Escherichia coli

Gene, 1995 Dec 29, 167(1-2), 69 - 74
The creation of a camptothecin-sensitive Escherichia coli based on the expression of the human topoisomerase I; Taylor ST et al.; The synthesis of the human topoisomerase I (Topo I) in Escherichia coli (Ec) results in phenotypes consistent with the appearance of a DNA-relaxing activity . High-level expression was problematic and recloning the gene in a reduced-copy-number plasmid under more stringently regulated control produced a stable plasmid . An Ec strain with an inducible sensitivity to the eukaryotic Topo I poison, camptothecin (CPT), was constructed by introducing a mutation (imp4312) known to enhance the permeability of Ec to a wide variety of compounds . We are able to infer that CPT sensitivity in Ec involves DNA damage by noting elevated sensitivity in a repair-deficient recA strain and by observing the induction of a sulA::lac fusion following exposure to the drug.

Gene, 1995 Dec 29, 167(1-2), 63 - 8
Rapid purification of recombinant proteins fused to chicken avidin; Airenne KJ et al.; A novel expression vector (pAVEX16C) has been constructed that directs the synthesis of desired polypeptides as fusions with the C terminus of chicken egg-white avidin (Avd) . With this and a commercial GST gene (encoding glutathione S-transferase) fusion vector (pGEX-3X, Pharmacia), we produced Avd as fusions C- and N-terminally linked to GST in Escherichia coli . By using the Avd tail and a simple affinity purification protocol, including biotin-agarose, we were able to obtain 1-2 micrograms/ml of highly purified Avd::GST and GST::Avd from crude bacterial lysates . The produced proteins were, to a great extent, in soluble fraction when the cells were grown at 22 degrees C and disrupted with a detergent, N-laurylsarcosine . The fusion proteins could also be affinity-purified with the GST tail using glutathione-Sepharose 4B, but the yield of GST::Avd was significantly lower than when using the Avd tail . Our results therefore indicate that it is possible to produce, in E . coli, biologically active fusion proteins consisting of Avd C- or N-terminally linked with the desired protein which then can easily be purified by a simple affinity chromatography procedure.

Gene, 1995 Dec 29, 167(1-2), 53 - 7
An improved TnMax mini-transposon system suitable for sequencing, shuttle mutagenesis and gene fusions; Kahrs AF et al.; A new collection of mini-transposons (mini-Tn) of the previously described TnMax series {Haas et al, Gene 130 (1993a) 23-31} has been constructed . The transposons (Tn) bear genes conferring resistance to either chloramphenicol (Cm) or kanamycin (Km) . Each member of the new series (TnMax5-TnMax11) contains the general M13 forward (M13-FP) and reverse (M13-RP1) sequencing primers close to the inverted repeats (IR), facilitating the rapid and convenient determination of the DNA sequences flanking the transposon insertion site . Furthermore, the mini-Tn possess the infrequently occurring NotI sites, allowing the localization of genes on macro-restriction maps of bacterial species . Some derivatives contain promoterless trp-lacZ (TnMax11), xylE (TnMax10), phoA (TnMax6) or blaM (TnMax7, TnMax9) genes next to the IR, suitable for the generation of in vivo gene- and operon fusions to study gene regulation, protein export, or to determine the topology of proteins in bacterial membranes . A set of conjugative minimal plasmid vectors (pMin1, pMin2) are used to select for TnMax insertions into the cloned insert, rather than the vector sequences . Due to the small size of the mini-Tn, and a simple and efficient mutagenesis procedure, the TnMax system is a useful tool for targeting and sequencing of cloned genes in Escherichia coli, and especially for shuttle mutagenesis of bacterial species which cannot be targeted by direct transposition.

Gene, 1995 Dec 29, 167(1-2), 227 - 31
The Caenorhabditis elegans rop-1 gene encodes the homologue of the human 60-kDa Ro autoantigen; Labbe JC et al.; As a first step toward establishing a genetic system for the elucidation of the cellular role(s) of the Ro ribonucleoproteins (RoRNP), we have cloned the gene encoding the homologue of the human 60-kDa Ro protein (Ro60) in Caenorhabditis elegans (Ce) . This Ce gene is present as a single copy and contains a 643-codon open reading frame interrupted by three introns . The encoded protein, Rop1p, shares 40% identity and 63% overall similarity with both the human and amphibian Ro60 . Recombinant protein has been produced in Escherichia coli and used to elicit anti-Rop1p antibodies . Immunological analysis indicated that the Ro60 epitopes have been poorly conserved . Gene-fusion expression studies in transgenic nematodes will provide a new avenue of research to shed light on the function of these particles.

Gene, 1995 Dec 29, 167(1-2), 17 - 24
Characterization of two members (ACS1 and ACS3) of the 1-aminocyclopropane-1-carboxylate synthase gene family of Arabidopsis thaliana; Liang X et al.; The nucleotide sequences of two highly homologous 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS; EC 4.4.1.14)-encoding genes, ACS1 and ACS3, of Arabidopsis thaliana (At) have been determined . The sequence analysis shows that ACS3 is a pseudogene representing a truncated version of ACS1 . The missing region of ACS3 corresponding to the fourth exon of ACS1 has been shown by Southern analysis to be absent in the At genome . The chromosomal locations of the five members of the At ACS multigene family have been determined . The results show that each family member resides on a different chromosome . This observation suggests that the ACS3 pseudogene originated by a partial inter-chromosomal gene duplication . The ACS1 polypeptide contains all the conserved and characteristic domains found in the ACC synthase isoenzymes from various plant species, but is unable to express ACS activity in Escherichia coli and yeast . The predicted amino-acid sequence of ACS1 is missing the highly conserved tripeptide, Thr-Asn-Pro (TNP), between Ile204 and Ser205 . Introduction of TNP into ACS1 restores the ACS activity, whereas its removal from the enzymatically active ACS2 results in a loss of activity . The results suggest that TNP is crucial for expression of ACS activity in E . coli.

Gene, 1995 Dec 29, 167(1-2), 147 - 9
A putative SOS repair gene (dinF-like) in a hyperthermophilic archaeon; Bouyoub A et al.; The hyperthermophilic archaeon, Pyrococcus (strain IFREMER AL585), contains an ORF that encodes a polypeptide with a high similarity to the Escherichia coli dinF (DNA damage-inducible) gene product . The conservation of this protein between Archaea and Bacteria suggests that a SOS repair system might operate in Archaea.

Gene, 1995 Dec 29, 167(1-2), 133 - 6
Identification of alcA, a Bordetella bronchiseptica gene necessary for alcaligin production; Giardina PC et al.; The alcA gene, essential for the production of the dihydroxamate siderophore, alcaligin, by Bordetella bronchiseptica, was cloned and sequenced . The alcA gene was identified on a 4.7-kb EcoRI genomic fragment adjacent to a Tn5lac transposon insertion that inactivated alcaligin production in strain MBORD846 . Analysis of the alcA nucleotide sequence revealed a putative Fur-binding site, suggesting that expression of this gene is repressed by iron . The deduced amino-acid sequence of this open reading frame had significant homology with the Escherichia coli iucD gene product, an enzyme required for biosynthesis of the dihydroxamate siderophore aerobactin.

Gene, 1995 Dec 29, 167(1-2), 127 - 32
Cloning, expression and sequence analysis of the genes encoding the heterodimeric methylmalonyl-CoA mutase of Porphyromonas gingivalis W50; Jackson CA et al.; Two genes that encode methylmalonyl-CoA mutase (MCM) have been characterised in Porphyromonas gingivalis W50 (Pg) . The genes, designated mcmA and mcmB are transcribed in an operon and encode the MCM small subunit (SS, 68,626 Da) and the MCM large subunit (LS, 78,703 Da), respectively . A recombinant Escherichia coli (Ec) clone harbouring the Pg mcmA and mcmB genes expressed MCM activity 280-times higher than that of the Ec control . The C terminus of the MCM LS has sequence homology to domains of a variety of enzymes that consume or produce methylmalonyl-CoA, suggesting that the MCM LS C-terminal domain is involved in substrate binding . The MCM LS C-terminal region also exhibits homology to other enzymes that have cobalamin-containing cofactors . It is likely, therefore, that the C terminus of the MCM LS is an important MCM domain involved in both substrate and cofactor binding.

Cell, 1995 Dec 29, 83(7), 1171 - 81
SecA membrane cycling at SecYEG is driven by distinct ATP binding and hydrolysis events and is regulated by SecD and SecF; Economou A et al.; The SecA subunit of E . coli preprotein translocase promotes protein secretion during cycles of membrane insertion and deinsertion at SecYEG . This process is regulated both by nucleotide binding and hydrolysis and by the SecD and SecF proteins . In the presence of associated preprotein, the energy of ATP binding at nucleotide-binding domain 1 (NBD1) drives membrane insertion of a 30 kDa domain of SecA, while deinsertion of SecA requires the hydrolysis of this ATP . SecD and SecF stabilize the inserted state of SecA . ATP binding at NBD2, though needed for preprotein translocation, is not needed for SecA insertion or deinsertion.

J Biol Chem, 1995 Dec 29, 270(52), 31397 - 404
An interaction between the Escherichia coli RecF and RecR proteins dependent on ATP and double-stranded DNA; Webb BL et al.; The DNA binding and ATPase activities of RecF protein are modulated by RecR protein . Stoichiometric amounts of RecF protein bind to double-stranded (ds) DNA (about 1 RecF monomer/4-6 base pairs) in the presence of adenosine 5'-O-(3-thio)triphosphate (ATP gamma S), forming a homogeneous protein coating on the DNA . Little or no cooperativity is evident in the binding process . In the presence of ATP, RecF binding to dsDNA is much weaker, and no RecF protein coating forms . Instead, small numbers of RecF protomers are interspersed randomly along the DNA . RecR protein does not bind appreciably to the dsDNA under these same conditions . However, a protein coating, similar to that which was observed with RecF protein alone in the presence of ATP gamma S, was produced when both RecF and RecR proteins were incubated with dsDNA in the presence of ATP . An interaction between RecF and RecR enables both proteins to bind tightly to the dsDNA in an approximately 1:1 molar ratio . We also report a weak ATP hydrolytic activity of RecF which is stimulated by RecR.

J Biol Chem, 1995 Dec 29, 270(52), 31345 - 52
Cloning and expression of a cDNA encoding the beta-subunit (30-kDa subunit) of bovine brain platelet-activating factor acetylhydrolase; Hattori M et al.; Bovine brain platelet-activating factor (PAF) acetylhydrolase isoform Ib is a heterotrimeric enzyme . Its gamma-subunit (which, formerly, we called the 29-kDa subunit) acts as a catalytic subunit, whereas the alpha-subunit (45 kDa) is the bovine homolog of the product of human LIS-1, the causative gene of Miller-Dieker lissencephaly, indicating that this intracellular PAF acetylhydrolase plays a key role in brain development . In the current study, we cloned the cDNA for the beta-subunit (30 kDa) of bovine brain PAF acetylhydrolase Ib . The predicted 229-amino acid sequence was homologous (63.2% identity) to that of the gamma-subunit, especially (86% identity) in the catalytic and PAF receptor homologous domains . The recombinant beta-protein produced in Escherichia coli showed significant PAF acetylhydrolase activity . A mutant protein, in which Ser48, which corresponds to the active serine residue of the gamma-subunit, was replaced with cysteine showed no enzymatic activity, suggesting Ser48 is the active serine residue . Although the beta- and gamma-subunits form a heterocomplex in the native enzyme, both recombinant beta- and gamma-proteins exist as a homodimer . The purified recombinant beta-protein was labeled readily with {1,3-H}diisopropyl fluorophosphate, whereas the beta-subunit in the native complex was only labeled with higher concentrations of {1,3-3H}diisopropyl fluorophosphate to a lesser extent than the gamma-subunit . Combined with our previous data, the present study demonstrated that bovine brain PAF acetylhydrolase Ib is a unique enzyme possessing two catalytic subunits and another, possibly regulatory, subunit.

J Biol Chem, 1995 Dec 29, 270(52), 31303 - 9
Presence of N-unsubstituted glucosamine units in native heparan sulfate revealed by a monoclonal antibody; van den Born J et al.; Immunohistochemical application of antibodies against heparan sulfate proteoglycan core protein and heparitinase-digested heparan sulfate stubs showed the presence of heparan sulfate proteoglycan in all basement membranes of the rat kidney . However, a monoclonal antibody (JM-403) against native heparan sulfate (van den Born, J., van den Heuvel, L . P . W . J., Bakker, M . A . H., Veerkamp, J . H., Assmann, K . J . M., and Berden, J . H . M . (1992) Kidney Int . 41, 115-123) largely failed to stain tubular basement membranes, suggesting the presence of heparan sulfate chains lacking the specific JM-403 epitope . Heparan sulfate preparations from various sources differed markedly with regard to JM-403 binding, as demonstrated by liquid phase inhibition in enzyme-linked immunosorbent assay, the interaction decreasing with increasing sulfate contents of the polysaccharide . Mapping of the JM-403 epitope indicated that it was dominated by one or more N-unsubstituted glucosamine unit(s), since treatments that destroyed or altered the structure of such units in heparan sulfate preparations (cleavage at N-unsubstituted glucosamine units with HNO2 at pH 3.9 and N-acetylation with acetic anhydride, respectively), abolished antibody binding . Conversely, immunoreactivity could be induced in a (D-glucuronyl-1,4-N-acetyl-D-glucosaminyl-1,4) polysaccharide by the generation of N-unsubstituted glucosamine N-unsubstituted glucosamine in a JM-403-binding heparan sulfate (preparation HS-II from human aorta) was demonstrated by an approximately 3-fold reduction in molecular size following HNO2 (pH 3.9) treatment . Further characterization of the epitope recognized by JM-403, based on enzyme-linked immunosorbent assay inhibition tests with chemically/enzymatically modified polysaccharides, indicated that one or more N-sulfated glucosamine units are invariable present, whereas L-iduronic acid and O-sulfate residues appear to inhibit JM-403 reactivity . It is concluded that the epitope contains one or more N-unsubstituted glucosamine and D-glucuronic acid units and is located in a region of the heparan sulfate chain composed of mixed N-sulfated and N-acetylated disaccharide units.

J Biol Chem, 1995 Dec 29, 270(52), 31262 - 8
Molecular characterization of Golgin-245, a novel Golgi complex protein containing a granin signature; Fritzler MJ et al.; The serum from a Sjogren's syndrome patient with anti-Golgi antibodies was used as a probe to isolate a 4.6-kilobase pair cDNA insert from a HeLa cDNA library . Expression of the cDNA in Escherichia coli and the in vitro translation products of the cDNA yielded a recombinant protein that migrated in SDS-polyacrylamide gel electrophoresis at 180 kDa . This protein was immuno-precipitated by the human anti-Golgi serum and by immune rabbit serum but not by normal human serum or preimmune rabbit serum . Western blot analysis showed that the prototype human and immune rabbit sera recognized a 245-kDa protein, suggesting that the isolated clone contained a partial cDNA . The 5'-upstream sequence obtained by the rapid amplification of cDNA ends methodology using human placental cDNA and the combined HeLa cDNA contained 6965 base pairs and combined HeLa cDNA contained 6965 base pairs and encoded a protein of 245 kDa and, like other Golgi autoantigens described earlier, is highly rich in coiled-coils . The deduced amino acid sequence included the decapeptide ESLALEELEL, which was identified as one of two signature sequences previously reported in a family of peptide hormones and neuropeptides known as "granins" . This is the first report of a Golgi complex autoantigen that bears structural similarities to the granin family of proteins.

J Biol Chem, 1995 Dec 29, 270(52), 31083 - 90
A clathrin-binding site in the hinge of the beta 2 chain of mammalian AP-2 complexes; Shih W et al.; The assembly of cytosolic clathrin into the cytoplasmic face of coated pits and coated vesicles appears to be driven by the clathrin-associated protein (AP) complexes . We have previously shown that one of the large chains of the AP complexes, the beta chain, is sufficient to drive coat assembly in vitro . This chain consists of two domains, the amino-terminal trunk and the carboxyl-terminal ear, linked by a "hinge." We report here that presence of the hinge in recombinant beta trunk or in recombinant beta ear fragments is essential for driving in vitro assembly of clathrin into coats . We have also used a binding assay to map the clathrin-binding site by nested deletion of hinge sequences to a 50-residue region in the center of the hinge . This sequence is conserved in all known beta sequences from multicellular organisms . The interaction of a single beta hinge with a clathrin triskelion is weak, and we propose that recruitment of cytosolic clathrin to a forming coated pit involves simultaneous contacts between the legs of single clathrin trimers and the beta hinges of two or three membrane-bound AP complexes . Uncoating is likely to require interruption of these contacts.

J Biol Chem, 1995 Dec 29, 270(52), 31071 - 6
Roles of amino acid residues surrounding phosphorylation site 1 of branched-chain alpha-ketoacid dehydrogenase (BCKDH) in catalysis and phosphorylation site recognition by BCKDH kinase; Hawes JW et al.; Branched-chain alpha-ketoacid dehydrogenase is regulated by reversible phosphorylation of serine 293 (site 1) on the E1 alpha subunit . Alanine-scanning mutagenesis was used to examine the roles of residues surrounding serine 293 in catalysis by the dehydrogenase and in substrate recognition by branched-chain alpha-ketoacid dehydrogenase kinase . Alanine substitution of serine 293 resulted in a 10-fold increased Km for alpha-ketoisovalerate, a less increased (2.8-fold) Km for alpha-ketoisocaproate, but no change in Vmax or the Km for thiamine pyrophosphate . Alanine substitutions of arginine 288, histidine 292, and aspartate 296, residues highly conserved among alpha-ketoacid dehydrogenases, resulted in inactive enzymes . Each of the inactive E1 mutants bound to the E2 core subunit with equal affinity as wild-type E1, and each produced circular dichroism spectra identical to that of wild-type E1 . Two mutations, H292A and S293E, abolished the ability of E1 apoenzyme to reconstitute with thiamine pyrophosphate . Each alanine-substituted E1 was phosphorylated at site 1 by branched-chain alpha-ketoacid dehydrogenase kinase with similar rates, with the exception of the R288A mutant, which displayed no detectable phosphorylation . Thiamine pyrophosphate inhibited the phosphorylation of all mutant enzymes with the exception of H292A, the mutant E1 that did not bind thiamine pyrophosphate.

J Biol Chem, 1995 Dec 29, 270(52), 30941 - 8
Mutations in the Escherichia coli Tus protein define a domain positioned close to the DNA in the Tus-Ter complex; Skokotas A et al.; A new genetic screen for mutations in the tus gene of Escherichia coli has been devised that selects for Tus proteins with altered ability to arrest DNA replication . We report here the characterization of three such mutants: TusP42S, TusE49K, and TusH50Y . TusP42S and TusE49K arrest DNA replication in vivo at 36% of the efficiency of wild-type Tus, whereas TusH50Y functions at 78% efficiency . The loss of replication arrest activity did not correlate with changes in the stability of the Tus-TerB complexes formed by the mutant proteins . TusE49K formed a more stable protein-DNA complex than wild-type Tus (t1/2 of 178 versus 149 min, respectively) and TusP42 had a 9-min half-life, yet these two mutants showed identical efficiencies for replication arrest . When tested in vitro using a helicase assay or an oriC replication system, we observed a general, but imperfect, correlation between the in vivo and in vitro assays . Finally, the half-lives of the mutant protein-DNA complexes suggested that the domain of Tus where these mutations are located is positioned close to the DNA in the Tus-Ter complex.

J Biol Chem, 1995 Dec 29, 270(52), 30927 - 32
The central aromatic residue in loop L2 of RecA interacts with DNA . Quenching of the fluorescence of a tryptophan reporter inserted in L2 upon binding to DNA; Maraboeuf F et al.; To determine the role of the central aromatic residue in one of the DNA binding domains in Escherichia coli RecA protein, we have constructed a protein in which a tryptophan fluorescence reporter is inserted in the place of phenylalanine residue 203 in loop L2, a putative DNA binding site, and measured its fluorescence . The modified protein is active both in vivo and in vitro . The binding of nucleotide cofactor (ATP or its analog adenosine 5'-O-3-thiotriphosphate) does not modify the fluorescence . By contrast, the binding of DNA, both in the absence and presence of cofactor, strongly decreases the fluorescence in intensity (40-65%) and shifts the emission peak from 344 to 337 nm . The change occurs both with single- and double-stranded DNA and also upon the binding of a second single-stranded DNA . The results indicate that the residue 203 is in fact close to the first and second DNA binding sites . However, the quenching is not total and depends only slightly on the nature of DNA bases, thus suggesting an indirect interaction with DNA bases.

J Biol Chem, 1995 Dec 29, 270(52), 30862 - 8
Stepwise movement of preproteins in the process of translocation across the cytoplasmic membrane of Escherichia coli; Uchida K et al.; Derivatives of proOmpA possessing the second cysteine residue at position +302 and the first one at different positions were constructed at the DNA level . They were oxidized to form disulfide-bridged loops of different sizes at different positions . In the presence of a protonmotive force, proOmpAs possessing a smaller loop could be translocated across the membrane in vitro, whereas ones possessing loops comprising more than 16 amino acid residues were hard to translocate . The sizes of polypeptide chains that had been translocated and had become protease-resistant were determined in both the presence and absence of the protonmotive force . The size was the same for all proOmpAs possessing the first cysteine residue between +244 (proOmpA L59) and +274 (proOmpA L29) . When the first cysteine residue was moved further away from the N terminus, a sudden increase in size, of approximately 30 amino acid residues, was observed, the size being the same for proOmpAs possessing the first cysteine residue between +278 (proOmpA L25) and +293 (proOmpA L10) . The shift in size between proOmpA L29 and proOmpA L25 was observed with different proteases exhibiting different substrate specificities . Treatment with these proteases resulted in complete digestion of SecA on everted membrane vesicles, whereas Sec proteins integrated into membranes were considerably resistant to the treatment . These results can be best interpreted as that the translocation of preproteins through the secretory machinery takes place in every 30 amino acid residues and that SecA is responsible for the stepwise movement.

FEBS Lett, 1995 Dec 27, 377(3), 481 - 4
Differential effects of molecular chaperones on refolding of homologous proteins; Widmann M et al.; Three homologous aspartate aminotransferases with virtually identical spatial structures and pairwise amino acid sequence identities of > 40% differ markedly with respect to the yield of renaturation upon dilution from 6 M guanidine hydrochloride (mitochondrial << cytosolic < Escherichia coli) . The enzymes also respond differently to molecular chaperones . GroEL/GroES, the Hsp60 homolog of E . coli, increased considerably the yield of renaturation of mitochondrial aspartate aminotransferase and to a lesser extent that of its cytosolic counterpart, but not that of the E . coli enzyme . DnaK/DnaJ/GrpE, the Hsp70 system of E . coli, also increased the yield of renaturation of mitochondrial aspartate aminotransferase . Apparently, specific features in the amino acid sequence or the folding pathway which are independent of the final secondary and tertiary structure determine the interactions of the folding proteins with the chaperone systems.

FEBS Lett, 1995 Dec 27, 377(3), 475 - 80
A new family of lipolytic plant enzymes with members in rice, arabidopsis and maize; Brick DJ et al.; We have noted a striking similarity between the sequences of proteins in a novel family of lipases we recently reported {Upton, C . and Buckley, J . T . (1995) Trends Biol . Sci . 20, 178-9} and more than 120 sequences from the database of Expressed Sequence Tags (dbEST) which correspond to at least 30 unique genes from arabidopsis, rice and maize . A cDNA (Arab-1) corresponding to one of these sequences was isolated, sequenced and translated . There was significant similarity to sequences in the new lipase family over the entire open reading frame of Arab-1 and when expressed in E . coli, the gene product was lipolytic . Arab-1 and genes for some of the other plant proteins appear to be differentially expressed . They may play a role in the regulation of lipid metabolism during plant development.

FEBS Lett, 1995 Dec 27, 377(3), 461 - 4
Induction of inducible nitric oxide synthase expression by neopterin in vascular smooth muscle cells; Schobersberger W et al.; The pteridine compounds neopterin and 7,8-dihydroneopterin serve as valuable indicators for the stimulation of the cellular immune system . Whether they exhibit distinct biochemical functions in the immunological process is at present under discussion . We show that neopterin, but not 7,8-dihydroneopterin, is a stimulus for iNOS gene expression in rat vascular smooth muscle cells in vitro . At a concentration of 20 microM, neopterin leads to an iNOS mRNA expression of 2.5 amol iNOS cDNA/micrograms total RNA . When cells were coincubated with 20 microM neopterin and 5 micrograms/ml lipopolysaccharide derived from Escherichia coli, at least an additive effect on iNOS mRNA expression could be detected (iNOS cDNA concentration was 5.0 amol/micrograms total RNA) . We speculate that neopterin enhances the macrophage-induced extracellular toxicity . This might be of relevance in situations associated with excessive release of cytokines, neopterin, and nitric oxide, as observed in septic shock.

FEBS Lett, 1995 Dec 27, 377(3), 421 - 5
5'-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase . Studies using bacterially expressed human protein phosphatase-2C alpha and native bovine protein phosphatase-2AC; Davies SP et al.; Human protein phosphatase-2C alpha (PP2C alpha) was purified to homogeneity after expression in Escherichia coli . AMP inhibited the dephosphorylation of AMP-activated protein kinase (AMPK), but not phosphocasein, by PP2C alpha . The concentration dependence and the effects of other nucleotides (ATP and formycin A-5'-monophosphate) suggest that AMP acts by binding to the same site which causes direct allosteric activation of AMPK . A similar, although less pronounced, effect was observed with another protein phosphatase (PP2AC) . We have now shown that AMPK activates the AMPK cascade by four mechanisms, which should make the system exquisitely sensitive to changes in AMP concentration.

FEBS Lett, 1995 Dec 27, 377(3), 295 - 300
Protein phosphatase 1 interacts with p53BP2, a protein which binds to the tumour suppressor p53; Helps NR et al.; The p53 binding protein, termed p53BP2, was identified as a protein interacting with protein phosphatase 1 (PP1) in the yeast two hybrid system . The interaction was confirmed by co-immunoprecipitation of p53BP2 with epitope-tagged PP1 in vitro . The p53BP2-PP1 complex was stable to NaCl at concentrations which dissociate the p53-p53BP2 complex, and the binding of PP1 and p53 to p53BP2 was mutually exclusive . The region required for interaction with PP1 was shown to be contained within amino acids 297-431 of p53BP2, which includes two ankyrin repeats . The phosphorylase phosphatase activity of PP1 was inhibited by p53BP2 at nanomolar concentrations . These results suggest that PP1 may be involved in dephosphorylation and regulation of p53 through interaction with p53BP2.

Biochim Biophys Acta, 1995 Dec 27, 1264(3), 388 - 96
Characterization of human E4BP4, a phosphorylated bZIP factor; Chen WJ et al.; In this report we described the isolation of transcription factor E4BP4 by lambda gt11 expression cloning using a probe containing the CRE/ATF-like sequence located between -2764 bp and -2753 bp in the upstream regulatory region for the human IL-1 beta gene . DNaseI protection, gel mobility shift analysis, and cotransfection studies were performed to investigate the binding and functional properties of E4BP4 using IL-1 beta promoter sequences . By DNaseI footprinting, a protection pattern was generated over the CRE/ATF-like site and the flanking sequences by bacterially produced E4BP4 . Competition experiment by gel shift assay indicated that E4BP4 bound specifically to CRE/ATF-like site, not NF kappa B-like site . In cotransfection studies, E4BP4 repressed promoter activity and this repression was mediated through the CRE/ATF-like site . Mutational analysis of E4BP4 suggested that the DNA binding as well as repression activities required leucine heptad repeat domain . Analysis of E4BP4 produced in Escherichia coli and Sf9 cells infected with recombinant baculovirus indicated that baculovirus produced protein showed enhanced binding to the CRE/ATF-like site compared to the E . coli-produced protein . Analysis of posttranslational modifications indicated that E4BP4 produced in Sf9 cells was phosphorylated and this phosphorylation was important for the DNA binding activity of E4BP4.

Biochim Biophys Acta, 1995 Dec 27, 1264(3), 347 - 56
Cloning, sequence analysis and expression of mammalian mitochondrial protein synthesis elongation factor Tu; Woriax VL et al.; The bovine liver mitochondrial protein synthesis elongation factor Tu.Ts complex (EF-TU.Tsmt) has been purified and partial peptide sequence information has been obtained for EF-Tumt . A complete cDNA has been obtained encoding bovine EF-Tumt and a nearly complete cDNA has been obtained for human EF-Tumt . The bovine cDNA has a 5' untranslated leader, an open reading frame of 1356 nucleotides and a 3' untranslated region of 189 base pairs . NH2-terminal sequencing of the mature protein indicates that the transit peptide for the mitochondrial localization of this protein is 43 amino acids in length . The human and bovine factors are 95% identical . The deduced protein sequences show considerable identity to bacterial and organellar EF-Tu sequences . At least two genes for EF-Tumt are present in the bovine system . Northern analysis indicates that EF-Tumt is synthesized in all tissues but that the level of expression varies over a wide range . EF-TUmt has been expressed in E . coli as a His-tagged protein and purified to near homogeneity . The expressed form of the factor is active in the poly(U)-directed polymerization of phenylalanine although it is less active than the native EF-Tu.Tsmt complex.

Biochim Biophys Acta, 1995 Dec 27, 1264(3), 330 - 6
Characterization of the binding of HU and IHF, homologous histone-like proteins of Escherichia coli, to curved and uncurved DNA; Shimizu M et al.; The binding of E . coli histone-like protein HU to curved and uncurved DNA fragments containing adenine tracts was characterized by relative binding affinity assay, and compared with that of other homologous histone-like protein integration host factor (IHF) . Both HU and IHF have about 3- to 5-fold higher affinity for overall curved DNA fragments such as (A6N4)11 and (A3T3N4)12 compared to a standard duplex fragment with mixed sequence . The binding manner of HU to the curved fragments was highly cooperative . However, loss of overall curvature for shorter fragments (< approximately 100 bp) reduced the preference of HU binding to curved (A3T3N4)n over uncurved (T3A3N4)n, indicating that the binding specificity of HU to curved DNA is length-dependent . Thus, the curved DNA configuration of the whole molecule facilitates the binding of several HU molecules to form the hierarchy of HU-DNA complex . Furthermore, it was shown that HU and IHF bind less well to (A6N9)n, which has a zig-zag straight structure, whereas they preferentially bind to uncurved (T3A3N4)14 . These results suggested that not only intrinsically overall curvature but also the preferred orientations for DNA bending in the protein-DNA complex are important factors for affinities of HU and IHF.

Biochim Biophys Acta, 1995 Dec 27, 1264(3), 323 - 9
Formation of a free radical of the sulfenylimine type in the mouse ribonucleotide reductase reaction with 2'-azido-2'-deoxycytidine 5'-diphosphate; Behravan G et al.; Mouse and Escherichia coli ribonucleotide reductases (RR) both belong to the same class of RR, where the enzyme consists of two non-identical subunits, proteins R1 and R2 . A transient free radical was observed by EPR spectroscopy in the mouse RR reaction with the suicidal inhibitor 2'-azido-2'-deoxycytidine 5'-diphosphate . The detailed hyperfine structure of the EPR spectrum of the transient radical is somewhat different for the mouse and previously studied E . coli enzymes . When the positive allosteric effector ATP was replaced by the negative effector dATP, no transient radical was observed, showing that 'normal' binding of the inhibitor to the substrate binding site is required . Using the mouse protein R2 mutants W103Y and D266A, where the mutations have been shown to specifically block long range electron transfer between the active site of the R1 protein to the iron/radical site in protein R2, no evidence of transient radical was found . Taken together, the data suggest that the radical is located at the active site in protein R1, and is probably of the sulfenylimine type.

Biochim Biophys Acta, 1995 Dec 27, 1264(3), 303 - 11
Expression of recombinant elongation factor 1 beta from rabbit in Escherichia coli . Phosphorylation by casein kinase II; Chen CJ et al.; The beta subunit of eukaryotic elongation factor 1 (EF-1) catalyzes the GDP/GTP exchange activity on EF-1 alpha . In these studies, two cDNAs for the beta subunit of EF-1 from rabbit are cloned and sequenced . The cDNAs consist of 808 and 798 bp and are identical except for the 5' leader sequences of 67 and 57 bp . Both cDNAs code for a protein of 225 amino acids . Using the pT7-7 expression vector, EF-1 beta was expressed in Escherichia coli and purified to apparent homogeneity by chromatography on DEAE-cellulose and FPLC on Superose 12 and Mono Q . EF-1 beta was highly phosphorylated by casein kinase II, with up to 1.3 mol of phosphate incorporated per mol protein . From microsequence analysis and manual Edman degradation, the majority of the phosphate was shown to be present in serine 106 in the peptide DLFGS106DDEEES112EEA . Serine 112 was also phosphorylated by casein kinase II, but to a lesser extent . Previously, little phosphorylation of the beta subunit by casein kinase II was observed in native EF-1 unless GDP was bound to the alpha subunit (Palen, E., Venema, R.C., Chang, Y-W.E . and Traugh, J.A . (1994) Biochemistry, 8515-8520) . In contrast, purified recombinant EF-1 beta was highly and specifically phosphorylated by casein kinase II; GDP and polylysine had little effect on the rate of phosphorylation of the purified subunit.

Biochem Biophys Res Commun, 1995 Dec 26, 217(3), 940 - 9
Sequence analysis of one major basic beta-crystallin (beta Bp) of amphibian lenses: evolutionary comparison and phylogenetic relatedness between beta- and gamma-crystallins; Pan FM et al.; beta Bp-Crystallin, a major basic beta-crystallin of vertebrate eye lens, is developmentally regulated during the process of amphibian metamorphosis . In order to facilitate the determination of the primary sequence of this ubiquitous crystallin present in all vertebrate species, cDNA mixture was synthesized from the poly(A)+ mRNA isolated from bullfrog eye lenses . A protocol of Rapid Amplification of cDNA Ends (RACE) was used to amplify cDNAs encoding beta Bp-crystallin by polymerase chain reaction (PCR) . PCR-amplified product corresponding to beta Bp-crystallin was then ligated into pGEM-T vector and then transformed into E . coli strain JM109 . One complete full-length reading frame of 615 base pairs, which covers a deduced protein sequence of 205 amino acids, including the universal initiating methionine, was revealed by automatic nucleotide sequencing with a fluorescence-based dideoxynucleotide chain-termination method . It shows 83, 74, 78 and 80 percent sequence similarity to the homologous beta 2 crystallins of chicken, rat, bovine, and human species, respectively, revealing the close structural relationship among beta Bp-crystallins even from remotely related species . In this study phylogenetic trees based on nucleotide and protein sequences of various beta- and gamma-crystallins from different vertebrate classes are constructed using a combination of distance matrix and approximate parsimony methods, which corroborate the previous supposition that beta- and gamma-crystallins form a superfamily with a common ancestry.

Biochem Biophys Res Commun, 1995 Dec 26, 217(3), 1223 - 30
Identification and localization of gene expression of a low M(r) GTP-binding protein, ram p25 in pituitary gland; Nagata K et al.; The localization of gene expression of a low M(r) GTP-binding protein, ram p25, was examined throughout the entire brain of adult rat by in situ hybridization . ram-mRNA was expressed predominantly in anterior and intermediate lobes of pituitary gland (hypophysis), and weakly in hippocampal formation . In other parts of brain, no significant expression of ram-mRNA was detected, indicating that ram p25 may have an important role(s) in adenohypophysis . In 15-day-fetal rat, the mRNA was observed to be expressed strongly in the primitive anterior lobe of pituitary gland . The ram p25 was also detected in bovine pituitary gland by a specific antibody and was purified to near homogeneity by stepwise column chromatographies . Biochemical characterization of the purified ram p25 revealed that the profile of {35S}GTP gamma S-binding activity was almost the same as that of recombinant ram p25 expressed in E . coli.

Biochem Biophys Res Commun, 1995 Dec 26, 217(3), 1177 - 84
P-azidoiodoacetanilide, a new short photocrosslinker that has greater cysteine specificity than p-azidophenacyl bromide and p-azidobromoacetanilide; Zhang J et al.; An important criterion for a protein modifying agent is its residue selectivity . We report the synthesis of a new photocrosslinking agent, p-azidoiodoacetanilide (AIA), which has a greater specificity to modify cysteine residues than the widely used p-azidophenacyl bromide (APB) . Crosslinking of UmuD protein, which only has one cysteine, in the homodimer using APB or AIA resulted in 39% and 30% crosslinking, respectively; however, crosslinking of UmuD/C24A, a derivative with no cysteines, resulted in 16% crosslinked dimer using APB but only 2% using AIA . In addition, incorporation of {2-14C}APB into UmuD/C24A was 43% the amount of incorporation into wildtype UmuD, whereas incorporation of {2-14C}AIA into UmuD/C24A was only 13% the amount incorporated into wildtype UmuD . We also examined the cysteine specificity of p-azidobromoacetanilide (ABA) and found it to be less cysteine specific than AIA.

Biochemistry, 1995 Dec 26, 34(51), 16852 - 9
Kinetic mechanism of chloramphenicol acetyltransferase: the role of ternary complex interconversion in rate determination; Ellis J et al.; Chloramphenicol acetyltransferase (CAT) catalyzes the acetyl-CoA-dependent acetylation of chloramphenicol (Cm) by a ternary complex mechanism and with a random order of addition of substrates . A closer examination of the mechanism of the reaction catalyzed by the type III CAT variant (CATIII) has included the measurement of the individual rate constants by stopped-flow fluorimetry at 5 degrees C . Under all conditions employed, product release from the binary complexes in both forward and reverse reactions was found to be too slow to account for the observed overall rate of turnover for the reaction . Additional, faster routes for product release are achieved via the formation of the nonproductive ternary complexes (CAT:3-acetyl-Cm:acetyl-CoA and CAT:CoA:Cm) . The release of 3-acetyl-Cm from the binary complex is 5-fold slower than kcat (135 s-1 at 5 degrees C), whereas the dissociation rate constants of 3-acetyl-Cm from the ternary complexes with CoA and acetyl-CoA are 120 and 200 s-1, respectively . Arrhenius plots of dissociation rate constants indicate a slow release of products over a broad temperature range . Computer simulations based on the rate constants of CATIII applied to a ternary complex mechanism, assuming random order of substrate addition and product release, yielded nonlinear initial rates of product formation unless both nonproductive ternary complexes were included in the model . Simulated steady-state kinetic analyses based on the latter assumption yielded kinetic parameters that compared favorably with those determined experimentally.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Dec 26, 34(51), 16770 - 80
Characterization of the active site cysteine residues of the thioredoxin-like domains of protein disulfide isomerase; Darby NJ et al.; The dithiol/disulfide active sites of each of the two isolated thioredoxin-like domains of protein disulfide isomerase (PDI) expressed in Escherichia coli have been characterized in order to understand their catalytic mechanisms and their functions in PDI . In each of the folded domains, as in other proteins of the thioredoxin family, only one of the cysteine residues of the active site sequence -Cys-Gly-His-Cys- is accessible, and its thiol group is highly reactive and has a low pKa value . The kinetics and equilibria have been measured of the reactions between the active site cysteine residues and glutathione, the predominant thiol/disulfide reagent of the endoplasmic reticulum . A disulfide bond can be formed very rapidly between the pair of cysteine residues of each domain, but each disulfide bond is very unstable and reacts rapidly with reduced glutathione . The very low stabilities of these disulfide bonds, which destabilize the protein structures, account for the efficiency with which PDI and each of the isolated domains can introduce disulfide bonds into proteins . These kinetics and equilibrium data go far in helping to understand the catalytic mechanism of PDI and its individual domains.

Biochemistry, 1995 Dec 26, 34(51), 16708 - 14
Magnesium acetate induces a conformational change in Escherichia coli primase; Urlacher TM et al.; Primase from Escherichia coli is a single-stranded DNA-dependent RNA polymerase . As such, it requires magnesium to carry out catalysis . Limited tryptic digestion was used to probe the conformations of primase as a function of magnesium acetate concentration . In the absence of magnesium, trypsin cleaved primase at three sites . Magnesium acetate induced a conformational change such that one of these sites became inaccessible to trypsin digestion and a new site became trypsin accessible . The conformational change was only induced by Mg(OAc)2 and not MnCl2, CaCl2, NaOAc or LiCl, indicating a clear magnesium acetate-dependent conformational change . The effect was slightly induced by MgSO4 and MgCl2 . An allosteric binding model indicates that primase binds at least two magnesiums in a cooperative manner . The data were best fit to a two-state model in which one conformation had a high affinity for magnesium, KR = 83.4 M-1, and the other state had virtually no affinity.

