1, 157(11), 4953 - 62
Molecular characterization of Bip 1, a monoclonal antibody that modulates IgE binding to birch pollen allergen, Bet v 1; Laffer S et al.; Bet v 1 and homologous proteins represent major cross-reactive allergens for more than 95% of tree pollen-, fruit-, and vegetable-allergic individuals . To study the interaction of Bet v 1 and the immune system, we characterized a Bet v 1-specific mAb, Bip 1 . Soluble rBip 1 Fabs were expressed in Escherichia coli and purified by affinity chromatography using immobilized Bet v 1 . Bip 1 Fabs displayed a cross-reactivity to homologous allergens comparable with that of IgE Abs from allergic patients . Preincubation of Bet v 1 with Bip 1 led to an up to fivefold increase of allergic patients' IgE binding to Bet v 1 . This enhancement in IgE binding may be interpreted as stabilization of a Bet v 1 state, in which certain IgE epitopes are better applicable . It also shows that allergic patients possess IgE Abs directed against different Bet v 1 conformations . The modulation of Ab binding to a given Ag by other Abs was observed also for human Bet v 1-specific IgG Abs, and may represent a novel mechanism for the regulation of specific humoral immune responses in a complex network.

Mol Cell Biol, 1996 Dec, 16(12), 7133 - 43
A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53; Lim DS et al.; RecA in Escherichia coli and its homolog, ScRad51 in Saccharomyces cerevisiae, are known to be essential for recombinational repair . The homolog of RecA and ScRad51 in mice, MmRad51, was mutated to determine its function . Mutant embryos arrested early during development . A decrease in cell proliferation, followed by programmed cell death and chromosome loss, was observed . Radiation sensitivity was demonstrated in trophectoderm-derived cells . Interestingly, embryonic development progressed further in a p53 null background; however, fibroblasts derived from double-mutant embryos failed to proliferate in tissue culture.

Virology, 1996 Dec 1, 226(1), 146 - 51
RNA polymerase activity catalyzed by a potyvirus-encoded RNA-dependent RNA polymerase; Hong Y et al.; We have expressed the putative RNA-dependent RNA polymerase encoded by the potyvirus tobacco vein mottling virus (TVMV) in Escherichia coli as a glutathione S-transferase fusion protein . As prepared, the fusion protein possessed the poly(U) polymerase activity that is a hallmark of other picornavirus-encoded polymerases . In addition, this protein was able to utilize full-length TVMV RNA as a template for RNA synthesis . A fusion protein containing a mutation in the highly conserved GDD motif of the polymerase (GDD-->ADD) possessed 7% of the activity of the wild type . Our results confirm that the presumed polymerase encoded by TVMV is in fact an RNA-dependent RNA polymerase and that the GDD motif so widely seen in viral polymerases has an important function in the TVMV protein.

Virology, 1996 Dec 1, 226(1), 127 - 31
Inducible gene expression of the human immunodeficiency virus LTR in a replication-incompetent herpes simplex virus vector; Warden MP et al.; Although replication-incompetent herpes simplex virus (HSV) vectors have the capability to express foreign genes, successful development of these vectors for gene delivery would require that expression of the foreign gene be regulated . To investigate the feasibility of obtaining inducible expression of a foreign gene in such a vector, a replication-incompetent HSV vector, vd120/LTR beta, was developed that used the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) to express the Escherichia coli lacZ gene . Examination of lacZ expression from the HIV-1 LTR in vd120/LTR beta-infected cells indicated that the LTR was active as a promoter under both replicating and nonreplicating conditions, although expression of lacZ under nonreplicating conditions was approximately 4-fold lower . In addition, the LTR expressed lacZ in a manner distinct from that of well-characterized HSV-1 promoters of each temporal class . The effect of the HIV-1 regulatory protein Tat on expression from the LTR in vd120/LTR beta was examined by infection of two different HeLa-derived cell lines that constitutively expressed Tat, HL2/3, and HLtat . Compared to infection of HeLa cells, lacZ expression from vd120/LTR beta-infected HL2/3 and HLtat cells increased from 4- to 24-fold, depending on the multiplicity of vector infection . Sustained expression of lacZ from the LTR in vd120/LTR beta-infected cells was not observed even in the continuous presence of Tat, although vector could be recovered for up to 5 days after infection . However, the amount of recoverable vector decreased during this time, suggesting that cellular cytotoxicity may account for some of the decrease in Tat-mediated expression from the LTR.

J Clin Microbiol, 1996 Dec, 34(12), 3233 - 4
Molecular epidemiology of Escherichia coli diarrhea in children in Hong Kong; Biswas R et al.; This pediatric hospital-based study of 388 diarrhea cases and 306 controls analyzed predominant E . coli colonies from primary culture (253 cases and 177 controls) with eight DNA probes for enteropathogenic, enterotoxigenic, enteroaggregative, and diffusely adherent E . coli . Only enteropathogenic E . coli adherence factor was identified significantly more frequently in cases (10) than in controls (0).

J Clin Microbiol, 1996 Dec, 34(12), 3160 - 4
Use of a recombinant Coccidioides immitis complement fixation antigen-chitinase in conventional serological assays; Johnson SM et al.; The coccidioidal complement fixation (CF) antigen has been cloned previously, and the fusion protein has been expressed in Escherichia coli . The recombinant CF (rCF) antigen was affinity purified by adsorption-desorption to chitin, and its reactivity was studied by using sera containing coccidioidal antibodies . The affinity-purified rCF antigen formed a line of identity with an immunodiffusion (ID) CF reference antigen (coccidioidin) derived from mycelial-phase Coccidioides immitis and was reactive with human, canine, and equine sera containing coccidioidal antibody . The affinity-purified rCF antigen yielded no detectable reaction with Blastomyces of Histoplasma antiserum by ID . The affinity-purified rCF antigen fixed complement with positive human sera and, even when used at lower concentrations, yielded titers comparable to those obtained with the coccidioidin . The reactivity of the affinity-purified rCF antigen was further evaluated by enzyme immunoassay, in which it manifested good sensitivity (96.9%) and specificity (100%) when evaluated with 43 human patients' sera . Thus, the affinity-purified rCF antigen has yielded reactions comparable to those of crude coccidioidal antigens in conventional CF, IDCF, and enzyme immunoassay.

J Clin Microbiol, 1996 Dec, 34(12), 3142 - 6
Specific amplification of Ehrlichia platys DNA from blood specimens by two-step PCR; Chang WL et al.; A two-step PCR method for diagnosis of canine infectious cyclic thrombocytopenia was established . Three primers derived from the 16S and rRNA gene sequence were used to amplify genomic DNA specifically from Ehrlichia platys . Two-round amplification with DNA templates prepared from E . platys-infected blood specimens produced 742- and 385-bp fragments, but these products were not found when an Ehrlichia canis-infected blood sample and Escherichia coli were used . This method, for which the minimum detectable copy number in the blood specimen was estimated to be five ehrlichia inclusion within platelets, is more sensitive than single PCR amplification . These results demonstrate that this two-step PCR is highly sensitive and efficient for detecting the etiologic agent of canine infectious cyclic thrombocytopenia in blood . The same technique was applied to blood specimens collected from a dog inoculated with E . platys . Amplification of the target DNA fragments was observed with blood collected on the fifth day after inoculation, which indicates that this method is also feasible for early diagnosis of E . platys infection.

J Clin Microbiol, 1996 Dec, 34(12), 2921 - 8
Rapid and specific detection of F17-related pilin and adhesin genes in diarrheic and septicemic Escherichia coli strains by multiplex PCR; Bertin Y et al.; The F17-related adhesins are prevalent in Escherichia coli strains isolated from calves with diarrhea or septicemia and from lambs with nephropathy . The F17 family includes the F17a, F17b, F17c, and F111 fimbriae produced by bovine E . coli strains and the G agglutinin produced by human uropathogenic E . coli strains . An easy and inexpensive multiplex PCR method was developed to detect all the F17-related fimbriae and to identify four subtypes of structural subunit genes and two distinct subfamilies of adhesin genes by only two runs of amplification . A strict correlation was observed between the phenotypic assays and the multiplex PCR method when 166 pathogenic E . coli strains isolated from intestinal content of calves or lambs were tested . Genes encoding the F17c structural subunit and the subfamily II adhesins were prominent among the bovine and ovine isolates, and the capsule-like CS31A antigen was strictly associated with the F17c fimbriae . The F17b subtype fimbriae were prominent among the bovine isolates producing the CNF2 toxin, whereas the F17a subtype fimbriae were associated with the bovine isolates producing neither the CS31A antigen nor the CNF2 toxin . Five bacterial strains possessed two distinct and complete F17-related fimbrial gene clusters, and two of them produced two F17-related fimbriae at the bacterial cell surface . The related fimbrial gene clusters are probably organized in mosaic operons consisting of F17-related pilin and adhesin genes, and horizontal gene transfer may occur among E . coli strains isolated form different animal species.

Endocrinology, 1996 Dec, 137(12), 5447 - 55
Molecular cloning of spermidine/spermine N1-acetyltransferase from the periimplantation porcine uterus by messenger ribonucleic acid differential display: temporal and conceptus-modulated gene expression; Green ML et al.; Using messenger RNA (mRNA) differential display, we isolated several putative differentially expressed complementary DNAs (cDNAs) from the periimplantation (days 11-12) endometrium of unilaterally pregnant pigs . Nucleotide sequence analysis revealed that one cDNA clone was 87% homologous to human spermidine/ spermine N1-acetyltransferase (SSAT) over a stretch of 201 bp and represents the porcine homologue of this cDNA . A second differentially expressed cDNA encoded the porcine equivalent of the human fragile X mental retardation gene (FMR1), whereas a third specified an open reading frame with significant homology to the Escherichia coli N-acetylglucosamine transfer protein . Because SSAT is the rate-limiting enzyme in polyamine metabolism and polyamines are required cytosolic components for cell growth and differentiation, we characterized the expression of the porcine SSAT gene as a potential marker for endometrial growth and/or differentiation during early pregnancy . Further, using the consensus sequence from human and mouse cDNAs, PCR primers were designed and used to generate a 568-bp cDNA fragment from gravid endometrium that encompassed the entire open reading frame for porcine SSAT and which was subsequently used for Northern hybridization analysis . Two distinct SSAT transcripts, a major species of 1.3 kilobase pairs (kb) and a minor species of 3.5 kb were detected in endometrium, each with similar temporal patterns of expression . The levels of SSAT mRNA were higher (P = 0.03) in gravid than in nongravid uterine endometrium of unilaterally pregnant pigs on days 11-12 . Similarly, SSAT mRNAs were more abundant (P = 0.0004) in day 12 pregnant than in day 12 cyclic, and in days 30, 60, 90, and 105 pregnant pig endometria . Uterine endometrial luminal epithelial (LE), glandular epithelial (GE), and stromal (ST) cells expressed the SSAT gene, but mRNA abundance varied among cell types (LE > GE > ST) . Expression of SSAT gene in ovariectomized gilts treated with estrogen (E2, 100 microg/day), progesterone (P4, 200 mg/day) or E2 + P4 for 11 days was highest (P = 0.03) in the endometria of the P4 group . In contrast, E2 (10 nM), P4 (10 nM) and E2 + P4 had no effect on SSAT mRNA abundance in uterine endometrial explants from day 12 pregnant pigs . However, steady-state SSAT mRNA levels were induced in day 12 pregnant uterine explants by conditioned medium from day 12 filamentous but not spherical conceptuses . These data demonstrate that the temporal induction of the endometrial SSAT gene during periimplantation is modulated by a factor(s) secreted by the periimplantation conceptus and suggest that this enzyme may have an important role in uterine endometrial growth, remodeling and/or differentiation during periimplantation.

Curr Genet, 1996 Dec, 30(6), 531 - 40
Molecular characterization of xyn3, a member of the endoxylanase multigene family of the rumen anaerobic fungus Neocallimastix frontalis; Durand R et al.; Different cDNAs designated xyn3 and xyn4 were isolated from an expression library of the anaerobic rumen fungus Neocallimastix frontalis . Xyn3 was further characterized and was shown to contain a single open reading frame of 1821 bp coding for a protein, XYN3, of 607 amino acids (Mr 66 000) . The predicted primary structure of XYN3 consisted of two large reiterated regions of 223 amino acids with a high degree of identity (88.3%) . Each domain of XYN3, XYN3A and XYN3B, showed significant homology with fungal and bacterial xylanases belonging to the endoxylanase family 11 . XYN3 and XYN3A were cloned in a bacterial expression plasmid harbouring a 6 His-C terminal tag and the recombinant proteins XYN3 and XYN3A were purified from Escherichia coli . The recombinant proteins had Mr of 66 800 and 34 000 respectively and hydrolysed xylan to xylo-oligosaccharides . Analysis of truncated forms of XYN3 confirmed that the full-length protein contained two catalytic domains displaying similar substrate specificity . Western-blot analysis using antiserum raised against XYN3A showed that the N . frontalis xylanase was not submitted to post-translational maturation . XYN3A antiserum recognized similar polypeptides in the culture medium of two other rumen fungi, Piromyces rhizinflata and Caecomyces communis.

Parasitology, 1996 Dec, 113 ( Pt 6), 519 - 26
Schistosoma mansoni elastase: an immune target regulated during the parasite life-cycle; Pierrot C et al.; Recombinant Schistosoma mansoni elastase was expressed in Escherichia coli and an antiserum raised against the recombinant protein was used to investigate stage-specific control of elastase in the parasite, and to determine whether the enzyme could form the basis of a strategy to prevent larval invasion of the host . Results showed that the expression of elastase is developmentally regulated, even if the basal promoter activity does not seem to be stage specific . The analysis of mRNA expression showed the presence of elastase transcript in adult worms although we could not detect the protein at this stage, suggesting that S . mansoni employs a form of translational control . The measurement of elastase levels in supernatants of culture schistosomula combined with the localization of elastase in cercarieae invading mouse skin showed that the enzyme is heavily released during penetration . Finally, we studied the cytotoxic activity of rat anti-elastase sera, and the analysis of the isotypic profile suggested that IgG2a anti-elastase may be responsible for the cytotoxic effect.

Hepatology, 1996 Dec, 24(6), 1468 - 74
Cholesterol 7alpha-hydroxylase activities from human and rat liver are modulated in vitro posttranslationally by phosphorylation/dephosphorylation; Nguyen LB et al.; Purified cholesterol 7alpha-hydroxylases (C7alphaH) from human and rat liver microsomes, and from transformed Escherichia coli expression systems, were incubated with 0.3 mmol/L {gamma-32P} adenosine triphosphate (ATP) in the presence and absence of bacterial alkaline phosphatase (AP) or rabbit muscle adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase . The amounts of 32P incorporation after separation of human and rat C7alphaH proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were related to C7alphaH catalytic activities (determined by a radioisotope incorporation method) and enzyme protein mass (determined by Western blotting and laser densitometry) . Both human and rat C7alphaH activities significantly decreased after dephosphorylation by AP (-57% - -72%) and increased up to twofold with phosphorylation by rabbit muscle cAMP-dependent protein kinase . The increases in C7alphaH activities were proportional to the amounts of cAMP-dependent protein kinase used, and were coupled to 32P incorporation into the purified enzymes . Both the activation of C7alphaH and the amounts of 32P incorporation were time-dependent and reached a maximum after 1 hour of incubation with 5 U of cAMP-dependent protein kinase . In a second set of experiments, purified human and rat liver C7alphaH were dephosphorylated by 30-minute incubation with AP, followed by inactivation of the phosphatase by the inhibitor NaF, and rephosphorylation of C7alphaH by 30-minute incubation with rabbit muscle cAMP-dependent protein kinase or bovine heart cAMP-independent protein kinase . Rephosphorylation of the dephosphorylated C7alphaH proteins by cAMP-dependent protein kinase increased C7alphaH catalytic activities up to fourfold, and the stimulation in catalytic activities paralleled the increases in 32P incorporation into the purified enzymes . Bovine heart protein kinase was as potent as rabbit muscle cAMP-dependent protein kinase in stimulating catalytic activity and 32P incorporation into the human C7alphaH protein . Because the protein mass of these purified enzymes did not change, the short-term regulation or catalytic efficiency of C7alphaH (activity per protein mass unit) is modulated, in vitro, posttranslationally by a phosphorylation/dephosphorylation mechanism in both the human and the rat enzymes.

Diabetes, 1996 Dec, 45(12), 1670 - 7
Heterologous expression and characterization of rat liver glucokinase regulatory protein; Mookhtiar KA et al.; Glucokinase is a critical component of the physiological glucose sensor found in cell types that are responsive to changes in plasma glucose levels . The acute regulation of glucokinase activity has been shown to occur via a regulatory protein found in liver parenchymal cells (Van Schaftingen E, Detheux M, Da Cunha MV . Faseb J 8:414-419, 1994) . The action of this protein is modulated by phosphate esters of fructose . In the presence of fructose-6-phosphate, the protein inhibits glucokinase in an allosteric competitive manner, while fructose-1-phosphate reverses this inhibition . A cDNA potentially encoding the rat liver regulatory protein has been cloned, but its identity is uncertain because of the small amounts of soluble protein obtained by expression in bacteria . We report the heterologous expression of the regulatory protein in Escherichia coli and its purification to homogeneity and high specific activity in a single chromatographic step . The properties of this recombinant protein are very similar to those of the liver protein . Direct demonstration of the binding of the recombinant protein to glucokinase has been obtained in vitro using coprecipitation experiments and in vivo, using the yeast two-hybrid system . These studies establish that the protein encoded by the cDNA is identical to the glucokinase regulatory protein and also validate tools with which to carry out structure-function studies on the interaction of the regulatory protein with glucokinase.

J Biotechnol, 1996 Nov 29, 52(1), 11 - 20
Cloning and expression of pyranose oxidase cDNA from Coriolus versicolor in Escherichia coli; Nishimura I et al.; Complementary DNA encoding pyranose oxidase (PROD) was cloned and sequenced for the first time from Coriolus versicolor . The nucleotide sequence revealed an open reading frame encoding a polypeptide composed of 623 amino acid residues . Compared with the experimentally determined N-terminal sequence of the PROD from C . versicolor . 38 amino acids from the N-terminus of the protein appeared to be eliminated during protein maturation . The cDNA was successfully expressed under the control of lacUV5 promoter in Escherichia coli at 25 degrees C, which will be beneficial in industrial production.

Cancer Lett, 1996 Nov 29, 108(2), 195 - 200
Helicobacter pylori extract stimulates inflammatory nitric oxide production; Tsuji S et al.; This study examined whether an extract of Helicobacter pylori had the ability to stimulate an inflammatory synthesis of nitric oxide, a mutagen and precursor of nitrosocompounds . Macrophages and neutrophils were prepared from rat and incubated with the Helicobacter pylori extract . L-Arginine-dependent nitric oxide production in these cells was significantly stimulated by the co-incubation with the Helicobacter pylori extract . This ability of the extract was strongly attenuated by protease digestion or heating . These results indicate that Helicobacter pylori induces production of nitric oxide and participates in development of gastritis and gastric carcinogenesis.

Biophys Chem, 1996 Nov 29, 62(1-3), 39 - 45
Study of tyrosine-containing mutants of ribosomal protein L7/L12 from Escherichia coli; Todorova RT et al.; Three mutant forms of the ribosomal protein L7/L12 with replacements of Ser1, Met14 and Met26 to Tyr were studied by the methods of fluorescence spectroscopy, circular dichroism and microcalorimetry . The amino-acid residue Tyr14 in the protein L7/L12 Tyr14 is located in a region with a more organized structure than Tyr26 in protein L7/L12 Tyr26 . The replacements Ser1-->Tyr1 and Met14-->Tyr14 do not affect the secondary structure of protein L7/L12 . The replacement Met26-->Tyr26 stabilizes the secondary structure of protein L7/L12 . A pH-induced temperature transition was observed in the pH range 5.0-7.3 in protein L7/L12 Tyr14 by tyrosine fluorescence . Analogous transitions were observed for protein L7/L12 Tyr26 by Tyr fluorescence and for the wild type protein L7/L12 by Phe fluorescence . Three pH-dependent states of protein L7/L12 and its mutant forms L7/L12 Tyr1 and L7/L12 Tyr14 were found on the microcalorimetric melting curves . The characteristics of protein L7/L12 Tyr14 are very close to the wild type protein L7/L12 and it is a suitable object for studying the structure of the N-terminal part of molecule by two-dimentional 1H-NMR.

J Mol Biol, 1996 Nov 29, 264(2), 364 - 76
Crystal structures and solution conformations of a dominant-negative mutant of Escherichia coli maltose-binding protein; Shilton BH et al.; A mutant of the periplasmic maltose-binding protein (MBP) with altered transport properties was studied . A change of residue 230 from tryptophan to arginine results in dominant-negative MBP: expression of this protein against a wild-type background causes inhibition of maltose transport . As part of an investigation of the mechanism of such inhibition, we have solved crystal structures of both unliganded and liganded mutant protein . In the closed, liganded conformation, the side-chain of R230 projects into a region of the surface of MBP that has been identified as important for transport while in the open form, the same side-chain takes on a different, and less ordered, conformation . The crystallographic work is supplemented with a small-angle X-ray scattering study that provides evidence that the solution conformation of unliganded mutant is similar to that of wild-type MBP . It is concluded that dominant-negative inhibition of maltose transport must result from the formation of a non-productive complex between liganded-bound mutant MBP and wild-type MalFGK2 . A general kinetic framework for transport by either wild-type MalFGK2 or MBP-independent MalFGK2 is used to understand the effects of dominant-negative MBP molecules on both of these systems.

Cell, 1996 Nov 29, 87(5), 893 - 903
Transcripts that increase the processivity and elongation rate of RNA polymerase; King RA et al.; Transcripts encoded by the cis-acting antitermination sites (put sites) of lambdoid phage HK022 promote readthrough of downstream transcription terminators . Proper conformation of the transcripts is essential for activity, since put mutations that prevent the formation of predicted RNA stems prevented antitermination, and suppressor mutations that restore the stems restored antitermination . Antitermination does not appear to require proteins other than RNA polymerase, since put-dependent readthrough of multiple sequential terminators was observed in a purified transcription system consisting of template, polymerase, substrates, and buffer . Transcription of put also increased the elongation rate of polymerase, very likely by suppressing pausing . A mutation that alters the zinc-finger region of the beta' subunit of polymerase specifically prevented the put-dependent increases in terminator readthrough and elongation rate . The simplicity of HK022 antitermination contrasts with that of other known antitermination pathways . We propose that the central effector is a transcript that directly alters the elongation properties of RNA polymerase.

Cell, 1996 Nov 29, 87(5), 881 - 91
Helicase-contrahelicase interaction and the mechanism of termination of DNA replication; Manna AC et al.; Termination of DNA replication at a sequence-specific replication terminus is potentiated by the binding of the replication terminator protein (RTP) to the terminus sequence, causing polar arrest of the replicative helicase (contrahelicase activity) . Two alternative models have been proposed to explain the mechanism of replication fork arrest . In the first model, the RTP-terminus DNA interaction simply imposes a polar barrier to helicase movement without involving any specific interaction between the helicase and the terminator proteins . The second model proposes that there is a specific interaction between the two proteins, and that the DNA-protein interaction both restricts the fork arrest to the replication terminus and determines the polarity of the process . The evidence presented in this paper strongly supports the second model.

J Biol Chem, 1996 Nov 29, 271(48), 30829 - 34
Efficient insertion of odd-numbered transmembrane segments of the tetracycline resistance protein requires even-numbered segments; Guo D et al.; Functional membrane insertion elements in the pBR322 tetracycline resistance protein were identified by comparing the ability of odd-numbered transmembrane segments and their attached periplasmic loops to insert into the membrane individually or when combined with the next even-numbered segment in the tetracycline resistance protein sequence . The efficiency with which individual odd-numbered segments and periplasmic loops inserted was probed by treating proteins truncated at the distal ends of periplasmic loops P2-P6 with carboxypeptidases and endoproteases in inside-out membrane vesicles . Insertion of odd-numbered segments and attached loops is inefficient when they occupy a C-terminal position in the protein . The C-terminal odd-numbered segment and loop sequences of 34-54% of the molecules of periplasmic loop truncation mutants could be removed by carboxypeptidase Y . In contrast, odd-numbered segments and loops insert efficiently if the next even-numbered segment in the sequence is present . In such cytoplasmic loop truncation mutants, only the cytoplasmic tail sequences of the proteins could be removed by carboxypeptidases . Remarkably, insertion of individual odd-numbered segments and loops is inefficient even though free energies for insertion of these sequences are highly favorable . The results indicate that pairs of adjacent segments, possibly "helical hairpins," are necessary for efficient membrane insertion of the tetracycline resistance protein.

J Biol Chem, 1996 Nov 29, 271(48), 30798 - 803
Role of the heat shock protein DnaJ in the lon-dependent degradation of naturally unstable proteins; Jubete Y et al.; We have investigated the role of DnaJ in protein degradation by examining the degradation of intrinsically unstable proteins by Lon protease in vivo . In Escherichia coli, Lon protease is responsible for the rate-limiting step in degradation of highly unstable proteins such as SulA, RcsA, and lambdaN protein, as well as for about 50% of the rapid degradation of abnormal proteins such as canavanine-containing proteins . We found that Lon-dependent degradation of both SulA and lambdaN protein was unaffected in cells lacking functional DnaJ, whereas Lon-dependent turnover of canavanine-containing proteins was slower in dnaJ mutant cells . DnaJ also affected the slow SulA degradation seen in the absence of Lon . The rate of degradation of RcsA was reduced in dnaJ mutants, but both Lon-dependent and Lon-independent degradation was affected; abnormal, canavanine-containing proteins were similarly affected . Both the RcsA that accumulated in dnaJ mutant cells and the SulA that accumulated in lon dnaJ mutant cells was aggregated . The abnormal proteins that partitioned to the insoluble pellet became solubilized over time in dnaJ+ cells but not in dnaJ- cells . Therefore, the co-chaperone DnaJ is not essential for Lon-dependent degradation and may act in protein turnover only as an accessory factor helping to maintain substrates in a soluble form . Such an accessory factor is apparently necessary for abnormal proteins and for RcsA . The relative rates of degradation and aggregation of specific protein targets may determine the importance of the chaperone systems in turnover of a given protein.

J Biol Chem, 1996 Nov 29, 271(48), 30731 - 5
Molecular cloning and expression of rat liver aminopeptidase B; Fukasawa KM et al.; We isolated, by immunological screening of a Uni-ZAP XR cDNA library constructed from rat liver mRNAs, a cDNA clone with 2212 base pairs encoding aminopeptidase B (EC 3.4.11.6) . The open reading frame encodes a 649-amino acid protein with a theoretical molecular mass of 72,545 Da and bears the consensus sequence of the zinc metalloexopeptidases, indicating that the enzyme belongs to this family, which includes aminopeptidase A, aminopeptidase N, and leukotriene-A4 hydrolase . Escherichia coli SOLR cells infected with the pBluescript phagemid excised from the Uni-ZAP XR vector containing the aminopeptidase B cDNA had a high L-arginyl-beta-naphthylamidase activity . The recombinant protein was purified to homogeneity from the recombinant E . coli extracts . The enzyme had Cl--dependent aminopeptidase activity specifically restricted to the Arg and Lys derivatives and contained 1 mol of zinc per mol of the enzyme.

J Biol Chem, 1996 Nov 29, 271(48), 30699 - 708
Dynamics of loading the beta sliding clamp of DNA polymerase III onto DNA; Bloom LB et al.; A "minimal" DNA primer-template system, consisting of an 80-mer template and 30-mer primer, supports processive DNA synthesis by DNA polymerase III core in the presence of the beta sliding clamp, gamma complex clamp loader, and single-stranded binding protein from Escherichia coli . This primer-template system was used to measure the loading of the beta sliding clamp by the gamma complex in an ATP-dependent reaction . Bound protein-DNA complexes were detected by monitoring fluorescence depolarization of DNA . Steady state and time-resolved anisotropies were measured, and stopped-flow pre-steady state fluorescence measurements allowed visualization of the loading reactions in real time . The rate of loading beta onto DNA was 12 s-1, demonstrating that clamp assembly is rapid on the time scale required for lagging strand Okazaki fragment synthesis . The association rate appears to be limited by an intramolecular step occurring prior to the clamp-loading reaction, possibly the opening of the toroidal beta dimer.

J Biol Chem, 1996 Nov 29, 271(48), 30672 - 6
Cleavage of insertion/deletion mismatches, flap and pseudo-Y DNA structures by deoxyinosine 3'-endonuclease from Escherichia coli; Yao M et al.; Deoxyinosine 3'-endonuclease, an Escherichia coli repair enzyme that recognizes and cleaves DNA containing deoxyinosine and base mismatches, can cleave heteroduplexes containing a hairpin or unpaired loop . These DNA structures, referred to as insertion/deletion mismatches (IDM), are abnormal intermediate structures generated during replication of repetitive DNA sequences . In addition, the enzyme also cleaved the 5'-single-stranded tails of flap and pseudo-Y DNA structures, suggesting that deoxyinosine 3'-endonuclease is a bacterial functional homologue of human FEN1 and yeast RTH1 nucleases . These biochemical properties suggest that deoxyinosine 3'-endonuclease might be important in the repair of IDM structures generated in lagging strand during DNA replication.

J Biol Chem, 1996 Nov 29, 271(48), 30548 - 53
The Escherichia coli pgpB gene encodes for a diacylglycerol pyrophosphate phosphatase activity; Dillon DA et al.; We provided genetic and biochemical evidence that supported the conclusion that the product of pgpB gene of Escherichia coli exhibited diacylglycerol pyrophosphate (DGPP) phosphatase activity . DGPP phosphatase activity was absent in pgpB mutant cells and was expressed at high levels in cells carrying the wild-type pgpB gene on a runaway replication plasmid . The pgpB mutant has been primarily characterized by a defect in phosphatidate (PA) phosphatase activity and also exhibits defects in lyso-PA phosphatase and phosphatidylglycerophosphate phosphatase activities . The defective PA phosphatase in the pgpB mutant was shown to be a Mg2+-independent PA phosphatase activity of the DGPP phosphatase enzyme . We characterized DGPP phosphatase activity in membranes from cells overproducing the pgpB gene product . DGPP phosphatase catalyzed the dephosphorylation of the beta phosphate of DGPP to form PA followed by the dephosphorylation of PA to form diacylglycerol . The specificity constant (Vmax/Km) for DGPP was 9.3-fold greater than that for PA . The pH optimum for the DGPP phosphatase reaction was 6 . 5 . Activity was independent of a divalent cation requirement, was potently inhibited by Mn2+ ions, and was insensitive to inhibition by N-ethylmaleimide . Pure DGPP phosphatase from Saccharomyces cerevisiae was shown to be similar to the E . coli DGPP phosphatase in its ability to utilize lyso-PA and phosphatidylglycerophosphate as substrates in vitro.

J Biol Chem, 1996 Nov 29, 271(48), 30426 - 35
The biogenetic anatomy of vitamin B6 . A 13C NMR investigation of the biosynthesis of pyridoxol in Escherichia coli; Hill RE et al.; It is shown by incorporation experiments with 13C bond-labeled substrates, followed by analysis by means of 13C NMR spectroscopy, that two compounds, 1-deoxy-D-xylulose (12) and 4-hydroxy-L-threonine (), serve as precursors of pyridoxol (vitamin B6) (1) in Escherichia coli . Together, these two compounds account for the entire C8N skeleton of the vitamin . 1-Deoxy-D-xylulose supplies the intact C5 unit, C-2',2,3,4,4' of pyridoxol . 4-Hydroxy-L-threonine undergoes decarboxylation in supplying the intact C3N unit, N-1,C-6,5,5' . Both precursors are ultimately derived from glucose . The C5 unit of pyridoxol that is derived from 1-deoxy-D-xylulose originates by union of a triose phosphate (yielding C-3,4,4') with pyruvic acid (which decarboxylates to yield C-2',2) . D-Erythroate (11) enters the C3 unit, C-6,5,5', and is therefore an intermediate on the route from glucose into 4-hydroxy-L-threonine.

J Biol Chem, 1996 Nov 29, 271(48), 30360 - 5
Molecular cloning of cDNA encoding rat very long-chain acyl-CoA synthetase; Uchiyama A et al.; The cDNA encoding rat very long-chain acyl-CoA synthetase (VLACS) was cloned, using degenerative primers synthesized according to the partial amino acid sequences of the peptide fragments of the purified rat liver enzyme . The longest cDNA insert was 2972 base pairs with a 1860-base pair open reading frame encoding 620 amino acids . The calculated molecular mass of 70,692 daltons was consistent with size of the purified enzyme . In Northern blot analysis, a single band was detected at the position of about 3 kilobases, corresponding to the size of the cloned cDNA . cDNA-directed expression in Escherichia coli resulted in accumulation of expressed protein, as an inclusion body . An antibody was raised using this expressed protein to characterize the cDNA and the enzyme . The subcellular localization of VLACS in peroxisomes and microsomes was demonstrated in Western blot analysis . The specific activity and the substrate specificity of the cDNA expressed enzyme in COS-1 cells were consistent with those of the purified rat enzyme . The predicted amino acid sequence of VLACS had a high sequence similarity to fatty acid transport protein (Schaffer, J . E., and Lodish, H . F . (1994) Cell 79, 427-436), and was considered to have domains for adenylation and thioester formation . The entire structure of VLACS was dissimilar to that of long-chain acyl-CoA synthetase (Suzuki, H., Kawarabayashi, Y., Kondo, Y., Abe, T., Nishikawa, K., Kimura, S., Hashimoto, T., and Yamamoto, T . (1990) J . Biol . Chem . 265, 8681-8685), except for the domains.

Eur J Pharmacol, 1996 Nov 28, 316(1), 111 - 21
Probing human beta 1- and beta 2-adrenoceptors with domain-specific fusion protein antibodies; Jahns R et al.; In order to generate antibodies suitable for immunological studies on beta-adrenoceptors constitutively expressed at low levels in cells or tissues we have produced fusion proteins of the amino- and carboxy-terminus, and the second extracellular loop of the human beta 1- or beta 2-adrenoceptors with bacterial glutathione-S-transferase in E . coli . Rabbit antibodies raised against these fusion proteins strongly reacted with intact human beta 1- or beta 2-adrenoceptors in a subtype- and domain-specific manner . Antibodies directed against the second extracellular loop of the beta 1-adrenoceptor reacted stronger with non-denatured receptors and decreased the affinity of the 3H-labelled antagonist (-)-4-(3-t-butylamino-2-hydroxypropoxy)-{5,7-3H}benzimidazol-2-one ({3H}CGP 12 177), indicating a specific interaction with the native receptor . In contrast, antibodies directed against carboxy- and amino-terminal receptor domains reacted strongly both with denatured and non-denatured receptors but did not interfere with binding of {3H}CGP 12 177 . Affinity purified antibodies were used for detecting the beta 1- or the beta 2-adrenoceptor subtype heterologously produced in Sf9 cells by enzyme-linked immunosorbent assay, Western blotting, immunoprecipitation, and indirect immunofluorescence microscopy . Moreover, we could demonstrate that avidity, titers, and specificity of these antibodies were high enough for studying beta-adrenoceptors constitutively expressed in human A431 cells, where we observed a patched membrane distribution of the receptors.

Gene, 1996 Nov 28, 181(1-2), 179 - 83
Nucleotide sequence of the Escherichia coli solA gene encoding a sarcosine oxidase-like protein and characterization of its product; Koyama Y et al.; An Escherichia coli gene, designated solA, was identified between pyrC and htrB around 24.2 min of the genetic map . The solA gene product (SolA, 372 aa) shows strong similarities (at most 42% identities over the entire length) to monomeric sarcosine (N-methylglycine) oxidases from other bacteria . However, only low levels of sarcosine oxidase activity were detected, even when SolA was overproduced . The SolA extracts exhibited much higher (about 200-fold) reactivity toward N-methyltryptophan than toward sarcosine . Thus, SolA appears to have a substrate specificity distinct from that of monomeric sarcosine oxidases, in spite of the strong similarities in the primary structure with such enzymes.

Gene, 1996 Nov 28, 181(1-2), 117 - 20
Hammerhead ribozyme structure probed by cell extracts; Wang JY et al.; To examine hammerhead ribozyme structure and vulnerability to cellular nucleases, ribozymes were incubated with soluble extracts from Escherichia coli, and cleavage sites were identified by primer extension analysis . Mapping of endonuclease-sensitive sites revealed that the most sensitive were in the 3'-substrate-binding region of the ribozyme . The catalytic domain was much less susceptible, although some cleavage preference was seen at two positions known to be twisted out of parallel stacking in a ribozyme-substrate analogue complex . Changes in substrate-binding domain nucleotide sequence had no effect on cleavage patterns of catalytic domains . Hammerhead ribozymes, in solution and free from substrate, appear to have structurally independent, asymmetrically arranged domains.

