Eur Surg Res, 1992, 24(5), 298 - 301 Effect of activated protein C on impaired fibrinolysis in rats with obstructive jaundice; Uchino R et al.; We studied the effect of activated protein C (APC) on impaired fibrinolysis using a rat model in which disseminated intravascular coagulation (DIC) is induced by the intravenous injection of endotoxin in rats with obstructive jaundice . An intravenous injection of endotoxin in rats with obstructive jaundice resulted in pulmonary hemorrhages and a marked increase in the plasma levels of tissue-type plasminogen activator (t-PA) antigen and plasminogen activator inhibitor activity . Prophylaxis with APC before the injection of endotoxin resulted in a decrease of the number of lung hemorrhages and an accelerated release of t-PA antigen . Thus, DIC in obstructive jaundice may be due to impairment of fibrinolysis and an increased susceptibility of endothelial cells to endotoxin . APC may be effective as a treatment for patients with obstructive jaundice associated with DIC. Environ Mol Mutagen, 1992, 20(4), 297 - 306 Assessment of oxidative DNA damage in the oxyR-deficient SOS chromotest strain Escherichia coli PQ300; Muller J et al.; The SOS chromotest is a simple short-term genotoxicity assay measuring the induction of gene sfiA in Escherichia coli K-12 . The recent availability of SOS tester strains with additional mutations in DNA repair or protection systems allows testing of DNA damaging compounds for genotoxic specificity . E . coli PQ300 differs from the standard SOS tester strain PQ37 in that it contains an additional mutation in gene oxyR that renders it more sensitive to oxidative genotoxins . The generation of reactive oxygen intermediates (ROI) by hydroperoxides (H2O2, t-butyl hydroperoxide, cumene hydroperoxide), gamma-radiation, glucose oxidase, and xanthine oxidase resulted in a more vigorous SOS response in strain PQ300 compared to strain PQ37 . PQ300 was also more sensitive than PQ37 for the detection of reducing agents such as ascorbic acid, cysteine, and glutathione, which also alter the redox status of the bacterial cells . However, intercalating agents (adriamycin, bleomycin, and mitomycin C) and the UV- and radiomimetic compound 4-nitroquinoline-1-oxide whose DNA damaging potential are known also to involve ROI did not show significant differences between strains PQ37 and PQ300 . It is concluded that the oxyR-deficient strain PQ300 is useful for detecting certain classes of genotoxins that change the oxidative/antioxidative balance of tester bacteria in the SOS chromotest. Curr Eye Res, 1992, 11 Suppl, 113 - 7 Pathogenicity and immunogenicity of recombinant human retinal S-antigen fusion protein; Kasp E et al.; A full-length cDNA clone to human S-antigen (HS-ag) was isolated from lambda gt 10 human retinal library and expressed as a fusion protein with glutathione S-transferase (GST) in E . Coli . Uveitogenicity and immunogenicity of recombinant GST-HS-ag fusion protein and native HS-ag were compared in EAU-susceptible Lewis rats . Recombinant HS-ag was found less uveitogenic than native HS-ag . Animals inoculated with recombinant HS-ag developed EAU on day 17, three days later than those inoculated with native HS-ag, the incidence of the disease was reduced from 80% to 58% and the score of clinical severity reduced from 2.2 to 1.3 points respectively . In contrast, rGST-HS-ag was more immunogenic than native HS-ag as it elicited four times higher levels of antibodies which reacted specifically with both antigens. J Biomol NMR, 1992 Jan, 2(1), 71 - 82 A 1H-15N NMR study of human c-Ha-ras protein: biosynthetic incorporation of 15N-labeled amino acids; Yamasaki K et al.; A 1H-15N NMR study was performed on the GDP-bound form of a truncated human c-Ha-ras oncogene product (171 amino acid residues) . Resonance cross peaks of the backbone amide 1H-15N nuclei of a uniformly 15N-labeled protein were observed with heteronuclear single-quantum coherence spectroscopy (HSQC) . In order to resolve overlapping cross peaks, selective 15N-labeling of one or two types of amino acid residues (Ala, Arg, Asx, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Tyr and/or Val) was carried out using appropriate E . coli mutant strains . By this procedure, all the backbone 1H-15N cross peaks were classified into amino acid types. Anim Genet, 1992, 23(5), 437 - 41 Characterization of a porcine variable number tandem repeat sequence specific for the glucosephosphate isomerase locus; Davies W et al.; A variable number of tandem repeat from a porcine glucosephosphate isomerase intron has been isolated and sequenced . The repeat has a unit size of 39 bp, is highly conserved and is present in at least 14 copies . Flanking sequences show a sequence periodicity of 53-54 bp and some sequence homology to the 39 bp repeat . A considerable part of the genomic DNA has been lost during subcloning and is considered to be deletion prone or refractory to propagation in E . coli . The tandem repeat is locus specific and detects at least six alleles in BamHI digested porcine DNA . No homology to other tandem repeat sequences has been found. Am J Nephrol, 1992, 12(1-2), 80 - 5 Inhibition of cytokine synthesis by peritoneal dialysate persists throughout the CAPD cycle; Jorres A et al.; The current study focused on the effect of continuous ambulatory peritoneal dialysis (CAPD) dialysate obtained following different intraperitoneal dwell periods on the release of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF alpha) from mononuclear leukocytes (PBMC) . Aliquots of 5 x 10(6)/ml healthy peripheral PBMC were exposed to fresh or spent CAPD dialysate (10-240 min of intra-peritoneal dwell) and stimulated with Escherichia coli endotoxin (10 micrograms/ml, 2h) . IL-6 and TNF alpha in cell supernatants were determined by specific enzyme immunoassays . Control PBMC in physiological buffer released 361 +/- 70 pg/ml IL-6 and 717 +/- 147 pg/ml TNF alpha (mean +/- SEM, n = 8), whereas exposure to fresh dialysis fluids severely suppressed cytokine release from PBMC (less than 30 pg/ml IL-6 and less than 15 pg/ml TNF alpha) . A significant inhibition of IL-6 and TNF alpha release was also observed in PBMC exposed to spent dialysate . The inhibitory capacity of the spent fluids was pronounced with increasing intra-peritoneal dwell time (10 min: 183 +/- 45 pg/ml IL-6 and 538 +/- 109 pg/ml TNF alpha; 240 min: 26 +/- 5 pg/ml IL-6 and 105 +/- 30 pg/ml TNF alpha; mean +/- SEM, n = 16) . These data indicate that the impairment of cell responsiveness following exposure of PBMC to peritoneal dialysate is not restricted to the unused fluids, but is also observed following intra-peritoneal equilibration . Moreover, our findings suggest the presence of cytokine inhibitory factors in the peritoneal dialysate of CAPD patients which appear to accumulate in the peritoneal effluent during the CAPD cycle. Zh Mikrobiol Epidemiol Immunobiol, 1992 Jan, (1), 8 - 11 {Sterol transformation by Escherichia}; Panchishina MV; The use of the Liebermann-Burhard reaction and the thin-layer chromatography of nonsaponifiable lipids of culture medium (donor blood serum) permitted the isolation of three biological variants of Escherichia in the process of their 48-hour cultivation in this medium . The cholesterol-destroying variant of Escherichia is characterized by a decrease in the content of total, free, esterified cholesterol and a decrease in the occurrence of fractions corresponding to cholesterol, delta 4-cholestenone-3, delta 5-cholestenone-3), as well as nonsaponifiable lipid, where Rf was equal to 0.36; two fractions of labeled nonsaponifiable lipids, not corresponding to cholesterol, appeared on plasma with sodium acetate-1-14C . Cholesterol-transforming biovars produced insignificant changes in the content of chemically determined cholesterol in the medium, but in plasma nonsaponifiable lipid with Rf = 0.26 and other less polar lipids were found . Escherichia strains increasing the amount of chemically determined cholesterol in the process of their growth more frequently transformed or used nonsaponifiable lipids with Rf = 0.26 and 0.42 . As a rule, the occurrence of cholesterol and less polar lipids increased . The sodium acetate-1-14C was incorporated into 3-4 fractions of nonsaponifiable lipids, one of them being identified as cholesterol. Ophthalmic Res, 1992, 24(3), 175 - 80 Aqueous humor polyamines and alkaline phosphatase activity in endotoxin-induced uveitis: correlations to diverse leukocyte subsets; Wickstrom K et al.; The polyamines putrescine, spermidine and spermine have been proposed to be a part of the acute phase inflammatory response . They have been shown to be useful markers for cellular kinetics and change with various pathological conditions . The hypothesis that aqueous humor polyamines could be used to follow the time course of an endotoxin-induced inflammation in the eye was investigated . Additional parameters studied were the amount of aqueous leukocytes, alkaline phosphatase activity, distribution of leukocyte subsets and breakdown of the blood-aqueous barrier . Aqueous leukocytes, protein, alkaline phosphatase activity, putrescine and acetylated spermidine increased significantly as a response to inflammation during the first days after uveitis induction . Spermidine decreased 24 h after injection and seemed to rise thereafter . The different polyamines, except spermidine, correlated to the diverse infiltrating leukocyte subsets . These observations indicate that aqueous polyamines may be applied as valid markers for inflammation in the eye. J Clin Lab Anal, 1992, 6(4), 232 - 8 A new sensitive microplate assay of plasma endotoxin; Tamura H et al.; We have developed a microplate method for determining endotoxin in platelet-rich plasma-using Endospecy, an endotoxin-specific chromogenic Limulus test reagent . Nonspecific activators and inhibitors of the test were eliminated by exposing samples (5 microliters) to the alkali reagent consisting of KOH, CaCl2, Triton X-100, ethyleniminepolymer and N,N-bis(2-hydroxyethyl)glycine . The recoveries of various endotoxins were almost complete and not enhanced by dilution . The dose-response curve was linear over endotoxin concentrations of 2-400 pg/ml with good precision (C.V . less than 5.0%) . Normal human plasmas (n = 30) contained less than 5.0 pg/ml of endotoxin in reference to that of Escherichia coli 0111: B4 . All plasma samples with high concentration of endotoxin by a conventional method showed high values by the microplate assay as well . Since it does not require centrifugation, the new treatment allows the whole reactions to proceed on the same microplate . This permits us to apply the Limulus test to an automated assay system, making plasma endotoxin determination simpler and more rapid than a conventional test tube method. Hereditas, 1992, 117(1), 1 - 9 RecA-like strand-transfer activity at the meiotic prophase in Bombyx mori; Wischmann B; An ATP-independent strand-transfer activity has been identified in nuclear extracts prepared from Drosophila tissue culture cells and isolated nuclei from Bombyx testes . Extraction of the activity from testes at larval stages where the majority of the cells were in meiotic prophase was only possible when the chromosome scaffold/synaptonemal complex was dissolved by addition of high concentrations of DTT (80 mM) . No cross reaction was detected when partly purified extracts were assayed with antibodies against E . coli RecA protein. Cytogenet Cell Genet, 1992, 61(2), 128 - 31 A chromosome 1-specific DNA library from the domestic pig (Sus scrofa domestica); Miller JR et al.; Chromosomes were prepared from lymphocytes of a male domestic pig and flow-sorted on a dual-laser FACS . Twenty spots were observed, corresponding to the known pig karyotype of 18 pairs of autosomes plus the X and Y . DNA was isolated from 10,000 copies of the presumed chromosome 1 spot, restricted with Sau3A, ligated into the vector pGEM4z, and PCR amplified using universal primers; the products were then re-ligated into pUC18 . After transformation into Escherichia coli, 210,000 independent colonies were obtained, 5% of which contained only vector DNA . The average insert size of the library was 405 bp . Southern blotting revealed that 36% of the clones contained single-copy DNA and that the remainder contained moderately or highly repetitive DNA . Screening with a (CA)n probe revealed that roughly 1% of the clones contained microsatellite sequences . A bulk insert of the library was biotinylated by PCR and used as a probe for chromosomal in situ suppression hybridization to pig chromosomes, which confirmed that the library is specific for chromosome 1 . However, sequences from the centromeric and telomeric regions seem to be underrepresented in the library. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 1992, 10(2), 82 - 5 {Chemical synthesis and cloning of Plasmodium falciparum 45 peptide antigen gene}; Qiu MY et al.; We have synthesized a 162 bp gene of human Plasmodium falciparum hybrid peptide antigen by the solid-phase phosphoramidite method with ABI 381A DNA synthesizer . The gene encodes three fragments of the relative molecules 83 kDa, 55 kDa and 35 kDa merozoite-specific proteins and two CS repeats or four peptides . The gene with the designed two cohesive ends was divided into 8 fragments to be synthesized . All synthetic fragments were annealed and ligated with T4 DNA ligase to form double DNA chain . This synthetic gene was recombined with P-Blue script as vector and transformed into E . coli JM109 . The positive recombinants were screened out by dot hybridization and enzyme analysis . The DNA sequence analysis showed that the synthesized human Plasmodium falciparum hybrid peptide antigen gene was identical with the designed one . (Figs . 