Escherichia coli

J Biol Chem, 1996 Mar 29, 271(13), 7559 - 67
Purification and characterization of intact and truncated forms of the Escherichia coli biotin carboxyl carrier subunit of acetyl-CoA carboxylase; Nenortas E et al.; Biotin biosynthesis and retention in Escherichia coli is regulated by the multifunctional protein, BirA . The protein acts as both the transcriptional repressor of the biotin biosynthetic operon and as a ligase for covalent attachment of biotin to a unique lysine residue of the acetyl-CoA carboxylase . Biotinyl-5'-AMP is the activated intermediate for the ligase reaction and the allosteric effector for DNA binding . We have purified and characterized apoBCCP and a truncated form containing the COOH-terminal 87 residues (apoBCCP87) . Molecular masses of the proteins measured using matrix-assisted laser desorption ionization time-of-flight mass spectrometry conformed to the expected values . The assembly states of apoBCCP and apoBCCP87 were determined using sedimentation equilibrium ultracentrifugation . Nearly quantitative enzymatic transfer of biotin from BirA-biotinyl-5'-AMP to the apoBCCP forms was assessed using two methods, mass spectrometric analysis of acceptor proteins after incubation with BirA-bio-5'-AMP and a steady state fluorescence assay . The BirA catalyzed rates of transfer of biotin from bio-5'-AMP to apoBCCP and apoBCCP87 were measured by stopped-flow fluorescence . Kinetic parameters estimated from these measurements indicate that the intact and truncated forms of the acceptor protein are functionally identical.

J Biol Chem, 1996 Mar 29, 271(13), 7535 - 43
Cloning and structure of delta-latroinsectotoxin, a novel insect-specific member of the latrotoxin family: functional expression requires C-terminal truncation; Dulubova IE et al.; The venom of the black widow spider (BWSV) (Latrodectus mactans tredecimguttatus) contains several potent, high molecular mass (>110 kDa) neurotoxins that cause neurotransmitter release in a phylum-specific manner . The molecular mechanism of action of these proteins is poorly understood because their structures are largely unknown, and they have not been functionally expressed . This study reports on the primary structure of delta-latroinsectotoxin (delta-LIT), a novel insect-specific toxin from BWSV, that contains 1214 amino acids . delta-LIT comprises four structural domains: a signal peptide followed by an N-terminal domain that exhibits the highest degree of identity with other latrotoxins, a central region composed of 15 ankyrin-like repeats, and a C-terminal domain . The domain organization of delta-LIT is similar to that of other latrotoxins, suggesting that these toxins are a family of related proteins . The predicted molecular mass and apparent mobility of the protein (approximately 130 kDa) encoded in the delta-LIT gene differs from that of native delta-LIT purified from BWSV (approximately 100 kDa), suggesting that the toxin is produced by proteolytic processing of a precursor . MALDI-MS of purified native delta-LIT revealed a molecular ion with m/z+ of 110916 +/- 100, indicating that the native delta-LIT is 991 amino acids in length . When the full-length delta-LIT cDNA was expressed in bacteria the protein product was inactive, but expression of a C-terminally truncated protein containing 991 residues produced a protein that caused massive neurotransmitter release at the locust neuromuscular junction at nanomolar concentrations . Channels formed in locust muscle membrane and artificial lipid bilayers by the native delta-LIT have a high Ca2+ permeability, whereas those formed by truncated, recombinant protein do not.

J Biol Chem, 1996 Mar 29, 271(13), 7494 - 500
Identification of the high affinity receptor binding region in human immunoglobulin E; Helm BA et al.; We have investigated the capacity of N- and C-terminally truncated and chimeric human (h) IgE-derived peptides to inhibit the binding of 125I-labeled hIgE, and to engage cell lines expressing high and low affinity receptors (Fc-epsilon-RI/II) . The peptide sequence Pro343-Ser353 of the hC-epsilon-3 domain is common to all h-epsilon-chain peptides that recognize hFc-epsilon-RI . This region in IgE is homologous to the A loop in C-gamma-2 that engages the rat neonatal IgG receptor . Optimum Fc-epsilon-RI occupancy by hIgE occurs at pH 6.4, with a second peak at 7.4 . N- or C-terminal truncation has little effect on the association rate of the ligands with this receptor . Dissociation markedly increases following C-terminal deletion, and hFc-epsilon-RI occupancy at pH 6.4 is diminished . His residue(s) in the C-terminal region of the epsilon-chain may thus contribute to the high affinity of interaction . Grafting the homologus rat epsilon-chain sequence into hIgE maintains hFc-epsilon-RI interaction without conferring binding to rat Fc-epsilon-RI . hFc-epsilon-RII interaction is lost, suggesting that these residues also contribute to hFc-epsilon RII binding . h-epsilon-chain peptides comprising only this sequence do not block hIgE/hFc-epsilon-RI interaction or engage the receptor . Therefore, sequences N- or C-terminal to this core peptide provide structures necessary for receptor recognition.

J Biol Chem, 1996 Mar 29, 271(13), 7423 - 8
Selective screening of a large phage display library of plasminogen activator inhibitor 1 mutants to localize interaction sites with either thrombin or the variable region 1 of tissue-type plasminogen activator; van Meijer M et al.; Phage display technology has been exploited to study in detail the interaction between plasminogen activator inhibitor 1 (PAI-1) and either thrombin or an essential positively charged "loop" of tissue-type plasminogen activator (t-PA), denoted variable region 1 (VR1) . For this purpose, a PAI-1 mutant phage library was used that served as a reservoir of PAI-1 proteins potentially deficient in the interaction with either VR1 or thrombin . A stringent two-step selection procedure was developed . (i) A negative selection was performed by incubating the pComb3/PAI-1 mutant library with an excess of a thrombin mutant with its VR1 domain substituted with that of t-PA (thrombin-VR1) . (ii) The remaining phages were complexed with t-PA (positive selection) and selected by panning with an immobilized anti-t-PA monoclonal antibody . Four consecutive panning rounds yielded an enrichment of pComb3/PAI-1 mutant phages of approximately 50-fold . Sequence analysis of 16 different cDNAs, encoding PAI-1 mutants that are hampered in the binding to thrombin-VR1, revealed the following mutations . Four independent variants share a mutation of the P4' residue (Glu350 --> Lys) . Nine independent PAI-1 variants share a substitution of P1' (Met347 --> Lys), whereas three others share a P2 substitution (Ala345 --> Asp) . Kinetic analysis of representative PAI-1 mutants provides evidence that the P4' residue is essential for the interaction with the VR1 domain, consistent with the data of Madison et al . (Madison, E.L., Goldsmith, E.J., Gething, M.J., Sambrook, J.F., and Gerard, R.D . (1990) J . Biol . Chem . 265, 21423-21426), whereas the P1' and P2 residues confer thrombin specificity . Concordant with the design of the selection procedure, mutants were obtained that inhibit thrombin-VR1 at least 100-fold slower than wild-type PAI-1, identifying residues that are central to the interaction with either thrombin or VR1 . This study demonstrates that phage technology can be used to analyze large numbers of mutants defective in their interaction with other (domains of) proteins, provided an adequate selection scheme is devised.

J Biol Chem, 1996 Mar 29, 271(13), 7404 - 11
A novel cytoplasmic hemimethylated oriC binding activity; Garwood J et al.; Using hemimethylated, fully methylated, and unmethylated oligonucleotide probes corresponding to part of the origin of Escherichia coli DNA replication, oriC (+81-136), we have characterized a novel hemimethylated DNA-specific protein binding activity . This activity appears to be located in the cytoplasm rather than in membrane fractions . It has been partially purified and, in DNase footprinting analysis, found to preferentially protect only a subset of the hemimethylated GATC sites present in the minimal oriC . These sites are found adjacent to the DnaA binding box, R1, and overlap the integration host factor binding site . The activity does not correspond to known hemimethylated binding proteins, although in the seqA deletion mutant, there is a 3-fold reduction of the activity . The stage of the cell cycle in synchronized PC2 cultures does not seem to significantly affect thte relative levels of this binding activity . A possible role in sequestration of the newly replicated hemimethylated origin is discussed.

J Biol Chem, 1996 Mar 29, 271(13), 7387 - 91
Site-directed mutagenesis of recombinant sulfite oxidase: identification of cysteine 207 as a ligand of molybdenum; Garrett RM et al.; Each of the four cysteines in rat sulfite oxidase was altered by site-directed mutagenesis to serine, and the mutant proteins were expressed in Escherichia coli . Three of the replacements proved to be silent mutations, while a single cysteine, Cys-207, was found to be essential for enzyme activity . The C207S mutation was also generated in cloned human sulfite oxidase . The mutant human enzyme also displayed severely attenuated activity but was expressed at higher levels allowing purification and spectroscopic analysis . The absorption spectrum of the isolated molybdenum domain of the human C207S mutant displayed marked attenuation of the peak at 350 nm and a lesser decrease in absorbance from 450-600 nm as compared with the native human molybdenum domain . The molybdenum and molybdopterin contents of the two samples were comparable . These data suggest that the major features in the absorption spectrum of the native molybdenum domain arise from the binding of Cys-207 to the molybdenum and indicate that this residue functions as a ligand of the metal.

J Biol Chem, 1996 Mar 29, 271(13), 7343 - 50
Adenovirus-mediated transfer of CCAAT/enhancer-binding protein-alpha identifies a dominant antiproliferative role for this isoform in hepatocytes; Diehl AM et al.; CCAAT/enhancer-binding protein (C/EBP) isoforms are thought to be important regulators of the hepatocyte phenotype . However, the specific physiological roles of different isoforms are poorly understood because hepatocytes express multiple C/EBPs, and various isoforms have overlapping functions . To identify the functions of C/EBPalpha in mature hepatocytes, replication-defective adenovirus vectors were used to efficiently and homogeneously overexpress the mouse C/EBPalpha gene in a SV40 virus-conditionally transformed rat hepatocyte line that can be induced to express C/EBPbeta and C/EBPdelta but that has little endogenous C/EBPalpha expression . Hepatocytes were infected with a recombinant adenovirus vector carrying the cDNA for C/EBPalpha driven by Rous sarcoma virus promoter elements (AdCEBPalpha) or a similar vector carrying the Escherichia coli lacZ gene (Adbetagal) . Staining for beta-galactosidase demonstrated an infection efficiency of 100% at a multiplicity of infection of 25 plaque-forming units/cell and persistence of foreign gene expression for at least 9 days . Cultures infected with AdCEBPalpha had 50-fold higher levels of C/EBPalpha mRNA and protein than those infected with Ad-beta-gal, but similar expression of C/EBP-beta . Infection with AdCEBPalpha inhibited proliferation in cells expressing little C/EBPbeta, even when proliferation was driven by the SV40 transforming antigen, and also blunted mitogenic induction of the c-myc proto-oncogene in nontransformed cells with high levels of C/EBPbeta . Although overexpression of C/EBPalpha consistently increased C/EBPalpha DNA binding activity, it was not sufficient for albumin expression . Infection with AdCEBPalpha only increased albumin mRNA levels in nontransformed cells that also expressed relatively high levels of C/EBPbeta . Thus, in hepatocytes, C/EBPalpha has a dominant antiproliferative function, but must interact with other factors to regulate hepatocyte-specific gene expression.

J Biol Chem, 1996 Mar 29, 271(13), 7269 - 72
Activation of SoxR-dependent transcription in vitro by noncatalytic or NifS-mediated assembly of {2Fe-2S} clusters into apo-SoxR; Hidalgo E et al.; SoxR is a transcriptional activator that senses superoxide and nitric oxide stress in Escherichia coli . The active protein isolated from E . coli contains a pair of {2Fe-2S} clusters per SoxR dimer . We previously demonstrated that the iron-free protein (apo-SoxR), isolated during purification in thiol-containing buffers, binds soxS promoter DNA with an affinity equal to that of the metalloprotein (Fe-SoxR), but lacks significant ability to activate transcription in vitro . Here we demonstrate the reversibility of this process: the full transcriptional activity of SoxR can be restored by in vitro assembly of iron-sulfur clusters into the apoprotein . Two methods were used to synthesize the metallocenters of SoxR: (i) nonenzymatic, in which apo-SoxR, incubated in the presence of iron, inorganic sulfide, and a reducing agent, regained full transcriptional activity in 5-6 h; (ii) enzymatic, in which NifS protein of Azotobacter vinelandii regenerated active Fe-SoxR in as little as 2 min . Analysis by electron paramagnetic resonance spectroscopy indicated that binuclear {2Fe-2S} clusters were restored by both the enzymatic and nonenzymatic reconstitutions . A mutant SoxR protein missing one of its four cysteine residues failed to undergo either transcriptional activation or the formation of {2Fe-2S} centers, even in the presence of NifS . Thus, only the presence of an iron-sulfur center is required to restore transcriptional activity to apo-SoxR . Moreover, the catalytic generation of {2Fe-2S} centers extends the known specificity of this enzyme beyond that already shown for {4Fe-4S} centers . Catalytic generation of {2Fe-2S}-containing SoxR could allow for rapid activation of this transcription factor in vivo.

J Mol Biol, 1996 Mar 29, 257(2), 233 - 45
Selection of Streptomyces griseus protease B mutants with desired alterations in primary specificity using a library screening strategy; Sidhu SS et al.; Streptomyces griseus protease B (SGPB) has primary specificity for large hydrophobic residues . The protease is secreted in a promature form, and autocatalytic removal of the propeptide is essential for activity . We genetically substituted the P1 Leu at the promature junction of SGPB with Phe, Met, or Val and monitored expression levels in Escherichia coli . Substitution with Phe had no effect on active SGPB production; substitution with Met or Val abolished proteolytic activity . An E . coli expression library containing 29,952 possible SGPB mutants was constructed with variations at seven sites involved in conferring primary specificity . A rapid, visual screening strategy was used to detect active protease secretion . The expression library was screened, in conjunction with the different promature junction sequences, for those variants producing increased proteolytic activity . The sequences of the isolated mutant genes were determined; the substrate specificities and thermostabilities of the corresponding protease were investigated . Mutants isolated from the screen with the wild-type promature junction exhibited substrate specificities and thermostabilities similar to wild-type . The screen with Phe at the promature junction P1 site resulted in the isolation of mutant proteases with increased thermostabilities (up to an order of magnitude increase in half-life at 55 degrees C), while a protease with broad substrate specificity was isolated from Val screen . Proteases isolated from the screen with Met at the promature junction P1 site exhibited dramatic increases in activity towards a synthetic substrate with Met at P1 site . The results suggests that the substrate specificity of recombinant SGPB is constrained by the sequence of the promature junction; active protease production is dependent on the efficiency of the self-processive promature junction cleavage . With an efficient screening strategy, this relationship can be used to isolate catalytically active proteases with desired specificities engineered at the promature junction.

Eur J Pharmacol, 1996 Mar 28, 299(1-3), 179 - 86
A glucocorticoid receptor-independent mechanism for neurosteroid inhibition of tumor necrosis factor production; Di Santo E et al.; We investigated the effect of two neurosteroids, pregnenolone and dehydroepiandrosterone sulfate on lipopolysaccharide-induced tumor necrosis factor (TNF) production in vivo and in vitro . Dehydroepiandrosterone sulfate (0.3-30 mg/kg, i.p.) inhibited serum TNF induced by lipopolysaccharide (2.5 micrograms/mouse, i.p.), without affecting the induction of serum corticosterone . Intracerebroventricular (i.c.v.) administration of dehydroepiandrosterone sulfate (0.2-5 micrograms/mouse) also inhibited brain TNF induced by i.c.v . lipopolysaccharide (2.5 micrograms/mouse) . Dehydroepiandrosterone sulfate and pregnenolone (10(-6)-10(-4) M) inhibited TNF production in vitro by lipopolysaccharide-stimulated human peripheral blood mononuclear cells or by the human THP-1 cell line, suggesting that this action might also be relevant in humans . We obtained two lines of evidence that neurosteroids do not inhibit TNF via the glucocorticoid receptor . (1) Dehydroepiandrosterone sulfate and pregnenolone did not activate the alpha 1-acid glycoprotein promoter, a typical effect of glucocorticoids mediated by the glucocorticoid receptor, while strong activation of this promoter was observed with dexamethasone . (2) The inhibitory effect of dehydroepiandrosterone sulfate and pregnenolone on TNF production was not reversed by the glucocorticoid receptor antagonist, mifepristone (RU38486) . On the contrary the inhibitory effect of dexamethasone, a classical glucocorticoid and inhibitor of TNF synthesis, was completely reversed by RU38486.

Eur J Pharmacol, 1996 Mar 28, 299(1-3), 153 - 9
Inhibition of lipopolysaccharide-induced bowel erythrocyte extravasation in rats, and of mesenteric hypoperfusion in dogs, by phosphodiesterase inhibitors; Cardelus I et al.; Sepsis is intricately associated with mesenteric ischemia . The remote complications of mesenteric ischemia are essentially those of sepsis, whether as a cause or as a consequence . Experimental endotoxic shock induces bowel hypoperfusion, erythrocyte extravasation and intestinal necrosis . The effects of pentoxifylline, rolipram and denbufylline, three phosphodiesterase inhibitors, were studied on endotoxin-induced bowel erythrocyte extravasation and intestinal and renal hypoperfusion, in conscious rats and anaesthetized dogs, respectively . Two hours after lipopolysaccharide i.v . injection in rats, erythrocyte extravasation was evident throughout the intestinal musculature and mucosa, apparently without affecting lungs, heart, kidneys, liver or pancreas . Pretreatment with the non-selective phosphodiesterase inhibitor, pentoxifylline, or selective phosphodiesterase IV inhibitors such as denbufylline or rolipram reduced intestinal haemoconcentration . In the anaesthetized dog, pentoxifylline and denbufylline both inhibited the E . coli lipopolysaccharide-induced mesenteric blood flow fall, without affecting renal blood flow or cardiac index . In conclusion, phosphodiesterase inhibitors protected from intestinal damage and bowel hypoperfusion after lipopolysaccharide challenge . This action may thus play a role in the protective effects against endotoxin-induced lethal toxicity previously described for phosphodiesterase inhibitors.

Eur J Pharmacol, 1996 Mar 28, 299(1-3), 119 - 26
A new and potent calmodulin antagonist, HF-2035, which inhibits vascular relaxation induced by nitric oxide synthase; Win NH et al.; HF-2035, 2-{N-(2-aminoethyl)-N-(2,4,5-trichlorobenzenesulfonyl)} amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, was synthesized and its effects on calmodulin-dependent enzymes were investigated . HF-2035 inhibited calmodulin kinase I, calmodulin kinase II and myosin light-chain kinase with IC50 values of 1.3 microM, 1.6 microM and 68 microM, respectively . HF-2035 also inhibited the activity of recombinant rat neuronal nitric oxide synthase, one of the calmodulin-dependent enzymes, with a Ki of 0.78 microM . Partially purified nitric oxide synthase of rat brain was also inhibited by HF-2035 with an IC50 of 3.2 microM . Kinetic analysis indicated that this inhibitory effect of HF-2035 was competitive with respect to calmodulin . We examined the effects of HF-2035 on constitutive nitric oxide synthase in a bioassay using vascular strips of rabbit carotid artery with and without endothelium . HF-2035 inhibited acetylcholine- and calcium ionophore, A23187 (6S-{6 alpha (2S*,3S*),8 beta (R*),9 beta, 11 alpha}-5- (methylamino)-2-{{3,9,11-trimethyl-8-{1-methyl-2-oxo-2-(1H-pyrrol-2-yl)- ethyl}-1,7-dioxaspiro{5.5}undec-2-yl}methyl}-4-benzoxazol ecarboxylic acid)-induced relaxation of endothelium-intact strips with an ED50 of 1.5 +/- 0.5 microM and 2.8 +/- 1 microM, respectively . This compound, however, did not inhibit N-nitroso-N-morpholinoaminoacetonitrile (SIN-1A), an exogenous nitric oxide donor, -induced relaxation of endothelium-denuded strips . W-7 (N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide) inhibited acetylcholine-induced relaxation with an ED50 of 46 +/- 7 microM, which was 30-fold less potent than HF-2035 . HF-2035 was unable to inhibit the activity of the inducible form of nitric oxide synthase in isolated thoracic aorta of rat treated with Escherichia coli lipopolysaccharide . These findings suggest that HF-2035 is a new and potent calmodulin antagonist, and may be used as a mother compound to develop more selective inhibitors of constitutive nitric oxide synthase.

Biochim Biophys Acta, 1996 Mar 28, 1273(3), 207 - 16
K+-dependent Na+ transport driven by respiration in Escherichia coli cells and membrane vesicles; Verkhovskaya ML et al.; Respiration-driven Na+ transport from Escherichia coli cells and right-side-out membrane vesicles is strictly dependent on K+ . Cells from an E . colic mutant deficient in three major K+ transport systems were incapable of accumulating K+ or expelling Na+ unless valinomycin was added . Membrane vesicles from an E . coli mutant from which the genes encoding the two known electrogenic Na+/nH+ antiporters nhaA and nhaB were deleted transported Na+ as well as did vesicles from wild-type cells . Quantitative analysis of Delta psi and Delta pH showed a high driving force for electrogenic Na+/nH+ antiport whether K+ was present or not, although Na+ transport occurred only in its presence . These results suggest that an Na+/nH+ antiporter is not responsible for the Na+ transport . Respiration-driven efflux of Na+ from vesicles was found to be accompanied by primary uphill efflux of K+ . Also, no respiration-dependent efflux of K+ was observed in the absence of Na+ . Such coupling between Na+ and K+ fluxes may be explained by the operation of an Na+, K+/H+ antiporter previously described in E . coli membrane vesicles (Verkhovskay, M.L., Verkhovsky, M.I . and Wikstrom, M . (1995) FEBS Lett . 363, 46-48) . Active Na+ transport is abolished when delta mu H+ is eliminated by a protonophore, but at low concentrations the protonophore actually accelerated Na+ transport . Such an effect may be expected if the Na+, K+/H+ antiporter normally operates in tight conjunction with respiratory chain complexes, thus exhibiting some phenomenological properties of a primary redox-linked sodium pump.

Biochim Biophys Acta, 1996 Mar 28, 1273(3), 191 - 4
Identification of an aspartic acid residue in the beta subunit which is essential for catalysis and proton pumping by transhydrogenase from Escherichia coli; Meuller J et al.; Based on the alignment of 7 unknown amino acid sequences, including the recently determined sequences for the mouse and human enzymes, a highly conserved acidic domain was identified which in the Escherichia coli enzyme is located close to the C-terminal end of the predicted NADP(H)-binding site of the beta subunit . The effect of replacing the four conserved acidic residues, betaE361, betaE374, betaD383 and betaD392, in this domain on catalytic and proton-pumping activity was tested by site-directed mutagenesis . In addition, betaE371, which is not conserved but located in the same domain, was also mutated . Of these residues, betaAsp 392 proved to be the only residue which is essential for both activities . However, two betaAsp 392 mutants were still partly active in catalyzing the cyclic reduction of 3-acetylpyridine-NAD+ by NADH in the presence of NADPH, suggesting that the mutations did not cause a global change but rather a subtle local change influencing the dissociation of NADP(H) . It is proposed that betaAsp 392 together with th previously identified betaHis91 form part of a proton wire in transhydrogenase.

J Immunol Methods, 1996 Mar 28, 190(1), 143 - 5
Comparison of protocols for depleting anti-E . coli antibody in immunoblotting of recombinant antigens; Covini G et al.; A common problem in immunoblotting for the detection of specific antibodies to recombinant proteins synthesized in E . coli is the presence of contaminating antibodies to E . coli proteins . Four protocols for the efficient depletion of anti-E . coli antibodies from human sera are provided and their uses are discussed.

Nature, 1996 Mar 28, 380(6572), 360 - 4
Structural basis of calcium-induced E-cadherin rigidification and dimerization; Nagar B et al.; The cadherins mediate cell adhesion and play a fundamental role in normal development . They participate in the maintenance of proper cell-cell contacts: for example, reduced levels of epithelial cadherin (E-cadherin) correlate with increased invasiveness in many human tumour cell types . The cadherins typically consist of five tandemly repeated extracellular domains, a single membrane-spanning segment and a cytoplasmic region . The N-terminal extracellular domains mediate cell-cell contact while the cytoplasmic region interacts with the cytoskeleton through the catenins . Cadherins depend on calcium for their function: removal of calcium abolishes adhesive activity, renders cadherins vulnerable to proteases (reviewed in ref . 4) and, in E-cadherin, induces a dramatic reversible conformational change in the entire extracellular region . We report here the X-ray crystal structure at 2.0 A resolution of the two N-terminal extracellular domains of E-cadherin in the presence of calcium . The structure reveals a two-fold symmetric dimer, each molecule of which binds a contiguous array of three bridged calcium ions . Not only do the bound calcium ions linearize and rigidify the molecule, they promote dimerization . Although the N-terminal domain of each molecule in the dimer is aligned in a parallel orientation, the interactions between them differ significantly from those found in the neural cadherin (N-cadherin) N-terminal domain (NCD1) structure . The E-cadherin dual-domain structure reported here defines the role played by calcium in the cadherin-mediated formation and maintenance of solid tissues.

Nature, 1996 Mar 28, 380(6572), 316 - 22
Structural similarity between TAFs and the heterotetrameric core of the histone octamer; Xie X et al.; A complex of two TFIID TATA box-binding protein-associated factors (TA FIIs) is described at 2.0A resolution . The amino-terminal portions of dTAFII42 and dTAFII62 from Drosophila adopt the canonical histone fold, consisting of two short alpha-helices flanking a long central alpha-helix . Like histones H3 and H4, dTAFII42 and dTAFII62 form an intimate heterodimer by extensive hydrophobic contacts between the paired molecules . In solution and in the crystalline state, the dTAFII42/dTAFII62 complex exists as a heterotetramer, resembling the (H3/H4)2 heterotetrameric core of the histone octamer, suggesting that TFIID contains a histone octamer-like substructure.

Biochem Biophys Res Commun, 1996 Mar 27, 220(3), 816 - 23
The dimerization domain upstream binding factor contains multiple helical structures; Lai YS et al.; The upstream binding factor, UBF, is an RNA polymerase I transcription factor which contains multiple DNA binding domains and a novel protein dimerization domain . Active UBF forms homodimers in vivo through the intramolecular interactions of its dimerization domain, which spans a hundred amino-terminal residues . In the presence of both UBF dimerization domain and its immediately adjacent lysine-rich basic DNA binding domain, the E . coli expressed recombinant polypeptide, dbUBF (dimerization plus basic motifs of UBF), forms homodimers in vitro and binds to double-stranded DNA nonselectively . In gel retardation assay, dbUBF dimers make multiple shift-ladders corresponding to numerous protein dimer-DNA complexes . The UBF dimerization domain contains multiple helical structures, as predicted by EMBO-PHD program . Most of hydrophobic residues in the dimerization domain are confined in the hydrophobic phase of these hypothetic helices . Mutating these hydrophobic residues to glutamate prohibits dbUBF association and gives a different shift pattern in gel retardation assay . The results we present here argue that UBF association is largely exerted by the hydrophobic interactions between the multiple helices to bring two molecules together.

Biochem Biophys Res Commun, 1996 Mar 27, 220(3), 633 - 6
Depth-targeted efficient gene delivery and expression in the skin by pulsed electric fields: an approach to gene therapy of skin aging and other diseases; Zhang L et al.; The ability to target genes to the various layers, cell types, and appendages of the skin could be used to correct disorders, including those of aging such as wrinkling, as well as utilize specific cell types for production molecules useful elsewhere in the body . However, the stratum corneum acts as a significant physical barrier to gene transfer into the skin . In this report we describe the ability to target and express the lacZ reporter gene to various depths of the dermis region in hairless mice . Skin-depth targeting is achieved by varying pulsed electrical fields and subsequent pressure from caliper-type electrodes on topically applied naked lacZ gene constructs . With electric pulses and extended pressure, the maximum depth of lacZ expression in the dermis and transfected cells was achieved at 370 micron and 457 cells/mm2, respectively . Gene expression was observed only the hair follicles in the case of the control.

Biochem Biophys Res Commun, 1996 Mar 27, 220(3), 496 - 501
Mutagenic specificity of ultraviolet light in the tonB gene on the chromosome of Escherichia coli uvrA cells; Kitamura K et al.; We have analyzed the DNA sequence changes in a total of 60 ultraviolet-induced mutations in the endogenous tonB gene of Escherichia coli uvrA strain . Of the mutations 82% were base substitutions among which G:C-->A:T transition predominated . Three GG-->AA tandem double-base substitutions, which are thought to originate from UV damage, were also observed . The sites where base substitutions occurred were correlated with sequences of adjacent pyrimidines, indicating mutation-targeted UV photoproducts . G:C-->A:T transition in the tonB gene mutation can be exclusively observed at either the 3' side of the TC site which is on the template for the lagging strand of DNA replication or the 5'/3' sides of the CC site on the template for the leading strand . We hypothesize that this extreme strand specificity is due to a difference in fidelity of DNA replication of the leading and the lagging strand.

Biochem Biophys Res Commun, 1996 Mar 27, 220(3), 1002 - 7
High reductase activity of recombinant NOS2 flavoprotein domain lacking the calmodulin binding regulatory sequence; Rafferty S et al.; Nitric oxide synthases (NOSs) represent a special case of the cytochrome P450: cytochrome P450 reductase system in which the two redox partners occur as distinct domains on the same polypeptide chain and are linked by a calmodulin binding regulatory sequence . We expressed the carboxy-terminal, flavoprotein domain (residues 527-1144) of murine NOS2 in E . coli . The UV-visible spectrum of this domain resembles those of other flavoproteins, and the protein catalyses the reduction of ferricytochrome c by NADPH {Vmax = 3.1 +/- .1 mol cytochrome reduced/minute/mmol flavoprotein domain, Km (cytochrome c) = 23 +/- 2 microM, Km (NADPH) 0.30 +/- .06 microM} . The high reductase activity of this NOS2 flavoprotein domain, which lacks a calmodulin binding site, precludes a role for this site in mediating electron transfer within the flavoprotein domain of the intact enzyme . This is in contrast to the case of NOS1 and suggests that electron transfer is regulated differently in the two isoforms.

Biochemistry, 1996 Mar 26, 35(12), 3837 - 44
Zinc stimulates Mg2+-dependent 3'-processing activity of human immunodeficiency virus type 1 integrase in vitro; Lee SP et al.; Human immunodeficiency virus type 1 integrase (HIV-1 IN) catalyzes both 3'-donor processing and strand transfer reactions . Previous studies have determined that the N-terminal region, a putative zinc finger, is capable of binding Zn2+ . The function of zinc coordination to this domain, however, is still unknown . In this report, we present evidence that Mg2+-dependent 3'-donor processing by HIV-1 IN is enhanced by the addition of Zn2+ in vitro . This activity is inhibited in the presence of the chelator 1,10-phenanthroline (OP) . In addition, the Mg2+-dependent 3'-donor processing activity is more sensitive to the concentration of IN than is the Mn2+-dependent activity . A combination of dimethyl sulfoxide (DMSO) and poly(ethylene glycol) (PEG) was found to further activate the Mg2+-dependent 3'-donor processing activity while diminishing the Mn2+-dependent activity . These results suggest factors such as substrate-length, concentration of IN, Zn2+ coordination, and protein-protein interactions are important for efficient and specific donor processing activity with Mg2+ in vitro.

Biochemistry, 1996 Mar 26, 35(12), 3735 - 45
Interactions between RNA polymerase and the positive and negative regulators of transcription at the Escherichia coli gal operon; Dalma-Weiszhausz DD et al.; The simultaneous binding of Gal repressor (GalR), catabolite activator protein (CAP or CRP), and RNA polymerase (RNAP) to the promoter region of the Escherichia coli gal operon has been analyzed thermodynamically, by quantitative DNase I "footprint" titration analysis, and structurally, by the use of hydroxyl radical (.OH) and 5-phenylphenanthroline (5OPP) "footprinting." In the absence of regulatory proteins, the preference of RNAP for one (P1) of the two gal operon overlapping promoters (P1 and P2) is -0.4 +/- 0.2 kcal/mol, indicating only a small energetic preference for P1 . The simultaneous binding of CAP and RNAP occurs with 10-fold cooperativity, with greater than 99% of the CAP-RNAP complex present at the P1 promoter . This cooperativity is inhibited by the binding of GalR to the upstream operator, OE, but does not result in the repartitioning of RNAP between the P1 and P2 promoters . These results suggest that the CAP-RNAP cooperativity and promoter partitioning are not linked and are consistent with a mechanism by which GalR binding to OE represses transcription by inhibiting the CAP-RNAP cooperativity . It is suggested that the CAP-RNAP cooperativity is dependent upon contacts made by the complex with the upstream DNA and that GalR binding to OE prevents these contacts from occurring . Changes in nuclease reactivity at the internal operator OI (centered at +53.5) take place upon RNAP binding . These changes are dependent on the DNA sequence present at OI and on the presence or absence of CAP . They are independent of the helical phasing between the promoters and OI and of the distance between them . These results suggest that RNAP can directly communicate with events occurring at both the external and the internal operator sequences without direct contact between repressor molecules bound at their cognate sites.

Biochemistry, 1996 Mar 26, 35(12), 3704 - 11
Phosphorylation of beta III-tubulin; Khan IA et al.; There is considerable evidence that mammalian beta-tubulin is phosphorylated . Specifically, of the seven beta isotypes, the phosphorylated one is beta III, the isotype found almost entirely in neurons . The phosphate is added at a serine and perhaps a tyrosine near the C-terminus . All the evidence to date has been gathered by growth of cells and tissues in the presence of radioactive inorganic phosphate followed by tubulin isolation and determination of the labeled tubulin; thus, the actual extent of phosphorylation of beta III is unknown . Nor is it known if alpha-tubulin and the other beta isotypes are phosphorylated by a mechanism which would not be revealed by previous experiments . In addition, the role of tubulin phosphorylation is unknown . We have purified the alpha beta II-, alpha beta III-, and alpha beta IV-tubulin dimers from bovine brain and have determined their phosphate content chemically . We have found that alpha-tubulin is not phosphorylated and neither are the beta II or beta IV isotypes . However, beta III is phosphorylated with a stoichiometry of about 1.52 mol/mol . We have found that the phosphate on beta III is resistant to a wide variety of phosphatases except for human erythrocyte phosphatase 2A and that removal of the phosphate inhibits microtubule assembly in vitro stimulated by microtubule-associated protein 2 (MAP 2) . However such an inhibition was not evident when microtubule assembly was induced in the absence of microtubule-associated proteins . Our results suggest the possibility that beta III phosphorylation may play a role in regulating microtubule assembly in vivo.

Mutat Res, 1996 Mar 26, 351(1), 33 - 43
Mutagenicity of acridines in a reversion assay based on tetracycline resistance in plasmid pBR322 in Escherichia coli; Hoffman GR et al.; The mutagenicity of a series of acridine compounds was studied in an assay based on the reversion of mutations in the tetracycline-resistance gene (tet) of plasmid pBR322 in Escherichia coli . Mutations that restore the tetracycline-resistant phenotype were detected in tetracycline-sensitive strains carrying mutant plasmids . Mutations that revert by +2, +1, -1 and -2 frameshift mutations and by base-pair substitutions were used to analyze the mutagenicity of two simple acridines, two acridine mustards, and a nitroacridine . The simple acridines (9-aminoacridine and quinacrine) effectively induced -1 frameshifts and weakly induced +1 frameshifts . The acridine mustards (quinacrine mustard and ICR-191) were more potent inducers of -1 and +1 frameshifts than the simple acridines . Reactive acridines, including both the mustards and the nitroacridine Entozon, were effective inducers of -2 frameshifts but the simple acridines were not . The two classes of reactive acridines differed from one another, in that the mustards were better inducers of +1 frameshifts than Entozon, whereas Entozon was a particularly potent inducer of -2 frameshifts . None of the compounds induced +2 frameshifts, and the induction of base-pair substitutions was negligible . These results confirm and extend studies showing that adduct-forming acridines are stronger frameshift mutagens than simple intercalating acridines and that the acridines differ from one another not only in overall mutagenic potency but also in the prevalence of different classes of frameshift mutations.

FEBS Lett, 1996 Mar 25, 383(1-2), 51 - 4
Potato yellow mosaic geminivirus AC2 protein is a sequence non-specific DNA binding protein; Sung YK et al.; The AC2 protein of potato yellow mosaic geminivirus (PYMV) is by analogy with related geminiviruses thought to be a transcriptional activator protein . We have over-expressed the AC2 open reading frame in E . coli and purified the protein from bacterial extracts to near homogeneity . We have studied the interaction of the AC2 protein with DNA and from gel retardation assays shown that it binds both double-stranded (ds) and single-stranded (ss) DNA non-specifically . The binding to PYMV intergenic region ds DNA appeared to be independent of the presence of zinc ions and did not require the protein to be phosphorylated.

FEBS Lett, 1996 Mar 25, 383(1-2), 124 - 8
Characterization of the dystrophin-syntrophin interaction using the two-hybrid system in yeast; Castello A et al.; The carboxy-terminal region of dystrophin has previously been shown to interact directly with alpha1 syntrophin, a cytoplasmic component of the dystrophin-glycoprotein complex, by in vitro biochemical studies such as overlay assay or immunoprecipitation . Using the two-hybrid system, we have isolated from a human heart cDNA library the entire coding sequence of human alpha1 syntrophin, therefore confirming for the first time this interaction via an in vivo approach . In addition, we have reduced the interaction domain to the distal half of alpha1 syntrophin.

FEBS Lett, 1996 Mar 25, 383(1-2), 119 - 23
Production and crystallization of MHC class I B allele single peptide complexes; Reid SW et al.; Major histocompatibility complex class I B alleles, HLA B8, B53 and B3501 have been cloned, expressed, refolded and crystallized in specific complexes with a number of different 8-mer and 9-mer peptides . For some of these crystallization was initiated by cross-seeding between different B allele complexes . All crystallize in the space group P212121, with similar unit cell dimensions of approximately 52 A X 81 A X 112 A, contain one complex per asymmetric unit and diffract to approximately 2.0 A resolution.

FEBS Lett, 1996 Mar 25, 383(1-2), 105 - 8
Conformational changes in subdomain I of actin induced by proteolytic cleavage within the DNase I-binding loop: energy transfer from tryptophan to AEDANS; Kuznetsova I et al.; Alteration of the actin polypeptide chain within the DNase I-binding loop by cleavage with E . coli A2 protease or subtilisin was shown to increase the efficiency of energy transfer from tryptophan residues to AEDANS attached to Cys-374 . Analysis of structural and fluorescence data suggested that only two of four actin tryptophan residues, namely, Trp-340 and/or Trp-356, can be energy transfer donors . It was also found that labelling with AEDANS induces perturbations in the environment of the tryptophan residues, these perturbations being smaller in the cleaved actin . These changes are consistent with a shift of the C-terminal segment of actin monomer upon cleavage and confirm the existence of high conformational coupling between subdomains 1 and 2 of actin monomer . We also suggest that tryptophan residues 340 and/or 356 are located in the focus of this coupling.

Mol Cell Biochem, 1996 Mar 23, 156(2), 117 - 24
Reduction of methylglyoxal in Escherichia coli K12 by an aldehyde reductase and alcohol dehydrogenase; Misra K et al.; Two enzymes, one NADPH-dependent and another NADH-dependent which catalyze the reduction of methylglyoxal to acetol have been isolated and substantially purified from crude extracts of Escherichia coli K12 cells . Substrate specificity and formation of acetol as the reaction product by both the enzymes, reversibility of NADH-dependent enzyme with alcohols as substrates and inhibitor study with NADPH-dependent enzyme indicate that NADPH-dependent and NADH-dependent enzymes are identical with an aldehyde reductase (EC 1.1.1.2) and alcohol dehydrogenase (EC 1.1.1.1) respectively . The K(m) for methylglyoxal have been determined to be 0.77 mM for NADPH-dependent and 3.8 mM for NADH-dependent enzyme . Stoichiometrically equimolar amount of acetol is formed from methylglyoxal by both NADPH- and NADH-dependent enzymes . In phosphate buffer, both the enzymes are active in the pH range of 5.8-6.6 with no sharp pH optimum . Molecular weight of both the enzymes were found to be 100,000 +/- 3,000 by gel filtration on a Sephacryl S-200 column . Both NADPH- and NADH-dependent enzymes are sensitive to sulfhydryl group reagents.

