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| J Biol Chem, 2000 Feb 11, 275(6), 4192 - 8 Inhibition of protein phosphatase-1 by clavosines A and B . Novel members of the calyculin family of toxins; McCready TL et al.; Site-directed mutagenesis was used to investigate the mechanism of interaction between the catalytic subunit of human protein phosphatase-1 (PP-1cgamma) and members of the calyculin family of toxins . Clavosines A and B are related to calyculins but are glycosylated with a trimethoxy rhamnose group . We provide experimental evidence implicating Tyr-134 as an important residue in PP-1cgamma that mediates interactions with the calyculins . Mutation of Tyr-134 to Phe, to prevent hydrogen bond formation, resulted in a slight increase in sensitivity of PP-1cgamma to clavosines A and B and calyculin A . In contrast, a Y134A mutant was 10-fold less sensitive to inhibition by all three inhibitors . The greatest effect on inhibition was found by substituting an Asp for Tyr-134 in the phosphatase . Clavosine B inhibited PP-1cgamma Y134D with a 310-fold decrease in potency . Clavosine A and calyculin A were also markedly poorer inhibitors of this mutant . These results suggest that a hydrogen bond between Tyr-134 and the calyculins is unlikely to be essential for inhibitor binding to the phosphatase . The clavosines and calyculin A were tested for their ability to inhibit other mutants of PP-1cgamma (including Ile-133, Val-223, and Cys-291) . Our mutagenesis studies provide an experimental basis for assessing models of calyculin binding found in the literature (Lindvall, M . K., Pihko, P . M., and Koskinen, A . M . (1997) J . Biol . Chem . 272, 23312-23316; Gupta, V., Ogawa, A . K., Du, X., Houk, K . N., and Armstrong, R . W . (1997) J . Med . Chem . 40, 3199-3206; Gauss, C . M., Sheppeck, I . J., Nairn, A . C., and Chamberlain, R . (1997) Bioorg . Med . Chem . 5, 1751-1773) . A new model for clavosine and calyculin A binding to PP-1c is presented that is consistent with previous structure-function experiments and which accommodates key structural features of the clavosines, including the novel rhamnose moiety. J Biol Chem, 2000 Feb 11, 275(6), 4183 - 91 Heparan sulfate proteoglycans as extracellular docking molecules for matrilysin (matrix metalloproteinase 7); Yu WH et al.; Many matrix metalloproteinases (MMPs) are tightly bound to tissues; matrilysin (MMP-7), although the smallest of the MMPs, is one of the most tightly bound . The most likely docking molecules for MMP-7 are heparan sulfate proteoglycans on or around epithelial cells and in the underlying basement membrane . This is established by extraction experiments and confocal microscopy . The enzyme is extracted from homogenates of postpartum rat uterus by heparin/heparan sulfate and by heparinase III treatment . The enzyme is colocalized with heparan sulfate in the apical region of uterine glandular epithelial cells and can be released by heparinase digestion . Heparan sulfate and MMP-7 are expressed at similar stages of the rat estrous cycle . The strength of heparin binding by recombinant rat proMMP-7 was examined by affinity chromatography, affinity coelectrophoresis, and homogeneous enzyme-based binding assay; the K(D) is 5-10 nM . Zymographic measurement of MMP-7 activity is greatly enhanced by heparin . Two putative heparin-binding peptides have been identified near the C- and N-terminal regions of proMMP-7; however, molecular modeling suggests a more extensive binding track or cradle crossing multiple peptide strands . Evidence is also found for the binding of MMP-2, -9, and -13 . Binding of MMP-7 and other MMPs to heparan sulfate in the extracellular space could prevent loss of secreted enzyme, provide a reservoir of latent enzyme, and facilitate cellular sensing and regulation of enzyme levels . Binding to the cell surface could position the enzyme for directed proteolytic attack, for activation of or by other MMPs and for regulation of other cell surface proteins . Dislodging MMPs by treatment with compounds such as heparin might be beneficial in attenuating excessive tissue breakdown such as occurs in cancer metastasis, arthritis, and angiogenesis. J Biol Chem, 2000 Feb 11, 275(6), 4112 - 7 Mutational analysis of Escherichia coli topoisomerase IV . III . Identification of a region of parE involved in covalent catalysis; Bahng S et al.; The products of three dominant-negative alleles of parE, encoding the ATP-binding subunit of topoisomerase IV (Topo IV), were purified and their activities characterized when reconstituted with ParC to form Topo IV . The ability of the ParE E418K, ParE G419D, and ParE G442D mutant Topo IVs to bind DNA, hydrolyze ATP, and close their ATP-dependent clamp was relatively unaffected . However, their ability to relax negatively supercoiled DNA was compromised significantly . This could be attributed to severe defects in covalent complex formation between ParC and DNA . Thus, these residues, which are far from the active site Tyr of ParC, contribute to covalent catalysis . This indicates that a dramatic conformational rearrangement of the protein likely occurs subsequent to the binding of the G segment at the DNA gate and prior to its opening. J Biol Chem, 2000 Feb 11, 275(6), 4104 - 11 Mutational analysis of Escherichia coli topoisomerase IV . II . ATPase negative mutants of parE induce hyper-DNA cleavage; Nurse P et al.; ParE is the ATP-binding subunit of topoisomerase IV (Topo IV) . During topoisomerization, the ATP-binding and hydrolysis cycle must be coordinated with the cycle of DNA cleavage and religation . We have isolated three dominant-negative mutant alleles of parE that encode ParE proteins that fail to hydrolyze ATP when reconstituted with ParC to form Topo IV . ParE G110S Topo IV and ParE S123L Topo IV failed to bind ATP at all, whereas ParE T201A could bind ATP . All three mutant Topo IV proteins exhibited an elevated level of spontaneous DNA cleavage that could be associated with a decreased rate of DNA resealing . In ParE T201A Topo IV, this defect appeared to result from an increased likelihood that the tetrameric enzyme would fall apart after DNA cleavage . Thus, while ATP is not required for DNA cleavage, the properties of these mutant enzymes suggests that ATP-hydrolysis informs DNA religation. J Biol Chem, 2000 Feb 11, 275(6), 4099 - 103 Mutational analysis of Escherichia coli topoisomerase IV . I . Selection of dominant-negative parE alleles; Mossessova E et al.; In order to define regions of ParE, one of the two subunits of topoisomerase IV, that are involved in catalysis during topoisomerization, we developed a selection procedure to isolate dominant-negative parE alleles . Both wild-type parC and mutagenized parE were expressed from a tightly-regulated lac promoter on a moderate-copy plasmid . Mutated parE alleles were rescued from those plasmids that caused IPTG-dependent cell death . The mutant ParE proteins could be divided into two groups when reconstituted with ParC to form topoisomerase IV, those that elicited hyper-DNA cleavage and those that affected covalent complex formation. J Biol Chem, 2000 Feb 11, 275(6), 4072 - 80 Characterization of a novel alanine-rich protein located in surface microdomains in Trypanosoma brucei; Nolan DP et al.; Heterologous expression in COS cells followed by orientation-specific polymerase chain reaction to select and amplify cDNAs encoding surface proteins in Trypanosoma brucei resulted in the isolation of a cDNA ( approximately 1.4 kilobase) which encodes an acidic, alanine-rich polypeptide that is expressed only in bloodstream forms of the parasite and has been termed bloodstream stage alanine-rich protein (BARP) . Analysis of the amino acid sequence predicted the presence of a typical NH(2)-terminal leader sequence as well as a COOH-terminal hydrophobic extension with the potential to be replaced by a glycosylphosphatidylinositol anchor . A search of existing protein sequences revealed partial homology between BARP and the major surface antigen of procyclic forms of Trypanosoma congolense . BARP migrated as a complex, heterogeneous series of bands on Western blots with an apparent molecular mass ( approximately 50-70 kDa) significantly higher than predicted from the amino acid sequence ( approximately 26 kDa) . Confocal microscopy demonstrated that BARP was present in small discrete spots that were distributed over the entire cellular surface . Detergent extraction experiments revealed that BARP was recovered in the detergent-insoluble, glycolipid-enriched fraction . These data suggested that BARP may be sequestered in lipid rafts. J Biol Chem, 2000 Feb 11, 275(6), 4060 - 5 The bifunctional active site of S-adenosylmethionine synthetase . Roles of the basic residues; Taylor JC et al.; S-adenosylmethionine (AdoMet) synthetase catalyzes a unique two-step enzymatic reaction leading to formation of the primary biological alkylating agent . The crystal structure of Escherichia coli AdoMet synthetase shows that the active site, which lies between two subunits, contains four lysines and one histidine as basic residues . In order to test the proposed charge and hydrogen bonding roles in catalytic function, each lysine has been changed to an uncharged methionine or alanine, and the histidine has been altered to asparagine . The resultant enzyme variants are all tetramers like the wild type enzyme; however, circular dichroism spectra show reductions in helix content for the K245*M and K269M mutants . (The asterisk denotes that the residue is in the second subunit.) Four mutants have k(cat) reductions of approximately 10(3)-10(4)-fold in AdoMet synthesis; however, the k(cat) of K165*M variant is only reduced 2-fold . In each mutant, there is a smaller catalytic impairment in the partial reaction of tripolyphosphate hydrolysis . The K165*A enzyme has a 100-fold greater k(cat) for tripolyphosphate hydrolysis than the wild type enzyme, but this mutant is not activated by AdoMet in contrast to the wild type enzyme . The properties of these mutants require reassessment of the catalytic roles of these residues. J Biol Chem, 2000 Feb 11, 275(6), 4055 - 9 Identification of a highly diverged class of S-adenosylmethionine synthetases in the archaea; Graham DE et al.; S-adenosylmethionine is the primary alkylating agent in all known organisms . ATP:L-methionine S-adenosyltransferase (MAT) catalyzes the only known biosynthetic route to this central metabolite . Although the amino acid sequence of MAT is strongly conserved among bacteria and eukarya, no homologs have been recognized in the completed genome sequences of any archaea . In this study, MAT has been purified to homogeneity from the archaeon Methanococcus jannaschii, and the gene encoding it has been identified by mass spectrometry . The peptide mass map identifies the gene encoding MAT as MJ1208, a hypothetical open reading frame . The gene was cloned in Escherichia coli, and expressed enzyme has been purified and characterized . This protein has only 22 and 23% sequence identity to the E . coli and human enzymes, respectively, whereas those are 59% identical to each other . The few identical residues include the majority of those constituting the polar active site residues . Each complete archaeal genome sequence contains a homolog of this archaeal-type MAT . Surprisingly, three bacterial genomes encode both the archaeal and eukaryal/bacterial types of MAT . This identification of a second major class of MAT emphasizes the long evolutionary history of the archaeal lineage and the structural diversity found even in crucial metabolic enzymes. J Biol Chem, 2000 Feb 11, 275(6), 3970 - 6 Rad51 uses one mechanism to drive DNA strand exchange in both directions; Namsaraev EA et al.; The Rad51 protein of Saccharomyces cerevisiae, like its bacterial counterpart RecA, promotes strand exchange between circular single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA) in vitro . However, the two proteins differ in the requirement for initiating joint molecules and in the polarity of branch migration . Whereas RecA initiates joint molecules from any type of ends on the dsDNA and branch migration proceeds exclusively in the 5'- to 3'-direction with respect to the single strand DNA substrate, initiation mediated by Rad51 requires a complementary 3' or 5' overhanging end of the linear dsDNA and branch migration proceeds in either direction . Here we report that the rates of Rad51-mediated branch migration in either the 5'- to 3'- or 3'- to 5'-directions are affected to the same extent by temperature and MgCl(2) . Furthermore, branch migration in both directions is equally impeded by insertions of non-homologous sequences in the dsDNA, inserts of 6 base pairs or more being completely inhibitory . We have also found that the preference of strand exchange in the 5'- to 3'-direction does not change if RPA is replaced by Escherichia coli SSB or T4 gene 32 proteins, suggesting that the preference for the direction of strand exchange is intrinsic to Rad51 . Based on these results, we conclude that Rad51-promoted branch migration in either direction occurs fundamentally by the same mechanism, quite probably by stabilizing successively formed heteroduplex base pair. J Biol Chem, 2000 Feb 11, 275(6), 3931 - 5 Invariance of the nucleoside triphosphate pools of Escherichia coli with growth rate; Petersen C et al.; The ATP and GTP pools of Escherichia coli have recently been reported to increase approximately 10-fold with increasing growth rates in the range from 0.4 to 1.4 generations/hour (Gaal, T., Bartlett, M . S., Ross, W., Turnbough, C . L., and Gourse, R . L . (1997) Science 278, 2092-2097) . Moreover, it was proposed that this variation of the nucleotide pools, particularly the ATP pool, might be responsible for the well known growth rate-dependent regulation of rRNA synthesis in E . coli . To test this hypothesis we have measured the nucleoside triphosphate pools as a function of growth rate for several E . coli strains . We found that the size of all four RNA precursor pools are essentially invariant with growth rate, in the range from 0.5 to 2.3 generations/hour . Nevertheless we observed the expected growth rate-dependent increase of RNA accumulation in these strains . In light of these results, it seems unlikely that nucleotide pool variations should be responsible for the growth rate-dependent regulation of rRNA synthesis. J Biol Chem, 2000 Feb 11, 275(6), 3873 - 8 The ATP hydrolytic activity of purified ZntA, a Pb(II)/Cd(II)/Zn(II)-translocating ATPase from Escherichia coli; Sharma R et al.; ZntA, a soft metal-translocating P1-type ATPase from Escherichia coli, confers resistance to Pb(II), Cd(II), and Zn(II) . ZntA was expressed as a histidyl-tagged protein, solubilized from membranes with Triton X-100, and purified to homogeneity . The soft metal-dependent ATP hydrolysis activity of purified ZntA was characterized . The activity was specific for Pb(II), Cd(II), Zn(II), and Hg(II), with the highest activity obtained when the metals were present as thiolate complexes of cysteine or glutathione . The maximal ATPase activity of ZntA was approximately 3 micromol/(mg x min) obtained with the Pb(II)-thiolate complex . In the absence of thiolates, Cd(II) inhibits ZntA above pH 6, whereas the Cd(II)-thiolate complexes stimulate activity, suggesting that a metal-thiolate complex is the true substrate in vivo . These results are consistent with the physiological role of ZntA as mediator of resistance to toxic concentrations of the divalent soft metals, Pb(II), Cd(II), and Zn(II), by ATP-dependent efflux . Our results confirm that ZntA is the first Pb(II)-dependent ATPase discovered to date. J Biol Chem, 2000 Feb 11, 275(6), 3737 - 40 Formation of the Ras dimer is essential for Raf-1 activation; Inouye K et al.; Although it is well established that Ras requires membrane localization for activation of its target molecule, Raf-1, the reason for this requirement is not fully understood . In this study, we found that modified Ras, which is purified from Sf9 cells, could activate Raf-1 in a cell-free system, when incorporated into liposome . Using a bifunctional cross-linker and a protein-fragmentation complementation assay, we detected dimer formation of Ras in the liposome and in the intact cells, respectively . These results suggest that dimerization of Ras in the lipid membrane is essential for activation of Raf-1 . To support this, we found that, when fused to glutathione S-transferase (GST), unprocessed Ras expressed in Escherichia coli could bypass the requirement for liposome . A Ras-dependent Raf-1 activator, which we previously reported (Mizutani, S., Koide, H., and Kaziro, Y . (1998) Oncogene 16, 2781-2786), was still required for Raf-1 activation by GST-Ras . Furthermore, an enforced dimerization of unmodified oncogenic Ras mutant in human embryonic kidney (HEK) 293 cells, using a portion of gyrase B or estrogen receptor, also resulted in activation of Raf-1 . From these results, we conclude that membrane localization allows Ras to form a dimer, which is essential, although not sufficient, for Raf-1 activation. Comp Immunol Microbiol Infect Dis, 2000 Jan, 23(1), 1 - 7 Serotypes of verotoxin-producing (Shiga toxin-producing) Escherichia coli isolated from healthy sheep; Bettelheim KA et al.; Using PCR techniques Shiga toxin-producing strains of Escherichia coli were isolated from the faeces of 45 out of 101 healthy sheep . These strains were serotyped and found to include O5:H-, O91:H- and O163:H19, which had previously been reported as being associated with human disease including haemolytic uraemic syndrome. APMIS, 1999 Dec, 107(12), 1069 - 78 Serological response to Helicobacter pylori recombinant antigens in Chilean infected patients with duodenal ulcer, non-ulcer dyspepsia and gastric cancer; Opazo P et al.; We have previously cloned 10 Helicobacter pylori antigen genes from a Chilean strain including: cytotoxin VacA, a truncated region of CagA (called A17), a species-specific protein (Ag26), urease subunits (UreA, UreB), a flagellin, (FlaB), heat shock proteins (HspA and HspB), an adhesin (HpaA) and a lipoprotein (Lpp20) . Immunogenicity of these antigens was tested by immunoblot with sera of Chilean infected patients, revealing that HpaA, A17, HspB and VacA were more frequently recognized (86%, 82%, 68% and 68%, respectively) . According to the clinical condition, it was determined that Lpp20 was preferentially recognized by sera from non-ulcer dyspepsia patients (80%), A17 and VacA by patients with duodenal ulcer (92% and 83% respectively), and HspB by patients with duodenal ulcer (83%) and gastric cancer (90%) . An ELISA was developed with a purified mixture of A17 and VacA antigens to test the different groups of patients . It was found that sera from duodenal ulcer patients showed higher values than those from non-ulcer dyspepsia patients, but this difference was not significant (p<0.2) . Moreover, sera from gastric cancer patients showed values lower than those from non-ulcer dyspepsia patients (p<0.019) . These results indicate that, in the Chilean population, antibodies raised against VacA and A 7 are not markers either for duodenal ulcer or for gastric cancer. Mol Gen Genet, 2000 Jan, 262(6), 1052 - 9 In vitro recruitment of the RfaH regulatory protein into a specialised transcription complex, directed by the nucleic acid ops element; Bailey MJ et al.; An unusual regulatory mechanism that controls transcription elongation in long fertility and virulence operons in bacteria is effected by two specialised components, the RfaH protein and the nucleic acid ops element . Without direct interaction, ops acts to reduce the concentration of RfaH required to stimulate distal gene transcription, and we have proposed that ops recruits RfaH to the transcription machinery . To provide direct experimental evidence for this view, we used gel fitration to identify potential RfaH complexes assembled in Escherichia coli cell extracts that carry out RfaH-dependent transcription . This novel molecular weight shift assay revealed that RfaH-dependent transcription elongation occurs concomitantly with recruitment of RfaH into a high molecular weight transcription complex, and that this recruitment is specifically directed by the ops element . Assembly of this complex required RNA polymerase and nucleotide hydrolysis, but not processive transcription . Neither assembly of the complex nor RfaH-dependent transcription was observed in in vitro reactions containing only ops, RfaH and purified core (alphabetabeta') RNA polymerase; both processes required the combination of subcellular fractions containing the RNA polymerase complex, the cytoplasmic membrane and ribosomes . The data confirm that the ops element directs recruitment of RfaH into a multi-component RNA polymerase complex that resists transcription termination. J Dairy Sci, 2000 Jan, 83(1), 48 - 51 Short communication: effects of isolation stress on mammary tight junctions in lactating dairy cows; Stelwagen K et al.; Eighteen cows had been selected for their responsiveness to psychological stress during the first lactation and were classified as having low (n = 10) or high (n = 8) cortisol concentrations in response to isolation-induced stress . In the present study these cows, now in their second lactation, were used to determine the effect of social isolation stress on the permeability of mammary tight junctions . During the experiment, each cow was isolated from the rest of the herd for 55 h . After the 1st h of isolation, each cow received a bolus infusion of endotoxin in one hind quarter in order to challenge tight junctions . Blood samples were taken throughout to measure lactose, which was used as an indicator of tight-junction leakiness . After 1 h of isolation, stress caused an increase in tight junction permeability in both groups, which was further enhanced by the endotoxin treatment . Although the permeability did not differ significantly between the two groups, it was consistently higher in the high-cortisol group, which was also the most stress-responsive group . Thus, psychological stress may adversely affect milk quality by allowing serum components to leak into milk. Nature, 2000 Jan 20, 403(6767), 335 - 8 A synthetic oscillatory network of transcriptional regulators; Elowitz MB et al.; Networks of interacting biomolecules carry out many essential functions in living cells, but the 'design principles' underlying the functioning of such intracellular networks remain poorly understood, despite intensive efforts including quantitative analysis of relatively simple systems . Here we present a complementary approach to this problem: the design and construction of a synthetic network to implement a particular function . We used three transcriptional repressor systems that are not part of any natural biological clock to build an oscillating network, termed the repressilator, in Escherichia coli . The network periodically induces the synthesis of green fluorescent protein as a readout of its state in individual cells . The resulting oscillations, with typical periods of hours, are slower than the cell-division cycle, so the state of the oscillator has to be transmitted from generation to generation . This artificial clock displays noisy behaviour, possibly because of stochastic fluctuations of its components . Such 'rational network design may lead both to the engineering of new cellular behaviours and to an improved understanding of naturally occurring networks. J Steroid Biochem Mol Biol, 1999 Dec 15, 71(3-4), 123 - 31 Correlation between PAP-dependent steroid binding activity and substrate specificity of mouse and human estrogen sulfotransferases; Qian Y et al.; Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes the sulfoconjugation and inactivation of estrogens using 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as an activated sulfate donor . A finding of undetermined significance in the study of EST has been that the guinea pig EST is able to bind pregnenolone and estradiol with high affinity in the presence of PAP, the reaction by-product of the sulfate donor PAPS . This finding has raised the possibility that EST may have other physiological functions independent of its enzymatic activity as a sulfotransferase . To determine if the PAP-dependent steroid binding activity is a common property shared by other estrogen sulfotransferases, we have expressed the mouse and human EST in bacteria and used the purified protein to address this question . We found that, in the presence of PAP, both recombinant mouse and human EST were able to bind estradiol with high affinity but only the human EST was able to bind pregnenolone . In addition, we show that human but not the mouse EST was also able to bind dehydroepiandrosterone, a property that was not described for the guinea pig EST . Furthermore, we demonstrate that the promiscuity of human EST in steroid binding is mirrored by a correspondingly low substrate specificity in its enzymatic activity as a sulfotransferase . Reversely, the lack of stable binding of pregnenolone and dehydroepiandrosterone by the mouse EST is paralleled by a lack of sulfotransferase activity of this enzyme toward these two steroids . Mutagenesis of mouse EST within a domain critical for PAPS binding abolished both its sulfotransferase and PAP-dependent estrogen binding activity . These data suggest that stable binding of steroids such as pregnenolone or estrogen is not an independent property of estrogen sulfotransferases but rather is related to their catalytic activity. Adv Exp Med Biol, 1999, 473, 163 - 71 Ultrastructure and DNA fragmentation analysis of arterioles in swine infected with Shiga toxin-producing Escherichia coli; Matise I et al.; Shiga toxins (Stx) produced by E . coli are potent cytotoxins that affect the vascular system . In humans, systemic toxemia causes renal glomerular damage manifested as hemolytic uremic syndrome . In swine, Stx-producing E . coli (STEC) cause edema disease that is characterized microscopically by segmental arteriolar smooth muscle cell (SMC) lesions . Our objectives were to characterize ultrastructurally and by TUNEL the type of death (apoptosis or necrosis) that occurs in SMCs during edema disease . Increased DNA fragmentation consistent with apoptosis was detected by TUNEL in arterioles of challenged pigs 14-15 days post inoculation . Ultrastructurally 3 grades of SMC lesions were distinguished: 1) Partial loss of SMCs, intercellular space filled with granular cellular debris admixed with membrane bound vacuoles; 2) Complete loss of SMCs; only granular cellular debris and clear vacuoles remained within basement membrane; 3) Inflammation of media; SMCs replaced by a rim of cellular debris located in the periphery of vessel wall . The most common lesion detected was grade 1 (9 ilea and 4 brains) . We did not find apoptotic nuclear changes in SMCs or apoptotic inclusion bodies within resident cells . Our study indicates, that (1) Stx produced during edema disease does not cause SMC apoptosis 14-15 dpi; (2) SMCs undergo an array of changes from degeneration to necrosis. Adv Exp Med Biol, 1999, 473, 147 - 54 K88 adhesins of enterotoxigenic Escherichia coli and their porcine enterocyte receptors; Francis DH et al.; The three antigenic variants of the K88 fimbrial adhesin (K88ab, K88ac, and K88ad) of enterotoxigenic Escherichia coli (ETEC) each exhibit unique specificity with regard to their hemagglutination characteristics . The variants are also unique in the specificity of their binding to the brush borders of enterocytes isolated from pigs with different genetic backgrounds . Diversity in enterocyte binding specificity suggests the existence of several K88 receptors, expressed individually or in various combinations on porcine enterocytes . Three candidate receptors have been identified that may explain the adhesion of K88 fimbrial variants to various porcine enterocytes . These receptors are an intestinal mucin-type sialoglycoprotein (IMTGP), an intestinal transferrin (GP74), and an intestinal neutral glycosphingolipid (IGLad) . The IMTGP binds K88ab and K88ac, but not K88ad . The GP74 binds K88ab, but not K88ac or K88ad, and the IGLad binds K88ad, but not K88ab or K88ac . Each of the candidate receptors has been found in brush borders that are adhesive for the fimbriae that bind the respective receptor . They have not been found in brush borders that are not adhesive for those same fimbriae . The presence of IMTGP was highly correlated with susceptibility of neonatal gnotobiotic pigs to ETEC expressing K88ab or K88ac. Adv Exp Med Biol, 1999, 473, 137 - 45 Age-dependent variation in the density and affinity of Escherichia coli heat-stable enterotoxin receptors in mice; al-Majali AM et al.; Enterotoxigenic strains of Escherichia coli that produce heat-stable enterotoxin (STa), are a major cause of diarrheal disease worldwide . Resistance to diarrheal disease in human infants and newborn animals has been attributed to a gradual turnover in the intestinal brush border membrane receptors to bacterial pili . In this study, we demonstrated age-dependent variation in the density and affinity of the mouse enterocyte receptors specific for STa . Flow cytometry and radiolabeled-STa (125I-STa) assays were used as more reliable quantitative measures for the characterization of STa-enterocyte receptor interaction . These assays indicated a stronger interaction of STa with its putative receptor on the enterocytes of the 2-day-old suckling mice than with enterocytes from 1-week, 2-week and 2-month-old mice . Scatchard plot analysis of 125I-STa-receptor interaction suggested that STa-receptors exist at a higher number on enterocytes from the 2-day-old mice than enterocytes of the older mice . Additionally, receptors from the 2-day-old mice had a greater affinity for STa ligand than receptors from the older mice . Density of STa receptors on enterocytes and their affinity to STa may determine the extent of binding and severity of secretory response . This may further explain the increased susceptibility of newborn animals and human infants to STa-mediated diarrheal disease. Adv Exp Med Biol, 1999, 473, 129 - 36 The locus for enterocyte effacement (LEE) of enteropathogenic Escherichia coli (EPEC) from dogs and cats; Goffaux F et al.; Enteropathogenic Escherichia coli (EPEC) produce attaching and effacing lesions . The genes responsible for this lesion are clustered on the chromosome forming a 35.5 kilobase pathogenesis island called LEE . The LEE was identified, characterized and completely sequenced from the human EPEC strain E2348/69 . The LEE carries genes coding for: a type III secretion system (genes esc and sep), the translocated intimin receptor (gene tir), the outer membrane protein intimin (gene eae) and the E . coli secreted proteins EspA, EspB, and EspD (genes esp) . In addition to man and farm animals, EPEC are also isolated from dogs and cats . We studied structurally and functionally the LEE of dog and cat EPEC . First, we used four probes scattered along the LEE to identify the presence of a LEE in canine and feline EPEC isolates . Second, by PCR, we checked the presence of genes homologous to eae, sep, esp, and tir genes in these strains . Third, since the four types of eae and tir genes were described, we developed a multiplex PCR in order to determine the type of eae and tir genes present in each strain . Fourth, we determined by PCR the site of the LEE insertion on the chromosome . Fifth, we tested several of the canine EPEC in their capacity to induce attaching and effacing lesions in the rabbit intestinal loop assay . We can conclude from this study: first, that the a LEE-like structure is present in all tested strains and that it contains genes homologous to esp, sep, tir, and eae genes; second, that there is some preferential associations between the type of eae gene and the type of tir gene present in a strain; third, that the majority of the tested strains contained a LEE located elsewhere on the chromosome in comparison to the human EPEC strain E2348/69; and fourth that dog EPEC were able to induce attaching and effacing lesions in rabbit ileal loop assay. Adv Exp Med Biol, 1999, 473, 125 - 8 Reproduction of lesions and clinical signs with a CNF2-producing Escherichia coli in neonatal calves; Van Bost S et al.; CNF2-producing necrotoxigenic E . coli (NTEC2) are associated with diarrhoea and septicaemia in calves . We orally inoculated neonatal calves with a NTEC2 strain in order to reproduce clinical signs and lesions . We observed diarrhoea in each inoculated calf, bacteraemia (80%), the presence of CNF2+ bacteria in the lungs (80%) and in the liver (20%) . The observed lesions were inflammation of the entire gut, hypertrophy of the mesenteric lymph nodes and hepatisation of the lungs . We were unable to detect characteristic lesions that are classical signs of septicaemia. Adv Exp Med Biol, 1999, 473, 113 - 23 Insulin modulates intestinal response of suckling mice to the Escherichia coli heat-stable enterotoxin; al-Majali AM et al.; Effect of insulin on the response of suckling mice to the enterotoxigenic Escherichia coli heat-stable enterotoxin (STa) was studied . Four groups (8-10 in each group) of two day old Swiss Webster suckling mice were used . Five, 10, 25, and 50 micrograms of insulin were given orally to half the mice in each group respectively . The rest of the mice in each group were given normal saline as intra-litter controls . After 7 days, the suckling mouse assay for STa was performed on three mice from each insulin-treated and control groups . Enterocyte suspensions were prepared from mice in all groups . Intestinal tissue samples were taken for electron microscopy . Interaction of STa with its putative receptor on the enterocytes was evaluated using indirect immunofluorescence and flow cytometry . The suckling mouse assay revealed a significant increase in the gut weight to body weight ratio in all mice in the insulin treated groups compared to control mice (p < 0.05) . Flow cytometry and indirect immunofluorescence analyses suggested that insulin had an upregulatory effect on the STa receptor level . Similarly, insulin was found to increase intestinal brush border membrane differentiation as indicated by the increase in the inward movement of milk particles through the intestinal mucosa . Insulin seems to modify the structure-function of the brush border membrane including the response of suckling mice to STa . This study may provide further insights into the mechanism of STa/receptor interaction in diarrhea in newborn animals and human infants. Microbiology, 2000 Jan, 146 ( Pt 1), 155 - 63 Elucidation of anthracyclinone biosynthesis by stepwise cloning of genes for anthracyclines from three different Streptomyces spp; Kantola J et al.; The anthracycline skeleton is biosynthesized by aromatic (type II) polyketide synthases . Furthermore, three post-polyketide steps are needed to form the basic aglycone of anthracyclines . Auramycinone was produced in Streptomyces lividans by introducing nine structural genes from three different anthracycline-producing Streptomyces species . The genes used to construct the auramycinone biosynthesis cluster were derived from nogalamycin-, daunomycin- and aclacinomycin-producing Streptomyces strains . The biosynthetic stages were divided into polyketide and post-polyketide steps on the assumption that the first stable intermediate would be nogalonic acid, named analogously to aklanonic acid, the precursor of several anthracyclines . Single genes were cloned in the expression construct in the order determined by the proposed biosynthetic pathway . This facilitated investigation of the products formed in the heterologous host after addition of each separate gene to the construct . The results thus elucidate the biosynthesis steps, products and the genes responsible for the reactions needed to build up an anthracyclinone. FASEB J, 2000 Feb, 14(2), 407 - 17 In situ detection of AP sites and DNA strand breaks bearing 3'-phosphate termini in ischemic mouse brain; Huang D et al.; Our aims were to examine whether oxidative DNA damage was elevated in brain cells of male C57BL/6 mice after oxidative stress, and to determine whether neuronal nitric oxide synthase (nNOS) was involved in such damage . Oxidative stress was induced by occluding both common carotid arteries for 90 min, followed by reperfusion . Escherichia coli exonuclease III (Exo III) removes apyrimidinic or apurinic (AP) sites and 3'-phosphate termini in single-strand breaks, and converts these lesions to 3'OH termini . These ExoIII-sensitive sites (EXOSS) can then be postlabeled using digoxigenin-11-dUTP and Klenow DNA polymerase-I, and detected using fluorescein isothiocyanate-IgG against digoxigenin . Compared with the non-ischemia controls, the density of EXOSS-positive cells was elevated at least 20-fold (P < 0.01) at 15 min of reperfusion, and remained elevated for another 30 min . EXOSS mainly occurred in the cell nuclei of the astrocytes and neurons . Signs of cell death were detected at 24 h of reperfusion and occurred mostly in the neurons . Both DNA damage and cell death in the cerebral cortical neurons were abolished by treatment with 3-bromo-7-nitroindazole (30 mg/kg, intraperitoneal), which specifically inhibited nNOS . Our results suggest that nNOS, its activator (calcium), and peroxynitrite exacerbate oxidative DNA damage after brain ischemia.-Huang, D., Shenoy, A., Cui, J., Huang, W., Liu, P . In situ detection of AP sites and DNA strand breaks bearing 3'-phosphate termini in ischemic mouse brain. Biochem J, 2000 Feb 15, 346 Pt 1, 223 - 32 Structure-function studies of tryptophan mutants of equinatoxin II, a sea anemone pore-forming protein; Malovrh P et al.; Equinatoxin II (EqtII) is a eukaryotic cytolytic toxin that avidly creates pores in natural and model lipid membranes . It contains five tryptophan residues in three different regions of the molecule . In order to study its interaction with the lipid membranes, three tryptophan mutants, EqtII Trp(45), EqtII Trp(116/117) and EqtII Trp(149), were prepared in an Escherichia coli expression system {here, the tryptophan mutants are classified according to the position of the remaining tryptophan residue(s) in each mutated protein} . They all possess a single intrinsic fluorescent centre . All mutants were less haemolytically active than the wild-type, although the mechanism of erythrocyte damage was the same . EqtII Trp(116/117) resembles the wild-type in terms of its secondary structure content, as determined from Fourier-transform infrared (FTIR) spectra and its fluorescent properties . Tryptophans at these two positions are buried within the hydrophobic interior of the protein, and are transferred to the lipid phase during the interaction with the lipid membrane . The secondary structure of the other two mutants, EqtII Trp(45) and EqtII Trp(149), was altered to a certain extent . EqtII Trp(149) was the most dissimilar from the wild-type, displaying a higher content of random-coil structure . It also retained the lowest number of nitrogen-bound protons after exchange with (2)H(2)O, which might indicate a reduced compactness of the molecule . Tryptophans in EqtII Trp(45) and EqtII Trp(149) were more exposed to water, and also remained as such in the membrane-bound form. Biochem J, 2000 Feb 15, 346 Pt 1, 163 - 8 Properties of Leishmania major dUTP nucleotidohydrolase, a distinct nucleotide-hydrolysing enzyme in kinetoplastids; Camacho A et al.; We have previously reported the presence, in the parasitic protozoan Leishmania major, of an enzyme involved in controlling intracellular dUTP levels . The gene encoding this enzyme has now been overexpressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity . Biochemical and enzymic analyses of the Leishmania enzyme show that it is a novel nucleotidohydrolase highly specific for deoxyuridine 5'-triphosphate . The enzyme has proved to be a dimer by gel filtration and is able to hydrolyse both dUTP and dUDP quite efficiently, acting as a dUTP nucleotidohydrolase (dUTPase)-dUDP nucleotidohydrolase but has a limited capacity to act upon other nucleoside di- or triphosphates . The reaction products are dUMP and PP(i) when dUTP is the substrate and dUMP and P(i) in the case of dUDP . The enzyme is sensitive to inhibition by the reaction product dUMP but not by PP(i) . dUTPase activity is highly dependent on Mg(2+) concentrations and markedly sensitive to the phosphatase inhibitor, NaF . In summary, Leishmania dUTPase appears to be markedly different to other proteins characterized previously that accomplish the same function. Biochem J, 2000 Feb 15, 346 Pt 1, 1 - 8 Thioredoxin reductase; Mustacich D et al.; The mammalian thioredoxin reductases (TrxRs) are a family of selenium-containing pyridine nucleotide-disulphide oxidoreductases with mechanistic and sequence identity, including a conserved -Cys-Val-Asn-Val-Gly-Cys- redox catalytic site, to glutathione reductases . TrxRs catalyse the NADPH-dependent reduction of the redox protein thioredoxin (Trx), as well as of other endogenous and exogenous compounds . The broad substrate specificity of mammalian TrxRs is due to a second redox-active site, a C-terminal -Cys-SeCys- (where SeCys is selenocysteine), that is not found in glutathione reductase or Escherichia coli TrxR . There are currently two confirmed forms of mammalian TrxRs, TrxR1 and TrxR2, and it is possible that other forms will be identified . The availability of Se is a key factor determining TrxR activity both in cell culture and in vivo, and the mechanism(s) for the incorporation of Se into TrxRs, as well as the regulation of TrxR activity, have only recently begun to be investigated . The importance of Trx to many aspects of cell function make it likely that TrxRs also play a role in protection against oxidant injury, cell growth and transformation, and the recycling of ascorbate from its oxidized form . Since TrxRs are able to reduce a number of substrates other than Trx, it is likely that additional biological effects will be discovered for TrxR . Furthermore, inhibiting TrxR with drugs may lead to new treatments for human diseases such as cancer, AIDS and autoimmune diseases. Nat Biotechnol, 2000 Feb, 18(2), 168 - 71 Trehalose expression confers desiccation tolerance on human cells; Guo N et al.; Many organisms that withstand desiccation express the disaccharide trehalose . We have now expressed the otsA and otsB genes of Escherichia coli, which encode trehalose biosynthetic enzymes, in human primary fibroblasts using a recombinant adenovirus vector . Infected cells produced increased amounts of trehalose with increasing multiplicity of infection (MOI) . Human primary fibroblasts expressing trehalose could be maintained in the dry state for up to five days . Fourier transform infrared spectroscopy indicated that dry, but viable, human cells contained no detectable water . This study shows that mammalian cells can be engineered to retain viability in the absence of water. J Mol Biol, 2000 Feb 11, 296(1), 57 - 86 Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides; Knappik A et al.; By analyzing the human antibody repertoire in terms of structure, amino acid sequence diversity and germline usage, we found that seven V(H) and seven V(L) (four Vkappa and three Vlambda) germline families cover more than 95 % of the human antibody diversity used . A consensus sequence was derived for each family and optimized for expression in Escherichia coli . In order to make all six complementarity determining regions (CDRs) accessible for diversification, the synthetic genes were designed to be modular and mutually compatible by introducing unique restriction endonuclease sites flanking the CDRs . Molecular modeling verified that all canonical classes were present . We could show that all master genes are expressed as soluble proteins in the periplasm of E . coli . A first set of antibody phage display libraries totalling 2x10(9) members was created after cloning the genes in all 49 combinations into a phagemid vector, itself devoid of the restriction sites in question . Diversity was created by replacing the V(H) and V(L) CDR3 regions of the master genes by CDR3 library cassettes, generated from mixed trinucleotides and biased towards natural human antibody CDR3 sequences . The sequencing of 257 members of the unselected libraries indicated that the frequency of correct and thus potentially functional sequences was 61 % . Selection experiments against many antigens yielded a diverse set of binders with high affinities . Due to the modular design of all master genes, either single binders or even pools of binders can now be rapidly optimized without knowledge of the particular sequence, using pre-built CDR cassette libraries . The small number of 49 master genes will allow future improvements to be incorporated quickly, and the separation of the frameworks may help in analyzing why nature has evolved these distinct subfamilies of antibody germline genes . J Mol Biol, 2000 Feb 11, 296(1), 19 - 31 Mapping RNA-protein interactions in ribonuclease P from Escherichia coli using disulfide-linked EDTA-Fe; Biswas R et al.; The protein subunit of Escherichia coli