J Bacteriol, 1999 Mar, 181(5), 1524 - 9 The Escherichia coli Ada protein can interact with two distinct determinants in the sigma70 subunit of RNA polymerase according to promoter architecture: identification of the target of Ada activation at the alkA promoter; Landini P et al.; The methylated form of the Ada protein (meAda) activates transcription from the Escherichia coli ada, aidB, and alkA promoters with different mechanisms . In this study we identify amino acid substitutions in region 4 of the RNA polymerase subunit sigma70 that affect Ada-activated transcription at alkA . Substitution to alanine of residues K593, K597, and R603 in sigma70 region 4 results in decreased Ada-dependent binding of RNA polymerase to the alkA promoter in vitro and impairs alkA transcription both in vivo and in vitro, suggesting that these residues define a determinant for meAda-sigma70 interaction . In a previous study (P . Landini, J . A . Bown, M . R . Volkert, and S . J . W . Busby, J . Biol . Chem . 273:13307-13312, 1998), we showed that a set of negatively charged amino acids in sigma70 region 4 is involved in meAda-sigma70 interaction at the ada and aidB promoters . However, the alanine substitutions of positively charged residues K593, K597, and R603 do not affect meAda-dependent transcription at ada and aidB . Unlike the sigma70 amino acids involved in the interaction with meAda at the ada and aidB promoters, K593, K597, and R603 are not conserved in sigmaS, an alternative sigma subunit of RNA polymerase mainly expressed during the stationary phase of growth . While meAda is able to promote transcription by the sigmaS form of RNA polymerase (EsigmaS) at ada and aidB, it fails to do so at alkA . We propose that meAda can activate transcription at different promoters by contacting distinct determinants in sigma70 region 4 in a manner dependent on the location of the Ada binding site. J Bacteriol, 1999 Mar, 181(5), 1515 - 23 SOS and UVM pathways have lesion-specific additive and competing effects on mutation fixation at replication-blocking DNA lesions; Rahman MS et al.; Escherichia coli cells have multiple mutagenic pathways that are induced in response to environmental and physiological stimuli . Unlike the well-investigated classical SOS response, little is known about newly recognized pathways such as the UVM (UV modulation of mutagenesis) response . In this study, we compared the contributions of the SOS and UVM pathways on mutation fixation at two representative noninstructive DNA lesions: 3,N4-ethenocytosine (epsilonC) and abasic (AP) sites . Because both SOS and UVM responses are induced by DNA damage, and defined UVM-defective E . coli strains are not yet available, we first constructed strains in which expression of the SOS mutagenesis proteins UmuD' and UmuC (and also RecA in some cases) is uncoupled from DNA damage by being placed under the control of a heterologous lac-derived promoter . M13 single-stranded viral DNA bearing site-specific lesions was transfected into cells induced for the SOS or UVM pathway . Survival effects were determined from transfection efficiency, and mutation fixation at the lesion was analyzed by a quantitative multiplex sequence analysis procedure . Our results suggest that induction of the SOS pathway can independently elevate mutagenesis at both lesions, whereas the UVM pathway significantly elevates mutagenesis at epsilonC in an SOS-independent fashion and at AP sites in an SOS-dependent fashion . Although mutagenesis at epsilonC appears to be elevated by the induction of either the SOS or the UVM pathway, the mutational specificity profiles for epsilonC under SOS and UVM pathways are distinct . Interestingly, when both pathways are active, the UVM effect appears to predominate over the SOS effect on mutagenesis at epsilonC, but the total mutation frequency is significantly increased over that observed when each pathway is individually induced . These observations suggest that the UVM response affects mutagenesis not only at class 2 noninstructive lesions (epsilonC) but also at classical SOS-dependent (class 1) lesions such as AP sites . Our results add new layers of complexity to inducible mutagenic phenomena: DNA damage activates multiple pathways that have lesion-specific additive as well as suppressive effects on mutation fixation, and some of these pathways are not directly regulated by the SOS genetic network. Biophys J, 1999 Mar, 76(3), 1706 - 19 Chemotactic responses of Escherichia coli to small jumps of photoreleased L-aspartate; Jasuja R et al.; Computer-assisted motion analysis coupled to flash photolysis of caged chemoeffectors provides a means for time-resolved analysis of bacterial chemotaxis . Escherichia coli taxis toward the amino acid attractant L-aspartate is mediated by the Tar receptor . The physiology of this response, as well as Tar structure and biochemistry, has been studied extensively . The beta-2, 6-dinitrobenzyl ester of L-aspartic acid and the 1-(2-nitrophenyl)ethyl ether of 8-hydroxypyrene-1,3,6-tris-sulfonic acid were synthesized . These compounds liberated L-aspartate and the fluorophore 8-hydroxypyrene 1,3,6-tris-sulfonic acid (pyranine) upon irradiation with near-UV light . Photorelease of the fluorophore was used to define the amplitude and temporal stability of the aspartate jumps employed in chemotaxis experiments . The dependence of chemotactic adaptation times on aspartate concentration, determined in mixing experiments, was best fit by two Tar aspartate-binding sites . Signal processing (excitation) times, amplitudes, and adaptive recovery of responses elicited by aspartate jumps producing less than 20% change in receptor occupancy were characterized in photorelease assays . Aspartate concentration jumps in the nanomolar range elicited measurable responses . The response threshold and sensitivity of swimming bacteria matched those of bacteria tethered to glass by a single flagellum . Stimuli of similar magnitude, delivered either by rapid mixing or photorelease, evoked responses of similar strength, as assessed by recovery time measurements . These times remained proportional to change in receptor occupancy close to threshold, irrespective of prior occupancy . Motor excitation responses decayed exponentially with time . Rates of excitation responses near threshold ranged from 2 to 7 s-1 . These values are consistent with control of excitation signaling by decay of phosphorylated pools of the response regulator protein, CheY . Excitation response rates increased slightly with stimulus size up to values limited by the instrumentation; the most rapid was measured to be 16 +/- 3 (SE) s-1 . This increase may reflect simultaneous activation of CheY dephosphorylation, together with inhibition of its phosphorylation. Biophys J, 1999 Mar, 76(3), 1241 - 9 Pathways of electron transfer in Escherichia coli DNA photolyase: Trp306 to FADH; Cheung MS et al.; We describe the results of a series of theoretical calculations of electron transfer pathways between Trp306 and *FADH . in the Escherichia coli DNA photolyase molecule, using the method of interatomic tunneling currents . It is found that there are two conformationally orthogonal tryptophans, Trp359 and Trp382, between donor and acceptor that play a crucial role in the pathways of the electron transfer process . The pathways depend vitally on the aromaticity of tryptophans and the flavin molecule . The results of this calculation suggest that the major pathway of the electron transfer is due to a set of overlapping orthogonal pi-rings, which starts from the donor Trp306, runs through Trp359 and Trp382, and finally reaches the flavin group of the acceptor complex, FADH. Antimicrob Agents Chemother, 1999 Mar, 43(3), 711 - 3 Genetic evidence that InhA of Mycobacterium smegmatis is a target for triclosan; McMurry LM et al.; Three Mycobacterium smegmatis mutants selected for resistance to triclosan each had a different mutation in InhA, an enoyl reductase involved in fatty acid synthesis . Two expressed some isoniazid resistance . A mutation originally selected on isoniazid also mediated triclosan resistance, as did the wild-type inhA gene on a multicopy plasmid . Replacement of the mutant chromosomal inhA genes with wild-type inhA eliminated resistance . These results suggest that M . smegmatis InhA, like its Escherichia coli homolog FabI, is a target for triclosan. Antimicrob Agents Chemother, 1999 Mar, 43(3), 697 - 8 Fosfomycin alters lipopolysaccharide-induced inflammatory cytokine production in mice; Matsumoto T et al.; To determine the mechanisms of immunomodulating action of fosfomycin (FOF), we examined its effect on the production of inflammatory cytokines in mice injected with lipopolysaccharide (LPS) . Treatment with FOF significantly lowered the peak serum levels of tumor necrosis factor alpha and interleukin-1 beta, indicating that FOF alters inflammatory cytokine production after LPS stimulation. Antimicrob Agents Chemother, 1999 Mar, 43(3), 616 - 22 Clindamycin suppresses endotoxin released by ceftazidime-treated Escherichia coli O55:B5 and subsequent production of tumor necrosis factor alpha and interleukin-1 beta; Kishi K et al.; Treatment of septicemia caused by Escherichia coli with ceftazidime (CAZ) may be associated with the development of septic shock due to the release of bacterial lipopolysaccharide . We examined the suppressive effect of clindamycin (CLDM) on CAZ-induced release of endotoxin by cultured E . coli and the subsequent production of inflammatory cytokines (tumor necrosis factor alpha {TNF-alpha} and interleukin-1 beta {IL-1 beta}) . E . coli ATCC 12014 was incubated in inactivated horse serum with or without CLDM for 1, 4, or 18 h, followed by the addition of CAZ and collection of the culture supernatant at 0, 1, and 2 h . The concentration of endotoxin in each sample was measured by a chromogenic Limulus test . Another portion of the culture supernatant was added to THP-1 cell culture and incubated for 4 h, and the concentrations of TNF-alpha and IL-1 beta in the supernatant were measured by an enzyme-linked immunosorbent assay . In the control group (no CLDM), CAZ administration resulted in significant increases in endotoxin, TNF-alpha, and IL-1 beta concentrations . Pretreatment of E . coli with CLDM for 4 or 18 h before the addition of CAZ significantly suppressed the concentrations of endotoxin, TNF-alpha, and IL-1 beta in a time-dependent manner . In addition, CAZ treatment transformed E . coli from rodshaped bacteria to filament-like structures, as determined by electron microscopy, while pretreatment with CLDM prevented these morphological changes . Our in vitro studies showed that CAZ-induced release of large quantities of endotoxin by E . coli could be suppressed by prior administration of CLDM. Antimicrob Agents Chemother, 1999 Mar, 43(3), 510 - 3 In vitro activities of cephalosporins and quinolones against Escherichia coli strains isolated from diarrheic dairy calves; Orden JA et al.; The in vitro activities of several cephalosporins and quinolones against 195 strains of Escherichia coli isolated from diary calves affected by neonatal diarrhea were determined . One hundred thirty-seven of these strains produced one or more potential virulence factors (F5, F41, F17, cytotoxic necrotizing factor, verotoxin, and the eae gene), but the remaining 58 strains did not produce any of these factors . From 11 to 18% of the E . coli strains were resistant to cephalothin, nalidixic acid, enoxacin, and enrofloxacin . However, cefuroxime, cefotaxime, and cefquinome were highly effective against the E . coli isolates tested . Some significant differences (P < 0.05) in resistance to quinolones between the strains producing potential virulence factors and nonfimbriated, nontoxigenic, eae-negative strains were found . Thus, eae-positive, necrotoxigenic, and verotoxigenic (except for nalidixic acid) E . coli strains were significantly more sensitive to nalidixic acid, enoxacin, and enrofloxacin than nonfimbriated, nontoxigenic, eae-negative strains . Moreover, eae-positive strains were significantly more sensitive to enoxacin and enrofloxacin than F5-positive strains . Thus, the result of this study suggest that the bovine E . coli strains that produce some potential virulence factors are more sensitive to quinolones than those that do not express these factors. Antimicrob Agents Chemother, 1999 Mar, 43(3), 447 - 53 RecA-Mediated gene conversion and aminoglycoside resistance in strains heterozygous for rRNA; Prammananan T et al.; Clinical resistance to aminoglycosides in general is due to enzymatic drug modification . Mutational alterations of the small ribosomal subunit rRNA have recently been found to mediate acquired resistance in bacterial pathogens in vivo . In this study we investigated the effect of 16S rRNA heterozygosity (wild-type {wt} and mutant {mut} operons at position 1408 {1408wt/1408mut}) on aminoglycoside resistance . Using an integrative vector, we introduced a single copy of a mutated rRNA operon (1408 A-->G) into Mycobacterium smegmatis, which carries two chromosomal wild-type rRNA operons; the resultant transformants exhibited an aminoglycoside-sensitive phenotype . In contrast, introduction of the mutated rRNA operon into an M . smegmatis rrnB knockout strain carrying a single functional chromosomal wild-type rRNA operon resulted in aminoglycoside-resistant transformants . Subsequent analysis by DNA sequencing and RNase protection assays unexpectedly demonstrated a homozygous mutant genotype, rRNAmut/rRNAmut, in the resistant transformants . To investigate whether RecA-mediated gene conversion was responsible for the aminoglycoside-resistant phenotype in the rRNAwt/rRNAmut strains, recA mutant strains were generated by allelic exchange techniques . Transformation of the recA rrnB M . smegmatis mutant strains with an integrative vector expressing a mutated rRNA operon (Escherichia coli position 1408 A-->G) resulted in transformants with an aminoglycoside-sensitive phenotype . Subsequent analysis showed stable heterozygosity at 16S rRNA position 1408 with a single wild-type allele and a single resistant allele . These results demonstrate