Mol Med, 1999 Aug, 5(8), 517 - 25 Cellular penetration of fluorescently labeled superoxide dismutases of various origins; Filipe P et al.; BACKGROUND: Using fluorescently labeled superoxide dismutase (SOD) and flow cytometry, we have shown previously that the enzyme CuZn SOD (EC 1.15.1.1) from bovine erythrocytes binds rapidly to the cell surface with slow uptake into the cell during the following hours . The degree of labeling was most important for monocytes in comparison to other blood cells (erythrocytes, lymphocytes, and neutrophils) and fibroblasts . In agreement with the flow-cytometric findings, the inhibition of superoxide production was more important for SOD-pretreated monocytes than for neutrophils, as demonstrated with the cytochrome c reduction assay . It was thus of interest to confirm the observed differences between monocytes and neutrophils with confocal laser microscopy, study in greater detail the kinetics of binding, penetration, and intracellular localization of the enzyme, and compare the results obtained with bovine CuZn SOD with those from SODs of other origins and carrying different active sites . MATERIALS AND METHODS: Recombinant human (rh), bovine, and equine CuZn SODs, as well as rh and E . coli Mn SODs, were studied before use with respect to specific activity and purity (HPLC, SDS-PAGE electrophoresis) . Fluorescein isothiocyanate was covalently conjugated to the various SODs for study with high-resolution confocal scanning laser microscopy . Superoxide production by monocytes and neutrophils was measured with the cytochrome c assay . RESULTS: As expected from our experiments with flow cytometry, only rare neutrophils were labeled with FITC-SOD, even with the longest incubation time of 3 hr and the highest dose of 1500 units/ml . In addition, they showed a localized fluorescence pattern that was quite different from the diffuse punctate fluorescence pattern of monocytes . Lymphocytes were not labeled at all . The rapid binding to the cellular surface of monocytes was confirmed, and even after 5 min of preincubation, FITC-SOD was found on a small percentage of monocytes . This was correlated with a reduction in superoxide release after phorbolmyristate acetate (PMA) stimulation by 40% . An interesting finding was the perinuclear accumulation of the penetrated SOD after the longest pretreatment of 3 hr, suggesting a barrier against further progression . Indeed, through confocal microscopy we were able to exclude any fluorescence at the nuclear level . While the fluorescence labeling patterns and the kinetics of penetration were quite similar for bovine, equine, and rh CuZn SOD, the Mn SODs showed poor labeling, correlated with a weak inhibitory effect on cytochrome c reduction, which was not statistically significant . CONCLUSIONS: The rapid binding of native CuZn SODs on the surface of monocytes, leading to reduced superoxide release by these cells, explains the observation that beneficial effects of injected SOD lasted for months despite rapid clearance of the enzyme from the bloodstream, according to pharmacodynamic studies . The preferential binding to monocytes, in contrast to neutrophils, may play a role in chronic inflammatory diseases in which the monocytes are in an activated state . The differences in binding capacity between CuZn SODs and Mn SODs, correlated with different inhibitory effects of superoxide production by monocytes, may also have therapeutic significance. J Gen Virol, 1999 Sep, 80 ( Pt 9), 2423 - 31 Identification and characterization of the UL14 gene product of herpes simplex virus type 2; Wada K et al.; The UL14 gene of herpes simplex virus type 2 (HSV-2) is predicted to encode a 219 amino acid protein with a molecular mass of 23 kDa . In this study, the HSV-2 UL14 gene product has been identified by using a rabbit polyclonal antiserum raised against a recombinant 6 x His-UL14 fusion protein expressed in E . coli . The antiserum reacted specifically with 34, 33 and 28 kDa proteins in HSV-2-infected cell lysates and also with a 34 kDa protein produced by in vitro transcription and translation reactions, suggesting that the 34 kDa protein is the primary translation product of the UL14 gene . The protein was synthesized at late times post-infection (p.i.) and was not detectable in the presence of the viral DNA synthesis inhibitor acycloguanosine . Indirect immunofluorescence studies localized the UL14 protein both to the nucleus and to perinuclear regions of the cytoplasm, and the nuclear UL14 protein was found to co-localize with the scaffolding protein ICP35 at 9 h p.i . However, the protein accumulated in a perinuclear region of the cytoplasm at 12 h p.i., while most of the ICP35 protein localized within assemblons in the nucleus . Although no detectable UL14 protein was associated with intracellular capsids isolated in the presence of 0.5 M NaCl, it was detected in purified virions . Furthermore, the UL14 protein expressed alone was detected both in the nucleus and in the cytoplasm at 24 h after transfection, but was mainly localized to the cytoplasm at later times. Vet Microbiol, 1999 Aug 16, 68(1-2), 171 - 7 Serological evidence of an Australian bovine lentivirus; Burkala EJ et al.; Recombinant 26 kDa capsid (CA) proteins of bovine lentiviruses, bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV), were expressed in Escherichia coli and utilised as antigens for an enzyme-linked immunosorbent assay (ELISA) and a western immunoblot (WIB) procedure for the detection of antibody in dairy cattle in Western Australia . A total of 690 serum samples, 30 from each of 23 farms, were tested by ELISA with a JDV CA protein antigen, and antibody was detected in 3.8% (p<0.05) of the sera . Nine sera from each farm were also tested by WIB with JDV CA protein antigens and antibody was detected in 15.9% of these samples . All ELISA-positive results were also WIB-positive, and all sera antibody-positive by WIB with JDV CA protein antigens were also antibody-positive by the WIB using recombinant BIV CA antigens . This study showed that recombinant protein antigens can be used for serological tests to detect bovine lentivirus infection in Australia. Biosci Biotechnol Biochem, 1999 Aug, 63(8), 1392 - 9 Structural properties of recombinant ovalbumin and its transformation into a thermostabilized form by alkaline treatment; Arii Y et al.; The recombinant ovalbumin produced in Escherichia coli was purified from the cytoplasmic fraction and analyzed for its chemical and conformational properties . The recombinant ovalbumin displayed almost exactly the same circular dichroism and intrinsic tryptophan fluorescence spectra as egg white ovalbumin . As in the egg white protein, four cysteine sulfhydryls and one cystine disulfide were contained in the recombinant protein, according to the results of amino acid analyses; the disulfide bond was found by a peptide mapping analysis to correspond to the native cystine, Cys73-Cys120 . According to a gel electrophoresis analysis, the presence of the disulfide bond was accounted for by specific oxidation of the corresponding cysteine residues during purification of the cytoplasmic protein . Unlike the identity in the conformational and peptide structures, none of the post-translational modifications (N-terminal acetylation, phosphorylation, and glycosylation) that are known with egg white ovalbumin were detected in the recombinant protein . The recombinant ovalbumin was transformed into a thermostabilized form in a similar manner to the transformation of egg white protein into S-ovalbumin; alkaline treatment increased the temperature for thermostability by 8.7 degrees C . These data strongly suggest that the post-translational modifications of ovalbumin are not related to the formation mechanism for S-ovalbumin. Biosci Biotechnol Biochem, 1999 Aug, 63(8), 1329 - 35 Characterization of chimeric enzymes constructed between two distinct alpha-amylase cDNAs from cultured rice cells; Abe R et al.; Cultured cells of rice (Oryza sativa cv Sasanishiki) produce two alpha-amylase isozymes, AMY-I and AMY-III . Using a bacterial expression system, eight chimeric genes constructed with various combination of AMY-I and AMY-III cDNA fragments were expressed, and each recombinant chimeric protein was characterized . Four of the eight recombinant enzymes having region c (one of the four regions having unconserved base sequences between AMY-I and AMY-III cDNAs) of AMY-I showed the same enzyme characteristics as that of native AMY-I, which had high temperature optimum at 50 degrees C . The other four chimeric proteins carrying region c of AMY-III showed the AMY-III type characteristics, which were a low temperature optimum at 25 degrees C and susceptibility to a higher maltooligosaccharide (G17) substrate . The unconserved region c is involved in the decision of the characteristic of AMY-I or AMY-III. Proc Natl Acad Sci U S A, 1999 Sep 28, 96(20), 11428 - 33 Hyperactive transposase mutants of the Himar1 mariner transposon; Lampe DJ et al.; Mariner-family transposable elements are active in a wide variety of organisms and are becoming increasingly important genetic tools in species lacking sophisticated genetics . The Himar1 element, isolated from the horn fly, Haematobia irritans, is active in Escherichia coli when expressed appropriately . We used this fact to devise a genetic screen for hyperactive mutants of Himar1 transposase that enhance overall transposition from approximately 4- to 50-fold as measured in an E . coli assay . Purified mutant transposases retain their hyperactivity, although to a lesser degree, in an in vitro transposition assay . Mutants like those described herein should enable sophisticated analysis of the biochemistry of mariner transposition and should improve the use of these elements as genetic tools, both in vivo and in vitro. Proc Natl Acad Sci U S A, 1999 Sep 28, 96(20), 11200 - 5 Verification of phylogenetic predictions in vivo and the importance of the tetraloop motif in a catalytic RNA; Pomeranz Krummel DA et al.; M1 RNA, the catalytic subunit of Escherichia coli RNase P, forms a secondary structure that includes five sequence variants of the tetraloop motif . Site-directed mutagenesis of the five tetraloops of M1 RNA, and subsequent steady-state kinetic analysis in vitro, with different substrates in the presence and absence of the protein cofactor, reveal that (i) certain mutants exhibit defects that vary in a substrate-dependent manner, and that (ii) the protein cofactor can correct the mutant phenotypes in vitro, a phenomenon that is also substrate dependent . Thermal denaturation curves of tetraloop mutants that exhibit kinetic defects differ from those of wild-type M1 RNA . Although the data collected in vitro underscore the importance of the tetraloop motif to M1 RNA function and structure, three of the five tetraloops we examined in vivo are essential for the function of E . coli RNase P . The kinetic data in vitro are not in total agreement with previous phylogenetic predictions but the data in vivo are, as only mutants in those tetraloops proposed to be involved in tertiary interactions fail to complement in vivo . Therefore, the tetraloop motif is critical for the stabilization of the structure of M1 RNA and essential to RNase P function in the cell. Proc Natl Acad Sci U S A, 1999 Sep 28, 96(20), 11178 - 82 Conformational flexibility at the substrate binding site in the lactose permease of Escherichia coli; Weinglass AB et al.; Glu-126 (helix IV) and Arg-144 (helix V) are charge paired and play a critical role in substrate binding in the lactose permease of Escherichia coli . When Glu-126 is replaced with Asp, the permease has relatively high activity, implying that helix V has sufficient flexibility to allow Arg-144 to accommodate the decreased length of the carboxylate-containing side chain of Asp-126 . Helices IV and V contain five Gly residues at positions 115, 121, 141, 147, and 150, all of which are conserved in the oligosaccharide/H(+) symport family of the major facilitator superfamily . To test the notion that these residues may contribute to conformational flexibility, each residue was replaced with Ala in either the wild type or the Glu-126 --> Asp mutant . Although the replacements are well tolerated in the wild type, the mutations severely inactivate substrate binding and transport in the Glu-126 --> Asp background, with the exception of Gly-121 --> Ala, which retains significant activity . Strikingly, moreover, in two instances (Gly-150 --> Ala and Gly-141 --> Ala), significant activity is recovered when Ala residues at approximately parallel positions in helix IV (Ala-122 or Ala-127, respectively) are replaced with Gly . In addition to providing further evidence that the major determinants for substrate binding in the permease are at the interface between helices IV and V, the findings indicate that the region is conformationally flexible. Proc Natl Acad Sci U S A, 1999 Sep 28, 96(20), 11151 - 5 A bicarbonate ion as a general base in the mechanism of peptide hydrolysis by dizinc leucine aminopeptidase; Strater N et al.; The active sites of aminopeptidase A (PepA) from Escherichia coli and leucine aminopeptidase from bovine lens are isostructural, as shown by x-ray structures at 2.5 A and 1.6 A resolution, respectively . In both structures, a bicarbonate anion is bound to an arginine side chain (Arg-356 in PepA and Arg-336 in leucine aminopeptidase) very near two catalytic zinc ions . It is shown that PepA is activated about 10-fold by bicarbonate when L-leucine p-nitroanilide is used as a substrate . No activation by bicarbonate ions is found for mutants R356A, R356K, R356M, and R356E of PepA . In the suggested mechanism, the bicarbonate anion is proposed to facilitate proton transfer from a zinc-bridging water nucleophile to the peptide leaving group . Thus, the function of the bicarbonate ion as a general base is similar to the catalytic role of carboxylate side chains in the presumed mechanisms of other dizinc or monozinc peptidases . A mutational analysis shows that Arg-356 influences activity by binding the bicarbonate