Protein Eng, 2001 Dec, 14(12), 949 - 52 Silent mutations in secondary Shine-Dalgarno sequences in the cDNA of human serum amyloid A4 promotes expression of recombinant protein in Escherichia coli; Hrzenjak A et al.; The serum amyloid A (SAA) superfamily comprises a number of differentially expressed genes with a high degree of homology in mammalian species . SAA4, an apolipoprotein constitutively expressed only in humans and mice, is associated almost entirely with lipoproteins of the high-density range . The presence of SAA4 mRNA and protein in macrophage-derived foam cells of coronary and carotid arteries suggested a specific role of human SAA4 during inflammation including atherosclerosis . Here we underline the importance of ribosome binding site (rbs)-like sequences (also known as Shine-Dalgarno sequences) in the SAA4 cDNA for expression of recombinant SAA4 protein in Escherichia coli . In contrast to rbs sequences coded by the expression vectors, rbs-like sequences in the cDNA of target protein(s) are known to interfere with protein translation via binding to the small 16S ribosome subunit, yielding low or even no expression . Here we show that PCR mutations of two rbs-like sequences in the human SAA4 cDNA promote expression of considerable amounts of recombinant SAA4 in E.coli. Nucleic Acids Res, 2002 Feb 1, 30(3), 818 - 22 The function of Asp70, Glu77 and Lys79 in the Escherichia coli MutH protein; Wu TH et al.; The MutH protein, which is part of the Dam-directed mismatch repair system of Escherichia coli, introduces nicks in the unmethylated strand of a hemi-methylated DNA duplex . The latent endonuclease activity of MutH is activated by interaction with MutL, another member of the repair system . The crystal structure of MutH suggested that the active site residues include Asp70, Glu77 and Lys79, which are located at the bottom of a cleft where DNA binding probably occurs . We mutated these residues to alanines and found that the mutant proteins were unable to complement a chromosomal mutH deletion . The purified mutant proteins were able to bind to DNA with a hemi-methylated GATC sequence but had no detectable endonuclease activity with or without MutL . Although the data are consistent with the prediction of a catalytic role for Asp70, Glu77 and Lys79, it cannot be excluded that they are also involved in binding to MutL. Nucleic Acids Res, 2002 Feb 1, 30(3), 685 - 94 Identification, cloning and characterization of a new DNA-binding protein from the hyperthermophilic methanogen Methanopyrus kandleri; Pavlov NA et al.; Three novel DNA-binding proteins with apparent molecular masses of 7, 10 and 30 kDa have been isolated from the hyperthermophilic methanogen Methanopyrus kandleri . The proteins were identified using a blot overlay assay that was modified to emulate the high ionic strength intracellular environment of M.kandleri proteins . A 7 kDa protein, named 7kMk, was cloned and expressed in Escherichia coli . As indicated by CD spectroscopy and computer-assisted structure prediction methods, 7kMk is a substantially alpha-helical protein possibly containing a short N-terminal beta-strand . According to analytical gel filtration chromatography and chemical crosslinking, 7kMk exists as a stable dimer, susceptible to further oligomerization . Electron microscopy showed that 7kMk bends DNA and also leads to the formation of loop-like structures of approximately 43.5 +/- 3.5 nm (136 +/- 11 bp for B-form DNA) circumference . A topoisomerase relaxation assay demonstrated that looped DNA is negatively supercoiled under physiologically relevant conditions (high salt and temperature) . A BLAST search did not yield 7kMk homologs at the amino acid sequence level, but based on a multiple alignment with ribbon-helix-helix (RHH) transcriptional regulators, fold features and self-association properties of 7kMk we hypothesize that it could be related to RHH proteins. Nucleic Acids Res, 2002 Feb 1, 30(3), 656 - 66 Supercoiling, knotting and replication fork reversal in partially replicated plasmids; Olavarrieta L et al.; To study the structure of partially replicated plasmids, we cloned the Escherichia coli polar replication terminator TerE in its active orientation at different locations in the ColE1 vector pBR18 . The resulting plasmids, pBR18-TerE@StyI and pBR18-TerE@EcoRI, were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis and electron microscopy . Replication forks stop at the Ter-TUS complex, leading to the accumulation of specific replication intermediates with a mass 1.26 times the mass of non-replicating plasmids for pBR18-TerE@StyI and 1.57 times for pBR18-TerE@EcoRI . The number of knotted bubbles detected after digestion with ScaI and the number and electrophoretic mobility of undigested partially replicated topoisomers reflect the changes in plasmid topology that occur in DNA molecules replicated to different extents . Exposure to increasing concentrations of chloroquine or ethidium bromide revealed that partially replicated topoisomers (CCCRIs) do not sustain positive supercoiling as efficiently as their non-replicating counterparts . It was suggested that this occurs because in partially replicated plasmids a positive DeltaLk is absorbed by regression of the replication fork . Indeed, we showed by electron microscopy that, at least in the presence of chloroquine, some of the CCCRIs of pBR18-Ter@StyI formed Holliday-like junction structures characteristic of reversed forks . However, not all the positive supercoiling was absorbed by fork reversal in the presence of high concentrations of ethidium bromide. Mol Pharmacol, 2002 Feb, 61(2), 400 - 6 Chimeric human immunodeficiency virus type 1 and feline immunodeficiency virus reverse transcriptases: role of the subunits in resistance/sensitivity to non-nucleoside reverse transcriptase inhibitors; Auwerx J et al.; Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are specific for human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and do not inhibit HIV-2 . Given that the amino acids lining the NNRTI-specific pocket of HIV-1 RT display higher similarity to the corresponding feline immunodeficiency virus (FIV) RT amino acids than to HIV-2 RT, the susceptibility of FIV RT and chimeric HIV-1/FIV RTs to NNRTIs and the role of the p51 subunit in the inhibitory action of NNRTIs were investigated . We found that the wild-type FIV RT and the FIVp66/HIVp51 chimeric enzyme showed no susceptibility for NNRTIs . On the other hand, the chimeric HIVp66/FIVp51 RT retained a sensitivity spectrum for NNRTIs similar to that of the wild-type HIV-1 RT . The noncompetitive nature of inhibition of HIV-1 RT by nevirapine was also observed with the HIVp66/FIVp51 chimeric enzyme . Inhibition of the chimeric RTs by nucleoside reverse transcriptase inhibitors and foscarnet was in the same range as observed for the corresponding HIVp66/HIVp51 and FIVp66/FIVp51 wild-type enzymes . The chimeric RTs had an affinity (K(m)) for their dNTP substrate and template/primer comparable with that of the wild-type HIV-1 and FIV RTs, but their catalytic efficacy (k(cat)) was markedly decreased . This decreased catalytic efficacy of the RT chimeras may suggest suboptimal interactions between p66 and p51 in the chimeric enzymes . Our results point to a minor role of the p51 subunit in the sensitivity to RT inhibitors. Mol Biol Cell, 2002 Jan, 13(1), 382 - 91 Pairwise assembly determines the intrinsic potential for self-organization and mechanical properties of keratin filaments; Yamada S et al.; Most type I and II keratin genes are spatially and temporally regulated in a pairwise manner in epithelial tissues, where they represent the major structural proteins . Epithelia can be partitioned into simple (single-layered) and complex (multilayered) types . We compared the structural and mechanical properties of natural keratin polymers occurring in complex (K5-K14) and simple (K8-K18) epithelia . The intrinsic properties of these distantly related keratin filaments, whether dispersed or bundled in vitro, were surprisingly similar in all respects when at high polymer concentration . When type I and II assembly partners were switched to give rise to mismatched polymers (K5-K18; K8-K14), the interfilament interactions, which determine the structural and mechanical properties of keratin polymers, were significantly altered . We also show that a K5-K16 polymer exhibits lesser elasticity than K5- K14, which may help explain the inability of K16 to fully rescue the skin blistering characteristic of K14 null mice . The property of self-interaction exhibited by keratin filaments is likely to assist their function in vivo and may account for the relative paucity of cytoplasmic and keratin-specific cross-linkers . Our findings underscore the fundamental importance of pairwise polymerization and have implications for the functional significance of keratin sequence diversity. Mol Biol Cell, 2002 Jan, 13(1), 146 - 57 Conserved domains of the Nullo protein required for cell-surface localization and formation of adherens junctions; Hunter C et al.; During cellularization, the Drosophila melanogaster embryo undergoes a transition from syncytial to cellular blastoderm with the de novo generation of a polarized epithelial sheet in the cortex of the embryo . This process couples cytokinesis with the establishment of apical, basal, and lateral membrane domains that are separated by two spatially distinct adherens-type junctions . In nullo mutant embryos, basal junctions fail to form at the onset of cellularization, leading to the failure of cleavage furrow invagination and the generation of multinucleate cells . Nullo is a novel protein that appears to stabilize the initial accumulation of cadherins and catenins as they form a mature basal junction . In this article we characterize a nullo homologue from D . virilis and identify conserved domains of Nullo that are required for basal junction formation . We also demonstrate that Nullo is a myristoylprotein and that the myristate group acts in conjunction with a cluster of basic amino acids to target Nullo to the plasma membrane . The membrane association of Nullo is required in vivo for its role in basal junction formation and for its ability to block apical junction formation when ectopically expressed during late cellularization. Mol Biol Cell, 2002 Jan, 13(1), 110 - 8 Aspergillus nidulans septin AspB plays pre- and postmitotic roles in septum, branch, and conidiophore development; Westfall PJ et al.; Members of the septin family of proteins act as organizational scaffolds in areas of cell division and new growth in a variety of organisms . Herein, we show that in the filamentous fungus Aspergillus nidulans, the septin AspB is important for cellular division, branching, and conidiation both pre- and postmitotically . AspB localizes postmitotically to the septation site with an underlying polarity that is evident as cytokinesis progresses . This localization at the septation site is dependent on actin and occurs before the cross-wall is visible . AspB localizes premitotically as a ring at sites of branching and secondary germ tube emergence . It is the only known branch site marker . In addition, AspB is found at several stages during the development of the asexual reproductive structure, the conidiophore . It localizes transiently to the vesicle/metula and metula/phialide interfaces, and persistently to the phialide/conidiospore interface . A temperature-sensitive mutant of AspB shows phenotypic abnormalities, including irregular septa, high numbers of branches, and immature asexual reproductive structures. Mol Biol Cell, 2002 Jan, 13(1), 52 - 70 Testing a mathematical model of the yeast cell cycle; Cross FR et al.; We derived novel, testable predictions from a mathematical model of the budding yeast cell cycle . A key qualitative prediction of bistability was confirmed in a strain simultaneously lacking cdc14 and G1 cyclins . The model correctly predicted quantitative dependence of cell size on gene dosage of the G1 cyclin CLN3, but it incorrectly predicted strong genetic interactions between G1 cyclins and the anaphase-promoting complex specificity factor Cdh1 . To provide constraints on model generation, we determined accurate concentrations for the abundance of all nine cyclins as well as the inhibitor Sic1 and the catalytic subunit Cdc28 . For many of these we determined abundance throughout the cell cycle by centrifugal elutriation, in the presence or absence of Cdh1 . In addition, perturbations to the Clb-kinase oscillator were introduced, and the effects on cyclin and Sic1 levels were compared between model and experiment . Reasonable agreement was obtained in many of these experiments, but significant experimental discrepancies from the model predictions were also observed . Thus, the model is a strong but incomplete attempt at a realistic representation of cell cycle control . Constraints of the sort developed here will be important in development of a truly predictive model. Mol Cell Biol, 2002 Feb, 22(4), 1079 - 93 Spt5 cooperates with human immunodeficiency virus type 1 Tat by preventing premature RNA release at terminator sequences; Bourgeois CF et al.; The human immunodeficiency virus type 1 (HIV-1) Tat protein activates transcription elongation by stimulating the Tat-activated kinase (TAK/p-TEFb), a protein kinase composed of CDK9 and its cyclin partner, cyclin T1 . CDK9 is able to hyperphosphorylate the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase during elongation . In addition to TAK, the transcription elongation factor Spt5 is required for the efficient activation of transcriptional elongation by Tat . To study the role of Spt5 in HIV transcription in more detail, we have developed a three-stage Tat-dependent transcription assay that permits the isolation of active preinitiation complexes, early-stage elongation complexes, and Tat-activated elongation complexes . Spt5 is recruited in the transcription complex shortly after initiation . After recruitment of Tat during elongation through the transactivation response element RNA, CDK9 is activated and induces hyperphosphorylation