Escherichia coli

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 Escherichia coli mediates an ATP-dependent, K+-independent electrogenic partial reaction; Fendler K et al.; Charge transport by the K+ transporting Kdp-ATPase from Escherichia coli was investigated using planar lipid membranes to which liposomes reconstituted with the enzyme were adsorbed . To study reactions in the absence of K+, given some contamination of solutions with K+, we used a mutant of Kdp whose affinity for K+ was 6 mM instead of the wild-type whose affinity is 2 microM . Upon rapid release of ATP from caged ATP, a transient current occurred in the absence of K+ . In the presence of K+, a stationary current was seen . On the basis of their structural similarity, we propose a kinetic model for the Kdp-ATPase analogous to that of the Na+K+-ATPase . In this model, the first, K+-independent step is electrogenic and corresponds to the outward transport of a negative charge . The second, K+-translocating step is probably also electrogenic and corresponds to transport of positive charge to the intracellular side of the protein.

Biochemistry, 1999 Feb 9, 38(6), 1838 - 49
Identification of microtubule binding sites in the Ncd tail domain; Karabay A et al.; Nonclaret disjunctional (Ncd) is a minus end-directed, C-terminal motor protein that is required for spindle assembly and maintenance during meiosis and early mitosis in Drosophila oocytes and early embryos . Ncd has an ATP-independent MT binding site in the N-terminal tail domain, and an ATP-dependent MT binding site in the C-terminal motor domain . The ability of Ncd to cross-link MTs through the action of these binding sites may be important for Ncd function in vivo . To identify the region(s) responsible for ATP-independent MT interactions of Ncd, 12 cDNAs coding various regions of Ncd tail domain were expressed in E . coli as C-terminal fusions to thioredoxin (Trx) . Ncd tail fusion proteins (TrxNT) were purified by ion exchange (S-Sepharose) and/or Talon metal affinity chromatography . Purified TrxNT and NT proteins were analyzed in microtubule (MT) cosedimentation and bundling assays to identify which tail proteins were able to bind and bundle MTs . Based on the results of these experiments, all TrxNT and NT proteins that showed MT binding activity also bundled MTs, and there are two ATP-independent MT interaction sites in the tail region: one within amino acids 83-100 that exhibits conformation-independent, high-affinity MT binding activity; and another within amino acids 115-187 that exhibits conformation-dependent, lower affinity MT binding activity . It is possible that both of these MT interacting sites combine in the native protein to form a single MT binding site that allows the Ncd tail to bind cargo MTs in vivo.

Biochemistry, 1999 Feb 9, 38(6), 1715 - 20
Site-directed chemical cross-linking demonstrates that helix IV is close to helices VII and XI in the lactose permease; Wu J et al.; The N-terminal six transmenbrane helices (N6) and the C-terminal six transmembrane helices (C6) of the lactose permease, each containing a single-Cys residue, were coexpressed, and proximity was studied . Paired Cys residues in helices IV (positions 114, 116, 119, 122, 125, or 129) and VII (227, 231, 232, 234, 235, 238, 239, 242, 243, 245, or 246) or XI (350, 353, 354, 357, 361, or 364) were tested for cross-linking in the presence of two rigid homobifunctional thiol-specific cross-linkers, N,N'-o-phenylenedimaleimide (o-PDM; 6 A) and N,N'-p-phenylenedimaleimide (p-PDM; 10 A) . Cys residues in the middle of helix IV (position 119 or 122) cross-link to Cys residues in the middle of helix VII (position 238, 239, 242, or 243) . In contrast, no cross-linking is evident with paired Cys residues at either end of helix IV (position 114, 116, 125, or 129) or helix VII (position 227, 231, 232, 234, 235, 245, or 246) . On the other hand, Cys residues in the cytoplasmic half of helix IV (position 125 or 129) cross-link with Cys residues in the cytoplasmic half of helix XI (position 350, 353, or 354), while paired Cys residues at the periplasmic ends of the two helices do not cross-link . The results indicate that helices IV and VII cross in a scissors-like manner with the cytoplasmic end of helix IV tilting toward helix XI.

Biochemistry, 1999 Feb 9, 38(6), 1705 - 14
Mapping RNA-protein interactions in ribonuclease P from Escherichia coli using electron paramagnetic resonance spectroscopy; Gopalan V et al.; Ribonuclease P (RNase P) is a catalytic ribonucleoprotein (RNP) essential for tRNA biosynthesis . In Escherichia coli, this RNP complex is composed of a catalytic RNA subunit, M1 RNA, and a protein cofactor, C5 protein . Using the sulfhydryl-specific reagent (1-oxyl-2,2,5, 5-tetramethyl-Delta3-pyrroline-3-methyl)methanethiosulfonate (MTSL), we have introduced a nitroxide spin label individually at six genetically engineered cysteine residues (i.e., positions 16, 21, 44, 54, 66, and 106) and the native cysteine residue (i.e., position 113) in C5 protein . The spin label covalently attached to any protein is sensitive to structural changes in its microenvironment . Therefore, we expected that if the spin label introduced at a particular position in C5 protein was present at the RNA-protein interface, the electron paramagnetic resonance (EPR) spectrum of the spin label would be altered upon binding of the spin-labeled C5 protein to M1 RNA . The EPR spectra observed with the various MTSL-modified mutant derivatives of C5 protein indicate that the spin label attached to the protein at positions 16, 44, 54, 66, and 113 is immobilized to varying degrees upon addition of M1 RNA but not in the presence of a catalytically inactive, deletion derivative of M1 RNA . In contrast, the spin label attached to position 21 displays an increased mobility upon binding to M1 RNA . The results from this EPR spectroscopy-based approach together with those from earlier studies identify residues in C5 protein which are proximal to M1 RNA in the RNase P holoenzyme complex.

Biochemistry, 1999 Feb 9, 38(6), 1676 - 84
Purification and reconstitution of an osmosensor: transporter ProP of Escherichia coli senses and responds to osmotic shifts; Racher KI et al.; The ProP protein of Escherichia coli is an osmoregulatory H+-compatible solute cotransporter . ProP is activated by an osmotic upshift in both whole cells and membrane vesicles . We are using biochemical and biophysical techniques to explore the osmosensory and catalytic mechanisms of ProP . We now report the purification and reconstitution of the active transporter . Protein purification was facilitated by the addition of six histidine (His) codons to the 3' end of proP . The recombinant gene was overexpressed from the E . coli galP promoter, and ProP-(His)6 was shown to be functionally equivalent to wild-type ProP by enzymatic assay of whole cells . ProP-(His)6, purified by Ni2+ (NTA) affinity chromatography, cross-reacted with antibodies raised against the ProP protein . ProP-(His)6 was reconstituted into Triton X-100 destabilized liposomes prepared with E . coli phospholipid . The reconstituted transporter mediated proline accumulation only if (1) a membrane potential was generated by valinomycin-mediated K+ efflux and (2) the proteoliposomes were subjected to an osmotic upshift (0.6 M sucrose) . Activity was also stimulated by DeltapH . Pure ProP acts, in the proteoliposome environment, as sensor, transducer, and respondent to a hyperosmotic shift . It is the first such osmosensor to be isolated.

J Cell Biochem, 1999 Jan 1, 72(1), 145 - 50
Kinetic comparison of the catalytic domains of SHP-1 and SHP-2; Niu T et al.; The phosphatase activity of SH2-containing protein tyrosine phosphatase (SHP) is inhibited by its SH2 domains and C-terminal tail . In order to determine the inhibitory effects of the SH2 domains and C-terminal tail, we have expressed and purified the catalytic domains of SHP-1 and SHP-2, and the SH2 domain truncated SHP-1 and SHP-2 . We have then measured their kinetic parameters using p-nitrophenyl phosphate (p-NPP) and phosphotyrosine (pY) as substrates under the same experimental conditions . The results indicate that the pH-dependent profiles of SHP-1 and SHP-2 are mainly determined by their catalytic domains . Both enzymes have maximum activity at pH 5.0 . In addition, the phosphatase activity of different forms of SHP-1 and SHP-2 decreases as the salt concentration increases . Without SH2 domains, both SHP-1 and SHP-2 are no longer inhibited by their C-terminal tails . However, the C-terminal tail of SHP-1 can further prevent the salt inhibition of the phosphatase activity . Under the same experimental conditions, the catalytic domain of SHP-1 is two times more active than the catalytic domain of SHP-2.

J Histochem Cytochem, 1999 Mar, 47(3), 373 - 82
Binding and selective detection of the secretory N-terminal domain of the alzheimer amyloid precursor protein on cell surfaces; Hoffmann J et al.; The secretory N-terminal domain of the Alzheimer amyloid precursor protein (sAPP) evokes specific responses in cells on binding to their surfaces . Because APP is expressed in a large variety of cell types, the localization of sAPP binding requires detection techniques that selectively recognize sAPP as a ligand . For this purpose, we prepared antibodies against recombinant sAPP695 (sAPPrec) previously expressed in E . coli . Such antibodies were found to distinguish between sAPPrec and cellular APP or sAPP, as shown by immunocytochemistry and by immunoblot . In addition, they allowed the selective localization of bound sAPPrec on cell surfaces without any signal from cellular APP or sAPP . Saturation of sAPPrec binding to cell surfaces, as determined radiometrically, was reached at 10 nM {125I}-sAPPrec . Binding was specific because it was almost completely inhibited by a 100-fold excess of unlabeled sAPPrec . This specificity of binding was confirmed by surface plasmon resonance spectroscopy . Binding of sAPPrec to cell surfaces occurred in patches and was dependent on the state of cell differentiation . The sAPPrec used in this study contains heparin binding sites, but enzymatic removal of cell surface associated heparin did not affect sAPPrec binding . Aldehyde fixation of cells strongly inhibited their ability to bind sAPPrec . The data point to a fixation-sensitive sAPPrec binding protein which is detectable in the form of patches and therefore is part of assembled cell surface microdomains.

J Biol Chem, 1999 Feb 26, 274(9), 5888 - 94
Human geranylgeranyl diphosphate synthase . cDNA cloning and expression; Kuzuguchi T et al.; Geranylgeranyl diphosphate (GGPP) synthase (GGPPSase) catalyzes the synthesis of GGPP, which is an important molecule responsible for the C20-prenylated protein biosynthesis and for the regulation of a nuclear hormone receptor (LXR.RXR) . The human GGPPSase cDNA encodes a protein of 300 amino acids which shows 16% sequence identity with the known human farnesyl diphosphate (FPP) synthase (FPPSase) . The GGPPSase expressed in Escherichia coli catalyzes the GGPP formation (240 nmol/min/mg) from FPP and isopentenyl diphosphate . The human GGPPSase behaves as an oligomeric molecule with 280 kDa on a gel filtration column and cross-reacts with an antibody directed against bovine brain GGPPSase, which differs immunochemically from bovine brain FPPSase . Northern blot analysis indicates the presence of two forms of the mRNA.

J Biol Chem, 1999 Feb 26, 274(9), 5407 - 14
A family of S-methylmethionine-dependent thiol/selenol methyltransferases . Role in selenium tolerance and evolutionary relation; Neuhierl B et al.; Several plant species can tolerate high concentrations of selenium in the environment, and they accumulate organoselenium compounds . One of these compounds is Se-methylselenocysteine, synthesized by a number of species from the genus Astragalus (Fabaceae), like A . bisulcatus . An enzyme has been previously isolated from this organism that catalyzes methyl transfer from S-adenosylmethionine to selenocysteine . To elucidate the role of the enzyme in selenium tolerance, the cDNA coding for selenocysteine methyltransferase from A . bisulcatus was cloned and sequenced . Data base searches revealed the existence of several apparent homologs of hitherto unassigned function . The gene for one of them, yagD from Escherichia coli, was cloned, and the protein was overproduced and purified . A functional analysis showed that the YagD protein catalyzes methylation of homocysteine, selenohomocysteine, and selenocysteine with S-adenosylmethionine and S-methylmethionine as methyl group donors . S-Methylmethionine was now shown to be also the physiological methyl group donor for the A . bisulcatus selenocysteine methyltransferase . A model system was set up in E . coli which demonstrated that expression of the plant and, although to a much lesser degree, of the bacterial methyltransferase gene increases selenium tolerance and strongly reduces unspecific selenium incorporation into proteins, provided that S-methylmethionine is present in the medium . It is postulated that the selenocysteine methyltransferase under selective pressure developed from an S-methylmethionine-dependent thiol/selenol methyltransferase.

J Biol Chem, 1999 Feb 26, 274(9), 5357 - 61
The contribution of adjacent subunits to the active sites of D-3-phosphoglycerate dehydrogenase; Grant GA et al.; D-3-Phosphoglycerate dehydrogenase (PGDH) from Escherichia coli is allosterically inhibited by L-serine, the end product of its metabolic pathway . Previous results have shown that inhibition by serine has a large effect on Vmax and only a small or negligible effect on Km . PGDH is thus classified as a V-type allosteric enzyme . In this study, the active site of PGDH has been studied by site-directed mutagenesis to assess the role of certain residues in substrate binding and catalysis . These consist of a group of cationic residues (Arg-240, Arg-60, Arg-62, Lys-39, and Lys-141') that potentially form an electrostatic environment for the binding of the negatively charged substrate, as well as the only tryptophan residue found in PGDH and which fits into a hydrophobic pocket immediately adjacent to the active site histidine residue . Interestingly, Trp-139' and Lys-141' are part of the polypeptide chain of the subunit that is adjacent to the active site . The results of mutating these residues show that Arg-240, Arg-60, Arg-62, and Lys-141' play distinct roles in the binding of the substrate to the active site . Mutants of Trp-139' show that this residue may play a role in stabilizing the catalytic center of the enzyme . Furthermore, these mutants appear to have a significant effect on the cooperativity of serine inhibition and suggest a possible role for Trp-139' in the cooperative interactions between subunits.

Biol Reprod, 1999 Mar, 60(3), 769 - 75
X inactive-specific transcript (Xist) expression and X chromosome inactivation in the preattachment bovine embryo; De La Fuente R et al.; Expression of the X inactive-specific transcript (Xist) is thought to be essential for the initiation of X chromosome inactivation and dosage compensation during female embryo development . In the present study, we analyzed the patterns of Xist transcription and the onset of X chromosome inactivation in bovine preattachment embryos . Reverse transcription-polymerase chain reaction (RT-PCR) revealed the presence of Xist transcripts in all adult female somatic tissues evaluated . In contrast, among the male tissues examined, Xist expression was detected only in testis . No evidence for Xist transcription was observed after a single round of RT-PCR from pools of in vitro-derived embryos at the 2- to 4-cell stage . Xist transcripts were detected as a faint amplicon at the 8-cell stage initially, and consistently thereafter in all stages examined up to and including the expanded blastocyst stage . Xist transcripts, however, were subsequently detected from the 2-cell stage onward after nested RT-PCR . Preferential {3H}thymidine labeling indicative of late replication of one of the X chromosomes was noted in female embryos of different developmental ages as follows: 2 of 7 (28.5%) early blastocysts, 6 of 13 (46.1%) blastocysts, 8 of 11 (72.1%) expanded blastocysts, and 14 of 17 (77.7%) hatched blastocysts . These results suggest that Xist expression precedes the onset of late replication in the bovine embryo, in a pattern compatible with a possible role of bovine Xist in the initiation of X chromosome inactivation.

FEBS Lett, 1999 Jan 29, 443(3), 367 - 9
R73A and H144Q mutants of the yeast mitochondrial cyclophilin Cpr3 exhibit a low prolyl isomerase activity in both peptide and protein-folding assays; Scholz C et al.; Previously we reported that the R73A and H144Q variants of the yeast cyclophilin Cpr3 were virtually inactive in a protease-coupled peptide assay, but retained activity as catalysts of a proline-limited protein folding reaction {Scholz, C . et al . (1997) FEBS Lett . 414, 69-73} . A reinvestigation revealed that in fact these two mutations strongly decrease the prolyl isomerase activity of Cpr3 in both the peptide and the protein-folding assay . The high folding activities found previously originated from a contamination of the recombinant Cpr3 proteins with the Escherichia coli protein SlyD, a prolyl isomerase that co-purifies with His-tagged proteins . SlyD is inactive in the peptide assay, but highly active in the protein-folding assay.

FEBS Lett, 1999 Jan 29, 443(3), 313 - 6
In vivo formation of Cu,Zn superoxide dismutase disulfide bond in Escherichia coli; Battistoni A et al.; We have found that the in vivo folding of periplasmic Escherichia coli Cu,Zn superoxide dismutase is assisted by DsbA, which catalyzes the efficient formation of its single disulfide bond, whose integrity is essential to ensure full catalytic activity to the enzyme . In line with these findings, we also report that the production of recombinant Xenopus laevis Cu,Zn superoxide dismutase is enhanced when the enzyme is exported in the periplasmic space or is expressed in thioredoxin reductase mutant strains . Our data show that inefficient disulfide bond oxidation in the bacterial cytoplasm inhibits Cu,Zn superoxide dismutase folding in this cellular compartment.

FEBS Lett, 1999 Jan 29, 443(3), 271 - 6
Insulin-like growth factors I and II are unable to form and maintain their native disulfides under in vivo redox conditions; Hober S et al.; Insulin-like growth factor (IGF) I does not quantitatively form its three native disulfide bonds in the presence of 10 mM reduced and 1 mM oxidized glutathione in vitro {Hober, S . et al . (1992) Biochemistry 31, 1749-1756} . In this paper, we show (i) that both IGF-I and IGF-II are unable to form and maintain their native disulfide bonds at redox conditions that are similar to the situation in the secretory vesicles in vivo and (ii) that the presence of protein disulfide isomerase does not overcome this problem . The results indicate that the previously described thermodynamic disulfide exchange folding problem of IGF-I in vitro is also present in vivo . Speculatively, we suggest that the thermodynamic disulfide exchange properties of IGF-I and II are biologically significant for inactivation of the unbound growth factors by disulfide exchange reactions to generate variants destined for rapid clearance.

Cell, 1999 Feb 5, 96(3), 341 - 52
Chaperone activity with a redox switch; Jakob U et al.; Hsp33, a member of a newly discovered heat shock protein family, was found to be a very potent molecular chaperone . Hsp33 is distinguished from all other known molecular chaperones by its mode of functional regulation . Its activity is redox regulated . Hsp33 is a cytoplasmically localized protein with highly reactive cysteines that respond quickly to changes in the redox environment . Oxidizing conditions like H2O2 cause disulfide bonds to form in Hsp33, a process that leads to the activation of its chaperone function . In vitro and in vivo experiments suggest that Hsp33 protects cells from oxidants, leading us to conclude that we have found a protein family that plays an important role in the bacterial defense system toward oxidative stress.

Mol Cell, 1999 Jan, 3(1), 77 - 86
Allosteric regulation of even-skipped repression activity by phosphorylation; Li C et al.; The Drosophila homeodomain protein Even-skipped (Eve) is a well characterized transcriptional repressor . Here, we show that Eve's ability to function in vitro is negatively regulated by phosphorylation . DNA-binding activity was unaffected by phosphorylation, but phosphorylated Eve was unable to interact with the TATA-binding protein (TBP), a known target for repression . Unexpectedly, phosphorylation of the Eve N terminus, which is dispensable for repression and TBP binding, was necessary and sufficient to inactivate Eve . LiCl, which specifically inhibits glycogen synthase kinase-3 (GSK-3), reduced Eve phosphorylation in nuclear extract and blocked inhibition of repression . In addition, Eve was phosphorylated and inactivated by purified GSK-3 beta plus casein kinase II . Our results suggest a novel mechanism of transcriptional control involving phosphorylation-induced allosteric interference with a repressive protein-protein interaction.

Mol Cell, 1999 Jan, 3(1), 33 - 42
Base excision repair of oxidative DNA damage activated by XPG protein; Klungland A et al.; Oxidized pyrimidines in DNA are removed by a distinct base excision repair pathway initiated by the DNA glycosylase--AP lyase hNth1 in human cells . We have reconstituted this single-residue replacement pathway with recombinant proteins, including the AP endonuclease HAP1/APE, DNA polymerase beta, and DNA ligase III-XRCC1 heterodimer . With these proteins, the nucleotide excision repair enzyme XPG serves as a cofactor for the efficient function of hNth1 . XPG protein promotes binding of hNth1 to damaged DNA . The stimulation of hNth1 activity is retained in XPG catalytic site mutants inactive in nucleotide excision repair . The data support the model that development of Cockayne syndrome in XP-G patients is related to inefficient excision of endogenous oxidative DNA damage.