ion but is not essential for activity . Mutation of the catalytic Lys-282 reduces k(cat)/K(m) about 10,000-fold. Proc Natl Acad Sci U S A, 1999 Sep 28, 96(20), 11054 - 61 Reverse biochemistry: use of macromolecular protease inhibitors to dissect complex biological processes and identify a membrane-type serine protease in epithelial cancer and normal tissue; Takeuchi T et al.; Serine proteases of the chymotrypsin fold are of great interest because they provide detailed understanding of their enzymatic properties and their proposed role in a number of physiological and pathological processes . We have been developing the macromolecular inhibitor ecotin to be a "fold-specific" inhibitor that is selective for members of the chymotrypsin-fold class of proteases . Inhibition of protease activity through the use of wild-type and engineered ecotins results in inhibition of rat prostate differentiation and retardation of the growth of human PC-3 prostatic cancer tumors . In an effort to identify the proteases that may be involved in these processes, reverse transcription-PCR with PC-3 poly(A)+ mRNA was performed by using degenerate oligonucleotide primers . These primers were designed by using conserved protein sequences unique to chymotrypsin-fold serine proteases . Five proteases were identified: urokinase-type plasminogen activator, factor XII, protein C, trypsinogen IV, and a protease that we refer to as membrane-type serine protease 1 (MT-SP1) . The cloning and characterization of the MT-SP1 cDNA shows that it encodes a mosaic protein that contains a transmembrane signal anchor, two CUB domains, four LDLR repeats, and a serine protease domain . Northern blotting shows broad expression of MT-SP1 in a variety of epithelial tissues with high levels of expression in the human gastrointestinal tract and the prostate . A His-tagged fusion of the MT-SP1 protease domain was expressed in Escherichia coli, purified, and autoactivated . Ecotin and variant ecotins are subnanomolar inhibitors of the MT-SP1 activated protease domain, suggesting a possible role for MT-SP1 in prostate differentiation and the growth of prostatic carcinomas. Genes Dev, 1999 Sep 15, 13(18), 2449 - 61 The Escherichia coli sigma(E)-dependent extracytoplasmic stress response is controlled by the regulated proteolysis of an anti-sigma factor; Ades SE et al.; The activity of the stress-responsive sigma factor, sigma(E), is induced by the extracytoplasmic accumulation of misfolded or unfolded protein . The inner membrane protein RseA is the central regulatory molecule in this signal transduction cascade and acts as a sigma(E)-specific anti-sigma factor . Here we show that sigma(E) activity is primarily determined by the ratio of RseA to sigma(E) . RseA is rapidly degraded in response to extracytoplasmic stress, leading to an increase in the free pool of sigma(E) and initiation of the stress response . We present evidence that the putative inner membrane serine protease, DegS, is responsible for this regulated degradation of RseA. Gastroenterology, 1999 Oct, 117(4), 893 - 9 Gastric ulcers in SCID mice induced by Helicobacter pylori infection after transplanting lymphocytes from patients with gastric lymphoma; Yokota K et al.; BACKGROUND & AIMS: Several studies have indicated that host factors are important in Helicobacter pylori-induced gastroduodenal diseases . We examined the pathological role of host immune responses in H . pylori infection by reconstituting components of the human immune system into severe combined immunodeficient (SCID) mice by transplantation of peripheral blood mononuclear cells (PBMCs) from H . pylori-infected patients . METHODS: PBMCs obtained from patients with mucosa-associated lymphoid tissue (MALT) lymphoma were injected intraperitoneally into SCID mice, designated MALToma-hu-SCID mice . One month after transplantation, H . pylori was administered orally to the mice . The mice were killed and examined for pathological changes and immunologic features . RESULTS: Human lymphocytes were detected in hu-SCID mice, and T- and B-cell functions were preserved for 1 month . Administration of H . pylori led to gastric ulcers with bleeding in the MALToma-hu-SCID mice . The gastric mucosa of control mice injected with Escherichia coli or transplanted with PBMCs from patients with peptic ulcers or gastritis or from healthy volunteers showed no pathological changes . CONCLUSIONS: Host immune responses against H . pylori appear to be involved in the development of gastric ulcers in patients who have MALT lymphoma. Electrophoresis, 1999 Sep, 20(12), 2400 - 6 Micellar electrokinetic chromatography as a complementary method to sodium dodecyl sulfate-polyacrylamide gel electrophoresis for studying limited proteolysis of proteins; Viglio S et al.; Micellar electrokinetic chromatography (MEKC) has been utilized as an analytical method to perform investigations on limited proteolysis of proteins . To this purpose partial proteolysis experiments with a series of proteinases were performed, utilizing as model protein pyruvate kinase (PK) from Escherichia coli, an enzyme that is regulated allosterically by fructose 1,6-bisphosphate (FBP) . Data obtained with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and MEKC were compared; the profiles generated by submitting digests of PK treated with different proteinases in the presence and absence of FBP to electrophoretic analysis provided a useful adjunct for a better understanding of the effects of the allosteric activator on the conformation of the model enzyme . MEKC was also found to be a convenient technique for determining the kinetics of substrate proteolysis. Clin Cancer Res, 1999 Sep, 5(9), 2399 - 408 A pilot study of interferon alpha-2a, fluorouracil, and leucovorin given with granulocyte-macrophage colony stimulating factor in advanced gastrointestinal adenocarcinoma; Shapiro JD et al.; We reported previously that the addition of recombinant Escherichia coli human granulocyte-macrophage colony stimulating factor (GM-CSF) to a 5-fluorouracil (5-FU) and leucovorin (LV) regimen seemed to ameliorate diarrhea and permit increased 5-FU dose intensity (J . L . Grem et al., J . Clin . Oncol., 12: 560-568, 1994) . We then tested the effect of GM-CSF given with a more toxic regimen of 5-FU/LV/IFN-alpha (IFN alpha-2a) . Thirty-one patients with a good performance status and no prior chemotherapy for systemic disease received IFN alpha(-2a (5 MU/m2 s.c., days 1-7), 5-FU (370 mg/m2 i.v., days 2-6), LV (500 mg/m2 i.v., days 2-6), and GM-CSF (Saccharomyces cerevisiae 250 microg/m2 s.c., days 7-18) every 3 weeks . Toxicities and 5-FU dose intensity were compared with that observed in our prior Phase II trial with 5-FU/LV/IFN alpha-2a (J . L . Grem et al., J . Clin . Oncol., 11: 1737-1745, 1993) . In comparison with the prior Phase II study, the WBC and granulocyte nadirs in the present trial were significantly higher . When trends in toxicity grades for all cycles were compared, stratifying for 5-FU dose, the incidence and severity of mucositis, skin rash, WBC toxicity, and granulocyte toxicity were significantly lower in the present trial, whereas nausea/vomiting and fatigue were significantly worse . The delivered 5-FU dose intensity for all cycles of therapy appeared to be significantly higher in the present trial . Six of 28 evaluable patients had a partial response (21.4%), and 13 (46%) had stable disease for > or =12 weeks . Despite treatment-related toxicity, patient quality of life did not worsen during the study . No correlation was observed between thymidylate synthase content in primary tumor specimens and response, time to treatment failure, or survival . The addition of GM-CSF appeared to decrease the severity of leukopenia, granulocytopenia, mucositis, and skin rash when compared with our prior experience with this regimen of 5-FU/LV/IFN alpha-2a, at the cost of greater nausea/vomiting and fatigue . The potential impact of increased 5-FU dose intensity on clinical response, however, remains to be determined. Virus Genes, 1999, 19(1), 33 - 43 Functional interactions between conserved motifs of the hepatitis C virus RNA helicase protein NS3; Min KH et al.; The hepatitis C virus NS3 gene encodes a RNA helicase with several sequence motifs conserved among the members of the DExH box protein family . The contributions of the sequence motifs to enzyme activity were assessed in this study by substitution of alanine for the Lys in the ATP binding motif GxGK (referred to as K1236A mutation), or for the Asp in the DExH motif (D1316A), or for the Arg in the middle of the QRxGRxGR motif known for RNA binding (R1490A) . Histidine-tagged recombinant proteins of Mr 54,000 were expressed in Escherichia coli and purified by chromatography on nickel agarose . All three mutants were severely defective in ATPase and RNA helicase activities, but loss of the ATPase activity was not dependent on polynucleotide cofactors . With the exception of R1490A mutant, a stable complex was formed between dsRNA substrates and recombinant proteins, indicating that the arginine-rich motif is required for efficient RNA binding . Complex formation was not affected by omission of ATP or substitution by a non-hydrolyzable analog AMP-PCP, suggesting that neither binding nor hydrolysis of ATP is required for RNA binding . Moreover, the K1236A mutant which was defective in binding ATP exhibited an unusually strong affinity for RNA duplex . These results suggest that the conserved motifs cooperatively constitute a large functional domain rather than act as individual domains with strictly independent functions, and that alteration of one motif affects functions of other motifs in a mutually interactive fashion. Eur J Pharmacol, 1999 Aug 20, 379(1), 73 - 80 The effects of bosentan, aminoguanidine and L-canavanine on mesenteric blood flow, spleen and liver in endotoxaemic mice; Iskit AB et al.; The modulatory effects of a non-selective endothelin receptor antagonist, bosentan, were investigated together with those of relatively selective inducible nitric oxide synthase inhibitors, aminoguanidine and L-canavanine, on mesenteric blood flow decrease, liver and spleen injury elicited by endotoxaemia . Swiss albino mice (20-40 g) were administered intraperitoneally bosentan (3, 10 or 30 mg kg(-1)), aminoguanidine (15 mg kg(-1)) or L-canavanine (20 or 100 mg kg(-1)) 10 min before they received saline or Escherichia coli endotoxin (10 mg kg(-1)) . After 4 h, the mice were anaesthetized, mesenteric blood flow values were measured, spleen and liver weight/body weight ratios were determined and the organs were examined histopathologically . Endotoxin decreased mesenteric blood flow (ml min(-1), saline: 3.0 +/- 0.2; endotoxin: 2.2 +/- 0.2: n = 10, P < 0.05), increased the weight of liver (g per kg body weight, saline: 47.5 +/- 2.0; endotoxin: 60.8 +/- 1.9: n = 10, P < 0.05) and spleen (g per kg body weight, saline: 3.9 +/- 0.5; endotoxin: 8.6 +/- 0.9; n = 10, P < 0.01) while it inflicted significant histopathological injury to both organs . Bosentan was ineffective at 3 mg kg(-1) but at 10 and 30 mg kg(-1) doses, it abolished all the deleterious effects of endotoxin without exception . Aminoguanidine blocked most of the effects of endotoxin except those on spleen . In contrast, L-canavanine blocked only the endotoxin-induced increase in liver weight but itself increased spleen weight and failed to block any other effects of endotoxin . Thus, it can be speculated that the beneficial effects of aminoguanidine are produced largely by mechanisms other than selective inducible nitric oxide synthase inhibition since L-canavanine was not fully effective . The beneficial effects of endothelin inhibition by using bosentan in endotoxaemia can be further exploited for the understanding and the therapy of sepsis-related syndromes. Appl Microbiol Biotechnol, 1999 Aug, 52(2), 221 - 5 An acetylxylan esterase and a xylanase expressed from genes cloned from the ruminal fungus Neocallimastix patriciarum act synergistically to degrade acetylated xylans; Cybinski DH et al.; A Neocallimastit patriciarum acetylxylan esterase (BnaA) was expressed from the cloned gene in Escherichia coli . Purified recombinant BnaA efficiently released acetate from soluble acetylated birchwood xylan (ABX), with a specific activity of 76 U mg-1 . In contrast, release of acetate was very inefficient from the insoluble substrates, spear grass and delignified spear grass . Addition of a recombinant xylanase, XynA, also expressed from a cloned N . patriciarum gene, had no effect on the release of acetate from ABX . However, the combination of recombinant BnaA and XynA released more acetate from spear grass and delignified spear grass than did BnaA alone . Significantly more reducing sugar was also released from all three substrates by the combination of recombinant XynA and BnaA than by XynA alone . Thus the extent of digestion of acetylated xylans by XynA appears to be limited by the acetylation . In this system BnaA does not appear to increase the rate of cleavage of insoluble substrates by XynA, but probably allows the release of shorter xylose oligomers from already solubilised acetylated xylan polymers. J Biol Inorg Chem, 1999 Apr, 4(2), 223 - 31 Iron coordination geometry in full-length, truncated, and dehydrated forms of human tyrosine hydroxylase studied by Mössbauer and X-ray absorption spectroscopy; Schunemann V et al.; Full-length human tyrosine hydroxylase 1 (hTH1) and a truncated enzyme lacking the 150 N-terminal amino acids were expressed in Escherichia coli and purified either with or without (6 x histidine) N-terminal tags . After reconstitution with 57Fe(II), the Mossbauer and X-ray absorption spectra of the enzymes were compared before and after dehydration by lyophilization . Before dehydration, > 90% of the iron in hTH1 had Mossbauer parameters typical for high-spin Fe(II) in a six-coordinate environment {isomer shift delta (1.8-77 K) = 1.26-1.24 mm s-1 and quadrupole splitting delta EQ = 2.68 mm s-1} . After dehydration, the Mossbauer spectrum changed and 63% of the area could be attributed to five-coordinate