of Spt5 in parallel to the hyperphosphorylation of the CTD of RNA polymerase II . However, immunodepletion experiments demonstrate that Spt5 is not required for Tat-dependent activation of the kinase . Chase experiments using the Spt5-depleted extracts demonstrate that Spt5 is not required for early elongation . However, Spt5 plays an important role in late elongation by preventing the premature dissociation of RNA from the transcription complex at terminator sequences and reducing the amount of polymerase pausing at arrest sites, including bent DNA sequences . This novel biochemical function of Spt5 is analogous to the function of NusG, an elongation factor found in Escherichia coli that enhances RNA polymerase stability on templates and shows sequence similarity to Spt5. J Biol Chem, 2002 Apr 12, 277(15), 13237 - 45 Epub 2002 Jan 24. Biochemical characterization of an invariant histidine involved in Escherichia coli DNA topoisomerase I catalysis; Perry K et al.; An invariant histidine residue, His-365 in Escherichia coli DNA topoisomerase I, is located at the active site of type IA DNA topoisomerases and near the active site tyrosine . Its ability to participate in the multistep catalytic process of DNA relaxation was investigated . His-365 was mutated to alanine, arginine, asparagine, aspartate, glutamate, and glutamine to study its ability to participate in general acid/base catalysis and bind DNA . The mutants were examined for pH-dependent DNA relaxation and cleavage, salt-dependent DNA relaxation, and salt-dependent DNA binding affinity . The mutants relax DNA in a pH-dependent manner and at low salt concentrations . The pH dependence of all mutants is different from the wild type, suggesting that His-365 is responsible for the pH dependence of the enzyme . Additionally, whereas the wild type enzyme shows pH-dependent oligonucleotide cleavage, cleavage by both H365Q and H365A is pH-independent . H365Q cleaves DNA with rates similar to the wild type enzyme, whereas H365A has a slower rate of DNA cleavage than the wild type but can cleave more substrate overall . H365A also has a lower DNA binding affinity than the wild type enzyme . The binding affinity was determined at different salt concentrations, showing that the alanine mutant displaces half a charge less upon binding DNA than an inactive form of topoisomerase I . These observations indicate that His-365 participates in DNA binding and is responsible for optimal catalysis at physiological pH. J Biol Chem, 2002 Apr 12, 277(15), 13246 - 56 Epub 2002 Jan 24. A three-domain structure for the delta subunit of the DNA polymerase III holoenzyme delta domain III binds delta' and assembles into the DnaX complex; Bullard JM et al.; Using psi-BLAST, we have developed a method for identifying the poorly conserved delta subunit of the DNA polymerase III holoenzyme from all sequenced bacteria . This approach, starting with Escherichia coli delta, leads not only to the identification of delta but also to the DnaX and delta' subunits of the DnaX complex and other AAA(+)-class ATPases . This suggests that, although not an ATPase, delta is related structurally to the other subunits of the DnaX complex that loads the beta sliding clamp processivity factor onto DNA . To test this prediction, we aligned delta sequences with those of delta' and, using the start of delta' Domain III established from its x-ray crystal structure, predicted the juncture between Domains II and III of delta . This putative delta Domain III could be expressed to high levels, consistent with the prediction that it folds independently . delta Domain III, like Domain III of DnaX and delta', assembles by itself into a complex with the other DnaX complex components . Cross-linking studies indicated a contact of delta with the DnaX subunits . These observations are consistent with a model where two tau subunits and one each of the gamma, delta', and delta subunits mutually interact to form a pentameric functional core for the DnaX complex. J Biol Chem, 2002 Apr 12, 277(15), 13074 - 81 Epub 2002 Jan 23. Substrate induced conformational changes in argininosuccinate synthetase; Lemke CT et al.; Argininosuccinate synthetase (AS) is the rate-limiting enzyme of both the urea and arginine-citrulline cycles . In mammals, deficiency of AS leads to citrullinemia, a debilitating and often fatal autosomal recessive urea cycle disorder, whereas its overexpression for sustained nitric oxide production via the arginine-citrulline cycle leads to the potentially fatal hypotension associated with septic and cytokine-induced circulatory shock . The crystal structures of Escherichia coli argininosuccinate synthetase (EAS) in complex with ATP and with ATP and citrulline have been determined at 2.0-A resolution . These are the first EAS structures to be solved in the presence of a nucleotide substrate and clearly identify the residues that interact with both ATP and citrulline . Two distinct conformations are revealed for ATP, both of which are believed to be catalytically relevant . In addition, comparisons of these EAS structures with those of the apoenzyme and EAS complexed with aspartate and citrulline (Lemke, C . T., and Howell, P . L . (2001) Structure (Lond.) 9, 1153-1164) provide structural evidence of ATP-induced conformational changes in the nucleotide binding domain . Combined, these structures also provide structural explanations of some of the observed kinetic properties of the enzyme and have enabled a detailed enzymatic mechanism of AS catalysis to be proposed. Cancer Lett, 2002 Mar 8, 177(1), 29 - 39 Efficient inhibition of human B-cell lymphoma xenografts with an anti-CD20 x anti-CD3 bispecific diabody; Xiong D et al.; Bispecific antibodies have been exploited both as cancer immunodiagnostics and as cancer therapeutics, and have shown promise in several clinical trials in cancer imaging and therapy . A number of bispecific antibodies against B-cell markers have been shown to be effective in vitro in mediating tumor cell lysis and in vivo in inhibiting tumor growth in animal models . We have constructed a bispecific diabody from the variable genes encoding two hybridoma-derived monoclonal antibodies directed against human CD20 on B cells and CD3 on T cells . The anti-CD20 x anti-CD3 diabody was expressed in a single Escherichia coli host and purified by a one-step affinity chromatography . The bispecific diabody bound as efficiently to both CD20- and CD3-positive cells as the respective parental antibodies, and was capable of cross-linking CD20-positive tumor cells and human T lymphocytes as shown by cellular rosetting . The diabody effectively lysed human B-lymphoma cells in the presence of T-enriched human peripheral blood lymphocytes (PBL) . Further, when combined with human PBL and interleukin-2, the diabody significantly prolonged the survival of nude mice inoculated with human B-lymphoma cells . Taken together, our results suggest that an anti-CD20 x anti-CD3 diabody may have significant clinical application in the treatment of human CD20-positive B-cell malignancies. J Photochem Photobiol B, 2001 Dec 31, 65(2-3), 145 - 50 Role of DNA polymerase II in the tolerance of thymine dimers remaining unexcised in UV-irradiated Escherichia coli exposed to pre-UV nutritional stress; Sedliakova M et al.; Nutritional stress applied prior to UV-irradiation to E . coli 15 555-7 reduced thymine dimer excision and inhibited post-UV incorporation of thymidine in polB(+) as well as in polB(-) cells . However, the pre-UV-stressed polB(+) cells were significantly more UV-resistant and after UV synthesized larger DNA molecules than the pre-UV-stressed polB(-) cells . The data suggest that DNA polymerase II is involved in the tolerance of unremoved thymine dimers. J Nat Prod, 2002 Jan, 65(1), 21 - 4 Isolation of four new cyclic depsipeptides, antanapeptins A-D, and dolastatin 16 from a Madagascan collection of Lyngbya majuscula; Nogle LM et al.; Examination of a Lyngbya majuscula collection from Antany Mora, Madagascar, led to the isolation of dolastatin 16 (5), a promising antineoplastic metabolite first reported from the marine mollusc Dolabella auricularia . In addition, a new series of depsipeptides, antanapeptins A-D (1-4), were discovered . Their structures were deduced by 2D NMR and mass spectrometry and are analogous to the molluscan kulomo'opunalides and the recently reported cyanobacterial metabolites, georgamide and the yanucamides . The antanapeptins were evaluated in several biological assays; however, this series did not exhibit activity. J Food Prot, 2002 Jan, 65(1), 192 - 5 A new technique for Escherichia coli testing of beef and pork carcasses; Erdmann JJ et al.; A novel technique has been developed to monitor Escherichia coli contamination on carcasses using membrane filtration and m-ColiBlue24 (mCB) . mCB is a membrane filtration medium that simultaneously detects total coliforms and E . coli (EC) in a period of 24 +/- 4 h . A study was conducted, using a sponge method to obtain samples from pork carcasses and the excision technique to remove samples from beef carcasses, that compared mCB to standard methods . On pork carcasses (n = 77), the mean values for mCB and violet red bile agar were 7.4 CFU/15 cm2 and 6.1 CFU/15 cm2, respectively . The paired t test (P > 0.05) indicated no significant difference between the two methods (t = 0.5; P = 0.6) . Samples from beef carcasses (n = 57) were used to compare mCB to both coliform count and EC Petrifilm . Of these samples, 27 were artificially inoculated with cattle manure . The mean total coliform count was 4.2 log CFU/cm2 and 4.0 log CFU/cm2 on mCB and coliform count Petrifilm, respectively . The mean EC count on mCB was 4.0 log CFU/cm2 and 3.5 log CFU/cm2 on EC Petrifilm . When comparing mCB to both coliform count (t = 2.4; P = 0.02) and EC (t = 3.5; P < 0.01) Petrifilm, paired t tests (P < or = 0.05) indicated significant differences. Biotechniques, 2002 Jan, 32(1), 110, 112 - 4, 116, 118-9 DNA array analysis in a Microsoft Windows environment; Conway T et al.; Microsoft Windows-based computers have evolved to the point that they provide sufficient computational and visualization power for robust analysis of DNA array data . In fact, smaller laboratories might prefer to carry out some or all of their analyses and visualization in a Windows environment, rather than alternative platforms such as UNIX . We have developed a series of manually executed macros written in Visual Basic for Microsoft Excel spreadsheets, that allows for rapid and comprehensive gene expression data analysis . The first macro assigns gene names to spots on the DNA array and normalizes individual hybridizations by expressing the signal intensity for each gene as a percentage of the sum of all gene intensities . The second macro streamlines statistical consideration of the confidence in individual gene measurements for sets of experimental replicates by calculating probability values with the Student's t test . The third macro introduces a threshold value, calculates expression ratios between experimental conditions, and calculates the standard deviation of the mean of the log ratio values . Selected columns of data are copied by a fourth macro to create a processed data set suitable for entry into a Microsoft Access database . An Access database structure is described that allows simple queries across multiple experiments and export of data into third-party data visualization software packages . These analysis tools can be used in their present form by others working with commercial E . coli membrane arrays, or they may be adapted for use with other systems . The Excel spreadsheets with embedded Visual Basic macros and detailed instructions for their use are available at http://www.ou.edu/microarray. Proteins, 2002 Feb 1, 46(2), 153 - 60 Design and initial characterization of a circular permuted variant of the potent HIV-inactivating protein cyanovirin-N; Barrientos LG et al.; A circular permuted variant of the potent human immunodeficiency virus (HIV)-inactivating protein cyanovirin-N (CV-N) was constructed . New N- and C-termini were introduced into an exposed helical loop, and the original termini were linked using residues of the original loop . Since the three-dimensional structure of wild-type cyanovirin-N is a pseudodimer, the mutant essentially exhibits a swap between the two pseudo-symmetrically related halves . The expressed protein, which accumulates in the insoluble fraction, was purified, and conditions for in vitro refolding were established . During refolding, a transient dimeric species is also formed that converts to a monomer . Similar to the wild-type CV-N, the monomeric circular permuted protein exhibits reversible thermal unfolding and urea denaturation . The mutant is moderately less stable than the wild-type protein, but it displays significantly reduced anti-HIV activity . Using nuclear magnetic resonance spectroscopy, we demonstrate that this circular permuted monomeric molecule adopts the same fold as the wild-type protein . Characterization of these two architecturally very similar molecules allows us to embark, for the first time, on a structure guided focused mutational study, aimed at delineating crucial features for the extraordinary difference in the activity of these molecules . Biopolymers, 2002 Apr 5, 63(4), 247 - 60 The association between a negatively charged ligand and the electronegative binding pocket of its receptor; Huang HC et al.; Many examples exist of charged amino acids that play a role in attracting or holding a charged ligand toward or inside an oppositely charged binding pocket of the protein . For example, the enzymes superoxide dismutase, triose-phosphate isomerase, and acetylcholinesterase can steer ligands toward their oppositely charged binding pockets or gorges . Interestingly, in our Brownian dynamics simulations of a phosphate-binding protein, we discovered that negatively charged phosphate (HPO(2-)(4)) could make its way into the negatively charged binding pocket . In fact, the phosphate-binding protein exhibits counterintuitive kinetics of association . That is, one would