J Biol Chem, 1999 Jul 9, 274(28), 19655 - 60 Identification and expression of the TREX1 and TREX2 cDNA sequences encoding mammalian 3'-->5' exonucleases; Mazur DJ et al.; The 3'-->5' exonucleases catalyze the excision of nucleoside monophosphates from the 3' termini of DNA . We have identified the cDNA sequences encoding two 3'-->5' exonucleases (TREX1 and TREX2) from mammalian cells . The TREX1 and TREX2 proteins are 304 and 236 amino acids in length, respectively . Analysis of the TREX1 and TREX2 sequences identifies three conserved motifs that likely generate the exonuclease active site in these enzymes . The specific amino acids in these three conserved motifs suggest that these mammalian exonucleases are most closely related to the proofreading exonucleases of the bacterial replicative DNA polymerases and the RNase T enzymes . Expression of TREX1 and TREX2 in Escherichia coli demonstrates that these recombinant proteins are active 3'-->5' exonucleases . The recombinant TREX1 protein was purified, and exonuclease activity was measured using single-stranded, partial duplex, and mispaired oligonucleotide DNA substrates . The greatest activity of the TREX1 protein was detected using a partial duplex DNA containing five mispaired nucleotides at the 3' terminus . No activity was detected using single-stranded RNA or an RNA-DNA partial duplex . Identification of the TREX1 and TREX2 cDNA sequences provides the genetic tools to investigate the physiological roles of these exonucleases in mammalian DNA replication, repair, and recombination pathways. J Biol Chem, 1999 Jul 9, 274(28), 19644 - 8 Specific DNA recognition by F Factor TraY involves beta-sheet residues; Lum PL et al.; The F Factor TraY protein is a sequence-specific DNA-binding protein required for efficient conjugal transfer . Genetic and biochemical studies indicate that TraY has two functional roles in conjugation . TraY binds to the PY promoter to up-regulate transcription of tra genes . TraY also binds to the plasmid origin of transfer (oriT), serving as an accessory protein in the nicking of F Factor in preparation for transfer . TraY is thought to belong to the ribbon-helix-helix family of transcription factors . These proteins contact DNA using residues of an antiparallel beta-sheet . We engineered and characterized six TraY mutants each having a single potential beta-sheet DNA contact residue replaced with Ala . Most TraY mutants had significantly reduced affinity for the TraY oriT binding site while possessing near wild-type stability and nonspecific DNA recognition . These results indicate that TraY beta-sheet residues participate in DNA recognition, and support inclusion of TraY in the ribbon-helix-helix family. J Biol Chem, 1999 Jul 9, 274(28), 19538 - 44 On the substrate specificity of DNA methyltransferases . adenine-N6 DNA methyltransferases also modify cytosine residues at position N4; Jeltsch A et al.; Methylation of DNA is important in many organisms and essential in mammals . Nucleobases can be methylated at the adenine-N6, cytosine-N4, or cytosine-C5 atoms by specific DNA methyltransferases . We show here that the M.EcoRV, M.EcoRI, and Escherichia coli dam methyltransferases as well as the N- and C-terminal domains of the M . FokI enzyme, which were formerly all classified as adenine-N6 DNA methyltransferases, also methylate cytosine residues at position N4 . Kinetic analyses demonstrate that the rate of methylation of cytosine residues by M.EcoRV and the M.FokI enzymes is reduced by only 1-2 orders of magnitude in relation to methylation of adenines . This result shows that although these enzymes methylate DNA in a sequence specific manner, they have a low substrate specificity with respect to the target base . This unexpected finding has implications on the mechanism of adenine-N6 DNA methyltransferases . Sequence comparisons suggest that adenine-N6 and cytosine-N4 methyltransferases have changed their reaction specificity at least twice during evolution, a model that becomes much more likely given the partial functional overlap of both enzyme types . In contrast, methylation of adenine residues by the cytosine-N4 methyltransferase M.BamHI was not detectable . On the basis of our results, we suggest that adenine-N6 and cytosine-N4 methyltransferases should be grouped into one enzyme family. J Biol Chem, 1999 Jul 9, 274(28), 19509 - 12 alpha-synuclein fibrillogenesis is nucleation-dependent . Implications for the pathogenesis of Parkinson's disease; Wood SJ et al.; Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies, the major components of which are filaments consisting of alpha-synuclein . Two recently identified point mutations in alpha-synuclein are the only known genetic causes of PD . alpha-Synuclein fibrils similar to the Lewy body filaments can be formed in vitro, and we have shown recently that both PD-linked mutations accelerate their formation . This study addresses the mechanism of alpha-synuclein aggregation: we show that (i) it is a nucleation-dependent process that can be seeded by aggregated alpha-synuclein functioning as nuclei, (ii) this fibril growth follows first-order kinetics with respect to alpha-synuclein concentration, and (iii) mutant alpha-synuclein can seed the aggregation of wild type alpha-synuclein, which leads us to predict that the Lewy bodies of familial PD patients with alpha-synuclein mutations will contain both, the mutant and the wild type protein . Finally (iv), we show that wild type and mutant forms of alpha-synuclein do not differ in their critical concentrations . These results suggest that differences in aggregation kinetics of alpha-synucleins cannot be explained by differences in solubility but are due to different nucleation rates . Consequently, alpha-synuclein nucleation may be the rate-limiting step for the formation of Lewy body alpha-synuclein fibrils in Parkinson's disease. Comp Immunol Microbiol Infect Dis, 1999 Jul, 22(3), 163 - 74 The use of polymerase chain reaction for determination of virulence factors of Escherichia coli strains isolated from pigs in Poland; Osek J et al.; E . coli strains isolated from pigs with postweaning diarrhea or edema disease were tested by phenotypic and genotypic methods for the presence of virulence antigens and genes, respectively . The slide agglutination and ELISA analyses were used for determination of F4, F5, F6, F17, and F41 fimbriae whereas the prevalence of fimbrial fedA and toxin eltI, estI, estII, stx1, stx2 and stx2e genes were recorded by the means of PCR . Only F4 antigen (ac variant) was found in strains of the serogroup O149:K91 isolated from pigs with diarrhea . PCR analyses showed that the fedA gene encoding F18 fimbriae was present in 61.9% of strains isolated from pigs with diarrhea and in 84.2% of strains isolated from pigs with edema disease . The eltI genes encoding heat-labile toxin I (LTI) were present only in 9 out of 21 strains recovered from pigs with diarrhea . Shiga toxin 2 variant (stx2e) genes were found in six isolates from edema disease and also in one strain from diarrhea . The PCR test used in the study was a sensitive and valuable method for determination of virulence factors of E . coli strains. Appl Microbiol Biotechnol, 1999 May, 51(5), 592 - 7 Functionality of biphenyl 2,3-dioxygenase components in naphthalene 1,2-dioxygenase; Barriault D et al.; Naphthalene 1,2-dioxygenase (Nap dox) and biphenyl 2,3-dioxygenase (Bph dox) are related enzymes that have differentiated during evolution as their specificity has changed . Although their component arrangement is similar, the structure of each component has been modified quite extensively . The purpose of this work was to determine the catalytic capacity of purified Nap dox toward chlorobiphenyls and to investigate the functionality of Bph dox components in the Nap dox system . Both enzyme systems were purified by affinity chromatography as histidine-tagged fused proteins . Data show for the first time that Nap dox can catalyze the oxygenation of all three monochlorobiphenyl isomers, but it is unable to hydroxylate 2,5-, 2,2'-, 3,3'-, 4,4'-di- and 2,2',5,5'-tetrachlorobiphenyl . The rates of cytochrome c reduction by the ferredoxin components of the two enzymes were identical when the Bph dox reductase component was used in the assay, showing an efficient electron transfer between the Bph dox reductase component and the Nap dox ferredoxin . However, when the Bph dox ferredoxin was used to reconstitute a hybrid Nap dox, the enzyme was only 22% as active as the parental enzyme . These data are discussed in terms of the potential use of Nap dox for the development of enhanced chlorobiphenyl-degrading dioxygenases. Nucleic Acids Res, 1999 Jul 15, 27(14), 2938 - 46 A dimer of the lymphoid protein RAG1 recognizes the recombination signal sequence and the complex stably incorporates the high mobility group protein HMG2; Rodgers KK et al.; RAG1 and RAG2 are the two lymphoid-specific proteins required for the cleavage of DNA sequences known as the recombination signal sequences (RSSs) flanking V, D or J regions of the antigen-binding genes . Previous studies have shown that RAG1 alone is capable of binding to the RSS, whereas RAG2 only binds as a RAG1/RAG2 complex . We have expressed recombinant core RAG1 (amino acids 384-1008) in Escherichia coli and demonstrated catalytic activity when combined with RAG2 . This protein was then used to determine its oligomeric forms and the dissociation constant of binding to the RSS . Electrophoretic mobility shift assays show that up to three oligomeric complexes of core RAG1 form with a single RSS . Core RAG1 was found to exist as a dimer both when free in solution and as the minimal species bound to the RSS . Competition assays show that RAG1 recognizes both the conserved nonamer and heptamer sequences of the RSS . Zinc analysis shows the core to contain two zinc ions . The purified RAG1 protein overexpressed in E.coli exhibited the expected cleavage activity when combined with RAG2 purified from transfected 293T cells . The high mobility group protein HMG2 is stably incorporated into the recombinant RAG1/RSS complex and can increase the affinity of RAG1 for the RSS in the absence of RAG2. J Mol Biol, 1999 Jul 9, 290(2), 565 - 79 On the structure and operation of type I DNA restriction enzymes; Davies GP et al.; Type I DNA restriction enzymes are large, molecular machines possessing DNA methyltransferase, ATPase, DNA translocase and endonuclease activities . The ATPase, DNA translocase and endonuclease activities are specified by the restriction (R) subunit of the enzyme . We demonstrate that the R subunit of the Eco KI type I restriction enzyme comprises several different functional domains . An N-terminal domain contains an amino acid motif identical with that forming the catalytic site in simple restriction endonucleases, and changes within this motif lead to a loss of nuclease activity and abolish the restriction reaction . The central part of the R subunit contains amino acid sequences characteristic of DNA helicases . We demonstrate, using limited proteolysis of this subunit, that the helicase motifs are contained in two domains . Secondary structure prediction of these domains suggests a structure that is the same as the catalytic domains of DNA helicases of known structure . The C-terminal region of the R subunit can be removed by elastase treatment leaving a large fragment, stable in the presence of ATP, which can no longer bind to the other subunits of Eco KI suggesting that this domain is required for protein assembly . Considering these results and previous models of the methyltransferase part of these enzymes, a structural and operational model of a type I DNA restriction enzyme is presented . J Mol Biol, 1999 Jul 9, 290(2), 559 - 64 Changing single side-chains can greatly enhance the resistance of a membrane protein to irreversible inactivation; Lau FW et al.; The thermal inactivation rates of a set of 20 cysteine-substituted variants of the integral membrane protein diacylglycerol kinase were measured . Two of the mutations, I53C and I70C, were found to significantly prolong the half-life of the enzyme in detergent solution . By combining the single mutants to create a double mutant, I53C/I70C, the half-life of the enzyme was improved from less than a minute at 70 degrees C to 51 minutes . These results demonstrate that individual side-chain substitutions can significantly improve the properties of membrane proteins in detergent solution . J Mol Biol, 1999 Jul 9, 290(2), 495 - 504 The N-terminal domain of the human Rad51 protein binds DNA: structure and a DNA binding surface as revealed by NMR; Aihara H et al.; Human Rad51 protein (HsRad51) is a homolog of Escherichia coli RecA protein, and functions in DNA repair and recombination . In higher eukaryotes, Rad51 protein is essential for cell viability . The N-terminal region of HsRad51 is highly conserved among eukaryotic Rad51 proteins but is absent from RecA, suggesting a Rad51-specific function for this region . Here, we have determined the structure of the N-terminal part of HsRad51 by NMR spectroscopy . The N-terminal region forms a compact domain consisting of five short helices, which shares structural similarity with a domain of endonuclease III, a DNA repair enzyme of E . coli . NMR experiments did not support the involvement of the N-terminal domain in HsRad51-HsBrca2 interaction or the self-association of HsRad51 as proposed by previous studies . However, NMR tiration experiments demonstrated a physical interaction of the domain with DNA, and allowed mapping of the DNA binding surface . Mutation analysis showed that the DNA binding surface is essential for double-stranded and single-stranded DNA binding of HsRad51 . Our results suggest the presence of a DNA binding site on the outside surface of the HsRad51 filament and provide a possible explanation for the regulation of DNA binding by phosphorylation within the N-terminal domain . J Mol Biol, 1999 Jul 9, 290(2), 391 - 409 The reliability of in vivo structure-function analysis of tRNA aminoacylation; McClain WH et al.; The G.U wobble base-pair in the