1-4). Cancer Immunol Immunother, 1992, 35(5), 307 - 14 Significant antitumor effect of a synthetic lipid A analogue, DT-5461, on murine syngeneic tumor models; Kumazawa E et al.; The antitumor effect of a synthetic lipid A analogue, DT-5461, was investigated using syngeneic tumor models in mice . Intravenous injection of DT-5461 into mice transplanted with solid tumors of MethA fibrosarcoma, MH134 hepatoma, MM46 mammary carcinoma, Lewis lung carcinoma (3LL), and colon adenocarcinomas 26 and 38 resulted in significant reductions in the weight of all tumors except Colon 26, with marked hemorrhagic necrosis of tumor tissues . Efficacy was almost equal to that of an Escherichia coli-type synthetic lipid A (compound 506), and also to those of some chemotherapeutics including Adriamycin, mitomycin C, fluorouracil and cisplatin . Furthermore, DT-5461 was more effective than other immunotherapeutics, including picibanil (OK-432) and lentinan . However, its antitumor effects were inferior to those of Adriamycin or OK-432 against the malignant ascites caused by intraperitoneal inoculation with MethA or with MH134 cells; life span was not prolonged by either intraperitoneal or intravenous administration . In addition, although DT-5461 showed direct inhibitory effects on the in vitro growth of MethA or MH134, these were much weaker than those of Adriamycin . These findings clearly indicated that DT-5461 with systemic administration is a highly effective antitumor agent on solid tumors, and suggest that the antitumor effect of DT-5461 with potent necrotizing activity might derive from indirect mechanisms related to the activation of host immune systems and not to the weak direct cytotoxicity. Biometals, 1992 Spring, 5(1), 37 - 46 Identification of the ferrioxamine B receptor, FoxB, in Escherichia coli K12; Nelson M et al.; The photoreactive p-azidobenzoyl analog of ferrioxamine B was used to show that ferrioxamine-B-mediated iron transport is separate and distinct from coprogen-mediated iron transport in Escherichia coli . Photolysis of this analog inhibited uptake of {59Fe}ferrioxamine B but not {59Fe}ferrichrome . Conversely, photolysis of the p-azidobenzoyl analog of coprogen B inhibited uptake of {59Fe}coprogen but not {59Fe}ferrioxamine B or {59Fe}ferrichrome . Photolabeling of outer membranes with p-azidobenzoyl-{59Fe}ferrioxamine B resulted in the labeling of two iron-regulated peptides with molecular masses of about 66 and 26 kDa . Expression of these peptides was increased when ferrioxamine B was the sole iron source . Both peptides were present in outer membrane preparations of the fhuF mutant H1717, but the 66 kDa peptide was not inducible . These results are evidence for an outer membrane receptor in E . coli unique for linear ferrioxamines. C R Acad Sci III, 1992, 315(1), 1 - 6 {Total chemical synthesis of natural transfer RNA}; Gasparutto D et al.; New improvements in the chemical synthesis of oligoribonucleotides are reported and they are applied to the first total chemical synthesis of a natural RNA . This E . coli K12 alanine tRNA contains in its sequence dihydrouridine, ribothymidine and pseudo-uridine . The synthetic tRNA was fully sequenced and showed a 42% aminoacyl acceptance activity . When tRNA was used as a template, reverse transcriptase directed the incorporation of adenine opposite dihydrouridine, ribothymidine and pseudouridine. Cancer Immunol Immunother, 1992, 35(6), 395 - 400 Effects of lipopolysaccharide on interleukin-2-induced cytotoxic activity of murine splenocyte cultures: role of prostaglandin E2 and interferons; Vaillier D et al.; Splenocytes cultured for 24 h in the presence of interleukin-2 (IL-2), lipopolysaccharide (LPS) or both together expressed a cytotoxic activity against the YAC-1 lymphoma cell line and to a lesser extent against P815 mastocytoma cells . The association of IL-2 and LPS had an additive effect on induction of cytotoxicity . The IL-2-induced cytotoxic activity lasted for the whole of the culture; however, the addition of LPS at the initiation of the culture increased the cytotoxic activity during its the early phase, the increment being followed by a fall of lytic activity after 72 h of culture . Assessment of interferon (IFN) in the culture supernatants showed (a) a production of IFN gamma by IL-2-supplemented cultures, (b) a more potent IFN production by cultures treated with IL-2 plus LPS (including 20% IFN alpha/beta, (c) and that indomethacin (1 microM) potentiated the effect of either IL-2 or LPS used alone but did not significantly increase the cytotoxic activity of cultures treated with IL-2 plus LPS (the one that produced a high level of IFN) . When cultures were treated by an anti-IFN gamma antibody we observed no change in the cytotoxic activity; however, in the presence of anti-IFN alpha/beta serum the cytotoxic activity of cultures treated with IL-2 plus LPS was inhibited after 24 h but stimulated after 72 h . When cultures treated with IL-2 plus LPS were supplemented with both indomethacin and anti-IFN alpha/beta the cytotoxic activity assessed after 72 h of culture was maintained at the same level as that of IL-2-treated cultures, hence the fall after 72 h of the cytotoxicity of cultures initiated in the presence of LPS alone was affected by both the immune serum and the cyclooxygenase inhibitor . Altogether these data show that when splenocytes are cultured for more than 72 h in the presence of IL-2 and LPS their cytotoxic activity decreases, and it is likely that this diminution is linked to the endogenous production of prostaglandin E2 and INF alpha/beta. Biomater Artif Cells Immobilization Biotechnol, 1992, 20(2-4), 1051 - 7 Inhibition of endotoxin-mediated activation of endothelial cells by a perfluorocarbon emulsion; Lane T et al.; Endothelial cell (EC) activation plays a key role in the inflammatory response by promoting the margination of leukocytes in inflamed loci . Augmented leukocyte margination to activated EC is mediated by the increased display of leukocyte adhesion molecules on EC surface membranes . The biocompatibility of synthetic oxygen-transport fluids is intimately linked to EC function, since one of the first tissues encountered by such fluids is the vascular endothelium . We investigated the effect of one such agent, a phospholipid-based perfluorocarbon emulsion containing 90% w/v perflurooctyl bromide (perflubron, PFOB) on EC activation . Human umbilical vein EC (HUVEC) were activated by 5 U/ml interleukin-1 (IL-1), 20 U/ml tumor necrosis factor (TNF), or 50 ng/ml E coli endotoxin (LPS) in the presence or absence of up to 20%, w/v perflubron . HUVEC activation was monitored by the extent of up-regulation of expression of intercellular adhesion molecule-1 (ICAM) and endothelial-leukocyte adhesion molecule-1 (ELAM) . Exposure of HUVEC to perflubron did not alter the up-regulation of ICAM or ELAM in response to IL-1 or TNF (n = 20) . However, at 10% perflubron ICAM up-regulation in response to LPS was inhibited by 95 +/- 6% (n = 9; p less than .05) . ELAM expression was similarly affected . The concentration of perflubron required to diminish LPS-induced up-regulation by 50% was 6.0 +/- 0.6% (n = 3) . The inhibitory effect of 10% perflubron was overcome by greater than 1 ug/ml LPS (n = 3) and the inhibitory effect was attenuated by adding perflubron to the cultures after LPS.(ABSTRACT TRUNCATED AT 250 WORDS) Annu Rev Biophys Biomol Struct, 1992, 21, 379 - 415 The single-nucleotide addition cycle in transcription: a biophysical and biochemical perspective; Erie DA et al.; This review has summarized the known features of the single-nucleotide addition reaction cycle in transcription . The reader will have noted that the information available is very incomplete, and that, in some cases, related experiments seem to lead to contradictory conclusions . We have tried to point out these discrepancies as they occur and to indicate areas where more experimentation is needed . We look forward to the day when all the microscopic steps of the single-nucleotide addition cycle can be identified and defined in thermodynamic, kinetic, and structural terms . At that point, we can begin to understand the principles that relate these parameters to template position and to the pathway of formation of a specific complex . It should be possible to provide specific molecular interpretations for observed effects on activation barrier heights to elongation and termination (154, 155) and to begin to understand the molecular bases of the regulation in these phases of transcription . Much work remains before this happy situation can be totally realized, but we feel that now the problem can at least be approached at this level . We hope that this review helps to illuminate the difficulties that remain. Bioorg Khim, 1992 Jan, 18(1), 71 - 7 {Chemical-enzymatic synthesis and cloning in Escherichia coli of a gene coding for human granulocyte-colony stimulating factor}; Kash'ian SK et al.; Two artificial genes, encoding two forms of human granulocyte colony stimulating factor as products of a normal and an alternative splicing, have been by a chemical-enzymatic way synthesized and cloned in Escherichia coli . The genes are supplied with recognition sites of restriction endonucleases to facilitate the further cassette mutagenesis. Arch Virol, 1992, 126(1-4), 321 - 8 Immunogenicity of recombinant core particles of hepatitis B virus containing epitopes of human immunodeficiency virus 1 core antigen; Ulrich R et al.; A Gag protein segment of human immunodeficiency virus 1 (HIV-1) has been fused to a C terminally truncated core antigen of hepatitis B virus (HBcAg) using an E . coli expression system . Fusion of 90 amino acids of HIV-1 Gag protein to HBcAg still allowed the formation of capsids presenting on their surface epitopes of HIV-1 core protein, whereas fusion of 317, 189, or 100 amino acids of Gag prevented self-assembly of chimeric particles . Mice immunized with recombinant particles emulsified with Freund's complete adjuvant (CFA) or aluminium hydroxide developed high anti-HBcAg titers . However, anti-HIVp24 antibodies were detected only in mice inoculated with immunogen emulsified with CFA. Mutat Res, 1992, 280(3), 205 - 14 Genotoxicity assessment of pirmenol, a new antiarrhythmic drug; Ciaravino V et al.; The genotoxicity of pirmenol was tested in the E . coli and S . typhimurium mutagenesis assay, an in vitro mammalian cell chromosome-aberration assay and an in vivo mouse micronucleus assay . The E . coli tester strain WP2s was exposed to concentrations of pirmenol as high as 10,000 micrograms/plate both in the absence (S9-) and presence (S9+) of metabolic activation . Five strains of S . typhimurium (TA98, TA100, TA1535, TA1537, TA1538) were exposed to concentrations of pirmenol as high as 5000 micrograms/plate in the absence and presence of S9 . Pirmenol was not mutagenic toward either E . coli or S . typhimurium . Chinese hamster lung V79 cell cultures were exposed to pirmenol at concentrations of 500-2500 micrograms/ml (S9-) and 500-3000 micrograms/ml (S9+) . Pirmenol increased the frequency of structural chromosome aberrations (SCAs) . The minimum clastogenic concentration was 1500 micrograms/ml (both S9- and S9+) with a peak clastogenic response of 6% (S9-) and 34% (S9+) cells with aberrations . Although there were statistically significant results in the S9- experiment, the percent cells with aberration values for treated groups were within the historical control range (0-6%) of this laboratory . The observed effects in both the absence and presence of S9 appear at high concentrations compared to human circulating plasma levels of 1-3 micrograms/ml and the clastogenicity was confined to chromosome gaps and breaks . Consequently, this in vitro effect would not be expected to be reflected by either in vivo clastogenic or carcinogenic activity . This was supported by findings in the mouse micronucleus study of pirmenol in which single oral doses administered to male CD-1 mice at 5, 55, or 115 mg/kg (80% LD50) produced no statistically significant increases in the frequency of micronucleated polychromatic erythrocytes in bone marrow at 24, 48 or 72 h postdosing . Additionally, no evidence of carcinogenicity was seen in a mouse or rat bioassay. Mutat Res, 1992, 280(3), 195 - 203 The genotoxicity of organotin compounds in SOS chromotest and rec-assay; Hamasaki T et al.; In these days pollution by organotin compounds in the environment extends widely and effects on human health are feared . We studied the genotoxicity of various organotin compounds (butyltins, phenyltins, methyltins) and inorganic tin (SnCl4), which are present in the environment, with the SOS chromotest and the rec-assay . Mono-n-butyltin oxide, n-butyltin trichloride and di-n-butyltin dichloride showed high SOS-inducing potency in the SOS chromotest with Escherichia coli PQ37 . Di-n-butyltin dichloride, tri-n-butyltin chloride, bis(tri-n-butyltin)oxide, dimethyltin dichloride and trimethyltin chloride were recognized as genotoxic chemicals by the rec-assay. Mol Biol (Mosk), 1992 Jan-Feb, 26(1), 70 - 82 {Cloning and expression in Escherichia coli of reverse transcriptase coded by the mobile genetic element jockey}; Ivanova VA et al.; The mobile element jockey is similar in structural organization and coding potential to the LINEs of various organisms . Current models of the mechanism of transposition involve reverse transcription of an RNA intermediate and utilization of element-encoded proteins . As it is demonstrated here, a 2.23 kb DNA fragment from the region of the jockey encoding the putative reverse transcriptase, was stably introduced into the expression system under inducible control of the Escherichia coli lac regulatory elements . We describe the expression of the 92 kDa protein and identify this polypeptide alone as authentic jockey reverse transcriptase based on some of its physical and enzymic properties . The jockey polymerase demonstrates RNA-directed and DNA-directed DNA polymerase activities, but lacks detectable RNase H, has a temperature optimum at 26 degrees C, requires Mg2+ or Mn2+ as a cofactor and is inactivated by sulfhydryl reagent . The enzyme prefers poly(rC) and poly(rA) as template and "activated" DNA is not effective . The results of this work suggest that the RNA-directed DNA polymerase coded by jockey elements may be involved in the transcription of the elements. Environ Mol Mutagen, 1992, 20(2), 127 - 33 Effects of Captan on DNA and DNA metabolic processes in human diploid fibroblasts; Snyder RD; The