Infect Immun, 1999 Mar, 67(3), 1439 - 44
Responses of human intestinal microvascular endothelial cells to Shiga toxins 1 and 2 and pathogenesis of hemorrhagic colitis; Jacewicz MS et al.; Endothelial damage is characteristic of infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) . Because Stx-mediated endothelial cell damage at the site of infection may lead to the characteristic hemorrhagic colitis of STEC infection, we compared the effects of Stx1 and Stx2 on primary and transformed human intestinal microvascular endothelial cells (HIMEC) to those on macrovascular endothelial cells from human saphenous vein (HSVEC) . Adhesion molecule, interleukin-8 (IL-8), and Stx receptor expression, the effects of cytokine activation and Stx toxins on these responses, and Stx1 and Stx2 binding kinetics and bioactivity were measured . Adhesion molecule and IL-8 expression increased in activated HIMEC, but these responses were blunted in the presence of toxin, especially in the presence of Stx1 . In contrast to HSVEC, unstimulated HIMEC constitutively expressed Stx receptor at high levels, bound large amounts of toxin, were highly sensitive to toxin, and were not further sensitized by cytokines . Although the binding capacities of HIMEC for Stx1 and Stx2 were comparable, the binding affinity of Stx1 to HIMEC was 50-fold greater than that of Stx2 . Nonetheless, Stx2 was more toxic to HIMEC than an equivalent amount of Stx1 . The decreased binding affinity and increased toxicity for HIMEC of Stx2 compared to those of Stx1 may be relevant to the preponderance of Stx2-producing STEC involved in the pathogenesis of hemorrhagic colitis and its systemic complications . The differences between primary and transformed HIMEC in these responses were negligible . We conclude that transformed HIMEC lines could represent a simple physiologically relevant model to study the role of Stx in the pathogenesis of hemorrhagic colitis.

Infect Immun, 1999 Mar, 67(3), 1131 - 8
Identification and characterization of a novel Ibe10 binding protein that contributes to Escherichia coli invasion of brain microvascular endothelial cells; Prasadarao NV et al.; The molecular basis of Escherichia coli traversal of the blood-brain barrier in the development of E . coli meningitis is not well understood . We have previously shown that a novel Ibe10 protein found in cerebrospinal fluid isolates of E . coli is necessary for invasion of the brain microvascular endothelial cells (BMEC) that constitute the blood-brain barrier both in vitro and in a newborn rat model of hematogenous meningitis . Here we identified a novel Ibe10 binding molecule/receptor (Ibe10R) on both bovine BMEC (HBMEC) and human BMEC (HBMEC) that is responsible for invasion by E . coli . Ibe10R, an approximately 55-kDa protein, was purified from BBMEC by Ibe10-Ni-Sepharose affinity chromatography . Bovine Ibe10R, as well as polyclonal antibodies to Ibe10R, blocked E . coli invasion of BBMEC very effectively . The N-terminal amino acid sequence of Ibe10R showed 75% homology to serum albumin . However, the amino acid sequence of an Ibe10R fragment generated by limited enzymatic digestion did not reveal homology to any other proteins, suggesting that Ibe10R represents a novel albumin-like protein . Immunocytochemical analysis of BBMEC using anti-Ibe10R antibody suggested that only a subset of cultured BBMEC express Ibe10R on their surface . Enrichment of Ibe10R-positive BBMEC by fluorescence-activated cell sorting with anti-Ibe10R antibody resulted in enhanced invasion by E . coli . The anti-Ibe10R antibody raised against bovine Ibe10R also blocked E . coli invasion of HBMEC very effectively . Interestingly, anti-Ibe10R antibody affinity chromatography of HBMEC membrane proteins revealed a smaller protein with an approximate molecular mass of 45 kDa . These results suggest that the Ibe10 of E . coli interacts with a novel BMEC surface protein, Ibe10R, for invasion of both BBMEC and HBMEC.

Biochem J, 1999 Mar 1, 338 ( Pt 2), 553 - 60
Identification and characterization of a recombinant metallothionein protein from a marine alga, Fucus vesiculosus; Morris CA et al.; A cDNA library was constructed from macroalgae adapted to prolonged elevated environmental copper levels . To investigate the possible existence of a metallothionein (MT) gene, the library was screened with degenerate probes designed using plant MT cysteine-rich motifs . A gene was identified (1229 bp) with a putative open reading frame (204 bp) encoding a 67-amino-acid protein exhibiting several characteristic features of MT proteins, including 16 cysteine residues (24%) and only one aromatic residue . Although the protein sequence showed high identity with plant and invertebrate MTs, it contained a unique 'linker' region (14 amino acid residues) between the two putative metal-binding domains which contained no cysteine residues . This extended linker is larger than the tripeptide found in archetypal vertebrate MTs, but does not conform either with the 40-amino-acid linkers commonly found in plant MT sequences . An S-peptide Fucus MT fusion protein expressed in Escherichia coli exhibited a relative molecular mass of approximately 14 kDa . The recombinant fusion bound seven Cd ions, of which 50% were dissociated at pH 4.1 . Under anaerobic conditions, the Cd ions were displaced by Cu(I), which associated with the protein at a ratio of 13:1 . Laboratory exposure of F . vesiculosus to elevated copper resulted in induction of the MT gene . Thus this paper describes, for the first time, an MT gene identified from macroalgae which is induced by copper exposure and whose encoded protein product binds cadmium and copper.

Biochem J, 1999 Mar 1, 338 ( Pt 2), 409 - 16
Regulation of 2-carboxy-D-arabinitol 1-phosphate phosphatase: activation by glutathione and interaction with thiol reagents; Heo J et al.; 2-Carboxy-D-arabinitol 1-phosphate (CA1P) phosphatase de- grades CA1P, an inhibitor associated with the regulation of ribulose bisphosphate carboxylase/oxygenase in numerous plant species . CA1P phosphatase purified from Phaseolus vulgaris was partially inactivated by oxidizing conditions during dialysis in air-equilibrated buffer . Phosphatase activity could then be stimulated 1.3-fold by dithiothreitol and also by addition of reduced thioredoxin from Escherichia coli . These effects were enhanced synergistically by the positive effector, fructose 1, 6-bisphosphate (FBP) . Most notably, CA1P phosphatase activity was stimulated up to 35-fold by glutathione, and was sensitive to the ratio of reduced (GSH) to oxidized (GSSG) forms . At concentrations of glutathione approximating measured levels in chloroplasts of P . vulgaris (5 mM total S), CA1P phosphatase exhibited >20-fold stimulation by a change in the redox status of glutathione from 60 to 100% GSH . This stimulation was augmented further by reduced E . coli thioredoxin . In contrast, FBP, which activates CA1P phosphatase under reducing conditions, was strongly inhibitory in the presence of GSSG . We propose that glutathione may have an appreciable role in the light/dark regulation of CA1P phosphatase in vivo . A model for the reversible activation of CA1P phosphatase by GSH was derived based upon the various responses of the enzyme's activity to a range of thiol reagents including N-ethylmaleimide, 5, 5'-dithiobis-(2-nitrobenzoic acid) and arsenite . These data indicate that the bean enzyme contains two physically distinct sets of thiol groups that are critical to its redox regulation.

Protein Expr Purif, 1999 Feb, 15(1), 146 - 54
Design and expression of a synthetic mucin gene fragment in Escherichia coli; Dolby N et al.; Adenocarcinomas of glandular tissues produce a hypoglycosylated form of a normal glycoprotein (mucin) that elicits an immune response . A tumor-specific epitope of mucin occurs in a 20-amino-acid, tandemly repeated domain of human MUC1 mucin . A synthetic gene encoding five tandem repeats of the tumor-specific epitope of human mucin (m5tr) was designed for efficient cloning and expression in Escherichia coli for subsequent use in preparing reagent quantities of the mucin 5 tandem repeat (mtr5) polypeptide . The synthetic gene was cloned in the correct reading frame into the maltose-binding protein (MBP) fusion expression vector pMAL-p2 . Bacterial clones containing the mucin synthetic gene (m5tr) were shown to produce the intended recombinant fusion protein, MBP-mtr5 . The fusion protein represents a significant fraction of the cell protein, 50% or more of which is secreted into the periplasm . The MBP-mtr5 protein is largely intact and easily prepared in sufficient quantity and purity for preliminary structure-function studies .

Protein Expr Purif, 1999 Feb, 15(1), 115 - 20
High-level expression and single-step purification of leucyl-tRNA synthetase from Escherichia coli; Chen J et al.; A T7 promoter-based His6-tagging vector has been constructed that directs the synthesis in Escherichia coli of fusion proteins containing a stretch of six histidine residues at the N terminus . The vector allows overproduction and single-step purification of His6-fusion protein by immobilized metal (Ni2+) chelate affinity chromatography . The gene encoding leucyl-tRNA synthetase (leuS) was cloned into this vector and expressed in high level . The specific activity of the synthetase in the crude extract of E . coli JM109(DE3) transformant containing the His6-tagging vector with the gene leuS was approximately 110 times that of JM109(DE3) (the host strain without the vector) . The overproduced His6-fusion leucyl-tRNA synthetase can be purified to homogeneity under native conditions within 2 h by one-step affinity chromatography with an overall yield of 55% . The His6-tag at the N terminus of leucyl-tRNA synthetase did not affect its aminoacylation activity or the secondary structure .

Protein Expr Purif, 1999 Feb, 15(1), 105 - 14
Empty and peptide-loaded class II major histocompatibility complex proteins produced by expression in Escherichia coli and folding in vitro; Frayser M et al.; The human class II major histocompatibility complex protein HLA-DR1 has been expressed in Escherichia coli as denatured alpha and beta subunits and folded in vitro to form the native structure . DR1 folding yields are 30-50% in the presence or absence of tight-binding antigenic peptides . The protein produced in this manner is soluble and monomeric with the expected apparent molecular weight . It reacts with conformation-sensitive anti-DR antibodies and exhibits peptide-dependent resistance to SDS-induced chain dissociation and to proteolysis as does the native protein . The observed peptide specificity and dissociation kinetics are similar to those of native DR produced in B-cells and finally the protein exhibits circular dichroism spectra and cooperative thermal denaturation as expected for a folded protein . We conclude that the recombinant DR1 has adopted the native fold . We have folded DR1 in the absence of peptide and isolated a soluble, peptide-free alphabeta-heterodimer . The empty DR1 can bind antigenic peptide but exhibits altered far UV-circular dichroism and thermal denaturation relative to the peptide-bound form .

Protein Expr Purif, 1999 Feb, 15(1), 92 - 8
Optimized heterologous expression of glutathione reductase from Cyanobacterium anabaena PCC 7120 and characterization of the recombinant protein; Jiang F et al.; Glutathione reductase (GR) from the cyanobacterium Anabaena PCC 7120 was heterologously expressed in Escherichia coli SG5 . Silent random mutations were introduced in the 5' region of DNA encoding the enzyme in order to generate a high-level expression clone . To maximize protein expression, the culture conditions were also optimized . In the high-level expression clones selected, E . coli-preferred codons were selectively used at certain positions . Under the optimal expression conditions, a yield of 17 mg recombinant protein per liter was obtained, which is about 10-fold higher than that of the wild-type enzyme . A hexahistidine tag was added at the C-terminal of the protein in order to allow IMAC affinity purification . This strategy simplified the purification process and provided a homogeneous enzyme for functional characterization . Anabaena GR uses NADPH as a coenzyme, like most of the GRs from other sources, but the KM values for NADPH and GSSG are higher than those of enzymes previously studied . The Anabaena enzyme also shows significant activity when NADH is used as a reductant .

Protein Expr Purif, 1999 Feb, 15(1), 77 - 82
Disulfide bond formation and folding of plant peroxidases expressed as inclusion body protein in Escherichia coli thioredoxin reductase negative strains; Teilum K et al.; Escherichia coli is widely used for the production of proteins, which are of interest in structure and function studies . The folding yield of inclusion body protein is, however, generally low (a few percent) for proteins such as the plant and fungal peroxidases, which contain four disulfide bonds, two Ca2+ ions, and a heme group . We have studied the expression yield and folding efficiency of (i) a novel Arabidopsis thaliana peroxidase, ATP N; and (ii) barley grain peroxidase, BP 1 . The expression yield ranges from 0 to 60 microgram/ml of cell culture depending on the peroxidase gene and the vector/host combination . The choice of E . coli strain in particular affects the yield of active peroxidase obtained in the folding step . Thus, the yield of active ATP N peroxidase can be increased 50-fold by using thioredoxin reductase negative strains, which facilitate the formation of disulfide bonds in inclusion body protein .

Protein Expr Purif, 1999 Feb, 15(1), 57 - 61
Purified recombinant Escherichia coli ketol-acid reductoisomerase is unsuitable for use in a coupled assay of acetohydroxyacid synthase activity due to an unexpected side reaction; Hill CM et al.; Ketol-acid reductoisomerase (EC 1.1.1.86) catalyzes the conversion of 2-aceto-2-hydroxyacids to 2-keto-3-hydroxyacids and their subsequent reduction by NADPH to 2,3-dihydroxyacids . The gene encoding the Escherichia coli enzyme was cloned and expressed as a hexahistidine-tagged fusion protein and the recombinant enzyme purified by metal-ligand affinity chromatography . The pure enzyme was tested for its ability to provide a sensitive and continuous coupled assay for acetohydroxyacid synthase (EC 4.1.3.18), the preceding enzyme in the pathway of branched-chain amino acid biosynthesis . An unexpected side reaction of ketol-acid reductoisomerase was observed in which it catalyzes the reduction of pyruvate . Although relatively slow, this side reaction is high enough to prohibit the use of this enzyme in a coupled assay for acetohydroxyacid synthase .

Protein Expr Purif, 1999 Feb, 15(1), 34 - 9
Overcoming expression and purification problems of RhoGDI using a family of "parallel" expression vectors; Sheffield P et al.; We describe the construction of expression vectors based on three of the most frequently used gene fusion affinity tags {glutathione S-transferase (GST), maltose binding protein (MBP), and the His6 peptide} . The polylinkers of pGEX4T1, pMal-c2, and a pET vector were replaced with the polylinker isolated from the baculovirus expression plasmid pFastBac . Once appropriate restriction sites have been introduced into a gene, it can be fused to all three affinity tags with little effort, allowing expression-screening experiments to be performed efficiently . We discuss the development and use of these vectors with respect to overcoming purification problems encountered for the RhoA GDP/GTP nucleotide dissociation inhibitor (RhoGDI) and their advantages over commercially available expression vectors .

Protein Expr Purif, 1999 Feb, 15(1), 24 - 33
Production of reagents and optimization of methods for studying calmodulin-binding proteins; Ulbricht B et al.; Owing to subtle but potentially crucial structural and functional differences between calmodulin (CaM) of different species, the biochemical study of low-affinity CaM-binding proteins from Dictyostelium discoideum likely necessitates the use of CaM from the same organism . In addition, most of the methods used for identification and purification of CaM-binding proteins require native CaM in nonlimiting biochemical quantities . The gene encoding D . discoideum CaM has previously been cloned allowing production of recombinant protein . The present study describes the expression of D . discoideum CaM in Escherichia coli and its straightforward and rapid purification . Furthermore, we describe the optimization of a complete palette of assays to detect as little as nanogram quantities of proteins binding CaM with middle to low affinities . Purified CaM was used to raise high-affinity polyclonal antibodies suitable for immunoblotting, immunofluorescence, and immunoprecipitation experiments . The purified CaM was also used to optimize a specific and sensitive nonradioactive CaM overlay assay as well as to produce a high-capacity CaM affinity chromatography matrix . The effectiveness of this methods is illustrated by the detection of potentially novel D . discoideum CaM-binding proteins and the preparatory purification of one of these proteins, a short tail myosin I .

Protein Expr Purif, 1999 Feb, 15(1), 8 - 15
Cloning, expression, and purification of cytidine deaminase from Arabidopsis thaliana; Vincenzetti S et al.; The complementary DNA (cDNA) coding for Arabidopsis thaliana cytidine deaminase 1 (AT-CDA1) was obtained from the amplified A . thaliana cDNA expression library, provided by R . W . Davis (Stanford University, CA) . AT-CDA1 cDNA was subcloned into the expression vector pTrc99-A and the protein, expressed in Escherichia coli following induction with isopropyl 1-thio-beta-d-galactopyranoside, showed high cytidine deaminase activity . The nucleotide sequence showed a 903-bp open reading frame encoding a polypeptide of 301 amino acids with a calculated molecular mass of 32,582 . The deduced amino acid sequence of AT-CDA1 showed no transit peptide for targeting to the chloroplast or mitochondria indicating that this form of cytidine deaminase is probably expressed in the cytosol . The recombinant AT-CDA1 was purified to homogeneity by a heat treatment followed by an ion-exchange chromatography . The final enzyme preparation was >98% pure as judged by SDS-PAGE and showed a specific activity of 74 U/mg . The molecular mass of AT-CDA1 estimated by gel filtration was 63 kDa, indicating, in contrast to the other eukaryotic CDAs, that the enzyme is a dimer composed of two identical subunits . Inductively coupled plasma-optical emission spectroscopy analysis indicated that the enzyme contains 1 mol of zinc atom per mole of subunit . The kinetic properties of AT-CDA1 both toward the natural substrates and with analogs indicated that the catalytic mechanism of the plant enzyme is probably very similar to that of the human the E . coli enzymes .

J Mol Biol, 1999 Feb 26, 286(3), 829 - 50
Crystal structure of phosphoserine aminotransferase from Escherichia coli at 2.3 A resolution: comparison of the unligated enzyme and a complex with alpha-methyl-l-glutamate; Hester G et al.; Phosphoserine aminotransferase (PSAT; EC 2.6.1.52), a member of subgroup IV of the aminotransferases, catalyses the conversion of 3-phosphohydroxypyruvate to l-phosphoserine . The crystal structure of PSAT from Escherichia coli has been solved in space group P212121 using MIRAS phases in combination with density modification and was refined to an R-factor of 17.5% (Rfree=20.1 %) at 2.3 A resolution . In addition, the structure of PSAT in complex with alpha-methyl-l-glutamate (AMG) has been refined to an R-factor of 18.5% (Rfree=25.1%) at 2.8 A resolution . Each subunit (361 residues) of the PSAT homodimer is composed of a large pyridoxal-5'-phosphate binding domain (residues 16-268), consisting of a seven-stranded mainly parallel beta-sheet, two additional beta-strands and seven alpha-helices, and a small C-terminal domain, which incorporates a five-stranded beta-sheet and two alpha-helices . A three-dimensional structural comparison to four other vitamin B6-dependent enzymes reveals that three alpha-helices of the large domain, as well as an N-terminal domain (subgroup II) or subdomain (subgroup I) are absent in PSAT . Its only 15 N-terminal residues form a single beta-strand, which participates in the beta-sheet of the C-terminal domain . The cofactor is bound through an aldimine linkage to Lys198 in the active site . In the PSAT-AMG complex Ser9 and Arg335 bind the AMG alpha-carboxylate group while His41, Arg42 and His328 are involved in binding the AMG side-chain . Arg77 binds the AMG side-chain indirectly through a solvent molecule and is expected to position itself during catalysis between the PLP phosphate group and the substrate side-chain . Comparison of the active sites of PSAT and aspartate aminotransferase suggests a similar catalytic mechanism, except for the transaldimination step, since in PSAT the Schiff base is protonated . Correlation of the PSAT crystal structure to a published profile sequence analysis of all subgroup IV members allows active site modelling of nifs and the proposal of a likely molecular reaction mechanism .

J Mol Biol, 1999 Feb 26, 286(3), 775 - 85
Involvement of the C-terminal region of yeast proteinase B inhibitor 2 in its inhibitory action; Kojima S et al.; Yeast proteinase B inhibitor 2 (YIB2), which is composed of 74 amino acid residues, is an unusual serine protease inhibitor, since it lacks disulfide bonds . To identify its reactive site for proteases, we constructed an expression system for a synthetic YIB2 gene and then attempted to change the inhibitory properties of YIB2 by amino acid replacements . The purified wild-type YIB2 inhibited the activity of subtilisin BPN', a protein homologous to yeast proteinase B, although its binding ability was not strong, and a time-dependent decrease in its inhibitory activity was observed, demonstrating that wild-type YIB2 behaves as a temporary inhibitor when subtilisin BPN' is the target protease . Since YIB2 exhibits sequence homology to the propeptide of subtilisin, which inhibits a cognate protease using its C-terminal region, we replaced the six C-termi nal residues of YIB2 with those of the propeptide of subtilisin BPN' to make the mutant YIB2m1 . This mutant exhibited markedly increased inhibitory activity toward subtilisin BPN' without a time-dependent decrease in its inhibitory activity . Replacement of only the C-terminal Asn of YIB2 by Tyr, or deletion of the C-terminal Tyr of YIB2m1, inhibited subtilisin, but the ability of these mutants to bind subtilisin and their resistance to proteolytic attack were weaker than those of YIB2m1, indicating that the C-terminal residue contributes to the interaction with the protease to a greater extent than the preceding five residues and that the resistance of YIB2 to proteolyic attack is closely related to its ability to bind a protease . These results demonstrate that YIB2 is a unique protease inhibitor that involves its C-terminal region in the interaction with the protease .

