Semin Thromb Hemost, 2001 Dec, 27(6), 619 - 31 Response of anticoagulant pathways in disseminated intravascular coagulation; Taylor FB Jr; This article describes the microvascular endothelium as both a target and a regulator of events after hemostatic and inflammatory stress . The first section describes the four-quadrant hemostatic system as consisting of the two coagulant and anticoagulant domains that control clot formation and the two fibrinolytic and antifibrinolytic domains that control clot removal . This section concentrates on the anticoagulant domain that operates from the microvascular endothelium and protects it from the effects of hemostatic and inflammatory stress . The next two sections present examples of pure hemostatic and inflammatory stress and illustrate how the four functional domains of the hemostatic systems respond as a unit to these stresses . The following section correlates the response of molecular markers that reflect the activity of the hemostatic system with markers of microvascular endothelial injury to increasing sublethal concentrations of Escherichia coli in the baboon model of E . coli sepsis . The final section correlates the response of these same markers to endotoxin in the human model of endotoxemia . Both sections emphasize that the hemostatic and inflammatory stress and injury to the microvascular endothelium occur at surprisingly low concentrations of E . coli and endotoxin, long before there is evidence of fibrinogen consumption and before the other standard criteria for disseminated intravascular coagulation (DIC) are met . We conclude that this represents a compensated response to stress that can be measured and can be designated as nonovert DIC . We further conclude that as bedside assays of these molecular markers become available they should be helpful in diagnosing and staging early nonovert DIC. Pharmacogenetics, 2001 Dec, 11(9), 793 - 801 Effect of two mutations of human CYP1B1, G61E and R469W, on stability and endogenous steroid substrate metabolism; Jansson I et al.; CYP1B1 is linked to normal eye development by the disease phenotype, primary congenital glaucoma (PCG) . CYP1B1 mRNA was expressed in a number of human fetal tissue cDNA libraries, supporting the suggestion of its involvement in tissue development . Highest expression levels were found in thymus and kidney, followed by spleen . A considerably lower level was observed in lung, cardiac and skeletal muscle . No expression was detected in liver or brain . CYP1B1 is able to metabolize steroid hormones . Testosterone was a poor substrate and activity with progesterone was 6-fold higher, but estradiol was the preferred substrate, exhibiting a greater metabolite profile with CYP1B1 than with CYP1A2 . Major metabolites were A-ring hydroxylations (75-80%) . Others were 15alpha-, 6alpha-, 16alpha- and 6beta-hydroxy metabolites . Two CYP1B1 mutations found in families with the PCG phenotype in which incomplete penetrance is seen were expressed in Escherichia coli . G61E, a hinge region mutation, and R469W, a heme region mutation, were shown to code for holoenzymes . G61E had greatly diminished stability, while the R469W holoenzyme, if anything, was stabilized . Both mutants showed compromised catalytic activity . The extents of isomeric site activity diminution were not proportional, resulting in alterations in the metabolite profiles . The results suggest that if a metabolite of CYP1B1 or elimination of a metabolite by CYP1B1 is necessary for normal embryonic or fetal tissue development, the appearance of these two mutations could result in developmental abnormalities . The altered activities of the mutants and ability of CYP1B1 to respond to external challenge may be the basis for the observed incomplete penetrance. Protein Eng, 2001 Oct, 14(10), 775 - 83 Polyionic fusion peptides function as specific dimerization motifs; Richter SA et al.; The de novo design of a molecular adapter for directed association and covalent linkage of two polypeptides is presented . Using peptides containing charged amino acid residues and an additional cysteine residue (AlaCysLys(8) and AlaCysGlu(8)) we demonstrate that the electrostatic interaction promotes the association of two synthetic peptides and, subsequently, disulfide bond formation . The reaction depends on both the redox potential and on the ionic strength of the buffer . Varying the redox potential, the interaction of the peptides was quantified by a Delta G(0') of 6.6 +/- 0.2 kcal/mol . Heterodimerization of the peptides is highly specific, a competition of association by other cysteine containing compounds could not be observed . Two proteins comprising cysteine-containing polyionic fusion peptides, a modified Fab fragment and an alpha-glucosidase fusion, could be specifically conjugated by directed association and subsequent disulfide bond formation . Both proteins retain their functional characteristics within the bifunctional conjugate: enzymatic activity of the alpha-glucosidase and antigen-binding capacity of the Fab fragment are equivalent to the non-conjugated components. Mol Biol Cell, 2001 Dec, 12(12), 3773 - 82 Divergent functional properties of the ribosome-associated molecular chaperone Ssb compared with other Hsp70s; Pfund C et al.; Ssbs of Saccharomyces cerevisiae are ribosome-associated molecular chaperones, which can be cross-linked to nascent polypeptide chains . Because Ssbs are members of a divergent subclass of Hsp70s found thus far only in fungi, we asked if the structural requirements for in vivo function were similar to those of "classic" Hsp70s . An intact peptide-binding domain is essential and an alteration of a conserved residue in the peptide-binding cleft (V442) affects function . However, Ssb tolerates a number of alterations in the peptide-binding cleft, revealing a high degree of flexibility in its functional requirements . Because binding of Ssb to peptide substrates in vitro was undetectable, we assessed the importance of substrate binding using the chimera BAB, in which the peptide binding domain of Ssb is exchanged for the analogous domain of the more "classical" Hsp70, Ssa . BAB, which binds peptide substrates in vitro, can substitute for Ssb in vivo . Alteration of a residue in the peptide-binding cleft of BAB creates a protein with a reduced affinity for peptide and altered ribosome binding that is unable to substitute for Ssb in vivo . These results indicate that Ssb's ability to bind unfolded polypeptides is likely critical for its function . This binding accounts, in part, for its stable interaction with translating ribosomes, even although it has a low affinity for peptides that detectably bind to other Hsp70s in vitro . These unusual properties may allow Ssb to function efficiently as a chaperone for ribosome-bound nascent chains. Microbiology, 2001 Dec, 147(Pt 12), 3353 - 8 Polyhydroxybutyrate biosynthesis in Caulobacter crescentus: molecular characterization of the polyhydroxybutyrate synthase; Qi Q et al.; Caulobacter crescentus was investigated with respect to polyhydroxybutyrate (PHB) biosynthesis . Polyhydroxyalkanoate (PHA) accumulation contributing to approximately 18% of the cell dry weight was obtained in the presence of glucose . Gas chromatography-mass spectrometry and gel permeation chromatography of the purified PHA showed that this polyester was solely composed of 3-hydroxybutyrate and had a weight average molar mass of 5.5 x 10(5) g mol(-1) and a polydispersity of 1.6 . An ORF encoding a conserved, hypothetical protein which shared approximately 47% identity with the PHB synthase from Azorhizobium caulinodans was identified within the complete C . crescentus genomic sequence . This putative C . crescentus PHB synthase gene, phaC, consisted of a 2019 nt stretch of DNA (encoding 673 aa residues), which encoded a PHB synthase with a molecular mass of approximately 73 kDa . This is currently the largest PHA synthase identified . The phaC coding region was subcloned into vector pBBR1-JO2 under lac promoter control . The resulting plasmid, pQQ4, mediated PHB accumulation in the mutant Ralstonia eutropha PHB(-)4 and recombinant Escherichia coli JM109(pBHR69), which produced the beta-ketothiolase and acetoacetyl-CoA reductase from R . eutropha, contributing to approximately 62% and 6% of cell dry weight, respectively . Functional expression of the coding region of phaC was confirmed by immunoblotting and in vitro PHB synthase activity. Microbiology, 2001 Dec, 147(Pt 12), 3345 - 52 Glucose-6-phosphate-dependent phosphoryl flow through the Uhp two-component regulatory system; Verhamme DT et al.; Expression of the UhpT sugar-phosphate transporter in Escherichia coli is regulated at the transcriptional level via the UhpABC signalling cascade . Sensing of extracellular glucose 6-phosphate (G6P), by membrane-bound UhpC, modulates a second membrane-bound protein, UhpB, resulting in autophosphorylation of a conserved histidine residue in the cytoplasmic (transmitter) domain of the latter . Subsequently, this phosphoryl group is transferred to a conserved aspartate residue in the response-regulator UhpA, which then initiates uhpT transcription, via binding to the uhpT promoter region . This study demonstrates the hypothesized transmembrane signal transfer in an ISO membrane set-up, i.e . in a suspension of UhpBC-enriched membrane vesicles, UhpB autophosphorylation is stimulated, in the presence of {gamma-(32)P}ATP, upon intra-vesicular sensing of G6P by UhpC . Subsequently, upon addition of UhpA, very rapid and transient UhpA phosphorylation takes place . When P approximately UhpA is added to G6P-induced UhpBC-enriched membrane vesicles, rapid UhpA dephosphorylation occurs . So, in the G6P-activated state, UhpB phosphatase activity dominates over kinase activity, even in the presence of saturating amounts of G6P . This may imply that maximal in vivo P approximately UhpA levels are low and/or that, to keep sufficient P approximately UhpA accumulated to induce uhpT transcription, the uhpT promoter DNA itself is involved in stabilization/sequestration of P approximately UhpA. Microbiology, 2001 Dec, 147(Pt 12), 3241 - 7 Homogeneous expression of the P(BAD) promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter; Khlebnikov A et al.; Genes placed under the control of the arabinose-inducible araBAD promoter (P(BAD)) of Escherichia coli are expressed in an all-or-none fashion, in which the percentage of induced cells in the population, rather than the degree of induction in individual cells, varies with the concentration of arabinose in the culture medium . Previous work showed that all-or-none gene expression from P(BAD) was due to the arabinose-dependent expression of the gene encoding the low-affinity high-capacity transporter (araE), and that expression of heterologous genes from P(BAD) in individual cells could be regulated by placing the araE gene under control of an arabinose-independent promoter . Based on these results, two expression systems were developed to allow regulatable control of genes under control of P(BAD) . In one system, the native araE promoter on the chromosome was replaced by constitutive promoters of different strengths . In the second system, the araE gene under control of the same constitutive promoters was placed on a medium-copy plasmid . Both systems allow regulatable expression of a plasmid-borne P(BAD)-controlled heterologous gene and a homogeneous population of cells over a wide range of arabinose concentrations . While the degree of induction varied slightly with the strength of the constitutive promoter, expression was affected most by the arabinose concentration. J Virol, 2002 Jan, 76(1), 364 - 78 The interacting UL31 and UL34 gene products of pseudorabies virus are involved in egress from the host-cell nucleus and represent components of primary enveloped but not mature virions; Fuchs W et al.; A 2.6-kbp fragment of the pseudorabies virus (PrV) genome was sequenced and shown to contain the homologues of the highly conserved herpesvirus genes UL31 and UL32 . By use of a monospecific antiserum, the UL31 gene product was identified as a nuclear protein with an apparent molecular mass of 29 kDa . For functional analysis, UL31 was deleted by mutagenesis in Escherichia coli of an infectious full-length clone of the PrV genome . The resulting virus mutants were deficient in plaque formation, and titers were reduced more than 100-fold from those of wild-type PrV . Ultrastructural analyses demonstrated that capsid maturation and DNA packaging were not affected . However, neither budding at the inner nuclear membrane nor cytoplasmic or extracellular virus particles were observed . These replication defects were similar to those of a UL34 deletion mutant (B . G . Klupp, H . Granzow, and T . C . Mettenleiter, J . Virol . 