fungicide Captan has been examined for its effects on DNA and DNA processing in order to better understand the genotoxicity associated with this agent . Captan treatment resulted in production of DNA single strand breaks and DNA-protein cross-links and elicited an excision repair response in human diploid fibroblasts . Captan was also shown to inhibit cellular DNA synthesis and to form stable adducts in herring sperm and human cellular DNA . Misincorporation of nucleotides into Captan-treated synthetic DNA templates was significantly elevated in an in vitro assay using E . coli DNA polymerase I, suggesting that DNA adduct formation by Captan could have mutagenic consequences . In sum, these studies demonstrate that Captan is capable of interacting with DNA at a number of levels and that these interactions could provide the basis for Captan's genotoxicity . The extreme cytotoxicity of this fungicide, however, could be due to other cellular effects since at the IC50 for cell killing, approximately 0.8 microM, none of the above genotoxic events could be detected by the methods employed. Mutagenesis, 1992 Jan, 7(1), 41 - 6 Mutagenesis of Escherichia coli: a method for determining mutagenic specificity by analysis of tRNA suppressors; Sledziewska-Gojska E et al.; A method for estimating mutagenic specificity in Escherichia coli (argE3, hisG4, thr-1, supE44), based upon the isolation of Arg+ or His+ revertants and identification of tRNA suppressors, is described . The method gives an insight not only into mutagenic pathways but also into the functioning of tRNA . With N-methyl-N'-nitro-N-nitrosoguanidine, 98% of mutations are GC----AT transitions . With N4-hydroxycytidine, 100% are AT----GC transitions . With hydroxylamine, apart from GC----AT transitions, approximately 30% of Arg+ revertants are formed by GC (or AT)----TA transversions . When the chemistry of the mutagenic attack is known, the method allows us to discriminate whether mutations occur on the transcribed or non-transcribed strands of DNA . It has been found that reversion of argE3 to Arg+ is a better monitor of mutagenic pathways than reversion of hisG4 to His+. Genetica, 1992, 85(2), 107 - 17 A nitrate reductase gene of the cyanobacterium Synechococcus PCC6301 inferred by heterologous hybridization, cloning and targeted mutagenesis; Lightfoot DA et al.; DNA probes from the narG gene of Escherichia coli, which encodes the large polypeptide of respiratory nitrate reductase, show cross-hybridization at low stringency to a single region of the genome of the cyanobacterium Synechococcus PCC6301 . This segment of cyanobacterial DNA was cloned as the insert of plasmid pDN1 and characterized . RNA complementary to pDN1 was shown to be substantially more abundant in nitrate grown cells of Synechococcus PCC6301 than in ammonium grown cells, thus parallelling the nitrate induction and ammonium repression of nitrate reductase activity in cultures of this cyanobacterium . A mutant of Synechococcus PCC6301 deficient in nitrate reductase activity was obtained after a potentially mutagenic transformation treatment using pDN1 as a donor . This mutant was restored to the wild type phenotype following stable integrative transformation with pDN1 DNA . Taken together these data suggest that pDN1 might encode a polypeptide of nitrate reductase . pDN1 is distinct from three clones of genes involved in nitrate assimilation that were isolated previously from the related cyanobacterium Synechococcus PCC7942 (Kuhlemeier et al., 1984a, J . Bact . 159, 36-41, and 1984b, Gene 31, 109-116). J Acquir Immune Defic Syndr, 1992, 5(7), 647 - 57 Epitope mapping of HIV-1 reverse transcriptase with monoclonal antibodies that inhibit polymerase and RNase H activities; Szilvay AM et al.; Lysates from E . coli expressing HIV-1 reverse transcriptase (RT) as a TrpE fusion protein were used for immunization of BALB/c mice . Twenty hybridomas producing monoclonal antibodies (MAbs) recognizing the RT part of the TrpE-RT fusion protein by Western blot analysis were isolated . Of these, 18 were reactive in immunofluorescence assays when tested on HIV-infected cells . Twelve MAbs were reactive with both the p66 and p51 fragments of RT, while 6 of the MAbs were reactive only with the p66 band, indicating specificity for the C-terminal (RNase H) region of RT . Mapping of the monoclonal antibody binding sites was performed using deletion and insertion mutants of recombinant RT . The antibodies bound to five distinct regions within amino acid sequences 190-560 of RT . In order to map functionally important regions of the RT molecule, the MAbs were tested for their ability to interfere with the polymerase and RNase H activities of the polypeptide . MAbs binding to two different epitopes in the polymerase domain were found to inhibit the polymerase activity . Of these, three MAbs also inhibited the RNase H activity . Two MAbs binding to the same epitope in the RNase H region inhibited RNase H activity and further mediated an effect on the polymerase activity. Autoimmunity, 1992, 11(3), 141 - 9 Mapping epitope specificities of monoclonal antibodies to thyroid peroxidase using recombinant antigen preparations; Ewins DL et al.; Five separate monoclonal antibodies (MoAbs) to human thyroid peroxidase (hTPO) were raised by immunising Balb/c mice with hTPO purified from detergent solubilised thyroid microsomes by high performance liquid chromatography (HPLC) . The epitope specificities of these MoAbs were determined by assessing their ability to bind to purified recombinant fusion protein fragments of human TPO (TPO(r)) generated in E . coli . A total of seven small overlapping fragments (averaging 104 amino acid residues) of hTPO, encompassing over 90% of the extracellular region of the molecule, were generated as glutathione S-transferase (GST) fusion proteins . The sequential epitopes on TPO(r) recognised by these MoAbs were analysed by both immunoblotting and enzyme linked immunosorbent assay (ELISA) . Two different MoAbs (A4 and A5) recognised sequential epitopes within the TPO(r) preparation termed R1a + b (residues 1-160) and more specifically, in the case of MoAb A4, within the subfragment R1b (residues 70-160) . The inability of the other MoAbs (A1-A3) to recognise recombinant fragments, suggests they either recognise conformational determinants on the TPO molecule or epitopes that are present on the small regions of the TPO molecule which have not been expressed as recombinant proteins. Mol Microbiol, 1992 Jan, 6(2), 247 - 55 Characterization of the antigenic and adhesive properties of FaeG, the major subunit of K88 fimbriae; Bakker D et al.; The two K88 serotypes, K88ab and K88ac, differ in terms of antigenic and adhesive properties . The structural determinants of the serotype-specific epitopes and the identify of the amino acid residues involved in fimbriae-receptor interaction were studied by the construction and analysis of K88 hybrid proteins in which various parts of the K88ab and K88ac fimbrial subunit FaeG were exchanged, and by in vitro mutagenesis of non-conserved amino acid residues . Using a set of monoclonal antibodies, several regions or amino acid residues involved in the formation of serotype-specific antigenic determinants were located . The haemagglutinating activity of the hybrid and mutant proteins revealed several amino acid residues involved in the formation of the receptor binding site . A clear correlation was found between the receptor binding site and the serotype-specific antigenic determinants. Am J Vet Res, 1992 Jan, 53(1), 36 - 43 Development of monoclonal antibody ELISA for simultaneous detection of bovine coronavirus, rotavirus serogroup A, and Escherichia coli K99 antigen in feces of calves; Thorns CJ et al.; A rapid ELISA was developed for simultaneous detection of bovine coronavirus (BCV), rotavirus (RV) serogroup A, and Escherichia coli K99 antigen in feces of calves . A mixture of 3 monoclonal antibodies specific for BCV, RV, or K99 was used successfully to capture the antigens; the same antibodies labeled with peroxidase were used to detect BCV, RV, or K99 . The triple ELISA was compared with standard reference diagnostic methods by examining feces from experimentally and naturally infected and healthy calves . All the components of the test were highly specific (greater than 90%) and sensitive (BCV, 77%; K99, 93%; RV, 100%) when used in a format requiring short incubation steps at 20 C and visual recording of results. Mutat Res, 1992 Jan, 281(1), 63 - 6 Inducible stable DNA replication in Escherichia coli uvr+ and uvr- cells, treated with genotoxic chemicals; Masek F et al.; Inducible stable DNA replication (iSDR) provoked by a damaging treatment with MMS, MNU, MNNG, NFAA, NFN, 4NQO, NAL or MMC, was followed in both repair-competent E . coli PQ35 and its uvrA derivative E . coli PQ37 . In contrast to SOS-inducible mutagenesis, which is more pronounced in excision-deficient cells, iSDR was more obvious in repair-competent cells . This may be due to special features of iSDR and need not indicate involvement of the uvrA gene product in it. Biotechniques, 1992 Jan, 12(1), 104 - 13 Sensitive chemiluminescent detection of digoxigenin-labeled nucleic acids: a fast and simple protocol and its applications; Holtke HJ et al.; A fast and simple protocol for the chemiluminescent detection of digoxigenin-labeled nucleic acids with anti-digoxigenin antibody Fab fragments coupled to alkaline phosphatase and 3-(4-methoxyspiro{1,2-dioxetane-3,2'-tricyclo-{3.3.1.1 (3,7)}decan}-4- yl)phenyl phosphate as substrate is described . The washing and blocking procedure was optimized to yield low background even on positively charged nylon membranes . The sensitivity of the system is equal or better than radioactive methods . Exposure to x-ray or Polaroid film for up to 30 minutes is sufficient for the detection of 70 femtograms of homologous DNA . Human single-copy genes are detected in Southern blots of as low as 0.3 microgram total placental DNA . Blots can be reprobed multiple times very easily . The advantages of the digoxigenin system are high sensitivity, absence of background and ease of reprobing and are illustrated by applications for single-copy gene detection in genomic blots of human DNA, Northern hybridizations to rare mRNA, detection of E . coli genes on blots of genomic digests after pulse field gel electrophoresis, as well as for nonradioactive DNA sequencing blots with digoxigenin-labeled primers. J Exp Med, 1992 Jan 1, 175(1), 245 - 55 The rat c-kit ligand, stem cell factor, induces c-kit receptor-dependent mouse mast cell activation in vivo . Evidence that signaling through the c-kit receptor can induce expression of cellular function; Wershil BK et al.; Interactions between products of the mouse W locus, which encodes the c-kit tyrosine kinase receptor, and the Sl locus, which encodes a ligand for c-kit receptor, which we have designated stem cell factor (SCF), have a critical role in the development of mast cells . Mice homozygous for mutations at either locus exhibit several phenotypic abnormalities including a virtual absence of mast cells . Moreover, the c-kit ligand SCF can induce the proliferation and maturation of normal mast cells in vitro or in vivo, and also can result in repair of the mast cell deficiency of Sl/Sld mice in vivo . We now report that administration of SCF intradermally in vivo results in dermal mast cell activation and a mast cell-dependent acute inflammatory response . This effect is c-kit receptor dependent, in that it is not observed when SCF is administered to mice containing dermal mast cells expressing functionally inactive c-kit receptors, is observed with both glycosylated and nonglycosylated forms of SCF, and occurs at doses of SCF at least 10-fold lower on a molar basis than the minimally effective dose of the classical dermal mast cell-activating agent substance P . These findings represent the first demonstration in vivo that a c-kit ligand can result in the functional activation of any cellular lineage expressing the c-kit receptor, and suggest that interactions between the c-kit receptor and its ligand may influence mast cell biology through complex effects on proliferation, maturation, and function. J Clin Invest, 1992 Jan, 89(1), 327 - 31 The susceptibility sequence to rheumatoid arthritis is a cross-reactive B cell epitope shared by the Escherichia coli heat shock protein dnaJ and the histocompatibility leukocyte antigen DRB10401 molecule; Albani S et al.; Immunological responses to bacterial heat shock proteins have been implicated in the pathogenesis of arthritis in animals and humans . The predicted amino acid sequence of dnaJ, a heat shock protein from Escherichia coli, contains an 11-amino acid segment that is homologous to the third hypervariable region of the human histocompatibility antigen (HLA) DRB10401 (formerly known as HLA Dw4), the part of the molecule that carries susceptibility to rheumatoid arthritis . To test the biological significance of this finding, we expressed and purified recombinant dnaJ (rdnaJ), and determined its immunologic cross-reactivity with HLA DRB10401 . A rabbit antipeptide antiserum raised against the sequence of the third hypervariable region of HLA DRB10401 specifically bound to 'dnaJ, thus confirming that a similar sequence is expressed on the bacterial protein . Of greater consequence, an antiserum to the 'dnaJ protein recognized not only a peptide from the third hypervariable region of HLA DRB10401, but also the intact HLA DRB10401 polypeptide . Furthermore, the antibody to 'dnaJ reacted with HLA DRB10401 homozygous B lymphoblasts, but not with HLA DRB11501, DRB10101, DRB10301, and DRB10701 (formerly known as HLA Dw2, DR 1, DR 3, and DR 7, in the same order) homozygous cells . These results demonstrate that exposure to a bacterial heat shock protein can elicit antibodies against the rheumatoid arthritis susceptibility sequence in the third hypervariable region of HLA DRB10401. J Bacteriol, 1992 Jan, 174(2), 627 - 9 The gene for a 4.5S RNA homolog from Mycoplasma pneumoniae: genetic selection, sequence, and transcription analysis; Simoneau P et al.; In an effort to make