Gene, 1996 Nov 28, 181(1-2), 103 - 8
Novel phosphotransferase-encoding genes revealed by analysis of the Escherichia coli genome: a chimeric gene encoding an Enzyme I homologue that possesses a putative sensory transduction domain; Reizer J et al.; Two genes (ptsI and ptsA) that encode homologues of the energy coupling Enzyme I of the phosphoenolpyruvate-dependent sugar-transporting phosphotransferase system (PTS) have previously been identified on the Escherichia coli chromosome . We here report the presence of a third E . coli gene, designated ptsP, that encodes an Enzyme I homologue, here designated Enzyme INtr . Enzyme INtr possesses an N-terminal domain homologous to the N-terminal domains of NifA proteins {(127 amino acids (aa)} joined via two tandem flexible linkers to the C-terminal Enzyme I-like domain (578 aa) . Structural features of the putative ptsP operon, including transcriptional regulatory signals, are characterized . We suggest that Enzyme INtr functions in transcriptional regulation of nitrogen-related operons together with previously described PTS proteins encoded within the rpoN operon . It may thereby provide a link between carbon and nitrogen assimilatory pathways.

Gene, 1996 Nov 28, 181(1-2), 89 - 94
Characterization of a Phytophthora infestans gene involved in vesicle transport; Chen Y et al.; Members of the Ras superfamily of monomeric GTP-binding proteins have been shown to be essential in specific steps of vesicle transport and secretion in widely divergent organisms . We report here the characterization of a gene from Phytophthora infestans encoding a deduced amino acid (aa) sequence belonging to the Ypt class of monomeric GTP-binding proteins, products shown in other organisms to be essential for vesicle transport between the endoplasmic reticulum and the cis-Golgi compartments . Analysis of genomic and cDNA sequences of this gene, Piypt1, indicates that it contains five introns, one in the 5'-untranslated region . All introns are typical in beginning with GT and ending with AG . The region of the transcription start point displays a number of features characteristic of fungi and other eukaryotes, but it does not contain TATA or CAAT motifs . A single transcript is produced from the gene, which is polyadenylated, but the gene does not contain a recognizable polyadenylation signal . Genomic DNA blots indicate that Piypt1 is a single-copy gene . Comparisons of Ypt1 aa sequences indicate that P . infestans is more closely related to algae and higher plants than to the true fungi . The protein product of the Piypt1 gene, expressed in Escherichia coli, cross-reacts with antiserum against yeast Ypt1 protein and binds GTP . Furthermore, the Piypt1 gene is able to functionally complement a mutant ypt1 gene in Saccharomyces cerevisiae . The aa sequence similarity, immunological cross-reactivity and functional attributes of Piypt1 make it likely that it is an authentic ypt1 gene which participates in vesicle transport in Phytophthora infestans.

Gene, 1996 Nov 28, 181(1-2), 85 - 8
Transcriptional pattern of Escherichia coli ihfB (himD) gene expression; Weglenska A et al.; Integration host factor (IHF) is a small heterodimer containing subunits encoded by the unlinked ihfA (himA) and ihfB (himD, hip) genes . The transcriptional pattern of ihfB expression in the logarithmic and stationary growth phases was investigated . The ihfB gene is expressed as both monocistronic and polycistronic (hybridizing also to an internal rpsA probe) transcript . The intensity of the polycistronic transcripts, initiated upstream of rpsA, decreased sharply upon growth cessation . In contrast, expression of the monocistronic ihfB transcript strongly increased when cells entered stationary growth phase . The observed growth rate-dependent regulation of the transcription of these transcripts is in agreement with the previously published data about the regulation of the rpsA and ihfB promoters (Pedersen et al., 1984; Aviv et al., 1994).

Gene, 1996 Nov 28, 181(1-2), 13 - 8
Stable nuclear transformation of Chlamydomonas reinhardtii with a Streptomyces rimosus gene as the selective marker; Sizova IA et al.; The aminoglycoside 3'-phosphotransferase type VIII (APHVIII) encoding gene (aphVIII) from Streptomyces rimosus was introduced by glass-bead high-efficiency transformation into the nuclear genome of green unicellular alga Chlamydomonas reinhardtii for induction of transformants resistant to aminoglycoside antibiotics . The aphVIII structural sequence was flanked by S . rimosus regulatory sequences which failed to direct expression in C . reinhardtii . The pSU937 plasmid containing these sequences was able to transform C . reinhardtii strain cw15 arg7-8 mt+ for paromomycin resistance (PmR) at a frequency (1.3-1.9) x 10(-7), probably as a result of in vivo gene fusion and expression of the aphVIII gene from regulatory elements of nuclear DNA . Evidence for the real C . reinhardtii transformation includes blot-hybridization with a probe specific for aphVIII and demonstration of APHVIII enzyme activity in crude cell extracts of transformants . Integrated Streptomyces DNA sequences, APHVIII enzyme activity and the aminoglycoside-resistance phenotype were stable through mitosis in the presence and absence of selection . The PmR phenotype was inherited by the meiotic progeny of transformants from crosses with wild-type strains.

Mol Gen Genet, 1996 Nov 27, 253(1-2), 198 - 204
Down regulation of cAMP production by cAMP receptor protein in Escherichia coli: an assessment of the contributions of transcriptional and posttranscriptional control of adenylate cyclase; Inada T et al.; Escherichia coli cells that are deficient in the cAMP receptor protein (CRP) overproduce cAMP . We and others have previously found that transcription of the adenylate cyclase gene (cya) is negatively regulated by the CRP-cAMP complex . Here, we have investigated the contribution of this transcriptional regulation to the control of cAMP levels . Several variants of the cya gene have been constructed and characterized with respect to their expression and their ability to produce cAMP . Overproduction of cAMP in a crp- background was reduced from 200-fold to 50-fold when transcriptional regulation by CRP-cAMP was eliminated by replacing the cya promoter with the constitutive bla promoter . When the C-terminal 48 amino acids of adenylate cyclase were deleted without changing the promoter, the degree of overproduction of cAMP was reduced to 4-fold . Finally, the increase in cAMP level observed in crp- cells was almost completely abolished when the truncated cyclase was expressed from the bla promoter . We conclude that transcriptional regulation of cya does indeed play a role in the down-regulation of cAMP production by CRP, although the major regulation is exerted at the posttranscriptional level . The C-terminal region comprising the last 48 amino acids of cyclase is responsible for the posttranscriptional regulation . A simple new method for the determination of cAMP is also described.

Mol Gen Genet, 1996 Nov 27, 253(1-2), 95 - 102
Effects of mutations in genes for proteins involved in disulphide bond formation in the periplasm on the activities of anaerobically induced electron transfer chains in Escherichia coli K12; Metheringham R et al.; The assembly of anaerobically induced electron transfer chains in Escherichia coli strains defective in periplasmic disulphide bond formation was investigated . Strains deficient in DsbA, DsbB or DipZ (DsbD) were unable to catalyse formate-dependent nitrite reduction (Nrf activity) or synthesize any of the known c-type cytochromes . The Nrf+ activity and cytochrome c content of mutants defective in DsbC, DsbE or DsbF were similar to those of the parental, wild-type strain . Neither DsbC expressed from a multicopy plasmid nor a second mutation in dipZ (dsbD) was able to compensate for a dsbA mutation by restoring nitrite reductase activity and cytochrome c synthesis . In contrast, only the dsbB and dipZ (dsbD) strains were defective in periplasmic nitrate reductase activity, suggesting that DsbB might fulfil an additional role in anaerobic electron transport . Mutants defective in dipZ (dsbD) were only slightly more sensitive to Cu++ ions at concentrations above 5 mM than the parental strain, but strains defective in DsbA, DsbB, DsbC, DsbE or DsbF were unaffected . These results are consistent with our earlier proposals that DsbA, DsbB and DipZ (DsbD) are part of the same pathway for ensuring that haem groups are attached to the correct pairs of cysteine residues of apocytochromes c in the E . coli periplasm . However, neither DsbE nor DsbF are essential for the reduction of DipZ (DsbD).

Mol Gen Genet, 1996 Nov 27, 253(1-2), 74 - 80
Comparative analysis of expression of the SalI restriction-modification system in Escherichia coli and Streptomyces; Alvarez MA et al.; The salIR and salIM genes encode the endonuclease and methyltransferase components of the SalI restriction-modification system from Streptomyces albus G . Expression of the salI genes in Escherichia coli was investigated and major differences with Streptomyces were found . In E . coli there is no detectable expression of the salI R gene due to inactivity of the sal-pR promoter region . In the natural host of the system this region directs transcription of the salI genes as a bicistronic message . In contrast to salIR, salIM is transcribed in the heterologous host from a promoter within the salI DNA . Since sal-pR is not active, the gene cannot be expressed as part of the salI operon . It is probably transcribed from sal-pM, a promoter internal to the operon which allows independent expression of the modification gene in Streptomyces . Replacement of sal-pR by the strong pLac promoter allows expression of salIR in E . coli and enhances expression of salIM . The resulting strain produces about 10 times more endonuclease than a Streptomyces clone containing the SalI system under the control of sal-pR.

Mol Gen Genet, 1996 Nov 27, 253(1-2), 42 - 9
The localized melting of mini-F origin by the combined action of the mini-F initiator protein (RepE) and HU and DnaA of Escherichia coli; Kawasaki Y et al.; Replication of mini-F plasmids requires the initiator protein RepE, which binds specifically to four iterons within the origin (ori2), as well as some host factors that are involved in chromosomal DNA replication . To understand the role of host factors and RepE in the early steps of mini-F DNA replication, we examined the effects of RepE and the Escherichia coli proteins DnaA and HU on the localized melting of ori2 DNA in a purified in vitro system . We found that the binding of RepE to an iteron causes a 50 degrees bend at or around the site of binding . RepE and HU exhibited synergistic effects on the localized melting within the ori2 region, as detected by sensitivity to the single-strand specific P1 endonuclease . This opening of duplex DNA occurred around the 13mer of ori2, whose sequence closely resembles the set of 13mers found in the chromosomal origin oriC . Further addition of DnaA to the reaction mixture increased the efficiency of melting and appeared to extend melting to the adjacent AT-rich region . Moreover, DNA melting with appreciably higher efficiencies was observed with mutant forms of RepE that were previously shown to be hyperactive both in DNA binding in vitro and in initiator activity in vivo . We propose that the binding of RepE to four iterons of ori2 causes bending at the sites of RepE binding and, with the assistance of HU, induces a localized melting in the 13mer region . The addition of DnaA extends melting to the AT-rich region, which could then serve as the entry site for the DnaB-DnaA complex, much as has been documented for oriC dependent replication.

Proc Natl Acad Sci U S A, 1996 Nov 26, 93(24), 14082 - 7
Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein; Kessler PD et al.; Somatic gene therapy has been proposed as a means to achieve systemic delivery of therapeutic proteins . However, there is limited evidence that current methods of gene delivery can practically achieve this goal . In this study, we demonstrate that, following a single intramuscular administration of a recombinant adeno-associated virus (rAAV) vector containing the beta-galactosidase (AAV-lacZ) gene into adult BALB/c mice, protein expression was detected in myofibers for at least 32 weeks . A single intramuscular administration of an AAV vector containing a gene for human erythropoietin (AAV-Epo) into mice resulted in dose-dependent secretion of erythropoietin and corresponding increases in red blood cell production that persisted for up to 40 weeks . Primary human myotubes transduced in vitro with the AAV-Epo vector also showed dose-dependent production of Epo . These results demonstrate that rAAV vectors are able to transduce skeletal muscle and are capable of achieving sustained expression and systemic delivery of a therapeutic protein following a single intramuscular administration . Gene therapy using AAV vectors may provide a practical strategy for the treatment of inherited and acquired protein deficiencies.

Proc Natl Acad Sci U S A, 1996 Nov 26, 93(24), 13925 - 30
Highly selective isolation of human DNAs from rodent-human hybrid cells as circular yeast artificial chromosomes by transformation-associated recombination cloning; Larionov V et al.; Transformation-associated recombination (TAR) can be exploited in yeast to clone human DNAs . TAR cloning was previously accomplished using one or two telomere-containing vectors with a common human repeat(s) that could recombine with human DNA during transformation to generate yeast artificial chromosomes (YACs) . On basis of the proposal that broken DNA ends are more recombinogenic than internal sequences, we have investigated if TAR cloning could be applied to the generation of circular YACs by using a single centromere vector containing various human repeats at opposite ends . Transformation with these vectors along with human DNA led to the efficient isolation of circular YACs with a mean size of approximately 150 kb . The circular YACs are stable and they can be easily separated from yeast chromosomes or moved into bacterial cells if the TAR vector contains an Escherichia coli F-factor cassette . More importantly, circular TAR cloning enabled the selective isolation of human DNAs from monochromosomal human-rodent hybrid cell lines . Although < 2% of the DNA in the hybrid cells was human, as much as 80% of transformants had human DNA YACs when a TAR cloning vector contained Alu repeats . The level of enrichment of human DNA was nearly 3000-fold . A comparable level of enrichment was demonstrated with DNA isolated from a radiation hybrid cell line containing only 5 Mb of human DNA . A high selectivity of human DNA cloning was also observed for linear TAR cloning with two telomere vectors . No human-rodent chimeras were detected among YACs generated by TAR cloning . The results with a circular TAR cloning vector or two vectors differed from results with a single-telomere vector in that the latter often resulted in a series of terminal deletions in linear YACs . This could provide a means for physical mapping of cloned material.

Proc Natl Acad Sci U S A, 1996 Nov 26, 93(24), 13919 - 24
Transcription-induced mutations: increase in C to T mutations in the nontranscribed strand during transcription in Escherichia coli; Beletskii A et al.; Cytosines in single-stranded DNA deaminate to uracils at 140 times the rate for cytosines in double-stranded DNA . If resulting uracils are not replaced with cytosine, C to T mutations occur . These facts suggest that cellular processes such as transcription that create single-stranded DNA should promote C to T mutations . We tested this hypothesis with the Escherichia coli tac promoter and found that induction of transcription causes approximately 4-fold increase in the frequency of C to U or 5-methylcytosine to T deaminations in the nontranscribed strand . Excess mutations caused by C to U deaminations were reduced, but not eliminated, by uracil-DNA glycosylase . Similarly, mutations caused by 5-methylcytosine to T deaminations were only partially reduced by the very short-patch repair process in E.coli . These effects are unlikely to be caused by differential repair of the two strands, and our results suggest that all actively transcribed genes in E . coli should acquire more C to T mutations in the nontranscribed strand.

Proc Natl Acad Sci U S A, 1996 Nov 26, 93(24), 13896 - 901
A positive genetic selection for disrupting protein-protein interactions: identification of CREB mutations that prevent association with the coactivator CBP; Shih HM et al.; The Escherichia coli tet-repressor (TetR) operator system was used to develop a variation of the yeast two-hybrid assay in which disruptions of protein-protein interactions can be identified by a positive selection . This assay, designated the "split-hybrid system," contains a two-component reporter . The first component contains LexA binding sites upstream of the TetR gene and the second contains TetR operator binding sites upstream of HIS3 . Interaction of one protein fused to the LexA DNA binding domain with a second protein fused to the VP16 activation domain results in TetR expression . TetR subsequently binds to the tet operators, blocking the expression of HIS3 and preventing yeast growth in media lacking histidine . The utility of the split-hybrid system was analyzed by examining the phosphorylation-dependent interaction of CREB and its coactivator CREB binding protein (CBP) . CREB and CBP associate through an interaction that depends upon CREB phosphorylation at Ser-133 . Mutation of this phosphorylation site prevents yeast growth in the standard two-hybrid assay but allows growth in the split-hybrid strains . The split-hybrid system was used to identify other CREB mutations that disrupt its association with CBP . These mutations localized around the site of CREB phosphorylation, indicating that only a small portion of the CREB activation domain is required for CBP interaction . The yeast split-hybrid system should be useful in identifying mutations, proteins, peptides, and drugs that disrupt protein-protein interactions.

Proc Natl Acad Sci U S A, 1996 Nov 26, 93(24), 13754 - 9
Metal binding properties and secondary structure of the zinc-binding domain of Nup475; Worthington MT et al.; Nup475 is a nuclear zinc-binding protein of unknown function that is induced in mammalian cells by growth factor mitogens . Nup475 contains two tandemly repeated sequences YKTELCX8CX5CX3H (Cys3His repeats) that are thought to be zinc-bindin domains . Similar sequences have been found in a number of proteins from various species of eukaryotes . To determine the metal binding properties and secondary structure of the putative zinc-binding domains of Nup475, we have used synthetic or recombinant peptides that contain one or two domain sequences . The peptide with a single domain bound 1.0 +/- 0.1 equivalents of Co2+, and the peptide with two domains bound 1.7 +/- 0.4 equivalents of Co2+ . Both peptides bound Co2+ and Zn2+ with affinities similar to those of classical zinc finger peptides . In each case, the Co2+ complex exhibited strong d-d transitions characteristic of tetrahedral coordination . For structural studies by nuclear magnetic resonance spectroscopy, we used a more soluble two-domain peptide that had a single amino acid substitution in a nonconserved amino acid residue in the second Cys3His repeat . The mutant peptide unexpectedly showed loss of one of its metal binding sites and displayed ordered structure for only the first Cys3His sequence . On the basis of the nuclear magnetic resonance data, we propose a structure for the Nup475 metal-binding domain in which the zinc ion is coordinated by the conserved cysteines and histidine, and the conserved YKTEL motif forms a parallel sheet-like structure with the C terminus of this domain . This structure is unlike that of any previously described class of metal binding domain.

Proc Natl Acad Sci U S A, 1996 Nov 26, 93(24), 13737 - 41
An unusual mechanism for the major human apurinic/apyrimidinic (AP) endonuclease involving 5' cleavage of DNA containing a benzene-derived exocyclic adduct in the absence of an AP site; Hang B et al.; The major human apurinic/apyrimidinic (AP) endonuclease (class II) is known to cleave DNA 5' adjacent to an AP site, which is probably the most common DNA damage produced hydrolytically or by glycosylase-mediated removal of modified bases . p-Benzoquinone (pBQ), one of the major benzene metabolites, reacts with DNA to form bulky exocyclic adducts . Herein we report that the human AP endonuclease directly catalyzes incision in a defined oligonucleotide containing 3,N4-benzetheno-2'-deoxycytidine (pBQ-dC) without prior generation of an AP site . The enzyme incises the oligonucleotide 5' to the adduct and generates 3'-hydroxyl and 5'-phosphoryl termini but leaves the pBQ-dC on the 5' terminus of the cleavage fragment . The AP function of the enzyme is not involved in this action, as no preexisting AP site is present nor is a DNA glycosylase activity involved . Nicking of the pBQ-dC adduct also leads to the same "dangling base" cleavage when two Escherichia coli enzymes, exonuclease III and endonuclease IV, are used . Our finding of this unusual mode of action used by both human and bacterial AP endonucleases raises important questions regarding the requirements for substrate recognition and catalytic active site(s) for this essential cellular repair enzyme . We believe this to be the first instance of the presence of a bulky carcinogen adduct leading to this unusual mode of action.

Proc Natl Acad Sci U S A, 1996 Nov 26, 93(24), 13635 - 40
Superoxide accelerates DNA damage by elevating free-iron levels; Keyer K et al.; Superoxide promotes hydroxyl-radical formation and consequent DNA damage in cells of all types . The long-standing hypothesis that it primarily does so by delivering electrons to adventitious iron on DNA was refuted by recent studies in Escherichia coli . Alternative proposals have suggested that superoxide may accelerate oxidative DNA damage by leaching iron from storage proteins or enzymic {4Fe-4S} clusters . The released iron might then deposit on the surface of the DNA, where it could catalyze the formation of DNA oxidants using other electron donors . The latter model is affirmed by the experiments described here . Whole-cell electron paramagnetic resonance demonstrated that the level of loose iron in superoxide-stressed cells greatly exceeds that of unstressed cells . Bacterial iron storage proteins were not the major source for free iron, since superoxide also increased iron levels in mutants lacking these iron storage proteins . However, overproduction of an enzyme containing a labile {4Fe-4S} cluster dramatically increased the free iron content of cells when they were growing in air . The rates of spontaneous mutagenesis and DNA damage from exogenous H2O2 increased commensurately . It is striking that both growth defects and DNA damage caused by superoxide ensue from its ability to damage a subset of iron-sulfur clusters.

Proc Natl Acad Sci U S A, 1996 Nov 26, 93(24), 13605 - 10
The role of apolipoprotein AI domains in lipid binding; Davidson WS et al.; Apolipoprotein AI (apoAI) is the principal protein constituent of high density lipoproteins and it plays a key role in human cholesterol homeostasis; however, the structure of apoAI is not clearly understood . To test the hypothesis that apoAI is organized into domains, three deletion mutants of human apo AI expressed in Escherichia coli were studied in solution and in reconstituted high density lipoprotein particles . Each mutant lacked one of three specific regions that together encompass almost the entire 243 aa sequence of native apoAI (apoAI delta 44-126, apoAI delta 139-170, and apoAI delta 190-243) . Circular dichroism spectroscopy showed that the alpha-helical content of lipid-free apoAI delta 44-126 was 27% while the other mutants and native apoAI averaged 55 +/- 2%, suggesting that the missing N-terminal portion contains most of the alpha-helical structure of lipid-free apoAI . ApoAI delta 44-126 exhibited the largest increase in alpha-helix upon lipid binding (125% increase versus an average of 25% for the others), confirming the importance of the C-terminal half of apoAI in lipid binding . Denaturation studies showed that the N-terminal half of apoAI is primarily responsible for alpha-helix stability in the lipid-free state, whereas the C terminus is required for alpha-helix stability when lipid-bound . We conclude that the N-terminal half (aa 44-126) of apoAI is responsible for most of the alpha-helical structure and the marginal stability of lipid-free apoAI while the C terminus (aa 139-243) is less organized . The increase in alpha-helical content observed when native apoAI binds lipid results from the formation of alpha-helix primarily in the C-terminal half of the molecule.

Proc Natl Acad Sci U S A, 1996 Nov 26, 93(24), 13565 - 70
A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal; Parks RJ et al.; Adenoviruses are attractive vectors for the delivery of foreign genes into mammalian cells for gene therapy . However, current vectors retain many viral genes that, when expressed at low levels, contribute to the induction of a host immune response against transduced cells . We have developed a helper-dependent packaging system for production of vectors that have large regions of the genome deleted . Helper viruses were constructed with packaging signals flanked by loxP sites so that in 293 cells that stably express the Cre recombinase (293Cre), the packaging signal was efficiently excised, rendering the helper virus genome unpackageable . However, the helper virus DNA was replicated at normal levels and could thus express all of the functions necessary in trans for replication and packaging of a vector genome containing the appropriate cis-acting elements . Serial passage of the vector in helper virus-infected 293Cre cells resulted in an approximately 10-fold increase in vector titer per passage . The vector could be partially separated from residual helper virus by cesium chloride buoyant density centrifugation . Large scale preparations of vector yielded semi-purified stocks of approximately 10(10) transducing virions/ml, with < 0.01% contamination by the E1-deleted helper virus . This system should have great utility for the generation of adenovirus-based vectors with increased cloning capacity, increased safety and reduced immunogenicity.

Biochemistry, 1996 Nov 26, 35(47), 15029 - 37
Functional interactions in cytochrome P450BM3 . Fatty acid substrate binding alters electron-transfer properties of the flavoprotein domain; Murataliev MB et al.; P450BM3 is a bacterial fusion protein between a cytochrome P450 fatty acid hydroxylase (CYP102) and an FAD- and FMN-containing flavoprotein homologous to NADPH: cytochrome P450 reductase . It has been shown that incubation of P450BM3 with NADPH in the absence of a fatty acid substrate results in inhibition of hydroxylase activity {Narhi, L . O., & Fulco, A . J . (1986) J . Biol . Chem . 261, 7160-7169} . We show that laurate-dependent oxidation of NADPH and oxygen consumption are also inhibited under those conditions . The inhibited enzyme is unable to transfer electrons to the heme iron, but reduces artificial electron acceptors such as cytochrome c, 2,6-dichlorophenolindophenol, or ferricyanide . Incubation with these acceptors rapidly restores hydroxylase activity of P450BM3 . The active enzyme is able to catalyze the reduction of cytochrome c and hydroxylation of laurate simultaneously . Cytochrome c has no effect on the K(m) and Vmax of laurate hydroxylation . Laurate and other substrates stimulate cytochrome c reduction by 50-70% . Carbon monoxide inhibits hydroxylase activity, but stimulates cytochrome c reduction 3-4 fold and has no effect on the K(m) for cytochrome c . This stimulation requires binding of a substrate at the heme catalytic site . Laurate binding induces conformational changes in the flavoprotein domain as shown by a 2-fold increase of the flavin fluorescence . Inactivation of P450BM3 by NADPH abolishes the stimulation of cytochrome c reduction by laurate and CO . Complete inhibition of hydroxylase activity correlates with complete lack of stimulation of cytochrome c reduction . The results suggest that a specific conformation of the two domains is maintained in the active P450BM3, ensuring high hydroxylase activity . Cytochrome c reductase and hydroxylase activities of P450BM3 involve different sites of interaction with the flavoprotein domain, different catalytic intermediates, and different rate-limiting steps.

Biochemistry, 1996 Nov 26, 35(47), 14984 - 91
Coupled folding and site-specific binding of the GCN4-bZIP transcription factor to the AP-1 and ATF/CREB DNA sites studied by microcalorimetry; Berger C et al.; The site-specific interaction of the basic leucine zipper protein C62GCN4, which corresponds to the C-terminal sequence 220-281 of the yeast transcription factor GCN4, with the AP-1 and ATF/ CREB DNA recognition sites was analyzed by isothermal titration microcalorimetry . Free C62GCN4 is a dimer composed of a C-terminal leucine zipper and a basic, mainly unstructured DNA binding domain . Upon association with the target DNA, C62GCN4 folds to a fully alpha-helical dimer {Ellenberger et al . (1992) Cell 71, 1223-1237; Konig and Richmond (1993) J . Mol . Biol . 233, 139-154} . The protein-bound AP-1 site is straight, and the protein-bound ATF/CREB site is bent by 20 degrees toward the leucine zipper domain . The coupling between protein folding and DNA association resulting in two conformationally different complexes with C62GCN4 poses interesting thermodynamic problems . The association was strongly exothermic for both DNA target sites . The free energies of binding were indistinguishable in buffers of low salt concentration, and no change of the protonation state of C62GCN4 and/or the DNA target site occurred on formation of the complexes . Both complexes exhibited large and negative heat capacity changes . The empirical correlation between buried nonpolar and polar surfaces and the reduction in heat capacity concomitant to complexation did hold for the reaction with the AP-1 site at low salt concentration . However, in the case of the ATF/CREB site, the change in heat capacity was larger than could be accounted for by the burial of solvent-accessible surface . Potential sources of the extra decrement in the heat capacity could be restrictions in the vibrational modes of polar groups and of bound water molecules at the protein-DNA interface, thought to result from the bending of the ATF/CREB site . In the presence of high concentrations of glutamate and NaCl, the complex with the ATF/CREB site was significantly weaker than the complex with the AP-1 site.

Biochemistry, 1996 Nov 26, 35(47), 14910 - 6
Preparation of an autolysis-resistant interleukin-1 beta converting enzyme mutant; Dang LC et al.; We describe the expression, purification, and characterization of human interleukin-1 beta converting enzyme (ICE) containing an affinity tag and modified to resist autoproteolysis . The point mutation Asp381 to Glu was added to eliminate the major site of autolytic degradation while maintaining catalytic activity, and an N-terminal polyhistidine tag was added in place of the ICE pro-region to facilitate purification . N-His (D381E) ICE was expressed in Escherichia coli and purified by nickel-chelating Sepharose and size-exclusion chromatography (SEC) . The enzyme was stabilized greater than 80-fold against autolytic degradation relative to wild-type N-His ICE . SDS-PAGE analysis with silver-staining revealed no impurities, and 85% of the protein was catalytically active as determined by titration with a novel titrant, PD 163594 (3-{2-(2-benzyloxycarbonylamino-3-methylbutyrylamino)prop ionylamino}-4- oxo-5-(2-oxo-2H-chromen-7-yloxypentanoic acid) . An oxidized adduct of ICE with glutathione, formed by disulfide rearrangement with oxidized glutathione to inhibit and stabilize the enzyme during purification, was rapidly reduced upon exposure to 5 mM DTT . One mole of glutathione was released per mole of active enzyme . Of the nine cysteines in ICE, eight were present in their reduced form in the glutathione adduct . N-His (D381E) ICE cleaved Ac-YVAD-Amc with the Michaelis-Menten parameters K(M) = 14 microM and Kcat = 0.7 s-1, values essentially identical to those reported for enzyme from natural sources.

Biochemistry, 1996 Nov 26, 35(47), 14876 - 81
Structural determinants of the catalytic reactivity of the buried cysteine of Escherichia coli thioredoxin; LeMaster DM; The structurally homologous thioredoxins and thioltransferases/glutaredoxins possess a solvent-exposed cysteine sulfur which carries out a nucleophilic attack on the target disulfide as well as a structurally adjacent solvent inaccessible thiol . The mechanistic basis of the essentially exclusive redox reactivity of the thioredoxins in contrast to the thiol-disulfide exchange reactions characteristic of the thioltransferases lies in the relative reactivity of the buried cysteine . A stable analog of the mixed disulfide state of Escherichia coli thioredoxin is used to demonstrate a pK value of 11.1 for the solvent inaccessible Cys 35 thiol . NMR chemical shift pH titration analysis indicates a very low dielectric surrounding the Cys 35 sulfur providing a basis for both the elevated pK and the enhanced apparent nucleophilicity . The buried Asp 26 likely serves as the proton sink for the (de)protonation of Cys 35 . Relevance to the reactivity of the mammalian protein isomerases is discussed.

Biochemistry, 1996 Nov 26, 35(47), 14725 - 33
Crystal structures of three misacylating mutants of Escherichia coli glutaminyl-tRNA synthetase complexed with tRNA(Gln) and ATP; Arnez JG et al.; Three previously described mutant Escherichia coli glutaminyl-tRNA synthetase (GlnRS) proteins that incorrectly aminoacylate the amber suppressor derived from tRNATyr (supF) with glutamine were cocrystallized with wild-type tRNAGln and their structures determined . In two of the mutant enzymes studied, Asp235, which contacts base pair G3-C70 in the acceptor stem, has been changed to asparagine in GlnRS7 and to glycine in GlnRS10 . These mutations result in changed interactions between Asn235 of GlnRS7 and G3-C70 of the tRNA and an altered water structure between Gly235 of GlnRS10 and base pair G3-C70 . These structures suggest how the mutant enzymes can show only small changes in their ability to aminoacylate wild-type cognate tRNA on the one hand and yet show a lack of discrimination against a noncognate U3-A70 base pair on the other . In contrast, the change of Ile129 to Thr in GlnRS15 causes virtually no change in the structure of the complex, and the explanation for its ability to misacylate supF is unclear.

Biochemistry, 1996 Nov 26, 35(47), 14671 - 8
Glu46 donates a proton to the 4-hydroxycinnamate anion chromophore during the photocycle of photoactive yellow protein; Xie A et al.; Photoactive yellow protein (PYP) is a photoreceptor containing a unique 4-hydroxycinnamic acid (pCA) chromophore . The trans to cis photoisomerization of this chromophore activates a photocycle involving first a short-lived red-shifted intermediate (pR), then a long-lived blue-shifted intermediate (pB), and finally recovery of the original receptor state (pG) . The pCA chromophore is deprotonated in pG and protonated in pB, but the proton donor for this process has not yet been identified . Here we report the first FTIR spectroscopic data on pG, pR, and pB . The IR difference signals in the carbonyl stretching region of COOH groups (1700-1800 cm-1) reveal that a buried carboxylic group close to the chromophore (i) is protonated in pG, (ii) develops a stronger hydrogen bonding in pR, and (iii) becomes deprotonated in pB . These signals are unambiguously assigned to Glu46, on the basis of the IR data and the 1.4 A X-ray structure of PYP {Borgstahl et al . (1995) Biochemistry 34, 6278-6287} . Our data demonstrate that in pR Glu46 remains in hydrogen bonding contact with the negatively charged phenolic oxygen of pCA after chromophore photoisomerization . This strongly implies that the chromophore is isomerized to the 7-cis 9-s-trans conformation in pR, resulting from co-isomerization of both the C7 = C8 and C9-C10 bonds . In the pR to pB transition, Glu46 becomes deprotonated, concomitant with chromophore protonation . Therefore, we conclude that Glu46 functions as the proton donor for the protonation of pCA during the PYP photocycle . We propose a molecular mechanism in which intramolecular proton transfer in PYP leads to global protein conformational changes involved in signal transduction.

Neuroreport, 1996 Nov 25, 7(18), 3105 - 8
HSV-1 vector mediated transfer of BDNF into cerebellar granule cells; Alonso MT et al.; Cerebellar granule cells offer a useful model system to study the effects of neurotrophins during development . We have used a defective herpes simplex virus type 1 (HSV-1) vector containing brain-derived neurotrophic factor (BDNF) to express this neurotrophin in aggregate cultures of granule cells . Viral infection led to easily detectable BDNF expression and neurite outgrowth of granule cells, expressing the high affinity receptor TrkB . Neurite elongation mediated by the HSV-1 vector producing BDNF was similar to that found after exposure to purified BDNF . This study demonstrates the efficacy of HSV-1 vectors for delivery and expression of neurotrophins in cerebellar granule cells . The biological responses measured indicate the effectiveness of HSV-1 vectors as potential therapeutic tools.

Mol Biochem Parasitol, 1996 Nov 25, 82(2), 245 - 55
Characterization of the Trypanosoma brucei homologue of a Trypanosoma cruzi flagellum-adhesion glycoprotein; Nozaki T et al.; An immunodominant 72-kDa surface glycoprotein (Gp72) of Trypanosoma cruzi is involved in adhesion of the flagellum to the cell body (Cooper, R, Ribeiro de Jesus, A and Cross, G.A.M (1993) J . Cell Biol . 122, 149-156) . We have characterized a gene, flagellum-adhesion glycoprotein genel (fla1), from Trypanosoma brucei that encodes a 546 amino-acid protein (Fla1) with high similarity to Gp72 . Their sequence similarity and cellular localization suggest that Fla1 and Gp72 have similar functions . We could disrupt individual fla1 alleles but not both, suggesting that fla1 is essential in T . brucei, in contrast to the situation for gp72 in T . cruzi . Using affinity-purified polyclonal antibody, raised against part of the amino-terminal domain of Fla1 expressed in Escherichia coli, we showed that Fla1 is concentrated along the flagellum and in the flagellar pocket in both bloodstream-form and procyclic trypanosomes . Fla1 from both life-cycle stages is N-glycosylated . Fla1 from bloodstream-form T . brucei contains additional glycans, which can be liberated by treatment with mild acid, suggestive of phosphodiester linkages.

J Mol Biol, 1996 Nov 22, 264(1), 179 - 90
Three-dimensional structure of the lipoyl domain from the dihydrolipoyl succinyltransferase component of the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli; Ricaud PM et al.; A sub-gene encoding the lipoyl domain of the dihydrolipoyl succinyltransferase polypeptide chain of the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli was over-expressed and the protein was purified uniformly labelled with 15N . The three-dimensional structure of the domain was determined by means of nuclear magnetic resonance spectroscopy, based on 905 nuclear Overhauser effect inter-proton distance restraints, 42 phi torsion angle restraints and hydrogen bond restraints from 24 slowly exchanging amide protons . The structure of the 80-residue domain is that of a flattened beta-barrel surrounding a hydrophobic core in which Trp22 plays a central role in anchoring two four-stranded sheets together . The polypeptide backbone exhibits a 2-fold axis of quasi-symmetry, with the lipoylation site, Lys43, located at the tip of an exposed beta-turn in one beta-sheet and the N and C-terminal residues close together in space in the other beta-sheet . The atomic r.m.s . distribution about the mean coordinate is 0.46 A for the backbone atoms in the highly structured region and 0.88 A along the entire backbone (residues Ser1 to Asn80), including a less well-defined surface loop and the lipoyl-lysine beta-turn . The structure closely resembles that of the lipoyl domains from pyruvate dehydrogenase complexes, in accord with the existence of strongly conserved residues at critical positions in the domains . The structures of the lipoyl domains throw light on the requirements for the specificity of reductive acylation of their pendant lipoyl groups in the parent 2-oxo acid dehydrogenase complexes; an important aspect of the mechanisms underlying active site coupling and substrate channelling.

