FEBS Lett, 1997 Sep 8, 414(2), 369 - 72 Generation of protonic potential by the bd-type quinol oxidase of Azotobacter vinelandii; Bertsova YV et al.; Inside-out subcellular vesicles of Azotobacter vinelandii are found to produce delta pH and delta psi (interior acidic and positive) when oxidising malate or menadiol . These effects are inherent in both Cyd+ Cyo- (lacking the o-type oxidase) and Cyd- Cyo+ (lacking the bd-type oxidase) strains . They appear to be myxothiazol-sensitive in the Cyd- Cyo+ strain but not in the Cyd+ Cyo- strain . The H+/e- ratio for the terminal part of respiratory chain of a bd-type oxidase overproducing strain is established as being close to 1 . It is also shown that NADH oxidation by the vesicles from the Cyd- Cyo+ strain is sensitive to low concentrations of myxothiazol and antimycin A whereas that of the Cyd+ Cyo- strain is resistant to these Q-cycle inhibitors . It is concluded that (i) the bd-type oxidase of A . vinelandii is competent in generating a protonic potential but its efficiency is lower than that of the o-type oxidase and (ii) Q-cycle does operate in the o-type cytochrome oxidase terminated branch of the A . vinelandii respiratory chain and does not in the bd-type quinol oxidase terminated branch . These relationships are discussed in the context of the respiratory protection function of the bd-type oxidase in A . vinelandii. FEBS Lett, 1997 Sep 8, 414(2), 362 - 4 Ribonucleolytic activities in the Escherichia coli in vitro translation system and in its separate components; Fuchs U et al.; mRNA stability is a limiting parameter for the efficiency of in vitro protein biosynthesis . In order to develop strategies to prolong the mRNA half-life, we investigated the ribonuclease activities in the complete Escherichia coli system, in the separate cell fractions 70S ribosomes and S-100 and in the non-cellular fraction . Our results imply that the amount of ribonucleolytic activities and the insensitivity to placental RNase inhibitor in the complete system are due to the 70S ribosome fraction, whereas the generation of small degradation products is due to the S-100 fraction . Remarkably, the human placental RNase inhibitor is able to reduce mRNA degradation in the bacterial S-100 fraction. FEBS Lett, 1997 Sep 8, 414(2), 327 - 32 Apo structure of the ligand-binding domain of aspartate receptor from Escherichia coli and its comparison with ligand-bound or pseudoligand-bound structures; Chi YI et al.; The aspartate receptor from E . coli is a dimeric transmembrane-signaling protein that mediates chemotaxis behavior and is the most studied system among the chemotaxis receptors to understand the molecular mechanism for transmembrane signaling . However, there is an unresolved issue for the structural event which initiates the transmembrane signal upon binding to the ligand . Biochemical and genetic evidence implies an intrasubunit mechanism (monomeric model) whereas crystallographic evidence implies an intersubunit mechanism (dimeric model) . Crystallographic evidence has been ambiguous because all the apo protein structures contained a pseudoligand sulfate, and a completely ligand-free structure has not been available thus far . Here we present the crystal structure of the ligand binding domain of the aspartate receptor free of the ligand aspartate or pseudoligand sulfate . The structural comparison of this structure with those of ligand-bound and pseudoligand-bound forms revealed that, on ligand or pseudoligand binding, the conformational change in the ligand-binding domain is relatively small, but there is a considerable rotation between two subunits, supporting the dimeric model. FEBS Lett, 1997 Sep 8, 414(2), 293 - 7 Identification of the 4-hydroxyphenylacetate transport gene of Escherichia coli W: construction of a highly sensitive cellular biosensor; Prieto MA et al.; The mechanism of uptake of 4-hydroxyphenylacetate (4-HPA) by Escherichia coli W was investigated . The 4-HPA uptake was induced by 4-HPA, 3-hydroxyphenylacetate (3-HPA) or phenylacetate (PA) and showed saturation kinetics with apparent Kt and Vmax values of 25 microM and 3 nmol/min per 10(9) cells, respectively . Transport of 4-HPA was resistant to N,N'-dimethylcarbodiimide (DCCD), but was completely inhibited by cyanide and 4-nitrophenol, and, to a lower extent, by arsenate and azide, suggesting that energy is required for the uptake process . Competition studies showed that 4-HPA uptake was inhibited by 3-HPA or 3,4-dihydroxyphenylacetate (3,4-DHPA) but not by 2-hydroxyphenylacetate (2-HPA), L-tyrosine or other structural analogues, indicating a narrow specificity of the transport system . We have demonstrated, using two experimental approaches, that the hpaX gene of the 4-HPA catabolic cluster, which encodes a protein of the superfamily of transmembrane facilitators, is responsible for 4-HPA transport . Aside from the aromatic amino acid transport systems, hpaX is the first transport gene for an aromatic compound of enteric bacteria that has been characterized . A highly sensitive cellular biosensor has been constructed by coupling the 4-HPA transport system to a regulatory circuit that controls the production of beta-galactosidase . This biosensor has allowed us to demonstrate that the transport system performs efficiently at very low external concentrations of 4-HPA, similar to levels that would be expected to occur in natural environments. FEBS Lett, 1997 Sep 8, 414(2), 275 - 80 Bound water in the proton translocation mechanism of the haem-copper oxidases; Riistama S et al.; We address the molecular mechanism by which the haem-copper oxidases translocate protons . Reduction of O2 to water takes place at a haem iron-copper (CuB) centre, and protons enter from one side of the membrane through a 'channel' structure in the enzyme . Statistical-mechanical calculations predict bound water molecules within this channel, and mutagenesis experiments show that breaking this water structure impedes proton translocation . Hydrogen-bonded water molecules connect the channel further via a conserved glutamic acid residue to a histidine ligand of CuB . The glutamic acid side chain may have to move during proton transfer because proton translocation is abolished if it is forced to interact with a nearby lysine or arginine . Perturbing the CuB ligand structure shifts an infrared mode that may be ascribed to the O-H stretch of bound water . This is sensitive to mutations of the glutamic acid, supporting its connectivity to the histidine . These results suggest key roles of bound water, the glutamic acid and the histidine copper ligand in the mechanism of proton translocation. FEBS Lett, 1997 Sep 8, 414(2), 271 - 4 dUTPase from the retrovirus equine infectious anemia virus: specificity, turnover and inhibition; Nord J et al.; The kinetic properties of dUTPase from equine infectious anemia virus (EIAV) were investigated . K(M) (1.1 +/- 0.1 microM) and k(cat) (25 s(-1)) were found to be independent of pH in the neutral pH range . Above pH 8.0, K(M) increases slightly . Below pH 6.0, the enzyme is rapidly deactivated . Detergent was found to enhance activity, leaving K(M) and k(cat) unaffected . Compared to the Escherichia coli dUTPase, the EIAV enzyme is equally potent in hydrolyzing dUTP, but less specific . Inhibition of the viral enzyme by the nucleotides dTTP, dUMP and a synthetic analogue, 2'-deoxyuridine 5'-(alpha,beta-imido)triphosphate, is stronger by one order of magnitude. FEBS Lett, 1997 Sep 8, 414(2), 219 - 20 Expression, purification, and co-crystallization of IRF-I bound to the interferon-beta element PRDI; Escalante CR et al.; Interferon regulatory factor 1 (IRF-1) is an essential factor involved in the regulation of type I interferon (IFN) and IFN-inducible genes . The protein consists of 329 amino acids that are highly conserved from mouse to human . Similar to other transcription factors, the protein is modular in nature with a basic N-terminal region involved in DNA binding and an acidic C-terminal region required for activation . We report here the expression, purification and co-crystallization of the minimal N-terminal region of IRF-1 involved in DNA binding (amino acids 1-113) with a 13 bp DNA fragment from the IFN-beta promoter . The crystals diffract to at least 3.0 A in resolution and belong to space group R3 with unit cell parameters of a = b = 84.8 A, c = 203.7 A. FEBS Lett, 1997 Sep 8, 414(2), 213 - 8 Decreased substrate affinity upon alteration of the substrate-docking region in cytochrome P450(BM-3); Maves SA et al.; A mutation at the surface of the substrate access channel which dramatically decreases the affinity for some fatty acids in P450(BM-3) was discovered by random mutagenesis . The mutation introduced, proline-25 to glutamine, is in close proximity to the arginine-47 residue thought to be responsible for the initial docking of fatty acid substrates . The P25Q mutant displays an affinity for palmitate which is approximately 100-fold weaker than the wild-type enzyme . In addition to its altered substrate affinity, P25Q also exhibits altered hydroxylation specificity and carbon monoxide recombination kinetics in the substrate-free form. Biomed Environ Sci, 1997 Sep, 10(2-3), 271 - 9 Selenium and the thioredoxin and glutaredoxin systems; Bjornstedt M et al.; Thioredoxin (Trx) is a small ubiquitous dithiol protein which together with the FAD-containing enzyme thioredoxin reductase (TR) and NADPH (the Trx system) is a hydrogen donor for ribonucleotide reductase essential for DNA synthesis and a general protein disulfide reductase involved in redox regulation . Selenite, selenodiglutathione (GS-Se-SG) and selenocystine are efficiently reduced by thioredoxins and also directly by NADPH and mammalian TR but not by the E . coli enzyme . Incubation of selenite or GS-Se-SG with the Trx system or with mammalian TR results in a rapid formation of selenide, which by redox cycling with oxygen may cause a large non-stoichiometric oxidation of NADPH . Selenocystine is efficiently reduced into two molecules of the selenol amino acid selenocysteine by mammalian TR with a K(m)-value (6 mumol.L-1) and a high turnover number (kappa cat 3200 min-1) almost identical to the natural substrate Trx-S2 . TR also directly reduces lipid hydroperoxides and this peroxidase reaction is strongly stimulated by the presence of catalytic amounts of free selenocysteine . Glutaredoxin (Grx) which catalyzes GSH-dependent disulfide reduction also via a redox-active disulfide and Trx are both efficient electron donors to the human plasma glutathione peroxidase providing a mechanism by which human plasma glutathione peroxidase may reduce hydroperoxides in an environment almost free from glutathione . Selenate is reduced by Grx and Trx in the presence of GSH . The DNA-binding of the transcription factor AP-1 is strongly inhibited by GS-Se-SG and selenite . Furthermore, selenide formed by TR-mediated reduction of selenite and GS-Se-SG inhibits lipoxygenase and changes the electron spin resonance spectrum of the active site iron . Mammalian TR with two subunits of 57 kDa has recently been cloned and shown to be homologous to glutathione reductase . The rat enzyme contains a selenocysteine residue in a unique Cterminal position and a conserved SECIS sequence directing insertion of the selenocysteine . The discovery of selenocysteine in mammalian TR may explain the broad substrate specificity of the enzyme and the requirement of selenium for cell proliferation. Biochem Mol Biol Int, 1997 Sep, 43(1), 47 - 52 Glycine68 to histidine73 has an important role in the function of human tumor necrosis factor alpha; Cen B et al.; Mutant human tumor necrosis factor alpha(hTNF alpha) genes have been constructed by in vitro mutagenesis and expressed in Escherichia coli . A deletion involving Gly68 to His73 in hTNF alpha remarkably decreased the solubility and biological activity of hTNF alpha . From the above and results of a molecular dynamics simulation it is proposed that the region of Gly68 to His73 in hTNF alpha has an important role in the maintenance of the 3-D structure and modulation of the biological activity of hTNF alpha. Biol Reprod, 1997 Oct, 57(4), 707 - 14 Cell-type expression, immunolocalization, and deoxyribonucleic acid-binding activity of basic transcription element binding transcription factor, an Sp-related family member, in porcine endometrium of pregnancy; Wang Y et al.; Basic transcription element binding (BTEB) protein is a newly identified member of the C2H2 zinc finger family that also includes the transcription factors, Sp1, Sp2, Sp3, and Sp4 . This family of proteins binds GC-rich motifs widely distributed in gene promoters, resulting in distinct activation or repression of transcriptional activities . Whereas Sp proteins are ubiquitously expressed, expression of BTEB appears more limited and has not been documented in the female reproductive tract of any mammalian species . This study was designed to identify and characterize the cellular distribution of BTEB in the porcine endometrium and placenta at known stages of pregnancy . Northern analysis of uterine endometrium detected BTEB mRNA that corresponds in size (5 kilobases) to that of the major BTEB transcript in rat brain . The steady-state levels of BTEB mRNA were higher (p < 0.05) in endometrium than placenta at corresponding days of pregnancy, although for each tissue, the levels did not change with pregnancy stage (p > 0.05) . Luminal epithelial (LE), glandular epithelial (GE), and stromal (ST) cells isolated from pregnancy endometrium expressed the BTEB gene, but mRNA abundance varied with cell type (LE, GE > ST) . Western blot analysis using an antiserum generated against the N-terminal region of a porcine BTEB fusion protein produced in Escherichia coli revealed the presence of BTEB protein only in endometrium, not in placenta . Immunohistochemical studies localized BTEB predominantly to the nuclei of endometrial GE and LE cells . Consistent with the presence of functional BTEB protein, binding to a double-stranded oligonucleotide containing multiple GC motifs was demonstrated in nuclear extracts prepared