IUBMB Life, 1999 Oct, 48(4), 397 - 404 Adeno-associated virus Rep78 binds to E2-responsive element 1 of bovine papillomavirus type 1; Chon SK et al.; Adeno-associated virus type-2 (AAV-2) is a helper-dependent parvovirus that has been implicated in the inhibition of replication and oncogenic transformation of bovine papillomavirus type-1 (BPV-1) and other transforming DNA viruses . Previous studies have suggested that the Rep78 protein of AAV-2 is a key player mediating this effect . In this report we have analyzed the effect of AAV-2 Rep78 protein on the regulation of gene expression of a reporter gene under the control of the long control region (LCR) of BPV-1 . Our results show that Rep78 is capable of down-regulating the promoter activity of the LCR in vivo in tissue culture cells . Inhibition of LCR activity in vivo suggested the need for Rep78 to bind to a region of the LCR promoter spanning the E2-responsive elements of BPV-1 . This observation was further confirmed in vitro with gel shift assays showing specific binding of Rep78 to DNA oligonucleotides containing E2-responsive element 1 (E2RE1) sequences of BPV-1 LCR . Our results expand the understanding of the mechanism of trans-regulation mediated by Rep78 and involving this protein and DNA sequences with complex secondary structure. J Pharm Pharmacol, 1999 Nov, 51(11), 1267 - 73 Immunomodulatory effect of arctigenin, a lignan compound, on tumour necrosis factor-alpha and nitric oxide production, and lymphocyte proliferation; Cho JY et al.; We have investigated the immunomodulatory effects of arctigenin, a dibenzyl butyrolactone lignan compound, on tumour necrosis factor (TNF)-alpha and nitric oxide (NO) production, and lymphocyte proliferation . Arctigenin inhibited strongly TNF-alpha production by lipopolysaccharide-stimulated murine macrophage RAW264.7 and differentiated human macrophage U937 with IC50 values of 5.0 and 3.9 microM, respectively, without displaying cytotoxicity . The TNF-alpha inhibitory effect of arctigenin in lipopolysaccharide-triggered RAW264.7 cells was increased by co-treatment with several known TNF-alpha inhibitors . It also potently attenuated T and B cell proliferation stimulated by concanavalin A and lipopolysaccharide in a dose-dependent manner with IC50 values of 2.9 and 14.6 microM, respectively . In contrast, the compound showed a different pattern in lipopolysaccharide- and interferon (IFN)-gamma-induced NO production from RAW264.7 cells . Arctigenin inhibited NO release by IFN-gamma signal, whereas it significantly enhanced lipopolysaccharide-triggered NO production in RAW264.7 cells . The results suggested that arctigenin may regulate immune responses in activated macrophages and lymphocytes including TNF-alpha and NO production and lymphocyte proliferation. J Am Soc Mass Spectrom, 2000 Jan, 11(1), 78 - 82 Proteome analysis using selective incorporation of isotopically labeled amino acids; Veenstra TD et al.; A method is described for identifying intact proteins from genomic databases using a combination of accurate molecular mass measurements and partial amino acid content . An initial demonstration was conducted for proteins isolated from Escherichia coli (E . coli) using a multiple auxotrophic strain of K12 . Proteins extracted from the organism grown in natural isotopic abundance minimal medium and also minimal medium containing isotopically labeled leucine (Leu-D10), were mixed and analyzed by capillary isoelectric focusing (CIEF) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR) . The incorporation of the isotopically labeled Leu residue has no effect on the CIEF separation of the protein, therefore both versions of the protein are observed within the same FTICR spectrum . The difference in the molecular mass of the natural isotopic abundance and Leu-D10 isotopically labeled proteins is used to determine the number of Leu residues present in that particular protein . Knowledge of the molecular mass and number of Leu residues present can be used to unambiguously identify the intact protein . Preliminary results show the efficacy of this method for unambiguously identifying proteins isolated from E . coli. Chem Biol, 1999 Dec, 6(12), 901 - 8 Structural elucidation of the binding and inhibitory properties of lanthanide (III) ions at the 3'-5' exonucleolytic active site of the Klenow fragment; Brautigam CA et al.; BACKGROUND: Biochemical and biophysical experiments have shown that two catalytically essential divalent metal ions (termed 'A' and 'B') bind to the 3'-5' exonuclease active site of the Klenow fragment (KF) of Escherichia coli DNA polymerase I . X-ray crystallographic studies have established the normal positions in the KF 3'-5' exonuclease (KF exo) active site of the two cations and the single-stranded DNA substrate . Lanthanide (III) luminescence studies have demonstrated, however, that only a single europium (III) ion (Eu3+) binds to the KF exo active site . Furthermore, Eu3+ does not support catalysis by KF exo or several other two-metal-ion phosphoryl-transfer enzymes . RESULTS: A crystal structure of KF complexed with both Eu3+ and substrate single-stranded oligodeoxynucleotide shows that a lone Eu3+ is bound near to metal-ion site A . Comparison of this structure to a relevant native structure reveals that the bound Eu3+ causes a number of changes to the KF exo active site . The scissile phosphate of the substrate is displaced from its normal position by about 1 A when Eu3+ is bound and the presence of Eu3+ in the active site precludes the binding of the essential metal ion B . CONCLUSIONS: The substantial, lanthanide-induced differences in metal-ion and substrate binding to KF exo account for the inhibition of this enzyme by Eu3+ . These changes also explain the inability of KF exo to bind more than one cation in the presence of lanthanides . The mechanistic similarity between KF exo and other two-metal-ion phosphoryl-transfer enzymes suggests that the principles of lanthanide (III) ion binding and inhibition ascertained from this study will probably apply to most members of this class of enzymes. Biophys Chem, 2000 Jan 10, 83(1), 73 - 8 Structural stability of ribosomes subjected to RNase treatment evidenced by dielectric spectroscopy and differential scanning microcalorimetry; Blasi M et al.; Previous studies from our laboratory demonstrated the existence of at least two levels of structural complexity in E . coli 70S ribosomes . Ribosomal RNA seems to be principally involved in the overall stability of these structures . In this paper we present an investigation of ribosomes subjected to treatment with RNase . The study is based on both differential scanning microcalorimetry and dielectric spectroscopy . In the thermograms obtained on treated ribosomes only the low temperature peak of the two typical denaturation events observed in native ribosomes, is promptly eliminated by the enzyme treatment . Dielectric spectroscopy measurements carried out on the same samples indicate an alteration of the dielectric behavior previously shown to consist of two subsequent relaxation processes . In fact, only the low frequency relaxation is affected by the treatment . The second one, observed at higher frequency, remains unaltered . The same effect on the dielectric parameters is observed if the ribosome particles are heated and then cooled prior to measurement . These results are consistent with the idea that two different structures are present within the ribosome . One is very stable and withstands both temperature and RNase treatment while the second is promptly abolished by both treatments . Data presented here strongly suggest that the RNA domains exposed to the solvent play a fundamental role in the stability of the 3-D structure of the ribosome particle. Mutat Res, 1999 Dec 6, 430(2), 221 - 8 Impact of microgravity on radiobiological processes and efficiency of DNA repair; Horneck G; To study the influence of microgravity on radiobiological processes in space, space experiments have been performed, using an on-board 1xg reference centrifuge as in-flight control . The trajectory of individual heavy ions was localized in relation to the biological systems by use of the Biostack concept, or an additional high dose of radiation was applied either before the mission or during the mission from an on-board radiation source . In embryonic systems, such as early developmental stages of Drosophila melanogaster and Carausius morosus, the occurrence of chromosomal translocations and larval malformations was dramatically increased in response to microgravity and radiation . It has been hypothesized that these synergistic effects might be caused by an interference of microgravity with DNA repair processes . However, recent studies on bacteria, yeast cells and human fibroblasts suggest that a disturbance of cellular repair processes in the microgravity environment might not be a complete explanation for the reported synergism of radiation and microgravity . As an alternative explanation, an impact of microgravity on signal transduction, on the metabolic/physiological state or on the chromatin structure at the cellular level, or modification of self-assembly, intercellular communication, cell migration, pattern formation or differentiation at the tissue and organ level should be considered. Biochemistry, 2000 Jan 18, 39(2), 348 - 55 Translesion replication by DNA polymerase delta depends on processivity accessory proteins and differs in specificity from DNA polymerase beta; Daube SS et al.; Mutations caused by DNA damage lead to the development of cancer . The critical step in the formation of these mutations is the replication of unrepaired lesions in DNA by DNA polymerases, a process termed translesion replication . Using a newly developed method for preparation of gapped plasmids, containing a site-specific synthetic abasic site, we analyzed translesion replication with purified mammalian DNA polymerases delta and beta . DNA polymerase delta was found to be unable to replicate through the abasic site . Addition of the sliding DNA clamp PCNA, the clamp loader RFC, and ATP caused a drastic 30-fold increase in translesion replication . Thus, similar to Escherichia coli DNA polymerase III, the processivity accessory proteins enable DNA polymerase delta to bypass blocking lesions . Under comparable conditions, DNA polymerase beta was unable to bypass the abasic site, unless its concentration was greatly increased . Analysis of translesion replication products revealed a marked difference in the specificity of bypass: whereas 90% of bypass events by DNA polymerase delta holoenzyme involved insertion of a dAMP residue opposite the abasic site, DNA polymerase beta tended to skip over the abasic site, producing mainly minus frameshifts (73%) . The significance of these results for in vivo translesion replication is discussed. Biochemistry, 2000 Jan 18, 39(2), 332 - 9 Identification of the magnesium ion binding site in the catalytic center of Escherichia coli primase by iron cleavage; Godson GN et al.; Magnesium is essential for the catalysis reaction of Escherichia coli primase, the enzyme synthesizing primer RNA chains for initiation of DNA replication . To map the Mg(2+) binding site in the catalytic center of primase, we have employed the iron cleavage method in which the native bound Mg(2+) ions were replaced with Fe(2+) ions and the protein was then cleaved in the vicinity of the metal binding site by adding DTT which generated free hydroxyl radicals from the bound iron . Three Fe(2+) cleavages were generated at sites designated I, II, and III . Adding Mg(2+) or Mn(2+) ions to the reaction strongly inhibited Fe(2+) cleavage; however, adding Ca(2+) or Ba(2+) ions had much less effect . Mapping by chemical cleavage and subsequent site-directed mutagensis demonstrated that three acidic residues, Asp345 and Asp347 of a conserved DPD sequence and Asp269 of a conserved EGYMD sequence, were the amino acid residues that chelated Mg(2+) ions in the catalytic center of primase . Cleavage data suggested that binding to D345 is significantly stronger than to D347 and somewhat stronger than to D269. Biochemistry, 2000 Jan 18, 39(2), 307 - 15 Quaternary structure of the HSC70 cochaperone HIP; Velten M et al.; HSC70 interacting protein (HIP) is an essential cytoplasmic cochaperone involved in the regulation of HSC70 chaperone activity and the maturation of progesterone receptor . To determine the quaternary structure and the gross conformation of the protein in solution, a wide array of biochemical and biophysical techniques has been used . Size-exclusion chromatography and sedimentation velocity indicate the presence of a single species with a Stokes radius, R(s), of 55 A and a sedimentation coefficient, s degrees (20,w), of 4.34 S . The combination of these data gives a molecular mass of 101 000 Da, a value close to that of the theoretical molecular mass of a dimer (87 090 Da) . Sedimentation equilibrium, performed at various protein concentrations and rotor speeds, gives a molecular mass of 88 284 Da, almost in exact agreement with the molecular mass of a dimer . On the basis of these data, a frictional ratio f/f(0) of 1.6 is obtained, suggesting an elongated shape for the HIP dimer . Secondary structure predictions, supported by circular dichroism experiments, indicate that HIP is an almost all alpha-protein, able to form extended coiled coils . Using threading and comparative model building methods, a structural model of a segment of HIP involved in HSC70 binding has been constructed and potential sites of interaction between HIP and HSC70 are proposed on the basis of electrostatic as well as shape complementarity . Altogether, these results indicate that HIP is an elongated dimer, able to bind two HSC70 molecules through its TPR regions, and suggest the existence of a versatile binding site on HSC70 that may be involved in the interaction of the chaperone with the cochaperones or other interacting proteins. J Med Virol, 2000 Mar, 60(3), 275 - 83 Cloning, sequencing, and expression of the hepatitis E virus (HEV) nonstructural open reading frame 1 (ORF1); Ansari IH et al.; Hepatitis E virus (HEV) causes enterically transmitted epidemic and