acceptor helix of Escherichia coli tRNAAlais critical for aminoacylation by the alanine synthetase . Previous work by several groups probed the mechanism of enzyme recognition of G.U by a structure-function analysis of mutant tRNAs using either a cell assay (amber suppressor tRNA) or a test tube assay (phage T7 tRNA substrate and purified enzyme) . However, the aminoacylation capacity of particular mutant tRNAs was about 10(4)-fold higher in the cell assay . This led us to scrutinize the cell assay to determine if any parameter exaggerates the extent of aminoacylation in mutants forming substantial amounts of alanyl-tRNAAla . In doing so, we have refined and developed experimental designs to analyze tRNA function . We examined the level of aminoacylation of amber suppressor tRNAAlawith respect to the method of isolating aminoacyl-tRNA, the rate of cell growth, the cellular levels of alanine synthetase and elongation factor TU (EF-Tu), the amount of tRNA and the characteristics of EF-Tu binding . Within the precision of our measurements, none of these parameters varied in a way that could significantly amplify cellular alanyl-tRNAAla . A key observation is that the extent of aminoacylation of tRNAAlawas independent of tRNAAlaconcentration over a 75-fold range . Therefore, the cellular assay of tRNAAlareflects the substrate quality of the molecule for formation of alanyl-tRNAAla . These experiments support the authenticity of the cellular assay and imply that a condition or factor present in the cell assay may be absent in the test tube assay . J Mol Biol, 1999 Jul 9, 290(2), 385 - 9 A set of plasmids constitutively producing different RNA levels in Escherichia coli; Gabriel K et al.; New plasmids were developed for the in vivo expression of RNA in Escherichia coli . These plasmids combine constitutive promoters of different strengths with different origins of replication to provide a 75-fold range of expression of amber suppressor tRNA . The plasmids are either pMB1, p15A or temperature-sensitive SC101 replicons, and can be used in two plasmid systems for studying RNA-protein interactions . The temperature-sensitive SC101 plasmids may be useful as gene replacement vectors . Another vector that is suitable for generating lethal mutations was constructed in a plasmid containing a regulatable phage T7 promoter . Int J Cancer, 1999 Jul 19, 82(2), 293 - 7 Adenoviral transduction of a cytosine deaminase/thymidine kinase fusion gene into prostate carcinoma cells enhances prodrug and radiation sensitivity; Blackburn RV et al.; Prostate tumor cells (PC-3) were transduced with defective, recombinant adenovirus containing a fusion gene encoding the Escherichia coli cytosine deaminase and herpes simplex virus type-1 thymidine kinase under the control of a cytomegalovirus promoter . Expression levels of the fusion protein were dependent on the multiplicity of infection used and incubation time following infection . PC-3 cells expressing this protein were sensitized to killing by the normally innocuous prodrugs 5-fluorocytosine and ganciclovir . In addition, radiation-induced killing was enhanced in virally infected cells in the presence of the prodrugs. Br J Cancer, 1999 Apr, 80(1-2), 279 - 85 The significance of measuring monocyte tissue factor activity in patients with breast and colorectal cancer; Lwaleed BA et al.; Monocytes express tissue factor (mTF) in several conditions including cancer where levels may be valuable in assessing tumour presence and progression . Using a two-stage kinetic chromogenic assay (KCA), mTF levels were measured in controls {normal subjects (n = 60) and patients undergoing hernia repair or cholecystectomy (n = 60)}, in patients with benign and malignant disease of the breast (n = 83) and of the large bowel (n = 62) . This was performed under fresh (resting) conditions and after incubation for 6 h without (unstimulated) and with (stimulated) Escherichia coli endotoxin . The malignant groups showed higher mTF levels than each of the three controls for resting (P < 0.05 breast, P < 0.05 colorectal) unstimulated (P < 0.05 breast, P < 0.05 colorectal) and stimulated cells (P < 0.001 breast, P < 0.01 colorectal) . Similarly, the benign inflammatory groups had higher mTF levels than controls for resting (P < 0.05 colorectal), unstimulated (P < 0.05 colorectal) and stimulated cells (P < 0.01 breast, P < 0.01 colorectal) . There was no significant difference between malignant and benign inflammatory groups in each organ . mTF levels showed an increase corresponding to that of histological tumour progression and were higher in non-surviving patients . In conclusion, mTF levels are raised in malignant and inflammatory disease compared to controls and patients with non-inflammatory conditions . Stimulated cells give better discrimination between the groups and may be of value in identifying high risk individuals . mTF levels showed an association with tumour grade or stage and the patients' survival time. Clin Cancer Res, 1999 Jun, 5(6), 1539 - 49 125I-labeled anti-epidermal growth factor receptor-vIII single-chain Fv exhibits specific and high-level targeting of glioma xenografts; Kuan CT et al.; A single-chain antibody fragment, MR1(scFv), with specific binding to epidermal growth factor receptor-vIII (EGFRvIII), was produced, radiolabeled, and evaluated for biodistribution in human glioma-bearing athymic mice . The mutant receptor EGFRvIII has a deletion in its extracellular domain that results in the formation of a new, tumor-specific antigen found in glioblastomas, breast carcinomas, and other tumors . The scFv molecule, designed as V(H)-(Gly4-Ser)3-V(L), was expressed in Escherichia coli in inclusion body form; recovered scFv fragments were properly refolded in redox-shuffling buffer . Size-exclusion chromatography of purified scFv demonstrated a protein monomer of Mr 26,000 . Labeling was performed using N-succinimidyl 5-{125I}iodo-3-pyridinecarboxylate (SIPC) or Iodogen to specific activities of 0.5-2.0 mCi/mg, with yields of 35-50% and 45-70%, respectively . The immunoreactive fraction (IRF) of the labeled MR1(scFv) was 65-80% when SIPC was used and 50-55% when Iodogen was used . The affinity (K(A)) of MRI(scFv) for EGFRvIII was 4.3 x 10(7) +/- 0.1 x 10(7) M(-1) by BIAcore analysis, and it was 1.0 x 10(8) +/- 0.1 x 10(8) M(-1) and by Scatchard analysis versus EGFRvIII-expressing cells . After incubation at 37 degrees C for 24 h, the binding affinity was maintained, and the IRF was maintained at 60-70% . The specificity of MR1(scFv) for EGFRvIII was demonstrated in vitro by incubation of radiolabeled MR1(scFv) with the EGFRvIII-expressing U87MG.deltaEGFR cell line in the presence or absence of competing unlabeled MR1(scFv) or anti-EGFRvIII MAbs L8A4 and H10 . In biodistribution studies using athymic mice bearing s.c . U87MG.deltaEGFR tumor xenografts, animals received intratumoral or i.v . infusions of paired-label {125I}SIPC-MR1(scFv) and {131I}SIPC-anti-Tac(scFv) as a control . When given by the intratumoral route, MR1(scFv) retained high tumor uptakes of 85% injected dose per gram of tissue at 1 h and 16% injected dose per gram of tissue at 24 h following administration . Specific: control scFv tumor uptake ratios of more than 20:1 at 24 h demonstrated specific localization of MR1(scFv) . The excellent tumor retention of MR1(scFv), combined with its rapid clearance from normal tissues, resulted in high tumor:normal organ ratios. Clin Cancer Res, 1999 Jun, 5(6), 1353 - 61 Immunogenicity of granulocyte-macrophage colony-stimulating factor (GM-CSF) products in patients undergoing combination therapy with GM-CSF; Wadhwa M et al.; In this study, we have assessed the development of neutralizing and nonneutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies in two groups of patients with metastatic colorectal carcinoma receiving two different GM-CSF products . Three clinical trials were carried out, and a combination of GM-CSF and a colon carcinoma-reactive antibody was used in the absence of any concomitant chemotherapy . Two different GM-CSF products, both rDNA-derived and produced in Escherichia coli, were used . Patients in Trial 1 received product X, and those in Trials 2 and 3 received product Y . Patients in Trial 2 also received interleukin 2 in an attempt to potentiate immune responses . After the first cycle of treatment, no GM-CSF antibodies were detected, but on subsequent therapy, 28 of the 38 patients tested receiving product Y (Trials 2 and 3) developed antibodies that bound to the GM-CSF product used for therapy . However, none of the patients developed antibodies that neutralized the biological activity of GM-CSF, as assessed using an in vitro bioassay . Furthermore, there was no in vivo impairment in GM-CSF-induced expansion of leukocytes, neutrophils, and eosinophils in the patients . In contrast, 19 of the 20 patients given product X (Trial 1) developed GM-CSF binding antibodies, and 9 of these patients were shown to develop antibodies that neutralized the biological activity of GM-CSF . The presence of the latter was associated with a significant reduction in GM-CSF-induced expansion of leukocytes, neutrophils, and eosinophils in patients . Therefore, product X appears to be more immunogenic than product Y . Immunochemical characterization confirmed that the specificity of the antibody responses varied depending on the product used for therapy . Whereas sera from Trial 1 patients treated with product X showed the presence of antibodies with strong recognition of GM-CSF proteins, sera from patients treated with product Y showed varied recognition of GM-CSF ranging from fairly strong to very weak but bound predominantly to two E . coli-derived, non-GM-CSF-related proteins of Mr approximately 20,000 and Mr approximately 30,000 . Therefore, in sera from patients receiving product Y, the antibody specificity appeared to be directed not only against GM-CSF but also against non-product-related host cell contaminants . This study shows that GM-CSF products used for therapy are potentially immunogenic and generate antibodies to GM-CSF and/or other non-product-related contaminants . However, only antibodies that neutralize the biological activity of GM-CSF compromise therapeutic efficacy of the cytokine. Indian J Public Health, 1998 Oct-Dec, 42(4), 138 - 40 Survival of enteropathogenic Escherichia coli exposed to adverse conditions; Vaishnavi C et al.; Enteropathogenic Escherichia coli (EPEC) was exposed to different adverse conditions for varying period of time to assess its survival under such circumstances . From the results it is extrapolated that EPEC survive for a very long time when shielded from sunlight and after several hours of exposure to UV irradiation. Zhonghua Yi Xue Za Zhi (Taipei), 1999 Jun, 62(6), 350 - 5 Detection of IgA against Epstein-Barr virus BZLF-1 replication activator (ZEBRA) in sera of nasopharyngeal carcinoma patients with a recombinant ZEBRA protein; Shu CH et al.; BACKGROUND: Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC) . An EBV-encoded immediate-early antigen, BZLF-1 replication activator (ZEBRA) initiates EBV replication and expression in all NPC tumors . In this study, we investigated whether immunoglobulin A (IgA) against ZEBRA is present in the sera of patients with NPC, and whether it was able to be determined by enzyme-linked immunosorbent assay (ELISA) using a recombinant ZEBRA prepared from Escherichia coli . METHODS: A polymerase chain reaction-amplified cDNA fragment of the ZEBRA gene was inserted into the expression vector of E coli under the control of an IpL promoter . E coli bacteria containing the CI857 gene served as host to overexpress the ZEBRA protein by heat induction . Recombinant ZEBRA was collected by mechanical disruption of the bacteria, purified by column chromatography, and analyzed by SDS-PAGE and Western blot assay using sera from NPC patients . The recombinant ZEBRA was used to develop the ELISA to detect IgA against ZEBRA . RESULTS: The amount of ZEBRA produced comprised 30% of total E coli protein . Western blot assay confirmed that affinity of the recombinant ZEBRA to IgA antibody was preserved . IgA against ZEBRA was shown to be positive by ELISA in 36 of 40 NPC sera, but in only nine of 55 patients with other head and neck malignancies, and two of 35 normal individuals . For serologic diagnosis of NPC, the sensitivity of IgA/ZEBRA detected by ELISA was 90% and the specificity was 87.4% . CONCLUSIONS: A recombinant ZEBRA was produced at high levels in E coli and retained affinity to IgA against ZEBRA . The recombinant ZEBRA was successfully used to develop an ELISA for the detection of IgA against ZEBRA . The high sensitivity and specificity of IgA against ZEBRA show that the ELISA is feasible for serologic diagnosis of NPC. Phytochemistry, 1999 Jun, 51(4), 551 - 4 Highly oxygenated bisabolenes and an acetylene from Matricaria aurea; Ahmed AA et al.; Reinvestigation of the aerial parts of Matricaria aurea led to the isolation of three new bisabolenes and a new acetylene . The structures of the four compounds, namely (1R*,2R*,3R*,6R*,7R*)1,2,3,6,7- pentahydroxy-bisabol-10(11)-ene, (1R*,2R*,3R*,6R*,7R*)1,2,3,6,7-pentahydroxy-1-acetoxy-bisabol-10(1 1)-ene, (1R*,2R*,3R*,6R*,7R*)1,2,3,6,7-pentahydroxy-2-acetoxy-bisabol-10(1 1)-ene and (3S*,4S*,5R*)-(E)-3,4-dihydroxy-2-(hexa-2,4-diynyliden)-1,6- dioxaspiro-(4,5)decane, were deduced from the high field NMR studies. Lett Appl Microbiol, 1999 Jun, 28(6), 457 - 60 Formation of a chiral acetoinic compound from diacetyl by Escherichia coli expressing meso-2,3-butanediol dehydrogenase; Ui S et al.; L-Acetoin (L-AC) was produced from diacetyl (DA) by Escherichia coli JM109/pBUD119 containing the meso-2,3-butanediol dehydrogenase (D-AC forming) gene . However, when the strain was cultured in the presence of isopropyl-beta-D-thiogalacto-pyranoside, the enzyme formed catalysed not only D-AC but also L-AC . L-AC was further reduced to L-2,3-butanediol (BD) . The yield of L-AC or L-BD from DA (3 gl-1) was about 70% (w/w). J Hosp Infect, 1999 Jun, 42(2), 119 - 23 The effect of poor handling procedures on enteral feeding