high-spin Fe(II) (delta = 1.07 mm s-1 and delta EQ = 2.89 mm s-1 at 77 K), whereas 28% of the iron remained as six-coordinate high-spin Fe(II) (delta = 1.24 mm s-1 and delta EQ = 2.87 mm s-1 at 77 K) . Similar changes upon dehydration were observed for truncated TH either with or without the histidine tag . After rehydration of hTH1 the spectroscopic changes were completely reversed . The X-ray absorption spectra of hTH1 in solution and in lyophilized form, and for the truncated protein in solution, corroborate the findings derived from the Mossbauer spectra . The pre-edge peak intensity of the protein in solution indicates six-coordination of the iron, while that of the dehydrated protein is typical for a five-coordinate iron center . Thus, the active-site iron can exist in different coordination states, which can be interconverted depending on the hydration state of the protein, indicating the presence or absence of a water molecule as a coordinating ligand to the iron . The present study explains the difference in iron coordination determined by X-ray crystallography, which has shown a five-coordinate iron center in rat TH, and by our recent spectroscopic study of human TH in solution, which showed a six-coordinated iron center. J Biol Inorg Chem, 1999 Apr, 4(2), 220 - 2 Expression of 15N-labeled eukaryotic cytochrome c in Escherichia coli; Morar AS et al.; We present a simple and inexpensive method for producing 15N-labeled Saccharomyces cerevisiae iso-1-cytochrome c in Escherichia coli . The labeled protein gives excellent NMR spectra. Biochemistry (Mosc), 1999 Aug, 64(8), 933 - 7 Isolation and characterization of Rous sarcoma virus recombinant reverse transcriptase dimers; Chernov AP et al.; Reverse transcriptase (RT) preparations containing various molecular forms of the enzyme consisting of alpha- and/or beta-subunits have been isolated from E . coli cells transformed with plasmid pMF14 containing the Rous sarcoma virus (RSV) pol gene . The three possible dimeric forms of the enzyme demonstrated DNA polymerase activity, the relative activities of the alphaalpha, betabeta, and alphabeta forms being about 1:3:4 . RNase H activity is associated with the betabeta and alphabeta dimers but not with the alphaalpha dimer . Comparison of the enzymic properties of the various dimers and dissociation--reassociation results suggest that the betabeta and alphabeta dimers of the RSV recombinant reverse transcriptase are similar to the corresponding virion RT forms. Bioinformatics, 1999 Sep, 15(9), 723 - 8 Neural network input representations that produce accurate consensus sequences from DNA fragment assemblies; Allex CF et al.; MOTIVATION: Given inputs extracted from an aligned column of DNA bases and the underlying Perkin Elmer Applied Biosystems (ABI) fluorescent traces, our goal is to train a neural network to determine correctly the consensus base for the column . Choosing an appropriate network input representation is critical to success in this task . We empirically compare five representations; one uses only base calls and the others include trace information . RESULTS: We attained the most accurate results from networks that incorporate trace information into their input representations . Based on estimates derived from using 10-fold cross-validation, the best network topology produces consensus accuracies ranging from 99.26% to >99.98% for coverages from two to six aligned sequences . With a coverage of six, it makes only three errors in 20 000 consensus calls . In contrast, the network that only uses base calls in its input representation has over double that error rate: eight errors in 20 000 consensus calls . CONTACT: allex@cs.wisc.edu J Bacteriol, 1999 Oct, 181(19), 6220 - 1 recD sbcB sbcD mutants are deficient in recombinational repair of UV lesions by RecBC; Seigneur M et al.; In recD sbcB sbcD mutants, repair of UV-irradiated DNA is strongly RecF dependent, indicating that RecBC is inactive . This finding suggests that exonuclease V, exonuclease I (SbcB), and the SbcCD nuclease play a redundant role in vivo, which is essential for the recombination activity of the RecBC complex during UV repair. J Bacteriol, 1999 Oct, 181(19), 6210 - 3 Functional expression of hMYH, a human homolog of the Escherichia coli MutY protein; Slupska MM et al.; We have previously described the hMYH cDNA and genomic clones (M . M . Slupska et al., J . Bacteriol . 178:3885-3892, 1996) . Here, we report that the enzyme expressed from an hMYH cDNA clone in Escherichia coli complements the mutator phenotype in a mutY mutant and can remove A from an A . 8-hydroxydeoxyguanine mismatch and to a lesser extent can remove A from an A . G mismatch in vitro. J Bacteriol, 1999 Oct, 181(19), 6179 - 83 Chromosome segregation and cell division defects in recBC sbcBC ruvC mutants of Escherichia coli; Zahradka D et al.; The RuvC protein is important for DNA recombination and repair in Escherichia coli . The present work shows that a ruvC null mutation introduced into a recBC sbcBC background causes severe defects in chromosome segregation and cell division . Both defects were found to result from abortive recombination initiated by the RecA protein. J Bacteriol, 1999 Oct, 181(19), 6108 - 13 Analysis of F factor TraD membrane topology by use of gene fusions and trypsin-sensitive insertions; Lee MH et al.; This report describes a procedure for characterizing membrane protein topology which combines the analysis of reporter protein hybrids and trypsin-sensitive 31-amino-acid insertions generated by using transposons ISphoA/in and ISlacZ/in . Studies of the F factor TraD protein imply that the protein takes on a structure with two membrane-spanning sequences and amino and carboxyl termini facing the cytoplasm . It was possible to assign the subcellular location of one region for which the behavior of fused reporter proteins was ambiguous, based on the trypsin cleavage behavior of a 31-residue insertion. J Bacteriol, 1999 Oct, 181(19), 6010 - 8 Plasmid RK2 ParB protein: purification and nuclease properties; Johnson EP et al.; The parCBA operon of the 3.2-kb stabilization region of plasmid RK2 encodes three cotranslated proteins . ParA mediates site-specific recombination to resolve plasmid multimers, ParB has been shown to be a nuclease, and the function of ParC is unknown . In this study ParB was overexpressed by cotranslation with ParC in Escherichia coli by using a plasmid construct that contained the parC and parB genes under the control of the T7 promoter . Purification was achieved by treatment of extracts with Polymin P, followed by ammonium sulfate precipitation and heparin and ion-exchange chromatography . Sizing-column analysis indicated that ParB exists as a monomer in solution . Analysis of the enzymatic properties of purified ParB indicated that the protein preferentially cleaves single-stranded DNA . ParB also nicks supercoiled plasmid DNA preferably at sites with potential single-stranded character, like AT-rich regions and sequences that can form cruciform structures . ParB also exhibits 5'-->3' exonuclease activity . This ParB activity on a 5'-end-labeled, double-stranded DNA substrate produces a 3', 5'-phosphorylated dinucleotide which is further cleaved to a 3', 5'-phosphorylated mononucleotide . The role of the ParB endonuclease and exonuclease activities in plasmid RK2 stabilization remains to be determined. J Bacteriol, 1999 Oct, 181(19), 5958 - 66 Essential role of the iron-regulated outer membrane receptor FauA in alcaligin siderophore-mediated iron uptake in Bordetella species; Brickman TJ et al.; Phenotypic analysis using heterologous host systems localized putative Bordetella pertussis ferric alcaligin transport genes and Fur-binding sequences to a 3.8-kb genetic region downstream from the alcR regulator gene . Nucleotide sequencing identified a TonB-dependent receptor family homolog gene, fauA, predicted to encode a polypeptide with high amino acid sequence similarity with known bacterial ferric siderophore receptors . In Escherichia coli, the fauA genes of both B . pertussis and Bordetella bronchiseptica directed the production of a 79-kDa polypeptide, approximating the predicted size of the mature FauA protein . B . bronchiseptica fauA insertion mutant BRM17 was unable to utilize ferric alcaligin, and in complementation analyses ferric alcaligin utilization was restored to this mutant by supplying the wild-type fauA gene in trans . Mutant BRM18, carrying a nonpolar in-frame fauA deletion mutation, was defective in ferric alcaligin utilization and (55)Fe-ferric alcaligin uptake and no longer produced a 79-kDa iron-regulated outer membrane protein . In complementation analyses, BRM18 merodiploids bearing the wild-type fauA gene in trans regained ferric alcaligin siderophore transport and utilization functions and produced the 79-kDa protein . Analysis of a plasmid-borne fauA-lacZ operon fusion confirmed that fauA is subject to iron regulation at the transcriptional level and that cis-acting transcriptional control elements mediating fauA iron repressibility reside within the 3.8-kb PstI fauA DNA region . Moreover, expression of the fauA-lacZ fusion gene under iron starvation conditions was shown to be alcR dependent . FauA is a 79-kDa iron-regulated outer membrane receptor protein required for transport and utilization of ferric alcaligin siderophore complexes by Bordetella species. J Bacteriol, 1999 Oct, 181(19), 5898 - 908 P1 ParB domain structure includes two independent multimerization domains; Surtees JA et al.; ParB is one of two P1-encoded proteins that are required for active partition of the P1 prophage in Escherichia coli . To probe the native domain structure of ParB, we performed limited proteolytic digestions of full-length ParB, as well as of several N-terminal and C-terminal deletion fragments of ParB . The C-terminal 140 amino acids of ParB form a very trypsin-resistant domain . In contrast, the N terminus is more susceptible to proteolysis, suggesting that it forms a less stably folded domain or domains . Because native ParB is a dimer in solution, we analyzed the ability of ParB fragments to dimerize, using both the yeast two-hybrid system and in vitro chemical cross-linking of purified proteins . These studies revealed that the C-terminal 59 amino acids of ParB, a region within the protease-resistant domain, are sufficient for dimerization . Cross-linking and yeast two-hybrid experiments also revealed the presence of a second self-association domain within the N-terminal half of ParB . The cross-linking data also suggest that the C terminus is inhibitory to multimerization through the N-terminal domain in vitro . We propose that the two multimerization domains play distinct roles in partition complex formation. Infect Immun, 1999 Oct, 67(10), 5463 - 9 Conformational nature of the Borrelia burgdorferi B31 outer surface protein C protective epitope; Gilmore RD Jr et al.; Active immunization with Escherichia coli-expressed recombinant outer surface protein C (OspC) of Borrelia burgdorferi has been demonstrated to confer protection against a tick-transmitted infection on laboratory animals . A previous study in this laboratory showed that OspC antibody raised against a denatured immunogen isolated from B . burgdorferi cells failed to provide protective immunity . Therefore, to determine whether the protective epitope of the recombinant antigen was sensitive to denaturation, recombinant OspC preparations were subjected to heat and chemical treatments prior to animal immunization . Following seroconversion to OspC, the animals were challenged with an infectious dose of B . burgdorferi B31 by tick bite . Whereas mice immunized with a soluble, nondenatured form continued to show protection rates close to 100%, mice that had been immunized with denatured antigen were not protected . Furthermore, mice that were immunized with an insoluble (rather than a soluble), nondenatured form of the recombinant OspC showed a protection rate of only 40% . Protective epitope localization experiments showed that either the amino or the carboxy end of the recombinant protein was required to react with a protective OspC-specific monoclonal antibody . The data from these experiments demonstrate that a conformational organization of the protein is essential for the protective capability of the strain B31 OspC immunogen. J Endocrinol, 1999 Oct, 163(1), 15 - 24 Pentoxifylline improves insulin action limiting skeletal muscle catabolism after infection; Vary T et al.; We investigated the ability of pentoxifylline (PTX) to modulate protein synthesis and degradation in the presence and absence of insulin during incubation of epitrochlearis muscle, 2 or 6 days after injection of Escherichia coli . On days 2 and 6 after infection, protein synthesis was inhibited by 25%, whereas proteolysis was enhanced by 75% . Insulin (2 nM) in vitro stimulated protein synthesis in muscles from infected rats to the same extent as in controls . The ability of insulin to limit protein degradation was severely blunted 48 h after infection . On day 6 after infection, insulin inhibited proteolysis to a greater extent than on day 2 . PTX suppressed the increase in plasma concentrations of tumor necrosis factor more than 600-fold after injection of bacteria, and partially prevented the inhibition of protein synthesis and stimulation of protein degradation during sepsis . Moreover, PTX administration maintained the responsiveness of protein degradation to insulin during sepsis . Thus cytokines may influence skeletal muscle protein metabolism during sepsis, both indirectly through inhibition of the effects of insulin on proteolysis, and directly on the protein synthesis and degradation machinery. J Gen Physiol, 1999 Oct, 114(4), 551 - 60 Single streptomyces lividans K(+) channels: functional asymmetries and sidedness of proton activation; Heginbotham L et al.; Basic electrophysiological properties of the KcsA K(+) channel were examined in planar lipid bilayer membranes . The channel displays open-state rectification and weakly voltage-dependent gating . Tetraethylammonium blocking