sporadic viral hepatitis affecting millions of people in the developing world . Different geographical isolates of HEV show a high degree of homology at the nucleotide and amino acid levels . The approximately 7.2 kb RNA genome has three open reading frames of which ORF1 is predicted to code for the viral nonstructural polyprotein . The expression, processing and properties of the nonstructural ORF1 polyprotein have not been reported so far . In this study, the complete HEV ORF1 was reconstructed from overlapping fragments amplified by polymerase chain reaction (PCR) of total RNA isolated from the bile fluid of a rhesus monkey experimentally infected with HEV isolate from an epidemic . The complete assembled ORF1 was sequenced using HEV specific primers . The ORF1 polyprotein was expressed in E . coli, in a cell free translation system and in HepG2 cells, and was characterized by western blotting and immunoprecipitation using acute phase patient serum as well as polyclonal antibodies raised against defined parts of the ORF1 polyprotein . The nonstructural polyprotein of HEV was expressed as a 186 kDa protein . No processing was observed into discrete units, either in-vitro based on a kinetic analysis, or in HepG2 cells based on immunoprecipitation . J Biochem Mol Toxicol, 2000, 14(2), 118 - 20 Flavin-containing monooxygenase isoform specificity for the N-oxidation of tamoxifen determined by product measurement and NADPH oxidation; Hodgson E et al.; The Km value for tamoxifen is 1.2 mM for mouse FMO1 (human FMO1 is not expressed in adults) and 1.4 mM for human FMO3, with no detectable activity being expressed toward tamoxifen by FMO5 from either mouse or human . These data are derived from experiments using 3H-tamoxifen as substrate in which the product, tamoxifen N-oxide, was measured directly . It was not possible to derive meaningful data from the measurement of NADPH consumption because Escherichia coli preparations, in the presence of tamoxifen, regardless of whether the E . coli was expressing an FMO isoform, consumed large amounts of NADPH without the appearance of tamoxifen N-oxide or other discernable product. Exp Mol Med, 1999 Dec 31, 31(4), 210 - 6 Characterization of yeast deoxyhypusine synthase: PKC-dependent phosphorylation in vitro and functional domain identification; Kang KR et al.; The biosynthesis of hypusine {Nepsilon-(4-amino-2-hydroxybutyl)-lysine} occurs in the eIF-5A precursor protein through two step posttranslational modification involving deoxyhypusine synthase which catalyzes transfer of the butylamine moiety of spermidine to the epsilon-amino group of a designated lysine residue and subsequent hydroxylation of this intermediate . This enzyme is exclusively required for cell viability and growth of yeast (Park, M.H . et al., J . Biol . Chem . 273: 1677-1683, 1998) . In an effort to understand structure-function relationship of deoxyhypusine synthase, posttranslational modification(s) of the enzyme by protein kinases were carried out for a possible cellular modulation of this enzyme . And also twelve deletion mutants were constructed, expressed in E . coli system, and enzyme activities were examined . The results showed that deoxyhypusine synthase was phosphorylated by PKC in vitro but not by p56lck and p60c-src . Treatment with PMA specifically increased the relative phosphorylation of the enzyme supporting PKC was involved . Phosphoamino acid analysis of this enzyme revealed that deoxyhypusine synthase is mostly phosphorylated on serine residue and weakly on threonine . Removal of Met1-Glu10 (deltaMet1-Glu10) residues from amino terminal showed no effect on the catalytic activity but further deletion (deltaMet1-Ser20) caused loss of enzyme activity . The enzyme with internal deletion, deltaGln197-Asn212 (residues not present in the human enzyme) was found to be inactive . Removal of 5 residues from carboxyl terminal, deltaLys383-Asn387, retained only slight activity . These results suggested that deoxyhypusine synthase is substrate for PKC dependent phosphorylation and requires most of the polypeptide chains for enzyme activity except the first 15 residues of N-terminal despite of N- and C-terminal residues of the enzyme consist of variable regions. Biol Bull, 1999 Dec, 197(3), 368 - 76 The role of latero-frontal cirri in particle capture by the gills of Mytilus edulis Silverman H, Lynn JW, Beninger PG, Dietz TH. In this study we examined the mechanism of particle capture in Mytilus edulis, using radioactive-label clearance studies, progressive fixation, and scanning electron microscopy to visualize in detail the cirri and their range of motion . Confocal laser scanning microscopy was used to observe the interaction of cirri with 1 mucrom fluorescent latex particles on live strips of control and serotonin-treated isolated gill tissue . The gills of M . edulis possess large, complex latero-frontal cirri composed of 18-26 pairs of cilia . Particles that were intercepted by the cirri were transferred to the water current on the frontal surface of the filament where they were propelled toward the ventral particle groove . Clearance studies demonstrated that M . edulis removed Escherichia coli from 5 degrees C seawater bathing medium at 4.9 ml g(-1) dry tissue min(-1) . When the gills were exposed to 10(-3) M serotonin, the latero-frontal cirri stopped moving and became fixed in a flexed position that partially blocked the frontal surface of the filament . Clearance studies demonstrated that removal of E . coli from the seawater bathing medium was reduced 90% to 0.5 ml g(-1) dry tissue min(-1) when 10(-3) M serotonin was present . These data demonstrated that for small particles (< 2 microm) in the near field, movement of cirri was essential for successful capture either by direct contact or with water acting as a hydromechanical coupler. Comp Biochem Physiol A Mol Integr Physiol, 1999 Oct, 124(2), 169 - 78 Effect of short-chain fatty acids on cyclic 3',5'-guanosine monophosphate-mediated colonic secretion; Charney AN et al.; Short chain fatty acids (SCFA) prevent and reverse cyclic 3',5'-adenosine monophosphate (cAMP) but not Ca(2+)-mediated Cl- secretion . Mucosal {HCO3-}i has an opposite effect on these secretagogues . We examined whether SCFA and {HCO3-}i affect cyclic 3',5'-guanosine monophosphate (cGMP)-induced secretion . Stripped segments of male Sprague-Dawley rat (Rattus norvegicus) proximal and distal colon, and cultured T84 cells were studied in Using chambers, and pHi and {HCO3-}i were determined . Mucosal {cGMP} was measured in proximal colon . In T84 cells, the increase in Cl- secretion (measured as Isc) induced by mucosal 0.25 microM Escherichia coli heat-stable enterotoxin (STa) was prevented/reversed by bilateral 50 mM Na+ butyrate (71%/73%), acetate (58%/76%), propionate (68%/73%) and (poorly metabolized) isobutyrate (80%/79%) . In proximal colon in HCO3- Ringer, basal Cl- secretion was not affected by {HCO3-}i or 25 mM butyrate . Mucosal 0.25 microM STa decreased net Na+ and Cl- absorption . Bilateral but not mucosal 25 mM SCFA reversed STa-induced effects on Na+ absorption and Cl- secretion . Bilateral and mucosal 25 mM SCFA but not {HCO3-}i prevented STa-induced Cl- secretion and increases in mucosal {cGMP} . STa did not produce Cl- secretion in distal colon . It was concluded that SCFA but not {HCO3-}i can prevent and reverse cGMP-induced colonic Cl- secretion. Carbohydr Res, 1999 Nov 23, 322(1-2), 57 - 66 Structural characterization of the antigenic O-chain of the lipopolysaccharide of Escherichia coli serotype O65; Perry MB et al.; The antigenic O-polysaccharide moiety of the lipopolysaccharide produced by Escherichia coli serotype O65 was investigated by composition, methylation, base hydrolysis, periodate oxidation, mass spectrometric methods, and by 1D and 2D NMR spectroscopy . The O-polysaccharide had {alpha}D + 108 degrees (water) and is a high-molecular-weight unbranched linear polymer of repeating pentasaccharide units composed of 1:1:1:1:1 D-galacturonic acid (D-GalA), D-galacturonamide (D-GalANH2), 2-acetamido-2-deoxy-D-glucose (D-GlcNAc), 2-acetamido-2-deoxy-D-galactose (D-GalNAc), and 3-acetamido-3,6-dideoxy-D-glucose (D-Qui3NAc), and has the following structure: {formula: see text} J Dairy Sci, 1999 Dec, 82(12), 2582 - 8 Efficacy of intramammary immunization with an Escherichia coli J5 bacterin; Smith JL et al.; Intramammary immunization was investigated as a procedure to reduce the clinical signs of coliform mastitis . Twenty-four cows were equally distributed to the following Escherichia coli J5 immunization schedules: 1) Subcutaneous injection 14 d prior to the end of lactation, intramammary immunization 7 d after drying off, and subcutaneous injection 30 d into the dry period; 2) subcutaneous injections at drying off, at 30 d into the dry period, and within 12 h after calving; and 3) unimmunized controls . Intramammary immunizations were the infusion of vaccine via the teat canal into each of the four mammary glands . Cows were challenged by infusion of E . coli 727 into one uninfected mammary quarter at approximately 30 d after calving . Intramammary immunization enhanced antibody titers against E . coli J5 and E . coli 727 compared with subcutaneous immunization . Immunoglobulin G titers against E . coli J5 and E . coli 727 in whey were greater at the time of challenge and 7 d after challenge for cows that received the intramammary immunization than for cows immunized by only subcutaneous injections . Serum IgG titers against E . coli 727 were enhanced at 7 d after challenge for cows receiving intramammary immunizations compared with conventionally immunized cows . Serum IgM titers against E . coli 727 were higher at calving for cows receiving intramammary immunization compared with conventionally immunized cows . Immunization schedule had minimal effect on systemic and local signs of clinical mastitis following challenge. J Dairy Sci, 1999 Dec, 82(12), 2574 - 81 Nitric oxide production during endotoxin-induced mastitis in the cow; Bouchard L et al.; Nitric oxide production was measured during endotoxin-induced mastitis . One hour after morning milking, the right hind quarters of 15 cows were infused with saline containing Escherichia coli endotoxin . Left hind control quarters were infused with saline only . At varying intervals before and after infusion, diagnostic markers of mastitis were recorded and nitric oxide production was evaluated by measuring nitrite plus nitrate levels in milk . In endotoxin-infused quarters, a significant increase in nitrite plus nitrate concentrations was observed 3 h postinfusion; concentrations decreased to preinfusion levels within 48 h . This change indicates that significant amounts of nitric oxide are released during endotoxin-induced mastitis . At 3 different time points, somatic cells were harvested from milk samples, plated, and maintained in culture for 24 h . The concentration of nitrite plus nitrate in medium from cells harvested 12 h postinfusion was increased, suggesting that nitric oxide is released, at least in part, by milk somatic cells . In a second set of experiments, we evaluated nitric oxide production when animals were infused with endotoxin and aminoguanidine, a specific inhibitor of the inducible form of nitric oxide synthase . In cows treated with aminoguanidine, the increase in nitrite plus nitrate observed after endotoxin infusion was prevented . These results suggest that nitric oxide production during endotoxin-induced mastitis resulted from the activity of the inducible form of nitric oxide synthase . They also support a possible involvement for nitric oxide in the inflammatory reaction observed during mastitis. Gynecol Obstet Invest, 2000, 49(1), 70 - 2 Xanthogranulomatous tubo-ovarian abscess resulting from chronic diverticulitis; Mesia AF et al.; We report a case of xanthogranulomatous tubo-ovarian abscess which was preoperatively suspected to be an adnexal neoplasm . With foreign body material found in the abscess wall and vegetable fiber in the tubal lumen, a previously treated chronic diverticulitis was the presumed cause . Culture studies showed polymicrobial isolates which included Escherichia coli, an enteric pathogen . After surgery, administration of antibiotics, and revision of delayed subcutaneous wound healing, the patient is reportedly well . J Bacteriol, 2000 Jan, 182(2), 536 - 9 The feedback response of Escherichia coli rRNA synthesis is not identical to the mechanism of growth rate-dependent control; Voulgaris J et al.; Growth rate-independent rrn P1 promoter mutants were tested for their ability to respond to changes in rrn gene dosage . Most were found to be normal for the feedback response . In addition, cellular levels of the initiating nucleoside triphosphates remained unchanged when the rrn gene dosage was altered . These results suggest that the feedback response cannot be the mechanism for growth rate-dependent control of rRNA synthesis and that the relationship between these two processes may be more complicated than is currently understood. J Bacteriol, 2000 Jan, 182(2), 529 - 31 Overexpression of the RNA polymerase alpha subunit reduces transcription of Bvg-activated virulence genes in Bordetella pertussis; Carbonetti NH et al.; Overexpression of the RNA polymerase alpha subunit in Bordetella pertussis reduces expression of the virulence factor pertussis toxin . Here we show that this reduction is at the level of transcription, is reversed by overexpression of the transcriptional activator BvgA, and is dependent on the C-terminal domain of alpha. J Bacteriol, 2000 Jan, 182(2), 498 - 503 The N-terminal region of the Escherichia coli WecA (Rfe) protein, containing three predicted transmembrane helices, is required for function but not for membrane insertion; Amer AO et al.; The correct site for translation initiation for Escherichia coli WecA (Rfe), presumably involved in catalyzing the transfer of N-acetylglucosamine 1-phosphate to undecaprenylphosphate, was determined by using its FLAG-tagged derivatives . The N-terminal region containing three predicted transmembrane helices was found to be necessary for function but not for membrane localization of this protein. J Bacteriol, 2000 