that rRNA-mediated mutational resistance to aminoglycosides is recessive. Gene, 1999 Jan 8, 226(1), 73 - 81 Caenorhabditis elegans ZC376.5 encodes a tRNA (m2/2G(26))dimethyltransferance in which (246)arginine is important for the enzyme activity; Liu J et al.; It has been estimated that eukaryotes carry more than 50 genes for tRNA modifying enzymes . Of the few so far identified most come from yeast, a lower eukaryote . In Saccharomyces cerevisiae, the TRM1 gene is a nuclear gene encoding the tRNA(m2/ 2G(26))dimethyltransferase, which catalyses the formation of the N2, N2-dimethylguanosine at position 26 in tRNA . We have isolated and characterized the corresponding gene ZC376.5 in Caenorhabditis elegans . Via RTPCR the cDNA sequence of the full length ZC376.5 has now been cloned, expressed in Escherichia coli and demonstrated to encode a tRNA(m2/2G(26))dimethyltransferase that produces dimethyl-G26 in vivo and in vitro with tRNA from yeast and bacteria as substrates . This is the first example of a complete gene sequence coding for a tRNA modifying enzyme from a multicellular organism . A point mutation in exon IV in the C . elegans genome sequence coding for the tRNA(m2/2G(26))methyltransferase that substituted arginine246 for glycine eliminated the modification activity . Exchanging the corresponding lysine residue in the yeast Trm1p for alanine caused a severe loss of activity, indicating that the identity of the amino acid at this position is important for enzyme activity. J Interferon Cytokine Res, 1999 Jan, 19(1), 27 - 32 Cloning of cDNA for canine interleukin-18 and canine interleukin-1beta converting enzyme and expression of canine interleukin-18; Okano F et al.; Cloning of canine interleukin-18 (IL-18) and canine interleukin-1beta converting enzyme (ICE) cDNA was carried out by using murine IL-18 cDNA and human ICE cDNA, respectively, as probes . Sequence homology to known sequences of human, mouse, or rat genes was noted at nucleotide and amino acid levels . Canine IL-18 mRNA was expressed in various canine organs, whereas canine ICE mRNA was expressed in only a few, particularly in the brain and testis . Cloned canine IL-18 cDNA was expressed in Escherichia coli . The resulting protein promoted induction of canine interferon-y (IFN-y) from stimulated canine lymphocytes . Canine IL-18 and canine IL-12 produced canine IFN-gamma synergistically . Canine IL-18 suppressed the growth of tumor cells transplanted in SCID mice . Cloned canine IL-18 should prove useful as an anticancer agent. DNA Res, 1998 Dec 31, 5(6), 341 - 8 Molecular cloning and characterization of three cDNAs encoding putative mitogen-activated protein kinase kinases (MAPKKs) in Arabidopsis thaliana; Ichimura K et al.; We isolated three Arabidopsis thaliana cDNA clones (ATMKK3, ATMKK4 and ATMKK5) encoding protein kinases with extensive homology to the mitogen-activated protein kinase kinases (MAPKKs) of various organisms in the catalytic domain . ATMKK3 shows high homology (85% identity) to NPK2, a tobacco MAPKK homologue . ATMKK4 and 5 are closely related to each other (84% identity) . Phylogenetic analysis showed that the plant MAPKKs constitute at least three subgroups . The recombinant ATMKK3 and ATMKK4 were expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli . Affinity purified GST-ATMKK3 and GST-ATMKK4 proteins contained phosphorylation activity, which shows that both the ATMKK3 and ATMKK4 genes encode functional protein kinases . Northern blot analysis revealed that the ATMKK3 gene expressed in all the organs . The levels of ATMKK4 and 5 mRNAs were relatively higher in steins and leaves than in flowers and roots . We determined the map positions of the ATMKK3, 4 and 5 genes on Arabidopsis chromosomes by RFLP mapping using P1 genomic clones. DNA Res, 1998 Dec 31, 5(6), 327 - 34 A novel plasmid recombination mechanism of the marine cyanobacterium Synechococcus sp . PCC7002; Akiyama H et al.; We describe a novel mechanism of site-specific recombination in the unicellular marine cyanobacterium Synechococcus sp . PCC7002 . The specific recombination sites on the smallest plasmid pAQ1 were localized by studying the properties of pAQ1-derived shuttle-vectors . We found that a palindromic element, the core sequence of which is G(G/A)CGATCGCC, functions as a resolution site for site-specific plasmid recombination . Furthermore, site-directed mutagenesis analysis of the element show that the site-specific recombination in the cyanobacterium requires sequence specificity, symmetry in the core sequence and, in part, the spacing between the elements . Interestingly, this element is over-represented not only in pAQ1 and in the genome of the cyanobacterium, but also in the accumulated cyanobacterial sequences from Synechococcus sp . PCC6301, PCC7942, vulcanus and Synechocystis sp . PCC6803 within GenBank and EMBL databases . Thus, these findings strongly suggest that the site-specific recombination mechanism based on the palindromic element should be common in these cyanobacteria. Protein Sci, 1999 Feb, 8(2), 443 - 6 Expression and characterization of the intact N-terminal domain of streptokinase; Azuaga AI et al.; Proteolytic studies have enabled two of the three putative domains of the fibrinolytic protein streptokinase to be isolated and characterized (Conejero-Lara F et al., 1996, Protein Sci 5:2583-2591) . The N-terminal domain, however, could not be isolated in these experiments because of its susceptibility to proteolytic cleavage . To complete the biophysical characterization of the domain structure of streptokinase we have overexpressed, purified, and characterized the N-terminal region of the protein, residues 1-146 . The results show this is cooperatively folded with secondary structure content and overall stability closely similar to those of the equivalent region in the intact protein. Protein Sci, 1999 Feb, 8(2), 430 - 4 Variants of ribonuclease inhibitor that resist oxidation; Kim BM et al.; Human ribonuclease inhibitor (hRI) is a cytosolic protein that protects cells from the adventitious invasion of pancreatic-type ribonucleases . hRI has 32 cysteine residues . The oxidation of these cysteine residues to form disulfide bonds is a rapid, cooperative process that inactivates hRI . The most proximal cysteine residues in native hRI are two pairs that are adjacent in sequence: Cys94 and Cys95, and Cys328 and Cys329 . A cystine formed from such adjacent cysteine residues would likely contain a perturbing cis peptide bond within its eight-membered ring, which would disrupt the structure of hRI and could facilitate further oxidation . We find that replacing Cys328 and Cys329 with alanine residues has little effect on the affinity of hRI for bovine pancreatic ribonuclease A (RNase A), but increases its resistance to oxidation by 10- to 15-fold . Similar effects are observed for the single variants, C328A hRI and C329A hRI, suggesting that oxidation resistance arises from the inability to form a Cys328-Cys329 disulfide bond . Replacing Cys94 and Cys95 with alanine residues increases oxidation resistance to a lesser extent, and decreases the affinity of hRI for RNase A . The C328A, C329A, and C328A/C329A variants are likely to be more useful than wild-type hRI for inhibiting pancreatic-type ribonucleases in vitro and in vivo . We conclude that replacing adjacent cysteine residues can confer oxidation resistance in a protein. Protein Sci, 1999 Feb, 8(2), 355 - 60 Thermodynamic analysis of halide binding to haloalkane dehalogenase suggests the occurrence of large conformational changes; Krooshof GH et al.; Haloalkane dehalogenase (DhlA) hydrolyzes short-chain haloalkanes to produce the corresponding alcohols and halide ions . Release of the halide ion from the active-site cavity can proceed via a two-step and a three-step route, which both contain slow enzyme isomerization steps . Thermodynamic analysis of bromide binding and release showed that the slow unimolecular isomerization steps in the three-step bromide export route have considerably larger transition state enthalpies and entropies than those in the other route . This suggests that the three-step route involves different and perhaps larger conformational changes than the two-step export route . We propose that the three-step halide export route starts with conformational changes that result in a more open configuration of the active site from which the halide ion can readily escape . In addition, we suggest that the two-step route for halide release involves the transfer of the halide ion from the halide-binding site in the cavity to a binding site somewhere at the protein surface, where a so-called collision complex is formed in which the halide ion is only weakly bound . No large structural rearrangements are necessary for this latter process. Protein Sci, 1999 Feb, 8(2), 343 - 54 Topology and dynamics of the 10 kDa C-terminal domain of DnaK in solution; Bertelsen EB et al.; Hsp70 molecular chaperones contain three distinct structural domains, a 44 kDa N-terminal ATPase domain, a 17 kDa peptide-binding domain, and a 10 kDa C-terminal domain . The ATPase and peptide binding domains are conserved in sequence and are functionally well characterized . The function of the 10 kDa variable C-terminal domain is less well understood . We have characterized the secondary structure and dynamics of the C-terminal domain from the Escherichia coli Hsp70, DnaK, in solution by high-resolution NMR . The domain was shown to be comprised of a rigid structure consisting of four helices and a flexible C-terminal subdomain of approximately 33 amino acids . The mobility of the flexible region is maintained in the context of the full-length protein and does not appear to be modulated by the nucleotide state . The flexibility of this region appears to be a conserved feature of Hsp70 architecture and may have important functional implications . We also developed a method to analyze 15N nuclear spin relaxation data, which allows us to extract amide bond vector directions relative to a unique diffusion axis . The extracted angles and rotational correlation times indicate that the helices form an elongated, bundle-like structure in solution. Mol Microbiol, 1999 Feb, 31(3), 983 - 93 Molecular characterization of Escherichia coli FtsE and FtsX; de Leeuw E et al.; The genes ftsE and ftsX are organized in one operon together with ftsY . FtsY codes for the receptor of the signal recognition particle (SRP) that functions in targeting a subset of inner membrane proteins . We have found no indications for a structural relationship between FtsE/X and FtsY . Evidence is presented that FtsE and FtsX form a complex in the inner membrane that bears the characteristics of an ATP-binding cassette (ABC)-type transporter . FtsE is a hydrophilic nucleotide-binding protein that has a tendency to dimerize and associates with the inner membrane through an interaction with the integral membrane protein FtsX . An FtsE null mutant showed filamentous growth and appeared viable on high salt medium only, indicating a role for FtsE in cell division and/or salt transport. Mol Microbiol, 1999 Feb, 31(3), 893 - 902 ZntR is a Zn(II)-responsive MerR-like transcriptional regulator of zntA in Escherichia coli; Brocklehurst KR et al.; We have identified the promoter/operator region of the zntA gene of Escherichia coli and shown that Zn(II) is the primary inducer of expression of this Zn(II)/Cd(II) export gene . The promoter PzntA shows sequence similarities to the promoters of mercury resistance (mer) operons, including a long spacer region containing an inverted repeat sequence . The gene encoding the transcriptional regulator of PzntA, designated zntR, has been identified from genome sequence data, by expression of the gene product and by insertional inactivation/complementation . The ZntR product is a member of the MerR family of transcriptional regulators and appears to act as a hypersensitive transcriptional switch . A hybrid MerR/ZntR protein has been constructed and indicates that the C-terminal region of ZntR recognizes Zn(II). Mol Microbiol, 1999 Feb, 31(3), 885 - 92 Localization and environmental regulation of MCP-like proteins in Rhodobacter sphaeroides; Harrison DM et al.; Chemotaxis to many compounds by Rhodobacter sphaeroides requires transport and at least partial metabolism of the chemoeffector . Previous investigations using phototrophically grown cells have failed to find any homologues of the MCP chemoreceptors identified in Escherichia coli . However, using an antibody raised against the highly conserved domain of E . coli Tsr, MCP-like proteins were identified in R . sphaeroides WS8N . Analysis using Western blotting and immunogold electron microscopy showed that expression of these MCP-like proteins is environmentally regulated and that receptors are targeted to two different cellular locations: the poles of the cells and the cytoplasm . In aerobically grown cells, these proteins were shown by immunoelectron microscopy to localize predominantly to the cell poles and to an electron-dense body in the cytoplasm . Western blot analysis indicated a 17-fold reduction in protein concentration when cells were grown in the light . The number of immunogold particles was also dramatically reduced in anaerobically light-grown cells and their cellular distribution was altered . Fewer receptors localized to the cell poles and more particles randomly distributed within the cell, but the cytoplasmic cluster remained . These trends were more pronounced in cells grown anaerobically under dim light than in those grown anaerobically under bright light, suggesting that expression is controlled by redox state and either light intensity or the extent of photosynthetic membrane synthesis . Recent work on E . coli chemosensing suggests that oligomerization of receptors and chemosensory proteins is important for sensory signalling . The data presented here suggest that this oligomerization can occur with cytoplasmic receptors and also provides an explanation for the multiple copies of chemosensory proteins in R . sphaeroides. Mol Microbiol, 1999 Feb, 31(3), 833 - 44 Balanced biosynthesis of major membrane components