J Mol Biol, 1999 Feb 26, 286(3), 637 - 43
Formation of knots in partially replicated DNA molecules; Sogo JM et al.; Bacterial plasmids with two origins of replication in convergent orientation are frequently knotted in vivo . The knots formed are localised within the newly replicated DNA regions . Here, we analyse DNA knots tied within replication bubbles of such plasmids, and observe that the knots formed show predominantly positive signs of crossings . We propose that helical winding of replication bubbles in vivo leads to topoisomerase-mediated formation of knots on partially replicated DNA molecules .

Calcif Tissue Int, 1999 Mar, 64(3), 214 - 8
Molecular chaperones stimulate bone resorption; Nair SP et al.; Molecular chaperones, also known as heat shock proteins (hsp), are intracellular proteins found in all cells that catalyze protein folding . We have discovered that one class of bacterial molecular chaperone, the chaperonins, are potent inducers of bone resorption . To address the question of whether the osteolytic activity of the chaperonins was unique to this protein class, or was a common attribute of molecular chaperones generally, we have examined a number of bacterial and mammalian molecular chaperones for activity in the murine calvarial bone resorption assay . All the Escherichia coli molecular chaperones (groEL, groES, and dnaK) were active . The osteolytic activity of groEL was inhibited by indomethacin and the natural antagonist of interleukin-1 receptor antagonist (IL-1ra) but was unaffected by neutralization of tumor necrosis factor (TNF) or inhibition of 5-lipoxygenase . Mammalian molecular chaperones of molecular mass 27, 47, 70, and 90 kDa were also tested and, with the exception of the 47 kDa protein, all showed activity in the murine calvarial assay . Molecular chaperones appear, therefore, to have the capacity to modulate the cellular processes in bone explant cultures, resulting in resorption of the calcified matrix . The possibility that these proteins could play a role in the normal or pathological remodeling of bone is discussed.

RNA, 1999 Feb, 5(2), 302 - 17
A novel nucleotide incorporation activity implicated in the editing of mitochondrial transfer RNAs in Acanthamoeba castellanii; Price DH et al.; In Acanthamoeba castellanii, most of the mtDNA-encoded tRNAs are edited by a process that replaces one or more of the first three nucleotides at their 5' ends . As a result, base pairing potential is restored at acceptor stem positions (1:72, 2:71, and/or 3:70, in standard tRNA nomenclature) that are mismatched according to the corresponding tRNA gene sequence . Here we describe a novel nucleotide incorporation activity, partially purified from A . castellanii mitochondria, that has properties implicating it in mitochondrial tRNA editing in this organism . This activity is able to replace nucleotides at the first three positions of a tRNA (positions 1, 2, and 3), matching the newly incorporated residues through canonical base pairing to the respective partner nucleotide in the 3' half of the acceptor stem . Labeling experiments with natural (Escherichia coli tRNATyr) and synthetic (run-off transcripts corresponding to A . castellanii mitochondrial tRNALeu1) substrates suggest that the nucleotide incorporation activity consists of at least two components, a 5' exonuclease or endonuclease and a template-directed 3'-to-5' nucleotidyltransferase . The nucleotidyltransferase component displays an ATP requirement and generates 5' pppN.. . termini in vitro . The development of an accurate and efficient in vitro system opens the way for detailed studies of the biochemical properties of this novel activity and its relationship to mitochondrial tRNA editing in A . castellanii . In addition, the system will allow delineation of the structural features in a tRNA that identify it as a substrate for the labeling activity.

RNA, 1999 Feb, 5(2), 188 - 94
Single atom modification (O-->S) of tRNA confers ribosome binding; Ashraf SS et al.; Escherichia coli tRNALysSUU, as well as human tRNALys3SUU, has 2-thiouridine derivatives at wobble position 34 (s2U*34) . Unlike the native tRNALysSUU, the full-length, unmodified transcript of human tRNALys3UUU and the unmodified tRNALys3UUU anticodon stem/loop (ASLLys3UUU) did not bind AAA- or AAG-programmed ribosomes . In contrast, the completely unmodified yeast tRNAPhe anticodon stem/loop (ASLPheGAA) had an affinity (Kd = 136+/-49 nM) similar to that of native yeast tRNAPheGmAA (Kd = 103+/-19 nM) . We have found that the single, site-specific substitution of s2U34 for U34 to produce the modified ASLLysSUU was sufficient to restore ribosomal binding . The modified ASLLysSUU bound the ribosome with an affinity (Kd = 176+/-62 nM) comparable to that of native tRNALysSUU (Kd = 70+/-7 nM) . Furthermore, in binding to the ribosome, the modified ASLLys3SUU produced the same 16S P-site tRNA footprint as did native E . coli tRNALysSUU, yeast tRNAPheGmAA, and the unmodified ASLPheGAA . The unmodified ASLLys3UUU had no footprint at all . Investigations of thermal stability and structure monitored by UV spectroscopy and NMR showed that the dynamic conformation of the loop of modified ASLLys3SUU was different from that of the unmodified ASLLysUUU, whereas the stems were isomorphous . Based on these and other data, we conclude that s2U34 in tRNALysSUU and in other s2U34-containing tRNAs is critical for generating an anticodon conformation that leads to effective codon interaction in all organisms . This is the first example of a single atom substitution (U34-->s2U34) that confers the property of ribosomal binding on an otherwise inactive tRNA.

Metabolism, 1999 Feb, 48(2), 190 - 3
Is the L-arginine-nitric oxide pathway involved in endotoxemia-induced muscular hypercatabolism in rats?
Pernet P, Coudray-Lucas C, Le Boucher J, Schlegel L, Giboudeau J, Cynober L, Aussel C.
We investigated the role of the nitric oxide (NO) synthase (NOS) pathway in muscular metabolism during endotoxemia in four groups of male Wistar rats . Two groups were injected with the lipopolysaccharide (LPS) of Escherichia coli (3 mg/kg), with one group treated using N(G)-nitro-L-arginine methylester ({L-NAME} 85 mg/kg/d) and the other not . The two control groups included one treated with L-NAME and the other not . After 24 hours of fasting, the rats were fed by controlled enteral nutrition and killed on day 3 . The results showed that (1) NOS inhibition was detrimental during endotoxemia, increasing lethality from 20% to 80.5%, and (2) NOS inhibition did not modify the hypercatabolic state consecutive to endotoxemia, particularly at the muscular level (nitrogen balance, total-body and muscular weight loss, and muscular protein and glutamine concentrations) . However, myofibrillar catabolism was delayed in the LPS-NAME group . In conclusion, NO production is of major importance for survival after an endotoxemic challenge, but contributes weakly to the metabolic response of muscle to injury.

Poult Sci, 1999 Jan, 78(1), 38 - 44
Sex differences in the resistance of turkeys to Escherichia coli challenge after immunosuppression with dexamethasone; Huff GR et al.; Five-week-old male and female commercial turkeys were immunosuppressed with two levels of dexamethasone (DEX) and challenged by airsac inoculation with 100 cfu of Escherichia coli . Mortality and airsacculitis (AS) scores were significantly higher in challenged birds treated with DEX and marginally higher in males than in females . Male mortalities had significantly higher AS scores than females . Recovery of E . coli from blood and tissues was significantly higher in challenged birds treated with DEX, marginally higher in males (P = 0.09), and significantly higher in male mortalities than in female mortalities . The low level of DEX seemed to have a protective effect against bacterial challenge in males, whereas the high level of DEX had a more adverse effect in males than in females . Body weights 2 wk postchallenge were significantly decreased by both DEX and E . coli, challenge . Relative liver and heart weights were increased by both DEX and E . coli, challenge, whereas bursal:spleen ratios were decreased by both treatments . Total leukocyte counts and relative heterophil counts from blood samples taken 24 h postinfection were significantly higher in DEX-treated birds and in unchallenged DEX-treated females than in males . The DEX treatment resulted in significantly higher heterophil:lymphocyte ratios, but there was no difference between sexes . Males had significantly lower serum levels of uric acid, total protein, albumin, aspartate aminotransferase, and lactate dehydrogenase than did females . Dexamethasone treatment also resulted in lower levels of total protein, albumin, and aspartate aminotransferase . These data suggest that male turkeys may be more susceptible to colisepticemia than female turkeys, especially when under severe stress.

Chirurg, 1998 Dec, 69(12), 1386 - 7
{Perforated appendiceal apex in an inguinal hernia as the cause of chronic abdominal wall abscess}; Schwonbeck M et al.; Perforated appendicitis within a hernia of any localisation is found in paediatric patients and in the elderly . Despite all modern diagnostic bias, it still may be found unexpectedly during operation . The fore most aim of surgical treatment has to be protection of the intra-abdominal cavity during appendectomy . Thus we provide it for contamination and avoid additional injury of the old and weak patient . The reported case fits in the history of unusual appendectomies.

Mutat Res, 1999 Feb 19, 439(2), 287 - 300
Escherichia coli MTC, a NADPH cytochrome P450 reductase competent mutagenicity tester strain for the expression of human cytochrome P450: comparison of three types of expression systems; Kranendonk M et al.; Currently three different methods have been taken to develop new mutagenicity tester strains containing human cytochrome P450s (CYPs) . Each of these use a single expression vector . In this paper we describe a fourth approach, i.e., the coexpression of a CYP and its electron-transfer flavoprotein, NADPH CYP reductase (RED), encoded by two different expression vectors . The Escherichia coli mutagenicity tester strain BMX100 has been expanded to a strain, MTC which stably expresses human RED . This new tester strain permits the biplasmid coexpression of human CYP1A2 and RED (MTC1A2) . This novel strain can be used for the determination of the mutagenicity of chemicals known to be procarcinogens and metabolized by CYP1A2 . The mutagenicity tester strain MTC1A2 was compared with: (i) BMX100 using the post-mitochondrial rat liver fraction (S9); (ii) BMX100 with expressing CYP1A2 alone (iii) or with expressing CYP1A2 fused to rat RED or (iv) with expressing CYP1A2, bicistronically coexpressed with rat RED . The biplasmid RED/CYP coexpression system generated a strain with the highest methoxy- and ethoxy-resorufin dealkylase activities and the highest mutagenic activities for the procarcinogens 2-aminoanthracene (2AA), aflatoxin B1 (AFB1) and 2-amino-3-methylimidazo(4,5-f)quinoline (IQ) . Furthermore, the metabolism of 2AA and IQ was detected more efficiently using the MTC1A2 strain than with the BMX100 strain plus the standard rodent liver S9 metabolic system .

Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Topics, 1994 Feb, 49(2), 1685 - 9
Mosaic organization of DNA nucleotides; Peng CK et al.; Long-range power-law correlations have been reported recently for DNA sequences containing noncoding regions . We address the question of whether such correlations may be a trivial consequence of the known mosaic structure ("patchiness") of DNA . We analyze two classes of controls consisting of patchy nucleotide sequences generated by different algorithms--one without and one with long-range power-law correlations . Although both types of sequences are highly heterogenous, they are quantitatively distinguishable by an alternative fluctuation analysis method that differentiates local patchiness from long-range correlations . Application of this analysis to selected DNA sequences demonstrates that patchiness is not sufficient to account for long-range correlation properties.

J Cell Biochem, 1999 Mar 1, 72(3), 435 - 44
Identification and characterization of a novel protein that regulates RNA-protein interaction; Hod Y et al.; In a previous study {Nachaliel et al., 1993}, we identified an RNA-binding protein (RBP) in FTO-2B rat hepatoma cells whose activity was stimulated upon the dissociation of a protein factor . We report in this article that the RBP is a complex protein of about 400 kDa, composed of RNA-binding subunit(s) (RBS), and regulatory subunit(s) (RS) . We purified the RS to near-homogeneity (Mr approximately 25,000) and determined the amino acid sequence of a peptide derived from RS . On the basis of this sequence information, the cDNA for RS was obtained . Recombinant RS protein expressed in Escherichia coli had the capacity to bind RBS and inhibit its RNA-binding activity . The cDNA contains the complete coding sequence because the recombinant protein has the same electrophoretic mobility as that of the native RS in SDS-polyacrylamide gels . Sequence comparison showed that RS is almost identical to DJ-1, a recently discovered protein with an oncogenic potential, and CAP1, a rat sperm protein . However, the protein does not contain any known motifs that can provide a clue as to its exact function . Indirect immunofluorescence analyses showed that in addition to the cytoplasm, where RS is associated with microtubular filaments, the polypeptide is localized to the cell nucleus . The possible role of RS is discussed.

Proteins, 1999 Feb 1, 34(2), 167 - 72
Strategy for membrane protein crystallization exemplified with OmpA and OmpX; Pautsch A et al.; The bacterial outer membrane proteins OmpA and OmpX were modified in such a manner that they yielded bulky crystals diffracting X-rays isotropically beyond 2 A resolution and permitting detailed structural analyses . The procedure involved semi-directed mutagenesis, mass production into inclusion bodies, and (re)naturation therefrom; it should be applicable for a broader range of membrane proteins.

Proteins, 1999 Feb 1, 34(2), 155 - 66
Structure of catalase HPII from Escherichia coli at 1.9 A resolution; Bravo J et al.; Catalase HPII from Escherichia coli, a homotetramer of subunits with 753 residues, is the largest known catalase . The structure of native HPII has been refined at 1.9 A resolution using X-ray synchrotron data collected from crystals flash-cooled with liquid nitrogen . The crystallographic agreement factors R and R(free) are respectively 16.6% and 21.0% . The asymmetric unit of the crystal contains a whole molecule that shows accurate 222-point group symmetry . The structure of the central part of the HPII subunit gives a root mean square deviation of 1.5 A for 477 equivalencies with beef liver catalase . Most of the additional 276 residues of HPII are located in either an extended N-terminal arm or in a C-terminal domain organized with a flavodoxin-like topology . A small number of mostly hydrophilic interactions stabilize the relative orientation between the C-terminal domain and the core of the enzyme . The heme component of HPII is a cis-hydroxychlorin gamma-spirolactone in an orientation that is flipped 180 degrees with respect to the orientation of the heme found in beef liver catalase . The proximal ligand of the heme is Tyr415 which is joined by a covalent bond between its Cbeta atom and the Ndelta atom of His392 . Over 2,700 well-defined solvent molecules have been identified filling a complex network of cavities and channels formed inside the molecule . Two channels lead close to the distal side heme pocket of each subunit suggesting separate inlet and exhaust functions . The longest channel, that begins in an adjacent subunit, is over 50 A in length, and the second channel is about 30 A in length . A third channel reaching the heme proximal side may provide access for the substrate needed to catalyze the heme modification and His-Tyr bond formation . HPII does not bind NADPH and the equivalent region to the NADPH binding pocket of bovine catalase, partially occluded in HPII by residues 585-590, corresponds to the entrance to the second channel . The heme distal pocket contains two solvent molecules, and the one closer to the iron atom appears to exhibit high mobility or low occupancy compatible with weak coordination.

J Comp Neurol, 1999 Mar 1, 405(1), 45 - 60
Early induction of Talpha1 alpha-tubulin transcription in neurons of the developing nervous system; Gloster A et al.; In this report, we have examined the relationship between the onset of neuronal gene transcription and neuronal development by characterizing expression of the early panneuronal Talpha1 alpha-tubulin promoter in developing neurons . In the peripheral nervous system, detectable expression of a beta-galactosidase transgene driven by the Talpha1 promoter (Talpha1:nlacZ) was coincident with neuronal birth dates, with the exception of sympathetic neuroblasts, which expressed the transgene prior to terminal mitosis . Similarly, in the central nervous system, the onset of beta-galactosidase expression was coincident with neuronal birth dates in most identifiable populations of central neurons . A small subpopulation of transgene-positive cells localized to ventricular zones, but the vast majority was observed in locations consistent with their identification as migrating and/or differentiating neurons . To determine more precisely the temporal relationship between transgene expression and terminal mitosis, we analyzed cultures of cortical progenitors that become postmitotic neurons in vitro . When initially plated, the vast majority of cells consisted of dividing, nestin-positive progenitors . Neurons differentiated from these progenitors as early as 1 day in vitro, as indicated by immunostaining for betaIII-tubulin, a neuron-specific tubulin isotype that is turned on shortly after terminal mitosis . Double-labeling studies showed that Talpha1:nlacZ expression was detectable in the same cells and at approximately the same time as was betaIII-tubulin, indicating that detectable transcription of the Talpha1 alpha-tubulin promoter commences at the time of terminal mitosis, at least in culture . This promoter, therefore, provides a valuable tool for genetic manipulation of early developing neurons in transgenic mice.

Mol Cell Biol, 1999 Mar, 19(3), 2212 - 9
A human sequence homologue of Staufen is an RNA-binding protein that is associated with polysomes and localizes to the rough endoplasmic reticulum; Marion RM et al.; In the course of a two-hybrid screen with the NS1 protein of influenza virus, a human clone capable of coding for a protein with high homology to the Staufen protein from Drosophila melanogaster (dmStaufen) was identified . With these sequences used as a probe, cDNAs were isolated from a lambda cDNA library . The encoded protein (hStaufen-like) contained four double-stranded RNA (dsRNA)-binding domains with 55% similarity and 38% identity to those of dmStaufen, including identity at all residues involved in RNA binding . A recombinant protein containing all dsRNA-binding domains was expressed in Escherichia coli as a His-tagged polypeptide . It showed dsRNA binding activity in vitro, with an apparent Kd of 10(-9) M . Using a specific antibody, we detected in human cells a major form of the hStaufen-like protein with an apparent molecular mass of 60 to 65 kDa . The intracellular localization of hStaufen-like protein was investigated by immunofluorescence using a series of markers for the cell compartments . Colocalization was observed with the rough endoplasmic reticulum but not with endosomes, cytoskeleton, or Golgi apparatus . Furthermore, sedimentation analyses indicated that hStaufen-like protein associates with polysomes . These results are discussed in relation to the possible functions of the protein.

Mol Cell Biol, 1999 Mar, 19(3), 2198 - 205
Hsp60 is targeted to a cryptic mitochondrion-derived organelle ("crypton") in the microaerophilic protozoan parasite Entamoeba histolytica; Mai Z et al.; Entamoeba histolytica is a microaerophilic protozoan parasite in which neither mitochondria nor mitochondrion-derived organelles have been previously observed . Recently, a segment of an E . histolytica gene was identified that encoded a protein similar to the mitochondrial 60-kDa heat shock protein (Hsp60 or chaperonin 60), which refolds nuclear-encoded proteins after passage through organellar membranes . The possible function and localization of the amebic Hsp60 were explored here . Like Hsp60 of mitochondria, amebic Hsp60 RNA and protein were both strongly induced by incubating parasites at 42 degreesC . 5' and 3' rapid amplifications of cDNA ends were used to obtain the entire E . histolytica hsp60 coding region, which predicted a 536-amino-acid Hsp60 . The E . histolytica hsp60 gene protected from heat shock Escherichia coli groEL mutants, demonstrating the chaperonin function of the amebic Hsp60 . The E . histolytica Hsp60, which lacked characteristic carboxy-terminal Gly-Met repeats, had a 21-amino-acid amino-terminal, organelle-targeting presequence that was cleaved in vivo . This presequence was necessary to target Hsp60 to one (and occasionally two or three) short, cylindrical organelle(s) . In contrast, amebic alcohol dehydrogenase 1 and ferredoxin, which are bacteria-like enzymes, were diffusely distributed throughout the cytosol . We suggest that the Hsp60-associated, mitochondrion-derived organelle identified here be named "crypton," as its structure was previously hidden and its function is still cryptic.