Jan, 182(2), 463 - 8 Recombination is essential for viability of an Escherichia coli dam (DNA adenine methyltransferase) mutant; Marinus MG; Double mutants of Escherichia coli dam (DNA adenine methyltransferase) strains with ruvA, ruvB, or ruvC could not be constructed, whereas dam derivatives with recD, recF, recJ, and recR were viable . The ruv gene products are required for Holliday junction translocation and resolution of recombination intermediates . A dam recG (Holliday junction translocation) mutant strain was isolated but at a very much lower frequency than expected . The inviability of a dam lexA (Ind(-)) host was abrogated by the simultaneous presence of plasmids encoding both recA and ruvAB . This result indicates that of more than 20 SOS genes, only recA and ruvAB need to be derepressed to allow for dam mutant survival . The presence of mutS or mutL mutations allowed the construction of dam lexA (Ind(-)) derivatives . The requirement for recA, recB, recC, ruvA, ruvB, ruvC, and possibly recG gene expression indicates that recombination is essential for viability of dam bacteria probably to repair DNA double-strand breaks . The effect of mutS and mutL mutations indicates that DNA mismatch repair is the ultimate source of most of these DNA breaks . The requirement for recombination also suggests an explanation for the sensitivity of dam cells to certain DNA-damaging agents. J Bacteriol, 2000 Jan, 182(2), 456 - 62 Constitutive expression of a transcription termination factor by a repressed prophage: promoters for transcribing the phage HK022 nun gene; King RA et al.; Lysogens of phage HK022 are resistant to infection by phage lambda . Lambda resistance is caused by the action of the HK022 Nun protein, which prematurely terminates early lambda transcripts . We report here that transcription of the nun gene initiates at a constitutive prophage promoter, P(Nun), located just upstream of the protein coding sequence . The 5' end of the transcript was determined by primer extension analysis of RNA isolated from HK022 lysogens or RNA made in vitro by transcribing a template containing the promoter with purified Escherichia coli RNA polymerase . Inactivation of P(Nun) by mutation greatly reduced Nun activity and Nun antigen in an HK022 lysogen . However, a low level of residual activity was detected, suggesting that a secondary promoter also contributes to nun expression . We found one possible secondary promoter, P(Nun)', just upstream of P(Nun) . Neither promoter is likely to increase the expression of other phage genes in a lysogen because their transcripts should be terminated downstream of nun . We estimate that HK022 lysogens in stationary phase contain several hundred molecules of Nun per cell and that cells in exponential phase probably contain fewer. J Bacteriol, 2000 Jan, 182(2), 371 - 6 Viability of an Escherichia coli pgsA null mutant lacking detectable phosphatidylglycerol and cardiolipin; Kikuchi S et al.; Phosphatidylglycerol, the most abundant acidic phospholipid in Escherichia coli, has been considered to play specific roles in various cellular processes and is believed to be essential for cell viability . It is functionally replaced in some cases by cardiolipin, another abundant acidic phospholipid derived from phosphatidylglycerol . However, we now show that a null pgsA mutant is viable, if the major outer membrane lipoprotein is deficient . The pgsA gene normally encodes phosphatidylglycerophosphate synthase that catalyzes the committed step in the biosynthesis of these acidic phospholipids . In the mutant, the activity of this enzyme and both phosphatidylglycerol and cardiolipin were not detected (less than 0.01% of total phospholipid, both below the detection limit), although phosphatidic acid, an acidic biosynthetic precursor, accumulated (4.0%) . Nonetheless, the null mutant grew almost normally in rich media . In low-osmolarity media and minimal media, however, it could not grow . It did not grow at temperatures over 40 degrees C, explaining the previous inability to construct a null pgsA mutant (W . Xia and W . Dowhan, Proc . Natl . Acad . Sci . USA 92:783-787, 1995) . Phosphatidylglycerol and cardiolipin are therefore nonessential for cell viability or basic life functions . This notion allows us to formulate a working model that defines the physiological functions of acidic phospholipids in E . coli and explains the suppressing effect of lipoprotein deficiency. J Bacteriol, 2000 Jan, 182(2), 337 - 47 Regulation of stalk elongation by phosphate in Caulobacter crescentus; Gonin M et al.; In Caulobacter crescentus, stalk biosynthesis is regulated by cell cycle cues and by extracellular phosphate concentration . Phosphate-starved cells undergo dramatic stalk elongation to produce stalks as much as 30 times as long as those of cells growing in phosphate-rich medium . To identify genes involved in the control of stalk elongation, transposon mutants were isolated that exhibited a long-stalk phenotype irrespective of extracellular phosphate concentration . The disrupted genes were identified as homologues of the high-affinity phosphate transport genes pstSCAB of Escherichia coli . In E . coli, pst mutants have a constitutively expressed phosphate (Pho) regulon . To determine if stalk elongation is regulated by the Pho regulon, the Caulobacter phoB gene that encodes the transcriptional activator of the Pho regulon was cloned and mutated . While phoB was not required for stalk synthesis or for the cell cycle timing of stalk synthesis initiation, it was required for stalk elongation in response to phosphate starvation . Both pstS and phoB mutants were deficient in phosphate transport . When a phoB mutant was grown with limiting phosphate concentrations, stalks only increased in length by an average of 1.4-fold compared to the average 9-fold increase in stalk length of wild-type cells grown in the same medium . Thus, the phenotypes of phoB and pst mutants were opposite . phoB mutants were unable to elongate stalks during phosphate starvation, whereas pst mutants made long stalks in both high- and low-phosphate media . Analysis of double pst phoB mutants indicated that the long-stalk phenotype of pst mutants was dependent on phoB . In addition, analysis of a pstS-lacZ transcriptional fusion showed that pstS transcription is dependent on phoB . These results suggest that the signal transduction pathway that stimulates stalk elongation in response to phosphate starvation is mediated by the Pst proteins and the response regulator PhoB. J Bacteriol, 2000 Jan, 182(2), 272 - 7 Anaerobic toluene catabolism of Thauera aromatica: the bbs operon codes for enzymes of beta oxidation of the intermediate benzylsuccinate; Leuthner B et al.; The pathway of anaerobic toluene oxidation to benzoyl coenzyme A (benzoyl-CoA) consists of an initial reaction catalyzed by benzylsuccinate synthase, a glycyl radical enzyme adding the methyl group of toluene to the double bond of a fumarate cosubstrate, and a subsequent beta-oxidation pathway of benzylsuccinate . Benzylsuccinate synthase has been studied in some detail, whereas the enzymes participating in beta oxidation of benzylsuccinate are unknown . We have investigated these enzymes by analyzing substrate-induced proteins in toluene-grown cells . Toluene-induced proteins were identified and N-terminally sequenced . Nine of these proteins are encoded by an 8.5-kb operon consisting of bbs (beta-oxidation of benzylsuccinate) genes whose products are apparently involved in the beta-oxidation pathway of benzylsuccinate . Two of the genes, bbsE and bbsF, code for the subunits of a succinyl-CoA:benzylsuccinate CoA-transferase whose activity was previously detected in toluene-grown Thauera aromatica . The bbsG gene codes for a specific benzylsuccinyl-CoA dehydrogenase, as confirmed by overexpression of the gene in Escherichia coli and detection of enzyme activity . The further enzymes of the pathway are probably encoded by bbsH (enoyl-CoA hydratase), bbsCD (3-hydroxyacyl-CoA dehydrogenase), and bbsB (3-oxoacyl-CoA thiolase) . The operon contains two additional genes, bbsA and bbsI, for which no obvious function could be derived . The bbs operon is expressed only in toluene-grown cells and is regulated at the transcriptional level . Promoter mapping revealed a transcription start site upstream of the bbsA gene . This represents the first known promoter site in Thauera spp. J Dairy Res, 2000 Nov, 67(4), 485 - 502 Treatment of acute Escherichia coli mastitis in cows with enrofloxacin: effect on clinical signs and chemiluminescence of circulating neutrophils; Hoeben BD et al.; We have studied the effect of treatment with enrofloxacin on local and general clinical signs and chemiluminescence of circulating polymorphonuclear leucocytes during experimentally induced Escherichia coli mastitis in cows immediately afer parturition . Twelve cows were infected with 10(4) cfu Esch . coli P4:032 into both left quarters . Six cows received an intravenous injection of 5 mg enrofloxacin/kg at 10 h after infection and a second enrofloxacin treatment administered subcutaneously at 30 h post infection . The other six cows were controls that received no treatment . General clinical signs (fever, tachycardia, loss of appetite, reduced rumen motility and depression) were similar in both groups . Local clinical signs, such as swelling, pain and firmness of the inflamed mammary quarters, were less severe in the treated cows . We saw no difference in the appearance of the milk: flecks and watery or purulent milk were observed in both groups . The beneficial effects of treatment with enrofloxacin were mainly on milk production and composition . The decline in milk production and the changes in milk concentrations of lactose, Na+ and bovine serum albumin were less pronounced in the treated cows . Treatment with enrofloxacin accelerated the clearance of bacteria from the infected quarters, but had no effect on the chemiluminescence response of isolated polymorphonuclear leucocytes . The changes in the number of circulating leucocytes and the appearance of immature neutrophils in the circulation of the treated cows indicated possible beneficial effects on migration of neutrophils into the inflamed glands . Higher milk somatic cell counts in the treated cows supported this hypothesis . The results of this study indicated that treating cows that have been experimentally infected with Esch . coli mastitis after parturition with enrofloxacin reduced the severity of the disease, especially the decline in milk production and the changes in milk composition. Arch Microbiol, 2000 Nov, 174(5), 297 - 306 Molecular characterization of the cyanophycin synthetase from Synechocystis sp . strain PCC6308; Aboulmagd E et al.; A 3878-bp genomic region from the cyanobacterium Synechocystis sp . strain PCC6308, amplified by inverse PCR, harbored the structural genes cphA (2625 bp) and cphB (819 bp) encoding cyanophycin synthetase and cyanophycinase, respectively . Both primary structures exhibited a high degree of similarity to the corresponding translational products from other cyanobacteria . Five regions were localized in the cyanophycin synthetase consensus sequence by their resemblance to conserved sites of ATP-dependent carboxylate-amine/thiol ligases and three substrate ligases . The functionality of cphA was proven by heterologous expression of active enzyme and synthesis of cyanophycin in Escherichia coli, which led to a maximum cyanophycin content of 26.6% (w/w) of cell dry mass . Furthermore, a modified radiometric enzyme assay for a more reliable and feasible measurement of cyanophycin synthetase activity was developed and applied to reveal the substrate specificity of the enzyme. Res Microbiol, 2000 Nov, 151(9), 755 - 68 Exploring the frontier between life and death in Escherichia coli: evaluation of different viability markers in live and heat- or UV-killed cells; Villarino A et al.; A number of methods have been proposed to assess the viability of cells without culture . Each method is based on criteria that reflect different levels of cellular integrity or functionality . As a consequence, the interpretation of viability is often ambiguous . The purposes of this work were to evaluate the capacity of current viability markers to distinguish between live and dead Escherichia coli K-12 cells . Methods that assess 'viability' by the demonstration of metabolic activities (esterase activity, active electron transport chain, transport of glucose), cellular integrity (membrane integrity, presence of nucleic acids) or the building up of cellular material (cell elongation) have been evaluated in live and UV- or heat-killed cells . With live cells, viability markers detected cells in counts similar to the colony count . However, these so-called viability markers could stain dead cells for some time after the lethal treatment . For the UV-killed cells, residual activities were detected even after 48 h of storage at 20 degrees C . However, for heat-treated cells, these activities disappeared within hours after heat treatment . Only a combination of fluorescence in situ hybridization with rRNA probes and cell elongation in response to nutrients (in the presence of an inhibitor of cell division) had the ability to differentiate live from dead cells . Problems in the definition of a viable but nonculturable state are in part due to the lack of a clear definition of bacterial death . We consider death as an irreversible state where no growth, cell elongation or protein synthesis may occur. Res Microbiol, 2000 Nov, 151(9), 727 - 38 Progressive loss of lambda prophage recombinogenicity in UV-irradiated Escherichia coli: the role of RecBCD enzyme; Vlahovic K et al.; RecBCD enzyme is involved in the radiation-induced process known as prophage inactivation . The process leads to the inability of lambda prophage to excise itself from the Escherichia coli chromosome via site-specific recombination . In this work we sought to further characterize the role of RecBCD enzyme in this process . In addition, we examined the ability of irradiated prophage to recombine with the infecting homologous phage . We used several E . coli mutants differentially altered in