Carbohydr Res, 1996 Mar 22, 283, 27 - 51
Syntheses of 1-O-carboxyalkyl GLA-60 analogues; Shiozaki M et al.; As part of our ongoing study to survey potent LPS antagonists, the following six compounds were synthesized in an efficient manner: 3-carboxypropyl and carboxymethyl 2-deoxy-2-(2,2-difluorotetradecanamido)-4-O-phosphono-3-O-{(R)-3- (tetradecanoyloxy)tetradecanoyl}-alpha- and beta-D-glucopyranosides (11 and 23; 32 and 36), as well as the non-fluorinated equivalents, carboxymethyl 2-deoxy-4-O-phosphono-2-tetradecanamido-3-O-{(R)-3-(tetradecano yloxy)- tetradecanoyl}-alpha-D-glucopyranoside (44) and carboxymethyl 2-deoxy-2-{(R)-3-(hydroxy)tetradecanamido}-4-O-phosphono-3-O-{(R)- 3- (tetradecanoyloxy)tetradecanoyl}-alpha-D-glucopyranoside (48) . Of these compounds, 32 was most pronounced in terms of LPS-antagonistic activity.

J Biol Chem, 1996 Mar 22, 271(12), 7212 - 7
GroEL binds to and unfolds rhodanese posttranslationally; Reid BG et al.; The Escherichia coli chaperone GroEL is a member of a class of molecular chaperones that possesses a stacked double ring structure containing seven subunits per ring, with approximately 60-kDa subunits . It has been suggested that newly synthesized proteins may interact with a eukaryotic homolog of GroEL co-translationally, thereby sequestering the unfolded protein from other proteins in the cell . To test whether it is essential for GroEL to form a stable interaction with a nascent polypeptide co-translationally, we translated the well studied GroEL substrate rhodanese in bacterial and wheat germ translation extracts . We found that rhodanese formed stable complexes with GroEL solely posttranslationally . Upon binding to GroEL, the protease resistant N-terminal domain of rhodanese unfolds . This interaction with GroEL leads to productive folding of the full-length rhodanese . We conclude that GroEL is able to assist in the folding of newly synthesized proteins following release from the ribosome and that GroEL can unfold a trapped protein folding intermediate of rhodanese.

J Biol Chem, 1996 Mar 22, 271(12), 7177 - 86
Analysis of incision sites produced by human cell extracts and purified proteins during nucleotide excision repair of a 1,3-intrastrand d(GpTpG)-cisplatin adduct; Moggs JG et al.; Nucleotide excision repair by mammalian enzymes removes DNA damage as part of approximately 30-mer oligonucleotides by incising phosphodiester bonds on either side of a lesion . We analyzed this dual incision reaction at a single 1,3-intrastrand d(GpTpG)-cisplatin cross-link in a closed circular duplex DNA substrate . Incisions were formed in the DNA with human cell extracts in which DNA repair synthesis was inhibited . The nicks were mapped by restriction fragment end labeling and primer extension analysis . Principal sites of cleavage were identified at the 9th phosphodiester bond 3' to the lesion and at the 16th phosphodiester bond 5' to the lesion . The predominant product was found to be a 26-mer platinated oligonucleotide by hybridization to a 32P-labeled complementary DNA probe . Oligonucleotides were formed at the same rate as the 3' cleavage, suggesting that both incisions are made in a near-synchronous manner . There was, however, a low frequency of 5' incisions in the absence of 3' cleavage . The dual incision reaction was reconstituted using the purified mammalian proteins XPA, RPA, XPC, TFIIH, XPG, and a fraction containing ERCC1-XPF and IF7 . All of these components were required in order to observe any cleavage.

J Biol Chem, 1996 Mar 22, 271(12), 7072 - 8
The plasmid RK2 initiation protein binds to the origin of replication as a monomer; Toukdarian AE et al.; The TrfA protein encoded by the broad host range bacterial plasmid RK2 specifically binds to eight direct repeats (iterons) present at the plasmid replication origin to initiate DNA replication . Purified TrfA protein is largely in the form of a dimer, and using a dimerization test system that involves the fusion of the amino-terminal domain of the lambda cI repressor protein to TrfA, we show that the TrfA protein forms dimers in vivo . Because of the high stability of the dimer form of TrfA, the formation of heterodimers between the wild-type and different sized TrfA proteins requires in vivo de novo folding of the primary protein sequence or in vitro denaturation and renaturation . The results of gel mobility shift assays using in vitro or in vivo formed heterodimers indicated that the TrfA protein binds to the iteron DNA as a monomer . Furthermore, when the monomeric and dimeric forms of TrfA are separated by gel filtration chromatography, only the protein in the chromatographic position of the monomeric form demonstrated significant DNA binding activity . These results indicate that only the monomer form of the TrfA protein is active for binding to the iterons at the RK2 replication origin.

J Biol Chem, 1996 Mar 22, 271(12), 7052 - 60
A novel plant calmodulin-binding protein with a kinesin heavy chain motor domain; Reddy AS et al.; Calmodulin, a ubiquitous calcium-binding protein, regulates many diverse cellular functions by modulating the activity of the proteins that interact with it . Here, we report isolation of a cDNA encoding a novel kinesin-like calmodulin-binding protein (KCBP) from Arabidopsis using biotinylated calmodulin as a probe . Calcium-dependent binding of the cDNA-encoded protein to calmodulin is confirmed by 35S-labeled calmodulin . Sequence analysis of a full-length cDNA indicates that it codes for a protein of 1261 amino acids . The predicted amino acid sequence of the KCBP has a domain of about 340 amino acids in the COOH terminus that shows significant sequence similarity with the motor domain of kinesin heavy chains and kinesin-like proteins and contains ATP and microtubule binding sites typical of these proteins . Outside the motor domain, the KCBP has no sequence similarity with any of the known kinesins, but contains a globular domain in the NH2 terminus and a putative coiled-coil region in the middle . By analyzing the calmodulin binding activity of truncated proteins expressed in Escherichia coli, the calmodulin binding region is mapped to a stretch of about 50 amino acid residues in the COOH terminus region of the protein . Using a synthetic peptide, the calmodulin binding domain is further narrowed down to a 23-amino acid stretch . The synthetic peptide binds to calmodulin with high affinity in a calcium-dependent manner as judged by electrophoretic mobility shift assay of calmodulin-peptide complex . The KCBP is coded by a single gene and is highly expressed in developing flowers and suspension cultured cells . Although many kinesin heavy chains and kinesin-like proteins have been extensively characterized at the biochemical and molecular level in evolutionarily distant organisms, none of them is known to bind calmodulin . The plant kinesin-like protein with a calmodulin binding domain and a unique amino-terminal region is a new member of the kinesin superfamily . The presence of a calmodulin-binding motif in a kinesin heavy chain-like protein suggests a role for calcium and calmodulin in kinesin-driven motor function(s) in plants.

J Biol Chem, 1996 Mar 22, 271(12), 7038 - 42
A mutation in which alanine 128 Is replaced by aspartic acid abolishes dimerization of the b-subunit of the F0F1-ATPase from Escherichia coli; Howitt SM et al.; Site-directed mutagenesis was used to investigate the roles of a short series of hydrophobic amino acids in the b-subunit of the Escherichia coli F0F1-ATPase . A mutation affecting one of these, G131D, had been previously characterized and was found to interrupt assembly of the F0F1-ATPase (Jans, D . A., Hatch, L., Fimmel, A . L., Gibson, D., and Cox, G . B . (1985) J . Bacteriol . 162, 420-426) . To extend this work, aspartic acid was substituted for each one of the residues from positions 124 to 132 . The properties of mutants in this series are consistent with the region from Val124 to Gly131 forming an alpha-helix . Two of the mutations, V124D and A128D, resulted in a similar phenotype to the G131D mutation . This suggested that Val124, Ala128, and Gly131 form a helical face which may have a role in inter- or intrasubunit interactions . This was tested by overexpressing and purifying the cytoplasmic domains of the wild type and A128D mutant b-subunits . Sedimentation equilibrium centrifugation indicated that the wild type domain formed a dimer whereas the mutant was present as a monomer.

J Biol Chem, 1996 Mar 22, 271(12), 6947 - 51
Activation of Gsalpha by the epidermal growth factor receptor involves phosphorylation; Poppleton H et al.; Previous studies from our laboratory have shown that epidermal growth factor (EGF) stimulates cAMP accumulation in the heart via a process involving Gsalpha and the EGF receptor (EGFR) protein tyrosine kinase activity (Nair, B . G., Parikh, B., Milligan, G., and Patel, T . B . (1990) J . Biol . Chem . 265, 21317-21322; Nair, B . G., and Patel, T . B . (1993) Biochem . Pharmacol . 46, 1239-1245) . Therefore, studies were performed to investigate the hypothesis that the EGFR protein tyrosine kinase phosphorylates Gsalpha and activates this protein . Employing purified EGFR and Gsalpha, we have demonstrated that the EGFR kinase phosphorylates Gsalpha in a time-dependent manner with a stoichiometry of 2 mol of phosphate incorporated/mol of Gsalpha . As determined by phosphoamino acid analysis, the phosphorylation of Gsalpha by the EGFR kinase was exclusively on tyrosine residues . Interestingly, GDP and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) inhibited the phosphorylation of Gsalpha without altering EGFR autophosphorylation . However, G protein betagamma subunits protected against GDP- and GTPgammaS-mediated inhibition of phosphorylation of Gsalpha . In functional studies, phospho-Gsalpha demonstrated a greater GTPase activity and also a greater capacity to bind GTPgammaS as compared to the nonphosphorylated Gsalpha . Moreover, the phospho-Gsalpha augmented adenylyl cyclase activity in S49 cyc- cell membranes to a greater extent than its nonphosphorylated counterpart . Therefore, we conclude that phosphorylation of Gsalpha on tyrosine residues by the EGFR kinase activates this G protein and increases its ability to stimulate adenylyl cyclase.

J Biol Chem, 1996 Mar 22, 271(12), 6895 - 902
Human stem cell factor dimer forms a complex with two molecules of the extracellular domain of its receptor, Kit; Philo JS et al.; Stem cell factor (SCF) is a cytokine that is active toward hematopoietic progenitor cells and other cell types, including germ cells, melanocytes, and mast cells, which express its receptor, the tyrosine kinase, Kit . SCF exists as noncovalently associated dimer at concentrations where it has been possible to study its quaternary structure; it stimulates dimerization and autophosphorylation of Kit at the cell surface . We have used recombinant versions of human SCF and human Kit extracellular domain (sKit) to study SCF-Kit interactions . By size exclusion chromatography, plus various physical chemical methods including light scattering, sedimentation equilibrium, and titration calorimetry, we demonstrate the formation of complexes containing a dimer of SCF (unglycosylated SCF1-165) plus two molecules of sKit . The concentrations of SCF and sKit in these studies were in the range of 0.35-16.2 microM . The data are analyzed and discussed in the context of several possible models for complex formation . In particular, the sedimentation data are not consistent with a model involving cooperative binding . The Kd estimate for SCF-sKit interaction, obtained by sedimentation equilibrium, is about 17 nm at 25 degrees C . With glycosylated SCF1-165, the Kd is considerably higher.

J Biol Chem, 1996 Mar 22, 271(12), 6827 - 31
The free radical of the anaerobic ribonucleotide reductase from Escherichia coli is at glycine 681; Sun X et al.; The anaerobic ribonucleoside triphosphate reductase of Escherichia coli is an iron-sulfur protein carrying an oxygen-sensitive organic radical, which is essential for catalysis . The radical was tentatively proposed to be on glycine 681, based on a comparison with the glycyl radical-containing enzyme pyruvate formate-lyase . By EPR spectroscopy of selectively 2H- and 13C-labeled anaerobic ribonucleotide reductase, the radical was now unambiguously assigned to carbon-2 of a glycine residue . The large 1H hyperfine splitting (1.4 millitesla) was assigned to the alpha-proton . Site-directed mutagenesis was used to change glycine 681 into an alanine residue . In separate experiments, the two adjacent residues, cysteine 680 and tyrosine 682, were changed into serine and phenylalanine, respectively . All mutated proteins were retained on dATP-Sepharose, indicating that the mutant proteins had intact allosteric sites . They also contained amounts of iron comparable with the wild type reductase and showed the same iron-sulfur-related spectrum, suggesting that the mutant proteins were properly folded . Of the three mutant proteins only the G681A protein completely lacked the detectable glycyl radical as well as enzyme activity . Our results identify glycine 681 as the stable free radical site in E . coli anaerobic ribonucleotide reductase.

J Biol Chem, 1996 Mar 22, 271(12), 6801 - 9
Wild-type Escherichia coli cells regulate the membrane lipid composition in a "window" between gel and non-lamellar structures; Morein S et al.; Escherichia coli strain K12 was grown at 17, 27, and 37 degrees C . The acyl chain composition of the membrane lipids varied with the growth temperature; the fraction of cis-vaccenoyl chains decreased, and the fraction of palmitoyl chains increased, when the growth temperature was increased . However, the polar head group composition did not change significantly . The equilibria between lamellar and reversed non-lamellar phases of lipids extracted from the inner membrane (IM), and from both the membranes (IOM), were studied with NMR and x-ray diffraction . At temperatures above the growth temperature the lipid extracts formed a reversed hexagonal phase, or a bicontinuous cubic phase, depending on the degree of hydration of the lipids . It was observed that: 1) at equal elevations above the growth temperature, IM lipid extracts, as well as IOM lipid extracts, have a nearly equal ability to form non-lamellar phases; 2) IM extracts have a stronger tendency than IOM extracts to form non-lamellar phases; 3) non-lamellar phases are formed under conditions that are relatively close to the physiological ones; the membrane lipid monolayers are thus "frustrated"; and 4) as a consequence of the change of the acyl chain structures, the temperature for the lamellar gel to liquid crystalline phase transition is changed simultaneously, and in the same direction, as the temperature for the lamellar to non-lamellar phase transition . With a too large fraction of saturated acyl chains the membrane lipids enter a gel state, and with a too large fraction of unsaturated acyl chains the lipids transform to non-lamellar phases . It is thus concluded that the regulation of the acyl chain composition in wild-type cells of E . coli is necessary for the organism to be able to grow in a "window" between a lamellar gel phase and reversed non-lamellar phases.

J Biol Chem, 1996 Mar 22, 271(12), 6789 - 93
Cloning, sequencing, and expression in escherichia coli of OxlT, the oxalate:formate exchange protein of Oxalobacter formigenes; Abe K et al.; OxlT is the oxalate/formate exchange protein that represents the vectorial component of a proton-motive metabolic cycle in Oxalobacter formigenes . Here we report the cloning and sequencing of OxlT and describe its expression in Escherichia coli . The OxlT amino acid sequence specifies a polytopic hydrophobic protein of 418 residues with a mass of 44,128 daltons . Analysis of hydropathy and consideration of the distribution of charged residues suggests an OxlT secondary structure having 12 transmembrane segments, oriented so that the N and C termini face the cytoplasm . Expression of OxlT in E . coli coincides with appearance of a capacity to carry out the self-exchange of oxalate and the heterologous, electrogenic exchange of oxalate with formate . The unusually high velocity of OxlT-mediated transport is also preserved in E . coli . We conclude that the essential features of OxlT are retained on its expression in E . coli.

J Biol Chem, 1996 Mar 22, 271(12), 6736 - 45
Glutaredoxin-3 from Escherichia coli . Amino acid sequence, 1H AND 15N NMR assignments, and structural analysis; Aslund F et al.; The primary and secondary structure of glutaredoxin-3 (Grx3), a glutathione-disulfide oxidoreductase from Escherichia coli, has been determined . The amino acid sequence of Grx3 consists of 82 residues and contains a redox-active motif, Cys-Pro-Tyr-Cys, typical of the glutaredoxin family . Sequence comparison reveals a homology (33% identity) to that of glutaredoxin-1 (Grx1) from E . coli as well as to other members of the thioredoxin superfamily . In addition to the active site cysteine residues, Grx3 contains one additional cysteine (Cys65) corresponding to one of the two non-active site (or structural) cysteine residues present in mammalian glutaredoxins . The sequence-specific 1H and 15N nuclear magnetic resonance assignments of reduced Grx3 have been obtained . From a combined analysis of chemical shifts, 3JHNalpha coupling constants, sequential and medium range NOEs, and amide proton exchange rates, the secondary structure of reduced Grx3 was determined and found to be very similar to that inferred from amino acid sequence comparison to homologous proteins . The consequences of the proposed structural similarity to Grx1 are that Grx3, while possessing a largely intact GSH binding cleft, would have a very different spatial distribution of charged residues, most notably surrounding the active site cysteine residues and occurring in the proposed hydrophobic protein-protein interaction area . These differences may contribute to the observed very low Kcat of Grx3 as a reductant of insulin disulfides or as a hydrogen donor for ribonucleotide reductase . Thus, despite an identical active site disulfide motif and a similar secondary structure and tertiary fold, Grx3 and Grx1 display large functional differences in in vitro protein disulfide oxido-reduction reactions.

J Biol Chem, 1996 Mar 22, 271(12), 6713 - 9
Equilibrium and kinetic measurements reveal rapidly reversible binding of Ras to Raf; Gorman C et al.; Raf is a serine/threonine kinase that binds through its amino-terminal regulatory domain to the GTP form of Ras and thereby activates the mitogen-activated protein kinase pathway . In this study, we have characterized the interaction of the Ras-binding domain of Raf with Ras using equilibrium binding methods (scintillation proximity assay and fluorescence anisotropy), rather than with more widely used nonequilibrium procedures (such as enzyme-linked immunosorbent assay and affinity precipitation) . Initial studies using glutathione S-transferase fusion proteins with either residues 1-257 or 1-190 of Raf showed that although it was possible to detect Ras binding using an enzyme-linked immunosorbent assay or affinity precipitation, it was substoichiometric; under equilibrium conditions with only a small excess of Raf almost no binding was detected . This difference was probably due to the presence of a high percentage of inactive Raf protein . Further studies used protein containing residues 51-131 of Raf, which expressed in Escherichia coli as a stable glutathione S-transferase fusion . With this protein, binding with Ras could readily be measured under equilibrium conditions . The catalytic domain of neurofibromin inhibited binding of Ras to Raf, and Raf inhibited the binding of Ras to neurofibromin showing that Raf and neurofibromin cannot be bound simultaneously to Ras . The affinities of interaction of neurofibromin and Raf with Harvey-RasLeu-61 were similar . The rate constant for dissociation of Raf from Ras was estimated to be >1 min-1, suggesting that Ras, Raf, and neurofibromin may be in rapid equilibrium in the cell . In contrast to previous reports, under equilibrium conditions there was no evidence for a difference in affinity between the minimal Ras binding domain of Raf (residues 51-131) and a region containing an additional 16 carboxyl-terminal amino acids, suggesting that residues 132-147 do not form a critical binding determinant.

J Biol Chem, 1996 Mar 22, 271(12), 6686 - 93
Selective sugar binding to the carbohydrate recognition domains of the rat hepatic and macrophage asialoglycoprotein receptors; Iobst ST et al.; Asialoglycoprotein receptors on the surfaces of both hepatocytes and peritoneal macrophages bind terminal galactose residues of desialylated glycoproteins and mediate endocytosis and eventual degradation of these ligands . The hepatic receptor binds oligosaccharides with terminal N-acetylgalactosamine residues more tightly than ligands with terminal galactose residues, but the macrophage receptor shows no such differential binding affinity . Carbohydrate recognition domains from the macrophage receptor and the major subunit of the hepatic receptor have been expressed in a bacterial system and have been shown to retain the distinct binding selectivities of the receptors from which they derive . Binding of a series of N-acyl derivatives of galactosamine suggests that the 2-substituent of these sugars interacts with the surface of the hepatic receptor with highest affinity binding observed for the N-propionyl derivative . Chimeric sugar-binding domains have been used to identify three regions of the hepatic receptor that are essential for establishing selectivity for N-acetylgalactosamine over galactose . Based on these results and the orientation of N-acetylgalactosamine when bound to an homologous galactose-binding mutant of rat serum mannose-binding protein, a fourth region likely to interact with N-acetylgalactosamine has been identified and probed by site-directed mutagenesis . The results of these studies define a binding pocket for the 2-substituent of N-acetylgalactosamine in the hepatic asialoglycoprotein receptor.

J Biol Chem, 1996 Mar 22, 271(12), 6611 - 7
The leucine-responsive regulatory protein (Lrp) from Escherichia coli . Stoichiometry and minimal requirements for binding to DNA; Cui Y et al.; Lrp (Leucine-responsive regulatory protein) regulates the expression of a number of operons in Escherichia coli . A recent study of DNA sequences recognized by Lrp established the consensus as a 15-bp sequence, YAGHAWATTWTDCTR (Y = C/T, H = "not G," W = A/T, D ="not C," R = A/G) (Cui, Y., Wang, Q., Stormo, G . D., and Calvo, J . M . (1995) J . Bacteriol . 177, 4872-4880) . Here we report the stoichiometry of Lrp binding (an Lrp dimer binds to a single binding site) and studies that define the minimal length of DNA required for binding . A double-stranded 15 mer having a sequence that closely matches the consensus does not show measurable binding to Lrp . One or two base pairs of DNA flanking each end are not sufficient for binding, but constructs having 3-5 additional base pairs (21 mer) show relatively strong binding . Single-stranded flanking DNA also contributes to strong binding . The extent of the contribution to binding is dependent upon whether the single strand is on the left or right of the double-stranded region and whether the polarity of the single-stranded DNA is 5' to 3' or 3' to 5'.

J Mol Biol, 1996 Mar 22, 257(1), 9 - 20
Identification of functional regions of the Nun transcription termination protein of phage HK022 and the N antitermination protein of phage lambda using hybrid nun-N genes; Henthorn KS et al.; Phages lambda and HK022 express proteins N and Nun, respectively, each of which acts with a number of Escherichia coli host Nus factors at lambda NUT RNA sites, to influence transcription elongation . The lambda nut sites, nearly identical sequences located downstream of the early promoters, pL and pR, were first identified as cis-acting signals required for the action of N in forming termination-resistant transcription complexes . Surprisingly, the Nun protein, resembling N and expressed by another lambdoid phage, HK022, also acts with Nus proteins to terminate specifically transcription initiating at pL and pR near the lambda nut sites . Based on structural considerations of the amino acid sequences, we have constructed nine hybrid N-nun genes and used these hybrids to identify functional regions of the N and Nun proteins . Three classes of hybrid gene products were identified: (1) those that, like N, support antitermination, (2) those that, like Nun, terminate transcription, and (3) those that block N action but do not terminate transcription . We find that, similar to N, the amino-terminal portion of Nun is involved in RNA recognition . The more carboxy portions influence transcription elongation, antitermination (N) and termination (Nun) . Depending on the derivations of the more carboxy regions, hybrids with either the N or Nun amino portions support either termination or antitermination . The activity of a hybrid protein may be influenced by the host strain depending on the nature of the rpoC locus, a locus encoding the beta' subunit of RNA polymerase . One of the hybrid proteins blocks antitermination when the rpoC locus is wild-type . The same hybrid in the presence of the rpoC100 mutation, which alters the beta' subunit, has antitermination activity . This result supports the argument that the beta' subunit plays an essential role in determining the progress of transcription elongation.

J Mol Biol, 1996 Mar 22, 257(1), 21 - 9
Repression of lac promoter as a function of distance, phase and quality of an auxiliary lac operator; Muller J et al.; The tetrameric Lac repressor can bind simultaneously to two lac operators on the same DNA molecule, thereby including the formation of a DNA loop . We investigated the phasing dependence of DNA loop formation between lac operator O1 and an auxiliary ideal lac operator (O(id)) on the bacterial chromosome, with inter-operator distances varying from 57.5 to 1493.5 bp . Repression of a CAP-independent lac UV5 promoter by O1 at its natural position increased up to 50-fold in the presence of an optimally positioned auxiliary O(id)) . Repression values alternated between local maxima and minima with a periodicity of 11.0 to 11.3 bp, suggesting that the chromosomal helical repeat is in this range in vivo . Repression increased significantly with decreasing inter-operator DNA length, indicating that the local Lac repressor concentration at O1 is crucial for tight repression . Maximal repression, attributed to stable DNA loop formation, was obtained at an operator spacing of 70.5 bp . Other repression maxima occurred at operator distances of 92.5 and 115.5 bp, corresponding to natural operator spacings in the lac and in the gal operon, respectively . Substitution of the auxiliary O(id) with the weaker binding lac operator O3 lowered repression efficiency, presumably due to the reduced local concentration of Lac repressor.

J Mol Biol, 1996 Mar 22, 257(1), 116 - 28
The three-dimensional structure of two mutants of the signal transduction protein CheY suggest its molecular activation mechanism; Bellsolell L et al.; The three-dimensional crystal structures of the single mutant M17G and the triple mutant F14G-S15G-M17G of the response regulator protein CheY have been determined to 2.3 and 1.9 angstrom, respectively . Both mutants bind the essential Mg2+ cation as determined by the changes in stability, but binding does not cause the intrinsic fluorescence quenching of W58 observed in the wild-type protein . The loop beta4-alpha4 appears to be very flexible in both mutants and helix alpha4, which starts at N94 in the native Mg2+-CheY and at K91 in the native apo-CheY, starts in both mutants at residue K92 . The side-chain of K109 appears to be more mobile because of the space freed by the M17G mutation . In the triple mutant the main chain of K109 and adjacent residues (loop beta5-alpha5) is displaced almost by 2 angstrom affecting the main chain at residues T87 to E89 (C terminus of beta4) . The triple mutant structure has a Mg2+ bound at the active site, but although the Mg2+ coordination is similar to that of the native Mg2+-CheY, the structural consequences of the metal binding are quite different . It seems that the mutations have disrupted the mechanism of movement transmission observed in the native protein . We suggest that the side-chain of K109, packed between V86, A88 and M17 in the native protein, slides forwards and backwards upon activation and deactivation dragging the main chain at the loop beta5-alpha5 and triggering larger movements at the functional surface of the protein.

Nature, 1996 Mar 21, 380(6571), 268 - 70
Kinetic trapping of oxygen in cell respiration; Verkhovsky MI et al.; Cell respiration in eukaryotes is catalysed by mitochondrial enzyme cytochrome c oxidase . In bacteria there are many variants of this enzyme, all of which have a binuclear haem iron-copper centre at which O2 reduction occurs, and a low-spin haem, which serves as the immediate electron donor to this centre . It is essential that the components of the cell respiratory system have a high affinity for oxygen because of the low concentration of dissolved O2 in the tissues; however, the binding of O2 to the respiratory haem-copper oxidases is very weak . This paradox has been attributed to kinetic trapping during fast reaction of O2 bound within the enzyme's binuclear haem iron-copper centre . Our earlier work indicated that electron transfer from the low-spin haem to the oxygen-bound nuclear centre may be necessary for such kinetic oxygen trapping . Here we show that specific decrease of the haem-haem electron transfer rate in the respiratory haem-copper oxidase from Escherichia coli leads to a corresponding decrease in the enzyme's operational steady-state affinity for O2 . This demonstrates directly that fast electron transfer between the haem groups is a key process in achieving the high affinity for oxygen in cell respiration.

Hum Gene Ther, 1996 Mar 20, 7(5), 589 - 93
In vivo gene transfer and expression in rat stomach by submucosal injection of plasmid DNA; Takehara T et al.; Gastrointestinal nonepithelial tissue is a useful target for in vivo gene transfer . The aim of this study was to investigate whether gene transfer into this organ could be achieved by submucosal injection of plasmid DNA . Plasmid DNA carrying either the firefly luciferase or Escherichia coli LacZ reporter gene was injected directly into the gastric submucosa of adult rats . Gene expression was characterized by quantitative luciferase assay and qualitative in situ beta-galactosidase (beta-Gal) staining . Luciferase activity was detected as early as 1 day after injection, increased markedly at 2 days, and then decreased . Some of the rats showed detectable levels of luciferase expression at 14 and 21 days postinjection . Histochemical staining for beta-Gal demonstrated that expression of the recombinant genes was localized in smooth muscle cells of the muscularis mucosae and the muscular layer and mesenchymal cells in the lamina propria . Our results indicate that gene transfer into the gastrointestinal tract can be achieved by simple needle insertion of naked plasmid DNA into the submucosa.

Mol Gen Genet, 1996 Mar 20, 250(5), 626 - 34
RNA facilitates RecA-mediated DNA pairing and strand transfer between molecules bearing limited regions of homology; Kotani H et al.; The RecA protein of Escherichia coli catalyzes homologous pairing and strand exchange between a wide range of molecules showing nucleotide sequence complementarity, including a linear duplex and a single-stranded DNA molecule . We demonstrate that RecA can promote formation of joint molecules when the duplex contains an RNA/DNA hairpin and a single-stranded circle serves as the pairing partner . A chimeric RNA/DNA hairpin can be used to form stable joint molecules with as little as 15 bases of shared homology as long as the RNA stretch contains complementarity to the circle . The joint molecule bears some resemblance to a triple helical structure composed of RNA residues surrounded by two DNA strands which are in a parallel orientation . Evidence is presented that supports the notion that short stretches of RNA can be used in homologous pairing reactions at lengths below that required for DNA-DNA heteroduplex formation.

Mol Gen Genet, 1996 Mar 20, 250(5), 593 - 600
Co-induction of DNA relaxation and synthesis of DnaK and GroEL proteins in Escherichia coli by expression of LetD (CcdB) protein, an inhibitor of DNA gyrase encoded by the F factor; Kaneko T et al.; We examined the influence of overexpression of LetD (CcdB) protein, an inhibitor of DNA gyrase encoded by the F factor of Escherichia coli, on DNA supercoiling and induction of heat shock proteins . Cells were transformed with a plasmid carrying the structural gene for LetD protein under control of the tac promoter, and LetD protein was induced by adding isopropyl beta-D-thiogalactopyranoside (IPTG) to the culture medium . Analysis by agarose gel electrophoresis in the presence of chloroquine revealed relaxation of plasmid DNA in cells depending on the concentration of IPTG employed for induction . Protein pulse-labeling experiments with {35S}methionine and cysteine revealed that synthesis of DnaK and GroEL proteins was also induced by IPTG, and concentrations necessary for DNA relaxation and induction of the heat shock proteins were much the same . Expression of mutant LetD protein lacking two amino acid residues at the C-terminus induced neither DNA relaxation nor the synthesis of DnaK and GroEL proteins . Induction of wild-type LetD protein but not mutant LetD protein markedly enhanced synthesis of sigma32 . We interpret these results to mean that DNA relaxation in cells caused by the expression of LetD protein induces heat shock proteins via increased synthesis of sigma32.

Mol Gen Genet, 1996 Mar 20, 250(5), 547 - 57
Self-incompatibility (S) alleles of the Rosaceae encode members of a distinct class of the T2/S ribonuclease superfamily; Sassa H et al.; Stylar ribonucleases (RNases) are associated with gametophytic self-incompatibility in two plant families, the Solanaceae and the Rosaceae . The self-incompatibility-associated RNases (S-RNases) of both the Solanaceae and the Rosaceae were recently reported to belong to the T2 RNase gene family, based on the presence of two well-conserved sequence motifs . Here, the cloning and characterization of S-RNase genes from two species of Rosaceae, apple (Malus x domestica) and Japanese pear (Pyrus serotina) is described and these sequences are compared with those of other T2-type RNases . The S-RNases of apple specifically accumulated in styles following maturation of the flower bud . Two cDNA clones for S-RNases from apple, and PCR clones encoding a further two apple S-RNases as well as two Japanese pear S-RNases were isolated and sequenced . The deduced amino acid sequences of the rosaceous S-RNases contained two conserved regions characteristic of the T2/S-type RNases . The sequences showed a high degree of diversity, with similarities ranging from 60.4% to 69.2% . Interestingly, some interspecific sequence similarities were higher than those within a species, possibly indicating that diversification of S-RNase alleles predated speciation in the Rosaceae . A phylogenetic tree of members of the T2/S-RNase superfamily in plants was obtained . The rosaceous S-RNases formed a new lineage in the tree that was distinct from those of the solanaceous S-RNases and the S-like RNases . The findings suggested that self-incompatibility mechanisms in Rosaceae and Solanaceae are similar but arose independently in the course of evolution.

Mol Gen Genet, 1996 Mar 20, 250(5), 523 - 32
Requirement for two conserved cysteine residues in the Ada protein of Escherichia coli for transactivation of the ada promoter; Taketomi A et al.; Cysteine residue 69 of the Escherichia coli Ada transcription factor, which accepts a methyl group from methylphosphotriester in methylated DNA, was substituted by each of 19 other amino acids . Only the mutant Ada (C69H), carrying a histidine substitution of Cys69, exhibited a limited degree of transactivating potential for the ada promoter in E . coli cells although the mutant protein was completely devoid of methylphosphotriester-DNA methyltransferase activity . Using a multicopy plasmid system for the expression of Ada protein, we have shown that Ada C69H has a transactivating capacity equivalent to that of wild-type Ada protein in the absence of an alkylating agent . This indicates that the zinc-binding capacity of histidine at residue 69 is likely to be sufficient for Ada to recognize and bind to the ada promoter . Furthermore, transactivation of the ada promoter by Ada C69H was enhanced up to 6-fold by treatment with methylating agents . An additional substitution was made with alanine in Ada C69H, replacing Cys321, the site for acceptance of a methyl group from O6-methylguanine and O4-methylthymine residues in DNA, with alanine . This renders the protein completely inactive as a methyltransferase but this derivative is constitutively active as a transactivator for the ada promoter . Therefore, acquisition of a methyl group at Cys321 apparently enhances the transactivating capacity of Ada protein on the ada promoter . We propose that the transcription-regulating function of Ada protein is under dual control by methylation of cysteine residues at positions 69 and 321; the former enhances DNA binding, while the latter enhances the transactivating capacity of the protein.

Biochemistry, 1996 Mar 19, 35(11), 3563 - 71
Differential proximity probing of two DNA binding sites in the Escherichia coli recA protein using photo-cross-linking methods; Wang Y et al.; The DNA strand-exchange reaction catalyzed by the Escherichia coli RecA protein occurs between the two DNA binding sites that are functionally distinct . Site I is the site to which a DNA molecule (normally single-stranded DNA) binds first; this first binding makes site II available for additional DNA-binding (normally double- stranded DNA) . Photo-cross linking was employed to identify the amino acid residues located close to the bound DNA molecule(s) . A ssDNA oligo containing multiple 5-iodouracil residues (IdU) was cross-linked to RecA by irradiation with a XeC1 pulse laser (308 nm), and the cross-linked peptides were purified and sequenced . To differentiate the two DNA binding sites, we used two protocols for making RecA-ssDNA complexes: (1) IdU-containing oligo was mixed with a stoichiometric excess of RecA, a condition which favors the binding of the oligo to site I, and (2) RecA was first allowed to bind to a nonphotoreactive oligo and then chased with the IdU-containing oligo, a condition which favors the binding of the IdU-oligo to site II . We observed that when RecA was in excess (site I probing), cross-linking occurred to Met-164 which is located in the disordered loop 1 of the RecA crystal structure {Story, R.M., Weber, I.T., & Steitz, T.A . (1992) Nature 355, 318-325} . When site II was probed, the majority of cross-linking occurred to Met-202 or Phe-203, located in loop 2 . These results support the idea that, as predicted by Story and co-workers (1992), the disordered loops are involved in DNA binding . The results also suggest that the two sites are not only functionally but also physically distinct.

Biochemistry, 1996 Mar 19, 35(11), 3525 - 33
Sequence-specific actinomycin D binding to single-stranded DNA inhibits HIV reverse transcriptase and other polymerases; Rill RL et al.; Primer extension assays using recombinant templates constructed to contain all 256 possible base quartets in a minimum length sequence were used to examine binding of the anticancer drug actinomycin D to single-stranded DNA . Single-stranded templates were generated by digestion of linearized plasmid with the double-strand-specific T7 gene 6 exonuclease . Actinomycin D formed high-affinity, kinetically stable complexes that paused primer elongation at specific sites by HIV-1 reverse transcriptase, Sequenase (modified T4 DNA polymerase), the Klenow fragment of Escherichia coli DNA polymerase, and Vent (exo-) DNA polymerase . Pauses occurred most commonly near G+C-rich nucleotide clusters, including GpC steps, the preferred sites of double-stranded DNA binding . Complexes were stable for several minutes at temperatures over 50 degrees C as determined by their abilities to pause Vent polymerase at elevated temperatures . Significant variations were noted in pause patterns of different polymerases, demonstrating differential responses of polymerases to a bound actinomycin . Covalent adducts formed on template DNA by a photoaffinity analog of actinomycin D completely stopped primer extension . These results support the possibility that actinomycin D inhibits transcription elongation by complexing single-stranded DNA in the open transcription complex . Single-stranded DNA binding by actinomycin D or analogs may also provide routes for combating HIV or other viruses which replicate through single-stranded intermediates.

Proc Natl Acad Sci U S A, 1996 Mar 19, 93(6), 2517 - 22
The translational function of nucleotide C1054 in the small subunit rRNA is conserved throughout evolution: genetic evidence in yeast; Chernoff YO et al.; Mutations at position C1054 of 16S rRNA have previously been shown to cause translational suppression in Escherichia coli . To examine the effects of similar mutations in a eukaryote, all three possible base substitutions and a base deletion were generated at the position of Saccharomyces cerevisiae 18S rRNA corresponding to E . coli C1054 . In yeast, as in E . coli, both C1054A (rdn-1A) and C1054G (rdn-1G) caused dominant nonsense suppression . Yeast C1054U (rdn-1T) was a recessive antisuppressor, while yeast C1054-delta (rdn-1delta) led to recessive lethality . Both C1054U and two previously described yeast 18S rRNA antisuppressor mutations, G517A (rdn-2) and U912C (rdn-4), inhibited codon-nonspecific suppression caused by mutations in eukaryotic release factors, sup45 and sup35 . However, among these only C1054U inhibited UAA-specific suppressions caused by a UAA-decoding mutant tRNA-Gln (SLT3) . Our data implicate eukaryotic C1054 in translational termination, thus suggesting that its function is conserved throughout evolution despite the divergence of nearby nucleotide sequences.

Proc Natl Acad Sci U S A, 1996 Mar 19, 93(6), 2488 - 92
The response regulator SprE controls the stability of RpoS; Pratt LA et al.; In Escherichia coli, the sigma factor, RpoS, is a central regulator in stationary-phase cells . We have identified a gene, sprE (stationary-phase regulator), as essential for the negative regulation of rpoS expression . SprE negatively regulates the rpoS gene product at the level of protein stability, perhaps in response to nutrient availability . The ability of SprE to destabilize RpoS is dependent on the ClpX/ClpP protease . Based on homology, SprE is a member of the response regulator family of proteins . SprE is the first response regulator identified that is implicated in the control of protein stability . Moreover, SprE is the first reported protein that appears to regulate rpoS in response to a specific environmental parameter.

Proc Natl Acad Sci U S A, 1996 Mar 19, 93(6), 2459 - 63
Non-iron porphyrins cause tumbling to blue light by an Escherichia coli mutant defective in hemG; Yang H et al.; Previously we showed that an Escherichia coli hemH mutant, defective in the ultimate step of heme synthesis, ferrochelatase, is somewhat better than 100-fold more sensitive than its wild-type parent in tumbling to blue light . Here we explore the effect of a hemG mutant, defective in the penultimate step, protoporphyrinogen oxidase . We found that a hemG mutant also is somewhat better than 100-fold more sensitive in tumbling to blue light compared to its wild-type parent . The amount of non-iron porphyrins accumulated in hemG or hemH mutants was more than 100-fold greater than in wild type . The nature of these accumulated porphyrins is described . When heme was present, as in the wild type, the non-iron (non-heme) porphyrins were maintained at a relatively low concentration and tumbling to blue light at an intensity effective for hemG or hemH did not occur . The function of tumbling to light is most likely to allow escape from the lethality of intense light.