an inventory of the tRNA genes of Mycoplasma pneumoniae, a DNA fragment was found to contain a sequence that can be folded into a hairpin structure very similar to that of the 4.5S RNA of Escherichia coli . Recombinant plasmids carrying this region were able to complement E . coli strains that were deficient in 4.5S RNA . S1 mapping showed that the mature transcript is only 79 nucleotides long. J Bacteriol, 1992 Jan, 174(1), 263 - 8 Location of the basal disk and a ringlike cytoplasmic structure, two additional structures of the flagellar apparatus of Wolinella succinogenes; Schuster SC et al.; The basal body of Wolinella succinogenes consists of a central rod, a set of two rings (L and P rings), a basal disk from 70 to 200 nm in diameter, and a terminal knob . In negatively stained preparations of flagellar hook-basal body complexes, some disks remain fixed perpendicularly to the grid and show that such a disk is located on the distal side of the P ring . The basal disks have been isolated with and without the P ring; in both cases there is a hole in the center of the disk . The diameter of the disk is smaller in the presence of the P ring . The L-P ring complex is therefore assumed to be a bushing for the rod . Thin sections of whole bacteria and spheroplasts reveal that the disk is attached to the inner surface of the outer membrane . At the insertions of the flagellar hook-basal body-basal disk complexes, depressions are visible in negatively stained preparations of whole bacteria and spheroplasts . A new ringlike structure is connected to an elongation of the basal body into the cytoplasm in both preparations . Its diameter (60 nm) is larger than that of the M ring . A heavily stained compartment can be seen in between the new ringlike structure and the basal disk, which may be formed by the energy transducing units. J Immunol, 1992 Jan 1, 148(1), 218 - 24 Epitopes on the outer surface protein A of Borrelia burgdorferi recognized by antibodies and T cells of patients with Lyme disease; Shanafelt MC et al.; We have characterized immunogenic epitopes of the 31-kDa outer surface protein A (OspA) protein of Borrelia burgdorferi, which is a major surface Ag of the spirochete causing Lyme disease . Full length and truncated forms of rOspA proteins were expressed in Escherichia coli, and their reactivities with antibodies and human T cell clones isolated from patients with Lyme disease were determined . The epitopes recognized by three of four OspA-reactive T cell clones are contained within the 60 COOH-terminal amino acids . Each of the four OspA-reactive T cell clones has a different HLA class II molecule involved in Ag recognition and recognizes a distinct epitope . One T cell clone promiscuously recognized an epitope in the context of different HLA-DQ molecules . In addition, the binding of a murine monoclonal anti-OspA antibody, as well as antibodies in sera of three of five patients with Lyme disease, was dependent upon the amino acids in the carboxy-terminal protion of this protein . Taken together, our results indicate that the 60 COOH-terminal amino acids of OspA contain epitopes recognized by human antibodies and T cells. J Virol, 1992 Jan, 66(1), 608 - 11 A CD4+ cytotoxic T-lymphocyte clone to a conserved epitope on human immunodeficiency virus type 1 p24: cytotoxic activity and secretion of interleukin-2 and interleukin-6; Littaua RA et al.; A CD4+ cytotoxic T-lymphocyte (CTL) clone, established from the peripheral blood of a human immunodeficiency virus (HIV)-seropositive donor, lysed autologous target cells that were infected with a recombinant vaccinia virus containing the gag gene of HIV type 1 and target cells pulsed with p24gag construct expressed in Escherichia coli . The recognition of the HLA-DQ-restricted epitope by this clone was further defined by using overlapping synthetic peptides . The epitope recognized by this CD4+ CTL clone (amino acids 140 to 148) overlaps with a CD8+ epitope and is highly conserved among all isolates of HIV type 1 that have been sequenced . Production and secretion of lymphokines such as interleukin-2 and interleukin-6 after specific antigenic stimulation were demonstrated by this gag-specific CD4+ CTL clone. J Virol, 1992 Jan, 66(1), 317 - 24 Colocalization of adeno-associated virus Rep and capsid proteins in the nuclei of infected cells; Hunter LA et al.; The mechanism of adeno-associated virus (AAV) DNA replication was characterized both genetically and biochemically . In this study, we used monoclonal and polyclonal antibodies to examine the AAV p5 (Rep78 and Rep68) and p19 (Rep52 and Rep40) proteins in infected cells . By overexpressing a truncated Rep78 protein in Escherichia coli, we obtained monoclonal antibody anti-78/68, which is specific for the p5 Rep proteins, and monoclonal antibody anti-52/40, which recognized both the p5 and p19 Rep proteins . In single-fluorochrome indirect immunofluorescence labeling experiments, the viral Rep proteins were localized in distinct intranuclear foci . Analysis of AAV proteins by double-fluorochrome indirect immunofluorescence experiments demonstrated that (i) all four AAV Rep proteins occupied the same intranuclear compartments and (ii) the Rep and capsid proteins colocalized in the nuclei of infected cells . These results suggest that replication centers similar to those established by other viruses exist for AAV . These reagents should provide a useful tool for further delineation of the mechanism of AAV replication in vitro. J Virol, 1992 Jan, 66(1), 106 - 14 The position of heterologous epitopes inserted in hepatitis B virus core particles determines their immunogenicity; Schodel F et al.; The nucleocapsid (HBcAg) of the hepatitis B virus (HBV) has been suggested as a carrier moiety for vaccine purposes . We investigated the influence of the position of the inserted epitope within hybrid HBcAg particles on antigenicity and immunogenicity . For this purpose, genes coding for neutralizing epitopes of the pre-S region of the HBV envelope proteins were inserted at the amino terminus, the amino terminus through a precore linker sequence, the truncated carboxy terminus, or an internal site of HBcAg by genetic engineering and were expressed in Escherichia coli . All purified hybrid HBc/pre-S polyproteins were particulate . Amino- and carboxy-terminal-modified hybrid HBc particles retained HBcAg antigenicity and immunogenicity . In contrast, insertion of a pre-S(1) sequence between HBcAg residues 75 and 83 abrogated recognition of HBcAg by 5 of 6 anti-HBc monoclonal antibodies and diminished recognition by human polyclonal anti-HBc . Predictably, HBcAg-specific immunogenicity was also reduced . With respect to the inserted epitopes, a pre-S(1) epitope linked to the amino terminus of HBcAg was not surface accessible and not immunogenic . A pre-S(1) epitope fused to the amino terminus through a precore linker sequence was surface accessible and highly immunogenic . A carboxy-terminal-fused pre-S(2) sequence was also surface accessible but weakly immunogenic . Insertion of a pre-S(1) epitope at the internal site resulted in the most efficient anti-pre-S(1) antibody response . Furthermore, immunization with hybrid HBc/pre-S particles exclusively primed T-helper cells specific for HBcAg and not the inserted epitope . These results indicate that the position of the inserted B-cell epitope within HBcAg is critical to its immunogenicity. Bioseparation, 1992-93, 3(6), 343 - 58 Mathematical modelling and simulation of aqueous two-phase continuous protein extraction; Mistry SL et al.; A mathematical model has been developed to describe the continuous, steady-state operation of an aqueous two-phase system for protein extraction . The model is based on steady-state mass balances of the main components and phase equilibrium data . Experimental data on the separation of thaumatin from contaminant proteins of an homogenate of E . coli in a PEG4000/Phosphate system was used . The data shows the effect of the presence and absence of NaCl which was used to carry out the extraction of thaumatin into the PEG phase and back into the PO4(-3) phase . Simulation results showing the sensitivity to key process parameters, and the effect of process variables on performance are presented and discussed . The model can be used to predict performance and thus 'robustness' of process conditions as well as predict protein recovery yield and purity . This model can also be used to implement a suitable control strategy to maintain process stability. Bioseparation, 1992-93, 3(5), 285 - 9 Shear thickening of DNA in SDS lysates; Stephenson D et al.; Cells of Escherichia coli were subjected to lysis by an alkali detergent treatment (sodium dodecyl sulphate) . The rate of detergent lysis was not first order . The rheological properties of the lysate were measured using a controlled stress rheometer . Detergent-lysed cells produced a lysate which showed marked non-Newtonian properties . The material showed a shear thickening at specific low shear stress conditions . The acceleration of the cone during the ascent stage of a flow curve, influenced the shape of the flow curves . These findings are discussed in relation to the form of bacterial DNA in solution. Cytotechnology, 1992, 8(2), 103 - 8 High level expression of a frog alpha-amidating enzyme, AE-II, in cultured cells and silkworm larvae using a Bombyx mori nuclear polyhedrosis virus expression vector; Kobayashi J et al.; A Xenopus laevis peptidyl C-terminal alpha-amidating enzyme (AE-II) gene, modified by deletion of a region encoding the putative membrane-spanning domain and the putative C-terminal cytosolic tail, was expressed in BoMo-15 AIIc insect cells and silkworm larvae using a Bombyx mori baculovirus expression vector system . The expressed enzyme was identified predominantly in the culture medium and the hemolymph of silkworm larvae, indicating successful secretion of the expressed AE-II . The level of recombinant enzyme in the larval hemolymph at 4 days post-infection (40 micrograms/ml) was more than 100-fold the peak levels found in the culture medium (250 ng/ml) . The enzyme activity in the larval hemolymph at 4 days post-infection was 3700 units/ml. Appl Microbiol Biotechnol, 1992 Jan, 36(4), 493 - 8 Promoter constructions for efficient secretion expression in Streptomyces lividans; Schmitt-John T et al.; Promoters from different Streptomyces genes were cloned in front of the Tendamistat gene from S . tendae, in order to study secretion-expression in S . lividans using a pIJ702 plasmid vector system . Besides the promoters we cloned a transcriptional terminator downstream of the Tendamistat gene to improve transcription efficiency . The promoters we selected were: (1) a synthetic Escherichia coli-like consensus promoter; (2) the aphI promoter of the neomycin resistance gene from S . fradiae; (3) an ermE-up promoter mutant from Saccharopolyspora erythraea; (4) the melC promoter of the tyrosinase operon from Streptomyces antibioticus . In addition, we tested the thiostrepton-inducible tipA promoter from S . lividans in our Tendamistat secretion system . The promoters were cloned upstream of the Tendamistat ribosome binding site in order to conserve the original translation initiation . The Tendamistat secretion mediated by the different promoter constructions above varied dramatically in up to 10 mg/l in the case of the synthetic promoter and the aph promoter, and up to 500 mg/l mediated by the ermE-up promoter . The melC promoter allowed about 200 mg/l Tendamistat secretion and the tipA promoter proved to be inducible from less than 0.5 mg/l up to 40 mg/l of Tendamistat secretion . Based on the amount of secreted Tendamistat and on the analysis of mRNA levels, we conclude that transcriptional activity regulates the efficiency of our secretion-expression system. Scanning Microsc Suppl, 1992, 6, 11 - 20; discussion 20-2 Alignment, classification, and three-dimensional reconstruction of single particles embedded in ice; Frank J et al.; Cryo-electron microscopy of single biological particles poses new challenges to digital image processing due to the low signal-to-noise ratio of the data . New tools have been devised to deal with important aspects of 3-D reconstruction following the random-conical data collection scheme: (a) a new shift-invariant function has been derived, which promises to facilitate alignment and classification of single particle projections; (b) a new method of orientation search is proposed, which makes it possible to relate random-conical data sets to one another prior to reconstruction; and (c) the foundation is laid for a 3-D variance estimation which utilizes the oversampling of 3-D angular space by projections in the random-conical reconstruction scheme. Biol Res, 1992, 25(2), 73 - 8 The separation and identification of picomole amounts of intermediates of glucose metabolism by high performance liquid chromatography on pellicular resins; Preller A et al.; A column (CarboPac PA1, Dionex) containing an anion-exchange pellicular resin was used for the separation of phosphoryl-hexoses derived from labeled glucose microinjected into individual frog oocytes or from cultures of Escherichia coli . Intermediates were identified by: a) comparison of retention times with those of authentic commercial compounds; b) the use of internal labeled standards; c) incubation of samples with specific enzymes and noting the disappearance of one radioactive peak and appearance of another at a new retention time. Protein Sci, 1992 Jan, 1(1), 145 - 50 Steric and hydrophobic determinants of the solubilities of recombinant sickle cell hemoglobins; Bihoreau MT et al.; Models for the structure of the fibers of deoxy sickle cell hemoglobin (Hb Hb S, beta 6 Glu-->Val) have been obtained from X-ray and electron microscopic studies . Recent molecular dynamics calculations of polymer formation give new insights on the various specific interactions between monomers . Site-directed mutagenesis with expression of the Hb S beta subunits in Escherichia coli provides the experimental tools to test these models . For Hb S, the beta 6 Val residue is intimately involved in a specific lateral contact, at