J Biol Chem, 1994 Apr 8, 269(14), 10201 - 4 Cloning and characterization of cDNA encoding rat hemin-sensitive initiation factor-2 alpha (eIF-2 alpha) kinase . Evidence for multitissue expression; Mellor H et al.; Reticulocytes contain an eukaryotic initiation factor-2 alpha (eIF-2 alpha) kinase that is negatively regulated by hemin . This protein kinase, which has been termed heme-regulated inhibitor and heme-controlled repressor (HCR), has a strong inhibitory effect on the initiation of protein synthesis and plays an important role in translational control in these cells . Previous evidence has suggested that HCR may be an erythroid-specific enzyme . As reported herein, we have cloned and characterized the cDNA encoding an eIF-2 alpha kinase from rat brain . The predicted amino acid sequence of the kinase from rat brain shows 82% homology to rabbit reticulocyte HCR with the greatest variation concentrated in a central region of approximately 135 amino acids located between protein kinase subdomains IV and VI . We have expressed the rat brain cDNA in Escherichia coli and have demonstrated that the recombinant enzyme is a hemin-sensitive eIF-2 alpha kinase . We have also shown that mRNA recognized by the rat brain cDNA is expressed in a wide range of non-erythroid rat tissues at a lower but significant level compared with reticulocytes . These findings raise the possibility of additional, uncharacterized roles for HCR in non-erythroid cells. Gene, 1994 Apr 8, 141(1), 17 - 23 IHF supresses the inhibitory effect of H-NS on HU function in the hin inversion system; Goshima N et al.; In the hin-mediated DNA inversion system, HU facilitates formation of the synaptic complex composed of two recombination sites spaced 996 bp apart and of the enhancer situated between them, by looping the DNA as to promote interaction of Hin invertase with the Fis enhancer factor {Johnson et al., Nature 329 (1987) 462-465} . The HU requirement for the in vivo hin-mediated inversion was demonstrated previously {Wada et al., Gene 76 (1989) 345-352; Hillyard et al., J . Bacteriol . 172 (1990) 5402-5407; Haykinson and Johnson, EMBO J . 12 (1993) 2503-2512} and in the current experiments . This HU action, however, required IHF when H-NS was present in the cell; i.e., the inversion reaction of the hin-invertible DNA fragment carried by the pKK1202R plasmid proceeded efficiently in host cells either deficient in H-NS or in the presence of both H-NS and IHF, but not in the cells depleted for IHF alone . The level of hin mRNA in mutant cells lacking HU or IHF, in which hin inversion did not occur, was normal or slightly increased . When IHF was absent, the stimulating effect of HU on in vitro DNA circle formation of a 125-bp hin fragment between hixL and the enhancer where Fis binds was inhibited by H-NS . The present study provides an example of a multi-component interaction between HU, H-NS and IHF on the hin DNA region, which contains three characteristic sites, a d(A/T)4 stretch and bent DNA site, and two putative IHF-binding sites. J Biol Chem, 1994 Apr 8, 269(14), 10797 - 803 Effects of nucleotide sequence on the specificity of rne-dependent and RNase E-mediated cleavages of RNA I encoded by the pBR322 plasmid; Lin-Chao S et al.; RNase E, an endoribonuclease encoded by the Escherichia coli ams/rne/hmp1 locus, cleaves RNA I, an antisense regulator of the replication of ColE1 type plasmids, in a single-stranded region near its 5' end . The rne-3071 mutation prolongs the RNA 1 half-life in cells cultured at an elevated temperature and imparts temperature sensitivity on RNase E isolated from the mutant strain . Here we report the effects of specific sequence changes introduced by site-directed mutagenesis on the location of ribonucleolytic cleavage near the 5' end of pBR322 RNA I in rne-3071 and congenic rne+ E . coli and on cleavage of RNA I by RNase E in vitro . Primer extension analyses showed that the occurrence and position of cleavages in vivo and in vitro are altered highly specifically by sequence changes but that the site of cleavage bears no simple relationship to a particular nucleotide order . Our results do not support either the notion that cleavage by RNase E is determined by a consensus sequence or the contrary view that RNase E is a virtually nonspecific single-stranded endonuclease with a preference for cutting 5' to an AU dinucleotide. J Biol Chem, 1994 Apr 8, 269(14), 10790 - 6 A+U content rather than a particular nucleotide order determines the specificity of RNase E cleavage; McDowall KJ et al.; Ribonuclease E has a central role in Escherichia coli mRNA decay and is dependent on a functional product of the rne (also called ams or hmp1) gene . We investigated the requirements for RNase E cleavage by introducing random mutations into the decanucleotide region at the 5' end of pACYC184 RNA I and studying the effects of these mutations on the position of rne-dependent cleavage in vivo and RNase E-mediated cutting in vitro . We find that the precise point of RNase E cleavage can be altered specifically and reproducibly by sequence changes in the region cleaved and, therefore, is not determined by a distance measured in nucleotides from any other sequence or region of secondary structure in RNA I . Although cleavage by RNase E occurs within sequences rich in A and/or U nucleotides and is affected by the extent of continuity of A and U nucleotides in the regions cleaved, there is no simple relationship between the order of nucleotides and the phosphodiester bond cleaved . Thus, our results are not consistent with either the notion that RNase E cleavages are determined by a simple consensus sequence or the contrary view that RNase E has few primary structural constraints other than a preference for cleaving 5' to an AU dinucleotide. Nature, 1994 Apr 7, 368(6471), 520 - 5 Promoter targeting by adenovirus E1a through interaction with different cellular DNA-binding domains; Liu F et al.; A puzzling property of the transcriptional activators encoded by several animal viruses is their ability to function promiscuously . The adenovirus E1a protein, for example, stimulates transcription of adenoviral genes as well as a wide variety of other viral and cellular genes . We show that E1a can interact with several classes of cellular DNA-binding domains and thereby be recruited to diverse promoters . Our results explain how a single protein can regulate transcription of multiple genes that lack a common promoter element. Biochemistry, 1994 Apr 5, 33(13), 3980 - 5 Dynamics of lactose permease of Escherichia coli determined by site-directed fluorescence labeling; Jung K et al.; Recently we described the use of site-directed pyrene labeling of engineered lactose permease containing paired Cys residues to obtain proximity relationships between helices in the C-terminal half of the molecule {Jung, K., Jung, H., Wu, J., Prive, G . G., & Kaback, H.R . (1993) Biochemistry 32, 12273} . Pyrene excimer fluorescence was detected for the double Cys mutants His322-->Cys/Glu325-->Cys, Arg302-->Cys/Glu325-->Cys, and Glu269-->Cys/His322-->Cys, indicating that helix X (His322-->Cys/Glu325-->Cys) is in an alpha-helical conformation and that helices VIII (Glu269-->Cys) and IX (Arg302-->Cys) are close to helix X (His322-->Cys and Glu325-->Cys) . In this report, these interactions are used to study dynamic aspects of the permease . Excimer fluorescence between helices VIII and X or helices IX and X is markedly diminished by sodium dodecyl sulfate, while the excimer observed within helix X is unaffected, suggesting that tertiary interactions are disrupted by the denaturant with little effect on secondary structure . Furthermore, excimer fluorescence observed between helices VIII (Glu269-->Cys) and helix X (His322-->Cys) is quenched by Tl+, and the effect is markedly and specifically attenuated by ligands of the permease, suggesting that the pyrene becomes less accessible to the aqueous phase . The reactivity of single Cys residues at positions 269 or 322 was also examined by studying the rate of increase in fluorescence with N-(l-pyrenyl)maleimide . With both mutants, ligands of the permease cause a dramatic increase in reactivity which is consistent with the notion that these positions are transferred into a more hydrophobic environment.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Apr 5, 33(13), 3949 - 58 Ecotin is a potent anticoagulant and reversible tight-binding inhibitor of factor Xa; Seymour JL et al.; Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, has been characterized as an extremely potent anticoagulant and reversible tight-binding inhibitor of human factor Xa (FXa) . The ecotin gene was cloned by PCR, highly expressed in E . coli, and purified from the E . coli periplasm . The binding of ecotin to FXa was stoichiometric with an equilibrium dissociation constant Ki of 54 pM . The association rate constant was 1.35 x 10(6) M-1 s-1, and the dissociation rate constant, measured in the presence of human leukocyte elastase (HLE) to prevent reassociation of ecotin with FXa, was 6.5 x 10(-5) s-1 . Ecotin prolonged clotting time ca . 10-fold at 0.3 microM and at 2 microM in activated partial thromboplastin time and prothrombin time assays, respectively . Ecotin did not effectively inhibit the human plasma proteases thrombin, tissue factor.factor VIIa, factor XIa, activated protein C, plasmin, or tissue plasminogen activator (t-PA); however, it did potently inhibit factor XIIa, plasma kallikrein, HLE, and bovine trypsin and chymotrypsin . Coincubation of ecotin and FXa at 10 microM each resulted in a (ecotin)2.(FXa)2 complex as determined by gel filtration . Dimerization of ecotin alone was measured by fluorescence titration which yielded a Kd of ca . 390 nM . FXa cleaved ecotin slowly at pH 4.0 between M84 and M85 . Replacement of the P1 Met84 residue with Arg and Lys led to FXa inhibitors with Ki values of 11 and 21 pM, respectively . The P1 Arg and Lys mutants also significantly inhibited thrombin, factor XIa, activated protein C, plasmin, factor XIIa, kallikrein, and bovine trypsin and chymotrypsin but did not inhibit tissue factor.factor VIIa, t-PA, or HLE. Biochemistry, 1994 Apr 5, 33(13), 3913 - 8 Involvement of the gamma-phosphate of UTP in the synergistic inhibition of Escherichia coli aspartate transcarbamylase by CTP and UTP; England P et al.; The allosteric control of Escherichia coli aspartate transcarbamylase (ATCase) involves synergistic feedback inhibition by CTP and UTP . Previously reported results {England, P., & Herve, G . (1992) Biochemistry 31, 9725-9732} suggest that this phenomenon relies entirely on interactions between the two neighboring allosteric sites, which belong to the same regulatory dimer . Furthermore, it has been demonstrated that UTP alone binds to the enzyme, but that it is only in the presence of CTP that this binding inhibits the catalytic activity . The properties of mutants in which the synergistic inhibition is totally abolished suggested that the terminal gamma-phosphate of the pyrimidine triphosphate nucleotides may play a crucial role in promoting site-site interactions within the regulatory dimer . In the present work, kinetic studies and binding experiments by continuous-flow dialysis were performed, using combinations of diphosphate and triphosphate nucleotides . The results obtained show that the gamma-phosphate lf UTP is indeed essential for synergistic inhibition to occur, as UDP is unable to inhibit ATCase activity, whether alone or in combination with CTP . On the contrary, the gamma-phosphate of CTP can be suppressed without modifying the inhibitory properties of this nucleotide and its synergy of action with UTP . These results indicate that the mutual effects of CTP and UTP on their respective binding are not symmetrical and that the signals emitted upon binding of the two triphosphate pyrimidine nucleotides to the regulatory sites do not follow the same pathway and involve different mechanisms. Biochemistry, 1994 Apr 5, 33(13), 3878 - 84 Cross-linking of mRNA analogues containing 4-thiouridine residues on the 3'- or 5'-side of the coding triplet to the mRNA binding center of the human ribosome; Graifer DM et al.; The interaction between mRNA and 18S rRNA within complexes of human placenta 80S ribosomes has been investigated by photochemical cross-linking experiments using mRNA analogues substituted with 4-thiouridine at specific locations . mRNA analogues 51 or 54 nucleotides long were prepared from synthetic DNA templates . These mRNA analogues contained either the sequence GGGACC (coding for glycine and threonine, respectively) or the single triplet GGG together with 2-4 4-thiouridine residues located at various positions with respect to the coding triplets . The products of cross-linking of the mRNA analogues to 18S rRNA within different model complexes without tRNA or in the presence of cognate tRNAs were analyzed by reverse transcription . Two cross-linking sites in the 18S rRNA were detected . The first site, U630, was cross-linked by mRNA 8' (s4U at +20, +22, +24, and +26), mRNA 9e' (s4U at -16, -18, and -20), and mRNA 10 (s4U at +4, +6, -1, and -3) but, unexpectedly, not with either mRNA 10b (s4U at +4 and +6) or mRNA 10c (s4U at -1 and -3) . The second site, U1111/A1112, was cross-linked by mRNA 10 and mRNA 10c but not by any of the other mRNA analogues . There is significant tRNA dependence on cross-linking only for mRNA analogue 9e' . Both of the sites detected correspond to sites of mRNA cross-linking in Escherichia coli 16S rRNA. Biochemistry, 1994 Apr 5, 33(13), 3872 - 7 Site-directed mutagenesis of kappa-bungarotoxin: implications for neuronal receptor specificity; Fiordalisi JJ et al.; Postsynaptic polypeptide neurotoxins isolated from the venoms of elapid and hydrophid snakes exhibit the ability to bind selectively to and inhibit different types of receptors that function in nerve signal transmission . On the basis of their amino acid sequences and three-dimensional structures, these neurotoxins are clearly related, but nothing is yet known about the basis for their physiological receptor specificity . In this report, site-directed mutants of kappa-bungarotoxin, produced by an Escherichia coli expression system, are tested to determine the function of selected amino acid side chains in the interaction between toxin and receptor . Highly conserved residues at the bottom of the second loop (a region that has been shown to be a major point of contact with the receptor), particularly those residues at the junction between the beta-sheet and the end of the loop, were selected . The results demonstrate that a single amino acid substitution of the invariant arginine residue (Arg-40 to Ala-40) renders the toxin unable to inhibit nerve transmission in the chick ciliary ganglion up to a concentration of 10 microM . Significantly, the results also show that conversion to alanine of the nearby proline residue (Pro-42) found to be invariant in all kappa-neurotoxins, but not found in any potent alpha-neurotoxin, produces a toxin with full inhibitory capacity . However, the introduction of a lysine residue at this position (P-42-K), like that found in alpha-bungarotoxin, reduces activity significantly.