Escherichia coli

Atherosclerosis, 1996 Sep 27, 126(1), 95 - 104
Synthesis of tissue inhibitor of metalloproteinase-1 (TIMP-1) in rabbit aortic neointima after selective de-endothelialization; Wang H et al.; Altered TIMP-1 synthesis in the arterial wall may be important for the balance between metalloproteinases and their inhibitors, and thus contribute to dysregulated extracellular matrix metabolism in atherosclerotic lesions . To examine this, we cloned the rabbit TIMP-1 gene from aortic neointima, developed in response to a balloon-catheter induced de-endothelialization . The apparent homology of cDNA with TIMP-1 genes from several sources suggested that it is a rabbit form of TIMP-1 . We examined the recombinant rabbit TIMP-1 expression in Escherichia coli using the pTrxFus expression system and the synthesis of the resulting soluble protein was confirmed by immunostaining with anti-TIMP-1 . The TIMP-1 concentration in normal and de-endothelialized rabbit aortas was compared using Northern blot, Western blot and mRNA in situ hybridization techniques . We observed a significant increase of TIMP-1 expression in neointimal SMCs at both nucleic acid and protein levels, suggesting a role of TIMP-1 in injury-induced atherogenesis.

Biochim Biophys Acta, 1996 Sep 27, 1303(2), 92 - 102
Human non-pancreatic (group II) secreted phospholipase A2 expressed from a synthetic gene in Escherichia coli: characterisation of N-terminal mutants; Othman R et al.; A gene coding for human non-pancreatic (group II) secreted phospholipase A2 (hnpsPLA2) has been constructed by the single-step ligation of twelve synthetic oligonucleotides . The gene has been cloned into a modification of the bacterial expression vector pET 11 which allows protein over-expression as inclusion bodies and enables about 3 mg/litre of pure refolded fully active enzyme to be obtained . The protein was expressed as a 1-Ala mutant (N1A) to allow removal of the initiator methionine by the Escherichia coli amino-peptidase . This mutant had very similar properties to the wild-type enzyme . A double mutant, N1A, V3W was also constructed and expressed in high yield . This tryptophan-containing mutant showed similar properties to the wild-type and N1A mutant but had about 40% of the activity under the assay conditions used . This tryptophan was used as a reporter group for interfacial binding and its properties were compared to those of the corresponding tryptophan in PLA2 from procine pancreas . Expression of the wild-type gene sequence for hnpsPLA2 in E . coli gave the expected mutant protein still with the initiator methionine and with much reduced activity . Interfacial binding of all hnpsPLA2 mutants to anionic phospholipids was very similar when assessed by fluorescence methods . Comparisons of these mutants with the pancreatic enzyme revealed significant differences in terms of the effect of calcium on interfacial binding . The ability to express reasonably large amounts of the N1A mutant in E . coli will provide a basis for future site directed mutagenesis studies of this important human enzyme.

J Mol Biol, 1996 Sep 27, 262(3), 375 - 86
A new subclass of the zinc metalloproteases superfamily revealed by the solution structure of peptide deformylase; Meinnel T et al.; Escherichia coli peptide deformylase, a member of the zinc metalloproteases family, is made up of an active core domain composed of 147 residues and of an additional and dispensable C-terminal tail of 21 residues . The three-dimensional structure of the catalytic core could be studied by NMR . 1H and 15N NMR resonances assignments were obtained by two-dimensional and three-dimensional heteronuclear spectroscopy . The structure could be calculated using a set of 1015 restraints for the 147 residues of the enzyme . The overall structure is composed of a series of antiparallel beta-strands which surround two perpendicular alpha-helices . The C-terminal helix contains the HEXXH motif, which is crucial for activity . This helical arrangement and the way the histidines bind the zinc ion clearly are structurally reminiscent of the other members of the metalloprotease family, such as thermolysin or metzincins . Nevertheless, the overall arrangement of secondary and tertiary structures of peptide deformylase and the positioning of its third zinc ligand (a cysteine) are quite different from those of the other members of the family . These discrepancies, together with several biochemical differences, lead us to propose that peptide deformylase is the first example of a new class of the zinc-metalloproteases family . Studies of the interaction of peptide deformylase with either an inhibitor of the reaction or a product of the catalysed reaction, Met-Ala-Ser, as well as comparisons with the structures of other enzymes of the family, have enabled us to delineate the area corresponding to their binding site . The structural basis of the specificity of recognition of the formyl group is discussed in the context of the protease superfamily.

J Biol Chem, 1996 Sep 27, 271(39), 24138 - 43
Catalytic mechanism and DNA substrate recognition of Escherichia coli MutY protein; Lu AL et al.; Escherichia coli MutY protein cleaves A/G- or a/7,8-dihydro-8-oxo-guanine (A/GO)-containing DNA on the A-strand by N-glycosylase and apurinic/apyrimidinic endonuclease or lyase activities . In this paper, we show that MutY can be trapped in a stable covalent enzyme-DNA intermediate in the presence of sodium borohydride, a new finding that supports the grouping of MutY in that class of DNA glycosylases that possess concomitant apurinic/apyrimidinic lyase activity . To potentially help determine the substrate recognition site of MutY, mutant proteins were constructed . MutY proteins with a Gly116 --> Ala (G116A) or Asp (G116D) mutation had reduced binding affinities for both A/G- and A/GO-containing DNA substrates . The catalytic parameters, however, were differentially affected . While A/G- and A/GO-containing DNA were cleaved by MutY with specificity constants (kcat/Km) of 10 and 3.3 min-1 microM-1, respectively, MutY(G116D) cleaved these DNAs 2, 300- and 9-fold less efficiently . The catalytic activities of MutY(G116A) with A/G- and A/GO-containing DNA were about the same as that of wild-type MutY . Both MutY(G116A) and MutY(G116D) could be trapped in covalent intermediates with A/GO-containing DNA, but with lower efficiencies than the wild-type enzyme in the presence of sodium borohydride . MutY(G116A) also formed a covalent intermediate with A/G-containing DNA, but MutY(G116D) did not . Since Gly116 of MutY lies in a region that is highly conserved among several DNA glycosylases, it is likely this conserved region is in the proximity of the substrate binding and/or catalytic sites.

J Biol Chem, 1996 Sep 27, 271(39), 24096 - 104
Functional dissection and site-directed mutagenesis of the structural gene for NAD(P)H-nitrite reductase in Neurospora crassa; Colandene JD et al.; Neurospora crassa NAD(P)H-nitrite reductase, encoded by the nit-6 gene, is a soluble, alpha2-type homodimeric protein composed of 127-kDa polypeptide subunits . This multicenter oxidation-reduction enzyme utilizes either NADH or NADPH as electron donor and possesses as prosthetic groups two iron-sulfur (Fe4S4) clusters, two siroheme groups, and two FAD molecules . The native activity of the enzyme is the NAD(P)H-dependent reduction of nitrite to ammonia . In addition, N . crassa nitrite reductase displays several partial activities in vitro, including a siroheme-independent NAD(P)H-cytochrome c reductase activity and an FAD-independent dithionite-nitrite reductase activity . These partial activities are presumed to be manifestations of discrete functional domains within the protein . A full-length nit-6 cDNA was constructed and used in developing an expression system within E . coli capable of yielding high levels of NADPH-nitrite reductase activity . Maximal expression was obtained in nirB- E . coli cells grown anaerobically at 22 +/- 1 degrees C, in conjunction with co-expression of a plasmid-borne cysG gene (encoding the rate-limiting enzyme in siroheme synthesis) and co-transformation with plasmid pGroESL (encoding bacterial chaperonins GroES and GroEL) . Dissection of gene segments encoding putative functional domains within the nit-6 gene was performed . Expression of a partial cDNA construct encoding the FAD-/NAD-binding domain yielded extracts with NADPH-cytochrome c reductase activity but no NADPH-nitrite reductase activity or dithionite-nitrite reductase activity . Expression of a cDNA construct encoding the (Fe4S4)-siroheme-binding domain resulted in extracts possessing dithionite-nitrite reductase activity but no NADPH-nitrite reductase or NADPH-cytochrome c reductase activity . Analysis of site-directed mutations altering amino acid residues Cys-331 within the FAD-/NAD-binding domain and Ser-755 within the (Fe4S4)-siroheme-binding domain of the nitrite reductase demonstrated that these residues were not essential for native or partial enzyme activity . Cys-757 within the (Fe4S4)-siroheme-binding domain was essential for native enzyme activity.

J Biol Chem, 1996 Sep 27, 271(39), 24010 - 6
Kinetic characterization of dUTPase from Escherichia coli; Larsson G et al.; The enzyme dUTPase catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, thereby preventing a deleterious incorporation of uracil into DNA . The best known dUTPase is that from Escherichia coli, which, like the human enzyme, consists of three identical subunits . In the present work, the catalytic properties of the E . coli dUTPase were investigated in the pH range 5-11 . The enzyme was found to be highly specific for dUTP and discriminated both base and sugar as well as the phosphate moiety (bound dUDP was not hydrolyzed) . The second best substrate among the nucleotides serving as building blocks for DNA was dCTP, which was hydrolyzed an astonishing 10(5) times less efficiently than dUTP, a decline largely accounted for by a higher Km for dCTP . With dUTP.Mg as substrate, kcat was found to vary little with pH and to range from 6 to 9 s-1 . Km passed through a broad minimum in the neutral pH range with values approaching 10(-7) M . It increased with deprotonation of the uracil moiety of dUTP and showed dependence on two ionizations in the enzyme, exhibiting pKa values of 5.8 and 10.3 . When excess dUTPase was reacted with dUTP middle dotMg at pH 8, the two protons transferred to the reaction medium were released in a concerted mode after the rate-limiting step . The Mg2+ ion enhances binding to dUTPase of dUTP by a factor of 100 and dUDP by a factor of 10 . Only one enantiomer of the substrate analog 2'-deoxyuridine-5'-(alpha-thio)-triphosphate was hydrolyzed by the enzyme . These results are interpreted to favor a catalytic mechanism involving magnesium binding to the alpha-phosphate, rate-limiting hydrolysis by a shielded and activated water molecule and a fast ordered desorption of the products . The results are discussed with reference to recent data on the structure of the E . coli dUTPase.UDP complex.

J Biol Chem, 1996 Sep 27, 271(39), 23954 - 9
Mechanism-based inactivation of dopa decarboxylase by serotonin; Bertoldi M et al.; Pig kidney dopa decarboxylase (DDC) expressed in Escherichia coli is a homodimeric enzyme containing one catalytically active pyridoxal 5'-phosphate active site per subunit . In addition to catalyzing the decarboxylation of -aromatic amino acids, DDC also reacts with 5-hydroxytryptamine (5-HT), converting it to 5-hydroxyindolacetaldehyde and ammonia . These products have been identified by means of the enzymes alcohol dehydrogenase and glutamate dehydrogenase, together with high performance liquid chromatographic and mass spectroscopic analysis . The Kcat and Km values of this reaction were determined to be 0.48 min-1 and 0.47 mM, respectively . The NaBH4-reduced enzyme does not catalyze this reaction . Concurrent with this reaction, 5-HT inactivates DDC in both a time- and concentration-dependent manner and exhibits saturation of the rate of inactivation at high concentrations, with Ki and Kinact values of 0.40 mM and 0.023 min-1, respectively . Protection from inactivation by 5-HT was observed in the presence of the active site-directed inhibitor 3,4-dihydroxy-D-phenylalanine . Inactivation with {2-14C}5-HT results in the incorporation of 1 mol of label/enzyme subunit . Taken together, these findings indicate that 5-HT is both a substrate and a mechanism-based inactivator with a partition ratio for product formation versus inactivation of 21 . The absorbance, CD, and fluorometric features of 5-HT-inactivated DDC have also been characterized . A speculative mechanism for the reaction and inactivation consistent with the experimental findings is presented.

J Biol Chem, 1996 Sep 27, 271(39), 23884 - 94
Transcriptional pausing of RNA polymerase in the presence of guanosine tetraphosphate depends on the promoter and gene sequence; Krohn M et al.; We have studied the response of the effector molecule guanosine 3',5'-bisdiphosphate (ppGpp) on RNA polymerase pausing during in vitro transcription elongation . Pausing was followed during single round extension of stalled ternary complexes excluding possible ppGpp effects on initiation . The ppGpp dependences of early pausing sites within different transcription systems controlled by promoters with known response to enhanced ppGpp levels in vivo were quantitatively characterized . Transcription of stable RNAs and mRNA genes were analyzed . In addition, the in vitro pausing behavior of two promoter variants directing the same sequence but differing in their in vivo ppGpp sensitivity were compared . In the presence of ppGpp we noted a slight general enhancement of specific pauses in all transcription systems . However, genes known to be under stringent or growth rate control in vivo revealed a notably stronger pausing enhancement . The sites of pausing are not changed by the presence of ppGpp but appear to be sequence-specific . The effect of ppGpp on the extent of pausing depends on the particular promoter and closely adjacent sequences that the RNA polymerase has passed during initiation . Pausing enhancement requires the presence of ppGpp during elongation but not during initiation . The results underline the importance of pausing for transcription regulation and offer a plausible explanation for inhibition of stable RNA expression under conditions of elevated concentrations of ppGpp.

J Biol Chem, 1996 Sep 27, 271(39), 23874 - 83
Interaction of Escherichia coli RecA protein with LexA repressor . II . Inhibition of DNA strand exchange by the uncleavable LexA S119A repressor argues that recombination and SOS induction are competitive processes; Harmon FG et al.; The Escherichia coli RecA protein is involved in SOS induction, DNA repair, and homologous recombination . In vitro, RecA protein serves as a co-protease to cleave LexA repressor, the repressor of the SOS regulon; in addition, RecA protein promotes homologous pairing and DNA strand exchange, steps important to homologous recombination and DNA repair . To determine if these two functions of RecA protein are competing or parallel, the effect of uncleavable LexA S119A repressor on RecA protein-dependent activities was examined . LexA S119A repressor inhibits both the single-stranded DNA (ssDNA)-dependent ATP hydrolysis and DNA strand exchange activities of RecA protein . As for wild-type LexA repressor (Rehrauer, W . M., Lavery, P . E., Palmer, E . L., Singh, R . N., and Kowalczykowski, S . C . (1996) J . Biol . Chem . 271, 23865-23873), inhibition of ATP hydrolysis is dependent upon the presence of E . coli single-stranded DNA binding (SSB) protein, arguing that LexA repressor affects the competition between RecA protein and SSB protein for ssDNA binding sites . In contrast, inhibition of DNA strand exchange activity is SSB protein-independent, suggesting that LexA S119A repressor blocks a site required for DNA strand exchange . These results imply that there is a common site on the RecA protein filament for secondary DNA and LexA repressor binding and raise the possibility that the recombination and co-protease activities of the RecA protein filament are competitive.

J Biol Chem, 1996 Sep 27, 271(39), 23865 - 73
Interaction of Escherichia coli RecA protein with LexA repressor . I . LexA repressor cleavage is competitive with binding of a secondary DNA molecule; Rehrauer WM et al.; Essential to the two distinct cellular events of genetic recombination and SOS induction in Escherichia coli, RecA protein promotes the homologous pairing and exchange of DNA strands and the proteolytic cleavage of the LexA repressor, respectively . Since both of these activities require single-stranded DNA (ssDNA) and ATP, the inter-relationship between these reactions was investigated and found to display many parallels . The extent of active complex formed between RecA protein and M13 ssDNA, as measured by both ATP hydrolysis and LexA proteolysis, is stimulated in a similar manner by either a reduction in magnesium ion concentration or the presence of single-stranded DNA binding (SSB) protein . However, unexpectedly, SSB protein inhibits both LexA proteolysis and ATP hydrolysis (in assays containing repressor) at concentrations of RecA protein that are substoichiometric to the ssDNA, arguing that LexA repressor affects the competition between RecA and SSB proteins for limited ssDNA binding sites . Additionally, attenuation of LexA repressor cleavage in the presence of double-stranded DNA or by an excess of ssDNA suggests that interaction of the RecA nucleoprotein filament with either LexA repressor or a secondary DNA molecule is mutually exclusive . The significance of these results is discussed in the context of both the regulation of inducible responses to DNA damage, and the competitive relationship between the processes of SOS induction and genetic recombination.

J Biol Chem, 1996 Sep 27, 271(39), 23615 - 8
High field EPR studies of mouse ribonucleotide reductase indicate hydrogen bonding of the tyrosyl radical; Schmidt PP et al.; Ribonucleotide reductase catalyzes by free radical chemistry the reduction of ribonucleotides to deoxyribonucleotides . The R2 protein of a class 1 ribonucleotide reductase contains a stable tyrosyl radical of neutral phenoxy character, which is necessary for normal enzymatic activity . Here we present the EPR spectra from the tyrosyl free radical in the R2 protein from mouse at 9.62, 115, and 245 GHz . We show that the g-value anisotropy of the mouse R2 radical, when precisely determined from high field EPR spectra, is similar to that of the hydrogen bonded dark stable YD middle dot tyrosyl radical of photosystem II and different from that of the Escherichia coli R2 radical . Because the g-value anisotropy is an important indicator of the hydrogen bonding status of the tyrosyl radical, this result suggests that the mouse R2 radical has its tyrosylate oxygen hydrogen bonded with a D2O exchangeable proton, whereas this hydrogen bond is absent in the E . coli enzyme . It is suggested that the observed proton may be derived from the tyrosine that will become a tyrosyl radical.

Gene, 1996 Sep 26, 174(1), 51 - 8
Construction and expression of a synthetic wheat storage protein gene; Anderson OD et al.; A synthetic wheat high-molecular-weight (HMW) glutenin storage protein gene analog was constructed for expression in E . coli . This first synthetic HMW-glutenin gene and future modifications are intended to allow systematic dissection of the molecular basis of HMW-glutenin role in the visco-elastic properties critical for wheat product processing and utilization . The design of the gene included four features: different construction strategies for the separate assembly of major polypeptide domains, the inclusion of convenient restriction sites for modifications, use of a codon selection similar to E . coli highly expressed genes, and the ability to produce repetitive sequence domains of exact numbers of defined repeats . The complete synthetic HMW-glutenin construct was 1908 bp, and contained 32 identical copies of one of the HMW-glutenin repetitive domain motifs . The gene expressed the novel HMW-glutenin protein to relatively high levels in bacterial cultures and the protein exhibited the known anomalous behavior of HMW-glutenins in SDS-PAGE.

Mol Gen Genet, 1996 Sep 25, 252(4), 398 - 403
Transcription-induced deletions in plasmid vectors: M13 DNA replication as a source of instability; Vilette D et al.; We have previously shown that concurrent progression of pBR322 replication and pTac-directed transcription in opposite orientations induces illegitimate recombination events . We tested here the effects of M13 rolling circle replication on the incidence of plasmid deletions . The progression of the M13 replication fork leads to an increase of more than 300-fold in the frequency of transcription-dependent deletion events . pBR322 derivatives carrying the M13 replication origin and a 511 bp transcribed region under the control of the pTac promoter were used . Up to 12% of the plasmid population has sustained deletions within 4 h following the induction of pTac-directed transcription and M13 DNA replication, provided that the two proceed in opposite orientations . We observed that induction of transcription of the whole Escherichia coli lacZ gene (3244 bp) in the direction opposite to M13 replication leads to a fivefold decrease in plasmid copy number within 2 h, which is consistent with the proposal that deletions arise because replication fork progression is impeded . This decrease in parental plasmid copy number leads in turn to an enrichment in deleted plasmid forms . Our data confirm and extend the notion that simultaneous transcription and replication in opposite directions can efficiently promote deletion formation . In addition, this instability may be amplified when the rearranged molecules acquire a replicative advantage.

Biochem Biophys Res Commun, 1996 Sep 24, 226(3), 822 - 9
Mutations in a putative zinc-binding domain inactivate the mitochondrial intermediate peptidase; Chew A et al.; The mitochondrial intermediate peptidase (MIP) cleaves characteristic octapeptides, (F/L/I)XX(T/S/ G)XXXX(decreases), from the N-terminus of many imported mitochondrial proteins . This leader peptidase is activated by divalent cations and inactivated by thiol-blocking agents, properties which are typical of metallo- and cysteine-proteases, respectively . To elucidate the mechanism of action of MIP, we analyzed by site-directed mutagenesis the functional role of a putative zinc-binding domain (F-H-E-X-G-H-(X)2-H-(X)12-G-(X)5-D-(X)2-E-X-P-S-(X)3-E) and two cysteine residues (C131 and C581), which are highly conserved in evolutionarily distant MIP sequences . We show that two histidines and a glutamic acid in the H-E-X-G-H motif and a glutamic acid 25 residues from the second histidine are essential for MIP function in vivo . In contrast, C131 and C581 are important for protein stability but are not required for activity in vivo or in vitro . These findings are consistent with MIP being a metallopeptidase.

Biochemistry, 1996 Sep 24, 35(38), 12623 - 8
Seeding of A beta fibril formation is inhibited by all three isotypes of apolipoprotein E; Wood SJ et al.; Apolipoprotein E is immunochemically localized to amyloid plaque in Alzheimer's brains, and the allelic distribution of ApoE in individuals is associated with a disposition toward Alzheimer's disease . We show here that all three ApoE isotypes exhibit a strong and specific ability to inhibit both nucleation and seeding of fibril formation by the A beta peptide in vitro . A beta (1-40) depleted of aggregates requires long incubation times before the onset of fibril formation, but addition of very low levels of A beta fibrils to such reactions is sufficient to reduce or eliminate this lag time . ApoE added to such seeded reactions extends the lag time in a dose-dependent manner, so that higher levels of seeding require higher levels of ApoE to achieve a given delay time to reaction onset . This effect is observed with all three isotypes produced in Escherichia coli, as well as with plasma-derived ApoE and the N-terminal domain of ApoE3 produced in E . coli . In contrast, bovine serum albumin and the four-helix bundle protein interleukin-4 are poor inhibitors of seeding . ApoE3 can also inhibit fibril formation by A beta (1-42) . The three full-length isotypes of ApoE produced in E . coli are equipotent at inhibition . It is therefore possible that the genetics of ApoE and AD may fundamentally depend on the ability of ApoE to inhibit seeding but that the trends in the genetics must be related to something other than the specific activities of the native ApoE isoforms used in these studies . The data show ApoE to be the first member of a new class of fibril formation inhibitor that acts by blocking the seeding of fibril growth.

Biochemistry, 1996 Sep 24, 35(38), 12526 - 31
Truncations of the C-terminus have different effects on the conformation and activity of phosphatidylinositol transfer protein; Voziyan PA et al.; Contributions of the C-terminus toward the conformation and activity of phosphatidylinositol transfer protein (PITP) were studied by comparing properties of the 271 amino acid, full-length protein, PITP(1-271), and two truncated species, PITP(1-259) and PITP(1-253) . Using recombinant proteins and an in vitro phospholipid transfer assay with phosphatidylcholine vesicles, the activities of PITP(1-271) and PITP(1-259) were identical, while the activity of PITP(1-253) was almost totally abolished . By most physical and chemical criteria, however, PITP(1-259) and PITP(1-253) were virtually indistinguishable and differed significantly from the full-length protein . Results of second derivative analysis of absorbance spectra were consistent with an additional two Tyr residues being exposed to the solvent in PITP(1-259) and PITP(1-253) in comparison to PITP(1-271) . Only one out of four Cys residues in PITP(1-271) reacted with dithiobisnitrobenzoic acid, while two Cys residues were accessible in both truncated species . Quenching of intrinsic Trp fluorescence by acrylamide demonstrated an increase in exposure of Trp residues in both PITP(1-259) and PITP(1-253); binding of the fluorescence probe 1,8-ANS to these proteins was also significantly higher compared to PITP(1-271) . These results describe a more relaxed overall tertiary structure brought about by the C-terminal truncations . This altered structure did not affect the stability of the truncated proteins, as indicated by equilibrium unfolding in guanidinium chloride . Refolding of the denatured PITP(1-259), however, was considerably slower than that of full-length PITP . Our study suggests a critical role of the C-terminal residues 254-259 in transfer activity of PITP . Residues 260-271, on the other hand, appear to be more important for the rapid folding and maintenance of a compact native conformation of the protein.

Biochemistry, 1996 Sep 24, 35(38), 12503 - 10
Internal mobility of reactive-site-hydrolyzed recombinant Cucurbita maxima trypsin inhibitor-V characterized by NMR spectroscopy: evidence for differential stabilization of newly formed C- and N-termini; Liu J et al.; The solution structure and internal dynamics of the reactive-site (Lys44-Asp45 peptide bond) hydrolyzed form of recombinant Cucurbita maxima trypsin inhibitor-V (rCMTI-V*) were characterized by the application of two-dimensional 1H-15N NMR methods to the uniformly 15N-labeled protein . The 1H-15N chemical shift correlation spectra of rCMTI-V* were assigned, and the chemical shift data were compared with those available for rCMTI-V {Liu, J., Prakash, O., Cai, M., Gong, Y., Huang, Y., Wen, L., Wen, J . J., Huang, J.-K., & Krishnamoorthi, R . (1996) Biochemistry 35, 1516-1524} and CMTI-V* {Cai, M., Gong, Y., Prakash, O., & Krishnamoorthi, R . (1995) Biochemistry 34, 12087-12094} for which three-dimensional solution structures have been determined . It was deduced that the solution structure of rCMTI-V* was almost the same as that of CMTI-V* . 15N spin-lattice and spin-spin relaxation rate constants (R1 and R2, respectively) and inverted question mark1H inverted question mark-15N steady-state heteronuclear Overhauser effects were measured for the peptide NH units and arginine and tryptophan N epsilon H groups in rCMTI-V*, and the model-free parameters {Lipari, G., & Szabo, A . (1982) J . Am . Chem . Soc . 104, 4546-4559, 4559-4570} were computed . Most of the backbone of rCMTI-V* is found to be highly constrained (S2 = 0.85), including the N-terminal residues 3-6 (S2 = 0.77) . Residues 39-44, forming the C-terminal fragment of the binding loop, exhibit increased mobility (S2 = 0.51); however, the N-terminal segment (residues 46-48) retains rigidity as in the intact form (S2 = 0.83) . The S2 values, 0.78 and 0.59, respectively, of Arg50 and Arg52 side chain NHs provide evidence not only for the conservation of the Arg hydrogen-bonds with the binding loop segments but also for the difference in strength between them . This is consistent with the earlier observation made from a study of rCMTI-V at two different pHs and its R50 and R52 mutants {Cai, M., Huang, Y., Prakash, O., Wen, L., Dunkelbarger, S . P., Huang, J.-K., Liu, J., & Krishnamoorthi, R . (1996) Biochemistry 35, 4784-4794} . The dynamical results suggest the mainchain oxygen atom of Asp45 as the hydrogen bond acceptor of Arg50 . Residues Trp9 and Trp54, which interact with many others in the protein scaffold and the binding loop region, respectively, remain rigid in the cleaved inhibitor with the S2 values of 0.84 and 0.71 determined for their respective N epsilon Hs . The internal dynamics of rCMTI-V* was compared with that of the noncovalent complex formed between the two fragments of reactive-site-hydrolyzed chymotrypsin inhibitor-2 from barley seeds {CI-2; Shaw, G . L., Davis, B., Keeler, J., & Fersht, A . R . (1995) Biochemistry 34, 2225-2233}, another potato I family inhibitor that lacks the Cys3-Cys48 disulfide present in rCMTI-V*.

Biochemistry, 1996 Sep 24, 35(38), 12487 - 94
Deacylation and reacylation for a series of acyl cysteine proteases, including acyl groups derived from novel chromophoric substrates; Doran JD et al.; In order to investigate structure-reactivity relationships within a series of acyl cysteine proteases {Doran, J . D., & Carey, P . R . (1996) Biochemistry 35, 12495-12502}, deacylation kinetics have been measured for a number of acyl intermediates involving members of the papain superfamily . Derivatives of the "simple" chromophoric ligand (5-methylthienyl)acrylate (5MTA) and those based on two chromophorically labeled derivatives of peptidyl substrates, viz., 2-{(N-acetyl-L-phenylalanyl)amino}-3-(5-methylthienyl)acrylate (Phe5MTA) and 2-{(N-acetyl-L-alanyl)amino}-3-(5-methylthienyl)acrylate (Ala5MTA), were used to create acyl enzyme adducts with papain, cathepsin B, and cathepsin L . The chromophoric specific substrates were designed to utilize hydrogen-bonding and hydrophobic interactions which are known to be important in promoting catalysis by papain . For cathepsins B and L, removing one of the hydrogen-bonding donors making up the putative oxyanion hole retards deacylation by 3-25-fold, demonstrating that the oxyanion hole has a modest effect on catalysis for these substrates . With the above substrates and the wild-type and oxyanion hole mutants, the values of the deacylation rate constants stretch over a 214-fold range, from 0.07 to 15 x 10(-3) s-1 . The pKa for deacylation of {(5-methylthienyl)-acryloyl}papain is 4.9, close to that reported for similar papain intermediates, while that for Ala5MTA-papain is at 3.5, which in the latter likely represents the effect of the P1-S1 and P2-S2 interactions on the environment of histidine-159 . For the Phe5MTA-papain the extent of deacylation was found to depend on the pH and the starting acyl enzyme concentration . A simple model has been derived which accounts quantitatively for this behavior, using the assumptions that the protonated form of the acyl product reacylates the enzyme and that in the pH range 5.0-7.5 the ionization of active-site groups has no effect on reacylation . The validity of the first assumption was demonstrated by following the deacylation of Phe5MTA-papain in the presence of the potent inhibitor E-64 {1-(L-trans-epoxysuccinyl-L-leucylamino)-4-guanidinobutane}, whereupon complete deacylation occurred at all pHs with a pKa identical to that for Ala5MTA-papain, viz., 3.5.

Biochemistry, 1996 Sep 24, 35(38), 12479 - 86
Factors influencing redox thermodynamics and electron self-exchange for the {Fe4S4} cluster in Chromatium vinosum high potential iron protein: the role of core aromatic residues in defining cluster redox chemistry; Soriano A et al.; The roles of aromatic core residues in regulating the reduction potential, the enthalpy and entropy of reduction, and the self-exchange rate constants for electron-transfer reactions for the prosthetic {Fe4S4}3+/2+ cluster of Chromatium vinosum high potential iron protein (HiPIP) have been addressed by a combination of site-directed mutagenesis, high field NMR (EXSY) experiments, and variable temperature spectrochemical redox titration measurements . Minimal changes are observed following nonconservative mutation of residues Tyr19, Phe48, and Phe66 . Apparently these hydrophobic residues play only a minor role in defining the electronic properties of the cluster . These data support a model, first defined from results obtained on Tyr19 mutant HiPIP's {Agarwal, A., Li, D., & Cowan, J.A . (1995) Proc . Natl . Acad . Sci . U.S.A . 92, 9440-9444}, in which the aromatic core restricts solvent accessibility and thereby stabilizes the oxidized {Fe4S4}3+ cluster.

Biochemistry, 1996 Sep 24, 35(38), 12455 - 63
Electrochemical measurement of second-order electron transfer rate constants for the reaction between cytochrome b5 and cytochrome c; Seetharaman R et al.; The second-order electron transfer reaction between cytochrome b5 and cytochrome c has been studied by cyclic voltammetry utilizing a gold electrode modified with beta-mercaptopropionate . When cyclic voltammetry is performed on a solution containing a mixture of cytochrome b5, cytochrome c and polylysine, cytochrome b5 undergoes reversible electrochemistry at the electrode surface while cytochrome c discriminates against the electrode surface . The selectivity of the modified electrode for negatively charged proteins makes it possible to selectively reduce a protein possessing a net negative charge and a relatively low reduction potential (outer mitochondrial membrane cytochrome b5, Eo = -102 mV; microsomal cytochrome b5, Eo = 3 mV) in the presence of another protein possessing a net positive charge and a relatively high reduction potential (cytochrome c, Eo = 265 mV) . The electrochemical reduction of ferricytochrome b5 at the electrode surface is followed by a second-order electron transfer reaction between ferrocytochrome b5 and ferricytochrome c that yields ferricytochrome b5 and ferrocytochrome c . This fast homogeneous electron transfer reaction which is preceded by a heterogeneous electron transfer reaction results in a characteristic cyclic voltammogram containing a pre-peak to the reduction current . The second-order rate constant for the homogeneous reaction was obtained by invoking the above reaction scheme for digital simulation of a cyclic voltammogram which was subsequently fitted to the experimental data . Second-order rate constants obtained with this method are 2.9 x 10(8) and 8.9 x 10(8) for the homogeneous electron transfer reactions between rat liver outer mitochondrial membrane (OM) ferrocytochrome b5 and beef liver microsomal ferrocytochrome b5, with horse heart ferricytochrome c, respectively . These values are in good agreement with second-order rate constants obtained for the same protein systems by flash photolysis . {Meyer, T . E., Rivera, M., Walker, F . A., Mauk, M . R., Mauk, A . G., Cusanovich, M . A., & Tollin, G . (1993) Biochemistry 32, 622-627}.

Biochemistry, 1996 Sep 24, 35(38), 12402 - 11
Medium-long-chain chimeric human Acyl-CoA dehydrogenase: medium-chain enzyme with the active center base arrangement of long-chain Acyl-CoA dehydrogenase; Nandy A et al.; The catalytically essential glutamate residue that initiates catalysis by abstracting the substrate alpha-hydrogen as H+ is located at position 376 (mature MCADH numbering) on loop JK in medium chain acyl-CoA dehydrogenase (MCADH) . In long chain acyl-CoA dehydrogenase (LCADH) and isovaleryl-CoA dehydrogenase (IVDH), the corresponding Glu carrying out the same function is placed at position 255 on the adjacent helix G . These glutamates thus act on substrate approaching from two opposite regions at the active center . We have implemented the topology of LCADH in MCADH by carrying out the two mutations Glu376Gly and Thr255Glu . The resulting chimeric enzyme, "medium-/long" chain acyl-CoA dehydrogenase (MLCADH) has approximately 20% of the activity of MCADH and approximately 25% that of LCADH with its best substrates octanoyl-CoA and dodecanoyl-CoA, respectively . MLCADH exhibits an enhanced rate of reoxidation with oxygen, however, with a much narrower substrate chain length specificity that peaks with dodecanoyl-CoA . This is the same maximum as that of LCADH and is thus significantly shifted from that of native MCADH (hexanoyl/octanoyl-CoA) . The putative, common ancestor of LCADH and IVDH has two Glu residues, one each at positions 255 and 376 . The corresponding MCADH mutant, Thr255Glu (glu/glu-MCADH), is as active as MCADH with octanoyl-CoA; its activity/chain length profile is, however, much narrower . The topology of the Glu as H+ abstracting base seems an important factor in determining chain length specificity and reactivity in acyl-CoA dehydrogenases . The mechanisms underlying these effects are discussed in view of the three-dimensional structure of MLCADH, which is presented in the accompanying paper {Lee et al . (1996) Biochemistry 35, 12412-12420}.

Biochemistry, 1996 Sep 24, 35(38), 12387 - 401
Structure-reactivity relationships for beta-galactosidase (Escherichia coli, lac Z) . 4 . Mechanism for reaction of nucleophiles with the galactosyl-enzyme intermediates of E461G and E461Q beta-galactosidases; Richard JP et al.; Second-order rate constants for transfer of the beta-D-galactopyranosyl group from the galactosyl-enzyme intermediates of the galactosyl transfer reactions catalyzed by E461G and E461Q beta-galactosidases to anionic nucleophiles have been determined . The second-order rate constant for reaction of the galactosylated E461G enzyme with azide ion is 4900 M-1 s-1 . By contrast, there is no detectable reaction of the galactosylated wild type enzyme with azide ion (Richard et al., 1995b), and the E461G mutation leads to a large decrease in the second-order rate constant kcat/Km for catalysis of cleavage of beta-D-galactopyranosyl azide, which is the microscopic reverse of the reaction of azide ion with the galactosyl-enzyme intermediate . These data show that the E461G mutation causes a more than 8000-fold increase in the equilibrium constant for transfer of the beta-D-galactopyranosyl group from beta-galactosidase to azide ion . We propose that this change represents the requirement for the coupling of galactosyl transfer from the native enzyme to the thermodynamically unfavorable protonation of the carboxylate group of Glu-461, but the expression of the full chemical affinity of azide ion for galactosyl transfer from the mutant enzyme which lacks this ionizable side chain at position 461 . The reactions of acetate, butyrate and methoxyacetate ions with the galactosylated E461G enzyme and of acetate with the galactosylated E461Q enzyme give both the corresponding beta-galactopyranosyl derivatives and D-galactose, and the formation of the latter represents formal catalysis of the reaction of water with the galactosylated enzyme . However, the reaction of formate ion with the galactosylated E461G enzyme gives only D-galactose . These results suggest that carboxylate anions can take the place of the excised propionate side chain of Glu-461 to provide general base catalysis of the reaction of water with the galactosyl-enzyme intermediates . The relative reactivity of anionic nucleophiles toward the covalent galactosyl-enzyme intermediate of the reactions catalyzed by the E461G enzyme is similar to that observed for partitioning of stable carbocations in water . This suggests that replacement of the anionic side chain of Glu-461 by a hydrogen exposes an enzyme-stabilized oxocarbenium ion intermediate to reaction with external nucleophilic reagents.

Biochemistry, 1996 Sep 24, 35(38), 12377 - 86
Structure-reactivity relationships for beta-galactosidase (Escherichia coli, lac Z) . 3 . Evidence that Glu-461 participates in Brønsted acid-base catalysis of beta-D-galactopyranosyl group transfer; Richard JP et al.; Experiments are reported to determine the role of Glu-461 in the beta-D-galactopyranosyl group transfer reaction catalyzed by beta-galactosidase . E461G beta-galactosidase catalyzes the hydrolysis of 4-nitrophenyl beta-D-galactopyranoside through a galactosyl-enzyme intermediate that shows a high reactivity toward the anionic nucleophile azide ion, but no detectable reactivity toward the neutral nucleophile trifluoroethanol . By contrast, the galactosylated wild type enzyme is reactive toward trifluoroethanol but not anions . The change in specificity observed for the E461G mutant can be rationalized by a mechanism in which Glu-461 participates in general acid-base catalysis at the leaving group/nucleophile . The observed low activity of E461G beta-galactosidase for hydrolysis of 2,2,2-trifluoroethyl beta-D-galactopyranoside is due entirely to a wild type enzyme contaminant in our preparation of the mutant enzyme, and the mutant enzyme itself has essentially no catalytic activity for cleavage of this substrate . The substitution of glutamate at position 461 by glycine leads to a more than 500 000-fold reduction in the rate constant for enzymatic cleavage of the glycosidic bond to the strongly basic trifluoroethoxide leaving group (pKa = 12.4), but to a smaller 1300-fold reduction in the rate constant for cleavage of the bond to the more weakly basic 4-nitrophenoxide leaving group (pKa = 7.1) . This corresponds to a more than 3.5 kcal/mol greater stabilization by Glu-461 of the transition state for the reaction of the substrate with the more basic trifluoroethoxide leaving group . These data are consistent with the conclusion that Glu-461 provides general acid catalysis of leaving group departure, which is most effective for cleavage of the relatively strong bonds to basic alkoxide leaving groups.

Biochemistry, 1996 Sep 24, 35(38), 12369 - 76
Trichodiene synthase . Probing the role of the highly conserved aspartate-rich region by site-directed mutagenesis; Cane DE et al.; Trichodiene synthase catalyzes the cyclization of farnesyl diphosphate to the sesquiterpene hydrocarbon trichodiene . The enzyme normally requires a divalent cation, Mg2+, which can be substituted by Mn2+ . Trichodiene synthase from Fusarium sporotrichioides has a highly conserved aspartate rich region, aa 100-104 (DDSKD) . Three mutants were constructed by site-directed mutagenesis in which each aspartate residue was individually replaced by glutamate . The mutants were each overexpressed and purified to homogeneity . The importance of Asp100 and Asp101 for catalysis was established by the observation of an increase in Km as well as a reduction in kcat in the corresponding Glu mutants . Replacement of the Asp104 residue with Glu had little effect on either Km or kcat . All three mutants produced anomalous sesquiterpene products in addition to trichodiene when incubated with farnesyl diphosphate . Interestingly, when Mg2+ was replaced by Mn2+ in the incubation buffer, the kcat/Km of both wild type trichodiene synthase and the D104E dropped significantly, while those of the other two mutants were not much affected . The proportion of anomalous products increased significantly when the D100E and D101E mutants were incubated in the presence of Mn2+ . These observations all lend weight to the proposal that the aspartate residues mediate substrate binding by chelation of the divalent metal ion . Asp100 and Asp101 appear to play a relatively more important role than Asp104.

Biochemistry, 1996 Sep 24, 35(38), 12354 - 62
Cleavage of DNA by the insulin-mimetic compound, NH4{VO(O2)2(phen)}; Hiort C et al.; The kinetics and mechanism of cleavage of DNA by the insulin-mimetic peroxo-vanadate NH4{VO(O2)2(phen)}, pV, are described . In the presence of low energy UV radiation or biologically common reducing agents, pV decomposes into the monomer, dimer, and tetramer of vanadate and an uncharacterized compound of V4+ as shown by 51V NMR, ESR, and absorption spectra . The rate of photodecomposition of pV is reduced in the presence of calf thymus DNA, indicating that a decomposition product of the peroxo-vanadate, that is important in the destruction pathway of the complex, is interacting with DNA . This species, probably a short-lived complex of V4+, may also be responsible for the observed catalytic decomposition of pV in the absence of DNA by ascorbate . If closed circular pBR322 DNA is present when the peroxo-vanadate is destroyed by either UV radiation or reducing agents, the polymer may have its sugar-phosphate backbone broken . Closed circular DNA (form I) is converted into nicked circular DNA (form II) and linear DNA (form III) . The amounts of the various forms produced as a function of irradiation time and peroxo-vanadate concentration were fit to a kinetic model to derive rate constants for the conversions . The kinetic analysis shows that pV is a single-strand nicking agent which exhibits some base and/or sequence preference . Furthermore, the pH dependences of the rates for conversion of form I to form II and for conversion of form II to form III are different, indicating that the nature of the chemistry at the site of cleavage on DNA influences further cutting by activated pV . Reduced amounts of DNA breakage in the presence of various salts and metal binding ligands indicate that a short-lived reactive complex of V4+, not the V4+ species detected by ESR at long irradiation times, is important in the cleavage process . The susceptibility of pV to decomposition by biologically common reducing agents suggests that metabolites of the agent, and not the compound itself, are responsible for its insulin-mimetic effects.