affinity depends on the side of the bilayer to which the blocker is added . Addition of Na(+) to the trans chamber causes block of open-channel current, while addition to the cis side has no effect . Most striking is the activation of KcsA by protons; channel activity is observed only when the trans bilayer chamber is at low pH . To ascertain which side of the channel faces which chamber, residues with structurally known locations were mapped to defined sides of the bilayer . Mutation of Y82, an external residue, results in changes in tetraethylammonium affinity exclusively from the cis side . Channels with cysteine residues substituted at externally exposed Y82 or internally exposed Q119 are functionally modified by methanethiosulfonate reagents from the cis or trans chambers, respectively . Block by charybdotoxin, known to bind to the channel's external mouth, is observed only when the toxin is added to the cis side of channels mutated to be toxin sensitive . These results demonstrate unambiguously that the protonation sites linked to gating are on the intracellular portion of the KcsA protein. J Trauma, 1999 Sep, 47(3), 492 - 8; discussion 498-9 Thermal injury alters endothelial vasoconstrictor and vasodilator response to endotoxin; Murphy JT et al.; BACKGROUND: The unique location of the endothelium makes it vulnerable to injury from circulating factors created at remote wounds . In this study, we examined the effect of a sequential burn and lipopolysaccharide (LPS) challenge on endothelial function in vitro . METHODS: Human umbilical vein endothelial cells treated with 20% human serum isolated from burn patients (>40% total burn surface area) at 2 and 24 hours postinjury . Cultures were subsequently treated with Escherichia coli LPS:0111:B4 (0.10-100ng/mL) . Endothelin-1 (ET-1), 6-ketoPGF1a, and NO2/NO3 were detected by using specific enzyme immunoassays . RESULTS: Burn serum did not alter endothelial ET-1, PGI2, or NO secretion compared with Control serum . LPS significantly enhanced 6-ketoPGF1a (54,242+/-14,466 pg/10(6) cells) and NO2/ NO3 (723+/-210 microM) secretion, but not ET-1 compared with Control serum alone (3,878+/-963 and 219+/-110) . Burn serum pretreatment significantly enhanced the ET-1 response to LPS (303+/-36 pg/10(6) cells vs . 193+/-47) . The 6-ketoPGF1a (16,509+/-3,785) and NO2/NO3 (354+/-98) responses to Burn/LPS were significantly diminished compared with Control/LPS . Although this level of 6-ketoPGF1a was elevated compared with Control alone (7,518+/-2,299), NO2/NO3 was unchanged (significance at p < 0.05) . CONCLUSION: Thermal injury may prime remote endothelium and alter the response to a septic focus with an enhanced vasoconstrictor (ET-1) and diminished vasodilator (PGI2/NO) response, a situation that may contribute to postburn distal organ injury. FEMS Immunol Med Microbiol, 1999 Sep, 25(4), 379 - 84 Immunological characterization of the sheep prion protein expressed as fusion proteins in Escherichia coli; Baron TG et al.; The prion protein (PrP) from sheep was produced in large quantities of entire protein in Escherichia coli after fusion with a carboxy-terminal hexahistidine sequence . In contrast, amino-terminal fusion with glutathione S-transferase (GST) revealed a high susceptibility toward cleavage of the protein . Both recombinant proteins were recognised, at variable levels, in Western blots using a panel of antibodies against the 40-56, 89-104, 98-113 and 112-115 sequences of the prion protein, similarly to the abnormal prion protein extracted from scrapie-infected sheep . Interestingly, monoclonal antibody 3F4 was found to react with these three proteins in Western blot. Can J Microbiol, 1999 Jul, 45(7), 632 - 7 Endonuclease III and endonuclease IV protect Escherichia coli from the lethal and mutagenic effects of near-UV irradiation; Serafini DM et al.; In contrast to the DNA damage caused by far-UV (lambda < 290 nm), near-UV (290 < lambda < 400 nm) induced DNA damage is partially oxygen dependent, suggesting the involvement of reactive oxygen species . To test the hypothesis that enzymes that protect cells from oxidative DNA damage are also involved in preventing near-UV mediated DNA damage, isogenic strains deficient in one or more of exonuclease III (xthA), endonuclease IV (nfo), and endonuclease III (nth) were exposed to increasing levels of far-UV and near-UV . All strains, with the exception of the nth single mutant, were found to be hypersensitive to the lethal effects of near-UV relative to a wild-type strain . A triple mutant strain (nth nfo xthA) exhibited the greatest sensitivity to near-UV-mediated lethality . The triple mutant was more sensitive than the nfo xthA double mutant to the lethal effects of near-UV, but not far-UV . A forward mutation assay also revealed a significantly increased sensitivity for the triple mutant compared to the nfo xthA deficient strain in the presence of near-UV . However, the triple mutant was no more sensitive to the mutagenic effects of far-UV than a nfo xthA double mutant . These data suggest that exonuclease III, endonuclease IV, and endonuclease III are important in protection against near-UV-induced DNA damage. Nucleic Acids Res, 1999 Oct 15, 27(20), 4077 - 82 The DNase activity of RNase T and its application to DNA cloning; Zuo Y et al.; RNase T is one of eight distinct 3'-->5' exoribonucleases present in Escherichia coli . The enzyme plays an important role in stable RNA metabolism, including tRNA end turnover and 3' maturation of most stable RNAs because it is the only RNase that can efficiently remove residues near a double-stranded (ds) stem . In the course of study of its specificity and mechanism, we found that RNase T also has single-strand-specific DNase activity . Purified RNase T degrades both single-stranded (ss)RNA and ssDNA in a non-processive manner . However, in contrast to its action on RNA, RNase T binds ssDNA much more tightly and shows less sequence specificity . As with RNA, DNA secondary structure strongly affects its degradation by RNase T . Thus, RNase T action on a dsDNA with a single-stranded 3'-extension efficiently generates blunt-ended DNA . This property of RNase T suggested that it might be a useful enzyme for blunt-ended DNA cloning . We show here that RNase T provides much higher cloning efficiency than the currently used mung bean nuclease. Nucleic Acids Res, 1999 Oct 15, 27(20), 4018 - 27 A phylogenetic approach to target selection for structural genomics: solution structure of YciH; Cort JR et al.; Structural genomics presents an enormous challenge with up to 100 000 protein targets in the human genome alone . At current rates of structure deter-mination, judicious selection of targets is necessary . Here, a phylogenetic approach to target selection is described which makes use of the National Center for Biotechnology Information database of Clusters of Orthologous Groups (COGS) . The strategy is designed so that each new protein structure is likely to provide novel sequence-fold information . To demonstrate this approach, the NMR solution structure of YciH (COG0023), a putative translation initiation factor from Escherichia coli, has been determined and its fold classified . YciH is an ortholog of eIF-1/SUI1, an integral component of the translation initiation complex in eukaryotes . The structure consists of two antiparallel alpha-helices packed against the same side of a five-stranded beta-sheet . The first 31 residues of the 11.5 kDa protein are unstructured in solution . Comparative analysis indicates that the folded portion of YciH resembles a number of structures with the alpha-beta plait topology, though its sequence is not homologous to any of them . Thus, the phylogenetic approach to target selection described here was used successfully to identify a new homologous superfamily within this topology. Nucleic Acids Res, 1999 Oct 15, 27(20), 4001 - 7 Excision of oxidatively damaged DNA bases by the human alpha-hOgg1 protein and the polymorphic alpha-hOgg1(Ser326Cys) protein which is frequently found in human populations; Dherin C et al.; We have investigated the substrate specificity of the major nuclear form of the human Ogg1 protein, referred as alpha-hOgg1, for excision of damaged bases from DNA exposed to gamma-irradiation . Excision products were identified and quantified using gas chromatography/isotope dilution mass spectrometry (GC/IDMS) . The GST-alpha-hOgg1 protein used in this study is a fusion of alpha-hOgg1 to the C-terminus of the GST protein . The results show that GST-alpha-hOgg1 protein excises 8-hydroxyguanine (8-OH-Gua) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from DNA exposed to gamma-irradiation in a solution saturated with N(2)O or air . Fourteen other lesions, including oxidised purines and pyrimidines, were not excised from these substrates . Catalytic constants were measured for the excision of 8-OH-Gua and FapyGua from DNA gamma-irradiated under N(2)O . The k (cat)/ K (m)values for excision of 8-OH-Gua and FapyGua were 4.47 x 10(-5)and 8.97 x 10(-5)(min(-1)nM(-1)), respectively . The substrate specificity and the catalytic parameters of the wild-type GST-alpha-hOgg1 protein were compared to that of a polymorphic form of alpha-hOgg1 harbouring a Ser-->Cys mutation at codon 326 . In the Japanese population, 47.6% of individuals possess both alleles coding for the wild-type alpha-hOgg1-Ser(326)and mutant alpha-hOgg1-Cys(326)proteins . The GST-alpha-hOgg1-Cys(326)protein was purified and its substrate specificity was determined by GC/IDMS analysis . The results show that the GST-alpha-hOgg1-Cys(326)protein efficiently excises 8-OH-Gua and FapyGua from gamma-irradiated DNA . The k (cat)/ K (m)values for excision of 8-OH-Gua and FapyGua were 2 . 82 x 10(-5)and 4.43 x 10(-5)(min(-1)nM(-1)), respectively . Furthermore, we compared the capacity of these two forms of alpha-hOgg1 to act on substrates containing 2,6-diamino-4-hydroxy-5- N -methylformamidopyrimidine (Me-FapyGua) . The k (cat)/ K (m)values for excision of Me-FapyGua were 278 x 10(-5)and 319 x 10(-5)(min(-1)nM(-1)), respectively . Cleavage of 34mer oligodeoxyribonucleotides containing 8-OH-Gua, 8-hydroxyadenine or an apurinic/apyrimidinic site paired with a cytosine was also investigated . The results show that both GST-alpha-hOgg1-Ser(326)and GST-alpha-hOgg1-Cys(326)catalyse the various cleavage reactions at very similar rates . Furthermore, both proteins efficiently complement the mutator phenotype of the fpg mutY mutant of Escherichia coli. Nucleic Acids Res, 1999 Oct 15, 27(20), 3953 - 63 Mutational analysis of Escherichia coli DNA ligase identifies amino acids required for nick-ligation in vitro and for in vivo complementation of the growth of yeast cells deleted for CDC9 and LIG4; Sriskanda V et al.; We report that the NAD-dependent Escherichia coli DNA ligase can support the growth of Saccharomyces cerevisiae strains deleted singly for CDC9 or doubly for CDC9 plus LIG4 . Alanine-scanning mutagenesis of E.coli DNA ligase led to the identification of seven amino acids (Lys115, Asp117, Asp285, Lys314, Cys408, Cys411 and Cys432) that are essential for nick-joining in vitro and for in vivo complementation in yeast . The K314A mutation uniquely resulted in accumulation of the DNA-adenylate intermediate . Alanine substitutions at five other positions (Glu113, Tyr225, Gln318, Glu319 and Cys426) did not affect in vivo complementation and had either no effect or only a modest effect on nick-joining in vitro . The E113A and Y225A mutations increased the apparent K (m)for NAD (to 45 and 76 microM, respectively) over that of the wild-type E . coli ligase (3 microM) . These results are discussed in light of available structural data on the adenylylation domains of ATP- and NAD-dependent ligases . We observed that yeast cells containing only the 298-amino acid Chlorella virus DNA ligase (a 'minimal' eukaryotic ATP-dependent ligase consisting only of the catalytic core domain) are relatively proficient in the repair of DNA damage induced by UV irradiation or treatment with MMS, whereas cells containing only E.coli ligase are defective in DNA repair . This suggests that the structural domains unique to yeast Cdc9p are not essential for mitotic growth, but may facilitate DNA repair. J Biol Chem, 1999 Oct 1, 274(40), 28751 - 61 Isolation and characterization of a split B-type DNA polymerase from the archaeon Methanobacterium thermoautotrophicum deltaH; Kelman Z et al.; We describe here the isolation and characterization of a B-type DNA polymerase (PolB) from the archaeon Methanobacterium thermoautotrophicum DeltaH . Uniquely, the catalytic domains of M . thermoautotrophicum PolB are encoded from two different genes, a feature that has not been observed as yet in other polymerases . The two genes were cloned, and the proteins were overexpressed in Escherichia coli and purified individually and as a complex . We demonstrate that both polypeptides are needed to form the active polymerase . Similar to other polymerases constituting the B-type family, PolB possesses both polymerase and 3'-5' exonuclease activities . We found that a homolog of replication protein A from M . thermoautotrophicum inhibits the PolB activity . The inhibition of DNA synthesis by replication protein A from M . thermoautotrophicum can be relieved by the addition of M . thermoautotrophicum homologs of replication factor C and proliferating cell nuclear antigen . The possible roles of PolB in M . thermoautotrophicum replication are discussed. J Biol Chem, 1999 Oct 1, 274(40), 28405 - 12 Intracellular localization of human cytidine deaminase . Identification of a functional nuclear localization signal; Somasekaram A et al.; The cytidine deaminases belong to the family of multisubunit enzymes that catalyze the hydrolytic deamination of their substrate to a corresponding uracil product . They play a major role in pyrimidine nucleoside and nucleotide salvage . The intracellular distribution of cytidine deaminase and related enzymes has previously been considered to be cytosolic . Here we show that human cytidine deaminase (HCDA) is present in the nucleus . A highly