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Apr 5, 33(13), 3848 - 54 Hybridization of a complementary ribooligonucleotide to the transcription start site of the lacUV-5-Escherichia coli RNA polymerase open complex . Potential for gene-specific inactivation reagents; Perrin DM et al.; An ribooligonucleotide, UGGAA, complementary to the template strand of the lacUV-5 promoter can hybridize to the transcription "bubble" of the open complex formed by Escherichia coli RNA polymerase . Its site-specific binding, measured by gel retardation, enzyme inhibition, and chemical nuclease footprinting, is dependent on catalysis by RNA polymerase and the sequence of the hybridizing ribooligonucleotide . When UGGAA is linked to the chemical nuclease 1,10-phenanthroline copper, site-specific scission of the template strand of the transcriptionally active gene is observed . The formation of single-stranded DNA at transcription start sites by RNA polymerases provides a target for antigene strategies. Biochemistry, 1994 Apr 5, 33(13), 3841 - 7 Xenopus laevis ovarian DNA helicase I: A 3' to 5' helicase that unwinds short duplexes; Poll EH et al.; A novel DNA helicase isolated from Xenopus laevis ovaries {Poll, E . H . A., & Benbow, R . M . (1988) Biochemistry 27, 8701-8706} was characterized biochemically . The directionality of DNA unwinding was determined to be 3' to 5' . A short 3' ssDNA tail adjacent to duplex DNA was required for DNA unwinding; the minimum length of this tail was between four and nine bases . Only short duplex DNA regions were unwound: duplex DNA of 16 base pairs was readily unwound, whereas a 26 base pair duplex was not . Longer duplex regions were unwound in the presence of Escherichia coli single-strand DNA binding protein if, in addition, the duplex region was flanked by an unpaired 3' or 5' tail and the substrate resembled a branched replicative intermediate . X . laevis DNA helicase I exhibited high affinity for ssDNA, moderate affinity for dsDNA, and no affinity for RNA . DNA unwinding activity was stimulated by monovalent cations, with an optimal concentration of 150 mM for NaCl or KCl or 125 mM for Na chi PO4 or K chi PO4 . The ATP analog ATP gamma S inhibited the DNA unwinding and copurifying DNA-dependent ATPase activity, whereas AMPPCP and AMPPNP moderately inhibited DNA unwinding activity and had little effect on the copurifying DNA-dependent ATPase activity . CTP was a relatively strong inhibitor of DNA unwinding activity, but GTP, UTP, dCTP, dGTP, or TTP showed moderate or no inhibition . The copurifying DNA-dependent ATPase activity was not inhibited by CTP, GTP, UTP, dCTP, dGTP, or TTP. Eur J Pharmacol, 1994 Apr 4, 270(2-3), 143 - 9 Lipopolysaccharide-induced pleural neutrophil accumulation depends on marrow neutrophils and platelet-activating factor; Bozza PT et al.; The involvement of platelet-activating factor (PAF) in lipopolysaccharide (LPS)-induced leukocyte accumulation in the rat pleural cavity was investigated . Intrathoracic (i.t.) injection of LPS (250 ng/cavity) induced a marked increase in the number of neutrophils at 1 h, which was maximum within 6-12 h, reducing after 24 h . In parallel, an increase in blood neutrophil counts within 1-6 h, concomitantly with a reduction in the number of these cells in the bone marrow, was observed . The number of eosinophils recovered from LPS-injected pleural cavity increased at 12 h and was maximum within 24-48 h . No change in blood or bone marrow eosinophil counts was detected . The pretreatment with WEB 2086 or PCA 4248 (20 mg/kg) significantly inhibited pleural neutrophil accumulation, blood neutrophilia and the decrease in the marrow neutrophil content, but not eosinophil accumulation . The blood neutrophilia and the decrease in marrow neutrophil counts induced by the intravenous (i.v.) injection of LPS (250 ng) were significantly lower than those observed after i.t . injection . Furthermore, WEB 2086 and PCA 4248 were ineffective against the systemic alteration induced by i.v . LPS . it was concluded that LPS-induced neutrophil, but not eosinophil, accumulation in the pleural cavity is related to the mobilization of neutrophils from the bone marrow and involves PAF dependent mechanisms. Proteins, 1994 Apr, 18(4), 394 - 403 Preliminary X-ray analysis of Escherichia coli GMP synthetase: determination of anomalous scattering factors for a cysteinyl mercury derivative; Tesmer JJ et al.; We have initiated a project to determine the three-dimensional structure of GMP synthetase (GMPS) from Escherichia coli . GMPS catalyzes the conversion of XMP to GMP in the final step of de novo guanine nucleotide biosynthesis, and is a member of the glutamine amidotransferase family: a group of enzymes responsible for the assimilation of nitrogen into compounds such as amino acids, purine and pyrimidine bases, amino sugars, and antibiotics . The E . coli guaA gene encoding GMPS was cloned into a tac expression vector, overexpressed, and its gene product purified . Conditions for the growth of protein crystals were developed using recombinant GMPS in the presence of MgCl2, ATP, and XMP . The crystals are monoclinic, space group P2(1), with cell parameters of a = 156.0 A, b = 102.0 A, c = 78.8 A, beta = 96.7 degrees . Diffraction data to 2.8 A spacings were collected on a Xuong-Hamlin area detector with an overall Rsym of 5.2% . Both the volume of the unit cell and the peaks in the self-rotation function are consistent with one GMPS tetramer of D2 symmetry in the crystallographic asymmetric unit . Previously, GMPS has been observed only as a dimer in solution . GMPS was covalently modified with p-chloromercuribenzylsulfonic acid (PCMBS), and its X-ray fluorescence spectrum was measured through the LIII absorption edge of mercury . Anomalous scattering factors for cysteinyl mercury were derived from this spectrum, and the feasibility of structure determination by multi-wavelength anomalous diffraction was evaluated . The optimal MAD dispersive signal is 4.5% of magnitude of F, and the optimal MAD Bijvoet signal is 7.5% of magnitude of F at a concentration of approximately 1 mercury per 10-kDa protein . The anomalous scattering factors tabulated here should be transferable to cysteinyl mercury in other proteins. Plant Cell, 1994 Apr, 6(4), 501 - 10 Phosphorylation and calcium binding properties of an Arabidopsis GF14 brain protein homolog; Lu G et al.; Arabidopsis GF14 omega was originally described because of its apparent association with a DNA-protein complex; it is a member of the 14-3-3 kinase regulatory protein family that is conserved throughout eukaryotes . Here, we demonstrated that recombinant GF14 omega is expressed in Escherichia coli as a dimer . Blot binding and electrophoretic mobility shift analyses indicated that GF14 omega binds calcium . Equilibrium dialysis further demonstrated that GF14 omega binds an equimolar amount of calcium with an apparent binding constant of 5.5 x 10(4) M-1 under physiological conditions . The C-terminal domain, which contains a potential EF hand motif, is responsible for the calcium binding . The C-terminal domain also cross-reacted with the anti-GF14 omega monoclonal antibody . In addition, GF14 omega is phosphorylated by Arabidopsis protein kinase activity at a serine residue(s) in vitro . Therefore, GF14 omega protein has biochemical properties consistent with potential signaling roles in plants . The presence of a potential EF hand-like motif in the highly conserved C terminus of 14-3-3 proteins together with the calcium-dependent multiple functions attributed to the 14-3-3 proteins indicate that the C terminus EF hand is a common functional element of this family of proteins. J Dairy Sci, 1994 Apr, 77(4), 930 - 9 Enterotoxin-binding glycoproteins in a proteose-peptone fraction of heated bovine milk; Shida K et al.; The binding of Escherichia coli heat-labile enterotoxin to caseins, whey proteins, milk fat globule membrane, and proteose-peptone fraction from bovine milk was studied by using the Western blot technique . Two toxin-binding glycoproteins, pp16k and pp20k, with molecular weights of 15,500 and 20,000, respectively, were detected only in a proteose-peptone fraction . These glycoproteins were partially purified by ammonium sulfate precipitation and Toyopearl HW 55 gel filtration chromatography . The binding ability to the toxin was destroyed by periodate treatment or beta-galactosidase treatment, indicating that a carbohydrate moiety, particularly a terminal galactosyl residue, was essential for the binding of the toxin . In contrast, the binding ability was not changed by mild acid treatment, and these glycoproteins did not bind cholera toxin, which can bind to ganglioside GM1, suggesting that the carbohydrate structure of the glycoproteins is different from that of GM1 . The N-terminal amino acid sequence and immunoblot analysis indicated that the protein moieties of pp16k and pp20k are identical to alpha-lactalbumin and beta-lactoglobulin, respectively . These toxin-binding glycoproteins were not detected in whey proteins isolated from unheated skim milk, suggesting that they are newly generated during heat treatment of skim milk before the preparation of a proteose-peptone fraction. Biotechnol Appl Biochem, 1994 Apr, 19 ( Pt 2), 217 - 31 Immobilization of beta-galactosidase on metal-chelate-substituted gels; Brena BM et al.; The use of copper, zinc, iron, nickel and calcium in three different chelating gels was investigated for preparing immobilized beta-galactosidase . The chelated ligands {Cu(2+)-iminodiacetate (IDA), Cu(2+)-Tris(carboxymethyl)ethylenediamine (TED), Ni(2+)-IDA and Fe(3+)-IDA} absorbed the protein so strongly that it can be considered a true immobilization . The obtained enzyme derivatives were investigated with regard to activity and stability . Enzymic activity was highly preserved in general for the TED derivates (90% when compared with that for Cu(2+)-TED) . The immobilized Ni2+ derivatives were more stable to high temperature and to storage than the Cu2+ derivatives . Temperature-stability of the immobilized enzyme was very much improved by adding a strong metal-chelating gel such as carboxymethylated tetraethylenepentamine-agarose . The gel could be re-used and reloaded after elution with chelator . beta-Galactosidase from Escherichia coli was purified using immobilized-metal-ion-chelate chromatography (i.m.a.c.) . The potential use of beta-galactosidase immobilized on i.m.a.c . gels for technical purposes is discussed. Mol Gen Genet, 1994 Apr, 243(1), 9 - 16 Multicopy suppressors, mssA and mssB, of an smbA mutation of Escherichia coli; Yamanaka K et al.; We have isolated and characterized two multicopy suppressors, mssA and mssB, which suppress the cold-sensitive growth phenotype of the smbA2 mutant of Escherichia coli . The mssA gene is located immediately upstream of the rpsA gene (20.5 min) . MssA protein was found to be related to nucleoside monophosphate kinases . The mssB gene was found to be identical to the deaD gene (69 min), which encodes a putative RNA helicase . The SmbA protein belongs to the aspartokinase family and probably represents a new, fourth aspartokinase species in E . coli . Expression of the smbA gene is essential for cell growth . The smbA2 mutant shows a pleiotropic phenotype characterized by cold-sensitive growth, hypersensitivity to the detergent sodium dodecyl sulfate, and formation of a translucent segment at midcell or at a pole of the cell when grown at 22 degrees C . In addition, some cellular proteins were either increased or decreased in amount in the smbA2 mutant . SmbA may be a regulatory factor in the expression of a battery of genes . MssA and MssB might also relate to the expression of some of these genes . Multiple copies mssA and mssB suppressed the various phenotypic features of the smbA2 mutant to various extents, suppressing the cold-sensitive growth completely. Nippon Seikeigeka Gakkai Zasshi, 1994 Apr, 68(4), 272 - 82 {Induction of arthritis in the ankles of rat immunized by intraperitoneally-injected lipopolysaccharide (LPS) or lipid A}; Suzuki K; The histological changes in the ankle joints were investigated in Sprague Dawley (SD) rat immunized through intraperitoneal injection of 10 micrograms or 100 micrograms of lipopolysaccharide(LPS; extracted from Escherichia coli), or Lipid A, for 10 or 15 weeks (short term) or for 30 weeks (long term) . The serum anti-IgM rheumatoid factor-like substance (RFLS) was detected by enzyme-linked immunosorbent assay (ELISA) . The macroscopic arthritic changes in the rat ankles, showing redness or swelling, were observed in 17 of 64 rats immunized by LPS and in 9 of 62 rats immunized by Lipid A . Rats immunized by 10 micrograms of LPS in the long term developed synovial lining cell hyperplasia in 12 of 28 ankles (12/28), lymphoid cell infiltration in 8/28, and pannus formation in 2/28 . Rats immunized by 100 micrograms of Lipid A in the long term developed synovial lining cell hyperplasia in 8/18, lymphoid cell infiltration in 7/18, and