through regulated degradation of the committed enzyme of lipid A biosynthesis by the AAA protease FtsH (HflB) in Escherichia coli; Ogura T et al.; The suppressor mutation, named sfhC21, that allows Escherichia coli ftsH null mutant cells to survive was found to be an allele of fabZ encoding R-3-hydroxyacyl-ACP dehydrase, involved in a key step of fatty acid biosynthesis, and appears to upregulate the dehydrase . The ftsH1(Ts) mutation increased the amount of lipopolysaccharide at 42 degrees C . This was accompanied by a dramatic increase in the amount of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase {the IpxC (envA) gene product} involved in the committed step of lipid A biosynthesis . Pulse-chase experiments and in vitro assays with purified components showed that FtsH, the AAA-type membrane-bound metalloprotease, degrades the deacetylase . Genetic evidence also indicated that the FtsH protease activity for the deacetylase might be affected when acyl-ACP pools were altered . The biosynthesis of phospholipids and the lipid A moiety of lipopolysaccharide, both of which derive their fatty acyl chains from the same R-3-hydroxyacyl-ACP pool, is regulated by FtsH. Mol Microbiol, 1999 Feb, 31(3), 773 - 83 Probing conserved surfaces on PapD; Hung DL et al.; PapD is the periplasmic chaperone required for the assembly of P pili in pyelonephritic strains of Escherichia coli . It consists of two immunoglobulin-like domains bisected by a subunit binding cleft . PapD is the prototype member of a super family of immunoglobulin-like chaperones that work in concert with their respective ushers to assemble a plethora of adhesive organelles including pilus- and non-pilus-associated adhesins . Three highly conserved residue clusters have been shown to play critical roles in the structure and function of PapD, as determined by site-directed mutagenesis . The in vivo stability of the chaperone depended on the formation of a buried salt bridge within the cleft . Residues along the G1 beta strand were required for efficient binding of subunits consistent with the crystal structure of PapD-peptide complexes . Finally, Thr-53, a residue that is part of a conserved band of residues located on the amino-terminal domain surface opposite the subunit binding cleft, was also found to be critical for pilus assembly, but mutations at Thr-53 did not interfere with chaperone-subunit complex formation. Mutat Res, 1999 Jan 26, 433(1), 59 - 66 Synergistic lethal effect between hydrogen peroxide and neocuproine (2,9-dimethyl 1,10-phenanthroline) in Escherichia coli; Almeida CE et al.; Despite 2,9-dimethyl 1,10-phenanthroline (NC) has been extensively used as a potential inhibitor of damage due to oxidative stress in biological systems, the incubation of E . coli cultures with the copper ion chelator NC prior to the challenge with hydrogen peroxide caused a lethal synergistic effect . The SOS response seems to be involved in the repair of the synergistic lesions through the recombination pathway . Furthermore, there is evidence for the UvrABC excinuclease participation in the repair of the synergistic lesions, and the base excision repair may also be required for bacterial survival to the synergistic effect mainly at high concentrations of H2O2, being the action of Fpg protein an important event . Incubation of lexA (Ind-) cultures with iron (II) ion chelator 2,2'-dipyridyl simultaneously with NC prevented the lethal synergistic effect . This result suggests an important role of the Fenton reaction on the phenomenon . NC treatment was able to increase the number of DNA strand breaks (DNAsb) induced by 10 mM of H2O2 in lexA (Ind-) strain and the simultaneous treatment with 2,2'-dipyridyl was able to block this effect. Curr Opin Struct Biol, 1999 Feb, 9(1), 135 - 41 Disassembly of intact multiprotein complexes in the gas phase; Rostom AA et al.; The observation of multiprotein complexes by mass spectrometry formerly relied upon chemical cross-linking to maintain interactions . Recent technological developments have enabled the observation of intact macromolecular complexes without modification . These assemblies, with masses far in excess of those measured previously, can be examined through controlled dissociation in the mass spectrometer, revealing information about their subunit interactions and topology. Curr Opin Microbiol, 1999 Feb, 2(1), 30 - 4 Cell biology of Legionella pneumophila; Vogel JP et al.; Legionella pneumophila is the causative agent of a potentially fatal form of pneumonia named Legionnaires' disease . L . pneumophila survives and replicates inside macrophages by preventing phagosome-lysosome fusion . A large number of L . pneumophila genes, called dot or icm, have been identified that are required for intracellular growth . It has recently been shown that the dot/icm genes code for a putative large membrane complex that forms a type IV secretion system used to alter the endocytic pathway. Curr Opin Microbiol, 1999 Feb, 2(1), 83 - 8 Enteropathogenic Escherichia coli: cellular harassment; DeVinney R et al.; The mechanisms by which enteropathogenic Escherichia coli (EPEC) mediates diarrhea remain a mystery . Recently a number of interesting and at times surprising results have come from studying EPEC interactions with host cells . Identification and characterization of bacterial factors, including Tir, EspA, EspB and EspD, and host responses have expanded our grasp of the diverse effects of EPEC on host cells. Curr Opin Biotechnol, 1999 Feb, 10(1), 71 - 5 Applications of peptide nucleic acids; Nielsen PE; Several exciting new developments in the applications of the DNA mimic peptide nucleic acid (PNA) have been published recently . A possible breakthrough may have come in efforts to develop PNA into gene therapeutic drugs . In eukaryotic systems, antisense activity of PNAs (as peptide conjugates) has been reported in nerve cells and even in rats upon injection into the brain, and antisense activity has also been demonstrated in Escherichia coli . PNA hybridization technology has developed rapidly within in situ hybridization, and exciting new methods based on MALDI-TOF detection have also been presented. J Mol Biol, 1999 Mar 5, 286(4), 1217 - 28 Serine 948 and threonine 1042 are crucial residues for allosteric regulation of Escherichia coli carbamoylphosphate synthetase and illustrate coupling effects of activation and inhibition pathways; Delannay S et al.; Escherichia coli carbamoylphosphate synthetase (CPSase) is a key enzyme in the pyrimidine nucleotides and arginine biosynthetic pathways . The enzyme harbors a complex regulation, being activated by ornithine and inosine 5'-monophosphate (IMP), and inhibited by UMP . CPSase mutants obtained by in vivo mutagenesis and selected on the basis of particular phenotypes have been characterized kinetically . Two residues, serine 948 and threonine 1042, appear crucial for allosteric regulation of CPSase . When threonine 1042 is replaced by an isoleucine residue, the enzyme displays a greatly reduced activation by ornithine . The T1042I mutated enzyme is still sensitive to UMP and IMP, although the effects of both regulators are reduced . When serine 948 is replaced by phenylalanine, the enzyme becomes insensitive to UMP and IMP, but is still activated by ornithine, although to a reduced extent . When correlating these observations to the structural data recently reported, it becomes clear that both mutations, which are located in spatially distinct regions corresponding respectively to the ornithine and the UMP/IMP binding sites, have coupled effects on the enzyme regulation . These results provide an illustration that coupling of regulatory pathways occurs within the allosteric subunit of E . coli CPSase.In addition, other mutants have been characterized, which display altered affinities for the different CPSase substrates and also slightly modified properties towards the allosteric effectors: P165S, P170L, A182V, P360L, S743N, T800F and G824D . Kinetic properties of these modified enzymes are also presented here and correlated to the crystal structure of E . coli CPSase and to the phenotype of the mutants . J Mol Biol, 1999 Mar 5, 286(4), 1197 - 215 Random circular permutation of DsbA reveals segments that are essential for protein folding and stability; Hennecke J et al.; One of the key questions in protein folding is whether polypeptide chains require unique nucleation sites to fold to the native state . In order to identify possible essential polypeptide segments for folding, we have performed a complete circular permutation analysis of a protein in which the natural termini are in close proximity . As a model system, we used the disulfide oxidoreductase DsbA from Escherichia coli, a monomeric protein of 189 amino acid residues . To introduce new termini at all possible positions in its polypeptide chain, we generated a library of randomly circularly permuted dsbA genes and screened for active circularly permuted variants in vivo . A total of 51 different active variants were identified . The new termini were distributed over about 70 % of the polypeptide chain, with the majority of them occurring within regular secondary structures . New termini were not found in approximately 30 % of the DsbA sequence which essentially correspond to four alpha-helices of DsbA . Introduction of new termini into these "forbidden segments" by directed mutagenesis yielded proteins with altered overall folds and strongly reduced catalytic activities . In contrast, all active variants analysed so far show structural and catalytic properties comparable with those of DsbA wild-type . We suggest that random circular permutation allows identification of contiguous structural elements in a protein that are essential for folding and stability . J Mol Biol, 1999 Mar 5, 286(4), 1025 - 32 Functional compensation by particular nucleotide substitutions of a critical G*U wobble base-pair during aminoacylation of transfer RNA; McClain WH et al.; Expression of the genetic code depends on precise tRNA aminoacylation by cognate aminoacyl-tRNA synthetase enzymes . The G.U wobble base-pair in the acceptor helix of Escherichia coli alanine tRNA is the primary aminoacylation determinant of this molecule . Previous work on the process of synthetase recognition of the G.U pair showed that replacing G.U by a G.C Watson-Crick base-pair inactivates alanine acceptance by the tRNA, but that C.A and G.A wobble pair replacements preserve acceptance . Work by another group reported that the effects of a G.C replacement were reversed by a distal wobble base-pair in the anticodon helix . This result is potentially interesting because it suggests that distant regions in alanine tRNA are functionally coupled during synthetase recognition and more generally because recognition determinants of many other tRNAs lie in both the acceptor helix and anticodon helix region . Here, we have conducted an extensive in vivo analysis of the distal wobble pair in alanine tRNA and report that it does not behave like a compensating mutation . Restoration of alanine acceptance was not detected even when the synthetase enzyme was overproduced . We discuss the previous experimental evidence and suggest how the distal wobble pair was incorrectly analyzed . The available data indicate that all principal recognition determinants of alanine tRNA lie in the molecule's acceptor helix . J Mol Biol, 1999 Mar 5, 286(4), 1009 - 24 The internal equilibrium of the hairpin ribozyme: temperature, ion and pH effects; Nesbitt SM et al.; The hairpin ribozyme reversibly cleaves phosphodiesters of RNA substrates to generate products with 5' hydroxyl and 2',3'-cyclic phosphate termini . We previously found that the rate constant for ligation is tenfold faster than the rate constant for cleavage under standard conditions . The hammerhead ribozyme catalyzes the same reactions but is reported to favor cleavage relative to ligation by more than 100-fold under the same conditions . To explore the basis for this difference, we examined the influence of temperature, ions and pH on the hairpin ribozyme internal equilibrium . Under the same conditions, the loss of entropy associated with ligation is less for the hairpin than for the hammerhead ribozyme, consistent with the notion that a more rigid hairpin structure undergoes a smaller decrease in dynamics upon ligation than the more flexible hammerhead structure . Increased salt and reduced temperature shift the equilibrium toward ligation while pH has little effect, suggesting that conditions that stabilize RNA structure tend to promote ligation . The hairpin ribozyme appears to take up at least one tri- or divalent cation or two monovalent cations upon ligation . The efficiency with which different cations promote ligation depends strongly on valence and, less strongly, on ionic radius or electronegativity . This pattern of cation selectivity suggests that cations promote ligation through delocalized electrostatic shielding, perhaps interacting with a region of especially high charge density in the ligated ribozyme . Changes in ionic conditions produce large but compensating changes in enthalpy and entropy for cleavage and ligation . Thus, in addition to any increase in ribozyme dynamics associated with cleavage, re-organization of associated cations contributes significantly to hairpin ribozyme thermodynamics . J Biol Chem, 1999 Mar 5, 274(10), 6776 - 82 An artificial transmembrane segment directs SecA, SecB, and electrochemical potential-dependent translocation of a long amino-terminal tail; McMurry JL et al.; Many integral membrane proteins contain an amino-terminal segment, often referred to as an N-tail, that is translocated across a membrane . In many cases, translocation of the N-tail is initiated by a cleavable, amino-terminal signal peptide . For N-tail proteins lacking a signal peptide, translocation is initiated by a transmembrane segment that is carboxyl to the translocated segment . The mechanism of membrane translocation of these segments, although poorly understood, has been reported to be independent of the protein secretion machinery . In contrast, here we describe alkaline phosphatase