ribonuclease P (which has a cysteine residue at position 113) and its single cysteine-substituted mutant derivatives (S16C/C113S, K54C/C113S and K66C/C113S) have been modified using a sulfhydryl-specific iron complex of EDTA-2- aminoethyl 2-pyridyl disulfide (EPD-Fe) . This reaction converts C5 protein, or its single cysteine-substituted mutant derivatives, into chemical nucleases which are capable of cleaving the cognate RNA ligand, M1 RNA, the catalytic RNA subunit of E . coli RNase P, in the presence of ascorbate and hydrogen peroxide . Cleavages in M1 RNA are expected to occur at positions proximal to the site of contact between the modified residue (in C5 protein) and the ribose units in M1 RNA . When EPD-Fe was used to modify residue Cys16 in C5 protein, hydroxyl radical-mediated cleavages occurred predominantly in the P3 helix of M1 RNA present in the reconstituted holoenzyme . C5 Cys54-EDTA-Fe produced cleavages on the 5' strand of the P4 pseudoknot of M1 RNA, while the cleavages promoted by C5 Cys66-EDTA-Fe were in the loop connecting helices P18 and P2 (J18/2) and the loop (J2/4) preceding the 3' strand of the P4 pseudoknot . However, hydroxyl radical-mediated cleavages in M1 RNA were not evident with Cys113-EDTA-Fe, perhaps indicative of Cys113 being distal from the RNA-protein interface in the RNase P holoenzyme . Our directed hydroxyl radical-mediated footprinting experiments indicate that conserved residues in the RNA and protein subunit of the RNase-P holoenzyme are adjacent to each other and provide structural information essential for understanding the assembly of RNase P . J Mol Biol, 2000 Jan 28, 295(4), 927 - 38 X-ray structure of yeast Hal2p, a major target of lithium and sodium toxicity, and identification of framework interactions determining cation sensitivity; Albert A et al.; The product of the yeast HAL2 gene (Hal2p) is an in vivo target of sodium and lithium toxicity and its overexpression improves salt tolerance in yeast and plants . Hal2p is a metabolic phosphatase which catalyses the hydrolysis of 3'-phosphoadenosine-5'-phosphate (PAP) to AMP . It is, the prototype of an evolutionarily conserved family of PAP phosphatases and the engineering of sodium insensitive enzymes of this group may contribute to the generation of salt-tolerant crops . We have solved the crystal structure of Hal2p in complex with magnesium, lithium and the two products of PAP hydrolysis, AMP and Pi, at 1.6 A resolution . A functional screening of random mutations of the HAL2 gene in growing yeast generated forms of the enzyme with reduced cation sensitivity . Analysis of these mutants defined a salt bridge (Glu238 ellipsis Arg152) and a hydrophobic bond (Va170 ellipsis Trp293) as important framework interactions determining cation sensitivity . Hal2p belongs to a larger superfamily of lithium-sensitive phosphatases which includes inositol monophosphatase . The hydrophobic interaction mutated in Hal2p is conserved in this superfamily and its disruption in human inositol monophosphatase also resulted in reduced cation sensitivity . J Mol Biol, 2000 Jan 28, 295(4), 831 - 52 Kinetic mechanism of the single-stranded DNA recognition by Escherichia coli replicative helicase DnaB protein . Application of the matrix projection operator technique to analyze stopped-flow kinetics; Bujalowski W et al.; Kinetics of the Escherichia coli primary replicative helicase DnaB protein binding to a single-stranded DNA, in the presence of the ATP non-hydrolyzable analog AMP-PNP, have been performed, using the fluorescence stopped-flow technique . This is the first direct determination of the mechanism of the ssDNA recognition by a hexameric helicase . Binding of the fluorescent etheno-derivative of a ssDNA to the enzyme is characterized by a strong increase of the nucleic acid fluorescence, which provides an excellent signal to quantitatively study the mechanism of ssDNA recognition by the helicase . The kinetic experiments have been performed with a ssDNA 20-mer, depsilonA(pepsilonA)(19), that encompasses the entire, total ssDNA-binding site of the helicase and with the 10-mer depsilonA(pepsilonA)(9), which binds exclusively to the ssDNA strong subsite within the total ssDNA-binding site . Association of the DnaB helicase with the 20-mer is characterized by three relaxation times, which indicates that the binding occurs by the minimum three-step mechanism where the bimolecular binding step is followed by two isomerization steps . This mechanism is described by the equation: Helicase+ssDNAk1/(k1)<-->(k-1)(H-ssDNA)1(k2)<-->(k-2)(H-ssDNA)2 (k3)<-->(k-3)(H-ssDNA)3 . The value of the bimolecular rate constant, k(1), is four to six orders of magnitude lower than the value expected for the diffusion-controlled reaction . Moreover, quantitative amplitude analysis suggests that the major conformational change of the ssDNA takes place in the formation of the (H-ssDNA)(1) . These results indicate that the determined first step includes formation of the collision and an additional transition of the protein-ssDNA complex, most probably the local opening of the protein hexamer . The data indicate that the binding mechanism reflects the interactions of the ssDNA predominantly through the strong ssDNA-binding subsite . The analysis of the stopped-flow kinetics has been performed using the matrix-projection operator technique, which provides a powerful method to address stopped-flow kinetics, particularly, the amplitudes . The method allowed us to determine the specific fluorescence changes accompanying the formation of all the intermediates . The sequential nature of the determined mechanism indicates the lack of the kinetically significant conformational equilibrium of the DnaB hexamer as well as a transient dissociation of the hexamer prior to the ssDNA binding . The significance of these results for the functioning of the DnaB helicase is discussed . J Mol Biol, 2000 Jan 28, 295(4), 815 - 29 Mutations influencing the frr gene coding for ribosome recycling factor (RRF); Janosi L et al.; A total of 52 null, six reversion, and five silent mutations of frr (the gene encoding for ribosome recycling factor (RRF)) of Escherichia coli are discussed along with 12 temperature-sensitive (ts) mutations and 14 intergenic suppressor strains of ts RRF . The null mutations were classified into six different categories . A computer-based secondary structure analysis showed three domains; domain A which has the N-terminal helix, domain B which contains coil, alpha-helix and beta-strand structure, and domain C which is a C-terminal helix . The ts mutations fell into domains A and C but not in domain B . More than a half of the null mutations fell into domain B while the silent mutations fell outside domain B . Substitution of Arg132 in domain C by other amino acids was observed among five independently isolated null mutants . It is suggested that domain B is important for maintaining the RRF structure, while the region including Arg132 is one of the active sites . A total of 14 intergenic suppressor strains of ts RRF were grouped into four categories, depending on which temperature-sensitive alleles were suppressed . J Mol Biol, 2000 Jan 28, 295(4), 803 - 14 DNA polymerase switching: II . Replication factor C abrogates primer synthesis by DNA polymerase alpha at a critical length; Mossi R et al.; A crucial event in DNA replication is the polymerase switch from the synthesis of a short RNA/DNA primer by DNA polymerase alpha/primase to the pro?cessive elongation by DNA polymerase delta . In order to shed light on the role of replication factor C (RF-C) in this process, the effects of RF-C on DNA polymerase alpha were investigated . We show that RF-C stalls DNA polymerase alpha after synthesis of approximately 30 nucleotides, while not inhibiting the polymerase activity per se . This suggested that RF-C and the length of the primer may be two important factors contributing to the polymerase switch . Furthermore the DNA binding properties of RF-C were tested . Band shift experiments indicated that RF-C has a preference for 5' recessed ends and double-stranded DNA over 3' ends . Finally PCNA can be loaded onto a DNA template carrying a RNA primer, suggesting that a DNA moiety is not necessarily required for the loading of the clamp . Thus we propose a model where RF-C, upon binding to the RNA/DNA primer, influences primer synthesis and sets the conditions for a polymerase switch after recruiting PCNA to DNA . J Mol Biol, 2000 Jan 28, 295(4), 777 - 89 Probing a tRNA core that contributes to aminoacylation; Hamann CS et al.; The contribution of the tRNA "core" to aminoacylation is beginning to be recognized . One example is the core region of Escherichia coli tRNA(Cys), which has been shown by biochemical studies to be important for aminoacylation . This core has several layers of unusual base-pairs, which are revealed by the recent crystal structure of the tRNA complexed with the elongation factor EF-Tu and an analog of GTP . One of these layers consists of a 9:{13:22} base-triple, rather than the 46:{13:22} or 45:{13:22} base-triple that is commonly observed in tRNA structure . Because 13:22 is an important element in aminoacylation of E . coli tRNA(Cys), a better understanding of its structure in the tRNA core will shed light on its role in aminoacylation . In this study, we used the phage T7 transcript of the tRNA as a substrate . We probed the structure of 13:22 by dimethyl sulfate and tested its partner in a base-triple by generating mutations that could be assayed for aminoacylation . The results of this study in general are in a better agreement with a 46:{13:22} base-triple that we previously proposed . Although these results are not interpreted as direct proof for the 46:{13:22} base-triple, they shed new light on features of the tRNA core that are important for aminoacylation . J Mol Biol, 2000 Jan 28, 295(4), 767 - 75 A DNA-binding domain swap converts the invertase gin into a resolvase; Schneider F et al.; DNA resolvases and invertases are closely related, yet catalyze recombination within two distinct nucleoprotein structures termed synaptosomes and invertasomes, respectively . Different protein-protein and protein-DNA interactions guide the assembly of each type of recombinogenic complex, as well as the subsequent activation of DNA strand exchange . Here we show that invertase Gin catalyzes factor for inversion stimulation dependent inversion on isolated copies of sites I from ISXc5 res, which is typically utilized by the corresponding resolvase . The concomitant binding of Gin to sites I and III in res, however, inhibits recombination . A chimeric recombinase, composed of the catalytic domain of Gin and the DNA-binding domain of ISXc5 resolvase, recombines two res with high efficiency . Gin must therefore contain residues proficient for both synaptosome formation and activation of strand exchange . Surprisingly, this chimera is unable to assemble a productive invertasome; a result which implies a role for the C-terminal domain in invertasome formation that goes beyond DNA binding . J Mol Biol, 2000 Jan 28, 295(4), 737 - 44 Localized, stereochemically sensitive hydrophobic packing in an early folding intermediate of dihydrofolate reductase from Escherichia coli; O'Neill JC Jr et al.; Mutational analysis was performed to probe the development of hydrophobic clusters during the early events in the folding of dihydrofolate reductase . Replacements were made in several hydrophobic subdomains to examine the roles of hydrophobicity and stereochemistry in the formation of kinetic intermediates . Amide protons in two of these clusters, including residues I91, I94, and I155, have been shown to be protected against solvent exchange within 13 ms of folding . Additional hydrophobic clusters were probed by substitutions at residues I2, I61, and L112; these residues are not protected from exchange until later in the folding reaction . Valine and leucine replacements at positions I91, I94, and I155 significantly diminish the formation of the burst phase kinetic intermediate, relative to the wild-type protein . In contrast, I2 and I61 are insensitive to these substitutions in the first 5 ms of the folding reaction, as is the replacement of L112 with either isoleucine or valine . These results demonstrate that the tightly packed core of dihydrofolate reductase is acquired in a non-uniform fashion, beginning in the submillisecond time frame . The progressive development of specific side-chain packing in localized hydrophobic clusters may be a common theme for complex protein folding reactions . Am Nat, 2000 Jan, 155(1), 24 - 35 Long-Term Experimental Evolution in Escherichia coli . VIII . Dynamics of a Balanced Polymorphism; Rozen DE et al.; We describe the short- and long-term dynamics of a phenotypic polymorphism that arose in a population of Escherichia coli while it was serially propagated for almost 20,000 generations in a glucose-limited minimal medium . The two types, designated L and S, differ conspicuously in the size of the colonies they form on agar plates as well as the size of their individual cells, and these differences are heritable . The S type reached a detectable frequency (>1%) at generation 6,000, and it remained above that frequency throughout the subsequent generations . In addition to morphological differences, L and S diverged in important ecological properties . With clones isolated at 18,000 generations, L has a maximal growth rate in fresh medium that is approximately 20% higher than that of S . However, experiments with conditioned media demonstrate that L and S secrete one or more metabolites that promote the growth of S but not of L . The death rate of L during stationary phase also increases when S is abundant, which suggests that S may either secrete a metabolite that is toxic to L or remove some factor that enables the survival of L . One-day competition experiments with the clones isolated at generation 18,000 show that their relative fitness is frequency dependent, with each type having an advantage when rare . When these two types are grown together for a period of several weeks, they converge on an equilibrium frequency that is consistent with the 1-d competition experiments . Over the entire 14,000-generation period of coexistence, however, the frequency of the S type fluctuated between approximately 10% and 85% . We offer several hypotheses that may explain the fluctuations in this balanced polymorphism, including the possibility of coevolution between the two types. Mech Ageing Dev, 1999 Nov, 111(2-3), 89 - 95 Role of superoxide, NO and oxygen in the regulation of energy metabolism and suppression of senile diseases; Inoue M et al.; Although nitric oxide (NO) rapidly reacts with molecular oxygen under air atmospheric conditions, thereby losing its biological functions, the lifetime of this gaseous radical increases under physiologically low intracellular oxygen tensions . To understand the pathophysiological roles of NO and related molecules in aerobic life, we analyzed the effect of oxygen tensions on the NO-dependent processes in resistance arteries, isolated mitochondria, intact cells and enteric bacteria . Kinetic analysis revealed that NO enhanced the generation of cGMP and induced vasorelaxation of resistance arteries more potently under physiologically low oxygen tensions than under hyperbaric conditions . NO reversibly inhibited the respiration of isolated mitochondria, intact cells and Escherichia coli; the inhibitory effect was more marked under hypoxic conditions than under hyperbaric conditions . Kinetic analysis revealed that NO has pivotal action to increase arterial supply of molecular oxygen for the generation of ATP in peripheral tissues and to suppress energy production in mitochondria and cells in an oxygen-dependent manner . These functions of NO are enhanced by decreasing oxygen tension in situ and suppressed by locally generated superoxide radicals . Thus, cross-talk of NO, superoxide and molecular oxygen constitutes a supersystem by which the energy metabolism in cells and tissues is beautifully regulated in a site-specific manner depending on the relative concentrations of these three radical species. Phytochemistry, 2000 Jan, 53(1), 55 - 8 Antifungal nitro compounds from skunk cabbage (Lysichitum americanum) leaves treated with cupric chloride; Hanawa F et al.; Two nitro compounds, 2-(4-methoxyphenyl)-1-nitroethane named as lysichitalexin and 2-(4-hydroxyphenyl)-1-nitroethane were isolated as stress metabolites from the leaves of Lysichitum americanum Hulten and St . John treated with cupric chloride . Their structures were determined by spectroscopic methods and chemical reactions . The former compound showed antifungal activities against Fusarium oxysporum and Cladosporium herbarum . Both compounds were isolated for the first time from this species and the former was isolated from natural sources for the first time . This is the first report on stress metabolites from a member of the Araceae. J Biotechnol, 2000 Jan 21, 76(2-3), 215 - 26 Integrated bioprocess for production of human proinsulin C-peptide via heat release of an intracellular heptameric fusion protein; Jonasson P et al.; An integrated bioprocess has been developed suitable for production of recombinant peptides using a gene multimerization strategy and site-specific cleavage of the resulting gene product . The process has been used for production in E . coli of the human proinsulin C-peptide via a fusion protein BB-C7 containing seven copies of the 31-residues C-peptide monomer . The fusion protein BB-C7 was expressed at high level, 1.8 g l(-1), as a soluble gene product in the cytoplasm . A heat treatment procedure efficiently released the BB-C7 fusion protein into the culture medium . This step also served as an initial purification step by precipitating the majority of the host cell proteins, resulting in a 70% purity of the BB-C7 fusion protein . Following cationic polyelectrolyte precipitation of the nucleic acids and anion exchange chromatography, native C-peptide monomers were obtained by enzymatic cleavage at flanking arginine residues . The released C-peptide material was further purified by reversed-phase chromatography and size exclusion chromatography . The overall yield of native C-peptide at a purity exceeding 99% was 400 mg l(-1) culture, corresponding to an overall recovery of 56% . The suitability of this process also for the production of other recombinant proteins is discussed. Free Radic Biol Med, 2000 Jan 1, 28(1), 55 - 63 DNA damage in arsenite- and cadmium-treated bovine aortic endothelial cells; Liu F et al.; Reactive oxygen species have been shown to be involved in the mutagenicity, clastogenicity, and apoptosis of mammalian cells treated with arsenic or cadmium . As these endpoints require several hours of cellular processing, it is not clear that reactive oxygen species damage DNA directly or interfere with DNA replication and repair . Using single-cell alkaline electrophoresis, we have detected DNA strand breaks (DSBs) in bovine aortic endothelial cells by a 4-h treatment with sodium arsenite (As) and cadmium chloride (Cd) in sublethal concentrations . As-induced DSBs could be decreased by nitric oxide (NO) synthase inhibitors, superoxide scavengers, and peroxynitrite scavengers and could be increased by superoxide generators and NO generators . Treatment with As also increased nitrite production . These results suggest that As-increased NO may react with O2*- to produce peroxynitrite and cause DNA damage . The results showing that Cd increased cellular H2O2 levels and that Cd-induced DSBs could be modulated by various oxidant modulators suggest that Cd may induce DSBs via O2*-, H2O2, and *OH . Nevertheless, the DSBs in both As- and Cd-treated cells seem to come from the excision of oxidized bases such as formamidopyrimidine and 8-oxoguanine, as the Escherichia coli enzyme formamidopyrimidine-DNA glycosylase (Fpg) increased DSBs in cells treated with As, 3-morpholinosydnonimine (a peroxynitrite-generating agent), Cd, or H2O2. Nat Struct Biol, 2000 Feb, 7(2), 108 - 13 Crystal structures of Escherichia coli phytase and its complex with phytate; Lim D et al.; Phytases catalyze the hydrolysis of phytate and are able to improve the nutritional quality of phytate-rich diets . Escherichia coli phytase, a member of the histidine acid phosphatase family has the highest specific activity of all phytases characterized . The crystal structure of E . coli phytase has been determined by a two-wavelength anomalous diffraction method using the exceptionally strong anomalous scattering of tungsten . Despite a lack of sequence similarity, the structure closely resembles the overall fold of other histidine acid phosphatases . The structure of E . coli phytase in complex with phytate, the preferred substrate, reveals the binding mode and substrate recognition . The binding is also accompanied by conformational changes which suggest that substrate binding enhances catalysis by increasing the acidity of the general acid. Proc Natl Acad Sci U S A, 2000 Feb 1, 97(3), 1062 - 7 Biosynthesis of terpenoids: YchB protein of Escherichia coli phosphorylates the 2-hydroxy group of 4-diphosphocytidyl-2C-methyl-D-erythritol; Luttgen H et al.; A comparative analysis of all published complete genomes indicated that the putative orthologs of the unannotated ychB gene of Escherichia coli follow the distribution of the dxs, dxr, and ygbP genes, which have been shown to specify enzymes of the deoxyxylulose phosphate pathway of terpenoid biosynthesis, thus suggesting that the hypothetical YchB protein also is involved in that pathway . To test this hypothesis, the E . coli ychB gene was expressed in a homologous host . The recombinant protein was purified to homogeneity and was shown to phosphorylate 4-diphosphocytidyl-2C-methyl-D-erythritol in an ATP-dependent reaction . The reaction product was identified as 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate by NMR experiments with various (13)C-labeled substrate samples . A (14)C-labeled specimen of this compound was converted efficiently into carotenoids by isolated chromoplasts of Capsicum annuum . The sequence of E . coli YchB protein is similar to that of the protein predicted by the tomato cDNA pTOM41 (30% identity), which had been implicated in the conversion of chloroplasts to chromoplasts. Proc Natl Acad Sci U S A, 2000 Feb 1, 97(3), 1002 - 7 Optimization of the helper-dependent adenovirus system for production and potency in vivo; Sandig V et al.; Helper-dependent (HD) adenoviral vectors devoid of all viral coding sequences provide for safe and highly efficient gene transfer with long-lasting transgene expression . High titer stocks of HD vectors can be generated by using the cre-recombinase system . However, we have encountered difficulties with this system, including rearranged HD vectors and variable efficiency of HD vector rescue . These problems represent a major hindrance, particularly with regard to large-scale production . To overcome these limitations, we have modified the system in two ways: We constructed a new helper virus with a modified packaging signal and enhanced growth characteristics . We also redesigned the vector backbones by including noncoding adenovirus sequences adjacent to the right inverted terminal repeat and by incorporated a number of different segments of noncoding DNA of human origin as "stuffer." Comparison of these vectors showed that the nature of the stuffer sequence affects replication of the HD vector . Optimization of the system resulted in a more robust and consistent production of HD vectors with low helper contamination and high in vivo potency. Genetics, 2000 Feb, 154(2), 533 - 42 Mechanisms of dinucleotide repeat instability in Escherichia coli; Bichara M et al.; The high level of polymorphism of microsatellites has been used for a variety of purposes such as positional cloning of genes associated with diseases, forensic medicine, and phylogenetic studies . The discovery that microsatellites are associated with human diseases, not only as markers of risk but also directly in disease pathogenesis, has triggered a renewed interest in understanding the mechanism of their instability . In this work we have investigated the role of DNA replication, long patch mismatch repair, and transcription on the genetic instability of all possible combinations of dinucleotide repeats in Escherichia coli . We show that the (GpC) and (ApT) self-complementary sequence repeats are the most unstable and that the mode of replication plays an important role in their instability . We also found that long patch mismatch repair is involved in avoiding both short deletion and expansion events and also in instabilities resulting from the processing of bulges of 6 to 8 bp for the (GpT/ApC)- and (ApG/CpT)- containing repeats . For each dinucleotide sequence repeat, we propose models for instability that involve the possible participation of unusual secondary structures. Genetics, 2000 Feb, 154(2), 513 - 22 Palindromes as substrates for multiple pathways of recombination in Escherichia coli; Cromie GA et al.; A 246-bp imperfect palindrome has the potential to form hairpin structures in single-stranded DNA during replication . Genetic evidence suggests that these structures are converted to double-strand breaks by the SbcCD nuclease and that the double-strand breaks are repaired by recombination . We investigated the role of a range of recombination mutations on the viability of cells containing this palindrome . The palindrome was introduced into the Escherichia coli chromosome by phage lambda lysogenization . This was done in both wt and sbcC backgrounds . Repair of the SbcCD-induced double-strand breaks requires a large number of proteins, including the components of both the RecB and RecF pathways . Repair does not involve PriA-dependent replication fork restart, which suggests that the double-strand break occurs after the replication fork has passed the palindrome . In the absence of SbcCD, recombination still occurs, probably using a gap substrate . This process is also PriA independent, suggesting that there is no collapse of the replication fork . In the absence of RecA, the RecQ helicase is required for palindrome viability in a sbcC mutant, suggesting that a helicase-dependent pathway exists to allow replicative bypass of secondary structures. Genetics, 2000 Feb, 154(2), 503 - 12 Antagonism of ultraviolet-light mutagenesis by the methyl-directed mismatch-repair system of Escherichia coli; Liu H et al.; Previous studies have demonstrated that the Escherichia coli MutHLS mismatch-repair system can process UV-irradiated DNA in vivo and that the human MSH2.MSH6 mismatch-repair protein binds more strongly in vitro to photoproduct/base mismatches than to "matched" photoproducts in DNA . We tested the hypothesis that mismatch repair directed against incorrect bases opposite photoproducts might reduce UV mutagenesis, using two alleles at E . coli lacZ codon 461, which revert, respectively, via CCC --> CTC and CTT --> CTC transitions . F' lacZ targets were mated from mut(+) donors into mutH, mutL, or mutS recipients, once cells were at substantial densities, to minimize spontaneous mutation prior to irradiation . In umu(+) mut(+) recipients, a range of UV fluences induced lac(+) revertant frequencies of 4-25 x 10(-8); these frequencies were consistently 2-fold higher in mutH, mutL, or mutS recipients . Since this effect on mutation frequency was unaltered by an Mfd(-) defect, it appears not to involve transcription-coupled excision repair . In mut(+) umuC122::Tn5 bacteria, UV mutagenesis (at 60 J/m(2)) was very low, but mutH or mutL or mutS mutations increased reversion of both lacZ alleles roughly 25-fold, to 5-10 x 10(-8) . Thus, at UV doses too low to induce SOS functions, such as Umu(2)'D, most incorrect bases opposite occasional photoproducts may be removed by mismatch repair, whereas in heavily irradiated (SOS-induced) cells, mismatch repair may only correct some photoproduct/base mismatches, so UV mutagenesis remains substantial. Br J Ophthalmol, 2000 Feb, 84(2), 205 - 11 Apoptosis is a prominent feature of acute anterior uveitis in the Fischer 344 rat; Smith JR et al.; AIMS: To examine the hypothesis that apoptosis of infiltrating cells contributes to spontaneous resolution of uveitis in clinically relevant rodent models . METHODS: Experimental melanin induced uveitis (EMIU) was induced in Fischer 344 rats by immunisation with 250 microg bovine ocular melanin . Endotoxin induced uveitis (EIU) was induced by injection of 200 microg Escherichia coli lipopolysaccharide . Formalin fixed, paraffin embedded ocular cross sections were stained by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate biotin nick end labelling (TUNEL) to identify apoptotic cells . Indirect immunoperoxidase staining of paraformaldehyde lysine periodate fixed tissue cross sections was used to demonstrate expression of inducible nitric oxide synthase (iNOS) . RESULTS: TUNEL positive mononuclear cells were observed in the anterior uvea during both EMIU and EIU at all selected time points . However, whereas the majority of mononuclear cells appeared apoptotic from the outset of disease, neutrophils were notably TUNEL negative at all time points examined . Many infiltrating neutrophils expressed iNOS . CONCLUSION: Apoptosis occurs early in the course of rat EMIU and EIU, and may contribute to resolution of these diseases . In general, infiltrating mononuclear cells die rapidly, while neutrophils survive, producing inducible nitric oxide synthase which may contribute to disease pathogenesis. Immunol Lett, 1999 Dec 1, 70(3), 151 - 5 Expression of an epitopic region of AspfI, an allergen/antigen/cytotoxin of Aspergillus fumigatus; Sarma PV et al.; The gene for an 18 kD allergen/cytotoxin of Aspergillus fumigatus was cloned in pUC-19 vector and expressed in Escherichia coli JM109 . Digestion of this gene with AluI resulted in four fragments of 216bp, 120bp, 39bp and 21bp . These fragments were cloned in the Sma-I site of pUC-19 . The recombinants thus, generated after transformation in E . coli JM109, were screened using monoclonal antibodies raised against the AspfI . The fusion protein containing 120 bp AluI fragment was recognised by the MoAb indicating presence of epitope(s) in the 120 bp region . The study indicates a viable strategy for identification and expression of an immunologically active domain of a major allergen/antigen of A . fumigatus for the first time. Nat Med, 2000 Feb, 6(2), 164 - 70 Protection from septic shock by neutralization of macrophage migration inhibitory factor; Calandra T et al.; Identification of new therapeutic targets for the management of septic shock remains imperative as all investigational therapies, including anti-tumor necrosis factor (TNF) and anti-interleukin (IL)-1 agents, have uniformly failed to lower the mortality of critically ill patients with severe sepsis . We report here that macrophage migration inhibitory factor (MIF) is a critical mediator of septic shock . High concentrations of MIF were detected in the peritoneal exudate fluid and in the systemic circulation of mice with bacterial peritonitis . Experiments performed in TNFalpha knockout mice allowed a direct evaluation of the part played by MIF in sepsis in the absence of this pivotal cytokine of inflammation . Anti-MIF antibody protected TNFalpha knockout from lethal peritonitis induced by cecal ligation and puncture (CLP), providing evidence of an intrinsic contribution of MIF to the pathogenesis of sepsis . Anti-MIF antibody also protected normal mice from lethal peritonitis induced by both CLP and Escherichia coli, even when treatment was started up to 8 hours after CLP . Conversely, co-injection of recombinant MIF and E . coli markedly increased the lethality of peritonitis . Finally, high concentrations of MIF were detected in the plasma of patients with severe sepsis or septic shock . These studies define a critical part for MIF in the pathogenesis of septic shock and identify a new target for therapeutic intervention. Nat Genet, 2000 Feb, 24(2), 188 - 91 Mutations in the gene encoding peroxisomal alpha-methylacyl-CoA racemase cause adult-onset sensory motor neuropathy; Ferdinandusse S et al.; Sensory motor neuropathy is associated with various inherited disorders including Charcot-Marie-Tooth disease, X-linked adrenoleukodystrophy/adrenomyeloneuropathy and Refsum disease . In the latter two, the neuropathy is thought to result from the accumulation of specific fatty acids . We describe here three patients with elevated plasma concentrations of pristanic acid (a branched-chain fatty acid) and C27-bile-acid intermediates . Two of the patients suffered from adult-onset sensory motor neuropathy . One patient also had pigmentary retinopathy, suggesting Refsum disease, whereas the other patient had upper motor neuron signs in the legs, suggesting adrenomyeloneuropathy . The third patient was a child without neuropathy . In all three patients we discovered a deficiency of alpha-methylacyl-CoA racemase (AMACR) . This enzyme is responsible for the conversion of pristanoyl-CoA and C27-bile acyl-CoAs to their (S)-stereoisomers, which are the only stereoisomers that can be degraded via peroxisomal beta-oxidation . Sequence analysis of AMACR cDNA from the patients identified two different mutations that are likely to cause disease, based on analysis in Escherichia coli . Our findings have implications for the diagnosis of adult-onset neuropathies of unknown aetiology. EMBO J, 2000 Feb 1, 19(3), 400 - 9 The organized chromatin domain of the repressed yeast a cell-specific gene STE6 contains two molecules of the corepressor Tup1p per nucleosome; Ducker CE et al.; In yeast alpha cells the a cell-specific genes STE6 and BAR1 are packaged as gene-sized chromatin domains of positioned nucleosomes . Organized chromatin depends on Tup1p, a corepressor that interacts with the N-terminal regions of H3 and H4 . If Tup1p functions to organize or stabilize a chromatin domain, the protein might be expected to be present at a level stoichiometric with nucleosomes . Chromatin immunoprecipitation assays using Tup1p antibodies showed Tup1p to be associated with the entire genomic STE6 coding region . To determine stoichiometry of Tup1p associated with the gene, a yeast plasmid containing varying lengths of the STE6 gene including flanking control regions and an Escherichia coli lac operator sequence was constructed . After assembly into chromatin in vivo in Saccharomyces cerevisiae, minichromosomes were isolated using an immobilized lac repressor . In these experiments, Tup1p was found to be specifically associated with repressed STE6 chromatin in vivo at a ratio of about two molecules of the corepressor per nucleosome . These observations strongly suggest a structural role for Tup1p in repression and constrain models for organized chromatin in repressive domains. Pediatr Nephrol, 2000 Jan, 14(1), 73 - 83 Enterohemorrhagic Escherichia coli infections: following transmission routes; Verweyen HM et al.; Infections with enterohemorrhagic Escherichia coli (EHEC) are the major cause of hemolytic-uremic syndrome (HUS), the most-common cause of acute renal failure in childhood . The mortality rate of HUS (0%-5% in most recent series and 10%-30% in individual reports) and residual chronic renal sequelae (in up to 50% of patients in long-term follow-up studies) emphasize the seriousness of HUS for public health . Several studies have described possible sources of EHEC infection . However, in the majority of cases the pathogen cannot be identified in food or animals and the routes of transmission remain unclear . In this review article the hypothesized routes of transmission are summarized . The medical data bases "Medline" and "Current contents" were screened for the years January 1966 through November 1998 . The difficulties in following the chain of EHEC infection are discussed . A precise evaluation of the environmental aspects of the patient is a precondition for further analysis. Biophys J, 2000 Feb, 78(2), 1036 - 41 Torque-speed relationship of the flagellar rotary motor of Escherichia coli; Chen X et al.; The output of a rotary motor is characterized by its torque and speed . We measured the torque-speed relationship of the flagellar rotary motor of Escherichia coli by a new method . Small latex spheres were attached to flagellar stubs on cells fixed to the surface of a glass slide . The angular speeds of the spheres were monitored in a weak optical trap by back-focal-plane interferometry in solutions containing different concentrations of the viscous agent Ficoll . Plots of relative torque (viscosity x speed) versus speed were obtained over a wide dynamic range (up to speeds of approximately 300 Hz) at three different temperatures, 22.7, 17.7, and 15.8 degrees C . Results obtained earlier by electrorotation (, Biophys . J . 65:2201-2216) were confirmed . The motor operates in two dynamic regimes . At 23 degrees C, the torque is approximately constant up to a knee speed of nearly 200 Hz, and then it falls rapidly with speed to a zero-torque speed of approximately 350 Hz . In the low-speed regime, torque is insensitive to changes in temperature . In the high-speed regime, it decreases markedly at lower temperature . These results are consistent with models in which torque is generated by a powerstroke mechanism (, Biophys . J . 76:580-587). Biochemistry, 2000 Feb 8, 39(5), 1169 - 79 Phosphorylation of recombinant human ATP:citrate lyase by cAMP-dependent protein kinase abolishes homotropic allosteric regulation of the enzyme by citrate and increases the enzyme activity . Allosteric activation of ATP:citrate lyase by phosphorylated sugars; Potapova IA et al.; Recombinantly expressed human ATP:citrate lyase was purified from E . coli, and its kinetic behavior was characterized before and after phosphorylation . Cyclic AMP-dependent protein kinase catalyzed the incorporation of only 1 mol of phosphate per mole of enzyme homotetramer, and glycogen synthase kinase-3 incorporated an additional 2 mol of phosphate into the phosphorylated protein . Isoelectric focusing revealed that all of the phosphates were incorporated into only one of the four enzyme subunits . Phosphorylation resulted in a 6-fold increase in V(max) and the conversion of citrate dependence from sigmoidal, displaying negative cooperativity, to hyperbolic . The phosphorylated recombinant enzyme is more similar to the enzyme isolated from mammalian tissues than unphosphorylated enzyme with respect to the K(m) for citrate, CoA, and ATP, and the specific activity . Fructose 6-phosphate was found to be a potent activator (60-fold) of the unphosphorylated recombinant enzyme, with half-maximal activation at 0.16 mM, which results in a decrease in the apparent K(m) for citrate and ATP, as well as an increase in the V(max) of the reaction . Thus, human ATP:citrate lyase activity is regulated in vitro allosterically by phosphorylated sugars as well as covalently by phosphorylation. J Mol Biol, 2000 Feb 4, 295(5), 1265 - 73 Orienting domains in proteins using dipolar couplings measured by liquid-state NMR: differences in solution and crystal forms of maltodextrin binding protein loaded with beta-cyclodextrin; Skrynnikov NR et al.; Protein function is often regulated by conformational changes that occur in response to ligand binding or covalent modification such as phosphorylation . In many multidomain proteins these conformational changes involve reorientation of domains within the protein . Although X-ray crystallography can be used to determine the relative orientation of domains, the crystal-state conformation can reflect the effect of crystal packing forces and therefore may differ from the physiologically relevant form existing in solution . Here we demonstrate that the solution-state conformation of a multidomain protein can be obtained from its X-ray structure using an extensive set of dipolar couplings measured by triple-resonance multidimensional NMR spectroscopy in weakly aligning solvent . The solution-state conformation of the 370-residue maltodextrin-binding protein (MBP) loaded with beta-cyclodextrin has been determined on the basis of one-bond (15)N-H(N), (15)N-(13)C', (13)C(alpha)-(13)C', two-bond (13)C'-H(N), and three-bond (13)C(alpha)-H(N) dipolar couplings measured for 280, 262, 276, 262, and 276 residues, respectively . This conformation was generated by applying hinge rotations to various X-ray structures of MBP seeking to minimize the difference between the experimentally measured and calculated dipolar couplings . Consistent structures have been derived in this manner starting from four different crystal forms of MBP . The analysis has revealed substantial differences between the resulting solution-state conformation and its crystal-state counterpart (Protein Data Bank accession code 1DMB) with the solution structure characterized by an 11(+/-1) degrees domain closure . We have demonstrated that the precision achieved in these analyses is most likely limited by small uncertainties in the intradomain structure of the protein (ca 5 degrees uncertainty in orientation of internuclear vectors within domains) . In addition, potential effects of interdomain motion have been considered using a number of different models and it was found that the structures derived on the basis of dipolar couplings accurately represent the effective average conformation of the protein . J Mol Biol, 2000 Feb 4, 295(5), 1237 - 49 High-resolution structure of bovine pancreatic trypsin inhibitor with altered binding loop sequence; Czapinska H et al.; A mutant of bovine pancreatic trypsin inhibitor (BPTI) has been constructed and expressed in Escherichia coli in order to probe the kinetic and structural consequences of truncating the binding loop residues to alanine . In addition to two such mutations (Thr11Ala and Pro13Ala), it has a conservative Lys15Arg substitution at position P(1) and an unrelated Met52Leu change . In spite of the binding loop modification, the affinity for trypsin is only 30 times lower than that of the wild-type protein . At pH 7.5 the protein can be crystallized on the time-scale of hours, yielding very stable crystals of a new (tetragonal) form of BPTI . Conventional source X-ray data collected to 1.4 A at room temperature allowed anisotropic structure refinement characterized by R=0.1048 . The structure reveals all 58 residues, including the complete C terminus, which is in a salt-bridge contact with the N terminus . The Cys14-Cys38 disulfide bridge is observed in two distinct chiralities . This bridge, together with an internal water molecule, contributes to the stabilization of the binding loop . The Ala mutations have only an insignificant and localized effect on the binding loop, which retains its wild-type conformation (maximum deviation of loop C(alpha) atoms of 0.7 A at Ala13) . Four (instead of the typical three) additional water molecules are buried in an internal cleft and connected to the surface via a sulfate anion . Three more SO(4)(2-) anions are seen in the electron density, one of them located on a 2-fold axis . It participates in the formation of a dimeric structure between symmetry-related BPTI molecules, in which electrostatic and hydrogen bonding interactions resulting from the mutated Lys15Arg substitution are of central importance . This dimeric interaction involves direct recognition loop-recognition loop contacts, part of which are hydrophobic interactions of the patches created by the alanine mutations . Another 2-fold symmetric interaction between the BPTI molecules involves the formation of an antiparallel intermolecular beta-sheet that, together with the adjacent intramolecular beta-hairpin loops, creates a four-stranded structure . J Mol Biol, 2000 Feb 4, 295(5), 1225 - 36 Structural comparison of the PhoB and OmpR DNA-binding/transactivation domains and the arrangement of PhoB molecules on the phosphate box; Okamura H et al.; PhoB is a transcriptional activator that binds to the phosphate box in the promoters of the phosphate genes of Escherichia coli . PhoB contains two functional domains, an N-terminal phosphorylation domain and a C-terminal DNA-binding/transactivation domain . Here, the three-dimensional structure of the DNA-binding/transactivation domain has been determined by NMR . It consists of an N-terminal four-stranded beta-sheet, a central three helical bundle and a C-terminal beta-hairpin . The second and third helices form a helix-turn-helix (HTH) variant containing a longer turn than the corresponding turn of the classical HTH motif . The overall architecture is very close to that of the OmpR DNA-binding/transactivation domain, however, the conformation of the long turn region of PhoB, a putative interaction site for the RNA polymerase sigma subunit, is entirely different from that of the corresponding turn of OmpR, which interacts with the alpha subunit . In addition, the third helix of PhoB is three amino acid residues longer than the corresponding helix of OmpR . The binding site of PhoB is a TGTCA sequence and the phospahte box contains the two binding sites . NMR studies of the complexes of the PhoB