Biochemistry, 1996 Sep 24, 35(38), 12228 - 34
Cobalamin-independent methionine synthase from Escherichia coli: a zinc metalloenzyme; Gonzalez JC et al.; Cobalamin-independent methionine synthase (MetE) from Escherichia coli catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine . Previous work had shown the existence of a reactive thiol group, cysteine 726, whose alkylation led to loss of all detectable enzymatic activity {Gonzalez, J.C., et al . (1992) Biochemistry 31, 6045-6056} . A site-directed mutation of MetE, Cys726Ser, was constructed to investigate the possible role of this cysteine . The Cys726Ser protein was purified to homogeneity, affording a protein with no detectable activity . To assess the possibility that cysteine726 functions as a metal ligand, inductively coupled plasma-atomic emission spectrometry was performed . The wild-type enzyme contains 1.02 equiv of zinc per subunit; the Cys726Ser mutant does not contain zinc, supporting the view that cysteine726 is required for metal binding . A loss of enzymatic activity is observed upon removal of zinc from the wild-type MetE by incubation in urea and EDTA; activity can subsequently be restored by zinc reconstitution, suggesting that zinc is required for catalysis . Circular dichroism measurements further suggest that there are no major differences in the secondary structures of the wild-type and the Cys726Ser mutant enzymes . Extended X-ray absorption fine structure analysis has established that the average zinc environment is different in the presence of homocysteine than in its absence and is consistent with the changes expected for displacement of an oxygen or nitrogen ligand by the sulfur of homocysteine . A possible model for zinc-dependent activation of homocysteine by MetE is presented.

FEBS Lett, 1996 Sep 23, 394(1), 71 - 5
5S rRNA sugar-phosphate backbone protection in complexes with specific ribosomal proteins; Shpanchenko OV et al.; 5S ribosomal RNA forms stable specific complexes with ribosomal proteins L18, L25 and L5 . In this work, interaction of phosphate residues of E . coli 5S rRNA within 5S rRNA-protein complexes has been studied . For this purpose 5S rRNA with statistically distributed phosphorothioate residues has been used for complex formation and the accessibility of phosphorothioates to iodine cleavage in the complex and in the free state has been studied . In free 5S rRNA, the phosphate residue at A73 was partially protected, probably due to being involved in the organization of the spatial structure of 5S rRNA . This protection is stronger in the complex with three proteins when the 5S rRNA structure is stabilized . In the 5S rRNA-L18 complex only two phosphate groups, G7 and A34, were protected . L25 in a complex with 5S rRNA protects large numbers of phosphorothioate groups concentrating in two clusters, indicating the possibility of two binding sites for this protein on 5S rRNA . The protection pattern differs from that for individual proteins because of the possible rearrangement of the structure.

FEBS Lett, 1996 Sep 23, 394(1), 66 - 70
Unusual enzyme characteristics of aspartyl-tRNA synthetase from hyperthermophilic archaeon Pyrococcus sp . KOD1; Fujiwara S et al.; The aspA gene, encoding the aspartyl-tRNA synthetase (AspRS) from the hyperthermophilic archaeon Pyrococcus sp . KOD1, was expressed in Escherichia coli . The KOD1 AspRS, which was purified to homogeneity and was shown to be functional in dimeric form, aminoacylated tRNA from KOD1 . The optimum temperature for this activity was 65 degrees C, which was lower than that for the cell growth of KOD1 (85 degrees C) . However, it increased to 75 degrees C by the addition of polyamine molecules, such as putrescine, spermine, and spermidine . Analysis of the thermal denaturations of the enzyme and of KOD1-tRNA indicated that neither of them was denatured at temperatures below 70 degrees C . These results suggest polyamine is one of the factors which are required to stabilize the AspRS-tRNA complex in vivo . In order to determine whether the nucleotide triphosphate (NTP) is required for Asp-tRNA synthesis, the aminoacylation was examined in the presence of each of the four NTPs . AspRS most effectively aminoacylated tRNA in the presence of ATP . However, we also found that the enzyme aminoacylated it even in the presence of GTP and UTP as well . Archaeon synthetase may have an interesting system to utilize other NTPs than ATP . The extreme conditions of early life may have given rise to these unique characteristics which then disappeared from developed organisms through evolution.

Carbohydr Res, 1996 Sep 23, 291, 127 - 39
Structural studies of the enteroinvasive Escherichia coli (EIEC) O28 O-antigenic polysaccharide; Rundlof T et al.; The structure of the O-specific side-chain of the lipopolysaccharide from Escherichia coli O28 has been investigated . NMR spectroscopy has been the main method used, complemented with sugar and methylation analyses . The polysaccharide contains one equivalent of O-acetyl groups per repeating unit . Selective cleavage of the O-deacetylated polymer was performed by treatment with aqueous hydrofluoric acid, and resulted in a trisaccharide-glycerol . The polysaccharide thus is of the teichoic acid type and composed of repeating units in which the trisaccharide-glycerol residues are joined by phosphodiester linkages . The O-antigen polysaccharide has the following structure . {sequence: see text} The absolute configuration of the glycerol moiety as R, )i.e., D-glycerol 1-phosphate) was determined by a new method based on TEMPO oxidation of the polysaccharide, followed by GLC analysis of the (+)-2-butyl ester of the resulting glyceric acid.

Carbohydr Res, 1996 Sep 23, 291, 43 - 52
Evaluation of C-(beta-D-galactosyl) and C-(2-deoxy-D-lyxo-hex-1-enopyranosyl) (D-galactal type) derivatives as inhibitors of beta-D-galactosidase from Escherichia coli; Kiss L et al.; C-(2-Deoxy-D-lyxo-hex-1-enopyranosyl)formamide was prepared from acetylated C-(beta-D-galactopyranosyl)formamide by a radical-mediated bromination-zinc/N-methylimidazole-induced reductive elimination-Zemplen deacetylation reaction sequence . The preparation of acetylated 5-(2-deoxy-D-lyxo-hex-1-enopyranosyl)tetrazole was improved . Methyl C-(2-deoxy-D-lyxo-hex-1-enopyranosyl)formimidate was transformed by benzylamine into N-benzyl-C-(2-deoxy-D-lyxo-hex-1-enopyranosyl)formamidine and, after hydrolysis to methyl C-(2-deoxy-D-lyxo-hex-1-enopyranosyl)formate, into N-benzyl-C-(2-deoxy-D-lyxo-hex-1-enopyranosyl)formamide . A series of C-(beta-D-galactopyranosyl) and C-(2-deoxy-D-lyxo-hex-1-enopyranosyl) derivatives was comparatively investigated for E . coli beta-D-galactosidase inhibitory activity . N-Benzyl-C-(2-deoxy-D-lyxo-hex-1-enopyranosyl)formamidine was the best inhibitor and had K(i) = 6 microM (on the basis of its free base concentration, K(i) = 8.3 nM was obtained) . Basicity and hydrophobicity of the aglycon proved to be more important factors for the inhibition than the conformation of the sugar ring.

Mutat Res, 1996 Sep 23, 356(2), 237 - 45
Nitro musks are cogenotoxicants by inducing toxifying enzymes in the rat; Mersch-Sundermann V et al.; In the present study, musk xylene (MX) and musk ketone (MK) were examined for their potency to induce toxifying enzymes in the liver of Sprague-Dawley rats, using an in vivo/in vitro model . After i.p . application of 10, 20 and 40 mg/day MX and MK over a period of 5 days, 9000 x g liver fractions (S9M) were used to study the toxification of a number of well-known pregenotoxicants in the SOS chromotest, i.e., benzo{a}pyrene (B{a}P), 2-aminoanthracene (2-AA), and aflatoxin B1 (AFB1) . The genotoxic potencies of B{a}P, 2-AA and AFB1 in the presence of S9M were compared to those obtained in the presence of S9 fractions of untreated animals (S9O, negative control) . S9M fractions derived from MK-treated rats showed an increased potency to toxify B{a}P, 2-AA and AFB1 in comparison to S9O fractions (for instance: TIP{toxifying induction potency} = 70 per nmol AFB1 using 10 mg MK treatment) . In comparison, S9M fractions from MX-pretreated rats exhibited an increased genotoxicity only when using 2-AA (TIP = 0.04) and AFB1 (TIP = 61) as pregenotoxicants, but not when using B{a}P . To summarize the results, both MX and MK were strong inducers of toxifying liver enzymes . Therefore, these compounds seem to be cogenotoxicants for a number of well-known pregenotoxicants . Synergistic effects were found when using inducers of toxifying enzymes and pregenotoxicants in the in vivo/in vitro induction model.

Mutat Res, 1996 Sep 23, 356(2), 229 - 35
Induction of SOS-independent mutations by benzo{a}pyrene treatment in Escherichia coli cells deficient in MutY or MutM DNA glycosylases: possible role of oxidative lesions; Urios A et al.; The induction of SOS-independent mutations by exposure to benzo{a}pyrene (BaP) was screened in Escherichia coli strains lacking SOS mutagenesis proteins and deficient in MutY or MutM glycosylases, which prevent mutations by 8-hydroxyguanine (GO lesion) . Mutagenicity assays, performed in the presence of S9 mix, indicated a great increase in the reversion of the trpE65 ochre mutation in both mutY and mutY mutM strains, whereas a lower increase was observed in a mutM strain . This mutability by BaP was observed in either uvr- or uvr+ strains . Moreover, it was increased when strains carried a deletion of the oxyR gene that abolished the OxyR response to oxidative stress, and reduced in the presence of the oxyR2 allele that rendered constitutive such response . It is suggested that SOS-independent mutations in cells treated with BaP arise from adenine/GO mispairs . The interaction of radical scavengers with BaP ultimate metabolites could cause oxidative stress capable of producing GO lesions . Strains lacking mutagenesis proteins and deficient in base excision repair systems, such as those dependent on MutY and MutM glycosylases, could be useful for screening the induction of SOS-independent mutations.

J Chromatogr B Biomed Appl, 1996 Sep 20, 684(1-2), 147 - 61
Purification methods of mammalian catechol-O-methyltransferases; Tilgmann C et al.; The protein purification strategies used for obtaining homogeneous rat and human soluble catechol-O-methyltransferase (S-COMT) polypeptides are reviewed . Expression and purification of recombinant rat and human S-COMT in Escherichia coli and for human S-COMT in baculevirus-infected insect cells made it possible to elucidate the S-COMT polypeptides in more detail . The application of these purification methods has allowed the crystallization of the rat S-COMT protein and the analysis of the kinetic properties of the enzyme in great detail . The availability of the pure S-COMT protein together with the structural data has also greatly enhanced the development of more potent COMT inhibitors.

J Mol Biol, 1996 Sep 20, 262(2), 225 - 42
The crystal structure of glutamine-binding protein from Escherichia coli; Hsiao CD et al.; The crystal structure of the glutamine-binding protein (GlnBP) from Escherichia coli in a ligand-free "open" conformational state has been determined by isomorphous replacement methods and refined to an R-value of 21.4% at 2.3 A resolution . There are two molecules in the asymmetric unit, related by pseudo 4-fold screw symmetry . The refined model consists of 3587 non-hydrogen atoms from 440 residues (two monomers), and 159 water molecules . The structure has root-mean-square deviations of 0.013 A from "ideal" bond lengths and 1.5 degrees from "ideal" bond angles . The GlnBP molecule has overall dimensions of approximately 60 A x 40 A x 35 A and is made up of two domains (termed large and small), which exhibit a similar supersecondary structure, linked by two antiparallel beta-strands . The small domain contains three alpha-helices and four parallel and one antiparallel beta-strands . The large domain is similar to the small domain but contains two additional alpha-helices and three more short antiparallel beta-strands . A comparison of the secondary structural motifs of GlnBP with those of other periplasmic binding proteins is discussed . A model of the "closed form" GlnBP-Gln complex has been proposed based on the crystal structures of the histidine-binding protein-His complex and "open form" GlnBP . This model has been successfully used as a search model in the crystal structure determination of the "closed form" GlnBP-Gln complex by molecular replacement methods . The model agrees remarkably well with the crystal structure of the Gln-GlnBP complex with root-mean-square deviation of 1.29 A . Our study shows that, at least in our case, it is possible to predict one conformational state of a periplasmic binding protein from another conformational state of the protein . The glutamine-binding pockets of the model and the crystal structure are compared and the modeling technique is described.

J Mol Biol, 1996 Sep 20, 262(2), 202 - 24
Crystal structure of the pyridoxal-5'-phosphate dependent cystathionine beta-lyase from Escherichia coli at 1.83 A; Clausen T et al.; Cystathionine beta-lyase (CBL) is a member of the gamma-family of PLP-dependent enzymes, that cleaves C beta-S bonds of a broad variety of substrates . The crystal structure of CBL from E . coli has been solved using MIR phases in combination with density modification . The structure has been refined to an R-factor of 15.2% at 1.83 A resolution using synchroton radiation diffraction data . The asymmetric unit of the crystal cell (space group C222(1)) contains two monomers related by 2-fold symmetry . A homotetramer with 222 symmetry is built up by crystallographic and non-crystallographic symmetry . Each monomer of CBL can be described in terms of three spatially and functionally different domains . The N-terminal domain (residues 1 to 60) consists of three alpha-helices and one beta-strand . It contributes to tetramer formation and is part of the active site of the adjacent subunit . The second domain (residues 61 to 256) harbors PLP and has an alpha/beta-structure with a seven-stranded beta-sheet as the central part . The remaining C-terminal domain (residues 257 to 395), connected by a long alpha-helix to the PLP-binding domain, consists of four helices packed on the solvent-accessible side of an antiparallel four-stranded beta-sheet . The fold of the C-terminal and the PLP-binding domain and the location of the active site are similar to aminotransferases . Most of the residues in the active site are strongly conserved among the enzymes of the transsulfuration pathway . Additionally, CBL is homologous to the mal gamma gene product indicating an evolutionary relationship between alpha and gamma-family of PLP-dependent enzymes . The structure of the beta, beta, beta-trifluoroalanine inactivated CBL has been refined at 2.3 A resolution to an R-factor of 16.2% . It suggests that Lys210, the PLP-binding residue, mediates the proton transfer between C alpha and S gamma.

J Mol Biol, 1996 Sep 20, 262(2), 151 - 67
Structural analysis of Sindbis virus capsid mutants involving assembly and catalysis; Choi HK et al.; Sindbis virus core protein (SCP) has been isolated from virus and crystallized . The X-ray crystallographic structure showed that the amino-terminal 113 residues appeared to be either disordered or truncated during crystallization and that the carboxy-terminal residues 114 to 264 had a chymotrypsin-like structure . The carboxy-terminal residues 106 to 264 and 106 to 266 of SCP have now been expressed in Escherichia coli . Most crystal forms of the truncated proteins were isomorphous with those of the virally extracted protein . There are only small structural differences between the truncated recombinant protein and the ordered part of the wild-type virus-extracted protein . Hence, E . coli-expressed SCP can be used to study proteolytic properties and the contribution of SCP to nucleocapsid assembly, interaction with the E2 glycoprotein and interaction with RNA . The same dimer that was found in two different crystal forms of the virus-extracted SCP was present also in some of the crystals of the truncated recombinant protein . The monomer-monomer interface is maintained by two pairs of hydrogen bonds and by hydrophobic interactions . Removal of the hydrogen bonds by single substitutions did not prevent dimer formation . However, a mutation that reduced the hydrophobic contacts did inhibit dimer formation . The wild-type truncated SCP is active in E . coli, as evidenced by proteolytic processing of a series of progressively longer precursors that extend beyond residue 264 . Unlike the virus-extracted capsid protein, the E . coli-expressed SCP described here is terminated following the carboxy-terminal residue and, therefore, does not require autocatalysis . Nevertheless, the E . coli-expressed protein folds with the carboxy-terminal tryptophan residue in the specificity pocket . Two crystallographically independent molecules of SCP(106 to 266), which had two additional downstream residues and had the essential S215 mutated to alanine, showed two distinct modes of binding the uncleaved carboxy-terminal residues . These may represent successive steps of binding substrate prior to catalytic cleavage . Refinement of the various crystal structures of SCP showed that the amino-terminal arm from residues 107 to 113 was not disordered, but is associated with neighboring molecules . Residues 108 to 111 bind into a hydrophobic pocket composed primarily of Y180, W247 and F166 . It had been shown that the double mutant (Y180S; E183G), with the Y180S substitution in this pocket, produced a large number of non-infectious virions, possibly because of modification in the interaction of the glycoprotein spikes with core proteins . The crystal structure of this double mutant showed that there was a large positional change in the side-chain of W247, which moved into the space created by the replacement of Y180 with serine . These conformational changes may alter the stability of the virion and, thus, regulate its functional requirements during cell entry.

J Mol Biol, 1996 Sep 20, 262(2), 140 - 50
Probing the structural role of an alpha beta loop of maltose-binding protein by mutagenesis: heat-shock induction by loop variants of the maltose-binding protein that form periplasmic inclusion bodies; Betton JM et al.; The maltose-binding protein (MBP) of Escherichia coli is the periplasmic receptor of the maltose transport system . Previous studies have identified amino acid substitutions in an alpha/beta loop of the structure of MBP that are critical for the in vivo folding . To probe genetically the structural role of this surface loop, we generated a library in which the corresponding codons 32 and 33 of malE were mutagenized . The maltose phenotype, which correlates with a biologically active structure of MBP in the periplasm, indicated a considerable variability in the loop residues compatible with a correct in vivo folding pathway of the protein . By the same genetic screens, we characterized loop-variant MBPs associated with a defective periplasmic folding pathway and aggregated into inclusion bodies . Heat-shock induction with production of misfolded loop variants was examined using both lon-lacZ and htrA-lacZ fusions . We found that the extent of formation of inclusion bodies in the periplasm of E . coli, from misfolded loop variant MBPs, correlated with the level of heat-shock response regulated by the alternate heat-shock sigma factor, sigma 24.

J Mol Biol, 1996 Sep 20, 262(2), 87 - 104
A tyrosyl-tRNA synthetase suppresses structural defects in the two major helical domains of the group I intron catalytic core; Myers CA et al.; The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase, the CYT-18 protein, functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron RNA . The group I intron catalytic core is thought to consist of two extended helical domains, one formed by coaxial stacking of P5, P4, P6, and P6a (P4-P6 domain) and the other consisting of P8, P3, P7, and P9 (P3-P9 domain) . To investigate how CYT-18 stabilizes the active RNA structure, we used an Escherichia coli genetic assay based on the phage T4 td intron to systematically test the ability of CYT-18 to compensate for structural defects in three key regions of the catalytic core: J3/4 and J6/7, connecting regions that form parts of the triple-helical-scaffold structure with the P4-P6 domain, and P7, a long-range base-pairing interaction that forms the guanosine-binding site and is part of the P3-P9 domain . Our results show that CYT-18 can suppress numerous mutations that disrupt the J3/4 and J6/7 nucleotide-triple interactions, as well as mutations that disrupt base-pairing in P7 . CYT-18 suppressed mutations of phylogenetically conserved nucleotide residues at all positions tested, except for the universally conserved G-residue at the guanosine-binding site . Structure mapping experiments with selected mutant introns showed that the CYT-18-suppressible J3/4 mutations primarily impaired folding of the P4-P6 domain, while the J6/7 mutations impaired folding of both the P4-P6 and P3-P9 domains to various degrees . The P7 mutations impaired the formation of both P7 and P3, thereby grossly disrupting the P3-P9 domain . The finding that the P7 mutations also impaired formation of P3 provides evidence that the formation of these two long-range pairings is interdependent in the td intron . Considered together with previous work, the nature of mutations suppressed by CYT-18 supports a model in which CYT-18 helps assemble the P4-P6 domain and then stabilizes the two major helical domains of the catalytic core in the correct relative orientation to form the intron's active site.

J Mol Biol, 1996 Sep 20, 262(2), 77 - 86
The "+70 pause": hypothesis of a translational control of membrane protein assembly; Kepes F; Sequences of 66 genes encoding bacterial or yeast membrane proteins have been examined for the respective positioning of putative transmembrane domains and translational pauses . The latter were operationally defined as clusters of at least 17 non-preferred codons along the mRNA . The putative transmembrane domains were defined as stretches of at least 17 hydrophobic amino acids in the encoded protein . For yeast non-mitochondrial membrane proteins, it was observed that clusters of non-preferred codons occur more frequently about 56 to 75 codons after a hydrophobic stretch in the encoded protein . About 40 amino acid residues are required to span the large ribosomal subunit . Such clusters were thus predicted to cause a severe slow-down in peptide elongation, just when the hydrophobic stretch fully protrudes from the ribosome . This transient slow-down of the ribosome pace has consequently been named the "+70 pause" . This pause was not observed for mitochondrial or bacterial membrane proteins, which are thought to insert post-translationally in their respective membranes . Because insertion of yeast proteins in the endoplasmic reticulum membrane is generally cotranslational instead, it is possible that the "+70 pause" reflects the coupling of translation, targeting, insertion and folding in this case . The pause may, for instance, give time for productive interaction of the newly synthesized hydrophobic domain with the proper targeting/insertion machineries . Thus, it would favor entrance of the stalled protein domain into the proper pathway.

J Mol Biol, 1996 Sep 20, 262(2), 71 - 6
Molecular symmetry of the ClpP component of the ATP-dependent Clp protease, an Escherichia coli homolog of 20 S proteasome; Shin DH et al.; The ClpP component Clp protease from Escherichia coli has been crystallized and examined by X-ray crystallography and self-rotation function calculations . The crystal belongs to the monoclinic space group P2(1) with unit cell dimensions of a = 196.9 A, b = 104.3 A, c = 162.4 A and beta = 98.3 degrees . The X-ray diffraction pattern extends at least to 2.5 A Bragg spacing when exposed to CuK alpha X-rays . Self-rotation function analyses indicate that the ClpP oligomer has 72-point group symmetry . This symmetry suggests that the ClpP oligomer is a tetradecamer, (ClpP)14, consisting of two heptamers, (ClpP)7 stacked on top of each other in a head-to-head fashion . The measurement of crystal density indicates that two independent copies of the ClpP oligomers are present in the asymmetric unit, giving a crystal volume per protein mass (VM) of 2.73 A3/Da and a solvent content of 54.9% (v/v) . Self-rotation function calculations are consistent with the presence of two ClpP tetradecamers in the asymmetric unit . The Patterson function suggests that a translation of x = 0.5 and y = 0.5 relates a pair of ClpP oligomers in one asymmetric unit to another pair in the other asymmetric unit . And the two independent tetradecamers in one asymmetric unit are related by a relative rotation of about 18 degrees around the 7-fold axis.

Cell, 1996 Sep 20, 86(6), 877 - 86
Replisome assembly reveals the basis for asymmetric function in leading and lagging strand replication; Yuzhakov A et al.; The E . coli replicase, DNA polymerase III holoenzyme, contains two polymerases for replication of duplex DNA . The DNA strands are antiparallel requiring different modes of replicating the two strands: one is continuous (leading) while the other is discontinuous (lagging) . The two polymerases within holoenzyme are generally thought to have asymmetric functions for replication of these two strands . This report finds that the two polymerases have equal properties, both are capable of replicating the more difficult lagging strand . Asymmetric action is, however, imposed by the helicase that encircles the lagging strand . The helicase contact defines the leading polymerase constraining it to a subset of actions, while leaving the other to cycle on the lagging strand . The symmetric actions of the two polymerases free holoenzyme to assemble into the replisome in either orientation without concern for a correct match to one or the other strand.

J Biol Chem, 1996 Sep 20, 271(38), 23235 - 8
The mechanism of velocity modulated allosteric regulation in D-3-phosphoglycerate dehydrogenase . Site-directed mutagenesis of effector binding site residues; Al-Rabiee R et al.; D-3-Phosphoglycerate dehydrogenase (EC 1.1.1.95) from Escherichia coli catalyzes the first committed step in serine biosynthesis and is allosterically regulated by L-serine, the end product of the pathway . Each subunit of the homotetramer is made up of three distinct domains with one of the intersubunit contacts being between adjacent regulatory domains . Each regulatory domain interface contains two symmetrical serine binding sites such that serine forms hydrogen bonds to both domains across the interface . Previous work (Al-Rabiee, R., Lee, E . J., and Grant, G . A . (1996) J . Biol . Chem . 271, 13013-13017) demonstrated that when adjacent regulatory domains are covalently linked to one another by engineered disulfide bonds, the enzyme was inactivated . Breaking the disulfide bonds by reduction restored enzymatic activity . This study demonstrates that the complementary situation is also true . Site-directed mutagenesis of three residues at the effector binding site, His344, Asn346, and Asn364', render the enzyme increasingly less susceptible to inhibition by the effector . When mutations result in a situation where it is no longer possible to establish a stable hydrogen bonding network across the regulatory domain interface, the inhibitory capacity of the effector is lost . Furthermore, mutations that produce as much as 5 orders of magnitude decrease in the ability of L-serine to inhibit the enzyme have no appreciable effect on the Km or kcat of the enzyme . These observations support the model that predicts that catalytic activity in D-3-phosphoglycerate dehydrogenase is regulated by the movement of adjacent regulatory domains about a flexible hinge and that effector binding tethers the regulatory domains together producing a state that results in a stable, open active site cleft that is no longer able to promote catalysis.

Nature, 1996 Sep 19, 383(6597), 279 - 82
Three-dimensional structure of human cytomegalovirus protease; Shieh HS et al.; Herpesviruses encode a serine protease that specifically cleaves assembly protein . This protease is critical for replication, and represents a new target for antiviral drug design . Here we report the three-dimensional structure of the protease from human cytomegalovirus (hCMV) at 2.27 angstroms resolution . The structure reveals a unique fold and new catalytic strategy for cleavage . The monomer fold of the enzyme, a seven-stranded beta-barrel encircled by a chain of helices that form the carboxy terminus of the molecule, is unrelated to those observed in classic serine proteases such as chymotrypsin and subtilisin . The serine nucleophile at position 132 is activated by two juxtaposed histidine residues at positions 63 and 157 . Dimerization, which seems to be necessary for activity, is observed in the crystals . Correlations of the structure with the sequences of herpesvirus proteases suggest that dimerization may confer specificity and recognition in substrate binding.

Nature, 1996 Sep 19, 383(6597), 272 - 5
A new serine-protease fold revealed by the crystal structure of human cytomegalovirus protease; Tong L et al.; Human cytomegalovirus (hCMV), a herpesvirus, infects up to 70% of the general population in the United States and can cause morbidity and mortality in immunosuppressed individuals (organ-transplant recipients and AIDS patients) and congenitally infected newborns . hCMV protease is essential for the production of mature infectious virions, as it performs proteolytic processing near the carboxy terminus (M-site) of the viral assembly protein precursor . hCMV protease is a serine protease, although it has little homology to other clans of serine proteases . Here we report the crystal structure of hCMV protease at 2.0 angstroms resolution, and show that it possesses a new polypeptide backbone fold . Ser 132 and His 63 are found in close proximity in the active site, confirming earlier biochemical and mutagenesis studies . The structure suggests that the third member of the triad is probably His 157 . A dimer of the protease with an extensive interface is found in the crystal structure . This structure information will help in the design and optimization of inhibitors against herpesvirus proteases.

Nature, 1996 Sep 19, 383(6597), 266 - 9
GTPase activity of Rab5 acts as a timer for endocytic membrane fusion; Rybin V et al.; The GTPase cycle is a versatile regulatory mechanism directing many cell functions, and Rab family members use it to regulate intracellular transport . Current models propose that GTP hydrolysis by Rab proteins is either required for membrane fusion or occurs afterwards to allow recycling of the protein . To measure the GTPase activity of Rab5 in endocytic membrane fusion, we engineered a mutant that preferentially binds xanthosine 5'-triphosphate (XTP),Rab5(D136N) and monitored the kinetics of {alpha(32)P}-XTP hydrolysis in situ during endosome fusion in vitro . Surprisingly, nucleotide hydrolysis occurred even in the absence of membrane fusion, indicating that membrane-bound Rab5 undergoes futile cycles of GTP(XTP) binding and hydrolysis . Nucleotide triphosphate hydrolysis by Rab5 is not conditional on membrane fusion and is reduced by its effector Rabaptin-5 . Our data reveal that the GTP cycle of Rab proteins differs from that of other GTPases (for example, EF-Tu) and indicate that GTP hydrolysis acts as a timer that determines the frequency of membrane docking/fusion events.

Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10291 - 6
Regulation of SOS mutagenesis by proteolysis; Frank EG et al.; DNA damage-inducible mutagenesis in Escherichia coli is largely dependent upon the activity of the UmuD (UmuD') and UmuC proteins . The intracellular level of these proteins is tightly regulated at both the transcriptional and the posttranslational levels . Such regulation presumably allows cells to deal with DNA damage via error-free repair pathways before being committed to error-prone pathways . We have recently discovered that as part of this elaborate regulation, both the UmuD and the UmuC proteins are rapidly degraded in vivo . We report here that the enzyme responsible for their degradation is the ATP-dependent serine protease, Lon . In contrast, UmuD' (the posttranslational product and mutagenically active form of UmuD) is degraded at a much reduced rate by Lon, but is instead rapidly degraded by another ATP-dependent protease, ClpXP . Interestingly, UmuD' is rapidly degraded by ClpXP only when it is in a heterodimeric complex with UmuD . Formation of UmuD/UmuD' heterodimers in preference to UmuD' homodimers therefore targets UmuD' protein for proteolysis . Such a mechanism allows cells to reduce the intracellular levels of the mutagenically active Umu proteins and thereby return to a resting state once error-prone DNA repair has occurred . The apparent half-life of the heterodimeric UmuD/D' complex is greatly increased in the clpX::Kan and clpP::Kan strains and these strains are correspondingly rendered virtually UV non-mutable . We believe that these phenotypes are consistent with the suggestion that while the UmuD/D' heterodimer is mutagenically inactive, it still retains the ability to interact with UmuC, and thereby precludes the formation of the mutagenically active UmuD'2C complex.

Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10228 - 33
Expression and characterization of glycogen synthase kinase-3 mutants and their effect on glycogen synthase activity in intact cells; Eldar-Finkelman H et al.; In these studies we expressed and characterized wild-type (WT) GSK-3 (glycogen synthase kinase-3) and its mutants, and examined their physiological effect on glycogen synthase activity . The GSK-3 mutants included mutation at serine-9 either to alanine (S9A) or glutamic acid (S9E) and an inactive mutant, K85,86MA . Expression of WT and the various mutants in a cell-free system indicated that S9A and S9E exhibit increased kinase activity as compared with WT . Subsequently, 293 cells were transiently transfected with WT GSK-3 and mutants . Cells expressing the S9A mutant exhibited higher kinase activity (2.6-fold of control cells) as compared with cells expressing WT and S9E (1.8- and 2.0-fold, respectively, of control cells) . Combined, these results suggest serine-9 as a key regulatory site of GSK-3 inactivation, and indicate that glutamic acid cannot mimic the function of the phosphorylated residue . The GSK-3-expressing cell system enabled us to examine whether GSK-3 can induce changes in the endogenous glycogen synthase activity . A decrease in glycogen synthase activity (50%) was observed in cells expressing the S9A mutant . Similarly, glycogen synthase activity was suppressed in cells expressing WT and the S9E mutant (20-30%, respectively) . These studies indicate that activation of GSK-3 is sufficient to inhibit glycogen synthase in intact cells, and provide evidence supporting a physiological role for GSK-3 in regulating glycogen synthase and glycogen metabolism.

Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10134 - 8
Human gamma-glutamyl hydrolase: cloning and characterization of the enzyme expressed in vitro; Yao R et al.; A cDNA encoding human gamma-glutamyl hydrolase has been identified by searching an expressed sequence tag data base and using rat gamma-glutamyl hydrolase cDNA as the query sequence . The cDNA encodes a 318-amino acid protein of Mr 35,960 . The deduced amino acid sequence of human gamma-glutamyl hydrolase shows 67% identity to that of rat gamma-glutamyl hydrolase . In both rat and human the 24 amino acids preceding the N terminus constitute a structural motif that is analogous to a leader or signal sequence . There are four consensus asparagine glycosylation sites in the human sequence, with three of them conserved in the rat enzyme . Expression of both the human and rat cDNA in Escherichia coli produced antigenically related proteins with enzyme activities characteristic of the native human and rat enzymes, respectively, when methotrexate di- or pentaglutamate were used as substrates . With the latter substrate the rat enzyme cleaved the innermost gamma-glutamyl linkage resulting in the sole production of methotrexate as the pteroyl containing product . The human enzyme differed in that it produced methotrexate tetraglutamate initially, followed by the triglutamate, and then the diglutamate and methotrexate . Hence the rat enzyme is an endopeptidase with methotrexate pentaglutamate as substrate, whereas the human enzyme exhibits exopeptidase activity . Another difference is that the expressed rat enzyme is equally active on methotrexate di- and pentaglutamate whereas the human enzyme has severalfold greater activity on methotrexate pentaglutamate compared with the diglutamate . These properties are consistent with the enzymes derived from human and rat sources.

Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10128 - 33
Interaction between human tRNA synthetases involves repeated sequence elements; Rho SB et al.; Aminoacyl-tRNA synthetases (tRNA synthetases) of higher eukaryotes form a multiprotein complex . Sequence elements that are responsible for the protein assembly were searched by using a yeast two-hybrid system . Human cytoplasmic isoleucyl-tRNA synthetase is a component of the multi-tRNA synthetase complex and it contains a unique C-terminal appendix . This part of the protein was used as bait to identify an interacting protein from a HeLa cDNA library . The selected sequence represented the internal 317 amino acids of human bifunctional (glutamyl- and prolyl-) tRNA synthetase, which is also known to be a component of the complex . Both the C-terminal appendix of the isoleucyl-tRNA synthetase and the internal region of bifunctional tRNA synthetase comprise repeating sequence units, two repeats of about 90 amino acids, and three repeats of 57 amino acids, respectively . Each repeated motif of the two proteins was responsible for the interaction, but the stronger interaction was shown by the native structures containing multiple motifs . Interestingly, the N-terminal extension of human glycyl-tRNA synthetase containing a single motif homologous to those in the bifunctional tRNA synthetase also interacted with the C-terminal motif of the isoleucyl-tRNA synthetase although the enzyme is not a component of the complex . The data indicate that the multiplicity of the binding motif in the tRNA synthetases is necessary for enhancing the interaction strength and may be one of the determining factors for the tRNA synthetases to be involved in the formation of the multi-tRNA synthetase complex.

Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10123 - 7
Site-directed spin labeling and chemical crosslinking demonstrate that helix V is close to helices VII and VIII in the lactose permease of Escherichia coli; Wu J et al.; Site-directed chemical cleavage of lactose permease indicates that helix V is in close proximity to helices VII and VIII . To test this conclusion further, permease containing a biotin-acceptor domain and paired Cys residues at positions 148 (helix V) and 228 (helix VII), 148 and 226 (helix VII), or 148 and 275 (helix VIII) was affinity purified and labeled with a sulfhydryl-specific nitroxide spin label . Spin-spin interactions are observed with the 148/228 and 148/275 pairs, indicating close proximity between appropriate faces of helix V and helices VII and VIII . Little or no interaction is evident with the 148/226 pair, in all likelihood because position 226 is on the opposite face of helix VII from position 228 . Broadening of the electron paramagnetic resonance spectra in the frozen state was used to estimate distance between the 148/228 and the 148/275 pairs . The nitroxides at positions 148 and 228 or 148 and 275 are within approximately 13-15 A . Finally, Cys residues at positions 148 and 228 are crosslinked by dibromobimane, a bifunctional crosslinker that is approximately 5 A . long, while no crosslinking is detected between Cys residues at positions 148 and 275 or 148 and 226 . The results provide strong support for a structure in which helix V is in close proximity to both helices VII and VIII and is oriented in such a fashion that Cys-148 is closer to helix VII.

Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10094 - 8
SoxR, a {2Fe-2S} transcription factor, is active only in its oxidized form; Gaudu P et al.; SoxR protein is known to function both as a sensor and as a transcriptional activator for a superoxide response regulon in Escherichia coli . The activity of SoxR was tested by its ability to enable the transcription of its target gene, soxS, in vitro . The activity of the oxidized form was lost when its {2Fe-2S} clusters were reduced by dithionite under anaerobic conditions, and it was rapidly restored by autooxidation . This result is consistent with the hypothesis that induction of the regulon is effected by the univalent oxidation of the Fe-S centers of SoxR . In vivo, this oxidation may be caused by an alteration of the redox balance of electron chain intermediates that normally maintains soxR in an inactive, reduced state . Oxidized SoxR was about twice as effective as reduced SoxR in protecting the soxS operator from endonucleolytic cleavage . However, this difference could not account for a greater than 50-fold difference in their activities and therefore could not support a model in which oxidation activates SoxR by enabling it to bind to DNA . NADPH, ferredoxin, flavodoxin, or ferredoxin (flavodoxin):NADP+ reductase could not reduce SoxR directly in vitro at a measurable rate . The midpoint potential for SoxR was measured at -283 mV.

Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10084 - 9
Behavioral responses of Escherichia coli to changes in redox potential; Bespalov VA et al.; Escherichia coli bacteria sensed the redox state in their surroundings and they swam to a niche that had a preferred reduction potential . In a spatial redox gradient of benzoquinone/benzoquinol, E . coli cells migrated to form a sharply defined band . Bacteria swimming out of either face of the band tumbled and returned to the preferred conditions at the site of the band . This behavioral response was named redox taxis . Redox molecules, such as substituted quinones, that elicited redox taxis, interact with the bacterial electron transport system, thereby altering electron transport and the proton motive force . The magnitude of the behavioral response was dependent on the reduction potential of the chemoeffector . The Tsr, Tar, Trg, Tap, and CheR proteins, which have a role in chemotaxis, were not essential for redox taxis . A cheB mutant had inverted responses in redox taxis, as previously demonstrated in aerotaxis . A model is proposed in which a redox effector molecule perturbs the electron transport system, and an unknown sensor in the membrane detects changes in the proton motive force or the redox status of the electron transport system, and transduces this information into a signal that regulates phosphorylation of the CheA protein . A similar mechanism has been proposed for aerotaxis . Redox taxis may play an important role in the distribution of bacterial species in natural environments.

Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10051 - 6
ATPase activity of Escherichia coli Rep helicase crosslinked to single-stranded DNA: implications for ATP driven helicase translocation; Wong I et al.; To examine the coupling of ATP hydrolysis to helicase translocation along DNA, we have purified and characterized complexes of the Escherichia coli Rep protein, a dimeric DNA helicase, covalently crosslinked to a single-stranded hexadecameric oligodeoxynucleotide (S) . Crosslinked Rep monomers (PS) as well as singly ligated (P2S) and doubly ligated (P2S2) Rep dimers were characterized . The equilibrium and kinetic constants for Rep dimerization as well as the steady-state ATPase activities of both PS and P2S crosslinked complexes were identical to the values determined for un-crosslinked Rep complexes formed with dT16 . Therefore, ATP hydrolysis by both PS and P2S complexes are not coupled to DNA dissociation . This also rules out a strictly unidirectional sliding mechanism for ATP-driven translocation along single-stranded DNA by either PS or the P2S dimer . However, ATP hydrolysis by the doubly ligated P2S2 Rep dimer is coupled to single-stranded DNA dissociation from one subunit of the dimer, although loosely (low efficiency) . These results suggest that ATP hydrolysis can drive translocation of the dimeric Rep helicase along DNA by a "rolling" mechanism where the two DNA binding sites of the dimer alternately bind and release DNA . Such a mechanism is biologically important when one subunit binds duplex DNA, followed by subsequent unwinding.

Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10034 - 9
Structure and inhibition of plasmepsin II, a hemoglobin-degrading enzyme from Plasmodium falciparum; Silva AM et al.; Plasmodium falciparum is the major causative agent of malaria, a disease of worldwide importance . Resistance to current drugs such as chloroquine and mefloquine is spreading at an alarming rate, and our antimalarial armamentarium is almost depleted . The malarial parasite encodes two homologous aspartic proteases, plasmepsins I and II, which are essential components of its hemoglobin-degradation pathway and are novel targets for antimalarial drug development . We have determined the crystal structure of recombinant plasmepsin II complexed with pepstatin A . This represents the first reported crystal structure of a protein from P . falciparum . The crystals contain molecules in two different conformations, revealing a remarkable degree of interdomain flexibility of the enzyme . The structure was used to design a series of selective low molecular weight compounds that inhibit both plasmepsin II and the growth of P . falciparum in culture.

Biochemistry, 1996 Sep 17, 35(37), 12164 - 74
Phospholipase A2 engineering . Deletion of the C-terminus segment changes substrate specificity and uncouples calcium and substrate binding at the zwitterionic interface; Huang B et al.; It has been suggested {Dijkstra, B . W., Drenth, J., & Kalk, K . H . (1981) Nature 289, 604-606} that the interfacial binding site of phospholipase A2 (PLA2) involves a large number of residues, including a cluster at the N-terminus and another cluster at the C-terminus . The approaches of multiple mutation and deletion were used to test the roles of the C-terminal residues of bovine pancreatic PLA2 overexpressed in Escherichia coli . A double mutant K120A/K121A and a deletion mutant delta 115-123/ C27A were constructed, and structural and functional analyses were performed on both mutants . The double mutant showed little perturbation in the global structure on the basis of proton NMR and X-ray crystallographic analyses . The proton NMR analysis of the deletion mutant suggested that a few residues at the active site, the hydrophobic channel, and the calcium binding loop are perturbed, but the global conformation is not changed . The mutants were then characterized for catalytic and binding properties by use of various kinetic and spectroscopic methods . The double mutant behaved in a manner similar to that of the wild type (WT) PLA2 in every property examined . The deletion mutant was found to show an interesting change of substrate specificity . The kcat,app of the zwitterionic DC8PC micelles but not the anionic DC8PM micelles decreased by a factor of > 100; however, the activity of DC8PC was restored upon addition of 4 M NaCl . The results of fluorescence spectroscopic studies indicate that the deletion mutant behaves in a manner similar to that of WT in the binding to anionic vesicles and to zwitterionic neutral diluent . Thus, the binding affinity of the enzyme to the interface (the E to E* step) should not be the main cause for the change in substrate specificity . The cause lies at least partially in the binding of substrate or inhibitor to the active site of the enzyme at the interface, i.e., the E* to E*L step, as revealed by the results of equilibrium binding studies . The equilibrium dissociation constants of ligands are generally higher for the deletion mutant (relative to WT) at the zwitterionic interface but not at the anionic interface . The cause for the low affinity of an active site-directed ligand to the active site at the zwitterionic interface could be related to the inability of Ca2+ to enhance ligand binding for the deletion mutant . This is in contrast to the WT PLA2 for which Ca2+ binding enhances binding of the substrate to the active site . Overall, the structural and functional perturbations caused by deleting the C-terminal segment are modest, but the changes in substrate specificity and the uncoupling between substrate and calcium binding are interesting and significant.