Mol Cell Biol, 1999 Mar, 19(3), 1686 - 94
Human topoisomerase I promotes initiation of simian virus 40 DNA replication in vitro; Trowbridge PW et al.; Addition of purified human topoisomerase I (topo I) to simian virus 40 T antigen-driven in vitro DNA replication reactions performed with topo I-deficient extracts results in a greater than 10-fold stimulation of completed molecules as well as a more than 3-fold enhancement of overall DNA replication . To further characterize this stimulation, we first demonstrate that bovine topo I but not Escherichia coli topo I can also enhance DNA replication . By using several human topo I mutants, we show that a catalytically active form of topo I is required . To delineate whether topo I influences the initiation or the elongation step of replication, we performed delayed pulse, pulse-chase, and delayed pulse-chase experiments . The results illustrate that topo I cannot promote the completion of partially replicated molecules but is needed from the beginning of the reaction to initiate replication . Competitive inhibition experiments with the topo I binding T antigen fragment 1-246T and a catalytically inactive topo I mutant suggest that part of topo I's stimulation of replication is mediated through a direct interaction with T antigen . Collectively, our data indicate that topo I enhances the synthesis of fully replicated DNA molecules by forming essential interactions with T antigen and stimulating initiation.

EMBO J, 1999 Feb 15, 18(4), 1049 - 58
Membrane deinsertion of SecA underlying proton motive force-dependent stimulation of protein translocation; Nishiyama K et al.; The proton motive force (PMF) renders protein translocation across the Escherichia coli membrane highly efficient, although the underlying mechanism has not been clarified . The membrane insertion and deinsertion of SecA coupled to ATP binding and hydrolysis, respectively, are thought to drive the translocation . We report here that PMF significantly decreases the level of membrane-inserted SecA . The prlA4 mutation of SecY, which causes efficient protein translocation in the absence of PMF, was found to reduce the membrane-inserted SecA irrespective of the presence or absence of PMF . The PMF-dependent decrease in the membrane-inserted SecA caused an increase in the amount of SecA released into the extra-membrane milieu, indicating that PMF deinserts SecA from the membrane . The PMF-dependent deinsertion reduced the amount of SecA required for maximal translocation activity . Neither ATP hydrolysis nor exchange with external SecA was required for the PMF-dependent deinsertion of SecA . These results indicate that the SecA deinsertion is a limiting step of protein translocation and is accelerated by PMF, efficient protein translocation thereby being caused in the presence of PMF.

Mol Cell Endocrinol, 1998 Nov 25, 146(1-2), 103 - 20
A novel piscine vitellogenin gene: structural and functional analyses of estrogen-inducible promoter; Teo BY et al.; The Oreochromis aureus vitellogenin, OaVtg, gene spans 9 kb and contains 34 exons . Its transcription start site is located 15 bp upstream of the translational start codon . Although the OaVtg promoter has a nonconsensus TATA, transient transfection assay showed that this promoter is capable of driving basal transcription . Two imperfect estrogen response elements: EREp (proximal) and EREd (distal) are located in the promoter at - 532 and - 1352, respectively . In competition gel mobility-shift assays, only EREp exhibited specific binding of the recombinant estrogen receptor protein, GST-C/D OaER . Another imperfect ERE (EREexon2) was detected within exon 2 of the OaVtg gene . This is a novel finding for a vitellogenin (Vtg) gene . EREexon2 similarly showed specific recognition of GST-C/D OaER . Both EREp and EREexon2 showed comparable binding affinities as consensus ERE . In transient transfections, the OaVtg promoter, EREp and EREd elicited significant increase in estrogen-dependent synthesis of CAT protein . Hence, we propose that the non-consensus OaVtg EREs contribute to the estrogen-dependent regulation of the OaVtg gene in vivo.

J Immunol Methods, 1999 Jan 1, 222(1-2), 65 - 72
Use of flow cytometry to measure the interaction between Escherichia coli heat-stable enterotoxin and its intestinal receptor in mice; Al-Majali AM et al.; Binding of Escherichia coli heat-stable enterotoxin (STa) to its putative receptor on the brush border membrane of enterocytes is a prerequisite for the induction of diarrhea in infected humans and animals . Humans and animals of different ages vary in their susceptibility to the effect STa, perhaps due to the difference in STa interaction with its intestinal receptor . Flow cytometry was compared to indirect immunofluorescence and 125I-STa binding assays to measure the STa-enterocytes receptor interaction in different age groups of Swiss Webster mice (2-, 7-, 14-day-old) . Flow cytometry indicated stronger interaction between STa and its putative receptor on enterocytes from the 2-day-old mice than enterocytes from older mice . 125I-STa-binding assay suggested that the stronger fluorescence intensity on enterocytes from younger mice is due to higher STa receptor density and higher receptor affinity to STa . Flow cytometry is more sensitive quantitative assay to measure the interaction between STa and its intestinal receptor than indirect immunofluorescence microscopy.

Philos Trans R Soc Lond B Biol Sci, 1998 Oct 29, 353(1376), 1701 - 5
Cytochrome P450 monooxygenases and insecticide resistance in insects; Berge JB et al.; Cytochrome P450 monooxygenases are involved in many cases of resistance of insects to insecticides . Resistance has long been associated with an increase in monooxygenase activities and with an increase in cytochrome P450 content . However, this increase does not always account for all of the resistance . In Drosophila melanogaster, we have shown that the overproduction of cytochrome P450 can be lost by the fly without a corresponding complete loss of resistance . These results prompted the sequencing of a cytochrome P450 candidate for resistance in resistant and susceptible flies . Several mutations leading to amino-acid substitutions have been detected in the P450 gene CYP6A2 of a resistant strain . The location of these mutations in a model of the 3D structure of the CYP6A2 protein suggested that some of them may be important for enzyme activity of this molecule . This has been verified by heterologous expression of wild-type and mutated cDNA in Escherichia coli . When other resistance mechanisms are considered, relatively few genetic mutations are involved in insecticide resistance, and this has led to an optimistic view of the management of resistance . Our observations compel us to survey in more detail the genetic diversity of cytochrome P450 genes and alleles involved in resistance.

J Clin Invest, 1999 Feb, 103(4), 571 - 7
Chlamydial and human heat shock protein 60s activate human vascular endothelium, smooth muscle cells, and macrophages; Kol A et al.; Both chlamydial and human heat shock protein 60s (HSP 60), which colocalize in human atheroma, may contribute to inflammation during atherogenesis . We tested the hypothesis that chlamydial or human HSP 60 activates human endothelial cells (ECs), smooth muscle cells (SMCs), and monocyte-derived macrophages . We examined the expression of adhesion molecules such as endothelial-leukocyte adhesion molecule-1 (E-selectin), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), and the production of the proinflammatory cytokine interleukin-6 (IL-6) . We also tested whether either HSP 60 induces nuclear factor-kappaB (NF-kappaB), which contributes to the gene expression of these molecules . Either chlamydial or human HSP 60 induced E-selectin, ICAM-1, and VCAM-1 expression on ECs similar to levels induced by Escherichia coli lipopolysaccharide (LPS) . Each HSP 60 also significantly induced IL-6 production by ECs, SMCs, and macrophages to an extent similar to that induced by E . coli LPS, as assessed by enzyme-linked immunosorbent assay (ELISA) . In ECs, either HSP 60 triggered activation of NF-kappaB complexes containing p65 and p50 Rel proteins . Heat treatment abolished all these effects, but did not alter the ability of E . coli LPS to induce these functions . Chlamydial and human HSP 60s therefore activate human vascular cell functions relevant to atherogenesis and lesional complications . These findings help to elucidate the mechanisms by which a chronic asymptomatic chlamydial infection might contribute to the pathophysiology of atheroma.

Curr Biol, 1999 Feb 11, 9(3), 151 - 4
Four dimers of lambda repressor bound to two suitably spaced pairs of lambda operators form octamers and DNA loops over large distances; Revet B et al.; Transcription factors that are bound specifically to DNA often interact with each other over thousands of base pairs {1} {2} . Large DNA loops resulting from such interactions have been observed in Escherichia coli with the transcription factors deoR {3} and NtrC {4}, but such interactions are not, as yet, well understood . We propose that unique protein complexes, that are not present in solution, may form specifically on DNA . Their uniqueness would make it possible for them to interact tightly and specifically with each other . We used the repressor and operators of coliphage lambda to construct a model system in which to test our proposition . lambda repressor is a dimer at physiological concentrations, but forms tetramers and octamers at a hundredfold higher concentration . We predict that two lambda repressor dimers form a tetramer in vitro when bound to two lambda operators spaced 24 bp apart and that two such tetramers interact to form an octamer . We examined, in vitro, relaxed circular plasmid DNA in which such operator pairs were separated by 2,850 bp and 2,470 bp . Of these molecules, 29% formed loops as seen by electron microscopy (EM) . The loop increased the tightness of binding of lambda repressor to lambda operator . Consequently, repression of the lambda PR promoter in vivo was increased fourfold by the presence of a second pair of lambda operators, separated by a distance of 3,600 bp.

Curr Biol, 1999 Feb 11, 9(3), R86 - 9
Insect evolution: Redesigning the fruitfly; Gibson G; Homeotic mutations in Drosophila can result in dramatic phenotypes that suggest the possibility for rapid morphological evolution, but dissection of the genetic pathway downstream of Ultrabithorax is beginning to reveal how wing morphology may have evolved by more gradual transformations.

Development, 1999 Mar, 126(6), 1247 - 58
Mrj encodes a DnaJ-related co-chaperone that is essential for murine placental development; Hunter PJ et al.; We have identified a novel gene in a gene trap screen that encodes a protein related to the DnaJ co-chaperone in E . coli . The gene, named Mrj (mammalian relative of DnaJ) was expressed throughout development in both the embryo and placenta . Within the placenta, expression was particularly high in trophoblast giant cells but moderate levels were also observed in trophoblast cells of the chorion at embryonic day 8.5, and later in the labyrinth which arises from the attachment of the chorion to the allantois (a process called chorioallantoic fusion) . Insertion of the ROSAbetageo gene trap vector into the Mrj gene created a null allele . Homozygous Mrj mutants died at mid-gestation due to a failure of chorioallantoic fusion at embryonic day 8.5, which precluded formation of the mature placenta . At embryonic day 8.5, the chorion in mutants was morphologically normal and expressed the cell adhesion molecule beta4 integrin that is known to be required for chorioallantoic fusion . However, expression of the chorionic trophoblast-specific transcription factor genes Err2 and Gcm1 was significantly reduced . The mutants showed no abnormal phenotypes in other trophoblast cell types or in the embryo proper . This study indicates a previously unsuspected role for chaperone proteins in placental development and represents the first genetic analysis of DnaJ-related protein function in higher eukaryotes . Based on a survey of EST databases representing different mouse tissues and embryonic stages, there are 40 or more DnaJ-related genes in mammals . In addition to Mrj, at least two of these genes are also expressed in the developing mouse placenta . The specificity of the developmental defect in Mrj mutants suggests that each of these genes may have unique tissue and cellular activities.

Development, 1999 Mar, 126(6), 1175 - 87
The Drosophila kismet gene is related to chromatin-remodeling factors and is required for both segmentation and segment identity; Daubresse G et al.; The Drosophila kismet gene was identified in a screen for dominant suppressors of Polycomb, a repressor of homeotic genes . Here we show that kismet mutations suppress the Polycomb mutant phenotype by blocking the ectopic transcription of homeotic genes . Loss of zygotic kismet function causes homeotic transformations similar to those associated with loss-of-function mutations in the homeotic genes Sex combs reduced and Abdominal-B . kismet is also required for proper larval body segmentation . Loss of maternal kismet function causes segmentation defects similar to those caused by mutations in the pair-rule gene even-skipped . The kismet gene encodes several large nuclear proteins that are ubiquitously expressed along the anterior-posterior axis . The Kismet proteins contain a domain conserved in the trithorax group protein Brahma and related chromatin-remodeling factors, providing further evidence that alterations in chromatin structure are required to maintain the spatially restricted patterns of homeotic gene transcription.

Genes Dev, 1999 Feb 1, 13(3), 345 - 56
Double-strand end repair via the RecBC pathway in Escherichia coli primes DNA replication; Kuzminov A et al.; To study the relationship between homologous recombination and DNA replication in Escherichia coli, we monitored the behavior of phage lambda chromosomes, repressed or not for lambda gene activities . Recombination in our system is stimulated both by DNA replication and by experimentally introduced double-strand ends, supporting the idea that DNA replication generates occasional double-strand ends . We report that the RecBC recombinational pathway of E . coli uses double-strand ends to prime DNA synthesis, implying a circular relationship between DNA replication and recombination and suggesting that the primary role of recombination is in the repair of disintegrated replication forks arising during vegetative reproduction.

FEMS Microbiol Rev, 1998 Dec, 22(5), 341 - 52
Oxygen sensing by the global regulator, FNR: the role of the iron-sulfur cluster; Kiley PJ et al.; FNR is a global regulator that controls transcription of genes whose functions facilitate adaptation to growth under O2 limiting conditions . It has long been appreciated that the activity of FNR must be regulated by O2 availability, since FNR dependent gene expression is observed in vivo only under anaerobic conditions, while similar levels of this protein are present in both aerobic and anaerobic grown cells . Recent progress in this field has shown that anaerobically purified FNR contains a {4Fe-4S}2+ cluster and that this {4Fe-4S}2+ cluster is sufficiently unstable toward O2 to make it suitable as an O2 sensor . The presence of the {4Fe-4S} cluster increases dimerization of FNR which is correlated with an increase in site-specific DNA binding of FNR, a property expected of transcription factors of the FNR/CRP family . According to Mossbauer spectroscopy on purified FNR and cells containing overexpressed FNR, the {4Fe-4S}2+ cluster of FNR is converted by O2 to a {2Fe-2S}2+ in high yield . The {2Fe-2S}2+ cluster can be reconverted to the {4Fe-4S}2+ cluster on reduction with dithionite in vitro raising the possibility that the {2Fe-2S}2+ cluster is a biologically inactive intermediate which may be more readily available for reconstitution into the {4Fe-4S}2+ form than the Fe-free apoform . The ability to observe, by Mossbauer spectroscopy, the Fe-S clusters of FNR in cells containing high levels of FNR should be of value in further unraveling how FNR functions in vivo . Attempts to reduce the {4Fe-4S}2+ cluster of FNR with dithionite indicated that the redox potential of the +1/+2 couple is < or = -650 mV and that the {4Fe-4S}+ cluster form is, therefore, not likely to occur in vivo.

Eur J Biochem, 1998 Dec 15, 258(3), 1014 - 21
The mammalian small heat-shock protein Hsp20 forms dimers and is a poor chaperone; van de Klundert FA et al.; Hsp20 is one of the newly described members of the mammalian small heat-shock protein (sHsp) family . It occurs most abundantly in skeletal muscle and heart . We isolated clones for Hsp20 from a rat heart cDNA library, and expressed the protein in Escherichia coli to characterize this little known sHsp . Recombinant Hsp20 displayed similar far-ultraviolet circular dichroism spectra as the most closely related sHsp, alpha B-crystallin, but was less heat stable, denaturing upon heating to 50 degrees C . While other mammalian recombinant sHsps form large multimeric complexes, Hsp20 occurs in two complex sizes, 43-kDa dimers and 470-kDa multimers . The ratio between the two forms depends on protein concentration . Moreover, Hsp20 has a much lower chaperone-like activity than alpha B-crystallin, as indicated by its relatively poor capacity to diminish the reduction-induced aggregation of insulin B chains . Hsp20 is considerably shorter at the C-terminus and less polar than other sHsps, but 1H-NMR spectroscopy reveals that the last 10 residues are flexible, as in the other sHsps . Our findings suggest that Hsp20 is a special member of the sHsp family in being less heat stable and tending to form dimers . These properties, together with the shorter and less polar C-terminal extension, may contribute to the less effective chaperone-like activity.

Eur J Biochem, 1998 Dec 15, 258(3), 1009 - 13
An apyrase from Mimosa pudica contains N5,N10-methenyl tetrahydrofolate and is stimulated by light; Ghosh R et al.; An apyrase (NTP/NDPase) implicated in the response of Mimosa pudica to stimuli, such as touch, has been cloned, sequenced and expressed in Escherichia coli . While purifying and characterizing this enzyme, it was observed that a chromophore is associated with it, having absorption in the ultraviolet-A/blue region of the spectrum . The absorbance maximum of the chromophore, purified from the enzyme complex by gel filtration and HPLC, is around 350 nm . The chromophore has been identified as N5,N10-methenyl tetrahydrofolate (MTHF) by comparing the excitation and emission spectra of synthetic MTHF and the isolated cofactor, and by reconstitution of the enzyme complex with synthetic MTHF . Upon excitation with light (350 nm), an increase of apyrase activity was observed in the purified or reconstituted holoenzyme but not in the apoenzyme . The wavelength dependence of the light stimulation matched well with the fluorescence excitation spectra of the cofactor, MTHF . Possible implications of the results for signal transduction in M . pudica have been discussed.

Eur J Biochem, 1998 Dec 15, 258(3), 1001 - 8
FhuF, an iron-regulated protein of Escherichia coli with a new type of {2Fe-2S} center; Muller K et al.; We previously used fhuF as a sensitive reporter gene of the iron status of Escherichia coli . In this report, the fhuF gene was identified as open reading frame f262b at 99.2 min on the genome sequence map of E . coli K-12 . The FhuF protein was labeled with a His-tag and then purified to electrophoretic homogeneity . Based on sulfur determinations and Mossbauer and EPR spectroscopy, FhuF was identified as a {2Fe-2S} protein . The g values (gx = 1.886, gy = 1.961, gz = 1.994) and some of the Mossbauer parameters of FhuF obtained {oxidized protein as isolated: delta EQ,4.2K = 0.474 mm s-1; Fe3+ (reduced protein): delta EQ = 0.978 mm s-1} are not typical of common {2Fe-2S} proteins and indicate that FhuF has unusual structural properties . The primary sequence of FhuF does not show any sequence similarities to known {2Fe-2S} proteins . By site-directed mutagenesis, each of the six cysteines of FhuF was replaced by serine . EPR of the six reduced mutant proteins revealed that the terminal cysteine residues 244, 245, 256, and 259 form the {2Fe-2S}Cys4 cluster . Mutants having the Cys-to-Ser replacement at positions 244, 245, 256, or 259 did not complement a fhuF mutant . The motif Cys-Cys-Xaa10-Cys-Xaa2-Cys in FhuF differs considerably from the motif Cys-Xaa2-Cys-Xaa9-15-Cys-Xaa2-Cys found in other {2Fe-2S} proteins . The unusual Cys-Cys terminal group of the cluster may explain the atypical EPR and Mossbauer spectroscopic properties of the FhuF protein; possibly the tetrahedral symmetry at the ferric ion site is distorted . The phenotype of fhuF mutants and the structural features of the FhuF protein suggest that FhuF is involved in the reduction of ferric iron in cytoplasmic ferrioxamine B.

Eur J Biochem, 1998 Dec 15, 258(3), 994 - 1000
A chicken homolog of mammalian interleukin-1 beta: cDNA cloning and purification of active recombinant protein; Weining KC et al.; Upon induction with lipopolysaccharide (LPS) the chicken macrophage cell line HD-11 secretes an activity that stimulates the synthesis of a CXC chemokine in the chicken fibroblast cell line CEC-32 . We used a cDNA expression cloning strategy in COS cells to characterize this activity . The isolated cDNA clone codes for a polypeptide of 267 amino acids which lacks a hydrophobic N-terminal domain that could serve as secretory signal . Sequence homology and structural features indicate that this protein is the chicken homolog of mammalian interleukin-1 beta (ChIL-1 beta) . Northern blot analysis showed that ChIL-1 beta RNA is quickly induced in blood monocyte-derived macrophages reaching maximal levels within one hour after onset of LPS treatment . To test for biological activity of putative mature ChIL-1 beta, a cDNA fragment comprising amino acids 106 to 267 of the open reading frame was expressed in Escherichia coli so that the resulting polypeptide carried a histidine tag at its N-terminus for easy purification by nickel chelate affinity chromatography . Purified His-ChIL-1 beta potently induced CXC chemokine RNA synthesis in CEC-32 cells . When injected intravenously into adult chickens, it quickly induced a transient increase in serum corticosterone levels.