from endometrium and from endometrial LE and GE, but not ST, cells by electrophoretic mobility shift assay . These results demonstrate the preferential endometrial cell-type expression of BTEB and suggest its regulatory role in pregnancy-associated endometrial epithelial gene expression. J Exp Med, 1997 Oct 6, 186(7), 1027 - 39 A critical role for Syk in signal transduction and phagocytosis mediated by Fcgamma receptors on macrophages; Crowley MT et al.; Receptors on macrophages for the Fc region of IgG (FcgammaR) mediate a number of responses important for host immunity . Signaling events necessary for these responses are likely initiated by the activation of Src-family and Syk-family tyrosine kinases after FcgammaR cross-linking . Macrophages derived from Syk-deficient (Syk-) mice were defective in phagocytosis of particles bound by FcgammaRs, as well as in many FcgammaR-induced signaling events, including tyrosine phosphorylation of a number of cellular substrates and activation of MAP kinases . In contrast, Syk- macrophages exhibited normal responses to another potent macrophage stimulus, lipopolysaccharide . Phagocytosis of latex beads and Escherichia coli bacteria was also not affected . Syk- macrophages exhibited formation of polymerized actin structures opposing particles bound to the cells by FcgammaRs (actin cups), but failed to proceed to internalization . Interestingly, inhibitors of phosphatidylinositol 3-kinase also blocked FcgammaR-mediated phagocytosis at this stage . Thus, PI 3-kinase may participate in a Syk-dependent signaling pathway critical for FcgammaR-mediated phagocytosis . Macrophages derived from mice deficient for the three members of the Src-family of kinases expressed in these cells, Hck, Fgr, and Lyn, exhibited poor Syk activation upon FcgammaR engagement, accompanied by a delay in FcgammaR-mediated phagocytosis . These observations demonstrate that Syk is critical for FcgammaR-mediated phagocytosis, as well as for signal transduction in macrophages . Additionally, our findings provide evidence to support a model of sequential tyrosine kinase activation by FcgammaR's analogous to models of signaling by the B and T cell antigen receptors. J Biol Chem, 1997 Sep 5, 272(36), 22960 - 5 Molecular cloning, primary structure, and properties of a new glycoamidase from the fungus Aspergillus tubigensis; Ftouhi-Paquin N et al.; A new glycoamidase, peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase (PNGase) At, was discovered in the eukaryote Aspergillus tubigensis . The enzyme was purified to homogeneity, and the DNA sequence was determined by cloning in Escherichia coli . Over 80% of the deduced amino acid sequence was verified independently by Edman analysis and/or electrospray ionization-mass spectrometry of protease fragments of native PNGase At . This glycoamidase contains 12 potential asparagine-linked glycosylation sites, of which at least 9 sites are occupied with typical high mannose oligosaccharides . PNGase At consists of two non-identical glycosylated subunits that are derived from a single polypeptide gene precursor . Evidence is presented suggesting that autocatalysis is involved in subunit formation . PNGase At is an important new tool for analysis of asparagine-linked glycans; it can hydrolyze a broad range of glycopeptides, including those with core-linked alpha1-->6 or alpha1-->3 fucose, under conditions not favorable with existing glycoamidases. Nature, 1997 Sep 25, 389(6649), 403 - 6 Visualization of elongation factor Tu on the Escherichia coli ribosome; Stark H et al.; The delivery of a specific amino acid to the translating ribosome is fundamental to protein synthesis . The binding of aminoacyl-transfer RNA to the ribosome is catalysed by the elongation factor Tu (EF-Tu) . The elongation factor, the aminoacyl-tRNA and GTP form a stable 'ternary' complex that binds to the ribosome . We have used electron cryomicroscopy and angular reconstitution to visualize directly the kirromycin-stalled ternary complex in the A site of the 70S ribosome of Escherichia coli . Electron cryomicroscopy had previously given detailed ribosomal structures at 25 and 23 A resolution, and was used to determine the position of tRNAs on the ribosome . In particular, the structures of pre-translocational (tRNAs in A and P sites) and post-translocational ribosomes (P and E sites occupied) were both visualized at a resolution of approximately 20 A . Our three-dimensional reconstruction at 18 A resolution shows the ternary complex spanning the inter-subunit space with the acceptor domain of the tRNA reaching into the decoding centre . Domain 1 (the G domain) of the EF-Tu is bound both to the L7/L12 stalk and to the 50S body underneath the stalk, whereas domain 2 is oriented towards the S12 region on the 30S subunit. Surgery, 1997 Sep, 122(3), 534 - 45 Effect of transfusion on physiologic changes after resuscitated trauma; Lyden SP et al.; BACKGROUND: The purpose of this experimental study was to test whether transfusion potentiated physiologic changes associated with fluid resuscitated trauma in controlled conditions . METHODS: Anesthetized and ventilated mongrel pigs were subjected to soft-tissue injury plus 35% hemorrhage and 1 hour shock and then were resuscitated with either autologous (shed) or heterologous (cross-transfused) fresh whole blood . Leukocyte differential counts, T-lymphocyte subsets, neutrophil adherence molecule (CD18) expression, granulocyte oxidative burst, plasma cortisol, and serum chemistries were monitored in awake animals with indwelling catheters on 3 consecutive days . Changes were referenced to preinjury baseline values and to a control group that received heterologous transfusion but no shock . To determine whether these changes might have influenced host defense, a low-dose challenge with Escherichia coli endotoxin (lipopolysaccharide {LPS}; 1 to 2 micrograms/kg for 30 minutes) was administered on day 4 . RESULTS: During recovery, neutrophil counts, neutrophil CD18 expression, and granulocyte oxidative burst were generally increased, but the changes were not potentiated by transfusion . Lymphocyte subpopulations remained relatively constant . Serum enzyme markers were elevated with trauma plus shed blood or trauma plus cross-transfusion, but they remained essentially unchanged after heterologous transfusion only . Plasma cortisol, a nonspecific index of stress, peaked at 3 to 6 times higher than baseline . The increases tended to be higher and later with heterologous transfusion only, relative to trauma plus shed blood or trauma plus cross-transfusion . The delayed LPS challenge evoked profound but transient pulmonary hypertension and leukopenia, followed by subsequent hypoxemia; the time courses and magnitude of these changes were similar in all groups . CONCLUSIONS: If these measured variables before and after LPS challenge are a valid index of host defense in this species, then a 35% transfusion does not potentiate the risk for posttrauma immune dysfunction when the magnitude of injury is constant . Thus the predisposition to infection after human trauma might be due to cold storage of blood; separation of blood into components, or other transfusion-related practices rather than to transfusion per se. Adv Enzyme Regul, 1997, 37, 95 - 109 Recent advances in the study of rTS proteins . rTS expression during growth and in response to thymidylate synthase inhibitors in human tumor cells; Dolnick BJ et al.; The rTS proteins have now been shown to be expressed in a variety of cell lines, with expression of rTS beta being found elevated in three cell lines which are resistant to TS inhibitors (3, 4) (Figure 1) . In one of these cell lines (K562 B1A), the cells were selected for resistance to MTX, which has a primary site of action on DHFR, but was found to be cross-resistant to FUdR (4) . The other two cell lines were selected for resistance to either 5-fluorouracil (H630-1) or a combination of ZD1694 and FU . In each case, elevation of rTS beta appears to be a selected response to thymidylate stress . In HCT-8 and HCT-8/DF2 cells, treatment of cells for a short period of time (2 hr) resulted in the elevation of rTS beta levels, again suggestive that expression of rTS beta is a response to thymidylate stress . rTS beta appears to be regulated with cell growth, its levels increasing at mid-log and at late-log/saturation phase in H630 and H630-1 cells (Fig . 2), and increasing with late-log in several other cell lines as well (Fig . 3) . The increase in rTS beta is suggestive of a cellular function associated with a state where growth is no longer desirable, reminiscent of the starvation-sensing protein homolog RSPA in E . coli (22) . While this relationship would not explain the spike in rTS beta levels in mid-log H630 and H630-1 cells, it does make sense if the rTS proteins (particularly rTS beta) are involved in down-regulating thymidylate biosynthesis . The potential mechanism of this down-regulation may be speculated to be the catabolism of some precursor for thymidylate biosynthesis or some direct effect upon TS through modulation by some other ligand, either a metabolite or another protein . Studies on the expression of rTS proteins in clinical specimens indicate that rTS beta is expressed at high levels in kidney and kidney tumor (Dolnick, unpublished results) . Given the physiologic role of the kidney, high level expression of rTS in this organ is consistent with a role in a catabolic pathway . Since down-regulation of TS activity is expected to increase sensitivity to TS inhibitors, a role for rTS beta in directly down-regulating TS activity in the biochemical sense would seem unlikely . However, the manner of biochemical TS down-regulation may make a difference . In the TS- Cl/Cl cell line, there are two mutations in TS which likely reduce affinity for N-5,10-methylene tetrahydrofolates (23) . This cell line is highly resistant to MTX, yet is still tumorigenic in vivo (24), and supplying the cells with high levels of exogenous folate can restore TS function (23) . Thus in TS- Cl/Cl cells, the TS phenotype is conditionally dependent upon the presence of high levels of exogenous folate . This suggests that a role of rTS proteins as conditional down-regulators of TS, perhaps through modulating folate binding, may be possible . Two cell lines (K562 B1A and H630-1) that overproduce rTS beta have altered sensitivity to TS inhibitors that differ depending upon the nature of the inhibitor . The K562 B1A cell line was found to be approximately 2000-fold resistant to ZD1694 and BW1843U89 (120 hr exposures), but only three-fold resistant to AG331 . The H630-1 cell line is approximately 30-fold resistant to BW1843U89 (120 hr exposure) and 40-fold resistant to ZD1694 (120 hr exposure), but only eight-fold resistant to AG331 . Since K562 B1A cells overproduce rTS beta (2), but have no significant alterations in FPGS activity, the possibility that rTS may affect folate binding remains a hypothesis worth examining . The recent discovery that TS is a phosphoprotein and that it is nuclear as well as cytoplasmic (21) raises the possibility that the phosphorylation state of TS may regulate one of its cellular functions, and that the subcellular localization of this enzyme is regulated as well . Since rTS proteins have HSP with proteins that participate in kinase/phosphatase reactions, this also seems to be an avenue worthy of future investigation . (ABSTRACT TRUNCATED Res Exp Med (Berl), 1997, 197(2), 91 - 9 Function of reduced-size liver transplant in the rat; Foss A et al.; We have studied the function of partial orthotopic liver transplantation in the rat by evaluating prothrombin time (PT), liver blood flow, basal and glucose-stimulated insulin secretion and glucose tolerance, and the reticuloendothelial function (RES) in hepatectomized rats subjected to partial liver transplantation . A graft corresponding to 68% of a normal liver was transplanted to totally hepatectomized rats . Comparison was made between control rats and rats subjected to 32% liver resection . PT was not significantly different in the transplanted group compared with liver-resected and control rats . Laser Doppler flowmetry showed that at 28 days after surgery, blood flow had increased in the transplanted livers . Furthermore, on the third day after transplantation, basal plasma insulin was increased and the plasma insulin response to glucose was exaggerated, suggesting reduced insulin action and impaired insulin degradation . Finally, uptake of radioactive-labeled E . coli bacteria, as a measure of RES function, was not compromised in transplanted animals . Based on these results, we conclude that reduced-size liver transplant in out-bred rats results in fast normalization of liver function after surgery although, immediately after surgery, glucose intolerance is seen. Proc Natl Acad Sci U S A, 1997 Sep 30, 94(20), 10583 - 7 Subunit rotation in Escherichia coli FoF1-ATP synthase during oxidative phosphorylation; Zhou Y et al.; We report evidence for proton-driven subunit rotation in membrane-bound FoF1-ATP synthase during oxidative phosphorylation . A betaD380C/gammaC87 crosslinked hybrid F1 having epitope-tagged betaD380C subunits (betaflag) exclusively in the two noncrosslinked positions was bound to Fo in F1-depleted membranes . After reduction of the beta-gamma crosslink, a brief exposure to conditions for ATP synthesis followed by reoxidation resulted in a significant amount of betaflag appearing in the beta-gamma crosslinked product . Such a reorientation of gammaC87 relative to the three beta subunits can only occur through subunit rotation . Rotation was inhibited when proton transport through Fo was blocked or when ADP and Pi were omitted . These results establish FoF1 as the second example in nature where proton transport is coupled to subunit rotation. Proc Natl Acad Sci U S A, 1997 Sep 30, 94(20), 10541 - 6 The Aer protein and the serine chemoreceptor Tsr independently sense intracellular energy levels and transduce oxygen, redox, and energy signals for Escherichia coli behavior; Rebbapragada A et al.; We identified a protein, Aer, as