pannus formation in 2/18 . SD rats immunized by LPS or Lipid A developed a significantly higher incidence rate of hyperplasia in the synovial lining cells, than controls . In each case of immunization by LPS or Lipid A, the serum RFLS levels at sacrifice were significantly higher than before immunization (p < 0.01) . These findings suggest that LPS and Lipid A played important roles as trigger substances causing arthritis with RFLS elevation in rats immunized with E . coli. Nippon Seikeigeka Gakkai Zasshi, 1994 Apr, 68(4), 221 - 33 {Pathologic findings of mouse air-pouches injected with Escherichia coli O: 14, lipopolysaccharide, or interleukin-1 beta}; Hirakawa K; Inflammation of the facsimile synovium was induced by injecting 4 mg of heat-killed Escherichia coli (E . coli) O: 14, 1 mg of lipopolysaccharide (LPS), or 100 U of recombinant interleukin-1 beta (IL-1 beta) into a 7-day-old subcutaneous air-pouch in C 57 Black mice . Saline was used for control animals . A total of 150 mice were used . Hyperplasia in the lining cells (lining greater than 5 cells thick) was induced in the inner layer of 18 of 20 air-pouches (in 9 of 10 mice) at 7 days after injection of E . coli or LPS . The results at 7 days after were significantly higher than those at 3 days after injection of E . coli (11/20 mice) or LPS (5/10) . The number of lining layers with neutrophil infiltration reached a maximum 3 days after injection of E . coli (3 days: 10/20, 7 days: 2/20, p < 0.01) or LPS (3 days: 7/10, 7 days: 2/10, p < 0.01) . There was a greater number of mononuclear cells in the sublining layers 7 days after injection of E . coli (9/20 mice) or LPS (7/10) than at 3 days (2/20, 3/10), (p < 0.01) . There was a higher incidence of mononuclear cells around the post-capillary venules (PCV) at 7 days after injection of E . coli (9/20 mice) or LPS (7/10) than at 1 day (0/20, 0/10), (p < 0.01) . There were significantly more mast cells around the PCV at 1 day after injection of E . coli (6.0 +/- 2.1) or LPS (5.0 +/- 1.8) than in the saline controls (1.0 +/- 0.4), (p < 0.01) . Immunohistochemical stains for IgG showed more positive cells at 7 days after injection of E . coli (4.0 +/- 1.7) or LPS (4.0 +/- 1.4) than in controls (0.5 +/- 0.2), (p < 0.05) . Significantly more exudate (0.5 +/- 0.3 mls) was found in the air-pouch at 1 day after injection with IL-1 beta than at 1 day after in the other groups . In conclusion, these results suggest that the air-pouch model may be very useful for investigating possible roles of arthritogenic agents or cytokines on the acute and/or chronic phases of inflammation in connective tissue. Carbohydr Res, 1994 Apr 1, 256(2), 289 - 301 Escherichia coli K48 capsular polysaccharide: a glycan containing a novel diacetamido sugar; Whittaker DV et al.; The structure of the exocellular capsular polysaccharide expressed by Escherichia coli O8:K48:H9 has been investigated by hydrolysis and methylation analysis, and by NMR spectroscopic studies of the polysaccharide and of the oligosaccharides generated by solvolysis with anhydrous HF . The capsular polysaccharide was shown to have the repeating unit: {formula: see text} where beta-L-Sug p represents 2,3-diacetamido-2,3,6-trideoxy-beta-L- mannopyranose. Am J Physiol, 1994 Apr, 266(4 Pt 2), H1581 - 7 High-energy phosphates in heart, liver, kidney, and skeletal muscle of endotoxemic rats; Van Lambalgen AA et al.; Endotoxemia can affect the storage of high-energy phosphates {ATP, creatine phosphate (CrP)} even in organs in which global blood flow does not fall . If a decrease in this storage is due to an inadequate oxygen supply-to-demand ratio, improving the perfusion should restore it . Therefore, in anesthetized endotoxemic rats we studied organ perfusion and the storage of high-energy phosphates of heart, liver, kidney, and skeletal muscle and measured the effects of improving cardiac output (CO) and organ blood flow with cardiostimulatory drugs {dopexamine (DX) and dobutamine (DB)} . Endotoxin (Escherichia coli O127.B8, 8 mg/kg) was infused from 0 to 60 min in three groups of anesthetized rats: one untreated (saline only) group (ES; n = 10), and two groups in which we infused DX (3 x 10(-8) mol.kg-1.min-1; n = 10) or DB (10(-7) mol.kg-1.min-1; n = 8) from 60 to 135 min . A fourth group served as time-matched controls (C; n = 8) . Organ blood flows at 0 and 135 min (end of experiment) were measured with radioactive microspheres . In biopsies (at 135 min) we measured lactate, ATP, and CrP concentrations . Endotoxemia decreased CO (45% at 135 min; P < 0.05), which could be restored by DX and DB . Myocardial and skeletal muscle blood flow and ATP did not differ in the groups at 135 min . Hepatic and renal blood flow decreased in the ES group 44 and 52%, respectively (P < 0.05); DX restored the fall of hepatic and DB of renal blood flow.(ABSTRACT TRUNCATED AT 250 WORDS) Am J Physiol, 1994 Apr, 266(4 Pt 2), H1558 - 64 Protective role of NO in the regional hemodynamic changes during acute endotoxemia in rats; Mulder MF et al.; The role of NO during the first hour of endotoxemia is still controversial . To evaluate whether NO is protective or detrimental to the regulation of systemic blood pressure, cardiac output (CO), and organ perfusion in rats during acute endotoxemia, we have studied the effects of inhibition of NO synthesis . Thirty minutes after 0.1 mg NG-nitro-L-arginine (L-NNA; group L, n = 7, partial inhibition), 1 mg L-NNA (group H, n = 6, complete inhibition), or saline (group E, n = 7) intravenous infusion, anesthetized volume-loaded rats were infused with endotoxin Escherichia coli O127:B8 (8 mg.kg-1 x h-1) from time (t) = 0 to 60 min . Organ blood flow was measured with radioactive microspheres . In group H, at time 0, CO was lower than in group E (by -29%; P < 0.05), and systemic vascular resistance (SVR) was higher than in groups E and L (by 72 and 51%, respectively; P < 0.05) . Perfusion of the pancreas, stomach, intestines, and kidney was lower (P < 0.05) and corresponding organ vascular resistance (OVR) higher (P < 0.05) in group H than in groups E and L (except kidney in group L) . At t = 60 min, in groups H and L, CO was lower (by -45 and -26%, respectively; P < 0.05) and SVR was higher (by 112 and 54%, respectively; P < 0.05) than in group E . In group L only blood flow to the heart, pancreas, intestines, and kidney was significantly lower than in group E, and corresponding OVR was higher.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Pharmacol, 1994 Apr, 45(4), 792 - 6 p-Alkoxyphenols, a new class of inhibitors of mammalian R2 ribonucleotide reductase: possible candidates for antimelanotic drugs; Potsch S et al.; The inhibition by different p-alkoxyphenol derivatives of the growth-regulating enzyme ribonucleotide reductase (RR) in purified Escherichia coli and mouse R2 protein preparations was studied by EPR spectroscopy . The inhibitor-induced inactivation of the catalytic subunit protein R2 was measured at 77 degrees K by observing the decrease of the typical EPR signal from the functionally essential protein-linked tyrosyl free radical . p-Methoxy-, p-ethoxy-, p-propoxy-, and p-allyloxyphenol were about 2 orders of magnitude more effective in inhibiting mouse R2, compared with E . coli R2 . Among the p-alkoxyphenols studied, p-propoxyphenol was the most effective inhibitor of mouse R2 (IC50, 0.7 microM) and p-methoxyphenol was the least effective (IC50, 11 microM); p-ethoxy- and p-allyloxyphenol were intermediate . The observed half-maximal inhibition values characterized p-alkoxyphenols as a new class of strong inhibitors of the R2 protein of mammalian RR . p-Propoxy-, p-ethoxy-, and p-allyloxyphenol could be considered as new candidates for anticancer drugs . A special cellular inhibition assay of RR in proliferating tumor cells, in which the tyrosyl radical of R2 at natural concentration was monitored by EPR, showed that the four para-substituted alkoxyphenols also inhibited the enzyme with high efficiency in tumor cells (IC50, between 0.5 microM and 5 microM) . Our results with inactivation of protein R2 of RR imply that the cytostatic effect of p-alkoxyphenols on melanoma cells, which has been hitherto explained by inhibition of tyrosinase {Melanoma Res . 2:295-304 (1992)}, may be caused at least partly by inhibition of RR . Protein R2 of RR may be considered as an additional target that could be used for future cancer chemotherapy. FEMS Microbiol Lett, 1994 Apr 1, 117(2), 225 - 9 Variations in flow cytometric forward scatter signals and cell size in batch cultures of Escherichia coli; Lopez-Amoros R et al.; Flow cytometry was used to study the lag, exponential, stationary and death phases of non-fixed cultures of Escherichia coli . Fluctuations in the forward angle scatter signal (FALS) were compared with cell size as measured by scanning electron microscopy at low temperature and image analysis . A correlation between FALS and cell size was not observed, although a correlation (r = -0.8) was obtained between FALS and the age of the culture for the first eleven days of incubation . Marked increases in FALS were observed during the lag phase, which were attributed both to changes in size and changes in structure or chemical composition . The distribution of FALS for all culture phases was asymmetric, and was associated with the cell size distribution. FEMS Microbiol Lett, 1994 Apr 1, 117(2), 217 - 23 The cydD gene product, component of a heterodimeric ABC transporter, is required for assembly of periplasmic cytochrome c and of cytochrome bd in Escherichia coli; Poole RK et al.; The cydD gene of Escherichia coli encodes a protein which, together with the CydC protein, probably constitutes a heterodimeric, ABC-family membrane transporter, necessary for biosynthesis of the cytochrome bd quinol oxidase . Here, we demonstrate that a cydD mutant also fails to synthesise periplasmic c-type cytochrome(s), suggesting that the transporter exports haem or some other component involved in assembly of cytochromes that are found in, or exposed to, the periplasm . The CydDC system appears to be the first example of a transporter required for periplasmic cytochrome assembly processes requiring more than one type of haem . A mutant defective in trxB (adjacent to the cydDC operon, and encoding thioredoxin reductase) was unaffected in cytochrome c or bd assembly. FEMS Microbiol Lett, 1994 Apr 1, 117(2), 181 - 7 Effects of starvation for exogenous carbon on functional mRNA stability and rate of peptide chain elongation in Escherichia coli; Albertson NH et al.; The decay rate of the potential to synthesize proteins after complete inhibition of transcription by rifampicin was analyzed to determine the functional mRNA stability of exponentially growing and glucose-starved Escherichia coli B and K12 cells . We found the following: (i) The half-life of the mRNA pool increased 2.2-fold during a period of 2 h of starvation (from 1.8 min in exponentially growing cells to 4.0 min for cells starved for 2 h); (ii) the effect on transcript stability appeared to be global since transcripts of genes that were induced, repressed or unaltered in their expression during starvation exhibited more or less the same increased stability; (iii) the rate of peptide chain elongation, as measured by the synthesis time for beta-galactosidase, decreased 1.9-fold during the starvation period studied and may, at least in part, account for the global stabilization of transcripts in starved cells. FEMS Microbiol Lett, 1994 Apr 1, 117(2), 169 - 74 Truncated mutants of the colicin A lysis protein are acylated and processed when overproduced; Cavard D; The lipid modification and processing of truncated mutants of the colicin A lysis protein were observed after overproduction by Escherichia coli bacteria, but at a rate far slower than that of the wild-type . The unmodified precursor form of the mutants was stable over hour(s) . The truncated mutants provoked lethality, but neither caused protein release nor quasi-lysis. Mol Gen Genet, 1994 Apr, 243(2), 244 - 8 A DNA repair process in Escherichia coli corrects U:G and T:G mismatches to C:G at sites of cytosine methylation; Gabbara S et al.; Escherichia coli contains a base mismatch correction system called VSP repair that is known to correct T:G mismatches to C:G when they occur in certain sequence contexts . The preferred sequence context for this process is the site for methylation by the E . coli DNA cytosine methylase (Dcm) . For this reason, VSP repair is thought to counteract potential mutagenic effects of deamination of 5-methylcytosine to thymine . We have developed a genetic reversion assay that quantitates the frequency of C to T mutations at Dcm sites and the removal of such mutations by DNA repair processes . Using this assay, we have studied the repair of U:G mismatches in DNA to C:G and have found that VSP repair is capable of correcting these mismatches . Although VSP repair substantially affects the reversion frequency, it may not be as efficient at correcting U:G mismatches as the uracil DNA glycosylase-mediated repair process. Mol Gen Genet, 1994 Apr, 243(2), 127 - 35 Membrane topology and assembly of the outer membrane protein OmpA of Escherichia coli K12; Ried G et al.; The 325-residue outer membrane protein OmpA of Escherichia coli has been proposed to consist of a membrane-embedded moiety (residues 1 to about 170) and a C-terminal periplasmic region . The former is thought to comprise eight transmembrane segments in the form of antiparallel beta-strands, forming an amphiphilic beta-barrel, connected by exposed turns . Several questions concerning this model were addressed . Thus no experimental evidence had been presented for the turns at the inner leaflet of the membrane and it was not known whether or not the periplasmic part of the polypeptide plays a role in the process of membrane incorporation . Oligonucleotides encoding trypsin cleavage sites were inserted at the predicted turn sites of the ompA gene and it was shown that the encoded proteins indeed become accessible to trypsin at the modified sites . Together with previous results, these data also show that the turns on both sides of the membrane do not possess specifically topogenic information . In two cases one of the two expected tryptic fragments was lost and could be detected at low concentration in only one case . Therefore, bilateral proteolytic digestion of outer membranes can cause loss of beta-strands and does not necessarily produce a reliable picture of protein topology . When ompA genes were constructed coding for proteins ending at residue 228 or 274, the membrane assembly of these proteins was shown to be partially defective with about 20% of the proteins not being assembled . No such defect was observed when, following the introduction of a premature stop codon, a truncated protein was produced ending with residue 171.