Mol Microbiol, 1994 Mar, 11(5), 875 - 84 A locus involved in the regulation of replication in plasmid pSC101; Manen D et al.; The origin of replication of plasmid pSC101 contains three directly repeated sequences RS1, RS2, and RS3 separated by 22 bp from two palindromic sequences, IR1 and IR2, which are partially homologous to the direct repeats . These inverted repeat (IR) sequences overlap the promoter of the repA gene which encodes a protein essential for plasmid replication . We have shown that RepA binds to the RS sites as a monomer and to the IR sites as a dimer . The influence of the IR1 site, and of the DNA segment that separates it from RS3, on plasmid copy number control has been studied in detail . We show that the integrity of IR1 is essential for efficient replication and plasmid stability, the critical site extending to the left of IR1 proper . We also show that the presence of IR1 modifies profoundly the binding properties of purified RepA protein to a segment of DNA containing the RS sequences . IR1 is separated from its homologous site on RS3 by approximately four turns of the DNA helix . Replication is abolished if this distance is increased by half a turn of the helix but it is restored if the distance is increased by a whole turn . These results suggest a DNA looping interaction, in the initiation of replication, between the RepA dimer that binds IR1 and the RepA monomers that bind the RS sequences. Mol Microbiol, 1994 Mar, 11(5), 833 - 9 Family of the major cold-shock protein, CspA (CS7.4), of Escherichia coli, whose members show a high sequence similarity with the eukaryotic Y-box binding proteins; Lee SJ et al.; The cspA is a gene of Escherichia coli, whose expression is specifically induced at low temperatures to a level of 13% of total protein synthesis . The CspA protein consisting of 70 amino acid residues has high sequence similarity with eukaryotic Y-box DNA-binding proteins . We found two independent clones from the Kohara miniset phage collection, which hybridized with a DNA fragment containing cspA . DNA sequencing of these clones confirmed that the two genes are highly homologous to cspA . One designated cspB is mapped at 35 min on the E . coli chromosome and encodes a 71-residue protein with 79% identity to CspA, while the other, cspC, is mapped at 40 min and encodes a 69-residue protein with 70% identity . In addition, a DNA sequence upstream of the clpA gene at 19 min published elsewhere contains an open reading frame for a 74-residue protein with 45% identity to CspA . All csp genes were fused in the coding regions with the lacZ gene, and the expression of beta-galactosidase was examined for these hybrid genes upon cold shock . A similar cold-shock induction to cspA was observed for cspB but not cspC and cspD . These results indicate that E . coli has a family of the cspA gene, some of which are induced by cold shock. Infect Dis Clin North Am, 1994 Mar, 8(1), 77 - 106 Acute gastroenteritis in Latin America; Prado V et al.; In Latin America, acute gastroenteritis remains to be an important cause of morbidity in adults and a major cause of morbidity and mortality in children . A child under 5 years of age belonging to a low income segment of the Latin American population will develop 5 to 10 bouts of diarrhea every year . The bacterial and viral enteropathogens associated with acute diarrhea are reviewed in this article . Updated information on epidemiologic, clinical, and pathogenic aspects for the relevant enteropathogens is discussed with special emphasis on regional data when available . The current diarrheogenic E . coli classification is particularly discussed . A diagnostic approach to acute gastroenteritis for both the clinical and research laboratories in Latin America as well as guidelines for treatment are proposed. J Magn Reson B, 1994 Mar, 103(3), 242 - 6 Conformation of dCDP bound to protein R1 of Escherichia coli ribonucleotide reductase; Allard P et al.; Deoxycytidine 5'-diphosphate (dCDP) is a product and competitive inhibitor of ribonucleoside-diphosphate reductase (EC 1.17.4.1) from Escherichia coli . Its conformation in the enzyme-bound state is of importance for understanding the reaction mechanism . Free and bound dCDP are in fast exchange and the transferred nuclear Overhauser effect in two-dimensional 1H NMR was used to obtain information about interproton distances within bound dCDP . The results are consistent with a model of dCDP with the base in anti conformation and the sugar in S-type puckering, when bound either to the complete enzyme complex or to the large protein subunit alone. Biol Pharm Bull, 1994 Mar, 17(3), 443 - 5 Silymarin and its components are inhibitors of beta-glucuronidase; Kim DH et al.; Silymarin, a commercial crude drug used as a hepatoprotective, was found to inhibit 53% of beta-glucuronidase activity at a final concentration of 0.8 mg/ml . Of three compounds A, silybin and C, which were isolated from silymarin, A and silybin potently inhibited the enzyme activity, followed by C . beta-Glucuronidases of intestinal bacteria, HGU-1 and HGU-2, and E . coli HB101 were noncompetitively inhibited by silybin . beta-Glucuronidase of the feces of a healthy human and of a human with colon cancer were also inhibited by silybin, silymarin and saccharic acid 1,4-lactone at 0.03-0.15 mg/ml . Silymarin and silybin protected the increase in enzyme activity in the serum of the rats treated with CCl4. Biol Pharm Bull, 1994 Mar, 17(3), 395 - 8 Lithium toxicity and Na+(Li+)/H+ antiporter in Escherichia coli; Inaba K et al.; The lithium ion (Li+) shows toxicity against Escherichia coli cells when present in a high concentration in the environment . Since Li+ is extruded from cells via a Na+(Li+)/H+ antiporter, this antiporter must be involved in the detoxification of Li+ . Two Na+(Li+)/H+ antiporters (NhaA system and NhaB system) are known to be present in E . coli . We investigated the properties of the antiporters and the participation of these systems in the detoxification of Li+ using mutants lacking one of the antiporters, or lacking both of them . Although the affinity for Li+ of the two systems was almost the same, the Vmax value for Li+ transport of the NhaA system was about 12 times larger than that of the NhaB system . Wild type cells were unable to grow in the presence of 0.7 M LiCl . Although a wild type cell and a mutant lacking the NhaB system grew in the presence of 0.6 M LiCl, a mutant lacking the NhaA system did not . This second mutant grew in the presence of 0.1 to 0.2 M LiCl . A mutant lacking both the NhaA and NhaB systems could not grow in the presence of 30 mM LiCl. Protein Sci, 1994 Mar, 3(3), 459 - 66 Residues 36-42 of liver RNase PL3 contribute to its uridine-preferring substrate specificity . Cloning of the cDNA and site-directed mutagenesis studies; Vicentini AM et al.; Within the superfamily of homologous mammalian ribonucleases (RNases) 4 distinct families can be recognized . Previously, representative members of three of these have been cloned and studied in detail . Here we report on the cloning of a cDNA encoding a member of the fourth family, RNase PL3 from porcine liver . The deduced amino acid sequence showed the presence of a signal peptide, confirming the notion that RNase PL3 is a secreted RNase . Expression of the cDNA in Escherichia coli yielded 1.5 mg of purified protein/liter of culture . The recombinant enzyme was indistinguishable from the enzyme isolated from porcine liver based on the following criteria: amino acid analysis, N-terminal amino acid sequence, molecular weight, specific activity toward yeast RNA, and kinetic parameters for the hydrolysis of uridylyl(3',5')adenosine and cytidylyl(3',5')adenosine . Interestingly, the kinetic data showed that RNase PL3 has a very low activity toward yeast RNA, i.e., 2.5% compared to pancreatic RNase A . Moreover, using the dinucleotide substrates and homopolymers it was found that RNase PL3, in contrast to most members of the RNase superfamily, strongly prefers uridine over cytidine on the 5' side of the scissile bond . Replacement, by site-directed mutagenesis, of residues 36-42 of RNase PL3 by the corresponding ones from bovine pancreatic RNase A resulted in a large preferential increase in the catalytic efficiency for cytidine-containing substrates . This suggests that this region of the molecule contains some of the elements that determine substrate specificity. Protein Sci, 1994 Mar, 3(3), 419 - 27 Characteristics of a de novo designed protein; Tanaka T et al.; A series of 204 amino acid proteins intended to form TIM (triose phosphate isomerase) barrel structures were designed de novo . Each protein was synthesized by expression of the synthetic gene as a fusion protein with a portion of human growth hormone in an Escherichia coli host . After BrCN treatment, the protein was purified to homogeneity . The refolded proteins are globular and exist as monomers . One of the designed proteins is stable toward guanidine hydrochloride (GuHCl) denaturation, with a midpoint of 2.6 M determined from CD and tryptophan fluorescence measurements . The GuHCl denaturation is well described by a 2-state model . The NMR spectra, the thermal denaturation curves, and the 1-anilino-8-naphthalene sulfonic acid binding imply that the stability of the protein arises mainly from hydrophobic interactions, which are probably of a nonspecific nature . The protein has a similar shape to that of rabbit triosephosphate isomerase, as determined by electron microscopy. Mol Endocrinol, 1994 Mar, 8(3), 325 - 32 Functional analysis of the cysteine residues of activin A; Mason AJ; Site-directed mutagenesis and mammalian cell expression was used to analyze the function of each of the 13 cysteine residues in the human activin A beta-subunit precursor . Substitution of the four cysteine residues in the proregion with alanine residues did not affect the function of the proregion in facilitating the dimerization and secretion of activin A homodimers . A series of activin mutants were constructed in which the nine cysteine residues (amino acids 4, 11, 12, 40, 44, 80, 81, 113, and 115) in the mature 116-amino acid beta-subunit were individually altered to alanine residues . Alanine substitution at either cysteine residues 4 or 12 did not interfere with homodimer formation, but the mutant activin A molecules had reduced biological and receptor binding activity (2- to 3-fold) . Activin A monomers were produced when cysteine mutants 44, 80, and 113 were expressed in tissue culture cells . Monomers of cys mutants 44 and 80 had approximately 2% of the biological and receptor binding activity of wild type activin A . Cys 113 monomers had undetectable levels of biological activity . No detectable activin monomers or dimers were secreted from cells transfected with plasmids containing cys mutants 11, 40, 81, and 115 . The data presented here suggest that a low level of noncovalent dimer formation of cysteine mutant monomers 44 and 80 may explain their low level of biological activity . Therefore, dimer formation is suggested to be an essential prerequisite for high affinity receptor binding and biological potency.(ABSTRACT TRUNCATED AT 250 WORDS) J Lipid Mediat Cell Signal, 1994 Mar, 9(2), 145 - 53 Essential fatty acid-deficient diet modifies PAF levels in stomach and duodenum of endotoxin-treated rats; Autore G et al.; Platelet-activating factor (PAF) is thought to play an important role in pathogenesis of endotoxin shock . Here, using essential fatty acid deficient (EFAD) rats, we have evaluated changes in mean arterial blood pressure, PAF levels and damage in both stomach and duodenum following intravenous administration of endotoxin (LPS) . In EFAD rats the second phase of LPS-induced hypotension was strongly reduced . Similarly, PAF levels in stomach and duodenum of EFAD rats were also reduced and correlated to the diminished damage . Our study confirms a direct involvement of PAF in LPS-induced gastrointestinal damage. Microbiology, 1994 Mar, 140 ( Pt 3), 651 - 7 A newly isolated lectin from the plant pathogenic fungus Sclerotium rolfsii: purification, characterization and role in mycoparasitism; Inbar J et al.; A novel lectin was isolated and purified from the culture filtrate of the soilborne plant pathogenic fungus Sclerotium rolfsii by anion-exchange chromatography using a DEAE-Sepharose column . The lectin came through the column with the flow-through, whereas all the non-agglutinating proteins present in the crude preparation remained bound to the column until elution in a NaCl gradient . SDS-PAGE analysis of the agglutinating fraction revealed a single band corresponding to a protein with a molecular mass of approximately 45 kDa . Agglutination of Escherichia coli cells by the purified lectin was not inhibited by any of the mono- or disaccharides tested, whereas the glycoproteins mucin and asialomucin did inhibit agglutination . Proteases, as well as 1,3-beta-glucanase, were found to be totally destructive to agglutination activity, indicating that both protein and 1,3-beta-glucan are necessary for agglutination . Using a biomimetic system based on binding of the lectin to the surface of inert nylon fibres revealed that the presence of the purified agglutinin on the surface of the fibres specifically induced mycoparasitic behaviour in Trichoderma harzianum . Trichoderma formed tightly adhering coils, which were significantly more frequent with the purified agglutinin-treated fibres than with untreated ones or with those treated with non-agglutinating extracellular proteins from S . rolfsil . Other mycoparasite-related structures, such as appressorium-like bodies and hyphal loops, were only observed in the interaction between T . harzianum and the purified agglutinin-treated fibres. Yonsei Med J, 1994 Mar, 35(1), 77 - 83 Adaptive response to ionizing radiation induced by low dose of gamma ray in human hepatoma cell lines; Seong J et al.; When cells are exposed to a low dose of a mutagenic or clastogenic agent, they often become less sensitive to the effects of a higher dose administered subsequently . Such adaptive responses were first described in Escherichia coli . Studies on mammalian cells have been limited to human lymphocytes exposed to low doses of an alkylating agent . In this study, the adaptive response to 1 cGy of gamma rays was investigated in human tumor cells using two human hepatoma cell lines, Hep G2 and Hep 3B . Experiments were carried out by delivering 1 cGy followed by 50 cGy of gamma radiation and chromatid breaks were scored as an endpoint . The results of this study indicate that prior exposure to 1 cGy of gamma rays reduces the number of chromatid breaks induced by subsequent higher doses (50 cGy) . The time necessary for the expression of the adaptive response was determined by varying the time interval between the two doses from 1 hour to 72 hours . In G2 chromatids, the adaptive response was observed both at short time intervals, as early as 1 hour, and at long time intervals . In S chromatids, however, the adaptive response was shown only at long time intervals . When 3-aminobenzamide, an inhibitor of poly (ADP-ribose) polymerase, was added after 50 cGy, adaptive responses were abolished in all the experimental groups . Therefore, it is suggested that the adaptive response can be observed in human hepatoma cell lines, which is first documented through this study.