expect that the rate of association would increase on increases to the ionic strength since the interaction between the ligand, with a charge of -2, and the electronegative binding pocket would be repulsive and greater screening should reduce this repulsion and increase the rate of association . However, the opposite is seen-i.e., the rate of association decreases on increases in the ionic strength . We used Brownian dynamics techniques to compute the diffusion limited association rate constants between the negatively charged phosphate ligand and several open forms of PBP (wild-type and several mutants based on an x-ray structure of open-form PBP, mutant T141D) . With the appropriate choices of reaction criteria and molecular parameters, the ligand was able to diffuse into the binding pocket . A number of residues influence binding of the ligand within the pocket via hydrogen bonds or salt bridges . Arg135 partially neutralizes the charges on the HPO(2-)(4) ligand in the binding pocket, allowing it to enter . It is also found that the positive electrostatic patches above and below the binding entrance of PBP contribute the major attractive forces that direct the ligand toward the surface of the protein near the binding site . Arch Microbiol, 2002 Feb, 177(2), 192 - 6 Epub 2001 Dec 01. Production of a monoclonal antibody specific to Escherichia coli H33 flagellin by using predetermined polypeptides; Seah JN et al.; The Escherichia coli H serogroup is determined by flagellin, which has both H-type-specific and cross-reactive epitopes . The cross-reactive epitopes are responsible for the cross-reaction found in agglutination . To identify the specific epitope in H33 flagellin, the H33 flagellin gene was sequenced and the encoded central variable region (CVR) was determined . Four overlapping fragments of the CVR were prepared and their specificity was verified using different H-type antisera . Short fragments carrying potential H-type-specific determinants were selected, and monoclonal antibodies (MAbs) against these fragments were prepared . A murine MAb of subtype IgG1 showing specificity to H33 flagellin was produced . The epitope of the MAb was mapped to amino acid residues 250-260. Ann Surg, 2002 Feb, 235(2), 285 - 91 A novel therapeutic strategy for attenuating neutrophil-mediated lung injury in vivo; Sookhai S et al.; OBJECTIVE: To evaluate the effect of inhalation of aerosolized opsonized dead Escherichia coli on inflammatory pulmonary neutrophil (PMN) apoptosis, lung injury, and survival in a PMN-mediated lung injury model in vivo . SUMMARY BACKGROUND DATA: Neutrophils that have transmigrated into an inflammatory focus display increased functional capacity and delayed apoptosis, resulting in an increased capacity to injure normal host tissue . The authors have previously shown that E . coli induces PMN apoptosis in vitro . METHODS: Lung injury mediated by PMNs was established by aortic occlusion and reperfusion . Adult male Sprague-Dawley rats were randomized into four groups: sham ischemia-reperfusion (I/R) treated with intratracheal inhalation of aerosolized normal saline, I/R treated with aerosolized normal saline intratracheally, I/R treated with aerosolized opsonized dead E . coli intratracheally, and I/R treated with aerosolized opsonized dead E . coli and the caspase inhibitor zVAD-FMK intratracheally 5 minutes before reperfusion . Both systemic and bronchoalveolar lavage PMNs were isolated and apoptosis was quantified at 0, 6, 12, 18, and 24 hours . Lung injury parameters including wet/dry lung weight ratio, histology, myeloperoxidase activity, and protein content were also assessed . In addition, a survival study was performed, both in a prophylactic and in a therapeutic setting . RESULTS: Administration of aerosolized dead E . coli before the reperfusion injury induced pulmonary PMN apoptosis and reversed the delayed apoptosis evident in the I/R plus normal saline group . There was also a significant improvement in lung injury parameters as well as in survival, both prophylactically as well as therapeutically . CONCLUSIONS: Directly modulating PMN cell death represents a novel mechanism for attenuating PMN-mediated lung injury and may ultimately benefit the outcome in patients with adult respiratory distress syndrome. J Biosci, 2001 Dec, 26(5), 595 - 606 MutS recognition: multiple mismatches and sequence context effects; Joshi A et al.; Escherichia coli MutS is a versatile repair protein that specifically recognizes not only various types of mismatches but also single stranded loops of up to 4 nucleotides in length . Specific binding, followed by the next step of tracking the DNA helix that locates hemi-methylated sites, is regulated by the conformational state of the protein as a function of ATP binding/hydrolysis . Here, we study how various molecular determinants of a heteroduplex regulate mismatch recognition by MutS, the critical first step of mismatch repair . Using classical DNase I footprinting assays, we demonstrate that the hierarchy of MutS binding to various types of mismatches is identical whether the mismatches are present singly or in multiples . Moreover, this unique hierarchy is indifferent both to the differential level of DNA helical flexibility and to the unpaired status of the mismatched bases in a heteroduplex . Surprisingly, multiple mismatches exhibit reduced affinity of binding to MutS, compared to that of a similar single mismatch . Such a reduction in the affinity might be due to sequence context effects, which we established more directly by studying two identical single mismatches in an altered sequence background . A mismatch, upon simply being flipped at the same location, elicits changes in MutS specific contacts, thereby underscoring the importance of sequence context in modulating MutS binding to mismatches. Acta Crystallogr D Biol Crystallogr, 2002 Feb, 58(Pt 2), 362 - 5 Epub 2002 Jan 24. Purification, crystallization and preliminary X-ray diffraction analysis of an archaeal ABC-ATPase; Verdon G et al.; In the archaeon Sulfolobus solfataricus glucose uptake is mediated by an ABC transport system . The ABC-ATPase of this transporter (GlcV) has been overproduced in Escherichia coli and purified . Crystals of GlcV suitable for data collection were obtained in the absence of nucleotide by microseeding combined with vapour diffusion from a mixture of PEG polymers and NaCl . Appearing under identical conditions, two crystal forms have been characterized by X-ray diffraction . Both forms diffract to high resolution using synchrotron radiation and both belong to space group P2(1)2(1)2(1) . The related crystal forms A (unit-cell parameters a = 47.0, b = 48.2, c = 182.1 A) and B (a = 47.0, b = 146.6, c = 178.5 A) feature one and three GlcV molecules in the asymmetric unit, respectively, with a solvent content of about 50% . Crystals have also been obtained in the presence of sodium iodide . From single-wavelength anomalous diffraction data extending to 2.1 A resolution, an iodide substructure could be resolved. Acta Crystallogr D Biol Crystallogr, 2002 Feb, 58(Pt 2), 359 - 61 Epub 2002 Jan 24. Crystallization and preliminary X-ray diffraction studies of Escherichia coli branching enzyme; Abad MC et al.; Branching enzyme catalyzes the formation of the branch points in glycogen and starch by cleavage of the alpha-1,4 link and its subsequent transfer to the alpha-1,6 position . This paper reports the crystallization and preliminary structural studies of an amino-terminally truncated branching enzyme from Escherichia coli . High-resolution diffracting crystals were obtained and a complete native data set to a resolution of 2.3 A was collected . These crystals belong to the P2(1) space group, with unit-cell parameters a = 91.44, b = 102.58, c = 185.41 A, beta = 91.38 degrees . A native data set with 99.6% completeness, an overall R(merge) of 0.086 and I/sigma(I) of 10.43 was obtained. Acta Crystallogr D Biol Crystallogr, 2002 Feb, 58(Pt 2), 355 - 8 Epub 2002 Jan 24. Crystallization and preliminary X-ray crystallographic studies on the parD-encoded protein Kid from Escherichia coli plasmid R1; Hargreaves D et al.; DNA replication in Escherichia coli and therefore bacterial proliferation relies upon the efficient functioning of the DnaB helicase . The toxin protein Kid from the plasmid-stability system parD encoded on plasmid R1 of E . coli is thought to target and block DnaB-dependent DNA replication . The toxicity of Kid is antagonized through interaction with the Kis antidote protein and the resultant complex can then act as a transcriptional regulator for the parD system . Crystals of selenomethionine-incorporated Kid have been obtained by the hanging-drop vapour-diffusion method using potassium phosphate as the precipitant . The crystals belong to the monoclinic system, space group P2(1), have unit-cell parameters a = 32.9, b = 45.0, c = 64.4 A, beta = 96.2 degrees and diffract to a d(min) of better than 1.8 A on a synchrotron-radiation source . The determination of the structure of Kid will permit a better understanding of its interactions with DnaB and Kis and allow the evolutionary relationships of Kid to other toxins of plasmid and chromosomal origin to be explored. Acta Crystallogr D Biol Crystallogr, 2002 Feb, 58(Pt 2), 352 - 4 Epub 2002 Jan 24. Purification, crystallization and preliminary X-ray analysis of aspartokinase III from Escherichia coli; Blanco J et al.; Aspartokinase III catalyzes the commitment step in the aspartate metabolism pathway, the phosphorylation of aspartic acid . The Escherichia coli enzyme has been crystallized in the presence of its natural substrate (aspartic acid) and Mg-ADP and diffraction data has been collected at a synchroton source . The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 60.44, b = 190.31, c = 99.55 A, and data 99.3% complete to 2.7 A . Solving the structure of AK III will provide the first structure of an aspartokinase from any organism. Acta Crystallogr D Biol Crystallogr, 2002 Feb, 58(Pt 2), 349 - 51 Epub 2002 Jan 24. Characterization of Escherichia coli OtsA, a trehalose-6-phosphate synthase from glycosyltransferase family 20; Gibson RP et al.; The Ots gene cluster of Escherichia coli encodes the synthetic apparatus for the formation of alpha,alpha-1,1-trehalose, a non-reducing glucose disaccharide . The otsA gene encodes a trehalose-6-phosphate synthase, a glycosyltransferase which catalyses the synthesis of alpha,alpha-1,1-trehalose-6-phosphate from glucose-6-phosphate using a UDP-glucose donor . It has been classified into glycosyltransferase family GT-20 based upon amino-acid sequence similarities . The otsA gene has been cloned and recombinant protein overexpressed using a pET-based system in E . coli BL21 cells . The recombinant protein (MW approximately 54.7 kDa) is active and has been crystallized in two forms suitable for X-ray diffraction analysis . The first is orthorhombic, P2(1)2(1)2(1), with unit-cell parameters a = 104.1, b = 127.8, c = 179.9 A . Data for this form have been collected to 3.0 A resolution at the CLRC Daresbury Synchrotron Radiation Source . The second form has unit-cell parameters a = b = 141.9, c = 317.8 A and displays the apparent space group P4(2) . These crystals diffract beyond 2 A resolution, but display merohedral twinning. Acta Crystallogr D Biol Crystallogr, 2002 Feb, 58(Pt 2), 330 - 2 Epub 2002 Jan 24. Crystallization and preliminary X-ray crystallographic studies on acyl-(acyl carrier protein) from Escherichia coli; Roujeinikova A et al.; Acyl carrier proteins carry the lipid substrate to the enzymes of the fatty acid synthase system . Crystals of Escherichia coli acyl carrier protein to which a butyryl group has been attached via a thioester link to the phosphopantetheine prosthetic arm have been obtained by the hanging-drop vapour-diffusion method . These crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 27.6, b = 41.6, c = 63.7 A . The asymmetric unit appears to contain one subunit, corresponding to a packing density of 2.1 A(3) Da(-1) . Crystals of the selenomethionine-substituted (SeMet) protein were obtained using different conditions and belong to space group P6(3), with unit-cell parameters a = b = 63.4, c = 37.0 A, alpha = beta = 90, gamma = 120 degrees and with a monomer in the asymmetric unit (V(M) = 2.5 A(3) Da(-1)) . Crystals of a SeMet butyryl-ACP I62M variant were obtained using the conditions for the native protein . Like the native crystals, these belong to space group P2(1)2(1)2(1) and have unit-cell parameters a = 27.3, b = 41.9, c = 64.5 A . A data set suitable for MAD phasing was collected from the crystals of the I62M variant to 1.8 A resolution on the ESRF beamline ID14-4. Acta Crystallogr D Biol Crystallogr, 2002 Feb, 58(Pt 2), 316 - 8 Epub 2002 Jan 24. Escherichia coli B gamma-glutamylcysteine synthetase: modification, purification, crystallization and preliminary crystallographic analysis; Hibi T et al.; Escherichia coli B gamma-glutamylcysteine synthetase (gammaGCS) catalyzes the ATP-dependent coupling of L-Glu and L-Cys to form the glutathione precursor gamma-L-Glu-Cys and is a target for development of potential therapeutic agents . By introducing four point mutations of surface-exposed cysteine residues to serine, the gammaGCS was purified to homogeneity; single crystals have been obtained using the hanging-drop vapour-diffusion method with sodium formate . The gammaGCS crystal diffracted to 2.8 A and belongs to space group R3, with unit-cell parameters a = b = 326.7, c = 103.9 A. Acta Crystallogr D Biol Crystallogr, 2002 Feb, 58(Pt 2), 303 - 5 Epub 2002 Jan 24. Crystallization and preliminary X-ray crystallographic analysis of the Rv2002 gene product from Mycobacterium tuberculosis, a beta-ketoacyl carrier protein reductase homologue; Yang JK et al.; A 260-residue protein (FabG3) encoded by the Rv2002 gene of Mycobacterium tuberculosis shows amino-acid sequence similarity to beta-ketoacyl carrier protein (ACP) reductase, FabG . A soluble mutant (I6T/V47M/T69M) was produced by the green fluorescent protein-based directed-evolution method . It was crystallized at 296 K using the