Biochemistry, 1995 Dec 26, 34(51), 16695 - 702
Effect of metal-ligand mutations on phosphoryl transfer reactions catalyzed by Escherichia coli glutamine synthetase; Abell LM et al.; Glutamine synthetase (GS) converts glutamate to glutamine in the presence of ATP and ammonia and requires two divalent metal ions, designated n1 and n2, for catalysis . The first intermediate, gamma-glutamyl phosphate, is formed during catalysis by the transfer of the gamma-phosphate of ATP to the gamma-carboxylate of glutamate . Efficient phosphoryl transfer between these two negatively charged moieties is thought to be mediated by the n2 metal . To explore the role of the n2 metal in catalysis, histidine 269, a ligand to the n2 metal, was changed to aspartate, asparagine, glutamate, and glutamine by site-directed mutagenesis . All of the mutants bind two manganese ions as determined by EPR titration . The mutations had little effect on the substrate Km's except in the case of H269E which exhibited a Km Glu = 92 mM, a 1000-fold increase compared to that for WT (Km Glu = 70 microM) . The ability of these mutants to catalyze phosphoryl transfer to glutamate or to the inhibitor phosphinothricin was examined by rapid quench kinetic experiments . Phosphorylated phosphinothricin was detected by 31P NMR and shown to be produced by both mutants and WT . The rate of phosphoryl transfer to PPT for H269E is reduced 100-fold (0.030 s-1) compared to WT (8 s-1) . The extra negative charge around the n2 metal ion contributed by glutamate 269 severely reduces the ability of the n2 metal to mediate efficient glutamate binding in the presence of negatively charged ATP and weakens the interactions between metal ion and the reactants in the transition state, thus resulting in a lower rate of phosphoryl transfer.

Biochemistry, 1995 Dec 26, 34(51), 16624 - 31
Evidence for distinct ligand-bound conformational states of the multifunctional Escherichia coli repressor of biotin biosynthesis; Xu Y et al.; The Escherichia coli repressor of biotin biosynthesis (BirA) is a unique transcriptional repressor which catalyzes synthesis of its own corepressor and catalyzes attachment of a cofactor to an essential metabolic enzyme . BirA both catalyzes synthesis of biotinyl-5'-AMP from the substrates ATP and biotin and transfer of the biotin moiety from the adenylate to a lysine residue of a subunit of the acetyl-CoA carboxylase . BirA-bio-5'-AMP, moreover, binds sequence specifically to the biotin operator to repress transcription of the biotin biosynthetic genes . Using a combination of kinetic measurements of binding of the two ligands, biotin and bio-5'-AMP, to BirA as well as proteolytic digestion experiments, we have found evidence for at least three discrete conformational states of BirA . Results of stopped-flow fluorescence measurements of association of both ligands with BirA indicate that the process involves initial formation of a collision complex followed by a slow conformational change . The kinetics of the conformational change are distinct for the two ligands and are the basis for the difference in the thermodynamic stabilities of the two protein-ligand complexes . Different rates of proteolytic digestion of apoBirA and complexes of BirA with the two ligands were also observed . Results of the combined approaches indicate that apoBirA, and the BirA-bio-5'-AMP and BirA-biotin complexes are conformationally distinct.

Biochemistry, 1995 Dec 26, 34(51), 16596 - 607
Structure of the A-domain of HMG1 and its interaction with DNA as studied by heteronuclear three- and four-dimensional NMR spectroscopy; Hardman CH et al.; HMG1 has two homologous, folded DNA-binding domains ("HMG boxes"), A and B, linked by a short basic region to an acidic C-terminal domain . Like the whole protein, which may perform an architectural role in chromatin, the individual boxes bind to DNA without sequence specificity, have a preference for distorted or prebent DNA, and are able to bend DNA and constrain negative superhelical turns . They show qualitatively similar properties with quantitative differences . We have previously determined the structure of the HMG box from the central B-domain (77 residues) by two-dimensional NMR spectroscopy, which showed that it contains a novel fold {Weir et al . (1993) EMBO J . 12, 1311-1319} . We have now determined the structure of the A-domain (as a Cys-->Ser mutant at position 22 to avoid oxidation, without effect on its DNA-binding properties or structure) using heteronuclear three- and four-dimensional NMR spectroscopy . The A-domain has a very similar global fold to the B-domain and the Drosophila protein HMG-D {Jones et al . (1994) Structure 2, 609-627} . There are small differences between A and B, in particular in the orientation of helix I, where the B-domain is more similar to HMG-D than it is to the A-domain; these differences may turn out to be related to the subtle differences in functional properties between the two domains {Teo et al . (1995) Eur . J . Biochem . 230, 943-950} and will be the subject of further investigation . NMR studies of the interaction of the A-domain of HMG1 with a short double-stranded oligonucleotide support the notion that the protein binds via the concave face of the L-shaped structure; extensive contacts with the DNA are made by the N-terminal extended strand, the N-terminus of helix I, and the C-terminus of helix II . These contacts are very similar to those seen in the LEF-1 and SRY-DNA complexes {Love et al . (1995) Nature 376, 791-795; Werner et al . (1995) Cell 81, 705-714}.

Biochemistry, 1995 Dec 26, 34(51), 16585 - 95
2 A resolution structure of DppA, a periplasmic dipeptide transport/chemosensory receptor; Nickitenko AV et al.; The family of about 50 periplasmic binding proteins, which exhibit diverse specificity (e.g., carbohydrates, amino acids, dipeptides, oligopeptides, oxyanions, metals, and vitamins) and range in size from 20 to 58 kDa, is a gold mine for an atomic-level investigation of structure and molecular recognition . These proteins serve as initial receptors for active transport systems or permeases . About six of these proteins, including the dipeptide-binding protein (DppA), are also primary receptors for chemotaxis . The structure of the unbound form of DppA (M(r) = 57,400) has been determined and refined to an R-factor of 0.169 to 2 A resolution . DppA consists of two distinct domains (I and II) connected by two "hinge" segments which form part of the base of the wide groove between the two domains . The relative orientation of the two domains gives the protein a pearlike shape, with domain I and domain II forming the larger and smaller apical ends, respectively . From the tip to the rounded bottom measures about 85 A, and the widest diameter is about 60 A . Domain I, which consists of two integrated subdomains, is folded from two separate polypeptide segments from the amino- and carboxyl-terminal ends . The more compact domain II is formed from the intervening segment . Comparison of the dipeptide-binding protein structure with that of the bound form of the similar oligopeptide-binding protein {Tame, J . R . H., Murshudov, G . N., Dodson, E . J., Neil, T . K., Dodson, G . G., Higgins, C . F., & Wilkinson, A . J . (1994) Science 264, 1578-1581} reveals the major features that differentiate the ligand specificity of the two proteins and describe the large hinge bending (about 55 degrees) between the two domains.

Biochemistry, 1995 Dec 26, 34(51), 16552 - 62
Folding pathway of Escherichia coli ribonuclease HI: a circular dichroism, fluorescence, and NMR study; Yamasaki K et al.; The unfolding and refolding processes of Escherichia coli ribonuclease HI at 25 degrees C, induced by concentration jumps of either guanidine hydrochloride (GuHCl) or urea, were investigated using stopped-flow circular dichroism (CD), stopped-flow fluorescence, and NMR spectroscopies . Only a single exponential process was detected for the fast time scale unfolding (rate constants from 0.014 to 0.54 s-1, depending on the final denaturant concentration) . For refolding, the far-UV CD value largely recovered within 50 ms of the stopped-flow mixing dead time (burst phase) . This phase was followed by either one or two phases, with rate constants from 0.035 to 2.45 s-1 as detected by CD and fluorescence, respectively . Although this protein has a single cis-Pro residue, a very slow phase due to proline isomerization was not observed, for either unfolding or refolding . The difference in the amplitudes of the burst phases for refolding in the far- and near-UV CD spectra revealed that an intermediate state exists, with the characteristics of a molten globule . Because the one-phased fast exponential process detected by CD corresponds to the slower of the two phases detected by fluorescence, the intermediate detected by CD might be the most stable . GuHCl denaturation experiments revealed that this intermediate cooperatively unfolds, with a transition midpoint of 1.33 +/- 0.03 M . The Gibbs free energy difference (delta G) between the intermediate and the unfolded states, under physiological conditions (25 degrees C, pH 5.5, and 0 M GuHCl), was estimated to be 20.0 +/- 2.3 kJ mol-1 . Therefore, it is reasonable to assume that the refolding intermediate, rather than the unfolded state, is the latent denatured state under physiological conditions . Approximately linear relationships between the GuHCl concentration and the logarithm of the microscopic rate constants determined by CD and fluorescence were also observed . By extrapolation to a GuHCl concentration of 0 M, activation Gibbs free energies of 98.5 +/- 1.1 kJ mol-1 for unfolding and 69.5 +/- 0.2 kJ mol-1 for refolding under physiological conditions were obtained . The hydrogen-exchange-refolding competition combined with two-dimensional NMR revealed that the amide protons of alpha-helix I are the most highly protected, suggesting that alpha-helix I is the initial site of protein folding . The CD and NMR data showed that the intermediate state has a structure similar to that of the acid-denatured molten globule.

Nucleic Acids Res, 1995 Dec 25, 23(24), 5006 - 11
RNA:DNA complex formation upon transcription of immunoglobulin switch regions: implications for the mechanism and regulation of class switch recombination; Daniels GA et al.; Central the regulation and mechanism of class switch recombination is the understanding of the relationship between transcription and DNA recombination . We demonstrated previously, using mini-chromosome substrates, that physiologically oriented transcription is required for recombination to occur between switch regions . In this report, we demonstrate the formation of an RNA:DNA complex under in vitro transcription conditions for these same and other switch DNA fragments . We find that cell-free transcription of repetitive murine switch regions (Smu, S gamma 2b and S gamma 3) leads to altered DNA mobility on agarose gels . These altered mobilities are resistant to RNase A but sensitive to RNase H . Transcription in the presence of labeled ribonucleotides demonstrates the stable physical association of the RNA with the DNA . Importantly, complex formation only occurs upon transcription in the physiologic orientation . Reaban and Griffin {1990 Nature, 348, 342-344} found an RNA:DNA hybrid structure that was limited to an atypical 143 nucleotide purine region within a 2.3 kb S alpha segment . Here we demonstrate RNA:DNA hybrid formation in more typical switch sequences (lacking the atypical 143 nucleotide purine tract) from a variety of switch regions that are only 60-70% purine on the non-template strand . These results suggest a general model involving an RNA:DNA complex as an intermediate during class switch recombination.

J Biol Chem, 1995 Dec 22, 270(51), 30804 - 12
Dissociation of hexameric Escherichia coli inorganic pyrophosphatase into trimers on His-136-->Gln or His-140-->Gln substitution and its effect on enzyme catalytic properties; Baykov AA et al.; Each of the five histidines in Escherichia coli inorganic pyrophosphatase (PPase) was replaced in turn by glutamine . Significant changes in protein structure and activity were observed in the H136Q and H140Q variants only . In contrast to wild-type PPase, which is hexameric, these variants can be dissociated into trimers by dilution, as shown by analytical ultracentrifugation and cross-linking . Mg2+ and substrate stabilize the hexameric forms of both variants . The hexameric H136Q- and H140Q-PPases have the same binding affinities for magnesium ion as wild-type, but their hydrolytic activities under optimal conditions are, respectively, 225 and 110% of wild-type PPase, and their synthetic activities, 340 and 140% . The increased activity of hexameric H136Q-PPase results from an increase in the rate constants governing most of the catalytic steps in both directions . Dissociation of the hexameric H136Q and H140Q variants into trimers does not affect the catalytic constants for PPi hydrolysis between pH 6 and 9 but drastically decreases their affinities for Mg2PPi and Mg2+ . These results prove that His-136 and His-140 are key residues in the dimer interface and show that hexamer formation improves the substrate binding characteristics of the active site.

J Biol Chem, 1995 Dec 22, 270(51), 30773 - 80
Phosphorylation of both serine residues in cardiac troponin I is required to decrease the Ca2+ affinity of cardiac troponin C; Zhang R et al.; The phosphorylation of cardiac muscle troponin I (CTnI) at two adjacent N-terminal serine residues by cAMP-dependent protein kinase (PKA) has been implicated in the inotropic response of the heart to beta-agonists . Phosphorylation of these residues has been shown to reduce the Ca2+ affinity of the single Ca(2+)-specific regulatory site of cardiac troponin C (CTnC) and to increase the rate of Ca2+ dissociation from this site (Robertson, S . P., Johnson, J . D., Holroyde, M . J., Kranias, E . G., Potter, J . D., and Solaro, R . J . (1982) J . Biol . Chem . 257, 260-263) . Recent studies (Zhang, R., Zhao, J., and Potter, J . D . (1995) Circ . Res . 76, 1028-1035) have correlated this increase in Ca2+ dissociation with a reduced Ca2+ sensitivity of force development and a faster rate of cardiac muscle relaxation in a PKA phosphorylated skinned cardiac muscle preparation . To further determine the role of the two PKA phosphorylation sites in mouse CTnI (serine 22 and 23), serine 22 or 23, or both were mutated to alanine . The wild type and the mutated CTnIs were expressed in Escherichia coli and purified . Using these mutants, it was found that serine 23 was phosphorylated more rapidly than serine 22 and that both serines are required to be phosphorylated in order to observe the characteristic reduction in the Ca2+ sensitivity of force development seen in a skinned cardiac muscle preparation . The latter result confirms that PKA phosphorylation of CTnI, and not other proteins, is responsible for this change in Ca2+ sensitivity . The results also suggest that one of the serines (23) may be constitutively phosphorylated and that serine 22 may be functionally more important.

J Biol Chem, 1995 Dec 22, 270(51), 30657 - 63
ARP is a plasma membrane-associated Ras-related GTPase with remote similarity to the family of ADP-ribosylation factors; Schurmann A et al.; The human and rat homologues of a novel Ras-related GTPase with unique structural features were cloned by polymerase chain reaction amplification and cDNA library screening . Their deduced amino acid sequences are highly homologous (97% identical amino acids; 88.3% identical nucleotides within the coding region) and comprise all six of the conserved motifs presumably involved in GTP binding . Because the sequences exhibit some similarity with members of the ADP-ribosylation factor (ARF) family (33% identity with ADP-ribosylation factor 1 (ARF1), 39% identity with ARF-like 3), the protein was designated ARP (ARF-related protein) . In contrast to all other members of the ARF family, ARP lacks the myristoylation site at position 2 and comprises an insertion of 8 amino acids in the region between PM1 and PM2 . mRNA was found in most rat tissues examined (skeletal muscle, fat, liver, kidney, spleen, testis, adrenals, ovary, thymus, intestine, and lung) . Western blot analysis with antiserum against recombinant ARP showed a 25-kDa protein in membranes from rat liver, testis, and kidney . Thus, the protein appears to be post-translationally modified for membrane anchoring . Unlike ARF, the ARP immunoreactivity was detected in plasma membranes but not in cytosol of fractionated 3T3-L1 cells . Recombinant ARP exhibited specific and saturable GTP gamma S (guanosine 5'-3-O-(thio)triphosphate) binding and, unlike ARF isotypes, GTPase activity in the absence of tissue extracts or phospholipids . Thus, the structural and functional characteristics of ARP indicate that it represents a novel subtype of GTPases, presumably exerting a unique function and possibly involved in plasma membrane-related signaling events.

J Biol Chem, 1995 Dec 22, 270(51), 30545 - 50
Site-directed mutagenic alteration of potential active-site residues of the A subunit of Escherichia coli heat-labile enterotoxin . Evidence for a catalytic role for glutamic acid 112; Cieplak W Jr et al.; Escherichia coli heat-labile enterotoxin (LT) and the related cholera toxin exert their effects on eukaryotic cells through the ADP-ribosylation of guanine nucleotide-binding proteins of the adenylate cyclase complex . The availability of the crystal structure for LT has permitted the tentative identification of residues that lie within or are vicinal to a presumptive NAD(+)-binding site and thus may play a role in substrate binding or catalysis . Using a plasmid clone encoding the A subunit of LT, we have introduced substitutions at such potential active-site residues and analyzed the enzymatic properties of the resultant mutant analogs . Enzymatic analyses, employing both transducin and agmatine as acceptor substrates, revealed that substitutions at serine 61, glutamic acid 110, and glutamic acid 112 resulted in reduction of enzyme activity to < 10% of wild-type levels . Kinetic analyses indicated that alteration of these sites affected the catalytic rate of the enzyme and had little or no effect on the binding of either NAD+ or agmatine . Of the mutant analogs analyzed, only glutamic acid 112 appeared to represent an essential catalytic residue as judged by the relative effects on kcat and kcat/Km . The results provide formal evidence that glutamic acid 112 of the A subunit of LT represents a functional homolog or analog of catalytic glutamic acid residues that have been identified in several other bacterial ADP-ribosylating toxins and that it may play an essential role in rendering NAD+ susceptible to nucleophilic attack by an incoming acceptor substrate.

J Biol Chem, 1995 Dec 22, 270(51), 30532 - 44
Spectroscopic and kinetic properties of unphosphorylated rat hepatic phenylalanine hydroxylase expressed in Escherichia coli . Comparison of resting and activated states; Kappock TJ et al.; The non-heme iron-dependent metalloenzyme, rat hepatic phenylalanine hydroxylase (EC 1.14.16.1; phenylalanine 4-monooxygenase (PAH) was overexpressed in Escherichia coli and purified to homogeneity, allowing a detailed comparison of the kinetic, hydrodynamic, and spectroscopic properties of its allosteric states . The homotetrameric recombinant enzyme, which is highly active and contains 0.7-0.8 iron atoms per subunit, is identical to the native enzyme in several properties: Km, 6-methyltetrahydropterin = 61 microM and L-Phe = 170 microM; Vmax = 9 s-1 (compared to 45 microM, 180 microM, and 13 s-1 for the rat hepatic enzyme) . L-Phe and lysolecithin treatment induce the rPAHT-->rPAHR (where r is recombinant) allosteric transformation necessary for rPAH activity . Characteristic changes in the fluorescence spectra, increased hydrophobicity, a large activation energy barrier, and a 10% volume increase of the tetrameric structure are consistent with a significant reorganization of the protein following allosteric activation . However, optical and EPR spectroscopic data suggest that only minor changes occur in the primary coordination sphere (carboxylate/histidine/water) of the catalytic iron center . Detailed steady state kinetic investigations, using 6-methyltetrahydropterin as cofactor and lysolecithin as activator, indicate rPAH follows a sequential mechanism . A catalytic Arrhenius Eact of 14.6 +/- 0.3 kcal/mol subunit was determined from temperature-dependent stopped-flow kinetics data . rPAH inactivates during L-Phe hydroxylation with a half-life of 4.3 min at 25 degrees C, corresponding to an Arrhenius Eact of 10 +/- 1 kcal/mol subunit for the inactivation process . Catechol binding (2.4 x 10(6) M-1) is shown to occur only at catalytically competent iron sites . Ferrous rPAH binds NO, giving rise to an ST = 3/2 spin system.

J Biol Chem, 1995 Dec 22, 270(51), 30508 - 15
The C-terminal region of the UvrB protein of Escherichia coli contains an important determinant for UvrC binding to the preincision complex but not the catalytic site for 3'-incision; Moolenaar GF et al.; The UvrABC endonuclease from Escherichia coli repairs damage in the DNA by dual incision of the damaged strand on both sides of the lesion . The incisions are in an ordered fashion, first on the 3'-side and next on the 5'-side of the damage, and they are the result of binding of UvrC to the UvrB-DNA preincision complex . In this paper, we show that at least the C-terminal 24 amino acids of UvrB are involved in interaction with UvrC and that this binding is important for the 3'-incision . The C-terminal region of UvrB, which shows homology with a domain of the UvrC protein, is part of a region that is predicted to be able to form a coiled-coil . We therefore propose that UvrB and UvrC interact through the formation of such a structure . The C-terminal region of UvrB only interacts with UvrC when present in the preincision complex, indicating that the conformational change in UvrB accompanying the formation of this complex exposes the UvrC binding domain . Binding of UvrC to the C-terminal region of UvrB is not important for the 5'-incision, suggesting that for this incision a different interaction of UvrC with the UvrB-DNA complex is required . Truncated UvrB mutants that lack up to 99 amino acids from the C terminus still give rise to significant incision (1-2%), indicating that this C-terminal region of UvrB does not participate in the formation of the active site for 3'-incision . This region, however, contains the residue (Glu-640) that was proposed to be involved in 3'-catalysis, since a mutation of the residue (E640A) fails to promote 3'-incision (Lin, J.J., Phillips, A.M., Hearst, J.E., and Sancar, A . (1992) J . Biol . Chem . 267, 17693-17700) . We have isolated a mutant UvrB with the same E640A substitution, but this protein shows normal UvrC binding and incision in vitro and also results in normal survival after UV irradiation in vivo . As a consequence of these results, it is still an open question as to whether the catalytic site for 3'-incision is located in UvrB, in UvrC, or is formed by both proteins.

J Biol Chem, 1995 Dec 22, 270(51), 30499 - 507
The Y-box motif mediates redox-dependent transcriptional activation in mouse cells; Duh JL et al.; We show here that the OxyR response element (ORE) in the bacterial oxyR promoter can also function as a redox-dependent enhancer in mammalian cells . Fusion of ORE to an SV40 basal promoter driving chloramphenicol acetyltransferase (CAT) expression confers H2O2 inducibility to expression of the cat gene in mouse Hepa-1 hepatoma cells . Nuclear extracts from these cells contain DNA-binding proteins that specifically interact with ORE DNA, cannot be completed by cognate oligonucleotides to AP-1 or NF kappa B, and are constitutively expressed, since treatment with H2O2 causes no detectable changes in binding activity or DNA-protein interaction . Recombinant cDNA clones that express ORE-binding proteins were isolated from a mouse hepatoma expression library and found to be representatives of two different members of the murine Y-box family of transcription factors . Canonical Y-box and ORE oligonucleotides compete with each other for binding to Y-box proteins in gel shift assays and antibodies to FRGY2, a Xenopus Y-box protein, supershift both Y-box and ORE DNA-protein complexes . In addition, antisense oligonucleotides to mouse YB-1 mRNA abolish induction of ORE-mediated cat expression by H2O2, and luciferase reporter constructs containing ORE, or the Y-box from the human MHC class II HLA-DQ gene, exhibit identical dose-dependent H2O2 inducibilities, which can be abolished by addition of 2-mercaptoethanol to the culture medium . These results suggest that the Y-box proteins may be an integral component of a eukaryotic redox signaling pathway.

J Biol Chem, 1995 Dec 22, 270(51), 30491 - 8
Processive post-translational modification . Vitamin K-dependent carboxylation of a peptide substrate; Morris DP et al.; Mass spectrometry has been used to demonstrate that vitamin K-dependent carboxylation is a processive post-translational modification (i.e . multiple carboxylations occur during a single association between enzyme and substrate) . Purified vitamin K-dependent carboxylase can carboxylate as many as 12 glutamate residues in FIXQ/S, a peptide substrate based on amino acids -18 to 41 of the human blood clotting enzyme factor IX . Mass spectrometry was used to determine the number of gamma-carboxyl groups added to FIXQ/S by the carboxylase during an in vitro reaction . Despite the fact that most substrate molecules in a reaction were uncarboxylated, almost all carboxylated FIXQ/S molecules were carboxylated many times . This observation can only be explained by two types of mechanisms . In a processive mechanism, multiple carboxylations could occur during a single substrate binding event . Alternatively, a distributive mechanism could result in the observed behavior if the initial carboxylation event results in a substrate that is additionally carboxylated far more efficiently than the uncarboxylated FIXQ/S . Kinetic experiments and arguments were used to show that the vitamin K-dependent carboxylase is not distributive but rather is one of the first well documented examples of an enzyme that catalyzes a processive post-translation modification.

J Biol Chem, 1995 Dec 22, 270(51), 30470 - 8
Purification, characterization, and molecular cloning of a novel rat liver Dopa/tyrosine sulfotransferase; Sakakibara Y et al.; A novel sulfotransferase was purified from the rat liver cytosol to electrophoretic homogeneity via five column chromatography steps (hydroxylapatite I, DEAE Bio-Gel, ATP-agarose I, hydroxylapatite II, and ATP-agarose II) . The minimum molecular weight of the purified enzyme was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis to be approximately 33,000 . Gel filtration chromatography revealed a native molecular weight of approximately 34,000, indicating the enzyme being present in the monomeric form . The purified sulfotransferase displayed enzymatic activities, with a pH optimum of 9.25, toward various tyrosine and 3,4-dihydroxyphenylalanine (Dopa) isomers, except DL-ortho-tyrosine . Thyroid hormones, as well as dopamine and p-nitrophenol, could also be used as substrates . The apparent Km value of the enzyme (designated the Dopa/tyrosine sulfotransferase) for L-Dopa, determined at a constant 14 microM of 3'-phosphoadenosine 5'-phosphosulfate, was 0.76 mM . The intact enzyme was found to be N-blocked when subjected to N-terminal sequencing . Three internal partial amino acid sequences, obtained by analyzing its proteolytic fragments, were found to be distinct from the homologous sequences of other known rat liver sulfotransferases . The deduced amino acid sequence of a full-length cDNA isolated from a rat liver cDNA library confirmed the identity of the Dopa/tyrosine sulfotransferase as a new type of aryl sulfotransferase . Upon transfection of COS-7 cells with an expression vector (pMSG-CMV) harboring the full-length cDNA, a 33-kDa protein displaying enzymatic and immunological properties similar to those of the purified Dopa/tyrosine sulfotransferase was expressed.

J Biol Chem, 1995 Dec 22, 270(51), 30458 - 63
Identification of amino acid residues critical for catalysis and cosubstrate binding in the flavonol 3-sulfotransferase; Marsolais F et al.; The comparison of the deduced amino acid sequences of plant and animal sulfotransferases (ST) has allowed the identification of four well conserved regions, and previous experimental evidence suggested that regions I and IV might be involved in the binding of the cosubstrate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS) . Moreover, region IV is homologous to the glycine-rich phosphate binding loop (P-loop) motif known to be involved in nucleotide phosphate binding in several protein families . In this study, the function of amino acid residues within these two regions was investigated by site-directed mutagenesis of the plant flavonol 3-ST . In region I, our results identify Lys59 as critical for catalysis, since replacement of this residue with alanine resulted in a 300-fold decrease in specific activity, while a 15-fold reduction was observed after the conservative replacement with arginine . Photoaffinity labeling of K59R and K59A with {35S}PAPS revealed that Lys59 is not required for cosubstrate binding . However, the K59A mutant had a reduced affinity for 3'-phosphoadenosine 5'-phosphate (PAP)-agarose, suggesting that Lys59 may participate in the stabilization of an intermediate during the reaction . In region IV, all substitutions of Arg276 resulted in a marked decrease in specific activity . Conservative and unconservative replacements of Arg276 resulted in weak photoaffinity labeling with {35S}PAPS and the R276A/T73A and R276E enzymes displayed reduced affinities for PAP-agarose, suggesting that the Arg276 side chain is required to bind the cosubstrate . The analysis of the kinetic constants of mutant enzymes at residues Lys277, Gly281, and Lys284 allowed to confirm that region IV is involved in cosubstrate binding.

J Biol Chem, 1995 Dec 22, 270(51), 30401 - 7
A mutation in the ATP binding domain of rho alters its RNA binding properties and uncouples ATP hydrolysis from helicase activity; Pereira S et al.; The Escherichia coli mutant rho201 was originally isolated in a genetic screen for defects in rho-dependent termination . Cloning and sequencing of this gene reveals a single phenylalanine to cysteine mutation at residue 232 in the ATP binding domain of the protein . This mutation significantly alters its RNA binding properties so that it binds trp t', RNA 100-fold weaker than the wild type protein, with a Kd of approximately 1.3 nM . Rho201 binds nonspecific RNA only 3-4-fold less tightly than it binds trp t', while the wild type differential for these same RNAs is 10-20-fold . Curiously, rho201 displays increased secondary site RNA activation, with a Km for ribo(C)10 of 0.6 microM, compared to the wild type value of 3-4 microM . Although rho201 and the wild type protein hydrolyze ATP similarly with poly(C), or trp t' RNA, as cofactors, rho201 has a higher ATPase activity when activated by nonspecific RNA . Physically, rho201 displays an abnormal conformation detectable by mild trypsin digestion . Despite effective ATP hydrolysis, the rho201 mutant is a poor RNA:DNA helicase and terminates inefficiently on trp t' . The single F232C mutation thus appears to uncouple the protein's ATPase activity from its helicase function, so rho can no longer harness available energy for use in subsequent reactions.

J Biol Chem, 1995 Dec 22, 270(51), 30392 - 400
The mechanism and substrate specificity of the NADPH:flavin oxidoreductase from Escherichia coli; Fieschi F et al.; The NAD(P)H:flavin oxidoreductase from Escherichia coli, Fre, is a monomer of 26.2 kDa that catalyzes the reduction of free flavins by NADPh or NADH . Overexpression in E . coli now allows the preparation of large amounts of pure protein . Structural requirements for recognition of flavins as substrates and not as cofactors were studied by steady-state kinetics with a variety of flavin analogs . The entire isoalloxazine ring was found to be the essential part of the flavin molecule for interaction with the polypeptide chain . Methyl groups at C-7 and C-8 of the isoalloxazine ring and the N-3 of riboflavin also play an important role in that interaction, whereas the ribityl chain of the riboflavin is not required for binding to the protein . On the other hand, the presence of the 2'-OH of the ribityl chain stimulates the NADPH-dependent reaction significantly . Moreover, a study of competitive inhibitors for both substrates demonstrated that Fre follows a sequential ordered mechanism in which NADPH binds first followed by riboflavin . Lumichrome, a very good inhibitor of Fre, may be used to inhibit flavin reductase in E . coli growing cells . As a consequence, it can enhance the antiproliferative effect of hydroxyurea, a cell-specific ribonucleotide reductase inactivator.

J Biol Chem, 1995 Dec 22, 270(51), 30384 - 91
The envA permeability/cell division gene of Escherichia coli encodes the second enzyme of lipid A biosynthesis . UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase; Young K et al.; The envA gene of Escherichia coli has been shown previously to be essential for cell viability (Beall, B . and Lutkenhaus, J . (1987) J . Bacteriol . 169, 5408-5415), yet it encodes a protein of unknown function . Extracts of strains harboring the mutant envA1 allele display 3.5-18-fold reductions in UDP-3-O-acyl-N-acetylglucosamine deacetylase specific activity . The deacetylase is the second enzymatic step of lipid A biosynthesis . The structural gene coding for the deacetylase has not been assigned . In order to determine if the envA gene encodes the deacetylase, envA was cloned into an isopropyl-1-thio-beta-D-galactopyranoside-inducible T7-based expression system . Upon induction, a protein of the size of envA was highly overproduced, as judged by SDS-PAGE . Direct deacetylase assays of cell lysates revealed a concomitant approximately 5,000-fold overproduction of activity . Assays of the purified, overproduced EnvA protein demonstrated a further approximately 5-fold increase in specific activity . N-terminal amino acid sequencing of the purified protein showed that the first 20 amino acids matched the predicted envA nucleotide sequence . Contaminating species were present at less than 1% of the level of the EnvA protein . Thus, envA is the structural gene for UDP-3-O-acyl-GlcNAc deacetylase . Based on its function in lipid A biosynthesis, we propose the new designation lpxC for this gene.

J Biol Chem, 1995 Dec 22, 270(51), 30274 - 7
Cysteine-rich region of Raf-1 interacts with activator domain of post-translationally modified Ha-Ras; Hu CD et al.; The interaction between "switch I/effector domain" of Ha-Ras and the Ras-binding domain (RBD, amino acid 51-131) of Raf-1 is essential for signal transduction . However, the importance of the "activator domain" (approximately corresponding to amino acids 26-28 and 40-49) of Ha-Ras and of the "cysteine-rich region" (CRR, amino acids 152-184) of Raf-1 have also been proposed . Here, we found that Raf-1 CRR interacts directly with Ha-Ras independently of RBD and that participation of CRR is necessary for efficient Ras-Raf binding . Furthermore, Ha-Ras carrying mutations (N26G and V45E) in the activator domain failed to bind CRR, whereas they bound RBD normally . On the contrary, Ha-Ras carrying mutations in the switch I/effector domain exhibited severely reduced ability to bind RBD, whereas their ability to bind CRR was unaffected . Mutants that bound to either RBD or CRR alone failed to activate Raf-1 . Ha-Ras without post-translational modifications, which lacks the ability to activate Raf-1, selectively lost the ability to bind CRR . These results suggest that the activator domain of Ha-Ras participates in activation of Raf-1 through interaction with CRR and that post-translational modifications of Ha-Ras are required for this interaction.

J Biol Chem, 1995 Dec 22, 270(51), 30268 - 70
The C-terminal sequence of the chaperonin GroES is required for oligomerization; Seale JW et al.; The Escherichia coli protein GroES is a co-chaperonin that is able to assist GroEL in the refolding of proteins . GroES is a heptamer of seven identical subunits . Recent work has focused on the structural aspects of GroES . We have investigated the role of the C-terminal portion of GroES on its oligomerization . Limited proteolysis of GroES by carboxypeptidase Y gives a product in which the C-terminal 7 amino acid residues have been removed . Sedimentation velocity analysis reveals that the truncated form of GroES is unable to reassemble . The results presented here implicate the C-terminal sequence in intermonomer actions within the GroES oligomer . In addition, this work provides experimental verification of predictions implied in the recent x-ray determination of the GroES structure (Hunt, J . F., Weaver, A . J., Landry, S . J., Gierasch, L . M., and Deisenhofer, J . Nature, in press).

Arch Biochem Biophys, 1995 Dec 20, 324(2), 391 - 400
Epitope mapping and tight-binding inhibition with monoclonal antibodies directed against Escherichia coli glucosamine 6-phosphate synthase; Cochet O et al.; In the present work, we attempt to identify inhibitory monoclonal antibodies directed against Escherichia coli glucosamine-6P synthase (GlmS) and to localize the corresponding epitopes to better understand the topology of the enzyme during catalysis . Four of the 15 monoclonal antibodies have been shown to be specific for the native form of the enzyme and 2 of them, 505.1 and 522.2, strongly inhibit the glucosamine synthase activity . Kinetic analysis of 505.1 antibody behavior revealed a tight-binding inhibition with a Ki = 40 +/- 20 pM, a value which is four orders of magnitude lower than the best active site-directed inhibitor reported so far . The reactivity of all the monoclonal antibodies with 601 overlapping octapeptides covering the entire sequence of GlmS was tested by enzyme-linked immunosorbent assay for precise epitope mapping . Four linear epitopes specific for the denatured protein and one present in both native and denatured enzyme were defined by this approach . Neither 505.1 nor 522.2 was directed against linear epitopes . However, evidence for the binding of 505.1 at the glutamine catalytic site was shown by using site-directed mutants of GlmS as well as by competition experiments with an irreversible inhibitor . The mAb 105.1, which recognizes the octapeptide containing the sequence RWATHG conserved among the six glucosamine-6P synthases reported so far, allowed the detection of the human enzyme.

Arch Biochem Biophys, 1995 Dec 20, 324(2), 379 - 84
Cloning, sequence, and expression of mouse protoporphyrinogen oxidase; Dailey TA et al.; Protoporphyrinogen oxidase (EC 1.3.3.4) is the penultimate enzyme in the heme biosynthetic pathway, catalyzing the six-electron oxidation of protoporphyrinogen to protoporphyrin . A dominantly inherited genetic deficiency in this enzyme results in the disease variegate porphyria . We now report the cloning, sequence, and expression of mouse protoporphyrinogen oxidase . The cDNA for mouse protoporphyrinogen oxidase was obtained by complementation of Escherichia coli SASX38, a protoporphyrinogen oxidase-deficient strain, with a mouse erythroleukemia (MEL) cell expression library . The sequence of this cDNA along with 5' untranslated sequence obtained by 5' rapid amplification of cDNA ends of MEL cell mRNA is 1814 bp in length and contains an open reading frame of 1431 bp . This encodes a protein of 477 amino acid residues with a calculated molecular weight of 50,870 . The protein as expressed in E . coli is sensitive to inhibition by the diphenyl ether herbicide acifluorfen . Northern blot analyses of RNA from uninduced and induced MEL cells as well as mouse hepatoma cells all show two major mRNA species of 1.8 and 3.6 kb.

Virology, 1995 Dec 20, 214(2), 439 - 44
Avian sarcoma leukemia virus protease linked to the adjacent Gag polyprotein is enzymatically active; Arad G et al.; The activity of avian sarcoma leukemia virus (ASLV) protease (PR) prior to its release from the precursor protein was determined by introducing mutations at the cleavage site between PR and the adjacent upstream nucleocapsid (NC) protein . Gag DNA fragments containing these mutations were cloned into expression vectors and introduced into Escherichia coli in which the ASLV proteins were expressed . The dipeptide NC-PR containing these mutations did not undergo autoprocessing when expressed in bacterial cells and the fused proteins were devoid of enzymatic activity . However, when the whole Gag polyprotein containing these mutations was expressed in bacterial cells, other PR cleavage sites in the viral Gag polyprotein underwent normal cleavage, indicating that the release of free PR is not a prerequisite for correct processing of the ASLV Gag precursor.

Mol Gen Genet, 1995 Dec 20, 249(6), 591 - 9
Mutation frequency decline in Escherichia coli . II . Kinetics support the involvement of transcription-coupled excision repair; Bockrath R et al.; Mutation frequency decline (MFD) in Escherichia coli was examined to demonstrate repair of targeting photoproducts during the post-UV incubation required in this process . Repair of mutation-targeting cyclobutane pyrimidine dimers (T < > C) was demonstrated when a correlation was established between the mutation frequency normally associated with these lesions and the rate of mutation production at these lesions by spontaneous deamination of cytosines and photoreversal in ung-defective cells . An incubation producing a decline in mutation frequency, i.e., MFD, also produces lower rates of mutation increase via the deamination mechanism . Since the latter assay involves processes entirely within the post-UV incubation period, the lower rates are attributed to rapid transcription-coupled nucleotide excision repair (TCR) that reduces the number of relevant T < > C dimers during this period . Rediscovery of the neglected fact that MFD can be stimulated by post-UV incubation in buffer alone is part of the analysis . Results presented here and a variety of others are discussed to support a model of MFD as a particular example of TCR: effective repair of photoproducts in the transcribed DNA strand that target glutamine tRNA suppressor mutations occurs during the appropriate post-UV incubation and is responsible for MFD.

Mol Gen Genet, 1995 Dec 20, 249(6), 585 - 90
Mutation frequency decline in Escherichia coli . I . Effects of defects in mismatch repair; Li BH et al.; Mutation frequency decline (MFD) in Escherichia coli was examined for effects associated with genetic defects in mismatch repair . The kinetics of MFD are slower when the B/r strain WU3610 carries the mutation mutS201::Tn5 or mutL::Tn10, both of which affect mismatch repair . Similar slow kinetics are produced by mutH34 but not by mutH471::Tn5; the latter has no apparent effect . Strain WU3610-45 (mfd-1) produces the slower kinetics if transcription is inhibited during the post-UV incubation, although it produces no decline in normal circumstances . The slower kinetics are therefore attributed to bulk excision repair that remains when rapid transcription-coupled repair (TCR) is eliminated by certain defects in mismatch repair . A model is proposed wherein mismatch repair defects are thought to slow the activity of TCR but, unlike an mfd defect, not to impede dissociation of stalled transcription complexes at lesions in the transcribed DNA strand.

