Escherichia coli

Biochemistry, 1997 May 20, 36(20), 6000 - 8
Solution structure of the actinorhodin polyketide synthase acyl carrier protein from Streptomyces coelicolor A3(2); Crump MP et al.; The solution structure of the actinorhodin acyl carrier protein (act apo-ACP) from the polyketide synthase (PKS) of Streptomyces coelicolor A3(2) has been determined using 1H NMR spectroscopy, representing the first polyketide synthase component for which detailed structural information has been obtained . Twenty-four structures were generated by simulated annealing, employing 699 distance restraints and 94 dihedral angle restraints . The structure is composed, principally, of three major helices (1, 2, and 4), a shorter helix (3) and a large loop region separating helices 1 and 2 . The structure is well-defined, except for a portion of the loop region (residues 18-29), the N-terminus (1-4), and a short stretch (57-61) in the loop connecting helices 2 and 3 . The RMS distribution of the 24 structures about the average structure is 1.47 A for backbone atoms, 1.84 A for all heavy atoms (residues 5-86), and 1.01 A for backbone atoms over the helical regions (5-18, 41-86) . The tertiary fold of act apo-ACP shows a strong structural homology with Escherichia coli fatty acid synthase (FAS) ACP, though some structural differences exist . First, there is no evidence that act apo-ACP is conformationally averaged between two or more states as observed in E . coli FAS ACP . Second, act apo-ACP shows a disordered N-terminus (residues 1-4) and a longer flexible loop (19-41 with 19-29 disordered) as opposed to E . coli FAS ACP where the N-terminal helix starts at residue 3 and the loop region is three amino acids shorter (16-35) . Most importantly, however, although the act apo-ACP structure contains a hydrophobic core, there are in addition a number of buried hydrophilic groups, principally Arg72 and Asn79, both of which are 100% conserved in the PKS ACPs and not the FAS ACPs and may therefore play a role in stabilizing the growing polyketide chain . The structure-function relationship of act ACP is discussed in the light of these structural data and recent genetic advances in the field.

FEBS Lett, 1997 May 19, 408(2), 182 - 6
Antibodies to rat soluble IL-6 receptor stimulate B9 hybridoma cell proliferation; Thibault V et al.; Interleukin-6 mediates its pleiotropic effects by interacting with its membrane bound receptor (gp80) or the soluble counterpart gp54, resulting in activation of a complex that includes the transducer protein gp130 . We have generated a polyclonal antibody against the rat soluble IL-6 receptor (anti-rat sIL-6R) in rabbits . By Western blot analysis we show that purified anti-rat sIL-6R IgG antibody reacts specifically with recombinant rat sIL-6R generated from E . coli, baculovirus or adenovirus expression systems . Anti-rat sIL-6R inhibited IL-6-induced acute phase protein synthesis in rat (H35) but not human (HepG2) hepatoma cells, and did not affect stimulation of those cells by Oncostatin-M . Conversely, on the mouse hybridoma B9 cell line, IgG anti-rat sIL-6R showed a dose-dependent stimulation of proliferation . Fab fragments of this antibody did not stimulate, but abrogated IL-6-mediated hepatoma cell stimulation and B9 cell proliferation . Gel shift analysis of STAT nuclear factors showed activation of STAT DNA binding in nuclei of B9 cells treated with IgG anti-rat sIL-6R, whereas in H35, NIH-3T3 and M1 cells, only IL-6 could trigger a similar STAT activation . Our data suggest that mechanisms of IL-6 receptor activation and signalling in mouse B9 hybridoma cells show subtle but important differences from other IL-6-responsive cells.

FEBS Lett, 1997 May 19, 408(2), 156 - 60
Purification and characterization of a fusion protein of plant acetohydroxy acid synthase and acetohydroxy acid isomeroreductase; Dumas R et al.; The nucleotide sequence coding for the Arabidopsis thaliana acetohydroxy acid synthase was genetically fused in frame with the nucleotide sequence coding for the Spinacia oleracea acetohydroxy acid isomeroreductase and expressed in Escherichia coli . This construction allowed the production of large amounts of soluble fusion protein . The pure chimeric enzyme exhibits high acetohydroxy acid synthase and acetohydroxy acid isomeroreductase specific activities . Fusion and native enzymes exhibit similar Km values for their substrates and for most cofactors . Furthermore, whereas native plant acetohydroxy acid synthase is highly unstable, the stability of this enzyme in the fusion has been increased . Thus, the chimeric enzyme appears to be a useful tool for the determination of kinetic and structural properties of plant acetohydroxy acid synthase.

Biochim Biophys Acta, 1997 May 17, 1346(1), 25 - 9
Nucleotide sequence of human alkyl-dihydroxyacetonephosphate synthase cDNA reveals the presence of a peroxisomal targeting signal 2; de Vet EC et al.; Two overlapping clones were isolated from a human liver cDNA library in lambda gt11 that coded for human alkyl-dihydroxyacetonephosphate synthase using guinea pig and PCR-derived human cDNA probes . The open reading frame encodes a protein of 658 amino acids that shows a homology of 92% with the guinea pig homolog and a similarity of 98% . The peroxisomal targeting signal 2 that was recently identified in the presequence of the guinea pig enzyme appeared to be completely preserved in the human enzyme . Supportive confirmation for parts of the sequence of the mature protein was obtained from the Expressed Sequence Tags database of the National Center for Biotechnology Information . This database contained nine cDNA sequences, derived from seven independent clones, that correspond exactly to parts of the cDNA of human alkyl-dihydroxyacetonephosphate synthase . One of these clones most likely represents a not fully processed RNA with a putative intron containing an Alu sequence . An unexpected homology with D-lactate dehydrogenase (cytochrome C) precursor from Saccharomyces cerevisiae and with glycolate oxidase subunit D from Escherichia coli was also revealed.

Carbohydr Res, 1997 May 16, 300(3), 259 - 64
Preparation and biological activity of manno- and galacto-validamines, new 5a-carba-glycosylamines as alpha-glycosidase inhibitors; Kameda Y et al.; Manno- and galacto-validamines, which are epimers of validamine, were semi-synthesized by the configurational inversion of validamine, a pseudo-sugar analogue of alpha-D-gluco-pyranose that has inhibitory activity for alpha-glucosidases . The inhibitory activities of these analogues were determined against several mannosidases and galactosidase . Manno-validamine shows potent inhibition for the alpha-mannosidases (competitive . K(i) = 4.6 x 10(-5) M for jack beans, and competitive, Ki = 2.8 x 10(-5) M for almonds), and galacto-validamine shows weak inhibition for the alpha-galactosidases (coffee bean and E . coli) . The inhibitory effect of the epimers on the N-linked oligosaccharide-processing mannosidases involved in glycoprotein biosynthesis and lysosomal mannosidase from rat liver were also examined . Mannovalidamine shows potent inhibition on the endoplasmic reticulumal alpha-mannosidase (competitive, K(i) = 1.2 x 10(-6) M), Golgi mannosidases IA, II (competitive, K(i) = 2.8 x 10(-5) M), and lysosomal alpha-mannosidase (competitive, Ki = 1.7 x 10(-5) M).

Carbohydr Res, 1997 May 16, 300(3), 251 - 8
Lectin-deficient ricin toxin intoxicates cells bearing the D-mannose receptor; Frankel AE et al.; Ricin toxin with genetic or chemical modification of lectin sites has been previously reported to show markedly reduced cytotoxicity to cells following uptake by several receptors including the mannose receptor . Investigators have hypothesized that an intracellular galactoside-binding function was required for optimal intracellular targeting of ricin for these receptors . We have prepared insect-derived mutant ricin toxin B chain (RTB) with modifications of three lectin side domains (1 alpha, 1 beta, and 2 gamma) yielding a 1000-fold reduced galactoside avidity . After reassociation with plant RTA, the recombinant heterodimer and plant ricin were tested for cytotoxicity on mammalian cells expressing (mouse peritoneal macrophages, J774E cells, and MMR61 cells) or not expressing (KB cells) the D-mannose receptor . Receptor expression was confirmed by immunofluorescence microscopy . Lactose was included in the media to block cell-surface galactoside binding, and mannan was added as a control in each experiment to confirm mannose receptor-specific targeting . Plant ricin A chain (RTA) and E . coli-derived RTA were also tested for cytotoxicity on J774E and KB cells . Both wild-type and lectin-deficient ricin displayed mannose-receptor mediated cell cytotoxicity . This is the first report of a genetically modified ricin showing that RTB intracellular galactose binding activity is not required for ricin cytotoxicity . Sensitivity of mannose-receptor bearing cells, but not control cells, to mannosylated RTA, but not unglycosylated RTA, confirmed these observations . These results imply fusion toxins employing ricin can be prepared with maximal reductions in normal tissue binding.

Biochim Biophys Acta, 1997 May 16, 1320(1), 83 - 94
The reduction of acetylpyridine adenine dinucleotide by NADH: is it a significant reaction of proton-translocating transhydrogenase, or an artefact?
Stilwell SN, Bizouarn T, Jackson JB.
Transhydrogenase is a proton pump . It has separate binding sites for NAD+/NADH (on domain I of the protein) and for NADP+/NADPH (on domain III) . Purified, detergent-dispersed transhydrogenase from Escherichia coli catalyses the reduction of the NAD+ analogue, acetylpyridine adenine dinucleotide (AcPdAD+), by NADH at a slow rate in the absence of added NADP+ or NADPH . Although it is slow, this reaction is surprising, since transhydrogenase is generally thought to catalyse hydride transfer between NAD(H)--or its analogues and NADP(H)--or its analogues, by a ternary complex mechanism . It is shown that hydride transfer occurs between the 4A position on the nicotinamide ring of NADH and the 4A position of AcPdAD+ . On the basis of the known stereospecificity of the enzyme, this eliminates the possibilities of transhydrogenation(a) from NADH in domain I to AcPdAD+ wrongly located in domain III; and (b) from NADH wrongly located in domain III to AcPdAD+ in domain I . In the presence of low concentrations of added NADP+ or NADPH, detergent-dispersed E . coli transhydrogenase catalyses the very rapid reduction of AcPdAD+ by NADH . This reaction is cyclic; it takes place via the alternate oxidation of NADPH by AcPdAD+ and the reduction of NADP+ by NADH, while the NADPH and NADP+ remain tightly bound to the enzyme . In the present work, it is shown that the rate of the cyclic reaction and the rate of reduction of AcPdAD+ by NADH in the absence of added NADP+/NADPH, have similar dependences on pH and on MgSO4 concentration and that they have a similar kinetic character . It is therefore suggested that the reduction of AcPdAD+ by NADH is actually a cyclic reaction operating, either with tightly bound NADP+/NADPH on a small fraction (< 5%) of the enzyme, or with NAD+/NADH (or AcPdAD+/AcPdADH) unnaturally occluded within the domain III site . Transhydrogenase associated with membrane vesicles (chromatophores) of Rhodospirillum rubrum also catalyses the reduction of AcPdAD+ by NADH in the absence of added NADP+/NADPH . When the chromatophores were stripped of transhydrogenase domain I, that reaction was lost in parallel with 'normal reverse' transhydrogenation (e.g., the reduction of AcPdAD+ by NADPH) . The two reactions were fully recovered upon reconstitution with recombinant domain I protein . However, after repeated washing of the domain I-depleted chromatophores, reverse transhydrogenation activity (when assayed in the presence of domain I) was retained, whereas the reduction of AcPdAD+ by NADH declined in activity . Addition of low concentrations of NADP+ or NADPH always supported the same high rate of the NADH-->AcPdAD+ reaction independently of how often the membranes were washed . It is concluded that, as with the purified E . coli enzyme, the reduction of AcPdAD+ by NADH in chromatophores is a cyclic reaction involving nucleotides that are tightly bound in the domain III site of transhydrogenase . However, in the case of R . rubrum membranes it can be shown with some certainty that the bound nucleotides are NADP+ or NADPH . The data are thus adequately explained without recourse to suggestions of multiple nucleotide-binding sites on transhydrogenase.

Int J Cancer, 1997 May 16, 71(4), 675 - 9
Transduction of cytosine deaminase gene makes rat glioma cells highly sensitive to 5-fluorocytosine; Ge K et al.; To investigate the potential use of E . coli cytosine deaminase (CD) gene instead of the commonly used HSV-TK gene in the gene therapy of brain tumors, we constructed a retrovirus vector carrying the CD gene . We then transduced a rat glioma cell line C6 with CD gene by the retrovirus vector . Transduction of the CD gene made C6 cells become highly sensitive to the anti-fungi drug 5-fluorocytosine (5FC) . IC50 for 5FC was 6,000 microM in CD-negative cells, while it was 3 microM in CD-positive cells . Mixed cellular assay showed that CD-positive cells had a strong "bystander effect" on CD-negative cells when exposed to 5FC . Significant anti-tumor effects were observed in nude mice bearing s.c . tumors derived from CD-positive cells when these animals were given 250 mg/kg 5FC twice a day for 20 consecutive days . A marked decrease in tumor weight occurred when a mixture containing 50% CD-positive and 50% CD-negative C6 cells was injected s.c., followed by 5FC treatment, suggesting the bystander effect in vivo . Concerning the pharmacokinetics of 5FC, especially its high oral bio-availability and good penetration into cerebrospinal fluid, we suppose that the combination of CD-gene transfer and 5FC oral administration may have potential use in the gene therapy of brain tumors.

Int J Cancer, 1997 May 16, 71(4), 638 - 44
Cloning and expression of the recombinant FAb fragment of monoclonal antibody K1 that reacts with mesothelin present on mesotheliomas and ovarian cancers; Brinkmann U et al.; The monoclonal antibody (MAb) K1 recognizes an approximate 40 kDa glycoprotein, mesothelin, that is present on the surface of human mesothelial cells, mesotheliomas and ovarian cancers . We have cloned the cDNAs encoding the variable regions of MAb K1 and constructed plasmids for expression of recombinant K1(FAb) . Recombinant FAb was produced in Escherichia coli in inclusion bodies that were solubilized and refolded to active protein . Binding of K1 MAb and FAb was compared by radioactive binding and competition assays and by surface plasmon resonance (BIAcore) . Recombinant K1(FAb) binds to cells expressing K1-antigen with a similar affinity as papain derived FAb from K1(IgG) and with a 4- to 10-fold reduced affinity compared with bivalent IgG . The cloned FAb can be used to make higher affinity antibodies and immunoconjugates that could be useful for various types of immunotherapies.

J Mol Biol, 1997 May 16, 268(4), 712 - 23
GroE modulates kinetic partitioning of folding intermediates between alternative states to maximize the yield of biologically active protein; Fedorov AN et al.; The central issue of chaperone function is the mechanism whereby partitioning of folding polypeptides along the productive pathway may be maximized, while non-productive folding pathways are minimized . We have found that the GroE chaperone is capable of accelerating the rate of the productive pathway of bacterial luciferase alphabeta heterodimer formation . At intermediate temperatures at which the productive pathway and non-productive pathways leading to dimerization-incompetent monomeric forms of the subunits coexist, GroE enhances the yield of native enzyme while minimizing the yield of misfolded protein . These results suggest that GroE releases the subunits in forms capable of achieving the native structure faster than the forms initially bound by the chaperone . At higher temperatures, at which the native enzyme is stable but the dimerization reaction is diminished, GroE is unable to force the productive folding reaction to occur . However, the chaperone decreases the rate of formation of the heterodimerization-incompetent species, thereby enhancing the final yield of active enzyme when the temperature is reduced to the permissive range . Our results suggest a mechanism by which the chaperone functions to maximize the yield of the biologically active form of the protein while maintaining or even accelerating the essential rapid kinetics of folding reactions.

J Biol Chem, 1997 May 16, 272(20), 13355 - 64
Interaction of MutS protein with the major and minor grooves of a heteroduplex DNA; Biswas I et al.; Thermus aquaticus MutS protein is a DNA mismatch repair protein that recognizes and binds to heteroduplex DNAs containing mispaired or unpaired bases . Using enzymatic and chemical probe methods, we have examined the binding of Taq MutS protein to a heteroduplex DNA having a single unpaired thymidine residue . DNase I footprinting identifies a symmetrical region of protection 24-28 nucleotides long centered on the unpaired base . Methylation protection and interference studies establish that Taq MutS protein makes contacts with the major groove of the heteroduplex in the immediate vicinity of the unpaired base . Hydroxyl radical and 1, 10-phenanthroline-copper footprinting experiments indicate that MutS also interacts with the minor groove near the unpaired base . Together with the identification of key phosphate groups detected by ethylation interference, these data reveal critical contact points residing in the major and minor grooves of the heteroduplex DNA.

J Biol Chem, 1997 May 16, 272(20), 13302 - 8
acrB mutation located at carboxyl-terminal region of gyrase B subunit reduces DNA binding of DNA gyrase; Funatsuki K et al.; Mutations that exhibit susceptibility to acriflavine have been isolated and classified as acr mutations in Escherichia coli . We cloned the acrB gene, which has been identified as a mutation of the gyrB gene, and found a double point mutation altering two consecutive amino acids (S759R/R760C) in the COOH-terminal region of the gyrase B subunit . The mutant B subunit was found to associate with the A subunit to make the quaternary structure, and the reconstituted gyrase showed an 80-fold reduction of specific activity in DNA supercoiling assay; the sensitivity to acriflavine was not different in the same unit of wild-type and mutant gyrases . The mutant enzyme retained intrinsic ATPase activity, but DNA-dependent stimulation was observed infrequently . A gel shift assay showed that acriflavine inhibited the DNA binding of gyrase . The acrB mutation also reduced significantly the DNA binding of gyrase but did not change the sensitivity to acriflavine . These results revealed that the acrB mutation is related to the inhibitory mechanism of acriflavine; and the acriflavine sensitivity of the mutant, at least in vitro, is caused mainly by reduction of the enzyme activity . Further, our findings suggest that the COOH-terminal region of the B subunit is essential for the initial binding of gyrase to the substrate DNA.

J Biol Chem, 1997 May 16, 272(20), 13171 - 9
Mutation of residue Phe97 to Leu disrupts the central allosteric pathway in Scapharca dimeric hemoglobin; Pardanani A et al.; Residue Phe97, which is thought to play a central role in the cooperative functioning of Scapharca dimeric hemoglobin, has been mutated to leucine to test its proposed role in mediating cooperative oxygen binding . This results in an 8-fold increase in oxygen affinity and a marked decrease in cooperativity . Kinetic measurements of ligand binding to the Leu97 mutant suggest an altered unliganded (deoxy) state, which has been confirmed by high resolution crystal structures in the unliganded and carbon monoxide-liganded states . Analysis of the structures at allosteric end points reveals them to be remarkably similar to the corresponding wild-type structures, with differences confined to the disposition of residue 97 side chain, F-helix geometry, and the interface water structure . Increased oxygen affinity results from the absence of the Phe97 side chain, whose tight packing in the heme pocket of the deoxy state normally restricts the heme from assuming a high affinity conformation . The absence of the Phe97 side chain is also associated with diminished cooperativity, since Leu97 packs in the heme pocket in both states . Residual cooperativity appears to be coupled with observed structural transitions and suggests that parallel pathways for communication exist in Scapharca dimeric hemoglobin.

J Biol Chem, 1997 May 16, 272(20), 13073 - 83
Regulation of substrate recognition by the MiaA tRNA prenyltransferase modification enzyme of Escherichia coli K-12; Leung HC et al.; We purified polyhistidine (His6)-tagged and native Escherichia coli MiaA tRNA prenyltransferase, which uses dimethylallyl diphosphate (DMAPP) to isopentenylate A residues adjacent to the anticodons of most tRNA species that read codons starting with U residues . Kinetic and binding studies of purified MiaA were performed with several substrates, including synthetic wild-type tRNAPhe, the anticodon stem-loop (ACSLPhe) of tRNAPhe, and bulk tRNA isolated from a miaA mutant . Gel filtration shift and steady-state kinetic determinations showed that affinity-purified MiaA had the same properties as native MiaA and was completely active for tRNAPhe binding . MiaA had a Kmapp (tRNA substrates) approximately 3 nM, which is orders of magnitude lower than that of other purified tRNA modification enzymes, a Kmapp (DMAPP) = 632 nM, and a kcatapp = 0.44 s-1 . MiaA activity was minimally affected by other modifications or nonsubstrate tRNA species present in bulk tRNA isolated from a miaA mutant . MiaA modified ACSLPhe with a kcatapp/Kmapp substrate specificity about 17-fold lower than that for intact tRNAPhe, mostly due to a decrease in apparent substrate binding affinity . Quantitative Western immunoblotting showed that MiaA is an abundant protein in exponentially growing bacteria (660 monomers per cell; 1.0 microM concentration) and is present in a catalytic excess . However, MiaA activity was strongly competitively inhibited for DMAPP by ATP and ADP (Kiapp = 0.06 microM), suggesting that MiaA activity is inhibited substantially in vivo and that DMAPP may bind to a conserved P-loop motif in this class of prenyltransferases . Band shift, filter binding, and gel filtration shift experiments support a model in which MiaA tRNA substrates are recognized by binding tightly to MiaA multimers possibly in a positively cooperative way (Kdapp approximately 0.07 microM).

J Biol Chem, 1997 May 16, 272(20), 13026 - 32
Characterization of TreR, the major regulator of the Escherichia coli trehalose system; Horlacher R et al.; The pathway of trehalose utilization in Escherichia coli is different at low and high osmolarity . The low osmolarity system takes up trehalose as trehalose 6-phosphate which is hydrolyzed to glucose and glucose 6-phosphate . treB and treC, the genes for the enzymes involved, form an operon that is controlled by TreR (encoded by treR), the repressor of the system, for which trehalose 6-phosphate is the inducer . We have cloned and sequenced treR . The protein contains 315 amino acids with a molecular weight of 34,508 . TreR was purified and shown to bind as a dimer trehalose 6-phosphate and trehalose with a Kd of 10 and 280 microM, respectively . The conformations of the protein differ from each other with either one or the other substrate-bound . Protease treatment removed the DNA-binding domain from the intact protein leaving the dimerization domain (a 29-kDa carboxyl-terminal fragment) intact . Nuclease protection experiments revealed a palindromic sequence located directly upstream of the -35 promoter sequence of treB that functions as the operator of the system.

J Biol Chem, 1997 May 16, 272(20), 13013 - 8
Expression and characterization of the small subunit of human DNA polymerase delta; Sun Y et al.; DNA polymerase delta is a heterodimer consisting of a catalytic subunit of 125 kDa and a small subunit of 50 kDa (p50) . We have overexpressed p50 in Escherichia coli and have characterized the recombinant protein . p50 was readily overexpressed using the pET vector as an insoluble protein . A procedure was developed for its purification and renaturation . Examination of the physicochemical properties of renatured p50 showed that it is a monomeric protein with an apparent molecular weight of 60,000, a Stokes radius of 34 A, and a sedimentation coefficient of 4.1 S . Its physical properties were indistinguishable from p50 expressed as a soluble protein using the pTACTAC vector . Examination of the effects of recombinant p50 on the activity of DNA polymerase delta showed that p50 is able to slightly stimulate (about 2-fold) the activity of the recombinant 125-kDa catalytic subunit using poly(dA).oligo(dT) as a template in the absence of proliferating cell nuclear antigen . In the presence of proliferating cell nulear antigen, activity is stimulated about 5-fold . Seven stable hybridoma cell lines were established that produced monoclonal antibodies against p50 . One of these antibodies (13D5) inhibited the activity of calf thymus DNA polymerase delta . This antibody, when coupled to a solid support, also was found to provide a method for the immunoafffinity purification of recombinant p50 and of DNA polymerase delta from calf thymus or HeLa extracts . Immunoprecipitation and enzyme-linked immunosorbent assays also confirmed that p50 interacts with the catalytic subunit of DNA polymerase delta.

J Biol Chem, 1997 May 16, 272(20), 12889 - 92
The yeast JEM1p is a DnaJ-like protein of the endoplasmic reticulum membrane required for nuclear fusion; Nishikawa S et al.; DnaJ-like proteins are functional partners for Hsp70 molecular chaperones . Complete nucleotide sequencing of yeast chromosome X has revealed that an open reading frame YJL073w encodes a novel member of the DnaJ-like protein family . The open reading frame represents a protein of 692 amino acids with a J-domain and one putative membrane-spanning segment . An epitope-tagged version of the protein was anchored in the endoplasmic reticulum (ER) membrane and its J-domain faced the ER lumen . We therefore propose to designate this gene JEM1 (DnaJ-like protein of the ER membrane) and to designate its gene product JEM1p . The JEM1 gene is not essential for cell growth, but double disruption of the JEM1 gene and the SCJ1 gene, which encodes another DnaJ-like protein in the ER lumen, causes growth arrest at elevated temperature . The Deltajem1 mutant is defective in nuclear fusion, karyogamy, during mating . A mutant JEM1p carrying a mutation in the highly conserved His-Pro-Asp sequence in the J-domain could not complement either temperature-sensitive growth of the Deltajem1 Deltascj1 double mutant or defects in karyogamy of the Deltajem1 mutant . JEM1p likely assists the functions of BiP, Hsp70 in the ER, including karyogamy.

Eur J Biochem, 1997 May 15, 246(1), 243 - 51
Domain-specific N-glycosylation of the membrane glycoprotein dipeptidylpeptidase IV (CD26) influences its subcellular trafficking, biological stability, enzyme activity and protein folding; Fan H et al.; Dipeptidyl peptidase IV (DPPIV, CD26) is an N-glycosylated type II plasma membrane protein . The primary structure of rat wild-type DPPIV contains eight potential N-glycosylation sites . To investigate the role of N-glycosylation in the function of DPPIV, three of its asparagine residues were separately converted to glutamine by site-directed mutagenesis . The resulting N-glycosylation mutants of rat DPPIV were studied in stable transfected Chinese hamster ovary cells . All three N-glycosylation mutants of DPPIV showed a reduced half-life, as well as differing degrees of inhibition of the processing of their N-glycans . Mutation of the first (Asn83-->Gln) or eighth (Asn686-->Gln) N-glycosylation site had only a small effect on its enzymatic activity, cell-surface expression and dimer formation, whereas the mutation of the sixth N-glycosylation site (Asn319-->Gln) abolished the enzymatic activity, eliminated cell-surface expression and prevented the dimerization of the DPPIV protein . The mutant {Gln319}DPPIV is retained in the cytoplasm and its degradation was drastically increased . Our data suggest that the N-glycosylation at Asn319 is involved in protein trafficking and correct protein folding.

Eur J Biochem, 1997 May 15, 246(1), 181 - 5
The complete cDNA sequence and expression of the first major allergenic protein of Malassezia furfur, Mal f 1; Schmidt M et al.; For the first time the complete cDNA encoding a major allergen and novel protein of the yeast Malassezia furfur, Mal f 1, has been sequenced and expressed . The amino acid sequences of nine tryptic peptides of the protein were determined . Oligonucleotides were designed from these amino acid sequences . The cDNA sequence was obtained by hybridizing these primers to mRNA and enhancement by reverse-transcriptase PCR techniques . The cDNA is 1176 bp in length . It shows an open reading frame of 1050 bp coding for a protein of 38178 Da and a deduced amino acid sequence containing 350 residues . The hydropathy plot and the tryptic digest indicate that the first 22 amino acids represent a leader sequence determining a mature protein of 35 988 Da . The complete encoding cDNA was expressed as a maltose-binding protein fusion protein in Escherichia coli . The recombinant fusion protein reacted with our specific monoclonal antibody and with IgE from patients with atopic dermatitis.

Eur J Biochem, 1997 May 15, 246(1), 166 - 72
The N-terminal region of alpha-dystroglycan is an autonomous globular domain; Brancaccio A et al.; The structure of the N-terminal region of mouse alpha-dystroglycan (DGN) was investigated by expression of two protein fragments (residues 30-180 and 30-438) in Escherichia coli cells . Trypsin susceptibility experiments show the presence of a stable alpha-dystroglycan N-terminal region (approximately from residue 30 to 315) . In addition, guanidinium hydrochloride (Gdn/HCl) denaturation of DGN-(30-438)-peptide, monitored by means of tryptophan fluorescence, produces a cooperative transition typical of folded protein structures . These results strongly suggest that the alpha-dystroglycan N-terminal is an autonomous folding unit preluding a flexible mucin-like region and that its folding is not influenced by the absence of glycosylation . In order to obtain more information on the structural features of the N-terminal domain we have also used circular dichroism, analytical sedimentation and electron microscopy analysis . Circular dichroic spectra show the absence of typical secondary structure (e.g . alpha-helix or beta-sheet) and closely resemble those recorded for loop-containing proteins . This is consistent with a sequence similarity of the alpha-dystroglycan domain with the loop-containing protein elastase . Analytical ultracentrifugation and electron microscopy analysis reveal that the N-terminal domain has a globular structure . DGN-(30-438)-peptide does not bind in the nanomolar range to an iodinated agrin fragment which binds with high affinity to tissue purified alpha-dystroglycan . No binding was detected also to laminin . This result suggests that the alpha-dystroglycan N-terminal domain does not contain the binding site to its extracellular matrix binding partners . It is less likely than the lack of glycosylation reduces its binding affinity, because the N-terminal globular domain only contains two glycosylation sites.

Eur J Biochem, 1997 May 15, 246(1), 127 - 32
Activation of partially folded mitochondrial malate dehydrogenase by thioredoxin; Li W et al.; CD spectroscopy reveals that mitochondrial malate dehydrogenase in 3M guanidinium chloride shows little residual secondary structure . Refolding of the denatured protein by dilution with buffer of pH 7.5 does not restore the CD spectrum of the native enzyme . A partially folded intermediate, possessing 25% of the alpha-helix content of the native enzyme, is formed upon dilution . The partially folded intermediate binds the extrinsic probe 1-anilinonaphtalene-8-sulfonate, and the increase in fluorescence (tenfold) is accompanied by a blue shift in the band position of the emission spectrum . Partially folded malate dehydrogenase is devoid of catalytic activity . In vitro refolding of the denatured protein takes place in the presence of dithiotreitol and thioredoxin . In the presence of micromolar concentrations of thioredoxin, a recovery of approximately 70% of the catalytic activity was observed . Emission-anisotropy titrations of oxidized thioredoxin, tagged with a fluorescent probe, revealed that the oxidoreductase recognizes partially folded intermediates of malate dehydrogenase with a dissociation constant of 6 microM . Moreover, a covalently linked complex formed by thioredoxin and monomeric malate dehydrogenase was detected by SDS/PAGE . A general mechanism is postulated for the reactivation of denatured proteins by thioredoxin.

Eur J Biochem, 1997 May 15, 246(1), 119 - 26
Characterisation of the molybdenum-responsive ModE regulatory protein and its binding to the promoter region of the modABCD (molybdenum transport) operon of Escherichia coli; Anderson LA et al.; Molybdenum-dependent repression of transcription of the Escherichia coli modABCD operon, which encodes the high-affinity molybdate transporter, is mediated by the ModE protein . This regulatory protein was purified as an N-terminal His6-tagged derivative and characterised both with and without the N-terminal oligohistidine extension . Equilibrium centrifugation showed that ModE is at least a 57-kDa homodimer . Circular dichroism spectroscopy indicated that when molybdate or tungstate bind to ModE there is little change in its alpha-helical content, but a major change in the environment of tryptophan and tyrosine residues occurs . Addition of molybdate or tungstate to the protein results in almost 50% quenching of the fluorescence attributed to tryptophan . Titration of fluorescence quenching showed that two molecules of molybdenum bind to each dimer of ModE with a Kd of 0.8 microM . DNA mobility-shift assays showed that ModE requires molybdenum, or tungstate, to bind with high affinity (approximate Kd of 30 nM ModE) to the modABCD promoter region . In accord with ModE's role as a molybdenum-dependent transcriptional repressor, DNase I footprinting experiments showed that the ModE-molybdenum complex binds to a single 31-bp region around the transcription start of the modABCD promoter . This region contains a 6-base palindromic sequence CGTTAT-N12-ATAACG.

Eur J Biochem, 1997 May 15, 246(1), 103 - 11
The nuclear ABC1 gene is essential for the correct conformation and functioning of the cytochrome bc1 complex and the neighbouring complexes II and IV in the mitochondrial respiratory chain; Brasseur G et al.; The nuclear ABC1 gene was isolated as a multicopy suppressor of a cytochrome b mRNA translation defect . Its inactivation leads to a respiratory deficiency suggesting a block in the bc1 segment of the respiratory chain {Bousquet, I., Dujardin, G . & Slonimski, P . P . (1991) EMBO J . 10, 2023-2031} . In the present study, we established that deleting the ABC1 chromosomal gene from Saccharomyces cerevisiae does not prevent the assembly of the bc1 complex (complex III) but markedly impairs the kinetics of its high-potential electron transfer pathway occurring on the positive, outer, side of the membrane, which results in reduced activity of the bc1 complex . In addition, the activity of complex II and its cytochrome b560 decrease drastically and complex IV activity is halved . It is also observed that the binding of the quinol to the bc1 complex ubiquinol oxidation site is affected and that adding exogenous quinones partially compensates for the respiratory deficiency in vitro, although the quinone content of mutant and wild-type mitochondria are similar . Lastly, complexes II, III and IV are found to be thermosensitive and the bc1 complex exhibits greater sensitivity than the wild-type strain to center N and P inhibitors, suggesting that the three multisubunit complexes have undergone structural modifications . The data suggest that the ABC1 gene product acts as a chaperone-like protein essential for the proper conformation and efficient functioning of the bc1 complex and the effects of the Abc1 protein on the complexes II and IV might result from interactions with the modified bc1 complex.

Eur J Biochem, 1997 May 15, 246(1), 38 - 44
The N-terminal Arg-rich region of human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus Nef is involved in RNA binding; Echarri A et al.; Comparison of the amino acid sequences of human immunodeficiency virus (HIV) Nef protein and several RNA-binding proteins shows similarities in some regions of these proteins . Thus, poliovirus protein 2C, an RNA-binding protein, shares with Nef the sequence YXQQ...MDD...DXXD . In addition, both proteins contain an Arg-rich motif that, in the case of poliovirus 2C, is involved in RNA-binding activity . Moreover, the RNA-binding, anti-terminator N proteins of lambda, phi21 and P22 phages show sequence similarities with HIV Nef at the Arg-rich motif . To assess the significance of this motif, native and deletion variants of Nef protein were assayed for RNA-binding activity . The N-terminal 35 amino acids of HIV-1 Nef that comprise the Arg-rich motif are sufficient for RNA binding . Point mutations engineered at the Arg-rich motif of HIV-1 Nef revealed that basic amino acid residues are essential for RNA-binding activity . The Nef proteins from HIV-2 and SIV can also interact with RNA, while the same proteins with the N-terminal Arg-rich domain truncated fail to interact with RNA . These findings indicate that all three Nef proteins from HIV-1, HIV-2 and simian immunodeficiency virus belong to the RNA-binding family of proteins . The three proteins contain an Arg-rich region at the N-terminus which is necessary to interact with RNA.

EMBO J, 1997 May 15, 16(10), 2968 - 74
Rescuing an essential enzyme-RNA complex with a non-essential appended domain; Whelihan EF et al.; Certain protein-RNA complexes, such as synthetase-tRNA complexes, are essential for cell survival . These complexes are formed with a precise molecular fit along the interface of the reacting partners, and mutational analyses have shown that amino acid or nucleotide substitutions at the interface can be used to disrupt functional or repair non-functional complexes . In contrast, we demonstrate here a feature of a eukaryote system that rescues a disrupted complex without directly re-engineering the interface . The monomeric yeast Saccharomyces cerevisiae glutaminyl-tRNA synthetase, like several other class I eukaryote tRNA synthetases, has an active-site-containing 'body' that is closely homologous to its Escherichia coli relative, but is tagged at its N-terminus with a novel and dispensable appended domain whose role has been obscure . Because of differences between the yeast and E . coli glutamine tRNAs that presumably perturb the enzyme-tRNA interface, E . coli glutaminyl-tRNA synthetase does not charge yeast tRNA . However, linking the novel appended domain of the yeast to the E . coli enzyme enabled the E . coli protein to function as a yeast enzyme, in vitro and in vivo . The appended domain appears to contribute an RNA interaction that compensates for weak or poor complex formation . In eukaryotes, extra appended domains occur frequently in these proteins . These domains may be essential when there are conditions that would otherwise weaken or disrupt formation of a critical RNA-protein complex . They may also be adapted for other, specialized RNA-related functions in specific instances.

EMBO J, 1997 May 15, 16(10), 2682 - 92
rqh1+, a fission yeast gene related to the Bloom's and Werner's syndrome genes, is required for reversible S phase arrest; Stewart E et al.; In eukaryotic cells, S phase can be reversibly arrested by drugs that inhibit DNA synthesis or DNA damage . Here we show that recovery from such treatments is under genetic control and is defective in fission yeast rqh1 mutants . rqh1+, previously known as hus2+, encodes a putative DNA helicase related to the Escherichia coli RecQ helicase, with particular homology to the gene products of the human BLM and WRN genes and the Saccharomyces cerevisiae SGS1 gene . BLM and WRN are mutated in patients with Bloom's syndrome and Werner's syndrome respectively . Both syndromes are associated with genomic instability and cancer susceptibility . We show that, like BLM and SGS1, rqh1+ is required to prevent recombination and that in fission yeast suppression of inappropriate recombination is essential for reversible S phase arrest.

Anal Biochem, 1997 May 15, 248(1), 94 - 101
Di-fluoresceinthiocarbamyl-insulin: a fluorescent substrate for the assay of protein disulfide oxidoreductase activity; Heuck AP et al.; We have developed a novel method for the continuous assay of protein disulfide oxidoreductase activity using as substrate bovine pancreas insulin in which both N-terminal amino groups are chemically modified with fluorescein isothiocyanate . The reduction of intercatenary disulfide bonds of di-fluoresceinthiocarbamyl-insulin with dithiothreitol initially lowers but subsequently enhances the emission intensity . In this biphasic kinetics, the rate of increase is sensitive enough for the estimation of Escherichia coli thioredoxin concentrations from 5 nM (0.06 microgram/ml) to 500 nM (6 micrograms/ml) . Neither changes of pH over a range of 6.2 to 8.4 nor neutral salts (K+, Mg2+, and Ca2+) at concentrations lower than 100 mM affect this simple reaction system . Moreover, the fluorometric method is functional for measuring the reductive capacity of Brassica napus protein disulfide isomerase . Hence, a highly reproducible and accurate one-state assay for protein disulfide oxidoreductase activity not only greatly improves the sensitivity compared to the commonly used turbidimetric assay but also represents a reliable alternative to assays based on accessory enzymes or radiolabeled substrates.

Anal Biochem, 1997 May 15, 248(1), 50 - 62
Accurate topological comparison of two recombinant human growth hormones by optical surface plasmon resonance; Mani JC et al.; A strategy for the comparison of two recombinant derived human growth hormones (r-hGH) has been developed using surface plasmon resonance (SPR) . Statistical analysis was systematically used on the results obtained with several batches derived from two different Escherichia coli strains . Monoclonal antibodies (MAb) directed against four different domains in the tertiary structure of natural human growth hormone were used to compare the epitopic maps of the three (two recombinant and one natural) hGH by SPR analysis . Topological studies show the homogeneity of the epitopic maps of the three hGH . The kinetic parameters, association rate, and dissociation rate constants were also analyzed for the binding of each hGH batch to all MAbs . They were found to be homogeneous between the three hormones . Furthermore, the two r-hGH were compared by more classical approaches examining recognition of lactogenic or somatogenic receptors using, respectively, a bioassay of Nb2 cell proliferation and binding to rat liver microsomes . Specific bioactivities and IC50 values calculated in radioreceptor assays did not significantly differ between different r-hGH . The method was sensitive enough to show slight differences on koff value for one MAb (3C11) between (natural) hormone and two r-hGH . These differences are discussed in relation to previous observation made in the literature and the presence of isoforms in the natural product . The strategy developed here was very useful as a new tool to establish the equivalence of the two r-hGH.

J Physiol, 1997 May 15, 501 ( Pt 1), 125 - 48
ATPase kinetics on activation of rabbit and frog permeabilized isometric muscle fibres: a real time phosphate assay; He ZH et al.; 1 . The rate of appearance of inorganic phosphate (Pi) and hence the ATPase activity of rabbit psoas muscle in single permeabilized muscle fibres initially in rigor was measured following laser flash photolysis of the P3-1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP) in the presence and absence of Ca2+ . Pi appearance was monitored from the fluorescence signal of a Pi-sensitive probe, MDCC-PBP, a coumarin-labelled A197C mutant of the phosphate-binding protein from Escherichia coli . Fibres were immersed in oil to optimize the fluorescence signal and to obviate diffusion problems . The ATPase activity was also measured under similar conditions from the rate of NADH disappearance using an NADH-linked coupled enzyme assay . 2 . On photolysis of NPE-caged ATP in the presence of Ca2+ at 20 degrees C, the fluorescence increase of MDCC-PBP was non-linear with time . ATPase activity was 41 s-1 in the first turnover based on a myosin subfragment 1 concentration of 150 microM . This was calculated from a linear regression of the fluorescence signal reporting 20-150 microM of Pi release . Tension was at 67% of its isometric level by the time 150 microM Pi was released . ATPase activities were 36 and 31 s-1 for Pi released in the ranges of 150-300 microM and 300-450 microM, respectively . The ATPase activity had a Q10 value of 2.9 based on measurements at 5, 12 and 20 degrees C . 3 . An NADH-linked assay showed the ATPase activity had a lower limit of 12.7 s-1 at 20 degrees C . The response to photolytic release of ADP showed that the rate of NADH disappearance was partially limited by the flux through the coupled reactions . Simulations indicated that the linked assay data were consistent with an initial ATPase activity of 40 s-1 . 4 . On photolysis of NPE-caged ATP in the absence of Ca2+ the ATPase activity was 0.11 s-1 at 20 degrees C with no discernible rapid transient phase of Pi release during the first turnover of the ATPase . 5 . To avoid the rigor state, the ATPase rate in the presence of Ca2+ was also measured on activation from the relaxed state by photolytic release of Ca2+ from a caged Ca2+ compound, nitrophenyl-EGTA . At 5 degrees C the ATPase rate was 5.8 and 4.0 s-1 in the first and second turnovers, respectively . These rates are comparable to those when NPE-caged ATP was used . 6 . The influence of ADP and Pi on the ATPase activities was measured using the MDCC-PBP and NADH-linked assays, respectively . ADP (0.5 mM) decreased the initial ATPase rate by 23% . Pi (10 mM) had no significant effect . Inhibition by ADP, formed during ATP hydrolysis, contributed to the decrease of ATPase activity with time . 7 . The MDCC-PBP assay and NPE-caged ATP were used to measure the ATPase rate in single permeabilized muscle fibres of the semitendinosus muscle of the frog . At 5 degrees C in the presence of Ca2+ the ATPase activity was biphasic being 15.0 s-1 during the first turnover (based on 180 microM myosin subfragment 1) . Tension was 74% of its isometric level by the time 180 microM Pi was released . During the third turnover the ATPase rate decreased to about 20% of that during the first turnover . 8 . ATPase activity in isometric rabbit muscle fibres during the first few turnovers is about an order of magnitude greater than that when a steady state is reached . Possible reasons and the consequences for understanding the mechanism of muscular contraction are discussed.