the donor site, that interacts with the acceptor site of an adjacent molecule composed predominantly of the hydrophobic residues Phe 85 and Leu 88 . Comparing natural and artificial mutants indicates that the solubility of deoxyHb decreases in relation to the surface hydrophobicity of the residue at the beta 6 position with Ile > Val > Ala . We also tested the role of the stereospecific adjustment between the donor and acceptor sites by substituting Trp for Glu at the beta 6 location . Among these hydrophobic substitutions and under our experimental conditions, only Val and Ile were observed to induce polymer formation . The interactions for the Ala mutant are too weak whereas a Trp residue inhibits aggregation through steric hindrance at the acceptor site of the lateral contact . Increasing the hydrophobicity at the axial contact between tetramers of the same strand also contributes to the stability of the double strand . This is demonstrated by associating the beta 23 Val-->Ile mutation at the axial contact with either the beta 6 Glu-->Val or beta 6 Glu-->Ile substitution in the same beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS) Chromosoma, 1992, 102(1 Suppl), S133 - 41 DNA polymerase delta and epsilon holoenzymes from calf thymus; Podust V et al.; Replication of singly-DNA primed M13 DNA by DNA polymerase (pol) delta completely relies on the simultaneous addition of proliferating cell nuclear antigen (PCNA), replication factor C (RF-C) and replication protein A (RP-A) (or E . coli single-strand DNA binding protein, SSB) . Pol epsilon core alone is able to synthesize the products on singly-primed ssDNA . However, DNA synthesis by pol epsilon was stimulated up to 10-fold upon addition of the three auxiliary proteins PCNA, RF-C and SSB . This stimulation of pol epsilon by PCNA/RF-C/SSB appears to be the superposition of two events: pol epsilon holoenzyme (pol epsilon, PCNA, RF-C) synthesized longer products than its pol epsilon core counterpart, but elongated less primers . Furthermore, we analyzed the cooperative action of pol alpha/primase with pol delta or pol epsilon holoenzymes on unprimed M13 DNA . While pol delta displayed higher dNMP incorporation than pol epsilon, when a single primer was preannealed to DNA, pol epsilon was more efficient in the utilization of the primers synthesized by pol alpha/primase . Under these conditions both longer products and a higher amount of dNMP incorporation was found for pol epsilon holoenzyme, than for pol delta . Our data support the hypothesis of pol delta as the leading and pol epsilon as the second lagging strand replication enzyme. DNA Seq, 1992, 2(4), 257 - 63 The complete nucleotide sequence of region 1 of the CFA/I fimbrial operon of human enterotoxigenic Escherichia coli; Jordi BJ et al.; The production of the plasmid-encoded fimbrial antigen CFA/I of enterotoxigenic Escherichia coli requires two DNA regions: CFA/I region 1 and CFA/I region 2 . These two regions are separated by about 40 kb on the wildtype plasmid . CFA/I region 1 contains the structural genes, whereas CFA/I region 2 contains a positive regulator . The first two genes (cfaA and cfaB) and the cfaD' sequence of region 1 have already been described . Here the total nucleotide sequence of region 1 is presented . Two new genes in region 1 are described, named cfaC and cfaE . The GC content of the genes in region 1 is 33.6% which is substantially lower than normally found in E . coli genes (50%) . The codon usage also differs from the standard codons used in E . coli. Z Naturforsch {C}, 1992 Jan-Feb, 47(1-2), 77 - 84 Characterization of plastid 5-aminolevulinate dehydratase (ALAD; EC 4.2.1.24) from spinach (Spinacia oleracea L.) by sequencing and comparison with non-plant ALAD enzymes; Schaumburg A et al.; We have sequenced 5-aminolevulinate dehydratase (ALAD; EC 2.4.1.24) of a plant . A full-length cDNA clone (1727 bp) encoding this enzyme has been identified by immunoscreening a lambda gt 11 cDNA library of spinach . ALAD is not a plant-specific enzyme; however, the plant enzyme differs from the well known ALAD enzymes of bacteria, yeast and animals in structural and biochemical properties and in that it is located in the plastid . Differences and homologies can be traced back to the molecular level . The mature ALAD subunit, whose N-terminus was determined by automatic Edman degradation, is a protein of 367 amino acid residues and has a Mr of 40,132 . This figure is in the range of molecular weights of non-plant ALADs . The active centre is highly conserved and the same is true for the ion-binding domain, except that 4 cysteines of the non-plant enzymes (binding Zn2+) have disappeared and a total of 6 aspartic acids meets the demands of Mg(2+)-binding . However, there are more distinct differences . Apart from a transit sequence of 56 amino acids targeting the plastid, the N-terminal part of the mature plant enzyme differs considerably from non-plant ALAD enzymes . It is rich in prolines and hydroxylated amino acids . The apparent Mr on SDS-PAGE is 45,000 or higher, but up to now posttranslational modifications have not been found. Microbiol Immunol, 1992, 36(3), 243 - 56 Establishment of a mouse model of cystitis and roles of type 1 fimbriated Escherichia coli in its pathogenesis; Liao J et al.; The role of type 1 fimbriae in promoting bladder colonization and the course of Escherichia coli cystitis were examined with type 1 fimbriated strains of clinically isolated E . coli . In the experiments of mice in vivo, intact bladder epithelium showed natural resistance to the adherence of type 1 fimbriated and non-fimbriated E . coli . However, the exfoliation of bladder superficial cells by trypsinization before the bacterial inoculation promoted the adhesion and colonization of type 1 fimbriated E . coli onto bladder epithelium . After colonization of E . coli, maximum numbers of E . coli and leukocytes were observed 3 days after inoculation . Nine days after inoculation, both of E . coli and leukocytes disappeared and the regeneration of superficial cells was observed . On the other hand, superficial cells in mice injected with phosphate-buffered saline or non-fimbriated E . coli regenerated 5 days after trypsinization . The present study demonstrated that the removal of superficial cells is essential for the adhesion and colonization of type 1 fimbriated E . coli onto bladder epithelium in vivo and a new model of E . coli cystitis in mice was established . The model which we established is valuable for histopathological, immunological, and therapeutic studies. Yi Chuan Xue Bao, 1992, 19(1), 55 - 60 {Location of ctDNA fragment related to CMS in Brassica napus L . var . xiangai}; Sun W et al.; The ctDNA of sterile line and its maintainer from Brassica napus L . var . xiangai were digested by restriction endonucleases EcoRI, BamHI, PstI and SmaI . Only one special fragment E3.2kb was observed in EcoRI restriction pattern of maintainer . After being eluted, this fragment was incubated with plasmid pUC9 and then transformed E . coli JM83 . Through colour screening, clony hybridization and electrophoretic analyses, the special clone carrying E3.2 was obtained . The rRNA gene probe was used to hybridize with the EcoRI restriction pattern . The result showed that E3.2kb fragment is homologous to rRNA gene . And then, we use E3.2 fragment as probe to hybridize with rRNA gene digested by BamHI, EcoRI, SalII, BglII, HindIII and PstI . According to rRNA gene map, E3.2 fragment was located at the leader sequence of 16S rRNA gene, from +2.0kb to +5.4kb . Because this 3.4kb region was in the inverted repeat region of ctDNA and it is homologous to E3.2kb that related to CMS . So probably CMS is related to this 3.4kb region in the inverted repeat region of ctDNA. Acta Virol, 1992 Jan, 36(1), 90 - 102 Cloning and expression in Escherichia coli of the 37-, 14-, and/or 16-kilodalton antigens genes from Rickettsia prowazekii strain E; Aniskovich LP et al.; A gene bank of Rickettsia prowazekii strain E constructed in the phage vector lambda EMBL4 was screened for antigen production with anti-R . prowazekii serum . One of the immunoreactive clones, grown at 37 degrees C exhibited the expression of at least two antigens of molecular weight (M(r)) 37 kD and 14 kD . Subcloning and further analysis revealed that the antigens (polypeptides) of Mr 37, 14, and/or 16 kD apparently represent structural units of the 138 kD complex antigen . Assembly of the above mentioned polypeptides was found to be thermosensitive as it took place at 30 degrees C but not at 37 degrees C and resulted in an oligomeric structure of M(r) 138 kD . The nucleotide sequence of the gene coding for a precursor of the mature polypeptides of Mr 14 and/or 16 kD was determined. Ultramicroscopy, 1992 Jan, 40(1), 13 - 32 Detection, classification and 3D reconstruction of biological macromolecules on hypercube computers; Carazo JM et al.; In this work we present results of the mapping on hypercube computers of some of the key steps involved in the procedure for 3D structural determination from transmission electron microscopy images . The goal is the introduction of parallel processing tools in the field of electron microscopy image processing . We show how the rich topology of the hypercube, combined with an efficient programming strategy, allows for order-of-magnitude increase in computational capacity for such time-consuming tasks as calculation of multidimensional FFT's, cross-correlation coefficients, fuzzy partitioning functionals and the filtered back-projection 3D reconstruction method. Vaccine, 1992, 10(5), 309 - 18 Induction of protective class I MHC-restricted CTL in mice by a recombinant influenza vaccine in aluminium hydroxide adjuvant; Dillon SB et al.; Induction of class I MHC-restricted cytotoxic T lymphocyte (CTL) responses by soluble proteins or peptides requires complex adjuvants or carrier systems which are not licensed for use with human vaccines . The data presented in this report show that vaccination with a highly purified recombinant influenza protein antigen in aluminium hydroxide adjuvant, the only adjuvant currently licensed for clinical use, elicited class I restricted CTL and protection from lethal challenge with H1N1 and H2N2 viruses . The antigen (D protein, SK&F 106160) is produced by expression of H1N1 influenza virus-derived cDNA (strain A/PR/8/34) in Escherichia coli, and is composed of the first 81 N-terminal amino acids (aa) of the non-structural protein 1 (NS1) fused via a nine nucleotide non-viral linker sequence to the 157 C-terminal aa of the haemagglutinin 2 subunit (HA2) . Previous work by Kuwano et al demonstrated that in vitro stimulation of spleen cells from influenza virus-primed mice, with a partially purified preparation of the D protein, selected for CD8+ CTL clones which facilitated lung clearance of H1N1 and H2N2 viruses . In the current study, these results were extended by studying the responses of mice actively immunized with highly purified D protein in the presence or absence of adjuvants . Vaccination of CB6F1 (H-2dxb) mice with D protein in aluminum hydroxide or Freund's complete adjuvant generated H1N1 cross-reactive, H-2d-restricted, CD8+ CTL directed against an immunodominant HA2 epitope (aa 189-199) . D protein without adjuvant did not elicit CTL, regardless of the route of injection . However, long-lived (greater than 6 months) splenic memory CTL were elicited by boosting mice intraperitoneally (i.p.) with the D protein in the absence of adjuvant . In mice injected subcutaneously with D protein in aluminium hydroxide at weeks 0 and 3, survival was increased relative to controls up to 16 weeks beyond the second vaccination, after which time additional boosting was required for protection . Studies in H-2b and H-2k mice vaccinated with the D protein showed that induction of CD4+ T-cell or antibody responses, in the absence of CD8+ CTL, did not correlate with protection . Passive transfer of immune sera from CB6F1 mice was also not protective . This prototype H1N1 recombinant subunit vaccine in aluminium adjuvant should directly address the feasibility of achieving a protective cell-mediated immune response in human influenza. Anim Genet, 1992, 23(1), 19 - 29 Evidence for linkage between the swine L blood group and the loci specifying the receptors mediating adhesion of K88 Escherichia coli pilus antigens; Vogeli P et al.; Brush borders or enterocytes obtained from the small intestine of 248 pedigreed pigs were tested by adhesion assay in vitro with enterotoxigenic Escherichia (E.) coli strains, each expressing one of the three K88 pilus variants K88ab, K88ac and K88ad . All pigs were classified as belonging to one of the four adhesion phenotypes: I--K88ab(-), ac(-), ad(-); II--K88ab(-), ac(-), ad(+); III--K88ab(+), ac(+), ad(-); and IV--K88ab(+), ac(+), ad(+) . Serum or red cells were typed for 15 blood group systems: A-O, B, C, D, E, F, G, H, I, J, K, L, M, N and O; for 11 biochemical polymorphisms: PI1, PI2, PO1A, A1BG, GPI, PGD, TF, HPX, ADA, PGM and AMY; the polymorphism at the IGHG1 locus . Linkage analysis was performed between the alleles at the locus (loci) specifying K88 receptors able to bind one or more different serological types of K88 E . coli and alleles for markers at other loci . Linkage was demonstrated between the locus for the L blood group system and the locus (loci) for K88 E . coli receptors (Z = 3.24), adding one locus (loci) to the previously identified linkage group IV (LGIV) {L-SLB} . The maximum likelihood estimate of the recombination fraction (theta) was 0.23 . No evidence was found for linkage between any of the other biochemical and immunogenetic markers and the receptor locus (loci) of K88 E . coli. Zentralbl Bakteriol, 1992 Jan, 276(2), 264 - 72 Restriction fragment length polymorphism and virulence pattern of the veterinary pathogen Escherichia coli O139:K82:H1; Tschape H et al.; Escherichia coli O139:K82:H1 strains originating from outbreaks and single cases of oedema disease in pigs were characterized by their genomic restriction fragment length polymorphism (RFLP), their virulence pattern, and by the occurrence as well as the