mutants containing artificial transmembrane segments that demonstrate that translocation of a long N-tail across the membrane is dependent upon SecA, SecB, and the electrochemical potential in the absence of a signal peptide . The corresponding mutants containing signal peptides also use the secretion machinery but are less sensitive to inhibition of its components . We present evidence that inhibition of SecA by sodium azide is incomplete even at high concentrations of inhibitor, which suggests why SecA-dependent translocation may not have been detected in other systems . Furthermore, by varying the charge around the transmembrane segment, we find that in the absence of a signal peptide, the orientation of the membrane-bound alkaline phosphatase is dictated by the positive inside rule . However, the presence of a signal peptide is an overriding factor in membrane orientation and renders all mutants in an Nout-Cin orientation. J Biol Chem, 1999 Mar 5, 274(10), 6763 - 9 Xer site-specific recombination . DNA strand rejoining by recombinase XerC; Grainge I et al.; Xer site-specific recombination functions in the stable maintenance of circular replicons in Escherichia coli . Each of two related recombinase proteins, XerC and XerD, cleaves a specific pair of DNA strands, exchanges them, and rejoins them to the partner DNA molecule during a complete recombination reaction . The rejoining activity of recombinase XerC has been analyzed using isolated covalent XerC-DNA complexes resulting from DNA cleavage reactions upon Holliday junction substrates . These covalent protein-DNA complexes are competent in the rejoining reaction, demonstrating that covalently bound XerC can catalyze strand rejoining in the absence of other proteins . This contrasts with a recombinase-mediated cleavage reaction, which requires the presence of both recombinases, the recombinase mediating catalysis at any given time requiring activation by the partner recombinase . In a recombining nucleoprotein complex, both cleavage and rejoining can occur prior to dissociation of the complex. J Biol Chem, 1999 Mar 5, 274(10), 6559 - 66 The biochemical role of glutamine 188 in human galactose-1-phosphate uridyltransferase; Lai K et al.; The substitution of arginine for glutamine at amino acid 188 (Q188R) ablates the function of human galactose-1-phosphate uridyltransferase (GALT) and is the most common mutation causing galactosemia in the white population . GALT catalyzes two consecutive reactions . The first reaction binds UDP-glucose (UDP-Glu), displaces glucose-1-phosphate (glu-1-P), and forms the UMP-GALT intermediate . In the second reaction, galactose-1-phosphate (gal-1-P) is bound, UDP-galactose (UDP-Gal) is released, and the free enzyme is recycled . In this study, we modeled glutamine, asparagine, and a common mutation arginine at amino acid 188 on the three-dimensional model of the Escherichia coli GALT-UMP protein crystal . We found that the amide group of the glutamine side chain could provide two hydrogen bonds to the phosphoryl oxygens of UMP with lengths of 2.52 and 2.82 A . Arginine and asparagine could provide only one hydrogen bond of 2 . 52 and 3.02 A, respectively . To test this model, we purified recombinant human Gln188-, Arg188-, and Asn188-GALT and analyzed the first reaction in the absence of gal-1-P by quantitating glu-1-P released using enzyme-linked methods . Gln188-GALT displaced 80 +/- 7 . 0 nmol glu-1-P/mg GALT/min in the first reaction . By contrast, both Arg188- and Asn188-GALT released more glu-1-P (170 +/- 8.0 and 129 +/- 28.4 nmol/mg GALT/min, respectively) . The overall, double displacement reaction was quantitated in the presence of gal-1-P . Gln188-GALT produced 80,030 +/- 5,910 nmol glu-1-P/mg GALT/min, whereas the mutant Arg188- and Asn188-GALT released only 600 +/- 71 . 2 and 2960 +/- 283.6 nmole glu-1-P/mg GALT/min, respectively . We conclude from these data that glutamine at position 188 stabilizes the UMP-GALT intermediate through hydrogen bonding and enables the double displacement of both glu-1-P and UDP-Gal . The substitution of arginine or asparagine at position 188 reduces hydrogen bonding and destabilizes UMP-GALT . The unstable UMP-GALT allows single displacement of glu-1-P with release of free GALT but impairs the subsequent binding of gal-1-P and displacement of UDP-Gal. J Biol Chem, 1999 Mar 5, 274(10), 6405 - 10 Solvent-exposed residues in the Tet repressor (TetR) four-helix bundle contribute to subunit recognition and dimer stability; Schnappinger D et al.; Dimerization specificity of Tet repressor (TetR) can be altered by changes in the core of the four-helix bundle that mediates protein-protein recognition . We demonstrate here that the affinity of subunit interaction depends also on the solvent-exposed residues at positions 128 and 179'-184', which interact across the dimerization surface . TetR(B) and (D), two naturally occurring sequence variants, differ at position 128 with respect to the monomer-monomer distances in the crystal structures and the charge of the amino acids, being glutamate in TetR(B) and arginine in TetR(D) . In vivo analysis of chimeric TetR(B/D) variants revealed that the single E128R exchange does not alter the dimerization specificity of TetR(B) to the one of TetR(D) . When combined with specificity mutations in alpha10, it is, however, able to increase dimerization efficiency of the TetR(B/D) chimera with TetR(D) . A loss of contact analysis revealed a positive interaction between Arg-128 and residues located at positions 179'-184' of the second monomer . We constructed a hyperstable TetR(B) variant by replacing residues 128 and 179-184 by the respective TetR(D) sequence . These results establish that in addition to a region in the hydrophobic core residues at the solvent-exposed periphery of the dimerization surface participate in protein-protein recognition in the TetR four-helix bundle. J Biol Chem, 1999 Mar 5, 274(10), 6350 - 9 Mapping of residues in the NADP(H)-binding site of proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli . A study of structure and function; Fjellstrom O et al.; Conformational changes in proton pumping transhydrogenases have been suggested to be dependent on binding of NADP(H) and the redox state of this substrate . Based on a detailed amino acid sequence analysis, it is argued that a classical betaalphabetaalphabeta dinucleotide binding fold is responsible for binding NADP(H) . A model defining betaA, alphaB, betaB, betaD, and betaE of this domain is presented . To test this model, four single cysteine mutants (cfbetaA348C, cfbetaA390C, cfbetaK424C, and cfbetaR425C) were introduced into a functional cysteine-free transhydrogenase . Also, five cysteine mutants were constructed in the isolated domain III of Escherichia coli transhydrogenase (ecIIIH345C, ecIIIA348C, ecIIIR350C, ecIIID392C, and ecIIIK424C) . In addition to kinetic characterizations, effects of sulfhydryl-specific labeling with N-ethylmaleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid, and diazotized 3-aminopyridine adenine dinucleotide (phosphate) were examined . The results are consistent with the view that, in agreement with the model, beta-Ala348, beta-Arg350, beta-Ala390, beta-Asp392, and beta-Lys424 are located in or close to the NADP(H) site . More specifically, beta-Ala348 succeeds betaB . The remarkable reactivity of betaR350C toward NNADP suggests that this residue is close to the nicotinamide moiety of NADP(H) . beta-Ala390 and beta-Asp392 terminate or succeed betaD, and are thus, together with the region following betaA, creating the switch point crevice where NADP(H) binds . beta-Asp392 is particularly important for the substrate affinity, but it could also have a more complex role in the coupling mechanism for transhydrogenase. J Biol Chem, 1999 Mar 5, 274(10), 6336 - 41 The interaction of the human MutL homologues in hereditary nonpolyposis colon cancer; Guerrette S et al.; Germline mutations in two human mismatch repair (MMR) genes, hMSH2 and hMLH1, appear to account for approximately 70% of the common cancer susceptibility syndrome hereditary nonpolyposis colorectal cancer (HNPCC) . Although the hMLH1 protein has been found to copurify with another MMR protein hPMS2 as a heterodimer, their function in MMR is unknown . In this study, we have identified the physical interaction regions of both hMLH1 with hPMS2 . We then examined the effects of hMLH1 missense alterations found in HNPCC kindreds for their interaction with hPMS2 . Four of these missense alterations (L574P, K616Delta, R659P, and A681T) displayed >95% reduction in binding to hPMS2 . Two additional missense alterations (K618A and K618T) displayed a >85% reduction in binding to hPMS2, whereas three missense alterations (S44F, V506A, and E578G) displayed 25-65% reduction in binding to hPMS2 . Interestingly, two HNPCC missense alterations (Q542L and L582V) contained within the consensus interaction region displayed no effect on interaction with hPMS2, suggesting that they may affect other functions of hMLH1 . These data confirm that functional deficiencies in the interaction of hMLH1 with hPMS2 are associated with HNPCC as well as suggest that other unknown functional alteration of the human MutL homologues may lead to tumorigenesis in HNPCC kindreds. J Biol Chem, 1999 Mar 5, 274(10), 6148 - 53 Altered substrate selectivity in a mutant of an intrahelical salt bridge in UhpT, the sugar phosphate carrier of Escherichia coli; Hall JA et al.; Site-directed and second site suppressor mutagenesis identify an intrahelical salt bridge in the eleventh transmembrane segment of UhpT, the sugar phosphate carrier of Escherichia coli . Glucose 6-phosphate (G6P) transport by UhpT is inactivated if cysteine replaces either Asp388 or Lys391 but not if both are replaced . This suggests that Asp388 and Lys391 are involved in an intrahelical salt bridge and that neither is required for normal UhpT function . This interpretation is strengthened by the finding that mutations at Lys391 (K391N, K391Q, and K391T) are recovered as revertants of the inactive D388C variant . Further work shows that although the D388C variant is null for G6P transport, movement of 32Pi by homologous Pi/Pi exchange is unaffected . This raises the possibility that this derivative may have latent function, a possibility confirmed by showing that D388C is a gain-of-function mutation in which phosphoenolpyruvate (PEP) is the preferred substrate . Added study of the Pi/Pi exchange shows that in wild type UhpT this partial reaction is readily blocked by G6P but not PEP . By contrast, in the D388C variant, Pi/Pi exchange is unaffected by G6P but is inhibited by both PEP and 3-phosphoglycerate . These latter substrates are used by PgtP, a related Pi-linked antiporter, which lacks the Asp388-Lys391 salt bridge but has instead an uncompensated arginine at position 391 . For this reason, we conclude that in both UhpT and PgtP position 391 can serve as a determinant of substrate selectivity by acting as a receptor for the anionic carboxyl brought into the translocation pathway by PEP. J Biol Chem, 1999 Mar 5, 274(10), 6114 - 21 Phosphotyrosine binding domains of Shc and insulin receptor substrate 1 recognize the NPXpY motif in a thermodynamically distinct manner; Farooq A et al.; Phosphotyrosine binding (PTB) domains of the adaptor protein Shc and insulin receptor substrate (IRS-1) interact with a distinct set of activated and tyrosine-phosphorylated cytokine and growth factor receptors and play important roles in mediating mitogenic signal transduction . By using the technique of isothermal titration calorimetry, we have studied the thermodynamics of binding of the Shc and IRS-1 PTB domains to tyrosine-phosphorylated NPXY-containing peptides derived from known receptor binding sites . The results showed that relative contributions of enthalpy and entropy to the free energy of binding are dependent on specific phosphopeptides . Binding of the Shc PTB domain to tyrosine-phosphorylated peptides from TrkA, epidermal growth factor, ErbB3, and insulin receptors is achieved via an overall entropy-driven reaction . On the other hand, recognition of the phosphopeptides of insulin and interleukin-4 receptors by the IRS-1 PTB domain is predominantly an enthalpy-driven process . Mutagenesis and amino acid substitution experiments showed that in addition to the tyrosine-phosphorylated NPXY motif, the PTB domains of Shc and IRS-1 prefer a large hydrophobic residue at pY-5 and a small hydrophobic residue at pY-1, respectively (where pY is phosphotyrosine) . These results agree with the calculated solvent accessibility of these two key peptide residues in the PTB domain/peptide structures and support the notion that the PTB domains of Shc and IRS-1 employ functionally distinct mechanisms to recognize tyrosine-phosphorylated receptors. J Biol Chem, 1999 Mar 5, 274(10), 6091 - 6 Mechanism of phosphoryl transfer in the dimeric IIABMan subunit of the Escherichia coli mannose transporter; Gutknecht R et al.; The mannose transporter of bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) mediates uptake of mannose, glucose, and related hexoses by a mechanism that couples translocation with phosphorylation of the substrate . It consists of the transmembrane IICMan.IIDMan complex and the cytoplasmic IIABMan subunit . IIABMan has two domains (IIA and IIB) that are linked by a 60-A long alanine-proline-rich linker . IIABMan transfers phosphoryl groups from the phospho-histidine-containing phospho-carrier protein of the PTS to His-10 on IIA, hence to His-175 on IIB, and finally to the 6'-OH of the transported hexose . IIABMan occurs as a stable homodimer . The subunit contact is mediated by a swap of beta-strands and an extensive contact area between the IIA domains . The H10C and H175C single and the H10C/H175C double mutants were used to characterize the phosphoryl transfer between IIA to IIB . Subunits do not exchange between dimers under physiological conditions, but slow phosphoryl transfer can take place between subunits from different dimers . Heterodimers of different subunits were produced in vitro by GuHCl-induced unfolding and refolding of