Biochemistry, 1996 Sep 17, 35(37), 12104 - 10
The role of the extrinsic 33 kDa protein in Ca2+ binding in photosystem II; Seidler A et al.; The role of the 33 kDa protein in Ca2+ binding was studied by comparing the EPR properties of photosystem II in the presence and absence of the 33 kDa protein and Ca2+ . When the removal of the 33 kDa protein was carried out in the dark, a normal manganese multiline EPR signal could be observed when the S2 state was generated . In addition, the split S3 signal could not be generated by illumination at 273 K . Exposure of the 33 kDa protein-less photosystem II to room light did not lead to any change in the EPR properties of the S2 state, but the split S3 state signal at around g = 2 could then be generated, indicating that Ca2+ was released from this preparation during the exposure to light . Treatment of photosystem II lacking the 33 kDa protein with EGTA in the light led to a modification of the S2 state characterized by a dark-stable multiline EPR signal . Much lower EGTA concentrations were required in order to obtain this modification in the absence of the 33 kDa protein than was required when the 33 kDa protein was present . This indicates that the manganese cluster was more accessible to chelator binding when the 33 kDa protein was absent . When 33 kDa protein-less photosystem II was treated with EGTA in the dark, no modification of the multiline EPR signal of the S2 state of the manganese cluster occurred, nor was Ca2+ released as monitored by the inability to generate the split S3 signal . These chelator- and Ca(2+)-binding properties occurring in PSII lacking the 33 kDa protein are very similar to those observed previously in NaCl-washed PSII in which the 33 kDa protein is present (reviewed in Boussac & Rutherford, 1994a) . It is concluded that the 33 kDa protein has little or no direct role in binding the Ca2+ ion which is required for oxygen evolution.

Biochemistry, 1996 Sep 17, 35(37), 12077 - 85
Alternative splicing determines the binding of platelet-derived growth factor (PDGF-AA) to glycosaminoglycans; Lustig F et al.; We have shown previously that the platelet-derived growth factor (PDGF) and a synthetic oligopeptide, corresponding to the basic carboxyl-terminal amino acid extension of the long PDGF-A isoform, bind to heparin . Here, we have expressed the long (rA125) and the short (rA109) variants of PDGF A-chains in Escherichia coli and produced the functional homodimers . Surface plasmon resonance analyses showed that while the dimeric rA125 bound with high affinity to low molecular weight heparin, the rA109, lacking the basic extension, did not . This strongly indicated that high affinity binding is due to the carboxyl-terminal extension . Investigations of kinetics and thermodynamics suggested an allosteric binding mechanism . Thus, dimeric rA125 contains two equivalent binding sites . Following low affinity binding of heparin to one binding site, the dimer undergoes a conformational change, increasing the affinity for heparin about 40 times . This positive cooperativity requires the basic amino acid extension in both monomers of the dimeric PDGF molecule . Thermodynamics of the reaction, showing an entropy-driven endothermic process, suggest the involvement of hydrophobic interactions in this rearrangement . Three amino acids in the basic carboxyl-terminal extension were essential for the interaction: the basic residues Arg111 and Lys116, and the polar Thr125 . We also found that other glycosaminoglycan species, corresponding to those produced by human arterial smooth muscle cells, bound to dimeric rA125 and that heparan sulfate showed the highest affinity.

Biochemistry, 1996 Sep 17, 35(37), 12053 - 60
Melibiose permease of Escherichia coli: structural organization of cosubstrate binding sites as deduced from tryptophan fluorescence analyses; Mus-Veteau I et al.; Binding of the coupling ion (Na+ or Li+) and sugars to the purified melibiose permease of Escherichia coli, reconstituted in proteoliposomes, produces selective and cooperative changes of the transporter tryptophan fluorescence . To assess the individual contribution of N- or C-terminal domains of the permease to these substrate-induced fluorescence variations, we replaced the two tryptophans located in its C-terminal half (W299 and W342) by a phenylalanine and compared the signal change in mutants and wild-type permease . None of the mutations significantly impairs transport activity . Persistence of the ion-induced signal quenching in a permease carrying only the six other tryptophans of the N-terminal domain is consistent with a previous suggestion that this domain accommodates the ion-binding site . On the other hand, the sugar-induced fluorescence increase varies from mutant to mutant in a sugar-specific fashion . While alpha-galactosides increase essentially the fluorescence of W299 and W342, beta-galactosides enhance the signal of W299 and of one (or more) of the N-terminal tryptophans but quench that of W342 . Moreover, addition of sugars producers a 10 nm blue shift of both W299 and W342 emission spectra, suggesting reduced accessibility of these residues to solvent following substrate binding . These data suggest that W299 and W342 are at or close to the sugar binding site and that this latter is lined by the C-terminal helices IX and X . Moreover, as sugars with the beta-configuration also enhance the fluorescence of the N-terminal tryptophans, it is suggested that one (or more) helix of the N-terminal half may be also at or near the sugar binding site . This implies close proximity and/or tight functional linkage between some N-terminal helices and helices IX and X of the C-terminal domain of the transporter.

Biochemistry, 1996 Sep 17, 35(37), 12046 - 52
Structural and functional properties of full-length and truncated human proapolipoprotein AI expressed in escherichia coli; Pyle LE et al.; Utilizing the Escherichia coli/pGex vector expression system incorporating a thrombin cleavage site, full-length (residues -6-243) and truncated forms of proapolipoprotein AI (proapoAI), terminating at amino acid residues 222, 210, 150, and 135, were purified to levels of at least 5 mg/L, after thrombin cleavage . Assessed by circular dichroism, the helical contents of L-alpha-dimyristoylphosphatidylcholine-associated forms of human plasma-derived apolipoprotein AI (apoAI) and recombinant proapoAI were comparable, being 69% and 65%, respectively . Circular dichroism measurements of the lipid-associated complexes of the truncated forms showed that between the sequence of residues 150-222 no additional helicity was gained until the carboxyl-terminal sequence was present in the molecule, indicating that the carboxyl terminus of the protein is required for the formation of helix within this central region . While tryptophan residues were more than 86% accessible, as assessed by iodide quenching, in the two truncated forms, proapoAI-6-135 and proapoAI-6-150, for both free and complexed protein, this figure fell to about 50% for full-length recombinant proapoAI, further indicating the influence of the carboxyl terminus on the structure of the whole protein . While cross-linking human plasma apoAI in solution with dithiobis-(succinimidyl propionate) revealed high molecular weight oligomers by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, recombinant proapoAI did not strongly form complexes larger than trimers . None of the truncated proapoAI molecules formed oligomers larger than trimers . The shortest form, proapoAI-6-135, only dimerized . Initial results from lecithin:cholesterol acyltransferase activation (apoAI peptide concentration 0.2 microM) indicated that truncation of the 21 carboxy-terminal amino acids resulted in a drop of approximately 53% in activation and 33 residues a drop of 67% relative to the full-length protein . Overall these results indicate the important influence of the carboxyl terminus on the structure of apoAI.

Biochemistry, 1996 Sep 17, 35(37), 12029 - 37
Intramolecular binding contributes to the activation of CDPK, a protein kinase with a calmodulin-like domain; Yoo BC et al.; The activity of calmodulin-like domain protein kinase (CDPK) is regulated by the direct binding of Ca2+ . Unmodified soybean CDPK alpha and a chimeric enzyme in which the calmodulin-like domain (CLD) was replaced by VU-1 calmodulin had similar values of Vmax(app) (3.19, 3.46, and 3.60, 3.93 mumol/ min/mg, respectively), and each was activated 30-70-fold by Ca2+ . To determine if activation results from the binding of the CLD to the autoinhibitory (junction) domain of CDPK alpha in a manner analogous to the activation of calmodulin-dependent enzymes by calmodulin, recombinant CLD and truncation mutants of CDPK alpha were expressed in bacteria and highly purified . In blot overlays, biotinylated CLD bound to mutants containing residues 312-328 of the junction domain . In an electrophoretic mobility shift assay CLD bound synthetic peptides containing residues 318-332 in a calcium-dependent manner, providing direct evidence for binding of CLD to a site in the junction domain . Mutants of CDPK alpha from which all or part of the CLD had been deleted were constitutively inactive . Addition of 20 microM CLD to these mutants in the presence, but not the absence, of calcium stimulated their activities, but to various degrees . His6-CDPK alpha (1-328), which contained none of the CLD, was activated only 5-fold, but the activity of His6-CDPK alpha (1-398), which retained nearly half of the CLD in its sequence, was stimulated 64-fold . The latter activity approached that of unmodified CDPK alpha and was half maximal at a CLD concentration of 7 microM . Our results suggest that binding of CLD to the junction domain contributes to, but is not sufficient for activation . Although calmodulin supported full activity of the chimeric enzyme, its addition to His6-CDPK alpha (1-398) resulted in activity that was only 6% of that of the unmodified enzyme and which was half-maximal at 20 microM Arabidopsis calmodulin . These results support the conclusion that simple binding of the calmodulin-like domain to the junction domain is not sufficient for activation.

Biochemistry, 1996 Sep 17, 35(37), 11994 - 2004
Domain interactions of the peripheral preprotein Translocase subunit SecA; den Blaauwen T et al.; The homodimeric SecA protein is the peripheral subunit of the preprotein translocase in bacteria . It binds the preprotein and promotes its translocation across the bacterial cytoplasmic membrane by nucleotide modulated coinsertion and deinsertion into the membrane . SecA has two essential nucleotide binding sites (NBS; Mitchell & Oliver, 1993): The high-affinity NBS-I resides in the amino-terminal domain of the protein, and the low-affinity NBS-II is localized at 2/3 of the protein sequence . The nucleotide-bound states of soluble SecA were studied by site directed tryptophan fluorescence spectroscopy, tryptic digestion, differential scanning calorimetry, and dynamic light scattering . A nucleotide-induced conformational change of a carboxy-terminal domain of SecA was revealed by Trp fluorescence spectroscopy . The Trp fluorescence of a single Trp SecA mutant containing Trp775 decreased and increased upon the addition of NBS-I saturating concentrations of ADP or AMP-PNP, respectively . DSC measurements revealed that SecA unfolds as a two domain protein . Binding of ADP to NBS-I increased the interaction between the two domains whereas binding of AMP-PNP did not influence this interaction . When both NBS-I and NBS-II are bound by ADP, SecA seems to have a more compact globular conformation whereas binding of AMP-PNP seems to cause a more extended conformation . It is suggested that the compact ADP-bound conformation resembles the membrane deinserted state of SecA, while the more extended ATP-bound conformation may correspond to the membrane inserted form of SecA.

Biochemistry, 1996 Sep 17, 35(37), 11985 - 93
Crystal structure of glycine N-methyltransferase from rat liver; Fu Z et al.; Glycine N-methyltransferase (GNMT) from rat liver is a tetrameric enzyme with 292 amino acid residues in each identical subunit and catalyzes the S-adenosylmethionine (AdoMet) dependent methylation of glycine to form sarcosine . The crystal structure of GNMT complexed with AdoMet and acetate, a competitive inhibitor of glycine, has been determined at 2.2 A resolution . The subunit of GNMT forms a spherical shape with an extended N-terminal region which corks the entrance of active site of the adjacent subunit . The active site is located in the near center of the spherical subunit . As a result, the AdoMet and acetate in the active site are completely surrounded by amino acid residues . Careful examination of the structure reveals several characteristics of GNMT . (1) Although the structure of the AdoMet binding domain of the GNMT is very similar to those of other methyltransferases recently determined by X-ray diffraction method, an additional domain found only in GNMT encloses the active site to form a molecular basket, and consequently the structure of GNMT looks quite different from those of other methyltransferases . (2) This unique molecular structure can explain why GNMT can capture folate and polycyclic aromatic hydrocarbons . (3) The unique N-terminal conformation and the subunit structure can explain why GNMT exhibits positive cooperativity in binding AdoMet . From the structural features of GNMT, we propose that the enzyme might be able to capture yet unidentified molecules in the cytosol and thus participates in various biological processes including detoxification of polycyclic aromatic hydrocarbons . In the active site, acetate binds near the S-CH3 moiety of AdoMet . Simple modeling indicates that the amino group of the substrate glycine can be placed close to the methyl group of AdoMet within 3.0 A and form a hydrogen bond with the carboxyl group of Glu15 of the adjacent subunit . On the basis of the ternary complex structure, the mechanism of the methyl transfer in GNMT has been proposed.

Biochemistry, 1996 Sep 17, 35(37), 11967 - 74
A pseudo-michaelis quaternary complex in the reverse reaction of a ligase: structure of Escherichia coli B glutathione synthetase complexed with ADP, glutathione, and sulfate at 2.0 A resolution; Hara T et al.; The crystal structure of glutathione synthetase from Escherichia coli B complexed with ADP, glutathione, and sulfate has been determined at 2.0 A resolution . Concerning the chemical similarity of sulfate and phosphate, this quaternary complex structure represents a pseudo enzyme-substrate complex in the reverse reaction and consequently allows us to understand the active site architecture of the E . coli glutathione synthetase . Two Mg2+ ions are coordinated with oxygen atoms from the alpha- and beta-phosphate groups of ADP and from the sulfate ion . The flexible loops, invisible in the unliganded or the binary and ternary complex structures, are fixed in the quaternary complex . The larger flexible loop (Ile226-Arg241) includes one turn of a 310-helix that comprises the binding site of the glycine moiety of GSH . The small loop (Gly164-Gly167) is involved in nucleotide binding and acts as a phosphate gripper . The side chains of Arg210 and Arg225 interact with the sulfate ion and the beta-phosphate moiety of ADP . Arg 210 is likely to interact with the carboxylate of the C-terminal gamma-glutamylcysteine in the substrate-binding form of the forward reaction . Other positively charged residues in the active site (Lys125 and Lys160) are involved in nucleotide binding, directing the phosphate groups to the right position for catalysis . Functional aspects of the active site architecture in the substrate-binding form are discussed.

Biochemistry, 1996 Sep 17, 35(37), 11951 - 8
Structure of the acid state of Escherichia coli ribonuclease HI; Dabora JM et al.; Under acidic conditions Escherichia coli ribonuclease HI* (RNase H*) adopts a partially folded state with all of the properties of a molten globule . Using amide hydrogen exchange carried out under acid state conditions, followed by quenching and NMR detection on the native state, we have determined the residues that are responsible for the observed structure of the acid state . Although RNase H* is a mixed alpha + beta protein, a helical subdomain (helices A, D, and B) defines the structure of the acid state . This structure correlates with the rare higher energy conformations detected under native conditions and with data for the earliest intermediates populated in the kinetic folding pathway of the protein.

Gene, 1996 Sep 16, 173(2), 271 - 4
Production of the beta-subunit of human chorionic gonadotropin in Escherichia coli and its export mediated by the heat-labile enterotoxin chain-B signal sequence; Pillai D et al.; The PCR-amplified beta-subunit of the human chorionic gonadotropin structural gene (betahCG) was cloned under the control of the tac promoter and the heat-labile enterotoxin chain B (LTB) signal sequence (LTBss) . BetahCG was successfully produced, processed and exported to the periplasmic space in Escherichia coli . Expression of betahCG was confirmed by immunoblot analysis using an anti-betahCG polyclonal antibody . The processing of the protein was very efficient, as only the processed band could be detected at all time points during the course of induction . Expression was evident soon after the addition of the lactose analogue, IPTG . These results demonstrate that E . coli cells can synthesize, process and export betahCG using the LTBss.

Gene, 1996 Sep 16, 173(2), 261 - 4
Cloning and sequence analysis of the human salivary peroxidase-encoding cDNA; Kiser C et al.; A cDNA (hSPO) encoding human salivary peroxidase (hSPO) was isolated and its nucleotide (nt) sequence determined . The coding sequence resembles those of other mammalian peroxidases and contains a 3' untranslated region of 538 nt . In a rabbit reticulocyte lysate transcription/translation system, the cDNA produces a major protein of approx . 67 kDa, which corresponds to the calculated molecular weight of unglycosylated hSPO.

Gene, 1996 Sep 16, 173(2), 147 - 54
Vectors for a 'double-tagging' assay for protein-protein interactions: localization of the CDK2-binding domain of human p21; Wang ZX et al.; We report the construction of three new vectors which can be used for the 'double-tagging' assay previously reported {Germino et al., Proc . Natl . Acad . Sci . USA 90 (1993) 933-937} . The vectors include two plasmids (pTrc.BCCP and pTrc.EZZ::BCCP) which encode different 'tags' for the capture of a target protein of interest on a filter coated with either avidin or IgG, respectively . The first plasmid (pTrc.BCCP) encodes the C terminus of the biotin carboxylase carrier protein (BCCP) under the control of the Ptac promoter, while the second produces fusions to an IgG-binding domain (EZZ) . The gene encoding a protein of interest can be inserted into these plasmids and thereby direct the production of a fusion protein which is biotinylated in vivo and can bind to avidin, or a fusion protein which can bind to IgG . The third is a positive-selection, phase lambda expression vector (lambdaFJG2) which permits the construction of lacZ::cDNA fusion proteins which retain beta-galactosidase activity . The insertion of an active ecoRVR gene between the cloning sites (EcoRI and HindII or NotI) permits the positive selection of inserts . The C-terminal two-thirds of the mouse retinoblastoma-encoding gene (containing the E1A-binding pocket) was cloned into pTrc.BCCP and pTrc.EZZ::BCCP, while the 13S E1a gene was cloned into lambdaFJG2 . We show that the interaction between these two proteins can be detected using the 'double-tagging' filter assay, and that this assay has high sensitivity and specificity for detecting this interaction . Finally, we have used these vectors to localize the CDK2-binding domain of the cyclin-dependent kinase inhibitor, p21 . These results closely correspond to those obtained using the yeast two-hybrid assay, as well as in vitro binding assays.

EMBO J, 1996 Sep 16, 15(18), 5104 - 11
Linear diffusion of the restriction endonuclease EcoRV on DNA is essential for the in vivo function of the enzyme; Jeltsch A et al.; Linear diffusion along DNA is a mechanism of enhancing the association rates of proteins to their specific recognition sites on DNA . It has been demonstrated for several proteins in vitro, but to date in no case in vivo . Here we show that the restriction endonuclease EcoRV slides along the DNA, scanning approximately 1000 bp in one binding event . This process is critically dependent on contacts between amino acid residues of the protein and the backbone of the DNA . The disruption of single hydrogen bonds and, in particular, the alteration of electrostatic interactions between amino acid side chains of the protein and phosphate groups of the DNA interfere with or abolish effective sliding . The efficiency of linear diffusion is dependent on salt concentration, having a maximum at 50 mM NaCl . These results suggest that a nonspecific and mobile binding mode capable of linear diffusion is dependent on a subtle balance of forces governing the interaction of the enzyme and the DNA . A strong correlation between the ability of EcoRV mutants to slide along the DNA in vitro and to protect Escherichia coli cells from phage infection demonstrates that linear diffusion occurs in vivo and is essential for effective phage restriction.

EMBO J, 1996 Sep 16, 15(18), 4970 - 80
Coordinated and differential expression of histone-like proteins in Escherichia coli: regulation and function of the H-NS analog StpA; Sonden B et al.; The histone-like protein H-NS has been shown to influence the regulation of gene expression at the transcriptional level in several Escherichia coli operons . We have examined the regulation of the stpA gene, which encodes a protein sharing 58% identity with H-NS, by mRNA analysis and by using stpA-lacZ operon fusions . The expression of stpA is temperature dependent, with 2-fold higher expression at 37 degrees C than at 26 degrees C . In addition, stpA expression is stimulated by the global regulator Lrp . In an hns mutant E.coli derivative stpA expression is derepressed, suggesting that regulation of the two genes is coupled . Overproduction of the StpA protein affects expression from at least four hns regulated operons (the papB, proU, bgl and hns operons), in both the presence and absence of H-NS . The construction of E.coli strains carrying mutations in both stpA and hns demonstrated that the absence of both proteins affects growth rate and viability of the cells . Our work establishes that E.coli can express two H-NS-like proteins with coordinated yet differential regulation . Evidently, these proteins have both overlapping and distinct functions in the cell, and they are both important for normal cell growth and gene control.

EMBO J, 1996 Sep 16, 15(18), 4798 - 805
Membrane topology and multimeric structure of a mechanosensitive channel protein of Escherichia coli; Blount P et al.; We have studied the membrane topology and multimeric structure of a mechanosensitive channel, MscL, which we previously isolated and cloned from Escherichia coli . We have localized this 15-kDa protein to the inner membrane and, by PhoA fusion, have shown that it contains two transmembrane domains with both the amino and carboxyl termini on the cytoplasmic side . Mutation of the glutamate at position 56 to histidine led to changes in channel kinetics which were dependent upon the pH on the periplasmic, but not cytoplasmic side of the membrane, providing additional evidence for the periplasmic positioning of this part of the molecule . Tandems of two MscL subunits expressed as a single polypeptide formed functional channels, suggesting an even number of transmembrane domains per subunit (amino and carboxyl termini on the same side of the membrane), and an even number of subunits per functional complex . Finally, cross-linking studies suggest that the functional MscL complex is a homohexamer . In summary, these data are all consistent with a protein domain assignment and topological model which we propose and discuss.

FEBS Lett, 1996 Sep 16, 393(2-3), 269 - 72
The C-terminal of rat 4-hydroxyphenylpyruvate dioxygenase is indispensable for enzyme activity; Lee MH et al.; We have cloned and overexpressed rat 4-hydroxyphenylpyruvate dioxygenase (4HPPD) in Escherichia coli . The soluble, active recombinant enzyme was shown to contain both 4HPPD and alpha-ketoisocaproate dioxygenase (alpha KICD) activity . However, upon truncation of the 14 amino acids at the C-terminus by site-directed mutagenesis, the resulting mutant enzyme (rat F antigen) exhibited complete loss of 4HPPD and alpha KICD activities . This finding suggests that the C-terminal extension domain plays an essential role in the catalytic activity of the enzyme.

FEBS Lett, 1996 Sep 16, 393(2-3), 205 - 10
High-level expression of Pinus sylvestris glutamine synthetase in Escherichia coli . Production of polyclonal antibodies against the recombinant protein and expression studies in pine seedlings; Canton FR et al.; In a previous work we reported the molecular characterization of a glutamine synthetase (GS; EC 6.3.1.2.) complementary DNA from a woody plant (Canton et al . (1993) Plant Mol . Biol . 22, 819-828) . The isolated cDNA (pGSP114) encoding a Scots pine (Pinus sylvestris) cytosolic subunit, has been subcloned into the expression vector pET3c to overproduce the GS polypeptide in Escherichia coli cells . The recombinant GS protein showed the same molecular size as a native Scots pine GS subunit . Antibodies against the pET3c-GSP114 encoded protein were raised in rabbits by injecting purified preparations and specificity was determined by immunoprecipitation of GS activity present in pine crude extracts . In spite of the antibodies were able to recognize both cytosolic and chloroplastic GS in tomato plants, they were unable to immunodetect chloroplastic GS in green cotyledons of pine seedlings and cytosolic GS was the unique recognized polypeptide . Unlike to that found in other plant species, cytosolic GS was strongly expressed in green tissues as determined by protein and Northern analysis . Our results suggest a key role for cytosolic GS in photosynthetic tissues of conifers.

Eur J Biochem, 1996 Sep 15, 240(3), 781 - 8
Kinetic evidence for protein complexes between thioredoxin and NADP-malate dehydrogenase and presence of a thioredoxin binding site at the N-terminus of the enzyme; Braun H et al.; The kinetics of activation of NADP-malate dehydrogenase (MDH; EC 1.1.1.82) from soybean and spinach leaves by the chloroplast thioredoxins isolated from the same plants, by the corresponding storage forms of the soybean chloroplast thioredoxins from soybean seeds, and by the bacterial Escherichia coli thioredoxin have been studied . The Hill equation has been applied to evaluate the saturation kinetics . The observed variable thioredoxin saturation characteristics (Vmax 0.37-14.5 mumol NADPH min-1 mg enzyme-1; K0.5 0.15-1.33 microM; Hill coefficient h 0.90-3.04) indicate that the activation of NADP-MDH depends strongly on the individual thioredoxin used . Thus, thioredoxin action is not solely due to simple reductive activation of the NADP-MDH . Specific thioredoxin complex formation between thioredoxin and NADP-MDH must be included into the mechanism of the activation process . To study the regulatory consequences of the specific thioredoxin/NADP-MDH complexes we investigated the saturation kinetics of the substrates NADPH and oxaloacetate in presence of different concentrations of each individual thioredoxin species . The kinetic characteristics of the substrates (S0.5, Vmax, and Hill coefficients h) varied individually in response to the different thioredoxin species substantiating our model of thioredoxin/ NADP-MDH complex formation . Aminopeptidase-K-truncated pea NADP-MDH has been used to demonstrate that the N-terminal 37 amino residues are involved in providing a specific thioredoxin binding site . The fact that the versatile light-dependent regulation of numerous enzyme activities by only two thioredoxin species in chloroplasts cannot be accomplished without the formation of thioredoxin/target enzyme complexes is discussed in detail.

Eur J Biochem, 1996 Sep 15, 240(3), 637 - 45
Different oligomeric states are involved in the allosteric behavior of uracil phosphoribosyltransferase from Escherichia coli; Jensen KF et al.; Uracil phosphoribosyltransferase, catalyzing the formation of UMP and pyrophosphate from uracil and 5-phosphoribosyl-alpha-1-diphosphate (PPRibP), was purified from an overproducing strain of Escherichia coli . GTP was shown to activate the enzyme by reducing K(m) for PPRibP by about fivefold without affecting Vmax . When started by addition of enzyme, the reactions accelerated over an extended period of time, while enzyme solutions incubated first with GTP and PPRibP displayed constant velocities . This indicated that PPRibP and GTP influenced the structure of the enzyme . Gel-filtration and sedimentation analyses showed that the apparent oligomeric state of uracil phosphoribosyltransferase is defined by a dynamic equilibrium between a slowly sedimenting form (dimeric or trimeric) that has only a little activity, and a more highly aggregated form (pentameric or hexameric), which is more active . It appears that the smaller form predominates in the absence of substrates, while the larger form predominates in the presence of GTP and PPRibP . Guanosine-3',5'-bis(diphosphate) was found to activate the enzyme much like GTP.

Eur J Biochem, 1996 Sep 15, 240(3), 615 - 21
Transient non-native interactions in early folding intermediates do not influence the folding kinetics of Escherichia coli tryptophan synthase beta 2 subunits; Planchenault T et al.; Local structures formed in early intermediates are thought to play a key role in orientating the protein folding pathway . Here, we test whether non-native interactions formed by eight N-terminal residues in early folding intermediates of tryptophan synthase beta chains {Navon, A., Schulze, A . J., Guillou, Y., Zylinksii, C . A., Baleux, F., Expert-Bezancon, N., Friguet, B., Djavadi-Ohaniance, L . & Goldberg, M . E . (1995) J . Biol . Chem . 270, 4255-4261} influence the folding kinetics . The kinetics of in vitro renaturation of wild-type beta chains and truncated beta chains lacking residues 2-9 were compared . Each step analyzed (molten globule formation, tryptophan shielding, closing up of distant residues, and complete renaturation) occurred with similar kinetics in the normal and truncated proteins . Thus, non-native interactions formed early during the folding of beta chains seem to influence neither the rate and efficiency of the complete renaturation, nor the kinetics of the early folding steps, which suggests that such non-native early intermediates are poorly stable and highly dynamic . This observation is consistent with the current view that productive folding should avoid the formation of stable intermediates.

Eur J Biochem, 1996 Sep 15, 240(3), 609 - 14
Soyacystatin, a novel cysteine proteinase inhibitor in soybean, is distinct in protein structure and gene organization from other cystatins of animal and plant origin; Misaka T et al.; Cystatins, cysteine proteinase inhibitors, deserve note because of their regulatory and protective functions in plant tissues . We isolated both genomic DNA and cDNA clones from soybean that encode a cystatin consisting of 245 amino acid residues (soyacystatin) . It is, while basically similar in sequence to known cystatins that are generally in the range of 12-15 kDa, characterized by having extremely large extension sequences in both its amino and carboxyl termini . The genomic DNA encoding soyacystatin is also unique in that it consists of four exons with three introns in its coding regions . The mRNA for soyacystatin is distinctly expressed in soybean seeds 2 weeks after flowering . Soyacystatin purified from mature soybean seeds had a molecular mass of about 26 kDa on SDS/PAGE which suggests that it contains the extension sequences . Papain-inhibition experiments demonstrate that this endogenous soyacystatin has almost the same inhibitory activity as that of its deletion mutant (102 amino acid residues) recombinantly produced by truncation of the amino and carboxyl terminal extensions, indicating that the occurrence of the extensions does not affect the cystatin activity . Immunohistochemical experiments reveal that soyacystatin is expressed nearly uniformly in the cotyledons . These results also suggest the possible occurrence of a cysteine proteinase as the target enzyme of soyacystatin.

Experientia, 1996 Sep 15, 52(9), 909 - 17
Splenic B-cell activation in lipopolysaccharide-non-responsive C3H/HeJ mice by lipopolysaccharide of Porphyromonas gingivalis; Shimauchi H et al.; Porphyromonas gingivalis 381 lipopolysaccharide (LPS) definitely exhibited mitogenic activity in purified B-cells, separated from spleens of LPS-responsive C3H/HeN mice and LPS-non-responsive C3H/HeJ mice by using a magnetic cell sorting system . The mitogenic activity induced by P . gingivalis LPS was incompletely inhibited by polymyxin B . P . gingivalis LPS also induced a higher production of interleukin-6 (IL-6) in splenic B-cells of C3H/HeN mice as compared with Escherichia coli LPS . Furthermore, P . gingivalis LPS, but not E . coli LPS, induced definite IL-6 production in C3H/HeJ mice . P . gingivalis LPS increased tyrosine, serine/threonine phosphorylation of proteins with various major induced bands in splenic B-cells of both C3H/HeN and C3H/HeJ mice . Additionally, radioiodinated P . gingivalis LPS, similarly to E . coli LPS, bound to a 73-kDa protein on C3H/HeJ as well as C3H/HeN B-cells . Thus P . gingivalis LPS may activate B-cells of C3H/HeJ as well as C3H/HeN mice via the LPS-specific binding protein on the cells.

Nucleic Acids Res, 1996 Sep 15, 24(18), 3601 - 6
Identification of a 4.5S-like ribonucleoprotein in maize mitochondria; Yang AJ et al.; Escherichia coli has a ribonucleoprotein complex that is composed of a 114 nucleotide 4.5S RNA and a 48 kDa polypeptide (P48) that has been demonstrated to function in translation and in the secretion of periplasmic polypeptides . A small RNA of approximately 220 nucleotides has been identified in maize mitochondria that includes sequence identity with the highly conserved domain of the bacterial 4.5S RNA . The transcript is mitochondrially encoded and maps to a region upstream of the gene for ATP synthase subunit I . The mitochondrial 4.5S-like RNA has 15 nucleotides of sequence identity with the highly conserved region of the bacterial 4.5S RNA . Sucrose density gradient centrifugation of a maize mitochondrial lysate demonstrated that the 4.5S RNA is a component of a high molecular weight complex under native conditions, and could be disrupted by phenol . Anti-P48 immune serum immuno-precipitated a mitochondrial protein of approximately 48 kDa, and RNA gel blot analysis of the immunoprecipitation reaction indicated that the 4.5S-like RNA co-immuno-precipitated with the 48 kDa polypeptide . The mitochondrial 4.5S ribonucleoprotein complex could function in translation or protein targeting.

Nucleic Acids Res, 1996 Sep 15, 24(18), 3527 - 32
The Escherichia coli Fis protein prevents initiation of DNA replication from oriC in vitro; Wold S et al.; Fis protein participates in the normal control of chromosomal replication in Escherichia coli . However, the mechanism by which it executes its effect is largely unknown . We demonstrate an inhibitory influence of purified Fis protein on replication from oriC in vitro . Fis inhibits DNA synthesis equally well in replication systems either dependent upon or independent of RNA polymerase, even when the latter is stimulated by the presence of HU or IHF . The extent of inhibition by Fis is modulated by the concentrations of DnaA protein and RNA polymerase; the more limiting the amounts of these, the more severe the inhibition by Fis . Thus, the level of inhibition seems to depend on the ease with which the open complex can be formed . Fis-mediated inhibition of DNA replication does not depend on a functional primary Fis binding site between DnaA boxes R2 and R3 in oriC, as mutations that cause reduced binding of Fis to this site do not affect the degree of inhibition . The data presented suggest that Fis prevents formation of an initiation-proficient structure at oriC by forming an alternative, initiation-preventive complex . This indicates a negative role for Fis in the regulation of replication initiation.

Biochem J, 1996 Sep 15, 318 ( Pt 3), 889 - 96
A novel nickel-containing superoxide dismutase from Streptomyces spp; Youn HD et al.; A novel type of superoxide dismutase (SOD) was purified to apparent homogeneity from the cytosolic fractions of Streptomyces sp . IMSNU-1 and Strep . coelicolor ATCC 10147 respectively . Both enzymes were composed of four identical subunits of 13.4 kDa, were stable at pH 4.0-8.0 and up to 70 degrees C, and were inhibited by cyanide and H2O2 but little inhibited by azide . The atomic absorption analyses revealed that both enzymes contain 0.74 g-atom of nickel per mol of subunit . Both enzymes were different from iron-containing SOD and manganese-containing SOD from Escherichia coli, and copper- and zinc-containing SODs from Saccharomyces cerevisiae and bovine erythrocytes, with respect to amino acid composition, N-terminal amino acid sequence and cross-reactivity against antibody . The absorption spectra of both enzymes were identical, exhibiting maxima at 276 and 378 nm, and a broad peak at 531 nm . The EPR spectra of both enzymes were almost identical with that of NiIII in a tetragonal symmetry of NiIII-oligopeptides especially containing histidine . The apoenzymes, lacking in nickel, had no ability to mediate the conversion of superoxide anion radical to hydrogen peroxide, strongly indicating that NiIII plays a main role in these enzymes.

Blood, 1996 Sep 15, 88(6), 2342 - 53
Anti-graft-versus-host disease effect of DT390-anti-CD3sFv, a single-chain Fv fusion immunotoxin specifically targeting the CD3 epsilon moiety of the T-cell receptor; Vallera DA et al.; In a recent study, we showed that an immunotoxin (IT) made with a conventional monoclonal antibody targeting the CD3 epsilon moiety of the T-cell receptor (TCR) had a potent, but partial, graft-versus-host disease (GVHD) effect (Vallera et al, Blood 86:4367, 1995) . Therefore, in this current study, we determined whether a fusion immunotoxin made with anti-CD3 single-chain Fv (sFv), the smallest unit of antibody recognizing antigen, would have anti-GVHD activity . A fusion protein was synthesized from a construct made by splicing sFv cDNA from the hybridoma 145-2C11 to a truncated form of the diphtheria toxin (DT390) gene . DT390 encodes a molecule that retains full enzymatic activity, but excludes the native DT binding domain . The DT390-anti-CD3sFv hybrid gene was cloned into a vector under the control of an inducible promoter . The protein was expressed in Escherichia coli and then purified from inclusion bodies . The DT390 moiety of the protein had full enzymatic activity compared with native DT and DT390-anti-CD3sFv, with an IC50 of 1 to 2 nmol/L against phytohemagglutinin-stimulated and alloantigen-stimulated T cells . Specificity was shown (1) by blocking the IT with parental anti-CD3 antibody, but not with a control antibody; (2) by failure of DT390-anti-CD3sFv to inhibit lipopolysaccharide-stimulated murine B cells; (3) by failure of an Ig control fusion protein, DT390-Fc, to inhibit T-cell responses; and (4) with in vivo immunohistochemisty studies . GVHD was studied in a model in which C57BL/6 (H-2b)-purified lymph node T cells were administered to major histocompatibility complex (MHC) antigen disparate unirradiated C.B.-17 scid (H-2d) mice to assess GVHD effects in the absence of irradiation toxicity . Flow cytometry studies showed that donor T cells were expanded 57-fold and histopathologic analysis showed the hallmarks of a lethal model of GVHD . Control mice receiving phosphate-buffered saline showed 17% survival on day 80 after bone marrow transplantation, and mice receiving 2 micrograms DT390-Fc fusion toxin control administered in 2 daily doses for 6 days (days 0 through 5) had a 43% survival rate . In contrast, 86% of mice receiving the same dose of DT390-anti-CD3sFv were survivors on day 80, a significant improvement, although survivors still showed histopathologic signs of GVHD . These findings suggest that new anti-GVHD agents can be genetically engineered and warrant further investigation of fusion proteins for GVHD treatment.

Arch Biochem Biophys, 1996 Sep 15, 333(2), 439 - 46
Heterologous expression and reconstitution of fungal Mn peroxidase; Whitwam R et al.; We have optimized the conditions under which recombinant Mn peroxidase from the white-rot fungus Phanerochaete chrysosporium can be expressed in Escherichia coli . A bacterial expression vector for the cDNA of Mn peroxidase isozyme H4 (lambda MP1) was constructed (R . E . Whitwam, I . G . Gazarian, and M . Tien, Biochem . Biophys . Res . Commun . 216, 1013-1017, 1995) whose expression in E . coli results in the formation of catalytically inactive polypeptide which can be refolded to active enzyme . The refolded enzyme was purified to homogeneity . Refolding was most efficient in 2 M urea, pH 8.0, and was absolutely dependent upon the presence of CaCl2, hemin, and oxidized glutathione . The recombinant enzyme had the same spectral and kinetic properties as the native fungal enzyme . The Km of recombinant Mn peroxidase for substrates H2O2 and the Mn2+/oxalate complex are 100 and 52 microM, respectively . The kcat as measured by Mn3+/oxalate formation is 450 s-1 . These are essentially the same values as seen with the native fungal enzyme . The rate of formation of compound I, the two-electron-oxidized state of the enzyme, is 4.0 x 10(6) M-1 s-1, identical to the rate of the native fungal Mn peroxidase . The reaction of compound I with Mn2+ is too fast to measure at pH 4.5 in the recombinant Mn peroxidase . At a suboptimal pH of 2.5 a rate of 4.2 x 10(4) M-1 s-1 is obtained for the recombinant enzyme . The reaction of compound II, the one-electron-oxidized state of the enzyme, with Mn2+/oxalate has a Kd of 13 microM and a first-order rate constant of 230 s-1 in the recombinant enzyme . These rates are essentially the same as those seen with the native fungal MnP . These results demonstrate that the bacterial expression of recombinant Mn peroxidase is a convenient and efficient system for the expression and characterization of Mn peroxidase.

FEMS Microbiol Lett, 1996 Sep 15, 143(1), 77 - 82
Comparative methodology to investigate the presence of Escherichia coli K-12 strains in environmental and human stool samples; Blum G et al.; This study was undertaken to evaluate the specificity and efficiency of different methods to detect Escherichia coli K-12 strains . Another aim was to determine the frequency of E . coli K-12 strains among wild-type E . coli isolates from different sources . The detection of K-12 strains was performed both genotypically by K-12 specific polymerase chain reaction (PCR) and on the basis of phenotypical tests . In addition, the genome structures of E . coli strains were characterized by pulsed-field gel electrophoresis (PFGE) . The most specific results could be obtained by the genotypical tests PCR and PFGE as well as by the K-12 specific phage assay . In total, 131 stool and 95 water isolates as well as 14 K-12 derivatives were examined by the different methods . No E . coli K-12 strains were detected among the wild-type isolates.

FEMS Microbiol Lett, 1996 Sep 15, 143(1), 63 - 7
Cloning and nucleotide sequence of the mono- and diacylglycerol lipase gene (mdlB) of Aspergillus oryzae; Tsuchiya A et al.; Aspergillus oryzae IFO4202 produces at least two extracellular lipolytic enzymes L1 and L2 (cutinase, and mono- and diacylglycerol lipase, respectively) . Southern hybridization of restriction enzyme-digested genomic DNA fragments with 23-mer oligonucleotides synthesized according to the amino acid sequence of the L2 as probe suggested the presence of the L2 gene (tentatively designated as mdlB) and an additional weakly hybridizing region . A fragment containing the genomic mdlB gene was cloned in Escherichia coli . Nucleotide sequencing of the fragment revealed an open reading frame, comprising 1021 nucleotides, which contains two introns (51 and 52 nucleotides) . Putative polyadenylation signals were found 182 and 287 bp downstream of the stop codon . The deduced amino acid sequence of the mdlB gene corresponds to 306 amino acid residues including a leader sequence of 28 amino acids and is highly similar to that of the mdlA gene of Penicillium camembertii . Three residues presumed to form the catalytic triad (serine, aspartic acid and histidine) of lipases were also conserved.

FEMS Microbiol Lett, 1996 Sep 15, 143(1), 47 - 55
Deletion of the putative effector region of Era, an essential GTP-binding protein in Escherichia coli, causes a dominant-negative phenotype; Pillutla RC et al.; Era is an essential gene in E . coli, encoding a GTP-binding protein of unknown function . In the present work, a mutant designated Era-dE, for deletion of effector region is described . This is the first and only known era allele that confers a dominant-negative phenotype . Phenotypic analysis of the mutant showed that overproduction of Era-dE caused a dominant inhibition of growth when TCA cycle intermediates such as succinate, pyruvate, malate, alpha-ketoglutarate, and fumarate were provided as the sole carbon source . Examination of the macromolecular composition of cells overexpressing the mutant showed protein, DNA, and ATP levels expected for cells growing at slow rates . The response of cells expressing Era-dE to different stress conditions was studied by examining the rates of synthesis of stress-inducible proteins . Interestingly, when subjected to succinate starvation, cells expressing Era-dE showed a defective carbon starvation response, whereas response to glucose starvation was similar to that seen in control cells . Taken together with previous results, these studies indicate that Era is perhaps involved in multiple cellular processes and Era-dE disrupts more than one of these functions . Furthermore, it appears that some possible functions of Era include regulation of the TCA cycle and response to carbon starvation.

Virology, 1996 Sep 15, 223(2), 396 - 400
Identification, molecular cloning, and transcription analysis of the Choristoneura fumiferana nuclear polyhedrosis virus spindle-like protein gene; Liu JJ et al.; The Choristoneura fumiferana nuclear polyhedrosis virus spindle-like protein (slp) gene has been identified and localized immediately downstream and in the same orientation as the CfMNPV DNA polymerase gene . The slp gene is 1101 bp long, predicted to code for a 366 amino acid (42.1 kDa) polypeptide . Transcriptional analysis revealed that the CfMNPV slp gene is expressed at late times postinfection, beginning at 24 hr postinfection and is most abundantly expressed after 36 hr . Transcription initiates within a single baculovirus consensus late start site sequence (GTAAG) at position -18 relative to the translation start codon . Based on amino acid comparisons, the CfMNPV gene is closely related to other similar baculovirus genes and distantly but recognizably related to the fusolin proteins of two entomopoxviruses . The conservation of amino acid sequence, glycosylation signals and specific domains throughout the protein suggest that this gene product may play an important role in insect DNA virus replication.