systems in Hong Kong; Lee CH et al.; Two enteral feeding systems commonly used in Hong Kong were evaluated for ease of bacterial entry during assembly and delivery of feeds . Wearing new, non-sterile disposable latex gloves during the assembly of the systems did not contaminate the feeds . The risk of contamination increased for systems assembled with bare hands . Systems assembled with hands experimentally contaminated with bacteria resulted in definite contamination of feeds. Protein Eng, 1999 Jun, 12(6), 515 - 21 Recombinant anti-polyamine antibodies: identification of a conserved binding site motif; Johnston JS et al.; Polyamines are small linear polycations found ubiquitously in eukaryotic cells . They are involved in nucleic acid and protein synthesis and rises in cellular polyamine levels have been correlated with cell proliferation . Antibodies to these molecules have potential as prognostic indicators of disease conditions and indicators of treatment efficacy . Antipolyamine monoclonal antibodies of differing but defined specificities have been generated in our laboratory using polyamine ovalbumin conjugates as immunogens . These antibodies show small but significant cross reactivities with other polyamine species; IAG-1 cross reacts with spermidine (8%), JAC-1 with spermine (6%) and JSJ-1 with both putrescine (11%) and spermine (6%) . We have rescued and sequenced the heavy and light chain variable regions of all three of these antibodies . While the light chains of two antibodies, IAG-1 and JSJ-1, were 93% homologous at the amino acid level, none of the heavy chains displayed any significant sequence homology . However, computer-generated models of all three antibody binding sites revealed a three-dimensionally conserved polyamine binding site motif . The polyamine appears to bind into a negatively charged cleft lined with acidic and polar residues . The cleft is partially or completely closed at one end and the specificity of the interaction is determined by placement of acidic residues in the cleft . Aromatic residues contribute to polyamine binding interacting with the carbon backbone . The polyamine-binding motif we have identified is very similar to that observed in the crystal structure of PotD, the primary receptor of the polyamine transport system in Escherichia coli. Protein Eng, 1999 Jun, 12(6), 497 - 503 Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K; Janssen MJ et al.; The catalytic contribution of His48 in the active site of porcine pancreatic phospholipase A2 was examined using site-directed mutagenesis . Replacement of His48 by lysine (H48K) gives rise to a protein having a distorted lipid binding pocket . Activity of this variant drops below the detection limit which is 10(7)-fold lower than that of the wild-type enzyme . On the other hand, the presence of glutamine (H48Q) or asparagine (H48N) at this position does not affect the structural integrity of the enzyme as can be derived from the preserved lipid binding properties of these variants . However, the substitutions H48Q and H48N strongly reduce the turnover number, i.e . by a factor of 10(5) . Residual activity is totally lost after addition of a competitive inhibitor . We conclude that proper lipid binding on its own accelerates ester bond hydrolysis by a factor of 10(2) . With the selected variants, we were also able to dissect the contribution of the hydrogen bond between Asp99 and His48 on conformational stability, being 5.2 kJ/mol . Another hydrogen bond with His48 is formed when the competitive inhibitor (R)-2-dodecanoylamino-hexanol-1-phosphoglycol interacts with the enzyme . Its contribution to binding of the inhibitor in the presence of an interface was found to be 5.7 kJ/mol. Protein Eng, 1999 Jun, 12(6), 491 - 6 A general method for relieving substrate inhibition in lactate dehydrogenases; Hewitt CO et al.; The mutation S163L in human heart lactate dehydrogenase removes substrate inhibition while only modestly reducing the turnover rate for pyruvate . Since this is the third enzyme to show this behaviour, we suggest that the S163L mutation is a general method for the removal of substrate inhibition in L-LDH enzymes . Engineering such enzymatic properties has clear industrial applications in the use of these enzymes to produce enantiomerically pure alpha-hydroxy acids . The mutation leads to two principal effects . (1) Substrate inhibition is caused by the formation of a covalent adduct between pyruvate and the oxidized form of the cofactor . The inability of S163L mutants to catalyse the formation of this inhibitory adduct is demonstrated . However, NMR experiments show that the orientation of the nicotinamide ring in the mutant NAD+ binary complex is not perturbed . (2) The mutation also leads to a large increase in the KM for pyruvate . The kinetic and binding properties of S163L LDH mutants are accounted for by a mechanism which invokes a non-productive, bound form of the cofactor . Molecular modelling suggests a structure for this non-productive enzyme-NADH complex. Biophys J, 1999 Jul, 77(1), 258 - 66 Catalytic activity of an isolated domain of Na,K-ATPase expressed in Escherichia coli; Tran CM et al.; Fusion proteins of glutathione-S-transferase and fragments from the large cytoplasmic domain of the sheep Na,K-ATPase alpha1-subunit were expressed in Escherichia coli . The Na,K-ATPase sequences begin at Ala345 and terminate at either Arg600 (DP600f), Thr610 (DP610f), Gly731 (DP731f), or Glu779 (DP779f) . After affinity purification on glutathione-Sepharose, the fusion proteins were labeled with {alpha-32P}-2-N3-ATP, and incorporation of the radiolabel into the fusion proteins was measured by scintillation counting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Kd values of 220-290 microM for 2-N3-ATP binding to the fusion proteins were obtained from the photolabeling experiments . Approximately 1 mol of 2-N3-ATP was calculated to be incorporated per mole of fusion protein after correction for photochemical incorporation efficiency . Labeling of all of the fusion proteins by 25 microM 2-N3-ATP was reduced in the presence of MgATP, Na2ATP, MgCl2, 2',3'-O-(2,4, 6-trinitrophenyl)-ATP, and p-nitrophenylphosphate, and Ki values of 2-11 mM for Na2ATP, 0.2-5 mM for MgCl2, 0.1-5 mM for MgATP, and 20-300 microM for p-nitrophenylphosphate were calculated for these ligands . All of the fusion proteins catalyze the hydrolysis of p-nitrophenylphosphate . The reaction requires MgCl2 and is inhibited by inorganic phosphate, which is similar to the hydrolysis of p-nitrophenylphosphate by native Na,K-ATPase . Based on these observations, it appears that the soluble fragments from the large cytoplasmic domain of Na,K-ATPase expressed in bacterial cells are folded in an E2-like conformation and are likely to retain much of the native structure. Appl Environ Microbiol, 1999 Jul, 65(7), 3129 - 33 Identification and characterization of a flagellin gene from the endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila; Millikan DS et al.; The bacterial endosymbionts of the hydrothermal vent tubeworm Riftia pachyptila play a key role in providing their host with fixed carbon . Results of prior research suggest that the symbionts are selected from an environmental bacterial population, although a free-living form has been neither cultured from nor identified in the hydrothermal vent environment . To begin to assess the free-living potential of the symbiont, we cloned and characterized a flagellin gene from a symbiont fosmid library . The symbiont fliC gene has a high degree of homology with other bacterial flagellin genes in the amino- and carboxy-terminal regions, while the central region was found to be nonconserved . A sequence that was homologous to that of a consensus sigma28 RNA polymerase recognition site lay upstream of the proposed translational start site . The symbiont protein was expressed in Escherichia coli, and flagella were observed by electron microscopy . A 30,000-Mr protein subunit was identified in whole-cell extracts by Western blot analysis . These results provide the first direct evidence of a motile free-living stage of a chemoautotrophic symbiont and support the hypothesis that the symbiont of R . pachyptila is acquired with each new host generation. Appl Environ Microbiol, 1999 Jul, 65(7), 3027 - 32 High-level production of human leptin by fed-batch cultivation of recombinant Escherichia coli and its purification; Jeong KJ et al.; Human leptin is a 16-kDa (146-amino-acid) protein that is secreted from adipocytes and influences body weight homeostasis . In order to obtain high-level production of leptin, the human obese gene coding for leptin was expressed in Escherichia coli BL21(DE3) under the strong inducible T7 promoter . The recombinant leptin was produced as inclusion bodies in E . coli, and the recombinant leptin content was as high as 54% of the total protein content . For production of recombinant human leptin in large amounts, pH-stat fed-batch cultures were grown . Expression of leptin was induced at three different cell optical densities at 600 nm (OD600), 30, 90, and 140 . When cells were induced at an OD600 of 90, the amount of leptin produced was 9.7 g/liter (37% of the total protein) . After simple purification steps consisting of inclusion body isolation, denaturation and refolding, and anion-exchange chromatography, 144.9 mg of leptin that was more than 90% pure was obtained from a 50-ml culture, and the recovery yield was 41.1%. J Struct Biol, 1999 Jun 15, 126(2), 145 - 55 An 8-A projected structure of FhuA, A "ligand-gated" channel of the Escherichia coli outer membrane; Lambert O et al.; The structure of FhuA, a siderophore and phage receptor in the outer membrane of Escherichia coli, has been investigated by electron crystallography . Bidimensional crystals of hexahistidine-tagged FhuA protein solubilized in N,N-dimethyldodecylamine-N-oxide were produced after detergent removal with polystyrene beads . Frozen-hydrated crystals (unit cell dimensions of a = 124 A, b = 98 A, gamma = 90 degrees ) exhibited a p22121 plane group symmetry . A projection map at 8 A resolution showed the presence of dimeric ring-like structures with an elliptical shape (48 x 40 A) . Each monomer was composed of a ring of densities with a radial width of 8-10 A corresponding to a cylinder of beta sheets . Few densities are present inside the barrel, leaving a central channel approximately 25 A in diameter . A projection map of FhuA at 15 A resolution, which was calculated from negatively stained preparations, demonstrated that most of the central channel was masked by extramembrane domains . This map also revealed an asymmetric distribution of extramembrane domains in FhuA, with large domains located mainly on one side of the molecule . Comparison with density maps derived from recent atomic structure allowed further interpretation of the electron microscopy projection structures with regard to long hydrophilic loops governing the selectivity and opening of the channel . J Mol Biol, 1999 Jul 2, 290(1), 347 - 61 Novel molecular architecture of the multimeric archaeal PEP-synthase homologue (MAPS) from Staphylothermus marinus; Cicicopol C et al.; The phosphoenolpyruvate (PEP)-synthases belong to the family of structurally and functionally related PEP-utilizing enzymes . The only archaeal member of this family characterized thus far is the Multimeric Archaeal PEP-Synthase homologue from Staphylothermus marinus (MAPS) . This protein complex differs from the bacterial and eukaryotic representatives characterized to date in its homomultimeric, as opposed to dimeric or tetrameric, structure . We have probed the molecular architecture of MAPS using limited proteolytic digestion in conjunction with electron microscopic, biochemical, and biophysical techniques . The 2.2 MDa particle was found to be organized in a concentric fashion . The 93.7 kDa monomers possess a pronounced tripartite domain structure and are arranged such that the N-terminal domains form an outer shell, the intermediate domains form an inner shell, and the C-terminal domains form a core structure responsible for the assembly into a multimeric complex . The core domain was shown to be capable of assembling into the native multimer by recombinant expression in Escherichia coli . Deletion mutants as well as a synthetic peptide were investigated for their state of oligomerization using native polyacrylamide gel electrophoresis, molecular sieve chromatography, analytical ultracentrifugation, circular dichroism (CD) spectroscopy, and chemical cross-linking . Our data confirmed the existence of a short C-terminal, alpha-helical oligomerization motif that had been suggested by multiple sequence alignments and secondary structure predictions . We propose that this motif bundles the monomers into six groups of four . An additional formation of 12 dimers between globular domains from different bundles leads to the multimeric assembly . According to our model, each of the six bundles of globular domains is positioned at the corners of an imaginary octahedron, and the helical C-terminal segments are oriented towards the centre of the particle . The edges of the octahedron represent the dimeric contacts . Phylogenetic analysis suggests that the ancient predecessor of this family of enzymes contained the C-terminal oligomerization motif as a feature that was preserved in some hyperthermophiles . J Mol Biol, 1999 Jul 2, 290(1), 331 - 45 Equilibrium folding properties of the yeast prion protein determinant Ure2; Perrett S et al.; The yeast non-Mendelian factor {URE3} propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2 . The {URE3} phenotype is equivalent to loss of function of Ure2, a protein involved in regulation of nitrogen metabolism . The prion-like behaviour of Ure2 in vivo is dependent on the first 65 amino acid residues of its N-terminal region which contains a highly repetitive sequence rich in asparagine . This region has been termed the prion-determining domain (PrD) . Removal of as little as residues 2-20 of the protein is sufficient to prevent occurrence of the {URE3} phenotype . Removal