Proc Natl Acad Sci U S A, 1996 Mar 19, 93(6), 2348 - 52
Baculovirus-mediated gene transfer into mammalian cells; Boyce FM et al.; This paper describes the use of the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) as a vector for gene delivery into mammalian cells . A modified AcMNPV virus was prepared that carried the Escherichia coli lacZ reporter gene under control of the Rous sarcoma virus promoter and mammalian RNA processing signals . This modified baculovirus was then used to infect a variety of mammalian cell lines . After infection of the human liver cell lines HepG2, >25% of the cells showed high-level expression of the transduced gene . Over 70% of the cells in primary cultures of rat hepatocytes showed expression of beta-galactosidase after exposure to the virus . Cell lines from other tissues showed less or no expression of lacZ after exposure to the virus . The block to expression in less susceptible cells does not appear to result from the ability to be internalized by the target cell but rather by events subsequent to viral entry . The onset of lacZ expression occurred within 6 hr of infection in HepG2 cells and peaked 12-24 hr postinfection . Because AcMNPV is able to replicate only in insect hosts, is able to carry large (>15 kb) inserts, and is a highly effective gene delivery vehicle for primary cultures of hepatocytes, AcMNPV may be a useful vector for genetic manipulation of liver cells.

Proc Natl Acad Sci U S A, 1996 Mar 19, 93(6), 2328 - 32
Genes encoding the same three subunits of respiratory complex II are present in the mitochondrial DNA of two phylogenetically distant eukaryotes; Burger G et al.; Although mitochondrial DNA is known to encode a limited number (<20) of the polypeptide components of respiratory complexes I, III, IV, and V, genes for components of complex II {succinate dehydrogenase (ubiquinone); succinate:ubiquinone oxidoreductase, EC 1.3.5.1} are conspicuously lacking in mitochondrial genomes so far characterized . Here we show that the same three subunits of complex II are encoded in the mitochondrial DNA of two phylogenetically distant eukaryotes, Porphyra purpurea (a photosynthetic red alga) and Reclinomonas americana (a heterotrophic zooflagellate) . These complex II genes, sdh2, sdh3, and sdh4, are homologs, respectively, of Escherichia coli sdhB, sdhC, and sdhD . In E . coli, sdhB encodes the iron-sulfur subunit of succinate dehydrogenase (SDH), whereas sdhC and sdhD specify, respectively, apocytochrome b558 and a hydrophobic 13-kDa polypeptide, which together anchor SDH to the inner mitochondrial membrane . Amino acid sequence similarities indicate that sdh2, sdh3, and sdh4 were originally encoded in the protomitochondrial genome and have subsequently been transferred to the nuclear genome in most eukaryotes . The data presented here are consistent with the view that mitochondria constitute a monophyletic lineage.

Biochem Biophys Res Commun, 1996 Mar 18, 220(2), 437 - 42
Cloning, sequencing, and expression of equinatoxin II; Anderluh G et al.; Equinatoxin II (EqtII), a basic protein of 179 amino acids lacking cysteine residues, is the most abundant cytolysin isolated from the sea anemone Actinia equina . Its mode of action is still poorly understood . In order to initiate further structure-function studies by protein engineering, cDNA library was prepared from the whole animal and hybridized with a PCR-derived probe, deduced from the EqtII primary structure . The longest positive clone of 899 bp was shown to encode a 214 residue precursor of EqtII . The mature protein region was amplified by PCR, cloned into a T7 RNA polymerase-based expression vector and expressed in Escherichia coli . Recombinant toxin was isolated by a simple, two-step isolation procedure including separation on CM-cellulose and gel filtration using an FPLC system . Its biochemical properties and hemolytic activity were practically indistinguishable from those of native toxin.

Biochem Biophys Res Commun, 1996 Mar 18, 220(2), 330 - 3
Site-directed mutagenesis of the conserved serine 138 of human placental NAD+-dependent 15-hydroxyprostaglandin dehydrogenase to an alanine results in an inactive enzyme; Ensor CM et al.; Human placental NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a member of the short-chain dehydrogenase family of enzymes . It has been proposed that a highly conserved serine residue (corresponding to serine 138 of 15-PGDH) may be involved in the catalytic mechanism of many of these enzymes . Site-directed mutagenesis was used to change serine 138 of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase to an alanine . The mutant protein was then expressed in E . coli . Western blot analysis indicated that the S138A mutant protein was expressed at levels similar to the wild type enzyme; however, the mutant protein was found to be inactive . These results support the proposed role of this highly conserved serine in enzyme activity.

Biochem Biophys Res Commun, 1996 Mar 18, 220(2), 280 - 4
A new plasmid-encoded proteic killer gene system: cloning, sequencing, and analyzing hig locus of plasmid Rts1; Tian QB et al.; A new proteic killer gene system, hig, was identified on the plasmid Rts1 . The hig locus consisting of a higA and higB is directly related to the temperature sensitive host cell growth conferred by Rts1 . We proved that higB encoding presumably a 92-amino-acid polypeptide inhibited segregation of plasmid free cells, and higA encoding a 104-amino-acid polypeptide suppressed the higB function both in cis and in trans.

Biochem Biophys Res Commun, 1996 Mar 18, 220(2), 255 - 63
Expression and functional characterization of a single chain Fv antibody directed against secretions involved in plant nematode infection process; Rosso MN et al.; Expression in plants of antibodies directed against proteins essential for pathogenesis could provide an alternative approach to engineer new resistance traits into crops . Salivary secretions of the root-knot nematode Meloidogyne incognita are known to play a key role during this nematode infection process . From a hybridoma cell line producing an IgM monoclonal antibody specific to these secretions, we have constructed a synthetic gene that encodes an antigen-binding single-chain Fv protein (scFv) . The scFv gene was created by polymerase chain reaction amplification of variable domain encoding regions from the IgM antibody . The cloned scFv was initially expressed in Escherichia coli as a 33-kDa protein which could be purified to near homogeneity by immobilized metal affinity chromatography . The produced scFv is fully functional since it shows the same specificity towards a crude extract of M . incognita infective larvae as the corresponding parental IgM . Transient expression assays with tobacco leaf protoplasts using different targeting signals resulted in a high intracellular accumulation of scFv, especially when fused to the tetrapeptide KDEL retention signal . Activity analysis and stability characterization of this scFv in tobacco protoplast represent the first step before plant transformation in order to test a new form of resistance to root-knot nematode in plants.

FEBS Lett, 1996 Mar 18, 382(3), 285 - 8
Cloning of rat 92-kDa type IV collagenase and expression of an active recombinant catalytic domain; Xia Y et al.; A full-length cDNA for rat 92-kDa type IV collagenase was isolated and sequenced . RNase protection assay revealed tissue specific differential expression of the 92-kDa type IV collagenase in the rat during development . Natural and modified forms of the 92-kDa type IV collagenase were expressed . One active protein, 92-CD, contained only the putative catalytic domain . Large quantities of the 92-CD were expressed in Escherichia coli, extracted from inclusion bodies, purified, and refolded to an active form . This recombinant protein was able to cleave denatured and native collagen and was inactivated by known MMP inhibitors.

FEMS Microbiol Lett, 1996 Mar 15, 137(1), 85 - 9
The structural gene for rusticyanin from Thiobacillus ferrooxidans: cloning and sequencing of the rusticyanin gene; Hall JF et al.; The rusticyanin gene from the acidophilic chemolithotroph Thiobacillus ferrooxidans has been cloned and sequenced . A central portion of the gene was identified by PCR reactions utilising primers optimised for codon bias followed by nested PCR with degenerate primers . The 5' and 3' ends of the rusticyanin gene were then cloned using degenerate primers to each end and anchor sequences to the known internal sequence . The entire gene was amplified using Tli DNA polymerase and specific primers to the 5' and 3' ends and the sequence confirmed after cloning into Bluescript and transformation of XL-1 Blue Escherichia coli.

FEMS Microbiol Lett, 1996 Mar 15, 137(1), 51 - 6
A putative signal recognition particle receptor alpha subunit (SR alpha) homologue is expressed in the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius; Moll R et al.; A 1.64 kb genomic DNA sequence from the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius is composed of two adjacent genes . The first functionally unassigned open reading frame (orf-1) comprises 450 base pairs . The second 1.1 kb large open reading frame encodes the putative signal recognition particle receptor alpha subunit (SR alpha) . Both genes are expressed under the heterotrophic growth conditions of the organism . The main transcript of orf-1 appears as a monocistronic RNA in Northern hybridization . With regard to SR alpha the transcription pattern was investigated by reverse transcription polymerase chain reaction and primer extension analysis . A polyclonal antiserum directed against E . coli lacZ'/Sulfolobus SR alpha fusion protein detects a 40.5 kDa protein (p41) in agreement with the 41.4 kDa as deduced from the nucleotide sequence.

Anal Biochem, 1996 Mar 15, 235(2), 195 - 201
Tetracycline-controlled gene expression system achieves high-level and quantitative control of gene expression; Yin DX et al.; The tetracycline-controlled gene expression system utilizes the control elements of the tetracycline resistance operon encoded in TnlO of Escherichia coli to control gene expression in eukaryotic cells . Here we demonstrate the quantitative control of the expression of the luciferase gene, dihydrofolate reductase gene, and bcl-2 gene in HeLa S3 or Chinese hamster ovary AA8 cells using the tetracycline-controlled gene expression system . Regardless of the host cell lines or the genes being expressed, there is a common range of tetracycline concentration within which the expression of genes is most sensitively regulated . In addition, the maximal gene expression level of the tetracycline-controlled gene expression system is higher than that of the wild-type CMV promoter/enhancer-driven system . Nonetheless, careful selection of stably transfected clones is necessary to achieve the optimally regulated gene expression using this system.

Structure, 1996 Mar 15, 4(3), 219 - 22
Lac repressor at last; Sauer RT; Crystal and cocrystal structures of the LacI and PurR repressors reveal a novel use of hinge alpha helices which bind in the minor groove of the operator and mediate transmission of the allosteric signals that modulate DNA-binding activity.

Eur J Biochem, 1996 Mar 15, 236(3), 991 - 5
Identification, nuclear localization, and binding activities of OZF, a human protein solely composed of zinc-finger motifs; Ferbus D et al.; The OZF cDNA was identified in a human mammary cell line and encodes a polypeptide solely composed of ten zinc-finger motifs which belongs to the Kruppel family of zinc-finger proteins . The OZF protein produced in Escherichia coli binds zinc ions, DNA and heparin . These binding activities are characteristic of zinc-finger proteins . Immunochemical analysis using antibodies produced against the recombinant protein detected its expression in human mammary epithelial cells but not in stroma cells, which is consistent with the pattern of expression observed at the RNA level in cell cultures . Western blot analysis demonstrated the expression of a 33-kDa nuclear protein similar in size to the predicted protein and therefore excluded the presence of an additional trans-acting domain . These data establish the unique structure of the OZF protein which is distinct from previously identified zinc-finger proteins . In addition, OZF protein overexpression was found in a tumor cell line, which suggests a possible involvement in carcinogenesis.

Eur J Biochem, 1996 Mar 15, 236(3), 937 - 46
Properties of the recombinant beta subunit of glutamate synthase; Vanoni MA et al.; Glutamate synthase is a complex iron-sulfur flavoprotein containing one molecule each of FAD and FMN and three distinct iron-sulfur centers/alpha beta protomer . Production of the beta subunit was observed in total extracts of Escherichia coli BL21 (DE) cells harbouring a pT7-7 derivative carrying gltD, the gene encoding the Azospirillum brasilense glutamate synthase beta subunit . The protein was soluble, and the identity of the purified protein with the Azospirillum glutamate synthase beta subunit was confirmed by N-terminal sequence analysis . The kinetic and spectroscopic characterization of the glutamate synthase beta subunit confirmed that it contains the NADPH binding site, but, in contrast with earlier proposals that assigned both FAD and FMN binding sites to the alpha subunit of glutamate synthase, the beta subunit was shown to contain stoichiometric amounts of FAD . No iron-sulfur centers were detected by EPR spectroscopy measurements of the recombinant beta subunit . Under steady-state conditions, the glutamate synthase beta subunit can catalyze the NADPH-dependent reduction of several synthetic electron acceptors but no glutamate synthase or glutamate dehydrogenase reactions in either direction . The results are in agreement with previous data from our laboratory and, together with the absence of amino acid sequence similarity between glutamate synthase beta subunit and glutamate dehydrogenases, are against the hypothesis that glutamate synthase is evolutionarily derived from the association of an ancestral glutamate dehydrogenase (the beta subunit) and an amidotransferase (the alpha subunit) . The protein-bound FAD is reduced by NADPH at a rate much faster than turnover with synthetic electron acceptors, leading to formation of a stable reduced flavin-NADP+ charge-transfer complex . The rate of reduction of the bound FAD by NADPH is also similar to the rate at which one of the flavins is reduced in the native glutamate synthase, as measured in a stopped-flow spectrophotometer under pre-steady-state conditions . The ability of FAD bound to the beta subunit of glutamate synthase to react with NADPH and the lack of reactivity with sulfite lead us to conclude that FAD is Flavin 1 of glutamate synthase {Vanoni, M.A., Edmondson, D.E., Zanetti, G . & Curti, B . (1992) Biochemistry 31, 4613-4623}.

Eur J Biochem, 1996 Mar 15, 236(3), 911 - 21
Solution structure of the DNA-binding domain of the tomato heat-stress transcription factor HSF24; Schultheiss J et al.; Two-dimensional-NMR and three-dimensional-NMR experiments were performed to determine the solution structure of the DNA-binding domain of the tomato heat-stress transcription factor HSF24 . Samples of uniformly 15N-labeled and 15N, 13C-labeled recombinant proteins were used in the investigation . A near-complete assignment of the backbone 1H, 15N, and 13C resonances was obtained by three-dimensional triple-resonance experiments, whereas three-dimensional 15N-TOCSY-heteronuclear-single-quantum-correlation-spectroscopy, HCCH-COSY and HCCH-TOCSY spectra were recorded for side-chain assignments, 885 non-redundant distance constraints from two-dimensional-homonuclear and three-dimensional-15N-edited and 13C-edited NOESY spectra and 40 hydrogen-bond constraints from exchange experiments were used for structure calculations . The resulting three-dimensional structure contains a three-helix bundle and a small four-stranded antiparallel beta-sheet that forms a hydrophobic core . The two C-terminal helices are parts of a highly conserved helix-turn-helix motif that is probably involved in DNA recognition and binding . In contrast to heat-stress factors from yeast and animals, the plant heat-stress factors lack a loop of 11 amino acid residues inserted between beta3 and beta4 . This leads to a tight turn between these beta-strands.

Cancer Res, 1996 Mar 15, 56(6), 1361 - 6
Treatment of microscopic pulmonary metastases with recombinant autologous tumor vaccine expressing interleukin 6 and Escherichia coli cytosine deaminase suicide genes; Mullen CA et al.; Poorly immunogenic tumor cells genetically transduced to simultaneously express the cytokine interleukin 6 (IL-6) and the bacterial metabolic suicide gene cytosine deaminase (205-IL6-CD) become highly immunogenic . They are rejected by normal mice without 5-fluorocytosine prodrug treatment . Mice with preexisting wild-type pulmonary micrometastases exhibit prolonged survival and an increased rate of cure when treated with live 205-IL6-CD cells as a therapeutic vaccine . Treatment with these autologous tumor cells producing both the cytokine and the bacterial protein was more effective than treatment with exogenous IL-6 and/or irradiated wild-type tumor cells . Irradiation of the 205-IL6-CD cells significantly reduced their therapeutic efficacy . Therapeutic vaccination with 205-IL6-CD was more effective in animals with wild-type 205 tumor than in animals bearing an unrelated syngeneic tumor . Vaccine efficacy was significantly reduced in animals pretreated with high-dose cyclophosphamide . The results indicate that genetically engineered autologous tumor vaccines may be capable of inducing significant antitumor immunity in hosts of preexisting micrometastatic disease.

Cancer Res, 1996 Mar 15, 56(6), 1315 - 23
Comparison of the effects of three different toxin genes and their levels of expression on cell growth and bystander effect in lung adenocarcinoma; Hoganson DK et al.; Transduction of malignant cells with toxin genes provides a novel means to promote tumor cell destruction . The efficacy of a toxin gene is dependent on the cell type targeted, the quantity of exogenous protein synthesized, and the mechanisms of growth inhibition and bystander killing . To develop gene therapy for targeting metastatic lung adenocarcinoma, the toxic activity of herpes simplex virus type 1-thymidine kinase, Escherichia coli cytosine deaminase, and human deoxycytidine kinase were investigated in metastatic human lung adenocarcinoma cell lines H1437 and H2122 . Cells were transduced stably with retroviral vectors containing the toxin gene cDNA under the control of either a strong {cytomegalovirus (CMV) immediate early promotor and enhancer} or an intermediate strength (Moloney murine leukemia virus long terminal repeat) promotor . A comparison of toxin gene efficacy was based on the level of specific enzyme activity, the concentration of prodrug required to inhibit cell growth by 50%, and the magnitude of the bystander effect . In lung adenocarcinoma cell lines, cytosine deaminase, driven by the CMV promoter, was superior to thymidine kinase and deoxycytidine kinase in its ability to achieve high levels of specific enzyme activity, to induce growth inhibition, and to affect neighboring cell growth . Therefore, cytosine deaminase expressed from the CMV promotor seems to be the most promising toxin gene for human lung adenocarcinoma gene therapy.

EMBO J, 1996 Mar 15, 15(6), 1340 - 9
Escherichia coli protein analogs StpA and H-NS: regulatory loops, similar and disparate effects on nucleic acid dynamics; Zhang A et al.; Expression of the Escherichia coli StpA protein was investigated and a functional comparison undertaken with the structurally analogous nucleoid protein H-NS . Analysis of stpA and hns expression indicated that although stpA transcript levels are much lower than those of hns, the two gene products are capable of both negative autogenous control and cross-regulation . Examination of cellular proteins in stpA, hns, or stpA-hns backgrounds revealed that StpA can repress and activate a subset of H-NS-regulated genes . Mechanistic parallels in regulation of gene expression are indicated by the ability of both proteins to inhibit transcription from promoters containing curved DNA sequences, and to form nucleoprotein structures that constrain DNA supercoils . Despite their functional similarities, each molecule is capable of independent activities . Thus, H-NS regulates a class of genes that are unaffected by StpA in vivo, whereas StpA has much stronger RNA chaperone activity in vitro . We therefore propose that in addition to its role as a molecular back-up of H-NS, StpA's superior effect on RNA may be exploited under some specific cellular conditions to promote differential gene expression.

EMBO J, 1996 Mar 15, 15(6), 1333 - 9
The response regulator RssB controls stability of the sigma(S) subunit of RNA polymerase in Escherichia coli; Muffler A et al.; The rpoS-encoded sigma(S) subunit of RNA polymerase is a central regulator in a regulatory network that governs the expression of many stationary phase-induced and osmotically regulated genes in Escherichia coli . sigma(S) is itself induced under these conditions due to an increase in rpoS transcription (only in rich media) and rpoS translation as well as a stabilization of sigma(S) protein which in growing cells is subject to rapid turnover . We demonstrate here that a response regulator, RssB, plays a crucial role in the control of the cellular sigma(S) content . rssB null mutants exhibit nearly constitutively high levels of sigma(S) and are impaired in the post-transcriptional growth phase-related and osmotic regulation of sigma(S) . Whereas rpoS translational control is not affected, sigma(S) is stable in rssB mutants, indicating that RssB is essential for sigma(S) turnover . RssB contains a unique C-terminal output domain and is the first known response regulator involved in the control of protein turnover.

EMBO J, 1996 Mar 15, 15(6), 1238 - 46
Linking microfilaments to intracellular membranes: the actin-binding and vesicle-associated protein comitin exhibits a mannose-specific lectin activity; Jung E et al.; Comitin is a 24 kDa actin-binding protein from Dictyostelium discoideum that is located primarily on Golgi and vesicle membranes . We have probed the molecular basis of comitin's interaction with both actin and membranes using a series of truncation mutants obtained by expressing the appropriate cDNA in Escherichia coli . Comitin dimerizes in solution; its principle actin-binding activity is located between residues 90 and 135 . The N-terminal 135 'core' residues of comitin contain a 3-fold sequence repeat that is homologous to several monocotyledon lectins and which retains key residues that determine these lectins' three-dimensional structure and mannose binding . These repeats of comitin appear to mediate its interaction with mannose residues in glycoproteins or glycolipids on the cytoplasmic surface of membrane vesicles from D.discoideum, and comitin can be released from membranes with mannose . Our data indicate that comitin binds to vesicle membranes via mannose residues and, by way of its interaction with actin, links these membranes to the cytoskeleton.

EMBO J, 1996 Mar 15, 15(6), 1203 - 10
Exploring the functional robustness of an enzyme by in vitro evolution; Martinez MA et al.; The evolution of natural proteins is thought to have occurred by successive fixation of individual mutations . In vitro protein evolution seeks to accelerate this process . RNA hypermutagenesis, cDNA synthesis in the presence of biased dNTP concentrations, delivers elevated mutant and mutation frequencies . Here lineages of active enzymes descended from the homotetrameric 78 residue dihydrofolate reductase (DHFR) encoded by the Escherichia coli R67 plasmid were generated by iterative RNA hypermutagenesis, resulting in >20% amino acid replacement . The 22 residue N-terminus could be deleted yielding a minimum functional entity refractory to further changes, designating it as a determinant of R67 robustness . Complete substitution of the segment still allowed fixation of mutations . By the facile introduction of multiple mutations, RNA hypermutagenesis allows the generation of active proteins derived from extant genes through a mode unexplored by natural selection.

J Med Chem, 1996 Mar 15, 39(6), 1293 - 302
Angular furoquinolinones, psoralen analogs: novel antiproliferative agents for skin diseases . Synthesis, biological activity, mechanism of action, and computer-aided studies; Rodighiero P et al.; With the aim of obtaining new potential photochemotherapeutic agents, having increased antiproliferative activity and decreased undesired effects, we have prepared some new furoquinolinones . Two of them have been studied in detail: 1,4,6,8-tetramethyl-2H-furo{2,3-h}-quinolin-2-one (8), and 4,6,8,9-tetramethyl-2H-furo{2,3-h}quinolin-2-one (10) . These compounds form a molecular complex with DNA, undergoing intercalation inside the duplex macromolecule, as shown by linear flow dichroism . The complexed ligands, by subsequent irradiation with UV-A light, photobind with the macromolecule forming only monocycloadducts with thymine with cis-syn configuration . In order to evaluate the electronic effects induced by the nitrogen atom in position 1 of 8, semiempirical calculations have been performed on both 4,6,4'-trimethylangelicin (TMA) and 8 . The results obtained do not clearly differentiate between the two molecules which, at this level of approximation, show the possibility of photoreaction with both the 3,4- and 8,9-olefinic bonds for 8 and the 3,4- and 4',5'-bonds for TMA . In the lower energy conformation of intercalated 8, the furan ring is turned toward the minor groove of the polynucleotide, in such a way that photoreaction of this ring with thymine is favored . These compounds unexpectedly inhibit DNA and RNA synthesis in Ehrlich cells, in the dark . They also show a strong photoantiproliferative activity, 2 orders of magnitude higher than 8-methoxypsoralen (8-MOP), the most used drug for photochemotherapy . Their mutagenic activity on Escherichia coli is similar to that of TMA and 8-MOP . On the basis of these results, the compounds should deserve evaluation of their activity in the treatment of hyperproliferative skin diseases.

Dev Biol, 1996 Mar 15, 174(2), 214 - 20
Initial cell type divergence in Dictyostelium is independent of DIF-1; Shaulsky G et al.; Prespore and prestalk cells can be distinguished within aggregates of Dictyostelium by the expression of well-characterized cell type-specific genes . Fusion of the tagB regulatory region to Escherichia coli beta-galactosidase revealed that this prestalk specific gene marks the differentiation of the initial prestalk cell population, PST-1 . The reporter gene was expressed normally in tagB- mutant cells despite the fact that they do not accumulate measurable levels of DIF-I, a morphogen that was previously implicated in prestalk differentiation . In an independent experimental system, wild-type cells respond to the addition of DIF-I by induction of the prestalk marker ecmA and repression of the prespore marker cotB . We found that DIF-1 did not affect the expression of the tagB or carB genes, both of which are prestalk specific and essential for PST-A cell differentiation . We conclude that the initiation of prestalk development is not dependent on DIF-1 and suggest that the morphogen participates mainly at later stages.

Blood, 1996 Mar 15, 87(6), 2337 - 44
Inhibition of factor XII in septic baboons attenuates the activation of complement and fibrinolytic systems and reduces the release of interleukin-6 and neutrophil elastase; Jansen PM et al.; In previous studies, we have shown that administration of monoclonal antibody (MoAb) C6B7 against human factor XII to baboons challenged with a lethal dose of Escherichia coli abrogates activation of the contact system and modulates secondary hypotension . To evaluate the contribution of activated contact proteases to the appearance of other inflammatory mediators in this experimental model of sepsis, we studied the effect of administration of MoAb C6B7 on activation of complement and fibrinolytic cascades, stimulation of neutrophil degranulation, and release of the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) . Activation of the complement system, as reflected by circulating C3b/c and C4b/c levels, was significantly reduced in five animals that had received MoAb C6B7 before a lethal dose of E coli as compared with five control animals that had been given a lethal challenge only . Inhibition of contact activation also modulated the fibrinolytic response, since the release of tissue-type plasminogen activator (t-PA) and the appearance of plasmin-alpha2-antiplasmin (PAP) complexes into the circulation was significantly attenuated upon pretreatment with anti-factor XII MoAb . In contrast, plasma levels of plasminogen activator inhibitor (PAI) were modestly enhanced in the treatment group . Degranulation of neutrophils, as assessed by circulating elastase-alpha1-protease inhibitor complexes, and release of IL-6 but not of TNF-alpha was decreased in anti-factor XII-treated animals . Observed differences in the inflammatory response between treatment and control groups were not likely due to different challenges, since the number of E coli that had been infused, as well as circulating levels of endotoxin after the challenge, were similar for both groups . These data suggest that activation of the contact system modulates directly or indirectly various mediator systems involved in the inflammatory response during severe sepsis in nonhuman primates.

J Biol Chem, 1996 Mar 15, 271(11), 6429 - 34
Different glycosylation requirements for the synthesis of enzymatically active angiotensin-converting enzyme in mammalian cells and yeast; Sadhukhan R et al.; For facilitating crystallization and structural studies of the testicular isozyme of angiotensin-converting enzyme (ACE,), we attempted the production of enzymatically active ACET proteins which are unglycosylated or underglycosylated . Expression in Escherichia coli of the rabbit ACET cDNA resulted in the synthesis of an unglycosylated but inactive protein . Similarly, unglycosylated ACET synthesized in HeLa cells, by using a cDNA in which all five potential N-glycosylation sites had been mutated, was inactive and rapidly degraded . Several ACET variants carrying mutations in one or more of the potential N-glycosylation sites were used to examine the role of glycosylation at specific sites on ACET synthesis, transport to the cell surface, cleavage processing, and enzyme activity . These experiments demonstrated that allowing glycosylation only at the first or the second site, as counted from the NH2 terminus, was sufficient for normal synthesis and processing of active ACET . In contrast, ACETg3, which had only the third glycosylation site available, was unglycosylated, enzymatically inactive and rapidly degraded . N-Glycosylated ACET could also be produced in yeast . Surprisingly, the mutant ACETg3 was synthesized, N-glycosylated, and properly transported in yeast . Wild type and mutant ACE proteins were cleavage-secreted from yeast and enzymatically active.

J Biol Chem, 1996 Mar 15, 271(11), 6313 - 21
Effects of conserved residues on the regulation of rabbit muscle pyruvate kinase; Cheng X et al.; A cDNA encoding the complete rabbit muscle pyruvate kinase isozyme (RMPK) was cloned using the method of rapid amplification of cDNA ends . The sequence encodes a polypeptide chain of 530 amino acids which differs in three amino acid residues from a sequence reported by Larsen et al . (Larsen, T.M., Laughlin, T., Holden, H.M., Rayment, L, and Reed, G.H . (1994) Biochemistry 33, 6301-6309) . Glu233-Gln234 and Ala400 were identified instead of Asp233-Glu234 and Ser400, respectively . The recombinant RMPK was overexpressed in the Escherichia coli JM 105 cells . Purified recombinant pyruvate kinase displayed identical physical and enzymatic properties as the authentic enzyme . Three point mutants of RMPK were constructed using site-directed mutagenesis . Like the wild type RMPK, sedimentation, and CD spectroscopic studies show that purified RI 19C and T340M are tetrameric proteins with similar secondary and tertiary structures . Mutant R119C enzyme exhibits 0.6% of the value of k(cat) and an order of magnitude decrease in the apparent affinity for ADP as compared to the wild type PK . The overall response to inhibitor and activator, Phe and FBP, respectively, were not affected by the R119C mutation . The T340M mutant enzyme is only half as active as the wild type PK . T340M is more susceptible to inhibition by Phe but apparently is not responsive to the activator FBP . The kinetic behavior of the Q377K mutant enzyme is in between that of the R119C and T340M mutants exhibiting 5% of the wild type enzymatic activity and an enhanced sensitivity to the inhibitor, Phe, while maintaining the same responsiveness to FBP and apparent affinities for substrates . The significant decrease in activity in all three mutants mimics the exact consequences of the same mutations in human erythrocyte PK from hemolytic anemia patients . Thus, this study demonstrates not only the effects of these conserved residues in the regulatory properties of mammalian PK . but also that the observed effects are most likely applicable to all isozymic forms of PK.

J Biol Chem, 1996 Mar 15, 271(11), 6241 - 4
A nascent secretory protein may traverse the ribosome/endoplasmic reticulum translocase complex as an extended chain; Whitley P et al.; We have measured the minimum number of residues in a translocating polypeptide required to bridge the distance between the P-site in endoplasmic reticulum-bound ribosomes and the lumenally disposed active site of the oligosaccharyl transferase . The results suggest that a nascent chain may traverse the ribosome/translocase complex in a largely extended conformation, and that hydrophobic stop-transfer segments have a more compact, possibly alpha-helical conformation in the translocase.

J Biol Chem, 1996 Mar 15, 271(11), 6137 - 43
Real time kinetics of the DnaK/DnaJ/GrpE molecular chaperone machine action; Banecki B et al.; Applying stopped-flow fluorescence spectroscopy for measuring conformational changes of the DnaK molecular chaperone (bacterial Hsp70 homologue) and its binding to target peptide, we found that after ATP hydrolysis, DnaK is converted to the DnaK*(ADP) conformation, which possesses limited affinity for peptide substrates and the GrpE cochaperone but efficiently binds the DnaJ chaperone . In the presence of DnaJ (bacterial Hsp40 homologue), the DnaK*(ADP) form is converted back to the DnaK conformation, and the resulting DnaJ-DnaK(ADP) complex binds to peptide substrates more tightly . Formation of the DnaJ(substrate-DnaK(ADP)) complex is a rate-limiting reaction . The presence of GrpE and ATP hydrolysis promotes the fast release of the peptide substrate from the chaperone complex and converts DnaK to the DnaK*(ADP) conformation . We conclude that in the presence of DnaJ and GrpE, the binding-release cycle of DnaK is stoichiometrically coupled to the adenosine triphosphatase activity of DnaK.

Arch Biochem Biophys, 1996 Mar 15, 327(2), 308 - 18
Elucidation of amino acid residues critical for unique activities of rabbit cytochrome P450 2B5 using hybrid enzymes and reciprocal site-directed mutagenesis with rabbit cytochrome P450 2B4; Szklarz GD et al.; The molecular basis for the unique activities of rabbit cytochrome P450 2B5, compared with the highly related rabbit 2B4, was investigated using hybrid enzymes and site-directed mutagenesis . Alterations in androstenedione hydroxylase profiles observed with 2B4-2B5 hybrids expressed in COS cells showed that key amino acids are present in both the N-terminal ApaI fragment (codons 1-122) and an internal SstI fragment (codons 220-393) . Based on these results, data obtained with other cytochromes P450 2B, and correlation to the six substrate recognition sites proposed by Gotoh (1992, J . Biol . Chem . 267, 83-90), reciprocal 2B4-2B5 mutants were constructed at positions 114, 294, 363, and 367 . Wild-type and mutant enzymes were expressed in Escherichia coli, and the oxidation of a number of substrates was analyzed . All residues studied were found to be important for regio- and stereospecificity of androstenedione hydroxylation . Mutations at these positions also caused alterations in the oxidation of progesterone, benzyloxyresorufin, pentoxyresorufin, ethoxycoumarin, and benzphetamine, with the magnitude and direction of the changes dependent upon the enzyme, residue, and substrate . Major changes in activity were consistently observed upon mutation of residues 114 and 294 in both enzymes, and some of these alterations were interpreted with the help of a 3-D model of P450 2B4 . For example, in the 2B4 Ile-114--> Phe mutant, Phe prevents androstenedione from assuming a 16 beta-binding orientation and also hinders binding of benzyloxyresorufin, leading to a loss of activity . Conversely, the presence of Phe-114 stabilizes a 16 alpha-binding orientation of androstenedione, resulting in an increase in this activity.

Arch Biochem Biophys, 1996 Mar 15, 327(2), 254 - 9
Coexpression of mammalian cytochrome P450 and reductase in Escherichia coli; Dong J et al.; cDNAs for human cytochrome P450 2E1 and rat NADPH-cytochrome-P450 reductase were cloned separately and in tandem into bacterial expression vectors, and expression of the two proteins in Escherichia coli was monitored by immunoblotting, spectroscopy, and catalytic assays . The cDNAs were separated on the coexpression plasmid by 22 nucleotides, with the P450 cDNA preceding the reductase cDNA . P450 content in solubilized cell membranes, whether expressed alone or coexpressed with P450 reductase, was approximately 0.11 nmol/mg of protein, and approximately 0.8 nmol could be obtained per liter of culture . Reductase content was five- to sixfold greater than P450 content when coexpressed, but severalfold less than that obtained when expressed without the upstream P450 cDNA, indicating differences in both stability and translatability between the two proteins . Solubilized membranes from cells expressing both proteins catalyzed aniline hydroxylation, p-nitrophenol hydroxylation, and N-nitrosodimethylamine demethylation at rates equivalent to those obtained by combining P450 and reductase preparations; addition of purified reductase to these membranes did not augment the activity . However, in contrast to results obtained with P450 2E1 expressed in other heterologous systems, addition of rabbit liver cytochrome b5 to preparations catalyzing p-nitrophenol or N-nitrosodimethylamine oxidation did not increase turnover, and, although activity could be shown with unsolubilized membranes, oxidation of these substrates in vivo could not be demonstrated . Nonetheless, the ability to coexpress P450 and reductase in E . coli so as to generate a functional monooxygenase system in vitro enhances the utility of this organism for the expression and characterization of cloned P450 isoforms.

Arch Biochem Biophys, 1996 Mar 15, 327(2), 249 - 53
Functional significance of the Cu,ZnSOD in Escherichia coli; Benov L et al.; Diethyl dithiocarbamate (DDC) was used to inhibit the copper- and zinc- containing superoxide dismutase (Cu,ZnSOD) of Escherichia coli in order to expose its physiological function . DDC inhibited the aerobic growth of a sodA sodB mutant much more than it did the growth of a SOD-replete parental strain and this inhibitory effect was oxygen-dependent . The SOD mimic MnTMPyP markedly diminished the growth inhibitory effect of DDC . Transfer of the sodA sodB strain from anaerobic to aerobic conditions induced fumarase C, which is a member of the soxRS regulon . DDC augmented this induction . These results indicate that the Cu,ZnSOD provides a defense against oxidative stress, which is more important to the sodA sodB mutant than to its SOD-replete parental strain.

Biochem J, 1996 Mar 15, 314 ( Pt 3), 827 - 32
Acidic phospholipids inhibit the intramolecular association between the N- and C-terminal regions of vinculin, exposing actin-binding and protein kinase C phosphorylation sites; Weekes J et al.; Chick vinculin polypeptides expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins have been used to identify the sites involved in the intramolecular association between the 90 kDa N-terminal head and the 30 kDa C-terminal tail region of the vinculin molecule . Fusion proteins spanning vinculin residues 1-258 and 1-398, immobilized on glutathione-agarose beads, were shown to bind a C-terminal vinculin polypeptide spanning residues 881-1066 (liberated from GST by thrombin cleavage) . However, the C-terminal polypeptide did not bind to a fusion protein spanning residues 399-881 or to itself . Binding was dependent on residues 167-207 within the N-terminal polypeptide, a sequence also essential for talin binding . Conversely, the 90 kDa head polypeptide was shown to bind to residues 1029-1036 in the tail region of vinculin . The association of the head and tail was inhibited by acidic, but not neutral, phospholipids . Pre-incubation of vinculin with acidic phospholipids exposed the binding site for F-actin and a phosphorylation site for protein kinase C . The phosphorylation site was located in the tail region of the vinculin molecule . These results raise the possibility that acidic phospholipids play a role in regulating the activity of vinculin and therefore the assembly of both cell-cell and cell-matrix adherens-type junctions.

Biochem J, 1996 Mar 15, 314 ( Pt 3), 787 - 91
The effect of different amino acid side chains on the stereospecificity and catalytic efficiency of the tryptophan synthase-catalysed exchange of the alpha-protons of amino acids; Milne JJ et al.; 1H-NMR has been used to follow the tryptophan synthase (EC 4.2.1.20) c catalysed hydrogen-deuterium exchange of the alpha-protons of L- and D-alanine and -tryptophan . The first-order and second-order rate constants for exchange have been determined at pH 7.8 in the presence and absence of the allosteric effector, DL-alpha-glycerol 3-phosphate . In the presence of DL-alpha-glycerol 3-phosphate the stereospecificity of the tryptophan synthase-catalyzed first-order exchange rates was in the order tryptophan > alanine > glycine . This increase in stereospecificity was largely due to the decrease in the magnitude of the first-order exchange rate of the slowly exchanged alpha-proton . A similar increase in the stereospecificity of the second-order exchange rates for alanine was also largely due to the decrease in the magnitude of the first-order exchange rate of the slowly exchanged alpha-proton of D-alanine . Adding DL-alpha-glycerol 3-phosphate produced an increase in the stereospecificity of the second-order exchange rate observed with alanine but no significant change in the stereospecificity of the first-order exchange rate with tryptophan . The alpha-subunits are shown to increase the exchange rates of the alpha-protons of L-alanine and L-tryptophan . We conclude that the contribution of the R-group of an amino acid to the stereospecificity of the exchange reactions of its alpha-proton can be similar to or larger than that of its alpha-carboxylate group . Possible mechanisms that could explain the stereospecificity of these exchange reactions are discussed.

Virology, 1996 Mar 15, 217(2), 607 - 12
DNA-binding activity of the C2 protein of tomato yellow leaf curl geminivirus; Noris E et al.; The in vitro DNA-binding activity of the C2 protein of tomato yellow leaf curl geminivirus (TYLCV) was studied following its expression in Escherichia coli as a fusion protein with an His tag N-terminal extension (His-C2) . Southwestern blotting experiments demonstrated that the C2 protein is able to bind both single-stranded and double-stranded DNA probes . In electrophoretic mobility shift assays performed using purified protein and single-stranded DNA probes several shifted complexes were formed . The presence of NaCl (up to 800 mM) did not substantially affect binding profiles, demonstrating a stable interaction . His-C2 appeared to bind single-stranded DNA in a sequence-nonspecific manner, with a preference for single-stranded compared to double-stranded DNA . Deletion mutants demonstrated that the central core of C2 (amino acids 33 to 104), which contains a Cys-His rich region, is sufficient for conferring binding activity . The potential significance of this DNA-binding activity with respect to possible biological functions of TYLCV C2 protein is discussed.

Virology, 1996 Mar 15, 217(2), 594 - 7
Disassembly of the coliphage lambda replication complex due to heat shock induction of the groE operon; Wegrzyn A et al.; We have found previously that, in contrast to the free O initiator protein of lambda phage or plasmid rapidly degraded by the Escherichia coli ClpP/ClpX protease, the lambda O present in the replication complex (RC) is protected from proteolysis . In amino acid-starved E . coli relA cells, a temperature shift from 30 to 43 degrees did not affect RC integrity, as judged from the unchanged level of stable lambda O observed; however, the same temperature shift in a complete medium resulted in the decay of this lambda O fraction, which suggested disassembly of the RC . Examination of this phenomenon revealed that for lambda RC disassembly, heat shock induction of the groE operon, coding for molecular chaperones of the Hsp60 class, is indispensable . Heat shock induction of the groE operon present on a multicopy plasmid inhibited the growth of infecting phage.