Virology, 1996 Sep 15, 223(2), 263 - 71
The ORF I and II proteins of Commelina yellow mottle virus are virion-associated; Cheng CP et al.; Antibodies were prepared against bacterially expressed Commelina yellow mottle badnavirus (CoYMV) proteins . Antiserum against purified virions and antiserum against the C-terminus of the putative coat protein-encoding region of ORF III detected the same virus-specific proteins, indicating that the CoYMV coat protein is encoded in ORF III . In addition to the two major forms of the coat protein (37 and 39 kDa), several high molecular weight virus-specific proteins were detected when virions were isolated without chloroform treatment . These proteins are possible ORF III polyprotein processing intermediates and might be associated with "immature" virions which are eliminated by chloroform treatment . As predicted by the genomic sequence, a 20-kDa virus-specific protein was detected by an antiserum raised against the C-terminus of the putative ORF I protein . Results of filtration experiments suggest that the ORF I protein is equally associated with virions and with plant component(s) . The association between the ORF I protein and the virions was further confirmed using immunosorbent electron microscopy and immunogold labeling . The ORF I protein was not detected in virus preparations treated with chloroform, and colocalized with virions containing immature coat protein on sucrose-cesium sulfate density gradients, suggesting that it is associated with immature virions . An antiserum raised against the putative ORF II gene product detected a 15-kDa virus-specific protein whose association with the virions was unaffected by chloroform treatment . The ORF II protein was found to be sensitive to some protease(s) that copurified with the virions, and protease inhibitors preventing this degradation have been identified.

Structure, 1996 Sep 15, 4(9), 1077 - 92
Human dUTP pyrophosphatase: uracil recognition by a beta hairpin and active sites formed by three separate subunits; Mol CD et al.; BACKGROUND . The essential enzyme dUTP pyrophosphatase (dUTPase) is exquisitely specific for dUTP and is critical for the fidelity of DNA replication and repair . dUTPase hydrolyzes dUTP to dUMP and pyrophosphate, simultaneously reducing dUTP levels and providing the dUMP for dTTP biosynthesis . A high cellular dTTP: dUTP ratio is essential to avoid uracil incorporation into DNA, which would lead to strand breaks and cell death . We report the first detailed atomic-resolution structure of a eukaryotic dUTPase, human dUTPase, and complexes with the uracil-containing deoxyribonucleotides, dUMP, dUDP and dUTP . RESULTS . The crystal structure reveals that each subunit of the dUTPase trimer folds into an eight-stranded jelly-roll beta barrel, with the C-terminal beta strands interchanged among the subunits . The structure is similar to that of the E . coli enzyme, despite low sequence homology between the two enzymes . The nucleotide complexes reveal a simple and elegant way for a beta hairpin to recognize specific nucleic acids: uracil is inserted into a distorted antiparallel beta hairpin and hydrogen bonds entirely to main-chain atoms . This interaction mimics DNA base pairing, selecting uracil over cytosine and sterically precluding thymine and ribose binding . Residues from the second subunit interact with the phosphate groups and a glycine-rich C-terminal tail of the third subunit caps the substrate-bound active site, causing total complementary enclosure of substrate . To our knowledge, this is the first documented instance of all three subunits of a trimeric enzyme supplying residues that are critical to enzyme function and catalysis . CONCLUSIONS . The dUTPase nucleotide-binding sites incorporate some features of other nucleotide-binding proteins and protein kinases, but seem distinct in sequence and architecture . The novel nucleic acid base recognition motif appears ancient; higher order structures, such as the ribosome, may have evolved from a motif of this kind . These uracil-beta-hairpin interactions are an obvious way for peptides to become early coenzymes in an RNA world, providing a plausible link to the protein-DNA world . Within the beta hairpin, there is a tyrosine corner motif that normally specifies beta-arch connections; this tyrosine motif was apparently recruited to discriminate against ribonucleotides, more recently than the evolution of the beta hairpin itself.

Structure, 1996 Sep 15, 4(9), 1053 - 64
Crystal structure of reduced protein R2 of ribonucleotide reductase: the structural basis for oxygen activation at a dinuclear iron site; Logan DT et al.; BACKGROUND . Ribonucleotide reductases (RNRs) catalyze the formation of the deoxyribonucleotides that are essential for DNA synthesis . The R2 subunit of Escherichia coli RNR is a homodimer containing one dinuclear iron centre per monomer . A tyrosyl radical is essential for catalysis, and is formed via a reaction in which the reduced, diferrous form of the iron centre activates dioxygen . To help understand the mechanism of oxygen activation, we examined the structure of the diferrous form of R2 . RESULTS . The crystal structures of reduced forms of both wild type R2 and a mutant of R2 (Ser211--> Ala) have been determined at 1.7 A and 2.2 A resolution, respectively . The diferrous iron centre was compared to the previously determined structure of the oxidized, diferric form of R2 . In both forms of R2 the iron centre is coordinated by the same carboxylate dominated ligand sphere, but in the reduced form there are clear conformational changes in three of the carboxylate ligands and the bridging mu-oxo group and two water molecules are lost . In the reduced form of R2 the coordination number decreases from six to four for both ferrous ions, explaining their high reactivity towards dioxygen . The structure of the mutant Ser211--> Ala, known to have impaired reduction kinetics, shows a large conformational change in one of the neighbouring helices although the iron coordination is very similar to the wild type protein . CONCLUSIONS . Carboxylate shifts are often important for carboxylate coordinated metal clusters; they allow the metals to achieve different coordination modes in redox reactions . In the case of reduced R2 these carboxylate shifts allow the formation of accessible reaction sites for dioxygen . The Ser211--> Ala mutant displays a conformational change in the helix containing the mutation, explaining its altered reduction kinetics.

Mol Gen Genet, 1996 Sep 13, 252(3), 332 - 41
The role of the genes nrf EFG and ccmFH in cytochrome c biosynthesis in Escherichia coli; Grovc J et al.; It has been suggested that two groups of Escherichia coli genes, the ccm genes located in the 47-min region and the nrfEFG genes in the 92-min region of the chromosome, are involved in cytochrome c biosynthesis during anaerobic growth . The involvement of the products of these genes in cytochrome c synthesis, assembly and secretion has now been investigated . Despite their similarity to other bacterial cytochrome c assembly proteins, NrfE, F and G were found not to be required for the biosynthesis of any of the c-type cytochromes in E . coli . Furthermore, these proteins were not required for the secretion of the periplasmic cytochromes, cytochrome C550 and cytochrome C552, or for the correct targeting of the NapC and NrfB cytochromes to the cytoplasmic membrane . NrfE and NrfG are required for formate-dependent nitrite reduction (the Nrf pathway), which involves at least two c-type cytochromes, cytochrome C552 and NrfB, but NrfF is not essential for this pathway . Genes similar to nrfE, nrfF and nrfG are present in the E . coli nap-ccm locus at minute 47 . CcmF is similar to NrfE, the N-terminal region of CcmH is similar to NrfF and the C-terminal portion of CcmH is similar to NrfG . In contrast to NrfF, the N-terminal, NrfF-like portion of CcmH is essential for the synthesis of all c-type cytochromes . Conversely, the NrfG-like C-terminal region of CcmH is not essential for cytochrome c biosynthesis . The data are consistent with proposals from this and other laboratories that CcmF and CcmH form part of a haem lyase complex required to attach haem c to C-X-X-C-H haem-binding domains . In contrast, NrfE and NrfG are proposed to fulfill a more specialised role in the assembly of the formate-dependent nitrite reductase.

Mol Gen Genet, 1996 Sep 13, 252(3), 266 - 74
Mini-TnhlyAs: a new tool for the construction of secreted fusion proteins; Gentschev I et al.; A simple and efficient procedure for the construction of secreted fusion proteins in Escherichia coli is described that uses a new minitransposon, termed TnhlyAs, carrying the secretion signal (HlyAs) of E . coli hemolysin (HlyA) . This transposon permits the generation of random gene fusions encoding proteins that carry the HlyAs at their C-termini . For the construction of model gene fusions we used lacZ, encoding the cytoplasmic beta-galactosidase (beta-Gal), and phoA, encoding the periplasmic alkaline phosphatase, as target genes . Our data suggest that all beta-Gal-HlyAs fusion proteins generated are secreted, albeit with varying efficiencies, by the HlyB/HlyD/TolC hemolysin secretion machinery under Sec-proficient conditions . In contrast, the PhoA-HlyAs fusion proteins are efficiently secreted in a secA mutant strain only under SecA-deficient conditions.

Biochim Biophys Acta, 1996 Sep 13, 1297(1), 90 - 8
In vivo incorporation of lipoic acid enantiomers and homologues in the pyruvate dehydrogenase complex from Escherichia coli; Loeffelhardt S et al.; The strain Escherichia coli JRG26, which has a defect in the lipoic acid biosynthesis, was cultivated in the presence of R-lipoic acid, S-lipoic acid, RS-dithiolane-3-caproic acid, RS-bisnorlipoic acid, and RS-tetranorlipoic acid, respectively . With the exception of the last compound the strain was able to grow with all these substances . R-lipoic acid was the most efficient factor, concentrations of 10 ng/l were sufficient to support growth of the cells, while 10(4)-fold to 10(7)-fold higher concentrations were necessary for the other compounds . The specific catalytic activity of the pyruvate dehydrogenase complex isolated from the cells grown on RS-dithiolane-3-caproic acid was only slightly lower than from cells grown on R-lipoic acid . With RS bisnorlipoic acid the specific activity was one third compared to that of the native enzyme complex . The incorporation of the RS-bisnorlipoic acid into the pyruvate dehydrogenase could directly be demonstrated by polyclonal antibodies directed against R-lipoic acid and RS-bisnorlipoic acid, both conjugated to BSA . Western blot analysis showed that the antibodies against the R-lipoic acid reacted specifically with the E2 component of pyruvate dehydrogenase complex purified from cells grown on this factor, while antibodies against RS-bisnorlipoic acid reacted with the enzyme complex isolated from cells grown in the presence of this compound.

Biochim Biophys Acta, 1996 Sep 13, 1297(1), 17 - 27
Analysis of wild-type and mutant aspartate aminotransferases using integrated rate equations; Schiller MR et al.; A general integrated rate equation was fit to reaction progress curves catalyzed by wild-type E . coli aspartate aminotransferase and the site-specific mutant enzymes, H193Q and Y70F . A nonlinear step-regression code, revised for this study selected from all kinetic constants in a general integrated rate equation for all unbranched enzyme mechanisms with stoichiometries upto two substrates and two products including terms for substrate inhibitions and that of an exogenous inhibitor . For each aspartate aminotransferase enzyme studied only kinetic constants consistent with a substituted enzyme mechanism were found statistically significant, thus the enzyme mechanism and sources of inhibition were determined objectively by statistics . The kinetic constants for wild-type and Y70F aspartate aminotransferase were similar to those previously reported indicating the validity of the integrated rate equation analysis . Minor changes in kinetic constants were observed for the H193Q mutant enzyme suggesting that the catalytic effects of the electrostatic hydrogen bonding network extending from the pyridine nitrogen of the cofactor through Asp-222, His-189 ends prior to His-193.

J Mol Biol, 1996 Sep 13, 262(1), 43 - 52
Three-dimensional reconstruction of the Escherichia coli 30 S ribosomal subunit in ice; Lata KR et al.; Three-dimensional (3D) reconstructions of both the heat-activated and non-activated 30 S subunit of the Escherichia coli 70 S ribosome were obtained from a frozen hydrated specimen preparation at 1/37 A-1 resolution . Well-characterized features that can be identified in both reconstructions are the head, the base, the platform and the cleft formed between the head and the platform . Comparisons between the 3D maps of 30 S subunits at 0 degree C, heat-activated at 37 degrees C, and the 30 S subunit portion identified in the cryo-3D map of 70 S ribosome reveal conformational changes the subunit probably undergoes during inactive-active transition and upon association with the 50 S subunit . These comparisons also allow us to localize the sites of association of 30 S and 50 S subunits.

J Mol Biol, 1996 Sep 13, 262(1), 12 - 20
Integration of foreign DNA in an intergenic region of the archaeon Methanosarcina mazei without effect on transcription of adjacent genes; de Macario EC et al.; Transformation systems for methanogenic archaea are scarce, none has been reported for the genus Methanosarcina, and plasmids useful as vectors for cloning foreign DNA into methanogens that stably replicate as extrachromosomal elements are not available . We developed an integration vector for transformation of a member of the genus Methanosarcina, i.e . Methanosarcina mazei, using a segment (Int alpha; 1015 bp) which encompasses the intergenic region (431 bp) between the stress (heat-shock) genes grpE and dnaK . This segment also includes the 3' end (270 bp) of the grpE protein-coding region and the 5' end (314 bp) of the dnaK protein-coding region . Int alpha has an EcoRI site, useful for cloning, situated in the 3' direction beyond the grpE transcription termination region, and far upstream from the dnaK promoter . This location of the site, and the monocistronic mode of transcription of grpE and dnaK in M . mazei, suggested to us that a foreign insert in the site would not affect transcription of either flanking gene . A puromycin-resistance cassette (pac cassette) was inserted in the EcoRI site of Int alpha already inserted in pUC18, to obtain a vector which integrated the pac cassette in the chromosome between grpE and dnaK . The pac gene was transcribed and the transformants acquired puromycin resistance . Constitutive and heat-shock-induced transcription of grpE and dnaK in the transformants was the same as in wild-type cells . The two vectors found with transforming ability differed in the orientation of the pac cassette but both had M . mazei's DNA on each flank of the cassette, with the same orientation as that of the homologous segments in the chromosome.

J Mol Biol, 1996 Sep 13, 262(1), 1 - 11
Two amino acid residues from the DNA-binding domain of MalT play a crucial role in transcriptional activation; Danot O et al.; MalT is the transcriptional activator of the Escherichia coli maltose regulon . Several lines of evidence suggest that MalT might act by interacting with RNA polymerase . Here, we show that inverted question markMalT, the DNA-binding domain of MalT, activates transcription . In order to identify amino acids of inverted question markMalT playing a specific role in activation, and therefore possibly involved in the putative contact(s) with RNA polymerase, we developed a double screen to isolate mutations of the inverted question markmalT gene affecting activation by inverted question markMalT without impairing its DNA-binding affinity . The effect of the mutations thus obtained on activation was assessed in vivo . This strategy essentially pointed to serine 834 and glutamine 876 of the MalT amino acid sequence as specifically involved in activation . Various inverted question markMalT derivatives substituted at positions 834 or 876 were purified and tested in vitro for their DNA-binding affinity, as well as for their activation ability . Together, the results obtained clearly show that serine 834 and glutamine 876 are important for activation by inverted question markMalT but not for DNA-binding . We argue that these amino acid residues are possibly solvent-exposed and propose that they act by contacting RNA polymerase.

Biochem Biophys Res Commun, 1996 Sep 13, 226(2), 431 - 8
Expression of thymopoietin beta/lamina-associated polypeptide 2 (TP beta/LAP2) and its family proteins as revealed by specific antibody induced against recombinant human thymopoietin; Ishijima Y et al.; An expression vector was constructed to produce a common region of human thymopoietin family proteins . The recombinant protein was expressed in Escherichia coli as a fusion protein with a biotinylated tag region and purified by affinity chromatography on a monomeric avidin resin . The thymopoietin family-specific antibody was induced in rabbits by immunization with the recombinant fusion protein . Western blotting analysis using the antibody revealed that the expression of thymopoietin family proteins was remarkably tissue specific . Among those, thymopoietin beta/lamina-associated polypeptide 2 appears to be specifically expressed in tissues with high proliferative activity.

Biochem Biophys Res Commun, 1996 Sep 13, 226(2), 420 - 5
Molecular cloning and expression of mouse procalcitonin; Rehli M et al.; The nucleotide sequence for mouse calcitonin was determined from a cDNA obtained using a polymerase chain reaction (PCR) based method, the rapid amplification of cDNA ends (RACE)-PCR . Primers designed from highly conserved regions in the coding sequences of known rat and human calcitonin cDNAs were used to amplify calcitonin cDNA as 5'-end and 3'-end fragments from mouse thyroid RNA . The obtained cDNA is 850 bp in length most probably representing the entire mouse calcitonin mRNA . It contains an open reading frame coding for a 136 amino acid protein with a calculated M(r) of 15,143 . Comparison of the deduced amino acid sequences of preprocalcitonin of mice with other species revealed highest homologies to the rat (93%) and human (77%) sequences . A recombinant form of mouse precalcitonin (rmPCT) of approximately 17 kDa was expressed as a fusion peptide in E.coli transformed with a PCR-cloned expression construct.

J Biol Chem, 1996 Sep 13, 271(37), 22831 - 8
Purification, kinetic properties, and cDNA cloning of mammalian betaine-homocysteine methyltransferase; Garrow TA; Porcine liver betaine-homocysteine methyltransferase (BHMT; EC) was purified to homogeneity, and the Michaelis constants for betaine, dimethylacetothetin, and L-homocysteine are 23, 155, and 32 microM, respectively . The maximum rate of catalysis is 47-fold greater using dimethylacetothetin as a methyl donor compared with betaine . Partial amino acid sequence of porcine BHMT was obtained, and inosine-containing redundant oligonucleotide primers were used to amplify an 815-base pair sequence of the porcine cDNA by polymerase chain reaction (PCR) . Nondegenerate oligonucleotide primers based on the porcine cDNA were synthesized and used to isolate a 463-base pair fragment of the human cDNA by PCR . The human PCR DNA product was then used to screen a cDNA library by plaque hybridization, and cDNAs encoding human BHMT were isolated . The primary structure of the human cDNA is reported here, and the open reading frame encodes a 406-residue protein of Mr 44,969 . The deduced amino acid sequence of human BHMT shows limited homology to bacterial vitamin B12-dependent methionine synthases (EC) . A plasmid containing the human BHMT cDNA fused in frame to the N terminus of beta-galactosidase was transformed into Escherichia coli, and transformants expressed BHMT activity, an activity that is absent from wild type E . coli.

J Biol Chem, 1996 Sep 13, 271(37), 22802 - 9
N-Acetylated domains in heparan sulfates revealed by a monoclonal antibody against the Escherichia coli K5 capsular polysaccharide . Distribution of the cognate epitope in normal human kidney and transplant kidney with chronic vascular rejection; Born J et al.; The Escherichia coli K5 capsular polysaccharide has the same (GlcUA-->GlcNAc)n structure as the nonsulfated heparan sulfate/heparin precursor polysaccharide . A monoclonal antibody (mAb 865) against the K5 polysaccharide has been described (Peters, H., Jurs, M., Jann, B., Jann, K., Timmis, K . N., and Bitter-Sauermann, D . (1985) Infect . Immun . 50, 459-466) . In this report, we demonstrate the binding of anti-K5 mAb 865 to N-acetylated sequences in heparan sulfates and heparan sulfate proteoglycans but not to heparin . This is shown by direct binding and fluid phase inhibition of mAb 865 in an enzyme-linked immunosorbent assay . In this system we found that the binding of the mAb decreased with increasing sulfate content of the polysaccharide . By testing chemically modified K5 and heparin polysaccharides, we found that each of the modifications that occur during heparan sulfate (HS) synthesis (N-sulfation, C-5 epimerization, and O-sulfation) prevents recognition by mAb 865 . Samples of heparan sulfate from human aorta (HS-II) were selectively degraded so as to allow the separate isolation of N-sulfated and N-acetylated block structures . N-Sulfated oligosaccharides (obtained after N-deacetylation by hydrazinolysis followed by nitrous acid deamination at pH 3.9) were not recognized by mAb 865, in contrast to N-acetylated oligosaccharides (obtained after nitrous acid deamination at pH 1.5), although the reactivity was lower than for intact HS-II . Analysis of the latter's pH 1.5 deamination products by gel filtration indicated that a minimal size of 18 saccharide units was necessary for antibody binding . These results lead us to propose bivalent antibody-heparan sulfate interaction, in which both F(ab) domains of the mAb interact with their epitopes, both of which are present in a single large (>/=18 saccharide units) N-acetylated domain and additionally with single epitopes present in two N-acetylated sequences (each <18 saccharide units) bridged by a short N-sulfated domain . Immunohistochemistry with mAb 865 on cryostat sections of normal human kidney tissue, revealed its binding to most but not all renal basement membranes . However, all renal basement membranes contain heparan sulfate, as shown by a mAb against heparitinase-digested heparan sulfate stubs (mAb 3G10) . This finding indicates that not all heparan sulfate chains present in basement membranes express the mAb 865 epitopes . Besides the normal distribution, mAb 865 staining was found in fibrotic and sclerotic lesions in vessels, interstitium, and mesangium in transplant kidneys with chronic vascular rejection . Occasionally, a decrease of staining was observed within tubulo-interstitium and glomeruli . These findings show that N-acetylated sequences in heparan sulfates can be demonstrated by anti-K5 mAb 865 in normal and diseased kidneys.

J Biol Chem, 1996 Sep 13, 271(37), 22462 - 9
Putidaredoxin reductase-putidaredoxin-cytochrome p450cam triple fusion protein . Construction of a self-sufficient Escherichia coli catalytic system; Sibbesen O et al.; Fusion proteins of cytochrome P450cam with putidaredoxin (Pd) and putidaredoxin reductase (PdR), the two proteins required to transfer electrons from NADH to P450cam, were constructed by fusing cDNAs encoding the three proteins in the expression vector pCWori+ . Several fusion proteins, in which the order of the three protein domains and the linkers between them were varied, were expressed in Escherichia coli, purified, and characterized . The highest activity (kcat = 30 min-1) was obtained with a PdR-Pd-P450cam construct in which the peptides TDGTASS and PLEL were used, respectively, to link the PdR to the Pd and the Pd to the P450cam domains . Oxygen and NADH consumption is tightly coupled to substrate oxidation in the fusion proteins . The rate-limiting step in the catalytic turnover of these fusion proteins is electron transfer from Pd to P450cam . This is indicated by high rates of electron transfer from the PdR and Pd domains to exogenous electron acceptors, by an increase in the activity of the P450cam domain upon addition of exogenous Pd, and by the high activity of wild-type P450cam when incubated with a PdR-Pd fusion protein . E . coli cells expressing the PdR-Pd-P450cam fusion protein efficiently oxidize camphor to 5-exo-hydroxycamphor and 5-oxocamphor . E . coli cells expressing the triple fusion protein thus constitute the first heterologous self-sufficient catalytic system for the oxidation of camphor and other substrates by P450cam.

J Biol Chem, 1996 Sep 13, 271(37), 22321 - 5
Interplay of methionine tRNAs with translation elongation factor Tu and translation initiation factor 2 in Escherichia coli; Guillon JM et al.; According to their role in translation, tRNAs specifically interact either with elongation factor Tu (EFTu) or with initiation factor 2 (IF2) . We here describe the effects of overproducing EFTu and IF2 on the elongator versus initiator activities of various mutant tRNAMet species in vivo . The data obtained indicate that the selection of a tRNA through one or the other pathway of translation depends on the relative amounts of the translational factors . A moderate overexpression of EFTu is enough to lead to a misappropriation of initiator tRNA in the elongation process, whereas overproduced IF2 allows the initiation of translation to occur with unformylated tRNA species . In addition, we report that a strain devoid of formylase activity can be cured by the overproduction of tRNAMetf . The present study brings additional evidence for the importance of formylation in defining tRNAMetf initiator identity, as well as a possible explanation for the residual growth of bacterial strains lacking a functional formylase gene such as observed in Guillon, J . M., Mechulam, Y., Schmitter, J.-M., Blanquet, S., and Fayat, G . (1992) J . Bacteriol . 174, 4294-4301.

Biochem Pharmacol, 1996 Sep 13, 52(5), 809 - 13
Increase in histamine synthesis by liver macrophages in CCl4-injured mast cell-deficient W/Wv mice; Suzuki M et al.; This study set out to examine the possible role of liver macrophages in histamine synthesis in the injured liver . The effects of the hepatotoxins Escherichia coli lipopolysaccharide (LPS) and CCl4 on histamine synthesis in the liver of mice were evaluated . C3H/HeJ mice were resistant to LPS in including histidine decarboxylase (HDC) in the liver compared with C3H/HeN mice and mast cell-deficient W/Wv mice . However, C3H/HeJ mice did respond strongly to another hepatotoxin, CCl4, leading to a significant increase in HDC activity . CCl4 also caused a marked increase in HDC activity and histamine levels in the liver of W/Wv mice . In addition, injection of CCl4 produced a large increase in the activity of HDC in the spleen and lung of W/Wv mice . HDC activity was confined to the nonparenchymal cells, with parenchymal cells expressing essentially no HDC activity . The CCl4-induced increase in HDC activity was confined, at least in part, to the liver macrophages . These results indicate that the macrophages are responsible for the increase in HDC-dependent histamine production in the liver caused by the injection of hepatotoxins . The possible role of histamine in liver regeneration after injury is discussed.

Biochim Biophys Acta, 1996 Sep 12, 1276(2), 154 - 60
The Escherichia coli F1-ATPase mutant beta Tyr-297-->Cys: functional studies and asymmetry of the enzyme under various nucleotide conditions based on reaction of the introduced Cys with N-ethylmaleimide and 7-chloro-4-nitrobenzofurazan; Haughton MA et al.; Conversion of residue beta Tyr-297 of the Escherichia coli F1-ATPase (ECF1) to a Cys in the mutant beta Y297C led to impaired oxidative phosphorylation based on growth curves . The ATPase activity of ECF1 isolated from the mutant beta Y297C was only 1% of wild-type activity, but the residual activity involves cooperative multi-site enzyme turnover based on inhibition by DCCD and azide . ATPase activity could be increased to 8%, and 13% of wild-type by reaction of the introduced Cys with N-ethyl maleimide (NEM), and 7-chloro-4-nitrobenzofurazan (NbfCl), respectively, suggesting that enzymatic function is improved by an increased hydrophobicity of residue beta Cys-297 . The mutation beta Tyr-297-->Cys had no effect on nucleotide binding in studies with the fluorescent analog lin-benzo-ADP . The asymmetry of ECF1 was investigated in the mutants beta Y297C and beta Y297C:E381C/epsilon S108C by examining the relative reactivity of Cys-297 in the three copies of the beta subunit under different nucleotide binding conditions . In agreement with a previous study (Haughton, M.A . and Capaldi, R.A . (1995) J . Biol . Chem., 270, 20568-20574), the asymmetry was maintained under all nucleotide conditions . The NbfCl reaction site was found to be beta free, which is also the site most reactive to NEM, beta epsilon is the second site which reacts with NbfCl or NEM, while the third site, beta gamma, is poorly reactive to either reagent.

Biochim Biophys Acta, 1996 Sep 11, 1308(3), 241 - 50
Enrichment for gene targeting in mammalian cells by inhibition of poly(ADP-ribosylation); Waldman BC et al.; Inhibition of poly(ADP-ribosylation) reduces random genomic integration of transfected DNA and mildly stimulates intrachromosomal homologous recombination in mammalian cells . We investigated the effect of inhibition of poly(ADP-ribosylation) on the efficiency of gene targeting in Chinese hamster ovary (CHO) cell line ATS-49tg . This cell line is hemizygous for a defective adenine phosphoribosyltransferase (aprt) gene and is hypoxanthine phosphoribosyltransferase (hprt) deficient . Plasmid pAG100 contains a portion of the CHO aprt gene sufficient to correct the defect in ATS-49tg cells via gene targeting; pAG100 also contains an Escherichia coli guanine phosphoribosyltransferase (gpt) gene . Following transfection of ATS-49tg cells with pAG100, selection for gpt-positive transfectants allowed recovery of cells that had randomly integrated pAG100 while selection for aprt-positive cells allowed recovery of cells that had undergone gene targeting at the endogenous aprt locus . Treatment of cells with 3 mM 3-methoxybenzamide (3-MB), an inhibitor of poly(ADP-ribose) polymerase, decreased random integration and gene targeting of electroporated pAG100 about 5-fold . In contrast, treatment with 3 mM 3-MB during calcium phosphate transfection could reduce random integration more than 150-fold while reducing gene targeting less than two-fold . Therefore, as much as a 100-fold enrichment for gene targeting was achieved with calcium phosphate transfection.

Hum Gene Ther, 1996 Sep 10, 7(14), 1735 - 42
A simple and efficient method for the concentration and purification of recombinant retrovirus for increased hepatocyte transduction in vivo; Bowles NE et al.; Although recombinant retroviruses have been widely used for the transduction of target organs in vivo, the viral titers achieved by current production methods are often too low to achieve therapeutic levels of gene expression . To overcome this limitation, a simple method for the efficient concentration and purification of amphotropic retrovirus particles was developed . After portal vein infusion into partially hepatectomized rats of 5.5 x 10(7) cfu of a beta-galactosidase (beta-gal)-expressing retrovirus (LX/beta geo) concentrated by this method, up to 25% of hepatocytes stained positive for beta-Gal activity . Measurement of human alpha 1-antitrypsin (hAAT) levels after infusion of various doses of a similarly concentrated retrovirus encoding hAAT (LX/hAAT) demonstrated that viral transduction increased proportionally with titer, up to a dose of 7.5 x 10(7) cfu per rat . The ability to concentrate retroviral virion efficiently from large volumes of supernatant has allowed the further purification of virus particles by sucrose banding ultracentrifugation . This procedure results in a greater than 50% recovery of infectious virus particles, with titers up to 500-fold higher than in the original supernatant . These methods may have significant utility in both ex vivo and in vivo retroviral applications in human gene therapy.

Hum Gene Ther, 1996 Sep 10, 7(14), 1719 - 26
Expression of the bcl-2 gene from a defective HSV-1 amplicon vector protects pancreatic beta-cells from apoptosis; Liu Y et al.; It has been suggested that the mechanism of pancreatic beta-cell death in autoimmune diabetes mellitus and in immunoisolated transplantation devices involves cytokine-induced apoptosis . To explore the feasibility of a gene transfer strategy to protect beta-cells, we evaluated the use of replication defective HSV-1 amplicon vectors as gene transfer vehicles . Post-mitotic murine and human beta-cells were efficiently transduced by a herpes simplex virus (HSV) vector that expresses the reporting gene Escherichia coli lacZ under the transcriptional control of a HSV promoter (HSVlac) both as islets and as single cells . Insulin secretion, a marker of beta-cell function, was unaffected by HSVlac transduction of a beta-cell line . A HSV amplicon vector that expressed bcl-2 (HSVbcl2) in beta-cells was constructed, and its effects on cytokine-mediated apoptosis in both a beta-cell line and primary murine beta-cells assessed by measuring internucleosomal fragmentation . beta-Cell apoptosis was blocked by transduction with HSVbcl2 but not HSVlac . The prevention of cytokine-induced apoptosis in beta-cells by bcl-2 expression has the potential both to ameliorate primary autoimmune beta-cell destruction as type I diabetes develops, and to prevent the destruction of transplanted beta-cells inside immunoisolation devices.

Biochemistry, 1996 Sep 10, 35(36), 11918 - 24
Allosteric effects of carbamoyl phosphate synthetase from Escherichia coli are entropy-driven; Braxton BL et al.; When catalyzing the formation of MgATP and carbamate from MgADP and carbamoyl phosphate, Escherichia coli carbamoyl phosphate synthetase (CPS) binds MgADP with a large negative change in heat capacity . The magnitude of this heat capacity change is not appreciably altered by the presence of a saturating concentration of either the allosteric activator ornithine or the inhibitor UMP despite the substantial and opposing effects these ligands have on the binding affinity for MgADP . By contrast, no detectable change in heat capacity is associated with the thermodynamic coupling between MgADP and either ornithine or UMP . The sign of the apparently constant enthalpic and entropic contributions to the coupling free energy for each of these ligands is opposite that of the coupling free energy, indicating that the observed allosteric phenomenology is in net opposed by the enthalpy of the interaction and instead arises from a change in entropy of the system . IMP produces only a very small allosteric effect as indicated by a near-zero value for the MgADP-IMP coupling free energy . However, the enthalpic and entropic contributions are individually larger in absolute value for the IMP coupling than for those pertaining to the other allosteric ligands, and entropy dominates the coupling free energy above 36 degrees C, causing IMP to become an activator at high temperature . In addition, the sign of the coupling enthalpy and entropy for IMP has the same sign as the coupling enthalpy and entropy produced by ornithine, suggesting that IMP and ornithine may similarly influence the enzyme at a molecular level despite binding to different allosteric sites on the enzyme . The data are consistent with a model in which the actions of the allosteric ligands arise primarily from changes in the conformational degeneracy introduced by each ligand . With this model, one can also rationalize the failure of these allosteric ligands to substantially influence kcat.

Biochemistry, 1996 Sep 10, 35(36), 11895 - 900
Na(+)-translocating cytochrome bo terminal oxidase from Vitreoscilla: some parameters of its Na+ pumping and orientation in synthetic vesicles; Park C et al.; Vitreoscilla cytochrome bo ubiquinol oxidase is similar in some properties to the Escherichia coli enzyme, but unlike the latter, the Vitreoscilla oxidase functions as a primary Na+ pump . When purified Vitreoscilla cytochrome bo is incorporated into liposomes made from Vitreoscilla phospholipids and energized with a quinol substrate, it translocates Na+, not H+, across the vesicle membrane . Since protonophores CCCP (carbonyl cyanide m-chlorophenylhydrazone) and DTHB (3,5-di-tert-butyl-4-hydroxybenzaldehyde) stimulated the Na+ pumping, it is unlikely that it is a secondary effect due to the presence of Na+/H+ antiporter activity in the preparations . The efficiency of the Na+ pumping was 3.93 Na+ pumped per O2 consumed when ascorbate/TMPD was used as the substrate . The cytochrome has a K(m) and Kcat for Na+ of 2.9 mM and 277 s-1, respectively . When ferricytochrome c was entrapped within liposomes prepared from Vitreoscilla phospholipids, it was reduced by Q1H2 (ubiquinol-1) but not by ascorbate/TMPD (N,N,N',N'-tetramethyl-1,4-phenylenediamine) . Although Q1H2 was oxidized by cytochrome bo in solution at a rate approximately 14 times that of the latter substrate, the rate of accumulation of Na+ within cytochrome bo vesicles driven by the membrane impermeable ascorbate/TMPD was 1.23 times that of the membrane permeable ubiquinol . These data allowed a calculation that in these synthetic proteoliposomes the cytochrome bo molecules are only 51% directed inward; a value of 61% inward-directed was estimated by measuring the ascorbate/TMPD oxidase activity of the proteoliposomes before and after disrupting them with Triton X-100 . A random orientation of the E . coli cytochrome bo oxidase in proteoliposomes has also been reported.

Biochemistry, 1996 Sep 10, 35(36), 11852 - 64
Nuclear magnetic resonance solution structure of the growth factor receptor-bound protein 2 Src homology 2 domain; Thornton KH et al.; A family of NMR solution structures of the growth factor receptor-bound protein 2 (Grb2) SH2 domain has been determined by heteronuclear multidimensional NMR . Proton, nitrogen, and carbon chemical shift assignments have been made for the SH2 domain of Grb2 . Assignments were made from a combination of homonuclear two-dimensional and 15N- and 13C-edited three-dimensional spectra at pH 6.2 and 298 K . Structure-induced proton and carbon secondary shifts were calculated and used to facilitate the spectral assignment process . NOE, scalar coupling, secondary chemical shift, and amide proton exchange data were used to characterize the secondary structural elements and hydrogen-bonding network in the Grb2 SH2 domain . The three-dimensional structure of the Grb2 SH2 domain was calculated using 1112 restraints obtained from NOE, coupling constant, and amide proton exchange data . The rmsd for the 24 calculated structures to the mean structure of the Grb2 SH2 domain was 0.75 A for backbone and 1.28 A for all heavy atoms . The three-dimensional fold of the Grb2 SH2 domain is similar to that observed for other SH2 domains and consists of two alpha-helical segments and eight beta-strands, six strands that make up two contiguous antiparallel beta-sheets, and two strands that form two short parallel beta-sheets . The structure of the phosphotyrosine binding pocket of Grb2 is similar to that observed for other SH2 domains . The hydrophobic binding pocket of Grb2 is similar to that observed for Src with the exception that tryptophan 121 of Grb2 occupies part of the pY+3 binding pocket . Structural implications for the Grb2 SH2 domain selectivity at the pY+2 and pY+3 sites are discussed.

Biochemistry, 1996 Sep 10, 35(36), 11756 - 62
Cloning and expression of salmon cardiac troponin C: titration of the low-affinity Ca(2+)-binding site using a tryptophan mutant; Moyes CD et al.; Activation of cardiac actomyosin ATPase requires the occupation of the single low-affinity Ca(2+)-binding site of troponin C (cTnC) . Previously, we demonstrated pronounced differences between mammals and cold-water salmonid fish in the Ca2+ sensitivity of cardiac preparations, particularly in relation to temperature {Churcotte, C., Moyes, C . D., Baldwin, K., Bressler, B., & Tibbits, G . F . (1994) Am . J . Physiol . 267, R62-R70} . In this study, we examine the extent to which cTnC structure could account for the observed differences in myofibrillar Ca2+ sensitivity . Salmonid (Oncorhynchus mykiss) cTnC was cloned, sequenced, and expressed in Escherichia coli as a maltose-binding protein fusion . The coding region has 87% homology with human cTnC cDNA and differs in 13 of 161 amino acid residues from the human/bovine/porcine isoform . The sequence corresponding to the single regulatory Ca(2+)-binding site II is completely homologous to that of mammals . The protein expressed exhibits optical properties similar (circular dichroism, intrinsic fluorescence) to those of cTnC purified from salmonid (Salmo salar) and bovine ventricle . A single tryptophan residue was introduced into the inactive Ca(2+)-binding site I (ScTnC-FW27) to facilitate Ca2+ titration . The Ca(2+)-binding constant (K1/2 = 5.33 pCa units) was within the range reported for the low-affinity sites of mammalian cTnC . Although differences in TnC primary structure are striking, Ca2+ affinity of intact cardiac myofibrils is likely influenced by interactions with other troponin proteins.

Biochemistry, 1996 Sep 10, 35(36), 11747 - 55
Characterization of SH2-ligand interactions via library affinity selection with mass spectrometric detection; Kelly MA et al.; Synthetic combinatorial libraries have proven to be a valuable source of diverse structures useful for large-scale biochemical screening . Their use has greatly facilitated the study of protein-protein interactions . We have developed a practical technique for screening such libraries by integrating affinity chromatography selection with electrospray ionization mass spectrometric detection, referred to as library affinity selection-mass spectrometry (LAS-MS) . The process allows for rapid and efficient screening of solution phase libraries and provides detailed information such as the relative affinities of substrates . The method is generally applicable to include nonpeptide libraries; moreover, electrospray tandem mass spectrometry (ES-MS/MS) yields sequence-specific identification of individual components without the need for chemical tags . This technique is demonstrated using the Src homology 2 (SH2) domain of phosphatidylinositol 3-kinase (PI 3-kinase) . The critical importance of methionine in the position +3 (relative to the phosphotyrosine position) is demonstrated in a library built with a phosphotyrosine mimic, (phosphonodifluoromethyl)phenylalanine . The described method has broad applicability to combinatorial library screening.

Biochemistry, 1996 Sep 10, 35(36), 11668 - 76
Kinetic analysis of human flap endonuclease-1 by flow cytometry; Nolan JP et al.; Human flap endonuclease-1 (FEN-1) is a structure-specific endonuclease and exonuclease which is essential for DNA replication and repair . We have cloned a human FEN-1 gene, overexpressed it in Escherichia coli, purified the recombinant protein to near homogeneity, and characterized its cleavage of a flap DNA structure using a novel analytical approach based on flow cytometry . With this approach, we were able to measure continuously the kinetics of DNA cleavage by FEN-1 and to separate experimentally the binding and catalysis functions of the enzyme . When the reaction was initiated by the addition of FEN-1, the cleavage kinetics were dependent on enzyme concentration and appeared to saturate at high concentrations . When enzyme and substrate were preincubated in the presence of EDTA and the reaction initiated by the addition of Mg2+, rapid kinetic flow cytometry measurements showed that cleavage is fast (t1/2 approximately 6 s, k = 0.10 s-1) . Using the single-turnover kinetics as a measure of the amount of enzyme-substrate complex present, we estimated the Kd for the FEN-1-flap DNA substrate to be 7.5 nM in the absence of Mg2+ and the rate constant for dissociation of the enzyme-substrate complex to be 0.07 s-1 . Computer fitting of the experimental data to a kinetic model confirms these estimates for the individual steps and suggests some interesting features of enzymology using a surface-bound substrate.

Biochemistry, 1996 Sep 10, 35(36), 11652 - 9
Recognition of the T-arm of tRNA by tRNA (m5U54)-methyltransferase is not sequence specific; Gu X et al.; tRNA (m5U54)-methyltransferase (RUMT) catalyzes the methylation of U54 of tRNAs . In contrast to enzymes which recognize a particular tRNA, RUMT recognizes features common to all tRNAs . We have shown that these features reside in the T-arm of tRNA and constructed a minimal consensus sequence for RUMT recognition and catalysis (Gu et al., 1991b) . Here, we have mutated each conserved T-loop residue and conserved T-stem base pair to bases or base pairs which are not observed in Escherichia coli tRNA . The substrate specificity of RUMT for 30 in vitro synthesized T-arm mutants of tRNAPhe and 37 mutants of the 17-mer analog of the T-arm derived from tRNA1Val was investigated . A 2-5 base pair stem was essential for recognition of the T-arm by RUMT, but the base composition of the stem was unimportant . The 7-base size of the T-loop maintained by the stem was essential for RUMT recognition . For tRNA, most base substitutions in the 7-base loop did not eliminate RUMT activity, except for any mutation of the methyl acceptor U54 and the C56G mutation . The effect of base and base pair mutations on Kcat or the rate of methylation by RUMT was more striking than the effect on the Kd for binding to RUMT . In comparison with mutations in the T-loop of intact tRNA, base mutation in the T-loop of the 17-mer T-arm had a more deleterious effect on binding and methylation . Surprisingly, recognition of tRNA by RUMT appears to reside in the three-dimensional structure of the seven-member T-loop rather than in its primary structure.