DNA-binding/transactivation domain bound to several different DNA molecules have revealed that two PhoB molecules bind in a tandem array on the phosphate box . In each complex of PhoB the third helix of the DNA-binding/transactivation domain is likely to recognize the TGTCA sequence from the major groove of DNA and the C-terminal beta-hairpin contacts on the minor groove of the 3' site out of the TGTCA sequence in a non-specific manner . The long turn region facing outward is likely to interact with the sigma subunit . J Mol Biol, 2000 Feb 4, 295(5), 1163 - 72 Two active-site tyrosyl residues of protein TrwC act sequentially at the origin of transfer during plasmid R388 conjugation; Grandoso G et al.; Protein TrwC is the relaxase-helicase responsible for the initiation and termination reactions of DNA processing during plasmid R388 conjugation . Site-directed mutagenesis was used to change to phenylalanine each of a set of four conserved tyrosyl residues in the sequence of the N-terminal relaxation domain of the protein . Simultaneous mutation of both Y18 and Y26 was required to abolish in vitro cleavage and strand-transfer reactions catalyzed by protein TrwC on oligonucleotides containing the nic site . Thus, both Y18 and Y26 could be involved independently in the formation of oligonucleotide-protein covalent complexes that constitute presumed intermediates of these reactions . This hypothesis was confirmed by the observation of Y18 and Y26-specific peptide-oligonucleotide adducts after protease digestion of TrwC and mutant derivatives . Finally mutation Y18F, but not mutation Y26F, abolished nic-cleavage of a supercoiled DNA containing the R388 origin of transfer (oriT) . These data allowed the construction of a model for conjugative DNA processing in which Y18 specifically catalyzes the initial cleavage reaction, while Y26 is used for the second strand-transfer reaction, which terminates conjugation . The model suggests a control mechanism that can be effective at each conjugative replication cycle . J Mol Biol, 2000 Feb 4, 295(5), 1113 - 8 Evidence for helical unwinding of an RNA substrate by the RNA enzyme RNase P: use of an interstrand disulfide crosslink in substrate; Pomeranz Krummel DA et al.; To gain an understanding of structural changes induced in substrates by Escherichia coli ribonuclease P (RNase P), we have incorporated an interstrand disulfide crosslink proximal to the cleavage site in a model substrate . RNase P is able to process the reduced, non-crosslinked form of this substrate as well as a substrate in which the free thiol molecules have been alkylated with iodoacetamide . However, the oxidized, crosslinked form is cleaved at a significantly lower rate . Therefore, helical unwinding of the analog of the aminoacyl stem of the substrate near its site of cleavage may be necessary for efficient processing by E . coli RNase P . Pflugers Arch, 2000, 439(3 Suppl), R119 - 21 Tissue expression and immunolocalization of a novel human cathepsin P; Pungercar J et al.; The mRNA of a novel human cathepsin P is expressed at high levels in lung, liver and heart . Using antibodies raised against recombinant cathepsin P produced in Escherichia coli, a single protein band of 33 kDa was detected by immunoblotting an extract of human liver . By immunofluorescence, positive signals were observed in hepatocytes and Kupffer cells of liver, and the distal tubule cells of kidney showing mainly perimembranous distribution, indicating a role, as yet unknown, for this novel putative protease that is distinct from other cathepsins of the papain family. Pflugers Arch, 2000, 439(3 Suppl), R113 - 5 Early events in TNFa-p55 receptor interations--experiments with TNF dimers; Menart V et al.; The first essential step in TNF signal transduction is believed to be clustering of the membrane bound receptors around the trimeric TNF molecule . To check if one receptor binding site would be enough to trigger the signal, we tried to prepare several types of TNF dimer . For this purpose, two TNF analogs bearing different cysteine mutations at the inner subunit binding surfaces were designed, expressed in E . coli and prepared in pure form . By mixing equimolar quantities of these analogs under appropriate conditions, two different types of dimer were prepared . The first, Dim/S2, proved to be composed mainly of a disulfide-linked dimer, which was still capable of trapping the third subunit of either of the precursor analogs, thus showing relatively high residual cytotoxicity . To avoid trimeric structures, Dim/S2 was further transformed into Dim/Iaa2 by alkylation of -SH groups of the newly introduced cysteines, allowing binding of only two TNF subunits through native contact surfaces . These dimers showed substantially reduced cytotoxicity on the L929 cell line . In addition, it appears that Dim/Iaa2 is able to competitively inhibit cytotoxicity of native TNF, as assessed on the L-M cell line. Anticancer Res, 1999 Jul-Aug, 19(4B), 2925 - 8 Viscotoxin-free aqueous extracts from European mistletoe (Viscum album L.) stimulate activity of human granulocytes; Stein GM et al.; BACKGROUND: Extracts from European mistletoe (Viscum album L., VAL) are used for complementary cancer treatment . Viscotoxins (VT) and whole plant extracts with high amounts of the VT have been shown to stimulate functional activity of granulocytes . MATERIALS AND METHODS: We stimulated neutrophils from healthy donors in vitro with aqueous VT-free VAL extracts and mistletoe lectins (ML) in the presence of E.coli and studied phagocytosis (via incorporation of FITC-labelled E.coli) and respiratory burst (via oxidation of dihydrorhodamine 123 to rhodamine 123) by flow cytometry . RESULTS: The VT-free VAL extract significantly stimulated granulocyte activity, and this effect correlated with the content of the ML, although the ML exerted no influence at relevant concentrations . Co-incubation of the cells with VAL in the presence of VT further increased granulocyte response . CONCLUSIONS: From these data it is suggested that (1) a non-VT non-ML component of the VAL extracts activated granulocytes and (2) different activation pathways may be involved in the stimulation by the whole plant extract and the VT. Anticancer Res, 1999 Jul-Aug, 19(4B), 2869 - 73 Recombinant vaccinia virus expressing cytokine GM-CSF as tumor vaccine; Chatterjee SK et al.; The efficacy of a recombinant vaccinia virus (rvv-GM-CSF) expressing the granulocyte macrophage colony stimulating factor (GM-CSF) as tumor vaccine was evaluated in the murine B16-F10 melanoma model . The vaccine was prepared by infection of irradiated tumor cells with rvv-GM-CSF . Control vaccine was B-16 cells infected with a recombinant vaccinia virus expressing Escherichia coli beta-galactosidase (rvv-lacZ) . Pre-vaccination of naive C57BL/6 mice later inoculated with tumor cells and treatment of mice bearing tumors with GM-CSF vaccine inhibited tumor development and prolonged survival . Lung metastasis of B-16 was also inhibited by treatment with GM-CSF vaccine . The vaccine effects appeared to be tumor cell specific . The efficacy of the vaccine was comparable to a retroviral vaccine (MFG-muGM-CSF) in this system . The vaccine was also effective when rvv-GM-CSF was directly injected into the tumor . These data suggest that this vaccine approach has potential for use in cancer treatment, especially for patients with easily accessible tumors. Trends Genet, 2000 Feb, 16(2), 88 - 92 Eukaryotic DNA replication: a model for a fixed double replisome; Falaschi A; To duplicate their genomes, eukaryotic cells have to overcome some formidable chemical and topological hurdles, considering the number of nucleotides that have to be polymerized faithfully and the sheer physical size of the DNA molecules that have to be disentangled and partitioned in an orderly way . This article tackles one particular aspect of the process: the organization of the apparatus that advances the replicative growing forks along the DNA molecule . Here, I suggest a solution to the difficulty of separating the daughter molecules in an orderly way and propose an alternative to the current models, which reconciles the use of a single polarity of synthesis by the DNA polymerases with the need for parallel polymerization of two strands of opposite polarity. J Biol Chem, 2000 Feb 4, 275(5), 3713 - 21 Elastase in intestinal mucus enhances the cytotoxicity of Shiga toxin type 2d; Kokai-Kun JF et al.; Shiga toxin variant type 2d (Stx2d) produced by some strains of Shiga toxin-producing Escherichia coli is composed of an enzymatically active A subunit and a B (binding) pentamer . The cytotoxicity of Stx2d is increased (activated) 10-1000-fold for Vero cells when the toxin is incubated with mucus obtained from the small intestine of mice . In this study we isolated an Stx2d activator and identified it as a mouse elastase with strong homology to human elastase IIIB . Moreover, commercially available porcine pancreatic elastase preparations also activated Stx2d cytotoxicity although with a lower specific activity than isolated mouse elastase . Elastase directly nicked the Stx2d A subunit to A(1) and A(2), an event that did not correlate with activation . However, elastase also reduced the size and changed the isoelectric point of the A(2) peptide, as determined by SDS-polyacrylamide gel electrophoresis and two-dimensional electrophoresis followed by Western immunoblot analysis . This elastase-mediated size and charge shift in the A(2) peptide of Stx2d occurred concurrently with activation of the toxin . Both the reduction in size of the Stx2d A(2) peptide by incubation with elastase as well as the associated activation of Stx2d cytotoxicity were fully inhibited by elastatinal, an elastase-specific inhibitor. J Biol Chem, 2000 Feb 4, 275(5), 3661 - 6 Identification of RNA polymerase beta' subunit segment contacting the melted region of the lacUV5 promoter; Brodolin K et al.; Identification of the RNA polymerase functional regions involved in interactions with promoter is a basis for understanding the mechanism of transcription initiation . We have used formaldehyde cross-linking to identify a region of Escherichia coli RNA polymerase beta' subunit contacting lacUV5 promoter in open complex . Treatment of open complex with formaldehyde results in cross-linking of beta' and sigma(70) subunits at positions -5 and -3 on the nontemplate strand of the promoter DNA . These cross-links reflect specific interactions between RNA polymerase and promoter established in open complex . The positions of formaldehyde cross-links in the beta' subunit were mapped to the N-terminal segment (Cys(198)-Met(237)), which is contiguous to the evolutionary conserved region B . The proximity of the beta' and sigma cross-links suggest that the N-terminal region of the beta' subunit, interacting with single-stranded promoter DNA, can cooperate with the sigma subunit in the process of open complex formation. J Biol Chem, 2000 Feb 4, 275(5), 3583 - 92 Mapping of subunit-subunit contact surfaces on the beta' subunit of Escherichia coli RNA polymerase; Katayama A et al.; The RNA polymerase core enzyme of Escherichia coli with the catalytic activity of RNA polymerization is assembled sequentially under the order: 2alpha --> alpha(2) --> alpha(2)beta --> alpha(2)betabeta' . The core enzyme gains the activities of promoter recognition and transcription initiation after binding the sigma subunit . The subunit-subunit contact surfaces of beta' subunit (1407 residues) were analyzed by testing complex formation between various beta' fragments and either the alpha(2)beta complex or the sigma(70) subunit . Results indicate that two regions, one central region between residues 515 and 842 and the other COOH-terminal proximal region downstream from residue 1141, are involved in binding the alpha(2)beta complex; and the NH(2)-terminal proximal region between residues 201 and 345 plays a major role in binding the sigma(70) subunit . However, both alpha(2)beta binding sites have weak activity of the sigma(70) subunit; likewise, the sigma(70) subunit-contact surface has weak binding activity of the alpha(2)beta complex . The sites involved in the catalytic function of RNA polymerization are all located within two spacer regions sandwiched between these three subunit-subunit contact surfaces. J Biol Chem, 2000 Feb 4, 275(5), 3360 - 4 Interactions of the sulfonylurea receptor 1 subunit in the molecular assembly of beta-cell K(ATP) channels; Mikhailov MV et al.; We have investigated protein interactions involved in pancreatic beta-cell ATP-sensitive potassium channel assembly . These channels, which are of key importance for control of insulin release, are a hetero-oligomeric complex of pore-forming Kir6.2 subunits and sulfonylurea receptor (SUR1) subunits with two nucleotide-binding domains (NBD1 and NBD2) . We divided SUR1 into two halves at Pro-1042 . Expression of either the individual N- or C-terminal domain in a baculovirus expression system did not lead to glibenclamide binding activity, although studies with green fluorescent protein fusion proteins showed that both half-molecules were inserted into the plasma membrane . However, significant glibenclamide binding activity was observed when the half-molecules were co-expressed (even when NBD2 was deleted from the C-terminal half-molecule) . Simultaneous expression of Kir6.2 resulted in enhanced glibenclamide binding activity . We conclude that the glibenclamide-binding site includes amino acid residues from both halves of the molecule, that there is strong interaction between different regions of SUR1, that NBD2 is not essential for glibenclamide binding, and that interactions between Kir6.2 and SUR1 participate in ATP-sensitive potassium channel assembly . Investigation of NBD1-green fluorescent protein fusion protein distribution inside insect cells expressing C-terminal halves of SUR1 demonstrated strong interaction between NBD1 and NBD2 . We also expressed and purified NBD1 from Escherichia coli . Purified NBD1 was found to exist as a tetramer indicating strong homomeric attractions and a possible role for NBD1 in SUR1 assembly. J Biol Chem, 2000 Feb 4, 275(5), 3201 - 5 The ferrous dioxygen complex of the oxygenase domain of neuronal nitric-oxide synthase; Couture M et al.; The mechanisms by which nitric-oxide synthases (NOSs) bind and activate oxygen at their P450-type heme active site in order to synthesize nitric oxide from the substrate L-arginine are mostly unknown . To obtain information concerning the structure and properties of the first oxygenated intermediate of the enzymatic cycle, we have used a rapid continuous flow mixer and resonance Raman spectroscopy to generate and identify the ferrous dioxygen complex of the oxygenase domain of nNOS (Fe(2+)O(2) nNOSoxy) . We detect a line at 1135 cm(-1) in the resonance Raman spectrum of the intermediate formed from 0.6 to 3.0 ms after the rapid mixing of the ferrous enzyme with oxygen that is shifted to 1068 cm(-1) with (18)O(2) . This line is assigned as the O-O stretching mode (nu(O-O)) of the oxygenated complex of nNOSoxy . Rapid mixing experiments performed with nNOSoxy saturated with L-arginine or N(omega)-hydroxy-L-arginine, in the presence or absence of (6R)-5,6, 7,8-tetrahydro-L-biopterin, reveal that the nu(O-O) line is insensitive to the presence of the substrate and the pterin . The optical spectrum of this ferrous dioxygen species, with a Soret band wavelength maximum at 430 nm, confirms the identification of the previously reported oxygenated complexes generated by stopped flow techniques. J Biol Chem, 2000 Feb 4, 275(5), 3192 - 200 Regulation of Pap phase variation . Lrp is sufficient for the establishment of the phase off pap DNA methylation pattern and repression of pap transcription in vitro; Weyand NJ et al.; The pyelonephritis-associated pili (pap) operon in Escherichia coli is regulated by an epigenetic mechanism involving the formation of specific DNA methylation patterns characteristic of transcriptionally active (phase ON) and inactive (phase OFF) cells . The formation of pap DNA methylation patterns in vivo was previously shown to require the leucine-responsive regulatory protein (Lrp) and DNA adenine methylase (Dam) . To monitor the binding of Lrp to pap DNA, an in vitro methylation protection assay was developed . Binding of Lrp to a Dam target site proximal to the papBA promoter (designated GATC(prox)) blocked methylation of this site and specifically repressed transcription . The DNA methylation pattern and transcription state are identical to those observed in vivo in phase OFF cells . To determine if binding of Lrp at GATC(prox) was necessary for repression of papBA transcription, we analyzed a pap mutation (pap-13) that reduced the affinity of Lrp for the GATC(prox) region . Binding of Lrp to pap-13 DNA was shifted to a promoter distal Dam target site (designated GATC(dist)) . Lrp blocked methylation of GATC(dist) in the pap-13 mutant, but did not repress papBA transcription . Together, these results show that binding of Lrp to the GATC(prox) region is sufficient for the establishment of the phase OFF DNA methylation pattern and repression of papBA transcription. J Biol Chem, 2000 Feb 4, 275(5), 3137 - 43 Molecular and biochemical evidence for the involvement of calcium/calmodulin in auxin action; Yang T et al.; The use of (35)S-labeled calmodulin (CaM) to screen a corn root cDNA expression library has led to the isolation of a CaM-binding protein, encoded by a cDNA with sequence similarity to small auxin up RNAs (SAURs), a class of early auxin-responsive genes . The cDNA designated as ZmSAUR1 (Zea mays SAURs) was expressed in Escherichia coli, and the recombinant protein was purified by CaM affinity chromatography . The CaM binding assay revealed that the recombinant protein binds to CaM in a calcium-dependent manner . Deletion analysis revealed that the CaM binding site was located at the NH(2)-terminal domain . A synthetic peptide of amino acids 20-45, corresponding to the potential CaM binding region, was used for calcium-dependent mobility shift assays . The synthetic peptide formed a stable complex with CaM only in the presence of calcium . The CaM affinity assay indicated that ZmSAUR1 binds to CaM with high affinity (K(d) approximately 15 nM) in a calcium-dependent manner . Comparison of the NH(2)-terminal portions of all of the characterized SAURs revealed that they all contain a stretch of the basic alpha-amphiphilic helix similar to the CaM binding region of ZmSAUR1 . CaM binds to the two synthetic peptides from the NH(2)-terminal regions of Arabidopsis SAUR-AC1 and soybean 10A5, suggesting that this is a general phenomenon for all SAURs . Northern analysis was carried out using the total RNA isolated from auxin-treated corn coleoptile segments . ZmSAUR1 gene expression began within 10 min, increased rapidly between 10 and 60 min, and peaked around 60 min after 10 microM alpha-naphthaleneacetic acid treatment . These results indicate that ZmSAUR1 is an early auxin-responsive gene . The CaM antagonist N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide hydrochloride inhibited the auxin-induced cell elongation but not the auxin-induced expression of ZmSAUR1 . This suggests that calcium/CaM do not regulate ZmSAUR1 at the transcriptional level . CaM binding to ZmSAUR1 in a calcium-dependent manner suggests that calcium/CaM regulate ZmSAUR1 at the post-translational level . Our data provide the first direct evidence for the involvement of calcium/CaM-mediated signaling in auxin-mediated signal transduction. J Biol Chem, 2000 Feb 4, 275(5), 3017 - 20 The DnaX-binding subunits delta' and psi are bound to gamma and not tau in the DNA polymerase III holoenzyme; Glover BP et al.; The DnaX complex subassembly of the DNA polymerase III holoenzyme is comprised of the DnaX proteins tau and gamma and the auxiliary subunits delta, delta', chi, and psi, which together load the beta processivity factor onto primed DNA in an ATP-dependent reaction . delta' and psi bind directly to DnaX whereas delta and chi bind to delta' and psi, respectively (Onrust, R., Finkelstein, J., Naktinis, V., Turner, J., Fang, L., and O'Donnell, M . (1995) J . Biol . Chem . 270, 13348-13357) . Until now, it has been unclear which DnaX protein, tau or gamma, in holoenzyme binds the auxiliary subunits delta, delta', chi,and psi . Treatment of purified holoenzyme with the homobifunctional cross-linker bis(sulfosuccinimidyl)suberate produces covalently cross-linked gamma-delta' and gamma-psi complexes identified by Western blot analysis . Immunodetection of cross-linked species with anti-delta' and anti-psi antibodies revealed that no tau-delta' or tau-psi cross-links had formed, suggesting that the delta' and psi subunits reside only on gamma within holoenzyme. Genes Dev, 2000 Jan 15, 14(2), 212 - 23 Dynamic organization of chromosomal DNA in Escherichia coli; Niki H et al.; We have revealed the subcellular localization of different DNA segments that are located at approximately 230-kb intervals on the Escherichia coli chromosome using fluorescence in situ hybridization (FISH) . The series of chromosome segments is localized within the cell in the same order as the chromosome map . The large chromosome region including oriC shows similar localization patterns, which we call the Ori domain . In addition, the localization pattern of the large segment including dif is characteristic of the replication terminus region . The segment also shows similar localization patterns, which we call the Ter domain . In newborn cells, Ori and Ter domains of the chromosome are differentially localized near opposite cell poles . Subsequently, in the B period, the Ori domain moves toward mid-cell before the initiation of replication, and the Ter domain tends to relocate at mid-cell . An inversion mutant, in which the Ter domain is located close to oriC, shows abnormal subcellular localization of ori and dif segments, resulting in frequent production of anucleate cells . These studies thus suggest that the E . coli chromosome is organized to form a compacted ring structure with the Ori and Ter domains; these domains participate in the cell cycle-dependent localization of the chromosome. Biochem Biophys Res Commun, 2000 Feb 5, 268(1), 136 - 40 Effects of C5 protein on Escherichia coli RNase P catalysis with a precursor tRNA(Phe) bearing a single mismatch in the acceptor stem; Park BH et al.; Escherichia coli RNase P, an RNA-processing enzyme that cleaves precursor tRNAs to generate the mature 5'-end, is composed of a catalytic component (M1 RNA) and a protein cofactor (C5 protein) . In this study, effects of C5 protein on the RNase P catalysis with a precursor E . coli tRNA(Phe) having a single mismatch in the acceptor stem were examined . This mutant precursor unexpectedly generated upstream cleavage products at the -8 position as well as normal cleavage products at the +1 position . The cleavage at the -8 position was essentially effective only in the presence of C5 protein . Possible secondary structures for cleavage at the -8 position deviate significantly from the structures of the known RNase P substrates, implying that C5 protein can allow the enzyme to broaden the substrate specificity more than previously appreciated . Biochem Biophys Res Commun, 2000 Feb 5, 268(1), 128 - 32 Activation of human progelatinase A/promatrix metalloproteinase 2 by Escherichia coli-derived serine proteinase; Takeda M et al.; Treatment of human uterine cervical fibroblasts with commercial lipopolysaccharide (LPS) preparations from different serotypes of Escherichia coli effectively augmented the processing of mammalian progelatinase A/promatrix metalloproteinase (proMMP)-2 to a 62-kDa form of MMP-2 . When purified proMMP-2 was incubated with LPS preparations, the proenzyme was similarly processed into the 62-kDa active MMP-2 in a time- and dose-dependent manner . By contrast, progelatinase B/proMMP-9 and prostromelysin 1/proMMP-3 were not activated . A serine proteinase inhibitor, phenylmethylsulfonyl fluoride, completely interfered with this LPS-mediated activation of proMMP-2 . This is novel evidence that E . coli serine proteinase is a specific activator of proMMP-2 . Thus, it is very likely that E . coli infection plays a crucial role in the degradation of connective tissues via the activation of proMMP-2, and the resultant active MMP-2 participates in the dysfunction of connective tissues such as in the preterm rupture of fetal membranes . Biochem Biophys Res Commun, 2000 Feb 5, 268(1), 118 - 23 Effects of terminal deletions in C5 protein on promoting RNase P catalysis; Kim M et al.; Deletion derivatives of C5 protein, the protein cofactor of Escherichia coli RNase P, were constructed as soluble MBP (maltose-binding protein) fusion proteins to assess the deletion effects on promoting RNase P catalysis and on binding to M1 RNA, the catalytic subunit of the enzyme . The C5 protein, with large terminal deletions, retained its promoting activity of RNase P catalysis under protein excess conditions in vitro . Some deletion derivatives complemented the temperature sensitive phenotype of E . coli A49 cells carrying the rnpA49 mutation . This ability also suggests that part of the C5 protein is enough to produce the catalytic activity of RNase P in vivo . Both the central conserved region, called the RNR motif, and the C-terminal region are essential for the binding of C5 protein to M1 RNA . Meanwhile, the N-terminal region contributes to promoting RNase P catalysis in ways other than binding to M1 RNA . Biochem Biophys Res Commun, 2000 Feb 5, 268(1), 73 - 7 Investigation of Fanconi anemia protein interactions by yeast two-hybrid analysis; Huber PA et al.; Fanconi anemia is a chromosomal breakage disorder with eight complementation groups (A-H), and three genes (FANCA, FANCC, and FANCG) have been identified . Initial investigations of the interaction between FANCA and FANCC, principally by co-immunoprecipitation, have proved controversial . We used the yeast two-hybrid assay to test for interactions of the FANCA, FANCC, and FANCG proteins . No activation of the reporter gene was observed in yeast co-expressing FANCA and FANCC as hybrid proteins, suggesting that FANCA does not directly interact with FANCC . However, a high level of activation was found when FANCA was co-expressed with FANCG, indicating strong, direct interaction between these proteins . Both FANCA and FANCG show weak but consistent interaction with themselves, suggesting that their function may involve dimerisation . The site of interaction of FANCG with FANCA was investigated by analysis of 12 mutant fragments of FANCG . Although both N- and C-terminal fragments did interact, binding to FANCA was drastically reduced, suggesting that more than one region of the FANCG protein is required for proper interaction with FANCA . J Cell Physiol, 2000 Mar, 182(3), 381 - 9 Differential induction of tumor necrosis factor alpha and manganese superoxide dismutase by endotoxin in human monocytes: role of protein tyrosine kinase, mitogen-activated protein kinase, and nuclear factor kappaB; White JE et al.; A mutant Escherichia coli lipopolysaccharide (LPS) lacking myristoyl fatty acid markedly stimulates the activity of manganese superoxide dismutase (MnSOD) without inducing tumor necrosis factor alpha (TNFalpha) production by human monocytes (Tian et al., 1998, Am J Physiol 275:C740.), suggesting that induction of MnSOD and TNFalpha by LPS are regulated through different signal transduction pathways . The protein tyrosine kinase (PTK)/mitogen-activated protein kinase (MAPK) pathway plays an important role in the LPS-induced TNFalpha production . In the current study, we determined the effects of PTK inhibitors, genistein and herbimycin A, on the induction of MnSOD and TNFalpha in human monocytes . Genistein (10 microg/ml) and herbimycin A (1 microg/ml) markedly inhibited LPS-induced protein tyrosine phosphorylation, phosphorylation and nuclear translocation of MAPK (p42 ERK, extracellular signal-regulated kinase), and increases in the steady state level of TNFalpha mRNA as well as TNFalpha production . In contrast, at similar concentrations, genistein and herbimycin A had no effect on the LPS-induced activation of nuclear factor kappaB (NFkappaB) and induction of MnSOD (mRNA and enzyme activity) in human monocytes . In addition, inhibition of NFkappaB activation by gliotoxin and pyrrodiline dithiocarbamate, inhibited LPS induction of TNFalpha and MnSOD mRNAs . These results suggest that (1) while PTK and MAPK are essential for the production of TNFalpha, they are not necessary for the induction of MnSOD by LPS, and (2) while activation of NFkappaB alone is insufficient for the induction of TNFalpha mRNA by LPS, it is necessary for the induction of TNFalpha as well as MnSOD mRNAs . Plant J, 1999 Dec, 20(6), 653 - 61 Chlorophyll breakdown by chlorophyllase: isolation and functional expression of the Chlase1 gene from ethylene-treated Citrus fruit and its regulation during development; Jacob-Wilk D et al.; We report on the isolation, functional expression and characterization of a cDNA encoding chlorophyllase, the enzyme catalyzing the first step in the chlorophyll breakdown pathway . The Chlase1 cDNA from Valencia Orange (Citrus sinensis cv . Valencia) was obtained by RT-PCR using degenerate primers based on the amino acid sequence of the previously purified protein . Chlase1 encodes a protein of 329 amino acids, including a sequence domain characterizing serine-lipases and a putative chloroplast-directing transit peptide . The Chlase1 gene encodes an active chlorophyllase enzyme which catalyzes the dephytylation of chlorophyll as shown by in vitro recombinant enzyme assays . Chlorophyllase expression at the transcript level in Valencia orange peel was found to be low and constitutive during natural fruit development without significant increase towards color-break and ripening . However, ethylene treatment induced an increase in chlorophyllase transcript at all stages of development . An enhanced response to ethylene treatment was observed during the months of October and November, corresponding to the time of natural color-break . The senescence-delaying regulator gibberellin-A3 (GA3) inhibited the effect of ethylene on chlorophyllase transcript accumulation . The data presented suggest that chlorophyllase may not be the regulator of chlorophyll breakdown during natural fruit ripening but is consistent with the notion that chlorophyll is gradually degraded during ripening due to a negative balance between synthesis and breakdown . According to this model, exogenous application of ethylene accelerates chlorophyll breakdown due to increased de novo synthesis of chlorophyllase . Further experimentation on the regulation and role of chlorophyllase in planta will be facilitated by the gene tools established in this work. Plant J, 1999 Dec, 20(5), 519 - 27 An Arabidopsis gene encoding a chloroplast-targeted beta-amylase; Lao NT et al.; beta-Amylase is one of the most abundant starch degrading activities found in leaves and other plant organs . Despite its abundance, most if not all of this activity has been reported to be extrachloroplastic and for this reason, it has been assumed that beta-amylases are not involved in the metabolism of chloroplast-localized transitory leaf starch . However, we have identified a novel beta-amylase gene, designated ct-Bmy, which is located on chromosome IV of Arabidopsis thaliana . Ct-Bmy encodes a precursor protein which contains a typical N-terminal chloroplast import signal and is highly similar at the amino acid level to extrachloroplastic beta-amylases of higher plants . Expression of the ct-Bmy cDNA in E . coli confirmed that the encoded protein possesses beta-amylase activity . CT-BMY protein, synthesized in vitro, was efficiently imported by isolated pea chloroplasts and shown to be located in the stroma . In addition, fusions between the predicted CT-BMY transit peptide and jellyfish green fluorescent protein (GFP) or the entire CT-BMY protein and GFP showed accumulation in vivo in chloroplasts of Arabidopsis . Expression of the GUS gene fused to ct-Bmy promoter sequences was investigated in transgenic tobacco plants . GUS activity was most strongly expressed in the palisade cell layer in the leaf blade and in chlorenchyma cells associated with the vascular strands in petioles and stems . Histochemical staining of whole seedlings showed that GUS activity was largely confined to the cotyledons during the first 2 weeks of growth and appeared in the first true leaves at approximately 4 weeks. Mol Microbiol, 2000 Jan, 35(2), 463 - 71 Two opposing effects of mismatch repair on CTG repeat instability in Escherichia coli; Schmidt KH et al.; The expansion of normally polymorphic CTG microsatellites in certain human genes has been identified as the causative mutation of a number of hereditary neurological disorders, including Huntington's disease and myotonic dystrophy . Here, we have investigated the effect of methyl-directed mismatch repair (MMR) on the stability of a (CTG)43 repeat in Escherichia coli over 140 generations and find two opposing effects . In contrast to orientation-dependent repeat instability in wild-type E . coli and yeast, we observed no orientation dependence in MMR- E . coli cells and suggest that, for the repeat that we have studied, orientation dependence in wild-type cells is mainly caused by functional mismatch repair genes . Our results imply that slipped structures are generated during replication, causing single triplet expansions and contractions in MMR- cells, because they are left unrepaired . On the other hand, we find that the repair of such slipped structures by the MMR system can go awry, resulting in large contractions . We show that these mutS-dependent contractions arise preferentially when the CTG sequence is encoded by the lagging strand . The nature of this orientation dependence argues that the small slipped structures that are recognized by the MMR system are formed primarily on the lagging strand of the replication fork . It also suggests that, in the presence of functional MMR, removal of 3 bp slipped structures causes the formation of larger contractions that are probably the result of secondary structure formation by the CTG sequence . We rationalize the opposing effects of MMR on repeat tract stability with a model that accounts for CTG repeat instability and loss of orientation dependence in MMR- cells . Our work resolves a contradiction between opposing claims in the literature of both stabilizing and destabilizing effects of MMR on CTG repeat instability in E . coli. Mol Microbiol, 2000 Jan, 35(2), 454 - 62 Mutant DnaA proteins defective in duplex opening of oriC, the origin of chromosomal DNA replication in Escherichia coli; Takata M et al.; We characterized three mutant DnaA proteins with an amino acid substitution of R334H, R342H and E361G that renders chromosomal replication cold (20 degrees C) sensitive . Each mutant DnaA protein was highly purified from overproducers, and replication activities were assayed in in vitro oriC replication systems . At 30 degrees C, all three mutant proteins exhibited specific activity similar to that seen with the wild-type protein, whereas at 20 degrees C, there was much less activity in a replication system using a crude replicative extract . Regarding the affinity for ATP, the dissociation rate of bound ATP and binding to oriC DNA, the three mutant DnaA proteins showed a capacity indistinguishable from that of the wild-type DnaA protein . Activity for oriC DNA unwinding of the two mutant DnaA proteins, R334H and R342H, was more sensitive to low temperature than that of the wild-type DnaA protein . We propose that R334H and R342H have a defect in their potential to unwind oriC DNA at low temperatures, the result being the cold-sensitive phenotype in oriC DNA replication . The two amino acid residues of DnaA protein, located in a motif homologous to that of NtrC protein, may play a role in the formation of the open complex . The E361 residue may be related to interaction with another protein present in a crude cell extract. Mol Microbiol, 2000 Jan, 35(2), 435 - 43 Emergency derepression: stringency allows RNA polymerase to override negative control by an active repressor; Kvint K et al.; The uspA promoter, driving production of the universal stress protein A in response to diverse stresses, is demonstrated to be under dual control . One regulatory pathway involves activation of the promoter by the alarmone guanosine 3',5'-bisphosphate, via the beta-subunit of RNA polymerase, whereas the other consists of negative control by the FadR repressor . In contrast to canonical dual control by activation and repression circuits, which depends on concomitant activation and derepression for induction to occur, the ppGpp-dependent activation of the uspA promoter overrides repression by an active FadR under conditions of severe cellular stress (starvation) . The ability of RNA polymerase to overcome repression during stringency depends, in part, on the strength of the FadR operator . This emergency derepression is operative on other FadR-regulated genes induced by starvation and is argued to be an essential regulatory mechanism operating during severe stress. Mol Microbiol, 2000 Jan, 35(2), 361 - 7 The linker peptide of the ArsA ATPase; Li J et al.; Plasmid R773 encodes an As(III)/Sb(III)-translocating ATPase that confers resistance to those metalloids in Escherichia coli . The catalytic subunit of the pump, the ArsA ATPase, consists of homologous N- and C-terminal nucleotide-binding domains connected by a 25-residue linker . The role of this linker sequence was examined by deletion of five, 10, 15 or 23 residues or insertion of five glycine residues . Cells expressing arsA with the 5-residue insertion had wild-type arsenite resistance . Resistance of cells expressing modified arsA genes with deletions was dependent on the linker length . Cells with five or 10 deleted residues exhibited slightly reduced resistance . Deletion of 15 or 23 residues resulted in further decreases in resistance . Each altered ArsA was purified . The enzyme with the 5-residue insertion had the same affinity for ATP and Sb(III) as the wild-type enzyme . Enzymes with 5-, 10-, 15- or 23-residue deletions exhibited decreased affinity for both Sb(III) and ATP . The enzyme with a 23-residue deletion exhibited only basal ATPase activity and was unable to be allosterically activated by Sb(III) . These results suggest that the linker has evolved to a length optimal for bringing the two halves of the protein into proper contact with each other, facilitating catalysis. Eur J Neurosci, 2000 Jan, 12(1), 381 - 4 Localization of the fragile X mental retardation 2 (FMR2) protein in mammalian brain; Miller WJ et al.; The transcriptional silencing of the FMR2 gene has been implicated in FRAXE mental retardation . FRAXE individuals have been shown to exhibit learning deficits, including speech delay, reading and writing problems . FMR2 encodes a large protein of 1311 amino acids and is a member of a gene family encoding proline-serine-rich proteins that have properties of nuclear transcription factors . To characterize the expression of the fragile X mental retardation 2 (FMR2) protein, polyclonal antibodies were raised against two regions of the human FMR2 protein and used in immunofluorescence experiments on mouse brain cryosections . Our results demonstrate for the first time that the FMR2 protein is localized in neurons of the neocortex, Purkinje cells of the cerebellum and the granule cell layer of the hippocampus . FMR2 staining is shown to colocalize with the nuclear stain 4,6-diamidino-2-phenylindole (DAPI) confirming that FMR2 is a nuclear protein . The localization of FMR2 protein to the mammalian hippocampus and other brain structures involved with cognitive function is consistent with the learning deficits seen in FRAXE individuals. Eur J Biochem, 2000 Feb, 267(3), 885 - 93 In vitro folding, purification and characterization of Escherichia coli outer membrane protease ompT; Kramer RA et al.; OmpT is a protease present in the outer membrane of Escherichia coli . The enzyme was overexpressed without its signal sequence in E . coli using a T7 system, resulting in the accumulation of OmpT as inclusion bodies . After solubilization of the inclusion bodies in urea, the protein could be folded in vitro by dilution in the presence of detergent n-dodecyl-N, N-dimethyl-1-ammonio-3-propanesulphonate . The addition of lipopolysaccharide to the protein was essential to obtain active enzyme . The correctly folded protein was purified to homogeneity by ion exchange chromatography with a 57% overall yield . Autoproteolysis between Lys217-Arg218 was a major problem during purification, but degradation could be abolished by introducing the mutations G216K and K217G . A novel fluorimetric assay using the internally quenched substrate Abz-Ala-Arg-Arg-Ala-Tyr(NO2)-NH2 (where Abz is o-aminobenzoyl and Tyr(NO2) is 3-nitrotyrosine) enabled the determination of the kinetic parameters . The wild-type enzyme has an affinity Km of 0.4 microM for the substrate and a turnover number kcat of 40 s-1 . The Km and kcat for the double variant were 1.1 microM and 1.6 s-1, respectively . The pH profiles of the wild type and variant were identical, showing optimal activity at pH 6.5 and pKa values of 5.6 and 7.5, respectively . Circular dichroism spectra of both enzymes indicated a high content of beta-strand conformation, and on that basis a beta-barrel topology model is proposed. Eur J Biochem, 2000 Feb, 267(3), 853 - 60 Site-directed mutagenesis of the active site serine290 in flavanone 3beta-hydroxylase from Petunia hybrida; Lukacin R et al.; Flavanone 3beta-hydroxylase (FHT) catalyzes a pivotal reaction in the formation of flavonoids, catechins, proanthocyanidins and anthocyanidins . In the presence of oxygen and ferrous ions the enzyme couples the oxidative decarboxylation of 2-oxoglutarate, releasing carbon dioxide and succinate, with the oxidation of flavanones to produce dihydroflavonols . The hydroxylase had been cloned from Petunia hybrida and expressed in Escherichia coli, and a rapid isolation method for the highly active, recombinant enzyme had been developed . Sequence alignments of the Petunia hydroxylase with various hydroxylating 2-oxoglutarate-dependent dioxygenases revealed few conserved amino acids, including a strictly conserved serine residue (Ser290) . This serine was mutated to threonine, alanine or valine, which represent amino acids found at the corresponding sequence position in other 2-oxoglutarate-dependent enzymes . The mutant enzymes were expressed in E . coli and purified to homogeneity . The catalytic activities of {Thr290}FHT and {Ala290}FHT were still significant, albeit greatly reduced to 20 and 8%, respectively, in comparison to the wild-type enzyme, whereas the activity of {Val290}FHT was negligible (about 1%) . Kinetic analyses of purified wild-type and mutant enzymes revealed the functional significance of Ser290 for 2-oxoglutarate-binding . The spatial configurations of the related Fe(II)-dependent isopenicillin N and deacetoxycephalosporin C synthases have been reported recently and provide the lead structures for the conformation of other dioxygenases . Circular dichroism spectroscopy was employed to compare the conformation of pure flavanone 3beta-hydroxylase with that of isopenicillin N synthase . A double minimum in the far ultraviolet region at 222 nm and 208-210 nm and a maximum at 191-193 nm which are characteristic for alpha-helical regions were observed, and the spectra of the two dioxygenases fully matched revealing their close structural relationship . Furthermore, the spectrum remained unchanged after addition of either ferrous ions, 2-oxoglutarate or both of these cofactors, ruling out a significant conformational change of the enzyme on cofactor-binding. Eur J Biochem, 2000 Feb, 267(3), 821 - 9 Trp scanning analysis of Tet repressor reveals conformational changes associated with operator and anhydrotetracycline binding; Kintrup M et al.; We analysed the conformational states of free, tet operator-bound and anhydrotetracycline-bound Tet repressor employing a Trp-scanning approach . The two wild-type Trp residues in Tet repressor were replaced by Tyr or Phe and single Trp residues were introduced at each of the positions 162-173, representing part of an unstructured loop and the N-terminal six residues of alpha-helix 9 . All mutants retained in vivo inducibility, but anhydrotetracycline-binding constants were decreased up to 7.5-fold when Trp was in positions 169, 170 and 173 . Helical positions (168-173) differed from those in the loop (162-167) in terms of their fluorescence emission maxima, quenching rate constants with acrylamide and anisotropies in the free and tet operator-complexed proteins . Trp fluorescence emission decreased drastically upon atc binding, mainly due to energy transfer . For all proteins, either free, tet operator bound or anhydrtetracycline-bound, mean fluorescence lifetimes were determined to derive quenching rate constants . Solvent-accessible surfaces of the respective Trp side