(ABSTRACT TRUNCATED AT 250 WORDS) Pediatr Pathol, 1994 Mar-Apr, 14(2), 207 - 12 Cutaneous bronchogenic cyst of the back: a case report and review of the literature; Tresser NJ et al.; Cutaneous and subcutaneous cysts with ciliated pseudostratified columnar (respiratory) epithelium present a diagnostic dilemma . We report a case of a bronchogenic cyst occurring on the back . The differential diagnosis includes branchial cleft cyst, thyroglossal duct cyst, cutaneous ciliated cyst, and mature cystic teratoma . We review reports of extrapulmonary bronchogenic cysts and discuss their possible embryology. J Rheumatol, 1994 Mar, 21(3), 484 - 8 Experimental induction of arthritis in rats immunized with Escherichia coli 0:14 lipopolysaccharide; Noyori K et al.; OBJECTIVE . Escherichia coli 0:14 (E . coli 0:14) induces arthritis in rabbits, mice and rats . This study was designed to investigate the effects of lipopolysaccharide (LPS) on the rat arthritis model induced by systemic injections of E . coli 0:14 . METHODS . The induction of arthritis in the ankles of rats immunized by subcutaneous injections with heat-killed E . coli 0:14 and its LPS was studied . The appearance and levels of serum IgM rheumatoid factor-like substance (RFLS) was also investigated . The localization of interleukin 1 (IL-1) and LPS in the ankle joints were investigated immunohistochemically . RESULTS . The induction rate of arthritis in rats immunized with LPS was the same as that in rats immunized with E . coli . LPS and IL-1 were detected in synovial cells, infiltrating cells and some cells on pannus in arthritic joints . Anti-LPS IgM levels in rats immunized with E . coli were as high as those in rats immunized with LPS . RFLS levels in rats immunized with LPS increased more gradually than those in rats immunized with E . coli . CONCLUSION . Our findings suggest that LPS induces arthritis resembling rheumatoid arthritis in rats . The detection of IL-1 in synovial cells in conjunction with LPS suggests that local stimulation of IL-1 production may play an important role in the induction of this experimental arthritis. J Appl Physiol, 1994 Mar, 76(3), 1285 - 92 Effects of hypercapnia on metabolism, temperature, and ventilation during heat and fever; Sachdeva U et al.; We wished to determine whether 1) acute hypercapnia in cats changes metabolic heat production to affect temperature regulation and 2) heat exposure and fever affect the normal response . The effects of breathing 4% CO2 on O2 consumption (VO2), rectal temperature (Tre), and ventilation (V) were measured in five conscious resting cats . Cats were exposed to a normal (24-27 degrees C) chamber temperature (Ta) and a warm (33-34 degrees C) chamber Ta . Fever was produced by intravenous injection of Escherichia coli endotoxin (0.02 microgram/kg) at a normal Ta . In normothermic cats, hypercapnia decreased VO2 by approximately 40%, despite an increased V (approximately 100%), but Tre decreased only transiently and slightly compared with studies in which air was breathed throughout . During heat studies, average V was elevated but VO2 was markedly lower than at the normal Ta; Tre gradually increased . Hypercapnia combined with heat did not cause additional increases in VO2, nor did it cause a decrease in VO2, as at a normal Ta; however, the rate of rise of Tre during heat was slowed by hypercapnia . During febrigenesis, hypercapnia prevented the transient increase in VO2 observed when air was breathed and delayed the rate of rise in Tre . As Tre was falling in fever, hypercapnia depressed VO2 but did not affect Tre compared with fever studies in which air was breathed . Unlike heat exposure, hypercapnia had a further additive stimulatory effect on the increase in V at the onset of fever, and it increased V during the falling phase of fever when V had returned to control levels.(ABSTRACT TRUNCATED AT 250 WORDS) Genetics, 1994 Mar, 136(3), 709 - 19 DNA sequence effects on single base deletions arising during DNA polymerization in vitro by Escherichia coli Klenow fragment polymerase; Wang FJ et al.; Most single base deletions detected after DNA polymerization in vitro directed by either Escherichia coli DNA polymerase I or its Klenow fragment are opposite Pu in the template . The most frequent mutations were previously found to be associated with the consensus template context 5'-PyTPu-3' . In this study, the predictive power of the consensus sequence on single base deletion frequencies was directly tested by parallel comparison of mutations arising in four related DNAs differing by a single base . G, a deletion hotspot within the template context 5'-TTGA-3', was substituted by each of the 3 other bases . Previous studies had shown that deletions opposite the G were frequent but that deletions opposite its neighboring A were never detected . Based on the predictions of the consensus, the substitution of T for G should produce frequent deletions opposite the neighboring A due to its new 5'-TTTA-3' template context . This prediction was fulfilled; no deletions of this A were detected in the other templates . The consensus further predicted that deletions opposite template C would be lower than those opposite either A or G at the same site and this prediction was also fulfilled . The C substitution also produced a new hotspot for 1 bp deletions 14 bp away . The new hotspot depends on quasi-palindromic misalignment of the newly synthesized DNA strand during polymerization; accurate, but ectopically templated synthesis is responsible for this mutagenesis . Mutations templated by quasi-palindromic misalignments have previously been recognized when they produced complex sequence changes; here we show that this mechanism can produce frequent single base deletions . The unique stimulation of misalignment mutagenesis by the C substitution in the template is consistent with the singular ability of C at that site to contribute to extended complementary pairing during the DNA misalignment that precedes mutagenesis. FEMS Immunol Med Microbiol, 1994 Mar, 8(3), 197 - 206 Efficient production of active and mutated ADP-ribosyltransferase (S1) of pertussis toxin using affinity expression cassette polymerase chain reaction; Raupach B et al.; We describe an efficient, general approach for cloning, expression and purification of heterologous proteins in Escherichia coli host strains . The affinity expression cassette polymerase chain reaction (AEC-PCR) allows the insertion of virtually any coding sequence in suitable expression vectors . For ease of purification of the (over)produced protein the gene expression cassettes are engineered by specifically designed oligonucleotide primers in the polymerase chain reaction (PCR) to contain either 3' or 5' additional nucleotides coding for a short amino acid sequence constituting an 'affinity block' fused to either end of the protein . This allows a one-step purification by affinity chromatography . In combination with PCR-mediated site-specific mutagenesis this approach is a highly efficient way for the expression and isolation of proteins and for the analysis and dissection of functional domains . The application of AEC-PCR is demonstrated by the cloning, production and purification of the native, active and the mutagenized, inactive ADP-ribosyltransferase (S1 subunit) of pertussis toxin . In this example, a string of six histidines has been engineered to either the N-terminal or the C-terminal end of the protein to serve as 'affinity block' for the isolation of the recombinant protein by immobilized metal ion affinity chromatography (IMAC) . Thus, the S1 subunit can now be produced in sufficient quantities to facilitate further studies on its immunological and molecular properties. Cell Mol Biol (Noisy-le-grand), 1994 Mar, 40(2), 147 - 57 Light and electron microscopic immunolocalization of L-asparaginase in the rat central nervous system; Martinez-Rodriguez R et al.; The investigation on the localization of L-asparaginase, the enzyme involved in the synthesis of L-aspartic acid, has been carried out using the immunohistochemical method . Antibodies against this enzyme were obtained immunizing BALB/c mice with purified Escherichia coli L-asparaginase . Light microscopic observation revealed positive immunoreactivity in the great majority of neurons and glial cells, and electron microscopic analysis demonstrated immunological localization of the enzyme in the cytosol . The ubiquitous distribution of L-asparaginase suggests its involvement in many important functions of the central nervous system. Berl Munch Tierarztl Wochenschr, 1994 Mar, 107(3), 82 - 5 {Therapy of edema disease/coli complex of weaned piglets--where do we stand today?}; Bolcskei A et al.; The paper gives an up to date review of our knowledge concerning etiology, clinics and pathogenesis of the edema disease . A therapy protocol is given, an extensive list of original literature for further study is presented. Zentralbl Veterinarmed B, 1994 Mar, 41(1), 49 - 59 Detection of fimbrial and toxin genes in Escherichia coli and their prevalence in piglets with diarrhoea . The application of colony hybridization assay, polymerase chain reaction and phenotypic assays; Ojeniyi B et al.; Two different genotypic methods, colony hybridization and polymerase chain reaction (PCR), were applied to detect enterotoxin, verotoxin and fimbrial genes in 708 Escherichia coli (E . coli) strains from piglets with diarrhoea . The results were compared with those obtained by phenotypic methods . DNA fragments specific for each of the enterotoxins LT, STa and STb, the verotoxins VT1, VT2 and VT2v, and for each of the fimbrial genes K88 (F4), K99 (F5), 987P (F6), F41 and F107, respectively, were used as probes in a colony hybridization assay of the E . coli strains . A PCR assay was used as genotypic test for the verotoxin gene . An Enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies was performed to test for the presence of K88 and K99 fimbriae, and a Vero cell test was performed to test for verotoxin production . Toxin detection kits were applied to detect the E . coli heat-labile enterotoxin (LT) and the heat-stable (STa) enterotoxin . The correlation between the results obtained by genotypic and phenotypic methods was 97.7-100% . The prevalence of the different fimbrial and toxin genes in E . coli strains from piglets with neonatal and postweaning diarrhoea were recorded . The verotoxin and the fimbrial F107 genes were found to be more frequent in postweaning E . coli strains than in neonatal E . coli strains. Curr Biol, 1994 Mar 1, 4(3), 249 - 51 DNA repair . DNA surveillance defect in cancer cells; Lindahl T; A defect in a protein involved in DNA-mismatch correction accounts for a common type of hereditary colon cancer, adding to the evidence that DNA repair is central to counteracting the transformation of human cells. Curr Biol, 1994 Mar 1, 4(3), 234 - 7 Signal transduction . Bringing the eukaryotes up to speed; Swanson RV et al.; 'Two-component' signalling systems have long been known to be used very generally in bacterial signal transduction; at last, related proteins have been discovered in eukaryotes. Bioorg Med Chem, 1994 Mar, 2(3), 153 - 68 Evidence for an intermediate in the enzymatic formation of uroporphyrinogen III; Pichon C et al.; Evidence for an azafulvene intermediate in the enzymatic formation of Uroporphyrinogen III has been obtained . Using conditions to slow down the enzyme activity (high pH, low temperature), the transient species was trapped with ammonium ions as aminomethylbilane and with sodium borohydride as methylbilane, and observed by 13C-NMR. Biochem Mol Biol Int, 1994 Mar, 32(3), 419 - 27 Inhibition of metal-catalyzed oxidation systems by a yeast protector protein in the presence of thiol; Kwon SJ et al.; A protector protein from Saccharomyces cerevisiae prevented the inactivation of enzyme and oxidative damage to protein and DNA caused by a thiol/Fe3+/O2 metal-catalyzed oxidation (MCO) system but not when thiol was replaced by ascorbate . In the presence of a reduced thiol such as dithiothreitol and reduced glutathione, however, the protector protein prevented inactivation of E . coli glutamine synthetase against a MCO system comprised of ascorbate and Fe3+ . The protector protein also inhibited the fragmentation of protein, incorporation of carbonyl groups into protein, strand breaks in pBluescript plasmid DNA, and the formation of 8-hydroxydeoxyguanosine in calf thymus DNA when induced by either the thiol/Fe3+ system or the ascorbate/Fe3+ system supplemented with dithiothreitol . These results suggest that antioxidant activity of protector protein against a MCO system requires thiol as a reducing equivalent to restore its catalytic activity. Protein Sci, 1994 Mar, 3(3), 476 - 81 Regeneration of catalytic activity of glutamine synthetase mutants by chemical activation: exploration of the role of arginines 339 and 359 in activity; Dhalla AM et al.; In order to understand the nature of ATP and L-glutamate binding to glutamine synthetase, and the involvement of Arg 339 and Arg 359 in catalysis, these amino acids were changed to cysteine via site-directed mutagenesis . Individual mutations (Arg-->Cys) at positions 339 and 359 led to a sharp drop in catalytic activity . Additionally, the Km values for the substrates ATP and glutamate were elevated substantially above the values for wild-type (WT) enzyme . Each cysteine was in turn chemically modified to an arginine "analog" to attempt to "rescue" catalytic activity by covalent modification; 2-chloroacetamidine (CA) (producing a thioether) and 2,2'-dithiobis (acetamidine)(DTBA) (producing a disulfide) were the reagents used to effect these chemical transformations . Upon reaction with CA, both R339C and R359C mutants showed a significant regain of catalytic activity (50% and 70% of WT, respectively) and a drop in Km value for ATP close to that for WT enzyme . With DTBA, chemically modified R339C had a greater kcat than WT glutamine synthetase, but chemically modified R359C only regained a small amount of activity . Modification with DTBA was quantitative for each mutant and each modified enzyme had similar Km values for both ATP and glutamate . The high catalytic activity of DTBA-modified R339C could be reversed to that of unmodified R339C by treatment with dithiothreitol, as expected for a modified enzyme containing a disulfide bond . Modification of each cysteine-containing mutant to a lysine "analog" was accomplished using 3-bromopropylamine (BPA).