74:10063-10073, 2000) and could be completely repaired in a cell line which constitutively expresses the UL31 protein . Yeast two-hybrid studies revealed that a UL31 fusion protein specifically interacts with plasmids of a PrV genome library expressing the N-terminal part of UL34 . Vice versa, UL34 selected UL31-encoding plasmids from the library . Immunofluorescence studies and immune electron microscopy demonstrated that in cells infected with wild-type PrV, both proteins accumulate at the nuclear membrane, whereas in the absence of UL34 the UL31 protein is dispersed throughout the nucleus . Like the UL34 protein, the UL31 gene product is a component of enveloped virus particles within the perinuclear space and absent from mature virions . Our findings suggest that physical interaction between these two virus proteins might be a prerequisite for primary envelopment of PrV at the inner nuclear membrane and that this envelope is removed by fusion with the outer nuclear membrane. J Virol, 2002 Jan, 76(1), 338 - 45 Biochemical characterization of Junonia coenia densovirus nonstructural protein NS-1; Ding C et al.; Junonia coenia densovirus (JcDNV) is an autonomous parvovirus that infects the larvae of the common buckeye butterfly, Junonia coenia . Unlike vertebrate parvoviruses, the genes encoding the structural protein and nonstructural (NS) proteins of JcDNV are in opposite orientations; thus, each strand contains a sense and antisense open reading frame (ORF) . The promoter at map position 93 controls expression of NS ORFs 2, 3, and 4, which encode three NS proteins, NS-1, NS-2, and NS-3 . These proteins are likely to be involved in viral DNA replication, among other functions . In contrast to the nonstructural proteins of the vertebrate parvoviruses, the NS proteins of the Densovirinae have not been characterized . Here, we describe biochemical properties of the NS-1 protein of JcDNV . The NS-1 ORF was cloned in frame with the Escherichia coli malE gene, which encodes the bacterial maltose binding protein (MBP) . Using electrophoretic mobility shift and DNase I protection assays, we identified the region of the JcDNV terminal sequence that is recognized specifically by the MBP-NS-1 fusion protein . The site consists of (GAC)4 and is located on the A-A' region of the terminal palindrome . In addition, the MBP-NS-1 fusion protein catalyzes the cleavage of single-stranded DNA (ssDNA) substrates derived from the JcDNV putative origin of replication, primarily at two sites in the motif 5'-G*TAT*TG-3' . One cleavage site is between the thymidine dinucleotide at positions 92 and 93 and the other site corresponds to thymidine at nucleotide 95; both sites are on the complementary strand of the sequence assigned GenBank accession number A12984 . Cleavage of ssDNA is dependent on the presence of a divalent metal cofactor but does not require nucleoside triphosphate hydrolysis . Parvovirus NS proteins contain the phylogenically conserved Walker A- and B-site ATPase motifs . These sites in JcDNV NS-1 diverge from the consensus, yet despite these atypical motifs our analyses support that MBP-NS-1 has ATP-dependent helicase activity . These results indicate that JcDNV NS-1 possesses activities common to the superfamily of rolling-circle replication initiator proteins in general and the parvovirus replication proteins in particular, and they provide a basis for comparative analyses of the structure and function relationships among the parvovirus NS-1 equivalents. J Virol, 2002 Jan, 76(1), 58 - 67 Woodchuck gamma interferon upregulates major histocompatibility complex class I transcription but is unable to deplete woodchuck hepatitis virus replication intermediates and RNAs in persistently infected woodchuck primary hepatocytes; Lu M et al.; Gamma interferon (IFN-gamma) is an important mediator with multiple functions in the host defense against viral infection . IFN-gamma, in concert with tumor necrosis factor alpha (TNF-alpha), leads to a remarkable reduction of intrahepatic replication intermediates and specific mRNAs of hepatitis B virus (HBV) by a noncytolytic mechanism in the transgenic mouse model . Thus, it is rational to evaluate the potential value of IFN-gamma for the treatment of chronic HBV infection . In the present study, we expressed recombinant woodchuck IFN-gamma (wIFN-gamma) in Escherichia coli and mammalian cells . wIFN-gamma protected woodchuck cells against infection of murine encephalomyocarditis virus in a species-specific manner . It upregulated the mRNA level of the woodchuck major histocompatibility complex class I (MHC-I) heavy chain in permanent woodchuck WH12/6 cells and regulated differentially the gene expression . However, the level of the replication intermediates and specific RNAs of woodchuck hepatitis virus (WHV) in persistently WHV-infected primary woodchuck hepatocytes did not change despite a treatment with 1,000 U of wIFN-gamma per ml or with a combination of wIFN-gamma and woodchuck TNF-alpha . Rather, hepatocytes derived from chronic carriers had an elevated level of the MHC-I heavy-chain mRNAs, most probably due to the exposure to inflammatory cytokines in vivo . Treatment with high doses of wIFN-gamma led to an abnormal cell morphology and loss of hepatocytes . Thus, wIFN-gamma regulates the gene expression in woodchuck hepatocytes but could not deplete WHV replication intermediates and mRNAs in persistently infected hepatocytes . The cellular response to wIFN-gamma may be changed in hepatocytes from chronically WHV-infected woodchucks . It should be clarified in the future whether the continuous exposure of hepatocytes to inflammatory cytokines or the presence of viral proteins leads to changes of the cellular response to wIFN-gamma. J Cell Sci, 2001 Nov, 114(Pt 22), 4025 - 31 Role of lipid rafts in Shiga toxin 1 interaction with the apical surface of Caco-2 cells; Kovbasnjuk O et al.; Enterohemorrhagic Escherichia coli producing Shiga toxins 1 and/or 2 have become major foodborne pathogens . The specific binding of Shiga toxin 1 B-subunit to its receptor, a neutral glycolipid globotriaosylceramide Gb(3), on the apical surface of colonic epithelium followed by toxin entry into cells are the initial steps of the process, which can result in toxin transcytosis and systemic effects of infection including hemolytic uremic syndrome . Understanding the complex mechanisms of Shiga toxin 1 binding and internalization may help to develop new strategies directed at preventing toxin internalization . Fluorescence resonance energy transfer microscopy revealed the clustering of Shiga toxin receptors Gb(3) in lipid rafts with another glycosphingolipid G(M1) on the apical surface of highly polarized intestinal epithelial Caco-2 cells . Lipid rafts disruption significantly decreased internalization of Shiga toxin 1 B-subunit . Although disruption of lipid rafts by cholesterol depletion did not affect the amount of bound Shiga toxin 1 B-subunit, lipid rafts are necessary for toxin uptake across the apical membrane of Caco-2 cells. J Biol Chem, 2002 Feb 15, 277(7), 5024 - 9 Epub 2001 Dec 05. Corneodesmosin, a component of epidermal corneocyte desmosomes, displays homophilic adhesive properties; Jonca N et al.; Corneodesmosomes, the modified desmosomes of the uppermost layers of the epidermis, play an important role in corneocyte cohesion . Corneodesmosin is a secreted glycoprotein located in the corneodesmosomal core and covalently linked to the cornified envelope of corneocytes . Its glycine- and serine-rich NH(2)-terminal domain may fold to give structural motifs similar to the glycine loops described in epidermal cytokeratins and loricrin and proposed to display adhesive properties . A chimeric protein comprising human corneodesmosin linked to the transmembrane and cytoplasmic domains of mouse E-cadherin was expressed in mouse fibroblasts to test the ability of corneodesmosin to promote cell-cell adhesion . Classic aggregation assays indicated that corneodesmosin mediates homophilic cell aggregation . Moreover, Ca(2+) depletion showed a moderate effect on aggregation . To assess the involvement of the glycine loop domain in adhesion, full-length corneodesmosin, corneodesmosin lacking this domain, or this domain alone were expressed as glutathione S-transferase fusion proteins and tested for protein-protein interactions by overlay binding assays . The results confirmed that corneodesmosin presents homophilic interactions and indicated that its NH(2)-terminal glycine loop domain is sufficient but not strictly necessary to promote binding . Altogether, these results provide the first experimental evidence for the adhesive properties of corneodesmosin and for the involvement of its glycine loop domain in adhesion. Blood, 2001 Dec 15, 98(13), 3817 - 22 Correction of cross-linker sensitivity of Fanconi anemia group F cells by CD33-mediated protein transfer; Holmes RK et al.; Studies have previously described the feasibility of receptor-mediated protein transfer in a cell culture model of Fanconi anemia (FA) group C . This study explores the versatility of this approach by using an antibody single-chain fusion protein to correct the phenotypic defect in FA group F cells . A 68.5-kd chimeric protein (His-M195FANCF) was expressed, consisting of a His tag, a single-chain antibody to the myeloid antigen CD33, and the FANCF protein, as well as a 43-kd His-FANCF fusion protein lacking the antibody motif, in Escherichia coli . The nickel-agarose-purified His-M195FANCF protein bound specifically to the surface of HeLa cells transfected with CD33 and internalized through vesicular structures . The fusion protein, but not CD33, sorted to the nucleus, consistent with the known nuclear localization of FANCF . No similar binding or internalization was observed with His-FANCF . Pretreatment of the transfected cells with chloroquine abolished nuclear accumulation, but there was little change with brefeldin A, indicating a minimal if any role for the Golgi apparatus in mediating transport from endosomes to the cytosol and the nucleus . The intracellular half-life of His-M195FANCF was approximately 160 minutes . Treatment of CD33-transfected FA group F lymphoblastoid cells with 0.1 mg/mL His-M195FANCF conferred resistance to mitomycin C . No similar protection was noted in CD33(-) parental cells or CD33(+) FA cells belonging to groups A and C . These results demonstrate that antibody-directed, receptor-mediated protein transfer is a versatile method for the delivery of biologically active proteins into hematopoietic cells. Mutat Res, 2001 Dec 19, 487(3-4), 173 - 90 Excision of uracil from DNA by the hyperthermophilic Afung protein is dependent on the opposite base and stimulated by heat-induced transition to a more open structure; Knaevelsrud I et al.; Hydrolytic deamination of DNA-cytosines into uracils is a major source of spontaneously induced mutations, and at elevated temperatures the rate of cytosine deamination is increased . Uracil lesions are repaired by the base excision repair pathway, which is initiated by a specific uracil DNA glycosylase enzyme (UDG) . The hyperthermophilic archaeon Archaeoglobus fulgidus contains a recently characterized novel type of UDG (Afung), and in this paper we describe the over-expression of the afung gene and characterization of the encoded protein . Fluorescence and activity measurements following incubation at different temperatures may suggest the following model describing structure-activity relationships: At temperatures from 20 to 50 degrees C Afung exists as a compact protein exhibiting low enzyme activity, whereas at temperatures above 50 degrees C, the Afung conformation opens up, which is associated with the acquisition of high enzyme activity . The enzyme exhibits opposite base-dependent excision of uracil in the following order: U>U:T>U:C>>U:G>>U:A . Afung is product-inhibited by uracil and shows a pronounced inhibition by p-hydroxymercuribenzoate, indicating a cysteine residue essential for enzyme function . The Afung protein was estimated to be present in A . fulgidus at a concentration of approximately 1000 molecules per cell . Kinetic parameters determined for Afung suggest a significantly lower level of enzymatic uracil release in A . fulgidus as compared to the mesophilic Escherichia coli. Mutat Res, 2001 Dec 19, 487(3-4), 149 - 56 Amplified UvrA protein can ameliorate the ultraviolet sensitivity of an Escherichia coli recA mutant; Kiyosawa K et al.; When a recA strain of Escherichia coli was transformed with the multicopy plasmid pSF11 carrying the uvrA gene of E . coli, its extreme ultraviolet (UV) sensitivity was decreased . The sensitivity of the lexA1 (Ind(-)) strain to UV was also decreased by pSF11 . The recA cells expressing Neurospora crassa UV damage endonuclease (UVDE), encoding UV-endonuclease, show UV resistance . On the other hand, only partial amelioration of UV sensitivity of the recA strain was observed in the presence of the plasmid pNP10 carrying the uvrB gene . Host cell reactivation of UV-irradiated lambda phage in recA cells with pSF11 was as efficient as that in wild-type cells . Using an antibody to detect cyclobutane pyrimidine dimers, we found that UV-irradiated recA cells removed dimers from their DNA more rapidly if they carried pSF11 than if they carried a vacant control plasmid . Using anti-UvrA antibody, we observed that the expression level of UvrA protein was about 20-fold higher in the recA strain with pSF11 than in the recA strain without pSF11 . Our results were consistent with the idea that constitutive level of UvrA protein in the recA cells results in constitutive levels of active UvrABC nuclease which is not enough to operate full nucleotide excision repair (NER), thus leading to extreme UV sensitivity. Mutat Res, 2001 Dec 19, 487(3-4), 109 - 19 A new assay to quantify in vivo repair of G:T mispairs by base excision repair; Waters SB et al.; The double mismatch reversion (DMR) assay quantifies the repair of G:T mispairs exclusively by base excision repair in vivo . Synthetic oligonucleotides containing two G:T mispairs on opposite strands were placed into the suppressor tRNA gene supF in the shuttle plasmid pDMR . Placement of two mispairs on opposite strands of supF creates a one to one correspondence between the number of correct repair events prior to replication in which G:T mispairs are converted to G:C base pairs and the number of post-replication progeny plasmids with functional supF . Replication of unrepaired or incorrectly repaired mispairs cannot produce progeny plasmids containing functional supF . Indeed, direct transformation of Escherichia coli strain MBL50, which reports the functional status of supF, with pDMR constructs containing two G:T or G:G mispairs yielded <0.5% wild-type supF-containing colonies . In contrast, passage of G:T mispair-containing pDMR constructs through human 5637 bladder carcinoma cells for 48h prior to plasmid recovery and transformation of the reporter E . coli strain MBL50 produced 47% wild-type supF-containing colonies . This finding was indicative of repair prior to the onset of replication in 5637 cells . However, passage of G:G mispair-containing pDMR constructs through 5637 cells yielded <0.5% wild-type supF-containing colonies . Moreover, no difference was observed in the rate of G:T mispair repair by HCT 116 colorectal carcinoma cells deficient in long-patch mismatch repair and a long-patch mismatch repair proficient HCT 116 subline . These data demonstrate that repair measured by the DMR assay is exclusively attributable to short-patch pathways . The