hanging-drop vapour-diffusion method . The diffraction quality of the crystal improved significantly after annealing/dehydration . X-ray diffraction data were collected to 1.8 A resolution using synchrotron radiation . The crystal belongs to the space group P3(1)21 (or P3(2)21), with unit-cell parameters a = b = 70.38, c = 148.93 A . The asymmetric unit contains two subunits, with a corresponding V(M) of 1.90 A(3) Da(-1) and a solvent content of 35.3%. Acta Crystallogr D Biol Crystallogr, 2002 Feb, 58(Pt 2), 296 - 8 Epub 2002 Jan 24. Crystallization and preliminary X-ray crystallographic analysis of p24, a component of the potato nuclear factor PBF-2; Desveaux D et al.; The Solanum tuberosum (potato) nuclear factor PBF-2 is implicated in pathogen-induced expression of the pathogenesis-related gene PR-10a . Crystals of the DNA-binding component of PBF-2, p24, have been obtained at 277 K in 20 mM Tris-HCl pH 8.0 . Recombinant protein with a His tag at its C-terminus was overexpressed in Escherichia coli in the presence and absence of selenomethionine and was purified using a combination of HiTrap affinity columns and gel-filtration chromatography . Crystals suitable for structural analysis were obtained for both native and selenomethionine-labelled proteins and yielded diffraction data at 100 K that were processed to 2.3 and 2.8 A resolution, respectively . The p24 protein crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 69.4 (69.1), b = 89.4 (90.5), c = 144.1 (144.3) A . The asymmetric unit contains four protomers, giving a crystal volume per protein mass (V(M)) of 2.23 A(3) Da(-1) and a solvent content of 45% by volume. J Gen Virol, 2002 Feb, 83(Pt 2), 471 - 7 Transcription and identification of an envelope protein gene (p22) from shrimp white spot syndrome virus; Zhang X et al.; White spot syndrome virus (WSSV) is one of the most virulent pathogens causing high mortality in shrimp . In the present study, an open reading frame (termed the p22 gene) was revealed from a WSSV cDNA library . The gene was expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli and purified . Specific antibody was raised using the purified fusion protein (GST-P22) . Temporal analysis showed that the p22 gene was a late gene . After binding between purified WSSV virions and anti-GST-P22 IgG followed by labelling with gold-labelled secondary antibody, the gold particles, under a transmission electron microscope, could be found along the outer envelope of WSSV virions . This experiment suggests that the p22 gene encodes an envelope protein of the virus. J Gen Virol, 2002 Feb, 83(Pt 2), 359 - 68 Hepatitis A virus polyprotein processing by Escherichia coli proteases; Pinto RM et al.; Hepatitis A virus (HAV) encodes a single polyprotein, which is post-translationally processed . This processing represents an essential step in capsid formation . The virus possesses only one protease, 3C, responsible for all cleavages, except for that at the VP1/2A junction region, which is processed by cellular proteases . In this study, data demonstrates that HAV polyprotein processing by Escherichia coli protease(s) leads to the formation of particulate structures . P3 polyprotein processing in E . coli is not dependent on an active 3C protease: the same processing pattern is observed with wild-type 3C or with several 3C mutants . However, this processing pattern is temperature-dependent, since it differs at 37 or 42 degrees C . The bacterial protease(s) cleave scissile bonds other than those of HAV; this contributes to the low efficiency of particle formation. J Bacteriol, 2002 Feb, 184(4), 1204 - 8 Genetic analysis of the RcsC sensor kinase from Escherichia coli K-12; Clarke DJ et al.; The Rcs two-component pathway is involved in the regulation of capsule production in Escherichia coli . RcsC is predicted to be the sensor component of this two-component pathway, and in this study we present the first genetic data that support the role of RcsC as a hybrid sensor kinase. J Bacteriol, 2002 Feb, 184(4), 1200 - 3 Mutations in the GTPase center of Escherichia coli 23S rRNA indicate release factor 2-interactive sites; Xu W et al.; Mutations in the GTPase center of Escherichia coli 23S rRNA were characterized in vivo as UGA-specific nonsense suppressors . Some site-directed mutations did not exhibit suppressor activity and were interspersed among suppressor mutations . Our results demonstrate the involvement of the two adjacent loops of this conserved rRNA structure in UGA-dependent translation termination and, taken with previous in vitro analyses and with consideration of the crystal structure of the GTPase center RNA, indicate that nucleotides 1067, 1093, 1094, and 1095 are sites of interaction with release factor 2. J Bacteriol, 2002 Feb, 184(4), 1196 - 9 In vitro identification of Rns-regulated genes; Munson GP et al.; To identify Rns-regulated genes, a maltose binding protein (MBP)-Rns fusion protein was used to isolate DNA fragments from enterotoxigenic Escherichia coli genomic DNA that carry Rns binding sites . In vivo transcription fusion analysis shows that Rns positively regulates the expression of the open reading frame of yiiS, which lies immediately downstream of one MBP-Rns-bound fragment. J Bacteriol, 2002 Feb, 184(4), 1192 - 5 Two "wild-type" variants of Escherichia coli sigma(70): context-dependent effects of the identity of amino acid 149; Baldwin NE et al.; The identity of amino acid 149 of Escherichia coli sigma(70) has been reported variably as either arginine or aspartic acid . We show that the behavior of both a region 1.2 deletion and a single-amino-acid substitution at position 122 are greatly affected by the identity of amino acid 149. J Bacteriol, 2002 Feb, 184(4), 1065 - 77 Identification and characterization of assembly proteins of CS5 pili from enterotoxigenic Escherichia coli; Duthy TG et al.; This study investigated the role of three genes comprising part of the operon which encodes CS5 pili from enterotoxigenic Escherichia coli . In-frame gene deletions were constructed, and the effects on biogenesis of the pili were examined . A deletion in csfB abolished CsfA major subunit accumulation in the periplasm, which could be restored by trans-complementation with a complete copy of the csfB gene . Localization studies using an antibody against CsfB showed that this protein was periplasmically located, and thus CsfB is likely to function as the specific chaperone for CsfA . An in-frame deletion mutation in the csfE gene resulted in pili approximately three times longer than those of the wild-type strain, thereby indicating a role for CsfE in pilus length regulation . Localization studies using an antibody generated against CsfE showed low-level CsfE accumulation in the outer membranes . Modulation of csfE expression in trans did not reduce the mean length of the pilus below that of the wild type, which indicated that CsfE is not rate-limiting for termination of pilus assembly . Interestingly, a deletion in the csfF gene also resulted in an elongated pilus morphology identical to that of the csfE deletion strain . However, unlike CsfE, CsfF was shown to be rate-limiting for termination of assembly, since overexpression of CsfF in a csfF deletion strain resulted in a significant decrease in the mean length of the pilus compared to that of the wild type . When the same construct was introduced into the wild-type strain, pilus expression was abolished . Since CsfF bears significant homology to the proposed CsfB chaperone, CsfF was predicted to act as the specific chaperone for CsfE . A double deletion in the csfB and csfF genes was shown to abolish the periplasmic accumulation of both CsfA and CsfD pilins, which could be restored individually only when the strain was trans-complemented with a wild-type copy of csfB or csfF, respectively . Therefore, CsfF may chaperone not only CsfE but also CsfD . A model for CS5 biogenesis is also proposed based on these and previous observations. Blood, 2002 Feb 1, 99(3), 1005 - 13 Adenosine deaminase deficiency with mosaicism for a "second-site suppressor" of a splicing mutation: decline in revertant T lymphocytes during enzyme replacement therapy; Arredondo-Vega FX et al.; Four patients from 3 Saudi Arabian families had delayed onset of immune deficiency due to homozygosity for a novel intronic mutation, g.31701T>A, in the last splice acceptor site of the adenosine deaminase (ADA) gene . Aberrant splicing mutated the last 4 ADA amino acids and added a 43-residue "tail" that rendered the protein unstable . Mutant complementary DNA (cDNA) expressed in Escherichia coli yielded 1% of the ADA activity obtained with wild-type cDNA . The oldest patient, 16 years old at diagnosis, had greater residual immune function and less elevated erythrocyte deoxyadenosine nucleotides than his 4-year-old affected sister . His T cells and Epstein-Barr virus (EBV) B cell line had 75% of normal ADA activity and ADA protein of normal size . DNA from these cells and his whole blood possessed 2 mutant ADA alleles . Both carried g.31701T>A, but one had acquired a deletion of the 11 adjacent base pair, g.31702-12, which suppressed aberrant splicing and excised an unusual purine-rich tract from the wild-type intron 11/exon 12 junction . During ADA replacement therapy, ADA activity in T cells and abundance of the "second-site" revertant allele decreased markedly . This finding raises an important issue relevant to stem cell gene therapy. Biophys J, 2002 Feb, 82(2), 1059 - 67 The effect of ligand dynamics on heme electronic transition band III in myoglobin; Nienhaus K et al.; Band III is a near-infrared electronic transition at ~13,000 cm(-1) in heme proteins that has been studied extensively as a marker of protein conformational relaxation after photodissociation of the heme-bound ligand . To examine the influence of the heme pocket structure and ligand dynamics on band III, we have studied carbon monoxide recombination in a variety of myoglobin mutants after photolysis at 3 K using Fourier transform infrared temperature-derivative spectroscopy with monitoring in three spectral ranges, (1) band III, the mid-infrared region of (2) the heme-bound CO, and (3) the photodissociated CO . Here we present data on mutant myoglobins V68F and L29W, which both exhibit pronounced ligand movements at low temperature . From spectral and kinetic analyses in the mid-infrared, a small number of photoproduct populations can be distinguished, differing in their distal heme pocket conformations and/or CO locations . We have decomposed band III into its individual photoproduct contributions . Each photoproduct state exhibits a different "kinetic hole-burning" (KHB) effect, a coupling of the activation enthalpy for rebinding to the position of band III . The analysis reveals that the heme pocket structure and the photodissociated CO markedly affect the band III transition . A strong kinetic hole-burning effect results only when the CO ligand resides in the docking site on top of the heme group . Migration of CO away from the heme group leads to an overall blue shift of band III . Consequently, band III can be used as a sensitive tool to study ligand dynamics after photodissociation in heme proteins. Biophys J, 2002 Feb, 82(2), 1004 - 16 Ultrafast dynamics of phytochrome from the cyanobacterium synechocystis, reconstituted with phycocyanobilin and phycoerythrobilin; Heyne K et al.; Femtosecond time-resolved transient absorption spectroscopy was employed to characterize for the first time the primary photoisomerization dynamics of a bacterial phytochrome system in the two thermally stable states of the photocycle . The 85-kDa phytochrome Cph1 from the cyanobacterium Synechocystis PCC 6803 expressed in Escherichia coli was reconstituted with phycocyanobilin (Cph1-PCB) and phycoerythrobilin (Cph1-PEB) . The red-light-absorbing form Pr of Cph1-PCB shows an approximately 150 fs relaxation in the S(1) state after photoexcitation at 650 nm . The subsequent Z-E isomerization between rings C and D of the linear tetrapyrrole-chromophore is best described by a distribution of rate constants with the first moment at (16 ps)(-1) . Excitation at 615 nm leads to a slightly broadened distribution . The reverse E-Z isomerization, starting from the far-red-absorbing form Pfr, is characterized by two shorter time constants of 0.54 and 3.2 ps . In the case of Cph1-PEB, double-bond isomerization does not take place, and the excited-state lifetime extends into the nanosecond regime . Besides a stimulated emission rise time between 40 and 150 fs, no fast relaxation processes are observed . This suggests that the chromophore-protein interaction along rings A, B, and C does not contribute much to the picosecond dynamics observed in Cph1-PCB but rather the region around ring D near the isomerizing C(15) {double bond} C(16) double bond . The primary reaction dynamics of Cph1-PCB at ambient temperature is found to exhibit very similar features as those described for plant type A phytochrome, i.e., a relatively slow Pr, and a fast Pfr, photoreaction . This suggests that the initial reactions were established already before evolution of plant phytochromes began. Biophys J, 2002 Feb, 82(2), 618 - 27 A dynamic model for determining the middle of Escherichia coli; Kruse K; Proper placement of the division septum is an essential part of bacterial cell division . In Escherichia coli, this process depends crucially on the proteins MinC, MinD, and MinE . The detailed mechanism by which these proteins determine the correct position of the division plane is currently unknown, but observed pole-to-pole oscillations of the corresponding distributions are thought to be of functional importance . Here, a theoretical approach toward an explanation of this dynamical behavior is reported . Emphasizing generic properties of the protein dynamics, two features are found to be sufficient for generating oscillations: first, a tendency of membrane bound MinD to cluster; and second, attachment to and detachment from the cell wall, which depends on the amount of molecules already attached . The model is in qualitative agreement with the presently