J Mol Biol, 1996 Nov 22, 264(1), 82 - 96
Relationship between Escherichia coli growth and deletions of CTG.CAG triplet repeats in plasmids; Bowater RP et al.; Instabilities that are intrinsic to natural repetitive DNA sequences produce high frequencies of length changes in vivo . Triplet repeats cloned in plasmids in Escherichia coli undergo expansions and deletions, and this instability is affected by multiple factors . We show that CTG-CAG repeats in plasmids can influence the growth of E . coli, which affects the observed stabilities . At extended growth periods, the observed frequencies of deletions were dramatically increased if the cells passed through stationary phase before subculturing . Deletions were particularly pronounced for a plasmid containing the longest repeat, 525 bp in total, with the CTG sequence as the lagging strand template for replication . Measurements of cell growth showed that the lag phase associated with E . coli growth was increased for cultures containing plasmids with long CTG-CAG repeats, particularly when the CTG-containing strand was the lagging template . High frequencies of deletions were observed because of a growth advantage of cells containing plasmids with deleted triplet repeats . Incubation conditions that reduced the bacterial growth-rate produced a decreased extent of deletions, presumably because they alleviated the growth advantage of cells harboring plasmids with deleted triplet repeats . The experimental observations were simulated by a model in which shorter triplet repeats provided a growth advantage due to a shorter lag phase . We demonstrate that the accumulation of deletions within repeating sequences during growth of E . coli can be prevented, and discuss these findings in relation to the studies of repetitive DNA sequences . These are the first observations to show a direct influence between a plasmid-based DNA sequence or structure and factors controlling bacterial growth.

J Mol Biol, 1996 Nov 22, 264(1), 68 - 81
IS911-mediated transpositional recombination in vitro; Polard P et al.; A cell-free system is described that accomplishes an unusual type of transposition/recombination involving the bacterial insertion sequence IS911 . Using a plasmid substrate carrying a derivative of IS911, we show that bacterial cell extracts enriched for the IS911 transposase, OrfAB, carry out a single-strand cleavage and transfer reaction . This results in the formation of a figure-eight molecule in which a single strand of the element is circularized, faithfully reproducing an event previously detected in vivo . Moreover, when presented with a figure-eight substrate, OrfAB is capable of "reversing" strand transfer . This activity is equivalent to the "disintegration" reaction carried out by retroviral integrases . We demonstrate that the domain of OrfAB responsible for this catalytic activity is located in the carboxy-terminal region of the protein, since a peptide composed of this region retains disintegration activity . The OrfAB-mediated excision-circularization process previously observed in vivo was proposed to proceed via a figure-eight intermediate by circularization of the second transposon strand . The absence of transposon circles in cell-free reaction suggests either that the figure-eight form is not an intermediate or that additional host factors are required that are eliminated from the cell extract . Two types of model, replicative and non-replicative, are discussed to explain how the figure-eight molecule could be processed into the transposon circle.

J Mol Biol, 1996 Nov 22, 264(1), 56 - 67
Functional domains in protein TrwC of plasmid R388: dissected DNA strand transferase and DNA helicase activities reconstitute protein function; Llosa M et al.; TrwC is a bifunctional enzyme that displays two biochemical activities essential for plasmid R388 conjugation: oriT-specific DNA strand-transferase and DNA helicase activities . We overproduced and purified different segments of the protein allowing us to map the relaxase and DNA helicase activities to separate regions of the protein . A peptide comprising the N-terminal 275 amino acid residues of the protein was able to catalyze DNA cleavage and strand-transfer reactions when using oligonucleotides encompassing the nic site, although a longer fragment of TrwC (348 amino acid residues) was required to produce the nick on a supercoiled double-stranded DNA substrate . The segment of the protein between amino acid residues 192 and 966 contained the ATPase and DNA helicase activities, while a peptide consisting of amino acid residues 346 to 966 lost both activities . The dimerization region lay in the 495 C-terminal amino acid residues . Two peptides containing the DNA strand-transferase and DNA helicase activities, respectively, could functionally substitute for TrwC in R388 conjugation although at a 10,000-fold lower efficiency . Thus, integrity of the covalent structure of the protein was required for efficient DNA transfer . It can be assumed that the covalent linkage increases the efficiency of conjugation by increasing the effective concentration of one component (presumably the DNA helicase) at its site of action.

J Mol Biol, 1996 Nov 22, 264(1), 32 - 45
Characterization of charge change super-repressor mutants of trp repressor: effects on oligomerization conformation, ligation and stability; Reedstrom RJ et al.; We have carried out a physical characterization of mutant repressor proteins of the trp repressor system of Escherichia coli by circular dichroism, chemical denaturation, and 8-anilino-1-naphthalenesulfonate binding . We have also probed the protein-protein interactions via fluorescence anisotropy and lifetime measurements and measured the thermodynamics of ligand (L-tryptophan) binding by isothermal titration calorimetry . Here, we present investigations of four charge change super-repressor mutants: EK13, EK18, DN46 and EK49, and compare these results with those previously obtained for wild-type trp repressor and the AV77 super-repressor mutant . These studies demonstrate that super-repressor phenotypes may result from changes in operator affinity (DN46, EK49), protein-protein interactions (EK18), as well as the coupling of folding to ligand binding (AV77, EK13, EK18) . Correlations between the oligomerization behavior and cooperativity of DNA binding for some of these mutants indicate that coupling of oligomerization to DNA binding modulates operator site occupation giving rise to the super-repressor phenotype . The present results underscore the complex interplay between the multiple equilibria in this system . Moreover, they provide insights into the structural basis for the mutational perturbation of the energetics of this classical allosterically controlled transcriptional regulator.

J Med Chem, 1996 Nov 22, 39(24), 4825 - 32
Solution of the conformation and alignment tensors for the binding of trimethoprim and its analogs to dihydrofolate reductase: 3D-quantitative structure-activity relationship study using molecular shape analysis, 3-way partial least-squares regression, and 3-way factor analysis; Dunn WJ 3rd et al.; Molecular recognition is the basis of rational drug design, and for this reason it has been extensively studied . However, the process by which a ligand recognizes and binds to its receptor is complex and not well understood . For the case in which the geometries (conformation and alignment) of the ligand and receptor are known from X-ray crystal structure data, the problem is simplified . The receptor-bound conformation and alignment of the ligand is assumed, and those of additional ligands are inferred . For the general case in which the geometries of the ligand(s) and receptor are unknown, no general treatment or solution is available and receptor-ligand geometries must be obtained indirectly from structure-activity studies or synthesis and evaluation of rigid analogs . A general treatment for solving for the receptor-bound geometry of a series of ligands is presented here . Using molecular shape analysis, for ligand description, tensor analysis of N-way arrays by partial least-squares (PLS) regression, and 3-way factor analysis, the receptor-bound geometries of trimethoprim and a series of trimethoprim-like dihydrofolate reductase inhibitors are correctly predicted.

J Biol Chem, 1996 Nov 22, 271(47), 30144 - 8
Two transcription factors related with the eucaryal transcription factors TATA-binding protein and transcription factor IIB direct promoter recognition by an archaeal RNA polymerase; Hausner W et al.; We reported previously that cell-free transcription in the Archaea Methanococcus and Pyrococcus depends upon two archaeal transcription factors, archaeal transcription factor A (aTFA) and archaeal transcription factor B (aTFB) . In the genome of Pyrococcus genes encoding putative homologues of eucaryal transcription factors TATA-binding protein (TBP) and TFIIB have been detected . Here, we report that Escherichia coli synthesized Pyrococcus homologues of TBP and TFIIB are able to replace endogenous aTFB and aTFA in cell-free transcription reactions . Antibodies raised against archaeal TBP and TFIIB bind to polypeptides of identical molecular mass in the aTFB and aTFA fraction . These data identify aTFB as archaeal TBP and aTFA as the archaeal homologue of TFIIB . At the Pyrococcus glutamate dehydrogenase (gdh) promoter these two bacterially produced transcription factors and endogenous RNA polymerase are sufficient to direct accurate and active initiation of transcription . DNase I protection experiments revealed Pyrococcus-TBP producing a characteristic footprint between position -20 and -34 centered around the TATA box of gdh promoter . Pyrococcus-TFIIB did not bind to the TATA box but bound cooperatively with Pyrococcus-TBP generating an extended DNase I footprinting pattern ranging from position -19 to -42 . These data suggest that the Pyrococcus homologue of TFIIB associates with the TBP-promoter binary complex as its eucaryal counterpart, but in contrast to eucaryal TFIIB, it causes an extension of the protection to the region upstream of the TATA box.

J Biol Chem, 1996 Nov 22, 271(47), 29897 - 902
Probing substrate binding site of the Escherichia coli quinol oxidases using synthetic ubiquinol analogues; Sakamoto K et al.; Substrate binding sites of the Escherichia coli bo- and bd-type quinol oxidases were probed with systematically synthesized ubiquinol analogues . The apparent Km values of ubiquinol-2 derivatives to the bo-type enzyme were much lower than that of the corresponding 6-n-decyl derivatives . The isoprenoid structure is less hydrophobic than the saturated n-alkyl group with the same carbon number; therefore, the native isoprenoid side chain appears to play a specific role in quinol binding besides simply increasing hydrophobicity of the molecule . The Vmax values of 2-methoxy-3-ethoxy analogues were greater than that of 2-ethoxy-3-methoxy analogues irrespective of the side chain structure . This result indicates not only that a methoxy group in the 2-position is recognized more strictly than the 3-position by the binding site but also that the side chain structure does not affect binding of the quinol ring moiety . Systematic analysis of the electron-donating activities of the analogues with different substituents in the 5-position revealed that the 5-methyl group is important for the activity . In the parallel studies with the bd-type enzyme, we obtained similar observations except that almost all quinol analogues, but not ubiquinol-1, elicited a remarkable substrate inhibition at higher concentrations . These results indicate that the two structurally unrelated terminal oxidases share common structural properties for the quinol-oxidation site.

J Biol Chem, 1996 Nov 22, 271(47), 29865 - 9
Engineered complementation in Escherichia coli aspartate transcarbamoylase . Heterotropic regulation by quaternary structure stabilization; Aucoin JM et al.; Escherichia coli aspartate transcarbamoylase regulates pyrimidine biosynthesis by altering its activity homotropically in response to one of its substrates and heterotropically in response to nucleotide effectors . The mechanism of this regulation involves two structurally and functionally different forms of the enzyme, one with low activity and low affinity for substrates (T state) and the other with high activity and high affinity for substrates (R state) . Heterotropic regulation may be due to the direct transmission of a regulatory "signal" between the regulatory site and the active site some 60 A away and/or may involve altering the relative stability of the two forms of the enzyme . By combining a T state-stabilized mutant version of the enzyme, previously thought to have a defect in a heterotropic transmission pathway, with a known R state-stabilized mutant enzyme, we were able to restore some properties of the wild-type enzyme . These data imply that the relative stabilization of the T and R states of the enzyme is in part responsible for the homotropic and heterotropic properties of aspartate transcarbamoylase and that direct pathways for transmission of the heterotropic signals are unlikely.

J Biol Chem, 1996 Nov 22, 271(47), 29752 - 8
Role of electrostatic interactions in defining the potency of neurotoxin B-IV from Cerebratulus lacteus; Wen PH et al.; Chemical modification implicates arginine residues of the Cerebratulus lacteus neurotoxin B-IV in biological activity . In the present study, we used site-directed mutagenesis to assess the functional contributions of each of these residues . Panels of mutants at each site have been constructed by polymerase chain reaction and recombinant proteins expressed and purified to homogeneity using an Escherichia coli expression system developed in this laboratory . All substitutions for Arg-17 (Gln, Ala, or Lys) yield proteins having undetectable levels of activity, while charge neutralizing replacement of Arg-25 (R25Q) causes a 400-fold reduction in specific toxicity . However, the R25K mutein is almost as active as natural toxin . Circular dichroism spectroscopy indicates that there are no major conformational changes in any of these muteins . These results therefore demonstrate the requirement for a guanidinium group at position 17, and a positive charge at position 25 . NMR analyses (Hansen, P . E., Kem, W . R., Bieber, A . L., and Norton, R . S . (1992) Eur . J . Biochem . 210, 231-240) reveal neurotoxin B-IV to contain two antiparallel alpha-helices, which together include 57% of the sequence . Both Arg-17 and Arg-25 lie on the same face of the N-terminal helix (residues 13-26), as do the carboxyl groups of Glu-13 and Asp-21 . However, charge neutralizing mutations of the latter two sites have no effects on biological activity . Arg-34, situated near the N terminus of helix 2 (residues 33-49) is also important for activity, as its replacement by Gln or Ala diminishes activity by 20- and 80-fold, respectively . However, unlike Arg-17 and Arg-25, thermal denaturation experiments suggest that R34Q may be structurally destabilized relative to wild-type B-IV.

J Biol Chem, 1996 Nov 22, 271(47), 29722 - 8
Subunit complementation of Escherichia coli adenylosuccinate synthetase; Kang C et al.; Data are presented, based upon subunit complementation experiments, that suggest that Escherichia coli adenylosuccinate synthetase contains two shared active sites between its dimeric interface . This conclusion was alluded to by use of mutant forms of adenylosuccinate synthetase previously prepared by site-directed mutagenesis . The experiments indicate that, although the R143L and D13A mutants have low or no activity independently, when they are mixed, a significant amount of activity was obtained . These results indicate that the subunits exchange with each other to form heterodimers with a single viable wild-type active site . The kcat value for the active hybrid active site in the R143L-D13A heterodimer is virtually identical to that observed with the wild-type enzyme, and the other kinetic parameters are very similar to those found for the wild-type enzyme . An analysis of the restoration of the activity in the presence of substrates suggests that GTP and IMP stabilize the dimeric structure of the protein . A comparison of the restoration of the activity using different combinations of mutants provides evidence indicating that some of the GTP binding elements, including the P-loop, in the protein are important for subunit integrity . Also, for the first time, a comprehensive analysis of subunit complementation is performed for the two inactive mutants (R143L and D13A) where the dissociation constants for the R143L-D13A heterodimer and the D13A homodimer were determined to be 21 and 2.9 microM, respectively . A concentration dependence of the specific activity of the wild-type protein in this study shows that the Kd for dimer dissociation is approximately 1 microM.

J Biol Chem, 1996 Nov 22, 271(47), 29698 - 706
A significant fraction of functional SecA is permanently embedded in the membrane . SecA cycling on and off the membrane is not essential during protein translocation; Chen X et al.; SecA has been suggested to cycle on and off the cytoplasmic membrane of Escherichia coli during protein translocation . We have reconstituted 35S-SecA onto SecA-depleted membrane vesicles and followed the fate of the membrane-associated 35S-SecA during protein translocation . Some 35S-SecA was released from the membranes in a translocation-independent manner . However, a significant fraction of 35S-SecA remained on the membranes even after incubation with excess SecA . This fraction of 35S-SecA was shown to be integrated into the membrane and was active in protein translocation, indicating that SecA cycling on and off membrane is not required for protein translocation . Proteolysis experiments did not support the model of SecA insertion and deinsertion during protein translocation; instead, a major 48-kDa domain was found persistently embedded in the membrane regardless of translocation status . Thus, in addition to catalyzing ATP hydrolysis, certain domains of SecA probably play an important structural role in the translocation machinery, perhaps forming part of the protein-conducting channels.

J Biol Chem, 1996 Nov 22, 271(47), 29561 - 8
Molecular identification and characterization of cytosolic isoforms of glutamine synthetase in maize roots; Sakakibara H et al.; In maize, a small multigene family encodes the cytosolic isoforms of glutamine synthetase (GS), and five cDNAs, designated pGS1a, pGS1b, pGS1c, pGS1d, and pGS1e, have been cloned (Sakakibara, H., Kawabata, S., Takahashi, H., Hase, T., and Sugiyama, T . (1992) Plant Cell Physiol . 33, 49-58; Li, M., Villemur, R., Hussey, P . J., Silflow, C . D., Gantt, J . S., and Snustad, D . P . (1993) Plant Mol . Biol . 23, 401-407) . This report describes the identification and enzymatic characterization of the cytosolic isoforms of GS in maize roots, namely GS1 and GSr . The purified isoforms, as well as recombinant enzymes that had been overexpressed in Escherichia coli, were analyzed by capillary liquid chromatography/electrospray ionization-mass spectrometry, and GS1 and GSr were identified as the products of the GS1a/GS1b and GS1c/GS1d genes, respectively . Upon the addition of ammonia to the culture medium, significant amounts of GSr accumulated and a preferential increase in GS synthetase activity, as compared to GS transferase activity, was found in the root extract . Assays with the purified recombinant enzymes confirmed that the specific biosynthetic and synthetase activities of GSr were 1.6-fold higher than those of GS1 . Marked differences in stability were also found between the two isoforms: GSr was more sensitive to heat than GS1 and octameric aggregates of the subunits of GSr were easily dissociated to monomers than those of GS1 at low concentrations of Mn2+ and Mg2+ ions . These characteristics of the ammonia-induced isoform of GS seem to be physiologically important for the primary assimilation of external ammonia by roots.

Gene, 1996 Nov 21, 180(1-2), 207 - 12
Molecular analysis of the gene encoding a protein component of the Eikenella corrodens adhesin complex that is close to the carbohydrate recognition domain; Azakami H et al.; A monoclonal antibody against a lectin-like substance (LS) of Eikenella corrodens (Ec) was used for screening the Ec DNA library . Three positive clones that carried an identical 12-kb segment were obtained . A 25-kDa protein, which specifically binds to the antibody, was overproduced in all of the Escherichia coli clones . Deletion analysis showed that the gene encoding the 25-kDa protein was located within a 1.2-kb segment . The nucleotide (nt) sequence of this segment contained an open reading frame encoding a protein of 24600 Da . We purified the 25-kDa protein from the cloned E . coli strain . The sequence of the first 10 amino acids(aa) from the N-terminus of the purified 25-kDa protein agreed with that deduced from the nt sequence . Since the monoclonal antibody used in this study inhibits the physiological activity of EcLS, we concluded that the 25-kDa protein is a component of the adhesin complex, which is located near the carbohydrate recognition domain of lectin in EcLS.

Gene, 1996 Nov 21, 180(1-2), 91 - 9
Bordetella pertussis adenylate cyclase toxin: proCyaA and CyaC proteins synthesised separately in Escherichia coli produce active toxin in vitro; Westrop GD et al.; Bordetella pertussis produces a cell-invasive adenylate cyclase toxin (CyaA) which is related to the RTX family of pore-forming toxins . Like all RTX toxins, CyaA is synthesised as a protoxin (proCyaA), encoded by the cyaA gene . Activation to the mature cell-invasive toxin involves palmitoylation of lysine 983 and is dependent on co-expression of cyaC . The role of the cyaC gene product in the acylation reaction has not been determined . We have developed an efficient T7 RNA polymerase system for over-expression of cyaA and cyaC separately in Escherichia coli . Each protein accumulated intracellularly in an insoluble form and could be collected by centrifugation of lysed cells . A single-step purification was achieved by extraction of the aggregated material with 8 M urea . Active cell-invasive CyaA was produced in vitro when the proCyaA and CyaC proteins were mixed with a cytosolic extract of either E . coli or B . pertussis . Activation was assumed to occur by an acylation reaction requiring acyl carrier protein (ACP) as cofactor, as the cytosolic factor required for toxin activation was lost if the S100 extract was dialysed before use and the cytosolic factor could be replaced in the in vitro reaction by ACP charged separately in vitro with palmitic acid, as reported previously for activation of the homologous E . coli haemolysin (HlyA) . The in vitro activation system may be used to investigate the mechanism of the CyaC-dependent acylation of proCyaA and the effect of variation of the modifying fatty acyl group on target cell specificity and toxic activity of CyaA.

Gene, 1996 Nov 21, 180(1-2), 81 - 9
The use of an improved transposon mutagenesis system for DNA sequencing leads to the characterization of a new insertion sequence of Streptomyces lividans 66; Fischer J et al.; A DNA sequencing strategy was developed based on the tetracycline resistance transposon Tn1721 . A universal M13 primer binding site (UP) for DNA sequencing and restriction sites for mapping were inserted near one end of Tn1721 and the new derivative, Tn5491, introduced onto a conjugative F' plasmid . The target sequence is inserted between two inverted resolution sites (res) of Tn1721 present on the high-copy plasmid pJOE2114 . Due to the inviability of long palindromic sequences in Escherichia coli insertions between the inversely orientated res sites of pJOE2114 are positively selected . Transposition of Tn5491 into the target sequence is selected by cointegrate formation of Tn5491 during transposition, mating and transfer of the nonconjugative sequencing vector . After cointegrate resolution, the additional res sites in the vector result in a second site-specific recombination removing most of the transposon (except of 136 bp) and part of the target sequence . The reduced plasmid sizes and the use of the universal primer improved the quality of the sequencing results obtained on an automated fluorescent sequencer . A 3.35-kb EcoRI fragment from the 30-kb terminal inverted repeats (TIR) of the Streptomyces lividans chromosome was sequenced by this method . A 1304-bp sequence was found on this fragment with the features of insertion elements . The element called IS1372 had 27-bp IR and two potential open reading frames . The predicted gene products had similar sizes and high similarity to gene products encoded by insertion sequences of the IS3 family . Furthermore, a potential signal stimulating ribosomal shifts and typical for members of the IS3 family was identified . Five to seven copies of IS1372 were found in different strains of S . lividans but none in other Streptomyces species tested.

Oncogene, 1996 Nov 21, 13(10), 2177 - 87
Rasp21 sequences opposite the nucleotide binding pocket are required for GRF-mediated nucleotide release; Leonardsen L et al.; The substrate requirements for the catalytic activity of the mouse Cdc25 homolog Guanine nucleotide Release Factor, GRF, were determined using the catalytic domain of GRF expressed in insect cells and E . coli expressed H-Ras mutants . We found a requirement for the loop 7 residues in Ras (amino acids 102 to 105) for stimulation of guanine nucleotide release . The dependence on the switch I and II regions of Rasp21 (encompassing the residues that shift position in the GTP- versus GDP-bound protein), which had been seen with Sdc25-mediated exchange, was also found for GRF . In addition, the sensitivity of H-Ras to GRF was abolished when residues 130-139 were replaced by proline-aspartic acid-glutamine, whereas substitution of the entire loop 8 (residues 123-130 replaced by leucine-isoleucine-arginine) had no effect on the stimulation of guanine nucleotide release by GRF . Substrate activity of Ras proteins were independent of their post-translational processing, GDP release was stimulated threefold more effectively by GRF than was GTP release, and no major differences were found between the mammalian N-, H- and K-Ras proteins . Examining the responsiveness of the Ras protein from S . pombe and the human Ras like proteins RhoA, Rap1A, Rac1 and G25K revealed a strict Ras specificity; of these only S . pombe Ras was GRF sensitive.

Biochem Biophys Res Commun, 1996 Nov 21, 228(3), 752 - 6
A novel heme protein that acts as a carbon monoxide-dependent transcriptional activator in Rhodospirillum rubrum; Aono S et al.; The gene coding for a carbon monoxide-dependent transcriptional activator (cooA) in Rhodospirillum rubrum has been expressed in E . coli, and the recombinant CooA has been purified . CooA contains b-type heme which may act as a CO sensor in vivo . CO-bound CooA was formed when reduced CooA was reacted with CO, but not in the case of oxidized CooA . CooA is the first example of the heme protein acting as a DNA-binding transcriptional activator.

Biochem Biophys Res Commun, 1996 Nov 21, 228(3), 724 - 32
Characterization of recombinant Eschericha coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase: analysis of enzymatic activity and substrate specificity; Cornell KA et al.; Recombinant E . coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (EC 3.2.2.9) was used to study the potential for this enzyme to serve as a target for chemotherapeutic intervention . An examination of the parameters required for enzymatic activity indicate that the nucleosidase functions over a broad range of pH and temperature, with acidic conditions and temperatures of 37-45 degrees C being optimal . Analogs of 5'-methylthioadenosine and adenosine were assessed as potential enzyme inhibitors and to provide details regarding substrate specificity and reaction mechanism . The 5'-arylthio analog, 5'-(p-nitrophenyl)thioadenosine, was the most potent enzyme inhibitor studied, with a Ki of 20nM . A mutant of the nucleosidase lacking the first 8 amino acids was engineered to determine the contribution of these conserved residues toward enzyme specificity . The truncated enzyme exhibited a K(m){MTA} of 1.43 microM, approximately 3 fold higher than the K(m) reported for the full-length nucleosidase.

Biochem Biophys Res Commun, 1996 Nov 21, 228(3), 704 - 8
Mutation of the cysteine residues in human initiation factor 4E: effects on mRNA cap binding ability; Teraoka Y et al.; To investigate the role of four Cys residues of eukaryotic initiation factor (eIF)-4E in the recognition of the mRNA cap structure, single, double and quadruple mutant genes which encoded the Ala residues in the place of the respective Cys residues were prepared using a synthetic human eIF-4E gene by the site-directed mutagenesis, and were expressed in E . coli with the same way as the wild type . The cap binding abilities of respective mutated eIF-4Es were compared with that of the wild-type by m7GTP affinity chromatography . The results suggest that, although all four of Cys residues participate in the recognition of the mRNA cap structure, they contribute indirectly to the stabilization of overall tertiary structure, especially of the cap binding pocket, rather than by direct interaction . A comparison among the cap binding abilities of single, double and quadruple mutants indicated no existence of internal disulfide bonds in eIF-4E.

FEBS Lett, 1996 Nov 18, 397(2-3), 303 - 7
Redox FTIR difference spectroscopy using caged electrons reveals contributions of carboxyl groups to the catalytic mechanism of haem-copper oxidases; Lubben M et al.; Redox spectra of the haem-copper oxidases cytochrome aa3 of Rhodobacter sphaeroides and cytochrome bo3 of Escherichia coli were recorded in the visible and infrared spectral regions . The reduction of oxidases was initiated after light activation of the 'caged electron' donor riboflavin . Infrared redox difference spectra exhibit absorbance changes in the amide I region, which are indicative of very small redox-linked conformational movements in the polypeptide backbone . A reproducible redox-dependent pattern of positive and negative absorption changes is found in the carbonyl region (1680-1750 cm(-1)) . The carbonyl bands shift to lower frequencies due to isotope exchange of the solvent H2O to D2O . This common feature of cytochrome c and quinol oxidases indicates that at least (i) one redox-sensitive carboxyl group is in the protonated state in the oxidized form and (ii) one carboxylic acid is involved at a catalytic step--presumably in proton translocation--of haem-copper oxidase.

FEBS Lett, 1996 Nov 18, 397(2-3), 283 - 9
The beta-tubulin monomer release factor (p14) has homology with a region of the DnaJ protein; Llosa M et al.; p14 is a molecular chaperone involved in beta-tubulin folding which catalyzes the release of beta-tubulin monomers from intermediate complexes . Here we demonstrate that active p14 protein which we have purified from an overproducing Escherichia coli strain can also release beta-tubulin monomers from tubulin dimers in the presence of an additional cofactor (Z) . Analysis of p14 secondary structure suggests that this protein may belong to a family of conserved proteins which share structural similarities with the J-domain of DnaJ . We have constructed deletions and site-directed mutations in the p14 gene . A single D to E mutation in the region shown in DnaJ to be an essential loop for its function affected the monomer-release activity of p14 . These results support the hypothesis that this p14 loop interacts with beta-tubulin in a similar fashion as DnaJ interacts with DnaK and suggest a possible role of p14 in the folding process.

FEBS Lett, 1996 Nov 18, 397(2-3), 277 - 82
The recombinant catalytic domain of membrane-type matrix metalloproteinase-1 (MT1-MMP) induces activation of progelatinase A and progelatinase A complexed with TIMP-2; Lichte A et al.; A truncated form of the membrane-type matrix metalloproteinase-1 {(Ala21-Ile318)proMT1-MMP} lacking the hemopexin-like and trans-membrane domain was produced in E . coli . We demonstrate that the recombinant proenzyme was autoproteolytically processed to a fully active catalytic domain with N-terminal Ile114 . The catalytic domain of MT1-MMP initiated the activation of progelatinase A and progelatinase A complexed with tissue inhibitor of metalloproteinases-2 (TIMP-2) . As a typical soluble metalloproteinase it was able to cleave physiologic as well as synthetic substrates . Our kinetic data demonstrate that TIMP-2 is a potent inhibitor for the recombinant enzyme.

FEBS Lett, 1996 Nov 18, 397(2-3), 273 - 6
Towards a mechanism of AMP-substrate inhibition in adenylate kinase from Escherichia coli; Sinev MA et al.; Crystallographic studies on adenylate kinase (AK) suggest that binding of ATP causes the LID domain of the enzyme to close over the ATP molecule (Schlauderer et al . (1996) J . Mol . Biol . 256, 223-227) . The method of time-resolved fluorescence resonance energy transfer was applied to study the proposed structural change in AK from Escherichia coli . Two active derivatives of the (C77S, A73C, V142C)-AK mutant containing the excitation energy donor attached to one of the two cysteine residues and the acceptor attached to the other cysteine were prepared to monitor displacements of the LID domain in response to substrate binding . Binding of either ATP or AMP was accompanied by an approximately 9 A decrease in the interprobe distances suggesting LID domain closure . Closure of the LID domain in response to AMP binding may be a possible reason for the strong AMP-substrate inhibition known for E . coli AK.

FEBS Lett, 1996 Nov 18, 397(2-3), 235 - 8
Further evidence that inhibitor-2 acts like a chaperone to fold PP1 into its native conformation; MacKintosh C et al.; The gamma1-isoform of protein phosphatase-1 expressed in Escherichia coli (PP1gamma) and the native PP1 catalytic subunit (PP1C) isolated from skeletal muscle dephosphorylated Ser-14 of glycogen phosphorylase at comparable rates . In contrast, PP1gamma dephosphorylated several tyrosine-phosphorylated proteins at similar rates to authentic protein tyrosine phosphatases (PTPases), but native PP1C was almost inactive towards these substrates . The phosphorylase phosphatase (PhP) and PTPase activities of PP1gamma were inhibited by vanadate with IC50 values (30-100 microM) comparable to authentic PTPases, whereas the PhP activity of native PP1C was insensitive to vanadate . PP1gamma lost its PTPase activity, and its PhP activity became insensitive to vanadate, after interaction with inhibitor-2, followed by the reversible phosphorylation of inhibitor-2 at Thr-72 . These findings support and extend the hypothesis that inhibitor-2 functions like a chaperone to fold PP1 into its native conformation, and suggest that the correct folding of PP1 may be critical to prevent the uncontrolled dephosphorylation of cellular phosphotyrosine residues.

FEBS Lett, 1996 Nov 18, 397(2-3), 210 - 4
Coexpression of a human P450 (CYP3A4) and P450 reductase generates a highly functional monooxygenase system in Escherichia coli; Blake JA et al.; The catalytic activities of recombinant cytochrome P450s expressed in E . coli have been impeded by the absence of endogenous P450 reductase . To solve this problem, we coexpressed P450 reductase with CYP3A4 . Membranes from this strain contained 215 pmol P450/mg protein and a reductase activity of 1315 nmol cytochrome c reduced/min per mg . We detected 6beta-hydroxylation of testosterone and oxidation of nifedipine in vivo with turnover numbers of 15.2 and 17.3 min(-1), respectively . These values compare favourably with those obtained using an optimally reconstituted system . Our data demonstrate that a catalytically efficient human P450 system can be generated in E . coli.

J Biotechnol, 1996 Nov 15, 51(3), 279 - 85
Sol-gel applications in environmental biotechnology; Armon R et al.; Sol gel process was applied for three different applications in environmental biotechnology: (1) thin, fluorescein diacetate-doped sol-gel film made possible epifluorescent microscopic examination of adsorbed Escherichia coli CN13 cells without additional staining: (2) Thiobacillus thiooxidans cell-free extract entrapped into sol-gel matrix displayed oxidative activity on H2S in liquid medium; and (3) two media (E . coli (EC) and sulfate-reducing bacteria (SRB)) were doped into sol-gel and used to enumerate environmental samples for E . coli and sulfate-reducing bacteria, by the most probable number (MPN) method . The comparison of the modified method with the standard enumeration method revealed very good correlation . The sol-gel MPN method is sensitive, saves times, and the substrate can be prepared and stored long-term at room temperature (up to 1 year).

Eur J Biochem, 1996 Nov 15, 242(1), 104 - 13
Isolation, expression and characterization of the gene for an ADP-ribosylation factor from the human malaria parasite, Plasmodium falciparum; Stafford WH et al.; We have isolated an ADP-ribosylation factor (ARF) gene from the human malarial parasite, Plasmodium falciparum . The gene (P . falciparum arf1) has four introns and the exons encode a protein of 181 amino acids with high similarity to the mammalian class I ARF proteins 1-3 (> or = 74% amino acid identity) . Southern hybridization suggests there is at least one additional arf in the P . falciparum genome . Northern analysis identified a single P . falciparum arf1 mRNA of 1.8 kb in the asexual blood stage form of the parasite . The P . falciparum arf1 mRNA levels are developmentally regulated, reaching a maximum during nuclear division towards the end of the intraerythrocytic cycle . P . falciparum arf1 cDNA was isolated by reverse-transcriptase polymerase chain reaction and used to express a recombinant protein in Escherichia coli . Recombinant P . falciparum ARF1 protein was purified with stoichiometric amounts of bound GDP, although intrinsic guanose triphosphatase activity of the protein could not be detected . The protein stimulated cholera-toxin-catalyzed ADP-ribosyltransferase activity in a reaction that was dependent upon the addition of both dimyristoylglycerophosphocholine and cholate . The protein bound GTP with first-order kinetics with an apparent rate constant, k', of 0.0145 (+/- 0.0019) min-1 . These results suggest that P . falciparum ARF1 is a member of the class 1 ARF family and provide additional evidence for the existence of a classical secretory pathway in P . falciparum.

Eur J Biochem, 1996 Nov 15, 242(1), 20 - 8
Locations of functional domains in the RecA protein . Overlap of domains and regulation of activities; Takahashi M et al.; We review the locations of various functional domains of the RecA protein of Escherichia coli, including how they have been assigned, and discuss the potential regulatory roles of spatial overlap between different domains . RecA is a multifunctional and ubiquitous protein involved both in general genetic recombination and in DNA repair: it regulates the synthesis and activity of DNA repair enzymes (SOS induction) and catalyses homologous recombination and mutagenesis . For these activities RecA interacts with a nucleotide cofactor, single-stranded and double-stranded DNAs, the LexA repressor, UmuD protein, the UmuD'2C complex as well as with RecA itself in forming the catalytically active nucleofilament . Attempts to locate the respective interaction sites have been advanced in order to understand the various functions of RecA . An intriguing question is how these numerous functional sites are contained within this rather small protein (38 kDa) . To assess more clearly the roles of the respective sites and to what extent the sites may be interacting with each other, we review and compare the results obtained from various biological, biochemical and physico-chemical approaches . From a three-dimensional model it is concluded that all sites are concentrated to one part of the protein . As a consequence there are significant overlaps between the sites and it is speculated that corresponding interactions may play important roles in regulating RecA activities.

J Biochem Biophys Methods, 1996 Nov 15, 33(2), 117 - 33
Novel fluorescence-photochrome labeling method in the study of biomembrane dynamics; Likhtenshtein GI et al.; A novel photochrome-fluorescence method (PFLM) based on monitoring fluorescence parameters and kinetics of photochrome photoisomerization of para-substituted stilbenes (PSS) has been proposed . It was shown that PSS exhibits fluorescence characteristics which are similar to ones of typical membrane fluorescence probes such as diphenylhexatriene (DPH) . A study of kinetics of PSS trans-cis and cis-trans photoisomerization makes it possible to estimate, under certain conditions, the rotational correlation time of the stilbene fragments in the excited state of PSS for the fixed angle 180 degrees . In viscous media this process is a rate-determining stage . Taken together, the both techniques, fluorescence and photochrome, make it possible to establish a detailed mechanism and measure quantitative parameters of stilbene probe (PSS) mobility in a membrane . The PFLM was applied to the study of E . coli membrane dynamics.

Biochim Biophys Acta, 1996 Nov 15, 1317(2), 149 - 54
A conformational epitope in the N-terminus of the Escherichia coli heat-stable enterotoxins is involved in receptor-ligand interactions; Garrett BM et al.; The heat-stable enterotoxins are a family of low molecular weight, cysteine rich peptide toxins which are one of the major causes of watery diarrhea in children and adults . These toxins bind to a cell surface receptor in intestinal cells and mediate their action through elevation of intracellular cyclic GMP . We have generated a monoclonal antibody to these peptide toxins which is able to neutralise the activity of the peptides in a human colonic cell line, the T84 cell line . The monoclonal antibody, ST:G8, appears to be directed to an epitope distinct from antibodies previously generated, and prior incubation of this antibody, or Fab generated from this antibody, with full length STh and STp peptides prevents cGMP accumulation in T84 cells . This inhibition is a direct result of the antibody preventing binding of the peptides to the receptor . ST:G8 Mab does not recognize a 13-mer biologically active analog of STp, comprising the core sequence of STp peptide, suggesting that it is directed to a region in the N-terminus of the peptides, which may modulate receptor interaction/activation . The antibody recognizes a conformational epitope in the ST peptides, since reduction and carboxyamidation of ST abolishes antibody cross-reactivity . Differential cross-reactivity of the Mab with STh and STp peptides which differ markedly only in their N-termini, suggests that this antibody recognizes a distinct conformation in the two peptides, which is essential for receptor interaction.