(ABSTRACT TRUNCATED AT 250 WORDS) Eur J Biochem, 1994 Apr 1, 221(1), 77 - 85 Conformational changes in proteins induced by dynamic associations . A tryptophan phosphorescence study; Gabellieri E et al.; Random collisions between macromolecules lead to dynamic associations (lengthy encounters) that in principle could affect their conformation and, in the case of enzymes, their binding and catalytic properties . Exploiting the unique sensitivity of the phosphorescence lifetime, tau, of Trp to the internal flexibility of globular proteins we probed the perturbations induced in the structure of the coenzyme-binding domain of alcohol dehydrogenase (LADH) and glyceraldehyde-3-phosphate dehydrogenase (GraPDH) by the presence in solution of other dehydrogenases and of functionally unrelated proteins . With Trp314 of LADH, the results emphasize that while tau is not affected by the concentration of LADH itself, the addition of micromolar quantities of other proteins causes a distinct reduction in it . From the linear increase of 1/tau with protein concentration one obtains values for the apparent second-order Stern-Volmer rate constant that range between 2-200 x 10(3) M-1 s-1, decreasing 2-3-fold when ternary complexes of LADH with NADH or NAD+ and inhibitors are involved . Similar effects were observed with Trp310 of GraPDH except that with sorbitol dehydrogenase as perturbant the increase of 1/tau is hyperbolic and governed by an apparent dissociation constant of about 1 microM . Finally, glycerol-3-phosphate dehydrogenase, the strongest perturber of both LADH and GraPDH, has either no effect on lactic dehydrogenase from pig heart or induces a moderate lengthening of the triplet lifetime of the rabbit muscle enzyme . Because Stern-Volmer behavior is typical also of diffusion-mediated quenching reactions, a parallel investigation with cysteine, cystine and N-acetyl-tryptophanamide demonstrated that among potential, protein-associated, quenching moieties namely, -SH, -S-S- and indole groups, only the latter has rate constants approaching the magnitude of protein perturbants . Since considerable evidence rules out the predominance of such quenching reactions, these findings confirm a subtle form of communication between protein molecules in solution . The lack of specificity and the similar effects between dehydrogenases with right and wrong stereospecificity for direct coenzyme transfer suggests that the perturbations monitored are unrelated to this function. Eur J Biochem, 1994 Apr 1, 221(1), 513 - 22 The kinetics and thermodynamics of the binding of cytochalasin B to sugar transporters; Walmsley AR et al.; The kinetics of the binding of cytochalasin B to the proton-linked L-arabinose (AraE) and D-galactose (GalP) symporters from Escherichia coli and to the human erythrocyte glucose transporter (GLUT1) have been investigated by exploiting the changes in protein fluorescence that occur upon binding the ligand . Steady-state measurements yielded Kd values of 1.1, 1.9 and 0.14 microM for the AraE, GalP and GLUT1 proteins, respectively . The association and dissociation rate constants for the binding of cytochalasin B have been determined by stopped-flow spectroscopy . In each case, the apparent Kd was calculated from the corresponding rate constants, yielding values of 1.5, 0.4 and 1.6 microM for AraE, GalP and GLUT1, respectively . The differences between these apparent Kd values and those measured by fluorescence titration is interpreted in terms of the following three step mechanism where CB represents cytochalasin B: {formula: see text} The transporter is proposed to alternate between two different conformational forms (T1 and T2), with cytochalasin B binding only to the T2 conformation, to induce a further conformational transition of the transporter to the T3 form . The values for the overall dissociation constants show that the T1 conformation is favoured by AraE and GalP in the absence of ligands, but the T2 conformation is favoured by GLUT1 . Thus, the binding of cytochalasin B to GLUT1 alters the equilibrium towards the T3(CB) conformational state, producing the observed tight binding, in contrast to the changes in the equilibrium observed with the binding of cytochalasin B to AraE and GalP . A thermodynamic analysis of these conformational transitions has been performed . The T1 and T2 conformations may represent transporter states in which the binding site is facing outwards and inwards, respectively. Eur J Biochem, 1994 Apr 1, 221(1), 435 - 43 Single-stranded-DNA-binding proteins from human mitochondria and Escherichia coli have analogous physicochemical properties; Curth U et al.; The gene for the mature human mitochondrial single-stranded-DNA binding protein (HsmtSSB) has been transferred into a protein-overproducing vector and expressed in Escherichia coli . The protein was purified to homogeneity and its physicochemical properties were investigated . From sequence comparison, HsmtSSB shows some similarities to the N-terminal part of the single-stranded DNA-binding protein (SSB) from E . coli (EcoSSB) . Hydrodynamic measurements show the protein to be tetrameric and give a sedimentation coefficient of 4.1 S corresponding to a C-terminally shortened EcoSSB . Electron-microscopic images of the free protein show a globular tetrahedral structure . Binding of poly(desoxythymidylic acid) {poly(dT)} leads to a reduction of the tryptophan fluorescence of the protein up to 96% . Fluorescence titrations with poly(dT) show apparent binding-site sizes of 50-70 nucleotides/tetramer between 0.05 M and 2 M NaCl . Binding to poly(dT) proceeds in a nearly diffusion-controlled reaction with an association-rate constant kass of 4 x 10(8) M-1s-1 . The rate-limiting step is the formation of a transient complex where less than four binding sites on the protein are involved and the reshuffling of the protein on the linear matrix is fast . Electron microscopy of the complex with poly(dT) using negative staining shows a nearly random distribution of the protein between the individual poly(dT) strands . This leads to the conclusion that the binding cooperativity is low (omega < 150) . The two tryptophans of HsmtSSB were replaced by threonine and tyrosine . The environment of both residues is influenced by nucleic acid binding with mutations of Trp68 strongly reducing the DNA-binding affinity of the protein. Eur J Biochem, 1994 Apr 1, 221(1), 151 - 7 Recombinant anti-sialidase single-chain variable fragment antibody . Characterization, formation of dimer and higher-molecular-mass multimers and the solution of the crystal structure of the single-chain variable fragment/sialidase complex; Kortt AA et al.; The single-chain antibody variable fragment (scFv), with a 15-residue polypeptide linker (Gly4Ser)3, of monoclonal antibody NC10 was expressed in Escherichia coli and purified to homogeneity . This scFv molecule, refolded from 6 M guanidine hydrochloride, was predominantly a monomer of 27 kDa and was stable on storage at 4 degrees and 20 degrees C . At higher protein concentrations (approximately 5 mg/ml) dimer and higher-molecular-mass multimers were formed and freezing enhanced this aggregation . The dimer was not stable and dissociated to monomer at 20 degrees C with a half-life of approximately 8 days . The higher-molecular-mass multimers and dimer dissociated to monomer in 60% ethylene glycol . Both the monomer and dimer were active and with tern N9 sialidase yielded complexes of 276 kDa and 569 kDa, respectively, indicating that four scFv molecules bound/sialidase tetramer and that the dimer was bivalent and cross-linked two sialidase tetramers . Binding studies at low concentrations and using radiolabelled scFv indicated that the binding affinity of the dimer was approximately twofold higher than that of the monomer, and the binding affinities of the scFv were similar to that of the parent NC10 antigen-binding fragment (Fab) molecule . A complex between tern N9 sialidase and NC10 scFv was crystallized and the structure of the complex was solved at 0.3-nm resolution by X-ray diffraction . Comparison of this scFv/sialidase structure with the parent Fab/sialidase structure revealed that the modes of attachment of scFv and Fab to sialidase were very similar . There was no discernible electron density for the peptide linker joining the variable heavy (VH) and variable light (VL) chains . A close interaction between two symmetry-related scFv suggests that they may have crystallized as dimers. Eur J Biochem, 1994 Apr 1, 221(1), 121 - 8 Overexpression in Escherichia coli, purification and characterization of the molecular chaperone HSC70; Benaroudj N et al.; The 70-kDa heat-shock cognate protein (HSC70), a constitutively expressed protein in mammalian cells, plays a major role in several cellular processes such as protein folding and assembly, uncoating of clathrin-coated vesicles and transport of protein through membranes . HSC70 has been overexpressed in Escherichia coli in a soluble form using a designed two-cistron expression vector, and purified to homogeneity in a two-step procedure involving ion-exchange and affinity chromatography . Up to 20 mg of pure protein could be obtained from 11 of cell culture . Amino-terminal sequencing of the recombinant protein gives the expected sequence, and non-denaturing gel electrophoresis as well as gel filtration analysis reveal the presence of self-associating species that could be dissociated by ATP . Crosslinking studies confirm the presence of multiple species and the dissociating effect of ATP . Temperatures above 42 degrees C induce the aggregation of HSC70; ATP shifts this effect to higher temperatures . The recombinant protein displays a low intrinsic ATPase activity that can be stimulated about threefold by binding to apocytochrome c, a permanently unfolded protein, while native cytochrome c has no effect on the ATPase activity indicating that recombinant HSC70 binds specifically unfolded protein but not their native counterpart . Thus, efficient production of recombinant HSC70 having structural and functional properties comparable to those of the natural protein could be achieved, thereby allowing the molecular basis of the chaperone function and its regulation through ATP hydrolysis to be probed. Biochem J, 1994 Apr 1, 299 ( Pt 1), 23 - 7 The potential dolichol recognition sequence of beta-1,4-mannosyltransferase is not required for enzymic activity using phytanyl-pyrophosphoryl-alpha-N,N'- diacetylchitobioside as acceptor; Revers L et al.; The ALG1 gene of Saccharomyces cerevisiae encodes beta-1,4-mannosyltransferase, an essential membrane-associated enzyme involved in the assembly of dolichyl-linked oligosaccharide precursors for N-glycosylation {Albright and Robbins (1990) J . Biol . Chem . 265, 7042-7049}, which catalyses the transfer of a mannose residue from GDP-mannose to dolichyl-pyrophosphoryl-alpha-N,N'- diacetylchitobioside; it also possesses a putative transmembrane domain, bearing an 11-amino-acid consensus sequence, which has been proposed to mediate dolichol recognition . Here we report the construction and bacterial expression of a mutant beta-1,4-mannosyltransferase derived from ALG1, which carries a 34-amino-acid deletion resulting in the absence of the entire N-terminal transmembrane domain . This truncated enzyme has an apparent Km value of 17 microM for phytanyl-pyrophosphoryl-alpha-N,N'-diacetylchitobioside, a known acceptor for beta-1,4-mannosyltransferase {Flitsch, Pinches, Taylor and Turner (1992) J . Chem . Soc., Perkin Trans . 