specific, affinity purified polyclonal antibody against HCDA was used to analyze the intracellular localization of native HCDA in a variety of mammalian cells by in situ immunochemistry . Native HCDA was found to be present in the nucleus as well as the cytoplasm in several cell types . Indirect immunofluorescence microscopy indicated a predominantly nuclear localization of FLAG-tagged HCDA overexpressed in these cells . We have identified an amino-terminal bipartite nuclear localization signal that is both necessary and sufficient to direct HCDA and a non-nuclear reporter protein to the nucleus . We also show HCDA binding to the nuclear import receptor, importin alpha . Similar putative bipartite nuclear localization sequences are found in other cytidine/deoxycytidylate deaminases . The results presented here suggest that the pyrimidine nucleotide salvage pathway may operate in the nucleus . This localization may have implications in the regulation of nucleoside and nucleotide metabolism and nucleic acid biosynthesis. J Biol Chem, 1999 Oct 1, 274(40), 28351 - 5 The epsilon subunit of the F(1)F(0) complex of Escherichia coli . cross-linking studies show the same structure in situ as when isolated; Schulenberg B et al.; Four double mutants in the epsilon subunit were generated, each containing two cysteines, which, based on the NMR structure of this subunit, should form internal disulfide bonds . Two of these were designed to generate interdomain cross-links that lock the C-terminal alpha-helical domain against the beta-sandwich (epsilonM49C/A126C and epsilonF61C/V130C) . The second set should give cross-linking between the two C-terminal alpha-helices (epsilonA94C/L128C and epsilonA101C/L121C) . All four mutants cross-linked with 90-100% efficiency upon CuCl(2) treatment in isolated Escherichia coli ATP synthase . This shows that the structure obtained for isolated epsilon is essentially the same as in the assembled complex . Functional studies revealed increased ATP hydrolysis after cross-linking between the two domains of the subunit but not after cross-linking between the C-terminal alpha-helices . None of the cross-links had any effect on proton pumping-coupled ATP hydrolysis, on DCCD sensitivity of this activity, or on ATP synthesis rates . Therefore, big conformational changes within epsilon can be ruled out as a part of the enzyme function . Protease digestion studies, however, showed that subtle changes do occur, since the epsilon subunit could be locked in an ADP or 5'-adenylyl-beta,gamma-imidodiphosphate conformation by the cross-linking with resulting differences in cleavage rates. J Biol Chem, 1999 Oct 1, 274(40), 28270 - 8 Properties of cloned and expressed human RNase H1; Wu H et al.; We have characterized cloned His-tag human RNase H1 . The activity of the enzyme exhibited a bell-shaped response to divalent cations and pH . The optimum conditions for catalysis consisted of 1 mM Mg(2+) and pH 7-8 . In the presence of Mg(2+), Mn(2+) was inhibitory . Human RNase H1 shares many enzymatic properties with Escherichia coli RNase H1 . The human enzyme cleaves RNA in a DNA-RNA duplex resulting in products with 5'-phosphate and 3'-hydroxy termini, can cleave overhanging single strand RNA adjacent to a DNA-RNA duplex, and is unable to cleave substrates in which either the RNA or DNA strand has 2' modifications at the cleavage site . Human RNase H1 binds selectively to "A-form"-type duplexes with approximately 10-20-fold greater affinity than that observed for E . coli RNase H1 . The human enzyme displays a greater initial rate of cleavage of a heteroduplex-containing RNA-phosphorothioate DNA than an RNA-DNA duplex . Unlike the E . coli enzyme, human RNase H1 displays a strong positional preference for cleavage, i.e . it cleaves between 8 and 12 nucleotides from the 5'-RNA-3'-DNA terminus of the duplex . Within the preferred cleavage site, the enzyme displays modest sequence preference with GU being a preferred dinucleotide . The enzyme is inhibited by single-strand phosphorothioate oligonucleotides and displays no evidence of processivity . The minimum RNA-DNA duplex length that supports cleavage is 6 base pairs, and the minimum RNA-DNA "gap size" that supports cleavage is 5 base pairs. J Biol Chem, 1999 Oct 1, 274(40), 28240 - 5 Functional linkage between the glutaminase and synthetase domains of carbamoyl-phosphate synthetase . Role of serine 44 in carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (cad); Hewagama A et al.; Mammalian carbamoyl-phosphate synthetase is part of carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (CAD), a multifunctional protein that also catalyzes the second and third steps of pyrimidine biosynthesis . Carbamoyl phosphate synthesis requires the concerted action of the glutaminase (GLN) and carbamoyl-phosphate synthetase domains of CAD . There is a functional linkage between these domains such that glutamine hydrolysis on the GLN domain does not occur at a significant rate unless ATP and HCO(3)(-), the other substrates needed for carbamoyl phosphate synthesis, bind to the synthetase domain . The GLN domain consists of catalytic and attenuation subdomains . In the separately cloned GLN domain, the catalytic subdomain is down-regulated by interactions with the attenuation domain, a process thought to be part of the functional linkage . Replacement of Ser(44) in the GLN attenuation domain with alanine increases the k(cat)/K(m) for glutamine hydrolysis 680-fold . The formation of a functional hybrid between the mammalian Ser(44) GLN domain and the Escherichia coli carbamoyl-phosphate synthetase large subunit had little effect on glutamine hydrolysis . In contrast, ATP and HCO(3)(-) did not stimulate the glutaminase activity, indicating that the interdomain linkage had been disrupted . In accord with this interpretation, the rate of glutamine hydrolysis and carbamoyl phosphate synthesis were no longer coordinated . Approximately 3 times more glutamine was hydrolyzed by the Ser(44) --> Ala mutant than that needed for carbamoyl phosphate synthesis . Ser(44), the only attenuation subdomain residue that extends into the GLN active site, appears to be an integral component of the regulatory circuit that phases glutamine hydrolysis and carbamoyl phosphate synthesis. J Biol Chem, 1999 Oct 1, 274(40), 28083 - 6 ClpB cooperates with DnaK, DnaJ, and GrpE in suppressing protein aggregation . A novel multi-chaperone system from Escherichia coli; Zolkiewski M; ClpB is a heat-shock protein from Escherichia coli with an unknown function . We studied a possible molecular chaperone activity of ClpB in vitro . Firefly luciferase was denatured in urea and then diluted into the refolding buffer (in the presence of 5 mM ATP and 0.1 mg/ml bovine serum albumin) . Spontaneous reactivation of luciferase was very weak (less than 0.02% of the native activity) because of extensive aggregation . Conventional chaperone systems (GroEL/GroES and DnaK/DnaJ/GrpE) or ClpB alone did not reactivate luciferase under those conditions . However, ClpB together with DnaK/DnaJ/GrpE greatly enhanced the luciferase activity regain (up to 57% of native activity) by suppressing luciferase aggregation . This coordinated function of ClpB and DnaK/DnaJ/GrpE required ATP hydrolysis, although the ClpB ATPase was not activated by native or denatured luciferase . When the chaperones were added to the luciferase refolding solutions after 5-25 min of refolding, ClpB and DnaK/DnaJ/GrpE recovered the luciferase activity from preformed aggregates . Thus, we have identified a novel multi-chaperone system from E . coli, which is analogous to the Hsp104/Ssa1/Ydj1 system from yeast . ClpB is the only known bacterial Hsp100 protein capable of cooperating with other heat-shock proteins in suppressing and reversing protein aggregation. Science, 1999 Sep 24, 285(5436), 2133 - 6 Identification of an RNA-protein bridge spanning the ribosomal subunit interface; Culver GM et al.; The 7.8 angstrom crystal structure of the 70S ribosome reveals a discrete double-helical bridge (B4) that projects from the 50S subunit, making contact with the 30S subunit . Preliminary modeling studies localized its contact site, near the bottom of the platform, to the binding site for ribosomal protein S15 . Directed hydroxyl radical probing from iron(II) tethered to S15 specifically cleaved nucleotides in the 715 loop of domain II of 23S ribosomal RNA, one of the known sites in 23S ribosomal RNA that are footprinted by the 30S subunit . Reconstitution studies show that protection of the 715 loop, but none of the other 30S-dependent protections, is correlated with the presence of S15 in the 30S subunit . The 715 loop is specifically protected by binding free S15 to 50S subunits . Moreover, the previously determined structure of a homologous stem-loop from U2 small nuclear RNA fits closely to the electron density of the bridge. Virology, 1999 Sep 1, 261(2), 216 - 26 Nucleoside triphosphatase and RNA helicase activities associated with GB virus B nonstructural protein 3; Zhong W et al.; GB virus B (GBV-B) is a positive-stranded RNA virus that belongs to the Flaviviridae family . This virus is closely related to hepatitis C virus (HCV) and causes acute hepatitis in tamarins (Saguinus species) . Nonstructural protein 3 (NS3) of GBV-B contains sequence motifs predictive of three enzymatic activities: serine protease, nucleoside triphosphatase (NTPase), and RNA helicase . The N-terminal serine protease has been characterized and shown to share similar substrate specificity with the HCV NS3 protease . In this report, a full-length GBV-B NS3 protein was expressed in Escherichia coli and purified to homogeneity . This recombinant protein was shown to possess polynucleotide-stimulated NTPase and double-stranded RNA (dsRNA) unwinding activities . Both activities were abolished by a single amino acid substitution, from the Lys (K) residue in the conserved walker motif A (or Ia) "AXXXXGK(210)S" to an Ala (A), confirming that they are intrinsic to GBV-B NS3 . Kinetic parameters (K(m) and k(cat)) for hydrolysis of various NTPs or dNTPs were obtained . The dsRNA unwinding activity depends on the presence of divalent metal ions and ATP and requires an RNA duplex substrate with 3' unpaired regions (RNAs with 5' unpaired regions only or with blunt ends are not suitable substrates for this enzyme) . This indicates that GBV-B NS3 RNA helicase unwinds dsRNA in the 3' to 5' direction . Direct interaction of the GBV-B NS3 protein with a single-stranded RNA was established using a gel-based RNA bandshift assay . Finally, a homology model of GBV-B NS3 RNA helicase domain based on the 3-dimensional structure of the HCV NS3 helicase that shows a great similarity in overall structure and surface charge distribution between the two proteins was proposed . Protein Expr Purif, 1999 Oct, 17(1), 169 - 81 Production of engineered human pancreatic ribonucleases, solving expression and purification problems, and enhancing thermostability; Canals A et al.; Human pancreatic ribonuclease, the homolog of bovine pancreatic ribonuclease, has a significant therapeutic potential . Its study has been hindered by the difficulty of obtaining the enzyme in a pure and homogeneous form, either from human source or using heterologous expression . Engineering of different variants of human pancreatic ribonuclease has allowed us to study and overcome some problems encountered during its heterologous production in an Escherichia coli system and its purification from inclusion bodies . The 5'-end region of the mRNA that encodes the enzyme is critical for obtaining high expression levels . The results also suggest the importance of the proline 50 residue in the recovery yields of human pancreatic ribonuclease . All the variants produced are pure and homogeneous . Their homogeneity has been demonstrated by cation-exchange and reversed-phase chromatography and by mass spectrometry analysis . Moreover, enhancement of human pancreatic ribonuclease thermal stability is observed when residues R4, K6, Q9, D16, and S17 are changed to the corresponding residues of bovine seminal ribonuclease . Protein Expr Purif, 1999 Oct, 17(1), 163 - 8 Cloning, Escherichia coli expression, and phase-transition chromatography-based purification of recombinant rabbit heart mitochondrial creatine kinase; Marcillat O et al.; A cDNA clone of the mitochondrial sarcomeric creatine kinase cDNA was obtained by screening a rabbit heart library . This cDNA is characterized by a 1257-nucleotide open reading frame encoding a 419-amino-acid protein with a cleavable 39-amino-acid mitochondrial presequence (Accession No . AJ011334) . This new member of the guanidino kinase family shows a high degree of sequence similarity with the other phosphagen kinases sequenced so far . The mature enzyme was efficiently expressed in Escherichia coli BL21(DE3) cells as a soluble octameric protein using the pET21 plasmid and purified by a three-step improved method including a final phase-transition chromatography . Protein Expr Purif, 1999 Oct, 17(1), 153 - 62 Expression, single-step purification, and matrix-assisted refolding of a maize cytokinin glucoside-specific beta-glucosidase; Zouhar J et al.; Availability of highly purified native beta-glucosidase Zm-p60.1 in milligram quantities was a basic requirement for analysis of structure-function relationships of the protein . Therefore, Zm-p60.1 was overexpressed to high levels as a fusion protein with a hexahistidine tag, (His)(6)Zm-p60.r, in Escherichia coli, resulting, however, in accumulation of most of the protein in insoluble inclusion bodies . Native (His)(6)Zm-p60.r was then purified either from the bacterial lysate soluble fraction or from inclusion bodies . In the first case, a single-step purification under native conditions based on immobilized metal affinity chromatography (IMAC) was developed . In the second case, a single-step purification protocol under denaturing conditions followed by IMAC-based matrix-assisted refolding was elaborated . The efficiency of the native protein purification from soluble fraction of bacterial homogenate was