Biochemistry, 1996 Sep 10, 35(36), 11642 - 51
DNA polymerase photoprobe 2-{(4-azidophenacyl)thio}-2'-deoxyadenosine 5'-triphosphate labels an Escherichia coli DNA polymerase I Klenow fragment substrate binding site; Moore BM 2nd et al.; The nucleotide photoprobe 2-{(4-azidophenacyl)thio}-2'-deoxyadenosine 5'-triphosphate (1) was evaluated as a photoaffinity label of the DNA polymerase I Klenow fragment . Photolabel {3H}-1 covalently labeled the Klenow fragment with photolysis at 300 nm, reaching saturation at an approximate 1:1 mole ratio at 5.7 microM and with an EC50 (the effective concentration at 50% maximum photoincorporation) of about 0.74 microM . Saturating concentrations of poly(dA).(T)10 protect the Klenow fragment from {3H}-1 photoincorporation, and TTP at a concentration approximately equal to its KD for the free enzyme form shifts the dose-response curve for photoincorporation of {3H}-1 into the Klenow fragment by a factor of 2, indicating a competitive relationship between TTP and 1 . Additionally, the photoincorporation of {3H}-1 into the Klenow fragment has an absolute requirement for magnesium, with no significant photoincorporation observed at concentrations of 1 up to 10 microM in the absence of magnesium . These results demonstrate that, as designed, photoprobe 1 binds to both the dNTP and a portion of the template-primer binding sites on the Klenow fragment . Photoaffinity labeling of the Klenow fragment by 1 yielded a single radiolabeled tryptic fragment which was isolated by HPLC; sequence analysis identified Asp732 in the peptide fragment Asp732-Ile733-His734-Arg735 as the site of covalent modification . Molecular modeling and complementary NMR analysis of the conformation of 1 indicated preferred C3'-exo and C2'-exo-C3'-endo symmetrical twist furanose ring puckers, with a high antibase conformation and a +sc C-5 torsional angle . Docking studies using Asp732 as an anchor point for the azide alpha-nitrogen on the photolabel indicate that the dNTP binding site is at the edge of the DNA binding cleft opposite the exonuclease site and that the template binding site includes helix O in the finger motif of the Klenow fragment.

Biochemistry, 1996 Sep 10, 35(36), 11634 - 41
Deoxyadenosine-based DNA polymerase photoprobes: design, synthesis, and characterization as inhibitors of the Escherichia coli DNA polymerase I Klenow fragment; Moore BM 2nd et al.; DNA polymerase photoprobes 2-{(4-azidophenacyl)thio}-2'-deoxyadenosine 5'-triphosphate (1), 2-{(4-azidophenylsulfenyl)thio}-2'-deoxyadenosine 5'-triphosphate (2), and 2-{(4-azido-2-nitrophenyl)-thio}-2'-deoxyadenosine 5'-triphosphate (3) were designed from a thermodynamic model of DNA polymerase 1-substrate interactions such that the triphosphate would anchor the inhibitor and allow the phenyl azide to interact with the complementary template binding site . Photoprobes 1-3 were synthesized by condensation of 2-thio-2'-deoxyadenosine or its phosphate with p-azidophenacyl bromide, N-(4-azidophenylsulfenyl)phthalimide, and 4-azido-1-fluoro-2-nitrobenzene, respectively, and characterized as reversible and photoinduced irreversible inhibitors of the DNA polymerase I Klenow fragment and HIV I reverse transcriptase . The aryl azides decomposed with irradiation at 300 and 350 nm with half-lives ranging from 0.98 to 2.33 min and 2.15 to 5.38 min, respectively, with quantum efficiencies ranging from 0.29 to 0.55 and no apparent photodecomposition of the 2-thio-2'-deoxyadenosine nucleotide . Photoprobes 1-3 showed mixed noncompetitive inhibition of the Klenow fragment polymerase activity versus poly(dA).(T)10 as variable substrate with apparent competitive inhibition constants of 2.1, 36, and 29 microM, respectively, evidence suggesting that these photoprobes bind to both the free enzyme form and the enzyme-template-primer binary complex . Of the three photoprobes, only nucleotide 1 photoinactivates the Klenow fragment; in the presence of a 200-fold excess of nitrene scavenger, photoprobe 1 inactivates 92% of the Klenow fragment polymerase activity with saturation observed at 9.7 microM and an IC50 of about 2 microM . This evidence demonstrates that photoprobe 1 does bind to the Klenow fragment in the absence of template-primer and that it is an efficient photoprobe.

Biochemistry, 1996 Sep 10, 35(36), 11605 - 11
Structure-based design of an intramolecular proton transfer site in murine carbonic anhydrase V; Heck RW et al.; Carbonic anhydrase V (CA V) is a mitochondrial enzyme that catalyzes the hydration of CO2 to produce bicarbonate and a proton . The catalytic properties of wild-type murine CA V suggest the presence of a proton shuttle residue having pKa = 9.2, the role of which is to transfer a proton from zinc-bound water to solution in the hydration direction to regenerate the zinc hydroxide form of the enzyme . Two likely candidates for shuttle residues are the tyrosines at positions 64 and 131 in the active site cavity . The crystal structure of wild-type carbonic anhydrase V {Boriack-Sjodin et al . (1995) Proc . Natl . Acad . Sci . U.S.A . 92, 10949-10953} shows that the side chain of Tyr 64 is forced into an orientation pointing away from the zinc by Phe 65, although Tyr 131 is oriented toward the zinc . We have prepared mutants of murine CA V replacing both Tyr 64 and Tyr 131 with His and Ala and investigated the proton shuttle mechanism using stopped-flow spectrophotometry and the depletion of 18O from CO2 measured by mass spectrometry . Experiments with both single and double mutations showed that neither position 64 nor position 131 was a prominent site for proton transfer . However, a double mutant of CA V containing the two replacements, Tyr 64-->His and Phe 65-->Ala, demonstrated enhanced proton transfer with an apparent pKa of 6.8 and maximal contribution to kcat of 2.2 x 10(5) s-1 . In addition to the altered catalytic properties, the crystal structure of the His 64/Ala 65 double mutant strongly suggested proton transfer by His 64 after removal of the steric hindrance of Phe 65 . This is the first structure-based design of an efficient proton transfer site in an enzyme.

Biochemistry, 1996 Sep 10, 35(36), 11560 - 9
The structure of nucleotidylated histidine-166 of galactose-1-phosphate uridylyltransferase provides insight into phosphoryl group transfer; Wedekind JE et al.; Galactose-1-phosphate uridylyltransferase catalyzes the reaction of UDP-glucose with galactose 1-phosphate to form UDP-galactose and glucose 1-phosphate during normal cellular metabolism . The reaction proceeds through a double displacement mechanism characterized by the formation of a stable nucleotidylated histidine intermediate . This paper describes the preparation of the uridylyl-enzyme complex on the crystalline enzyme from Escherichia coli and its subsequent structure determination by X-ray crystallography . The refined structure has an R-factor of 19.6% (data between 65 and 1.86 A resolution) and reveals modest conformational changes at the active site compared to the inactive UMP/UDP-enzyme complex reported previously {Wedekind, J.E., Frey, P.A., & Rayment, I . (1995) Biochemistry 34, 11049-11061} . In particular, positions of the respective UMP alpha-phosphoryl groups differ by approximately 4 A . Well-defined electron density for the nucleotidylated imidazole supports the existence of a covalent bond between N epsilon 2 of the nucleophile and the alpha-phosphorus of UMP . A hydrogen bond that is conserved in both complexes between His 166 N delta 1 and the carbonyl O of His 164 serves to properly orient the nucleophile and electrostatically stabilize the positively charged imidazolium that results from nucleotidylation . Hydrogen bonds from side-chain Gln 168 to the nonbridging phosphoryl oxygens of the nucleotidyl intermediate appear crucial for the formation and reaction of the uridylyl-enzyme complex as well . The significance of the latter interaction is underscored by the fact that the predominant cause of the metabolic disease galactosemia is the mutation of the corresponding Gln (Gln 188 in humans) to Arg . A comparison to other phosphohistidyl enzymes is described, as well as a revised model for the mechanism of the uridylyltransferase.

Biochemistry, 1996 Sep 10, 35(36), 11529 - 35
Fhit, a putative tumor suppressor in humans, is a dinucleoside 5',5"'-P1,P3-triphosphate hydrolase; Barnes LD et al.; Human Fhit (fragile histidine triad) protein, encoded by the FHIT putative tumor suppressor gene, is a typical dinucleoside 5',5"'-P1,P3-triphosphate (Ap3A) hydrolase (EC 3.6.1.29) on the basis of its enzymatic properties we report here . Ap3A is the preferred substrate among ApnA (n = 3-6), and AMP is always one of the reaction products . Mn2+ and Mg2+ are equally stimulatory, while Zn2+ is inhibitory with Ap3A as the substrate . Values of the K(m) for Ap3A and Ap4A are 1.3 and 4.6 microM, respectively . Values of the specificity constant, kcat/K(m), for Ap3A and Ap4A are 2.0 x 10(6) and 6.7 x 10(3) s-1 M-1, respectively, for a glutathione S-transferase (GST)-Fhit fusion protein . Site-directed mutagenesis of FHIT demonstrated that all four conserved histidines are required for full activity, and the central histidine of the triad is absolutely essential for Ap3A hydrolase activity . This putative tumor suppressor is the first evidence for a connection between dinucleotide oligophosphate metabolism and tumorigenesis . Also, Fhit is the first HIT protein in which the histidine residues have been demonstrated by mutagenesis to be critical for function.

FEBS Lett, 1996 Sep 9, 393(1), 101 - 4
Activation of a recombinant membrane type 1-matrix metalloproteinase (MT1-MMP) by furin and its interaction with tissue inhibitor of metalloproteinases (TIMP)-2; Sato H et al.; Membrane type 1-matrix metalloproteinase (MT1-MMP) initiates the activation of the zymogen progelatinase A/ 72-kDa type IV collagenase by cleavage of the Asn66-Leu peptide bond . We previously pointed out that MT1-MMP possesses a unique amino acid sequence Arg-Arg-Lys-Arg111 which is a potential recognition sequence for furin-like proteases (Nature, 370 (1994) 61-65) . Here, using a recombinant MT1-MMP expressed in Escherichia coli we demonstrated that furin specifically cleaves MT1-MMP between Arg111-Tyr in vitro, which resulted in a stimulation of progelatinase A-activation function . Tissue inhibitor of metalloproteinases (TIMP)-2 inhibited activation of progelatinase A by forming a stable complex with activated MT1-MMP.

J Mol Biol, 1996 Sep 6, 261(5), 614 - 9
Autonomous and reversible folding of a soluble amino-terminally truncated segment of the mouse prion protein; Hornemann S et al.; Prion diseases are assumed to be caused by the infectious isoform, PrPsc, of a single cellular surface protein, PrPc . PrPsc is an insoluble form of PrPc and is believed to possess a different three-dimensional fold . It may propagate by causing PrPc to adopt its own infectious conformation by an unknown mechanism . Studies on folding and thermodynamic stability of prion proteins are essential for understanding the processes underlying the conversion from PrPc to PrPsc, but have so far been hampered by the low solubility of prion proteins in the absence of detergents . Here, we show that the amino-terminally truncated segment of mouse PrP comprising residues 121 to 231 is an autonomous folding unit . It consists predominantly of alpha-helical secondary structure and is soluble at high concentrations up to 1 mM in distilled water . PrP(121-231) undergoes a cooperative and completely reversible unfolding/refolding transition in the presence of guanidinium chloride with a free energy of folding of -22 kJ/mol at pH 7 . The intrinsic stability of segment 121-231 is not in accordance with present models of the structure of PrPc and PrPsc PrP(121-231) may represent the only part of PrPc with defined three-dimensional structure.

Cell, 1996 Sep 6, 86(5), 823 - 34
The RNA world of the nucleolus: two major families of small RNAs defined by different box elements with related functions; Balakin AG et al.; We have discovered that all known yeast and vertebrate small nucleolar RNAs (snoRNAs), except for the MRP/7-2 RNA, fall into two major classes . One class is defined by conserved boxes C and D and the other by a novel element: a consensus ACA triplet positioned 3 nt before the 3' end of the RNA . A role for the ACA box is snoRNA stability has been established by mutational analysis of a yeast ACA snoRNA (snR 11) . Full function of the box depends on the integrity of an adjacent upstream stem . All members of the yeast ACA family are associated with the GAR1 protein . Binding of this or another common small nucleolar ribonucleoprotein particle protein is predicted to be a critical entry point to snoRNA posttranscriptional life, including precise formation of the snoRNA 3' end.

Cell, 1996 Sep 6, 86(5), 719 - 29
Nitrosative stress: activation of the transcription factor OxyR; Hausladen A et al.; Hydrogen peroxide (H2O2) imposes an oxidative stress to Escherichia coli that is manifested by oxidation of glutathione and related redox-sensitive targets . OxyR is a thiol-containing transcriptional activator whose oxidation controls the expression of genes involved in H2O2 detoxification . Here we report that certain S-nitrosothiols (RSNOs) impose what we term a "nitrosative stress" to E . coli, evidenced by lowering of intracellular thiol and the transcriptional activation of OxyR by S-nitrosylation . This cellular and genetic response determines the metabolic fate of RSNOs and thereby contributes to bacterial rescue from stasis . Our studies reveal that signaling by S-nitrosylation can extend to the level of transcription and describe a metabolic pathway that constitutes an adaptation to nitrosative stress.

J Biol Chem, 1996 Sep 6, 271(36), 22256 - 61
Chaperonin-promoted post-translational membrane insertion of a multispanning membrane protein lactose permease; Bochkareva E et al.; Using an in vitro membrane-free translation system from Escherichia coli, it is shown that chaperonin GroEL added cotranslationally interacts with newly synthesized lactose permease (LacY), a polytopic membrane protein, thereby preventing aggregation . Subsequently, when the isolated GroEL-LacY complex is incubated with inverted membrane vesicles, the permease is inserted into the membrane in a MgATP-dependent manner . Post-translational membrane insertion is also observed when aggregation of newly synthesized LacY is prevented by addition of the nonionic detergent n-dodecyl-beta,D-maltoside during translation in place of GroEL . No membrane integration occurs with right-side-out vesicles, indicating that LacY interacts specifically only with the cytosolic face of the membrane . Ligand thiodigalactoside protection against alkylation of the Cys-148 residue in the permease shows proper post-translational insertion . Moreover, limited proteolysis of soluble LacY either complexed with GroEL or in detergent indicates that the newly synthesized protein assumes a conformation that is comparable to that of native, membrane-embedded permease prior to insertion into the membrane.

J Biol Chem, 1996 Sep 6, 271(36), 22196 - 202
Poly(3-hydroxybutyrate) is associated with specific proteins in the cytoplasm and membranes of Escherichia coli; Huang R et al.; Poly(3-hydroxybutyrate) (PHB) is well-known as a high molecular weight homopolymer of R-3-hydroxybutyrate which accumulates in storage granules within the cytosols of certain bacteria . Escherichia coli does not amass these granules; however, small amounts of low molecular weight PHB (<0.02% of dry weight) have been found in the plasma membranes in complexes with calcium polyphosphate; the complexes serve as voltage-activated calcium channels . Here we report that polyphosphate-complexed PHB is only a minor fraction of the polyester in E . coli . PHB comprises 0.36 to 0 . 55% of the dry weight of log-phase cells, depending on culture medium, and this amount increases by 15 to 20% when the cells are made genetically competent . The PHB is widely distributed throughout the cell, wherein it is primarily associated with proteins . The identity of protein-associated PHB was established by antibody reaction, chemical assay, and 1H NMR spectroscopy . As expected, the physical and chemical properties of protein-associated PHB were found to be considerably different from those of the bulk polymer or granule PHB, e.g . protein-PHB complexes are normally insoluble in chloroform, soluble in water and alkaline hypochlorite, and are converted to crotonic acid more slowly on heating in concentrated sulfuric acid . Our studies indicate that the majority of cellular PHB (over 80%) is located in cytoplasmic proteins, especially proteins of the ribosomal fraction . Western immunoblots, probed with polyclonal anti-PHB IgG, revealed a number of PHB-polypeptides having a wide range of molecular weights in all cell fractions . These results suggest that PHB is a fundamental constituent of cells that may have physiological functions in addition to facilitating ion transmembrane transport or serving as a carbon reserve.

J Biol Chem, 1996 Sep 6, 271(36), 22052 - 7
Cloning and characterization of a novel membrane-associated lymphocyte NAD:arginine ADP-ribosyltransferase; Okazaki IJ et al.; Mono-ADP-ribosylation is a post-translational modification of proteins in which the ADP-ribose moiety of NAD is transferred to proteins and is responsible for the toxicity of some bacterial toxins (e.g . cholera toxin and pertussis toxin) . NAD:arginine ADP-ribosyltransferases cloned from human and rabbit skeletal muscle and from mouse lymphoma (Yac-1) cells are glycosylphosphatidylinositol-anchored and have similar enzymatic and physical properties; transferases cloned from chicken heterophils and red cells have signal peptides and may be secreted . We report here the cloning and characterization of an ADP-ribosyltransferase (Yac-2), also from Yac-1 lymphoma cells, that differs in properties from the previously identified eukaryotic transferases . The nucleotide and deduced amino acid sequences of the Yac-1 and Yac-2 transferases are 58 and 33% identical, respectively . The Yac-2 protein is membrane-bound but, unlike the Yac-1 enzyme, appears not to be glycosylphosphatidylinositol-anchored . The Yac-1 and Yac-2 enzymes, expressed as glutathione S-transferase fusion proteins in Escherichia coli, were used to compare their ADP-ribosyltransferase and NAD glycohydrolase activities . Using agmatine as the ADP-ribose acceptor, the Yac-1 enzyme was predominantly an ADP-ribosyltransferase, whereas the transferase and NAD glycohydrolase activities of the recombinant Yac-2 protein were equivalent . The deduced amino acid sequence of the Yac-2 transferase contained consensus regions common to several bacterial toxin and mammalian transferases and NAD glycohydrolases, consistent with the hypothesis that there is a common mechanism of NAD binding and catalysis among ADP-ribosyltransferases.

J Biol Chem, 1996 Sep 6, 271(36), 21927 - 32
Structural topology of transmembrane helix 10 in the lactose permease of Escherichia coli; Goswitz VC et al.; In the lactose permease of Escherichia coli, transmembrane helix 10 has been shown to be functionally important . The structure of this helix has been examined in greater detail in this study . A total of 46 substitution and 8 insertional mutants were constructed and analyzed along the entire length of transmembrane helix 10 . The results identified amino acids that are tolerant of substitutions by a variety of amino acids . Since a number of these amino acids (Thr-320, Val-331, Phe-325, and Ile-317) are clustered in one region in a helical wheel projection of transmembrane helix 10, it seems likely that this face of helix 10 is interacting with the membrane . The channel lining domain is thought to consist of the helical face containing Glu-325, Leu-318, Leu-329, His-322, Val-315, Cys-333, Val-326, and Lys-319 based on the results here and from earlier findings . Deleterious mutations along this face tended to greatly increase the Km value for lactose transport with only minor effects on the Vmax . Analysis of insertional mutants revealed that perturbation of the spatial relationship between amino acids at the periplasmic edge is less deleterious than perturbation in the center of the helix or the cytoplasmic edge . Using all of the above information, a detailed structural topology of transmembrane helix 10 is proposed.

Brain Res Mol Brain Res, 1996 Sep 5, 41(1-2), 200 - 9
Inducible gene expression from defective herpes simplex virus vectors using the tetracycline-responsive promoter system; Ho DY et al.; Herpes simplex virus-based amplicon vectors have been used for gene transfer into cultured neurons and the adult CNS . Since constitutive expression of a foreign gene or overexpression of an endogenous gene may have deleterious effects, the ability to control temporal expression would be advantageous . To achieve inducible gene expression, we have incorporated the tetracycline-responsive promoter system into amplicon vectors and showed, both in vitro and in vivo, that expression can be modulated by tetracycline . Using the firefly luciferase as the reporter gene, maximal repression by tetracycline in hippocampal cultures was about 50-fold . Withdrawal of tetracycline derepressed gene expression, reaching maximal levels within 10-12 h . In contrast, addition of tetracycline to cultures without prior tetracycline exposure inhibited gene expression rapidly; luciferase activity was reduced to less than 8% within 24 h . In adult rat hippocampus, vectors expressing luciferase or the Escherichia coli lacZ were repressed by tetracycline 9- and 60-fold, respectively . Maximum gene expression from the vectors occurred 2-3 days post-infection and declined thereafter . Such decline impeded further induction of expression by withdrawing tetracycline . This study demonstrates the feasibility of incorporating a powerful inducible promoter system into HSV vectors . The development of such an inducible viral vector system for gene transfer into the adult CNS might prove to be of experimental and therapeutic value.

Biochim Biophys Acta, 1996 Sep 5, 1296(2), 159 - 66
Site-directed mutagenesis of rat hepatic hydroxysteroid sulfotransferases; Homma H et al.; Two cDNA clones of rat hepatic hydroxysteroid sulfotransferase (ST) (ST-40 and ST-20) were isolated and expressed in Escherichia coli cells . Several histidine residues in their coding regions are highly conserved in the ST superfamily, and histidine mutants were constructed by site-directed mutagenesis . The substitution of alanine or lysine for the histidine at position 98 in the ST-40 enzyme resulted in a loss of ST activities toward dehydroepiandrosterone (DHEA), androsterone (AD) and cortisol (CS) . The mutation of histidine 98 into alanine abolished the specific binding to 3'-phosphoadenosine 5'-phosphate agarose, suggesting that the residue is located at a critical position in the 3'-phosphoadenosine 5'-phosphosulfate (PAPS) binding site . In the ST-20 enzyme, the replacement of histidine 98 with alanine also resulted in the loss of ST activity toward its preferential substrate, CS . In the ST-40 enzyme, the mutation at histidine 256 into alanine markedly reduced CS-ST activity, but DHEA-ST activity was not changed . Furthermore, selective decrease in CS-ST activity was also observed in the alanine mutant at lysine 254 or at asparagine 255 of the ST-40 enzyme . Kinetic analysis on the ST-40 and its mutant at asparagine 255 indicated that the Km value for CS was significantly increased in the mutant without any change in the Km values for 3'-phosphoadenosine 5'-phosphosulfate and DHEA . Inhibition studies demonstrated that DHEA-ST activity was competitively inhibited by AD, but not by CS in the ST-40 enzyme, whereas triethylamine, a noncompetitive inhibitor of hydroxysteroid ST, inhibited DHEA-ST activity in the ST-40 enzyme but did not inhibit CS-ST activity in either ST-40 or ST-20 enzymes . These data provide evidence that DHEA and CS bind to different sites, which probably function in a different manner in the ST-40 enzyme.

Anal Biochem, 1996 Sep 5, 240(2), 289 - 97
The utility of FK506-binding protein as a fusion partner in scintillation proximity assays: application to SH2 domains; Sonatore LM et al.; Methodology has been developed which gives a specific measure of the interaction of an SH2 domain with a phosphopeptide ligand using scintillation proximity assay (SPA) technology . Recombinant SH2 domains were expressed from a T7 RNA polymerase-based vector in Escherichia coli as fusions to the C-terminus of the FK506-binding protein (FKBP) and purified from freeze-thaw lysates in high yield by affinity chromatography using immobilized phosphopeptides . For binding assays the phosphopeptide ligands were synthesized with a biotin tag and the FKBP fusion proteins were noncovalently radiolabeled with commercially available {3H}dihydroFK506 . Complexes of tritiated SH2 fusion protein and biotinyl-phosphopeptide were then captured on streptavidin-coated SPA beads and counted . The modular protocol is an equilibrium technique that does not employ washing steps or specialized radiochemical syntheses required in other binding assays . The utility of the assay has been demonstrated in an examination of the ligand specificity of the SH2 domains of the tyrosine kinases ZAP70, Syk, and Lck . The methodology is potentially generalizable to any receptor-ligand interaction in which one component can be expressed as a fusion partner with FKBP and the other component can be captured on a SPA bead.

Biochem Biophys Res Commun, 1996 Sep 4, 226(1), 237 - 41
Specific interaction of pRB with a rat genomic DNA fragment, REC11; Ozaki T et al.; The retinoblastoma gene product (pRB) has been known to contain a sequence-nonspecific DNA binding activity . It is unknown whether pRB can recognize and bind to specific DNA sequences . We have recently identified a rat genomic DNA fragment, termed REC11, which can interact with rat Cdc37-related protein (RCdc37) . In this study, we have found that pRB could interact with the REC11, DNA in a sequence-specific manner . A series of GST-RB deletion mutants was used in the gel shift assays to define the domain of pRB responsible for this interaction . GST-RB (385-611) and GST-RB (612-928) completely lost the binding activity, while GST-RB (555-682) retained an activity to associate with the REC11 DNA, indicating that the continuous spacer region of pRB might be important for this sequence-specific DNA binding activity.

Biochem Biophys Res Commun, 1996 Sep 4, 226(1), 42 - 50
High-level expression in Escherichia coli and purification of recombinant plant profilins: comparison of IgE-binding capacity and allergenic activity; Vrtala S et al.; Because of their structural similarity and ubiquitous distribution as actin binding proteins, plant profilins represent important cross-reactive allergens for almost 20% of patients suffering from Type I allergy to pollen and other plant products . The cDNAs coding for three birch profilin variants (Tyr44, Glu47, and Asn47), timothy grass profilin, and three tobacco profilin isoforms (ntprof1-3) were expressed at high levels in Escherichia coli as non-fusion proteins . The recombinant plant profilins were purified to homogeneity by poly (L-proline) affinity chromatography and showed comparable capacity to bind IgE-antibodies from profilin allergic patients . All recombinant plant profilins elicited dose-dependent histamine release from basophils of a profilin allergic patient and induced immediate type skin reactions . It is concluded that profilins from different plant species share IgE-epitopes and allergenic properties . Plant profilins therefore constitute a family of functional pan-allergens which may substitute each other for diagnosis and specific immunotherapy.

Proc Natl Acad Sci U S A, 1996 Sep 3, 93(18), 9742 - 7
Differential inhibition of HIV-1 preintegration complexes and purified integrase protein by small molecules; Farnet CM et al.; To replicate, HIV-1 must integrate a cDNA copy of the viral RNA genome into a chromosome of the host . The integration system is a promising target for antiretroviral agents, but to date no clinically useful integration inhibitors have been identified . Previous screens for integrase inhibitors have assayed inhibition of reactions containing HIV-1 integrase purified from an Escherichia coli expression system . Here we compare action of inhibitors in vitro on purified integrase and on subviral preintegration complexes (PICs) isolated from lymphoid cells infected with HIV-1 . We find that many inhibitors active against purified integrase are inactive against PICs . Using PIC assays as a primary screen, we have identified three new anthraquinone inhibitors active against PICs and also against purified integrase . We propose that PIC assays are the closest in vitro match to integration in vivo and, as such, are particularly appropriate for identifying promising integration inhibitors.

Proc Natl Acad Sci U S A, 1996 Sep 3, 93(18), 9670 - 5
Random mutagenesis of Thermus aquaticus DNA polymerase I: concordance of immutable sites in vivo with the crystal structure; Suzuki M et al.; Expression of Thermus aquaticus (Taq) DNA polymerase I (pol I) in Escherichia, coli complements the growth defect caused by a temperature-sensitive mutation in the host pol I . We replaced the nucleotide sequence encoding amino acids 659-671 of the O-helix of Taq DNA pol I, corresponding to the substrate binding site, with an oligonucleotide containing random nucleotides . Functional Taq pol I mutants were selected based on colony formation at the nonpermissive temperature . By using a library with 9% random substitutions at each of 39 positions, we identified 61 active Taq pol I mutants, each of which contained from one to four amino acid substitutions . Some amino acids, such as alanine-661 and threonine-664, were tolerant of several or even many diverse replacements . In contrast, no replacements or only conservative replacements were identified at arginine-659, lysine-663, and tyrosine-671 . By using a library with totally random nucleotides at five different codons (arginine-659, arginine-660, lysine-663, phenylalanine-667, and glycine-668), we confirmed that arginine-659 and lysine-663 were immutable, and observed that only tyrosine substituted for phenylalanine-667 . The two immutable residues and the two residues that tolerate only highly conservative replacements lie on the side of O-helix facing the incoming deoxynucleoside triphosphate, as determined by x-ray analysis . Thus, we offer a new approach to assess concordance of the active conformation of an enzyme, as interpreted from the crystal structure, with the active conformation inferred from in vivo function.

Proc Natl Acad Sci U S A, 1996 Sep 3, 93(18), 9612 - 7
Split invertase polypeptides form functional complexes in the yeast periplasm in vivo; Schonberger O et al.; The assembly of functional proteins from fragments in vivo has been recently described for several proteins, including the secreted maltose binding protein in Escherichia coli . Here we demonstrate for the first time that split gene products can function within the eukaryotic secretory system . Saccharomyces cerevisiae strains able to use sucrose produce the enzyme invertase, which is targeted by a signal peptide to the central secretory pathway and the periplasmic space . Using this enzyme as a model we find the following: (i) Polypeptide fragments of invertase, each containing a signal peptide, are independently translocated into the endoplasmic reticulum (ER) are modified by glycosylation, and travel the entire secretory pathway reaching the yeast periplasm . (ii) Simultaneous expression of independently translated and translocated overlapping fragments of invertase leads to the formation of an enzymatically active complex, whereas individually expressed fragments exhibit no activity . (iii) An active invertase complex is assembled in the ER, is targeted to the yeast periplasm, and is biologically functional, as judged by its ability to facilitate growth on sucrose as a single carbon source . These observation are discussed in relation to protein folding and assembly in the ER and to the trafficking of proteins through the secretory pathway.

Proc Natl Acad Sci U S A, 1996 Sep 3, 93(18), 9477 - 82
An RNA topoisomerase; Wang H et al.; A synthetic strand of RNA has been designed so that it can adopt two different topological states (a circle and a trefoil knot) when ligated into a cyclic molecule . The RNA knot and circle have been characterized by their behavior in gel electrophoresis and sedimentation experiments . This system allows one to assay for the existence of an RNA topoisomerase, because the two RNA molecules can be inter-converted only by a strand passage event . We find that the interconversion of these two species can be catalyzed by Escherichia coli DNA topoisomerase III, indicating that this enzyme can act as an RNA topoisomerase . The conversion of circles to knots is accompanied by a small amount of RNA catenane generation . These findings suggest that strand passage must be considered a potential component of the folding and modification of RNA structures.

Proc Natl Acad Sci U S A, 1996 Sep 3, 93(18), 9449 - 53
Glutathione-mediated destabilization in vitro of {2Fe-2S} centers in the SoxR regulatory protein; Ding H et al.; SoxR is a transcription factor that governs a global defense against the oxidative stress caused by nitric oxide or excess superoxide in Escherichia coli . SoxR is a homodimer containing a pair of {2Fe-2S} clusters essential for its transcriptional activity, and changes in the stability of these metal centers could contribute to the activation or inactivation of SoxR in vivo . Herein we show that reduced glutathione (GSH) in aerobic solution disrupts the SoxR {2Fe-2S} clusters, releasing Fe from the protein and eliminating SoxR transcriptional activity . This disassembly process evidently involves oxygen-derived free radicals . The loss of {2Fe-2S} clusters does not occur in anaerobic solution and is blocked in aerobic solution by the addition of superoxide dismutase and catalase . Although H2O2 or xanthine oxidase and hypoxanthine (to generate superoxide) were insufficient on their own to cause {2Fe-2S} cluster loss, they did accelerate the rate of disassembly after GSH addition . Oxidized GSH alone was ineffective in disrupting the clusters, but the rate of {2Fe-2S} cluster disassembly was maximal when reduced and oxidized GSH were present at a ratio of approximately 1:3, which suggests the critical involvement of a GSH-based free radical in the disassembly process . Such a reaction might occur in vivo: we found that the induction by paraquat of SoxR-dependent soxS transcription was much higher in a GSH-deficient E . coli strain than in its GSH-containing parent . The results imply that GSH may play a significant role during the deactivation process of SoxR in vivo . Ironically, superoxide production seems both to activate SoxR and, in the GSH-dependent disassembly process, to switch off this transcription factor.

Proc Natl Acad Sci U S A, 1996 Sep 3, 93(18), 9431 - 6
The mode of action and the structure of a herbicide in complex with its target: binding of activated hydantocidin to the feedback regulation site of adenylosuccinate synthetase; Fonne-Pfister R et al.; (+)-Hydantocidin, a recently discovered natural spironucleoside with potent herbicidal activity, is shown to be a proherbicide that, after phosphorylation at the 5' position, inhibits adenylosuccinate synthetase, an enzyme involved in de novo purine synthesis . The mode of binding of hydantocidin 5'-monophosphate to the target enzyme was analyzed by determining the crystal structure of the enzyme-inhibitor complex at 2.6-A resolution . It was found that adenylosuccinate synthetase binds the phosphorylated compound in the same fashion as it does adenosine 5'-monophosphate, the natural feedback regulator of this enzyme . This work provides the first crystal structure of a herbicide-target complex reported to date.

Proc Natl Acad Sci U S A, 1996 Sep 3, 93(18), 9425 - 30
A thermodynamic coupling mechanism for GroEL-mediated unfolding; Walter S et al.; Chaperonins prevent the aggregation of partially folded or misfolded forms of a protein and, thus, keep it competent for productive folding . It was suggested that GroEL, the chaperonin of Escherichia coli, exerts this function 1 unfolding such intermediates, presumably in a catalytic fashion . We investigated the kinetic mechanism of GroEL-induced protein unfolding by using a reduced and carbamidomethylated variant of RNase T1, RCAM-T1, as a substrate . RCAM-T1 cannot fold to completion, because the two disulfide bonds are missing, and it is, thus, a good model for long-lived folding intermediates . RCAM-T1 unfolds when GroEL is added, but GroEL does not change the microscopic rate constant of unfolding, ruling out that it catalyzes unfolding . GroEL unfolds RCAM-T1 because it binds with high affinity to the unfolded form of the protein and thereby shifts the overall equilibrium toward the unfolded state . GroEL can unfold a partially folded or misfolded intermediate by this thermodynamic coupling mechanism when the Gibbs free energy of the binding to GroEL is larger than the conformational stability of the intermediate and when the rate of its unfolding is high.

Proc Natl Acad Sci U S A, 1996 Sep 3, 93(18), 9408 - 13
Distortion in the spacer region of Pm during activation of middle transcription of phage Mu; Artsimovitch I et al.; Transcription from the middle promoter, Pm, of phage Mu is initiated by Escherichia coli RNA polymerase holoenzyme (E sigma 70; RNAP) and the phage-encoded activator, Mor . Point mutations in the spacer region between the -10 hexamer and the Mor binding site result in changes of promoter activity in vivo . These mutations are located at the junction between a rigid T-tract and adjacent, potentially deformable G + C-rich DNA segment, suggesting that deformation of the spacer region may play a role in the transcriptional activation of Pm . This prediction was tested by using dimethyl sulfate and potassium permanganate footprinting analyses . Helical distortion involving strand separation was detected at positions -32 to -34, close to the predicted interface between Mor and RNAP . Promoter mutants in which this distortion was not detected exhibited a lack of melting in the -12 to -1 region and reduced promoter activity in vivo . We propose that complexes containing the distortion represent stressed intermediates rather than stable open complexes and thus can be envisaged as a transition state in the kinetic pathway of Pm activation in which stored torsional energy could be used to facilitate melting around the transcription start point.

Biochemistry, 1996 Sep 3, 35(35), 11512 - 6
Effect on DNA topology by DnaA protein, the initiation factor of chromosomal DNA replication in Escherichia coli; Mizushima T et al.; We examined effects on supercoiled DNA topology of DnaA protein, the initiator protein of chromosomal DNA replication in Escherichia coli . The activity was identified in an analysis of plasmid DNA incubated with DnaA protein and DNA topoisomerase I . In Superose 12 gel filtration chromatography, the activity coeluted with DnaA protein . Incubation of DnaA protein with DNA at temperatures over 24 degrees C was required for this activity, which was observed with either oriC plasmid or the replicative form I of phi X174 with no DnaA box . As binding of ATP or ADP to DnaA protein prevented the activity of DnaA protein on DNA topology, binding of the adenine nucleotide may regulate the activity.

Biochemistry, 1996 Sep 3, 35(35), 11487 - 92
1,N6-ethenodeoxyadenosine, a DNA adduct highly mutagenic in mammalian cells; Pandya GA et al.; 1,N6-Ethenodeoxyadenosine (epsilon dA) is one of four exocyclic DNA adducts produced by chloroethylene oxide and chloroacetaldehyde, reactive metabolites of vinyl chloride, a human carcinogen . epsilon dA has also been detected in DNA of the liver of humans and untreated animals, suggesting its formation from endogenous sources . The mutagenic potential of epsilon dA was studied using a single-stranded shuttle vector system in several E . coli strains and in simian kidney cells (COS7) . This vector system enables quantitative analysis of translesional synthesis past a site-specifically placed DNA adduct in both hosts owing to the lack of the complementary strand . In experiments with five strains of E . coli, a very limited number of targeted mutations (one epsilon dA-->T, one epsilon dA-->dC, and two epsilon dA-->single base deletion) were observed among 756 transformants in hosts preirradiated with UV; no targeted mutations were observed among 563 transformants in nonirradiated hosts . These results indicate that nonmutagenic base pairings of epsilon dA:T are the almost exclusive events in E . coli . In COS7 cells, the frequency of targeted mutations was 70%, consisting of epsilon dA-->dG (63%), epsilon dA-->T (6%), and epsilon dA-->dC (1%), indicating that the insertion of dCMP opposite the adduct is predominant . When compared with the results for 3,N4-ethenodeoxycytidine (epsilon dC), which was studied previously in the same system {Moriya et al . (1994) Proc . Natl . Acad . Sci . U.S.A . 91, 11899-11903}, the results of this study indicate that the intrinsic mutagenic potency of epsilon dA is comparable to that of epsilon dC in mammalian cells.

Biochemistry, 1996 Sep 3, 35(35), 11435 - 46
Alanine point-mutations in the reactive region of bovine pancreatic trypsin inhibitor: effects on the kinetics and thermodynamics of binding to beta-trypsin and alpha-chymotrypsin; Castro MJ et al.; In an effort to relate structural, kinetic, and thermodynamic features in a model macromolecular recognition process, the amino acid residues in the reactive surface of bovine pancreatic trypsin inhibitor (BPTI) and surrounding residues were substituted individually by alanine, and the effects of the point-mutations on the kinetics and thermodynamics of inhibition by BPTI toward trypsin and chymotrypsin were investigated . Fifteen alanine mutants were produced . The majority of the BPTI mutants exhibited a binding affinity similar to that of the wild-type protein . The exceptions were the primary specificity site (PI) mutant and those mutants that seem to have nonlocal perturbations of structure, as revealed by circular dichroism and thermostability measurements . The mutation at the P1 site caused a reduction in the binding free energy of 10 and 1.8 kcal mol-1 for trypsin and chymotrypsin, respectively . The losses in binding affinity were determined almost exclusively by an increase in the dissociation rate constant . However, the rate of association of the P1 mutant, Lys-15-Ala, with trypsin was also drastically reduced (> 200-fold) . Calorimetric measurements of the heats of binding for the association of chymotrypsin with the wild-type inhibitor and its alanine mutants allowed determination of the relative contributions of the changes in enthalpy and entropy to the free energy of binding . Compensatory changes in the two parameters were observed in several cases, which were attributed to desolvation effects at the binding interface.

Biochemistry, 1996 Sep 3, 35(35), 11369 - 78
Conformational stability of the Escherichia coli HPr protein: test of the linear extrapolation method and a thermodynamic characterization of cold denaturation; Nicholson EM et al.; The conformational stability of the histidine-containing phosphocarrier protein (HPr) from Escherichia coli has been determined using a combination of thermal unfolding and urea denaturation experiments . The analysis of the denaturation data provides a measure of the changes in conformational free energy, enthalpy, entropy, and heat capacity that accompany the equilibrium folding of HPr over a wide range of temperature and urea concentrations . In moderate concentrations of urea, HPr undergoes both high- and low-temperature unfolding, allowing for a reliable determination of the change in heat capacity for the conformational transition . The data are consistent with the linear free energy relationship commonly employed to analyze protein denaturation data, even over a relatively large temperature and urea concentration range . Furthermore, we find that a temperature-independent delta Cp is adequate to describe HPr stability over the accessible temperature range . Finally, our data allow us to evaluate the energetics of the urea-protein interaction . For HPr, the changes in excess enthalpy and entropy of the denaturant-protein interaction(s) make only minor contributions to the observed delta H and delta S terms, presumably due in some part to the small size of the HPr protein.

Biochemistry, 1996 Sep 3, 35(35), 11286 - 92
Solution structure of the IIB domain of the glucose transporter of Escherichia coli; Eberstadt M et al.; The structure of the IIBGlc domain of the Escherichia coli transporter for glucose was determined by multidimensional heteronuclear NMR . The glucose transporter (IICBGlc) belongs to the bacterial phosphotransferase system . It mediates uptake with concomittant phosphorylation of glucose . The N-terminal IICGlc domain spans the membrane, the C-terminal IIBGlc domain (residues 386-477) contains the phosphorylation site, Cys421 . The structure of the subclonal IIB domain was determined based on 927 conformational constraints, including 744 NOE derived upper bounds, 43 constraints of ranges of dihedral angles based on measurements of vicinal coupling constants, and 70 upper and lower bound constraints associated with 35 hydrogen bonds . The distance geometry interpretation of the NMR data is based on the previously published sequence-specific 1H, 15N, and 13C resonance assignments {Golic Grdadolnik et al . (1994) Eur . J . Biochem . 219, 945-952} . The sequence of the secondary structure elements of IIB is alpha 1 beta 1 beta 2 alpha 2 beta 3 beta 4 alpha 3 . The basic fold consists of a split alpha/beta-sandwich composed of an antiparallel sheet with strand order beta 1 beta 2 beta 4 beta 3 and three alpha-helices superimposed onto one side of the sheet . The hydrophobic helix alpha 1 is packed against helices alpha 2, alpha 3, and the beta-sheet . The phosphorylation site (Cys421) is at the end of beta 1 on the solvent-exposed face of the sheet surrounded by Asp419, Thr423 Arg424, Arg426, and Gln456 which are invariant in 15 homologous IIB domains from other PTS transporters.

EMBO J, 1996 Sep 2, 15(17), 4767 - 74
Intrinsic termination of T7 RNA polymerase mediated by either RNA or DNA; Hartvig L et al.; Intrinsic termination of T7 RNA polymerase transcription occurs at different signals in vitro . One type of signal is similar to that mediating factor-independent termination of Escherichia coli RNA polymerase, whereas the other type does not involve RNA hairpin formation . By examining the termination behaviour of T7 RNA polymerase at the E.coli rrnB operon t1 terminator, at the T7-t(phi) terminator, at the human preproparathyroid hormone gene terminator on both single- and double-stranded templates, and in the presence of GTP or ITP during transcription, we show that the termination event can be mediated by either RNA or DNA structural features . Moreover, by using co-transcriptional probing with potassium permanganate, we present evidence for the presence of transcription-induced hyperreactive thymidines on the non-template strand in the DNA-mediated event, and a putative sequence motif is identified . We conclude that intrinsic termination of T7 RNA polymerase transcription in vitro can be mediated either by a hairpin in the nascent RNA or by a sequence motif including hyperreactive thymidines in the non-template DNA strand.