Nucleic Acids Res, 1996 Nov 15, 24(22), 4594 - 6
Rapid construction in yeast of complex targeting vectors for gene manipulation in the mouse; Storck T et al.; Targeting vectors for embryonic stem (ES) cells typically contain a mouse gene segment of >7 kb with the neo gene inserted for positive selection of the targeting event . More complex targeting vectors carry additional genetic elements (e.g . lacZ, loxP, point mutations) . Here we use homologous recombination in yeast to construct targeting vectors for the incorporation of genetic elements (GEs) into mouse genes . The precise insertion of GEs into any position of a mouse gene segment cloned in an Escherichia coli/yeast shuttle vector is directed by short recombinogenic arms (RAs) flanking the GEs . In this way, complex targeting vectors can be engineered with considerable ease and speed, obviating extensive gene mapping in search for suitable restriction sites.

Nucleic Acids Res, 1996 Nov 15, 24(22), 4577 - 83
In vitro transcription of a poly(dA) x poly(dT)-containing sequence is inhibited by interaction between the template and its transcripts; Kiyama R et al.; Transcription of poly(dA) x poly(dT)-containing sequences was investigated in vitro using plasmids carrying a (dA)34 x (dT)34 tract in the coding region of the lacZ gene . The efficiency of transcription of the (dT)34 sequence on the transcribing strand by Escherichia coli RNA polymerase was substantially lower (approximately 60%) than that of the (dA)34 sequence or of the control lacZ gene . Analysis of the transcription process of the (dT)34 sequence by T3 RNA polymerase showed that the transcription was frequently arrested or terminated at the middle as well as immediately proximal of the (dA)34 x (dT)34 tract, and it occurred more prominently following accumulation of transcription products . This inhibition was strongly enhanced by the addition of the oligonucleotide (dT)34 or poly(U) to the reaction mixture, while (dA)34 and the duplex (dA)34 x (dT)34 suppressed the inhibition . A similar transcriptional inhibition was also observed in transcription mediated by T7 RNA polymerase and eukaryotic RNA polymerase II . We also demonstrated RNA x DNA complex formation of the (dA)34 x (dT)34 tract with poly(U), but not with poly(A) . These findings strongly suggest that poly(dT)-containing template sequences interact and form a complex with its transcription products, possibly an RNA x DNA triplex, which blocks further transcription . This would explain the instability of the plasmids transcribing mRNAs with poly(U) but not poly(A) tracts and the underrepresentation of poly(U) but not poly(A) tracts in mRNAs.

Nucleic Acids Res, 1996 Nov 15, 24(22), 4572 - 6
Cleavage of single- and double-stranded DNAs containing an abasic residue by Escherichia coli exonuclease III (AP endonuclease VI); Shida T et al.; The Escherichia coli exonuclease III (AP endonuclease VI) is a DNA-repair enzyme that hydrolyzes the phosphodiester bond 5' to an abasic site in DNA . To study how the enzyme recognizes the abasic site, we used oligonucleotides containing a synthetic abasic site at any desired position in the sequence . We prepared oligonucleotides containing an abasic residue such as 2'-deoxyribosylformamide, 2'-deoxyribose, 1',2'-dideoxy ribofuranose or propanediol . Duplex oligonucleotides containing an abasic residue used in this study were cleaved on the 5' side of the abasic site by exonuclease III in spite of the varieties of the bases opposite and adjacent to the abasic site . In addition, we observed that the enzyme cleaved single-stranded oligonucleotides containing an abasic site on the 5' side of the abasic site . These findings suggest that the enzyme may principally recognize the DNA-pocket formed at an abasic site . The indole ring of the tryptophan 212 residue of the exonuclease III is probably intercalated to the abasic site . The tryptophan in the vicinity of the catalytic site is conserved in the type II AP endonuclease from various organisms.

Nucleic Acids Res, 1996 Nov 15, 24(22), 4487 - 94
DNA binding of Jun and Fos bZip domains: homodimers and heterodimers induce a DNA conformational change in solution; John M et al.; We constructed plasmids encoding the sequences for the bZip modules of c-Jun and c-Fos which could then be expressed as soluble proteins in Escherichia coli . The purified bZip modules were tested for their binding capacities of synthetic oligonucleotides containing either TRE or CRE recognition sites in electrophoretic mobility shift assays and circular dichroism (CD) . Electrophoretic mobility shift assays showed that bZip Jun homodimers and bZip Jun/Fos heterodimers bind a collagenase-like TRE (CTGACTCAT) with dissociation constants of respectively 1.4 x 10(-7) M and 5 x 10(-8) M . As reported earlier {Patel et al . (1990) Nature 347, 572-575}, DNA binding induces a marked change of the protein structure . However, we found that the DNA also undergoes a conformational change . This is most clearly seen with small oligonucleotides of 13 or 14 bp harboring respectively a TRE (TGACTCA) or a CRE (TGACGTCA) sequence . In this case, the positive DNA CD signal at 280 nm increases almost two-fold with a concomitant blue-shift of 3-4 nm . Within experimental error the same spectral changes are observed for TRE and CRE containing DNA fragments . The spectral changes observed with a non-specific DNA fragment are weaker and the signal of free DNA is recovered upon addition of much smaller salt concentrations than required for a specific DNA fragment . Surprisingly the spectral changes induced by Jun/Jun homodimers are not identical to those induced by Jun/Fos heterodimers . However, in both cases the increase of the positive CD band and the concomitant blue shift would be compatible with a B to A-transition of part of the binding site or a DNA conformation intermediate between the canonical A and B structures.

J Mol Biol, 1996 Nov 15, 263(5), 789 - 99
Pressure effects on the structure of oligomeric proteins prior to subunit dissociation; Cioni P et al.; In studies of pressure-induced subunit dissociation of protein aggregates, now widely used to evaluate the association free energy, entropy and enthalpy of very stable complexes, it is assumed that high pressure does not influence their structure/thermodynamic parameters and that some peculiarities of these equilibria, such as the decrease in subunit affinity at larger degrees of dissociation (alpha) and hysteresis in alpha/pressure diagrams are imputable to the slow conformational drift of isolated subunits . To test this premise, the conformation of dimeric alcohol dehydrogenase from horse liver and alkaline phosphatase from Escherichia coli was monitored as a function of pressure (up to 3 kbar) and temperature (0 to 50 degrees C) by means of the intrinsic Trp fluorescence and phosphorescence emission and binding of the 1-anilinonaphatalene-8-sulphonic acid (ANS) fluorophore . The results show a distinct influence of high pressure on the native dimers whose changes in conformation may, depending on whether or not these alterations are promptly reversed, be distinguished in elastic and inelastic changes . Elastic changes are ubiquitous and refer to pronounced modulations of the phosphorescence lifetime which is a monitor of the internal flexibility of the macromolecules . They attest to a tightening of the globular structure in the lower pressure range (below 1.5 kbar) as opposed to an increased fluidity in the higher range . The trend is similar between the two proteins and the tightening/loosening effect is fully consistent with the role that internal cavities and hydration of polypeptide is expected to play in determining the compressibility of these biopolymers . Inelastic perturbations reveal a more profound loosening of the globular fold and were observed only with alcohol dehydrogenase under conditions (low temperature (t < 10 degrees C) and high pressure (p > 2.5 kbar)) that favour protein hydration . They involve slow consecutive reactions that produce drastic reductions in phosphorescence lifetime, spectral red shifts, quenching of fluorescence and phosphorescence emission and modulation of ANS binding . Judging from the full protection afforded by glycerol as cosolvent, or the remarkable enhancement caused by modest concentrations of urea, the driving force of these perturbations appears to be pressure-induced hydration of the protein . Inelastic conformational changes are accompanied by a slow and often incomplete recovery of enzymatic activity . The characteristic times of these processes, their pressure dependence and the slow, thermally activated, reversibility are discussed in the light of hysteresis phenomena and changes of subunit affinity in dissociation equilibria.

J Mol Biol, 1996 Nov 15, 263(5), 699 - 706
Interaction of the basic protrusion of Escherichia coli ribonuclease HI with its substrate; Iwai S et al.; In order to determine the actual distance between the active site and the substrate binding site, termed the basic protrusion, of Escherichia coli ribonuclease HI, synthetic oligonucleotide duplexes with gradually extended overhangs were used, in which the enzymatic cleavage was restricted to a single site with 2'-O-methylnucleosides . The affinity of the enzyme for each substrate was determined by kinetic analysis . It was found that the affinity increased markedly when one nucleotide was attached to the 3' end of the DNA strand of the nine-base-pair hybrid duplex and then increased slightly as the DNA strand was extended further, whereas elongation of the strand in the other direction caused no change . When a mutant enzyme, in which three lysine residues in the basic protrusion were altered to alanine, was used, no increase in the kcat/K(m) value was observed . The results indicate that, for the productive binding, the axis from the 3' to the 5' end of the RNA strand of the substrate duplex must be oriented in agreement with the direction from the active site to the basic protrusion of the enzyme . The distance between the active site and the basic protrusion in the enzyme-substrate complex was shorter than that anticipated in modeling studies . A dynamic structure refinement, referred to as the normal mode analysis, was carried out in order to simulate the fluctuations of the basic protrusion.

J Mol Biol, 1996 Nov 15, 263(5), 685 - 98
Residues in Escherichia coli RNase P RNA important for cleavage site selection and divalent metal ion binding; Kufel J et al.; We have used genetics as a tool to study the importance of an internal loop (P7) of Escherichia coli RNase P RNA (M1 RNA) in cleavage site selection and the binding of a divalent metal ion(s) . The preferred cleavage site on a model tRNA precursor substrate shifted as a result of base-substitutions and deletions within this loop, in particular when changes were introduced at positions directly involved in base-pairing with the 3'-terminal RCCA motif of the substrate . Additionally, these changes in M1 RNA resulted in alterations in the binding of a divalent metal ion(s) in the vicinity of this internal loop as revealed by lead(II)-induced cleavage . From these data we conclude that the structural integrity of the P7 loop is important for both cleavage site selection and divalent metal ion binding . Cross-linking experiments using precursors carrying a 4-thioU immediately 5' of two independent cleavage sites suggest that close contact points between M1 RNA and nucleotides at these cleavage sites depend on the interaction between M1 RNA and the 3'-terminal RCCA motif of the substrate . Our findings further support the view that there are at least two different ways for the tRNA domain of a tRNA precursor to interact with M1 RNA . These results support a model in which base-pairing between M1 RNA and its substrate results in a re-coordination of a divalent metal ion(s) such that cleavage at the correct position is accomplished.

J Mol Biol, 1996 Nov 15, 263(5), 671 - 84
The effect of self-association on the interaction of the Escherichia coli regulatory protein TyrR with DNA; Bailey MF et al.; The interaction of the Escherichia coli regulatory protein TyrR, with a 42 bp oligonucleotide (42A/42B) containing a centrally located recognition sequence (TyrR box), was examined by analytical ultracentrifugation . The stoichiometry of the binding of oligonucleotide to dimeric TyrR was determined by equilibrium centrifugation of a mixture of fluorescein-5-isothiocyanate-labelled 42A/42B (F-42A/42B) in the presence of an eightfold molar excess of TyrR . The molecular mass (M) of the labelled oligonucleotide was estimated as 148,000, indicating a 1:1 complex composed of oligonucleotide (M = 27,000) and TyrR dimer (M = 113,000) . The association constant (Ko,d = 2.8(+/- 0.1) x 10(6) M-1) was determined by a global analysis of sedimentation data, collected at multiple wavelengths between 230 and 285 nm . The presence of 30 microM ATP gamma S enhanced the affinity of TyrR for DNA approximately 3.5-fold, (Ko,d = 9.9(+/- 0.3) x 10(6) M-1) . The effect of dimer to hexamer self-association of TyrR on the binding of 42A/42B was also examined . Multiple wavelength sedimentation data fitted a model in which the oligonucleotide could bind to one site on the dimer (Ko,d = 9.9 x 10(6) M-1), and to either one or three sites on the hexamer (Ko,h) = 2.0(+/- 0.1) x 10(6) M-1 and 3.8(+/- 0.1) x 10(6) M-1, respectively) . Competitive sedimentation equilibrium and fluorescence anisotropy titrations were performed under stoichiometric conditions to resolve the number of oligonucleotide binding sites per hexamer . In these experiments, 42A/42B was used as a competitor to displace F-42A/42B from the hexamer, which was found to bind the 42mer with a 1:1 stoichiometry . Our data support a model in which ATP increases the affinity of TyrR for the DNA recognition sequence, and tyrosine induced self-association of TyrR generates a hexameric species with a single binding site for the 42A/42B oligonucleotide.

J Mol Biol, 1996 Nov 15, 263(5), 657 - 70
The second step of ATP binding to DnaK induces peptide release; Theyssen H et al.; The interaction of the nucleotide-free molecular chaperone DnaK (Hsp70) from Escherichia coli with nucleotides was studied under equilibrium and transient kinetic conditions . These studies used the intrinsic fluorescence signal of the single tryptophan residue (Trp102) of DnaK, or of novel fluorescent nucleotide analogs of ADP and ATP, N8-(4-N'-methylanthraniloylaminobutyl)-8-aminoadenosine 5'-di- or triphosphate (MABA-ADP and MABA-ATP) as spectroscopic probes . Titration of MABA-ADP with DnaK resulted in a 2.3-fold increase of the fluorescence signal, from which a binding stoichiometry of 1:1, and a dissociation constant (Kd) of 0.09 microM were derived . The intrinsic rate constant of hydrolysis of ATP or MABA-ATP in single turnover experiments was found to be 1.5 x 10(-3) s-1 and 1.6 x 10(-3) s-1, identical with the catalytic rate constant of 1.5(+/- 0.17) x 10(-3) s-1 obtained under steady-state conditions . The dissociation rate constant of ADP was measured to be 35(+/- 7) x 10(-3) s-1 in the absence or 15(+/- 5) x 10(-3) in the presence of 2 mM inorganic phosphate (Pi) and is therefore 10 to 20 times faster than the rate of hydrolysis . These results demonstrated that processes governing ATP hydrolysis are rate-limiting in the DnaK ATPase reaction cycle . The three observed different fluorescent states of the single tryptophan residue were investigated . The binding of ATP gave a decrease of 15% in fluorescence intensity compared with the nucleotide-free state . Subsequent ATP hydrolysis, or the simultaneous addition of ADP and Pi, increased the fluorescence 7% above the fluorescence intensity of the nucleotide-free protein . Changes in the tryptophan fluorescence could not be detected when ADP, Pi or the non-hydrolyzable nucleotide analogs AMPPNP (Kd = 1.62(+/- 0.1) microM) or ATP gamma S (Kd = 0.044(+/- 0.003) microM) were added . These data suggested that DnaK exists in at least three different conformational states, depending on nucleotide site occupancy . The fluorescence increase of DnaK upon ATP binding was resolved into two steps; a rapid first step (Kd 1 = 7.3 microM) is followed by a second slow step (k+2 = 1.5 s-1 and k-2 < or = 1.5 x 10(-3) s-1) that causes the decrease in the tryptophan fluorescence signal . The addition of ATP also resulted in the release of DnaK-bound peptide substrate with koff = 3.8 s-1, comparable with the rate of the second step of nucleotide binding . AMPPNP or ATP gamma S were not able to change the fluorescence signal nor to release the peptide . We therefore conclude that the second step of ATP binding, and not the 1000-fold slower ATP hydrolysis is coupled to peptide release.

J Mol Biol, 1996 Nov 15, 263(5), 637 - 47
Domain organization of the Escherichia coli RNA polymerase sigma 70 subunit; Severinova E et al.; We used limited trypsin digestion to determine the domain organization of the Escherichia coli RNA polymerase sigma 70 subunit . Trypsin-resistant fragments containing sigma 70 conserved region 2 (sigma 70(2)), and carboxy-terminal fragments containing conserved regions 3 and 4 (sigma 70(3-4)) were identified by a combination of amino acid sequencing and mass spectrometry . The domains were studied for partial biochemical functions of sigma 70.sigma 70(2) bound core RNA polymerase competitively with intact sigma 70 . In contrast to sigma 70(2) alone, the RNA polymerase holoenzyme formed with sigma 70(2) specifically bound a single-stranded DNA oligomer with a sequence corresponding to the non-template strand of the -10 promoter element (the Pribnow box) . Sigma 70(2) also forms crystals that are suitable for X-ray analysis . Sigma 70(3-4) bound the T4 AsiA protein with high affinity . The epitope for T4 AsiA on sigma 70 was further localized to within sigma 70{551-608}, comprising sigma conserved region 4.2.

Biochem J, 1996 Nov 15, 320 ( Pt 1), 93 - 9
Interaction of plant lipids with 14 kDa phospholipase A2 enzymes; Vishwanath BS et al.; Several structurally related plant lipids were isolated and their effect was assessed on the enzyme activity of group I (pancreatic and Naja mocambique venom) and group II (Crotalus atrox venom) phospholipase A2 (PLA2) enzymes, with labelled Escherichia coli as an enzyme substrate . The neutral monogalactosyldiacylglycerol (MGDG) and negatively charged diacylglyceryl alpha-D-glucuronide (DGGA) did not influence the enzyme activity of either group . Digalactosyldiacylglycerol (DGDG), another uncharged glycolipid, inhibited PLA2 activity in a dose-dependent manner to 60-70% of the control . Sulphoquinovosyldiacylglycerol (SQDG), which is also anionic, activated both groups of PLA2 enzyme . A similar activation was observed with the zwitterionic diacylglyceryl-O-(N,N,N-trimethylhomoserine) (DGTS) and diacylglyceryl-O-(hydroxymethyl)(N,N, N-trimethyl)-beta-alanine (DGTA) . DGDG, SQDG and DGTS are dispersed homogeneously with low critical micelle concentrations (CMCs) . The hydrodynamic radius of neutral DGDG is an order of magnitude larger than the charged lipids SQDG and DGTS . The inhibition of pig pancreatic PLA2 by DGDG was dependent on substrate concentration . The intrinsic fluorescence spectra of the enzyme was not changed in the presence of native or hydrogenated DGDG . Thus the inhibition is most probably due to a non-specific interaction of plant lipids with the substrate . Different lengths and saturations of the fatty acyl chains of DGDG did not alter the inhibition of PLA2, whereas deacylation abrogated the inhibitory effect . Both SQDG and DGTS activated pig pancreatic PLA2 in a dose-dependent manner . Saturation of the double bonds of these lipids decreased the activating effect . The fluorescence of pig pancreatic PLA2 incubated with SQDG and DGTS was enhanced by 2-fold and 3-fold respectively, suggesting the formation of a complex between enzyme and lipids . In conclusion, the effect of different plant lipids on PLA2 activity depends on different structural elements of the polar head group and their charge as well as the degree of unsaturation of the fatty acyl chains.

EMBO J, 1996 Nov 15, 15(22), 6335 - 47
Repercussions of DNA tracking by the type IC restriction endonuclease EcoR124I on linear, circular and catenated substrates; Szczelkun MD et al.; Type I restriction endonucleases such as EcoR124I cleave DNA at undefined loci, distant from their recognition sequences, by a mechanism that involves the enzyme tracking along the DNA between recognition and cleavage sites . This mechanism was examined on plasmids that carried recognition sites for EcoR124I and recombination sites for resolvase, the latter to create DNA catenanes . Supercoiled substrates with either one or two restriction sites were linearized by EcoR124I at similar rates, although the two-site molecule underwent further cleavage more readily than the one-site DNA . The catenane from the plasmid with one EcoR124I site, carrying the site on the smaller of the two rings, was cleaved by EcoR124I exclusively in the small ring, and this underwent multiple cleavage akin to the two-site plasmid . Linear substrates derived from the plasmids were cleaved by EcoR124I at very slow rates . The communication between recognition and cleavage sites therefore cannot stem from random looping . Instead, it must follow the DNA contour between the sites . On a circular DNA, the translocation of non-specific DNA past the specifically bound protein should increase negative supercoiling in one domain and decrease it in the other . The ensuing topological barrier may be the trigger for DNA cleavage.

EMBO J, 1996 Nov 15, 15(22), 6122 - 31
A protease complex in the Escherichia coli plasma membrane: HflKC (HflA) forms a complex with FtsH (HflB), regulating its proteolytic activity against SecY; Kihara A et al.; Escherichia coli FtsH (HflB), a membrane-bound ATPase is required for proteolytic degradation of uncomplexed forms of the protein translocase SecY subunit . We have now isolated SecY-stabilizing mutations that cause an amino acid substitution in the HflK-HflC membrane protein complex . Although HflKC protein was believed to have a proteolytic activity against lambda cII protein, deletion of hflK-hflC did not stabilize SecY . Instead, the mutant alleles were partially dominant and overexpression of ftsH suppressed the mutational effects, suggesting that the mutant proteins antagonized the degradation of SecY . These results raise the possibility that even the wild-type HflKC protein acts to antagonize FtsH . Consistent with this notion, the hflkC null mutation accelerated degradation of the SecY24 protein . Furthermore cross-linking, co-immunoprecipitation, histidine-tagging and gel filtration experiments all indicated that FtsH and HflKC form a complex in vivo and in vitro . Finally, purified HflKC protein inhibited the SecY-degrading activity of purified FtsH protein in vitro . These results indicate that the proteolytic activity of FtsH is modulated negatively by its association with HflKC.

EMBO J, 1996 Nov 15, 15(22), 6111 - 21
Mechanism of chaperonin action: GroES binding and release can drive GroEL-mediated protein folding in the absence of ATP hydrolysis; Hayer-Hartl MK et al.; As a basic principle, assisted protein folding by GroEL has been proposed to involve the disruption of misfolded protein structures through ATP hydrolysis and interaction with the cofactor GroES . Here, we describe chaperonin subreactions that prompt a re-examination of this view . We find that GroEL-bound substrate polypeptide can induce GroES cycling on and off GroEL in the presence of ADP . This mechanism promotes efficient folding of the model protein rhodanese, although at a slower rate than in the presence of ATP . Folding occurs when GroES displaces the bound protein into the sequestered volume of the GroEL cavity . Resulting native protein leaves GroEL upon GroES release . A single-ring variant of GroEL is also fully functional in supporting this reaction cycle . We conclude that neither the energy of ATP hydrolysis nor the allosteric coupling of the two GroEL rings is directly required for GroEL/GroES-mediated protein folding . The minimal mechanism of the reaction is the binding and release of GroES to a polypeptide-containing ring of GroEL, thereby closing and opening the GroEL folding cage . The role of ATP hydrolysis is mainly to induce conformational changes in GroEL that result in GroES cycling at a physiologically relevant rate.

EMBO J, 1996 Nov 15, 15(22), 6075 - 83
Activation of Dictyostelium myosin light chain kinase A by phosphorylation of Thr166; Smith JL et al.; Phosphorylation of the regulatory light chain is an important mechanism for the activation of myosin in non-muscle cells . Unlike most myosin light chain kinases (MLCKs), MLCK-A from Dictyostelium is not activated by Ca2+/calmodulin . Autophosphorylation increases activity, but only to a low level, suggesting that there is an additional activation mechanism . Here, we show that MLCK-A is autophosphorylated on Thr289, which is C-terminal to the catalytic domain . Phosphorylation of MLCK-A increases in response to concanavalin A (conA) treatment of cells, which was previously shown to activate MLCK-A . However, a mutant kinase with an alanine at position 289 (T289A) is also phosphorylated in vivo, indicating that there is an additional phosphorylated residue . Based on comparisons with other protein kinases, we tested whether phosphorylation of Thr166 drives activation of MLCK-A . Our data indicate that phosphorylation of Thr289 occurs in vivo, but is not associated with conA-induced activation, whereas phosphorylation of Thr166 by some as yet unidentified kinase is associated with activation . Replacement of Thrl66 with glutamate results in a 12-fold increase in activity as compared with the wild-type enzyme, supporting the idea that phosphorylation of Thr166 increases MLCK-A activity.

J Clin Invest, 1996 Nov 15, 98(10), 2364 - 72
Systemic hypertension induced by hepatic overexpression of human preproendothelin-1 in rats; Niranjan V et al.; Endothelin-1 (ET-1) has been implicated in the regulation of vascular tone in various pathological conditions . To examine the effect of in vivo overexpression of the peptide in rats, we prepared recombinant adenovirus stocks encoding the human preproET-1 cDNA (Ad.ET-1) or Escherichia coli lacZ (Ad.betaGal), each driven by cytomegalovirus early promoter . Ad.ET-1 or Ad.betaGal was injected into the caudal vein of rats and the animals were studied under anesthesia 96 h later . Hepatic overexpression of the virus-derived human ET-1 mRNA was accompanied by a 13-fold elevation of liver ET-1 content in the Ad.ET-1 group . Circulating plasma ET-1 levels in the Ad.ET-1 group were sixfold higher than those in the Ad.betaGal group . Mean arterial blood pressure was increased by 28 mmHg in the Ad.ET-1 group as compared with the Ad.betaGal group . In the Ad.ET-1 group, intravenous infusion of the ET(A) receptor antagonist FR 139317 reduced the blood pressure to levels seen in the Ad.betaGal group, whereas the same antagonist did not significantly alter the blood pressure in the Ad.betaGal group . Intravenous infusion of the ET(B) receptor antagonist BQ-788 caused a small but significant increase in blood pressure in both groups . These findings demonstrate that endogenous overexpression of preproET-1, accompanied by an elevation of plasma ET-1 concentrations to the levels seen in pathophysiological states, can cause systemic hypertension through the activation of the ETA receptor.

Structure, 1996 Nov 15, 4(11), 1235 - 8
Sigma domain structure: one down, one to go; Chan CL et al.; The recent publication of the 2.6 A crystal structure of a portion of sigma70 provides insight into the role of sigma during transcription initiation . This high resolution picture unveils novel questions.

Structure, 1996 Nov 15, 4(11), 1303 - 15
The crystal structure of a class II fructose-1,6-bisphosphate aldolase shows a novel binuclear metal-binding active site embedded in a familiar fold; Cooper SJ et al.; BACLGROUND: Aldolases catalyze a variety of condensation and cleavage reactions, with exquisite control on the stereochemistry . These enzymes, therefore, are attractive catalysts for synthetic chemistry . There are two classes of aldolase: class I aldolases utilize Schiff base formation with an active-site lysine whilst class II enzymes require a divalent metal ion, in particular zinc . Fructose-1,6-bisphosphate aldolase (FBP-aldolase) is used in gluconeogenesis and glycolysis; the enzyme controls the condensation of dihydroxyacetone phosphate with glyceraldehyde-3-phosphate to yield fructose-1,6-bisphosphate . Structures are available for class I FBP-aldolases but there is a paucity of detail on the class II enzymes . Characterization is sought to enable a dissection of structure/activity relationships which may assist the construction of designed aldolases for use as biocatalysts in synthetic chemistry . RESULTS: The structure of the dimeric class II FBP-aldolase from Escherichia coli has been determined using data to 2.5 A resolution . The asymmetric unit is one subunit which presents a familiar fold, the (alpha/beta)8 barrel . The active centre, at the C-terminal end of the barrel, contains a novel bimetallic-binding site with two metal ions 6.2 A apart . One ion, the identity of which is not certain, is buried and may play a structural or activating role . The other metal ion is zinc and is positioned at the surface of the barrel to participate in catalysis . CONCLUSIONS: Comparison of the structure with a class II fuculose aldolase suggests that these enzymes may share a common mechanism . Nevertheless, the class II enzymes should be subdivided into two categories on consideration of subunit size and fold, quaternary structure and metal-ion binding sites.

Anal Biochem, 1996 Nov 15, 242(2), 228 - 33
Equilibrium constants for nonspecific binding of proteins to DNA may be obtained by quantitative zonal DNA affinity chromatography; Jenuwine ES et al.; Quantitative zonal DNA affinity chromatography may be used to determine accurate equilibrium constants for the binding of proteins nonspecifically to DNA . Zonal quantitative affinity chromatography has not previously been applied to the determination of binding constants of proteins to DNA, although its use is quite commonplace for determination of affinity constants for protein-protein or protein-small ligand interactions . Equilibrium constants were measured for the nonspecific binding of bovine pancreatic ribonuclease A and Escherichia coli lac repressor to double-stranded DNA immobilized on cellulose . The equilibrium constants determined agree with literature values evaluated using other techniques . The experimental advantages of the zonal technique, when it can be applied, are that collection of data is fast and data analysis is simple . Detection of the protein elution profile by absorbance at 220 nm with an in-line detector can provide adequate sensitivity when binding constants are in the range 10(2)-10(4) M-1.

FEMS Microbiol Lett, 1996 Nov 15, 145(1), 117 - 23
The Escherichia coli modE gene: effect of modE mutations on molybdate dependent modA expression; McNicholas PM et al.; The Escherichia coli modABCD operon, which encodes a high-affinity molybdate uptake system, is transcriptionally regulated in response to molybdate availability by ModE . Here we describe a highly effective enrichment protocol, applicable to any gene with a repressor role, and establish its application in the isolation of transposon mutations in modE . In addition we show that disruption of the ModE C-terminus abolishes derepression in the absence of molybdate, implying this region of ModE controls the repressor activity . Finally, a mutational analysis of a proposed molybdate binding motif indicates that this motif does not function in regulating the repressor activity of ModE.

FEMS Microbiol Lett, 1996 Nov 15, 145(1), 95 - 100
Molecular cloning and expression of the glucose/xylose isomerase gene from Streptomyces sp . NCIM 2730 in Escherichia coli; Bhosale SH et al.; A partial genomic library of Streptomyces sp . NCIM 2730 was constructed in Escherichia coli using pUC8 vector and screened for the presence of the D-glucose/xylose isomerase (GXI) gene using an 18-mer mixed oligonucleotide probe complementary to a highly conserved six-amino acid sequence of GXI from actinomycetes . Eight clones which hybridized with the radiolabelled oligoprobe showed the ability to complement xylose isomerase-defective E . coli mutants . The restriction map of the insert from one (pMSG27) of the eight GXI-positive clones showing detectable GXI activity was constructed . GXI-deficient strains of E . coli were able to utilize xylose as the sole carbon source for their growth upon transformation with pMSG27 . E . coli JM105 (pMSG27) and E . coli JC1553 (pMSG27) were inducible by IPTG suggesting that the expression of the cloned gene was under the control of the lacZ promoter . Western blot analysis revealed that the cloned gene is expressed as a fusion protein of M(r) 110 . This is the first report of expression of a catalytically active GXI from Streptomyces in Escherichia coli.

FEMS Microbiol Lett, 1996 Nov 15, 145(1), 49 - 54
Pyrophosphate is a source of phosphoryl groups for Escherichia coli protein phosphorylation; Duclos B et al.; Pyrophosphate can serve as a source of phosphoryl groups for the phosphorylation of E . coli proteins . The main target of such phosphorylation is a 49-kDa protein which is covalently modified at serine . The same phosphorylation pattern is obtained in the presence of ATP, which suggests that pyrophosphate can substitute for ATP for bacterial protein phosphorylation.

Cell, 1996 Nov 15, 87(4), 757 - 66
Human Rad51 protein promotes ATP-dependent homologous pairing and strand transfer reactions in vitro; Baumann P et al.; The human testis Rad51 protein, a structural homolog of E . coli RecA, binds single- and double-stranded DNA and exhibits DNA-dependent ATPase activity . Using circular ssDNA and linear dsDNA (3.0 kb in length), we demonstrate that hRad51 promotes homologous pairing and strand exchange reactions in vitro . Joint molecule formation was dependent upon ATP hydrolysis and DNA homology and was stimulated by the single-strand DNA-binding protein RP-A . In these reactions, the 5' terminus of the complementary strand of the linear duplex was efficiently transferred to the ssDNA . However, under standard conditions, extensive strand exchange was not observed . These results establish hRad51 as a functional homolog of RecA, but indicate that the human protein and its bacterial counterpart differ in their ability to promote extensive strand transfer . It is proposed that hRad51 mediates homology recognition and initiates strand exchange, but that extensive heteroduplex formation in higher organisms requires the actions of additional proteins.

Virology, 1996 Nov 15, 225(2), 328 - 38
Enhancement of hepatitis C virus NS3 proteinase activity by association with NS4A-specific synthetic peptides: identification of sequence and critical residues of NS4A for the cofactor activity; Butkiewicz NJ et al.; The NS3 proteinase of hepatitis C virus utilizes NS4A as a cofactor for cleavages at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the nonstructural region of the viral polyprotein . To characterize NS4A for its role in modulating the NS3 proteinase activity at various cleavage sites, synthetic peptides spanning various parts of NS4A were synthesized and tested in a cell-free trans-cleavage reaction using purified NS3 proteinase domain and polyprotein substrates . The NS3 proteinase domain was expressed in Escherichia coli, purified, denatured, and refolded to an enzymatically active form . We found that a 12-amino-acid peptide containing amino acid residues 22 to 33 in NS4A (CVVIVGRIVLSG) was sufficient for cofactor activity in NS3-mediated proteolysis . The peptide enhanced the cleavage at the NS5A/5B site and was necessary for NS3-mediated cleavage at NS4A/4B and NS4B/5A . Sequential amino acid substitution within the designated peptide identified residues I29 and I25 as critical for potential cofactor activity . We provide evidence that the NS4A peptide and the NS3 catalytic domain form an enzymatically active complex . These data suggest that the central 12-amino-acid peptide (aa 22-33) of NS4A is primarily important for the cofactor activity through complex formation with NS3, and the interaction may represent a new target for antiviral drug development.

Genes Dev, 1996 Nov 15, 10(22), 2903 - 11
SREBP transcriptional activity is mediated through an interaction with the CREB-binding protein; Oliner JD et al.; The sterol regulatory element binding proteins (SREBP-1 and -2) activate transcription of genes whose products are involved in the cellular uptake and synthesis of cholesterol . Although considerable effort has been exerted to define the events regulating the levels of active SREBP, little is known about the transcriptional cofactors mediating SREBP function . In an unbiased search for potential coactivators of SREBP, we isolated a protein of 265 kD from HeLa cells that directly bound SREBP-1 and SREBP-2 . Peptide sequencing and Western blot analysis established that the 265-kD protein was CBP (CREB-binding protein), a recently identified transcriptional coactivator . The putative activation domain of SREBP was shown to bind specifically to amino-terminal domains of recombinant CBP and p300 (a CBP-related protein) . Moreover, transfection studies demonstrated that CBP enhances the ability of SREBP to activate transcription of reporter genes in HeLa cells . Together, these data suggest that CBP mediates SREBP transcriptional activity, thus revealing a new step in the biochemical pathway regulating cholesterol metabolism.

Anal Chem, 1996 Nov 15, 68(22), 4044 - 51
Confirmation by mass spectrometry of a trisulfide variant in methionyl human growth hormone biosynthesized in Escherichia coli; Canova-Davis E et al.; A sulfur-containing compound found in acid hydrolysates of proteins was identified 30 years ago as a trisulfide: bis-(2-amino-2-carboxyethyl) trisulfide (cysteine2S3) . At that time, studies concerning the chemistry of sulfur-transferring enzyme systems suggested that cysteine2S3 also existed in biological systems . Two decades later, a cystine trisulfide structure was postulated in the regulator protein molecule for the activation of delta-aminolevulinate synthetase . Recently, a trisulfide bond was reported to occur in the minor loop disulfide at Cys182-Cys189 in human growth hormone . We have detected a trisulfide structure in methionyl human growth hormone in the major loop disulfide Cys53-Cys165 . The development of mass spectral analyses of high molecular weight molecules, such as proteins, led to the eventual identification of the modification . A tandem mass spectral analysis on a Sciex electrospray instrument localized an addition of 32 Da to the Cys53-Cys165 fragment . Elemental composition was determined by accurate mass measurement obtained by peak matching to a synthetic peptide and established that an extra sulfur atom was involved.