DMR assay proved useful in the analysis of the effect of the base 5' to a mispaired G on the rate of G:T base excision repair by 5637 cells, indicating the sequence preference CpG approximately 5mCpG>TpG>GpG approximately ApG, and in the comparison of G:T base excision repair rates between cell lines. Gene, 2001 Dec 12, 280(1-2), 153 - 62 A nonspecific nucleoside hydrolase from Leishmania donovani: implications for purine salvage by the parasite; Cui L et al.; In contrast to their mammalian hosts, protozoan parasites do not synthesize purines de novo, but depend on preformed nucleotides that they purportedly obtain by salvage pathways . Nucleoside hydrolases may play a crucial role in that salvage process . By screening Leishmania donovani libraries with polyclonal antibodies against promastigote soluble exo-antigens, we have identified a cDNA encoding a protein with significant homology to nonspecific and uridine-inosine-preferring nucleoside hydrolases . Sequence comparison demonstrated that all the residues involved in Ca(2+)-binding and substrate recognition in the active site are conserved among the characterized protozoan nucleoside hydrolases . Genomic analysis suggests that it is a single copy gene in L . donovani, and its homologues are present in members representing other Leishmania species complexes . Both Northern blot and immunoblot analyses indicate that it is constitutively expressed in L . donovani promastigotes . The recombinant enzyme overexpressed in and purified from bacteria showed significant activity with all naturally occurring purine and pyrimidine nucleosides, and efficient utilization of p-nitrophenyl-beta-D-ribofuranoside as a substrate . Altogether, the sequence comparison and substrate specificity data identify this L . donovani nucleoside hydrolase as a nonspecific nucleoside hydrolase . Further, the nucleoside hydrolase was localized to specific foci in L . donovani promastigotes by immunofluorescent assays . Although the conservation of the nucleoside hydrolases among protozoan parasites offers promise for the design of broad-spectrum anti-parasitic drugs, the existence of multiple and distinct nucleoside hydrolases in a single species demands special consideration. Gene, 2001 Dec 12, 280(1-2), 145 - 51 An in vitro transposon system for highly regulated gene expression: construction of Escherichia coli strains with arabinose-dependent growth at low temperatures; Grant AJ et al.; Placing a gene of interest under the control of an inducible promoter greatly aids the purification, localization and functional analysis of proteins but usually requires the sub-cloning of the gene of interest into an appropriate expression vector . Here, we describe an alternative approach employing in vitro transposition of Tn Omega P(BAD) to place the highly regulable, arabinose inducible P(BAD) promoter upstream of the gene to be expressed . The method is rapid, simple and facilitates the optimization of expression by producing constructs with variable distances between the P(BAD) promoter and the gene . To illustrate the use of this approach, we describe the construction of a strain of Escherichia coli in which growth at low temperatures on solid media is dependent on threshold levels of arabinose . Other uses of the transposable promoter are also discussed. Vaccine, 2001 Dec 12, 20(5-6), 934 - 42 Use of fusion protein constructs to generate potent immunotherapy and protection against scorpion toxins; Legros C et al.; We report the use of recombinant scorpion toxins in the form of fusion proteins as antigens for immunisation in rabbits and mice: the aim was to produce in these animal models protective antisera against the most lethal alpha-type toxins in the venom from the North African scorpion Androctonus australis . The cDNAs encoding AaH I, AaH II and AaH III (the three major alpha-type toxins acting on voltage-sensitive sodium channels) were fused to the sequence encoding the maltose binding protein (MBP) . The constructs (MBP-AaH I, MBP-AaH II, MBP-AaH I+II and MBP-AaH III) were expressed in Escherichia coli, and resulting fusion proteins were translocated to the periplasmic space . The recombinant fusion proteins were characterised and used as antigens to generate antibodies in rabbits . These antibodies raised specifically recognised their corresponding radiolabelled-toxin with affinities in the 0.1nM range . In vitro neutralisation assays indicated that 1ml of serum raised against a mixture of fusion proteins was able to neutralise 15 LD(50) of the toxic fraction (AaH-G50) purified from the crude venom by molecular filtration through Sephadex G50 . In vivo, the fusion proteins induced a long-term protection in mice against the lethal effects of AaH-G50 or of the native toxins . Ten weeks after the beginning of the immunisation programme, mice were challenged with various toxins or AaH-G50 doses . Mice were fully protected against three LD(50) of AaH-G50 . Our work shows that fusion protein constructs can be used as a vaccine providing efficient immune protection against A . australis venom. Bioorg Med Chem, 2002 Jan, 10(1), 97 - 101 Rapid photolytic release of cytidine 5'-diphosphate from a coumarin derivative: a new tool for the investigation of ribonucleotide reductases; Schonleber RO et al.; In order to study the long-range radical transfer in the Escherichia coli ribonucleotide reductase (RNR), caged cytidine 5'-diphosphate (CDP) 1 was synthesized, which contains the photolabile (7-diethylaminocoumarin-4-yl)methyl moiety . The caged CDP 1 triggers the release of CDP when irradiated at wavelengths between 365 and 436 nm . The rate constant of the formation of alcohol 2 and cytidine 5'-diphosphate 3 is 2x10(8) s(-1) and the quantum efficiency for the disappearance of caged CDP 1 is 2.9%. Biochim Biophys Acta, 2001 Nov 26, 1550(1), 27 - 36 Cloning, characterisation and crystallisation of a diadenosine 5',5"'-P(1),P(4)-tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans; Abdelghany HM et al.; Asymmetrically cleaving diadenosine 5',5"'-P(1),P(4)-tetraphosphate (Ap4A) hydrolase activity has been detected in extracts of adult Caenorhabditis elegans and the corresponding cDNA amplified and expressed in Escherichia coli . As expected, sequence analysis shows the enzyme to be a member of the Nudix hydrolase family . The purified recombinant enzyme behaves as a typical animal Ap4A hydrolase . It hydrolyses Ap4A with a K(m) of 7 microM and k(cat) of 27 s(-1) producing AMP and ATP as products . It is also active towards other adenosine and diadenosine polyphosphates with four or more phosphate groups, but not diadenosine triphosphate, always generating ATP as one of the products . It is inhibited non-competitively by fluoride (K(i)=25 microM) and competitively by adenosine 5'-tetraphosphate with Ap4A as substrate (K(i)=10 nM) . Crystals of diffraction quality with the morphology of rectangular plates were readily obtained and preliminary data collected . These crystals diffract to a minimum d-spacing of 2 A and belong to either space group C222 or C222(1) . Phylogenetic analysis of known and putative Ap4A hydrolases of the Nudix family suggests that they fall into two groups comprising plant and Proteobacterial enzymes on the one hand and animal and archaeal enzymes on the other . Complete structural determination of the C . elegans Ap4A hydrolase will help determine the basis of this grouping. Biochim Biophys Acta, 2001 Nov 26, 1550(1), 1 - 5 Kinetic constants of signal peptidase I using cytochrome b5 as a precursor substrate; Gallagher J et al.; A procedure is described for measuring Escherichia coli signal peptidase I activity which exploits an intact precursor protein composed of the alkaline phosphatase signal peptide fused to the full length mammalian cytochrome b5 . This cytochrome b5 precursor protein has been extensively characterised and shown to be processed accurately by purified signal peptidase I {Protein Expr . Purif . 7 (1996) 237} . The amphipathic, chimaeric cytochrome b5 precursor was isolated in mg quantities in a highly homogeneous state under non-denaturing conditions . The processing of the cytochrome b5 precursor by signal peptidase displayed Michaelis-Menten kinetics with K(m)=50 microM and k(cat)=11 s(-1) . The K(m) was 20-fold lower than that obtained with signal peptide substrates and 3-fold higher than that reported for pro-OmpA-nuclease A precursor fusion . The corresponding turnover number, k(cat), was four orders of magnitude greater than the peptide substrates but was 2-fold lower than pro-OmpA-nuclease A precursor fusion . These results confirm that both the affinities and the catalytic power of the signal peptidase are significantly higher for macromolecular precursor substrates than for the shorter signal peptide substrates. Structure (Camb), 2001 Dec, 9(12), 1225 - 36 Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage; Blaszczyk J et al.; BACKGROUND: Aquifex aeolicus Ribonuclease III (Aa-RNase III) belongs to the family of Mg(2+)-dependent endonucleases that show specificity for double-stranded RNA (dsRNA) . RNase III is conserved in all known bacteria and eukaryotes and has 1-2 copies of a 9-residue consensus sequence, known as the RNase III signature motif . The bacterial RNase III proteins are the simplest, consisting of two domains: an N-terminal endonuclease domain, followed by a double-stranded RNA binding domain (dsRBD) . The three-dimensional structure of the dsRBD in Escherichia coli RNase III has been elucidated; no structural information is available for the endonuclease domain of any RNase III . RESULTS: We present the crystal structures of the Aa-RNase III endonuclease domain in its ligand-free form and in complex with Mn(2+) . The structures reveal a novel protein fold and suggest a mechanism for dsRNA cleavage . On the basis of structural, genetic, and biological data, we have constructed a hypothetical model of Aa-RNase III in complex with dsRNA and Mg(2+) ion, which provides the first glimpse of RNase III in action . CONCLUSIONS: The functional Aa-RNase III dimer is formed via mainly hydrophobic interactions, including a "ball-and-socket" junction that ensures accurate alignment of the two monomers . The fold of the polypeptide chain and its dimerization create a valley with two compound active centers at each end of the valley . The valley can accommodate a dsRNA substrate . Mn(2+) binding has significant impact on crystal packing, intermolecular interactions, thermal stability, and the formation of two RNA-cutting sites within each compound active center. Structure (Camb), 2001 Dec, 9(12), 1191 - 9 Specificity in Trk receptor:neurotrophin interactions: the crystal structure of TrkB-d5 in complex with neurotrophin-4/5; Banfield MJ et al.; BACKGROUND: The binding of neurotrophin ligands to their respective Trk cellular receptors initiates intracellular signals essential for the growth and survival of neurons . The site of neurotrophin binding has been located to the fifth extracellular domain of the Trk receptor, with this region regulating both the affinity and specificity of Trk receptor:neurotrophin interaction . Neurotrophin function has been implicated in a number of neurological disorders, including Alzheimer's disease and Parkinson's disease . RESULTS: We have determined the 2.7 A crystal structure of neurotrophin-4/5 bound to the neurotrophin binding domain of its high-affinity receptor TrkB (TrkB-d5) . As previously seen in the interaction of nerve growth factor with TrkA, neurotrophin-4/5 forms a crosslink between two spatially distant receptor molecules . The contacts formed in the TrkB-d5:neurotrophin-4/5 complex can be divided into a conserved area similar to a region observed in the TrkA-d5:NGF complex and a second site-unique in each ligand-receptor pair-formed primarily by the ordering of the neurotrophin N terminus . CONCLUSIONS: Together, the structures of the TrkB-d5:NT-4/5 and TrkA-d5:NGF complexes confirm a consistent pattern of recognition in Trk receptor:neurotrophin complex formation . In both cases, the N terminus of the neurotrophin becomes ordered only on complex formation . This ordering appears to be directed largely by the receptor surface, with the resulting complementary surfaces providing the main determinant of receptor specificity . These features provide an explanation both for the limited crossreactivity observed between the range of neurotrophins and Trk receptors and for the high-affinity binding associated with respective ligand-receptor pairs. Plant J, 2001 Nov, 28(4), 455 - 64 Involvement of TAAAG elements suggests a role for Dof transcription factors in guard cell-specific gene expression; Plesch G et al.; Due to their unique structure and function, guard cells have attracted much attention at the physiological level . Very little, however, is known about the molecular events involved in the determination and maintenance of guard cell specificity . The KST1 gene encodes a K+ influx channel of guard cells in potato, and was therefore chosen as a model to study regulation of guard cell-specific gene expression . Transgenic potato plants carrying a fusion between the KST1 promoter and the E . coli uidA (beta-glucuronidase) reporter gene revealed promoter activity in guard cells and in flowers . A detailed dissection of the KST1 promoter led to the discovery of two independent small TATA box-proximal regulatory units, each of which was sufficient to direct guard cell-specific gene transcription . Both fragments contain the sequence motif, 5'-TAAAG-3', which is related to known target sites for a novel class of zinc finger transcription factors, called Dof proteins . Block mutagenesis of these Dof target sites in the context of different promoter constructs dramatically reduced guard cell promoter activity . A Dof gene, StDof1, was cloned and shown to be expressed in epidermal fragments highly enriched for guard cells . In gel retardation experiments, the StDof1 protein interacted in a sequence-specific manner with a KST1 promoter fragment containing the TAAAG motif . These results provide evidence that TAAAG elements are target sites for trans-acting Dof proteins controlling guard cell-specific gene