FEMS Microbiol Lett, 1997 May 15, 150(2), 297 - 301
Immotile phenotype of an Escherichia coli mutant lacking the histone-like protein HU; Nishida S et al.; The histone-like protein HU in Escherichia coli are encoded by the hupA and hupB genes . A hupA-hupB double deletion mutant has now been shown to express an immotile phenotype . The motility of hupA or hupB single mutants was similar to that of wild-type cells . SDS-polyacrylamide gel electrophoresis revealed that the amount of flagellin in the hupA-hupB double deletion mutant was markedly reduced compared with the wild-type strain suggesting that the immotile phenotype of the double deletion mutant is caused by a loss of flagella.

FEMS Microbiol Lett, 1997 May 15, 150(2), 283 - 8
Nucleotide sequence of a nicking site of the Streptomyces plasmid pSN22 replicating by the rolling circle mechanism; Suzuki I et al.; A putative nicking site in the double strand origin (DSO) of the Streptomyces plasmid pSN22 was identified by comparing the nucleotide sequence of the DSO region with those of two other Streptomyces plasmids, pIJ101 and pJVI . A 7-bp sequence of this putative nicking site, 5'-CTTGGGA-3', was similar to the consensus sequence of the nicking site of the pC194 group of plasmids . When several point mutations were introduced into this 7-bp sequence, the transformation abilities of the mutant plasmid molecules for Streptomyces lividans were either reduced or lost . Southern hybridization analysis indicated that these mutant plasmids could not replicate in S . lividans, but were integrated into the chromosomal DNA.

FEMS Microbiol Lett, 1997 May 15, 150(2), 239 - 47
Characterization of dnaA gene expression in Mycoplasma capricolum; Seto S et al.; Expression of the dnaA gene in Mycoplasma capricolum was studied . The transcriptional start site was located 10 bp upstream from the putative translational initiation codon . Immunoblotting analysis revealed that DnaA protein was expressed at the levels significantly larger than those in Escherichia coli, and was localized mainly in the membrane . The transcriptional level of dnaA gene was reduced by inhibition of DNA synthesis with methyl methanesulfonate or mitomycin C, while the level of DnaA protein did not change . Reduction of protein synthesis did not significantly affect the total amount of DnaA protein, suggesting that the rates of synthesis and degradation of the protein are slow . These observations showed that the expression pattern of dnaA gene in M . capricolum is different from those of walled bacteria in some aspects.

Arch Biochem Biophys, 1997 May 15, 341(2), 353 - 9
Selenophosphate synthetase: enzyme labeling studies with {gamma-32P}ATP, {beta-32P}ATP, {8-14C}ATP, and {75Se}selenide; Liu SY et al.; Selenophosphate synthetase catalyzes a reaction in which ATP and selenide are converted to H3SeP03, H3P04, and AMP in a 1:1:1 ratio . Selenophosphate is derived from the gamma phosphoryl group and orthophosphate from the beta phosphoryl group of ATP . In the absence of selenide, a slow reaction in which ATP is converted quantitatively to 2 H3P04 and AMP occurs . Labeling experiments carried out to detect a putative enzyme-bound pyrophosphate intermediate in the overall reaction showed that up to 0.6 equivalent of the 32P label from {gamma-32P}ATP was bound to protein under enzyme turnover conditions, but only a negligible amount of 32P from {beta-32P}ATP was present . Thus, no Enz-PP intermediate was present in a detectable amount under the experimental conditions used . Isolated enzyme samples contained 75Se from 75Se-labeled selenide and {14C}AMP from {8-14C}ATP in amounts similar to the bound 32P from {gamma-32P}ATP, suggesting that two of the final products, selenophosphate and AMP, were the radioactive compounds detected in these experiments.

Arch Biochem Biophys, 1997 May 15, 341(2), 329 - 36
Mapping the mechanism-based modification sites in L-aspartase from Escherichia coli; Giorgianni F et al.; Inactivation of the enzyme L-aspartase from Escherichia coli by the substrate analog aspartate beta-semialdehyde has previously been shown to occur by the mechanism-based conversion to the corresponding product aldehyde, followed by covalent modification of cysteine-273 (F . Giorgianni et al . (1995) Biochemistry 34, 3529) . Inactivation by the product analog, fumaric acid aldehyde (FAA), has now been examined directly by adding a reduction step to the modification protocol in order to stabilize the resulting enzyme-FAA derivative(s) . HPLC and mass spectrometric analyses of proteolytic digests of inactivated L-aspartase have confirmed the modification at cysteine-273, and have also identified an additional modified peptide . The inactivation at this additional site involves a crosslink between cysteine-140 and an adjacent lysine . Site-directed mutagenesis studies have shown that cysteine-140 is a very reactive and accessible nucleophile that is not, however, directly involved in enzyme activity . The adjacent lysine-139 that is modified does appear to play a role in substrate binding . A double mutant in which both of the reactive cysteines have been replaced is almost completely insensitive to modification by these substrate and product analogs.

Arch Biochem Biophys, 1997 May 15, 341(2), 309 - 14
Construction and expression of chimeric rat liver hydroxysteroid sulfotransferase isozymes; Tamura H et al.; The St-20 and ST-40 cDNAs encode rat liver hydroxysteroid sulfotransferases (HS-ST) that are 90% identical in amino acid sequence but exhibit different substrate preferences for dehydroepiandrosterone (DHEA), androsterone (AD), and cortisol (CS) . ST-40 is active for all three substrates, whereas ST-20 is mainly active for cortisol . To determine the domain responsible for the substrate preferences of the HS-STs, 20 chimeric HS-STs were constructed by reciprocal exchanges of DNA fragments derived from the cDNAs and were expressed in Escherichia coli . Some chimeric enzymes were enzymatically active for all three substrates, and some displayed reduced or lost CS-ST activity, with retention of DHEA- and AD-ST activities . Others lost all HS-ST activity . Analysis revealed that a central region (region III spanning amino acids 102-164 with five amino acid differences between ST-20 and ST-40) is essential for HS-ST activity, whereas regions II (amino acids 65-101) and IV (amino acids 165-219) are unimportant with regard to substrate preference . It was also shown that the parental combination of regions I (amino acids 1-64) and V (amino acids 220-284) is essential for CS-ST activity . Photoaffinity labeling with {35S}3'-phosphoadenosine 5'-phosphosulfate (PAPS) revealed that some inactive chimeras lost affinity for PAPS . These results suggested that an ordered structure formed by regions I, III, and V is required for HS-ST activity, especially for substrate preference and PAPS binding.

Biochem J, 1997 May 15, 324 ( Pt 1), 273 - 81
Biochemical characteristics of a rice (Oryza sativa L., IR36) G-protein alpha-subunit expressed in Escherichia coli; Seo HS et al.; A cDNA encoding the alpha-subunit of the heterotrimeric G-protein in rice (RGA1) was overexpressed in Escherichia coli and then isolated by Ni2+-nitrilotriacetic acid affinity chromatography . The molecular mass of RGA1 bearing a His tag was approx . 49 kDa . Immunoblot analysis using anti-RGA1 revealed that the RGA1 protein is most abundant in seedling leaves and least abundant in mature roots . It exists at particularly high levels in the immature embryo after pellicle extrusion . In addition, the RGA1 antiserum exhibited a difference in binding affinity for Galpha proteins from monocots (maize and rice) and dicots (Arabidopsis, pea, soya bean and tomato); whereas it cross-reacted with Galpha proteins of monocots, it did not with those of dicot plants . When bound to guanosine 5'-(gamma-thio)triphosphate (GTP{S}), the RGA1 protein was partially protected from tryptic proteolysis . In the presence of GTP{S}, trypsin cleaved the RGA1 protein into four fragments 24, 14, 11 and 5 kDa in size . When RGA1 was bound to GDP, only the 5 kDa polypeptide was seen on SDS/PAGE after trypsin digestion . Photoaffinity labelling with {alpha-32P}GTP and a GTP{S}-binding assay revealed that RGA1 incorporated 32P and showed specific binding to a guanine nucleotide . Guanidine binding of RGA1 was affected by the concentration of MgCl2 (maximum at 2 mM) . The rate of guanine nucleotide binding of RGA1 (kon,GTP{S}=0.0141+/-0.0014 min-1) and, at steady state, the kcat value for GTP hydrolysis (0.0075+/-0.0001 min-1) were very low even at 2 mM MgCl2 . The binding affinity for the nucleotides examined was in the order GTP-S- >/= GTP > GDP > CTP > ATP >/= dTTP.

Biochem J, 1997 May 15, 324 ( Pt 1), 97 - 102
Cloning and characterization of two glutathione S-transferases from a DDT-resistant strain of Anopheles gambiae; Ranson H et al.; Two cDNA species, aggst1-5 and aggst1-6, comprising the entire coding region of two distinct glutathione S-transferases (GSTs) have been isolated from a 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) resistant strain (ZANDS) of Anopheles gambiae . The nucleotide sequences of these cDNA species share 80.2% identity and their derived amino acid sequences are 82.3% similar . They have been classified as insect class I GSTs on the basis of their high sequence similarity to class I GSTs from Drosophila melanogaster and Musca domestica and they are localized to a region of an An . gambiae chromosome known to contain further class I GSTs . The genes aggst1-5 and aggst1-6 were expressed at high levels in Escherichia coli and the recombinant GSTs were purified by affinity chromatography and characterized . Both agGST1-5 and agGST1-6 showed high activity with the substrates 1-chloro-2,4-dinitrobenzene and 1, 2-dichloro-4-nitrobenzene but negligible activity with the mammalian theta class substrates, 1,2-epoxy-3-(4-nitrophenoxy)propane and p-nitrophenyl bromide . Despite their high level of sequence identity, agGST1-5 and agGST1-6 displayed different kinetic properties . Both enzymes were able to metabolize DDT and were localized to a subset of GSTs that, from earlier biochemical studies, are known to be involved in insecticide resistance in An . gambiae . This subset of enzymes is one of three in which the DDT metabolism levels are elevated in resistant insects.

Biochem J, 1997 May 15, 324 ( Pt 1), 25 - 8
Glutathione transferases catalyse the detoxication of oxidized metabolites (o-quinones) of catecholamines and may serve as an antioxidant system preventing degenerative cellular processes; Baez S et al.; o-Quinones are physiological oxidation products of catecholamines that contribute to redox cycling, toxicity and apoptosis, i.e . the neurodegenerative processes underlying Parkinson's disease and schizophrenia . The present study shows that the cyclized o-quinones aminochrome, dopachrome, adrenochrome and noradrenochrome, derived from dopamine, dopa, adrenaline and noradrenaline respectively, are efficiently conjugated with glutathione in the presence of human glutathione transferase (GST) M2-2 . The oxidation product of adrenaline, adrenochrome, is less active as a substrate for GST M2-2, and more efficiently conjugated by GST M1-1 . Evidence for expression of GST M2-2 in substantia nigra of human brain was obtained by identification of the corresponding PCR product in a cDNA library . Glutathione conjugation of these quinones is a detoxication reaction that prevents redox cycling, thus indicating that GSTs have a cytoprotective role involving elimination of reactive chemical species originating from the oxidative metabolism of catecholamines . In particular, GST M2-2 has the capacity to provide protection relevant to the prevention of neurodegenerative diseases.

Cancer Res, 1997 May 15, 57(10), 2035 - 41
The human ALL-1/MLL/HRX antigen is predominantly localized in the nucleus of resting and proliferating peripheral blood mononuclear cells; Ennas MG et al.; The ALL-1 gene is an important regulator of embryonal and hematopoietic development, and structural variants of the human gene generated by chromosomal translocations and other genomic alterations presumably act as oncogenes in the pathogenesis of acute leukemias and other hematological malignancies . Antisera against two different epitopes of the human ALL-1 protein (anti-ALL1-N and anti-ALL1-C) were produced . Both sera revealed indistinguishable patterns of antigen localization in human peripheral blood mononuclear cells (PBMCs) . In resting PBMCs, the antigen was distributed in a speckled pattern across the nuclei, with an increased density at the nuclear envelope and the nuclear indentation . In mitotically stimulated PBMCs, the antigen surrounded the condensing chromosomes but did not colocalize with chromatin or the nuclear scaffold . The antigen is considered a marker for a novel nuclear subcompartment, a perichromosomal area termed the "chromosomal envelope." In Western blot experiments, the anti-ALL1-N serum reacted with a polypeptide corresponding to the expected full-length 430-kDa ALL-1 protein . Recombinant proteins representing the AT-hook and zinc binding subdomains of the ALL-1 protein interacted in vitro with a degenerate mixture of double-stranded oligodeoxynucleotides . Thus, the ALL-1 protein probably is a DNA-binding protein with both a sequence-unspecific (AT-hook) and a sequence-specific (zinc binding subdomains) double-stranded DNA binding mode.

Cancer Res, 1997 May 15, 57(10), 2007 - 12
Creation of human alkyltransferases resistant to O6-benzylguanine; Christians FC et al.; O6-benzylguanine (BG), an inhibitor of O6-alkylguanine-DNA alkyltransferase, is being tested clinically for its ability to chemosensitize tumors to alkylating agents . Although this drug may increase the killing of tumors that express high levels of alkyltransferase, it would also be expected to reduce the already low alkyltransferase levels of hematopoietic stem cells and, thus, exacerbate the dose-limiting side effect of myelosuppression . One way to overcome this problem would be to transduce hematopoietic stem cells with a gene encoding a BG-resistant alkyltransferase prior to BG/alkylation treatment . We used the technique of random mutagenesis followed by positive genetic selection to create such a mutant gene . A pool of 6.5 x 10(6) human alkyltransferases that were randomly mutated at six amino acids near the alkyl-accepting cysteine was transformed into alkyltransferase-deficient Escherichia coli . Five mutants were selected based on their ability to provide the bacteria with resistance to both N-methyl-N'-nitro-N-nitrosoguanidine and BG . One mutant, V139F/P140R/L142M, not only had the highest BG resistance (50% inhibitory concentration, >500 microM) but also offered E . coli the best protection from N-methyl-N'-nitro-N-nitrosoguanidine and, thus, is a promising gene therapy candidate.

Nucleic Acids Res, 1997 May 15, 25(10), 2025 - 9
Angle and locus of the bend induced by the msp I DNA methyltransferase in a sequence-specific complex with DNA; Dubey AK et al.; Bending of DNA induced by M.Msp I, one of the m5C-DNA methyltransferases, has been investigated using circular permutation analysis . The M.Msp I MTase induced sharp bends in DNA containing its recognition sequence 5'-CCGG-3'which was estimated to be 142+/-4 degrees and 132+/-4 degrees for circularly permuted DNA fragments of 127 and 1459 bp respectively . The bend centre was found to be asymmetric with respect to the CCGG sequence and appeared to exclude the 'target cytosine' . An estimate of approximately 15 kcal/mol was obtained for the free energy associated with M.Msp I-induced DNA bending.

Nucleic Acids Res, 1997 May 15, 25(10), 2012 - 9
Transcription factor Sp3 antagonizes activation of the ornithine decarboxylase promoter by Sp1; Kumar AP et al.; Ornithine decarboxylase (ODC) expression is important for proliferation and is elevated in many tumor cells . We previously showed that Sp1 is a major positive regulator of ODC transcription . In this paper we have investigated transcriptional regulation of rat ODC by the closely related factor Sp3 . While over-expression of Sp1 caused a dramatic activation of the ODC promoter, over-expression of Sp3 caused little or no activation in either Drosophila SL2 cells (lacking endogenous Sp1 or Sp3) or in H35 rat hepatoma cells . Furthermore, co-transfection studies demonstrated that Sp3 abolished trans -activation of the ODC promoter by Sp1 . DNase I footprint studies and electrophoretic mobility shift assays demonstrated that both recombinant Sp1 and Sp3 bind specifically to several sites within the ODC promoter also protected by nuclear extracts, including overlapping GC and CT motifs located between -116 and -104 . This CT element is a site of negative ODC regulation . Mutation of either element reduced binding, but mutation of both sites was required to eliminate binding of either Sp1 or Sp3 . These results demonstrate that ODC is positively regulated by Sp1 and negatively regulated by Sp3, suggesting that the ratio of these transcription factors may be an important determinant of ODC expression during development or transformation.

Nucleic Acids Res, 1997 May 15, 25(10), 2005 - 11
Analyses of frameshifting at UUU-pyrimidine sites; Schwartz R et al.; Others have recently shown that the UUU phenylalanine codon is highly frameshift-prone in the 3'(rightward) direction at pyrimidine 3'contexts . Here, several approaches are used to analyze frameshifting at such sites . The four permutations of the UUU/C (phenylalanine) and CGG/U (arginine) codon pairs were examined because they vary greatly in their expected frameshifting tendencies . Furthermore, these synonymous sites allow direct tests of the idea that codon usage can control frameshifting . Frameshifting was measured for these dicodons embedded within each of two broader contexts: the Escherichia coli prfB (RF2 gene) programmed frameshift site and a 'normal' message site . The principal difference between these contexts is that the programmed frameshift contains a purine-rich sequence upstream of the slippery site that can base pair with the 3'end of 16 S rRNA (the anti-Shine-Dalgarno) to enhance frameshifting . In both contexts frameshift frequencies are highest if the slippery tRNAPhe is capable of stable base pairing in the shifted reading frame . This requirement is less stringent in the RF2 context, as if the Shine-Dalgarno interaction can help stabilize a quasi-stable rephased tRNA:message complex . It was previously shown that frameshifting in RF2 occurs more frequently if the codon 3'to the slippery site is read by a rare tRNA . Consistent with that earlier work, in the RF2 context frameshifting occurs substantially more frequently if the arginine codon is CGG, which is read by a rare tRNA . In contrast, in the 'normal' context frameshifting is only slightly greater at CGG than at CGU . It is suggested that the Shine-Dalgarno-like interaction elevates frameshifting specifically during the pause prior to translation of the second codon, which makes frameshifting exquisitely sensitive to the rate of translation of that codon . In both contexts frameshifting increases in a mutant strain that fails to modify tRNA base A37, which is 3'of the anticodon . Thus, those base modifications may limit frameshifting at UUU codons . Finally, statistical analyses show that UUU Ynn dicodons are extremely rare in E.coli genes that have highly biased codon usage.

Nucleic Acids Res, 1997 May 15, 25(10), 1984 - 90
A novel genetic system to isolate a dominant negative effector on DNA-binding activity of Oct-2; Terunuma A et al.; Recent studies have revealed that interactions between transcription factors play an important role in regulation of gene expression in eukaryotic cells . To isolate cDNA clones that dominantly inhibit the DNA-binding activity of Oct-2, chosen as a representative factor, we have developed a novel screening system . This employs an Escherichia coli tester strain carrying a modified lac operon as a reporter gene, with the lac operator sequence replaced by an octamer sequence . Oct-2 expressed in this tester strain represses the expression of the reporter gene and changes the phenotype of the cell from Lac+to Lac- . Introduction of a cDNA expression library prepared from a human T-cell line into the Oct-2-harboring tester strain allowed selection of three Lac+clones out of 1 x 10(5) transformants . One of them, hT86, encoding a putative zinc finger protein was found to derepress beta-galactosidase activity in the Oct-2-harboring tester strain at the transcriptional level . In gel mobility shift assays, hT86 attenuated the intensity of the retarded band composed of the octamer probe and Oct-2, suggesting a dominant negative effect on the DNA-binding activity of Oct-2 . The strategy described here provides a new approach for studying protein-protein interactions that govern the complex regulation of gene expression.

Nucleic Acids Res, 1997 May 15, 25(10), 1920 - 9
A truncation in the 14 kDa protein of the signal recognition particle leads to tertiary structure changes in the RNA and abolishes the elongation arrest activity of the particle; Thomas Y et al.; The signal recognition particle (SRP) provides the molecular link between synthesis of polypeptides and their concomitant translocation into the endoplasmic reticulum . During targeting, SRP arrests or delays elongation of the nascent chain, thereby presumably ensuring a high translocation efficiency . Components of the Alu domain, SRP9/14 and the Alu sequences of SRP RNA, have been suggested to play a role in the elongation arrest function of SRP . We generated a truncated SRP14 protein, SRP14-20C, which forms, together with SRP9, a stable complex with SRP RNA . However, particles reconstituted with SRP9/14-20C, RC(9/14-20C), completely lack elongation arrest activity . RC(9/14-20C) particles have intact signal recognition, targeting and ribosome binding activities . SRP9/14-20C therefore only impairs interactions with the ribosome that are required to effect elongation arrest . This result provides evidence that direct interactions between the Alu domain components and the ribosome are required for this function . Furthermore, SRP9/14-20C binding to SRP RNA results in tertiary structure changes in the RNA . Our results strongly indicate that these changes account for the negative effect of SRP14 truncation on elongation arrest, thus revealing a critical role of the RNA in this function.

Nucleic Acids Res, 1997 May 15, 25(10), 1875 - 82
Strong, specific, monodentate G-C base pair recognition by N7-inosine derivatives in the pyrimidine.purine-pyrimidine triple-helical binding motif; Marfurt J et al.; The nucleoside analogs 7-(2'-deoxy-alpha-D-ribofuranosyl)hypoxanthine (alpha7H,1), 7-(2'-deoxy-beta-D-ribofuranosyl)hypoxanthine (beta7H,2) and 7-7-(2'-O-methyl-beta-D- ribofuranosyl)hypoxanthine (beta7HOMe,3) were prepared and incorporated into triplex forming oligodeoxynucleotides, designed to bind to DNA in the parallel (pyrimidine.purine-pyrimidine) motif . By DNase I footprinting techniques and UV-melting curve analysis it was found that, at pH 7 . 0, the 15mer oligonucleotides d(TTTTTMeCTXTMeCTMeCTMeCT) (MeC = 5-methyl-deoxycytidine, X =beta7H,beta7HOMe) bind to a DNA target duplex forming a H.G-C base triple with equal to slightly increased (10-fold) stability compared to a control oligodeoxynucleotide in which the hypoxanthine residue is replaced by MeC . Remarkably, triple-helix formation is specific to G-C base pairs and up to 40 microM third strand concentration, no stable triplex exhibiting H.A-T, H.T-A or H.C-G base arrangements could be found (target duplex concentration approximately 0.1 nM) . Multiply substituted sequences containing beta7H residues either in an isolated {d(TTTTTbeta7HTbeta7HTbeta7HTbeta7HTbeta7HT)} or in a contiguous {d(TTTbeta7Hbeta7Hbeta7Hbeta7HTTTTbeta7HTTT)} manner still form triplexes with their targets of comparable stability as the control (MeC-containing) sequences at pH 7.0 and high salt or spermine containing buffers . General considerations lead to a structural model in which the recognition of the G-C base pair by hypoxanthine takes place via only one H-bond of the N-H of hypoxanthine to N7 of guanine . This model is supported by a molecular dynamics simulation . A general comparison of the triplex forming properties of oligonucleotides containing beta7H with those containing MeC or N7-2'-deoxyguanosine (N7G) reveals that monodentate recognition in the former case can energetically compete with bidentate recognition in the latter two cases.

Biochemistry, 1997 May 13, 36(19), 5884 - 92
A method for assessing the stability of a membrane protein; Lau FW et al.; The integral membrane protein diacylglycerol kinase (DGK) from Escherichia coli has been reversibly unfolded in a protein/detergent/mixed micelle system by varying the molar ratio of n-decyl beta-D-maltoside (DM) and sodium dodecyl sulfate (SDS) . Unfolding was monitored by circular dichroism (CD) and ultraviolet (UV) absorbance spectroscopy . When unfolding is monitored by measuring changes in absorbance at 294 nm, two distinct denaturation phases are observed, indicative of a stable intermediate . When CD is used as a conformational probe, the resulting denaturation curve contains only one major transition, which corresponds to the first unfolding phase observed by absorbance changes . The unfolding behavior of several mutant proteins in which the tryptophan residues were selectively replaced made it possible to assign the first unfolding phase to a denaturation event in a cytoplasmic domain and the second phase to denaturation of the membrane-embedded portion of the protein . The denaturation curves fit well to a model which assumes two cooperative transitions and a linear relationship between unfolding free energy and SDS concentration . Extrapolation back to zero denaturant indicates an unfolding free energy of 6 kcal/mol for the cytoplasmic domain and 16 kcal/mol for the transmembrane domain . The high apparent stability of the transmembrane domain could explain the high degree of tolerance to amino acid substitutions seen for DGK and other membrane proteins . The approach described in this paper may be applicable to other membrane protein systems.

Biochemistry, 1997 May 13, 36(19), 5846 - 52
Two reactive site locations and structure-function study of the arrowhead proteinase inhibitors, A and B, using mutagenesis; Xie ZW et al.; The arrowhead (Sagittaria sagittifolia, Linn.) proteinase inhibitor A and B are double-headed and multifunctional, consisting of 179 amino acid residues with three disulfide bridges . Both their primary structures and cDNA sequences have been elucidated {Yang, H . L., Luo, R . S., Wang, L . X., Zhu, D . X., & Chi, C . W . (1992) J . Biochem . 111, 537; Xu, W . F., Tao, W . K., Gong, Z . Z., & Chi, C . W . (1993) J . Biochem . 113, 153; Luo, M . J., Lu, W . Y., & Chi, C . W . (1997) J . Biochem . (in press)} . Though they share 91% homology, they are different in inhibitory activities . Sequence analysis of their full-length cDNAs showed that there are seven extra residues in the C-terminal part which might be cleaved off by proteinase post-processing . To locate the reactive sites and study the structure-function relationship of the two forms A and B, the genes coding for the mature inhibitor B and its extended form were respectively cloned into the secretion expression vector, pVT102U/alpha, and expressed in Saccharomyces cerevisiae strain S-78 . Both of the gene products were purified and characterized to have the same inhibitory activities as the natural one . The gene product of the extended form was a mixture with the extended C-terminal part of the inhibitor either completely or partially removed . The two previously predicted reactive site residues, Lys-44 and Arg-76 of inhibitor B, were then respectively substituted with Ala by site-directed mutagenesis and expressed . As compared with the natural inhibitor, each of the mutants could only inhibit one molecule, instead of two molecules of trypsin, and displayed an inhibitory activity against elastase, thus confirming the location of the two reactive sites in the inhibitors . The gene coding for inhibitor A, which for some reason could not be expressed in S . cereviciae, was successfully expressed in the reconstructed plasmid pET-1522bx in Escherichia coli strain BL21 with the expressed product existing in the inclusion body . After denaturation and renaturation, the active inhibitor A was obtained and purified by anhydrotrypsin affinity chromatography . Using site-directed mutagenesis, two residues of inhibitor A, namely, Ser-82 and Leu-87, prominently different from Leu-82 and Arg-87 in inhibitor B, were replaced by these two corresponding residues, respectively . As compared with the natural inhibitor A, its S82L mutant showed a lower inhibitory activity toward trypsin, whereas a higher activity was found in the L87R mutant . Meanwhile, both of their chymotrypsin inhibitory activities became weaker than the natural one . The important accessary role of the residue of position 87 in causing the difference in inhibitory properties between inhibitor A and B was discussed.

Biochemistry, 1997 May 13, 36(19), 5769 - 76
Specific recognition of O6-methylguanine in DNA by active site mutants of human O6-methylguanine-DNA methyltransferase; Hazra TK et al.; O6-Methylguanine-DNA methyltransferase (MGMT), a ubiquitous DNA repair protein, acts as a monomer in removing the mutagenic DNA adduct O6-alkylguanine (induced by alkylating carcinogens) via a stoichiometric reaction . The alkyl group is transferred without a cofactor to a specific cysteine acceptor residue of MGMT, Cys-145 in the case of human MGMT, containing 207 amino acid residues and thereby inactivates the protein . As a prelude to the investigation of the reaction mechanism of human MGMT by elucidation of its structure in free and substrate-bound forms via NMR spectroscopy and X-ray crystallography, two types of MGMT mutants were generated and characterized . First, systematic deletion analysis of the protein was carried out to determine the smallest size at which it is active or inactive but forms a stable complex with the substrate and so may be useful for NMR spetroscopic analysis . Deletion of more than 8 or 31 residues from the amino or carboxyl terminus, respectively, led to the loss of both activity and substrate binding . Removal of Arg-9 or Leu-176 and distal residues inactivated the protein, presumably by altering its tertiary structure . On the basis of the criteria of bacterial overexpression and solubility, the mutant MGMT with deletion of 28 residues at the carboxyl terminus should be suitable for NMR studies . In the second approach, we examined mutants at the active site (Cys-145) that retain substrate binding . Inactive C145A and C145S substitution mutants were found to form specific and stable complexes with an O6-methylguanine (m6G)-containing oligonucleotide substrate . Wild type MGMT also formed a similar complex, but only as a transient intermediate . Footprinting studies indicated a strong discriminatory effect of the base adduct on the binding of C145A to substrate DNA; 17-18 nucleotides on the m6G-containing strand and 13-14 nucleotides in the complementary strand spanning the base adduct were protected from DNase I digestion by the mutant protein . These results, as well as the identical protease sensitivity of the wild type and mutant proteins, suggest minimal structural change due to conservative mutations at the active site . Thus, the mutant proteins may be utilized for solving the structure and mechanism of human MGMT.

Biochemistry, 1997 May 13, 36(19), 5612 - 23
Urea and thermal equilibrium denaturation studies on the dimerization domain of Escherichia coli Trp repressor; Gloss LM et al.; The urea-induced equilibrium unfolding of the Escherichia coli Trp repressor (TR) is a two-state process, involving the native dimeric and unfolded monomeric species . Kinetic studies, however, reveal the presence of transient intermediates that appear only during the folding of the 107-residue protein {Gittelman, M . G., & Matthews, C . R . (1990) Biochemistry 29, 7011-7020} . In order to gain insight into the complex kinetic folding mechanism, the sequence of TR was reduced to the amino-terminal 66 residues, corresponding to the dimerization domain . Two polypeptides, 2-66 and NHis-7-66, were shown to be dimeric at 25 degrees C by size exclusion chromatography and to retain native-like spectroscopic features as evidenced by near- and far-UV circular dichroism and fluorescence spectroscopy . The equilibrium properties of the urea-induced folding of these core fragments were examined by intrinsic tryptophan fluorescence and circular dichroism and found to be well described by a two-state model . At 25 degrees C, the stabilities of both fragments are 14 kcal mol(-1), as compared to the 24 kcal mol(-1) observed for full-length TR . In contrast, the thermal denaturation of {2-66}2 and full-length TR are three-state processes; the midpoint of the transition monitored by absorbance at 292 nm precedes that monitored by circular dichroism at 222 nm . Global analysis of the thermal data as a function of monomer concentration suggests that both the full-length and {2-66}2 TR variants unfold via a dimeric intermediate . Taken together, these results demonstrate that the {2-66}2 fragment constitutes a well-structured, independently folding subdomain of TR that may be useful in elucidating the properties of the transient intermediates observed in the folding of the full-length protein . The dimeric intermediate observed in the thermal denaturation of {2-66}2 suggests that it may be possible to further reduce the core sequence while maintaining the ability to dimerize.

Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 5461 - 6
A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine; Li L et al.; S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin . The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants . In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied . However, little is known about lignin pathway OMTs in gymnosperms . We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT) . The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences . However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis . The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem . Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem.

Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 5417 - 21
Novel form of crosstalk between G protein and tyrosine kinase pathways; Diverse-Pierluissi M et al.; Neuronal Ca2+ channels are inhibited by a variety of transmitter receptors coupled to Go-type GTP-binding proteins . Go has been postulated to work via a direct interaction between an activated G protein subunit and the Ca2+ channel complex . Here we show that the inhibition of sensory neuron N-type Ca2+ channels produced by gamma-aminobutyric acid involves a novel, rapidly activating tyrosine kinase signaling pathway that is mediated by Galphao and a src-like kinase . In contrast to other recently described G protein-coupled tyrosine kinase pathways, the Galphao-mediated modulation requires neither protein kinase C nor intracellular Ca2+ . The results suggest that this pathway mediates rapid receptor-G protein signaling in the nervous system and support the existence of a previously unrecognized form of crosstalk between G protein and tyrosine kinase pathways.

Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 5405 - 10
Interaction of the synprint site of N-type Ca2+ channels with the C2B domain of synaptotagmin I; Sheng ZH et al.; N-type Ca2+ channels mediate Ca2+ influx, which initiates fast exocytosis of neurotransmitters at synapses, and they interact directly with the SNARE proteins syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) through a synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their alpha1B subunits . Introduction of peptides containing the synprint site into presynaptic neurons reversibly inhibits synaptic transmission, confirming the importance of interactions with this site in synaptic transmission . Here we report a direct interaction of the synprint peptide from N-type Ca2+ channels with synaptotagmin I, an important Ca2+ sensor for exocytosis, as measured by an affinity-chromatography binding assay and a solid-phase immunoassay . This interaction is mediated by the second C2 domain (C2B) of synaptotagmin I, but is not regulated by Ca2+ . Using both immobilized recombinant proteins and native presynaptic membrane proteins, we found that the synprint peptide and synaptotagmin competitively interact with syntaxin . This interaction is Ca2+-dependent because of the Ca2+ dependence of the interactions between syntaxin and these two proteins . These results provide a molecular basis for a physical link between Ca2+ channels and synaptotagmin, and suggest that N-type Ca2+ channels may undergo a complex series of Ca2+-dependent interactions with multiple presynaptic proteins during neurotransmission.

Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 5290 - 5
Prevention of autoimmune disease due to lymphocyte modulation by the B-subunit of Escherichia coli heat-labile enterotoxin; Williams NA et al.; We demonstrate that the receptor binding moiety of Escherichia coli heat-labile enterotoxin (EtxB) can completely prevent autoimmune disease in a murine model of arthritis . Injection of male DBA/1 mice at the base of the tail with type II collagen in the presence of complete Freund's adjuvant normally leads to arthritis, as evidenced by inflammatory infiltration and swelling of the joints . A separate injection of EtxB at the same time as collagen challenge prevented leukocyte infiltration, synovial hyperplasia, and degeneration of the articular cartilage and reduced clinical symptoms of disease by 82% . The principle biological property of EtxB is its ability to bind to the ubiquitous cell surface receptor GM1 ganglioside, and to other galactose-containing glycolipids and galactoproteins . The importance of receptor interaction in mediating protection from arthritis was demonstrated by the failure of a non-receptor-binding mutant of EtxB to elicit any protective effect . Analysis of T cell responses to collagen, in cultures of draining lymph node cells, revealed that protection was associated with a marked increase in interleukin 4 production concomitant with a reduction in interferon gamma levels . Furthermore, in protected mice there was a significant reduction in anti-collagen antibody levels as well as an increase in the IgG1/IgG2a ratio . These observations show that protection is associated with a shift in the Th1/Th2 balance as well as a general reduction in the extent of the anti-type II collagen immune response . This suggests that EtxB-receptor-mediated modulation of lymphocyte responses provides a means of preventing autoimmune disease.

Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 5278 - 83
High-affinity binding of bioactive glycosylation-inhibiting factor to antigen-primed T cells and natural killer cells; Sugie K et al.; High-affinity binding was demonstrated between suppressor-T-cell-derived bioactive glycosylation-inhibiting factor (GIF) and helper T hybridomas and natural killer cell line cells . Inactive GIF present in cytosol of suppressor T cells and Escherichia coli-derived recombinant human GIF (rhGIF) failed to bind to these cells . However, affinity of rhGIF for the target cells was generated by replacement of Cys-57 in the sequence with Ala or of Asn-106 with Ser or binding of 5-thio-2-nitrobenzoic acid to Cys-60 in the molecule . Such mutations and the chemical modification of rhGIF synergistically increased the affinity of GIF molecules for the target cells . The results indicated that receptors on the target cells recognize conformational structures of bioactive GIF . Equilibrium dissociation constant (Kd) of the specific binding between bioactive rGIF derivatives and high-affinity receptors was 10-100 pM . Receptors for bioactive GIF derivatives were detected on Th1 and Th2 T helper clones and natural killer NK1.1(+) cells in normal spleen but not on naive T or B cells . Neither the inactive rGIF nor bioactive rGIF derivatives bound to macrophage and monocyte lines or induced macrophages for tumor necrosis factor alpha production.

Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 4982 - 7
RNA polymerase sigma factor determines start-site selection but is not required for upstream promoter element activation on heteroduplex (bubble) templates; Fredrick K et al.; Sequence-selective transcription by bacterial RNA polymerase (RNAP) requires sigma factor that participates in both promoter recognition and DNA melting . RNAP lacking sigma (core enzyme) will initiate RNA synthesis from duplex ends, nicks, gaps, and single-stranded regions . We have used DNA templates containing short regions of heteroduplex (bubbles) to compare initiation in the presence and absence of various sigma factors . Using bubble templates containing the sigmaD-dependent flagellin promoter, with or without its associated upstream promoter (UP) element, we demonstrate that UP element stimulation occurs efficiently even in the absence of sigma . This supports a model in which the UP element acts primarily through the alpha subunit of core enzyme to increase the initial association of RNAP with the promoter . Core and holoenzyme do differ substantially in the template positions chosen for initiation: sigmaD restricts initiation to sites 8-9 nucleotides downstream of the conserved -10 element . Remarkably, sigmaA also has a dramatic effect on start-site selection even though the sigmaA holoenzyme is inactive on the corresponding homoduplexes . The start sites chosen by the sigmaA holoenzyme are located 8 nucleotides downstream of sequences on the nontemplate strand that resemble the conserved -10 hexamer recognized by sigmaA . Thus, sigmaA appears to recognize the -10 region even in a single-stranded state . We propose that in addition to its described roles in promoter recognition and start-site melting, sigma also localizes the transcription start site.

Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 4978 - 81
Trigger factor is induced upon cold shock and enhances viability of Escherichia coli at low temperatures; Kandror O et al.; Trigger factor (TF) in Escherichia coli is a molecular chaperone with remarkable properties: it has prolyl-isomerase activity, associates with nascent polypeptides on ribosomes, binds to GroEL, enhances GroEL's affinity for unfolded proteins, and promotes degradation of certain polypeptides . Because the latter effects appeared larger at 20 degrees C, we studied the influence of temperature on TF expression . Unlike most chaperones (e.g., GroEL), which are heat-shock proteins (hsps), TF levels increased progressively as growth temperature decreased from 42 degrees C to 16 degrees C and even rose in cells stored at 4 degrees C . Upon temperature downshift from 37 degrees C to 10 degrees C or exposure to chloramphenicol, TF synthesis was induced, like that of many cold-shock proteins . We therefore tested if TF expression might be important for viability at low temperatures . When stored at 4 degrees C, E . coli lose viability at exponential rates . Cells with reduced TF content die faster, while cells overexpressing TF showed greater viability . Although TF overproduction protected against cold, it reduced viability at 50 degrees C, while TF deficiency enhanced viability at this temperature . By contrast, overproduction of GroEL/ES, or hsps generally, while protective against high temperatures, reduced viability at 4 degrees C, which may explain why expression of hsps is suppressed in the cold . Thus, TF represents an example of an E . coli protein which protects cells against low temperatures . Moreover, the differential induction of TF at low temperatures and hsps at high temperatures appears to provide selective protection against these opposite thermal extremes.

Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 4913 - 8
Structural basis of DNA bending and oriented heterodimer binding by the basic leucine zipper domains of Fos and Jun; Leonard DA et al.; Interactions among transcription factors that bind to separate sequence elements require bending of the intervening DNA and juxtaposition of interacting molecular surfaces in an appropriate orientation . Here, we examine the effects of single amino acid substitutions adjacent to the basic regions of Fos and Jun as well as changes in sequences flanking the AP-1 site on DNA bending . Substitution of charged amino acid residues at positions adjacent to the basic DNA-binding domains of Fos and Jun altered DNA bending . The change in DNA bending was directly proportional to the change in net charge for all heterodimeric combinations between these proteins . Fos and Jun induced distinct DNA bends at different binding sites . Exchange of a single base pair outside of the region contacted in the x-ray crystal structure altered DNA bending . Substitution of base pairs flanking the AP-1 site had converse effects on the opposite directions of DNA bending induced by homodimers and heterodimers . These results suggest that Fos and Jun induce DNA bending in part through electrostatic interactions between amino acid residues adjacent to the basic region and base pairs flanking the AP-1 site . DNA bending by Fos and Jun at inverted binding sites indicated that heterodimers bind to the AP-1 site in a preferred orientation . Mutation of a conserved arginine within the basic regions of Fos and transversion of the central C:G base pair in the AP-1 site to G:C had complementary effects on the orientation of heterodimer binding and DNA bending . The conformational variability of the Fos-Jun-AP-1 complex may contribute to its functional versatility at different promoters.

Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 4907 - 12
A sigma32 mutant with a single amino acid change in the highly conserved region 2.2 exhibits reduced core RNA polymerase affinity; Joo DM et al.; sigma32, the product of the rpoH gene in Escherichia coli, provides promoter specificity by interacting with core RNAP . Amino acid sequence alignment of sigma32 with other sigma factors in the sigma70 family has revealed regions of sequence homology . We have investigated the function of the most highly conserved region, 2.2, using purified products of various rpoH alleles . Core RNAP binding analysis by glycerol gradient sedimentation has revealed reduced core RNAP affinity for one of the mutant sigma32 proteins, Q80R . This reduced core interaction is exacerbated in the presence of sigma70, which competes with sigma32 for binding of core RNAP . When a different but more conserved amino acid was introduced at this position by site-directed mutagenesis (Q80N), this mutant sigma factor still displayed a significant reduction in its core RNAP affinity . Based on these results, we conclude that at least one specific amino acid in region 2.2 is involved in core RNAP interaction.

Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 4872 - 7
Redesign of soluble fatty acid desaturases from plants for altered substrate specificity and double bond position; Cahoon EB et al.; Acyl-acyl carrier protein (ACP) desaturases introduce double bonds at specific positions in fatty acids of defined chain lengths and are one of the major determinants of the monounsaturated fatty acid composition of vegetable oils . Mutagenesis studies were conducted to determine the structural basis for the substrate and double bond positional specificities displayed by acyl-ACP desaturases . By replacement of specific amino acid residues in a Delta6-palmitoyl (16:0)-ACP desaturase with their equivalents from a Delta9-stearoyl (18:0)-ACP desaturase, mutant enzymes were identified that have altered fatty acid chain-length specificities or that can insert double bonds into either the Delta6 or Delta9 positions of 16:0- and 18:0-ACP . Most notably, by replacement of five amino acids (A181T/A200F/S205N/L206T/G207A), the Delta6-16:0-ACP desaturase was converted into an enzyme that functions principally as a Delta9-18:0-ACP desaturase . Many of the determinants of fatty acid chain-length specificity in these mutants are found in residues that line the substrate binding channel as revealed by x-ray crystallography of the Delta9-18:0-ACP desaturase . The crystallographic model of the active site is also consistent with the diverged activities associated with naturally occurring variant acyl-ACP desaturases . In addition, on the basis of the active-site model, a Delta9-18:0-ACP desaturase was converted into an enzyme with substrate preference for 16:0-ACP by replacement of two residues (L118F/P179I) . These results demonstrate the ability to rationally modify acyl-ACP desaturase activities through site-directed mutagenesis and represent a first step toward the design of acyl-ACP desaturases for the production of novel monounsaturated fatty acids in transgenic oilseed crops.

Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 4866 - 71
Crystal structure of glutamate-1-semialdehyde aminomutase: an alpha2-dimeric vitamin B6-dependent enzyme with asymmetry in structure and active site reactivity; Hennig M et al.; The three-dimensional structure of glutamate-1-semialdehyde aminomutase (EC 5.4.3.8), an alpha2-dimeric enzyme from Synechococcus, has been determined by x-ray crystallography using heavy atom derivative phasing . The structure, refined at 2.4-A resolution to an R-factor of 18.7% and good stereochemistry, explains many of the enzyme's unusual specificity and functional properties . The overall fold is that of aspartate aminotransferase and related B6 enzymes, but it also has specific features . The structure of the complex with gabaculine, a substrate analogue, shows unexpectedly that the substrate binding site involves residues from the N-terminal domain of the molecule, notably Arg-32 . Glu-406 is suitably positioned to repel alpha-carboxylic acids, thereby suggesting a basis for the enzyme's reaction specificity . The subunits show asymmetry in cofactor binding and in the mobilities of the residues 153-181 . In the unliganded enzyme, one subunit has the cofactor bound as an aldimine of pyridoxal phosphate with Lys-273 and, in this subunit, residues 153-181 are disordered . In the other subunit in which the cofactor is not covalently bound, residues 153-181 are well defined . Consistent with the crystallographically demonstrated asymmetry, a form of the enzyme in which both subunits have pyridoxal phosphate bound to Lys-273 through a Schiff base showed biphasic reduction by borohydride in solution . Analysis of absorption spectra during reduction provided evidence of communication between the subunits . The crystal structure of the reduced form of the enzyme shows that, despite identical cofactor binding in each monomer, the structural asymmetry at residues 153-181 remains.

J Immunol Methods, 1997 May 12, 204(1), 51 - 6
A flow cytometric method for the rapid detection of beta-galactosidase transfected cells: an in vitro and in vivo study; Mouawad R et al.; A flow cytometric method has been developed for the rapid analysis of lacZ transduced cells . The method described is based on an indirect immunofluorescence staining procedure using a monoclonal antibody which binds specifically to beta-galactosidase from E . coli and to beta-galactosidase fusion proteins . This technique was used for the quantification in vitro as well as in vivo of beta-galactosidase expression in B16 melanoma cells . The described method is appropriate for a variety of cell types (species, lineage), is simple, quantitative, reliable, rapid and applicable to all constructs containing the lacZ selectable markers . It should prove to be very helpful (1) for the quantification of cells expressing the lacZ reporter gene and (2) for studying gene regulation, including transfection modality, promoter efficacy, enhancer activity, and other regulatory factors.

FEBS Lett, 1997 May 12, 408(1), 99 - 104
Regulation of the DNA binding of p53 by its interaction with protein kinase CK2; Prowald A et al.; Some of the numerous functions of the growth suppressor protein p53 are regulated by its interaction with viral and cellular proteins . C-terminal sequences of p53 are implicated in binding to the regulatory beta-subunit of protein kinase CK2 . Using a p53-specific DNA binding element we found that the beta-subunit of CK2 inhibited the DNA binding of p53 whereas the alpha-subunit had no influence . The CK2 holoenzyme consisting of two alpha- and two beta-subunits led to a supershift in DNA binding of p53 similar to the p53-specific monoclonal antibody PAb421 as well as the C-terminus of p53 . Thus, our results showed an individual role of the free beta-subunit of CK2 on the DNA binding activity of p53.

FEBS Lett, 1997 May 12, 408(1), 62 - 6
Cloning, expression and characterisation of murine procathepsin E; Tatnell PJ et al.; The cDNA encoding murine procathepsin E was isolated and sequenced and recombinant enzyme was produced in Escherichia coli . The activity of the purified recombinant mouse cathepsin E was characterised quantitatively using two synthetic peptide substrates and naturally occurring inhibitors . The majority of the recombinant enzyme was present as a homodimer (Mr approximately 80) in which the two monomers were linked by an intermolecular disulfide bond . By analogy to previous studies with human cathepsin E, this is most likely a consequence of the presence of a unique cysteine residue near the N-terminus of the mature proteinase . The availability of (i) recombinant murine enzyme in reasonable quantities and (ii) a full-length cDNA now enables structural investigations and attempts to generate 'knock-out' mice deficient in this important aspartic proteinase to be undertaken.

FEBS Lett, 1997 May 12, 408(1), 52 - 6
Molecular heterogeneity of the cDNA encoding a 74-kDa regulatory subunit (B" or delta) of human protein phosphatase 2A; Tanabe O et al.; Two cDNAs for possible splicing variants of a 74-kDa regulatory subunit (B" or delta) of human protein phosphatase 2A, were isolated . These variants were identified from human cerebral cortex by library screening and PCR, and designated delta1 and delta3 isoforms, while the previously reported isoform {Tanabe et al . (1996) FEBS Lett . 379, 107-1111 was designated delta2 . Compared with the delta2 isoform, the delta1 isoform contained a 32-residue insertion beginning at residue 84, and consisted of 602 amino acids in all . The delta3 isoform lacked a 74-residue sequence corresponding to residues 1083 of the delta2 isoform, and consisted of 496 amino acids . Using isoform-specific antipeptide antisera, the 74-kDa subunit (B" or delta) originally purified from human erythrocytes was identified as the delta1 isoform.

FEBS Lett, 1997 May 12, 408(1), 25 - 9
DNA binding sites recognised in vitro by a knotted class 1 homeodomain protein encoded by the hooded gene, k, in barley (Hordeum vulgare); Krusell L et al.; The homeodomain of the knotted classes of transcription factors from plants differs from the well characterized Antp/En type homeodomains from Drosophila at key amino acid residues contributing to the DNA binding . A cDNA, Hvh21, derived from the hooded gene and encoding a full length homolog of knotted1 from maize was isolated from barley seedlings and expressed as a maltose binding protein fusion in E . coli . The purified HvH21-fusion protein selected DNA fragments with 1-3 copies of the sequence TGAC . Gel shift experiments showed that the TGAC element was required for binding and the results further indicate that the HvH21-fusion protein binds DNA as a monomer.

FEBS Lett, 1997 May 12, 408(1), 11 - 5
Subunit interactions in the Escherichia coli protein translocase: SecE and SecG associate independently with SecY; Homma T et al.; We used hexahistidine-tagged SecE and SecY to study how the core subunits (SecY, SecE and SecG) of Escherichia coli protein translocase interact with each other . Detergent extracts were prepared from the plasma membranes and fractionated by Ni2+-NTA agarose affinity binding . Although His6-SecE, expressed in wild-type cells, brought down both SecY and SecG, neither of them was brought down when the same protein was expressed in the secY24 mutant cells . His6-SecY brought down both SecE and SecG, as expected . Interestingly, His6-SecY24 was able to bring down SecG but not SecE . These results confirm our previous conclusion that the secY24 alteration impairs the SecY-SecE interaction, and demonstrate that SecY and SecG can form a complex that does not contain SecE . Likewise, SecY-SecE complex could be isolated from the secG-deleted strain . The trimeric complex, in detergent extracts, dissociated at a critical temperature between 23 and 26 degrees C, whereas the SecY-SecE complex without SecG dissociated at a slightly lower temperature (20-23 degrees C) . We conclude that each of SecE and SecG independently binds to SecY, the central subunit of protein translocase, although the trimeric complex is more stable than the binary complexes.

Biochem Pharmacol, 1997 May 9, 53(9), 1323 - 32
Compartmentalisation and characteristics of a Ca2+-dependent phospholipase A2 in human colon mucosa; Lamura E et al.; The biochemical properties of the phospholipase A2 (PLA2) found in the 100,000 x g centrifugate cytosol or particulate fractions of human colonic mucosa have been investigated using both deoxycholate-solubilized and Escherichia coli (E . coli) phospholipids as substrates . PLA2 activity was present in both subcellular fractions and the profiles of biochemical activites were similar . Activity in the particulate fraction was approximately twofold greater than the cytosol fraction when expressed on the basis of protein concentration . The PLA2 is Ca2+ dependent and using EGTA-regulated buffers cytosolic or particulate fraction activity was similar at both 10 microm or 10 mm Ca2+ concentrations . Using deoxycholate-phospholipid micelles as substrate a small but statistically significant twofold preference for glycero-phosphatidylcholine bearing sn-2-arachidonate compared with sn-2-oleate was seen, but this preference was not noted using arachidonate or oleate labelled E . coli membranes . Dithiothreitol (10 mM) reduced colon mucosal cytosol PLA2 activity significantly by 63.5 +/- 1.90% in cytosol and by 30.54 +/- 1.27% in microsomes using micelles as substrate or by 84.3 +/- 2.30% in cytosol and by 69.33 +/- 11.30% in microsomes using oleate-labelled E . coli as substrates . Warming at 57 degrees C reduced activity significantly by 35.0 +/- 5.80% in microsomes and by 40.0 +/- 7.08% in cytosol . Acid treatment increased PLA2 activity to 148 +/- 16.3% in microsomes and 145 +/- 18.6% in cytosol . When mucosal preparations were subjected to heparin-Sepharose chromatography, it bound tightly and eluted in the same position on a salt gradient as authentic human group II PLA2 . Further purification by gel-permeation chromatography gave activity in the 14 kDa region of the elution profile . These features have many of the characteristics expected of a 14 kDa isoform of PLA2 but exhibit activity at concentrations of Ca2+ that are relevant in the intracellular environment and may participate in cellular lipid metabolism.

J Biol Chem, 1997 May 9, 272(19), 12847 - 53
Unique structural features of a novel class of small heat shock proteins; Leroux MR et al.; Small heat shock proteins (smHSPs) and alpha-crystallins constitute a family of related molecular chaperones that exhibit striking variability in size, ranging from 16 to 43 kDa . Structural studies on these proteins have been hampered by their tendency to form large, often dynamic and heterogeneous oligomeric complexes . Here we describe the structure and expression of HSP12.6, a member of a novel class of smHSPs from the nematode Caenorhabditis elegans . Like other members of its class, HSP12.6 possesses a conserved alpha-crystallin domain but has the shortest N- and C-terminal regions of any known smHSP . Expression of HSP12.6 is limited to the first larval stage of C . elegans and is not significantly up-regulated by a wide range of stressors . Unlike other smHSPs, HSP12.6 does not form large oligomeric complexes in vivo . HSP12.6 was produced in Escherichia coli as a soluble protein and purified . Cross-linking and sedimentation velocity analyses indicate that the recombinant HSP12.6 is monomeric, making it an ideal candidate for structure determination . Interestingly, HSP12.6 does not function as a molecular chaperone in vitro, since it is unable to prevent the thermally induced aggregation of a test substrate . The structural and functional implications of these findings are discussed.

J Biol Chem, 1997 May 9, 272(19), 12816 - 23
DNA topoisomerases regulate R-loop formation during transcription of the rrnB operon in Escherichia coli; Masse E et al.; Recent in vivo and in vitro studies have suggested an important role for DNA topoisomerases in regulating R-loop formation during transcription in Escherichia coli . In the present report we present genetic and biochemical evidence strongly suggesting that R-loop formation can occur during transcription of a portion of the rrnB operon and that it is regulated by DNA topoisomerase activity . We found that a multicopy plasmid (pBR322) carrying an heavily transcribed portion of the rrnB operon cannot be transformed in topA mutants unless RNase H is overproduced . Transcription of the 567-base pair HindIII fragment from the rrnB operon allows the extraction of large amount of R-looped plasmid DNAs from a topA mutant, in a manner that depends on the intracellular level of RNase H activity . When DNA gyrase is sufficiently active, hypernegatively supercoiled plasmid DNA is produced if the same DNA fragment is transcribed in a topA mutant . The formation of such topoisomers most likely reflect the presence of extensive R-loops since it is sensitive to the intracellular level of RNase H activity . Finally, the formation of R-looped plasmid DNAs in an in vitro transcription system using phage RNA polymerases is also detected when the 567-base pair HindIII fragment is transcribed on a negatively supercoiled DNA template.

J Biol Chem, 1997 May 9, 272(19), 12771 - 7
The DNA binding pattern of the retinoid X receptor is regulated by ligand-dependent modulation of its oligomeric state; Kersten S et al.; The retinoid X receptor (RXR) regulates target gene transcription via its association with cognate DNA response elements either as a homodimer or as a heterodimer with a number of other nuclear receptors . We previously demonstrated that, in solution, RXR forms tetramers with a high affinity and that ligand binding leads to dissociation of receptor tetramers to smaller species . Here it is shown that RXR tetramers form stable complexes with direct repeats (DR-1 or DR-5) or palindromic (TREpal) response elements . Binding of RXR tetramers to cognate DNA occurs with a significantly higher affinity as compared with dimers . Ligand binding by DNA-bound RXR tetramers results in their dissociation to DNA-bound dimers, a process that is completely reversed upon removal of the ligand . Formation of stable tetramer-DNA complexes requires binding of two oligonucleotides/tetramer . It is proposed that ligand-dependent modulation of the oligomeric state of RXR is a regulatory feature of this nuclear receptor.

J Biol Chem, 1997 May 9, 272(19), 12723 - 9
Cloning, expression, and catalytic mechanism of murine lysophospholipase I; Wang A et al.; A lysophospholipase (LysoPLA I) has been purified and characterized from the mouse macrophage-like P388D1 cell line (Zhang, Y . Y, and Dennis, E . A . (1988) J . Biol . Chem . 263, 9965-9972) . This enzyme has now been sequenced, cloned, and expressed in Escherichia coli cells . The enzyme contains 230 amino acid residues with a calculated molecular mass of 24.7 kDa . It has a high helical content in its predicated secondary structure, which is also indicated in its CD spectrum . The cloned LysoPLA I was purified to homogeneity from the transformed E . coli cells by a gel filtration column and an ion exchange column . The specific activity of the purified protein is 1 . 47 micromol/min.mg toward 1-palmitoyl-sn-glycero-3-phosphorylcholine at pH 8.0 and 40 degrees C, corresponding to the reported value of 1.3-1.7 micromol/min.mg for the protein purified from the P388D1 cells . In addition, the cloned protein cross-reacted with an antibody raised against LysoPLA I also purified from the P388D1 cells . The deduced LysoPLA I sequence contains a well conserved GXSXG motif found in the active site of many serine enzymes, and the activity of the LysoPLA I was irreversibly inhibited by the classical serine protease inhibitor diisopropyl fluorophosphate . Furthermore, site-directed mutagenesis was employed to change Ser-119 in the GXSXG motif to an Ala . The resulting mutant protein lost all of its lysophospholipase activity, even though it had the same overall protein conformation as that of the wild-type LysoPLA I . Therefore, LysoPLA I has been demonstrated to be a serine enzyme with Ser-119 at the active site.

J Biol Chem, 1997 May 9, 272(19), 12568 - 74
Heme oxygenase-2 is a hemoprotein and binds heme through heme regulatory motifs that are not involved in heme catalysis; McCoubrey WK Jr et al.; The heme oxygenase (HO) system degrades heme to biliverdin and CO and releases chelated iron . In the primary sequence of the constitutive form, HO-2, there are three potential heme binding sites: two heme regulatory motifs (HRMs) with the absolutely conserved Cys-Pro pair, and a conserved 24-residue heme catalytic pocket with a histidine residue, His151 in rat HO-2 . The visible and pyridine hemochromogen spectra suggest that the Escherichia coli expressed purified HO-2 is a hemoprotein . The absorption spectrum, heme fluorescence quenching, and heme titration analysis of the wild-type protein versus those of purified double cysteine mutant (Cys264/Cys281 --> Ala/Ala) suggest a role of the HRMs in heme binding . While the His151 --> Ala mutation inactivates HO-2, Cys264 --> Ala and Cys281 --> Ala mutations individually or together (HO-2 mut) do not decrease HO activity . Also, Pro265 --> Ala or Pro282 --> Ala mutation does not alter HO-2 activity . Northern blot analysis of ptk cells indicates that HO-2 mRNA is not regulated by heme . The findings, together with other salient features of HO-2 and the ability of heme-protein complexes to generate oxygen radicals, are consistent with HO-2, like five other HRM-containing proteins, having a regulatory function in the cell.

J Biol Chem, 1997 May 9, 272(19), 12508 - 12
Antisense RNA control of plasmid R1 replication . The dominant product of the antisense rna-mrna binding is not a full RNA duplex; Malmgren C et al.; The replication frequency of plasmid R1 is controlled by an antisense RNA (CopA) that binds to its target site (CopT) in the leader region of repA mRNA and inhibits the synthesis of the replication initiator protein RepA . Previous studies on CopA-CopT pairing in vitro revealed the existence of a primary loop-loop interaction (kissing complex) that is subsequently converted to an almost irreversible duplex . However, the structure of more stable binding intermediates that lead to the formation of a complete duplex was speculative . Here, we investigated the interaction between CopA and CopT by using Pb(II)-induced cleavages . The kissing complex was studied using a truncated antisense RNA (CopI) that is unable to form a full duplex with CopT . Furthermore, RNase III, which is known to process the CopA-CopT complex in vivo, was used to detect the existence of a full duplex . Our data indicate that the formation of a full CopA-CopT duplex appears to be a very slow process in vitro . Unexpectedly, we found that the loop-loop interaction persists in the predominant CopA-CopT complex and is stabilized by intermolecular base pairing involving the 5'-proximal 30 nucleotides of CopA and the complementary region of CopT . This almost irreversible complex suffices to inhibit ribosome binding at the tap ribosome binding site and may be the inhibitory complex in vivo.

J Biol Chem, 1997 May 9, 272(19), 12505 - 7
Molecular cloning and expression of amadoriase isoenzyme (fructosyl amine:oxygen oxidoreductase, EC 1.5.3) from Aspergillus fumigatus; Takahashi M et al.; Amadoriase is an enzyme catalyzing the oxidative deglycation of Amadori products to yield corresponding amino acids, glucosone, and H2O2 . We previously reported the purification and characterization of two amadoriase isozymes from Aspergillus sp . that degrade both glycated low molecular weight amines and amino acids (Takahashi, M., Pischetsrieder, M., and Monnier, V . M . (1997) J . Biol . Chem . 272, 3437-3443) . To identify the primary structure of the enzymes, we have prepared a cDNA library from Aspergillus fumigatus induced with fructosyl propylamine and isolated a clone using polyclonal anti-amadoriase II antibody . The primary structure of the enzyme deduced from the nucleotide sequence comprises 438 amino acid residues with a predicted molecular mass of 48,798 Da . The deduced primary structure exhibits the presence of an ADP-binding motif near the NH2 terminus . The identity of the amadoriase II cDNA was further confirmed by expression in Escherichia coli cells with an inducible expression system . Northern blotting analysis revealed that amadoriase II was induced by fructosyl propylamine in a dose-dependent manner.

J Biol Chem, 1997 May 9, 272(19), 12384 - 92
Mutagenesis and chemical rescue indicate residues involved in beta-aspartyl-AMP formation by Escherichia coli asparagine synthetase B; Boehlein SK et al.; Site-directed mutagenesis and kinetic studies have been employed to identify amino acid residues involved in aspartate binding and transition state stabilization during the formation of beta-aspartyl-AMP in the reaction mechanism of Escherichia coli asparagine synthetase B (AS-B) . Three conserved amino acids in the segment defined by residues 317-330 appear particularly crucial for enzymatic activity . For example, when Arg-325 is replaced by alanine or lysine, the resulting mutant enzymes possess no detectable asparagine synthetase activity . The catalytic activity of the R325A AS-B mutant can, however, be restored to about 1/6 of that of wild-type AS-B by the addition of guanidinium HCl (GdmHCl) . Detailed kinetic analysis of the rescued activity suggests that Arg-325 is involved in stabilization of a pentacovalent intermediate leading to the formation beta-aspartyl-AMP . This rescue experiment is the second example in which the function of a critical arginine residue that has been substituted by mutagenesis is restored by GdmHCl . Mutation of Thr-322 and Thr-323 also produces enzymes with altered kinetic properties, suggesting that these threonines are involved in aspartate binding and/or stabilization of intermediates en route to beta-aspartyl-AMP . These experiments are the first to identify residues outside of the N-terminal glutamine amide transfer domain that have any functional role in asparagine synthesis.

Biochem Biophys Res Commun, 1997 May 8, 234(1), 283 - 7
Expression of the Solfolobus acidocaldarius Rieske iron sulfur protein II (SOXF) with the correctly inserted {2FE-2S} cluster in Escherichia coli; Schmidt CL et al.; The Rieske protein II (Schmidt et al., 1996, FEBS Lett . 388, 43-46) from the thermoacidophilic crenarcheon Sulfolobus acidocaldarius (DSM 639) was expressed in E . coli cells . The full length protein was strictly bound to the E . coli membranes and could only be removed by detergent treatment indicating the presence of a membrane anchor . The iron sulfur cluster was correctly inserted into a fraction of the full length protein and much more effectively into a soluble form created by the deletion of the 45 N-terminal amino acids . The soluble form of the protein displayed the typical spectroscopic properties of a respiratory Rieske protein . The midpoint potential was +375 mV determined by CD redox potentiometry . The presented data demonstrate that the structure of the recombinant protein is very similar or identical to the authentic protein making this a powerful model system for the studies of Rieske proteins by site directed mutagenesis.

Biochem Biophys Res Commun, 1997 May 8, 234(1), 183 - 9
Membrane binding and enzymatic activation of a Dbl homology domain require the neighboring pleckstrin homology domain; Wang DS et al.; Dbl-homology (DH) domains are invariably located immediately N-terminal to a pleckstrin homology (PH) domain . To understand the functional relationship between these two domains we expressed the DH domain alone, the PH domain alone, and the DH-PH combination of the invasion inducing protein Tiam-1 fused to glutathione-S-transferase (GST) or green fluorescent protein (GFP) . We found that the GST-DH-PH and the GST-PH constructs bind to preparations of brain membranes and to the beta gamma subunits of trimeric G proteins in vitro, while the GST-DH and GST control do not . The GFP-DH-PH and GFP-PH constructs are localized to peripheral membranes of COS-7 cells in vivo, while GFP and GFP-DH domain constructs are found diffusely in the cytoplasm . The DH-PH domain combination activates Jun N-terminal kinase (JNK) strongly, but the DH domain alone and the PH domain alone have little effect . We conclude that membrane localization and enzymatic activation of the DH domain require the adjacent PH domain.

Biochem Biophys Res Commun, 1997 May 8, 234(1), 173 - 7
Functional expression and characterization of frog photoreceptor-specific calcium-binding proteins; Hisatomi O et al.; S-modulin (sensitivity-modulating protein) is a photoreceptor-specific calcium-binding protein which plays an important role in the light adaptation process by controlling rhodopsin phosphorylation in rods . S-modulin and its cone homologue, s26, were expressed at high level (more than 30% of total protein) in Escherichia coli and then purified . They both inhibited rhodopsin phosphorylation in a calcium dependent manner . Myristoylated recombinants of S-modulin and s26 showed calcium-dependent changes in tryptophan emission spectra with half-maxima at about 0.7 microM free calcium concentration . However, the spectral changes are distinctive from each other, suggesting that there is some difference in the structural change between S-modulin and s26.

Biochem Biophys Res Commun, 1997 May 8, 234(1), 101 - 6
Molecular cloning and characterization of a cDNA for the highly conserved HMG-like protein (Pf16) gene of Plasmodium falciparum; Nambiar A et al.; A cDNA clone (PfHB3-2-4) of 1538 bp corresponding to the highly conserved HMG-like protein (Pf16) was isolated . However, northern analysis suggests that the mRNA is about 2.2 to 2.3 kb . Analysis by RT-PCR indicated that the 0.6 to 0.7 kb sequence missing in the cDNA maps to the 3' end, suggesting that the cDNA is terminated within the 26 adenosine residues that are in the middle of the Pf16 sequence . The most unique feature about this cDNA is the presence of two open reading frames (ORF), one from nucleotides 91 to 927 and the other starting from 1421 . The second ORF corresponds to Pf16 . Expression of the cDNA clones in Escherichia coli and translation in rabbit reticulocytes of RNA transcribed from the T7 promoter of the cDNA clones revealed that only the 3' end Pf16 is translated from this mRNA . Further experiments with antisense oligonucleotides specific for Pf16 indicated that the Pf16 protein serves an important function in the life cycle of the parasite.

Biochem Biophys Res Commun, 1997 May 8, 234(1), 48 - 53
Cloning of a cDNA encoding a novel importin-alpha homologue, Qip1: discrimination of Qip1 and Rch1 from hSrp1 by their ability to interact with DNA helicase Q1/RecQL; Seki T et al.; We isolated two cDNA clones encoding human proteins which interact with DNA helicase Q1/RecQL, a human homologue of Eschelichia coli RecQ protein, by two-hybrid screening . One of these proteins, named Qip1, was a novel protein homologous to the nuclear localization signal (NLS) receptor importin-alpha, and the other was the known protein Rch1, which is also a homologue of importin-alpha . DNA helicase Q1 in human cell lysates was coprecipitated with bacterially expressed Qip1 and Rch1 fused with glutathione-S-transferase with glutathione Sepharose beads, confirming the interaction between these proteins and DNA helicase Q1 . Two-hybrid experiments revealed that Qip1 interacted with the NLS of SV40 T antigen similar to Rch1 and hSrp1 . In addition, interaction of the putative NLS in DNA helicase Q1 with Qip1 and Rch1 but not with hSrp1 was confirmed by the two-hybrid system.

J Theor Biol, 1997 May 7, 186(1), 65 - 74
Quantitative analysis of binding protein-mediated ABC transport systems; Bohl E et al.; We present a mathematical proof for the applicability of the Michaelis-Menten theory to the analysis of binding protein-dependent transport systems . Fitting the rate equation for transport of substrate to experimental data obtained with the Escherichia coli maltose system under conditions where the concentrations of the binding protein was varied, it was possible to determine without any further assumptions: (1) K1, the affinity constant of the binding protein towards its substrate; (2) K3, the affinity constant of the unloaded binding protein towards the membrane components; and (3) R, the concentration of the membrane component . This analysis allows determination of the alteration of KM and Vmax of the system at varying concentrations of binding protein.

J Theor Biol, 1997 May 7, 186(1), 7 - 15
Cross-species protein identification using amino acid composition, peptide mass fingerprinting, isoelectric point and molecular mass: a theoretical evaluation; Wilkins MR et al.; Proteins can be identified by rapid techniques that do not involve Edman degradation sequencing . These approaches entail the matching of amino acid compositions or tryptic peptide masses of proteins against databases, often in conjunction with estimated protein molecular weight and isoelectric point . As genome sequencing projects progress, proteins from poorly molecularly defined organisms will increasingly be identified by cross-species comparison to proteins from well-defined organisms . To investigate the application of rapid techniques for cross-species protein identification, a total of 65 theoretical cross-species comparisons involving 21 proteins (nine human and 12 E . coli) were undertaken . The degree of conservation of amino acid composition, tryptic peptides, protein isoelectric point and mass was established . Protein amino acid composition was well conserved across species boundaries, whilst tryptic peptides were poorly conserved . The molecular weight of proteins was generally well conserved, but protein isoelectric point was not . These results suggest that cross-species protein identification by rapid techniques will be done best by protein amino acid composition and protein molecular weight.

Biochemistry, 1997 May 6, 36(18), 5560 - 5
Nonsequential unfolding of the alpha/beta barrel protein indole-3-glycerol-phosphate synthase; Sanchez del Pino MM et al.; The folding of the enzyme indole-3-glycerol-phosphate synthase (IGPS), a member of the (alpha/beta)8 fold family, has been studied . At least two folding intermediates have been detected using spectroscopic and activity measurements in combination with gel filtration chromatography . These two intermediates are produced by parallel pathways of a nonsequential unfolding mechanism rather than being consecutive steps in a sequential process . One intermediate can be detected in unfolding experiments because it is kinetically trapped in that conformation, but it is not observed in refolding experiments . It has spectroscopic and hydrodynamic properties very similar to those of the native protein, but it is inactive . The other intermediate could not be characterized because it either aggregates or unfolds under our experimental conditions and could not be isolated chromatographically.

Biochemistry, 1997 May 6, 36(18), 5425 - 31
Oxidation of ubiquinol by cytochrome bo3 from Escherichia coli: kinetics of electron and proton transfer; Svensson Ek M et al.; In this study we have used the so-called flow-flash technique to investigate electron and proton transfer during the reaction between cytochrome bo3 with bound ubiquinol (QH2) and dioxygen . The results are compared to those from the well-characterized mitochondrial cytochrome alpha alpha3 . Qualitatively, the same type of absorbance changes associated with electron transfer were observed in both enzymes whereas the protonation reactions were markedly different . In the bacterial QH2-bound enzyme, three kinetic phases with time constants of approximately 45 micros, approximately 700 micros, and approximately 4 ms associated with electron-transfer reactions were observed . The first phase is attributed to oxidation of hemes b and o3 and formation of the "peroxy" intermediate . The second and third phases were not observed after addition of the herbicide HQNO, which displaces QH2 from its binding site . They are attributed to electron transfer from QH2 to heme b and from heme b to the binuclear center, respectively . In both enzymes, the initial electron transfer was followed by a slower uptake of 0.9 +/- 0.3 proton per enzyme molecule (tau approximately 90 micros), previously attributed to protonation of a group near the binuclear center . Only in the bacterial enzyme, the second electron-transfer reaction was accompanied by a net release of 1.1 +/- 0.3 H+, which is attributed to proton release during oxidation of QH2 . It was followed by a slower uptake of 1.2 +/- 0.4 H+ during transfer of the fourth electron to the binuclear center . The two slowest protonation reactions were not observed in the presence of HQNO.

Biochemistry, 1997 May 6, 36(18), 5393 - 401
Structural basis for the broad substrate specificity of fiddler crab collagenolytic serine protease 1; Tsu CA et al.; Crab collagenolytic serine protease 1 efficiently cleaves peptide bonds directly C-terminal to basic, polar, and hydrophobic amino acids . The crystal structure of this enzyme complexed to the protein inhibitor ecotin at 2.5 A resolution reveals a large primary binding pocket punctuated on one wall by the side chain of aspartate-226 . Removal or relocation of this negatively charged group by site-directed mutagenesis generates variant enzymes which retain very high activities toward selected substrates . Full retention of activity toward hydrophobic substrates in collagenase D226G is accompanied by a 10-100-fold reduction in k(cat)/Km toward basic residues . In contrast, restoration of the negative charge in a trypsin-like position in collagenase D226G/G189D regenerates nearly full activity toward basic substrates while introducing a 5-fold decrease in k(cat)/Km toward hydrophobic amino acids . These results imply that the collagenase S1 pocket has multiple distinct binding sites for different amino acid side chains, a suggestion supported by molecular modeling studies based on the crystal structure . The ease of specificity modification in the primary binding site of this serine protease parallels similar observations with the bacterial enzymes alpha-lytic protease and subtilisin, and stands in sharp distinction to the extensive mutagenesis required to alter specificity in trypsin.

Biochemistry, 1997 May 6, 36(18), 5381 - 92
Crystal structure of an ecotin-collagenase complex suggests a model for recognition and cleavage of the collagen triple helix; Perona JJ et al.; The crystal structure of fiddler crab collagenase complexed with the dimeric serine protease inhibitor ecotin at 2.5 A resolution reveals an extended cleft providing binding sites for at least 11 contiguous substrate residues . Comparison of the positions of nine intermolecular main chain hydrogen bonding interactions in the cleft, with the known sequences at the cleavage site of type I collagen, suggests that the protease binding loop of ecotin adopts a conformation mimicking that of the cleaved strand of collagen . A well-defined groove extending across the binding surface of the enzyme readily accommodates the two other polypeptide chains of the triple-helical substrate . These observations permit construction of a detailed molecular model for collagen recognition and cleavage by this invertebrate serine protease . Ecotin undergoes a pronounced internal structural rearrangement which permits binding in the observed conformation . The capacity for such rearrangement appears to be a key determinant of its ability to inhibit a wide range of serine proteases.

Biochemistry, 1997 May 6, 36(18), 5353 - 62
Guinea pig and bovine zeta-crystallins have distinct functional characteristics highlighting replacements in otherwise similar structures; Rao PV et al.; zeta-Crystallin, a major cytosolic protein of guinea pig lens, has been characterized as an NADPH:quinone oxidoreductase (EC 1.6.5.5) {Rao et al . (1992) J . Biol . Chem . 267, 97-103} . A bovine lens homolog with 83% sequence identity was found to have very different functional characteristics . While the bovine lens zeta-crystallin exhibits similar physicochemical properties, such as molecular weight, hydropathy profile, and predicted secondary structure, and exhibits strong immunological cross-reactivity with the guinea pig and human lens zeta-crystallins, it shows minimal quinone oxidoreductase activity . On the other hand, bovine lens zeta-crystallin, but not guinea pig zeta-crystallin, showed a strong binding affinity to single-stranded DNA (ssDNA) that could be competed with NADPH, the specific cofactor of zeta-crystallin . NADH and dextran sulfate did not affect this characteristic of bovine zeta-crystallin and the enzyme showed no binding affinity for the heparin-Ultragel A4R . Two-dimensional electrophoresis of bovine lens zeta-crystallin showed a distinct pattern of posttranslational charge modification as compared to the guinea pig protein . Alignment of eight zeta-crystallin sequences, and computer modelling of the bovine and human forms based on the crystallographically analyzed Escherichia coli form, suggest that if loss of a functional residue accounts for the lowered catalytic activity of the bovine protein, Tyr 52 of the E . coli enzyme, and the equivalent Tyr present in all known mammalian forms except the bovine, is the likely candidate . In the bovine form this tyrosine is replaced by histidine.

Biochemistry, 1997 May 6, 36(18), 5323 - 35
"Designing out" disulfide bonds: thermodynamic properties of 30-51 cystine substitution mutants of bovine pancreatic trypsin inhibitor; Liu Y et al.; We have used a combination of spectroscopic and calorimetric techniques to assess the thermodynamic and extrathermodynamic consequences of paired amino acid substitutions at positions 30 and 51 in bovine pancreatic trypsin inhibitor (BPTI) . Correctly folded, wild type BPTI contains a disulfide at the 30-51 positions, with the nonbackbone atoms of this cystine being relatively solvent inaccessible . Mutants missing this buried 30-51 disulfide adopt a conformation very similar to that of the native state of wild type BPTI (Eigenbrot et al., 1990, 1992), although they are severely destabilized relative to the wild type molecule (Hurle et al., 1990) . We have conducted a systematic effort to find the energetically most favorable substitution for this buried 30-51 disulfide in BPTI . To this end, we have studied and characterized the thermally induced and guanidine hydrochloride-induced denaturation transitions for a family of mutants in which the amino acid residue(s) at positions 30 and/or 51 have been systematically altered . Specifically, we studied the unfolding transitions of the following series of residue 30/residue 51 paired substitution mutants: C30A/C51A, C30V/C51A, C30G/C51A, C30S/C51A, C30T/C51A, C30A/C51S, C30S/C51S, and C30G/C51M . For this series of mutants, comparisons between the relative stabilization free energies, derived from analysis of the denaturation profiles, allow us to reach the following conclusions: (a) side chains containing polar moieties (Ser and Thr) are destabilizing, with this effect being position dependent (i.e., a serine substitution is more destabilizing at position 51 than at position 30); (b) the destabilizing effects of two serine substitutions are approximately additive, suggesting that side chain-side chain hydrogen bonds between the two serine hydroxyl groups probably are weak or nonexistent; (c) the thermodynamic impact of a Gly30 substitution is consistent with a glycine-induced increase in the configurational entropy of the unfolded state; (d) the C30G/C51M mutant is highly destabilized relative to the C30A/C51A mutant despite the fact that, based on considerations of hydrophobicity and steric fit, substitution of a buried disulfide by Gly30 and Met51 would be expected to be optimal . Methionine may be particularly ill-suited as a buried disulfide substitute due to the large loss of side chain conformational entropy it undergoes during the transition from the unfolded to the native state . In the aggregate, our data provide insight into the residue-, position-, and context-dependent consequences on protein stability of "designing out" the buried 30-51 disulfide bond in the BPTI molecule . These data also suggest that a previously unrecognized component of disulfide bridge stabilization of proteins is the relatively minor penalty in side chain conformational entropy incurred by cystine residues during folding due to their severely restricted rotation even in the unfolded state.

Biochemistry, 1997 May 6, 36(18), 5311 - 22
Combinations of the alpha-helix-turn-alpha-helix motif of TetR with respective residues from LacI or 434Cro: DNA recognition, inducer binding, and urea-dependent denaturation; Backes H et al.; We constructed 10 different variants of TetR by substituting all or some of the residues in the alpha-helix-turn-alpha-helix (HTH) operator binding motif with the respective amino acids from LacI or 434Cro . The variants were soluble, negative transdominant over tetR in vivo, and as active as wild-type TetR in tetracycline binding in vitro . The urea-induced denaturation of the 10 variants occurs in single reversible transitions, which are centered around 4.3 M urea . Denaturation is concentration-dependent, supporting a simple two-state mechanism in which the folded dimeric protein is in equilibrium with unfolded monomers . An analysis according to the two-state model yields a Gibbs free energy of stabilization (at 0 M urea, 25 degrees C) of about 75 kJ/mol, typical for dimeric proteins of this size . Even a deletion of 24 residues from the reading head decreased the stability by only 2.7 kJ/mol . These results suggest that the DNA reading head of Tet repressor is a thermodynamically independent domain and that the thermodynamic stability of the Tet repressor dimer is determined by the association of the dimerization domains of the individual monomers . Variants containing replacements in the first alpha-helix of HTH did not show any DNA binding activity whatsoever . We attribute this to the alteration of the two N-terminal residues in this alpha-helix . TetR variants were active in nonspecific DNA binding, when either all or only the solvent-exposed residues in the recognition alpha-helix of HTH were exchanged to the respective LacI sequence . Replacement of the same residues by the respective amino acids from 434Cro yielded hybrid proteins that specifically recognize tetO in vitro . Taken together, these results establish that the similarity of operator recognition between 434Cro and TetR is greater than between TetR and LacI and confirm that prediction of the recognized DNA sequence is not obvious from the sequence of the respective HTH or recognition alpha-helix.

Biochemistry, 1997 May 6, 36(18), 5293 - 9
Quantitative determination of conformational, dynamic, and kinetic parameters of a ligand-protein/DNA complex from a complete relaxation and conformational exchange matrix analysis of intermolecular transferred NOESY; Moseley HN et al.; We report a quantitative analysis of the 13C-edited intermolecular transferred NOESY (inter-TrNOESY) spectrum of the trp-repressor/operator complex (trp-rep/op) with {ul-13C/15N}-L-tryptophan corepressor using a computer program implementing complete relaxation and conformational exchange matrix (CORCEMA) methodology {Moseley et al . (1995) J . Magn . Reson . 108B, 243-261} . Using complete mixing time curves of three inter-TrNOESY peaks between the tryptophan and the Trp-rep/op, this self-consistent analysis determined the correlation time of the bound species (tauB = 13.5 ns) and the exchange off-rate (k(off) = 3.6 s(-1)) of the corepressor . In addition, the analysis estimated the correlation time of the free species (tauF approximately 0.15 ns) . Also, we demonstrate the sensitivity of these inter-TrNOESY peaks to several factors including the k(off) and orientation of the tryptophan corepressor within the binding site . The analysis indicates that the crystal structure orientation for the corepressor is compatible with the solution NMR data.