Anal Chem, 1996 Nov 15, 68(22), 3939 - 44
Bifunctional fusion proteins of calmodulin and protein A as affinity ligands in protein purification and in the study of protein-protein interactions; Hentz NG et al.; An affinity chromatography system is described that incorporates a genetically designed bifunctional affinity ligand . The utility of the system in protein purification and in the study of protein-protein interactions is demonstrated by using the interaction between protein A and the heat shock protein DnaK as a model system . The bifunctional affinity ligand was developed by genetically fusing calmodulin (CaM) to protein A (ProtA) . The dual functionality of protein A-calmodulin (ProtA-CaM) stems from the molecular recognition properties of the two components of the fusion protein . In particular, CaM serves as the anchoring component by virtue of its binding properties toward phenothiazine . Thus, the ProtA-CaM can be immobilized on a solid support containing phenothiazine from the C-terminal domain of the fusion protein . Protein A is at the N-terminal domain of the fusion protein and serves as the affinity site for DnaK . While DnaK binds specifically to the protein A domain of the bifunctional ligand, it is released upon addition of ATP and under very mild conditions (pH 7.0) . In addition to obtaining highly purified DnaK, this system is very rugged in terms of its performance . The proteinaceous bifunctional affinity ligand can be easily removed by addition of EGTA, and fresh ProtA-CaM can be easily reloaded onto the column . This allows for a facile regeneration of the affinity column because the phenothiazine-silica support matrix is stable for long periods of time under a variety of conditions . This study also demonstrates that calmodulin fusions can provide a new approach to study protein-protein interactions . Indeed, the ProtA-CaM fusion protein identified DnaK as a cellular component that interacts with protein A from among the thousands of proteins present in Escherichia coli.

Cancer Res, 1996 Nov 15, 56(22), 5253 - 9
The NAD(P)H:quinone oxidoreductase locus in human colon carcinoma HCT 116 cells resistant to mitomycin C; Hu LT et al.; Previously, we reported an association of mitomycin C resistance and a deficiency of NAD(P)H:quinone oxidoreductase (NQO1) in HCT 116-R30A cells, a subline derived from mitomycin C-sensitive HCT 116 cells . In HCT 116 cells, we found two mRNAs coding full-length cDNAs of NQO1 differing at codon 139, one with arginine (wild type), and one with tryptophan . Only the tryptophan 139 form of mRNA was detected in HCT 116-R30A cells . In addition, an exon 4 deleted mRNA of NQO1, a product of alternative splicing, was detected in both cell lines . Analysis by semiquantitative reverse transcription-PCR showed that NQO1 mRNA coding full-length cDNAs in HCT 116-R30A cells was 15% of that present in HCT 116 cells . A Mr 26,000 protein, representing the exon 4 deleted mRNA, was not detected by polyclonal anti-NQO1 in HCT 116 sublines . Recombinant plasmids of exon 4 deleted cDNA generated a Mr 26,000 protein without enzymatic activity in Escherichia coli but not in Cos7 cells . The function of exon 4 deleted mRNA is yet unknown . The rates of decay of all NQO1 mRNAs in HCT 116 and HCT 116-R30A cells were similar . DNA sequences of the promoter regions of the NQO1 gene (-837 bp) from both cell lines did not differ from each other or from the same region of the human liver NQO1 gene . Sequences of cis elements in the 837-bp region and mRNA stability could not account for the low expression of full-length mRNA in HCT 116-R30A cells . Southern blot analysis showed the size and the intensity of the NQO1 gene in the two cell lines to be similar . This result was confirmed by semiquantitative PCR analysis of a 450-bp fragment in the NQO1 gene containing codon 139 and the exon 4 region . Digestion of this PCR-amplified fragment by restriction enzyme MspI revealed that HCT 116 cells have two heterozygous NQO1 alleles, a wild-type and a tryptophan 139 form . The functional wild-type NQO1 allele was not detected in HCT 116-R30A cells . Sensitive and resistant cell lines each contained one normal and one abnormal chromosome 16 . Loss of the wild-type NQO1 allele in HCT 116-R30A cells did not result from a loss of chromosome 16 or copies of the NQO1 gene . Alteration of factor(s) such as trans-acting factors and DNA methylation may be involved in the down-regulation of NQO1 in the mitomycin C-resistant HCT 116-R30A cells.

J Biol Chem, 1996 Nov 15, 271(46), 29473 - 82
(+)-Pinoresinol/(+)-lariciresinol reductase from Forsythia intermedia . Protein purification, cDNA cloning, heterologous expression and comparison to isoflavone reductase; Dinkova-Kostova AT et al.; Lignans are a widely distributed class of natural products, whose functions and distribution suggest that they are one of the earliest forms of defense to have evolved in vascular plants; some, such as podophyllotoxin and enterodiol, have important roles in cancer chemotherapy and prevention, respectively . Entry into lignan enzymology has been gained by the approximately 3000-fold purification of two isoforms of (+)-pinoresinol/(+)-lariciresinol reductase, a pivotal branchpoint enzyme in lignan biosynthesis . Both have comparable ( approximately 34.9 kDa) molecular mass and kinetic (Vmax/Km) properties and catalyze sequential, NADPH-dependent, stereospecific, hydride transfers where the incoming hydride takes up the pro-R position . The gene encoding (+)-pinoresinol/(+)-lariciresinol reductase has been cloned and the recombinant protein heterologously expressed as a functional beta-galactosidase fusion protein . Its amino acid sequence reveals a strong homology to isoflavone reductase, a key branchpoint enzyme in isoflavonoid metabolism and primarily found in the Fabaceae (angiosperms) . This is of great evolutionary significance since both lignans and isoflavonoids have comparable plant defense properties, as well as similar roles as phytoestrogens . Given that lignans are widespread from primitive plants onwards, whereas the isoflavone reductase-derived isoflavonoids are mainly restricted to the Fabaceae, it is tempting to speculate that this branch of the isoflavonoid pathway arose via evolutionary divergence from that giving the lignans.

J Biol Chem, 1996 Nov 15, 271(46), 29312 - 20
Transmembrane topology of alpha- and beta-subunits of Na+,K+-ATPase derived from beta-galactosidase fusion proteins expressed in yeast; Fiedler B et al.; Various models of the transmembrane topology of the Na+,K+-ATPase predict either 8 or 10 membrane spans for the alpha-subunit and one to three membrane spans for the beta-subunit . Structure/function analysis, however, requires precise knowledge about the folding of enzymes . Therefore, the intention of this work was to establish a transmembrane topology model for the subunits of Na+,K+-ATPase . The bacterial enzyme beta-galactosidase was fused to the C termini of truncated alpha- and beta-subunits of Na+,K+-ATPase . Fusions were generated at Arg60 (LTTAR60), Glu116 (AATEE116), Ala247 (VEGTA247), Leu311 (YTWEL311), Ala444 (VAGDA444), Ala789 (IFIIA789), Met809 (LGTDM809), Asp884 (RVTWD884), Ile946 (MKNKI946), and Arg972 (GVALR972) of the sheep alpha1-subunit and at Pro236 (LGGYP236) of the dog beta-subunit . The fusion constructs were expressed in yeast cells for studies on the localization of the fused reporter enzyme . Activity measurements of the reporter enzyme revealed that only intracellular fusion sites lead to active beta-galactosidase . Indirect immunofluorescence microscopy with cells expressing alpha1/beta-galactosidase and beta/beta-galactosidase hybrid proteins demonstrated that inactive beta-galactosidase is associated with the yeast plasma membrane and can be detected from the extracellular side . The data obtained suggest that Pro236 of the beta-subunit is located on the extracellular surface, corresponding to a model with one transmembrane segment, and that the alpha-subunit of the Na+,K+-ATPase consists of 10 membrane-associated spans . They also suggest that a stretch of the alpha1-subunit between membrane spans M7 and M8 might be hidden within the membrane, surrounded by the other hydrophobic spans, in analogy to the P-loop of Na+ or K+ channels and to the "hourglass" structure of water channels.

J Biol Chem, 1996 Nov 15, 271(46), 29121 - 5
Carboxymethylation of MutS-cysteine-15 specifically inactivates adenosylcobalamin-dependent glutamate mutase . Examination of the role of this residue in coenzyme-binding and catalysis; Holloway DE et al.; The sensitivity of adenosylcobalamin (AdoCbl)-dependent glutamate mutase toward thiol-directed reagents has been investigated . Iodoacetate specifically alkylates one cysteine residue, Cys-15, in MutS with concomitant irreversible loss of enzyme activity . Cys-15 lies between the conserved residues Asp-14 and His-16, that are believed to coordinate cobalt to form a Co-His-Asp hydrogen-bonded "triad" when AdoCbl is bound by the enzyme . Although inactive, carboxymethylated MutS still bound AdoCbl with only a 5-fold increase in apparent Kd . To determine whether Cys-15 plays an essential role in catalysis, it was mutated to serine and to alanine . These mutants were active, but both exhibited decreased Vmax and increased apparent Km and Kd for AdoCbl . To mimic the effect of carboxymethylation, Cys15 was mutated to aspartate and, as an isosteric control, to asparagine . Neither of these mutants was active: MutS-C15N bound AdoCbl approximately 10-fold weaker than wild type, whereas MutS-C15N bound AdoCbl over 100 times less strongly than wild type . The results demonstrate both coenzyme-binding and catalysis to be very sensitive to mutations at position 15 that could potentially perturb the Co-His-Asp hydrogen-bonding network.

J Biol Chem, 1996 Nov 15, 271(46), 28861 - 7
Xanthophyll biosynthesis . Cloning, expression, functional reconstitution, and regulation of beta-cyclohexenyl carotenoid epoxidase from pepper (Capsicum annuum); Bouvier F et al.; Pepper (Capsicum annuum) beta-cyclohexenyl xanthophyll epoxidase cDNA was cloned and the corresponding enzyme overexpressed and purified from Escherichia coli, for investigation of its catalytic activity . The recombinant protein did not directly accept NADPH for epoxidation of cyclohexenyl carotenoids, nor did it operate according to a peroxygenase-based mechanism . Instead, the reducing power of NADPH was transferred to the epoxidase via reduced ferredoxin as shown by reconstitution of epoxidase activity in the presence of NADPH, ferredoxin oxidoreductase, and ferredoxin . Bacterial rubredoxin could be substituted for ferredoxin . The pepper epoxidase acted specifically on the beta-ring of xanthophylls such as beta-cryptoxanthin, zeaxanthin, and antheraxanthin . The proposed reaction mechanism for epoxidation involves the formation of a transient carbocation . This characteristic allows selective inhibition of the epoxidase activity by different nucleophilic diethylamine derivatives, p-dimethylaminobenzenediazonium fluoroborate and N,N-dimethyl-2-phenylaziridinium . It was also shown that the epoxidase gene was up-regulated during oxidative stress and when chloroplasts undergo differentiation into chromoplasts in pepper fruit.

J Biol Chem, 1996 Nov 15, 271(46), 28844 - 52
Membrane targeting and determination of transmembrane topology of the human vasopressin V2 receptor; Schulein R et al.; The human vasopressin V2 receptor belongs to the large family of G-protein-coupled receptors, which possess seven transmembrane helices, an extracellular N terminus and an intracellular C terminus . We have determined the sequence requirements of the V2 receptor for membrane insertion and correct topology for the inner membrane of Escherichia coli with the PhoA/LacZ gene fusion system . In addition, we have studied the signals for its membrane insertion and correct topology for the membrane of the endoplasmic reticulum of the authentic eucaryotic transport system . To this end, we have extended the PhoA/LacZ gene fusion system for the first time to eucaryotic cells, i.e . transiently transfected COS.M6 cells . Truncated V2 receptor sequences were fused to PhoA and LacZ and expressed in both E . coli and COS.M6 cells . Cells were fractionated, and LacZ/PhoA activity assays and immunoblots were performed . We show here that a V2 receptor fragment consisting of the N terminus, the first transmembrane segment and the first cytoplasmic loop (71 amino acids) provided sufficient information for membrane insertion and correct orientation (extracellular N terminus) in both procaryotic and eucaryotic cells . Our data differ substantially from those obtained for the human beta2-adrenergic receptor expressed in E . coli (Lacatena, R . M., Cellini, A., Scavizzi, F., and Tocchini-Valentini, G . P . (1994) Proc . Natl . Acad . Sci . U . S . A . 91, 10521-10525) . To establish correct topology, the beta2-adrenergic receptor requires a larger receptor portion, including the three N-terminal transmembrane segments and/or parts of the second cytoplasmic loop . The present data show that the observations made for the beta2-adrenergic receptor cannot be applied to G-protein-coupled receptors generally.

J Biol Chem, 1996 Nov 15, 271(46), 28784 - 91
Subunit association and DNA binding activity of the heterotrimeric transcription factor NF-Y is regulated by cellular redox; Nakshatri H et al.; NF-Y is a heterotrimeric transcription factor that specifically recognizes a CCAAT box motif found in a variety of eukaryotic promoter and enhancer elements . The subunit association and DNA binding properties of the NF-Y complex were examined as a function of redox state using recombinant NF-YA, NF-YB, and NF-YC subunits . Reduction of NF-YB by dithiothreitol (DTT) was essential for reconstitution of specific NF-Y CCAAT box DNA binding activity in vitro . Approximately 30% of the Escherichia coli-derived NF-YB subunit existed as intermolecular disulfide-linked dimers . NF-YB mutants in which the highly conserved cysteine residues at positions 85 and 89 had been converted to serines existed only as monomers and did not require DTT for functional NF-Y DNA binding activity . DTT was required, however, for the functional association of NF-YC with wild-type NF-YB but not with the NF-YB cysteine mutants . The cellular redox factors Ref-1 and adult T-cell leukemia-derived factor stimulated the DNA binding activity of recombinant NF-Y in the absence of DTT . Cells treated with 1-chloro-2,4-dinitrobenzene, an irreversible inhibitor of thioredoxin reductase, exhibited reduced endogenous NF-Y DNA binding activity . Together these results suggest that the cellular redox environment of mammalian cells is an important posttranscriptional regulator of NF-Y subunit association and DNA binding activities.

J Immunol, 1996 Nov 15, 157(10), 4554 - 67
Chlamydia trachomatis major outer membrane protein (MOMP) epitopes that activate HLA class II-restricted T cells from infected humans; Ortiz L et al.; We localized peptide epitopes within the Chlamydia trachomatis (Ct) major outer membrane protein (MOMP) that activate human T cells . T-MOMP' cells were prepared by culturing PBL from 38 humans who had Ct infections in the presence of Ct serovar E MOMP . Some epitopes were first localized by quantifying proliferative responses of T-MOMP' cells to overlapping MOMP segments (sixths) that were produced in Escherichia coli . Further localization was achieved by using overlapping synthetic 16-22 mers that spanned stimulatory MOMP sixths as Ags . The APCs used were human B cell lines (LCL) that were matched or mismatched with respect to HLA class II alleles of the T-MOMP' cells . T cell epitopes were detected in 18 Ct serovar E MOMP 16-22 mers and were presented in association with HLA-DR1, -7, -13, -17, HLA-DRw52, HLA-DQ3 and at least two from among HLA-DR4, -8, -11, -14, -18, in probable addition to HLA-DP4 . Peptide 249-265 stimulated T-MOMP' cells from 83% of the subjects; studies with overlapping 11-13 mers spanning peptide 249-265 revealed at least six epitopes presented with different HLA-class II allotypes . Diverse T-MOMP' cultures responded to between 2 and 7 MOMP epitopes . All but 1 of the 23 epitopes localized to date are distributed among four MOMP constant segments . T-MOMP' cells from subjects infected with serovars other than E also responded.

Gene, 1996 Nov 14, 179(2), 263 - 70
Isolation of a mRNA instability sequence that is cis-dominant to the ompA stability determinant in Escherichia coli; Meyer BJ et al.; Transcriptional fusions with ompA and bla have been used to identify a novel mRNA instability element . A 287-nucleotide (nt) sequence containing a repetitive extragenic palindrome (REP) from the chloramphenicol acetyltransferase (cat) gene was inserted into the 3' untranslated region (UTR) of the ompA gene . In one orientation, the insert had no effect on the half-life of the ompA-cat chimeric transcript . In the other orientation, however, the sequence functioned as a destabilizing element and was dominant to the 5'-UTR ompA and REP stability elements . The orientation-dependent effect of the instability sequence suggests that sequence rather than structure alone is important to the function of the instability determinant . In addition, the instability sequence also destabilized an ompA-bla fusion construct when fused to its 3'-UTR region . A sensitive RNA ligation/PCR amplification technique was developed and used to analyze RNA decay intermediates . The results indicated that degradation of the chimeric transcript initiated within the 287-nt inserted sequence.

Gene, 1996 Nov 14, 179(2), 211 - 8
The major subunit ClpG of Escherichia coli CS31A fibrillae as an expression vector for different combinations of two TGEV coronavirus epitopes; Mechin MC et al.; Previously, two B-cell epitopes from the entero-pathogenic transmissible gastroenteritis virus (TGEV), namely the C epitope (TGEV-C) amino acids (aa) 363-371 and the A epitope (TGEV-A) aa 522-531 of the spike S protein (TGEV-S), have been separately expressed on the CS31A fibrillae at the surface of Escherichia coli following insertion into a same region of ClpG . However, the resulting chimeras induced a marginal TGEV-neutralizing antibody (Ab) response in mice . Here, with the view to improving this response, we introduced TGEV-C alone or in different tandem association with TGEV-A (A::C or C::A) in twelve putatively exposed regions of ClpG . Among the 28 resulting engineered proteins only 15, carrying up to 51 extra aa, had not essentially disturbed the correct CS31A fibrillae formation process . Six partially permissive sites accepting only TGEV-C and three highly permissive sites tolerating A::C or C::A tandem peptide, were identified throughout ClpG . Intact bacteria or extracted CS31A hybrid fibrillae expressing TGEV epitopes at any of the permissive sites, were recognized by Ab directed against the foreign parent protein, providing a direct argument for exposure of the corresponding CIpG region at the cell surface and for antigenicity of the epitopes in the polymeric CS31A fibrillae context . The potential of CS31A fibrillae as carriers of the TGEV peptides indicates that there may be three positions (N terminus, aa 202-204 and 202-218) in ClpG which may turn out to be important fusion sites and therefore be relevant for the eventual design of TGEV vaccines . Unexpectedly, TGEV-A, whatever its position in ClpG, mediated the partial proteolytic degradation of the hybrid proteins, suggesting that it functions as a substrate for a cellular protease, and thereby that its suitability as a vaccine antigen candidate is doubtful.

Nature, 1996 Nov 14, 384(6605), 176 - 9
Biochemical evidence that patched is the Hedgehog receptor; Marigo V et al.; The protein Sonic hedgehog (Shh) is essential for a variety of patterning events during development . It is the signal from the notochord that induces ventral cell fate in the neural tube and somites, and is the polarizing signal for patterning of the anterior-posterior axis of the developing limb bud . Because of these and other inductive functions of Shh, it is important to understand how the Hedgehog (Hh) signal is received by the target cells . Here we describe binding studies using labelled Shh that strongly suggest that the Hh receptor is encoded by patched (ptc), a gene first identified in genetic screens in Drosophila.

J Immunol Methods, 1996 Nov 13, 198(2), 113 - 8
Particle concentration fluorescence as an alternative to radioactivity for cytokine receptor binding assays; Sarubbi E; To detect inhibitors of the type-I interleukin-1 receptor in a safe and cost-effective nonisotopic environment, the use of particle concentration fluorescence (PCF) as a detection method for a high volume receptor binding assay (RBA) has been explored . A cell-free PCF-RBA has been developed using a soluble form of the type-I IL-1 receptor (sIL-1R), a nonneutralizing anti-sIL-1R monoclonal antibody and two fluorescein-labelled tracers: IL-1 alpha and IL-1ra fused to the Escherichia coli maltose binding protein (MBP-IL-1ra) . Both ligands showed saturable specific binding (KA values in the 10(9) M-1 range), although signal intensity was about 3-fold higher with MBP-IL-1ra . The data presented here suggest that the combined use of recombinant DNA and PCF technologies can replace the traditional cell-based, radioactive RBA when screening for inhibitors of cytokine receptors.

Proc Natl Acad Sci U S A, 1996 Nov 12, 93(23), 12926 - 31
Development of an in vitro mRNA decay system for Escherichia coli: poly(A) polymerase I is necessary to trigger degradation; Ingle CA et al.; Using a novel Escherichia coli in vitro decay system in which polysomes are the source of both enzymes and mRNA, we demonstrate a requirement for poly(A) polymerase I (PAP I) in mRNA turnover . The in vitro decay of two different mRNAs (trxA and lpp) is triggered by the addition of ATP only when polysomes are prepared from s strain carrying the wild-type gene for PAP I (pcnB+) . The relative decay rates of these two messages are similar in vitro and in vivo . Poly(A) tails are formed on both mRNAs, but no poly(A) are detected on the 3' end of mature 23S rRNA . The size distribution of poly(A) tails generated in vitro, averaging 50 nt in length, is comparable to that previously reported in vivo . PAP I activity is associated exclusively with the polysomes . Exogenously added PAP I does not restore mRNA decay to PAP I-polysomes, suggesting that, in vivo, PAP I may be part of a multiprotein complex . The potential of this in vitro system for analyzing mRNA decay in E . coli is discussed.

Proc Natl Acad Sci U S A, 1996 Nov 12, 93(23), 13048 - 53
An in vivo pathway for disulfide bond isomerization in Escherichia coli; Rietsch A et al.; Biochemical studies have shown that the periplasmic protein disulfide oxidoreductase DsbC can isomerize aberrant disulfide bonds . Here we present the first evidence for an in vivo role of DsbC in disulfide bond isomerization . Furthermore, our data suggest that the enzymes DsbA and DsbC play distinct roles in the cell in disulfide bond formation and isomerization, respectively . We have shown that mutants in dsbC display a defect in disulfide bond formation specific for proteins with multiple disulfide bonds . The defect can be complemented by the addition of reduced dithiothreitol to the medium, suggesting that absence of DsbC results in accumulation of misoxidized proteins . Mutations in the dipZ and trxA genes have similar phenotypes . We propose that DipZ, a cytoplasmic membrane protein with a thioredoxin-like domain, and thioredoxin, the product of the trxA gene, are components of a pathway for maintaining DsbC active as a protein disulfide bond isomerase.

Proc Natl Acad Sci U S A, 1996 Nov 12, 93(23), 12902 - 7
Direct physical interaction between DnaG primase and DnaB helicase of Escherichia coli is necessary for optimal synthesis of primer RNA; Lu YB et al.; The primase DnaG of Escherichia coli requires the participation of the replicative helicase DnaB for optimal synthesis of primer RNA for lagging strand replication . However, previous studies had not determined whether the activation of the primase or its loading on the template was accomplished by a helicase-mediated structural alteration of the single-stranded DNA or by a direct physical interaction between the DnaB and the DnaG proteins . In this paper we present evidence supporting direct interaction between the two proteins . We have mapped the surfaces of interaction on both DnaG and DnaB and show further that mutations that reduce the physical interation also cause a significant reduction in primer synthesis . Thus, the physical interaction reported here appears to be physiologically significant.

Proc Natl Acad Sci U S A, 1996 Nov 12, 93(23), 12828 - 33
A common mechanism for the biosynthesis of methoxy and cyclopropyl mycolic acids in Mycobacterium tuberculosis; Yuan Y et al.; Mycobacterium tuberculosis produces three classes of mycolic acids that differ primarily in the presence and nature of oxygen-containing substituents in the distal portion of the meromycolate branch . The methoxymycolate series has a methoxy group adjacent to a methyl branch, in addition to a cyclopropane in the proximal position . Using the gene for the enzyme that introduces the distal cyclopropane (cma1) as a probe, we have cloned and sequenced a cluster of genes coding for four highly homologous methyl transferases (mma1-4) . When introduced into Mycobacterium smegmatis, this gene cluster conferred the ability to synthesize methoxymycolates . By determining the structure of the mycolic acids produced following expression of each of these genes individually and in combination, we have elucidated the biosynthetic steps responsible for the production of the major series of methoxymycolates . The mma4 gene product (MMAS-4) catalyzes an unusual S-adenosyl-L-methionine-dependent transformation of the distal cis-olefin into a secondary alcohol with an adjacent methyl branch . MMAS-3 O-methylates this secondary alcohol to form the corresponding methyl ether, and MMAS-2 introduces a cis-cyclopropane in the proximal position of the methoxy series . The similarity of these reactions and the enzymes that catalyze them suggests that some of the structural diversity of mycolic acids results from different chemical fates of a common cationic intermediate, which in turn results from methyl group addition to an olefinic mycolate precursor.

Biochemistry, 1996 Nov 12, 35(45), 14405 - 12
Mutational analysis of a leucine heptad repeat motif in a class I aminoacyl-tRNA synthetase; Ohannesian DW et al.; Aminoacyl-tRNA synthetases activate amino acids with ATP to form aminoacyl adenylates as the essential intermediates for aminoacylation of their cognate tRNAs . The class I Escherichia coli cysteine tRNA synthetase contains an N-terminal nucleotide binding fold that provides the catalytic site of adenylate synthesis . The C-terminal domain of the cysteine enzyme is predominantly alpha-helical and contains a leucine heptad repeat motif . We show here that specific substitutions of leucines in the leucine heptad repeats reduced tRNA aminoacylation . In particular, substitution of Leu316 with phenylalanine reduced the catalytic efficiency of aminoacylation by 1000-fold . This deleterious effect was partially alleviated by a more conservative substitution of leucine with valine . Filter binding assays show that neither the phenylalanine nor the valine substitution at Leu316 had a major effect on the ability of the cysteine enzyme to bind tRNA(Cys) . In contrast, pyrophosphate exchange assays show that both substitutions decreased the adenylate synthesis activity of the enzyme . Analysis of these results suggests that the primary defect of the valine substitution is executed at adenylate synthesis while that of the phenylalanine substitution is at both adenylate synthesis and the transition state of tRNA aminoacylation . Thus, although Leu316 is located in the C-terminal domain of the cysteine enzyme, it may modulate the capacity of the N-terminal domain for amino acid activation and tRNA aminoacylation through a domain-domain interaction.

Biochemistry, 1996 Nov 12, 35(45), 14362 - 9
Comparison of the functional differences for the homologous residues within the carboxy phosphate and carbamate domains of carbamoyl phosphate synthetase; Javid-Majd F et al.; Carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from two molecules of MgATP, bicarbonate, and glutamine . It has been previously shown that the amino- and carboxy-terminal halves of the large subunit of this protein are homologous . A working model for the active site structure of the carboxy-terminal domain of the large subunit of CPS was constructed based upon amino acid sequence alignments and the previously determined three-dimensional structures of two mechanistically related proteins, biotin carboxylase and D-alanine:D-alanine ligase . The model was tested by mutation of ten amino acid residues predicted to be important for binding and/or catalysis . The mutated residues were as follows: R571, R675, R715, D753, E761, N827, Q829, E841, N843, and R845 . The mutant proteins were expressed, purified to homogeneity and the catalytic properties determined for a variety of assay formats . The mutants E761A, E841Q, N843D, and R845Q were diminished in their ability to synthesize carbamoyl phosphate . The R715A, Q829A, and R675A mutants displayed elevated Michaelis constants for MgADP in the partial back reaction . The mutants E761A, N827A, E841Q, N843D, and R845Q showed significant increases in the Michaelis constants for either bicarbonate or carbamoyl phosphate . No significant alterations were noted upon mutation of either R571 or D753 to an alanine residue and thus these amino acids do not appear essential for structure or catalytic activity . These results have been utilized to further support the proposal that the C-terminal half of the large subunit of CPS is primarily responsible for the phosphorylation of the carbamate intermediate during the final formation of carbamoyl phosphate . The measured effects on the catalyic activities displayed by these mutations were found to be comparable to the previously determined effects after mutation of the homologous residues located on the N-terminal half of CPS and also for those residues mutated within D-alanine:D-alanine ligase {Shi, Y., & Walsh, C.T . (1995) Biochemistry 34, 2768-2776}.

Biochemistry, 1996 Nov 12, 35(45), 14352 - 61
Role of conserved residues within the carboxy phosphate domain of carbamoyl phosphate synthetase; Stapleton MA et al.; Carbamoyl phosphate synthetase (CPS) catalyzes the formation of carbamoyl phosphate from glutamine, bicarbonate, and 2 mol of MgATP . The heterodimeric protein is composed of a small amidotransferase subunit and a larger synthetase subunit . The synthetase subunit contains a large tandem repeat for each of the nucleotides used in the overall synthesis of carbamoyl phosphate . A working model for the three-dimensional fold of the carboxy phosphate domain of CPS was constructed on the basis of amino acid sequence alignments and the X-ray crystal structure coordinates for biotin carboxylase and D-alanine:D-alanine ligase . This model was used to select ten residues within the carboxy phosphate domain of CPS for modification and subsequent characterization of the kinetic constants for the mutant proteins . Residues R82, R129, R169, D207, E215, N283, and Q285 were changed to alanine residues; residues E299 and R303 to glutamine; and residue N301 to aspartate . No significant changes in the catalytic constants were observed upon mutation of either R82 or D207, and thus these residues appear to be nonessential for binding and/or catalytic activity . The Michaelis constant for ATP was most affected by modification of residues R129, R169, Q285, and N301 . The binding of bicarbonate was most affected by the mutagenesis of residues E215, E299, N301, and R303 . The mutation of residues E215, N283, E299, N301, and R303 resulted in proteins which were unable to synthesize carbamoyl phosphate at a significant rate . All of the mutations, with the exception of the N301D mutant, primarily affected the enzyme by altering the step for the phosphorylation of bicarbonate . However, mutation of N301 to aspartic acid also disrupted the catalytic step involved in the phosphorylation of carbamate . These results are consistent with a role for the N-terminal half of the synthetase subunit of CPS that is primarily directed at the initial phosphorylation of bicarbonate by the first ATP utilized in the overall synthesis of carbamoyl phosphate . The active site structure appears to be very similar to the ones previously determined for D-alanine:D-alanine ligase and biotin carboxylase.

Mutat Res, 1996 Nov 11, 372(1), 97 - 105
Towards validation of the Big Blue transgenic mouse mutagenesis assay: the mutational spectrum of ex vivo pinpoint mutant plaques; Nishino H et al.; To explore further the origin of spontaneous mutations recovered with the Big Blue transgenic mouse mutagenesis assay, the spectrum of ex vivo mutations from pinpoint mutant plaques was determined and compared with the spectrum of putatively mouse-derived mutations from circular, mutant plaques . The entire lacI gene and lacZ operator region from 62 pinpoint blue plaques was sequenced . The observed mutational spectrum of pinpoint mutants differed significantly from that seen in circular mutants (p < 0.0001) . Only four percent of the mutations were transitions at CpG sites whereas this type of mutation was the most common (35%) in circular mutants . Microdeletions/microinsertions were seen more frequently in pinpoint mutants relative to circular mutants . Four base pair deletion/insertion events at the E . coli hotspot tandem repeats were seen in 10 of 62 (16%) pinpoint mutants and minor hotspots of mutation were observed at bp 141 and 1110 . The mutational spectrum of pinpoint mutants provides further evidence that most circular mutants originate in mouse.

Mutat Res, 1996 Nov 11, 372(1), 87 - 96
MutS interaction with mismatch and alkylated base containing DNA molecules detected by optical biosensor; Babic I et al.; An optical biosensor was used to monitor interactions between the Escherichia coli DNA mismatch repair molecule MutS and various immobilized oligonucleotides . While associating poorly with single-stranded DNA, MutS was capable of rapid association/dissociation from homoduplex DNA . The interaction of MutS with oligonucleotide 30-mers containing single site mismatches demonstrated that during the dissociation phase, MutS binding was greatest to a G-G mismatch, followed by G-T > A-A > C-T, A-C . Binding to A-G, T-T and C-C mispairs was marginally higher than that seen between MutS and homoduplex DNA . The ability of MutS to interact with 30-mers containing alkylated bases was also tested . While binding to O6-methyl-G-C, or to O4-methyl-T-A base pairs was similar to that of homoduplex DNA, strong binding was seen to a O6-methyl-G-T mispair . O4-methyl-T-G, however, was poorly recognized by MutS, with relative binding affinity similar to homoduplex DNA, predicting poor in vivo recognition of O4-methyl-T-G by MutS . Interestingly, MutS demonstrated a relatively high affinity for an 1,N6-etheno-A-T containing homoduplex . Thus, in allowing rapid evaluation of interactions between such molecules, the biosensor will be useful to structure-function analyses.

Mutat Res, 1996 Nov 11, 372(1), 53 - 64
Molecular analysis of lacI mutations in Rat2 cells exposed to 7,12-dimethylbenz{a}anthracene: evidence for DNA sequence and DNA strand biases for mutation; Manjanatha MG et al.; The Rat2 cell line carries 50-70 stably integrated copies per cell of a lambda/lacI shuttle vector as a target for mutagenicity testing . Rat2 cells were exposed to 1 and 10 micrograms/ml of 7,12-dimethylbenz{a}anthracene (DMBA) for 24 h at 37 degrees C in the presence of primary rat hepatocytes, and grown to confluence . The shuttle vector was rescued from untreated and mutagen-treated cells and mutant frequencies were determined . The low and high doses of DMBA induced mutant frequencies that were 7-fold (25 +/- 4.9 x 10(-5)) and 33-fold (127 +/- 19.9 x 10(-5)) higher, respectively, than the spontaneous mutant frequency (3.8 +/- 0.7 x 10(-5)) . DNA sequence analysis of the DMBA-induced lacI- mutants indicated that they contained mainly basepair substitution mutations at A:T and G:C, and that A:T-->T:A and G:C-->T:A transversions were the predominant types . In addition, 23 of 28 (82%) A:T basepair substitution mutations occurred with the mutated dA, the putatively adducted base, on the coding strand . Furthermore, 20 of the 28 (71%) A:T mutations had the mutated dA flanked 5' by a dC, and 17 of these were A:T-->T:A transversions, suggesting a sequence preference for this mutation . Except for a higher proportion of G:C-->A:T transitions in the low dose data, the mutational profiles from low and high doses of DMBA were similar . These results indicate that DMBA mutagenesis in the lacI gene of Rat2 cells displays distinct DNA sequence and DNA strand preferences.

Mutat Res, 1996 Nov 11, 372(1), 23 - 31
A 3 milliTesla 60 Hz magnetic field is neither mutagenic nor co-mutagenic in the presence of menadione and MNU in a transgenic rat cell line; Suri A et al.; The mechanisms by which an electromagnetic field (EMF) influences biological material are poorly understood . One potentially important model suggests that a magnetic field can stabilize free radicals in such a way as to permit their dispersement rather than their return to the ground state (Okazaki et al., 1988; Scaiano, 1995) . We have tested this hypothesis by examining mutagenesis in the E . coli lacI gene target carried in the Big Blue rat embryo fibroblast cell line, R2 lambda LIZ . Mutant frequencies were determined in cells exposed to a magnetic field, cells pretreated with the mutagens N-methylnitrosourea (MNU) or 2-methyl-1,4-naphthoquinone (menadione), prior to being held in a 60 Hz 3 milliTesla (mT) magnetic field and cells concurrently exposed to the mutagens and the magnetic field . Menadione was selected because its mutagenic mechanism involves the formation of free radicals, while MNU is an alkylating agent not thought to act through radical formation . According to the radical stabilization hypothesis the application of a magnetic field to menadione treated cells would accentuate the mutagenic effects . Our results failed to indicate that the magnetic field affects mutagenesis by the oxygen-radical mediated mutagen, menadione.

Biochim Biophys Acta, 1996 Nov 11, 1309(1-2), 109 - 21
Expression of glutamyl-tRNA reductase in Escherichia coli; Chen W et al.; The biosynthesis of the hemes, chlorophylls, corrins and other tetrapyrroles begins with the synthesis of 5-aminolevulinic acid (ALA) . The pathway is highly conserved except for the synthesis of ALA which is derived from glycine and succinyl CoA (C4) in most eukaryotes and from glutamate (C5) in most bacteria and in green plants . In C5, glutamyl-tRNA synthetase (GTS) converts glutamate to glutamyl-tRNA (glu-tRNA), which is reduced by glutamyl-tRNA reductase (GTR) to glutamyl-1-semialdehyde (GSA), which is converted by aminotransferase (GSA-AT) to ALA . Since GTS is also involved in protein synthesis and GSA can be converted to ALA non-enzymatically, it is highly probable that control of ALA synthesis and thus of the whole pathway resides in the GTR step . In Escherichia coli, GTR is the gene product of hemA . BL21(DE3), a protease-deficient strain which contains the T7 RNA polymerase gene in front of a lac promoter, was transformed with a pET14b-based vector, pWC01, harboring hemA in front of a T7 promoter and ORF1 which is transcribed in the opposite direction . The transformed strain, WC1201, secreted ALA and porphyrins into the medium . Induction of expression of hemA by WC1201 was optimized for concentration of inducer (IPTG, 5 mM), temperature (37 degrees C), presence of betaine and sorbitol (no change) and time of induction (2h) . GTR was observable as a 46 kDa band by Brilliant blue G staining of SDS-PAGE gels . Sonicates of the induction mixture exhibited strong ALA synthesis activity which was enhanced by tRNAglu . Most of the activity was in the supernatant of the sonicate indicating that GTR is a soluble enzyme . The induced strain had more GTS activity than the uninduced strain which had more GTS activity than its parent wild-type strain . Autoradiography on native gradient PAGE showed that GTR expressed in vivo by induction of WC1201 had a molecular weight of approx . 117 kDa . Gel filtration of the induced sonicate showed a peak of enzymatic activity at about 126 kDa . When pET14b- or pUC19-based plasmids harboring hemA and ORF1, or importantly, a pUC19-based plasmid harboring only hemA and not ORF1, were expressed in an in vitro transcription-translation system, native gradient PAGE showed a product with a molecular weight of approximately 175 kDA . This expression was higher in the presence of tRNAglu . When the 117 kDa and 175 kDa proteins were excised from their native gels respectively, and run on SDS PAGE, autoradiography showed bands at 46 kDa . We conclude that GTR is present in both high molecular weight species . Since overexpression of hemA from pET14b-based plasmids is associated with increased glutamyl-tRNA synthetase activity, the 175 kDa species may represent different complexes of GTR, GTS and glutamyl-tRNA as observed in Chlamydomonas and the 117-126 kDa species may be an dimer of GTR associated with glu-tRNA or a complex of GTR, GTS and glu-tRNA . These possibilities are being investigated.