of the PrD does not affect the regulatory activity of Ure2 . The C-terminal portion of the protein has homology to glutathione S -transferases, which are dimeric proteins . We have produced the Ure2 protein to high yield in Escherichia coli from a synthetic gene . The recombinant purified protein is shown to be a dimer . The stability, folding and oligomeric state of Ure2 and a series of N-terminally truncated or deleted variants were studied and compared . The stability of Ure2, DeltaGD-N, H2O, determined by chemical denaturation and monitored by fluorescence, is 12.1(+/-0.4) kcal mol-1at 25 degrees C and pH 8.4 . A range of structural probes show a single, coincident unfolding transition, which is invariant over a 550-fold change in protein concentration . The stability is the same within error for Ure2 variants lacking all or part of the prion-determining domain . The data indicate that in the folded protein the PrD is in an unstructured conformation and does not form specific intra- or intermolecular interactions at micromolar protein concentrations . This suggests that the C-terminal domain may stabilise the PrD against prion formation by steric means, and implies that the PrD does not induce prion formation by altering the thermodynamic stability of the folded protein . J Mol Biol, 1999 Jul 2, 290(1), 49 - 60 The McrBC endonuclease translocates DNA in a reaction dependent on GTP hydrolysis; Panne D et al.; McrBC specifically recognizes and cleaves methylated DNA in a reaction dependent on GTP hydrolysis . DNA cleavage requires at least two recognition sites that are optimally separated by 40-80 bp, but can be spaced as far as 3 kb apart . The nature of the communication between two recognition sites was analyzed on DNA substrates containing one or two recognition sites . DNA cleavage of circular DNA required only one methylated recognition site, whereas the linearized form of this substrate was not cleaved . However, the linearized substrate was cleaved if a Lac repressor was bound adjacent to the recognition site . These results suggest a model in which communication between two remote sites is accomplished by DNA translocation rather than looping . A mutant protein with defective GTPase activity cleaved substrates with closely spaced recognition sites, but not substrates where the sites were further apart . This indicates that McrBC translocates DNA in a reaction dependent on GTP hydrolysis . We suggest that DNA cleavage occurs by the encounter of two DNA-translocating McrBC complexes, or can be triggered by non-specific physical obstacles like the Lac repressor bound on the enzyme's path along DNA . Our results indicate that McrBC belongs to the general class of DNA "motor proteins", which use the free energy associated with nucleoside 5'-triphosphate hydrolysis to translocate along DNA . J Mol Biol, 1999 Jul 2, 290(1), 37 - 48 DNA-induced conformational changes in cyclic AMP receptor protein: detection and mapping by a protein footprinting technique using multiple chemical proteases; Baichoo N et al.; Cyclic AMP receptor protein (CRP) is a regulator of transcription in Escherichia coli which mediates its activity by binding specific DNA sequences in a cyclic AMP-dependent manner . The interaction of CRP with specific DNA was probed by a protein footprinting technique using chemical proteases of different charge, size, and hydrophobicity . The experimental data were compared with known crystal structures of cAMP-CRP and cAMP-CRP-DNA complexes to determine a correlation between the structure of the complexes, the nature of the chemical protease and protein cleavage patterns . In addition, such comparison allowed us to determine if DNA binding in solution induced conformational changes in the protein not apparent in the crystal structure . In the cAMP-CRP-DNA complex, both the protections and the enhancements of proteolytic cleavage were observed outside of the known CRP-DNA interface, suggesting that CRP undergoes a conformational change upon binding DNA . Among the observed changes, the most interesting were those around the B alpha-helix and beta-strand 8, since this region overlaps with the activation region 2 which CRP uses for protein-protein interactions with RNA polymerase . DNA-induced changes were observed also in the region involved in CRP-CytR interaction and in CRP intersubunit contact regions . These data suggest that binding of DNA in solution induces conformational changes in CRP which can be transmitted via intersubunit contacts to regions of the protein involved in interactions with other members of transcriptional machinery . Biochemistry, 1999 Jun 29, 38(26), 8590 - 7 Estimating loop-helix interfaces in a polytopic membrane protein by deletion analysis; Wolin CD et al.; Insertions of amino acids into transmembrane helices of polytopic membrane proteins disrupt helix-helix interactions with loss of function, while insertions into loops have little effect on transmembrane helices and therefore little effect on activity {Braun, P., Persson, B., Kaback, H . R., and von Heijne, G . (1997) J . Biol . Chem . 272, 29566-29571} . Here the inverse approach, amino acid deletion, is utilized systematically to approximate loop-helix boundaries in the lactose permease of Escherichia coli . Starting with deletion mutants in the periplasmic loop between helices VII and VIII (loop VII/VIII), which has been defined by immunological analysis and nitroxide-scanning electron paramagnetic resonance spectroscopy, it is shown that mutants with single or multiple deletions in the central portion of the loop retain significant transport activity, while deletion of amino acid residues near the loop-helix boundaries or within the flanking helices leads to complete inactivation . Results consistent with hydropathy analysis are obtained with loops VI/VII, VIII/IX, and IX/X and the flanking helices . In contrast, deletion analysis of loops III/IV, IV/V, and V/VI and the flanking helices indicates that this region of the permease differs from hydropathy predictions . More specifically, evidence is presented supporting the contention that Glu126 and Arg144 which are charge paired and critical for substrate binding are within helices IV and V, respectively. Biochemistry, 1999 Jun 29, 38(26), 8359 - 66 Nonaggregating mutant of recombinant human hexokinase I exhibits wild-type kinetics and rod-like conformations in solution; Aleshin AE et al.; Hexokinase I governs the rate-limiting step of glycolysis in brain tissue, being inhibited by its product, glucose 6-phosphate, and allosterically relieved of product inhibition by phosphate . On the basis of small-angle X-ray scattering, the wild-type enzyme is a monomer in the presence of glucose and phosphate at protein concentrations up to 10 mg/mL, but in the presence of glucose 6-phosphate, is a dimer down to protein concentrations as low as 1 mg/mL . A mutant form of hexokinase I, specifically engineered by directed mutation to block dimerization, remains monomeric at high protein concentration under all conditions of ligation . This nondimerizing mutant exhibits wild-type activity, potent inhibition by glucose 6-phosphate, and phosphate reversal of product inhibition . Small-angle X-ray scattering data from the mutant hexokinase I in the presence of glucose/phosphate, glucose/glucose 6-phosphate, and glucose/ADP/Mg2+/AlF3 are consistent with a rodlike conformation for the monomer similar to that observed in crystal structures of the hexokinase I dimer . Hence, any mechanism for allosteric regulation of hexokinase I should maintain a global conformation of the polypeptide similar to that observed in crystallographic structures. Biochemistry, 1999 Jun 29, 38(26), 8299 - 303 The HELLGH motif of rat liver dipeptidyl peptidase III is involved in zinc coordination and the catalytic activity of the enzyme; Fukasawa K et al.; The role of the HELLGH (residues 450-455) motif in the sequence of rat dipeptidyl peptidase III (EC 3.4.14.4) was investigated by replacing Glu451 with an alanine or an aspartic acid residue and by replacing His450 and His455 with a tyrosine residue by site-directed mutagenesis . Mutated cDNAs were expressed three or four times in Escherichia coli, and the resulting proteins were purified to apparent homogeneity . None of the expressed mutated proteins exhibited DPP III activity . The mutants of Glu451 contained 1 mol of zinc per mole of protein, but mutants His450 and His455 did not contain significant amounts of zinc as determined by atomic absorption spectrometry . The Leu453-deleted enzyme (having the zinc aminopeptidase motif HExxH-18-E) had almost the same order of binding affinity (for Arg-Arg-2-naphthylamide) as the wild-type enzyme, but the specificity constant was about 10% . These results provide evidence that the suitable number of amino acids included between Glu451 and His455 is three residues for the enzyme activity and confirm that residues His450, His455, and Glu451 are involved in zinc coordination and catalytic activity. Biochemistry, 1999 Jun 29, 38(26), 8217 - 27 The active site base controls cofactor reactivity in Escherichia coli amine oxidase: x-ray crystallographic studies with mutational variants; Murray JM et al.; Amine oxidases utilize a proton abstraction mechanism following binding of the amine substrate to the C5 position of the cofactor, the quinone form of trihydroxyphenylalanine (TPQ) . Previous work {Wilmot, C . M., et al . (1997) Biochemistry 36, 1608-1620} has shown that Asp383 in Escherichia coliamine oxidase (ECAO) is the catalytic base which performs the key step of proton abstraction . This paper explores in more depth this and other roles of Asp383 . The crystal structures of three mutational variants are presented together with their catalytic properties, visible spectra, and binding properties for a substrate-like inhibitor, 2-hydrazinopyridine (2-HP), in comparison to those of the wild type enzyme . In wild type ECAO, the TPQ is located in a wedge-shaped pocket which allows more freedom of movement at the substrate binding position (C5) than for TPQ ring carbons C1-C4 . A role of Asp383, whose carboxylate is located close to O5, is to stabilize the TPQ in its major conformation in the pocket . Replacement of Asp383 with the isostructural, but chemically distinct, Asn383 does not affect the location or dynamics of the TPQ cofactor significantly, but eliminates catalytic activity and drastically reduces the affinity for 2-HP . Removal of the side chain carboxyl moiety, as in Ala383, additionally allows the TPQ the greater conformational flexibility to coordinate to the copper, which demonstrates that Asp383 helps maintain the active site structure by preventing TPQ from migrating to the copper . Glu383 has a greatly decreased catalytic activity, as well as a decreased affinity for 2-HP relative to that of wild type ECAO . The electron density reveals that the longer side chain of Glu prevents the pivotal motion of the TPQ by hindering its movement within the wedge-shaped active site pocket . The results show that Asp383 performs multiple roles in the catalytic mechanism of ECAO, not only in acting as the active site base at different stages of the catalytic cycle but also in regulating the mobility of the TPQ that is essential to catalysis. Biochemistry, 1999 Jun 22, 38(25), 8179 - 88 Phosphate release during microtubule assembly: what stabilizes growing microtubules? Vandecandelaere A, Brune M, Webb MR, Martin SR, Bayley PM. The molecular mechanism underlying microtubule dynamic instability depends on the relationship between the addition of tubulin-GTP to a growing microtubule and its hydrolysis in the microtubule lattice to tubulin-GDP, with release of inorganic phosphate (Pi) . Since this relationship remains controversial, we have re-examined the release of Pi upon microtubule assembly using a fluorometric assay for Pi, based on the phosphate-binding protein of Escherichia coli {Brune M., Hunter, J . L., Corrie, J . E . T., and Webb, M . R . (1994) Biochemistry 33, 8262-8271} . Microtubule assembly and Pi release were monitored simultaneously in a standard fluorimeter as an increase in the turbidity and fluorescence, respectively, in tubulin-GTP solutions assembled under conditions supporting dynamic instability . At the steady state of assembly, Pi release is nonlinear with respect to time, proceeding at a rate determined by the following: (a) the intrinsic GTPase activity of the nonpolymerized tubulin-GTP, and (b) the microtubule number concentration, which decreases progressively . Direct observation of the time course of nucleated microtubule assembly indicates that Pi release is closely coupled to microtubule elongation, even during the initial stages of assembly when uncoupling of tubulin-GTP addition and GTP hydrolysis would be most evident . Studies of the inhibition and reversal of the growth phase by cytostatic drugs show no evidence of a burst of Pi release . We conclude that nucleotide hydrolysis can keep pace with tubulin-GTP addition rates of 200 molecules per second per microtubule and that extended caps of tubulin-GTP or tubulin-GDP-Pi are not generated in normal assembly, nor are they required to stabilize growing microtubules or to support the phenomenon of dynamic instability of microtubules at the steady state. Biochemistry, 1999 Jun 22, 38(25), 8088 - 93 Misacylation of tRNALys with noncognate amino acids by lysyl-tRNA synthetase; Jakubowski H; Lysyl-tRNA synthetase (LysRS), a class II enzyme whose major function is to provide Lys-tRNALys for protein synthesis, also catalyzes aminoacylation of tRNALys with arginine, threonine, methionine, leucine, alanine, serine, and cysteine . The limited selectivity in the tRNA aminoacylation reaction appears to be due to inefficient editing of some amino acids (Met, Leu, Cys, Ala, Thr) by a pre-transfer mechanism or the absence of editing of other amino acids (Arg and Ser) . Purified Arg-tRNALys, Thr-tRNALys, and Met-tRNALys were essentially not deacylated by LysRS, indicating that the enzyme does not possess a post-transfer editing mechanism . However, LysRS possesses an efficient pre-transfer