existing experimental results and further tests of the underlying model assumptions are suggested . Finally, based on the analysis of the model a simple mechanism is proposed on how these proteins might initiate septal growth . In addition, to ensure correct positioning of the septum, the MinCDE complex could therefore also play an important role in cell cycle control. Angiogenesis, 2001, 4(2), 103 - 12 Ectodomain shedding of VEGF183, a novel isoform of vascular endothelial growth factor, promotes its mitogenic activity in vitro; Jingjing L et al.; We have previously reported the discovery of VEGF183, a novel isoform of vascular endothelial growth factor (VEGF), whose nucleotide sequence revealed an 18-bp deletion in the exon 6A-encoded region of VEGF . The following study was done to characterize VEGF183 and to determine its biological activity in vitro and in vivo . Recombinant human VEGF183 was expressed in Escherichia coli (rhVEGF183) or in human 293 embryonic kidney cells (293-VEGF183) and tested for stimulation of permeability of dermal vessels in normal rats as well as for mitogenic activity and phosphorylation of mitogen-activated protein kinases (MAPK) in cultured human umbilical vein endothelial cells (HUVECs) . While small amounts of VEGF183 were secreted into the conditioned media (CM) of 293 cells expressing VEGF183 (293-VEGF183 cells), most of the VEGF183 remained cell surface-bound and could be released into the CM following treatment with plasmin or heparin . CM from 293-VEGF183 cells treated with heparin or plasmin induced about a twofold increase in cell numbers and stimulated MAPK phosphorylation in HUVECs as compared with CM from untreated 293-VEGF183 cells or from heparin- or plasmin-treated 293 cells containing the vector alone . Intradermal injections of rhVEGF183 promoted increased permeability of dermal vessels to Evan's blue dye . Our study shows that VEGF183 is predominantly a cell-anchored protein that promotes increased vascular permeability in vivo but requires extracellular cleavage or release by heparin or plasmin to promote its mitogenic activity in vitro. Lipids, 2001 Nov, 36(11), 1183 - 93 Influence of dietary supplementation with long-chain n-3 or n-6 polyunsaturated fatty acids on blood inflammatory cell populations and functions and on plasma soluble adhesion molecules in healthy adults; Thies F et al.; Greatly increasing the amounts of flaxseed oil {rich in alpha-linolenic acid (ALNA)} or fish oil (FO); {rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)} in the diet can decrease inflammatory cell functions and so might impair host defense . The objective of this study was to determine the effect of dietary supplementation with moderate levels of ALNA, gamma-linolenic acid (GLA), arachidonic acid (ARA), DHA, or FO on inflammatory cell numbers and functions and on circulating levels of soluble adhesion molecules . Healthy subjects aged 55 to 75 yr consumed nine capsules per day for 12 wk . The capsules contained placebo oil (an 80:20 mix of palm and sunflowerseed oils) or blends of placebo oil with oils rich in ALNA, GLA, ARA, or DHA or FO . Subjects in these groups consumed 2 g ALNA; approximately 700 mg GLA, ARA, or DHA; or 1 g EPA plus DHA (720 mg EPA + 280 mg DHA) daily from the capsules . Total fat intake from the capsules was 4 g per day . None of the treatments affected inflammatory cell numbers in the bloodstream; neutrophil and monocyte phagocytosis or respiratory burst in response to E . coli; production of tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 in response to bacterial lipopolysaccharide; or plasma concentrations of soluble intercellular adhesion molecule-1 . In contrast, the ALNA and FO treatments decreased the plasma concentrations of soluble vascular cell adhesion molecule-1 (16 and 28% decrease, respectively) and soluble E-selectin (23 and 17% decrease, respectively) . It is concluded that, in contrast to previous reports using higher amounts of these fatty acids, a moderate increase in consumption of long-chain n-6 or n-3 polyunsaturated fatty acids does not significantly affect inflammatory cell numbers or neutrophil and monocyte responses in humans and so would not be expected to cause immune impairment . Furthermore, we conclude that moderate levels of ALNA and FO, which could be incorporated into the diet, can decrease some markers of endothelial activation and that this mechanism of action may contribute to the reported health benefits of n-3 fatty acids. Curr Genet, 2001 Dec, 40(4), 260 - 7 Deletion analysis of the enolase gene (enoA) promoter from the filamentous fungus Aspegillus oryzae; Toda T et al.; The enolase gene (enoA) is one of the most strongly expressed genes in Aspergillus oryzae . To elucidate the transcription regulatory element for this strong expression and the process of glucose induction, the transcription activity of a series of truncated enoA promoters was measured by using the Escherichia coli uidA gene as a reporter . Deletion of a 104-bp region located -224 nt to -121 nt upstream of the translation initiation site caused both a drastic decrease in the beta-glucuronidase (GUS) activity and a loss of glucose induction . Northern blot analysis confirmed that the decrease in GUS activity was achieved at the transcriptional level . In addition, electrophoretic gel mobility shift assays indicated that the 104-bp region contained a 15-bp element, to which one or more A . oryzae cellular factors specifically bind . These results suggest that the 15-bp element between -195 nt and -181 nt includes the sequence essential for the transcription regulation of the A . oryzae enoA gene. Anal Chem, 2002 Jan 1, 74(1), 158 - 62 Enzyme-amplified amperometric sandwich test for RNA and DNA; Campbell CN et al.; A one-step enzyme-amplified amperometric sandwich hybridization test for RNA and DNA is described . The test utilizes a carbon electrode, modified with a film of co-electrodeposited avidin and redox polymer; the redox polymer electrically "wiring" horseradish peroxidase (HRP) reaction centers upon contact . The film is made specific for the particular RNA or DNA sequence tested by conjugating its avidin with a biotinylated oligonucleotide, complementary to the assayed sequence . This oligonucleotide-modified redox polymer film, prepared prior to the test, forms the base of the sandwich . The center layer of the sandwich, added in the test, is the analyte RNA or DNA; its top is a second complemetary oligonucleotide, which is HRP-labeled, and is cohybridized in the test . The test consists of mixing the analyte DNA or RNA solution, the HRP-labeled oligonucleotide solution, and a hydrogen peroxide solution, immersing the base-layer carrying electrode applying a potential of 0 V versus Ag/AgCl, and measuring the H2O2 electroreduction current . Completion of the sandwich brings the HRP label into electrical contact with the redox polymer, converting the nonelectrocatalytic base layer into an electrocatalyst for the electroreduction of H2O2 to water . Flow of H2O2 electroreduction current when the electrode is poised near Ag/AgCl potential indicates the presence of the analyte RNA or DNA . The current density for the maximally sandwich-covered electrode was 250 microA cm(-2), exceeding more than a 100-fold the current density flowing upon nonspecific binding of the HRP-labeled oligonucleotide . High concentrations of irrelevant DNA and diluted serum did not interfere with the assay . When the electrodes were rotated in order to make the solution-phase mass transport rapid, the test was completed in approximately 30 min . The test was applied in probing for the presence of a 60-base E . coli mRNA sequence. Shock, 2002 Jan, 17(1), 77 - 80 Lipopolysaccharide-induced gastrointestinal injury in rats: role of surface hydrophobicity and bile salts; Dial EJ et al.; Sepsis of gastrointestinal origin can lead to life-threatening complications in vital organs due to bacterial overgrowth and/or translocation from the lumen into the blood . In a rat model of endotoxemia, changes in surface hydrophobicity (associated with barrier integrity) of the gastrointestinal mucosa were examined . Rats were treated with Escherichia coli lipopolysaccharide (LPS), and gastric and ileal tissue were collected for determination of surface hydrophobicity by contact angle analysis . A role for bile salts in hydrophobicity changes was tested by quantifying bile salts in the lumen of both the stomach and ileum after LPS and by the administration of LPS to bile duct-ligated rats . A single intraperitoneal dose of LPS induced a dose- and time-dependent reduction in hydrophobicity of both the stomach and ileum, with the stomach showing greater sensitivity at an earlier time than the ileum . LPS also induced gastric bleeding, reflux of bile acid into the gastric lumen, and decreased levels of bile salt in the ileum . The LPS-induced reductions in surface hydrophobicity of the stomach were prevented by prior bile duct ligation . We conclude that LPS disrupts gastrointestinal barrier integrity, in part by mechanisms involving bile constituents and an attenuation in the mucosa's hydrophobic characteristics. Shock, 2002 Jan, 17(1), 70 - 6 Differential involvement of guanylate cyclase and potassium channels in nitric oxide-induced hyporesponsiveness to phenylephrine in endotoxemic rats; da Silva-Santos JE et al.; This study evaluated the involvement of nitric oxide (NO), guanylate cyclase, and potassium channels in the long-lasting vascular hyporesponsiveness to phenylephrine induced by Escherichia coli lipopolysaccharide (LPS) in vitro and in vivo . Experiments in rat aorta rings with endothelium incubated with LPS (10 microg/mL) for 12 h showed that the hyporesponsiveness depends on guanylate cyclase activity and tetraethylammonium-sensitive, but not voltage- or ATP-dependent, potassium channels . Pressor responses to phenylephrine were reduced by 50% in rats injected 8 and 24 h before with LPS (10 mg/kg, intraperitoneally) . Pretreatment with NO synthase inhibitors (iNOS; Nomega-nitro-L-arginine methyl ester {L-NAME}, 55 micromol/kg or aminoguanidine, 244 micromol/kg, intraperitoneally) fully prevented LPS-induced hyporesponsiveness . When administered just before phenylephrine, L-NAME (11 micromol/kg, intravenously) reversed the hyporesponsiveness in rats injected 8 h, but not in those injected 24 h before with LPS, whereas 1H-{1,2,4}-oxadiazolo-{4,3-a}-quinoxalin-1 (ODQ, 11 micromol/kg, intravenously) reversed the hyporesponsiveness in animals injected 24 h, but not in those injected 8 h before with LPS . Tetraethylammonium (360 micromol/kg, intravenously) reestablished normal responses to phenylephrine in rats injected 8 and 24 h before with LPS . Again, neither voltage- nor ATP-dependent potassium channels appears to be involved . Western blot showed that iNOS expression peaked at 8 h, decreasing to low levels 24 h after LPS injection . Therefore, NO is important in initiating LPS-induced hyporesponsiveness to vasoconstrictors, but not in maintaining it for long periods . Once NO has exerted its effects and even when iNOS expression is minimal, the long-lasting hyporesponsiveness appears to depend on a complex interplay between guanylate cyclase and potassium channel activation. Ann N Y Acad Sci, 2001 Apr, 928, 121 - 31 Premature aging and predisposition to cancers caused by mutations in RecQ family helicases; Furuichi Y; DNA helicases, because they unwind duplex DNA, have important roles in cellular DNA events such as replication, recombination, repair, and transcription . Multiple DNA helicase families with seven consensus motifs have been found, and members within each helicase family also share sequence homologies between motifs . The RecQ helicase family includes helicases that have extensive amino acid sequence homologies to the E . coli DNA helicase RecQ, which has been implicated in double-strand break repair and suppression of illegitimate recombination . To date, five RecQ helicase species exist in humans, but their exact biological functions remain unknown . In this paper, on the basis of five years of work, I overview the updated molecular biology of five human RecQ helicases; genetic diseases such as Werner's, Bloom's, and Rothmund-Thomson's syndromes caused by helicase mutations; the associated premature aging phenotype; and an increased risk of neoplasms . I also describe a hypothesis of "tissue-specific genomic instability" that accounts for the pathology behind multisymptomatic RecQ helicase syndromes. Adv Nutr Res, 2001, 10, 389 - 404 Milk banking: the influence of storage procedures and subsequent processing on immunologic components of human milk; Lawrence RA; The immunoprotective constituents of human milk are stable when stored at room temperature for 8 hours, when stored at 0 degree-4 degrees C for three days, or when frozen at -20 degrees C for 12 months . They are also stable during pasteurization at 56 degrees C for 30 minutes . Sonification may reduce levels of sIgA and lysozyme and the ability of milk to inhibit growth of E coli . The number of cells in human milk is reduced by storage, freezing, pasteurizing, microwaving and sonification, and the functional capacity of surviving cells is also reduced. Nature, 2002 Jan 10, 415(6868), 192 - 5 The motor domain determines the large step of myosin-V; Tanaka H et al.; Class-V myosin proceeds along actin filaments with large ( approximately 36 nm) steps . Myosin-V has two heads, each of which consists of a motor domain and a long (23 nm) neck domain . In accordance with the widely accepted lever-arm model, it was suggested that myosin-V steps to successive (36 nm) target zones along the actin helical repeat by tilting its long neck (lever-arm) . To test this hypothesis, we measured the mechanical properties of single molecules of myosin-V truncation mutants with neck domains only one-sixth of the native length . Our results show that the processivity and step distance along actin are both similar to those of full-length myosin-V . Thus, the long neck domain is not essential for either the large steps or processivity of myosin-V . These results challenge the lever-arm model . We propose that the motor domain and/or the actomyosin interface enable myosin-V to produce large processive steps during translocation along actin. Nature, 2002 Jan 10, 415(6868), 183 - 7 Alternative nucleotide incision repair pathway for oxidative DNA damage; Ischenko AA et al.; The DNA glycosylase pathway, which requires the sequential action of two enzymes for the incision of DNA, presents a serious problem for the efficient repair of oxidative DNA damage, because it generates genotoxic intermediates such as abasic sites and/or blocking 3'-end groups that must be eliminated by additional steps before DNA repair synthesis can be initiated . Besides the logistical problems, biological evidence hints at the existence of an alternative repair pathway . Mutants of Escherichia coli and mice (ref . 