Eur J Biochem, 1998 Dec 15, 258(3), 923 - 8
Importance of heptameric ring integrity for activity of Escherichia coli ClpP; Thompson MW et al.; Radiation target analysis has been used to identify the minimal functional unit for expression of activity of ClpP, the proteolytic component of the ATP-dependent ClpAP protease . Radiation target sizes determined for small peptide hydrolysis, for ClpA activated and nucleotide-activated oligopeptide cleavage, and for ClpA-activated ATP-dependent protein degradation were 154, 118, and 160 kDa, respectively . Thus, the hydrolytic activity of ClpP, subunit M, 21,500, is dependent on the native oligomeric structure . The quaternary structure of ClpP determined by electron microscopy and hydrodynamic studies consists of two face-to-face seven-membered rings . The radiation target sizes are consistent with a requirement for conformational integrity of an entire ring for expression of hydrolytic activity . Radiation damage led to disruption of inter-ring contacts, giving rise to isolated rings of ClpP . Thus, contacts between rings of ClpP are less stable and more easily disrupted than contacts between subunits within the rings . Our data suggest that cooperative interactions between subunits within the ClpP rings are important for maintaining the active conformation of the proteolytic active site.

Biochim Biophys Acta, 1999 Jan 11, 1448(3), 463 - 72
Targeted delivery of multivalent phage display vectors into mammalian cells; Ivanenkov VV et al.; Novel peptide motives targeting endocytosing receptors were isolated from phage display libraries of random peptides by recovering internalized phage from mammalian cells . The peptide-presenting phage selected by internalization in HEp-2 and ECV304 human cells were taken up 1000- to 100,000-fold more efficiently than their parent libraries, and from 10 to 100 times faster than phage particles displaying integrin-binding peptides . A high degree of selectivity of phage uptake was observed in these cells: phage selected in ECV304 cells were internalized approximately 100-fold more efficiently in ECV304 cells than in HEp-2 cells . Likewise, phage selected in HEp-2 cells were subsequently taken up approximately 40-fold more efficiently by HEp-2 cells than by ECV304 cells . In multiple independent trials using a cyclic peptide library, an identical peptide sequence displayed on phage was internalized by and recovered from ECV304 cells . These findings indicate that the internalization process is highly selective, and is capable of capturing a specific peptide from 2 x 10(7) peptide variants . Immunofluorescence microscopy showed juxtanuclear localization of internalized phage . These results demonstrate the feasibility of using multivalent phage-display libraries to identify new targeting ligands for the intracellular delivery of macromolecules.

Biochim Biophys Acta, 1999 Jan 11, 1448(3), 450 - 62
Uptake and intracellular fate of phage display vectors in mammalian cells; Ivanenkov V et al.; Receptor-mediated endocytosis is exploited in experimental systems for selective delivery of genes and drugs into specific cells . To improve targeting efficiency of delivery vectors, we have used phage display technology to isolate novel ligands for endocytosed receptors . We show here that phage vectors internalized by mammalian cells via integrin-mediated endocytosis can be rescued by cell lysis and quantitated by infection of bacteria . Immediately following uptake, phage enter an intracellular compartment where they remain intact, with phage titer unaffected by the addition of chloroquine . Phage are then translocated to a second intracellular compartment in which they are inactivated and their titer affected by chloroquine . Immunofluorescence microscopy showed an association of the second compartment with supranuclear organelles . The ability to recover internalized phage in an infectious form from two distinctive intracellular compartments provides a means to select novel ligands from phage libraries for targeted delivery of macromolecules into mammalian cells.

J Biochem (Tokyo), 1999 Feb, 125(2), 422 - 9
Molecular structure of the amyloid-forming protein kappa I Bre; Steinrauf LK et al.; The molecular structure of the amyloid-forming Bence-Jones protein kappa I Bre has been determined by X-ray crystallography at 2.0 A resolution . The fragment from the kappa chain of immunoprotein contains 107 amino acid residues, and polymerizes in the crystal form into a giant helical spiral, surrounding a cylinder of water 50 A in diameter with a repeat of 77.56 A, containing 12 kappa molecules, plus another 12 molecules from neighboring parallel spirals . The resulting structure has many features which have been found or suggested from studies on the protein fibrils found in amyloid deposits . From the results of the X-ray crystal structure a hypothesis is presented for the structure and formation of the amyloid fibril.

J Biochem (Tokyo), 1999 Feb, 125(2), 399 - 405
The stabilized structural array of two HMG1/2-boxes endowed by a linker sequence between them is requisite for the effective binding of HMG1 with DNA; Saito K et al.; High mobility group (HMG) protein 1 contains two DNA binding motifs, called HMG1/2-boxes, linked with a linker region . The functional relationships between the two boxes and the mechanism of involvement of the linker region for effective binding of HMG1 were examined . The binding analyses of truncated HMG1 peptides with DNA indicated that the structural array of two boxes stabilizes the interaction of HMG1 with DNA . The mutation analyses of the linker region suggested that the region is equipped with tolerance for the deletion of a few amino acid residues to allow appropriate binding of the two boxes with DNA, and that the basic cluster in the linker sequence is in a position to interact with DNA . The existence of tolerance for the linker sequence was found to be conserved during the evolution of HMG1 protein homologues . A structural model for array of two boxes associating with DNA minor groove was constructed on the basis of the experimental results and energy minimization . The model proposes that the DNA binding region in HMG1 covers an 18 bp DNA region and induces its bending by about 140 degrees . The linker region may function to maintain the structural array of two HMG1/2-boxes by direct interaction with DNA.

J Biochem (Tokyo), 1999 Feb, 125(2), 348 - 53
Human renin-binding protein is the enzyme N-acetyl-D-glucosamine 2-epimerase; Takahashi S et al.; The existence of human renin-binding protein (RnBP) in the kidney has been shown by the isolation and characterization of a complex of porcine renin-human RnBP {S . Takahashi et al . (1985) J . Biochem . 97, 671-677} . However, the properties of the free form of human RnBP had not been understood, because of the limitation of materials . In the present study, we have expressed human RnBP in Escherichia coli JM 109 cells under the transcriptional control of taq promoter and purified it by conventional column chromatographies . The purified recombinant human RnBP (rhRnBP) exists as a dimer and inhibits porcine renin activity through formation of a complex of porcine renin with rhRnBP, the so-called high-molecular-weight renin . Moreover, the rhRnBP catalyzes the interconversion between N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-Mannosamine (ManNAc) with the apparent Km values of 21.3 mM for GlcNAc and 12.8 mM for ManNAc, and 0.13 mM for effector ATP . ATP is essential for the GlcNAc 2-epimerase activity of human RnBP . These results indicate that the human RnBP is a GlcNAc 2-epimerase.

J Biochem (Tokyo), 1999 Feb, 125(2), 328 - 33
Cell-free expression of two single-chain monoclonal antibodies against lysozyme: effect of domain arrangement on the expression; Merk H et al.; Single-chain antibodies (scFv), which can be produced in Escherichia coli cells, have been shown in numerous cases to be active in antigen binding . In the case of the two anti-lysozyme single-chain antibodies, scFvLH and scFvHL, which have the reversed arrangement of the variable domains of the heavy and light chains of the corresponding monoclonal antibodies, the expression level differs greatly when they are produced in Escherichia coli {Tsumoto et al . (1995) Biochem . Biophys . Res . Commun . 201, 546-551} . Although the expression level of scFvLH is high in vivo, the single chain antibody with the reversed orientation (scFvHL) was synthesized in a very low yield and no active product could be obtained . We report here the synthesis of these two anti-lysozyme single-chain antibodies in high yields and with high biological activities in a cell-free E . coli expression system in the presence of reduced and oxidized glutathione, protein disulfide isomerase (PDI), and chaperones . In immunological blotting assays, the synthesized scFvs with both arrangements exhibit specific binding activity to the corresponding antigens, hen egg-white lysozyme, and in an activity assay both inhibited the action of lysozyme . scFvLH is synthesized mainly as a product with the expected molecular weight, whereas scFvHL is produced with additional shorter fragments, suggesting that the low yield isolation through the expression in vivo is due to mistranslation or ribonucleolytic cleavage of the transcript . In the cell-free expression of scFv a certain amount of the product is precipitated but in the presence of chaperones the amount of soluble protein increased from 25 to 90% (PDI and chaperones) . The overall expression level and the specific biological activity, however, were hardly influenced . The system reported here can provide significant amounts of various scFv fragments regardless of the order of variable regions, including those which are hardly expressed in vivo.

J Biochem (Tokyo), 1999 Feb, 125(2), 297 - 304
Influence of chondroitin sulfate charge density, sulfate group position, and molecular mass on Cu2+-mediated oxidation of human low-density lipoproteins: effect of normal human plasma-derived chondroitin sulfate; Volpi N et al.; The effects of chondroitin sulfate samples with decreasing charge densities, different 4-sulfate/6-sulfate ratios, and various molecular masses on Cu2+-induced oxidation of human low-density lipoprotein (LDL) were evaluated by monitoring conjugated diene formation and the tryptophan fluorescence kinetics . Low-sulfated chondroitin sulfate (CS) from beef trachea had a very strong protective antioxidant effect . Quite similar behavior was observed for CS from pig trachea, and a fructose-containing polysaccharide with a chondroitin backbone from Escherichia coli was also strongly protective as to LDL oxidation . CS samples with decreasing charge densities proved effective in inhibiting LDL oxidation . A totally desulfated sample still exhibited a great capacity to protect LDL against oxidation . CS-4-sulfate samples (sulfate to carboxyl ratio of 0.62, about 65% 4-sulfate groups and 5% 6-sulfate groups) retained great ability to inhibit the Cu2+-mediated human LDL oxidation . CS fractions with different molecular masses were examined as possible inhibitors of LDL oxidation . Samples with molecular masses lower than about 8,570 (13-15 disaccharide units) were unable to protect human LDL from Cu2+-induced oxidation . Similar results were obtained on studying the degradation of tryptophan residues of the LDL protein moiety resulting from Cu2+ complexation through amino acid residues . A low-sulfated CS (sulfate to carboxyl ratio of 0.41, a molecular mass of about 15,600) having effective anti-oxidant properties as to metal-induced LDL oxidation was isolated from normal human plasma . The protective capacity as to Cu2+-mediated LDL oxidation of CS is discussed in relation to its structure, also considering the physiological role of plasma CS in relation to factors that can alter its properties.

Proc Natl Acad Sci U S A, 1999 Feb 16, 96(4), 1257 - 61
Crystal structure of a dimeric chymotrypsin inhibitor 2 mutant containing an inserted glutamine repeat; Chen YW et al.; We have constructed mutants of chymotrypsin inhibitor 2 with short glutamine repeats inserted into its inhibitory loop . These mutants oligomerize when expressed in Escherichia coli . The dimer of a mutant with four glutamines now has been crystallized, and its structure has been solved by molecular replacement by using the wild-type monomer as a search model . The structure of each half of the dimer is found to be the same as that of the wild-type monomer, except around the glutamine insertion . It was proposed that the components of the oligomers are held together by hydrogen bonds between the main-chain and side-chain amides of the glutamine repeats . Instead, they appear to form by swapping domains on folding in E . coli, and the glutamine repeats connecting the components of the dimers are disordered.

Arch Biochem Biophys, 1999 Feb 15, 362(2), 363 - 75
Inhibition of 3C protease from human rhinovirus strain 1B by peptidyl bromomethylketonehydrazides; Kati WM et al.; The gene coding for the 3C protease from human rhinovirus strain 1B was efficiently expressed in an Escherichia coli strain which also overexpressed the rare argU tRNA . The protease was isolated from inclusion bodies, refolded, and exhibited a kcat/Km = 3280 M-1 s-1 using an internally quenched peptidyl fluorogenic substrate . This continuous fluorogenic assay was used to measure the kinetics of 3C protease inhibition by several conventional peptidyl chloromethylketones as well as a novel series of compounds, the bromomethylketonehydrazides . Compounds containing the bromomethylketonehydrazide backbone and a glutamine-like side chain at the P1 position were potent, time-dependent inhibitors of rhinovirus 3C protease with kinact/Kinact values as high as 23,400 M-1 s-1 . The inhibitory activity of compounds containing modified P1 side chains suggests that the interactions between the P1 carboxamide group and the 3C protease contributes at least 30-fold to the kinact/Kinact rate constants for bromomethylketonehydrazide inhibition of 3C protease . Electrospray ionization mass spectrometry measurements of the molecular weights of native and inhibited 3C protease have established an inhibitory mechanism involving formation of a covalent adduct between the enzyme and the inhibitor with the loss of a bromide ion from the bromomethylketonehydrazide . Tryptic digestion of bromomethylketonehydrazide-inhibited 3C protease established adduct formation to a peptide corresponding to residues 145-154, a region which contains the active site cysteine-148 residue . The bromomethylketonehydrazides were fairly weak inhibitors of chymotrypsin, human elastase, and cathepsin B and several of these compounds also showed evidence for inhibition of human rhinovirus 1B replication in cell culture .

Nature, 1999 Feb 4, 397(6718), 417 - 20
Hybrid hydrogels assembled from synthetic polymers and coiled-coil protein domains; Wang C et al.; Stimuli-sensitive polymer hydrogels, which swell or shrink in response to changes in the environmental conditions, have been extensively investigated and used as 'smart' biomaterials and drug-delivery systems . Most of these responsive hydrogels are prepared from a limited number of synthetic polymers and their derivatives, such as copolymers of (meth)acrylic acid, acrylamide and N-isopropyl acrylamide . Water-soluble synthetic polymers have also been crosslinked with molecules of biological origin, such as oligopeptides and oligodeoxyribonucleotides, or with intact native proteins . Very often there are several factors influencing the relationship between structure and properties in these systems, making it difficult to engineer hydrogels with specified responses to particular stimuli . Here we report a hybrid hydrogel system assembled from water-soluble synthetic polymers and a well-defined protein-folding motif, the coiled coil . These hydrogels undergo temperature-induced collapse owing to the cooperative conformational transition of the coiled-coil protein domain . This system shows that well-characterized water-soluble synthetic polymers can be combined with well-defined folding motifs of proteins in hydrogels with engineered volume-change properties.

FEBS Lett, 1999 Jan 25, 443(2), 167 - 9
Chaperone-assisted expression of authentic bovine adrenodoxin reductase in Escherichia coli; Vonrhein C et al.; Adrenodoxin reductase is an essential component of the mitochondrial monooxygenase systems that are involved in the synthesis of steroid hormones and related compounds . After removing by mutagenesis a secondary ribosome binding site and an mRNA loop formed between the gene and the vector, large amounts of the enzyme could be produced in Escherichia coli by coexpression with the HSP60-chaperone system . The purified protein was homogeneous enough for reproducible crystallization . The crystals diffracted X-rays isotropically beyond 1.7 A resolution permitting a structure analysis.

FEBS Lett, 1999 Jan 25, 443(2), 85 - 8
Reactivity of glutaredoxins 1, 2 and 3 from Escherichia coli and protein disulfide isomerase towards glutathionyl-mixed disulfides in ribonuclease A; Lundstrom-Ljung J et al.; We have examined the activity of protein disulfide isomerase (PDI) and glutaredoxin (Grx) 1, 2 and 3 from Escherichia coli to catalyze the cleavage of glutathionylated ribonuclease A (RNase-SG) by 1 mM GSH to yield reduced RNase . Apparent Km values for RNase-SG were similar, 2-10 microM, for Grx 1, 3 and PDI but Grx I and Grx 3 showed 500-fold higher turnover numbers than PDI . The atypical Grx 2 also catalyzed deglutathionylation by GSH, but had higher Km and apparent turnover number values compared to the two classical Grx . Refolding of RNase in a glutathione redox buffer was catalyzed by PDI . However, it could be measured only after a characteristic lag phase that was shortened by all three E . coli Grxs in a concentration-dependent manner . A role of the glutaredoxin mechanism in the endoplasmic reticulum is suggested.

Cell, 1999 Jan 8, 96(1), 153 - 63
Trading places on DNA--a three-point switch underlies primer handoff from primase to the replicative DNA polymerase; Yuzhakov A et al.; This study reports a primase-to-polymerase switch in E . coli that closely links primase action with extension by DNA polymerase III holoenzyme . We find that primase tightly grips its RNA primer, protecting it from the action of other proteins . However, primase must be displaced before the beta sliding clamp can be assembled on the primed site . A single subunit of the holoenzyme, chi, is dedicated to this primase displacement task . The displacement mechanism depends on a third protein, SSB . Primase requires contact to SSB for its grip on the primed site . The chi subunit also binds SSB, upon which the primase-to-SSB contact is destabilized leading to dissociation of primase and assembly of beta onto the RNA primer . The conservation of this three-point switch, in which two proteins exchange places on DNA via mutually exclusive interaction with a third protein, is discussed.

Anal Chem, 1999 Feb 1, 71(3), 589 - 95
A dynamical investigation of acrylodan-labeled mutant phosphate binding protein; Lundgren JS et al.; The static and dynamical behavior of a fluorescently labeled mutant of the Escherichia coli periplasmic phosphate binding protein (PBP) was investigated through steady-state and time-resolved fluorescence spectroscopy . As a means of developing a biorecognition element for inorganic phosphate (P(i)), alanine-197 of PBP was replaced with a cysteine . This site was then labeled with an environmentally sensitive fluorophore . The fluorescence emission of the mutant PBP labeled with acrylodan (MPBP-AC) proved to be sensitive to micromolar concentrations of P(i), as indicated by a 50% increase in the steady-state emission intensity . Steady-state results indicated that the labeling protocol was specific for cys-197 only and did not label the wild-type PBP; thus, a site-selective labeling protocol was developed . Time-resolved measurements were used to determine the influence of the dynamics of MPBP-AC on the process of signal transduction . Time-resolved anisotropy measurements revealed that rotational dynamics were best described by a model with two independent motions: the global motion of the protein and the local motion of the acrylodan probe . The rates of both global and local rotational reorientation of MPBP-AC were faster when the protein was P(i)-bound rather than P(i)-free . This was a result of structural changes involving or surrounding both the P(i)-binding site (global changes) and the residues in near proximity to the fluorescent reporter group (local changes) . Recovery of the semiangle (theta) indicated that local structural changes in MPBP-AC took place when P(i) was bound to the protein . Acrylodan gained mobility when MPBP-AC bound P(i), as indicated by the fact that theta increased by approximately 5 degrees . In addition, dynamic quenching measurements confirmed that structural changes occurred locally near the cys-197 . Acrylodan became more accessible to iodide when MPBP-AC bound P(i), as demonstrated by the 35% increase in the value of the bimolecular quenching constant.

Biochim Biophys Acta, 1999 Jan 4, 1436(3), 616 - 27
Study on the activation of phospholipases A2 by purinergic agonists in rat submandibular ductal cells; Kabre E et al.; Extracellular ATP and benzoyl-ATP (Bz-ATP) increased the release of {3H}arachidonic acid ({3H}AA) from prelabeled rat submandibular gland (RSMG) ductal cells respectively two- and threefold . Both agonists also increased the release of {3H}AA from acini but at a lower level (+50% and +100% respectively) . Carbachol had no significant effect on either cellular population . In ductal cells phorbol myristate acetate, an activator of protein kinase C, slightly increased the basal release of {3H}AA but did not affect the release of {3H}AA in response to ATP . Staurosporine, an inhibitor of protein kinases, inhibited the response to the purines . The removal of calcium from the extracellular medium decreased the response to ATP and Bz-ATP . Only barium could partly substitute for calcium to restore the purinergic response . Zinc inhibited the release of {3H}AA . Permeabilization of the cells with streptolysin O (SLO) activated the calcium-independent phospholipase A2 activity (iPLA2) . The iPLA2, not the calcium-dependent PLA2 (cPLA2), released {3H}oleic acid ({3H}OA) from RSMG ductal cells . It is concluded that RSMG ducts have a higher PLA2 activity when compared to acini . This activity is accounted for by iPLA2 and cPLA2 . Both enzymes are activated by P2X agonists by a staurosporine-sensitive mechanism . Cells permeabilized with SLO or membranes from Escherichia coli as a substrate are not good models to study the regulation of these enzymes . In intact RSMG ductal cells the two activities can be distinguished by rather specific inhibitors, by different ionic conditions and also by the fatty acid used to label the cells.