Virology, 1996 Mar 15, 217(2), 527 - 31
Mutagenesis of the Sindbis virus nsP1 protein: effects on methyltransferase activity and viral infectivity; Wang HL et al.; It has been suggested that four amino acids which are absolutely conserved in th nsP1 nonstructural proteins encoded by togaviruses and in the homologous proteins encoded by plant viruses in the Sindbis virus (SV) superfamily may constitute a "methyltransferase motif." In the Sindbis virus nsP1 protein (540 amino acids) these four amino acids are represented by His39, Arg91, Asp94, and Tyr249 . Earlier, in assays of methyltransferase (MTase) activity generated in SV-infected cells, we had shown that amino acid changes at positions 87 and 88 of SV nsP1 resulted in a 10-fold lower Km for S-adenosyl methionine, the methyl donor in MTase reactions . Using site-directed mutagenesis we now report the expression of nsP1 in Escherichia coli, and in the infectious clone of Sindbis virus, Toto/1101, in which His39, Arg91, Asp94, and Tyr249 were changed one at a time to Ala . We also expressed nsP1 with C-terminal deletions of varying size, as well as with internal deletions in the C-terminal portion of the protein, in E . coli . Changing His39, Arg91, Asp94, or Tyr249 to Ala led to a loss of both MTase activity and viral infectivity; however, changing Ile369 to Val, a conservative change in the carboxy-terminal half of nsP1, had no effect on either MTase activity or viral infectivity . With respect to the deleted forms of nsP1, a carboxy-terminal deletion of 48 amino acids was still compatible with MTase activity in vitro . However, larger deletions including those in which the amino acids between positions 442 and 492 were deleted abolished MTase activity.

Nucleic Acids Res, 1996 Mar 15, 24(6), 1112 - 8
Orientation of functional activating regions in the Escherichia coli CRP protein during transcription activation at class II promoters; Williams RM et al.; At class II CRP-dependent promoters the DNA site for CRP overlaps the DNA site for RNA polymerase, covering the -35 region . Transcription activation at class II CRP- dependent promoters requires a contact between an activating region in the upstream subunit of the bound CRP dimer and a contact site in the C-terminal domain of the alpha-subunit of RNA polymerase . Transcription activation is suppressed by amino acid substitutions in the activating region, but activation can be restored by second site substitutions at K52 or E96 . These substitutions identify two separate regions on the surface of CRP that appear to be able to interact with RNA polymerase specifically at class II promoters . Using the method of 'oriented heterodimers' we show that these alternative activating regions are functional in the downstream subunit of the bound CRP dimer.

Nucleic Acids Res, 1996 Mar 15, 24(6), 1073 - 9
Conformational flexibility in RNA: the role of dihydrouridine; Dalluge JJ et al.; In order to further understand the structural role of the modified nucleoside dihydrouridine in RNA the solution conformations of Dp and ApDpA were analyzed by one- and two-dimensional proton NRM spectroscopy and compared with those of the related uridine-containing compounds . The analyses indicate that dihydrouridine significantly destabilizes the C3'-endo sugar conformation associated with base stacked, ordered, A-type helical RNA . Equilibrium constants (Keq = {C2'-endo}/{C3'-endo}) for C2'-endo-C3'-endo interconversion at 25 degrees C for Dp, the 5'-terminal A of ApDpA and D in ApDpA are 2.08, 1.35 and 10.8 respectively . Stabilization of the C2'-endo form was shown to be enhanced at low temperature, indicating that C2'-endo is the thermodynamically favored conformation for dihydrouridine . DeltaH values show that for Dp the C2'-endo sugar conformation is stabilized by 1.5 kcal/mol compared with Up . This effect is amplified for D in the oligonucleotide ApDpA and propagated to the 5'-neighboring A, with stabilization of the C2'-endo form by 5.3 kcal/mol for D and 3.6 kcal/mol for the 5'-terminal A . Post-transcriptional formation of dihydrouridine therefore represents a biological strategy opposite in effect to ribose methylation, 2-thiolation or pseudouridylation, all of which enhance regional stability through stabilization of the C3'-endo conformer . Dihydrouridine effectively promotes the C2'-endo sugar conformation, allowing for greater conformational flexibility and dynamic motion in regions of RNA where tertiary interactions and loop formation must be simultaneously accommodated.

Nucleic Acids Res, 1996 Mar 15, 24(6), 1059 - 64
Interaction of tRNA (uracil-5-)-methyltransferase with NO2Ura-tRNA; Gu X et al.; tRNA in which uracil is completely replaced by 5-nitro-uracil was prepared by substituting 5-nitro-UTP for UTP in an in vitro transcription reaction . The rationale was that the 5-nitro substituent activates the 6-carbon of the Ura heterocycle towards nucleophiles, and hence could provide mechanism-based inhibitors of enzymes which utilize this feature in their catalytic mechanism . When assayed shortly after mixing, the tRNA analog, NO2Ura-tRNA, is a potent competitive inhibitor of tRNA-Ura methyl transferase (RUMT) . Upon incubation, the analog causes a time-dependent inactivation of RUMT which could be reversed by dilution into a large excess of tRNA substrate . Covalent RUMT-NO2Ura-tRNA complexes could be isolated on nitrocellulose filters or by SDS-PAGE . The interaction of RUMT and NO2Ura-tRNA was deduced to involve formation of a reversible complex, followed by formation of a reversible covalent complex in which Cys 324 of RUMT is linked to the 6-position of NO2Ura 54 in NO2Ura-tRNA.

J Neurosci, 1996 Mar 15, 16(6), 1975 - 81
Localization of synaptotagmin-binding domains on syntaxin; Kee Y et al.; Synaptotagmin, an abundant calcium- and phospholipid-binding protein of synaptic vesicles, has been proposed to regulate neurotransmitter release at the nerve terminal . To understand better the biochemical mechanism of neurotransmitter release, we have investigated the calcium-dependent and -independent protein-protein interactions between synaptotagmin I and syntaxin 1a, a subunit of the receptor for synaptic vesicles on the presynaptic plasma membrane . Soluble syntaxin 1a binds to synaptotagmin glutathione S-transferase (GST) fusion protein, and the binding was decreased in the presence of calcium . A synaptotagmin fragment containing the second C2 repeat (Syt3-5) had the same binding profile as the whole cytoplasmic domain; however, fragments containing the first C2 repeat (Syt1-3 and Syt2-3) showed calcium-dependent binding to syntaxin . In addition, the soluble full-length cytoplasmic domain of synaptotagmin binds to a syntaxin GST fusion protein in a calcium-dependent manner . Syntaxin domains required for calcium-dependent and -independent synaptotagmin-binding were localized using syntaxin deletion mutants . Amino acids 241-266 of the syntaxin C terminus were required for calcium-independent binding of synaptotagmin . The minimal domain required for calcium-dependent binding of synaptotagmin to syntaxin was localized to amino acids 220-266 . The syntaxin domains required for synaptotagmin binding overlap with the domains for vesicle-associated membrane protein (or VAMP) and alpha-soluble N-ethyl-maleimide-sensitive fusion protein attachment protein (or alphaSNAP) interactions . The data suggest both calcium-dependent and -independent roles of synaptotagmin in regulating synaptic vesicle release and/or recycling.

J Mol Biol, 1996 Mar 15, 256(5), 897 - 908
The substrate-binding site in Escherichia coli cyclophilin A preferably recognizes a cis-proline isomer or a highly distorted form of the trans isomer; Konno M et al.; The three-dimensional structure of Escherichia coli cytosolic cyclophilin A (CyPA) complexed with a tripeptide (succinyl-Ala-Pro-Ala-p-nitroanilide) was refined at 1.8 A resolution by the multiple isomorphous replacement method to a crystallographic R-factor of 17.6% . As in human CyPA, the peptide binding site in E . coli enzyme is in a cleft created on the surface of the upper sheet of two orthogonal beta-sheets . In this cleft, the walls of the hydrophobic pocket are formed by the side-chains of five non-polar residues, Phe48, Met49, Phe107, Leu108, and Try120, with Phe99 at the bottom . When the cis isomer of the tripeptide binds to the enzyme, a cis-proline ring is inserted into the hydrophobic pocket . Since the binding pocket of CyPAs are largely hydrophobic, the cis isomer of a peptide can be bound more firmly than the trans isomer . Distortion of the trans isomer could lead to better binding, but at an energetic cost of the distortion energy . At the periphery of the upper beta-sheet in E . coli CyPA, conformations of loops L1, L3, and L4 and the segment connecting alpha1 and beta3 with deletions or insertions against human CyPA differ significantly from those in human CyPA . The refined model also shows that steric hindrance to attachment of cyclosporin A (CsA) prevents E . coli CyPA forming a complex with CsA . Thus, the extra amino acid residue of E . coli CyPA, polar Gln89, lies along the pathway to the hydrophobic pocket of CyPA and seems to prevent the access hydrophobic part of CsA to the cleft of CyPA.

J Mol Biol, 1996 Mar 15, 256(5), 889 - 96
In vivo interaction between mutated tryptophan repressors of Escherichia coli; Storbakk N et al.; By expressing a mutant trpR gene in an Escherichia coli strain that is trpR and has beta-galactosidase activity fused to the trp promoter/operator, thus putting the beta-galactosidase activity under the control of the Trp repressor, we can determine quantitatively the relative repression activity of such mutant(s) . We used this technique to analyse the biological consequences of substituting certain amino acid residues in only one of the two corepressor binding pockets . By combining two compatible plasmids in this strain, one expressing the mutant T44M and the other expressing only one substitution at a time at position 85, we analysed the repression activity of the resulting interactions in vivo . This approach allowed us to engineer active dimer repressors made of two inactive or partially active monomers . Amino acid substitutions at position 85 with a positive or with an indole ring (W) appeared to complement T44M, which amino acids with a negative charge did not . Only L substitution at position 85 appeared to restore activity among the hydrophobic amino acids tested . Similar to the wild-type repressor activity, the successful mutant-mutant interactions were L-tryptophan dependent . In vivo regulation by three known L-tryptophan analogues demonstrated the same trend of regulation among the wild-type repressors and the active mutant-mutant combinations.

J Mol Biol, 1996 Mar 15, 256(5), 878 - 88
Identification of local carboxy-terminal hydrophobic interactions essential for folding or stability of chloramphenicol acetyltransferase; Van der Schueren J et al.; The role of the carboxy terminal in folding and stabilization of type I chloramphenicol acetyltransferase (CAT1) has been studied by mutagenesis and Fourier transform infrared analysis . We have shown that a CAT mutant truncated by seven amino acid residues folds into active protein . In this study, the last three residues of this truncated CAT mutant were randomized to detect structural information required for achieving a native enzyme conformation . Statistical analysis of sequencing data from randomly chosen mutants revealed that the amino-terminal CAT fragment of 212 amino acid residues is the shortest deletion mutant able to adopt a soluble, enzymatically active structure . This minimal length corresponds to a protein with full-length alpha5-helix in the three-dimensional crystal structure of CAT type III . The amino acid preferences at the carboxy terminal in the randomization experiments suggest that this helix also forms completely in the shortened CAT mutants . In addition correct folding and/or stabilization requires the formation of a hydrophobic + microdomain at the end of the alpha5-helix . The role of this hydrophobic interaction in CAT folding and structure stabilization is discussed.

J Mol Biol, 1996 Mar 15, 256(5), 859 - 69
Specificity and binding properties of a single-chain T cell receptor; Schlueter CJ et al.; The specificity of a T cell is dictated by an alpha beta T cell receptor (TCR) that recognizes a complex of peptide and a product of the major histocompatibility complex (MHC) . Recent studies have begun to characterize the affinities and kinetics of these interactions, but details of the alpha beta TCR structure and function are not known . To examine some of these issues we focus in this report on a TCR derived from the T cell clone 2C . This TCR binds to a complex of the nonapeptide QL9 and the class I MHC product Ld with the highest affinity of any known TCR/ligand interaction (KD approximately 10 (-7) M) . Circular dichroism showed that a single-chain TCR (scTCR) containing linked V alpha and V beta regions from T cell 2C and refolded from Escherichia coli inclusion bodies exhibited the characteristic beta-sheet structure of immunoglobulins . A sensitive assay that is capable of detecting the interaction of soluble scTCR with peptide /MHC ligand on the surface of target cells was used to demonstrate that the peptide specificity of this scTCR reflects that of the TCR found on the surface of 2C . Analysis of several scTCR V alpha region mutants confirmed that the V alpha domain is critical for the specificity of scTCR binding . Finally, we identified some notable differences in the complementarity determining regions (CDR) of the 2C TCR compared to the CDR of previously characterized, cytochrome- specific TCR . These differences are discussed in the light of what is known about antibody binding sites, the high affinity of the 2C TCR, and the nature of the residues on QL9 that are predicted to interact with the TCR.

J Mol Biol, 1996 Mar 15, 256(5), 818 - 28
Functional connectivity between tRNA binding domains in glutaminyl-tRNA synthetase; Sherman JM et al.; The structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) in complex with tRNAGln and ATP has identified a number a sequence-specific protein-tRNA interactions . The contribution to glutamine identity has previously been determined for the nucleotides in tRNAGln . Here, we report the mutational analysis of residues in all three tRNA recognition domains of GlnRS, thus completing a survey of the major sequence-specific contacts between GlnRS and tRNAGln . Specifically, we analyzed the GlnRS determinants involved in recognition of the anticodon which is essential for glutamine identity and in the communication of anticodon recognition to the acceptor binding domain in GlnRS . A combined in vivo and in vitro approach has demonstrated that Arg341, which makes a single sequence-specific hydrogen bond with U35 in the anticodon of tRNAGln, is involved in initial RNA recognition and is an important positive determinant for this base in both cognate and non- cognate tRNA contexts . However, Arg341, as well as Arg402, which interacts with G36 in the anticodon, are negative determinants for non-cognate nucleotides at their respective positions . Analysis of acceptor-anticodon binding double mutants and of a mutation of Glu323 in the loop-strand-helix connectivity subdomain in GlnRS has further implicated this domain in the functional communication of anticodon recognition . The better than expected activity (anticooperativity) of these double mutants has led us to propose an "anticodon-independent" mechanism, in which the removal of certain synthetase interactions with the anticodon eliminates structural constraints, thus allowing the relaxed specificity mutants in the acceptor binding domain ot make more productive interactions.

Exp Cell Res, 1996 Mar 15, 223(2), 348 - 56
Immunocytochemical characterization and subcellular localization of human myristoyl-CoA: protein N-myristoyltransferase in HeLa cells; McIlhinney RA et al.; Antisera have been raised to three synthetic peptides based on the sequence of human myristoyl-CoA:protein N-myristoyl transferase (NMT) and to the purified enzyme following its expression in Escherichia coli . These antisera have been affinity purified and shown to react both with the E . coli expressed human NMT, and specifically with a protein of molecular weight of 63 kDa in immunoblots of the human cell line HeLa . The affinity purified antibodies have also been used to localize NMT in methanol/acetone permeabilized HeLa cells by immunofluorescent staining . The immunofluorescence showed a diffuse staining pattern throughout the cell, suggesting that the enzyme is predominantly cytosolic . This was confirmed by determining the distribution of NMT activity in different subcellular fractions of HeLa cells . Over 90% of NMT enzymatic activity was released from cell lysates during either hypotonic or isotonic homogenization . However, a small amount of enzymatic activity remained associated with cell membranes, despite extensive washing, and this was confirmed by immunoblot analysis of these membranes for NMT . In comparison, over 99.5% of lactate dehydrogenase activity was released under the same conditions, which suggests that the NMT was genuinely associated with the cell membranes . The membrane-bound enzyme behaved like a peripheral membrane protein . Permeabilization HeLa cells with 50 microM digitonin resulted in the release of 90-93% of lactate dehydrogenase compared to 73-85% of NMT, again suggesting that the majority of the enzyme is cytosolic, but that some may be associated with cell membranes or organelles.

Exp Cell Res, 1996 Mar 15, 223(2), 203 - 14
A keratin antibody recognizing a heterotypic complex: epitope mapping to complementary locations on both components of the complex; Waseem A et al.; Keratin filaments in simple epithelial cells are heteropolymers of keratin 8 (K8) and keratin 18 (K18), which can be stained by the monoclonal antibody (MAb) LE61 . This antibody has been widely used to study keratin expression in normal and neoplastic tissues . In this study we have found that MAb LE61 does not react with individual keratin polypeptides either derived from natural sources or expressed as recombinant proteins in Escherichia coli . However, when K8 or K18 bound to nitrocellulose were incubated with complementary keratin they became reactive with this antibody . A mixture of K8 and K18 in solution also reacted strongly with the MAb LE61 in ELISA . These observations suggest that the antibody recognizes a discontinuous epitope on the keratin complex . The antibody also reacted with complexes of K8 and K18 with other keratins . To locate the epitope of this antibody we have expressed K8 and K18 fragments, deleted from the amino- and carboxyl-termini, as fusion proteins with glutathione S-transferase . These fragments were able to form a heterotypic complex with the complementary keratin . Binding of the MAb LE61 to these complexes mapped the two halves of the epitope on K8, between residues 353 and 367, and on K18, between residues 357 and 385 . The two halves of the epitope appear to be in close association in the heterotypic complex since deletions from the amino-terminus did not influence the antibody binding . The highly conserved nature of this epitope in both type I and type II keratins could explain the MAb LE61 reactivity with complexes of K8 or K18 with other keratins.

Genes Dev, 1996 Mar 15, 10(6), 762 - 73
Determinants of selectivity in Xer site-specific recombination; Blakely G et al.; A remarkable property of some DNA-binding proteins that can interact with and pair distant DNA segments is that they mediate their biological function only when their binding sites are arranged in a specific configuration . Xer site-specific recombination at natural plasmid recombination sites (e.g., cer in ColE1) is preferentially intramolecular, converting dimers to monomers . In contrast, Xer recombination at the Escherichia coli chromosomal site dif can occur intermolecularly and intramolecularly . Recombination at both types of site requires the cooperative interactions of two related recombinases, XerC and XerD, with a 30-bp recombination core site . The dif core site is sufficient for recombination when XerC and XerD are present, whereas recombination at plasmid sites requires approximately 200 bp of adjacent accessory sequences and accessory proteins . These accessory factors ensure that recombination is intramolecular . Here we use a model system to show that selectivity for intramolecular recombination, and the consequent requirement for accessory factors, can arise by increasing the spacing between XerC- and XerD-binding sites from 6 to 8 bp . This reduces the affinity of the recombinases for the core site and changes the geometry of the recombinase/DNA complex . These changes are correlated with altered interactions of the recombinases with the core site and a reduced efficiency of XerC-mediated cleavage . We propose that the accessory sequences and proteins compensate for these changes and provide a nucleoprotein structure of fixed geometry that can only form and function effectively on circular molecules containing directly repeated sites.

Genes Dev, 1996 Mar 15, 10(6), 755 - 61
Transposase is the only nematode protein required for in vitro transposition of Tc1; Vos JC et al.; The Tc1 element of Caenorhabditis elegans is a member of the most widespread class of DNA transposons known in nature . Here, we describe efficient and precise transposition of Tc1 in a cell-free system . Tc1 appears to jump by a cut-and-paste mechanism of transposition . The terminal 26 bp of the Tc1 terminal repeats together with the flanking TA sequence are sufficient for transposition . The target site choice in vitro is similar to that in vivo . Transposition is achieved with an extract prepared from nuclei of transgenic nematodes that overexpress Tc1 transposase but also by recombinant transposase purified from Escherichia coli . The simple reaction requirements explain why horizontal spread of Tc1/mariner transposons can occur . They also suggest that Tcl may be a good vector for transgenesis of diverse animal species.

Biochemistry, 1996 Mar 12, 35(10), 3328 - 34
Intersubunit communication in tryptophan synthase by carbon-13 and fluorine-19 REDOR NMR; McDowell LM et al.; The beta subunits of the 143-kDa alpha2beta2 tetrameric enzyme tryptophan synthase have been labeled by L-{ring-4-19F}phenylalanine and L-{phenol-4-13C}tyrosine in an effort to monitor the positions of these residues on ligand binding . Of the 13 phenylalanine and 11 tyrosine residues in the beta subunit, only three pairs have labels with 13C-19F separations of less than 6 angstrom . The beta subunit residues Tyr279 and Phe280 (each members of one of the three Tyr-Phe proximate pairs) have been suggested as possible conformational gates on ligand binding . The 188-MHz 19F NMR spectrum of the microcrystalline, double-labeled enzyme complex has five resolved lines under 5-kHz magic-angle spinning and 80-kHz proton dipolar decoupling . The distribution of beta-subunit 19F isotropic shifts is altered by addition of L-{3-13C}-serine to the mother liquor in contact with the microcrystals, consistent with a conformational rearrangement . The 13C label from serine is detected at 28 ppm as a methyl tautomer of bound aminoacrylate . The change in aromatic 19F chemical shifts on binding of serine indicates an alteration in local electric field gradients within the beta subunit . However, rotational-echo double-resonance 13C NMR (with 19F dephasing) shows that the average 13C-19F distance for the three phenylalanine-tyrosine proximate pairs in the beta subunit is changed by less than 1 angstrom.

Biochemistry, 1996 Mar 12, 35(10), 3297 - 308
Conformational characterization of DnaK and its complexes by small-angle X-ray scattering; Shi L et al.; DnaK, a member of the 70 kDa heat shock protein (hsp70) family, and its complexes with substrate proteins and nucleotides were characterized by small-angle X-ray scattering (SAXS) and size-exclusion chromatography (SEC) techniques . The SAXS data indicated that DnaK has a dumbbell-shaped structure with a maximum dimension (dmax) of 112 angstrom, which is consistent with the reported two major functional domains {Chappell et al . (1987) J . Biol . Chem . 268, 12730-12735; Flaherty et al . (1990) Nature 346, 623-628} . The data were best fit by a model in which the two domains either are connected by a short hinge region or are just in contact with each other . The radius of gyration (Rg) of DnaK was determined as 37.5 +/- 1.0 angstrom in the absence of nucleotide . Binding of ATP induces a conformational change in DnaK as reflected by the changes in its P(r) function and Kratky plot, the increases (1-2 angstrom) in both its radius of gyration (Rg) and its Stokes radius (Rs), and the increase in its dmax (5-10 angstrom ) . SAXS and SEC-HPLC results indicate that the association state of DnaK is very sensitive to the buffer concentration and the presence of substrates, as well as the protein concentration . At high buffer and protein concentrations, DnaK dimerizes, resulting in an increase in its apparent Rg and dmax values . The addition of substrate (unfolded protein or ATP) results in a return to the Rg value of monomeric DnaK, due to the dissociation of DnaK multimers induced by the substrate binding and resultant conformational changes . The DnaK-substrate protein complex gives a smaller Rg than expected, suggesting that the substrate protein binds to a cavity or cleft on DnaK rather than the exterior of the chaperone . The Kratky plot of the Gdn.HCl-induced unfolding intermediate state of DnaK is consistent with a compact, molten globule-like conformation, as previously suggested based on CD, fluorescence, and SEC-HPLC results {Palleros et al . (I 993) Biochemistry 32, 4314-4321}.

Biochemistry, 1996 Mar 12, 35(10), 3270 - 6
Complexation of the tissue plasminogen activator protease with benzamidine-type inhibitors: interference by the kringle 2 module; Hu CK et al.; Well-resolved high-field 1H NMR signals between -0.1 and -0.7 ppm afford convenient probes to monitor the conformational state of the tissue plasminogen activator (tPA) protease, modulated by covalent inhibitor binding or activation cleavage {Hu, C.-K., Kohnert, U., Wilhelm, O., Fischer, S., & Llinas, M . (1994) Biochemistry 33, 11760-11766} . We have investigated recombinant BM 06.022 (a domain-deletion variant mutant from Escherichia coli comprising the kringle 2 and protease modules) and protease constructs of tPA in both single-chain (sc) and two-chain (tc) forms . The two proteins were studied when confronted with the noncovalent (i.e., reversible) active site inhibitors benzamidine and a series of bisbenzamidine derivatives: 2,5-bis(4-amidinobenzylidene)cyclopentanone, 2,6-bis(4-amidinobenzylidene)cyclohexanone, 2,7-bis(4-amidinobenzylidene)cycloheptanone, and 2,8-bis(4-amidino- benzylidene)cyclooctanone . At pH* 4.6, the 1H NMR spectrum is sensitive to complexation of the protease module with the various effectors . The amplitude of the inhibitor-shifted resonances is more pronounced for the tc-protease than for the sc-protease, suggesting that access of inhibitors to the protease catalytic site is facilitated upon conversion to the tc form . The effects detected by the NMR spectrum suggest a biphasic process, involving stronger (primary) and weaker (secondary) bindings to a single protease active site . Binding to the protease module in tc-BM 06.022 essentially generates the same spectral characteristics as detected upon binding to the isolated tc-protease construct . In contrast, a negligible perturbation by the inhibitors is observed on the (sc) BM 06.022 . Hence, in the intact BM 06.022 the kringle 2 is structurally coupled to the protease module thus interfering with inhibitor molecules from accessing the protease active site . These domain-domain interactions relax upon conversion to the catalytically active tc form, thus decoupling the kringle 2 from the protease module in BM 06.022 while simultaneously exposing the active site to become accessible to effectors or substrates.

Biochemistry, 1996 Mar 12, 35(10), 3247 - 57
Interaction of an engineered {3Fe-4S} cluster with a menaquinol binding site of Escherichia coli DMSO reductase; Rothery RA et al.; We have characterized by EPR the interaction of the Em,7 = -50 mV {4Fe-4S} cluster of Escherichia coli DMSO reductase (DmsABC) with a menaquinol (MQH2) binding site . Potentiometric titrations indicate that in DmsAB(C102S)C, the Em,7 = -50 mV {4Fe-4S} cluster is replaced by an Em,7 = +260 mV {3Fe-4S} cluster . The Q-pool coupling assay in combination with the MQH2 analog HOQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide) was used to examine the effect of the DmsB(Cl02S) mutation on physiological electron transfer through DmsABC . Forward electron transfer through the mutant (MQH2 to DmsA) is blocked in the Q-pool coupling assay, but reverse electron transfer (DmsA to MQ) is not . HOQNO elicits a significant change in the EPR line shape of the oxidized DmsAB(Cl02S)C {3Fe-4S} cluster but has no effect on the line shape of the reduced {4Fe-4S} clusters . We have identified a residue in DmsC involved in MQH2 oxidation . DmsC(H65), and in a double mutant, DmsAB(C102S)C(H65R), the DmsC mutation blocks the HOQNO effect on the {3Fe-4S} EPR line shape, suggesting, that the DmsC(H65R) mutation either blocks HOQNO binding or blocks a conformational link between a HOQNO binding site and the DmsB(C102S) {3Fe-4S} cluster . These results suggest that the MQH2 binding site of DmsC is conformationally and functionally linked to the Em,7 = -50 mV {4Fe-4S} cluster of DmsB.

Biochemistry, 1996 Mar 12, 35(10), 3213 - 21
An intracellular adenine nucleotide binding site inhibits guanyly cyclase C by a guanine nucleotide-dependent mechanism; Parkinson SJ et al.; Guanylyl cyclase C (GCC), the receptor for the Escherichia coli heat-stable enterotoxin (ST), is inhibited by 2-substituted adenine nucleotides in an allosteric fashion . In confluent cultures of Caco-2 intestinal epithelial cells, extracellular 2-methylthioadenosine triphosphate (2MeSATP) had no effect on basal or ST-stimulated cyclic GMP (cGMP) accumulation . However, this nucleotide inhibited cGMP accumulation in digitonin-permeabilized Caco-2 human colon carcinoma cells, demonstrating that allosteric inhibition of GCC by adenine nucleotides is mediated by an intracellular adenine nucleotide binding site rather than purinergic receptors . The role of guanine nucleotides in the regulation of GCC by adenine nucleotides was examined . Increasing GTP concentrations from 5 to 100 microM increased the potency of 2MeSATP inhibition of GCC 20-fold, with a shift in the Ki from 447 to 22 microM, respectively . Also, the hydrolysis-resistant analogue, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), supported 2MeSATP inhibition of GCC with a potency which was 10-fold greater than GTP . In addition, GTP alone, in the absence of adenine nucleotides and at concentrations greater than 1 mM, inhibited GCC through a mechanism convergent with 2MeSATP . Guanine nucleotides supported adenine nucleotide inhibition of GCC at low concentrations and directly inhibited this enzyme at high concentrations when these studies were conducted with receptors expressed in Caco-2 cells, native rat intestine, or cloned rat GCC heterologously expressed in 293 monkey kidney cells . These observations demonstrate that adenine nucleotide inhibition of GCC is mediated through an intracellular mechanism which is guanine nucleotide-dependent.

Brain Res, 1996 Mar 11, 712(1), 153 - 8
Regionally specific induction of ICE mRNA and enzyme activity in the rat brain and adrenal gland by LPS; Tingsborg S et al.; Pro interleukin-1 beta converting enzyme (ICE) activity in the pituitary was found to be significantly increased 4 h after intraperitoneal injection of E . coli lipopolysaccharides, when distribution and inducibility of the enzyme was studied in the adult rat brain and the adrenal gland, using an artificial fluorescence peptide substrate . The same lipopolysaccharide treatment induced ICE mRNA levels in the pituitary, adrenal gland and hypothalamus as studied by reverse transcript-polymerase chain reaction.

FEBS Lett, 1996 Mar 11, 382(1-2), 79 - 83
Comparative studies of gamma-interferon receptor-like proteins of variola major and variola minor viruses; Seregin SV et al.; To study specific properties of the human gamma-interferon (gamma-IFN) receptor-like proteins of the highly virulent and low virulent strains of variola (smallpox) virus (VAR) recombinant plasmids determining synthesis of these proteins in E . coli cells have been constructed . The recombinant viral gamma-IFN receptor-like proteins have been found to have high interferon-neutralising activity with regard to human gamma-IFN but not murine gamma-IFN and human alpha-IFN . The variola major and variola minor proteins under study do not differ in the efficiency of human gamma-IFN antiviral activity inhibition.

FEBS Lett, 1996 Mar 11, 382(1-2), 6 - 10
Expression of recombinant pro-neuropeptide Y, proopiomelanocortin, and proenkephalin: relative processing by 'prohormone thiol protease' (PTP); Schiller MR et al.; The preference of the 'prohormone thiol protease' (PTP), a candidate prohormone processing enzyme, for different peptide precursors was assessed in vitro with recombinant prohormones near estimated in vivo levels . Pro-neuropeptide Y (pro-NPY), proopiomelanocortin (POMC), and proenkephalin (PE) were expressed at high levels in E . coli . Purification of prohormones utilized a combination of DEAE-Sepharose, Mono Q, and preparative electrophoresis . PTP cleaved PE most readily, and also cleaved pro-NPY . The processing of POMC by PTP was minimal . These results demonstrate PTP's preference for certain prohormone substrates.

FEBS Lett, 1996 Mar 11, 382(1-2), 218 - 9; discussion 220-1
Redox control of gene expression involving iron-sulfur proteins . Change of oxidation-state or assembly/disassembly of Fe-S clusters?
Beinert H, Kiley P.
Attention is drawn to a mechanism of redox control of gene expression involving Fe-S proteins which depends on the disassembly and reassembly of Fe-S clusters rather than a change in oxidation state . Iron Regulatory Protein (IRP)/aconitase and FNR are discussed as examples for such a mechanism.

FEBS Lett, 1996 Mar 11, 382(1-2), 21 - 5
Solubility of artificial proteins with random sequences; Prijambada ID et al.; A library of artificial random proteins of 141 amino acid residues of which 95 are random and which includes the 20 kinds of amino acids was prepared . Out of the 25 identified random proteins, 5 were soluble in the cell lysate, indicating that about 20% of the random proteins expressed in Escherichia coli are expected to be soluble . The soluble random proteins RP3-42 and RP3-45 and insoluble RP3-70 were purified . The solubility of the purified form is the same as that in the cell lysate.

FEBS Lett, 1996 Mar 11, 382(1-2), 153 - 8
Identification of a third yeast mitochondrial Tom protein with tetratrico peptide repeats; Bomer U et al.; The mitochondrial outer membrane contains a protein complex with at least eight subunits responsible for recognition and translocation of preproteins synthesized in the cytosol . Two subunits, the receptors Tom20 and Tom70, contain tetratrico peptide repeats that are thought to be involved in protein-protein interactions . We have identified Saccharomyces cerevisiae Tom72, a new Tom protein expressed at a low level . Tom72 is homologous to Tom 70, including seven tetratrico peptide repeats . Tom72 is targeted to the mitochondrial outer membrane, forms a large domain exposed to the cytosol and loosely associates with the translocase complex of the outer membrane . These results suggest that Tom72 represents a ninth, weakly expressed component of the preprotein translocase of the mitochondrial outer membrane.

FEBS Lett, 1996 Mar 11, 382(1-2), 141 - 4
The flavohaemoglobin (HMP) of Escherichia coli generates superoxide in vitro and causes oxidative stress in vivo; Membrillo-Hernandez J et al.; Purified flavohaemoglobin (HMP) of Escherichia coli reduces Fe(III) in a superoxide dismutase (SOD)-sensitive reaction, demonstrating superoxide anion generation during aerobic NADH oxidation . In vivo, sodA-lacZ fusion activity was increased 3-fold by introducing plasmid pPL341, containing the hmp gene, or by growth with paraquat . The effects were additive and SOXS-dependent . Thus HMP activity causes oxidative stress in vivo . Activities of sodA-lacZ and hmp-lacZ fusions were stimulated in a himA mutant, demonstrating repression of both promoters by integration host factor (IHF), but the effects of pPL341 on sodA-lacZ activity were not due to titration of IHF by the hmp promoter.

Gene, 1996 Mar 9, 169(2), 251 - 5
Synthesis of a modified gene encoding human ornithine transcarbamylase for expression in mammalian mitochondrial and universal translation systems: a novel approach towards correction of a genetic defect; Wheeler VC et al.; The mitochondrial (MT) genome is a potential means of gene delivery to human cells for therapeutic expression . As a first step towards this, we have synthesized a gene coding for mature human ornithine transcarbamylase (OTC) by recursive PCR using 18 oligodeoxyribonucleotides, each 70-80 nucleotides in length, using codons which should allow translation in accordance with both mammalian mt and universal codon usage . Flanking mt DNA sequences were incorporated which are designed to facilitate site-specific cloning into the mt genome . Expression of this human gene in Escherichia coli leads to an immunoreactive OTC product of the correct size and N-terminal amino-acid sequence, but which forms inclusion bodies and lacks enzymatic activity.

Gene, 1996 Mar 9, 169(2), 165 - 71
Cloning, sequencing and expression of the bovine CD3 epsilon and TCR-zeta chains, two invariant components of the T-cell receptor complex; Hagens G et al.; CD3 epsilon and the zeta-chain of the bovine T-cell receptor (TCR) are two invariant molecules with an important role in signal transduction via the TCR/CD3 complex . The nucleotide sequence of a bovine CD3 epsilon cDNA clone containing the complete coding sequence was determined and the deduced amino acid (aa) sequence compared to that of other species . The cytoplasmic domains of the different CD3 epsilon clearly show a higher degree of conservation than the extracellular domains . Bovine CD3 epsilon produced in Escherichia coli using different bacterial expression vectors was recognised by antibodies (Ab) directed against the intracytoplasmic domain of human CD3 epsilon . A partial bovine TCR zeta-chain cDNA was generated by the polymerase chain reaction (PCR) using primers that were based on sequences that are conserved between different species; 3' and 5' RACE-PCR were carried out to obtain the complete TCR zeta-chain cDNA sequence . A comparison of the predicted TCR zeta-chain aa sequence reveals that the GDP/GTP-binding motif, which is conserved in other species, shows marked differences in the bovine and ovine TCR zeta-chains . In contrast to CD3 epsilon, the short extracellular domain of the TCR zeta-chain is 100% conserved between the different species and the transmembrane domain also shows a high degree of identity . Ab were raised against the TCR zeta-chain, produced as a glutathione S-transferase fusion protein in E . coli, and were used in Western blot analysis to further characterise TCR zeta-chain expression in T-cells . The regents provide valuable tools for the study of signal transduction pathways in normal and transformed bovine T-cells.

J Biotechnol, 1996 Mar 8, 45(3), 235 - 41
Investigating the use of the chymosin-sensitive sequence of kappa-casein as a cleavable linker site in fusion proteins; Walsh MK et al.; The chymosin-sensitive sequence of bovine k-casein A (kappa-CN A) was investigated as a cleavable linker site between the two domains of a streptavidin-chloramphenicol acetyltransferase fusion protein . Two DNA sequences were synthesized which encode the amino acids from 101 to 107 and from 97 to 113 of bovine kappa-CN A . These sequences were separately cloned in-frame to a streptavidin expression vector used for fusion protein construction . The gene for chloramphenicol acetyltransferase (CAT) was then cloned in-frame to a streptavidin-chymosin-sensitive linker vector forming plasmids pStCL1CAT and pStCL2CAT . The fusion protein was expressed in Escherichia coli and SDS-PAGE and Western blot analysis of chymosin-treated cell lysates showed a pH-dependent cleavage of the fusion proteins . Fusion proteins were also bioselectively immobilized onto biotinylated controlled-pore glass beads and treated with chymosin . CAT was specifically released by chymosin treatment and was identified by SDS-PAGE.

J Biotechnol, 1996 Mar 8, 45(3), 211 - 6
Overexpression and purification of human immunodeficiency virus type 1 env derived epitopes in Escherichia coli; Sohn MJ et al.; In order to develop a reliable and inexpensive serodiagnostic method, a part of envelope gene of HIV-1, gp120' and gp41' (HIV-1 env a.a . 295-474 and a.a . 556-647) was cloned into a T7 expression vector (pET3d) . The fusion protein (gp120'-gp41') was overexpressed in Escherichia coli, then purified to homogeneity by a simple gel filtration chromatography . Western blot analysis and enzyme-linked immunosorbent assay (ELISA) using the purified fusion protein showed a high sensitivity and a specificity for the detection of anti HIV-1 antibodies in testing human plasma . These results suggest that the expression scheme employing a direct expression vector and the rapid purification method are reliable and applicable for obtaining a large quantity of HIV-1 env protein for diagnoses of HIV-1 infections.

J Mol Biol, 1996 Mar 8, 256(4), 685 - 700
Major identity determinants in the "augmented D helix" of tRNA(Glu) from Escherichia coli; Sekine S et al.; By a kinetic analysis of 59 variant transcripts of Escherichia coli tRNA(Glu) with glutamyl-tRNA synthetase (GluRS), the U11.A24 base-pair, the U13.G22..A46 base-triple, and the lack of residue 47 (delta47) were found to serve as major determinants for tRNA(Glu) identity . This is the first system for which major identity determinants are reported to be clustered in the "augmented D helix", consisting of the D stem with some neighboring residues and the variable loop . Other identity determinants are U34, U35, C36 and A37 in the anticodon loop, and G1.C72 and U2.A71 in the acceptor stem . Phosphate-group protection by GluRS from ethylnitrosourea was observed most strongly for the minor groove side of D-stem helix, indicating that GluRs tightly binds to the D stem for recognition, on the minor groove side, of the potent identity-determinant groups of the U11.A24 and U13.G22 base-pairs . A46 is not involved in direct recognition by GluRS; the U13.G22..A46 base-triple is required probably for formation of the structural features that are recognized by GluRS . In this context, the essential role of characteristic delta47 in tRNA(Glu) identity may be to maintain the U13.G22..A46 base-triple.

J Biol Chem, 1996 Mar 8, 271(10), 5805 - 11
Expression, purification, and mechanistic studies of bovine mitochondrial translational initiation factor 2; Ma J et al.; A complete cDNA clone encoding bovine mitochondrial translational initiation factor 2 (IF-2mt) has been obtained . The regions of the cDNA corresponding to mature IF-2mt and several of its functional domains have been expressed in Escherichia coli as histidine-tagged proteins . The precursor (approximately 90 kDa) and mature (approximately 85 kDa) forms of IF-2mt are toxic to E . coli and can only be expressed at low levels . Shorter forms of this factor (approximately 80 and approximately 72 kDa) are also found during the expression of mature IF-2mt . The various forms of IF-2mt can be separated by high performance liquid chromatography . All of these forms are active in promoting the GTP-dependent binding of formyl-Met-tRNA to the small subunit of either E . coli or bovine mitochondrial ribosomes . IF-2mt can bind to mitochondrial ribosomes in the absence of GTP, initiator tRNA, or messenger RNA . The presence of GTP stimulates IF-2mt binding to ribosomes about 3-fold . IF-2mt interacts only weakly with GTP or with the initiator tRNA in the absence of ribosomes . Molecular dissection of IF-2mt shows that a long deletion (approximately 150 amino acid residues) from the NH2-terminal region does not affect its activity in vitro . The COOH domain of IF-2mt (amino acid residues 332-727) can bind to ribosomes even though it does not promote initiator-tRNA binding.