RecBCD's activities . The results showed that in the mutants carrying either recB2109 or recD1903, which do not exhibit significant nuclease activities, the prophage progressively loses its capacity for both site-specific and general recombination . In the recB268 null mutant, however, prophage recombinogenicity remained fully preserved . We also showed that the prophage unable to recombine retained its ability to complement the mutant infecting phage and that the recombination frequencies in phage x phage crosses were not affected by postirradiation incubation . Our results suggest that the helicase activity of RecBCD is responsible for the progressive loss of prophage recombinogenicity . This loss is most probably a consequence of the unsuccessful RecBCD-dependent recombinational repair of double-stranded breaks in the cell chromosome, during which some structures unsuitable for further recombination reactions may be produced. Res Microbiol, 2000 Nov, 151(9), 721 - 5 Molecular characterization of the hlyX-like gene of Actinobacillus actinomycetemcomitans Y4; Kokeguchi S et al.; We isolated and characterized a possible regulatory gene, designated actX gene, from Actinobacillus actinomyctemcomitans Y4, which defined the Actinobacillus pleuropneumoniae hlyX-like regulatory gene . DNA sequence analysis for plasmid clone pKM317 containing a 1.6-kb DNA insert indicated an open reading frame encoding a polypeptide of 257 amino acid residues . Analysis of the deduced amino acid sequence showed the presence of five characteristic cysteine residues in the N-terminal region and a putative DNA binding residue in the C-terminal region, indicating that actX might belong to a regulatory gene family . Escherichia coli DH5alpha and a mutant strain JRG1728 transformed by plasmid carrying actX manifested apparent hemolytic activity on sheep blood agar and grew anaerobically, although the original strains did not. Comp Biochem Physiol B Biochem Mol Biol, 2000 Sep, 127(1), 65 - 73 GTP cyclohydrolase I from Tetrahymena pyriformis: cloning of cDNA and expression; Tazawa M et al.; A full-length cDNA clone for GTP cyclohydrolase I (EC 3.5.4.16) was isolated from a Tetrahymena pyriformis cDNA library by plaque hybridization . The nucleotide sequence determination revealed that the length of the cDNA insert was 1516 bp . The coding region encoded a protein of 223 amino acid residues with a calculated molecular mass of 25 416 Da . The deduced amino acid sequence of Tetrahrymena GTP cyclohydrolase I showed sequence identity with that of Escherichia coli (55%) . The identity of T . pyriformis GTP cyclohydrolase I with sequences of Dictyostelium discoideum, Saccharomyces cerevisiae, Drosophila melanogaster, mouse, rat, and human enzymes was less marked and was 30, 30, 25, 28, 28, and 27%, respectively . RNA blot analysis showed a single mRNA species of 2.1 kb in this protozoan . The mRNA level of GTP cyclohydrolase I increased during synchronous cell division induced by intermittent heat treatment . The results suggest that the mRNA expression is associated with the cell cycle of T . pyriformis. Comp Biochem Physiol B Biochem Mol Biol, 2000 Sep, 127(1), 31 - 44 An 18.5 kDa protein from the amebocyte of Limulus polyphemus, homologous to the previously described amebocyte aggregation factor, expresses alternative phospholipase A2 activity; MacPherson JC et al.; A protein expressing phospholipase A2 activity was purified from the granular amebocyte of the horseshoe crab, Limulus polyphemus by cation-exchange, size-exclusion chromatography and semi-preparative reverse-phase-high pressure liquid chromatography (RP-HPLC) . The protein had an apparent mass of 17.7 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), but a more accurate estimate of 18.5 kDa was assigned by electrospray ionization-mass spectrometry (ESI-MS) . A partial sequence of this protein demonstrated total sequence homology with an 18.5 kDa protein with cell aggregating properties from Limulus reported by Fujii et al . {J . Biol . Chem . 267:22452.} . In these studies, the Limulus protein demonstrated a positive cross-reaction to polyclonal anti-human recombinant phospholipase A2 (group II, 14 kDa) . The protein did not display a significant loss of biological activity after boiling, but all enzymatic activity was lost after boiling in the presence of the reducing agent betamercaptoethanol (beta-mercaptoethanol) . The Limulus protein was inhibited by manoalide, a covalent irreversible phospholipase A2 inhibitor, in a dose-dependent fashion with 50% inhibition occurring at a concentration of 0.48 microM . The Limulus protein displayed no activity in a triglyceride lipase assay . These studies characterize an alternative phospholipase A2 activity for the previously described 18.5 kDa protein from the L . polyphemus amebocyte. Acta Vet Scand, 2000, 41(3), 249 - 59 Identification of Escherichia coli recovered from milk of sows with coliform mastitis by random amplified polymorphic DNA (RAPD) using standardized reagents; Ramasoota P et al.; A standardized-reagents commercial kit for random amplified polymorphic DNA (RAPD) analysis was used for typing 58 Escherichia coli strains that were recovered from the milk of sows, having coliform mastitis, within a single swineherd in Sweden . Previously, the 58 E . coli strains were characterized serologically and profiled biochemically . They were also evaluated for their serum resistance and their ability to adhere to fibronectin and bovine fetal fibroblasts . The RAPD analysis was fast, easily performed, and required only a nanogram of DNA . The indistinguishable banding patterns obtained with repeated analyses of 2 isolates from each strain demonstrated that RAPD analysis using standardized beads is a technique that provides reproducible results for typing E . coli strains that cause mastitis in sows . The results of the RAPD analyses demonstrated that E . coli sow mastitis strains are highly variable in serotype, biochemical profiles, virulence factors, and RAPD type, and that all 58 strains can be differentiated by means of the RAPD technique . The strains grouped into 24 RAPD types by combining the results of 2 primers, and into 38 groups by combining the results of serotype and RAPD type . No relationship between serotypes, virulence factors and RAPD types was found. Ultramicroscopy, 2000 Dec, 85(4), 225 - 34 A robust algorithm for the reconstruction of helical filaments using single-particle methods; Egelman EH; Some of the earliest methods for three-dimensional reconstruction from electron microscopic images were developed for helical objects . Single-particle methods have been used with great success for the three-dimensional reconstruction of macromolecular assemblies that have no internal symmetry or closed point group symmetries . An approach is presented for the application of single-particle methods to helical filaments that surmounts many of the difficulties of helical image analysis, including indexing, unbending and the need to find long helically symmetric filament segments . It is shown using both human Rad51 and E . coli RecA nucleoprotein filaments that this approach converges without user intervention to a stable solution, and that it has the potential to overcome many of the problems associated with image analysis of disordered helical polymers . The method can be applied transparently to structures where Bessel overlap would greatly complicate helical analysis . In addition, the procedure allows for the ab initio determination of helical symmetry, when no prior knowledge exists. Nucleic Acids Res . 2001 Jan 1;29(1):277. PromEC: An updated database of Escherichia coli mRNA promoters with experimentally identified transcriptional start sites; Hershberg R et al.; PromEC is an updated compilation of Escherichia coli mRNA promoter sequences . It includes documentation on the location of experimentally identified mRNA transcriptional start sites on the E . coli chromosome, as well as the actual sequences in the promoter region . The database was updated as of July 2000 and includes 472 entries . PromEC is accessible at il/marg/promec Nucleic Acids Res, 2001 Jan 1, 29(1), 72 - 4 RegulonDB (version 3.2): transcriptional regulation and operon organization in Escherichia coli K-12; Salgado H et al.; RegulonDB is a database on mechanisms of transcription regulation and operon organization in Escherichia coli K-12 . The current version has considerably increased numbers of regulatory elements such as promoters, binding sites and terminators . The complete repertoire of known and predicted DNA-binding transcriptional regulators can be considered to be included in this version . The database now distinguishes different allosteric conformations of regulatory proteins indicating the one active in binding and regulating the different promoters . A new set of operon predictions has been incorporated . The relational design has been modified accordingly . Furthermore, a major improvement is a graphic display enabling browsing of the database with a Java-based graphic user interface with three zoom-levels connected to properties of each chromosomal element . The purpose of these modifications is to make RegulonDB a useful tool and control set for transcriptome experiments . RegulonDB can be accessed on the web at the URL: +regulondb/ Nucleic Acids Res, 2000 Dec 15, 28(24), 4912 - 8 Adenine excisional repair function of MYH protein on the adenine:8-hydroxyguanine base pair in double-stranded DNA; Shinmura K et al.; Adenine paired with 8-hydroxyguanine (oh(8)G), a major component of oxidative DNA damage, is excised by MYH base excision repair protein in human cells . Since repair activity of MYH protein on an A:G mismatch has also been reported, we compared the repair activity of His(6)-tagged MYH proteins, expressed in Spodoptera frugiperda Sf21 cells, on A:oh(8)G and A:G mismatches by DNA cleavage assay and gel mobility shift assay . We also compared the repair ability of type 1 mitochondrial protein with type 2 nuclear protein, as well as of polymorphic type 1-Q(324) and 2-Q(310) proteins with type 1-H(324) and 2-H(310) proteins by DNA cleavage assay and complementation assay of an Escherichia coli mutM mutY strain . In a reaction buffer with a low salt (0-50 mM) concentration, adenine DNA glycosylase activity of type 2 protein was detected on both A:oh(8)G and A:G substrates . However, in a reaction buffer with a 150 mM salt concentration, similar to physiological conditions, the glycosylase activity on A:G, but not on A:oh(8)G, was extremely reduced and the binding activity of type 2 protein for A:G, but not for A:oh(8)G, was proportionally reduced . The glycosylase activity on A:oh(8)G and the ability to suppress spontaneous mutagenesis were greater for type 2 than type 1 enzyme . There was apparently no difference in the repair activities between the two types of polymorphic MYH proteins . These results indicate that human MYH protein specifically catalyzes the glycosylase reaction on A:oh(8)G under physiological salt concentrations. Genetics, 2000 Jan, 154(1), 39 - 48 Prophage lambda induces terminal recombination in Escherichia coli by inhibiting chromosome dimer resolution . An orientation-dependent cis-effect lending support to bipolarization of the terminus; Corre J et al.; A prophage lambda inserted by homologous recombination near dif, the chromosome dimer resolution site of Escherichia coli, is excised at a frequency that depends on its orientation with respect to dif . In wild-type cells, terminal hyper- (TH) recombination is prophage specific and undetectable by a test involving deletion of chromosomal segments between repeats identical to those used for prophage insertion . TH recombination is, however, detected in both excision and deletion assays when Deltadif, xerC, or ftsK mutations inhibit dimer resolution: lack of specialized resolution apparently results in recombinogenic lesions near dif . We also observed that the presence near dif of the prophage, in the orientation causing TH recombination, inhibits dif resolution activity . By its recombinogenic effect, this inhibition explains the enhanced prophage excision in wild-type cells . The primary effect of the prophage is probably an alteration of the dimer resolution regional control, which requires that dif is flanked by suitably oriented (polarized) stretches of DNA . Our model postulates that the prophage inserted near dif in the deleterious orientation disturbs chromosome polarization on the side of the site where it is integrated, because lambda DNA, like the chromosome, is polarized by sequence elements . Candidate sequences are oligomers that display skewed distributions on each oriC-dif chromosome arm and on lambda DNA. Anticancer Res, 1999 Sep-Oct, 19(5B), 3907 - 14 Influence of polysaccharides from Viscum album L . on human lymphocytes, monocytes and granulocytes in vitro; Stein GM et al.; BACKGROUND: An acidic arabinogalactan from European mistletoe (Viscum album L, VAL; 1.34 x 10(6) Dalton) was studied in detail because its immunological properties are poorly characterised . MATERIALS AND METHODS: Flow cytometric studies focussed on PS-activated proliferation of human lymphocytes measured via incorporation of bromo-deoxyuridine (BrdU), granulocyte phagocytosis via ingestion of FITC-labelled E.coli, and respiratory burst via oxidation of dihydrorhodamine 123 to rhodamine 123 . Cytokines were detected in the cell culture supernatants by ELISA . RESULTS: PS, in contrast to mistletoe lectins (ML), significantly stimulated proliferation of CD4+ T-cells but not CD8+ and CD19+ cells . However, ML influenced PS-mediated stimulation, with a synergistic effect in one and an inhibitory effect in another individual . Furthermore, IFN-gamma release was significantly enhanced by PS, favouring a T-helper cell type-1 cytokine pattern, further IL-6 was significantly stimulated, while granulocyte activity was not affected . CONCLUSIONS: VAL-PS exert yet unknown stimulatory activities, especially on specific CD4+ T-cells which may be influenced by other extract components like the ML . These components may contribute to the anti-tumour effect of VAL. Mol Gen Genet, 1999 Dec, 262(4-5), 