1, 2087-2093} . The intact enzyme, expressed in the same system, has an apparent Km value of 25 microM . These figures are in good agreement with previously reported values for wild-type beta-1,4-mannosyl-transferase incubated with the natural dolichyl-linked substrate . Gel-filtration chromatography (before and after beta-mannosidase digestion) of the products of both forms of the enzyme verifies the formation of Man beta 1-->4GlcNAc beta 1-->4GlcNAc . We therefore conclude that the putative dolichol recognition sequence is not necessary for recognition of the phytanyl analogue of its natural dolichol substrate and suggest it probably also is not needed for its natural substrate. Invest Ophthalmol Vis Sci, 1994 Apr, 35(5), 2535 - 42 Adenovirus vector-mediated in vivo gene transfer into adult murine retina; Bennett J et al.; PURPOSE . To determine whether a reporter gene can be introduced into the adult mammalian retina in vivo through means of a recombinant replication-deficient adenovirus . METHODS . A dilution series of purified Ad.CMVlacZ ranging from 10(5) to 10(11) pfu/ml was prepared and microinjected into the subretinal space of adult CD-1 mice . This virus contained the cytomegalovirus (CMV)-promoted Escherichia coli reporter gene, lacZ . LacZ expression was assessed in enucleated eyes from 0 to 95 days after injection by beta-galactosidase (beta-Gal) assay . RESULTS . The efficiency of transfection increased as a function of concentration of recombinant virus injected . Eyes injected with greater than 10(7) pfu of Ad.CMVlacZ demonstrated beta-Gal activity lasting at least 95 days . LacZ expression was apparent only in those cells directly exposed to the adenovirus . LacZ expression was observed in the retinal pigment epithelium (RPE) at high efficiency at 48 hours after exposure . By 2 weeks after injection of > 10(7) pfu, lacZ was also expressed in photoreceptors, but at lower density . CONCLUSIONS . These results demonstrate that high efficiency stable transfer of functional genes can be achieved in vivo in post-mitotic mammalian retina using recombinant adenoviral vectors . Adenovirus vectors appear to be a promising means for delivering therapeutic genes in vivo to the mammalian neural retina and particularly to the RPE. Chest, 1994 Apr, 105(4), 1241 - 5 Effect of recombinant IL-1 beta and recombinant gamma interferon on septic acute lung injury in mice; Torre D et al.; The effect of recombinant human interleukin 1B (IL-1B) and recombinant human gamma interferon (IFN-g), when given prophylactically, in a mouse model of septic acute lung injury was studied . Mice were treated with various doses of IL-1B and IFN-g for 3 consecutive days prior to administration of lipopolysaccharide of Escherichia coli (1 mg/kg given intraperitoneally) . To determine the histologic changes occurring after prophylactic administration of such cytokines, a scoring system was assessed . A significant reduction of edema and neutrophil accumulation into the lungs of mice was observed, especially at doses of 100 U per mouse and 10,000 U per mouse of IL-1B and IFN-g, respectively . Prophylactic administration of IL-1B or IFN-g caused histologic changes, including marked reduction of edema and neutrophil accumulation in the interstitial and alveolar spaces . Combined prophylactic administration of IL-1B and IFN-g provoked a marked decrease of neutrophil accumulation into the lungs, but was not accompanied by significant reduction of edema or hemorrhage . These results provide evidence for the beneficial role of IL-1B and IFN-g in the abnormality of septic acute lung injury by reducing inflammatory lesions. Arch Biochem Biophys, 1994 Apr, 310(1), 172 - 9 Identification of chicken liver glucose transporter; Wang MY et al.; A cDNA, Ch-GT2, encoding a glucose transporter was cloned by screening a chicken liver lambda gt11 cDNA library and by modified polymerase chain reaction (PCR) techniques . The encoded protein is highly homologous to rat and human GLUT 2 . Within the predicted amino acid sequence, there are 12 putative transmembrane helices with a relatively large exofacial hydrophilic loop between transmembrane segments 1 and 2, which is characteristic of mammalian GLUT 2 . Expression of Ch-GT2 gene in E . coli results in sixfold increase in the uptake of 2-deoxy-D-glucose uptake, and this increment can be reduced by incubation with phloretin . Thus, Ch-GT2 is a bona fide chicken liver glucose transporter . A deletion mutant generated by PCR was expressed in bacteria and shown to be functional, indicating that the N-terminal seven amino acid residues of Ch-GT2 is not essential for transport of glucose . From Northern blot analysis, Ch-GT2 is prominently expressed in chicken liver with a transcript of 4.0 kb, but not in chicken brain and heart, suggesting that distinct types of glucose transporters are present in various chicken tissues. Arch Biochem Biophys, 1994 Apr, 310(1), 158 - 66 Cloning and expression of pigeon liver cytosolic NADP(+)-dependent malic enzyme cDNA and some of its abortive mutants; Chou WY et al.; A full-length 1927-base-pair cDNA of pigeon liver malic enzyme was obtained by utilizing the screening of the cDNA library and polymerase chain reaction techniques . The cDNA contained one open reading frame coding for 557 amino acid residues, flanked by 86 and 167 nucleotides at the 5' and 3' termini, respectively, and was successfully cloned and expressed in Escherichia coli cells . Functionally active recombinant malic enzyme was purified from the cells . This recombinant enzyme has a Km value for L-malate of 160 +/- 30 microM, which is almost identical to that for the natural enzyme (150 +/- 17 microM) . The Km value for Mn2+ (4.2 +/- 0.3 microM) is higher than that for the natural pigeon malic enzyme (1.4 +/- 0.2 microM), while the Km value for NADP+ (3.8 +/- 0.3 microM) is lower than that for the natural enzyme (10.8 +/- 0.1 microM) . The catalytic constant (kcat) for the recombinant enzyme is decreased by 3.6-fold, but the substrate inhibition constant for L-malate is increased by about 40-fold . Change in the quaternary structure of the recombinant enzyme was revealed in the pH perturbation examination . A truncated pigeon liver malic enzyme, lacking the first 13 amino acid residues, and a recombinant protein, mutated at F19S, N250S, and L353Q, showed no enzymatic activity . Both abortive recombinant mutant proteins were still able to bind with 2',5'-ADP agarose; however, the fluorescence emission spectrum of the protein bound NADPH did not show a blue shift as the natural enzyme . In accordance with these observations, we suggest that the adenosine 2',5'-bisphosphate binding domain of the NADP+ binding site in the beta alpha beta motif may still be retained in these mutant proteins . However, the local hydrophobic environment for the binding of the nicotinamide moiety of the coenzyme molecule may be altered . Therefore, the lack of catalytic activity of the mutant proteins could be attributed to an improper orientation of the bound NADP+. Am Heart J, 1994 Apr, 127(4 Pt 2), 1073 - 80 Complications of pacemaker-defibrillator devices: diagnosis and management; Pfeiffer D et al.; Treatment of resuscitated patients with implantable cardioverter defibrillators has become increasingly more common as a method for the prevention of sudden cardiac death . Major complications such as perioperative death (incidence 2% to 8%), infection (2% to 11%); and lead-related problems (3% to 27%) have been described in previous trials . In our experience with 140 patients, problems were related to leads (n = 11), the device (n = 2), pacing (n = 1), sensing (n = 13), and defibrillation function (n = 5) . Additional problems that occurred during the perioperative period included infection (n = 11), hematoma, and seroma (n = 2) . Thrombus formation along endocardial leads was observed in 13 of 62 (21%) patients . Different arrhythmias (n = 10), such as sinus tachycardia, atrial fibrillation, and nonsustained, slow or incessant ventricular tachycardia with shock delivery, were also detected . Surgical management (predominantly for the major problems) was used in 31 (48%) patients, drug treatment in 25 (39%), and reprogramming of the device in 24 (38%) patients . All of these problems can result in an increase in mortality rates . This article provides an overview of the complications of cardioverter defibrillator treatment and is based on both published data and our series. J Bacteriol, 1994 Apr, 176(8), 2415 - 26 Cell cycle regulation of the Escherichia coli nrd operon: requirement for a cis-acting upstream AT-rich sequence; Sun L et al.; The expression of the nrd operon encoding ribonucleotide reductase in Escherichia coli has been shown to be cell cycle regulated . To identify the cis-acting elements required for the cell cycle regulation of the nrd promoter, different 5' deletions as well as site-directed mutations were translationally fused to a lacZ reporter gene . The expression of beta-galactosidase from these nrd-lacZ fusions in single-copy plasmids was determined with synchronously growing cultures obtained by repeated phosphate starvation as well as with exponentially growing cultures by flow cytometry analysis . Although Fis and DnaA, two regulatory proteins that bind at multiple sites on the E . coli chromosome, have been found to regulate the nrd promoter, the results in this study demonstrated that neither Fis nor DnaA was required for nrd cell cycle regulation . A cis-acting upstream AT-rich sequence was found to be required for the cell cycle regulation . This sequence could be replaced by a different sequence that maintained the AT richness . A flow cytometry analysis that combined specific immunofluorescent staining of beta-galactosidase with a DNA-specific stain was developed and employed to study the nrd promoter activity in cells at specific cell cycle positions . The results of the flow cytometry analysis confirmed the results obtained from studies with synchronized cells. J Bacteriol, 1994 Apr, 176(8), 2393 - 7 Characterization of the interaction of the glp repressor of Escherichia coli K-12 with single and tandem glp operator variants; Zhao N et al.; The glp operons of Escherichia coli are negatively controlled by the glp repressor . Comparison of the repressor-binding affinities for consensus and altered consensus operators in vivo showed that all base substitutions at positions 3, 4, 5, and 8 from the center of the palindromic operator caused a striking decrease in repressor binding . Substitutions at other positions had a severe to no effect on repressor binding, depending on the base substitution . The results obtained indicate that the repressor binds with highest affinity to operators with the half-site WATKYTCGWW, where W is A or T, K is G or T, and Y is C or T . Strong cooperative binding of the repressor to tandem operators was demonstrated in vivo . Cooperativity was maximal when two 20-bp operators were directly repeated or when 2 bp separated the two operators . Cooperativity decreased with the deletion of 2 bp or the addition of 4 bp between the individual operators . Cooperativity was eliminated with a 6-bp insertion between the operators. J Bacteriol, 1994 Apr, 176(8), 2326 - 38 Role of the TonB amino terminus in energy transduction between membranes; Jaskula JC et al.; Escherichia coli TonB protein is an energy transducer, coupling cytoplasmic membrane energy to active transport of vitamin B12 and iron-siderophores across the outer membrane . TonB is anchored in the cytoplasmic membrane by its hydrophobic amino terminus, with the remainder occupying the periplasmic space . In this report we establish several functions for the hydrophobic amino terminus of TonB . A G-26-->D substitution in the amino terminus prevents export of TonB, suggesting that the amino terminus contains an export signal for proper localization of TonB within the cell envelope . Substitution of the first membrane-spanning domain of the cytoplasmic membrane protein TetA for the TonB amino terminus eliminates TonB activity without altering TonB export, suggesting that the amino terminus contains sequence-specific information . Detectable TonB cross-linking to ExbB is also prevented, suggesting that the two proteins interact primarily through their transmembrane domains . In vivo cleavage of the amino terminus of TonB carrying an engineered leader peptidase cleavage site eliminates (i) TonB activity, (ii) detectable interaction with a membrane fraction having a density intermediate to those of the cytoplasmic and outer membranes, and (iii) cross-linking to ExbB . In contrast, the amino terminus is not required for cross-linking to other proteins with which TonB can form complexes, including FepA . Additionally, although the amino terminus clearly is a membrane anchor, it is not the only means by which TonB associates with the cytoplasmic membrane . TonB lacking its amino-terminal membrane anchor still remains largely associated with the cytoplasmic membrane. J Bacteriol, 1994 Apr, 176(8), 2300 - 7 Induction of Escherichia coli hydroperoxidase I by acetate and other weak acids; Mukhopadhyay S et al.; Escherichia coli produces two independently regulated hydroperoxidases (catalases) that protect the cell from toxic concentrations of hydrogen peroxide . Hydroperoxidase I (HPI) is induced by hydrogen peroxide in an OxyR-dependent manner, while hydroperoxidase II (HPII) synthesis is regulated by an alternative sigma factor called RpoS (KatF) . The activities of both hydroperoxidases increase as exponentially growing cells enter stationary phase . In this study, we examined the growth phase-dependent expression of HPI . Treatment of early-exponential-phase cells with spent culture supernatant resulted in induction of HPI synthesis . Extracellular levels of hydrogen peroxide, accumulating in the culture supernatant during late exponential phase, were found to be lower than the concentrations normally required to induce OxyR-dependent synthesis of HPI . This finding suggested that factors other than hydrogen peroxide may play a role in HPI expression . Weak acids such as acetate, which accumulate in culture supernatant and have been implicated in the regulation of HPII, caused a sixfold increase in HPI expression . Increases in HPI synthesis, mediated by weak acids and spent culture fluid supernatant, could be prevented by chloramphenicol, indicating that de novo protein synthesis is required for induction . Expression studies using a plasmid-borne lacZ transcriptional fusion to katG, the structural gene for HPI, indicated that growth phase-dependent regulation of HPI occurs primarily at the level of transcription and is dependent on RpoS . These results suggest that there may be a common regulatory mechanism of HPI and HPII expression in addition to previously described independent control mechanisms. J Bacteriol, 1994 Apr, 176(8), 2259 - 64 Assembly of LamB and OmpF in deep rough lipopolysaccharide mutants of Escherichia coli K-12; Laird MW et al.; Assembly of the OmpF and LamB proteins was kinetically retarded in deep rough lipopolysaccharide mutants of Escherichia coli K-12 . OmpF assembly was affected at the step of conversion of metastable trimers to stable trimers, whereas LamB assembly was influenced both at the monomer-to-metastable trimer and metastable-to-stable trimer steps . These assembly defects were reversed in the presence of the sfaA1 and sfaB3 suppressor alleles, which were isolated by using ompF assembly mutants. J Bacteriol, 1994 Apr, 176(8), 2210 - 5 The sphR product, a two-component system response regulator protein, regulates phosphate assimilation in Synechococcus sp . strain PCC 7942 by binding to two sites upstream from the phoA promoter; Nagaya M et al.; In the photosynthetic cyanobacterium Synechococcus sp . strain PCC 7942, the sphS and sphR genes were previously suggested to encode a typical pair of two-component signal transduction proteins . A deletion mutant strain lacking these genes failed to exhibit induction of alkaline phosphatase, the phoA gene product, in response to phosphate limitation in the medium . The SphR protein was overexpressed in Escherichia coli and then purified to near homogeneity . A truncated form of the SphS polypeptide (named SphS*) was also isolated . Here, we demonstrate that purified SphR is phosphorylated by phosphotransfer from SphS and binds to two distinct sites upstream from the phoA promoter . From these results, we conclude that the SphS and SphR proteins are directly involved in the regulation of phoA transcription in response to phosphate limitation in Synechococcus species. J Bacteriol, 1994 Apr, 176(8), 2194 - 9 De novo synthesis of thymidylate via deoxycytidine in dcd (dCTP deaminase) mutants of Escherichia coli; Weiss B et al.; dcd (dCTP deaminase) mutants of Escherichia coli were reported not to require thymidine for growth even though most of the thymidylate that is synthesized de novo arises from cytosine nucleotides through a pathway involving dCTP deaminase . We found, however, that the fresh introduction of dcd mutations into many strains of E . coli produced a requirement for thymidine for optimum aerobic growth, but the mutants readily reverted to prototrophy via mutations in other genes . One such mutation was in deoA, the gene for deoxyuridine phosphorylase . However, a dcd deo mutant became thymidine dependent once again if a cdd mutation (affecting deoxycytidine deaminase) were introduced . The results indicate that dcd mutants utilize an alternative pathway of TMP synthesis in which deoxycytidine and deoxyuridine are intermediates . A cdd mutation blocks the pathway by preventing the conversion of deoxycytidine to deoxyuridine, whereas a deoA mutation enhances it by sparing deoxyuridine from catabolism . The deoxycytidine must arise from dCTP or dCDP via unknown steps . It is not known to what extent this pathway is utilized in wild-type cells, which, unlike the dcd mutants, do not accumulate dCTP. EMBO J, 1994 Apr 1, 13(7), 1752 - 9 Mutations that alter the transmembrane signalling pathway in an ATP binding cassette (ABC) transporter; Covitz KM et al.; The maltose transport system of Escherichia coli is a well-characterized member of the ATP binding cassette transporter superfamily . Members of this family share sequence similarity surrounding two short sequences (the Walker A and B sequences) which constitute a nucleotide binding pocket . It is likely that the energy from binding and hydrolysis of ATP is used to accomplish the translocation of substrate from one location to another . Periplasmic binding protein-dependent transport systems, like the maltose transport system of E.coli, possess a water-soluble ligand binding protein that is essential for transport activity . In addition to delivering ligand to the membrane-bound components of the system on the external face of the membrane, the interaction of the binding protein with the membrane complex initiates a signal that is transmitted to the ATP binding subunit on the cytosolic side and stimulates its hydrolytic activity . Mutations that alter the membrane complex so that it transports independently of the periplasmic binding protein also result in constitutive activation of the ATPase . Genetic analysis indicates that, in general, two mutations are required for binding protein-independent transport and constitutive ATPase . The mutations alter residues that cluster to specific regions within the membrane spanning segments of the integral membrane components MalF and MalG . Individually, the mutations perturb the ability of MBP to interact productively with the membrane complex . Genetic alteration of this signalling pathway suggests that other agents might have similar effects . These could be potentially useful for modulating the activities of ABC transporters such as P-glycoprotein or CFTR, that are implicated in disease. EMBO J, 1994 Apr 1, 13(7), 1541 - 8 Conjugative transposition: Tn916 integrase contains two independent DNA binding domains that recognize different DNA sequences; Lu F et al.; Transposition of the conjugative transposon Tn916 requires the activity of a protein, called Int, which is related to members of the integrase family of site-specific recombinases . This family includes phage lambda integrase as well as the Cre, FLP and XerC/XerD recombinases . Different proteins, consisting of fragments of Tn916 Int protein fused to the C-terminal end of maltose binding protein (MBP) were purified from Escherichia coli . DNase I protection experiments showed that MBP-INT proteins containing the C-terminal end of Int bound to the ends of the transposon and adjacent plasmid DNA . MBP-INT proteins containing the N-terminal end of Int bound to sequences within the transposon close to each end . Competition binding experiments showed that the sites recognized by the C- and N-terminal regions of Int did not compete with each other for binding to MBP-INT . We suggest that Tn916 and related conjugative transposons are unique among members of the integrase family of site-specific recombination systems because the presence of two DNA binding domains in the Int protein might allow Int to bridge recombining sites, and this bridging seems to be the sole mechanism ensuring that only correctly aligned molecules undergo recombination. EMBO J, 1994 Apr 1, 13(7), 1534 - 40 The HimA and HimD subunits of integration host factor can specifically bind to DNA as homodimers; Zulianello L et al.; Integration host factor (IHF) is a heterodimeric protein from Escherichia coli which specifically binds to an asymmetric consensus sequence . We have isolated the individual subunits of IHF, HimA and HimD, and show that an active IHF protein can be reconstituted from these subunits . The HimA and HimD polypeptides alone are capable of specifically recognizing the same ihf sequence . The mobilities of the protein-DNA complexes in a gel-retardation assay suggest that the proteins bind as homodimers . The stability of the HimD-DNA complex is approximately 100-fold lower than that of the IHF-DNA complex . The HimA-DNA complex is even less stable and is only observed when a large excess of HimA is used . This instability is possibly due to the inability of HimA to form stable homodimers . By domain swapping between HimA and HimD, we have constructed an IHF fusion protein which has the putative DNA-binding domains of only HimA . This fusion protein forms stable dimers and makes specific protein-DNA complexes with a high efficiency . A comparable fusion protein with only the DNA-binding domains of HimD forms less stable complexes, suggesting that sequence-specific contacts between IHF and the ihf consensus are mainly provided by the HimA subunit. EMBO J, 1994 Apr 1, 13(7), 1502 - 7 Macromolecular chelation as an improved mechanism of protease inhibition: structure of the ecotin-trypsin complex; McGrath ME et al.; The 2.4 A crystal structure (R = 0.180) of the serine protease inhibitor ecotin was determined in a complex with trypsin . Ecotin's dimer structure provides a second discrete and distal binding site for trypsin and, as shown by modelling experiments, other serine proteases . The second site is approximately 45 A from the reactive/active site of the complex and features 13 hydrogen bonds, including six that involve carbonyl oxygen atoms and four bridged by water molecules . Contacts ecotin makes with trypsin's active site are similar to, though more extensive than, those found between trypsin and basic pancreatic trypsin inhibitor . The side chain of ecotin Met84 is found in the substrate binding pocket of trypsin where it makes few contacts, but also does not disrupt the solvent structure or cause misalignment of the scissile bond . This first case of protein dimerization being used to augment binding energy and allow chelation of a target protein provides a new model for protein-protein interactions and for protease inhibition. Gan To Kagaku Ryoho, 1994 Apr, 21(5), 719 - 24 {Interleukin-2 (IL-2)}; Taguchi T; Interleukin-2, a polypeptide lymphokine that induces proliferation of antigen- or mitogen-stimulated T cells, was first described as "T-cell growth factor" by Morgan et al . in 1976 . IL-2 is one of several lymphocyte-produced messenger regulatory molecules that modulate immunocyte function . The main secretory source of IL-2 is the T-helper cell . In 1983, Taniguchi and colleagues isolated a human IL-2 complementary DNA clone from a high-producer Jurkat leukemic cell line, and established its nucleotide sequence . In 1984, Rosenberg et al . described the isolation of cDNA clones of the gene for IL-2 from the Jurkat cell line, its expression in Escherichia coli and its biological characteristics . The mature secreted protein contains 133 amino acids, constituting a calculated molecular weight of 15,420 . Since the discovery of IL-2 and its T-cell growth-promoting activity, extensive research has revealed the complex nature of its immunologic effects, both in vitro and in vivo . The immunopotentiating activities, encouraging in vitro results, plus successful therapy of animal tumors in preclinical studies provided the rationale for investigation of IL-2 in patients with advanced malignancy and immunodeficiencies . The IL-2 receptor has been found to have an unexpected by unusual structure in that it is composed of two separate chains designated alpha (p75) and beta (p55) . Recently, it has been discovered the 3rd gamma-chain by Sugamura et al . Clinical trials of IL-2 in patients with cancer have been done by many researchers . The clinical trials has reviewed briefly. J Med Microbiol, 1994 Apr, 40(4), 229 - 35 Genetic relationships and virulence factors among classical enteropathogenic Escherichia coli serogroup O126 strains; Yam WC et al.; Thirty-nine Escherichia coli strains of the enteropathogenic (EPEC) serogroup O126 isolated from sporadic and outbreak cases of infantile diarrhoea between 1982 and 1988 were studied . These strains consisted of four serotypes showing close genetic relationships between their virulence markers, outer-membrane protein and lipopolysaccharide profiles, and electrophoretic types by multilocus enzyme electrophoresis . None of these strains exhibited localised adherence to HEp-2 cells or the attaching-effacing properties of classical type I EPEC . Of the 39 strains, 31 were of serotype O126:H12 and enterotoxigenic; one strain was serotype O126:H10 and enteroaggregative . The remaining six strains of serotype O126:H21 and one strain of serotype O126:H8 harboured no known virulence factors for diarrhoeagenic E . coli. J Pediatr, 1994 Apr, 124(4), 561 - 5 Immune responses to the Escherichia coli dnaJ heat shock protein in juvenile rheumatoid arthritis and their correlation with disease activity; Albani S et al.; Patients with juvenile rheumatoid arthritis frequently have abnormal immune responses to the hsp65 class of bacterial heat shock proteins . However, lymphocytes from children with other inflammatory diseases may also recognize hsp65, and the role of these antigens in juvenile rheumatoid arthritis remains controversial . We have studied humoral and cellular immune responses to a distinct, recently described bacterial heat shock protein, designated dnaJ . The Escherichia coli dnaJ gene was cloned and expressed, and the purified recombinant protein was used as an antigen . Neither normal children nor children with various chronic inflammatory diseases had lymphocyte proliferative responses to recombinant dnaJ . However, lymphocytes from patients with polyarticular, pauciarticular, and systemic manifestations of juvenile rheumatoid arthritis responded strongly to the antigen . Cellular immune responses to dnaJ were higher in synovial fluid than in blood and higher in children with active disease than in children in remission . These data show that increased immune reactivity to dnaJ is characteristic of juvenile rheumatoid arthritis and that the magnitude of the immune response is linked to disease activity . The results suggest that an abnormal immune response to antigens on commensal gut bacteria may contribute to the generation of chronic inflammation in juvenile rheumatoid arthritis. J Invest Dermatol, 1994 Apr, 102(4), 415 - 21 Ex vivo and in vivo gene transfer to the skin using replication-deficient recombinant adenovirus vectors; Setoguchi Y et al.; The skin has the potential for a variety of gene therapy applications . In addition to local delivery, it is the largest organ of the body, and highly vascular, and thus is an ideal site for systemic delivery of gene products . To evaluate the potential for adenovirus-mediated skin gene transfer, the replication-deficient recombinant adenovirus vectors Ad.RSV beta gal (coding for Escherichia coli beta-galactosidase) and Ad alpha 1AT (coding for human alpha 1-antitrypsin) were used in both ex vivo and in vivo approaches . Following in vitro infection with Ad.RSV beta gal, murine keratinocytes expressed beta-galactosidase . Parallel in vitro studies with Ad alpha 1AT documented de novo synthesis and secretion of human alpha 1AT as shown by {35S}methionine labeling and immunoprecipitation . Quantification of human alpha 1AT in the culture supernatants demonstrated 0.1-0.3 microgram human alpha 1AT secreted/ml-24 h . Evaluation of the serum of mice receiving transplants (10(5) cells/mouse) of Ad alpha 1AT-infected syngeneic keratinocytes demonstrated human alpha 1AT for at least 14 d with maximum levels of 41 ng/ml . To demonstrate the feasibility of direct adenovirus-mediated in vivo transfer of genes to the skin, Ad.RSV beta gal or Ad alpha 1AT were administered subcutaneously to mice . Histologic evaluation after 4 d demonstrated expression of beta-galactosidase in various types of skin cells . Quantification of human alpha 1AT in serum of animals infected subcutaneously with Ad alpha 1AT showed levels of 53 ng/ml at day 4, with human alpha 1AT detectable for at least 14 d . These observations support the feasibility of ex vivo and in vivo gene transfer to the skin mediated by replication-deficient