a signal transducer that senses intracellular energy levels rather than the external environment and that transduces signals for aerotaxis (taxis to oxygen) and other energy-dependent behavioral responses in Escherichia coli . Domains in Aer are similar to the signaling domain in chemotaxis receptors and the putative oxygen-sensing domain of some transcriptional activators . A putative FAD-binding site in the N-terminal domain of Aer shares a consensus sequence with the NifL, Bat, and Wc-1 signal-transducing proteins that regulate gene expression in response to redox changes, oxygen, and blue light, respectively . A double mutant deficient in aer and tsr, which codes for the serine chemoreceptor, was negative for aerotaxis, redox taxis, and glycerol taxis, each of which requires the proton motive force and/or electron transport system for signaling . We propose that Aer and Tsr sense the proton motive force or cellular redox state and thereby integrate diverse signals that guide E . coli to environments where maximal energy is available for growth. Proc Natl Acad Sci U S A, 1997 Sep 30, 94(20), 10506 - 11 A thymidine triphosphate shape analog lacking Watson-Crick pairing ability is replicated with high sequence selectivity; Moran S et al.; Compound 1 (F), a nonpolar nucleoside analog that is isosteric with thymidine, has been proposed as a probe for the importance of hydrogen bonds in biological systems . Consistent with its lack of strong H-bond donors or acceptors, F is shown here by thermal denaturation studies to pair very poorly and with no significant selectivity among natural bases in DNA oligonucleotides . We report the synthesis of the 5'-triphosphate derivative of 1 and the study of its ability to be inserted into replicating DNA strands by the Klenow fragment (KF, exo- mutant) of Escherichia coli DNA polymerase I . We find that this nucleotide derivative (dFTP) is a surprisingly good substrate for KF; steady-state measurements indicate it is inserted into a template opposite adenine with efficiency (Vmax/Km) only 40-fold lower than dTTP . Moreover, it is inserted opposite A (relative to C, G, or T) with selectivity nearly as high as that observed for dTTP . Elongation of the strand past F in an F-A pair is associated with a brief pause, whereas that beyond A in the inverted A-F pair is not . Combined with data from studies with F in the template strand, the results show that KF can efficiently replicate a base pair (A-F/F-A) that is inherently very unstable, and the replication occurs with very high fidelity despite a lack of inherent base-pairing selectivity . The results suggest that hydrogen bonds may be less important in the fidelity of replication than commonly believed and that nucleotide/template shape complementarity may play a more important role than previously believed. Mol Microbiol, 1997 Aug, 25(4), 707 - 16 Messenger RNA release from ribosomes during 5'-translational blockage by consecutive low-usage arginine but not leucine codons in Escherichia coli; Gao W et al.; In '5'-translational blockage', significantly reduced yields of proteins are synthesized in Escherichia coli when consecutive low-usage codons are inserted near translation starts of messages (with reduced or no effect when these same codons are inserted downstream) . We tested the hypothesis that ribosomes encountering these low-usage codons near the translation start prematurely release the mRNA . RNA from polysome gradients was fractionated into pools of polysomes and monosomes and a ribosome-free pool . New hybridization probes, called 'molecular beacons', and standard slot blots were used to detect test messages containing either consecutive low-usage AGG (arginine) or synonymous high-usage CGU insertions near the 5' end . The results show an approximately twofold increase in the ratio of free to bound mRNA when the low-usage codons were present in the message compared with when high-usage codons were present . In contrast, there was no difference in the ratio of free to bound mRNA when consecutive low-usage CUA or high-usage CUG (leucine) codons were inserted or when the arginine codons were inserted near the 3' end . These data indicate that at least some mRNA is released from ribosomes during 5'-translational blockage by arginine but not leucine codons, and they support proposals that premature termination of translation can occur in some conditions in vivo in the absence of a stop codon. Mol Microbiol, 1997 Aug, 25(4), 661 - 70 Overinitiation of chromosome replication in the Escherichia coli dnaAcos mutant depends on activation of oriC function by the dam gene product; Katayama T et al.; The activity of DnaA protein, the initiator of chromosome replication in Escherichia coli, is regulated by adenine nucleotide binding; the ATP-bound form, not the ADP-bound form, is active . DnaAcos is a mutant protein that is insensitive to negative regulation by ADP . Initiation of chromosome replication occurs excessively in the dnaAcos mutant at 30 degrees C, a restrictive temperature for growth . To determine the control factors that act independently of adenine nucleotide binding of DnaA, we analysed suppressors from the dnaAcos mutant isolated by Tn5 insertion mutagenesis . Three of the suppressors carried Tn5 in the aroK or aroB gene, the first two cistrons in the dam operon . Complementation tests revealed that the dam gene is responsible for the suppression . Over-replication of the chromosome was inhibited in the dnaAcos aroK::Tn5 double mutant, and initiation of chromosome replication in the dnaA+ aroK::Tn5 mutant was partially inhibited . The aroK(or B)::Tn5 cells contained DnaA molecules at a level similar to that in the parental aroBK+ strain . Moreover, dnaAcos suppression depended on the function of the seqA gene . Thus, Dam activity positively regulates initiation of chromosome replication in vivo . SeqA function seems to be distinguished from the control of DnaA protein by adenine nucleotide binding. J Biochem (Tokyo), 1997 Aug, 122(2), 430 - 7 A novel chloride-binding site modulates the heme-copper binuclear center of the Escherichia coli bo-type ubiquinol oxidase; Hirano T et al.; Cytochrome bo-type ubiquinol oxidase in Escherichia coli belongs to a superfamily of the heme-copper respiratory oxidases and catalyzes the redox-coupled proton pumping . Previous studies {Y . Orii, T . Mogi, M . Sato-Watanabe, T . Hirano, and Y . Anraku (1995) Biochemistry 34, 1127-1132} suggest that it requires chloride ions for the facilitated heme b-to-heme o intramolecular electron transfer . To extend our previous studies on chloride binding by bo-type ubiquinol oxidase, we prepared two kinds of chloride-bound enzymes, UQO-412 and UQO-409, and a chloride-depleted enzyme, UQO-407, and examined their spectroscopic and enzymatic properties . UQO-412, which exhibits the Soret peak at 412 nm in the air-oxidized state, was obtained by purification with anion-exchange liquid chromatography, and UQO-409 was derived from UQO-412 by extensive washing and showed a 3-nm blue shift . UQO-407 was obtained from UQO-409 by omitting chloride ions from buffers throughout purification and showed a further blue shift in the Soret peak and the pronounced chloride-sensitive EPR signals at g=6 and g=3.15, which are attributable to spin-spin exchange interaction at the binuclear center . Kinetic studies on chloride binding by UQO-407 revealed the presence of a chloride-binding site with a K(d) value of 3.5 mM . Flow-flash experiments demonstrated that the heme b-to-heme o electron transfer was perturbed in both UQO-409 and UQO-407, although steady state enzyme activities of three UQOs were indistinguishable . The present studies demonstrated that the E . coli bo-type ubiquinol oxidase is endowed with a novel chloride-binding site which controls the electromagnetic state of the heme-copper binuclear center . Further, we suggest that the intramolecular electron transfer in the enzyme requires diffusible molecules other than the bound chloride ion. J Biochem (Tokyo), 1997 Aug, 122(2), 422 - 9 Substitutions of charged amino acid residues conserved in subunit I perturb the redox metal centers of the Escherichia coli bo-type ubiquinol oxidase; Kawasaki M et al.; Cytochrome bo is a four-subunit quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as a redox-coupled proton pump . Subunit I binds all the redox metal centers, low-spin heme b, high-spin heme o, and Cu(B), and serves as a reaction center of the oxidase complex . This work focuses on the functional and structural roles of 14 charged amino acid residues that are conserved in subunit I of the heme-copper terminal oxidases . Substitutions of Lys55, Tyr173, Asp188, Asp256, Arg481, and Arg482 by neutral amino acid residues did not affect the catalytic activity and spectroscopic properties of the cytoplasmic membranes . In contrast, genetic complementation tests indicated that replacements of Arg80, Asp135, Arg257, Glu286, Tyr288, Lys362, Asp407, and Glu540 resulted in nonfunctional enzymes . The R80Q mutation caused loss of a diagnostic peak for low-spin heme b in the 77 K redox difference spectrum . The K362Q, D407N, and E540Q mutations affected the CO-binding by the heme-copper binuclear center . The D135N, R257Q, E286Q, and Y288F mutations specifically eliminated the Cu(B) center from the oxidase complex, whereas the E286D mutant did not show significant perturbations on the redox metal centers even though it was still inactive . Based on these findings and recent crystallographic studies on cytochrome c oxidases, we discuss the possible roles of the conserved charged amino acid residues in subunit I of the heme-copper terminal oxidases. J Biochem (Tokyo), 1997 Aug, 122(2), 415 - 21 Assignment and functional roles of the cyoABCDE gene products required for the Escherichia coli bo-type quinol oxidase; Nakamura H et al.; Cytochrome bo from Escherichia coli belongs to the heme-copper terminal oxidase superfamily and functions as a redox-driven proton pump . In the present study, we examined the functional roles of the cyoABCDE genes, which encode cytochrome bo . We expressed the cyoABCDE genes in minicells using pTTQ18 derivatives and identified subunits II, I, III, and IV of the oxidase complex and heme O synthase as polypeptides with molecular weights of 33,500, 75,000, 20,500, 12,000, and 28,000, respectively . The expression level of heme O synthase (CyoE) was much lower than those of the oxidase subunits and seems to be controlled just tightly enough for the incorporation of heme O into the oxidase complex . To facilitate functional analysis of the gene products, we developed a single copy expression vector pHNF2, a derivative of the F-sex factor . Genetic complementation tests showed that deletions in each gene resulted in nonfunctional enzymes . Western blotting analysis indicated that the expression levels of subunits I and II were not affected by the deletions in the other cyo gene products . However, spectroscopic analyses of the mutant membranes revealed that all the deletions perturbed or eliminated the redox metal centers in subunit I . Present findings suggest that subunits II, III, and IV of the oxidase complex are required for the assembly of the metal centers in subunit I. J Biochem (Tokyo), 1997 Aug, 122(2), 309 - 13 Characterization of molecularly cloned human 5-aminoimidazole-4-carboxamide ribonucleotide transformylase; Sugita T et al.; The cDNA encoding human 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase has been cloned from a placenta cDNA library, utilizing a PCR-derived probe . It encodes a peptide of 592 amino acids . The amino (N)-terminal sequence of this enzyme, purified from HeLa cells and CCRF-CEM cells, was found to be APGQLALF- . Both sequencing results revealed a difference of six N-terminal residues when compared to the reported sequence of cloned cDNA from a hepatoma cDNA library . Northern-blot analysis of human AICAR transformylase mRNA showed the expression of a single 2.0 kb mRNA in all tissues examined . With the cloned cDNA fragment, we constructed expression vectors for mature and GST-fused AICAR transformylase . Both recombinant molecules possessing AICAR transformylase activity were overproduced in Escherichia coli . GST-AICAR transformylase can be purified to homogeneity by a single-step affinity procedure with glutathione Sepharose . Mutational analysis, utilizing this expression system, showed that His213 and His267 were essential for AICAR transformylase activity. J Allergy Clin Immunol, 1997 Sep, 100(3), 365 - 72 Cloning and expression of the panallergen profilin and the major allergen (Ole e 1) from olive tree pollen; Asturias JA et al.; BACKGROUND: Olive tree (Olea europaea) pollen allergy is one of the main causes of allergy in Mediterranean countries and some areas of North America . OBJECTIVE: To clone olive allergens and to characterize immunologically the purified recombinant allergens . METHODS: Full-length complementary deoxyribonucleic acid (cDNA) strands encoding olive allergens (Ole e 1) were cloned by polymerase chain reaction amplification and sequenced . Recombinant proteins were produced in Escherichia coli by the use of two different expression systems . Immunoreactivity of the recombinant proteins was tested by ELISA and Western blot with serum from patients with allergy to olive . RESULTS: Significant sequence polymorphism was found in both allergens . The panallergen profilin was expressed as a nonfusion protein and was purified to homogeneity after a single step of affinity chromatography with a poly-L-proline Sepharose column . One cDNA encoding an Ole e 1 isoform was expressed as a fusion protein consisting of the glutathione S-transferase of Schistosoma japonicum and Ole e 1 . The fusion protein was purified to homogeneity by gel filtration chromatography and affinity chromatography with a glutathione-Sepharose column, and digested with thrombin . Both recombinant allergens shared B cell epitopes with the corresponding natural allergens . CONCLUSION: IgE-reactive Ole e 1 and olive profilin expressed in bacteria were purified after simple chromatographic procedures