expression . Our data will add to the design of tailor-made guard cell promoters as a further tool in molecular engineering of guard cell function and, hence, control of stomatal carbon dioxide (CO2) uptake and water loss in crop plants. Mol Microbiol, 2001 Nov, 42(4), 1065 - 73 Novel deoxynucleoside-phosphorylating enzymes in mycoplasmas: evidence for efficient utilization of deoxynucleosides; Wang L et al.; Mycoplasmas are unable to synthesize purine and pyrimidine bases de novo . Therefore, salvage of existing nucleosides and bases is essential for their survival . Four mycoplasma species were studied with regard to their ability to phosphorylate deoxynucleosides . High levels of thymidine kinase (TK), deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK) and deoxyadenosine kinase (dAK) activities were detected in extracts from Mycoplasma pneumoniae, Mycoplasma mycoides subsp . mycoides SC (M . mymySC), Acholeplasma laidlawii (A . laidlawii) and Mycoplasma arginini (M . arginini) . Nucleoside phosphotransferase activities were found at high levels in A . laidlawii and low levels in M . arginini . Pyrophosphate-dependent deoxynucleoside kinase activities were detected mainly in A . laidlawii and M . mymySC extracts . Two open reading frames were identified in the M . mymySC genome; one showed 25% sequence identity to human dGK and the other one had about 26% sequence identity to human TK1 . The M . mymySC dGK-like enzyme was cloned, expressed in Escherichia coli and affinity-purified . This enzyme phosphorylated dAdo, dGuo and dCyd, and the highest catalytic rate was with dAdo as substrate . Therefore, we suggest that this enzyme should be named deoxyadenosine kinase . The physiological role of mycoplasma dAK and TK may be to support the unusually large dATP and dTTP pools required for replication of mycoplasma genomes. Mol Microbiol, 2001 Nov, 42(4), 939 - 54 Promoter recognition and discrimination by EsigmaS RNA polymerase; Gaal T et al.; Although more than 30 Escherichia coli promoters utilize the RNA polymerase holoenzyme containing sigmaS (EsigmaS), and it is known that there is some overlap between the promoters recognized by EsigmaS and by the major E . coli holoenzyme (Esigma70), the sequence elements responsible for promoter recognition by EsigmaS are not well understood . To define the DNA sequences recognized best by EsigmaS in vitro, we started with random DNA and enriched for EsigmaS promoter sequences by multiple cycles of binding and selection . Surprisingly, the sequences selected by EsigmaS contained the known consensus elements (-10 and -35 hexamers) for recognition by Esigma70 . Using genetic and biochemical approaches, we show that EsigmaS and Esigma70 do not achieve specificity through 'best fit' to different consensus promoter hexamers, the way that other forms of holoenzyme limit transcription to discrete sets of promoters . Rather, we suggest that EsigmaS-specific promoters have sequences that differ significantly from the consensus in at least one of the recognition hexamers, and that promoter discrimination against Esigma70 is achieved, at least in part, by the two enzymes tolerating different deviations from consensus . DNA recognition by EsigmaS versus Esigma70 thus presents an alternative solution to the problem of promoter selectivity. Mol Microbiol, 2001 Nov, 42(4), 903 - 17 Requirement for the molecular adapter function of StpA at the Escherichia coli bgl promoter depends upon the level of truncated H-NS protein; Free A et al.; Truncated derivatives of the Escherichia coli nucleoid-associated protein H-NS that lack the DNA-binding domain remain competent for silencing of the cryptic bgl operon in vivo . Previous studies have provided evidence for the involvement of either the homologous nucleoid protein StpA or the alternative sigma factor RpoS in this unusual silencing mechanism . Here, we rationalize this apparent discrepancy . We show that two hns alleles (hns-205::Tn10 and hns60), which produce virtually identical amino-terminal fragments of H-NS, have very different requirements for StpA to mediate bgl silencing . The hns60 allele produces a high level of truncated H-NS, which can overcome the absence of StpA, whereas the lower level expressed by hns-205::Tn10 requires StpA for silencing . Reversing the relative levels of the two H-NS fragments reverses their requirement for StpA to silence bgl transcription . This suggests that the amino-terminal fragment of H-NS can be targeted to DNA to mediate silencing by multiple protein-protein interactions . The high-specificity interaction with StpA can function at low levels of truncated H-NS, whereas an alternative mechanism, perhaps involving lower specificity interactions with another protein(s), is only functional when truncated H-NS is abundant . These findings have important implications for the involvement of other proteins in H-NS-dependent transcriptional repression. Mol Microbiol, 2001 Nov, 42(4), 887 - 901 The basis of asymmetry in IS2 transposition; Lewis LA et al.; In the first step of IS2 transposition, the formation of an IS2 minicircle, the roles of the two IS ends differ . Terminal cleavage initiates exclusively at the right inverted repeat (IRR) - the donor end - whereas IRL is always the target . At the resulting minicircle junction, the two abutted ends are separated by a spacer of 1 or 2 basepairs . In this study, we have identified the determinants of donor and target function . The inability of IRL to act as a donor results largely from two sequence differences between IRL and IRR - an extra basepair between the conserved transposase binding sequences and the end of the element, and a change of the terminal dinucleotide from CA-3' to TA-3' . These two changes also impose a characteristic size on the minicircle junction spacer . The only sequences required for the efficient target function of IRL appear to be contained within the segment from position 11-42 . Although IRR can function as a target, its shorter length and additional contacts with transposase (positions 1-7) result in minicircles with longer, and inappropriate, spacers . We propose a model for the synaptic complex in which the terminus of IRL makes different contacts with the transposase for the initial and final strand transfer steps . The sequence differences between IRR and IRL, and the behavioural characteristics of IRL that result from them, have probably been selected because they optimize expression of transposase from the minicircle junction promoter, Pjunc. Kidney Int, 2001 Dec, 60(6), 2299 - 310 Renal cortical cholesterol accumulation is an integral component of the systemic stress response; Zager RA et al.; BACKGROUND: Direct tubular injury (such as ischemia or myohemoglobinuria) increases renal cortical cholesterol content . This study explored whether systemic forms of stress (such as heat shock or sepsis) can trigger renal cholesterol accumulation, and if so, whether increased 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGCR) expression might be involved . METHODS: Male CD-1 mice were subjected to glycerol-induced myohemoglobinuria (MH), systemic heat shock (HS), or E . coli sepsis . Free cholesterol (FC), cholesteryl esters (CE), and HMGCR (Western blot) levels were assessed 18 hours later . Statin effects on renal cholesterol levels and on the severity of MH-acute renal failure (ARF) were also determined . RESULTS: Sepsis and HS each induced dramatic FC and CE increments, comparable to those observed with myohemoglobinuria, and without inducing acute tubular necrosis (ATN) . Part of the cholesterol increments was localized within plasma membrane (detergent resistant) microdomains (for example, rafts/caveolae) . HS and MH each increased renal HMGCR, as well as HS protein (HSP-72) expression . Oxidant stress (Fe) imposed on cultured proximal tubule (HK-2) cells also enhanced HMGCR content . Conversely, sepsis did not raise renal HMGCR or HSP-72 levels . Statin therapy decreased the severity of MH-ARF and renal cholesterol content . However, this appeared to arise from a statin-mediated decrease in glycerol-induced extrarenal tissue damage (myolysis/LDH release) . CONCLUSIONS: Cholesterol appears to be a renal 'acute phase reactant' with tissue levels increasing with either systemic stress (such as, heat shock, sepsis), or direct tissue damage (such as ATN) . Increased HMGCR expression can contribute to this result . Mechanisms other than HMGCR induction also can mediate stress-induced cholesterol increments (for example, in the case of sepsis), and statins can mitigate MH-ARF . However, systemic anti-inflammatory effects, rather than a primary renal action, appear more likely to be involved. Eur J Biochem, 2001 Dec, 268(24), 6607 - 15 Structure-function analysis of CYP27B1 and CYP27A1 . Studies on mutants from patients with vitamin D-dependent rickets type I (VDDR-I) and cerebrotendinous xanthomatosis (CTX); Sawada N et al.; We have determined eight types of missense mutants of CYP27B1 from Japanese vitamin D-dependent rickets type I (VDDR-I) patients {Kitanaka, S., Takeyama, K., Murayama, A., Sato, T., Okumura, K., Nogami, M., Hasegawa, Y., Niimi, H., Yanagisawa, J., Tanaka, T . & Kato, S . (1998) New England J . Med., 338, 653-661 and Kitanaka, S., Murayama, A., Sakaki, T., Inouye, K., Seino, Y., Fukumoto, S., Shima, M., Yukizane, S., Takayanagi, M., Niimi, H., Takeyama, K . & Kato, S . (1999) J . Clin . Endocrine Metab., 84, 4111-4117} . None of the CYP27B1 mutants showed 1alpha-hydroxylase activity towards 25-hydroxyvitamin D3 . Thus, it was assumed that the mutated amino-acid residues play important roles in the 1alpha-hydroxylase activity, such as substrate binding, activation of molecular oxygen, interaction with adrenodoxin, and folding of the cytochrome P450 structure . To examine our hypothesis, we generated various mutants of CYP27B1 and studied their enzymatic properties . In addition, the corresponding mutations were introduced to CYP27A1, which belongs to the same family as CYP27B1 . As CYP27A1 showed much higher expression level than CYP27B1 in Escherichia coli, further analysis including heme-binding and substrate-binding was performed with CYP27A1 in place of CYP27B1 . Western blot analysis, spectral analysis including reduced CO-difference spectra and substrate-induced difference spectra, and enzymatic analysis of the mutant CYP27A1 gave information on the structure-function relationships of both CYP27A1 and CYP27B1 . Although the sequence alignment suggested that Arg107, Gly125, and Pro497 of CYP27B1 might be involved in substrate binding, the experimental data strongly suggested that mutations of these amino-acid residues destroyed the tertiary structure of the substrate-heme pocket . It was also suggested that Arg389 and Arg453 of CYP27B1 were involved in heme-propionate binding, and Asp164 stabilized the four-helix bundle consisting of D, E, I and J helices, possibly by forming a salt bridge . Thr321 was found to be responsible for the activation of molecular oxygen. Eur J Biochem, 2001 Dec, 268(24), 6508 - 25 l-Threonine aldolase, serine hydroxymethyltransferase and fungal alanine racemase . A subgroup of strictly related enzymes specialized for different functions; Contestabile R et al.; Serine hydroxymethyltransferase (SHMT) is a member of the fold type I family of vitamin B6-dependent enzymes, a group of evolutionarily related proteins that share the same overall fold . The reaction catalysed by SHMT, the transfer of Cbeta of serine to tetrahydropteroylglutamate (H4PteGlu), represents in the cell an important link between the breakdown of amino acids and the metabolism of folates . In the absence of H4PteGlu and when presented with appropriate substrate analogues, SHMT shows a broad range of reaction specificity, being able to catalyse at appreciable rates retroaldol cleavage, racemase, aminotransferase and decarboxylase reactions . This apparent lack of specificity is probably a consequence of the particular catalytic apparatus evolved by SHMT . An interesting question is whether other fold type I members that normally catalyse the reactions which for SHMT could be considered as 'forced errors', may be close relatives of this enzyme and have a catalytic apparatus with the same basic features . As shown in this study, l-threonine aldolase from Escherichia coli is able to catalyse the same range of reactions catalysed by SHMT, with the exception of the serine hydroxymethyltransferase reaction . This observation strongly suggests that SHMT and l-threonine aldolase are closely related enzymes specialized for different functions . An evolutionary analysis of the fold type I enzymes revealed that SHMT and l-threonine aldolase may actually belong to a subgroup of closely related proteins; fungal alanine racemase, an extremely close relative of l-threonine aldolase, also appears to be a member of the same subgroup . The construction of three-dimensional homology models of l-threonine aldolase from E . coli and alanine racemase from Cochliobolus carbonum, and their comparison with the SHMT crystal structure, indicated how the tetrahydrofolate binding site might have evolved and offered a starting point for further investigations. Acta Anaesthesiol Scand, 2001 Nov, 45(10), 1246 - 54 Continuously infused methylene blue modulates the early cardiopulmonary response to endotoxin in awake sheep; Evgenov OV et al.; BACKGROUND: In endotoxemia and septic shock, enhanced generation of endogenous nitric oxide (NO) contributes to myocardial depression, hypotension, and derangement of gas exchange . We hypothesized that continuous infusion of methylene blue (MB), an inhibitor of the NO pathway, would counteract these effects in endotoxemic sheep . METHODS: Twenty-one sheep were anesthetized and instrumented for a chronic study with vascular catheters . On the day of the experiment, 18 conscious animals randomly received either an intravenous injection of MB 10 mg x kg(-1) or isotonic saline . Thirty minutes later, sheep received a 20-min intravenous infusion of Escherichia coli endotoxin 1 microg x kg(-1) and either an intravenous infusion of MB 2.5 mg x kg(-1) x h(-1) or isotonic saline, respectively, for 5 h . In