J Natl Cancer Inst, 1995 Dec 20, 87(24), 1853 - 61
Induction of T-cell immunity against Ras oncoproteins by soluble protein or Ras-expressing Escherichia coli; Fenton RG et al.; BACKGROUND: Point mutations in the ras proto-oncogene that activate its oncogenic potential occur in approximately 30% of human cancers . Previous studies have demonstrated that T-cell immunity against some forms of mutant Ras proteins could be elicited, and some effectiveness against tumors expressing activated Ras has been reported . PURPOSE: The goal of this study was to determine if immunization of mice with two forms of mutant Ras protein can induce high levels of Ras mutation-specific T-cell immunity in vitro and tumor regression in vivo . METHODS: Mice (BALB/c or C3H/HeJ) were immunized subcutaneously at 2-week intervals with purified Ras oncoproteins mixed with the immunologic adjuvants Antigen Formulation or QS-21, both of which have been shown to enhance the induction of T-cell-mediated immunity when included as components of soluble protein vaccines . In some experiments, mice were immunized directly with heat-killed Escherichia coli that had been induced to express one of the mutant Ras proteins . Spleen cells plus lymph node cells from Ras-immunized mice were tested in vitro for lysis of syngeneic Ras-expressing tumor cells and proliferation in response to mutant Ras peptides . For some of the cytolytic activity experiments, the spleen cells were grown under TH1 conditions (growth in presence of interleukin 2, interferon gamma, and an antibody directed against interleukin 4 to stimulate a cell-mediated immune response) or TH2 conditions (growth in presence of interleukins 2 and 4 to stimulate a humoral immune response) . The specificity of immunity was examined in vivo by challenge of Ras-immunized mice with syngeneic tumor cells expressing mutant Ras oncoproteins (HaBalb, i.e., BALB/c mouse cells expressing Ras with arginine substituted at amino acid position 12 {Arg 12 Ras}; C3HL61, i.e., C3H/HeJ mouse cells expressing Ras with leucine substituted at position 61 {Leu 61 Ras}) . Ten mice per group were used in each experiment . RESULTS: Proliferative and cytolytic T-cell responses directed against the Arg 12 Ras protein were generated in BALB/c mice, resulting in protection against challenge with cells expressing Arg 12 Ras and therapeutic benefit in mice bearing established tumors expressing this protein . In C3H/HeJ mice, high levels of cytolytic and proliferative responses were induced against Leu 61 Ras . Immunization with heat-killed E . coli genetically engineered to express Leu 61 Ras also led to the induction of anti-Ras T-cell immunity . T cells grown under TH1 conditions were cytolytic against Ras-transformed tumor cells, whereas those grown under TH2 conditions were not . CONCLUSIONS: Immunization as described here leads to Ras mutation-specific antitumor immunity in vitro and in vivo, with therapeutic efficacy in an established tumor model.

Biochemistry, 1995 Dec 19, 34(50), 16359 - 64
Effect of hemimethylation and methylation of adenine on the structure and stability of model DNA duplexes; Guo Q et al.; Enzymatic methylation of adenine underlies a variety of biological regulatory mechanisms in Escherichia coli . We present here structural and thermodynamic characterization of a non-self-complementary DNA decamer duplex containing the dam sequence 5'-GATC in the unmethylated, hemimethylated (both forms), and methylated states . Differential scanning calorimetry measurements show that the free energies for adenine methylation of the decamer duplex are +1.1 and +2.0 kcal/mol for hemimethylation, respectively, and +3.3 kcal/mol for full methylation . In all cases, a large unfavorable enthalpy change is partially compensated by a favorable entropy term . CD spectroscopy indicates an overall conformational difference between the unmethylated decamer duplex and its methylated analogs . Reaction with diethyl pyrocarbonate (DEPC), a purine-specific probe sensitive to conformation, is enhanced in the vicinity of the methylation site of the duplex, consistent with loosening of base pairing at this site . Comparison of the scission patterns of these decamer duplexes by the reactive probes methidiumpropyl-EDTA.FeII {MPE.FeII} and CuI(o-phenanthroline)2 {(OP)2CuI} indicates that the methylation site of the decamer duplex represents a site of enhanced reactivity for these agents . On the basis of these thermodynamics and structural features, we suggest that the methylated base pair exists in two different helical states, which require local transient opening of the duplex for interconversion.

Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12309 - 13
UGA suppression by a mutant RNA of the large ribosomal subunit; Jemiolo DK et al.; A role for rRNA in peptide chain termination was indicated several years ago by isolation of a 168 rRNA (small subunit) mutant of Escherichia coli that suppressed UGA mutations . In this paper, we describe another interesting rRNA mutant, selected as a translational suppressor of the chain-terminating mutant trpA (UGA211) of E . coli . The finding that it suppresses UGA at two positions in trpA and does not suppress the other two termination codons, UAA and UAG, at the same codon positions (or several missense mutations, including UGG, available at one of the two positions) suggests a defect in UGA-specific termination . The suppressor mutation was mapped by plasmid fragment exchanges and in vivo suppression to domain II of the 23S rRNA gene of the rrnB operon . Sequence analysis revealed a single base change of G to A at residue 1093, an almost universally conserved base in a highly conserved region known to have specific interactions with ribosomal proteins, elongation factor G, tRNA in the A-site, and the peptidyltransferase region of 23S rRNA . Several avenues of action of the suppressor mutation are suggested, including altered interactions with release factors, ribosomal protein L11, or 16S rRNA . Regardless of the mechanism, the results indicate that a particular residue in 23S rRNA affects peptide chain termination, specifically in decoding of the UGA termination codon.

Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12300 - 3
Distance determination in proteins using designed metal ion binding sites and site-directed spin labeling: application to the lactose permease of Escherichia coli; Voss J et al.; As shown in the accompanying paper, the magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances in T4 lysozyme, a protein of known structure . To evaluate this potentially powerful method for general use, particularly with membrane proteins that are difficult to crystallize, both a paramagnetic metal ion binding site and a nitroxide side chain were introduced at selected positions in the lactose permease of Escherichia coli, a paradigm for polytopic membrane proteins . Thus, three individual cysteine residues were introduced into putative helix IV of a lactose permease mutant devoid of native cysteine residues containing a high-affinity divalent metal ion binding site in the form of six contiguous histidine residues in the periplasmic loop between helices III and IV . In addition, the construct contained a biotin acceptor domain in the middle cytoplasmic loop to facilitate purification . After purification and spin labeling, electron paramagnetic resonance spectra were obtained with the purified proteins in the absence and presence of Cu(II) . The results demonstrate that positions 103, 111, and 121 are 8, 14, and > 23 A from the metal binding site . These data are consistent with an alpha-helical conformation of transmembrane domain IV of the permease . Application of the technique to determine helix packing in lactose permease is discussed.

Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12225 - 9
Purification, cDNA cloning, functional expression, and characterization of a 26-kDa endogenous mammalian carboxypeptidase inhibitor; Normant E et al.; The recent demonstration of the occurrence in rat brain and other nonpancreatic tissues of carboxypeptidase A (CPA) gene transcripts without associated catalytic activity could be ascribed to the presence of a soluble endogenous protein inhibitor . This tissue carboxypeptidase inhibitor (TCI), detected by the inhibition of added bovine pancreatic CPA, was purified from rat brain . Peptides were obtained by partial proteolysis of purified TCI, a protein of approximately 30 kDa, and starting from their sequences, a full-length cDNA encoding a 223-amino acid protein containing three potential phosphorylation sites was cloned from a cDNA library . Its identity with TCI was shown by expression in Escherichia coli of a recombinant protein recognized by antibodies raised against native TCI and display characteristic CPA-inhibiting activity . TCI appears as a hardly reversible, non-competitive, and potent inhibitor of CPA1 and CPA2 (Ki approximately 3 nM) and mast-cell CPA (Ki = 16 nM) and inactive on various other proteases . This pattern of selectivity might be attributable to a limited homology of a 11-amino acid sequence with sequences within the activation segments of CPA and CPB known to interact with residues within their active sites . The widespread expression of TCI in a number of tissues (e.g., brain, lung, or digestive tract) and its apparently cytosolic localization point to a rather general functional role, e.g., in the control of cytosolic protein degradation.

Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12205 - 9
A soluble domain of the membrane-anchoring chain of influenza virus hemagglutinin (HA2) folds in Escherichia coli into the low-pH-induced conformation; Chen J et al.; The extensive refolding of the membrane-anchoring chain of hemagglutinin (HA) of influenza virus (termed HA2) in cellular endosomes, which initiates viral entry by membrane fusion, suggests that viral HA is meta-stable . HA2 polypeptide residues 38-175 expressed in Escherichia coli are reported here to fold in vivo into a soluble trimer . The structure appears to be the same as the low-pH-induced conformation of viral HA2 by alpha-helical content, thermodynamic stability, protease dissection, electron microscopy, and antibody binding . These results provide evidence that the structure of the low-pH-induced fold of viral HA2 (TBHA2) observed crystallographically is the lowest-energy-state fold of the HA2 polypeptide . They indicate that the HA2 conformation in viral HA before low pH activation of its fusion potential is metastable and suggest that removal of the receptor-binding chain (HA1) is enough to allow HA2 to adopt the stable state . Further, they provide direct evidence that low pH is not required to form the membrane-fusion conformation but acts to make this state kinetically accessible in viral HA.

Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12120 - 5
Active site topology and reaction mechanism of GTP cyclohydrolase I; Nar H et al.; GTP cyclohydrolase I of Escherichia coli is a torus-shaped homodecamer with D5 symmetry and catalyzes a complex ring expansion reaction conducive to the formation of dihydroneopterin triphosphate from GTP . The x-ray structure of a complex of the enzyme with the substrate analog, dGTP, bound at the active site was determined at a resolution of 3 A . In the decamer, 10 equivalent active sites are present, each of which contains a 10-A deep pocket formed by surface areas of 3 adjacent subunits . The substrate forms a complex hydrogen bond network with the protein . Active site residues were modified by site-directed mutagenesis, and enzyme activities of the mutant proteins were measured . On this basis, a mechanism of the enzyme-catalyzed reaction is proposed . Cleavage of the imidazole ring is initiated by protonation of N7 by His-179 followed by the attack of water at C8 of the purine system . Cystine Cys-110 Cys-181 may be involved in this reaction step . Opening of the imidazole ring may be in concert with cleavage of the furanose ring to generate a Schiff's base from the glycoside . The gamma-phosphate of GTP may be involved in the subsequent Amadori rearrangement of the carbohydrate side chain by activating the hydroxyl group of Ser-135.

Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12017 - 20
Adaptive mutation sequences reproduced by mismatch repair deficiency; Longerich S et al.; Adaptive reversions of a lac frameshift mutation in Escherichia coli are -1 deletions in small mononucleotide repeats, whereas growth-dependent reversions are heterogeneous . The adaptive mutations resemble instability of simple repeats, which, in hereditary colon cancer, in yeast, and in E . coli occurs in the absence of mismatch repair . The postulate that mismatch repair is disabled transiently during adaptive mutation in E . coli is supported here by the demonstration that the growth-dependent mutation spectrum can be made indistinguishable from adaptive mutations by disallowing mismatch repair during growth . Physiologically induced mismatch repair deficiency could be an important mutagenic mechanism in cancers and in evolution.

Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 11955 - 9
Crystal structure of the membrane-exposed domain from a respiratory quinol oxidase complex with an engineered dinuclear copper center; Wilmanns M et al.; Cytochrome oxidase is a membrane protein complex that catalyzes reduction of molecular oxygen to water and utilizes the free energy of this reaction to generate a transmembrane proton gradient during respiration . The electron entry site in subunit II is a mixed-valence dinuclear copper center in enzymes that oxidize cytochrome c . This center has been lost during the evolution of the quinoloxidizing branch of cytochrome oxidases but can be restored by engineering . Herein we describe the crystal structures of the periplasmic fragment from the wild-type subunit II (CyoA) of Escherichia coli quinol oxidase at 2.5-A resolution and of the mutant with the engineered dinuclear copper center (purple CyoA) at 2.3-A resolution . CyoA is folded as an 11-stranded mostly antiparallel beta-sandwich followed by three alpha-helices . The dinuclear copper center is located at the loops between strands beta 5-beta 6 and beta 9-beta 10 . The two coppers are at a 2.5-A distance and symmetrically coordinated to the main ligands that are two bridging cysteines and two terminal histidines . The residues that are distinct in cytochrome c and quinol oxidases are around the dinuclear copper center . Structural comparison suggests a common ancestry for subunit II of cytochrome oxidase and blue copper-binding proteins.

FEBS Lett, 1995 Dec 18, 377(2), 249 - 52
Human ClpP protease: cDNA sequence, tissue-specific expression and chromosomal assignment of the gene; Bross P et al.; We identified three overlapping human expressed sequence tags with significant homology to the E . coli ClpP amino sequence by screening the EMBL nucleotide database . With this sequence information we applied 5' and 3'-rapid amplification of cDNA ends (RACE) to amplify and sequence human clpP cDNA in two overlapping fragments . The open reading frame encodes a 277 amino acid long precursor polypeptide . Two ClpP specific motifs surrounding the active site residues are present and extensive homology to ClpP's from other organisms was observed . Northern blotting showed high relative expression levels of clpP mRNA in skeletal muscle, intermediate levels in heart, liver and pancreas, and low levels in brain, placenta, lung and kidney . By analysis of human/rodent cell hybrids the human clpP gene was assigned to chromosome 19.

FEBS Lett, 1995 Dec 18, 377(2), 221 - 6
A novel approach for expression cloning of small GTPases: identification, tissue distribution and chromosome mapping of the human homolog of rheb; Gromov PS et al.; We report a novel approach for identifying monomeric GTP-binding proteins that is based on probing cDNA expression libraries with {alpha-32P}GTP . In short, a nitrocellulose replica from a plated cDNA expression library is treated with 2% SDS to block the GTP-binding activity of various G proteins expressed by E . coli, thus allowing the direct identification of positive clones . Using this procedure we have cloned several small GTP-binding proteins from human keratinocytes including the human homolog of rheb, a novel member of the ras-related GTP-binding proteins . Human rheb cDNA shares 90% identity with the rat counterpart and it is highly upregulated in transformed human cells of various origin . Northern analysis showed that human rheb is ubiquitously expressed, with the highest levels observed in skeletal and cardiac muscle, and not in brain, as it is the case for rat rheb . The human RHEB gene was mapped to chromosome 10q11.

FEBS Lett, 1995 Dec 18, 377(2), 193 - 6
Characterization of functionally independent domains in the human ubiquitin conjugating enzyme UbcH2; Kaiser P et al.; UbcH2 encodes a human ubiquitin conjugating enzyme (E2) able to conjugate ubiquitin to histone H2A in an E3 independent manner in vitro, which indicates that UbcH2 directly interacts with its substrates . To identify parts of the enzyme that are capable of binding H2A, we expressed several deletion mutants of UbcH2 in E . coli and tested the ability of the affinity purified mutant proteins to ubiquitinate H2A in the presence of bacterial expressed E1 and ubiquitin . With this in vitro assay we identified a C-terminal part of UbcH2 to be important for the interaction with H2A . Transfer of this C-terminal domain to another human E2, which is unable to catalyze ubiquitination of histones, leads to a fully active hybrid human ubiquitin conjugating enzyme capable of H2A ubiquitination . These results demonstrate that UbcH2 consists of two functionally independent domains . A N-terminal core domain with ubiquitin conjugating activity, and a C-terminal domain which interacts with substrate proteins.

FEBS Lett, 1995 Dec 18, 377(2), 172 - 4
Synthesis, cloning and expression in Escherichia coli of a gene coding for the Met8-->Leu CMTI I--a representative of the squash inhibitors of serine proteinases; Bolewska K et al.; A chemically synthesized gene coding for a Cucurbita maxima trypsin inhibitor modified at position P'3 (Met8-->Leu CMTI I), i.e . at the third position downstream of the reactive site bond (Arg5-Ile), was cloned into a derivative of the plasmid pAED4 that utilizes a T7 expression system . The gene was expressed in Escherichia coli as a fusion protein that accumulates in inclusion bodies . After reduction and CNBr cleavage of the fusion protein followed by oxidative refolding and reverse-phase HPLC, about 5 mg of pure protein was obtained per 1 of cell culture . Association constants of recombinant Leu-8-CMTI I with bovine beta-trypsin and human cathepsin G are the same, within experimental error, as for CMTI I isolated from a natural source.

FEBS Lett, 1995 Dec 18, 377(2), 155 - 8
Precise mapping of the tms1 binding site on p53; Wagner P et al.; Originally identified as multicopy suppressor of a lethal growth arrest caused by expression of a tumour mutant cDNA of p53 in fission yeast the tms1 gene product was found to form stable complexes with p53 in yeast . By using purified recombinant proteins multimeric complexes of tms1 and p53 could be demonstrated and recently the p53 binding site on the tms1 protein was established to the sequence YYITTEDFCT (aa 116-125) in the vicinity of a well conserved cell division motif . Here we report the precise mapping of the tms1 binding site on the p53 protein to the sequence LQIRGRERFE (aa 330-339) which defines a new functional domain on the p53 protein.

FEBS Lett, 1995 Dec 18, 377(2), 123 - 7
Molecular cloning and functional expression of a recombinant 72.5 kDa fragment of the 110 kDa regulatory subunit of smooth muscle protein phosphatase 1M; Haystead CM et al.; We have cloned a partial rat kidney cDNA that encodes a 72.5 kDa N terminal fragment of a third isoform of the M110 subunit of phosphatase 1 . This new isoform contains an insert in the 542-597 position not present in the M110 previously cloned (Chen et al . (1994) FEBS Lett . 356, 51-55) from the same species . The encoded cDNA was expressed as a soluble GST-fusion protein in E . coli, and its ability to interact with native PP-1C was measured both in vitro and in permeabilized smooth muscle . In vitro, the fusion protein was capable of selectively binding PP-1C and increasing the substrate specificity of the phosphatase towards myosin 13.2 +/- 3.5-fold (S.E . of the mean, n = 3) . In permeabilized smooth muscle pretreated with microcystin, the recombinant protein alone (1.0 microM) did not cause relaxation, but did significantly enhance the ability of PP-1C (0.3 microM) to relax the muscle . These findings show that the N terminal domain of the M110 subunit is the primary site for both PP-1C and myosin binding, and thereby determines myosin specificity . The presence of isoformic variation within this sequence may permit organ/cell specific regulation of phosphorylation sites.

Structure, 1995 Dec 15, 3(12), 1407 - 19
Structure of the biotinyl domain of acetyl-coenzyme A carboxylase determined by MAD phasing; Athappilly FK et al.; BACKGROUND: Acetyl-coenzyme A carboxylase catalyzes the first committed step of fatty acid biosynthesis . Universally, this reaction involves three functional components all related to a carboxybiotinyl intermediate . A biotinyl domain shuttles its covalently attached biotin prosthetic group between the active sites of a biotin carboxylase and a carboxyl transferase . In Escherichia coli, the three components reside in separate subunits: a biotinyl domain is the functional portion of one of these, biotin carboxy carrier protein (BCCP) . RESULTS: We have expressed natural and selenomethionyl (Se-met) BCCP from E . coli as biotinylated recombinant proteins, proteolyzed them with subtilisin Carlsberg to produce the biotinyl domains BCCP and Se-met BCCPsc, determined the crystal structure of Se-met BCCPsc using a modified version of the multiwavelength anomalous diffraction (MAD) phasing protocol, and refined the structure for the natural BCCPsc at 1.8 A resolution . The structure may be described as a capped beta sandwich with quasi-dyad symmetry . Each half contains a characteristic hammerhead motif . The biotinylated lysin is located at a hairpin beta turn which connects the two symmetric halves of the molecule, and its biotinyl group interacts with a non-symmetric protrusion from the core . CONCLUSIONS: This first crystal structure of a biotinyl domain helps to unravel the central role of such domains in reactions catalyzed by biotin-dependent carboxylases . The hammerhead structure observed twice in BCCPsc may be regarded as the basic structural motif of biotinyl and lipoyl domains of a superfamily of enzymes . The new MAD phasing techniques developed in the course of determining this structure enhance the power of the MAD method.

Structure, 1995 Dec 15, 3(12), 1323 - 32
Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 A resolution; Oliva G et al.; BACKGROUND: Glucosamine 6-phosphate deaminase from Escherichia coli is an allosteric hexameric enzyme which catalyzes the reversible conversion of D-glucosamine 6-phosphate into D-fructose 6-phosphate and ammonium ion and is activated by N-acetyl-D-glucosamine 6-phosphate . Mechanistically, it belongs to the group of aldoseketose isomerases, but its reaction also accomplishes a simultaneous amination/deamination . The determination of the structure of this protein provides fundamental knowledge for understanding its mode of action and the nature of allosteric conformational changes that regulate its function . RESULTS: The crystal structure of glucosamine 6-phosphate deaminase with bound phosphate ions is presented at 2.1 A resolution together with the refined structures of the enzyme in complexes with its allosteric activator and with a competitive inhibitor . The protein fold can be described as a modified NAD-binding domain . CONCLUSIONS: From the similarities between the three presented structures, it is concluded that these represent the enzymatically active R state conformer . A mechanism for the deaminase reaction is proposed . It comprises steps to open the pyranose ring of the substrate and a sequence of general base-catalyzed reactions to bring about isomerization and deamination, with Asp72 playing a key role as a proton exchanger.

Structure, 1995 Dec 15, 3(12), 1307 - 14
X-ray structure of human nucleoside diphosphate kinase B complexed with GDP at 2 A resolution; Morera S et al.; BACKGROUND: Nucleoside diphosphate (NDP) kinases provide precursors for DNA and RNA synthesis . In mammals, these enzymes are also involved in cell regulations . Human NDP kinase B, product of the human nm23-H2 gene, is both an enzyme and a transcription factor . It activates transcription of the c-myc oncogene independently of its catalytic function, by binding to its promoter DNA . How do the two functions coexist? RESULTS: Recombinant human NDP kinase B was co-crystallized with GDP . The X-ray structure was solved at 2.0 A resolution by molecular replacement from the homologous Drosophila Awd protein . Both enzymes are homo-hexamers with a characteristic beta alpha beta beta alpha beta fold . GDP binds near the active site His118 . The guanine base is in a surface cleft and interacts with the C terminus of another subunit . CONCLUSIONS: The beta alpha beta beta alpha beta fold, also present in the 'palm' domain of Escherichia coli DNA polymerase I and HIV reverse transcriptase, is both a mononucleotide- and a polynucleotide-binding fold . If NDP kinase B binds DNA in the same way as the polymerases, the enzyme must undergo a conformation change in order to carry out gene activation.

Structure, 1995 Dec 15, 3(12), 1277 - 9
How to make my blood boil; Goldman A; Two recent papers comparing the structure of a hyperthermophilic protein with its mesophilic counterpart both conclude that large networks of ion-pairs are important for hyperthermostability . How and why is not yet clear.

J Biotechnol, 1995 Dec 15, 43(3), 195 - 204
Response of guanosine tetraphosphate to glucose fluctuations in fed-batch cultivations of Escherichia coli; Neubauer P et al.; A rapid transient increase of guanosine 3'-diphosphate 5'-diphosphate (ppGpp) in Escherichia coli was found in response to short-term glucose fluctuations that may occur in large-scale fed-batch cultivations . The concentration of ppGpp was measured in laboratory-scale glucose limited fed-batch cultivations . Starvation zones were imitated by using an intermittent feeding scheme or a two-compartment reactor system . The cellular concentration of ppGpp per biomass increased from 80 nmol to 300-600 nmol per g cell dry weight within only 1 min after consumption of the residual glucose in dependence on the test system, which is much faster than earlier described in literature . Readdition of glucose caused immediate reduction of the ppGpp to the basic level which did not differ in cultivations with simulated starvation zones from control cultivations . Possible physiological consequences by an enhanced stringent response in cultivations with limited mass transfer have to be considered.

FEMS Microbiol Lett, 1995 Dec 15, 134(2-3), 153 - 8
Transfer of the IncJ plasmid R391 to recombination deficient Escherichia coli K12: evidence that R391 behaves as a conjugal transposon; Murphy DB et al.; A study of the IncJ plasmid R391 confirmed a low frequency of transfer between recombination proficient (recA+) Escherichia coli (10(-5) donor -1) . Reanalysis of its transfer to recombination deficient (recA) E . coli revealed an equivalent transfer frequency to and from all mutants tested . Extrachromosomal DNA could not be detected in either recA+ or recA transconjugants, while R391 proved refractory to curing in both backgrounds implying a high degree of stability . The integration of R391 into a specific region of the chromosome was demonstrated by its transfer as part of the exogenote mobilised from the transfer origins of Hfr strains BW6165 and JC158 . Transfer of R391 coupled to recA independent chromosomal integration has significant implications as to the nature and classification of the element . We propose that R391 behaves like a conjugal transposon.

FEMS Microbiol Lett, 1995 Dec 15, 134(2-3), 143 - 6
Expression of the cyanide hydratase enzyme from Fusarium lateritium in Escherichia coli and identification of an essential cysteine residue; Brown DT et al.; The filamentous fungus Fusarium lateritium is cyanide tolerant, due partly to the induction of the enzyme cyanide hydratase in the presence of cyanide . This enzyme catalyses the hydration of cyanide to formamide . The expression in Escherichia coli of a cDNA clone encoding cyanide hydratase is described . The cDNA cloned was expressed as a transcriptional fusion in the expression vector pKK233-2 and a high level of activity of cyanide hydratase was detected in E . coli . Site-directed mutagenesis of the cys-163 residue inactivated the enzyme.

Eur J Biochem, 1995 Dec 15, 234(3), 947 - 52
Lipid interaction of the 37-kDa and 58-kDa fragments of the Helicobacter pylori cytotoxin; Moll G et al.; Helicobacter pylori cytotoxin vacA (95 kDa) causes a vacuolar degeneration of epithelial cells . There is evidence that this protein toxin acts inside cells, and hence has to cross a cell membrane . This cytotoxin is frequently obtained as two fragments of 58 kDa (p58) and 37 kDa (p37) and it is available only in minute amounts . Here, its membrane interaction was studied with the two fragments, produced in Escherichia coli . Light scattering and energy transfer experiments show that p37 and p58 cause aggregation and fusion of small unilamellar lipid vesicles; only a reversible aggregation is induced at neutral pH, whereas at acid pH fusion also takes place . p58, but not p37, causes potassium efflux from liposomes and this occurs only at acid pH . Hydrophobic photolabelling with photoactivatable phosphatidylcholines inserted into liposomes shows that both fragments are labelled at neutral pH . The amount of labelling of the two fragments is much higher at acid pH, consistent with a further penetration into the hydrophobic core of the lipid bilayer . Tryptophan fluorescence measurements indicate that the two fragments undergo a pH-driven conformational change . These data are consistent with cytotoxin entry in the cell cytosol via an intracellular acidic compartment.

Eur J Biochem, 1995 Dec 15, 234(3), 910 - 20
The tungsten formylmethanofuran dehydrogenase from Methanobacterium thermoautotrophicum contains sequence motifs characteristic for enzymes containing molybdopterin dinucleotide; Hochheimer A et al.; Formylmethanofuran dehydrogenases are molybdenum or tungsten iron-sulfur proteins containing a pterin dinucleotide cofactor . We report here on the primary structures of the four subunits FwdABCD of the tungsten enzyme from Methanobacterium thermoautotrophicum which were determined by cloning and sequencing the encoding genes fwdABCD . FwdB was found to contain sequence motifs characteristic for molybdopterin-dinucleotide-containing enzymes indicating that this subunit harbors the active site . FwdA, FwdC and FwdD showed no significant sequence similarity to proteins in the data bases . Northern blot analysis revealed that the four fwd genes form a transcription unit together with three additional genes designated fwdE, fwdF and fwdG . A 17.8-kDa protein and an 8.6-kDa protein, both containing two {4Fe-4S} cluster binding motifs, were deduced from fwdE and fwdG . The open reading frame fwdF encodes a 38.6-kDa protein containing eight binding motifs for {4Fe-4S} clusters suggesting the gene product to be a novel polyferredoxin . All seven fwd genes were expressed in Escherichia coli yielding proteins of the expected size . The fwd operon was found to be located in a region of the M . thermoautotrophicum genome encoding molybdenum enzymes and proteins involved in molybdopterin biosynthesis.

Eur J Biochem, 1995 Dec 15, 234(3), 861 - 70
The interaction between lipoamide dehydrogenase and the peripheral-component-binding domain from the Azotobacter vinelandii pyruvate dehydrogenase complex; Westphal AH et al.; The sensitivity of lipoamide dehydrogenase (dihydrolipoamide:NAD+ oxidoreductase E3) from Azotobacter vinelandii to inhibition by NADH requires measurement of the activity in the initial phase of the reaction . Stopped-flow turnover experiments show that kcat is 830 s-1 compared with 420 s-1 found in standard steady-state experiments . Mutations at the si-side of the flavin prosthetic group that cause severe inhibition by NADH were studied . Tyr16 was replaced by phenylalanine and serine, which causes the loss of two intersubunit H-bonds . {F16}E3 shows only 5.7% of wild-type activity in the standard assay procedure, but analyzed by stopped-flow the activity is 70% of the wild-type enzyme . The NADH-->Cl2Ind (dichloroindophenol) activity was normal or slightly increased . The inhibition by NADH is competitive with respect to NAD+, Ki = 50 microM . Spectral analysis show that electrons readily pass over from the disulfide to the FAD, indicating an increase in the redox potential of the flavin . It is concluded that subunit interaction plays an important role in the protection of the enzyme against over-reduction by decreasing the redox potential of the flavin . The interaction of wild-type or mutant enzymes with the core component of the pyruvate (E2p) or oxoglutarate (E2o) dehydrogenase multienzyme complex relieves the inhibition to a large extent . In the mutant enzymes, the mechanism of inhibition changes from competitive to the mixed-type inhibition observed for the wild-type enzyme . The stabilizing effect of E2 on {F16}E3 was used as an assay to analyze the stoichiometry of interaction of E3 with E2p as well as E2o . 1 mol E2p monomer was sufficient to saturate 1 mol E3 dimer with a Kd of about 1 nM . Similarly, 1 mol E2o saturated the E3 dimer with a Kd of 30 nM . From these experiments it is concluded that the E3-binding domain of E2 interacts with the subunit interface of E3 near the dyad axis, thus preventing sterically the interaction with a second molecule of the binding domain . This mode of interaction, which causes asymmetry in the complex, explains the stabilization against over-reduction by tightening the subunit interaction . Subgene cloning of the E2p component of the pyruvate dehydrogenase complex is described in order to obtain a complex between the lipoamide dehydrogenase component (E3) and the binding domain of E2p . A unique restriction site in the DNA encoding the flexible linker between the third lipoyl domain and the binding domain combined with timed digestion with exonuclease Bal31 was used to create a set of deletion mutants in the N-terminal region of the binding-catalytic didomain, fused to six N-terminal amino acids from beta-galactosidase . The expressed proteins, selected for E2p activity, were analyzed for binding of E3 and E1p . The shortest fusion protein containing a functional binding domain was expressed and purified . {F16}E3 was combined with this fusion protein in a stoichiometric ratio and the resulting complex was subjected to limited proteolysis to remove the catalytic domain . The resulting {F16}E3-binding domain preparation was purified to homogeneity.

Eur J Biochem, 1995 Dec 15, 234(3), 855 - 60
Synthesis and characterization of a monomeric mutant Cu/Zn superoxide dismutase with partially reconstituted enzymic activity; Banci L et al.; A monomeric analog of human Cu/Zn superoxide dismutase (F50E/G51E SOD), previously characterized and found to have reduced enzymic activity, was here further modified by replacing Glu133 with Gln . This substitution does not dramatically affect the coordination geometry at the active site, but enhances enzymic activity, and also increases the affinity for anions at the active site . This behavior parallels earlier published results in which this point mutation was made in the dimeric wild-type enzyme . The analog described here has afforded for the first time a monomeric superoxide dismutase with substantial activity . This point mutation does not significantly influence the protein structure but interactions with anions, including superoxide, are altered with respect to the monomeric form . The present monomeric Glu133Gln mutant has partially restored enzymic activity . The diminished activity of the monomeric analogs is discussed in the light of possible minor structural changes and some of their characteristics are compared with those of naturally occurring mutants associated with various neurological diseases.

Eur J Biochem, 1995 Dec 15, 234(3), 811 - 8
Expression and characterization of the reverse transcriptase enzyme from type 1 human immunodeficiency virus using different baculoviral vector systems; Pekrun K et al.; To produce the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in amounts required to study its structure and function, the p66 enzyme subunit was expressed using two different baculovirus vectors in Sf158 insect host cells . Both vectors permitted an efficient HIV-1 RT expression . The resulting products were purified up to 90% homogeneity, characterized, and investigated for their susceptibility to digestion with various proteases . The recombinant baculoviral RT obtained with the pAc373 expression vector was purified as a p66/p60 heterodimer . The recombinant His-RT was expressed with the pBlueBacHis vector . Thereby, the protein was tagged with an N-terminal hexahistidine peptide and it was purified as a p70/p70 homodimer . The two enzymes differed in their specific activity, kinetic properties, and in vitro activation by viral and non-viral proteases . The recombinant His-RT exhibited a lower specific activity than the recombinant RT . The latter yielded enzyme activities as high as an Escherichia coli-expressed RT . Removal of the hexahistidine tag from the recombinant His-RT by digestion with enterokinase resulted in a complete loss of enzyme activity . Thus, the hexahistidine tag might be an intrinsic part of the active recombinant His-RT.

Eur J Biochem, 1995 Dec 15, 234(3), 766 - 72
Kinetic studies of a soluble alpha beta complex of nitrate reductase A from Escherichia coli . Use of various alpha beta mutants with altered beta subunits; Buc J et al.; A soluble alpha beta complex of nitrate reductase can be obtained from a strain of Escherichia coli that lacks the narI gene and expresses only the alpha and beta subunits . The beta subunit contains four Fe-S centres and the alpha subunit contains the molybdenum cofactor, which is the site at which nitrate is reduced . Despite the lack of the gamma subunit of the complete enzyme, this complex can still catalyse the reduction of nitrate with artificial electron donors such as benzyl viologen, so that it is suitable for studying the transfer of electrons between these two types of redox centre . To examine whether the electrons from reduced benzyl viologen are initially delivered to the Fe-S centres, or directly to the molybdenum cofactor, or both, we have studied the steady-state kinetics and the binding of benzyl viologen to the alpha beta complex and mutants alpha beta* with altered beta subunits . Reduction of the enzyme by reduced benzyl viologen in the absence of nitrate showed that all four Fe-S centres and the molybdenum cofactor could be reduced . Two classes of site with different equilibrium constants could be distinguished . The kinetic results suggest that benzyl viologen supplies its electrons directly to the molybdenum cofactor, at a rate showing a hyperbolic dependence on the square of the concentration of the electron donor . A reaction mechanism is proposed for the reduction of nitrate catalysed by the alpha beta complex of nitrate reductase with artificial electron donors.

Eur J Biochem, 1995 Dec 15, 234(3), 706 - 13
Synthesis and characterization of a homogeneous chemical conjugate between basic fibroblast growth factor and saporin; Buechler YJ et al.; Basic fibroblast growth factor (FGF-2) and saporin were chemically conjugated using the crosslinker, N-succinimidyl-3(2-pyridyldithio)-propionate . When purified, the conjugate was found to be heterogeneous as analyzed by SDS/PAGE, size-exclusion HPLC and reverse-phase HPLC . Therefore, we sought to synthesize a molecule that would be homogeneous and thus easier to characterize and evaluate its efficacy and toxicity for pharmaceutical drug development . A homogeneous chemical conjugate was successfully synthesized by using a mutant FGF-2 with Cys96 replaced by Ser ({S96}FGF-2) and a recombinant saporin mutant containing a single Cys at the -1 position (C-SAP) . The latter was expressed in Escherichia coli and isolated to 99% purity by expanded-bed adsorption chromatography followed by cation-exchange chromatography . The cysteine in C-SAP was activated by Ellman's reagent and then reacted with the only available cysteine (position 78) in {S96}FGF-2 to produce the homogeneous conjugate, designated as FGF2-C-SAP . The purified FGF2-C-SAP was more than 98% pure as judged by HPLC . In vitro biological assays indicated that FGF2-C-SAP was a potent inhibitor of protein synthesis in a cell-free system and was cytotoxic to FGF-receptor-bearing cells.

EMBO J, 1995 Dec 15, 14(24), 6292 - 300
Comparison of protein binding to DNA in vivo and in vitro: defining an effective intracellular target; Yang SW et al.; We have quantitatively evaluated the affinity of a set of target sites for the integration host factor (IHF) protein of Escherichia coli by their performance as competitors in an electrophoretic mobility shift assay . We also determined how well each of these sites is filled by IHF in vivo . The data show that several natural sites have an affinity not much greater than that required for intracellular occupancy . The data also indicate that very little of the IHF in a cell is present as free protein available for binding, suggesting that binding to non-specific targets dominates the operation of this system . The correlation between in vitro affinity and in vivo occupancy provides a ready means to assess the likely physiological significance of putative IHF sites . It also provides a general method to assess the importance of non-specific interactions by DNA binding proteins inside a cell.

EMBO J, 1995 Dec 15, 14(24), 6280 - 91
Structure of the even-skipped homeodomain complexed to AT-rich DNA: new perspectives on homeodomain specificity; Hirsch JA et al.; even-skipped is a homeobox gene important in controlling segment patterning in the embryonic fruit fly . Its homeobox encodes a DNA binding domain which binds with similar affinities to two DNA consensus sequences, one AT-rich, the other GC-rich . We describe a crystallographic analysis of the Even-skipped homeodomain complexed to an AT-rich oligonucleotide at 2.0 A resolution . The structure reveals a novel arrangement of two homeodomains bound to one 10 bp DNA sequence in a tandem fashion . This arrangement suggests a mechanism for the homeoproteins' regulatory specificity . In addition, the functionally important residue Gln50 is observed in multiple conformations making direct and water-mediated hydrogen bonds with the DNA bases.

Biochem J, 1995 Dec 15, 312 ( Pt 3), 971 - 7
Catalytic consequences of experimental evolution: catalysis by a 'third-generation' evolvant of the second beta-galactosidase of Escherichia coli, ebgabcde, and by ebgabcd, a 'second-generation' evolvant containing two supposedly 'kinetically silent' mutations; Krishnan S et al.; The kinetics of hydrolysis of a series of synthetic substrates by two experimentally evolved forms ('evolvants'), ebgabcd and ebgabcde, of the second beta-galactosidase of Escherichia coli have been measured . The ebgabcd enzyme differs from the wild-type (ebgo) enzyme by Asp92-->Asn (a) and Trp977-->Cys (b) changes in the large subunit, as well as two changes hitherto considered to have no kinetic effect, Ser979-->Gly in the large subunit (c) and Glu122-->Gly in the small subunit (d) . The enzyme ebgabcde contains in addition a Glu93-->Lys change in the large subunit (e) . Comparison of ebgabcd with ebgab {Elliott, K, Sinnott, Smith, Bommuswamy, Guo, Hall and Zhang (1992) Biochem . J . 282, 155-164} indicates that the c and d changes in fact accelerate the hydrolysis of the glycosyl-enzyme intermediate by a factor of 2.5, and also decrease the charge on the aglycone oxygen atom at the first transition state; the charge on the glycone, however, is unaltered {see K, Konstantinidis, Sinnott and Hall (1993) Biochem . J . 291, 15-17} . The e mutation causes a fall in the degalactosylation rate of about a factor of 3, and its occurrence only together with c and d mutations {Hall, Betts and Wootton (1989) Genetics 123, 635-648} suggests that degalactosylation of a hypothetical ebgabe enzyme would be so slow that the enzyme would have no biological advantage over the ancestral ebgab . The transfer products from galactosyl-ebgabcd and galactosyl-ebgabcde to high concentrations to glucose have been measured; the predominant product is allolactose, but significant quantities of lactose are also formed; however, at apparent kinetic saturation of the galactosyl-enzyme, hydrolysis rather than transfer is the preponderant pathway . A knowledge of the rates of enzyme-catalysed exchange of 18O from {1-18O}galactose to water permits the construction of the free-energy profiles for hydrolysis of lactose by begabcd and ebgabcde . As with the other evolvants, changes in the profile away from the rate-determining transition state are essentially random, and there is no correlation between the changes in the free energies of intermediates and of their flanking transition states . We consider the aggregate of our kinetic data on the ebg system to be telling experimental support for the theoretical objections of Pettersson {Pettersson (1992) Eur . J . Biochem . 206, 289-295 and previous papers} to the Albery-Knowles theory of the evolution of enzyme kinetic activity.