editing mechanism which prevents misacylation of tRNALys with ornithine . A novel feature of this editing reaction is that ornithine lactam is formed by the facile cyclization of ornithyl adenylate. Biochemistry, 1999 Jun 22, 38(25), 8032 - 7 A novel, definitive test for substrate channeling illustrated with the aspartate aminotransferase/malate dehydrogenase system; Geck MK et al.; A novel method is presented that establishes definitively the existence or nonexistence of direct metabolite transfer between consecutive enzymes in a metabolic sequence . The procedure is developed with the specific example of channeling of oxaloacetate between Escherichia coli aspartate aminotransferase (AATase) and malate dehydrogenase (MDH) . The assay is carried out in the presence of a large excess of inactive variants of AATase . These mutants would outcompete the much smaller quantities of wild-type AATase for any docking sites on MDH and thus decrease the rate of the coupled L-aspartate to oxaloacetate to malate sequence only if the direct metabolite transfer mechanism is operative . The results show that oxaloacetate is not transferred directly from AATase to MDH because no decrease in rate was observed in the presence of approximately 100 microM inactive mutants . This concentration is 10 times the physiological AATase concentration, which was determined in this work . The methodology can be applied generally. Biochemistry, 1999 Jun 22, 38(25), 7891 - 9 The amidotransferase family of enzymes: molecular machines for the production and delivery of ammonia; Raushel FM et al.; The amidotransferase family of enzymes utilizes the ammonia derived from the hydrolysis of glutamine for a subsequent chemical reaction catalyzed by the same enzyme . The ammonia intermediate does not dissociate into solution during the chemical transformations . A well-characterized example of the structure and mechanism displayed by this class of enzymes is provided by carbamoyl phosphate synthetase (CPS) . Carbamoyl phosphate synthetase is isolated from Escherichia coli as a heterodimeric protein . The smaller of the two subunits catalyzes the hydrolysis of glutamine to glutamate and ammonia . The larger subunit catalyzes the formation of carbamoyl phosphate using 2 mol of ATP, bicarbonate, and ammonia . Kinetic investigations have led to a proposed chemical mechanism for this enzyme that requires carboxy phosphate, ammonia, and carbamate as kinetically competent reaction intermediates . The three-dimensional X-ray crystal structure of CPS has localized the positions of three active sites . The nucleotide binding site within the N-terminal half of the large subunit is required for the phosphorylation of bicarbonate and subsequent formation of carbamate . The nucleotide binding site within the C-terminal domain of the large subunit catalyzes the phosphorylation of carbamate to the final product, carbamoyl phosphate . The three active sites within the heterodimeric protein are separated from one another by about 45 A . The ammonia produced within the active site of the small subunit is the substrate for reaction with the carboxy phosphate intermediate that is formed in the active site found within the N-terminal half of the large subunit of CPS . Since the ammonia does not dissociate from the protein prior to its reaction with carboxy phosphate, this intermediate must therefore diffuse through a molecular tunnel that connects these two sites with one another . Similarly, the carbamate intermediate, initially formed at the active site within the N-terminal half of the large subunit, is the substrate for phosphorylation by the ATP bound to the active site located in the C-terminal half of the large subunit . A molecular passageway has been identified by crystallographic methods that apparently facilitates diffusion between these two active sites within the large subunit of CPS . Synchronization of the chemical transformations is controlled by structural perturbations among the three active sites . Molecular tunnels between distant active sites have also been identified in tryptophan synthase and glutamine phosphoribosyl pyrophosphate amidotransferase and are likely architectural features in an expanding list of enzymes. Biochemistry, 1999 Jun 15, 38(24), 7847 - 55 The functional role of positively charged amino acid side chains in alpha-bungarotoxin revealed by site-directed mutagenesis of a His-tagged recombinant alpha-bungarotoxin; Rosenthal JA et al.; A polyhistidine tag was added to the N-terminus of alpha-bungarotoxin (Bgtx) recombinantly expressed in E . coli . The His-tagged Bgtx was identical to native, venom-derived Bgtx in its apparent affinity for the nicotinic acetylcholine receptor (nAChR) in Torpedo electric organ membranes . Furthermore, in a physiological assay involving mouse muscle nAChR expressed in Xenopus oocytes, the His-tagged Bgtx was as effective as authentic Bgtx at blocking acetylcholine-evoked currents . Ala-substitution mutagenesis of His-tagged Bgtx was used to evaluate the functional contribution of Arg36, a residue that is invariant among all alpha-neurotoxins . Replacement with Ala resulted in a 90-fold decrease in the apparent affinity for the Torpedo nAChR and a corresponding 150-fold increase in the IC50 for block of heterologously expressed mouse muscle nAChR, demonstrating the critical importance of this positive charge for the binding and functional activity of a long alpha-neurotoxin . The observed decrease in affinity corresponds to a DeltaDeltaG of 2.7 kcal/mol and indicates that Arg36 makes a major contribution to complex formation . This finding is consistent with the proposal that Arg36 mimics the positive charge found on acetylcholine and directs the toxin to interact with receptor sites normally involved in acetylcholine recognition . In comparison, Ala-substitution of the highly conserved Lys26 resulted in only a 9-fold decrease in apparent affinity . Truncation of the His-tagged Bgtx following residue 67 produces a toxin lacking the seven C-terminal residues including the two positively charged residues Lys70 and Arg72 . Truncation leads to a 7-fold decrease in apparent binding affinity. Biochemistry, 1999 Jun 15, 38(24), 7791 - 802 Stages in iron storage in the ferritin of Escherichia coli (EcFtnA): analysis of Mössbauer spectra reveals a new intermediate; Bauminger ER et al.; Iron uptake into the nonheme ferritin of Escherichia coli (EcFtnA) and its site-directed variants have been investigated by Mossbauer spectroscopy . EcFtnA, like recombinant human H chain ferritin (HuHF), oxidized Fe(II) at a dinuclear ferroxidase center situated at a central position within each subunit . As with HuHF, Mossbauer subspectra observed between 1 min and 24 h after Fe(II) addition were assigned to Fe(III) monomers, "c", mu-oxo-bridged dimers, "b", and clusters, "a", the latter showing magnetically split spectra, "d", at 4.1 K . Like those of HuHF, the mu-oxo-bridged dimers were formed at the ferroxidase centers . However, the analysis also revealed the presence of a new type of dimer, "e" (QS1 = 0.38 mm/s, IS1 = 0.51 mm/s and QS2 = 0.72 mm/s, IS2 = 0.50 mm/s), and this was also assigned to the ferroxidase center . Dimers "b" appeared to be converted to dimers "e" over time . Subspectra "e" became markedly asymmetric at temperatures above 90 K, suggesting that the two Fe(III) atoms of dimers "e" were more weakly coupled than in the mu-oxo-bridged dimers "b", possibly due to OH- bridging . Monomeric Fe(III), giving relaxation spectra "c", was assigned to a unique site C that is near the dinuclear center . In EcFtnA all three iron atoms seemed to be oxidized together . In contrast to HuHF, no Fe(III) clusters were observed 24 h after the aerobic addition of 48 Fe(II) atoms/molecule in wild-type EcFtnA . This implies that iron is more evenly distributed between molecules in the bacterial ferritins, which may account for its greater accessibility. Biochemistry, 1999 Jun 15, 38(24), 7670 - 7 The role of beta-Arg-182, an essential catalytic site residue in Escherichia coli F1-ATPase; Nadanaciva S et al.; Beta-Arg-182 in Escherichia coli F1-ATPase (beta-Arg-189 in bovine mitochondrial F1) is a residue which lies close to catalytic site bound nucleotide (Abrahams et al . (1994) Nature 370, 621-628) . Here we investigated the role of this residue by characterizing two mutants, betaR182Q and betaR182K . Oxidative phosphorylation and steady-state ATPase activity of purified F1 were severely impaired by both mutations . Catalytic site nucleotide-binding parameters were measured using the fluorescence quench of beta-Trp-331 that occurred upon nucleotide binding to purified F1 from betaR182Q/betaY331W and betaR182K/betaY331W double mutants . It was found that (a) beta-Arg-182 interacts with the gamma-phosphate of MgATP, particularly at catalytic sites 1 and 2, (b) beta-Arg-182 has no functional interaction with the beta-phosphate of MgADP or with the magnesium of the magnesium-nucleotide complex in the catalytic sites, and (c) beta-Arg-182 is directly involved in the stabilization of the catalytic transition state . In these features the role of beta-Arg-182 resembles that of another positively charged residue in the catalytic site, the conserved lysine of the Walker A motif, beta-Lys-155 . A further role of beta-Arg-182 is suggested, namely involvement in conformational change at the catalytic site beta-alpha subunit interface that is required for multisite catalysis. Protein Sci, 1999 Jun, 8(6), 1332 - 41 A novel recombinant single-chain hepatitis C virus NS3-NS4A protein with improved helicase activity; Howe AY et al.; Hepatitis C virus (HCV) nonstructural protein 3 (NS3) has been shown to possess protease and helicase activities and has also been demonstrated to spontaneously associate with nonstructural protein NS4A (NS4A) to form a stable complex . Previous attempts to produce the NS3/NS4A complex in recombinant baculovirus resulted in a protein complex that aggregated and precipitated in the absence of nonionic detergent and high salt . A single-chain form of the NS3/NS4A complex (His-NS4A21-32-GSGS-NS3-631) was constructed in which the NS4A core peptide is fused to the N-terminus of the NS3 protease domain as previously described (Taremi et al., 1998) . This protein contains a histidine tagged NS4A peptide (a.a . 21-32) fused to the full-length NS3 (a.a . 3-631) through a flexible tetra amino acid linker . The recombinant protein was expressed to high levels in Escherichia coli, purified to homogeneity, and examined for NTPase, nucleic acid unwinding, and proteolytic activities . The single-chain recombinant NS3-NS4A protein possesses physiological properties equivalent to those of the NS3/NS4A complex except that this novel construct is stable, soluble and sixfold to sevenfold more active in unwinding duplex RNA . Comparison of the helicase activity of the single-chain recombinant NS3-NS4A with that of the full-length NS3 (without NS4A) and that of the helicase domain alone suggested that the presence of the protease domain and at least the NS4A core peptide are required for optimal unwinding activity. Protein Sci, 1999 Jun, 8(6), 1305 - 13 The 80s loop of the catalytic chain of Escherichia coli aspartate transcarbamoylase is critical for catalysis and homotropic cooperativity; Macol C et al.; The X-ray structure of the Escherichia coli aspartate transcarbamoylase with the bisubstrate analog phosphonacetyl-L-aspartate (PALA) bound shows that PALA interacts with Lys84 from an adjacent catalytic chain . To probe the function of Lys84, site-specific mutagenesis was used to convert Lys84 to alanine, threonine, and asparagine . The K84N and K84T enzymes exhibited 0.08 and 0.29% of the activity of the wild-type enzyme, respectively . However, the K84A enzyme retained 12% of the activity of the wild-type enzyme . For each of these enzymes, the affinity for aspartate was reduced 5- to 10-fold, and the affinity for carbamoyl phosphate was reduced 10- to 30-fold . The enzymes K84N and K84T exhibited no appreciable cooperativity, whereas the K84A enzyme exhibited a Hill coefficient of 1.8 . The residual cooperativity and enhanced activity of the K84A enzyme suggest that in this enzyme another mechanism functions to restore catalytic activity . Modeling studies as well as molecular dynamics simulations suggest that in the case of only the K84A enzyme, the lysine residue at position 83 can reorient into the active site and complement for the loss of Lys84 . This hypothesis was tested by the creation and analysis of the K83A enzyme and a double mutant enzyme (DM) that has both Lys83 and Lys84 replaced by alanine . The DM enzyme has no cooperativity and exhibited 0.18% of wild-type activity, while the K83A enzyme exhibited 61% of wild-type activity . These data suggest that Lys84 is not only catalytically important, but is also essential for binding both substrates and creation of the high-activity, high-affinity active site . Since low-angle X-ray scattering demonstrated that the mutant enzymes can be converted to the R-structural state, the loss of cooperativity must be related to the inability of these mutant enzymes to form the high-activity, high-affinity active site characteristic of the R-functional state of the enzyme. Protein Sci, 1999 Jun, 8(6), 1276 - 85 Probing the role of water in the tryptophan repressor-operator complex; Brown MP et al.; The Escherichia coli tryptophan repressor protein (TR) represses the transcription of several genes in response to the concentration of tryptophan in the environment . In the co-crystal structure of TR bound to a DNA fragment containing its target very few direct contacts between TR and the DNA were observed . In contrast, a number of solvent mediated contacts were apparent . NMR solution structures, however, did not resolve any solvent mediated bonds at the complex interface . To probe for the role of water in TR operator recognition, the effect of osmolytes on the interactions between TR and a target oligonucleotide bearing the operator site was examined . In the absence of specific solvent mediated hydrogen bonding interactions between