Biochim Biophys Acta, 1999 Jan 4, 1436(3), 299 - 306
Characterization of recombinant guinea pig alkyl-dihydroxyacetonephosphate synthase expressed in Escherichia coli . Kinetics, chemical modification and mutagenesis; de Vet EC et al.; A recombinant form of guinea pig alkyl-dihydroxyacetonephosphate synthase, a key enzyme in the biosynthesis of ether phospholipids, was characterized . Kinetic analysis yielded evidence that the enzyme operates by a ping-pong rather than a sequential mechanism . Enzyme activity was irreversibly inhibited by N-ethylmaleimide, p-bromophenacylbromide and 2,4-dinitrofluorobenzene . The enzyme could be protected against the inactivation by either of these three compounds by the presence of saturating amounts of the substrate palmitoyl-dihydroxyacetonephosphate . The rate of inactivation of the enzyme by p-bromophenacylbromide was strongly pH dependent and the highest at alkaline conditions . Collectively, these results are indicative of cysteine, histidine and lysine residues, respectively, at or close to the active site . The divalent cations Mg2+, Zn2+ and Mn2+ were found to be inhibitors of enzymatic activity, whereas Ca2+ had no effect . Mutational analysis showed that histidine 617 is an essential amino acid for enzymatic activity: replacement of this residue by alanine resulted in complete loss of enzymatic activity . A recombinant enzyme with the C-terminal five amino acids deleted was shown to be inactive, indicating an important role of the C-terminus for catalytic activity.

Biochim Biophys Acta, 1999 Jan 11, 1429(2), 512 - 5
cDNA sequence and overexpression of chloroplast chaperonin 21 from Arabidopsis thaliana; Hirohashi T et al.; Higher plant chloroplasts contain a 21-kDa protein, chaperonin 21 (Cpn21), that is a functional homolog of the chaperonin 10 (Cpn10) . The chloroplast Cpn21 polypeptide consists of two Cpn10-like domains fused together in tandem . We describe here the cDNA sequence of the Cpn21 (AtCpn21) precursor protein from Arabidopsis thaliana . The deduced amino acid sequence of the AtCpn21 precursor protein, 253 amino acids long, shows 61% identity with the spinach Cpn21 protein . The AtCpn21 precursor protein contains the typical chloroplast transit peptide of 51 amino acids at its aminoterminus and the two Cpn10-like domains which exhibits 46% sequence identity to each other . The predicted mature-sized polypeptide of AtCpn21 was expressed in Escherichia coli as a soluble 21-kDa protein . Gel-filtration and chemical cross-linking analyses showed that the recombinant mature AtCpn21 protein forms a stable homo-oligomer composed of three or four polypeptides.

Biochim Biophys Acta, 1999 Jan 11, 1429(2), 401 - 10
High expression and steady-state kinetic characterization of methionine site-directed mutants of Escherichia coli methionyl- and selenomethionyl-dihydrofolate reductase; Shaw D et al.; A high expression system that produces Escherichia coli dihydrofolate reductase (DHFR) at 30% total cellular protein was constructed . This expression vector, named pCOCK, allowed for the purification of nearly 100 mg of homogeneous DHFR from a 11 bacterial culture . A simple, single Q-Sepharose anion exchange column purification was developed on an FPLC instrument . Methionine site-directed mutants were constructed in DHFR to assess the role of Met within the enzymes . These mutants consisted of a Met16leucine (Leu), Met20Leu, Met42Leu, Met92Leu, Met16,20Leu and Met16,20,42Leu . Steady-state kinetic studies showed that the Met16Leu, Met42Leu and Met92Leu mutants possessed essentially the same kcat, Km(DHF) and Km(NADPH) as that of wild-type (wt) DHFR (13.7 s-1, 0.97 microM and 2.52 microM, respectively) . Mutants which contained a Leu at position 20 possessed substantially elevated specific activity and kcat values . The specific activity and kcat of wt, Met20Leu, Met16,20Leu and Met16,20,42Leu were 45.9, 92.7, 90.2 and 172 mumol/min/mg and 13.7, 24.6, 25.2 and 52.7 s-1, respectively . Upon substitution of Met by selenomethionine (SeMet) in the aforementioned mutants, further information as to the effect of SeMet incorporation into proteins was ascertained . Steady-state kinetic parameters of the SeMet substituted Met16Leu, Met20Leu, Met42Leu and Met92Leu mutants were nearly identical to those of their Met containing counterparts . These data indicate that Met apparently has a limited role in the protein structure and function of DHFR and that SeMet incorporation has no effect on the steady-state kinetic constants of DHFR.

Biochim Biophys Acta, 1999 Jan 11, 1429(2), 331 - 41
Cloning of a cDNA encoding SjIrV1, a Schistosoma japonicum calcium-binding protein similar to calnexin, and expression of the recombinant protein in Escherichia coli; Hooker CW et al.; Membrane-associated proteins were isolated from adult Philippine strain Schistosoma japonicum by partitioning into the detergent phase of Triton X-114 . A rabbit polyclonal antiserum raised against these proteins was used to screen an S . japonicum expression cDNA library . Positive clones were identified which encoded the species orthologue of SmIrV1, a Schistosoma mansoni protein which was initially identified by screening with sera from mice protectively vaccinated with irradiated cercariae {Hawn et al., J . Biol . Chem . 268 (1993) 7692-7698} . The S . japonicum molecule, which we term SjIrV1, is 83% identical to SmIrV1 at the predicted amino acid level and is a member of the calreticulin family of non-EF-hand, calcium-binding proteins . The Chinese strain S . japonicum orthologue of SjIrV1 was obtained by screening with the radiolabelled insert of the Philippine strain clone . Northern blot analysis revealed a single message of around 2.4 kb and gave no indication of alternative splicing . Southern blot analysis gave a simple pattern, indicating a single-copy gene, and showed a single restriction fragment length polymorphism between the genomes of Chinese and Philippine strains of S . japonicum . Recombinant, full-length SjIrV1 was expressed with a hexahistidine tag in Escherichia coli and the recombinant protein isolated by nickel-chelate chromatography . Recombinant SjIrV1 was shown to exhibit calcium-dependent, differential electrophoretic migration and to bind ruthenium red in the absence but not in the presence of calcium ions . The presence of conserved Ca(2+)-binding motifs predicted from the primary sequence, together with the Ca(2+)-dependent electrophoretic mobility of recombinant SjIrV1, confirmed that SjIrV1 was a functional calcium-binding protein.

Biochim Biophys Acta, 1999 Jan 29, 1423(1), R45 - 51
LXIII Cold Spring Harbor Symposium on Quantitative Biology: Mechanisms of Transcription, 3-8 June 1998; Emerson BM et al.; A new perspective is emerging in the transcription field towards understanding gene regulation not only at its most fundamental level but also in the context of chromatin, nuclear compartmentalization, and physiological processes . This direction is being fueled by several key observations . Among them is the discovery of multi-protein complexes whose components reveal a link between gene activity, nuclear structure, and cellular signaling pathways . This information will no doubt be extended by identifying expanded regulatory circuitry using the microchip oligonucleotide array technology . In addition to elucidating the regulatory consequences of these intricate connections, another frontier will be to analyze gene expression within chromosomes . This requires deciphering the mechanism of action of a variety of DNA elements that create a genetic domain such as locus control regions, distal enhancers, insulators, silencers, and matrix attachment regions . Hopefully, with the development of new assays these elements can be as rigorously defined as promoters have been . We can also look forward to capturing critical transcriptional processes by increasingly refined structural analyses . Thus, the scope of problems being addressed in gene regulation has been greatly expanded and the opportunity exists to answer very sophisticated questions in the future.

Zentralbl Bakteriol, 1998 Dec, 288(4), 501 - 8
Adhesion of Helicobacter pylori to yeast cells; Ansorg R et al.; In order to get information as to whether direct interaction of H . pylori and yeasts may modulate the course of H . pylori infections, the adhesion of H . pylori to C . albicans, C . glabrata, C . guilliermondii, C . krusei, C . parapsilosis, C . tropicalis and S . cereviseae was investigated . H . pylori adhered significantly more frequently to C . tropicalis (adhesion ratio > 10%) than to the other yeasts (adhesion ratios < 5%) . On an average, no significant difference to the adhesion ratios of E . coli and S . aureus was found . Electron microscopic examinations showed that H . pylori cells contacted the cells of C . tropicalis either by knob-like structures or by close surface-to-surface adhesion . Cholesterol-depleted H . pylori cells adhered to the yeast no more than cholesterol-carrying cells . There was no indication that a direct cooperation with yeasts plays a role in H . pylori infections.

J Clin Microbiol, 1999 Mar, 37(3), 548 - 52
The Borrelia burgdorferi 37-kilodalton immunoblot band (P37) used in serodiagnosis of early lyme disease is the flaA gene product; Gilmore RD Jr et al.; The 37-kDa protein (P37) of Borrelia burgdorferi is an antigen that elicits an early immunoglobulin M (IgM) antibody response in Lyme disease patients . The P37 gene was cloned from a B . burgdorferi genomic library by screening with antibody from a Lyme disease patient who had developed a prominent humoral response to the P37 antigen . DNA sequence analysis of this clone revealed the identity of P37 to be FlaA, an outer sheath protein of the periplasmic flagella . Recombinant P37 expression was accomplished in Escherichia coli by using a gene construct with the leader peptide deleted and fused to a 38-kDa E . coli protein . The recombinant antigen was reactive in IgM immunoblots using serum samples from patients clinically diagnosed with early Lyme disease that had been scored positive for B . burgdorferi anti-P37 reactivity . Lyme disease patient samples serologically negative for the B . burgdorferi P37 protein did not react with the recombinant . Recombinant P37 may be a useful component of a set of defined antigens for the serodiagnosis of early Lyme disease . This protein can be utilized as a marker in diagnostic immunoblots, aiding in the standardization of the present generation of IgM serologic tests.

Am J Obstet Gynecol, 1999 Feb, 180(2 Pt 1), 429 - 34
Differential fetal and maternal contributions to the cytokine milieu in a murine model of infection-induced preterm birth; Hirsch E et al.; OBJECTIVE: The aim of the study was to determine the relative productions by maternal and fetal tissues of proinflammatory and anti-inflammatory cytokines in a murine model of infection-induced preterm delivery . STUDY DESIGN: The right uterine horns of CD-1 female mice at 14.5 days of a 19- to 20-day gestation were inoculated with either sterile media or live Escherichia coli . The concentrations of cytokines within uteri, placentas, membranes, and fetal lower body segments were determined by enzyme-linked immunosorbent assay at various times after inoculation . RESULTS: All infected tissues showed large, time-dependent increases in interleukin 1alpha, interleukin 1beta, and interleukin 6 . These increases were maximal 13 hours after infection and were highest in uteri (15-60 times levels in uninfected tissues) . Increases in tumor necrosis factor alpha and interleukin 1 receptor antagonist were much smaller (3 to 5 times) and were confined to the uterus . Although the uterus contained the greatest concentrations of interleukin 1alpha, interleukin 1beta, interleukin 6, and tumor necrosis factor alpha, fetal bodies and placentas contained the highest levels of interleukin 1 receptor antagonist . CONCLUSIONS: Time-dependent increases in maternal and fetal cytokines occurred after acute bacterial infection in this murine model . The fetus and placenta may be the most significant sources of the anti-inflammatory cytokine interleukin 1 receptor antagonist during pregnancy, whereas the uterus appears to be a more important source of interleukin 1, interleukin 6, and tumor necrosis factor alpha . Interleukin 1 receptor antagonist levels within uteri were insufficiently high to effectively inhibit interleukin 1 activity during infection.

J Biol Chem, 1999 Feb 19, 274(8), 5245 - 51
Activation of Rho-dependent transcription termination by NusG . Dependence on terminator location and acceleration of RNA release; Burns CM et al.; There is a kinetic limitation to Rho function at the first intragenic terminator in the lacZ gene (tiZ1) which can be overcome by NusG: Rho can terminate transcription with slowly moving, but not rapidly moving, RNA polymerase unless NusG is also present . Here we report further studies with two other Rho-dependent terminators that are not kinetically limited (tiZ2 and lambda tR1) which show that the requirement for NusG depends on the properties of the terminator and its location in the transcription unit . NusG is also shown to increase the rate of Rho-mediated dissociation of transcription complexes arrested at a specific termination stop point in the tiZ1 region and the rates of dissociation with three different Rho factors and two different terminators correlated with their sensitivity to RNA polymerase elongation kinetics . These results suggest a model of NusG function which involves an alteration in the susceptibility of the transcription complex to Rho action which allows termination to occur within the short kinetic window when RNA polymerase is traversing the termination region.

J Biol Chem, 1999 Feb 19, 274(8), 5114 - 9
Non-lamellar structure and negative charges of lipopolysaccharides required for efficient folding of outer membrane protein PhoE of Escherichia coli; de Cock H et al.; Lipopolysaccharides (LPS) are amphiphilic molecules in the outer leaflet of the bacterial outer membrane . Recently, an early role for LPS in the folding of outer membrane porin PhoE was demonstrated in vitro . In order to elucidate the molecular mechanism of LPS-protein interactions, folding of PhoE protein was studied with a large set of well characterized LPS chemotypes . We demonstrate that negative charges in the inner core region contribute to the high efficiency of folding of PhoE protein . In addition, the supramolecular structure of the LPS aggregate seems to be important . LPS with a lipid A part that prefers a lamellar or a direct micellar structure and a high state of order of its acyl chains is much less efficient to support folding as compared with LPS with lipid A that prefers a non-lamellar structure and a low acyl chain order . These in vitro data indicate that extensive interactions between the core and lipid A region of LPS with the protein are required to support protein folding . The LPS-PhoE binding might be promoted by the presence of hydroxy fatty acids in the lipid A moiety of LPS.

J Biol Chem, 1999 Feb 19, 274(8), 5078 - 82
Presence of a structurally novel type ribulose-bisphosphate carboxylase/oxygenase in the hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1; Ezaki S et al.; We have characterized the gene encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of the hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1 . The gene encoded a protein consisting of 444 amino acid residues, corresponding in size to the large subunit of previously reported Rubiscos . Rubisco of P . kodakaraensis KOD1 (Pk-Rubisco) showed only 51.4% similarity with the large subunit of type I Rubisco from spinach and 47.3% with that of type II Rubisco from Rhodospirillum rubrum, suggesting that the enzyme was not a member of either type . Active site residues identified from type I and type II Rubiscos were conserved . We expressed the gene in Escherichia coli, and we obtained a soluble protein with the expected molecular mass and N-terminal amino acid sequence . Purification of the recombinant protein revealed that Pk-Rubisco was an L8 type homo-octamer . Pk-Rubisco showed highest specific activity of 19.8 x 10(3) nmol of CO2 fixed per min/mg, and a tau value of 310 at 90 degreesC, both higher than any previously characterized Rubisco . The optimum pH was 8.3, and the enzyme possessed extreme thermostability, with a half-life of 15 h at 80 degreesC . Northern blot analysis demonstrated that the gene was transcribed in P . kodakaraensis KOD1 . Furthermore, Western blot analysis with cell-free extract of P . kodakaraensis KOD1 clearly indicated the presence of Pk-Rubisco in the native host cells.

J Biol Chem, 1999 Feb 19, 274(8), 4977 - 84
Mutagenesis of the conserved residue Glu259 of Gsalpha demonstrates the importance of interactions between switches 2 and 3 for activation; Warner DR et al.; We previously reported that substitution of Arg258 within the switch 3 region of Gsalpha impaired activation and increased basal GDP release due to loss of an interaction between the helical and GTPase domains (Warner, D . R., Weng, G., Yu, S., Matalon, R., and Weinstein, L . S . (1998) J Biol . Chem . 273, 23976-23983) . The adjacent residue (Glu259) is strictly conserved in G protein alpha-subunits and is predicted to be important in activation . To determine the importance of Glu259, this residue was mutated to Ala (Gsalpha-E259A), Gln (Gsalpha-E259Q), Asp (Gsalpha-E259D), or Val (Gsalpha-E259V), and the properties of in vitro translation products were examined . The Gsalpha-E259V was studied because this mutation was identified in a patient with Albright hereditary osteodystrophy . S49 cyc reconstitution assays demonstrated that Gsalpha-E259D stimulated adenylyl cyclase normally in the presence of GTPgammaS but was less efficient with isoproterenol or AlF4- . The other mutants had more severely impaired effector activation, particularly in response to AlF4- . In trypsin protection assays, GTPgammaS was a more effective activator than AlF4- for all mutants, with Gsalpha-E259D being the least severely impaired . For Gsalpha-E259D, the AlF4--induced activation defect was more pronounced at low Mg2+ concentrations . Gsalpha-E259D and Gsalpha-E259A purified from Escherichia coli had normal rates of GDP release (as assessed by the rate GTPgammaS binding) . However, for both mutants, the ability of AlF4- to decrease the rate of GTPgammaS binding was impaired, suggesting that they bound AlF4- more poorly . GTPgammaS bound to purified Gsalpha-E259D irreversibly in the presence of 1 mM free Mg2+, but dissociated readily at micromolar concentrations . Sucrose density gradient analysis of in vitro translates demonstrated that all mutants except Gsalpha-E259V bind to beta gamma at 0 degreesC and were stable at higher temperatures . In the active conformation Glu259 interacts with conserved residues in the switch 2 region that are important in maintaining both the active state and AlF4- in the guanine nucleotide binding pocket . Although both Gsalpha Arg258 and Glu259 are critical for activation, the mechanisms by which these residues affect Gsalpha protein activation are distinct.

J Biol Chem, 1999 Feb 19, 274(8), 4715 - 21
Distinct "assisted" and "spontaneous" mechanisms for the insertion of polytopic chlorophyll-binding proteins into the thylakoid membrane; Kim SJ et al.; The biogenesis of several bacterial polytopic membrane proteins has been shown to require signal recognition particle (SRP) and protein transport machinery, and one such protein, the major light-harvesting chlorophyll-binding protein (LHCP) exhibits these requirements in chloroplasts . In this report we have used in vitro insertion assays to analyze four additional members of the chlorophyll-a/b-binding protein family . We show that two members, Lhca1 and Lhcb5, display an absolute requirement for stroma, nucleoside triphosphates, and protein transport apparatus, indicating an "assisted" pathway that probably resembles that of LHCP . Two other members, however, namely an early light-inducible protein 2 (Elip2) and photosystem II subunit S (PsbS), can insert efficiently in the complete absence of SRP, SecA activity, nucleoside triphosphates, or a functional Sec system . The data suggest a possibly spontaneous insertion mechanism that, to date, has been characterized only for simple single-span proteins . Of the membrane proteins whose insertion into thylakoids has been analyzed, five have now been shown to insert by a SRP/Sec-independent mechanism, suggesting that this is a mainstream form of targeting pathway . We also show that PsbS and Elip2 molecules are capable of following either "unassisted" or assisted pathways, and we discuss the implications for the mechanism and role of SRP in chloroplasts.

J Biol Chem, 1999 Feb 19, 274(8), 4613 - 9
The Aspergillus nidulans GATA factor SREA is involved in regulation of siderophore biosynthesis and control of iron uptake; Haas H et al.; A gene encoding a new GATA factor from Aspergillus nidulans, sreA, was isolated and characterized . SREA displays homology to two fungal regulators of siderophore biosynthesis: about 30% overall identity to SRE from Neurospora crassa and about 50% identity to URBS1 from Ustilago maydis over a stretch of 200 amino acid residues containing two GATA-type zinc finger motifs and a cysteine-rich region . This putative DNA binding domain, expressed as a fusion protein in Escherichia coli, specifically binds to GATA sequence motifs . Deletion of sreA results in derepression of L-ornithine-N5-oxygenase activity and consequently in derepression of the biosynthesis of the hydroxamate siderophore N,N',N"-triacetyl fusarinine under sufficient iron supply in A . nidulans . Transcription of sreA is confined to high iron conditions, underscoring the function of SREA as a repressor of siderophore biosynthesis under sufficient iron supply . Nevertheless, overexpression of sreA does not result in repression of siderophore synthesis under low iron conditions, suggesting additional mechanisms involved in this regulatory circuit . Consistent with increased sensitivity to the iron-activated antibiotics phleomycin and streptonigrin, the sreA deletion mutant displays increased accumulation of 59Fe . These results demonstrate that SREA plays a central role in iron uptake in addition to siderophore biosynthesis.