Biochem J, 1995 Dec 15, 312 ( Pt 3), 839 - 45
Lactoferrin-lipopolysaccharide interaction: involvement of the 28-34 loop region of human lactoferrin in the high-affinity binding to Escherichia coli 055B5 lipopolysaccharide; Elass-Rochard E et al.; The ability of lactoferrin (Lf), an iron-binding glycoprotein that is also called lactotransferrin, to bind lipopolysaccharide (LPS) may be relevant to some of its biological properties . A knowledge of the LPS-binding site on Lf may help to explain the mechanism of its involvement in host defence . Our report reveals the presence of two Escherichia coli 055B5 LPS-binding sites on human Lf (hLf): a high-affinity binding site (Kd 3.6 +/- 1 nM) and a low-affinity binding site (Kd 390 +/- 20 nM) . Bovine Lf (bLf), which shares about 70% amino acid sequence identity with hLf, exhibits the same behaviour towards LPS . Like hLf, bLf also contains a low- and a high-affinity LPS-binding site . The Kd value (4.5 +/- 2 nM) corresponding to the high-affinity binding site is similar to that obtained for hLf . Different LPS-binding sites for human serum transferrin have been suggested, as this protein, which is known to bind bacterial endotoxin, produced only 12% inhibition of hLf-LPS interaction . Binding and competitive binding experiments performed with the N-tryptic fragment (residues 4-283), the C-tryptic fragment (residues 284-692) and the N2-glycopeptide (residues 91-255) isolated from hLf have demonstrated that the high-affinity binding site is located in the N-terminal domain I of hLf, and the low-affinity binding site is present in the C-terminal lobe . The inhibition of hLf-LPS interaction by a synthetic octadecapeptide corresponding to residues 20-37 of hLf and lactoferricin B (residues 17-41), a proteolytic fragment from bLf, revealed the importance of the 28-34 loop region of hLf and the homologous region of bLf for LPS binding . Direct evidence that this amino acid sequence is involved in the high-affinity binding to LPS was demonstrated by assays carried out with EGS-loop hLf, a recombinant hLf mutated at residues 28-34.

Biochem J, 1995 Dec 15, 312 ( Pt 3), 739 - 47
The first putative transmembrane helix of the 16 kDa proteolipid lines a pore in the Vo sector of the vacuolar H(+)-ATPase; Jones PC et al.; The 16 kDa proteolipid is the major component of the vacuolar H(+)-ATPase membrane sector, responsible for proton translocation . Expression of a related proteolipid from the arythropod Nephrops norvegicus in a Saccharomyces strain in which the VMA3 gene for the endogenous proteolipid has been disrupted results in restored vacuolar H(+)-ATPase function . We have used this complementation system, coupled to cysteine substitution mutagenesis and protein chemistry, to investigate structural features of the proteolipid . Consecutive cysteines were introduced individually into putative transmembrane segment 1 of the proteolipid, and at selected sites in extramembranous regions and in segment 3 and 4 . Analysis of restored vacuolar H(+)-ATPase function showed that segment 1 residues sensitive to mutation to cysteine were clustered on a single face, but only if the segment was helical . Only residues insensitive to mutation could be covalently modified by the cysteine-specific reagent fluorescein 5-maleimide . A cysteine introduced into segment 3 was the only residue accessible to a relatively hydrophobic reagent, suggesting accessibility to the lipid phase . Analysis of disulphide bond formation between introduced cysteines indicates that the first transmembrane alpha-helices of each monomer are adjacent to each other at the centre of the proteolipid multimeric complex . The data are consistent with a model in which the fluorescein maleimide-accessible face of helix I lines a pore at the centre of a hexameric complex formed by the proteolipid, with the mutationally sensitive face oriented into the protein core . The implications for ion-transport function in this family of proteins are discussed in the context of this structural model.

Biochem J, 1995 Dec 15, 312 ( Pt 3), 679 - 85
Structure of the quinoprotein glucose dehydrogenase of Escherichia coli modelled on that of methanol dehydrogenase from Methylobacterium extorquens; Cozier GE et al.; The structure of methanol dehydrogenase (MDH) at 0.194 nm (1.94 A) has been used to provide a model structure for part of a membrane quinoprotein glucose dehydrogenase (GDH) . The basic superbarrel structure is retained, along with the tryptophan-docking motifs . The active-site regions are similar, but there are important differences, the most important being that GDH lacks the novel disulphide ring structure formed from adjacent cysteines in MDH; in GDH the equivalent region is occupied by His-262 . Because of the overall similarities in the active-site region, the mechanism of action of GDH is likely to be similar to that of MDH . The differences in co-ordination to the cation and bonding to the pyrrolo-quinoline quinone (PQQ) in the active site may explain the relative ease of dissociation of the prosthetic group from the holo-GDH . There are considerable differences in the external loops, particularly those involved in formation of the shallow funnel leading to the active site, the configuration of which influences substrate specificity . The proposed model is consistent in many respects with previous proposals for the active-site structure based on the effects of chemical modification on binding of PQQ and enzymic activity.

Mol Gen Genet, 1995 Dec 15, 249(5), 545 - 56
Characterization of an EcR/USP heterodimer target site that mediates ecdysone responsiveness of the Drosophila Lsp-2 gene; Antoniewski C et al.; The Larval serum protein-2 gene (Lsp-2) of Drosophila melanogaster is uniquely expressed in the fat body tissue from the beginning of the third instar to the end of adult life . Accumulation of the larval Lsp-2 transcript is enhanced by 20-hydroxyecdysone . To study the molecular basis for ecdysone regulated Lsp-2 activity, deletion mutants of the Lsp-2 5'-flanking region were constructed by fusion to either the Escherichia coli chloramphenicol acetyltransferase (CAT) gene or to an hsp70-lacZ hybrid gene encoding beta-galactosidase . Constructs transfected into Drosophila S2/M3 cells were shown to confer transient ecdysone inducibility on the reporter genes . A single functional ecdysone response element (EcRE) was localized at position -75 relative to the Lsp-2 transcription initiation site . In gel mobility shift assays using fat body nuclear extracts or nuclear receptors synthesized in vitro, a 27-bp sequence harboring the EcRE bound both the Drosophila ecdysone receptor and the Drosophila retinoid-X homologue, Ultraspiracle, in a cooperative manner . Competition experiments indicate that the affinity of the Lsp-2 EcRE for the ecdysone receptor complex is comparable to that of the canonical EcRE of the hsp27 gene and is at least 4-fold greater than that of Fbp1, another fat body-specific Drosophila gene . Our results suggest that structural features of this EcRE determine its ability to induce ecdysone responsiveness at a lower ligand concentration and may form the basis for differential hormone responsiveness within the fat body.

Mol Gen Genet, 1995 Dec 15, 249(5), 498 - 506
Multicopy plasmid suppression of stationary phase chaperone toxicity in Escherichia coli by phosphogluconate dehydratase and the N-terminus of DnaK; Rockabrand D et al.; Overproduction of DnaK in Escherichia coli results in a bacteriocidal effect . This effect is most acute in stationary phase cells . A selection scheme was developed to isolate multicopy suppressors from an E . coli plasmid expression library, which overcome the stationary phase toxicity of excess DnaK . Two suppressor plasmids were recovered which contained inserts of 1.85 kb and 2.69 kb, respectively . Rearranged and deleted plasmid derivatives were constructed and used to further localize the suppressors . DNA sequence analysis demonstrated that one suppressor encoded phosphogluconate dehydratase (Edd) while the other suppressor encoded the N-terminal 237 amino acids of DnaK itself (DnaK') . Strains bearing the suppressor plasmids constitutively overproduced proteins with apparent masses of 66 kDa (Edd) and 37 kDa (DnaK') as determined by gel electrophoresis . Western blot analysis using polyclonal antisera specific for either Edd or DnaK confirmed the identity of these overproduced proteins . Suppression of DnaK toxicity was eliminated by the introduction of a + 1 frameshift mutation early in the respective coding regions of either of the two suppressors . These results suggest that suppressor gene translation plays a role in the mechanism of DnaK suppression.

Mol Gen Genet, 1995 Dec 15, 249(5), 465 - 73
Molecular cloning and functional characterization of a cDNA encoding nucleosome assembly protein 1 (NAP-1) from soybean; Yoon HW et al.; NAP-1, a protein first isolated from mammalian cells, can introduce supercoils into relaxed circular DNA in the presence of purified core histones . Based on its in vitro activity, it has been suggested that NAP-1 may be involved in nucleosome assembly in vivo . We isolated a cDNA clone encoding a soybean NAP-1 homolog, SNAP-1 . The SNAP-1 cDNA contains an open reading frame of 358 amino acids residues with a calculated molecular weight of 41 kDa . The deduced amino acid sequence of SNAP-1 shares sequence similarity with yeast NAP-1 (38%) and human hNRP (32%) . Notable features of the deduced sequence are two extended acidic regions thought to be involved in histone binding . SNAP-1 expressed in Escherichia coli induces supercoiling in relaxed circular DNA, suggesting that SNAP-1 may have nucleosome assembly activity . The specific activity of SNAP-1 is comparable to that of HeLa NAP-1 in an in vitro assay . Western analysis reveals that SNAP-1 is expressed in the immature and young tissues that were examined, while mature tissues such as old leaves and roots, show very little or no expression . NAP-1 homologs also appear to be present in other plant species.

Blood, 1995 Dec 15, 86(12), 4553 - 8
Verotoxin-1 promotes leukocyte adhesion to cultured endothelial cells under physiologic flow conditions; Morigi M et al.; Hemolytic uremic syndrome (HUS), which is the most common cause of acute renal failure in infants and small children, is caused by verotoxin (VT)-producing Escherichia coli infection . Endothelial injury determines microvascular thrombosis and evidence is available from recent studies that suggests that leukocyte activation participates in endothelial damage . We studied here the effect of VT-1 on leukocyte adhesion to vascular endothelium under physiologic flow conditions . Human umbilical vein endothelial cells (HUVECs) were incubated for 24 hours with VT-1 (0.1, 1, and 10 pmol/L) and then exposed to a total leukocyte suspension in a parallel plate flow chamber under laminar flow conditions (1.5 dynes/cm2) . Adherent cells were counted by digital image processing . Results showed that VT-1 dose-dependently increased the number of adhering leukocytes to HUVECs as compared with unstimulated cells . The adhesive response elicited by VT-1 was comparable to that of interleukin-1 beta (IL-1 beta), one of the most potent inducers of endothelial cell adhesiveness . Exposure of HUVECs to VT-1 did not affect the number of rolling leukocytes, which was similar to that of control values . To examine the role of adhesion molecules in VT-1-induced leukocyte adhesion, HUVECs were incubated with mouse monoclonal antibodies against E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) before adhesion assay . Functional blocking of E-selectin, ICAM-1, and VCAM-1 on endothelial cells significantly inhibited VT-1-induced increase in leukocyte adhesion . In some experiments, before VT-1 incubation, HUVECs were pretreated for 24 hours with tumor necrosis factor alpha (TNF alpha; 100 U/mL), which is known to increase VT receptor expression on HUVECs . The number of adhering leukocytes on HUVECs exposed to TNF alpha and VT-1 significantly increased as compared with HUVECs incubated with VT-1 and TNF alpha alone . These results suggest that VT-1 modulates leukocyte-endothelium interaction, thus increasing leukocyte adhesion and upregulating adhesive proteins on endothelial surface membrane.

J Biol Chem, 1995 Dec 15, 270(50), 30230 - 3
Photocross-links between single-stranded DNA and Escherichia coli RecA protein map to loops L1 (amino acid residues 157-164) and L2 (amino acid residues 195-209); Malkov VA et al.; To function as a repair and recombination protein, RecA has to be assembled as an active filament on single-stranded DNA in the presence of ATP or its analogs . We have identified amino acids in the primary DNA binding site of RecA that interact with single-stranded DNA by photocross-linking . A nucleoprotein complex consisting of RecA protein bound to a monosubstituted oligonucleotide bearing a 5-iododeoxyuracil cross-linking moiety was irradiated with long wavelength ultraviolet radiation to effect cross-linking with RecA protein . Subsequent trypsin digestion, followed by purification and peptide sequencing, revealed the cross-linking of two independent peptides, amino acid residues 153-169 and 199-216 . Met164 from loop L1 and Phe203 from loop L2 were determined to be the exact points of cross-linking . Thus, our data confirm and extend predictions about the DNA binding domain of RecA protein based on the molecular structure of RecA (Story, R . M., Weber, I . T., and Steitz, T . A . (1992) Nature 355, 318-325).

J Biol Chem, 1995 Dec 15, 270(50), 30051 - 9
Analysis of three DnaK mutant proteins suggests that progression through the ATPase cycle requires conformational changes; Kamath-Loeb AS et al.; DnaK, the bacterial homolog of the eukaryotic hsp70 proteins, is an ATP-dependent chaperone whose basal ATPase is stimulated by synthetic peptides and its cohort heat shock proteins, DnaJ and GrpE . We have used three mutant DnaK proteins, E171K, D201N, and A174T (corresponding to Glu175, Asp206, and Ala179, respectively, in bovine heat stable cognate 70) to probe the ATPase cycle . All of the mutant proteins exhibit some alteration in basal ATP hydrolysis . However, they all exhibit more severe defects in the regulated activities . D201N and E171K are completely defective in all regulated activities of the protein and also in making the conformational change exhibited by the wt protein upon binding ATP . We suggest that the inability of D201N and E171K to achieve the ATP activated conformation prevents both stimulation by all effectors and the ATP-mediated release of GrpE . In contrast, the defect of A174T is much more specific . It exhibits normal binding and release of GrpE and normal stimulation of ATPase activity by DnaJ . However, it is defective in the synergistic activation of its ATPase by DnaJ and GrpE . We suggest that this mutant protein is specifically defective in a DnaJ/GrpE mediated conformational change in DnaK necessary for the synergistic action of DnaJ+GrpE.

J Biol Chem, 1995 Dec 15, 270(50), 29959 - 66
The role of threonine 54 in adrenodoxin for the properties of its iron-sulfur cluster and its electron transfer function; Uhlmann H et al.; The amino acid in position 54 of adrenodoxin is strongly conserved among ferredoxins, consisting of a threonine or serine . Its role was studied by analyzing mutants T54S and T54A of bovine adrenodoxin . Absorption, circular dichroism, fluorescence, and electron paramagnetic resonance spectra of mutant T54S show that this substitution has no influence on the formation and stability of the ferredoxin . The redox potential of this mutant, however, was lowered by 55 mV as compared with native adrenodoxin, indicating a role for this residue in redox potential modulation . Incorporation of the iron-sulfur cluster was not impaired in the T54A mutant, although structural features of the oxidized protein were considerably changed . The decreased stability of the T54A mutant as compared with the wild type and mutant T54S indicates that a hydrogen bond donor at this position stabilizes the protein . Both mutants have been shown to be functionally active . Replacement of threonine 54 by serine or alanine, however, leads to rearrangements at the recognition sites for its redox partners . This is reflected by decreased Km and Kd values of both mutants for the cytochromes P450, whereas only T54A displayed a decreased Km value in cytochrome c reduction . Substrate conversion was accelerated (2.2- and 2.4-fold for mutants T54A and T54S, respectively) in the CYP11B1-, but not in the CYP11A1-dependent reaction.

J Biol Chem, 1995 Dec 15, 270(50), 29953 - 8
Kinetics of acid-mediated disassembly of the B subunit pentamer of Escherichia coli heat-labile enterotoxin . Molecular basis of pH stability; Ruddock LW et al.; The B-subunit pentamer of Escherichia coli heat-labile enterotoxin (EtxB) is highly stable, maintaining its quaternary structure in a range of conditions that would normally be expected to cause protein denaturation . In this paper the structural stability of EtxB has been studied as a function of pH by electrophoretic, immunochemical, and spectroscopic techniques . Disassembly of the cyclic pentameric structure of human EtxB occurs only below pH 2 . As determined by changes in intrinsic fluorescence this process follows first-order kinetics, with the rate constant for disassembly being proportional to the square of the H+ ion concentration, and with an activation energy of 155 kJ mol-1 . A C-terminal deletion mutant, hEtxB214, similarly shows first-order kinetics for disassembly but with a higher pH threshold, resulting in disassembly being seen at pH 3.4 and below . These findings are consistent with the rate-limiting step for disassembly of human EtxB being the simultaneous disruption of two interfaces by protonation of two C-terminal carboxylates . By comparison, disassembly of the B-subunit of cholera toxin (CtxB), a protein which shows 80% sequence identity with EtxB, exhibits a much lower stability to acid conditions; with disassembly of CtxB occurring below pH 3.9, with an activation energy of 81 kJ mol-1 . Reasons for the observed differences in acid stability are discussed, and the implications of these findings to the development of oral vaccines using EtxB and CtxB are considered.

J Biol Chem, 1995 Dec 15, 270(50), 29904 - 9
Interaction of wheat germ protein synthesis initiation factor eIF-(iso)4F and its subunits p28 and p86 with m7GTP and mRNA analogues; Sha M et al.; The binding of p28, p86, and native wheat germ eIF-(iso)4F with m7GTP and oligonucleotides was measured and compared . The purified subunits (p28, 28 kDa and p86, 86 kDa) of wheat germ protein synthesis initiation factor eIF-(iso)4F have been obtained from Escherichia coli expression of the cloned DNA (van Heerden, A., and Browning, K . S . (1994) J . Biol . Chem . 269, 17454-17457) . The binding of the 5'-terminal cap analogue m7GTP to the small subunit (p28) of eIF-(iso)4F as a function of pH, temperature, and ionic strength is described . The mode of binding of p28 to cap analogues is very similar to the intact protein . Assuming that all tryptophan residues contribute to p28 and eIF-(iso)4F fluorescence, iodide quenching shows that all 9 tryptophan residues in p28 are solvent-accessible, while only 6 out of 16 tryptophan residues are solvent-accessible on the intact eIF-(iso)4F . The fluorescence stopped-flow studies of eIF-(iso)4F and p28 with cap show a concentration-independent conformational change . The rate of this conformational change was approximately 10-fold faster for the isolated p28 compared with the native eIF-(iso)4F . From these studies it appears that cap recognition resides in the p28 subunit . However, p86 enhances the interaction with capped oligonucleotides and probably is involved in protein-protein interactions as well . Both subunits are required for helicase activity.

J Biol Chem, 1995 Dec 15, 270(50), 29831 - 5
SecA-independent translocation of the periplasmic N-terminal tail of an Escherichia coli inner membrane protein . Position-specific effects on translocation of positively charged residues and construction of a protein with a C-terminal translocation signal; Whitley P et al.; We have shown previously that the 100-residue-long periplasmic N-terminal tail of the Escherichia coli inner membrane protein ProW can be translocated across the inner membrane in a sec-independent manner and that its translocation is blocked by the introduction of three positively charged residues near its C-terminal end (Whitley, P., Zander, T., Ehrmann, M., Haardt, M., Bremer, E., and von Heijne, G . (1994) EMBO J . 13, 4653-4661) . We have now further analyzed the requirements for translocation of the N-terminal tail and found that the introduction of even a single arginine can block translocation . Position-specific differences in the effects on translocation of arginine insertions suggest that the C-terminal end of the N-terminal tail is more critical for translocation than the central and N-terminal regions . We also show that the N-terminal tail is translocated in a truncation mutant where a stop codon is placed immediately after the first transmembrane segment, provided that the transmembrane segment is flanked on its C-terminal end by positively charged residues . Thus, sec-independent translocation of a relatively large domain can be induced by a translocation signal located at the extreme C terminus of a protein.

J Biol Chem, 1995 Dec 15, 270(50), 29825 - 30
Substrate specificities of the insulin and insulin-like growth factor 1 receptor tyrosine kinase catalytic domains; Xu B et al.; To compare the substrate specificities of the insulin and insulin-like growth factor 1 (IGF-1) receptor tyrosine kinases, the catalytic domains of the enzymes have been expressed in Escherichia coli as fusion proteins . The purified proteins have kinase activity, demonstrating that the catalytic domain of IGF-1 receptor, like that of insulin receptor, is active independent of its ligand-binding and transmembrane domains . The specificities of the two enzymes for the divalent cations Mg2+ and Mn2+ are indistinguishable . A series of peptides has been prepared that reproduces the major phosphorylation sites of insulin receptor substrate-1, a common substrate for the two receptor tyrosine kinases in vivo . Insulin and IGF-1 receptors show distinct preferences for these peptides; whereas insulin receptor prefers peptides based on Tyr-987 or Tyr-727 of insulin receptor substrate-1, the IGF-1 receptor preferentially recognizes the Tyr-895 site . The latter site, when phosphorylated, is a binding site for the SH2 domain-containing adapter protein Grb2 . The ability of the two receptor tyrosine kinases to be phosphorylated and activated by v-Src has also been examined . The catalytic activity of IGF-1 receptor is stimulated approximately 3.4-fold by treatment with purified v-Src, while insulin receptor shows very little effect of Src phosphorylation under these conditions . This observation is relevant to recent findings of IGF-1 receptor activation in Src-transformed cells, and may represent one method by which Src amplifies its mitogenic signal . Collectively the data suggest that the catalytic domains of the two receptor kinases possess inherently different substrate specificities and signaling potentials.

J Biol Chem, 1995 Dec 15, 270(50), 29799 - 805
Purification and characterization of protease Ci, a cytoplasmic metalloendoprotease in Escherichia coli; Kim KI et al.; Protease Ci, a cytoplasmic metalloprotease in Escherichia coli, has been purified to apparent homogeneity by conventional chromatographic procedures using 125I-labeled oxidized insulin B-chain as a substrate . The purified enzyme behaves as a 54-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consists of a single polypeptide chain . It is inhibited by metal-chelating agents, including o-phenanthroline and NaCN, but not by inhibitors of serine proteases or thiol-blocking agents . Furthermore, protease Ci was found to contain 1.1 mol of zinc per mol of the enzyme upon analysis by HR ICP mass spectroscopy . Thus, protease Ci must be a zinc metalloprotease . Among the polypeptides tested as substrates, oxidized insulin B-chain and glucagon are most rapidly hydrolyzed . Intact insulin is a much poorer substrate than oxidized insulin B-chain, even though the affinity of the enzyme to intact insulin is approximately 100-fold greater than that to the B-chain . Since unlabeled oxidized insulin A-chain is capable of inhibiting the hydrolysis of 125I-labeled insulin B-chain, it also appears to be a substrate . Protease Ci also degrades lysozyme and lactalbumin, although to a much lesser extent than oxidized insulin B-chain . However, it shows little or no activity against proteins larger than 15 kDa (e.g . ovalbumin and denatured bovine serum albumin) . Hydrolysis of oxidized insulin B-chain followed by amino acid composition analyses of the cleavage products reveals that as many as 10 of its 29 peptide bonds are hydrolyzed by protease Ci . This ability to hydrolyze relatively small polypeptides suggests that protease Ci may catalyze the later steps in the pathway for intracellular protein breakdown.

J Biol Chem, 1995 Dec 15, 270(50), 29652 - 5
The inhibition mechanism of serpins . Evidence that the mobile reactive center loop is cleaved in the native protease-inhibitor complex; Wilczynska M et al.; Inhibitors that belong to the serine protease inhibitor or serpin family have reactive centers that constitute a mobile loop with P1-P1' residues acting as a bait for cognate protease . Current hypotheses are conflicting as to whether the native serpin-protease complex is a tetrahedral intermediate with an intact inhibitor or an acyl-enzyme complex with a cleaved inhibitor P1-P1' peptide bond . Here we show that the P1' residue of the plasminogen activator inhibitor type 1 mutant (P1' Cys) became more accessible to radiolabeling in complex with urokinase-type plasminogen activator (uPA) compared with its complex with catalytically inactive anhydro-uPA, indicating that complex formation with cognate protease leads to a conformational change whereby the P1' residue becomes more accessible . Analysis of chemically blocked NH2 termini of serpin-protease complexes revealed that the P1-P1' peptide bonds of three different serpins are cleaved in the native complex with their cognate protease . Complex formation and reactive center cleavage were found to be rapid and coordinated events suggesting that cleavage of the reactive center loop and the subsequent loop insertion induce the conformational changes required to lock the serpin-protease complex.

Science, 1995 Dec 15, 270(5243), 1828 - 31
Control of cell fate by a deubiquitinating enzyme encoded by the fat facets gene; Huang Y et al.; Ubiquitin is a highly conserved polypeptide found in all eukaryotes . The major function of ubiquitin is to target proteins for complete or partial degradation by a multisubunit protein complex called the proteasome . Here, the Drosophila fat facets gene, which is required for the appropriate determination of particular cells in the fly eye, was shown to encode a ubiquitin-specific protease (Ubp), an enzyme that cleaves ubiquitin from ubiquitin-protein conjugates . The Fat facets protein (FAF) acts as a regulatory Ubp that prevents degradation of its substrate by the proteasome . Flies bearing fat facets gene mutations were used to show that a Ubp is cell type--and substrate-specific and a regulator of cell fate decisions in a multicellular organism.

Science, 1995 Dec 15, 270(5243), 1825 - 8
DNA template and activator-coactivator requirements for transcriptional synergism by Drosophila bicoid; Sauer F et al.; The template and coactivator requirements for synergistic transcription directed by a single activator, Bicoid (BCD), bound to multiple sites have been determined . Mutagenesis studies in combination with protein binding experiments and reconstituted transcription reactions identified two independent activation domains of BCD that target different coactivator subunits (TAFII110 and TAFII60) of the basal transcription factor IID (TFIID) . The presence of both coactivators is required for BCD to recruit the TATA binding protein (TBP)-TAF complex to the promoter and direct synergistic activation of transcription . Thus, contact between multiple activation domains of BCD and different targets within the TFIID complex can mediate transcriptional synergism.

Science, 1995 Dec 15, 270(5243), 1811 - 5
Identification of RANTES, MIP-1 alpha, and MIP-1 beta as the major HIV-suppressive factors produced by CD8+ T cells; Cocchi F et al.; Evidence suggests that CD8+ T lymphocytes are involved in the control of human immunodeficiency virus (HIV) infection in vivo, either by cytolytic mechanisms or by the release of HIV-suppressive factors (HIV-SF) . The chemokines RANTES, MIP-1 alpha, and MIP-1 beta were identified as the major HIV-SF produced by CD8+ T cells . Two active proteins purified from the culture supernatant of an immortalized CD8+ T cell clone revealed sequence identity with human RANTES and MIP-1 alpha . RANTES, MIP-1 alpha, and MIP-1 beta were released by both immortalized and primary CD8+ T cells . HIV-SF activity produced by these cells was completely blocked by a combination of neutralizing antibodies against RANTES, MIP-1 alpha, and MIP-1 beta . Recombinant human RANTES, MIP-1 alpha, and MIP-1 beta induced a dose-dependent inhibition of different strains of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) . These data may have relevance for the prevention and therapy of AIDS.

Cancer Res, 1995 Dec 15, 55(24), 6045 - 52
Mch3, a novel human apoptotic cysteine protease highly related to CPP32; Fernandes-Alnemri T et al.; Recent evidence suggests that mammalian cysteine proteases related to Caenorhabditis elegans CED-3 are key components of mammalian programmed cell death or apoptosis . We have shown recently that the CPP32 and Mch2 alpha cysteine proteases cleave the apoptotic markers poly(ADP-ribose) polymerase (PARP) and lamins, respectively . Here we report the cloning of a new Ced-3/interleukin 1 beta-converting enzyme-related gene, designated Mch3, that encodes a protein with the highest degree of homology to CPP32 compared to other family members . An alternatively spliced isoform, named Mch3 beta, was also identified . Bacterially expressed recombinant Mch3 has intrinsic autocatalytic/autoactivation activity . The specific activity of Mch3 alpha toward the peptide substrate DEVD-7-amino-4-methylcoumarin and PARP resembles that of CPP32 . Like interleukin 1 beta-converting enzyme and CPP32, the active Mch3 alpha is made of two subunits derived from a precursor (proMch3 alpha) . It was of interest that recombinant CPP32-p17 subunit can form an active heteromeric enzyme complex with recombinant Mch3 alpha-p12 subunit and vice versa, as determined by the ability of the heteromeric complexes to induce apoptosis in Sf9 cells . These data suggest that proMch3 alpha and proCPP32 can interact to form an active Mch3 alpha/CPP32 heteromeric complex . We also provide evidence that CPP32 can efficiently cleave proMch3 alpha, but not the opposite, suggesting that Mch3 alpha activation in vivo may depend in part on CPP32 activity . The high degree of conservation in structure and specific activity and the coexistence of Mch3 alpha and CPP32 in the same cell suggests that the PARP cleavage activity observed during apoptosis cannot solely be attributed to CPP32 but could also be an activity of Mch3 alpha.

J Mol Biol, 1995 Dec 15, 254(5), 848 - 55
The action of pokeweed antiviral protein and ricin A-chain on mutants in the alpha-sarcin loop of Escherichia coli 23S ribosomal RNA; Marchant A et al.; The alpha-sarcin loop of Escherichia coli 23S rRNA is a universally-conserved structure involved in the binding of elongation factors Tu and G and is the site of action of the ribosome-inactivating proteins (RIPs) . One such group, the N-glycosidase RIPs, act by the removal of a single adenine residue (A2660) believed to exist in a GAGA-containing tetraloop structure . The action of two RIPs, the catalytic A-chain from the heterodimeric toxic lectin ricin (RTA) and the single-chain RIP pokeweed antiviral protein (PAP), which are known to be highly homologous in tertiary structure, was determined on native ribosomes or naked 23S rRNA containing mutations designed to affect the structure of the GAGA tetraloop . One such mutant rRNA containing G2663C, which abolishes the potential tetraloop by disrupting the Watson-Crick base-pair involved in closing it, resulted in a loss of depurination by RTA, but not by PAP . A similar result was observed for mutant G2661A . The double mutant C2658G + G2663C, which restores the tetraloop-closing base-pair but in the reverse orientation, resulted in sensitivity to both PAP and RTA, as in the wild-type . Thus, the tetraloop structure is required for the action of RTA, but not of PAP, and unlike RTA, PAP does not require G at position 2661 . RNA containing the G2664C mutation, which lies outside the tetraloop, served as a substrate for both PAP and RTA . The comparison of the recognition elements for PAP and RTA was made with naked (deproteinised) rRNA, because RTA does not act on E . coli ribosomes . However, PAP is active on E . coli ribosomes, and it was found that the action of PAP on ribosomes containing the above mutations paralleled exactly that on the corresponding naked rRNAs . It is concluded that the recognition elements for PAP and RTA differ and may account, at least in part, for the fact that PAP but not RTA catalyses the depurination of E . coli ribosomes.

J Mol Biol, 1995 Dec 15, 254(5), 838 - 47
The involvement of two distinct regions of 23 S ribosomal RNA in tRNA selection; O'Connor M et al.; The role of ribosomal RNA in maintaining the accuracy of translation has been investigated genetically by selecting for rRNA mutations that promoted frameshifting at a specific site in a reporter gene in Escherichia coli . Mutations were recovered in two different regions of 23 S rRNA and each promoted readthrough of stop codons as well as increasing the levels of frameshifting . The first group of mutations was in a small stem loop (the 1916 loop) in domain IV of 23 S rRNA . This stem-loop has been mapped to the subunit interface of the ribosome, close to the decoding center on the 30 S subunit . The second group of mutations was in helix 89, one of the helices emerging from the central loop of domain V . Helix 89 has been implicated in subunit-subunit interactions and peptidyltransferase activity, and it is proposed that mutations in helix 89 influence the accuracy of decoding by affecting the interaction of the CCA end of the tRNA with the peptidyltransferase center.

J Mol Biol, 1995 Dec 15, 254(5), 815 - 37
Structural and functional dissections of transcription termination factor rho by random mutagenesis; Miwa Y et al.; Transcription termination factor rho from Escherichia coli is a homohexamer of 419 amino acid subunits and catalyzes an ATP-dependent release of nascent RNA transcripts . A rho monomer has three distinct domains functioning independently at the first approximation: the amino-terminal one quarter containing a primary RNA-binding site, the central 270-amino acids region constituting an ATP-binding domain with homologies to F1-ATPase, and the carboxy-terminal remainder with unknown function(s) . To further delineate the structural and functional organizations of rho protein, we undertook its random mutagenesis using error-prone polymerase chain reactions with the carboxy-terminal 100-amino acid region chosen as the initial target . From 14 mutants identified, rho protein was purified and characterized in vitro . Of these, 11 mutants are defective in termination in vivo and show decreased activities in various partial functions examined: ATP binding; RNA binding; and ATPase activities dependent on three cofactors with decreasing efficacies, poly(C), lambda cro RNA and poly(U) . A few of them are also affected in the putative secondary RNA-binding site that is functionally coupled to ATP hydrolysis . By contrast, the three other mutants are hyperactive in termination, poly(U)-dependent ATPase activity, and RNA interaction at the primary site . In these properties, the hyper-terminating mutants strikingly resemble the "super rho" mutant formerly found in the amino-terminal domain . Taken together, these findings indicate that the carboxy-terminal region plays a pivotal role in functionally coupling the RNA and ATP-binding domains, plausibly by acting as an interface for their interaction within or across individual subunits . In light of the reported X-ray crystallographic structure of F1-ATPase, we propose a model for the tertiary and quaternary structure of rho that is consistent with the observed mutational effects as well as a number of structural and functional properties characteristic of rho.

J Immunol, 1995 Dec 15, 155(12), 5711 - 8
Inhibition of vaccinia virus DNA replication by inducible expression of nitric oxide synthase; Melkova Z et al.; Nitric oxide (NO) exerts multiple biologic roles in animal cells through differential regulation of three distinct forms of NO synthase encoded by separate genes . Macrophage-inducible nitric oxide synthase (iNOS) has been correlated with inhibition of viral growth, but little is known about the mechanism of this effect . To study the antiviral role of NO, we have generated a vaccinia virus (VV) recombinant expressing iNOS under the control of Escherichia coli LacI operator/repressor elements . When cultured cells of various origins are infected with this recombinant virus, there is inducible expression of iNOS in the presence of isopropylthio-beta-galactoside, as determined by Western blot and by detection of nitrite, a NO oxidation product . The levels of nitrite increase with time after infection, correlating with marked inhibition of VV DNA and late protein synthesis . Expression of VV early proteins is not affected by NO . Inhibition of VV DNA synthesis is likely to be in part a consequence of NO-mediated inhibition of viral ribonucleotide reductase, as this inhibition can be partially overcome by addition of deoxyribonucleosides . Inhibition of the essential viral functions by NO results in a reduction of virus yields by 50 to 90%, depending on the cell line . Thus, our results demonstrate a direct antiviral effect of NO, with inhibition of VV replication occurring at the level of DNA synthesis.

Biochim Biophys Acta, 1995 Dec 14, 1245(3), 397 - 401
Control by phosphatidylglycerol of expression of the flhD gene in Escherichia coli; Mizushima T et al.; We reported elsewhere that mutation in the pgsA gene, responsible for the synthesis of phosphatidylglycerol, repressed the synthesis of flagellin and caused the loss of motility of Escherichia coli (Tomura et al., FEBS Letters 329, 287-290, 1993) . We now describe evidence for a decrease in promoter activity of the flhD gene, a master gene for flagellum synthesis, in the pgsA3 mutant . We constructed a plasmid with a promoter region of the flhD gene connected with the structure region of the lacZ gene . The activity of beta-galactosidase in the extract prepared from the pgsA3 mutant harboring the fusion plasmid was 30% of that in the wild type cells . This result means that phosphatidylglycerol is likely to be required for the initiation of transcription of the flhD gene . We also found that the motility-less phenotype of the mutant was partially suppressed by elevating incubation temperature . This suppression is caused by restoration of transcription of the flhD gene by high temperature . As the content of phosphatidylglycerol did not increase by elevating incubation temperature, we proposed that this suppression is caused by alternation of a physical structure of phospholipid bilayers in cytoplasmic membranes.

Biochem Biophys Res Commun, 1995 Dec 14, 217(2), 575 - 83
The pulmonary epithelial cell line L 2 as a new model for an inducible nitric oxide synthase expressing distal airway epithelial cell; Hoffmann G et al.; Nitric oxide released in large amounts by inducible nitric oxide synthase (iNOS)-containing pulmonary cells plays an important role in many aspects of lung function in health and disease . The aim of this study was to establish a permanent non tumor-derived alveolar epithelial cell line that exhibits the typical characteristics of an iNOS-expressing cell . Therefore, the pulmonary epithelial cell line L2 (adult rat) was incubated with lipopolysaccharide derived from Escherichia coli (serotype 0111:B4) and different cytokines . The strongest effect on iNOS gene expression and nitric oxide release could be detected when L2 cells were coincubated with interferon-gamma + tumor necrosis factor-alpha . iNOS complementary DNA concentration was 25 amol/microliters at 9h, and nitrite/nitrate levels were 99.43 +/- 3.97 nmol/10(6) cells at 24h, respectively . Our results show that L2 cells can be regarded as an appropriate model for investigating iNOS gene expression and nitric oxide functions in alveolar epithelial cells.

Biochem Biophys Res Commun, 1995 Dec 14, 217(2), 482 - 7
Functional expression of two forms of rat acyl-CoA oxidase and their substrate specificities; Setoyama C et al.; Using the reverse transcription of RNA followed by the polymerase chain reaction, we cloned the cDNAs for the rat acyl-CoA oxidases I and II, which are produced by alternative splicing from a single gene, and developed a system for their expression in Escherichia coli . The homogeneous preparations of these enzymes, without proteolytic procession, showed oxidase activity with acyl-CoAs having various acyl-chain lengths . The two types of the enzyme exhibited different substrate specificities with respect to the acyl-chain length, acyl-CoA oxidase I showing the optimum activity at shorter chain-length relative to acyl-CoA oxidase II.

Biochem Biophys Res Commun, 1995 Dec 14, 217(2), 428 - 36
Characterization of chimeric heme-copper respiratory oxidases using subunits I of Escherichia coli cytochrome b o and Halobacterium salinarium cytochrome aa3; Denda K et al.; We constructed chimeric enzymes with the Escherichia coli cytochrome bo and the Halobacterium salinarium cytochrome aa3 through recombinant DNA techniques and investigated their spectroscopic and biochemical properties . Although most of the chimeras could not retain hemes in the molecule, the chimeric enzyme containing helix VII of subunit I of the H . salinarium cytochrome aa3 showed the spectral properties similar to those of the native E . coli oxidase, suggesting that both the low-spin heme b and the high-spin heme o are associated with the chimeric subunit I . However, CuB was absent in the chimera . Helix VII of subunit I of the H . salinarium cytochrome aa3 is 70% similar to the counterpart of the E . coli cytochrome bo and further contains two invariant histidines which serve as the CuB ligands . These results indicate that helix VII must be arranged properly relative to helix VI which provides the third CuB ligand.

Nature, 1995 Dec 14, 378(6558), 690 - 7
A structural role for hormone in the thyroid hormone receptor; Wagner RL et al.; The crystal structure of the rat alpha 1 thyroid hormone receptor ligand-binding domain bound with a thyroid hormone agonist reveals that ligand is completely buried within the domain as part of the hydrophobic core . In addition, the carboxy-terminal activation domain forms an amphipathic helix, with its hydrophobic face constituting part of the hormone binding cavity . These observations suggest a structural role for ligand, in establishing the active conformation of the receptor, that is likely to underlie hormonal regulation of gene expression for the nuclear receptors.

Gene, 1995 Dec 12, 166(2), 237 - 42
Isolation and sequence determination of the cDNA encoding DNA polymerase delta from Drosophila melanogaster; Chiang CS et al.; The cDNA encoding the catalytic subunit of Drosophila melanogaster (Dm) DNA polymerase delta (Pol delta) was isolated by a combination of PCR amplification and cDNA library screening . The cDNA is 3457 nucleotides in length and contains an open reading frame (ORF) that encodes a protein of 1092 amino acids (124,799 Da) . The ORF contains the sequence that was determined for a peptide from the purified catalytic subunit of Dm Pol delta . Polyclonal antibodies raised against Dm Pol delta specifically recognize a protein of the expected size when the cDNA is expressed in either Escherichia coli or insect cells . Comparison of the deduced aa sequence with other Pol delta sequences demonstrates that Pol delta is one of the most highly conserved of the DNA polymerases.