(ABSTRACT TRUNCATED AT 250 WORDS) Genetics, 1994 Mar, 136(3), 721 - 30 Insertion sequence-related genetic variation in resting Escherichia coli K-12; Naas T et al.; Bacterial subclones recovered from an old stab culture of Escherichia coli K-12 revealed a high degree of genetic diversity, which occurred in spite of a very reduced rate of propagation during storage . This conclusion is based on a pronounced restriction fragment length polymorphism (RFLP) detected upon hybridization with internal fragments of eight resident insertion sequences (IS) . Genetic diversity was dependent on the IS considered and, in many cases, a clear consequence of IS transposition . IS5 was particularly active in the generation of variation . All subclones in which IS30 had been active testify to a burst of IS30 transposition . This was correlated with a loss of prototrophy and a reduced growth on rich media . A pedigree of the entire clone could be drawn from the RFLP patterns of the subclones . Out of 118 subclones analyzed, 68 different patterns were found but the putative ancestral population had disappeared . A few patterns were each represented by several subclones displaying improved fitness . These results offer insights into the role of IS elements in the plasticity of the E . coli genome, and they further document that enzyme-mediated DNA rearrangements do occur in resting bacterial cultures. J Nat Prod, 1994 Mar, 57(3), 339 - 47 Further butenolides from the Caribbean octocoral Pterogorgia citrina; Rodriguez AD et al.; Seven fatty acid derivatives containing one or two butenolide moieties along with ancepsenolide {1}, a known bis-butenolide of marine origin, were isolated from the Caribbean gorgonian Pterogorgia citrina . The structures of the new metabolites were elucidated on the basis of spectroscopic data and were confirmed upon chemical conversion to either ancepsenolide or to its homolog, homoancepsenolide {4}. Vet Immunol Immunopathol, 1994 Mar, 40(3), 253 - 74 Protective antibody titres and antigenic competition in multivalent Dichelobacter nodosus fimbrial vaccines using characterised rDNA antigens; Raadsma HW et al.; The relationship between K-agglutination antibody titres and protection against experimental challenge with Dichelobacter nodosus, the effect of increasing the number of D . nodosus fimbrial antigens, and the importance of the nature of additional antigens in multivalent vaccines on antibody response and protection against experimental challenge with D . nodosus were examined in Merino sheep . A total of 204 Merino sheep were allocated to one of 12 groups, and vaccinated with preparations containing a variable number of rDNA D . nodosus fimbrial antigens . The most complex vaccine contained ten fimbrial antigens from all major D . nodosus serogroups, while the least complex contained a single fimbrial antigen . In addition to D . nodosus fimbrial antigens, other bacterial rDNA fimbrial antigens (Moraxella bovis Da12d and Escherichia coli K99), and bovine serum albumin (BSA) were used in some vaccines . Antibody titres to fimbrial antigens and BSA were measured by agglutination and ELISA tests, respectively . Antibody titres were determined on five occasions (Weeks 0, 3, 6, 8, and 11 after primary vaccination) . All sheep were exposed to an experimental challenge with virulent isolates of D . nodosus from either serogroup A or B, 8 weeks after primary vaccination . For D . nodosus K-agglutinating antibody titres, a strong negative correlation between antibody titre and footrot lesion score was observed . This relationship was influenced by the virulence of the challenge strain . Increasing the number of fimbrial antigens in experimental rDNA D . nodosus fimbrial vaccines resulted in a linear decrease in K-agglutinating antibody titres to individual D . nodosus serogroups . Similarly, a linear decrease in protection to challenge with homologous serogroups was observed as the number of D . nodosus fimbrial antigens represented in the vaccine increased . The reduction in antibody titres in multicomponent vaccines is thought to be due to antigenic competition . The level of competition between individual antigens is not constant and appears to be related to the immunodominance (nature) of the competing antigens . Both BSA ELISA, and M . bovis K-agglutinating antibody titres were adversely affected by the presence of two D . nodosus fimbrial preparations, whereas the antigenicity of E . coli K99 was unchanged by the presence of two additional D . nodosus antigens . Further studies are required to determine the step(s) in the immune response which are influenced by antigenic competition . Our results suggest that antigen presentation, particularly following primary vaccination, is the step most strongly influenced by antigenic competition. Biochem Cell Biol, 1994 Mar-Apr, 72(3-4), 132 - 42 Analysis of the lipoprotein binding site of rat liver membranes; Adam L et al.; Intermediate density lipoproteins (IDL) were shown to bind to high- and low-affinity binding sites on rat liver membranes . The low-affinity sites were named lipoprotein binding sites (LBS), since they bind all classes of lipoproteins . This study was undertaken to further characterize the interaction of 125I-labelled IDL with the LBS of rat liver membranes to determine the chemical nature of the LBS . We found that the binding of IDL to the LBS is insensitive to EDTA and sensitive to heparin and that it is present on plasma membranes . Also, membranes were pretreated with various enzymes that have an effect on the membrane constituents, and the activity of the LBS on these treated membranes was determined . Our results reveal that the LBS of rat liver membranes is insensitive to heparinase I, chondroitinase ABC, and phospholipase C, while it is partially sensitive to phospholipase A2 and sensitive to proteases and heat . Rat liver membrane proteins were solubilized with Triton X-100, reconstituted in liposomes, and analyzed for their ability to bind lipoproteins . 125I-labelled IDL were shown to bind to high- and low-affinity sites that are similar, in affinity and specificity, to the ones observed with intact rat liver membranes, indicating that a LBS activity is detectable on these liposomes . We found that the binding capacity of low-affinity sites in liposomes containing either no protein or containing proteins solubilized from Escherichia coli membranes is five times weaker than low-affinity sites in liposomes containing liver membrane proteins . Thus, a protein solubilized from rat liver membranes has LBS activity when reconstituted in liposomes . Taken altogether our results provide new information on the binding of IDL to the LBS and indicate that the LBS activity is in part mediated by a protein . Thus, the LBS appears to be a bona fide receptor. Fiziol Zh, 1994 Mar-Apr, 40(2), 100 - 3 {Morphological and functional status of mast cells in the late stage of acute infectious inflammation}; Tatarko SV et al.; The model of acute E . coli-induced infectious peritonitis in rats has been used to show that the late reparative stage of acute inflammation is followed by repopulation and regranulation of mast cells . The number of mast cells in the peritoneal fluid and mesrnterium vas resuscitated at the 90th day . At the same time regranulation of peritoneal mast cells was retarded in comparison with that of mast cells of the mesenterium and was not yet over by the 120th day . The increased degranulation of mast cells and free histamine content in the peritoneal fluid and mesenterium and histamine concentration in the blood testified to probable active role of mast cells in lste reparative events on the site of scute inflammation. Shock, 1994 Mar, 1(3), 217 - 20 The catabolism of apolipoprotein B from very low density lipoprotein and triglyceride-rich lipoprotein remnants in fasted septic rats; Phetteplace H et al.; The catabolism of apolipoprotein B (apo B) from very low density lipoproteins (VLDL) and triglyceride-rich lipoprotein (TRL) remnants in fasted control (C) and Escherichia coli-treated septic (S) rats was measured . In Experiment 1, {125I}VLDL was injected into rats from C and S groups . Blood was collected from 0 to 60 min postdose and apo B was isolated and counted for radioactivity . No significant difference in the half-life (t1/2) or fractional catabolic rate (FCR) of apo B was observed in the VLDL fraction . In Experiment 2, {125I}TRL remnants were injected into rats from C and S groups . Blood was collected from 0 to 90 min postdose and apo B isolated . The t1/2 was 2.5 times longer in the S compared with the C and the FCR was slower in the S rats . These experiments suggest that S rats do not clear triglyceride-rich lipoproteins as easily as normal-sized lipoproteins. Shock, 1994 Mar, 1(3), 196 - 200 Effects of sodium bicarbonate on striated muscle metabolism and intracellular pH during endotoxic shock; Bollaert PE et al.; The effects of HCO3Na load on acid-base balance and muscle intracellular bioenergetics have been investigated using 31P-magnetic resonance spectroscopy in an experimental model of endotoxinic shock . Anesthetized, mechanically ventilated, and paralyzed rats (n = 16) were given an intravenous bolus of Escherichia coli lipopolysaccharide (15 mg/kg) . When shock was established they were randomly assigned to receive either HCO3Na intravenously (2 mmol/kg in 2 min) or an equimolar saline injection . Lipopolysaccharide induced a significant decrease in the levels of mean arterial pressure (58 +/- 6 vs . 120 +/- 8 mmHg), arterial pH (7.20 +/- .03 vs . 7.35 +/- .01), intracellular pH (6.86 +/- .04 vs . 7.08 +/- .01), a marked hyperlactatemia (7 +/- 3 vs . 1.2 +/- .2 mmol/L) and a drop in the phosphocreatine-inorganic phosphate ratio . In the bicarbonate-loaded rats, mean arterial pressure further decreased whereas it remained unchanged in the saline group . Bicarbonate increased arterial pH and PaCO2 transiently . In the saline group, arterial pH decreased and PaCO2 remained stable . In both groups, intracellular pH and high energy phosphates had a similar evolution . In this model of septic shock, partial correction of arterial pH using HCO3Na did not reduce the metabolic cellular injury in skeletal muscle . Based on these results, HCO3Na may be of limited therapeutic value in severe septic metabolic acidosis. Shock, 1994 Mar, 1(3), 184 - 7 Endothelin-stimulated monocyte supernatants enhance neutrophil superoxide production; Huribal M et al.; Endothelin-1 (ET-1) is a vasoconstrictive peptide released by ischemic/injured endothelium which increases intracellular ionized calcium {Ca2+}i in vascular smooth muscle . Previous work from this lab has shown that ET-1 also increases human peripheral blood monocyte {Ca2+}i, and that 24 h incubation of monocytes with 10(-9) M ET-1 causes production of prostaglandin E2 and interleukin-6 . In these studies, ET-1-stimulated monocyte supernatants were evaluated for their effect on neutrophil superoxide production . While ET-1 alone had no direct effect, incubation of neutrophils for 20 min in ET-1-stimulated monocyte supernatants resulted in a 10-fold increase in superoxide production over basal levels, 44% as much superoxide production as induced by peptide N-formyl-methionyl-leucyl-phenylalanine (N = 6, p < .001) . Monocyte supernatants were analyzed for interleukin-8 (IL-8 or neutrophil activation protein) content by radioimmunoassay . ET-1-stimulation resulted in production of 54% as much IL-8 as lipopolysaccharide controls (N = 6, p < .001) . While a number of monokines can activate neutrophils, IL-8 has been shown to be a potent neutrophil activator as well as a superoxide primer . Therefore, ET-1-treated monocytes probably upregulate neutrophil superoxide production via a mechanism which includes IL-8. Nat Struct Biol, 1994 Mar, 1(3), 186 - 94 Structure of and kinetic channelling in bifunctional dihydrofolate reductase-thymidylate synthase; Knighton DR et al.; The bifunctional enzyme dihydrofolate reductase-thymidylate synthase catalyses both the reductive methylation of 2'-deoxyuridylate and the subsequent reduction of dihydrofolate to yield 2'-deoxythymidylate and tetrahydrofolate at two spacially discrete sites situated on different protein domains . The X-ray structure of dihydrofolate reductase-thymidylate synthase from Leishmania