chains were calculated and compared with the quenching rate constants in the anhydrotetracycline-bound complexes . The results support a model, in which residues in the loop become more exposed, whereas residues in alpha-helix 9 become more buried upon the induction of TetR by anhydrotetracycline. Eur J Biochem, 2000 Feb, 267(3), 780 - 7 Characterization of a nif-regulated flavoprotein (FprA) from Rhodobacter capsulatus . Redox properties and molecular interaction with a {2Fe-2S} ferredoxin; Jouanneau Y et al.; A flavoprotein from Rhodobacter capsulatus was purified as a recombinant (His)6-tag fusion from an Escherichia coli clone over-expressing the fprA structural gene . The FprA protein is a homodimer containing one molecule of FMN per 48-kDa monomer . Reduction of the flavoprotein by dithionite showed biphasic kinetics, starting with a fast step of semiquinone (SQ) formation, and followed by a slow reduction of the SQ . This SQ was in the anionic form as shown by EPR and optical spectroscopies . Spectrophotometric titration gave a midpoint redox potential for the oxidized/SQ couple of Em1 = +20 mV (pH 8.0), whereas the SQ/hydroquinone couple could not be titrated due to the thermodynamic instability of SQ associated with its slow reduction process . The inability to detect the intermediate form, SQ, upon oxidative titration confirmed this instability and led to an estimate of Em2 - Em1 of > 80 mV . The reduction of SQ by dithionite was significantly accelerated when the {2Fe-2S} ferredoxin FdIV was used as redox mediator . The midpoint redox potential of this ferredoxin was determined to be -275 +/- 2 mV at pH 7.5, consistent with FdIV serving as electron donor to FprA in vivo . FdIV and FprA were found to cross-react when incubated together with the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, giving a covalent complex with an Mr of approximately 60 000 . Formation of this complex was unaffected by the redox states of the two proteins . Other {2Fe-2S} ferredoxins, including FdV and FdVI from R . capsulatus, were ineffective as electron carriers to FprA, and cross-reacted poorly with the flavoprotein . The possible function of FprA with regard to nitrogen fixation was investigated using an fprA-deleted mutant . Although nitrogenase activity was significantly reduced in the mutant compared with the wild-type strain, nitrogen fixation was apparently unaffected by the fprA deletion even under iron limitation or microaerobic conditions. Eur J Biochem, 2000 Feb, 267(3), 728 - 36 Spectroscopic characterization of the conformational adaptability of Bombyx mori apolipophorin III; Narayanaswami V et al.; Apolipophorin III (apoLp-III) from the silkmoth, Bombyx mori, has been over-expressed in Escherichia coli, purified and characterized . Far-UV CD spectroscopic analysis revealed 65% alpha-helix secondary structure . Near-UV CD spectra obtained in buffer or complexed with dimyristoylglycerophosphocholine (DMPC), provided evidence that apoLp-III alpha-helices reorient upon interaction with lipid, indicative of a protein conformational change . In guanidine hydrochloride (GdnHCl) denaturation studies, a transition midpoint of 0.33 M was observed, corresponding to a DeltaGDH2O = 2.46 kcal . mol-1 . Fluorescence studies of the sole tryptophan residue (Trp40) in apoLp-III revealed an emission lambdamax = 327 nm . Compared to free tryptophan, Stern-Volmer constants (KSV) for acrylamide and KI quenching of Trp40 fluorescence were decreased by 20-fold and sevenfold, respectively . In studies of apoLp-III-DMPC disc complexes, far-UV CD spectroscopy revealed an increase in alpha-helix content to approximately 85% and a ninefold increase in the GdnHCl-induced denaturation transition midpoint to 3 M . In studies of lipid interaction, apoLp-III was shown to disrupt both negatively charged and zwitterionic phospholipid bilayer vesicles, transforming them into discoidal complexes . Characterization of apoLp-III-DMPC discs, using 5-doxyl or 12-doxyl stearic acid as lipid-based quenching agents, revealed that Trp40 localizes near the phospholipid polar head groups . KSV values for acrylamide and KI quenching of intrinsic fluorescence of apoLp-III-DMPC discs indicate that Trp40 is embedded in the lipid milieu, with little or no accessibility to the aqueous quenchers . Given the large amount of alpha-helix in apoLp-III, the data presented support a model in which amphipathic alpha-helical segments are stabilized by helix-helix interactions and lipid association induces a protein conformational change which results in substitution of helix-helix interactions for helix-lipid contacts. Eur J Biochem, 2000 Feb, 267(3), 627 - 33 Production of recombinant human beta2-microglobulin for scintigraphic diagnosis of amyloidosis in uremia and hemodialysis; Linke RP et al.; Amyloid of beta2-microglobulin (beta2m) origin can be diagnosed using 131I-radiolabelled-beta2m scintigraphy in patients with uremia and hemodialysis treatment . As the tracer beta2m is isolated from another patient, it carries the common risks, including viral infections such as Hepatitis B, C and HIV, which are associated with human plasma products . In order to exclude these risks we have produced recombinant human beta2m (rhbeta2m) in Escherichia coli . The expression vector pASK40DeltaLbeta2m(His)5 contains a C-terminal (His)5-tag for purification via immobilized metal ion affinity chromatography (IMAC) . Size exclusion chromatography on a Superose 12 column represents the second step of purification . The isolated rhbeta2mH5 reacted in an immunochemically identical manner to native human beta2m, and showed a single band of approximately 11.8 kDa in Western blot analysis and revealed a single spot in two-dimensional gel electrophoresis . Mass spectrometry analysis revealed a single peak at the expected molecular mass of 12 415.8 Da . Uniformity was further proven by crystallization and N-terminal amino-acid sequence analysis . The rhbeta2mH5 protein was then produced under conditions that allow the intravenous use in humans . Intraveneously applied indium-111-labelled rhbeta2mH5 was monitored in hemodialysed patients with and without known beta2m-amyloidosis . The tracer was localized specifically to particular areas known to contain amyloid . Thus, this rhbeta2mH5 preparation is suitable for detecting amyloid-containing organs of the beta2m-class in vivo and fulfils the requirements of a tracer for common use . Finally, the use of indium-111 instead of iodine-131 has reduced the radioactive load and resulted in higher resolution. Biochemistry, 2000 Feb 1, 39(4), 763 - 72 DNA base excision repair in human malaria parasites is predominantly by a long-patch pathway; Haltiwanger BM et al.; Mammalian cells repair apurinic/apyrimidinic (AP) sites in DNA by two distinct pathways: a polymerase beta (pol beta)-dependent, short- (one nucleotide) patch base excision repair (BER) pathway, which is the major route, and a PCNA-dependent, long- (several nucleotide) patch BER pathway . The ability of a cell-free lysate prepared from asexual Plasmodium falciparum malaria parasites to remove uracil and repair AP sites in a variety of DNA substrates was investigated . We found that the lysate contained uracil DNA glycosylase, AP endonuclease, DNA polymerase, flap endonuclease, and DNA ligase activities . This cell-free lysate effectively repaired a regular or synthetic AP site on a covalently closed circular (ccc) duplex plasmid molecule or a long (382 bp), linear duplex DNA fragment, or a regular or reduced AP site in short (28 bp), duplex oligonucleotides . Repair of the AP sites in the various DNA substrates involved a long-patch BER pathway . This biology is different from mammalian cells, yeast, Xenopus, and Escherichia coli, which predominantly repair AP sites by a one-nucleotide patch BER pathway . The apparent absence of a short-patch BER pathway in P . falciparum may provide opportunities to develop antimalarial chemotherapeutic strategies for selectively damaging the parasites in vivo and will allow the characterization of the long-patch BER pathway without having to knock-out or inactivate a short-patch BER pathway, which is necessary in mammalian cells. Biochemistry, 2000 Feb 1, 39(4), 745 - 52 DnaB helicase affects the initiation specificity of Escherichia coli primase on single-stranded DNA templates; Bhattacharyya S et al.; The effect of DnaB helicase on the initiation specificity of primase was studied biochemically using a series of single-stranded DNA templates in which each nucleotide of the trinucleotide d(CTG) initiation sequence was systematically varied . DnaB helicase accelerated the rate of primer syntheisis, prevented "overlong" primers from forming and decreased the initiation specificity of primase . In the presence of DnaB helicase, all trinucleotides could serve as the primer initiation site although there was a distinct preference for d(CAG) . These data may explain the high chromosomal prevalence of octanucleotides containing CTG on the leading strand and its complement CAG on the lagging strand . The specificity of DnaB helicase places it on the lagging strand template where it stimulates the initiation of Okazaki fragment synthesis . In the absence of DnaB helicase, primase preferentially primed the d(CTG) template . In the presence of DnaB helicase, the initiation preference was not only altered but also the preferred initiating nucleotide was found to be GTP rather than ATP, for both the d(CTG) and the d(CAG) templates . This suggested that the specificity of primase for the d(CTG) initiation trinucleotide was predominantly unaffected in the absence of DnaB helicase on short ssDNA templates, whereas in conjunction with DnaB helicase, the specificity was altered and this alteration has significant implications in the replication of Escherichia coli chromosome in vivo. Biochemistry, 2000 Feb 1, 39(4), 736 - 44 DnaB helicase stimulates primer synthesis activity on short oligonucleotide templates; Johnson SK et al.; DnaB helicase stimulated the second-order RNA primer synthesis activity of primase by over 5000-fold on DNA templates that were 23 nucleotides long . This template length is the same as the DnaB helicase thermodynamic binding site size {Jezewska, M . J., and Bujalowski, W . (1996) Biochemistry 35, 2117-2128} . This phenomenal stimulation was achieved by increasing the template affinity of primase by over 300-fold and increasing the catalytic rate by over 15-fold . It was necessary to determine the optimal amount of DnaB helicase to achieve this stimulation because helicase stimulation was cooperative at low concentration and inhibitory at high helicase concentration . The cooperative stimulation at low concentration indicated the presence of a time-dependent assembly step that preceded the active state . Besides stimulating primase activity, DnaB helicase also prevented primase from synthesizing RNA primers that were longer than the template sequence . In the absence of DnaB helicase, the majority of primers synthesized by primase were longer than the template and were named "overlong primers" {Swart, J . R., and Griep, M . A . (1995) Biochemistry 34, 16097-16106} . In contrast, the helicase-stimulated RNA primers were from 10 to 14 nucleotides in length with the 12-mer representing the majority of the total RNA primers produced . It was shown that DnaB helicase stabilized the open or single-stranded conformation of the template, which favored the synthesis of the template-length-dependent primers . In contrast, when primase acted alone, it stabilized the 3'-end hairpin conformation of the template so that the template's 3'-hydroxyl served as a "DNA primer" from which primase elongated to create the overlong primers. Biochemistry, 2000 Feb 1, 39(4), 658 - 66 Structure-function relationships in sorcin, a member of the penta EF-hand family . Interaction of sorcin fragments with the ryanodine receptor and an Escherichia coli model system; Zamparelli C et al.; Sorcin, a 21.6 kDa cytosolic EF-hand protein which undergoes a Ca(2+)-induced translocation from cytoplasm to membranes, has been assigned to the newly defined penta EF-hand family . A molecular model of the C-terminal Ca(2+)-binding domain has been generated using as a template the X-ray coordinates of the corresponding domain in the calpain light subunit, the family prototype {Lin, G., et al . (1997) Nat . Struct . Biol . 4, 539-546} . The model indicates that in sorcin the three-dimensional structure is conserved and in particular that of EF1, the novel EF-hand motif characteristic of the family . On this basis, two stable fragments have been obtained and characterized . Just like the native protein, the sorcin Ca(2+)-binding domain (residues 33-198) is largely dimeric, interacts with the ryanodine receptor at physiological calcium concentrations, and undergoes a reversible, Ca(2+)-dependent translocation from cytosol to target proteins on Escherichia coli membranes . In contrast, the 90-198 fragment (residues 90-198), which lacks EF1 and EF2, does not bind Ca(2+) with high affinity and is unable to translocate . Binding of calcium to the EF1-EF2 pair is therefore required for the activation of sorcin which uses the C-terminal calcium-binding domain for interaction with the ryanodine receptor, a physiological target in muscle cells. Proteins, 1999 Dec 1, 37(4), 729 - 42 Insights into the mechanisms of catalysis and heterotropic regulation of Escherichia coli aspartate transcarbamoylase based upon a structure of the enzyme complexed with the bisubstrate analogue N-phosphonacetyl-L-aspartate at 2.1 A; Jin L et al.; A high-resolution structure of Escherichia coli aspartate transcarbamoylase has been determined to 2.1 A; resolution in the presence of the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA) . The structure was refined to a free R-factor of 23.4% and a working R-factor of 20.3% . The PALA molecule is completely saturated with interactions to side chain and backbone groups in the active site, including two interactions that are contributed from the 80s loop of the adjacent catalytic chain . The charge neutralization of the bound PALA molecule (and presumably the substrates as well) induced by the electrostatic field of the highly positively charged active site is an important factor in the high binding affinity of PALA and must be important for catalysis . The higher-resolution structure reported here departs in a number of ways from the previously determined structure at lower resolution . These modifications include alterations in the backbone conformation of the C-terminal of the catalytic chains, the N- and C-termini of the regulatory chains, and two loops of the regulatory chain . The high-resolution of this structure has allowed a more detailed description of the binding of PALA to the active site of the enzyme and has allowed a detailed model of the tetrahedral intermediate to be constructed . This model becomes the basis of a description of the catalytic mechanism of the transcarbamoylase reaction . The R-structural state of the enzyme-PALA complex is an excellent representation of the form of the enzyme that occurs at the moment in the catalytic cycle when the tetrahedral intermediate is formed . Finally, improved electron density in the N-terminal region of the regulatory chain (residues 1 to 7) has allowed tracing of the entire regulatory chain . The N-terminal segments of the R1 and R6 chains are located in close proximity to each other and to the regulatory site . This portion of the molecule may be involved in the observed asymmetry between the regulatory binding sites as well as in the heterotropic response of the enzyme. Pflugers Arch, 1999 Dec, 439(1-2), 67 - 75 Investigation of a genetically engineered mutant of barnacle troponin C containing a central helix deletion; Allhouse LD et al.; To examine the importance of the central alpha-helix of troponin C (TnC) we have bacterially expressed one of the isoforms of barnacle TnC (BTnC2), BTnCWT, but without the aspartate residue at position 80 in the central helix (BTnC80-) . This manipulation is expected to produce an approximately 100 degrees axial rotation of the C-domain with respect to the N-domain, and a net charge change of -1 . BTnC80- mutant was able to restore force to TnC-depleted skinned barnacle myofibrillar bundles to a greater extent than wild-type protein (approximately = 170%) . Competition experiments between BTnC80- and BTnC2-4-, a mutant lacking both of the calcium-specific sites (sites II and IV), shows that deletion of a single amino acid in the central helix results in a protein with increased affinity for the thin filament and one that is bound preferentially compared to BTnC2-4- when at equimolar concentrations. FEMS Microbiol Lett, 2000 Feb 1, 183(1), 183 - 9 An efficient approach for cloning the dNDP-glucose synthase gene from actinomycetes and its application in Streptomyces spectabilis, a spectinomycin producer; Hyun C et al.; Specifically designed PCR primers were applied to amplify a segment of dTDP-glucose synthase gene from six actinomycete strains . About 300-bp or 580-bp DNA fragments were obtained from all the organisms tested . By DNA sequence analysis, seven amplified fragments showed high homology with dTDP-glucose synthase genes that participate in the biosynthesis of secondary metabolites or in deoxy-sugar moieties in lipopolysaccharides . In addition, we have cloned a 45-kb region of DNA from Streptomyces spectabilis ATCC27741, a spectinomycin producer which contained the dTDP-glucose synthase and dTDP-glucose 4,6-dehydratase genes named spcD and spcE, respectively . The spcE gene was expressed in Escherichia coli and the activity was assayed in cell extracts . The enzyme showed substrate specificity only to dTDP-glucose. FEMS Microbiol Lett, 2000 Feb 1, 183(1), 115 - 7 A colicin-tolerant Escherichia coli mutant that confers hfl phenotype carries two mutations in the region coding for the C-terminal domain of FtsH (HflB); Teff D et al.; An Escherichia coli mutant, ER437, which was originally isolated for colicin tolerance, was found to carry two amino acid changes in the C-terminal portion of FtsH (HflB) . These mutations were demonstrated to reduce the ability of FtsH to degrade the phage lambda CII protein in vivo and in vitro, providing a rationalization for the mutant Hfl phenotype. FEMS Microbiol Lett, 2000 Feb 1, 183(1), 37 - 42 Transcriptional analysis of a superoxide dismutase gene of Borrelia burgdorferi; Nichols TL et al.; A single superoxide dismutase (Sod) gene was identified in Borrelia burgdorferi strains, Borrelia afzelii Ple and Borrelia garinii Pbi . Recombinant enzymatic activity was detected only when sod expression was controlled by the lacZ promoter in the cloning vector . Northern blot analysis with sod- or secA-specific probes identified a common 3.7-kb transcript . Reverse transcriptase-PCR analysis confirmed that secA and sod constitute a single transcriptional unit in B . burgdorferi . A transcriptional start site of this operon, containing -10 and -35 regions of a sigma(70)-type promoter, was mapped to 100 bp upstream of the ATG start codon of secA. FEMS Microbiol Lett, 2000 Feb 1, 183(1), 15 - 21 Cloning, sequencing and variability analysis of the gap gene from Mycoplasma hominis; Mygind T et al.; The gap gene encodes the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) . The gene was cloned and sequenced from the Mycoplasma hominis type strain PG21(T) . The intraspecies variability was investigated by inspection of restriction fragment length polymorphism (RFLP) patterns after polymerase chain reaction (PCR) amplification of the gap gene from 15 strains and furthermore by sequencing of part of the gene in eight strains . The M . hominis gap gene was found to vary more than the Escherichia coli counterpart, but the variation at nucleotide level gave rise to only a few amino acid substitutions . To verify that the gene was expressed in M . hominis, a polyclonal antibody was produced and tested against whole cell protein from 15 strains . The enzyme was expressed in all strains investigated as a 36-kDa protein . All strains except type strain PG21(T) showed reaction to a 104-kDa band in addition to the expected 36-kDa band . The protein reacting at 104 kDa is a M . hominis protein with either an epitope similar to one on GAPDH, or it is an immunoglobulin binding protein. Chem Res Toxicol, 2000 Jan, 13(1), 18 - 25 Mutagenic potential of guanine N2 adducts of butadiene mono- and diolepoxide; Carmical JR et al.; To explore the role of guanine N(2) adducts of stereoisomeric butadiene metabolites in butadiene-induced mutagenesis, 11-mer deoxyoligonucleotides were prepared containing adducts of (R)- and (S)-monoepoxide and (R,R)- and (S,S)-diolepoxide . These adducted oligonucleotides were utilized in both in vivo and in vitro experiments designed to examine the mutagenic potency of each and their replication by Escherichia coli polymerases . Each of the four adducted deoxyoligonucleotides was ligated into a single-stranded M13mp7L2 vector and transfected into E . coli . The resulting plaques were screened for misincorporation at position 2 of the N-ras 12 codon . Although the mutagenic frequencies were low, different relative mutagenicities of the various stereoisomers were discernible . In addition, the biological effects of each adduct on the three major E . coli polymerases were determined via primer extension assays . The adducted 11-mers were ligated into a 60-mer linear DNA molecule to provide a sufficiently long template for primer elongation . All four guanine adducts were determined to be blocking to each of the three polymerases via primer extension assays. Biotechniques, 2000 Jan, 28(1), 156 - 60 Identification of proteins in cell-free extracts using liquid chromatography-electrospray ionization mass spectrometry; Dalluge JJ et al.; A rapid method for the identification and characterization of proteins in bacterial cell-free extracts has been developed using directly combined liquid chromatography-electrospray mass spectrometry . The usefulness of this technique is demonstrated for monitoring the expression and chemical modification of phosphoenolpyruvate-sugar phosphotransferase system (PTS) proteins from E . coli with molecular masses ranging from 9-65 kDa . The technique is characterized by minimal sample preparation, remarkable mass accuracy and resolution, reproducibility and the ability, unlike gel electrophoresis, to directly identify posttranslational modifications . The advantages of this technique over analogous matrix-assisted laser desorption ionization mass spectrometry approaches and its potential as a standard tool in the biomolecular research laboratory are discussed. J Cell Biochem, 2000 Jan, 76(3), 499 - 508 Protein-protein interaction of FHL2, a LIM domain protein preferentially expressed in human heart, with hCDC47; Chan KK et al.; In the yeast two-hybrid library screening, the heart-specific FHL2 protein was found to interact with hCDC47 . In vitro interaction study between FHL2 protein and hCDC47 was demonstrated . From the results of domain studies by the yeast two-hybrid assay, the second and third LIM domains in conjunction with the first half LIM domain of FHL2 were identified to be important in binding with hCDC47 . Besides, in Northern blot hybridization of human cancer cell lines, the highest FHL2 mRNA expression was detected in colorectal adenocarcinoma SW480 and HeLa cell S3 . Our results imply that FHL2 protein may associate with cancer development and may act as a molecular adapter to form a multicomplex with hCDC47 in the nucleus, thus it plays an important role in the specification or maintenance of the terminal differentiated phenotype of heart muscle cells . J Cell Biochem, 2000 Jan, 76(3), 368 - 75 Mutagenesis in the switch IV of the helical domain of the human Gsalpha reduces its GDP/GTP exchange rate; Echeverria V et al.; The Galpha subunits of heterotrimeric G proteins are constituted by a conserved GTPase "Ras-like" domain (RasD) and by a unique alpha-helical domain (HD) . Upon GTP binding, four regions, called switch I, II, III, and IV, have been identified as undergoing structural changes . Switch I, II, and III are located in RasD and switch IV in HD . All Galpha known functions, such as GTPase activity and receptor, effector, and Gbetagamma interaction sites have been found to be localized in RasD, but little is known about the role of HD and its switch IV region . Through the construction of chimeras between human and Xenopus Gsalpha we have previously identified a HD region, encompassing helices alphaA, alphaB, and alphaC, that was responsible for the observed functional differences in their capacity to activate adenylyl cyclase (Antonelli et al . {1994}: FEBS Lett 340:249-254) . Since switch IV is located within this region and contains most of the nonconservative amino acid differences between both Gsalpha proteins, in the present work we constructed two human Gsalpha mutant proteins in which we have changed four and five switch IV residues for the ones present in the Xenopus protein . Mutants M15 (hGsalphaalphaS133N, M135P, P138K, P143S) and M17 (hGsalphaalphaS133N, M135P, V137Y, P138K, P143S) were expressed in Escherichia coli, purified, and characterized by their ability to bind GTPgammaS, dissociate GDP, hydrolyze GTP, and activate adenylyl cyclase . A decreased rate of GDP release, GTPgammaS binding, and GTP hydrolysis was observed for both mutants, M17 having considerably slower kinetics than M15 for all functions tested . Reconstituted adenylyl cyclase activity with both mutants showed normal activation in the presence of AlF(4)(-), but a decreased activation with GTPgammaS, which is consistent with the lower GDP dissociating rate they displayed . These data provide new evidence on the role that HD is playing in modulating the GDP/GTP exchange of the Gsalpha subunit . Biotechnol Bioeng, 2000 Mar 5, 67(5), 565 - 74 Observations of green fluorescent protein as a fusion partner in genetically engineered Escherichia coli: monitoring protein expression and solubility; Cha HJ et al.; We have constructed three plasmid vectors for the expression of green fluorescent protein (GFP) fusion proteins using the following motif: (His)(6)-GFP-EK-X, where X represents chloramphenicol acetyl-transferase (CAT), human interleukin-2 (hIL-2), and organophosphorous hydrolase (OPH), respectively, (His)(6) represents a histidine affinity ligand for purification, and EK represents an enterokinase cleavage site for recovering the protein-of-interest from the fusion . The CAT and OPH fusion products ( approximately 63 kDa GFP/CAT and approximately 70 kDa GFP/OPH) were expressed at 4.85 microg/mL (19.9 microg/mg-total protein) and 1.42 microg/mL (4.2 microg/mg-total protein) in the cell lysis supernatant, and, in both cases, enzymatic activity was retained while coupled to GFP . In the case of hIL-2 fusion ( approximately 52 kDa), however, the GFP fluorescence was significantly reduced and most of the fusion was retained in the cell pellet . Linear relationships between GFP fluorescence and CAT or OPH concentration, and with enzymatic activity of CAT or OPH, indicated, for the first time, that in vivo noninvasive quantification of proteins-of-interest, was made possible by simple measurement of GFP fluorescence intensity . The utility of GFP as a reporter was not realized without disadvantages however, in particular, an incremental metabolic cost of GFP was found . This could be offset by many benefits foreseen in expression and purification efficiencies . Biochemistry (Mosc), 1999 Dec, 64(12), 1354 - 9 Ribosomal RNAs in translation termination: facts and hypotheses; Arkov AL et al.; It is now well established that ribosomal RNAs (rRNAs) play an active role in every aspect of translation . This review focuses on recent evidence for the involvement of rRNAs from both subunits of the ribosome in translation termination . This evidence comprises data obtained with rRNA mutants both in vivo and in vitro . In particular, mutations in specific regions of rRNAs caused readthrough of nonsense codons in vivo . Consistent with their in vivo characteristics, the mutations decreased the productive association of the ribosome with release factor 2 (RF2) and the efficiency of catalysis of peptidyl-tRNA hydrolysis in the presence of RF2 in realistic in vitro termination systems . It is now evident that genetic selections for termination-defective mutants in vivo and their characterization in realistic in vitro termination assays will rapidly advance our understanding of the mechanism of termination. J Immunol Methods, 1999 Dec 10, 231(1-2), 105 - 17 Selection of a human anti-progesterone antibody fragment from a transgenic mouse library by ARM ribosome display; He M et al.; In antibody-ribosome-mRNA complex (ARM) ribosome display, stable complexes of nascent protein, mRNA and ribosomes are produced in a eukaryotic in vitro expression system, through coupled transcription and translation of DNA lacking a 3' stop codon . Selection of the protein simultaneously captures the relevant mRNA, which is recovered as DNA by coupled reverse transcription-polymerase chain reaction (RT-PCR) performed on the intact complexes . Here, we describe the use of ARM display to select a specific human antibody fragment from a transgenic mouse library . The mice carry unrearranged gene segments of the human heavy (H) and kappa light (L) chain loci, while the endogenous murine H and kappa loci are functionally silenced; they respond to immunisation by production of fully human IgM antibodies . A library encoding human single-chain (sc) antibody (V(H)/K) fragments, in which V(H) domains and kappa light chains were combined at random by PCR, was prepared from spleen cells of transgenic mice immunised with progesterone-bovine serum albumin (BSA) . Library diversity was demonstrated by sequencing . Progesterone-binding fragments were selected over five cycles of ARM display and the selected DNA cloned and expressed in Escherichia coli . Soluble V(H)/K fragments obtained in periplasmic extracts had the same specificity as ribosome-bound V(H)/K, supporting the view that folding and specificity of the displayed and soluble proteins are equivalent . The affinity of the expressed V(H)/K was approximately 10(-8) M . Sequencing showed that ARM display selected a single V(H)/V(L) combination (V(H)1-2, Vkappa4-1) and rearrangement, with a few mutational differences between clones . Monoclonal antibodies against progesterone-BSA obtained from hybridomas were encoded by the same V(H) and V(L) segments and had similar properties to the fragments obtained in vitro . The combination of ribosome display and transgenic mouse technologies is a rapid means of generating fully human antibody fragments in vitro for expression and further manipulation. J Immunol Methods, 2000 Jan 13, 233(1-2), 131 - 40 Analysis of immune system gene expression in small rheumatoid arthritis biopsies using a combination of subtractive hybridization and high-density cDNA arrays; Zanders ED et al.; Subtractive hybridization of cDNAs generated from synovial RNA which had been isolated from patients with rheumatoid arthritis (RA) or normal controls was used in conjunction with high-density array hybridization to identify genes of immunological interest . The method was designed to detect gene expression in small needle biopsy specimens by means of a prior amplification of nanogram amounts of total RNA to full-length cDNA using PCR . The latter was cut with Rsa I, ligated with adapters, hybridized with unmodified driver cDNA, and subjected to suppression subtraction PCR . Differentially expressed products were cloned into E . coli and picked into 384 well plates . Inserts were obtained by PCR across the multiple cloning site, and the products arrayed at high density on nylon filters . The subtracted cDNAs were also labelled by random priming for use as probes for library screening . The libraries chosen were the subtracted one described above and a set of 45,000 ESTs from the I.M . A.G.E consortium . Clones showing positive hybridization were identified by sequence analysis and homology searching . The results showed that the subtracted hybridization approach could identify many gene fragments expressed at different levels, the most abundant being immunoglobulins and HLA-DR . The expression profile was characteristic of macrophage, B cell and plasma cell infiltration with evidence of interferon induction . In addition, a significant number of sequences without matches in the nucleotide databases were obtained, this demonstrates the utility of the method in finding novel gene fragments for further characterisation as potential members of the immune system . Although RA was studied here, the technology is applicable to any disease process even in cases where amounts of tissue may be limited. FEBS Lett, 2000 Jan 21, 466(1), 192 - 6 Cloning, expression and characterization of a novel four EF-hand Ca(2+)-binding protein from olive pollen with allergenic activity; Ledesma A et al.; A novel allergenic member of the family of Ca(2+)-binding proteins has been cloned from olive tree pollen . The isolated DNA codes for a protein of 171 amino acid residues, which displays four EF-hand sequence motifs . The encoded protein was overproduced in Escherichia coli and purified . The protein (18 inverted question mark omitted inverted question mark795 Da), which binds Ca(2+) and IgE antibodies from patients allergic to olive pollen, undergoes Ca(2+)-dependent conformational changes . It is retained on a phenyl-Sepharose column, which indicates the existence of regulatory EF-hand domains . This fact suggests its involvement in Ca(2+)-dependent signal transduction events of the pollen grain . This allergen could be considered as a member of a new subfamily of EF-hand Ca(2+)-binding proteins since it displays a low amino acid sequence similarity with the so far known proteins. FEBS Lett, 2000 Jan 21, 466(1), 107 - 11 Molecular cloning and characterization of a rice dehydroascorbate reductase; Urano J et al.; Plant dehydroascorbate reductase (DHAR), which re-reduces oxidized ascorbate to maintain an appropriate level of ascorbate, is very important, but no gene or cDNA for plant DHAR has been cloned yet . Here, we describe a cDNA for a rice glutathione-dependent DHAR (designated DHAR1) . A recombinant Dhar1p produced in Escherichia coli was functional . The expression sequence tag database suggests that Dhar1p homologs exist in various plants . Furthermore, the rice Dhar1p has a low similarity to rat DHAR, although the rice enzyme has a considerably higher specific activity than the mammalian one . The mRNA level of DHAR1, the protein level of Dhar1p and the DHAR activity in rice seedlings were elevated by high temperature, suggesting the protection role of DHAR at high temperature. FEBS Lett, 2000 Jan 21, 466(1), 87 - 90 Single amino acid substitutions affecting the specificity of the fungal ribotoxin mitogillin; Kao R et al.; Mitogillin and related fungal ribotoxins are small basic ribonucleolytic proteins that inhibit protein synthesis by specifically hydrolyzing a single phosphodiester bond in the universally conserved alpha-sarcin/ricin loop (SRL) of large subunit ribosomal RNAs . It was previously shown that mitogillin is a natural derivative of a T1/U2-like ribonuclease with inserted domains that are involved in target selection and specificity . Site-directed mutagenesis was used to substitute single amino acids in the previously identified functional domains Ala1-Tyr24 (B1-L1-B2 domain) and Lys106-Lys113 (L4 region) . Examination of the activities of the mutants in the digestion of polyinosinic acid (a ribonuclease substrate) and specific cleavage of the SRL shows that Asn7Ala and Lys111Gln substitutions lead to altered ribonuclease activity and diminished substrate specificity consistent with the proposed functions of these domains. FEBS Lett, 2000 Jan 21, 466(1), 75 - 9 The affinity of the GroEL/GroES complex for peptides under conditions of protein folding; Preuss M et al.; The affinity of four short peptides for the Escherichia coli molecular chaperone GroEL was studied in the presence of the co-chaperone GroES and nucleotides . Our data show that binding of GroES to one ring enhances the interaction of the peptides with the opposite GroEL ring, a finding that was related to the structural readjustments in GroEL following GroES binding . We further report that the GroEL/GroES complex has a high affinity for peptides during ATP hydrolysis when protein substrates would undergo repeated cycles of assisted folding . Although we could not determine at which step(s) during the cycle our peptides interacted with GroEL, we propose that successive state changes in GroEL during ATP hydrolysis may create high affinity complexes and ensure maximum efficiency of the chaperone machinery under conditions of protein folding. FEBS Lett, 2000 Jan 21, 466(1), 67 - 9 Functional expression of His-tagged sensory rhodopsin I in Escherichia coli; Schmies G et al.; Sensory rhodopsin I (SRI) from Halobacterium salinarum was functionally expressed in Escherichia coli and subsequently purified to homogeneity using a C-terminal His-tag anchor . Yields of 3-4 mg SRI/l cell culture can be obtained . The absorption and photocycle properties of SRI were similar if not indistinguishable from those of the homologously expressed SRI . A global fit analysis of the photocycle data and the calculation of the spectra of states provided strong evidence for the existence of an N-like intermediate. FEBS Lett, 2000 Jan 21, 466(1), 45 - 8 Modification of Cys-418 of pyruvate formate-lyase by methacrylic acid, based on its radical mechanism; Plaga W et al.; The recently determined crystal structure of pyruvate formate-lyase (PFL) suggested a new view of the mechanism of this glycyl radical enzyme, namely that intermediary thiyl radicals of Cys-418 and Cys-419 participate in different ways {Becker, A . et al . (1999) Nat . Struct . Biol . 6, 969-975} . We report here a suicide reaction of PFL that occurs with the substrate-analog methacrylate with retention of the protein radical (K(I)=0.42 mM, k(i)=0.14 min(-1)) . Using {1-(14)C}methacrylate (synthesized via acetone cyanhydrin), the reaction end-product was identified by peptide mapping and cocrystallization experiments as S-(2-carboxy-(2S)-propyl) substituted Cys-418 . The stereoselectivity of the observed Michael addition reaction is compatible with a radical mechanism that involves Cys-418 thiyl as nucleophile and Cys-419 as H-atom donor, thus supporting the functional assignments of these catalytic amino acid residues derived from the protein structure. FEBS Lett, 2000 Jan 21, 466(1), 1 - 5 Progress in understanding structure-function relationships in respiratory chain complex II; Ackrell BA; Complex II (succinate:quinone oxidoreductase) of aerobic respiratory chains oxidizes succinate to fumarate and passes the electrons directly into the quinone pool . It serves as the only direct link between activity in the citric acid cycle and electron transport in the membrane . Finer details of these reactions and interactions are but poorly understood . However, complex II has extremely similar structural and catalytic properties to quinol:fumarate oxidoreductases of anaerobic organisms, for which X-ray structures have recently become available . These offer new insights into structure-function relationships of this class of flavoenzymes, including evidence favoring protein movement during catalysis. Mol Pharmacol, 2000 Feb, 57(2), 359 - 66 Drug-resistant variants of Escherichia coli thymidylate synthase: effects of substitutions at Pro-254; Fantz C et al.; Drug-resistant variants of thymidylate synthase (TS) can potentially be used in gene therapy applications to decrease the myelosuppressive side effects of TS-directed anticancer agents or to select genetically modified cells in vivo . Mutations of proline 303 of human TS confer resistance to TS-directed fluoropyrimidines and antifolates () . We generated the corresponding variants in Escherichia coli TS (ecTS), position 254, to better understand the mechanism by which mutations at this residue confer resistance . In addition, because ecTS is intrinsically resistant to several antifolates when compared with human TS, we suspected that greater resistance could be achieved with the bacterial enzyme . The P254L enzyme conferred >100-fold resistance to both raltitrexed and 5-fluoro-2'-deoxyuridine (FdUrd) compared with wild-type ecTS . Four additional mutants (P254F, P254S, P254G, and P254D), each of which complemented growth of a TS-deficient cell line, were generated, isolated, and characterized . Steady-state values of K(m) for dUMP and k(cat) were not substantially different among the variants and were comparable with the wild-type values, but K(m) for methylenetetrahydrofolate (CH(2)H(4)PteGlu) was >10-fold higher for P254D . Values of k(on) and k(off) for nucleotide binding, which were obtained by stopped-flow spectroscopy, were virtually unchanged among the mutants . Drastic differences were observed for CH(2)H(4)PteGlu binding, with K(d) values >15-fold higher than observed with the wild-type enzyme; surprisingly, the proposed isomerization reaction that is very evident for the wild-type enzyme is not observed with P254S . The decrease in affinity for CH(2)H(4)PteGlu correlates well with K(i) values obtained for three TS-directed inhibitors . These results show that mutations at Pro-254 specifically affect the initial binding interactions between enzyme and cofactor and also alter the ability of the mutant enzymes to undergo conformational changes that occur on ternary complex formation . The crystal structure of P254S was determined at 1.5 A resolution and is the most precise structure of TS available . When compared with wild-type TS, the structure shows local conformational changes affecting mostly Asp-253; its carbonyl is rotated approximately 40 degrees, and the side chain forms an ion pair with Arg-225. J Bacteriol, 2000 Feb, 182(4), 1181 - 4 Two extracytoplasmic function sigma subunits, sigma(E) and sigma(FecI), of Escherichia coli: promoter selectivity and intracellular levels; Maeda H et al.; The promoter selectivity of two extracytoplasmic function (ECF) subfamily sigma subunits, sigma(E) (sigma(24)) and sigma(FecI) (sigma(18)), of Escherichia coli RNA polymerase was analyzed by using an in vitro transcription system and various promoters . The Esigma(E) holoenzyme recognized only the known cognate promoters, rpoEP2, rpoHP3, and degP, and the Esigma(FecI) recognized only one known cognate promoter, fecA . The strict promoter recognition properties of sigma(E) and sigma(FecI) are similar to those of other minor sigma subunits . Transcription by Esigma(E) and Esigma(FecI) was enhanced by high concentrations of glutamate, as in the case of other minor sigma subunits . The optimum temperature for transcription by Esigma(FecI) was low, around 25 degrees C, apparently in agreement with the high rate of iron sequestration by E . coli at low temperatures . By quantitative Western blot analysis, the intracellular levels of sigma(E) and sigma(FecI) in the uninduced steady-state culture of E . coli W3110 (type A) were determined to be 0.7 to 2.0 and 0.1 to 0.2 fmol per microg of total proteins (or 3 to 9 and 0.4 to 0.9 molecules per cell), respectively, and less than 1% of the level of the major sigma(70) subunit. J Bacteriol, 2000 Feb, 182(4), 1176 - 80 Native plasmids of Fusobacterium nucleatum: characterization and use in development of genetic systems; Haake SK et al.; Three native plasmids of Fusobacterium nucleatum were characterized, including DNA sequence analysis of one plasmid, pFN1 . A shuttle plasmid, pHS17, capable of transforming Escherichia coli and F . nucleatum ATCC 10953 was constructed with pFN1 . pHS17 was stably maintained in the F . nucleatum transformants, and differences in the transformation efficiencies suggested the presence of a restriction-modification system in F . nucleatum. J Bacteriol, 2000 Feb, 182(4), 1172 - 5 Visualization of phospholipid domains in Escherichia coli by using the cardiolipin-specific fluorescent dye 10-N-nonyl acridine orange; Mileykovskaya E et al.; Cardiolipin (CL)-specific fluorescent dye 10-N-nonyl-acridine orange (NAO) was used to visualize CL distribution in Escherichia coli cells of different phospholipid compositions . In a filamentous mutant containing only anionic phospholipids, green fluorescent spots were observed along the filaments at approximately regular intervals . Three-dimensional image reconstruction obtained by optical sectioning and a deconvolution algorithm revealed NAO-binding domains in the plane of the cell membrane . Substantial red fluorescence emission of bound NAO supported labeling of CL-containing domains . These structures were not found in mutants deficient in CL biosynthesis . The domains were also observed mostly in the septal region and on the poles in cells of normal size with wild-type phospholipid composition. J Bacteriol, 2000 Feb, 182(4), 967 - 73 Polar clustering of the chemoreceptor complex in Escherichia coli occurs in the absence of complete CheA