876 - 83 Growth phase-dependent regulation of nuoA-N expression in Escherichia coli K-12 by the Fis protein: upstream binding sites and bioenergetic significance; Wackwitz B et al.; The expression of the nuoA-N operon of Escherichia coli K-12, which encodes the proton-pumping NADH dehydrogenase I is modulated by growth phase-dependent regulation . Under respiratory growth conditions, expression was stimulated in early exponential, and to a lesser extent in late exponential and stationary growth phases . The stimulation in the early exponential growth phase was not observed in fis mutants, which are deficient for the growth phase-responsive regulator Fis . Neither the alternative sigma factor RpoS nor the integration host factor (IHF) are involved in growth phase-dependent regulation of this operon . When incubated with nuo promoter DNA, isolated Fis protein formed three retarded complexes in gel mobility experiments . DNase I footprinting identified three distinct binding sites for Fis, 237 bp (fis1), 197 bp (fis2) and 139 bp (fis3) upstream of the start of the major transcript of nuoA-N, T1 . The protein concentrations required for half-maximal binding to fis1, fis2 and fis3 were about 20 nM, 40 nM and 100 nM Fis, respectively . The IHF protein bound 82 bp upstream of the start of transcript T2 with a half-maximal concentration for binding of 50 nM . Due to the growth phase-dependent regulation by Fis, the synthesis of the coupling NADH dehydrogenase I is increased relative to that of the noncoupling NADH dehydrogenase II during early exponential growth . This ensures higher ATP yields under conditions where large amounts of ATP are required. Mol Gen Genet, 1999 Dec, 262(4-5), 815 - 21 The tryptophan biosynthesis gene cluster trpCDEGFBA from Pyrococcus kodakaraensis KOD1 is regulated at the transcriptional level and expressed as a single mRNA; Tang X et al.; The entire gene cluster encoding enzymes involved in biosynthesis of L-tryptophan in the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 has been cloned and sequenced . Seven ORFs, which encode indole-3-glycerol phosphate synthase (trpC), anthranilate phosphoribosyltransferase (trpD), the two subunits of anthranilate synthase (trpEG), phosphoribosyl anthranilate isomerase (trpF) and the two subunits of tryptophan synthase (trpAB), were identified . The gene order is trpCDEGFBA, covering a region of 6045 bp . In order to confirm the function of the gene products, we expressed the first gene, Pk-trpC, in Escherichia coli . The protein product was purified, and was found to show the expected indole-3-glycerol phosphate synthase activity, with a temperature optimum of 85 degrees C . We could clearly identify a single mRNA transcript by Northern analysis using probes in the central and 3'-regions of the gene cluster, indicating that the gene cluster is transcribed as an operon . A significant increase in trp mRNA level was observed in cells grown in medium depleted of L-tryptophan, compared to cells grown in medium supplemented with L-tryptophan, indicating that expression of the gene cluster is regulated at the transcriptional level. Mol Gen Genet, 1999 Dec, 262(4-5), 800 - 6 Genetic probing of the interaction between the translation factor SelB and its mRNA binding element in Escherichia coli; Kromayer M et al.; Decoding of the UGA codon in mRNAs for selenoproteins as selenocysteine requires interaction of the translation factor SelB with an mRNA structure, the SECIS element . A genetic analysis of this interaction was performed by selecting for intergenic suppressor mutations in selB which counteracted the detrimental effect of defined mutations in the SECIS element . Both allele-nonspecific and allele-specific mutations, as judged by readthrough of the UGA into the LacZ-encoding segment of fdhF'-'lacZ fusions and by incorporation of selenium, were isolated . selB genes from ten suppressor mutants were sequenced and the corresponding mutations were localized to five positions within the protein . Four of the suppressors had amino acid exchanges within a 23-amino acid stretch in domain 4b of SelB, which probably represent sites of contact between the protein and the mRNA . A fifth mutation was localized in domain 4a of SelB; it promoted allele-nonspecific readthrough . Since a truncated SelB species lacking domain 4b did not show complex formation with the SECIS element, we speculate that the latter mutation affects the interaction between the tRNA-binding and the mRNA-binding domains . None of the SelB variants was able to promote UGA readthrough when major structural changes that altered the length of the helical part or enlarged the apical loop were introduced into the SECIS element . The results obtained also show that novel pairs of SelB/SECIS derivatives can be generated which may be useful for the targeted insertion of selenocysteine into proteins. Mol Gen Genet, 1999 Dec, 262(4-5), 790 - 9 A GntR-like negative regulator of the biphenyl degradation genes of the transposon Tn4371; Mouz S et al.; Tn4371, a 55-kb catabolic transposon originally isolated from Ralstonia eutropha A5, carries genes for the degradation of biphenyl/4-chlorobiphenyl, which are clustered on a 13-kb DNA segment located in the middle of the element . DNA sequencing revealed that two potential regulatory genes, bphR and bphS, border this region . Transcriptional fusion experiments using bphC as a reporter gene, Northern hybridization and primer extension analysis led to the conclusion that the transposon-encoded genes bphEFGA1A2A3BCD form an operon transcribed from a sigma70 promoter, pE . Transcription from pE was not influenced by deletion of the bphR gene of Tn4371, which should encode a LysR-like regulator . The bphS gene product negatively regulated pE, and displayed significant similarity to GntR-like regulators . This is the first reported example of a GntR-like regulator involved in the control of an aromatic degradation pathway. Mol Gen Genet, 1999 Dec, 262(4-5), 781 - 9 Isolation of a LIM15/DMC1 homolog from the basidiomycete Coprinus cinereus and its expression in relation to meiotic chromosome pairing; Nara T et al.; The Escherichia coli gene recA is essential for homologous recombination and DNA repair, and homologs have been identified in eukaryotes . A basidiomycete, Coprinus cinereus, which has many advantages for the study of meiosis, was recently reported to have a homolog of one of these, RAD51 . In the yeast Saccharomyces, mutations in the RAD51 gene cause defects in both somatic and meiotic cells . Based on this finding, we screened for a meiosis-specific homolog of recA, equivalent to Lilium LIM15 or Saccharomyces DMC1, in C . cinereus, and isolated a clone containing a 1.2-kb DNA fragment from a cDNA library constructed with Coprinus poly(A)+ RNA isolated from cells undergoing meiosis . The predicted amino acid sequence was 52% identical to the putative gene product of the lily cDNA clone LIM15 and 61% identical to Saccharomyces DMC1, and showed limited sequence similarity to the products of RAD52, 55, and 57 . The synchrony of meiosis in Coprinus provides an ideal system for the investigation of differential gene expression in relation to meiosis and fruiting body development . Northern analysis indicated that Coprinus LIM15/DMC1 was expressed at meiotic prophase within 8 h after the onset of karyogamy, suggesting that the gene functions mostly at the stage at which the homologous chromosomes pair, but may not be essential at the point at which they recombine . The gene is not expressed in somatic cells. Mol Gen Genet, 1999 Dec, 262(4-5), 768 - 71 Transcripts containing the 5' untranslated regions of the plastid genes psbA and psbB from higher plants are unstable in Chlamydomonas reinhardtii chloroplasts; Nickelsen J; The 5' regions of chloroplast genes contain cis-acting regulatory elements including promoters, and determinants of RNA stability and translation . In this work I examined whether the 5' regions of the spinach psbB and the wheat psbA genes can drive the expression of an aadA reporter gene in chloroplasts of the unicellar green alga Chlamydomonas reinhardtii . Both plant 5' sequences confer aadA-dependent, spectinomycin resistance on Escherichia coli, but not on the alga following integration into its chloroplast genome . Northern and run-on transcription analyses reveal that the plant promotors are active in C . reinhardtii but that the resulting chimeric transcripts are unstable . Therefore, the data suggest differences between higher plants and green algae with respect to the molecular mechanisms underlying plastid RNA metabolism. Mol Gen Genet, 1999 Dec, 262(4-5), 677 - 82 Upregulation of stress response genes and ABC transporters in anaerobic stationary-phase Mycobacterium smegmatis; Murugasu-Oei B et al.; Oxygen starvation triggers an adaptive stationary-phase response in Mycobacterium smegmatis . During this anaerobic stationary phase, RNA synthesis continues at a low but significant level . Employing a modified expressed-sequence-tag (EST) approach, in combination with the M . tuberculosis genome data and comparative Northern analysis, we have identified the first genes that show an increase in transcription in M . smegmatis cells that have entered anaerobic stationary phase . One gene encodes the counterpart of the M . tuberculosis NifS-like protein Rv1464 . Two genes are homologues of M . tuberculosis Rv1460 and Rv3368c, of unknown function . Strikingly, several genes induced by oxygen starvation encode putative stress protection proteins (counterparts of M . tuberculosis DnaK, Rv0350; betaine-aldehyde dehydrogenase, Rv0768; thioredoxin reductase, Rv3913) and ABC transporters (counterparts of M . tuberculosis Rv1463, Rv1473, Rv3197) . We conclude that development of general stress resistance and certain active transport processes might play a role in the survival of oxygen-starved M . smegmatis. Mol Gen Genet, 1999 Dec, 262(4-5), 633 - 42 Isolation and characterization of AtMLH1, a MutL homologue from Arabidopsis thaliana; Jean M et al.; DNA mismatch repair systems play an essential role in the maintenance of genetic information in living organisms and are also implicated in genetic recombination and genome stability . Using degenerate primers, we have cloned the first plant homologue of the E . coli MutL gene, which we have called AtMLH1 for Arabidopsis thaliana MutL-homologue 1 . AtMLH1 is present as a single-copy gene in the Arabidopsis genome and is located on the top arm of chromosome 4 . Sequence analysis revealed that the product of this gene shows extensive sequence homology with other eukaryotic MLH1 proteins . As mlh1-deficient lines would be useful for studying the biological function of this gene, several populations that had been mutagenized using T-DNA and transposon insertions were screened to identify such mutants . One line that carries a T-DNA insertion in the promoter region of the AtMLH1 gene was isolated . Surprisingly, although the insertion occurred only approximately 80 bp upstream of the putative transcription start site, Northern analyses revealed very low but similar amounts of AtMLH1 transcript in both the wild type and the T-DNA insertion lines . RT-PCR analyses suggest, however, that transcription is initiated further upstream in the insertion line and that the T-DNA may supply this novel initiation site . Finally, no increase in microsatellite instability - a phenotype often associated with mutations in mismatch repair genes - was observed in plants homozygous for this insertion. Vet Immunol Immunopathol, 1999 Dec 30, 72(3-4), 315 - 24 Molecular cloning of the canine nicotinic acetylcholine receptor alpha-subunit gene and development of the ELISA method to diagnose myasthenia gravis; Yoshioka T et al.; We investigated the molecular structure of canine nicotinic acetylcholine receptor (AChR) alpha-subunit gene and developed an enzyme-linked immunosorbent assay (ELISA) as an immunological method to diagnose myasthenia gravis (MG) . Canine AChR alpha-subunit cDNA was constructed from mRNA isolated from skeletal muscle of five dogs using the reverse transcriptase-polymerase chain reaction and its molecular structure was determined . The canine AChR alpha-subunit gene had 1371 base pairs encoding 457 amino acids and had a 96.1% homology to the human AChR alpha-subunit gene at the amino acid level . From the results of sequencing the DNA, specific antibodies to the acetylcholine binding domain of the canine AChR alpha-subunit were produced by immunizing rabbits with synthetic oligopeptides (alpha-subunit 183-200 amino acids) . The specificity of the rabbit anti-oligopeptide serum was examined by Western blot analysis using an E . coli-expressed AChR alpha-subunit protein and an AChR alpha-subunit protein fraction prepared from canine skeletal muscle as an antigen . An ELISA assay was developed using oligopeptides corresponding to the binding domain to diagnose canine MG; specific antibodies were detected from two dogs with MG, one diabetic dog and two healthy dogs among 25 dogs examined . Further examinations of the ELISA using a large number of samples of clinically MG-positive and MG-negative dogs are needed to establish its usefulness in MG diagnosis. Crit Care Med, 1999 Dec, 27(12), 2735 - 40 Vascular hyporesponsiveness of the renal circulation during endotoxemia in anesthetized pigs; Pastor CM; OBJECTIVE: To compare the vascular reactivity of the renal circulation in control and septic conditions . DESIGN: Prospective, randomized, controlled animal study . SETTING: University research laboratory . SUBJECTS: Anesthetized pigs (n = 17) . INTERVENTIONS: Ten pigs received a continuous intravenous infusion of endotoxin from Escherichia coli (160 ng x kg(-1) x hr(-1)) during 18 hrs, whereas seven control animals received a saline infusion . To test the vascular reactivity, norepinephrine (NE) (1 microg x kg(-1)), acetylcholine (10 microg x kg(-1)), and sodium nitroprusside (10 microg x kg(-1)) were intravenously injected for 20 secs and changes of mean arterial pressure and renal blood flow were observed during the 200 secs after the drug administration . To compare the evolution of the vascular reactivity over time, three tests were