major indicates that transfer of dihydrofolate between these sites does not occur by transient binding at both sites but rather by movement of dihydrofolate across the surface of the protein . The enzyme has an unusual surface charge distribution that could account for this channelling of dihydrofolate between active sites. Gene Ther, 1994 Mar, 1(2), 122 - 6 In vivo adenovirus-mediated gene transfer into ocular tissues; Mashhour B et al.; Replication-deficient adenoviruses have been successfully used to transfer foreign DNA into a variety of cells including post-mitotic cells, in vivo . In the eyes, most of the cells are quiescent or slowly dividing . They constitute the obligatory targets of gene transfer for a number of ocular diseases that have been elucidated at the molecular level and are potential targets for gene therapy . We have therefore analysed the ability of an adenovirus vector to transfer in vivo the Escherichia coli lacZ gene into ocular cells of mice . Injection of up to 3 x 10(7) p.f.u . into the vitreous body, the anterior chamber or the peribulbar space, did not result in any detectable cytopathic effect and was associated with endocytosis of viral particles in corneal endothelial, photoreceptor, bipolar, ganglionic and oculomotor muscle cells, depending on the administration route . At the viral titer used (3 x 10(7) p.f.u.), the expression was detected for at least 50 days . These results open new prospects for the treatment of some retinal hereditary disorders and acquired corneal or retinal alterations due to inflammatory disease. Shock, 1994 Mar, 1(3), 159 - 65 Comparative effects of 7% NaCl in 6% dextran 70 and 0.9% NaCl on oxygen transport in endotoxemic dogs; Weeren FR et al.; We compared the effects of 7% NaCl in 6% dextran 70 (HSD) and 0.9% NaCl (IS) resuscitation of endotoxic dogs on hemodynamic and cardiorespiratory parameters and the oxygen consumption-delivery relationship . Escherichia coli endotoxin (3 mg.kg-1, intravenously) was infused over 5 min into 13 paralyzed, chloralose-anesthetized, splenectomized dogs . Six additional dogs received a sham endotoxin infusion (saline) and served as controls . After 30 min, the endotoxic animals were resuscitated to 150% of their baseline cardiac output (CO) and maintained at this CO for 30 min using 7% NaCl in 6% dextran 70 (HSD at 1 ml.kg-1.min-1; n = 7) or 0.9% NaCl (IS at 4 ml.kg-1.min-1; n = 6) . Oxygen consumption (VO2), measured by indirect calorimetry, hemodynamic parameters, and oxygen delivery (DO2), improved in similar temporal patterns in both groups during resuscitation and VO2 reached steady-state values . Oxygen delivery, VO2, mean arterial pressure, and cardiac output did not significantly differ between groups at the end of resuscitation, but VO2 increased significantly from baseline values only in the HSD group . The total volume of HSD administered averaged 10.0 +/- 0.2 ml.kg-1 which was significantly less than the volume of IS, which averaged 67.2 +/- 9.3 ml.kg-1 . Incremental hemorrhages (2-5 ml.kg-1) were then performed in all dogs to determine the oxygen consumption-delivery relationship and the critical level of oxygen delivery (DO2Crit) . The average DO2Crit values of the HSD, IS, and control groups were 9.42, 9.15, and 6.82 ml.min-1.kg-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) Bioorg Med Chem, 1994 Mar, 2(3), 169 - 79 Sequence specific cleavage of messenger RNA by a modified ribonuclease H; Ma WP et al.; Ribonuclease H (RNase H) is an endonuclease that cleaves only the RNA strand of an RNA-DNA hybrid to produce 5'-phosphate and 3'-hydroxy termini and lacks useful sequence specific recognition properties . A mutant form of the E . coli enzyme has been prepared that is suited for selective chemical modification at a site proximal to the substrate binding region . The chemical derivatization involves the formation of a disulfide linkage to a modified octadeoxyribonucleotide . The conjugate retains only 0.3% of the normal sequence independent RNase H activity demonstrating that substrate recognition can be modulated by a covalent appendage . A beta-globin RNA transcript containing a sequence complementary to that of the octadeoxyribonucleotide was cleaved in a catalytic fashion to two products upon treatment with the conjugate . The selectivity in the phosphodiester bond cleavage mediated by the conjugate was found to be different than that displayed by the nonderivatized enzyme . These results demonstrate the potential of semi-synthetic RNase H conjugates for mechanistic studies and their application as RNA targeted diagnostic or therapeutic agents. J Physiol Pharmacol, 1994 Mar, 45(1), 41 - 53 Generation of nitric oxide from nitrovasodilators modulates the release of histamine from mast cells; Masini E et al.; The effect of organic and inorganic nitrovasodilators (sodium nitroprusside; 3-morpholinosydnonimine; glyceryl trinitrate; isosorbide dinitrate; sodium nitrite, was studied on the release of histamine evoked by compound 48/80 and calcium ionophore A 23187 in isolated purified rat serosal mast cells . All the compounds tested were capable of significantly reducing the release of histamine in a concentration-dependent fashion, at different levels of potency . This effect was reverted by oxyhaemoglobin . The inhibitory effect of glyceryl trinitrate on the release of histamine was potentiated in cells taken from animals pretreated with Escherichia coli lipopolysaccharide, and decreased by NG-nitro-L-arginine methyl ester . Glyceryl trinitrate and isosorbide dinitrate concentration-dependently increase the generation of nitric oxide by rat serosal mast cells . The inhibitory effect of glyceryl trinitrate and isosorbide dinitrate on the release of histamine from mast cells was potentiated by N-acetylcysteine, which significantly increases the generation of nitric oxide by mast cells . It is concluded that nitrovasodilators inhibit the release of mast cell histamine through the generation of nitric oxide . The effect may be relevant in considering the perivascular location of mast cells and the role played by these cells in cardiovascular pathophysiology. Mol Microbiol, 1994 Mar, 11(6), 1085 - 98 Interaction of the Chlamydia trachomatis histone H1-like protein (Hc1) with DNA and RNA causes repression of transcription and translation in vitro; Pedersen LB et al.; The 18 kDa histone H1-like protein from Chlamydia trachomatis (Hc1) is a DNA-binding protein thought to be involved in condensation of the chlamydial chromosome during late stages in the chlamydial life cycle . Expression of Hc1 in Escherichia coli results in an overall relaxation of DNA and severely affects DNA, RNA and protein synthesis . We have analysed the interaction of Hc1 with single-stranded DNA and RNA by Southwestern and Northwestern blotting . Furthermore, we show that purified, recombinant Hc1 dramatically affects transcription and translation in vitro at physiologically relevant concentrations . These results were found to coincide with the formation of condensed Hc1-DNA and Hc1-RNA complexes as revealed by agarose gel electrophoresis and electron microscopy . The implications of these results for possible functions of Hc1 in vivo are discussed. Mol Microbiol, 1994 Mar, 11(5), 919 - 31 Coupling between mRNA synthesis and mRNA stability in Escherichia coli; Chow J et al.; Transiently stable products derived from the endonuclease cleavage of transcripts from the secEnusG and rplKAJLrpoBC operons have been identified . Cleavage sites for RNase III occur in the leader of the secEnusG transcript and in the L12-beta intercistronic space of the rplKAJLrpoBC transcript . A single RNase E cleavage site was located in the L1-L10 intergenic space . Inactivation of RNase III and RNase E results respectively in a one- to twofold and a greater than 10-fold stabilization of five mRNA sequences from within the secE, nusG, L11-L1, L10 and beta encoding cistrons . The relative amounts of each of these five mRNA sequences were found to be nearly constant when measured either in the presence or absence of cleavage by RNase III or RNase E . This clearly implies that any increases in the stability of these mRNA sequences resulting from the inactivation of processing by RNase III or RNAase E are counterbalanced by changes in the mRNA synthesis rates . The mechanism that links mRNA synthesis to mRNA decay is not known. Mol Microbiol, 1994 Mar, 11(5), 849 - 59 Cloning and characterization of the RNase P RNA genes from two porcine mycoplasmas; Svard SG et al.; We report the cloning of the RNase P RNA genes from the primary aetiological agent of porcine pneumonia, Mycoplasma hyopneumoniae, and the closely related commensal, Mycoplasma flocculare . The monocistronic genes each have promoters with AT-rich -35 regions and Rho-independent-like transcription terminators which are retained in the RNase P RNA . Both of these RNase P RNA variants are shown to be catalytically active in vitro in spite of a low overall GC content (30%) . Our results suggest a new example of a stable mini-helix in the conserved core of the mycoplasmal RNase P RNAs . Deletion of the corresponding structural element in Escherichia coli RNase P RNA (M1 RNA) generated an RNase P RNA with an impaired substrate interaction . Displacement of this structural element with the mycoplasmal mini-helix resulted in an enzyme with a phenotype similar to that of wild-type M1 RNA . In addition, this structural element is important for lead ion-induced cleavage at specific sites in M1 RNA. AIDS Res Hum Retroviruses, 1994 Mar, 10(3), 235 - 43 Multiepitope polypeptide of the HIV-1 envelope induces neutralizing monoclonal antibodies against V3 loop; Duarte CA et al.; A gene encoding a multiepitope polypeptide (MEP) has been synthesized . It contains the information for (1) an 11-amino acid (aa) epitope from the C1 region of gp120 of HIV-1 and (2) 3 epitopes of 15 amino acids each, from the central part of the V3 loop of isolates MN, SC, and WMJII . These four segments are linked by the short spacer peptide AGGGA . This gene was cloned in a plasmid vector and expressed in Escherichia coli as a fusion product with a 62-aa fragment of human IL-2 . The recombinant protein TAB1 was purified by washed pellet procedures and reversed-phase HPLC . TAB1 was recognized in ELISAs by 25 of 27 sera from seropositive individuals . Mice were immunized and several hybridomas were obtained . Two of them secrete monoclonal antibodies that react with synthetic peptides from isolates MN, WMJI, WMJIII, and SC with an affinity constant in the range of 10(8) M-1 . They also recognized peptides from isolates SF2 and WMJII, but at much lower affinity . The results obtained from peptide ELISAs indicate that the putative epitope recognized by these MAbs lies within the sequence IHIGPGRAFYT . Classic neutralization assays demonstrated that MAb 2C4 neutralizes 50% of the MN isolate at 0.6 micrograms/ml but fails to neutralize IIIB and SF2 strains . The presence of antibodies directed against every one of the component peptides in the sera of rabbits immunized with TAB1 was also documented. Vopr Virusol, 1994 Mar-Apr, 39(2), 59 - 62 {The determination of the antigenic activity of recombinant virus-specific polypeptides from the herpes simplex virus types 1 and 2 and their use in immunoenzyme analysis}; Zvonarev AIu et al.; The structural and nonstructural recombinant proteins of HSV type 1 and type 2 were expressed in E . coli by fusion with cro-beta-galactosidase proteins . These proteins containing amino acid sequences of ICP4, ICP27, ICP47, ICP22, major capsid protein and major DNA-binding proteins of HSV types 1 and 2 reacted in immunoblotting with the corresponding anti-HSV sera from hyperimmune rabbits . The nonstructural recombinant proteins can be used for enzyme immunoassay detection of HSV1 or HSV2 type-specific antibodies in sera from patients with acute HSV infection. Vopr Virusol, 1994 Mar-Apr, 39(2), 56 - 9 {The antiviral action of recombinant forms of the human T-lymphocyte CD4 receptor}; Zverev VV et al.; A recombinant protein containing the first 179 N-terminus amino acids of human T-lymphocyte CD4-receptor was synthesized in E . coli cells . This recombinant protein was shown to interact with OKT4A and Leu3a monoclonal antibodies competing with HIV gp120 glycoprotein for binding with the native CD4 receptor . Experiments in vitro in human T-lymphocyte cultures showed that the recombinant CD4-protein in concentrations of 1 to 10 micrograms/ml inhibited the virus-induced syncytium formation, HIV replication in cell culture, synthesis of HIV reverse transcriptase and other virus-specific proteins, that is, behaved as a HIV inhibitor. Mol Biol (Mosk), 1994 Mar-Apr, 28(2), 464 - 7 {Cloning, determination of primary structure, and expression of the C-terminal segment of human cholesterol-esterase/lipase, containing the antigenic determinant of the protein, in Escherichia coli}; Chekhranova MK et al.; Clone pHICE0.9 was selected from human insulinoma cDNA library by immunoscreening with antibodies against total human insulinoma proteins . This clone contains a 0.9 kb cDNA insert and expresses a fusion protein with beta-galactosidase . Nucleotide sequences of 5'- and 3'-terminal regions of this cDNA insert show that clone pHICE0.9 expresses a protein which is identical to the C-terminal fragment (amino acids 483 to 745) of human pancreatic cholesterol esterase and Homo sapiens bile-acid-salt-stimulated lipase from milk . It is concluded that the protein fragment contains the antigenic determinant of human cholesterol esterase/lipase, and can be used for lipase determination in blood. Mol Biol (Mosk), 1994 Mar-Apr, 28(2), 374 - 82 {Biogenesis and secretion of alkaline phosphatase and its mutants in Escherichia coli . III . Substitution of N-terminal amino acids of alkaline phosphatase affect its biogenesis}; Karamyshev AL et al.; The effect of the N-terminal amino acid substitution on E . coli alkaline phosphatase biogenesis has been studied . The substitutions of Ser, Gln, Tyr, Leu, Gly, Ala, Glu, Phe, His, Cys, Lys