addition, 3 animals were exposed to the same dose of MB alone . RESULTS: MB reduced the early endotoxin-induced declines in stroke volume, left ventricular stroke work and cardiac indices, and prevented mean arterial pressure from falling . Moreover, MB ameliorated the increases in pulmonary arterial pressure and pulmonary vascular resistance index . In addition, MB reduced the increments in venous admixture and AaPO2, decreased the falls in PaO2, SaO2, and oxygen delivery, and maintained oxygen consumption . MB also prevented the rises in body temperature and plasma nitrites and nitrates, and delayed the elevation of plasma lactate . When given alone to healthy sheep, MB transiently reduced plasma lactate and PaO2, and increased AaPO2 . CONCLUSION: In ovine endotoxemia, continuously infused MB counteracts the early myocardial dysfunction and derangement of hemodynamics and gas exchange. Biochem J, 2001 Dec 15, 360(Pt 3), 699 - 706 Determination of the native form of FadD, the Escherichia coli fatty acyl-CoA synthetase, and characterization of limited proteolysis by outer membrane protease OmpT; Yoo JH et al.; Several studies have described FadD, the Escherichia coli fatty acyl-CoA synthetase {also known as fatty acid:CoA ligase (AMP-forming); EC 6.2.1.3}, as a 42-50 kDa enzyme . Based on sequencing and expression data from the fadD gene, other reports have suggested that FadD is a 62 kDa protein and represents the sole fatty acyl-CoA synthetase in E . coli . We report that the 62 kDa FadD enzyme is a substrate for the outer membrane protease OmpT in vitro, producing a 43 kDa C-terminal fragment and a 19 kDa N-terminal fragment . Immunoblotting with a FadD antibody revealed that only the 62 kDa form of the enzyme is present in vivo, but we utilized the proteolytic sensitivity of FadD to investigate its structure . Photoaffinity labelling experiments revealed that both intact FadD and the 43 kDa fragment bound a long-chain fatty acid . Intact and cleaved FadD were also purified to determine the effect of cleavage on function . When using oleate as a substrate, cleaved FadD displayed 2-fold higher K(m) and V(max) values compared with intact FadD, but the catalytic efficiencies (k(cat)/K(m)) of the two forms were similar . This indicated that cleavage did not adversely affect enzyme activity . Proteolysis of FadD by OmpT was altered by the presence of oleate or ATP, both of which are ligands for the fatty acyl-CoA synthetase . This suggested that FadD undergoes ligand-induced conformational changes and implies that the region surrounding the cleavage site is mobile, a common characteristic of linker domains. Biochem J, 2001 Dec 15, 360(Pt 3), 657 - 65 Identification of essential active-site residues in ornithine decarboxylase of Nicotiana glutinosa decarboxylating both L-ornithine and L-lysine; Lee YS et al.; The cDNA encoding ornithine decarboxylase (ODC; EC 4.1.1.17), a key enzyme in putrescine and polyamine biosynthesis, has been cloned from Nicotiana glutinosa (GenBank AF 323910), and was expressed in Escherichia coli . The amino acid sequence of N . glutinosa ODC showed 90% identity with Datura stramonium ODC, and 44% identity with human ODC . N . glutinosa ODC did not possess the PEST sequence {a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues} found in mammalian ODCs, which are thought to be involved in rapid degradation of the protein . The purified ODC was a homodimeric protein, having a native M(r) of 92000 . Kinetic studies of ODC showed that N . glutinosa ODC decarboxylated both l-ornithine and l-lysine with K(m) values of 562 microM and 1592 microM at different optimal pH values of 8.0 and 6.8 respectively . ODC activity was completely and irreversibly inhibited by alpha-difluoromethylornithine (K(i) 1.15 microM), showing a competitive inhibition pattern . Site-directed mutagenesis was performed on ODC to introduce mutations at conserved lysine (Lys(95)) and cysteine (Cys(96), Cys(338) and Cys(377)) residues, chosen by examination of the conserved sequence, which were proven by chemical modification to be involved in enzymic activity . Except for Cys(96), each mutation caused a substantial loss in enzyme activity . Most notably, Lys(95) increased the K(m) for l-ornithine by 16-fold and for l-lysine by 3-fold, with 100-fold and 2.8-fold decreases in the k(cat) for ODC and lysine decarboxylase (LDC) activity respectively . The Cys(377)-->Ala mutant possessed a k(cat) that was lowered by 23-fold, and the K(m) value was decreased by 1.4-fold for l-ornithine . The three-dimensional model of ODC protein constructed on the basis of the crystal structure of Trypanosoma brucei, mouse and human ODCs localized the four residues in the active-site cleft . This is the first work carried out on active-site residues of plant ODC, where ODC and LDC activities occur in the same catalytic site. Biochem J, 2001 Dec 15, 360(Pt 3), 617 - 23 Kynurenine aminotransferase and glutamine transaminase K of Escherichia coli: identity with aspartate aminotransferase; Han Q et al.; The present study describes the isolation of a protein from Escherichia coli possessing kynurenine aminotransferase (KAT) activity and its identification as aspartate aminotransferase (AspAT) . KAT catalyses the transamination of kynurenine and 3-hydroxykynurenine to kynurenic acid and xanthurenic acid respectively, and the enzyme activity can be easily detected in E . coli cells . Separation of the E . coli protein possessing KAT activity through various chromatographic steps led to the isolation of the enzyme . N-terminal sequencing of the purified protein determined its first 10 N-terminal amino acid residues, which were identical with those of the E . coli AspAT . Recombinant AspAT (R-AspAT), homologously expressed in an E . coli/pET22b expression system, was capable of catalysing the transamination of both l-kynurenine (K(m)=3 mM; V(max)=7.9 micromol.min(-1).mg(-1)) and 3-hydroxy-dl-kynurenine (K(m)=3.7 mM; V(max)=1.25 micromol.min(-1).mg(-1)) in the presence of pyruvate as an amino acceptor, and exhibited its maximum activity at temperatures between 50-60 degrees C and at a pH of approx . 7.0 . Like mammalian KATs, R-AspAT also displayed high glutamine transaminase K activity when l-phenylalanine was used as an amino donor (K(m)=8 mM; V(max)=20.6 micromol.min(-1).mg(-1)) . The exact match of the first ten N-terminal amino acid residues of the KAT-active protein with that of AspAT, in conjunction with the high KAT activity of R-AspAT, provides convincing evidence that the identity of the E . coli protein is AspAT. Biochem J, 2001 Dec 15, 360(Pt 3), 563 - 7 Peroxynitrite-induced nitration of tyrosine-34 does not inhibit Escherichia coli iron superoxide dismutase; Soulere L et al.; The peroxynitrite anion is a potent oxidizing agent, formed by the diffusion-limited combination of nitric oxide and superoxide, and its production under physiological conditions is associated with the pathologies of a number of inflammatory and neurodegenerative diseases . Nitration of Escherichia coli iron superoxide dismutase (Fe-SOD) by peroxynitrite was investigated, and demonstrated by spectral changes and electrospray mass spectroscopic analysis . HPLC and mass studies of the tryptic digests of the mono-nitrated Fe-SOD indicated that tyrosine-34 was the residue most susceptible to nitration by peroxynitrite . Exclusive nitration of this residue occurred when Fe-SOD was exposed to a cumulative dose of 0.4 mM peroxynitrite . Unlike with human Mn-SOD, this single modification did not inactivate E . coli Fe-SOD at pH 7.4 . When Fe-SOD was exposed to higher concentrations of peroxynitrite (7 mM), eight tyrosine residues per subunit of the protein, of the nine available, were nitrated without loss of catalytic activity of the enzyme . The pK(a) of nitrated tyrosine-34 was determined to be 7.95+/-0.15, indicating that the peroxynitrite-modified enzyme appreciably maintains its protonation state under physiological conditions. Biochemistry (Mosc), 2001 Oct, 66(10), 1067 - 76 Study of the properties of phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase; Nagradova NK; The properties of the active center of phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are considered with emphasis on the structure of anion-binding sites and their role in catalysis . The results of studies on the molecular mechanism of the effect of NAD+ on the enzyme conformation are discussed . Experimental evidence is presented supporting the idea that negative cooperativity of NAD+ binding and half-of-the-sites reactivity exhibited by GAPDH are generated by different mechanisms . Data obtained with rabbit muscle and Escherichia coli GAPDH point to preexisting asymmetry in these tetramers . Structural determinants that can control the transition of the tetramer from the symmetric to the asymmetric state were found. Biotechnol Prog, 2001 Nov-Dec, 17(6), 1107 - 13 Selective precipitation of DNA by spermine during the chemical extraction of insoluble cytoplasmic protein; Choe WS et al.; The direct chemical extraction of recombinant L1 protein (the major capsid protein of human papillomavirus type 16) from the cytoplasm of E . coli HMS174(DE3) has recently been demonstrated at high cell density (to OD(600) = 160) without the use of reducing agent (1) . Coextraction of DNA at high concentration prevents direct coupling to postextraction recovery operations including expanded bed adsorption . In this study, spermine is used to selectively precipitate DNA during chemical extraction . Highly efficient and selective DNA precipitation was achieved . An approximate 10-fold increase in the specific spermine concentration (mg of spermine/mg of DNA) was required to precipitate DNA when 8 M urea was added to the extraction buffer . EDTA (3 mM), required for effective chemical extraction, does not significantly inhibit DNA precipitation . Precipitation selectivity was demonstrated in a bovine serum albumin spiking test, with almost complete recovery of the spiked protein . During studies on the direct extraction of L1 protein from cells at OD(600) = 80, high DNA removal efficiency (>85%) and negligible L1 protein coprecipitation were achieved . This selective precipitation technique simply requires the addition of spermine to the chemical extraction buffer and therefore does not increase technique complexity . This modification enhances the method's general applicability and enables direct coupling to downstream recovery units following chemical extraction at high cell and product concentrations. Biochemistry, 2001 Dec 18, 40(50), 15362 - 8 Tryptophan and tyrosine radicals in ribonucleotide reductase: a comparative high-field EPR study at 94 GHz; Bleifuss G et al.; Tryptophan radicals, which are generated in the reconstitution reaction of mutants Y122F and Y177W of subunit R2 apoprotein of E . coli and mouse ribonucleotide reductase (RNR), respectively, with Fe(2+) and oxygen, are investigated by high-field EPR at 94 GHz and compared with the tyrosine radicals occurring in the respective wild-type proteins . For the first time, accurate g-values are obtained for protein-associated neutral tryptophan free radicals, which show only a small anisotropy . The apparent hyperfine patterns observed in frozen solutions are very similar for tryptophan and tyrosine radicals in mouse subunit R2 at conventional X-band EPR . The radicals can, however, be discriminated by their different g-tensors using high-field EPR . Tryptophan radicals were postulated as reaction intermediates in the proposed radical transfer pathway of RNR . Furthermore, the data obtained here for the electronic structure of protein-associated tryptophan neutral free radicals are important for identification and understanding of the functional important tryptophan radicals which occur in other enzymes, e.g., DNA photolyase and cytochrome c peroxidase, where they are magnetically coupled to other radicals or to a metal center. Biochemistry, 2001 Dec 18, 40(50), 15341 - 8 Characterization of hK4 (prostase), a prostate-specific serine protease: activation of the precursor of prostate specific antigen (pro-PSA) and single-chain urokinase-type plasminogen activator and degradation of prostatic acid phosphatase; Takayama TK et al.; hK4 (prostase, KLK4), a recently cloned prostate-specific serine protease and a member of the tissue kallikrein family, is a zymogen composed of 228 amino acid residues including an amino-terminal propiece, Ser-Cys-Ser-Gln- . A chimeric form of hK4 (ch-hK4) was constructed in which the propiece of hK4 was replaced by that of prostate-specific antigen (PSA) to create an activation site susceptible to trypsin-type proteases . ch-hK4 was expressed in Escherichia coli, isolated from inclusion bodies, refolded, and purified with an overall yield of 25% . The zymogen was readily self-activated during the refolding process to generate an active form (21 kDa) of hK4 (rhK4) . rhK4 cleaved the chromogenic substrates Val-Leu-Arg-pNA (S-2266), Pro-Phe-Arg-pNA (S-2302), Ile-Glu-Gly-Arg-pNA (S-2222), and Val-Leu-Lys-pNA (S-2251), indicating that rhK4 has a trypsin-type substrate specificity . The rhK4 was inhibited by aprotinin (6 kDa), forming an equimolar 27 kDa complex . rhK4 readily activated both the precursor of PSA (pro-PSA) and single chain urokinase-type plasminogen activator (scuPA, pro-uPA) . rhK4 also completely degraded prostatic acid phosphatase but failed to cleave serum albumin, another protein purified from human seminal plasma . These results indicate that hK4 may have a role in the physiologic processing of seminal plasma proteins such as pro-PSA, as well as in the pathogenesis of prostate cancer through its activation of pro-uPA. Biochemistry, 2001 Dec 18, 40(50), 15224 - 33 Role of SRP RNA in the GTPase cycles of Ffh and FtsY; Peluso P et al.; The bacterial homologues of the signal recognition particle (SRP) and its receptor, the Ffh*4.5S RNA ribonucleoprotein complex and the FtsY protein, respectively, form a unique complex in which both Ffh and FtsY act as GTPase activating proteins for one another, resulting in the mutual stimulation of GTP hydrolysis by both proteins . Previous work showed that 4.5S RNA enhances the GTPase