4 and M . Takao et al., personal communication) that are deficient in DNA glycosylases that remove oxidized bases are not sensitive to reactive oxygen species, and the E . coli triple mutant nei, nth, fpg is more radioresistant than the wild-type strain . Here we show that Nfo-like endonucleases nick DNA on the 5' side of various oxidatively damaged bases, generating 3'-hydroxyl and 5'-phosphate termini . Nfo-like endonucleases function next to each of the modified bases that we tested, including 5,6-dihydrothymine, 5,6-dihydrouracil, 5-hydroxyuracil and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine residues . The 3'-hydroxyl terminus provides the proper end for DNA repair synthesis; the dangling damaged nucleotide on the 5' side is then a good substrate for human flap-structure endonuclease and for DNA polymerase I of E . coli. Intervirology, 2001, 44(6), 350 - 4 Characterization of DNA binding protein of porcine adenovirus type 3; Zhou Y et al.; To identify and characterize the protein encoded by the E2A region of porcine adenovirus (PAV)-3, DNA sequence coding for a portion (amino acids 102-457) of the DNA binding protein (DBP) open reaching frame was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase protein of Schistosoma japonica . The affinity-purified fusion protein was used to immunize rabbits . Immunoprecipitation/Western blot analysis demonstrated that the antisera specifically recognized a protein of 50 kD in PAV-3-infected cells . Immunoperoxidase staining detected the DBP protein predominantly in the nucleus of the cells . Western blot analysis demonstrated that DBP was detected as early as 6 h after infection and remained detectable throughout the infection . Based on these results, a novel assay for quantitation of PAV-3 was established . The assay is less time consuming and can be performed in different porcine cells . In addition, virus titers determined by this assay are comparable to the standard plaque assay . Proc Natl Acad Sci U S A, 2002 Jan 22, 99(2), 679 - 84 Synchronizing genetic relaxation oscillators by intercell signaling; McMillen D et al.; The ability to design and construct synthetic gene regulatory networks offers the prospect of studying issues related to cellular function in a simplified context; such networks also have many potential applications in biotechnology . A synthetic network exhibiting oscillatory behavior has recently been constructed {Elowitz, M . B . & Leibler, S . (2000) Nature (London) 403, 335-338} . It has also been shown that a natural bacterial quorum-sensing mechanism can be used in a synthetic system to communicate a signal between two populations of cells, such that receipt of the signal causes expression of a target gene {Weiss, R . & Knight, T . F . (2000) in DNA6: Sixth International Meeting on DNA-Based Computers, June 13-17, 2000, Leiden, The Netherlands} . We propose a synthetic gene network in Escherichia coli which combines these two features: the system acts as a relaxation oscillator and uses an intercell signaling mechanism to couple the oscillators and induce synchronous oscillations . We model the system and show that the proposed coupling scheme does lead to synchronous behavior across a population of cells . We provide an analytical treatment of the synchronization process, the dominant mechanism of which is "fast threshold modulation." Proc Natl Acad Sci U S A, 2002 Feb 5, 99(3), 1473 - 8 Epub 2002 Jan 22. HemK, a class of protein methyl transferase with similarity to DNA methyl transferases, methylates polypeptide chain release factors, and hemK knockout induces defects in translational termination; Nakahigashi K et al.; HemK, a universally conserved protein of unknown function, has high amino acid similarity with DNA-(adenine-N6) methyl transferases (MTases) . A certain mutation in hemK gene rescues the photosensitive phenotype of a ferrochelatase-deficient (hemH) mutant in Escherichia coli . A hemK knockout strain of E . coli not only suffered severe growth defects, but also showed a global shift in gene expression to anaerobic respiration, as determined by microarray analysis, and this shift may lead to the abrogation of photosensitivity by reducing the oxidative stress . Suppressor mutations that abrogated the growth defects of the hemK knockout strain were isolated and shown to be caused by a threonine to alanine change at codon 246 of polypeptide chain release factor (RF) 2, indicating that hemK plays a role in translational termination . Consistent with such a role, the hemK knockout strain showed an enhanced rate of read-through of nonsense codons and induction of transfer-mRNA-mediated tagging of proteins within the cell . By analysis of the methylation of RF1 and RF2 in vivo and in vitro, we showed that HemK methylates RF1 and RF2 in vitro within the tryptic fragment containing the conserved GGQ motif, and that hemK is required for the methylation within the same fragment of, at least, RF1 in vivo . This is an example of a protein MTase containing the DNA MTase motif and also a protein-(glutamine-N5) MTase. J Pharmacol Exp Ther, 2002 Feb, 300(2), 460 - 7 Underlying endotoxemia augments toxic responses to chlorpromazine: is there a relationship to drug idiosyncrasy? Buchweitz JP, Ganey PE, Bursian SJ, Roth RA. Idiosyncratic reactions occur in a small fraction (typically <5%) of the population taking therapeutic drugs . Chlorpromazine (CPZ) is a phenothiazine, antipsychotic drug that has caused several idiosyncratic responses during its therapeutic use . Clinical evidence suggests that conditions associated with inflammation are risk factors for the appearance of these responses . Accordingly, we tested the hypothesis that an inflammatory stimulus, bacterial lipopolysaccharide (LPS), renders animals susceptible to CPZ-induced idiosyncratic reactions seen in humans . Male Sprague-Dawley rats (200-250 g) were fasted for 24 h . A small dose of LPS (7.4 x 10(6) EU/kg from Escherichia coli) or its vehicle (saline) was administered by tail vein 2 h before an intraperitoneal injection of CPZ (70 mg/kg) or its vehicle (saline) . Cholestasis and hepatocellular necrosis were evaluated as increased concentrations of serum bile acids and bilirubin and increased activities of alkaline phosphatase, gamma-glutamyltransferase, alanine aminotransferase, and aspartate aminotransferase . With the exception of bile acids, these serum markers were elevated in animals treated with LPS/CPZ . Histopathological lesions in liver sections were consistent with these findings . Elevated serum creatine kinase activity, which is associated with human idiosyncratic responses to phenothiazines, was also found in animals treated with LPS/CPZ, but not with either LPS or CPZ alone . These results raise the possibility that concurrent, modest inflammation may underlie susceptibility of individuals to certain idiosyncratic reactions and may form the basis for an animal model with which to understand and predict drug idiosyncrasy. J Biol Chem, 2002 Apr 5, 277(14), 12253 - 62 Epub 2002 Jan 22. Regulation of protein kinase C in Escherichia coli K1 invasion of human brain microvascular endothelial cells; Sukumaran SK et al.; Escherichia coli is one of the most important pathogens involved in the development of neonatal meningitis in many parts of the world . Traversal of E . coli across the blood-brain barrier is a crucial event in the pathogenesis of E . coli meningitis . Our previous studies have shown that outer membrane protein A (OmpA) expression is necessary in E . coli for a mechanism involving actin filaments in its passage through the endothelial cells . Focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K) have also been activated in host cells during the process of invasion . In an attempt to elucidate the mechanisms leading to actin filament condensation, we have focused our attention on protein kinase C (PKC), an enzyme central to many signaling events, including actin rearrangement . In the current study, specific PKC inhibitors, bisindolmaleimide and a PKC-inhibitory peptide, inhibited E . coli invasion of human brain microvascular endothelial cells (HBMEC) by more than 75% in a dose-dependent manner, indicating a significant role played by this enzyme in the invasion process . Our results further showed that OmpA+ E . coli induces significant activation of PKC in HBMEC as measured by the PepTag nonradioactive assay . In addition, we identified that the PKC isoform activated in E . coli invasion is a member of the conventional family of PKC, PKC-alpha, which requires calcium for activation . Immunocytochemical studies have indicated that the activated PKC-alpha is associated with actin condensation beneath the bacterial entry site . Overexpression of a dominant negative mutant of PKC-alpha in HBMEC abolished the E . coli invasion without significant changes in FAK phosphorylation or PI3K activity patterns . In contrast, in HBMEC overexpressing the mutant forms of either FAK or PI3K, E . coli-induced PKC activation was significantly blocked . Furthermore, our studies showed that activation of PKC-alpha induces the translocation of myristoylated alanine-rich protein kinase C substrate, an actin cross-linking protein and a substrate for PKC-alpha, from the membrane to cytosol . This is the first report of FAK- and PI3K-dependent PKC-alpha activation in bacterial invasion related to cytoskeletal reorganization. Genetics, 2002 Jan, 160(1), 13 - 23 The roles of Klenow processing and flap processing activities of DNA polymerase I in chromosome instability in Escherichia coli K12 strains; Nagata Y et al.; The sequences of spontaneous mutations occurring in the endogenous tonB gene of Escherichia coli in the DeltapolA and polA107 mutant strains were compared . Five categories of mutations were found: (1) deletions, (2) minus frameshifts, (3) plus frameshifts, (4) duplications, and (5) other mutations . The DeltapolA strain, which is deficient in both Klenow domain and 5' --> 3' exonuclease domain of DNA polymerase I, shows a marked increase in categories 1-4 . The polA107 strain, which is deficient in the 5' --> 3' exonuclease domain but proficient in the Klenow domain, shows marked increases in categories 3 and 4 but not in 1 or 2 . Previously, we reported that the polA1 strain, which is known to be deficient in the Klenow domain but proficient in the 5' --> 3' exonuclease domain, shows increases in categories 1 and 2 but not in 3 or 4 . The 5' --> 3' exonuclease domain of DNA polymerase I is a homolog of the mammalian FEN1 and the yeast RAD27 flap nucleases . We therefore proposed the model that the Klenow domain can process deletion and minus frameshift mismatch in the nascent DNA and that flap nuclease can process plus frameshift and duplication mismatch in the nascent DNA. Mutat Res, 2002 Jan 29, 499(1), 97 - 101 DNA polymerase V-dependent mutator activity in an SOS-induced Escherichia coli strain with a temperature-sensitive DNA polymerase III; Timms AR et al.; The temperature-sensitive DNA polymerase III (Pol III) encoded by the dnaE486 allele confers a spontaneous mutator activity in SOS-induced bacteria that is largely dependent upon DNA polymerase V (Pol V), encoded by umuD, C . This mutator activity is influenced by the defective proof-reading sub-unit of Pol III encoded by the dnaQ905 (mutD5) allele arguing that Pol V is most likely fixing mutations arising from mismatched primer termini produced by Pol III(486) . The size of the dnaQ effect is, however, modest leaving open the possibility that Pol V may be responsible for some of the mutator effect by engaging in bursts of processive activity. Mutat Res, 2002 Jan 29, 499(1), 85 - 95 Sequence analysis and phenotypes of five temperature sensitive mutator alleles of dnaE, encoding modified alpha-catalytic subunits of Escherichia coli DNA polymerase III holoenzyme; Vandewiele D et al.; In the 1970s, several thermosensitive alleles of dnaE (encoding the alpha-catalytic subunit of pol III) were isolated . Genetic characterization of these dnaE mutants revealed that some are mutator alleles at permissive temperature . We have determined the nucleotide changes of five such temperature sensitive mutator alleles (dnaE9, dnaE74, dnaE486, dnaE511, and dnaE1026) and find that most are single missense mutations . The exception is dnaE1026 which is a compound allele consisting of multiple missense mutations . When the previously characterized mutator alleles were moved into a lexA51(Def) recA730 strain, dnaE486, dnaE1026 and dnaE74 conferred a modest approximately two-six-fold increase in spontaneous mutagenesis when grown at the permissive temperature of 28 degrees C, while dnaE9 and dnaE511 actually resulted in a slight decrease in spontaneous mutagenesis . In isogenic DeltaumuDC derivatives, the level of spontaneous mutagenesis dropped significantly, although in each case, the overall mutator effect conferred by the dnaE allele was relatively larger, with all five dnaE alleles conferring an increased spontaneous mutation rate approximately 5-22-fold over the isogenic dnaE+ DeltaumuDC strain . Interestingly, the temperature sensitivity conferred by each allele varied considerably in the lexA51(Def) recA730 background and in many cases, this phenotype was dependent upon the presence of functional pol V (UmuD'2C) . Our