and may be useful for diagnostic purposes. J Biol Chem, 1997 Oct 3, 272(40), 25296 - 303 Interactions between brain-derived neurotrophic factor and the TRKB receptor . Identification of two ligand binding domains in soluble TRKB by affinity separation and chemical cross-linking; Haniu M et al.; The extracellular domain of the human neurotrophin TRKB receptor expressed in Chinese hamster ovary cells is a highly glycosylated protein, possessing binding ability for brain-derived neurotrophic factor (BDNF) . Two distinct ligand binding domains of TRKB were isolated from proteolytic digests of the receptor by affinity separation on immobilized BDNF . One of these domains consists of amino acid residues 103-181 and contains both the third leucine-rich motif and the second cysteine cluster domain . The second domain is close to the second immunoglobulin-like domain (amino acid residues 342-394) . Each of these two domains can bind BDNF independently . Disulfide linkages present in the first domain are necessary for BDNF binding, probably because of preservation of the native conformation . To study the second domain in greater detail, a truncated form of TRKB containing the second immunoglobulin-like domain (residues 248-398) was expressed in Escherichia coli . This domain was cross-linked to BDNF through a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide coupling reaction . Several synthetic peptides corresponding to amino acid residues 343-379 were able to bind immobilized BDNF . Amino acid substitution and cross-linking analysis indicated that amino acids Phe347, Asp354, and Tyr361 are intimately involved in BDNF binding . These results, obtained from a variety of experimental techniques, highlight the importance of two distinct regions of the extracellular domain of the TRKB receptor in binding BDNF. J Biol Chem, 1997 Oct 3, 272(40), 25224 - 8 The open reading frame 1 of the L1Tc retrotransposon of Trypanosoma cruzi codes for a protein with apurinic-apyrimidinic nuclease activity; Olivares M et al.; The deduced amino acid sequence of the open reading frame 1 (ORF1) of the L1Tc non-site-specific non-long terminal repeat retrotransposon of Trypanosoma cruzi exhibits a significant homology with the consensus sequence of the class II family of the endonuclease apurinic-apyrimidinic (AP) proteins . The analysis of the activity of the 40-kDa recombinant protein, named NL1Tc, obtained from the expression of the L1Tc ORF1 in an Escherichia coli "in vitro" expression system revealed that the sequence codes for a protein with endonuclease activity specific for apurinic-apyrimidinic (AP) sites . Data are also presented showing that in vivo expression of the NL1Tc protein conferred viability by complementation to E . coli exonuclease III deletion mutants (BW286 strain) . We propose that the biological function of the AP endonuclease activity of the NL1Tc protein may be connected with the introduction into the DNA of free 3' ends that could be used as primers for the integration, along the T . cruzi genome, of the L1Tc element and that the nicking could be a general mechanism for the retrotransposition of non-site-specific non-long terminal repeat retrotransposons. J Biol Chem, 1997 Oct 3, 272(40), 25149 - 56 Analysis of factor XIII substrate specificity using recombinant human factor XIII and tissue transglutaminase chimeras; Hettasch JM et al.; Human factor XIII (FXIII) and tissue transglutaminase (tTG) are homologous proteins . FXIII requires thrombin for activation and cross-links the gamma chains of fibrin(ogen) more efficiently than the Aalpha chains . On the other hand, tTG is thrombin-independent and forms predominantly Aalpha and Aalpha-gamma chain complexes . Previous work from this laboratory demonstrated that amino acid residues within exon 7 of FXIII were important for catalysis (Hettasch, J . M., and Greenberg, C . S . (1994) J . Biol . Chem . 269, 28309-28313) . To determine to what extent the primary amino acid sequence within exon 7 defines substrate specificity, exon 7 of FXIII was replaced with the corresponding exon of tTG using gene splicing by overlap extension . Other work from this laboratory (Achyuthan, K . E., Slaughter, T . F., Santiago, M . A., Enghild, J . J., and Greenberg, C . S . (1993) J . Biol . Chem . 268, 21284-21292) using synthetic peptides identified two other domains that might play a role in substrate recognition (located in exons 3 and 5) . Therefore, recombinant chimeras of FXIII/tTG were also created in which these two exons were exchanged . FXIII, tTG, and chimeras 3, 5, and 7 were expressed in Escherichia coli, purified, and the nature of the fibrin cross-linking pattern of these five proteins was determined by immunoblot analysis . FXIII preferentially formed the gamma-gamma dimer, whereas tTG formed Aalpha-gamma complexes . Chimera 7 formed Aalpha-gamma complexes that resembled the cross-linking pattern of tTG . This finding demonstrates that the primary amino acid sequence of exon 7 of tTG confers some of the specificity for the Aalpha and Aalpha-gamma cross-link pattern characteristic of tTG . Chimera 5 exhibited reduced cross-linking activity (50% of FXIII activity) but still retained preference for formation of the gamma-gamma dimer, whereas chimera 3 was not active . In conclusion, exchanging the primary amino acid sequence of the active site exon of human FXIII with that of human tTG modifies the enzyme such that the fibrin cross-linking pattern more closely resembles that of tTG (Aalpha and Aalpha-gamma complexes) instead of FXIII (gamma-gamma dimers). J Biol Chem, 1997 Oct 3, 272(40), 25128 - 34 A new function of p120-GTPase-activating protein . Prevention of the guanine nucleotide exchange factor-stimulated nucleotide exchange on the active form of Ha-ras p21; Giglione C et al.; This work studies the coordination of the action of GTPase-activating protein (GAP) and guanine nucleotide exchange factor (GEF) on activated human c-Ha-Ras p21 . Purified human p120-GAP was obtained with a new efficient procedure . To distinguish the GTPase-activating effect of p120-GAP from other effects dependent on the interaction with activated Ha-Ras, the nonhydrolyzable GTP analogue guanosine 5'-O-(thiotriphosphate) (GTPgammaS) was used . The results showed that the GTPgammaS/GTPgammaS exchange enhanced by the C-terminal catalytic domain of the yeast GEF Sdc25p (C-Sdc25p) is prevented by p120-GAP . This effect is strictly specific for the activated form of Ha-Ras, the target of GAP; no effect on Ha-Ras.GDP was detectable . The GAP catalytic domain also inhibited C-Sdc25p but to a lower extent . The interfering effect by p120-GAP was also evident in a homologous mammalian system, using full-length mouse RasGEF, its C-terminal half-molecule, or C-terminal catalytic domain . As a consequence of this inhibition, presence of p120-GAP enhanced the regeneration of Ha-Ras.GTPgammaS by GEF at a GDP:GTPgammaS ratio mimicking the in vivo GDP:GTP ratio . Our work describes a novel function of p120-GAP and suggests a mechanism by which GAP protects Ha-Ras.GTP in vivo against unproductive exchanges . This constrain is likely involved in the regulation of the physiological GDP/GTP cycle of Ras and in the action of p120-GAP as downstream effector of Ras . Helix alpha3 is proposed as a Ras element playing a key-role in the interference between GAP and GEF on Ras. J Biol Chem, 1997 Oct 3, 272(40), 25091 - 8 Identification of shallow and deep membrane-penetrating forms of diphtheria toxin T domain that are regulated by protein concentration and bilayer width; Wang Y et al.; The alpha-helix-rich, hydrophobic transmembrane (T) domain of diphtheria toxin is believed to play a central role in membrane insertion by the toxin and in the translocation of its catalytic domain across membranes . In this report, T domain structure was studied using site-directed single-Cys mutants . The residues chosen, 322 (near the amino-terminal end of helix TH8), 333 (within helix TH8), and 356 (within helix TH9) were substituted with Cys and labeled with the fluorescent probe bimane . (Residues 333 and 356 should be located within the bilayer in the transmembrane state, and residue 322 should not penetrate the bilayer.) After insertion of T domain into model membrane vesicles, the location of bimane label relative to the lipid bilayer was characterized by its fluorescence emission and by its quenching with nitroxide-labeled phospholipids . It was found that when the T domain is added to dioleoylphosphatidylcholine-containing vesicles, all three residues reside close to the outer surface . However, at high T domain concentration or in thinner dimyristoleoylphosphatidylcholine-containing vesicles, a large fraction of residues 333 and 356 penetrate deeply into the membrane . In contrast, residue 322 remains exposed to aqueous solution under these conditions . These conclusions were confirmed by a novel antibody binding method . Antibodies that quench the fluorescence of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacene++ + (BODIPY) groups were used to evaluate the exposure of BODIPY-labeled 322, 333, and 356 . Maximum exposure of residues 333 and 356 to externally added antibody was only observed under conditions in which bimane fluorescence showed that these residues do not penetrate the bilayer . In contrast, residue 322 remained exposed under all conditions . We propose that the deeply penetrating T domain conformation represents a transmembrane or near-transmembrane state . The regulation of the transmembrane/nontransmembrane equilibrium should be a key to understanding diphtheria toxin membrane insertion and translocation . Our results suggest that toxin-toxin interactions may play an important role in regulating this behavior. J Biol Chem, 1997 Oct 3, 272(40), 25077 - 82 Characterization of a GDP dissociation inhibitory region of ADP-ribosylation factor domain protein ARD1; Vitale N et al.; ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to stimulate cholera toxin ADP-ribosyltransferase activity and later recognized as critical components in intracellular vesicular transport and phospholipase D activation . ARF domain protein 1 (ARD1) is a member of the ARF family that differs from other ARFs by the presence of a 46-kDa amino-terminal extension . We previously reported that this extension acts as a GTPase-activating protein for the ARF domain of ARD1 (Vitale, N., Moss, J., and Vaughan, M . (1996) Proc . Natl . Acad . Sci . U . S . A . 93, 1941-1944) . Both GTP binding and GTP hydrolysis are necessary for physiological function of guanine nucleotide-binding proteins, and the rates of GDP/GTP exchange and GTPase activity are critical in the activation/deactivation cycle . Dissociation of GDP from the ARF domain of ARD1 was faster than from ARD1 itself (both proteins synthesized in Escherichia coli) . Using deletion mutations, it was demonstrated that the 15 amino acids directly preceding the ARF domain were responsible for decreasing the rate of GDP dissociation but not guanosine 5-{gamma-thio}triphosphate dissociation . By site-specific mutagenesis it was shown that hydrophobic amino acids in this region were particularly important in stabilizing the GDP-bound form of ARD1 . It is suggested that, like the amino-terminal segment of ARF, the equivalent region in ARD1, located between the GTPase-activating protein and ARF domains, may act as a GDP dissociation inhibitor. J Biol Chem, 1997 Oct 3, 272(40), 25071 - 6 Nitric oxide sensitivity of the aconitases; Gardner PR et al.; Aconitases are important cellular targets of nitric oxide (NO.) toxicity, and NO.-derived species, rather than NO . per se, have been proposed to mediate their inactivation . NO.-mediated inactivation of the Escherichia coli aconitase and the porcine mitochondrial aconitase was investigated . In E . coli, aconitase activity decreased by approximately 70% during a 2-h exposure to an atmosphere containing 120 ppm NO . in N2 . The NO.-inactivated aconitase reactivated poorly in E . coli under anaerobic or aerobic conditions . Elevated superoxide dismutase activity did not affect the aerobic inactivation of aconitase by NO., thus indicating a limited role of the NO.- and superoxide-derived species peroxynitrite . Glutathione-deficient and glutathione-containing E . coli were comparably sensitive to NO.-mediated aconitase inactivation, thus excluding the participation of S-nitrosoglutathione or more oxidizing NO.-derived species . NO . progressively decreased aconitase activity in extracts in the presence of substrates, and inactivation was greatest at an acidic pH with cis-aconitate . The porcine mitochondrial aconitase was sensitive to NO . when exposed at pH 6.5, but not at pH 7.5, and irreversible inactivation occurred during catalysis . The requirement of an acidic pH or substrates for sensitivity may explain the reported resistance of aconitases to NO . in vitro (Castro, L., Rodriguez, M., and Radi, R . (1994) J . Biol . Chem . 269, 29409-29415; Hausladen, A., and Fridovich, I . (1994) J . Biol . Chem . 