adenovirus vectors. Int J Cancer, 1994 Apr 1, 57(1), 90 - 7 Molecular cloning and expression of a cDNA encoding a protein detected by the K1 antibody from an ovarian carcinoma (OVCAR-3) cell line; Chang K et al.; MAb KI recognizes a cell-surface glycoprotein (MW approximately 40 kDa) present in ovarian carcinomas, malignant mesotheliomas, squamous-cell carcinomas and normal mesothelial cells . In this study, expression screening was used to isolate cDNA clones encoding an antigen recognized by MAb KI from a cDNA library made from a human ovarian carcinoma cell line (OVCAR-3) . Subsequently, other clones were isolated by DNA hybridization using a cDNA probe derived from one of the initial clones . The sequence of all the clones was similar . The longest cDNA contains 2,444 base pairs, and encodes a polypeptide of 263 amino acids with a calculated molecular weight of 30,511 daltons . The nucleotide sequence and deduced amino-acid sequence of the protein show no homology to other sequences in current data bases . In vitro translation of RNA transcripts from the cDNA inserts yielded polypeptides of 29 and 30 kDa . Similar-sized proteins were obtained upon expression of the cDNA in Escherichia coli, and these proteins were reactive with MAb KI . The protein(s) expressed in E . coli were purified and used to make rabbit or mouse antisera . These antisera reacted strongly with a soluble cytosolic protein in OVCAR-3 cells, but not with the membrane-bound antigen . Soluble cytosolic proteins of a similar size, recognized with MAb KI, were found in OVCAR-3 and N87 (gastric cancer) cells but not in 10 other cancer cell lines . These data indicate that the cloned cDNA encodes a cytosolic protein that reacted with MAb KI . This soluble protein is expressed only in cells containing the CAKI surface glycoprotein, suggesting that the 2 proteins could be structurally related. Int J Cancer, 1994 Apr 1, 57(1), 67 - 72 Production and tumour-binding characterization of a chimeric anti-CEA Fab expressed in Escherichia coli; Chester KA et al.; A recombinant chimeric Fab (rcFab), with Fv derived from the monoclonal A5B7 antibody to carcinoembryonic antigen (CEA), and with human CHI and C kappa was cloned into pUC 19 and expressed in Escherichia coli . rcFab (10 to 12 mg per litre) was produced in bacterial culture fluid, and functional purified rcFab was isolated by affinity chromatography (using antibody to human C kappa) and size-exclusion gel filtration . The rcFab did not show reduced affinity for CEA, and reacted with human colorectal tumours showing a typical anti-CEA pattern by immunocytochemistry; it was also stable after iodination . Biodistribution studies in nude mice bearing human tumour xenografts showed no toxicity and good tumour localization . Therapeutic ratios at early time points were better than those obtained with whole murine antibody . The results demonstrate that bacterially produced anti-CEA Fab is of use for tumour targeting. Carcinogenesis, 1994 Apr, 15(4), 627 - 33 A unique structural feature of rabbit DNA repair methyltransferase as revealed by cDNA cloning; Iyama A et al.; cDNA encoding rabbit O6-methylguanine-DNA methyltransferase that repairs DNA damaged by alkylating agents was isolated, using as a probe a fragment of mouse cDNA coding for a region containing the active site of the enzyme . The nucleotide sequence of the cDNA revealed that the rabbit methyltransferase is a 181-amino acid polypeptide with a mol . wt of 19,385 . Expression of the cDNA in a methyltransferase-deficient Escherichia coli mutant resulted in appearance of a 23 kDa polypeptide with methyltransferase activity, and this rendered the E.coli cells resistant to N-methyl-N'-nitro-N-nitrosoguanidine, in terms of both cell killing and induction of mutation . The rabbit methyltransferase is highly homologous to this enzyme in human, mouse, rat and hamster, but is 26-30 amino acid residues shorter as compared with methyltransferases in other mammalian species . Based on a comparison of the nucleotide sequences for the C-terminal regions of these proteins, we propose that a single base substitution, which would generate a TGA termination codon, was introduced into the sequence for the rabbit enzyme during the process of evolution . Existence of the naturally occurring truncated form of methyltransferase suggests that the longer C-terminal tails of other mammalian methyltransferases may have no significant role in exerting functions of the enzyme in vivo. Biometals, 1994 Apr, 7(2), 149 - 54 HPLC separation of enterobactin and linear 2,3-dihydroxybenzoylserine derivatives: a study on mutants of Escherichia coli defective in regulation (fur), esterase (fes) and transport (fepA); Winkelmann G et al.; Reversed-phase HPLC separation of enterobactin and its 2,3-dihydroxybenzoylserine derivatives was used for a comparative analysis of mutants of Escherichia coli, defective in the regulation of enterobactin biosynthesis (fur), enterobactin transport (fepA) and enterobactin esterase (fes) . A complete separation of all 2,3-dihydroxybenzoylserine compounds was achieved: the monomer (DHBS), the linear dimer (DHBS)2 and trimer (DHBS)3, the cyclic trimer, enterobactin, as well as 2,3-dihydroxybenzoic acid . The production of all these compounds was followed after ethylacetate extraction from acidified culture fluids . Enterobactin was found to be the predominant product in all mutant strains . The mutant strains behaved differently with regard to the breakdown products . All degradation products, such as DHBS, (DHBS)2 and (DHBS)3, were detected in the overproducing fur mutant where both transport and esterase are still functioning, while only the monomer, DHBS, was detected in the fepA mutant and no degradation was found in the esterase-deficient fes mutant . From the pattern of breakdown products it may be inferred that the esterase acts in two different ways, depending on whether transport is functioning or not . Thus, esterolytic cleavage of ferric enterobactin after entering the cells results in a mixture of all three hydrolysis products, i.e . DHBS, (DHBS)2 and (DHBS)3, while cleavage of iron-free enterobactin subsequent to its biosynthesis yields only the monomer . Thus, the results of quantitative HPLC analysis of enterobactin and its breakdown products show that different enterobactin esterase products arise, depending on whether iron is bound to enterobactin or not. Mol Carcinog, 1994 Apr, 9(4), 200 - 9 Influence of DNA repair by ada and ogt alkyltransferases on the mutational specificity of alkylating agents; Roldan-Arjona T et al.; We investigated the influence of the alkyltransferases (ATases) encoded by the ada and ogt genes of Escherichia coli on the mutational specificity of alkylating agents . A new mutational assay for selection of supF- mutations in shuttle-vector plasmids was used . Treating plasmid-bearing bacteria with N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), and ethyl methanesulfonate (EMS) dramatically increased the mutation frequency (from 33-fold to 789-fold) . The vast majority of mutations (89-100%) were G:C-->A:T transitions . This type of mutation increased in ada- (MNU) or ogt- (ENU) bacteria, suggesting that repair of O6-methylguanine by ada ATase and repair of O6-ethylguanine by ogt ATase contribute mainly to the decrease in G:C-->A:T transitions . The analysis of neighboring base sequences revealed an overabundance of G:C-->A:T transitions at 5'-GG sequences . The 5'-PuG bias increased in ATase-defective cells, suggesting that these sequences were not refractory to repair . G:C-->A:T transitions occurred preferentially in the untranscribed strand after in vivo exposure . That this strand specificity was detected even in bacteria devoid of ATase activity (ada- ogt-) and not after in vitro mutagenesis suggests a bias for damage induction rather than for DNA repair . Highly significant differences were found between the in vivo and in vitro incidences of G:C-->A:T substitutions at the two major hotspots, positions 123 (5'-GGG-3'; antisense strand) and 168 (5'-GGA-3'; sense strand) . These results are explained by differences in the probability of formation of stem-loop structures in vivo and in vitro. J Mol Biol, 1994 Apr 1, 237(3), 266 - 74 The Escherichia coli prr region encodes a functional type IC DNA restriction system closely integrated with an anticodon nuclease gene; Tyndall C et al.; The prr locus was originally described as coding a ribonuclease that is activated after phage T4 infection to cut within the anticodon of a specific tRNA, inactivating protein synthesis and thus blocking phage development . Wild-type T4 phage has two genes coding the enzymes polynucleotide kinase and RNA ligase, whose only function seems to be to repair the damage done by the anticodon nuclease . As the only apparent function of the prr ribonuclease is to combat phage infection, it can be considered as an RNA-based restriction enzyme . In non-infected cells, the prr enzyme is kept inactive in a complex with three other proteins which were predicted on the basis of DNA homologies to be the subunits of a type IC DNA restriction and modification system . Unlike other type IC systems so far characterized, prr is chromosomally rather than plasmid coded . However, sequences upstream from prr also have homology with sequences from the plasmid R124 and the prophage P1 . We have now investigated the prr system and shown that it is indeed a bona fide type IC system which we call EcoprrI, and which is active both in vivo and in vitro . The system is fully functional even in the absence of the anticodon nuclease and seems to be a typical type I enzyme . EcoprrI recognizes the sequence CCA(N7)RTGC . One peculiarity is that, with low efficiency, EcoprrI will recognize and methylate variants of its recognition sequence such as CCT(N7)ATGC, which is methylated in one strand of the DNA only. J Mol Biol, 1994 Apr 1, 237(3), 255 - 65 Deletion analysis of the lambda tR1 termination region . Effect of sequences near the transcript release sites, and the minimum length of rho-dependent transcripts; Hart CM et al.; In order to determine how much of the natural sequence is required for function of the lambda tR1 rho-dependent terminator, and to determine the minimum length required, we made two deletion series of a tR1 derivative that contains mostly foreign DNA, encoding C-rich RNA, substituted for the natural upstream sequences of tR1 . We find the minimum transcript length to be 85 to 90 nucleotides, although an additional 10 to 25 nucleotides provide more efficient termination . Sequences as close as ten nucleotides to the release sites could be replaced without destroying termination, although termination efficiency was reduced by substitution of these proximal regions with DNA encoding the cytidine-rich RNA that is active at upstream sites . These results suggest that sequences proximal and distal to release sites have different functions and optimal structures for rho activation . We also show that two potential stem-loop structures in the tR1 region are not essential for terminator function, or for pausing at the release region . The results are consistent with the model that any pause site downstream of DNA encoding unstructured C-rich RNA is a potential rho-dependent terminator. J Exp Med, 1994 Apr 1, 179(4), 1253 - 9 Elimination of interleukin 6 attenuates coagulation activation in experimental endotoxemia in chimpanzees; van der Poll T et al.; The role of interleukin 6 (IL-6) in the toxic sequelae of sepsis is controversial . To assess the part of IL-6 in inflammatory responses to endotoxin, we investigated eight chimpanzees after either a bolus intravenous injection of Escherichia coli endotoxin (n = 4; 4 ng/kg) or after the same dose of endotoxin with a simultaneous bolus intravenous injection of an anti-IL-6 mAb (30 mg; n = 4) . Anti-IL-6 did not affect the induction of the cytokine network (tumor necrosis factor {TNF}, soluble TNF receptors types I and II, and IL-8) by endotoxin, nor did it influence the occurrence of a neutrophilic leukocytosis and neutrophil degranulation, as monitored by the measurement of elastase-alpha 1-antitrypsin complexes . In contrast, anti-IL-6 markedly attenuated endotoxin-induced activation of coagulation, monitored with the plasma levels of the prothrombin fragment F1+2 and thrombin-antithrombin III complexes, whereas activation of fibrinolysis, determined with the plasma concentrations of plasmin-alpha 2-antiplasmin complexes, remained unaltered . We conclude that IL-6 does not have a feedback effect on the release of other cytokines after injection of endotoxin, and that it is not involved in endotoxin-induced neutrophilia or neutrophil degranulation . IL-6 is, however, an important intermediate factor in activation of coagulation in low grade endotoxemia in chimpanzees. J Biol Chem, 1994 Apr 1, 269(13), 9993 - 9 Role of histidine 35 of the S1 subunit of pertussis toxin in the ADP-ribosylation of transducin; Xu Y et al.; Molecular modeling of the S1 subunit (S1) of pertussis toxin with other ADP-ribosylating bacterial exotoxins predicted that histidine 35 (His-35) would residue within the active site of S1 . Recombinant derivatives of S1 (rS1 and the C180 peptide) which contained either a H35Q or H35P mutation were analyzed to determine the role of His-35 in ADP-ribosylation . C180 peptide is a recombinant peptide composed of the amino-terminal 180 amino acids of S1 . Under linear velocity conditions, C180H35Q possessed 2% of wild type C180 peptide activity and C180H35P possessed no detectable activity in the ADP-ribosylation of transducin . The H35Q mutation did not change the affinity of recombinant peptides for NAD or two targets for ADP-ribosylation, transducin, or alpha i3C20, but did lower the kcat in the NAD glycohydrolase and ADP-ribosyltransferase reactions . Neither the H35Q nor H35P mutation reduced the ability of recombinant proteins to be photocross-linked with NAD which was consistent with the His-35 mutations not reducing the affinity for NAD . These data indicate that His-35 does not reduce the affinity of S1 