FEBS Lett, 1997 May 5, 407(3), 353 - 6
Function of the repeated sequence in the 3' flanking region of the Escherichia coli rnpB gene on transcription termination and RNA processing; Kim S et al.; The 3' flanking region of the Escherichia coli rnpB gene-encoding M1 RNA, the RNA component of RNase P, contains a 113 bp repeated sequence . This sequence, successively reiterating 3.5 times, includes the region for intrinsic termination . In vivo termination of transcription occurs mostly at the first terminator (T1) . Analysis of deletions at the 3' flanking region revealed that the second terminator (T2) and third (T3) are functional in vivo and that the sequences preceding the region coding for an RNA-terminator hairpin and U-rich 3' tail are essential for efficient termination . Transcripts terminating at T2 and T3 were also processed at the 3' end in a manner similar to those terminating at T1.

FEBS Lett, 1997 May 5, 407(3), 337 - 42
Demonstration that the BchH protein of Rhodobacter capsulatus activates S-adenosyl-L-methionine:magnesium protoporphyrin IX methyltransferase; Hinchigeri SB et al.; The bchH gene of Rhodobacter capsulatus has been cloned into an expression strain of Escherichia coli . Following induction of expression of the BchH protein, it was found that the E . coli strain also accumulated porphyrins with the fluorescence properties of protoporphyrin and zinc protoporphyrin . It was also found that the soluble BchH protein increased the activity of S-adenosyl-L-methionine:magnesium protoporphyrin IX methyltransferase, when mixed with membranes of an expression strain of E . coli into which the bchM gene (which encodes the methyltransferase) had been cloned, as well as membranes of a bchH mutant of R . capsulatus.

FEBS Lett, 1997 May 5, 407(3), 325 - 8
Periodicity in recA protein-DNA complexes; Volodin AA et al.; The reaction of guanine residues with dimethylsulfate was studied for complexes of recA protein with fluorescent dye tagged double stranded oligonucleotides . The patterns of dimethylsulfate modification obtained demonstrate a similarity of DNA states in the complexes with recA protein formed as a result of recA promoted strand exchange and renaturation reactions . The guanine modification efficiency varies periodically as a function of the base position along the oligonucleotide axis, with a period of 3 nucleotides . This effect suggests that the arrangement of recA monomers along the oligonucleotide is strictly ordered, and the dimethylsulfate reactivity of a guanine residue depends on the site of its binding in a recA monomer.

FEBS Lett, 1997 May 5, 407(3), 303 - 8
A novel model for the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator; Annereau JP et al.; Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene . The most frequent mutation is the deletion of F508 in the first nucleotide binding fold (NBF1) . It induces a perturbation in the folding of NBF1, which impedes posttranslational maturation of CFTR . Determination of the three-dimensional structure of NBF1 would help to understand this defect . We present a novel model for NBF1 built from the crystal structure of bovine mitochondrial F1-ATPase protein . This model gives a reasonable interpretation of the effect of mutations on the maturation of the protein and, in agreement with the CD data, leads to reconsideration of the limits of NBF1 within CFTR.

FEBS Lett, 1997 May 5, 407(3), 291 - 6
Interaction between cellohexaose and cellulose binding domains from Trichoderma reesei cellulases; Mattinen ML et al.; Most Trichoderma reesei cellulases consist of a catalytic and a cellulose binding domain (CBD) joined by a linker . We have used cellohexaose as a model compound for the glucose chain to investigate the interaction between the soluble enzyme and cellulose . The binding of cellohexaose to family I CBDs was studied by NMR spectroscopy . CBDs cause line broadening effects and decreasing T2 relaxation times for certain cellohexaose resonances, whereas there are no effects in the presence of a mutant which binds weakly to cellulose . Yet it remains uncertain how well the soluble cellooligosaccharide mimics the binding of CBD to the cellulose.

FEBS Lett, 1997 May 5, 407(3), 280 - 4
Studies of high thermostability and peroxidase activity of recombinant antibody L chain-porphyrin Fe(III) complex; Kohda K et al.; A complex of an independent L chain from anti-mesotetrakis (4-carboxyphenyl) porphyrin (TCPP) monoclonal antibody 13-1 and TCPP Fe(III) was designated as L-zyme and shown to exhibit high peroxidase activity and high optimal reaction temperature (90 degrees C) . Heat denaturation study and circular dichroism (CD) spectra analysis suggested that refolded structure of 13-1 L chain exhibited significantly reduced inactivation rate after heat treatment . The secondary structure of 13-1 L chain changed slightly by the encapsulation of TCPP Fe(III) and the complex was found to be less thermostable than the L chain alone . Furthermore, by characterization of truncated forms of the L chain, it was revealed that the hydrophobic region (115-146) and hydrophilic region (147-189) in CL are important for thermostability and activity, respectively . Tertiary structure of L-zyme was predicted by AbM . Comparison of residues of L-zyme with those in the active centre of known structure of the peroxidase from Arthromyces ramosus (ARP) indicated that His38(CDR1), His94(CDR3), Arg96(CDR3) of L-zyme are important residues for peroxidase activity . Moreover, the steric arrangements of these residues in both L-zyme and ARP are similar, respectively . Distance between proximal His and distal His in L-zyme is 9.09 A, whereas that of ARP is 7.8 A.

FEBS Lett, 1997 May 5, 407(3), 275 - 9
Cytotoxic activity of ribonucleolytic toxin restrictocin-based chimeric toxins targeted to epidermal growth factor receptor; Rathore D et al.; Targeted toxins represent a new approach to specific cytocidal therapy . The ribonucleolytic protein toxin restrictocin is a potent protein synthesis inhibitor produced by the fungus Aspergillus restrictus . In the present study we have constructed two restrictocin based chimeric toxins where human transforming growth factor alpha (TGF alpha) has been used as a ligand . TGF alpha is a single chain polypeptide, which binds to epidermal growth factor receptor (EGFR) and causes proliferation in a large number of cancers . The ligand has been separately fused either at the amino terminus or carboxyl terminus of restrictocin, giving rise to TGF alpha-restrictocin and restrictocin-TGF alpha respectively . The fusion proteins were overexpressed in Escherichia coli and purified from inclusion bodies by a denaturation-renaturation protocol . Both the chimeric toxins actively inhibited eukaryotic protein synthesis in a cell free in vitro translation assay system . These chimeric toxins selectively killed human epidermal growth factor receptor positive target cells in culture . Among the two proteins, restrictocin-TGF alpha was more active than TGF alpha-restrictocin on all the cell lines studied.

FEBS Lett, 1997 May 5, 407(3), 253 - 6
The L7/L12 ribosomal domain of the ribosome: structural and functional studies; Gudkov AT; The L7/L12 protein forms a functionally important domain in the ribosome . This domain is involved in interaction with translation factors during protein biosynthesis . The tertiary and quaternary structure of the L7/L12 protein was established as a result of intensive studies in solution and in the ribosome . The conformational changes of L7/L12, the elongation factors Tu and G and other ribosomal proteins were traced by different experimental techniques . These changes occur upon interaction of the ribosome with the elongation factors and depend on GTP hydrolysis in accordance with the functional states of the ribosome.

Biochim Biophys Acta, 1997 May 2, 1352(1), 85 - 90
Molecular cloning of a cDNA encoding the nucleosome core histone H3 from Dictyostelium discoideum by genetic screening in yeast; Bukenberger M et al.; The one-hybrid method for genetic screening in yeast was used to search a Dictyostelium discoideum cDNA library for DNA-binding proteins that interact with the C-module of the Dictyostelium repetitive element . The C-module was formerly shown to contain two high affinity, sequence-specific binding sites for a nuclear protein factor of unknown function (CMBF) . The bait DNA sequence was bound in vivo by a cDNA-encoded protein whose derived amino acid sequence showed high homology to nucleosome core histone H3, but not to partially available CMBF sequences . The D . discoideum histone H3 homolog is encoded by a single gene and shows significant sequence variation at the amino terminus of the protein, including a triple-serine insertion not found in any other histone H3.

Biochim Biophys Acta, 1997 May 2, 1352(1), 48 - 55
Cloning of two splicing variants of the novel Ras-related GTPase Rab29 which is predominantly expressed in kidney; Massmann S et al.; cDNA of a novel Ras-related GTP-binding protein was isolated from rat tissue by a PCR-based cloning approach, and was designated Rab29 because its deduced amino acid sequence (204 aa) is remotely similar to that of members of the Rab family (30% identity with Rab1) . mRNA of Rab29 was found predominately in kidney . Recombinant Rab29 exhibited rapid exchange of bound guanine nucleotides for radiolabeled GTP but lacked a detectable intrinsic GTPase activity . A second cDNA clone was isolated which contained a 287 bp in-frame insertion with characteristics of an intron sequence; this insertion introduces a stop codon after arginine 167 . The recombinant protein (Rab29delta37) derived from the cDNA carrying the insertion was loaded with GTP during biosynthesis, but showed almost no exchange of the nucleotide for radiolabeled GTP . Thus, the C-terminus of Rab29 appears to harbor a structural element which is essential for the nucleotide exchange of the protein.

J Mol Biol, 1997 May 2, 268(2), 512 - 25
GroEL-mediated folding of structurally homologous dihydrofolate reductases; Clark AC et al.; Using stopped-flow fluorescence techniques, we have examined both the refolding and unfolding reactions of four structurally homologous dihydrofolate reductases (murine DHFR, wild-type E . coli DHFR, and two E . coli DHFR mutants) in the presence and absence of the molecular chaperonin GroEL . We show that GroEL binds the unfolded conformation of each DHFR with second order rate constants greater than 3 x 10(7) M(-1)s(-1) at 22 degrees C . Once bound to GroEL, the proteins refold with rate constants similar to those for folding in the absence of GroEL . The overall rate of formation of native enzyme is decreased by the stability of the complex between GroEL and the last folding intermediate . For wild-type E . coli DHFR, complex formation is transient while for the others, a stable complex is formed . The stable complexes are the same regardless of whether they are formed from the unfolded or folded DHFR . When complex formation is initiated from the native conformation, GroEL binds to a pre-existing non-native conformation, presumably a late folding intermediate, rather than to the native state, thus shifting the conformational equilibrium toward the non-native species by mass action . The model presented here for the interaction of these four proteins with GroEL quantitatively describes the difference between the formation of a transient complex and a stable complex as defined by the rate constants for release and rebinding to GroEL relative to the rate constant for the last folding step . Due to this kinetic partitioning, three different mechanisms can be proposed for the formation of stable complexes between GroEL and either murine DHFR or the two E . coli DHFR mutants . These data show that productive folding of GroEL-bound proteins can occur in the absence of nucleotides or the co-chaperonin GroES and suggest that transient complex formation may be the functional role of GroEL under normal conditions.

J Mol Biol, 1997 May 2, 268(2), 261 - 72
Analysis of the in vivo decay of the Escherichia coli dicistronic pyrF-orfF transcript: evidence for multiple degradation pathways; Arraiano CM et al.; Messenger RNA decay in Escherichia coli is slowed in pnp-7 (PNPase) rnb-500 (RNase II) rne-1(RNase E) multiple mutants . We have used Northern blots, S1 nuclease protection and primer extension analysis to map 18 endonucleolytic cleavage sites within the pyrF-orfF dicistronic transcript . Although examination of a total of 27 cleavage sites including those determined for the monocistronic trxA transcript revealed a complex pattern, the central four nucleotides within a cluster of 12 residues encompassing the cleavage sites showed a definite A/U preference . Also of interest was the processing of the dicistronic transcript to remove the downstream orfF sequence as a stable but untranslated RNA fragment . The data provide further support for the hypothesis that multiple decay pathways are involved in the decay of a single transcript . In particular, the pyrF-orfF transcript apparently can be degraded either in the 5' to 3' or the 3' to 5' direction . Our results are discussed in light of current models of mRNA decay involving polyadenylation and multiprotein decay complexes.

Cell, 1997 May 2, 89(3), 373 - 80
Nuclear receptor repression mediated by a complex containing SMRT, mSin3A, and histone deacetylase; Nagy L et al.; The transcriptional corepressors SMRT and N-CoR function as silencing mediators for retinoid and thyroid hormone receptors . Here we show that SMRT and N-CoR directly interact with mSin3A, a corepressor for the Mad-Max heterodimer and a homolog of the yeast global-transcriptional repressor Sin3p . In addition, we demonstrate that the recently characterized histone deacetylase 1 (HDAC1) interacts with Sin3A and SMRT to form a multisubunit repressor complex . Consistent with this model, we find that HDAC inhibitors synergize with retinoic acid to stimulate hormone-responsive genes and differentiation of myeloid leukemia (HL-60) cells . This work establishes a convergence of repression pathways for bHLH-Zip proteins and nuclear receptors and suggests this type of regulation may be more widely conserved than previously suspected.

J Biol Chem, 1997 May 2, 272(18), 12189 - 94
Hydrolysis of a broad spectrum of extracellular matrix proteins by human macrophage elastase; Gronski TJ Jr et al.; Macrophage elastase (ME) was originally named when metal-dependent elastolytic activity was detected in conditioned media of murine macrophages . Subsequent cDNA cloning of the mouse and human enzyme demonstrated that ME is a distinct member of the matrix metalloproteinase family . To date, the catalytic parameters that describe the hydrolysis of elastin by ME have not been quantified and its activity against other matrix proteins have not been described . In this report, we have examined the action of purified recombinant human ME (rHME), produced in Escherichia coli, on elastin and other extracellular matrix proteins . On a molar basis, rHME is approximately 30% as active as human leukocyte elastase in solubilizing elastin . rHME also efficiently degrades alpha1-antitrypsin (alpha1-AT), the primary physiological inhibitor of human leukocyte elastase . In addition, rHME efficiently degrades fibronectin, laminin, entactin, type IV collagen, chondroitan sulfate, and heparan sulfate . These results suggest that HME may be required for macrophages to penetrate basement membranes and remodel injured tissue during inflammation . Moreover, abnormal expression of HME may contribute to destructive processes such as pulmonary emphysema and vascular aneurysm formation . To further understand the specificity of HME, the initial cleavage sites in alpha1-AT have been determined . In addition, the hydrolysis of a series of synthetic peptides with different P'1 residues has been determined . rHME can accept large and small amino acids at the P'1 site, but has a preference for leucine.

J Biol Chem, 1997 May 2, 272(18), 12138 - 43
Characterization of P0, a ribosomal phosphoprotein of Plasmodium falciparum . Antibody against amino-terminal domain inhibits parasite growth; Goswami A et al.; A cDNA expression clone of the human malarial parasite Plasmodium falciparum, lambdaPf4, which was reactive only to the immune sera and not to the patient sera, has recently been found to be the P . falciparum homologue of the P0 ribosomal phosphoprotein gene . A Northern analysis of the P0 gene revealed the presence of two transcripts, both present in all the different intraerythrocytic stages of the parasite life cycle . A 138-base pair amino-terminal domain of this gene was expressed as a fusion protein with glutathione S-transferase in Escherichia coli . Polyclonal antibodies raised against this domain immunoprecipitated the expected 38-kDa P0 protein from the 35S-labeled as well as 32P-labeled P . falciparum cultures . Monospecific human immune sera affinity-purified using the expression clone lambdaPf4 also immunoprecipitated the same size protein from {35S}methionine-labeled P . falciparum protein extract . Purified IgG from polyclonal antibodies raised against the amino-terminal domain of P0 protein completely inhibited the growth of P . falciparum in vitro . This inhibition appears to be mainly at the step of erythrocyte invasion by the parasites.

J Biol Chem, 1997 May 2, 272(18), 12116 - 21
Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase bind in the ATP site; Young PR et al.; The site of action of a series of pyridinyl imidazole compounds that are selective inhibitors of p38 mitogen-activated protein kinase in vitro and block proinflammatory cytokine production in vivo has been determined . Using Edman sequencing, 125I-SB206718 was shown to cross-link to the nonphosphorylated Escherichia coli-expressed p38 kinase at Thr175, which is proximal to the ATP binding site . Titration calorimetric studies with E . coli-expressed p38 kinase showed that SB203580 bound with a stoichiometry of 1:1 and that binding was blocked by preincubation of p38 kinase with the ATP analogue, FSBA (5'-{p-(fluorosulfonyl)benzoyl}adenosine), which covalently modifies the ATP binding site . The intrinsic ATPase activity of the nonphosphorylated enzyme was inhibited by SB203580 with a Km of 9.6 mM . Kinetic studies of active, phosphorylated yeast-expressed p38 kinase using a peptide substrate showed that SB203580 was competitive with ATP with a Ki of 21 nM and that kinase inhibition correlated with binding and biological activity . Mutagenesis indicated that binding of 125I-SB206718 was dependent on the catalytic residues K53 and D168 in the ATP pocket . These findings indicate that the pyridinyl imidazoles act in vivo by inhibiting p38 kinase activity through competition with ATP and that their selectivity is probably determined by differences in nonconserved regions within or near the ATP binding pocket.

J Biol Chem, 1997 May 2, 272(18), 11975 - 8
Alteration of the substrate specificity of the malonyl-CoA/acetyl-CoA:acyl carrier protein S-acyltransferase domain of the multifunctional fatty acid synthase by mutation of a single arginine residue; Rangan VS et al.; The structural basis for the dual specificity of the malonyl-CoA/acetyl-CoA:acyl carrier protein S-acyltransferase associated with the multifunctional animal fatty acid synthase has been investigated by mutagenesis . Arginine 606, which is positionally conserved in the transacylase domains of all multifunctional fatty acid and polyketide synthases, was replaced by alanine or lysine in the context of the isolated transacylase domain, and the mutant proteins were expressed in Escherichia coli . Malonyl transacylase activity of the Arg-606 --> Ala and Arg-606 --> Lys mutant enzymes was reduced by 100- and 10-fold, respectively . In contrast, acetyl transacylase activity was increased 6.6-fold in the Arg-606 --> Ala mutant and 1.7-fold in the Arg-606 --> Lys mutant . Kinetic studies revealed that selectivity of the enzyme for acetyl-CoA was increased >16,000-fold by the Ala mutation and 16-fold by the Lys mutation . Activity toward medium chain length acyl thioesters was also increased >3 orders of magnitude by mutation of Arg-606, so that the Ala-606 enzyme is an effective medium chain length fatty acyl transacylase . These results indicate that Arg-606 plays an important role in the binding of malonyl moieties to the transacylase domain but is not required for binding of acetyl moieties; these results are also consistent with a mechanism whereby interaction between the positively charged guanidinium group of Arg-606 and the free carboxylate anion of the malonyl moiety serves to position this substrate in the active site of the enzyme.

J Biol Chem, 1997 May 2, 272(18), 11908 - 15
Convenient isolation and kinetic mechanism of glutathionylspermidine synthetase from Crithidia fasciculata; Koenig K et al.; Trypanothione, the essential metabolite in the oxidant defense system of trypanosomatids, is synthesized by two distinct proteins, glutathionylspermidine synthetase and trypanothione synthetase . Glutathionylspermidine synthetase was purified to homogeneity from the trypanosomatid Crithidia fasciculata by aqueous two-phase systems and chromatography . The enzyme showed a specific activity of 38 micromol of glutathionylspermidine formed per min per mg of protein . Its molecular mass was 78 kDa in SDS-polyacrylamide gel electrophoresis, and it appeared predominantly monomeric in native polyacrylamide gel electrophoresis and gel filtration . The isoelectric point was at pH 4.6, and the pH optimum was near 7.6 . Partial amino acid sequencing revealed homology with, but low similarity to, the glutathionylspermidine synthetase/amidase of Escherichia coli, and amidase activity was not detected in glutathionylspermidine synthetase of C . fasciculata . The kinetics of trypanosomatid glutathionylspermidine synthetase revealed a rapid equilibrium random mechanism with limiting Km values for Mg2+-ATP, GSH, and spermidine of 0.25 +/- 0.02, 2.51 +/- 0.33, and 0.47 +/- 0 . 09 mM, respectively, and a kcat of 415 +/- 78 min-1 . Partial reactions at restricted cosubstrate supply were not detected by 31P NMR, supporting the necessity of a quarternary complex formation for catalysis . ADP inhibited competitively with respect to ATP (Ki = 0 . 08 mM) and trypanothione exerted a feedback inhibition competitive with GSH (Ki = 0.48 mM).

J Biol Chem, 1997 May 2, 272(18), 11895 - 901
Isolation and characterization of the cDNA encoding bovine poly(ADP-ribose) glycohydrolase; Lin W et al.; The synthesis and rapid turnover of ADP-ribose polymers is an immediate cellular response to DNA damage . We report here the isolation and characterization of cDNA encoding poly(ADP-ribose) glycohydrolase (PARG), the enzyme responsible for polymer turnover . PARG was isolated from bovine thymus, yielding a protein of approximately 59 kDa . Based on the sequence of oligopeptides derived from the enzyme, polymerase chain reaction products and partial cDNA clones were isolated and used to construct a putative full-length cDNA . The cDNA of approximately 4.1 kilobase pairs predicted expression of a protein of approximately 111 kDa, nearly twice the size of the isolated protein . A single transcript of approximately 4 . 3 kilobase pairs was detected in bovine kidney poly(A)+ RNA, consistent with expression of a protein of 111 kDa . Expression of the cDNA in Escherichia coli resulted in an enzymatically active protein of 111 kDa and an active fragment of 59 kDa . Analysis of restriction endonuclease fragments from bovine DNA by Southern hybridization indicated that PARG is encoded by a single copy gene . Taken together, the results indicate that previous reports of multiple PARGs can be explained by proteolysis of an 111-kDa enzyme . The deduced amino acid sequence of the bovine PARG shares little or no homology with other known proteins . However, it contains a putative bipartite nuclear location signal as would be predicted for a nuclear protein . The availability of cDNA clones for PARG should facilitate structure-function studies of the enzyme and its involvement in cellular responses to genomic damage.

J Biol Chem, 1997 May 2, 272(18), 11881 - 5
Residues essential for catalysis and stability of the active site of Escherichia coli adenylosuccinate synthetase as revealed by directed mutation and kinetics; Kang C et al.; Examined here by directed mutation, circular dichroism spectroscopy, and kinetics are the relationships of five residues, Asp13, Glu14, Lys16, His41, and Arg131, to the catalytic function and structural organization of adenylosuccinate synthetase from Escherichia coli . The D13A mutant has no measurable activity . Mutants E14A and H41N exhibit 1% of the activity of the wild-type enzyme and 2-7-fold increases in the Km of substrates . The mutant K16Q has 34% of the activity of wild-type enzyme and Km values for substrates virtually unchanged from those of the wild-type system . Mutation of Arg131 to leucine caused only a 4-fold increase in the Km for aspartate relative to the wild-type enzyme . The dramatic effects of the D13A, E14A, and H41N mutations on kcat are consistent with the putative roles assigned to Asp13 (catalytic base), His41 (catalytic acid), and Glu14 (structural organization of the active site) . The modest effect of the R131L mutation on the binding of aspartate is also in harmony with recent crystallographic investigations, which suggests that Arg131 stabilizes the conformation of the loop that binds the beta-carboxylate of aspartate . The modest effect of the K16Q mutation, however, contrasts with significant changes brought about by the mutation of the corresponding lysines in the P-loop of other GTP- and ATP-binding proteins . Crystallographic structures place Lys16 in a position of direct interaction with the gamma-phosphate of GTP . Furthermore, lysine is present at corresponding positions in all known sequences of adenylosuccinate synthetase . We suggest that along with a modest role in stabilizing the transition state of the phosphotransfer reaction, Lys16 may stabilize the enzyme structurally . In addition, the modest loss of catalytic activity of the K16Q mutant may confer such a selective disadvantage to E . coli that this seemingly innocuous mutation is not tolerated in nature.

J Biol Chem, 1997 May 2, 272(18), 11869 - 73
Identification of an unusual intein in chloroplast ClpP protease of Chlamydomonas eugametos; Wang S et al.; The proteasome-like ClpP protease is widely distributed and structurally conserved among bacteria and eukaryotic cell organelles . In Chlamydomonas eugametos, however, the chloroplast clpP gene predicted a much larger ClpP protein containing large insertion sequences (ISs) . One insertion sequence, IS2, is 456 amino acid residues long and not similar to known proteins . Here we show that IS2 is an unusual intein, and its protein splicing activity in Escherichia coli cells can be activated by a single amino acid substitution . Analysis of IS2 sequence revealed short sequence motifs that are similar to known intein motifs, including putative LAGLI-DADG endonuclease motifs . But a histidine residue conserved at the C terminus of known inteins is replaced in the IS2 sequence by a glycine residue (Gly455), rendering the IS2 sequence incapable of detectable protein splicing when tested in E . coli cells . Changing Gly455 to histidine activated the ability of IS2 to undergo protein splicing in E . coli cells . The IS2 sequence (intein) was precisely excised from a precursor protein, with the flanking sequences (exteins) joined together by a normal peptide bond.

Genes Cells, 1997 May, 2(5), 303 - 14
The duplex DNA is very underwound in the three-stranded RecA protein-mediated synaptic complex; Voloshin ON et al.; BACKGROUND: The RecA protein is a central player in bacterial homologous recombination . It promotes two key events: the search for homology between two DNA molecules and the subsequent formation of the synaptic complex composed of RecA and three DNA strands (two from one duplex and one single strand) . In spite of numerous studies, the architecture of the synaptic complex is still far from clear . RESULTS: We have exploited two approaches to study the structure of Escherichia coli RecA protein-mediated DNA synapsis: chemical modification with potassium permanganate and treatment of synaptic complexes of different lengths with topoisomerase I . The linking number difference values, obtained after separation of the individual sets of topoisomers in an agarose gel, were used to determine the number of bases per helical turn . We were able to show that the topology of the three-stranded complexes containing RecA is quite different from that expected for deproteinized D-loops . The original duplex in the synaptic complex is unwound, but not necessarily unpaired, to a structure topologically equivalent to DNA with approximately 27 bp per turn . Despite the fact that the patterns of reactivity towards potassium permanganate cannot be interpreted unambiguously, the results of chemical footprinting can be explained in terms of a synaptic complex as an extended and unwound three-stranded helical structure . CONCLUSIONS: This work provides the first quantitative topological parameters for the RecA protein-mediated three-stranded synaptic complex . Furthermore, the structure of synaptic complexes is different from that of a simple D-loop.

Biochemistry (Mosc), 1997 May, 62(5), 480 - 4
Effects of penetrating and non-penetrating oxidants on Escherichia coli; Smirnova GV et al.; Treatment of Escherichia coli K-12 cells aerobically grown in M9 glucose salt medium (H2O2) and non-penetrating (ferricyanide) oxidants resulted in similar inhibition of growth and decrease in intracellular K+ pool by 15% . Only H2O2 inhibited growth of auxotrophic strains grown in M9 medium supplemented with protein hydrolysate . Ferricyanide reduction was associated with decrease in low-molecular-weight thiols, whereas the treatment of cells with H2O2 increased their level . Pretreatment of cells with ferricyanide enhanced the H2O2-induced expression of katG gene encoding for catalase HPI; this gene is a member of the gene family controlled by the oxyR gene . Pretreatment with ferricyanide inhibited H2O2-induced expression of the sfiA gene which is the member of the gene family controlled by the recA and lexA genes . Glutathione is the major low-molecular-weight thiol in E . coli, and it can play different roles in cellular responses to H2O2 and ferricyanide.

Gene Ther, 1997 May, 4(5), 465 - 72
Efficient muscle-specific transgene expression after adenovirus-mediated gene transfer in mice using a 1.35 kb muscle creatine kinase promoter/enhancer; Larochelle N et al.; Replication-defective (E1-E3-deleted) human adenovirus vectors are a promising means of therapeutic gene delivery to skeletal muscle cells . Since the tropism of adenovirus is nonselective, muscle-specific expression of systemically administered vectors can only be achieved by the use of a tissue-specific promoter/enhancer that is small enough to fit the insert capacity of the vector . We have generated two replication-defective adenovirus recombinants (AV) in which the reporter gene (either firefly luciferase or E . coli beta-galactosidase) was driven by a truncated (1.35 kb) muscle creatine kinase (MCK) promoter/enhancer or by the fast troponin I (TnI) promoter/enhancer . Highly efficient and muscle-specific transgene expression was demonstrated in immunodeficient mice after local injection of AV into muscles at an early age . In nonmuscle tissues (brain, liver, kidney, lung), the transgene expression was extremely low even though in these tissues in situ polymerase chain reaction showed as high an infectivity of the cells by the AV as in muscle . The relatively small size, the good efficiency and the muscle specificity of the MCK promoter would make it ideal to drive the 6.3 kb (truncated) dystrophin cDNA in first generation AV (with a limited (8 kb) insert capacity) designed for gene therapy of Duchenne muscular dystrophy.

Mikrobiologiia, 1997 May-Jun, 66(3), 329 - 34
{Exchange of putrescine and potassium between cells and media as a factor in the adaptation of Escherichia coli to hyperosmotic shock}; Tkachenko AG et al.; Putrescine/potassium exchange in response to hyperosmotic stress was studied . The addition of 0.3 M NaCl or 0.44 M sucrose to an exponentially growing E . coli culture induced potassium uptake and putrescine release from the cell . Potassium added to an osmotically stressed potassium-deficient culture was readily absorbed by cells; this was accompanied by the loss of intracellular putrescine, both free and bound . Since DNA is the main binding site of putrescine, the loss of bound putrescine caused a relaxation of DNA supercoiling . The increase in the intracellular content of potassium not only restored but also enhanced DNA supercoiling as compared to the initial level . In vitro experiments showed the degree of plasmid DNA supercoiling to rise drastically at potassium concentrations of 300-500 mM, while different putrescine concentrations affected this parameter differently . Thus, the physiological concentrations of putrescine (below 1 mM) greatly augmented DNA supercoiling, whereas higher concentrations (5-10 mM) exerted a relaxing effect . A change in DNA supercoiling in vivo in response to osmotic stress is the result of competition between biogenic and abiogenic cations for the sites of binding to polyanionic DNA structures . A change in DNA topology serves as the regulatory factor controlling the expression of genes responsible for cell adaptation to osmotic stress.

Am J Perinatol, 1997 May, 14(5), 289 - 91
Emphysematous chorioamnionitis diagnosed by ultrasonography; Wein P et al.; Preterm labor, cervical cerclage (especially when performed as an emergency procedure), and diabetes mellitus are all associated with an increased risk of chorioamnionitis . It might be expected that the combination of all 3 could lead to especially severe infection . We report such a case . A woman with a history of two spontaneous midtrimester abortions had had cervical cerclage performed at 13 weeks . She was referred at 24 weeks' gestation with preterm labor, and the cervix was found to be dilated . An emergency repeat cerclage was performed . The following day, ultrasonography revealed the presence of intra-amniotic gas . Infection was confirmed by the presence of a purulent cervical discharge, a neutrophilia with a left shift, and an elevated C-reactive protein level . The cervical stitch was removed and labor induced . The infant was liveborn, but succumbed to the complications of prematurity and sepsis . E . coli was isolated . In her subsequent pregnancy, severe gestational diabetes was diagnosed and following pregnancy, permanent diabetes mellitus was confirmed . The combination of infection, diabetes, and intact membranes may lead to a particularly severe form of chorioamnionitis, with the production of gas within the amniotic cavity . Infection should be excluded before emergency cervical cerclage, especially in the woman with diabetes mellitus.

Biochimie, 1997 May, 79(5), 303 - 8
Heterologous expression in Escherichia coli of the gene encoding an archaeal thermoacidophilic elongation factor 2 . Properties of the recombinant protein; de Vendittis E et al.; The gene encoding the elongation factor 2 from the hyperthermophilic archaeon Sulfolobus solfataricus (SsEF-2) was expressed in Escherichia coli using the pT7-7 expression vector . The synthesis of the heterologous product did not increase upon addition of isopropyl-beta-thiogalactopyranoside . The amount of purified intact recombinant SsEF-2 (SsEF-2rec) was about 3 mg from 60 g of transformed wet cells . Recombinant and naturally occurring SsEF-2 showed identical electrophoretic mobility, immunological properties and the N-terminal amino acid sequence; both were lacking the initial methionine . Differently from SsEF-2, SsEF-2rec did not undergo post-translational modification of His603 into diphthamide, as indicated by its inability to be ADP-ribosylated . SsEF-2rec appeared indistinguishable from SsEF-2 in the fulfillment of its biological functions; in fact, it was fully capable to support poly(Phe) synthesis, to bind GDP and to display either the intrinsic or the ribosome-dependent GTPase . Finally, SsEF-2rec was endowed with the same heat stability as SsEF-2 . Altogether these findings proved that SsEF-2rec was functionally active as SsEF-2 . The used expression system could allow to produce mutated forms of SsEF-2 obtained by mutagenesis of the corresponding gene.

Biochimie, 1997 May, 79(5), 247 - 59
Phosphorylation of Srp1p, the yeast nuclear localization signal receptor, in vitro and in vivo; Azuma Y et al.; Srp1p, the protein encoded by SRP1 of the yeast Saccharomyces cerevisiae, is a yeast nuclear localization signal (NLS) receptor protein . We have previously reported isolation of a protein kinase from yeast extracts that phosphorylates Srp1p complexed with NLS peptides/proteins . From partial amino acid sequences of the four subunits of the purified kinase, we have now identified this protein kinase to be identical to yeast casein kinase II (CKII) . It was previously thought that autophosphorylation of the 36 kDa subunit of the yeast enzyme was stimulated by the substrate, GST-Srp1p . However, with the use of a more refined system, no stimulation of autophosphorylation of the 36 kDa subunit of yeast CKII was observed . Biochemical and mutational analyses localized the in vitro phosphorylation site of Srp1p by CKII to serine 67 . It was shown that, in the absence of NLS peptides/proteins, phosphorylation of the intact Srp1p protein is very weak, but deletion of the C-terminal end causes great stimulation of phosphorylation without NLS peptides/proteins . Thus, the CKII phosphorylation site is apparently masked in the intact protein structure by the presence of a C-terminal region, probably between amino acids 403 and 516 . Binding of NLS peptides/proteins most likely causes a change in protein conformation, exposing the CKII phosphorylation site . Mutational alterations of serine 67, the CKII phosphorylation site, to valine (S67V) and aspartic acid (S67D) were not found to cause any significant deleterious effects on cell growth . Analysis of in vivo phosphorylation showed that at least 30% of the wild type Srp1p molecules are phosphorylated in growing cells, and that the phosphorylation is mostly at the serine 67 CKII site . The ability of Srp1p purified from E coli and treated with calf intestinal phosphatase to bind a SV40 T-antigen NLS peptide was compared with that of Srp1p which was almost fully phosphorylated by CKII . No significant difference was observed . It appears that NLS binding does not require any phosphorylation of Srp1p, either by CKII or by some other protein kinase.

Biochimie, 1997 May, 79(5), 243 - 6
Purification of active Escherichia coli ribosome recycling factor (RRF) from an osmo-regulated expression system; MacDougall J et al.; Ribosome release factor (RRF) from Escherichia coli was overproduced from an osmo-expression vector . More than 40% of cell protein was RRF after 6 h of induction . A purification scheme is described that produced 50 mg of RRF from an initial culture of 2 L . The recycling time for ribosomes synthesising the tripeptide fMet-Phe-Leu in vitro in the absence of RF3 was reduced from 40 to 15 s by the addition of purified 1.5 microM RRF.

Int J Biochem Cell Biol, 1997 May, 29(5), 841 - 8
Generation of chromophore Tyr-containing mutants of the ribosomal protein L7/L12 from Escherichia coli by site-directed mutagenesis and characterization; Todorova R; The ribosomal protein L7/L12 from Escherichia coli has two domains with different structure-a globular C-terminal domain and a non-globular elongated N-terminal domain . The N-terminal domain of the protein has been subjected to site-directed mutagenesis to introduce the chromophore Tyr and to study its structure by spectroscopic methods . The mutant proteins S1Y, M14Y and M26Y were expressed at high levels in Escherichia coli and purified to homogeneity by ion-exchange chromatography and gel-filtration . Growth and purification protocols were optimized to allow reproducible and efficient production of mutant proteins . The effects of the replacements were assessed by UV, far-UV circular dichroism (CD), DSC and 1H-NMR studies . The spectroscopic characteristics (far-UV CD and 1H-NMR) and the thermal unfolding (far-UV CD and DSC) of the mutants have shown that these single mutations in the N-terminal region of the protein have no appreciable effect on its secondary and tertiary structures . 1H-NMR spectroscopy was used to show that the Tyr mutants retain their dimer structure . The initiating Met is the first amino acid in the mutant protein S1Y . Y2(S1) is located in a structurally disordered region of the N-terminal domain of the protein S1Y and does not seem to have close amino acid neighbours . Y14(M14) and Y26(M26) participate in the structurally ordered regions of the molecule . Phe30 is situated in the surroundings of Y14 . The unchanged structure resulting from the mutations makes these proteins highly suitable for structural studies by multidimentional NMR to determine the structure of the N-terminal domain of protein L7/L12.

Int J Biochem Cell Biol, 1997 May, 29(5), 815 - 28
Effect of sphingomyelin and its metabolites on the activity of human recombinant PLC delta 1; Matecki A et al.; In an attempt to obtain sufficient quantities of pure phospholipase C delta 1 (PLC delta 1) necessary for structural and kinetic studies, human fibroblast PLC delta 1 was cloned in the pPROEX-1 vector, expressed in E . coli cells as a (6xHis) fusion protein and purified to homogeneity . From 11 of E . coli culture 21 mg of pure PLC delta 1 was obtained by a two-step purification procedure, which includes Ni(2+)-NAT agarose and Mono S cation exchange chromatography . Catalytic properties of recombinant PLC delta 1 with respect to activation by spermine and calcium ions and inhibition by sphingomyelin were similar to or identical to PLC delta 1 purified from rat liver . Calcium activation of PLC delta 1 was dependent on the presence of spermine . Half-maximal activity was attained at 250 and 170 nM of free Ca2+ in the presence and absence of spermine, respectively . Sphingomyelin and lysosphingomyelin were mixed type inhibitors with respect to PIP2 . Ceramide inhibits PLC delta 1 very weakly . GM1, which is a ceramide bound glucosidically to the oligosaccharide moiety, was a strong non-competitive inhibitor of PLC delta 1 . In the absence of spermine, sphingosine and phytosphingosine weakly activated PLC delta 1 . The results indicate that the effect of sphingomyelin and its metabolites on PLC delta 1 activity depends on the presence of spermine . It is postulated that, among other factors, in vivo, activity of PLC delta 1 may depend on the turnover of sphingomyelin.

Can J Physiol Pharmacol, 1997 May, 75(5), 407 - 13
Submandibular glands: novel structures participating in thermoregulatory responses; Mathison RD et al.; Since submandibular glands participate in the regulation of cardiovascular and immunological responses to bacterial endotoxin, we examined their role as modulators of endotoxin-induced fever . Core body temperatures were measured by telemetry in rats that had either a sham operation or a sialadenectomy 1 week previously and that were maintained at 23-24 degrees C . The sialadenectomized rats showed a circadian variation in body temperature similar to sham-operated rats, although their daytime body temperature was 0.24 +/- 0.01 degree C lower . The fever elicited by intraperitoneal injection of Escherichia coli endotoxin was biphasic, with an initial phase occurring between 2 and 3 h, and a broader second phase peaking between 4 and 8 h after endotoxin injection . The initial fever was similar in the two groups of rats, but the second phase of fever was significantly higher by 0.28 +/- 0.09 and 0.26 +/- 0.07 degree C in sialadenectomized rats receiving 50 or 150 micrograms/kg of endotoxin, respectively . Intravenous treatment with a novel peptide, submandibular gland peptide-T (SGP-T; 100 micrograms/kg), 30 min before endotoxin injection did not affect the early fever response, but significantly suppressed by 0.37 +/- 0.10 degrees C the late-phase fever provoked by 150 micrograms/kg of endotoxin . These results suggest that the submandibular glands modulate thermogenic responses to inflammatory stimuli possibly through the endocrine release of hormones, such as SGP-T.

Appl Biochem Biotechnol, 1997 May, 66(2), 147 - 58
Construction and characterization of a biotin-regulated gene expression system in Escherichia coli; Chang YS et al.; An autoregulated gene expression system in Escherichia coli was designed such that the cloned genes on the vector were not expressed until biotin was depleted during cell growth . The expression vectors were constructed by assembling the DNA fragments containing the regulatory region of the E . coli biotin operon (bio operon), the universal ribosome-binding site (RBS) and the strong transcription terminator rrnBT1T2 . The promoter region was further modified by site-directed mutagenesis to create promoters of varied strength . The feasibility of this system was examined in E . coli strain R901 (with bio operon deleted) using various marker genes, including the E . coli birA gene, T7 RNA polymerase gene and yellowfin-porgy growth-hormone gene . The results demonstrated that the induction of marker-gene expression can be triggered as the biotin concentration drops to a threshold value of approximately 2 ng/mL by metabolic utilization.

C R Acad Sci III, 1997 May, 320(5), 349 - 58
Active rat aromatic-L-amino acid decarboxylase as a fusion protein in Escherichia coli; Jebai F et al.; The DNA sequence encoding rat aromatic-L-amino acid decarboxylase (AADC) was inserted into the Escherichia coli (E . coli) expression vector pMAL-c2 . This clone produced a fusion protein able to catalyze the conversion of L-DOPA to dopamine . After purification and treatment of the fusion protein by factor Xa (FXa), an enzymatically active form of the enzyme resistant to FXa was isolated . It showed kinetic constants, Vmax, K(m) and enzymatic properties very similar to those obtained previously for the mammalian enzyme . This method for obtaining active AADC appears to be useful for initiating the study of the catalytic activity of this protein because it permitted the rapid isolation and the stabilization of an active form of the enzyme.