genomic distribution of the determinants for hemolysin (hly) and verotoxins (shiga-like toxins; sltI, sltII) . Whereas the RFLPs revealed considerable variation among the E . coli O139:K82:H1 isolates depending the origin and epidemic source of the strains, the virulence gene slt II was found to be present in nearly all strains in a particular chromosomal region . Similar to RFLPs, the plasmid profiles are useful for epidemiological analysis. Zentralbl Bakteriol, 1992 Jan, 276(2), 152 - 64 Restriction fragment length polymorphisms associated with alpha-hemolysin determinants are correlating with the expression of alpha-hemolysin in strains of Escherichia coli; Prada J et al.; Three different phenotypes of hemolysis on blood-agar were detected when 58 isolates of Escherichia coli were investigated for alpha-hemolysin synthesis . In strains with chromosomally encoded alpha-hly genes, phenotype I (large and clear hemolysis zones) corresponded with high hemolytic activity and with a 17.2 kb size BamHI restriction fragment hybridizing with an alpha-hly-specific gene probe . Phenotype II (small and turbid hemolysis zones) was associated with low hemolytic activity and generally with a 12.8 kb size hybridizing BamHI restriction fragment . An intermediate phenotype (type I/II) was found in a few strains with moderate hemolytic activity . This correlation between hemolytic phenotype and activity was not found in E . coli carrying alpha-hly plasmids . Type I strains differed from all others in the promotor region of their 17.2 kb size BamHI fragment associated chromosomal alpha-hly determinant . The relation between hemolytic phenotype and DNA hybridization pattern was found in unrelated E . coli isolates of human and animal origin . An association of alpha-hemolysin with other virulence factors was found in most strains of all three phenotypes. Vaccine, 1992, 10(1), 10 - 3 Preparation of candidate vaccinia-vectored vaccines for haemorrhagic fever with renal syndrome; Schmaljohn CS et al.; Two vaccinia-vectored candidate vaccines for haemorrhagic fever with renal syndrome were prepared by inserting cDNA, representing the medium (M) genome segment, or the M and the small (S) genome segments of Hantaan virus into the thymidine kinase gene of the Connaught vaccine strain of vaccinia virus . In the single recombinant, the M segment was placed under control of the vaccinia virus 7.5 kDa promoter . In the double recombinant, the M and S segments were placed under control of the vaccinia virus 7.5 kDa and 11 kDa promoters, respectively . An immunoplaque assay technique was developed to select recombinants without the need for expression of irrelevant genes or use of potential mutagens . Proteins indistinguishable from authentic viral envelope glycoproteins and nucleocapsid protein were observed by immunoprecipitation with antibodies to Hantaan virus . The recombinant expressing both the M and the S segments was selected for further development and testing as a human vaccine. Plant Mol Biol, 1992 Jan, 18(2), 327 - 36 Heat shock protein synthesis of the cyanobacterium Synechocystis PCC 6803: purification of the GroEL-related chaperonin; Lehel C et al.; Synechocystis PCC 6803 cells could be induced to synthesize four major HSPs with apparent molecular sizes of 70, 64, 15 and 14 kDa . Heat stress at 42.5 degrees C appeared to be the optimum temperature for HSP formation in cells grown at 30 degrees C . The relative rate of synthesis of HSP70 and HSP15 reached a maximum at 30 min after the temperature shift-up whereas the capability of cells to accumulate HSP64 and HSP14 continued through 2 h . The two most abundant HSPs, HSP70 and HSP64, were recognized on western blots by antibodies raised against authentic DnaK and GroEL from Escherichia coli . To furnish sufficient evidence for the assumption that HSP64 is a GroEL-related chaperonin, this protein was purified to homogeneity . There was a 76% sequence identity between the amino acid sequence of HSP64 and the corresponding protein in Synechococcus PCC 7942 . Moreover, the purified HSP64 cross-reacted to anti-E . coli GroEL antibody . To our knowledge, this is the first report about the purification and partial protein sequencing of a cyanobacterial chaperonin. J Bacteriol, 1992 Jan, 174(2), 633 - 7 Mutations at Glu-32 and His-39 in the epsilon subunit of the Escherichia coli F1F0 ATP synthase affect its inhibitory properties; LaRoe DJ et al.; A collection of amino acid substitutions at residues Glu-32 and His-39 in the epsilon subunit of the Escherichia coli F1F0 ATP synthase has been constructed by cassette mutagenesis . Substitutions for residue Glu-32 appeared to cause abnormal inhibition of membrane-bound F1 ATPase activity, and replacement of His-39 by Arg, Val, and Pro affected F1F0 interactions. Infect Immun, 1992 Jan, 60(1), 206 - 12 Mucosal immune response to RDEC-1 infection: study of lamina propria antibody-producing cells and biliary antibody; McQueen CE et al.; Infection of rabbits with Escherichia coli RDEC-1 is a useful model for diarrheal disease caused by mucosally attaching E . coli . Understanding of the protective immunity induced by RDEC-1 infection in rabbits should provide information useful in the design of vaccines for protection against this infection and other mucosally attaching organisms as well . Thus, to define the time course and location of specific immunoglobulin A secretion in relation to bacterial colonization during primary RDEC-1 infection, we infected rabbits with RDEC-1, which express AF/R1 adherence pili, and compared sites of anti-AF/R1 antibody-containing cells in the intestinal mucosa with the sites of luminal colonization and mucosal attachment of RDEC-1 . Also, anti-AF/R1 antibodies in intestinal fluids and bile were measured by enzyme-linked immunosorbent assay, and attachment sites of RDEC-1 to the intestinal epithelium were determined by immunohistochemical examination . Anti-AF/R1 pilus antibody-containing cells were most numerous in the proximal intestine (duodenum and jejunum) . In contrast, both luminal colonization and attachment of RDEC-1 to epithelial cells were densest in the distal intestine (cecum and colon) . Anti-AF/R1 antibodies were present in approximately equal amounts in fluids collected from all levels of the gut after week 1 postinfection . Anti-AF/R1 antibody levels in undiluted bile exceeded those in gut flushes by at least 2 orders of magnitude . Loss of RDEC-1 attachment to epithelial cells preceded resolution of diarrheal illness despite the presence of large numbers of organisms in the intestinal lumen . Our studies indicate that during RDEC-1 infection (i) sites of greatest mucosal anti-AF/R1 antibody secretion are proximal to sites of maximal RDEC-1 luminal colonization and attachment, (ii) bile is a major source of specific antibodies in the intestinal lumen, and (iii) interference with RDEC-1 attachment to epithelial cells may permit resolution of disease. Second Messengers Phosphoproteins, 1992-93, 14(3), 163 - 71 Lipopolysaccharide stimulates phosphorylation of eukaryotic initiation factor-4F in macrophages and tumor necrosis factor participates in this event; Haas DW et al.; Bacterial lipopolysaccharide (LPS) produces rapid changes in macrophage protein synthesis and function . Phosphorylation of the 25 kDa mRNA cap-binding protein (eIF-4E) in model systems regulates the efficiency of protein synthesis . We report that both LPS and tumor necrosis factor-alpha (TNF-alpha) stimulate phosphorylation of eIF-4E and the p220 component of eIF-4F in bone marrow-derived macrophages . Moreover, anti-TNF-alpha antibodies inhibit LPS-stimulated phosphorylation of eIF-4E and p220 by 43% (+/- 6%) and 50% (+/- 5%), respectively . Our results indicate that LPS stimulates eIF-4F phosphorylation by a TNF-alpha-dependent mechanism, and suggest that phosphorylation of eIF-4F might play a role in the post-transcriptional regulation of gene expression in macrophages exposed to LPS. Second Messengers Phosphoproteins, 1992-93, 14(3), 127 - 37 Selection of cDNAs for phosphodiesterases that hydrolyze guanosine 3';5'-monophosphate in Escherichia coli; Van Lookeren Campagne MM et al.; A genetic selection procedure has been developed which makes the growth of E . coli dependent on expression of a cGMP phosphodiesterase cDNA . E . coli does not contain a cGMP phosphodiesterase, and guanine auxotrophs cannot extract the guanine from cGMP . If a functional cGMP phosphodiesterase is introduced, then guaA auxotrophs will grow on cGMP as a guanine source . The method also selects GMP synthetase cDNAs, which complement the guanine auxotrophy directly . Expression of a Dictyostelium discoideum or human heart cyclic nucleotide phosphodiesterase cDNA permits growth of the E . coli guaA auxotroph in the presence of cGMP . Several commercial cDNA libraries were corrupt and contained phosphodiesterase and/or GMP synthetase sequences that were from a contaminating DNA source. Rev Cubana Med Trop, 1992, 44(1), 50 - 4 {The study and evaluation of a method for identifying Escherichia coli by using fluorescent disks}; Niebla Perez A et al.; A study was carried out with 101 strains, 79 of Escherichia coli and 22 of other genera isolated from clinical samples at several hospitals in Havana form October 1989 to January 1990 . In all the strains, beta-glycuronidase enzyme was detected in conditions established by our laboratory and was compared with the results reached by the ROCHE enterotube method . Of the 79 Escherichia coli strains, 74 were positive to the beta-glycuronidase detection test . Sensitivity was 94% and specificity, was 100%. Insect Mol Biol, 1992, 1(1), 37 - 43 A Drosophila metallothionein promoter is inducible in mosquito cells; Kovach MJ et al.; Expression from a Drosophila metallothionein promoter (Mtn) was investigated in mosquito cells . Recombinant plasmids carrying a transcription unit comprised of the Escherichia coli beta-galactosidase gene (lacZ) fused to the metallothionein promoter were stably introduced into Aedes albopictus C6/36 cells . A low copy transformant containing approximately 60 copies of plasmid per cell, and a high copy transformant (1-2 x 10(4) copies/cell) were characterized . The expression of beta-galactosidase from the metallothionein promoter could be induced and controlled in this heterologous system, even when the copy number of introduced plasmid was several thousand. Mem Inst Oswaldo Cruz, 1992, 87 Suppl 3, 413 - 22 Protection of Aotus monkeys after immunization with recombinant antigens of Plasmodium falciparum; Enders B et al.; The genus Aotus spp . (owl monkey) is one of the WHO recommended experimental models for Plasmodium falciparum blood stage infection, especially relevant for vaccination studies with asexual blood stage antigens of this parasite . For several immunization trials with purified recombinant merozoite/schizont antigens, the susceptible Aotus karyotypes II, III, IV and VI were immunized with Escherichia coli derived fusion proteins containing partial sequences of the proteins MSAI (merozoite surface antigen I), SERP (serine-stretch protein) and HRPII (histidine alanine rich protein II) as well as with a group of recombinant antigens obtained by an antiserum raised against a protective 41 kD protein band . The subcutaneous application (3x) of the antigen preparations was carried out in intact animals followed by splenectomy prior to challenge, in order to increase the susceptibility of the experimental hosts to the parasite . A partial sequence of HRPII, the combination of three different fusion proteins of the 41 kD group and a mixture of two sequences of SERP in the presence of a modified Al(OH)3 adjuvant conferred significant protection against a challenge infection with P . falciparum blood stages (2-5 x 10(6)) i . RBC) . Monkeys immunized with the MS2-fusion protein carrying the N-terminal part of the 195 kD precursor of the major merozoite surface antigens induced only marginal protection showing some correlation between antibody titer and degree of parasitaemia . Based on the protective capacity of these recombinant antigens we have expressed two hybrid proteins (MS2/SERP/HRPII and SERP/MSAI/HRPII) in E . coli containing selected partial sequences of SERP, HRPII and MSAI . Antibodies raised against both hybrid proteins in rabbits and Aotus monkeys recognize the corresponding schizont polypeptides . In two independent immunization trials using 13 animals (age 7 months to 3 years) we could show that immunization of Aotus monkeys with either of the two hybrid proteins administered in an oil-based well tolerated formulation protected the animals from a severe experimental P . falciparum (strain Palo Alto) infection. Mem Inst Oswaldo Cruz, 1992, 87 Suppl 3, 179 - 84 A recombinant hybrid protein as antigen for an anti-blood stage malaria vaccine: a study on the conservation of a protective component; Knapp B et al.; Recently we have shown that two hybrid proteins expressed in Escherichia coli confer protective immunity to Aotus monkeys against an experimental Plasmodium falciparum infection (Knapp et al., 1992) . Both hybrid proteins carry a sequence containing amino acids 631 to 764 of the serine stretch protein SERP (Knapp et al., 1989b) . We have studied the diversity of this SERP region in field isolates of P . falciparum . Genomic DNA was extracted from the blood of six donors from different endemic areas of Brazil and West Africa . The SERP region encoding amino acids 630 to 781 was amplified by polymerase chain reaction (PCR) and sequenced . Only conserved amino acid substitutions in maximally two positions of the analyzed SERP fragment could be detected which supports the suitability of this SERP region as a component of an anti-blood stage malaria vaccine. Acta Physiol Hung, 1992, 79(4), 399 - 408 Influence of endotoxin on experimental post-alcoholic liver injury; Boron-Kaczmarska A et al.; The morphological and biochemical changes of the liver after endotoxin intake were analyzed in rats receiving 20% ethanol during 60 days . Besides morphological changes, concentration of serotonin and histamine in liver homogenates, the activity of asparagine and