performed 5 hrs, 11 hrs, and 17 hrs after initial endotoxin or saline administration . MEASUREMENTS AND MAIN RESULTS: Endotoxin infusion induced a hypotensive and hypokinetic syndrome with renal hypoperfusion . The mean arterial pressure increase after NE injection and the mean arterial pressure decrease after acetylcholine and nitroprusside were lower in endotoxin than in control pigs . In the renal circulation, the increase of resistance after NE injection and the decrease of renal resistance after acetylcholine and nitroprusside injections were lower in endotoxin than in control pigs . CONCLUSIONS: This study shows a hyporesponsiveness of the renal circulation to vasoactive agents during endotoxemia . Vasoconstriction to NE, endothelium-dependent as well as endothelium-independent relaxations are altered during endotoxemia but not abolished, and despite the continuous infusion of endotoxin for 18 hrs, no recovery was observed over time. Domest Anim Endocrinol, 1999 Nov, 17(4), 345 - 60 Challenge differentially affects cytokine production and metabolic status of growing and finishing swine; Myers MJ et al.; Growing (35 kg body weight) and finishing (85 kg body weight) swine challenged with endotoxin (Escherichia coli O55:B5) at a dose of either 2 or 20 microg/kg produced tumor necrosis factor (TNF)alpha in a dose-response relationship as measured by bioassay . Peak TNFalpha plasma levels were observed 1-2 hr post-challenge, returning to basal values 4 hr post-challenge . However, both an enzyme-linked immunosorbent assay specific for swine TNFalpha and total human TNFalpha demonstrated no dose-response relationship; peak plasma levels of immunoreactive TNFalpha were also observed 1-2 hr post-challenge . Maximal plasma interleukin-6 levels occurred 1-2 hr post-challenge and remained elevated through 8 hr post-challenge; there was no effect of lipopolysaccharide dose or metabolic status . Although the metabolic status of the animals also affected glucose levels, with growing animals exhibiting greater sensitivity compared with finishing animals, endotoxin-induced decreases in blood glucose levels were primarily dose-dependent . In contrast, changes in plasma urea nitrogen and free fatty acid (FFA) levels were strictly related to the metabolic status . Urea nitrogen levels were unchanged in growing swine, whereas they were increased in finishing swine and remained elevated 24 hr post-challenge . FFA levels in growing and finishing swine increased 3-6 hr post-challenge . FFA levels returned to basal values for finishing swine 24 hr post challenge, but in growing swine remained elevated 24 hr post-challenge . Plasma aspartate transaminase levels were increased through 24 hr post-challenge; animals given a dose of 20 microg/kg exhibited the greatest increase . Similarly, swine challenged with a dose of 20 microg/kg also exhibited the greatest increase in levels of conjugated bilirubin; there was no effect on unconjugated (free) bilirubin . These results demonstrate that endotoxin challenge of swine result in a pattern of changes that are dependent on both the dose of endotoxin used and the metabolic status of the animal examined. Eye, 1999 Jun, 13 ( Pt 3b), 403 - 8 Lens alpha-crystallin: function and structure; Horwitz J et al.; alpha-Crystallin is a major lens protein, comprising up to 40% of total lens proteins, where its structural function is to assist in maintaining the proper refractive index in the lens . In addition to its structural role, it has been shown to function in a chaperone-like manner . The chaperone-like function of alpha-crystallin will help prevent the formation of large light-scattering aggregates and possibly cataract . In the lens, alpha-crystallin is a polydisperse molecule consisting of a 3:1 ratio of alpha A to alpha B subunits . In this study, we expressed recombinant alpha A- and alpha B-crystallin in E . coli and compared the polydispersity, structure and aggregation state between each other and native bovine lens alpha-crystallin . Using gel permeation chromatography to assay for polydispersity, we found native alpha-crystallin to be significantly more polydisperse than either recombinant alpha A- or alpha B-crystallin, with alpha B-crystallin having the most homogeneous structure of the three . Reconstructed images of alpha B-crystallin obtained with cryo-electron microscopy support the concept that alpha B-crystallin is an extremely dynamic molecule and demonstrated that it has a hollow interior . Interestingly, we present evidence that native alpha-crystallin is significantly more thermally stable than either alpha A- or alpha B-crystallin alone . In fact, our experiments suggest that a 3:1 ratio of alpha A to alpha B subunit composition in an alpha-crystallin molecule is optimal in terms of thermal stability . This fascinating result explains the stoichiometric ratios of alpha A- and alpha B-crystallin subunits in the mammalian lens. Ann Rheum Dis, 2000 Jan, 59(1), 64 - 6 No serological indications that systemic lupus erythematosus is linked with exposure to human parvovirus B19; Bengtsson A et al.; OBJECTIVES: Infectious agents like parvovirus have been implicated as exogenous factors that could trigger onset of systemic lupus erythematosus (SLE) . A number of case reports describing a SLE-like presentation of acute human parvovirus B19 infection have been published, but no systematic investigation of the actual seroprevalence in epidemiologically defined SLE populations has previously been reported . METHODS: Sera from 99 SLE patients from a defined area in Southern Sweden, representing 88% of all new SLE cases 1981-1995 within the Lund-Orup Health Care district with 175 000 adult inhabitants (> 15 years of age), and sera from 99 age and sex matched healthy controls were investigated for the presence of IgG parvovirus antibodies . Two different commercially available EIA kits were used; one using E coli synthesised parvovirus VP1/VP2 antigen, and one using baculovirus derived parvovirus VP2 antigen . RESULTS: The EIA using baculovirus derived antigen was more sensitive and surprisingly the controls were more often positive than the SLE patients were (79% versus 65%, chi(2) p=0.027) . No difference between the groups was seen with the EIA using E coli derived antigen (46% versus 49%) . Titration experiments indicated that the discordance between the two tests was a matter of sensitivity rather than specificity . CONCLUSION: No evidence was found of human parvovirus B19 infection being more prevalent among SLE patients . On the contrary, in one of the parvovirus EIAs the controls were more often positive than the SLE patients were. Biochim Biophys Acta, 2000 Jan 10, 1456(2-3), 121 - 37 Reaction of Escherichia coli cytochrome bo(3) and mitochondrial cytochrome bc(1) with a photoreleasable decylubiquinol; Hansen KC et al.; In order to probe the reaction chemistry of respiratory quinol-oxidizing enzymes on a rapid time scale, a photoreleasable quinol substrate was synthesized by coupling decylubiquinol with the water-soluble protecting group 3',5'-bis(carboxymethoxy)benzoin (BCMB) through a carbonate linkage . The resulting compound, DQ-BCMB, was highly soluble in aqueous detergent solution, and showed no reactivity with quinol-oxidizing enzymes prior to photolysis . Upon photolysis in acetonitrile, 5, 7-bis(carboxymethoxy)-2-phenylbenzofuran, carbon dioxide, and decylubiquinol were formed . In aqueous media, free 3', 5'-bis(carboxymethoxy)benzoin was also produced . Photolysis of DQ-BCMB with a 308 nm excimer laser led to the release of the BCMB group in less than 10(-6) s . Decylubiquinol was released in the form of a carbonate monoester, which decarboxylated with an observed first-order rate constant of 195-990 s(-1), depending on the reaction medium . Yields of decylubiquinol as high as 35 microM per laser pulse were attained readily . In the presence of Escherichia coli cytochrome bo(3), photolysis of DQ-BCMB led to the oxidation of quinol by the enzyme with a rate that was limited by the rate of the decylubiquinol release . Mitochondrial cytochrome bc(1) reacted with photoreleased decylubiquinol with distinct kinetic phases corresponding to rapid b heme reduction and somewhat slower c heme reduction . Oxidation of photoreleased ubiquinol by this enzyme showed saturation kinetics with a K(m) of 3.6 microM and a k(cat) of 210 s(-1) . The saturation behavior was a result of decylubiquinol being released as a carbonate monoester during the photolysis of DQ-BCMB and interacting with cytochrome bc(1) before decarboxylation of this intermediate yielded free decylubiquinol . The reaction of cytochrome bc(1) and photoreleased decylubiquinol in the presence of antimycin A led to monophasic b heme reduction, but also yielded slower quinol oxidation kinetics . The discrimination of kinetic phases in the reaction of cytochrome bc(1) with ubiquinol substrates has provided a means of exploring the bifurcation of electron transfer that is central to the operation of the Q-cycle in this enzyme. Microbiology, 1999 Dec, 145 ( Pt 12), 3353 - 63 Family 19 chitinases of Streptomyces species: characterization and distribution; Watanabe T et al.; Chitinase C from Streptomyces griseus HUT6037, described in 1997, is the first family 19 chitinase found in an organism other than higher plants . In this study, some properties of chitinase C were compared with those of family 18 bacterial chitinases, and the distribution of family 19 chitinases in Streptomyces species was investigated . The specific hydrolysing activity of chitinase C against soluble and insoluble chitinous substrates was markedly higher than those of bacterial family 18 chitinases . Chitinase C exhibited marked antifungal activity, whereas the other bacterial chitinases examined had no antifungal activity . Chitinase C was insensitive to allosamidin, whereas the family 18 bacterial chitinases were sensitive . Taking advantage of this insensitivity to allosamidin, a search was made for family 19 chitinases in various Streptomyces species . Chitinases insensitive to allosamidin were detected in the culture supernatants of all tested Streptomyces species . Southern hybridization analysis using a labelled DNA fragment corresponding to the catalytic domain of chitinase C strongly suggested that these species have genes similar to the chiC gene of S . griseus HUT6037 . DNA fragments corresponding to the major part of the catalytic domains were amplified by PCR . The amplified fragments encoded amino acid sequences very similar to that of the corresponding region of chitinase C . Therefore, it was concluded that Streptomyces species generally possess family 19 chitinases which are very similar to chitinase C . Comparison of their amino acid sequences with those of plant family 19 chitinases revealed that Streptomyces family 19 chitinases are class IV type in terms of the presence and positions of deletions of amino acid sequences which are characteristic of plant class IV chitinases. Microbiology, 1999 Dec, 145 ( Pt 12), 3331 - 41 AUD4, a new amplifiable element from Streptomyces lividans; Schmid E et al.; After transformation of the Streptomyces lividans chloramphenicol-sensitive, arginine-auxotrophic mutant strain AJ100 with a derivative of plasmid SCP2, some of the regenerated protoplasts contained an 8.2 kb DNA sequence amplified to several hundred copies per chromosome . The corresponding non-amplified sequence, called AUD4, was isolated from a lambda phage genomic library of S . lividans 1326 . Two cytosine residues were the only directly repeated nucleotides at the ends of the element, indicating that AUD4 is a class I amplifiable sequence . The element mapped in the AseI-D fragment of the S . lividans chromosome, where other class I amplifications have been described . The complete element was sequenced and 10 ORFs were identified . Some of the deduced proteins are highly conserved in other organisms but a putative function could be attributed to only a few of them . Duplication of AUD4 by integration of an Escherichia coli plasmid carrying various parts of AUD4 and a thiostrepton-resistance gene in S . lividans AJ100, ZX7 or TK64 induced amplification of the integrated plasmid, AUD4 or both at high frequency. IARC Sci Publ, 1999, (150), 279 - 93 Localization of chloroacetaldehyde-induced DNA damage in human p53 gene by DNA polymerase fingerprint analysis; Tudek B et al.; Chloroacetaldehyde (CAA) reacts with DNA bases, forming hydroxyethano derivatives of different stability, which are subsequently converted into etheno (epsilon) adducts: epsilon A, epsilon C, epsilon G . DNA polymerase fingerprint analysis was used to study the distribution of CAA-induced modifications in the p53 sequence . A plasmid bearing cDNA containing the human p53 gene was reacted in vitro with CAA, then dehydrated for conversion of hydroxyethano into etheno adducts, and primer extension by T7 DNA polymerase in the presence of four dNTPs was performed . The DNA repair enzymes methylpurine-DNA glycosylase and Escherichia coli exonuclease III were used to convert epsilon A residues in the template into DNA strand breaks, which enabled precise localization of the epsilon A residues within the p53 gene . Hydroxyethano derivatives of adenine and cytosine in a template blocked T7 DNA polymerase and caused premature chain termination opposite adenine or one base before cytosine . After dehydration, both epsilon A and epsilon C were much more easily by-passed by T7 DNA polymerase . Formation of epsilon G was identified as 'stop bands' one base before guanine residues . Modification of cytosine and guanine was additionally recognized by weakening or disappearance of non-specific stops on an undamaged template, probably due to steric hindrance by the tertiary DNA structure for polymerase . Etheno adduction of cytosine and guanine relaxed the compact DNA structure and enabled DNA polymerase to by-pass . In exons 5-8 of p53, 143 out of 500 sites appeared to be damaged by CAA, with four particularly densely modified regions between codons 135-147, 218-222, 234-255 and 284-292 . The pattern of modification followed the pattern of p53 mutations found in vinyl chloride-associated liver angiosarcomas