Z Naturforsch {C}, 1997 May-Jun, 52(5-6), 364 - 72
Induction of single- and double-strand breaks in plasmid DNA by monoenergetic alpha-particles with energies below the Bragg-maximum; Scholz V et al.; The yield of single-strand breaks (ssb) and double-strand breaks (dsb) produced by alpha-particles at the end of their track in DNA-films was determined experimentally . Helium nuclei were accelerated to 600 keV in the 400 kV ion accelerator and scattered at a carbon target . The elastically scattered alpha-particles with energies of 344 keV and 485 keV were used to irradiate supercircular plasmid DNA in vacuo . For the dosimetry of the alpha-particles a surface barrier detector was used and the energy distribution of the alpha-particles determined . The energy loss of the particles in the DNA-layer was calculated . DNA samples were separated into the three conformational isomers using agarose gel electrophoresis . After fluorochromation the number of ssb and dsb per plasmid DNA molecule was established from the band intensities assuming the validity of Poisson statistics . Linear dose effect correlations were found for ssb and dsb per plasmid molecule . In the case of 344 keV-alpha-particles the yield of dsb was (8.6 +/- 0.9) x 10(-11) breaks/Gy x dalton . The ratio of ssb/dsb was 0.5 +/- 0.2 . This is at least a factor of six larger than the ratio found in experiments with higher energy alpha-particles and from model calculations . Similar experiments with protons yielded a relative biological effectiveness (rbe) value of 2.8 for the induction of double-strand breaks by track end alpha-particles.

Burns, 1997 May, 23(3), 218 - 24
Evolution and significance of circulating procalcitonin levels compared with IL-6, TNF alpha and endotoxin levels early after thermal injury; Carsin H et al.; To determine the evolution and significance of circulating procalcitonin (ProCT), IL-6 TNF alpha and endotoxin levels early after thermal injury, we performed a prospective, single unit, longitudinal study . Forty burn patients with total body surface area (TBSA) > 30 per cent were studied, of whom 33 suffered an inhalation injury . Blood samples were taken on the day of admission, every 4 h during the first day and daily during the first week . All patients had increased ProCT and IL-6 levels without any proven infection . Endotoxin and TNF alpha levels remained very low or undetectable . ProCT and IL-levels correlated well with the severity of skin burn injury (respectively, p < 0.006 and p < 0.028, using the non-parametric Kruskal-Wallis test) . ProCT levels are not associated with smoke inhalation . ProCT and IL6 are prognostic factors of mortality at the time of admission but less reliable than the clinical UBS (unit burn standard) score . Endotoxin and TNF alpha were undetectable, suggesting that the problem of the early gut bacterial translocation remains to be proven.

Vet J, 1997 May, 153(3), 321 - 7
Endotoxaemia in dairy cattle: mechanism of reticulorumen stasis; Eades SC; The objective of this study was to determine whether blockade of alpha(-2) adrenergic receptors would restore reticulorumen motility during toxaemia in cows . Reticulorumen contractions were measured via a water-filled balloon connected to a pressure transducer . Intravenous infusion of endotoxin (100 ng kg-1 over 30 min) significantly decreased the number of reticulorumen contractions . Intravenous infusion of yohimbine (125 micrograms kg-1 over 30 min) alone did not affect reticulorumen contractions . However, when yohimbine (125 micrograms kg-1 over 30 min) was infused concurrently with endotoxin (100 ng kg-1 over 30 min), the effects of endotoxin on reticulorumen contraction frequency decreased, suggesting that endotoxaemia causes reticulorumen stasis via a mechanism that involves alpha(-2) adrenergic receptors.

J Surg Res, 1997 May, 69(2), 408 - 12
Macrophage TNF mRNA expression is modulated by protease inhibitors; Lo CJ et al.; BACKGROUND: Nuclear factor kappa B (NF kappa B) is an important transcriptional activator protein and is a crucial component of the host's response to infection . The activation of NF kappa B is correlated with the phosphorylation of inhibitory kappa B (I kappa B) and its subsequent degradation . We hypothesized that protease inhibitors which prevented I kappa B degradation could inhibit the macrophage gene activation and reduce the production of inflammatory cytokines . METHODS: Rabbit alveolar macrophages (M phi) were obtained by bronchoalveolar lavage . M phi were exposed to Escherichia coli lipopolysaccharide (LPS) (10 ng/ml) in the presence of various concentrations of protease inhibitors, either N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) or N-benzoyl-L-tyrosine ethyl ester (BTEE) . Total RNA was extracted for Northern blot assay of tumor necrosis factor (TNF) mRNA expression using a rabbit genomic DNA probe . Total nuclear extracts were also obtained for the measurement of the NF kappa B activity with the electrophoretic mobility shift assay . The TNF production in the M phi supernatant was measured by L929 bio-assays . RESULTS: NF kappa B activity induced by LPS was inhibited by either BTEE or TPCK . Inhibition of NF kappa B activity by these agents also prevented TNF mRNA expression and TNF production induced by LPS . The cellular mechanism leading to NF kappa B activation was further studied . TNF mRNA expression and NF kappa B activation were inhibited by D609, a phospholipase C (PLC) inhibitor, as well as by protein kinase C (PKC) inhibitors . In addition, direct stimulation of PKC led to NF kappa B activation and TNF mRNA expression . CONCLUSIONS: These data suggest that TNF mRNA expression of LPS-stimulated M phi is mediated through NF kappa B, NF kappa B activation is intimately regulated by the PLC signaling pathway.

J Surg Res, 1997 May, 69(2), 325 - 30
Starvation enhances hepatic free radical release following endotoxemia; Robinson MK et al.; Although it is well known that malnourished patients who become septic have an increased risk of organ failure and death compared to normally nourished individuals, the pathological processe(s) underlying this observation are unknown . To evaluate one possible explanation for this finding, we tested the hypothesis that malnutrition depresses hepatic antioxidant stores and accelerates hepatic release of oxygen free radicals in an animal model of sepsis . Male rats were either fasted (n = 14) or fed (n = 14) for 3 days prior to receiving lipopolysaccharide (LPS, 17 mg/kg intraperitoneally) . Animals were weighed daily and then sacrificed 6 and 24 hr after LPS administration to determine hepatic superoxide anion (an oxygen free radical) release and liver glutathione (GSH, an antioxidant) content . Fasted rats were severely malnourished as indicated by a 23% decrease in body weight compared to fed rats, which gained 11% (P < 0.05) . Liver GSH was depressed by 30% (P < 0.05) and 20% (P = 0.066) in the fasted compared to fed animals 6 and 24 hr after LPS administration . In addition, hepatic superoxide anion release was 210 and 75% higher in the fasted animals 6 and 24 hr after LPS injection (P < 0.05 at both time points) . Liver superoxide anion release and GSH content were negatively correlated (P < 0.001, R = - 0.73) indicating that superoxide anion release increased as GSH content fell . Malnutrition leads to depletion of liver antioxidant stores with accelerated release of hepatic oxygen free radicals . Oxidant-mediated organ damage may be one cause of increased morbidity and mortality in malnourished, systemically infected patients.

J Surg Res, 1997 May, 69(2), 277 - 82
Expression of two novel recombinant proteins from aortic adventitia (kappafibs) sharing amino acid sequences with cytomegalovirus; Ozsvath KJ et al.; We have recently purified and partially sequenced a microfibrillar protein from human aortic adventitia (aneurysm-associated antigenic protein, 40 kDa {AAAP-40}) that is immunoreactive with immunoglobulin (IgG) from the wall of abdominal aortic aneurysms (AAAs) . It shares motifs with Ig kappa (which may act as a binding site for interaction with integrins), cytomegalovirus (which may be a molecular mimic in the pathogenesis of AAA), and vitronectin and the fibrinogens . A cDNA library was constructed from the aortic adventitia of a AAA . The library was screened with either rabbit anti-vitronectin antibody or rabbit anti-fibrinogen antibody . Positive plaques were purified and expressed in a strain of Escherichia coli . The clone sequences were analyzed . The expressed proteins were separated by SDS/PAGE and the immunoblots were probed with either AAA IgG or anti-human Ig kappa antibody . Experimental cell lines, transfected with the clones (clones 1 and 5), synthesized recombinant proteins (rAAAP-CL1 and rAAAP-CL5), detectable in Western immunoblots with AAA IgG . A prediction of the tertiary structure resembles well-characterized cell adhesion molecules . These findings suggest that there is a novel family of matrix proteins that may use immunoglobulin motifs as binding sites for cellular integrins and that there are matrix proteins in addition to AAAP-40 that may serve as autoantigens in the pathogenesis of AAA disease.

Vet Immunol Immunopathol, 1997 May, 56(3-4), 221 - 31
Molecular cloning and functional expression of equine interleukin-1 receptor antagonist; Kato H et al.; Equine interleukin-1 receptor antagonist (IL-1ra) was molecularly cloned to establish a basis for cytokine therapy of acute and chronic inflammatory diseases in the horse . cDNA clones encoding the whole coding sequence of equine IL-1ra were isolated from equine peripheral blood mononuclear cells (PBMC) that had been stimulated with lipopolysaccharide (LPS) . The equine IL-1ra cDNA obtained in this study contained an open reading frame encoding 177 amino acid residues . The predicted amino acid sequence of equine IL-1ra shared 75.7, 75.3 and 76.3% similarity with sequences of human, murine and rabbit IL-1ras, respectively . An N-glycosylation site and five cysteine residues conserved in human, murine and rabbit IL-1ras were also found at the corresponding positions in equine IL-1ra . Recombinant glutathione S-transferase (GST)-equine IL-1ra fusion protein produced by Escherichia coli was purified . This protein was shown to inhibit the cytostatic or cytotoxic activity of IL-1 on A375S2 cells, indicating that the equine IL-1ra cDNA obtained in this study encodes biologically active equine IL-1ra.

Hepatogastroenterology, 1997 May-Jun, 44(15), 664 - 6
Centrocytic lymphoma presenting as a subphrenic abscess and a solitary liver nodule; Sousa AE et al.; Non Hodgkin's lymphoma revealed by hepatic manifestations is extremely rare . We describe here a 82-year old male patient who presented with a right subphrenic abscess and a solitary liver tumour that was shown to be a centrocytic lymphoma . Furthermore, asymptomatic cryptogenic liver cirrhosis was diagnosed . This previously unreported form of clinical presentation of a non Hodgkin's lymphoma as well as the association with liver cirrhosis are discussed in the context of the recent literature.

Vet Immunol Immunopathol, 1997 May, 56(1-2), 107 - 17
Expression of ovine interleukin-2 cDNA in Escherichia coli; Seow HF et al.; The expression plasmids pGEX-2T and pT7-7 were used to express ovine (Ov) IL-2 cDNA in Escherichia coli . The pGEX-2T vector contained glutathione-S-transferase (GST) as the affinity handle and resulted in high level expression of the GST-IL-2 fusion protein . However, only a small proportion of this fusion protein was present in the soluble fraction . The insoluble fraction was extracted with a detergent, sarkosyl, and even though a large amount of fusion product was obtained, it would not bind to glutathione beads efficiently . Thus, only low yields of biologically active rOvIL-2 were obtained . The yields were not significantly improved when other detergents were used for extraction except for a non-ionic detergent, Zwittergent 3-14, where there was a two- to three-fold increase compared with extraction with sarkosyl . An alternative vector, pT7-7 was used with a 6 x histidine tag followed by a thrombin cleavage site at the amino terminus of the mature ovine IL-2 protein to allow affinity purification by Ni-NTA resin . A large proportion of the rOvIL-2 was partitioned to the insoluble fraction . This expression system was more useful than the pGEX-2T as large quantities of biologically active rOvIL-2 of at least 10 mg l-1 were obtained . The presence of the six histidine residues at the amino end of rOvIL-2 did not reduce its biological activity . Both systems yielded rOvIL-2 with a high specific activity of about 1 x 10(7) U mg-1 as measured by the ability to maintain proliferation of ovine ConA lymphoblasts . Recombinant OvIL-2 was active on bovine but not porcine ConA lymphoblasts.

Insect Biochem Mol Biol, 1997 May, 27(5), 405 - 12
Expression and characterization of a lepidopteran general odorant binding protein; Feng L et al.; Olfaction in months involves the transport of volatile, hydrophobic odorant molecules through the aqueous interior of the antennal sensory hairs by soluble odorant binding proteins . Two subfamilies of the 17 kDa general odorant binding proteins, GOBP1 and GOBP2, are 47-57% identical to each other and 21-57% to the pheromone binding proteins (PBPs); identity within a GOBP subfamily exceeds 78% in all lepidopteran species examined . However, the ligands for GOBPs are unknown . In order to investigate odorant specificities of GOBPs, recombinant proteins were expressed in Escherichia coli using PCR-prepared expression cassettes based on the cDNA sequences of GOBP1 and GOBP2 from Manduca sexta . Both soluble and insoluble recombinant GOBPs (rGOBPs) were obtained, and the inclusion body GOBPs were solubilized, refolded and purified . The soluble and refolded rGOBPs were purified by preparative isoelectric focusing (IEF), gel filtration, and finally by ion-exchange fast protein liquid chromatography (FPLC) . Only rGOBP2, but not rGOBP1, was crossreactive with an anti-GOBP2 (Antheraea polyphemus) antiserum . rGOBP2, but not rGOBP1, could be photoaffinity labelled by the diazoacetate pheromone analog {3H}-6E, 11Z, 16:Dza . For rGOBP2, plant odors such as (Z)-3-hexen-1-ol (3Z-6:OH), geraniol, geranyl acetate, and limonene showed significant competition for binding; binding specificity was sensitive to pH and to salt concentrations . Circular dichroism (CD) confirms that, as with the pheromone binding proteins, GOBP2 is predominantly alpha-helical . Although the characterization of rGOBP1 has resisted analysis, rGOBP2 is readily prepared and studied . We suggest that GOBP2 may be broadly tuned to a class of "green" and floral odors.

FEMS Immunol Med Microbiol, 1997 May, 18(1), 39 - 46
Ehrlichia sennetsu groE operon and antigenic properties of the GroEL homolog; Zhang Y et al.; A clone expressing an immunoreactive 55-kilodalton (kDa) protein of Ehrlichia sennetsu, the causative agent of human Sennetsu ehrlichiosis, was isolated from a gene library of this organism . Sequence analysis of the DNA insert revealed two open reading frames, encoding proteins of 10,620 and 58,225 kDa, respectively . These deduced amino acid sequences were homologous to those of the GroES and GroEL heat shock proteins (HSP) of other bacteria, respectively . Phylogenetic trees based on GroES and GroEL homologs of several bacteria including E . sennetsu showed a relationship similar to that based on 16S rRNA gene sequences . The recombinant and native 55-kDa proteins of E . sennetsu, GroEL homolog, reacted with a monoclonal antibody (SPA807) which recognizes a homologous sequence between human and mycobacterial HSP60 and a polyclonal antibody (SPA804) to cyanobacteria HSP60, but not with antibodies to HSP60 of several other organisms used . Furthermore, anti-recombinant E . sennetsu 55-kDa protein antibody prepared in a rabbit was reactive to HSP60 antigens of other Ehrlichia and Rickettsia species, but not GroEL of E . coli . The recombinant 55-kDa protein would be a useful tool for studying the role of this antigen in the immune response to E . sennetsu infection.

Protein Eng, 1997 May, 10(5), 601 - 6
Heterogeneity in recombinant HIV-1 integrase corrected by site-directed mutagenesis: the identification and elimination of a protease cleavage site; Hickman AB et al.; Purified recombinant human immunodeficiency virus type 1 (HIV-1) integrase and certain deletion mutants exhibit heterogeneity consistent with proteolysis at a site close to the C-terminus . Electrospray ionization mass spectrometric analysis indicated that proteolytic cleavage generated a protein missing five residues from the C-terminus . PCR mutagenesis of amino acids on either side of the cleavage site identified two changes which were subsequently shown to prevent clipping when proteins were expressed and purified from Escherichia coli: the substitution of Arg284, the residue on the C-terminal side of the cleavage site, by either glycine or lysine . The introduction of either of these mutations into full-length integrase did not affect in vitro 3' processing or strand transfer activities . Thus, the incorporation of either of these mutations is likely to be beneficial when homogeneity of HIV-1 integrase is a concern, as in crystallographic or nuclear magnetic resonance spectroscopic experiments.

Protein Eng, 1997 May, 10(5), 593 - 9
Recombinant human TIMP-3 from Escherichia coli: synthesis, refolding, physico-chemical and functional insights; Negro A et al.; Matrix metalloproteinases are inhibited by a growing family of specific tissue inhibitors, TIMPs . The cDNA of the third member of the family, TIMP-3, was obtained by using a reverse transcription-polymerase chain reaction (RT-PCR) to amplify the corresponding mRNA from human placenta . Cloning and expression of the TIMP-3 were performed in Escherichia coli as a fusion protein with a 36 amino acid N-tail containing a His cluster . In the host vector system, rhTIMP-3 was stored intracellularly in its denatured, insoluble form in inclusion bodies . Slow dilution of denaturing and reducing agents, from rhTIMP-3 His bound to a metal affinity solid phase, was followed by partial acid removal of the N-tail, which leaves a residue of four amino acids . Circular dichroism, fluorescence and second-derivative UV spectroscopic analyses supported correct refolding of the recombinant and zymography showed inhibition of both MMP-2 and MMP-9 gelatinolytic activities . The role of the C-terminus, which has closer homology with TIMP-2 than TIMP-1, was also investigated: a C-truncated mutant, similarly cloned and expressed in E . coli, shows complete lack of inhibitory activity on MMP-9, still retaining some on MMP-2 . The described protein engineering shows high yield of active inhibitor, unglycosylated as in the native form.

Protein Eng, 1997 May, 10(5), 567 - 73
Construction of a homodimeric dihydrofolate reductase-thymidylate synthase bifunctional enzyme; Trujillo M et al.; A gene encoding a bifunctional homodimeric dihydrofolate reductase-thymidylate synthase (DHFR-TS) was constructed by destroying the stop codon of Escherichia coli dihydrofolate reductase (DHFR) and joining the coding sequences of the monofunctional enzymes by a five amino acid linker . The protein was designed to mimic features of active site proximity and electrostatics in the protozoan DHFR-TSs which are believed to be important in channeling of the DHFR substrate, H2folate, to TS . The genetically engineered catalytically active homodimeric bifunctional DHFR-TS was expressed, purified and characterized . The component activities of the purified bifunctional enzyme had kinetic properties similar to those of the monofunctional TS and DHFR, but unlike the authentic bifunctional enzymes from protozoa this enzyme did not kinetically channel dihydrofolate from DHFR to TS.

Protein Eng, 1997 May, 10(5), 561 - 6
Characterization of the malonyl-/acetyltransacylase domain of the multifunctional animal fatty acid synthase by expression in Escherichia coli and refolding in vitro; Rangan VS et al.; cDNAs of various lengths encoding the second domain of the multifunctional fatty acid synthase (FAS) have been expressed in Escherichia coli and the recombinant proteins refolded in vitro to catalytically active monomeric malonyl-/acetyltransacylases . FAS residues 428-487, previously thought to represent the amino terminus of the malonyl-/acetyltransacylase, can be omitted from the recombinant enzyme with no loss in catalytic activity . This shortened transacylase, consisting of FAS residues 488-809, can be repeatedly denatured and renatured in vitro with reproducibly high recovery and no loss in specific activity . When expressed as a soluble enzyme in Spodoptera frugiperda cells, this transacylase has the same specific activity as the enzyme that has been refolded in vitro . The refolded transacylase consisting of FAS residues 488-809, but not the longer enzyme consisting of residues 428-815, can be crystallized readily . These results suggest that FAS residues 428-487, previously thought to represent the amino terminus of the malonyl-/acetyltransacylase, are not required for catalysis of the transacylase reaction . This region of the FAS is less well conserved than the transacylase catalytic domain and may constitute an extended structural linker that facilitates the functional interaction between the transacylase and acyl carrier protein domains.

Protein Eng, 1997 May, 10(5), 551 - 60
Regulation of trypsin activity by Cu2+ chelation of the substrate binding site; Briand L et al.; Lysine 188 of trypsin was replaced with histidine in order to create a metal chelation site in the substrate binding pocket of this protease, built in a metal binding 'switch,' and to be able to modulate its activity at lower pH . The catalytic properties of wild-type and mutant trypsin were measured with tetrapeptide substrates containing a nitroanilide leaving group and whole native protein substrate: beta-casein . The results obtained reveal that K188H mutation does not affect catalytic efficiency, raising only slightly (from 6 to 8) the arginine/lysine preference of the mutant and increasing 1.8- and 1.2-fold the second-order rate constant k(cat)/Km for arginine- and lysine-containing substrates, respectively . Compared with wild-type trypsin, K188H mutant shows, in the absence of Cu2+, a different catalytic activity pattern as a function of pH . The addition of Cu2+ to trypsin K188H induces a 30-100-fold increase in Km, while k(cat) is scarcely decreased . The hydrolytic activity of this mutant can be fully restored by addition of EDTA . In contrast to a chelating active site, a novel mode of metal-dependent inhibition activity of trypsin with copper is presented . As suggested by molecular modelling studies, the substrate binding pocket of the protease is considerably perturbed by vicinal chelation . More generally, this type of transition metal chelate may present wider possibilities of trypsin activity and specificity modulation than the previously accomplished chelation of a histidine moiety from a catalytic triad.

Plant Cell Physiol, 1997 May, 38(5), 558 - 67
Nucleotide sequence and transcriptional analysis of the flanking region of the gene (spb) for the trans-acting factor that controls light-mediated expression of the puf operon in Rhodobacter sphaeroides; Mizoguchi H et al.; We recently reported the existence of a trans-acting factor (SPB) in Rhodobacter sphaeroides that repressed the expression of the puf operon during illumination . SPB was somewhat homologous to HvrA of Rhodobacter capsulatus, but these proteins appear to have functionally different properties . We now report an analysis of the flanking region of spb in the genome of R.sphaeroides, and we show that spb is a positional counterpart of hvrA of R . capsulatus . The region directly downstream of spb was found to contain three genes, two of which were highly homologous to orf5 and ahcY in R . capsulatus . However, a gene corresponding to hvrB, which controls the expression of orf5 and ahcY in R . capsulatus, was absent in R . sphaeroides . The level of the transcript of ahcY did not change in cells grown under photosynthetic and by respiratory conditions . By contrast, orf5 was transcribed at a higher rate in photosynthetically grown cells under high-intensity light than under low-intensity light, indicating features of transcription different from those in R . capsulatus . A third gene, orf318, which was absent in the corresponding region of R . capsulatus, encoded an amino acid sequence that was significantly homologous to the consensus sequence of RfaI and RfaJ of E.coli, which are glycosyl transferases involved in the synthesis of lipopolysaccharide . orf318 was transcribed in the opposite direction to ahcY, and at only a low level, under all conditions tested.

Vet Res, 1997 May-Jun, 28(3), 231 - 8
Sensitization of the bovine mammary gland to Escherichia coli endotoxin; Rainard P et al.; The effect of repeated infusions of Escherichia coli endotoxin on the acute phase response in the bovine mammary gland was assessed through the concentrations of tumor necrosis factor alpha (TNF-alpha) in milk . Four clinically normal lactating cows received two intramammary infusions of E coli endotoxin (33 micrograms) 24 h apart in the same mammary quarter . Along with the second infusion, the cows received one dose of endotoxin in the contralateral quarter . Milk was collected at varying intervals before and after infusion and TNF-alpha concentrations were determined by ELISA . Following the first infusion at 0 h, the mean concentrations of TNF-alpha augmented from undetectable concentrations to a maximum of 0.4 ng/mL at 4 h and declined to below 0.04 ng/mL at 24 h, the time of the second infusion . In the quarters challenged twice, the increase in TNF-alpha concentrations was abrupt, culminating at 11.7 ng/mL 6 h later (at 30 h) . The increases in TNF-alpha concentrations were similar in the contralateral quarters infused once . TNF-alpha concentrations in the control, uninfused quarters of infused cows remained undetectable (< 0.04 ng/mL) . Despite the low TNF-alpha response following the first infusion, mean somatic cell counts increased markedly, being only slightly lower than after the second infusion (10(7)/mL and 5 x 10(7)/mL at 8 h and 32 h, respectively) in the quarters challenged twice . After the first infusion, none of the cows developed fever, but following the second infusion, rectal temperature increased markedly, culminating 6 h after the second infusion . These results show that an infusion in one quarter of an amount of endotoxin sufficient to induce a pronounced cell recruitment but insufficient to induce a marked TNF-alpha secretion following the first infusion sensitized not only that quarter but also the contralateral one to a second infusion with the endotoxin . It is thus possible that sensitization of the whole udder follows a first contact with a moderate dose of endotoxin in one quarter.

Allergol Immunopathol (Madr), 1997 May-Jun, 25(3), 127 - 34
Recombinant DNA technology in allergology: cloning and expression of plant profilins; Asturias JA et al.; Profilin, an ubiquitous protein involved in eukaryotic cytoskeleton regulation, has been previously described as allergen in grasses, weeds and trees and in many fruits and vegetables, and it is in part responsible for cross-reactivities pollen and food allergic patients . Completed cDNA clones coding for Phleum pratense, Olea europaea, Cynodon dactylon, Parietaria judaica and Helianthus annuus pollen profilins were isolated and sequenced . The deduced amino acid sequences share high identity with other plant profilins . Recombinant profilins were produced in Escherichia coli as non-fusion proteins . Induced cells produced high amounts of recombinant profilin, and after a single purification step on poly-(L-proline)-Sepharose, up to 45 mg of pure allergen per liter culture could be obtained . Recombinant profilins have similar allergenic determinants to their natural counterparts . The tertiary structure of Phleum pratense profilin described here showed three regions important for antibody recognition . The availability of a plant profilin tertiary structure opens future ways on the study of structure/antigenity relationships of these important allergens.

Plant Mol Biol, 1997 May, 34(2), 339 - 43
DNA binding and bending by a chloroplast-encoded HU-like protein overexpressed in Escherichia coli; Wu H et al.; The Guillardia theta chloroplast hlpA gene encodes a protein resembling bacterial histone-like protein HU . This gene was cloned and overexpressed in Escherichia coli cells, and the resulting protein product, HlpA, was purified and characterized in vitro . In addition to exhibiting a general DNA-binding activity, the chloroplast HlpA protein also strongly facilitated cyclization of a short DNA fragment in the presence of T4 DNA ligase, indicating its ability to mediate very tight DNA curvatures.

Plant Mol Biol, 1997 May, 34(2), 295 - 306
The ribosomal protein P0 of soybean (Glycine max L . Merr.) has antigenic cross-reactivity to soybean seed lectin; Wycoff KL et al.; Soybean (Glycine max L . Merr.) mutants lacking the ability to produce the lectin normally found in soybean seeds (SBL) are designated Le- . A protein of higher molecular weight that cross-reacts with antibodies raised to SBL was found at nearly equivalent levels in roots, hypocotyls, and leaves, and at lower levels in cotyledons and dry seeds of both Le+ and Le- soybean cultivars . Earlier work suggested that this protein was a novel lectin . Clones isolated from a Le- soybean root cDNA library produced a cross-reacting protein of the same size in Escherichia coli . Sequence analysis of these clones revealed a high degree of similarity to the ribosomal protein P0 . The cross-reacting protein co-purified with ribosomes, and a monoclonal antibody raised to purified brine shrimp P0 cross-reacted to the same protein . The protein showed no lectin activity in a hemagglutination assay, nor did it bind to an N-acetyl-D-galactosamine affinity column . On the basis of this evidence, we conclude that the SBL-cross-reacting protein is not a lectin but a homologue of the ribosomal protein P0 . Consequently, Le- soybeans must produce a lectin that is dissimilar to SBL at both the DNA and amino acid levels and we suggest that it is this lectin which is involved in nodulation.

Plant Mol Biol, 1997 May, 34(2), 287 - 93
Cloning and expression of an Arabidopsis thaliana cDNA encoding a monofunctional aspartate kinase homologous to the lysine-sensitive enzyme of Escherichia coli; Tang G et al.; As in many bacterial species, the first enzymatic reaction of the aspartate-family pathway in plants is mediated by several isozymes of aspartate kinase (AK) that are subject to feedback inhibition by the end-product amino acids lysine or threonine . So far, only cDNAs and genes encoding threonine-sensitive AKs have been cloned from plants . These were all shown to encode polypeptides containing two linked activities, namely AK and homoserine dehydrogenase (HSD), similar to the Escherichia coli thrA gene encoding a threonine-sensitive bifunctional AK/HSD isozyme . In the present report, we describe the cloning of a new Arabidopsis thaliana cDNA that is relatively highly homologous to the E . coli lysC gene encoding the lysine-sensitive AK isozyme . Moreover, similar to the bacterial lysine-sensitive AK, the polypeptide encoded by the present cDNA is monofunctional and does not contain and HSD domain . These observations imply that our cloned cDNA encodes a lysine-sensitive AK . Southern blot hybridization detected a single gene highly homologous to the present cDNA, plus an additional much less homologous gene . This was confirmed by the independent cloning of an additional Arabidopsis cDNA encoding a lysine-sensitive AK (see accompanying paper) . Northern blot analysis suggested that the gene encoding this monofunctional AK cDNA is abundantly expressed in most if not all tissues of Arabidopsis.

Mol Gen Genet, 1997 May, 254(6), 654 - 64
Cloning, sequencing, disruption and phenotypic analysis of uvsC, an Aspergillus nidulans homologue of yeast RAD51; van Heemst D et al.; We have cloned the uvsC gene of Aspergillus nidulans by complementation of the A . nidulans uvsC114 mutant . The predicted protein UVSC shows 67.4% sequence identity to the Saccharomyces cerevisiae Rad51 protein and 27.4% sequence identity to the Escherichia coli RecA protein . Transcription of uvsC is induced by methyl-methane sulphonate (MMS), as is transcription of RAD51 of yeast . Similar levels of uvsC transcription were observed after MMS induction in a uvsC+ strain and the uvsC114 mutant . The coding sequence of the uvsC114 allele has a deletion of 6 bp, which results in deletion of two amino acids and replacement of one amino acid in the translation product . In order to gain more insight into the biological function of the uvsC gene, a uvsC null mutant was constructed, in which the entire uvsC coding sequence was replaced by a selectable marker gene . Meiotic and mitotic phenotypes of a uvsC+ strain, the uvsC114 mutant and the uvsC null mutant were compared . The uvsC null mutant was more sensitive to both UV and MMS than the uvsC114 mutant . The uvsC114 mutant arrested in meiotic prophase-I . The uvsC null mutant arrested at an earlier stage, before the onset of meiosis . One possible interpretation of these meiotic phenotypes is that the A . nidulans homologue of Rad51 of yeast has a role both in the specialized processes preceding meiosis and in meiotic prophase I.

Mol Gen Genet, 1997 May, 254(6), 643 - 53
Two uvs genes of Aspergillus nidulans with different functions in error-prone repair: uvsI, active in mutation-specific reversion, and uvsC, a recA homolog, required for all UV mutagenesis; Chae SK et al.; Two genes of Aspergillus nidulans are known to function in UV mutagenesis, but have been assigned to different epistasis groups: uvsC, which is also required for meiosis and mitotic recombination, and uvsI, which may have no other function . To clarify their role in error-prone repair and to investigate their interaction, uvsI and uvsC single and uvsI;uvsC double mutant strains were further tested for mutagen sensitivities and characterized for effects on mutation . Spontaneous and induced frequencies were compared in forward and reverse mutation assays . All results confirmed that uvsI and uvsC are members of different epistasis groups, and demonstrated that these uvs mutants have very different defects in UV mutagenesis . The uvsI strains showed wild-type frequencies in all forward mutation tests, but greatly reduced spontaneous and UV-induced reversion of some, but not other, point mutations . In contrast, uvsC had similar effects in all assay systems: namely pronounced mutator effects and greatly reduced UV mutagenesis . Interestingly, the uvsI;uvsC double mutant strains differed from both single mutants; they clearly showed synergism for all types of reversion tested: none were ever obtained spontaneously, nor after induction by UV or EMS (ethylmethane sulfonate) . Based on these results, we conclude that uvsI is active in a mutation-specific, specialized error-prone repair process in Aspergillus . In contrast, uvsC, which is now known to show sequence homology to recA, has a basic function in mutagenic UV repair in addition to recombinational repair, similar to recA of Escherichia coli.

Mol Gen Genet, 1997 May, 254(6), 635 - 42
Gene cloning, sequencing and enzymatic properties of glutamate synthase from the hyperthermophilic archaeon Pyrococcus sp . KOD1; Jongsareejit B et al.; The gltA gene encoding a glutamate synthase (GOGAT) from the hyperthermophilic archaeon Pyrococcus sp . KOD1 was cloned as a 6.6 kb HindIII-BamHI fragment . Sequence analysis indicates that gltA encodes a 481- amino acid protein (53,269 Da) . The deduced amino acid sequence of KOD1-GltA includes conserved regions that are found in the small subunits of bacterial GOGAT: two cysteine clusters, an adenylate-binding consensus sequence and an FAD-binding consensus sequence . However, no sequences homologous to the large subunit of bacterial GOGAT were found in the upstream or downstream regions . In order to examine whether GltA alone can act as a functional GOGAT, GltA was overexpressed in Escherichia coli BL21 (DE3) cells using an expression plasmid . GltA was purified to homogeneity and shown to be functional as a homotetramer of approximately 205 kDa, which is equivalent to the molecular weight of the native GOGAT from KOD1, thus indicating that KOD1-GOGAT is the smallest known active GOGAT . GltA is capable of both glutamine-dependent and ammonia-dependent synthesis of glutamate . Synthesis of glutamate by KOD1-GltA required NADPH, indicating that this enzyme is an NADPH-GOGAT (EC 1.4.1.13) . The optimum pH for both activities was 6.5 . However, GltA exhibited different optimum temperatures for activity depending on the reaction assayed (glutamine-dependent reaction, 80 degrees C; ammonia-dependent reaction, 90 degrees C).

Mol Microbiol, 1997 May, 24(4), 793 - 801
Involvement of the ribulosephosphate epimerase gene in the dnaA and dnaR functions for initiation of chromosome replication in Escherichia coli; Sakakibara Y; Initiation of replication of the Escherichia coli chromosome is rendered temperature-sensitive by the dnaR130 mutation in the prs gene that encodes phosphoribosylpyrophosphate synthetase . The thermosensitivity of the dnaR mutant is suppressed by a spontaneous mutation in rpe, the gene encoding ribulosephosphate epimerase . Disruption of the rpe gene reverses the thermosensitive growth of the dnaR mutant . The rpe-disrupted dnaR mutant exhibits extensive DNA synthesis at low and high temperatures, as does the dnaR+ rpe disruptant and the dnaR+ rpe+ strain . The thermoresistant DNA synthesis in the rpe dnaR double mutant is dnaA dependent, because the dnaA167 mutation renders the synthesis thermosensitive . The rpe-knockout mutation abolishes the ability of the dnaA115 allele to complement the defect of the dnaA167 mutant with or without the dnaR mutation and diminishes the dnaR-complementing ability of the dnaR224 allele . These results show that the rpe product is involved in the functions of the products of both dnaA and dnaR for initiation of replication of the bacterial chromosome.

Mol Microbiol, 1997 May, 24(4), 737 - 45
In vivo mobility of a group I twintron in nuclear ribosomal DNA of the myxomycete Didymium iridis; Johansen S et al.; DiSSU1 is an optional group I twintron present in the nuclear extrachromosomal ribosomal DNA of the myxomycete Didymium iridis . DiSSU1 appears to be complex both in structure and function . At the RNA level it has a twin-ribozyme organization composed of two group I ribozymes with different functions, separated by an open reading frame . Here, we show that DiSSU1 is mobile when haploid intron-containing and intron-less amoebae are mated . The mobility process is fast, being completed in 5-10 nuclear cycles after mating in the developing zygote and plasmodia . Analyses of progeny from genetic crosses confirm intron mobility . DiSSU1 is the first example of a mobile group I twintron . The intron-encoded protein was expressed in Escherichia coli and found to be an endonuclease, I-DirI, that cleaves an intron-less ribosomal DNA allele at the intron-insertion site, and is probably involved in intron homing . The endonuclease I-DirI seems to be a rare example of a protein that is expressed from a ribozyme-processed RNA polymerase I transcript in vivo.

Mol Microbiol, 1997 May, 24(4), 723 - 35
Inactivation of the replication-termination system affects the replication mode and causes unstable maintenance of plasmid R1; Krabbe M et al.; Two so-called Ter sites, which bind the Escherichia coli Tus protein, are located near the replication origin of plasmid R1 . Inactivation of the tus gene caused a large decrease in the stability of maintenance of the R1 mini-derivative pOU47 despite the presence of a functional partition system on the plasmid . Deletion of the right Ter site caused a drop in stability similar to that observed after inactivation of the tus gene . Substitution of 2bp required for Tus binding also caused unstable plasmid maintenance, whereas no effects on stability were observed when the left Ter site was deleted . Inactivation of the tus gene was coupled to an increased occurrence of multimeric plasmid forms as shown by gel electrophoresis of pOU47 DNA . Inactivation of the recA gene did not increase plasmid stability, suggesting that the multimerization was not mediated by RecA . Plasmid DNA was isolated from the tus strain carrying plasmid pOU47 and from a wild-type strain carrying pOU47 in which the right Ter site had been inactivated; in both cases, electron microscopy revealed the presence of multimers as well as rolling-circle structures with double-stranded tails . Thus, the right Ter site in plasmid R1 appears to stabilize the plasmid by preventing multimerization and shifts from theta to rolling-circle replication.

Electrophoresis, 1997 May, 18(5), 762 - 6
Characterization of thioredoxins by sodium dodecyl sulfate-slab gel electrophoresis and high performance capillary electrophoresis; Bunik V et al.; Disulfide containing proteins--thioredoxins from E . coli and pig heart mitochondria--were characterized by sodium dodecyl sulfate (SDS)-electrophoresis and high performance capillary electrophoresis (HPCE) . Following the mitochondrial thioredoxin samples at different stages of purification, we found that their electrophoretic patterns vary, dependent on the redox condition of isolation, preparation of the samples for SDS-electrophoresis, and sample storage . All these factors influenced the relative intensities of several protein bands with thioredoxin-like mobility, whereas the sample storage also resulted in the appearance of SDS- and dithiothreitol (DTT)-resistant high molecular mass forms, probably thioredoxin dimers . The multiple forms of the thioredoxin from pig heart mitochondria in SDS-electrophoresis might be dependent on the oxidation state of the protein cysteine residues . A commercial preparation of the thioredoxin from E . coli did not exhibit any changes in mobility in SDS gels whether the sample was prepared with or without DTT . After the final purification step no correlation was found between mitochondrial thioredoxin activity, determined in the insulin assay, and its purity in SDS-electrophoresis . A correlation was, however, found when analyzing the thioredoxin by HPCE . The latter approach demonstrated the heterogeneity of the thioredoxin samples homogeneous on SDS electrophoresis, only one of the several HPCE peaks being active in the insulin assay . Also, thioredoxin from E . coli, homogeneous on SDS-electrophoresis, was found heterogeneous on HPCE . The peak corresponding to the insulin-dependent thioredoxin activity was split into two by DTT treatment, suggesting that redox transformations of thioredoxin could be followed by HPCE.

Plant J, 1997 May, 11(5), 993 - 1005
Development of necrosis and activation of disease resistance in transgenic tobacco plants with severely reduced catalase levels; Takahashi H et al.; Numerous studies argue that salicylic acid (SA) is an important component of the plant signal transduction pathway(s) leading to disease resistance . The discovery that the SA-binding protein is a catalase, whose activity is blocked by SA, led to the proposal that one of SA's modes of action is to inhibit this H2O2-degrading enzyme and thus elevate H2O2 levels . To test this model, an attempt was made to mimic the action of SA by reducing the synthesis of catalase using antisense RNA technology . Analyses of transgenic tobacco plants that expressed the tobacco catalase 1 (cat1) or catalase 2 (cat2) gene in an antisense orientation indicate that there is no correlation between modest to high levels of reduction in catalase activity and activation of plant defenses such as pathogenesis-related (PR)-1 protein synthesis . However, three independent antisense catalase transgenic plants (ASCAT1 Nos 16, 17, and 28), which exhibited the most severe reduction in catalase activity (approximately 90% or more), developed chlorosis or necrosis on some of their lower leaves . These same leaves accumulated very high levels of PR-1 proteins and showed enhanced resistance to tobacco mosaic virus . Necrosis and elevated SA, which appear to result from severe depression of catalase levels, may be responsible for the induction of these defense responses.

J Biochem (Tokyo), 1997 May, 121(5), 868 - 75
Isolation and molecular cloning of epidermal- and hair follicle-specific peptidylarginine deiminase (type III) from rat; Nishijyo T et al.; Peptidylarginine deiminase (PAD) is a post-translational modification enzyme that catalyzes deimination of arginine residues of proteins in the presence of calcium ions . There are three types of PAD in rodent tissues: PAD types I, II, and III {Terakawa et al . (1991) J . Biochem . 110, 661-666} . Type III enzyme was detected only in the epidermis and in hair follicles . In this study, we have purified PAD type III from 2-day-old rat epidermis by a four-step procedure that included soybean trypsin inhibitor-affinity chromatography . The enzyme was purified about 600-fold from the crude extract and the recovery was 23% . The final preparation of the enzyme gave only a single protein band on SDS-PAGE and showed an apparent molecular weight of 76,000 . Subsequently, we cloned and sequenced the full-length cDNA encoding rat PAD type III by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate oligonucleotide primers designed from the internal amino acid sequences and by the rapid amplification of the cDNA ends method . The composite cDNA sequence contained a 5' untranslated region of 42 bp, an open reading frame of 1,995 bases that encoded 664 amino acids (Mr=75,036), a 3' untranslated region of 1,063 bp, and part of a poly(A)+ tail . The entire reading frame sequence of rat PAD type III showed 51% homology with that of rat PAD type II, and the C-terminal region is highly conserved between the two types . The cloned gene was expressed in Escherichia coli cells to produce PAD type III, which had not only enzymatic activity, but also immunoreactivity against specific antibodies toward PAD type II . Furthermore, the specific expression of the enzyme in the epidermis and hair follicles was confirmed by RT-PCR assays of mRNAs from several tissues.