alanine aminotransferases (AspAT, ALAT), gamma-glutamyltranspeptidase (GGTP) and alcohol dehydrogenase (ADH) in blood serum were determined, too . The most extensive morphologic changes of the liver were seen in group of animals intoxicated with 20% ethanol during 60 days and single dose of endotoxin E . coli 0127:B8 intraperitoneally . These changes included necrosis most hepatocytes, focal steatosis of liver parenchyma, considerable hyperemia and parenchymatous degeneration of the liver cells . The cells lining liver sinuses showed considerable swelling as well as necrotic changes . Figures of cell division and haemorrhagic focuses were seen, too . The clusters of mononuclear cells, surrounding necrotically changed hepatocytes were seen in the central part of the liver lobule . Among the inflammatory mediators estimated in liver homogenate only serotonin reached a high level in the group of experimental animals receiving only endotoxin . Increased activity of aminotransferases AspAt and ALAT were associated with these morphologic and biochemical changes in liver tissue observed in animals receiving ethanol and endotoxin. Braz J Med Biol Res, 1992, 25(8), 809 - 12 Inhibition of enteropathogenic Escherichia coli adherence to HeLa cells by immune rabbit sera; Barros HC et al.; We have studied the effect of immune rabbit sera on the localized (LA) and diffuse (DA) adherence to HeLa cells of 10 enteropathogenic Escherichia coli (EPEC) strains belonging to serogroups O55, O86, O111, O119, and O142 . Anti-La1 serum, obtained by rabbit immunization with an E . coli strain harboring a cloned DNA fragment from an EPEC LA plasmid, strongly inhibited the adherence of all serogroups but one (O142) . Similar results were obtained with anti-LA2 serum, which is anti-O111 serum absorbed with a non-adherent O111:H- EPEC strain . In contrast, non-absorbed anti-O55 and anti-O111 sera showed an inhibitory effect mainly on the adherence of homologous strains . Except for one experiment diffuse adherence was not inhibited by any antiserum used . The inhibitory effect of immune sera on localized adherence does not seem to be correlated with plasmid curing since adherence plasmid pMS49 proved to be stable after treatment with anti-O55 and anti-O111 sera . The cross-inhibition of adherence by anti-LA sera suggests that localized adherence-related adhesins of the O55, O86, O111, and O119 strains share similar antigens. Braz J Med Biol Res, 1992, 25(8), 761 - 76 In vitro activities of a recombinant foot-and-mouth disease virus replicase expressed in Escherichia coli; Pacheco AB et al.; 1 . The replicase gene of foot-and-mouth disease virus (FMDV) was expressed in Escherichia coli under the control of a tac promoter . The recombinant enzyme was purified by inclusion body precipitation, elution, and poly(U) Sepharose chromatography . 2 . The enzyme exhibits poly(A)-dependent oligo(U)-primed poly(U) polymerase activity . The specific activity of the purified replicase is 1.3 x 10(5) . The recombinant replicase synthesizes RNA using FMDV RNA as template, as well as heterologous RNAs, such as globin RNA and synthetic RNAs, polyadenylated or not . In all polymerization reactions, RNA products twice the size of the template are formed, both in the presence and absence of an oligo(U) primer . The enzyme is also capable of incorporating {alpha 32P}UTP in all RNAs tested except the viral template . This activity does not seem to be related to the primer independent polymerization activity . 3 . The products from polymerization reactions were characterized by hybridization . In the absence of primer they consist of the template and a complementary strand covalently attached, while in the presence of primer they consist of two complementary strands synthesized de novo . 4 . We propose mechanisms of RNA synthesis by the recombinant FMDV replicase in the absence and presence of primer . These mechanisms are discussed in terms of models for in vitro RNA synthesis of other picornaviruses. Braz J Med Biol Res, 1992, 25(7), 667 - 72 Use of plasmid profiles to differentiate strains within specific serotypes of classical enteropathogenic Escherichia coli; Fernandes RM et al.; 1 . The usefulness of plasmid profile analysis to differentiate strains of enteropathogenic Escherichia coli (EPEC) was evaluated by studying 123 strains of the most prevalent serotypes causing infant diarrhea in the city of Sao Paulo, Brazil, i.e., O111ab:H-, O111ab:H2 and O119:H6 . 2 . No common profiles were found among strains of distinct serotypes . However, within each serotype, most of the strains were grouped within a few major profiles . More than 68% of the strains of serotypes O111ab:H- and O111ab:H2 were included in 6 and 9 major profiles, respectively . In serotype O119:H6, about 48% of the strains were included in 3 major profiles . 3 . This analysis suggests that only a few EPEC clones are causing infant diarrhea in Sao Paulo and revealed that the distribution of serotypes O111ab:H- and O111ab:H2 during the one-year study was at least partly determined by small outbreaks of the most common profiles . 4 . We conclude that plasmid profile analysis is very useful to differentiate strains within specific EPEC serotypes. Braz J Med Biol Res, 1992, 25(7), 659 - 66 The N-terminal amino acid sequence is essential for foot-and-mouth disease virus replicase activity; Pacheco AB et al.; 1 . Foot-and-mouth disease virus replicase was expressed by fusing its cDNA to the OmpA signal peptide coding sequence present in the pIN-III ompA series vectors . 2 . Two constructions were developed to express either a full-length or truncated enzyme lacking the 20 amino acids at the N-terminal end . Bacterial extracts expressing the recombinant proteins were submitted to SDS-PAGE and the presence of the replicase was revealed by immunoblotting . The truncated form exhibited a higher mobility and the relative positions of the proteins show that the signal peptide was removed . 3 . The biological activity of these two molecules was tested using a poly(A)-dependent oligo(U)-primed poly(U)-polymerase assay . The full-length replicase is active . The aminoterminal truncated enzyme had 0.02% activity of the intact one . 4 . This result indicates the importance of the twenty N-terminal amino acids for the activity of FMDV RNA-dependent RNA polymerase. Braz J Med Biol Res, 1992, 25(5), 467 - 75 Chemical and biological properties of endotoxin from Leptospira interrogans serovars canicola and icterohaemorrhagiae; De-Souza L et al.; 1 . Endotoxin-like activity was extracted with phenol-chloroform-petroleum either (PCP) from Leptospira interrogans serovars icterohaemorrhagiae and canicola . Chemical analysis of leptospiral cells obtained from the PCP extract indicated the following distribution of lipopolysaccharide (LPS), protein and polysaccharide in mg/ml: 3.0, 4.5 and 1.0 for icterohaemorrhagiae and 3.3, 5.6 and 1.5 for canicola . 2 . The preparations presented several biological activities: positive Limulus test (1.0 pg/ml) for icterohaemorrhagiae and canicola PCP extract and 0.5 pg/ml for E . coli O111:B4 LPS, lethality for chicken embryos (LD50 45, 25 and 1.0) for icterohaemorrhagiae, canicola and E . coli O111:B4 LPS, pyrogenicity in rabbits with an average increase in rectal temperature of 0.6 degrees C, 0.9 degrees C and 2.2 degrees C for canicola, icterohaemorrhagiae and E . coli O111:B4 LPS, reacted with complement inhibiting the lysis of sheep red blood cells, 62%, 75% and 90% for 2.0 micrograms/ml of icterohaemorrhagiae, canicola PCP extract and E . coli O111:B4 LPS . The PCP extract showed no cytotoxicity on chicken embryo fibroblasts and epithelial cells . 3 . These results demonstrate that Leptospira endotoxin activity is similar to E . coli O111:B4 lipopolysaccharide. Brain Pathol, 1992 Jan, 2(1), 31 - 7 Integration site-dependent transgene expression used to mark subpopulations of cells in vivo: an example from the neuromuscular junction; Weis J et al.; To produce transgenic mice, an exogenous gene is inserted into the germ line, usually by injection of the DNA construct into a pronucleus of a fertilized egg . In most cases the transgene is integrated into the genome at a single random site . Frequently, the transgenes are combinations of regulatory elements from one gene and protein coding sequences from another gene, the reporter . As expected, the promoter in the construct usually controls the expression pattern of the reporter . In some cases, however, transgenes have been constructed with regulatory elements that are not able to direct transcription on their own . In animals containing such transgenes, the expression of the reporter is dependent on endogenous regulatory elements near the chromosomal site of transgene integration . In the present study, an Escherichia coli beta-galactosidase (lacZ) reporter gene linked to a weak promoter was selectively expressed in discrete subpopulations of cells in each of eight independently derived lines of mice . In one line (line 42), which we analyzed in detail, a subset of cells in skeletal muscle were lacZ-positive . Specifically, fibroblasts close to neuromuscular junctions expressed the lacZ-protein, whereas skeletal muscle fibroblasts far from synaptic sites and fibroblasts in other organs were lacZ-negative . Moreover, Schwann cells at nerve terminals were lacZ-positive, whereas Schwann cells in extramuscular nerves were lacZ-negative . These results indicate the existence of differences between perisynaptic and extrasynaptic fibroblasts in normal skeletal muscle . They also demonstrate how such transgenic mice can be used to identify and mark discrete cell populations. Zhen Ci Yan Jiu, 1992, 17(3), 212 - 6 {The effect of acupuncture on rabbits with fever caused by endotoxin}; Kuang X et al.; Forty-eight rabbits were used to investigate the effect of puncturing GV14, LI11 on the change of temperature and the level of plasma endotoxin . The animals were divided into 5 groups: reinforcing manipulation, reducing manipulation, electroacupuncture, endotoxin-control, N.S.-control group . The result shows that there is no influence on the fever caused by endotoxin and the level of plasma endotoxin in the treatments by the different acupuncture techniques. Protein Sci, 1992 Jan, 1(1), 10 - 21 Differences in hydrogen exchange behavior between the oxidized and reduced forms of Escherichia coli thioredoxin; Kaminsky SM et al.; Amide proton exchange of thioredoxin is used to monitor the structural effects of reduction of its single disulfide . An effective 3-5-proton difference between the oxidized and reduced protein form is observed early in proton out-exchange of the whole protein, which is independent of temperature in the range of 5-45 degrees C, indicating that redox-sensitive changes are probably not due to low-energy structural fluctuations . Medium resolution hydrogen exchange experiments have localized the redox-sensitive amide protons to two parts of the sequence that are distant from each other in the three-dimensional structure: the active-site turn and the first beta-strand . The sum of the proton differences observed in the peptides from these regions is equal to that of the whole protein, indicating that all redox-sensitive hydrogen exchange effects are observed in the peptide experiments . A model combining structural changes within the protein matrix with changes in the surface hydration properties is proposed as a mechanism for the communication between distant sites within the protein . Sound velocity and density measurements of reduced and oxidized thioredoxin are presented in the accompanying paper (Kaminsky, S.M . & Richards, F.M., 1992, Protein Sci . 1, 22-30). J Nutr Sci Vitaminol (Tokyo), 1992, Spec No, 114 - 7 Serum B12 binding proteins versus seminal plasma binder; Fukuda M et al.; It was well confirmed that B12 binding protein in human serum and body fluids largely consists of two kinds of binding proteins, physiologically B12 transporting transcobalamin and haptocorrins which does not involve in B12 transport . In seminal plasma, very potent B12 binding protein, in quantity as much as 23 times of normal serum, was identified . Separation characteristics, gel filtration, CM cellulose column chromatography and column IEF all agreed as one of the haptocorrins . One peculiar feature of seminal plasma binder is marked heterogeneity of much lower pI regions suggesting the presence of increased amount of sialic residues on the molecule . The preliminary data from this laboratory support the view, however, physiological role such as the relationship to the spermatogenesis is not known . The most recent findings in clinical observation on B12 is the treatment of male infertility using methycobalamin . The detailed analysis of seminal binders may open up new arena of B12 bioeffect. DNA Seq, 1992, 3(4), 233 - 5 A rapid PCR dependent microtitre plate screening method for DNA sequences altered by site-directed mutagenesis; Trower MK; A simple and rapid method for identifying DNA sequences altered by site-directed mutagenesis is described . The procedure requires that a restriction endonuclease recognition sequence is either created or removed from the target DNA sequence by the site-directed mutagenesis reaction . In the screening method presented, transformant colonies or M13 plaques are directly subjected to PCR using universal primers that flank the region containing the site-directed changes . The double stranded DNA products generated are then digested, with no purification, by the restriction enzyme whose site is modified, allowing mutant clones to be differentiated from those carrying wild type sequences . The protocol also provides for recovery of the bacterial cultures harbouring the mutated plasmid or M13 for further characterisation . All the procedures are carried out in small volumes in microtitre plates, thereby lowering costs and enabling the investigation of large numbers of clones . The technique allows a considerable amount of effort to be saved compared to conventional screening practices by circumventing time consuming DNA template preparations. Eur Surg Res, 1992, 24(6), 363 - 71 Superoxide production