EMBO J, 1996 Sep 2, 15(17), 4734 - 9
Ribosome-initiator tRNA complex as an intermediate in translation initiation in Escherichia coli revealed by use of mutant initiator tRNAs and specialized ribosomes; Wu XQ et al.; For functional studies of mutant Escherichia coli initiator tRNAs in vivo, we previously described a strategy based on the use of tRNA genes carrying an anticodon sequence change from CAU to CUA along with a mutant chloramphenicol acetyltransferase (CAT) gene carrying an initiation codon change from AUG to UAG . Surprisingly, under conditions where the mutant initiator tRNA is optimally active, the CAT gene with the UAG initiation codon produced more CAT protein (3- to 9-fold more depending on the conditions) than the wild-type CAT gene . Here we show that two new mutant CAT genes having GUC and AUC initiation codons also produce more of the CAT protein in the presence of the corresponding mutant initiator tRNAs . These results are most easily understood if assembly of the 30S ribosome-initiator tRNA-mRNA initiation complex in vivo proceeds with the 30S ribosome binding first to the initiator tRNA and then to the mRNA . In cells overproducing the mutant initiator tRNAs, most ribosomes would carry the mutant initiator tRNA and these ribosomes would select the mutant CAT mRNA over the other mRNAs.

Carbohydr Res, 1996 Sep 2, 290(2), 209 - 16
A direct enzymatic synthesis of beta-D-galactopyranosyl-D-xylopyranosides and their use to evaluate rat intestinal lactase activity in vivo; Aragon JJ et al.; By enzymatic beta-D-galactosylation of D-xylose a mixture of 4-, 3-, and 2-O-beta-D-galactopyranosyl-D-xyloses (1, 4, and 7, respectively) was obtained in 50% isolated yield . Disaccharides 1, 4, and 7 are substrates of intestinal lactase isolated from lamb small intestine with K(m) values of 250.0, 4.5, and 14.0 mM, respectively . The mixture was used to monitor the normal decline in lactase activity in rats that takes place after weaning . The data obtained by this method correlated with the levels of intestinal lactase activity in the same animals after being sacrificed.

Mutat Res, 1996 Sep 2, 364(1), 43 - 9
UV-induced mutations of supF gene on a shuttle vector plasmid in p53-deficient mouse cells are qualitatively different from those in wild-type cells; Ishizaki K et al.; To verify the genetic instability of p53-deficient cells, UV-induced mutation of the supF gene on a shuttle vector was analyzed . UV-irradiated or non-irradiated shuttle vector plasmid carrying the supF gene as a target of mutation (pYZ289) was introduced into p53-deficient and p53-proficient mouse embryonic fibroblasts, and then the plasmid DNA replicated in mouse cells was recovered . Survival of UV-irradiated plasmid was almost equivalent in both p53-deficient and p53-proficient cells . The frequencies of UV-induced mutation of the supF gene were also the same in both types of cells . However, the distributions of base change mutations in the supF sequence were different between p53-deficient and p53-proficient cells; especially the locations of tandem CpC to TpT changes exhibited a marked difference . Since DNA repair activities of these two types of cell were almost the same, these qualitative differences in UV-induced mutations were probably caused by as yet unidentified differences in other than DNA repair activity.

FEBS Lett, 1996 Sep 2, 392(3), 285 - 8
Site-directed mutagenesis of Flaveria trinervia phosphoenolpyruvate carboxylase: Arg450 and Arg767 are essential for catalytic activity and Lys829 affects substrate binding; Gao Y et al.; Phosphoenolpyruvate carboxylases (PEPC) of all known sequences contain 11 conserved arginine and two lysine residues located in highly conservative regions . Previous chemical modifications show that arginine and lysine residues are essential for catalytic activity . Three conserved residues, Arg450, Arg767 and Lys829, in PEPC of Flaveria trinervia were converted to glycine . All three mutant PEPC proteins were similarly expressed in Escherichia coli . However, mutant Gly450 and Gly767 PEPCs had no catalytic activity and Gly829 PEPC showed increased Km for PEP and Mg2+ . It seems that Arg450 and Arg767 are essential for PEPC function while Lys829 might be associated with PEP and/or Mg2+ binding domain.

FEBS Lett, 1996 Sep 2, 392(3), 281 - 4
Quo vadis photorespiration: a tale of two aldolases; Hixon M et al.; An O2-consuming side reaction of D-ribulose 1,5-bisphosphate carboxylase causes photorespiration in plants . This reaction may be an inevitable consequence of the enzyme's inability to protect its ene-diolate reaction intermediate from O2, a notion that is supported by the failure of persistent efforts to eliminate selectively its oxygenase activity by genetic manipulation . We have examined two a1dolases with similar ene-diolate intermediates, L-rhamnulose 1-phosphate aldolase and L-fuculose 1-phosphate aldolase . The former enzyme has an oxygenase activity, while the latter does not, suggesting that the reaction with O2 is not inevitable.

FEBS Lett, 1996 Sep 2, 392(3), 259 - 62
Expression of ribonucleolytic toxin restrictocin in Escherichia coli: purification and characterization; Rathore D et al.; Restrictocin is a toxin produced by the fungus Aspergillus restritus . The DNA coding for restrictocin was isolated from the host by polymerase chain reaction and cloned into a T7 promoter-based expression vector . The protein was overproduced in Escherichia coli and remained insoluble in the cell in the form of inclusion bodies . Recombinant restrictocin was purified in large amounts, by a simple denaturation-renaturation protocol involving a redox system, with typical yields of 45 mg/l of original culture . Restrictocin could be secreted into the bacterial medium using ompA, pelB and LTB signal sequences . Among the three signal sequences, ompA was found to be the most efficient in secreting the recombinant protein . The protein secreted into the extracellular medium was properly processed as evident by the amino-terminal sequencing . Recombinant restrictocin was readily purified to homogeneity from either the medium or inclusion bodies by simple chromatographic techniques and was found to be functionally as active as the native fungal protein in inhibiting the eukaryotic translation.

Cell Stress Chaperones, 1996 Sep, 1(3), 177 - 87
Identification of the C-terminal region of 70 kDa heat shock protein from Leishmania (Viannia) braziliensis as a target for the humoral immune response; Amorim AG et al.; A Leishmania (Viannia) braziliensis (Lb) promastigote cDNA library in lambda gt11 was screened with patients' sera with the aim of identifying antigens specifically related to mucocutaneous leishmaniasis (MCL) . One of the clones isolated, 133P, consistently reacted with MCL sera; it was sequenced and found to encode the C-terminal three-quarters of a protein belonging to the highly conserved Hsp70 family . Since Hsp70 proteins from different species tend to be less conserved through the C-terminal end, it was predicted that this region would be more antigenic and was likely to bear the discriminatory epitopes . In order to test this hypothesis, the N- and C-terminal halves of the polypeptide encoded by 133P, 133P-N and 133P-C, respectively, were expressed in Escherichia coli . Immunoblotting analysis demonstrated that 133P-C reacted more strongly with a pool of MCL sera than 133P-N, and both recombinant proteins reacted faintly with pools of cutaneous (CL) and visceral (VL) leishmaniasis sera . These results confirmed the predicted epitope location in the C-terminal region . The 133P-C fragment was also expressed as a fusion protein with glutathione-S-transferase (GST-133P-C), affinity-purified with glutathione-agarose and tested by ELISA with individual sera . From 46 Lb-infected patients, 41 sera (89%) were positive, no cross-reactivity was observed with healthy, Trypanosoma cruzior L . amazonensis-infected individuals . Despite a relatively high cross-reactivity with VL sera, the enhanced humoral response of MCL as compared with CL patients might be interesting for studies on disease aggravation.

J Mol Recognit, 1996 Sep-Dec, 9(5-6), 683 - 90
Oriented immobilization of restriction endonuclease EcoRI; Bircakova M et al.; Two activated matrices have been developed to determine whether immobilization chemistry can be used to orient proteins on a support . Restriction endonuclease EcoRI from Escherichia coli RY13 (E.C.3.1.23.13) was used as a model in these studies . Thiol-activated Sephadex G-10 was used to couple the EcoRI endonuclease through its free sulfhydryl, while amino-activated Sephadex G-10 was used to couple it randomly through its free carboxyl groups . To determine whether the enzyme was immobilized randomly or specifically, both lower and higher molecular weight substrates were used . The polymerase chain reaction amplified multiplied cloning site region of pBluescript KS obtained using T3 and T7 primers was considered as the small substrate . The plasmid SP64 containing firefly luciferase gene was the large substrate . Immobilized EcoRI preparations were characterized with respect to repeated usage and storage stability . The EcoRI immobilized on thiopropyl-Sepharose 4B could be stored for over 14 days at 4 degrees C without observable loss of activity . In an independent experiment the same gel was used thrice repeatedly without any discernible loss of activity.

J Mol Recognit, 1996 Sep-Dec, 9(5-6), 543 - 8
Dihydrofolate reductase synthesis in the presence of immobilized methotrexate . An approach to a continuous cell-free protein synthesis system; Marszal E et al.; Dihydrofolate reductase was synthesized in a batch system in the presence of the affinity ligand methotrexate, bound to various matrices . Two types of gel were used: commercial methotrexate-agarose with pores inaccessible for translation machinery and methotrexate-POROS with pores easily accessible for translation reaction mixture components . The transcription/translation reaction was not inhibited by either the immobilized methotrexate or the matrix . The enzyme was synthesized with a high yield and could simultaneously be removed from the reaction mixture by the affinity matrix during the synthesis . With methotrexate-POROS present the reaction probably proceeded mainly in the pores of the gel . Kinetic limitations to the reaction in the presence of the gel were not observed . Active dihydrofolate reductase was eluted from methotrexate-POROS . The activity recovered was higher than dihydrofolate reductase activity synthesized in free solution system . The influence of the presence of immobilized methotrexate on dihydrofolate reductase synthesis will be further studied in a novel type of a continuous protein synthesis system.

J Mol Recognit, 1996 Sep-Dec, 9(5-6), 524 - 7
Single nucleotide modulation of uridine to pseudouridine rearrangement in transfer RNA catalyzed by pseudouridine synthase I; Chihade JW et al.; E . coli pseudouridine synthase I (PSUI) catalyzes the rearrangement of uridine residues in positions 38, 39 and 40 of tRNA transcripts to pseudouridine . These positions are located in the anticodon stem-loop of the tRNA molecule . Fourteen different E . coli tRNAs are substrates for the enzyme, whereas four other tRNAs which contain uridine in position 38 are not . Investigations were focused on the basis of enzyme differentiation between substrate and non-substrate tRNAs . Comparison of modification reactions with mutant and wild-type tRNA transcripts demonstrates that the presence of a G36 residue modulates modification by PSUI at position 38 . In addition to local sequence effects, steady-state kinetic analyses suggest the existence of other recognition elements distinct from the immediate vicinity of modification.

J Mol Recognit, 1996 Sep-Dec, 9(5-6), 468 - 73
Binding of Escherichia coli LexA repressor to the RecA operator; Shaner SL et al.; Equilibrium binding of Escherichia coli LexA repressor to the recA operator was studied by the polyacrylamide gel mobility shift assay as a function of solution conditions . In the presence of NaCl at 20 degrees C, there was a significant salt dependence in binding to the recA operator, typical for protein-nucleic acid interactions with some electrostatic contribution to the binding free energy . In preliminary experiments in which the anion of the Na+ salt was changed from chloride to fluoride, little change was found with anion identity . This indicates that the salt effect on the binding interaction arises solely from the polyelectrolyte effect, not from anion binding or release by the protein upon complex formation . Increasing the temperature to 37 degrees C changed the binding affinity for complex formation at any given salt concentration and resulted in a change in the sensitivity of complex formation to NaCl concentration . Quantitative analysis of the data to obtain equilibrium binding constants is discussed.

Genes Cells, 1996 Sep, 1(9), 829 - 41
Recognition mechanisms of the minus-strand origin of phage f1 by Escherichia coli RNA polymerase; Higashitani N et al.; BACKGROUND: The primer RNA for the synthesis of the minus strand of filamentous coliphages is produced by host RNA polymerase at a specific site on the plus strand template . The mechanism used by the enzyme in recognizing the origin is unknown, but minus strand replication requires the holoenzyme form of RNA polymerase . The origin contains two inverted repeats which can form hairpins . RESULTS: When the origin sequence is drawn arranging the two hairpins horizontally, it resembles a stretch of a mostly double-stranded molecule . The nucleotides protected from nucleases by the holoenzyme were found to be located in two regions on this drawing: one around the 35th nucleotide upstream of the RNA start site, and the other ranging from the 10th nucleotide upstream to the 10th downstream, of the start site . The core enzyme did not show any protection . Mutational analyses of the origin indicated that the base-paired structure in the former region was important for origin activity . In the region around the 10th nucleotide upstream of the start site, specific base(s) in the non-template strand were required for origin activity, while the base sequence of the template strand was irrelevant . CONCLUSIONS: It is likely that the recognition mechanism of the origin by RNA polymerase shares common features with that of transcriptional promoters.

Biol Chem, 1996 Sep, 377(9), 555 - 61
The recombinant dehydrin-like desiccation stress protein from the resurrection plant Craterostigma plantagineum displays no defined three-dimensional structure in its native state; Lisse T et al.; Dehydration stress in the drought-tolerant resurrection plant Craterostigma plantagineum is accompanied by the accumulation of a large number of desiccation stress proteins (Dsp) . One abundant class of these is represented by the dehydrin-related Dsp16 protein which contains 15 amino acid conserved lysine-rich repeats and a stretch of eight serine residues providing extremely hydrophilic characteristics . Recombinant Dsp16 from Craterostigma plantagineum has been cloned and expressed in Escherichia coli . The protein was purified and characterized regarding its physicochemical properties . Irrespective of successful crystallization experiments, dilute aqueous buffer solutions do not display a well-defined three-dimensional structure in terms of the canonical secondary structural elements . 1H-NMR (nuclear magnetic resonance) spectra in aqueous solution are characterized by a small chemical shift dispersion typical for an unfolded protein; however, the observed line-widths are not typical for a highly mobile random coil structure . Instead they indicate an equilibrium between conformational states with preferentially extended substructures . As a consequence of its loose structure, Dsp16 is extremely sensitive towards proteolysis unless its structure is stabilized by structure-making additives such as trifluoroethanol . Denaturants such as guanidinium chloride do not induce cooperative structural transitions . pH-dependent fluorescence changes reflect protonation/deprotonation rather than conformational changes . Sedimentation/diffusion experiments confirm the predicted molecular mass of 16 kDa . Due to the high serine/threonine content and its loose structure, Dsp16 is accessible to phosphorylation, supporting the idea that in situ the structurally relatively undefined protein may be involved in both water binding and phosphorylation.

Biol Chem, 1996 Sep, 377(9), 549 - 54
Protease Ti (Clp), a multi-component ATP-dependent protease in Escherichia coli; Chung CH et al.; The ATP-dependent protease Ti(Clp) consists of two different multimeric components: ClpA containing ATP-cleaving sites and ClpP, with serine active sites for proteolysis . Here we summarize the most recent results on the structure and function of protease Ti . (1) The clpA gene has dual translational initiation sites and therefore encodes two polypeptides with sizes of 84 and 65 kDa . The abbreviated form of ClpA may play an important role in regulation of the ATP-dependent proteolysis, since it inhibits the ability of the 84-kDa ClpA in supporting the ClpP-mediated protein breakdown and the autodegradation of the 84-kDa ClpA . (2) ClpA contains two highly conserved sequences for ATP-binding: the first site is essential for oligomerization and the second site is responsible for ATP hydrolysis . (3) ATP hydrolysis by ClpA is required not only for assembly of the ClpA/ClpP complex but also for its rapid dissociation.

Somat Cell Mol Genet, 1996 Sep, 22(5), 383 - 92
Genes transfected into embryonal carcinoma stem cells are both lost and inactivated at high frequency; Schmidt-Kastner PK et al.; Embryonal carcinoma (EC) cells can be efficiently transfected with cloned DNAs but there is a strong tendency for expression from transfected genes to be lost from stably transformed cells . To investigate the mechanism responsible for this loss of expression, we transfected P19 EC cells with a gene encoding the E . coli beta-galactosidase and examined expression of this gene in clonal populations of cells . Cells that carry and express the beta-galactosidase gene give rise to cells that do not express at a rate of about 0.02 events per cell per cell division . These non-expressing cells were of two types, some had lost the transfected genes while others had inactivated them . In those cells that retained but inactivated the transfected genes, the inactive state was stable and suppression was at the level of transcription initiation but not associated with increased DNA methylation . Because transfected DNAs integrate into the genome as tandem arrays, the gene loss and inactivation seen in EC cells may be analogous to the repeat-induced gene inactivation seen in lower eukaryotes.

Radiats Biol Radioecol, 1996 Sep-Oct, 36(5), 731 - 3
{Radiation protective effects of laser irradiation with wavelength 532 nm}; Voskanian KSh et al.; The combined effect of 532 nm laser radiation and alpha-particles on survival of Escherichia coli (AB 1157) has been investigated . The sensitivity of cells to alpha-particles was decreased by pre- and post-irradiation with laser.

Biofizika, 1996 Sep-Oct, 41(5), 1075 - 81
{Electron spin resonance determination of copper binding site on Escherichia coli cell membrane}; Volodina LA et al.; The electron-spin relaxation of Escherichia coli cytoplasmic membrane strong binding Cu-centers was investigated by means of the microwave power saturation of the electronic spin resonance signal . It has been established, that copper centres in the strong binding sites of the cytoplasmic membrane E.coli may be represented in the following way: 1) isolate copper complexes with small speed of the spin-lattice relaxation; 2) isolate copper complexes with increased speed of spin-lattice relaxation by means of interaction with rapidly relaxation centres; 3) dipol-binding clasters; 4) ESR-nondetectable at T = 40 K, exchange-binding clasters, which cause increasing of the spin-lattice relaxation speed for isolate copper complexes.

Biofizika, 1996 Sep-Oct, 41(5), 1033 - 7
{Plasmid DNA strand breaks induced by laser irradiation of 193 nm wavelength}; Gurzadian GG et al.; DNA of plasmid pBR322 irradiated with laser at a wavelength of 193 mm was treated with an extract containing proteins from E.coli K12 AB1157 (wild-type) . The enzymes were found to produce single- and double-strand DNA breaks, which was interpreted as a transformation of a portion of cyclobutane pyrimidine dimers and (6-4) photoproducts into nonrepairable single-strand DNA breaks . The products resulted from ionization of DNA, in particular, single-strand breaks, transform to double-strand breaks . A comparison of these data with the data on survival of plasmid upon transformation of E.coli K12 AB1157 enables one to assess the biological significance of single- and double-strand breaks . The inactivation of the plasmid (in AB1157) is mainly determined by the number of directly formed laser-induced single-strand breaks, whereas the contribution of enzymatically produced single- and double-strand breaks is insignificant.

Mol Immunol, 1996 Sep, 33(13), 1049 - 58
Human recombinant antibody fragments specific for a rye-grass pollen allergen: characterization and potential applications; de Lalla C et al.; One of the major allergens from the pollen of perennial rye grass (Lolium perenne), Lol pII, was used to isolate specific antibody fragments from a random combinatorial library displaying a large repertoire of human Fab on filamentous phages . After five panning cycles on recombinant Lol pII immunotubes, phage binders were isolated and the antibody fragments expressed as soluble Fab molecules in the Escherichia coli periplasm . The DNA sequencing of the clones producing antibodies with the highest binding activity showed three of them to be identical, while one differed by two amino acid substitutions in the heavy chain . The antibody fragments were produced in milligram amounts, affinity-purified and further characterized . They bound the natural allergen as well as the recombinant one, with no cross-reactivity with other allergens contained in the pollen extract of L . perenne . One antibody bound the allergen with Kd = 2.63 x 10(-9) M, as demonstrated by the surface plasmon resonance technique, and was able to compete with a fraction of serum IgE . Epitope mapping using synthetic peptides revealed that antigenic domains, located between amino acids 39 and 51 of Lol pII, are recognized by Fab and polyclonal IgE from sera of allergic donors . The Fab fragments inhibited the binding of serum IgE to the allergen . In vitro experiments on whole blood from allergic subjects showed that recombinant Fab fragments had a blocking activity on histamine release from cells challenged with recombinant Lol pII allergen . Thus, serum IgE and recombinant Fab fragments recognize common epitopes, although they represent the outcome of different maturation and/or selection processes . Our molecular and functional findings altogether indicate that allergen-specific human antibodies may be useful for the characterization of the antigenic structure of allergens . We conclude that a phage library is a powerful source of anti-allergen human antibodies with high affinity and high specificity . Moreover, these molecules may be potentially innovative reagents for the treatment of atopic allergy.

Mol Immunol, 1996 Sep, 33(13), 1017 - 24
Recombinant fusion peptides containing single or multiple repeats of a ubiquitous T-helper epitope are highly immunogenic; Astori M et al.; Two recombinant mouse mammary tumor virus (MMTV) subunit vaccines have been constructed, in which linear sequences of the envelope gp 52 glycoprotein (EP3) or the superantigen (SAg) have been fused to single or multiple repeats of a T-cell epitope (P30) from tetanus toxin . Histidine tags or glutathione-S-transferase (GST) sequences have been included in the recombinant peptides in order to facilitate their purification by affinity chromatography . The EP3 or SAg recombinant polypeptides with four, one, or no T-cell epitopes were expressed in Escherichia coli, purified and injected intramuscularly, with non-ionic block copolymers as an adjuvant, into BALB/c mice . Following one or two boosts, the B- and T-cell responses against the recombinant proteins were analysed . The addition of T-cell epitopes considerably increased the immunogenicity of the subunit vaccines . The anti-EP3 response was maximum with four T-cell epitopes, while a single T epitope was optimal for the anti-SAg response.

Biokhimiia, 1996 Sep, 61(9), 1637 - 47
{Crystallization of a covalently-linked complex of adrenodoxin and adrenodoxin reductase}; Lapko AG et al.; Two forms of mitochondrial adrenodoxin reductase from bovine adrenals and recombinant bovine adrenodoxin and adrenodoxin reductase expressed in Escherichia coli were isolated, purified to homogeneity and biochemically characterized . Recombinant adrenodoxin reductase was expressed as a single polypeptide; its retention time on DEAE-Fractogel coincides with the second form (F2) of the mitochondrial reductase . Two enzyme forms have similar adrenodoxin reductase activities in two types of systems comprising either cytochrome c or cytochrome P-450 (11 beta) as the terminal electron acceptor . Adrenodoxin and each of two reductase forms were cross-linked using 1-ethyl-3-(dimethyl-amino-propyl)carbodiimide . An effective two-step method for the purification of the active heterologous cross-linked complexes is suggested that enables purification of the functional complexes to homogeneity . The cross-linked bimolecular complex of adrenodoxin and adrenodoxin reductase was crystallized for the first time.

J Radiat Res (Tokyo), 1996 Sep, 37(3), 171 - 6
Induction of repair capacity for oxidatively damaged DNA as a component of peroxide stress response in Escherichia coli; Zhang QM et al.; We examined whether or not peroxide stress induces a repair capacity for oxidatively damaged DNA in Escherichia coli cells . Peroxide stress was brought about by adding 30 microM hydrogen peroxide (H2O2) to exponentially growing cells . The following results were obtained . (1) After exposure to H2O2, E . coli resistance to X-rays was enhanced . The acquisition of resistance was inhibited by rifampicin and chloramphenicol . (2) The response was acquired in mutants defective in the katG and oxyR genes, as well as in the wild-type strain . (3) Lambda phages damaged by exposure to H2O2 showed higher survival on H2O2-treated cells than on untreated cells . (4) The peroxide stress did not render E . coli cells resistant to UV and mitomycin C . These suggest that peroxide stress induces a repair capacity against oxidative DNA damage and that this response must be regulated by a different mechanism from oxyR(+)-mediated regulatory system.

Biosci Biotechnol Biochem, 1996 Sep, 60(9), 1548 - 50
Analysis of the Escherichia coli gntT and gntU genes and comparison of the products with their homologues; Yamada M et al.; The Escherichia coli gluconate permease genes, gntT ant gntU, were cloned and characterized . At least four homologues to GntT were found in E . coli by database searching . These proteins including GntT and GntU appear to have similar topological structures with 14 membrane-spanning segments, suggesting that they constitute a GntP family.

Biosci Biotechnol Biochem, 1996 Sep, 60(9), 1492 - 4
Inhibition of Escherichia coli heat-labile enterotoxin by an amino carbonyl product, lactose-alpha-lactalbumin; Shida K et al.; The inhibition by lactose-alpha-lactalbumin amino carbonyl product of Escherichia coli heat-labile enterotoxin was studied by GM1-ELISA and by assay with CHO-K1 cells . The product dose-dependently inhibited the binding of the enterotoxin to GM1 ganglioside and decreased the morphological change of CHO-K1 cells caused by this toxin . The results suggest that this product may be a receptor analogue in the intestine.

Biosci Biotechnol Biochem, 1996 Sep, 60(9), 1469 - 73
Molecular cloning of cDNA that encodes chymotrypsin inhibitor ECI from Erythrina variegata seeds and its expression in Escherichia coli; Kuramitsu J et al.; Synthetic oligonucleotides representing all possible sequences of the N-terminal and internal amino acid sequences of the chymotrypsin inhibitor ECI from Erythrina variegata seeds were used to generate a probe specific for ECI-related sequences by the polymerase chain reaction on the E . variegata genomic DNA . A lambda phage cDNA library constructed from poly(A+) RNA from maturing seeds was screened with the ECI gene thus obtained as a probe and characterized by DNA sequencing . The cloned ECI cDNA comprised 737 nucleotides and one open reading frame that encoded a polypeptide chain of 203 amino acids including a signal peptide composed of 24 amino acids . An expression plasmid was designed for export of the recombinant inhibitor into the periplasm . For this purpose, the cDNA fragment encoding matured ECI was ligated into the NcoI and BamHI sites following the pel B signal sequence in the expression vector pET-22b and expressed in Escherichia coli BL21 (DE3) . However, this attempt failed as the recombinant inhibitor caused the formation of inclusion bodies in E . coli cells as a heterologous preprotein (SR-ECI), with the pel B upstream leader . SR-ECI was made soluble and renatured by refolding and reoxidation, and subsequently processed with pronase to give rise to recombinant ECI (R-ECI) that had an extra methionine residue attached to the N-terminal amino acid of ECI . Purified R-ECI inhibited chymotrypsin almost as strongly as authentic ECI.

Biosci Biotechnol Biochem, 1996 Sep, 60(9), 1401 - 5
Cloning and nucleotide sequence of ApaLI restriction-modification system from Acetobacter pasteurianus IFO 13753; Suzuki T et al.; The ApaLI restriction-modification system from Acetobacter pasteurianus IFO 13753 recognizes the nucleotide sequence GTGCAC . The gene coding for the ApaLI methylase (M.ApaLI) was cloned into Escherichia coli DH5 alpha MCR, and the nucleotide sequence of the gene was analyzed . The M.ApaLI gene coded for a protein of 429 amino acid residues (molecular mass, 46,554 daltons) . The ApaLI restriction endonuclease (R.ApaLI) gene was analyzed by inverse polymerase chain reaction . The R.ApaLI gene coded for a protein of 375 amino acid residues (molecular mass, 42,143 daltons) . The two genes had the same orientation separated by two base pairs . The deduced amino acid sequence of M.ApaLI shows significant similarities to the family of cytosine-5 methylases . However, the deduced amino acid sequence of R.ApaLI did not have as much relatedness in the nucleotide sequence, when compared with those of the other restriction endonucleases already reported.

Acta Trop, 1996 Sep, 62(1), 45 - 56
During canine leishmaniasis a protein belonging to the 83-kDa heat-shock protein family elicits a strong humoral response; Angel SO et al.; By screening of a Leishmania infantum expression library with the serum from a dog affected with visceral leishmaniasis, a cDNA clone with sequence homology to the Hsp83 gene family was isolated . From analysis of the genomic distribution of the cDNA sequence, it was estimated that the L . infantum genome contains 7 Hsp83 genes tandemly organized . The full-length coding region of the Hsp83 gene located at the 5'-end of the cluster was determined . The deduced amino acid sequence of the L . infantum Hsp83 shows a high level of sequence identity with members of the Hsp83's protein family from other eukaryotic organisms . The complete protein (LiHsp83) and 4 subfragments (LiA1, LiB1, LiC1 and LiD1) were expressed in Escherichia coli as recombinant proteins and used as target antigens in FAST-ELISA assays against a collection of sera from dogs with visceral leishmaniasis . Ninety percent of the sera recognized the recombinant LiHsp83, indicating that L . infantum Hsp83 is a potent immunogen during canine leishmaniasis . Serological analysis of the recombinant subfragments identified the LiB1 subfragment, from amino acid 156 to 283, as the immunodominant region of the protein . This region, which is the less evolutionary conserved region of the protein, was recognized by 88% of the visceral leishmaniasis sera . The results suggest that L . infantum Hsp83 and particular protein subfragments may be useful in serodiagnostic assays for canine leishmaniasis.

Vaccine, 1996 Sep, 14(13), 1273 - 9
Production of a genetically engineered inhibin vaccine; Geary TW et al.; The goal of this research was to synthesize a chimeric ovalbumin/inhibin antigen using recombinant techniques . Antigenic epitopes of the translated product from an ovalbumin cDNA were mapped using computer modeling techniques . The corresponding nucleotide sequences of ovalbumin epitopes were examined for unique restriction sites to allow insertion of a synthetic bovine inhibin alpha gene fragment which encoded the first 26 amino acids . A plasmid (pETOI-8) was synthesized which contained the chimeric ovalbumin/inhibin alpha 1-26 gene . A 46 kDa recombinant protein (ovalin) was produced from BL21 (DE3) Escherichia coli cells containing pETOI-8 that was identified with anti-ovalbumin and anti-inhibin alpha 1-26 antisera . Rabbits were immunized subcutaneously in four sites along the back against ovalbumin (n = 3), crude ovalin (n = 3), or gel-purified ovalin (n = 3) at week 0, 4, 7, and 18 . Primary and booster immunizations contained ca 300 micrograms of antigen emulsified in complete Freund's adjuvant and incomplete Freund's adjuvant, respectively . Blood samples collected at week 0, 6, 8, and 19 were evaluated for their ability to bind ovalbumin, 32 kDa bovine inhibin and bovine inhibin alpha 1-26 using ELISA . Mean anti-ovalbumin titers at week 19 were 1:100000 in rabbits immunized against ovalbumin or crude ovalin, and 1:23333 in rabbits immunized against gel-purified ovalin . Anti-inhibin and anti-inhibin alpha 1-26 titers were nonexistent in antisera from pre-immunized rabbits and rabbits immunized against ovalbumin . Mean anti-inhibin titers at week 19 were 1:833 and 1:10000 in rabbits immunized against crude ovalin or gel-purified ovalin, respectively . Mean anti-inhibin alpha 1-26 titers at week 19 were 1:3333 and 1:6666 in rabbits immunized against crude or gel-purified ovalin, respectively . We conclude that genetic engineering of inhibin alpha 1-26 into the antigenic epitopes of ovalbumin provides potential for the development of an anti-inhibin vaccine.

Plasmid, 1996 Sep, 36(2), 86 - 94
Efficient circularization in Escherichia coli of linear plasmid multimers from Dictyostelium discoideum genomic DNA; Barth C et al.; Transformation of Escherichia coli with Dictyostelium discoideum genomic DNA containing integrated shuttle vectors in multicopy, tandemly duplicated format resulted in the establishment of the linear plasmid molecules as circular monomeric replicons . The transformation efficiencies were comparable to those obtained with circular plasmid DNA and the recovered plasmids were free of deletions and rearrangements . Digestion of the genomic DNA prior to the transformation using restriction enzymes that cut within the inserted plasmids reduced the transformation efficiency dramatically and a high proportion of the recovered plasmids carried deletions . Our results provide evidence that the linear plasmid multimers cyclize in E . coli by homologous recombination in order to be established as autonomously replicated plasmids . The efficiency of recircularization was found to be independent of the recA gene product but dramatically reduced in the absence of recB recC or sbcB gene products . However, the paradoxically high efficiency of transformation with plasmid multimers of a recB recC sbcB mutant indicated the presence of an additional pathway for recombinational recircularization independent of these gene products . Unlike previous studies using as a DNA source linearized plasmid monomers and dimers that were created in vitro, the use of linear plasmid multimers integrated into the D . discoideum genome ensured that none of the E . coli transformants we obtained could be attributed to low levels of uncut circular plasmid molecules . The efficient recovery of the plasmid monomers faithfully reflects the structure of the insertion and thus provides a useful tool in the characterization of such plasmid insertions in the genome of D . discoideum.

Chem Biol, 1996 Sep, 3(9), 739 - 46
Recognition of tetrahedral 1,10-phenanthroline-cuprous chelates by transcriptionally active complexes does not depend on the sequence of the promoter; Gallagher J et al.; BACKGROUND: The open complex formed at the initiation site of transcription within the active site of RNA polymerase is unique to actively transcribing genes and is thus an ideal target for the design of transcription inhibitors . Many redoxactive tetrahedral cuprous chelates of 1,10-phenanthroline (OP) or derivatives cleave the single-stranded template, principally at sequence positions -7 to -3, whereas the redox-inactive tetrahedral cuprous chelate of 2, 9-dimethyl-OP (neocuproine) blocks transcription, but does not cleave . The octahedral (OP)3-Fe2+ chelate has no effect . Different promoters can give different cleavage patterns . We therefore searched for structural determinants of the open complex that are important in the cleavage reaction . RESULTS: Using site-directed mutagenesis, we systematically altered the nucleotides at the cleavage sites of the Escherichia coli lac UV-5-RNA polymerase open complex (positions -6 to -4), which are highly variable in E . coli promoters . Surprisingly, these changes had little effect on catalytic activity, on transcription inhibition by the cuprous complex of neocuproine and on the cleavage patterns generated by the cuprous chelates of OP derivatives . The scission pattern of a lac UV-5 promoter mutant in which the cleavage sites have the sequence of the trp EDCBA promoter is that of the lac UV-5 promoter, not the trp EDCBA promoter . CONCLUSIONS: Nucleotide-specific interactions are not responsible for the observed cleavage patterns . The recognition of the tetrahedral OP chelate must be due to a specific structure of the single-stranded regions, determined by RNA polymerase-DNA interactions in the upstream regulatory region.

Jpn J Antibiot, 1996 Sep, 49(9), 892 - 8
{Isolation and characterization of isogenic transductants related with the resistance from in vitro fosfomycin resistant strains}; Tsuruoka T et al.; The mutation sites in FR182 and FR190, which were selected as fosfomycin (FOM) resistant mutants in vitro from an Escherichia coli (E . coli) K-12 derivative, were found to be close to the 52 min . and the 85 min., respectively, on linkage map of E . coli K-12 . Therefore, the site of mutation in FR182 appears to be ptsI and that in FR190 to be cyaA based on the physiological and biochemical characteristics . We isolated isogenic transductants from the FOM resistant strains (from FR182, resistant strain AMG3 and sensitive strain AMG4, and from FR190, resistant strain AMI5 and sensitive strain AMI6) by transducing the genes of the resistant strains into recipients that were deficient in alkaline phosphatase . Under anaerobic conditions, MICs in the isogenic transductants were all decreased and the resistant transductants showed similar FOM sensitivities to the respective sensitive transductants . The FOM resistant strains were clearly lysed by much lower concentrations of FOM in the presence of cyclic adenosine-3',5'-monophosphate (cAMP) or glucose 6-phosphate (G6P) than in their absence, but the extent of lysis was more pronounced when both cAMP and G6P were present together, resembling the lysis of sensitive strains in same condition.

Appl Biochem Biotechnol, 1996 Sep, 60(3), 251 - 301
On the topological features of optimal metabolic pathway regimes; See SM et al.; In this work, the stoichiometric metabolic network of Escherichia coli has been formulated as a comprehensive mathematical programming model, with a view to identifying the optimal redirection of metabolic fluxes so that the yield of particular metabolites is maximized . Computation and analysis has shown that the over-production of a given metabolite at various cell growth rates is only possible for a finite ordered set of metabolic structures which, in addition, are metabolite-specific . Each regime has distinct topological features, although the actual flux values differ . Application of the model to the production of 20 amino acids on four carbon sources (glucose, glycerol, lactate, and citrate) has also indicated that, for fixed cell composition, the maximum amino acid yield decreases linearly with increasing cell growth rate . However, when the cell composition varies with cell growth rate, the amino-acid yield varies in a nonlinear manner . Medium optimization studies have also demonstrated that, of the above substrates, glucose and glycerol are the most efficient from the energetic viewpoint . Finally, model predictions are analyzed in the light of experimental data.

Appl Biochem Biotechnol, 1996 Sep, 60(3), 189 - 202
Detection of endotoxin using an evanescent wave fiber-optic biosensor; James EA et al.; The lipopolysaccharide endotoxin is the most powerful immune stimulant known and a causative agent in the clinical syndrome known as sepsis . Sepsis is responsible for more than 100,000 deaths annually, in large part due to the lack of a rapid, reliable, and sensitive diagnostic technique . This study describes the detection of LPS from E . coli at concentrations as low as 10 ng/mL, in 30 s using an evanescent wave fiber-optic biosensor . Polymyxin B, covalently immobilized onto the surface of the fiber-optic probe, selectively bound fluorescently labeled LPS . Unlabeled LPS was detected in a competitive assay format using labeled LPS for signal generation . The competitive assay format worked in both buffer and plasma with similar sensitivities . This method can be used with other LPS capture molecules such as antibodies, lectins, or antibiotics, to simultaneously detect LPS and to determine the LPS serotype . The LPS assay using the fiber-optic biosensor is applicable to both clinical and environmental testing.

Cytokine, 1996 Sep, 8(9), 686 - 97
Immunoregulatory cytokines modify Escherichia coli induced uroepithelial cell IL-6 and IL-8 responses; Hedges SR et al.; This study analysed the effects of immunoregulatory cytokines on uroepithelial cell cytokine responses . The A-498 human kidney cell line was treated with the interleukins IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, interferon gamma (IFN-alpha) and transforming growth factor beta (TGF-beta 1) . Secreted IL-6 and IL-8 were quantitated by enzyme-linked immunoabsorbent assay (ELISA) and bioassay; IL-6 and IL-8 mRNA species were quantitated by reverse transcriptase polymerase chain reaction (RT-PCR) . IL-4, IL-13, IFN-gamma, and TGF-beta 1, but not IL-2, IL-5, IL-10 or IL-12, stimulated IL-6 secretion . At high concentrations, IL-4 and IL-13 stimulated low levels of IL-8 secretion . Immunoregulatory cytokines were analysed for their ability to modify the A-498 cells' IL-6 and IL-8 secretion in response to Escherichia coli . IL-5, IL-12, IL-13 and TGF-beta 1 additively enhanced the bacterially induced IL-6 secretion, but they did not affect IL-8 secretion . The strongest affects on uroepithelial cell IL-6 and IL-8 responses in the presence of bacteria were observed in conjunction with IL-4 and IFN-alpha . IL-4 induced IL-6 production in synergy with E . coli . IFN-alpha both enhanced and inhibited IL-6 and IL-8 responses in combination with E . coli, depending on the order of stimulant addition . The results demonstrate that immunoregulatory cytokines can modify the uroepithelial cell responses to bacteria in vitro . In this way, T cells may regulate the cytokine responses of uroepithelial and possibly other mucosal epithelial cells in vivo.

Int J Biochem Cell Biol, 1996 Sep, 28(9), 1031 - 43
Renaturation of SPARC expressed in Escherichia coli requires isomerization of disulfide bonds for recovery of biological activity; Bassuk JA et al.; SPARC (secreted protein acidic and rich in cysteine, also known as osteonectin and BM-40) belongs to a group of secreted macromolecules that modulate cellular interactions with the extracellular matrix . During vertebrate embryogenesis, as well as in tissues undergoing remodeling and repair, the expression pattern of SPARC is consistent with a fundamental role for this protein in tissue morphogenesis and cellular differentiation . Human SPARC was cloned by the polymerase chain reaction from an endothelial cell cDNA library and was expressed in Escherichia coli as a biologically active protein . Two forms of recombinant SPARC (rSPARC) were recovered from BL21(DE3) cells after transformation with the plasmid pSPARCwt: a soluble, monomeric form that is biologically active (Bassuk et al., 1996, Archiv . Biochem . Biophys . 325, 8-19), and an insoluble form sequestered in inclusion bodies . Aggregated rSPARC was unfolded by urea treatment, purified by nickel-chelate affinity chromatography, and renatured by gradual removal of the denaturant . Proper isomerization of the disulfide bonds was achieved in the presence of a glutathione redox couple . After final purification by high resolution gel filtration chromatography, a monomeric form of rSPARC displaying biological activity was obtained . The recombinant protein inhibited the spreading and synthesis of DNA by endothelial cells, two properties characteristic of the native protein . We conclude that the information for the correct folding of rSPARC resides in the primary structure of the protein, and suggest that post-translational modifications are required neither for folding nor for biological activity.

Int J Biochem Cell Biol, 1996 Sep, 28(9), 975 - 82
Isolation, renaturation and partial characterization of recombinant human transferrin and its half molecules from Escherichia coli; Hoefkens P et al.; Recombinant human transferrin as well as N- and C-terminal half-transferrins, produced in Escherichia coli, are deposited in inclusion bodies by the bacteria . The isolation and purification of the recombinant proteins from these inclusion bodies are described here . The amino acid compositions and N-terminal sequences of the proteins were determined, and found to be in agreement with the known protein structure of human serum transferrin . Renaturation of the recombinant proteins is described, resulting in water-soluble iron-binding molecules . Iron binding was confirmed by 59Fe labelling, absorption spectrophotometry and EPR spectrometry.