J Biochem (Tokyo), 1997 May, 121(5), 842 - 8
Purification and characterization of glutaredoxin (thioltransferase) from rice (Oryza sativa L.); Sha S et al.; We purified and characterized glutaredoxin (thioltransferase), which catalyzes thiol/disulfide exchange reaction, for the first time in plants . The purification procedure employed an immunoabsorbent, antiglutaredoxin-Sepharose . Glutaredoxin was purified about 2,200-fold from rice bran and it appeared to be homogeneous on SDS-PAGE . MALDI-TOF mass spectrometry revealed that the protein has a molecular mass of 11,097.9 Da . Rice glutaredoxin consists of 105 amino acid residues, containing the tetrapeptide -Cys-Phe-Pro (Tyr)-Cys-, which constitutes the active site of Escherichia coli and mammalian glutaredoxins . Inactivation assay also indicated that cysteine residues are responsible for enzyme activity . Kinetic analyses revealed that the enzyme did not exhibit normal Michaelis-Menten kinetics . The enzyme has an optimum pH of about 8.7 with 2-hydroxyethyl disulfide as a substrate . In addition, rice glutaredoxin has dehydroascorbate reductase activity, like mammalian glutaredoxin.

J Vet Med Sci, 1997 May, 59(5), 401 - 3
A novel developmental process of intestinal epithelial lesions in a calf infected with attaching and effacing Escherichia coli; Wada Y et al.; A comparative study on the adhesion of attaching and effacing Escherichia coli (AEEC) to the enterocytes between the colon of a calf and the jejunum of a piglet showed differences in the developmental process of attaching and effacing (AE) lesions . In the calf, pedestals consisted of fused microvilli, while in the piglet they developed from the apical epithelial cell membranes after effacing microvilli . Microvilli adjacent to the AEEC attachment site were atrophic in the calf, whereas they were elongated in the piglet . The production of AE lesions in the calf may be indicative of a novel developmental process with AEEC infection.

Biomed Chromatogr, 1997 May-Jun, 11(3), 164 - 6
Removal of endotoxin from aqueous solutions by affinity membrane; Guo W et al.; Endotoxin was removed by affinity membranes with histidine immobilized as affinity ligand . Macropore cellulose membrane was prepared from filter paper by alkaline treatment and chemical crosslinking, and was used as matrix for the immobilization of affinity ligand . The matrix membrane was derived by hexamethylenediamine and activated by glutaraldehyde before histidine was immobilized . Membrane cartridges containing 40 or 80 sheets of affinity membrane were also prepared, which can be used to remove pyrogen from aqueous solutions . Using a cartridge with 40 sheets of affinity membrane, the endotoxin content in solution can be reduced to a minimum of 0.12 EU/mL.

J Cell Sci, 1997 May, 110 ( Pt 10), 1215 - 26
Molecular cloning, over-expression, developmental regulation and immunolocalization of fragminP, a gelsolin-related actin-binding protein from Physarum polycephalum plasmodia; T'Jampens D et al.; FragminP is a Ca2+-dependent actin-binding and microfilament regulatory protein of the gelsolin family . We screened a Physarum polycephalum cDNA library with polyclonal fragminP antibodies and isolated a cDNA clone of 1,104 bp encoding 368 amino acids of fragminP, revealing two consensus phosphatidylinositol 4,5 bisphosphate-binding motifs in the central part of the protein . The first methionine is modified by an acetyl group, and three amino acids were missing from the protein coded for by the cDNA clone . Full-length recombinant fragminP was generated by PCR, purified after over-expression from Escherichia coli and displayed identical properties to native Physarum fragminP . Northern blot analysis against RNA, isolated from cultures at various stages of development, indicated that fragminP is absent from amoebae and that expression is initiated at an early stage during apogamic development, in a similar way to that observed for the profilin genes . In situ immunolocalization of fragminP in Physarum microplasmodia revealed that the protein is localized predominantly at the plasma membrane, suggesting a role in the regulation of the subcortical actin meshwork . Our data indicate that we have isolated the plasmodium-specific fragminP cDNA (frgP) and suggest that, in each of its two vegetative cell types, P . polycephalum uses a different fragmin isoform that performs different functions.

Biol Chem, 1997 May, 378(5), 407 - 15
Dam methyltransferase from Escherichia coli: kinetic studies using modified DNA oligomers: nonmethylated substrates; Thielking V et al.; Steady-state kinetics of the N6-adenine Dam methyltransferase have been measured using as substrates non-self-complementary tetradecanucleotide duplexes that contain the GATC target sequence . Modifications in the GATC target sequence of one or both of the strands included substitution of guanine by hypoxanthine, thymine by uracil or 5-ethyl-uracil and adenine by diamino-purine (2-amino-adenine) . Thermodynamic parameters for the 14-mer duplexes were also determined . DNA methylation of duplexes containing single dl for dG substitution of the Dam recognition site was little perturbed compared with the canonical substrate . Replacement of dG residues by dl in both strands resulted in a decrease of the specificity constant . Substitution in both strands appears to be cumulative . Substitution of the methyl-accepting adenine residues by 2-amino-adenine resulted in surprisingly little perturbation . Dam methyltransferase is rather tolerant to different substitutions . The results show much less spread than those for the analogous hemimethylated substrates studied previously (Marzabal et al., 1995) . The absence of the methylation marker appears to be deleterious to the specificity of the transition state of the active complex, while the binding of the DNA substrate to the enzyme appears to be mostly determined by the thermodynamic stability of the DNA duplex.

Crit Care Med, 1997 May, 25(5), 858 - 63
N-acetylcysteine attenuates endotoxin-induced leukocyte-endothelial cell adhesion and macromolecular leakage in vivo; Schmidt H et al.; OBJECTIVE: To determine the influence of N-acetylcysteine on endotoxin-induced leukocyte-endothelial cell adhesion, vascular leakage, and venular microhemodynamics . DESIGN: Randomized, blinded, controlled trial . SETTING: Experimental laboratory . SUBJECTS: Thirty male Wistar rats . INTERVENTIONS: After pretreatment with N-acetylcysteine (150 mg/kg; n = 40; group A) or 0.9% saline solution (n = 10; group B) animals were given an intravenous infusion of endotoxin (Escherichia coli lipopolysaccharide 026:B6; 2 mg/kg/hr) over 120 mins . Animals in the control group (n = 10; group C) received a volume-equivalent infusion of 0.9% saline solution . MEASUREMENTS AND MAIN RESULTS: Leukocyte adherence, red cell velocity (VRBC), vessel diameters, venular wall shear rate, and macromolecular leakage were determined in mesenteric postcapillary venules using in vivo videomicroscopy at baseline and at 30, 50, 90, and 120 mins after the start of the endotoxin challenge . Endotoxin exposure induced a marked increase in adherent leukocytes (group B: baseline, 391 +/- 24 cells/mm2; 120 mins, 1268 +/- 131 cells/mm2; p < .01) . N-acetylcysteine pretreatment attenuated the adherence of leukocytes during endotoxemia (baseline, 366 +/- 28 cells/mm2; 120 mins, 636 +/- 49 cells/mm2; p < .01 vs . baseline; p < .01 vs . group B) . Leukocyte adherence in control animals (group C) did not increase significantly . Administration of N-acetylcysteine did not influence the decrease in VRBC observed during endotoxemia . In group B1 VRBC decreased during the infusion of endotoxin from 2.0 +/- 0.2 mm/sec at baseline to 1.1 +/- 0.2 mm/ sec after 120 mins (p < .01 vs . baseline; p < .05 vs . group C), and in group A from 2.2 +/- 0.2 mm/sec to 1.1 +/- 0.1 mm/sec after 120 mins (p < .01 vs . baseline; p < .05 vs . group C) . In group C, VRBC remained unchanged (baseline, 1.7 +/- 0.2 mm/sec; at 120 mins, 1.5 +/- 0.2 mm/sec) . The venular diameters remained unchanged in all groups during the entire study period . After 120 mins, the venular wall shear rate decreased from 502 +/- 62 secs-1 at baseline to 272 +/- 46 sec-1 in group B (p < .01), and from 563 +/- 45 secs-1 at baseline to 283 +/- 31 secs-1 in group A (p < .01) . No differences in venular wall shear rate were observed between these groups . In group C, the venular wall shear rate remained unchanged (baseline, 457 +/- 54 secs-1; at 120 mins, 409 +/- 51 secs-1) . Macromolecular leakage, expressed as perivenular/intravenular fluorescence intensity after injection of fluorescence-labeled albumin, increased from 0.29 +/- 0.03 to 0.58 +/- 0.03 (p < .01) during the infusion of endotoxin in group B . In contrast, pretreatment with N-acetylcysteine diminished the extravasation of albumin (baseline, 0.27 +/- 0.01; at 120 mins, 0.37 +/- 0.02; p < .01 vs . baseline; p < .01 vs . group B) . CONCLUSION: These results demonstrate that N-acetylcysteine attenuates endotoxin-induced alterations in leukocyte-endothelial cell adhesion and macromolecular leakage, suggesting N-acetylcysteine might be therapeutic in the prevention of endothelial damage in sepsis.

Crit Care Med, 1997 May, 25(5), 848 - 57
The endothelin receptor antagonist, bosentan, in combination with the cyclooxygenase inhibitor, diclofenac, counteracts pulmonary hypertension in porcine endotoxin shock; Wanecek M et al.; OBJECTIVE: To prevent endotoxin-induced pulmonary hypertension in the pig with a combination of a nonpeptide mixed endothelin receptor antagonist, bosentan, and a cyclooxygenase inhibitor, diclofenac . DESIGN: Prospective, controlled trial . SETTING: Animal laboratory at a large university medical center . SUBJECTS: Twelve domestic pigs, weighing 17.5 to 27 kg . INTERVENTIONS: Endotoxin shock was induced by intravenous infusion of Escherichia coli lipopolysaccharide endotoxin (15 micrograms/kg/hr) . Six pigs receiving only endotoxin served as controls . Six pigs were pretreated with intravenous bolus injections of bosentan (5 mg/kg) and diclofenac (3 mg/kg) followed by a continuous bosentan infusion (2.5 mg/kg/hr) . MEASUREMENTS AND MAIN RESULTS: Systemic hemodynamics and regional circulation were measured using ultrasonic flow probes . Arterial and mixed venous blood samples were collected regularty for determination of Big endothelin-1-like immunoreactivity, endothelin-1-like immunoreactivity, norepinephrine, and blood gases . The bosentan/diclofenac pretreatment per se significantly decreased mean pulmonary arterial pressure (p < .001), pulmonary vascular resistance index (p < .001), and mean arterial blood pressure (p < .001), but cardiac index did not change . Splenic blood flow increased (p < .01) while renal blood flow decreased (p < .001) . In addition, intestinal blood flow decreased slightly (p < .05) . In the control group, only three animals survived the 3 hrs of endotoxin infusion, while all pretreated animals survived . The biphasic increase in mean pulmonary arterial pressure and pulmonary vascular resistance index seen in control animals during endotoxemia was markedly attenuated in animals pretreated with the bosentan/diclofenac combination . The pretreated group generally showed a favorable hemodynamic course, with a relatively higher cardiac index, stroke volume index, and splenic and renal blood flow . In control animals, a pronounced metabolic acidosis developed during endotoxin infusion . A relatively higher arterial plasma concentration of endothelin-1-like immunoreactivity was reached in pretreated animals, while the Big endothelin-1-like immunoreactivity plasma increase was similar in both groups . Arterial concentrations of norepinephrine were significantly (p < .01) higher in control animals when compared with diclofenac/bosentan-treated animals . CONCLUSIONS: The combination of bosentan and diclofenac induced systemic and pulmonary vasodilation in the intrinsic state . During endotoxin shock, this drug combination efficiently counteracts pulmonary hypertension and improves cardiac performance and splenic and renal blood flow . These favorable circulatory effects may have resulted in a reduction of both sympathetic nervous system activation and metabolic acidosis . Thus, we conclude that the endothelin receptors participate in intrinsic regulation of vascular tone in the anesthetized pig . During endotoxin shock, blockade of these receptors, as well as inhibition of the cyclooxygenase enzymes, contributes to a less adverse effect on the systemic and pulmonary circulation.

Eur J Biochem, 1997 May 1, 245(3), 738 - 44
Characterization of the molecular-chaperone function of the heat-shock-cognate-70-interacting protein; Bruce BD et al.; A histidine-tagged form of the recently discovered molecular chaperone, 70-kDa heat-shock cognate (Hsc70)-interacting protein (Hip), has been expressed in Escherichia coli and purified to near homogeneity . This protein remains soluble when expressed in E . coli . Several important properties of this chaperone have been investigated . HPLC size-exclusion chromatography indicates that the chaperone forms a tetramer similar to what has been reported for the native protein from rat liver cytosol . The recombinant form of Hip did not catalyze the hydrolysis of ATP and ATP analogs, although fluorescence measurements indicated that the chaperone recognizes anthraniloyl-dATP, anthraniloyl-ADP, and 2'-O-trinitrophenyl-ATP . The role of Hip as a molecular chaperone has been confirmed by its ability to strongly bind to the reduced, carboxymethylated form of alpha-lactalbumin . This interaction is specific for non-native domains since native alpha-lactalbumin fails to interact with Hip . Fluorescence-anisotropy measurements indicate that reduced, carboxymethylated lactalbumin binds Hip with a Kd of 5 microM . Although Hip appears to be able to bind nucleotides and non-native proteins, it is unable to facilitate the refolding of two denatured proteins, E . coli alkaline phosphatase and mitochondrial malate dehydrogenase . Hip inhibited the refolding of alkaline phosphatase and malic dehydrogenase . Inhibition occurred at near stoichiometric levels of Hip and could not be reversed by the addition of ATP . These results suggest that Hip may regulate the function of the Hsp70 molecular chaperone complex in vivo and play a critical role in protein folding in the eukaryotic cytoplasm.

Eur J Biochem, 1997 May 1, 245(3), 715 - 9
Thermochemical and kinetic evidence for nucleotide-sequence-dependent RecA-DNA interactions; Wittung P et al.; RecA catalyses homologous recombination in Escherichia coli by promoting pairing of homologous DNA molecules after formation of a helical nucleoprotein filament with single-stranded DNA . The primary reaction of RecA with DNA is generally assumed to be unspecific . We show here, by direct measurement of the interaction enthalpy by means of isothermal titration calorimetry, that the polymerisation of RecA on single-stranded DNA depends on the DNA sequence, with a high exothermic preference for thymine bases . This enthalpic sequence preference of thymines by RecA correlates with faster binding kinetics of RecA to thymine DNA . Furthermore, the enthalpy of interaction between the RecA x DNA filament and a second DNA strand is large only when the added DNA is complementary to the bound DNA in RecA . This result suggests a possibility for a rapid search mechanism by RecA x DNA filaments for homologous DNA molecules.

Eur J Biochem, 1997 May 1, 245(3), 600 - 7
Characterization of human and pig kidney long-chain-acyl-CoA dehydrogenases and their role in beta-oxidation; Eder M et al.; Long-chain-acyl-CoA dehydrogenase (LCADH) has been produced by recombinant techniques from the human cDNA and purified after expression in Escherichia coli . Pig kidney LCADH was purified using an optimized method which also produces apparently pure short-chain-acyl-CoA dehydrogenase (SCADH) and medium-chain-acyl-CoA dehydrogenase (MCADH) in good yields . LCADH from both sources has a maximal turnover rate (Vmax of 650-700 min(-1) at pH 7.6) with the best substrates, which is approximately fivefold higher than reported previously . The human enzyme has an approximately fivefold higher Km compared with the pig kidney enzyme with substrates of chain length from C10 to C18 and a significantly different dependence of Vmax on the chain length . Pig kidney LCADH has a similar Vmax/Km with C10 to C14 substrates as MCADH does with C6 to C10 substrates . Recombinant human LCADH, however, is significantly less efficient (approximately fourfold with C12) than purified pig kidney enzyme . We conclude that human LCADH is either quantitatively less important in beta-oxidation than in the pig, or that post-translational modifications, not present in the recombinant human enzyme, are required to optimize human LCADH activity . Our results demonstrate that LCADH is as important as the other acyl-CoA dehydrogenases in fatty acid oxidation at physiological, mitochondrial pH with optimal substrates of chain length C10-C14 . The extent of the LCADH-flavin cofactor reduction observed with most substrates and the rate of the subsequent reoxidation with oxygen are markedly different from those found with human medium chain acyl-CoA dehydrogenase . Both LCADH are inactivated by the substrate analogue 2-octynoyl-CoA, possibly via covalent modification of Glu261, the active-site residue involved in deprotonation of the substrate (alpha)C-H.

Mol Carcinog, 1997 May, 19(1), 39 - 45
Mutation spectra analysis suggests that N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-induced lesions are subject to transcription-coupled repair in Escherichia coli; Iannone R et al.; To determine the influence of some bacterial DNA repair pathways on the mutagenic and the lethal effects of N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU), pZ189 plasmids treated in vitro with 2 mM CCNU were transfected into Escherichia coli strains with different repair capacities (uvr+ada+ogt+, uvr-ada+ogt+, and uvr-ada-ogt-) . Despite the differences in repair capacities, no statistically significant difference in survival and mutability was observed among the tested strains . One hundred and sixty-six CCNU-induced supF mutants were isolated and sequenced . All mutants were characterized by single base-pair substitutions, most of which (more than 96%) were GC-->AT transitions (the mutated G being almost exclusively preceded 5' by a purine) . Mutation distribution was not random . Position 160 (5'-GGT-3', nontranscribed (NT) strand) was a uvr+ada+ogt(+)-specific hot-spot . Position 123 (5'-GGG-3', NT strand) was a common hot-spot but significantly more mutable in repair-proficient strains than in repair-deficient strains . Conversely, position 168 (5'-GGA-3', transcribed (T) strand) was significantly more mutable in repair-deficient strains than in repair-proficient strains . By applying a computer program for comparison of mutational spectra, we found that the uvr+ mutational spectrum was significantly different from those obtained in uvr- strains, whereas in the uvr- background, no difference was observed between mutation spectra in ada+ogt+ versus ada-ogt- strains . Our results are consistent with the hypothesis that O6-alkylguanine is responsible for most mutations observed in all strains . The results also indicate that excision repair modulates the distribution of GC-->AT transitions . The fact that mutations at G lesions on the T strand were significantly less frequent in uvr+ than in uvr- strains suggests that CCNU-induced premutational lesions are susceptible to strand-preferential repair in E . coli.

Comp Biochem Physiol B Biochem Mol Biol, 1997 May, 117(1), 135 - 42
Kinetic characteristics of nucleoside mono-, di- and triphosphatase activities of the periplasmic 5'-nucleotidase of Escherichia coli; Garcia L et al.; Periplasmic 5'-nucleotidase from Escherichia coli, in addition to the monophosphoesterase activity has a diphosphohydrolase activity, acting on nucleoside di- and triphosphates . We proposed that the monophosphoesterase and diphosphohydrolase activities have their own active site . This proposal is based on the different types of bonds being broken . Chemical modification with selective group reagents did not show differences in the essentiality of some residues, like histidyl, carboxyl and arginyl groups, of these two hydrolytic activities . While kinetic approaches employing the competition plot and unidirectional substrate inhibition point to that diphosphohydrolase activity (ATPase-ADPase) do not share the same active site with monophosphoesterase activity . Western blotting developed with polyclonal anti-placental apyrase antibody revealed a single protein in the periplasmic fraction of 66.5 kDa similar to the Mr of the purified enzyme by isoelectrofocusing.

Mol Microbiol, 1997 May, 24(3), 643 - 51
The sigma S level in starving Escherichia coli cells increases solely as a result of its increased stability, despite decreased synthesis; Zgurskaya HI et al.; The sigma S level in starving (stationary phase) Escherichia coli cells increases four-to sixfold following growth in a defined or a complex medium . Chemostat-grown cells, subjected to increasing carbon starvation, also become progressively richer in sigma S content . These increases occur despite reduced transcription of the sigma S-encoding gene, rpoS, and translation of rpoS mRNA, and result solely from a large increase in the stability of the sigma protein . Previous results, based on rpoS::lacZ transcriptional and translational fusions, and on methionine incorporation in sigma S, had suggested increased synthesis of sigma S in starving cells . Alternative explanations for these results consistent with the conclusions of this paper are discussed.

Mol Microbiol, 1997 May, 24(3), 617 - 27
Characterization of the negative elements involved in silencing the bgl operon of Escherichia coli: possible roles for DNA gyrase, H-NS, and CRP-cAMP in regulation; Mukerji M et al.; The bgl operon of Escherichia coli is rendered cryptic and uninducible in wild-type cells by the presence of DNA structural elements that negatively regulate transcription . We have carried out a detailed analysis of the sequences implicated in negative regulation . Fine-structure deletion analysis of the upstream sequences showed the presence of at least two elements involved in silencing the promoter . Chemical probing of genomic DNA in vivo showed that a region of dyad symmetry, present upstream of the promoter, is hypersensitive to KMnO4 . The hypersensitive region detected corresponds to the potential cruciform structure implicated earlier in negative regulation . Enhancement of transcription from the wild-type promoter, observed in the presence of the gyrase inhibitor novobiocin, was absent in a mutant that carried point mutations in the inverted repeat . This observation suggests that the activation seen in a gyrase mutant is mediated by destabilization of the cruciform because of reduced supercoiling . Deletion of sequences downstream of the potential cruciform also resulted in an increase in transcription, indicating the presence of a second regulatory element . Measurement of transcription from the bgl promoter carrying the deletion, in a strain that has a mutation in the hns gene, indicated that this region is likely to be involved in binding to H-NS or a protein regulated by H-NS, which acts as a non-specific repressor . We also provide evidence which suggests that transcriptional activation by mutations at the cAMP receptor protein (CRP)-binding site is mediated partly by antagonization of the negative effect of H-NS by CRP-cAMP as a result of its increased affinity for the mutant site.

Mol Microbiol, 1997 May, 24(3), 593 - 605
HlyX, the FNR homologue of Actinobacillus pleuropneumoniae, is a {4Fe-4S}-containing oxygen-responsive transcription regulator that anaerobically activates FNR-dependent class I promoters via an enhanced AR1 contact; Green J et al.; The hlyX gene of the pig pathogen Actinobacillus pleuropneumoniae encodes HlyX, a homologue of FNR, the anaerobic transcription regulator of Escherichia coli . The hlyX gone complements the anaerobic respiratory deficiencies of E . coli fnr mutants but also induces the expression of an otherwise latent haemolysin . Therefore, FNR and HlyX have distinct but overlapping regulons . The hlyX gene has been overexpressed as a gst::hlyX fusion and the HlyX protein purified . Similar to FNR, HlyX can acquire a {4Fe-4S} cluster, which promotes binding to the FNR box (Kd of 20-30 nM) under anaerobic conditions . Expression of hlyX in E . coli induced the anaerobic production of at least five polypeptides, including the yfiD gene product, which were not induced by fnr . Analysis of the yfiD promoter region revealed the presence of two FNR boxes situated at -61.5 and -114.5 . Consistent with this observation, expression from the semi-synthetic Class I promoter FF + 20pmelR was efficiently activated by HlyX but not by FNR . The weaker level of FNR-mediated activation of Class I promoters suggests that there is a poorer activating contact (activating region 1 (AR1) equivalent) between FNR and RNA polymerase at these promoters and that HlyX possesses an additional or improved AR1 . The AR1 of HlyX is partially characterized by a surface-exposed region around amino acid A187, which confers the altered specificity and provides an explanation for the existence of distinct but overlapping HlyX and FNR regulons.

Mol Microbiol, 1997 May, 24(3), 523 - 34
Ffh and FtsY in a Mycoplasma mycoides signal-recognition particle pathway: SRP RNA and M domain of Ffh are not required for stimulation of GTPase activity in vitro; Macao B et al.; Mycoplasma mycoides contains a signal-recognition particle (SRP) composed of an RNA molecule and an SRP54 homologue (Ffh) . We have now identified a mycoplasma homologue to the alpha subunit of the mammalian SRP receptor and Escherichia coli FtsY . The protein (MmFtsY) was expressed in E . coli and purified to homogeneity . MmFtsY has a weak intrinsic GTPase activity but GTP hydrolysis was markedly stimulated when it was combined with mycoplasma Ffh (MmFfh) and SRP RNA . Also, in the absence of SRP RNA GTPase activity was significantly enhanced . Furthermore, GTP hydrolysis was stimulated when MmFtsY was combined with the N-terminal GTPase domain (N+G) of MmFfh . These findings indicate that basic features of the GTPase activation mechanism are independent of the C-terminal M domain of the MmFfh protein . We propose that the activation is mediated to a large extent by contacts between the GTPase domains of the mycoplasma Ffh and FtsY proteins and that the contribution of the M domain and SRP RNA in the activation mechanism is mainly for modifying the conformation of the MmFfh GTPase domain.

Mol Microbiol, 1997 May, 24(3), 511 - 21
Action at a distance for glp repressor control of glpTQ transcription in Escherichia coli K-12; Yang B et al.; The adjacent, divergently transcribed glpACB and glpTQ operons of Escherichia coli encode the anaerobic glycerol 3-phosphate dehydrogenase and glycerol 3-phosphate transporter/phosphodiesterase, respectively . These operons are negatively controlled by glp repressor binding to operators that overlap the glpA promoter elements . Using DNase I footprinting, three additional operators (OT1-3) were identified at positions +307 to +359 within the glpT coding region . To assess a potential regulatory role for these remote operators in vivo, a glpT-lacZ transcriptional fusion containing all of the glpA and glpT operators was constructed . The response of this fusion to the glp repressor was compared to fusion constructs in which OT1 and OT3 were inactivated, either by deletion or by site-directed mutagenesis . It was found that repression of glpT conferred by binding of glp repressor to glpA operators was increased about three- to fourfold upon introduction of the remote glpT operators . In addition, two integration host factor (IHF) binding sites were identified downstream of the glpT transcriptional start site at positions +15 to +51 and +193 to +227 . A regulatory role for IHF was demonstrated by showing that repression of glpT mediated by GlpR was decreased about twofold in strains deficient in IHF and that mutations in IHF1 and/or IHF2 decreased repression about two- to threefold . The effect of IHF was apparent only when the remote operators were present . All of the results are consistent with a model of repression involving GlpR binding simultaneously to the glpA and remote glpT operators, with intervening DNA forming a loop.

Mol Microbiol, 1997 May, 24(3), 457 - 64
Energy requirement for pullulanase secretion by the main terminal branch of the general secretory pathway; Possot OM et al.; The energy requirement for the second step in pullulanase secretion by the general secretory pathway was studied in Escherichia coli . In order to uncouple the two steps in the secretion pathway (across the cytoplasmic and outer membranes, respectively) and to facilitate kinetic analysis of secretion, a variant form of pullulanase lacking its N-terminal fatty acid membrane anchor was used . The transport of the periplasmic secretion intermediate form of this protein across the outer membrane was not inhibited by concentrations of sodium arsenate in excess of those required to reduce ATP levels to < or = 10% of their normal value . Pullulanase secretion was inhibited by the protonophore carbonyl cyanide m-chlorophenyl hydrazone at concentrations which were similar to those reported by others to be required to prevent solute uptake or the export and processing of preproteins across the cytoplasmic membrane, but which were in excess of those required to fully dissipate the proton-motive force and to reduce lactose uptake to a significant extent.

Biol Pharm Bull, 1997 May, 20(5), 556 - 9
Biochemical and immunochemical properties of modified human neuron-specific enolase; Kaneta M et al.; 125I-Labeled recombinant human neuron-specific enolase (R-NSE) was inadequate for RIA as a labeled antigen . The binding activity of labeled R-NSE to the antibody was markedly decreased . To supplement this defect and facilitate purification, we constructed two R-NSE derivatives, Y-NSE (one tyrosine residue was added at the N-terminal of R-NSE) and Y-NSE.H6 (six histidine residues were further added at the C-terminal of Y-NSE) . The biochemical and immunochemical characteristics of these R-NSE derivatives were essentially the same to those of R-NSE . These derivatives were useful not only as standards for enzyme immunoassay (EIA), but also as labeled antigens for RIA . These results clearly indicate that the reactivity of these modified NSEs to anti-NSE antibody is almost equivalent to that of human brain gammagamma-enolase (B-NSE), and that even if the modified NSEs are labeled, they retain their binding affinities to antibodies in contrast to R-NSE.

Biol Pharm Bull, 1997 May, 20(5), 479 - 81
Alteration of fatty acid composition in a pgsA3 mutant of Escherichia coli; Suzuki E et al.; We examined the fatty acid composition in an Escherichia coli pgsA3 mutant lacking the potential to synthesize phosphatidylglycerolphosphate, a precursor of phosphatidylglycerol . The contents of C18:1cis-9 (oleic acid) and C18:1cis-11 (cis-vaccenic acid) in the total phospholipids extracted from the pgsA3 mutant growing at 37 degrees C were higher and that of C14:0 was lower than the wild type cells, resulting in a higher level of unsaturation of fatty acids (ratio of unsaturated fatty acids to saturated ones) in the mutant . The higher level of the unsaturated fatty acids in the pgsA3 mutant was more obvious in cardiolipin than in phosphatidylethanolamine . On the other hand, at 28 degrees C, at which the pgsA3 mutant shows limited cell growth, the content of unsaturated fatty acids in cardiolipin decreased in the pgsA3 mutant compared with the wild type . We consider that the pgsA3 mutant maintains cellular homeostasis by altering the level of unsaturated fatty acids in cardiolipin, and the mechanism is influenced by temperature.

Biol Pharm Bull, 1997 May, 20(5), 467 - 70
Transient DNA relaxation in Escherichia coli induced by nalidixic acid; Ohtsuka Y et al.; We report here the transient relaxation of plasmid DNA by inhibitors of DNA gyrase in Escherichia coli . Relaxation of plasmid DNA in the presence of nalidixic acid, an inhibitor of the A subunit of DNA gyrase, lasted less than 60 min . The effect of nalidixic acid was not observed in a nalA26 mutant, a strain resistant to nalidixic acid due to a mutation in the gyrA gene . Novobiocin, whose target is the B subunit of DNA gyrase, also caused transient DNA relaxation . Resupercoiling of relaxed DNA in the presence of nalidixic acid was inhibited by pretreatment of the cells with chloramphenicol . As mutations of both the rpoH and recA genes did not affect the resupercoiling reaction, this step seems to require protein synthesis that is independent of both heat shock and SOS responses.

Mol Biochem Parasitol, 1997 May, 86(1), 85 - 94
Molecular biology of the hexokinase isoenzyme pattern that distinguishes pathogenic Entamoeba histolytica from nonpathogenic Entamoeba dispar; Ortner S et al.; The electrophoretic patterns of hexokinase and phosphoglucomutase have been widely used to distinguish Entamoeba histolytica from Entamoeba dispar isolates . Although E . histolytica and E . dispar, previously called pathogenic and nonpathogenic Entamoeba histolytica, differ clearly in sequences of many homologous genes, a conversion between the two has been reported by several laboratories, in each case showing the conversion of hexokinase (ATP, D-hexose 6-phosphotransferase, EC 2.7.1.1) isoenzyme patterns . An apparent mobility shift of this enzyme may either be due to posttranslational modification or processing, or to the appearance of a new isoform encoded by a second gene . In this study we observed that the four observed bands in the isoenzyme patterns of pathogenic and nonpathogenic forms of Entamoeba were correlated with four different cDNAs, and that the four recombinant hexokinases produced in Escherichia coli comigrated with their natural counterparts . Polymerase chain reaction (PCR) experiments did not reveal hidden genes which might be responsible for conversion phenomena . These results strongly support the redefinition of pathogenic and nonpathogenic Entamoeba histolytica as two closely related species Entamoeba histolytica and Entamoeba dispar.

Mol Biochem Parasitol, 1997 May, 86(1), 29 - 36
Expression of Toxoplasma gondii genes in the closely-related apicomplexan parasite Neospora caninum; Howe DK et al.; We have established the feasibility of using Neospora caninum as a heterologous system for the expression of genes from the closely-related parasite Toxoplasma gondii . Plasmid construct containing the lacZ gene from Escherichia coli driven by T . gondii promoters were electroporated into N . caninum parasites, and expression of beta-galactosidase (beta-Gal) activity was assayed enzymatically . In transient assays, expression of beta-Gal driven by the GRA1 promoter was approximately 10-fold higher than the expression obtained with the SAG1 promoter . Enzyme activity was not detected when N . caninum parasites were transfected with a promoterless lacZ construct . Transfection of N . caninum with complete genomic clones of SAG1 or GRA2 from T . gondii yielded parasites that transiently expressed the respective gene products, as detected by immunofluorescence and Western blot . Additionally, these transiently expressed T . gondii proteins appeared by immunofluorescence localization and Triton X-114 partitioning to be correctly targeted in both extracellular and intracellular N . caninum parasites . Heterologous gene expression should be useful for studying the function of specific gene products and may facilitate the identification of genes responsible for the phenotypic differences observed between these two closely-related apicomplexan parasites.

Anal Biochem, 1997 May 1, 247(2), 279 - 86
Use of thermostable and Escherichia coli RNase H in RNA mapping studies; Porter D et al.; A recently introduced thermostable RNase H was tested to determine its effectiveness in RNase H mapping reactions . Procedures are described which should have general use with both the thermostable and the Escherichia coli RNase H enzymes . Using the thermostable RNase H at higher temperatures extends the range of oligodeoxyribonucleotide/RNA combinations that yield satisfactory results . Northern blot analyses of total RNA was used to demonstrate that native RNAs can be analyzed by oligodeoxyribonucleotide directed RNase H digestion with minimal sample processing as long as care is taken to maintain thermal stringency both during reaction assembly and termination . Increased thermal stringency allows for higher DNA concentrations to ensure complete site-specific digestion of target RNAs or to permit simultaneous cleavage with multiple oligodeoxyribonucleotides . Partial digests can also be controlled by manipulating oligodeoxyribonucleotide concentrations . In addition, the thermostable RNase H was shown to be active at magnesium ion concentrations as low as 0.1 mM . This allows for optimization of Mg2+ effects on overall sample integrity and DNA/RNA interactions over at least a 20-fold range (2.0-0.1 mM).

Plant Mol Biol, 1997 May, 34(1), 169 - 74
RAP-1 is an Arabidopsis MYC-like R protein homologue, that binds to G-box sequence motifs; de Pater S et al.; An Arabidopsis cDNA clone encoding a DNA-binding protein, RAP-1, was isolated by southwestern screening of an Escherichia coli cDNA expression library . The protein contains a bHLH DNA-binding domain and is homologous to R proteins, regulating anthocyanin biosynthesis . RAP-1 binds to the sequence CACNTG . It is encoded by a single gene, which is expressed to high levels in root and stem and to low levels in leaf and flower . No expression could be detected in siliques . Rap-1 does not correspond to one of the known loci involved in anthocyanin biosynthesis, since it is located at a different map position . In contrast to the maize R protein Lc, RAP-1 did not induce anthocyanin biosynthesis in pea cotyledons . Thus, RAP-1 is a novel member of the bHLH class of DNA-binding proteins.

Plant Mol Biol, 1997 May, 34(1), 151 - 61
Role of PSII-L protein (psbL gene product) on the electron transfer in photosystem II complex . 1 . Over-production of wild-type and mutant versions of PSII-L protein and reconstitution into the PSII core complex; Ozawa S et al.; To establish a system for over-production of PSII-L protein which is a component of photosystem II (PSII) complex, a plasmid designated as pMAL-psbL was constructed and expressed in Escherichia coli JM109 . A fusion protein of PSII-L and maltose-binding proteins (53 kDa on SDS-PAGE) was accumulated in E . coli cells to a level of 10% of the total protein upon isopropyl-beta-D-thiogalactopyranoside (IPTG) induction . The carboxyl-terminal part of 5.0 kDa was cleaved from the fusion protein and purified by an anion exchange column chromatography in the presence of detergents . This 5.0 kDa protein was identified as PSII-L by amino-terminal amino acid sequence analysis and the chromatographic behavior on an anion exchange gel . A few types of mutant PSII-L were also prepared by the essentially same procedure except for using plasmids which contain given mutations in psbL gene . Plastoquinone-9 (PQ-9) depleted PSII reaction center core complex consisting of D1, D2, CP47, cytochrome b-559 (cyt b-559), PSII-I and PSII-W was reconstituted with PQ-9 and digalactosyldiglyceride (DGDG) together with the wild-type or mutant PSII-L produced in E . coli or isolated PSII-L from spinach . Significant difference between the wild-type PSII-L proteins from E . coli and spinach was not recognized in the effectiveness to recover the photo-induced electron transfer activity in the resulting complexes . The analysis of stoichiometry of PQ-9 per reaction center in the PQ-9 reconstituted PS II revealed that two molecules of PQ-9 were reinserted into a reaction center independent of the presence or absence of PSII-L . These results suggest that PSII-L recovers the electron transfer activity in the reconstituted RC by a mechanism different from the stabilization of PQ-9 in the Q(A) site of PSII . Ubiquinone-10 (UQ-10), but not plastoquinone-2 (PQ-2), substituted PQ-9 for recovering the PSII-L supported electron transfer activity in the reconstituted PSII reaction center complexes . The results obtained with the mutant PSII-L proteins revealed that the carboxyl terminal part rather than amino terminal part of PSII-L is crucial for recovering the electron transfer activity in the reconstituted complexes.

Plant Mol Biol, 1997 May, 34(1), 15 - 29
Expression of de novo high-lysine alpha-helical coiled-coil proteins may significantly increase the accumulated levels of lysine in mature seeds of transgenic tobacco plants; Keeler SJ et al.; We have designed protein molecules based on an alpha-helical coiled-coil structure . These proteins can be tailored to complement nutritionally unbalanced seed meals . In particular, these proteins may contain up to 43% mol/mol of the essential amino acid lysine . Genes encoding such proteins were constructed using synthetic oligonucleotides and the protein stability was tested for in vivo by expression in an Escherichia coli model system . A protein containing 31% lysine and 20% methionine (CP 3-5) was expressed in transgenic tobacco seeds utilizing the seed specific bean phaseolin and soybean beta-conglycinin promoters . Both promoters provided a level of expression in the mature transgenic tobacco seeds which resulted in a significant increase in the total lysine content of the seeds . Several of these transgenic lines were analyzed for three generations to determine the stability of gene expression . Plants transformed with the soybean beta-conglycinin promoter/CP 3-5 gene consistently expressed the high-lysine phenotype through three generations . However, expression of the high-lysine phenotype in plants transformed with the bean phaseolin/CP 3-5 was variable . This is the first report of a significant increase in seed lysine content due to the seed-specific expression of a de novo protein sequence.

Cell Mol Life Sci, 1997 May, 53(5), 459 - 65
Effects of amino acid replacements on cadmium binding of metallothionein alpha-fragment; Yamasaki F et al.; To evaluate whether only 20 cysteine residues at invariant positions are needed to bind and coordinate the metals in metallothioneins (MTs), and whether changing the positions of cysteine residues in the sequence affects the metal-binding capacity and the coordination of MTs, we examined the cadmium-binding affinities of seven mutant MT alpha s using an Escherichia coli expression system . Five mutant MT alpha s in which the constitutive amino acid residues other than cysteines of the alpha-fragment were replaced with glycine residues, and the remaining two mutant MT alpha s in which the invariant positions of the cysteine residues of the alpha-fragment were shifted, were analysed for their ability to be expressed as cadmium-binding forms and for their biochemical properties . The results showed that extreme alteration of the constitutive amino acid residues other than cysteines in the MT alpha-fragment leads to disruption of their cadmium-binding abilities and of their structure . However, mutant MT alpha s containing changes of the invariant positions of the cysteine residues were expressed in a cadmium-binding form in Escherichia coli, although the invariant positions of 20 cysteine residues in the MTs are thought to be important for their metal-binding abilities . These results suggest that the position of cysteine residues and the chemical nature of the other amino acids in the amino acid sequence of MTs are less critical than expected.

Am J Physiol, 1997 May, 272(5 Pt 2), R1525 - 31
Ventromedial hypothalamic lesions impair glucoregulation in response to endotoxin; Lynch JP et al.; The purpose of the present study was to determine the role of the ventromedial hypothalamus (VMH) in regulating counter-regulatory hormone release and the increase in glucose flux that is observed after injection of endotoxin {lipopolysaccharide (LPS)} . Bilateral lesions of the VMH were produced electrolytically 2 wk before the experiment; sham-operated rats served as controls . {3-3H}glucose was infused to assess whole body glucose flux before and for 4 h after intravenous injection of Escherichia coli LPS . In control rats, LPS increased the plasma concentrations of glucose and lactate and the rates of glucose appearance and disappearance . In these animals, LPS also produced sustained elevations in corticosterone, glucagon, and catecholamines . In contrast, the glucose metabolic response to LPS was attenuated by > 50% in VMH-lesioned rats . These changes were associated with a blunted increase in the plasma concentration of glucagon, epinephrine, and norepinephrine in VMH-lesioned rats compared with control animals . There was no difference in the plasma concentrations of corticosterone or TNF-alpha between the two groups after LPS or the responsiveness of sham- and VMH-lesioned rats to an infusion of either glucagon or epinephrine . These data indicate that the VMH plays a central role in regulating the secretion of glucagon and catecholamines and the stimulation of glucose flux after LPS.

Am J Physiol, 1997 May, 272(5 Pt 1), G1230 - 5
Regulation of superoxide dismutase in primary cultures of rat colonic smooth muscle cells; Tannahill CL et al.; We have observed a rapid induction of manganese superoxide dismutase (MnSOD) in epithelial, neuronal, and smooth muscle cells (SMC) after acetic acid-induced colitis . To examine the regulation of MnSOD in the SMC more specifically, primary cultures of colonic SMC were developed by enzymatic digestion of the circular muscle layer from an adult rat . SMC were treated for 2-72 h with 0.5 microgram/ml Escherichia coli endotoxin {lipopolysaccharide (LPS)}, 10 ng/ml tumor necrosis factor (TNF)-alpha, or 2 ng/ml interleukin-1 beta (IL-1 beta) . Cotreatments were performed with IL-1 beta and 4 microM actinomycin or 50 microM cycloheximide . Northern analysis demonstrated 23-fold, 8-fold, and 6-fold inductions of MnSOD mRNA by IL-1 beta, LPS, and TNF-alpha, respectively . Induction of MnSOD by IL-1 beta was eliminated by actinomycin but not by cycloheximide, implicating a requirement for de novo transcription . Western analysis resulted in a 23.7-fold induction of MnSOD protein after 48-h treatment with IL-1 beta . Induction of MnSOD by IL-1 beta and other inflammatory mediators may serve as a protective mechanism to reduce oxygen free radical- and nitric oxide-mediated cell damage during inflammation.