function; Skidmore JM et al.; Bacterial chemotaxis requires a phosphorelay system initiated by the interaction of a ligand with its chemoreceptor and culminating in a change in the directional bias of flagellar rotation . Chemoreceptor-CheA-CheW ternary complexes mediate transduction of the chemotactic signal . In vivo, these complexes cluster predominantly in large groups at the cell poles . The function of chemoreceptor clustering is currently unknown . To gain insight into the relationship between signaling and chemoreceptor clustering, we examined these properties in several Escherichia coli mutant strains that produce CheA variants altered in their ability to mediate chemotaxis, autophosphorylate, or bind ATP . We show here that polar clustering of chemoreceptor complexes does not require functional CheA protein, although maximal clustering occurred only in chemotactically competent cells . Surprisingly, in cells containing a minimum of 13 gold particles at the cell pole, a significant level of clustering was observed in the absence of CheA, demonstrating that CheA is not absolutely essential for chemoreceptor clustering . Nonchemotactic cells expressing only CheA(S), a C-terminal CheA deletion, or CheA bearing a mutation in the ATP-binding site mediated slightly less than maximal chemoreceptor clustering . Cells expressing only full-length CheA (CheA(L)) from either a chromosomal or a plasmid-encoded allele displayed a methyl-accepting chemotaxis protein localization pattern indistinguishable from that of strains carrying both CheA(L) and CheA(S), demonstrating that CheA(L) alone can mediate polar clustering. J Bacteriol, 2000 Feb, 182(4), 961 - 6 The TorR high-affinity binding site plays a key role in both torR autoregulation and torCAD operon expression in Escherichia coli; Ansaldi M et al.; In the presence of trimethylamine N-oxide (TMAO), the TorS-TorR two-component regulatory system induces the torCAD operon, which encodes the TMAO respiratory system of Escherichia coli . The sensor protein TorS detects TMAO and transphosphorylates the response regulator TorR which, in turn, activates transcription of torCAD . The torR gene and the torCAD operon are divergently transcribed, and the short torR-torC intergenic region contains four direct repeats (the tor boxes) which proved to be TorR binding sites . The tor box 1-box 2 region covers the torR transcription start site and constitutes a TorR high-affinity binding site, whereas box 3 and box 4 correspond to low-affinity binding sites . By using torR-lacZ operon fusions in different genetic backgrounds, we showed that the torR gene is negatively autoregulated . Surprisingly, TorR autoregulation is TMAO independent and still occurs in a torS mutant . In addition, this negative regulation involves only the TorR high-affinity binding site . Together, these data suggest that phosphorylated as well as unphosphorylated TorR binds the box 1-box 2 region in vivo, thus preventing RNA polymerase from binding to the torR promoter whatever the growth conditions . By changing the spacing between box 2 and box 3, we demonstrated that the DNA motifs of the high- and low-affinity binding sites must be close to each other and located on the same side of the DNA helix to allow induction of the torCAD operon . Thus, prior TorR binding to the box 1-box 2 region seems to allow cooperative binding of phosphorylated TorR to box 3 and box 4. J Bacteriol, 2000 Feb, 182(4), 891 - 7 Cloning and characterization of 1-deoxy-D-xylulose 5-phosphate synthase from Streptomyces sp . Strain CL190, which uses both the mevalonate and nonmevalonate pathways for isopentenyl diphosphate biosynthesis; Kuzuyama T et al.; In addition to the ubiquitous mevalonate pathway, Streptomyces sp . strain CL190 utilizes the nonmevalonate pathway for isopentenyl diphosphate biosynthesis . The initial step of this nonmevalonate pathway is the formation of 1-deoxy-D-xylulose 5-phosphate (DXP) by condensation of pyruvate and glyceraldehyde 3-phosphate catalyzed by DXP synthase . The corresponding gene, dxs, was cloned from CL190 by using PCR with two oligonucleotide primers synthesized on the basis of two highly conserved regions among dxs homologs from six genera . The dxs gene of CL190 encodes 631 amino acid residues with a predicted molecular mass of 68 kDa . The recombinant enzyme overexpressed in Escherichia coli was purified as a soluble protein and characterized . The molecular mass of the enzyme was estimated to be 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 130 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a dimer . The enzyme showed a pH optimum of 9.0, with a V(max) of 370 U per mg of protein and K(m)s of 65 microM for pyruvate and 120 microM for D-glyceraldehyde 3-phosphate . The purified enzyme catalyzed the formation of 1-deoxyxylulose by condensation of pyruvate and glyceraldehyde as well, with a K(m) value of 35 mM for D-glyceraldehyde . To compare the enzymatic properties of CL190 and E . coli DXP synthases, the latter enzyme was also overexpressed and purified . Although these two enzymes had different origins, they showed the same enzymatic properties. Blood, 2000 Feb 1, 95(3), 1093 - 9 Cloning of the cellular receptor for feline leukemia virus subgroup C (FeLV-C), a retrovirus that induces red cell aplasia; Quigley JG et al.; Feline leukemia virus-C (FeLV-C) causes red cell aplasia in cats, likely through its interaction with its cell surface receptor . We identified this receptor by the functional screening of a library of complementary DNAs (cDNA) from feline T cells . The library, which was cloned into a retroviral vector, was introduced into FeLV-C-resistant murine (NIH 3T3) cells . The gene conferring susceptibility to FeLV-C was isolated and reintroduced into the same cell type, as well as into FeLV-C-resistant rat (NRK 52E) cells, to verify its role in viral infection . The receptor cDNA is predicted to encode a protein of 560 amino acids with 12 membrane-spanning domains, termed FLVCR . FLVCR has significant amino acid sequence homology with members of the major facilitator superfamily and especially D-glucarate transporters described in bacteria and in C . elegans . As FeLV-C impairs the in vivo differentiation of burst-forming unit-erythroid to colony-forming unit-erythroid, we hypothesize that this transporter system could have an essential role in early erythropoiesis . In further studies, a 6-kb fragment of the human FLVCR gene was amplified by polymerase chain reaction from genomic DNA, using homologous cDNA sequences identified in the human Expressed Sequence Tags database . By radiation hybrid mapping, the human gene was localized to a 0.5-centiMorgan region on the long arm of chromosome 1 at q31.3. Development, 2000 Feb, 127(4), 907 - 20 A novel hydra matrix metalloproteinase (HMMP) functions in extracellular matrix degradation, morphogenesis and the maintenance of differentiated cells in the foot process; Leontovich AA et al.; As a member of Cnidaria, the body wall of hydra is structurally reduced to an epithelial bilayer with an intervening extracellular matrix (ECM) . Biochemical and cloning studies have shown that the molecular composition of hydra ECM is similar to that seen in vertebrates and functional studies have demonstrated that cell-ECM interactions are important to developmental processes in hydra . Because vertebrate matrix metalloproteinases (MMPs) have been shown to have an important role in cell-ECM interactions, the current study was designed to determine whether hydra has homologues of these proteinases and, if so, what function these enzymes have in morphogenesis and cell differentiation in this simple metazoan . Utilizing a PCR approach, a single hydra matrix metalloproteinase, named HMMP was identified and cloned . The structure of HMMP was similar to that of vertebrate MMPs with an overall identity of about 35% . Detailed structural analysis indicated some unique features in (1) the cysteine-switch region of the prodomain, (2) the hinge region preceding the hemopexin domain, and (3) the hemopexin domain . Using a bacterial system, HMMP protein was expressed and folded to obtain an active enzyme . Substrate analysis studies indicated that recombinant HMMP could digest a number of hydra ECM components such as hydra laminin . Using a fluorogenic MMP substrate assay, it was determined that HMMP was inhibited by peptidyl hydroxamate MMP inhibitors, GM6001 and matlistatin, and by human recombinant TIMP-1 . Whole-mount in situ studies indicated that HMMP mRNA was expressed in the endoderm along the entire longitudinal axis of hydra, but at relatively high levels at regions where cell-transdifferentiation occurred (apical and basal poles) . Functional studies using GM6001 and TIMP-1 indicated that these MMP inhibitors could reversibly block foot regeneration . Blockage of foot regeneration was also observed using antisense thio-oligo nucleotides to HMMP introduced into the endoderm of the basal pole using a localized electroporation technique . Studies with adult intact hydra found that GM6001 could also cause the reversible de-differentiation or inhibition of transdifferentiation of basal disk cells of the foot process . Basal disk cells are adjacent to those endoderm cells of the foot process that express high levels of HMMP mRNA . In summary, these studies indicate that hydra has at least one MMP that is functionally tied to morphogenesis and cell transdifferentiation in this simple metazoan. Protein Expr Purif, 2000 Feb, 18(1), 100 - 10 Molecular cloning and high-level expression of human polymerase beta cDNA and comparison of the purified recombinant human and rat enzymes; Patterson TA et al.; The cDNA encoding the human polymerase beta from HeLa cells was PCR amplified and cloned, and its nucleotide sequence determined . The DNA sequence is identical to the polymerase beta cDNA sequence from Tera-2 cells . Three expression strategies were employed that were designed to maximize translation initiation of the polymerase beta mRNA in Escherichia coli and all yielded a high level of human polymerase beta . The recombinant protein was purified and its properties were compared with those of the recombinant rat enzyme . The domain structure and kinetic parameters (k(cat) and K(m)) were nearly identical . A mouse IgG monoclonal antibody to the rat enzyme (mAb-10S) was approximately 10-fold less reactive with the human enzyme than with the rat enzyme as determined by ELISA . Protein Expr Purif, 2000 Feb, 18(1), 95 - 9 Purification of his-tagged proteins by immobilized chelate affinity chromatography: the benefits from the use of organic solvent; Franken KL et al.; Recombinant proteins overexpressed in and purified from Escherichia coli contain impurities that are toxic in biological assays . The application of affinity purification procedures is often not sufficient to remove these toxic components . We here describe a simple and fast, one-step protocol to remove these impurities highly efficiently . Four recombinant proteins were overexpressed in E . coli as His-tagged fusion proteins and purified by immobilized metal chelate affinity chromatography on Ni-NTA beads . Depending on the protein, the composition of the lysis buffer, and the washing protocol, various impurities appeared to be present in the purified protein preparations . Here we show how the use of 60% isopropanol during washing steps removed most of these contaminants from the end products . In addition to the removal of proteins that aspecifically adhere to the beads or to the tagged protein, this procedure was particularly useful in removing endotoxins . Moreover, we show that detergents such as NP-40, that are necessarily employed during lysis, are also efficiently removed . Finally, we show that proteins are able to refold correctly after isopropanol treatment . Thus, the resulting end products contain significantly less contaminating E . coli proteins, endotoxins, and detergents . Protein Expr Purif, 2000 Feb, 18(1), 71 - 6 Overproduction in Escherichia coli and purification of the hemolytic protein sticholysin II from the sea anemone Stichodactyla helianthus; de los Rios V et al.; The cDNA coding for the cytolytic toxins sticholysin I and sticholysin II from the sea anemone Stichodactyla helianthus has been isolated, cloned in pUC18, and sequenced . A 6His-tagged version of sticholysin II has been overproduced in Escherichia coli and purified to homogeneity in milligram amounts . Conformational and functional analyses of recombinant sticholysin II do not reveal any significant difference when compared to the natural cytolysin . Protein Expr Purif, 2000 Feb, 18(1), 64 - 70 Cloning, overexpression, and purification of Escherichia coli quinolinate synthetase; Ceciliani F et al.; Quinolinate synthetase catalyzes the second step of the de novo biosynthetic pathway of pyridine nucleotide formation . In particular, quinolinate synthetase is involved in the condensation of dihydroxyacetone phosphate and iminoaspartate to form quinolinic acid . To study the mechanism of action, the specificity of the enzyme and the interaction with l-aspartate oxidase, the other component of the so-called "quinolinate synthetase complex," the cloning, the overexpression, and the purification to homogeneity of Escherichia coli quinolinate synthetase were undertaken . The results are presented in this paper . Since the overexpression of the enzyme resulted in the formation of inclusion bodies, a procedure of renaturation and refolding had to be set up . The overexpression and purification procedure reported in this paper allowed the isolation of 12 mg of electrophoretically homogeneous quinolinate synthetase from 1 liter of E . coli culture . A new, continuous, method for the evaluation of quinolinate synthetase activity was also devised and is presented . Finally, our data definitely exclude the possibility that other enzymes are involved in the biosynthesis of quinolinic acid in E . coli, since it is possible to synthesize quinolinic acid from l-aspartate, dihydroxyacetone phosphate, and O(2) by using only nadA and nadB gene overexpressed products . Protein Expr Purif, 2000 Feb, 18(1), 1 - 10 Improved expression characteristics of single-chain Fv fragments when fused downstream of the Escherichia coli maltose-binding protein or upstream of a single immunoglobulin-constant domain; Hayhurst A; The expression of single-chain Fv fragments (scFv) targeted to the periplasm of Escherichia coli often results in very low yields of soluble protein frequently accompanied by host cell growth arrest and sometimes lysis . Single-chain antibody fragments (scAb) are scFv with a human kappa light chain constant (HuCkappa) domain attached C-terminally and share similar problems of expression . By fusing the E . coli maltose-binding protein (mbp) gene either 3' or 5' to a scAb specific for the herbicide atrazine, a reduction in growth arrest was observed that was dependent on the order of gene fusion . The scAb-mbp fusion delayed the onset of growth arrest following induction while the mbp-scAb fusion appeared to ablate growth arrest completely . Cell fractionation revealed barely detectable levels of scAb-mbp in the periplasm while mbp-scAb was detected at equivalent levels as scAb in the periplasmic compartment, indicating that periplasmic scAb solubility is unrelated to propensity to cause growth arrest . IMAC purification of scAb and mbp-scAb proteins followed by liquid competition ELISA revealed the IC(50) for atrazine to be approximately 1 nM for both proteins demonstrating that 5'-mbp fusion does not alter antigen binding . The equivalent scFv and mbp-scFv vectors expressed far less material in both periplasmic and insoluble fractions indicating that the HuCkappa domain can have a positive effect on scFv expression when expressed either alone or as a mbp fusion . The ablation of growth arrest by a 5'-mbp fusion and enhancement of expression by a 3'-HuCkappa domain fusion were extended to a second scFv specific for the herbicide diuron . Therefore, by expressing scFv as tripartite fusions (mbp-scFv-HuCkappa) enhanced levels of soluble periplasmic expression can be achieved without causing growth arrest of the host cell, realizing the potential for constitutive expression of hapten-binding scFv in the E . coli periplasm . Indoor Air, 1999 Dec, 9(4), 219 - 25 House dust induces IL-6 and IL-8 response in A549 epithelial cells; Saraf A et al.; The in vitro potency of house dust to induce cytokine response in A549 lung epithelial cells was studied . Dusts collected from carpet, bed, shelf and floor of a villa and an apartment by vacuuming were found to trigger the production of interleukin-8 (IL-8) and interleukin-6 (IL-6) in a dose-dependent manner, and the interleukin production was several-fold higher than of swine dust (used as a positive control) . The IL-8 and IL-6 production of pure Escherichia coli lipopolysaccharide was significantly lower than of the dusts and a peptidoglycan-polysaccharide complex did not show any stimulatory effect at all . The lipopolysaccharide and peptidoglycan contents of the samples were determined by gas chromatography-tandem mass spectrometry analysis of, respectively, 3-hydroxy fatty acids and muramic acid; in addition, ergosterol was monitored for fungal biomass . The inflammatory properties of house dust upon inhalation may be reflected in its high potency to induce cytokine response in lung epithelial cells. Angew Chem Int Ed Engl, 1999 Dec 16, 38(24), 3654 - 3657 Tightening the Belt on Polymerases: Evaluating the Physical Constraints on Enzyme Substrate Size; Frieden M et al.; To test the limits of polymerase enzyme activity on geometrically constrained DNAs, four very small synthetic circular DNAs were constructed by using newly developed methods . Surprisingly, even a 13-nucleotide circular DNA (1) can be copied successfully by both DNA and RNA polymerases, despite the very small diameter and large degree of distortion in this synthetic DNA . The picture shows models to indicate the relative sizes of 1 and the Klenow fragment of the DNA polymerase I from E . coli. Nucleic Acids Res, 2000 Feb 15, 28(4), 853 - 61 ATP-hydrolysis-dependent conformational switch modulates the stability of MutS-mismatch complexes; Joshi A et al.; The mismatch repair pathway in Escherichia coli has been extensively studied in vitro as well as in vivo . The molecular mechanisms by which nucleotide cofactors regulate the whole process constitute an area of active debate . Here we demonstrate that nucleotide (ADP or ATP) binding to MutS mediates a switch in protein conformation . However, in MutS that is DNA bound, this switch ensues only with ATP and not with ADP and is similar, irrespective of whether it is bound to a homo- or a heteroduplex . The results envisage a minimal model of three confor-mational states of MutS as reflected in: (i) a specific and highly stable MutS-mismatch complex in the absence of a nucleotide; (ii) a specific but less stable complex in the presence of ATP hydrolysis; and (iii) an irreversibly dissociated complex in the presence of ATP binding (ATPgammaS) . Such transitions are of relevance to the protein's function in vivo where it has to first recognize a mismatch, followed by a search for hemimethylated sites. Structure Fold Des, 1999 Dec 15, 7(12), 1567 - 73 Direct three-dimensional localization and positive identification of RNA helices within the ribosome by means of genetic tagging and cryo-electron microscopy; Spahn CM et al.; BACKGROUND: Ribosomes are complex macromolecular machines that perform the translation of the genetic message . Cryo-electron microscopic (cryo-EM) maps of the Escherichia coli 70S ribosome are approaching a resolution of 10 A and X-ray maps of the 30S and 50S subunits are now available at 5 A . These maps show a lot of details about the inner architecture of the ribosome and ribosomal RNA helices are clearly visible . However, in the absence of further biological information, even at the higher resolution of the X-ray maps many rRNA helices can be placed only tentatively . Here we show that genetic tagging in combination with cryo-EM can place and orient double-stranded RNA helices with high accuracy . RESULTS: A tRNA sequence inserted into the E . coli 23S ribosomal RNA gene, at one of the points of sequence expansion in eukaryotic ribosomes, is visible in the cryo-EM map as a peripheral 'foot' structure . By tracing its acceptor-stem end, the location of helix 63 in domain IV and helix 98 in domain VI of the 50S subunit could be precisely determined . CONCLUSIONS: Our study demonstrates for the first time that features of a three-dimensional cryo-EM map of an asymmetric macromolecular complex can be interpreted in terms of secondary and primary structure . Using the identified helices as a starting point, it is possible to model and interpret, in molecular terms, a larger portion of the ribosome . Our results might be also useful in interpreting and refining the current X-ray maps. Structure Fold Des, 1999 Dec 15, 7(12), 1483 - 92 The solution structure of Lac repressor headpiece 62 complexed to a symmetrical lac operator; Spronk CA et al.; BACKGROUND: Lactose repressor protein (Lac) controls the expression of the lactose metabolic genes in Escherichia coli by binding to an operator sequence in the promoter of the lac operon . Binding of inducer molecules to the Lac core domain induces changes in tertiary structure that are propagated to the DNA-binding domain through the connecting hinge region, thereby reducing the affinity for the operator . Protein-protein and protein-DNA interactions involving the hinge region play a crucial role in the allosteric changes occurring upon induction, but have not, as yet, been analyzed in atomic detail . RESULTS: We have used nuclear magnetic resonance (NMR) spectroscopy and restrained molecular dynamics (rMD) to determine the structure of the Lac repressor DNA-binding domain (headpeice 62; HP62) in complex with a symmetrized lac operator . Analysis of the structures reveals specific interactions between Lac repressor and DNA that were not found in previously investigated Lac repressor-DNA complexes . Important differences with the previously reported structures of the HP56-DNA complex were found in the loop following the helix-turn-helix (HTH) motif . The protein-protein and protein-DNA interactions involving the hinge region and the deformations in the DNA structure could be delineated in atomic detail . The structures were also used for comparison with the available crystallographic data on the Lac and Pur repressor-DNA complexes . CONCLUSIONS: The structures of the HP62-DNA complex provide the basis for a better understanding of the specific recognition in the Lac repressor-operator complex . In addition, the structural features of the hinge region provide detailed insight into the protein-protein and protein-DNA interactions responsible for the high affinity of the repressor for operator DNA. Nature, 1999 Nov 11, 402(6758), 147 - 54 Identification of in vivo substrates of the chaperonin GroEL; Houry WA et al.; The chaperonin GroEL has an essential role in mediating protein folding in the cytosol of Escherichia coli . Here we show that GroEL interacts strongly with a well-defined set of approximately 300 newly translated polypeptides, including essential components of the transcription/translation machinery and metabolic enzymes . About one third of these proteins are structurally unstable and repeatedly return to GroEL for conformational maintenance . GroEL substrates consist preferentially of two or more domains with alphabeta-folds, which contain alpha-helices and buried beta-sheets with extensive hydrophobic surfaces . These proteins are expected to fold slowly and be prone to aggregation . The hydrophobic binding regions of GroEL may be well adapted to interact with the non-native states of alphabeta-domain proteins. Nature, 2000 Jan 13, 403(6766), 223 - 6 Bioorganic synthesis of lipid-modified proteins for the study of signal transduction; Bader B et al.; Biological membranes define the boundaries of the cellular compartments in higher eukaryotes and are active in many processes such as signal transduction and vesicular transport . Although post-translational lipid modification of numerous proteins in signal transduction is crucial for biological function, analysis of protein-protein interactions has mainly focused on recombinant proteins in solution under defined in vitro conditions . Here we present a new strategy for the synthesis of such lipid-modified proteins . It involves the bacterial expression of a carboxy-terminally truncated non-lipidated protein, the chemical synthesis of differently lipidated peptides representing the C terminus of the proteins, and their covalent coupling . Our technique is demonstrated using Ras constructs, which exhibit properties very similar to fully processed Ras, but can be produced in high yields and are open for selective modifications . These constructs are operative in biophysical and cellular assay systems, showing specific recognition of effectors by Ras lipoproteins inserted into the membrane surface of biosensors and transforming activity of oncogenic variants after microinjection into cultured cells. Nature, 2000 Jan 13, 403(6766), 175 - 9 DNA computing on surfaces; Liu Q et al.; DNA computing was proposed as a means of solving a class of intractable computational problems in which the computing time can grow exponentially with problem size (the 'NP-complete' or non-deterministic polynomial time complete problems) . The principle of the technique has been demonstrated experimentally for a simple example of the hamiltonian path problem (in this case, finding an airline flight path between several cities, such that each city is visited only once) . DNA computational approaches to the solution of other problems have also been investigated . One technique involves the immobilization and manipulation of combinatorial mixtures of DNA on a support . A set of DNA molecules encoding all candidate solutions to the computational problem of interest is synthesized and attached to the surface . Successive cycles of hybridization operations and exonuclease digestion are used to identify and eliminate those members of the set that are not solutions . Upon completion of all the multistep cycles, the solution to the computational problem is identified using a polymerase chain reaction to amplify the remaining molecules, which are then hybridized to an addressed array . The advantages of this approach are its scalability and potential to be automated (the use of solid-phase formats simplifies the complex repetitive chemical processes, as has been demonstrated in DNA and protein synthesis) . Here we report the use of this method to solve a NP-complete problem . We consider a small example of the satisfiability problem (SAT), in which the values of a set of boolean variables satisfying certain logical constraints are determined. J Neurochem, 2000 Feb, 74(2), 763 - 9 Effect of alpha-phenyl-N-tert-butylnitrone on brain cell membrane function and energy metabolism in experimental Escherichia coli meningitis in the newborn piglet; Park WS et al.; We evaluated the efficacy of alpha-phenyl-N-tertbutylnitrone as an adjunctive therapy in experimental bacterial meningitis in the newborn piglet . Meningitis was induced by intracisternal injection of 10(8) colony-forming units of Escherichia coli in 100 microl of saline . Alpha-Phenyl-N-tert-butylnitrone 100 mg/kg was given as a bolus intravenous injection 30 min before induction of meningitis . Although it completely abolished the elevated CSF tumor necrosis factor-a level observed in the meningitis group, alpha-phenyl-N-tert-butylnitrone did not down-modulate parameters of inflammatory responses such as increased intracranial pressure, hypoglycorrhachia, elevated CSF lactate level, and CSF leukocytosis observed in this group . However, alpha-phenyl-N-tert-butylnitrone treatment mitigated alterations in brain cell membrane structure and function during meningitis, evidenced by amelioration of increased brain cell membrane lipid peroxidation products (conjugated dienes) and decreased Na+, K+-ATPase activity . Reduced mean arterial blood pressure, cerebral perfusion pressure, brain glucose concentration, and cerebral energy stores and marginally increased brain lactate level observed in the meningitis group were also ameliorated . These results suggest that although it failed to attenuate the inflammatory responses, alpha-phenyl-N-tert-butylnitrone was effective in ameliorating brain injury in neonatal bacterial meningitis. J Gen Virol, 2000 Feb, 81(Pt 2), 461 - 9 Identification of a human epitope in hepatitis C virus (HCV) core protein using a molecularly cloned antibody repertoire from a non-symptomatic, anti-HCV-positive patient; Barban V et al.; Healthy carriers of hepatitis C virus (HCV) infection exhibit a specific antibody response against all HCV antigens, which could play a role in disease control . Generation of panels of human antibodies may permit a thorough characterization of this response and further identify particular antibodies with potential clinical value . To this effect, we have established a human phage-display antibody library from a patient exhibiting a high antibody response against HCV antigens and no clinical symptoms of disease . This library was screened against a recombinant core antigen {amino acids (aa) 1-119} produced in E . coli . Two recombinant Fab-carrying phages (rFabCs) were isolated and characterized . Both rFabC3 and rFabC14 recognize aa 1-48 on core antigen, but rFabC14 is competed out by a synthetic peptide, C(2-20) (aa 1-20), at much lower concentrations than rFabC3 . In order to identify more precisely the recognition sites of these antibodies, we produced soluble forms of the rFabs (sFabs), and used them to pan a random phage-display peptide library . A single peptide sequence, QLITKPL, was identified with sFabC3, while two equally represented sequences, HAFPHLH and SAPSSKN, were isolated using sFabC14 . The QLITKPL sequence was partially localized between aa 8 and 14 of core protein, but no clear homology was found for the two sFabC14 peptides . However, we confirmed the specificity of these peptides by competition experiments with sFabC14. J Biol Chem, 2000 Jan 28, 275(4), 3006 - 15 A model for Escherichia coli DNA polymerase III holoenzyme assembly at primer/template ends . DNA triggers a change in binding specificity of the gamma complex clamp loader; Ason B et al.; The gamma complex of the Escherichia coli DNA polymerase III holoenzyme assembles the beta sliding clamp onto DNA in an ATP hydrolysis-driven reaction . Interactions between gamma complex and primer/template DNA are investigated using fluorescence depolarization to measure binding of gamma complex to different DNA substrates under steady-state and presteady-state conditions . Surprisingly, gamma complex has a much higher affinity for single-stranded DNA (K(d) in the nM range) than for a primed template (K(d) in the microM range) under steady-state conditions . However, when examined on a millisecond time scale, we find that gamma complex initially binds very rapidly and with high affinity to primer/template DNA but is converted subsequently to a much lower affinity DNA binding state . Presteady-state data reveals an effective dissociation constant of 1.5 nM for the initial binding of gamma complex to DNA and a dissociation constant of 5.7 microM for the low affinity DNA binding state . Experiments using nonhydrolyzable ATPgammaS show that ATP binding converts gamma complex from a low affinity "inactive" to high affinity "active" DNA binding state while ATP hydrolysis has the reverse effect, thus allowing cycling between active and inactive DNA binding forms at steady-state . We propose that a DNA-triggered switch between active and inactive states of gamma complex provides a two-tiered mechanism enabling gamma complex to recognize primed template sites and load beta, while preventing gamma complex from competing with DNA polymerase III core for binding a newly loaded beta.DNA complex. J Biol Chem, 2000 Jan 28, 275(4), 2938 - 42 Distinct protein domains are responsible for the interaction of Hrs-2 with SNAP-25 . The role of Hrs-2 in 7 S complex formation; Tsujimoto S et al.; Regulated secretion of neurotransmitter at the synapse is likely to be mediated by dynamic protein interactions involving components of the vesicle (vesicle-associated membrane protein; VAMP) and plasma membrane (syntaxin and synaptosomal associated protein of 25 kDa (SNAP-25)) along with additional molecules that allow for the regulation of this process . Recombinant Hrs-2 interacts with SNAP-25 in a calcium-dependent manner (they dissociate at elevated calcium levels) and inhibits neurotransmitter release . Thus, Hrs-2 has been hypothesized to serve a negative regulatory role in secretion through its interaction with SNAP-25 . In this report, we show that Hrs-2 and SNAP-25 interact directly through specific coiled-coil domains in each protein . The presence of syntaxin enhances the binding of Hrs-2 to SNAP-25 . Moreover, while both Hrs-2 and VAMP can separately bind to SNAP-25, they cannot bind simultaneously . Additionally, the presence of Hrs-2 reduces the incorporation of VAMP into the syntaxin.SNAP-25.VAMP (7 S) complex . These findings suggest that Hrs-2 may modulate exocytosis by regulating the assembly of a protein complex implicated in membrane fusion. J Biol Chem, 2000 Jan 28, 275(4), 2924 - 30 Thioredoxin-dependent hydroperoxide peroxidase activity of bacterioferritin comigratory protein (BCP) as a new member of the thiol-specific antioxidant protein (TSA)/Alkyl hydroperoxide peroxidase C (AhpC) family; Jeong W et al.; Escherichia coli bacterioferritin comigratory protein (BCP), a putative bacterial member of the TSA/AhpC family, was characterized as a thiol peroxidase . BCP showed a thioredoxin-dependent thiol peroxidase activity . BCP preferentially reduced linoleic acid hydroperoxide rather than H(2)O(2) and t-butyl hydroperoxide with the use of thioredoxin as an in vivo immediate electron donor . The value of V(max)/K(m) of BCP for linoleic acid hydroperoxide was calculated to be 5-fold higher than that for H(2)O(2), implying that BCP has a selective capability to reduce linoleic acid hydroperoxide . Replacement of Cys-45 with serine resulted in the complete loss of thiol peroxidase activity, suggesting that BCP is a new bacterial member of TSA/AhpC family having a conserved cysteine as the primary site of catalysis . BCP exists as a monomer, and its functional Cys-45 appeared to exist as cysteine sulfenic acid . The expression level of BCP gradually elevated during exponential growth until mid-log phase growth, beyond which the expression level was decreased . BCP was induced 3-fold by the oxidative stress given by changing the growth conditions from the anaerobic to aerobic culture . Bcp null mutant grew more slowly than its wild type in aerobic culture and showed the hypersensitivity toward various oxidants such as H(2)O(2), t-butyl hydroperoxide, and linoleic acid hydroperoxide . The peroxide hypersensitivity of the null mutant could be complemented by the expression of bcp gene . Taken together, these data suggest that BCP is a new member of thioredoxin-dependent TSA/AhpC family, acting as a general hydroperoxide peroxidase. J Biol Chem, 2000 Jan 28, 275(4), 2745 - 55 A developmentally regulated aconitase related to iron-regulatory protein-1 is localized in the cytoplasm and in the mitochondrion of Trypanosoma brucei; Saas J et al.; Mitochondrial energy metabolism and Krebs cycle activities are developmentally regulated in the life cycle of the protozoan parasite Trypanosoma brucei . Here we report cloning of a T . brucei aconitase gene that is closely related to mammalian iron-regulatory protein 1 (IRP-1) and plant aconitases . Kinetic analysis of purified recombinant TbACO expressed in Escherichia coli resulted in a K(m) (isocitrate) of 3 +/- 0.4 mM, similar to aconitases of other organisms . This was unexpected since an arginine conserved in the aconitase protein family and crucial for substrate positioning in the catalytic center and for activity of pig mitochondrial aconitase (Zheng, L., Kennedy, M . C., Beinert, H., and Zalkin, H . (1992) J . Biol . Chem . 267, 7895-7903) is substituted by leucine in the TbACO sequence . Expression of the 98-kDa TbACO was shown to be lowest in the slender bloodstream stage of the parasite, 8-fold elevated in the stumpy stage, and increased a further 4-fold in the procyclic stage . The differential expression of TbACO protein contrasted with only minor changes in TbACO mRNA, indicating translational or post-translational mechanisms of regulation . Whereas animal cells express two distinct compartmentalized aconitases, mitochondrial aconitase and cytoplasmic aconitase/IRP-1, TbACO accounts for total aconitase activity in trypanosomes . By cell fractionation and immunofluorescence microscopy, we show that native as well as a transfected epitope-tagged TbACO localizes in both the mitochondrion (30%) and in the cytoplasm (70%) . Together with phylogenetic reconstructions of the aconitase family, this suggests that animal IRPs have evolved from a multicompartmentalized ancestral aconitase . The possible functions of a cytoplasmic aconitase in trypanosomes are discussed. J Biol Chem, 2000 Jan 28, 275(4), 2713 - 20 Combinatorial analysis of the structural requirements of the Escherichia coli hemolysin signal sequence; Hui D et al.; We have investigated the substrate specificity of the Escherichia coli hemolysin transporter system . Translocation of hemolysin is dependent on a C-terminal signal sequence located within the last 60 amino acids of this protein . Previous comparative studies of the signal sequence have revealed a conserved helix(alpha1)-linker-helix(alpha2) motif, suggesting that secondary structure is important for transport . In this study, we generated three random libraries in the alpha1, linker, and alpha2 regions, as well as an alpha1-amphiphilic helical library to identify features buried within the structural motif that contribute to transport . Combinatorial variants were generated by altering the primary sequence of specific regions, and correlation between the genotype and phenotype of the mutant populations allowed us to objectively identify any functional features involved . It was found that the alpha1-amphiphilic helix and the linker are both important for function . To our surprise, the second helix of the conserved structural motif was not essential for transport . The finding that a predicted amphiphilic helix and hydrophobicity, rather than primary sequence, contribute to transport in the alpha1 region allows us to speculate on the mechanism of multiple substrate recognition . This may have implications for understanding the broad substrate specificity common among other ATP-binding cassette transporters. J Biol Chem, 2000 Jan 28, 275(4), 2545 - 53 Engineering microsomal cytochrome P450 2C5 to be a soluble, monomeric enzyme . Mutations that alter aggregation, phospholipid dependence of catalysis, and membrane binding; Cosme J et al.; Deletion of the N-terminal membrane-spanning domain from microsomal P450s 2C5 and 2C3 generates the enzymes, 2C5dH and 2C3dH, that exhibit a salt-dependent association with membranes indicating that they retain a monofacial membrane interaction domain . The two proteins are tetramers and dimers, respectively, in high salt buffers, and only 2C5dH requires phospholipids to reconstitute fully the catalytic activity of the enzyme . Amino acid residues derived from P450 2C3dH between residues 201 and 210 were substituted for the corresponding residues in P450 2C5 to identify those that would diminish the membrane interaction, the phospholipid dependence of catalysis, and aggregation of 2C5dH . Each of four substitutions, N202H, I207L, S209G, and S210T, diminished the aggregation of P450 2C5dH and produced a monomeric enzyme . The N202H and I207L mutations also diminished the stimulation of catalytic activity by phospholipid and reduced the binding of P450 2C5dH to phospholipid vesicles . The modified enzymes exhibit rates of progesterone 21-hydroxylation that are similar to that of P450 2C5dH . These conditionally membrane-bound P450s with improved solubility in high salt buffers are suitable for crystallization and structural determination by x-ray diffraction studies. J Biol Chem, 2000 Jan 28, 275(4), 2505 - 12 Thioredoxin 2 is involved in the oxidative stress response in Escherichia coli; Ritz D et al.; Two genes encoding thioredoxin are found on the Escherichia coli genome . Both of them are capable of reducing protein disulfide bonds in vivo and in vitro . The catalytic site contains a Cys-X(1)-X(2)-Cys motif in a so-called thioredoxin fold . Thioredoxin 2 has two additional pairs of cysteines in a non-conserved N-terminal domain . This domain does not appear to be important for the function of thioredoxin 2 in donating electrons to ribonucleotide reductase, 3'-phosphoadenylsulfate-reductase, or the periplasmic disulfide isomerase DsbC . Our results suggests that the two thioredoxins are equivalent for most of the in vivo functions that were tested . On the other hand, transcriptional regulation is different . The expression of trxC is regulated by the transcriptional activator OxyR in response to oxidative stress . Oxidized OxyR binds directly to the trxC promoter and induces its expression in response to elevated hydrogen peroxide levels or the disruption of one or several of the cytoplasmic redox pathways . Mutants lacking thioredoxins 1 and 2 are more resistant to high levels of hydrogen peroxide, whereas they are more sensitive to diamide, a disulfide bond-inducing agent. J Biol Chem, 2000 Jan 28, 275(4), 2359 - 66 Cloning, expression, and functional characterization of the beta regulatory subunit of human methionine adenosyltransferase (MAT II); LeGros HL Jr et al.; MAT II, the extrahepatic form of methionine adenosyltransferase (MAT), consists of catalytic alpha(2)/alpha(2') subunits and a noncatalytic beta subunit, believed to have a regulatory function . The full-length cDNA that encodes the beta subunit of human MAT II was cloned and found to encode for a 334-amino acid protein with a calculated molecular weight of 37,552 . Analysis of sequence homology showed similarity with bacterial enzymes that catalyze the reduction of TDP-linked sugars . The beta subunit cDNA was cloned into the pQE-30 expression vector, and the recombinant His tagged protein, which was expressed in Escherichia coli, was recognized by antibodies to the human MAT II, to synthetic peptides copying the sequence of native beta subunit protein, and to the rbeta protein . There is no cross-reactivity between the MAT II alpha(2) or beta subunits . None of the anti-beta subunit antibodies reacted with protein extracts of E . coli host cells, suggesting that these bacteria have no beta subunit protein . Interestingly, the rbeta subunit associated with E . coli as well as human MAT alpha subunits . This association changed the kinetic properties of both enzymes and lowered the K(m) of MAT for L-methionine . Together, the data show that we have cloned and expressed the human MAT II beta subunit and confirmed its long suspected regulatory function . This knowledge affords a molecular means by which MAT activity and consequently the levels of AdoMet may be modulated in mammalian cells. J Biol Chem, 2000 Jan 28, 275(4), 2335 - 41 Inteins of Thermococcus fumicolans DNA polymerase are endonucleases with distinct enzymatic behaviors; Saves I et al.; The DNA polymerase gene of Thermococcus fumicolans harbors two intein genes . Both inteins have been produced in Escherichia coli and purified either as naturally spliced products from the expression of the complete DNA polymerase gene or directly from the cloned inteins genes . Both recombinant inteins exhibit endonuclease activity, with an optimal temperature of 70 degrees C . The Tfu pol-1 intein, which belongs to the Psp KOD pol-1 allelic family, recognizes and cleaves a minimal sequence of 16 base pairs (bp) on supercoiled DNA with either Mn(2+) or Mg(2+) as cofactor . It cleaves linear DNA only with Mn(2+) and requires a 19-bp minimal recognition sequence . The Tfu pol-2 intein, which belongs to the Tli pol-2 allelic family, is a highly active homing endonuclease using Mg(2+) as cofactor . Its minimal recognition and cleavage site is 21 bp long either on linear or circular DNA substrates . Its endonuclease activity is strongly inhibited by the 3' digestion product, which remains bound to the enzyme after the cleavage reaction . According to current nomenclature, these endonucleases were named PI-TfuI and PI-TfuII . These two inteins thus exhibit different requirements for metal cofactor and substrate topology as well as different mechanism of action. Am J Physiol Lung Cell Mol Physiol, 2000 Jan, 278(1), L33 - 41 Increased elastase release by CF neutrophils is mediated by tumor necrosis factor-alpha and interleukin-8; Taggart C et al.; Cystic fibrosis (CF) is a lethal, hereditary disorder characterized by a neutrophil-dominated