activity in the presence of both Ffh and FtsY, but it was not clear how this was accomplished . In this work, kinetic and thermodynamic analyses of the GTPase reactions of Ffh and FtsY have provided insights into the role of 4.5S RNA in the GTPase cycles of Ffh and FtsY . We found that 4.5S RNA accelerates the association between Ffh and FtsY 400-fold in their GTP-bound form, analogous to its 200-fold catalytic effect on Ffh*FtsY association previously observed with the GppNHp-bound form {Peluso, P., et al . (2000) Science 288, 1640-1643} . Further, Ffh-FtsY association is rate-limiting for the observed GTPase reaction with subsaturating Ffh and FtsY, thereby accounting for the apparent stimulatory effect of 4.5S RNA on the GTPase activity observed previously . An additional step, GTP hydrolysis from the Ffh*FtsY complex, is also moderately facilitated by 4.5S RNA . These results suggest that 4.5S RNA modulates the conformation of the Ffh*FtsY complex and may, in turn, regulate its GTPase activity during the SRP functional cycle. Biochemistry, 2001 Dec 18, 40(50), 15203 - 14 Evidence of powerful substrate electric fields in DNA photolyase: implications for thymidine dimer repair; MacFarlane AW 4th et al.; DNA photolyase is a flavoprotein that repairs cyclobutylpyrimidine dimers by ultrafast photoinduced electron transfer . One unusual feature of this enzyme is the configuration of the FAD cofactor, where the isoalloxazine and adenine rings are nearly in vdW contact . We have measured the steady-state and transient absorption spectra and excited-state decay kinetics of oxidized (FAD-containing, folate-depleted) Escherichia coli DNA photolyase with and without dinucleotide and polynucleotide single-stranded thymidine dimer substrates . The steady-state absorption spectrum for the enzyme-polynucleotide substrate complex showed a blue shift, as seen previously by Jorns et al . (1) . No shift was observed for the dinucleotide substrate, suggesting that there are significant differences in the binding geometry of dinucleotide versus polynucleotide dimer lesions . Evidence was obtained from transient absorption experiments for a long-lived charge-transfer complex involving the isoalloxazine of the FAD cofactor . No evidence of excited-state quenching was measurable upon binding either substrate . To explain these data, we hypothesize the existence of a large substrate electric field in the cavity containing the FAD cofactor . A calculation of the magnitude and direction of this dipolar electric field is consistent with electrochromic band shifts for both S(0) --> S(1) and S(0) --> S(2) transitions . These observations suggest that the substrate dipolar electric field may be a critical component in its electron-transfer-mediated repair by photolyase and that the unique relative orientation of the isoalloxazine and adenine rings may have resulted from the consequences of the dipolar substrate field. Biochemistry, 2001 Dec 18, 40(50), 15176 - 83 Translesional synthesis past acetylaminofluorene-derived DNA adducts catalyzed by human DNA polymerase kappa and Escherichia coli DNA polymerase IV; Suzuki N et al.; Human DNA polymerase kappa (pol kappa) has a sequence significantly homologous with that of Escherichia coli DNA polymerase IV (pol IV) . We used a truncated form of human pol kappa (pol kappaDeltaC) and full-length pol IV to explore the miscoding properties of these enzymes . Oligodeoxynucleotides, modified site-specifically with N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-AF), were used as DNA templates in primer extension reactions that included all four dNTPs . Reactions catalyzed by pol kappaDeltaC were partially blocked one base prior to dG-AAF or dG-AF, and also opposite both lesions . At higher enzyme concentrations, a significant fraction of primer was extended . Analysis of the fully extended reaction product revealed incorporation of dTMP opposite dG-AAF, accompanied by much smaller amounts of dCMP, dAMP, and dGMP and some one- and two-base deletions . The product terminating 3' to the adduct site contained AMP misincorporated opposite dC . On templates containing dG-AF, dAMP, dTMP, and dCMP were incorporated opposite the lesion in approximately equal amounts, together with some one-base and two-base deletions . Steady-state kinetics analysis confirmed the results obtained from primer extension reactions catalyzed by pol kappa . In contract, primer extension reactions catalyzed by pol IV were blocked effectively by dG-AAF and dG-AF . At high concentrations of pol IV, full-length products were formed containing primarily one- or two-base deletions with dCMP, the correct base, incorporated opposite dG-AF . The miscoding properties of pol kappa observed in this study are consistent with mutational spectra observed when plasmid vectors containing dG-AAF or dG-AF are introduced into simian kidney cells {Shibutani, S., et al . (2001) Biochemistry 40, 3717-3722}, supporting a model in which pol kappa plays a role in translesion synthesis past acetylaminofluorene-derived lesions in mammalian cells. Mol Ther, 2001 Dec, 4(6), 603 - 13 Absence of germline infection in male mice following intraventricular injection of adenovirus; Peters AH et al.; The possibility of inadvertent exposure of gonadal tissue to gene therapy vectors has raised safety concerns about germline infection . We show here that the receptor for coxsackie B viruses and adenoviruses 2 and 5 (CXADR) is expressed in mouse germ cells, suggesting the possibility that these viruses could infect germ cells . To directly assess the risk of germline infection in vivo, we injected an adenovirus carrying the germ-cell-specific protamine promoter fused to the bacterial lacZ reporter gene into the left ventricular cavity of mice and then monitored expression of the reporter gene in germ cells . To differentiate between infection of stem cells and differentiating spermatogenic cells, we analyzed expression of the reporter cassette at different times after viral delivery . Under all conditions tested, mice did not express the Escherichia coli beta-galactosidase protein in developing spermatids or in mature epididymal spermatozoa . Primary germ cells cultured in vitro were also refractory to adenoviral infection . Our data suggest that the chance of vertical germline transmission and insertional mutagenesis is highly unlikely following intracoronary adenoviral delivery. J Surg Res, 2001 Dec, 101(2), 232 - 7 TNF receptor I mediates chemokine production and neutrophil accumulation in the lung following systemic lipopolysaccharide; Calkins CM et al.; BACKGROUND: Tumor necrosis factor (TNF)-alpha is a critical effector of lipopolysaccharide (LPS)-induced acute lung injury, and its effects are mediated by two structurally related receptors, RI and RII . Cellular adhesion molecules and C-X-C chemokines (Keratinocyte chemoattractant (KC) and macrophage inflammatory protein {MIP}-2) regulate tissue neutrophil polymorphonuclear neutrophil (PMN) accumulation in a multitude of inflammatory states . We hypothesized that TNFRI signaling dictates PMN accumulation in the lung via regulation of chemokine molecule production . Therefore, the purposes of this study were to (1) delineate LPS-induced lung TNF-alpha production and (2) characterize the contribution of both TNF receptors to lung chemokine production and neutrophil influx following systemic LPS . METHODS: Wild-type or TNFRI and TNFRII knockout (KO) mice were injected with vehicle (saline) or LPS (Escherichia coli 0.5 mg/kg intraperitoneally) . After 2, 4, 6, or 24 h, lungs were analyzed for TNF-alpha and chemokine (KC and MIP-2) protein expression (enzyme-linked immunosorbent assay) and PMN accumulation (myeloperoxidase assay) . RESULTS: There was an increase in total lung TNF-alpha (vehicle, 5.0 +/- 1.2 pg/mg total protein vs LPS, 950 +/- 318; P < 0.05) after LPS . Lung chemokine production and PMN accumulation were also increased compared to vehicle-injected mice . Lung chemokine production and PMN accumulation were significantly lower in TNFRI KO, but not TNFRII KO, mice, despite no difference in TNF-alpha production (TNFRI KO, 925 +/- 301 vs TNFRII KO, 837 +/- 267, P = 0.82) . CONCLUSIONS: Acute lung injury following systemic LPS administration is characterized by increased lung (1) TNF-alpha production, (2) C-X-C chemokine production, and (3) neutrophil accumulation . The maximal effect of LPS-induced lung neutrophil accumulation appears to be dependent upon the TNFRI receptor but not the TNFRII receptor . . J Surg Res, 2001 Dec, 101(2), 210 - 5 Inhibition of cyclic-3',5'-nucleotide phosphodiesterase abrogates the synergism of hypoxia with lipopolysaccharide in the induction of macrophage TNF-alpha production; Meng X et al.; BACKGROUND: Local tumor necrosis factor (TNF)-alpha production by resident macrophages (M phi) contributes to posttraumatic tissue injury . Hypoxia decreases cellular cyclic adenosine monophosphate (cAMP) levels and enhances M phi secretion of TNF-alpha following lipopolysaccharide (LPS) stimulation . Thus, tissue hypoxia associated with trauma likely synergizes with proinflammatory mediators in the induction of M phi TNF-alpha production through an influence on cAMP generation or degradation . It is unclear whether elevation of cellular cAMP inhibits LPS-stimulated TNF-alpha production by hypoxic M phi . Moreover, it is unknown whether the synergism of hypoxia with LPS can be abrogated by promotion of cAMP generation or inhibition of cAMP degradation . METHODS: Rat peritoneal M phi were stimulated with Escherichia coli LPS (20 ng/ml) in a normoxic (room air with 5% CO(2)) or hypoxic (95% N(2) with 5% CO(2)) condition . TNF-alpha levels in cell-free supernatants were measured by enzyme-linked immunoassay . The beta-adrenoceptor agonist isoproterenol (ISP; 5.0 microM) and the adenylate cyclase activator forskolin (FSK; 50 microM) were applied to promote cAMP generation . The nonselective cyclic-3',5'-nucleotide phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1.0 mM) and the PDE III-specific inhibitor milrinone (200 microM) were used to inhibit cAMP degradation . The nondegradable cAMP analogue dibutyryl cAMP (dbcAMP; 100 microM) was applied to further determine the role of PDE . RESULTS . Although hypoxia alone had a minimal effect on TNF-alpha production, it dramatically enhanced LPS-stimulated TNF-alpha production (4.08 +/- 0.28 ng/10(6) cells in hypoxia plus LPS vs 1.63 +/- 0.26 ng/10(6) cells in LPS, 2.5-fold, P < 0.01) . Promotion of cAMP generation by either ISP or FSK reduced TNF-alpha production by hypoxic cells . However, neither of these two agents abolished the synergism of hypoxia with LPS (1.68 +/- 0.13 ng/10(6) cells in ISP plus hypoxia plus LPS vs 0.55 +/- 0.04 ng/10(6) cells in ISP plus LPS, threefold; 1.17 +/- 0.03 ng/10(6) cells in FSK plus hypoxia plus LPS vs 0.33 +/- 0.02 ng/10(6) cells in FSK plus LPS, 3.5-fold; both P < 0.01) . Inhibition of cAMP degradation with IBMX reduced TNF-alpha production in hypoxic cells and abrogated the synergism (0.31 +/- 0.11 ng/10(6) cells in IBMX plus hypoxia plus LPS vs 0.27 +/- 0.04 ng/10(6) cells in IBMX plus LPS, P > 0.05), and the PDE III inhibitor milrinone had a comparable effect . Moreover, dbcAMP also attenuated TNF-alpha production with abrogation of the synergistic effect of hypoxia (0.56 +/- 0.08 ng/10(6) cells in dbcAMP plus hypoxia plus LPS vs 0.46 +/- 0.04 ng/10(6) cells in dbcAMP plus LPS, P > 0.05) . CONCLUSIONS: The results show that elevation of cellular cAMP, either by promotion of generation or by inhibition of degradation, suppresses LPS-stimulated TNF-alpha production in hypoxic M phi . It appears that hypoxia synergizes with LPS in the induction of M phi TNF-alpha production through PDE-mediated cAMP degradation . Inhibition of PDE may be a therapeutic approach for suppression of synergistic induction of M phi TNF-alpha production by hypoxia and LPS in posttraumatic tissue. Biochem Biophys Res Commun, 2001 Dec 14, 289(4), 832 - 7 Kinetic determinants of the interaction of enoyl-ACP reductase from Plasmodium falciparum with its substrates and inhibitors; Kapoor M et al.; We have recently demonstrated that Plasmodium falciparum, unlike its human host, has the type II fatty acid synthase, in which steps of fatty acid biosynthesis are catalyzed by independent enzymes . This difference could be successfully exploited in the design of drugs specifically targeted at the different enzymes of this pathway in P . falciparum, without affecting the corresponding enzymes in humans . The importance of enoyl-ACP reductase (FabI) in the fatty acid biosynthesis pathway makes it an important target in antimalarial therapy . We report here the initial characterization of Plasmodium FabI expressed in Escherichia coli . The K(m) values of the enzyme for crotonyl-CoA and NADH were derived as 165 and 33 microM, respectively . Triclosan shows competitive kinetics with respect to NADH but is uncompetitive with respect to NAD(+), which shows that the binding of triclosan to the enzyme is facilitated in the presence of NAD(+) . (c)2001 Elsevier Science. Semin Hematol, 2001 Oct, 38(4 Suppl 12), 35 - 8 Tissue factor in experimental acute lung injury; Welty-Wolf KE et al.; Acute lung injury (ALI) is characterized by fibrin deposition in the tissue and vascular spaces . Coagulation is activated after exposure to endotoxin or bacteria, and a procoagulant environment rapidly develops in the vascular, interstitial, and alveolar spaces of the lung . These changes are tissue factor (TF)-dependent and associated with increases in inflammatory cytokines . Procoagulant changes also occur in the lungs of patients with the acute respiratory distress syndrome (ARDS), suggesting that epithelial inflammation activates the extrinsic pathway . Many inflammatory mediators have specific effects on coagulation; however, the role of TF in regulation of pulmonary inflammatory responses is less clear . Here we report initial data on blockade of TF-initiated coagulation in baboons with Escherichia coli sepsis-induced ALI, using active site-inactivated FVIIa (FVIIai ASIS) . Treatment with FVIIai prevented plasma fibrinogen depletion and attenuated fibrin