and Pro for Arg(+1) were obtained by creating amber mutation at the corresponding position within phoA gene and expressing this mutated gene in E . coli strains that produce the amber-suppressor tRNAs . All mutant proteins were shown to translocate across the cytoplasmic membrane and possess enzyme activity . The introduction of Pro in +1 position disturbs the cleavage of signal peptide whereas the insertion of the other amino acids does not change the rates of processing in comparison with wild-type protein . All amino acid substitutions affect alkaline phosphatase isoenzyme composition . Some experimental evidence were also obtained on the specificity of protease, which split off N-terminal Arg during alkaline phosphatase maturation. Biotechnol Prog, 1994 Mar-Apr, 10(2), 160 - 4 Effect of glucose on the expression parameters of recombinant protein in Escherichia coli during batch growth in complex medium; Li X et al.; The expression parameters were determined at different phases of batch growth of Escherichia coli JM101/pYEJ001 in complex medium and at different conditions of glucose addition . Thus the plasmid content, the RNA content, the RNA synthesis rate, the specific recombinant mRNA content, the specific recombinant mRNA synthesis rate, the recombinant chloramphenicol acetyltransferase content, and the overall protein synthesis rate were determined during growth with no glucose, with initial glucose, with glucose feeding during stationary phase, and with initial glucose plus glucose feeding during stationary phase . The results show that the specific rate of total RNA synthesis was enhanced in the presence of glucose at both exponential and stationary phases, while recombinant mRNA synthesis was enhanced only at stationary phase by glucose feeding . However, the steady-state level of the recombinant mRNA was not changed under the same conditions . In addition, although the overall protein synthesis rate at exponential phase was enhanced in the presence of glucose, the specific recombinant protein level was unaffected . The specific synthesis rate of recombinant mRNA varied inversely with the plasmid content during exponential and stationary phases . Furthermore, changes in the specific activity of the recombinant protein were not correlated with either the changes in the specific synthesis rate of the recombinant mRNA or the overall protein synthesis rate . Therefore, the specific activity of the recombinant protein is not universally limited by its gene transcription rate or the overall protein synthesis capacity. Trends Microbiol, 1994 Mar, 2(3), 91 - 4 Gates of Janus: cystic fibrosis and diarrhea; Guggino SE; Heat-stable enterotoxin, produced by Escherichia coli, binds to particulate guanylate cyclase to increase cyclic GMP in intestinal cells . This in turn stimulates the cyclic-GMP- or cyclic-AMP-dependent protein kinase, activating the same chloride channel that is defective in cystic fibrosis . It is possible that the relatively high prevalence of cystic fibrosis in humans results from its protective effect against diarrhea. J Allergy Clin Immunol, 1994 Mar, 93(3), 614 - 27 Molecular characterization of dog albumin as a cross-reactive allergen; Spitzauer S et al.; Indoor allergens comprise a group of allergenic proteins that are commonly derived from house dust mite and cat and dog dander . In addition to the two major dog allergens (molecular weights: 19 and 23 kd), dog albumin represents an important allergen for up to 35% of patients who are allergic to dogs . In IgE immunoblot inhibition studies and histamine release tests it has been demonstrated that patients who react to dog albumin exhibit IgE reactivity with purified albumins from cat, mouse, chicken, and rat . The proportion of dog-specific IgE directed against dog albumin was determined for patients allergic to dog albumin, and it ranges from 70% to 90% . By IgE immunoscreening of a lambda gt11 expression library from a dog salivary gland, we identified a number of reactive complementary DNA clones . All patients with IgE reactivity against natural dog albumin displayed IgE reactivity to the beta-galactosidase fusion protein encoded by clone 54c, which was therefore assumed to contain major IgE epitopes of dog albumin . The deduced amino acid sequence of clone 54c was compared with the Swiss-Prot library, and significant sequence homologies were found with albumins from different species (human: 82.6%, pig: 81.8%, cattle: 77.3%, sheep: 78.8%, mouse: 75.8%, and rat: 76.2%) . Several other IgE-positive clones hybridized with oligonucleotides that were prepared according to this sequence . Partial complementary DNA coding for dog albumin fragments may be considered a useful tool for further characterization of major IgE epitopes of dog albumin. J Bacteriol, 1994 Mar, 176(6), 1787 - 9 NusA changes the conformation of Escherichia coli RNA polymerase at the binding site for the 3' end of the nascent RNA; Zhang Y et al.; A conformational change in Escherichia coli RNA polymerase induced by NusA was detected by utilizing photocrosslinking . A change in the binding site for the 3' end of the RNA occurred, and NusA increased interactions of the RNA with the beta subunit of the polymerase . NusA was not contacted by the 3' end of the RNA. J Bacteriol, 1994 Mar, 176(6), 1683 - 8 An efficient Shine-Dalgarno sequence but not translation is necessary for lacZ mRNA stability in Escherichia coli; Wagner LA et al.; The 5' ends of many bacterial transcripts are important in determining mRNA stability . A series of Shine-Dalgarno (SD) sequence changes showed that the complementarity of the SD sequence to the anti-SD sequence of 16S rRNA correlates with lacZ mRNA stability in Escherichia coli . Several initiation codon changes showed that an efficient initiation codon is not necessary to maintain lacZ mRNA stability . A stop codon in the 10th codon of lacZ increased mRNA stability . Therefore, ribosomal binding via the SD sequence but not translation of the coding region is necessary to maintain lacZ mRNA stability. Mutat Res, 1994 Mar 1, 305(2), 119 - 26 The SOS function-inducing activity of the new nitrothiophenic derivatives in Escherichia coli; da Silva KV et al.; Genotoxicity and cytotoxic effects of eight new nitrothiophenic compounds with trypanocidal activity were determined by means of the SOS Chromotest assay . Our results indicate that all nitro compounds with one aromatic ring in the R group were genotoxic without and with metabolic activation . The compounds with two aromatic rings in the R group, except for indazol-1-yl, were strongly cytotoxic but they were unable to induce SOS functions without metabolic activation . However, after metabolic activation no cytotoxic effect was observed for these compounds . The role of the nitroreductases to explain the different genotoxic responses of these nitrothiophenic derivatives is discussed. Mol Gen Genet, 1994 Mar, 242(5), 595 - 601 The Tn5 bleomycin resistance gene confers improved survival and growth advantage on Escherichia coli; Blot M et al.; The bleomycin resistance gene (ble) of transposon Tn5 is known to decrease the death rate of Escherichia coli during stationary phase . Bleomycin is a DNA-damaging agent and bleomycin resistance is produced by improved DNA repair which also requires the host genes aidC and polA coding, respectively, for an alkylation-inducible gene product and DNA polymerase I . In the absence of the drug, this DNA repair system is believed to cause the slower death rate of bleomycin-resistant bacteria . In this study, the effect of ble and aidC genes on the viability of bacteria and their growth rate in chemostat competitions was studied . The results indicate, that bleomycin-resistant bacteria display greater fitness under these conditions . Another beneficial effect of transposon Tn5 had been previously attributed to the insertion sequence IS 50 R . We were not able to reproduce this result with IS 50 R, however, the complete transposon was beneficial under similar conditions . Moreover, we showed the Tn5 fitness effect to be aidC-dependent . The ble gene was discovered after the fitness effect of IS 50 R had been established; it has not previously been considered to mediate the beneficial effect of Tn5 . This possibility is discussed based on the molecular mechanism of bleomycin resistance. Mol Cell Biol, 1994 Mar, 14(3), 1764 - 75 The Drosophila micropia retrotransposon encodes a testis-specific antisense RNA complementary to reverse transcriptase; Lankenau S et al.; The micropia transposable element of Drosophila hydei is a long terminal repeat-containing retrotransposon present in both the autosomes and the Y chromosome . micropia expression gives rise to a complex set of sense and antisense RNAs transcribed primarily during spermatogenesis . The most abundant sense RNAs constitute an assortment of heterogeneous high-molecular-weight transcripts expressed as constituents of the Y-chromosomal lampbrush loops of primary spermatocytes . In addition, micropia encodes a full-length RNA that extends between the two long terminal repeats of the element . The major 1.0-kb antisense RNA characterized is complementary to the reverse transcriptase and RNase H coding regions of micropia . It is expressed from a testis-specific promoter during the primary spermatocyte stages and is detectable until spermatid elongation stages . Sequence comparison of this promoter with the 5' region of other testis-specific genes allows the conception of a conserved sequence that is responsible for this pattern of expression . A 284-bp fragment containing this sequence is able to drive testis-specific expression of the Escherichia coli lacZ gene in Drosophila melanogaster . This sequence is conserved in the micropia elements present in other Drosophila species that also encode an antisense RNA . The evolutionary conservation of micropia antisense RNA expression and the sequences responsible for its testis-specific transcription suggests a role for this antisense RNA in the control of germ line expression of the full-length transcript or transposon-encoded proteins. J Bacteriol, 1994 Mar, 176(5), 1242 - 50 Translation through an uncDC mRNA secondary structure governs the level of uncC expression in Escherichia coli; Dallmann HG et al.; Escherichia coli expresses the beta and epsilon subunits of F1F0-ATP synthase at relative levels consistent with the 3:1 (beta/epsilon) stoichiometry in the holoenzyme . The mechanism of translational control of expression of the uncC gene (epsilon subunit) relative to the immediately 5' uncD gene (beta subunit) was examined . Previous expression studies and a computer analysis suggested the presence of an RNA secondary structure including the 3' end of uncD, the uncDC intergenic region, and the uncC Shine-Dalgarno sequence (S . D . Dunn and H . G . Dallmann, J . Bacteriol . 172:2782-2784, 1990) . Analysis of in vitro-transcribed RNA by cleavage with RNases T1, V1, and CL3 and by chemical modification with dimethyl sulfate and diethyl pyrocarbonate confirmed a predicted structure . Introduction of premature uncD stop codons inserted 5' of the secondary structure strongly reduced epsilon expression, whereas stop codons inserted at positions within the secondary structure showed smaller effects, indicating that translational control of epsilon synthesis involves partial coupling to beta synthesis . Possible mechanisms by which the RNA secondary structure and the unfolding of this structure by translation of uncD may govern the level of uncC expression are discussed. Infect Immun, 1994 Mar, 62(3), 1070 - 8 Epitopes shared by unrelated antigens of Borrelia burgdorferi; Anda P et al.; An immunoglobulin M kappa-chain murine monoclonal antibody (CAB) reacted in a Western blot (immunoblot) with approximately 30 polypeptides from a whole-cell lysate of several American and European Borrelia burgdorferi strains . The reactive antigen with the highest M(r) was measured at 93 kDa (p93) and had an NH2-terminal sequence identical to the one previously reported for this antigen . The lowest reactive antigen had an M(r) of 16,000 . All antigens recognized by CAB had isoelectric points within a narrow acidic range, between 5.4 and 6.2 . Thus, the objective of this study was to determine whether the broad reactivity of CAB could be due to degradation of the antigen with the highest M(r), since such spontaneous degradation of p93 has already been reported, and to determine whether CAB could recognize shared epitopes in different antigens . Treatment of B . burgdorferi with protease inhibitors did not result in changes in CAB reactivity, indicating that if such degradation existed, it was most likely not due to the action of endogenous proteases . Likewise, protease treatment of intact organisms and recovery of the antigens in the insoluble fraction of a Triton X-114 partition indicated that they were internal and thus less likely to be degraded by experimental procedures . Amino-terminal sequences of other reactive polypeptides showed one approximately 72-kDa polypeptide to be identical to the DnaK homolog of B . burgdorferi . Two other antigens at approximately 49 and 47 kDa were blocked to Edman degradation . Finally, one sequenced polypeptide with a molecular mass of approximately 38.5 kDa had a strong identity with glyceraldehyde-3-phosphate dehydrogenase of other bacteria and vertebrates . Thus, while it cannot be ruled out that some of the CAB reactivity may be due to fragmentation of p93, there is strong evidence to indicate the presence of a shared epitope in at least three, possibly five, unrelated antigens of B . burgdorferi . A linear epitope within amino acid residues 357 to 371 of p93 was identified . Evidence is presented for a discontinuous epitope in the carboxy-terminal region of the DnaK homolog, which bears strong amino acid identity with the p93 epitope . The conserved amino acid sequences necessary for these shared epitopes indicate possible genetic and/or functional relatedness among these various antigens. Mutat Res, 1994 Mar, 323(3), 105 - 12 Evidence for nontargeted mutagenesis in a monkey kidney cell line and analysis of its sequence specificity using a shuttle-vector plasmid; Zhang X et al.; Intact pZ189 DNA was allowed to replicate in monkey kidney vero cells that had been pretreated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . The E . coli MBM7070 was transfected with replicated plasmid, and those with mutations in the supF gene were identified . The frequency of mutants that did not contain recognizable changes in the electrophoretic mobility of the plasmid DNA was scored . The frequency of such mutants was 12.2 x 10(-4) (43/35376) and 6.2 x 10(-4) (22/35712) in mutants derived from cells pretreated with 0.2 mumoles/l and 2 mumoles/l MNNG respectively; these values represent an increase of 5.8- and 2.9-fold over the spontaneous mutation frequency of 2.1 x 10(-4) (10/47741) (p < 0.01) . Sequence analysis of the supF genes of these mutants showed that 89% (24/27) of base substitutions occurred at G.C base pairs; 59% of the base substitutions (16/27) were transversions, and 41% (11/27) were transitions . The types of base substitutions were predominantly G.C-->T.A and G.C-->A.T . 