J Biol Chem, 1996 Mar 8, 271(10), 5725 - 32
Evidence for the coupling of ATP hydrolysis to the final (extension) phase of RecA protein-mediated DNA strand exchange; Bedale WA et al.; RecA protein promotes a limited DNA strand exchange reaction, without ATP hydrolysis, that typically results in formation of short (1-2 kilobase pairs) regions of hybrid DNA . This nascent hybrid DNA is extended in a reaction that can be coupled to ATP hydrolysis . When ATP is hydrolyzed, the extension phase is progressive and its rate is 380 +/- 20 bp min-1 at 37 degrees C . A single RecA nucleoprotein filament can participate in multiple DNA strand exchange reactions concurrently (involving duplex DNA fragments that are homologous to different segments of the DNA within a nucleoprotein filament), with no effect on the observed rate of ATP hydrolysis . The ATP hydrolytic and hybrid DNA extension activities exhibit a dependence on temperature between 25 and 45 degrees C that is, within experimental error, identical . This provides new evidence that the two processes are coupled . Arrhenius activation energies derived from the work are 13.3 +/- 1.1 kcal mole-1 for DNA strand exchange, and 14.4 +/- 1.4 kcal mole-1 for ATP hydrolysis during strand exchange . The rate of branch movement in the extension phase (base pair min-1) is related to the kcat for ATP hydrolysis during strand exchange (min-1) by a factor equivalent to 18 bp throughout the temperature range examined . The 18-base pair factor conforms to a quantitative prediction derived from a model in which ATP hydrolysis is coupled to a facilitated rotation of the DNA substrates . RecA filaments possess an intrinsic capacity for DNA strand exchange, mediated by binding energy rather than ATP hydrolysis, that is augmented by an ATP-dependent molecular motor.

J Biol Chem, 1996 Mar 8, 271(10), 5712 - 24
DNA strand exchange promoted by RecA K72R . Two reaction phases with different Mg2+ requirements; Shan Q et al.; Replacement of lysine 72 in RecA protein with arginine produces a mutant protein that binds but does not hydrolyze ATP . The protein nevertheless promotes DNA strand exchange (Rehrauer, W . M., and Kowalczykowski, S . C . (1993) J . Biol . Chem . 268, 1292-1297) . With RecA K72R protein, the formation of the hybrid DNA product of strand exchange is greatly affected by the concentration of Mg2+ in ways that reflect the concentration of a Mg.dATP complex . When Mg2+ is present at concentrations just sufficient to form the Mg.dATP complex, substantial generation of completed product hybrid DNAs over 7 kilobase pairs in length is observed (albeit slowly) . Higher levels of Mg2+ are required for optimal uptake of substrate duplex DNA into the nucleoprotein filament, indicating that the formation of joint molecules is facilitated by Mg2+ levels that inhibit the subsequent migration of a DNA branch . We also show that the strand exchange reaction promoted by RecA K72R, regardless of the Mg2+ concentration, is bidirectional and incapable of bypassing structural barriers in the DNA or accommodating four DNA strands . The reaction exhibits the same limitations as that promoted by wild type RecA protein in the presence of adenosine 5'-O-(3-thio)triphosphate . The Mg2+ effects, the limitations of RecA-mediated DNA strand exchange in the absence of ATP hydrolysis, and unusual DNA structures observed by electron microscopy in some experiments, are interpreted in the context of a model in which a fast phase of DNA strand exchange produces a discontinuous three-stranded DNA pairing intermediate, followed by a slow phase in which the discontinuities are resolved . The mutant protein also facilitates the autocatalytic cleavage of the LexA repressor, but at a reduced rate.

J Biol Chem, 1996 Mar 8, 271(10), 5617 - 22
Requirements for calcium and calmodulin in the calmodulin kinase activation cascade; Tokumitsu H et al.; We have previously purified and cloned rat brain Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK), and the 68-kDa recombinant CaM-KK activates in vitro both CaM-kinase IV (CaM-K IV) and CaM-K I (Tokumitsu, H., Enslen, H., and Soderling, T . R . (1995) J . Biol . Chem . 270, 19320-19324) . In the present study we have determined that activation of CaM-K IV through phosphorylation of Thr196 by CaM-KK is triggered by elevated intracellular Ca2+ in intact cells and requires binding of Ca2+/CaM to both enzymes . An expressed fragment of CaM-K IV (CaM-K IV178-246), which contains the activating phosphorylation site (Thr196) but not the autoinhibitory domain or the CaM-binding domain, still required Ca2+/CaM for phosphorylation by wild-type CaM-KK . A truncated mutant of CaM-KK (CaM-KK1-434) phosphorylated CaM-K IV178-246 in a Ca2+/CaM-independent manner, but this constitutively active CaM-KK1 434 required Ca2+/CaM for phosphorylation and activation of wild-type CaM-K IV . These results demonstrate that binding of Ca2+/CaM to both CaM-K IV and CaM-KK is required for the CaM-kinase cascade . Both CaM-KK and CaM-K IV appear to have similar Ca2+/CaM requirements with EC50 values of approximately 100 nM . Studies using co-expression of CaM-K IV with CaM-KK in COS-7 cells demonstrated that CaM-KK rapidly activated both total and Ca2+/CaM-independent activities of wild-type CaM-K IV, but not the Thr196 --> Ala mutant, upon ionomycin stimulation.

J Biol Chem, 1996 Mar 8, 271(10), 5580 - 8
Intermediates in the catalytic cycle of copper-quinoprotein amine oxidase from Escherichia coli; Steinebach V et al.; Investigations on the reduction of copper quinoprotein amine oxidases (EC 1.4.3.6) by substrate indicate that the nature of the reduced enzyme species formed varies, as judged from the spectroscopic data reported in the literature for different enzymes and substrates . The availability of substantial amounts of overproduced, homogeneous Escherichia coli amine oxidase (ECAO) enabled us to investigate this aspect with a number of different approaches: quantitative titration of enzyme with substrate, stopped-flow kinetic spectrophotometry (anaerobic and semianaerobic), EPR spectroscopy of stable intermediates in the catalytic cycle, and conversions with H2O2 as the oxidant . Reduction of ECAO by a variety of substrates led to spectra (UV/Vis, EPR) identical to those that have been ascribed to the semiquinone form of the topaquinone cofactor . The extent of semiquinone formation was enhanced in the presence of KCN, but the properties of the artificially induced semiquinone were different from those of the spontaneously induced one, as shown by the spectroscopic data and the reactivity toward O2 and H2O2 . On titrating ECAO at high concentrations with substrate, evidence was obtained that disproportionation takes place of the semiquinone formed, the reaction most probably proceeding via intermolecular electron transfer, leading to a topaquinone- and Cu1+-containing enzyme species that is able to perform substrate conversion . The latter, as well as OH*, is probably also formed when H2O2 replaces O2 as oxidant, explaining why substrate conversion with concomitant enzyme inactivation occurs under this condition . Formation of the semiquinone was always preceded by that of a hitherto unknown species with an absorbance maximum at 400 nm . The structure proposed for this species is a protonated form of the aminoquinol cofactor, the Zwitter ionic structure being stabilized by amino acid residues in the active site having opposite charges . Based on the properties observed and the moment of appearance during conversions, a proposal is made for the sequence in which the three reduced enzyme species convert into each other.

J Biol Chem, 1996 Mar 8, 271(10), 5558 - 64
Selective modification of recombinant bovine placental lactogen by site-directed mutagenesis at its C terminus; Vashdi-Elberg D et al.; Five recombinant analogues of bovine placental lactogen (bPL) ((bPL(S184H), bPL(S187A), bPL(S187F), bPL(T188F), bPL(T188F,I190F)) were prepared, expressed in Escherichia coli, and purified to homogeneity . Circular dichroism analysis revealed no or minor structural changes, except in bPL(T188F,I190F) . Binding and biological activities of bPL(T188F,I190F) were almost completely abolished, whereas bPL analogues mutated at position 187 retained their full activity . Point mutation T188F resulted in selective modification; binding to somatogenic receptors, their extracellular domains (ECDs), and to bPLR in the endometrium as well as somatogenic receptor-mediated biological activities were reduced or abolished, whereas binding to lactogenic receptors, their ECDs, and subsequent biological activity was fully or almost fully retained . This selective modification most likely results from a steric hindrance induced by a bulky Phe-188 chain of bPL which interacts with the Arg-43 of the human or Leu-43 of the non-human GHRs . Point mutation S184H abolished the interaction with hGHR, most likely due to the unfavorable charge-charge interaction, possibly accompanied by steric hindrance between Arg-43 of the receptor and the newly introduced His-184 and possible interference with the putative interaction between the alkyl portion of Thr-188 and Lys-185 of bPL with Trp-104 of hGHR . In contrast, bPL(S184H) retained its capacity to interact with nonhuman GHRs . Decrease in the biological activity of bPL(S184H) was also observed in two lactogenic receptor-mediated bioassays most likely due to the elimination of the intermolecular hydrogen bond of Ser-184 with a side chain of Tyr-127, which appears in all lactogenic receptors.

J Biol Chem, 1996 Mar 8, 271(10), 5438 - 42
Porcine 80-kDa protein reveals intrinsic 17 beta-hydroxysteroid dehydrogenase, fatty acyl-CoA-hydratase/dehydrogenase, and sterol transfer activities; Leenders F et al.; Four types of 17beta-hydroxysteroid dehydrogenases have been identified so far . The porcine peroxisomal 17beta-hydroxysteroid dehydrogenase type IV catalyzes the oxidation of estradiol with high preference over the reduction of estrone . A 2.9-kilobase mRNA codes for an 80-kDa (737 amino acids) protein featuring domains which are not present in the other 17beta-hydroxysteroid dehydrogenases . The 80-kDa protein is N terminally cleaved to a 32-kDa fragment with 17beta-hydroxysteroid dehydrogenase activity . Here we show for the first time that both the 80-kDa and the N-terminal 32 kDa (amino acids 1-323) peptides are able to perform the dehydrogenase reaction not only with steroids at the C17 position but also with 3-hydroxyacyl-CoA . The central part of the 80-kDa protein (amino acids 324-596) catalyzes the 2-enoyl-acyl-CoA hydratase reaction with high efficiency . The C-terminal part of the 80-kDa protein (amino acids 597-737) is similar to sterol carrier protein 2 and facilitates the transfer of 7-dehydrocholesterol and phosphatidylcholine between membranes in vitro . The unique multidomain structure of the 80-kDa protein allows for the catalysis of several reactions so far thought to be performed by complexes of different enzymes.

J Biol Chem, 1996 Mar 8, 271(10), 5422 - 9
Adriamycin-induced DNA adducts inhibit the DNA interactions of transcription factors and RNA polymerase; Cutts SM et al.; Adriamycin is known to specifically induce DNA interstrand cross-links at 5'-GC sequences . Because 5'-GC sequences are a predominant feature of 5'-untranslated regions (transcription factor-binding sites, promoter, and enhancer regions), it is likely that adriamycin adducts at GC sites would affect the binding of DNA-interacting proteins . Two model systems were chosen for the analysis: the octamer-binding proteins Oct-1, N-Oct-3 and N-Oct-5, which bind to ATGCAAAT and TAATGARAT recognition sites, and Escherichia coli RNA polymerase binding to the lac UV5 promoter . Electrophoretic mobility shift studies showed that adriamycin adducts at GC sites inhibited the binding of octamer proteins to their consensus motifs at drug levels as low as 1 micoM, but no effect was observed with a control sequence lacking a GC site . Adriamycin adducts at GC sites also inhibited the binding of RNA polymerase to the lac UV5 promoter . Adriamycin may therefore function by down-regulating the expression of specific genes by means of inactivation of short but critical motifs containing one or more GC sites.

J Biol Chem, 1996 Mar 8, 271(10), 5338 - 46
Comparative properties of the single chain antibody and Fv derivatives of mAb 4-4-20 . Relationship between interdomain interactions and the high affinity for fluorescein ligand; Mallender WD et al.; Recombinant Fv derivative of the high affinity murine anti-fluorescein monoclonal antibody 4-4-20 was constructed and expressed in high yields, relative to the single chain antibody (SCA) derivative (2 3-fold), in Escherichia coli . Both variable heavy (VH) and variable light (VL) domains, that accumulated as insoluble inclusion bodies, were isolated, denatured, mixed, refolded, and affinity-purified to yield active Fv 4-4-20 . Affinity-purified Fv 4-4-20 showed identical ligand binding properties compared with the SCA construct, both were slightly lower than the affinities expressed by Fab or IgG 4-4-20 . Proper protein folding was shown to be domain-independent by in vitro mixing of individually refolded variable domains to yield functional Fv protein . In solid phase and solution phase assays, Fv 4-4-20 closely approximated the SCA derivative in terms of both idiotype and metatype, confirming identical active site structures and conformations . The equilibrium dissociation constant (Kd) for the VL/VH association (1.43 x 10(-7) M), which was determined using the change in fluorescein spectral properties upon ligand binding, was relatively low considering the high affinity displayed by the Fv protein for fluorescein (Kd, 2.9 x 10(-10) M) . Thus, domain-domain stability in the Fv and SCA 4-4-20 proteins cannot be the sole cause of reduced affinity (2-3-fold) for fluorescein as compared with the Fab or IgG form of 4-4-20 . With their identical ligand binding and structural properties, the decreased SCA or Fv affinity for fluorescein must be an ultimate consequence of deletion of the CH1 and CL constant domains . Collectively, these results verify the importance of constant domain interactions in antibody variable domain structure-function analyses and future antibody engineering endeavors.

J Biol Chem, 1996 Mar 8, 271(10), 5313 - 6
The nuclear transport factor karyopherin beta binds stoichiometrically to Ran-GTP and inhibits the Ran GTPase activating protein; Floer M et al.; The heterodimeric karyopherin functions in targeting a nuclear localization sequence (NLS)-containing protein to the nuclear pore complex followed by Ran-GTP and p10-mediated translocation of the NLS protein into the nucleoplasm . It was shown recently that Ran-GTP dissociated the karyopherin heterodimer and, in doing so, associated with karyopherin beta (Rexach, M., and Blobel, G . (1995) Cell 83, 683-692) . We show here, using all recombinant yeast proteins expressed in Escherichia coli, that karyopherin beta binds to Ran-GTP and inhibits GTP hydrolysis stimulated by RanGAP (the Ran-specific GTPase activating protein) . Inhibition of RanGAP-stimulated GTP hydrolysis by karyopherin beta was dependent on karyopherin beta concentration relative to Ran-GTP . Complete inhibition of RanGAP was observed at karyopherin beta concentrations that were equimolar to Ran-GTP . In gel filtration experiments, we found Ran-GTP and karyopherin beta to form a stoichiometric complex . Ran-GDP bound only weakly to karyopherin beta . We propose that stoichiometric complex formation between karyopherin beta and Ran-GTP renders Ran-GTP inaccessible to RanGAP.

J Biol Chem, 1996 Mar 8, 271(10), 5297 - 300
ADP-ribosylation factor-1 stimulates formation of nascent secretory vesicles from the trans-Golgi network of endocrine cells; Chen YG et al.; ADP-ribosylation factor (ARF) is a small GTP-binding protein that has been implicated in intracellular vesicular transport . ARF regulates the budding of vesicles that mediate endoplasmic reticulum to Golgi and intra-Golgi transport . It also plays an important role in maintaining the function and morphology of the Golgi apparatus . Using a permeabilized cell system derived from GH3 cells, we provide evidence that ARF-1 regulates the formation of nascent secretory vesicles from the trans-Golgi network . Both myristoylated and non-myristoylated forms of recombinant human ARF-1 enhanced secretory vesicle budding about 2-fold . A mutant lacking the first 17 N-terminal residues, as well as one that preferentially binds GDP (T31N) did not stimulate vesicle formation . In contrast, a mutant defective in GTP hydrolysis (Q71L) promoted vesicle budding . Strikingly, a peptide corresponding to the N terminus of human ARF-1 (amino acids 2-17) also stimulated vesicle budding from the trans-Golgi network, in marked contrast to its inhibitory effect on vesicular transport from the endoplasmic reticulum to Golgi . These data demonstrate that in endocrine cells, ARF-1 and in particular its N terminus play an essential role in the formation of secretory vesicles.

Biochim Biophys Acta, 1996 Mar 7, 1293(1), 90 - 6
Synthesis, characterization and properties of sialylated catalase; Fernandes AI et al.; Colominic acid (CA), an alpha-(2-->8) N-acetylneuraminic acid (sialic acid) polymer (average molecular weight of 10 kDa) was activated by periodate oxidation of carbon 7 at the non-reducing end of the saccharide . The oxidized CA was then coupled to catalase by reductive amination in the presence of sodium cyanoborohydride . The extent of sialylation of catalase, estimated by ammonium sulfate precipitation as 3.8+/-0.4 (mean+/-S.D.) moles of CA per mole of catalase, did not improve significantly when depolymerized CA was used in the coupling reaction . At the end of the coupling reaction, sialylated catalase exhibited a two-fold (70%) retention of initial activity compared to enzyme controls (29-35%) subjected to the same conditions . Formation of sialylated catalase was confirmed by ammonium sulfate or trichloroacetic acid precipitation, molecular sieve chromatography and SDS-PAGE electrophoresis . Enzyme kinetics studies revealed an increase in the apparent Km of the enzyme from 70.0 (native) to 122.9 mmol l-1 H2O2 (sialylated catalase) indicating a reduction of enzyme affinity for the substrate (hydrogen peroxide) on sialylation . Compared to native enzyme, sialylated catalase was much more stable in the presence of specific proteinases, completely resisting degradation by chymotrypsin and losing only some of its activity in the presence of trypsin . The increased stability conferred to catalase by sialylation agrees with similar observations on enzymes modified by other hydrophilic molecules (e.g., monomethoxypoly(ethyleneglycol)) and suggests that steric stabilization with the biodegradable polysialic acid may prove an alternative means to render therapeutic proteins more effective in vivo.

Biochim Biophys Acta, 1996 Mar 7, 1293(1), 31 - 8
Evidence that human milk isolated cyclophilin B corresponds to a truncated form; Mariller C et al.; Cyclophilin B (CyPB) is a member of the cyclophilin family (cyclosporin A-binding proteins) with specific N- and C-terminal extensions . In contrast to cyclophilin A, CyPB owns a signal sequence leading to its translocation in the endoplasmic reticulum . CyPB was reported to be present in human blood and milk, suggesting it is secreted . For this purpose, CyPB was purified to homogeneity from human milk and compared to recombinant CyPB expressed in E . coli . Ion spray mass spectrometry revealed that CyPB secreted in human milk exhibits a lower molecular mass than the one expected . Identification of phenylalanine as the C-terminus amino-acid residue of human milk CyPB indicates that the difference in molecular mass may be explained by the absence of the five C-terminal amino-acid residues AIAKE . These results suggest that in the sequence VEKPFAIAKE known to be responsible for retention of CyPB in the endoplasmic reticulum, the sequence AIAKE is more particularly necessary . Our findings raise the possibility that the CyPB may be processed to promote its release . As recombinant CyPB was shown to bind specifically to Jurkat cells, a lymphoblastic T-cell line, we then wanted to investigate the binding of human milk CyPB to these cells . Despite lacking the five C-terminal amino-acid residues, human milk CyPB is able to inhibit the binding of recombinant CyPB to Jurkat T cells.

Biochim Biophys Acta, 1996 Mar 7, 1293(1), 154 - 60
Physical and kinetic effects on induction of various linker regions in beta-galactosidase/galactose dehydrogenase fusion enzymes; Carlsson H et al.; To examine the role of the connecting region in the artificial bifunctional enzyme beta-galactosidase/galactose dehydrogenase, linkers of different length were inserted between the catalytic units . The specific activity of the galactose dehydrogenase part of the complex was increased when longer linkers (9 and 13 amino acids) were used as connectors . These bifunctional enzymes were predominantly found to comprise hexamers, however, complexes of higher molecular weight were also formed . The sequential reaction was carried out more efficiently when hybrid enzymes with the longer linkers were used as demonstrated both in vitro by using purified protein preparations as well as in vivo by determining the growth rates of recombinant E . coli cells on a minimal medium containing lactose.

Biochem Biophys Res Commun, 1996 Mar 7, 220(1), 94 - 7
An attempt to convert noncatalytic nucleotide binding site of F1-ATPase to the catalytic site: hydrolysis of tethered ATP by mutated alpha subunits in the enzyme; Matsui T et al.; The alpha and beta subunits of F1-ATPase are homologous in primary structure and have similar folding topologies . The position of the essential Glu residue in the catalytic sites which reside in the beta subunits is occupied by a Gln residue in the noncatalytic nucleotide binding sites which reside in the alpha subunits . To test if an exchange of catalytic and noncatalytic binding sites is possible, we have replaced the Gln-Lys sequence in the noncatalytic binding site of the alpha subunit with Glu-Arg and, reciprocally, the Glu in the catalytic site of the beta subunit with Gln . The resultant mutant alpha3beta3gamma complex lost steady-state ATPase activity . However, HPLC analysis of tryptic digests of the mutant alpha3beta3gamma complex which had been photolabeled with 2-N3-{8-3H}ATP revealed that ATP tethered to the noncatalytic binding side was hydrolyzed, indicating that a primitive catalytic ability was generated at the alpha subunit by the introduced Glu.

Biochem Biophys Res Commun, 1996 Mar 7, 220(1), 53 - 6
Selection of trypsin inhibitors in phage peptide library; Fang R et al.; The newly developed techniques of peptide libraries have become a conventional and efficient method in screening ligands of proteins of interest . We present here the successful results of selection of trypsin inhibitors in a phage hexapeptide library . After affinity selection and activity assay, peptide sequences, deduced from DNA sequencing of the phage peptides with the most striking trypsin activity, share some common features with trypsin inhibitors reported . All of the phage peptides selected out and those native and synthetic trypsin inhibitors reported are composed of three parts: (a) positively charged part (Arg, Lys or their analogs); (b) polar part that may form hydrogen bonds with Ser in the active site of trypsin; (c) hydrophobic part that interacts with the nonpolar region of trypsin active site.

Biochem Biophys Res Commun, 1996 Mar 7, 220(1), 160 - 5
Inhibition of protein serine/threonine phosphatases by fumonisin B1, a mycotoxin; Fukuda H et al.; Fumonisin B1 (FB1), a mycotoxin produced by the fungus Fusarium moniliforme, which is a common contaminant of corn, is suspected to be a cause of human esophageal cancer . FB1 is hepatotoxic and hepatocarcinogenic in rats, and although the mechanisms involved have not been clarified, the latter is associated with a weak initiating activity . The effects of FB1 on the activity of protein serine/threonine phosphatases (PPs) (PP1, PP2A, PP2B, PP2C and PP5/T/K/H) were investigated in the present study . Inhibition of dephosphorylation was noted for all five PPs with IC50 values of 80 microM-3000 microM . Among the five PPs examined, PP5 was most sensitive with an IC50 of 80 microM . This concentration is comparable to that estimated to be reached in the rat body by feeding FB1 to obtain hepatic tumors . Inhibition of PP5 could thus play important roles in the toxicity and carcinogenic action of FB1.

Biochem Biophys Res Commun, 1996 Mar 7, 220(1), 131 - 6
Renaturation of recombinant human pro-urokinase expressed in Escherichia coli; Hua ZC et al.; A synthetic gene encoding human pro-urokinase (pro-UK) with E . coli-favored codon usage was cloned into plasmid pET-3d and expressed in E . coli BL21(DE3) LysS strain . The expressed products, which accumulated as inactive inclusion bodies, were denatured and renatured in vitro . A broad range of parameters such as pH, protein concentration, denaturant concentration, the use of cosolvent polyethylene glycol and presence of basic or acidic amino acid was examined . At optimal renaturation condition, pro-UK activity of more than 1000I.U was obtained from 1 milliliter cell culture.

Mol Gen Genet, 1996 Mar 7, 250(4), 466 - 76
Differential mRNA decay within the transfer operon of plasmid R1: identification and analysis of an intracistronic mRNA stabilizer; Koraimann G et al.; Processing of the transfer operon mRNA of the conjugative resistance plasmid R1-19 results in the accumulation of stable traA mRNAs . The stable traA transcripts found in vivo have identical 3' ends within downstream traL sequences, but vary at their 5' ends . The 3' ends determined coincide with the 3' base of a predicted large clover-leaf-like RNA secondary structure . Here we demonstrate that this putative RNA structure, although part of a coding sequences, stabilizes the upstream traA mRNA very efficiently . We also show that the 3' ends of the stable mRNAs are formed posttranscriptionally and not by transcription termination . Half-life determinations reveal the same half-lives of 13 +/- 2 min for the traA mRNAs transcribed from hybrid lac-traAL-cat test plasmids, the R1-19 plasmid, and the F plasmid . Protein expression experiments demonstrate that the processed stable traA mRNA is translationally active . Partial deletions of sequences corresponding to the predicted secondary structure within the traL coding region drastically reduce the chemical and functional half-life of the traA mRNA . The results presented here unambiguously demonstrate that the proposed secondary structure acts as an efficient intracistronic mRNA stabilizer.

Mol Gen Genet, 1996 Mar 7, 250(4), 455 - 65
Surface signaling in transcriptional regulation of the ferric citrate transport system of Escherichia coli: mutational analysis of the alternative sigma factor FecI supports its essential role in fec transport gene transcription; Ochs M et al.; Ferric citrate induces transcription of the ferric citrate transport genes (fec) in escherichia coli by binding to the outer membrane receptor protein FecA without entering the cell . The signal elicited by ferric citrate crosses the outer membrane via TonB, ExbB, and ExbD . FecR transmits the signal across the cytoplasmic membrane and activates FecI located in the cytoplasm . FecI belongs to a subgroup of sigma factors that respond to extracytoplasmic stimuli . Chromosomal insertion and deletion mutations were generated in fecI; the resulting mutants were totally devoid of FecA production and fecB-lacZ expression . Iron starvation did not derepress fec transport gene transcription in fecI mutants . Missense point mutations were generated in the predicted helix-turn-helix motif of FecI to examine its role in transcription initiation . Replacement of glutamate by alanine (E141A) at the third position in the first helix reduced the residual activity of FecI in the absence of ferric citrate to 30% of the wild-type level, but induced fec transcription almost normally n the presence of ferric citrate . Mutant FecI(K145E) displayed 156% of the activity of wild-type FecI in the absence of ferric citrate and conferred full induction by ferric citrate . Mutant FecI(K155E), which has a mutation in the second helix, showed 9% of the wild-type activity in the presence of ferric citrate and 78% in the absence of ferric citrate . The reduced activity of FecI(K155E) was also shown in vitro by DNA binding assays with cell lysates; in gel retardation experiments FecI(K155E) reduced the electrophoretic mobility of fecA promoter-containing DNA less than did wild-type FecI . fecI is not autoregulated, as demonstrated by the lack of FecI-induced fecI-lacZ expression in vivo and by the lack of specific fecI transcription in vitro . Instead, formation of fecI mRNA requires sigma 70 . We conclude that transcription of the fec transport genes is regulated by FecI, which responds to ferric citrate via FecR . fecI and fecR co-transcription is inhibited by the iron-loaded Fur repressor, which then results in a low level of transcription of the fec transport genes.

Proc Natl Acad Sci U S A, 1996 Mar 5, 93(5), 2100 - 4
Inhibition of G-protein betagamma-subunit functions by phosducin-like protein; Schroder S et al.; Phosducin is a cytosolic protein predominantly expressed in the retina and the pineal gland that can interact with the betagamma subunits of guanine nucleotide binding proteins (G proteins) and thereby may regulate transmembrane signaling . A cDNA encoding a phosducin-like protein (PhLP) has recently been isolated from rat brain {Miles, M . F., Barhite, S., Sganga, M . & Elliott, M . (1993) Proc . Natl . Acad . Sci . USA 90, 10831-10835 . Here we report the expression of PhLP in Escherichia coli and its purification . Recombinant purified PUP inhibited multiple effects of G-protein betagamma subunits . First, it inhibited the betagamma-subunit-dependent ADP-ribosylation of purified alpha(o) by pertussis toxin . Second, it inhibited the GTPase activity of purified G(o) . The IC50 value of PhLP in the latter assay was 89 nM, whereas phosducin caused half-maximal inhibition at 17 nM . And finally, PhLP antagonized the enhancement of rhodopsin phosphorylation by purified betagamma subunits . The N terminus of PhLP shows no similarity to the much longer N terminus of phosducin, the region shown to be critical for phosducin-betagamma-subunit interactions . Therefore, PhLP appears to bind to G-protein betagamma subunits by an as yet unknown mode of interaction and may represent an endogenous regulator of G-protein function.

Proc Natl Acad Sci U S A, 1996 Mar 5, 93(5), 2095 - 9
Cloning the expression of a mammalian gene involved in the reduction of methionine sulfoxide residues in proteins; Moskovitz J et al.; An enzyme that reduces methionine sulfoxide {Met(O)} residues in proteins {peptide Met(O) reductase (MsrA), EC 1.8.4.6; originally identified in Escherichia coli} was purified from bovine liver, and the cDNA encoding this enzyme was cloned and sequenced . The mammalian homologue of E . coli msrA (also called pmsR) cDNA encodes a protein of 255 amino acids with a calculated molecular mass of 25,846 Da . This protein has 61% identity with the E . coli MsrA throughout a region encompassing a 199-amino acid overlap . The protein has been overexpressed in E . coli and purified to homogeneity . The mammalian recombinant MsrA can use as substrate, proteins containing Met(O) as well as other organic compounds that contain an alkyl sulfoxide group such as N-acetylMet(O), Met(O), and dimethyl sulfoxide . Northern analysis of rat tissue extracts showed that rat msrA mRNA is present in a variety of organs with the highest level found in kidney . This is consistent with the observation that kidney extracts also contained the highest level of enzyme activity.

Proc Natl Acad Sci U S A, 1996 Mar 5, 93(5), 2065 - 70
Two-hybrid system as a model to study the interaction of beta-amyloid peptide monomers; Hughes SR et al.; The kinetics of amyloid fibril formation by beta-amyloid peptide (Abeta) are typical of a nucleation-dependent polymerization mechanism . This type of mechanism suggests that the study of the interaction of Abeta with itself can provide some valuable insights into Alzheimer disease amyloidosis . Interaction of Abeta with itself was explored with the yeast two-hybrid system . Fusion proteins were created by linking the Abeta fragment to a LexA DNA-binding domain (bait) and also to a B42 transactivation domain (prey) . Protein-protein interactions were measured by expression of these fusion proteins in Saccharomyces cerevisiae harboring lacZ (beta-galactosidase) and LEU2 (leucine utilization) genes under the control of LexA-dependent operators . This approach suggests that the Abeta molecule is capable of interacting with itself in vivo in the yeast cell nucleus . LexA protein fused to the Drosophila protein bicoid (LexA-bicoid) failed to interact with the B42 fragment fused to Abeta, indicating that the observed Abeta-Abeta interaction was specific . Specificity was further shown by the finding that no significant interaction was observed in yeast expressing LexA-Abeta bait when the B42 transactivation domain was fused to an Abeta fragment with Phe-Phe at residues 19 and 20 replaced by Thr-Thr (AbetaTT), a finding that is consistent with in vitro observations made by others . Moreover, when a peptide fragment bearing this substitution was mixed with native Abeta-(1-40), it inhibited formation of fibrils in vitro as examined by electron microscopy . The findings presented in this paper suggest that the two-hybrid system can be used to study the interaction of Abeta monomers and to define the peptide sequences that may be important in nucleation-dependent aggregation.

Proc Natl Acad Sci U S A, 1996 Mar 5, 93(5), 1977 - 81
Nucleotide binding-promoted conformational changes release a nonnative polypeptide from the Escherichia coli chaperonin GroEL; Lin Z et al.; The Escherichia coli chaperonins GroEL and GroES facilitate the refolding of polypeptide chains in an ATP hydrolysis-dependent reaction . The elementary steps in the binding and release of polypeptide substrates to GroEL were investigated in surface plasmon resonance studies to measure the rates of binding and dissociation of a normative variant of subtilisin . The rate constants determined for GroEL association with and dissociation from this variant yielded a micromolar dissociation constant, in agreement with independent calorimetric estimates . The rate of GroEL dissociation from the nonnative chain was increased significantly in the presence of 5'-adenylylimidodiphosphate (AMP-PNP), ADP, and ATP, yielding maximal values between 0.04 and 0.22 s(-1) . The sigmoidal dependence of the dissociation rate on the concentration of AMP-PNP and ADP indicated that polypeptide dissociation is limited by a concerted conformational change that occurs after nucleotide binding . The dependence of the rate of release on ATP exhibited two sigmoidal transitions attributable to nucleotide binding to the distal and proximal toroid of a GroEL-polypeptide chain complex . The addition of GroES resulted in a marked increase in the rate of nonnative polypeptide release from GroEL, indicating that the cochaperonin binds more rapidly than the dissociation of polypeptides . These data demonstrate the importance of nucleotide binding-promoted concerted conformational changes for the release of chains from GroEL, which correlate with the sigmoidal hydrolysis of ATP by the chaperonin . The implications of these findings are discussed in terms of a working hypothesis for a single cycle of chaperonin action.

Proc Natl Acad Sci U S A, 1996 Mar 5, 93(5), 1962 - 6
Single-amino acid substitutions eliminate lysine inhibition of maize dihydrodipicolinate synthase; Shaver JM et al.; Dihydrodipicolinate synthase (DHPS; EC 4.2.1.52) catalyzes the first step in biosynthesis of lysine in plants and bacteria . DHPS in plants is highly sensitive to end-product inhibition by lysine and, therefore, has an important role in regulating metabolite flux into lysine . To better understand the feedback inhibition properties of the plant enzyme, we transformed a maize cDNA for lysine-sensitive DHPS into an Escherichia coli strain lacking DHPS activity . Cells were mutagenized with ethylmethanesulfonate, and potential DHPS mutants were selected by growth on minimal medium containing the inhibitory lysine analogue S-2-aminoethyl-L-cysteine . DHPS assays identified surviving colonies expressing lysine-insensitive DHPS activity . Ten single-base-pair mutations were identified in the maize DHPS cDNA sequence; these mutations were specific to one of three amino acid residues (amino acids 157, 162, and 166) localized within a short region of the polypeptide . No other mutations were present in the remaining DHPS cDNA sequence, indicating that altering only one of the three residues suffices to eliminate lysine inhibition of maize DHPS . Identification of these specific mutations that change the highly sensitive maize DHPS to a lysine-insensitive isoform will help resolve the lysine-binding mechanism and the resultant conformational changes involved in inhibition of DHPS activity . The plant-derived mutant DHPS genes may also be used to improve nutritional quality of maize or other cereal grains that have inadequate lysine content when fed to animals such as poultry, swine, or humans.

Proc Natl Acad Sci U S A, 1996 Mar 5, 93(5), 1941 - 4
ARD1, a 64-kDa bifunctional protein containing an 18-kDa GTP-binding ADP-ribosylation factor domain and a 46-kDa GTPase-activating domain; Vitale N et al.; The alpha subunits of the heterotrimeric guanine nucleotide-binding proteins (G proteins) hydrolyze GTP at a rate significantly higher than do most members of the Ras family of approximatelly 20-kDa GTP-binding proteins, which depend on a GTPase-activating protein (GAP) for acceleration of GTP hydrolysis . It has been demonstrated that an inserted domain in the G-protein alpha subunit, not present in the much smaller Ras-like proteins, is responsible for this difference {Markby, D . W., Onrust, R . & Bourne, H . R . (1993) Science 262, 1895-1900} . We report here that ARD1, a 64-kDa protein with an 18-kDa carboxyl-terminal ADP-ribosylation factor (ARF) domain, exhibited significant GTPase activity, whereas the ARF domain, expressed as a recombinant protein in Escherichia coli, did not . Addition of the 46-kDa amino-terminal extension (similarly synthesized in E . coli) to the GTP-binding ARF-domain of ARD1 enhanced GTPase activity and inhibited GDP dissociation . The kinetic properties of mixtures of the ARF and non-ARF domains were similar to those of an intact recombinant ARD1 . Physical association of the two proteins was demonstrated directly by gel filtration and by using the immobilized non-ARF domain . Thus, like the alpha subunits of heterotrimeric G proteins, ARD1 appears to consist of two domains that interact to regulate the biological activity of the protein.

Proc Natl Acad Sci U S A, 1996 Mar 5, 93(5), 1776 - 80
Peroxynitrite-mediated nitration of tyrosine residues in Escherichia coli glutamine synthetase mimics adenylylation: relevance to signal transduction; Berlett BS et al.; Treatment of Escherichia coli glutamine synthetase (GS) with peroxynitrite leads to nitration of some tyrosine residues and conversion of some methionine residues to methionine sulfoxide (MSOX) residues . Nitration, but not MSOX formation, is stimulated by Fe-EDTA . In the absence of Fe-EDTA, nitration of only one tyrosine residue per subunit of unadenylylated GS leads to changes in divalent cation requirement, pH-activity profile, affinity for ADP, and susceptibility to feedback inhibition by end products (tryptophan, AMP, CTP), whereas nitration of one tyrosine residue per subunit in the adenylylated GS leads to complete loss of catalytic activity . In the presence of Fe-EDTA, nitration is a more random process: nitration of five to six tyrosine residues per subunit is needed to convert unadenylylated GS to the adenylylated configuration . These results and the fact that nitration of tyrosine residues is an irreversible process serve notice that the regulatory function of proteins that undergo phosphorylation or adenylylation in signal transduction cascades might be seriously compromised by peroxynitrite-promoted nitration.

Biochemistry, 1996 Mar 5, 35(9), 3133 - 9
X-ray absorption spectroscopy of the zinc site in tRNA-guanine transglycosylase from Escherichia coli; Garcia GA et al.; A key step in the post-transcriptional modification of tRNA with queuine in Escherichia coli is the exchange of the queuine precursor, preQ1 into tRNA . This reaction is catalyzed by tRNA-guanine transglycosylase (TGT) . We have previously shown that the E . coli TGT is a zinc metalloprotein {Chong et al . (1995) Biochemistry 34, 3694-3701} . Site-directed mutagenesis studies indicated that cysteines 302, 304, 307 and histidine 317 constitute the four ligands to the zinc . The involvement of histidine 317 is somewhat confounded by the presence of histidine 316 . We have examined the zinc site in TGT (wt) and TGT (H317C) by X-ray absorption spectroscopy . The TGT (wt) data are most consistent with a tetracoordinate zinc with one nitrogen and three sulfur ligands . Interestingly, the data for TGT (H317C) are also consistent with a tetracoordinate zinc with one nitrogen and three sulfur ligands . The outer shell imidazole scattering for TGT (H317C) appears to be somewhat more ordered than that for TGT (wt), consistent with our previous suggestion that the wild-type enzyme may exist in two conformations the predominant one involving histidine 317 liganding to the zinc and the minor conformer involving histidine 316 liganding to the zinc . The minor conformer, with histidine 316 coordinating the zinc, appears to have an overall conformation that is subtly different from that of the wild-type enzyme . While TGT (H317C) has kinetic parameters very similar to the wild-type, it does not form the homotrimer quaternary structure of the wild-type . TGT (H317A) has previously {Chong et al . (1995) Biochemistry 34, 3694-3701} been found to contain a significant amount of zinc, but is essentially inactive . This suggests that careful analysis of EXAFS data can reveal subtle conformational changes in metal binding sites that are not observed in more common probes of protein conformation such as CD spectroscopy.