by liver macrophages in a septic rat model--relation to arterial ketone body ratio; Iimuro Y et al.; The relationship between superoxide production by liver macrophages and arterial ketone body ratio (AKBR), which reflects the oxidation-reduction state in the mitochondrial compartment of hepatocytes, was studied in rats with lethal and sublethal septicemia induced by intravenous injection of live Escherichia coli 014 . In the sublethal model, AKBR decreased transiently (p < 0.01) and superoxide production by isolated liver macrophages increased significantly after opsonized zymosan (OZ) stimulation (p < 0.05) . On the other hand, in the lethal model, AKBR decreased markedly (p < 0.01) to below 0.4 without recovery, and superoxide production was not activated by OZ stimulation . Thus, when AKBR decreases to an irreversible level, below about 0.4, superoxide production by liver macrophages is impaired, while as long as AKBR remains reversible, more than about 0.4, it is enhanced . It is suggested that superoxide production by the Kupffer cells is related to the intrahepatic oxidation-reduction state in the septic model. Folia Microbiol (Praha), 1992, 37(5), 347 - 52 Electron microscopic analysis of two nonconjugative derivatives of plasmid R1drd-19Km- from Escherichia coli; Benada O et al.; The plasmids pON5300 and pON5304, nonconjugative variants of the plasmid R1drd-19Km, were analyzed by electron microscopy . It was found by heteroduplex mapping that a 1.4 kb DNA segment was inserted into EcoRI E fragment of both plasmids, where some tra-genes and oriT are localized . Although this DNA segment was mapped to the same region its orientation was different in each of the two plasmids . The inserted DNA segment was identified as an IS10R sequence on the basis of analysis of self-annealed molecules of pON5304 and their cleavage with EcoRV restriction enzyme . These methods enable us not only to map IS10R sequences on 87 kb pON5300 and 65 kb pON5304 molecules, respectively, but also to define their orientation. J Basic Microbiol, 1992, 32(5), 309 - 15 The metabolism of gluconate in Escherichia coli: a study in continuous culture; Coello NB et al.; The gluconate metabolism in Escherichia coli involves duplicate activities of transport and phosphorylation for gluconate . In both cases, these activities can be differentiated in vitro by their different affinities for the substrate . In addition, the two gluconokinases can be differentiated by their heat sensitivities . The technique of continuous culture was used to investigate the influence of the growth rate on this metabolism in an E . coli HfrG6 strain during gluconate-limited growth under conditions of high and low oxygen concentrations . The transport and phosphorylation for gluconate, induced when the cells are cultivated in media with gluconate were differently influenced by the culture dilution rate . These activities were induced under the two conditions investigated; however, the low affinity transport system for gluconate and the thermosensitive gluconokinase were not detected under conditions of high and low oxygen concentrations, respectively . The induction of the dehydratase was favoured under conditions of low oxygen concentration . The experimental data suggest that induction and repression work together to regulate the levels of these activities during gluconate-limited growth conditions . Furthermore, that an effector molecule distinct from gluconate might be involved in the induction of the dehydratase. J Ocul Pharmacol, 1992 Winter, 8(4), 295 - 307 Effects of steroids and immunosuppressive drugs on endotoxin-uveitis in rabbits; Ohia EO et al.; Anti-inflammatory actions of dexamethasone (DEXA), Cyclosporin A (CSA) and Rapamycin (RAPA) were assessed on uveitis induced by intravitreal E-coli Endotoxin (100ng) in rabbits at 24 hrs . In this model, endotoxin caused a breakdown of the blood-aqueous barrier (BAB) and polymorphonuclear neutrophils (PMN) infiltration into the aqueous humor (AH) and iris-ciliary body (ICB) . Intramuscular (I.M.) DEXA (2mg/kg) but not topical DEXA (0.1% 6 x daily) inhibited AH leukocytes and protein level . However, both routes caused an inhibition of AH Prostaglandin E2 (PGE2) and Leukotriene B4 (LTB4) . In the ICB, I.M . DEXA significantly inhibited PGE2 synthesis and myeloperoxidase (MPO) activity . I.M . CSA (25mg/kg) and I.M . RAPA (10mg/kg) inhibited the AH leukocytes and protein content and MPO activity in the ICB . RAPA also inhibited AH protein and eicosanoid (except AH LTB4) levels in both the AH and ICB . Interestingly, castor oil, a vehicle of CSA, also inhibited AH leukocytes and the release of PGE2 into AH and from ICB . In summary, systemic administration of DEXA and other immunosuppressive drugs CSA and RAPA significantly inhibited endotoxin-induced uveitis in rabbits. Vaccine, 1992, 10 Suppl 1, S48 - 52 Possible approaches to develop vaccines against hepatitis A; D'Hondt E; More than a decade ago, successful replication of hepatitis A virus (HAV) in cell culture opened the way to the development of live attenuated and inactivated vaccine candidates . Serial passages of HAV in cell culture led to attenuation as demonstrated by experiments in non-human primates . Several live vaccine candidates obtained through serial passages have been evaluated in volunteers . Significant improvements in the yield of viral antigen from infected cell cultures stimulated the development of killed vaccine candidates . These formalin-inactivated vaccines contain the viral capsid antigens assembled into viral particles . The immunogenic potential of the vaccine candidates depends strongly on the preservation of the configuration of the capsid proteins . Synthetic peptides covering immunogenic sequences of VP1 as well as soluble capsid proteins expressed as fusion proteins in Escherichia coli were therefore only weakly immunogenic when injected at high concentrations in rabbits . On the other hand, tamarin monkeys immunized with a live recombinant vaccinia expressing P1 were protected against virulent challenge . There are, however, considerable drawbacks related to the use of live vaccinia as a carrier virus . Chimeric polio-HAV VP1 viruses have been constructed . These hybrid viruses were not able to induce an immune response, probably because of configurational constraints of poliovirus on the inserted HAV epitopes . More recently, encouraging data on empty virus particles expressed in baculovirus and vaccinia virus systems have been reported. Agents Actions Suppl, 1992, 38 ( Pt 1), 340 - 8 Characterization of two members (CST4 and CST5) of the cystatin gene family and molecular evolution of cystatin genes; Saitoh E et al.; Two members (CST4 and CST5) of the cystatin gene family have been characterized partially by DNA analysis . The CST4 clone contained the gene coding for the precursor form(141 amino acids) of cystatin S, and its exon-intron organization is the same as that of other members (the cystatin SN gene at the CST1 locus, the cystatin SA gene at the CST2 locus, the cystatin C gene at the CST3 locus and a cystatin pseudogene at the CSTP1 locus) . The second cystatin pseudogene was elucidated in the clone, CST5, and it was assigned to the CSTP2 locus . Alignment of DNA sequences of cystatin genes with other genes suggested that the genes for cystatins, kininogens, and Bowman-Birk type inhibitors have evolved from an ancient ribonuclease-like gene. Acta Physiol Scand Suppl, 1992, 607, 23 - 9 Mitochondrial F-type ATPases: the glycine-rich loop of the beta-subunit is a pyrophosphate binding domain; Thomas PJ et al.; The beta-subunit of the mitochondrial ATP synthase complex comprises the bulk, if not all, of the catalytic nucleotide binding site on the enzyme . A region of homologous sequence rich in glycines (G) and containing a basic lysine (K) and a threonine (T) is found in the beta-subunit as well as many other purine nucleotide binding proteins . The consensus sequence of this region is Gx4GKT, where x represents any amino acid, and is called the A region or glycine-rich loop . The related function of these proteins implies that the glycine-rich loop is directly involved in nucleotide binding . Here we directly test the involvement of the beta-subunit's glycine-rich region in adenine nucleotide binding using two independent approaches . A synthetic fifty amino acid peptide, PP-50, containing the glycine-rich region and the surrounding sequence was assessed for secondary structure and interaction with potential ligands . Circular dichroism spectropolarimetry indicates that PP-50 assumes a predominantly beta-sheet conformation in solution . Significantly, the peptide precipitates from solution when ATP, ADP, GTP, ITP, and pyrophosphate are added, but not when AMP or phosphate are included . Magnesium is not required for the interaction with the purine nucleotides . Complimentary to these studies, the sequence around the Gx4GKT motif was deleted from a recombinant rat liver beta-subunit overexpressed in E . coli . While the wild type beta-subunit showed specificity for the tri- and diphosphonucleotides, the deletion mutant bound tri-, di-, and monophosphate nucleotides with equal affinity.(ABSTRACT TRUNCATED AT 250 WORDS) Antisense Res Dev, 1992 Summer, 2(2), 111 - 8 Translation inhibition by phosphorothioate oligodeoxynucleotides in cell-free systems; Ghosh MK et al.; A detailed comparison was made of the concentration dependence of translation inhibition by phosphorothioate and phosphodiester oligodeoxynucleotides of the same anti-beta-globin sequence in cell-free systems using beta-globin mRNA and unrelated mRNAs as controls . The results confirm that at low concentrations the phosphorothioate oligomer is more potent as an antisense compound, while at higher concentration (greater than 4 microM) it exhibits more nonspecific inhibition than the phosphodiester oligomer for RNase H-mediated translation inhibition. Mol Carcinog, 1992, 6(2), 140 - 7 Kinds of mutations induced by aflatoxin B1 in a shuttle vector replicating in human cells transiently expressing cytochrome P4501A2 cDNA; Trottier Y et al.; Transient expression of rat liver cytochrome P450lA2 cDNA was combined with the use of a shuttle vector as a mutational target to determine the frequency and types of mutation caused by the conversion of aflatoxin B1 into genotoxic metabolites within human cells . Ad293 cells were first transfected with p91-lA2, a rat liver P450lA2 cDNA expression vector, or with p91-lA2(i) (a control vector that has the P450 cDNA in the inverted orientation) and incubated for 24 h to permit P450lA2 accumulation . Cells were then transfected with the pS189 shuttle-vector plasmid, which carries the Escherichia coli supF gene as a mutational target, and incubated for a further 24 h in the presence of aflatoxin B1 to permit promutagen activation and pS189 replication . In shuttle vectors replicated in p91-lA2-transfected cells, the supF point-mutation frequency increased with increasing concentration of aflatoxin B1 . This frequency was nine to 23 times greater than the background point-mutation frequency obtained with aflatoxin B1-treated control (p91-lA2(i)-transfected) cells . The large majority of the aflatoxin B1-induced supF point mutations were base substitutions, mostly G:C----T:A transversions . This mutagenesis system permits the molecular analysis of mutations induced by specific P450/promutagen pairs in a shuttle vector replicating in human cells and will permit the investigation of host cell mechanisms involved in the generation of these mutations. Protein Eng, 1992 Jan, 5(1), 93 - 100 Biochemical properties of the kringle 2 and protease domains are maintained in the refolded t-PA deletion variant BM 06.022; Kohnert U et al.; BM 06.022 is a t-PA deletion variant which comprises the kringle 2 and the protease domain . Production of BM 06.022 in Escherichia coli leads to the formation of inactive inclusion bodies, which have to be refolded by an in vitro refolding process to achieve activity and proper structure of the domains . We analysed the biochemical properties of BM 06.022 to obtain some information about the structure of kringle 2 and the protease as compared with the structure of these domains in the intact t-PA molecule . The kinetic analysis of the amidolytic activity of BM 06.022 and CHO-t-PA yielded similar values for kcat (13.9 s-1 and 11.4 s-1 for the single chain forms and 33.9 s-1 and 27.1 s-1 for the two chain forms of BM 06.022 and CHO-t-PA, respectively) and for Km (2.5 mM and 2.1 mM for the single chains forms and 0.5 mM and 0.3 mM for the two chain forms of BM 06.022 and CHO-t-PA, respectively) . BM 06.022 and CHO-t-PA have the same plasminogenolytic activity in the absence of CNBr fragments of fibrinogen . However, BM 06.022 has a lower plasminogenolytic activity in the presence of CNBr fragments of fibrinogen and a lower affinity to fibrin as compared with CHO-t-PA . The affinity of BM 06.022 for fibrin is completely suppressed by 0.3 mM epsilon-aminocaproic acid, while the intact t-PA has a residual affinity of approximately 30% . The dissociation constants for the interaction with the lysine analogue epsilon-aminocaproic acid are 0.10 mM and 0.09 mM for BM 06.022 and the intact t-PA, respectively . Furthermore, BM 06.022 and CHO-t-PA are inhibited by PAI-1 in a similar manner. Kurume Med J, 1992, 39(1), 9 - 12 Construction of an expression vector for human papillomavirus type 16 E7-glutathione S transferase fusion protein; Chinami M et al.; An expression vector for human papillomavirus type 16 (HPV 16) E7-glutathione S transferase fusion protein was constructed . The E7 gene was confirmed by southern blot analysis with a digoxygenin 11-dUTP labeled and simultaneously amplified E7 DNA probe by polymerase chain reaction (PCR) . The fusion protein was expressed in E . coli, recovered as inclusion bodies, and was confirmed by western blot analysis using a monoclonal antibody against HPV 16-E7 protein . A polyclonal antibody against the HPV 16-E7 protein was raised in the serum of mice hyper-immuned with the fusion protein . This antibody will be available for screening of patients with cerv