the protein and the DNA, increasing osmolyte concentration is expected to strongly stabilize the TR operator interaction due to the large amount of macromolecular surface area buried upon complexation . The results of our studies indicate that xylose did not alter the binding affinity significantly, while glycerol and PEG had a small stabilizing effect . A study of binding as a function of betaine concentration revealed that this osmolyte at low concentration results in a stabilization of the 1:1 TR/operator complex, but at higher concentrations leads to a switching between binding modes to favor tandem binding . Analysis of the effects of betaine on the 1:1 complex suggest that this osmolyte has about 78% of the expected effect . If one accepts the analysis in terms of the number of water molecules excluded upon complexation, these results suggest that about 75 water molecules remain at the interface of the 1:1 dimer/DNA complex . This value is consistent with the number of water molecules found at the interface in the crystallographically determined structure and supports the notion that interfacial waters play an important thermodynamic role in the specific complexation of one TR dimer with its target DNA . However, the complexity of the effects of betaine and the small or negligible effects of the other osmolytes could also arise from osmolyte induced competition between antagonistic coupled reactions. Protein Sci, 1999 Jun, 8(6), 1200 - 9 Identifying the structural boundaries of independent folding domains in the alpha subunit of tryptophan synthase, a beta/alpha barrel protein; Zitzewitz JA et al.; Two equilibrium intermediates have previously been observed in the urea denaturation of the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, an eight-stranded beta/alpha barrel protein . In the current study, a series of amino-terminal fragments were characterized to probe the elementary folding units that may be in part responsible for this complex behavior . Stop-codon mutagenesis was used to produce eight fragments ranging in size from 105-214 residues and containing incremental elements of secondary structure . Equilibrium studies by circular dichroism indicate that all of these fragments are capable of adopting secondary structure . All except for the shortest fragment fold cooperatively . The addition of the fourth, sixth, and eighth beta-strands leads to distinct increases in structure, cooperativity, and/or stability, suggesting that folding involves the modular assembly of betaalphabeta supersecondary structural elements . One-dimensional NMR titrations at high concentrations of urea, probing the environment around His92, were also performed to test for the presence of residual structure in the fragments . All fragments that contained the first four betaalpha units of structure exhibited a cooperative unfolding transition at high concentrations of urea with significant but reduced stability relative to the full-length protein . These results suggest that the residual structure in alphaTS requires the participation of hydrophobic residues in multiple beta-strands that span the entire sequence. J Interferon Cytokine Res, 1999 May, 19(5), 505 - 13 Structure and expression of the human small cytokine B subfamily member 11 (SCYB11/formerly SCYB9B, alias I-TAC) gene cloned from IFN-gamma-treated human monocytes (THP-1); Laich A et al.; Among CXC chemokines, monokine induced by interferon-gamma (IFN-gamma) (MIG) and IGN-gamma-inducible protein, 10 kDa (INP10), constitute a distinct group because of their sequence and function . We studied genomic structure and expression of a third, recently identified member of this group named small inducible cytokine B subfamily member 11 (SCYB11, formerly SCYB9B) or IFN-inducible T cell alpha chemoattractant (I-TAC) . The cDNA (1445 bp) for this 94 amino acid protein (Mr 10,364) was cloned from IFN-gamma-treated human myelomonocytic cells (THP-1) . The reading frame of SCYB11 is distributed to 4 exons spanning 1197 bp of the genomic sequence . In vitro transcription/translation yielded a single protein of about 10 kDa, indicating that the deduced reading frame is translated by eukaryotic ribosomes . The recombinant 73 amino acid mature protein overexpressed in Escherichia coli was chemotactic for interleukin-2 (IL-2)-selected T memory cells . Studying various cytokines and lipopolysaccharide in THP-1 cells identified IFN-gamma as the major stimulus for SCYB11 mRNA expression, followed by IFN-alpha and IFN-beta, which were about 25 times less effective . Of a panel of different human cells tested, SCYB11 mRNA was also induced in umbilical vein endothelial cells, dermal fibroblasts, and tumor cell lines from various organs, whereas it was not found in T lymphocytes activated via anti-CD3 antibodies or via IL-2. J Interferon Cytokine Res, 1999 May, 19(5), 479 - 85 Augmentation of verotoxin-induced cytotoxicity/apoptosis by interferon is repressed in cells persistently infected with mumps virus; Hariya Y et al.; Verotoxin type 2 (VT2) produced by enterohemorrhagic Escherichia coli (EHEC) has been shown to have high cytotoxic potency toward several human B lymphoid cell lines with and without Epstein-Barr virus (EBV) . Cell death, apoptosis induced by VT2, is closely correlated with the expression of receptor molecule Gb3/CD77, recognized by the toxin, but not with the infection or presence of EBV . Pretreatment of cells with interferon-alpha (IFN-alpha) for 24 h resulted in augmentation of apoptosis by VT2 . Pretreatment within 8 h, however, was not effective . It has been reported that IFN-alpha-induced apoptosis is correlated with the induction of the 2',5'-OAS/RNase L system or dsRNA-activated protein kinase (PKR) or both . We have established persistent infection in both Akata and P3HR-1 cells with mumps virus . The persistently infected cell lines, P3HR-MP2 and Akata-MP2, showed poor induction of 2',5'-OAS and PKR in response to IFN-alpha . Augmentation of VT2-induced apoptosis by IFN-alpha was not found in the cell lines P3HR-MP2 and Akata-MP2 . Therefore, these findings were interpreted to indicate that augmentation of VT2-induced apoptosis by IFN-alpha may be mediated by PKR and the 2',5'-OAS/RNaseL system . It is also suggested that mumps virus can suppress apoptosis and establish persistent infection. Cell Mol Biol (Noisy-le-grand), 1999 May, 45(3), 347 - 52 Overexpression, purification and photoaffinity labeling with a 3H-analogue of norfloxacin, of the GyrA and GyrB subunits of the DNA gyrase; Hombrouck C et al.; In spite of much work on DNA gyrase and quinolones for many years, our knowledge of the molecular basis of quinolone-gyrase action is still incomplete . We designed a photoaffinity labeling reagent for the quinolone target, and synthesized a norfloxacin analogue with an azide function which, under UV irradiation, becomes covalently linked to its target . For that, a large amount of purified gyrase was needed . Both subunits were purified using exclusion and affinity chromatography . A plasmid was used that allowed the overproduction of GyrA as a fusion-protein with six histidine residues at its carboxy-terminal domain . GyrA-(His)6 was purified after chromatography on a nickel-containing column, and native GyrB after chromatography on immobilized novobiocin . Reconstituted DNA gyrase (A2B2) had supercoiling activity . Photoaffinity labeling showed covalent binding of the 3H-photoaffinity analogue of norfloxacin to the gyrase-DNA complex, and mainly to the GyrA . The specific binding site remains to be explored. FEBS Lett, 1999 Jun 11, 452(3), 375 - 8 Cell-free synthesis of the Ras-binding domain of c-Raf-1: binding studies to fluorescently labelled H-ras; Sydor JR et al.; It has previously been shown that the transient kinetics of the interaction between the Ras-binding domain of c-Raf-1 and the proto-oncoprotein Ras can be followed by stopped-flow measurements using the 2',3'-(N-methylanthraniloyl) fluorescence of 2',3'-(N-methylanthraniloyl) guanyl-5'-yl-imidodiphosphate-labelled Ras . In continuation of this work, we demonstrate that the His-tagged Ras-binding domain of c-Raf-1 can also be synthesized in a cell-free expression system . After purification by Ni2+ affinity chromatography, His-tagged Ras-binding domain of c-Raf-1 could be isolated in sufficient amounts for biochemical and biophysical investigations . The results obtained describe the first example of a cell-free synthesized protein which has been used for stopped-flow measurements to determine the transient kinetics of protein-protein interactions with an effector. FEBS Lett, 1999 Jun 11, 452(3), 323 - 7 PCR random mutagenesis into Escherichia coli serine acetyltransferase: isolation of the mutant enzymes that cause overproduction of L-cysteine and L-cystine due to the desensitization to feedback inhibition; Takagi H et al.; PCR random mutagenesis in the cysE gene encoding Escherichia coli serine acetyltransferase was employed to isolate the mutant enzymes that, due to a much less feedback inhibition by L-cysteine, cause overproduction of L-cysteine and L-cystine in the recombinant strains . The L-cysteine auxotrophic and non-utilizing E . coli strain was transformed with plasmids having the altered cysE genes . Then, several transformants overproducing L-cysteine were selected by detecting the halo formation of the L-cysteine auxotroph . The production test of amino acids and analysis of the catalytic property on the mutant enzymes suggest that the carboxy-terminal region of serine acetyltransferase plays an important role in the desensitization to feedback inhibition and the high level production of L-cysteine and L-cystine. FEBS Lett, 1999 Jun 11, 452(3), 319 - 22 Effect of mutations found in carbohydrate-deficient glycoprotein syndrome type IA on the activity of phosphomannomutase 2; Pirard M et al.; Seven mutant forms of human phosphomannomutase 2 were produced in Escherichia coli and purified . These mutants had a Vmax of 0.2-50% of the wild enzyme and were unstable . The least active protein (R141H) bears a very frequent mutation, which has never been found in the homozygous state whereas the second least active protein (D188G) corresponds to a mutation associated with a particularly severe phenotype . We conclude that total lack of phosphomannomutase 2 is incompatible with life . Another conclusion is that the elevated residual phosphomannomutase activity found in fibroblasts of some patients is contributed by their mutated phosphomannomutase 2. FEMS Microbiol Lett, 1999 Jun 15, 175(2), 247 - 53 Non-curliation of Escherichia coli O78:K80 isolates associated with IS1 insertion in csgB and reduced persistence in poultry infection; La Ragione RM et al.; The elaboration of curli fimbriae by Escherichia coli is associated with the development of a lacy colony morphology when grown on colonisation factor antigen agar at 25 degrees C . Avian colisepticaemia E . coli isolates screened for curliation by this culture technique showed lacy and smooth colonial morphologies and the genetic basis of the non-curliated smooth colonial phenotype was analysed . Two smooth E . coli O78:K80 isolates possessed about 40 copies of the IS1 element within their respective genomes of which one copy insertionally inactivated the csgB gene, the nucleator gene for curli fibril formation . One of these two isolates also possessed a defective rpoS gene which is a known regulator of curli expression . In the day-old chick model, both smooth isolates were as invasive as a known virulent O78:K80 isolate as determined by extent of liver and spleen colonisation post oral inoculation but were less persistent in terms of caecal colonisation. Phytochemistry, 1999 Mar, 50(6), 1021 - 5 Immunological and molecular comparison of polyphenol oxidase in Rosaceae fruit trees; Haruta M et al.; An antibody raised against apple polyphenol oxidase (PPO) cross-reacted with PPOs from Japanese pear (Pyrus pyrifolia), pear (Pyrus communis), peach (Prunus persica), Chinese quince (Pseudocydonia sinensis) and Japanese loquat (Eriobotrya japonica) . Core fragments (681 bp) of the corresponding PPO genes were amplified and characterized . The deduced protein sequences showed identities of 85.3 to 97.5% . Chlorogenic acid oxidase activity of these PPOs showed higher activities when assayed at pH 4 than at pH 6 . These results indicate that PPOs in Rosaceae plants are structurally and enzymatically similar. Hepatology, 1999 Jul, 30(1), 21 - 6 Characterization of the autoantibody responses to recombinant E3 binding protein (protein X) of pyruvate dehydrogenase in primary biliary cirrhosis; Palmer JM et al.; Autoantibodies to the pyruvate dehydrogenase complex (PDC) are present in the serum of more than 95% of patients with primary biliary cirrhosis (PBC), the major epitope being the inner lipoyl domain of the E2 component . Immunoblotting suggests a similar prevalence of antibodies to a tightly associated lipoic acid-containing protein, E3 binding protein (E3BP) . Attempts to resolve E3BP from E2 have been unsuccessful, restricting study of the nature and significance of antibody responses to the individual proteins . In particular, it is unclear (1) whether there is true cross-reactivity between E3BP and E2 and, if so, which is the originating response and (2) whether autoantibodies preferentially bind a lipoylated epitope on E3BP as is the case with PDC-E2 . In this study, complementary DNAs encoding rE2, full-length rE3BP, its single lipoyl domain (rLip), and core domain (rE3BPCore) were cloned, and the proteins were expressed in Escherichia coli . Sera from 47 PBC patients were studied by immunoblotting and enzyme-linked immunosorbent assay (ELISA) against rE2, rE3BP, rE3BPCore, and both unlipoylated (U) and lipoylated (L) rLip . All sera were reactive by ELISA to some degree with all recombinant proteins except rE3BPCore, to which only 6 of 47 showed any reactivity . Significant correlations (P <.0001) were observed when comparing absorbance values for rE3BP with both rLip (U) (r = 0.793) and (L) (r = 0.963) . The mean absorbance for rLip (U, 0.26 +/- 0.05) was, however, significantly