269, 29405-29408) . An S-nitrosation of the aconitase {4Fe-4S} center catalyzed by the solvent-exposed electron withdrawing iron atom (Fea) is proposed. J Biol Chem, 1997 Oct 3, 272(40), 24942 - 7 Mutations in the glutathione-gated KefC K+ efflux system of Escherichia coli that cause constitutive activation; Miller S et al.; The kefC gene of Escherichia coli encodes a potassium efflux system that is gated by glutathione (GSH) and by GSH adducts . Independently isolated kefC mutations that result in spontaneous activation of the efflux system have been analyzed . Three mutations affect residues located adjacent to the conserved Rossman fold in the carboxyl-terminal domain . Two mutations lie in a sequence predicted to form a cytoplasmically located loop in the membrane domain of KefC . All of the mutants retain normal regulation by the YabF protein and by GSH adducts . A mutation in the Rossman fold, R416S, alters the normal regulation of KefC by GSH . In contrast to the wild-type protein, which is inactive in the presence of GSH, the R416S mutant is only active in the presence of GSH or its analogue, ophthalmic acid . Other mutations in this region or elsewhere in the protein have their spontaneous activity augmented by depletion of the GSH pool . These data identify a specific role for the carboxyl-terminal domain of KefC in regulating KefC activity and are discussed in the light of recent data that suggest that GSH adducts can bind within a Rossman fold. J Biol Chem, 1997 Oct 3, 272(40), 24825 - 31 A mutational study of the peptide-binding domain of Hsc70 guided by secondary structure prediction; Boice JA et al.; The abundant, cytoplasmic molecular chaperones of eukaryotic cells, of which mammalian Hsc70 is a member, have central roles in protein folding pathways in cells . Although substantial information is now available on substrate interactions and ATPase activity, neither the crystal structure of the intact Hsc70 molecule nor its isolated peptide-binding domain is known . Recently, the crystal structure of the isolated peptide-binding domain of an evolutionary relative of mammalian Hsc70, the DnaK protein of Escherichia coli, was solved . We have generated several rat Hsc70 mutants using site-directed and cassette mutagenesis guided by secondary structure predictions to test the hypothesis that the peptide-binding domains of mammalian Hsc70 and DnaK have similar molecular structures . Biochemical properties along with the ATPase and peptide binding activities of the resulting recombinant proteins were determined . Biochemical analyses included one- and two-dimensional gel electrophoresis, electrospray mass spectrometry, and N-terminal amino acid sequencing . The results of our study suggest that the DnaK molecular structure is a useful working model for the mammalian Hsc70 peptide-binding domain . Evidence is provided that (i) small additions to the N terminus of Hsc70 alter the function of the peptide-binding domain, (ii) alterations in the C-terminal tetrapeptide EEVD result in dramatic increases in basal ATPase activity, (iii) polyalanine substitution of a helical connector segment compensates for changes at the C terminus to restore near-normal function, (iv) specific side chain interactions involving this connector segment are not required for peptide-stimulated ATPase activity, and (v) disruption of the cap homologue region inhibits peptide binding by Hsc70. EMBO J, 1997 Oct 1, 16(19), 5922 - 9 The Na+-specific interaction between the LysR-type regulator, NhaR, and the nhaA gene encoding the Na+/H+ antiporter of Escherichia coli; Carmel O et al.; We used partially purified NhaR and a highly purified His-tagged NhaR derivative to identify the cis-regulatory sequences of nhaA recognized by NhaR and to study the specific effect of Na+ on this interaction . Gel retardation assay with DNase I footprinting analysis showed that NhaR binds a region of nhaA which spans 92 bp and contains three copies of the conserved LysR-binding motif . Na+, up to 100 mM, had no effect on the binding of NhaR to nhaA . The dimethylsulfate methylation protection assay in vivo and in vitro, showed that bases G-92, G-60, G-29 and A-24 form direct contacts with NhaR; in the absence of added Na+ in vivo, these bases were protected but became exposed to methylation in a DeltanhaR strain; accordingly, these bases were protected in vitro by the purified His-tagged NhaR . 100 mM Na+, but not K+, removed the protection of G-60 conferred by His-tagged NhaR in vitro . Exposure of intact cells to 100 mM Na+, but not K+, exposed G-60 . The maximal effect of Na+ in vitro was observed at 20 mM and was pH dependent, vanishing below pH 7.5 . In contrast to G-60, G-92 was exposed to methylation by the ion only in vivo, suggesting a requirement for another factor existing only in vivo for this interaction . We suggest that NhaR is both sensor and transducer of the Na+ signal and that it regulates nhaA expression by undergoing a conformational change upon Na+ binding which modifies the NhaR-nhaA contact points. EMBO J, 1997 Oct 1, 16(19), 5819 - 26 The three-dimensional solution structure of the matrix protein from the type D retrovirus, the Mason-Pfizer monkey virus, and implications for the morphology of retroviral assembly; Conte MR et al.; The Mason-Pfizer monkey virus (M-PMV) is the prototype of the type D retroviruses . In type B and D retroviruses, the Gag protein pre-assembles before association with the membrane, whereas in type C retroviruses (lentiviruses, BLV/HTLV group) Gag is targeted efficiently to the plasma membrane, where the particle formation occurs . The N-terminal domain of Gag, the matrix protein (MA), plays a critical role in determining this morphogenic difference . We have determined the three-dimensional solution structure of the M-PMV MA by heteronuclear nuclear magnetic resonance . The protein contains four alpha-helices that are structurally similar to the known type C MA structures . This similarity implies possible common assembly units and membrane-binding mechanisms for type C and B/D retroviruses . In addition to this, the interpretation of mutagenesis data has enabled us to identify, for the first time, the structural basis of a putative intracellular targeting motif. EMBO J, 1997 Sep 15, 16(18), 5582 - 91 Crystal structures of the small G protein Rap2A in complex with its substrate GTP, with GDP and with GTPgammaS; Cherfils J et al.; The small G protein Rap2A has been crystallized in complex with GDP, GTP and GTPgammaS . The Rap2A-GTP complex is the first structure of a small G protein with its natural ligand GTP . It shows that the hydroxyl group of Tyr32 forms a hydrogen bond with the gamma-phosphate of GTP and with Gly13 . This interaction does not exist in the Rap2A-GTPgammaS complex . Tyr32 is conserved in many small G proteins, which probably also form this hydrogen bond with GTP . In addition, Tyr32 is structurally equivalent to a conserved arginine that binds GTP in trimeric G proteins . The actual participation of Tyr32 in GTP hydrolysis is not yet clear, but several possible roles are discussed . The conformational changes between the GDP and GTP complexes are located essentially in the switch I and II regions as described for the related oncoprotein H-Ras . However, the mobile segments vary in length and in the amplitude of movement . This suggests that even though similar regions might be involved in the GDP-GTP cycle of small G proteins, the details of the changes will be different for each G protein and will ensure the specificity of its interaction with a given set of cellular proteins. EMBO J, 1997 Sep 15, 16(18), 5562 - 71 The two opposing activities of adenylyl transferase reside in distinct homologous domains, with intramolecular signal transduction; Jaggi R et al.; Adenylyl transferase (ATase) is the bifunctional effector enzyme in the nitrogen assimilation cascade that controls the activity of glutamine synthetase (GS) in Escherichia coli . This study addresses the question of whether the two antagonistic activities of ATase (adenylylation and deadenylylation) occur at the same or at different active sites . The 945 amino acid residue ATase has been truncated in two ways, so as to produce two homologous polypeptides corresponding to amino acids 1-423 (AT-N) and 425-945 (AT-C) . We demonstrate that ATase has two active sites; AT-N carries a deadenylylation activity and AT-C carries an adenylylation activity . Glutamine activates the adenylylation reaction of the AT-C domain, whereas alpha-ketoglutarate activates the deadenylylation reaction catalysed by the AT-N domain . With respect to the regulation by the nitrogen status monitor PII, however, the adenylylation domain appears to be dependent on the deadenylylation domain: the deadenylylation activity of AT-N depends on PII-UMP and is inhibited by PII . The adenylylation activity of AT-C is independent of PII (or PII-UMP), whereas in the intact enzyme PII is required for this activity . The implications of this intramolecular signal transduction for the prevention of futile cycling are discussed. EMBO J, 1997 Sep 15, 16(18), 5491 - 500 Pex14p is a member of the protein linkage map of Pex5p; Brocard C et al.; To identify members of the translocation machinery for peroxisomal proteins, we made use of the two-hybrid system to establish a protein linkage map centered around Pex5p from Saccharomyces cerevisiae, the receptor for the C-terminal peroxisomal targeting signal (PTS1) . Among the five interaction partners identified, Pex14p was found to be induced under conditions allowing peroxisome proliferation . Deletion of the corresponding gene resulted in the inability of yeast cells to grow on oleate as well as the absence of peroxisomal structures . The PEX14 gene product of approximately 38 kDa was biochemically and ultrastructurally demonstrated to be a peroxisomal membrane protein, despite the lack of a membrane-spanning domain . This protein was shown to interact with itself, with Pex13p and with both PTS receptors, Pex5p and Pex7p, indicating a central function for the import of peroxisomal matrix proteins, either as a docking protein or as a releasing factor at the organellar membrane. EMBO J, 1997 Sep 15, 16(18), 5483 - 90 Mammalian protein RAP46: an interaction partner and modulator of 70 kDa heat shock proteins; Zeiner M et al.; A ubiquitously expressed nuclear receptor-associating protein of approximately 46 kDa (RAP46) was identified recently . Interaction experiments with in vitro-translated proteins and proteins contained in cell extracts revealed that a great variety of cellular regulators associate with RAP46 . However, in direct interaction tests by the far-Western technique, only 70 kDa proteins showed up and were identified as members of the 70 kDa heat shock protein (hsp70) family . Interaction is specific since not all members of the hsp70 family bind to RAP46; interaction occurs through their ATP-binding domain . RAP46 forms complexes with hsp70 in mammalian cells and interacts with hsp70 in the yeast two-hybrid system . Consistent with the fact that hsp70 can bind a multitude of proteins, we identified heteromeric complexes of RAP46-hsp70 with some selected proteins, most notably c-Jun . Complex formation is increased significantly by pre-treatment with alkaline phosphatase, thus suggesting modulation of interactions by protein phosphorylation . We observed that RAP46 interferes with efficient refolding of thermally denatured luciferase . Moreover, ATP-dependent binding of misfolded proteins to hsp70 was greatly inhibited by RAP46 . These data suggest that RAP46 functions as a regulator of hsp70 in higher eukaryotes. EMBO J, 1997 Sep 1, 16(17), 5260 - 72 Transfer of Tat and release of TAR RNA during the activation of the human immunodeficiency virus type-1 transcription elongation complex; Keen NJ et al.; The HIV-1 trans-activator protein, Tat, is a potent activator of transcriptional elongation . Tat is recruited to the elongating RNA polymerase during its transit through the trans-activation response region (TAR) because of its ability to bind directly to TAR RNA expressed on the nascent RNA chain . We have shown that transcription complexes that have acquired Tat produce 3-fold more full-length transcripts than complexes not exposed to Tat . Western blotting experiments demonstrated that Tat is tightly associated with the paused polymerases . To determine whether TAR RNA also becomes attached to the transcription complex, DNA oligonucleotides were annealed to the nascent chains on the arrested complexes and the RNA was cleaved by RNase H . After cleavage, the 5' end of the nascent chain, carrying TAR RNA, is quantitatively removed, but the 3' end of the transcript remains associated with the transcription complex . Even after the removal of TAR RNA, transcription complexes that have been activated by Tat show enhanced processivity . We conclude that Tat, together with cellular co-factors, becomes attached to the transcription complex and stimulates processivity, whereas TAR RNA does not play a direct role in the activation of elongation and is used simply to recruit Tat and cellular co-factors. EMBO J, 1997 Sep 1, 16(17), 5235 - 46 RNA recognition by the joint action of two nucleolin RNA-binding domains: genetic analysis and structural modeling; Bouvet P et al.; The interaction of nucleolin with a short stem-loop structure (NRE) requires two contiguous RNA-binding domains (RBD 1+2) . The structural basis for RNA recognition by these RBDs was studied using a genetic system in Escherichia coli . Within each of the two domains, we identified several mutations that severely impair interaction with the RNA target . Mutations that alter RNA-binding specificity were also isolated, suggesting the identity of specific contacts between RBD 1+2 amino acids and nucleotides within the NRE stem-loop . Our data indicate that both RBDs participate in a joint interaction with the NRE and that each domain uses a different surface to contact the RNA . The constraints provided by these genetic data and previous mutational studies have enabled us to propose a three-dimensional model of nucleolin RBD 1+2 bound to the NRE stem-loop. Science, 1997 Oct 3, 278(5335), 128 - 30 Counteraction by MutT protein of transcriptional errors caused by oxidative damage; Taddei F et al.; Oxidized guanine (8-oxo-7,8-dihydroguanine; 8-oxo-G) is a potent mutagen because of its ambiguous pairing with cytosine and adenine . The Escherichia coli MutT protein specifically hydrolyzes both 8-oxo-deoxyguanosine triphosphate (8-oxo-dGTP) and 8-oxo-guanosine triphosphate (8-oxo-rGTP), which are otherwise incorporated in DNA and RNA opposite template A . In vivo, this cleaning of the nucleotide pools decreases both DNA replication and transcription errors . The effect of mutT mutation on transcription fidelity was shown to depend on oxidative metabolism . Such control of transcriptional fidelity by the ubiquitous MutT function has implications for evolution of RNA-based life, phenotypic expression, adaptive mutagenesis, and functional maintenance of nondividing cells. J Virol, 1997 Oct, 71(10), 7841 - 50 Toward a poliovirus-based simian immunodeficiency virus vaccine: correlation between