inflammation of the lung . We sought to determine whether neutrophils from individuals with CF release more neutrophil elastase (NE) than neutrophils from normal subjects . Our results showed that peripheral blood neutrophils (PBNs) from normal subjects and individuals with CF contained similar amounts of NE, but after preincubation with CF bronchoalveolar lavage (BAL) fluid, significantly more NE was released by CF PBNs, a release that was amplified further by incubation with opsonized Escherichia coli . To determine which components of CF BAL fluid stimulated this excessive NE release from CF PBNs, we repeated the experiments after neutralization or immunoprecipitation of tumor necrosis factor (TNF)-alpha and interleukin (IL)-8 in CF BAL fluid . We found that subsequent NE release from CF PBNs was reduced significantly when TNF-alpha and IL-8 were removed from CF BAL fluid . When TNF-alpha and IL-8 were used as activating stimuli, CF PBNs released significantly greater amounts of NE compared with PBNs from control subjects and individuals with bronchiectasis . These results indicate that CF PBNs respond abnormally to TNF-alpha and IL-8 in CF BAL fluid and react to opsonized bacteria by releasing more NE . This may help explain the increased NE burden seen in this condition. Microb Pathog, 2000 Feb, 28(2), 71 - 80 The 120 kDa outer membrane protein of Ehrlichia chaffeensis: preferential expression on dense-core cells and gene expression in Escherichia coli associated with attachment and entry; Popov VL et al.; The immunodominant 120 kDa protein (p120) of Ehrlichia chaffeensis was demonstrated to be exposed on the surface of purified whole ehrlichial cells examined by immunoelectron microscopy with a rabbit antibody against a portion of the domain containing tandem repeat units . In the intracellular location, the 120 kDa protein was detected by immunoelectron microscopy in the outer membrane of the cell wall of dense-core forms of the ehrlichiae in infected canine macrophage-like cells and as a component of the intramorular fibrillary matrix . No 120 kDa protein was detected in the cell wall of ehrlichial reticulate cells . Recombinant Escherichia coli with a plasmid containing the entire 120 kDa protein gene, but no bacteria with non-recombinant plasmid, attached to the surface of HeLa cells as visualized by electron microscopy . Some of the recombinant 120 kDa protein expressing E . coli invaded the HeLa cells as determined by gentamicin protection assays and by intravacuolar localization ultrastructurally . J Virol Methods, 2000 Jan, 84(1), 59 - 63 A strategy for rapid cDNA cloning from double-stranded RNA templates isolated from plants infected with RNA viruses by using Taq DNA polymerase; Zhang YP et al.; A fast and efficient cDNA cloning procedure for plant RNA viruses was developed . In this procedure, double-stranded RNA (dsRNA) was used as a template source . Standard cDNA synthesis reagents and random hexamers were then used for making cDNAs . Taq DNA polymerase was used to add additional (A) at the ends of cDNAs, a TA cloning kit to ligate the cDNAs to vectors, and an electroporator to transform the DNAs to E . coli cells . dsRNAs were extracted from grapevine tissues infected with four different viruses and used for cloning . These viruses included grapevine rupestris stem pitting associated virus, grapevine leafroll associated virus 5, and two uncharacterized grapevine viruses, one each closely related to marafivirus and vitivirus groups . Selected cDNA clones were sequenced and PCR primers were developed for RT-PCR detection of these viruses in host plants. J Food Prot, 2000 Jan, 63(1), 126 - 30 Comparison of membrane filtration rates and hydrophobic grid membrane filter coliform and Escherichia coli counts in food suspensions using paddle-type and pulsifier sample preparation procedures; Sharpe AN et al.; Food suspensions prepared by Pulsifier contained less debris and filtered 1.3x to 12x faster through hydrophobic grid membrane filters (HGMFs) than those prepared by Stomacher 400 . Coliform and Escherichia coli counts made by an HGMF method yielded 84 and 36 paired samples, respectively, positive by both suspending methods . Overall counts of pulsificates and stomachates did not differ significantly for either analysis, though coliform counts by Pulsifier were significantly higher in mushrooms and significantly lower in ground pork (P = 0.05) . Regression equations for log10 counts of coliform and E . coli by Pulsifier and Stomacher were: Pulsifier = 0.12 + 0.97 x Stomacher, and Pulsifier = 0.01 + 1.01 x Stomacher, respectively. Mikrobiol Z, 1999 Sep-Oct, 61(5), 28 - 32 {The use of gamma-interferon for the prevention and treatment of infectious diseases in piglets and calves}; Kishko IaH et al.; Administration of the homologous natural gamma-interferon for prophylaxis of infectious diseases showed that 2-3 injections of gamma-interferon prevented infectious morbidity of more than 90% of sucking pigs and new-born calves, sharply increased their preservation to 90% and above, increased their productivity by 1/3 . In conditions of industrial growing of young animals therapy by this preparation of pigs and calves with colibacteriosis and parainfluenza in comparison with control significantly decreased mortality of morbid young animals, accelerated their recovery and promoted the weight gain in pigs and calves. Glycobiology, 2000 Feb, 10(2), 159 - 71 Biosynthesis of heparin/heparan sulfate: kinetic studies of the glucuronyl C5-epimerase with N-sulfated derivatives of the Escherichia coli K5 capsular polysaccharide as substrates; Hagner-McWhirter A et al.; The D-glucuronyl C5-epimerase involved in the biosynthesis of heparin and heparan sulfate was investigated with focus on its substrate specificity, its kinetic properties, and a comparison of epimerase preparations from the Furth mastocytoma and bovine liver, which synthesize heparin and heparan sulfate, respectively . New substrates for the epimerase were prepared from the capsular polysaccharide of Escherichia coli K5, which had been labeled at C5 of its D-glucuronic and N-acetyl-D-glucosamine moieties by growing the bacteria in the presence of D-{5-(3)H}glucose . Following complete or partial ( approximately 50%) N-deacetylation of the polysaccharide by hydrazinolysis, the free amino groups were sulfated by treatment with trimethylamine.SO(3)complex, which yielded products that were recognized as substrates by the epimerase and released tritium from C5 of the D-glucuronyl residues upon incubation with the enzyme . Comparison of the kinetic properties of the two substrates showed that the fully N-sulfated derivative was the best substrate in terms of its K(m)value, which was significantly lower than that of its partially N-acetylated counterpart . The V(max)values for the E.coli polysaccharide derivatives were essentially the same but were both lower than that of the O-desulfated {(3)H}heparin used in our previous studies . Surprisingly, the apparent K(m)values for all three substrates increased with increasing enzyme concentration . The reason for this phenomenon is not entirely clear at present . Partially purified C5-epimerase preparations from the Furth mastocytoma and bovine liver, respectively, behaved similarly in terms of their reactivity towards the various substrates, but the variation in apparent K(m)values with enzyme concentration precluded a detailed comparison of their kinetic properties. Glycobiology, 2000 Feb, 10(2), 121 - 30 Characterization of high affinity monoclonal antibodies specific for chlamydial lipopolysaccharide; Muller-Loennies S et al.; Pathogens belonging to the genus Chlamydia contain lipopolysaccharide with a 3-deoxy-D- manno- oct-2-ulosonic acid (Kdo) trisaccharide of the sequence alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo . This lipopolysaccharide is recognized in a genus-specific pattern by murine monoclonal antibodies (mAbs), S25-23 and S25-2 (both IgG1kappa), which bind as the minimal structures the trisaccharide and the terminal Kdo-disaccharide, respectively . The variable domains of these mAbs were reverse transcribed from mRNA which was isolated from hybridomas and cloned as single-chain variable fragments (scFvs) in E.coli TG1 . The kinetics of binding of whole antibodies, Fab fragments and scFvs to natural and synthetically modified ligands were determined by surface plasmon resonance (SPR) using synthetic neoglycoconjugates . As examples of an antibody-carbohydrate interaction involving anionic carboxyl groups on the ligand, we report that the affinities of these antibodies are higher than usually observed in carbo-hydrate-protein interactions (K(D)of 10(-3)to 10(-5)M) . SPR analy-ses of monovalent Fab and scFv binding to the natural trisaccharide epitope gave dissociation constants of 770 nM for S25-2 and 350 nM for S25-23, as determined by global fitting (simultaneous fitting of several measurements at different antibody concentrations) of sensorgram data to a one-to-one interaction model . Local fitting (separate fitting of individual sensorgram data at different antibody concentrations) and Scatchard analysis of the data gave kinetic and affinity constants that were in good agreement with those obtained by global fitting . The SPR data also showed that while S25-2 bound well to several Kdo disaccharides and carboxyl-reduced Kdo ligands, S25-23 did not . Identification of amino acids in the complementarity determining regions revealed the presence of a large number of positively charged amino acids which were located towards the center of the combining site, thus suggesting a different recognition mechanism than that observed for neutral ligands . The latter mainly involves aromatic amino acids for hydrophobic stacking inter-actions and hydrogen bonds. Biol Reprod, 2000 Feb, 62(2), 390 - 7 Characterization of ovarian carbonyl reductase gene expression during ovulation in the gonadotropin-primed immature Rat; Espey LL et al.; In this differential-display polymerase chain reaction-based study, four different primer sets generated cDNA fragments of ovarian carbonyl reductase genes that were uniquely expressed during the ovulatory process in eCG-primed immature rats . The temporal pattern of expression of this aldo-keto reductase gene was delineated by extracting ovarian RNA at 0, 2, 4, 8, 12, and 24 h after induction of ovulation via injection of the primed animals with hCG . The results showed that at least four homologous forms of this gene were transcribed during ovulation . Northern blot analyses indicated a 14-fold increase in ovarian mRNA for carbonyl reductase, with expression reaching a peak at 8 h after hCG treatment and then declining to negligible levels during the next 16 h . In situ hybridization revealed that most of the transcription was in the thecal connective tissue of the ovary and was absent from the granulosa layer of ovarian follicles . Treatment of the animals with ovulation-blocking doses of epostane (an inhibitor of progesterone synthesis) or indomethacin (an inhibitor of prostanoid synthesis) did not reduce the expression of ovarian carbonyl reductase . Nevertheless, the temporal pattern of expression of carbonyl reductase after the induction of ovulation suggests that this enzyme activity is at least indirectly associated with the ovulatory process. J Virol, 2000 Feb, 74(4), 1658 - 62 Mechanism of assembly of recombinant murine polyomavirus-like particles; Schmidt U et al.; VP1 is the major viral coat protein of murine polyomavirus and can be used for the generation of virus-like particles in vitro . Here, we demonstrate that capsid assembly is an equilibrium reaction followed by oxidation of intracapsomere disulfide bonds, which are not essential for the formation of virus-like particles but enable complete particle assembly and prevent capsid disassembly. Science, 2000 Jan 21, 287(5452), 479 - 82 One polypeptide with two aminoacyl-tRNA synthetase activities; Stathopoulos C et al.; The genome sequences of certain archaea do not contain recognizable cysteinyl-transfer RNA (tRNA) synthetases, which are essential for messenger RNA-encoded protein synthesis . However, a single cysteinyl-tRNA synthetase activity was detected and purified from one such organism, Methanococcus jannaschii . The amino-terminal sequence of this protein corresponded to the predicted sequence of prolyl-tRNA synthetase . Biochemical and genetic analyses indicated that this archaeal form of prolyl-tRNA synthetase can synthesize both cysteinyl-tRNA(Cys) and prolyl-tRNA(Pro) . The ability of one enzyme to provide two aminoacyl-tRNAs for protein synthesis raises questions about concepts of substrate specificity in protein synthesis and may provide insights into the evolutionary origins of this process. Biochemistry, 2000 Jan 25, 39(3), 575 - 83 Aggregation events occur prior to stable intermediate formation during refolding of interleukin 1beta; Finke JM et al.; A point mutation, lysine 97 --> isoleucine (K97I), in a surface loop in the beta-sheet protein interleukin 1beta (IL-1beta), exhibits increased levels of inclusion body (IB) formation relative to the wild-type protein (WT) when expressed in Escherichia coli . Despite the common observation that less stable proteins are often found in IBs, K97I is more stable than WT . We examined the folding pathway of the mutant and wild-type proteins at pH 6.5 and 25 degrees C with manual-mixing and stopped-flow optical spectroscopy to determine whether changes in the properties of transiently populated species in vitro correlate with the observation of increased aggregation in vivo . The refolding reactions of the WT and K97I proteins are both described by three exponential processes . Two exponential processes characterize fast events (0.1-1.0 s) in folding while the third exponential process correlates with a slow (70 s) single pathway to and from the native state . The K97I replacement affects the earlier steps in the refolding pathway . Aggregation, absent in the WT refolding reaction, occurs in K97I above a critical protein concentration of 18 microM . This observation is consistent with an initial nucleation step mediating protein aggregation . Stopped-flow kinetic studies of the K97I aggregation process demonstrate that K97I aggregates most rapidly during the earliest refolding times, when unfolded protein conformers remain highly populated and the concentration of folding intermediates is low . Folding and aggregation studies together support a model in which the formation of stable folding intermediates afford protection against further K97I aggregation. Biochemistry, 2000 Jan 25, 39(3), 516 - 28 Mechanism of 8-amino-7-oxononanoate synthase: spectroscopic, kinetic, and crystallographic studies; Webster SP et al.; 8-Amino-7-oxononanoate synthase (also known as 7-keto-8-aminopelargonate synthase, EC 2.3.1.47) is a pyridoxal 5'-phosphate-dependent enzyme which catalyzes the decarboxylative condensation of L-alanine with pimeloyl-CoA in a stereospecific manner to form 8(S)-amino-7-oxononanoate . This is the first committed step in biotin biosynthesis . The mechanism of Escherichia coli AONS has been investigated by spectroscopic, kinetic, and crystallographic techniques . The X-ray structure of the holoenzyme has been refined at a resolution of 1.7 A (R = 18.6%, R(free) = 21 . 2%) and shows that the plane of the imine bond of the internal aldimine deviates from the pyridine plane . The structure of the enzyme-product external aldimine complex has been refined at a resolution of 2.0 A (R = 21.2%, R(free) = 27.8%) and shows a rotation of the pyridine ring with respect to that in the internal aldimine, together with a significant conformational change of the C-terminal domain and subtle rearrangement of the active site hydrogen bonding . The first step in the reaction, L-alanine external aldimine formation, is rapid (k(1) = 2 x 10(4) M(-)(1) s(-)(1)) . Formation of an external aldimine with D-alanine, which is not a substrate, is significantly slower (k(1) = 125 M(-)(1) s(-)(1)) . Binding of D-alanine to AONS is enhanced approximately 2-fold in the presence of pimeloyl-CoA . Significant substrate quinonoid formation only occurs upon addition of pimeloyl-CoA to the preformed L-alanine external aldimine complex and is preceded by a distinct lag phase ( approximately 30 ms) which suggests that binding of the pimeloyl-CoA causes a conformational transition of the enzyme external aldimine complex . This transition, which is inferred by modeling to require a rotation around the Calpha-N bond of the external aldimine complex, promotes abstraction of the Calpha proton by Lys236 . These results have been combined to form a detailed mechanistic pathway for AONS catalysis which may be applied to the other members of the alpha-oxoamine synthase subfamily. Cancer, 2000 Jan 15, 88(2), 454 - 60 Short term treatment with Escherichia coli recombinant human granulocyte-macrophage-colony stimulating factor prior to chemotherapy for Hodgkin disease; Aglietta M et al.; BACKGROUND: Granulocyte-macrophage-colony stimulating factor (GM-CSF) administration stimulates the proliferation of hemopoietic progenitors . Shortly (48-96 hours) after its discontinuation, feedback phenomena occur and the progenitor proliferation rate drops below baseline levels . As the quiescence of hyperplastic bone marrow suggests that hemopoietic cells may be refractory to the toxic effects of cytostatic drugs, the decision was made to test the hypothesis that GM-CSF given before chemotherapy may be myeloprotective . METHODS: Fifty-six patients with newly diagnosed Stage II-IV Hodgkin disease, ages 18-77 years, were randomized to receive GM-CSF (5 microg/kg subcutaneously) or placebo from Day 7 to Day 4 before each chemotherapy administration (6 cycles of a hybrid of mechlorethamine, vincristine, procarbazine, and prednisone with doxorubicin, bleomycin, vinblastine, and dacarbazine) . The treatment was considered a success if the delivery rate of chemotherapy was >90% after 3 cycles and >80% after 6 cycles . RESULTS: Thirty patients received GM-CSF and 26 placebo . The dose intensity (85.2% vs . 79.6%) and the overall success in terms of delivery rate (56.7% vs . 50%) were higher in the GM-CSF group, although these differences were not statistically significant . The neutrophil nadirs were higher in the GM-CSF group during the first three cycles and subsequently similar in both groups . CONCLUSIONS: No significant differences in terms of myelotoxicity or drug delivery were observed between the two treatment arms . Although the myeloprotective effect of the prechemotherapy administration of GM-CSF seems to be minimal, the data indicate a safe timing between GM-CSF discontinuation and further chemotherapy . Because cumulative myelotoxicity has been observed with other growth factors, given in the interval between the chemotherapy cycles, this may be relevant to the planning of rapid cycling . Brain Res Mol Brain Res, 1999 Dec 10, 74(1-2), 126 - 34 Interaction of PKN with a neuron-specific basic helix-loop-helix transcription factor, NDRF/NeuroD2; Shibata H et al.; By the yeast two-hybrid screening of a human brain cDNA library with the amino-terminal regulatory region of PKN as a bait, a clone encoding a neuron-specific basic Helix-Loop-Helix (bHLH) transcription factor, NDRF/NeuroD2 was isolated . NDRF/NeuroD2 was co-precipitated with PKN from the lysate of COS-7 cells transfected with both expression constructs for NDRF/NeuroD2 and PKN . In vitro binding studies using the deletion mutants of NDRF/NeuroD2 synthesized in a rabbit reticulocyte lysate indicated that the internal region containing the bHLH domain of NDRF/NeuroD2 was necessary and sufficient for the interaction with PKN . In addition, recombinant NDRF/NeuroD2 purified from Escherichia coli could bind PKN, suggesting the direct interaction between NDRF/NeuroD2 and PKN . Transient transfection assays using P19 cells revealed that expression of NDRF/NeuroD2 increased the transactivation of the rat insulin promoter element 3 (RIPE3) enhancer up to approximately 12-fold and that co-expression of catalytically active form of PKN, but not kinase-deficient derivative, resulted in a further threefold increase of NDRF/NeuroD2-mediated transcription . These findings suggest that PKN may contribute to transcriptional responses through the post-translational modification of the NDRF/NeuroD2-dependent transcriptional machinery. J Control Release, 2000 Feb 14, 64(1-3), 155 - 66 DepoFoam technology: a vehicle for controlled delivery of protein and peptide drugs; Ye Q et al.; A major challenge in the development of sustained-release formulations for protein and peptide drugs is to achieve high drug loading sufficient for prolonged therapeutic effect coupled with a high recovery of the protein/peptide . This challenge has been successfully met in the formulation of several peptide and protein drugs using the DepoFoam, multivesicular lipid-based drug delivery system . DepoFoam technology consists of novel multivesicular liposomes characterized by their unique structure of multiple non-concentric aqueous chambers surrounded by a network of lipid membranes . The objective of this paper is to demonstrate that DepoFoam technology can be used to develop sustained-release formulations of therapeutic proteins and peptides with high loading . DepoFoam formulations of a protein such as insulin, and peptides such as leuprolide, enkephalin and octreotide have been developed and characterized . The data show that these formulations have high drug loading, high encapsulation efficiency, low content of free drug in the suspension, little chemical change in the drug caused by the formulation process, narrow particle size distribution, and spherical particle morphology . Drug release assays conducted in vitro in biological suspending media such as human plasma indicate that these formulations provide sustained release of encapsulated drug over a period from a few days to several weeks, and that the rate of release can be modulated . In vivo pharmacodynamic studies in rats also show a sustained therapeutic effect over a prolonged period . These results demonstrate that the DepoFoam system is capable of efficiently encapsulating therapeutic proteins and peptides and effectively providing controlled delivery of these biologically active macromolecules. J Gen Virol, 2000 Jan, 81(Pt 1), 181 - 8 High affinity interaction between nucleocapsid protein and leader/intergenic sequence of mouse hepatitis virus RNA; Nelson GW et al.; The nucleocapsid (N) protein of mouse hepatitis virus (MHV) is the major virion structural protein . It associates with both viral genomic RNA and subgenomic mRNAs and has structural and non-structural roles in replication including viral RNA-dependent RNA transcription, genome replication, encapsidation and translation . These processes all involve RNA-protein interactions between the N protein and viral RNAs . To better understand the RNA-binding properties of this multifunctional protein, the N protein was expressed in Escherichia coli as a chimeric protein fused to glutathione-S-transferase (GST) . Biochemical analyses of RNA-binding properties were performed on full-length and partial N protein segments to define the RNA-binding domain . The full-length N protein and the GST-N protein fusion product had similar binding activities with a dissociation constant (K(d)) of 14 nM when the MHV 5'-leader sequence was used as ligand . The smallest N protein fragment which retained RNA-binding activity was a 55 aa segment containing residues 177-231 which bound viral RNA with a K(d) of 32 nM . A consensus viral sequence recognized by the N protein was inferred from these studies; AAUCYAAAC was identified to be the potential minimum ligand for the N protein . Although the core UCYAA sequence is often tandemly repeated in viral genomes, ligands containing one or more repeats of UCYAA showed no difference in binding to the N protein . Together these data demonstrate a high-affinity, specific interaction between the N protein and a conserved RNA sequence present at the 5'-ends of MHV mRNA. J Gen Virol, 2000 Jan, 81(Pt 1), 59 - 65 Characterization of the african swine fever virus protein p49: a new late structural polypeptide; Galindo I et al.; The open reading frame B438L, located within the EcoRI B fragment of the African swine fever virus genome, is predicted to encode a protein of 438 amino acids with a molecular mass of 49.3 kDa . It presents a cell attachment RGD (Arg-Gly-Asp) motif but no other significant similarity to protein sequences in databases . Northern blot and primer extension analysis showed that B438L is transcribed only at late times during virus infection . The B438L gene product has been expressed in Escherichia coli, purified and used as an antigen for antibody production . The rabbit antiserum specific for pB438L recognized a protein of about 49 kDa in virus-infected cell extracts . This protein was synthesized late in infection by all the virus strains tested, was located in cytoplasmic virus factories and appeared as a structural component of purified virus particles. Arch Biochem Biophys, 2000 Feb 1, 374(1), 73 - 8 Slow-binding inhibition of branching enzyme by the pseudooligosaccharide BAY e4609; Binderup K et al.; Branching enzyme from Escherichia coli is shown to be inhibited by the pseudooligosaccharide BAY e4609 . The mechanism of binding is studied in detail by kinetics using reduced amylose as substrate . Lineweaver-Burk plots suggest the mechanism of a noncompetitive or slow-binding inhibitor . Further studies by progress curves and rate of loss of branching activity allows us to conclude BAY e4609 as being a slow-binding inhibitor of branching enzyme . We discuss how these results parallel the inhibition of alpha-amylase by acarbose and the significance of branching enzyme as belonging to the amylolytic family . Anal Biochem, 2000 Feb 1, 278(1), 39 - 45 A high-throughput assay to identify compounds that can induce dimerization of the erythropoietin receptor; Biazzo DE et al.; Erythropoietin induces dimerization of the erythropoietin receptor on the surface of erythroid progenitor cells, promoting the differentiation of these cells into mature red blood cells . To facilitate screening of large chemical collections for identification of compounds that can dimerize erythropoietin receptor, we have developed a novel, high-throughput in vitro assay to detect compounds that can cause dimerization of the erythropoietin receptor in solution . To develop this assay, amino acid sequences corresponding to the extracellular domain of erythropoietin receptor were expressed in Escherichia coli as erythropoietin-binding protein (rEBP) . A modified version of this protein ((33)P-rEBP) containing a protein kinase A substrate site incorporated into the rEBP was also expressed in E . coli and labeled in vitro using protein kinase A and inverted question markgamma-(33)PATP . An erythropoietin mimetic peptide (EMP-1), that induces dimerization of rEBP in solution was used to demonstrate dimerization of (33)P-rEBP and rEBP in a 96-well microtiter plate format . EMP-1 induced dimerization of rEBP in this assay with an EC(50) of approximately 245 nM and had a maximal effect at 0.5-2 microM and required the presence of rEBP immobilized on the plate capable of binding EMP-1 . EMP-1-induced dimerization of (33)P-rEBP and rEBP was reversed by excess unlabeled rEBP and was not masked by complex mixtures such as whole cell fungal extracts . These data demonstrate the ability of (33)P-rEBP to dimerize with rEBP in vitro in a format that is fully compatible with high-throughput screening . Exp Hematol, 1999 Dec, 27(12), 1746 - 56 Use of combinatorial mutagenesis to select for multiply substituted human interleukin-3 variants with improved pharmacologic properties; Klein BK et al.; A combinatorial mutagenesis strategy was used to create a collection of nearly 500 variants of human interleukin 3 (IL-3), each with four to nine amino acid substitutions clustered within four linear, nonoverlapping regions of the polypeptide . The variants were secreted into the periplasm of Escherichia coli and supernatants were assayed for IL-3 receptor-dependent cell proliferation activity . Sixteen percent of the variants, containing "region-restricted" substitutions, retained substantial proliferative activity through two rounds of screening . A subset of these was combined to yield variants with substitutions distributed through approximately half of the polypeptide . With one exception, "half-substituted" variants exhibited proliferative activity within 3.5-fold of native IL-3 . A subset of the "half-substituted" variants was combined to yield "fully substituted" IL-3 variants having 27 or more substitutions . The combination of the substitutions resulted in a set of polypeptides, some of which exhibit increased proliferative activity relative to native IL-3 . The elevated hematopoietic potency was confirmed in a methylcellulose colony-forming unit assay using freshly isolated human bone marrow cells . A subset of the multiply substituted proteins exhibited only a modest increase in inflammatory mediator (leukotriene C4) release . The molecules also exhibited 40- to 100-fold greater affinity for the alpha subunit of the IL-3 receptor and demonstrated a 10-fold faster association rate with the alpha-receptor subunit . The multiply substituted IL-3 variants described in this study provide a unique collection of molecules from which candidates for clinical evaluation may be defined and selected. Acta Vet Hung, 1999, 47(4), 481 - 92 Induction of protective immunity in chickens immunised with plasmid DNA encoding infectious bursal disease virus antigens; Fodor I et al.; Direct DNA inoculations were used to determine the efficacy of gene immunisation of chickens to elicit protective immune responses against infectious bursal disease virus (IBDV) . The vp2 gene of IBDV strains GP40 and D78, and the vp2-vp4-vp3 encoding segment of strain D78 were cloned in an expression vector which consisted of human cytomegalovirus (HCMV) immediate early enhancer and promoter, adenovirus tripartite leader sequences and SV40 polyadenylation signal . For purification of vaccine-quality plasmid DNA from E . coli, an effective method was developed . Chickens were vaccinated by inoculation of DNA by two routes (intramuscular and intraperitoneal) . Two weeks later, chickens were boosted with DNA, and at 2 weeks post-boost, they were challenged with virulent IBDV strain . Low to undetectable levels of IBDV-specific antibodies and no protection were observed with DNA encoding VP2 . However, plasmids encoding VP2-VP4-VP3 induced IBDV-specific antibodies and protection in the chickens . DNA immunisation opens a new approach to the development of gene vaccines for chickens against infectious diseases. Indian J Pathol Microbiol, 1999 Apr, 42(2), 135 - 43 Cloning and expression of a cell wall associated protein gene of Mycobacterium tuberculosis; Mohamed AJ et al.; Genomic library of Mycobacterium tuberculosis (SIHV) was constructed into T7 promoter based vector pBluescript II KS (+/-) in Escherichia coil . The expression of mycobacterial antigens from recombinants was detected by immunoscreening using E-coli-preabsorbed hyperimmune rabbit anti-M tuberculosis cell wall associated proteins serum . Among 980 clones tested by immunoscreening, a clone pJJ5 was found to produce mycobacterial antigen . This clone was further characterized by immunoblotting and found to produce a 36 kDa of cell wall associated antigen Detection of antibodies response to this antigen in the serum of the majority of the individuals with tuberculosis indicates that this antigen is capable of stimulating a humoral immune response. Int J Oncol, 2000 Feb, 16(2), 267 - 76 Cancer immunomodulation by carbohydrate ligands for the lymphocyte receptor NKR-P1; Pospisil M et al.; Natural killer (NK) and T cells express a superfamily of proteins with structural features of C-type lectins . Recombinantly prepared soluble form of rat NKR-P1 (CD161) recognized carbohydrate GalNac and GlcNac moieties . Ganglioside GM2 and heparin related-IS oligosaccharides representing the high affinity ligands for this receptor, increased the sensitivity of targets for killing by the rat effectors isolated from blood and spleen in vitro . Based on these results, we investigated in vivo the possible therapeutic effect of GM2 and IS carried by liposomes during induced rat colorectal carcinogenesis . The reduction of cancer incidence versus the controls (50% vs 88.88%), approached the 5-fluorouracil treatment (41.66%). Virology, 2000 Jan 20, 266(2), 264 - 74 Mutagenesis of murine cytomegalovirus using a Tn3-based transposon; Zhan X et al.; A transposon derived from Escherichia coli Tn3 was introduced into the genome of murine cytomegalovirus (MCMV) to generate a pool of viral mutants . We analyzed three of the constructed recombinant viruses that contained the transposon within the M25, M27, and m155 open reading frames . Our studies provide the first direct evidence to suggest that M25 and M27 are not essential for viral replication in mouse NIH 3T3 cells . Studies in cultured cells and Balb/c mice indicated that the transposon insertion is stable during viral propagation both in vitro and in vivo . Moreover the virus that contained the insertion mutation in M25 exhibited a titer similar to that of the wild-type virus in the salivary glands, lungs, livers, spleens, and kidneys of the Balb/c mice that were intraperitoneally infected with these viruses . These results suggest that M25 is dispensable for viral growth in these organs and the presence of the transposon sequence in the viral genome does not significantly affect viral replication in vivo . The Tn3-based system can be used as a mutagenesis approach for studying the function of MCMV genes in both tissue culture and in animals . Infect Immun, 2000 Feb, 68(2), 871 - 6 GAA trinucleotide repeat region regulates M9/pMGA gene expression in Mycoplasma gallisepticum; Liu L et al.; Mycoplasma gallisepticum, the cause of chronic respiratory infections in the avian host, possesses a family of M9/pMGA genes encoding an adhesin(s) associated with hemagglutination . Nucleotide sequences of M9/pMGA gene family members indicate extensive sequence similarity in the promoter regions of both the transcribed and silent genes . The mechanism that regulates M9/pMGA gene expression is unknown, but studies have revealed an apparent correlation between gene expression and the number of tandem GAA repeat motifs located upstream of the putative promoter . In this study, transposon Tn4001 was used as a vector with the Escherichia coli lacZ gene as the reporter system to examine the role of the GAA repeats in M9/pMGA gene expression in M . gallisepticum . A 336-bp M9 gene fragment (containing the GAA repeat region, the promoter, and the translation start codon) was amplified by PCR, ligated with a lacZ gene from E . coli, and inserted into the Tn4001-containing plasmid pISM2062 . This construct was transformed into M . gallisepticum PG31 . Transformants were filter cloned on agar supplemented with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to monitor lacZ gene expression on the basis of blue/white color selection . Several cycles of filter cloning resulted in cell lineages in which lacZ gene expression alternated between the On and Off states in successive generations of progeny clones . The promoter regions of the M9-lacZ hybrid genes of individual progeny clones were amplified by PCR and sequenced . The only differences between the promoter regions of the blue and white colonies were in the number of GAA repeats . Clones that expressed lacZ had exactly 12 tandem copies of the GAA repeat . Clones that did not express lacZ invariably had either more than 12 (14 to 16) or fewer than 12 (5 to 11) GAA repeats . Southern analysis of M . gallisepticum chromosomal DNA confirmed that the phase-variable expression of the lacZ reporter gene was not caused by Tn4001 transposition . These data strongly indicate that changes in the length of the GAA repeat region are responsible for regulating M9/pMGA gene expression. Infect Immun, 2000 Feb, 68(2), 809 - 14 Priming of a beta-galactosidase (beta-GAL)-specific type 1 response in BALB/c mice infected with beta-GAL-transfected Leishmania major; Chakkalath HR et al.; To determine whether an ongoing response to Leishmania major would affect the response to a non-cross-reacting, non-leishmanial antigen, susceptible BALB/c mice and resistant C3H mice were infected with L . major parasites expressing Escherichia coli beta-galactosidase (beta-GAL); this parasite was designated L . major-betaGAL . BALB/c and C3H mice responded to infection with L . major-betaGAL by mounting a CD4 T-cell response to both parasite antigens and to the reporter antigen, beta-GAL . The phenotypes of these T cells were characterized after generating T-cell lines from infected mice . As expected, BALB/c mice responded to infection with L . major-betaGAL by producing interleukin 4 in response to the parasite and C3H mice produced gamma interferon (IFN-gamma) in response to the parasite and beta-GAL . Interestingly, however, BALB/c mice produced IFN-gamma in response to beta-GAL . Taken together, these results demonstrate that priming of IFN-gamma-producing cells can occur in BALB/c mice despite the fact the animals are simultaneously mounting a potent Th2 response to L . major. Infect Immun, 2000 Feb, 68(2), 716 - 24 Enzymatic properties of dipeptidyl aminopeptidase IV produced by the periodontal pathogen Porphyromonas gingivalis and its participation in virulence; Kumagai Y et al.; Porphyromonas gingivalis is a major pathogen associated with adult periodontitis . We cloned and sequenced the gene (dpp) coding for dipeptidyl aminopeptidase IV (DPPIV) from P . gingivalis W83, based on the amino acid sequences of peptide fragments derived from purified DPPIV . An Escherichia coli strain overproducing P . gingivalis DPPIV was constructed . The enzymatic properties of recombinant DPPIV purified from the overproducer were similar to those of DPPIV isolated from P . gingivalis . The three amino acid residues Ser, Asp, and His, which are thought to form a catalytic triad in the C-terminal catalytic domain of eukaryotic DPPIV, are conserved in P . gingivalis DPPIV . When each of the corresponding residues of the enzyme was substituted with Ala by site-directed mutagenesis, DPPIV activity significantly decreased, suggesting that these three residues of P . gingivalis DPPIV are involved in the catalytic reaction . DPPIV-deficient mutants of P . gingivalis were constructed and subjected to animal experiments . Mice injected with the wild-type strain developed abscesses to a greater extent and died more frequently than those challenged with mutant strains . Mice injected with the mutants exhibited faster recovery from the infection, as assessed by weight gain and the rate of lesion healing . This decreased virulence of mutants compared with the parent strain suggests that DPPIV is a potential virulence factor of P . gingivalis and may play important roles in the pathogenesis of adult periodontitis induced by the organism. Infect Immun, 2000 Feb, 68(2), 564 - 9 Isolation, affinity purification, and identification of piglet small intestine mucosa receptor for enterotoxigenic Escherichia coli k88ac+ fimbriae; Fang L et al.; An affinity chromatography technique was utilized to isolate and purify the receptors of Escherichia coli K88ac(+) fimbriae from the mucus of the small intestines of newborn piglets . Purified K88ac+ fimbriae were covalently immobilized onto a beaded agarose matrix (Sepharose 4B) . The immobilized fimbriae were used for the affinity purification of the K88ac+ receptors . Only two major proteins were tightly and specifically bound to the immobilized fimbriae after the column containing bound receptor was washed exhaustively with a buffer containing a high concentration of salt and a detergent . The receptors were eluted as a single component at a low pH . The isolated proteins were then subjected to enzyme-linked immunosorbent assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot (immunoblot) analyses . The two proteins were of high purity, were responsible for nearly all of the fimbrial binding capacity of the crude mucus, and had molecular masses of 26 and 41 kDa . The method for isolation of E . coli binding proteins is simple and yields purified intestinal receptors in a single chromatographic run . The intestinal mucus of different piglets has different proportions of the two receptor proteins. Proc Natl Acad Sci U S A, 2000 Jan 18, 97(2), 783 - 6 Drug target validation: lethal infection blocked by inducible peptide; Tao J et al.; Genome projects are generating large numbers of potential new targets for drug discovery . One challenge is target validation, proving the usefulness of a specific target in an animal model . In this paper, we demonstrate a new approach to validation and assay development . We selected in vitro specific peptide binders to a potential pathogen target . By inducing the expression of a selected peptide in pathogen cells causing a lethal infection in mice, the animals were rescued . Thus, by combining in vitro selection methods for peptide binders with inducible expression in animals, the target's validity was rigorously tested and demonstrated . This approach to validation can be generalized and has the potential to become a valuable tool in the drug discovery process. Proc Natl Acad Sci U S A, 2000 Jan 18, 97(2), 646 - 51 RNA folding energy landscapes; Chen SJ et al.; Using a statistical mechanical treatment, we study RNA folding energy landscapes . We first validate the theory by showing that, for the RNA molecules we tested having only secondary structures, this treatment (i) predicts about the same native structures as the Zuker method, and (ii) qualitatively predicts the melting curve peaks and shoulders seen in experiments . We then predict thermodynamic folding intermediates . For one hairpin sequence, unfolding is a simple unzipping process . But for another sequence, unfolding is more complex . It involves multiple stable intermediates and a rezipping into a completely non-native conformation before unfolding . The principle that emerges, for which there is growing experimental support, is that although protein folding tends to involve highly cooperative two-state thermodynamic transitions, without detectable intermediates, the folding of RNA secondary structures may involve rugged landscapes, often with more complex intermediate states. Proc Natl Acad Sci U S A, 2000 Jan 18, 97(2), 605 - 10 In vivo selection of functional ribosomes with variations in the rRNA-binding site of Escherichia coli ribosomal protein S8: evolutionary implications; Moine H et al.; The highly conserved nature of rRNA sequences throughout evolution allows these molecules to be used to build philogenic trees of different species . It is unknown whether the stability of specific interactions and structural features of rRNA reflects an optimal adaptation to a functional task or an evolutionary trap . In the work reported here, we have applied an in vivo selection strategy to demonstrate that unnatural sequences do work as a functional replacement of the highly conserved binding site of ribosomal protein S8 . However, growth competition experiments performed between Escherichia coli isolates containing natural and unnatural S8-binding sites showed that the fate of each isolate depended on the growth condition . In exponentially growing cells, one unnatural variant was found to be equivalent to wild type in competition experiments performed in rich media . In culture conditions leading to slow growth, however, cells containing the wild-type sequence were the ultimate winner of the competition, emphasizing that the wild-type sequence is, in fact, the most fit solution for the S8-binding site. Proc Natl Acad Sci U S A, 2000 Jan 18, 97(2), 583 - 7 Molecular cloning of a 10-deacetylbaccatin III-10-O-acetyl transferase cDNA from Taxus and functional expression in Escherichia coli; Walker K et al.; The cDNA clone for a 10-deacetylbaccatin III-10-O-acetyl transferase, which catalyzes formation of the last diterpene intermediate in the Taxol biosynthetic pathway, has been isolated from Taxus cuspidata . By using consensus sequences from an assembly of transacylases of plant origin and from many deduced proteins of unknown function, a homology-based PCR cloning strategy was employed to amplify initially a 911-bp gene fragment of the putative taxane C-10 hydroxyl acetyl transferase from Taxus . This amplicon was used to screen a cDNA library constructed from mRNA isolated from methyl jasmonate-induced Taxus cells, from which the full-length 10-deacetylbaccatin III-10-O-transacetylase sequence was obtained . Expression of the ORF from pCWori(+) in Escherichia coli JM109 afforded a functional enzyme, as determined by (1)H-NMR and MS verification of the product baccatin III derived from 10-deacetylbaccatin III and acetyl CoA . The full-length cDNA has an ORF of 1,320 bp corresponding to a deduced protein of 440 residues with a calculated molecular weight of 49,052, consistent with the size of the operationally soluble, monomeric, native acetyl transferase . The recombinant acetyl transferase has a pH optimum of 7.5, has K(m) values of 10 microM and 8 microM for 10-deacetylbaccatin III and acetyl CoA, respectively, and is apparently regiospecific toward the 10-hydroxyl group of the taxane ring . Amino acid sequence comparison of 10-deacetylbaccatin III-10-O-acetyl transferase with taxadienol-5-O-acetyl transferase and with other known acyl transferases of plant origin indicates a significant degree of similarity between these enzymes (80% and 64-67%, respectively). Proc Natl Acad Sci U S A, 2000 Jan 18, 97(2), 565 - 70 Highly mutagenic replication by DNA polymerase V (UmuC) provides a mechanistic basis for SOS untargeted mutagenesis; Maor-Shoshani A et al.; When challenged by DNA-damaging agents, Escherichia coli cells respond by inducing the SOS stress response, which leads to an increase in mutation frequency by two mechanisms: translesion replication, a process that causes mutations because of misinsertion opposite the lesions, and an inducible mutator activity, which acts at undamaged sites . Here we report that DNA polymerase V (pol V; UmuC), which previously has been shown to be a lesion-bypass DNA polymerase, was highly mutagenic during in vitro gap-filling replication of a gapped plasmid carrying the cro reporter gene . This reaction required, in addition to pol V, UmuD', RecA, and single-stranded DNA (ssDNA)-binding protein . pol V produced point mutations at a frequency of 2.1 x 10(-4) per nucleotide (2.1% per cro gene), 41-fold higher than DNA polymerase III holoenzyme . The mutational spectrum of pol V was dominated by transversions (53%), which were formed at a frequency of 1.3 x 10(-4) per nucleotide (1 . 