Gene, 1995 Dec 12, 166(2), 187 - 95
Epitope mapping of anti-HIV and anti-HCV monoclonal antibodies and characterization of epitope mimics using a filamentous phage peptide library; Grihalde ND et al.; A large filamentous phage library (1 x 10(9) clones) displaying random 30-amino-acid (aa) sequences on the N terminus of the pIII coat protein was constructed and characterized . Clones in the library were affinity selected for binding to monoclonal antibodies (mAb) against two viral antigens, the HIV gp120 protein and the HCV core protein . The obtained aa sequences precisely identified the epitopes recognized by the mAb . Binding of peptide-carrying phages to the Ab was demonstrated by ELISA, Western blot and the surface plasmon resonance (SPR) method . The mAb-specific peptides were transferred via genetic techniques onto the N terminus of Escherichia coli alkaline phosphatase (AP) . When fused to the enzyme, the peptides maintained their ability to bind their respective mAb, indicating that the peptides contained the necessary contact residues for binding . The affinity of the peptides was estimated to be 100 nM by SPR . A comparison is presented of the relative affinities of phage-derived peptides to the native viral epitopes also displayed on the AP scaffold . The approach of transferring epitopes from phage to AP for further evaluation should be applicable to many other mAb or receptors.

Biochemistry, 1995 Dec 12, 34(49), 16228 - 34
The pleckstrin homology domain of phospholipase C-delta 1 binds with high affinity to phosphatidylinositol 4,5-bisphosphate in bilayer membranes; Garcia P et al.; The pleckstrin homology (PH) domain of phospholipase C-delta 1 (PLC-delta 1) binds to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in phospholipid membranes with an affinity (Ka approximately 10(6) M-1) and specificity comparable to those of the native enzyme . PLC-delta 1 and its PH domain also bind inositol 1,4,5-trisphosphate, the polar head group of PI(4,5)P2, with comparable affinity and approximately 1:1 stoichiometry . A peptide corresponding to amino acids 30-43 of the PLC-delta 1 PH domain contains several basic residues predicted to bind PI(4,5)P2, but binds weakly and with little specificity for PI(4,5)P2; hence the tertiary structure of the isolated PH domain is required for high affinity PI(4,5)P2 binding . Our PI-(4,5)P2 binding results support the hypothesis that the intact PH domain, serving as a specific tether, directs PLC-delta 1 to membranes enriched in PI(4,5)P2 and permits the active site, located elsewhere in the protein, to hydrolyze multiple substrate molecules before this enzyme dissociates from the membrane surface.

Biochemistry, 1995 Dec 12, 34(49), 16186 - 93
Proton-translocating carboxyl of subunit c of F1Fo H(+)-ATP synthase: the unique environment suggested by the pKa determined by 1H NMR; Assadi-Porter FM et al.; Subunit c of the H(+)-transporting F1Fo ATP synthase (EC 3.6.1.34) is thought to fold across the membrane as a hairpin of two alpha helices with a conserved Asp/Glu residue, centered in the second membrane-spanning helix, which is thought to function in H+ translocation . NMR studies indicate that the purified subunit c from Escherichia coli is also folded as a hairpin in a chloroform/methanol/H2O (4:4:1) solvent mixture {Girvin, M . E., & Fillingame, R . H . (1993) Biochemistry 32, 12167-12177} and that the conserved Asp remains uniquely reactive in this solvent mixture {Girvin, M . E., & Fillingame, R . H . (1994) Biochemistry 33, 665-674} . The pKa of Asp61 is of interest because of its unique reactivity and because it is thought to protonate and deprotonate during each proton translocation cycle . We have determined the pKa value of the carboxyl group of the functional Asp in wild type and two functional, mutant subunit c proteins, i.e . the Ala24-->Asp (D24D61) and the Ala24-->Asp/Asp61-->Asn (D24N61) mutant proteins . The pKa values were determined by 1H NMR spectroscopy by measuring changes in the alpha and beta proton chemical shifts by constant time two-dimensional (2D) correlated spectroscopy . The pKa of Asp61 in the purified wild type protein was 7.1 . This pKa was significantly higher than the pKa of the other two Asp residues, i.e . Asp7 and Asp44 which were 5.4 and 5.6, respectively . The pKa of the two Glu residues in the protein were determined by 2D total correlation spectroscopy and found to be approximately 5.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Dec 12, 34(49), 16097 - 106
Primer synthesis kinetics by Escherichia coli primase on single-stranded DNA templates; Swart JR et al.; The kinetics of primer RNA initiation and elongation by Escherichia coli primase were measured . The single-stranded DNA template that was used to develop the system, d(CAGA-(CA)5CTGCAAAGC), contained: (1) the preferred initiating trinucleotide d(CTG); (2) five residues 3' to the d(CTG), the minimum required for efficient primer synthesis; and (3) a single guanine placed near the 5'-end to facilitate study of cytidine triphosphate analog incorporation at a unique site . The assay monitored radiolabeled nucleotide incorporation into the RNA primers . The various primers were separated by length using denaturing polyacrylamide gel electrophoresis . Different types of primers were observed when synthesis was monitored using gamma- versus alpha-radiolabeled nucleotides as the probe . When {gamma-32P}-ATP incorporation was the probe, only primers initiated with ATP from the unique template thymine were observed . The sequences of these primers were complementary to that of the template . No primers shorter than a 12-mer accumulated, demonstrating that formation of the first phosphodiester bond was much slower than that of the next 10 phosphodiester bonds . The longest primer observed when monitoring {gamma-32P}ATP incorporation was 16 nucleotides long, the correct length for a primer completely template-directed and initiated at the unique thymine . Misinsertion of a noncognate nucleotide at the template's guanine indicated very poor nucleotide discrimination by this enzyme . When {alpha-32P}UTP was the probe for primer synthesis, all primers synthesized were observed whether or not they were initiated with ATP . Under these conditions, "overlong" primers and the above-described template length-dependent primers were observed . The template length-dependent primers accumulated faster than the overlong primers, but, at long incubation times, the overlong primers became the dominant species . The overlong primers were not fully related to the template length-dependent primers since they were not initiated complementary to the template d(CTG) . Nevertheless, the overlong primers did appear to arise as a consequence of the template length-dependent species since their length was double and they arose in the time course after the length-dependent species.

Biochemistry, 1995 Dec 12, 34(49), 15918 - 24
Characterization of the different spectral forms of glutamate 1-semialdehyde aminotransferase by mass spectrometry; Brody S et al.; Glutamate 1-semialdehyde aminotransferase produces delta-aminolevulinate for the synthesis of chlorophyll, heme, and other tetrapyrrole pigments . The native enzyme from Synechococcus is pale yellow and has absorption maxima at 338 and 418 nm from vitamin B6 . Yellow, colorless, and pink forms of the protein are obtained by treatment with 4,5-dioxovalerate, 4,5-diaminovalerate, and acetylenic GABA, respectively . Compared to the native enzyme, the 418 nm absorption maximum in the yellow enzyme is enhanced and the 338 nm maximum reduced while the colorless enzyme has a heightened maximum at 338 nm and a barely detectable peak at 418 nm . The pink enzyme has an absorption maximum at 560 nm . When the native and colorless enzymes are repeatedly diluted in 0.5 M Na2HPO4, pH 7.0, and reconcentrated, pyridoxamine 5'-phosphate is released and the 338 nm maximum lost . Thus the 338 nm absorption maximum is associated with noncovalently bound pyridoxamine 5'-phosphate . NaBH4 reduction proved that the absorbance at 418 nm is from pyridoxal 5'-phosphate cofactor bound by a Schiff base to the protein . When the native, colorless, and yellow enzymes were subjected to electrospray ionization mass spectrometry, the B6 cofactor dissociated from the protein and gave a molecular weight of 46,401-46,418 . Acetylenic GABA and NaBH4 were used for protein modification, and they reacted with the native and yellow enzymes but had no effect on the colorless enzyme . Pyridoxal 5'-phosphate bound covalently to the protein after NaBH4 reduction . Acetylenic GABA attached covalently to the enzyme produced an additional mass peak, 123-126 mass units higher, in the electrospray ionization spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Dec 12, 34(49), 15886 - 9
The other function of DNA photolyase: stimulation of excision repair of chemical damage to DNA; Ozer Z et al.; DNA photolyase is a light-dependent DNA repair enzyme . It binds to cyclobutane pyrimidine dimers <PyrPyr> in DNA and upon excitation with a blue light photon splits the cyclobutane ring and restores the pyrimidines to native forms . The enzyme is specific for pyrimidine dimers, and it is not known to catalyze any other reaction either in ground or in excited state . However, when photolyase binds to <PyrPyr> but cannot catalyze repair because of lack of photoreactivating light, it still aids DNA repair by stimulating the nucleotide excision repair system . Recently, it was found that yeast photolyase binds to other lesions in DNA . In particular, the binding to cisplatin damaged DNA was highly specific . However, in vivo experiments revealed that this binding, in contrast to <PyrPyr> binding, did not stimulate but actually inhibited the removal of cisplatin damage by excision repair and hence photolyase sensitized cells to killing by cisplatin . In the present study, it is demonstrated that Escherichia coli DNA photolyase binds specifically to cisplatin 1,2-d(GpG) intrastrand cross-link and stimulates the removal of the lesion by E . coli excision nuclease in vitro . In agreement with the in vitro data, in vivo experiments revealed that photolyase makes cells more resistant to cisplatin killing.

FEBS Lett, 1995 Dec 11, 377(1), 62 - 6
Cloning and expression in Escherichia coli of cDNA encoding house dust mite allergen Der f 3, serine protease from Dermatophagoides farinae; Nishiyama C et al.; Der f 3 is one of the allergens produced by house dust mite Dermatophagoides farinae showing serine protease activity . Based on its amino acid sequence, a cDNA clone encoding Der f 3 was isolated from a cDNA library of D . farinae . Sequencing analysis of the clone revealed the presence of an open reading frame of 780 bp, which encodes a mature protein of 232 amino acids with 27 amino acids of pre-pro sequence at the N-terminus . When proDer f 3 was produced in Escherichia coli as a fused protein with glutathione-S-transferase, the fused protein was accumulated as inclusion bodies . The protein purified with 8 M urea and glutathione-affinity column chromatography, however, did not show protease activity . When an arginine residue was introduced at the C-terminus of the pro-region in place of threonine, removal of the pro-region to produce an active mature protease was observed . The specificity and the activity of this recombinant protease were almost the same as those of native Der f 3.

FEBS Lett, 1995 Dec 11, 377(1), 44 - 6
Mg2+ activation of Escherichia coli inorganic pyrophosphatase; Avaeva SM et al.; Further refinement of X-ray data on Escherichia coli inorganic pyrophosphatase {Oganessyan et al . (1994) FEBS Lett . 348, 301-304} to 2.2 A reveals a system of noncovalent interactions involving Tyr55 and Tyr141 in the active site . The pKa for one of the eight Tyr residues in wild-type pyrophosphatase is as low as 9.1 and further decreases to 8.1 upon Mg2+ binding, generating characteristic changes in the absorption spectrum . These effects are lost in a Y55F but not in a Y141F variant . It is suggested that the lower-affinity site for Mg2+ in the enzyme is formed by Tyr55 and Asp70, which are in close proximity in the apo-enzyme structure.

FEBS Lett, 1995 Dec 11, 377(1), 41 - 3
The 65-kDa protein derived from the internal translational start site of the clpA gene blocks autodegradation of ClpA by the ATP-dependent protease Ti in Escherichia coli; Seol JH et al.; The ATP-dependent protease Ti consists of two different components: ClpA containing ATP-cleaving sites and ClpP having serine active sites for proteolysis . The clpA gene has dual translational start sites and therefore encodes two polypeptides with sizes of 84 and 65 kDa (referred to as ClpA84 and ClpA65, respectively) . Here we show that ClpA84, but not ClpA65, is degraded in vitro by ClpP in the presence of ATP . The ClpP-mediated hydrolysis of ClpA84 could be prevented by casein, which is an excellent substrate of protease Ti (i.e . ClpA84/ClpP complex) . Thus, it appears that free form of ClpA84 competes with casein for the degradation by ClpA/ClpP complex . Furthermore, ClpA65 inhibited the auto-degradation of ClpA84 by the complex . These results suggest that ClpA65 may play an important role in the control of the ClpA84 level and in turn in the regulation of ATP-dependent protein breakdown in E . coli.

FEBS Lett, 1995 Dec 11, 377(1), 37 - 40
A rainbow trout SRY-type gene expressed in pituitary glands; Ito M et al.; A Sox (SRY-type HMG box) gene, designated SoxP1, was isolated from a cDNA library made from pituitaries of immature rainbow trout . Sequence analysis indicated that the cDNA had an open reading frame encoding 467 amino acid residues containing a DNA binding motif, known as the high mobility group (HMG) box . Northern blot analysis showed trout SoxP1 mRNA was detected in pituitaries and gonadal tissues, but not in liver, spleen, and heart . In pituitaries, trout SoxP1 mRNA was more abundant in immature fish than in mature fish . Gel shift retardation analysis indicated that the recombinant HMG box protein of SoxP1 produced in E . coli had a DNA binding property for an AACAAT or AACAAAG sequence . These findings suggest that the trout SoxP1 protein may play certain roles in growth or maturation in pituitaries as a transcription factor.

Nucleic Acids Res, 1995 Dec 11, 23(23), 4864 - 71
Homodimerization of the human U1 snRNP-specific protein C; Gunnewiek JM et al.; The U1 snRNP-specific protein C contains an N-terminal zinc finger-like CH motif which is required for the binding of the U1C protein to the U1 snRNP particle . Recently a similar motif was reported to be essential for in vivo homodimerization of the yeast splicing factor PRP9 . In the present study we demonstrate that the human U1C protein is able to form homodimers as well . U1C homodimers are found when (i) the human U1C protein is expressed in Escherichia coli, (ii) immunoprecipitations with anti-U1C antibodies are performed on in vitro translated U1C, and when (iii) the yeast two hybrid system is used . Analyses of mutant U1C proteins in an in vitro dimerization assay and the yeast two hybrid system revealed that amino acids within the CH motif, i.e . between positions 22 and 30, are required for homodimerization.

Nucleic Acids Res, 1995 Dec 11, 23(23), 4850 - 6
A new vector for recombination-based cloning of large DNA fragments from yeast artificial chromosomes; Bradshaw MS et al.; The functional analysis of genes frequently requires manipulation of large genomic regions embedded in yeast artificial chromosomes (YACs) . We have designed a yeast-bacteria shuttle vector, pClasper, that can be used to clone specific regions of interest from YACs by homologous recombination . The important feature of pClasper is the presence of the mini-F factor replicon . This leads to a significant increase in the size of the plasmid inserts that can be maintained in bacteria after cloning by homologous recombination in yeast . The utility of this vector lies in its ability to maintain large fragments in bacteria and yeast, allowing for mutagenesis in yeast and simplified preparation of plasmid DNA in bacteria . Using PCR-generated recombinogenic fragments in pClasper we cloned a 27 kb region from a YAC containing the Hoxc cluster and a 130 kb region containing the entire Hoxb cluster . No rearrangements were seen when the recombinants in the shuttle vector were transferred to bacteria . We outline the potential uses of pClasper for functional studies of large genomic regions by transgenic and other analyses.

Nucleic Acids Res, 1995 Dec 11, 23(23), 4836 - 43
Characterization of the XRCC1-DNA ligase III complex in vitro and its absence from mutant hamster cells; Caldecott KW et al.; The human DNA repair protein XRCC1 was overexpressed as a histidine-tagged polypeptide (denoted XRCC1-His) in Escherichia coli and purified in milligram quantities by affinity chromatography . XRCC1-His complemented the mutant Chinese hamster ovary cell line EM9 when constitutively expressed from a plasmid or when introduced by electroporation . XRCC1-His directly interacted with human DNA ligase III in vitro to form a complex that was resistant to 2 M NaCl . XRCC1-His interacted equally well with DNA ligase III from Bloom syndrome, HeLa and MRC5 cells, indicating that Bloom syndrome DNA ligase III is normal in this respect . Detection of DNA ligase III on far Western blots by radiolabelled XRCC1-His indicated that the level of the DNA ligase polypeptide was reduced approximately 4-fold in the mutant EM9 and also in EM-C11, a second member of the XRCC1 complementation group . Decreased levels of polypeptide thus account for most of the approximately 6-fold reduced DNA ligase III activity observed previously in EM9 . Immunodetection of XRCC1 on Western blots revealed that the level of this polypeptide was also decreased in EM9 and EM-C11 (> 10-fold), indicating that the XRCC1-DNA ligase III complex is much reduced in the two CHO mutants.

Nucleic Acids Res, 1995 Dec 11, 23(23), 4793 - 8
Transition state stabilization by the 'high' motif of class I aminoacyl-tRNA synthetases: the case of Escherichia coli methionyl-tRNA synthetase; Schmitt E et al.; Methionyl-tRNA synthetase belongs to the class I aminoacyl-tRNA synthetase family characterized both by a catalytic center built around a Rossmann Fold and by the presence of the two peptidic marker sequences HIGH and KMSKS . In this study, the role of the 21HLGH24 motif of Escherichia coli methionyl-tRNA synthetase was studied in a systematic fashion by site-directed mutagenesis . It is shown that the two histidine residues play a crucial role in the catalysis of the methionyl adenylate formation by participating in the stabilisation of the ATP phosphate chain during the transition state . Moreover, the results suggest the involvement of the epsilon-imino group of histidine 21 and of the delta-imino group of histidine 24 . Notably, the substitution of either the leucine or the glycine residue of the HLGH motif by alanine had no effect on the catalysis . From the data and from other studies with class I aminoacyl-tRNA synthetases, concomitant positive contributions of the HIGH and KMSKS sequences to reach the transition state of aminoacyl adenylate formation can be envisaged.

Anal Biochem, 1995 Dec 10, 232(2), 180 - 9
Fluorescence recovery assay: a continuous assay for processive DNA polymerases applied specifically to DNA polymerase III holoenzyme; Griep MA; A continuous assay was developed for processive DNA polymerases . The specific enzyme used to develop the assay was the most processive polymerase known, Escherichia coli DNA polymerase III holoenzyme . The assay was based upon the recovery of the intrinsic fluorescence of single-stranded DNA binding protein (SSB) as it was displaced from the DNA template during DNA synthesis . The intrinsic fluorescence of SSB was quenched by as much as 80% when it bound to single-stranded DNA . As the DNA was replicated, SSB was displaced and recovered its fluorescence . The amount of fluorescence recovered was directly proportional to the amount of DNA synthesized and was used to quantitate the rate of DNA synthesis . However, since 50 to 60 nucleotides must be replicated for every SSB tetramer released, the assay is expected to work best for processive DNA polymerases . The only requirement for using this assay with other DNA polymerases is that they be able to synthesize DNA on a template coated with SSB . The replication SSBs do not pose an obstacle to the assay because they all appear to have intrinsic fluorescence that is sensitive to their ssDNA-bound state.

Mol Gen Genet, 1995 Dec 10, 249(4), 456 - 64
Functional analysis by site-directed mutagenesis of individual amino acid residues in the flavin domain of Neurospora crassa nitrate reductase; Gonzalez C et al.; Nitrate reductase of Neurospora crassa is a complex multi-redox protein composed of two identical subunits, each of which contains three distinct domains, an amino-terminal domain that contains a molybdopterin cofactor, a central heme-containing domain, and a carboxy-terminal domain which binds a flavin and a pyridine nucleotide cofactor . The flavin domain of nitrate reductase appears to have structural and functional similarity to ferredoxin NADPH reductase (FNR) . Using the crystal structure of FNR and amino acid identities in numerous nitrate reductases as guides, site-directed mutagenesis was used to replace specific amino acids suspected to be involved in the binding of the flavin or pyridine nucleotide cofactors and thus important for the catalytic function of the flavin domain . Each mutant flavin domain protein was expressed in Escherichia coli and analyzed for NADPH: ferricyanide reductase activity . The effect of each amino acid substitution upon the activity of the complete nitrate reductase reaction was also examined by transforming each manipulated gene into a nit-3- null mutant of N . crassa . Our results identify amino acid residues which are critical for function of the flavin domain of nitrate reductase and appear to be important for the binding of the flavin or the pyridine nucleotide cofactors.

Science, 1995 Dec 8, 270(5242), 1653 - 7
Transcription against an applied force; Yin H et al.; The force produced by a single molecule of Escherichia coli RNA polymerase during transcription was measured optically . Polymerase immobilized on a surface was used to transcribe a DNA template attached to a polystyrene bead 0.5 micrometer in diameter . The bead position was measured by interferometry while a force opposing translocation of the polymerase along the DNA was applied with an optical trap . At saturating nucleoside triphosphate concentrations, polymerase molecules stalled reversibly at a mean applied force estimated to be 14 piconewtons . This force is substantially larger than those measured for the cytoskeletal motors kinesin and myosin and exceeds mechanical loads that are estimated to oppose transcriptional elongation in vivo . The data are consistent with efficient conversion of the free energy liberated by RNA synthesis into mechanical work.

J Mol Biol, 1995 Dec 8, 254(4), 552 - 65
Regulation of codBA operon expression in Escherichia coli by UTP-dependent reiterative transcription and UTP-sensitive transcriptional start site switching; Qi F et al.; Reiterative transcription is the repetitive addition of nucleotides to the 3' end of a nascent transcript due to slippage between the transcript and DNA template . Recently, we showed that pyrimidine-mediated regulation of pyrBI operon expression in Escherichia coli occurs, in part, through a mechanism in which induction of UTP-dependent reiterative transcription within the initially transcribed region prevents downstream extension of the nascent transcript to include structural gene sequences . In this study we demonstrate that pyrimidine-mediated regulation of codBA operon expression in E . coli also involves UTP-dependent reiterative transcription during initiation; however, the mechanism is different from that of the pyrBI operon . The initially transcribed region of the codBA promoter contains the sequence GATTTTTTG (non-template strand) . Our results show that transcription is initiated primarily at the first two bases designated G7 and A8 (counting from the -10 region) . When transcripts are initiated at position A8, UTP-dependent reiterative transcription always occurs within the run of T residues in the initially transcribed region . The AUUUUn (where n = 1 to > 15) transcripts produced by this reaction are not extended productively to include downstream codBA sequences . In contrast, most transcripts initiated at position G7 do not engage in reiterative transcription and can be elongated normally . Characterization of a codBA promoter mutation that prevents reiterative transcription showed that this reaction is required for virtually all pyrimidine-mediated regulation of operon expression and that UTP levels control the selection of the G7 and A8 transcriptional start sites . These results suggest a model for regulation in which high intracellular levels of UTP favor transcriptional initiation at position A8 and thus the accompanying reiterative transcription, which together preclude initiation at position G7 . Low levels of UTP inhibit initiation at position A8 and the associated reiterative transcription, thereby allowing high levels of initiation at position G7 and operon expression . Our results also indicate critical sequence requirements for reiterative transcription, which are important for understanding the mechanism of this reaction as well as for identifying other promoters at which this reaction may occur . Of particular interest is the indication that an RNA:DNA hybrid forms during transcriptional initiation and the strength of this hybrid controls the extent of reiterative transcription.

J Mol Biol, 1995 Dec 8, 254(4), 538 - 43
The mitochondrial ClpB homolog Hsp78 cooperates with matrix Hsp70 in maintenance of mitochondrial function; Moczko M et al.; The mitochondrial heat shock protein Hsp78 is a member of the Hsp104/Clp family with unknown function . Saccharomyces cerevisiae deletion mutants of HSP78 show wild-type like growth . We report that deletion of the HSP78 gene in yeast strains with point mutations in the SSC1 gene (encoding matrix Hsp70) led to loss of mitochondrial DNA, indicating that at least one of the heat shock proteins Hsp78 and mt-Hsp70 is needed to maintain a rho+ state of the mitochondrial genome . Mitochondria isolated from these double mutants had a strongly reduced membrane potential, explaining defects in the rate of preprotein import . The lack of Hsp78 led to aggregation of the mutant mt-Hsp70 while other matrix chaperones stayed soluble . We conclude that Hsp78 is required to keep mutant forms of mt-Hsp70 soluble and suggest a cooperation of Hsp78 and mt-Hsp70 in maintenance of essential mitochondrial functions.

J Biol Chem, 1995 Dec 8, 270(49), 29570 - 7
DnaX complex of Escherichia coli DNA polymerase III holoenzyme . The chi psi complex functions by increasing the affinity of tau and gamma for delta.delta' to a physiologically relevant range; Olson MW et al.; An artificial operon that contains tandem holC-holD genes was used to overproduce a complex of the chi and psi subunits of the DNA polymerase III holoenzyme . Normally insoluble by itself, psi forms a tight soluble complex with chi . A purification procedure that yields pure, active chi psi complex in 100-mg quantities suitable for biophysical studies is reported . Sedimentation equilibrium studies demonstrate that chi psi is a 1:1 heterodimer . The presence of chi psi dramatically lowers the level of delta.delta' required to reconstitute holoenzyme to levels expected in vivo . That chi psi accomplishes this by binding to gamma or tau and increasing their affinity for delta.delta' was demonstrated by surface plasmon resonance using a Pharmacia BIA-core instrument . In the absence of delta.delta', chi psi binds to either the gamma or tau DnaX protein with Kd = 2 nM.

J Biol Chem, 1995 Dec 8, 270(49), 29563 - 9
DnaX complex of Escherichia coli DNA polymerase III holoenzyme . Physical characterization of the DnaX subunits and complexes; Dallmann HG et al.; A physical characterization of the tau and gamma subunits of the Escherichia coli DNA polymerase III holoenzyme and their complexes with the delta, delta', chi, and psi subunits is presented . The native molecular mass of the tau and gamma subunits was determined to be 255,000 and 189,000 Da, respectively, by sedimentation equilibrium analytical ultracentrifugation . Both values indicate a tetrameric quaternary structure . The tau and gamma complexes were reconstituted and purified using two different methods . Both complexes assembled readily and were reconstituted at subunit concentrations approaching physiological levels . The stoichiometries of the tau and gamma complexes, as determined by quantitative densitometry of SDS-polyacrylamide gels, were found to be tau 4 delta 1 delta' 1 chi 1 psi 1 and gamma 4 delta 1 delta' 1 chi 1 psi 1 . BIAcore analysis demonstrated that the formation of large multiprotein complexes of holoenzyme subunits depends on the presence of the tau subunit; gamma could not substitute . We present a model for a gamma-less form of DNA polymerase III holoenzyme that has asymmetrical structural features that may be responsible for the functional asymmetry observed in holoenzyme . The stoichiometry of the reconstituted DNA polymerase III* component of holoenzyme in this model is (alpha epsilon theta)2DnaX4 delta 1 delta' 1 chi 1 psi 1.

J Biol Chem, 1995 Dec 8, 270(49), 29555 - 62
DnaX complex of Escherichia coli DNA polymerase III holoenzyme . Central role of tau in initiation complex assembly and in determining the functional asymmetry of holoenzyme; Dallmann HG et al.; The alternative forms of the DnaX protein found in Escherichia coli DNA polymerase III holoenzyme, tau and gamma, were purified from extracts of strains carrying overexpressing plasmids mutated in their frameshifting sequences such that they produced only one subunit or the other . The purified subunits were used to reconstitute the tau and gamma complexes which were characterized by functional assays . The gamma complex-reconstituted holoenzyme required a stoichiometric excess of DNA polymerase III core, beyond physiological levels, for activity . The tau subunit stimulated the gamma complex 2-fold, but could not be used to reconstitute a holoenzyme with gamma complex and stoichiometric quantities of core . In the presence of adenosine 5'-O-(3'-thiotriphospate) (ATP gamma S), the DNA polymerase III holoenzyme behaves as an asymmetric dimer; it can form only initiation complexes with primed DNA in one-half of the enzyme (Johanson, K . O., and McHenry, C . S . (1984) J . Biol . Chem . 259, 4589-4595) . An asymmetric distribution of two products of the dnaX gene, gamma and tau, has been postulated to underlie the asymmetry of holoenzyme . To provide a direct test for this hypothesis, we reconstituted holoenzyme containing only the gamma or tau DnaX proteins . We observed that, although gamma could function in the presence of ATP and high concentrations of DNA polymerase III core, it was nearly inert in the presence of ATP gamma S . In contrast, tau-containing holoenzyme behaved exactly like native holoenzyme in the presence of ATP gamma S . These results implicate tau as a key component required to reconstitute holoenzyme with native behavior and show that tau plays a key role in initiation complex formation . These results also show that gamma is not a necessary component, since all of the known properties of native holoenzyme can be reproduced with a 9-subunit tau-holoenzyme.

J Biol Chem, 1995 Dec 8, 270(49), 29428 - 32
The beta subunit Rif-cluster I is only angstroms away from the active center of Escherichia coli RNA polymerase; Severinov K et al.; Ribonucleotide analogs bound in the initiating site of Escherichia coli RNA polymerase-promoter complex were cross-linked to the beta subunit . Using limited proteolysis and chemical degradation, the cross-link was mapped to a segment of beta between amino acids Val516 and Arg540 . This region (Rif-cluster I) is known to harbor many rifampicin-resistant (RifR) mutations . The results demonstrate that Rif-culster I is part of the "5'-face" of the active center and provide structural basis for the long-known effects of RifR mutations on transcription initiation, elongation, and termination.

J Biol Chem, 1995 Dec 8, 270(49), 29407 - 12
The essential arginine residue at position 210 in the alpha subunit of the Escherichia coli ATP synthase can be transferred to position 252 with partial retention of activity; Hatch LP et al.; The substitution of arginine at position 210 in the alpha subunit of Escherichia coli F0F1-ATPase by either lysine or alanine causes dominance in complementation tests with a chromosomal c subunit mutation . Reversal of dominance was achieved for the alpha R210K mutation but not for the alpha R210A mutation by the presence of an aspartic acid residue at position 50 or at position 252 in the alpha subunit . It was concluded that position 210 in putative helix 4 of a previously proposed model of the alpha subunit is close to position 252 in putative helix 5 and to position 50 in putative helix 1 . The juxtaposition of residues 252 and 210 was also indicated by the observation that the double mutant alpha R210Q/Q252R was partially functional . A revertant of the partially functional double mutant, isolated on succinate medium, was found to contain a third mutation resulting in Pro-204 in the alpha subunit being replaced by threonine . That the revertant phenotype was due to the alpha P204T change was confirmed by site-directed mutagenesis . ATP synthesis in the revertant strain was at near normal levels as judged by growth yield experiments, but the revertant strain was unable to pump protons in response to ATP hydrolysis.

J Biol Chem, 1995 Dec 8, 270(49), 29217 - 23
Biosynthesis of Azorhizobium caulinodans Nod factors . Study of the activity of the NodABCS proteins by expression of the genes in Escherichia coli; Mergaert P et al.; By in vitro and in vivo studies with Escherichia coli expressing different combinations of the nodABCS genes of Azorhizobium caulinodans, Nod factor intermediates were identified and their structures determined using mass spectrometry . Substrate-product relationships were studied by time course experiments, and the Nod factor biosynthetic pathway was partially resolved . E . coli strains, harboring nodA and/or nodB, did not produce Nod metabolites, whereas the strain expressing nodC produced chitooligosaccharides . Thus, the first committed step was the production of the carbohydrate backbone . Bacitracin and tunicamycin did not affect this step, suggesting that undecaprenyl pyrophosphate-linked intermediates were not involved . The second step was the deacetylation of chitooligosaccharides by NodB since the E . coli strain expressing nodBC produced chitooligosaccharides, deacetylated at the non-reducing end and since the NodC products were precursors of the NodBC products . A strain expressing nodBCS produced N-methylated oligosaccharides, whereas a strain expressing nodCS produced unmethylated oligosaccharides . Time course experiments showed that methylation occurred after deacetylation . Thus, NodS acted after NodB . The NodBCS metabolites were partially converted to lipo-chitooligosaccharides when the nodABCS genes were expressed, showing that NodA was involved in the acylation and acted after NodS.

J Biol Chem, 1995 Dec 8, 270(49), 29105 - 10
A rice HAL2-like gene encodes a Ca(2+)-sensitive 3'(2'),5'-diphosphonucleoside 3'(2')-phosphohydrolase and complements yeast met22 and Escherichia coli cysQ mutations; Peng Z et al.; A plant homolog of yeast HAL2 gene (RHL) was cloned from rice (Orizya sativa L.) . The RHL cDNA complemented an Escherichia coli cysteine auxotrophic mutant, cysQ, and the yeast HAL2 mutant, met22 . The latter is a methionine auxotroph and cannot use sulfate, sulfite, or sulfide as sulfur sources but exhibits wild-type activities of the enzymes necessary to assimilate sulfate and has normal sulfur uptake system . These results demonstrated that HAL2, cysQ, and RHL genes encode proteins with similar function in sulfur assimilatory pathway . The RHL cDNA expressed a 40-kDa protein that was shown to catalyze the conversion of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) to adenosine 5'-phosphosulfate (APS) and 3'(2')-phosphoadenosine 5'-phosphate (PAP) to AMP . The enzyme activity is Mg(2+)-dependent, sensitive to Ca2+, Li+, and Na+ and activated by K+ . The inhibition by Ca2+ depends on the Mg2+/Ca2+ ratio and is reversible by high Mg2+ concentration . The substrate specificity and kinetics of RHL enzyme are very similar to the Chlorella 3'(2'),5'-diphosphonucleoside 3'(2')-phosphohydrolase (DPNPase) . Our evidence suggests that this enzyme regulates the flux of sulfur in the sulfur-activation pathway by converting PAPS to APS . Several residues that are essential for the activity of this enzyme were identified by site-directed mutagenesis, and the possible role of DPNPase in salt tolerance is discussed.

J Biol Chem, 1995 Dec 8, 270(49), 29096 - 104
Pleiotropic regulation of central carbohydrate metabolism in Escherichia coli via the gene csrA; Sabnis NA et al.; The carbon storage regulator gene csrA has been shown previously to dramatically affect the biosynthesis of intracellular glycogen in Escherichia coli through its negative control of the expression of two glycogen biosynthetic operons and the gluconeogenic gene pckA (Romeo, T., Gong, M., Liu, M . Y., and Brun-Zinkernagel, A . M . (1993) J . Bacteriol . 175, 4744-4755) . Examination of the effects of csrA on several enzymes, genes, and metabolites of central carbohydrate metabolism now establishes a more extensive role for csrA in directing intracellular carbon flux . Phosphoglucomutase and the gluconeogenic enzymes fructose-1,6-bisphosphatase and phosphoenolpyruvate synthetase were found to be under the negative control of csrA, and these enzyme activities were maximal during the early stationary phase of growth . The enzymes glucose-6-phosphate isomerase, triose-phosphate isomerase, and enolase were positively regulated by csrA . Thus, csrA exerts reciprocal effects on glycolysis versus gluconeogenesis and glycogen biosynthesis . The glycolytic isozymes pyruvate kinase F and A (encoded by pykF and pykA, respectively) and phosphofructokinase I and II (pfkA and pfkB, respectively) exhibited differential regulation via csrA . Since the individual members of these isozyme pairs are allosterically regulated by different cellular metabolites, csrA is also capable of fine-tuning the allosteric regulation of glycolysis . In contrast, the expression of genes of the pentose phosphate pathway was weakly or negligibly affected by csrA.

J Biol Chem, 1995 Dec 8, 270(49), 29063 - 6
Molecular cloning and characterization of AqpZ, a water channel from Escherichia coli; Calamita G et al.; The aquaporin family of molecular water channels is widely expressed throughout the plant and animal kingdoms . No bacterial aquaporins are known; however, sequence-related bacterial genes have been identified that encode glycerol facilitators (glpF) . By homology cloning, a novel aquaporin-related DNA (aqpZ) was identified that contained no surface N-glycosylation consensus . The aqpZ RNA was not identified in mammalian mRNA by Northern analysis and exhibited bacterial codon usage preferences . Southern analysis failed to demonstrate aqpZ in mammalian genomic DNA, whereas a strongly reactive DNA was present in chromosomal DNA from Escherichia coli and other bacterial species and did not correspond to glpF . The aqpZ DNA isolated from E . coli contained a 693-base pair open reading frame encoding a polypeptide 28-38% identical to known aquaporins . When compared with other aquaporins, aqpZ encodes a 10-residue insert preceding exofacial loop C, truncated NH2 and COOH termini, and no cysteines at known mercury-sensitive sites . Expression of aqpZ cRNA conferred Xenopus oocytes with a 15-fold increase in osmotic water permeability, which was maximal after 5 days of expression, was not inhibited with HgCl2, exhibited a low activation energy (Ea = 3.8 kcal/mol), and failed to transport nonionic solutes such as urea and glycerol . In contrast, oocytes expressing glpF transported glycerol but exhibited limited osmotic water permeability . Phylogenetic comparison of aquaporins and homologs revealed a large separation between aqpZ and glpF, consistent with an ancient gene divergence.

Eur J Pharmacol, 1995 Dec 7, 293(4), 327 - 34
Lipopolysaccharide-induced hepatotoxicity is inhibited by the antioxidant melatonin; Sewerynek E et al.; Oxidative damage to the liver of lipopolysaccharide-treated rats was evaluated using four parameters: level of lipid peroxidation, changes in total GSH and GSSG concentrations and hepatic morphology . Bacterial lipopolysaccharide (10 mg/kg b.w.) was injected i.p . either at 6, 16 or 24 h before animals were killed . Lipopolysaccharide increased lipid peroxidation most dramatically when it is injected 6 h before killing . Hepatic total GSH increased after lipopolysaccharide in a time-dependent manner . The highest level of GSSG and largest GSSG/total GSH ratio were also observed in the group of animals injected with lipopolysaccharide 6 h before tissue collection . In a second study, lipopolysaccharide was injected 6 h before the animals were killed, with or without 1 mg/kg b.w . melatonin . Melatonin totally abolished lipopolysaccharide-induced increase in lipid peroxidation, exaggerated the rise in total GSH and reversed the increase in GSSG concentration . The liver showed obvious histological degenerative changes after lipopolysaccharide, effects that were counteracted by melatonin administration . The protection conferred by melatonin is presumably due to its antioxidant activity.

Oncogene, 1995 Dec 7, 11(11), 2255 - 65
B-ATF: a novel human bZIP protein that associates with members of the AP-1 transcription factor family; Dorsey MJ et al.; A new member of the ATF/CREB family of transcription factors, called B-ATF, has been isolated from a cDNA library prepared from Epstein-Barr virus stimulated human B cells . B-ATF is a 125 amino acid nuclear protein possessing a basic leucine zipper domain that is most similar to the basic leucine zipper of ATF-3 . Northern blot analysis of polyadenylated mRNA isolated from a variety of human tissues and established cell lines indicates that the 1.0 kilobase B-ATF mRNA is expressed differentially, with the strongest hybridization detected in lung and in Raji Burkitt's lymphoma . Efficient homodimerization of the B-ATF protein cannot be detected using the yeast two hybrid system or using in vitro binding assays with glutathione-s-transferase-B-ATF and maltose binding protein-B-ATF fusion proteins produced in E . coli . However, a yeast two hybrid library screen has identified the human oncoprotein JunB as a specific binding partner for B-ATF . Glutathione-s-transferase-B-ATF heterodimerizes efficiently with in vitro translated JunB, c-Jun, and JunD, but only weakly associates with c-Fos . In addition, electrophoretic mobility shift assays demonstrate that a B-ATF/c-Jun protein complex can interact with DNA containing a consensus binding site for AP-1, suggesting that B-ATF functions as a tissue-specific modulator of the AP-1 transcription complex in human cells.

Biochim Biophys Acta, 1995 Dec 7, 1259(3), 245 - 53
Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein; Rolf B et al.; The coding part of the cDNA for bovine liver-type fatty acid binding protein (L-FABP) has been amplified by RT-PCR, cloned and used for the construction of an Escherichia coli (E . coli) expression system . The recombinant protein made up to 25% of the soluble E . coli proteins and could be isolated by a simple two step protocol combining ion exchange chromatography and gel filtration . Dissociation constants for binding of oleic acid, arachidonic acid, oleoyl-CoA, lysophosphatidic acid and the peroxisomal proliferator bezafibrate to L-FABP have been determined by titration calorimetry . All ligands were bound in a 2:1 stoichiometry, the dissociation constants for the first ligand bound were all in the micro molar range . Oleic acid was bound with the highest affinity and a Kd of 0.26 microM . Furthermore, binding of cholesterol to L-FABP was investigated with the Lipidex assay, a liposome binding assay and a fluorescence displacement assay . In none of the assays binding of cholesterol to L-FABP was observed.