48% of base substitutions occurred at 6 sites of the supF gene; 4 of these sites consist of 5'-TTNN where N is G or C . Base substitutions never previously reported were found, namely, T-->C at 61, G-->T at 70, G-->T at 99, and G-->C at 103 were found; these have never been reported up to now . In addition, 2 of the 5 frameshifts occurred in the region 99-105 of the supF gene (GGTGGGG), suggesting that this region is a hot spot for nontargeted frameshifts . These results strongly suggest that nontargeted mutagenesis can occur in mammalian cells and shows that the spectrum of mutations induced differs from that of spontaneous and targeted mutations. J Virol, 1994 Mar, 68(3), 1970 - 1 Redesignation of the RNase D activity associated with retroviral reverse transcriptase as RNase H; Hostomsky Z et al.; In the presence of Mn2+, reverse transcriptase of both human immunodeficiency virus and murine leukemia virus hydrolyzes duplex RNA . However, designating this novel activity RNase D conflicts with Escherichia coli RNase D, which participates in tRNA processing . On the basis of its location in the RNase H domain, we propose that this novel retroviral activity be redesignated RNase H*. Biochem Biophys Res Commun, 1994 Feb 28, 199(1), 106 - 18 cDNA cloning and expression of timothy grass (Phleum pratense) pollen profilin in Escherichia coli: comparison with birch pollen profilin; Valenta R et al.; Profilin, an actin-binding protein, was previously described as a ubiquitous allergen which is responsible for cross-reactivities in about 20% of pollen and food allergic patients . A complete cDNA clone coding for timothy grass (Phelum pratense) pollen profilin was isolated using allergic patients IgE . The deduced amino acid sequence of timothy grass profilin shares a sequence identity of 79% with birch profilin and other plant profilins and a lower average sequence identity of 35% with other eukaryotic profilins . The high degree of homology among different plant profilins at the DNA and protein level explains the extensive cross-reactivities observed in profilin allergic patients . Recombinant timothy grass pollen profilin was expressed in Escherichia coli as a beta-galactosidase fusion protein and shown to bind IgE from profilin allergic patients similar to recombinant birch profilin . Slight differences regarding the IgE-binding capacity of birch and timothy grass profilin indicate that not all IgE-epitopes of the two profilins are conserved . It is speculated that profilin allergic patients were initially sensitized against a certain profilin and then cross-react with the homologous proteins. J Biotechnol, 1994 Feb 28, 32(3), 289 - 98 Effect of growth rate on stability and gene expression of recombinant plasmids during continuous and high cell density cultivation of Escherichia coli TG1; Hellmuth K et al.; Continuous and fed-batch cultures of recombinant Escherichia coli TG1 were carried out in order to study plasmid stability and recombinant product formation at different specific growth rates . The aprotinin::beta-galactosidase gene (Ap::lacZ) was placed under the control of two different promoter/repressor systems, the PLac/lacI (pPLac8) and the lambda PL/cIts857 (pPL6) system . The chemically (0.5 mM IPTG) induced gene expression exhibited higher product activity and plasmid stability than the thermally (40 degrees C) induced expression . In fed-batch cultivations with the more stable E . coli TG1(pPLac8) a special feeding strategy allowed bacterial growth with a constant growth rate mu for several hours up to high cell densities . The cloned gene product activity was noticeably effected by the specific growth rate and the cell density at the moment of induction . In particular, the enzyme activities passed a pronounced maximum value in dependence of the set growth rate . The results indicate that fed-batch cultivation strategies are well suited to produce recombinant gene products. J Biotechnol, 1994 Feb 28, 32(3), 273 - 81 A novel procedure for the preparation of biologically active recombinant peptides using a cyanylation reaction; Koyama N et al.; A cysteine specific cleavage reaction was used for the preparation of biologically active peptides from recombinant fusion proteins . The fusion protein through cysteine was prepared by a recombinant DNA technology and then treated with cyanylating reagents such as 2-nitro-5-thiocyanatobenzoic acid (NTCB) and 1-cyano-4-(dimethylamino) pyridinium tetrafluoroborate (DMAP-CN) to release the desired product . As an example, we have selected a glucagon-like peptide 1 (7-37) (termed insulinotropin) . We constructed an expression vector for a fusion protein in which insulinotropin and human basic fibroblast growth factor (hbFGF) mutein (abbreviated as CS 23) are connected by cysteine and then expressed it in Escherichia coli cells . The fusion protein, after refolding, was purified by heparin affinity chromatography, since CS23 has a strong affinity for heparin . The affinity-purified fusion protein was treated with NTCB or DMAP-CN to give crude insulinotropin, which was then purified by reversed phase (rp) high-performance liquid chromatography (HPLC) . From various criteria such as amino acid analysis, amino acid sequence and the biological activity, the purified material obtained was found to be methionylated insulinotropin (Met-insulinotropin) with full activity . The specificity and simplicity of the present method make it versatile and convenient for the preparation of biologically active peptides. Nucleic Acids Res, 1994 Feb 25, 22(4), 576 - 82 Most compact hairpin-turn structure exerted by a short DNA fragment, d(GCGAAGC) in solution: an extraordinarily stable structure resistant to nucleases and heat; Hirao I et al.; The three-dimensional structure of a short DNA fragment, d(GCGAAGC) exhibiting an extraordinarily stable hairpin structure was determined by nuclear magnetic resonance spectroscopy . Two possible models were obtained by molecular modelling using distance and torsion constraints . Only one of these two models is the correct structure, which can clearly explain all the 1H chemical shifts . d(GCGAAGC) is folded back on itself between A4 and A5, and all the sugars in the fragment adopt the C2'-endo conformation . This compact molecule is stabilized by regular extensive base-stacking interaction within each B-form helical strand of G1C2G3A4 and A5G6C7, and by two G-C and one G3-A5 base pairs . The molecule is hard to differentiate into stem and loop regions, so that we classify it as a turn (hairpin-turn) structure experted by a single-stranded DNA . This highly stacked structure shows high thermostability and strong resistance against nucleases contained in E . coli extracts and in human serum. J Biol Chem, 1994 Feb 25, 269(8), 6058 - 63 Primase couples leading- and lagging-strand DNA synthesis from oriC; Hiasa H et al.; Coupling of leading- and lagging-strand DNA synthesis at replication forks formed at Escherichia coli oriC has been studied in vitro using a replication system reconstituted with purified proteins . At low concentrations of primase (8 nM), the major replication products were multigenome-length molecules, generated by a rolling circle-type mechanism, and unit-length molecules . Rolling circle DNA replication was inhibited at high concentrations of primase (80 nM) and the major replication products were half-unit-length leading strands and a distinct population of short Okazaki fragments . At low primase concentrations, an asymmetric mode of DNA synthesis occurred . Each strand was made independently and initiation could occur outside of oriC . At high primase concentrations, initiation occurred exclusively at oriC and two coupled replication forks proceeded bidirectionally around the plasmid . Presumably, at low concentrations of primase, DnaB (the replication fork helicase) unwound the plasmid DNA before replication forks could form, leading to initiation at sites other than oriC . On the other hand, high concentrations of primase resulted in successful capture of the helicase leading to the formation at oriC of coupled replication forks capable of coordinated leading- and lagging-strand synthesis. J Biol Chem, 1994 Feb 25, 269(8), 6001 - 10 Characterization of the signal peptide at the amino terminus of the rat peroxisomal 3-ketoacyl-CoA thiolase precursor; Tsukamoto T et al.; The amino-terminal presequences of rat peroxisomal 3-ketoacyl-CoA thiolase precursors (types A and B) were reported to be cleavable signal peptides for peroxisomal protein translocation . In the present study, this was proven by immunoelectron microscopy of the cultured Chinese hamster ovary cells stably expressing fusion proteins of the amino-terminal sequences of the thiolase precursor and Escherichia coli dihydrofolate reductase . The fusion proteins were processed into mature forms of the apparently correct sizes . Site-directed mutagenesis studies of the charged residues in the B-type presequence (26 amino acid residues) revealed that arginine at position -24 and histidine at position -17 were both indispensable . Even replacement of these residues with other basic amino acids abolished the import activity . Both Arg-24 and His-17 were also required in a longer presequence (36 amino acid residues) of the thiolase A, thereby suggesting that the signal can function in an internal position . When glutamic acid at position -11 was changed to amino acids other than aspartic acid, the signal peptide became apparently effective in both peroxisomal and mitochondrial targeting . All of these data indicate that the thiolase signal peptide is a newly defined type of peroxisomal targeting signal recognized by a mechanism presumably different from that for a known peroxisomal signal, the carboxy-terminal Ser-Lys-Leu-COOH motif. J Biol Chem, 1994 Feb 25, 269(8), 5958 - 62 Uncoupling of peptide-stimulated ATPase and clathrin-uncoating activity in deletion mutant of hsc70; Tsai MY et al.; The recombinant 60-kDa fragment of rat hsc70 has been overexpressed in Escherichia coli . The recombinant protein is not capable of disassembling clathrin from coated vesicles . However, the affinity for peptides and the peptide-stimulated ATPase activity of the intact protein are retained in the 60-kDa fragment . The dissociation constants of peptide P3a (the recognition sequence of clathrin light chain LCa by hsc70) and S peptide of ribonuclease for 60-kDa protein are 13 and 7 microM, respectively . The maximal velocities of stimulated ATPase activity by peptides P3a and GT4 are 0.25 and 0.31 nmol/h/microgram of protein, respectively, and the EC50 values (the concentration of peptides that brought about half-maximum hydrolysis) for peptides P3a and GT4 are 0.56 and 0.30 mM, respectively . These results indicate that peptide-stimulated ATPase activity of hsc70 is not sufficient for clathrin uncoating . We suggest that other activities or cellular components as yet unidentified associated with the C-terminal 10-kDa fragment of hsc70 are required for clathrin uncoating. J Biol Chem, 1994 Feb 25, 269(8), 5890 - 6 Differential expression of bovine adseverin in adrenal gland revealed by in situ hybridization . Cloning of a cDNA for adseverin; Nakamura S et al.; Adseverin is a Ca(2+)-dependent actin filament-severing protein that is presumed to have a regulatory function in exocytosis by affecting the organization of the microfilament network underneath the plasma membrane . As the first step to investigate whether adseverin has the expected function in vivo, we have cloned a cDNA for bovine adseverin . Analysis of the nucleotide sequence revealed the following points . 1) Adseverin consisted of 715 amino acid residues, and its predicted molecular mass was 80.5 kDa . 2) Adseverin belonged to the gelsolin family of proteins, which consists of six homologous segments . 