Biochemistry, 1996 Mar 5, 35(9), 3115 - 21
Site-directed mutagenesis of lysine382, the activator-binding site, of ADP-glucose pyrophosphorylase from Anabaena PCC 7120; Sheng J et al.; Previous studies have shown that a highly conserved lysyl residue (Lys 419) near the C-terminus of Anabaena ADP-glucose pyrophosphorylase is involved in the binding of 3-P-glycerate, the allosteric activator {Charng, Y., Iglesias, A . A., & Preiss, J . (1994) J . Biol . Chem . 269, 24107-24113} . Phosphopyridoxylation of the K419R mutant enzyme modified another conserved lysyl residue (Lys382), suggesting that this residue might be also located within the activator-binding site {Charng, Y., Iglesias, AA., & Preiss, J . (1994) J . Biol . Chem . 269, 24107-24113} . Site-directed mutagenesis of Lys382 of the Anabaena enzyme was performed to determine the role of this residue . Replacing Lys382 with either arginine, alanine, or glutamine produced mutant enzymes with apparent affinities for 3-P-glycerate 10-160-fold lower than that of the wild-type enzyme . The glutamic acid mutant enzyme was inhibited by 3-P-glycerate . These mutations had lesser impact on the kinetic constants for the substrates and inhibitor, P(i), and on the thermal stability . These results indicate that both the charge and size of the residue at position 382 influence the binding of 3-P-glycerate . Site-directed mutagenesis was also performed to obtain a K382R-K419R double mutant . The apparent affinity for 3-P-glycerate of this double-mutant enzyme was 104-fold lower than that of the wild-type enzyme, and the specificity for activator of this mutant enzyme was altered . The K382R-K419R enzyme could not be phosphopyridoxylated, suggesting that other lysine residues are not involved in the binding of 3-P-glycerate.

Biochemistry, 1996 Mar 5, 35(9), 3108 - 14
Transient-state kinetic analysis of the oxidative decarboxylation of D-malate catalyzed by tartrate dehydrogenase; Tipton PA; The oxidative decarboxylation of D-malate catalyzed by tartrate dehydrogenase has been analyzed by transient-state kinetic methods and kinetic isotope effect measurements . The reaction time courses show a burst of NADH formation prior to the attainment of the steady-state velocity . The binding of the inhibitor tartronate to the enzyme was examined by monitoring the quenching of the protein's intrinsic fluorescence; the tartronate concentration dependence of the observed rate constant for association was hyperbolic, supporting a two-step model for inhibitor binding . Analysis of the time courses for D-malate oxidation yielded values for many of the microscopic rate constants governing the reaction . The range of possible solutions for the microscopic rate constants was constrained by comparison of the time course for oxidation of unlabeled malate with that of deuterated malate; this analysis relied on the determination of the intrinsic isotope effect on hydride transfer via measurement of D(V/K), T(V/K), and the oxaloacetate partition ratio . The results of the transient-state kinetic analyses suggest that the rate of D-malate oxidation is largely limited by the rate of decarboxylation of the intermediate oxaloacetate which occurs at 11 s-1 . Hydride transfer from D-malate to NAD+ occurs with a rate constant of 300 s-1, and (D)k for this step is 5.5 . The agreement between experimentally measured steady-state kinetic parameters and kinetic isotope effects and their values calculated from the microscopic rate constants derived from the transient-state kinetic analyses was quite good.

Biochemistry, 1996 Mar 5, 35(9), 3031 - 7
Glutamic acid gamma-monohydroxamate and hydroxylamine are alternate substrates for Escherichia coli asparagine synthetase B; Boehlein SK et al.; Escherichia coli asparagine synthetase B (AS-B) catalyzes the synthesis of asparagine from aspartic acid and glutamine in an ATP-dependent reaction . The ability of this enzyme to employ hydroxylamine and L-glutamic acid gamma-monohydroxamate (LGH) as alternative substrates in place of ammonia and L-glutamine, respectively, has been investigated . The enzyme is able to function as an amidohydrolase, liberating hydroxylamine from LGH with high catalytic efficiency, as measured by k(cat)/K(M) . In addition, the kinetic parameters determined for hydroxylamine in AS-B synthetase activity are very similar to those of ammonia . Nitrogen transfer from LGH to yield aspartic acid beta-monohydroxamate is also catalyzed by AS-B . While such an observation has been made for a few members of the trpG amidotransferase family, our results appear to be the first demonstration that nitrogen transfer can occur from glutamine analogs in a purF amidotransferase . However, k(cat)/K(M) for the ATP-dependent transfer of hydroxylamine from LGH to aspartic acid is reduced 3-fold relative to that for glutamine-dependent asparagine synthesis . Further, the AS-B mutant in which asparagine is replaced by alanine (N74A) can also use hydroxylamine as an alternate substrate to ammonia and catalyze the hydrolysis of LGH . The catalytic efficiencies (k(cat)/K(M)) of nitrogen transfer from LGH and L-glutamine to beta-aspartyl-AMP are almost identical for the N74A AS-B mutant . These observations support the proposal that Asn-74 plays a role in catalyzing glutamine-dependent nitrogen transfer . We interpret our kinetic data as further evidence against ammonia-mediated nitrogen transfer from glutamine in the purF amidotransferase AS-B . These results are consistent with two alternate chemical mechanisms that have been proposed for this reaction {Boehlein, S . K., Richards, N . G . J., Walworth, E . S., & Schuster, S . M . (1994) J . Biol . Chem . 269, 26789-26795}.

Biochemistry, 1996 Mar 5, 35(9), 3024 - 30
Probing the mechanism of nitrogen transfer in Escherichia coli asparagine synthetase by using heavy atom isotope effects; Stoker PW et al.; In experiments aimed at determining the mechanism of nitrogen transfer in purF amidotransferase enzymes, 13C and 15N kinetic isotope effects have been measured for both of the glutamine-dependent activities of Escherichia coli asparagine synthetase B (AS-B) . For the glutaminase reaction catalyzed by AS-B at pH 8.0, substitution heavy atom labels in the side chain amide of the substrate yields observed values of 1.0245 and 1.0095 for the amide carbon and amide nitrogen isotope effects, respectively . In the glutamine-dependent synthesis of asparagine at pH 8.0, the amide carbon and amide nitrogen isotope effects have values of 1.0231 and 1.0222, respectively . We interpret these results to mean that nitrogen transfer does not proceed by the formation of free ammonia in the active site of the enzyme and probably involves a series of intermediates in which glutamine becomes covalently attached to aspartate . While a number of mechanisms are consistent with the observed isotope effects, a likely reaction pathway involves reaction of an oxyanion with beta-aspartyl-AMP . This yields an intermediate in which C-N bond cleavage gives an acylthioenzyme and a second tetrahedral intermediate . Loss of AMP from the latter gives asparagine . An alternate reaction mechanism in which asparagine is generated from an imide intermediate also appears consistent with the observed kinetic isotope effects.

Biochemistry, 1996 Mar 5, 35(9), 2978 - 84
Topography of the Escherichia coli initiation factor 2/fMet-tRNA(f)(Met) complex as studied by cross-linking; Yusupova G et al.; trans-Diamminedichloroplatinum(II) was used to induce reversible cross-links between Escherichia coli initiation factor 2 (IF-2) and fMet-tRNA(f)(Met) . Two distinct cross-links between IF-2 and the initiator tRNA were produced . Analysis of the cross-linking regions on both RNA and protein moieties reveals that the T arm of the tRNA is in the proximity of a region of the C-terminal domain of IF-2 (residues Asn611-Arg645) . This cross-link is well-correlated with the fact that the C-domain of IF-2 contains the fMet-tRNA binding site and that the cross-linked RNA fragment precisely maps in a region which is protected by IF-2 from chemical modification and enzymatic digestion . Rather unexpectedly, a second cross-link was characterized which involves the anticodon arm of fMet-tRNA(f)(Met) and the N-terminal part of IF-2 (residues Trp215-Arg237).

Biochemistry, 1996 Mar 5, 35(9), 2894 - 900
Expression, purification, and ligand-binding analysis of recombinant keratinocyte lipid-binding protein (MAL-1), an intracellular lipid-binding found overexpressed in neoplastic skin cells; Kane CD et al.; The keratinocyte lipid-binding protein (KLBP) has been identified on the basis of nucleotide sequence analysis of its cloned cDNA as a new member of the intracellular lipid-binding protein (iLBP) multigene family . To characterize KLBP and determine its ligand-binding properties, its cDNA was subcloned into Escherichia coli, and the protein was overexpressed and purified to homogeneity by a combination of acid extraction, gel permeation, and ion-exchange chromatographies . Purified KLBP exhibited high-affinity binding of the fluorescent hydrophobic probe 1-anilinonaphthalene-8-sulfonate (1,8-ANS), displaying an apparent dissociation constant of 390 +/- 90 nM (n = 0.74 +/- 0.2) . Using an assay based upon displacement of the bound fluorophore, KLBP was found to bind long chain fatty acids most avidly; oleic acid (18:1) bound with an apparent Kd of 248 +/- 12 nM, and arachidonic acid (20:4) exhibited a dissociation constant of 318 +/- 14 nM . As the length of the fatty acid decreased, the binding affinity was reduced; myristic acid (14:0) bound with a K(d) of 1409 +/- 423 nM, but medium-chain (decanoic acid, 10:0) and short-chain (octanoic acid, 8:0) lipids were not bound at all . The protein did not bind prostaglandin E2 with any measurable affinity but did associate with eicosanoids such as 5-hydroperoxyeicosatetraenoic acid (5-HPETE; K(d) of 848 +/- 211 nM) and 15-HPETE (Kd of 463 +/- 243 nM) and to a lesser extent their hydroxy derivatives, 5-HETE and 15-HETE (Kd of 1560 +/- 115 nM and greater than 4 microM, respectively) . all-trans-Retinoic acid was a weak ligand for KLBP, binding with a Kd of 3600 nM, and all-trans-retinol did not displace 1,8-ANS . Molecular modeling of the KLBP sequence upon the X-ray crystal structures of several iLBP's suggested that the side chains of one or more cysteine residues may reside within the putative ligand-binding cavity . Consistent with this, sulfhydryl titration of purified KLBP with 5,5'-dithiobis(2-nitrobenzoic acid) at pH 8.0 in the presence and absence of oleic acid revealed that at least one residue was protected from modification by the fatty acid . These results describe the first purification and characterization of the ligand-binding properties of KLBP and indicate that the protein is a fatty acid binding protein with a tertiary structure likely to be similar to other members of the iLBP multigene family.

FEBS Lett, 1996 Mar 4, 381(3), 217 - 21
Expression of Zm13, a pollen specific maize protein, in Escherichia coli reveals IgE-binding capacity and allergenic potential; Heiss S et al.; Plant proteins belong to the most frequent elicitors of type I allergic symptoms in industrialized countries . Several relevant plant allergens have been found to be either specifically expressed or highly upregulated in mature pollen . The cDNA coding for a pollen specific maize protein, Zm13, shows significant sequence homology with a number of pollen or anther specific proteins from monocot and dicot plants as well as with recently described allergens from olive and rye grass . To test whether the Zm13 protein might possess IgE-binding capacity, Zm13 was expressed in E . coli . The coding region of Zm13 was PCR amplified from a genomic clone and expressed as a glutathione-S-transferase fusion protein . The recombinant Zm13 fusion protein bound a Zm13 specific rabbit antiserum and reacted with serum IgE from grass pollen allergic patients indicating that Zm13 and homologous proteins represent a family of conserved plant allergens.

J Lab Clin Med, 1996 Mar, 127(3), 253 - 62
Molecular cloning and expression of a recombinant Aspergillus fumigatus protein Asp f II with significant immunoglobulin E reactivity in allergic bronchopulmonary aspergillosis; Banerjee B et al.; The cDNA of Aspergillus fumigatus encoding an allergen was cloned and expressed in Escherichia coli . The 987 bp long cDNA clone expressed a recombinant protein Asp f II of 34 kd . This protein exhibited binding to immunoglobulin E (IgE) in the serum samples from patients with allergic bronchopulmonary aspergillosis (ABPA) . The patients with ABPA and central bronchiectasis demonstrated high levels of serum IgE antibodies, whereas patients with ABPA without central bronchiectasis, patients with asthma and Aspergillus skin test reactivity but no evidence of ABPA, and patients with aspergilloma showed only low levels of IgE antibody to Asp f II . In two-dimensional electrophoresis, a native antigen electroeluted from an A . fumigatus culture filtrate antigen preparation showed an isoelectric point and molecular weight similar to that of Asp f II . In a competitive enzyme-linked immunosorbent assay (ELISA), the IgE antibody reactivity of Asp f II with patient serum samples could be significantly inhibited by culture filtrate antigens of A . fumigatus . These results indicate that Asp f II has immunologic reactivities comparable to those of native A . fumigatus antigens . The recombinant Asp f II can be expressed in E . coli in large quantities and should prove useful as a standardized allergen for sensitive and specific immunodiagnosis of ABPA, especially in patients with central bronchiectasis.

Zhongguo Zhong Xi Yi Jie He Za Zhi, 1996 Mar, 16(3), 160 - 3
{Mechanism of injury of intestine, liver and lung in acute cholangitis of severe type model and the protective effect of huoxue qingjieling}; Xu F et al.; Lung capillary permeability was measured with 125I labelled bovine serum albumin, the progression of hepato-cellular injury was quantitatively assessed using atriphenyltetrazolium chloride assay, injury of small intestinal mucosa was determined by the changes of diamine oxidase (DAO) level, content of oxygen free radicals (OFR) was measured by electron spin resonance spectrometer and levels of phospholipase A2(PLA2) of intestine, hepatocellular viability were observed by bioassay methods respectively . The results showed that function of lung, liver and intestine was seriously injuried in ACST rats . These injuries demonstrated by increase of capillary permeability and decrease of hepatocellular viability as well as DAO in intestinal mucosa (P < 0.01) in comparing with control, while the levels of OFR and PLA2 in the above-mentioned 3 organs were elevated simultaneously (P < 0.01) . As for the comparison of efficacy of different treatment, Huoxue Qingjieling was the best one, the bile duct decompression or ampicillin showed little benefit on injury protection even aggravated in some tissues, they displayed a close relationship with their influence on the OFR and PLA2 . Besides, the levels of OFR or PLA2 in various tissues were different from each other, therefore, the serum levels of OFR or PLA2 can not reflect the real levels occurred in organ or tissue level.

Genes Cells, 1996 Mar, 1(3), 293 - 301
Mechanism responsible for glucose-lactose diauxie in Escherichia coli: challenge to the cAMP model; Inada T et al.; BACKGROUND: The inhibition of beta-galactosidase expression in glucose-lactose diauxie is a typical example of the glucose effect in Escherichia coli . It is generally believed that glucose exerts its effect at least partly by reducing the intracellular cAMP level . However, there is no direct evidence that the inhibitory effect of glucose on the expression of the lac operon is mediated by a reduction of the cAMP level in the glucose-lactose system . RESULTS: To examine the roles of cAMP and the cAMP receptor protein (CRP) in the glucose effect, the intracellular levels of these factors were determined during diauxic growth in a glucose-lactose medium . We found that the levels of cAMP and CRP in a lactose-grown phase were not higher than those in a glucose-grown phase, although the cAMP levels increased transiently during the lag phase . The addition of exogenous cAMP eliminated diauxic growth but did not eliminate glucose repression . Glucose repression and diauxie were observed in cells which lack cAMP but produce a cAMP-independent CRP . In addition, inactivation of the lac repressor by the disruption of the lacI gene or the addition of IPTG, eliminated glucose repression . CONCLUSION: We conclude that the repression of beta-galactosidase expression by glucose is not due to the reduction of the cAMP-CRP level but due to an inducer exclusion mechanism which is mediated by the phosphoenolpyruvate-dependent sugar phosphotransferase system.

Genes Cells, 1996 Mar, 1(3), 285 - 91
The sbcC and sbcD genes of Escherichia coli encode a nuclease involved in palindrome inviability and genetic recombination; Connelly JC et al.; BACKGROUND: Long DNA palindromes have the potential to adopt hairpin or cruciform secondary structures that inhibit DNA replication . In Escherichia coli, this palindrome-mediated inviability results from the activity of the sbcC and sbcD genes, and genetic observations have suggested that they may encode a nuclease . Mutations in these genes also restore the defect in genetic recombination associated with recBC sbcB mutants . RESULTS: We have purified the E . coli SbcCD protein from an overexpressing strain and have shown that it has an ATP-dependent DNA double-strand exonuclease activity . Co-purification of nuclease with protein, antibody inhibition and the absence of activity in extracts lacking the sbcCD genes confirm that the activity is intrinsic to SbcCD . The purified protein also has an ATP-independent single-strand DNA endonuclease activity . CONCLUSIONS: We have shown that sbcCD encodes a nuclease . The purified protein has double-strand DNA exonuclease and single-strand DNA endonuclease activities . We propose that SbcCD cleaves secondary structures formed at replication forks and that the broken forks can be repaired by homologous recombination.

Genes Cells, 1996 Mar, 1(3), 277 - 84
Regulation and conservation of the heat-shock transcription factor sigma32; Yura T; Heat shock and other stresses induce a set of genes encoding molecular chaperones and proteases through increase in the level of transcription factor sigma32 in Escherichia coli . Some of the cis- and trans-acting factors involved in the control of synthesis (translation), degradation and activity of sigma32 have been identified and characterized . These studies contribute to our understanding of early events of the heat-shock response, including modes of interaction between chaperones and sigma32 . Besides, analysis of sigma32 homologues from diverse bacteria reveals new insights into evolution of the regulatory mechanisms.

Microbiologia, 1996 Mar, 12(1), 99 - 106
A second Escherichia coli gene with similarity to gapA; Hidalgo E et al.; An open reading frame has been found downstream of the ald gene at 31 min in the Escherichia coli chromosome and has been designated gapC because of its high similarity with gapA (min 39, encoding glyceraldehyde-3-phosphate dehydrogenase), and with gapB (min 62, a gene with high similarity to gapA, encoding erythrose-4-phosphate dehydrogenase) . The gapC gene (min 31) encodes a polypeptide of 204 amino acids, 126 residues shorter than glyceraldehyde-3-phosphate dehydrogenase . In this 204-codon open reading frame several amino acids important for catalysis are conserved . However, the cofactor binding site is lost . The results illustrate a case of a gene, encoding a glycolytic enzyme, for which at least three copies maintaining a certain degree of similarity are apparent in the E . coli genome . It seems likely that the genes encode products with different cellular functions . The origin of these three copies of the gap gene by horizontal transfer or by duplication of an ancestral gene is discussed.

Arch Pharm (Weinheim), 1996 Mar, 329(3), 161 - 8
New antifolate 4,4'-diaminodiphenyl sulfone substituted 2,4-diamino-5-benzylpyrimidines . Proof of their dual mode of action and autosynergism; Wiese M et al.; New 4,4'-diaminodiphenylsulfone substituted 2,4-diamino-5-benzylpyrimidines were synthesized . These compounds are highly active inhibitors of both bacterial dihydrofolate reductase (DHFR) and dihydropteroic acid synthase (SYN) . The simultaneous inhibition of both enzymes leads to autosynergism in whole cells in the same way as known for combinations of sulfonamides with trimethoprim . The inhibitory activity is demonstrated in cell-free systems of DHFR and SYN derived from various species (M . lufu, E . coli, C . albicans) and in whole cell systems of the mycobacterial strain M . lufu . The compounds are rare examples for the combination of two mechanisms of action in one molecule.

Ukr Biokhim Zh, 1996 Mar-Apr, 68(2), 37 - 41
{The effect of 2,4-dinitrophenol and glucose on the utilization of succinate, lactate, pyruvate and formate by Escherichia coli K12}; Dyl'ovyi MV; The effect of protonophore 2,4-dinitrophenol (5-25 microM) in lithium chloride acidic medium (pH 4.5) on utilization of acidic energetic substrates was studied in the presence and absence of glucose . The rate of succinate, lactate and pyruvate utilization by AB 1157 strains was two times lower, than by the KS 400 strain . Isomolar glucose concentration (0.5 mM) decreased the utilization rate of succinate by 60% pyruvate by 30% the AB 1157 strain being much more sensitive than the KS 400 strain both to the action of glucose and to the reaction to glucose and 2,4-dinitrophenol . Acidification of the medium by bacteria has been observed, which shows changes in energy metabolism (glycolysis takes place instead of oxidation of substrates) under conditions of intensive aeration . The results obtain permit assuming that the AB 1157 strain has a certain defect of the membrane which causes increased proton conduction.

Appl Environ Microbiol, 1996 Mar, 62(3), 942 - 6
Phylogenetic position of the spirochetal genus Cristispira; Paster BJ et al.; Comparative sequence analysis of 16S rRNA genes was used to determine the phylogenetic relationship of the genus Cristispira to other spirochetes . Since Cristispira organisms cannot presently be grown in vitro, 16S rRNA genes were amplified directly from bacterial DNA isolated from Cristispira cell-laden crystalline styles of the oyster Crassostrea virginica . The amplified products were then cloned into Escherichia coli plasmids . Sequence comparisons of the gene coding for 16S rRNA (rDNA) insert of one clone, designated CP1, indicated that it was spirochetal . The sequence of the 16S rDNA insert of another clone was mycoplasmal . The CP1 sequence possessed most of the individual base signatures that are unique to 16S rRNA (or rDNA) sequences of known spirochetes . CP1 branched deeply among other spirochetal genera within the family Spirochaetaceae, and accordingly, it represents a separate genus within this family . A fluorescently labeled DNA probe designed from the CP1 sequence was used for in situ hybridization experiments to verify that the sequence obtained was derived from the observed Cristispira cells.

Appl Environ Microbiol, 1996 Mar, 62(3), 898 - 906
A novel family 9 endoglucanase gene (celD), whose product cleaves substrates mainly to glucose, and its adjacent upstream homolog (celE) from Fibrobacter succinogenes S85; Malburg LM Jr et al.; Two adjacent, highly homologous endoglucanase genes, celD and celE from Fibrobacter succinogenes S85, which were separated by an AT-rich 223-nucleotide intergenic region were characterized . The celD gene codes for endoglucanase D (EGD), a protein of 668 residues with a molecular mass of 71.7 kDa, while the celE gene encodes endoglucanase E, a protein of 467 amino acids with a molecular mass of 50.7 kDa . Both gene products belong to family 9 of glycosyl hydrolases . EGD displays an array of serine-rich periodic sequences (SRPS) near its C terminus which separate the catalytic domain from a basic terminal domain (BTD) rich in positively charged amino acids . Endoglucanase E has a BTD which is homologous to that of EGD, but it lacks the SRPS and 151 residues present at the N terminus of EGD . The SRPS structures may function as flexible linkers which facilitate interactions between the BTDs and acidic membrane proteins from F . succinogenes S85 . The recombinant EGD showed pH and temperature optima of 5.5 and 35 degrees C, respectively . The enzyme cleaved barley-beta-glucan, carboxymethyl cellulose, and acid-swollen cellulose with specific activities of 19.1, 11.5 and 1.7 micromol x min-1 x mg of protein-1, respectively . There was a rapid drop in viscosity during hydrolyses of carboxymethyl cellulose, which is characteristic of an endoglucanase . Glucose was the main hydrolysis product of acid-swollen cellulose . Monospecific polyclonal antibodies against EGD detected the expression of a 68-kDa cellulose-inducible protein corresponding in size to the recombinant EGD in the culture fluid of F . succinogenes S85 and several larger proteins . The celE gene appeared to have little activity when expressed from the beta-galactosidase promoter in pBluescript in Escherichia coli; however, reverse transcriptase PCR analysis with internal primers for the gene revealed that a cellulose-inducible message was made in F . succinogenes, thereby documenting expression of the gene.

Appl Microbiol Biotechnol, 1996 Mar, 45(1-2), 240 - 4
Detection of endogenous beta-glucuronidase activity in Aspergillus niger; Gottschalk TE et al.; An endogenous beta-glucuronidase that hydrolyses the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-gluc) in Aspergillus niger is reported . The activity was induced when the fungus was grown in media containing xylan, but was either very low, or absent, when grown on glucose . Endogenous beta-glucuronidase was primarily located in newly formed hyphae, and was apparent at pH values between 3 and 6 . Hydrolysis of X-gluc was sensitive to the inhibitor D-saccharic acid 1,4-lactone and was irreversibly inactivated by heating . The bacterial uid A beta-glucuronidase reporter gene was strongly expressed in the hyphae of transformed A . niger but, in contrast to the endogenous activity, the enzyme was also active at pH 7-8.5 . Histochemical localization of uidA expression in A . niger, without interference from the endogenous beta-glucuronidase activity, was achieved by staining at this pH.

J Virol Methods, 1996 Mar, 57(1), 71 - 85
Expression and purification of an Epstein-Barr virus encoded 23-kDa protein and characterization of its immunological properties; Reischl U et al.; Serodiagnosis of Epstein-Barr virus (EBV) infection is currently based on the detection of antibodies to distinct EBV antigens by immunofluorescence and enzyme-linked immunosorbent assay-based tests, or in part on the detection of heterophile antibodies by the Paul-Bunnell-Davidson heterophile assay . In the past few years, the specificity and the sensitivity of serodiagnostic assay systems has been improved considerably by the use of purified recombinant EBV antigens . Screening of EBV-positive sera for antigenic reactivities by immunoprecipitation with extracts of EBV-positive cells revealed a 23-kDa protein (p23) that was recognized by antibodies from all EBV carriers tested . Open reading frame BLRF2 was identified as the coding region for this protein . After cloning and high-level expression of the BLRF2 open reading frame as DHFR fusion protein in Escherichia coli, the recombinant protein was purified to near homogeneity with the help of continuous elution electrophoresis . Sera from both EBV-positive and -negative donors were screened by immunoblot analysis and enzyme-linked immunosorbent assay for IgM and IgG antibodies against the EBV-encoded protein p23 . Since anti-p23 antibodies were not detectable in 30 of 30 EBV-negative sera, and 294 of 302 EBV-positive sera had either IgM and/or IgG antibody responses to this protein, recombinant p23 seems to be a useful diagnostic marker for EBV-infection.

J Virol Methods, 1996 Mar, 57(1), 61 - 70
Rapid, efficient, large-scale purification of unfused, non-denatured E7 protein of cottontail rabbit papillomavirus; Sundaram P et al.; Cottontail rabbit papillomavirus (CRPV) E7 protein is one of the :high risk' papillomavirus E7 oncoproteins that are partially insoluble in aqueous solution . An Escherichia coli expression system was used for purification of CRPV E7 protein in quantities sufficient for immunologic studies and structural analysis . A glutathione S-transferase (GST)-CRPV E7 fusion protein was solubilized in the presence of non-ionic and ionic detergents, and isolated on an affinity column of glutathione Sepharose beads . The CRPV E7 portion was cleaved from the column with thrombin at a thrombin cleavage site between the fused partners . Thrombin was removed subsequently by adsorption to benzamidine . This method is rapid, requiring just one week, and efficient, yielding 3 mg of pure CRPV E7 protein per liter of bacterial culture . It produced a protein product that was about 95% pure . High-titered polyclonal antisera generated to the product recognized CRPV E7 but not GST . Purified CRPV E7 protein exhibited the ability to bind pRB, making it the first unfused, non-denatured CRPV E7 product reported to do so . This attribute could facilitate structure-function studies of CRPV E7-pRB interactions.

Q Rev Biol, 1996 Mar, 71(1), 37 - 78
Models of parasite virulence; Frank SA; Several evolutionary processes influence virulence, the amount of damage a parasite causes to its host . For example, parasites are favored to exploit their hosts prudently to prolong infection and avoid killing the host . Parasites also need to use some host resources to reproduce and transmit infections to new hosts . Thus parasites face a tradeoff between prudent exploitation and rapid reproduction-a life history tradeoff between longevity and fecundity . Other tradeoffs among components of parasite fitness also influence virulence . For example, competition among parasite genotypes favors rapid growth to achieve greater relative success within the host . Rapid growth may, however, lower the total productivity of the local group by overexploiting the host, which is a potentially renewable food supply . This is a problem of kin selection and group selection . I summarize models of parasite virulence with the theoretical tools of life history analysis, kin selection, and epidemiology . I then apply the theory to recent empirical studies and models of virulence . These applications, to nematodes, to the extreme virulence of hospital epidemics, and to bacterial meningitis, show the power of simple life history theory to highlight interesting questions and to provide a rich array of hypotheses . These examples also show the kinds of conceptual mistakes that commonly arise when only a few components of parasite fitness are analysed in isolation . The last part of the article connects standard models of parasite virulence to diverse topics, such as the virulence of bacterial plasmids, the evolution of genomes, and the processes that influenced conflict and cooperation among the earliest replicators near the origin of life.

J Clin Microbiol, 1996 Mar, 34(3), 717 - 9
Controlled study of cytolethal distending toxin-producing Escherichia coli infections in Bangladeshi children; Albert MJ et al.; The role of cytolethal distending toxin (CDT)-producing Escherichia coli, a newly described category of E . coli, in the causation of diarrhea was studied by screening E . coli isolates from 546 children < 5 years of age with diarrhea and 215 matched controls without diarrhea by using a specific DNA probe . Although CDT-positive E . coli strains were isolated from more children with diarrhea than from healthy controls (3.1 versus 0.93%), this difference did not reach statistical significance (P = 0.082) . All CDT-positive strains also possessed the virulence factors of enteropathogenic E . coli or enteroaggregative E . coli isolates.

J Crit Care, 1996 Mar, 11(1), 27 - 36
A comparison between the acute effects of nitric oxide synthase inhibition and fluid resuscitation on myocardial function and metabolism in entotoxemic dogs; Cohen RI et al.; PURPOSE: Nitric oxide (NO) synthase inhibitors increase mean arterial pressure (MAP) and systemic vascular resistance (SVR) in animal models of sepsis and in humans with septic shock . However, NO synthase inhibitors may cause coronary vessel constriction leading to myocardial ischemia and increased mortality in endotoxemic animals . This study was designed to test the acute effect of NG-nitro-L-arginine (L-NAME) on left ventricular (LV) function and coronary blood flow in a dog model of endotoxemia . METHODS: In open chest, anesthetized dogs endotoxemia was induced intravenously (IV) by Escherichia coli lipopolysaccharide at 2 mg/kg for 60 minutes . This resulted in hypotension, acidosis, and decreased SVR while cardiac index (CI) was maintained . When MAP was < or = 60 mm Hg, animals were resuscitated with either dextran (group I), or L-NAME 30 mg/kg IV bolus (group II) . Group III received L-NAME only . A fourth group of dogs was given endotoxin and not resuscitated . Animals were followed up for 30 minutes after intervention . Animals in the fourth group were followed up until the MAP was approximately 30 mm Hg . Heart rate, CI, MAP, LV end systolic and diastolic pressures, dP/dt at a pressure of 40 mm Hg, left anterior descending artery coronary blood flow, regional LV contraction (sonomicrometer crystals), coronary pressures, gas tension, and lactates were continuously recorded . A catheter placed in the coronary sinus allowed measurement of coronary sinus pressure, as well as coronary sinus lactate and gas tensions . Stroke volume index, stroke work index, systemic vascular resistance index (SVRI), coronary vascular resistance, percent myocardial shortening, myocardial oxygen consumption (Mvo2) and net myocardial lactate production were calculated . RESULTS: In Group I, fluid administration increased MAP, stroke work index, coronary blood flow, percent myocardial shortening, and Mvo2 . In Group II, L-NAME increased MAP to the same extent as fluid administration without evidence of coronary ischemia or myocardial dysfunction . L-NAME did not alter Mvo2 in either endotoxemic or nonendotoxemic animals . In group III, L-NAME alone resulted in a significant increase in MAP and SVRI, but its effects on coronary blood flow and LV function were not significant . We did not observe net lactate production in any of the groups . Coronary blood flow increased out of proportion to Mvo2 in group I animals . CONCLUSIONS: We conclude that although L-NAME at 30 mg/kg causes vasoconstriction, its effects on coronary blood flow and LV function were not significant.

Genetica, 1996 Mar, 97(2), 153 - 63
Identification of the native NIT2 major nitrogen regulatory protein in nuclear extracts of Neurospora crassa; Xiao X et al.; The nit-2 gene of Neurospora crassa encodes the major nitrogen regulatory protein which acts in a positive fashion to activate the expression of many different structural genes during conditions of nitrogen limitation . An E . coli-expressed NIT2/beta-Gal fusion protein binds specifically to DNA in vitro by recognizing GATA core elements . Nuclear extracts prepared from a wild-type N . crassa strain contain a protein factor which displays all of the properties expected for the native NIT2 protein . The native NIT2 protein in nuclear extracts binds with high affinity to DNA fragments which contain two GATA elements, weakly to fragments with a single GATA element, and fails to bind to DNAs which lack these sequences . The DNA binding ability of the protein factor in nuclear extracts is efficiently blocked by a polyclonal antibody developed against the zinc-finger region of NIT2 protein . Western blot analysis with the anti-NIT2 antiserum revealed a specific protein with a size of approximately 110,000 daltons, in excellent agreement with the predicted size of NIT2 . Both the specific NIT2 DNA binding activity and the protein detected by Western blot are totally lacking in nuclear extracts of a nit-2 rip mutant strain . These results all support the conclusion that the native NIT2 protein in Neurospora cells has been identified . The NIT2 protein is localised in nuclei and could not be detected in the cytoplasmic fraction of cells subjected to nitrogen derepression or nitrogen repression, indicating that the nuclear import of NIT2 is not regulated.

Biosci Biotechnol Biochem, 1996 Mar, 60(3), 472 - 5
The integrative transformation of Pleurotus ostreatus using bialaphos resistance as a dominant selectable marker; Yanai K et al.; A plasmid pLC-bar containing the bialaphos resistance gene derived from Streptomyces hygroscopicus between the Lentinus edodes ras gene promoter and priA gene terminator was constructed . When protoplasts of Pleurotus ostreatus were mixed with the plasmid DNA in the presence of polyethylene glycol and CaCl2, bialaphos-resistant colonies were obtained . This indicated that transformation was successful . Southern blot analysis of total DNAs from transformants showed that the introduced plasmid DNA was integrated into the host chromosome and partly rearranged . A plasmid, pLC-GUS, containing the Escherichia coli beta-glucuronidase (GUS) gene under the control of the L . edodes ras gene promoter and priA gene terminator was constructed and introduced into protoplasts of P . ostreatus with pLC-bar by co-transformation . Two of 5 transformants obtained as bialaphos-resistant colonies showed two to twenty times higher specific activity of GUS than the recipient . Southern blot analysis of total DNAs from transformants indicated the presence of the GUS gene only in the two transformants . These results indicated that co-transformation of P . ostreatus was successful, and that the GUS gene was expressed in P . ostreatus . This transformation system will enable us to breed commercial strains of P . ostreatus at the molecular level.

Biosci Biotechnol Biochem, 1996 Mar, 60(3), 383 - 9
Construction of a promoter probe vector autonomously maintained in Aspergillus and characterization of promoter regions derived from A . niger and A . oryzae genomes; Ozeki K et al.; We used a plasmid carrying a sequence for autonomous maintenance in Aspergillus (AMA1) and the E . coli uidA gene as a reporter gene to search the A . oryzae and A . niger genomes for DNA fragments having strong promoter activity . Beta-glucuronidase (GUS)-producing A . oryzae transformants containing the No . 8AN derived from A . niger, or the No . 9AO derived from A . oryzae, were constitutive for the expression of the uidA gene when cultivated in the presence of a variety of carbon and nitrogen sources . When the GUS-producing transformants were grown in liquid culture, the No . 8AN showed an increase of approximately 3-fold in GUS activity compared to the amyB (alpha-amylase encoding gene) promoter . There was also a corresponding increase in the amount of GUS gene-specific mRNA . When these transformants were grown as rice-koji, the No . 8AN showed an increase of approximately 6-fold compared to the amyB promoter, and the amount of GUS protein produced also increased . These strong promoter regions might be applicable to the production of other heterologous proteins in Aspergillus species.

Br J Pharmacol, 1996 Mar, 117(6), 1053 - 8
Functional effects of econazole on inducible nitric oxide synthase: production of a calmodulin-dependent enzyme; Bogle RG et al.; 1 . We performed experiments to examine the effects of an anti-fungal imidazole compound, econazole, on the regulation and effects of lipopolysaccharide-inducible nitric oxide synthase (iNOS) activity in rat aortic rings and cultured J774 murine macrophage cells . 2 . In endothelium-intact rings of thoracic aorta, phenylephrine caused a concentration-dependent contraction with EC50 of 1.9 +/- 0.15 x 10(-8) M (n = 5) . Following incubation with lipopolysaccharide (LPS, 5 micrograms ml-1) for 8 h there was a right-shift in the concentration-response curve (EC50 3.1 +/- 0.28 x 10(-7) M, P < 0.05) with a depression in the maximum contraction from 1.44 +/- 0.25 g to 0.86 +/- 0.26 g (n = 4) . Co-incubation of rings with econazole (1 x 10(-5) M) partially inhibited the LPS-induced loss of reactivity to phenylephrine (EC50 6.5 +/- 0.72 x 10(-8) M) and fully inhibited the reduction in maximum tension (1.49 +/- 0.19 g; n = 5) . 3 . In J774 cells, incubation with LPS (10 micrograms ml-1, 24 h) resulted in significant nitrite production that was inhibited by co-incubation with econazole (IC50 5.0 +/- 0.9 x 10(-6) M; n = 5) . In cells stimulated with LPS, production of L-{3H}-citrulline from L-{3H}-arginine was 6.41 +/- 0.22 pmol mg-1 protein min-1 (n = 3) . This was inhibited by 92 +/- 6% by addition of NG-monomethyl-L-arginine (L-NMMA, 1 x 10(-3) M; n = 3) to the homogenate but not by econazole (1 x 10(-5) M; n = 3) . In contrast pretreatment of cells with econazole (1 x 10(-5) M) markedly reduced the LPS-induced {3H}-citrulline production (0.86 +/- 0.053 pmol mg-1 protein min-1; P < 0.01; n = 3) . 4 . In cells treated with LPS and econazole, L-{3H}-citrulline production was restored in a concentration-dependent manner by addition of calmodulin (1 x 10(-8)-3 x 10(-7) M) with an IC50 of 4.2 +/- 0.9 x 10(-8) M . 5 . We have shown that econazole inhibits the functional and biochemical activity of iNOS in rat aortic rings and cultured J774 cells . Treatment of cells with econazole renders the NO synthase functionally inactive . In econazole-treated cells enzyme activity is restored by calmodulin suggesting that econazole may inhibit the binding of this essential co-factor to the enzyme following its production . These studies may have implications for the design of novel anti-inflammatory agents working through the L-arginine-nitric oxide pathway.

Trends Biochem Sci, 1996 Mar, 21(3), 107 - 11
Processing the holliday junction in homologous recombination; Shinagawa H et al.; The Holliday junction is a well-known intermediate of homologous recombination . Recently, the proteins involved in the correct processing of the Holliday structure into mature recombinant molecules, namely RuvA, RuvB, RuvC and RecG have been isolated and characterized . This has culminated in a model for their synergistic mechanism of action and the solving of the RuvC crystal structure.

Protein Sci, 1996 Mar, 5(3), 542 - 5
A large compressibility change of protein induced by a single amino acid substitution; Gekko K et al.; The adiabatic compressibility (beta s) was determined, by means of the precise sound velocity and density measurements, for a series of single amino acid substituted mutant enzymes of Escherichia coli dihydrofolate reductase (DHFR) and aspartate aminotransferase (AspAT) . Interestingly, the beta s values of both DHFR and AspAT were influenced markedly by the mutations at glycine-121 and valine-39, respectively, in which the magnitude of the change was proportional to the enzyme activity . This result demonstrates that the local change of the primary structure plays an important role in atomic packing and protein dynamics, which leads to the modified stability and enzymatic function . This is the first report on the compressibility of mutant proteins.

Protein Sci, 1996 Mar, 5(3), 535 - 7
Ca(2+)-binding domain VI of rat calpain is a homodimer in solution: hydrodynamic, crystallization and preliminary X-ray diffraction studies; Blanchard H et al.; The 21-kDa calcium-binding domain (VI) of the small subunit of rat calpain II has been expressed in Escherichia coli, purified, and crystallized . Two orthorhombic crystal forms have been obtained: space group P2(1)2(1)2(1) with a = 50.3, b = 56.5, c = 141.3 A; and space group C222(1) with a = 69.4, b = 73.9, c = 157.4 A . Diffraction data have been collected to 2.4 A . Sedimentation equilibrium, dynamic light scattering, and gel-permeation chromatography indicate that domain VI exists as a homodimer in solution . In accordance with the protein's behavior in solution, each crystal form contains two molecules per asymmetric unit . Screening for heavy-atom derivatives is in progress . To decrease the sensitivity to mercurials and to aid in the search for useful derivatives, Cys-to-Ser mutants have been prepared, expressed, and crystallized.