J Biol Chem, 1999 Feb 19, 274(8), 4570 - 6
Cloning and characterization of human MMP-23, a new matrix metalloproteinase predominantly expressed in reproductive tissues and lacking conserved domains in other family members; Velasco G et al.; A cDNA encoding a new human matrix metalloproteinase (MMP), tentatively called MMP-23, has been cloned from an ovary cDNA library . This protein exhibits sequence similarity with MMPs, but displays a different domain structure . Thus, MMP-23 lacks a recognizable signal sequence and has a short prodomain, although it contains a single cysteine residue that can be part of the cysteine-switch mechanism operating for maintaining enzyme latency . The C-terminal domain is considerably shortened and shows no sequence similarity to hemopexin, whereas all human MMPs, with the exception of matrilysin, contain four hemopexin-like repeats . Furthermore, MMP-23 is devoid of structural features distinctive of the diverse MMP subclasses, including the specific residues located close to the zinc-binding site in collagenases, the transmembrane domain of membrane-type MMPs, or the fibronectin-like domain of gelatinases . Fluorescent in situ hybridization experiments showed that the human MMP-23 gene maps to 1p36, a location which differs from all MMP genes mapped to date . Recombinant MMP-23 produced in Escherichia coli exhibits low, but significant proteolytic activity against a synthetic substrate commonly used for assaying MMPs . Northern blot analysis demonstrated that MMP-23 is predominantly expressed in ovary, testis, and prostate, suggesting that this new MMP may play a specialized role in reproductive processes.

J Biol Chem, 1999 Feb 19, 274(8), 4532 - 6
Proline isomerization is unlikely to be the cause of slow annealing and reactivation during the folding of alkaline phosphatase; Dirnbach E et al.; The in vitro folding of Escherichia coli alkaline phosphatase (AP) from the guanidine hydrochloride (GdnHCl) denatured state is characterized by a significant slow phase in the post activational recovery of native protein lability (probed by the susceptibility to GdnHCl denaturation and occurring on the time scale of days) as well as a slow phase in the recovery of activity (on the time scale of minutes) . Slow folding events have often been attributed to cis-trans isomerizations of X-Pro peptide bonds, a plausible explanation for AP, which contains 21 prolines per subunit . To investigate the role of proline isomerization in the two measures of refolding mentioned above, we have performed "double-jump" GdnHCl denaturation/renaturation experiments, with a third jump, where the rate of unfolding of refolded protein upon exposure to denaturant was added to assess the rate of change of lability . Our measurements of the time evolution of both the lability and the reactivation of refolded AP as a function of denaturation time show that proline isomerization is unlikely to be the cause of either of these slow events in the refolding of AP . The conclusions are further confirmed by the absence of proline isomerization effects when AP is refolded in the presence of human and periplasmic E . coli peptidyl-prolyl isomerase.

J Protein Chem, 1998 Nov, 17(8), 757 - 63
Interactions between NFkappaB and its inhibitor ikappaB: biophysical characterization of a NFkappaB/ikappaB-alpha complex; Li T et al.; The N-terminal domain (1-318 amino acids) of mouse NFkappaB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein . Its complex with a full-length ikappaB-alpha (MAD3, 1-317 amino acids) molecule was generated by binding the E . coli-derived ikappaB-alpha to the purified NFkappaB and purifying the complex by sequential chromatography . The stoichiometry of NFkappaB to ikappaB in the complex was determined to be 2 to 1 by light scattering and SDS-polyacrylamide gel electrophoresis . The secondary structure of the NFkappaB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFkappaB occurs upon binding of DNA . The FTIR spectrum of the NFkappaB/ikappaB complex indicates that its secondary structure is composed of 17% alpha-helix, 39% beta-strand, 18% irregular structures, and 26% beta-turns and loops . By comparing these data to the FTIR data for NFkappaB alone, it is concluded that the ikappaB (MAD3) in the complex contains 35% alpha-helix, 27% beta-strand, 22% irregular structures, and 16% beta-turns and loops . Circular dichroism (CD) analysis of a shorter form of ikappaB (pp40) indicates that it contains at least 20% alpha-helix and that the ikappaB subunit accounts for nearly all of the alpha-helix present in the NFkappaB/ikappaB complex, consistent with the FTIR results . The stabilities of NFkappaB, ikappaB, and their complex against heat-induced denaturation were investigated by following changes in CD signal . The results indicate that the thermal stability of ikappaB is enhanced upon the formation of the NFkappaB/ikappaB complex.

J Protein Chem, 1998 Nov, 17(8), 745 - 55
The sequential mechanism of guanidine hydrochloride-induced denaturation of cAMP receptor protein from Escherichia coli . A fluorescent study using 8-anilino-1-naphthalenesulfonic acid; Malecki J et al.; cAMP receptor protein (CRP) regulates expression of a number of genes in Escherichia coli . The protein is a homodimer and each monomer is folded into two structural domains . The biological activation of CRP upon cAMP binding may involve the subunit realignment as well as reorientation between the domains within each subunit . In order to study the interactions between the subunits or domains, we performed stopped-flow measurements of the guanidine hydrochloride (GuHCI)-induced denaturation of CRP . The changes in CRP structure induced by GuHCl were monitored using both intrinsic Trp fluorescence as well as the fluorescence of an extrinsic probe, 8-anilino-1-Naphthalenesulfonic acid (ANS) . Results of CRP denaturation using Trp fluorescence detection are consistent with a two-step model {Malecki, and Wasylewski, (1997), Eur . J . Biochem . 243, 660}, where the dissociation of dimer into subunits is followed by the monomer unfolding . The denaturation of CRP monitored by ANS fluorescence reveals the existence of two additional processes . One occurs before the dissociation of CRP into subunits, whereas the second takes place after the dissociation, but prior to proper subunit unfolding . These additional processes suggest that CRP denaturation is described by a more complicated mechanism than a simple three-state equilibrium and may involve additional changes in both inter- and intrasubunit interactions . We also report the effect of cAMP on the kinetics of CRP subunit unfolding and refolding.

Mol Microbiol, 1998 Dec, 30(5), 1113 - 22
Promoter element spacing controls basal expression and light inducibility of the cyanobacterial secA gene; Mazouni K et al.; The Synechocystis PCC6803 secA gene was found to be essential for cell viability and to be transcriptionally controlled by the redox state of the cells . The basic promoter (BP, -71 to +47 relative to the transcription start site) is controlled by three cis-acting elements, which together mediate the fourfold light induction of BP activity . The positively acting element (PE1, -361 to -71) upstream of BP exerts a twofold stimulation of BP; the negative element (NE, +47 to +104) downstream of BP decreases BP strength about sixfold . The PE2 element (+104 to +175) lying in the coding sequence overcomes NE-dependent downregulation of BP . BP harbours Escherichia coli sigma70-like promoter elements -35 (5'-TTGAat-3') and -10 (5'-TAagAT-3') . The -10 motif, which has the features of an 'extended -10' box, is absolutely essential to promoter activity . The -35 hexamer is critical to the enhancement of promoter strength above BP level and to light inducibility, both features involving regulatory elements flanking BP . Most interestingly, reducing the length of the 30 bp spacing between the -35 and -10 boxes down to 17 bp was found to increase promoter activity and to confer light inducibility to BP . This demonstrates that promoter element spacing controls basal expression and light inducibility of the secA gene.

Mol Microbiol, 1998 Dec, 30(5), 1067 - 79
Conversion to bidirectional replication after unidirectional initiation from R1 plasmid origin integrated at oriC in Escherichia coli; Maisnier-Patin S et al.; The cell division phenotypes of Escherichia coli with its chromosome replication driven by oriR (from plasmid R1) were examined by fluorescence microscopy and flow cytometry . Chromosome replication patterns in these strains were followed by marker frequency analyses . In one of the strains, the unidirectional oriR was integrated so that the replication fork moved clockwise from the oriC region, and bacterial growth and division were similar to those of the wild-type parent . The bacteria were able to convert the unidirectional initiation from oriR into bidirectional replication . The site for conversion of uni- to bidirectional replication seemed to be localized and could be mapped genetically within 6 min to the immediate right of the minimal oriC . Replication starting in the counterclockwise direction from the R1 replicon integrated at the same site in the opposite orientation could not be described as either bi- or unidirectional, as no single predominant origin could be discerned from the more or less flat marker frequency pattern . These strains also showed extensive filamentation, irregular nucleoid distribution and the presence of anucleate cells, indicative of segregation and division defects . Comparison among intR1 derivatives differing in the position of the integrated oriR relative to the chromosome origin suggested that the oriC sequence itself was dispensable for the conversion to bidirectionality . However, passage of the replication fork over the 6 min region to the right of oriC seemed important for the bidirectional replication pattern and normal cell division phenotype.

Mol Microbiol, 1998 Dec, 30(5), 911 - 21
Enteropathogenic and enterohaemorrhagic Escherichia coli: more subversive elements; Frankel G et al.; Enteropathogenic (EPEC) and enterohaemorrhagic Escherichia coli (EHEC) constitute a significant risk to human health worldwide . Both pathogens colonize the intestinal mucosa and, by subverting intestinal epithelial cell function, produce a characteristic histopathological feature known as the 'attaching and effacing' (A/E) lesion . Although EPEC was the first E . coli to be associated with human disease in the 1940s and 1950s, it was not until the late 1980s and early 1990s that the mechanisms and bacterial gene products used to induce this complex brush border membrane lesion and diarrhoeal disease started to be unravelled . During the past few months, there has been a burst of new data that have revolutionized some basic concepts of the molecular basis of bacterial pathogenesis in general and EPEC pathogenesis in particular . Major breakthroughs and developments in the genetic basis of A/E lesion formation, signal transduction, protein translocation, host cell receptors and intestinal colonization are highlighted in this review.

Mol Microbiol, 1998 Dec, 30(5), 905 - 10
SOS and Mayday: multiple inducible mutagenic pathways in Escherichia coli; Humayun MZ; Environmental and physiological stress conditions can transiently alter the fidelity of DNA replication . The DNA damage-mediated SOS response in Escherichia coli is the best-known example of such an 'inducible mutagenesis' or 'transient mutator' pathway . Emerging evidence suggests the existence of a number of other stress-inducible pathways that also affect the fidelity of replication . Among the more provocative recent findings are UVM, an SOS-independent damage-inducible mutagenic pathway, and a new recA-dependent but umuD/C-independent pathway that appears to be provoked by translational stress . These findings alter our view of inducible mutagenesis, and anticipate the existence of previously unrecognized links between protein synthesis and DNA replication.

FEMS Microbiol Lett, 1999 Feb 1, 171(1), 37 - 42
A consensus sequence for the Rhodospirillaceae SOS operators; Labazi M et al.; The sequences controlling the expression of the Rhodobacter capsulatus recA and uvrA genes belonging to the SOS DNA repair system have been identified by PCR mutagenesis . Data obtained demonstrated that the GTTCN7GTAC and GAACN7GAAC motifs present upstream of the recA gene and the GTTCN7GTTC motif found upstream of the uvrA gene are required for their respective DNA damage-mediated induction . Alignment of recA promoters of R . capsulatus, Rhodobacter sphaeroides and Rhodopseudomonas viridis with the uvrA promoters of R . capsulatus and R . sphaeroides has identified the consensus sequence GTTCVYVYTWTGTTC as the SOS operator site of the Rhodospirillaceae family.

Vaccine, 1999 Jan 28, 17(4), 353 - 61
Immunogenicity of recombinant Escherichia coli expressing the omp31 gene of Brucella melitensis in BALB/c mice; Guilloteau LA et al.; BALB/c mice were immunized with recombinant Escherichia coli expressing the omp31 gene of Brucella melitensis, a gene coding for a major outer membrane protein . Immunization resulted in the production of specific antibodies to B . melitensis in the serum, the production of which was considerably increased after boosting with a dose ten times lower than the first . A significant specific proliferative response of immune spleen cells to B . melitensis was observed 5 weeks after the first immunization but this response did not persist . Despite the induction of systemic humoral and cellular immune responses by recombinant E . coli expressing the B . melitensis omp31 gene, no significant protection against a challenge with smooth B . melitensis H38S was observed in immunized mice . These results demonstrate that despite the strong antibody response induced in mice, immunization with the recombinant Omp31 of B . melitensis does not confer any protective effect against a virulent smooth B . melitensis . However, its potential protective effect for protection against rough Brucella would be worth testing.

Mol Microbiol, 1999 Jan, 31(1), 381 - 92
Characterization of the C-terminal domain essential for toxic activity of adenylate cyclase toxin; Bejerano M et al.; Adenylate cyclase toxin (CyaA) of Bordetella pertussis belongs to the RTX family of toxins . These toxins are characterized by a series of glycine- and aspartaterich nonapeptide repeats located at the C-terminal half of the toxin molecules . For activity, RTX toxins require Ca2+, which is bound through the repeat region . Here, we identified a stretch of 15 amino acids (block A) that is located C-terminally to the repeat and is essential for the toxic activity of CyaA . Block A is required for the insertion of CyaA into the plasma membranes of host cells . Mixing of a short polypeptide composed of block A and eight Ca2+ binding repeats with a mutant of CyaA lacking block A restores toxic activity fully . This in vitro interpolypeptide complementation is achieved only when block A is present together with the Ca2+ binding repeats on the same polypeptide . Neither a short polypeptide composed of block A only nor a polypeptide consisting of eight Ca2+ binding repeats, or a mixture of these two polypeptides, complement toxic activity . It is suggested that functional complementation occurs because of binding between the Ca2+ binding repeats of the short C-terminal polypeptide and the Ca2+ binding repeats of the CyaA mutant lacking block A.

Mol Microbiol, 1999 Jan, 31(1), 261 - 70
Involvement of the FtsH (HflB) protease in the activity of sigma 54 promoters; Carmona M et al.; The effect of FtsH, an essential inner membrane-bound protease, in the regulation of the sigma 54-dependent Pu promoter has been examined in vivo . Escherichia coli cells lacking FtsH failed to activate a Pu-lacZ fusion in response to the cognate enhancer-binding protein XylR . However, the intracellular concentrations of XylR and sigma 54, as well as their apparent physical integrity were the same regardless of the presence or absence of the protease . The loss of Pu activity in FtsH-minus cells was not due to the imbalance between sigma factors caused by the lack of the protease . ftsH mutants could not grow in media with glutamine as the only nitrogen source and failed also to induce the sigma 54 promoters PnifH by NifA and PpspA by PspF . These lesions were fully complemented by a ftsH+ plasmid . Therefore, part of the pleiotropic phenotype of FtsH-less cells corresponded to the lack of sigma 54 activity . Overproduction of sigma 54, however, restored both transcriptional activity of Pu and growth in glutamine of a ftsH strain . These observations suggested that the activity of sigma 54 is checked in vivo by an interplay of factors that ultimately determine the performance of cognate promoters under given physiological conditions.

Mol Microbiol, 1999 Jan, 31(1), 167 - 75
Hemi-methylated oriC DNA binding activity found in non-specific acid phosphatase; Reshetnyak E et al.; The lacZ-hobH fusion clone, containing an Escherichia coli DNA segment located at 92 min on the chromosomal map, was screened as a producer of E . coli oriC hemi-methylated binding activity . We have purified the protein encoded by this locus to near homogeneity . The protein corresponds to the monomeric form of a non-specific acid phosphatase (NAP) whose gene has been designated aphA . oriC DNA footprinting experiments showed protection of hemi-methylated probe by partially purified NAP, but not by purified preparations . Yet, gel retardation experiments with an oriC oligonucleotide demonstrated DNA binding activity of purified NAP in the presence of Mg2+ . This experiment also showed an increased affinity of the protein for the hemi-methylated probe compared with the fully or unmethylated form . Indirect immunofluorescene microscopy revealed the existence of discrete NAP foci at mid-cell in cells with two nucleoids, but at cell poles in those with one nucleoid.

Mol Microbiol, 1999 Jan, 31(1), 157 - 66
On the mechanism of FtsH-dependent degradation of the sigma 32 transcriptional regulator of Escherichia coli and the role of the Dnak chaperone machine; Blaszczak A et al.; The Escherichia coli sigma 32 transcriptional regulator has been shown to be degraded both in vivo and in vitro by the FtsH (HflB) protease, a member of the AAA protein family . In our attempts to study this process in detail, we found that two sigma 32 mutants lacking 15-20 C-terminal amino acids had substantially increased half-lives in vivo or in vitro, compared with wild-type sigma 32 . A truncated version of sigma 32, sigma 32 C delta, was purified to homogeneity and shown to be resistant to FtsH-dependent degradation in vitro, suggesting that FtsH initiates sigma 32 degradation from its extreme C-terminal region . Purified sigma 32 C delta interacted with the DnaK and DnaJ chaperone proteins in a fashion similar to that of wild-type sigma 32 . However, in contrast to wild-type sigma 32, sigma 32 C delta was largely deficient in its in vivo and in vitro interaction with core RNA polymerase . As a consequence, the truncated sigma 32 protein was completely non-functional in vivo, even when overproduced . Furthermore, it is shown that wild-type sigma 32 is protected from degradation by FtsH when complexed to the RNA polymerase core, but sensitive to proteolysis when in complex with the DnaK chaperone machine . Our results are in agreement with the proposal that the capacity of the DnaK chaperone machine to autoregulate its own synthesis negatively is simply the result of its ability to sequester sigma 32 from RNA polymerase, thus making it accessible to degradation by the FtsH protease.

Mol Microbiol, 1999 Jan, 31(1), 67 - 77
Translation initiation factor 3 antagonizes authentic start codon selection on leaderless mRNAs; Tedin K et al.; In this study, we have examined the influence of initiation factors on translation initiation of leaderless mRNAs whose 5'-terminal residues are the A of the AUG initiating codon . A 1:1 ratio of initiation factors to ribosomes abolished ternary complex formation at the authentic start codon of different leaderless mRNAs . Supporting this observation, in vitro translation assays using limiting ribosome concentrations with competing leaderless lambda cl and Escherichia coli ompA mRNAs, the latter containing a canonical ribosome binding site, revealed reduced cl synthesis relative to OmpA in the presence of added initiation factors . Using in vitro toeprinting and in vitro translation assays, we show that this effect can be attributed to IF3 . Moreover, in vivo studies revealed that the translational efficiency of a leaderless reporter gene is decreased with increased IF3 levels . These studies are corroborated by the observed increased translational efficiency of a leaderless reporter construct in an infC mutant strain unable to discriminate against non-standard start codons . These results suggest that, in the absence of a leader or a Shine-Dalgarno sequence, the function(s) of IF3 limits stable 30S ternary complex formation.

Mol Microbiol, 1999 Jan, 31(1), 19 - 30
The long-term cytoskeletal rearrangement induced by rabbit enteropathogenic Escherichia coli is Esp dependent but intimin independent; Nougayrede JP et al.; Attaching and effacing rabbit enteropathogenic Escherichia coli (REPEC) of the O103 serogroup adhere diffusely on HeLa cells and trigger a slow progressive cytopathic effect (CPE) characterized by the recruitment of vinculin and the assembly of actin stress fibres . In contrast to REPEC O103, the reference human EPEC strain E2348/69 is unable to trigger the CPE . In this study, we have shown first that the fimbrial adhesin AF/R2, which mediates the diffuse adhesion of REPEC O103, was not sufficient to induce the CPE capability upon E2348/69 . Non-polar mutants of REPEC O103 for espA, espB, espD and eae were then constructed . The four mutants were unable to induce attaching and effacing lesions in the rabbit ileal loop model . The esp mutants were no longer able to induce the CPE, whereas the eae mutant still induced the CPE . Each espA, -B, -D mutant could be fully complemented in trans by the corresponding cloned esp genes from both the parental strain and the CPE-negative E2348/69 strain, indicating that no single esp encodes the information needed to confer the CPE phenotype . In conclusion, the CPE is the first example of an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton by certain EPEC strains.

Kidney Int, 1999 Feb, 55(2), 554 - 61
Recruitment of renal tubular epithelial cells expressing verotoxin-1 (Stx1) receptors in HIV-1 transgenic mice with renal disease; Liu XH et al.; BACKGROUND: Human immunodeficiency virus (HIV)-infected children are at risk of developing several renal parenchymal diseases, including hemolytic uremic syndrome (HUS) . HUS is most frequently caused by infection with enteric Escherichia coli producing Shiga-like toxins (Stxs) . In vitro studies have shown that cytokines known to be present at high systemic levels in HIV-1-infected children up-regulate the expression of the Stx glycolipid receptor (Gb3) in cultured endothelial cells . Thus, we studied whether HIV-1 or the HIV-associated "cytokine milieu" could modulate the expression of renal Stxs receptors in vivo . METHODS: We used HIV-1 transgenic mice (HIV-Tg) expressing a deletion mutant of HIV-1 (pNL4-3) . These mice develop renal disease similar to that of HIV-1-infected children . The expression of Gb3 was studied in renal sections from control and HIV-Tg mice by histochemistry, thin layer chromatography overlay studies, and high-pressure liquid chromatography . RESULTS: By histochemistry, we found a significant recruitment of renal tubular epithelial cells expressing Gb3 in HIV-Tg mice with nephropathy, whereas kidneys from control mice showed limited staining in renal tubules . Gb3 was not found in glomeruli of either control or HIV-Tg mice . Thin layer chromatography overlay studies with Stxs and high-pressure liquid chromatography studies confirmed the marked elevation of Gb3 in HIV-Tg kidneys with renal disease . CONCLUSIONS: These results suggest that the presence of HIV-associated nephropathy is associated with the recruitment of renal tubular epithelial cells expressing Stx1 receptors . The up-regulation of Stx1 receptors in HIV-diseased kidneys may increase the sensitivity of these cells to the cytotoxic effects of Stxs.