lower than the absorbance for rLip (L) (0.78 +/- 0.12; P <.0001) . After probing by immunoblotting and elution of antibodies from rE2 and rE3BP, subsequent reprobing against the components in whole PDC revealed true cross-reactivity . In summary, the response to E3BP is primarily directed against the lipoylated domain of the protein . It still remains unclear, however, whether the initial breakdown of tolerance is to E2 or E3BP. Nature, 1999 Jun 17, 399(6737), 704 - 8 A specific partner for abasic damage in DNA; Matray TJ et al.; In most models of DNA replication, Watson-Crick hydrogen bonding drives the incorporation of nucleotides into the new strand of DNA and maintains the complementarity of bases with the template strand . Studies with nonpolar analogues of thymine and adenine, however, have shown that replication is still efficient in the absence of hydrogen bonds . The replication of base pairs might also be influenced by steric exclusion, whereby inserted nucleotides need to be the correct size and shape to fit the active site against a template base . A simple steric-exclusion model may not require Watson-Crick hydrogen bonding to explain the fidelity of replication, nor should canonical purine and pyrimidine shapes be necessary for enzymatic synthesis of a base pair if each can fit into the DNA double helix without steric strain . Here we test this idea by using a pyrene nucleoside triphosphate (dPTP) in which the fluorescent 'base' is nearly as large as an entire Watson-Crick base pair . We show that the non-hydrogen-bonding dPTP is efficiently and specifically inserted by DNA polymerases opposite sites that lack DNA bases . The efficiency of this process approaches that of a natural base pair and the specificity is 10(2)-10(4)-fold . We use these properties to sequence abasic lesions in DNA, which are a common form of DNA damage in vivo . In addition to their application in identifying such genetic lesions, our results show that neither hydrogen bonds nor purine and pyrimidine structures are required to form a base pair with high efficiency and selectivity . These findings confirm that steric complementarity is an important factor in the fidelity of DNA synthesis. Nature, 1999 Jun 17, 399(6737), 700 - 4 The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta; Masutani C et al.; Xeroderma pigmentosum variant (XP-V) is an inherited disorder which is associated with increased incidence of sunlight-induced skin cancers . Unlike other xeroderma pigmentosum cells (belonging to groups XP-A to XP-G), XP-V cells carry out normal nucleotide-excision repair processes but are defective in their replication of ultraviolet-damaged DNA . It has been suspected for some time that the XPV gene encodes a protein that is involved in trans-lesion DNA synthesis, but the gene product has never been isolated . Using an improved cell-free assay for trans-lesion DNA synthesis, we have recently isolated a DNA polymerase from HeLa cells that continues replication on damaged DNA by bypassing ultraviolet-induced thymine dimers in XP-V cell extracts . Here we show that this polymerase is a human homologue of the yeast Rad30 protein, recently identified as DNA polymerase eta . This polymerase and yeast Rad30 are members of a family of damage-bypass replication proteins which comprises the Escherichia coli proteins UmuC and DinB and the yeast Rev1 protein . We found that all XP-V cells examined carry mutations in their DNA polymerase eta gene . Recombinant human DNA polymerase eta corrects the inability of XP-V cell extracts to carry out DNA replication by bypassing thymine dimers on damaged DNA . Together, these results indicate that DNA polymerase eta could be the XPV gene product. J Cell Biol, 1999 Jun 28, 145(7), 1341 - 54 Large-scale chromatin unfolding and remodeling induced by VP16 acidic activation domain; Tumbar T et al.; Analysis of the relationship between transcriptional activators and chromatin organization has focused largely on lower levels of chromatin structure . Here we describe striking remodeling of large-scale chromatin structure induced by a strong transcriptional activator . A VP16-lac repressor fusion protein targeted the VP16 acidic activation domain to chromosome regions containing lac operator repeats . Targeting was accompanied by increased transcription, localized histone hyperacetylation, and recruitment of at least three different histone acetyltransferases . Observed effects on large-scale chromatin structure included unfolding of a 90-Mbp heterochromatic chromosome arm into an extended 25-40-micrometers chromonema fiber, remodeling of this fiber into a novel subnuclear domain, and propagation of large-scale chromatin unfolding over hundreds of kilobase pairs . These changes in large-scale chromatin structure occurred even with inhibition of ongoing transcription by alpha-amanitin . Our results suggest a functional link between recruitment of the transcriptional machinery and changes in large-scale chromatin structure . Based on the observed long-range propagation of changes in large-scale chromatin structure, we suggest a possible rationale for the observed clustering of housekeeping genes within Mbp-sized chromosome bands. Endocrinology, 1999 Jul, 140(7), 3140 - 6 FKHR binds the insulin response element in the insulin-like growth factor binding protein-1 promoter; Durham SK et al.; The insulin response element (IRE) in the IGFBP-1 promoter, and in other gene promoters, contains a T(A/G)TTT motif essential for insulin inhibition of transcription . Studies presented here test whether FKHR may be the transcription factor that confers insulin inhibition through this IRE motif . Immunoblots using antiserum to the synthetic peptide FKHR413-430, RNase protection, and Northerns blots show that FKHR is expressed in HEP G2 human hepatoma cells . Southwestern blots, electromobility shift assays, and DNase I protection assays show that Escherichia coli-expressed GST-FKHR binds specifically to IREs from the IGFBP-1, PEPCK and TAT genes; however, unlike HNF3beta, another protein proposed to be the insulin regulated factor, GST-FKHR does not bind the insulin unresponsive G/C-A/C mutation of the IGFBP-1 IRE . When HEP G2 cells were cotransfected with FKHR expression vectors and with IGFBP-1 promoter plasmids containing either native or mutant IREs, FKHR expression induced a 5-fold increase in activity of the native IGFBP-1 promoter but no increase in activity of promoter constructs containing insulin unresponsive IRE mutants . These data suggest that FKHR, and/or a related family member, is the important T(G/A)TTT binding protein that confers the inhibitory effect of insulin on gene transcription. Microbiol Immunol, 1999, 43(4), 331 - 7 In vitro assessment of a chemically synthesized Shiga toxin receptor analog attached to chromosorb P (Synsorb Pk) as a specific absorbing agent of Shiga toxin 1 and 2; Takeda T et al.; A synthetic analog of Shiga toxin (Stx) receptor (Synsorb Pk) was quantitatively assessed to determine whether it can protect human renal adenocarcinoma cells (ACHN cells) from the cytotoxicity of Stx1 and Stx2 by coincubation experiments . Coincubation of 100 and 20 ng of Stxl and Stx2 with 50 mg of Synsorb Pk for 1 hr at 37 C in 1 ml of Eagle's Minimum Essential Medium supplemented with 1% (v/v) non-essential amino acid and 10% (v/v) fetal calf serum protected 50% of the cells from the cytotoxic effect . Chromosorb P, an inert matrix control, did not absorb the Stxs at all . Heat-treatment (boiled for 10 min) to Synsorb Pk caused a 50% decrease in Stx2-binding activity, but did not effect the Stx1 binding . Further, Stxs bound to Synsorb Pk could be demonstrated . When 20 mg of Synsorb Pk was coincubated for 30 min at 37 C in 1 ml of phosphate-buffered saline with 1 and 10 ng or more of Stx1 or Stx2, respectively, the toxins could be detected on the surface when the bound toxins on Synsorb Pk were used as the solid phase in enzyme immunoassay . The amount of 100 ng/ml of both Stxl and Stx2 appeared to saturate 20 mg/ml of Synsorb Pk after coincubating for 30 min at 37 C . While assessing the Stxs' binding activity to Synsorb Pk, it was demonstrated that Stxl had a higher affinity to Pk trisaccharide than Stx2 . These observations provide useful information on the effectiveness of Synsorb Pk to trap and eliminate free Stxs produced in the gut of patients infected by Stx-producing Escherichia coli, and to prevent the progression of hemorrhagic colitis to hemolytic uremic syndrome. Respir Physiol, 1999 Apr 1, 115(2), 119 - 33 Models for oxygen sensing in yeast: implications for oxygen-regulated gene expression in higher eucaryotes; Poyton RO; Adaptation to changes in oxygen tension in cells, tissues, and organisms depends on changes in the level of expression of a large and diverse set of proteins . It is likely that most cells and tissues possess an oxygen sensing apparatus and signal transduction pathways for regulating expression of oxygen-responsive genes . Although progress has been made in understanding the transcriptional machinery involved in oxygen-regulated gene expression of eucaryotic genes the underlying mechanism(s) of oxygen sensing and the signaling pathways that connect oxygen sensor(s) to the transcription machinery of eucaryotes are still poorly understood . The yeast Saccharomyces cerevisiae is ideal for addressing these problems . Indeed, it is well-suited for broadly based studies on oxygen sensing at the cellular level because it lends itself well to genetic and biochemical studies and because its genome has been completely sequenced . This review focuses on oxygen-regulated gene expression and current models for oxygen sensing in this yeast and then considers their applicability for understanding oxygen sensing in mammals. Biochimie, 1999 Mar, 81(3), 245 - 53 Pyrophosphate increases the efficiency of enterobactin-dependent iron uptake in Escherichia coli; Perrotte-Piquemal M et al.; Exogenous inorganic pyrophosphate increases the biomass yield of Escherichia coli . In this report, we show that the effect of pyrophosphate is related to iron uptake . We have found that addition of pyrophosphate, ammonium iron (III) citrate or iron (III) chloride, in M63 minimal medium containing 1.7 microM of iron, causes an increase in growth yield . In contrast to iron chloride or ammonium iron (III) citrate, exogenous pyrophosphate is deleterious to strains unable to synthesize enterobactin . Thus the positive effect of pyrophosphate is related to the enterobactin uptake system expressed in a low iron content medium . Pyrophosphate in minimal medium has a repressing effect on the expression of Fur-regulated genes . In iron rich medium where enterobactin synthesis is strongly decreased, addition of pyrophosphate increases expression of Fur-regulated genes . Furthermore, this latter regulatory effect of pyrophosphate in iron-rich medium is enhanced in the absence of enterobactin synthesis . It has also been shown that addition of pyrophosphate protects the cell against the oxidative stress caused by the presence of hydrogen peroxide in an iron-rich containing medium . These results indicate that pyrophosphate acts as an iron-chelating agent, could trigger the enterobactin-dependent iron uptake system and could promote an increased binding of iron to enterobactin. Biochimie, 1999 Mar, 81(3), 197 - 200 Detection of an extended-10 element in the promoter region of the pckA gene encoding phosphoenolpyruvate carboxykinase in Escherichia coli; Prost JF et al.; The regulation of transcription of the pckA gene coding for phosphoenolpyruvate carboxykinase in Escherichia coli was analysed by site-directed mutagenesis of the promoter region and measurement of in vitro transcription initiation . Mutation of the guanine residue at position -14 to either cytosine or thymine was found to result in a drastic decrease of transcription, even in the presence of the natural -35 hexamer TTTCCA that differs by only two nucleotides from the consensus sequence TTGACA . It was concluded that the promoter region of pckA contains an extended -10 module, 5'-TG-3', located one base upstream of the -10 hexamer, which is crucial for transcription. Biol Chem, 1999 May, 380(5), 531 - 40 On the role of symmetrical and asymmetrical chaperonin complexes in assisted protein folding; Hayer-Hartl MK et al.; The cylindrical chaperonin GroEL of E . coli and its ring-shaped cofactor GroES cooperate in mediating the ATP-dependent folding of a wide range of polypeptides in vivo and in vitro . By binding to the ends of the GroEL cylinder, GroES displaces GroEL-bound polypeptide into an enclosed folding cage, thereby preventing protein aggregation during folding . The dynamic interaction of GroEL and GroES is regulated by the GroEL ATPase and involves the formation of asymmetrical GroEL:GroES1 and symmetrical GroEL: GroES2 complexes . The proposed role of the symmetrical complex as a catalytic intermediate of the chaperonin mechanism has been controversial . It has also been suggested that the formation of GroEL:GroES2 complexes allows the folding of two polypeptide molecules per GroEL reaction cycle, one in each ring of GroEL . By making use of a procedure to stabilize chaperonin complexes by rapid crosslinking for subsequent analysis by native PAGE, we have quantified the occurrence of GroEL:GroES1 and GroEL:GroES2 complexes in active refolding reactions under a variety of conditions using mitochondrial malate dehydrogenase (mMDH) as a substrate . Our results show that the symmetrical complexes are neither required for chaperonin function nor does their presence significantly increase the rate of mMDH refolding . In contrast, chaperonin-assisted folding is strictly dependent on the formation of asymmetrical GroEL:GroES1 complexes . These findings support the view that GroEL:GroES2 complexes have no essential role in the chaperonin mechanism. Cold Spring Harb Symp Quant Biol, 1998, 63, 1 - 9 Transcription regulation by repressosome and by RNA polymerase contact; Adhya S et al.; The original model of repression of transcription initiation is steric interference of RNA