Protein Sci, 1996 Mar, 5(3), 495 - 506
Three-dimensional solution structure of the HIV-1 protease complexed with DMP323, a novel cyclic urea-type inhibitor, determined by nuclear magnetic resonance spectroscopy; Yamazaki T et al.; The three-dimensional solution structure of the HIV-1 protease homodimer, MW 22.2 kDa, complexed to a potent, cyclic urea-based inhibitor, DMP323, is reported . This is the first solution structure of an HIV protease/inhibitor complex that has been elucidated . Multidimensional heteronuclear NMR spectra were used to assemble more than 4,200 distance and angle constraints . Using the constraints, together with a hybrid distance geometry/simulated annealing protocol, an ensemble of 28 NMR structures was calculated having no distance or angle violations greater than 0.3 A or 5 degrees, respectively . Neglecting residues in disordered loops, the RMS deviation (RMSD) for backbone atoms in the family of structures was 0.60 A relative to the average structure . The individual NMR structures had excellent covalent geometry and stereochemistry, as did the restrained minimized average structure . The latter structure is similar to the 1.8-A X-ray structure of the protease/DMP323 complex (Chang CH et al., 1995, Protein Science, submitted); the pairwise backbone RMSD calculated for the two structures is 1.22 A . As expected, the mismatch between the structures is greatest in the loops that are disordered and/or flexible . The flexibility of residues 37-42 and 50-51 may be important in facilitating substrate binding and product release, because these residues make up the respective hinges and tips of the protease flaps . Flexibility of residues 4-8 may play a role in protease regulation by facilitating autolysis.

Protein Sci, 1996 Mar, 5(3), 488 - 94
Electrospray mass spectrometric investigation of the chaperone SecB; Smith VF et al.; Electrospray ionization mass spectrometry was used to investigate the structure of the Escherichia coli chaperone protein SecB . It was determined that the N-terminal methionine of SecB has been removed and that more than half of all SecB monomers are additionally modified, most likely by acetylation of the N-terminus or a lysine . The use of gentle mass spectrometer interface conditions showed that the predominant, oligomeric form of SecB is a tetramer that is stable over a range of solution pH conditions and mass spectrometer interface heating (i.e., inlet capillary temperatures) . At very high pH, SecB dimers are observed . SecB contains a region that is hypersensitive to cleavage by proteinase K and is thought to be involved in conformational changes that are crucial to the function of SecB . We identified the primary site of cleavage to be between Leu 141 and Gln 142 . Fourteen amino acids are removed, but the truncated form remains a tetramer with stability similar to that of the intact form.

Protein Sci, 1996 Mar, 5(3), 478 - 87
Influence of the GroE molecular chaperone machine on the in vitro refolding of Escherichia coli beta-galactosidase; Ayling A et al.; We have studied the effect of the components of the GroE molecular chaperone machine on the refolding of the Escherichia coli enzyme beta-galactosidase, a tetrameric protein whose 116-kDa promoters should not completely fit within the central cavity of the GroEL toroid . In the absence of other additives, GroEL formed a weak complex with chemically denatured beta-galactosidase, reduced its propensity to aggregate, and increased the recovery yields of active enzyme twofold without altering its folding pathway . When present together with the chaperonin, ATP--and to a lesser extent AMP-PNP--reduced the recovery yields and led to the resumption of aggregation . The use of the complete chaperonin system (GroEL, GroES, and ATP) eliminated the GroEL-mediated increase in recovery and folding proceeded less efficiently than in buffer alone . This unusual behavior can be explained in terms of a chaperonin "buffering" effect and the different affinities of GroE complexes for denatured beta-galactosidase.

Protein Sci, 1996 Mar, 5(3), 456 - 67
Overexpression of bacterio-opsin in Escherichia coli as a water-soluble fusion to maltose binding protein: efficient regeneration of the fusion protein and selective cleavage with trypsin; Chen GQ et al.; Bacteriorhodopsin (bR) is a light-driven proton pump from Halobacterium salinarium and is a model system for studying membrane protein folding, stability, function, and structure . bR is composed of bacterio-opsin (bO), the 248-amino acid apo protein, and all-trans retinal, which is linked to lysine 216 via a protonated Schiff base . A bO gene (sbOd) possessing 29 unique restriction sites and a carboxyl-terminal purification epitope (1D4, nine amino acids) has been designed and synthesized . Overexpression of bO was achieved by fusion to the carboxyl terminus of maltose binding protein (MBP) . The expressed fusion protein (MBP-sbO-1D4) formed inclusion bodies in Escherichia coli and, following solubilization with urea and removal of the urea by dialysis, approximately 170 mg of approximately 75% pure MBP-sbO-1D4 was obtained from 1 L of culture . MBP-sbO-1D4 formed high molecular weight (> or = 2,000 kDa) oligomers that were water-soluble . The synthetic bO with the 1D4 tag (sbO-1D4) was separated from MBP by trypsin cleavage at the factor Xa site between the MBP and sbO-1D4 domains . Selective trypsin cleavage at the factor Xa site, instead of at the 14 other potential trypsin sites within bO, was accomplished by optimization of the digestion conditions . Both MBP-sbO-1D4 and sbO-1D4 were regenerated with all-trans retinal and purified to homogeneity . In general, 6-10 mg of sbR-1D4 and 52 mg of MBP-sbR-1D4 were obtained from 1 L of cell culture . No significant differences in terms of UV/vis light absorbance, light/dark adaptation, and photocycle properties were observed among sbR-1D4, MBP-sbR-1D4, and bR from H . salinarium.

Microbiology, 1996 Mar, 142 ( Pt 3), 575 - 83
Regulation of Escherichia coli adenylate cyclase activity during hexose phosphate transport; Dumay V et al.; In Escherichia coli, cAMP levels vary with the carbon source used in the culture medium . These levels are dependent on the cellular concentration of phosphorylated EnzymeIIAglc, a component of the glucose-phosphotransferase system, which activates adenylate cyclase (AC) . When cells are grown on glucose 6-phosphate (Glc6P), the cAMP level is particularly low . In this study, we investigated the mechanism leading to the low cAMP level when Glc6P is used as the carbon source, i.e . the mechanism preventing the activation of AC by phosphorylated EnzymeIIAglc . Glc6P is transported via the Uhp system which is inducible by extracellular Glc6P . The Uhp system comprises a permease UhpT and three proteins UhpA, UhpB and UhpC which are necessary for uhpT gene transcription . Controlled expression of UhpT in the absence of the regulatory proteins (UhpA, UhpB and UhpC) allowed us to demonstrate that (i) the Uhp regulatory proteins do not prevent the activation of AC by direct interaction with EnzymeIIAglc and (ii) an increase in the amount of UhpT synthesized (corresponding to an increase in the amount of Glc6P transported) correlates with a decrease in the cAMP level . We present data indicating that Glc6P per se or its degradation is unlikely to be responsible for the low cAMP level . It is concluded that the level of cAMP in the cell is determined by the flux of Glc6P through UhpT.

J Cell Biochem, 1996 Mar 1, 60(3), 400 - 10
Molecular cloning of the cDNA of mouse mitochondrial NADP-dependent isocitrate dehydrogenase and the expression of the gene during lymphocyte activation; Yang L et al.; The current report documents the molecular cloning of the mouse mitochondrial NADP-dependent isocitrate dehydrogenase (mNADP-IDH) cDNA . The cDNA was 1,863 bp in length and contained one open reading frame encoding a 523-residue polypeptide with a predicted molecular weight of 58 kDa . The cDNA and the deduced amino acid (AA) sequence of the mouse mNADP-IDH had a high degree of homology with those of porcine, bovine, alfalfa, and yeast . The recombinant mNADP-IDH expressed in Escherichia coli had active enzymatic function, as well as an expected molecular weight . The heart had the highest constitutive expression of the steady-state mNADP-IDH mRNA, followed by the kidney, while the expression of the gene in other tissues was low . The enzymatic activity of different tissues was in agreement with their mNADP-IDH mRNA levels . The resting lymphocytes had low constitutive expression of the gene, but the steady-state mRNA could be induced 48 h after mitogen stimulation . At the protein level, the resting lymphocytes had low enzymatic activity of mNADP-IDH, but the activity was augmented fivefold after mitogen stimulation . The cytosolic NADP-IDH, on the contrary, remained low or undetectable before and after the mitogen stimulation . Based on our current findings as well as the known roles of the mNADP-IDH in anabolism and in the isocitrate shuttle, it is conceivable that the mNADP-IDH is necessary for optimizing proliferation in lymphocytes.

Chemosphere, 1996 Mar, 32(5), 959 - 65
Inactivation of Escherichia coli by photocatalytic oxidation; Bekbolet M et al.; The inactivation of Escherichia coli (E.coli) was studied in presterilized surface water sample using titanium dioxide as the photocatalyst under irradiation of BLF Fluorescent lamps . Inactivation of E.coli (10(3) CFU/mL) was achieved in 60 min in the presence of 1.0 mg TiO2/mL . Photocatalytic inactivation data was evaluated in terms of first order rate equation N/N0 = e (-kIt) . The reaction rate constant k, 1.22*10(-2)(mW min/cm2)-1 was calculated.

FEMS Immunol Med Microbiol, 1996 Mar, 13(3), 253 - 56
Cryptic plasmid pFNL10 from Francisella novicida-like F6168: the base of plasmid vectors for Francisella tularensis; Pavlov VM et al.; The plasmid pFNL1OO was created by ligation of Escherichia coli plasmid pBR328 and plasmid pFNL1O from Francisella novicida-like strain F6168 . This plasmid was able to replicate and to express the genes for chloramphenicol and tetracycline resistance in both E . coli and F . tularensis . The origin of replication of pFNL1O, needed for the replication of pFNL1OO in F . tularensis, was mapped . A Sau3A-deletion derivative of pFNL1OO, designated pFNL2OO, was constructed . This plasmid could replicate only in F . tularensis and was found to be stably inherited during cultivation both on solid medium and in liquid cultures.

Microbiol Res, 1996 Mar, 151(1), 99 - 103
Influence of relA locus on electrophoretic mobility of a stringent/relaxed Escherichia coli K 12 strain pair; Gitter B et al.; Stringent (relA+) and relaxed (rel A-) controlled Escherichia coli cells differ in their regulation of many biochemical pathways such as phospholipid and lipopolysaccharide metabolism (LPS) after amino acid limitation . Because such differences could result in various cell envelopes, cells of stringent controlled E . coli strain CP78 (relA+) and relaxed controlled E . coli strain CP79 (relA) were studied regarding their electrophoretic mobility . The graphs of the mobility distributions of both strains were different: cells of strain CP79 caused secondary peaks in addition to the main peaks whereas the mobility distributions of cells of strain CP78 showed only one maximum . In the pH range from 6.0 to 8.0 the location of the main peaks of cells of strain CP79 were changed to less negative values after induction of relaxed response . In contrast to this the stringent response in strain CP78 caused no change of the mobility distributions.

Biotechnol Prog, 1996 Mar-Apr, 12(2), 201 - 4
Optimization study of Escherichia coli TB1 cell disruption for cytochrome b5 recovery in a small-scale bead mill; Belo I et al.; The recovery of a recombinant intracellular protein, cytochrome b5, from Escherichia coli TB1 cells was carried out by bead mill disintegration in a discontinuous small-scale instrument . This process was optimized by the use of experimental factorial design . Several parameters were studied: operating time, amount and size of beads, cellular suspension concentration, and presence of toluene and lysozyme . For the experimental conditions used, only the time of treatment and bead load had significant effects . The optimal values of these variables were found by applying the response surface methodology.

Biotechnol Prog, 1996 Mar-Apr, 12(2), 190 - 5
Cell segregation and lysis have profound effects on the growth of Escherichia coli in high cell density fed batch cultures; Andersson L et al.; Cell segregation into nondividing states and lysis was found to dominate the growth behavior of high cell density fed batch cultures of Escherichia coli . When the specific growth rate declined below a critical value, the biomass production, oxygen consumption, and carbon dioxide formation rates declined sharply . Concomitantly, an extensive loss of colony-forming ability (cfu) and accumulation of extracellular proteins was observed . A segregated model that considered different physiological states, including dividing, nondividing, and lysed cells, was developed and applied to experimental data from high cell density cultures of E . coli.

Hum Mol Genet, 1996 Mar, 5(3), 331 - 7
Chaperonin-mediated assembly of wild-type and mutant subunits of human propionyl-CoA carboxylase expressed in Escherichia coli; Kelson TL et al.; We developed a bacterial expression system for the human alpha and beta cDNAs of propionyl-CoA carboxylase (PCC) . These cDNAs (less the putative mitochondrial matrix targeting presequences) were co-expressed in Escherichia coli on one plasmid vector with each cDNA having its own IPTG-inducible promoter . Only negligible amounts of active PCC were measured despite the presence of both alpha and beta subunits as indicated by Western blot analysis and the almost complete biotinylation of the alpha subunit . Co-expression of this plasmid with a second plasmid vector over-expressing the E . coli chaperonin proteins, groES and groEL, resulted in a several hundred-fold increase in PCC specific activity, to a level comparable with that found in crude human liver extracts . PCC was partially purified on monomeric avidin affinity resin and the presence of both alpha and beta subunits was demonstrated, thereby confirming the assembly of both subunits into an active enzyme . Deficiency of either alpha PCC or beta PCC results in propionic acidemia, an autosomal recessive disorder . We used this expression system to characterize one missense mutation previously described in five Japanese alleles, namely C1283T (Thr428lle) in beta PCC . This mutation, when expressed in E.coli under the same conditions as that of wild-type PCC, had null activity, despite the presence of assembled alpha PCC and beta PCC subunits . This bacterial expression system can be useful for analysis of either alpha PCC or beta PCC mutations . Our findings indicated that the groES and groEL chaperonin proteins were essential for folding and assembly of the human PCC heteromeric subunits.

Lett Appl Microbiol, 1996 Mar, 22(3), 189 - 91
Sensitization of Escherichia coli to nisin by maltol and ethyl maltol; Schved F et al.; When used separately, 20 mmol l-1 maltol or 1600 AU ml-1 nisin resulted in a 0-0.6 log10 reduction in viable counts of Escherichia coli in a buffer system . However, when added in combination they yielded a 1.8-5.5-log-cycle reduction in viable counts of E . coli at pH 5.0 and 6.8 respectively . It is postulated that maltol (and ethyl maltol) destabilizes the cell outer membrane by chelation of Mg2+ and/or Ca2+, thus permeabilizing the E . coli cell to nisin.

Antimicrob Agents Chemother, 1996 Mar, 40(3), 727 - 33
Identification of a class of sulfonamides highly active against dihydropteroate synthase form Toxoplasma gondii, Pneumocystis carinii, and Mycobacterium avium; Chio LC et al.; Sulfanilanilides with 3',5'-halogen substitutions had Ki values 6- to 57-fold lower than the Ki of sulfamethoxazole when tested against dihydropteroate synthase from Toxoplasma gondii . The compounds acted as competitive inhibitors . These compounds were also active against dihydropteroate synthase from Pneumocystis carinii, Mycobacterium avium, and Escherichia coli but were not significantly more active than sulfamethoxazole . The compounds were significantly more active in culture than were standard agents . Against T . gondii in culture, 50% inhibitory concentrations were 7- to 30-fold lower than that of sulfadiazine; against P . carinii in culture, a concentration of 100 microM caused 33 to 95% inhibition of growth, compared with 9% inhibition with 100 microM sulfamethoxazole.

Antimicrob Agents Chemother, 1996 Mar, 40(3), 710 - 14
Quinolone-resistant mutants of escherichia coli DNA topoisomerase IV parC gene; Kumagai Y et al.; Escherichia coli quinolone-resistant strains with mutations of the parC gene, which codes for a subunit of topoisomerase IV, were isolated from a quinolone-resistant gyrA mutant of DNA gyrase . Quinolone-resistant parC mutants were also identified among the quinolone-resistant clinical strains . The parC mutants became susceptible to quinolones by introduction of a parC+ plasmid . Introduction of the multicopy plasmids carrying the quinolone-resistant parC mutant gene resulted in an increase in MICs of quinolones for the parC+ and quinolone-resistant gyrA strain . Nucleotide sequences of the quinolone-resistant parC mutant genes were determined, and missense mutations at position Gly-78, Ser-80, or Glu-84, corresponding to those in the quinolone-resistance-determining region of DNA gyrase, were identified . These results indicate that topoisomerase IV is a target of quinolones in E . coli and suggest that the susceptibility of E . coli cells to quinolones is determined by sensitivity of the targets, DNA gyrase and topoisomerase IV.

Genetics, 1996 Mar, 142(3), 705 - 16
The yeast HRS1 gene encodes a polyglutamine-rich nuclear protein required for spontaneous and hpr1-induced deletions between direct repeats; Santos-Rosa H et al.; The hrs1-1 mutation was isolated as an extragenic suppressor of the hyperrecombination phenotype of hpr1 delta cells . We have cloned, sequenced and deleted from the genome the HRS1 gene . The DNA sequence of the HRS1 gene reveals that it is identical to PGD1, a gene with no reported function, and that the Hrs1p protein contains polyglutamine stretches typically found in transcription factors . We have purified a His(6) tagged version of Hrs1p protein from E . coli and have obtained specific anti-Hrs1p polyclonal antibodies . We show that Hrs1p is a 49-kD nuclear protein, as determined by indirect immunofluorescence microscopy and Western blot analysis . The hrs1 delta null mutation reduces the frequency of deletions in wild-type and hpr1 delta backgrounds sevenfold below wild-type and rad52 levels . Furthermore, hrs1 delta cells show reduced induction of the GAL1,10 promoter relative to wild-type cells . Our results suggest that Hrs1p is required for the formation of deletions between direct repeats and that it may function in gene expression . This suggests a connection between gene expression and direct repeat recombination . In this context, we discuss the possible roles of Hrs1p and Hpr1p in initiation of direct-repeat recombination.

Genetics, 1996 Mar, 142(3), 681 - 91
Opposing roles of the holliday junction processing systems of Escherichia coli in recombination-dependent adaptive mutation; Harris RS et al.; Aspects of the molecular mechanism of "adaptive" mutation are emerging from one experimental system: reversion of an Escherichia coli lac frameshift mutation carried on a conjugative plasmid . Homologous recombination is required and the mutations resemble polymerase errors . Reports implicating a role for conjugal transfer proteins suggested that the mutation mechanism is ordinary replication error occurring during transfer synthesis, followed by conjugation-like recombination, to capture the replicated fragment into an intact replicon . Whereas conjugational recombination uses either of two systems of Holliday junction resolution, we find that the adaptive lac reversions are inhibited by one resolution system and promoted by the other . Moreover, temporary absence of both resolution systems promotes mutation . These results imply that recombination intermediates themselves promote the mutations.

Neuroscience, 1996 Mar, 71(1), 221 - 30
Analysis of neurotrophin-3 expression using the lacZ reporter gene suggests its local mode of neurotrophic activity; Tojo H et al.; We replaced the mouse neurotrophin-3 gene with the Escherichia coli-derived lacZ gene by means of homologous recombination . The mice with this mutation were useful models for studying the distribution of neurotrophin-3 expression in vivo, because visualization by 5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside (X-Gal) staining was simple and rapid compared with in situ hybridization or immunohistochemistry . Whole-mount staining of mutant embryos at embryonic day 10 revealed that lacZ, a reporter for the neurotrophin-3 gene, was expressed in the mesencephalon, mandibular arch and somites . In the embryos at days 13-17, lacZ was markedly expressed in the peripheral target tissues of sensory and sympathetic neurons . We also found that spinal motor neurons and sensory neurons in trigeminal and dorsal root ganglia express lacZ . Some of these X-Gal staining regions overlapped with the sites expressing trkC, a high-affinity receptor for neurotrophin-3 . The distribution of X-Gal staining in heterozygotes and homozygotes was similar to that of neurotrophin-3 messenger RNA detected by in situ hybridization . However, there was less lacZ expression in the dorsal root ganglia of homozygotes than neurotrophin-3 expression in wild-type mice . These results suggest that the neurotrophin-3 produced in the dorsal root ganglia also plays a role in the survival of some of the neurotrophin-3-positive neurons and that the local mode of neurotrophic activity is widely distributed.

Cytokine, 1996 Mar, 8(3), 222 - 6
Differential changes in the concentrations of cytokine-induced neutrophil chemoattractant (CINC)-1 and CINC-2 in exudate during rat lipopolysaccharide-induced inflammation; Shibata F et al.; Recently we have purified four neutrophil chemotactic factors from conditioned medium of rat granulation tissue . Two chemokines besides cytokine-induced neutrophil chemoattractant-1 (CINC-1, formerly called CINC) and CNC-3/macrophage inflammatory protein-2 (MIP-2) were novel chemoattractants, designated as CINC-2 alpha and CINC-2 beta . In the present report, we developed an enzyme-linked immunosorbent assay (ELISA) specific for CINC-2 . The biotin-streptavidin sandwich ELISA for CINC-2 beta detected CINC-2 alpha and CINC- 2 beta equally well between 1 ug/mI and 300 ng/ml, but did not show cross-reactivity with CINC-1 and CINC-3 . The concentrations of CINC-1 and CINC-2 in the exudate during rat lipopolysaccharide (LPS)-induced inflammation were determined . The CINC-1 concentration in the exudate reached a maximum at 4 h after LPS injection, whereas the CINC-2 level steadily increased up to 8 h . The number of infiltrated cells in the exudate increased linearly until 6 h and gradually up to 8 h . Increase in the cell number was correlated with total concentrations of CINC-1 and CINC-2 . The results suggest that CINC-2 as well as CINC-1 plays an important role in accumulation of neutrophils into the inflammatory lesion of LPS- induced inflammation in rats.

Cytokine, 1996 Mar, 8(3), 214 - 21
A fusion protein of IL-8 and a Fab antibody fragments binds to IL-8 receptors and induces neutrophil activation; Holzer W et al.; A fusion protein was generated by genetic engineering which combined a Fab fragment of a monoclonal antibody directed to the human epidermal growth factor receptor with the biologically active N-terminally truncated 2-72 amino acid form of the human chemokine IL-8 . The Fab IL-8 fusion protein was expressed in E . coli and antibody binding and IL-8 activity were determined . Our data indicate that the N-terminus of IL-8 remains functional for receptor interaction . The fusion protein showed specific binding to IL-8 receptors, induced IL-8 mediated chemotactic activity, and the release of MPO activity . However, N-terminal fusion of IL-8 to the carboxyl terminus of the Fab fragment resulted in reduced binding to IL-8 receptors and consequently to reduced biologic activity of IL-8 . The affinity of the antibody arm for EGF-R was improved when compared to a monovalent Fab . Fusion proteins as described herein may represent improved therapeutics for cancer therapy based on their potential to selectively increase and prolong cytokine concentration in the tumour . Since chemokines such as IL-8 recruit effector cells and stimulate effector cell function in situ, a lymphocyte-independent anti-tumour activity followed by tumour-specific immunity could be proposed.

Mol Microbiol, 1996 Mar, 19(5), 997 - 1005
Polynucleotide phosphorylase is required for the rapid degradation of the RNase E-processed rpsO mRNA of Escherichia coli devoid of its 3' hairpin; Braun F et al.; The monocistronic transcript of rpsO undergoes an endonucleolytic cleavage downstream of the coding sequence, which removes the hairpin of the transcription terminator and initiates the rapid degradation of the message . We demonstrate here that the two rne-dependent cleavages, on both sides of the transcription terminator, are catalysed by RNase E in vitro and that the RNase E-processed rpsO message is rapidly degraded by polynucleotide phosphorylase, while RNase II produces stable decay intermediates . Moreover, we show that RNase E cuts in vitro the coding sequence of the rpsO mRNA at several sites which are not detected in vivo.

Mol Microbiol, 1996 Mar, 19(5), 985 - 96
Three novel plasmid R6K proteins act in concert to distort DNA within the alpha and beta origins of DNA replication; Flashner Y et al.; Three novel R6K genes which are responsible for expression of DNA distortion polypeptides (DDP) were identified . The DDPs act in vivo in concert to induce similar stepwise DNA helix distortions within two long inverted repeats (alpha LIR and beta LIR), which are essential elements for the two distally located R6K alpha and beta DNA replication origins . DDP1 and DDP2 are encoded by two tandem genes located at the 5' end of alpha LIR, whereas a gene coding for DDP3 is located at the 3' end of beta LIR . DDP1 and DDP2 are required for primary DNA distortion within alpha LIR or beta LIR, while DDP3 is essential for generation of secondary DNA distortion in these LIR sequences . Creation of DNA distortion within alpha LIR depends on its specific interaction with DDP1 and on the presence of the R6K primase DNA-binding site . The possible relevance of these findings to R6K replication is discussed.

Mol Microbiol, 1996 Mar, 19(5), 965 - 75
Antagonistic involvement of FIS and H-NS proteins in the transcriptional control of hns expression; Falconi M et al.; Gel shift and DNase I footprinting experiments showed that Escherichia coli FIS (factor for inversion stimulation) protein binds to at least seven sites in the promoter region of hns . These sites extend from -282 to +25 with two sites, closely flanking the DNA bend located at -150 from the transcriptional startpoint, partly overlapping the H-NS binding sites involved in the transcriptional autorepression of hns . The interplay between FIS, H-NS and the hns promoter region were studied by examining the effects of FIS and H-NS on in vitro transcription of hns-cat fusions, as well as looking at the effect of FIS on preformed complexes containing H-NS and a DNA fragment derived from the hns promoter region . Taken together, our data suggest that in the cell, FIS and H-NS interact with the promoter region of hns and influence their respective interactions (possibly competing for the same binding site), eliciting antagonistic effects so that an interplay between these proteins might contribute to the transcriptional control of hns.

Mol Microbiol, 1996 Mar, 19(5), 1137 - 47
A novel DnaA protein-binding site at 94.7 min on the Escherichia coli chromosome; Kitagawa R et al.; There is a DNA sequence which has unusually high affinity for the DnaA protein of Escherichia coli between the glyV and amiB-mutL operons at 94.7 min on the genetic map . Affinity of DnaA protein for DNA was measured in vivo as the activity of beta-galactosidase produced from the lacZ gene on a plasmid fused to the 5'-terminal portion of the mioC gene, which is under the control of the DnaA protein . The chromosomal DNA segment between the two operons, carried on a compatible plasmid, derepressed the beta-galactosidase activity by titrating DnaA protein . Derepression occurred on the chromosomal dnaA gene as well, since it is autoregulated . Hence, as measured by immunoassays, one plasmid molecule carrying the DnaA-binding region titrated 370 dnaA molecules, which is a value eightfold higher than that for a plasmid containing the oriC region . We estimate that about 60% of the total cellular DnaA molecules are bound to this site . Four DnaA-binding sequences (DnaA boxes) and a DnaA-regulated promoter directing transcription of two small genes were present within a 250 bp stretch in this region but additional long DNA regions, including the fifth DnaA box located about 650 bp downstream, were required for maximum binding . A role for the DnaA-binding site in controlling DnaA-protein concentration in the cell cycle is discussed.

Mol Microbiol, 1996 Mar, 19(5), 1127 - 35
Plasmid transfer and expression of the transfer (tra) gene product of plasmid pIJ101 are temporally regulated during the Streptomyces lividans life cycle; Pettis GS et al.; We previously have shown that a chromosomally integrated copy of the tra gene of plasmid pIJ101 under the control of the KorA repressor protein, which regulates transcription of tra as well as its own synthesis, can promote the conjugal transfer of both chromosomal and plasmid genes in Streptomyces lividans . Using an antibody generated against a fusion protein containing the C-terminal portion of Tra, we show here that this essential conjugation protein is present in membrane fractions of both surface-grown S . lividans, which mate readily, and of cells grown in liquid culture, where mating has not been found . Expression of Tra during the S . lividans life cycle was temporally regulated and was reduced late during vegetative growth so that little or no Tra protein was detected in cells as they began to differentiate morphologically and produce secondary metabolites . Comparison of the membrane concentration of Tra protein with tra mRNA concentration during the S . lividans life cycle indicated that the disappearance of Tra is post-transcriptionally controlled and thus is not mediated by KorA . The results of 'interrupted mating' experiments, together with the time of appearance of Tra in S . lividans membranes, indicate that the intermycelial transfer of pIJ101 in S . lividans is complete by the onset of cellular differentiation.

Mol Microbiol, 1996 Mar, 19(5), 1085 - 93
Expression in Mycoplasma pneumoniae of the recombinant gene encoding the cytadherence-associated protein HMW1 and identification of HMW4 as a product; Hahn TW et al.; Mycoplasma pneumoniae is a major cause of tracheobronchitis and pneumonia in older children and young adults . The lack of adequate tools for genetic analysis has hindered the elucidation of function and regulation of mycoplasma virulence determinants . We describe here the use of a transposon vector to deliver the cloned gene for the cytadherence-associated protein HMW1 in M . pneumoniae . A 4.95 kbp BamHI fragment encoding all but the C-terminal end of HMW1 was cloned into a modified Tn4001 and transformed into wild-type M . pneumoniae and into a non-cytadhering mutant lacking HMW1-HMW5 . Southern blot hybridizations confirmed insertion of the transposon and the presence of both the resident and recombinant hmw1 alleles . Analysis by Western immunoblotting revealed a truncated HMW1 (HMW1') in the transformants, the level of HMW1' being dependent upon the orientation of the hmw1 gene in the transposon and the site of insertion . Similar expression patterns were noted in wild-type and mutant backgrounds . However, expression of wild-type levels of HMW1' in the mutant did not restore adherence . Finally, HMW4 and HMW1 were shown to be products of the same gene, HMW4 being a heat-modified derivative of HMW1.

J Biochem (Tokyo), 1996 Mar, 119(3), 559 - 64
Purification in an active state and properties of the 3-step phytoene desaturase from Rhodobacter capsulatus overexpressed in Escherichia coli; Raisig A et al.; The phytoene desaturase gene from Rhodobacter capsulatus was expressed in Escherichia coli and the resulting protein was purified . The purification steps involved were ammonium sulfate precipitation and ion exchange chromatography, leading to a homogenous protein of 57 kDa with high specific enzymatic activity . The purified enzyme was characterized with respect to substrate specificity and product formation . In addition to phytoene, the intermediates, phytofluene and zeta-carotene, were both converted to neurosporene, the end product of the reaction . Furthermore, 1,2-epoxy phytoene was a suitable substrate whereas the C30 diapophytoene was not . The Km values for phytoene and zeta-carotene were determined to be 33.3 and 16.6 microM, respectively . The desaturation reaction is dependent on the cofactor FAD . Oxidized nicotine nucleotides or ATP had no positive effect . The Km value for FAD was 4.9 microM . Inhibition of the desaturation reaction was observed with diphenylamine.

J Biochem (Tokyo), 1996 Mar, 119(3), 500 - 5
Molecular cloning, sequencing, and expression of a cDNA encoding alpha-glucosidase from Mucor javanicus; Sugimoto M et al.; A cDNA encoding Mucor javanicus alpha-glucosidase was cloned and sequenced by the reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends methods . The cDNA comprised 2,751 bp, and included an open reading frame which encodes a polypeptide of 864 amino acid residues with a molecular mass of 98,759 Da . The deduced amino acid sequence showed homology of fungal and mammalian alpha-glucosidases and related enzymes, and the sequence around the putative active site was well conserved among these enzymes . The cloned gene was expressed in Escherichia coli cells to produce the alpha-glucosidase, which hydrolyzes not only maltose, but also soluble starch.

J Biochem (Tokyo), 1996 Mar, 119(3), 463 - 7
10Sa RNA is associated with 70S ribosome particles in Escherichia coli; Komine Y et al.; The intracellular distribution of 10Sa RNA in Escherichia coli was investigated in cell extracts . Northern hybridization revealed that a large fraction of 10Sa RNA cosediments with 70S ribosomes . When 70S ribosomes were dissociated into 50S and 30S subunits in the presence of low levels of Mg2+ ions, almost all of the 10Sa RNA disappeared from both subunits . The extent of the association of the 10Sa RNA with ribosomes was much enhanced during the growth phase of the cells . These results suggest the possibility that 10Sa RNA might function on the ribosomes in E . coli cells.

J Biochem (Tokyo), 1996 Mar, 119(3), 414 - 20
Stability and reversibility of thermal denaturation are greatly improved by limiting terminal flexibility of Escherichia coli dihydrofolate reductase; Iwakura M et al.; Short peptides which contained a single Cys residue were introduced into both N- and C-termini of the Cys-free mutant of DHFR (Cys85 --> Ala, Cys152 --> Ser double mutant) by a recombinant DNA method, then the terminal regions were connected through a disulfide bond by oxidation . The oxidized form and reduced form proteins have as high enzymatic activity as wild-type DHFR . There is no detectable difference between the CD spectra of the reduced and oxidized forms at low (15 degrees C, native condition) and high temperature (80 degrees C, unfolded condition) . The thermal transition of the oxidized proteins at the concentration of 0.15 mg/ml (8.5 microM) is completely reversible as demonstrated by the CD spectra . No aggregated materials were detected in the oxidized protein on gel-filtration HPLC after heat treatment up to the protein concentration of 0.5 mg/ml . The reduced protein, however, even in the presence of reducing agent, showed only partial reversibility, with as much as 55 and 95% of the heat-treated protein at the concentrations of 0.15 and 0.5 mg/ml being eluted as the high molecular aggregated form, respectively . The apparent transition temperatures (Tm) of the oxidized forms were 5-7 degrees C higher than those of the reduced counterparts . The oxidized protein that had been denatured with guanidine-HCl was eluted later than the denatured reduced protein on gel-filtration HPLC in the presence of 5 M guanidine-HCl . The limitation of spatial movement of the termini may prevent intermolecular interaction of exposed domains during denaturation-renaturation process, giving rise to the irreversible denaturation . The flexibility of the terminal is also suggested to be an important factor for improving thermal stability of proteins.

Res Commun Mol Pathol Pharmacol, 1996 Mar, 91(3), 303 - 18
Local and systemic factors in periodontal disease increase matrix-degrading enzyme activities in rat gingiva: effect of micocycline therapy; Chang KM et al.; We previously reported that both local and systemic factors relevant to the pathogenesis of periodontal disease can increase gingival collagenase activity in rats . Since the degradation of extracellular matrix is an essential feature of periodontal disease and this tissue breakdown requires multiple enzyme interactions, the current study was carried out to determine the effects of bacterial endotoxin (LPS) (a local factor) and diabetes (a systemic factor) on a panel of matrix-degrading enzymes (collagenase, gelatinase, elastase, and beta-glucuronidase) in the gingiva of rats . In addition, the effects of therapy with a semisynthetic tetracycline (minocycline) were investigated . Ten male, Sprague-Dawley rats were made diabetic by IV injection of streptozotocin . Four of the ten rats then received minocycline (10 mg/day) by oral gavage on a daily basis for 3 weeks . Nineteen nondiabetic rats served as controls and 9 of them received 10 microliters of E . coli LPS (10 mg/ml) by injection into the labial gingiva every other day during the last week of the study . The other 10 nondiabetic rats were sham injected with saline into the gingiva . At the end of the 3 week experimental period, gingival tissue and skin were dissected from each rat and extracted for enzyme analysis . Our results showed that diabetes markedly increased the four matrix-degrading enzyme activities in both gingiva and skin . In contrast, local LPS injection increased these enzyme activities in the gingiva alone . Systemic therapy with minocycline completely ameliorated these elevated enzyme levels in diabetic rats in both gingiva and skin . Minocycline added in vitro to the enzyme assay systems containing skin extract from diabetic rats also inhibited collagenase and gelatinase activities, but no inhibition was observed for elastase and beta-glucuronidase activities, indicating that the MMPs and other enzymes were inhibited by minocycline, during diabetes, by indirect and indirect mechanisms, respectively.

J Pineal Res, 1996 Mar, 20(2), 84 - 9
Melatonin as a therapeutic agent in experimental endotoxic shock; Maestroni GJ; We demonstrated that the pineal neurohormone melatonin exerts immunoregulatory effects via T-helper 2 (Th2) cell products . Th2 products may modulate the secretion and/or action of inflammatory cytokines, which play an important role in the development of septic shock associated with endotoxemia . Here we report that a single melatonin injection protects mice treated with a lethal dose of lipolysaccharide (LPS) especially when melatonin was injected 3 to 6 hr after LPS . This effect did not apparently involve Th cells or inhibition of inflammatory cytokines or macrophage nitric oxide (NO) generation . Nevertheless, plasma nitrate concentration, which reflects the rate of NO synthesis, showed a significant reduction at 18 and 24 hr after LPS administration . Melatonin is being studied in humans for cancer immunotherapy . The data presented here identify melatonin as potential therapy for septic shock.

J Struct Biol, 1996 Mar-Apr, 116(2), 330 - 4
Crystallization and preliminary X-ray crystallographic studies of Escherichia coli xanthine phosphoribosyltransferase; Vos S et al.; Xanthine phosphoribosyltransferase (XPRT; EC 2.4.2.22) from Escherichia coli is a purine salvage enzyme which synthesizes the nucleotides GMP, XMP, and IMP . A mutant C59A, which is more stable than wild-type XPRT while retaining high activity, has been prepared and crystallized to give three different crystal forms (A, B, and C) . Form A crystals are orthorhombic (P21212), with unit cell dimensions a = 59.2 A, b = 92.9 A, c = 53.2 A . Form B crystals are monoclinic (C2) with unit cell dimensions a = 84.4 A, b = 70.8 A, c = 54.1 A, and beta = 113.4 degrees, and form C crystals are tetragonal (P41212 or P43212) with unit cell dimensions a,b = 94 A, c = 167.5 A . Wild-type XPRT and a selenomethionine derivative of C59A XPRT have also been crystallized in the orthorhombic form . The selenomethionine derivative was prepared by expressing XPRT in the usual E . coli strain without the need for a methionine auxotroph . Cells were grown in a methionine-deficient medium supplemented with selenomethionine which gave >95% incorporation . Both the wild-type and selenomethionine C59A XPRT crystals are isomorphous with C59A form A crystals.

J Struct Biol, 1996 Mar-Apr, 116(2), 326 - 9
Crystallization and preliminary X-ray diffraction analysis of nuclear transport factor 2; Kent HM et al.; We have cloned and expressed in Escherichia coli cDNA for rat nuclear transport factor 2 (NTF2), a key cytoplasmic factor that facilitates the import of proteins into the nucleus through nuclear pores . We have used this bacterially expressed material to produce orthorhombic crystals suitable for high-resolution X-ray diffraction structure determination . The crystals have P212121 symmetry with a = 55.9 A; b = 56.7 A; c = 88.3 A and diffract past 2 A on laboratory X-ray sources . The asymmetric unit of these crystals contains two NTF2 polypeptide chains, consistent with the reported dimeric structure of the molecule.

J Struct Biol, 1996 Mar-Apr, 116(2), 320 - 5
Crystallization and preliminary X-ray diffraction studies of human cytochrome P450 reductase; Zhao Q et al.; The two functional domains of a cloned human fibroblast NADPH-cytochrome P450 reductase have been expressed in Escherichia coli and purified on the milligram scale for crystallization studies . One domain contains the cofactor FMN-binding site and the other contains the binding sites for cofactor FAD and substrate NADPH . Crystals of both domains have been obtained by the microbatch method . The crystals of the FMN domain belong to the monoclinic space group P21, with unit cell dimensions of a = 39.3 A, b = 51.5 A, c = 47.8 A, and beta = 105.7 degrees and have one molecule in the asymmetric unit . Diffraction data up to 2.3 A were collected with a merging residual on intensity of 9.3% . The crystals of the FAD/NADPH domain belong to the ortho-rhombic space group P212121 with unit cell dimensions of a = 55.9 A, b = 58.6 A, c = 131.1 A and have one molecule in the asymmetric unit . Diffraction data up to 2.6 A were collected with a merging residual on intensity of 8.0%.