for NAD or transducin, but functions as a catalytic residue in the ADP-ribosylation reaction possibly in a hydrogen bonding capacity. J Biol Chem, 1994 Apr 1, 269(13), 9974 - 85 The HBP-1 family of wheat basic/leucine zipper proteins interacts with overlapping cis-acting hexamer motifs of plant histone genes; Mikami K et al.; The type I element (CCACGTCANCGATCCGCG) is a cis-acting element that is essential for the transcriptional regulation of the wheat histone H3 (TH012) gene . The sequence CCACGTCA in the type I element resembles various plant regulatory elements that share an ACGT core sequence, which can be recognized by different basic/leucine zipper (bZIP) proteins . Here we describe the isolation and characterization of wheat cDNA clones encoding three novel bZIP proteins, designated HBP (histone promoter-binding protein)-1a(1), HBP-1a(c14), and HBP-1b(c1) . These proteins specifically bind to the ACGT core sequence and, together with previously identified HBP-1a(17) and HBP-1b(c38), constitute a protein family, named the HBP-1 family . Based on their structural characteristics and DNA binding specificities, members of the HBP-1 family can be grouped into HBP-1a and HBP-1b subfamilies . The HBP-1a isoforms are characterized by their N-terminal proline-rich domain and a C-terminal bZIP domain, which binds to the CCACGT motif . In contrast, the HBP-1b isoforms have a bZIP domain at the N terminus, which binds to the ACGTCA motif, and a glutamine-rich domain at the C terminus . All members of both subfamilies interact with the CCACGTCA sequence, but their DNA binding specificities and affinities differ . Since HBP-1a isoforms form heterodimers in all pairwise combinations, heterodimer formation among these bZIP proteins may generate an expanded repertoire of regulatory potential for gene expression in plants. J Biol Chem, 1994 Apr 1, 269(13), 9798 - 804 Differential regulation of Hsp70 subfamilies by the eukaryotic DnaJ homologue YDJ1; Cyr DM et al.; In Saccharomyces cerevisiae Ydj1p, a DnaJ homolog, is localized to the cytosol with the Ssa and Ssb Hsp70 proteins . Ydj1p helps facilitate polypeptide translocation across mitochondrial and endoplasmic reticulum membranes (Caplan, A . J., Cyr, D . M., and Douglas, M . G . (1992) Cell 71, 1143-1155) and can directly interact with Ssa1p to regulate chaperone activity (Cyr, D . M., Lu, X., and Douglas, M . G . (1992) J . Biol . Chem . 267, 20927-20931) . In this study, the role of Ydj1p in modulating ATP-dependent reactions catalyzed by Ssa and Ssb Hsp70 proteins has been examined using purified components and compared with that of other Hsp70 homologs BiP and DnaK . Ssa1p, Ssa2p, and Ssb1/2p all formed stable complexes with the mitochondrial presequence peptide, F1 beta(1-51) . ATP alone had only modest effects on polypeptide complex formation with Ssa1p and Ssa2p, but prevented the majority of polypeptide binding to BiP and DnaK . ATP by itself also reduced polypeptide binding to Ssb1/2p to a level that was intermediate between that observed for the Ssa Hsp70 proteins tested and BiP and DnaK . ATP hydrolysis by Ssa1p, Ssa2p, and Ssb1/2p occurred at similar rates . Ydj1p was a potent modulator of the both the ATPase and polypeptide binding activities of Ssa1p and Ssa2p . In contrast, Ydj1p had little effect on the ATPase and polypeptide binding activity of Ssb1/2p . Therefore the chaperone-related activities of Ssa and Ssb Hsp70 proteins exhibit significant differences in sensitivity to ATP and YDJ1p . These data indicate that regulation of Hsp70 activity by DnaJ homologs can be specific . The specificity of interactions between Ydj1p and the Ssa and Ssb Hsp70 proteins observed could contribute in determining the functional specificity of these chaperones in the cytosol . In related experiments, F1 beta(1-51) was found to reduce the extent to which Ydj1p stimulated Ssa1p ATPase activity . This effect correlated with the formation of F1 beta(1-51).Ssa1p complexes . We propose that intramolecular communication between the polypeptide binding, ATPase and DnaJ regulatory domains on Ssa1p plays a role in the regulation of chaperone activity. J Biol Chem, 1994 Apr 1, 269(13), 9714 - 20 Role of folylpolyglutamate synthetase in the metabolism and cytotoxicity of 5-deazaacyclotetrahydrofolate, an anti-purine drug; Kim JS et al.; Chinese hamster ovary (CHO) cells expressing human and Escherichia coli folylpolyglutamate synthetase (FPGS) activities were used as models to study factors regulating the cytotoxicity and metabolism of 5-deazaacyclotetrahydrofolate (DATHF), an anti-purine agent that requires conversion to polyglutamate forms to be a potent inhibitor of its target enzymes . The sensitivity of cells continuously exposed to DATHF was not influenced by FPGS activity . After short term exposure to DATHF, cells expressing higher levels of human FPGS were more sensitive to the drug while cells expressing low levels were resistant . Cells expressing E . coli FPGS solely in the cytosol were resistant to DATHF and accumulated only low levels of the drug . Expression of E . coli FPGS in the mitochondria of these cells restored drug sensitivity and cytosolic drug accumulation . DATHF is extensively metabolized in the mitochondria of mammalian cells and, despite an inability to enter the mitochondria, folylpolyglutamates and DATHF polyglutamates formed in the mitochondria are released into the cytosol without prior hydrolysis . DATHF was not solely an inhibitor of de novo purine biosynthesis but also inhibited glycine synthesis in the cell. J Biol Chem, 1994 Apr 1, 269(13), 9705 - 13 Expression of Escherichia coli folylpolyglutamate synthetase in the Chinese hamster ovary cell mitochondrion; Lin BF et al.; Chinese hamster ovary (CHO) cell transfectants expressing Escherichia coli folylpoly-gamma-glutamate synthetase (FPGS) activity solely in their cytosol lack mitochondrial folylpolyglutamates and are auxotrophic for glycine . Addition of a mammalian mitochondrial leader sequence targeted E . coli FPGS to the mitochondria of these cells . Mitochondrial expression of FPGS restored mitochondrial folylpolyglutamate pools and overcame the glycine requirement . Pteroyltriglutamates functioned as effectively as the longer glutamate chain length folates found in wild type CHO cells in the metabolic cycle of glycine synthesis provided they were located in the mitochondria . Although folypolyglutamates cannot enter the mitochondria, mitochondrial folylpolyglutamates can be released without prior hydrolysis and CHO transfectants expressing E . coli FPGS activity solely in the mitochondria possessed normal cytosolic folylpolyglutamate pools . The proportion of cellular folate in the mitochondrion is governed by competition between mitochondrial and cytosolic FPGS activities. J Biol Chem, 1994 Apr 1, 269(13), 9698 - 704 In vitro assembly of an anion-stimulated ATPase from peptide fragments; Kaur P et al.; The oxyanion-translocating ATPase encoded by the ars operon of plasmid R 773 confers resistance to antimonials and arsenicals in Escherichia coli by extrusion of the oxyanions from the cells . The catalytic subunit, the ArsA protein, is an oxyanion-stimulated ATPase with two nucleotide binding consensus sequences, one in the N-terminal half and one in the C-terminal half of the protein . In this report subclones of the arsA gene were constructed to produce polypeptide fragments of the ArsA protein . By themselves none of the fragments exhibited anion-stimulated ATPase activity . Denaturation and renaturation of mixtures of N- and C-terminal polypeptides that between them comprised an entire ArsA protein resulted in active ATPase complexes. J Biol Chem, 1994 Apr 1, 269(13), 9644 - 50 Gelonin analogs with engineered cysteine residues form antibody immunoconjugates with unique properties; Better M et al.; We engineered the ribosome inactivating-protein gelonin (Gel) to generate a family of Gel analogs, each with a single unpaired cysteine residue . The cysteine sites coincide with surface-accessible loops in the probable three-dimensional structure of Gel, or with the positions of endogenous cysteine residues . In most cases, enzymatic activity in vitro was unaltered by this modification . The rGel analogs were conjugated via their unpaired cysteine residue to the anti-CD5 antibody H65, or to H65 Fab and F(ab')2 . Several rGel analogs formed immunoconjugates that were up to 6-fold more cytotoxic to antigen-bearing cells than those made with linker-modified rGel, whereas others were less potent . In the rat, the in vivo clearance rates of whole antibody conjugates correlated with their relative in vitro disulfide bond stability, and deconjugation to intact antibody and rGel was the predominant clearance mechanism . Fab conjugates to rGel analogs which differed in their in vitro disulfide bond stability had similar serum clearance rates, suggesting that clearance occurs mainly by removal of intact immunoconjugate from the serum, and is less dependent on deconjugation . Our results demonstrate that rGel analogs with a single cysteine at various positions on the solvent exposed surface are produced efficiently in Escherichia coli (>1 g/liter), and that the position of the cysteine greatly influences the potency and stability of the resulting immunoconjugates. J Biol Chem, 1994 Apr 1, 269(13), 9627 - 31 Single amino acid substitutions of alpha 1-antitrypsin that confer enhancement in thermal stability; Kwon KS et al.; A recombinant alpha 1-antitrypsin variant which increased thermal stability was obtained from random mutagenesis followed by screening . The clone was identified as having a single mutation of Phe51-->Cys . Heat deactivation of purified recombinant alpha 1-antitrypsin produced in Escherichia coli revealed that the mutation slowed down the deactivation rate 10-fold at 57 degrees C, increasing thermal stability of recombinant protein to almost that of natural glycosylated plasma form . The mutant protein also exhibited increased stability against denaturant . The urea-induced unfolding monitored by the changes in fluorescence intensity at 360 nm showed that the mutation shifted midpoint of the transition from 1.9 M to 2.8 M . The mutation site is particularly interesting in that some genetic variants mapped at adjacent positions were shown previously to cause aggregation of the polypeptides, while the Phe51-->Cys mutation decreased aggregation rate significantly during heat deactivation . The association rate constant with porcine pancreatic elastase revealed that the mutation did not affect inhibitory activity significantly . The site identified may be critical for regulating stability of alpha 1-antitrypsin . Characterization of various single amino acid substitutions at position 51 suggests that volume and flexibility of hydrophobic side chain at the site are critical factors for enhancing the stability of alpha 1-antitrypsin. J Biol Chem, 1994 Apr 1, 269(13), 9539 - 46 The NH2-terminal fibrin-binding site of fibronectin is formed by interacting fourth and fifth finger domains . Studies with recombinant finger fragments expressed in Escherichia coli; Matsuka YV et al.; The NH2-terminal 29-kDa Fib-1 fragment consisting of the first five finger modules of fibronectin (F1-5) binds reversibly to fibrin and facilitates cross-linking by Factor XIII . To narrow down the fibrin-binding site within this region, we have used recombinant technology to express a number of individual fingers, rF1, rF2, rF3, rF4, and rF5, and their pairs, rF1-2 rF2-3, and rF4-5, as fusion proteins in Escherichia coli . These recombinant fragments were separated from the carrier maltose-binding protein by digestion with human factor Xa or other proteases, and their structural integrity was confirmed by spectroscopic and calorimetric methods . The recombinant F1 and F4-5 exhibited fluorescence-detected melting transitions of the same magnitude and with the same midpoint (Tm) as their natural analogues prepared from Fib-1 by proteolysis . Differential scanning calorimetry experiments further demonstrated that these fragments are properly folded and have compact structures identical to the natural ones . Isolated rF4 melts at a much lower temperature than rF5 or the bimodular fragment rF4-5, indicating the loss of a stabilizing interaction between fingers 4 and 5 . Comparison of fluorescence spectra of individual rF4 and rF5 with that of rF4-5 was also consistent with an interaction that affects the environment of Trp residue(s) . rF2 also melts at a lower temperature than rF3 or rF2-3, suggesting a stabilizing interaction between the second and third fingers as well . When tested on fibrin-Sepharose, only the bimodular fragment rF4-5 was able to bind . All other fragments, including individual fingers 4 and 5, failed to bind . Thus, fibrin binding is not a common property of all fingers . The results indicate that a recognition site for fibrin is located within fingers 4 and 5 . The interaction between these neighboring domains may play an important role in proper orientation of the residues forming this site. J Biol Chem, 1994 Apr 1, 269(13), 9436 - 44 Isolation and characterization of cDNA clones for chloroplast translational initiation factor-3 from Euglena gracilis; Lin Q et al.; A complete cDNA clone encoding Euglena gracilis chloroplast translational initiation factor 3 (IF-3chl) has been obtained . Analysis of the sequence indicates that the IF-3chl mRNA contains the spliced leader found at the 5' end of nuclear encoded mRNAs in E . gracilis . The open reading frame for IF-3chl encodes a 537-amino acid protein . IF-3chl appears to be divided into four domains . The first 140 amino acids correspond to a transit peptide required for the import of IF-3chl into the chloroplast . The mature form of IF-3chl encompasses domains 2-4 and is about twice the size of Escherichia coli IF-3 . The second domain has no homology to other known proteins . It begins with a stretch of 35 residues, of which about 30% are proline . Downstream from this region is a stretch of about 25 amino acids with a repeating (GX)n motif followed by a very acidic region .