compared to the feasibility of purification and renaturation of the protein from inclusion bodies . Gain of authentic biological activity and quaternary structure after the refolding process was confirmed by K(m) determination and electrophoretic mobility under native conditions . The yield of properly refolded protein was assessed based on the specific activity of the refolded product . Protein Expr Purif, 1999 Oct, 17(1), 139 - 45 Avidin is a promising tag for fusion proteins produced in baculovirus-infected insect cells; Airenne KJ et al.; The baculovirus expression vector system (BEVS) has become one of the most versatile and powerful eukaryotic systems for recombinant protein expression . We have constructed a novel baculovirus transfer vector (pbacAVs+C) which allows for the efficient production, detection, and single-step purification of the desired molecule as a secretion-compatible avidin fusion protein in insect cells . It also enables fast construction of the baculoviruses by site-specific transposition in Escherichia coli . To demonstrate the power of this vector, we report here on the production of immunologically intact hevein, a major cysteine-rich latex allergen, as avidin fusion protein . Our results indicate that avidin is a stable and versatile tag in the BEVS . It retains its extraordinarily high biotin-binding activity and also enables independent folding of the fusion partner . The versatility with which avidin fusion proteins can be detected, purified, and immobilized is the basis for the use of our system as a useful alternative in eukaryotic fusion protein production . Protein Expr Purif, 1999 Oct, 17(1), 128 - 38 Production of "authentic" poliovirus RNA-dependent RNA polymerase (3D(pol)) by ubiquitin-protease-mediated cleavage in Escherichia coli; Gohara DW et al.; The first amino acid of "authentic" poliovirus RNA-dependent RNA polymerase, 3D(pol), is a glycine . As a result, production of 3D(pol) in Escherichia coli requires addition of an initiation codon; thus, a formylmethionine is added to the amino terminus . The formylmethionine should be removed by the combined action of a cellular deformylase and methionine aminopeptidase . However, high-level expression of 3D(pol) in E . coli yields enzyme with a heterogeneous amino terminus . To preclude this problem, we developed a new expression system for 3D(pol) . This system exploits the observation that proteins fused to the carboxyl terminus of ubiquitin can be processed in E . coli to produce proteins with any amino acid as the first residue when expressed in the presence of a ubiquitin-specific, carboxy-terminal protease . By using this system, authentic 3D(pol) can be obtained in yields of 30-60 mg per liter of culture . While addition of a single glycine, alanine, serine, or valine to the amino terminus of 3D(pol) produced derivatives with a specific activity reduced by at least 25-fold relative to wild-type enzyme, addition of a methionine to the amino terminus resulted in some processing to yield enzyme with a glycine amino terminus . Addition of a hexahistidine tag to the carboxyl terminus of 3D(pol) had no deleterious effect on the activity of the enzyme . The utility of this expression system for production of other viral polymerases and accessory proteins is discussed . Protein Expr Purif, 1999 Oct, 17(1), 123 - 7 Expression and biotinylation of a mutant of the transcarboxylase carrier protein from Propioni shermanii; Jank MM et al.; A deletion mutant (residues 10 to 48 cut) of the biotinyl subunit (tcc) from the enzyme transcarboxylase (EC 2.1.3.1) of Propioni shermanii was overexpressed in Escherichia coli . Complete biotinylation of the protein was achieved by addition of exogenous biotin and coexpression of the biotin holoenzyme synthetase (EC 6.3 . 4.15.) from E . coli . The transcription of both genes was put under control of different operators/promoters, thus achieving independent control of expression levels and optimized yields of the holo-tcc . Bacteria were grown in a biotin-supplemented minimal medium (M9) that contained {(13)C}glucose as the carbon source and {(15)N}NH(4)Cl as the sole nitrogen source . The target protein could be purified to homogeneity by ion-exchange chromatography and concentrated to NMR-suitable concentrations (2 mM) without aggregation . Protein Expr Purif, 1999 Oct, 17(1), 105 - 12 Use of silent mutations in cDNA encoding human glutathione transferase M2-2 for optimized expression in Escherichia coli; Johansson AS et al.; Heterologous expression of human glutathione transferase M2-2 (GST M2-2) using Escherichia coli was improved 140-fold by mutating the cDNA expressing the enzyme . Expression of GST M2-2 from this cDNA clone, pKHXhGM2, generated approximately 190 mg protein per liter of bacterial culture, corresponding to approximately 12% of the total amount of soluble protein . The high-level-expressing cDNA was generated by oligonucleotide-directed mutagenesis introducing alternative silent mutations into the third nucleotide of codons 2, 4-7, and 10-14 in the 5' end of the cDNA coding region . The choice of alternative codons was restricted to those naturally occurring in highly biased genes in E . coli . Furthermore, the wild-type TAG stop codon at the 3' end was replaced with the two stop codons TAA and TGA in tandem to increase translation termination efficiency . The resulting partially randomized cDNA library was assayed for high-level expression using immunoscreening . Sequence similarities between the constructed high-level-expressing GST M2-2 cDNA and a similarly designed cDNA encoding the closely related human GST M1-1 suggest that the codons in the region immediately following the start codon are influential in achieving high-level expression . Pyrimidines seem to be more favorable than purines in the third position of codons in optimizing the expression of these enzymes in E . coli . Protein Expr Purif, 1999 Oct, 17(1), 64 - 73 Heterologous expression of the avirulence gene product, NIP1, from the barley pathogen Rhynchosporium secalis; Gierlich A et al.; NIP1, the product of the avirulence gene AvrRrs1 from Rhynchosporium secalis, a fungal pathogen of barley, is a small secreted cysteine-rich protein . This protein is essential for the specific recognition of the fungus by host plants carrying the complementary resistance gene Rrs1 . Different heterologous expression systems were tested to produce sufficient quantities of NIP1 to allow its utilization in receptor identification and isolation . In addition, protein amounts higher than those produced in fungal cultures are required to determine its 3D structure and to analyze its interaction with a receptor . The most efficient method, the synthesis of a His-tag fusion protein in Escherichia coli combined with a refolding procedure, yielded up to 3 mg of recombinant NIP1 from a 1-liter bacterial culture . After removal of the His-tag, the recombinant protein showed the same physicochemical characteristics as the native NIP1 and, most importantly, full biological activity . Protein Expr Purif, 1999 Oct, 17(1), 57 - 63 Cloning, expression, and purification of the functional 2,4-dienoyl-CoA reductase from rat liver mitochondria; Fillgrove KL et al.; The mitochondrial 2,4-dienoyl-CoA reductase (EC 1.3.1.34) is an auxiliary enzyme for the beta-oxidation of unsaturated fatty acids . Import of this enzyme into the mitochondria requires a mitochondrial signal sequence at the amino terminus of the polypeptide chain which is processed/removed once inside the mitochondria . The cDNA of the full-length 2,4-dienoyl-CoA reductase was previously cloned as pRDR181 . PCR methodologies were used to subclone the gene encoding the functional 2,4-dienoyl-CoA reductase from pRDR181 . The PCR product was inserted into a pET15b expression vector and overexpressed in Escherichia coli . The soluble expressed protein can be separated into high- and low-activity fractions . The low-activity fraction can be converted to the high specific activity form by thermal annealing, suggesting it is a metastable misfolded form of the enzyme . Using ion-exchange and affinity chromatography, the enzyme has been purified to homogeneity and exhibits a single band on Coomassie blue-stained SDS-PAGE . The molecular mass of 32,413 Da determined by electrospray ionization-mass spectrometry indicates that the amino-terminal methionine had been removed . The Michaelis constants for trans-2, trans-4-hexadienoyl-CoA and NADPH were determined to be 0.46 and 2.5 microM, respectively; a turnover number of 2.1 s(-1) was calculated . Protein Expr Purif, 1999 Oct, 17(1), 49 - 56 Expression and purification of recombinant mouse fibrillarin; Pearson DL et al.; Fibrillarin is a 34-kDa nucleolar protein associated with many of the small nucleolar ribonucleoprotein (snoRNP) particles and plays a role in ribosomal RNA processing . A subset of patients with the systemic autoimmune disease Scleroderma produce autoantibodies against fibrillarin and it is a genetically restricted target of murine mercury-induced autoimmunity . To aid in characterizing the antigenicity of fibrillarin, we have constructed two forms of mouse fibrillarin . The wild-type clone contains two cysteine residues that enable the protein to form an intramolecular disulfide bond, whereas the mutant clone contains alanine replacements which cannot form the disulfide bond . We have successfully expressed and purified both wild-type and mutant recombinant mouse fibrillarin using nickel-chelation chromatography . The combination of T7 promoter-driven expression vector pET28 and Escherichia coli strain JM109(DE3) induced at 25 degrees C yielded up to 19 mg of 94% pure recombinant protein per liter of culture . As the antigenicity of fibrillarin requires the full-length protein, the purification protocol was optimized for isolation of the full-length protein by the addition of N- and C-terminal T7 Tag and FLAG epitope sequences to the fibrillarin sequence . Anti-peptide antibodies were used in immunoblot to identify conditions favoring minimal proteolysis of recombinant protein . Both wild-type and mutant recombinant fibrillarin, purified under denaturing conditions and in the presence of 2-mercaptoethanol, were recognized by anti-fibrillarin antibodies from Scleroderma patients and exhibited structural similarities to eukaryotic and in vitro translated fibrillarin . Protein Expr Purif, 1999 Oct, 17(1), 41 - 8 Functional analysis of affinity-purified polyhistidine-tagged DnaA protein; Li Z et al.; DnaA protein initiates DNA replication at the Escherichia coli chromosomal origin . We describe a system for efficient production and purification of replicatively active DnaA protein . The dnaA gene was cloned in-frame with a sequence encoding a polyhistidine tag and expressed from a T7 promoter regulated by the lac operator . DnaA with the amino terminal polyhistidine tag was isolated using immobilized metal-ion affinity chromatography . Immunoblot analysis indicated that the tagged protein was intact and migrated with the expected molecular weight . The yield of purified protein was greater than 10 mg per liter of cell culture . The polyhistidine-tagged DnaA protein was comparable to nontagged DnaA protein for initiating in vitro DNA replication, binding to oriC DNA, binding of allosteric effector adenine nucleotides, and interaction with membrane acidic phospholipids . This system for rapid and high-yield generation of replication-active DnaA protein should facilitate structure-function studies and mutagenic analyses of this initiator protein . Protein Expr Purif, 1999 Oct, 17(1), 26 - 32 Refolding and characterization of recombinant human soluble CTLA-4 expressed in Escherichia coli; Cox GN et al.; CD28 and CTLA-4 are homologous cell surface proteins expressed by T cells . CD28 is constitutively expressed by most T cells, whereas CTLA-4 is expressed by activated T cells . Both proteins are ligands for the costimulatory molecules CD80 and CD86 expressed by activated B cells, macrophages, and dendritic cells . A fusion protein comprising the CTLA-4 extracellular domain joined to a human immunoglobulin heavy chain constant region (CTLA4Ig) binds CD80 and CD-86 with high affinity and inhibits CD80/CD86-dependent immune responses in vitro and in vivo . Attempts at producing the CTLA-4 extracellular domain as an unfused protein have met with limited success . Here we describe the expression and purification of the CTLA-4 extracellular domain as a nonfused protein in Escherichia coli . The 12.5-kDa CTLA-4 extracellular domain was insoluble when expressed in E . coli and required denaturation, reduction, and refolding steps to become soluble and assume its proper conformation . The protein refolded into a mixture of monomers, disulfide-linked dimers, and higher order disulfide-linked aggregates . sCTLA-4 dimers were the predominant refold form when air was used as the oxidizing agent during the refold procedure . Purified sCTLA-4 dimers were 10- to 50-fold more potent than sCTLA-4 monomers at inhibiting T cell activation using a CD80-dependent in vitro bioassay . J Mol Biol, 1999 Sep 24, 292(3), 707 - 16 Hyperthermostable protein structure maintained by intra and inter-helix ion-pairs in archaeal O6-methylguanine-DNA methyltransferase; Hashimoto H et al.; The crystal structure of O6-methylguanine-DNA methyltransferase (EC 2.1.1.63) of hyperthermophilic archaeon Pyrococcuskodakaraensis strain KOD1 (Pk -MGMT) was determined by single isomorphous replacement method with anomalous scattering (SIRAS) at 1.8 A resolution . The archaeal protein is extremely thermostable and repairs alkylated DNA by suicidal alkyl transfer from guanine O6 to its own cysteine residue . Archaea constitute the third primary kingdom of living organisms, sharing characteristics with procaryotic and eucaryotic cells . They live in various extreme environments and are thought to include the most ancient organisms on the earth . Structural studies on hyperthermophilic archaeal proteins reveal the structural features essential for stability under the extreme environments in which these organisms