Protein Expr Purif, 1996 Mar, 7(2), 147 - 54
Hyperexpression of chicken riboflavin carrier protein: antibodies to the recombinant protein curtail pregnancy in rodents; Sooryanarayana et al.; Chicken riboflavin carrier protein (RCP) is a phosphoglycoprotein present in the egg white and yolk of egg-laying animals and in the sera of laying hens and of estrogenized chicks . The RCP cDNA, encoding a protein of predicted M(r) 27,000, has been cloned into a T7 polymerase-driven vector, and high-level expression was observed on induction with IPTG in Escherichia coli . The protein was largely localized in inclusion bodies when expressed at 37 degrees C but was present in the cytosolic fraction when induced at 22 degrees C . At 37 degrees C, two major bands were detected in whole-cell lysates of the strain expressing the protein . N-terminal sequence analysis indicated that the two proteins represented translated products with and without the pelB leader sequence encoded in the pET20b vector, but both included an additional 10 amino acids generated during cloning procedures . The inclusion body obtained at 37 degrees C, on extraction with detergent, led to preferential solubilization of the protein without the pelB signal sequence . The solubilized recombinant RCP was recognized by polyclonal antisera to native RCP but radioimmunoassay revealed quantitative differences in the epitopes exhibited by the recombinant protein . Thus, sequence-specific monoclonal antibodies to chicken RCP also cross-reacted with the recombinant protein with almost equal efficiency, but antibodies which recognize conformation-dependent epitopes showed relatively reduced cross-reactivity with the recombinant protein . Polyclonal antibodies to recombinant RCP were able to recognize both the native and the denatured RCP . Administration of recombinant RCP antisera to pregnant mice led to embryonic resorption leading to early pregnancy termination . These findings reveal that the recombinant protein will be useful for investigations related to the mechanism of pregnancy termination on immunoneutralization of RCP in mammals, as well as in unraveling folding properties of RCP in terms of its ligand binding and antigenetic determinants exposed at its surface.

J Viral Hepat, 1996 Mar, 3(2), 67 - 73
The expression of hepatitis B virus polymerase in hepatocytes during chronic HBV infection; McGarvey MJ et al.; A recombinant protein corresponding to part of the amino-terminal domain of hepatitis B virus (HBV) polymerase was expressed in Escherichia coli . Antisera raised against this protein stained hepatocytes, from human liver biopsies, predominantly in the nucleus but some cytoplasmic staining was also observed . No staining was observed in hepatocytes from uninfected patients . Liver biopsies, taken from patients who were infected with human immunodeficiency virus (HIV) as well as HBV showed more intense staining with these antisera than that seen in patients who were infected with HBV alone . The staining pattern suggests that either the whole HBV polymerase protein, or a portion encoding the amino-terminal domain, is translocated to the nucleus . This event may be an important early step in replication of the HBV genome.

Curr Biol, 1996 Mar 1, 6(3), 315 - 24
Codon usage limitation in the expression of HIV-1 envelope glycoprotein; Haas J et al.; BACKGROUND . The expression of both the env and gag gene products of human immunodeficiency virus type 1 (HIV-1) is known to be limited by cis elements in the viral RNA that impede egress from the nucleus and reduce the efficiency of translation . Identifying these elements has proven difficult, as they appear to be disseminated throughout the viral genome . RESULTS . Here, we report that selective codon usage appears to account for a substantial fraction of the inefficiency of viral protein synthesis, independent of any effect on improved nuclear export . The codon usage effect is not specific to transcripts of HIV-1 origin . Re-engineering the coding sequence of a model protein (Thy-1) with the most prevalent HIV-1 codons significantly impairs Thy-1 expression, whereas altering the coding sequence of the jellyfish green fluorescent protein gene to conform to the favored codons of highly expressed human proteins results in a substantial increase in expression efficiency . CONCLUSIONS . Codon-usage effects are a major impediment to the efficient expression of HIV-1 genes . Although mammalian genes do not show as profound a bias as do Escherichia coli genes, other proteins that are poorly expressed in mammalian cells can benefit from codon re-engineering.

Curr Biol, 1996 Mar 1, 6(3), 272 - 5
Chaperones get Hip . Protein folding; Ziegelhoffer T et al.; The discovery of a new co-chaperone, Hip, that interacts with Hsp70 underscores the complexity of the Hsp70 'chaperone machine' that mediates early steps of protein folding in cells.

J Med Virol, 1996 Mar, 48(3), 253 - 61
Analysis of the human serological immune response to a variable region of the attachment (G) protein of respiratory syncytial virus during primary infection; Cane PA et al.; The serum antibody responses of babies to the variable carboxy-terminal region of the attachment (G) protein of respiratory syncytial virus (RSV) have been analysed using paired acute and convalescent sera from infants experiencing their first RSV infection with viruses of known genotype . The variable 84-85 carboxy-terminal amino acids of the G protein of six recent isolates of group A RSV were expressed in Escherichia coli as fusion proteins with glutahione S-transferase . About half the infants developed antibodies which recognised these fusion proteins . The patterns of response obtained in enzyme linked immunosorbant assays and immunoblotting assays were closely related to the infecting genotype.

Nutrition, 1996 Mar, 12(3), 189 - 94
Supplementation of oral nutrition with pancreatic enzymes improves the nutritional status of aged endotoxemic rats; Farges MC et al.; Malnutrition is a common problem in elderly people . The association of malnutrition and physical illness or injury leads to both localized and general complications . In particular, impairment of the adaptive response of pancreatic function to undernutrition and refeeding may adversely affect nutritional status and elicit morbidity and mortality . Aged rats (24 mo old) were treated with lipopolysaccharide (LPS) from E . Coli (3 mg/kg body weight) . Six days later, survivors were randomized to receive, for 7 days, an oral chow diet enriched with either a pancreatic extract (PE) (2.4 mg/day) or an isonitrogenous supply of casein (CAS) . Endotoxemia induced a catabolic state, with a body weight loss of 7.6 +/- 1.1% on day two after LPS treatment . Mean food intake from day 6 to day 13 was similar in LPS-PE and LPS-CAS groups (19.0 +/- 5.6 versus 19.7 +/- 6.9 g) . The metabolic response varied according to the type of muscle studied . In fast (white) muscle, the protein content and the glutamine pool remained markedly depleted in endotoxemic rats receiving casein supplementation . In contrast, enrichment of nutrition with PE significantly limited the LPS-induced muscle wasting and increased the muscle glutamine content . As in previous observations, no significant change occurred in slow (red) muscle . These results could indicate that PE supplementation counteracts pancreatic deficiency caused by aging and worsened by stress and this, in turn, could improve the efficiency of nutrition, to support the hypermetabolism of aged injured rats.

Proteins, 1996 Mar, 24(3), 404 - 6
Purification, crystallization, and preliminary crystallographic analysis of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli; Shumilin IA et al.; The phenylalanine-regulated isozyme of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) from Escherichia coli, its binary complexes with either substrate, phosphoenolpyruvate (PEP), or feedback inhibitor, Phe, and its ternary complexes with either PEP or Phe plus metal cofactor (either Mn2+, Cd2+, or Pb2+) were crystallized from polyethylglycol (PEG) solutions . All crystals of the DAHPS without Phe belong to space group C2, with cell parameters a = 213.5 A, b = 54.3 A, c = 149.0 A, beta = 116.6 degrees . All crystals of the enzyme with Phe also belong to space group C2, but with cell parameters a = 297.1 A, b = 91.4 A, c = 256.5 A, and beta = 148.2 degrees.

Proteins, 1996 Mar, 24(3), 402 - 3
Crystallization and preliminary X-ray analysis of the Escherichia coli replication terminator protein complexed with DNA; Kamada K et al.; Crystals of the Escherichia coli replication terminator protein (Tus) complexed with its binding site DNA were obtained by a microdialysis method using PEG 4000 . They belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 with the unit cell parameter: a = 68.1 A, c = 230.7 A and contain one protein-DNA complex in an asymmetric unit . The native data set has been collected to 2.7 A resolution.

Proteins, 1996 Mar, 24(3), 314 - 21
Structural investigation of the complexation properties between horse spleen apoferritin and metalloporphyrins; Michaux MA et al.; Crystallographic studies of L-chain horse spleen apoferritin (HSF) co-crystallized with Pt-hematoporphyrin IX and Snprotoporphyrin IX have brought significant new insights into structure-function relationships in ferritins . Interactions of HSF with porphyrins are discussed . Structural results show that the nestling properties into HSF are dependent on the porphyrin moiety . (Only protoporphyrin IX significantly interacts with the protein, whereas hematoporphyrin IX does not.) These studies additionally point out the L-chain HSF ability to demetalate metalloporphyrins, a result which is of importance in looking at the iron storage properties of ferritins . In both compound investigated (whether the porphyrin reaches the binding site or not), the complexation appears to be concomitant with the extraction of the metal from the porphyrin . To analyze further the previous results, a three-dimensional alignment of ferritin sequences based on available, crystallographic coordinates, including the present structures, is given . It confirms a high degree of homology between these members of the ferritin family and thus allows us to emphasize observed structural differences: 1) unlike L-chain HSF, H-chain human ferritin presents no preformed binding site; and 2) despite the absence of axial ligands, and due to the demetalation, L-chain HSF is able to host protoporphyrin at a similar location to that naturally found in bacterioferritin.

J Vet Med Sci, 1996 Mar, 58(3), 219 - 224
Production and characterization of polyclonal anti-canine interleukin-8 antibodies; Mohamed A et al.; Polyclonal anti-canine interleukin-8 (cIL-8) antibodies were raised in rabbits immunized with cIL-8 expressed by E . coli . Polyclonal antibodies were purified by affinity chromatography . In the enzyme linked immunosorbent assay (ELISA), the resulting anti- cIL-8 antibodies showed relatively high reactivities with cIL-8 in the fusion proteins of glutathione-S-transferase/cIL-8 (GST/cIL-8) and maltose binding protein/cIL-8 (MBP/cIL-8), but negligible ones with MBP . Furthermore, Western blotting analysis using these polyclonal antibodies showed distinct bands for cIL-8, GST/cIL-8, and MBP/cIL-8 . These antibodies also bound to recombinant human IL-8 (rhIL-8) in ELISA but not in Western immunoblotting . The rHIL-8 (50-800 ng/ml) was chemoattractant for canine neutrophils in a dose dependent manner, but the anti-cIL-8 antibodies did not show the inhibitory effect on the chemotactic activity of rhIL-8 of canine neutrophils, when tested by the chemotaxis assay using Boyden chambers . In addition, GST/cIL-8 and rhIL-8 induced strong and rapid shape change responses of canine neutrophils . However, the anti-cIL-8 antibodies inhibited shape change responses induced by GST/cIL-8 but not by rhIL-8.

J Neurochem, 1996 Mar, 66(3), 908 - 14
Identification of Gln313 and Pro327 as residues critical for substrate inhibition in tyrosine hydroxylase; Quinsey NS et al.; Rat tyrosine hydroxylase was expressed in Escherichia coli . High-level expression was obtained after incubation at 27 degrees C for 18 h . The smallest fragment of tyrosine hydroxylase that gave a soluble active molecule was from Leu188 to Phe456 . This fragment corresponds directly to the section of phenylalanine hydroxylase that had previously been shown to be this enzyme's catalytic core region . It has been shown that Glu288 plays a critical role in pterin function in phenylalanine hydroxylase . The corresponding residue in tyrosine hydroxylase (Glu332) has no significant role in pterin function . Substitution of a leucine for a proline at position 327 in tyrosine hydroxylase produces a molecule with a K(m) for tetrahydrobiopterin 20-fold higher than that of the wild-type molecule, whereas the same substitution at the corresponding residue in phenylalanine hydroxylase (pro281) has no effect on the kinetic constant for the cofactor . This suggests that corresponding residues in phenylalanine hydroxylase and tyrosine hydroxylase can have different roles in pterin function . Substitution of a leucine for a proline at position 281 in phenylalanine hydroxylase increases the K(m) for phenylalanine > 20-fold over that of the wild-type . Substitution of leucine or alanine for Pro327 or a glutamic acid for Gln313 in tyrosine hydroxylase eliminates the substrate inhibition shown by wild-type tyrosine hydroxylase.

Bull Acad Natl Med, 1996 Mar, 180(3), 645 - 57
{Gene therapy in ophthalmology}; Mashhour B; Replication-deficient adenoviral vectors have been used to transfer foreign DNA into a variety of cells including post-mitotic cells, in vivo . They constitute the obligatory targets of gene transfer for a number of ocular diseases that have been elucidated at the molecular level and are potential targets for gene therapy . We have therefore analysed the ability of an adenoviral vector to transfer in vivo the E . coli LacZ gene into ocular cells of mice and rabbits . Injection of up to 3 10(7) pfu in mice and 10(9) pfu in rabbits, into the vitreous cavity, the anterior chamber or the peribulbar space did not result in any detectable cytopathic effect and was associated with endocytosis of viral particles in corneal endothelial, photoreceptor, bipolar, ganglionic and oculomotor muscle cells, depending on the administration route . At the viral titer used (3 10(7) or 10(9) pfu), the expression was detected for at least 50 days . These results open new prospects for the treatment of some retinal hereditary disorders and acquired corneal or retinal alterations due to inflammatory disease.

Res Microbiol, 1996 Mar-Apr, 147(3), 175 - 82
Characterization of enterotoxigenic Escherichia coli by random amplification of polymorphic DNA; Pacheco AB et al.; Two enterotoxigenic Escherichia coli (ETEC) strains (H10407 and 4011-1) were characterized by random amplification of polymorphic DNA (RAPD) profiles using 10-mer oligonucleotides with diverse GC content . All tested primers yielded arrays of amplified DNA products ranging in size from 200 to 3000 bp . The effects of annealing temperature, template concentration and GC content of the primers were evaluated and an optimal reaction procedure was established . Application of the RAPD analysis to ten ETEC strains belonging to five different serotypes showed that strains of the same serotype shared identical or almost identical band profiles, suggesting a similar genetic composition . The use of RAPD profiles as a tool in epidemiological analysis of ETEC is discussed.

Res Microbiol, 1996 Mar-Apr, 147(3), 145 - 57
Overproduction of the Brucella melitensis heat shock protein DnaK in Escherichia coli and its localization by use of specific monoclonal antibodies in B . melitensis cells and fractions; Cloeckaert A et al.; The Brucella melitensis dnaK gene was amplified by the polymerase chain reaction using primers chosen according to the published sequence of B . ovis and cloned in multiple copy plasmids enabling expression under the control of the Plac promoter . Monoclonal antibodies (mAb) obtained by immunizing mice with B . melitensis B115 cell wall (CW) fraction or by infecting mice with virulent B . melitensis strain H38 and recognizing a 73-kDa band in immunoblotting of the B . melitensis CW fraction reacted with the cloned dnaK gene product and were thus shown to be specific for the heat shock protein DnaK . The anti-Dnak protein mAbs did not react with Escherichia coli control cells or cell lysates and could therefore be specific to Brucella DnaK protein epitopes . These mAbs were further used to study overproduction of the DnaK protein . B . melitensis DnaK overproduction in E . coli resulted in a defect in cell septation and formation of cell filaments . Immunogold labelling with the mAbs and electron microscopy localized the DnaK protein inside as well as outside the E . coli cells, probably resulting from lysis due to toxicity of the overproduced DnaK protein . These results indicated that overproduction of the B . melitensis DnaK protein in E . coli had similar physiological consequences as that of E . coli overproduced in E . coli . The DnaK protein localization in B . melitensis cells was essentially cytoplasmic, as shown by immunoelectron microscopy . Heat shock treatment of these cells resulted in increased binding of mAbs and labelling in the cytoplasm . However, in subcellular fractions the DnaK protein was predominantly found in the cell envelope fraction of B . melitensis, which could perhaps be due to interaction of the DnaK protein with membrane proteins.

Vaccine, 1996 Mar, 14(4), 277 - 84
Asymmetric deletion of the junction between the short unique region and the inverted repeat does not affect viral growth in culture and vaccine-induced immunity against Marek's disease; Sonoda K et al.; To construct an effective recombinant Marek's disease virus type 1 (MDV1), we localized a stable insertion site for expression of the Escherichia coli lacZ gene near or within the short inverted repeats of MDV1 strain K554 DNA . A stable recombinant MDV1 was obtained by deleting the junction region between the short unique sequence (Us) and the internal short inverted repeat (IRs) . The recombinant MDV1 replicated in cultured cells as well as the parental viral DNA . Antibodies against both MDV1 antigen and beta-galactosidase encoded by the lacZ gene were detected in the sera of chickens immunized with the virus, and persisted for at least 16 weeks . Moreover, the recombinant virus conferred protection upon chickens against a challenge with virulent MDV1 . These results demonstrated that the Us-IRs junction region is an effective site for the insertion of foreign genes from which to construct a polyvalent live vaccine for poultry . Analysis of the Us-IRs junction region which was deleted from the parental MDV1 indicated that there is a tandem direct repeat of a 220-bp exists within the short internal and terminal inverted repeats of avirulent MDV1 K554 strain DNA . The 220-bp sequence was well conserved among DNAs from various strains . The number of the repeat units may differ between the IRs and TRs or among various MDV1 strain DNAs.

Vaccine, 1996 Mar, 14(4), 260 - 6
Mucosal immunogenicity of the Escherichia coli heat-labile enterotoxin: role of the A subunit; de Haan L et al.; The Escherichia coli heat-labile enterotoxin (LT) is a potent mucosal immunogen, inducing high secretory as well as systemic antibody responses upon oral or intranasal (i.n.) administration . In addition, LT has the capacity to act as an adjuvant in antibody responses against coadministered other antigens . To investigate the role of the individual subunits of LT in the mucosal immunogenicity and adjuvanticity of LT, the LT holotoxin and the non-toxic B subunit (LTB) were cloned separately and purified from overproducing E . coli cultures . Mice were immunized i.n . with the recombinant LT, LTB and combinations of the two and the induction of LTB-specific serum IgG and IgA as well as mucosal S-IgA was monitored . Intranasal administration of 2 micrograms LTB by itself induced a moderate systemic and a low mucosal antibody response, the latter being restricted to the site of immunization . However, addition of a trace amount (50 ng) of LT holotoxin to LTB strongly stimulated both serum antibody and mucosal S-IgA responses to LTB: the antibody levels induced by 2 micrograms LTB supplemented with 50 ng LT were similar to those seen after immunization with 2.9 micrograms of the LT holotoxin alone (representing an amount of 2 micrograms LTB) . Furthermore, immunization with LT-supplemented LTB or with LT holotoxin alone, but not immunization with LTB alone, induced an S-IgA response in distant mucosal tissues including the lung, intestine and the urogenital system . Nicking of the LTA chain with trypsin did not enhance the immunogenicity of LT . These results indicate that, although the LTA chain plays an important role in the mucosal immunogenicity of LT including priming of the common mucosal immune system, extremely low amounts of the LT holotoxin suffice for the induction of high antibody responses to LTB, the trace LT and LTB acting in a synergistic fashion.

Electrophoresis, 1996 Mar, 17(3), 547 - 55
Two-dimensional gel electrophoresis of Escherichia coli homogenates: the Escherichia coli SWISS-2DPAGE database; Pasquali C et al.; Numerous Escherichia coli proteins have already been characterized by two-dimensional gel electrophoresis (2-D PAGE), using carrier ampholytes in the first dimension (VanBogelen, R . A., Sankar, P., Clark, R . L., Bogan, J . A . and Neidhardt, F . C., Electrophoresis 1992, 13, 1014-1054) . We present here a reference protein map of E . coli obtained with immobilized pH gradients (IPG) and available in a SWISS-2DPAGE format . Out of the protein spots identified in the E . coli gene protein database by Neidhardt's group, 153 have been identified in the E . coli gene protein database by Neihardt's group, 153 have been identified on the E . coli SWISS-2DPAGE database map by gel comparison and most of them were confirmed either by the analysis of amino acid composition (AAC) and/or N-terminal microsequencing . Additionally, five as yet unsequenced proteins were found . The E . coli SWISS-2DPAGE database is part of the ExPASy molecular biology server accessible through the Word Wide Web network.

Protein Eng, 1996 Mar, 9(3), 291 - 8
Engineering the aggregation properties of dodecameric glutamine synthetase: a single amino acid substitution controls 'salting out'; Dabrowski MJ et al.; Escherichia coli glutamine synthetase (GS) is a dodecamer of identical subunits which are arranged as two face-to-face hexameric rings . In the presence of 10% ammonium sulfate, wild type GS exhibits a pH-dependent "salting out' with a pKa of 4.51 . Electron micrographs indicate that the pH-dependent aggregation corresponds to a highly specific self-assembly of GS tubules, which result from stacking of individual dodecamers . This stacking of dodecamers is similar to the metal ion-induced GS tubule formation previously described . Site-directed mutagenesis experiments indicate that the N-terminal helix of each subunit is involved in the salting out reaction, as it is in the metal-induced stacking . A single substitution of alanine for His4 completely abolishes the (NH4)2SO4-induced aggregation . However, the H4C mutant protein does nearly completely precipitate under the same salting out conditions . Mutations at other residues within the helix have no effect on the stacking reaction . Differential catalytic activity of unadenylylated GS versus adenylylated GS has been used to determine whether wild type dodecamers "complement' the H4A mutant in the stacking reaction . The complementation experiments indicate that His4 residues on both sides of the dodecamer-dodecamer interfaces are not absolutely required for salting out, although the wild type dodecamers clearly stack preferentially with other wild type dodecamers . Approximately 20% of the protein precipitated from the mixtures containing the wild type GS and the H4A mutant is the mutant . The implications of these results for protein engineering are discussed.

Protein Eng, 1996 Mar, 9(3), 253 - 63
Recognition of transmembrane alpha-helical segments with environmental profiles; Efremov RG et al.; A method for assessing the environmental properties of membrane-spanning alpha-helical peptides in proteins has been proposed . The algorithm employs a set of environmental preference parameters derived for amino acid residues based on the analysis of the 3-D structures of membrane domains in bacteriorhodopsin and photoreaction centers Rhodopseudomonas viridis and Rhodobacter sphaeroides . The resulting 3-D-1-D scores for transmembrane segments are significantly different from those derived for alpha-helices in globular proteins . The parameters obtained have been used to construct environmental profiles for membrane alpha-helices in bacteriorhodopsin and photoreaction centers . The profiles successfully recognize their own sequences in several specially designed large databases . The method has been applied to several membrane proteins with unknown spatial structures . Most of their membrane-spanning peptides were efficiently recognized by the profiles . The predicted environment of the residues in the membrane segments fits the experimental data well . The approach is independent of any homology data and can be employed to delineate the membrane segments of a protein with environmental characteristics close to those of bacteriorhodopsin and photoreaction centers . The alignment of these segments with the reference profiles provides a considerable amount of data about their lipid and protein exposure.

Braz J Med Biol Res, 1996 Mar, 29(3), 381 - 8
Differential production of nitric oxide by endotoxin-stimulated rat and mouse neutrophils; Tavares-Murta BM et al.; There is controversy regarding the evidence for the production of nitric oxide (NO) by neutrophils (PMNs) . The present study investigates NO production, as assessed by the biosynthesis of the end products, nitrite and nitrate, in the pellets and supernatants of rat and mouse peripheral blood neutrophils obtained during endotoxemia and of peritoneal carrageenin-elicited PMNs stimulated in vitro with E . coli lipopolysaccharide (LPS) . We also investigated the induction of NO synthase by rat and mouse peritoneal cells . The intraperitoneal (ip) administration of LPS to mice (10 mg/kg) and rats (5 mg/kg) significantly increased plasma nitrate concentration by six and 23-fold, respectively . In vivo pretreatment with L-NGmonomethyl arginine (L-NMMA) significantly inhibited this production . Compared to animals injected with PBS, the cell pellets of blood PMNs obtained from mice, but not rats, 2 or 6 h after LPS administration produced significant amounts of nitrite (14 +/- 3 and 18 +/- 2 nmol/mg protein, respectively) . Little or no nitrite was found in the incubating medium . In contrast, 6 h after a carrageenin challenge (700 micrograms) peritoneal neutrophils obtained from rats, but not mice, released high concentrations of nitrite into the supernatant during a 24-h period of incubation (34 +/- 0.8 microM) . The nitrite concentration of the pellet of these cells was negligible . In contrast to the lack of increase in the amount of nitrite released into the supernatants, the in vitro stimulation of rat PMNs with LPS (10 micrograms/ml) for 24 h did increase intracellular nitrite concentration (from 0.8 +/- 0.07 to 8 +/- 0.3 nmol/mg protein) . In mouse PMNs, LPS treatment caused only a small release of nitrite into the incubation medium (14 +/- 1 microM) . There was no significant change in nitrite concentration in the cell pellets . These data suggest that rat and mouse neutrophils differ in their ability to produce nitric oxide following stimulation with endotoxin.

Blood Coagul Fibrinolysis, 1996 Mar, 7(2), 183 - 6
Expression, purification and characterisation of recombinant pallidipin, a novel platelet aggregation inhibitor from the haematophageous triatomine bug Triatoma pallidipennis; Haendler B et al.; Pallidipin is a platelet aggregation inhibitor protein originating from the saliva of the haematophageous triatomine bug Triatoma pallidipennis . Its inhibitory effects are specific for collagen-induced platelet aggregation . The recombinant form of the protein was expressed in the periplasmic space of transformed Escherichia coli using a vector based on the alkaline phosphatase gene promoter and leader peptide . Recombinant pallidipin was purified in three chromatographic steps including cation exchange, anion exchange and size exclusion gel chromatography . SDS/PAGE and N-terminal amino acid sequencing showed that recombinant pallidipin had a molecular weight similar to that of the salivary protein (approximately 19 kDa) and had been correctly processed . The yield was 864 micrograms of pure protein from one litre of bacterial culture . The biological activity of recombinant pallidipin was assessed in a platelet aggregation assay using collagen at a concentration of 2 micrograms/ml as inducer, and the IC50 found to be 33 nM, similar to that determined for the native protein . When the collagen concentration used for induction was increased, higher pallidipin concentrations were also needed to achieve a comparable inhibition of platelet aggregation.

Mol Microbiol, 1996 Mar, 19(6), 1373 - 84
Mutational analysis of the Escherichia coli spoT gene identifies distinct but overlapping regions involved in ppGpp synthesis and degradation; Gentry DR et al.; The spoT gene of Escherichia coli encodes a guanosine 3',5'-bis(diphosphate) 3'-pyrophosphohydrolase (ppGppase) as well as an apparent guanosine 3',5'-bis(diphosphate) synthetase (designated PSII) . To determine the regions of the SpoT protein that are required for these two competing activities, we analysed plasmid-borne deletion mutations for their ability to complement chromosomal mutations defective in each activity . We found that a region containing the first 203 amino acids of the 702-amino-acid SpoT protein was sufficient for ppGppase activity while an overlapping region containing residues 67-374 was sufficient for PSII activity . These data indicate that the catalytic sites involved in the two activities are separate but closely linked in the primary sequence of the SpoT protein . A ppGppase-defective delta 1-58 deletion mutant strain failed to synthesize ppGpp in response to nutrient limitation, also supporting the notion that PSII activity from wild-type SpoT does not increase in response to nutrient limitation . Using a strain lacking PSII activity but retaining ppGppase activity, we determined the contribution of the RelA protein (ppGpp synthetase I, PSI) to ppGpp synthesis following glucose starvation . We found that the RelA protein activity accounts for the initial burst of ppGpp synthesis at the onset of glucose starvation but that this source of synthesis is absent when amino acids are present during glucose starvation.

Mol Microbiol, 1996 Mar, 19(6), 1319 - 30
The Escherichia coli ribosomal protein S16 is an endonuclease; Oberto J et al.; The histone-like protein HU isolated from Escherichia coli exhibited, after several purification steps, a Mg(2+)-dependent nuclease activity . We show here that this activity can be dissociated from HU by a denaturation-renaturation step, and is due to a small fraction of ribosomal protein S16 co-purifying with HU . S16 is an essential component of the 30S ribosomal particles . We have cloned, overproduced, and purified a histidine-tagged S16 and shown that this protein is a DNA-binding protein carrying a Mg(2+)-Mn(2+)-dependent endonuclease activity . This is an unexpected property for a ribosomal protein.

Mol Microbiol, 1996 Mar, 19(6), 1287 - 94
A periplasmic protein (Skp) of Escherichia coli selectively binds a class of outer membrane proteins; Chen R et al.; A search was performed for a periplasmic molecular chaperone which may assist outer membrane proteins of Escherichia coli on their way from the cytoplasmic to the outer membrane . Proteins of the periplasmic space were fractionated on an affinity column with sepharose-bound outer membrane porin OmpF . A 17 kDa polypeptide was the predominant protein retained by this column . The corresponding gene was found in a gene bank; it encodes the periplasmic protein Skp . The protein was isolated and it could be demonstrated that it bound outer membrane proteins, following SDS-PAGE, with high selectivity . Among these were OmpA, OmpC, OmpF and the maltoporin LamB . The chromosomal skp gene was inactivated by a deletion causing removal of most of the signal peptide plus 107 residues of the 141-residue mature protein . The mutant was viable but possessed much-reduced concentrations of outer membrane proteins . This defect was fully restored by a plasmid-borne skp gene which may serve as a periplasmic chaperone.

Mol Microbiol, 1996 Mar, 19(6), 1277 - 86
Effects of F-encoded components and F-pilin domains on the synthesis and membrane insertion of TraA'-'PhoA fusion proteins; Paiva WD et al.; F-pilin, the 70-amino-acid F-pilus subunit, accumulates in the cell envelope of F+ strains in a process that requires interactions between its precursor (the traA gene product) and other host and F-encoded proteins . Here, we have used a set of phi(traA-phoA) genes to explore the effects of different TraA domains on the synthesis and membrane insertion of TraA-PhoA fusion proteins, particularly in relation to other F-encoded gene products . The 51-amino-acid TraA leader peptide fused directly to alkaline phosphatase was synthesized at comparable rates and incorporated rapidly and efficiently into the inner membrane in F' and F- cells . A second fusion gene encoded the TraA leader peptide and the first 51 amino acids of F-pilin itself fused to PhoA (TraA'-'PhoA-102 polypeptide) . Alkaline phosphatase activities and patterns of pulse-labelled polypeptides indicated that TraA'-'PhoA-102 was synthesized at comparable rates in F' and F- cells, but in neither was the TraA'-'PhoA-102 polypeptide efficiently processed as a membrane protein . A third gene encoded the entire 121-amino-acid TraA polypeptide fused to PhoA (TraA-'PhoA-121 polypeptide) . About 70% of the pulse-labelled TraA-'PhoA-121 polypeptide was rapidly processed in F' cells, where it accumulated in the cell envelope as active alkaline phosphatase, whereas in F- cells, < 5% of the pulse-labelled polypeptide was processed . Additionally, the apparent rate of TraA-'PhoA-121 polypeptide synthesis was threefold higher in F' cells . The traQ gene alone could not substitute for F in restoring TraA-'PhoA-121 (or wild-type F-pilin) accumulation.

Mol Microbiol, 1996 Mar, 19(6), 1205 - 14
Delta mu H+ dependency of in vitro protein translocation into Escherichia coli inner-membrane vesicles varies with the signal-sequence core-region composition; Nouwen N et al.; Signal sequences frequently contain alpha-helix-destabilizing amino acids in the hydrophobic core . Nuclear magnetic resonance studies on the conformation of signal sequences in membrane mimetic environments revealed that these residues cause a break in the alpha-helix . In the precursor of the Escherichia coli outer membrane protein PhoE (pre-PhoE), a glycine residue at position -10 (Gly -10) is thought to be responsible for the break in the alpha-helix . We investigated the role of this glycine residue in the translocation process by employing site-directed mutagenesis . SDS-PAGE analysis showed drastic variations in the electrophoretic mobilities of the mutant precursor proteins, suggesting an important role of the glycine residue in determining the conformation of the signal sequence . In vivo, no drastic differences in the translocation kinetics were observed as compared with wild-type PhoE, except when a charged residue (Arg) was substituted for Gly -10 . However, the in vitro translocation of all mutant proteins into inverted inner-membrane vesicles was affected . Two classes of precursors could be distinguished . Translocation of one class of mutant proteins (Ala, Cys and Leu for Gly -10) was almost independent of the presence of a delta mu H+, whereas translocation of the other class of precursors (wild type or Ser) was strongly decreased in the absence of the delta mu H+ . Apparently, the delta mu H+ dependency of in vitro protein translocation varies with the signal-sequence core-region composition . Furthermore, a proline residue at position -10 resulted in a signal sequence that did not prevent the folding of the precursor in an in vitro trimerization assay.

Parasitology, 1996 Mar, 112 ( Pt 3), 331 - 8
Cloning and expression of DiT33 from Dirofilaria immitis: a specific and early marker of heartworm infection; Hong XQ et al.; Dirofilaria immitis is an important filarial parasite of dogs and cats, and a useful model for human filariasis . Current diagnostic tests for heartworm infection in animals rely on the presence of fecund female worms (usually found 6.5 months post-infection or later) and therefore fail to detect pre-patent infections . Putative pepsin inhibitors from 2 filarial parasites of humans namely Onchocerca volvulus (Ov33, Oc3.6, OvD5B) and Brugia malayi (Bm33), have been shown to be useful in diagnosis of onchocerciasis and lymphatic filariasis, respectively . Previous studies have suggested that a homologue exists in D . immitis (DiT33), which may have potential in diagnosis of heartworm infection . In this study, the isolation and characterization of a cDNA clone encoding DiT33 is described . This cDNA contains 12 bases of the nematode-specific 22 nucleotide spliced leader sequence and encodes a 26.4 kDa-protein with a high level of similarity (87-89%) to other filarial members of the family . DiT33 was over-expressed in E . coli as a fusion with the maltose-binding protein and serological analysis was performed using a panel of clinically defined dog sera . The findings of this study indicate that DiT33 is a promising antigen for the early detection of D . immitis and may be a valuable tool in the control and management of heartworm infection.

Parasitology, 1996 Mar, 112 ( Pt 3), 277 - 84
During canine viscero-cutaneous leishmaniasis the anti-Hsp70 antibodies are specifically elicited by the parasite protein; Quijada L et al.; A Leishmania infantum cDNA library was screened with sera from dogs with viscero-cutaneous leishmaniasis . Sequence analysis of a positive clone isolated from the library revealed that it coded for the carboxyl-terminal region of a member of the 70-kDa heat-shock protein family . The full-length sequence of the L . infantum hsp70 gene was determined after isolation of genomic clones . This protein shows a high degree of sequence conservation with the homologous protein from other organisms . To test its antigenicity a recombinant Hsp70 protein fused to the maltose-binding protein was produced in Escherichia coli using the expression vector pMAL-cRI . By FAST-ELISA assays it was observed that while the complete recombinant protein was recognized by 100% of the sera, the 20 carboxyl-terminal amino acids of the protein were only recognized by 30% of those sera . Thus, although a B-cell epitope must be present within the carboxyl terminal end of the protein other antigenic determinant(s) must reside out of this region . The analysis of the cross-reactivity with mouse Hsp70 by Western blotting strongly suggests that the anti-Hsp70 antibodies generated by infection with L . infantum are directed at specific determinants of the L . infantum Hsp70 . Thus, our results indicate that anti-Hsp70 autoantibodies are not induced during Leishmania infection.

Intensive Care Med, 1996 Mar, 22(3), 252 - 8
A new side effect of inhaled nitric oxide in neonates and infants with pulmonary hypertension: functional impairment of the neutrophil respiratory burst; Gessler P et al.; INTRODUCTION: Inhaled nitric oxide (NO) may be beneficial in the treatment of pulmonary hypertension, both of the newborn and in the adult respiratory distress syndrome . Up to now, serious systemic side effects have not been reported . OBJECTIVE: The effect of inhaled NO on superoxide anion production by neutrophils . DESIGN: Prospective study of a consecutive series of 15 neonates and infants . SETTING: Neonatal and paediatric ICUs with a total of 17 beds (university hospital) . MEASUREMENTS AND RESULTS: Superoxide anion production was determined by a flow cytometric method using dihydrorhodamine 123 (DHR) as an oxidative probe after the priming of neutrophils with N-formyl-methionyl- leucylphenylalanine (fMLP) or with Escherichia coli . The generated fluorescence was expressed as relative fluorescence intensity (RFI) . Inhalation of NO for more than 24 h reduced the superoxide anion production by neutrophils stimulated with E . coli to below baseline values before NO inhalation (mRFI = 158 +/- 25 vs 222 +/- 24; P = 0.03) . This decrease was more pronounced after more than 72 h (mRFI = 133 +/- 17) . At this time, superoxide anion production by fMLP-stimulated neutrophils was also decreased (mRFI = 40 +/- 3, vs 57 +/- 5; P = 0.03) . The reduced capacity of superoxide production persisted throughout therapy with NO and lasted up to more than 4 days after the end of NO inhalation . CONCLUSION: The results suggest that inhalation of NO in patients with pulmonary hypertension causes reduced superoxide anion production by neutrophils stimulated with E . coli or with fMLP . To determine the clinical importance of this systemic side effect with respect to bacterial infections, a randomized controlled study is necessary.

Mol Med, 1996 Mar, 2(2), 204 - 10
Parathyroid hormone-related protein is induced during lethal endotoxemia and contributes to endotoxin-induced mortality in rodents; Funk JL et al.; BACKGROUND: Parathyroid hormone-related protein (PTHrP) is a ubiquitous and highly conserved vasoactive peptide whose role and regulation in normal physiology remain an enigma . Recently, we demonstrated that low-dose endotoxin (LPS) induces intrasplenic, but not systemic, levels of PTHrP; and that tumor necrosis factor, a pro-inflammatory cytokine, is the major mediator of this effect . We have therefore hypothesized that, with higher, lethal doses of endotoxin, PTHrP could be induced in multiple tissues to such a degree that it could contribute to the lethality of septic shock . MATERIALS AND METHODS: Northern blot analysis was used to measure PTHrP mRNA levels in vital organs of rats after administration of a near lethal dose (5 mg/250 g) of LPS (or vehicle alone) . Plasma levels of PTHrP were also measured by immunoradiometric assay . The ability of the immunoglobulin fraction of two different PTHrP(1-34) antisera to protect from LPS-induced lethality was also studied in mice using survival analysis . RESULTS: In response to a near-lethal dose of endotoxin, PTHrP mRNA levels increased acutely in every vital organ examined (spleen, lung, heart, kidney, and liver) . Circulating levels of PTHrP also increased, peaking 2 hr after administration of high-dose endotoxin . Passive immunization of mice with anti-PTHrP(1-34) antibody 6 hr prior to administration of a lethal dose of LPS protected mice from endotoxin-induced death (p < 0.00005) . CONCLUSIONS: These results suggest that PTHrP belongs to the cascade of pro-inflammatory cytokines induced during lethal endotoxemia that is responsible for the toxic effects of LPS.

Virus Res, 1996 Mar, 41(1), 11 - 23
The effect of transcription on genetic recombination in poxvirus-infected cells; Parks RJ et al.; We have examined the effects of transcription on recombination frequencies in poxvirus-infected cells . A synthetic poxviral promoter was shown to function as a hybrid early/late transcription element when fused to a luciferase reporter gene, and then cloned into genetically-marked recombination substrates . These lambda DNA substrates were transfected into cells infected with Shope fibroma virus (SFV) and the recombinants detected by recovering the transfected DNA, packaging it in vitro into infectious particles, and then assaying the yield of recombinants on Escherichia coli . Controls showed that the poxviral promoter conferred no replicative advantage, or disadvantage, on molecules encoding the promoter . Furthermore, the promoter had no detectable effect on the recombination frequency when recombination was measured in the interval immediately adjacent to the promoter-insertion site . However, the promoter did appear to stimulate recombination at a distance, in a manner that appeared to be dependent on the level of transcription, and the effect was observed regardless of whether or not the promoter was present on one or both of the recombinational substrates . The peak of recombinational enhancement was centered about 500 bp away from the promoter element, where the frequency of recombination was 30-50% higher than that seen when the recombinational substrates lacked the promoter . Possible explanations for these observations are discussed.

Biokhimiia, 1996 Mar, 61(3), 559 - 66
{Major p50 protein of the somatic cell cytoplasmic mRNP: expression in Escherichia coli, isolation, and some properties of the recombinant protein}; Ustinov VA et al.; The major mRNP protein of rabbit reticulocytes, p50, belonging to the family of Y-box transcription factors has been expressed in E . coli . The isolation procedure of the recombinant protein has been described . The recombinant protein is similar to the protein isolated from rabbit mRNPs in SDS-PAGE mobility and interaction with specific antibodies . Similar to the natural protein, the recombinant protein forms homooligomeric complexes with a molecular mass of about 800 kDa, binds to the alpha-globin RNA and double-stranded DNA containing the Y-box . Both proteins can be phosphorylated in vitro.

Biokhimiia, 1996 Mar, 61(3), 555 - 8
{Stimulating effect of sodium ions on Escherichia coli growth in the presence of protonophore uncoupler}; Avetisian AV; The increase of Na+ concentration from the submillimolar level to 50 mM stimulates growth of the Escherichia coli cells in the presence of the protonophorous uncoupler CCCP . The half-maximal effects is observed at 1 mM Na+ . The decrease of inorganic phosphate concentration from tens of millimoles to 0.5 mM inhibits cell growth under same conditions.

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