Biochim Biophys Acta, 1996 Nov 11, 1309(1-2), 21 - 4
Cloning and expression of the cDNA for mouse NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase; Matsuo M et al.; The cDNA for mouse NAD+ dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) was isolated from a lung cDNA library . The cDNA contains a 798 bp open reading frame that codes for a protein of 266 amino acids (M(r) 28775) which shares 87% identity with the human 15-PGDH protein . The regions that are believed to form the NAD+ binding site and the active site are conserved in the mouse and human enzymes . The authenticity of the mouse cDNA was confirmed by expression of an active 15-PGDH in Escherichia coli . Northern blot analysis demonstrated that 15-PGDH mRNA is expressed primarily in lung, intestine, stomach and liver.

FEBS Lett, 1996 Nov 11, 397(1), 127 - 9
Different efficiencies of the Tag and AlkA DNA glycosylases from Escherichia coli in the removal of 3-methyladenine from single-stranded DNA; Bjelland S et al.; Escherichia coli possesses two different DNA repair glycosylases, Tag and AlkA, which have similar ability to remove the alkylation product 3-methyladenine from double-stranded DNA . In this study we show that these enzymes have quite different activities for the excision of 3-methyladenine from single-stranded DNA, AlkA being 10-20 times more efficient than Tag . We propose that AlkA and perhaps other glycosylases as well may have an important role in the excision of base damage from single-stranded regions transiently formed in DNA during transcription and replication.

FEBS Lett, 1996 Nov 11, 397(1), 93 - 6
Mechanism of hydride transfer during the reduction of 3-acetylpyridine adenine dinucleotide by NADH catalyzed by the pyridine nucleotide transhydrogenase of Escherichia coli; Bragg PD; The pyridine nucleotide transhydrogenase is a proton pump which catalyzes the reversible transfer of a hydride ion equivalent between NAD+ and NADP+ coupled to translocation of protons across the cytoplasmic membrane . The enzyme also catalyzes the reduction of the NAD+ analog 3-acetylpyridine adenine dinucleotide (AcPyAD+) by NADH . It has been proposed (Hutton et al . (1994) Eur . J . Biochem . 219, 1041-1051) that this reaction requires NADP(H) as an intermediate . Thus, NADP+ bound at the NADP(H)-binding site on the transhydrogenase would be reduced by NADH and reoxidized by AcPyAD+ binding alternately to the NAD(H)-binding site . The reduction of AcPyAD+ by NADPH would be a partial reaction in the reduction of AcPyAD+ by NADH . Using cytoplasmic membrane vesicles from mutants having elevated activities for transhydrogenation of AcPyAD+ by NADH in the absence of added NADP(H), the kinetics of reduction of AcPyAD+ by NADH and NADPH have been compared . The Km values for the reductants NADPH and NADH over a range of mutants, and for the non-mutant enzyme, differed to a much lesser degree than the Km for AcPyAD+ in the two reactions . The Km(AcPyAD) values for the transhydrogenation of AcPyAD+ by NADH were over an order of magnitude greater than those for the transhydrogenation of AcPyAD+ by NADPH . It is unlikely that AcPyAD+ binds at the same site in both reactions . A plausible explanation is that this substrate binds to the NADP(H)-binding site for transhydrogenation by NADH . Thus, a hydride equivalent can be transferred directly between NADH and AcPyAD+ under these conditions.

FEBS Lett, 1996 Nov 11, 397(1), 75 - 8
Expression and biochemical analyses of the recombinant potato virus X 25K movement protein; Kalinina NO et al.; The 25K movement protein (MP) of potato virus X (PVX) is encoded by the 5'-proximal gene of three overlapping MP genes forming a 'triple gene block' . The PVX 25K MP (putative NTPase-helicase) has been synthesized in Escherichia coli as a recombinant containing a six-histidine tag at the amino terminus . The His-tagged 25K protein was purified in a one-column Ni-chelate affinity chromatography procedure . In the absence of any other viral factors, this protein had obvious Mg2+-dependent ATPase activity, which was stimulated slightly (1.7-1.9-fold) by various polynucleotides . Like other viral proteins possessing ATPase-helicase motifs and many plant viral movement proteins, the PVX 25K MP was able to bind nucleic acids in vitro . The RNA binding activity of the 25K MP was pronounced only at very low salt concentrations and was independent of its ATPase activity.

FEBS Lett, 1996 Nov 11, 397(1), 30 - 4
Topographical structure of membrane-bound Escherichia coli F1F0 ATP synthase in aqueous buffer; Singh S et al.; Scanning force microscope images of membrane-bound Escherichia coli ATP synthase F0 complexes have been obtained in aqueous solution . The images show a consistent set of internal features: a ring structure which surrounds a central dimple and contains an asymmetric lateral mass . Images of trypsin-treated F0 complexes, which have lost part of their b subunits, show a reduced asymmetric mass, while images of c-subunit oligomers, which lack both the a and b subunits, show a ring and dimple but do not have an asymmetric mass . These results support models in which the F0 complex contains a ring of 9-12 c subunits with the b subunits located outside this ring, and show that scanning force microscopy is able to provide structural information on membrane proteins of molecular mass less than 200 000 Da.

Biochim Biophys Acta, 1996 Nov 8, 1314(1-2), 157 - 66
Generation of antibodies specific for the RalA and RalB GTP-binding proteins and determination of their concentration and distribution in human platelets; Jilkina O et al.; Peptide specific polyclonal antibodies directed against C-termini of ras p21 related GTP-binding proteins, ralA and ralB, were generated . To assess antibody specificity, cDNAs coding for full length ralA and ralB were expressed in Escherichia coli as GST fusion proteins . Western blotting analysis using enhanced chemiluminescence technique confirmed that ralA and ralB antibodies were specific for their respective protein . To determine the concentration and distribution, varying amounts of GST-ralA and GST-ralB and, human platelet particulate and cytosolic proteins were loaded during Western blotting . The amount of ralA and ralB proteins in the platelet particulate fraction was determined to be 0.16 +/- 0.017 microgram/mg protein (n = 3) and 0.15 +/- 0.009 microgram/mg protein (n = 3) respectively . In the cytosol, only ralB protein was detected and its concentration was estimated to be 0.03 +/- 0.009 microgram/mg protein (n = 3) . Both ralA and ralB proteins were isoprenylated in the presence of {3H} mevalonolactone plus rabbit reticulocyte lysate although radioactivity incorporated into ralA was three times higher than that associated with the ralB protein . Addition of geranylgeranyl pyrophosphate to the reaction mixture inhibited incorporation of radioactivity into ralA and ralB but not cH-ras suggesting that both ralA and ralB proteins are geranylgeranylated . Differential distribution of ralA and ralB GTP-binding proteins in human platelets suggests a distinct role for each of these proteins in platelet function.

Biochim Biophys Acta, 1996 Nov 8, 1314(1-2), 120 - 4
Isolation and characterization of a human heart cDNA encoding a new member of the small heat shock protein family--HSPL27; Lam WY et al.; A novel cDNA clone was isolated from a human adult heart cDNA library . This cDNA clone is similar to the small heat shock protein (smhsp) in both DNA and amino acid sequences, especially in the conserved region . Sequence analysis has shown that the putative novel smhsp, named 27 kDa heat-shock-protein-like protein (HSPL27) is a protein of 241 amino acids with a deduced molecular mass of 26.7 kDa and a deduced pI of 8.0 . We have expressed the HSPL27 in E . coli and the expressed protein was found to be present in the soluble fraction of the bacterial cell lysate . Chromosomal mapping data shows that the HSPL27 gene is located at human chromosome 5q11.2.

Biochem Pharmacol, 1996 Nov 8, 52(9), 1447 - 51
Effect of septicaemia on the plasma levels of biopterin and nitric oxide metabolites in rats and rabbits; van Amsterdam JG et al.; Live Escherichia coli decreased mean arterial blood pressure in rabbits from 67 to 20 mmHg . E . coli did not affect blood pressure in rats but did significantly increase heart rate by 29% . To related the cardiovascular effects with putative relevant biochemical pathways, the plasma levels of nitrate + nitrite (NOx) and biopterin, representing the main metabolites of nitric oxide and tetrahydrobiopterin, respectively, were determined in conscious rats and rabbits after treatment with live E . coli . In rats, E . coli induced a rapid 43% increase in the plasma level of biopterin preceding the 7- to 26-fold increase in NOx level . In rabbits, no increase in the NOx level was observed despite a 3- to 5-fold increase in the biopterin level at 6-10 hr posttreatment . It is concluded that the synthesis of tetrahydrobiopterin precedes nitric oxide synthesis after induction of septicaemia in the rat . After the induction of septicaemia, rabbits show a clear hypotensive response and an increase in biopterin level but no concomitant increase in NOx . Biopterin apparently represents a more appropriate biochemical marker of septic shock than does NOx.

Biochem Pharmacol, 1996 Nov 8, 52(9), 1365 - 74
Stable expression of human inducible nitric oxide synthase in V79 Chinese hamster cells; Schmalix WA et al.; A recombinant expression vector containing the full-length cDNA for human inducible nitric oxide (NO) synthase was constructed for constitutive expression in V79 Chinese hamster cells . Expression was followed by Western analyses using three different NO synthase antisera . Activity remained stable during 4 months of continued cultivation . Activities were 25 pmol min-1 mg-1 cytosolic protein with L-arginine and 47 pmol min-1 mg-1 cytosolic protein with NG-hydroxy-L-arginine as substrates . Activity was concentration-dependently inhibited by inhibitors such as NG-methyl-L-arginine, NG-nitro-L-arginine, NG-nitro-L-arginine methyl ester, aminoguanidine, and S-methyl-isothiourea . The rank order of inhibitor potencies was different from published results obtained with rodent inducible NOS . Parental V79 cells do not express and cannot be induced for NO synthase activity . Therefore, the genetically engineered V79 cell line is defined for the cDNA-encoded human inducible NO synthase . The new cell line may serve as a useful tool to study human inducible NO synthase.

J Mol Biol, 1996 Nov 8, 263(4), 582 - 96
Bypass of DNA heterologies during RuvAB-mediated three- and four-strand branch migration; Adams DE et al.; During general genetic recombination and recombinational DNA repair, DNA damages and heterologies are often encountered which must be efficiently processed by the cellular recombination machinery . In RecA-mediated three-strand exchange reactions between single-stranded circular and linear duplex DNA, or four-strand exchange reactions between gapped circular and linear duplex DNA, heterologies can only be bypassed in vitro when they are short in length and are followed by homologous DNA downstream . Larger DNA inserts block RecA-mediated strand exchange, indicating that effective bypass requires other components of the recombination machinery . The RuvA and RuvB proteins of Escherichia coli form an important part of this machinery . In this work, we have analysed the ability of RuvA and RuvB to bypass large tracts of DNA heterology in both three- and four-strand exchange reactions, using recombination intermediates made by the E . coli RecA protein . Under optimal reaction conditions for RuvAB, up to 1000 bp of DNA heterology can by bypassed in three-strand reactions and 300 bp of DNA heterology can be bypassed in four-strand reactions . Whereas high concentrations of RuvB (in the absence of RuvA) can promote homologous branch migration, we find that RuvB alone is unable to catalyse heterologous bypass, indicating an essential role for both proteins in homologous recombination and recombinational DNA repair processes . Under certain conditions, the bypass of heterology is stimulated by the single-strand binding protein SSB.

J Biol Chem, 1996 Nov 8, 271(45), 28558 - 66
Effects of site-directed mutations on the chaperone-like activity of alphaB-crystallin; Plater ML et al.; Recombinant alphaB-crystallin has been shown to exhibit chaperone-like activity, suppressing the thermal aggregation of gamma-crystallin and aggregation of the reduced insulin B chain conferring thermotolerance to Escherichia coli BL21(DE3) cells . Mutations were made in three specific areas of the alphaB-crystallin, the N terminus D2G, the conserved phenylalanine-rich region, F24R, F27R, F27A, and the two C-terminal lysines K174L/K175L, K174G/K175G . Biophysical characterization of the mutant alphaB-crystallins using far-UV CD revealed no change in secondary structural elements . Tryptophan fluorescence demonstrated global structural changes . Heat stability of the mutant alphaB-crystallins was not significantly affected as indicated by tryptophan fluorescence of heat-treated proteins . Mutations within the phenylalanine-rich region abolish the chaperone-like activity as measured by both in vivo and in vitro assays . Proteins with mutations at the C terminus demonstrated no significant chaperone-like activity, failing to confer thermotolerance on E . coli and demonstrating no significant inhibition of protein aggregation in either gamma-crystallin or reduced insulin B chain assays . The N-terminal mutation D2G demonstrated a significant reduction in efficiency of the chaperone-like activity although some thermotolerance was conferred in the E . coli assay . In vitro assays showed that complete inhibition of aggregation was only achieved at 10-fold higher concentrations of D2G than that required by the native alphaB-crystallin . Consistent changes in the chaperone-like activity of the site-directed mutants were demonstrated by the three assays . The results suggested that both charge-charge and hydrophobic interactions are important in protein binding by alphaB-crystallin and that the conserved RLFDQFF region is vital for chaperone-like activity.

J Biol Chem, 1996 Nov 8, 271(45), 28541 - 8
Identification of a cysteine residue essential for activity of protein farnesyltransferase . Cys299 is exposed only upon removal of zinc from the enzyme; Fu HW et al.; Protein farnesyltransferase (FTase) is a zinc metalloenzyme that performs a post-translational modification on many proteins that is critical for their function . The importance of cysteine residues in FTase activity was investigated using cysteine-specific reagents . Zinc-depleted FTase (apo-FTase), but not the holoenzyme, was completely inactivated by treatment with N-ethylmaleimide (NEM) . Similar effects were detected after treatment of the enzyme with iodoacetamide . The addition of zinc to apo-FTase protects it from inactivation by NEM . These findings indicated the presence of specific cysteine residue(s), potentially located at the zinc binding site, that are required for FTase activity . We performed a selective labeling strategy whereby the cysteine residues exposed upon removal of zinc from the enzyme were modified with {3H}NEM . The enzyme so modified was digested with trypsin, and four labeled peptides were identified and sequenced, one peptide being the major site of labeling and the remaining three labeled to lesser extents . The major labeled peptide contained a radiolabeled cysteine residue, Cys299, that is in the beta subunit of FTase and is conserved in all known protein prenyltransferases . This cysteine residue was changed to both alanine and serine by site-directed mutagenesis, and the mutant proteins were produced in Escherichia coli and purified . While both mutant proteins retained the ability to bind farnesyl diphosphate, they were found to have lost essentially all catalytic activity and ability to bind zinc . These results indicate that the Cys299 in the beta subunit of FTase plays a critical role in catalysis by the enzyme and is likely to be one of the residues that directly coordinate the zinc atom in this enzyme.

J Biol Chem, 1996 Nov 8, 271(45), 28509 - 15
Different collagenase gene products have different roles in degradation of type I collagen; Krane SM et al.; Vertebrate collagenases, matrix metalloproteinases (MMPs), cleave type I collagen at a single helical locus . We show here that rodent interstitial collagenases (MMP-13), but not human fibroblast collagenase (MMP-1), cleave type I collagen at an additional aminotelopeptide locus . Collagenase cDNAs and chimeric constructs in pET-3d, juxtaposing MMP-13 sequences amino-terminal to the active site in the catalytic domain and MMP-1 sequences carboxyl-terminal and vice versa, were expressed in Escherichia coli . Assays utilized collagen from wild type (+/+) mice or mice that carry a targeted mutation (r/r) that encodes substitutions in alpha1(I) chains that prevent collagenase cleavage at the helical locus . MMP-13 and chimeric molecules that contained the MMP-13 sequences amino-terminal to the active site cleaved (+/+) collagen at the helical locus and cleaved cross-linked (r/r) collagen in the aminotelopeptide (beta components converted to alpha chains) . Human MMP-1 and chimeric MMP-1/MMP-13 with MMP-1 sequences amino-terminal to the active site cleaved collagen at the helical locus but not in the aminotelopeptide . All activities were inhibited by TIMP-1, 1,10-phenanthroline, and EDTA . Sequences in the distal two-thirds of the catalytic domain determine the aminotelopeptide-degrading capacity of MMP-13.

J Biol Chem, 1996 Nov 8, 271(45), 28485 - 91
Osteopontin N-terminal domain contains a cryptic adhesive sequence recognized by alpha9beta1 integrin; Smith LL et al.; Osteopontin is an adhesive glycoprotein implicated in numerous diseases associated with inflammation and remodeling . There are several structural domains in osteopontin that are of particular interest . The RGD motif is a cell attachment sequence shown to be critical for cell adhesion through alphav-containing integrins . In close proximity to the RGD domain is the thrombin cleavage site . Previous observations suggest that thrombin cleavage of osteopontin occurs in vivo and may be physiologically important . To study the functional significance of osteopontin cleavage by thrombin, we made glutathione S-transferase-osteopontin fusion proteins . These proteins contain either the N- or C-terminal domains expected to be formed following thrombin cleavage at the Arg169-Ser170 peptide bond . We compared these osteopontin fragments with native osteopontin in their ability to support adhesion of several different cell lines and identified the receptors mediating these interactions . Our data show that the N-terminal osteopontin fragment, which contains the RGD domain, supports adhesion of a melanoma cell line that is unable to bind native osteopontin . This suggests that osteopontin adhesive interactions may be regulated by thrombin cleavage . We also demonstrate that osteopontin contains a cryptic binding activity, which can be recognized by a novel osteopontin receptor . This receptor has been identified as the alpha9beta1 integrin.

J Biol Chem, 1996 Nov 8, 271(45), 28391 - 8
Replication across O6-methylguanine by human DNA polymerase beta in vitro . Insights into the futile cytotoxic repair and mutagenesis of O6-methylguanine; Singh J et al.; Replication in vivo across unrepaired O6-methylguanine (m6dG) lesions by mammalian DNA polymerase beta (pol beta) during short patch repair may contribute to the cytotoxicity and mutagenesis of m6dG . We have employed in vitro steady state kinetic analysis to investigate the replication of oligonucleotide templates containing site-specific m6dG by human pol beta . Our results show that m6dG is a strong but not absolute block to replication by pol beta . pol beta exhibits mixed kinetic discrimination during overall replication across dG and m6dG . pol beta preferentially inserts dTMP rather than dCMP opposite m6dG . However, pol beta extends from the dC-m6dG base pair more efficiently than from the dT-m6dG base pair . This is in strong contrast to other polymerases such as the exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I (exo-KF) that preferentially extends dT-m6dG by a factor of 10 over dC-m6dG . When both insertion and extension are considered, pol beta has a 15-fold overall preference for incorporation of the mutagenic substrate dTTP rather than the nonmutagenic substrate dCTP during replication across m6dG . This suggests that pol beta, in concert with the T:G-specific thymine DNA glycosylase, may be intricately involved in the futile cytotoxic repair induced by m6dG . Our results also suggest that replication across m6dG by pol beta may contribute to m6dG-induced G --> A transition mutations.

J Biol Chem, 1996 Nov 8, 271(45), 28341 - 7
The stalk region of the Escherichia coli ATP synthase . Tyrosine 205 of the gamma subunit is in the interface between the F1 and F0 parts and can interact with both the epsilon and c oligomer; Watts SD et al.; The soluble portion of the Escherichia coli F1F0 ATP synthase (ECF1) and E . coli F1F0 ATP synthase (ECF1F0) have been isolated from a novel mutant gammaY205C . ECF1 isolated from this mutant had an ATPase activity 3.5-fold higher than that of wild-type enzyme and could be activated further by maleimide modification of the introduced cysteine . This effect was not seen in ECF1F0 . The mutation partly disrupts the F1 to F0 interaction, as indicated by a reduced efficiency of proton pumping . ECF1 containing the mutation gammaY205C was bound to the membrane-bound portion of the E . coli F1F0 ATP synthase (ECF0) isolated from mutants cA39C, cQ42C, cP43C, and cD44C to reconstitute hybrid enzymes . Cu2+ treatment or reaction with 5,5'-dithio-bis(2-nitro-benzoic acid) induced disulfide bond formation between the Cys at gamma position 205 and a Cys residue at positions 42, 43, or 44 in the c subunit but not at position 39 . Using Cu2+ treatment, this covalent cross-linking was obtained in yields as high as 95% in the hybrid ECF1 gammaY205C/cQ42C and in ECF1F0 isolated from the double mutant of the same composition . The covalent linkage of the gamma to a c subunit had little effect on ATPase activity . However, ATP hydrolysis-linked proton translocation was lost, by modification of both gamma Cys-205 and c Cys-42 by bulky reagents such as 5,5'-dithio-bis (2-nitro-benzoic acid) or benzophenone-4-maleimide . In both ECF1 and ECF1F0 containing a Cys at gamma 205 and a Cys in the epsilon subunit (at position 38 or 43), cross-linking of the gamma to the epsilon subunit was induced in high yield by Cu2+ . No cross-linking was observed in hybrid enzymes in which the Cys was at position 10, 65, or 108 of the epsilon subunit . Cross-linking of gamma to epsilon had only a minimal effect on ATP hydrolysis . The reactivity of the Cys at gamma 205 showed a nucleotide dependence of reactivity to maleimides in both ECF1 and ECF1F0, which was lost in ECF1 when the epsilon subunit was removed . Our results show that there is close interaction of the gamma and epsilon subunits for the full-length of the stalk region in ECF1F0 . We argue that this interaction controls the coupling between nucleotide binding sites and the proton channel in ECF1F0.

J Biol Chem, 1996 Nov 8, 271(45), 28038 - 44
Kinetic and thermodynamic characterizations of yeast guanylate kinase; Li Y et al.; Yeast guanylate kinase was expressed at high level in Escherichia coli using pET-17b vector . It was purified to homogeneity by a simple two-column procedure with an average yield of approximately 100 mg/liter . The steady-state kinetic parameters for both forward and reverse reactions were determined by initial velocity measurements . The turnover numbers (kcat) were 394 s-1 for the forward reaction (formation of ADP and GDP) and 90 s-1 for the reverse reaction (formation of ATP and GMP) . Km values were 0.20, 0 . 091, 0.017, and 0.097 mM for MgATP, GMP, MgADP, and GDP, respectively . Analysis of the initial velocity patterns indicated a sequential mechanism . GMP was found to have partial substrate inhibition . The substrate inhibition was not competitive with MgATP and could be attributed to formation of the abortive complex guanylate kinase.MgADP.GMP . The equilibrium constant of the reaction was measured under various conditions by NMR and a radiometric assay . The results showed that the steady-state kinetic parameters were consistent with the thermodynamic constant . NMR titration and equilibrium dialysis showed that both substrates and products could bind to free guanylate kinase . The dissociation constants were 0.090, 0.18, 0.029, 0.084, and 0.12 mM for MgATP, ATP, GMP, MgADP, and GDP, respectively . Viscosity-dependent kinetics was used to identify the rate-limiting steps of the reaction . The results indicated that the reaction rate is largely controlled by the chemical step.

Gene, 1996 Nov 7, 179(1), 181 - 8
A broad-host-range in vivo pop-out and amplification system for generating large quantities of 50- to 100-kb genomic fragments for direct DNA sequencing; Wild J et al.; A prerequisite for sequencing large genomes is to obtain 30- to 150-kb genomic DNA fragments in adequate quantity . Previously, we developed a system which enables one to excise and amplify in vivo such segments directly from the Escherichia coli genome . This system, which employed the yeast Flp/FRT elements for excision and the plasmid R6K-based replication machinery for DNA amplification, permits one to bypass conventional cloning {Posfai et al . (1994) Nucleic Acids Res . 22, 2392-2398} . To extend the applicability of such a system to many species, we describe here a broad-host-range (bhr) system in which the amplification of the excised DNA fragment depends on the oriV element and the Rep (TrfA) protein from the promiscuous RK2/RP4 plasmid . We have constructed insertion plasmids which carry the FRT and oriV sites . To introduce such plasmids into the appropriate position in the host genome, a short genomic sequence homologous to this position was cloned into the multiple cloning site (MCS) of the FRT/oriV insertion plasmid and then recombined into this position in the genome by RecA-mediated recombination . In such a manner, many strains with single FRT/oriV insertions at various positions could be generated . Subsequent genetic crosses or phage transduction allow two neighboring FRT/oriV sites (less than 150 kb apart) to be brought into a single genome . In the present report, the lacZ and phoB sites, which are 51 kb apart in the E . coli genome, were used for the introduction of the FRT/oriV sites . To deliver the Flp (excision) and Rep (amplification) functions in trans, the yeast FLP and RK2 plasmid trfA genes were placed under the control of the Ptet promoter/operator which is tightly regulated by the TetR repressor . The addition of heated chlortetracycline (cTc) inactivates TetR, turning on the synthesis of Flp and TrfA, which respectively, execute (i) excision of the 51-kb genomic segment between the two FRT sites (in lacZ and in phoB), and (ii) its amplification.

Gene, 1996 Nov 7, 179(1), 129 - 32
Finding new mutator strains of Escherichia coli--a review; Miller JH et al.; Mutations in the lacZ gene were constructed that allowed the detection of mutators that caused specific transversion mutations . This allowed the detection of two new mutator genes, mut Y and mut M, which are involved in the repair of the 8-oxodguanine (8oxodG) lesion.

Gene, 1996 Nov 7, 179(1), 53 - 7
Redox signaling and gene control in the Escherichia coli soxRS oxidative stress regulon--a review; Demple B; The soxRS regulon of Escherichia coli coordinates the induction of at least twelve genes in response to superoxide or nitric oxide . This review describes recent progress in understanding the signal transduction and transcriptional control mechanisms that activate the soxRS regulon, and some aspects of the physiological functions of this system . The SoxS protein represents a growing family of transcription activators that stimulate genes for resistance to oxidative stress and antibiotics . SoxR is an unusual transcription factor whose activity in vitro can be switched off by the removal of {2Fe-2S} centers, and activated by their reinsertion . The activated form of SoxR remodels the structure of the soxS promoter to activate transcription . When the soxRS system is activated, bacteria gain resistance to oxidants, antibiotics and immune cells that generate nitric oxide . The latter features could increase the success (virulence) of some bacterial infections.

Gene, 1996 Nov 7, 179(1), 39 - 44
Probing the structure of catalase HPII of Escherichia coli--a review; Loewen P; Escherichia coli produces two catalases or hydroperoxidases, HPI and HPII . HPI is a bifunctional catalase-peroxidase active as a tetramer of identical 80049-Da subunits encoded by katG . The expression of katG is controlled at the basal level by sigma s (KatF), and its induction by H2O2 is regulated by OxyR . HPII is a monofunctional catalase active as a tetramer of identical 84118-Da subunits encoded by katE . The induction of katE expression in the stationary phase is controlled by sigma s . The core of HPII is similar in sequence to other catalases including the conservation of several residues that have been implicated as playing a catalytic role, His128, Asn201, Ser167 and Tyr415 . These residues have served as targets for site-directed mutagenesis in a study that has demonstrated their role in the catalytic mechanism of HPII . In addition, the two Cys residues in HPII have been targeted in a similar study revealing that they do not have a catalytic role, but that Cys438 is blocked by a novel modification . Despite many structural similarities to bovine liver catalase, the heme component of HPII has proved to be quite different . The presence of a cis heme d was determined spectrally and chromatographically, and the inability of certain mutants to generate the modified heme revealed that it was HPII itself that was catalysing the oxidation of heme b to heme d . The recent solution of the crystal structure of HPII and mass spectrometry have revealed that the heme d bound to HPII is a spirolactone structure with a cis orientation of the oxygens on the proximal side of the heme . This has created the problem of explaining how the oxidation of the heme can occur on the opposite side of the heme ring, remote from the catalytic residues.

Biochemistry, 1996 Nov 5, 35(44), 13955 - 65
Crystallographic studies of the catalytic and a second site in fumarase C from Escherichia coli; Weaver T et al.; Fumarase C catalyzes the stereospecific interconversion of fumarate to L-malate as part of the metabolic citric acid or Kreb's cycle . The recent three-dimensional structure of fumarase C from Escherichia coli has identified a binding site for anions which is generated by side chains from three of the four subunits within the tetramer (Weaver et al., 1995) . These same side chains are found in the three most highly conserved regions within the class II fumarase superfamily . The site was initially characterized by crystallographic studies through the binding of a heavy atom derivative, tungstate . A number of additional crystallographic structures using fumarase crystals with bound inhibitors and poor substrates have now been studied . The new structures have both confirmed the originally proposed active site, site A, and led to the discovery of a novel second binding site that is structurally nearby, site B . Site A utilizes a combination of residues, including H188, T187, K324, N326, T100, N141, S139, and S140, to form direct hydrogen bonds to each of the inhibitors . The A-site has been demonstrated by studying crystalline fumarase with the bound competitive inhibitors-citrate and 1,2,4,5-benzenetetracarboxylic acid . The crystal structure of fumarase C with beta-(trimethylsilyl)maleate, a cis substrate for fumarase, has led to the discovery of the second site or B-site . Sites A and B have different properties in terms of their three-dimensional structures . Site B, for example, is formed by atoms from only one of the subunits within the tetramer and mainly by atoms from a pi-helix between residues H129 through N135 . The crystal structures show that the two locations are separated by approximately 12 A . A highly coordinated buried water molecule is also found at the active or A-site . The high-resolution crystal structures describe both sites, and atoms near the A-site are used to propose a likely enzyme/substrate complex.

FEBS Lett, 1996 Nov 4, 396(2-3), 181 - 8
Isolation and characterisation of a cDNA encoding rat mitochondrial GrpE, a stress-inducible nucleotide-exchange factor of ubiquitous appearance in mammalian organs; Naylor DJ et al.; In contrast to the E . coli chaperones DnaK, GroEL and GroES, cDNAs encoding mitochondrial homologues of DnaJ and GrpE from higher eukaryotes have yet to be reported . Based on peptide sequences, we have isolated a cDNA encoding a 217 residue nuclear encoded precursor of rat mitochondrial GrpE (mt-GrpE) including a typical mitochondrial presequence of 27 residues . Western blotting revealed that the 21 kDa GrpE homologue is present exclusively in the mitochondrial fraction where it comprises only approximately 0.03% of the total soluble protein, while Northern blotting showed that the mt-GrpE transcript is present in most if not all organs . By contrast to other mitochondrial chaperones, the levels of mt-GrpE and its transcript in cultured cells are only marginally increased in response to the proline analog L-azetidine 2-carboxylic acid but not by heat shock . Furthermore, members of the GrpE family exhibit a much lower degree of sequence identity than do the well studied members of the Hsp70, Hsp60 and Hsp10 families.

FEBS Lett, 1996 Nov 4, 396(2-3), 172 - 6
Two-dimensional crystals of the Kdp-ATPase of Escherichia coli; Iwane AH et al.; A variant form of the Kdp-ATPase of Escherichia coli was overproduced to a level approaching 37% of the protein in the inner membrane of this organism . Membranes from overproducing cells were prepared with an inside-out orientation . Incubation of the membranes on ice for 1-2 weeks in the presence of sodium vanadate resulted in the formation of two-dimensional crystals of the Kdp-ATPase . The calculated projection map of the p1 crystal form showed three prominent density peaks at a resolution of 22 A . This technique is a useful and simple method to obtain low-resolution structures of membrane proteins.

FEBS Lett, 1996 Nov 4, 396(2-3), 152 - 6
Identification of the amino acid residues responsible for cold tolerance in Flaveria brownii pyruvate,orthophosphate dikinase; Ohta S et al.; Pyruvate,orthophosphate dikinase (PPDK), an enzyme important in C4 photosynthesis, is typically a cold-sensitive enzyme . However, a cold-tolerant form of the enzyme has been isolated from the leaves of Flaveria brownii . Using an Escherichia coli expression system and the PPDK cDNAs from F . brownii (cold-tolerant), F . bidentis (cold-sensitive) and maize (intermediate cold tolerance), site-directed mutagenesis studies indicated that as few as three amino acids residues (of 880 residues) strongly influence the cold sensitivity of Flaveria PPDK . Gel filtration analysis of the PPDK expressed in E . coli showed that subunit association and cold tolerance are closely linked.

FEBS Lett, 1996 Nov 4, 396(2-3), 147 - 51
Plant calcium-dependent protein kinase-related kinases (CRKs) do not require calcium for their activities; Furumoto T et al.; In plants, calcium-dependent protein kinases (CDPKs) make up a large family that is characterized by a C-terminal calmodulin(CaM)-like domain . Recently, a novel carrot cDNA clone encoding an atypical CDPK, which has a significantly degenerate sequence in the CaM-like domain, was found and named CDPK-related protein kinase (CRK) {Lindzen, E . and Choi, J.H . (1995) Plant Mol . Biol . 28, 785-797} . We obtained two different cDNA clones from maize which encode CRKs . For the first enzymatic characterization of CRK, a maize cDNA clone was expressed in E . coli . The recombinant protein efficiently phosphorylated casein, a conventional protein substrate . Notably, in this in vitro phosphorylation assay, the kinase activity did not require calcium as an activator . Thus, CRKs were suggested to be novel calcium-independent protein kinases having a degenerate CaM domain, the function of which remains to be elucidated.

Zhonghua Yi Xue Za Zhi, 1996 Nov, 76(11), 813 - 7
{Effects of selective nitric oxide synthase inhibitor in sheep with endotoxic shock}; Liu D et al.; OBJECTIVE: To investigate the effects of S-methylisothiourea sulfate (SMT), a selective inducible nitric oxide synthase (iNOS) inhibitor, on hyperdynamic endotoxic shock sheep . METHODS: Endotoxic shock was induced by Escherichia coli endotoxin in both control (n = 8) and SMT groups (n = 8) . SMT was given intravenously . Hemodynamic data, oxygen delivery derived parameters and intramucosal pH (pHi) were measured . RESULTS: The control group had a hyperdynamic state, similar to that of human septic shock . In the SMT group, blood pressure was maintained at baseline, and cardiac index (CI) was lower than that in the control group (P < 0.05) . Oxygen extraction ratio (O2 ext) was increased up to 40% +/- 5% and was much higher than that of the control group (P < 0.01) . Pulmonary artery pressure (PAP) was higher than that of the control group (P < 0.01), and pHi decreased gradually similarly to the control group . CONCLUSION: SMT restored the blood pressure and increased O2 extespecially in the gut, but decreased CI and oxygen delivery and increased PAP . So over inhibition of iNOS should be cautiously considered.

Acta Virol, 1996 Nov-Dec, 40(5-6), 273 - 9
Hepatitis B virus core-preS2 particles expressed by recombinant vaccinia virus; Nemeckova S et al.; Vaccinia virus (VV) recombinants expressing hepatitis B virus (HBV) surface (HBsAg) or core (HBcAg) antigens (Kunke et al., Virology 195, 132 - 139 (1993)} have been shown to raise specific antibodies in mice, nevertheless the levels of antibodies reactive with the preS2 and S antigens were low . In an attempt to enhance the immunogenicity of HBsAg-preS2, a fused C-preS2 gene was constructed . The fusion protein was expressed in E . coli and displayed both HBcAg and preS2 antigen as demonstrated by enzyme-linked immunosorbent assay (ELISA) . The same gene was then expressed using recombinant VV and chimerical particles whose size and density were similar to those of native HBV core particles produced in CV-1 cells infected with recombinant VV . Unlike HBcAg, preS2 antigen could not be detected on these particles by ELISA but was revealed by immunoblot analysis only . The immunogenicity of the recombinant VV was evaluated in mice . Antibodies to HBcAg and VV antigen but not to preS2 antigen were found in sera of animals inoculated with 10(7) PFU of the recombinant VV . Presumably, HBcAg-preS2 particles produced in E . coli and in eukaryotic cells have a different conformation, and the presence of preS2 antigen on the surface of chimerical particle might be necessary for a pronounced antibody response.