mixtures of two different homodimers . With respect to wild-type homodimers, the heterodimers have the following activities: wild-type.H10C, 50%; wild-type.H175C 45%; H10C.H175C, 37%; and wild-type.H10C/H175C (double mutant), 29% . Taken together, this indicates that both cis and trans pathways contribute to the maximal phosphotransferase activity of IIABMan . A phosphoryl group on a IIA domain can be transferred either to the IIB domain on the same or on the second subunit in the dimer, and interruption of one of the two pathways results in a reduction of the activity to 70-80% of the control. J Biol Chem, 1999 Mar 5, 274(10), 6074 - 9 In vitro study of two dominant inhibitory GTPase mutants of Escherichia coli translation initiation factor IF2 . Direct evidence that GTP hydrolysis is necessary for factor recycling; Luchin S et al.; We have recently shown that the Escherichia coli initiation factor 2 (IF2) G-domain mutants V400G and H448E do not support cell survival and have a strong negative effect on growth even in the presence of wild-type IF2 . We have isolated both mutant proteins and performed an in vitro study of their main functions . The affinity of both mutant proteins for GTP is almost unchanged compared with wild-type IF2 . However, the uncoupled GTPase activity of the V400G and H448E mutants is severely impaired, the Vmax values being 11- and 40-fold lower, respectively . Both mutant forms promoted fMet-tRNAfMet binding to 70 S ribosomes with similar efficiencies and were as sensitive to competitive inhibition by GDP as wild-type IF2 . Formation of the first peptide bond, as measured by the puromycin reaction, was completely inhibited in the presence of the H448E mutant but still significant in the case of the V400G mutant . Sucrose density gradient centrifugation revealed that, in contrast to wild-type IF2, both mutant proteins stay blocked on the ribosome after formation of the 70 S initiation complex . This probably explains their dominant negative effect in vivo . Our results underline the importance of GTP hydrolysis for the recycling of IF2. Environ Mol Mutagen, 1999, 33(1), 75 - 85 Base-substitution profiles of externally activated polycyclic aromatic hydrocarbons and aromatic amines determined in a lacZ reversion assay; Garganta F et al.; Using an improved set of lactose-auxotrophic Escherichia coli tester strains, the proportion of the six possible transitions and transversions after mutagen exposure was assessed . Mutagenic specificity was determined in plate-incorporation assays using lactose-containing minimal medium for the selection of revertants, either after application of directly acting mutagens or by including a metabolic activation system with rat liver S9-extract . The differential and dose-dependent response of the six tester strains was shown by treating the bacteria with described diagnostic mutagens and other directly DNA damaging substances, e.g., N-methyl-N-nitrosoguanidine (MNNG) and benzo{a}pyrene-diolepoxide (BPDE) . Polycyclic aromatic hydrocarbons and aromatic amines were investigated in the presence of an external metabolic activation system . Benzo{a}pyrene (BaP) yielded similar mutation profiles as its ultimate mutagen BPDE, if 100-fold increased doses were applied . In contrast to the mutation profile of BaP, which was dominated by G:C-T:A transversions, mutagenesis with benzo{c}phenanthrene (BcPh) produced predominantly A:T-T:A transversions . The same base change was observed with 5-methylchrysene and found to be missing with 5,6-dimethylchrysene, while both compounds caused G:C-A:T transitions . The aromatic amines 4-aminobiphenyl (4-ABP), 2-aminoanthracene (2-AA) and 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhlP) yielded similar yet distinguishable mutation profiles . Base-substitution reversion profiles of the chemical mutagens were in agreement with those obtained in other systems and with molecular analysis of mutants induced by these agents. Microbiol Immunol, 1998, 42(12), 845 - 9 Relationship between O-serogroup and presence of pathogenic factor genes in Escherichia coli; Sunabe T et al.; A total of 383 isolates of serogroup-based enteropathogenic and enteroinvasive Escherichia coli (310 strains of EPEC and 73 strains of EIEC) were examined for the presence of corresponding pathogenic genes . The serogroup-based EPEC consisted of 232 strains isolated from diarrhea patients and of 78 strains from healthy carriers . The gene encoding intimin, eaeA, was detected in 42 of the 232 EPEC strains from patients (18.1%) and 9 of the 78 strains from carriers (11.5%) . The difference was not significant . The bfp gene on the EAF plasmid was detected in 7 of the 42 eaeA-positive EPEC strains from patients but was not detected in the 9 strains from carriers . In serogroup-based EIEC, a chromosomal ipaH gene encoding one of the invasive plasmid antigens was detected in 4 of the 60 strains from patients (6%) but not in the 13 strains from carriers . The 4 ipaH-positive strains possessed the invasive plasmid . These results suggested that the serogroup-based diagnosis of EPEC and EIEC is not sufficient for identifying strains carrying the eaeA or ipaH gene. FEBS Lett, 1999 Feb 5, 444(1), 65 - 70 Distinct sensitivities of OmpF and PhoE porins to charged modulators; Samartzidou H et al.; The inhibition of the anion-selective PhoE porin by ATP and of the cation-selective OmpF porin by polyamines has been previously documented . In the present study, we have extended the comparison of the inhibitor-porin pairs by investigating the effect of anions (ATP and aspartate) and positively charged polyamines (spermine and cadaverine) on both OmpF and PhoE with the patch-clamp technique, and by comparing directly the gating kinetics of the channels modulated by their respective substrates . The novel findings reported here are (1) that the activity of PhoE is completely unaffected by polyamines, and (2) that the kinetic changes induced by ATP on PhoE or polyamines on OmpF suggest different mechanisms of inhibition . ATP induces a high degree of flickering in the PhoE-mediated current and appears to behave as a blocker of ion flow during its presumed transport through PhoE . Polyamines modulate the kinetics of openings and closings of OmpF, in addition to promoting a blocker-like flickering activity . The strong correlation between sensitivity to inhibitors and ion selectivity suggests that some common molecular determinants are involved in these two properties and is in agreement with the hypothesis that polyamines bind inside the pore of cationic porins. J Biotechnol, 1999 Feb 5, 68(1), 29 - 35 Overexpression and simple purification of human superoxide dismutase (SOD1) in yeast and its resistance to oxidative stress; Yoo HY et al.; The structural gene of human Cu/Zn superoxide dismutase (hSOD1) was cloned into a yeast expression vector containing the promoter of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene . The recombinant plasmid produced hSOD1 (20 kDa), about 6% of the total cellular protein, and the expressed hSOD1 was enzymatically active . The hSOD1 was purified from the cultured yeast by ammonium sulfate-methanol extraction and DEAE-cellulose column chromatography . This relatively simple purification method produced a single band on analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . The amount of hSOD1 appeared to be considerably increased in cultures of higher cell density . The yeast overexpressing hSOD1 appeared to be more resistant to oxidative stresses such as paraquat, menadione and heat shock. Biochim Biophys Acta, 1999 Feb 24, 1453(2), 254 - 60 Characterisation of copper-binding to the second sub-domain of the Menkes protein ATPase (MNKr2); Harrison MD et al.; The Menkes ATPase (MNK) has an essential role in the translocation of copper across cellular membranes . In a complementary manner, the intracellular concentration of copper regulates the activity and cellular location of the ATPase through its six homologous amino-terminal domains . The roles of the six amino-terminal domains in the activation and cellular trafficking processes are unknown . Understanding the role of these domains relies on the development of an understanding of their metal-binding properties and structural properties . The second conserved sub-domain of MNK was over-expressed, purified and its copper-binding properties characterised . Reconstitution studies demonstrate that copper binds to MNKr2 as Cu(I) with a stoichiometry of one copper per domain . This is the first direct evidence of copper-binding to the MNK amino-terminal repeats . Circular dichroism studies suggest that the binding or loss of copper to MNKr2 does not cause substantial changes to the secondary structure of the protein. J Cell Sci, 1999 Mar, 112 ( Pt 6), 957 - 65 RhoA activity is required for fibronectin assembly and counteracts beta1B integrin inhibitory effect in FRT epithelial cells; Cali G et al.; FRT thyroid epithelial cells synthesize fibronectin and organize a network of fibronectin fibrils at the basal surface of the cells . Fibronectin fibril formation is enhanced by the overexpression of the ubiquitous beta1A integrin and is inhibited by the expression of the dominant-negative beta1B subunit . We tested the hypotheses that RhoA activity might mediate the integrin-dependent fibronectin fibrillogenesis and might counteract beta1B integrin inhibitory effect . FRT-beta1A cells were transfected with a vector carrying a dominant negative form of RhoA (RhoAN19) or treated with the C3 transferase exoenzyme . Both treatments inhibited fibronectin assembly and caused loss of actin microfilaments and adhesion plaques . On the other hand, FRT-beta1B cells were transfected with the constitutively activated form of RhoA (RhoAV14) or treated with the E . coli cytotoxic necrotizing factor 1, which directly activates RhoA . Either treatment restored microfilament and adhesion plaque assembly and promoted fibronectin fibril organization . A great increase in fibronectin fibril assembly was also obtained by treatment of FRT-beta1B cells with TGF-beta . Our data indicate that RhoA is required to promote fibronectin matrix assembly in FRT cells and that the activation of the signal transduction pathway downstream of RhoA can overcome the inhibitory effect of beta1B integrin. J Theor Biol, 1999 Mar 7, 197(1), 63 - 76 Deviations from Chargaff's second parity rule correlate with direction of transcription; Bell SJ et al.; The distribution of deviations from Chargaff's second parity rule was examined for overlapping sequence windows of a length (1 kb) predicted to be suitable for detecting correlations with functional features of DNA . For long genomic segments from E . coli, Saccharomyces cerevisiae, and Vaccinia virus, Chargaff differences for the W bases and/or for the S bases correlate with transcription direction and gene location . For W-rich genomes, the mRNA-synonymous strand contains regions which, if extruded from negatively supercoiled DNA, would fold to generate stem-loop structures with A-rich loops . Similarly, for S-rich genomes the loops would be G-rich . We suggest that the disposition of genes in nucleic acid sequences arises from their having to adapt to a preexisting mosaic of genomic regions, each distinguished by its potential to extrude single-strand loops enriched for a particular base (or two non-Watson-Crick pairing bases) . The mosaic would have facilitated the intrastrand and interstrand accounting required for correction of mutations, and would have evolved in the early RNA world before the emergence of protein-encoding capacity . The preexisting mosaic would have determined transcription direction since there is pressure for all mRNAs of a cell to have purine-rich loops, thus decreasing loop-loop interactions which might lead to formation of "self" sense-antisense RNA duplexes . J Theor Biol, 1999 Mar 7, 197(1), 51 - 61 Accounting units in DNA; Bell SJ et al.; Chargaff's first parity rule (%A=%T and %G=%C) is explained by the Watson-Crick model for duplex DNA in which complementary base pairs form individual accounting units . Chargaff's second parity rule is that the first rule also applies to single strands of DNA . The limits of accounting units in single strands were examined by moving windows of various sizes along sequences and counting the relative proportions of A and T (the W bases), and of C and G (the S bases) . Shuffled sequences account, on average, over shorter regions than the corresponding natural sequence . For an E . coli segment, S base accounting is, on average, contained within a region of 10 kb, whereas W base accounting requires regions in excess of 100 kb . Accounting requires the entire genome (190 kb) in the case of Vaccinia virus, which has an overall "Chargaff difference" of only 0.086% (i.e . only one in 1162 bases does not have a potential pairing partner in the same strand) . Among the chromosomes of Saccharomyces cerevisiae, the total Chargaff differences for the W bases and for the S bases are usually correlated . In general, Chargaff differences for a natural sequence and its shuffled counterpart diverge maximally when 1 kb sequence windows are employed . This should be the optimum window size for examining correlations between Chargaff differences and sequence features which have arisen through natural selection . We propose that Chargaff's second parity rule reflects the evolution of genome-wide stem-loop potential as part of short- and long-range accounting processes which work together to sustain the integrity of various levels of information in DNA . Anal Biochem, 1999 Feb 15, 267(2), 319 - 30 Cytochrome P450c17-expressing Escherichia coli as a first-step screening system for 17alpha-hydroxylase-C17,20-lyase inhibitors; Grigoryev DN et al.; We have designed and synthesized a number of cytochrome P450 17alpha-hydroxylase-C17,20-lyase (P450c17) inhibitors with the aim of inhibiting androgen synthesis . To select the most potent inhibitors, we initially used human testicular microsomes, which have a high level of expression of this enzyme . However, due to lack of availability of human tissue and variability among the