Am J Physiol, 1997 May, 272(5 Pt 1), C1475 - 81
A carboxy-terminal peptide of the alpha 1-subunit of the dihydropyridine receptor inhibits Ca(2+)-release channels; Slavik KJ et al.; Excitation-contraction coupling in skeletal muscle is thought to involve a physical interaction between the alpha 1-subunit of the dihydropyridine receptor (DHPR) and the sarcoplasmic reticulum (SR) Ca(2+)-release channel (also known as the ryanodine receptor) . Considerable evidence has accumulated to suggest that the cytoplasmic loop between domains II and III of the DHPR alpha 1-subunit is at least partially responsible for this interaction . Other parts of this subunit or other subunits may, however, contribute to the functional and/or structural coupling between these two proteins . A synthetic peptide corresponding to a conserved sequence located between amino acids 1487 and 1506 in the carboxy terminus of the alpha 1-subunit inhibits both {3H}ryanodine binding to skeletal and cardiac SR membranes and the activity of skeletal SR Ca(2+)-release channels reconstituted into planar lipid bilayers . A second, multiantigenic peptide synthesized to correspond to the same sequence inhibits both binding and channel activity at lower concentrations than the linear peptide . These peptides slow the rate at which {3H}ryanodine binds to its high-affinity binding site and decrease the rate at which {3H}ryanodine dissociates from this site . A third polypeptide synthesized in Escherichia coli and corresponding to amino acids 1381-1627 and encompassing the above sequence has similar effects . This portion of the alpha 1-subunit of the transverse tubule DHPR is therefore a candidate for contributing to the interaction of this protein with the Ca(2+)-release channel.

Lett Appl Microbiol, 1997 May, 24(5), 319 - 28
Regulatory components, including integration host factor, CysB and H-NS, that influence pH responses in Escherichia coli; Rowbury RJ; This review describes a range of pH responses . Some are only induced if relevant DNA is brought to an appropriately supercoiled configuration by DNA gyrase and bent by the action of, for example, integration host factor (IHF) . Bending may allow transcription by bringing activators into juxtaposition with RNA polymerase, which is CysB-associated in several of the responses . Control of arginine decarboxylase (AdiA) synthesis at acid pH is of the above type, with dependence on the presence of gyrase, H-NS, IHF and CysB; acid induction of LysU has similar requirements but also needs Lrp; lysine decarboxylase (CadA) formation at acid pH is controlled quite differently, needing the CadC activator and interaction of lysine/lysine permease; H-NS probably reverses induction by CadC . The Hyd components of formic hydrogenlyase are induced by acid under anaerobiosis; a transcriptional activator is involved and Fur may also function in regulation . Acid tolerance induced at low pH in log-phase cells needs CysB and PhoE but not DNA gyrase; tolerance is reduced by NaCl but not affected by Fe3+, Fe2+, glucose/cAMP or by lrp, him, fur, hns or nhaA/B lesions . Alkali tolerance (habituation), induced at pH0 8.5-9.0, probably involves DNA supercoiling and bending; the induction process needs IHF, CysB, PhoE, NhaA, TonB and Fur and is glucose-repressed; tolerance may result from Na+ efflux catalysed by the NhaA antiporter, which is induced at pH0 9.0 . Alkali sensitivity induced at pH0 5.5 also requires gyrase, IHF and CysB, but H-NS, Lrp, NhaA and OmpC are also needed and induction is abolished by NaCl . Salt-induced acid sensitivity results from PhoE formation and is blocked by glucose (reversed by cAMP), FeCl3 and hns and relA lesions, the effect of relA being envZ-suppressed . Acid sensitivity induction (ASI) at pH0 9.0 needs H-NS, is inhibited by FeCl3 and amiloride, and is associated with alkyl hydroperoxide reductase synthesis . Leucine-induced acid sensitivity needs gyrase, CysB, H-NS, Fur, OmpA and RelA, is inhibited by Fe3+, Fe2+, tetracycline, glucose and nalidixic acid, but not by chloramphenicol; increased outer membrane proton passage may result from OmpA modification.

EMBO J, 1997 May 1, 16(9), 2535 - 44
Recombinational repair in yeast: functional interactions between Rad51 and Rad54 proteins; Clever B et al.; Rad51p is a eukaryotic homolog of RecA, the central homologous pairing and strand exchange protein in Escherichia coli . Rad54p belongs to the Swi2p/Snf2p family of DNA-stimulated ATPases . Both proteins are also important members of the RAD52 group which controls recombinational DNA damage repair of double-strand breaks and other DNA lesions in Saccharomyces cerevisiae . Here we demonstrate by genetic, molecular and biochemical criteria that Rad51 and Rad54 proteins interact . Strikingly, overexpression of Rad54p can functionally suppress the UV and methyl methanesulfonate sensitivity caused by a deletion of the RAD51 gene . However, no suppression was observed for the defects of rad51 cells in the repair of gamma-ray-induced DNA damage, mating type switching or spontaneous hetero-allelic recombination . This suppression is genetically dependent on the presence of two other members of the recombinational repair group, RAD55 and RAD57 . Our data provide compelling evidence that Rad51 and Rad54 proteins interact in vivo and that this interaction is functionally important for recombinational DNA damage repair . As both proteins are conserved throughout evolution from yeasts to humans, a similar protein-protein interaction may be expected in other organisms.

EMBO J, 1997 May 1, 16(9), 2188 - 96
The protease-protected 30 kDa domain of SecA is largely inaccessible to the membrane lipid phase; Eichler J et al.; SecA binds to the inner membrane of Escherichia coli through low affinity lipid interactions or with high affinity at SecYEG, the integral domain of preprotein translocase . Upon addition of preprotein and nucleotide, a 30 kDa domain of SecYEG-bound SecA is protected from proteolysis via membrane insertion . Such protection could result from some combination of insertion into the lipid phase, into a proteinaceous environment or across the membrane . To assess the exposure of SecYEG-bound SecA to membrane lipids, a radiolabeled, photoactivatable and lipid-partitioning crosslinker, 3-trifluoromethyl-3-(m{125I}iodophenyl) diazirine benzoic acid ester, was incorporated into inner membrane vesicles . The 30 kDa domain of SecYEG-bound SecA, inserted into the membrane in response to translocation ligands, is 18-fold less labeled than SecY, which is labeled effectively . In contrast, incorporation of the purified 30 kDa SecA fragment into crosslinker-containing detergent micelles or addition of detergent to crosslinker-containing membranes bearing the protease-protected SecA domain readily allows for labeling of this domain . We propose that the protease-inaccessible 30 kDa SecA domain is shielded from the fatty acyl membrane phase by membrane-spanning SecYEG helices and/or is largely exposed to the periplasm.

EMBO J, 1997 May 1, 16(9), 2171 - 8
Function of the Greek key connection analysed using circular permutants of superoxide dismutase; Boissinot M et al.; Human Cu,Zn superoxide dismutase (SOD) is a single domain all beta-sheet protein with its eight beta-strands arranged as a Greek key beta-barrel or immunoglobulin fold . Three circularly permuted variants of SOD were made by joining the native amino- and carboxy-termini, and introducing new termini at sites originally within connections between beta-strands . The locations of the new termini were chosen to interrupt beta-turns between the two N-terminal beta-hairpins and the short cross-barrel Greek key connection . Expression levels in the Escherichia coli periplasm were indistinguishable from that of native SOD . Reaction rates for the purified proteins were similar to those of the native enzyme, indicating that the permutants are correctly folded . Interrupting the covalent cross-bracing provided by the Greek key connection reduced the stability of the protein by approximately 1.0 kcal/mol, indicating only a slight contribution to conformational stability . The experiments test and eliminate two hypotheses for folding pathways for Greek key beta-barrels that require N-terminal beta-hairpins or covalent attachment across the short Greek key connection.

Biochem J, 1997 May 1, 323 ( Pt 3), 741 - 7
Phosphorylation of tau by glycogen synthase kinase 3beta affects the ability of tau to promote microtubule self-assembly; Utton MA et al.; To study the effects of phosphorylation by glycogen synthase kinase-3beta (GSK-3beta) on the ability of the microtubule-associated protein tau to promote microtubule self-assembly, tau isoform 1 (foetal tau) and three mutant forms of this tau isoform were investigated . The three mutant forms of tau had the following serine residues, known to be phosphorylated by GSK-3, replaced with alanine residues so as to preclude their phosphorylation: (1) Ser-199 and Ser-202 (Ser-199/202-->Ala), (2) Ser-235 (Ser-235-->Ala) and (3) Ser-396 and Ser-404 (Ser-396/404-->Ala) . Wild-type tau and the mutant forms of tau were phosphorylated with GSK-3beta, and their ability to promote microtubule self-assembly was compared with the corresponding non-phosphorylated tau species . In the non-phosphorylated form, wild-type tau and all of the mutants affected the mean microtubule length and number concentrations of assembled microtubules in a manner consistant with enhanced microtubule nucleation . Phosphorylation of these tau species with GSK-3beta consistently reduced the ability of a given tau species to promote microtubule self-assembly, although the affinity of the tau for the microtubules was not greatly affected by phosphorylation since the tau species remained largely associated with the microtubules . This suggests that the regulation of microtubule assembly can be controlled by phosphorylation of tau at sites accessible to GSK-3beta by a mechanism that does not necessarily involve the dissociation of tau from the microtubules.

Biochem J, 1997 May 1, 323 ( Pt 3), 661 - 9
Escherichia coli L-aspartate-alpha-decarboxylase: preprotein processing and observation of reaction intermediates by electrospray mass spectrometry; Ramjee MK et al.; The Escherichia coli panD gene, encoding l-aspartate-alpha-decarboxylase, was cloned by PCR, and shown to complement a panD mutant defective in beta-alanine biosynthesis . Aspartate decarboxylase is a pyruvoyl-dependent enzyme, and is synthesized initially as an inactive proenzyme (the pi-protein), which is proteolytically cleaved at a specific X-Ser bond to produce a beta-subunit with XOH at its C-terminus and an alpha-subunit with a pyruvoyl group at its N-terminus, derived from the serine . The recombinant enzyme, as purified, is a tetramer, and comprises principally the unprocessed pi-subunit (of 13.8 kDa), with a small proportion of the alpha- and beta-subunits (11 kDa and 2.8 kDa respectively) . Incubation of the purified enzyme at elevated temperatures for several hours results in further processing . Using fluorescein thiosemicarbazide, the completely processed enzyme was shown to contain three pyruvoyl groups per tetrameric enzyme . The presence of unchanged serine at the N-terminus of some of the alpha-subunits was confirmed by electrospray mass spectrometry (ESMS) and N-terminal amino acid sequencing . A novel HPLC assay for aspartate decarboxylase was established and used to determine the Km and kcat for l-aspartate as 151+/-16 microM and 0.57 s-1 respectively . ESMS was also used to observe substrate and product adducts trapped on the pyruvoyl group by sodium cyanoborohydride treatment.

Microbiology, 1997 May, 143 ( Pt 5), 1701 - 8
Highly thermostable endo-1,3-beta-glucanase (laminarinase) LamA from Thermotoga neapolitana: nucleotide sequence of the gene and characterization of the recombinant gene product; Zverlov VV et al.; The nucleotide sequence of clone pTT26 (3786 bp), containing the gene for 1,3-beta-glucanase LamA (laminarinase) from Thermotoga neapolitana, was determined . It contains an ORF encoding a protein of 646 aa (73328 Da) . The central part of the protein is homologous to the complete catalytic domain of bacterial and some eukaryotic endo-1,3-beta-D-glucanases and belongs to family 16 of glycosyl hydrolases . This domain is flanked on both sides by one copy on each side of a substrate binding domain homologue (family II) . The recombinant laminarinase protein was purified from Escherichia coli host cells in two forms, a 73 kDa and a processed 52 kDa protein, both having high specific activity towards laminarin (3100 and 2600 U mg-1, respectively) and K(m) values of 2.8 and 2.2 mg ml-1, respectively . Limited activity on 1,3-1,4-beta-glucan (lichenan) was detected (90 U mg-1) . Laminarin was degraded in an endoglucanase modus, yielding glucose, laminaribiose and -triose as end products . Thus LamA classifies as an endo-1,3(4)-beta-glucanase (EC 3.2.1.6) . The optimum temperature of the enzymes was 95 degrees C (73 kDa) and 85 degrees C (52 kDa) at an optimum pH of 6.2 . The superior thermostability of the 73 kDa enzyme is demonstrated by incubation without substrate at 100 degrees C, where 57% of the initial activity remained after 30 min (82% at 95 degrees C) . Thus, LamA is the most thermostable 1,3-beta-glucanase described to date.

Microbiology, 1997 May, 143 ( Pt 5), 1567 - 74
Use of a glycerol-limited, long-term chemostat for isolation of Escherichia coli mutants with improved physiological properties; Weikert C et al.; The evolution of Escherichia coli MG1655 mutants was followed over 126 d in a glycerol-limited chemostat at a dilution rate of 0.05 h-1 . This corresponds to a total of 217 generations at a doubling time of 13.9 h . After this time, nearly 90% of the chemostat population consisted of evolved mutant strains as determined by their altered colony morphologies on plates . Two mutants were isolated that exhibited generally improved growth phenotypes in batch cultivations on glycerol, glucose or the gluconeogenic substrate acetate . Higher specific growth rates and increased biomass yields were found for both mutants . For one mutant, this behaviour was combined with significantly reduced secretion of overflow metabolites when either glycerol or glucose was the carbon source . Additionally, during all growth phases of a batch cultivation, this mutant exhibited increased resistance to a variety of adverse conditions including heat shock, osmotic stress and nutrient deprivation . It also displayed significantly shorter lag phases.

Microbiology, 1997 May, 143 ( Pt 5), 1549 - 55
Brucella abortus strain 2308 putative glucose and galactose transporter gene: cloning and characterization; Essenberg RC et al.; The gene for the putative transporter for glucose and galactose from Brucella abortus strain 2308 was isolated by functional complementation of Escherichia coli strains lacking either glucose or galactose transport systems . The same two plasmid clones were isolated from each screen . These clones restored glucose and galactose transport to the respective E . coli strains . The sequence of the 1806 bp overlap between these two plasmids was determined . A 1242 bp ORF whose disruption eliminated complementation of both E . coli strains showed 36% identity with the E . coli fucP gene encoding a fucose transporter . These two transporters are members of the major facilitator superfamily, in which they represent a previously undescribed family . In addition, an incomplete gene similar to E . coli hisG was found . One of the plasmids complemented E . coli hisG mutants.

Microbiology, 1997 May, 143 ( Pt 5), 1521 - 32
FNR-dependent repression of ndh gene expression requires two upstream FNR-binding sites; Meng W et al.; The ndh gene of Escherichia coli encodes a non-proton-translocating NADH dehydrogenase (NdhII) that is anaerobically repressed by the global transcription regulator, FNR . FNR binds at two sites (centred at -50.5 and -94.5) in the ndh promoter but the mechanism of FNR-mediated repression appears not to be due to promoter occlusion . This mechanism has been investigated using an aerobically active derivative of FNR, FNR* (FNR-D154A), with ndh promoters containing altered FNR-binding sites . FNR* repressed ndh gene expression both aerobically and anaerobically in vivo . Gel retardation analysis and DNase I footprinting with purified FNR* protein confirmed that FNR interacts at two sites in the ndh promoter, and that FNR and RNA polymerase (RNAP) can bind simultaneously . Studies with three altered ndh promoters, each containing an impaired or improved FNR-site, indicated that both FNR-sites are needed for efficient repression in vivo . The alpha-subunit of RNAP interacted with two regions (centred at -105 and -46), each overlapping one of the FNR-sites in the ndh promoter . Footprints of the FNR*-RNAP-ndh ternary complex indicated that FNR*-binding at -50.5 prevents the alpha-subunit of RNAP from docking with the DNA just upstream of the -35 element . Binding of a second FNR* molecule at the -105 site likewise prevents binding of the alpha-subunit at its alternative site, thus providing a plausible mechanism for FNR-mediated repression based on displacement of the alpha-subunit of RNAP.

Microbiology, 1997 May, 143 ( Pt 5), 1503 - 12
Isolation and characterization of a strong promoter element from the Streptomyces ghanaensis phage I19 using the gentamicin resistance gene (aacC1) of Tn 1696 as reporter; Labes G et al.; A promoter-probe shuttle plasmid (pGL7011) containing the promoterless aminoglycoside-O-acetyltransferase I gene (aacC1) of Tn1696 was used to isolate DNA fragments from Streptomyces ghanaensis phage I19 that possessed promoter activity in Streptomyces lividans TK23 . Analysis of gentamicin (Gm) resistance levels in Escherichia coli and in S . lividans TK23, and of aacC1 mRNA levels in S . lividans, identified a fragment (F14) that exhibited a high level of promoter activity in both species . Subsequent analysis revealed that the promoter activity of SF14 (a subcloned fragment of F14) was about twice that of ermEp*, one of the strongest characterized actinomycete promoters . SF14 contained two tandemly arranged promoters, 14-Ip and p14-IIp, with overlapping and adjacent -10 and -35 regions, respectively . Both promoters appear to be recognized with different efficiencies by the major RNA polymerase holoenzyme (E sigma hrdB) of Streptomyces coelicolor A3(2).

Chem Res Toxicol, 1997 May, 10(5), 568 - 74
Frameshift mutagenesis induced in Escherichia coli after in vitro treatment of double-stranded DNA with methylene blue plus white light: evidence for the involvement of lesion(s) other than 8-oxo-7,8-dihydro-2'-deoxyguanosine; Wagner J et al.; By means of specific mutation assays, we show here that in vitro treatment of double-stranded plasmid DNA with methylene blue and white light efficiently promotes frameshift mutagenesis in Escherichia coli . The assays detect either -1 or -2 frameshift mutations within previously characterized hot spot sequences for frameshift mutagenesis induced by the chemical carcinogen N-2-acetylaminofluorene, namely, short runs of contiguous guanines and alternating GpC sequences, respectively . The SOS and umuDC dependences of these mutagenic processes have been investigated . Both -1 and -2 frameshift mutagenesis are increased when the host SOS functions are induced . However, and although functional UmuDC proteins are required for maximal mutation induction, the inducibility of both -1 and -2 frameshift mutagenesis is partially independent upon the integrity of the umuDC operon . In addition, results obtained using plasmids with a site specifically located 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo) residue show that this lesion, the major methylene blue plus light induced lesion characterized so far, is inefficient in promoting frameshift mutagenesis . Together, these results led us to conclude that methylene blue plus light treatment of DNA induces, at relatively high rates, lesion(s) other than 8-oxo-dGuo, that efficiently promote(s) frameshift mutagenesis in E . coli.

Shock, 1997 May, 7(5), 383 - 8
Cytosolic Ca2+ concentration and contraction-relaxation properties of ventricular myocytes from Escherichia coli endotoxemic guinea pigs: effect of fluid resuscitation; Zhong J et al.; Hearts isolated from a guinea pig model of Escherichia coli endotoxemia exhibit decreased systolic contractile function and reduced diastolic compliance of the left ventricle within 4 h after injection of endotoxin . Fluid resuscitation prevented the endotoxin-induced decrease in diastolic compliance without affecting systolic contractile depression . Because intrinsic myocardial dysfunction after endotoxemia may result from alterations in intracellular Ca2+ handling, we tested the hypothesis that in vivo fluid resuscitation improved diastolic function by altering Ca2+ handling of the myocardium . We tested this hypothesis by measuring cell shortening and intracellular Ca2+ of ventricular myocytes isolated from endotoxemic guinea pigs . E . coli endotoxin (LPS, 1 mg/kg)-injected guinea pigs were divided into resuscitated and nonresuscitated groups . Fluid resuscitated animals received a Ringer's infusion (8 mL.kg-1.h-1) intravenously (i.v.) beginning immediately after endotoxin injection . Four hours later, ventricular myocytes were isolated enzymatically and loaded with fura-2/AM . When myocytes were field stimulated at .8 Hz, peak systolic Ca2+ transients of LPS-resuscitated (619 +/- 75 nM) and LPS-nonresuscitated (599 +/- 60 nM) myocytes were not significantly different from each other, but both were significantly less than values from control myocytes (1187 +/- 118 nM, p < .05) . The percentage of cell shortening of LPS-resuscitated (6.2 +/- .9%) and LPS-nonresuscitated (6.2 +/- .3%) myocytes were also less than control (11.8 +/- .5%, p < .05) . In contrast to improved diastolic compliance of fluid-resuscitated hearts, diastolic {Ca2+}i of myocytes (at .8 Hz) from LPS-resuscitated animals (138 +/- 47 nM) was not statistically different from LPS-nonresuscitated animals (129 +/- 19 nM) . Diastolic values of both LPS groups were consistently lower than control value (251 +/- 38 nM, p < .05) . These data suggest that improved diastolic compliance of LPS hearts following fluid resuscitation is not associated with improved myocyte contractility or myoplasmic Ca2+ handling.

Shock, 1997 May, 7(5), 332 - 8
Induction of muscle glutamine synthetase gene expression during endotoxemia is adrenal gland dependent; Lukaszewicz GC et al.; Skeletal muscle plays a crucial role in maintaining nitrogen homeostasis during health and critical illness by exporting glutamine, the most abundant amino acid in the blood . We hypothesized that induction of glutamine synthetase (GS) expression, the principal enzyme of de novo glutamine biosynthesis, in skeletal muscle after endotoxin administration was adrenal gland dependent . We studied the expression of GS in normal and adrenalectomized rats after intraperitoneal administration of Escherichia coli lipopolysaccharide (LPS) . Treatment of normal rats with LPS resulted in a marked increase in GS mRNA that was dose and time dependent, and preceded the increase in GS protein and specific activity . The increase in muscle GS mRNA observed in normal rats in response to LPS was abrogated in adrenalectomized rats at 3 h after high dose LPS treatment and markedly attenuated at 5.5 h after low dose LPS treatment . These and other studies implicate glucocorticoid hormones as a key, but not exclusive, regulator of skeletal muscle GS expression after a catabolic insult.

Mutat Res, 1997 May 1, 383(3), 231 - 42
Photoreversal of UV-potentiated glutamine tRNA suppressor mutations in excision proficient Escherichia coli; Bockrath R et al.; UV-irradiated excision proficient Escherichia coli were exposed to light for photoenzymatic reversal (PR) of cyclobutane pyrimidine dimers (CPD) and assayed for reversion mutation (glutamine tRNA suppressor mutations) on semi-enriched medium or on the same medium containing acriflavine to inhibit excision repair . The initial mutation frequency without PR was relatively greater when assayed with acriflavine, and this difference increased as larger UV fluences were used . The PR kinetics were first order and about the same or slightly faster when cells were assayed with acriflavine (after 15, 30 or 45 J/m2, respectively) . The results indicated mutation targeting by CPD in excision proficient cells . These results and conclusion contrast sharply with the original study of this type done several years ago . PR kinetics were considerably slower with assays containing acriflavine, sustaining the idea that PR causes repair of non-dimer targeting lesions by enhancing excision repair . To explain this contrast we devised a fluence-decrement rate for estimating the effectiveness of PR and measured PR-dependent excision repair (PER) as the difference in the fluence-decrement rate with excision proficient and deficient cells . PER was more evident when cells were prepared as in the original study but was still an insufficient factor . More importantly, the original study included a component of indirect photoreactivation or photoprotection (using unfiltered PR light) which accentuated the role of excision repair . Taking these factors into account, the original data also are consistent with the model that glutamine tRNA suppressor mutations produced by UV-mutagenesis in excision proficient E . coli result from targeting by CPD just as in excision defective cells . Thus, with regards to a common UV mutation assay, there does not appear to be two types of targeting lesion depending on excision proficiency.

Mutat Res, 1997 May 1, 383(3), 223 - 30
An Escherichia coli topB mutant increases deletion and frameshift mutations in the supF target gene; Uematsu N et al.; We have improved a system to examine forward mutations that occurred in the supF gene of Escherichia coli placed on a multicopy plasmid . Using this system, we determined the mutational specificity for a topB deletion mutator in which topoisomerase III is hampered . The frequency of supF- mutations in topB strain was 4.9 x 10(-7), that is essentially the same as that in wild-type strain, 3.1 x 10(-7) . Half the number of the supF- mutations were large deletions, where a specific deletion among a 10-base pair direct repeat dominated, but other types of deletions were also found . Most of the deletions were associated with the presence of directly repeated sequences capable of accounting for their endpoints . Frameshift mutations in topB strain also significantly increased compared with those of wild-type (17 vs . 2%) . Base substitutions comprised 27% of the events, specificity of which in topB strain was the same as that in wild-type strain . The present data suggest that topB is a novel class of mutator that strongly induces repeated sequence dependent deletion mutagenesis and high frequencies of frameshift mutagenesis.

FEMS Microbiol Lett, 1997 May 1, 150(1), 165 - 71
Yeast linear plasmids with T2AG3 telomeres: TEL+CEN antagonism and genetic and molecular stability; Guerrini AM et al.; A linear plasmid containing ARS1, CEN4, and 48 bp of vertebrate (T2AG3) telomeric sequences at each end was used to transform Saccharomyces cerevisiae . Only circular plasmids that had lost the centromere and had retained the T2AG3 sequences were obtained, indicating that the vertebrate T2AG3 sequences and the yeast CEN4 could not be simultaneously present in this vector . This hypothesis was verified by removing the CEN4 sequence from the construct . In fact, the resulting transformants contained two classes of efficiently replicating linear plasmids: one of the expected size and one about twice as large . During subsequent growth, plasmids of the former, but not latter, class were subjected to concatemer formation . This can best be explained by recombination events involving the T2AG3 sequences at the ends of the molecule, since very similar centric and acentric linear plasmids bearing Tetrahymena telomeric ends replicated faithfully.

FEMS Microbiol Lett, 1997 May 1, 150(1), 157 - 64
Cloning and sequencing of a species-specific nucleotide fragment of Borrelia burgdorferi sensu stricto, which is repeated in several plasmids of the species; Misonne MC et al.; Among the etiological agents of Lyme disease, Borrelia burgdorferi sensu stricto strains carry a 16 kb plasmid, which did not hybridize to plasmids of B . garinii and B . afzelii strains . A 1271 bp DNA fragment of the 16 kb plasmid was cloned . It hybridized to several plasmids of this species (16, 27 and 55 kb) . Sequencing of the cloned insert revealed a 327 bp ORF coding for a 14 kDa protein of unknown function, which could be expressed in E . coli . This ORF, conserved among B . burgdorferi sensu stricto strains, was carried by the same three plasmids.

FEMS Microbiol Lett, 1997 May 1, 150(1), 33 - 41
Molecular cloning and expression studies of two divergent alpha-tubulin genes in Neurospora crassa; Monnat J et al.; Three alpha-tubulin isoforms were previously detected in Neurospora crassa . We have cloned and analysed two alpha-tubulin cDNAs, Tub alpha A and Tub alpha B that encode polypeptides of 453 and 451 amino acids, respectively . The encoded amino acids exhibit an unusual divergence of 35% . This is the highest divergence ever observed between alpha-tubulins from the same species . The expression of the two genes is developmentally regulated . We did not detect any transcription of the Tub alpha A gene in dormant macroconidia and during the first 30 min of development even though the alpha-tub A protein is already present in the early stage of germination . In contrast, the Tub alpha B gene is continuously transcribed during the vegetative cycle and the expression profile of the protein follows the ones of its mRNA.

Carcinogenesis, 1997 May, 18(5), 1045 - 8
Relative mutagenicities of gaseous nitrogen oxides in the supF gene of pSP189; Kelman DJ et al.; Gaseous nitric oxide (NO), an environmental pollutant found in cigarette smoke and diesel exhaust, has been shown to generate mutations in aerobic in vitro assays . The objective of this study was to identify which oxides of nitrogen, formed in the gaseous phase from NO, possess mutagenic activity . Samples of the plasmid pSP189, in 1 M HEPES buffer, pH 7.4, were exposed to preparations of nitrogen dioxide (NO2), dinitrogen trioxide (N2O3) or an air control . The gas mixtures were formed in a gas-tight syringe and were then introduced into 1 l flasks . The plasmid solution was introduced immediately afterwards . Transformation of Escherichia coli strain MBM7070 with the treated plasmids allowed analysis of mutation frequencies and the types of mutations induced in the target supF gene . The mutation frequency resulting from NO2 exposure was not different from that of the control . However, N2O3 produced a substantial number of mutations . The mutation frequency and the types of mutations were found to depend on the length of time that the gases were allowed to incubate in the syringe prior to introduction into the 1 l flasks (mutation frequency was maximal at approximately 2 min) . The most prevalent mutations were AT-->GC transitions (68%), followed by GC-->AT transitions (30%), similar to previous findings when pure NO was bubbled through pSP189 solutions . These results suggest that it is N2O3, rather than NO2, that is the most likely source of mutagenic potential from gaseous nitrogen oxides.

Carcinogenesis, 1997 May, 18(5), 1035 - 8
Fpg protein releases a ring-opened N-7 guanine adduct from DNA that has been modified by sulfur mustard; Li Q et al.; Transfection of the Escherichia coli fpg gene into Chinese hamster ovary cells has been reported to enhance survival after exposure to aziridine (C.Cussac and F.Laval, 1996, Nucleic Acids Res., 24, 1742-1746) . This result suggests that Fpg protein protects cells from toxicity by removing ring-opened N-7 guanine adducts from DNA, and raises the possibility that Fpg protein would offer protection from other agents that alkylate the N-7 position of guanine . Since the major adduct formed by sulfur mustard in DNA is 7-hydroxyethyl-thioethylguanine (HETEG), we have investigated the action of Fpg protein on the ring-opened form of this adduct (ro-HETEG) . A substrate containing ro-HETEG was prepared by alkaline treatment of DNA modified by {14C}sulfur mustard . Fpg protein purified from an over-producing strain of E . coli released ro-HETEG from this substrate in an enzyme- and time-dependent manner, and at a rate that is similar to that at which it releases ring-opened 7-methylguanine . Thus, Fpg protein acts efficiently on ro-HETEG, and may offer some protection against the toxic action of sulfur mustard.

Biochem Mol Biol Int, 1997 May, 41(6), 1191 - 9
Denaturation of uridine phosphorylase from Escherichia coli K-12 by guanidine hydrochloride; Kurganov BI et al.; Denaturation of uridine phosphorylase from Escherichia coli K-12 by guanidine hydrochloride results in red shift of the maximum in the protein fluorescence spectrum, dissociation of the hexameric enzyme molecule into monomers, and the loss of the enzymatic activity . The initial rate of the enzyme reactivation after the dilution of the enzyme preincubated with guanidine hydrochloride has the second order with respect to protein . It is assumed that the rate of the reactivation process is limited by the reassociation of monomers possessing low enzymatic activity to dimers followed by the rapid step of hexamer formation.

Biol Reprod, 1997 May, 56(5), 1336 - 42
Molecular cloning of sheep and goat ferredoxin reductase messenger ribonucleic acids, and identification of an alternatively spliced form of sheep ferredoxin reductase; Furukawa A et al.; Complementary DNA clones of mRNAs for sheep and goat NADPH-ferredoxin reductases (ferredoxin NADP+ oxidoreductase, EC 1.18.1.2) were isolated by the reverse transcriptase polymerase chain reaction method, and the complete nucleotide sequences of the coding and 3'-flanking regions of these cDNA clones were determined . Comparative analysis using the deduced amino acid sequences of NADPH-ferredoxin reductases clarified the interspecific conservation of the ferredoxin-binding and flavin adenine dinucleotide (FAD)-binding regions, confirming the results reported previously . During this study, we happened to identify an alternatively spliced mRNA that completely lacks exon 3, just adjacent to the FAD-binding region of the sheep NADPH-ferredoxin reductase cDNA clone . In the screening of other alternatively spliced mRNAs of NADPH-ferredoxin reductases derived from several steroidogenic organs, such as adrenocortices, testes, and ovaries of sheep and goats, only one kind of alternatively spliced mRNA as described above was detected in sheep adrenocortices . Then, we constructed Escherichia coli expression systems for these two forms of mRNA and analyzed their enzymatic properties . We found that the ability of the alternatively spliced NADPH-ferredoxin reductase protein to transfer electrons to ferredoxin is completely abolished because FAD binding is inhibited.

Plant Physiol, 1997 May, 114(1), 265 - 73
Cloning and overexpression of two cDNAs encoding the low-CO2-inducible chloroplast envelope protein LIP-36 from Chlamydomonas reinhardtii; Chen ZY et al.; Chlamydomonas reinhardtii, a unicellular green alga, grows photoautotrophically at very low concentrations of inorganic carbon due to the presence of an inducible CO2-concentrating mechanism . During the induction of the CO2-concentrating mechanism at low-CO2 growth conditions, at least five polypeptides that are either absent or present in low amounts in cells grown on high-CO2 concentrations are induced . One of these induced polypeptides with a molecular mass of 36 kD, LIP-36, has been localized to the chloroplast envelope . The protein was purified and the partial internal amino acid sequences were obtained through lys-C digestion . Two cDNAs encoding LIP-36 have been cloned using degenerate primers based on the amino acid sequences . The two genes encoding LIP-36 are highly homologous in the coding region but are completely different in the 5'-end and 3'-end untranslated regions . The deduced protein sequences show strong homology to the mitochondrial carrier protein superfamily, suggesting that LIP-36 is a chloroplast carrier protein . The regulation of the expression of these two genes at high- and low-CO2 growth conditions is also different . Both genes were highly expressed under low-CO2 growth conditions, with the steady-state level of LIP-36 G1 mRNA more abundant . However, neither gene was expressed at high-CO2 growth conditions . The gene products of both clones expressed in Escherichia coli were recognized by an antibody raised against LIP-36, confirming that the two cDNAs indeed encode the C . reinhardtii chloroplast envelope carrier protein LIP-36.

Plant Physiol, 1997 May, 114(1), 29 - 37
Abundant accumulation of the calcium-binding molecular chaperone calreticulin in specific floral tissues of Arabidopsis thaliana; Nelson DE et al.; Calreticulin (CRT) is a calcium-binding protein in the endoplasmic reticulum (ER) with an established role as a molecular chaper-one . An additional function in signal transduction, specifically in calcium distribution, is suggested but not proven . We have analyzed the expression pattern of Arabidopsis thaliana CRTs for a comparison with these proposed roles . Three CRT genes were expressed, with identities of the encoded proteins ranging from 54 to 86% . Protein motifs with established functions found in CRTs of other species were conserved . CRT was found in all of the cells in low amounts, whereas three distinct floral tissues showed abundant expression: secreting nectaries, ovules early in development, and a set of subepidermal cells near the abaxial surface of the anther . Localization in the developing endosperm, which is characterized by high protein synthesis rates, can be reconciled with a specific chaperone function . Equally, nectar production and secretion, a developmental stage marked by abundant ER, may require abundant CRT to accommodate the traffic of secretory proteins through the ER . Localization of CRT in the anthers, which are degenerating at the time of maximum expression of CRT, cannot easily be reconciled with a chaperone function but may indicate a role for CRT in anther maturation or dehiscence.

Mol Biol Evol, 1997 May, 14(5), 578 - 88
Evolution of the large-subunit ribosomal RNA binding site for protein L23/25; Chenuil A et al.; The region of the large-subunit rRNA encompassing the D7 divergent domain is organized within eukaryotes in a patchwork of short conservative secondary-structure features interspersed with more rapidly evolving sequences . It contains the attachment site of protein L25 (E . coli L23), which binds rRNA in the first stages of ribosome assembly, suggesting a crucial importance of this region in ribosome elaboration and functioning . A better understanding of its roles requires a good knowledge of its mode of structural variation during the course of evolution . With this aim, we sequenced the D7 region for 24 new invertebrate species belonging to annelids, molluscs, arthropods, and eight other deep-branching invertebrate phyla . Their comparison allowed us to propose refinements in previous eukaryotic folding models . A detailed analysis of the pattern of variation at each position both within the D7 region and along the L23/25 sequence by reference to previous heterologous binding experiments gives new insight into the rRNA-protein contacts . We identified in the D7 region and L23/25, respectively, six and five positions presenting a pattern of variation compatible with experimental results, three of which show coincident variations which support their possible involvement in the rRNA-L23/25 binding.

Genes Dev, 1997 May 1, 11(9), 1183 - 93
The sigma(E) and the Cpx signal transduction systems control the synthesis of periplasmic protein-folding enzymes in Escherichia coli; Danese PN et al.; In Escherichia coli, the heat shock-inducible sigma-factor sigma(E) and the Cpx two-component signal transduction system are both attuned to extracytoplasmic stimuli . For example, sigma(E) activity rises in response to the overproduction of various outer-membrane proteins . Similarly, the activity of the Cpx signal transduction pathway, which consists of an inner-membrane sensor (CpxA) and a cognate response regulator (CpxR), is stimulated by overproduction of the outer-membrane lipoprotein, NlpE . In response to these extracytoplasmic stimuli, sigma(E) and CpxA/CpxR stimulate the transcription of degP, which encodes a periplasmic protease . This suggests that CpxA/CpxR and sigma(E) both mediate protein turnover within the bacterial envelope . Here, we show that CpxA/CpxR and sigma(E) also control the synthesis of periplasmic enzymes that can facilitate protein-folding reactions . Specifically, sigma(E) controls transcription of fkpA, which specifies a periplasmic peptidyl-prolyl cis/trans isomerase . Similarly, the Cpx system controls transcription of the dsbA locus, which encodes a periplasmic enzyme required for efficient disulfide bond formation in several extracytoplasmic proteins . Taken together, these results indicate that sigma(E) and CpxA/CpxR are involved in regulating both protein-turnover and protein-folding activities within the bacterial envelope.

Genes Dev, 1997 May 1, 11(9), 1169 - 82
Regulation of Escherichia coli cell envelope proteins involved in protein folding and degradation by the Cpx two-component system; Pogliano J et al.; We show that the two-component signal transduction system of Escherichia coli, CpxA-CpxR, controls the expression of genes encoding cell envelope proteins involved in protein folding and degradation . These findings are based on three lines of evidence . First, activation of the Cpx pathway induces 5- to 10-fold the synthesis of DsbA, required for disulfide bond formation, and DegP, a major periplasmic protease . Second, using electrophoretic mobility shift and DNase I protection assays, we have shown that phosphorylated CpxR binds to elements upstream of the transcription start sites of dsbA, degP, and ppiA (rotA), the latter coding for a peptidyl-prolyl cis/trans isomerase . Third, we have demonstrated increased in vivo transcription of all three genes, dsbA, degP, and ppiA, when the Cpx pathway is activated . We have identified a putative CpxR consensus binding site that is found upstream of a number of other E . coli genes . These findings suggest a potentially extensive Cpx regulon including genes transcribed by sigma70 and sigma(E), which encode factors involved in protein folding as well as other cellular functions.

Genes Dev, 1997 May 1, 11(9), 1111 - 21
Yeast Rad55 and Rad57 proteins form a heterodimer that functions with replication protein A to promote DNA strand exchange by Rad51 recombinase; Sung P; Saccharomyces cerevisiae RAD51, RAD55, and RAD57 genes, required for genetic recombination and DNA double-strand-break repair, encode proteins homologous to one another and to the Escherichia coli RecA protein . Rad51 protein catalyzes the DNA strand-exchange reaction with a dependence on ATP and on the heterotrimeric single-strand DNA (ssDNA) binding factor replication protein A (RPA) . By several independent criteria, RAD55- and RAD57-encoded products are shown here to exist as a stable heterodimer, with a dissociation constant of <2 x 10(-10) M . In strand exchange, the reaction proceeds efficiently if RPA is incorporated after nucleation of Rad51 onto ssDNA, but if RPA is present during the nucleation phase, as is likely the case in vivo, the amount of strand-exchange products becomes relatively insignificant . Inclusion of the Rad55-Rad57 heterodimer with Rad51 and RPA results in a marked stimulation of strand exchange, providing evidence for a role of the Rad55-Rad57 heterodimer in overcoming the inhibitory effect of RPA.

J Anim Sci, 1997 May, 75(5), 1324 - 31
Pancreatic exocrine secretion during the first days after weaning in pigs; Rantzer D et al.; Feed replacement at weaning plays an important role in the induction of pancreatic maturation . To understand the changes in the exocrine pancreas at weaning and the relation to postweaning problems, we studied the function of the exocrine pancreas and changes of intestinal hemolytic Escherichia coli in four pigs . The pigs were chronically fitted with pancreatic duct catheters and T-shaped cannula inserted into the duodenum for reintroduction of pancreatic juice . One day before weaning (at 30 d of age), pancreatic juice was collected for 1 h before and 1 h after a morning and an evening suckling . The pigs were not creep fed, but from weaning the pigs received a standard weaning diet ad libitum . On d 1, 2, 3, and 5 after weaning, pancreatic juice was collected continuously for the 24-h period . The total pancreatic secretion was measured at hourly intervals, 1.5-mL samples were taken for analysis, and the remaining juice was returned to the animal . On these days, samples from the duodenum, ileum, and rectum were also taken for analyses of hemolytic E . coli . From the day before to 5 d after weaning, a gradual increase in pancreatic secretion was observed concerning volume (P < .001) and protein (P < .01) and trypsin (P < .02) levels . An increase (P < .01) in hemolytic E . coli in the duodenal contents was also documented during this period . We assume that the gradual increase in the measured variables of pancreatic secretion is related to the increasing consumption of solid feed . However, the appearance of E . coli and disappearance of milk components from the gastrointestinal tract could be other factors stimulating the exocrine pancreas.