in humans and rats, but only in regions that showed 100% homology with the human sequence . The factors that influence DNA damage and induction of mutations in the p53 gene by CAA and vinyl chloride are discussed. IARC Sci Publ, 1999, (150), 271 - 7 Role of base excision repair in protecting cells from the toxicity of chloroethylnitrosoureas; Ludlum DB et al.; The chloroethylnitrosoureas react extensively with cellular DNA to produce a variety of DNA adducts, including a deoxycytidine-deoxyguanosine (dC-dG) cross-link that is clearly cytotoxic . It is now well established that O6-alkylguanine-DNA-alkyltransferase can prevent formation of this dC-dG cross-link and thereby diminish the toxicity of the chloroethylnitrosoureas . Besides alkyltransferase, DNA glycosylases from various species can also contribute to cellular resistance to the chloroethylnitrosoureas, but the mechanism for this increased resistance has not been established . It is known, however, that several chloroethylnitrosoureas-modified DNA bases, including the exocyclic adduct, N2,3-ethanoguanine, are released by Escherichia coli 3-methyladenine DNA glycosylase II . In the study described here, we examined the possibility that this enzyme might act on the exocyclic intermediate in dC-dG formation, 1,O6-ethanodeoxyguanosine, and prevent-dC-dG cross-linking in this way . However, the presence of E . coli 3-methyladenine DNA glycosylase II does not decrease the amount of dC-dG cross-link formed when chloroethylnitrosourea reacts with DNA, and we conclude that this enzyme does not recognize 1,O6-ethanodeoxyguanosine . Therefore, its contribution to resistance probably resides in its action on other nitrosourea-induced DNA modifications. IARC Sci Publ, 1999, (150), 263 - 70 Cellular response to exocyclic DNA adducts; Moriya M et al.; The mutagenic potential of three exocyclic DNA adducts was studied in Escherichia coli and simian kidney cells by incorporating them into single-stranded DNA . Differences in the mutagenic potency of the adducts were observed between hosts: 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine were more mutagenic in simian cells, whereas 1,N2-(1,3-propan-1,3-diyl)-2'-deoxyguanosine was more mutagenic in E . coli . To investigate the cellular response to DNA adducts, a double-stranded DNA vector system was developed . Use of this system showed that 1,N6-ethenodeoxyadenosine blocks DNA synthesis strongly, and DNA synthesis past this adduct was highly accurate in E . coli . The blockage of DNA synthesis was overcome in an error-free manner by the recombination repair mechanism (daughter-strand gap repair). IARC Sci Publ, 1999, (150), 249 - 61 Enzymology of the repair of etheno adducts in mammalian cells and in Escherichia coli; Saparbaev M et al.; Exocyclic adducts are generated in cellular DNA by reaction with epoxides that are formed metabolically from various industrial pollutants and by reaction with activated aldehydes that arise during membrane lipid peroxidation . The etheno (epsilon) derivatives of purine and pyrimidine bases, e.g . 3,N4-ethenocytosine, 1,N6-ethenoadenine, N2,3-ethenoguanine and 1,N2-ethenoguanine, are probably involved in carcinogenesis because they are highly mutagenic and genotoxic . Therefore, the repair processes that eliminate exocyclic adducts from DNA should play a crucial role in maintaining the stability of the genetic information . The DNA glycosylases implicated in the repair of etheno adducts have been identified . Human and Escherichia coli 3-methyladenine-DNA-glycosylases excise 1,N6-ethenoadenine residues . We have identified two homologous proteins present in human cells and E . coli that remove 3,N4-ethenocytosine residues by DNA glycosylase activity . The human enzyme is an activity of the mismatch-specific thymine-DNA glycosylase, while the bacterial enzyme is an activity of the double-stranded uracil-DNA glycosylase, i.e., the homologue of the human enzyme . The fact that 1,N6-ethenoadenine and 3,N4-ethenocytosine are recognized and efficiently excised by DNA glycosylases in vitro suggests that these enzymes may be responsible for the repair of these mutagenic lesions in vivo and may contribute importantly to genetic stability. IARC Sci Publ, 1999, (150), 137 - 45 Formation of etheno adducts and their effects on DNA polymerases; Guengerich FP et al.; Etheno (epsilon) and related DNA adducts are formed from the reaction of certain bifunctional electrophiles with DNA . Our interest has been focused on oxiranes substituted with leaving groups, e.g . 2-chlorooxirane, the epoxide derived from the carcinogen vinyl chloride . The chemical mechanisms of the formation of the major etheno products derived from adenine, cytosine and guanine have been elucidated by nuclear magnetic resonance analysis and 13C-labelled precursors . The amounts of all major etheno adducts have been quantified in DNA treated with 2-chlorooxirane by coupled high-performance liquid chromatography of nucleoside and base products . 1,N2-epsilon-Gua, its formally hydrated but stable hemiaminal HO-ethanoGua (5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo{1,2-a}purine) and 1,N2-ethanoGua have all been inserted at a single site in oligonucleotides . All three of these bases block polymerases, cause misincorporations and produce some mutations in bacteria . The patterns of blockage and substitution vary among polymerases . In nucleotide excision repair-deficient Escherichia coli, 1,N2-epsilon-Gua yielded a calculated 16% mutation frequency (base-pair substitutions) when the results were corrected for strand usage . 1,N2-epsilon-Gua was also examined in Chinese hamster ovary cells with a stable integration system; the mutants are more complex than observed in bacteria and include rearrangements, deletions and base-pair substitutions other than at the adduct site. J Biol Chem, 2000 Jan 14, 275(2), 1485 - 94 Vaccinia virus gene A18R DNA helicase is a transcript release factor; Lackner CA et al.; Prior phenotypic analysis of a vaccinia virus gene A18R mutant, Cts23, showed the synthesis of longer than wild type (Wt) length viral transcripts during the intermediate stage of infection, indicating that the A18R protein may act as a negative transcription elongation factor . The purpose of the work described here was to determine a biochemical activity for the A18R protein . Pulse-labeled transcription complexes established from intermediate virus promoters on bead-bound DNA templates were assayed for transcript release during an elongation step that contained nucleotides and various proteins . Pulse-labeled transcription complexes elongated in the presence of only nucleotides were unable to release nascent RNA . The addition of Wt extract during the elongation phase resulted in release of the nascent transcript, indicating that additional factors present in the Wt extract are capable of inducing transcript release . Extract from Cts23 or mock-infected cells was unable to induce release . The lack of release upon addition of Cts23 extract suggests that A18R is involved in release of nascent RNA . By itself, purified polyhistidine-tagged A18R protein (His-A18R) was unable to induce release; however, release did occur in the presence of purified His-A18R protein plus extract from either Cts23 or mock-infected cells . These data taken together indicate that A18R is necessary but not sufficient for release of nascent transcripts . We have also demonstrated that the combination of A18R protein and mock extract induces transcript release in an ATP-dependent manner, consistent with the fact that the A18R protein is an ATP-dependent helicase . Further analysis revealed that the release activity is not restricted to a vaccinia intermediate promoter but is observed using pulse-labeled transcription complexes initiated from all three viral gene class promoters . Therefore, we conclude that A18R and an as yet unidentified cellular factor(s) are required for the in vitro release of nascent RNA from a vaccinia virus transcription elongation complex. J Biol Chem, 2000 Jan 14, 275(2), 1433 - 8 Identification and modification of the uridine-binding site of the UDP-GalNAc (GlcNAc) pyrophosphorylase; Wang-Gillam A et al.; UDP-GalNAc pyrophosphorylase (UDP-GalNAcPP; AGX1) catalyzes the synthesis of UDP-GalNAc from UTP and GalNAc-1-P . The 475-amino acid protein (57 kDa protein) also synthesizes UDP-GlcNAc at about 25% the rate of UDP-GalNAc . The cDNA for this enzyme, termed AGX1, was cloned in Escherichia coli, and expressed as an active enzyme that cross-reacted with antiserum against the original pig liver UDP-HexNAcPP . In the present study, we incubated recombinant AGX1 with N(3)-UDP-{(32)P}GlcNAc and N(3)-UDP-{(32)P}GalNAc probes to label the nucleotide-binding site . Proteolytic digestions of the labeled enzyme and analysis of the resulting peptides indicated that both photoprobes cross-linked to one 24-amino acid peptide located between residues Val(216) and Glu(240) . Four amino acids in this peptide were found to be highly conserved among closely related enzymes, and each of these was individually modified to alanine . Mutation of Gly(222) to Ala in the peptide almost completely eliminated UDP-GlcNAc and UDP-GalNAc synthesis, while mutation of Gly(224) to Ala, almost completely eliminated UDP-GalNAc synthesis, but UDP-GlcNAc was only diminished by 50% . Both of these mutations also resulted in almost complete loss of the ability of the mutated proteins to cross-link N(3)-UDP-{(32)P}GlcNAc or N(3)-UDP-{(32)P}GalNAc . On the other hand, mutations of either Pro(220) or Tyr(227) to Ala did not greatly affect enzymatic activity, although there was some reduction in the ability of these proteins to cross-link the photoaffinity probes . We also mutated Gly(111) to Ala since this amino acid was reported to be necessary for catalysis (Mio, T., Yabe, T., Arisawa, M., and Yamada-Okabe, H . (1998) J . Biol . Chem . 273, 14392-14397) . The Gly(111) to Ala mutant lost all enzymatic activity, but interestingly enough, this mutant protein still cross-linked the radioactive N(3)-UDP-GlcNAc although not nearly as well as the wild type . On the other hand, mutation of Arg(115) to Ala had no affect on enzymatic activity although it also reduced the amount of cross-linking of N(3)-UDP-{(32)P}GlcNAc . These studies help to define essential amino acids at or near the nucleotide-binding site and the catalytic site, as well as peptides involved in binding and catalysis. J Biol Chem, 2000 Jan 14, 275(2), 1421 - 32 Dynamics of beta and proliferating cell nuclear antigen sliding clamps in traversing DNA secondary structure; Yao N et al.; Chromosomal replicases of cellular organisms utilize a ring shaped protein that encircles DNA as a mobile tether for high processivity in DNA synthesis . These "sliding clamps" have sufficiently large linear diameters to encircle duplex DNA and are perhaps even large enough to slide over certain DNA secondary structural elements . This report examines the Escherichia coli beta and human proliferating cell nuclear antigen clamps for their ability to slide over various DNA secondary structures . The results show that these clamps are capable of traversing a 13-nucleotide ssDNA loop, a 4-base pair stem-loop, a 4-nucleotide 5' tail, and a 15-mer bubble within the duplex . However, upon increasing the size of these structures (20-nucleotide loop, 12-base pair stem-loop, 28-nucleotide 5' tail, and 20-nucleotide bubble) the sliding motion of the beta and proliferating cell nuclear antigen over these elements is halted . Studies of the E . coli replicase, DNA polymerase III holoenzyme, in chain elongation with the beta clamp demonstrate that upon encounter with an oligonucleotide annealed in its path, it traverses the duplex and resumes synthesis on the 3' terminus of the oligonucleotide . This sliding and resumption of synthesis occurs even when the oligonucleotide contains a secondary structure element, provided the beta clamp can traverse the structure . However, upon encounter with a downstream oligonucleotide containing a large internal secondary structure, the holoenzyme clears the obstacle by strand displacing the oligonucleotide from the template . Implications of these protein dynamics to DNA transactions are discussed. J Biol Chem, 2000 Jan 14, 275(2), 1287 - 93 Identification of amino acid determinants of the positional specificity of mouse 8S-lipoxygenase and human 15S-lipoxygenase-2; Jisaka M et al.; Phorbol ester-inducible mouse 8S-lipoxygenase (8-LOX) and its human homologue, 15S-lipoxygenase-2 (15-LOX-2), share 78% identity in amino acid sequences, yet there is no overlap in their positional specificities . In this study, we investigated the determinants of positional specificity using a random chimeragenesis approach in combination with site-directed mutagenesis . Exchange of the C-terminal one-third of the 8-LOX with the corresponding portion of 15-LOX-2 yielded a chimeric enzyme with exclusively 15S-lipoxygenase activity . The critical region was narrowed down to a cluster of five amino acids by expression of multiple cDNAs obtained by in situ chimeragenesis in Escherichia coli . Finally, a pair of amino acids, Tyr(603) and His(604), was identified as the positional determinant by site-directed mutagenesis . Mutation of both of these amino acids to the corresponding amino acids in 15-LOX-2 (Asp and Val, respectively) converted the positional specificity from 8S to 90% 15S without yielding any other by-products . Mutation of the corresponding residues in 15-LOX-2 to the 8-LOX sequence changed specificity to 50% oxygenation at C-8 for one amino acid substitution and 70% at C-8 for the double mutant . Based on the crystal structure of the reticulocyte 15-LOX, these two amino acids lie opposite the open coordination position of the catalytic iron in a likely site for substrate binding . The change from 8 to 15 specificity entails a switch in the head to tail binding of substrate . Enzymes that react with substrate "head first" (5-LOX and 8-LOX) have a bulky aromatic amino acid and a histidine in these positions, whereas lipoxygenases that accept substrates "tail first" (12-LOX and 15-LOX) have an aliphatic residue with a glutamine or