samples, we utilized recombinant human enzyme expressed in Escherichia coli . We designed a simple and economical protocol based on the report that recombinant bovine P450c17 can be functionally active in live bacteria . In the assay we report here, we substituted high-performance liquid chromatography product isolation with a rapid biochemical acetic acid releasing assay and utilized intact P450c17-expressing E . coli for the source of the enzyme . Enzymatic parameters of the bacterial system (Km = 5.1 x 10(-7) M, Vmax = 15.0 pmol/min/mg) were similar to those of human testicular microsomes (Km = 4.8 x 10(-7) M, Vmax = 40.0 pmol/min/mg), and our compounds displayed a similar pattern of inhibition in both systems . This new system is a fast, reliable, and reproducible method for screening P450c17 inhibitors . Furthermore, it eliminates our dependence on human tissue and potential data fluctuations caused by variations in enzymatic activity between donors . Shock, 1999 Feb, 11(2), 120 - 6 Carnitine deprivation adversely affects cardiac performance in the lipopolysaccharide- and hypoxia/reoxygenation-stressed piglet heart; Penn D et al.; Sepsis and hypoxia are important stressors for the neonate . Newborn infants receiving total parenteral nutrition are routinely deprived of carnitine and develop low carnitine plasma and tissue levels . Because of its high metabolic rate and dependence on fatty acids for energy, the newborn heart may be particularly vulnerable to stress in the face of an inadequate carnitine supply . To investigate whether carnitine deprivation affects cardiac performance under stress, 23 neonatal piglets received parenteral nutrition for 2-3 weeks that was either carnitine free (CARN -) or supplemented (CARN +) with L-carnitine (400 mg/L) . Bacterial endotoxin (lipopolysaccharide (LPS), 250 microg/kg intravenous bolus) or saline vehicle was administered to anesthetized piglets 3 h prior to study of isolated perfused hearts . Left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure, and left ventricular developed pressure (LVDP) were measured in vitro under aerobic, hypoxic, and reoxygenation conditions in all animals . Plasma and tissue carnitine values were lower in CARN - than in CARN + piglets . In hearts from LPS-treated animals prior to hypoxia, there was no difference in ventricular compliance between CARN - and CARN + groups . LVSP and LVDP were lower in CARN - than CARN + hearts . During hypoxia, LVSP and LVDP fell, but left ventricular end diastolic pressure increased in hearts from both LPS- and saline- treated piglets . Reoxygenation led to poorer recovery in CARN - than CARN + hearts from LPS-treated animals, but not from saline controls . During hypoxia/reoxygenation, lactate efflux initially rose and then fell, while carnitine efflux increased continually . Acetyl- and medium-chain acylcarnitines were detected in the coronary effluent . Our findings suggest that carnitine deprivation diminishes heart carnitine concentrations and impairs cardiac recovery from combined endotoxic and hypoxic stress . Possible mechanisms include reduced acyl buffering and/or impaired transport of fatty acyl groups into mitochondria. Shock, 1999 Feb, 11(2), 115 - 9 Oral spermine administration inhibits nitric oxide-mediated intestinal damage and levels of systemic inflammatory mediators in a mouse endotoxin model; ter Steege JC et al.; Enhanced intestinal nitric oxide production observed during sepsis is thought to play a central role in lipopolysaccharide-induced intestinal damage . In contrast intestinal polyamines, both from endogenous and exogenous origin, are essential for the maintenance of mucosal integrity . Polyamines have been shown to inhibit lipopolysaccharide-induced nitric oxide release in vitro and have been claimed to exert additional antiinflammatory actions . In this study, the effect of the polyamine spermine on the release of the proinflammatory mediators nitric oxide and tumor necrosis factor-alpha by a murine macrophage cell line was investigated . Furthermore, we investigated whether oral spermine administration inhibits lipopolysaccharide-induced intestinal inducible nitric oxide synthase and nitrotyrosine expression and modulates the release of inflammatory mediators . Our results show that although spermine inhibited lipopolysaccharide-induced nitric oxide release in a murine macrophage cell line, no effect on tumor necrosis factor-alpha release was observed . In addition, oral spermine administration inhibited intestinal inducible nitric oxide synthase and nitrotyrosine expression suggesting a protective effect of spermine on lipopolysaccharide-induced intestinal damage . In parallel a decrease in serum levels of the proinflammatory mediators nitrate, nitrite, and interferon-gamma and an increase in the antiinflammatory cytokine interleukin-10 was observed, although tumor necrosis factor-alpha levels were unaffected . These results indicate that spermine inhibits lipopolysaccharide-induced nitric oxide release in vitro as well as in vivo . Further, intraluminally derived polyamines modulate the systemic immune response . It is concluded that oral spermine administration might have therapeutic perspectives for several disorders characterized by systemic inflammation and intestinal damage. Shock, 1999 Feb, 11(2), 93 - 7 Role of tumor necrosis factor and interferon gamma in endotoxin-induced E-selectin expression; Eppihimer MJ et al.; Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), potent inflammatory cytokines, are released by macrophages during endotoxin shock . However, the contribution of these cytokines to endotoxin-induced inflammation has not been defined . The expression of E-selectin, measured using the dual radiolabeled monoclonal antibody (mAb) technique, was monitored in different tissues of endotoxin-challenged wild-type and IFN-gamma-deficient mice receiving a mAb to TNF-alpha (TN3) . A significant elevation in E-selectin expression occurred in all tissues of wild-type mice challenged with endotoxin . Injection of TN3 in wild-type mice significantly attenuated the endotoxin-induced up-regulation of E-selectin in all tissues (p < .05) except the pancreas . The level of reduction in endotoxin-induced E-selectin expression ranged between 30% in the stomach to 60% in the small intestine . E-selectin expression in endotoxin-challenged, IFN-gamma-deficient mice was significantly reduced in the small and large intestines, when compared with endotoxin-challenged wild-type mice . Although IFN-gamma deficiency had no effect on E-selectin expression in other tissues, administration of TN3 to endotoxin-challenged, IFN-gamma-deficient mice significantly reduced E-selectin expression to levels observed in endotoxin-challenged, wild-type mice that received TN3 . These findings indicate that TNF-alpha is essential for achievement of maximal E-selectin expression in most vascular beds during endotoxemia, whereas the contribution of IFN-gamma is largely confined to the small intestine. Epidemiol Infect, 1998 Dec, 121(3), 599 - 608 P fimbriae and other adhesins enhance intestinal persistence of Escherichia coli in early infancy; Adlerberth I et al.; Resident and transient Escherichia coli strains were identified in the rectal flora of 22 Pakistani infants followed from birth to 6 months of age . All strains were tested for O-antigen expression, adhesin specificity (P fimbriae, other mannose-resistant adhesins or type 1 fimbriae) and adherence to the colonic cell line HT-29 . Resident strains displayed higher mannose-resistant adherence to HT-29 cells, and expressed P fimbriae (P = 0.0036) as well as other mannose-resistant adhesins (P = 0.012) more often than transient strains . In strains acquired during the first month of life, P fimbriae were 12 times more frequent in resident than in transient strains (P = 0.0006) . The O-antigen distribution did not differ between resident and transient strains, and none of the resident P-fimbriated strains belonged to previously recognized uropathogenic clones . The results suggest that adhesins mediating adherence to intestinal epithelial cells, especially P fimbriae, enhance the persistence of E . coli in the large intestine of infants. Vet J, 1999 Jan, 157(1), 69 - 77 Serological evidence of spirochaetal infections associated with digital dermatitis in dairy cattle; Demirkan I et al.; A potentially infectious aetiology for digital dermatitis in dairy cattle was investigated and centred on the possible involvement of spirochaetes . An enzyme-linked immunosorbent assay (ELISA) was developed to detect bovine anti-Borrelia burgdorferi (B31) and anti-Treponeme (USA bovine isolates) antibodies in the sera of cows; sera were further tested for antigen specificity by Western blotting . Compared to normal cows, those with digital dermatitis had a much higher seropositivity rate to B . burgdorferi and the treponemes . Significant correlations were shown between antibodies to B . burgdorferi and to Treponemes (P < 0.001), suggesting strong cross-reacting epitopes shared by these spirochaetes . In Western blotting of B . burgdorferi antigens, the main band detected by ELISA positive sera was the 41 kDa flagellar protein; lesser frequency of staining was seen with 34 (OspB), 39 and 55 kDa bands . For the USA treponeme antigens, ELISA positive sera gave reactions to the 34-kDa band and also bands at 41 and 55 kDa . Polyclonal antibodies to Treponema denticola and T . vincentii showed reactions with the bovine treponemes which were predominantly to the 34-kDa antigen . Monoclonal antibodies to B . burgdorferi flagella (41 kDa) antigen and OspA (31 kDa) did not detect any treponeme bands in Western blotting . The study has provided serological evidence that spirochaetes (which are related to human treponemes) may be involved in the pathogenesis of digital dermatitis. Can J Microbiol, 1998 Nov, 44(11), 1106 - 9 Isolation and characterization of an Escherichia coli B mutant strain defective in uracil catabolism; West TP; A reductive pathway of uracil catabolism was shown to be functioning in Escherichia coli B ATCC 11303 by virtue of thin-layer chromatographic and enzyme analyses . A mutant defective in uracil catabolism was isolated from this strain and subsequently characterized . The three enzyme activities associated with the reductive pathway of pyrimidine catabolism were detectable in the wild-type E . coli B cells, while the mutant strain was found to be deficient for dihydropyrimidine dehydrogenase activity . The dehydrogenase was shown to utilize NADPH as its nicotinamide cofactor . Growth of ATCC 11303 cells on uracil or glutamic acid instead of ammonium sulfate as a nitrogen source increased the reductive pathway enzyme activities . The mutant strain exhibited increased catabolic enzyme activities after growth on ammonium sulfate or glutamic acid. Mutat Res, 1999 Jan 25, 423(1-2), 73 - 7 Stabilization of the intermediate in frameshift mutation; Sagher D et al.; A mismatch repair, proofreading deficient mutant of Escherichia coli lost a C from a C8 run at a rate 10 times higher than the loss of A from an A8 sequence in the same double mutant . This greater frameshift instability of a homopolymeric run of C's may be due to stabilization of a stacked intermediate . Gain of a (CA) unit in a similarly constructed (CA)15 sequence occurred at a rate about 1/3 that previously reported for a (CA)14 construct losing a (CA) repeat unit . Biochemistry, 1999 Feb 23, 38(8), 2542 - 50 The conformations of a substrate and a product bound to the active site of S-adenosylmethionine synthetase; Schalk-Hihi C et al.; S-Adenosylmethionine (AdoMet) is the most widely used alkyl group donor in biological systems . The formation of AdoMet from ATP and L-methionine is catalyzed by S-adenosylmethionine synthetase (AdoMet synthetase) . Elucidation of the conformations of enzyme-bound substrates, product, and inhibitors is important for the understanding of the catalytic mechanism of the enzyme and the design of new inhibitors . To obtain structural data for enzyme-bound substrates and product, we have used two-dimensional transferred nuclear Overhauser effect spectroscopy to determine the conformation of enzyme-bound AdoMet and 5'-adenylyl imidodiphosphate (AMPPNP) . AMPPNP, an analogue of ATP, is resistant to the ATP hydrolysis activity of AdoMet synthetase because of the presence of a nonhydrolyzable NH-link between the beta- and gamma-phosphates but is a substrate for AdoMet formation during which tripolyphosphate is produced . AdoMet and AMPPNP both bind in an anti conformation about the glycosidic bond . The ribose rings are in C3'-exo and C4'-exo conformations in AdoMet and AMPPNP, respectively . The differences in ribose ring conformations presumably reflect the different steric requirements of the C5' substituents in AMPPNP and AdoMet . The NMR-determined conformations of AdoMet and AMPPNP were docked into the E . coli AdoMet synthetase active site taken from the enzyme.ADP . Pi crystal structure . Since there are no nonexchangeable protons either in the carboxy-terminal end of the methionine segment of AdoMet or in the tripolyphosphate segment of AMPPNP, these portions of the molecules were modeled into the enzyme active site . The interactions of AdoMet and AMPPNP with the enzyme predict the location of the methionine binding site and suggest how the positive charge formed on the sulfur during AdoMet synthesis is stabilized. Biochemistry, 1999 Feb 23, 38(8), 2435 - 43 Mutational analysis of amino acid residues involved in argininosuccinate lyase activity in duck delta II crystallin; Chakraborty AR et al.; Delta-crystallins are the major structural eye lens proteins of most birds and reptiles and are direct homologues of the urea cycle enzyme argininosuccinate lyase . There are two isoforms of delta-crystallin, delta Iota and delta IotaIota, but only delta IotaIota crystallin exhibits argininosuccinate lyase (ASL) activity . At the onset of this study, the structure of argininosuccinate lyase/delta IotaIota crystallin with bound inhibitor or substrate analogue was not available . Biochemical and X-ray crystallographic studies had