polymerase binding to a promoter by its repressor protein bound to a DNA site that overlaps the promoter . From the results described here, we propose two other mechanisms of repressor action, both of which involve formation of higher-order DNA-multiprotein complexes . These models also explain the problem of RNA polymerase gaining access to a promoter in the condensed nucleoid in response to an inducing signal to initiate transcription. J Bacteriol, 1999 Jul, 181(13), 3994 - 4003 Type 2 NADH dehydrogenases in the cyanobacterium Synechocystis sp . strain PCC 6803 are involved in regulation rather than respiration; Howitt CA et al.; Analysis of the genome of Synechocystis sp . strain PCC 6803 reveals three open reading frames (slr0851, slr1743, and sll1484) that may code for type 2 NAD(P)H dehydrogenases (NDH-2) . The sequence similarity between the translated open reading frames and NDH-2s from other organisms is low, generally not exceeding 30% identity . However, NAD(P)H and flavin adenine dinucleotide binding motifs are conserved in all three putative NDH-2s in Synechocystis sp . strain PCC 6803 . The three open reading frames were cloned, and deletion constructs were made for each . An expression construct containing one of the three open reading frames, slr1743, was able to functionally complement an Escherichia coli mutant lacking both NDH-1s and NDH-2s . Therefore, slr0851, slr1743, and sll1484 have been designated ndbA, ndbB, and ndbC, respectively . Strains that lacked one or more of the ndb genes were created in wild-type and photosystem (PS) I-less backgrounds . Deletion of ndb genes led to small changes in photoautotrophic growth rates and respiratory activities . Electron transfer rates into the plastoquinone pool in thylakoids in darkness were consistent with the presence of a small amount of NDH-2 activity in thylakoids . No difference was observed between wild-type and the Ndb-less strains in the banding patterns seen on native gels when stained for either NADH or NADPH dehydrogenase activity, indicating that the Ndb proteins do not accumulate to high levels . A striking phenotype of the PS I-less background strains lacking one or more of the NDH-2s is that they were able to grow at high light intensities that were lethal to the control strain but they retained normal PS II activity . We suggest that the Ndb proteins in Synechocystis sp . strain PCC 6803 are redox sensors and that they play a regulatory role responding to the redox state of the plastoquinone pool. J Bacteriol, 1999 Jul, 181(13), 3904 - 11 Effects of ethyl and benzyl analogues of spermine on Escherichia coli peptidyltransferase activity, polyamine transport, and cellular growth; Karahalios P et al.; Various ethyl and benzyl spermine analogues, including the anticancer agent N1,N12-bis(ethyl)spermine, were studied for their ability to affect the growth of cultured Escherichia coli cells, to inhibit {3H}putrescine and {3H}spermine uptake into cells, and to modulate the peptidyltransferase activity (EC 2 . 3 . 2 . 12) . Relative to other cell lines, growth of E . coli was uniquely insensitive to these analogues . Nevertheless, these analogues conferred similar modulation of in vitro protein synthesis and inhibition of {3H}putrescine and {3H}spermine uptake, as is seen in other cell types . Thus, both ethyl and benzyl analogues of spermine not only promote the formation and stabilization of the initiator ribosomal ternary complex, but they also have a sparing effect on the Mg2+ requirements . Also, in a complete cell-free protein-synthesizing system, these analogues at low concentrations stimulated peptide bond formation, whereas at higher concentrations, they inhibited the reaction . The ranking order for stimulation of peptide-bond formation by the analogues was N4,N9-dibenzylspermine > N4, N9-bis(ethyl)spermine congruent with N1-ethylspermine > N1, N12-bis(ethyl)spermine, whereas the order of analogue potency regarding the inhibitory effect was inverted, with inhibition constant values of 10, 3.1, 1.5, and 0.98 microM, respectively . Although the above analogues failed to interact with the putrescine-specific uptake system, they exhibited high affinity for the polyamine uptake system encoded by the potABCD operon . Despite this fact, none of the analogues could be internalized by the polyamine transport system, and therefore they could not influence the intracellular polyamine pools and growth of E . coli cells. Carcinogenesis, 1999 Jul, 20(7), 1351 - 6 Tamoxifen induces G:C-->T:A mutations in the cII gene in the liver of lambda/lacI transgenic rats but not at 5'-CpG-3' dinucleotide sequences as found in the lacI transgene; Davies R et al.; Tamoxifen, a rat liver carcinogen, can induce mutations in the lacI gene in the livers of lambda/lacI transgenic rats . However, the presence of persistent tamoxifen adducts on the liver DNA raises the possibility that some contribution to the mutagenesis from ex vivo mutations during the in vitro lacI assay cannot be ruled out . To address this issue, mutagenesis at the cII gene of the transgenic shuttle vector was determined using a selection based assay which is unaffected by the presence of tamoxifen-DNA adducts . Female lambda/lacI transgenic rats were dosed orally with tamoxifen (20 mg/kg body wt) daily for 6 weeks, causing a 3.2-fold increase in the mutant frequency (MF) in the cII gene compared with that obtained with solvent treated animals . This was similar to the MF found previously at the lacI gene and confirms that tamoxifen is mutagenic in vivo . The major class of mutation induced by tamoxifen in the cII gene was G:C-->T:A transversions as was found previously in the lacI gene . However, in the one unreplicated study of mutations in the p53 gene of liver tumours induced by tamoxifen, no G:C-->T:A transversions were found; possible differences between mutagenesis in normal and tumour tissues are explored . The major proportion of the G:C-->T:A transversions occurred at 5'-CpG-3' dinucleotide (CpG) sites in the lacI gene, but not at such sites in the cII gene . The methylation of CpG sites greatly enhances the targeting of deoxyguanosine by carcinogens, thus this finding might be explained by differences in the methylation patterns at their respective CpG sites; however, nothing is known about the methylation status of either the lacI nor the cII gene in this transgenic rat . This study raises the important issue of which target genes (mammalian or transgenic) should be used as endpoints in mammalian mutagenesis assays. Carcinogenesis, 1999 Jul, 20(7), 1315 - 21 Oxazepam is mutagenic in vivo in Big Blue transgenic mice; Shane BS et al.; Although oxazepam (Serax), a widely used benzodiazepine anxiolytic, does not induce gene mutations in vitro or chromosomal aberrations in vivo, it was found to be a hepatocarcinogen in a 2 year bioassay in B6C3F1 mice . Thus, it was of interest to determine whether this carcinogen is mutagenic in vivo . Male B6C3F1 Big Blue transgenic mice were fed 2500 p.p.m . oxazepam or control diet alone for 180 days and killed on the next day . The mutant frequency (MF) of lacI in control mice was 5.02 +/- 2.4x10(5), whereas the MF in the oxazepam-treated mice was 9.17 +/- 4.82x10(-5), a significant increase (P < 0.05) . Correction of the mutant frequency of lacI from the oxazepam-treated mice for clonality resulted in a decrease in the mean mutant frequency to 8.15 +/- 2 . 54x10(-5) . Although the mutant frequency difference was small, sequencing of a random collection of the mutants from each oxazepam-exposed mouse showed a significant difference (P < 0.015) in the mutation spectrum compared with that from control mice . In the oxazepam-exposed mice, an increase in G:C-->T:A and G:C-->C:G transversions and a concomitant decrease in G:C-->A:T transitions were observed . Clonal expansion of mutations at guanines in 5'-CpG-3' sequencing contexts at three sites was noted . It is postulated that some of the mutations found in the oxazepam-derived spectrum were due to oxidative damage elicited by induction of CYP2B isozymes as the result of chronic oxazepam administration . This study demonstrates that the in vivo Big Blue transgenic rodent mutation assay can detect mutations derived from a carcinogen that did not induce gene mutations in vitro or micronuclei in mouse bone marrow . Moreover, the sequencing of the recovered mutants can distinguish between the mutation spectrum from treated mice compared with that from control mice, thereby confirming the genotoxic consequences. Carcinogenesis, 1999 Jul, 20(7), 1215 - 23 cDNA cloning, expression and activity of a second human aflatoxin B1-metabolizing member of the aldo-keto reductase superfamily, AKR7A3; Knight LP et al.; The aflatoxin B1 (AFB1) aldehyde metabolite of AFB1 may contribute to the cytotoxicity of this hepatocarcinogen via protein adduction . Aflatoxin B1 aldehyde reductases, specifically the NADPH-dependent aldo-keto reductases of rat (AKR7A1) and human (AKR7A2), are known to metabolize the AFB1 dihydrodiol by forming AFB1 dialcohol . Using a rat AKR7A1 cDNA, we isolated and characterized a distinct aldo-keto reductase (AKR7A3) from an adult human liver cDNA library . The deduced amino acid sequence of AKR7A3 shares 80 and 88% identity with rat AKR7A1 and human AKR7A2, respectively . Recombinant rat AKR7A1 and human AKR7A3 were expressed and purified from Escherichia coli as hexa-histidine tagged fusion proteins . These proteins catalyzed the reduction of several model carbonyl-containing substrates . The NADPH-dependent formation of AFB1 dialcohol by recombinant human AKR7A3 was confirmed by liquid chromatography coupled to electrospray ionization mass spectrometry . Rabbit polyclonal antibodies produced using recombinant rat AKR7A1 protein were shown to detect nanogram amounts of rat and human AKR7A protein . The amount of AKR7A-related protein in hepatic cytosols of 1, 2-dithiole-3-thione-treated rats was 18-fold greater than in cytosols from untreated animals . These antibodies detected AKR7A-related protein in normal human liver samples ranging from 0.3 to 0.8 microg/mg cytosolic protein . Northern blot analysis showed varying levels of expression of AKR7A RNA in human liver and in several extrahepatic tissues, with relatively high levels in the stomach, pancreas, kidney and liver . Based on the kinetic parameters determined using recombinant human AKR7A3 and AFB1 dihydrodiol at pH 7.4, the catalytic efficiency of this reaction (k2/K, per M/s) equals or exceeds those reported for other enzymes, for example cytochrome P450s and glutathione S-transferases, known to metabolize AFB1 in vivo . These findings indicate that, depending on the extent of AFB1 dihydrodiol formation, AKR7A may contribute to the protection against AFB1-induced hepatotoxicity. Eur J Immunol, 1999 Jun, 29(6), 1955 - 65 Structure-activity relationships within the N-terminal short consensus repeats (SCR) of human CR1 (C3b/C4b receptor, CD35): SCR 3 plays a critical role in inhibition of the classical and alternative pathways of complement activation; Mossakowska D et al.; Genes coding for between one and four short consensus repeats (SCR) of the N-terminal region of human complement receptor 1 (CR1) were synthesized from oligonucleotides and those encoding SCR(1-2), SCR(1-3), SCR(1-4), SCR3 and SCR(3-4) were expressed as inclusion bodies in Escherichia coli . Following solubilization in urea, the proteins were partially purified and refolded and the activity of each protein was assessed in both classical and alternative pathway complement assays . All fragments showed a varying degree of activity with the general order being SCR(1-3) = SCR(1-4) > SCR(1-2) . Addition of SCR3 to SCR(1-2) significantly improved potency, whereas the addition of SCR4 conferred no additional benefit . This observation, coupled with the ability of the single-domain SCR3 to inhibit classical pathway mediated lysis with an IH50% (inhibition of hemolysis by 50%) of 4.8 microM, demonstrates that SCR3 provides key binding interactions with activated complement components . SCR(1-3) was able to inhibit both classical and alternative pathways of complement activation, showing that the N-terminal SCR of CR1 retain the ability to interact with C3b . Assays for CR1-like cofactor activity for factor I using C4b-like C4 or C3b-like C3 as substrates showed that SCR(1-3) possessed such cofactor activity and that C4b-like C4 was a better substrate . When compared to full-length (30 SCR) soluble CR1 (sCR1), SCR(1-3) was significantly less potent in accord with a model involving multi-valent binding of C3b/C4b to CR1. Mol Microbiol, 1999 Jun, 32(6), 1296 - 304 Antiterminator-dependent modulation of transcription elongation rates by NusB and NusG; Zellars M et al.; Ribosomal RNA is transcribed about twice as fast as messenger RNA in vivo, and this increased transcription rate requires the rrn boxA antitermination system . Because several Nus factors have been implicated in rrn antitermination, we have examined the role of NusB, NusE and NusG in controlling the rate of rrn boxA-mediated transcript elongation . In vivo RNA polymerase transcription rates were determined by measuring the rate of appearance of lacZ transcript using a plasmid that contained an inducible T7 promoter fused to the rrn boxA sequence followed by the lacZ gene . This plasmid was introduced into Escherichia coli mutant strains that can be conditionally depleted of NusG, or that carry a deficient nusB gene or a nusE mutation . We found that, in addition to the rrn boxA antiterminator sequence, both NusG and NusB were required to maintain the high transcription rate . The nusE mutation used in this study may be specific for lambda antitermination, as it did not influence the boxA-mediated increase in transcription rate. Mol Microbiol, 1999 Jun, 32(6), 1226 - 38 Molecular analysis of the cytolytic protein ClyA (SheA) from Escherichia coli; Oscarsson J et al.; Escherichia coli K-12 carries a gene for a protein denoted ClyA or SheA that can mediate a cytolytic phenotype . The ClyA protein is not expressed at detectable levels in most strains of E . coli,