Plant Mol Biol, 1996 Sep, 31(6), 1141 - 51
Characterization of pectinases and pectin methylesterase cDNAs in pods of green beans (Phaseolus vulgaris L.); Ebbelaar ME et al.; Tomato fruit maturation is accompanied by a depolymerization of cell wall pectins which is due to the action of endopolygalacturonase (endoPG) preceded by pectin methylesterase (PE) activity . To investigate the role of endoPG and PE in determining the structure of green bean (Phaseolus vulgaris L.) pectins, these pectinases were studied during pod development . Early developmental stages displayed low endoPG or exoPG activities while PE activities were measurable during all stages of pod and seed development . These results do not favour a possible synergistic action of PE and PG . For seeds, the relatively high PE activities concurred with relatively low levels of pectin methyl esterification . At a molecular level, one partial chromosomal clone of 210 bp (PE1V), two partial PE cDNA clones of 660 bp (PE2V and PE3V) from cv . verona and one full-length PE cDNA clone of 1990 bp (PE3M), from cv . Masai were isolated . The identity of the CDNA clones was confirmed by expression in Escherichia coli and immunodetection with antibodies directed towards a tomato fruit PE . Transcripts corresponding with the genomic clone PE1V were not detected but both PE2 and PE3 cDNAs corresponded with mRNAs 1.8 kb in length . In contrast to PE2, PE3 gene expression levels varied significantly in pods from different cultivars suggesting an involvement in determining pod morphology.

J Biomol NMR, 1996 Sep, 8(2), 184 - 92
Genetic tools for selective labeling of proteins with alpha-15N-amino acids; Waugh DS; A collection of genetic tools that can be used to manipulate amino acid metabolism in Escherichia coli is described . The set comprises 21 strains of bacteria, each containing a different genetic defect that is closely linked to a selectable transposon marker . These tools can be used to construct strains of E . coli with ideal genotypes for residue-specific, selective labeling of proteins with nearly any 15N-amino acid . By using strains which have been modified to contain the appropriate genetic lesions to control amino acid biosynthesis, dilution of the isotope by endogenous amino acid biosynthesis and scrambling of the label to other types of residues can be avoided.

J Biomol NMR, 1996 Sep, 8(2), 136 - 46
The new program OPAL for molecular dynamics simulations and energy refinements of biological macromolecules; Luginbuhl P et al.; A new program for molecular dynamics (MD) simulation and energy refinement of biological macromolecules, OPAL, is introduced . Combined with the supporting program TRAJEC for the analysis of MD trajectories, OPAL affords high efficiency and flexibility for work with different force fields, and offers a user-friendly interface and extensive trajectory analysis capabilities . Salient features are computational speeds of up to 1.5 GFlops on vector supercomputers such as the NEC SX-3, ellipsoidal boundaries to reduce the system size for studies in explicit solvents, and natural treatment of the hydrostatic pressure . Practical applications of OPAL are illustrated with MD simulations of pure water, energy minimization of the NMR structure of the mixed disulfide of a mutant E . coli glutaredoxin with glutathione in different solvent models, and MD simulations of a small protein, pheromone Er-2, using either instantaneous or time-averaged NMR restraints, or no restraints.

Vet Microbiol, 1996 Sep, 52(1-2), 153 - 64
The enterohemolysin phenotype of bovine Shiga-like toxin-producing Escherichia coli (SLTEC) is encoded by the EHEC-hemolysin gene; Wieler LH et al.; Naturally occurring enterohemolysin negative variants were observed during studies on bovine Shiga-like toxin-producing E . coli (SLTEC) . Examination of three strains (413/89-1 and 332, 026:H-, and 570/89, O111:H-) and their isogenic variants (413/89-6, 332-I and 570/89-I, respectively) showed, that in each strain loss of the enterohemolytic phenotype correlated with the loss of a large plasmid ranging from 94 to 104 kb in size . The hemolysin determinant present on the 94 kb plasmid of strain 413/89-1 was cloned and discovered by DNA and N-terminal aminoacid sequence analysis to be highly homologous to the recently published EHEC-hemolysin (HlyEHEC; Schmidt et al., 1994; 1995) . When a recombinant plasmid harboring this determinant was reintroduced into the enterohemolysin negative isogenic mutant 413/89-6, the enterohemolytic phenotype was restored . Southern blot hybridization analysis was used to demonstrate that the HlyEHEC is plasmid-borne in SLTEC-strains . Our cumulative data suggest that the enterohemolytic phenotype of SLTEC is encoded by the plasmid-borne HlyEHEC . These results further demonstrate the close similarity between SLTEC-isolates from bovine and human.

J Biochem (Tokyo), 1996 Sep, 120(3), 608 - 15
Properties of H(+)-ATPase from rat liver lysosomes as revealed by reconstitution into proteoliposomes; Okamoto M et al.; The properties of H(+)-ATPase from rat liver lysosomes were analyzed by reconstituting proton pump activity from solubilized enzyme and Escherichia coli phospholipids in proteoliposomes devoid of anion-channels . The reconstitution procedure involved solubilization of the ATPase with n-octyl-beta-D-thioglucoside in the presence of asolectin, and incorporation of the solubilized enzyme into E . coli phospholipid liposomes by dilution, freeze-thawing, and sonication . Proton pump activity of reconstituted H(+)-ATPase as detected by the ATP-dependent quenching of acridine orange fluorescence indicated that ATP can be replaced with dATP and to a lesser extent with GTP, but not with any other nucleotide, that Mg2+ can be replaced with Mn2+, but not with Ca2+, Sr2+, or Ba2+, that Zn2+, Pd2+, Cd2+, and Hg2+ were inhibitory, and that the enzyme was sensitive to inhibitors of v-type H(+)-ATPase, including bafilomycin A1, N-ethylmaleimide, DCCD, DIDS, and tri-n-butyltin . The enzyme showed unique sensitivity to anions and was activated by chloride, fluoride, and bromide from inside, but not from outside the vesicles . It was inhibited by sulfate, sulfite, and thiocyanate from outside the vesicles, and by nitrate from both inside and outside the vesicles.

J Biochem (Tokyo), 1996 Sep, 120(3), 494 - 97
Cloning and expression of cDNA encoding a human ubiquitin-conjugating enzyme similar to the Drosophila bendless gene product; Yamaguchi T et al.; cDNA encoding a novel human ubiquitin-conjugating enzyme has been cloned from an epidermoid carcinoma KB cDNA library . This clone encodes a protein of 152 amino acids with a calculated M(r) of 17,137 . The amino acid sequence showed 80% identity with the Drosophila's bendless gene product (ubiquitin-conjugating enzyme E2) . The corresponding transcripts are highly expressed in heart, skeletal muscle, and testis . The product expressed in Escherichia coli exhibited the ability to form a thiol ester linkage with ubiquitin in a ubiquitin-activating enzyme E1-dependent manner . These results suggest that the obtained cDNA encodes a novel human E2 which may be involved in protein degradation mainly in the muscles and testis.

J Biochem (Tokyo), 1996 Sep, 120(3), 488 - 93
Phorbol ester, but not endotoxin, desensitizes mannan-induced glycogenolysis in the perfused rat liver; Kimura K et al.; Mannan, a ligand for the mannose/N-acetylglucosamine (GlcNAc) receptor, induces suppression of oxygen consumption and increases glucose production in the perfused rat liver, and repeated infusion of mannan causes desensitization of the responses . In this study, we examined whether activation of Kupffer cells by endotoxin and phorbol ester alters the glycogenolytic responses to mannan . Infusion of lipopolysaccharide (LPS, 10 micrograms/ ml) in the perfusate failed to inhibit the responses to mannan . Intravenous administration of LPS (1 mg/kg) 6 and 24 h before perfusion did not desensitize the responses to mannan, suggesting that the responses through mannose/GlcNAc receptors in the liver are retained even after activation of Kupffer cells by LPS . In contrast, prior infusion of phorbol 12-myristate 13-acetate (PMA, 100 nM) in vitro abolished the glycogenolytic responses to subsequently infused mannan, but not that to norepinephrine (100 nM), while prior infusions of 4-alpha-phorbol 12,13-didecanoate (100 nM), A23187 (50 nM), or forskolin (1 microM) had no effect on the mannan-induced responses . H-7, an inhibitor of protein kinase C, reduced the glycogenolytic responses to mannan, while it failed to restore the desensitization . These results suggest that protein kinase C may be involved in the process of glycogenolysis by mannan, but is unlikely to be involved in the homologous desensitization of the responses.

J Biochem (Tokyo), 1996 Sep, 120(3), 481 - 2
Crystallization and preliminary X-ray diffraction studies of N-acyl-D-glucosamine 2-epimerase from porcine kidney; Maru I et al.; N-Acyl-D-glucosamine 2-epimerase from porcine kidney, which was cloned and expressed in Escherichia coli, was crystallized by the vapor-diffusion method, using polyethylene glycol and ammonium acetate as precipitants . The crystals were resistant to X-ray radiation damage and diffracted to more than 2.0 A resolution . The diffraction pattern indicated that the crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit-cell dimensions of a = 78.1, b = 97.2, and c = 100.7 A . It is supposed that the asymmetric unit consists of two N-acyl-D-glucosamine 2-epimerase molecules . Collection of data on the native crystals indicated that they are suitable for X-ray structural analysis.

Mol Microbiol, 1996 Sep, 21(6), 1273 - 81
Carbon catabolite repression of xylanase I (xyn1) gene expression in Trichoderma reesei; Mach RL et al.; The filamentous fungus Trichoderma reesei forms two specific, xylan-inducible xylanases encoded by xyn1 and xyn2 to degrade the beta-1,4-D-xylan backbone of hemicelluloses . This enzyme system is formed in the presence of xylan, but not glucose . The molecular basis of the absence of xylanase I formation on glucose was the purpose of this study . Northern blotting of the xyn1 transcript as well as the use of the Escherichia coli hygromycin B phosphotransferase-encoding gene (hph) as a reporter consistently showed that the basal expression of xyn1 was affected by glucose, whereas its induction by xylan remained uninfluenced . The repression of basal xyn1 transcription is mediated by the carbon catabolite repressor protein Cre1, which in vivo binds to two of four consensus sites (5'-SYG-GRG-3') in the xyn1 promoter, which occurred in the form of an inverted repeat . T . reesei strains, bearing a xyn1::hph reporter construct, in which four nucleotides from the middle of the inverted repeat had been removed, expressed hph on glucose at a level comparable to that observed during growth on a carbon catabolite derepressing carbon source . Northern analysis of xyn1 expression in a T . reesei mutant strain (RUT C-30), which contains a truncated, non-functional cre1 gene, also confirmed basal transcription of xyn1 . In this strain, xyn1 transcription was still inducible by xylose or xylan to an even higher degree than in the wild-type strain, suggesting that induction overcomes glucose repression at the level of xyn1 expression . Based on these data, we postulate that basal transcription of xyn1 is repressed by glucose and mediated by an inverted repeat of the consensus motif for Cre1-mediated carbon catabolite repression.

Mol Microbiol, 1996 Sep, 21(6), 1207 - 18
RbfA, a 30S ribosomal binding factor, is a cold-shock protein whose absence triggers the cold-shock response; Jones PG et al.; The cold-shock response, characterized by a specific pattern of gene expression, is induced upon a downshift in temperature and in the presence of inhibitors of ribosomal function . Here, we demonstrate that RbfA of Escherichia coli, considered to be involved in ribosomal maturation and/or initiation of translation, is a cold-shock protein . Shifting the rbfA mutant to a lower temperature resulted in a constitutive induction of the cold-shock response accompanied by slower growth at low temperatures, while shifting the rbfA mutant that overproduces wild-type RbfA resulted in an increase in total protein synthesis accompanied by faster growth adaptation to the lower temperature . Furthermore, the cold-shock response was also constitutively induced in a cold-sensitive 16S rRNA mutant at low temperatures . Accompanying the transient induction of the cold-shock response, we also report that shifting E . coli from 37 degrees C to 15 degrees C resulted in a temporary inhibition of initiation of translation, as evidenced by the transient decrease in polysomes accompanied by the transient increase in 70S monosomes . The accumulative data indicate that the inducing signal for the cold-unadapted non-translatable ribosomes which are converted to cold-adapted translatable ribosomes by the association of cold-shock proteins such as RbfA . Therefore, the expression of the cold-shock response, and thus cellular adaptation to low temperature, is regulated at the level of translation . The data also indicate that cold-shock proteins can be translated by ribosomes under conditions that are not translatable for most mRNAs.

Mol Microbiol, 1996 Sep, 21(6), 1185 - 96
Protein folding in the cytoplasm of Escherichia coli: requirements for the DnaK-DnaJ-GrpE and GroEL-GroES molecular chaperone machines; Thomas JG et al.; We have systematically investigated the influence of mutations in the sigma(32) heat-shock transcription factor and the DnaK-DnaJ-GrpE and GroEL-GroES molecular chaperone machines on the folding of preS2-beta-galactosidase . This 120 kDa fusion protein between the hepatitis B surface antigen preS2 sequence and beta-galactosidase was synthesized in a highly soluble and enzymatically active form in wild-type Escherichia coli cells cultured at temperatures between 30 degrees C and 42 degrees C, but aggregated extensively in an rpoH165 (Am) mutant . Proper folding was partially restored upon co-overexpression of the dnaKJ operon, but not when the groE operon or dnaK alone were overproduced . The enzymatic activities in dnaK103, dnaJ259 and grpE280 mutants were 40-60% lower relative to a dnaK756 mutant or isogenic wild-type cells at 30 degrees C and 37 degrees C . At 42 degrees C, only 10-40% of the wild-type activity was present in each of the early-folding-factor mutants . Although the synthesis levels of preS2-beta-galactosidase were reduced in the dnaK103, dnaJ259 and grpE280 genetic backgrounds, aggregation was primarily responsible for the loss of activity when the cells were grown at 37 degrees C or 42 degrees C . By contrast, the groEL140, groES30 and groES619 mutations, which induced the aggregation of homodimeric ribulose bisphosphate carboxylase (Rubisco), did not affect the solubility of preS2-beta-galactosidase at temperatures up to 42 degrees C . Our results are discussed in terms of the current understanding of the E . coli protein-folding cascade . The potential usefulness of heat-shock protein mutants for the production of soluble proteins in an inclusion-body form is addressed.

Mol Microbiol, 1996 Sep, 21(6), 1161 - 73
Multicopy fimB gene expression in Escherichia coli: binding to inverted repeats in vivo, effect on fimA gene transcription and DNA inversion; Dove SL et al.; Transcription of fimA, the Escherichia coli gene encoding the type 1 fimbrial subunit protein, is driven by a promoter carried on a 314 bp segment of invertible DNA . We have discovered that overexpression of fimB, one of the genes required for inversion of this DNA element, results in transcriptional repression of fimA . Furthermore, under these conditions inversion ceases to be dependent on the integration host factor (IHF) or the leucine-responsive regulatory protein (LRP), cofactors hitherto considered to be essential for inversion . Inversion will even occur (albeit at a very low level) in the absence of both cofactors . The interaction of the fimB gene product with the invertible element was studied in vivo in the presence of single- and multicopy fimB genes . Dimethyl sulphoxide (DMS)-mediated methylation of DNA at the 9 bp inverted repeats, which flank the invertible element, was found to vary in the presence and absence of functional fimB . The DMS reactivity profile at the left-hand inverted repeat was similar with single or multicopy fimB . The corresponding profile at the right-hand inverted repeat varied with fimB copy number . As this repeat lies between the fimA promoter and open reading frame, FimB binding here is likely to modulate fimA transcription and vice versa.

Mol Microbiol, 1996 Sep, 21(6), 1147 - 60
A cyclophilin-like peptidyl-prolyl cis/trans isomerase from Legionella pneumophila--characterization, molecular cloning and overexpression; Schmidt B et al.; Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires' disease) . A major virulence factor, the Mip protein (FK506-binding protein, FKBP25mem), belongs to the enzyme family of peptidyl-prolyl cis/trans isomerases (PPlases) . Here we show that L . pneumophila Philadelphia I possesses an additional cytoplasmic PPlase at a level of enzyme activity comparable to that of FKBP25mem . The N-terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPlases . The Icy gene (Legionella cyclophilin) was cloned and sequenced . It encodes a putative 164-amino-acid protein with a molecular mass of 17968 Da called L . pneumophila cyclophilin 18 (L.p.Cyp18) . Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coli with 60.5% and 51.5% identity, respectively . The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins . An L . pneumophila Cyp18 derivative with a 19-amino-acid polypeptide extension including a 6-histidine tag and an enterokinase cleavage site exhibits PPlase activity when produced at high levels in E . coli K-12 . After removal of the extension by enterokinase, the properties of the recombinant Cyp18 were indistinguishable from those of the authentic enzyme . In order to investigate the influence of Cyp18 on intracellular survival of L . pneumophila an Icy-negative L . pneumophila strain was constructed . Compared with the wild-type strain, the mutant did not exhibit a significant phenotype but was 10-fold less invasive for Acanthamoeba castellanii . Like human cyclophilin, the L . p . Cyp18 exhibits nuclease activity, but this enzymatic activity does not appear to be linked with the native structure of the protein.

Mol Microbiol, 1996 Sep, 21(6), 1125 - 35
Effects of systematic variation of the minimal Escherichia coli met consensus operator site: in vivo and in vitro met repressor binding; Wild CM et al.; We have produced a set of sequence variants based upon the idealized, minimal Escherichia coli met operator in which each position within the basic recognition unit, the 8 bp met box (dAGACGTCT), has been changed to all other possible sequences containing single symmetrical base substitutions . The effects of these sequence variations have been assayed in vivo by monitoring the production of beta-galactosidase from a standard promoter regulated by the operator variants, and in vitro by gel-retardation assay . The two sets of data are consistent and correlate well with expectations based on the three-dimensional structure of the holorepressor bound to a minimal idealized operator and the results of in vitro evolution experiments . Comparison with two natural operators, metA and metC, suggests that in vivo, with non-consensus operators, the repressor binds to at least four consecutive met boxes.

Int J Pept Protein Res, 1996 Sep, 48(3), 215 - 9
Inhibition of prophage induction by synthetic pentapeptide; Abdel-Rahman S et al.; Six peptide chains different in length and number of sequences were synthesized by the continuous-flow solid-phase method . Their effects on prophage induction were tested . It was found that pentapeptide 1 H-Val-Val-Asn-Asp-Leu-OH, at a concentration of 25 micrograms/mL inhibited the spontaneous induction of prophage Lambda in E . coli strain W 3110(lambda) and phi RLZLS in R . leguminosarum (lysogenic local isolate) by 99.5% and 65.7%, respectively . Moreover, this concentration was also able to inhibit the replication of the developed phages in the indicator strains . It was suggested that this peptide may block the attachment site of the induced phages or inhibit Rec A Protease.

Yeast, 1996 Sep, 12(10B Suppl), 1005 - 11
Sequencing analysis of a 4.1 kb subtelomeric region from yeast chromosome IV identifies HXT15, a new member of the hexose transporter family; Bargues M et al.; The DNA sequence of a 4.1 kb region of Saccharomyces cerevisiae chromosome IV was determined . This region contains a single open reading frame which codes for a member of the hexose transporter family . This new gene has been named HXT15 according to yeast gene data bases.

Equine Vet J, 1996 Sep, 28(5), 382 - 9
Endotoxin induced expression of tumour necrosis factor, tissue factor and plasminogen activator inhibitor activity by peritoneal macrophages; Barton MH et al.; Peritoneal fluid was collected aseptically from 30 healthy adult horses and 115 horses with acute gastrointestinal disease and supernatant was separated from cells by centrifugation followed by freezing until assayed for endotoxin and tumour necrosis factor activity . Peritoneal macrophages obtained from healthy horses were incubated in vitro for 3, 6, 12 or 24 h in the absence (media control) or presence of Escherichia coli 055:B5 endotoxin (final concentrations of 1, 10, 100 or 1000 ng/ml) . Macrophages obtained from horses with acute gastrointestinal disease were incubated for 12 h in the absence (media control) or presence of 100 ng endotoxin/ml . At the conclusion of the incubation, macrophage supernatants were collected and frozen at -70 degrees C until analysed for tumour necrosis factor activity . Macrophage membranes were lysed and frozen at -70 degrees C until assayed for tissue factor and plasminogen activator inhibitor type 2 activity . Compared to cells incubated with media, incubation of macrophages, obtained from healthy horses, with endotoxin significantly increased tumour necrosis factor, tissue factor and plasminogen activator inhibitor type 2 activity . These increases were dependent on the endotoxin concentration and the duration of incubation . Compared to cells incubated with media alone, incubation of macrophages, obtained from horses with acute gastrointestinal disease with endotoxin, significantly increased tumour necrosis factor and tissue factor activity . Endotoxin induced tumour necrosis factor activity in vitro was significantly less for macrophages from horses with acute gastrointestinal disease, as compared to that produced by similarly treated cells obtained from healthy horses . For those horses with acute gastrointestinal disease, macrophages obtained from horses with either endotoxin or tumour necrosis factor activity in the peritoneal fluid supernatant had significantly less endotoxin induced tumour necrosis factor in vitro, as compared to similarly treated cells obtained from horses without endotoxin or tumour necrosis factor activity in the peritoneal fluid supernatant . The results of this study indicate that exposure of equine peritoneal macrophages to endotoxin results in a significant increase in tumour necrosis factor, tissue factor and plasminogen activator inhibitor type 2 activity . After in vitro exposure to endotoxin, there is significant down-regulation of inflammatory mediator production by peritoneal macrophages obtained from endotoxaemic horses . These results suggest that these macrophages may exhibit early endotoxin tolerance.

Cancer Gene Ther, 1996 Sep-Oct, 3(5), 345 - 51
Use of bicistronic retroviral vectors encoding the LacZ gene together with a gene of interest: a method to select producer cells and follow transduced target cells; Staal FJ et al.; The coordinate expression of a marker gene and a therapeutic gene in one retroviral vector has considerable advantages . High-titer producer lines can potentially be selected on the basis of marker gene expression, and the expression of transduced genes in target cells can readily be followed . Moreover, target cells with stable high expression can be selected before use in therapeutic protocols or research questions . We used internal ribosomal entry site (IRES) sequences to express two genes in the same retroviral vector . We used the LacZ gene as the marker gene and the cytokine interleukin (IL)-7 or dominant negative (dn) forms of the T-cell tyrosine kinases ZAP-70 and lck as genes of interest . Amphotropic packaging cells transfected with MFG-IL-7-IRES-LacZ, MFG-dnZAP-70-IRES-LacZ, or MFG-dnlck-IRES-LacZ were sorted on the basis of beta-galactosidase expression . These LacZ-positive producer cells also expressed the gene of interest, produced high-titer retrovirus, and were capable of efficiently transducing Jurkat T cells and T-cell clones . When MFG-IL-7-IRES-LacZ-transduced Jurkat T cells were sorted on the basis of LacZ expression, a positive correlation with the amount of IL-7 produced by these cells was found . This demonstrates that selection of the LacZ marker gene also selects for cells that express the gene of interest at high levels . Moreover, T cells transduced with the dn tyrosine kinases and selected on the basis of LacZ expression showed functional alterations after T-cell receptor stimulation, demonstrating that retrovirally transduced signaling molecules can alter the function of T cells.

Br Poult Sci, 1996 Sep, 37(4), 779 - 86
Immunoresponsiveness of fast-growing chickens as influenced by feeding regimen; Praharaj NK et al.; 1 . Immunoresponsiveness and disease resistance were measured in broiler males maintained on ad libitum feeding throughout or on alternate-day feeding . Alternate-day restrictions were started 1 and 2 d after hatch so that on any one day there were chicks fed and fasted . 2 . Severity of response to E . coli challenge as measured by lesion scores, and mortality was greater for chicks fed ad libitum than those fed on alternate days . For chicks fed on alternate days, lesion scores were lower for those without access to feed for the 24-h period immediately after challenge . 3 . Spleen weights, the indicator of response to marble spleen disease virus challenge, were higher for chicks fed ad libitum than those fed on alternate days . 4 . Antibody response to sheep red blood cell antigen was not affected by feeding regimen . 5 . Ratios of heterophils to lymphocytes were higher for chicks given access to feed for the previous 24-h period than for those fasted during the previous 24-h or those that had been fed ad libitum . 6 . Results of this experiment suggest that for alternate-day feeding programs, vaccination be administered on the day that chicks are not fed.

Pharm Res, 1996 Sep, 13(9), 1389 - 92
Liposomal induction of a heat-stable macrophage priming factor to induce nitric oxide in response to LPS; Aramaki Y et al.; PURPOSE: The effects of liposomes on nitric oxide (NO) production from mouse peritoneal macrophages following intraperitoneal injection of liposomes were investigated . METHODS: Mouse peritoneal macrophages were collected following intraperitoneal injection of liposomes and cultured with and without lipopolysaccharide (LPS) . Peritoneal washing fluid was also collected from the mice injected with liposomes . NO production was evaluated by measuring the concentration of nitrite in the macrophage culture supernatant by Griess reagent . RESULTS: NO production stimulated by LPS was observed in peritoneal macrophages obtained from the liposome-treated mice, but liposomes did not active macrophages directly to induce NO in response to LPS . NO production was higher in the liposomes composed of phosphatidylcholine than that of negatively charged liposomes composed of phosphatidylserine . Peritoneal washing fluid obtained from mice injected with liposomes has a capacity to induce NO production in the macrophages from naive mice . This capacity was not diminished by heat-treatment at 100 degrees C for 5 min . CONCLUSIONS: Peritoneal macrophages were activated to produce NO in response to LPS following intraperitoneal injection of liposomes . They did not activate macrophages directly, and the induction of heat-stable macrophage priming factor, but not cytokines, is suggested.

Alcohol Clin Exp Res, 1996 Sep, 20(6), 1065 - 70
Nitric oxide and liver injury in alcohol-fed rats after lipopolysaccharide administration; Chamulitrat W et al.; Earlier studies showed that alcohol-fed animals were more susceptible than controls to injurious effects of endotoxin . Increased superoxide radical production by hepatocyte organelles, Kupffer cells, and neutrophils from alcohol-fed animals has been well documented . In this study, electron paramagnetic resonance spectroscopy was used to detect nitrosyl protein complexes indicating nitric oxide (.NO) production . We showed that the concentrations of nitrosyl complexes in whole blood and in liver tissues of alcohol-fed rats treated with lipopolysaccharide (alc + LPS), increased 3-fold, compared with those from rats on control diet treated with LPS (con+LPS) . Electron paramagnetic resonance spectra of whole blood and liver tissues from the alc + LPS-treated group exhibited features characteristic of hemoglobin nitrosyl complexes . Plasma levels of the hepatic ASTs and ALTs from the alc + LPS-treated group were increased 2- to 3-fold, compared with those from the con+LPS-treated group . Inhibition of .NO production of aminoguanidine treatment attenuated plasma hepatic enzyme levels in the alc + LPS-treated group . Thus, under the conditions of elevated inflammatory oxidative states caused by chronic alcohol feeding, endotoxin treatment enhanced liver injury as a result of the actions of .NO, and/or the cytotoxic species derived from .NO.

Eur J Clin Chem Clin Biochem, 1996 Sep, 34(9), 765 - 70
A principle of quality assessment using a competitive polymerase chain reaction assay for the detection of Chlamydia trachomatis in cervical specimens; Lichtinghagen R et al.; The polymerase chain reaction (PCR)-based identification of Chlamydia trachomatis in clinical specimens should include a built-in control to assess the quality of the whole assay from DNA isolation to detection . For this purpose we established a competitive PCR assay with the following design . A 215 base-pair DNA fragment from a Chlamydia trachomatis plasmid sequence was amplified in a polymerase chain reaction . An internal control DNA was coamplified in the same reaction . The differentiation between the amplified C . trachomatis DNA and the internal control is based on hybridisation against two different probes using Enzymun Test DNA detection (Boehringer Mannheim) . The internal control capture probe recognizes an additional 20 base-pair DNA sequence in the competitor DNA construct . Cervical swabs from 65 C . trachomatis positive patients were used . We examined the influence of different DNA isolation methods on the sensitivity of the assay . Detection of C . trachomatis from positive cervical swabs was compared using PCR and competitive PCR assays . The advantage of the competitive assay was a better assessment of reduced sensitivity arising from inhibitory effects or mistakes during the DNA preparation or amplification.

Eur J Clin Chem Clin Biochem, 1996 Sep, 34(9), 691 - 6
Preparation and characterization of human recombinant protein 1/Clara cell M(r) 10,000 protein; Okutani R et al.; Protein 1, which is identical to human Clara cell M(r) 10(4) protein, is a homodimeric, low molecular mass protein (M(r) 14,000) and an effective inhibitor of phospholipase A2 activity . We have expressed this protein in E . coli and characterized its physiochemical and biological properties . Using a pET expression system, about 1.7 mg of purified recombinant protein 1 was obtained from 250 ml of E . coli culture . The amino-terminal sequence of recombinant protein 1 up to the 20th residue was identical to that of native protein 1 except for an extra methionine at the amino-terminus . On reversed-phase HPLC, recombinant protein 1 eluted at the same retention time as native protein 1 . The dose-response curves of recombinant protein 1 and native protein 1 in an enzyme-linked immunosorbent assay for protein 1 were identical . Recombinant protein 1 inhibited both porcine pancreas and cobra venom phospholipase A2 activities . These results indicated that recombinant protein 1 is structurally and biologically identical to native protein 1 . We found that recombinant protein 1 also inhibits phosphatidylinositol-specific phospholipase C activity.

Nippon Rinsho, 1996 Sep, 54(9), 2551 - 60
{Hemolytic uremic syndrome}; Arai T et al.; Hemolytic uremic syndrome (HUS) was first reported by Gasser in 1966 as a syndrome characterized by triadic symptomatology of microangiopathic hemolytic anemia (MAHA), thrombocytopenia and acute renal failure, affecting young children more than adults . Its causes include hemorrhagic colitis, cancer, chemotherapy, pregnancy, transplantation and so on . Considering a variety of causes for HUS, it is obvious that adults may be also affected by this syndrome . Since a pathogenetic relationship between HUS and verotoxin producing Escherichia coli (VTEC) infection was pointed out in 1983, there have been substantial progresses in understanding of the VTEC infection, a leading cause for HUS among the causes described above, is now emerging as one of national crisis-management matters in the health and welfare administration in our country . Therefore, in this review, we discuss a current understanding of the pathophysiology and reevaluation of recommended therapeutic modalities for HUS mainly in the case of VTEC infection induced HUS.

Int J Food Sci Nutr, 1996 Sep, 47(5), 427 - 36
The effect of microwave heating on vitamins B1 and E, and linoleic and linolenic acids, and immunoglobulins in human milk; Ovesen L et al.; Breast milk was treated with (1) conventional heating (in water bath) vs microwave heating; (2) microwave heating at two power levels (30% and 100%); (3) increasing final temperatures; and (4) microwave thawing vs refrigerator thawing and examined for changes in specific immunoglobulins to a pool of E . coli and poliovirus type 1 antigens, vitamins E and B1, and the polyunsaturated fatty acids linoleic and linolenic acid . Immunoglobulin activities were stable until final milk temperatures of around 60-65 degrees C were reached, and total inactivation occurred at 77 degrees C . Heating even to high final temperatures did not change contents of vitamins and polyunsaturated fatty acids . No differences in immunoglobulins and nutrients were demonstrated between microwave heating and conventional heating, and between power levels or thawing methods . The study shows that microwave heating of human milk can be performed without significant losses of examined immunoglobulins and nutrients, provided that final temperatures are below 60 degrees C.

J Interferon Cytokine Res, 1996 Sep, 16(9), 745 - 9
Comparative study on the effect of signal peptide codons and arginine codons on the expression of human interferon-alpha 1 gene in Escherichia coli; Saraffova A et al.; Human interferon-alpha 1 (HuIFN-alpha 1) gene containing signal peptide codons is poorly expressed in bacteria, and this is explained by the presence of clusters of rare (AGG) arginine codons in its structure . In this study, we have constructed a series of modified HuIFN-alpha 1 genes to study the effect of both residual signal peptide codons and clusters of AGG codons on gene expression in Escherichia coli cells . Our results showed that substitution of preferential for rare arginine codons in two clusters did not affect the yield, whereas deletion of the signal peptide codons led to a 10-fold increase in the yield of recombinant protein . To understand the mechanism of interference of gene structure on the expression of the HuIFN-alpha 1 gene in vivo, both the level and stability of HuIFN-alpha 1 mRNA were measured . The amount of HuIFN mRNA increased almost five times on deletion of the signal peptide codons from HuIFN-alpha 1 gene constructs (containing AGG clusters or not) . The stability of mRNA obtained from all gene constructs was shown to be the same (half-life of 60 +/- 5 secs), indicating that the signal peptide codons interfere with both the efficiency of transcription of the HuIFN-alpha 1 gene and translation of its mRNA.

Eur J Radiol, 1996 Sep, 23(2), 130 - 4
Percutaneous catheter drainage of tuberculous and nontuberculous psoas abscesses; Dinc H et al.; OBJECTIVE: To assess the utility of percutaneous catheter drainage in the management of tuberculous and nontuberculous psoas abscesses associated without any bony involvement or with minimal bony lesions that could not cause vertebral instability . MATERIALS AND METHOD: Eleven patients with psoas, iliopsoas and pelvic abscesses were drained under computed tomography and ultrasonography guidance . RESULTS: There were 15 (10 tuberculous, 5 pyogenic) abscesses in 11 patients . Six of the tuberculous abscesses and one of the pyogenic abscess were associated with vertebral involvement . Vertebral lesions were located in one or two vertebrae without causing any serious disturbance in the vertebral stabilization . In one case, the abscess was bilateral . Nine cases were drained under computed tomography guidance, while two cases were drained under both computed tomography and ultrasonography guidance . One session drainage was sufficient for abscess resolution in uniloculated cases . In the two of four multiloculated cases, catheter drainage was performed twice . Relapse of the abscess was found in only one patient . The mean abscess volume was 520 ml and mean drainage duration was 12 days . None of the cases required surgery . CONCLUSION: Percutaneous drainage, chemotherapy and additional external brace application with the cases associated with bony lesion may be used for treatment of tuberculous and nontuberculous unilocule and multiloculated abscesses.

Biochem Mol Biol Int, 1996 Sep, 40(1), 93 - 100
Conformational change of cyclic AMP receptor protein by the binding of cyclic nucleotide; Park SH et al.; The cAMP Receptor Protein (CRP) of Escherichia coli requires a conformational change induced by the binding of cAMP in order to function as a site-specific DNA-binding protein . An intrinsic fluorescence study showed that the tryptophan residues at position 13 and 85 within CRP were located in an internal, nonpolar environment, and the conformational change induced by the binding of cAMP occurred around the tryptophan residue NMR experiment has manifested that the comformational change around the tryptophan residue at position 85 and histidine residue at position 159 of CRP is induced by the binding of cAMP . An extrinsic fluorescence study showed that the distance between the two cysteine 178 residues in the dimer was within about 3.5 A . This distance didn't exhibit a large change upon cAMP binding.

Arch Androl, 1996 Sep-Oct, 37(2), 73 - 8
In vitro effect of Escherichia coli on human sperm acrosome reaction; el-Mulla KF et al.; Escherichia coli are known to reduce human sperm motility . The purpose of the present study was to examine whether these bacteria may also affect the acrosome reaction, which is another important sperm function . The acrosome reaction was determined in spermatozoa from 29 fertile men by triple stain after 3-h incubation at 37 degrees C with 2 x 10(6) E . coli/mL or without bacteria (control) . Each sample was treated with 0.1% DMSO (spontaneous acrosome reaction) or calcium ionophore A23187 (induced acrosome reaction) for 1 h at 37 degrees C . The inducibility of the acrosome reaction was significantly lower in semen samples pretreated with E . coli than in the control samples (9.8 +/- 4.2% vs . 12.7 +/- 5.3%; p < .05) . The results demonstrate that E . coli affect the inducibility of the acrosome reaction in vitro and may impair the fertilizing capacity of human spermatozoa.

Mol Microbiol, 1996 Sep, 21(5), 1037 - 47
Nucleotide sequence and role in DNA amplification of the direct repeats composing the amplifiable element AUD1 of Streptomyces lividans 66; Volff JN et al.; The amplifiable unit of DNA no . 1 (AUD1) of Streptomyces lividans consists of three 1 kb repeats (left direct repeat, LDR; middle direct repeat, MDR; and the slightly different right direct repeat, RDR) and two 4.7 kb repeats alternately arranged in identical orientation to each other . Both 4.7 kb repeats have been sequenced . They are identical and contain one open reading frame (orf4.7) . The deduced amino acid sequence has a low similarity to chitinases, and two amino acid repeats present high similarities to fibronectin type III modules . Sequencing had previously shown that the ORF corresponding to each 1 kb repeat encodes a putative DNA-binding protein . Crude extracts of Escherichia coli overexpressing the orfRDR-encoded protein and of S . lividans Jni1, having a high amplification of AUD1 and therefore orfMDR, were used in gel retardation assays . The orfRDR- and probably the orfMDR-encoded proteins can bind to an imperfect palindromic sequence upstream from MDR and RDR and to another sequence downstream from RDR . An extrachromosomal DNA amplification system was constructed containing different combinations of the sequences composing AUD1 . In mutants having a deletion of the chromosomal AUD1, the 4.7 kb repeats could be reduced in size, mutated or replaced by E . coli DNA without altering the ability to amplify when RDR was present . Therefore, the only function of the 4.7 kb repeats in amplification is to provide directly repeated DNA sequences . When RDR was lacking or mutated, no amplification was observed . This strongly suggests that the DNA-binding protein encoded by orfRDR is required for AUD1 amplification.

Mol Microbiol, 1996 Sep, 21(5), 1029 - 36
The P1 ParA protein and its ATPase activity play a direct role in the segregation of plasmid copies to daughter cells; Davis MA et al.; The P1 ParA protein is an ATPase that recognizes the parA promoter region where it acts to autoregulate the P1 parA-parB operon . The ParB protein is essential for plasmid partition and recognizes the cis-acting partition site parS . The regulatory role of ParA is also essential because a controlled level of ParB protein is critical for partition . However, we show that this regulatory activity is not the only role for ParA in partition . Efficient partition can be achieved without autoregulation as long as Par protein levels are kept within a range of low values . The properties of ParA mutants in these conditions showed that ParA is essential for some critical step in the partition process that is independent of par operon regulation . The putative nucleotide-binding site for the ParA ATPase was identified and disrupted by mutation . The resulting mutant was substantially defective for autoregulation and completely inactive for partition in a system in which the need for autoregulation is abolished . Thus, the ParA nucleotide-binding site appears to be necessary both for the repressor activity of ParA and for some essential step in the partition process itself . We propose that the nucleotide-bound form of the enzyme adopts a configuration that favours binding to the operator, but that the ATPase activity of ParA is required for some energetic step in partition of the plasmid copies to daughter cells.

Mol Microbiol, 1996 Sep, 21(5), 963 - 75
Cloning and characterization of bfpTVW, genes required for the transcriptional activation of bfpA in enteropathogenic Escherichia coli; Tobe T et al.; Expression of the bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is regulated at the transcriptional level by growth phase, temperature, calcium and ammonium . Genes required for the transcriptional activation of bfpA were localized to a 1.8 kb fragment of the enteroadherent factor (EAF) plasmid of EPEC that is separated from the bfp operon by 6 kb . Within this fragment three identically oriented and closely spaced open reading frames (ORFs) were identified and designated bfpT, bfpV and bfpW . bfpT is predicted to encode a 31.8 kDa protein that shares homology with the AraC family of transcriptional regulators, including the presence of a conserved C-terminal DNA-binding helix-turn-helix motif . Insertional inactivation of bfpT led to the loss of bfpA transcription, BfpA protein production and the localized adherence (LA) phenotype; this mutant phenotype could be complemented by introduction of bfpTVW and, on separate plasmids, bfpT + bfpW . However, introduction of bfpT + bfpV, bfpV alone, bfpW alone, or bfpV + bfpW did not enable recovery of the wild-type phenotype . Maximal efficiency of bfpA transcription required all three genes, but bfpV and bfpW each enhanced transcription providing bfpT was also present . A series of deletions of the bfpA upstream promoter region was prepared; with respect to the bfpA transcription start site, sequence between nucleotides -94 and -55 was found to bind bfpT . BfpT also bound a DNA fragment containing the eaeA promoter region on the EPEC chromosome . From these results we conclude that bfpTV W causes transcriptional activation of bfpA, and possibly eaeA, by a trans-acting mechanism that may co-ordinately regulate the expression of EPEC virulence determinants.

Mol Microbiol, 1996 Sep, 21(5), 895 - 9
The Clp ATPases define a novel class of molecular chaperones; Wawrzynow A et al.; The Clp ATPases were originally identified as a regulatory component of the bacterial ATP-dependent Clp serine proteases . Proteins homologous to the Escherichia coli Clp ATPases (ClpA, B, X or Y) have been identified in every organism examined so far . Recent data suggest that the Clp ATPases are not only specificity factors which help to 'present' various protein substrates to the ClpP or other catalytic proteases, but are also molecular chaperones which can function independently of ClpP . This review discusses the recent evidence that the Clp ATPases are indeed molecular chaperones capable of either repairing proteins damaged during stress conditions or activating the initiation proteins for Mu, lambda or P1 DNA replication . A mechanism is suggested to explain how the Clp ATPases 'decide' whether to repair or destroy their protein substrates.

Mol Microbiol, 1996 Sep, 21(5), 887 - 93
Back to log phase: sigma S as a global regulator in the osmotic control of gene expression in Escherichia coli; Hengge-Aronis R; It is now well established that the sigma S subunit of RNA polymerase is a master regulator in a complex regulatory network that governs the expression of many stationary-phase-inducible genes in Escherichia coli . In this review, more recent findings will be summarized that demonstrate that sigma S also acts as a global regulator for the osmotic control of gene expression, and actually does so in exponentially growing cells . Thus, many sigma S-dependent genes are induced during entry into stationary phase as well as in response to osmotic upshift . K+ glutamate, which accumulates in hyperosmotically stressed cells, seems to specifically stimulate the activity of sigma S-containing RNA polymerase at sigma S-dependent promoters . Moreover, osmotic upshift results in an elevated cellular sigma S level similar to that observed in stationary-phase cells . This increase is the result of a stimulation of rpoS translation as well as an inhibition of the turnover of sigma S, which in exponentially growing non-stressed cells is a highly unstable protein . Whereas the RNA-binding protein HF-I, previously known as a host factor for the replication of phage Q beta RNA, is essential for rpoS translation, the recently discovered response regulator RssB, and ClpXP protease, have been shown to be required for sigma S degradation . The finding that the histone-like protein H-NS is also involved in the control of rpoS translation and sigma S turnover, sheds new light on the function of this protein in osmoregulation . Finally, preliminary evidence suggests that additional stresses, such as heat shock and acid shock, also result in increased cellular sigma S levels in exponentially growing cells . Taken together, sigma S function is clearly not confined to stationary phase . Rather, sigma S may be regarded as a sigma factor associated with general stress conditions.