data suggest that pol V can compete effectively with the impaired alpha-subunit for a 3' primer terminus and as a result, a large proportion of the phenotypic effects observed with strains carrying missense temperature sensitive mutations in dnaE can, in fact, be attributed to the actions of pol V rather than pol III. Mol Cell, 2002 Jan, 9(1), 11 - 22 The Sm-like Hfq protein increases OxyS RNA interaction with target mRNAs; Zhang A et al.; The Escherichia coli host factor I, Hfq, binds to many small regulatory RNAs and is required for OxyS RNA repression of fhlA and rpoS mRNA translation . Here we report that Hfq is a bacterial homolog of the Sm and Sm-like proteins integral to RNA processing and mRNA degradation complexes in eukaryotic cells . Hfq exhibits the hallmark features of Sm and Sm-like proteins: the Sm1 sequence motif, a multisubunit ring structure (in this case a homomeric hexamer), and preferential binding to polyU . We also show that Hfq increases the OxyS RNA interaction with its target messages and propose that the enhancement of RNA-RNA pairing may be a general function of Hfq, Sm, and Sm-like proteins. Mol Cell, 2002 Jan, 9(1), 7 - 8 A BLAST from the past: ancient origin of human Sm proteins; Donahue WF et al.; In this issue of Molecular Cell, Zhang et al . and Moller et al . independently report studies of the E . coli Hfq protein, revealing significant sequence similarities with human Sm proteins . Their findings suggest that Hfq, and the Sm and Sm-like (Lsm) proteins, may function in stabilizing interactions between RNA molecules. Mol Cell, 2002 Jan, 9(1), 3 - 5 Caught in the act: how ATP binding triggers cooperative conformational changes in a molecular machine; Gierasch LM; A paper recently published in Cell describes ATP-triggered conformational changes in the GroEL folding machine deciphered by use of cryo-electron microscopy, molecular engineering, and X-ray crystallographic data . Mechanistically crucial allosteric effects of ATP binding arise from rearrangement of interdomain electrostatic contacts. J Am Chem Soc, 2002 Jan 30, 124(4), 672 - 8 Top down characterization of larger proteins (45 kDa) by electron capture dissociation mass spectrometry; Ge Y et al.; The structural characterization of proteins expressed from the genome is a major problem in proteomics . The solution to this problem requires the separation of the protein of interest from a complex mixture, the identification of its DNA-predicted sequence, and the characterization of sequencing errors and posttranslational modifications . For this, the "top down" mass spectrometry (MS) approach, extended by the greatly increased protein fragmentation from electron capture dissociation (ECD), has been applied to characterize proteins involved in the biosynthesis of thiamin, Coenzyme A, and the hydroxylation of proline residues in proteins . With Fourier transform (FT) MS, electrospray ionization (ESI) of a complex mixture from an E . coli cell extract gave 102 accurate molecular weight values (2-30 kDa), but none corresponding to the predicted masses of the four desired enzymes for thiamin biosynthesis (GoxB, ThiS, ThiG, and ThiF) . MS/MS of one ion species (representing approximately 1% of the mixture) identified it with the DNA-predicted sequence of ThiS, although the predicted and measured molecular weights were different . Further purification yielded a 2-component mixture whose ECD spectrum characterized both proteins simultaneously as ThiS and ThiG, showing an additional N-terminal Met on the 8 kDa ThiS and removal of an N-terminal Met and Ser from the 27 kDa ThiG . For a second system, the molecular weight of the 45 kDa phosphopantothenoylcysteine synthetase/decarboxylase (CoaBC), an enzyme involved in Coenzyme A biosynthesis, was 131 Da lower than that of the DNA prediction; the ECD spectrum showed that this is due to the removal of the N-terminal Met . For a third system, viral prolyl 4-hydroxylase (26 kDa), ECD showed that multiple molecular ions (+98, +178, etc.) are due to phosphate noncovalent adducts, and MS/MS pinpointed the overall mass discrepancy of 135 Da to removal of the initiation Met (131 Da) and to formation of disulfide bonds (2 x 2 Da) at C32-C49 and C143-C147, although 10 S-S positions were possible . In contrast, "bottom up" proteolysis characterization of the CoaBC and the P4H proteins was relatively unsuccessful . The addition of ECD substantially increases the capabilities of top down FTMS for the detailed structural characterization of large proteins. Mol Cells, 2001 Dec 31, 12(3), 321 - 8 Specific detection of cell surface-displayed human melanocortin 4 receptors with antibodies generated in mice; Ju SK et al.; The melanocortin-4 receptor (MC-4R) is a 7-transmembrane protein, which is involved in the central regulation of appetite and obesity . Despite the great interest in this protein, tools for detecting this molecule (as expressed on the cell surface in its native state) have been unavailable . Radioactive- or otherwise labeled ligands showed low receptor specificity to this particular melanocortin receptor isotype . Also, the antibodies were only available for epitopes that were displayed in the cytoplasm . To produce antibodies that enable the detection of this receptor (as expressed on the cell surface without disruption of the target cells), a candidate epitope was selected from the extracellular domains by a computer-aided analysis of the IC-4R secondary structure . This particular region was then recombinantly expressed in E . coli . Immunization of BALB/c mice with the recombinant proteins induced a specific immune reaction, which resulted in the production of MC-4R-specific antibodies . Enzyme-linked immunosorbent assays confirmed the specificity of these antibodies . To examine whether this tool also reacts with native cell surface-displayed MC-4R, HEK-293 cells were transfected with the human MC-4R cDNA . They were analyzed with these antibodies using Western blot and flow cytometry . Specificity and exclusion of cross-reactivity of these antibodies to other MC receptors were further confirmed by an immunofluorescence analysis of the HEK-293 cells that were transfected with other MC receptor isotypes . It is evident that with the availability of this tool, studies on the cell- and tissue-specificity, as well as the regulation mechanism of the MC-4 receptor, will be largely facilitated. Curr Opin Mol Ther, 2001 Dec, 3(6), 526 - 32 Computational aspects of protein identification by mass spectrometry; Gras R et al.; Recent developments in proteomics and genomics provide huge quantities of data to analyze . Automatic interpretation of mass spectrometry data has become essential for high-throughput processes aiming to study complete proteomes . There exist two main sources of mass spectrometric data: peptide mass fingerprint and fragmentation spectra, both of which require specific bioinformatic algorithms . We present a survey of these algorithms and discuss the efficiency of the different approaches and the possible improvements that may lead to a complete automatic high-throughput identification process. J Bioenerg Biomembr, 2001 Dec, 33(6), 459 - 68 Structure-function analysis of the ArsA ATPase: contribution of histidine residues; Bhattacharjee H et al.; The ArsA ATPase is the catalytic subunit of the ArsAB oxyanion pump in Escherichia coli that is responsible for extruding arsenite or antimonite from inside the cell, thereby conferring resistance . Either antimonite or arsenite stimulates ArsA ATPase activity . In this study, the role of histidine residues in ArsA activity was investigated . Treatment of ArsA with diethyl pyrocarbonate (DEPC) resulted in complete loss of catalytic activity . The inactivation could be reversed upon subsequent incubation with hydroxylamine, suggesting specific modification of histidine residues . ATP and oxyanions afforded significant protection against DEPC inactivation, indicating that the histidines are located at the active site . ArsA has 13 histidine residues located at position 138, 148, 219, 327, 359, 368, 388, 397, 453, 465, 477, 520, and 558 . Each histidine was individually altered to alanine by site-directed mutagenesis . Cells expressing the altered ArsA proteins were resistant to both arsenite and antimonite . The results indicate that no single histidine residue plays a direct role in catalysis, and the inhibition by DEPC may be caused by steric hindrance from the carbethoxy group. J Perinatol, 2001 Dec, 21 Suppl 1, S56 - 8; discussion S59-62 Cytokine- and endotoxin-enhanced bilirubin cytotoxicity; Yeung CY et al.; AIM: To determine the effects of endotoxin and cytokines on the cytotoxic effects of bilirubin . METHODS: A cell-culture model was developed to simulate the effect of an infection by adding endotoxin from E . coli (LPS) and pro-inflammatory cytokines (TNF-alpha, IL-Ialpha, IL-1beta, and IL-6) to the medium . The cytotoxic effects were measured by a modified MTT method . Four cell lines were tested; they were neuroblastoma, glioblastoma, liver, and endothelial cells . RESULTS: Both endotoxin and pro-inflammatory cytokines were demonstrated to enhance bilirubin cytotoxicity on all the cell lines tested, as illustrated by endothelial cell from umbilical vein . Endotoxin and TNF-alpha also showed an additive effect . TNF-alpha concentrations at much lower than clinical sepsis levels have been shown to produce significant cytotoxic effects . CONCLUSION: We speculate that in the jaundiced neonate, infection may increase the risk of tissue damage or kernicterus. Vaccine, 2002 Jan 15, 20(7-8), 1019 - 29 Mutant Escherichia coli heat-labile enterotoxin {LT(R192G)} enhances protective humoral and cellular immune responses to orally administered inactivated influenza vaccine; Lu X et al.; Influenza vaccines capable of inducing both systemic and mucosal antibody responses are highly desirable . Optimal induction of mucosal IgA is accomplished by mucosal delivery of vaccine . Mucosal adjuvants may improve the immunogenicity and efficacy of vaccines delivered by this route . Here, we compare the adjuvant activities of a mutant of heat-labile enterotoxin from Escherichia coli {LT(R192G)} with those of the wildtype LT (wtLT) for oral vaccination with inactivated influenza vaccine in BALB/c mice . Compared with administration of oral influenza vaccine alone, co-administration of vaccine with LT(R192G) provided enhanced protection from infection in the upper and lower respiratory tract equivalent to and at similar doses as that obtained with wtLT . Likewise, LT(R192G) augmented virus-specific IgG and IgA responses in serum, lung and nasal washes and the numbers of virus-specific antibody-forming cells in spleen, lung and Peyer's patches in a manner comparable to wtLT . Virus-specific splenic CD4(+) cells from mice administered oral vaccine with either adjuvant produced a mixed Th1- and Th2-type cytokine response pattern . Taken together, these results indicate that LT(R192G), like wtLT, is a potent adjuvant for oral vaccination of mice with influenza vaccine. Biochim Biophys Acta, 2002 Jan 17, 1553(1-2), 171 - 6 Purification, crystallisation and preliminary crystallographic studies of succinate:ubiquinone oxidoreductase from Escherichia coli; Tornroth S et al.; A membrane protein complex, succinate dehydrogenase (SQR) from Escherichia coli has been purified and crystallised . This enzyme is composed of four subunits containing FAD, three iron-sulphur clusters and one haem b as prosthetic groups . The obtained crystals belong to the hexagonal space group P6(3) with the unit-cell dimensions of a=b=123.8 A and c=214.6 A . An asymmetric unit of the crystals contains one SQR monomer (M(r) 120 kDa) . A data set is now available at 4.0 A resolution with 88.1% completeness and 0.106 R(merge) . We have obtained a molecular replacement solution that shows sensible molecular packing, using the soluble domain of E . coli QFR (fumarate reductase) as a search model . The packing suggests that E . coli SQR is a crystallographic trimer rather than a dimer as observed for the E . coli QFR. Biochim Biophys Acta, 2002 Jan 17, 1553(1-2), 140 - 57 Succinate dehydrogenase and fumarate reductase from Escherichia coli; Cecchini G et al.; Succinate-ubiquinone oxidoreductase (SQR) as part of the trichloroacetic acid cycle and menaquinol-fumarate oxidoreductase (QFR) used for anaerobic respiration by Escherichia coli are structurally and functionally related membrane-bound enzyme complexes . Each enzyme complex is composed of four distinct subunits . The recent solution of the X-ray structure of QFR has provided new insights into the function of these enzymes . Both enzyme complexes contain a catalytic domain composed of a subunit with a covalently bound flavin cofactor, the dicarboxylate binding site, and an iron-sulfur subunit which contains three distinct iron-sulfur clusters . The catalytic domain is bound to the cytoplasmic membrane by two hydrophobic membrane anchor subunits that also form the site(s) for interaction with quinones . The membrane domain of E . coli SQR is also the site where the heme b556 is located . The structure and function of SQR and QFR are briefly summarized in this communication and the similarities and differences in the membrane domain of the two enzymes are discussed. Biochem J, 2002 Feb 1, 361(Pt 3), 641 - 51 Human Hand1 basic helix-loop-helix (bHLH) protein: extra-embryonic expression pattern, interaction partners and identification of its transcriptional repressor domains; Knofler M et al.; The basic helix-loop-helix (bHLH) transcription factor, Hand1, plays an important role in the development of the murine extra-embryonic trophoblast cell lineage . In the present study, we have analysed the expression of Hand1 in human extra-embryonic cell types and determined its binding specificity and transcriptional activity upon interaction with different class A bHLH factors . Northern blotting and in situ hybridization showed that Hand1 mRNA is specifically expressed in amnion cells at different stages of gestation . Accordingly, we demonstrate that the protein is exclusively produced