genetic stability and immunogenicity; Tang S et al.; Recombinant polioviruses expressing foreign antigens may provide a convenient vaccine vector to engender mucosal immunity . Replication-competent chimeric viruses can be constructed by fusing foreign antigenic sequences to several positions within the poliovirus polyprotein . Artificial cleavage sites ensure appropriate proteolytic processing of the recombinant polyprotein, yielding mature and functional viral proteins . To study the effect of the position of insertion, two different recombinant polioviruses were examined . A small amino-terminus insertion delayed virus maturation and produced a thermosensitive particle . In contrast, insertion at the junction between the P1 and P2 regions yielded a chimeric poliovirus that replicated like the wild type . Eight different chimeras were constructed by inserting simian immunodeficiency virus (SIV) sequences at the P1/P2 junction . All recombinant viruses replicated with near-wild-type efficiency in tissue culture cells and expressed high levels of the SIV antigens . One of the inserted fragments corresponding to gp41 envelope protein was N-glycosylated but was not secreted . Inserted sequences were only partially retained after few rounds of replication in HeLa cells . This problem could be remedied to some extent by altering the sequences flanking the insertion point . Reducing the homology of the direct repeats by 37% decrease the propensity of the recombinant viruses to delete the insert . To determine the immunogenic potential of the recombinants, mice susceptible to poliovirus infection were inoculated intraperitoneally . The antibody titers elicited against Gag p17 depended on the viral doses and the number of inoculations . In addition, recombinants which display higher genetic stability were more effective in inducing an immune response against the SIV antigens, and inoculation with a mix of recombinants carrying different SIV antigens (a cocktail of recombinants) elicited humoral responses against each of the individual SIV sequences. J Virol, 1997 Oct, 71(10), 7807 - 13 Repression of gene expression upon infection of cells with herpes simplex virus type 1 mutants impaired for immediate-early protein synthesis; Preston CM et al.; Herpes simplex virus type 1 (HSV-1) mutants defective in immediate-early (IE) gene expression do not readily enter productive replication after infection of tissue culture cells . Instead, their genomes are retained in a quiescent, nonreplicating state in which the production of viral gene products cannot be detected . To investigate the block to virus replication, we used the HSV-1 triple mutant in1820K, which, under appropriate conditions, is effectively devoid of the transactivators VP16 (a virion protein), ICP0, and ICP4 (both IE proteins) . Promoters for the HSV-1 IE ICP0 gene or the human cytomegalovirus (HCMV) major IE gene, cloned upstream of the Escherichia coli lacZ coding sequences, were introduced into the in1820K genome . The regulation of these promoters and of the endogenous HSV-1 IE promoters was investigated upon conversion of the virus to a quiescent state . Within 24 h of infection, the ICP0 promoter became much less sensitive to transactivation by VP16 whereas the same element, when used to transform Vero cells, retained its responsiveness . The HCMV IE promoter, which is not activated by VP16, also became less sensitive to the HCMV functional homolog of VP16 . Both elements remained available for transactivation by HSV-1 IE proteins at 24 h postinfection, showing that the in1820K genome was not irreversibly inactivated . The promoters controlling the HSV-1 ICP4, ICP22, and ICP27 genes also became essentially unresponsive to transactivation by VP16 . The ICP0 promoter was induced when hexamethylene bisacetamide was added to cultures at the time of infection, but the response to this agent was also lost by 24 h after infection . Therefore, promoter elements within the HSV-1 genome are actively repressed in the absence of IE gene expression, and repression is not restricted specifically to HSV-1 IE promoters. J Virol, 1997 Oct, 71(10), 7657 - 62 Foot-and-mouth disease virus and poliovirus particles contain proteins of the replication complex; Newman JF et al.; Nonstructural proteins 2C, 3CD, 3C, and 3D, and the cellular protein actin, are present in highly purified preparations of foot-and-mouth disease virus (FMDV) and poliovirus . They remain bound in variable amounts to the RNAs when the RNAs are extracted from the viruses with phenol or phenol-sodium dodecyl sulfate (SDS) and, for FMDV, when the RNA is released from the particles by a lowering of the pH below 7 . RNA prepared by these methods is rapidly degraded at 37 degrees C, particularly in the presence of NH4+ ions, but hydrolysis can be prevented by antibody against Escherichia coli-expressed 3D, indicating that it is the RNA polymerase that has nuclease activity . In contrast, virion RNA from which the nonstructural proteins and actin have been removed by extraction with guanidine thiocyanate-phenol-chloroform or proteinase K-phenol is stable at 37 degrees C, although its specific infectivity is lower than that of the RNA extracted with phenol or phenol-SDS . The possible implications of the close association of replication complex proteins with the RNA in virus particles are discussed. J Virol, 1997 Oct, 71(10), 7258 - 65 The N-terminal region of the luteovirus readthrough domain determines virus binding to Buchnera GroEL and is essential for virus persistence in the aphid; van den Heuvel JF et al.; Luteoviruses and the luteovirus-like pea enation mosaic virus (PEMV; genus Enamovirus) are transmitted by aphids in a circulative, nonreplicative manner . Acquired virus particles persist for several weeks in the aphid hemolymph, in which a GroEL homolog, produced by the primary endosymbiont of the aphid, is abundantly present . Six subgroup II luteoviruses and PEMV displayed a specific but differential affinity for Escherichia coli GroEL and GroEL homologs isolated from the endosymbiotic bacteria of both vector and nonvector aphid species . These observations suggest that the basic virus-binding capacity resides in a conserved region of the GroEL molecule, although other GroEL domains may influence the efficiency of binding . Purified luteovirus and enamovirus particles contain a major 22-kDa coat protein (CP) and lesser amounts of an approximately 54-kDa readthrough protein, expressed by translational readthrough of the CP into the adjacent open reading frame . Beet western yellows luteovirus (BWYV) mutants devoid of the readthrough domain (RTD) did not bind to Buchnera GroEL, demonstrating that the RTD (and not the highly conserved CP) contains the determinants for GroEL binding . In vivo studies showed that virions of these BWYV mutants were significantly less persistent in the aphid hemolymph than were virions containing the readthrough protein . These data suggest that the Buchnera GroEL-RTD interaction protects the virus from rapid degradation in the aphid . Sequence comparison analysis of the RTDs of different luteoviruses and PEMV identified conserved residues potentially important in the interaction with Buchnera GroEL. Am J Trop Med Hyg, 1997 Sep, 57(3), 368 - 74 Rio Mamore virus: genetic characterization of a newly recognized hantavirus of the pygmy rice rat, Oligoryzomys microtis, from Bolivia; Bharadwaj M et al.; Human hantavirus disease occurs throughout much of South America . The rodent hosts and the specific etiologic agent(s) are largely unknown, but many reported cases occurred within the habitation ranges of oryzomine rodents (rice rats) . We have identified a genetically novel hantavirus (Rio Mamore virus {RM}) of the pygmy rice rat Oligoryzomys microtis in Bolivia . The complete sequence of the small (S) genome and the partial sequence of the medium (M) genome are described . This virus is closely related to the newly identified human pathogen Andes virus from Patagonia . To facilitate improved diagnosis of hantavirus infections in South America, we have expressed the complete nucleocapsid protein of RM in Escherichia coli and affinity-purified it for use in an ELISA and Western blot assays for antibodies to RM. Ryumachi, 1997 Aug, 37(4), 556 - 63 {Novel autoantibodies that recognize native transfer RNAs in patients with systemic rheumatic diseases}; Matsumura M et al.; Autoantibodies directed against aminoacyl-tRNA synthetases are well described in adult polymyositis/dermatomyositis . However, the characteristics of antibodies against other tRNA and tRNA-associated proteins have not been well defined . In this study, we identified novel autoantibodies to total tRNAs in patients with systemic rheumatic diseases and characterized the structure that was recognized by anti-tRNA antibodies . We identified fifteen patients sera that immunoprecipitated the deproteinized cognate tRNA . Two of them were identified as having previously well-defined anti-PL-12 antibodies . Three of the remaining 13 patient sera immunoprecipitated naked tRNA of E . coli . To investigate the antibody binding site on tRNAs, we have used in vitro transcripts from cDNAs encoding wild type tRNAs of E . coli and their synthesized mutants . These sera revealed different reactivity to mutated tRNAs . Eleven of these 13 patients met disease specific criteria for: SLE (3 patients), Sjogren's syndrome (6), or SLE/Sjogren's syndrome overlap (2) . In addition, fever, Raynaud's phenomenon, and non-erosive polyarthritis, were frequently found in these patients . In summary, we have identified novel antibodies to tRNAs which appear to be quite common, and distinct from previously described anti-aminoacyl-tRNA synthetase antibodies. Phytochemistry, 1997 Sep, 46(2), 283 - 7 Anthocyanin-producing dandelion callus as a chalcone synthase source in recombinant polyketide reductase assay; Akashi T et al.; Purple-coloured dandelion (Taraxacum officinale) callus cultures producing anthocyanin pigments were established on a cytokinin-rich medium under the light . When the cells were placed in the dark, only grey cells proliferated . Anthocyanin productivity of these cells was partially restored in the light . The major pigment was identified as cyanidin 3-(6"-malonylglucoside) . The lower stem of the original plant contained the same pigment . Chalcone synthase (CHS) activity was detected in the extracts of these purple cells, whereas no activity was observed in grey cells propagated in the dark . When the CHS-active cell-free extract was combined with the extract of Escherichia coli over expressing polyketide reductase (PKR) cDNA of licorice (Glycyrrhiza echinata), isoliquiritigenin (a 6'-deoxychalcone), in addition to naringenin (a 5-hydroxyflavanone), was detected as the reaction product from 4-coumaroyl-CoA, malonyl-CoA and NADPH . This result confirms the catalytic function of the PKR gene product. FEMS Microbiol Lett, 1997 Sep 15, 154(2), 415 - 21 Bovine attaching and effacing Escherichia coli possess a pathogenesis island related to the LEE of the human enteropathogenic Escherichia coli strain E2348/69; Goffaux F et al.; Attaching and effacing Escherichia coli (AEEC) has been described as a cause of diarrhea in calves . The molecular pathogenesis of AEEC was mainly studied in human enteropathogenic E . coli strain E2348/69 in which the virulence correlated with the presence of a 35.4 kb pathogenesis island called LEE . We showed that several strains isolated from calves with diarrhea were able to produce attaching and effacing lesions in a rabbit ileal loop model and that they possess a pathogenesis island related to the LEE . Moreover, we showed that the LEE from bovine strains was inserted mainly at a different position in the chromosome compared to the human enteropathogenic E . coli strain E2348/69. FEMS Microbiol Lett, 1997 Sep 15, 154(2), 283 - 8 Flow cytometric analysis of chlorhexidine action; Sheppard FC et al.; The mechanism by which chlorhexidine kills bacteria is still ill defined . We have investigated the action of chlorhexidine on Escherichia coli JM101/psb311 using a combination of flow cytometry and traditional methods . Chlorhexidine-induced uptake by E . coli cells of bis-(1,3-dibutylbarturic acid) trimethine oxonol and propidium iodide, which monitor membrane potential and membrane integrity respectively, was shown to be concentration dependent for the range 0.003-0.3 mmol-1 . In addition, cells in log phase growth were more susceptible to 0.03 mmol-1 chlorhexidine than those in stationary phase . There was, however, no direct correlation between dye uptake and decline in colony forming units. FEMS Microbiol Lett, 1997 Sep 15, 154(2), 271 - 6 In vitro plasmid-encoded resistance to quinolones; Gomez-Gomez JM et al.; The possibility of plasmid-encoded quinolone resistance is explored in two model systems . In the first, increasing amounts of wild-type gyrA allele moderately increased minimum inhibitory concentrations to quinolone antibiotics . In the second model, a mutant gyrA allele encoded by a multicopy plasmid produced a quinolone resistance phenotype upon its expression in a quinolone-susceptible Escherichia coli strain. FEMS Microbiol Lett, 1997 Sep 15, 154(2), 219 - 22 An Escherichia coli gene divergently transcribed from a promoter overlapping the metA promoter; Gery S et al.; The upstream region of the metA gene in Escherichia coli contains two promoters . We have identified by lacZ fusion an additional promoter in this region, and showed that it is transcribed in the opposite orientation from the metA gene . The putative translation product corresponds to a peptide of 147 amino acids-ORF19 by molecular mass . This peptide is probably not essential for growth, as an insertion mutant is viable. FEMS Microbiol Lett, 1997 Sep 15, 154(2), 195 - 200 Aggregation of yeast cells induced by the Arg-Gly-Asp motif-containing fragment of high-molecular-mass cell-adhesion protein MFBA, derived from the basidiomycetous mushroom Lentinus edodes; Yasuda T et al.; A fruiting body-specific cDNA mfbAc, derived from the basidiomycete Lentinus edodes, has been shown to