3) Adseverin had two putative polyphosphoinositide binding sequences, very similar to but distinct from those in gelsolin . Adseverin was produced in Escherichia coli and purified to homogeneity . The adseverin reacted with adseverin-specific antibody and showed Ca(2+)-dependent actin filament severing activity like authentic adseverin . In situ hybridization demonstrated that adseverin mRNA was differentially expressed in the bovine adrenal gland; it was expressed in the adrenal medulla but not in most parts of the adrenal cortex, although both have a secretory function . The observation strongly suggested that adseverin is not involved in secretory processes in general but is specifically involved in exocytosis. J Biol Chem, 1994 Feb 25, 269(8), 5874 - 80 Colicin A and the Tol proteins involved in its translocation are preferentially located in the contact sites between the inner and outer membranes of Escherichia coli cells; Guihard G et al.; Colicin A is a bacterial toxin which forms channels in the cytoplasmic membrane of Escherichia coli . Its translocation through the envelope requires the participation of bacterial proteins encoded by the tolQ, -R, -A, and -B genes . Overproduction of the Tol proteins decreased the time needed for colicin A translocation and increased the number of channels formed in vivo . Cells overproducing radioactively labeled Tol proteins and containing or not colicin A were fractionated . The Tol proteins were mainly recovered in the inner membrane and in the contact sites between the two membranes . The presence of colicin A increased the specific radioactivity of the Tol proteins in the contact sites . Our data suggest that the Tol proteins form a complex of definite stoichiometry in the membranes and that colicin A is associated to this complex upon channel formation . We discuss the possibility that the channel activity determined in vivo is due to the colicin A-Tol proteins complex. J Biol Chem, 1994 Feb 25, 269(8), 5781 - 7 Structure and expression of the cDNA for the C isozyme of phosphofructo-1-kinase from rabbit brain; Li Y et al.; Rabbit brain contains three phosphofructo-1-kinase (PFK) isozymic subunits designated A, B, and C . The primary structures of the first of these two isozyme types have been determined previously . The isozyme C of rabbit brain was isolated by immunoaffinity chromatography and subjected to proteolytic and chemical digestion . A large number of peptides were sequenced, the total number of amino acids identified being equal to about 80% of the total structure . The sequence of the cDNA derived from brain mRNA for C isozyme was determined from polymerase chain reaction fragments synthesized using oligonucleotides designed on the basis of the peptide sequences . The deduced size of the C isozyme was 86,371 Da, slightly larger than PFKs described previously . The amino acid sequence identity with the rabbit A isozyme was 68.9% and a range of identity to other sequenced mammalian PFKs was 67-69% . Using these data plus previously published data on chemical modification, assignments of the 6 organic ligand binding sites of PFK were inferred . The full-length cDNA was cloned into and expressed in Escherichia coli . Phosphofructokinase C was purified to homogeneity from the bacterial extracts. J Biol Chem, 1994 Feb 25, 269(8), 5720 - 4 Site-specific alteration of arginine 376, the unique positively charged amino acid residue in the mid-membrane-spanning regions of the proline carrier of Escherichia coli; Yamato I et al.; An alignment of 5 amino acids in the Escherichia coli proline carrier (G328-A366-L371-GR376) is common in the amino acid sequences of several Na+ symport carriers, and it has been proposed as the putative sodium binding motif (Deguchi, Y., Yamato, I., and Anraku, Y . (1990) J . Biol . Chem . 265, 21704-21708) . To determine whether these amino acids are essential for Na+ symport activity as the Na+ binding site, one of the amino acids in this alignment, Arg-376, which is the only positively charged amino acid in the innermost part of the predicted membrane-spanning regions, was changed to either lysine, glutamine, or glutamic acid by oligonucleotide dependent site-specific mutagenesis . The transport and binding activities of the proline of the R376K mutant carrier were not detected at all . The activities of the other mutant carriers for uptake and binding of proline were as high as those of the wild-type carrier . These two mutant carriers were as sensitive to the proline analogue azetidine-2-carboxylate and to N-ethylmaleimide as the wild-type carrier, indicating that they have the same properties as the wild-type . The amounts of the carrier proteins expressed from these mutated putP genes were similar to that from the wild-type gene . These results imply that the Arg-376 in the proline carrier does not reside at the sodium binding site, suggesting that the similar alignment found in the amino acid sequences of several Na+ symport carriers is not essential for the transport or binding activities, although this similar alignment may have some relevance to the structure of the Na+ symporter . Furthermore, that the only Arg residue in the middle part of the predicted membrane-spanning regions is dispensable for the energy coupling activity indicates a unique difference of the coupling mechanism from the other secondary active transport systems, such as that of the lactose permease and the tetracycline/H+ antiporter. J Biol Chem, 1994 Feb 25, 269(8), 5602 - 11 Characterization of protein tyrosine phosphatase SH-PTP2 . Study of phosphopeptide substrates and possible regulatory role of SH2 domains; Dechert U et al.; The src homology 2 (SH2) domain containing protein-tyrosine-phosphatase SH-PTP2, was over-expressed in Escherichia coli for a kinetic study employing a set of synthetic 13- to 14-mer phosphopeptide substrates . The full-length SH-PTP2 protein, as well as a truncated form, lacking the two amino terminus SH2 domains (SH-PTP2(delta SH2)), exhibited Michaelis-Menten kinetics, and demonstrated striking substrate preferences on phosphopeptides having sequences based on sites of intracellular protein tyrosine phosphorylation . For example, while a KM of 59 microM and kcat/KM of 1.1 x 10(5) were obtained using SH-PTP2(delta SH2) and PDGFRY1021, a phosphorylation site within the platelet-derived growth factor receptor, other peptides revealed no detectable phosphate release . PDGFRY1009, modeled after a sequence identified as an in vivo binding site for SH-PTP2, was also a good substrate for this enzyme . The truncated form, lacking the SH2 domains demonstrated higher catalytic efficiency than the full-length enzyme . Interestingly, soluble SH2 domains were found to inhibit the catalytic activity of SH-PTP2 in a concentration-dependent manner . There was also evidence of a non-phosphotyrosine-mediated association between the two domains . These observations suggested that the SH2 domains have a direct role in regulating the catalytic activity of SH-PTP2. J Biol Chem, 1994 Feb 25, 269(8), 5566 - 73 Novel FMN-binding protein from Desulfovibrio vulgaris (Miyazaki F) . Cloning and expression of its gene in Escherichia coli; Kitamura M et al.; A gene encoding a novel FMN-binding protein from Desulfovibrio vulgaris (Miyazaki F) was cloned, and its expression system was constructed in Escherichia coli . The 1.4-kilobase pair DNA fragment isolated from D . vulgaris (Miyazaki F) by double digestion with KpnI and SmaI was found to express a protein binding FMN as a prosthetic group under control of the lac promoter in E . coli . This DNA fragment contained several putative open reading frames . The partial amino acid sequence of the polypeptide portion of the purified FMN-binding protein and its tryptic peptides were completely consistent with those deduced from the nucleotide sequence of the third open reading frame in the cloned SmaI-SmaI fragment of D . vulgaris (Miyazaki F) DNA, which may include promoter and regulatory sequences . The nucleotide sequence of FMN-binding protein indicated that the protein is composed of 122 amino acids including an initiator Met residue and lacks a signal peptide for secretion . The main redox potential of the FMN-binding protein was measured as -325 mV using direct current cyclic and differential pulse voltammetric techniques and an electroreflectance method, suggesting that this FMN-binding protein functions as a redox protein like other FMN-binding proteins . Immunoblot analysis of the whole proteins from D . vulgaris (Miyazaki F) clearly indicated that this protein is expressed in this bacteria . However, the protein was found to have a primary structure distinct from those of other FMN-binding proteins and to be the smallest FMN-binding protein yet reported. J Biol Chem, 1994 Feb 25, 269(8), 5548 - 53 A role for the carboxyl terminus of human granulocyte-macrophage colony-stimulating factor in the binding of ligand to the alpha-subunit of the high affinity receptor; Seelig GF et al.; A synthetic segment (110-127) of the carboxyl terminus of recombinant human granulocyte-macrophage colony-stimulating factor (rh-GM-CSF) was used to generate a rabbit polyclonal antibody (345-6), which recognized both peptide and full-length Escherichia coli-derived rh-GM-CSF in a direct enzyme-linked immunosorbent assay . Antibody 345-6 was shown to antagonize the binding of 125I-labeled rh-GM-CSF to its receptor on the KG-1 cell line and to inhibit human GM-CSF-dependent proliferation of the AML-193 cell line . The purified IgG fraction of neutralizing antibody 345-6 was used as immunogen to obtain sheep anti-serum 1418 . Antibody 1418 recognized antibody 345-6 on direct enzyme-linked immunosorbent assay but did not recognize rh-GM-CSF or the peptide 110-127 to which antibody 345-6 was raised . Antiserum 1418, as well as a purified IgG fraction of this serum, inhibited both rh-GM-CSF-stimulated cell proliferation and 125I-labeled rh-GM-CSF receptor binding but not 125I-labeled recombinant human interleukin-4 receptor binding . The anti-idiotypic antibody response derived from the anti-(110-127) antibody strongly suggests that the carboxyl-terminal region of rh-GM-CSF may be directly involved in the receptor-ligand interaction of this protein . The high affinity receptor consists of two different components (GM-R alpha beta) a cytokine-specific alpha-subunit and a beta-subunit that is shared by human GM-CSF, interleukin-3, and interleukin-5 . In an effort to localize the epitope of antibody 1418 to either GMR alpha or GMR beta, several cell lines containing high, low, or both high and low affinity receptors were examined . Each was specifically and completely inhibited by antibody 1418 . Interleukin-3-dependent cell proliferation of the AML-193 cell line was found to be unaffected by the antibody 1418 . Thus, the carboxyl-terminal region of rh-GM-CSF is likely to be involved in the interaction of the ligand with the alpha-subunit of the high affinity receptor. J Biol Chem, 1994 Feb 25, 269(8), 5523 - 6 Product-induced stabilization of tertiary and quaternary structure in Escherichia coli dehydroquinase; Reilly A et al.; The effects of irreversibly attaching product at the active site of type I dehydroquinase from Escherichia coli have been investigated at the level of the secondary, tertiary, and quaternary structure of the protein by spectroscopic and hydrodynamic techniques . The results agree with the previously identified stabilization of the enzyme but show for the first time that the protein dimer is also stabilized and suggest that the E . coli dehydroquinase subunit may be bilobal. J Biol Chem, 1994 Feb 25, 269(8), 5493 - 6 Protein EnvM is the NADH-dependent enoyl-ACP reductase (FabI) of Escherichia coli; Bergler H et al.; The EnvM protein was purified from an overproducing Escherichia coli strain . It showed NADH-dependent enoyl-acyl carrier protein (ACP) reductase activity using both crotonyl-ACP and crotonyl-CoA as substrates . The protein bound a radioactive diazaborine derivative in the presence of NAD+ and radioactive NAD+ in the presence of the drug . Based on these data, it is concluded that EnvM is the NADH-dependent enoyl-ACP reductase (EC 1.3.1.9) of E . coli and we propose to rename the corresponding gene fabI. J Mol Biol, 1994 Feb 25, 236(3), 844 - 61 Folding the main chain of small proteins with the genetic algorithm; Dandekar T et al.; Grid-free protein folding simulations were effected using the genetic algorithm, a backbone representation and standard dihedral angular conformations . The topological folding of idealized four-helix bundles was investigated in detail to differentiate among the important protein folding forces used as fitness criteria . Hydrophobic interactions were the most significant while local forces and hydrogen bonds were far less effective in promoting folding . Stable secondary structural regions were also important as nucleating centers . Using the fitness parameters optimized in idealized simulations together with standard secondary structure predictions derived from the amino acid sequence alone, the proper main-chain folding of the four-helix bundle proteins cytochrome b562, cytochrome c' and hemerythrin was achieved . In addition the backbone topology as predicted by the genetic algorithm for crambin, a mixed helix/strand protein with known structure, is presented and discussed. J Mol Biol, 1994 Feb 25, 236(3), 800 - 16 Crystal structure of Escherichia coli thioredoxin reductase refined at 2 A resolution . Implications for a large conformational change during catalysis; Waksman G et al.; The crystal structures of three forms of Escherichia coli thioredoxin reductase have been refined: the oxidized form of the wild-type enzyme at 2.1 A resolution, a variant containing a cysteine to serine mutation at the active site (Cys138Ser) at 2.0 A resolution, and a complex of this variant with nicotinamide adenine dinucleotide phosphate (NADP+) at 2.3 A resolution . The enzyme mechanism involves the transfer of reducing equivalents from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to a disulfide bond in the enzyme, via a flavin adenine dinucleotide (FAD) . Thioredoxin reductase contains FAD and NADPH binding domains that are structurally similar to the corresponding domains of the related enzyme glutathione reductase . The relative orientation of these domains is, however, very different in the two enzymes: when the FAD domains of thioredoxin and glutathione reductases are superimposed, the NADPH domain of one is rotated by 66 degrees with respect to the other . The observed binding mode of NADP+ in thioredoxin reductase is non-productive in that the nicotinamide ring is more than 17 A from the flavin ring system . While in glutathione reductase the redox active disulfide is located in the FAD domain, in thioredoxin reductase it is in the NADPH domain and is part of a four-residue sequence (Cys-Ala-Thr-Cys) that is close in structure to the corresponding region of thioredoxin (Cys-Gly-Pro-Cys), with a root-mean-square deviation of 0.22 A for atoms in the disulfide bonded ring . There are no significant conformational differences between the structure of the wild-type enzyme and that of the Cys138Ser mutant, except that a disulfide bond is not present in the latter . The disulfide bond is positioned productively in this conformation of the enzyme, i.e . it stacks against the flavin ring system in a position that would facilitate its reduction by the flavin . However, the cysteine residues are relatively inaccessible for interaction with the substrate, thioredoxin . These results suggest that thioredoxin reductase must undergo conformational changes during enzyme catalysis . All three structures reported here are for the same conformation of the enzyme and no direct evidence is available as yet for such conformational changes . The simplest possibility is that the NADPH domain rotates between the conformation observed here and an orientation similar to that seen in glutathione reductase . This would alternately place the nicotinamide ring and the disulfide bond near the flavin ring, and expose the cysteine residues for reaction with thioredoxin in the hypothetical conformation.