live, and will provide the structural basis required for stabilizing various mesophilic proteins for industrial applications . Here, we report the crystal structure of Pk-MGMT and structural comparison of Pk-MGMT and methyltransferase homologue from Escherichia coli (AdaC, C-terminal fragment of Ada protein) . Analyses of solvent-accessible surface area (SASA) reveals a large discrepancy between Pk-MGMT and AdaC with respect to the property of the ASA . In the Pk-MGMT structure, the intra-helix ion-pairs contribute to reinforce stability of alpha-helices . The inter-helix ion-pairs exist in the interior of Pk-MGMT and stabilize internal packing of tertiary structure . Furthermore, structural features of helix cappings, intra and inter-helix ion-pairs are found around the active-site structure in Pk-MGMT . J Mol Biol, 1999 Sep 24, 292(3), 619 - 32 Ligand-linked structural changes in the Escherichia coli biotin repressor: the significance of surface loops for binding and allostery; Streaker ED et al.; The Escherichia coli repressor of biotin biosynthesis (BirA) is an allosteric site-specific DNA-binding protein . BirA catalyzes synthesis of biotinyl-5'-AMP from substrates biotin and ATP and the adenylate serves as the positive allosteric effector in binding of the repressor to the biotin operator sequence . Although a three-dimensional structure of the apo-repressor has been determined by X-ray crystallographic techniques, no structures of any ligand-bound forms of the repressor are yet available . Results of previously published solution studies are consistent with the occurrence of conformational changes in the protein concomitant with ligand binding . In this work the hydroxyl radical footprinting technique has been used to probe changes in reactivity of the peptide backbone of BirA that accompany ligand binding . Results of these studies indicate that binding of biotin to the protein results in protection of regions of the central domain in the vicinity of the active site and the C-terminal domain from chemical cleavage . Biotin-linked changes in reactivity constitute a subset of those linked to adenylate binding . Binding of both bio-5'-AMP and biotin operator DNA suppresses cleavage at additional sites in the amino and carboxy-terminal domains of the protein . Varying degrees of protection of the five surface loops on BirA from hydroxyl radical-mediated cleavage are observed in all complexes . These results implicate the C-terminal domain of BirA, for which no function has previously been known, in small ligand and site-specific DNA binding and highlight the significance of surface loops, some of which are disordered in the apoBirA structure, for ligand binding and transmission of allosteric information in the protein . J Mol Biol, 1999 Sep 24, 292(3), 467 - 83 Singly and bifurcated hydrogen-bonded base-pairs in tRNA anticodon hairpins and ribozymes; Auffinger P et al.; The tRNA anticodon loops always comprise seven nucleotides and is involved in many recognition processes with proteins and RNA fragments . We have investigated the nature and the possible interactions between the first (32) and last (38) residues of the loop on the basis of the available sequences and crystal structures . The data demonstrate the conservation of a bifurcated hydrogen bond interaction between residues 32 and 38, located at the stem/loop junction . This interaction leads to the formation of a non-canonical base-pair which is preserved in the known crystal structures of tRNA/synthetase complexes . Among the tRNA and tDNA sequences, 93 % of the 32.38 oppositions can be assigned to two families of isosteric base-pairs, one with a large (86 %) and the other with a much smaller (7 %) population . The remainder (7 %) of the oppositions have been assigned to a third family due to the lack of evidence for assigning them into the first two sets . In all families, the Y32.R38 base-pairs are not isosteric upon reversal (like the sheared G.A or wobble G.U pairs), explaining the strong conservation of a pyrimidine at position 32 . Thus, the 32.38 interaction extends the sequence signature of the anticodon loop beyond the conserved U-turn at position 33 and the usually modified purine at position 37 . A comparison with other loops containing both a singly hydrogen-bonded base-pair and a U-turn suggests that the 32.38 pair could be involved in the formation of a base triple with a residue in a ribosomal RNA component . It is also observed that two crystal structures of ribozymes (hammerhead and leadzyme) present similar base-pairs at the cleavage site . Arch Biochem Biophys, 1999 Oct 1, 370(1), 1 - 8 Structure-function studies of adenylosuccinate synthetase from Escherichia coli; Honzatko RB et al.; Adenylosuccinate synthetase catalyzes the first committed step in the de novo biosynthesis of AMP, thermodynamically coupling the hydrolysis of GTP to the formation of adenylosuccinate from l-aspartate and IMP . The enzyme from Esherichia coli undergoes a ligand-induced dimerization, which leads to the assembly of a complete active site . The binding of IMP causes conformational changes over distances of 30 A, the end result of which is the activation of essential catalytic elements and the organization of the binding pocket for Mg(2+)-GTP . The enzyme promotes first a phosphoryl transfer from GTP to the 6-oxygen atom of IMP, by way of a transition state that has characteristics of both associative and dissociative reaction pathways . Following the formation of 6-phosphoryl-IMP, the enzyme then catalyzes the nucleophilic displacement of the 6-phosphoryl group by the alpha-amino group of l-aspartate in a transition state, which requires two metal cations . Infect Immun, 1999 Oct, 67(10), 5538 - 40 Type III secretion-dependent hemolytic activity of enteropathogenic Escherichia coli; Warawa J et al.; Enteropathogenic Escherichia coli (EPEC) was found to exhibit a type III secretion-dependent, contact-mediated, hemolytic activity requiring the EspA, EspB, and EspD secreted proteins . EspB and EspD display homology to pore-forming molecules . Our data suggest a mechanism to explain the requirement for all three Esp proteins in the transfer of EPEC proteins, such as Tir, into target cells. Infect Immun, 1999 Oct, 67(10), 5522 - 5 Escherichia coli binding to and invasion of brain microvascular endothelial cells derived from humans and rats of different ages; Stins MF et al.; Escherichia coli meningitis commonly occurs in the neonatal period, but the basis of this age dependency is unclear . We have previously identified two types of E . coli-brain microvascular endothelial cell (BMEC) interactions contributing to E . coli traversal of the blood-brain barrier (i.e., binding and invasion) . The present study examined whether the age dependency of E . coli meningitis stemmed from differences in the capacities of neonatal and adult BMECs to interact with E . coli . BMECs were isolated from rats of different ages (10 days, 20 days and 3 months) as well as from humans of different ages (fetuses, 4- to 7-year-old children, and a 35-year-old adult, and 60- to 85-year-old geriatrics) . The bindings of E . coli to young and old rat BMECs were similar . Also, the abilities of E . coli to invade BMECs were similar for BMECs derived from young and old rats and from human fetuses, children, adults, and geriatrics . These findings suggest that the predominance of E . coli meningitis in neonates is not likely due to greater binding and invasion capacities of newborn compared to adult BMECs. Infect Immun, 1999 Oct, 67(10), 5455 - 62 Complete DNA sequence and structural analysis of the enteropathogenic Escherichia coli adherence factor plasmid; Tobe T et al.; The complete nucleotide sequence and organization of the enteropathogenic Escherichia coli (EPEC) adherence factor (EAF) plasmid of EPEC strain B171 (O111:NM) were determined . The EAF plasmid encodes two known virulence-related operons, the bfp operon, which is composed of genes necessary for biosynthesis of bundle-forming pili, and the bfpTVW (perABC) operon, composed of regulatory genes required for bfp transcription and also for transcriptional activation of the eae gene in the LEE pathogenicity island on the EPEC chromosome . The 69-kb EAF plasmid, henceforth designated pB171, contains, besides the bfp and bfpTVW (perABC) operons, potential virulence-associated genes, plasmid replication and maintenance genes, and many insertion sequence elements . Of the newly identified open reading frames (ORFs), two which comprise a single operon had the potential to encode proteins with high similarity to a C-terminal region of ToxB whose coding sequence is located on pO157, a large plasmid harbored by enterohemorrhagic E . coli . Another ORF, located between the bfp and bfpTVW operons, showed high similarity with trcA, a bfpT-regulated chaperone-like protein gene of EPEC . Two sites were found to be putative replication regions: one similar to RepFIIA of p307 or F, and the other similar to RepFIB of R100 (NR1) . In addition, we identified a third region that contains plasmid maintenance genes . Insertion elements were scattered throughout the plasmid, indicating the mosaic nature of the EAF plasmid and suggesting evolutionary events by which virulence genes may have been obtained. Infect Immun, 1999 Oct, 67(10), 5338 - 44 Involvement of the enteroaggregative Escherichia coli plasmid-encoded toxin in causing human intestinal damage; Henderson IR et al.; Enteroaggregative Escherichia coli (EAEC) strains have been shown to adhere to human intestinal tissue in an in vitro organ culture (IVOC) model, and certain strains manifest mucosal toxicity . We have recently described the EAEC plasmid-encoded toxin (Pet), a member of a specific serine protease subclass of the autotransporter proteins . When injected into rat ileal loops, Pet both elicited fluid accumulation and had cytotoxic effects on the mucosa . Furthermore, the Pet protein caused rises in short circuit current from rat jejunal tissue mounted in a Ussing chamber and rounding of intestinal epithelial cells in culture . We therefore hypothesized that the mucosal pathology induced by EAEC strains in the IVOC model was related to expression of the Pet protein . Here, we have examined the effects of EAEC strain 042 and its isogenic pet mutant in the IVOC model . 042-infected colonic explants exhibited dilation of crypt openings, increased cell rounding, development of prominent intercrypt crevices, and absence of apical mucus plugs . Colonic tissue incubated with the pet mutant exhibited significantly fewer mucosal abnormalities both subjectively and as quantitated morphometrically by measurement of crypt aperture diameter . Mucosal effects were restored upon complementation of the pet mutation in trans . Interestingly, we found that the ability of 042 to damage T84 cells was not dependent upon Pet . The data suggest that the Pet toxin is active on the human intestinal mucosa but that EAEC may have other mechanisms of eliciting mucosal damage. Infect Immun, 1999 Oct, 67(10), 5243 - 6 Serological investigation of Chlamydia trachomatis heat shock protein 10; Betsou F et al.; The humoral immune response to Chlamydia trachomatis 10-kDa heat shock protein (Chsp10) in populations of Russian and French origin was studied by using a recombinant Chsp10 enzyme-linked immunosorbent assay . A physiological but not a serological correlation of Chsp10 exposure with Chsp60 exposure was observed in the Russian population . In the French population studied, there was a significant association between detection of anti-r-Chsp10 immunoglobulin G (IgG) antibodies and chronic genital tract infections . Chsp10 residues 50 to 67 were found to contain an immunodominant although not universal B epitope . Cross-reactions with Chlamydia pneumoniae or Escherichia coli GroES protein are limited but may occur . Our study suggests that detection of anti-Chsp10 IgG antibodies is associated with chronicity of C . trachomatis genital tract infection and does not parallel that of anti-Chsp60 IgG antibodies. RNA, 1999 Sep, 5(9), 1210 - 21 Effects of oligonucleotide length and atomic composition on stimulation of the ATPase activity of translation initiation factor elF4A; Peck ML et al.; Eukaryotic translation initiation factor 4A (elF4A) has been proposed to use the energy of ATP hydrolysis to remove RNA structure in the 5' untranslated region (UTR) of mRNAs, helping the 43S ribosomal complex bind to an mRNA and scan to find the 5'-most AUG initiator codon . We have examined the effect of changing the atomic composition and length of single-stranded oligonucleotides on binding to elF4A and on stimulation of its ATPase activity once bound . Substitution of 2'-OH groups with 2'-H or 2'-OCH3 groups reduces ATPase stimulation at least 100-fold, to background levels, without significantly affecting oligonucleotide affinity . These effects suggest that 2'-OH groups participate in an elF4A conformational change that occurs subsequent to oligonucleotide binding and is required for ATPase stimulation . Replacing nonbridging oxygen atoms in phosphodiester linkages with sulfur atoms to make phosphorothioate linkages has no significant effect on stimulation, while substantially increasing affinity . Extending the length of an RNA oligonucleotide from 4 to approximately 15 nt gradually increases oligonucleotide affinity and ATPase stimulation . Consistent with this observation, the increase in affinity and stimulation provided by phosphorothioate linkages and 2'-OH groups is proportional to the number of these groups present within larger oligonucleotides . Further, changing the position of blocks of phosphorothioate linkages or 2'-OH groups within a larger oligonucleotide does not affect affinity and has only a small effect on stimulation . These observations suggest that numerous interactions between the oligonucleotide and elF4A contribute individually to binding and ATPase stimulation . Nevertheless, significant stimulation is observed with as few as four RNA residues . These properties may allow elF4A to operate within regions of 5' UTRs