encode a high-molecular-mass (2157 amino acids) cell-adhesion protein MFBA containing the Arg-Gly-Asp (RGD) motif . A 425-amino-acid fragment containing the RGD motif of MFBA (designated MFBA(582-1006) peptide) produced in Escherichia coli exhibited cell-adhesion and spreading activity toward mammalian cells and cell-aggregation activity toward basidiomycetous hyphal cells via the RGD sequence . Here we investigated the biological activity of MFBA(582-1006) peptide in Saccharomyces cerevisiae . The DNA sequence encoding MFBA(582-1006) peptide, introduced into the yeast using an expression vector, resulted in a marked aggregation of the yeast cells . The aggregation was almost completely abolished by replacement of the RGD motif by an RGE motif in the peptide sequence. Microbiol Immunol, 1997, 41(8), 633 - 6 Involvement of Ca(2+)-calmodulin-dependent protein kinase II in the intestinal secretory action of Escherichia coli heat-stable enterotoxin II; Fujii Y et al.; Treating the mouse intestine with the calmodulin antagonist W-7 and KN-93, an inhibitor of Ca(2+)-calmodulin-dependent protein kinase II (CaMK II), reduced the sensitivity of the host to the action of Escherichia coli heat-stable enterotoxin II (STII) . CaMK II activity in mouse intestinal cells increased after exposure to STII . These results indicate that CaMK II is involved in the mechanism of action of STII. Blood, 1997 Sep 15, 90(6), 2482 - 91 Spread of human cytomegalovirus (HCMV) after infection of human hematopoietic progenitor cells: model of HCMV latency; Zhuravskaya T et al.; Clinical experience and laboratory data suggest that human cytomegalovirus (HCMV) is present in peripheral blood of seropositive individuals in a latent or persistent state and can be transmitted via blood products and be reactivated in seropositive immunocompromised patients . The pathophysiology of HCMV latency and the nature of HCMV interaction with hematopoietic cells remains unknown . In this study, we investigated the infection of bone marrow (BM) progenitor cells and their progeny as a model of HCMV latency . A clinical isolate and the recombinant laboratory strain Towne/lox containing the Escherichia coli beta galactosidase (beta-gal) gene regulated by immediately early (IE) HCMV promoter were used to infect highly purified CD34+ cells . Although the infection of these cells with a clinical isolate was associated with an inhibition of proliferation by 59%, an expansion of progeny derived from these cells was possible . Polymerase chain reaction analysis and staining for beta-gal have shown that HCMV persisted in infected BM CD34+ cells and their progeny for up to 4 weeks . However, IE and late gene products (mRNA and protein) were detected only late in the course of infection and their expression correlated with terminal macrophage differentiation of the CD34+-derived progeny . Although early infection of CD34+ progenitor cells was not productive (as shown by the plaque assay), infectious virus could be recovered from the terminally differentiated cultures . BM progenitor cells may serve as a reservoir of the latent virus with limited transcription . Proliferation and monocytic maturation of infected progenitors may lead to the numerical expansion of HCMV-infected cells, which serve as a source of HCMV dissemination and reactivation. Eur J Biochem, 1997 Aug 15, 248(1), 200 - 8 Modulation of the enzymatic properties of protein phosphatase 2A catalytic subunit by the recombinant 65-kDa regulatory subunit PR65alpha; Turowski P et al.; All protein phosphatase 2A (PP2A) holoenzymes contain a 36-kDa catalytic subunit (PP2Ac) and a regulatory subunit of 65 kDa (PR65) . We have studied the interaction between PP2Ac and PR65 in an in vitro system, using PP2Ac isolated from rabbit skeletal muscle and recombinant PR65alpha expressed in bacteria or insect cells . Bacterially expressed PR65alpha exhibited identical biochemical properties to the protein expressed and isolated from the baculoviral expression system . The association of recombinant PR65 with PP2Ac was very tight (K(D)app = 85 pM) and led to a suppression of PP2A activity, which was maximal (70-80%) when phosphoproteins were used as substrates . When less-structured or smaller substrates (such as phosphopeptides) were used, this inhibition was only 30% . PR65 stimulated PP2Ac activity when the assays were performed in the presence of polycations . This indicates that the PR65 not only serves the previously predicted structural role as a molecular scaffold, but also allosterically modulates the enzymatic properties of PP2Ac . Furthermore, we identified a site of interaction between PP2Ac and PR65alpha by disruption of a stretch of basic amino acids by introduction of a glutamate at position 416 . This produced an almost 100-fold reduced affinity for PP2Ac and indicated that this basic motif is an important determinant for the interaction of PR65 and PP2Ac. Eur J Biochem, 1997 Aug 15, 248(1), 156 - 62 Conformational studies of human vitamin-D receptor by antipeptide antibodies, partial proteolytic digestion and ligand binding; Vaisanen S et al.; We have studied conformational changes of human vitamin-D receptor by using antipeptide antibodies, partial proteolytic digestion and binding of the natural ligand calcitriol or its synthetic analogs . Before exposing either {35S}methionine-labelled in vitro translated human vitamin-D receptor or a recombinant human vitamin-D receptor produced either in Escherichia coli or in Sf9 insect cells to limited proteolysis by trypsin or chymotrypsin, the proteins were treated with calcitriol or its synthetic analogs . The digestion products were analyzed by SDS/PAGE, immunoblotting with polyclonal antipeptide antibodies targeted against different domains of the receptor, and Edman N-terminal sequencing . After limited proteolysis with trypsin, two fragments of Mr 21,000 and Mr 34,000 could be localized into N-terminus and C-terminus of the receptor, respectively, by antipeptide antibodies . We found that treatment with calcitriol or its synthetic analogs leads to differential resistance of the ligand-binding domain of the recombinant receptor to partial proteolysis in vitro . We suggest that this is due to distinct conformational changes in the domain induced by the different ligands . The short N-terminal region and the Zn-finger domain form, however, a protease-resistant structure which is independent on the presence or absence of the ligand . When the C-terminal fragment of Mr 34,000 was further analyzed by Edman N-terminal sequencing, the major cleavage site in the receptor between amino acids Arg173 and His174 was revealed. Eur J Biochem, 1997 Aug 15, 248(1), 72 - 9 Inactivation of the regulatory protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath) by proteolysis can be overcome by a Gly to Gln modification; Lloyd JS et al.; The regulatory protein B of soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), exists as a mixture of the full-length active form and truncated forms, B' and B" . Electrospray ionisation mass spectrometry (ESI-MS) was used to identify a cleavage site between Met12 and Gly13, such that 12 amino acids were lost from the N-terminus of protein B . This truncate was designated B' and molecular masses were assigned to proteins B and B' of 15,852.6+/-0.4 Da and 14,629.5+/-0.3 Da, respectively . A cleavage site between Gln29 and Val30 was also identified such that 29 amino acids were lost from the N-terminus of protein B . This truncate was designated B" and had a molecular mass of 12,709.93+/-0.02 Da . Proteins B' and B" were found to be inactive in the sMMO system . Addition of protease inhibitors or the heterologous expression of protein B in various strains of lon-deficient or ompT-deficient Escherichia coli, did not inhibit B' formation . Expression of protein B as a glutathione S-transferase fusion protein and subsequent purification of protein B from E . coli using affinity chromatography resulted in preparations of protein B with higher enzyme activities than that of wild-type protein B . However, ESI-MS confirmed that protein B' was still present . Alteration of the Met12-Gly13 cleavage site to Met12-Gln13 revealed that the stability of G13Q at 20 degrees C and 37 degrees C was higher than that of wild-type preparations . ESI-MS indicated that protein B' was absent and could only be identified after prolonged incubation at room temperature . The amount of active protein B present in the cell may be controlled by protein B cleavage, thereby regulating electron transfer . Alternatively, it may allow protein B to maintain a certain conformation necessary for enzyme activity and this may control the activity of sMMO in response to the supply of methane to the cell. Eur J Biochem, 1997 Aug 15, 248(1), 10 - 4 Gene expression evidence indicates that nucleotides 507-513 and 1434-1440 in 16S rRNA are organized in close proximity on the Escherichia coli 30S ribosomal subunit; Kaloyanova D et al.; A non-Shine-Dalgamo translational initiator is identified in Escherichia coli . The nucleotide sequence ACCUACUCGAGUUAG, designated as PL, is capable of initiating translation of pokeweed antiviral protein (PAP) and human calcitonin (hCT) mRNAs in E . coli cells . The yield of recombinant protein was double that obtained with the consensus Shine-Dalgarno-sequence-(SD)-driven translation . The PL sequence is composed of two heptanucleotides (ACCUACU, box I and GAGUUAG, box II) which are complementary to nucleotides 1434-1440 and 507-513, respectively, in 16S rRNA . Mutational analysis shows that the translation initiation efficiency with either box alone is much lower than that obtained with the entire PL sequence, indicating that the boxes interact simultaneously with both complementary regions in 16S rRNA during the translation initiation step . Based on these results, we propose that the two widely separated regions 507-513 (part of helical domain 18) and 1434-1440 (belonging to helical domain 44) are organized in close proximity to each other and to the ribosome decoding center on the surface of the E . coli 30S ribosomal subunit. Biochimie, 1997 Jun, 79(6), 365 - 72 Localization of two domains of a mutant form of Escherichia coli protein L7/L12 that binds the large ribosomal subunit as a single dimer; Wiggers RJ et al.; Escherichia coli ribosomal protein L7/L12 occurs on the large subunit as two dimers: one dimer is extended and comprises the stalk, while the second dimer is folded and occupies a site on the subunit body . A variant protein, in which all 18 amino acids of the flexible hinge region that links separate N-terminal and C-terminal domains of L7/L12 has been deleted, binds the subunit as a single dimer and does not generate stalks that are visible in electron micrographs . Monoclonal antibodies directed against each domain of the protein have been used to localize the variant in electron micrographs of 50S subunits . Both C-terminal domains are seen at a shoulder of the subunit, near its edge as viewed in the most common quasisymmetric projection . N-terminal domains are placed on the subunit body, about 50 A from the C-terminal domains . The antibody to the N-terminal domain also causes dissociation of the variant dimer from the particle and the formation of oligomeric antibody-protein dimer complexes . Similar complexes were seen previously (Olson HM et al (1986) J Biol Chem 261, 6924-6936) when this antibody induced dissociation of one dimer of the native protein . We conclude that the shortened variant most probably occupies the lower-affinity site on the subunit that is normally filled by the stalk dimer. Biochimie, 1997 Jun, 79(6), 359 - 64 Role of SOS and OxyR systems in the repair of Escherichia coli submitted to hydrogen peroxide under low iron conditions; Asad LM et al.; There are at least two mechanisms by which H2O2 induces DNA lesions in Escherichia coli: one in the presence of physiological iron levels and the other in low iron conditions . The survival as well as the induction of SOS response in different DNA repair mutant strains of E coli was evaluated after H2O2 treatment under low iron conditions (pretreatment with an iron chelator) . Our results indicate that, in normal iron conditions RecA protein has a relevant role in recombination repair events, while in low iron conditions RecA protein is important as a positive regulator of the SOS response . On the other hand, the oxy delta R mutant is sensitive to the lethal effects of H2O2 only in low iron conditions and this sensitivity cannot be correlated with DNA strand breaks. Proc Natl Sci Counc Repub China B, 1997 Jul, 21(3), 100 - 5 The seventh copy of IS1 in Escherichia coli W3110 belongs to the IS1 A (IS1E) type which is the only IS1 type that transposes from chromosome to plasmids; Chen JH et al.; In the Escherichia coli K-12 chromosome, six copies of IS1 (IS1A - IS1F) have been identified and characterized . According to their nucleotide (nt) sequences, the six IS1 copies can be classified into four types, IS1A(IS1E), IS1B(IS1C), IS1D and IS1F type . Zuber and Schumann (1993) identified the seventh IS1 copy at 49.6 minutes on the E . coli W3110 genetic map . Unfortunately, only the end 21-bp sequence as well as the neighboring 120-bp E . coli sequence were reported . We therefore designed two oligonucleotide primers to specifically amplify the seventh IS1 copy by PCR . One primer is homologous to the first sixteen bases of the IRL sequence of IS1A(IS1E), IS1B(IS1C) and IS1D . The other primer is complementary to the eighteen bases of E . coli sequence adjacent to IRR of the seventh IS1 copy . An 800-bp PCR fragment was obtained and its nt sequence determined, revealing an identical nucleotide sequence to that of IS1A(IS1E) . A plasmid system was then used to isolate insertion mutations caused by insertions of the chromosomal insertion sequences . Of the 142 plasmid insertion mutants isolated, thirty-eight were insertions of chromosomal IS5, ten were IS30, and ninety-four were IS1 . Detailed restriction mapping indicates that all ninety-four plasmid IS1 insertions were insertions of IS1 of the IS1A(IS1E) type. Proc