(ABSTRACT TRUNCATED AT 400 WORDS) J Mol Biol, 1994 Feb 25, 236(3), 738 - 48 Escherichia coli seryl-tRNA synthetase recognizes tRNA(Ser) by its characteristic tertiary structure; Asahara H et al.; To investigate the sequence requirements of Escherichia coli tRNA(Ser) for recognition by seryl-tRNA synthetase, various mutants of unmodified tRNA(Ser) were constructed . Substitution of G2.C71 by C2.G71, but not by A2.U71 or U2.A71, impaired the serine-accepting activity, indicating that this position is not involved in recognition by seryl-tRNA synthetase, but contributes to discrimination from other tRNAs processing C2.G71 such as tRNA(Leu) . Other nucleotides characteristic of tRNA(Ser), including the discriminator base, were not involved in recognition by seryl-tRNA synthetase . The anticodon was not involved, as suggested by its sequence variety within the isoacceptors . The long variable arm composed of over ten nucleotides, which is a characteristic feature of tRNA(Ser) together with tRNA(Leu) and tRNA(Tyr), was stem-length-specifically, but not sequence-specifically, important for recognition . In order to introduce a sufficient serine-accepting activity to a tRNA(1LEU) transcript in vitro, besides the change from C2.G71 to G2.C71, the following elements had to be changed to those characteristic of tRNA(Ser): the sequence in the D-loop, the stem pairing pattern of the variable arm, the tertiary base-pair 15.48 and the nucleotide at position 59 in the T psi C-loop . None of the nucleotides at these changed positions was involved in base-specific recognition, indicating that seryl-tRNA synthetase selectively recognizes tRNA(Ser) on the basis of its characteristic tertiary structure rather than the nucleotides specific to tRNA(Ser). J Mol Biol, 1994 Feb 25, 236(3), 679 - 84 Use of an inducible site-specific recombinase to probe the structure of protein-DNA complexes involved in F plasmid partition in Escherichia coli; Lynch AS et al.; The induced expression of a tightly regulated site-specific recombinase is shown to efficiently form intracellular DNA rings of well-defined nucleotide sequences in Escherichia coli . To provide information on the organization of an intracellular protein-DNA complex, the linking number distributions of excised DNA rings containing cognate binding sites for the protein can be measured after their isolation . Application of this approach to the partition system of the E . coli F plasmid suggests that the SopB protein and the sopC locus, the latter being composed of 12 tandemly joined imperfect repeats of a 43 base-pair motif, form a complex in which the DNA is wrapped right-handedly around a multimeric protein core; the presence of a single copy of a 43 base-pair motif on a DNA appears to be sufficient to nucleate the formation of this nucleoprotein complex. Gene, 1994 Feb 25, 139(2), 281 - 6 Production of human matrix metalloproteinase 3 (stromelysin) in Escherichia coli; Rosenfeld SA et al.; Full-length human matrix metalloproteinase 3 (prostomelysin or proMMP-3) was produced in Escherichia coli as an intracellular insoluble aggregate that could be solubilized and refolded to yield an activatable proenzyme . The refolded protein was purified to > 95% homogeneity . The recombinant proMMP-3 (re-proMMP-3) could be activated by agents known to stimulate self-catalyzed cleavage of native fibroblast proMMP-3 . The N-terminal amino-acid sequence of the re-proMMP-3 and its activation products indicated that they were the same as those obtained with the natural material. Nucleic Acids Res, 1994 Feb 25, 22(4), 613 - 8 One of two Ets-binding sites in the cytokeratin EndoA enhancer is essential for enhancer activity and binds to Ets-2 related proteins; Fujimura Y et al.; Expression of the mouse cytokeratin EndoA gene is restricted in endodermal and epithelial cells, and is regulated by an enhancer that is located 1 kilobase (kb) 3' downstream from the gene . The enhancer consists of six direct repeats, of which each contains two predicted Ets binding sites (EBS1 and EBS2) containing GGAA as a core . Mutation analysis showed that EBS1 is essential for the enhancer activity and additional effects of EBS2, suggesting that some Ets-related proteins bind and activate the enhancer through EBS1 . We also showed that Ets-2 mRNA is expressed in PYS-2 cells and that Ets-2 protein produced by E . coli interacts with EBS1 but not with EBS2 . Using co-transfection assays, we showed that Ets-2 can trans-activate the enhancer in PYS-2 cells . Mutations that impair Ets-2 binding abolished the activity of the EndoA enhancer . The results obtained from the binding competition assay using an Ets-2 specific antibody, however, suggest that EBS1 binds to an Ets protein which is distinct from Ets-2 . These data show that Ets-2 related protein binds and activates the EndoA enhancer in a sequence-specific fashion. J Biol Chem, 1994 Feb 25, 269(8), 6064 - 71 The role of individual exoribonucleases in processing at the 3' end of Escherichia coli tRNA precursors; Li Z et al.; We have used an in vitro Escherichia coli tRNA processing system to investigate the specific role of individual exoribonucleases in the 3' maturation of tRNA precursors . The processing of pre-tRNA(Tyr)su3+ and pre-tRNA(2Arg) was studied using extracts from cells lacking one or multiple exoribonucleases or using purified RNases . Earlier genetic studies had suggested that multiple exoribonucleases contributed to the maturation of tRNA precursors, and this was proven directly in the studies described here . Complete 3' processing required the combined action of multiple exoribonucleases, and each RNase showed distinct specificities for maturation of the different parts of the 3' precursor segment . RNase II and polynucleotide phosphorylase were most effective in shortening long 3' trailer sequences to intermediates with 2-4 extra 3' residues . Final trimming of the last few 3' nucleotides of these precursors was carried out most efficiently by RNases T and PH, but the two enzymes differed in their specificity for individual nucleotide positions . Depending on the tRNA precursor, the relative importance of the various RNases to the overall maturation process differed . We also showed that purified exoribonucleases can completely complement mutant extracts and that tRNA maturation can be totally reconstructed in vitro using purified enzymes . These studies provide the first detailed information about the specific role of individual exoribonucleases in tRNA processing, and bring us closer to defining a complete E . coli tRNA maturation pathway. Biochim Biophys Acta, 1994 Feb 23, 1190(1), 91 - 8 Arrangement of phosphatidylethanolamine molecular species in Escherichia coli membranes and reconstituted lipids as determined by dimethyl suberimidate cross-linking of nearest neighbor lipids; Roth MR et al.; Dimethylsuberimidate cross-linking has been used to determine the arrangement of phosphatidylethanolamine (PE) molecular species in Escherichia coli membranes . No large deviations from random mixing were found in wild-type strain AB1623, either in whole cells or in extracted lipids which were reconstituted into multilamellar vesicles . These results suggest, first, that there is little difference in the PE molecular species composition of the three lipid monolayers (the inner and outer monolayers of the inner membrane and the inner monolayer of the outer membrane) which contain significant amounts of PE . Secondly, the results suggest that the molecular species within each monolayer and in the extracted lipids are arranged close to randomly with no tendency for like molecular species to cluster . E . coli strain L8-2, which has a defect in beta-oxidation and a temperature-sensitive mutation in total fatty acid synthesis, was grown on cis-vaccenate (cis-11,12- octadecenate) to enrich the cells in divaccenoyl PE . Again, in whole cells or in lipids extracted from whole cells and reconstituted into multilamellar vesicles, the species were close to randomly arranged . However, a consistent, slight tendency of divaccenoyl species to pair with like species as compared to pairing with the second most common species, vaccenoyl, palmitoleoyl PE, was noted in both extracted lipids and in whole cells. Biochim Biophys Acta, 1994 Feb 22, 1225(3), 259 - 63 5-Aminolevulinic acid induces single-strand breaks in plasmid pBR322 DNA in the presence of Fe2+ ions; Onuki J et al.; 5-Aminolevulinic acid (ALA), a heme precursor accumulated in chemical and inborn porphyrias, has been demonstrated to produce reactive oxygen species upon metal-catalyzed aerobic oxidation and to cause oxidative damage to proteins, liposomes and subcellular structures . Exposure of plasmid pBR322 DNA to ALA (0.01-3 mM) in the presence of 10 microM Fe2+ ions causes DNA single-strand breaks (ssb), revealed by agarose gel electrophoresis as an increase in the proportion of the open circular form (75 +/- 7.5% at 3 mM ALA) at the expense of the supercoiled form . Addition of either anti-oxidant enzymes such as superoxide dismutase (10 micrograms/ml) and catalase (20 micrograms/ml), or a metal chelator (DTPA, 2.5 mM), or a HO . scavenger (mannitol, 100 mM) inhibited the damage (by 30, 45, 55, and 81%, respectively), evidencing the involvement of O2-., H2O2 and HO . (by the Haber-Weiss reaction) in this process . Hydrogen peroxide (100 microM) or Fe2+ (10 microM) alone were of little effect on the extent of DNA ssb . The present data may shed light on the correlation reported between primary liver-cell carcinoma and intermittent acute porphyria. Proc R Soc Lond B Biol Sci, 1994 Feb 22, 255(1343), 153 - 7 Adenine transport in Escherichia coli; Burton K; The purP gene for energized high-affinity adenine uptake in Escherichia coli is localized to a restriction fragment between tnaB and bglR . Comparison with DNA sequence data (Burland et al . 1993) indicates the involvement of the open-reading frame f445 (86003 < 84666) . A non-energized transport route of lower affinity also occurs . The purP system is known to require metabolic removal of transported adenine which would diminish futile cycling by the two transport systems and toxicity by external adenine . Analysis of a conventional kinetic model for permeases suggests that internal adenine might inhibit transport sufficiently to account for the lack of accumulation. Biochemistry, 1994 Feb 22, 33(7), 1951 - 60 Pre-steady-state kinetics of the microtubule-kinesin ATPase; Gilbert SP et al.; The pre-steady-state kinetics of the microtubule-kinesin ATPase were investigated by chemical-quench flow methods using the Drosophila kinesin motor domain (K401) expressed in Escherichia coli {Gilbert, S . P., & Johnson, K . A . (1993) Biochemistry 32, 4677-4684} . The results define a minimal mechanism: M.K + ATP in equilibrium with (M).K.ATP in equilibrium with (M).K.ADP.Pi in equilibrium with M.K.ADP + Pi in equilibrium with M.K + ADP, where M, K, and Pi represent microtubules, kinesin, and inorganic phosphate, respectively, with k+1 = 0.8-3 microM-1 s-1, k-1 = 100-300 s-1, k+2 = 70-120 s-1, k+4 = 10-20 s-1, and k+3 >> k-2 and k+3 >> k+4 . Conditions were as follows: 25 degrees C, 20 mM HEPES, pH 7.2 with KOH, 5 mM magnesium acetate, 0.1 mM EDTA, 0.1 mM EGTA, 50 mM potassium acetate, 1 mM DTT . The experiments presented do not determine the step in the cycle where kinesin dissociates from the microtubule or the step at which kinesin reassociates with the microtubule; therefore, the steps that may represent kinesin as the free enzyme are indicated by (M) . A burst of ADP product formation was observed during the first turnover of the enzyme in the acid-quench experiments that define the ATP hydrolysis transient . The observation of the burst demonstrates that product release is rate limiting even in the presence of saturating microtubule concentrations . The pulse-chase experiments define the time course of ATP binding to the microtubule-K401 complex . At low ATP concentrations, ATP binding limits the rate of the burst . However, at high concentrations of ATP, ATP binding is faster than the rate of ATP hydrolysis with k+2 = 70-120 s-1 . The amplitude of the burst of the ATP binding transient reached a maximum of 0.7 per site at saturating concentrations of ATP and microtubules . The amplitude of less than 1 is attributed to the fast k(off) for ATP (k-1 = 100-300 s-1) that leads to a partitioning of the M.K.ATP complex between ATP hydrolysis (k+2) and ATP release (k-1) . These results indicate that ATP binds weakly to the M.K complex (Kd,ATP app approximately 100 microM) . ADP release (k+4 = 10-20 s-1) is rate limiting during steady-state turnover, indicating that microtubules activate the kinesin ATPase by increasing k(off),ADP from 0.01 s-1 in the absence of microtubules to 10-20 s-1 at saturating microtubule concentrations. Biochemistry, 1994 Feb 22, 33(7), 1907 - 14 Replacement of the active-site cysteine residues of DsbA, a protein required for disulfide bond formation in vivo; Zapun A et al.; DsbA is a periplasmic protein of Esch