Biochim Biophys Acta, 1995 Dec 6, 1253(2), 169 - 74
Completion of the thioredoxin reaction mechanism: kinetic evidence for protein complexes between thioredoxin and fructose 1,6-bisphosphatase; Haberlein I et al.; The activation of chloroplast fructose 1,6-bisphosphatase from spinach and soybean leaves by the two chloroplast thioredoxins isolated from the same plants has been studied . The thioredoxin saturation characteristics (Vmax: 0.15-103.2 mumol Pi/min per mg enzyme; K0.5: 0.0048-0.84 microM; Hill coefficient n: 1.02-3.80) indicate that in addition to the reductive activation by thioredoxin specific complex formation between thioredoxin and fructose 1,6-bisphosphatase is responsible for fine regulation of the enzyme activity . This complex formation has been inserted into the thioredoxin mechanism and the physiological consequences discussed . Obviously, physiologically relevant investigations of the thioredoxin-dependent regulation of fructose 1,6-bisphosphatase activity can only be performed in homologous enzyme-thioredoxin combinations . Dithiothreitol and E . coli thioredoxin are no complete substitutes in regulatory studies.

Biochem Biophys Res Commun, 1995 Dec 5, 217(1), 41 - 51
Requirement of ATP hydrolysis for assembly of ClpA/ClpP complex, the ATP-dependent protease Ti in Escherichia coli; Seol JH et al.; The ATP-dependent protease Ti (Clp) consists of two distinct components, ClpP containing the serine active sites for proteolysis and ClpA having two ATP-binding sites . A ClpA variant (ClpAT) carrying Thr in place of Met169 is highly soluble but indistinguishable from the wild-type ClpA in its ability to hydrolyze ATP and to support the ClpP-mediated proteolysis . Here we show that ATP hydrolysis is essential for assembly of ClpAT/ClpP complex upon analysis of the mixture of its components by gel filtration followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate . Either ADP or adenosine 5'-(beta,gamma-imido)-triphosphate could not support the complex formation . Furthermore, ClpAT/K501T which carries a mutation in the second ATP-binding site and therefore is unable to cleave ATP could not interact with ClpP . On the other hand, ClpAT/K220T carrying a mutation in the first site and ClpP could be assembled into a complex at 2 mM ATP but not at 0.5 mM, at which concentration the trimeric mutant protein can not form a hexamer . These results indicate that assembly of protease Ti requires hydrolysis of ATP by ClpA in addition to its binding for hexamer formation.

Biochem Biophys Res Commun, 1995 Dec 5, 217(1), 217 - 23
Preferential interaction of Sec-G with Sec-E stabilizes an unstable Sec-E derivative in the Escherichia coli cytoplasmic membrane; Nishiyama K et al.; SecY, SecE and SecG form a membrane part of the protein translocation machinery . A SecG-overproducing plasmid was constructed by placing the secG gene under the control of the tac promoter . From the extent of SecG overproduction, the number of SecG molecules in one normal cell was estimated to be about 1,000, which is similar to those of SecY and SecE . Overproduction of SecG stabilized the overproduction of SecE-C, an unstable truncated derivative of SecE, as effectively as SecY does . SecG overproduction also stabilized the overproduction of SecY . However, the SecG-dependent stabilization of SecY was less potent than the SecE-dependent stabilization . These results indicate that SecG preferentially interacts with SecE, which associates with SecY, the SecG-SecE-SecY complex thus being formed in the cytoplasmic membrane.

Proc Natl Acad Sci U S A, 1995 Dec 5, 92(25), 11921 - 5
Sequence similarity analysis of Escherichia coli proteins: functional and evolutionary implications; Koonin EV et al.; A computer analysis of 2328 protein sequences comprising about 60% of the Escherichia coli gene products was performed using methods for database screening with individual sequences and alignment blocks . A high fraction of E . coli proteins--86%--shows significant sequence similarity to other proteins in current databases; about 70% show conservation at least at the level of distantly related bacteria, and about 40% contain ancient conserved regions (ACRs) shared with eukaryotic or Archaeal proteins . For > 90% of the E . coli proteins, either functional information or sequence similarity, or both, are available . Forty-six percent of the E . coli proteins belong to 299 clusters of paralogs (intraspecies homologs) defined on the basis of pairwise similarity . Another 10% could be included in 70 superclusters using motif detection methods . The majority of the clusters contain only two to four members . In contrast, nearly 25% of all E . coli proteins belong to the four largest superclusters--namely, permeases, ATPases and GTPases with the conserved "Walker-type" motif, helix-turn-helix regulatory proteins, and NAD(FAD)-binding proteins . We conclude that bacterial protein sequences generally are highly conserved in evolution, with about 50% of all ACR-containing protein families represented among the E . coli gene products . With the current sequence databases and methods of their screening, computer analysis yields useful information on the functions and evolutionary relationships of the vast majority of genes in a bacterial genome . Sequence similarity with E . coli proteins allows the prediction of functions for a number of important eukaryotic genes, including several whose products are implicated in human diseases.

Proc Natl Acad Sci U S A, 1995 Dec 5, 92(25), 11810 - 3
Human neoplasms elicit multiple specific immune responses in the autologous host; Sahin U et al.; Expression of cDNA libraries from human melanoma, renal cancer, astrocytoma, and Hodgkin disease in Escherichia coli and screening for clones reactive with high-titer IgG antibodies in autologous patient serum lead to the discovery of at least four antigens with a restricted expression pattern in each tumor . Besides antigens known to elicit T-cell responses, such as MAGE-1 and tyrosinase, numerous additional antigens that were overexpressed or specifically expressed in tumors of the same type were identified . Sequence analyses suggest that many of these molecules, besides being the target of a specific immune response, might be of relevance for tumor growth . Antibodies to a given antigen were usually confined to patients with the same tumor type . The unexpected frequency of human tumor antigens, which can be readily defined at the molecular level by the serological analysis of autologous tumor cDNA expression cloning, indicates that human neoplasms elicit multiple specific immune responses in the autologous host and provides diagnostic and therapeutic approaches to human cancer.

Proc Natl Acad Sci U S A, 1995 Dec 5, 92(25), 11806 - 9
Recovery of respiration following the SOS response of Escherichia coli requires RecA-mediated induction of 2-keto-4-hydroxyglutarate aldolase; Cayrol C et al.; Agents that damage DNA in Escherichia coli or interfere with its replication induce DNA repair and mutagenesis via the SOS response . This well-known activity is regulated by the RecA protein and the LexA repressor . Following repair or bypass of the DNA lesion, the cell returns to its resting state by a largely unknown process . We found that 2-keto-4-hydroxyglutarate aldolase (4-hydroxy-2-oxoglutarate aldolase; EC 4.1.3.16) is necessary for the recovery of respiration and that it is regulated by the SOS response . This protein was induced by DNA-damaging agents . Induction required RecA activation . When the LexA regulon was repressed, activation of RecA was not sufficient for induction, indicating the requirement for an additional protein under LexA control . Finally, a mutant in the corresponding hga gene was UV sensitive . 2-Keto-4-hydroxyglutarate aldolase also plays a role in respiratory metabolic pathways, which suggests a mechanism for respiration resumption during the termination of the SOS response.

Proc Natl Acad Sci U S A, 1995 Dec 5, 92(25), 11701 - 5
Mapping by multifragment cloning in vivo; Marykwas DL et al.; An efficient method for mapping mutations is described in which hybrid genes, derived partly from mutant and partly from wild-type DNA, are obtained in vivo by homologous recombination of multiple fragments . The recombinants are formed in a strain in which their phenotypes are immediately apparent . This method was developed to identify changes that disrupt protein-protein interactions demonstrable by the two-hybrid system in yeast . However, it can be extended to any system where recombination is possible, provided an assay is available to distinguish between mutant and wild-type phenotypes.

Proc Natl Acad Sci U S A, 1995 Dec 5, 92(25), 11681 - 5
The "DEAD box" protein DbpA interacts specifically with the peptidyltransferase center in 23S rRNA; Nicol SM et al.; The Escherichia coli DEAD (Asp-Glu-Ala-Asp) box protein DbpA is a putative RNA helicase and established RNA-dependent ATPase and is the only member of the DEAD box protein family for which a specific RNA substrate, bacterial 23S rRNA, has been identified . We have investigated the nature of this specificity in depth and have localized by deletion mutagenesis and PCR a single region of 93 bases (bases 2496-2588) in 23S rRNA that is both necessary and sufficient for complete activation of ATPase activity of DbpA . This target region forms part of the peptidyltransferase center and includes many bases involved in interaction with the 3' terminal adenosines of both A- and P-site tRNAs . Deletion of stem loops within the 93-base segment abolished ATPase activation . Similarly, point mutations that disrupt base pairing within stem structures ablated stimulation of ATPase activity . These data are consistent with roles for DbpA either in establishing and/or maintaining the correct three-dimensional structure of the peptidyltransferase center in 23S rRNA during ribosome assembly or in the peptidyltransferase reaction.

Proc Natl Acad Sci U S A, 1995 Dec 5, 92(25), 11666 - 70
A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity; Chen R et al.; The isocitrate dehydrogenase of Escherichia coli, which lacks the Rossmann fold common to other dehydrogenases, displays a 7000-fold preference for NADP over NAD (calculated as the ratio of kcat/Km) . Guided by x-ray crystal structures and molecular modeling, site-directed mutagenesis has been used to introduce six substitutions in the adenosine binding pocket that systematically shift coenzyme preference toward NAD . The engineered enzyme displays an 850-fold preference for NAD over NADP, which exceeds the 140-fold preference displayed by a homologous NAD-dependent enzyme . Of the six mutations introduced, only one is identical in all related NAD-dependent enzyme sequences--strict adherence to homology as a criterion for replacing these amino acids impairs function . Two additional mutations at remote sites improve performance further, resulting in a final mutant enzyme with kinetic characteristics and coenzyme preference comparable to naturally occurring homologous NAD-dependent enzymes.

Proc Natl Acad Sci U S A, 1995 Dec 5, 92(25), 11588 - 92
Escherichia coli transcript cleavage factors GreA and GreB stimulate promoter escape and gene expression in vivo and in vitro; Hsu LM et al.; The process of RNA chain initiation by RNA polymerases plays a central role in the regulation of transcription . In this complex phase of transcription, short oligomers are synthesized and released from the enzyme-promoter complex in a reaction termed abortive initiation . The polymerase undergoes many cycles of abortive initiation prior to completion of the initiation process, which is signaled by the translocation of the enzyme away from the promoter, release of sigma factor, and formation of an elongation complex in which the RNA is stably bound . We have studied the parameters that affect escape from the promoter by Escherichia coli RNA polymerase for the phage T7 A1 promoter, the phage T5 N25 promoter, and the chimeric promoter T5 N25antiDSR . The latter site contains a synthetic initial transcribed region that reduces its ability to synthesize RNA both in vivo and in vitro . Clearance from T5 N25antiDSR can be stimulated up to 10-fold in vitro by addition of the E . coli transcript cleavage factor GreA or GreB, but these factors have little effect on transcription from the normal T7 A1 or T5 N25 promoters . Using an E . coli strain lacking GreA and GreB, we were also able to show stimulation of transcription by the Gre factors from the T5 N25antiDSR promotor in vivo . The stimulation of RNA chain initiation by Gre factors, together with their known biochemical properties in the transcription elongation reaction, suggests some specific models for steps in the transcription initiation reaction.

Proc Natl Acad Sci U S A, 1995 Dec 5, 92(25), 11583 - 7
Coupling the phosphotransferase system and the methyl-accepting chemotaxis protein-dependent chemotaxis signaling pathways of Escherichia coli; Lux R et al.; Chemotactic responses in Escherichia coli are typically mediated by transmembrane receptors that monitor chemoeffector levels with periplasmic binding domains and communicate with the flagellar motors through two cytoplasmic proteins, CheA and CheY . CheA autophosphorylates and then donates its phosphate to CheY, which in turn controls flagellar rotation . E . coli also exhibits chemotactic responses to substrates that are transported by the phosphoenolpyruvate (PEP)-dependent carbohydrate phosphotransferase system (PTS) . Unlike conventional chemoreception, PTS substrates are sensed during their uptake and concomitant phosphorylation by the cell . The phosphoryl groups are transferred from PEP to the carbohydrates through two common intermediates, enzyme I (EI) and phosphohistidine carrier protein (HPr), and then to sugar-specific enzymes II . We found that in mutant strains HPr-like proteins could substitute for HPr in transport but did not mediate chemotactic signaling . In in vitro assays, these proteins exhibited reduced phosphotransfer rates from EI, indicating that the phosphorylation state of EI might link the PTS phospho-relay to the flagellar signaling pathway . Tests with purified proteins revealed that unphosphorylated EI inhibited CheA autophosphorylation, whereas phosphorylated EI did not . These findings suggest the following model for signal transduction in PTS-dependent chemotaxis . During uptake of a PTS carbohydrate, EI is dephosphorylated more rapidly by HPr than it is phosphorylated at the expense of PEP . Consequently, unphosphorylated EI builds up and inhibits CheA autophosphorylation . This slows the flow of phosphates to CheY, eliciting an up-gradient swimming response by the cell.

Proc Natl Acad Sci U S A, 1995 Dec 5, 92(25), 11470 - 4
Enhanced somatic mutation rates induced in stem cells of mice by low chronic exposure to ethylnitrosourea; Shaver-Walker PM et al.; We have found that the somatic mutation rate at the Dlb-1 locus increases exponentially during low daily exposure to ethylnitrosourea over 4 months . This effect, enhanced mutagenesis, was not observed at a lacI transgene in the same tissue, although the two loci respond very similarly to acute doses . Since both mutations are neutral, the mutant frequency was expected to increase linearly with time in response to a constant mutagenic exposure, as it did for lacI . Enhanced mutagenesis does not result from an overall sensitization of the animals, since mice that had first been treated with a low daily dose for 90 days and then challenged with a large acute dose were not sensitized to the acute dose . Nor was the increased mutant frequency due to selection, since animals that were treated for 90 days and then left untreated for up to 60 days showed little change from the 90-day frequency . The effect is substantial: about 8 times as many Dlb-1 mutants were induced between 90 and 120 days as in the first 30 days . This resulted in a reverse dose rate effect such that 90 mg/kg induced more mutants when delivered at 1 mg/kg per day than at 3 mg/kg per day . We postulate that enhanced mutagenesis arises from increased stem cell proliferation and the preferential repair of transcribed genes . Enhanced mutagenesis may be important for risk evaluation, as the results show that chronic exposures can be more mutagenic than acute ones and raise the possibility of synergism between chemicals at low doses.

Biochemistry, 1995 Dec 5, 34(48), 15872 - 9
The contribution of the conserved hinge region residues of alpha1-antitrypsin to its reaction with elastase; Hopkins PC et al.; The hinge region of serpins is a conserved sequence of 8 amino acids located 7 residues away from the scissile bond at P8 to P15, on the edge of the protease-binding domain . In the inhibitory serpins the P8 to P12 residues of this motif are usually small side-chain amino acids, most commonly alanine . Each of these residues in alpha1-antitrypsin was mutated to a glutamate, and the effect of a hinge-region glutamic acid substitution was found . While substitutions at positions P10 and P12 affected the inhibitory characteristics of alpha1-antitrypsin, substitutions at positions P7, P8, P9, and P11 had no effect on inhibition . Thus, the conservation of residues with small side chains at the latter positions does not appear to be related to an essential function in the inhibitory mechanism . Following the glutamate substitution at P10, alpha1-antitrypsin remained a rapid inhibitor of elastase, but the elastase--serpin complex slowly broke down to yield active elastase and cleaved alpha1-antitrypsin . The glutamate substitution at P12 caused the resultant molecule (P12 Ala-->Glu) to become a partial substrate of elastase such that four moles of inhibitor were required to inhibit one mole of enzyme, and led to a 12-fold decrease in the association rate constant . The data could be interpreted in terms of the suicide substrate inhibition model for serpin-protease interactions and allowed a further refinement of the role of the hinge region in this process.

Biochemistry, 1995 Dec 5, 34(48), 15802 - 12
Interaction between the Escherichia coli Regulatory protein TyrR and DNA: a fluorescence footprinting study; Bailey M et al.; The Escherichia coli regulatory protein TyrR controls the expression of eight transcription units that encode proteins involved in the biosynthesis and transport of aromatic amino acids . It is a homodimer of 57 600 subunit molecular weight and has a binding site for ATP and weak ATPase activity . In the presence of ATP, TyrR binds tyrosine, which induces self-association of TyrR from a dimer to a hexamer . This report examines the interaction of TyrR with a 42 bp DNA oligonucleotide containing a centrally located binding site for TyrR (TyrR box) . Replacement of a thymidine residue with an aminouridine residue at positions 7, 9, 13, 15, 19, 22, and 26 from one end of the 42mer enables labeling with fluorescein and successive placement of the label along the major groove of the DNA . The fluorescence footprinting of the oligonucleotide was followed using steady-state and time-resolved fluorescence methods . Binding of the TyrR dimer caused significant changes in the fluorescent properties of the labels attached to positions 13, 15, and 26, suggesting the involvement of these bases in the binding of the protein . Except for the position 15 conjugate, binding of the TyrR dimer caused little change in fluorescence intensity . Therefore, fluorescence anisotropy was used to follow the binding equilibrium . The fluorescence of the position 15 conjugate increased 1.6-fold on binding TyrR, suggesting that the fluorophore was in close contact with the protein . For all conjugates, the addition of tyrosine at the end of the titration with TyrR increased the anisotropy markedly, suggesting that the hexameric form of TyrR could bind the oligonucleotide . Two rotational correlation times were found for the labeled conjugates: one reflecting the motion of the probe at its point of attachment to the DNA (220-290 ps), the other reflecting the global tumbling of the labeled oligonucleotide (14-21 ns) . On binding TyrR, changes in the correlation times and their associated amplitudes and changes in the range of angular motion of the probe depended on the position of the label . Evidence is presented that the binding of the TyrR hexamer, but not the TyrR dimer, affects regions that flank the binding sequence . The results support the hypothesis that the binding of the TyrR hexamer is responsible for interaction between tandem TyrR boxes in the tyrR regulon.

Biochemistry, 1995 Dec 5, 34(48), 15681 - 8
Crucial role of an idiosyncratic insertion in the Rossman fold of class 1 aminoacyl-tRNA synthetases: the case of methionyl-tRNA synthetase; Fourmy D et al.; A few aminoacyl-tRNA synthetases are characterized by their ability to tightly bind a zinc atom . In the case of Escherichia coli methionyl-tRNA synthetase, a peptide of 21 residues (138--163) having a stable 3-D structure in solution is responsible for zinc binding {Fourmy, D., Meinnel, T., Mechulam, Y., & Blanquet, S . (1993) J . Mol . Biol . 231, 1066--1077; Fourmy, D., Dardel, F., & Blanquet, S . (1993) J . Mol . Biol . 231, 1078--1089} . This peptide, which belongs to a region connecting the two halves of the nucleotide-binding domain of methionyl-tRNA synthetase, is likely to form a modular domain close to the active center of the enzyme . In this study, two residues of the zinc-binding module, Asp138 and Arg139, are shown to contribute to the stabilization of the transition state of the reaction leading to the activation of methionine . Moreover, another residue, Phe135, located at the surface of the zinc-binding domain, is found to possibly guide the tRNA acceptor stem toward the active site of the enzyme during catalysis . The available data indicate an important functional role for the zinc-binding module of methionyl-tRNA synthetase, as well as for other modules connecting conserved secondary structure elements in the aminoacyl-tRNA synthetase family . The relation between the occurrence of such variable peptide modules and the expression of both substrate specificity and catalytic efficiency is discussed.

Biochemistry, 1995 Dec 5, 34(48), 15671 - 80
Molecular basis for nonadditive mutational effects in Escherichia coli dihydrofolate reductase; Wagner CR et al.; Recently, two sets of single, double, and quadruple residue changes within the hydrophobic substrate binding pocket of Escherichia coli dihydrofolate reductase (5,6,7,8-tetrahydrofolate+ oxidoreductase, EC 1.5.1.3) were shown to exhibit nonadditive mutational effects {Huang, Z., Wagner, C . R., & Benkovic, S . J . (1994) Biochemistry 33, 11576--11585} . In particular, the analysis of data for the L28Y, L54F, and L28Y-L54F mutations revealed nonadditive changes in the free energy associated with the substrate and cofactor binding, hydride transfer, and product release steps . Construction of a related set of mutant proteins including L28F and L28F-L54F permits a comparison of similar energy changes and provides a means for assessing differences in the interactions of Phe28 and Tyr28 with both the ligands and the side chains at residue 54 . We find a single functional group change, from Phe C4-H to Tyr C4-OH, can influence the additivity of mutational effects and serve as a probe to monitor the appearance of differing enzyme conformations along the reaction pathway through changes in the interaction energy (delta GI) . The comparison of additivity/nonadditivity in free energy changes for three interrelated double mutational cycles (WT --> L28F-L54F, WT --> L28Y-L54F, and L28F --> l28Y-L54F) demonstrates that the side chains of positions 28 and 54 interact cooperatively to facilitate hydride transfer by preferentially influencing the enzyme--substrate ground-state complexes . The delta GI data for individual steps also provide evidence for multiple conformations of the enzyme operating during the catalytic cycle . The fact that there are no published examples of the synergistic enhancement of favorable mutational effects is consistent with the expectation that the binding/active site surface of wild-type dihydrofolate reductase has been optimized.

Biochemistry, 1995 Dec 5, 34(48), 15667 - 70
Use of designed metal-binding sites to study helix proximity in the lactose permease of Escherichia coli . 2 . Proximity of helix IX (Arg302) with helix X (His322 and Glu325); He MM et al.; Engineering divalent metal-binding sites into the lactose permease of Escherichia coli by introducing bis-His residues has been utilized to confirm the proximity of helices VIII (Glu269 --> His) and X (His322) {Jung, K., Voss, J., He, M., Hubbell, W . L., & Kaback, H . R . (1995) Biochemistry 34, 6272} and helices VII (Asp237 --> His) and XI (Lys358 --> His) {He, M . M., Voss, J., Hubbell, W . L., & Kaback, H.R . (1995) Biochemistry 34, 00000--00000} . In this paper, the approach is used to confirm and extend the relationship between helices IX (Arg302) and X (His322 and Glu325) {Jung, K., Jung, H., Wu, J., Prive, G . G., l& Kaback, H . R . (1993) Biochemistry 32, 12273} . Thus, mutants Arg302 --> His, Glu325 --> His, and Arg302 --> His/Glu325 --> His were constructed, and Mn2+ binding was assayed by electron paramagnetic resonance . Mutant Arg302 --> His binds Mn2+ with a KD of about 24 microM and a stoichiometry approximating unity in all likelihood because the His residue at position 302 forms a metal-binding site in conjunction with the native His residue at position 322 . Mutant Arg302 --> His/Glu325 --> His also binds Mn2+ with a 1:1 stoichiometry, but the KD is decreased to about 13 microM . The results suggest that Arg302 is sufficiently close to both Glu325 and His322 to form a tridentate metal-binding site in mutant Arg302 --> His/Glu325 --> His . In contrast, replacement of Glu325 with His in permease with a native His residue at position 322 does not lead to Mn2+ binding . The results provide strong support for the helix packing model proposed.

Biochemistry, 1995 Dec 5, 34(48), 15661 - 6
Use of designed metal-binding sites to study helix proximity in the lactose permease of Escherichia coli . 1 . Proximity of helix VII (Asp237 and Asp240) with helices X (Lys319) and XI (Lys358); He MM et al.; The lactose permease of Escherichia coli contains two pairs of oppositely charged residues that interact functionally, Asp240 (helix VII)/Lys319 (helix X) and Asp237 (helix VII)/Lys358 (helix XI) . Single- and double-His replacement mutants at these positions have been constructed and characterized with respect to transport activity and Mn2+ binding . The following results confirm the functional interactions between both sets of residues: (i) At pH 7.5, where the imidazole is likely to be unprotonated, the double-His mutants Asp237 --> His/Lys358 --> His and Asp240 --> His/Lys319 --> His exhibit significant transport activity while the single-His mutants Lys319 --> His and Lys358 --> His are inactive . (ii) At pH 5.5, where the imidazole is likely to be protonated, the double-His mutants Asp240 --> His/Lys319 --> His and Asp237 --> His/Lys358 --> His are inactive; however, the single-His mutant Lys319 --> His exhibits significant activity . (iii) The single-His mutant Asp237 --> His or ASP240 --> His is inactive at all pH values tested . In addition, a pH titration of Asp237 --> His/Lys358 --> His permease activity exhibits a midpoint at about 6.2 . Finally, the purified mutant proteins Asp237 --> His/Lys358 --> His and Asp240 --> His/Lys319 --> His were assayed for Mn2+ binding by electron paramagnetic resonance spectroscopy . Asp237 --> His/Lys358 --> His permease binds Mn2+ with a stoichiometry of unity at pH 7.5, but much less binding is observed at pH 5.5, demonstrating directly that helix VII (Asp237) is in close proximity to helix XI (Lys358).(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Dec 5, 34(48), 15654 - 60
Unlike the quaternary structure transition, the tertiary structure change of the 240s loop in allosteric aspartate transcarbamylase requires active site saturation by substrate for completion; Fetler L et al.; The quaternary structural change associated with the homotropic cooperative interactions in Escherichia coli aspartate transcarbamylase (ATCase) is accompanied by various tertiary structural modifications; the most notable one involves the 240s loop formed by residues 230--245 of the catalytic chain . In order to monitor local conformational changes in this region by fluorescence spectroscopy, Tyr-240 has been replaced by a Trp residue, in a mutant enzyme, in which both naturally occurring Trp residues in positions 209 and 284 of the catalytic chains had previously been substituted by Phe residues . This F209F284W240-ATCase still displays homotropic cooperativity for aspartate and undergoes the same T to R quaternary structure change as does the wild-type enzyme . Upon binding of the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate, the fluorescence emission spectrum of this mutant shows a red shift directly proportional to the fraction of catalytic sites occupied by this compound, a maximum value of 4 nm being attained when all six active sites are ligated . An identical shift is observed with the catalytic subunits of this modified enzyme, when all three active sites are occupied . In contrast, the quaternary structural change of the F209F284W240-ATCase, monitored by small-angle X-ray scattering, is complete when only four out of six catalytic sites are occupied . Thus, the 240s loop adopts its final conformation only when the neighboring active site is bound.

Biochemistry, 1995 Dec 5, 34(48), 15633 - 7
Identification of a "peroxy" intermediate in cytochrome bo3 of Escherichia coli; Morgan JE et al.; The respiratory heme-copper oxidases catalyze the reduction of dioxygen to water and link this chemistry to proton translocation . The main subgroups of the enzyme family are the cytochrome c oxidases and the quinol oxidases . For the cytochrome c oxidases, several key intermediates have been described in the oxygen reaction . Two of these (suggested to be "peroxy" and "ferryl" species) are also produced in the reaction of the oxidized enzyme with hydrogen peroxide . However, only a single product (a "ferryl" species) has been reported for the reaction of hydrogen peroxide with the quinol oxidase cytochrome bo3 from Escherichia coli . The same "ferryl" species has also been reported to be produced when two-electron reduced cytochrome bo3 reacts with oxygen, whereas this reaction leads to the "peroxy" intermediate in the cytochrome c oxidases . Consequently, the oxygen reaction has been considered to be different in the two enzyme subgroups . Here we show that both the peroxide reaction and the reaction of the two-electron reduced enzyme with oxygen actually result in primary formation of a hitherto unreported "peroxy" species in cytochrome bo3 . This intermediate subsequently relaxes into the "ferryl" species which has been described previously . We conclude that the oxygen reaction is similar in the cytochrome c and quinol oxidases.

Biochemistry, 1995 Dec 5, 34(48), 15624 - 32
HO . and DNase I probing of E sigma 70 RNA polymerase--lambda PR promoter open complexes: Mg2+ binding and its structural consequences at the transcription start site; Craig ML et al.; Chemical and enzymatic probing (footprinting) of the reactivity of the promoter DNA backbone is applied to characterize two binary open complexes RPo1 (-Mg2+) and RPo2 (+Mg2+), formed by Escherichia coli RNA polymerase (E sigma 70) at the lambda PR promoter . We report that HO . detects major differences in backbone reactivity between RPo1 and RPo2 in the open region from -4 to +1 relative to the transcription start site . Deoxyribose sugars at positions -4 to +1 of the t (template) strand react with HO . in RPo2 but are relatively protected in RPo1 . Binding of Mg2+ to convert RPo1 to RPo2 therefore increases the reactivity of two negatively charged footprinting agents {MnO4-; Suh, W.-C., Ross, W., & Record, M . T., Jr., (1993) Science 259, 358-361; and Fe(EDTA)2-/HO.} at the start site and is required for binding of the negatively-charged initiating nucleotides to the polymerase and the t strand at the start site . We propose that these effects result from binding of two Mg2+ ions to the catalytic carboxyls in the nucleotide binding sites . Except for the key region on the t strand at the start site, the promoter DNA of both RPo1 and RPo2 is continuously protected from DNase I and hydroxyl radical (HO.) cleavage between the -12 and +25 promoter positions . Protection in the upstream region, extending from -13 to about -70, is periodic, with an 11 base pair periodicity indicative of binding of polymerase to a single face of the DNA helix.(ABSTRACT TRUNCATED AT 250 WORDS)

FEBS Lett, 1995 Dec 4, 376(3), 229 - 32
The N-terminal, dehydrogenase/cyclohydrolase domain of yeast cytoplasmic trifunctional C1-tetrahydrofolate synthase requires the C-terminal, synthetase domain for the catalytic activity in vitro; Song JM et al.; The yeast ADE3(1-333) gene which encodes a truncated protein containing the N-terminal 5,10-methylene-tetrahydrofolate (THF) dehydrogenase (D)/5,10-methyl-THF cyclohydrolase (C) domain of cytoplasmic trifunctional C1-THF synthase is able to complement all the phenotypes associated with ade3 mutations in vivo . However, expression of the ADE3(1-333) gene in an ade3 strain does not retain any D activity in vitro . Expression in a yeast ade3 strain of the ADE3(1-333) fused to the Escherichia coli lacZ gene or to the yeast SER2 gene allows detection of D and C activities in vitro . These results indicate that the N-terminal D/C domain of C1-THF synthase requires the C-terminal 10-formyl-THF synthetase domain for stable catalytic activity in vitro.

J Neurovirol, 1995 Dec, 1(5-6), 359 - 68
Dendritic morphology of cardiac related medullary neurons defined by circuit-specific infection by a recombinant pseudorabies virus expressing beta-galactosidase; Standish A et al.; The transneuronal herpesvirus tracer, pseudorabies virus (PRV) was used to determine the dendritic architecture of cardiac-related neurons . We constructed a derivative of the Bartha strain of PRV called PRV-BaBlu, that carries the lacZ gene of E . coli . Expression of beta-galactosidase by this recombinant virus enabled us to define the dendritic morphology of motoneurons and interneurons that innervate the heart . beta-galactosidase antigen filled dendritic processes that were clearly revealed by antibodies to beta-galactosidase . In contrast, the standard enzymatic reaction for detection of beta-galactosidase activity stained the cell soma well, but was inferior for labeling dendrites . Following PRV-BaBlu cardiac injection, infected neurons were clearly defined and labeled dendrites could be traced for long distances, sometimes greater than 800 microns from the cell body . Labeled dendrites of cardiomotor neurons primarily located in the nucleus ambiguus (NA) were extensive and sometimes intertwined with dendrites from other labeled motoneurons . Dendrites of labeled neurons in the dorsal motor nucleus of the vagus (DMV) typically extended in the mediolateral direction in the transverse plane . Transynaptically labeled interneurons interposed between the cardiorespiratory region of the nucleus tractus solitarius (NTS) and the NA were primarily located in the NA region and the reticular arc, the area between the DMV and NA . These interneurons had long dendrites extending along the reticular arc in the transverse plane . The dendritic arborizations of infected cardiac-related neurons in the NTS were variable in extent . We conclude that antibody detection of beta-galactosidase expressed by PRV-BaBlu after infection of neural cardiac circuits provides a superior method to define the dendrites and dendritic fields of cardiac-related motoneurons and interneurons.

Biosci Rep, 1995 Dec, 15(6), 469 - 79
Cellular responses to enteropathogenic Escherichia coli infection; Knutton S; Enteropathogenic Escherichia coli (EPEC), first described in the 1940's and 1950's, remain an important cause of severe infantile diarrhoea in many parts of the developing world . EPEC do not produce enterotoxins and are not invasive; instead their virulence depends upon exploitation of host cell signalling pathways and the host cell cytoskeleton both as a means of colonizing mucosal surfaces of the small intestine and causing diarrhoea . Following initial mucosal attachment, EPEC secrete 'signalling' proteins and express a surface adhesin, intimin, to produce 'attaching & effacing' lesions in the enterocyte brush border membrane characterised by localised destruction of brush border microvilli, intimate bacterial adhesion and cytoskeletal reorganisation and accretion beneath attached bacteria . The pathophysiology of EPEC diarrhoea is also complex and probably results from a combination of epithelial cell responses including both electrolyte secretion and structural damage.

Cell Growth Differ, 1995 Dec, 6(12), 1643 - 50
Cell growth stimulation by the redox protein thioredoxin occurs by a novel helper mechanism; Gasdaska JR et al.; Thioredoxins are a class of low molecular weight redox proteins that undergo reversible reduction-oxidation of two active-site cysteine residues with reduction catalyzed by the NADPH-dependent flavoenzyme thioredoxin reductase . Human thioredoxin has been shown to be identical to a previously reported leukemic cell growth factor . We now report that recombinant human thioredoxin added to minimal culture medium in the absence of serum stimulates the proliferation of a number of human solid tumor cell lines measured over several days . The concentration of thioredoxin producing half-maximal stimulation of MCF-7 breast cancer cell proliferation was 350 nM, and the maximum stimulation occurred at 5 microM . The maximum increase in cell proliferation caused by thioredoxin was up to 90% of that seen with 10% bovine serum in the medium . There was a positive correlation between the ability of cell lines to proliferate in minimal medium, presumably, due to the autocrine production of growth factors by the cells, and the stimulation of proliferation by thioredoxin . Neither a redox inactive, mutant human thioredoxin, C32S/C35S, nor reduced Escherichia coli thioredoxin were able to stimulate MCF-7 cell proliferation . MCF-7 cell proliferation caused by human thioredoxin was completely abolished if the culture medium was changed each day . Antibody to thioredoxin blocked the cell proliferation caused by thioredoxin . Studies with 125I-labeled thioredoxin showed time-dependent binding to the surface of MCF-7 cells, but the binding was not saturable, indicating the absence of specific binding of thioredoxin to a cell surface receptor . Most of the thioredoxin associated with the cell could be released by trypsinization, and relatively little intact thioredoxin was taken up by the cell . The results of the study suggest that thioredoxin acts by a novel helper, redox mechanism to increase the cell proliferation response to growth factor(s) produced by the cell itself.

Brain Res, 1995 Dec 1, 701(1-2), 19 - 27
Demonstration of deacetylated hippocampal cholinergic neurostimulating peptide and its precursor protein in rat tissues; Ojika K et al.; This report concerns the demonstration of hippocampal cholinergic neurostimulating peptide (HCNP), its deacetylated analogue (free HCNP) and HCNP precursor protein in rat tissues . To avoid possible enzymatic degradation during sample manipulation, tissue extracts were prepared under acidic conditions using trifluoroacetic acid . The tissue contents of free HCNP and of precursor protein were determined by radioimmuno-assay (RIA) using two antibodies with different specificities, and by a combination of HPLC and RIA . Free HCNP was detected in neuronal and renal tissues, but not in liver . All tissues examined had measurable amounts of HCNP precursor protein . The concentrations of free HCNP and precursor in neuronal tissues were inversely related to the age . These results suggest that the deacetylated analogue of HCNP and its precursor protein may have significant physiological functions, especially in the central nervous system of young animals.

Minerva Anestesiol, 1995 Dec, 61(12), 509 - 13
{Antioxidant action of gabexate mesilate (Foy) in an experimental model of endotoxic shock}; Novelli GP et al.; BACKGROUND: Circulatory shock, especially endotoxin shock, is characterized by the release of a large number of mediators, among which proteases play a key role . The production of oxygen free radicals into the extracellular space and the increase of capillary permeability is one of the most important consequences of that phenomenon . In order to evaluate the efficacy of gabexate mesilate (Foy) in preventing such increase of microvascular permeability, an experimental model of endotoxin shock was used . MATERIALS AND METHODS: Experiments were performed on the mesocecum of male Wistars rats, fluorescent labeled bovine albumine was injected intrarterially to evaluate the capillary permeability and the mesocecum microcirculation was observed by fluorescent light . The control group received saline i.v.; the II group received a DL 100 of E . coli endotoxin (DIFCO 0111: B4); the III and the IV group received a continuous infusion or topical application of gabexate mesilate respectively, before the administration of endotoxin . To evaluate capillary permeability and to quantify the degree of extravasion by counting the number of leaky sites, fluorescent labelled bovine albumin was injected i.v . and mesocecum was observed with fluorescent microscopy for 2 hours . RESULTS AND CONCLUSIONS: Capillary permeability did not increase in control rats; it largely increased in rats receiving endotoxin i.v . but it did not almost increased in rats receiving gabexate mesilate (Foy) that prevents the increase of capillary permeability that was observed in the group treated with endotoxin alone.

Protein Eng, 1995 Dec, 8(12), 1317 - 21
Genetic construction and characterization of the diphtheria toxin-related interleukin 15 fusion protein DAB389 sIL-15; vanderSpek JC et al.; A gene fusion encoding DAB389 sIL-15 was constructed in which the catalytic and transmembrane domains of native diphtheria toxin (DAB389) are genetically linked to the N-terminus of simian interleukin 15 (sIL-15) . It was demonstrated that the cytotoxic action of DAB389 sIL-15 is mediated through the IL-15 receptor . Since toxicity may be blocked with chloroquine, it was concluded that following binding to the IL-15 receptor, the fusion toxin is internalized by receptor-mediated endocytosis and must pass through an acidic compartment in order to facilitate the delivery of the catalytic domain to the cytosol of target cells . As a non-toxic control, the ADP-ribosyltransferase defective mutant DA(E149S)B389 sIL-15 was constructed . It was demonstrated that both sIL-15 and DA(E149S)B389 sIL-15 stimulate protein and DNA synthesis in IL-15 receptor-positive CTLL-2 cells in vitro.

Protein Eng, 1995 Dec, 8(12), 1309 - 16
Regulation of protein tyrosine phosphatase 1C: opposing effects of the two src homology 2 domains; Pregel MJ et al.; The regulatory roles of the two src homology 2 (SH2) domains of protein tyrosine phosphatase 1C were investigated by comparing recombinant full-length PTP1C with mutants in which either the N-terminal SH2 (N-SH2) domain (PTP1C delta NSH2), the C-terminal SH2 (C-SH2) domain (PTP1C delta CSH2) or both SH2 domains were deleted (PTP1C delta NSH2 delta CSH2) . This revealed that the SH2 domains have opposing and independent effects on activity: strong inhibition by N-SH2 (42-fold) and weak activation by C-SH2 (2.1-fold) . C-SH2 caused activation across a wide pH range while N-SH2 inhibited most at neutral and high pH through a shift of the basic limb of the pH profile of kcat/Km, apparently via perturbation of an active-site pKa value . A phosphotyrosyl peptide derived from the erythropoietin receptor caused an approximately 30-fold activation of PTP1C and PTP1C delta CSH2 but had no effect on PTP1C delta NSH2 or PTP1C delta NSH2 delta CSH2, indicating that binding of this peptide to N-SH2 abolished its inhibition . Since C-SH2 separates N-SH2 from the catalytic domain in full-length PTP1C and activation is observed for PTP1C delta CSH2, it appears that the inhibitory effect of N-SH2 is independent of the position in the sequence and that intermolecular interactions may also be possible.

Protein Eng, 1995 Dec, 8(12), 1303 - 7
Involvement of the NH2-terminal region of oryzacystatin-I in cysteine proteinase inhibition; Urwin PE et al.; Cystatins are small protein inhibitors of cysteine proteinases . The relative importance of the N-terminal region of cystatins, and of a conserved glycine within this region, remains unclear despite several studies . It was found that deletion of the N-terminal 21 amino acids abolishes the inhibitory capacity of oryzacystatin-I . The importance of a conserved glycine residue (Gly10) was also examined by replacing it with 11 other amino acids . Three further glycine residues (Gly5, -6 and -11) in this N-terminal region of oryzacystatin-I were similarly mutated . Only those variants in which Gly10 was substituted show any significant change in inhibitory capacity compared with wild-type oryzacystatin-I . The inhibitory characteristics of hybrid cystatin molecules comprising regions of chicken egg white cystatin and oryzacystatin were also examined . It is suggested that in common with animal cystatins, the N-terminal region of the plant cystatin, oryzacystatin-I, and in particular the highly conserved Gly10 residue are important for effective inhibition of papain.