containing only short stretches of exposed single-stranded RNA . As stimulation increases when longer stretches of single-stranded RNA are available, it is possible that the accessibility of single-stranded RNA in a 5' UTR influences translation efficiency. RNA, 1999 Sep, 5(9), 1180 - 90 In vitro selection of RNA aptamers that bind special elongation factor SelB, a protein with multiple RNA-binding sites, reveals one major interaction domain at the carboxyl terminus; Klug SJ et al.; The SelB protein of Escherichia coli is a special elongation factor required for the cotranslational incorporation of the uncommon amino acid selenocysteine into proteins such as formiate dehydrogenases . To do this, SelB binds simultaneously to selenocysteyl-tRNA(Sec) and to an RNA hairpin structure in the mRNA of formiate dehydrogenases located directly 3' of the selenocysteine opal (UGA) codon . The protein is also thought to contain binding sites allowing its interaction with ribosomal proteins and/or rRNA . SelB thus includes specific binding sites for a variety of different RNA molecules . We used an in vitro selection approach with a pool completely randomized at 40 nt to isolate new high-affinity SelB-binding RNA motifs . Our main objective was to investigate which of the various RNA-binding domains in SelB would turn out to be prime targets for aptamer interaction . The resulting sequences were compared with those from a previous SELEX experiment using a degenerate pool of the wild-type formiate dehydrogenase H (fdhF) hairpin sequence (Klug SJ et al., 1997, Proc . Natl . Acad . Sci . USA 94:6676-6681) . In four selection cycles an enriched pool of tight SelB-binding aptamers was obtained; sequencing revealed that all aptamers were different in their primary sequence and most bore no recognizable consensus to known RNA motifs . Domain mapping for SelB-binding aptamers showed that despite the different RNA-binding sites in the protein, the vast majority of aptamers bound to the ultimate C-terminus of SelB, the domain responsible for mRNA hairpin binding. RNA, 1999 Sep, 5(9), 1167 - 79 A selection system for functional internal ribosome entry site (IRES) elements: analysis of the requirement for a conserved GNRA tetraloop in the encephalomyocarditis virus IRES; Robertson ME et al.; Picornavirus internal ribosome entry site (IRES) elements direct cap-independent internal initiation of protein synthesis within mammalian cells . These RNA elements (about 450 nt) contain extensive secondary structure including a hairpin loop with a conserved GNRA motif . Such loops are important in RNA-RNA and RNA-protein interactions . Plasmids that express dicistronic mRNAs of the structure GUS/IRES/HOOK have been constructed . The HOOK sequence encodes a cell-surface-targeted protein (sFv); the translation of this open reading frame within mammalian cells from these dicistronic mRNAs requires a functional IRES element . Cells that express the sFv can be selected from nonexpressing cells . A pool of up to 256 mutant encephalomyocarditis virus IRES elements was generated by converting the wild-type hairpin loop sequence (GCGA) to NNNN . Following transfection of this pool of mutants into COS-7 cells, plasmids were recovered from selected sFv-expressing cells . These DNAs were amplified in Escherichia coli and transfected again into COS-7 cells for further cycles to enrich for plasmids encoding functional IRES elements . The sequence of individual selected IRES elements was determined . All functional IRES elements had a tetraloop with a 3' terminal A residue . Optimal IRES activity, assayed in vitro and within cells, was obtained from plasmids encoding an IRES with the hairpin loop sequence fitting a RNRA consensus . In contrast, IRES elements containing YCYA tetraloops were severely defective. J Hum Genet, 1999, 44(5), 337 - 42 Cloning and characterization of human and mouse PROSC (proline synthetase co-transcribed) genes; Ikegawa S et al.; Large-scale DNA sequencing, coupled with in silico gene trapping, is a robust approach to identifying unknown genes in selected genomic regions . Using this approach we have isolated a novel human gene, PROSC (for proline synthetase co-transcribed {bacterial homolog}), from human chromosome 8p11.2, and its mouse counterpart . The human PROSC gene spanned 17 kb of genomic DNA; its cDNA was 2530 bp long, with 8 exons that included an open reading frame of 825 bp (275 amino acids) . The mouse cDNA (Prosc), 1995 bp long, was predicted to encode 274 amino acids . PROSC is ubiquitously expressed in human tissues and has been highly conserved among divergent species from bacteria to mammals, suggesting its important cellular function . The gene product is likely to be a soluble cytoplasmic protein, but its function remains to be determined. Mol Gen Mikrobiol Virusol, 1999, (3), 17 - 21 {Effect of simple repeats (AC)n and (AT)n on chloramphenicol acetyltransferase gene expression in Escherichia coli}; Pisarchik AV et al.; According to some published reports, simple repeats can modulate gene transcription . The ability of (AC)n and (AT)n sequences to modulate gene expression in E . coli was investigated . These repeats were inserted in the leader sequence, in its 3' uncoding region, and in both these sites . When simple sequences were inserted in the leader sequence, gene expression was the highest for constructions containing 40 bp insertion . An increase in the insertion length led to a decrease in the gene expression . The (AC)20 sequence insertions in a 3' uncoding site of cat gene did not notably change its expression . Gene expression was much more modified by inserting the simple sequences on both sites of cat gene . Cat gene expression depended on the ratio of simple sequence lengths in its 5' and 3' uncoding sites . It was the highest when (AC)n sequence in the gene leader sequence or its 3' uncoding region was 40 bp . Gene expression modulation by simple sequence is assumed to be associated with the DNA lability increase. Growth Factors, 1999, 17(1), 49 - 61 Induction of anchorage independent growth by heparin-binding EGF-like growth factor (HB-EGF); Harding PA et al.; Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is initially synthesized as a membrane bound protein that is subsequently processed to yield an approximately 74 amino acid secreted product . To investigate the biological activities of HB-EGF and its role(s) in tumor formation, the full-length HB-EGF cDNA was cloned under the regulation of the mouse metallothionein promoter and stably expressed in HB-EGF deficient mouse L cells . HB-EGF immunoreactive proteins of 21 and 24 kDa were observed from transfected MLC lysates, and these lysates exhibited the ability to bind to the EGF receptor, stimulate 3H-thymidine uptake in BALB/c-3T3 cells, and induce anchorage independent growth (AIG) of normal rat kidney (NRK) cells . Furthermore, NRK cells treated with either E . coli-derived or vaccinia virus-derived HB-EGF, as well as NRK cells directly transfected with the HB-EGF construct, demonstrated AIG . We conclude that HB-EGF is a potent growth factor capable of stimulating altered cell growth and anchorage independence. Genetika, 1999 May, 35(5), 574 - 86 {The plasmid carrying the temperature-sensitive mutation in the DNa-methylase gene of the PStI system: effect on host cells at nonpermissive temperature}; Danilevich VN et al.; Temperature-sensitive (ts) derivatives of plasmid pRMP1, the derivative of PBR322 containing restriction and modification (RM) genes of the PstI system, were obtained using hydroxylamine mutagenesis . One of the isolated plasmids responsible for the inhibition of Escherichia coli cell growth at 42 degrees C, pRMPts, was analyzed in this work . Cells of Rec+ strains carrying this plasmid were unable to divide at 42 degrees C and formed long non-septated filaments that died upon prolonged cultivation . Cells of the RecA- strains carrying pRMPts did not form filaments at 42 degrees C and rapidly disappeared . On agar media with or without ampicillin, Rec+ and RecA- strains with this plasmid formed colonies of temperature-resistant (tr) derivatives with frequencies ranging from 1.5 x 10(-4) to 4 x 10(-6) in independent clones . The structure of plasmids from cells of tr-derivatives of Rec+ and RecA- strains carrying plasmid pRMPts was analyzed by the set of restriction enzymes . Reversions to the temperature-resistant phenotype were shown to result from the following events: (1) the insertional inactivation of the PstI restriction enzyme gene in pRMPts (the insertion of the IS1 element); (2) deletions in plasmid DNA fragments that partially or completely cover the restriction enzyme gene; (3) point mutations; and (4) others . The effect of the chromosomal sulA mutation on the maintenance of the ts-plasmid in bacterial cells was studied at 42 degrees C . High efficiency loss of the plasmid was detected in pRMPts-carrying Rec+ cells with the sulA::Tn5 mutation grown in liquid and solid nutrient media at this temperature . Under similar conditions, plasmid loss was not detected in SulA+ cells . On the basis of the data obtained, it is concluded that the ts-mutation is located in the DNA-methylase gene of plasmid pRMPts . Mutant DNA methylase was unable to methylate all sites in the chromosomal DNA at 42 degrees C . Some of the unmethylated sites can be digested with the PstI enzyme, which leads to the induction of SOS response in Rec+ cells or to total mortality in cells with the recA phenotype. Genetika, 1999 Feb, 35(2), 187 - 92 {Mutational analysis of the CytR protein binding site within the regulatory region of Escherichia coli udp gene}; Domakova EV et al.; Site-specific mutagenesis of the pentameric motif TGCAA within the regulatory region of the udp gene with coordinates -68 and -64 relative to the transcription initiation site was performed . Nine mutant promoters containing multiple nucleotide base-pair substitutions in this pentameric motif were isolated and characterized . One mutant contained a deletion of the C/G nucleotide pair in the -66 position . Isolated mutant promoters were cloned into a low-copy-number expression vector pJEL250 to determine the level of their expression, depending on the allelic state of cytR and cya genes . The level of CytR-dependent regulation of the udp gene and the ability to titrate the CytR repressor in vivo were shown to be drastically decreased in all mutant promoters isolated . On the basis of these results, it is concluded that the pentameric motif TGCAA plays a key role in binding the CytR repressor protein to the udp gene promoter. Bioorg Khim, 1999 May, 25(5), 398 - 400 {Hot start of the polymerase chain reaction using DNA helicase}; Kaboev OK et al.; A novel method for the hot start of PCR using DNA helicases is developed . The addition of a DNA helicase prevents the random annealing of primers and synthesis of nonspecific products during the preparation of the reaction mixture and initial heating . The hot start of PCR occurs automatically after inactivation of the DNA helicase upon heating of the reaction mixture. Ann Surg, 1999 Sep, 230(3), 352 - 60; discussion 360-1 Regional versus systemic delivery of recombinant vaccinia virus as suicide gene therapy for murine liver metastases; Gnant MF et al.; OBJECTIVE: Specific and efficient tumor-targeted gene delivery is the major goal for successful cancer gene therapy . SUMMARY BACKGROUND DATA: A recombinant thymidine kinase-deleted vaccinia virus (vv) encoding the firefly luciferase (luc) reporter gene or the prodrug converter gene cytosine deaminase (CD) was constructed . The authors compared the extent, duration, and pattern of transgene (luc) expression in vivo after portal venous, intraperitoneal, or intravenous virus administration and survival after treatment with the vv containing CD followed by the prodrug 5-fluorocytosine (5-FC) in a murine model of disseminated liver metastases from colon cancer . METHODS: Recombinant vv containing the luc transgene within the thymidine kinase locus was administered to mice with isolated liver metastases from an MC38 adenocarcinoma . Transgene expression was determined in tumor and organs at various time points . Tumor-bearing mice were treated with recombinant vv containing CD and 5-FC or with appropriate controls and followed for survival . RESULTS: Tumor-specific gene delivery was achieved irrespective of administration route, with gene expression in tumors increased by up to 100,000-fold compared with normal tissues . There was significantly increased transgene expression in tumor after portal venous or intraperitoneal virus administration (p = 0.001 vs . systemic) . Treatment using a CD-expressing vv and systemic 5-FC resulted in a significant survival benefit in all treatment groups compared with controls (p < 0.007); there was no additional benefit for portal venous or intraperitoneal virus administration . CONCLUSIONS: Suicide gene therapy using vv with the CD/5-FC system leads to tumor-specific gene expression and improved survival and can result in cure of established liver metastases. Electrophoresis, 1999 Aug, 20(11), 2181 - 95 Enrichment of low abundance proteins of Escherichia coli by hydroxyapatite chromatography; Fountoulakis M et al.; Visualization of low-copy-number gene products is essential for the detection of novel drug targets by differential protein expression studies . We investigated the enrichment of low-abundance proteins of Escherichia coli by hydroxyapatite chromatography . The proteins of the various pools collected from a ceramic hydroxyapatite column were analyzed by two-dimensional electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry . Approximately 800 spots corresponding to 296 different proteins were identified in the hydroxyapatite eluate . About 130 proteins that had not been detected in the two-dimensional gels of the total extract were identified . Hydroxyapatite chromatography enriched low-abundance but also major components of the E . coli protein extract . In particular, it enriched many low-molecular-mass proteins, such as cold-shock proteins . The proteins bound to