1% per cro gene), 74-fold higher than with pol III holoenzyme . The prevalence of transversions and the protein requirements of this system are similar to those of in vivo untargeted mutagenesis (SOS mutator activity) . This finding suggests that replication by pol V, in the presence of UmuD', RecA, and ssDNA-binding protein, is the basis of chromosomal SOS untargeted mutagenesis. Nature, 2000 Jan 6, 403(6765), 112 - 5 Three-dimensional structure of the ion-coupled transport protein NhaA; Williams KA; Ion-coupled membrane-transport proteins, or secondary transporters, comprise a diverse and abundant group of membrane proteins that are found in all organisms . These proteins facilitate solute accumulation and toxin removal against concentration gradients using energy supplied by ion gradients across membranes . NhaA is a Na+/H+ antiporter of relative molecular mass 42,000, which is found in the inner membrane of Escherichia coli, and which has been cloned and characterized . NhaA uses the H+ electrochemical gradient to expel Na+ from the cytoplasm, and functions primarily in the adaptation to high salinity at alkaline pH . Most secondary transporters, including NhaA, are predicted to have 12 transmembrane helices . Here we report the structure of NhaA, at 7 A resolution in the membrane plane and at 14 A vertical resolution, determined from two-dimensional crystals using electron cryo-microscopy . The three-dimensional map of NhaA reveals 12 tilted, bilayer-spanning helices . A roughly linear arrangement of six helices is adjacent to a compact bundle of six helices, with the density for one helix in the bundle not continuous through the membrane . The molecular organization of NhaA represents a new membrane-protein structural motif and offers the first insights into the architecture of an ion-coupled transport protein. IUBMB Life, 1999 Nov, 48(5), 525 - 9 Phosphoserine aminotransferase, the second step-catalyzing enzyme for serine biosynthesis; Basurko MJ et al.; As a step toward analyzing the serine biosynthetic pathway in mammals, we have studied the properties of phosphoserine aminotransferase, the second step-catalyzing enzyme . The K(m) values for 3-phosphohydroxypyruvate and L-phosphoserine are 5 and 35 microM, respectively, and those for glutamate and alpha-ketoglutarate are 1.2 and 0.8 mM, respectively . The product inhibition studies strengthened the support for a ping-pong mechanism and allowed evaluation of Ki values for the four substrates . The equilibrium constant evaluated from the kinetic parameters is approximately 40 . Additionally, some physical properties relative to the bound coenzyme and the secondary structure were investigated . The results are consistent with a structural relationship between the Escherichia coli enzyme and the mammalian enzyme . The mammalian enzyme has specific kinetic parameters, the determination of which is a prerequisite to analyzing the serine biosynthetic pathway in mammals. IUBMB Life, 1999 Nov, 48(5), 513 - 7 Construction of murine phage antibody library and selection of ricin-specific single-chain antibodies; Gao X et al.; A murine phage antibody library containing 1.7 x 10(8) independent clones was constructed and then screened by affinity panning of anti-ricin antibody fragments . First, mRNA was purified from the total RNA extracted from fresh spleens of nonimmunized mice of Kunming, and then the total antibody variable region cDNA was amplified via reverse transcription-polymerase chain reaction . Gene fragments encoding VH and VL were amplified and assembled into a single gene by using a DNA linker encoding a pentadecapeptide (Gly4Ser)3 through primary PCR . Finally the recombinant DNA fragments were cloned into the phagemid pCANTAB 5E vector and electroporated into Escherichia coli TG1 . The recombinant phage particles displaying functional single-chain fragment variable regions (scFvs) were rescued by reinfection of helper phage M13KO7, thus constructing a murine antibody library . Two ricin-specific scFvs strains were selected from the phage antibody library by using affinity panning . The target antigen, ricin, was biotinylated with photobiotin and the biotin-labeled ricin interacted with the phage antibody library . Subsequent addition of streptavidincoated paramagnetic beads isolated the biotinylated ricin-binding phage particle complex . Washing, elution, and reinfection of the isolated complex then proceeded sequentially . After six rounds of panning, 2 strains of the 34 clones were verified to show greater specific affinity to ricin. Trends Biochem Sci, 2000 Jan, 25(1), 24 - 8 GHKL, an emergent ATPase/kinase superfamily; Dutta R et al.; An interesting recent development is the recognition of a novel ATP-binding superfamily that includes diverse protein families such as DNA topoisomerase II, molecular chaperones Hsp90, DNA-mismatch-repair enzymes MutL and histidine kinases . The most singular unifying feature of this superfamily is the unconventional Bergerat ATP-binding fold . The far-reaching significance of this commonality is still in the process of being explored. Gene Ther, 1999 Dec, 6(12), 1995 - 2004 Comparison between cationic polymers and lipids in mediating systemic gene delivery to the lungs; Bragonzi A et al.; Airway inflammation frequently found in congenital and acquired lung diseases may interfere with gene delivery by direct administration through either instillation or aerosol . Systemic delivery by the intravenous administration represents an alternative route of delivery that might bypass this barrier . A nonviral approach for transfecting various airway-derived cell lines in vitro showed that cationic polymers (PEI 22K and 25K) and lipids (DOTAP, GL-67/DOPE) are able to transfect with high efficiency the reporter genes firefly luciferase and E . coli lacZ . Notably, two properties predicted that cationic vectors would be useful for a systemic gene delivery approach to the lung: (1) transfection was not inhibited or increased when cells were incubated with cationic lipids or polymers in the presence of serum; and (2) cationic vectors protected plasmid DNA from DNase degradation . A single injection of DNA complexed to the cationic polymer PEI 22K into the tail vein of adult mice efficiently transfected primarily the lungs and to a lesser extent, heart, spleen, kidney and liver . The other vectors mediated lower to undetectable levels of luciferase expression in the lungs, with DOTAP > GL67/DOPE > PEI 25K > DOTMA/DOPE . A double injection protocol with a 15-min interval between the two doses of DOTAP/DNA complexes was investigated and showed a relevant role of the first injection in transfecting the lungs . A two log increase in luciferase expression was obtained either when the two doses were comprised of luciferase plasmid or when an irrelevant plasmid was used in the first injection . The double injection of luciferase/PEI 22K complexes determined higher transgene levels than a single dose, but a clear difference using an irrelevant plasmid as first dose was not observed . Using lacZ as a reporter gene, it was shown that only cells in the alveolar region, including type II penumocytes, stained positively for the transgene product. Gene Ther, 1999 Dec, 6(12), 1960 - 71 A sequence-specific gene correction by an RNA-DNA oligonucleotide in mammalian cells characterized by transfection and nuclear extract using a lacZ shuttle system; Igoucheva O et al.; The variability in gene conversion frequency by an RNA-DNA oligonucleotide (RDO) prompted us to develop a system as a means of measuring the conversion frequency rapidly and reproducibly . A shuttle vector was constructed to measure the frequency of targeted gene correction by RDO of the E . coli beta-galactosidase gene containing a single point mutation (G --> A), that resulted in inactivation of enzymatic activity . An RDO corrected the point mutation and restored the enzymatic activity, approximately 1%, determined by a histochemical staining in mammalian cells and by a color selection (blue or white) of bacteria transformed with Hirt DNA . In addition, we established an in vitro system capable of gene correction using nuclear extracts . CHO-K1 nuclear extracts corrected the point mutation approximately 0.1%, determined by bacterial transformation . Using the in vitro reaction, frequency of gene conversion in different cell types was measured . The embryonic fibroblasts from p53-/- mouse showed higher gene correction than that of the isogenic p53+/+ cells . Nuclear extracts from DT40 cells, which have a higher homologous recombination rate than any other mammalian cells exhibited 0.1-0.6% of gene correction . These results indicated that recombination may be rate-limiting in gene conversion by RDO in cells with competent mismatch repair activities . Utilizing transfection and in vitro reaction, we demonstrated that such a shuttle system might be useful in comparing the frequency of targeting among different cell types and to investigate the mechanism of gene conversion by RDO. EMBO J, 2000 Jan 17, 19(2), 282 - 94 Feedback-regulated degradation of the transcriptional activator Met4 is triggered by the SCF(Met30 )complex; Rouillon A et al.; Saccharomyces cerevisiae SCF(Met30) ubiquitin-protein ligase controls cell cycle function and sulfur amino acid metabolism . We report here that the SCF(Met30 )complex mediates the transcriptional repression of the MET gene network by triggering degradation of the transcriptional activator Met4p when intracellular S-adenosylmethionine (AdoMet) increases . This AdoMet-induced Met4p degradation is dependent upon the 26S proteasome function . Unlike Met4p, the other components of the specific transcriptional activation complexes that are assembled upstream of the MET genes do not appear to be regulated at the protein level . We provide evidence that the interaction between Met4p and the F-box protein Met30p occurs irrespective of the level of intracellular AdoMet, suggesting that the timing of Met4p degradation is not controlled by its interaction with the SCF(Met30) complex . We also demonstrate that Met30p is a short-lived protein, which localizes within the nucleus . Furthermore, transcription of the MET30 gene is regulated by intracellular AdoMet levels and is dependent upon the Met4p transcription activation function . Thus Met4p appears to control its own degradation by regulating the amount of assembled SCF(Met30) ubiquitin ligase. EMBO J, 2000 Jan 17, 19(2), 234 - 40 A membrane-embedded glutamate is required for ligand binding to the multidrug transporter EmrE; Muth TR et al.; EmrE is an Escherichia coli multidrug transporter that confers resistance to a variety of toxins by removing them in exchange for hydrogen ions . The detergent-solubilized protein binds tetraphenylphosphonium (TPP(+)) with a K(D) of 10 nM . One mole of ligand is bound per approximately 3 mol of EmrE, suggesting that there is one binding site per trimer . The steep pH dependence of binding suggests that one or more residues, with an apparent pK of approximately 7.5, release protons prior to ligand binding . A conservative Asp replacement (E14D) at position 14 of the only membrane-embedded charged residue shows little transport activity, but binds TPP(+) at levels similar to those of the wild-type protein . The apparent pK of the Asp shifts to <5.0 . The data are consistent with a mechanism requiring Glu14 for both substrate and proton recognition . We propose a model in which two of the three Glu14s in the postulated trimeric EmrE homooligomer deprotonate upon ligand binding . The ligand is released on the other face of the membrane after binding of protons to Glu14. Clin Immunol, 2000 Feb, 94(2), 140 - 51 Cell-free recombination of immunoglobulin switch-region DNA with nuclear extracts; Zhang K et al.; We have developed an in vitro recombination system employing cell-free nuclear extracts from human B lymphocytes capable of detecting the recombination between human mu switch (Smu) and Sepsilon sequences in a model plasmid . Nuclear extracts from CD40-stimulated B lymphocytes gave a higher frequency of recombination in the assay than the unstimulated B cells . Recombination between Smu and Sepsilon was mediated by the nuclear extracts as the recombinational products could be amplified prior to bacterial transformation . Characterization of the recombination products demonstrated that the recombination process had the characteristics of immunoglobulin (Ig) isotype switching, as it was (i) switch-region-sequence specific, (ii) nonhomologous recombination, and (iii) enhanced by CD40 stimulation . Transcription through the S region DNA was not required for recombination in the system . These results demonstrate that Ig switch-region DNA recombination can be accomplished in vitro by cell-free nuclear extracts . This in vitro system for Ig switch-region DNA recombination using cell-free nuclear extracts will permit the dissection of the events involved in IgE class switch recombination, a critical event in the development of allergic diseases . Shock, 2000 Jan, 13(1), 46 - 51 Prevention of early myocardial depression in hyperdynamic endotoxemia in dogs; Wolfard A et al.; In our study the pathomechanism of sepsis-induced early myocardial depression was investigated . We determined the effects of the inducible nitric oxide synthase inhibitor and free radical scavenger mercaptoethylguanidine (MEG) on the myocardial contractility, the endothelial and inducible nitric oxide synthase (eNOS and iNOS) activities, and the activation and tissue accumulation of polymorphonuclear leukocytes in hyperdynamic endotoxemia in dogs . Group 1 served as endotoxemic control . Mean arterial pressure and cardiac output were measured, myocardial contractility was estimated from the end-systolic pressure-diameter relationship . The eNOS, iNOS and myeloperoxidase activities were determined on myocardial biopsy samples, and the free radical-producing capacity of granulocytes was measured from separated cells . The effect of MEG on the in vitro free radical production of isolated granulocytes was measured by chemiluminometry . Endotoxin induced a hyperdynamic circulatory reaction and significant myocardial depression . The myocardial eNOS activity was significantly increased 4 h after induction of endotoxemia and remained elevated, the iNOS activity was increased only 8 h after endotoxemia induction . The free radical-producing capacity and the myocardial accumulation of the granulocytes were significantly increased . In group 2, MEG treatment selectively inhibited the iNOS activity, prolonged the hyperdynamic circulatory reaction, prevented myocardial depression and decreased the activation and tissue accumulation of granulocytes . The compound dose-dependently decreased the in vitro activation of previously resting granulocytes . Our study demonstrates that iNOS do not contribute to the early cardiac failure in endotoxemia . MEG selectively inhibits iNOS in vivo, but its beneficial effects are rather related to the decreases in leukocyte and free radical-mediated myocardial dysfunction during early endotoxemia. Shock, 2000 Jan, 13(1), 41 - 5 Cyclooxygenase 2 (COX-2) gene activation is regulated by cyclic adenosine monophosphate; Lo CJ et al.; Prostaglandin E2 production by tissue-fixed macrophages (Mphi) after severe injury contributes to an enhanced susceptibility to infection and sepsis . The purpose of this study was to investigate the effect of cyclic adenosine monophosphate (cAMP) on prostaglandin (PGE2) production and cyclooxygenase II (COX-2) gene activation in LPS-stimulated macrophages (Mphi) . RAW264.7 cells, a mouse Mphi cell line, were exposed to various concentrations of dibutyryl cAMP +/- lipopolysaccharide (10 microg/mL) stimulation . Total Mphi ribonucleic acid (RNA) was harvested for the determination of COX-2 messenger RNA (mRNA) with mouse complementary deoxyribonucleic acid (cDNA) by Northern blot assay . Mphi supernatant was collected for the measurement of tumor necrosis factor (TNF) by L929 bioassay and PGE2 by enzyme-linked immunosorbent assay (ELISA), respectively . Mphi NFkappaB activity was determined by electrophoretic mobility shift assays (EMSA) . Dibutyryl cAMP significantly inhibited TNF production by LPS-stimulated Mphi . Dibutyryl cAMP (1 mM) alone induced PGE2 production . Dibutyryl cAMP (100 microM and 1 mM) also augmented PGE2 production by LPS-stimulated Mphi . Dibutyryl cAMP had similar effect on Mphi COX-2 mRNA expression and NFkappaB activity . Our data demonstrate that cAMP modulates Mphi TNF production and upregulates COX-2 gene and PGE2 production. Carbohydr Res, 1999 Dec 12, 322(3-4), 219 - 27 Chemical and immunochemical studies of the O-antigen from enteropathogenic Escherichia coli O158 lipopolysaccharide; Datta AK et al.; The O-specific polysaccharide isolated from Escherichia coli O158 smooth lipopolysaccharide contains L-rhamnose, D-glucose and 2-acetamido-2-deoxy-D-galactose in the molar ratios 1:2:2 . Studies on composition, methylation analysis and specific degradations together with a 1H and 13C NMR spectral study established that the O-antigen is built up from a pentasaccharide repeating unit having the following structure: {formula: see text} The most effective inhibitory part of the oligosaccharide from E . coli O158 lipopolysaccharide has been serologically characterized by an ELISA-inhibition study using different sugars . The results showed that methyl alpha- and beta-D-GalpNAc are the most effective inhibitors among the monosaccharides tested, while the main antibody specificity lies on the main-chain trisaccharide repeating unit. Nucleic Acids Res, 2000 Feb 1, 28(3), 762 - 9 The genes encoding endonuclease VIII and endonuclease III in Escherichia coli are transcribed as the terminal genes in operons; Gifford CM et al.; Escherichia coli endonuclease VIII and endo-nuclease III are oxidative base excision repair DNA glycosylases that remove oxidized pyrimidines from DNA . The genes encoding these proteins, nei and nth, are both co-transcribed as the terminal genes in operons . nei is the terminal gene in an operon with four open reading frames that encode proteins of unknown function . This operon has two confirmed transcription initiation sites upstream of the first open reading frame and two transcript termination sites downstream of nei . nth is the terminal gene in an operon with seven open reading frames that encode proteins of unknown function . The six open reading frames immediately upstream of nth show homology to the genes rnfA, rnfB, rnfC, rnfD, rnfG and rnfE from Rhodobacter capsulatis . The rnf genes are required for nitrogen fixation in R.capsulatis and have been predicted to make up a membrane complex involved in electron transport to nitrogenase . The nth operon has transcription initiation sites upstream of the first and second open reading frames and a single transcript termination site downstream of nth . The order of genes in these operons has been conserved or partially conserved in other bacteria, although it is not known whether the genes are co-transcribed in these other organisms. Mol Biol Cell, 2000 Jan, 11(1), 325 - 37 Identification of filamin as a novel ligand for caveolin-1: evidence for the organization of caveolin-1-associated membrane domains by the actin cytoskeleton; Stahlhut M et al.; Reports on the ultrastructure of cells as well as biochemical data have, for several years, been indicating a connection between caveolae and the actin cytoskeleton . Here, using a yeast two-hybrid approach, we have identified the F-actin cross-linking protein filamin as a ligand for the caveolae-associated protein caveolin-1 . Binding of caveolin-1 to filamin involved the N-terminal region of caveolin-1 and the C terminus of filamin close to the filamin-dimerization domain . In in vitro binding assays, recombinant caveolin-1 bound to both nonmuscle and muscle filamin, indicating that the interaction might not be cell type specific . With the use of confocal microscopy, colocalization of caveolin-1 and filamin was observed in elongated patches at the plasma membrane . Remarkably, when stress fiber formation was induced with Rho-stimulating Escherichia coli cytotoxic necrotizing factor 1, the caveolin-1-positive structures became coaligned with stress fibers, indicating that there was a physical link connecting them . Immunogold double-labeling electron microscopy confirmed that caveolin-1-labeled racemose caveolae clusters were positive for filamin . The actin network, therefore, seems to be directly involved in the spatial organization of caveolin-1-associated membrane domains. J Biol Chem, 2000 Jan 21, 275(3), 2191 - 8 Lipid-dependent targeting of G proteins into rafts; Moffett S et al.; Domains rich in sphingolipids and cholesterol, or rafts, may organize signal transduction complexes at the plasma membrane . Raft lipids are believed to exist in a state similar to the liquid-ordered phase . It has been proposed that proteins with a high affinity for an ordered lipid environment will preferentially partition into rafts (Melkonian, K . A., Ostermeyer, A . G., Chen, J . Z., Roth, M . G., and Brown, D . A . (1999) J . Biol . Chem . 274, 3910-3917) . We investigated the possibility that lipid-lipid interactions between lipid-modified proteins and raft lipids mediate targeting of proteins to these domains . G protein monomers or trimers were reconstituted in liposomes, engineered to mimic raft domains . Assay for partitioning of G proteins into rafts was based on Triton X-100 insolubility . Myristoylation and palmitoylation of Galpha(i) were necessary and sufficient for association with liposomes and partitioning into rafts . Strikingly, the amount of fatty-acylated Galpha(i) in rafts was significantly reduced when myristoylated Galpha(i) was thioacylated with cis-unsaturated fatty acids instead of saturated fatty acids such as palmitate . Prenylated betagamma subunits were excluded from rafts, whether reconstituted alone or with fatty-acylated alpha subunits . These results suggest that the structural difference between lipids that modify proteins is one basis for the selectivity of protein targeting to rafts. J Biol Chem, 2000 Jan 21, 275(3), 2174 - 84 DNA polymerase III proofreading mutants enhance the expansion and deletion of triplet repeat sequences in Escherichia coli; Iyer RR et al.; The influence of mutations in the 3' to 5' exonucleolytic proofreading epsilon-subunit of Escherichia coli DNA polymerase III on the genetic instabilities of the CGG.CCG and the CTG.CAG repeats that cause human hereditary neurological diseases was investigated . The dnaQ49(ts) and the mutD5 mutations destabilize the CGG.CCG repeats . The distributions of the deletion products indicate that slipped structures containing a small number of repeats in the loop mediate the deletion process . The CTG.CAG repeats were destabilized by the dnaQ49(ts) mutation by a process mediated by long hairpin loop structures (>/=5 repeats) . The mutD5 mutator strain stabilized the (CTG.CAG)(175) tract, which contained two interruptions . Since the mutD5 mutator strain has a saturated mismatch repair system, the stabilization is probably an indirect effect of the nonfunctional mismatch repair system in these strains . Shorter uninterrupted tracts expand readily in the mutD5 strain, presumably due to the greater stability of long CTG.CAG tracts (>100 repeats) in this strain . When parallel studies were conducted in minimal medium, where the mutD5 strain is defective in exonucleolytic proofreading but has a functional MMR system, both CTG.CAG and CGG.CCG repeats were destabilized, showing that the proofreading activity is essential for maintaining the integrity of TRS tracts . Thus, we conclude that the expansion and deletion of triplet repeats are enhanced by mutations that reduce the fidelity of replication. J Biol Chem, 2000 Jan 21, 275(3), 2080 - 6 Mutation in the magnesium binding site of hMSH6 disables the hMutSalpha sliding clamp from translocating along DNA; Iaccarino I et al.; In human cells, binding of base/base mismatches and small insertion/deletion loops is mediated by hMutSalpha, a heterodimer of hMSH2 and hMSH6 . In the presence of ATP and magnesium, hMutSalpha dissociates from the mismatch by following the DNA contour in the form of a sliding clamp . This process is enabled by a conformational change of the heterodimer, which is driven by the binding of ATP and magnesium in the Walker type A and B motifs of the polypeptides, respectively . We show that a purified recombinant hMutSalpha variant, hMutSalpha 6DV, which contains an aspartate to valine substitution in the Walker type B motif of the hMSH6 subunit, fails to undergo the conformational change compatible with translocation . Instead, its direct dissociation from the mismatch-containing DNA substrate in the presence of ATP and magnesium precludes the assembly of a functional mismatch repair complex . The "translocation-prone" conformation of wild type hMutSalpha could be observed solely under conditions that favor hydrolysis of the nucleotide and mismatch repair in vitro . Thus, whereas magnesium could be substituted with manganese, ATP could not be replaced with its slowly or nonhydrolyzable homologues ATP-gammaS or AMPPNP, respectively . The finding that ATP induces different conformational changes in hMutSalpha in the presence and in the absence of magnesium helps explain the functional differences between hMutSalpha variants incapable of binding ATP as compared with those unable to bind the metal ion. J Biol Chem, 2000 Jan 21, 275(3), 1897 - 901 Primary structure and function analysis of the Abrus precatorius agglutinin A chain by site-directed mutagenesis . Pro(199) Of amphiphilic alpha-helix H impairs protein synthesis inhibitory activity; Liu CL et al.; Abrus agglutinin (AAG), a low-toxicity protein from the plant Abrus precatorius, is less lethal than abrina (ABRa) in mice (LD(50) = 5 mg/kg versus 20 microg/kg of body weight) . Nucleotide sequence analysis of a cDNA clone encoding full-length AAG showed an open reading frame with 1641 base pairs, corresponding to a 547-amino acid residue preproprotein containing a signal peptide and a linker region (two amino acid residues) between the AAG-A and AAG-B subunits . AAG had high homology to ABRa (77.8%) . The 13 amino acid residues involved in catalytic function, which are highly conserved among abrins and ricins, were also conserved within AAG-A . The protein synthesis inhibitory activity of AAG-A (IC(50) = 3.5 nM) was weaker than that of ABRa-A (0.05 nM) . Molecular modeling followed by site-directed mutagenesis showed that Pro(199) of AAG-A, located in amphiphilic helix H and corresponding to Asn(200) of ABRa-A, can induce bending of helix H . This bending would presumably affect the binding of AAG-A to its target sequence, GpApGpAp, in the tetraloop structure of the 28 S rRNA subunit and could be one of the major factors contributing to the relatively weak protein synthesis inhibitory activity and toxicity of AAG. J Biol Chem, 2000 Jan 21, 275(3), 1864 - 72 Binding of flavin adenine dinucleotide to molybdenum-containing carbon monoxide dehydrogenase from Oligotropha carboxidovorans . Structural and functional analysis of a carbon monoxide dehydrogenase species in which the native flavoprotein has been replaced by its recombinant counterpart produced in Escherichia coli; Gremer L et al.; The carbon monoxide (CO) dehydrogenase of Oligotropha carboxidovorans is composed of an S-selanylcysteine-containing 88 . 7-kDa molybdoprotein (L), a 17.8-kDa iron-sulfur protein (S), and a 30.2-kDa flavoprotein (M) in a (LMS)(2) subunit structure . The flavoprotein could be removed from CO dehydrogenase by dissociation with sodium dodecylsulfate . The resulting M(LS)(2)- or (LS)(2)-structured CO dehydrogenase species could be reconstituted with the recombinant apoflavoprotein produced in Escherichia coli . The formation of the heterotrimeric complex composed of the apoflavoprotein, the molybdoprotein, and the iron-sulfur protein involves structural changes that translate into the conversion of the apoflavoprotein from non-FAD binding to FAD binding . Binding of FAD to the reconstituted deflavo (LMS)(2) species occurred with second-order kinetics (k(+1) = 1350 M(-1) s(-1)) and high affinity (K(d) = 1.0 x 10(-9) M) . The structure of the resulting flavo (LMS)(2) species at a 2.8-A resolution established the same fold and binding of the flavoprotein as in wild-type CO dehydrogenase, whereas the S-selanylcysteine 388 in the active-site loop on the molybdoprotein was disordered . In addition, the structural changes related to heterotrimeric complex formation or FAD binding were transmitted to the iron-sulfur protein and could be monitored by EPR . The type II 2Fe:2S center was identified in the N-terminal domain and the type I center in the C-terminal domain of the iron-sulfur protein. J Biol Chem, 2000 Jan 21, 275(3), 1829 - 38 CD44 interaction with tiam1 promotes Rac1 signaling and hyaluronic acid-mediated breast tumor cell migration; Bourguignon LY et al.; In this study we have explored the interaction between CD44 (the hyaluronic acid (HA)-binding receptor) and Tiam1 (a guanine nucleotide exchange factor) in metastatic breast tumor cells (SP1 cell line) . Immunoprecipitation and immunoblot analyses indicate that both the CD44v3 isoform and the Tiam1 protein are expressed in SP1 cells and that these two proteins are physically associated as a complex in vivo . Using an Escherichia coli-derived calmodulin-binding peptide-tagged Tiam1 fragment (i.e . the NH(2)-terminal pleckstrin homology (PHn) domain and an adjacent protein interaction domain designated as PHn-CC-Ex, amino acids 393-738 of Tiam1) and an in vitro binding assay, we have detected a specific binding interaction between the Tiam1 PHn-CC-Ex domain and CD44 . Scatchard plot analysis indicates that there is a single high affinity CD44 binding site in the PHn-CC-Ex domain of Tiam1 with an apparent dissociation constant (K(d)) of 0.2 nM, which is comparable with CD44 binding (K(d) = approximately 0.13 nM) to intact Tiam1 . These findings suggest that the PHn-CC-Ex domain is the primary Tiam1-binding region for CD44 . Most importantly, the binding of HA to CD44v3 of SP1 cells stimulates Tiam1-catalyzed Rac1 signaling and cytoskeleton-mediated tumor cell migration . Transfection of SP1 cells with Tiam1cDNA promotes Tiam1 association with CD44v3 and up-regulates Rac1 signaling as well as HA/CD44v3-mediated breast tumor cell migration . Co-transfection of SP1 cells with PHn-CC-Ex cDNA and Tiam1 cDNA effectively inhibits Tiam1 association with CD44 and efficiently blocks tumor behaviors . Taken together, we believe that the linkage between CD44v3 isoform and the PHn-CC-EX domain of Tiam1 is required for HA stimulated Rac1 signaling and cytoskeleton-mediated tumor cell migration during breast cancer progression. J Biol Chem, 2000 Jan 21, 275(3), 1781 - 6 Fluorophores at the N terminus of nascent chloramphenicol acetyltransferase peptides affect translation and movement through the ribosome; Ramachandiran V et al.; Structurally different fluorescent probes were covalently attached to methionyl-tRNA(f) and tested for their incorporation into nascent peptides and full-length protein using an Escherichia coli cell-free coupled transcription/translation system . Bovine rhodanese and bacterial chloramphenicol acetyltransferase (CAT) were synthesized using derivatives of cascade yellow, eosin, pyrene, or coumarin attached to {(35)S}Met-tRNA(f) . All of the probes tested were incorporated into polypeptides, although less efficiently when compared with formyl-methionine . Eosin, the largest of the fluorophores used with estimated dimensions of 20 x 11 A, caused the largest reduction in product formed . The rate of initiation was reduced with the fluorophore-Met-tRNA(f) compared with fMet-tRNA(f) with pyrene having the least and eosin the biggest effect . Analysis of the nascent polypeptides showed that the modifications at the N terminus affected the rate at which nascent CAT peptides were elongated causing accumulation of peptides of about 4 kDa, possibly by steric hindrance inside the tunnel within the 50 S ribosomal subunit . Fluorescence measurements indicate that the probe at the N terminus of nascent pyrene-CAT peptides is in a relatively hydrophilic environment . This finding is in agreement with recent data showing cross-linking of the N terminus of nascent peptides to nucleotides of the 23 S ribosomal RNA. J Biol Chem, 2000 Jan 21, 275(3), 1594 - 600 Refolded outer membrane protein A of Escherichia coli forms ion channels with two conductance states in planar lipid bilayers; Arora A et al.; Outer membrane protein A (OmpA), a major structural protein of the outer membrane of Escherichia coli, consists of an N-terminal 8-stranded beta-barrel transmembrane domain and a C-terminal periplasmic domain . OmpA has served as an excellent model for studying the mechanism of insertion, folding, and assembly of constitutive integral membrane proteins in vivo and in vitro . The function of OmpA is currently not well understood . Particularly, the question whether or not OmpA forms an ion channel and/or nonspecific pore for uncharged larger solutes, as some other porins do, has been controversial . We have incorporated detergent-purified OmpA into planar lipid bilayers and studied its permeability to ions by single channel conductance measurements . In 1 M KCl, OmpA formed small (50-80 pS) and large (260-320 pS) channels . These two conductance states were interconvertible, presumably corresponding to two different conformations of OmpA in the membrane . The smaller channels are associated with the N-terminal transmembrane domain, whereas both domains are required to form the larger channels . The two channel activities provide a new functional assay for the refolding in vitro of the two respective domains of OmpA . Wild-type and five single tryptophan mutants of urea-denatured OmpA are shown to refold into functional channels in lipid bilayers. Biosci Biotechnol Biochem, 1999 Nov, 63(11), 2023 - 4 Effects of a feedback-resistant aspartokinase III gene on L-isoleucine production in Escherichia coli K-12; Hashiguchi K et al.; An L-isoleucine-overproducing recombinant strain of E . coli, TVD5, was also found to overproduce L-valine . The L-isoleucine productivity of TVD5 was markedly decreased by addition of L-lysine to the medium . Introduction of a gene encoding feedback-resistant aspartokinase III increased L-isoleucine productivity and decreased L-valine by-production . The resulting strain accumulated 12 g/l L-isoleucine from 40 g/l glucose, and suppression of L-isoleucine productivity by L-lysine was relieved. Biosci Biotechnol Biochem, 1999 Nov, 63(11), 2001 - 4 The trichothecene biosynthesis regulatory gene from the type B producer Fusarium strains: sequence of Tri6 and its expression in Escherichia coli; Matsumoto G et al.; A genomic DNA fragment containing Tri6, a transcription activator gene of trichothecene biosynthesis, was cloned by vectorette PCR from Fusarium graminearum F15, which produces type B trichothecene, deoxynivalenol . The nucleotide sequence of the gene showed 84% of identity to that of the type A trichothecene producer Fusarium sporotrichioides NRRL 3299, but the sequence around the initiation codon was not highly conserved between these producers . Based on the upstream and downstream sequences of the coding region of F . graminearum, Tri6 could be amplified by PCR from other type B trichothecene producers . Tri6 appeared to be expressed for only a limited period prior to the toxin production. Vopr Med Khim, 1999 Sep-Oct, 45(5), 416 - 29 {Expression constructs of inducible isoform of nitric oxide synthase in Escherichia coli cells}; Gervaziev IuV et al.; In the present work we describe the construction of expression system for inducible murine macrophage nitric oxide synthase (iNOS) in E.coli . For this purpose a framework of translation iNOS was cloned in the expression vector pCWori + . As biosynthesis of active iNOS requires coexpression of calmodulin (CaM), for obtaining functional expression of this protein we conducted amplification of an appropriate site of the library total cDNA a frog Xenopus laevis, then plasmids for coexpression of calmodulin were constructed under a control tac and T7 promotors . Recombinant iNOS was functionally active as revealed by the analysis of CO-reduced spectrums, detection of derivation NO with the help of reaction conversion HbO2 in metHb, and also identification of a molecule NO by EPR method . The output of recombinant iNOS at usage of different constructions varied from 10 up to 22 mg/l culture, and specific activity was from 0.42 up to 0.64 U/mg of protein . These data coincide with the earlier published results of other investigators . It was established, that the expressed iNOS is associated to a membrane fraction of cells, thus in the 105,000 g-supernatant the activity of an enzyme is not detected . The data on membrane localization iNOS are inconsistent with general notion this enzyme is soluble. Biochim Biophys Acta, 2000 Jan 17, 1483(2), 263 - 74 A second Escherichia coli protein with CL synthase activity; Guo D et al.; The Escherichia coli open reading frame f413, which has the potential to code for a polypeptide homologous to cardiolipin (CL) synthase, has been cloned . Its polypeptide product has a molecular mass of 48 kDa, is membrane-bound, and catalyzes CL formation but does not hydrolyze CL . A comparison of the sequences predicted for the polypeptides encoded by f413 and cls indicates that the N-terminal residues specified by cls may be unnecessary for CL synthase activity . Construction of a truncated cls gene and characterization of its polypeptide product have confirmed this conclusion. Plant Cell, 2000 Jan, 12(1), 137 - 50 Evidence for a role of ClpP in the degradation of the chloroplast cytochrome b(6)f complex; Majeran W et al.; In the green alga Chlamydomonas reinhardtii, the ClpP protease is encoded by an essential chloroplast gene . Mutating its AUG translation initiation codon to AUU reduced ClpP accumulation to 25 to 45% of that of the wild type . Both the mature protein and the putative precursor containing its insertion sequence were present in reduced amounts . Attenuation of ClpP did not affect growth rates under normal conditions but restricted the ability of the cells to adapt to elevated CO(2) levels . It also affected the rate of degradation of the cytochrome b(6)f complex of the thylakoid membrane in two experimental situations: (1) during nitrogen starvation, and (2) in mutants deficient in the Rieske iron-sulfur protein . The ClpP level also controls the steady state accumulation of a mutated version of the Rieske protein . In contrast, attenuation of ClpP did not rescue the fully unassembled subunits in other cytochrome b(6)f mutants . We conclude that proteolytic disposal of fully or partially assembled cytochrome b(6)f is controlled by the Clp protease. Plant Cell, 2000 Jan, 12(1), 81 - 96 Cryptochrome nucleocytoplasmic distribution and gene expression are regulated by light quality in the fern Adiantum capillus-veneris; Imaizumi T et al.; Numerous cellular responses are reportedly regulated by blue light in gametophytes of lower plants; however, the molecular mechanisms of these responses are not known . Here, we report the isolation of two blue light photoreceptor genes, designated cryptochrome genes 4 and 5 (CRY4 and CRY5), from the fern Adiantum capillus-veneris . Because previously we identified three cryptochrome genes, this fern cryptochrome gene family of five members is the largest identified to date in plants . The deduced amino acid sequences of the five genes show remarkable similarities with previously identified cryptochromes as well as class I photolyases . Like the other plant cryptochromes, none of the cryptochromes of this fern possesses photolyase activity . RNA gel blot analysis and competitive polymerase chain reaction analysis indicate that the expression of the newly identified CRY4 and CRY5 genes is regulated by light and is under phytochrome control . The intracellular distribution of reporter beta-glucuronidase (GUS)-CRY fusion proteins indicates that GUS-CRY3 and GUS-CRY4 localize in fern gametophyte nuclei . The nuclear localization of GUS-CRY3 is regulated in a light-dependent manner . Together with our physiological knowledge, these results suggest that CRY3, CRY4, or both might be the photoreceptor that mediates inhibition of spore germination by blue light. Plant Cell, 2000 Jan, 12(1), 5 - 21 The late developmental pattern of Mu transposon excision is conferred by a cauliflower mosaic virus 35S -driven MURA cDNA in transgenic maize; Raizada MN et al.; The MuDR element responsible for Mutator activities in maize encodes two genes, mudrA and mudrB . Each encodes multiple transcripts hypothesized to regulate, directly or indirectly, the unique late timing and switch in transposition mechanism during maize development . mudrA, which encodes the MURA transposase, is unstable in bacterial plasmids, a technical problem solved by using phage M13 as a vector to prepare DNA for biolistic transformation . In transgenic maize, a single 2.7-kb mudrA cDNA predicted to encode an 823-amino acid protein is sufficient to catalyze late somatic excisions, despite removal of the native promoter, alternative transcription start sites, known introns, polymorphic 5' and 3' untranslated sequences, and the mudrB gene . These results suggest that post-translational regulation confers Mu excision timing . The transgene is active in lines containing silencing MuDR elements . This suggests that endogenous MuDR transposons do not measurably immunize the host against expression of a homologous transgene. J Mol Neurosci, 1999 Feb, 12(1), 35 - 51 PhosphoCREB and CREM/ICER: positive and negative regulation of proenkephalin gene expression in the paraventricular nucleus of the hypothalamus; Borsook D et al.; In the hypothalamic paraventricular nucleus (PVN), the proenkephalin gene may be upregulated by lipopolysaccharide (LPS) and downregulated by the GABA-A agonist muscimol . Candidate transcription factors regulating the proenkephalin gene in opposite directions are cAMP-response-element-binding protein (CREB) (when phosphorylated, a positive regulator) and cAMP-responsive modulatory inducible cAMP early repressor (CREM/ICER) (a negative regulator) . Our results demonstrate that CREM alpha,beta,gamma transcripts and ICER are induced in the PVN by LPS and remain elevated for periods of up to 12 h . PhosphoCREB is elevated after LPS administration, peaking at 8 h, but remaining elevated over control levels at 12 h . Phospho-CREB induction by LPS is also seen in primary hypothalamic cultures . Cotransfection of ICER with ENK-CAT12 into primary hypothalamic cultures produced a decrease in chloramphenicol acetyl transferase (CAT) levels following cAMP or LPS stimulation . PhosphoCREB is downregulated and CREM/ICER is upregulated in the PVN by muscimol, suggesting that the regulation of these transcription factors may underlie the inhibitory effect of muscimol on target genes in the PVN. Mutat Res, 1999 Dec 13, 446(2), 205 - 13 Mutagenicity of thiol compounds in Escherichia coli WP2 tester strain IC203, deficient in OxyR: effects of S9 fractions from rat liver and kidney; Martinez A et al.; Low doses of L-cysteine (CYS), cysteinyl-glycine (CYSGLY) and reduced glutathione (GSH) activated by gamma-glutamyl transpeptidase (GGT) were mutagenic in strain IC203 (oxyR), whereas higher doses were required to observe a weak mutagenicity in the oxyR+ strain WP2 uvrA/pKM101 (denoted IC188) . This indicates that thiol mutagenesis is suppressed by OxyR-regulated antioxidant defenses and confirms its oxidative character . The mutagenesis by low doses of CYS, CYSGLY and GSH + GGT detected in IC203 was abolished by rat liver S9, through the activity of catalase, as well as by the metal chelator diethyldithiocarbamate (DETC), supporting the dependence of this mutagenesis on H2O2 production, probably in thiol autoxidation reactions in which transition metals are involved . Surprisingly, low DETC concentrations greatly potentiate the mutagenicity of low CYS doses . Mutagenesis by high doses of CYS and CYSGLY occurred in both IC203 and IC188 in the presence of liver S9, and was resistant to inhibition by catalase, although it was prevented by DETC . Mutagenesis by GSH activated by rat kidney S9, rich in GGT, was detected in IC203 and IC188 only at high doses since catalase and glutathione peroxidase, both present in kidney S9, might inhibit its induction by low GSH doses . In the presence of liver S9, almost deficient in GGT, GSH was not mutagenic . The mutagenicity of a high GSH dose occurring in the presence either of GGT plus liver S9 or of kidney S9 was weakly prevented by DETC. Mol Cell, 1999 Dec, 4(6), 961 - 9 An early developmental transcription factor complex that is more stable on nucleosome core particles than on free DNA; Cirillo LA et al.; In vivo footprinting studies have shown that transcription factor binding sites for HNF3 and GATA-4 are occupied on the albumin gene enhancer in embryonic endoderm, prior to the developmental activation of liver gene transcription . We have investigated how these factors can stably occupy silent chromatin . Remarkably, we find that HNF3, but not GATA-4 or a GAL4 control protein, binds far more stably to nucleosome core particles than to free DNA . In the presence of HNF3, GATA-4 binds stably to an HNF3-positioned nucleosome . Histone acetylation does not affect HNF3 binding . This is evidence for stable nucleosome binding by a transcription factor and shows that a winged helix protein is sufficient to initiate the assembly of an enhancer complex on nonacetylated nucleosomes. Mol Cell, 1999 Dec, 4(6), 949 - 59 Reciprocal control of catalysis by the tyrosine recombinases XerC and XerD: an enzymatic switch in site-specific recombination; Hallet B et al.; In Xer site-specific recombination, sequential DNA strand exchange reactions are catalyzed by a heterotetrameric complex composed of two recombinases, XerC and XerD . It is demonstrated that XerC and XerD catalytic activity is controlled by an interaction involving the C-terminal end of each protein (the donor region) and an internal region close to the active site (the acceptor region) . Mutations in these regions reciprocally alter the relative activity of XerC and XerD, with their combination producing synergistic effects on catalysis . The data support a model in which C-terminal intersubunit interactions contribute to coupled protein-DNA conformational changes that lead to sequential activation and reciprocal inhibition of pairs of active sites in the recombinase tetramer during recombination. Arterioscler Thromb Vasc Biol, 2000 Jan, 20(1), 210 - 6 A single amino acid deletion in the carboxy terminal of apolipoprotein A-I impairs lipid binding and cellular interaction; Huang W et al.; The carboxy-terminal region of apolipoprotein (apo) A-I has been shown by mutagenesis or synthetic peptides to play an important role in lipid binding . However, the precise functional domain of the C-terminal remains to be defined . In this study, apoA-I Nichinan, a naturally occurring human apoA-I variant with a deletion of glutamic acid 235, was expressed in Escherichia coli to examine the effect of this mutation on the functional domain of apoA-I for lipid binding and related consequences . A dimyristoyl phosphatidylcholine binding study with recombinant (r-) proapoA-I Nichinan showed a significantly slow initial rate of lipid binding . On preincubation with human plasma lipoprotein fractions (d<1.225 g/mL) at 37 degrees C for 1 hour, (125)I-labeled normal r-proapoA-I was chromatographed as a single peak at the high density lipoprotein (HDL) fraction, whereas (125)I-labeled r-proapoA-I Nichinan was chromatographed into the HDL fraction as well as the free r-proapoA-I fraction (23% of radioactivity) . Circular dichroism measurements showed that the alpha-helix content of lipid-bound r-proapoA-I Nichinan was reduced, being 62% (versus 73%) of normal r-proapoA-I . Nondenaturing gradient gel electrophoresis of reconstituted HDL particles assembled with r-proapoA-I Nichinan and normal r-proapoA-I showed similar particle size . To study cholesterol efflux, human skin fibroblasts were labeled with {(3)H}cholesterol, followed by incubation with either lipid-free r-proapoA-I or DMPC/r-proapoA-I complex . Fractional cholesterol efflux from {(3)H}cholesterol-labeled fibroblasts to lipid-free r-proapoA-I Nichinan or DMPC/r-proapoA-I Nichinan complexes was significantly reduced relative to that of normal r-proapoA-I or DMPC/r-proapoA-I during the 6-hour incubation . Binding assays of human skin fibroblasts by lipid-free r-proapoA-I showed that r-proapoA-I Nichinan was 32% less bound to fibroblasts than was normal r-proapoA-I . Our data demonstrate that the deletion of glutamic acid 235 at the C-terminus substantially reduces the lipid-binding properties of r-proapoA-I Nichinan, which may cause a reduction in its capacity to interact with plasma membranes as well as to promote cholesterol efflux from cultured fibroblasts. Mol Biol Rep, 1999 Dec, 26(4), 269 - 76 Influence of highly curved DNA segments on in vivo topology of plasmids; Ohyama T et al.; Recombinant plasmids carrying a highly curved DNA structure are sometimes unstable in Escherichia coli . In order to know the underlying mechanism, several plasmids carrying one or two highly bent DNA segment(s) from the human adenovirus type 2 (Ad2) enhancer and/or origin region of phage lambda replication were systematically constructed and propagated in E . coli . The highly bent DNA segments disturbed the action of DNA topoisomerases: i.e . they were shown to be able to produce an anomalously wide spectrum of linking number topoisomers that tails toward lower supercoiling with a little of the DNA actually positively supercoiled . Furthermore, bent DNA caused multimeric plasmid formation . The linking number topoisomers and multimers seemed to be intermediate topological states of the bent DNA-containing plasmids that would lead to the deletion occurring in them . The nucleotide sequence of a deletion product of a bent DNA-containing plasmid showed that the putative source of the severe topological constraint was entirely eliminated from the plasmid. J Struct Biol, 1999 Dec 30, 128(3), 287 - 96 Oligomerization state of MIP proteins expressed in Xenopus oocytes as revealed by freeze-fracture electron-microscopy analysis; Bron P et al.; The MIP (major intrinsic protein) family is a widespread family of membrane proteins exhibiting two major types of channel properties: aquaporins and solute facilitators . In the present study, freeze-fracture electron microscopy was used to investigate the oligomerization state of two MIP proteins heterologously expressed in the plasma membrane of Xenopus laevis oocytes: AQPcic, an aquaporin from the insect Cicadella viridis, and GlpF, a glycerol facilitator from Escherichia coli . Swelling assays performed on oocytes 48 and 72 h following cRNA microinjections showed that these proteins were functionally expressed . Particle density determinations indicated that expression of proteins is related to an increase in particle density on the P fracture face of oocyte plasma membranes . Statistical analysis of particle sizes was performed on protoplasmic fracture faces of the plasma membrane of oocytes expressing AQPcic and GlpF 72 h after cRNA microinjections . Compared to control oocytes, AQPcic-expressing oocytes exhibited a specific population of particles with a mean diameter of 8.7 +/- 0.1 nm . This value is consistent with the previously reported tetrameric organization of AQPcic . In addition, AQPcic particles aggregate and form orthogonal arrays similar to those observed in native membranes of C . viridis, consisting of homotetramers of AQPcic . On the protoplasmic fracture face of oocytes expressing GlpF, the particle density is increased by 4.1-fold and the mean diameter of specifically added particles is 5.8 +/- 0.1 nm . This value fits with a monomer of the 28-kDa GlpF protein plus the platinum-carbon layer . These results clearly demonstrate that GlpF is a monomer when functionally expressed in plasma membranes of Xenopus oocytes and therefore emphasize the key role of the oligomerization state of MIP proteins with respect to their function . J Struct Biol, 1999 Dec 30, 128(3), 237 - 42 Crystals of acetylated SecB diffract to 2.3-A resolution; Dekker C et al.; The molecular chaperone SecB is part of the protein translocation pathway in Escherichia coli . SecB was purified from an overproducing strain and crystallized, resulting in crystals diffracting to 2.3-A resolution . The analysis of electrospray ionization mass spectra of dissolved crystals of SecB indicated that we have crystallized an acetylated form of SecB . Sequence analysis suggests that the protein is fully acetylated at its N-terminus in vivo, indicating that potential deacetylation is artificially introduced by purification methods . The high degree of acetylation that we observed might account for the fact that the crystals obtained as described in this study diffract to higher resolution than those in previously reported trials . Nat Struct Biol, 2000 Jan, 7(1), 23 - 7 The structural basis for tRNA recognition and pseudouridine formation by pseudouridine synthase I; Foster PG et al.; Pseudouridine synthases catalyze the isomerization of specific uridines to pseudouridine in a variety of RNAs, yet the basis for recognition of the RNA sites or how they catalyze this reaction is unknown . The crystal structure of pseudouridine synthase I from Escherichia coli, which, for example, modifies positions 38, 39 and/or 40 in tRNA, reveals a dimeric protein that contains two positively charged, RNA-binding clefts along the surface of the protein . Each cleft contains a highly conserved aspartic acid located at its center . The structural domains have a topological similarity to those of other RNA-binding proteins, though the mode of interaction with tRNA appears to be unique . The structure suggests that a dimeric enzyme is required for binding transfer RNA and subsequent pseudouridine formation. Nat Biotechnol, 2000 Jan, 18(1), 71 - 4 Selection and application of peptide-binding peptides; Zhang Z et al.; Peptide-binding ligands would be useful for directing reagents to particular epitopes in a protein, the detection of peptide hormones, and many other applications . Here we show that peptides of modest size isolated from a library using a simple genetic assay can act as specific receptors for other peptides . The equilibrium dissociation constants of these peptide-peptide complexes are higher than those of typical monoclonal antibody-epitope complexes . Nonetheless, as shown here, these peptide-binding peptides can be used to detect or purify proteins containing the partner peptide. Nat Biotechnol, 2000 Jan, 18(1), 62 - 5 Transgenic zebrafish for detecting mutations caused by compounds in aquatic environments; Amanuma K et al.; We have established a transgenic zebrafish line carrying a shuttle vector plasmid (pML4) for detecting mutagens in aquatic environments . The plasmid contains the rpsL gene of Escherichia coli as a mutational target gene, and the kanamycin-resistance gene for recovering the plasmid from the chromosomal DNA . To evaluate the system, we treated embryos of the transgenic fish with N-ethyl-N-nitrosourea (ENU), which induces a dose-dependent increase in the mutation frequency of the target gene . The mutation spectrum was consistent with the proposed mechanism of ENU mutagenesis . Similarly, treating the embryos with benzo{a}pyrene or 2-amino-3, 8-dimethylimidazo{4,5- f}quinoxaline, which are found in naturally polluted water, significantly increased the frequency of mutations in the target gene. Pharmacogenetics, 1999 Dec, 9(6), 763 - 71 Characterization of a novel variant (S145C/L311V) of 3alpha-hydroxysteroid/dihydrodiol dehydrogenase in human liver; Kume T et al.; Human liver 3alpha-hydroxysteroid/dihydrodiol dehydrogenase (DD) is involved in the metabolism of steroid hormones and polycyclic aromatic hydrocarbons, and is also responsible for the reduction of ketone-containing drugs . To account for the interindividual difference in the activity, we isolated and characterized clones for the human liver enzymes . The sequence of the cDNA clone coding for the variant differed from that coding for the wild-type DD by two nucleotides (substitutions of C with G at positions 434 and 931) which caused two amino acid replacements, Ser145 to Cys (S145C) and Leu311 to Val (L311V) . The heterologous expression of the variant mRNA was confirmed in four of 31 liver samples from Japanese by an allele-specific polymerase chain reaction . The effects of the mutations on the catalytic properties were examined with the recombinant enzymes expressed in Escherichia coli . The introduction of S145C/L311V double mutations resulted in three- to five-fold decreased activities for xenobiotic and steroidal substrates, whereas no significant change was observed by an introduction of the S145C mutation alone . The results substantiate the existence of polymorphic forms for human liver DD, and also suggest the importance of the residue at position 311 for substrate binding to the enzyme. J Bacteriol, 2000 Feb, 182(3), 855 - 8 Posttranslational processing of Methanococcus voltae preflagellin by preflagellin peptidases of M . voltae and other methanogens; Correia JD et al.; Methanococcus voltae is a mesophilic archaeon with flagella composed of flagellins that are initially made with 11- or 12-amino-acid leader peptides that are cleaved prior to incorporation of the flagellin into the growing filament . Preflagellin peptidase activity was demonstrated in immunoblotting experiments with flagellin antibody to detect unprocessed and processed flagellin subunits . Escherichia coli membranes containing the expressed M . voltae preflagellin (as the substrate) were combined in vitro with methanogen membranes (as the enzyme source) . Correct processing of the preflagellin to the mature flagellin was also shown directly by comparison of the N-terminal sequences of the two flagellin species . M . voltae preflagellin peptidase activity was optimal at 37 degrees C and pH 8.5 and in the presence of 0.4 M KCl with 0.25% (vol/vol) Triton X-100. J Bacteriol, 2000 Feb, 182(3), 842 - 7 Towards single-copy gene expression systems making gene cloning physiologically relevant: lambda InCh, a simple Escherichia coli plasmid-chromosome shuttle system; Boyd D et al.; We describe a simple system for reversible, stable integration of plasmid-borne genes into the Escherichia coli chromosome . Most ordinary E . coli strains and a variety of pBR322-derived ampicillin-resistant plasmids can be used . A single genetic element, a lambda phage, is the only specialized vector required . The resultant strains have a single copy of the plasmid fragment inserted stably at the lambda attachment site on the chromosome, with nearly the entire lambda genome deleted. J Bacteriol, 2000 Feb, 182(3), 829 - 32 Increased sensitivity to oxidative challenges associated with topA deletion in Escherichia coli; Tse-Dinh YC; Deletion of topA in Escherichia coli was found to result in a higher level of killing after treatment with either hydrogen peroxide or N-ethylmaleimide . This effect on oxidative challenge response represents a new role for E . coli DNA topoisomerase I in addition to prevention of excessive negative supercoiling of DNA. J Bacteriol, 2000 Feb, 182(3), 723 - 7 On the functional interchangeability, oxidant versus reductant, of members of the thioredoxin superfamily; Debarbieux L et al.; Escherichia coli thioredoxin 1 has been characterized in vivo and in vitro as one of the most efficient reductants of disulfide bonds . Nevertheless, under some conditions, thioredoxin 1 can also act in vivo as an oxidant, promoting formation of disulfide bonds in the cytoplasm (E . J . Stewart, F . Aslund, and J . Beckwith, EMBO J . 17:5543-5550, 1998) . We recently showed that when a signal sequence is attached to thioredoxin 1 it is exported to the periplasm, where it can also act as an oxidant, replacing the normal periplasmic catalyst of disulfide bond formation, DsbA, in oxidizing cell envelope proteins (L . Debarbieux and J . Beckwith, Proc . Natl . Acad . Sci . USA 95:10751-10756, 1998) . Here we report pulse-chase studies of the efficiency of disulfide bond formation in strains exporting thioredoxin 1 and more-oxidizing variants of it . While the exported thioredoxin 1 itself substantially speeds up the kinetics of disulfide bond formation, a version of this protein containing the DsbA active site exhibits kinetics that are indistinguishable from those of the DsbA protein itself . Further, we confirm the findings of Jonda et al . (S . Jonda, M . Huber-Wunderlich, R . Glockshuber, and E . Mossner, EMBO J . 18:3271-3281, 1999), who found that DsbB is responsible for the oxidation of exported thioredoxin 1, and we report the detection of a disulfide-bonded DsbB-thioredoxin 1 complex . Finally, we have found that under conditions of high-level expression of exported thioredoxin 1, the protein can act as both an oxidant and a reductant. J Bacteriol, 2000 Feb, 182(3), 655 - 63 Cloning and characterization of a family B DNA polymerase from the hyperthermophilic crenarchaeon Pyrobaculum islandicum; Kahler M et al.; In order to extend the limited knowledge about crenarchaeal DNA polymerases, we cloned a gene encoding a family B DNA polymerase from the hyperthermophilic crenarchaeon Pyrobaculum islandicum . The enzyme shared highest sequence identities with a group of phylogenetically related DNA polymerases, designated B3 DNA polymerases, from members of the kingdom Crenarchaeota, Pyrodictium occultum and Aeropyrum pernix, and several members of the kingdom Euryarchaeota . Six highly conserved regions as well as a DNA-binding motif, indicative of family B DNA polymerases, were identified within the sequence . Furthermore, three highly conserved 3'-5' exonuclease motifs were also found . The gene was expressed in Escherichia coli, and the DNA polymerase was purified to homogeneity by heat treatment and affinity chromatography . Activity staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an active polypeptide of approximately 90 kDa . For the recombinant DNA polymerase from P . islandicum, activated calf thymus DNA was used as a substrate rather than primed single-stranded DNA . The enzyme was strongly inhibited by monovalent cations and N-ethylmaleimide; it is moderately sensitive to aphidicolin and dideoxyribonucleoside triphosphates . The half-life of the enzyme at 100 and 90 degrees C was 35 min and >5 h, respectively . Interestingly, the pH of the assay buffer had a significant influence on the 3'-5' exonuclease activity of the recombinant enzyme . Under suitable assay conditions for PCR, the enzyme was able to amplify lambda DNA fragments of up to 1,500 bp. J Bacteriol, 2000 Feb, 182(3), 637 - 46 Surface signaling in ferric citrate transport gene induction: interaction of the FecA, FecR, and FecI regulatory proteins; Enz S et al.; In Escherichia coli, transcription of the ferric citrate transport genes fecABCDE is controlled by a novel signal transduction mechanism that starts at the cell surface . Binding of ferric citrate to the outer membrane protein FecA initiates a signal that is transmitted by FecR across the cytoplasmic membrane into the cytoplasm where FecI, the sigma factor, is activated . Interaction between the signaling proteins was demonstrated by utilizing two methods . In in vitro binding assays, FecR that was His tagged at the N terminus {(His)(10)-FecR} and bound to a Ni-nitrilotriacetic acid agarose column was able to retain FecA, and FecR that was His tagged at the C terminus {FecR-(His)(6)} retained FecI on the column . An N-terminally truncated, induction-negative but transport-active FecA protein did not bind to (His)(10)-FecR . The in vivo assay involved the determination of the FecA, FecR, and FecI interacting domains with the bacterial two-hybrid Lex-based system . FecA(1-79) interacts with FecR(101-317) and FecR(1-85) interacts with FecI(1-173) . These data clearly support a model that proposes interaction of the periplasmic N terminus of FecA with the periplasmic C-terminal portion of FecR and interaction of the cytoplasmic N terminus of FecR with FecI, which results in FecI activation. J Bacteriol, 2000 Feb, 182(3), 613 - 9 Membrane redistribution of the Escherichia coli MinD protein induced by MinE; Rowland SL et al.; Escherichia coli cells contain potential division sites at midcell and adjacent to the cell poles . Selection of the correct division site at midcell is controlled by three proteins: MinC, MinD, and MinE . It has previously been shown (D . Raskin and P . de Boer, Cell 91:685-694, 1997) that MinE-Gfp localizes to the midcell site in an MinD-dependent manner . We use here Gfp-MinD to show that MinD associates with the membrane around the entire periphery of the cell in the absence of the other Min proteins and that MinE is capable of altering the membrane distribution pattern of Gfp-MinD . Studies with the isolated N-terminal and C-terminal MinE domains indicated different roles for the two MinE domains in the redistribution of membrane-associated MinD. Pathol Int, 1999 Dec, 49(12), 1067 - 77 Novel human PDCD4 (H731) gene expressed in proliferative cells is expressed in the small duct epithelial cells of the breast as revealed by an anti-H731 antibody; Yoshinaga H et al.; The novel gene H731 (approved name: PDCD4 (programmed cell death 4)) has been isolated as an antigen gene of the monoclonal antibody Pr-28 which recognized a nuclear antigen in proliferating cells . The gene is homologous to the mouse gene (MA-3/Pdcd4/A7-1) which was associated with apoptosis and was shown to suppress tumor promoter-induced neoplastic transformation . A polyclonal antibody against H731-protein derived from an extract of Escherichia coli transformed with an H731 expression plasmid was prepared, and the H731-protein expression in human normal and tumor cells using the antibody was studied . The staining patterns of asynchronous cultures of human normal fibroblasts (MRC-5) were heterogeneous but the antigen was accumulated in the nuclei at the G0 phase . On the contrary, the antigen was overproduced and localized in the cytoplasm during the cell cycle in tumor cell lines . Immunohistological studies revealed that the H731-protein was highly expressed in bladder carcinoma and breast carcinoma tissues compared with the normal tissues so far tested . These results indicated that expression of the H731-protein was up-regulated or induced in the proliferative cells . Immunohistological studies also revealed that the protein was abundantly expressed in the small duct epithelial cells of the normal mammary gland. Mol Microbiol, 2000 Jan, 35(1), 204 - 10 UvrD-dependent replication of rolling-circle plasmids in Escherichia coli; Bruand C et al.; The Escherichia coli UvrD helicase (or helicase II) is known for its involvement in DNA repair . We report that UvrD is required for DNA replication of several different rolling-circle plasmids in E . coli, whereas its homologue, the Rep helicase, is not . Lack of UvrD helicase does not impair the first step of plasmid replication, nicking of the double-stranded origin by the plasmid initiator protein . However, replication proceeds no further without UvrD . Indeed, the nicked plasmid molecules accumulate to a high level in uvrD mutants . We conclude that UvrD is the replicative helicase of various rolling-circle plasmids . This is the first description of a direct implication of UvrD in DNA replication in vivo. Mol Microbiol, 2000 Jan, 35(1), 113 - 22 Protection of the DNA during the exposure of Escherichia coli cells to a toxic metabolite: the role of the KefB and KefC potassium channels; Ferguson GP et al.; The effect of the toxic metabolite methylglyoxal on the DNA of Escherichia coli cells has been investigated . Exposure of E . coli cells to methylglyoxal reduces the transformability of plasmid DNA and results in the degradation of genomic DNA . The activity of the KefB and KefC potassium channels protects E . coli cells against methylglyoxal and limits the amount of DNA damage . In mutants lacking KefB and KefC, methylglyoxal-induced DNA damage was reduced by incubation with a weak acid that lowers the pHi to the same extent as through KefB and KefC activation . This provides evidence that acidification of the cytoplasm protects E . coli DNA against methylglyoxal . By the analysis of cells lacking UvrA, we demonstrate that this repair protein is required for the degradation of the DNA upon methylglyoxal exposure . However, protection by KefB and KefC occurred independently of UvrA . Although we present evidence that exposure of E . coli cells to methylglyoxal results in DNA degradation, our results suggest this event is not essential for methylglyoxal-induced death . The implications of these findings will be discussed. Mol Microbiol, 2000 Jan, 35(1), 58 - 68 Isolation of the topB gene encoding DNA topoisomerase III as a multicopy suppressor of topA null mutations in Escherichia coli; Broccoli S et al.; One major function of DNA topoisomerase I in Escherichia coli is to repress R-loop formation during transcription elongation, which may otherwise inhibit cell growth . We have previously shown that the growth problems of topA mutants can be corrected by overproducing RNase H, an enzyme that degrades the RNA moiety of an R-loop . The goal of the present study was to identify other potential regulators of R-loop formation . To this end, we have screened for multicopy suppressors of topA null mutations . As expected using this procedure, we cloned the rnhA gene encoding RNase H . In addition, we also identified the topB gene encoding DNA topoisomerase III as an efficient suppressor of topA null mutations and, hence, of R-loop formation . We show that DNA topoisomerase III is able to relax transcription-induced negative supercoiling both in vitro and in vivo . An R-loop is also shown to be a hot-spot for relaxation by DNA topoisomerase III, and we found that R-loop-dependent hypernegative supercoiling can be prevented by the activity of this topoisomerase in vivo . It is also shown that the topB gene can act synergistically with the rnhA gene to correct the growth defect of topA null mutants efficiently . This synergistic effect can be explained by the fact that some R-loops must not be degraded in order for the RNA to be available for protein synthesis . Topoisomerase III can presumably repress the formation of such R-loops or cause their destabilization to prevent RNA degradation . This is supported by the fact that overproduction of this topoisomerase corrects the negative effect of overexpressing RNase H activity on the growth of topA null mutants at low temperatures . Moreover, the fact that DNA topoisomerase III does not relax global supercoiling supports our previous conclusion that R-loop formation, and therefore the essential function of DNA topoisomerase I, involves local, rather than global, supercoiling. Eur J Biochem, 2000 Jan, 267(2), 606 - 15 Cloning of the guanylate kinase homologues AGK-1 and AGK-2 from Arabidopsis thaliana and characterization of AGK-1; Kumar V et al.; Guanylate kinase is an essential enzyme for nucleotide metabolism, phosphorylating GMP to GDP or dGMP to dGDP . The low molecular mass cytosolic forms of guanylate kinase are implicated primarily in the regulation of the supply of guanine nucleotides to cell signalling pathways . The high molecular mass and membrane-associated forms of guanylate kinase homologues, notably found in neuronal tissues, are assigned roles in cell junction organization and transmembrane regulation . Here, we describe the first plant guanylate kinase-encoding genes, AGK1 and AGK2, from Arabidopsis thaliana . The nucleotide sequences of their genomic and cDNA clones predict proteins that carry N-terminal and C-terminal extensions of the guanylate kinase-like domain . The amino acid sequences of this domain share 46-52% identity with guanylate kinases from yeast, Escherichia coli, human, mouse and Caenorhabditis elegans . Arabidopsis guanylate kinases (AGKs) exhibit a high degree of conservation of active site residues and sequence motifs in common with other nucleoside monophosphate kinases, which suggests overall structural similarity of the plant proteins . Although bacterially expressed AGK-1 is enzymatically much less active than yeast guanylate kinase, its kinase domain is shown to complement yeast GUK1 recessive lethal mutations . AGKs are expressed ubiquitously in plant tissues with highest transcriptional activity detected in roots . The identification of AGKs provides new perspectives for understanding the role of guanylate kinases in plant cell signalling pathways. Eur J Biochem, 2000 Jan, 267(2), 498 - 506 The N-terminal region of the heme-regulated eIF2alpha kinase is an autonomous heme binding domain; Uma S et al.; The N-terminal domain (NTD) of the heme-regulated eukaryotic initiation factor (eIF)2alpha kinase (HRI) was aligned to sequences in the NCBI data base using ENTREZ and a PAM250 matrix . Significant similarity was found between amino acids 11-118 in the NTD of rabbit HRI and amino acids 16-120 in mammalian alpha-globins . Several conserved amino acid residues present in globins are conserved in the NTD of HRI . His83 of HRI was predicted to be equivalent to the proximal heme ligand (HisF8) that is conserved in all globins . Molecular modeling of the NTD indicated that its amino acid sequence was compatible with the globin fold . Recombinant NTD (residues 1-159) was expressed in Escherichia coli . Spectral analysis of affinity purified recombinant NTD indicated that the NTD contained stably bound hemin . Mutational analysis indicated that His83 played a critical structural role in the stable binding of heme to the NTD, and was required to stabilize full length HRI synthesized de novo in the rabbit reticulocyte lysate . These results indicate that the NTD of HRI is an autonomous heme-binding domain, with His83 possibly serving as the proximal heme binding ligand. Eur J Biochem, 2000 Jan, 267(2), 321 - 8 Novel prenyltransferase gene encoding farnesylgeranyl diphosphate synthase from a hyperthermophilic archaeon, Aeropyrum pernix . Molecularevolution with alteration in product specificity; Tachibana A et al.; Prenyltransferases catalyse sequential condensations of isopentenyl diphosphate with allylic diphosphates . Previously, we reported the presence of farnesylgeranyl diphosphate (FGPP) synthase activity synthesizing C25 isoprenyl diphosphate in Natronobacterium pharaonis which is a haloalkaliphilic archaeon having C20-C25 diether lipids in addition to C20-C20 diether lipids commonly occurring in archaea {Tachibana, A . (1994) FEBS Lett . 341, 291-294} . Recently, it was found that a newly isolated aerobic hyperthermophilic archaeon, Aeropyrum pernix, had only C25-C25 diether lipids, not the usual C20-containing lipids {Morii, H., Yagi, H., Akutsu, H., Nomura, N., Sako, Y . & Koga, Y . (1999) Biochim . Biophys . Acta 1436, 426-436} . In this report, we describe the isoloation from A . pernix of the novel prenyltransferase gene, fgs, encoding FGPP synthase . The protein encoded by fgs was expressed in Escherichia coli as a glutathione S-transferase fusion protein and produced FGPP as a final product . Phylogenetic analysis of fgs with other prenyltransferases revealed that the short-chain prenyltransferase family is divided into three subfamilies: bacterial subfamily I, eukaryotic subfamily II, and archaeal subfamily III . fgs is clearly contained within the archaeal geranylgeranyl diphosphate (GGPP) synthase group (subfamily III), suggesting that FGPP synthase evolved from an archaeal GGPP synthase with an alteration in product specificity. Protein Sci, 1999 Dec, 8(12), 2791 - 805 Function of the central domain of streptokinase in substrate plasminogen docking and processing revealed by site-directed mutagenesis; Chaudhary A et al.; The possible role of the central beta-domain (residues 151-287) of streptokinase (SK) was probed by site-specifically altering two charged residues at a time to alanines in a region (residues 230-290) previously identified by Peptide Walking to play a key role in plasminogen (PG) activation . These mutants were then screened for altered ability to activate equimolar "partner" human PG, or altered interaction with substrate PG resulting in an overall compromised capability for substrate PG processing . Of the eight initial alanine-linker mutants of SK, one mutant, viz . SK(KK256.257AA) (SK-D1), showed a roughly 20-fold reduction in PG activator activity in comparison to wild-type SK expressed in Escherichia coli (nSK) . Five other mutants were as active as nSK, with two {SK(RE248.249AA) and SK(EK281.282AA), referred to as SK(C) and SK(H), respectively} showing specific activities approximately one-half and two-thirds, respectively, that of nSK . Unlike SK(C) and SK(H), however, SK(D1) showed an extended initial delay in the kinetics of PG activation . These features were drastically accentuated when the charges on the two Lys residues at positions 256 and 257 of nSK were reversed, to obtain SK(KK256.257EE) {SK(D2)} . This mutant showed a PG activator activity approximately 10-fold less than that of SK(D1) . Remarkably, inclusion of small amounts of human plasmin (PN) in the PG activation reactions of SK(D2) resulted in a dramatic, PN dose-dependent rejuvenation of its PG activation capability, indicating that it required pre-existing PN to form a functional activator since it could not effect active site exposure in partner PG on its own, a conclusion further confirmed by its inability to show a "burst" of p-nitrophenol release in the presence of equimolar human PG and p-nitrophenyl guanidino benzoate . The steady-state kinetic parameters for HPG activation of its 1:1 complex with human PN revealed that although it could form a highly functional activator once "supplied" with a mature active site, the Km for PG was increased nearly eightfold in comparison to that of nSK-PN . SK mutants carrying simultaneous two- and three-site charge-cluster alterations, viz., SK(RE24249AA:EK281.282AA) {SK(CH)}, SK(EK272.273AA;EK281.282AA) {SK(FH)}, and SK(RE248.249AA;EK272.273AA:EK281.282AA+ ++) {SK(CFH)}, showed additive/synergistic influence of multiple charge-cluster mutations on HPG activation when compared to the respective "single-site" mutants, with the "triple-site" mutant {SK(CFH)} showing absolutely no detectable HPG activation ability . Nevertheless, like the other constructs, the double- and triple-charge cluster mutants retained a native like affinity for complexation with partner PG . Their overall structure also, as judged by far-ultraviolet circular dichroism, was closely similar to that of nSK . These results provide the first experimental evidence for a direct assistance by the SK beta-domain in the docking and processing of substrate PG by the activator complex, a facet not readily evident probably because of the flexibility of this domain in the recent X-ray crystal structure of the SK-plasmin light chain complex. Protein Sci, 1999 Dec, 8(12), 2686 - 96 Inhibition of the HIV-1 and HIV-2 proteases by a monoclonal antibody; Lescar J et al.; The monoclonal antibody 1696, directed against the HIV-1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates . This antibody cross-reacts with peptides that include the N-terminus of the enzyme, a region that is well conserved in sequence among different viral strains and which, furthermore, is crucial for homodimerization to the active enzymatic form . This observation, as well as antigen-binding studies in the presence of an active site inhibitor, suggest that 1696 inhibits the HIV protease by destabilizing its active homodimeric form . To characterize further how the antibody 1696 inhibits the HIV-1 and HIV-2 proteases, we have solved the crystal structure of its Fab fragment by molecular replacement and refined it at 3.0 A resolution . The antigen binding site has a deep cavity at its center, which is lined mainly by acidic and hydrophobic residues, and is large enough to accommodate several antigen residues . The structure of the Fab 1696 could form a starting basis for the design of alternative HIV protease-inhibiting molecules of broad specificity. Protein Sci, 1999 Dec, 8(12), 2655 - 62 The mechanism of aconitase: 1.8 A resolution crystal structure of the S642a:citrate complex; Lloyd SJ et al.; The crystal structure of the S642A mutant of mitochondrial aconitase (mAc) with citrate bound has been determined at 1.8 A resolution and 100 K to capture this binding mode of substrates to the native enzyme . The 2.0 A resolution, 100 K crystal structure of the S642A mutant with isocitrate binding provides a control, showing that the Ser --> Ala replacement does not alter the binding of substrates in the active site . The aconitase mechanism requires that the intermediate product, cis-aconitate, flip over by 180 degrees about the C alpha-C beta double bond . Only one of these two alternative modes of binding, that of the isocitrate mode, has been previously visualized . Now, however, the structure revealing the citrate mode of binding provides direct support for the proposed enzyme mechanism. Protein Sci, 1999 Dec, 8(12), 2562 - 9 Changing the surface of a virus shell fusion of an enzyme to polyoma VP1; Gleiter S et al.; Recent developments on virus-like particles have demonstrated their potential in transfecting eucaryotic cells . In the case of particles based on the major coat protein VP1 of polyoma virus, transfection occurs via binding of VP1 to sialic acids . Since sialic acid is present on almost every eucaryotic cell line, this results in an unspecific cell targeting . Generation of a cell-type specificity of this system would imply the presentation of a new function on the surface of VP1 . To analyze whether a new functional protein can be placed on VP1, we inserted dihydrofolate reductase from Escherichia coli as a model protein . The effect of such an insertion on both VP1 and the inserted protein was investigated, respectively . The function of VP1, like the formation of pentameric capsomers and its ability to assemble into capsids, was not influenced by the insertion . The inserted dihydrofolate reductase showed major changes when compared to the wild-type form . The thermal stability of the enzyme was dramatically reduced in the fusion protein; nevertheless, the dihydrofolate reductase proved to be a fully active enzyme with only slightly increased K(M) values for its substrates . This model system provides the basis for further modifications of the VP1 protein to achieve an altered surface of VP1 with new properties. Comp Biochem Physiol B Biochem Mol Biol, 1999 Nov, 124(3), 269 - 80 Anaerobiosis in the nematode Caenorhabditis elegans; Foll RL et al.; In Caenorhabditis elegans, mortality rates and changes in concentrations of carbohydrate stores and anaerobic end products were determined in anoxic (test) and normoxic (control) animals at two different temperatures (10 and 20 degrees C) . The anoxic tolerance of the free-living nematode proved to be well-developed: at 10 degrees C, about 50% of animals had survived a period of 50 h of anoxia . The carbohydrate stores (approximately 30 mmol glycosyl units kg-1 freshweight (FW)) were reduced by two-thirds within 24 h of anoxia at both temperatures . L-lactate, acetate, succinate, and propionate were identified as the main anaerobic end products . The amounts and proportions of the end products were dependent on temperature . They did not accumulate very much in the tissues, but were mainly excreted . During anoxia, the metabolism of C . elegans was depressed to 3-4% of the aerobic value . The food-source Escherichia coli was found to be at least partly alive in the gut of the animals . To separate between anaerobiosis in animals and bacteria, cleaning procedures were applied, and additional control measurements were made: anaerobic end products produced either by E . coli alone or by bacteria-free (axenic) bred nematodes were quantified at identical incubation conditions. FEBS Lett, 2000 Jan 14, 465(2-3), 161 - 4 Crystal structure of Escherichia coli UvrB C-terminal domain, and a model for UvrB-uvrC interaction; Sohi M et al.; A crystal structure of the C-terminal domain of Escherichia coli UvrB (UvrB') has been solved to 3.0 A resolution . The domain adopts a helix-loop-helix fold which is stabilised by the packing of hydrophobic side-chains between helices . From the UvrB' fold, a model for a domain of UvrC (UvrC') that has high sequence homology with UvrB' has been made . In the crystal, a dimerisation of UvrB domains is seen involving specific hydrophobic and salt bridge interactions between residues in and close to the loop region of the domain . It is proposed that a homologous mode of interaction may occur between UvrB and UvrC . This interaction is likely to be flexible, potentially spanning > 50 A. FEBS Lett, 2000 Jan 14, 465(2-3), 157 - 60 Biosynthesis of terpenoids: 1-deoxy-D-xylulose-5-phosphate reductoisomerase from Escherichia coli is a class B dehydrogenase; Radykewicz T et al.; 1-Deoxy-D-xylulose-5-phosphate is converted into 2-C-methyl-D-erythritol-4-phosphate by the catalytic action of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr protein) using NADPH as cofactor . The stereochemical features of this reaction were investigated in in vitro experiments with the recombinant Dxr protein of Escherichia coli using (4R)- or (4S)-{4-(2)H(1)}NADPH as coenzyme . The enzymatically formed 2-C-methyl-D-erythritol-4-phosphate was isolated and converted into 1,2:3,4-di-O-isopropylidene-2-C-methyl-D-erythritol; NMR spectroscopic investigation of this derivative indicated that only (4S)-{4-(2)H(1)}NADPH affords 2-C-methyl-D-erythritol-4-phosphate labelled exclusively in the H(Re) position of C-1 . Stereospecific transfer of H(Si) from C-4 of the cofactor identifies the Dxr protein of E . coli as a class B dehydrogenase. Biochem Biophys Res Commun, 2000 Jan 19, 267(2), 588 - 92 Deamidation of RhoA glutamine 63 by the Escherichia coli CNF1 toxin requires a short sequence of the GTPase switch 2 domain; Flatau G et al.; CNF1, a toxin produced by pathogenic Escherichia coli strains, deamidates the RhoA GTP-binding protein glutamine 63 and impairs RhoGAP-mediated GTP hydrolysis resulting in RhoA permanent activation . Using peptides derived from the RhoA sequence, we found that DTAGQEDYDRL (corresponding to RhoA 59-69 residues) was the minimum RhoA-derived peptide which could be deamidated in vitro by the CNF1 catalytic domain (CNF1-Cter) . Site-directed mutagenesis outside the RhoA 59-69 sequence had no influence on glutamine 63 deamidation by CNF1-Cter . RhoA proteins with substitutions L57G, D65G, Y66G, or R70G were not affected in their ability to be deamidated by CNF1-Cter, whereas this was abolished by the R68G substitution . Arginine 68 is part of the DYDRL motif that is strictly conserved in Rho, Rac, and Cdc42 but not in other small GTP-binding proteins consistent with the observation that only Rho, Rac, and Cdc42 can be modified by CNF1 . Biochem Biophys Res Commun, 2000 Jan 19, 267(2), 558 - 64 A novel gene family with a developmentally regulated expression in Xenopus laevis; Shim S et al.; We have isolated a new maternal gene called 4G2 . 4G2 cDNA encodes a predicted protein of 501 amino acids, and its apparent molecular mass of 61 kDa was determined by SDS-PAGE of 4G2 recombinant protein expressed in E . coli or in vitro translated in rabbit reticulocyte lysate . Amino acid analysis of 4G2 revealed the RGD and LDV motif with a potential cell attachment activity . The open reading frames (ORF) also contained a consensus bipartite nuclear localization signal (NLS) . There were number of expressed tag sequences (ESTs) from Drosophila, zebrafish, chicken, mouse, and human origin that encode a high degree of identity to the predicted 4G2 protein, thereby suggesting that 4G2 may constitute a novel gene family whose function has not been elucidated . We also present evidence that 4G2 transcript is maternally synthesized in stage IV oocyte, localized to animal hemisphere of egg, and zygotically reactivated in mid-neurula stage . Copyright 2000 Academic Press.
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