deposition in the tissues . The drug also decreased systemic cytokine responses and inflammatory changes in the lung, including neutrophil infiltration, and decreased edema . Coagulation blockade with FVIIai improved lung function by preserving gas exchange and compliance, decreased pulmonary hypertension, and enhanced renal function . These results show that TF-FVIIa complex is an important regulatory site for the pathologic response of the lung to sepsis . Proc Natl Acad Sci U S A, 2001 Dec 4, 98(25), 14202 - 7 Pattern formation in Escherichia coli: a model for the pole-to-pole oscillations of Min proteins and the localization of the division site; Meinhardt H et al.; Proper cell division requires an accurate definition of the division plane . In bacteria, this plane is determined by a polymeric ring of the FtsZ protein . The site of Z ring assembly in turn is controlled by the Min system, which suppresses FtsZ polymerization at noncentral membrane sites . The Min proteins in Escherichia coli undergo a highly dynamic localization cycle, during which they oscillate between the membrane of both cell halves . By using computer simulations we show that Min protein dynamics can be described accurately by using the following assumptions: (i) the MinD ATPase self-assembles on the membrane and recruits both MinC, an inhibitor of Z ring formation, and MinE, a protein required for MinC/MinD oscillation, (ii) a local accumulation of MinE is generated by a pattern formation reaction that is based on local self-enhancement and a long range antagonistic effect, and (iii) it displaces MinD from the membrane causing its own local destabilization and shift toward higher MinD concentrations . This local destabilization results in a wave of high MinE concentration traveling from the cell center to a pole, where it disappears . MinD reassembles on the membrane of the other cell half and attracts a new accumulation of MinE, causing a wave-like disassembly of MinD again . The result is a pole-to-pole oscillation of MinC/D . On time average, MinC concentration is highest at the poles, forcing FtsZ assembly to the center . The mechanism is self-organizing and does not require any other hypothetical topological determinant. J Lipid Res, 2001 Dec, 42(12), 2084 - 91 Preparation and incorporation of probe-labeled apoA-I for fluorescence resonance energy transfer studies of rHDL; Li HH et al.; Apolipoprotein A-I (apoA-I), the major constituent of HDL, plays an essential role in regulating cholesterol metabolism, acting as the physiological activator of lecithin: cholesterol acyltransferase, which converts cholesterol to cholesterol ester . Thiol-reactive fluorescent probes attached to cysteine-containing apoA-I mutants are currently being used to investigate the "LCAT active" conformation of lipid-bound apoA-I . Herein, we report new methodologies allowing rapid expression, fluorescent labeling, and recombinant HDL (rHDL) preparation for use in apoA-I in fluorescence resonance energy transfer (FRET) studies . Cysteine-containing mutant forms of human apoA-I were cloned into the pTYB12 vector containing a T7 promoter, a modified self-splicing protein element (intein), and a small affinity tag {chitin binding domain (CBD)} . The fusion proteins were expressed in Escherichia coli, isolated from cell lysates, and bound to a chitin-affinity column . Release of mature human apoA-I was initiated by the addition of DTT, which induced self-cleavage at the COOH terminus of the intein - CBD fusion protein . ApoA-I was further purified by Q-sepharose and then used for fluorescent probe labeling . Discoidal rHDL were then prepared with donor and/or acceptor labeled apoA-I and characterized with respect to their size, composition and ability to activate LCAT. J Biol Chem, 2002 Feb 15, 277(7), 5265 - 74 Epub 2001 Dec 04. Efficiency and accuracy of SOS-induced DNA polymerases replicating benzo{a}pyrene-7,8-diol 9,10-epoxide A and G adducts; Shen X et al.; Nucleotide incorporation fidelity, mismatch extension, and translesion DNA synthesis efficiencies were determined using SOS-induced Escherichia coli DNA polymerases (pol) II, IV, and V to copy 10R and 10S isomers of trans-opened benzo{a}pyrene-7,8-diol 9,10-epoxide (BaP DE) A and G adducts . A-BaP DE adducts were bypassed by pol V with moderate accuracy and considerably higher efficiency than by pol II or IV . Error-prone pol V copied G-BaP DE-adducted DNA poorly, forming A*G-BaP DE-S and -R mismatches over C*G-BaP DE-S and -R correct matches by factors of approximately 350- and 130-fold, respectively, even favoring G*G-BaP DE mismatches over correct matches by factors of 2-4-fold . In contrast, pol IV bypassed G-BaP DE adducts with the highest efficiency and fidelity, making misincorporations with a frequency of 10(-2) to 10(-4) depending on sequence context . G-BaP DE-S-adducted M13 DNA yielded 4-fold fewer plaques when transfected into SOS-induced DeltadinB (pol IV-deficient) mutant cells compared with the isogenic wild-type E . coli strain, consistent with the in vitro data showing that pol IV was most effective by far at copying the G-BaP DE-S adduct . SOS polymerases are adept at copying a variety of lesions, but the relative contribution of each SOS polymerase to copying damaged DNA appears to be determined by the lesion's identity. Hum Mol Genet, 2001 Nov 15, 10(24), 2775 - 81 Molecular effects of Eya1 domain mutations causing organ defects in BOR syndrome; Buller C et al.; Eya1 is a critical gene for mammalian organogenesis . Mutations in human EYA1 cause branchio-oto-renal (BOR) syndrome, an autosomal dominant disorder characterized by varying combinations of branchial, otic and renal anomalies, whereas deletion of mouse Eya1 results in the absence of multiple organ formation . Eya1 and other Eya gene products share a highly conserved 271 amino acid Eya domain that is required for protein-protein interaction . Recently, several point mutations that result in single amino acid substitutions in the conserved Eya domain region of EYA1 have been identified in BOR patients; however, the molecular and developmental basis of organ defects that occurred in BOR syndrome is unclear . To understand how these point mutations cause disease, we have analyzed the functional importance of these Eya domain missense mutations with respect to protein complex formation and cellular localization . We have demonstrated that these point mutations do not alter protein localization . However, four mutations are crucial for protein-protein interactions in both yeast and mammalian cells . Our results provide insights into the molecular mechanisms of organ defects detected in human syndromes. FEBS Lett, 2001 Nov 30, 509(1), 111 - 4 Engineering of the H2O2-binding pocket region of a recombinant manganese peroxidase to be resistant to H2O2; Miyazaki C et al.; The manganese peroxidase produced by Phanerochaete chrysosporium, which catalyzes the oxidation of Mn(2+) to Mn(3+), is easily inactivated by the hydrogen peroxide (H2O2) presented in the reaction . We attempted to increase H2O2 resistance by the conformational stabilization around the H2O2-binding pocket . Based on its structural model, engineering of oxidizable Met273 located near the pocket to a non-oxidizable Leu showed a great improvement . Furthermore, after treatment at 1 mM H2O2 where the wild-type is completely inactivated, full activity can be retained by engineering the Asn81, which might have conformational changes due to the environment of the pocket, to a non-bulky and non-oxidizable Ser. FEBS Lett, 2001 Nov 30, 509(1), 53 - 8 Both N-terminal catalytic and C-terminal RNA binding domain contribute to substrate specificity and cleavage site selection of RNase III; Conrad C et al.; The double-stranded RNA-specific endoribonuclease III (RNase III) of bacteria consists of an N-terminal nuclease domain and a double-stranded RNA binding domain (dsRBD) at the C-terminus . Analysis of two hybrid proteins consisting of the N-terminal half of Escherichia coli RNase III fused to the dsRBD of the Rhodobacter capsulatus enzyme and vice versa reveals that both domains in combination with the particular substrate determine substrate specificity and cleavage site selection . Extension of the spacer between the two domains of the E . coli enzyme from nine to 20 amino acids did not affect cleavage site selection. J Mol Biol, 2001 Dec 7, 314(4), 709 - 16 Expansion/contraction of mammalian mitochondrial DNA repeats in Escherichia coli mimics the mitochondrial heteroplasmy; Pfeuty A et al.; Length polymorphism due to tandem repeats is a common feature in animal mitochondrial DNA . The rabbit mitochondrial genome contains a 20 bp repeat domain, which generates a general heteroplasmic state . The observed polymorphic patterns suggest a dynamic equilibrium between gain and loss of units that maintains the copy number in the range 3-19 repeat units . In the apparent absence of recombination, slipped-strand mispairing during replication appears to be the primary cause of additions and deletions . To investigate this hypothesis we have set up a plasmid assay in Escherichia coli . A variable number of repeat units was inserted into a plasmid in both orientations relative to the colE1 origin of replication . Our data show that (i) a minimum unit number (>3) is necessary to generate length polymorphs, (ii) the number of events increases with the length tract, (iii) an excess of additions over deletions is found when the copy number is less than 10 and the trend is reversed when it is over 10, (iv) the frequency of deletions-additions is dependent on the orientation, (v) the polymorphism patterns are different according to the orientation . The length polymorphic pattern generated in the bacteria, in one orientation, mimics that observed in the mitochondria, suggesting that slipped mispairing between repeated sequences during DNA replication is responsible for the mitochondrial heteroplasmic state . Nat Rev Genet, 2001 Dec, 2(12), 919 - 29 Non-coding RNA genes and the modern RNA world; Eddy SR; Non-coding RNA (ncRNA) genes produce functional RNA molecules rather than encoding proteins . However, almost all means of gene identification assume that genes encode proteins, so even in the era of complete genome sequences, ncRNA genes have been effectively invisible . Recently, several different systematic screens have identified a surprisingly large number of new ncRNA genes . Non-coding RNAs seem to be particularly abundant in roles that require highly specific nucleic acid recognition without complex catalysis, such as in directing post-transcriptional regulation of gene expression or in guiding RNA modifications. J Microbiol Methods, 2002 Jan, 48(1), 43 - 51 Green fluorescent protein-based biosensor for detecting SOS-inducing activity of genotoxic compounds; Kostrzynska M et al.; Increasing levels of environmental pollution demand specific and sensitive methods for detection of genotoxic agents in water, food products and environmental samples . Tests for genotoxicity assessment are often based on biosensor strains that respond to DNA damage induced by chemicals . In the present study, fluorescent reporter Escherichia coli strains have been developed, which contain a plasmid-borne transcriptional fusion between the DNA-damage inducible recA promoter and the green fluorescent protein gene (gfp) or a gene encoding a red-shifted, higher intensity GFP variant (mutant 3) . GFP-based biosensors allowed the detection of a dose-dependent response to genotoxic agents such as mitomycin C (MMC), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and nalidixic acid (NA) . A reporter strain carrying recA'-gfp mutant 3 fusion gave more dramatic and sensitive response than a strain containing the wild-type gfp . These results indicate that recA'-gfp mutant 3-based biosensor is potentially useful for detection of genotoxins. Mutat Res, 2001 Dec 12, 484(1-2), 77 - 86 Induction of lacI mutations in Big Blue Rat-2 cells treated with 1-(2-hydroxyethyl)-1-nitrosourea: a model system for the analysis of mutagenic potential of the hydroxyethyl adducts produced by 1,3-bis (2-chloroethyl)-1-nitrosourea; Sprung CN et al.; We have investigated the genotoxic effects of 1-(2-hydroxyethyl)-1-nitrosourea (HENU) . We have chosen this agent because of its demonstrated ability to produce N7-(2-hydroxyethyl) guanine (N7-HOEtG) and O(6)-(2-hydroxyethyl) 2'-deoxyguanosine (O(6)-HOEtdG); two of the DNA alkylation products produced by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) . For these studies, we have used the Big Blue Rat-2 cell line that contains a lambda/lacI shuttle vector . Treatment of these cells with HENU produced a dose dependent increase in the levels of N7-HOEtG and O(6)-HOEtdG as quantified by HPLC with electrochemical detection . Treatment of Big Blue Rat-2 cells with either 0, 1 or 5mM HENU resulted in mutation frequencies of 7.2+/-2.2x10(-5), 45.2+/-2.9x10(-5) and 120.3+/-24.4x10(-5), respectively . Comparison of the mutation frequencies demonstrates that 1 and 5mM HENU treatments have increased the mutation frequency by 6- and 16-fold, respectively . This increase in mutation frequency was statistically significant (P<0.001) . Sequence analysis of HENU-induced mutations have revealed primarily G:C-->A:T transitions (52%) and a significant number of A:T-->T:A transversions (16%) . We propose that the observed G:C-->A:T transitions are produced by the DNA alkylation product O(6)-HOEtdG . These results suggest that the formation of O(6)-HOEtdG by BCNU treatment contributes to its observed mutagenic properties. Mutat Res, 2001 Dec 12, 484(1-2), 19 - 48 Cellular determinants of the mutational specificity of 1-nitroso-6-nitropyrene and 1-nitroso-8-nitropyrene in the lacI gene of Escherichia coli; Lambert IB et al.; We have characterized 202 lacI(-) mutations, and 158 dominant lacI(-d) mutations following treatment of Escherichia coli strains NR6112 and EE125 with 1-nitroso-6-nitropyrene (1,6-NONP), an activated metabolite of the carcinogen 1,6-dinitropyrene . In all, 91% of the induced point mutations occurred at G:C residues . The -(G:C) frameshifts were the dominant mutational class in the lacI(-) collections