aspartate . Thus, this positional determinant of the 8-LOX and 15-LOX-2 may have significance for other lipoxygenases. J Biol Chem, 2000 Jan 14, 275(2), 1209 - 15 Canstatin, a novel matrix-derived inhibitor of angiogenesis and tumor growth; Kamphaus GD et al.; We isolated and identified an endogenous 24-kDa human basement membrane-derived inhibitor of angiogenesis and tumor growth, termed canstatin . Canstatin, a fragment of the alpha2 chain of type IV collagen, was produced as a recombinant molecule in Escherichia coli and 293 embryonic kidneys cells . Canstatin significantly inhibited human endothelial cell migration and murine endothelial cell tube formation . Additionally, canstatin potently inhibited 10% fetal bovine serum-stimulated endothelial cell proliferation and induced apoptosis, with no inhibition of proliferation or apoptosis observed on non-endothelial cells . Inhibition of endothelial proliferation was not concomitant with a change in extracellular signal-regulated kinase activation . We demonstrate that apoptosis induced by canstatin was associated with a down-regulation of the anti-apoptotic protein, FLIP . Canstatin also suppressed in vivo growth of large and small size tumors in two human xenograft mouse models with histology revealing decreased CD31-positive vasculature . Collectively, these results suggest that canstatin is a powerful therapeutic molecule for suppressing angiogenesis. J Biol Chem, 2000 Jan 14, 275(2), 1145 - 51 Expression, purification, and characterization of natural mutants of human aldolase B . Role of quaternary structure in catalysis; Rellos P et al.; Fructaldolases (EC 4.1.2.13) are ancient enzymes of glycolysis that catalyze the reversible cleavage of phosphofructose esters into cognate triose (phosphates) . Three vertebrate isozymes of Class I aldolase have arisen by gene duplication and display distinct activity profiles with fructose 1,6-bisphosphate and with fructose 1-phosphate . We describe the biochemical and biophysical characterization of seven natural human aldolase B variants, identified in patients suffering from hereditary fructose intolerance and expressed as recombinant proteins in E . coli, from which they were purified to homogeneity . The mutant aldolases were all missense variants and could be classified into two principal groups: catalytic mutants, with retained tetrameric structure but altered kinetic properties (W147R, R303W, and A337V), and structural mutants, in which the homotetramers readily dissociate into subunits with greatly impaired enzymatic activity (A149P, A174D, L256P, and N334K) . Investigation of these two classes of mutant enzyme suggests that the integrity of the quaternary structure of aldolase B is critical for maintaining its full catalytic function. J Biol Chem, 2000 Jan 14, 275(2), 1119 - 27 Transcription activation mediated by the carboxyl-terminal domain of the RNA polymerase alpha-subunit . Multipoint monitoring using a fluorescent probe; Ozoline ON et al.; Conformational changes within the carboxyl-terminal domain of the Escherichia coli RNA polymerase alpha-subunit (alpha-CTD) upon interaction with the DNA UP element or the transcription factor cAMP receptor protein (CRP) were studied by monitoring the spectral parameters of a fluorescent dye, fluorescein mercuric acetate, conjugated to various positions of alpha-CTD . When fluorescein mercuric acetate was conjugated to Cys located on helix I and the loop between helices III and IV, the spectral changes typical for DNA interaction were observed for the RNA polymerase-promoter binary complex with UP element-dependent rrnBP1 and the ternary complex with the CRP-dependent uxuAB promoter in the presence of cAMP/CRP . Likewise, the chemical nuclease iron-(p-bromoacetamidobenzyl)-EDTA conjugated to Cys-269 or Cys-272 introduced CRP-dependent cleavage of the uxuAB promoter, as in the case of rrnBP1 (Murakami, K., Owens, J . T., Belyaeva, T . A., Meares, C . F., Busby, S . J . W., and Ishihama, A . (1997) Proc . Natl . Acad . Sci . U . S . A . 94, 11274-11278), indicating that CRP rearranges the topology of the DNA contact surface in alpha-CTD . Conformational changes in alpha-CTD were also observed upon formation of a binary complex with the uxuAB (in the absence of CRP) and factor-independent T7D promoters . The spectral changes suggested that helix IV of alpha-CTD approaches the negatively charged phosphate moiety of DNA . In agreement with this prediction, iron-(p-bromoacetamidobenzyl)-EDTA conjugated to Cys-309 induced extensive DNA cleavage upstream from the uxuAB promoter -35 element . We propose that helix IV of alpha-CTD is involved in direct interaction with some promoters. J Biol Chem, 2000 Jan 14, 275(2), 1050 - 6 Glycine betaine-assisted protein folding in a lysA mutant of Escherichia coli; Bourot S et al.; Osmoprotectants exogenously supplied to a hyperosmotic culture medium are efficiently imported and amassed by stressed cells of Escherichia coli . In addition to their evident role in the recovery and maintenance of osmotic balance, these solutes should play an important role on the behavior of cellular macromolecules, for example in the process of protein folding . Using a random chemical mutagenesis approach, a conditional lysine auxotrophic mutant was obtained . The growth of this mutant was restored by addition of either lysine or osmoprotectants including glycine betaine (GB) in the minimal medium . The growth rate increased proportionally with the augmentation of the intracellular GB concentration . The mutation was located in the lysA gene and resulted in the substitution of the Ser at position 384 by Phe of the diaminopimelate decarboxylase (DAPDC), which catalyzes the conversion of meso-diaminopimelate to L-lysine . We purified both the wild type DAPDC and the mutated DAPDC-sf and demonstrated that GB was capable of activating DAPDC-sf in vitro, thus confirming the in vivo results . Most importantly, we showed that the activation was correlated with a conformational change of DAPDC-sf . Taken together, these results show, for the first time, that GB may actively assist in vivo protein folding in a chaperone-like manner. J Biol Chem, 2000 Jan 14, 275(2), 1015 - 22 Contributions of the different extramembranous domains of the mechanosensitive ion channel MscL to its response to membrane tension; Ajouz B et al.; MscL is a mechanosensitive channel that is gated by tension in the membrane bilayer alone . It is a homo-oligomer of a protein comprising two transmembrane segments connected by an external loop, with the NH(2) and COOH termini located in the cytoplasm . The contributions of the extramembranous domains of the channel to its activity were investigated by specific proteolysis during patch-clamp experiments . Limited proteolysis of the COOH terminus or the NH(2) terminus increased the mechanosensitivity of the channel without changing its conductance . Strikingly, after cleavage of the external loop of each monomer, the channel was still functional, and its mechanosensitivity was increased dramatically, indicating that the loop acts as a spring that resists the opening of the channel and promotes its closure when it is open . These results indicate that the integrity of most of the extramembranous domains is not essential for mechanosensitivity . They suggest that these domains counteract the movement of the transmembrane helices to which they are connected, thus setting the level of sensitivity of the channel to tension. J Biol Chem, 2000 Jan 14, 275(2), 959 - 68 Holo-(acyl carrier protein) synthase and phosphopantetheinyl transfer in Escherichia coli; Flugel RS et al.; Holo-(acyl carrier protein) synthase (AcpS) post-translationally modifies apoacyl carrier protein (apoACP) via transfer of 4'-phosphopantetheine from coenzyme A (CoA) to the conserved serine 36 gamma-OH of apoACP . The resulting holo-acyl carrier protein (holo-ACP) is then active as the central coenzyme of fatty acid biosynthesis . The acpS gene has previously been identified and shown to be essential for Escherichia coli growth . Earlier mutagenic studies isolated the E . coli MP4 strain, whose elevated growth requirement for CoA was ascribed to a deficiency in holoACP synthesis . Sequencing of the acpS gene from the E . coli MP4 strain (denoted acpS1) showed that the AcpS1 protein contains a G4D mutation . AcpS1 exhibited a approximately 5-fold reduction in its catalytic efficiency when compared with wild type AcpS, accounting for the E . coli MP4 strain phenotype . It is shown that a conditional acpS mutant accumulates apoACP in vivo under nonpermissive conditions in a manner similar to the E . coli MP4 strain . In addition, it is demonstrated that the gene product, YhhU, of a previously identified E . coli open reading frame can completely suppress the acpS conditional, lethal phenotype upon overexpression of the protein, suggesting that YhhU may be involved in an alternative pathway for phosphopantetheinyl transfer and holoACP synthesis in E . coli. J Biol Chem, 2000 Jan 14, 275(2), 937 - 41 Structural characterization of the inflammatory moiety of a variable major lipoprotein of Borrelia recurrentis; Scragg IG et al.; Louse-borne relapsing fever, caused by Borrelia recurrentis, provides one of the best documented examples of the causative role of tumor necrosis factor (TNF) in the pathology of severe infection in humans . We have identified the principal TNF-inducing factor of B . recurrentis as a variable major lipoprotein (Vmp) . Here we report the complete gene sequence of Vmp, including its lipoprotein leader sequence . Using metabolically labeled forms of the native Vmp we confirm that the TNF inducing properties are associated with the lipid portion of the molecule . Quadrupole orthogonal time of flight mass spectrometry unequivocally locates the lipidic moiety at the NH(2)-terminal cysteine of the native polypeptide, and indicates the existence of three forms which are consistent with the structures C16:0, C16:0, C16:0 glyceryl cysteine; C18:1, C16:0, C16:0 glyceryl cysteine; and C18:0, C16:0, C16:0 glyceryl cysteine . These data provide the first direct evidence that the TNF inducing lipid modification of native Borrelia lipoproteins is a structural homologue of the murein lipoprotein of Escherichia coli. J Biol Chem, 2000 Jan 14, 275(2), 833 - 9 Locations of the regulatory sites for isocitrate dehydrogenase kinase/phosphatase; Miller SP et al.; Isocitrate dehydrogenase (IDH)(1) of Escherichia coli is regulated by a bifunctional protein, IDH kinase/phosphatase . In this paper, we demonstrate that the effectors controlling these activities belong to two distinct classes that differ in mechanism and in the locations of their binding sites . NADPH and isocitrate are representative members of one of these effector classes . NADPH inhibits both IDH kinase and IDH phosphatase, whereas isocitrate inhibits only IDH kinase . Isocitrate can "activate" IDH phosphatase by reversing product inhibition by dephospho-IDH . Mutations in icd, which encodes IDH, had parallel effects on the binding of these ligands to the IDH active site and on their effects on IDH kinase and phosphatase, indicating that these ligands regulate IDH kinase/phosphatase through the IDH active site . Kinetic analyses suggested that isocitrate and NADPH prevent formation of the complex between IDH kinase/phosphatase and its protein substrate . AMP, 3-phosphoglycerate, and pyruvate represent a class of regulatory ligands that is distinct from that which includes isocitrate and NADPH . These ligands bind directly to IDH kinase/phosphatase, a conclusion which is supported by the observation that they inhibit the IDH-independent ATPase activity of this enzyme . These effector classes can also be distinguished by the observation that mutant derivatives of IDH kinase/phosphatase expressed from aceK3 and aceK4 exhibited dramatic changes in their responses to AMP, 3-phosphoglycerate, and pyruvate but not to NADPH and isocitrate. J Biol Chem, 2000 Jan 14, 275(2), 752 - 8 Three of the six possible intersubunit stabilizing interactions involving Glu-239 are sufficient for restoration of the homotropic and heterotropic properties of Escherichia coli aspartate transcarbamoylase; Sakash JB et al.; A hybrid version of Escherichia coli aspartate transcarbamoylase was investigated in which one catalytic subunit has the wild-type sequence, and the other catalytic subunit has Glu-239 replaced by Gln . Since Glu-239 is involved in intersubunit interactions, this hybrid could be used to evaluate the extent to which T state stabilization is required for homotropic cooperativity and for heterotropic effects . Reconstitution of the hybrid holoenzyme (two different catalytic subunits with three wild-type regulatory subunits) was followed by separation of the mixture by anion-exchange chromatography . To make possible the resolution of the three holoenzyme species formed by the reconstitution, the charge of one of the catalytic subunits was altered by the addition of six aspartic acid residues to the C terminus of each of the catalytic chains (AT-C catalytic subunit) . Control experiments indicated that the AT-C catalytic subunit as well as the holoenzyme formed with AT-C and wild-type regulatory subunits had essentially the same homotropic and heterotropic properties as the native catalytic subunit and holoenzyme, indicating that the addition of the aspartate tail did not influence the function of either enzyme . The control reconstituted holoenzyme, in which both catalytic subunits have Glu-239 replaced by Gln, exhibited no cooperativity, an enhanced affinity for aspartate, and essentially no heterotropic response identical to the enzyme isolated without reconstitution . The hybrid containing one normal and one mutant catalytic subunit exhibited homotropic cooperativity with a Hill coefficient of 1.4 and responded to the nucleotide effectors at about 50% of the level of the wild-type enzyme . Small angle x-ray scattering experiments with the hybrid enzyme indicated that in the absence of ligands it was structurally similar, but not identical, to the T state of the wild-type enzyme . In contrast to the wild-type enzyme, addition of carbamoyl phosphate induced