Cell Adhes Commun, 1996 Nov, 4(4-5), 317 - 25
The role of carboxy-terminal glycosaminoglycan-binding domain of vitronectin in cytoskeletal organization and migration of endothelial cells; Thiagarajan P et al.; Vitronectin is a major cell adhesion molecule present in the subendothelial matrix that mediates the attachment and spreading of a variety of cells . The carboxy-terminal end of vitronectin has a consensus sequence for glycosaminoglycan-binding . To define the functional role of this domain, we generated fragments of vitronectin that lack the glycosaminoglycan-binding domain by formic acid cleavage of plasma-derived vitronectin . In addition, we also generated similar recombinant fragments of vitronectin as glutathione S-transferase fusion proteins in E . coli . These fragments were tested for their ability to support the adhesion of human umbilical vein endothelial cells . These fragments promoted endothelial cell adhesion, reaching half maximal activity at 2-5 micrograms/well compared to plasma-derived vitronectin which reached at 0.2 micrograms/well . However, the cells that adhered to these fragments did not develop well-formed focal adhesion plaques and actin stress fibers . In addition, these fragments were poorly chemotactic for endothelial cell migration when compared to intact plasma-derived vitronectin in a modified Boyden chamber assay . The present studies show that carboxy-terminal glycosaminoglycan-binding domain of vitronectin is essential for proper cytoskeletal organization and migration of endothelial cells on vitronectin substratum.

Mikrobiologiia, 1996 Nov-Dec, 65(6), 749 - 52
{Multiple protective effect of an exometabolite (exometabolites) released by Escherichia coli during treatment with tetracycline}; Nikolaev IuA; Exponentially growing tetracycline-treated Escherichia coli cells release (an) as yet unidentified compound(s) into the medium . These compounds produce a protective effect on E . coli cells under conditions of heat, oxidative, and osmotic shocks, or in the presence of tetracycline . The protective effect is manifested as an acceleration of growth in the presence of tetracycline or under conditions of heat shock or as a shortening of the period of adaptation to other stresses.

Mikrobiologiia, 1996 Nov-Dec, 65(6), 740 - 4
{Role of putrescine and potassium transport in the adaptation of Escherichia coli to ammonium starvation}; Tkachenko AG et al.; Ammonium depletion in the nutrient medium induced an active transport of putrescine into the cell, which was not associated with its utilization as a nitrogen source . The uptake of putrescine was accompanied by a proportional release of potassium from the cells . Quantitative analysis of free as well as weakly and tightly bound pools of putrescine showed that this diamine was bound mainly to DNA . Topological studies by the plasmid method indicated an increase in the DNA supercoiling in response to the putrescine transport . In vitro experiments made it possible to establish an ambiguous dependence of DNA topology on putrescine content--its physiological concentrations (0.5-1.0 mM) enhanced DNA supercoiling, while higher concentrations (2-10 mM) caused gradual relaxation of DNA . A possible physiological significance of these effects in adaptive response of cells to ammonium deficiency is discussed.

Genes Cells, 1996 Nov, 1(11), 953 - 63
Protein inheritance: lambda plasmid replication perpetuated by the heritable replication complex; Wegrzyn A et al.; BACKGROUND: Replication of a plasmid derived from the Escherichia coli phage lambda initiates by binding of the lambda O protein initiator to the origin of lambda DNA replication, ori lambda . The lambda P protein participates in subsequent steps of assembly of the lambda replication complex . A function of lambda P required for replication complex assembly is inactivated at 43 degrees C by the ts1 mutation . RESULTS: We found that the lambda replication complex assembled at 30 degrees C survives the temperature upshift in lambda crotsPts1 plasmid-harbouring bacteria . We present several lines of evidence that in this system (in which the replication complex assembly does not occur), the replication complex assembled prior to the temperature upshift is inherited by one of two daughter plasmid copies at each replication round for more than 30 cell generations . The 'old' replication complex-driven replication is chloramphenicol-resistant and rifampicin-sensitive . This replication is dependent on lambda O and host dnaK, dnaJ and grpE chaperone gene functions . CONCLUSIONS: The lambda O-containing replication complex is inherited together with DNA and bears information how to initiate the next round of replication at ori lambda; thus, we consider that this phenomenon deserves to be called protein inheritance.

Mikrobiol Z, 1996 Nov-Dec, 58(6), 67 - 70
{The production of HIV recombinant proteins and their immunochemical characteristics}; Martynenko DL et al.; Several Escherichia coli strains producing HIV-specific recombinant proteins including env-1, env-2, and gag sequences have been obtained using different gene engineering techniques . These proteins have been isolated and purified from bacterial lysates by ion exchange chromatography on DEAE-Toyopearl and Streamline-SP columns . Polyacrylamide gel electrophoresis and immunoblotting have been used to prove recombinant proteins purity and to identify them . These proteins have been shown to be adequate as antigen preparations for enzyme immunoassay test-systems aimed at anti-HIV antibodies detection.

Gene Ther, 1996 Nov, 3(11), 1026 - 31
The use of cationic liposomes DC-CHOL/DOPE and DDAB/DOPE for direct transfer of Escherichia coli cytosine deaminase gene into growing melanoma tumors; Szala S et al.; An attempt was made to use simple cationic liposomes DC-Chol/DOPE and DDAB/DOPE (DC-Chol is 3 beta (N(N',N-dimethylaminoethane) carbamoyl) cholesterol, DDAB is dimethyldioctadecyl ammonium bromide and DOPE is dioleoylphosphatidylethanolamine) for transfer of Escherichia coli cytosine deaminase 'suicide' gene under the control of tissue-specific tyrosinase gene promoter directly into the murine melanoma B16(F10) tumor . Several repeated intratumoral injections of DNA-liposome complexes followed by intraperitoneal administrations of 5-fluorocytosine, which is converted to 5-fluorouracil, caused strong retardation of murine melanoma B16(F10) tumor growth and, in some cases, rejection of the pre-established tumor . The inhibition of tumor growth expressed as the increased survival of mice is better seen in the case of using DNA-DDAB/DOPE complexes as compared to DNA-DC-Chol/DOPE ones . It seems that the observed therapeutic effect appears to result from several factors: 5-fluorouracil generation by transfected cells, liposome toxicity (DDAB is more toxic than DC-Chol and hence more tumor cells are killed), increased transfection efficiency of surviving cancer cells (in this case DDAB is a better transfection agent than DC-Chol) and, finally, the bystander effect which causes destruction of cells untransfected with CD gene by easily diffusible 5-fluorouracil.

Anticancer Res, 1996 Nov-Dec, 16(6B), 3551 - 6
Molecular cloning and primary characterization of a single-chain antibody against human sarcoma-associated antigen p200; Chen H et al.; BACKGROUND: Monoclonal Antibody (MAb) 29-13 reacts with the human sarcoma-associated antigen (SAA) p200 . We report here engineering and primary characterization of a single chain antibody (scFV2913) . MATERIALS AND METHODS: The scFV2913 recombinant gene, consisting of VH-linker-VK, was constructed with RT-PCR . This gene was cloned and expressed in E . coli . The renatured scFV2913 was used in the immunostaining study . RESULTS: Consistent with its parent MAb 29-13, purified and renatured scFV2913 showed affinity and specificity to the SAA p200 according to the immuno-histochemical staining study of 99 specimens of human sarcomas and other tissues . CONCLUSIONS: Due to its retained specificity and affinity, scFV2913 may be useful in immunodiagnosis and immunotherapy for sarcoma patients.

PDA J Pharm Sci Technol, 1996 Nov-Dec, 50(6), 360 - 5
Enhancing effect of non-ionic surfactant on the inactivation of lipopolysaccharide by steam-heat treatment I; Bamba T et al.; Polyoxyethylene (20) sorbitan mono-fatty acid esters strongly enhanced the inactivation of lipopolysaccharide (LPS) by steam-heat treatment at 121 degrees C, as assayed by using the Limulus amebocyte lysate (LAL) and the pyrogen test . In an aqueous solution containing 0.1% surfactant, the decrease of LPS (1 microgram/ml) from E . coli 055:B5 at 121 degrees C followed first-order kinetics . Based on the LAL assay, 0.1% surfactant was essential to achieve 3-log cycle reduction of LPS with concomitant loss of pyrogenicity by steam-heat treatment for 20 min at 121 degrees C . Steam-heat treatment for 20 min at 121 degrees C in the absence of surfactant was insufficient to achieve depyrogenation . Polyoxyethylene (9) lauryl ether and decaglycerin mono-laurate similarly enhanced depyrogenation by steam-heat treatment . The effects of all the surfactants were concentration-dependent for all of the six kinds of LPS examined.

Toxicon, 1996 Nov-Dec, 34(11-12), 1243 - 56
Binding of native kappa-neurotoxins and site-directed mutants to nicotinic acetylcholine receptors; Chiappinelli VA et al.; The kappa-neurotoxins are useful ligands for the pharmacological characterization of nicotinic acetylcholine receptors because they are potent antagonists at only a subgroup of these receptors containing either alpha 3- or alpha 4-subunits (IC50 < or = 100 nM) . Four of these highly homologous, 66 amino acid peptides have been purified from the venom of Bungarus multicinctus (kappa-bungarotoxin (kappa-Bgt), kappa 2-Bgt, kappa 3-Bgt} and Bungarus flaviceps {kappa-Fvt)} . Two approaches were taken to examine the binding of these toxins to nicotinic receptors . First, venom-derived kappa-Fvt and kappa-Bgt were radioiodinated and the specific binding was measured of these toxins to overlapping synthetic peptides (16-20 amino acids in length) prepared based on the known sequence of the nicotinic receptor alpha 3-subunit . At least two main regions of interaction between the toxins and the receptor subunit were identified, both lying in the N-terminal region of the subunit that is exposed to the extracellular space . The second approach examined the importance of several sequence position in kappa-Bgt for binding to alpha 3-containing receptors in autonomic ganglia and alpha 1-containing muscle receptors . This was done using site-directed mutants of kappa-Bgt produced by an Escherichia coli expression system . Arg-34 and position 36 were important for binding to both receptor subtypes, while replacing Gln-26 with Trp-26 (an invariant in alpha-neurotoxins) increased affinity for the muscle receptor by 8-fold . The results confirm that kappa-neurotoxins bind potently to the alpha 3-subunit and bind with considerably reduced affinity (Kd approximately 10 microM) to muscle receptors . Site-directed mutagenesis of recombinant kappa-Bgt is thus an important approach for the study of structure-function relationships between kappa-Bgt and nicotinic receptors.

Int J Biochem Cell Biol, 1996 Nov, 28(11), 1241 - 7
Overexpression and functional analysis of a mitogen-inducible nuclear GTPase activating protein, Spa-1; Nur-e-Kamal MS; The two Ras-related GTPases called Rap1 and Rsr1, which share 50% sequence identity with Ras GTPases are known to be activated by two distinct mammalian GAPs, i.e . cytosolic GAP3c of 55 kDa and membrane-bound GAP3m of 85 kDa . Recently we have cloned a gene encoding a 68 kDa (p68) protein product, which is associated with chromosomes during interphase . The N-terminal 190 amino acids share 43% sequence identity with the second half of the GTPase activating domain (residues 210-397) of GAP3m . The N-terminal fragment of 209 amino acids of Spa-1 (called Span-N) was overproduced in E . coli as a glutathione S-transferase (GST) fusion protein and affinity purified . Rap1 and Rsr1 GTPase stimulatory activity of Spa-1 was tested and compared with GAP3m . Spa-1 preferentially stimulates Rsr1 GTPase rather than Rap1 GTPase, while GAP3m has a preference for Rap1 GTPase . This suggests that although Spa-1 and GAP3m stimulate GTPase of Rap1 family members, they differ in affinity for them . By mutational analysis it was also found that amino acid residues 10-183 are enough for Rap GAP activity of Spa-1.

Int J Biochem Cell Biol, 1996 Nov, 28(11), 1233 - 40
Gastrin and gastrin receptor antagonists bind to both N- and C-terminal halves of the 78 kDa gastrin-binding protein; Murphy VJ et al.; A 78 kDa gastrin-binding protein (GBP) has previously been identified as the target of the anti-proliferative effects of non-selective gastrin/cholecystokinin receptor antagonists on colorectal carcinoma cell lines . The GBP was related in sequence to a family of fatty acid oxidation enzymes possessing enoyl CoA hydratase and 3-hydroxyacyl CoA dehydrogenase activity . This study aims to define the binding site for gastrin and gastrin antagonists in greater detail . The N- and C-terminal halves of the porcine GBP were expressed independently as glutathione S-transferase fusion proteins in E . coli . Affinities of gastrin and gastrin antagonists for the fusion proteins were measured by competition for 125I-{Nle15}-gastrin binding in a covalent cross-linking assay . The N- and C-terminal fusion proteins bound gastrin with affinities of 9.9 +/- 6.1 and 71 +/- 48 microM, respectively (n = 3) . These values were 40-fold and 300-fold lower than the affinity of the full-length GBP for gastrin (0.23 +/- 0.15 microM) . In contrast, the affinities of the N- and C-terminal halves for the antagonists proglumide (22 +/- 13 and 10 +/- 4 mM, respectively) and benzotript (350 +/- 90 and 400 +/- 160 micro M, respectively) were similar to each other and to the affinities of proglumide and benzotript for the full-length GBP (5.1 +/- 3.6 mM and 200 +/- 120 microM, respectively) . It is concluded that proglumide and benzotript bind independently to both the hydratase and dehydrogenase active sites of the GBP, while a single molecule of gastrin may bind simultaneously to both active sites . A model is proposed which is consistent with these data, and which will assist in the development of more potent and selective GBP antagonists.

Gac Med Mex, 1996 Nov-Dec, 132(6), 611 - 5
{Infections caused by enteropathogenic Escherichia coli}; Cravioto A et al.; Escherichia coli strains currently recognized to cause human diarrhea can be distinguished on the basis of pathogenic mechanism and separated into five categories: enterotoxigenic (ETEC), enteroinvasive (EIEC), enterohemorrhagic (EHEC), enteroaggregative (EAggEC) and enteropathogenic (EPEC) . EPEC is a bacterial pathogen that causes diarrhea in infants, particularly in developing countries . The purpose of this review is to summarize recent advances in the understanding of EPEC pathogenesis and the contribution of Mexican investigators to the knowledge of this pathogen.

Br J Surg, 1996 Nov, 83(11), 1569 - 73
Fast-to-slow muscle conversion by chronic electrostimulation: effects on mitochondrial respiratory chain function with possible implications for the gracilis neosphincter procedure; Altomare DF et al.; The effects of chronic, around the clock, low-frequency electrostimulation on the respiratory chain activity and cytochrome content of freshly isolated mitochondria were evaluated in rabbit skeletal muscle before and after 30 days of continuous or cyclical electrostimulation using a totally implantable system and a training programme now used in humans . The respiratory activity measured in state III increased strongly after electrostimulation . The efficiency of the respiratory chain increased significantly after electrostimulation but the activity of complex {(reduced nicotinamide adenine dinucleotide dehydrogenase) did not increase . The amount of cytochromes a and a3, b562, and c and c1 increased clearly after electrostimulation . The respiratory activity rate of mitochondria obtained after continuous electrostimulation was apparently higher than after cyclical electrostimulation . Chronic uninterrupted low-frequency electrostimulation, using a clinical training programme, induces an increase in mitochondrial respiratory chain activity in purified mitochondria of skeletal muscle . These changes are the basis of induced resistance to fatigue in fast-to-slow muscle conversion by chronic electrostimulation.

Vaccine, 1996 Nov, 14(16), 1560 - 8
Immunogenicity of a fusion protein linking the beta subunit carboxyl terminal peptide (CTP) of human chorionic gonadotropin to the B subunit of Escherichia coli heat-labile enterotoxin (LTB); Rock EP et al.; Human chorionic gonadotropin (hCG) is currently under investigation as an antigenic target in both anti-cancer and anti-fertility vaccines . Formulations studied to date show promise in clinical trials for both applications yet are expensive to produce and require frequented administration in order to maintain an effective antibody titer . We have engineered a fusion protein consisting of Escherichia coli heat-labile enterotoxin subunit B (LTB) genetically linked at its C terminus via a nine amino acid linker to the 37 amino acid carboxyl terminal peptide (CTP) of the hCG beta chain . This LTB-CTP fusion protein is stably expressed in bacteria and forms pentamers of full-length protein subunits . Purified LTB-CTP protein hCG-specific antibodies in mice without additional adjuvants.

Int J Dev Neurosci, 1996 Nov, 14(7-8), 853 - 66
Early olfactory fiber projections and cell migration into the rat telencephalon; De Carlos JA et al.; The formation and development of primary olfactory axons was studied in the rat embryo using acetylcholinesterase histochemistry, immunocytochemistry for neuron-specific beta-tubulin (TuJ1) and growth associated protein 43 (GAP43), and a fluorescent tracer DiI . Olfactory axons extend from the olfactory receptor neurons localized in the olfactory epithelium . These fibers grow to reach and enter the olfactory bulbs, where they form the first relay and integrative synaptic station in the olfactory system: the olfactory glomerulus . In this communication we address the development of primary olfactory fibers: first from the olfactory placode and later from the olfactory epithelium . Olfactory fibers enter the olfactory bulbs apparently in a disordered manner but soon arrange themselves in hook shaped aggregates of fibers, with many boutons (immature synaptic terminals), to form the glomeruli . We detected this kind of structure for the first time at embryonic day 16 . The olfactory receptor cells are usually anchored in the basal lamina of the olfactory epithelium but some of them, after reaching their targets, lose their epithelial attachment, leave the olfactory epithelium and migrate to and enter the olfactory bulbs . The traffic of cells between the olfactory epithelium and the brain lasts late into embryonic development . We describe four types of migratory mechanism used by different populations of cells to reach their targets in the telencephalic vesicle and propose the existence of migrating cells that enter the telencephalon . These data were corroborated by injections into the olfactory epithelium a of murine retrovirus carrying the Escherichia coli lac-Z gene.

J Steroid Biochem Mol Biol, 1996 Nov, 59(3-4), 243 - 50
Induction of cell-free, in vitro transcription by recombinant androgen receptor peptides; Snoek R et al.; An in vitro, cell-free transcription system, based on prostate-derived transcriptional machinery and very powerful androgen response elements (AREs), has been developed . Multiple (p(ARR3)LovTATA) AREs from the androgen-regulated probasin gene were linked to G-free cassettes and used in nuclear extracts prepared from prostate carcinoma cell lines (PC3 and LNCaP cells) to test specific induction of transcription by full-length AR and by glutathione-S-transferase (GST)-fusion peptides in which the androgen receptor (AR) DNA-binding domain alone (AR524-649), or together with the ligand-binding domain (AR524-902), or a portion of the NH2-terminal domain (AR232-649) were incorporated . In the presence of AR, nuclear extracts from PC3 cells had greater activity in supporting transcription than those from LNCaP cells; and lower background activity than those from HeLa cells . All of the AR forms correctly initiated in vitro transcription of ARE-templates in an androgen-independent manner . The amount of specific, inducible transcript was dependent on the concentration of AR peptide present . AR524-902 was the most potent transactivator tested, with the maximal level of specific transcript over 900-fold higher than the minimal level . At all concentrations this peptide was three to four times more active than either AR524-649 or AR232-649 . In conclusion, we have developed a very specific and sensitive cell-free transcription system for delineating trans-activational regions of the AR.

J Protein Chem, 1996 Nov, 15(8), 731 - 6
Stability and folding of a mutant ribose-binding protein of Escherichia coli; Kim JS et al.; A mature mutant ribose-binding protein (RBP) of Escherichia coli was obtained by site-directed mutagenesis, replacing Thr-3 in the N-domain of wild-type mature RBP (WT-mRBP) with a Trp residue (N-Trp-mRBP) . The equilibrium unfolding properties and the refolding kinetics of this protein were monitored by fluorescence and circular dichroism (CD) . The stability of N-Trp-mRBP appears to be the same as that of C-Trp-mRBP, another mutant obtained by replacing Phe-187 with a Trp, and lower than that of WT-mRBP . The overall refolding rate of N-Trp-mRBP is much smaller than that of C-Trp-mRBP, which, in turn, is similar to that of WT-mRBP . For the case of WT-mRBP, the rate constant obtained by Tyr fluorescence is identical to the value obtained by CD . But with C-Trp-mRBP, the rate constant from CD is smaller than the value from the Trp fluorescence and this difference in the rate constants is much greater with the N-Trp-mRBP.

Biokhimiia, 1996 Nov, 61(11), 1928 - 30
{Topography of ribosomal proteins: reconsideration of of protein map of small ribosomal subunit}; Spirin AS et al.; Exposure of proteins on the surface of the small (30S) ribosomal subunit of Escherichia coli was studied by the hot tritium bombardment technique . Eight of 21 proteins of the 30 S subunit (S3, S8, S10, S12, S15, S16, S17, and S19) had virtually no groups exposed on the surface of the particle, i.e., they were mainly hidden inside . Seven proteins (S1, S4, S5, S7, S18, S20, and S21) were all well exposed on the surface of the particle, thus being outside proteins . The remaining proteins (S2, S6, S9 and/or S11, S13, and S14) were partially exposed . On the basis of these results a reconcilement of the three-dimensional protein map of the small ribosomal subunit has been done and corrected model is proposed.

Berl Munch Tierarztl Wochenschr, 1996 Nov-Dec, 109(11-12), 431 - 3
{Utilization of nylon membranes for specific isolation and characterization of verotoxin-producing Escherichia coli using DNA probes}; Gallien P et al.; A method for specific isolation of VT(+)-strains in raw milk is given . DNA-hybridization technique with DIG-labeled PCR-amplificates as probes are the basis . No background is seen by using "DIG Easy Hyb" solution and nylon membranes for colony- and plaque-hybridization (Boehringer Mannheim GmbH) . Marked colonies are visible on the membranes after detection . So it is possible to select these colonies from a masterplate . The results are available within one day (without enrichment and membrane preparation) . After stripping the membranes can be used for a new hybridisation to detect another factor of virulence.

Biosci Biotechnol Biochem, 1996 Nov, 60(11), 1899 - 901
Intracellular fate of 2-NBDG, a fluorescent probe for glucose uptake activity, in Escherichia coli cells; Yoshioka K et al.; A fluorescent derivative of D-glucose, 2-NBDG, which was previously developed for the evaluation of glucose uptake activity by living cells, was used on Escherichia coli cells and its fate after incorporation in the cells was investigated . 2-NBDG was converted to another fluorescent derivative (2-NBDG metabolite) immediately after it was taken by E . coli cells . This 2-NBDG metabolite was then decomposed to non-fluorescent forms . 2-NBDG metabolite was decomposed into the original 2-NBDG by G6Pase with concurrent liberation of inorganic phosphate . Furthermore, FAB/MS analysis showed that its molecular weight was 420, the same value as that of 2-NBDG 6-phosphate . These indicate 2-NBDG metabolite should be 2-NBDG 6-phosphate . Based on these results, the feasibility of 2-NBDG as a fluorescent non-toxic probe for glucose uptake activity and its application to viability assessment of various living systems are discussed.

Biosci Biotechnol Biochem, 1996 Nov, 60(11), 1882 - 5
Gene cloning and expression of new trehalose-producing enzymes from the hyperthermophilic archaeum Sulfolobus solfataricus KM1; Kobayashi K et al.; The genes encoding for trehalose-producing enzymes, a glycosyl-trehalose-producing enzyme (glycosyltransferase) and a gylcosyl-trehalose-hydrolyzing enzyme (alpha-amylase), from Sulfolobus solfataricus KM1 were cloned and expressed in E . coli . The nucleotide sequence of the glycosyltransferase gene and the alpha-amylase gene indicated proteins with lengths of 728 and 558 amino acids and molecular masses of 86-kDa and 65-kDa, respectively . Regions highly conserved in the alpha-amylase family exist in the amino acid sequences of these enzymes.

Biosci Biotechnol Biochem, 1996 Nov, 60(11), 1790 - 4
Cloning and sequence analysis of a cDNA encoding salmon (Onchorhynchus keta) liver transglutaminase; Sano K et al.; We isolated cDNA clones encoding a transglutaminase (TGase: EC 2.3.2.13) from a salmon (Onchorhynchus keta) cDNA library prepared from the liver . In the cDNA sequence combined, an open reading frame coding for a protein of 680 aa was found . The deduced sequence showed a considerable similarity (62.4%) to that of red sea bream TGase . By comparison of sequence similarity to other TGases, the structure of salmon TGase was like tissue type TGases, rather than membrane-associated type or plasma type TGases . As a structural feature of salmon TGase, 3 aa residues were substituted in the 25 aa sequence around the active site Cys residue, which is conserved among several tissue type TGases . The critical residues thought to form the catalytic-center triad (Cys272, His331, and Asp301) were found in the highly conserved region, but the region surrounding Tyr511, which corresponds to the residue participates in hydrogen-bond interactions of active center domain, was less similar to other TGases, except for red sea bream TGase . These finding suggests that the overall structure of fish TGase resembles tissue-type TGases, but has some unique structure.

Biosci Biotechnol Biochem, 1996 Nov, 60(11), 1776 - 9
Sequence-specific binding sites in the Taka-amylase A G2 promoter for the CreA repressor mediating carbon catabolite repression; Kato M et al.; The N-terminal part of the CreA protein encompassing two zinc fingers was expressed in Escherichia coli as a fusion protein with the maltose binding protein (MalE) of E . coli . Our results show that CreA binds to the promoter of the Taa-G2 gene encoding Taka-amylase A of Aspergillus oryzae . DNase I footprinting experiments showed that CreA bound to three sites with high affinity and to one site with low affinity within the first 401-bp region upstream of the transcription initiation site . All of the sites contained sequences related to the CreA consensus binding site (5'-SYGGRG-3'), and are suggested to participate in repression of the Taa-G2 gene in response to glucose.

J Biochem (Tokyo), 1996 Nov, 120(5), 974 - 81
Characterization of the S1 subsite specificity of aspergillopepsin I by site-directed mutagenesis; Shintani T et al.; The structural determinants of S1 substrate specificity of aspergillopepsin I (API; EC 3.4.23.18), an aspartic proteinase from Aspergillus saitoi, were investigated by site-directed mutagenesis . Aspartic proteinases generally favor hydrophobic amino acids at P1 and P1' . However, API accommodates a Lys residue at P1, which leads to activation of trypsinogen . On the basis of amino acid sequence alignments of aspartic proteinases, Asp-76 and Ser-78 of API are conserved only in fungal enzymes with the ability to activate trypsinogen, and are located in the active-site flap . Site-directed mutants (D76N, D76E, D76S, D76T, S78A, and delta S78) were constructed, overexpressed in Escherichia coli cells and purified for comparative studies using natural and synthetic substrates . Substitution of Asp-76 to Ser or Thr and deletion of Ser-78, corresponding to the mammalian aspartic proteinases, caused drastic decreases in the activities towards substrates containing a basic amino acid residue at P1 . In contrast, substrates with a hydrophobic residue at P1 were effectively hydrolyzed by each mutant enzyme . These results demonstrate that Asp-76 and Ser-78 residues on the active site flap play important roles in the recognition of a basic amino acid residue at the P1 position.

J Biochem (Tokyo), 1996 Nov, 120(5), 969 - 73
Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding the Candida utilis urate oxidase (uricase); Koyama Y et al.; A urate oxidase (uricase) gene was cloned from Candida utilis with an oligonucleotide probe based on the amino acid sequence of cyanogen bromide-cleaved uricase . The uricase gene contains 909 base pairs and encodes a protein with a predicted mass of 34,193 Da . Candida uricase was similar (49% match in amino acid sequence) to the uricase from Aspergillus flavus . The uricase from Candida utilis has four cysteines and one of them, Cys168, participates in the enzyme activity . This enzyme was expressed to a level of about 20% of total cellular protein in an Escherichia coli cell as a soluble and functional form.

J Biochem (Tokyo), 1996 Nov, 120(5), 946 - 53
Acid and thermal unfolding of Escherichia coli dihydrofolate reductase; Ohmae E et al.; The acid and thermal unfolding of Escherichia coli dihydrofolate reductase (DHFR) were studied by means of circular dichroism (CD) and fluorescence spectroscopy . There existed at least one intermediate around pH 4 in the acid unfolding process at 15 degrees C, in which the tertiary structure was disrupted before unfolding of the secondary structure . The fluorescence energy transfer from intrinsic tryptophan residues to 1-anilinonaphthalene-8-sulfonate suggested the disruption of the tertiary structure around some tryptophan residues of the intermediate . The thermal unfolding process at pH 7.0 also involved at least one intermediate having a disrupted tertiary structure and a folded secondary structure . The three-state thermodynamic analysis showed that the intermediate in thermal unfolding was less stable by 1.8 kcal/mol than the native state . The similarity of the far-ultraviolet CD spectra of acid and thermally unfolded forms suggests that both types of unfolding produce the same structure, which may be a molten globule intermediate such as that in the folding kinetics of DHFR . The acid and thermal unfolding were depressed in the presence of KCl due to stabilization of the native form.

Electrophoresis, 1996 Nov, 17(11), 1787 - 96
Expression and divalent cation binding properties of the novel chemotactic inflammatory protein psoriasin; Vorum H et al.; Psoriasin is a novel chemotactic inflammatory protein that possesses weak similarity to the S100 family members of Ca(2+)-binding proteins, and that is highly up-regulated in hyperproliferative psoriatic keratinocytes . Here we have used the psoriasin cDNA to express recombinant human (rh) psoriasin in Escherichia coli as a fusion protein containing a hexa His tag and a factor Xa cleavage site in the NH2-terminus . The protein was purified by affinity chromatography on Ni(2+)-nitrilotriacetic acid agarose, digested with factor Xa, further purified by ion-exchange chromatography and characterized by two-dimensional (2-D) gel electrophoresis and NH2-terminal sequencing . The ability of rh psoriasin to bind Ca2+, Zn2+, and Mg2+ was determined by dialysis experiments . We found that rh psoriasin may bind at least seven molecules of Ca2+ in KCl and several molecules in NaCl, with an affinity for the first bound molecule of 1.3-1.6 x 10(4) M-1 . This indicates that psoriasin may cooperatively bind several molecules of Ca2+ when present in the extracellular space, or putatively, if localized in subcellular compartments where the concentration of Ca2+ is relatively high . At least eight molecules of Zn2+ were bound in KCl and four in NaCl, with an affinity just below 1 x 10(4) M-1 for the first molecule . Thus psoriasin does not bind significant amounts of Zn2+ at physiological concentrations . Mg2+ and Ca2+ are bound anti-cooperatively and binding of each of the ions (Ca2+, Zn2+, or Mg2+), is accompanied by conformational changes that move tyrosine residues to more hydrophobic areas.

Plant Mol Biol, 1996 Nov, 32(4), 751 - 7
Isolation of cDNAs encoding two purine biosynthetic enzymes of soybean and expression of the corresponding transcripts in roots and root nodules; Schnorr KM et al.; Soybean nodule cDNA clones encoding glycinamide ribonucleotide (GAR) synthetase (GMpurD) and GAR transformylase (GMpurN) were isolated by complementation of corresponding Escherichia coli mutants . GAR synthetase and GAR transformylase catalyse the second and the third steps in the de novo purine biosynthesis pathway, respectively . One class of GAR synthetase and three classes of GAR transformylase cDNA clones were identified . Northern blot analysis clearly shows that these purine biosynthetic genes are highly expressed in young and mature nodules but weakly expressed in roots and leaves . Expression levels of GMpurD and GMpurN mRNAs were not enhanced when ammonia was provided to non-nodulated roots.

Plant Mol Biol, 1996 Nov, 32(4), 727 - 34
Lysine and threonine metabolism are subject to complex patterns of regulation in Arabidopsis; Ben-Tzvi Tzchori I et al.; To study the regulation of lysine and threonine metabolism in plants, we have transformed Arabidopsis thaliana with chimeric genes encoding the two bacterial enzymes dihydrodipicolinate synthase (DHPS) and aspartate kinase (AK) . These bacterial enzymes are much less sensitive to feedback inhibition by lysine and threonine than their plant counterparts . Transgenic plants expressing the bacterial DHPS overproduced lysine, but lysine levels were quite variable within and between transgenic genotypes and there was no direct correlation between the levels of free lysine and the activity of DHPS . The most lysine-overproducing plants also exhibited abnormal phenotypes . However, these phenotypes were detected only at early stages of plant growth, while at later stages, new buds emerged that looked completely normal and set seeds . Wild-type plants exhibited relatively high levels of free threonine, suggesting that in Arabidopsis AK regulation may be more relaxed than in other plants . This was also supported by the fact that expression of the bacterial AK did not cause any dramatic elevation in this amino acid . Yet, the relaxed regulation of threonine synthesis in Arabidopsis was not simply due to a reduced sensitivity of the endogenous AK to feedback inhibition by lysine and threonine because growth of wild-type plants, but not of transgenic plants expressing the bacterial AK, was arrested in media containing these two amino acids . The present results, combined with previous studies from our laboratory, suggest that the regulation of lysine and threonine metabolism is highly variable among plant species and is subject to complex biochemical, physiological and environmental controls . The suitability of these transgenic Arabidopsis plants for molecular and genetic dissection of lysine and threonine metabolism is also discussed.

Plant Mol Biol, 1996 Nov, 32(4), 653 - 62
A novel plasma membrane-bound thioredoxin from soybean; Shi J et al.; Two thioredoxin cDNAs from soybean were isolated by screening an expression library using an anti-(plasma membrane) serum . The nucleotide sequences of the two cDNAs were found to be 89% identical . The polypeptides encoded by the two cDNAs, designated TRX1 and TRX2, contain a disulfide active site, as found in other thioredoxins . TRX1 was expressed as a fusion protein in Escherichia coli and shown to possess thiol-disufide interchange activity . Unlike other eukaryotic thioredoxins, these two soybean thioredoxins contain a putative transmembrane domain in their N-terminal regions . To determine subcellular location, the TRX1 was fused with a reporter epitope at its C-terminus and expressed in transgenic tobacco plants . The fusion protein was co-purified with plasma membrane markers 1,3 beta-glucan synthase and vanadate-sensitive ATPase, indicating the plasma membrane location of TRX1 . When the reporter epitope was inserted between the start codon and the transmembrane domain in the N-terminus, the fusion protein was found in the soluble fraction, possibly due to disruption of the transmembrane domain by the highly hydrophilic epitope sequence . Taken together, our results demonstrate that soybean TRX1 is a plasma membrane-bound thioredoxin, which is most likely anchored to the membrane through the N-terminal transmembrane domain . It is known that plant plasma membranes contain various proteins with thiol-disulfide interchange activity . The soybean thioredoxins reported here are the first group of such proteins to be characterized at the molecular level . However, the biological function of the plasma membrane-bound thioredoxin remains to be determined.

Plant Mol Biol, 1996 Nov, 32(3), 505 - 13
Functional studies of chloroplast glyceraldehyde-3-phosphate dehydrogenase subunits A and B expressed in Escherichia coli: formation of highly active A4 and B4 homotetramers and evidence that aggregation of the B4 complex is mediated by the B subunit carboxy terminus; Baalmann E et al.; Chloroplast glyceraldehyde-3-phosphate dehydrogenase (phosphorylating, E.C . 1.2.1.13) (GAPDH) of higher plants exists as an A2B2 heterotetramer that catalyses the reductive step of the Calvin cycle . In dark chloroplasts the enzyme exhibits a molecular mass of 600 kDa, whereas in illuminated chloroplasts the molecular mass is altered in favor of the more active 150 kDa form . We have expressed in Escherichia coli proteins corresponding to the mature A and B subunits of spinach chloroplast GAPDH (GapA and GapB, respectively) in addition to a derivative of the B subunit lacking the GapB-specific C-terminal extension (CTE) . One mg of each of the three proteins so expressed was purified to electrophoretic homogeneity with conventional methods . Spinach GapA purified from E . coli is shown to be a highly active homotetramer (50-70 U/mg) which does not associate under aggregating conditions in vitro to high-molecular-mass (HMM) forms of ca . 600 kDa . Since B4 forms of the enzyme have not been described from any source, we were surprised to find that spinach GapB purified from E . coli was active (15-35 U/mg) . Spinach GapB lacking the CTE purified from E . coli is more highly active (130 U/mg) than GapB with the CTE . Under aggregating conditions, GapB lacking the CTE is a tetramer that does not associate to HMM forms whereas GapB with the CTE occurs exclusively as an aggregated HMM form . The data indicate that intertetramer association of chloroplast GAPDH in vitro occurs through GapB-mediated protein-protein interaction.