suggested that H162 may function as the catalytic base in the argininosuccinate lyase/delta IotaIota crystallin reaction mechanism, either directly or indirectly through the activation of a water molecule . The identity of the catalytic acid was unknown . In this study, the argininosuccinate substrate was modeled into the active site of duck delta IotaIota crystallin, using the coordinates of an inhibitor-bound Escherichia coli fumarase C structure to orient the fumarate moiety of the substrate . The model served as a means of identifying active site residues which are positioned to potentially participate in substrate binding and/or catalysis . On the basis of the results of the modeling, site-directed mutagenesis was performed on several amino acids, and the kinetic and thermodynamic properties of each mutant were determined . Kinetic studies reveal that five residues, R115, N116, T161, S283, and E296, are essential for catalytic activity . Determination of the free energy of unfolding/refolding of wild-type and mutant delta II crystallins revealed that all constructs exhibit similar thermodynamic stabilities . During the course of this work, the structure of an inactive delta IotaIota crystallin mutant with bound substrate was solved {Vallee et al . (1999) Biochemistry 38, 2425-2434}, which has allowed the kinetic data to be interpreted on a structural basis. Biochemistry, 1999 Feb 23, 38(8), 2347 - 57 Carbamoyl phosphate synthetase: closure of the B-domain as a result of nucleotide binding; Thoden JB et al.; Carbamoyl phosphate synthetase (CPS) catalyzes the production of carbamoyl phosphate which is subsequently employed in the metabolic pathways responsible for the synthesis of pyrimidine nucleotides or arginine . The catalytic mechanism of the enzyme occurs through three highly reactive intermediates: carboxyphosphate, ammonia, and carbamate . As isolated from Escherichia coli, CPS is an alpha, beta-heterodimeric protein with its three active sites separated by nearly 100 A . In addition, there are separate binding sites for the allosteric regulators, ornithine, and UMP . Given the sizable distances between the three active sites and the allosteric-binding pockets, it has been postulated that domain movements play key roles for intramolecular communication . Here we describe the structure of CPS from E . coli where, indeed, such a domain movement has occurred in response to nucleotide binding . Specifically, the protein was crystallized in the presence of a nonhydrolyzable analogue, AMPPNP, and its structure determined to 2.1 A resolution by X-ray crystallographic analysis . The B-domain of the carbamoyl phosphate synthetic component of the large subunit closes down over the active-site pocket such that some atoms move by more than 7 A relative to that observed in the original structure . The trigger for this movement resides in the hydrogen-bonding interactions between two backbone amide groups (Gly 721 and Gly 722) and the beta- and gamma-phosphate groups of the nucleotide triphosphate . Gly 721 and Gly 722 are located in a Type III' reverse turn, and this type of secondary structural motif is also observed in D-alanine:D-alanine ligase and glutathione synthetase, both of which belong to the "ATP-grasp" superfamily of proteins . Details concerning the geometries of the two active sites contained within the large subunit of CPS are described. Biochemistry, 1999 Feb 23, 38(8), 2320 - 5 Tertiary contacts of helix V in the lactose permease determined by site-directed chemical cross-linking in situ; Wu J et al.; The six N-terminal transmembrane helices (N6) and the six C-terminal transmembrane helices (C6) in lactose permease, each containing a single Cys residue, were coexpressed, and cross-linking was studied . The proximity of paired Cys residues in helices V and VII, VIII, or X was studied by thiol-specific chemical cross-linking . The results demonstrate that Cys residues in the periplasmic half of helix V cross-link with Cys residues in the periplasmic half of helix VII . In contrast, no cross-linking is evident with paired Cys residues in the cytoplasmic halves of helices V and VII . Moreover, Cys residues on one entire face of helix V cross-link with Cys residues on one face of helix VIII . Finally, paired Cys residues at the cytoplasmic ends of helices V and X cross-link, but no cross-linking is observed when paired Cys residues are placed at the periplasmic ends of the two helices . Taken together, the results indicate that the periplasmic halves of helices V and VII are in close proximity and that the two helices tilt away from one another toward the cytoplasmic side of the membrane . Furthermore, helices V and VIII are in close proximity throughout their lengths and do not tilt appreciably with respect to one another, and helices V and X are in close proximity at the cytoplasmic but not at the periplasmic face of the membrane. Biochemistry, 1999 Feb 23, 38(8), 2259 - 71 Kinetic characterization of CheY phosphorylation reactions: comparison of P-CheA and small-molecule phosphodonors; Mayover TL et al.; In the chemotaxis system of Escherichia coli, phosphorylation of the CheY protein plays an important role in regulating the swimming pattern of the cell . In vitro, CheY can be phosphorylated either by phosphotransfer from phospho-CheA or by acquiring a phosphoryl group from any of a variety of small, high-energy phosphodonor molecules such as acetyl phosphate . Previous work explored the rapid kinetics of CheY phosphorylation by CheA . Here we extend that work and examine the kinetics of CheY phosphorylation by several small-molecule phosphodonors, including acetyl phosphate, benzoyl phosphate, carbamoyl phosphate, 2-methoxybenzoyl phosphate, and phosphoramidate . Our results indicate that these phosphodonors bind to CheY with relatively low affinity (Ks values ranging from 10 to 600 mM) and that the rate constant (kphos) for phosphotransfer at saturating phosphodonor concentrations is relatively slow (values ranging from 0.05 to 0.5 s-1) . By contrast, under identical conditions, phosphorylation of CheY by phospho-CheA occurs much more rapidly (kphos approximately 800 s-1) and reflects CheY binding to phospho-CheA considerably more tightly (Ks approximately 60 microM) than it does to the small-molecule phosphodonors . In comparing CheA-mediated phosphorylation of CheY to small-molecule-mediated phosphorylation of CheY, the large difference in kphos values suggests that phospho-CheA makes significant contributions to the catalysis of CheY phosphorylation . The effects of pH and ionic strength on CheY phosphorylation kinetics were also investigated . For CheA-->CheY phosphotransfer, increasing ionic strength resulted in increased Ks values while kphos was unaffected . For CheY phosphorylation by small-molecule phosphodonors, increasing ionic strength resulted in decreasing Ks values and increasing kphos values . The significance of these effects is discussed in relation to the catalytic mechanism of CheY phosphorylation by phospho-CheA and small-molecule phosphodonors. Mol Biochem Parasitol, 1999 Jan 5, 98(1), 43 - 51 Characterisation and expression of the carbamate kinase gene from Giardia intestinalis; Minotto L et al.; The arginine dihydrolase pathway in Giardia intestinalis produces energy via the carbamate kinase (CBK, ATP:carbamate phosphotransferase, EC 2.7.2.2) reaction . Characterisation of the CBK gene from the Portland 1 strain indicated that it is located on either chromosome 3 or 4, does not appear to contain introns and is expressed in both the trophozoite and early cyst stages . Heterologous expression of CBK in Escherichia coli, using the pQE-30 expression system (QIAGEN), enabled a one-step purification of the recombinant enzyme via affinity chromatography . The expressed protein was identified by enzyme assay and mass spectrometry . The native and recombinant forms of the enzyme have similar physical properties and the recombinant enzyme appears to be active as the homodimer. Mol Biochem Parasitol, 1999 Jan 5, 98(1), 29 - 41 Purification and characterization of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase and comparison with the human enzyme; Keough DT et al.; The human malaria parasite Plasmodium falciparum is auxotrophic for purines and relies on the purine salvage pathway for the synthesis of its purine nucleotides . Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) is a key purine salvage enzyme in P . falciparum, making it a potential target for chemotherapy . Previous attempts to purify this enzyme have been unsuccessful because of the difficulty in obtaining cultured parasite material and because of the inherent instability of the enzyme during purification and storage . Other groups have tried to express recombinant P . falciparum HGXPRT but only small amounts of activity were obtained . The successful expression of recombinant P . falciparum HGXPRT in Escherichia coli has now been achieved and the enzyme purified to homogeneity in mg quantities . The measured molecular mass of 26 229+/-2 Da is in excellent agreement with the calculated value of 26232 Da . A method to stabilise the activity and to reactivate inactive samples has been developed . The subunit structure of P . Jilciparum HGXPRT has been determined by ultracentrifugation in the absence (tetramer) and presence (dimer) of KC1 . Kinetic constants were determined for 5-phospho-alpha-D-ribosyl-1-pyrophosphate, for the three naturally-occurring 6-oxopurine bases guanine, hypoxanthine, and xanthine and for the base analogue, allopurinol . Differences in specificity between the purified P . falciparum HGXPRT and human hypoxanthine guanine phosphoribosyltransferase enzymes were detected which may be able to be exploited in rational drug design. Mol Biochem Parasitol, 1999 Jan 5, 98(1), 17 - 28 Molecular cloning of p67, a lysosomal membrane glycoprotein from Trypanosoma brucei; Kelley RJ et al.; We have previously characterized a highly glycosylated membrane protein (p67) in Trypanosoma brucei spp that is apparently targeted to lysosomes in a developmentally regulated manner . Antibody to native p67 identified a partial cDNA clone from a T . b . rhodesiense expression library and RT-PCR was used to complete the sequence of the cDNA . Equal levels of p67 transcript are detected in both procyclic and bloodstream stages of the life cycle . The 2771 nt cDNA contains a 1980 nt orf encoding a 659 amino acid polypeptide (72,567 Da) . Hydropathy analysis predicts a Type I membrane topology (N to C): an N-terminal signal sequence, a large hydrophilic lumenal domain with 14 N-glycosylation sites, a trans-membrane domain (19 aa), and a short (24 aa) cytoplasmic domain . Peptide microsequencing of purified p67 identified nine residues identical to the deduced amino acid sequence, confirming the identity of the cDNA and defining the signal sequence cleavage site . Antibody to p67 protein produced in E . coli recognizes the same spectrum of native p67 glycoforms as the antibody used to clone the cDNA . All features of the deduced amino acid sequence are consistent with the known properties of the native protein and suggest a structure similar to mammalian LAMPS . The cytoplasmic domain contains two putative di-leucine targeting motifs similar to those involved in lysosomal targeting in vertebrate cells . Our results suggest that a single p67 polypeptide, or a group of highly related polypeptides, is synthesized in both bloodstream and procyclic trypanosomes and that subsequent post-translational processing and lysosomal targeting is subject to stage-specific regulation. J Trauma, 1999 Feb, 46(2), 280 - 5 The effect of hypoxia/reoxygenation on the cellular function of intestinal epithelial cells; Xu DZ et al.; BACKGROUND: Previously, using in vivo models, we have demonstrated that ischemia/reperfusion can increase intestinal mucosal permeability, promote bacterial translocation, and induce gut cytokine production . Because of the cellular heterogeneity of the gut, however, studies investigating the direct effects of hypoxia/reoxygenation on intestinal epithelial cells are confounded in in vivo model systems . Consequently, this study examines oxidant-mediated enterocyte injury using an in vitro intestinal enterocyte hypoxia/reoxygenation model system . METHODS: Two intestinal epithelial cell lines, IEC-6 and Caco-2, were seeded onto 3-microm filters in a Transwell bicameral system and grown until tight junction integrity was established . Cells were subjected to hypoxia in a sealed chamber with 95% nitrogen and 5% carbon dioxide and incubated at 37 degrees C for 60 or 90 minutes . Reoxygenation was initiated by replacing the media and putting the cells in an environment of room air plus 5% carbon dioxide . Permeability and bacterial translocation were assayed by measuring the phenol red concentration and culturing the bacteria that crossed the cell monolayer and reached the basal chamber of the bicameral system . Monolayer tight junction integrity was monitored by serial measurements of transepithelial electrical resistance (TEER), and cell viability was assessed by trypan blue dye . RESULTS: IEC-6 cell monolayers subjected to 60 or 90 minutes of hypoxia showed significantly higher permeability to phenol red, with 54+/-5% and 57+/-5% of the dye crossing the monolayers, respectively, compared with normoxic control (38+/-2%; p < 0.01) . Caco-2 cell monolayers also had increased permeability to phenol red, with 24+/-6% and 20+/-4% of the phenol red crossing the monolayer after 60 or 90 minutes of hypoxia, respectively, compared with 8+/-3% in the normoxic controls (p < 0.01) . At 3 hours after challenge with Escherichia coli, the monolayers subjected to 60 or 90 minutes of hypoxia had significantly increased bacterial translocation (IEC-6 cells, p < 0.05; Caco-2 cells, p < 0.01) compared with controls . The increased permeability of the hypoxic Caco-2 and IEC-6 monolayers was associated with a decrease in TEER beginning as early as 1 hour after reoxygenation (p < 0.01) . Cell viability, however, was not decreased . CONCLUSION: These results indicate that hypoxia/reoxygenation can directly impair cellular