Natl Sci Counc Repub China B, 1997 Jul, 21(3), 96 - 9 Identification of a measles virus isolate from a recent outbreak in northern Taiwan; Kuan MM et al.; Specimens were collected during a recent outbreak . Those specimens displaying both CPE positive in B95-8 lymphocyte cell culture and positive by IFA were checked by a RT-PCR with a specific set of measles virus primers derived from the C-terminal of the nucleoprotein . Such RT-PCR method was found ideal for routine diagnostic purposes . Product from this RT-PCR was treated for plasmid construction before transformed into E . coli . One of those transformed clones, i . e . T94, was further studied for its DNA sequence . Since T94 is found to bear several evident different characteristics from those ever published, we conclude that this isolate is neither a vaccine derived strain nor one of those reported previously with specific amino acid residues, but unique in its own right . This isolate can well be a local lineage of wild measles virus in Taiwan. Structure, 1997 Aug 15, 5(8), 997 - 1015 Extensive features of tight oligosaccharide binding revealed in high-resolution structures of the maltodextrin transport/chemosensory receptor; Quiocho FA et al.; BACKGROUND: Active-transport processes perform a vital function in the life of a cell, maintaining cell homeostasis and allowing access of nutrients . Maltodextrin/maltose-binding protein (MBP; M(r) = 40k) is a receptor protein which serves as an initial high-affinity binding component of the active-transport system of maltooligosaccharides in bacteria . MBP also participates in chemotaxis towards maltooligosaccharides . The interaction between MBP and specific cytoplasmic membrane proteins initiates either active transport or chemotaxis . In order to gain new understanding of the function of MBP, especially its versatility in binding different linear and cyclic oligosaccharides with similar affinities, we have undertaken high-resolution X-ray analysis of three oligosaccharide-bound structures . RESULTS: The structures of MBP complexed with maltose, maltotriose and maltotetraose have been refined to high resolutions (1.67 to 1.8 A) . These structures provide details at the atomic level of many features of oligosaccharide binding . The structures reveal differences between buried and surface binding sites and show the importance of hydrogen bonds and van der Waals interactions, especially those resulting from aromatic residue stacking . Insights are provided into the structural plasticity of the protein, the binding affinity and the binding specificity with respect to alpha/beta anomeric preference and oligosaccharide length . In addition, the structures demonstrate the different conformations that can be adopted by the oligosaccharide within the complex . CONCLUSIONS: MBP has a two-domain structure joined by a hinge-bending region which contains the substrate-binding groove . The bound maltooligosaccharides have a ribbon-like structure: the edges of the ribbon are occupied by polar hydroxyl groups and the flat surfaces are composed of nonpolar patches of the sugar ring faces . The polar groups and nonpolar patches are heavily involved in forming hydrogen bonds and van der Waals contacts, respectively, with complimentary residues in the groove . Hinge-bending between the two domains enables the participation of both domains in the binding and sequestering of the oligosaccharides . Changes in the subtle contours of the binding site allow binding of maltodextrins of varying length with similarly high affinities . The fact that the three bound structures are essentially identical ensures productive interaction with the oligomeric membrane proteins, which are distinct for transport and chemotaxis. Curr Genet, 1997 Jul, 32(1), 41 - 51 Bleomycin hydrolase (Blh1p), a multi-sited thiol protease in search of a distinct physiological role; Niemer I et al.; Bleomycin hydrolase, Blh1p, from yeast was co-purified with Gce1p, a cAMP-binding ectoprotein, anchored to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) anchor . Blh1p is a hydrophilic thiol protease lacking transmembrane domains . We have used polyclonal antibodies to study the topology of the over-expressed protein in yeast and have found that it is amphitropic . Part of Blh1p is associated with plasma membranes, and most of the rest occurs in the cytosol . Both the growth conditions and calcium were found to have minor influences on the topology of Blh1p, in that glucose and the earth-alkali ion slightly enhanced recruitment to the membrane . We have examined the possibility that co-purification of Blh1p with Gce1p has a functional basis, and have observed that over-expression of BLH1 in yeast leads to an acceleration of the glucose-induced amphiphilic to hydrophilic conversion of Gce1p, wherein Blh1p could either directly catalyse the proteolytic removal of the polar head-group of the GPI anchor subsequent to an initial lipolytic cleavage by a GPI-specific phospholipase C or indirectly modulate the reaction . The data show that a thiol protease is involved, but point to an indirect role of Blh1p in GPI processing . Proteases with similar or overlapping substrate specificity are likely to exist, since deletion of BLH1 neither entails a growth-defect on any carbon source tested, nor the loss of proteolytic processing of the GPI anchor of Gce1p . Reduced proteolytic GPI processing is, however, observed in the blh1 mutant and the corresponding acceleration in the respective BLH1 multi-copy transformant. Gan To Kagaku Ryoho, 1997 Sep, 24(11), 1392 - 400 {Mismatch repair protein}; Ikejima M et al.; A DNA mismatch repair system exists that repairs mispaired bases formed during DNA replication and genetic recombination . Genetic defects in this mismatch repair system are known to increase the rate of spontaneous mutation in Escherichia coli . Some cases of inherited cancer are associated with inherited defects of mismatch repair genes, showing the importance of the mismatch repair system in maintenance of genetic stability and avoidance of cancer susceptibility . This review focused on what is known about the mechanisms of mismatch repair in human cells and the relationship between defects in mismatch repair and carcinogenesis. Microbiology, 1997 Sep, 143 ( Pt 9), 2931 - 8 Identification of residues in the putative TolA box which are essential for the toxicity of the endonuclease toxin colicin E9; Garinot-Schneider C et al.; E colicins are plasmid-coded, protein antibiotics which bind to the BtuB outer membrane receptor of Escherichia coli cells and are then translocated either to the outer surface of the cytoplasmic membrane in the case of the pore-forming colicin E1, or to the cytoplasm in the case of the enzymic colicins E2-E9 . Translocation has been proposed to be dependent on a putative TolA box; a pentapeptide (DGSGW) located in the N-terminal 39 residues of several Tol-dependent colicins . In this study, site-directed mutagenesis was used to change each of the residues of the putative TolA box of colicin E9 to alanines . In the case of the two glycine residues, the resulting mutant proteins were indistinguishable from the native colicin E9 protein in a biological assay; whereas the other three residues when mutated to alanines resulted in a complete loss of biological activity . Mutagenesis of two serine residues flanking the putative TolA box, Ser34 and Ser40, to alanines did not abolish the biological activity of the mutant colicin E9, although the zones of growth inhibition were hazy and slow to form . The size of the zone of inhibition was significantly smaller than the control in the case of the Ser40Ala mutant . The ColE9/Im9 complex was isolated from the three biologically inactive TolA box alanine mutants . In competition assays all three mutant protein complexes were capable of protecting sensitive E . coli cells against killing by the native ColE9/Im9 complex . On removal of the Im9 protein from the three mutant ColE9/Im9 complexes, all three mutant colicins exhibited DNase activity . These results confirm the importance of the putative TolA box in the biological activity of colicin E9, and suggest that the TolA box has a role in the translocation of colicin E9. Microbiology, 1997 Sep, 143 ( Pt 9), 2883 - 90 The gshB gene in the cyanobacterium Synechococcus sp . PCC 7942 encodes a functional glutathione synthetase; Okumura N et al.; The gene homologous to glutathione synthetase of Escherichia coli was inactivated in the cyanobacterium Synechococcus sp . PCC 7942 . The region of genomic DNA including the mutation site was isolated from the mutant by plasmid rescue and the native gene of the wild-type was cloned from a genomic DNA library of the wild-type using the flanking DNA as a probe . The wild-type gene, designated gshB, encodes a polypeptide of 323 amino acids with a molecular mass of 35 kDa . The deduced amino acid sequence resembles glutathione synthetases of bacteria, but not those of higher organisms . When gshB was overexpressed in E . coli, glutathione synthetase activity was increased markedly in the E . coli extract . In addition, the Synechococcus sp . PCC 7942 gshB mutants had lost their ability to synthesize glutathione . These findings demonstrate that the gshB gene of Synechococcus sp . PCC 7942 is a structural gene for glutathione synthetase and is involved in the biosynthesis of glutathione. Microbiology, 1997 Sep, 143 ( Pt 9), 2865 - 75 Regulation of the ndh gene of Escherichia coli by integration host factor and a novel regulator, Arr; Green J et al.; The ndh gene of Escherichia coli encodes the non-proton-translocating NADH dehydrogenase II . Expression of the ndh gene is subject to a complex network of regulatory controls at the transcriptional level . Under anaerobic conditions ndh is repressed by the regulator of fumarate and nitrate reduction (FNR) . However, in the absence of FNR, ndh expression is activated by the amino acid response regulator (Arr) during anaerobic growth in rich medium . Expression of the ndh gene varies during the growth cycle in response to the intracellular concentration of the heat-stable DNA-binding protein, Fis . In this work two additional heat-stable proteins, integration host factor (IHF) and the histone-like protein HU were found to interact with the ndh promoter . IHF was shown to bind at three sites centred at +26, -17 and -58 in the ndh promoter (Kd = 10(-8) M), to prevent open-complex formation and to repress ndh transcription in vitro . Studies with an ndh-lacZ fusion confirmed that IHF represses ndh expression in vivo . Two putative binding sites for Arr, which overlap the two FNR boxes in the ndh promoter, were identified . Studies with the FNR-activated and amino-acid-inducible asparaginase II gene (ansB) showed that IHF and a component of the Arr-containing fraction (but not HU) interact with the corresponding ansB promoter. Lipids, 1997 Sep, 32(9), 935 - 42 The effect of highly purified eicosapentaenoic and docosahexaenoic acids on monocyte phagocytosis in man; Halvorsen DS et al.; The n-3 fatty acids (FA) from marine sources are known to exert antiinflammatory effects on monocyte function . There is still controversy whether n-3 FA may increase the susceptibility to infections . The present study was designed to assess the effect of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on monocyte phagocytosis and respiratory burst activity . Fifty-eight healthy men were randomized to take a daily supplement of 3.8 g highly purified EPA (n = 20), 3.6 g DHA (n = 19), or corn oil (n = 19) for 7 wk . Mononuclear leukocytes were collected, isolated, and cryopreserved prior to and after dietary supplementation . Paired samples were analyzed in the presence of autologous serum in a crossover design . Monocyte phagocytosis and respiratory burst activity were measured by flow cytometry after ingestion of Escherichia coli . Monocytes retained their phagocytic ability and respiratory burst activity after supplementation . No reduction in internalization of bacteria was registered . Dietary n-3 FA and particularly EPA improved bacterial adherence to the monocyte surface . In the crossover experiments, there was an adverse effect of serum enriched with n-3 FA on bacterial adherence . We conclude that monocytes retain their phagocytic potential after supplementation with purified EPA and DHA. Curr Microbiol, 1997 Oct, 35(4), 244 - 8 Phylogenetic and narG analysis of a Hyphomicrobium isolate; Tuhela L et al.; An obligately methylotrophic organism was isolated from a water well that manifested symptoms of biofouling . The isolate was appendaged and utilized methylamine, dimethylamine, trimethylamine, or methanol as the sole carbon and energy source . The isolate exhibited hydroxypyruvate reductase activity, suggesting C1-assimilation via the serine pathway . Fatty acid profiling indicated the predominance of 18:1 cis-fatty acids . The isolate did not grow anaerobically with nitrate as the final electron acceptor . Genomic DNA from the isolate did not hybridize against the narG gene, which encodes the alpha subunit of dissimilatory nitrate reductase in Escherichia coli . The phenotypic data suggested the assignment of the isolate to the genus Hyphomicrobium . The identification was supported by phylogenetic characterization based on 16S rRNA sequence comparisons of the isolate. Curr Microbiol, 1997 Oct, 35(4), 228 - 32 A gene encoding an exo-beta-glucosidase from Cellvibrio mixtus; Sakellaris H et al.; Cellvibrio mixtus produces an array of endohydrolytic enzymes involved in the initial phases of beta-glycan polysaccharide degradation in the soil . These enzymes convert complex, high-molecular-weight, insoluble polysaccharides into low-molecular-weight, soluble oligosaccharides which must be further degraded for cellular uptake and catabolism . Little is known about the enzymes involved in this latter process in C . mixtus . In this paper we report the cloning of the lam2 gene, which encodes an exohydrolase of low-molecular-weight, soluble beta-glucans and which may be involved in the latter stages of beta-glucan degradation in C . mixtus . T