of both NR6112 and EE125, and in the lacI(-d) collection of NR6112 . Frameshift mutations occurred preferentially in runs of guanine residues, and their frequency increased with the length of the reiterated sequence . In strain EE125, which contained the plasmid pKM101, there was a marked stimulation in the frequency of base substitution mutations that was particularly apparent in the lacI(-d) collection . This study completes a comprehensive analysis of 1194 lacI(-) and 348 lacI(-d) mutations induced by either 1,6-NONP or its positional isomer 1-nitroso-8-nitropyrene (1,8-NONP) in strains of E . coli that differ with regard to their ability to carry out nucleotide excision repair and/or their ability to express the translesion synthesis DNA polymerase RI (MucAB) encoded by plasmid pKM101 . Among the mutations are 763 frameshift mutations, 367 base substitutions and 47 deletions; these mutations have been characterized at more than 300 distinct sites in the lacI gene . Our studies provide detailed insight into the DNA sequence alterations and mutational mechanisms associated with dinitropyrene mutagenesis . We review the mutational spectra, and discuss cellular lesion repair or tolerance mechanisms that modulate the observed mutational specificity. Eur J Biochem, 2001 Dec, 268(23), 6207 - 13 Shifted positioning of the anticodon nucleotide residues of amber suppressor tRNA species by Escherichia coli arginyl-tRNA synthetase; Kiga D et al.; Cytidine in the anticodon second position (position 35) and G or U in position 36 of tRNAArg are required for aminoacylation by arginyl-tRNA synthetase (ArgRS) from Escherichia coli . Nevertheless, an arginine-accepting amber suppressor tRNA with a CUA anticodon (FTOR1Delta26) exhibits suppression activity in vivo {McClain, W.H . & Foss, K . (1988) Science, 241, 1804-1807} . By an in vitro kinetic study with mutagenized tRNAs, we showed that the arginylation of FTOR1Delta26 involves C34 and U35, and that U35 can be replaced by G without affecting the activity . Thus, the positioning of the essential nucleotides for the arginylation is shifted to the 5' side, by one residue, in the suppressor tRNAArg . We found that the shifted positioning does not depend on the tRNA sequence outside the anticodon . Furthermore, by a genetic method, we isolated a mutant ArgRS that aminoacylates FTOR1Delta26 more efficiently than the wild-type ArgRS . The isolated mutant has mutations at two nonsurface amino-acid residues that interact with each other near the anticodon-binding site. Eur J Biochem, 2001 Dec, 268(23), 6140 - 5 Comparison of starch branching enzyme I and II from potato; Rydberg U et al.; The in vitro activities of purified potato starch branching enzyme (SBE) I and II expressed in Escherichia coli were compared using several assay methods . With the starch-iodine method, it was found that SBE I was more active than SBE II on an amylose substrate, whereas SBE II was more active than SBE I on an amylopectin substrate . Both enzymes were stimulated by the presence of phosphate . On a substrate consisting of linear dextrins (chain length 8-200 glucose residues), no significant net increase in molecular mass was seen on gel-permeation chromatography after incubation with the enzymes . This indicates intrachain branching of the substrate . After debranching of the products, the majority of dextrins with a degree of polymerization (dp) greater than 60 were absent for SBE I and those with a dp greater than 70 for SBE II . To study the shorter chains, the debranched samples were also analysed by high-performance anion-exchange chromatography . The products of SBE I showed distinct populations at dp 11-12 and dp 29-30, whereas SBE II products had one, broader, population with a peak at dp 13-14 . An accumulation of dp 6-7 chains was seen with both isoforms. Eur J Biochem, 2001 Dec, 268(23), 6105 - 13 Immunohistochemical localization of guinea-pig leukotriene B4 12-hydroxydehydrogenase/15-ketoprostaglandin 13-reductase; Yamamoto T et al.; We have cloned cDNA for leukotriene B4 12-hydroxydehydrogenase (LTB4 12-HD)/15-ketoprostaglandin 13-reductase (PGR) from guinea-pig liver . LTB4 12-HD catalyzes the conversion of LTB4 into 12-keto-LTB4 in the presence of NADP+, and plays an important role in inactivating LTB4 . The cDNA contained an ORF of 987 bp that encodes a protein of 329 amino-acid residues with a 78% identity with porcine LTB4 12-HD . The amino acids in the putative NAD+/NADP+ binding domain are well conserved among the pig, guinea-pig, human, rat, and rabbit enzymes . The guinea-pig LTB4 12-HD (gpLTB4 12-HD) was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli, which exhibited similar enzyme activities to porcine LTB4 12-HD . We examined the 15-ketoprostaglandin 13-reductase (PGR) activity of recombinant gpLTB4 12-HD, and confirmed that the Kcat of the PGR activity is higher than that of LTB4 12-HD activity by 200-fold . Northern and Western blot analyses revealed that gpLTB4 12-HD/PGR is widely expressed in guinea-pig tissues such as liver, kidney, small intestine, spleen, and stomach . We carried out immunohistochemical analyses of this enzyme in various guinea-pig tissues . Epithelial cells of calyx and collecting tubules in kidney, epithelial cells of airway, alveoli, epithelial cells in small intestine and stomach, and hepatocytes were found to express the enzyme . These findings will lead to the identification of the unrevealed roles of PGs and LTs in these tissues. Biochemistry, 2001 Dec 11, 40(49), 15024 - 30 Characterization of the tryptophan residues of Escherechia coli alkaline phosphatase by phosphorescence and optically detected magnetic resonance spectroscopy; Ghosh S et al.; The phosphorescence and zero field optically detected magnetic resonance (ODMR) of the tryptophan (Trp) residues of alkaline phosphatase from Escherechia coli are examined . Each Trp is resolved optically and identified with the aid of the W220Y mutant and the terbium complex of the apoenzyme . Trp(109), known from earlier work to be the source of room-temperature phosphorescence (RTP), emits a highly resolved low-temperature phosphorescence (LTP) spectrum and has the narrowest ODMR bands observed thus far from any protein site, revealing a uniquely homogeneous local environment . The decay kinetics of Trp(109) at 1.2 K reveals that the major triplet population (70%) undergoes inefficient crystallike spin-lattice relaxation by direct interaction with lattice phonons, the remainder being relaxed efficiently by local disorder modes . The latter population is smaller than is typical for protein sites, suggesting an unusual degree of local rigidity and order consistent with the long-lived RTP . Trp(220) emits a broader LTP spectrum originating to the blue of Trp(109) . It has typically broad ODMR bands consistent with local heterogeneity . The LTP of Trp(268) has an ill-defined origin blue shifted relative to Trp(220) and ODMR frequencies consistent with a greater degree of solvent exposure . Trp(268) has noticeable dispersion of its decay kinetics, consistent with quenching at the triplet level by a nearby disulfide residue. Biochemistry, 2001 Dec 11, 40(49), 15009 - 16 Product release during the first turnover of the ATP sulfurylase-GTPase; Sukal S et al.; ATP sulfurylase, from Escherichia coli Kappa-12, is a GTPase target complex that catalyzes and couples the chemical potentials of two reactions: GTP hydrolysis and activated sulfate (APS) synthesis . Previous work suggested that the product release branch of the GTPase mechanism might include rate-determining release and/or isomerization step(s) . Such steps are known to couple chemical potentials in other energy transducing systems . Rate-determining, product release step(s) were confirmed in the ATP sulfurylase-GTPase reaction by a burst of product in pre-steady-state, rapid-quench experiments . Classical rapid-quench experiments, which measure total product formation, do not allow the slow steps to be assigned to the release of a specific product, or to slow isomerization, because they do not distinguish solution-phase from enzyme-bound product . Assay systems that exclusively monitor solution-phase P(i) and GDP were used to obtain free product progress curves during the first turnover of ATP sulfurylase . Together, the free and total product data describe how the products partition between the enzyme surface and solution during the first turnover . In combination, the data provide the time dependence of the concentrations of specific product intermediates, AMP.PP(i).E.GDP.P(i) and AMP.PP(i).E.GDP, the rate constants for the release of P(i) (4.2 s(-1)) and GDP (4.8 s(-1)) from these complexes, respectively, and the equilibrium constant for the enzyme-bound, beta,gamma-bond cleavage reaction: {AMP.PP(i).E.GTP'}/{AMP.PP(i).E.GDP.P(i)} = 0.7 . The data are fit, using global analysis, to obtain a complete kinetic and energetic description of this GTPase reaction. Biochemistry, 2001 Dec 11, 40(49), 14968 - 75 Effect of sequence context on O(6)-methylguanine repair and replication in vivo; Delaney JC et al.; Understanding the origins of mutational hotspots is complicated by the intertwining of several variables . The selective formation, repair, and replication of a DNA lesion, such as O(6)-methylguanine (m(6)G), can, in principle, be influenced by the surrounding nucleotide environment . A nearest-neighbor analysis was used to address the contribution of sequence context on m(6)G repair by the Escherichia coli methyltransferases Ada or Ogt, and on DNA polymerase infidelity in vivo . Sixteen M13 viral genomes with m(6)G flanked by all permutations of G, A, T, and C were constructed and individually transformed into repair-deficient and repair-proficient isogenic cell strains . The 16 genomes were introduced in duplicate into 5 different cellular backgrounds for a total of 160 independent experiments, for which mutations were scored using a recently developed assay . The Ada methyltransferase demonstrated strong 5' and 3' sequence-specific repair of m(6)G in vivo . The Ada 5' preference decreased in the general order: GXN > CXN > TXN > AXN (X = m(6)G, N = any base), while the Ada 3' preference decreased in the order: NX(T/C) > NX(G/A), with mutation frequencies (MFs) ranging from 35% to 90% . The Ogt methyltransferase provided MFs ranging from 10% to 25% . As was demonstrated by Ada, the Ogt methyltransferase repaired m(6)G poorly in an AXN context . When both methyltransferases were removed, the MF was nearly 100% for all sequence contexts, consistent with the view that the replicative DNA polymerase places T opposite m(6)G during replication irrespective of the local sequence environment. Biochemistry, 2001 Dec 11, 40(49), 14932 - 41 Electron-transfer reactions of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath); Kopp DA et al.; Soluble methane monooxygenase (sMMO) catalyzes the hydroxylation of methane by dioxygen to afford methanol and water, the first step of carbon assimilation in methanotrophic bacteria . This enzyme comprises three protein components: a hydroxylase (MMOH) that contains a dinuclear nonheme iron active site; a reductase (MMOR) that facilitates electron transfer from NADH to the diiron site of MMOH; and a coupling protein (MMOB) . MMOR uses a noncovalently bound FAD cofactor and a {2Fe-2S} cluster to mediate electron transfer . The gene encoding MMOR was cloned from Methylococcus capsulatus (Bath) and expressed in Escherichia coli in high yield . Purified recombinant MMOR was indistinguishable from the native protein in all aspects examined, including activity, mass, cofactor content, and EPR spectrum of the {2Fe-2S} cluster . Redox potentials for the FAD and {2Fe-2S} cofactors, determined by reductive titrations in the presence of indicator dyes, are FAD(ox/sq), -176 +/- 7 mV; FAD(sq/hq), -266 +/- 15 mV; and {2Fe-2S}(ox/red), -209 +/- 14 mV . The midpoint potentials of MMOR are not altered by the addition of MMOH, MMOB, or both MMOH and MMOB . The reaction of MMOR with NADH was investigated by stopped-flow UV-visible spectroscopy, and the kinetic and spectral properties of intermediates are described . The effects of pH on the redox properties of MMOR are described and exploited in pH jump kinetic studies to measure the rate constant of 130 +/- 17 s(-)(1) for electron transfer between the FAD and {2Fe-2S} cofactors in two-electron-reduced MMOR . The thermodynamic and kinetic parameters determined significantly extend our understanding of the sMMO system. Biochemistry, 2001 Dec 11, 40(49), 14891 - 7 Effects of phospholipid headgroup and phase on the activity of diacylglycerol kinase of Escherichia coli; Pilot JD et al.; Diacylglycerol kinase (DGK) of Escherichia coli has been reconstituted into a variety of phospholipid bilayers and its activity determined as a function of lipid headgroup structure and phase preference . The anionic phospholipids dioleoylphosphatidic acid, dioleoylphosphatidylserine, and cardiolipin were all found to support activities lower than that supported by dioleoylphosphatidylcholine . In mixtures of dioleoylphosphatidylcholine and 20 mol % anionic phospholipids, the presence of anionic phospholipids all resulted in lower activities than in dioleoylphosphatidylcholine, except for dioleoylphosphatidylglycerol whose presence had little effect on activity . In some cases, the low activity in the presence of anionic phospholipid followed from a decrease in v(max); in some cases, it followed from an increase in the K(m) for diacylglycerol, and in the case of dioleoylphosphatidic acid, it followed from both . Activities in mixtures containing 80 mol % dioleoylphosphatidylethanolamine were lower than in dioleoylphosphatidylcholine at temperatures where both lipids adopted a bilayer phase; at higher temperatures where dioleoylphosphatidylethanolamine preferred a hexagonal H(II) phase, the differences in activity were greater . These experiments suggest that the presence of lipids preferring a hexagonal H(II) phase leads to low activities . Activities of DGK are low in a gel phase lipid. Biochemistry, 2001 Dec 11, 40(49), 14855 - 61 In vitro reconstitution and analysis of the chain initiating enzymes o