Protein Eng, 1995 Dec, 8(12), 1267 - 73
Site-directed mutagenesis of Arg58 and Asp86 of elongation factor Tu from Escherichia coli: effects on the GTPase reaction and aminoacyl-tRNA binding; Knudsen CR et al.; Elongation factor Tu from Escherichia coli was mutated separately at positions Asp86 and Arg58, in order to shed light both on the GTPase mechanism of elongation factor Tu and on the binding of aminoacyl-tRNA . In addition, the binding of guanine nucleotides was investigated by determination of the dissociation and association rate constants . The results imply that Arg58 is unimportant for the intrinsic GTPase mechanism and the binding of guanine nucleotides, whereas it is strongly involved in the binding of aminoacyl-tRNA and of the ribosome . Asp86 appears to be essential for the regulation of guanine-nucleotide affinities, and it may also play a role in the intrinsic GTPase mechanism.

Protein Eng, 1995 Dec, 8(12), 1205 - 13
Designing amino acid sequences to fold with good hydrophobic cores; Sun S et al.; We present two methods for designing amino acid sequences of proteins that will fold to have good hydrophobic cores . Given the coordinates of the desired target protein or polymer structure, the methods generate sequences of hydrophobic (H) and polar (P) monomers that are intended to fold to these structures . One method designs hydrophobic inside, polar outside; the other minimizes an energy function in a sequence evolution process . The sequences generated by these methods agree at the level of 60-80% of the sequence positions in 20 proteins in the Protein Data Bank . A major challenge in protein design is to create sequences that can fold uniquely, i.e . to a single conformation rather than to many . While an earlier lattice-based sequence evolution method was shown not to design unique folders, our method generates unique folders in lattice model tests . These methods may also be useful in designing other types of foldable polymer not based on amino acids.

Protein Eng, 1995 Dec, 8(12), 1189 - 95
The evolution of sugar isomerases; Banerjee S et al.; L-Arabinose isomerase (EC 5.3.1.4) catalyzes the isomerization of L-arabinose to L-ribulose . Here we report on the purification, kinetic mechanism and chemical mechanism of L-arabinose isomerase from Escherichia coli . The enzyme catalyzes the isomerization of L-arabinose to L-ribulose by a proton transfer mechanism, in contrast to xylose isomerase which uses a hydride transfer mechanism to perform a similar isomerization . Arabinose isomerase activity is metal dependent, although the enzyme can catalyze the exchange of the proton attached to carbon 2 of arabinose with the solvent in the absence of metal ion . Manganese(II) is the only metal ion which renders the enzyme active for the isomerization reaction . Arabinose isomerase has high substrate specificity for L-arabinose . The difference in chemical mechanism between xylose isomerase and arabinose isomerase suggests that these enzymes are not related by convergent evolution . This work also suggests that unless convergent evolution has been demonstrated, the mechanism of one enzyme may not give any insight into the mechanism of a second enzyme catalyzing the same reaction.

Mol Pharmacol, 1995 Dec, 48(6), 1087 - 98
An acidic amino acid in the N-methyl-D-aspartate receptor that is important for spermine stimulation; Williams K et al.; The polyamine spermine has multiple effects on N-methyl-D-aspartate (NMDA) receptors, including "glycine-independent" stimulation, which is seen in the presence of saturating concentrations of glycine; "glycine-dependent" stimulation, which is due to an increase in the affinity of the receptor for glycine; and voltage-dependent block . These effects may involve three separate polyamine binding sites on the receptor . To identify amino acid residues that are important for spermine binding, we used site-directed mutagenesis to alter amino acids in and around a region of the NR1 subunit of the NMDA receptor that shows homology with PotD, a polyamine binding protein from Escherichia coli . Mutated subunits, expressed in heteromeric and homomeric NMDA receptors, were studied by voltage-clamp recording in Xenopus oocytes . Mutation of two acidic residues (E339-E342) to neutral amino acids reduced or abolished glycine-independent stimulation by spermine without affecting glycine-dependent stimulation or voltage-dependent block by spermine . Mutation of these residues also had modest effects on sensitivity to protons and to ifenprodil but did not alter sensitivity to glutamate and glycine or to voltage-dependent block by Mg2+ . Residue E342 in NR1 appears to be critical for glycine-independent spermine stimulation . Mutations at equivalent positions in NR2A(E352Q) or NR2B(E353Q) had no effect on sensitivity to spermine, pH, or ifenprodil . Residue E342 in NR1 may form part of a discrete spermine binding site on the NMDA receptor or be involved in the mechanism of modulation by polyamines . This residue may also be involved in modulation by protons and ifenprodil.

J Gen Virol, 1995 Dec, 76 ( Pt 12), 3137 - 43
Expression of the herpes simplex virus type 1 glycoprotein E in human cells and in Escherichia coli: protection studies against lethal viral infection in mice; Miriagou V et al.; The objective of this study was to examine the protective efficacy of purified recombinant herpes simplex virus type 1 (HSV-1) glycoprotein E (gE-1) in the mouse lethal challenge model . A secreted form of gE-1 (hgE-1s) protein, containing amino acids 1-406, was produced in human cells by using the episomal replicating pRP-RSV expression vector . In addition, a portion of the gE-1 (bgE-1t) protein corresponding to amino acids 90-406, was expressed in Escherichia coli as a fusion protein with maltose binding protein using the pMAL-c2 expression vector . Mice vaccinated with hgE-1s developed high serum titres of HSV-1-neutralizing antibodies and were significantly protected from intraperitoneal lethal HSV-1 challenge, whereas mice vaccinated with bgE-1t exhibited only moderate levels of protective immunity . These results demonstrate that the expression of gE-1 in human cells has a strong impact on its protective efficacy and that most importantly the hgE-1s protein could be of values as a component of an HSV multi-subunit vaccine.

J Gen Virol, 1995 Dec, 76 ( Pt 12), 2999 - 3008
Mutations within conserved motifs in the 3'-5' exonuclease domain of herpes simplex virus DNA polymerase; Hall JD et al.; We investigated mutations within the presumed 3'-5' exonuclease domain of the DNA polymerase from herpes simplex virus type 1 . The mutation sites correspond to residues in DNA polymerase I (Escherichia coli) which bind two metal ions that are required for exonuclease function . To evaluate the effect of the herpesvirus mutations on enzymatic activity, we overexpressed the wild-type DNA polymerase and one mutant enzyme using a baculovirus expression system . Both proteins exhibited DNA polymerase activity after partial purification, but the mutant protein was drastically deficient in exonuclease activity . This finding suggests that the herpesvirus exonuclease may utilize the same metal-ion-mediated mechanism employed by DNA polymerase I . We also attempted to transfer each of the mutations into the herpesvirus genome using a marker rescue protocol . Although wild-type sequences could be transferred readily, recombinant viruses carrying mutant sequences were not recovered . We discuss the possibility that the mutations are lethal and suggest mechanisms by which a deficiency in 3'-5' exonuclease might cause loss of viability.

J Appl Physiol, 1995 Dec, 79(6), 2094 - 100
Short exposure to endotoxin increases the constriction induced by angiotensin II in rat aorta; Rasetti C et al.; The contraction elicited by angiotensin II (ANG II) was studied by using standard isometric tension techniques in aortic rings exposed for 1 h to 1 or 10 micrograms/ml Escherichia coli lipopolysaccharide endotoxin (LPS) . This contraction was 18 and 71% greater for the two doses of LPS, respectively, than in unexposed control rings . In endothelium-denuded rings, the LPS-induced increase in contraction in response to ANG II was completely abolished . Because the contraction induced by ANG II is modulated by the simultaneous release of prostaglandins, we tested the hypothesis that LPS interferes with this modulation . We found that the LPS-induced increase in contraction to ANG II was inhibited in the presence of the cyclooxygenase inhibitor indomethacin (10(-5) M) or the prostaglandin H2/thromboxane A2-receptor antagonist SQ-29548 (2 x 10(-7) M) . Conversely, the LPS-induced increase in contraction in response to ANG II was not inhibited by the presence of dexamethasone (10(-6) M), which inhibits new protein synthesis . In addition, there was no loss of vasodilator response to the endothelium-dependent receptor agonist acetylcholine (10(-8)-10(-4) M) or in the constrictor responses to norepinephrine (10(-9)-10(-5) M) and KCl (20-100 mM) . We conclude that short exposure to LPS produces a specific increase in the constrictor response to ANG II via mechanisms mediated by prostaglandin H2/thromboxane A2 . This effect could be a LPS-induced shift in favor of constrictor prostanoids in the balance of dilator/constrictor prostanoids, the release of which is associated with stimulation by ANG II.

J Appl Physiol, 1995 Dec, 79(6), 2021 - 8
Alveolar endotoxin increases alveolar liquid clearance in rats; Garat C et al.; Under some pathological conditions, ion transport across alveolar epithelial cells is downregulated, whereas under other pathological conditions, it may be upregulated . Because endotoxin is a biologically relevant pathological stimulus, we investigated the effect of endotoxin on alveolar epithelial liquid clearance in vivo . Escherichia coli endotoxin (220 micrograms/kg) was instilled into the lungs via the trachea of rats . Then, 24 or 40 h after endotoxin instillation, alveolar and lung liquid clearances were studied over 1 h by instillation of a 5% albumin solution with 1.5 microCi of 125I-labeled albumin (6 ml/kg into both lungs) . Alveolar liquid clearance was significantly greater at 24 h (36 +/- 5%) and 40 h (38 +/- 7%) after endotoxin exposure than in saline-instilled controls (27 +/- 6%) . Although there was an influx of neutrophils into the air space, there was no increase in lung epithelial permeability to protein at 24 or 40 h . Amiloride (2 x 10(-3) M), a sodium channel inhibitor, significantly reduced alveolar liquid clearance in the rats exposed to endotoxin . However, the increase in alveolar liquid clearance was not inhibited when propranolol (2 x 10(-5) M) was added to the 5% albumin solution . Thus exposure to alveolar endotoxin upregulates net alveolar fluid clearance in vivo for up to 40 h, a potentially important mechanism for accelerating alveolar fluid clearance under some pathological conditions . The increase in alveolar liquid clearance 24 and 40 h after instillation of endotoxin into the air spaces is mediated by an increased uptake of sodium through amiloride-sensitive sodium channels.

EMBO J, 1995 Dec 1, 14(23), 6021 - 7
Trigger factor is involved in GroEL-dependent protein degradation in Escherichia coli and promotes binding of GroEL to unfolded proteins; Kandror O et al.; In Escherichia coli, the molecular chaperones of hsp60/hsp10 (GroEL/GroES) families are required not only for protein folding but also for the rapid degradation of certain abnormal proteins . The rate-limiting step in the degradation of the fusion protein CRAG by protease ClpP appears to be the formation of a complex with GroEL . We have isolated these complexes and found that each GroEL 14mer contained a short-lived fragment of CRAG plus a 50 kDa polypeptide, which we identified by sequencing and immunological methods as Trigger Factor (TF) . Upon ATP addition, GroEL and TF dissociated together from CRAG but remained tightly associated with each other even upon gel filtration . TF was originally proposed to function in protein translocation across membranes but altering cellular content of TF did not affect this process in vivo . By contrast, low levels of TF expression markedly reduced CRAG degradation, while an overproduction of TF greatly stimulated this process . Furthermore, in extracts of cells expressing high levels of TF, the capacity of GroEL to bind to CRAG is greatly increased . Overproduction of TF also stimulated GroEL's ability to bind to other unfolded proteins (fetuin and histone) . Thus, TF is a rate-limiting factor for CRAG degradation; it appears to regulate GroEL function and to promote the formation of TF-GroEL-CRAG complexes which are critical for proteolysis.

EMBO J, 1995 Dec 1, 14(23), 5833 - 41
Cell cycle-specific changes in nucleoprotein complexes at a chromosomal replication origin; Cassler MR et al.; Initiation of DNA synthesis is triggered by the binding of proteins to replication origins . However, little is known about the order in which specific proteins associate with origin sites during the cell cycle . We show that in cycling cells there are at least two different nucleoprotein complexes at oriC . A factor for inversion stimulation (FIS)-bound nucleoprotein complex, present throughout the majority of the cell cycle, switches to an integration host factor (IHF)-bound form as cells initiate DNA replication . Coincident with binding of IHF, initiator DnaA binds to its previously unoccupied R3 site . In stationary phase, a third nucleoprotein complex forms . FIS is absent and inactive oriC forms a nucleoprotein structure containing IHF that is not observed in cycling cells . We propose that interplay between FIS and IHF aids assembly of initiation nucleoprotein complexes during the cell cycle and blocks initiation at inappropriate times . This exchange of components at replication origins is reminiscent of switching between pre- and post-replicative chromatin states at yeast ARS1.

EMBO J, 1995 Dec 1, 14(23), 5785 - 97
Differential binding of Lrp to two sets of pap DNA binding sites mediated by Pap I regulates Pap phase variation in Escherichia coli; Nou X et al.; Pyelonephritis-associated pili (Pap) expression in Escherichia coli is subject to a phase variation control mechanism that is regulated by the leucine-responsive regulatory protein (Lrp), PapI, and deoxyadenosine methylase (Dam) . In previous work, we found that the differential Dam methylation of two target sites in pap regulatory DNA, GATC-I and GATC II, is essential for the transition between active and inactive pap transcriptional states . Here, we identify six Lrp binding sites within the pap regulatory DNA, each separated by about three helical turns . Lrp binds with highest affinity to three sites (1, 2 and 3) proximal to the papBAp promoter . A mutational analysis indicates that the binding of Lrp to sites 2 and 3 inhibits pap transcription, which is consistent with the fact that Lrp binding site 3 is located between the --35 and --10 RNA polymerase binding region of papBAp . The addition of PapI decreases the affinity of Lrp for sites 1, 2 and 3 and increases its affinity for the distal Lrp binding sites 4 and 5 . Mutations within Lrp binding sites 4 and 5 shut off pap transcription, indicating that the binding of Lrp to this pap region activates pap transcription . The pap GATC-I and GATC-II methylation sites are located within Lrp binding sites 5 and 2, respectively, providing a mechanism by which Dam controls Lrp binding and Pap phase variation.

Nat Struct Biol, 1995 Dec, 2(12), 1095 - 101
Ligand-induced distortion of an active site in thymidylate synthase upon binding anticancer drug 1843U89; Weichsel A et al.; The anticancer drug 1843U89 inhibits thymidylate synthase (TS) at sub-nanomolar concentrations and is undergoing clinical trial . The 1.95 A crystal structure of Escherichia coli TS bound to the drug and dUMP reveals that the 1843U89 binding surface includes a hydrophobic patch that is normally buried . To reach this patch, 1843U89 inserts into the wall of the TS active site, resulting in a severe local distortion of the protein . In this new conformation, active-site groups that normally bind to the catalytic cofactor methylene-tetrahydrofolate instead bind to 1843U89 in new ways . This structure provides a rare example of a protein that can bind tightly to distinct substances using a single, flexible, binding surface . This has implications for drug design, as 1843U89 could not have been obtained from current structure-based approaches.

Nat Struct Biol, 1995 Dec, 2(12), 1083 - 94
Conformational variability in the refined structure of the chaperonin GroEL at 2.8 A resolution; Braig K et al.; Improved refinement of the crystal structure of GroEL from Escherichia coli has resulted in a complete atomic model for the first 524 residues . A new torsion-angle dynamics method and non-crystallographic symmetry restraints were used in the refinement . The model indicates that conformational variability exists due to rigid-body movements between the apical and intermediate domains of GroEL, resulting in deviations from strict seven-fold symmetry . The regions of the protein involved in polypeptide and GroES binding show unusually high B factors; these values may indicate mobility or discrete disorder . The variability of these regions may play a role in the ability of GroEL to bind a wide variety of substrates.

J Diarrhoeal Dis Res, 1995 Dec, 13(4), 232 - 4
Intracellular distribution of radio-labelled enterotoxigenic Escherichia coli heat-stable toxin (STa) in the intestine of suckling rat; Aye-Kyaw et al.; The chloramine T-procedure was employed to radio-iodinate Escherichia coli heat-stable toxin (STa) . The percentage iodination yield was 53.14% with a specific activity of 25.9 microCi/micrograms . Intragastric injections of both labelled and unlabelled toxin administered to suckling rats produced significant increase of fluid accumulation indicating that the biological activity of STa was not lost during radio-iodination . The uptake of the labelled toxin by subcellular particles of the intestinal cells in the suckling rats indicated that the toxin uptake was maximum in mitochondrial-lysosomal fraction . This confirms our previous observation on lysosomal involvement in digestion of the toxin . Results of the study also seem to be consistent with the short-lived hyper-secretory action of the toxin.

Mol Microbiol, 1995 Dec, 18(5), 953 - 62
Phosphotransfer circuitry of the putative multi-signal transducer, ArcB, of Escherichia coli: in vitro studies with mutants; Tsuzuki M et al.; Recently we demonstrated the occurrence of a novel device of signal transducers in Escherichia coli . This class of bacterial sensory kinases, typified by ArcB and BarA, possesses two phospho-donor (His) sites, together with a phospho-accepting (Asp) site . These multi-phosphorylation sites were suggested to make a phosphotransfer circuit . To clarify this complex circuitry, we carried out a series of in vitro assays involving a set of ArcB mutant proteins which have an amino acid substitution at each putative phosphorylation site (His-292, Asp-576 and His-717) . By these in vitro phosphorylation and/or phosphotransfer assays, the followings were assessed: (i) ArcB autophosphorylation; (ii) ArcB-mediated phosphorylation of the cognate response regulator, ArcA; (iii) ArcB-mediated phosphorylation of its truncated form (ArcBc) encompassing only the C-terminal phosphorylation site (His-717); (iv) phosphotransfer from ArcBc to ArcA; and (v) phosphotransfer from ArcBc to ArcB . On the basis of these in vitro results, a complex circuitry was revealed for the signal transducer ArcB . This evidence obtained in vitro supports the view that ArcB can serve as a powerful device for not only propagating multi-signals, but also making up signalling networks, in ways more sophisticated than previously thought.

Mol Microbiol, 1995 Dec, 18(5), 943 - 52
A missense mutation in rpoD results in an A-signalling defect in Myxococcus xanthus; Davis JM et al.; The Myxococcus xanthus asg genes (asgA, asgB, and asgC) are necessary for production of extracellular A-signal, which is thought to function as a cell-density signal . Previous analyses of the asgA and asgB genes suggest that they perform regulatory functions . In this work, we localized asgC to a region that contains genes homologous to rpsU, dnaG, and rpoD of the Escherichia coli macromolecular synthesis (MMS) operon . Surprisingly, asgC767 was found to be a mutant allele of rpoD, the gene encoding the major sigma factor of M . xanthus . The mutation in asgC767 results in a glutamate to lysine substitution at amino acid 598, which lies within conserved region 3.1 of the major sigma factors . Previous studies have shown that the asg mutants share a number of growth and developmental phenotypes . We found that A-signal restores developmental expression of an A-signal-dependent gene (omega 4521) in the asgC767 (rpoDEK598) mutant background in a manner similar to that seen in the asgA and asgB mutants . Because the asg mutants have very similar phenotypes and the asg genes encode proteins that appear to have regulatory functions, we hypothesize that the asg gene products function together in a regulatory pathway that is required for extracellular A-signal production.

Mol Microbiol, 1995 Dec, 18(5), 841 - 50
Mode of promoter recognition by the Escherichia coli RNA polymerase holoenzyme containing the sigma S subunit: identification of the recognition sequence of the fic promoter; Hiratsu K et al.; Transcription of a number of genes during stationary phase in Escherichia coli requires the alternative sigma factor sigma S, encoded by the rpoS gene . No consensus sequence for the promoters recognized by the RNA polymerase holoenzyme containing the sigma S subunit (E sigma S) is known . To identify the E sigma S recognition sequence, we analysed the promoter region of the fic gene, whose expression depends on E sigma S both in vivo and in vitro . By random mutagenesis, we isolated a large number of the fic promoter mutants that had defective promoter activities in an rpoS+ strain . All such mutants contained alterations within the -10 region, TATACT, whereas no mutant was isolated with alterations in the -35 region . Based on further analyses including scanning mutagenesis and DNase I footprinting assays of the fic promoter region, and in vitro transcription assays, we conclude that the -10 hexamer is essential to transcription initiation from the fic promoter by E sigma S.

Mol Microbiol, 1995 Dec, 18(5), 813 - 20
Characterization of Escherichia coli DnaAcos protein in replication systems reconstituted with highly purified proteins; Katayama T et al.; Excessive initiation of chromosomal replication occurs in the dnaAcos mutant at 30 degrees C . Whereas purified wild-type DnA protein binds ATP and ADP tightly, DnaAcos protein is defective for such nucleotide binding . As initiation is a multistep reaction and DnaA protein functions at each step, activities of DnaAcos protein need to be examined precisely . DnaAcos protein specifically bound a DNA fragment containing the chromosomal replication origin with an affinity similar to that seen with the wild-type protein . In a system reconstituted with purified proteins at 30 degrees C, the mutant protein initiated replication of single-stranded DNA that contains a DnA-binding hairpin structure . Thus, DnaAcos protein basically sustains affinity to a DnaA-binding sequence and functions in the loading of DnaB helicase onto single-stranded DNA . Thermal stabilities of wild-type DnA and DnaAcos activities were comparable . Unlike wild-type DnaA protein, DnaAcos protein was inactive for minichromosomal replication in systems reconstituted with purified proteins in which the ATP-bound form of DnaA protein is required for initiation . Taken together, the data indicate that the prominent defect in DnaAcos protein appears to be the inability to bind nucleotide.

Biochem Mol Med, 1995 Dec, 56(2), 172 - 5
Cell targeting for gene delivery: use of fusion protein containing the modified human receptor for ecotropic murine leukemia virus; Ohno K et al.; We have previously cloned a human gene (H13) homologous to the murine ecotropic retrovirus (E-MuLV) receptor, which, however, does not confer susceptibility to E-MuLV infection . The extracellular domain 3 (ECD3) of H13 contains amino acid residues critical for E-MuLV binding in that the modified H13 gene (mH13), substituted with amino acids from the actual receptor, has the ability to bind E-MuLV . Here we have expressed a fusion protein consisting of mH13/ECD3 and transforming growth factor-alpha in Escherichia coli and demonstrated its binding activity to both ecotropic AKR virus and the epidermal growth factor receptor expressed on the cell surface . Fusion proteins of mH13/ECD3 and ligands to cell surface molecules might be useful for specific cell targeting in E-MuLV-based gene delivery systems.

J Autoimmun, 1995 Dec, 8(6), 931 - 45
Autoreactive epitopes within the human alpha-enolase and their recognition by sera from patients with endometriosis; Walter M et al.; Patients with endometriosis significantly develop autoantibodies directed against endometrial proteins, which may be involved in the aetiology of this gynaecological disease . Based on standard Western blot analysis, a 48 kDa protein was localized in the soluble protein extract of endometrial adenocarcinoma cells using sera from patients with clinically staged endometriosis and identified as the glycolytic enzyme alpha-enolase . The corresponding cDNA coding for the human alpha-enolase was isolated from a human endometrial cDNA library and cloned into the vector pH6EX3, allowing the efficient expression of recombinant human alpha-enolase with an N-terminal histidine-hexapeptide as affinity ligand in Escherichia coli . The purified recombinant human alpha-enolase was evaluated as a specific antigenic tool for the diagnostic measurement of antiendometrial antibodies in sera from patients with endometriosis . With selected endometriosis sera, two linear autoreactive epitopes were localized within the recombinant human alpha-enolase using epitope mapping techniques, and they were characterized.

Genome Res, 1995 Dec, 5(5), 474 - 82
A novel in vivo method to detect DNA sequence variation; Faham M et al.; Mismatch repair detection (MRD) is an in vivo method that uses a change in bacterial colony color to detect DNA sequence variation . DNA fragments to be screened for variation are cloned into two MRD plasmids, and bacteria are transformed with heteroduplexes of these constructs . The resulting colonies are blue in the absence of a mismatch and white in the presence of a mismatch . MRD is capable of detecting a single mismatch in a DNA fragment as large as 10 kb in size . In addition, MRD has the potential for analyzing many fragments simultaneously, offering a powerful method for high-throughput genotyping and mutation detection in a large genomic region.

J Comput Aided Mol Des, 1995 Dec, 9(6), 521 - 31
Hydration in drug design . 3 . Conserved water molecules at the ligand-binding sites of homologous proteins; Poornima CS et al.; Water molecules are known to play an important role in mediating protein-ligand interactions . If water molecules are conserved at the ligand-binding sites of homologous proteins, such a finding may suggest the structural importance of water molecules in ligand binding . Structurally conserved water molecules change the conventional definition of 'binding sites' by changing the shape and complementarity of these sites . Such conserved water molecules can be important for site-directed ligand/drug design . Therefore, five different sets of homologous protein/protein-ligand complexes have been examined to identify the conserved water molecules at the ligand-binding sites . Our analysis reveals that there are as many as 16 conserved water molecules at the FAD binding site of glutathione reductase between the crystal structures obtained from human and E . coli . In the remaining four sets of high-resolution crystal structures, 2-4 water molecules have been found to be conserved at the ligand-binding sites . The majority of these conserved water molecules are either bound in deep grooves at the protein-ligand interface or completely buried in cavities between the protein and the ligand . All these water molecules, conserved between the protein/protein-ligand complexes from different species, have identical or similar apolar and polar interactions in a given set . The site residues interacting with the conserved water molecules at the ligand-binding sites have been found to be highly conserved among proteins from different species; they are more conserved compared to the other site residues interacting with the ligand . These water molecules, in general, make multiple polar contacts with protein-site residues.

Neuropharmacology, 1995 Dec, 34(12), 1635 - 45
Localization of the 5-hydroxytryptamine2C receptor protein in human and rat brain using specific antisera; Abramowski D et al.; The mouse 5-HT2C receptor and its third and fourth (C-terminal) cytoplasmic domain have been expressed as fusion proteins in bacteria . After purification antisera were generated against the fusion proteins . Characterization by immunoblotting using eukaryotic cells expressing the 5-HT2C and 5-HT2A receptors showed that high titer antibodies could be obtained only against the third and fourth cytoplasmic domain but not the entire receptor . Affinity purified antibodies were used to study the location of 5-HT2C receptors in rat and human brain sections . This distribution was compared with the location of 5-HT2C receptor binding sites as determined by {3H}mesulergine, a 5-HT2C receptor radioligand . The antibodies recognized sites in the rat choroid plexus, hippocampus, cerebral cortex, striatum and substantia nigra with a similar distribution as the 5-HT2C binding sites . One antiserum directed against the 5-HT2C receptor C-terminus crossreacted with the human receptor protein in immunoblots . In human brain sections it labelled sites including cerebral cortex, substantia nigra and cerebellum . Our results demonstrate that the antibodies are suitable to identify 5-TH2C receptors in rat and human brain . They visualize a protein distribution which correlates well with the location of the 5-HT2C receptor binding sites as would be expected if affinity states do not influence the binding pattern.

Biol Pharm Bull, 1995 Dec, 18(12), 1700 - 4
Detoxification of paraquat poisoning: effects of carbohydrate sulfate, alkylsulfate and alkylsulfonate on active oxygen; Tsuchiya T et al.; The potency of carbohydrate sulfate (dextran sulfate, DS; glucose sulfate, GS), alkyldisulfonate (butane-disulfonate, BDS; propanedisulfonate, TDS) and polyvinyl sulfate (PVP) as the quencher of active oxygen species was studied . Co-incubation of GS, BDS or TDS and tert-butylhydroperoxide together with human erythrocytes resulted in a marked decrease in hemolysis relative to the hemolysis induced by peroxide alone . DS and PVP exhibited no suppressive effect . When hemolysis was induced by a singlet oxygen derived from a photosensitizer-coupled reaction, PVP, BDS and TDS lowered its extent by 30-40% . DS and GS did not exhibit any effect on singlet oxygen-induced hemolysis . The quenching effect towards hydroxyl radical was assessed by investigating the protective effect on DNA strand breakage which was introduced by the Fenton reaction and enzymatic reduction of paraquat (PQ) . The result showed that BDS and TDS are potent hydroxyl radical scavengers . All compounds examined here failed to reduce PQ toxicity assessed by the inhibition of E . coli cell growth . In addition, all compounds had no effect on the magnitude of PQ-induced induction of superoxide dismutase in E . coli . These results strongly suggest that low-molecular weight alkyldisulfonates, BDS and TDS, are potent scavengers of peroxyl, alkoxyl and hydroxyl radicals, and singlet oxygen although these reagents cannot interact with superoxide anion radicals.

Pharm Res, 1995 Dec, 12(12), 1889 - 95
Systemic absorption and activity of recombinant consensus interferons after intratracheal instillation and aerosol administration; Niven RW et al.; PURPOSE: The pulmonary pharmacokinetics and bioactivity of E . coli derived recombinant consensus interferon (CIFN) and a modified lactose-conjugated consensus interferon (LacCIFN) were evaluated in animals . METHODS: Estimated doses of 20 and 100 micrograms/kg of the interferons were administered to anesthetized rats by aerosol via ultrasonic nebulizer as well as intravenous injection . Rats also received nominal doses of 50 and 200 micrograms/kg via intratracheal instillation (IT) . Hamsters were treated with interferon via various routes including IT . The effectiveness of treatment was assessed by the resistance to development of hind leg paralysis following infection with encephalomyocarditis virus . RESULTS: Significant amounts of CIFN and LacCIFN were found in rat plasma after aerosol administration . Peak plasma levels occurred approximately 25-30 minutes with estimated bioavailabilities approaching 70% . Absorption of CIFN was rate limiting and plasma levels were detectable 12 hr post-dose . The CIFN at IT doses as low as 5 micrograms/kg was effective at reducing paralysis in hamsters but protection was variable and doses of up to 100 micrograms/kg were not 100% effective . CONCLUSIONS: Despite the incomplete protection, the results demonstrate the feasibility of treating systemic viral infections with interferon administered directly to the lungs.

Matrix Biol, 1995 Dec, 14(9), 765 - 71
Involvement of osteopontin in egg shell formation in the laying chicken; Pines M et al.; Expression of the osteopontin (OPN) gene in the oviduct of the laying hen was studied . It was detected only in the egg shell gland (ESG), where massive calcification occurs . No OPN gene expression was detected in any other part of the oviduct, such as the magnum and isthmus . The OPN gene was expressed in a circadian fashion during the daily egg cycle only during the period of egg shell calcification . No OPN gene expression was detected in the ESG of a pre-laying hen before the onset of reproduction, or after forced removal of the egg close to its entrance into the ESG . OPN was found to be synthesized by the epithelial cells of the ESG lining the lumen . Upon synthesis, OPN is immediately secreted out of cells and accumulates in the egg shell . These findings demonstrate for the first time temporal and spatial association of OPN with egg shell calcification . OPN, which was found to be part of the organic matrix of the egg shell, may play an important role in egg shell calcification.

Thromb Haemost, 1995 Dec, 74(6), 1578 - 82
Endotoxin-induced intravascular coagulation in rabbits: effect of tissue plasminogen activator vs urokinase of PAI generation, fibrin deposits and mortality; Paloma MJ et al.; We have evaluated the effect of plasminogen activators (t-PA and urokinase) on an experimental model of disseminated intravascular coagulation (DIC) in rabbits by injection of 20 micrograms/kg/h of E . coli lipopolysaccharide during 6 h t-PA (0.2 mg/kg and 0.7 mg/kg), urokinase (3000 U/kg/h) and saline (control) were given simultaneously with endotoxin . Results indicated that urokinase and low dose of t-PA significantly reduced the increase of plasminogen activator inhibitor (PAI) activity observed 2 h after endotoxin (p < 0.001) . High t-PA dose also diminished the PAI levels at 6 h (p < 0.001) . A significant reduction of fibrin deposits in kidneys was observed din both t-PA treated groups as compared with findings in the group of rabbits infused with saline solution (p < 0.005), whereas urokinase had no significant effect on the extent of fibrin deposition . Finally, the mortality rate in the control group (70%) was reduced to 50% in rabbits receiving high doses of t-PA . In conclusion, treatment with t-PA resulted in reduced PAI generation, fibrin deposits and mortality in endotoxin-treated rabbits.

Bioorg Med Chem, 1995 Dec, 3(12), 1685 - 92
New EPSP synthase inhibitors: synthesis and evaluation of an aromatic tetrahedral intermediate mimic containing a 3-malonate ether as a 3-phosphate surrogate; Miller MJ et al.; A new analog of the EPSP synthase enzyme reaction intermediate 1, containing a 3-malonate ether moiety in place of the usual 3-phosphate group, was synthesized from 3,5-dihydroxybenzoic acid . This simple, synthetically accessible aromatic compound (5) is an effective competitive inhibitor versus S3P with an apparent Ki of 1.3 +/- 0.22 microM . This result demonstrates that a simple benzene ring can be a suitable achiral substitute for the more complex shikimate ring in the design of EPSP synthase inhibitors . Furthermore, the greater potency of 5 versus the phenol 6, glycolate 7 and the gallic acid analog 8 demonstrates the requirement for multiple anionic charges at the dihydroxybenzoate 5-position in order to attain effective inhibition of this enzyme . However, this 3-malonate ether substituted compound was at least 10-fold less effective as a bisubstrate inhibitor than the corresponding 3-phosphate . This suggests that tetrahedral intermediate mimics possessing a 3-malonate ether moiety are less effective than their corresponding 3-phosphates in accessing the optimal enzyme conformation stabilizing 1.

Bioorg Med Chem, 1995 Dec, 3(12), 1631 - 6
Synthesis of N-acetylglucosaminyl asparagine-substituted puromycin analogues; Kirsch P et al.; As part of our project aimed to introduce specifically glycosylated amino acids into proteins, new glycosylated puromycin analogues were chemically synthesized . Introduction of a free N-acetylglucosaminyl asparaginyl side chain abolished the activity of puromycin completely, but when the sugar OH groups were rendered increasingly hydrophobic by acetylation or benzylation, up to 8% of the activity was recovered . The results of our preliminary inhibition tests suggest that the interaction of puromycin analogues and therefore also of glycosylated aminoacyl tRNA, with the ribosomal A site increase with hydrophobicity of the modifying protecting groups.

Nihon Kyobu Shikkan Gakkai Zasshi, 1995 Dec, 33 Suppl, 231 - 8
{Acute lung injury and chemical mediators}; Kubo K et al.; The essential abnormality in acute lung injury is an increase in pulmonary permeability . The mechanisms involved are complex, and may include direct effects on endothelial cells; the actions of neutrophils, macrophages, and lymphocytes; and chemical mediators generated by these cells . Neutrophils are known to be important in acute lung injury, and the mediators they produce, especially protease and oxidants, may be responsible for endothelial cell injury . We studied the roles of neutrophil elastase and oxygen radicals in acute lung injury by Escherichia coli endotoxin in sheep with lung lymph fistulae . The results of these and other studies indicate that these substances are related to the increase in pulmonary permeability during the late phase of injury.

J Neurooncol, 1995 Dec, 26(3), 243 - 50
Gene therapeutic approach to primary and metastatic brain tumors: I . CD44 variant pre-RNA alternative splicing as a CEPT control element; Asman DC et al.; Our laboratory and others have shown alternative splicing of up to ten exons at a discrete extracellular site to be primarily responsible for the generation of CD44 variant (CD44v) isoforms . Based on clear differences in the expression of these CD44v isoforms between normal and malignant tissues, we believe that elucidation of the mechanisms underlying the regulation of CD44 alternative splicing may provide a new gene therapeutic targeting approach based on CD44 pre-mRNA processing in vivo . This strategy incorporates utilization of CD44 alternative splicing control elements into a chimeric enzyme/prodrug therapy (CEPT), a novel modification of the virus-directed enzyme/prodrug therapy (VDEPT) approach for the treatment of brain metastases from tumors of systemic origin . As initial steps towards the development of a gene therapeutic approach based on targeting tumor cell expression of specific CD44v alternatively spliced isoforms, we have: (1) developed a novel in vivo assay system that allows the rapid analyses of potentially therapeutic CD44 alternative splicing minigene constructs; and (2) cloned the E . coli cytosine deaminase (CD) gene and fused its enzymatically active domain to alternatively spliced CD44 exons (CD44/CD) . Deamination of cytosine by this CD44/CD chimeric fusion protein is demonstrated in E . coli cell lysates to be equal to that of wild type cytosine deaminase.

Proteins, 1995 Dec, 23(4), 598 - 603
Preliminary X-ray crystallographic analysis of Tritrichomonas foetus inosine-5'-monophosphate dehydrogenase; Whitby FG et al.; Inosine-5'-monophosphate dehydrogenase (IMPDH) from the protozoan parasite Tritrichomonas foetus has been expressed in E . coli and crystallized . Crystals were grown to 0.1 mm in each dimension in 18 to 72 h using ammonium sulfate and low-molecular-weight polyethylene glycols . The crystals belong to the cubic space group P432 with unit cell edge = 157.25 A . The enzyme is a homotetramer with each monomer having a molecular weight of 55,534 Da . There is one monomer per asymmetric unit, based on a volume/mass ratio of 2.7 A3/Da and self-rotation analysis . The crystals are adequately stable to allow a complete data set to be collected from a single crystal . Complete native data sets have been collected to 2.3 A resolution at 4 degrees C using synchrotron radiation . High-quality complete data extending to 3.0 A resolution have been collected from crystals of four putative derivatives, and the data appear to be isomorphous with that of the native crystals in each case . Efforts to solve the derivatives for use in MIR phasing are underway.

Proteins, 1995 Dec, 23(4), 595 - 7
Crystallization and preliminary crystallographic analysis of an amylopullulanase from the hyperthermophilic archaeon Pyrococcus woesei; Knapp S et al.; The thermostable amylopullulanase from Pyrococcus woesei was crystallized . Crystals, suitable for a crystallographic analysis up to a size of 0.6 mm in their longest dimension, have been obtained by the vapor diffusion method in a solution containing polyethyleneglycol 4000 (PEG 4000), isopropanol, and Tris/Cl- buffer pH 7.5 . Crystals grown under these conditions form hexagonal rods and diffract to a maximum resolution of 3 A . The crystals belong to the trigonal lattice type with the spacegroup P3(1)21 or P3(2)21, respectively, have the cell dimensions a = b = 96.8 A, c = 196.2 A, alpha = beta = 90 degrees, gamma = 120 degrees . The crystals have a theoretical packing density of 2.7 A3/Da, assuming one molecule with a molecule weight of 88.8 kDa in the asymmetric unit . Furthermore the self-rotation analysis of the dataset revealed only crystallographic symmetries . The merged native data of two crystals resulted in a 88% complete dataset.

Vet Microbiol, 1995 Dec, 47(3-4), 229 - 33
Aerobactin production by enterotoxigenic Escherichia coli of porcine intestine; Suarez S et al.; Of 135 strains of Escherichia coli from the intestines of healthy and diseased piglets screened, 16 (12%) were positive in a bioassay for aerobactin production . Of these, 15 also carried genetic determinants for heat stable or heat labile enterotoxins.

Glycoconj J, 1995 Dec, 12(6), 865 - 78
Functional domains of bovine beta-1,4 galactosyltransferase; Boeggeman EE et al.; A number of N- and C-terminal deletion and point mutants of bovine beta-1,4 galactosyltransferase (beta-1,4GT) were expressed in E . coli to determine the binding regions of the enzyme that interact with N-acetylglucosamine (NAG) and UDP-galactose . The N-terminal truncated forms of the enzyme between residues 1-129, do not show any significant difference in the apparent Kms towards NAG or linear oligosaccharide acceptors e.g . for chitobiose and chitotriose, or for the nucleotide donor UDP-galactose . Deletion or mutation of Cys 134 results in the loss of enzymatic activity, but does not affect the binding properties of the protein either to NAG- or UDP-agarose . From these columns the protein can be eluted with 15 mM NAG and 50 mM EDTA, like the enzymatically active protein, TL-GT129, that contains residues 130-402 of bovine beta-1,4GT . Also the N-terminus fragment, TL-GT129NAG, that contains residues 130-257 of the beta-1,4GT, binds to, and elutes with 15 mM NAG and 50 mM EDTA from the NAG-agarose column as efficiently as the enzymatically active TL-GT129 . Unlike TL-GT129, the TL-GT129NAG binds to UDP-columns less efficiently and can be eluted from the column with only 15 mM NAG . The C-terminus fragment GT-257UDP, containing residues 258-402 of beta-1,4GT, binds tightly to both NAG- and UDP-agarose columns . A small fraction, 5-10% of the bound protein, can be eluted from the UDP-agarose column with 50 mM EDTA alone . The results show that the binding behaviour of N- and C-terminal fragments of beta-1,4GT towards the NAG- and UDP-agarose columns differ, the former binds preferentially to NAG-columns, while the latter binds to UDP-agarose columns via Mn2+.