function as manifested by increased monolayer permeability to phenol red, increased E . coli bacterial translocation, and a decrease in TEER values. J AOAC Int, 1999 Jan-Feb, 82(1), 73 - 8 Dry rehydratable film method for enumerating confirmed Escherichia coli in poultry, meats, and seafood: collaborative study; Gangar V et al.; A rehydratable dry-film plating method for Escherichia coli, the Petrifilm E . coli/Coliform (EC) Count Plate in foods, has been compared with the AOAC INTERNATIONAL most probable number (MPN) method . Eleven laboratories participated in the collaborative study . Three E . coli levels in 8 samples each of frozen raw ground turkey, frozen raw ground beef, and frozen cooked fish were tested in duplicate . Mean log counts for the Petrifilm plate procedure were not significantly different from those for the MPN procedure for cooked fish samples inoculated with low or high inocula levels, for samples of raw turkey inoculated at medium level, and for beef inoculated at low, medium, and high levels . Repeatability and reproducibility variances of the Petrifilm EC Plate method recorded at 24 h were as good as or better than those of the MPN method . The dry rehydratable film method for enumerating confirmed E . coli in poultry, meats, and seafood has been adopted first action by AOAC INTERNATIONAL. Mol Microbiol, 1999 Jan, 31(2), 725 - 37 Localization of FtsL to the Escherichia coli septal ring; Ghigo JM et al.; In Escherichia coli, nine gene products are known to be essential for assembly of the division septum . One of these, FtsL, is a bitopic membrane protein whose precise function is not understood . Here we use fluorescence microscopy to study the subcellular localization of FtsL, both in a wild-type strain and in a merodiploid strain that expresses a GFP-FtsL fusion protein . We show that FtsL localizes to the cell septum where it forms a ring analogous to the cytoplasmic FtsZ ring . FtsL localization is dependent upon the function of FtsZ, FtsA and FtsQ, but not FtsI . In a reverse approach, we use fusions of green fluorescent protein (GFP) to FtsZ, FtsA and ZipA to show that these proteins localize to the division site in an FtsL-independent fashion . We propose that FtsL is a relatively late recruit to the ring structure that mediates septation. Mol Microbiol, 1999 Jan, 31(2), 641 - 50 The DNA binding protein Tfx from Methanobacterium thermoautotrophicum: structure, DNA binding properties and transcriptional regulation; Hochheimer A et al.; In Methanobacterium thermoautotrophicum, the fmdECB operon encoding the molybdenum formyl-methanofuran dehydrogenase is directly preceded by an open reading frame tfx predicted to encode a DNA binding protein . The 16.1 kDa protein has an N-terminal basic domain with a helix-turn-helix motif for DNA binding and a C-terminal acidic domain possibly for transcriptional activation . We report here on the DNA binding properties of the Tfx protein heterologously overproduced in Escherichia coli . Tfx was found to bind specifically to a DNA sequence downstream of the promoter of the fmdECB operon, as shown by electrophoretic mobility shift assays and DNase I footprint analysis . Northern blot hybridizations revealed that transcription of tfx is repressed during the growth of M . thermoautotrophicum in the presence of tung-state . Based on its structure and properties, the DNA binding protein Tfx is proposed to be a transcriptional regulator composed of a basic DNA binding domain and an acidic activation domain. Mol Microbiol, 1999 Jan, 31(2), 579 - 83 The cytoplasmic domain of FtsK protein is required for resolution of chromosome dimers; Steiner W et al.; Chromosome dimers, formed by homologous recombination between sister chromosomes, normally require cell division to be resolved into monomers by site-specific recombination at the dif locus of Escherichia coli . We report here that it is not in fact cell division per se that is required for dimer resolution but the action of the cytoplasmic domain of FtsK, which is a bifunctional protein required both for cell division and for chromosome partition. Mol Microbiol, 1999 Jan, 31(2), 451 - 61 Role of HU and DNA supercoiling in transcription repression: specialized nucleoprotein repression complex at gal promoters in Escherichia coli; Lewis DE et al.; Efficient repression of the two promoters P1 and P2 of the gal operon requires the formation of a DNA loop encompassing the promoters . In vitro, DNA looping-mediated repression involves binding of the Gal repressor (GalR) to two gal operators (OE and OI) and binding of the histone-like protein HU to a specific locus (hbs) about the midpoint between OE and OI, and supercoiled DNA . Without DNA looping, GalR binding to OE partially represses P1 and stimulates P2 . We investigated the requirement for DNA supercoiling and HU in repression of the gal promoters in vivo in strains containing a fusion of a reporter gene, gusA or lacZ, to each promoter individually . While the P1 promoter was found to be repressible in the absence of DNA supercoiling and HU, the repression of P2 was entirely dependent upon DNA supercoiling in vivo . The P2 promoter was fully derepressed when supercoiling was inhibited by the addition of coumermycin in cells . P2, but not P1, was also totally derepressed by the absence of HU or the OI operator . From these results, we propose that the repression of the gal promoters in vivo is mediated by the formation of a higher order DNA-multiprotein complex containing GalR, HU and supercoiled DNA . In the absence of this complex, P1 but not P2 is still repressed by GalR binding to OE . The specific nucleoprotein complexes involving histone-like proteins, which repress promoter activity while remaining sensitive to inducing signals, as discussed, may occur more generally in bacterial nucleoids. Mol Microbiol, 1999 Jan, 31(2), 443 - 50 Molecular genetic analysis of enoyl-acyl carrier protein reductase inhibition by diazaborine; de Boer GJ et al.; Diazaborine and isoniazid are, at first sight, unrelated anti-bacterial agents that inhibit the enoyl-ACP reductase (ENR) of Escherichia coli and Mycobacterium tuberculosis respectively . The crystal structures of these enzymes including that of the diazaborine-inhibited E . coli ENR have been obtained at high resolution . Site-directed mutagenesis was used to study the importance of amino acid residues in diazaborine susceptibility and enzyme function . The results show that drug binding and inhibition require the presence of a glycine residue at position 93 of E . coli ENR or at the structurally equivalent position in the plant homologue, which is naturally resistant to the drug . The data confirm the hypothesis that any amino acid side-chain other than hydrogen at this position within the three-dimensional structure of these enzymes will affect diazaborine resistance by encroaching into the drug binding site . Substitutions of Gly-93 by amino acids with small side-chains, such as serine, alanine, cysteine and valine, hardly affected the catalytic parameters and rendered the bacterial host resistant to the drug . Larger amino acid side-chains, such as that of arginine, histidine, lysine and glutamine, completely inactivated the activity of the enzyme. Chem Res Toxicol, 1999 Feb, 12(2), 144 - 50 Sequence context modulation of translesion synthesis at a single N-2-acetylaminofluorene adduct located within a mutation hot spot; Burnouf DY et al.; Oligonucleotides containing a single N-(deoxyguanosin-8-yl)acetylaminofluorene lesion (dGuo-C8-AAF) at each guanine residue of the sequence (5'-G1G2G3) have been used as templates for in vitro primer extension reactions by several DNA polymerases {Escherichia coli DNA polymerase III holoenzyme, its alpha subunit, DNA polymerase I Klenow fragment proficient (exo+) or deficient (exo-) in its 3' --> 5' exonuclease activity, and Sequenase} . The dGuo-C8-AAF lesion appears to be a strong block for all DNA polymerases: exo+ DNA polymerases stop one nucleotide before encountering the lesion, while partial incorporation opposite the lesion is observed only with enzymes devoid of the exonuclease activity . The efficiency of incorporation across from the adduct depends on both the DNA polymerase and the position of the lesion . When polymerase I Klenow fragment exo- is used, translesion synthesis (TLS) is observed with efficiencies varying according to the position of the adduct (G2 > G1 > G3) . Sequencing of the TLS products shows that error-free TLS is observed only when the AAF lesion is bound to G1, while all TLS events occurring at G2- or G3-AAF adducts are mutagenic . The major mutational event is a G deletion (27, 76, and 55% of the events for G1, G2, and G3, respectively), while two-G deletions occur to a lesser extent (17-30%) . These results are discussed in view of the slippage model developed for frameshift mutagenesis occurring during translesion synthesis at replication blocking lesions. Life Sci, 1999, 64(1), 37 - 43 Role of the solitary tract nucleus and caudal ventrolateral medulla in temperature responses in endotoxemic rats; Koulchitsky SV et al.; In experiments on conscious rats it was found that preliminary microinjection of 100 nl 100 microM glutamic acid to the rostral commissural part of the solitary tract nucleus or to the caudal ventrolateral medulla increased a rise in colonic temperature induced by systemically applied endotoxin (3 microg/kg Escherichia coli lipopolysaccharide, i.p.) as compared to animals with intrabulbar injection of vehicle (control group) . Preliminary microinjection of glutamate to the caudal commissural part of the solitary tract nucleus levelled the endotoxin-induced temperature response . After glutamate treatment of the caudal ventrolateral medulla there was a significant decrease in the noradrenaline content and decrease in the adrenaline level in the caudal (not significant) and rostral ventrolateral medulla (significant), as well as a small rise in noradrenergic activity at the solitary tract nucleus as compared to control animals . The post-mortem measurement of the optical density of brainstem tissues revealed its significant attenuation at the solitary tract nucleus and caudal ventrolateral medulla after glutamate as compared with these structures after vehicle . The involvement of monoaminergic systems of both structures under study in the initiation and control of temperature responses during endotoxemia is suggested. Neuron, 1999 Jan, 22(1), 43 - 52 even-skipped determines the dorsal growth of motor axons in Drosophila; Landgraf M et al.; Axon pathfinding and target choice are governed by cell type-specific responses to external cues . Here, we show that in the Drosophila embryo, motorneurons with targets in the dorsal muscle field express the homeobox gene even-skipped and that this expression is necessary and sufficient to direct motor axons into the dorsal muscle field . Previously, it was shown that motorneurons projecting to ventral targets express the LIM homeobox gene islet, which is sufficient to direct axons to the ventral muscle field . Thus, even-skipped complements the function of islet, and together these two genes constitute a bimodal switch regulating axonal growth and directing motor axons to ventral or to dorsal regions of the muscle field. Infection, 1999 Jan-Feb, 27(1), 12 - 5 Thrombotic microangiopathies and HIV infection: report of two typical cases, features of HUS and TTP, and review of the literature; Sutor GC et al.; Haemolytic uraemic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) are thrombotic microangiopathies increasingly reported in patients with HIV infection . However, characteristic features of thrombotic microangiopathies associated with HIV disease have not been defined yet . The typical courses of HUS and TTP in two patients are presented . The data as well as the analysis of cases published in the literature demonstrate the association of thrombotic microangiopathies with late-stage HIV disease . Moreover, differences between HUS and TTP can be detected . Patients with HUS present with more severe immunologic deterioration . Although clinical symptoms are fewer, HUS implicates a very poor prognosis . Life expectancy rarely exceeded 1 year after diagnosis . HUS and TTP should therefore be added to the international AIDS classification. Biochemistry, 1999 Feb 16, 38(7), 2048 - 56 Electron transfer induces side-chain conformational changes of glutamate-286 from cytochrome bo3; Lubben M et al.; Heme-copper oxidases have two putative proton channels, the so-called K-channel and the membrane-spanning D-channel . The latter contains a number of polar groups with glutamate-286 located in its center, which could-together with bound water-contribute to a transmembrane hydrogen-bonded network . Protonation states of carboxyl groups from cytochrome bo3 of Escherichia coli were studied by redox Fourier transform infrared (FTIR) difference spectroscopy . A net absorbance increase in the carboxyl region was observed upon reduction . The band signature typically found in heme-copper oxidases comprises an absorbance decrease (reduced-minus-oxidized difference spectra) at 1745 cm-1 and increase at 1735 cm-1 . No significant changes in the carboxyl region were found in the site-specific mutants D135E and D407N . The difference bands were lacking in redox spectra of mutants at position 286; they could clearly be related to Glu-286 . In wild-type oxidase, the pK of Glu-286 appears to be higher than 9.8 . Upon solvent isotope exchange from H2O to D2O, the band at 1745 cm-1 shifts more readily than the one at 1735 cm-1, indicating dissimilar accessibility of the carboxyl side chain to the hydrogen-bonded network in both redox states . The data are consistent with a redox-triggered conformational change of Glu-286, which attributes to the carboxyl group an orientation toward the interior of the D-channel for the oxidized form . The change of Glu-286 is retained in cyanide complexes of cytochrome bo3 and of cytochrome c oxidase; therefore it should be related to oxidoreduction of the heme b and/or CuB metal centers. Biochemistry, 1999 Feb 9, 38(6), 1850 - 6 The Kdp-ATPase of