Shock, 1996 Sep, 6(3), 188 - 93
Splanchnic homeostasis during endotoxin challenge in the pig as assessed by microdialysis and tonometry; Oldner A et al.; The splanchnic region is particularly susceptible to shock . The purpose of this study was to evaluate microdialysis of the liver and small intestine as a monitor of splanchnic metabolic deterioration (elevation of lactate and hypoxanthine) in porcine endotoxic shock . Tonometry of the small intestine was used as a reference . Microdialysis probes (liver, ileum, and artery), tonometer, and pulmonary artery catheter were inserted . Eight animals were given Escherichia coli lipopolysaccharide endotoxin (20 micrograms kg-1 h-1 for 2 h) . Five animals served as controls . Measurements were made every half-hour . Three hours after onset of endotoxin challenge, there were significant differences between endotoxin and control groups in intestinal lactate and hypoxanthine, as well as liver lactate, in addition to mucosal pH obtained by tonometry . Lactate elevation in blood was first seen at 4 h, while there was no significant hypoxanthine elevation in arterial blood over 5 h . Hence, data obtained from the splanchnic region became significantly different early, when compared with data obtained from arterial blood . Microdialysis of liver and small intestine as well as intestinal tonometry are sensitive tools for detection of splanchnic metabolic deterioration during endotoxin shock.

Shock, 1996 Sep, 6(3), 164 - 70
Endotoxin-induced alterations in insulin-stimulated phosphorylation of insulin receptor, IRS-1, and MAP kinase in skeletal muscle; Fan J et al.; Sepsis and endotoxin (LPS) have been demonstrated to impair insulin-mediated glucose uptake in skeletal muscle . However, the intracellular mechanism responsible for this defect is not fully defined . The purpose of the present study was to determine whether specific elements of the insulin receptor (IR) signaling pathway in skeletal muscle are altered by LPS . In vivo injection of Escherichia coli LPS resulted in a 44% reduction in whole body glucose disposal under euglycemic hyperinsulinemic conditions, which was largely accounted for by a decreased rate of glycogen synthesis . Scatchard analysis indicated that the number and affinity of the high-affinity insulin binding sites in muscle were similar between control and LPS-treated rats . Western blot analysis indicated that under basal conditions, the levels of total and phosphorylated IR, insulin receptor substrate (IRS)-1, and mitogen-activated protein (MAP) kinase were not significantly different between control and endotoxic rats . In control animals, muscle obtained 2 min after intravenous injection of a maximally stimulating dose of insulin demonstrated a marked increase in the amount of phosphorylated IR (approximately 5-fold), IRS-1 (approximately 10-fold), and MAP kinase (approximately 10-fold) . Insulin-stimulated phosphorylation of IR, IRS-1, and MAP kinase was markedly diminished (approximately 75%, 90%, and 78%, respectively) in LPS-treated rats . However, there was no concomitant reduction in the total abundance of these proteins under hyperinsulinemic conditions . These data demonstrate that LPS alters multiple steps in the insulin signal transduction pathway, but not insulin binding, in skeletal muscle that may mediate the observed impairment in glucose uptake.

Thromb Haemost, 1996 Sep, 76(3), 361 - 8
Activation of factor X by factor VIIa complexed with human-mouse tissue factor chimeras requires human exon 3; Fang CH et al.; In an attempt to define sequence elements in human and mouse tissue factor (TF) that are responsible for the species specificity observed in their interaction with human factor VIIa (HVIIa), we constructed human-mouse chimeric TF cDNAs, inserted them into plasmid vectors, and induced their expression in E . coli . Assays for procoagulant activity were carried out with the resulting E . coli lysates using (HVIIa) human and mouse (MVIIa) . The ratio of the procoagulant activities, HVIIa/MVIIa, revealed that human TF exon 3 was essential for activity when the TF:VIIa complex was formed with HVIIa . By ligating the maltose binding protein (MBP) gene to TF cDNAs it was possible to construct, express and purify MBP-TF chimeras as well as to estimate their specific activities . With selected MBP-TF chimeras and HVIIa we determined kinetic parameters for the activation of human factor X . Replacement of exon 3 in human TF cDNA with the corresponding exon from mouse TF cDNA resulted in both lower affinity for HVIIa and failure to convert bound HVIIa into a potent protease.

J Virol Methods, 1996 Sep, 61(1-2), 113 - 25
A two plasmid co-expression system in Escherichia coli for the production of virion-like reverse transcriptase of the human immunodeficiency virus type 1; Jonckheere H et al.; Many bacterial expression systems have been developed to study the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) . This enzyme exists in the virions as a heterodimer of a 66 kDa (p66) subunit and a 51 kDa (p51) subunit, originating through proteolytic maturation of the p66 subunit . Most expression systems rely on the processing of p66 by bacterial proteases, this results in a p51 subunit with a non-authentic carboxy-terminus . In contrast, the expression system described produces an RT with an authentic carboxy-terminus . This was achieved by the co-expression of the two subunits of HIV-1 RT, which were each cloned on a different, compatible plasmid in Escherichia coli, and by the use of protease inhibitors during cell lysis . This approach enabled us not only to obtain virion-like RT, as verified by mass spectrometry, but also to monitor the effect of mutations in one or both subunits on the activity of RT and on its sensitivity towards RT inhibitors . The co-expression system described represents a useful method to produce HIV-1 RT, both authentic and mutated, in quantities that allow large-scale studies on the functional organisation of the RT-subunits and the sensitivity of the enzyme to RT inhibitors.

Proteins, 1996 Sep, 26(1), 115 - 7
Crystallization and preliminary crystallographic analysis of tetrahydrodipicolinate-N-succinyltransferase; Binder DA et al.; Crystals of tetrahydrodipicolinate-N-succinyltransferase have been obtained from solutions containing 2-propanol and polyethylene glycol 4,000 . These crystals belong to the monoclinic space group P2(1), diffract X-rays to a resolution of 2.2 A, and contain one trimer per asymmetric unit.

Proteins, 1996 Sep, 26(1), 108 - 14
Purification and crystallization of a complex between human interferon gamma receptor (extracellular domain) and human interferon gamma; Windsor WT et al.; X-ray diffraction quality crystals have been obtained from a complex between interferon gamma and the extracellular domain of its high-affinity cell surface receptor . The crystals were obtained from interferon gamma/interferon gamma receptor complexes purified by size exclusion chromatography . Diffraction quality crystals required analyzing these complex samples by isoelectric focusing gels to select purified complex fractions devoid of unbound interferon gamma . These studies used interferon gamma receptor engineered with an eight amino acid N-terminal deletion to eliminate heterogeneity generated due to proteolytic cleavage . In addition, the receptor was expressed in an E . coli secretion cell line which eliminated the need to refold the protein . Hexagonal crystals were grown from 1.6 M ammonium phosphate solutions and belong to a spacegroup of P6(5)22 with unit cell dimensions a = 145.9 A and c = 180.3 A . These crystals diffract to at least 2.9 A resolution when exposed to synchrotron radiation . SDS PAGE analysis of the crystals demonstrated that both interferon gamma and the receptor were present . Analysis of the x-ray diffraction data revealed that the crystals contain complexes with a stoichiometry of 2:1 receptor: ligand within the crystallographic asymmetric unit and consist of approximately 55% solvent.

Proteins, 1996 Sep, 26(1), 95 - 107
Requirements for perpendicular helix pairing; Beasley JR et al.; Cassette mutagenesis was used to produce a library of mutations at the interface of the N- and C-terminal helices of Saccharomyces cerevisiae iso-1-cytochrome c . The library is random and comprises > 98% mutations . Over 11,000 candidates were assayed for function by selecting for the ability of yeast, with the mutated gene as their sole cytochrome c source, to grow on nonfermentable carbon sources . We estimate that approximately 0.5% of the 160,000 total amino acid combinations at these four residues result in a functional cytochrome c . Significant correlations are found between the phenotype of yeast harboring the alleles and both the Dayhoff mutation matrix and transfer free energies (cyclohexane-to-water and n-octanol-to-water) . Similar correlations are observed with respect to growth rate . Finally, sequences that are consistent with function follow a binary amino acid pattern.

Protein Sci, 1996 Sep, 5(9), 1928 - 30
Purification and crystallization of cyclin-dependent kinase inhibitor p21; Mayrose DR et al.; p21, a universal inhibitor of mammalian cyclin-dependent kinases (CDK), regulates cell cycle progression by forming various distinct protein complexes with cyclins, CDKs, and the proliferating cell nuclear antigen . We have overexpressed recombinant human p21 in E . coli and purified active p21 to near homogeneity on a large scale . Crystals of recombinant p21 have been grown in the space group P2(1) a = 157.4, b = 152.7, c = 90.6 A, and beta = 92.7 degrees . The diffraction data of the recombinant p21 have been collected to 2.5 and 3.5 A resolution for the native crystal and two heavy atom derivatives of mercury and iridium.

Protein Sci, 1996 Sep, 5(9), 1844 - 51
Circularly permuted dihydrofolate reductase possesses all the properties of the molten globule state, but can resume functional tertiary structure by interaction with its ligands; Uversky VN et al.; It is obvious that functional activity of a protein molecule is closely related to its structure . On the other hand, the understanding of structure-function relationship still remains one of the intriguing problems of molecular biology . There is widespread belief that mutagenesis presents a real way to solve this problem . Following this assumption, we have investigated the effect of circular permutation in dihydrofolate reductase from E . coli on protein structure and functioning . It has been shown that in the absence of ligands two circularly permuted variants of dihydrofolate reductase possess all the properties of the molten globule state . However, after addition of ligands they gain the native-like structural properties and specific activity . This means that the in vitro folding of permuted dihydrofolate reductase is terminated at the stage of the molten globule formation . Interaction of permuted protein with ligands leads to the structural adjustment and formation of active protein molecules.

Protein Sci, 1996 Sep, 5(9), 1776 - 84
Structural characterization of the tumor suppressor p16, an ankyrin-like repeat protein; Boice JA et al.; The p16 protein has been identified as a tumor suppressor that functions by inhibiting the cyclin-dependent kinases CDK4 and CDK6 . Deletions or point mutations in the p16 gene have been found in a number cancers, emphasizing its importance in regulating cell cycle progression . Inhibition by p16 occurs through protein-protein interactions with its targets . This is not surprising, since p16 is thought to contain ankyrin-like repeats, motifs implicated in protein-protein interactions . Our goal was to identify structural characteristics of p16 not only as an important step towards understanding CDK4 inhibition but also to explore the role of ankyrin repeats in the p16 structure, as no detailed structure of any protein containing these motifs has been reported . We have expressed, refolded, and purified p16 from E . coli and have shown it to be functionally active by specific binding to CDK4 . Analytical ultracentrifugation has shown that p16 weakly self-associates to form dimers with a Kd = 270 microM . The CD spectrum indicates that the protein is composed of 33% alpha-helix, 22% beta-sheet, 19% beta-turn, and 27% other (which includes aromatic and random coil contributions) . Further CD experiments suggest that p16 exhibits low structural stability with a delta G of -2.3 kcal/mol . This weak stability is a consequence of a highly dynamic structure as measured by ANS-binding, NMR hydrogen-deuterium exchange, and fluorescence . It is possible that a well-defined tertiary structure is imparted upon the binding of p16 to CDK4.

Biotechniques, 1996 Sep, 21(3), 492 - 7
Efficiency of transduction by recombinant Sindbis replicon virus varies among cell lines, including mosquito cells and rat sensory neurons; Corsini J et al.; Recombinant alphaviruses have been used as vehicles for delivery and expression of heterologous genes in mammalian, avian and insect cell lines . We have used a Sindbis replicon virus (Sinreplac) able to express the E . coli lacZ gene to compare the efficiency of transduction in one insect, six mammalian cell lines and cultured rat dorsal neurons which apparently express beta-galactosidase over a 30-day time period . Results show that different cell lines were transduced with varying degrees of efficiency and that this efficiency could be improved in some cell lines by packaging the replicon with a helper derived from a more neurovirulent strain of Sindbis.

Biotechniques, 1996 Sep, 21(3), 453 - 7
Automated DNA sequencing requiring no DNA template purification; Chen Q et al.; A critical component in automated fluorescent DNA sequencing is good quality of DNA template . In an effort to reduce the dependence of sequencing success on DNA template quality, the effect of heat-soaked PCR on automated sequencing reactions has been examined . We have found that the heat-soaked PCR protocol considerably improves the overall quality of sequence data and significantly reduces the dependence on the quality of DNA templates . The improvement is corroborated by our ability to obtain over 500 bp of readable sequence per reaction using DNA from E . coli lysates as template obviating DNA purification.

Biotechnol Prog, 1996 Sep-Oct, 12(5), 723 - 7
Polylinker-encoded peptides can confer toxicity to recombinant proteins produced in Escherichia coli; Viaplana E et al.; Three DNA segments encoding fragments of the p60 capsid protein of rabbit haemorrhagic disease virus (RHDV) have been cloned and expressed in Escherichia coli . The cDNAs were placed under the control of the T7 promoter in a pET-derived expression vector designed to produce C-terminal histidine tail fusions . By using different cloning strategies, cell toxicity exhibited by some of the expressed products was dramatically affected, coincident with minor modifications in the carboxy terminus sequences of the recombinant proteins . In particular, the presence of a nonhydrophobic peptide encoded by polylinker sequences promotes cell death and a reduced yield after induction of gene expression, whereas a histidine tail has no detectable effect . These data point out the critical role that needless peptide tails, accidentally introduced into recombinant proteins by nonrelevant DNA stretches, can have on protein expression and final yield of a production process.

Antimicrob Agents Chemother, 1996 Sep, 40(9), 2226 - 8
Glycylcyclines bind to the high-affinity tetracycline ribosomal binding site and evade Tet(M)- and Tet(O)-mediated ribosomal protection; Bergeron J et al.; N,N-dimethylglycylamido (DMG) derivatives of 6-demethyl-6-deoxytetracycline and doxycycline bind 5-fold more effectively than tetracycline to the tetracycline high-affinity binding site on the Escherichia coli 70S ribosome, which correlates with a 10-fold increase in potency for inhibition of E . coli cell-free translation . The potencies of DMG-doxycycline and DMG-6-demethyl-6-deoxytetracycline were unaffected by the ribosomal tetracycline resistance factors Tet(M) and Tet(O) in cell-free translation assays and whole-cell bioassays with a conditional Tet(M)-producing E . coli strain.

Microb Pathog, 1996 Sep, 21(3), 205 - 13
Detection and localization of the EaeA protein of attaching and effacing Escherichia coli O45 from pigs using a monoclonal antibody; Zhu C et al.; The eaeA-positive, attaching and effacing (A/E) O45 E . coli isolates from pigs express an EaeA protein with an estimated molecular weight of 97 kDa . In the present study, a monoclonal antibody was raised against the EaeA protein of an A/E O45 isolate . Cross reaction of the monoclonal antibody with the EaeA protein of A/E strain of the rabbit (RDEC-1), but not with those of A/E strains of the human (E2348/69) and dog (89-4221), was observed . Reactions of the monoclonal antibody to A/E isolates in the O45 serogroup on the ELISA varied among isolates and appeared to be correlated with in vivo A/E capacity of these isolates . The EaeA protein of A/E O45 E . coli has an apparent isoelectric point of 8.4 and is exposed on the bacterial surface . The monoclonal antibody provides a useful tool for characterization of the EaeA protein of E . coli isolates from pigs.

Microb Pathog, 1996 Sep, 21(3), 183 - 92
Characterization of F18 fimbrial genes fedE and fedF involved in adhesion and length of enterotoxemic Escherichia coli strain 107/86; Imberechts H et al.; Infection of susceptible weaned pigs with oedema disease strains of E . coli is associated with bacterial adhesion to the small intestine . F18 fimbria (previously named F107) was the first colonisation factor described on oedema disease strains, and its genetic determinant was cloned . In the present study, genes fedE and fedF were positioned in the F18 gene cluster, downstream of the major structural subunit gene fedA . Two fedE and two fedF mutants were identified that had lost their capacity to adhere to isolated porcine villi . Moreover, these mutants produced significantly longer fimbriae . In vitro adhesion tests, electron microscopy study, transcomplementation tests, and nucleotide sequence analysis indicated that proteins FedE and FedF are F18 minor subunits essential for fimbrial adhesion and effecting fimbrial length.

Microb Pathog, 1996 Sep, 21(3), 157 - 71
Attaching and effacing of host cells by enteropathogenic Escherichia coli in the absence of detectable tyrosine kinase mediated signal transduction; Rabinowitz RP et al.; An unusual mutant of enteropathogenic E . coli (EPEC), deficient in its ability to invade host cells, was evaluated . The gene interrupted by the transposon in this mutant was located within a region of the EPEC chromosome devoted to secretion of proteins required for signal transduction . The mutant did not secrete detectable levels of the EspB protein, previously shown to be required for attaching and effacing, and did not induce detectable tyrosine phosphorylation of a 90 kDa host cell protein, previously associated with attaching and effacing and invasion . No quantitative or qualitative defect in the ability of the mutant to induce attaching and effacing effects was observed . Moreover, attaching and effacing by wild-type EPEC was unaffected by high doses of the tyrosine kinase inhibitor genistein . These results indicate that attaching and effacing activity can occur in the absence of detectable EspB secretion and tyrosine kinase mediated signal transduction.

Clin Diagn Lab Immunol, 1996 Sep, 3(5), 523 - 6
Mapping of the H7-serospecific domain of Escherichia coli flagellin; Kwang J et al.; The amino acid sequences responsible for H7 and H23 flagellum serology have been identified by using a genetic approach . The H7-specific domain was located between amino acids 352 and 374 of the H7 flagellin . The sequencing data also demonstrated that the difference between the H7 and H23 flagellins in this region results from a single substitution at amino acid 366 (Ser-->Thr) . The common epitopes for H7 and H23 were located between amino acids 284 and 366.

Gene Ther, 1996 Sep, 3(9), 825 - 8
In vivo gene transfer to the human biliary tract; Vickers SM et al.; The human biliary tract offers an excellent model for gene transfer studies for a variety of diseases localized to the liver . The aim of this study was to determine if a viable liver might be employed to study viral transfection of the human biliary system in order to mimic in vivo human experiments . Using a normal human liver initially procured for transplantation, but subsequently found unsuitable, and with an intact biliary tree, the hepatic vascular supply was accessed for continuous perfusion . The common and left hepatic biliary system was isolated by balloon catheterization . A replication defective adenoviral vector containing the Escherichia coli beta-galactosidase (lac Z) reporter gene (AdCMVLacZ) was injected into the catheter-isolated left and common bile duct lumen . Viral exposure to the right duct system was prevented by ligation . The bile duct segments were excised and prepared for enzymatic (X-gal) staining . Intense staining was observed in the biliary epithelium exposed to the adenoviral vector . No evidence of beta-galactosidase staining was noted in the unexposed biliary mucosa . We report direct transfection of biliary epithelial cells from normal human liver with a recombinant adenovirus . Our data suggest potential therapeutic applications for gene therapy of hepatobiliary disorders.

Dev Dyn, 1996 Sep, 207(1), 75 - 88
Spatial and temporal activity of the alpha B-crystallin/small heat shock protein gene promoter in transgenic mice; Haynes JI 2nd et al.; In order to study the spatial and temporal activity of the mouse alpha B-crystallin/small heat shock gene promoter during embryogenesis, we generated mice harboring a transgene consisting of approximately 4 kbp of alpha B-crystallin promoter sequence fused to the Escherichia coli lacZ reporter gene . beta-galactosidase activity was first observed in the heart rudiment of 8.5 days post coitum (d.p.c.) embryos . An identical expression pattern was obtained for the endogenous alpha B-crystallin gene by whole mount in situ hybridization . At 9.5 d.p.c., beta-galactosidase activity was detected in the lens placode, in the myotome of the somites, in Rathke's pouch (future anterior pituitary), and in some regions of oral ectoderm . We also examined the stress inducibility of the alpha B-crystallin promoter in vivo . Injection of sodium arsenite into mice resulted in increased endogenous alpha B-crystallin expression in the adrenal gland and possibly the liver . Our results indicate that visualization of beta-galactosidase activity provides an accurate reflection of endogenous alpha B-crystallin expression and demonstrate that the complex developmental pattern of mouse alpha B-crystallin gene expression is regulated at the transcriptional level . This expression pattern, coupled with the present literature which addresses functions of the protein, suggests a role for the alpha B-crystallin/small heat shock protein in intermediate filament turnover and cellular remodeling which occur during normal development and differentiation.

Can J Microbiol, 1996 Sep, 42(9), 934 - 43
Endoglucanase G from Fibrobacter succinogenes S85 belongs to a class of enzymes characterized by a basic C-terminal domain; Iyo AH et al.; A 3.6-kb fragment of the Fibrobacter succinogenes S85 DNA was sequenced and found to contain two open reading frames (ORFs) on the same strand separated by 242 nucleotide bases . The translated protein from ORF1 had a predicted mass of 52.3 kDa . In a region of 320 amino acid overlap, it shares a 35% identity with the b-chain of the glutamate synthase of Escherichia coli . The ORF2 protein encodes a 519 residue protein designated CelG . It consists of an ORF of 1557 bp, encoding a polypeptide of 54.5 kDa . The N-terminal region, which contains the catalytic domain, is linked to a C-terminal basic domain, which has a predicted isoelectric point of 10.8 . The catalytic domain in endoglucanase G (CelG) is homologous to the family 5 (A) cellulases . The enzyme has an apparent mass of 55 kDa, a pH optimum of 5.5, and temperature optimum of 25 degrees C . It had a specific activity of 16.5 mumols x min(-1) x mg-1 on barley b-glucan and produced a mixture of cellooligosaccharides from the hydrolysis of acid swollen cellulose and cellooligosaccharides . Antiserum raised against the purified form of CelG in E . coli failed to react with proteins from the native organism when grown on either glucose or crystalline cellulose, but reverse transcription and polymerase chain reaction techniques using RNA from the native organism demonstrated that the celG gene was expressed constitutively . Its distribution amongst subspecies of Fibrobacter was restricted to F . succinogenes S85.

Am J Physiol, 1996 Sep, 271(3 Pt 2), H891 - 5
Cardiorespiratory and tissue oxygen dose response to rat endotoxemia; Rosser DM et al.; The dose response to endotoxin (Escherichia coli serotype 127:B8) was assessed in a spontaneously breathing, halothane-anesthetized, Sprague-Dawley rat model monitoring blood pressure, aortic and renal blood flows, blood gases, and bladder epithelial PO2, a marker of organ perfusion . The animals received either saline or endotoxin at doses of 1, 10 and 100 mg/kg body wt . Blood pressure changed significantly in all three endotoxin groups, though only the 100 mg/kg group showed significant changes in arterial PCO2, arterial PO2, and body temperature compared with controls . Whereas aortic and renal blood flow rose significantly in the two lower-dose groups, an approximate one-third fall occurred in the 100 mg/kg group (P < 0.001) . Notwithstanding these macrocirculatory hemodynamic changes, both bladder epithelial PO2 and arterial base deficit rose significantly in all groups, though only the base deficit showed a progressive dose response . This model illustrates that responses to endotoxin are dose dependent but with changing patterns for different variables . The consistent finding of an elevated tissue PO2 in endotoxemia, regardless of dose, is suggestive of defective cellular oxygen metabolism.

Am J Physiol, 1996 Sep, 271(3 Pt 1), C825 - 32
Amino-terminal processing of the catalytic subunit from Na(+)-K(+)-ATPase; Pressley TA et al.; The first five amino acids of the catalytic alpha 1-subunit predicted from its cDNA are not found in purified mammalian Na(+)-K(+)-ATPase, suggesting co- or posttranslational cleavage . To facilitate evaluation of amino-terminal structure and the cleavage process, we developed a site-directed antibody (anti-VGR) specific for the first nine residues of nascent alpha 1 from rat . In immunoblots of polypeptides generated by in vitro translation, anti-VGR detected a prominent band with a mobility appropriate for the alpha 1-subunit (100 kDa) . Immunoblots of total protein from various rat organs, however, revealed no significant binding, implying that virtually all the alpha 1-subunit expressed in vivo was modified . We also assessed amino-terminal structure in various heterologous expression systems . Binding of anti-VGR was observed in Escherichia coli transformed with a vector containing an alpha 1/troponin fusion protein and in insect cells infected with baculovirus containing full-length alpha 1 or alpha 1T . This suggests that modification of the introduced alpha 1 in these expression systems was absent or different from that in mammals . In contrast, green monkey kidney cells (COS-1) transfected with alpha 1 did not reveal significant binding of the antibody, indicating that the introduced isoform was processed appropriately . These results demonstrate that the structure of the alpha 1-subunit's amino terminus differs among various expression systems . The results further imply that efficient co- or posttranslational processing of nascent alpha 1 is conserved among various organs within the rat, yet the required modification enzymes are not present in distant phyla.

Eur J Biochem, 1996 Sep 1, 240(2), 454 - 60
The effects of calcium and other polyvalent cations on channel formation by Escherichia coli alpha-hemolysin in red blood cells and lipid bilayer membranes; Dobereiner A et al.; Channel formation by Escherichia coli alpha-hemolysin (HlyA) was studied in lipid bilayer membranes and in erythrocytes as a function of the concentration of divalent and trivalent cations . Hemolysin showed full channel-forming activity in artificial lipid bilayers, even in the presence of 5 mM EDTA and when the E . coli cells were grown in calcium-depleted media (< 1 microM Ca2+) . The addition of divalent cations decreased the single-channel conductance by about 50% with half-saturation constants of 5 mM and less, while the mean lifetime of the HlyA channel was not affected . The addition of trivalent cations, such as Fe3+ or La3+, had a similar effect on the channel conductance, but the half-saturation constant was 1 microM or below . These effects may be caused by the binding of the cations to negatively charged groups at the channel mouth and have probably nothing to do with the possible binding of these cations to the repeat domain of the toxin, which is essential for target cell recognition . When cells were grown in calcium-depleted media, the supernatants showed absolutely no hemolytic activity . Addition of small amounts of Ca2+ to the supernatant led to toxin-mediated hemolysis . Its half-saturation constant was 120 microM . Of the other earth alkaline cations only strontium (Sr2+), which has an ion radius similar to Ca2+, led to full activation of HlyA with a K(m) of 1.5 mM . Ba2+ induced only weak hemolytic activity, while Mg2+ and several heavy metal cations had no effect . These results led to the conclusion that the target cell recognition sites formed by the repeat region of HlyA have defined sizes and bind only ions with defined radii.

Plant Cell, 1996 Sep, 8(9), 1641 - 50
Two members of the thioredoxin-h family interact with the kinase domain of a Brassica S locus receptor kinase; Bower MS et al.; To determine potential targets of the S locus receptor kinase (SRK) during the Brassica self-incompatibility response, a yeast two-hybrid library was screened with the SRK-910 protein kinase domain . Two thioredoxin-h-like clones, THL-1 and THL-2, were found to interact specifically with the SRK-910 protein kinase domain and not to interact with the protein kinase domains from the Arabidopsis receptor-like protein kinases (RLK) RLK4 and RLK5 . The interaction between THL-1 and the SRK-910 protein kinase domain was confirmed using coimmunoprecipitation experiments with fusion proteins produced in Escherichia coli . THL-1 has thioredoxin activity based on an insulin reduction assay, and THL-1 is weakly phosphorylated by the SRK-910 protein kinase domain . THL-1 and THL-2 are both expressed in a variety of tissues but show some differences in steady state mRNA levels, with THL-2 being preferentially expressed in floral tissues . This indicates a more general biological function for these thioredoxins in addition to a potential role as effector molecules in the self-incompatibility signal cascade.

Plant Cell, 1996 Sep, 8(9), 1613 - 26
Functional analysis of the beta and epsilon lycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation; Cunningham FX Jr et al.; Carotenoids with cyclic end groups are essential components of the photosynthetic membranes in all plants, algae, and cyanobacteria . These lipid-soluble compounds protect against photooxidation, harvest light for photosynthesis, and dissipate excess light energy absorbed by the antenna pigments . The cyclization of lycopene (psi, psi-carotene) is a key branch point in the pathway of carotenoid biosynthesis . Two types of cyclic end groups are found in higher plant carotenoids: the beta and epsilon rings . Carotenoids with two beta rings are ubiquitous, and those with one beta and one epsilon ring are common; however, carotenoids with two epsilon rings are rare . We have identified and sequenced cDNAs that encode the enzymes catalyzing the formation of these two rings in Arabidopsis . These beta and epsilon cyclases are encoded by related, single-copy genes, and both enzymes use the linear, symmetrical lycopene as a substrate . However, the epsilon cyclase adds only one ring, forming the monocyclic delta-carotene (epsilon, psi-carotene), whereas the beta cyclase introduces a ring at both ends of lycopene to form the bicyclic beta-carotene (beta, beta-carotene) . When combined, the beta and epsilon cyclases convert lycopene to alpha-carotene (beta, epsilon-carotene), a carotenoid with one beta and one epsilon ring . The inability of the epsilon cyclase to catalyze the introduction of a second epsilon ring reveals the mechanism by which production and proportions of beta,beta- and beta, epsilon-carotenoids may be controlled and adjusted in plants and algae, while avoiding the formation of the inappropriate epsilon,epsilon-carotenoids.

Plant Cell, 1996 Sep, 8(9), 1545 - 53
Physical association of KAB1 with plant K+ channel alpha subunits; Tang H et al.; K+ channel proteins contain four alpha subunits that align along a central axis perpendicular to membranes and form an ion-conducting pore . Recent work with K+ channels native to animal membranes has shown that at least some members of this protein family also have four beta subunits . These structural components of the holoenzyme each form tight associations with the cytoplasmic portion of an alpha subunit . We have cloned an Arabidopsis cDNA (KAB1) that encodes a polypeptide sharing 49% amino acid identity with animal K+ channel beta subunits . In this study, we provide experimental evidence that the KAB1 polypeptide forms a tight physical association with the Arabidopsis K+ channel alpha subunit, KAT1 . An affinity-purified KAB1 fusion protein was immobilized to a support resin and shown to sequester selectively the KAT1 polypeptide . In addition, polyclonal antibodies raised against KAB1 were shown to immunoprecipitate the KAT1 polypeptide as a KAT1-KAB1 protein complex . Immunoblot analysis demonstrated that KAB1 is expressed in Arabidopsis seedings and is present in both membrane and soluble protein fractions . The presence of KAB1 (a soluble polypeptide) in both soluble and membrane protein fractions suggests that a portion of the total amount of native KAB1 is associated with an integral membrane protein, such as KAT1 . The presence of KAB1 in crude protein fractions prepared from different Arabidopsis plant organs was evaluated . High levels of KAB1 protein were present in flowers, roots, and leaves . Immunoblot analysis of protein extracts prepared from broad bean leaves indicated that the KAB1 expression level was 80-fold greater in guard cells than in mesophyll cells . Previous studies of the in situ transcription pattern of KAT1 in Arabidopsis indicated that this alpha subunit is abundantly present in leaves and, within the leaf, exclusively present in guard cells . Thus, KAB1 was determined to be expressed in plant organs (leaves) and cell types (guard cells) that are sites of KAT1 expression in the plant . The in situ expression pattern of KAB1 suggests that it may associate with more than one type of K+ channel alpha subunit . Sequence analysis indicates that KAB1 may function in plant K+ channels as an oxidoreductase . It is postulated that beta subunits native to animal K+ channels act as regulatory subunits through pyridine nucleotide-linked reduction of alpha polypeptides . Although the KAB1 primary structure is substantially different from that of animal beta subunits, amino acid motifs critical for this catalytic activity are retained in the plant beta subunit.

Plant Cell, 1996 Sep, 8(9), 1519 - 32
Apparent redundancy in myb gene function provides gearing for the control of flavonoid biosynthesis in antirrhinum flowers; Moyano E et al.; Two Myb-related transcription factors, Myb305 and Myb340, are expressed specifically in flowers of Antirrhinum . The proteins are structurally very similar throughout their DNA binding domains, implying that they bind to common target motifs . This binding has been demonstrated experimentally . Myb305 has been shown to activate the gene encoding the first enzyme of phenylpropanoid metabolism, phenylalanine ammonia-lyase . We show that Myb340 can also activate transcription from the phenylalanine ammonia-lyase gene promoter and that both transcription factors can activate two other genes involved in flavonoid metabolism, thereby linking early and later steps in plant secondary metabolism . Myb340 is a stronger activator than Myb305, but relatively more Myb305 than Myb340 protein is able to bind to target promoters when both proteins are synthesized in yeast or Escherichia coli, probably as a result of inhibition of Myb340 binding by phosphorylation . This means that Myb305 can compete with Myb340 to reduce its effective transcriptional activation when both transcription factors are expressed in the same cell . This competitive interaction has been demonstrated in plant cells . Expression patterns determined by in situ hybridization showed that the two transcription factors are expressed within the same cells of the flower and imply that the detailed specializations in function of these two apparently redundant transcription factors may be used to provide gears that adjust the rate of induction of secondary metabolism to floral development.

Bioessays, 1996 Sep, 18(9), 757 - 65
Unraveling the late stages of recombinational repair: metabolism of DNA junctions in Escherichia coli; Kuzminov A; DNA junctions are by-products of recombinational repair, during which a damaged DNA sequence, assisted by RecA filament, invades an intact homologous DNA to form a joint molecule . The junctions are three-strand or four-strand depending on how many single DNA strands participate in joint molecules . In E . coli, at least two independent pathways to remove the junctions are proposed to operate . One is via RuvAB-promoted migration of four-strand junctions with their subsequent resolution by RuvC . In vivo, RuvAB and RuvC enzymes might work in a single complex, a resolvasome, to clean DNA from used RecA filaments and to resolve four-strand junctions . An alternative pathway for junction removal could be via RecG-promoted branch migration of three-strand junctions, provided that an as yet uncharacterized endonuclease activity incises one of the strands in the joint molecules.

J Cell Biol, 1996 Sep, 134(6), 1573 - 82
Separable cis-regulatory elements that contribute to tissue- and site-specific alpha 2(XI) collagen gene expression in the embryonic mouse cartilage; Tsumaki N et al.; Type XI collagen is a structural component of the cartilage extracellular matrix and plays an important role in skeletal morphogenesis . As a step toward defining the molecular mechanisms responsible for the regulation of type XI collagen expression, we characterized the promoter region of the mouse alpha 2(XI) collagen gene (Coll1a2) . We also generated transgenic mice harboring various fragments of the promoter and the first intron of Coll1a2 linked to the Escherichia coli beta-galactosidase gene to identify the cis-acting elements responsible for tissue- and site-specific expression during development . Cloning and sequence analysis of the 5' flanking region of Coll1a2 showed that the putative 3' end of the retinoid X receptor beta gene was located 742 bp upstream of the Coll1a2 start site . This suggested that the promoter region of Coll1a2 was localized within this 742-bp sequence, which contained multiple consensus regulatory elements . Examination of the transgenic mice revealed that the longest DNA construct (containing the entire promoter and first intron sequences) directed lacZ expression in the notochord as well as in the primordial cartilage throughout the body, with the pattern of expression mimicking that of endogenous Coll1a2 transcripts . On the other hand, deletion of the upstream approximately 290 bp resulted in the elimination of lacZ expression in the primordial cartilage of the carpals, tarsals, and vertebral bodies, whereas lacZ expression in the notochord and in the other primordial cartilage elsewhere was not affected . Deletion of the first intron sequence also resulted in the loss of lacZ expression in the primordial cartilage of the carpals, tarsals, and vertebral bodies, as well as in the notochord . These results demonstrate that the upstream 742-bp and first intron segments of the mouse Coll1a2 gene contain the necessary information to confer high level tissue-specific expression in mouse embryos . In addition, our observations suggest the presence of site-specific cis-acting elements that control Coll11a2 gene expression in different cartilaginous components of the skeleton.

J Cell Biol, 1996 Sep, 134(6), 1387 - 99
Dynamic measurement of the pH of the Golgi complex in living cells using retrograde transport of the verotoxin receptor; Kim JH et al.; The B subunit of verotoxin (VT1B) from enterohemorrhagic Escherichia coli is responsible for the attachment of the holotoxin to the cell surface, by binding to the glycolipid, globotriaosyl ceramide . After receptor-mediated endocytosis, the toxin is targeted to the Golgi complex by a process of retrograde transport . We took advantage of this unique property of VT1B to measure the pH of the Golgi complex in intact live cells . Purified recombinant VT1B was labeled with either rhodamine or fluorescein for subcellular localization by confocal microscopy . After 1 h at 37 degrees C, VT1B accumulated in a juxtanuclear structure that colocalized with several Golgi markers, including alpha-mannosidase II, beta-COP, and NBD-ceramide . Moreover, colchicine and brefeldin A induced dispersal of the juxtanuclear staining, consistent with accumulation of VT1B in the Golgi complex . Imaging of the emission of fluorescein-labeled VT1B was used to measure intra-Golgi pH (pHG), which was calibrated in situ with ionophores . In intact Vero cells, pHG averaged 6.45 +/- 0.03 (standard error) . The acidity of the Golgi lumen dissipated rapidly upon addition of bafilomycin A1, a blocker of vacuolar-type ATPases, pHG remained constant despite acidification of the cytosol by reversal of the plasmalemmal Na+/H+ antiport . Similarly, pHG was unaffected by acute changes in cytosolic calcium . Furthermore, pHG recovered quickly toward the basal level after departures imposed with weak bases . These findings suggest that pHG is actively regulated, despite the presence of a sizable H+ "leak" pathway . The ability of VT1B to target the Golgi complex should facilitate not only studies of acid-base regulation, but also analysis of other ionic species.

Blood, 1996 Sep 1, 88(5), 1718 - 24
Peripheral blood eosinophilia in owl monkeys is associated with increased eosinophilopoiesis yet depressed recruitment kinetics to the Chemokine RANTES; Albert DL et al.; New world nonhuman primates of the genus Aotus (owl monkeys) can be categorized by 11 distinct karyotypes (K) . It has been demonstrated that monkeys of K-VI persistently have one order of magnitude more eosinophils (EOS) in the peripheral blood than K-I monkeys . The purpose of this study was to investigate the basis for this difference and examine EOS recruitment using two cutaneous models of inflammation . Peripheral blood EOS were isolated on metrizamide gradients to > or = 95% purity and then used for phenotypic studies . There were no significant differences when comparing karyotypes in the ratio of normodense (K-I, 6.4% +/- 3.8%; K-VI, 21.1% +/- 8.8%) EOS or their survival in culture (K-I, 5.3% +/- 2.9% at 72 hours; K-VI, 2.8% +/- 0.7% at 72 hours) (P > .05) . Examination of bone marrow revealed that K-VI monkeys had greater than fivefold more EOS and EOS precursors than K-I animals . To examine EOS function in recruitment, monkeys of each karyotype were given a single intradermal injection of Escherichia coli lipopolysaccharide (LPS) or human recombinant (PMN) and mononuclear cells occurred in response to LPS as early as 4 hours after injection; the severity of infiltration was similar in both karyotypes and at all time points up to 24 hours . In contrast, by 8 hours after intradermal injection of RANTES, leukocyte infiltration in K-I monkeys consisted mostly of PMN (94.8% +/- 0.7%) that were predominantly EOS . In comparison, there was essentially no infiltrate in K-VI animals at all time points . There was no difference in VCAM-1 expression in response to intradermal LPS or RANTES between the two karyotypes . These results suggest that the genetic basis of peripheralblood eosinophilia in K-VI owl monkeys is likely a function of heightened eosinophilopoiesis and depressed recruitment kinetics from the peripheral circulatory pool in response to RANTES.

J Allergy Clin Immunol, 1996 Sep, 98(3), 652 - 8
Comparison of recombinant timothy grass pollen allergens with natural extract for diagnosis of grass pollen allergy in different populations; Laffer S et al.; BACKGROUND: Complementary DNAs coding for the major timothy grass pollen (Phleum pratense) allergens Phl p 1, Phl p 2, and Phl p 5 and birch profilin were isolated, expressed as recombinant nonfusion proteins in Escherichia coli, and purified . OBJECTIVE: In this study the in vitro IgE-binding capacity of recombinant Phl p 1, Phl p 2, Phl p 5, and birch profilin and their IgE recognition frequencies were investigated by using sera from different populations . METHODS: One hundred eighty-three sera from patients allergic to grass pollen were obtained from different populations in Europe, Japan, and Canada . The sera were selected according to clinical criteria, skin testing, and RAST (CAP system; Pharmacia, Uppsala, Sweden) and then tested for IgE reactivity with natural and purified recombinant timothy grass pollen allergens by ELISA and Western blot . RESULTS: Most (94.5%) of the patients allergic to grass pollen could be diagnosed with a combination of recombinant Phl p 1, Phl p 2, Phl p 5, and profilin by means of ELISA . Sera that did not react with the recombinant allergens contained low levels of timothy grass pollen-specific IgE . Although considerable variability in IgE recognition frequency of the recombinant allergens was observed in certain populations, a good correlation was found between natural timothy CAP results and the combination of recombinant allergens in all 183 tested sera (r = 0.87) . CONCLUSIONS: Despite considerable variability in the IgE recognition frequency, purified recombinant timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 5) and profilin permitted successful in vitro diagnosis of grass pollen allergy in 94.5% of allergic individuals from different populations . The addition of other recombinant allergens (e.g., recombinant Phl p 4) would only slightly improve the in vitro test sensitivity.

Am J Obstet Gynecol, 1996 Sep, 175(3 Pt 1), 713 - 8
Usefulness of a new tactile sensor for measurement of uterine cervical ripening in mice in a quantitative and noninvasive manner; Kaga N et al.; OBJECTIVE: The purpose of this study was to establish a method with a new tactile sensor for determining in a quantitative and noninvasive manner the extent of uterine cervical ripening . STUDY DESIGN: We used a newly designed tactile sensor to measure the softness of the cervix in untreated nonpregnant, pregnant, parturient, and nursing mice and then on day 15 of gestation in pregnant mice treated with dehydroepiandrosterone sulfate or Escherichia coli lipopolysaccharide . Additionally, to elucidate the correlation between the extent of cervical softness and its morphologic changes, we observed microscopically the uterine cervices . RESULTS: The hardness of the murine cervix decreased with the progression of pregnancy and became minimal at delivery . We demonstrated for the first time with a tactile sensor for measuring hardness that dehydroepiandrosterone sulfate and lipopolysaccharide significantly decreased the stiffness of the murine cervix . These findings were supported by the morphologic observations on the cervices . CONCLUSIONS: These results show that the tactile sensor for measuring hardness makes it possible to determine the extent of cervical ripening quantitatively rather than qualitatively . We consider that cervical ripening determined objectively in this manner is a good parameter to predict the onset of preterm delivery.

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