Glycoconj J, 1995 Dec, 12(6), 802 - 12
Murine monoclonal antibody recognizing human alpha(1,3/1,4)fucosyltransferase; Kimura H et al.; We prepared a mouse monoclonal antibody, FTA1-16, that specifically recognizes human alpha(1,3/1,4)fucosyltransferase without crossreactivity to any other members of the alpha(1,3)fucosyltransferase family . The specificity was confirmed by both immunofluorescense staining of native antigens in the Golgi apparatus and Western blotting analysis, using stable transformant cells transfected with each gene of the alpha(1,3)fucosyltransferase family . Western blotting analysis on a series of human tumour cell lines from various tissues revealed that some epithelial cancer cell lines from digestive organs expressed an amount of alpha(1,3/1,4)fucosyltransferase in good correlation with expression of sialyl Lewis a antigen . Immunohistochemical staining by FTA1-16 on colon cancer tissues revealed enhanced expression of the enzyme in cancer cells in comparison to normal cells . Finally, the antigenic epitope recognized by FTA1-16 was determined using truncated recombinant peptides which were expressed in E . coli . A minimal length determined was a fragment, amino acid positions 132-153, of the alpha(1,3/1,4)fucosyltransferase.

Vet Parasitol, 1995 Dec, 60(3-4), 199 - 212
Vaccination of vervet monkeys against cutaneous leishmaniosis using recombinant Leishmania 'major surface glycoprotein' (gp63); Olobo JO et al.; Vervet monkeys (Cercopithicus aethiops) were shown to give a positive delayed-type hypersensitivity (DTH) reaction to gp63, a major surface glycoprotein of Leishmania parasites, and also produce antibodies to the molecule following a triple vaccination with a total dose of 150 micrograms of recombinant gp63 mixed with Bacille Calmette Guerin (BCG) . However, peripheral blood leucocytes (PBL) from these animals neither proliferated nor produced any interferon-gamma (IFN-gamma) following in vitro stimulation with the antigen . Analysis of lymphocyte subsets following vaccination did not reveal any striking phenotypic alteration of cellular sub-populations in PBL . When vaccinated animals were rechallenged, via the needle, with virulent Leishmania major promastigotes containing salivary gland extracts from vector sandflies, only partial protection was achieved . We concluded from these studies that rgp63 produced in Escherichia coli is a safe vaccine molecule which gives only partial protection following vaccination in the vervet monkey host . The molecule requires further improvement for vaccine and/or immunodiagnosis application.

J Membr Biol, 1995 Dec, 148(3), 277 - 85
Reconstitution of a passive Ca(2+)-transport pathway from the basolateral plasma membrane of rat parotid gland acinar cells; Lockwich T et al.; We have previously reported that rat parotid gland basolateral plasma membrane vesicles (BLMV) have a relatively high affinity Ca2+ transport pathway and an unsaturable Ca2+ flux component (Lockwich et al., 1994 . J . Membrane Biol . 141:289-296) . In this study, we have solubilized BLMV with octylglucoside (1.5%) and have reconstituted the solubilized proteins into proteoliposomes (PrL) composed of E . coli bulk phospholipids, by using a detergent dilution method . PrL exhibited 3-5-fold higher 45Ca2+ influx than control liposomes (without protein) . Ca2+ uptake into PrL was dependent on the {protein} in PrL and steady state {Ca2+} in PrL was in equilibrium with external {Ca2+} . These data demonstrate that a passive, protein-mediated Ca2+ transport has been reconstituted from BLMV into PrL . 45Ca2+ influx into liposomes did not saturate with increasing {Ca2+} in the assay medium . In contrast, PrL displayed saturable 45Ca2+ influx and exhibited a single Ca2+ flux component with an apparent KCa = 242 +/- 50.9 microM and Vmax = 13.5 +/- 1.14 nmoles Ca2+/mg protein/ minute . The KCa of Ca(2+)-transport in PrL was similar to that of the high affinity Ca2+ influx component in BLMV while the Vmax was about 4-fold higher . The unsaturable Ca2+ flux component was not detected in PrL . 45Ca2+ influx in PrL was inhibited by divalent cations in the order of efficacy, Zn2+ > Mn2+ > Co2+ = Ni2+, and appeared to be more sensitive to lower concentrations of Zn2+ than in BLMV . Consistent with our observations with BLMV, the carboxyl group reagent N,N'-dicyclohexylcarbodiimide (DCCD) inhibited the reconstituted Ca2+ transport in PrL . Importantly, in both BLMV and PrL, DCCD induced a 40-50% decrease in Vmax of Ca2+ transport without an alteration in KCa . These data strongly suggest that the high affinity, passive Ca2+ transport pathway present in BLMV has been functionally reconstituted into PrL . We suggest that this approach provides a useful experimental system towards isolation of the protein(s) involved in mediating Ca2+ influx in the rat parotid gland basolateral plasma membrane.

Biochem Mol Biol Int, 1995 Dec, 37(6), 1163 - 71
A rapid single-step purification method for human interferon-gamma from isolated Escherichia coli inclusion bodies; Haelewyn J et al.; A fast purification method for recombinant human interferon-gamma, produced in E . coli, was elaborated . Human IFN-gamma accumulated in the cytoplasm of E . coli cells as inclusion bodies (IB) . After lysis, the IB were isolated from the cell debris by means of a density gradient ultracentrifugation, and solubilized in 6 M guanidine hydrochloride . The subsequent refolding step was optimized for a maximal recovery of the biologically active dimer . Refolded IFN-gamma was then purified to homogeneity in a single cation exchange chromatographic step, yielding 12.5 mg protein per liter E . coli culture . The dimeric nature of the refolded protein was visualized by means of interchain cross-linking . In a subsequent Western blot the resulting derivative was recognized by a panel of five monoclonal antibodies, indicating that those epitopes on the protein surface remained unaffected upon cross-linking.

Pediatr Nephrol, 1995 Dec, 9(6), 700 - 4
Escherichia coli verotoxin binding to human paediatric glomerular mesangial cells; Robinson LA et al.; Haemolytic uraemic syndrome (HUS) remains the leading cause of acute renal failure in children . Although an Escherichia coli-produced verotoxin (VT) has been implicated in the pathogenesis of HUS, the precise mechanisms of disease are not well defined . We hypothesise that the pathogenesis of renal failure in HUS includes the binding of E . coli VT to the glomerular mesangial cell, with consequent effects on renal function . Using human paediatric mesangial cells, we studied the binding and biological effects of the purified verotoxin VT-1 . We isolated, purified and characterised paediatric glomerular mesangial cells . The mesangial cells were characterised by their immunoreactivity with both smooth muscle actin and vimentin antibodies, and lack of immunoreactivity with cytokeratin or factor VIII antibodies . Using an fluorescein isothiocyanate-conjugated VT (10(-7)-10(-8) M), we demonstrated specific binding to the mesangial cell membrane by immunofluorescence microscopy . We also demonstrated a dose-dependent inhibition of mesangial cell mitogenesis at concentrations from 10(-9) to 10(-17) M . Our data demonstrate that VT-1 binds to paediatric human glomerular mesangial cells and this binding results in specific biological actions, including an inhibition of cell mitogenesis.

J Bioenerg Biomembr, 1995 Dec, 27(6), 543 - 54
Experimental determination of control by the H(+)-ATPase in Escherichia coli; Jensen PR et al.; Strains carrying deletions in the atp genes, encoding the H(+)-ATPase, were unable to grow on nonfermentable substrates such as succinate, whereas with glucose as the substrate the growth rate of an atp deletion mutant was surprisingly high (some 75-80% of wild-type growth rate) . The rate of glucose and oxygen consumption of these mutants was increased compared to the wild-type rates . In order to analyze the importance of the H(+)-ATPase at its physiological level, the cellular concentration of H(+)-ATPase was modulated around the wild-type level, using genetically manipulated strains . The control coefficient by the H(+)-ATPase with respect to growth rate and catabolic fluxes was measured . Control on growth rate was absent at the wild-type concentration of H(+)-ATPase, independent of whether the substrate for growth was glucose or succinate . Control by the H(+)-ATPase on the catabolic fluxes, including respiration, was negative at the wild-type H(+)-ATPase level . Moreover, the turnover number of the individual H(+)-ATPase enzymes increased as the H(+)-ATPase concentration was lowered . The negative control by the H(+)-ATPase on catabolism may thus be involved in a homeostatic control of ATP synthesis and, to some extent, explain the zero control by the H(+)-ATPase on E . coli growth rate.

Protein Expr Purif, 1995 Dec, 6(6), 821 - 5
Expression of a highly unstable and insoluble transcription factor in Escherichia coli: purification and characterization of the fork head homolog HNF3 alpha; Zaret KS et al.; The HNF3 alpha transcription factor is highly enriched in adult hepatocytes and activates many liver-specific genes; its expression in hepatic primordia and neurogenic tissue implies a developmental role . HNF3 alpha is a member of a family of proteins, including fork head in Drosophila, which bind DNA at specific sites using a newly defined "winged helix" structural motif . We describe here the expression and purification of full-length HNF3 alpha containing an amino-terminal histidine tag and show that the protein binds DNA with dissociation constant in the sub-nanomolar range . The techniques used to isolate HNF3 alpha should be applicable to other members of the HNF3/fork head family which appear to be generally unstable and insoluble in Escherichia coli.

Protein Expr Purif, 1995 Dec, 6(6), 799 - 805
Expression, purification, and characterization of acylphosphatase muscular isoenzyme as fusion protein with glutathione S-transferase; Modesti A et al.; A genetic construct consisting of the synthetic gene coding for human muscle acylphosphatase linked to the gene for glutathione S-transferase has been prepared . This gene was transformed into and expressed by the Escherichia coli strains DB1035 and TB1, respectively . The fusion protein was purified by affinity chromatography and subsequently cleaved to the fully active acylphosphatase, which was further purified by gel filtration chromatography . Such a purification procedure is very rapid and suitable for obtaining considerable amounts of enzyme at a very high yield . The purified human muscle acylphosphatase was fully active and showed structural features, as well as kinetic and stability parameters, identical to those of the native enzyme.

Protein Expr Purif, 1995 Dec, 6(6), 771 - 9
Purification of recombinant shiga-like toxin type I B subunit; Austin PR et al.; Shiga-like toxin type I (SLT-I) is a cytotoxin produced by certain strains of Escherichia coli . SLT-I is a bipartite molecule comprised of A (SLT-IA) and B (SLT-IB) subunits . In holotoxin, five B subunits are arranged in a pentameric ring and bind to globotriaosylceramide receptors on the surface of susceptible eukaryotic cells . The SLT-IB pentamer is noncovalently associated with one A subunit that has N-glycosidase activity and ultimately causes the death of targeted cells . Using a previously described overexpression vector, plasmid SBC32, we developed a two-step procedure for the purification of biologically active recombinant SLT-IB . Periplasmic proteins were extracted from E . coli JM105(pSBC32), fractionated by ammonium sulfate precipitation, and separated by isoelectric focusing in a pH 3-10 gradient . SLT-IB was present in fractions with pH values between 5.0 and 6.0, consistent with its reported pI of 5.8 . SLT-IB was purified to homogeneity in a second step by native polyacrylamide gel electrophoresis . Purified SLT-IB was characterized for biological and biochemical activity . When analyzed by native polyacrylamide gel electrophoresis, the majority of SLT-IB had an apparent molecular weight of 38,900, consistent with a pentameric subunit association . Chemical cross-linking of SLT-IB with disuccinimidyl suberate resulted in species with molecular weights corresponding to dimeric, trimeric, tetrameric, and pentameric forms of B subunit . SLT-IB was not cytotoxic to Vero cells at concentrations as high as 10 micrograms/ml and protected Vero cells from native SLT-I . Purified SLT-IB maintained its ability to associate with SLT-IA to form holotoxin that exhibited toxicity similar to native toxin.

Protein Expr Purif, 1995 Dec, 6(6), 763 - 70
Overexpression and rapid purification of biologically active yeast proliferating cell nuclear antigen; Biswas EE et al.; Yeast proliferating cell nuclear antigen (yPCNA) is a cell-cycle-regulated protein that has been shown to be required for the efficient elongation of primed DNA templates by DNA polymerase delta in vitro . We have expressed yPCNA to a high level (> or = 30% of the total cellular protein) with and without a six-residue histidine tag at its amino-terminus . Both forms of the recombinant protein were found to be biologically active and no significant differences were observed between the two forms . In this report we describe an efficient method of extraction of DNA binding proteins, such as yPCNA, overexpressed in Escherichia coli . The presence of a (His) delta tag on the polypeptide permitted rapid and high-yield single-step purification of the protein (approximately 60 mg of purified yPCNA per liter of induced cell culture) by immobilized metal affinity chromatography using an imidazole gradient elution procedure . The purified yPCNA was used to characterize the biological activity and tertiary structure of the protein . Chemical crosslinking and size-exclusion FPLC studies indicated that both forms of the protein have a trimeric-oligomeric structure in solution . Taken together these results indicate that both forms of the recombinant yPCNA were similar to the endogenous protein in their biochemical properties . The strategies presented here are designed to maximize the yield of recombinant protein and should prove useful to the purification of other recombinant DNA binding proteins.

Protein Expr Purif, 1995 Dec, 6(6), 757 - 62
Overproduction and one-step purification of Escherichia coli UDP-N-acetylglucosamine enolpyruvyl reductase; Tayeh MA et al.; The Escherichia coli gene murB, encoding the enzyme uridine-5'-diphospho-N-acetyl-2-amino-2-deoxy-3-O-lactylglucosenicoti namide adenine dinucleotide phosphate oxidoreductase (EC 1.1.1.158) (EP-reductase), the second enzyme in the peptidoglycan biosynthetic pathway, has been amplified using PCR technology with the Kohara recombinant lambda phage E11C11 (534) as template . The synthetic gene was subcloned into the NdeI and BamHI restriction sites of the expression vector pT7-7, designed to utilize T7 RNA polymerase to direct transcription of the target gene, in a two-step procedure . The first step involved the directional insertion of the 590-bp NdeI to BamHI restriction fragment of murB into the pT7-7 vector to give the plasmid pT7-7-murB-590 . The construction of the desired overproducing plasmid was completed by the bidirectional insertion of the 442-bp BamHI to BamHI restriction fragment of murB into a similarly restricted pT7-7-murB-590 plasmid followed by restriction digestion to select the properly oriented insert, pT7-7-murB . Overexpression of EP-reductase from the E . coli strain BL 21 (DE 3) containing the pT7-7-murB gene, after induction, allowed the production of 36 mg of target protein per 3 wet grams of E . coli cells . The EP-reductase was purified in a single step utilizing dye-ligand chromatography to yield 30 mg of pure protein . The availability of these levels of reductase will allow the mechanism of this pivotal enzyme to be thoroughly studied as a potential target for the design of a new generation of antibiotics . In addition, the EP-reductase generated in this study has been utilized as a coupling enzyme to assay the first enzyme in the peptidoglycan biosynthetic pathway, UDP-N-acetylglucosamine enolpyruvyl transferase, and these results are also presented.

Protein Expr Purif, 1995 Dec, 6(6), 748 - 56
Identification of an inhibitory element within the human 68-kDa (U1) ribonucleoprotein antigen; Northemann W et al.; Various nuclear proteins are the major targets of autoimmune responses in various rheumatic disorders . In particular, autoantibodies directed against a 68-kDa protein associated with the (U1) RNA-containing small nuclear ribonucleoprotein complexes typically occur in sera of patients with mixed connective tissue disease and related rheumatic disorders, such as systemic lupus erythematosus, and therefore are very useful as a serological marker . For establishing powerful immunoassays, it was necessary to generate recombinant human P68 antigen as the antigenic target . In this study we demonstrated that the cDNA coding for the full-length human P68 antigen could not be expressed by a traditional bacterial vector system due to a putative inhibitory sequence designated as inhibitory sequence X which is located between the autoreactive domains C' and D' of the human P68 antigen . The construction of corresponding hybrid plasmids carrying two functional and independent gene blocks indicated the trans-active function of the inhibitory sequence X, which could be localized by expression studies of various deletion constructs . Comparable Northern blot analysis clearly demonstrated that the inhibitory sequence X could act on the translation of the P68 mRNA . After excision of the inhibitory sequence X a dramatic increase in the production of recombinant human P68 antigen was observed.

Protein Expr Purif, 1995 Dec, 6(6), 737 - 47
Expression of soluble human interleukin-2 receptor alpha-chain in Escherichia coli; Dracheva S et al.; A soluble, biologically active form of IL-2R alpha known as delta MST and consisting of the 178 N-terminal amino acid residues of the mature protein was directly expressed in the cytoplasm and the periplasm of Escherichia coli . Because it was not glycosylated, the E . coli protein was substantially less heterogeneous than delta MST expressed in insect cells . Nevertheless, it manifested equivalent biological activity in an IL-2 binding assay . The level of active delta MST production was higher when the protein was expressed in secretable form with a bacterial signal peptide than when it was produced in the cytoplasm, probably because the oxidizing environment and the presence of disulfide isomerases in the periplasm facilitated the correct folding of delta MST.

Protein Expr Purif, 1995 Dec, 6(6), 727 - 36
Overexpression in Escherichia coli, folding, purification, and characterization of the first three short consensus repeat modules of human complement receptor type 1; Dodd I et al.; We have developed a simple expression, isolation, and folding protocol for an SCR oligomer comprising the first three SCRs of complement receptor Type 1 (C3b/C4b receptor, CD35) . A T7 RNA polymerase expression system in Escherichia coli was used to express the oligomer as inclusion bodies . The oligomer was recovered from solubilized inclusion bodies using batch adsorption on SP-Sepharose . The oligomer was folded by one-step dilution in 20 mM ethanolamine/1 mM EDTA supplemented with 1 mM GSH/0.5 mM GSSG . The folded material was processed to a concentrated (> 20 mg/ml), usable product of greater than 98% purity using a combination of ultrafiltration, ammonium sulfate treatment, hydrophobic interaction, and size-exclusion chromatography . The yield of folded material varied between 6 and 15 mg/liter culture . The oxidation states of the 12 cysteine residues in SCR(1-3) were identified by HPLC of peptide fragments from a tryptic digest using dual UV/fluorescence detection, collection of selected peaks, and N-terminal sequencing . This methodology confirmed the expected location of disulfide bridges . Equilibrium and velocity sedimentation studies are interpreted in terms of a single sedimenting species with molecular weights of 21,629 and 21,063 by these respective techniques . These values compare to the predicted molecular weight, from amino acid composition, of 21,817 . The hydrodynamic properties of the molecule indicate that it is asymmetric with an axial ratio of 1:5.2 or equivalent dimensions of 21 x 110 A . SCR(1-3) has an unusual CD spectrum exhibiting a broad maximum at 220-230 nm and a minimum at 190 nm . There was little evidence of classical secondary structure . The product exhibited concentration-dependent inhibition of complement-mediated lysis of sensitized sheep red blood cells.

Protein Expr Purif, 1995 Dec, 6(6), 722 - 6
Expression and purification of a synthetic human obese gene product; Altmann SW et al.; Human obese (hOB) protein was recently identified as a secreted hormone-like factor that is exclusively produced by adipose tissue and appears to regulate the size of the body's fat stores . We describe a rapid and efficient repetitive extension PCR method for the construction of a 453-bp synthetic hOB gene with high-frequency codons to optimize expression in Escherichia coli . The use of a bacterial signal sequence fused to hOB together with expression of bacteriocin release protein resulted in efficient release of hOB into the culture medium . The protein was readily purified to homogeneity from the culture medium using a two-step procedure.

Hua Xi Yi Ke Da Xue Xue Bao, 1995 Dec, 26(4), 357 - 60
{Construction of genomic library of L . interrogans serovar lai and preliminary study of recombinant plasmid pDC38}; Chen Z et al.; A genomic library, consisting of approximate 12 000 recombinants, has been constructed for the Leptospira interrogans serovar lai in E . coli . using pUC18 as the vector . Hybridization analysis with the DNA fragment containing OmpL1 gene was performed and 10 positive clones were screened from the genomic library . One of the positive clones, designated pDC38, showed hybridization signal with the DNA of 7 serovars 8 strains of pathogenic leptospires, but not with the DNA of nonpathogenic leptospires (L . biflexa), Leptonama illini, E . coli., and 2 strains of L . interrongans.

Mol Biochem Parasitol, 1995 Dec, 75(1), 99 - 111
Characterization of a Schistosoma mansoni cDNA encoding a B-like cyclophilin and its expression in Escherichia coli; Klinkert MQ et al.; A cDNA encoding a Schistosoma mansoni cyclophilin (SmCyP) has been cloned by polymerase chain reaction amplification using degenerate oligonucleotides based on known conserved cyclophilin (CyP) sequences and by screening an expression cDNA library . The cDNA sequence encodes a 21.5-kDa protein, which shares 59% sequence identity with human CyP B . The SmCyP protein was expressed in Escherichia coli with a hexahistidine affinity tag at its amino terminus and antibodies to the purified (His6)-SmCyP fusion protein were raised in a rabbit . Fractionation of parasite material followed by immunoblot analysis revealed that schistosome CyP is a soluble protein . The N-terminus of the predicted protein contains a hydrophobic region, suggestive of a signal sequence . Accordingly, a recombinant SmCyP protein, lacking the first 23 amino acids was found to share the same gel electrophoretic mobility as the parasite-derived CyP protein, suggesting cleavage of a leader sequence . Hybridization of genomic DNA to a full-length cDNA probe indicates that the SmCyP gene is present as a single copy . Immunohistological experiments in conjunction with confocal scanning laser microscopy and immune electron microscopy show that SmCyP is present in abundance in the adult worm as well as in the schistosomula . The function of CyP in the schistosome is presently unclear, but since its ligand, cyclosporin A, has antischistosomal activity, its function is expected to be a vital one.

Mol Biochem Parasitol, 1995 Dec, 75(1), 33 - 42
Recombinant Pfs230, a Plasmodium falciparum gametocyte protein, induces antisera that reduce the infectivity of Plasmodium falciparum to mosquitoes; Williamson KC et al.; Six regions of malaria transmission-blocking target antigen, Pfs230, encoding 80% of the 363-kDa protein, were expressed as recombinant proteins in E . coli as fusions with maltose-binding protein (MBP) . Antisera generated against amylose-purified recombinant Pfs230/MBP fusion proteins (r230/MBP.A-r230/MBP.F) all recognized the 360-kDa form of parasite-produced Pfs230 by immunoblot . However, only antisera against the four carboxy regions (C-F) of Pfs230 and not the two amino regions (A and B) recognized the 310-kDa form of Pfs230, the form expressed on the surface of gametes . The data suggest that the 310-kDa form of Pfs230 arises from the cleavage of 50 kDa from the amino terminus of the 360-kDa form . Furthermore, antisera against r230/MBP.C bound to the surface of intact gametes and significantly reduced (by 71.2-89.8% (rank sum analysis, P < 0.01)) the infectivity of P . falciparum parasites to mosquitoes . This is the first report of a recombinant form of a P . falciparum gametocyte protein capable of inducing antisera that reduce malaria parasite infectivity to mosquitoes.

J Biochem (Tokyo), 1995 Dec, 118(6), 1216 - 23
Subunit association of gamma-glutamyltranspeptidase of Escherichia coli K-12; Hashimoto W et al.; gamma-Glutamyltranspeptidase {EC 2.3.2.2} of Escherichia coli K-12 consists of one large subunit and one small subunit, which can be separated from each other by high-performance liquid chromatography . Using ion spray mass spectrometry, the masses of the large and the small subunit were determined to be 39,207 and 20,015, respectively . The large subunit exhibited no gamma-glutamyltranspeptidase activity and the small subunit had little enzymatic activity, but a mixture of the two subunits showed partial recovery of the enzymatic activity . The results of native-polyacrylamide gel electrophoresis suggested that they could partially recombine, and that the recombined dimer exhibited enzymatic activity . The gene of gamma-glutamyltranspeptidase encoded a signal peptide, and the large and small subunits in a single open reading frame in that order . Two kinds of plasmid were constructed encoding the signal peptide and either the large or the small subunit . A gamma-glutamyltranspeptidase-less mutant of E . coli K-12 was transformed with each plasmid or with both of them . The strain harboring the plasmid encoding each subunit produced a small amount of the corresponding subunit protein in the periplasmic space but exhibited no enzymatic activity . The strain transformed with both plasmids together exhibited the enzymatic activity, but its specific activity was approximately 3% of that of a strain harboring a plasmid encoding the intact structural gene . These results indicate that a portion of the separated large and small subunits can be reconstituted in vitro and exhibit the enzymatic activity, and that the expressed large and small subunits independently are able to associate in vivo and be folded into an active structure, though the specific activity of the associated subunits was much lower than that of native enzyme . This suggests that the synthesis of gamma-glutamyltranspeptidase in a single precursor polypeptide and subsequent processing are more effective to construct the intact structure of gamma-glutamyltranspeptidase than the association of the separated large and small subunits.

J Biochem (Tokyo), 1995 Dec, 118(6), 1184 - 91
Sequence-specific DNA recognition of the Escherichia coli Ada protein associated with the methylation-dependent functional switch for transcriptional regulation; Sakashita H et al.; The Escherichia coli Ada protein, a suicidal DNA methyltransferase, is converted into a transcriptional regulator for methylation-resistance genes by the transfer of a methyl group from a DNA methylphosphotriester to its own Cys69 residue . Here, we report the DNA recognition mode and the functional switch mechanism of the N-terminal 16 kDa fragment of the Ada protein . NMR analysis has revealed that the segment from residues 102 to 123 forms a helix-turn-helix structure . A site-directed mutagenesis study has shown that the second helix in the helix-turn-helix structure plays a crucial role in specific recognition of DNA . These results imply that the sequence-specific interaction of the Ada protein with DNA occurs through the helix-turn-helix motif . NMR experiments on the methylated protein-DNA complex showed line broadening for the amide proton signals from the helix-turn-helix motif and for the protons in the vicinity of Cys69 . In the case of the nonmethylated protein-DNA complex, signal broadening was observed only for protons from the helix-turn-helix . These findings suggest that the residues in the vicinity of Cys69 come into direct contact with the cognate DNA after methylation . We propose that the direct contact of this region is a major factor for the "switch" that converts the Ada protein from a nonspecific DNA binding form to a transcription factor.

J Biochem (Tokyo), 1995 Dec, 118(6), 1131 - 7
Expression of human metallothionein-2 in Escherichia coli: cadmium tolerance of transformed cells; Odawara F et al.; A genetic approach was undertaken to investigate the physiological roles of human metallothionein-2 . A constructed expression plasmid, pEXPMTII, in which human metallothionein-IIA cDNA was inserted downstream of a tryptophan-lactose promoter, was used to transform Escherichia coli JM105 strain . Cadmium-binding metallothionein was successfully expressed in E . coli in the medium containing cadmium, while copper and zinc-metallothioneins were scarcely observed in copper- or zinc-containing medium . The amino acid composition and sequence of the biosynthesized cadmium-metallothionein were analyzed . The selectivity of metals bound to metallothionein and the stability of metal-binding forms of metallothionein in E . coli were discussed . In addition, cadmium, zinc, or copper resistance of the cells expressing metallothionein was examined . Cells transformed with the plasmid pEXPMTII and cultured in a medium containing cadmium exhibited tolerance only to cadmium . It was demonstrated that human metallothionein-2 functioned for cadmium detoxification in E . coli.

Glycobiology, 1995 Dec, 5(8), 807 - 11
Identification of O-sulphate substituents on D-glucuronic acid units in heparin-related glycosaminoglycans using novel synthetic disaccharide standards; Razi N et al.; The two disaccharides, methyl 4-O-(2-O-sulpho-beta-D-glucopyranosyl-uronic acid)-2-deoxy-2-amino-alpha-D-glucopyranoside and methyl 4-O-(3-O-sulpho-beta-D-glucopyranosyluronic acid)-2-deoxy-2-amino-alpha-D-glucopyranoside, were prepared by de novo synthesis, and converted to the corresponding 2,5-anhydro-D-{1-3H}mannitol derivatives by deamination with nitrous acid followed by reduction with NaB3H4 . The resultant labelled products were used as standards in the identification, by anion-exchange high-performance liquid chromatography (HPLC), of disaccharides generated by HNO2/NaB3H4 treatment of heparan sulphate isolated from human brain . The two standards, containing 2-O- and 3-O-sulphated glucuronic acid, respectively, were clearly separated by the HPLC procedure . Comparison with the deamination products derived from heparan sulphate showed that the mono-O-sulphated disaccharide species containing a sulphated glucuronic acid unit co-eluted with the 2-O-sulphated standard . The corresponding component isolated from other heparan sulphate preparations, or from heparin, also eluted at the same position . No disaccharide derived from heparin or heparan sulphate appeared at the elution position of the 3-O-sulphated standard . It is concluded that D-glucuronic acid units in heparin-related glycosaminoglycans may be sulphated at C2, whereas no evidence has been found for sulphation at C3 . By contrast, analysis of mono-O-sulphated disaccharides derived from a chemically sulphated, bacterial capsular polysaccharide (generated by Escherichia coli K5) clearly demonstrated the occurrence of O-sulphate groups at C-3 of D-glucuronic acid units.

J Vet Med Sci, 1995 Dec, 57(6), 1089 - 91
Effects of oil adjuvant on systemic response to Escherichia coli lipopolysaccharide in swine; Norimatsu M et al.; Intramuscular injection of 0.1 mg/kg of Escherichia coli lipopolysaccharide (LPS) mixed with Freund's complete adjuvant (LPS+FCA) in piglets mitigated the leukopenia and TNF-alpha and cortisol levels in the serum compared with that of LPS suspended in LPS-free saline . The endotoxin level in the serum of the LPS+FCA was remarkably reduced . These results suggest that the addition of oil adjuvant mitigate the systemic toxicity of LPS.

J Vet Med Sci, 1995 Dec, 57(6), 1041 - 4
Changes in the behavioral parameters following the lipopolysaccharide administration in goats; Takeuchi Y et al.; The present study was aimed at the establishment of an experimental model for the numerical assessment of sick animal behavior . Four goats were given bolus injections of 200 ng/kg of lipopolysaccharide (LPS) or vehicle (0 hr) under non-restrained conditions, and observed for behavioral changes and clinical symptoms during the period between -1 and 10 hr . The apparent clinical symptoms of miosis and shivering were observed during the period from 39.5 +/- 3.1 to 296.5 +/- 9.9 min and from 46.0 +/- 2.3 to 251.0 +/- 15.5 min after the LPS administration, respectively . As to the general behaviors, the total length for standing and sternum lying during the period from 0 to 5 hr after LPS administration showed no change, however, that for feeding and rumination, and the cumulative number of grooming episode were significantly reduced as compared to the control period . On the other hand, the cumulative numbers of urination, defecation and yawning showed a tendency to increase but not significantly . These results suggest that stereotyped behavioral responses, which are typically seen in acute phase of sickness, can be transiently induced in goats by treating them with LPS.

Br J Pharmacol, 1995 Dec, 116(8), 3316 - 22
The effect of endotoxin on sympathetic responses in the rat isolated perfused mesenteric bed; involvement of nitric oxide and cyclo-oxygenase products; Fatehi-Hassanabad Z et al.; 1 . The effects of endotoxin on the vasoconstrictor responses to sympathetic nerve stimulation (SNS) were investigated in the rat isolated perfused mesenteric bed . 2 . Rats received either saline (0.1 ml h-1) or endotoxin (2.5 mg kg-1 h-1) intravenously for 4 h; the mesenteric beds were then isolated, perfused with Krebs and prepared for SNS (50 V, 3 ms, 7-40 Hz) . 3 . SNS caused a frequency-dependent vasoconstrictor response which was abolished by either tetrodotoxin (10(-7) M), prazosin (2.4 x 10(-7) M) or guanethidine (2.4 x 10(-7) M) . 4 . In mesenteric vascular beds removed from rats infused with endotoxin, there were markedly impaired vasoconstrictor responses to SNS, although responses to noradrenaline were not modified . 5 . Removal of the endothelium with distilled water prevented endotoxin-induced impairment of vasoconstrictor responses to SNS, without modifying these responses in preparations from control rats . 6 . Pretreatment with dexamethasone (3 mg kg-1 i.p . 1h before commencing endotoxin or saline infusions) did not modify responses to SNS in control rats but prevented the effects of endotoxin . 7 . Both L-NAME (10(-3) M) and indomethacin (10(-5) M) restored responses to SNS in preparations from endotoxin-treated rats without modifying these responses in control preparations . However, co-administration of L-NAME and indomethacin markedly augmented responses in both control and endotoxin-treated preparations . 8 . The effects of L-NAME were reversed by addition of L-arginine (10(-3) M) . 9 . The data suggest that endotoxin impairs the release of noradrenaline and that this effect is secondary to increased production of nitric oxide and prostanoids, possibly by the endothelium.

Br J Pharmacol, 1995 Dec, 116(8), 3309 - 15
The contribution of tumour necrosis factor-alpha and endothelin-1 to the increase of coronary resistance in hearts from rats treated with endotoxin; Hohlfeld T et al.; 1 . Inflammatory disease states predispose to myocardial infarction . Here we have investigated the effects of a systemic inflammatory response syndrome, i.e . lipopolysaccharide (LPS)-induced circulatory shock in rats, on coronary vascular tone . 2 . Anaesthetized rats were given LPS (10 mg kg-1, i.v.) and the hearts excised 180 min later for isolated perfusion at constant flow by the Langendorff technique . Once the ex vivo perfusion was started, the perfusion pressure strongly increased in these hearts compared to hearts from control rats (130 +/- 3 vs . 49 +/- 3 mmHg after 10 min) . This increase in coronary resistance was not associated with a reduction in endothelial cell function, for the vasodilator responses to bradykinin were unchanged . 3 . When hearts were removed 30 min after the injection of LPS, the LPS-induced rise in perfusion pressure was delayed . No changes in perfusion pressure were seen when the hearts were removed 15 min after the injection of LPS . Pre-treatment with cycloheximide or an anti-tumour necrosis factor-alpha (TNF-alpha) antibody or continuous infusion in vivo and in vitro of the endothelin ETA receptor selective antagonist FR 139317, greatly decreased the increase in coronary vascular resistance induced by LPS . 4 . These data suggest that TNF-alpha may induce the release of endothelin-1 (ET-1) and that this mediates at least part of the coronary vasoconstriction . This hypothesis is supported by the demonstration that LPS administration increased the circulating levels of both TNF-alpha and ET-1 . 5 . We conclude, therefore, that in inflammatory disease states, such as LPS-induced septic shock, there is the sequential release of TNF-alpha and endothelin-1 which leads to an increase in coronary vascular tone and so a predisposition to myocardial ischaemia . Inactivation of TNF-alpha by an antibody as well as ETA receptor blockade by a selective antagonist may effectively interfere with this pathway.

Immunol Lett, 1995 Dec, 48(2), 103 - 7
Glutathione S-transferase a major allergen of the house dust mite, Dermatophagoides pteronyssinus; O'Neill GM et al.; Recently, we cloned a new Dermatophagoides pteronyssinus allergen homologous with glutathione S-transferase . IgE radioimmunoassays against the Escherichia coli lysate of the recombinant clone reveal that 40% of mite allergic subjects recognize the mite glutathione S-transferase allergen . Of these sera, greater that 50% have reactivity with the recombinant mite glutathione S-transferase that is greater than 10 times the result observed with a normal control . Immunoblotting studies with sera from patients that recognize the recombinant protein reveal IgE binding to a 26-kDa protein on immunoblots of reduced mite protein extracts . The 26-kDa IgE-binding band observed on immunoblots of reduced mite proteins, corresponding to the cloned protein, is a separate and unique allergen from the 25-kDa Der p I as the apparent electrophoretic molecular mass of Der p I shifts from 25 to 30 kDa under conditions of reduction.

Plant Cell, 1995 Dec, 7(12), 2101 - 14
Tobacco mosaic virus movement protein associates with the cytoskeleton in tobacco cells; McLean BG et al.; Tobacco mosaic virus movement protein P30 complexes with genomic viral RNA for transport through plasmodesmata, the plant intercellular connections . Although most research with P30 focuses on its targeting to and gating of plasmodesmata, the mechanisms of P30 intracellular movement to plasmodesmata have not been defined . To examine P30 intracellular localization, we used tobacco protoplasts, which lack plasmodesmata, for transfection with plasmids carrying P30 coding sequences under a constitutive promoter and for infection with tobacco mosaic virus particles . In both systems, P30 appears as filaments that colocalize primarily with microtubules . To a lesser extent, P30 filaments colocalize with actin filaments, and in vitro experiments suggested that P30 can bind directly to actin and tubulin . This association of P30 with cytoskeletal elements may play a critical role in intracellular transport of the P30-viral RNA complex through the cytoplasm to and possibly through plasmodesmata.

Indian J Biochem Biophys, 1995 Dec, 32(6), 361 - 7
Cloning and characterization of mycobacteriophage I3 promoters; Ramesh G et al.; Restriction fragments of mycobacteriophage I3 DNA capable of initiating transcription have been cloned into a promoter selection vector of Escherichia coli, and selected on the basis of development of resistance to chloramphenicol . The growth pattern of these 'promoter clones' on a concentration gradient of chloramphenicol and the biochemical assays of the chloramphenicol acetyl transferase have permitted the assessment of their relative promoter strengths . DNA sequence analysis revealed significant homology of these promoters to the -35 regions of the mycobacterial--and E . coli promoter consensus, but less so the -10 region . Based on the sequence of phage I3 promoters identified here and the reported sequences of mycobacterial promoters, a promoter consensus for mycobacteria has been generated.

Zhongguo Zhong Yao Za Zhi, 1995 Dec, 20(12), 725 - 8, 762
{Technology of processing carbonized root of Sanguisorba of ficinalis L . and the quality standards for its prepared pieces}; Ding A et al.; The technology of processing carbonized root of Sanguisorba of ficinalis was selected using orthogonal experiment design . The result shows that the best way is to stir-fry the drug in a pan for 7.5 min at 250 degrees C . The contents of trace elements in the prepared piece are significantly increased, and the prepared piece have marked hemostatic and bacteriostastic effects.

Vaccine, 1995 Dec, 13(18), 1754 - 8
Optimization of the intestinal lavage procedure for determination of intestinal immune responses; Ahren C et al.; Optimal conditions to process, concentrate and store intestinal lavage fluid were studied in samples collected from volunteers before and after oral immunization with a prototype vaccine against enterotoxigenic Escherichia coli (ETEC) diarrhoea . Total IgA and specific IgA antibody titres against enterotoxin and colonization factor antigen were determined in 22 lavage samples which were either enzyme-inhibited or heat-inactivated and then subjected to different long-term storage conditions . Samples were analysed within 1 month of collection and also after 3, 6 and 24 months of storage . Total IgA concentrations and specific IgA antibody levels were higher in lavage samples treated with enzyme inhibitors (soybean trypsin inhibitor and phenylmethylsulfonyl fluoride) than in those heat-inactivated . Similarily, concentration of the lavage fluid by freeze-drying was superior to concentration against polyethylene glycol . Specific antibody titres remained elevated after storage for at least 6 months but declined after 2 years in frozen compared with freeze-dried samples.

Mol Biotechnol, 1995 Dec, 4(3), 247 - 58
One-step purification of recombinant proteins with the 6xHis tag and Ni-NTA resin; Crowe J et al.; The 6xHis/Ni-NTA system allows rapid and efficient affinity purification of recombinant proteins from virtually any expression system . Protocols and tips for purification under both native and denaturing conditions are provided, as well as a rapid spin procedure for protein minipreps.

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