Plant Mol Biol, 1996 Nov, 32(3), 493 - 504
A novel plant peptidyl-prolyl-cis-trans-isomerase (PPIase): cDNA cloning, structural analysis, enzymatic activity and expression; Blecher O et al.; A novel cDNA encoding for a peptidyl-prolyl-cis-trans-isomerase (PPIase) belonging to the FK506-binding protein (FKBP) family was isolated from wheat . It contains an open reading frame of 559 amino acids and it represents the first plant FKBP-PPIase to be cloned . It possesses a unique sequence which is composed of three FKPB-like domains, in addition to a putative tetratricopeptide repeat (TPR) motif and a calmodulin-binding site . The recombinant FKBP-PPIase expressed in and purified from Escherichia coli exhibits PPIase activity that is efficiently inhibited by the immunosuppressive drugs FK506 and rapamycin . Northern blot analysis showed that wheat FKBP was found mainly in young tissues . Polyclonal antibodies revealed the presence of cross-reacting proteins in embryos, roots and shoots . The unique structural features, the enzymatic activity and the presence of putative isoforms in wheat tissues indicate the possibility of the involvement of wheat PPIase in essential biological functions, similar to other members of the FKBP gene family.

Plant Mol Biol, 1996 Nov, 32(3), 447 - 52
Cloning and expression of transaldolase from potato; Moehs CP et al.; We have isolated a cDNA encoding transaldolase, an enzyme of the pentose-phosphate pathway, from potato (Solanum tuberosum) . The 1.5 kb cDNA encodes a protein of 438 amino acid residues with a molecular mass of 47.8 kDa . When the potato cDNA was expressed in Escherichia coli a 45 kDa protein with transaldolase activity was produced . The first 62 amino acids of the deduced amino acid sequence represent an apparent plastid transit sequence . While the potato transaldolase has considerable similarity to the enzyme from cyanobacteria and Mycobacterium leprae, similarity to the conserved transaldolase enzymes from humans, E . coli and Saccharomyces cerevisiae is more limited . Northern analysis indicated that the transaldolase mRNA accumulated in tubers in response to wounding . Probing the RNA from various potato tissues indicated that the transaldolase mRNA accumulation to higher levels in the stem of mature potato plants than in either leaves or tubers . These data are consistent with a role for this enzyme in lignin biosynthesis.

Br Vet J, 1996 Nov, 152(6), 699 - 708
Evaluation of a nutritive oral rehydration solution for the treatment of calf diarrhoea; Brooks HW et al.; The essential constituents of a conventional oral rehydration solution (ORS) are sodium, glucose and a bicarbonate precursor . The glucose promotes sodium uptake but because these solutions are isotonic, it is insufficient to sustain calorie requirements . This paper examines the performance of a novel ORS with over three times the conventional glucose concentration, by comparing it with two leading commercial ORSs in calves with induced Escherichia coli diarrhoea . This solution showed greater ability than the current market-leading ORS to repair extracellular fluid and plasma volume and to correct both hyponatraemia and metabolic acidosis, especially in more severely affected calves . In acidotic calves it was more effective in correcting hyperkalaemia, probably by supplying glucose to promote cellular potassium uptake as well as by correcting the acidosis . It therefore appears possible to depart from the traditional isotonic formulations for calf ORSs and gain significant nutritional support while retaining effective rehydration and correction of acid-base and electrolyte disturbances . This seems especially important in young animals where energy deprivation imposes a particular penalty; the use of hypertonic ORSs should not, however, be extended to other species without further research.

Endocr Res, 1996 Nov, 22(4), 665 - 71
The use of electrochemistry for the synthesis of 17 alpha-hydroxyprogesterone by a fusion protein containing P450c17; Estabrook RW et al.; A method has been developed for the commercial application of the unique oxygen chemistry catalyzed by various cytochrome P450s . This is illustrated here for the synthesis of hydroxylated steroids . This method requires the preparation of large amounts of enzymatically functional P450 proteins that can serve as catalysts and a technique for providing electrons at an economically acceptable cost . To generate large amounts of enzymatically active recombinant P450s we have engineered the cDNAs for various P450s, including bovine adrenal P450c17, by linking them to a modified cDNA for rat NADPH-P450 reductase and placing them in the plasmid pCWori+ . Transformation of E . coli results in the high level expression of an enzymatically active protein that can be easily purified by affinity chromatography . Incubation of the purified enzyme with steroid in a reaction vessel containing a platinum electrode and a Ag/AgCl electrode couple poised at -650 mV, together with the electromotively active redox mediator, cobalt sepulchrate, results in the 17 alpha-hydroxylation of progesterone at rates as high as 25 nmoles of progesterone hydroxylated/min/nmole of P450 . Thus, high concentrations of hydroxylated steroids can be produced with incubation conditions of hours duration without the use of costly NADPH . Similar experiments have been carried out for the generation of the 6 beta-hydroxylation product of testosterone (using a fusion protein containing human P450 3A4) . It is apparent that this method is applicable to many other P450 catalyzed reactions for the synthesis of large amounts of hydroxylated steroid metabolites . The electrochemical system is also applicable to drug discovery studies for the characterization of drug metabolites.

Endocr Res, 1996 Nov, 22(4), 631 - 9
Adrenocortical-specific transgene expression directed by steroid hydroxylase gene promoters; Morley SD et al.; The 5'-flanking regions of genes for three mouse adrenal steroid hydroxylases were analyzed for their ability to direct adrenal cortex-specific beta-galactosidase (beta-gal) reporter expression both in cell culture and transgenic mice . The 5'-flanking regions chosen were from the genes for steroid 21-hydroxylase (21-OHase), expressed throughout the adrenal cortex and mediating both glucocorticoid and mineralocorticoid synthesis, and aldosterone synthetase (AS) and steroid 11 beta-hydroxylase (11 beta-OHase), which catalyze respectively the terminal steps of mineralocorticoid synthesis in the zona glomerulosa and glucocorticoid synthesis in the zona fasciculata/reticularis . While 5.0 kb of 11 beta-OHase gene 5'-flanking region and 5.4 kb of the AS gene 5'-flanking region mediated respectively moderate and low levels of beta-gal reporter expression in Y1 adrenocortical tumor cells, neither of these 5'-flanking regions was able to direct reporter expression to the appropriate adrenocortical zone of transgenic mice . This suggests that additional regulatory elements, lying outside these 5'-flanking regions, are required for 11 beta-OHase and AS gene expression in the intact mouse . In contrast, 6.4 kb of the mouse 21-OHase A gene 5' flanking region was able to direct specific beta-galactosidase reporter expression, in both Y1 cells and transgenic mice, indicating that elements directing adrenal cortex-specific gene expression in vivo are located not more than 6.4 kb 5' of the 21-OHase gene transcription start site.

Microbiology, 1996 Nov, 142 ( Pt 11), 3231 - 9
Kinetic analysis and substrate specificity of Escherichia coli dimethyl sulfoxide reductase; Simala-Grant JL et al.; We have characterized the substrate specificity of dimethyl sulfoxide reductase (DmsABC) of Escherichia coli by determining Km and Kcat values for 22 different substrates . The enzyme has a very broad substrate specificity . The Km values varied 470-fold, while Kcat values varied only 20-fold, implicating Km as the major determinant of Kcat/Km values . Sulfoxides and pyridine N-oxide exhibited the lowest Km values, followed by aliphatic N-oxides . The Kcat values for these compounds also followed the same pattern . Substitution at the 2 or 3 position of the pyridine N-oxide ring had little effect on Km while substitution at the 4 position had a greater effect, and increased Km . Negatively charged substrates were poorly accepted . A few compounds that are not S- or N-oxides were also reduced by the enzyme . Most compounds reduced by DmsABC were not toxic to E . coli under anaerobic growth conditions, and E . coli was able to use many of these compounds anaerobically as terminal electron acceptors in the presence of glycerol . Anaerobic growth on sulfoxides is solely due to DmsABC expression . However, there appears to be another as yet unidentified terminal reductase capable of using pyridine N-oxides as terminal electron acceptors.

Microbiology, 1996 Nov, 142 ( Pt 11), 3219 - 30
The purB gene of Escherichia coli K-12 is located in an operon; Green SM et al.; The structural gene (purB) for succinyl-AMP (S-AMP) lyase and three additional ORFs are on the same DNA strand of the chromosome of Escherichia coli . Cassette mutagenesis and primer extension mapping demonstrated that purB is co-transcribed with an upstream gene (ORF23, or ycfC) encoding a 22.9 kDa membrane-associated protein of non-essential, but unknown, function unrelated to purine biosynthesis . The purB operon lies between phoP and an ORF expressing an essential function which may correspond to asuE (trmU) . S-AMP lyase was purified to near homogeneity . The purified enzyme is a homotetramer of 50 kDa subunits, has a K(m) for S-AMP of 3.7 microM and a pH optimum of 7.4-7.6.

Microbiology, 1996 Nov, 142 ( Pt 11), 3211 - 8
hymA (hypha-like metulae), a new developmental mutant of Aspergillus nidulans; Karos M et al.; Asexual fruiting body development in Aspergillus nidulans requires a precise spatial and temporal coordinated expression of many genes . Insertional mutagenesis was used to isolate and characterize a new mutant of A . nidulans in which hyphal growth was slightly reduced and conidiophore development was specifically blocked at the metula stage . In contrast to the uninucleate metulae of the wild-type, in the mutant these structures were elongated, multinucleate and septate . Further differentiation and production of phialides by a budding-like process was not observed . The mutant metulae thus resembled hyphae rather than metulae and the gene was therefore named hypha-like metulae (hymA) . The hymA gene was mapped to linkage group VI . The integrated vector was rescued with border sequences from the integration site . The border sequences were used to isolate a cosmid from a wild-type library which was subcloned to a 5 kb fragment able to complement the mutation in trans . This fragment encoded a 1.8 kb transcript expressed in hyphae and throughout development . It is proposed that hymA is involved in budding processes, and is required for the formation of metulae and for their further differentiation.

Carcinogenesis, 1996 Nov, 17(11), 2347 - 56
Species-specific differences in hepatic mutant frequency and mutational spectrum among lambda/lacI transgenic rats and mice following exposure to aflatoxin B1; Dycaico MJ et al.; In vivo mutations were studied in lambda/lacI (Big Blue) transgenic C57BL/6 mice and F344 rats following exposure to either AFB1 (aflatoxin B1) or DMSO vehicle . Fourteen days after exposure, livers were removed for DNA extraction and subsequent mutational analysis of the lacI gene . Mice injected with a single i.p . dose of AFB1 at 2.5 mg/kg did not show a significant increase in liver mutant frequency relative to vehicle-treated controls . DNA sequence analysis of lacI mutations collected from the AFB1-treated mice showed a pattern of mutation similar to that of the previously observed spontaneous mouse liver mutational spectrum . In contrast, rats subjected to one-tenth the mouse AFB1 dosage responded with an approximate 20-fold induction in liver mutant frequency over background . Sequencing of lacI mutations also revealed spectral differences between vehicle- and AFB1-treated rats . A large increase in G:C-->T:A transversions was observed among lacI mutations isolated from the AFB1-treated rats . This work is among the first multi-species in vivo mutagenicity studies using transgenic rodents harboring the same shuttle vector . Such multi-species in vivo assays may prove to be valuable in the areas of mechanistic analysis and risk assessment.

Protein Eng, 1996 Nov, 9(11), 1029 - 42
Engineering a de novo-designed coiled-coil heterodimerization domain off the rapid detection, purification and characterization of recombinantly expressed peptides and proteins; Tripet B et al.; Using the techniques of genetic engineering and the principles of protein de novo design, we have developed a unique affinity matrix protein tag system as a rapid, convenient and sensitive method to detect, purify and characterize newly expressed recombinant peptides or proteins from cell extracts . The method utilizes two de novo-designed linear peptide sequences that can selectively dimerize to form the stable protein motif, the two-stranded alpha-helical coiled-coil . In this method, a recombinant bacterial expression vector pRLDE has been engineered so that one of the dimerization strands (E-coil) is expressed as a C-terminal fusion tag on newly expressed peptides or proteins, while the other (K-coil) is either biotin-labeled for detection in a Western blot-type format or immobilized on an insoluble silica support for selective dimerization affinity chromatography . Recombinantly expressed peptides from Escherichia coli containing the dimerization tag have been produced, detected and purified using this method . The recombinant peptides were easily and clearly identified using the biotin-labeled coil, while the single-step affinity purification results indicated the purity of the affinity purified expressed peptides to be > 95%, as assessed by reversed-phase chromatography . The stability of the dimerization domain also allows for the purified peptide to be left attached to the matrix, thus creating a new peptide-bound column that can be used to study peptide-protein or peptide-ligand interactions . Therefore this system offers a new alternative to existing peptide or protein fusion tags and demonstrates the utility of a de novo-designed system.

Eur J Clin Chem Clin Biochem, 1996 Nov, 34(11), 873 - 88
Analysis of eukaryotic DNA topoisomerases and topoisomerase-directed drug effects; Boege F; DNA topoisomerases are enzymes which control DNA topology by cleaving and rejoining DNA strands and passing other DNA strands through the transient gaps . Consequently these enzymes play a crucial role in the regulation of the physiological function of the genome . Beyond their normal functions, topoisomerases are important cellular targets in the treatment of human cancers . Some of the most powerful anti-cancer drugs used clinically stabilize the catalytic topoisomerase-DNA intermediates and, thus, cause DNA disorders that will induce apoptosis in proliferating cells . This review summarizes current protocols for measuring the catalytic activity of topoisomerases and for monitoring the molecular effects of topoisomerase-directed antitumour drugs in living cells and in cell-free assays . Furthermore, preanalytical factors are discussed, such as enzyme stability, methods for extracting DNA topoisomerases from cells, and protocols for separating subtypes and isoforms of these enzymes.

Biol Chem, 1996 Nov, 377(11), 731 - 9
The antitumor agent cisplatin inhibits DNA gyrase and preferentially induces gyrB gene expression in Escherichia coli; Neumann S et al.; Cisplatin is a widely used anticancer agent that exerts its biological activity principally by damaging DNA . Although detailed knowledge exists concerning mechanisms that lead to cisplatin adducts in DNA, there are few insights into the processes that result in its antitumor action . To explore some of the cellular responses elicited by cisplatin treatment, we studied its influence on DNA supercoiling and DNA gyrase gene expression in E . coli . We found that cisplatin inhibits DNA gyrase in a concentration-dependent manner leading to a transient alteration of DNA supercoiling and to an induction of gyrase gene expression . The induction effect was asymmetrical, affecting gyrB stronger than gyrA . Furthermore, we studied the influence of cisplatin on the supercoiling activity of purified DNA gyrase in vitro and found that cisplatin was an efficient inhibitor of DNA gyrase in the standard assay . However, cisplatin was an excellent inhibitor when added to DNA gyrase before it could interact with its substrate . In this assay GyrB was also more affected by cisplatin than GyrA . This strongly suggests that cisplatin inhibits DNA gyrase primarily by direct interaction with the enzyme . The data from this work present evidence that further cellular responses following cisplatin treatment include DNA gyrase inhibition, altered DNA supercolling and enhanced DNA gyrase gene expression . This suggests an important role of DNA topology in the induction of defense mechanisms against the action of cisplatin in addition to the processes related to DNA damage and repair.

Oncology, 1996 Nov-Dec, 53(6), 488 - 93
A clinical study of 130 patients with biliary tract cancers and periampullary tumors; Su WC et al.; A retrospective review of 130 patients with peripheral-type cholangiocarcinomas (PTCC), hilar-type cholangiocarcinomas (HTCC), extrahepatic cholangiocarcinomas (EHCC), gallbladder cancers (GBCA), and periampullary cancers (PACA), seen at National Cheng Kung University Hospital and Tainan Municipal Hospital from June 1987 to July 1993 was performed . There were 47 (36%) HTCC, 32 (25%) PACA, 24 (19%) PTCC, 17 (13%) GBCA, and 10 (8%) EHCC patients . The distribution is completely different from that reported in western countries . These cancers mainly occur in elderly patients . HTCC and GBCA were predominantly noted in female patients . Biliary cancers in Taiwan were not related to liver fluke infestation, inflammatory bowel disease or hepatitis B virus infection . However, a close association with biliary lithiasis was found . The incidence of gallstones was 67, 39, 20, 29 and 19% for PTCC, HTCC, EHCC, GBCA and PACA, respectively . The most common presentation for PTCC and GBCA was abdominal pain, or jaundice for HTCC, EHCC and PACA . These symptoms correlate well with the location of the tumors . Among serum tumor markers, the elevation of CA19-9 was most frequent, occurring in 86% of the patients while CA125 and CEA occurred in 47% and 30% of the patients, respectively . During the course of disease, infection developed in 61% of the patients and was the main cause of death in 25% . Biliary tract infection and sepsis were the two leading manifestations and occurred in 49% and 32% of the patients, respectively . Overall survival was poor except in patients whose tumor could be completely resected.

Resuscitation, 1996 Nov, 33(1), 63 - 8
Significance of elevated cytochrome aa3 in a state of endotoxemia in dogs; Ogata H et al.; It is now possible to detect quantitative changes in cytochrome aa3 by means of near-infrared spectrophotometry . This technique is also suitable for determining oxidised hemoglobin (HbO2), reduced hemoglobin (Hb), cerebral blood volume, and the redox state of cytochrome aa3 (cyt aa3) in the tissues . The significance of elevated cyt aa3, measured by near-infrared spectrophotometry, is still unclear, so we investigated this question using both near-infrared spectrophotometry and oxygen saturation meters in endotoxemic dogs . Ten anaesthetised mongrel dogs were injected with endotoxin (E . coli 0111: B4 Difco 2 mg/kg i.v.) and the redox state of Hb and cyt aa3 was determined in real time by near-infrared spectrophotometry . The levels of arterial and cisternal venous oxygen saturation were recorded simultaneously by two Oximetrix 3 saturation meters to calculate the cerebral arterial and venous oxygen saturation difference (Sata-vO2D) in real time . HbO2 decreased along with the fall in mean arterial pressure and remained at a low level, while Hb increased and remained at a high level . The cerebral blood volume decreased in the endotoxic early stage and then returned gradually towards baseline . Cyt aa3 showed an increase following endotoxin injection and maintained an oxidised form . The cerebral Sata-vO2D rose to about three times the control level . From these observations, an increase of oxidised cytochrome aa3 after endotoxin administration seems to be a compensatory protective effect in response to the cerebral oxygen demand rather than over-oxygenation or hyperoxia.

J Vet Med Sci, 1996 Nov, 58(11), 1145 - 7
A new adherent form of an attaching and effacing Escherichia coli (eaeA+, bfp-) to the intestinal epithelial cells of chicks; Sueyoshi M et al.; The adherent site of "attaching and effacing Escherichia coli" (AEEC; O103: H-, SK-1 strain) on the intestinal epithelial cells of chicks infected naturally and experimentally was ultrastructurally investigated . The eaeA gene was detected by polymerase chain reaction in the SK-1 strain of E . coli isolated from the intestinal content of a chick infected naturally, however, the bundle-forming pilus (bfp) gene could not be detected . The SK-1 strain (bfp-) of AEEC could attach to the intestinal epithelial cell and induce attaching-effacing lesions in the intestine of chicks . Transmission electron microscopy revealed numerous pilus-like microfilaments in the space between colibacilli and the membranes of the intestinal epithelial cells . The present study suggests that SK-1 strain (eaeA+, bfp-) may attach closely to the intestinal epithelial cells by a novel adhesion different from bfp.

Drug Metab Rev, 1996 Nov, 28(4), 625 - 58
Pharmacokinetics and pharmacodynamics of a recombinant human granulocyte colony-stimulating factor; Kuwabara T et al.; Granulocyte colony-stimulating factor (G-CSF), a hematopoietic growth factor, is a clinically effective drug used to promote neutrophil recovery in patients with chemo- or radiotherapy-induced neutropenia . We have reviewed the pharmacokinetic and pharmacodynamic properties of three kinds of G-CSFs: E . coli derived G-CSF, CHO-derived G-CSF, and mutein G-CSF . The clearances of G-CSFs are saturable and autoinducible in experimental animals and humans . That is, the systemic clearances of G-CSFs decrease as the dose injected increases and approaches a constant value . Both saturable and nonsaturable processes are involved in G-CSF elimination . Also, the systemic clearances of G-CSFs are increased by repeated administration of G-CSF . Although the relative bioavailability of G-CSFs after subcutaneous administration is approximately 60%, the increase in peripheral white blood cells or neutrophils is greater than that after intravenous administration at the same dose . The effects of G-CSFs seem to be time dependent rather than AUC dependent, considering that mean residence time of G-CSFs in the plasma is longer after subcutaneous administration than that after intravenous administration . There is a slight difference in the pharmacokinetics of E-coli- and CHO-G-CSF although they seem to be pharmacologically equivalent . The correlation between G-CSF clearance and peripheral neutrophil counts in the patients suggests that G-CSF receptors contribute to G-CSF clearance . Quantitative pharmacokinetic analysis using mutein G-CSF shows that the G-CSF receptor plays a major role in saturable G-CSF clearance, and that this saturable process accounts for approximately 80% of the total clearance at low doses . That is, the degradation following the receptor-mediated endocytosis in bone marrow might be a major clearance system of G-CSF at a physiological blood level . The G-CSF receptor in bone marrow might work not only as a signal transducer for differentiation and proliferation of granulopoietic precurcer cells but as a regulator of G-CSF levels in blood . In addition, at high doses, glomerular filtration in the kidneys is the major process for nonsaturable G-CSF clearance . At present, polyethylene glycol derivatives of G-CSF are being developed to reduce the frequency of G-CSF administration.

Biochem Mol Biol Int, 1996 Nov, 40(5), 965 - 74
Structural and functional roles of the amino-terminal region and collagen-like domain of human serum mannan-binding protein; Ma Y et al.; The serum mannan-binding protein (S-MBP) is a Ca(2+)-dependent C-type animal lectin specific for mannose and N-acetylglucosamine, which plays an important role in first-line host defense . To study the structure and function relationship of the lectin, a full-length human S-MBPcDNA was expressed in Sf9 insect cells using a baculovirus expression system, and a cDNA encoding the carbohydrate recognition domain (CRD) of human S-MBP was expressed in E . coli . The properties of the recombinant S-MBP and recombinant S-MBP-CRD were compared with those of the native human S-MBP and the CRD of the native S-MBP . The results indicated that functional human S-MBP can be successfully expressed in Sf9 cells and functional S-MBP-CRD in E . coli . In addition, the amino-terminal region and collagen-like domain are required for higher oligomer formation and play important roles in complement activation.

Biochem Mol Biol Int, 1996 Nov, 40(5), 939 - 45
C-terminal peptide of streptokinase, Met369-Pro373, is important in plasminogen activation; Kim IC et al.; Streptokinase(SK), a plasminogen activator, is known to have multi-domain structure . The function of the C-terminal region of streptokinase was investigated with SK mutants constructed by truncating 26, 33, 37, 40, 41, 46, 47, 70 or 97 amino acid residues from the C-terminus . The truncated SKs were expressed in E . coli and purified . The 41 residue deletion (SKP373) from the C-terminus had not effect on the plasminogen activation activity . However, the deletion of 46 amino acid residues (SKP368) resulted in the dramatic reduction of the plasminogen activation efficiency . The result suggests that the C-terminal peptide from Met369 to Pro373 of SK may play an important role on the plasminogen activation.

Acta Crystallogr A, 1996 Nov 1, 52 ( Pt 6), 937 - 46
Direct methods in protein electron crystallography: the ab initio structure determination of two membrane protein structures in projection using maximum entropy and likelihood; Gilmore CJ et al.; Using maximum entropy and likelihood, an ab initio phase determination was carried out in projection at ca 6-10 A resolution for two dissimilar membrane proteins: the Omp F porin from the outer membrane of E . coli (largely beta-sheet) and halorhodopsin (largely alpha-helix) . Accurate phase information found for the most likely solutions enabled potential maps to be calculated that contained most of the essential structural details of these macromolecules without the need for any image-derived phases as a starting set for phase extension or the necessity to use envelopes or electron-density histograms . A comparison with earlier calculations using the Sayre-Hughes equation coupled with phase annealing and the Luzzati flatness criterion used as a figure of merit is made.

Proteins, 1996 Nov, 26(3), 358 - 60
Crystallization and preliminary X-ray investigation at 2.0 A resolution of Bet v 1, a birch pollen protein causing IgE-mediated allergy; Spangfort MD et al.; The 17 kDa protein from Betula verrucosa (White Birch) pollen, Bet v 1, is the clinically most important birch pollen allergen causing immediate Type I IgE-mediated allergy . The three-dimensional structure of Bet v 1 and its IgE-binding epitopes are at present not known . In addition, the biological function of Bet v 1 in birch pollen is not fully established . In this work, Bet v 1 has been expressed in Escherichia coli as a recombinant protein, purified and crystallized . The space group of recombinant Bet v 1 crystals is orthorhombic C2221 with unit cell parameters a = 32.13 A, b = 74.22 A, and c = 118.60 A . There is one Bet v 1 molecule per asymmetric unit and the water content is 41% . Crystals diffract to 2.0 A resolution and a complete native data set was collected from a single crystal using CuK alpha X-rays from a rotating anode.

Proteins, 1996 Nov, 26(3), 353 - 7
Crystallization of the receptor binding domain of vascular endothelial growth factor; Christinger HW et al.; Vascular endothelial growth factor (VEGF) is a potent angiogenic factor with a unique specificity for vascular endothelial cells . In addition to its role in vasculogenesis and embryonic angiogenesis, VEGF is implicated in pathologic neovascularization associated with tumors and diabetic retinopathy . Four different constructs of a short variant of VEGF sufficient for receptor binding were overexpressed in Escherichia coli, refolded, purified, and crystallized in five different space groups . In order to facilitate the production of heavy atom derivatives, single cysteine mutants were designed based on the crystal structure of platelet-derived growth factor . A construct consisting of residues 8 to 109 was crystallized in space group P2(1), with cell parameters a = 55.6 A, b = 60.4 A, c = 77.7 A, beta = 90.0 degrees, and four monomers in the asymmetric unit . Native and derivative data were collected for two of the cysteine mutants as well as for wild-type VEGF.

Bull Math Biol, 1996 Nov, 58(6), 1171 - 85
A novel intact circular dsDNA supercoil; Wu R et al.; A novel intact circular dsDNA supercoil is proposed as an alternative to the conventional DNA supercoil, so that the two complementary strands of ssDNA circles are separable without any covalent bond breakage . This new structure can be visualized by using two tubings: one black and one clear . Twist the black tubing a number of times and connect its two ends . Do the same for the clear tubing . Then wrap the two tubings together . This forms the separable or novel supercoil . On the other hand, the conventional supercoil can be modeled by twisting the black and clear tubings together and then connect their respective ends, so that the two tubings are not separable unless one of them is cut . Experimentally, in the absence of any enzyme, many intact plasmid dsDNA circles give two bands on agarose gel electrophoresis under a certain given condition, while the same plasmid molecules after cutting once by a restriction enzyme give only one band under the same condition . In the case of intact pUC19 plasmids, these two bands can then be recovered and sequenced separately, using two primers in opposite directions . Each band gives mostly one sequence which is complementary to that of the other band . The combination of the above theoretical model and experimental results strongly suggests that there is an alternative structure of DNA which does not have the usual difficulty of unwinding, rewinding and requiring numerous covalent bond breakages and ligations during semiconservative.

Plant J, 1996 Nov, 10(5), 883 - 92
Isolation and characterization of a cDNA that encodes maize uroporphyrinogen III methyltransferase, an enzyme involved in the synthesis of siroheme, which is prosthetic group of nitrite reductase; Sakakibara H et al.; A full-length cDNA clone, pZmSUMT1, encoding an S-adenosyl-L-methionine-dependent uroporphyrinogen III C-methyltransferase (SUMT; EC 2.1.1.107) of maize was isolated from a root cDNA library . pZmSUMT1 had an insert of 1.7 kb and the amino acid sequence deduced from the open reading frame of the cDNA was similar to that of SUMT from various bacteria and also to the SUMT catalytic region of siroheme synthase (cysG) from Escherichia coli . Overproduction of ZmSUMT1 in a cysG mutant of E . coli eliminated the requirement of the strain for cysteine . Transcripts for ZmSUMT1 accumulated rapidly in both rots and leaves in response to the addition of nitrate to the culture medium . The effects of biochemical inhibitors on the nitrate-dependent induction of the gene for ZmSUMT1 coincided with the effects on the genes for other nitrate-assimilatory enzymes, nitrate reductase and nitrite reductase . An import experiment in vitro suggested that the gene product might be located in plastids . The results together indicate that ZmSUMT1 might be involved in the synthesis of siroheme, a prosthetic group of nitrite reductase, and that the expression of its gene is co-regulated with that of other nitrate-assimilatory genes.

Plant J, 1996 Nov, 10(5), 815 - 21
Cytosolic and plastidic chorismate mutase isozymes from Arabidopsis thaliana: molecular characterization and enzymatic properties; Eberhard J et al.; The three aromatic amino acids phenylalanine, tyrosine, and tryptophan are synthesized in the plastids of higher plants . There is, however, biochemical evidence that a cytosolic isoform exists of the enzyme catalysing the first step of that branch of the pathway which is specific for the synthesis of phenylalanine and tyrosine, i.e . chorismate mutase (CM) . We now report on the isolation of a cDNA clone encoding a cytosolic CM isozyme from Arabidopsis thaliana that was identified by complementing a CM-deficient Escherichia coli strain . The deduced amino acid sequence of this isozyme was 50% identical to that of a previously isolated plastidic CM, and 41% identical to that of yeast CM . The organ-specific expression patterns of the two CM genes were rather similar, but only the gene encoding the plastidic isozyme was elicitor- and pathogen-inducible . The plastidic CM expressed in E . coli was activated by tryptophan and inhibited by phenylalanine and tyrosine, whereas the cytosolic isozyme was insensitive . The existence of a cytosolic CM isozyme implies that either a cytosolic pathway (partial or complete) for the biosynthesis of phenylalanine and tyrosine exists, or that prephenate, originating from chorismate in the cytosol, is utilized for the synthesis of metabolites other than these two aromatic amino acids.

Mol Microbiol, 1996 Nov, 22(4), 747 - 55
Effect of UVM induction on mutation fixation at non-pairing and mispairing DNA lesions; Rahman MS et al.; Mutation fixation at an ethenocytosine (epsilon C) residue borne on transfected M13 single-stranded DNA is significantly enhanced in response to pretreatment of Escherichia coli cells with UV, alkylating agents or hydrogen peroxide, a phenomenon that we have called UVM for UV modulation of mutagenesis . The UVM response does not require the E . coli SOS or adaptive responses, and is observed in cells defective for oxyR, an oxidative DNA damage-responsive regulatory gene . UVM may represent either a novel DNA-repair phenomenon, or an unrecognized feature of DNA replication in damaged cells that affects a specific class of non-coding DNA lesions . To explore the range of DNA lesions subject to the UVM effect, we have examined mutation fixation at 3,N4-ethenocytosine and 1,N6-ethenoadenine, as well as at O6-methylguanine (O6mG) . M13 viral single-stranded DNA constructs bearing a single mutagenic lesion at a specific site were transfected into cells pretreated with UV or 1-methyl-3-nitro-1-nitroso-guanidine (MNNG) . Survival of transfected viral DNA was measured as transfection efficiency, and mutagenesis at the lesion site was analysed by a quantitative multiplex sequence analysis technology . The results suggest that the UVM effect modulates mutagenesis at the two etheno lesions, but does not appear to significantly affect mutagenesis at O6mG . Because the modulation of mutagenesis is observed in cells incapable of the SOS response, these data are consistent with the notion that UVM may represent a previously unrecognized DNA damage-inducible response that affects the fidelity of DNA replication at certain mutagenic lesions in Escherichia coli.

Mol Microbiol, 1996 Nov, 22(4), 619 - 29
SecA is an intrinsic subunit of the Escherichia coli preprotein translocase and exposes its carboxyl terminus to the periplasm; van der Does C et al.; SecA is the dissociable ATPase subunit of the Escherichia coli preprotein translocase, and cycles in a nucleotide-modulated manner between the cytosol and the membrane . Overproduction of the integral subunits of the translocase, the SecY, SecE and SecG polypeptides, results in an increased level of membrane-bound SecA . This fraction of SecA is firmly associated with the membrane as it is resistant to extraction with the chaotropic agent urea, and appears to be anchored by SecYEG rather than by lipids . Topology analysis of this membrane-associated form of SecA indicates that it exposes a carboxy-terminal domain to the periplasmic face of the membrane.

Biol Pharm Bull, 1996 Nov, 19(11), 1490 - 3
Effects of a dosing solution on the nasal absorption of non-glycosylated recombinant human granulocyte colony-stimulating factor in rats; Nomura H et al.; The nasal absorption of non-glycosylated recombinant human granulocyte colony-stimulating factor produced by Esherichia coli (E . coli-rhG-CSF) was studied in rats . We investigated the effects of the basic conditions of the dosing solution and the enhancing effects of various substances to find means to improve the nasal absorption of E . coli-rhG-CSF . Relative bioavailability following the nasal administration of E.coli-rhG-CSF solution to subcutaneous administration was 3.6% . Osmotic pressure of the dosing solution had virtually no effect on the nasal absorption of E . coli-rhG-CSF . Absorption was enhanced by a decrease in pH, an increase in buffer concentration at pH2 and an increase in drug concentration . Sodium cholate, ascorbic acid, tartaric acid, beta-cyclodextrin and dimethyl-beta-cyclodextrin in remarkably increased the nasal absorption of E . coli-rhG-CSF . Condition adjustments to the dosing solution and the use of several enhancers were significantly concluded to increase the intranasal absorption of E . coli-rhG-CSF.

Biol Pharm Bull, 1996 Nov, 19(11), 1401 - 6
Different effects of carboxy-terminal deletion in the adrenodoxin molecule on cytochrome c and acetylated cytochrome c reductions; Sagara Y et al.; In immunoblotting analysis using a rabbit antibody to bovine adrenodoxin, the total proteins of the bovine adrenal cortex gave two bands, suggesting the presence of two forms of adrenodoxin in vivo: full-length and carboxy-terminal deleted adrenodoxins . To examine the effect of the carboxy-terminal deletion of adrenodoxin on its activity, cDNAs for Arg115stop mutant adrenodoxin and for Asp113stop mutant adrenodoxin were constructed . The wild type {Ad(2-128)} and carboxy-terminal deleted {Ad(2-114) and Ad(2-112)} recombinant adrenodoxins expressed in Escherichia coli were purified to give a single band on SDS-PAGE . They showed an A414/A276 value of 0.92 . In an NADPH-cytochrome c reduction assay, the Km values for cytochrome c in the reconstituted system with AD(2-128), Ad(2-114) and Ad(2-112) were 39, 235 and 618 mM, respectively . The Vmax values were 638, 700 and 898 mol/min/mol flavin, respectively . In an NADPH-acetylated cytochrome c reduction assay, the maximum activity of Ad(2-128) was obtained at 50 mM NaCl, while the maximum activities of Ad(2-114) and Ad(2-112) were obtained at 100 mM NaCl; the latter values were 4-times higher than that of Ad(2-128) . In the presence of 100 mM NaCl, the Km values for acetylated cytochrome c in the system reconstituted with Ad(2-128), Ad(2-114) and Ad(2-112) were 220, 33 and 22 microM, respectively . The Vmax values were 352, 305 and 382 mol/min/mol flavin, respectively . These results indicate that the effects of the carboxy-terminal deletion of adrenodoxin on NADPH-cytochrome c and acetylated cytochrome c reductions are different; the carboxy-terminal region (residues 113-128) of adrenodoxin largely contributes to the binding with cytochrome c but disturbs the binding with acetylated cytochrome c.

Genetics, Food Microbiology, Functional Genomics, Microbe, Genetic Engineering, Microorganism, Staphylococcus Aureus, Microbiology, Salmonella, Microbes, Yeast, Bacterium, Growth Medium, Bacteria, d, Biofilm, l, Microorganisms, i, Dna, a, Bacteriology, m, Protein, Pcr, Fermentations, a, E. coli, c, Prokaryotes, w, Gram positive, k, Escherichia coli, e, Pathogenic bacterium, m, Escherichia coli, e, Neisseria, r, Staphylococcus, s, Listeriosis, a, Bacillus, c, S. aureus, y, Schizosaccharomyces, a, Typhus, b, Saccharomyces cerevisiae, m, Escherichia coli, s, Bioremediation, a, Campylobacter, b, Agrobacterium, d, Microorganism, s, Bacteriological, e, Bacterium, e, Pichia, a, Yeasts, s, Pseudomonas, s, Escherichia coli, a, B. anthracis, h, Streptococcus, l, Microorganism, Streptococcal,

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005