Br Poult Sci, 1997 May, 38(2), 195 - 8
Spread of an enteric 'marker' organism during evisceration of New York dressed poultry in a simulated kitchen environment; Mead GC et al.; 1 . Five volunteers, with experience of eviscerating poultry or game birds in the home, each eviscerated three New York dressed chicken carcases that had been artificially inoculated in the colon with a readily identifiable "marker' strain of Escherichia coli . 2 . In all cases, evisceration resulted in breakage of the intestines at one or more sites, often with leakage of gut contents, or extrusion of faeces from the cloaca because of pressure on the colon during the manipulations involved . 3 . The marker organism was detected in the vent region and abdominal cavity of each carcase and sometimes on the breast and back, which appeared to reflect the degree of handling during evisceration, because the hands of each individual became heavily contaminated . 4 . With some individuals, the marker also spread beyond the immediate preparation area and was detected on exposure plates . 5 . Results support the view that evisceration of poultry in a domestic environment could lead to cross-contamination of other foods with any food-borne human pathogens present.

Hum Mol Genet, 1997 May, 6(5), 695 - 707
The molecular basis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency in compound heterozygous patients: is there correlation between genotype and phenotype?
Andresen BS, Bross P, Udvari S, Kirk J, Gray G, Kmoch S, Chamoles N, Knudsen I, Winter V, Wilcken B, Yokota I, Hart K, Packman S, Harpey JP, Saudubray JM, Hale DE, Bolund L, Kolvraa S, Gregersen N.
Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most commonly recognized defect of mitochondrial beta-oxidation . It is potentially fatal, but shows a wide clinical spectrum . The aim of the present study was to investigate whether any correlation exists between MCAD genotype and disease phenotype . We determined the prevalence of the 14 known and seven previously unknown non-G985 mutations in 52 families with MCAD deficiency not caused by homozygosity for the prevalent G985 mutation . This showed that none of the non-G985 mutations are prevalent, and led to the identification of both disease-causing mutations in 14 families in whom both mutations had not previously been reported . We then evaluated the severity of the mutations identified in these 14 families . Using expression of mutant MCAD in Escherichia coli with or without co-overexpression of the molecular chaperonins GroESL we showed that five of the missense mutations affect the folding and/or stability of the protein, and that the residual enzyme activity of some of them could be modulated to a different extent depending on the amounts of available chaperonins . Thus, some of the missense mutations may result in relatively high levels of residual enzyme activity, whereas the mutations leading to premature stop codons will result in no residual enzyme activity . By correlating the observed types of mutations identified to the clinical/biochemical data in the 14 patients in whom we identified both disease-causing mutations, we show that a genotype/phenotype correlation in MCAD deficiency is not straightforward . Different mutations may contribute with different susceptibilities for disease precipitation, when the patient is subjected to metabolic stress, but other genetic and environmental factors may play an equally important role.

Clin Exp Immunol, 1997 May, 108(2), 181 - 90
The systemic lupus erythematosus (SLE) disease autoantigen-calreticulin can inhibit C1q association with immune complexes; Kishore U et al.; Following its release from cells during infection and inflammation, calreticulin (CRT) can act as an autoantigen in diseases such as SLE . Why CRT is a target of protective immunity and whether it may interfere with innate immunity once released from cells during inflammation is unclear . In the present study, we found that CRT was detected more frequently in SLE sera and in higher amounts than found in control sera . Approximately 40% of SLE sera tested contained autoantibodies against CRT as detected by ELISA and immunoblotting . CRT was found to be predominantly in the sera of SLE patients associated with immune complexes and C1q, and only bound to the surfaces of neutrophils in the presence of low levels of calcium and magnesium . In order to further investigate the C1q-CRT interaction, recombinant CRT and its discrete domains (N-, P-, and C-domains) were produced in Escherichia coli . CRT binds to globular head region of C1q primarily via its N- and P-domains . The N-domain was shown to be the most autoantigenic region of CRT, as the anti-CRT autoantibodies from most patients reacted against this region . CRT also altered C1q-mediated immune functions . The P-domain of CRT bound to C1q and reduced the binding of immune complexes in SLE sera to immobilized C1q . Full length CRT and its N- and P-domains were able to reduce the C1q-dependent binding of immune complexes to neutrophils and solid-phase bound C1q . We conclude that CRT, once released from leucocytes during inflammation, may not only induce an antigenic reaction, but also interfere with C1q-mediated inflammatory processes.

Am J Respir Crit Care Med, 1997 May, 155(5), 1643 - 8
L-canavanine improves organ function and tissue adenosine triphosphate levels in rodent endotoxemia; Liaudet L et al.; Overproduction of NO by an inducible NO synthase (iNOS) plays a major role in the pathophysiology of septic shock, and selective inhibition of iNOS in this setting could be of great therapeutic value . In the present study, we evaluated the effects of L-canavanine, a selective iNOS inhibitor, in an animal model of septic shock, with a particular focus on tissue oxidative metabolism and organ functions . Anesthetized rats challenged intravenously with lipopolysacharide (LPS) were treated after 1 h by a continuous infusion of either L-canavanine (20 mg/kg/h; n = 11) or an equivalent volume of saline (2 ml/kg/h; n = 17) given for 4 h . A third group (sham rats; n = 9) did not receive LPS and was treated with a continuous infusion of saline (2 ml/kg/h) . At the end of experiments, biopsies were taken from the liver, the kidney, and the small intestine for the measurement of tissue ATP . LPS induced a progressive fall in blood pressure, accompanied by biologic signs of liver and kidney failure, concomitant with a marked decrease in tissue ATP stores . L-canavanine largely prevented hypotension and significantly increased tissue ATP while reducing the signs of organ dysfunction . These effects were associated with a significant improvement in survival during the 5 h of study . We conclude that L-canavanine not only reduces hypotension in endotoxin shock but also largely prevents the detrimental consequences of LPS on tissue oxidative metabolism and major organ functions, allowing a decrease in endotoxin lethality.

Ren Fail, 1997 May, 19(3), 475 - 9
Early gestational hemolytic uremic syndrome: case report and review of literature; Ribeiro FM et al.; Hemolytic uremic syndrome (HUS) is a rare condition which most frequently follows gastrointestinal or respiratory infection episodes in young children, but it can also occur in other settings such as the postpartum period and during use of drugs such as oral contraconceptives, immunosuppressors, and antineoplastics . In early pregnancy, however, its frequency is thought to be very low . The authors report a case of a 30-year-old woman who developed HUS early in her first pregnancy . She had persistent aqueous diarrhea from the beginning of the pregnancy . At the 21st week she developed hypertension which in 2 weeks was followed by seizures, oliguria, and acute pulmonary edema despite intensive medical efforts to control her blood pressure . Surgical intervention for fetal delivery was performed . The patient was initially kept on continuous hemodialysis (CVVHD) followed by an alternate-day conventional hemodialysis schedule . A peripheral blood analysis showed a microangiopathic hemolytic anemia with thrombocytopenia; blood coagulation tests were completely normal . A brain CT scan and an abdominal MRI showed no major abnormalities . HUS was confirmed by a percutaneal kidney biopsy, performed at the 21st day of anuria . Techniques for identification of verotoxin-producing E . coli were not available . Renal function did not recover and the patient has been undergoing regular maintenance hemodialysis for a year.

Proc Assoc Am Physicians, 1997 May, 109(3), 245 - 53
Delivery of primary autologous skeletal myoblasts into rabbit heart by coronary infusion: a potential approach to myocardial repair; Taylor DA et al.; Myocardial repair after injury is limited because the adult heart cannot regenerate . We propose using autologous skeletal muscle cells (myoblasts) as a source of reserve cells for repair of regions of damaged myocardium . This report examines two potential methods for the transfer of cells to the myocardium: selective coronary catheterization, and myoblast infusion or myoblast injection directly into the left ventricular wall . Autologous, primary rabbit skeletal myoblasts were harvested, were transduced ex vivo with adenoviruses expressing the Escherichia coli beta-galactosidase (beta-gal) gene, and were infused selectively into the coronary circulation or injected directly into the myocardial wall . After either delivery method, beta-gal expression was detectable at the earliest times examined (3 days) and persisted for several weeks . The method of delivery influenced the spatial pattern of beta-gal expression . After direct injection, a localized concentration of myoblasts that decreased with distance from the injection site was visible primarily in the myocardial layer of the ventricle, although occasional staining could be detected in other layers . After coronary infusion, discrete punctate or linear foci of beta-gal expression were found throughout the distribution of the left coronary circulation in all cardiac layers . After infusion or injection, beta-gal-positive cells were seen in direct physical apposition to cardiocytes; interestingly, beta-gal could be detected also in some branched cells with clear cross-striations . Autologous myoblasts survived with no obvious dysrhythmic effects despite their presence in extensive or discrete loci in the myocardium . These observations provide the first evidence that myoblast transfer is possible by catheter-based methods, and they create the basis for studies to investigate the functional consequences of myoblast infusion in damaged heart.

Br J Pharmacol, 1997 May, 121(2), 296 - 302
Central involvement of kinin B1 and B2 receptors in the febrile response induced by endotoxin in rats; Coelho MM et al.; 1 . The effect of central injection of selective kinin B1 and B2 receptor antagonists on the febrile response induced by endotoxin (E . coli lipopolysaccharide, LPS) in rats was investigated . 2 . Intracerebroventricular (i.c.v.) injection of a selective B2 receptor antagonist (Hoe-140, 8 nmol) reduced the early (0-2 h), but increased the late phase (4-6 h) of the febrile response induced by intravenous (i.v.) injection of LPS (0.5 microgram kg-1) . 3 . Co-administration of Hoe-140 (8 nmol, i.c.v.) with LPS (0.5 microgram kg-1, i.v.), followed 2.5 h later by the i.c.v . injection of a selective B1 receptor antagonist {des-Arg9-Leu8}-bradykinin (BK, 8 nmol), significantly reduced the febrile response induced by LPS throughout the whole experimental period . 4 . Intravenous injection of Hoe-140 (1 mg kg-1) significantly reduced the febrile response induced by LPS (0.5 microgram kg-1, i.p.) . 5 . Pretreatment (24 h) with LPS (0.5 microgram kg-1, i.v.) reduced the febrile response induced by BK or {Tyr8}-BK (both, 5 nmol, i.c.v.), but markedly increased the febrile response induced by {des-Arg9}-BK (5 nmol, i.c.v.) . The response induced by {des-Arg9}-BK in LPS-pretreated rats was significantly inhibited by co-injection of {des-Arg9-Leu8}-BK (15 nmol, i.c.v.) . 6 . The results suggest that kinins are involved in the induction of LPS-induced fever and that central B2 and B1 receptors are activated during the initial and late phase of this response, respectively . The results also suggest that downregulation and/or desensitization of B2 receptors and induction and/or upregulation of B1 receptors in LPS-pretreated animals may have a significant pathophysiological role in the induction and maintenance of fever . These observations may be specifically important in the case of chronic inflammatory conditions, because the BK metabolite {des-Arg9}-BK, so far considered an inactive metabolite, acquires an active and relevant role with the progressive expression of B1 receptors that occurs in such states.

Ann Otol Rhinol Laryngol, 1997 May, 106(5), 394 - 8
Horseradish peroxidase permeation from the capillaries of the stria vascularis after inoculation of endotoxin into the middle ear; Watanabe K et al.; Escherichia coli-derived endotoxin was inoculated in the middle ear of guinea pigs 24 hours after being injected intraperitoneally . Twenty-four hours after the middle ear inoculation, horseradish peroxidase (HRP) was injected via the femoral vein and the permeability of HRP through the capillaries of the stria vascularis and the destination of the leaked HRP were examined . A large amount of HRP leaked out of the capillary through he opened endothelial cell junctions and penetrated the enlarged intercellular spaces . Leaked HRP entered the pinocytotic vesicles of the intermediate cells . Even slightly degenerated intermediate cells retained this function . The HRP penetrated the spongelike structure of the marginal cells leading to the intercellular space . This structure was not observed without endotoxin . The HRP could not pass the cochlear duct through the tight junctions between marginal cells . Blood sludging was observed in the strial capillaries . It appeared more frequently in the upper three turns than in the basal turn . The HRP leakage out of the capillaries was observed not only in the upper three turns but also in the basal turn.

Drug Metab Dispos, 1997 May, 25(5), 617 - 22
Oxidation of xenobiotics by recombinant human cytochrome P450 1B1; Shimada T et al.; Human cytochrome P450 (P450) 1B1 (CYP1B1) has recently been shown to be an important enzyme in the activation of diverse procarcinogens such as arylarenes, nitroarenes, and arylamines to reactive metabolites that cause DNA damage in the cells . However, it is not known whether this P450 enzyme also plays roles in the oxidation of certain drugs or model substrates commonly used in P450 assays . We examined the substrate oxidation activities of recombinant human CYP1B1 in yeast microsomes and compared these activities with those catalyzed by reconstituted systems containing recombinant CYP1A1 and CYP1A2 which were isolated from membranes of Escherichia coli in which respective cDNAs have been expressed . Catalytic activities towards some of the model substrates of other human P450 enzymes including CYP2A6, 2C9, 2C19, 2D6, 2E1, and 3A4 were also determined and compared . CYP1B1 catalyzed benzo{a}pyrene 3-hydroxylation at rates lower than those of CYP1A1 but higher than those of CYP1A2 . The activity towards 7-ethoxyresorufin O-deethylation catalyzed by CYP1B1 was about one-tenth of that of CYP1A1, but the Km values were lower for CYP1B1 than those for CYP1A1 and CYP1A2 . CYP1B1 was also able to catalyze the oxidation of theophylline and caffeine, two prototypic substrates for CYP1A2 . CYP1B1 did not oxidize other typical P450 substrates such as coumarin, tolbutamide, S-mephenytoin, chlorzoxazone, nifedipine, and testosterone, while low rates of oxidation of bufuralol and 7-ethoxycoumarin were found for CYP1B1 . These results indicate that CYP1B1 has catalytic activities overlapping CYP1A1 and CYP1A2 with respect to the oxidation of drugs and model P450 substrates, although the relative catalytic roles in these three P450 enzymes differ depending upon the substrates examined . A distinct marker activity of CYP1B1 has not been identified.

J Gen Virol, 1997 May, 78 ( Pt 5), 1175 - 9
Subcellular localization of the 28 kDa protein of the triple-gene-block of bamboo mosaic potexvirus; Chang BY et al.; Open reading frame 2 of the bamboo mosaic potexvirus (BaMV) genome encodes a 28 kDa protein, the first of the "triple-gene-block' of BaMV which is believed to play a role in cell-to-cell movement of the virus in host plants . The 28 kDa protein was expressed in Escherichia coli and polyclonal antiserum was raised in a rabbit . Western blot analyses showed that the 28 kDa protein was associated mainly with components in the cell wall and 30000 g pellet fractions of a BaMV-infected leaf homogenate . Immunogold electron microscopy of infected leaf tissues revealed that the 28 kDa protein was associated with electron-dense crystal-line bodies (EDCBs) in the cytoplasm and nuclei . Nuclear EDCBs were found closely associated with nucleoli . Gold-labelled EDCB-like structures were also detected in the cytoplasm, but not within nuclei, in protoplasts up to 48 h post-inoculation . No specific labelling of the 28 kDa protein was found within any cytoplasmic structures or within cell walls.

J Med Microbiol, 1997 May, 46(5), 403 - 6
Role of type-1 fimbriae in the pathogenesis of chronic pyelonephritis in relation to reactive oxygen species; Gupta R et al.; The role of type-1 fimbriae in the pathogenesis of chronic pyelonephritis was studied for two Escherichia coli strains . Although both strains produced a similar total oxidative burst of chemiluminescence in macrophages from uninfected mice, the extracellular oxidative burst was greater with the non-fimbriate mutant E . coli BH-5 than its type-1 fimbriate parent E . coli 31-B . Moreover, macrophages from mice infected with the non-fimbriate mutant gave a much greater oxidative burst when stimulated with latex particles than that given by macrophages from mice infected with the type-1 fimbriate parent . These results correlated with the degree of renal inflammation and scarring as measured by malondialdehyde formation . Hence, the role of type-1 fimbriae in the pathogenesis of chronic UTI although documented does not appear to be significant.

Hum Genet, 1997 May, 99(5), 692 - 5
Identification of an HD patient with a (CAG)180 repeat expansion and the propagation of highly expanded CAG repeats in lambda phage; Sathasivam K et al.; The Huntington's disease mutation has been identified as a CAG/polyglutamine repeat expansion in a large gene of unknown function . In order to develop the transgenic systems necessary to uncover the molecular pathology of this disorder, it is necessary to be able to manipulate highly expanded CAG repeats in a cloned form . We have identified a patient with an expanded allele of greater than 170 repeat units and have cloned the mutant allele in the lambda zap vector . The recovery of highly expanded repeats after clone propagation was more efficient when repeats were maintained as lambda phage clones rather than as the plasmid counterparts . Manipulation of the repeats as phage clones has enabled us to generate Huntington's disease transgenic mice that contain highly expanded (CAG)115-(CAG)150 repeats and that develop a progressive neurological phenotype.

Hum Genet, 1997 May, 99(5), 616 - 23
Isolation of a human gene (HES1) with homology to an Escherichia coli and a zebrafish protein that maps to chromosome 21q22.3; Scott HS et al.; Exon trapping was performed with chromosome 21 cosmids to identify those that may be involved in the pathogenesis of Down syndrome, or several of the genetic diseases that map to chromosome 21 . BLASTX analysis revealed two exons with significant homology to a zebrafish protein (ES1) and an Escherichia coli protein (sigma cross-reacting protein 27A), both of unknown function . The exons also showed identity with several expressed sequence tags (ESTs) . Sequences from all ESTs derived from this gene and reverse transcription-polymerase chain reaction (RT-PCR) analysis were used to determine the full cDNA sequence, which corresponded to an mRNA of 1.7 kb with an open reading frame of 268 amino acids . The mRNA from this gene, termed HES1, is ubiquitously expressed, but strongly so in heart and skeletal muscle . Potential mitochondrial targeting signals were found in both the human and zebrafish proteins, consistent with the high expression levels in muscle tissues . The strong homology between the E . coli, zebrafish and HES1 proteins suggests an important biological role . Hybridization of RT-PCR products to a cosmid contig in chromosome 21q22.3, mapped HES1 just proximal to D21S25, a critical mapping region for several genetic diseases . Given the mapping position, this gene is a candidate for involvement in these disorders, including autoimmune polyglandular disease type I and the autosomal nonsyndromic deafness loci, DFNB8 and DFNB10 . In addition, the initial method of EST identification for gene isolation presented here is valid for many genes and can be used to obtain initial sequence contigs without cloning or library screening.

Hum Genet, 1997 May, 99(5), 590 - 5
Alternative splicing of hMSH2 in normal human tissues; Mori Y et al.; hMSH2 is a homolog of bacterial mutS and yeast Msh2, a member of the group of mismatch repair genes whose products bind to mismatched regions of double-stranded DNA . We analyzed expression of hMSH2 in normal human organs by the polymerase chain reaction coupled with reverse transcription and found two novel types of alternatively spliced mRNAs that were expressed in normal human organs . One lacked exon 13, and the other lacked a portion from the second nucleotide of codon 633 to the second nucleotide of codon 719 . In the latter transcript, intro 12 started with TA and ended with TT (TA-TT intron) which did not meet the GT-AG rule . Both types of transcript resulted in frameshifts which generated truncated hMSH2 proteins lacking the main part of the highly conserved region . The biological significance of the alternative splicing remains to be elucidated.

J Bacteriol, 1997 May, 179(10), 3365 - 7
Antimonite is accumulated by the glycerol facilitator GlpF in Escherichia coli; Sanders OI et al.; In a search for genes responsible for the accumulation of antimonite in Escherichia coli, TnphoA was used to create a pool of random insertional mutants, from which one antimonite-resistant mutant was isolated . Sequence analysis showed that the TnphoA insertion was located in the glpF gene, coding for the glycerol facilitator GlpF . The mutant was shown to be defective in polyol transport by GlpF . These results suggest that in solution Sb(III) is recognized as a polyol by the glycerol facilitator.

J Bacteriol, 1997 May, 179(10), 3354 - 7
Cloning and insertional inactivation of Streptomyces argillaceus genes involved in the earliest steps of biosynthesis of the sugar moieties of the antitumor polyketide mithramycin; Lombo F et al.; Two genes (mtmD and mtmE) were cloned and sequenced from the mithramycin producer Streptomyces argillaceus . Comparison with proteins in databases and enzymatic assays after expression in Escherichia coli showed that they encode a glucose-1-phosphate:TTP thymidylyl transferase and a TDP-D-glucose 4,6-dehydratase, respectively . The mtmD gene was inactivated by gene replacement, generating a nonproducing mutant that accumulates a tetracyclic compound designated premithramycinone . The identification of premithramycinone reveals new aspects of the mithramycin biosynthetic pathway and suggests that at least some glycosylations occur before breakage of the fourth ring.

J Bacteriol, 1997 May, 179(10), 3317 - 23
A topological model for the general aromatic amino acid permease, AroP, of Escherichia coli; Cosgriff AJ et al.; The general aromatic amino acid permease, AroP, of Escherichia coli is responsible for the active transport of phenylalanine, tyrosine, and tryptophan . A proposed topological model for the AroP permease, consisting of 12 hydrophobic transmembrane spans connected by hydrophilic loops, is very similar to that of the closely related phenylalanine-specific permease . The validity of this model and its similarity to that of the PheP permease were investigated by studying fusion proteins of AroP permease and alkaline phosphatase . Based on the results obtained from the AroP-alkaline phosphatase sandwich fusions, we have significantly revised the proposed topological model for AroP in two regions . In this modified AroP topological model, the three charged residues E151, E153, and K160 are repositioned within the membrane in span 5 . These three residues are conserved in a large family of amino acid transport proteins, and site-directed mutagenesis identifies them as being essential for transport activity . It is postulated that these residues together with E110 in transmembrane span 3 may be involved in a proton relay system.

J Bacteriol, 1997 May, 179(10), 3284 - 92
Two divergent catalase genes are differentially regulated during Aspergillus nidulans development and oxidative stress; Kawasaki L et al.; Catalases are ubiquitous hydrogen peroxide-detoxifying enzymes that are central to the cellular antioxidant response . Of two catalase activities detected in the fungus Aspergillus nidulans, the catA gene encodes the spore-specific catalase A (CatA) . Here we characterize a second catalase gene, identified after probing a genomic library with catA, and demonstrate that it encodes catalase B . This gene, designated catB, predicts a 721-amino-acid polypeptide (CatB) showing 78% identity to an Aspergillus fumigatus catalase and 61% identity to Aspergillus niger CatR . Notably, similar levels of identity are found when comparing CatB to Escherichia coli catalase HPII (43%), A . nidulans CatA (40%), and the predicted peptide of a presumed catA homolog from A . fumigatus (38%) . In contrast, the last two peptides share a 79% identity . The catalase B activity was barely detectable in asexual spores (conidia), disappeared after germination, and started to accumulate 10 h after spore inoculation, throughout growth and conidiation . The catB mRNA was absent from conidia, and its accumulation correlated with catalase activity, suggesting that catB expression is regulated at the transcription level . In contrast, the high CatA activity found in spores was lost gradually during germination and growth . In addition to its developmental regulation, CatB was induced by H2O2, heat shock, paraquat, or uric acid catabolism but not by osmotic stress . This pattern of regulation and the protective role against H2O2 offered by CatA and CatB, at different stages of the A . nidulans life cycle, suggest that catalase gene redundancy performs the function of satisfying catalase demand at the two different stages of metabolic and genetic regulation represented by growing hyphae versus spores . Alternative H2O2 detoxification pathways in A . nidulans were indicated by the fact that catA/catB double mutants were able to grow in substrates whose catabolism generates H2O2.

J Bacteriol, 1997 May, 179(10), 3260 - 9
Defective export in Escherichia coli caused by DsbA'-PhoA hybrid proteins whose DsbA' domain cannot fold into a conformation resistant to periplasmic proteases; Guigueno A et al.; The disulfide bond-forming factor DsbA and the alkaline phosphatase are stable in the Escherichia coli periplasmic space and can be overproduced without significant perturbation of the cell's physiology . By contrast, DsbA'-PhoA hybrid proteins resulting from TnphoA insertions into different regions of a plasmid-borne dsbA gene could become toxic (lethal) to bacteria . Toxicity was concomitant with an impairment of some step of the export mechanism and depended on at least three parameters, i.e., (i) the rate of expression of the hybrid protein, (ii) the ability of the amino-terminal DsbA' domain of the hybrid protein to fold into a protease-resistant conformation in the periplasmic space, and (iii) the activity of the DegP periplasmic protease . Even under viable conditions of low expression, DsbA' folding-deficient hybrid proteins accumulated more than the folding-proficient ones in the insoluble material and this was aggravated in a strain lacking the DegP protease . When production was more elevated, the folding-deficient hybrid proteins became lethal, but only in strains lacking the DegP activity, while the folding-proficient ones were not . Under conditions of very high production by degP+ or degP strains, both types of hybrid proteins accumulated as insoluble preproteins . Meanwhile, the export machinery was dramatically handicapped and the cells lost viability . However, the folding-deficient hybrid proteins had a higher killing efficiency than the folding-proficient ones . Free DsbA'-truncated polypeptides, although not toxic, were processed more slowly when they could not fold into a protease-resistant form in the periplasmic space . This provides indications in E . coli for a direct or indirect influence of the folding of a protein in the periplasmic environment on export efficiency.

J Bacteriol, 1997 May, 179(10), 3232 - 8
Polymorphism, duplication, and IS1-mediated rearrangement in the chromosomal his-rfb-gnd region of Escherichia coli strains with group IA and capsular K antigens; Drummelsmith J et al.; Individual Escherichia coli strains produce several cell surface polysaccharides . In E . coli E69, the his region of the chromosome contains the rfb (serotype O9 lipopolysaccharide O-antigen biosynthesis) and cps (serotype K30 group IA capsular polysaccharide biosynthesis) loci . Polymorphisms in this region of the Escherichia coli chromosome reflect extensive antigenic diversity in the species . Previously, we reported a duplication of the manC-manB genes, encoding enzymes involved in GDP-mannose formation, upstream of rfb in strain E69 (P . Jayaratne et al., J . Bacteriol . 176:3126-3139, 1994) . Here we show that one of the manC-manB copies is flanked by IS1 elements, providing a potential mechanism for the gene duplication . Adjacent to manB1 on the IS1-flanked segment is a further open reading frame (ugd), encoding uridine-5'-diphosphoglucose dehydrogenase . The Ugd enzyme is responsible for the production of UDP-glucuronic acid, a precursor required for K30 antigen synthesis . Construction of a chromosomal ugd::Gm(r) insertion mutation demonstrated the essential role for Ugd in the biosynthesis of the K30 antigen and confirmed that there is no additional functional ugd copy in strain E69 . PCR amplification and Southern hybridization were used to examine the distribution of IS1 elements and ugd genes in the vicinity of rfb in other E . coli strains, producing different group IA K antigens . The relative order of genes and, where present, IS1 elements was established in these strains . The regions adjacent to rfb in these strains are highly variable in both size and gene order, but in all cases where a ugd homolog was present, it was found near rfb . The presence of IS1 elements in the rfb regions of several of these strains provides a potential mechanism for recombination and deletion events which could contribute to the antigenic diversity seen in surface polysaccharides.

J Bacteriol, 1997 May, 179(10), 3213 - 21
Regions of Escherichia coli TonB and FepA proteins essential for in vivo physical interactions; Larsen RA et al.; The transport of Fe(III)-siderophore complexes and vitamin B12 across the outer membrane of Escherichia coli is an active transport process requiring a cognate outer membrane receptor, cytoplasmic membrane-derived proton motive force, and an energy-transducing protein anchored in the cytoplasmic membrane, TonB . This process requires direct physical contact between the outer membrane receptor and TonB . Previous studies have identified an amino-terminally located region (termed the TonB box) conserved in all known TonB-dependent outer membrane receptors as being essential for productive energy transduction . In the present study, a mutation in the TonB box of the ferric enterochelin receptor FepA resulted in the loss of detectable in vivo chemical cross-linking between FepA and TonB . Protease susceptibility studies indicated this effect was due to an alteration of conformation rather than the direct disruption of a specific site of physical contact . This suggested that TonB residue 160, implicated in previous studies as a site of allele-specific suppression of TonB box mutants, also made a conformational rather than a direct contribution to the physical interaction between TonB and the outer membrane receptors . This possibility was supported by the finding that TonB carboxyl-terminal truncations that retained Gln-160 were unable to participate in TonB-FepA complex formation, indicating that this site alone was not sufficient to support the physical interactions involved in energy transduction . These studies indicated that the final 48 residues of TonB were essential to this physical interaction . This region contains a putative amphipathic helix which could facilitate TonB-outer membrane interaction . Amino acid replacements at one site in this region were found to affect energy transduction but did not appear to greatly alter TonB conformation or the formation of a TonB-FepA complex . The effects of amino acid substitutions at several other TonB sites were also examined.

J Bacteriol, 1997 May, 179(10), 3164 - 70
Paraquat regulation of hmp (flavohemoglobin) gene expression in Escherichia coli K-12 is SoxRS independent but modulated by sigma S; Membrillo-Hernandez J et al.; We report the first example of a gene, hmp, encoding a soluble flavohemoglobin in Escherichia coli K-12, which is up-regulated by paraquat in a SoxRS-independent manner . Unlike what is found for other paraquat-inducible genes, high concentrations of paraquat (200 microM) were required to increase the level of hmp expression, and maximal induction was observed only after 20 min of exposure to paraquat . Neither a mutation in soxS nor one in soxR prevented the paraquat-dependent increase in phi(hmp-lacZ) expression, but either mutant allele delayed full expression of phi(hmp-lacZ) activity after paraquat addition . Induction of hmp by paraquat was demonstrated in aerobically grown cultures during exponential growth and the stationary phase, thus revealing two Sox-independent regulatory mechanisms . Induction of hmp by paraquat in the stationary phase was dependent on the global regulator of stationary-phase gene expression, RpoS (sigma S) . However, a mutation in rpoS did not prevent an increase in hmp expression by paraquat in exponentially growing cells . Induction of sigma S in the exponential phase by heat shock also induced phi(hmp-lacZ) expression in the presence of paraquat, supporting the role of sigma S in one of the regulatory mechanisms . Mutations in oxyR or rob, known regulators of several stress promoters in E . coli, had no effect on the induction of hmp by paraquat . Other known superoxide-generating agents (plumbagin, menadione, and phenazine methosulfate) were not effective in inducing hmp expression.

J Bacteriol, 1997 May, 179(10), 3139 - 45
An alkB gene homolog is differentially transcribed during the Caulobacter crescentus cell cycle; Colombi D et al.; A Caulobacter crescentus alkB gene homolog was identified in a clone previously shown to contain the heat shock genes dnaK and dnaJ; the homolog is located upstream of dnaK and is transcribed in the opposite orientation . An analysis of the alkB gene has shown that the deduced amino acid sequence is that of a 21-kDa protein, which is 42% identical and 78% similar to Escherichia coli AlkB . Furthermore, an alkB-null mutant was constructed by gene disruption and was shown to be highly sensitive to the alkylating agent methyl methanesulfonate (MMS) . However, the alkB gene of C . crescentus, unlike its E . coli counterpart, is not located downstream of the ada gene, and its transcription is not induced by alkylating agents . In addition, no acquired enhanced resistance to MMS toxicity by treatment with low MMS doses was observed, suggesting that no adaptive response occurs in C . crescentus . Nevertheless, transcription of the alkB gene is cell cycle controlled, with a pattern of expression similar to that of several Caulobacter genes involved in DNA replication.

J Bacteriol, 1997 May, 179(10), 3133 - 8
Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and characterization of a new isochorismate synthase from Escherichia coli; Daruwala R et al.; The first committed step in the biosynthesis of menaquinone (vitamin K2) is the conversion of chorismate to isochorismate, which is mediated by an isochorismate synthase encoded by the menF gene . This isochorismate synthase (MenF) is distinct from the entC-encoded isochorismate synthase (EntC) involved in enterobactin biosynthesis . MenF has been overexpressed under the influence of the T7 promoter and purified to homogeneity . The purified protein was found to have a molecular mass of 98 kDa as determined by gel filtration column chromatography on Sephacryl S-200 . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a molecular mass of 48 kDa . Thus, the enzyme is a homodimer . The purified enzyme showed a pH optimum of 7.5 to 8.0 and a temperature optimum of 37 degrees C . The enzyme carries out the irreversible conversion of chorismate to isochorismate in the presence of Mg2+ . The enzyme was found to have a Km of 195 +/- 23 microM and a k(cat) of 80 min(-1) . In the presence of 30 mM beta-mercaptoethanol (BME), the k(cat) increased to 176 min(-1) . The reducing agents BME and dithiothreitol stimulated the enzymatic activity more than twofold . Treatment of the enzyme with the cysteine-specific modifying reagent N-ethylmaleimide (NEM) resulted in the complete loss of activity . Preincubation of the enzyme with the substrate, chorismate, before NEM treatment resulted in complete protection of the enzyme from inactivation.

J Bacteriol, 1997 May, 179(10), 3095 - 102
The RIB element in the goaG-pspF intergenic region of Escherichia coli; Jovanovic G et al.; The sequence (2,700 bp) between the aldH and pspF genes of Escherichia coli was determined . The pspF gene encodes a sigma54 transcriptional activator of the phage shock protein (psp) operon (pspA to pspE) . Downstream of the pspF transcribed region are two open reading frames (ORFs), ordL and goaG, convergently oriented with respect to pspF . These two ORFs, together with the adjacent aldH gene, may constitute a novel operon (aldH-ordL-goaG) . The goaG-pspF intergenic region contains a complex extragenic mosaic element, RIB . The structure of this RIB element, which belongs to the BIME-1 family, is Y(REP1) > 16 < Z1(REP2), where Y and Z1 are palindromic units and the central 16 bases contain an L motif with an ihf consensus sequence . DNA fragments containing the L motif of the psp RIB element effectively bind integration host factor (IHF), while the Y palindromic unit (REP1) of the same RIB element binds DNA gyrase weakly . Computer prediction of the pspF mRNA secondary structure suggested that the transcribed stem-loop structures formed by the 3'-flanking region of the pspF transcript containing the RIB element can stabilize and protect pspF mRNA . Analysis of pspF steady-state mRNA levels showed that transcripts with an intact RIB element are much more abundant than those truncated at the 3' end by deletion of either the entire RIB element or a single Z1 sequence (REP2) . Thus, the pspF 3'-flanking region containing the RIB element has an important role in the stabilization of the pspF transcript.

Biotechniques, 1997 May, 22(5), 982 - 7
The PerFect lipid optimizer kit for maximizing lipid-mediated transfection of eukaryotic cells; Griffiths T et al.; The transfection efficiencies of a panel of eight uniquely different lipid reagents has been evaluated with two other commercially available lipids for use in transfecting a diversity of eukaryotic cell lines . The PerFect lipids are available individually or together in an optimization panel format that can be tested in any given cell line, enabling one to evaluate the optimal lipid for transfecting each individual cell line . Our results demonstrate that no single lipid is optimal for plasmid transfection over a broad range of cell types, thus emphasizing the need for multiple unique lipid reagents and a simple format for testing their transfection efficiency on a given cell type.

Biotechniques, 1997 May, 22(5), 912 - 5
Cloning DNA fragments between two adjacent/overlapping restriction sites using a "positive stuffer"; Loukianov E et al.; Here we describe a solution to a common problem encountered in recombinant DNA cloning when directional cloning of a DNA fragment into a predetermined plasmid requires the use of restriction enzymes with adjacent or overlapping recognition sites . In preparing the double-digested plasmid, only one enzyme will often cut, whereas the second will not because of the lack of a sufficiently long stretch of double-stranded DNA at its recognition site . The problem can be solved by construction of a "user-friendly" intermediary plasmid in which the desired restriction sites are separated by a positively selectable stuffer with resistance to neomycin . This approach is particularly useful in cases where the choices of restriction sites are severely limited, for example, when it is necessary to clone an additional piece of DNA into a complex vector already containing multiple gene cassettes.

Biotechniques, 1997 May, 22(5), 897 - 904
Agglutination-inhibition assay for the detection of recombinant proteins tagged with peptide epitopes; Dolezal O et al.; We have demonstrated that the expression of recombinant proteins labeled with an immunoreactive epitope can be rapidly assessed and quantitated using a modified haemagglutination inhibition assay in enzyme-linked immunosorbent assay (ELISA) trays . The agglutination of erythrocytes from a droplet of whole blood provided a simple visual assay . The additional reagents required for the assay were a recombinant anti-human erythrocyte Fab fragment fused to a peptide epitope and a bivalent antibody with specificity to the same epitope . In this report, we found that a convenient and sensitive epitope was the octapeptide FLAG in conjunction with the M2 anti-FLAG antibody, which had affinity to FLAG incorporated either at the C-terminus or N-terminus of the recombinant protein . The agglutination-inhibition (AI) assay was configured to detect as little as 1 mg/L of soluble recombinant protein in a 30-min assay . Since the AI assay was substantially more rapid and convenient than dot-blot or Western blot analyses, our laboratory now uses this method routinely for the assay of FLAG-labeled recombinant products following protein expression and subsequent small- and large-scale purification procedures.

Parasitology, 1997 May, 114 ( Pt 5), 447 - 53
Cloning, heterologous expression and antigenicity of a schistosome cercarial protease; Price HP et al.; A gene coding for the 30 kDa Schistosoma mansoni cercarial protease was amplified using the polymerase chain reaction (PCR) from genomic DNA templates . Cloning and sequencing of several independent PCR clones revealed the presence of an intron additional to the one described in the original cloning of the gene . The 3 exons were cloned into expression vectors so that they could be expressed as separate glutathione-S-transferase (GST) translational fusions . Recombinant bacteria carrying these expression plasmids expressed the fusion proteins at high levels . Western blotting of bacterial lysates with sera raised against the native S . mansoni cercarial protease showed that all 3 exons were recognized . Thus we have produced recombinant bacteria capable of providing large amounts of an S . mansoni antigen for immunological studies and evaluation as a candidate vaccine.

Physiol Behav, 1997 May, 61(5), 659 - 60
Selected temperature correlates with intensity of fever in rats; Briese E; Fever is considered due to an elevation of the setpoint of body temperature . The temperature is regulated at a higher level and the higher temperature is established by activation of the heat-seeking thermoeffectors . However, it is surprising that, for this widely accepted hypothesis, there is little experimental evidence of the setpoint shifting to a higher level . The present study shows, for the first time, a significant correlation between the magnitude of the temperature rise in fever and the ambient temperature selected by rats in a thermal gradient.

Antimicrob Agents Chemother, 1997 May, 41(5), 992 - 8
Glycosylated flavones as selective inhibitors of topoisomerase IV; Bernard FX et al.; Three flavonoids which promoted Escherichia coli topoisomerase IV-dependent DNA cleavage were isolated from cottonseed flour and identified as quercetin 3-O-beta-D-glucose-{1,6}-O-alpha-L-rhamnose (rutin), quercetin 3-O-beta-D-galactose-{1,6}-O-alpha-L-rhamnose, and quercetin 3-O-beta-D-glucose (isoquercitrin) . The most active one (rutin) also inhibited topoisomerase IV-dependent decatenation activity (50% inhibitory concentration, 64 microg/ml) and induced the SOS response of a permeable E . coli strain . Derivatives of quercetin glycosylated at position C-3 were shown to induce two site-specific DNA cleavages of pBR322 DNA, which were mapped by DNA sequence analysis to the gene encoding resistance to tetracycline . Cleavage at these sites was hardly detectable in cleavage reactions with quercetin or fluoroquinolones . None of the three flavonoids isolated from cottonseeds had any stimulatory activity on E . coli DNA gyrase-dependent or calf thymus topoisomerase II-dependent DNA cleavage, and they were therefore specific to topoisomerase IV . These results show that selective inhibitors of topoisomerase IV can be derived from the flavone structure . This is the first report on a DNA topoisomerase inhibitor specific for topoisomerase IV.

Nat Struct Biol, 1997 May, 4(5), 405 - 12
Oligosaccharide substrate binding in Escherichia coli maltodextrin phosphorylase; O'Reilly M et al.; The crystal structure of E . coli maltodextrin phosphorylase co-crystallized with an oligosaccharide has been solved at 3.0 A resolution, providing the first structure of an oligosaccharide bound at the catalytic site of an alpha-glucan phosphorylase . An induced fit mechanism brings together two domains across the catalytic site tunnel . A stacking interaction between the glucosyl residue and the aromatic group of a tyrosine residue at a sub-site remote (8 A) from the catalytic site provides a key element in substrate recognition; mutation of this residue to Ala decreases the Kcat/Km by 10(4) . Extrapolation of the results to substrate binding across the site of attack by phosphorolysis indicates a likely alteration in the glycosidic torsion angles from their preferred values, an alteration that appears to be important for the catalytic mechanism.

Nat Struct Biol, 1997 May, 4(5), 342 - 9
Interaction of Hsp70 chaperones with substrates; Rudiger S et al.; Determination of the structure of the substrate binding domain of the Escherichia coli Hsp70 chaperone, DnaK, and the biochemical characterisation of the motif it recognizes within substrates provide insights into the principles governing Hsp70 interaction with polypeptide chains . DnaK recognizes extended peptide strands composed of up to five consecutive hydrophobic residues within and positively charged residues outside the substrate binding cavity.

What Is Activated Sludge?, What Is Functional Genomics?, What is Food Microbiology?, What Is Genetic Engineering?, What Is Nitrification?, c, Microbe, s, Bacterium, a, Microorganism, s, Microbiology, c, Bacteria, a, Functional genomics, i, Meningococcus, e, Escherichia coli, n, B. anthracis, e, Sepsis, c, Sepsis, o, Ps. fluorescens, a, Antibiotics, r, Yeast, o, Growth media, r, Lactobacillus, i, Fermentations, n, Sepsis, a, Pseudomonas aeruginosa, o, Streptococci, e, Escherichia coli, o, Streptococci, o, O157, r, S. cerevisiae, r, Streptococcal, r, Fermentations

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