in the amniotic epithelium in vivo and in purified amnion cells in vitro using a novel polyclonal Hand1 antiserum . Reverse transcriptase-PCR and immunohistochemical staining of blastocysts revealed the production of Hand1 mRNA and polypeptide in the trophectodermal cell layer . In the presence of E12/E47, Hand1 stimulated the transcription of luciferase reporters harbouring degenerate E-boxes, suggesting that E-proteins are potential dimerization partners in trophoblastic tumour and amnion cells . In contrast, Hand1 diminished E12/E47-dependent transcription of reporters containing perfect E-boxes by inhibiting the interaction of Hand1/E-protein heterodimers with the palindromic cognate sequence . Furthermore, we show that Hand1 down-regulated GAL-E12-dependent reporter expression, indicating that the protein can also act directly as a transcriptional repressor . Mutational analyses of GAL-Hand1 suggested that two protein regions located within its N-terminal portion mainly confer the repressing activity . In conclusion, human Hand1 may play an important role in the differentiation of the amniotic membrane and the pre-implanting trophoblast . Furthermore, the data suggest that Hand1 can act as a repressor by two independent mechanisms; sequestration of class A bHLH factors from E-boxes and inhibition of their transcriptional activity. Biochem J, 2002 Feb 1, 361(Pt 3), 557 - 66 Inhibition of the integrases of human immunodeficiency viruses type 1 and type 2 by reverse transcriptases; Oz I et al.; We present evidence that the integrases (INs) of HIV types 1 and 2 are inhibited in vitro by the reverse transcriptases (RTs) of HIV-1, HIV-2 and murine leukaemia virus . Both 3'-end processing and 3'-end joining (strand transfer) activities of IN were affected by the RTs . Full inhibitions were accomplished with most RT and IN combinations tested at around equimolar RT/IN ratios . The disintegration activity of IN was also inhibited by RTs . Neither DNA synthesis nor the ribonuclease H (RNase H) domain of RT were involved in IN inhibition, since specific DNA polymerase inhibitors did not affect the level of IN inhibition, and the p51 isoform of HIV-1 RT (which lacks the RNase H domain) is as effective in inhibiting IN as the heterodimeric p66/p51 isoform . On the other hand, the catalytic activities of HIV RTs were not affected by the INs, showing that RTs can inhibit IN activities, whereas INs do not inhibit RTs . We postulate that sequences and/or three-dimensional protein structures common to RTs interact with INs and inhibit their activities . We show evidence for this hypothesis and discuss the possible sites of IN involved in this interaction. Biochemistry, 2002 Jan 29, 41(4), 1343 - 50 The ribonucleolytic activity of angiogenin; Leland PA et al.; Angiogenin (ANG), a homologue of bovine pancreatic ribonuclease A (RNase A), promotes the growth of new blood vessels . The biological activity of ANG is dependent on its ribonucleolytic activity, which is far lower than that of RNase A . Here, the efficient heterologous production of human ANG in Escherichia coli was achieved by replacing two sequences of rare codons with codons favored by E . coli . Hypersensitive fluorogenic substrates were used to determine steady-state kinetic parameters for catalysis by ANG in continuous assays . The ANG pH-rate profile is a classic bell-shaped curve, with pK(1) = 5.0 and pK(2) = 7.0 . The ribonucleolytic activity of ANG is highly sensitive to Na(+) concentration . A decrease in Na(+) concentration from 0.25 to 0.025 M causes a 170-fold increase in the value of k(cat)/K(M) . Likewise, the binding of ANG to a tetranucleotide substrate analogue is dependent on {Na(+)} . ANG cleaves a dinucleotide version of the fluorogenic substrates with a k(cat)/K(M) value of 61 M(-1) s(-1) . When the substrate is extended from two nucleotides to four or six nucleotides, values of k(cat)/K(M) increase by 5- and 12-fold, respectively . Together, these data provide a thorough picture of substrate binding and turnover by ANG. Biochemistry, 2002 Jan 29, 41(4), 1149 - 55 Comparative analyses of the lysine binding site properties of apolipoprotein(a) kringle IV types 7 and 10; Rahman MN et al.; Apolipoprotein(a) {apo(a)} shares extensive sequence similarity with plasminogen and consists of multiple tandem repeats of domains similar to plasminogen kringle IV (KIV), followed by domains homologous to the plasminogen KV and protease domains . The apo(a) KIV domains can be classified into 10 types on the basis of amino acid sequence (KIV(1)-KIV(10)) of which KIV(10) contains a canonical lysine binding site (LBS); KIV(10) mediates the lysine-dependent interaction of Lp(a) with certain biological substrates . Molecular modeling studies indicated the presence of weak LBS in each of KIV(5)-KIV(8), and subsequent biochemical studies have revealed contributions of these kringles to lysine-mediated interactions involving apo(a) . The present study describes the direct demonstration of a weak LBS within KIV(7), as well as the first characterization of the ligand specificity of an LBS outside that of KIV(10) . We have expressed both KIV(7) and KIV(10) from bacterial cells and purified them to homogeneity from cell lysates . Equilibrium binding analyses of the KIV(7) LBS using intrinsic fluorescence revealed an affinity for L-lysine and its analogues approximately 10-fold weaker (K(D) = 230 +/- 42 microM for epsilon-aminocaproic acid) than that of KIV(10) (K(D) = 33 +/- 4 microM for epsilon-aminocaproic acid) . Moreover, we demonstrated differences in specificity of the LBS of KIV(7) in comparison with KIV(10) in that KIV(7) preferentially bound L-proline . Both kringles bind 4-aminobutyric acid with similar affinities albeit with apparently different mechanisms . Key Phe(62) --> Tyr and Asp(56) --> Glu substitutions in the KIV(7) LBS result in alterations in the size of the LBS and in the spatial relationship between the cationic and anionic centers in the LBS and thus account for the differences in the binding properties of KIV(7) and KIV(10). J Basic Microbiol, 2001, 41(6), 329 - 37 The role of the intracellular inhibitor of periplasmic UDP-sugar hydrolase (5'-nucleotidase) in Escherichia coli: cytoplasmic localisation of 5'-nucleotidase is conditionally lethal; Innes D et al.; E . coli UshA, a bifunctional enzyme with UDP-sugar hydrolase and 5'-nucleotidase activities, is secreted to the periplasm but has a specific protein inhibitor located in the cytoplasm . It has been previously suggested that some 5'-nucleotidase, or a folded domain of this enzyme, may be active in the cytoplasm prior to export . If true, the intracellular inhibitor may have a role in protecting the cell from the likely deleterious effects of any intracellular UshA activity . Using deletion mutagenesis to remove the UshA signal peptide, we have shown that the resulting UshA derivative is an active cytoplasmic 5'-nucleotidase, and causes conditional lethality . Our results support the hypothesis that the physiological role of the UshA inhibitor is to protect the intracellular nucleotide pool from any cytoplasmic 5'-nucleotidase activity. Crit Care Med, 2001 Dec, 29(12), 2371 - 3276 Effects of adenosine on extravascular lung water content in endotoxemic pigs; Kutzsche S et al.; OBJECTIVE: To investigate whether adenosine protects against endotoxin-induced increments in extravascular lung water content . DESIGN: Prospective, randomized, animal study . SETTING: University research laboratory . SUBJECTS: Twenty-one anesthetized juvenile pigs . INTERVENTIONS: The animals were divided into two groups subjected to endotoxin infusion: Endotoxin alone (n = 7), or endotoxin combined with adenosine infusion (n = 7) administered during the whole experimental period . Two other groups were exposed to anesthesia alone (n = 4) or adenosine infusion alone (n = 3), respectively . MEASUREMENTS AND MAIN RESULTS: Central hemodynamic variables and extravascular lung water, as assessed by the thermal dye dilution double indicator technique, were monitored . Plasma endothelin-1 concentrations were measured hourly . Extravascular lung water increased significantly in response to endotoxemia (p <.001) along with an increase in pulmonary microvascular pressure (P(mv) {p <.01}) . Although the Pmv increased less in endotoxemic animals exposed to adenosine infusion, no intergroup difference was found . From 4 through 6 hrs, adenosine-treated pigs displayed only half of the extravascular lung water content of nontreated animals (p <.01) . The latter did not differ from that of anesthetized controls receiving anesthesia or adenosine alone . Adenosine administered alone had no effect on P(mv) . In pigs receiving adenosine alone, extravascular lung water content reached nadir after 3 hrs . In both endotoxin groups, plasma endothelin-1 concentration increased two-fold, peaking 4-6 hrs after the start of endotoxin infusion (p <.001) . CONCLUSIONS: The endotoxin-induced increase in lung extravascular water was hampered by intravenously infused adenosine in the presence of a nonsignificantly reduced microvascular pressure . This leaves reduced microvascular permeability the most likely reason for the beneficial effect of adenosine. Crit Care Med, 2001 Dec, 29(12), 2245 - 50 Effects of burn with and without Escherichia coli infection in rats on intestinal vs . splenic T-cell responses; Ravindranath T et al.; OBJECTIVE: To evaluate the effect of burn injury with and without an Escherichia coliseptic complication on T-cell proliferation, interleukin-2 production, and Ca(2+) signaling responses in intestinal Peyer's patch and splenic T cells . DESIGN: Prospective, randomized, sham-controlled animal study . SETTING: University medical center research laboratory . SUBJECTS: Adult male Sprague-Dawley rats . INTERVENTIONS: Rats were subjected to a 30% total body surface area, full skin thickness burn . Infection in rats was induced via intraperitoneal inoculation of E . coli, 10(9) colony forming units/kg, with or without a prior burn . MEASUREMENTS AND MAIN RESULTS: Rat Peyer's patch and splenic T lymphocytes were isolated by using a nylon wool cell purification protocol . T-cell proliferation, interleukin-2 production, and Ca(2+) signaling responses were measured after stimulation of cells with the mitogen, concanavalin A . T-cell proliferation was determined by measuring incorporation of (3)H-thymidine into T-cell cultures . Interleukin-2 production by T-cell cultures was measured by using enzyme-linked immunosorbent assay . Intracellular T-cell Ca2(+ )concentration, {Ca(2+)}(i), was measured by the use of Ca(2+)-specific fluorescent label, fura-2, and its fluorometric quantification . {Ca(2+)}(i) was also evaluated by the use of digital video imaging of fura-2 loaded individual T cells . T-cell proliferation and interleukin-2 production were suppressed substantially in both Peyer's patch and splenic T cells 3 days after either the initial burn alone or burn followed by the E . coli inoculation at 24 hrs after the initial burn . There seemed to be no demonstrable additive effects of E . coli infection on the effects produced by burn injury alone . The T-cell proliferation and interleukin-2 production suppressions with burn or burn-plus-infection insults were correlated with attenuated Ca(2+) signaling . E . coli infection alone suppressed T-cell proliferation in Peyer's patch but not in splenic T cells at 2 days postbacterial inoculation; E . coli infection had no effect on Peyer's patch or splenic T cells at 1 day postinjury . On the other hand, burn injury alone caused a substantial T-cell proliferative suppression at 2 days postburn in both Peyer's patch and splenic cells and a significant suppression in T-cell proliferation on day 1 postburn in Peyer's patch but not in the spleen . CONCLUSION: An initial burn injury suppressed T-cell proliferation at a level that it would not be further affected by a subsequent infection even if the infection by itself has the potential of suppressing T-cell proliferation . An earlier onset of T-cell suppression in Peyer's patch cells than in the spleen with burn could be attributable to an initial hypoperfusion-related intestinal mucosal tissue injury . Overall, our study supports the concept that burn injury per se can significantly suppress T-cell mediated immunity and that the intestine is an early tissue site of such suppression. J Biol Chem, 2002 Apr 5, 277(14), 12275 - 9 Epub 2002 Jan 18. SMAC negatively regulates the anti-apoptotic activity of melanoma inhibitor of apoptosis (ML-IAP); Vucic D et al.; Inhibitors of apoptosis (IAPs) physically interact with a variety of pro-apoptotic proteins and inhibit apoptosis induced by diverse stimuli . X-linked IAP (X-IAP) is a prototype IAP family member that inhibits several caspases, the effector proteases of apoptosis . The inhibitory activity of X-IAP is regulated by SMAC, a protein that is processed to its active form upon receipt of a death stimulus . Cleaved SMAC binds X-IAP and antagonizes its anti-apoptotic activity . Here we show that melanoma IAP (ML-IAP), a potent anti-cell death protein and caspase inhibitor, physically interacts with SMAC through its BIR (baculovirus IAP repeat) domain . In addition to binding full-length SMAC, ML-IAP BIR associates with SMAC peptides that are derived from the amino terminus of active, processed SMAC . This high affinity interaction is very specific and can be completely abolished by single amino acid mutations either in the amino terminus of active SMAC or in the BIR domain of ML-IAP . In cells expressing ML-IAP and X-IAP, SMAC coexpression or addition of SMAC peptides abrogates the ability of the IAPs to inhibit cell death . These results demonstrate the feasibility of using SMAC peptides as a way to sensitize IAP-expressing cells to pro-apoptotic stimuli such as chemotherapeutic agents. J Biol Chem, 2002 Mar 29, 277(13), 11135 - 42 Epub 2002 Jan 18. Human MutY homolog, a DNA glycosylase involved in base excision repair, physically and functionally interacts with mismatch repair proteins human MutS homolog 2/human MutS homolog 6; Gu Y et al.; A