J Clin Microbiol, 1999 Mar, 37(3), 649 - 52
Laboratory diagnosis of Toscana virus infection by enzyme immunoassay with recombinant viral nucleoprotein; Soldateschi D et al.; A recombinant enzyme immunoassay (rEIA) to detect serum immunoglobulin M (IgM) and IgG to Toscana virus (TOSV) was developed with the aim of establishing a simple and easily available assay for diagnosing acute and/or previous infections . The rEIA, based on the recombinant nucleoprotein of TOSV expressed in Escherichia coli, was evaluated with 97 serum samples collected in an area where TOSV is endemic and compared to an analogous assay based on cell-derived TOSV . Discordant results were resolved by immunoblotting (IB) . Twenty-two of these samples, obtained from subjects hospitalized during the summer season with meningitis of suspected TOSV etiology, were further characterized by indirect immunofluorescence and IB, and detection of specific TOSV RNA sequences in the cerebrospinal fluid of these patients was attempted by nested PCR . The results indicated that rEIA was able to diagnose acute TOSV infection by detection of specific serum IgM in all of the subjects with TOSV meningitis confirmed by nested PCR or serology . The overall sensitivity and specificity of rEIA were both 100% for IgM detection and 100 and 96.6%, respectively, for IgG detection . Thus, rEIA appears to be a simple and reliable laboratory test for the diagnosis of acute TOSV infection and for the assessment of immune status.

J Clin Microbiol, 1999 Mar, 37(3), 497 - 503
Associations between virulence factors of Shiga toxin-producing Escherichia coli and disease in humans; Boerlin P et al.; Associations between known or putative virulence factors of Shiga toxin-producing Escherichia coli and disease in humans were investigated . Univariate analysis and multivariate logistic regression analysis of a set of 237 isolates from 118 serotypes showed significant associations between the presence of genes for intimin (eae) and Shiga toxin 2 (stx2) and isolates from serotypes reported in humans . Similar associations were found with isolates from serotypes reported in hemorrhagic colitis and hemolytic-uremic syndrome . The enterohemorrhagic E . coli (EHEC) hemolysin gene was significantly associated with isolates from serotypes found in severe diseases in univariate analysis but not in multivariate logistic regression models . A strong association between the intimin and EHEC-hemolysin genes may explain the lack of statistical significance of EHEC hemolysin in these multivariate models, but a true lack of biological significance of the hemolysin in humans or in disease cannot be excluded . This result warrants further investigations of this topic . Multivariate analysis revealed an interaction between the eae and stx2 genes, thus supporting the hypothesis of the synergism between the adhesin intimin and Shiga toxin 2 . A strong statistical association was observed between the stx2 gene and severity of disease for a set of 112 human isolates from eight major serotypes . A comparison of 77 isolates of bovine origin and 91 human isolates belonging to six major serotypes showed significant associations of the genes for Shiga toxin 1 and EspP protease with bovine isolates and an increased adherence on HEp-2 cell cultures for human isolates, particularly from diarrheic patients and healthy persons.

Arterioscler Thromb Vasc Biol, 1999 Feb, 19(2), 220 - 8
Oleic acid inhibits endothelial activation : A direct vascular antiatherogenic mechanism of a nutritional component in the mediterranean diet; Carluccio MA et al.; Because oleic acid is implicated in the antiatherogenic effects attributed to the Mediterranean diet, we investigated whether this fatty acid can modulate endothelial activation, ie, the concerted expression of gene products involved in leukocyte recruitment and early atherogenesis . We incubated sodium oleate with human umbilical vein endothelial cells for 0 to 72 hours, followed by coincubation of oleate with human recombinant tumor necrosis factor, interleukin (IL)-1alpha, IL-1beta, IL-4, Escherichia coli lipopolysaccharide (LPS), or phorbol 12-myristate 13-acetate for a further 6 to 24 hours . The endothelial expression of vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and intercellular adhesion molecule-1 was monitored by cell surface enzyme immunoassays or flow cytometry, and steady-state levels of VCAM-1 mRNA were assessed by Northern blot analysis . At 10 to 100 micromol/L for >24 hours, oleate inhibited the expression of all adhesion molecules tested . After a 72-hour incubation with oleate and a further 16-hour incubation with oleate plus 1 microg/mL LPS, VCAM-1 expression was reduced by >40% compared with control . Adhesion of monocytoid U937 cells to LPS-treated endothelial cells was reduced concomitantly . Oleate also produced a quantitatively similar reduction of VCAM-1 mRNA levels on Northern blot analysis and inhibited nuclear factor-kappaB activation on electrophoretic mobility shift assays . Incubation of endothelial cells with oleate for 72 hours decreased the relative proportions of saturated (palmitic and stearic) acids in total cell lipids and increased the proportions of oleate in total cell lipids without significantly changing the relative proportions of polyunsaturated fatty acids . Although less potent than polyunsaturated fatty acids in inhibiting endothelial activation, oleic acid may contribute to the prevention of atherogenesis through selective displacement of saturated fatty acids in cell membrane phospholipids and a consequent modulation of gene expression for molecules involved in monocyte recruitment.

Nucleic Acids Res, 1999 Mar 1, 27(5), 1369 - 76
Probing the environment of nascent RNA in Escherichia coli transcription elongation complexes utilizing a new fluorescent ribonucleotide analog; Hanna MM et al.; We report the synthesis and characterization of 5-thioacetamidofluorescein-uridine 5'-triphosphate (5-SF-UTP), and its application to the characterization of the environment of the nascent RNA during trans-cription . This analog specifically replaced UTP as a transcription substrate for Escherichia coli and T7 RNA polymerases, and yeast RNA polymerase III . Escherichia coli transcription complexes containing analog incorporated at only position +21 of the RNA were prepared . The RNA was then elongated in the absence of analog, moving the fluorescent group further away from the enzyme active site, and the fluorescence polarization was measured . Analog positioned near the 3' end of the transcript exhibited significantly increased polarization relative to that of free probe, consistent with the constrained environment of the RNA in the DNA-RNA hybrid . Analog positioned 14 nucleotides from the 3' end exhibited significantly decreased polarization relative to that at the 3' end of the RNA, but only slightly above that of free RNA, suggesting that the probe was on the solvent-exposed surface of the polymerase . Molecular modeling of these analog-substituted RNAs produced structures consistent with the experimental data . The excellent substrate properties of this analog make it useful for the characterization of the environment of RNA not only during transcription and translation, but in any type of ribonucleoprotein complex.

Nucleic Acids Res, 1999 Mar 1, 27(5), 1331 - 7
Cloning and characterization of the p42 subunit of mammalian translation initiation factor 3 (eIF3): demonstration that eIF3 interacts with eIF5 in mammalian cells; Bandyopadhyay A et al.; Eukaryotic translation initiation factor 3 (eIF3) is a large multisubunit protein complex that plays an essential role in the binding of the initiator methionyl-tRNA and mRNA to the 40S ribosomal subunit to form the 40S initiation complex . cDNAs encoding all the subunits of mammalian eIF3 except the p42 subunit have been cloned in several laboratories . Here we report the cloning and characterization of a human cDNA encoding the p42 subunit of mammalian eIF3 . The open reading frame of the cDNA, which encodes a protein of 320 amino acids (calculated Mr35 614) has been expressed in Escherichia coli and the recombinant protein has been purified to homogeneity . The purified protein binds RNA in agreement with the presence of a putative RNA binding motif in the deduced amino acid sequence . The protein shows 33% identity and 53% similarity with the Tif35p subunit (YDR 429C) of yeast eIF3 . Transfection experiments demonstrated that polyhistidine-tagged p42 protein, transiently expressed in human U20S cells, was incorporated into endogenous eIF3 . Furthermore, eIF3 isolated from transfected cell lysates contains bound eIF5 indicating that a specific physical interaction between eIF5 and eIF3 may play an important role in the function of eIF5 during translation initiation in eukaryotic cells.

Nucleic Acids Res, 1999 Mar 1, 27(5), 1323 - 30
Targeted gene repair directed by the chimeric RNA/DNA oligonucleotide in a mammalian cell-free extract; Cole-Strauss A et al.; Chimeric oligonucleotides consisting of RNA and DNA residues have been shown to catalyze site-directed genetic alteration in mammalian cells both in vitro and in vivo . Since the frequency of these events appears to be logs higher than the rates of gene targeting, a process involving homologous recombination, we developed a system to study the mechanisms of chimera-directed gene conversion . Using a mammalian cell-free extract and a genetic readout in Escherichia coli, we find that point mutations and single base deletions can be corrected at frequencies of approximately 0.1% and 0.005%, respectively . The reaction depends on an accurately designed chimera and the presence of functional hMSH2 protein . The results of genetic and biochemical studies reported herein suggest that the process of mismatch repair functions in site-directed gene correction.

Nucleic Acids Res, 1999 Mar 1, 27(5), 1296 - 9
Gene replacement with linear DNA in electroporated wild-type Escherichia coli; El Karoui M et al.; Gene replacement using linear double-stranded DNA fragments in wild-type Escherichia coli transformation is generally inefficient due to exonucleolytic degradation of incoming DNA . Recombination-proficient strains, in which the exonucleolytic activity of RecBCD is inactivated, have been used as transformation recipients to overcome this difficulty . Here we report that gene replacements using linear double-stranded donor DNA can be achieved in wild-type E.coli if electrocompetent cells are used . Using a plasmid target, we obtained 10(2)-10(3) gene replacement events/microgram linear DNA . Using an independent chromosomal target, approximately 60 gene replacement events/microgram linear DNA were obtained . The presence of Chi sites on the linear DNA, which are known to block DNA degradation and stimulate recombination in E.coli, had no effect on gene replacement efficiency in either case . RecBCD-mediated exonucleolytic activity was found to be diminished in electroporated cells . Electrotransformation thus provides a simple way to perform gene replacements in many E.coli strains.

Nucleic Acids Res, 1999 Mar 1, 27(5), 1275 - 82
Escherichia coli RuvBL268S: a mutant RuvB protein that exhibits wild-type activities in vitro but confers a UV-sensitive ruv phenotype in vivo; Mezard C et al.; The RuvABC proteins of Escherichia coli process recombination intermediates during genetic recombination and DNA repair . RuvA and RuvB promote branch migration of Holliday junctions, a process that extends heteroduplex DNA . Together with RuvC, they form a RuvABC complex capable of Holliday junction resolution . Branch migration by RuvAB is mediated by RuvB, a hexameric ring protein that acts as an ATP-driven molecular pump . To gain insight into the mechanism of branch migration, random mutations were introduced into the ruvB gene by PCR and a collection of mutant alleles were obtained . Mutation of leucine 268 to serine resulted in a severe UV-sensitive phenotype, characteristic of a ruv defect . Here, we report a biochemical analysis of the mutant protein RuvBL268S . Unexpectedly, the purified protein is fully active in vitro with regard to its ATPase, DNA binding and DNA unwinding activities . It also promotes efficient branch migration in combination with RuvA, and forms functional RuvABC-Holliday junction resolvase complexes . These results indicate that RuvB may perform some additional, and as yet undefined, function that is necessary for cell survival after UV-irradiation.

J Mol Biol, 1999 Feb 19, 286(2), 541 - 52
NMR structure of Escherichia coli glutaredoxin 3-glutathione mixed disulfide complex: implications for the enzymatic mechanism; Nordstrand K et al.; Glutaredoxins (Grxs) catalyze reversible oxidation/reduction of protein disulfide groups and glutathione-containing mixed disulfide groups via an active site Grx-glutathione mixed disulfide (Grx-SG) intermediate . The NMR solution structure of the Escherichia coli Grx3 mixed disulfide with glutathione (Grx3-SG) was determined using a C14S mutant which traps this intermediate in the redox reaction . The structure contains a thioredoxin fold, with a well-defined binding site for glutathione which involves two intermolecular backbone-backbone hydrogen bonds forming an antiparallel intermolecular beta-bridge between the protein and glutathione . The solution structure of E . coli Grx3-SG also suggests a binding site for a second glutathione in the reduction of the Grx3-SG intermediate, which is consistent with the specificity of reduction observed in Grxs . Molecular details of the structure in relation to the stability of the intermediate and the activity of Grx3 as a reductant of glutathione mixed disulfide groups are discussed . A comparison of glutathione binding in Grx3-SG and ligand binding in other members of the thioredoxin superfamily is presented, which illustrates the highly conserved intermolecular interactions in this protein family .

J Mol Biol, 1999 Feb 19, 286(2), 521 - 40
Three-dimensional placement of the conserved 530 loop of 16 S rRNA and of its neighboring components in the 30 S subunit; Wang R et al.; Nucleotides 518-533 form a loop in ribosomal 30 S subunits that is almost universally conserved . Both biochemical and genetic evidence clearly implicate the 530 loop in ribosomal function, with respect both to the accuracy control mechanism and to tRNA binding . Here, building on earlier work, we identify proteins and nucleotides (or limited sequences) site-specifically photolabeled by radioactive photolabile oligoDNA probes targeted toward the 530 loop of 30 S subunits . The probes we employ are complementary to 16 S rRNA nucleotides 517-527, and have aryl azides attached to nucleotides complementary to nucleotides 518, 522, and 525-527, positioning the photogenerated nitrene a maximum of 19-26 A from the complemented rRNA base . The crosslinks obtained are used as constraints to revise an earlier model of 30 S structure, using the YAMMP molecular modeling package, and to place the 530 loop region within that structure .

J Mol Biol, 1999 Feb 19, 286(2), 447 - 64
Two distinct mechanisms operate in the reactivation of heat-denatured proteins by the mitochondrial Hsp70/Mdj1p/Yge1p chaperone system; Kubo Y et al.; The yeast mitochondrial Hsp70, Ssc1p, functions as a molecular chaperone with its partner proteins, Mdj1p (DnaJ homologue) and Yge1p (GrpE homologue) . We have purified a mature form of Ssc1p from yeast mitochondria and those of Mdj1p and Yge1p from Escherichia coli overexpresser cells . With these purified components of the mitochondrial Hsp70 chaperone system, we have succeeded in reconstituting their chaperone functions in the protection of firefly luciferase against thermal damage in vitro . Heat-denatured luciferase is prevented from irreversible aggregation and is maintained in a refolding-competent state by Ssc1p and/or Mdj1p at 42 degreesC . Luciferase denatured at 42 degreesC is actively reactivated by Ssc1p, Mdj1p and/or Yge1p after lowering the temperature to 25 degreesC . The reactivation process of heat-denatured luciferase shows two-phase kinetics . The slow refolding process requires either Ssc1p or Mdj1p at 42 degreesC but the presence of Ssc1p, Mdj1p and Yge1p, and ATP hydrolysis, is essential at 25 degreesC . The slow refolding of luciferase involves multiple rounds of formation and dissociation of the complex between luciferase and Mdj1p/Ssc1p . On the other hand, the fast refolding process is most enhanced when luciferase is incubated with Ssc1p alone at 42 degreesC, and it requires neither the assistance of Mdj1p and Yge1p nor ATP hydrolysis . We have observed a similar two-pathway reactivation of heat-denatured luciferase by the bacterial Hsp70 and the yeast cytosolic Hsp70 systems .

J Mol Biol, 1999 Feb 19, 286(2), 437 - 45
On the in vivo function of the RecA ATPase; Campbell MJ et al.; The Escherichia coli RecA protein is the prototype of the RecA/RAD51/DMC1 family of strand transferases acting in genetic recombination . The E96D mutant was previously isolated in a screen for toxic recA mutants and was found to constitutively derepress the SOS genes and inhibit chromosome segregation in E . coli . Here, we have found that the E96D mutation lowers the RecA kcat value for ATP hydrolysis 100-fold . Use of this mutant reveals that the ATPase and branch migration activities of RecA are not necessarily required for catalyzing in vivo recombinational pairing and LexA cleavage . In addition to its effect on ATP hydrolysis, the mutation causes ATP to more strongly promote the transition to the biologically active, extended conformation of the RecA enzyme . The enhanced ATP binding is apparently the cause for a broader nucleic acid ligand specificity . The use of RNA and double-stranded DNA as cofactors for LexA cleavage could give rise to the inappropriate, constitutive derepression of the SOS genes . This underscores the need for the ATP affinity to be optimized so that RecA becomes selectively activated only during DNA repair and recombination through binding single-stranded DNA .

J Mol Biol, 1999 Feb 19, 286(2), 355 - 64
Probing the rRNA environment of ribosomal protein S5 across the subunit interface and inside the 30 S subunit using tethered Fe(II); Culver GM et al.; A newly developed 30 S subunit reconstitution system using a complete set of recombinant proteins was used to study the ribosomal RNA (rRNA) neighborhood of ribosomal protein S5 in 30 S subunits and 70 S ribosomes by directed hydroxyl radical probing . Using three cysteine-containing mutant S5 proteins derivatized with 1-(p-bromoacetamidobenzyl)-Fe(II)-EDTA, we expanded on experiments carried out earlier using a natural protein reconstitution system . Natural 16 S rRNA, Fe(II)-S5, and the other recombinant ribosomal proteins were reconstituted into 30 S subunits . Both 30 S subunits and 70 S ribosomes containing Fe(II)-S5 were purified, and hydroxyl radicals were generated in situ from the tethered Fe(II) . In 30 S subunits, 16 S rRNA nucleotides targeted by two positions on S5, C21 and C99, were virtually identical to those observed in the previous work, supporting the validity of the recombinant protein reconstitution system for probing studies . Interestingly, new cleavages were detected using Fe(II)-C129-S5, possibly reflecting incorporation of more derivatized protein into 30 S subunits due to the increased reconstitution efficiency of the recombinant protein system . These newly targeted positions overlap, but are distinct from, those observed using Fe(II) tethered to C21, which is near C129 in the S5 structure.In 70 S ribosomes, the cleavage pattern of 16 S rRNA was very similar to that observed in 30 S subunits for all target sites except for the absence of those at the extreme 5' end of 16 S rRNA . Additionally, probing of 70 S ribosomes from Fe-C99-S5 results in cleavage of 23 S rRNA in the 1690-1770 region of domain IV . These data provide constraints for the three-dimensional location of nucleotides within domain IV of 23 S ribosomal RNA relative to known features of the 30 S subunit .

J Immunol, 1999 Feb 1, 162(3), 1795 - 801
Signs of immaturity of splenic dendritic cells from the autoimmune prone biobreeding rat: consequences for the in vitro expansion of regulator and effector T cells; Delemarre FG et al.; From the biobreeding-diabetic prone (BB-DP) rat, an animal model for endocrine autoimmunity, phenotype and function of splenic dendritic cells (DC) were studied . Furthermore, the suppressive effect of peritoneal macrophages (pMphi) from the BB-DP rat in the MLR was investigated . Lower numbers of splenic DC were isolated from BB-DP rats than from control Wistar rats . In the preautoimmune phase, DC of the BB-DP rat had a lower surface MHC class II expression (and in preliminary data, a lower CD80 expression), ingested more bacteria, and had a lower stimulatory potency in the syngeneic (syn)MLR as compared with control DC . During disease development, the MHC class II expression further decreased, and a low stimulatory activity became evident in the allogeneic (allo)MLR . With regard to the expansion of suppressor/regulatory T cells, a lower percentage of RT6+ T cells but higher percentages of CD45RClow T cells were induced by BB-DP DC in synMLR, but not in alloMLR . An increase in the CD4/CD8 T cell ratio was observed in both the syn- and alloMLR due to a relative weak expansion of CD8+ T cells with DC of the BB-DP rat . Resident pMphi isolated from BB-DP or Wistar rats were equally effective in suppressing the DC-driven synMLR . In conclusion, splenic DC from the BB-DP rat have a lower accessory cell function already at young age, before the development of disease, and expanded different subsets of effector/suppressor T cells in vitro as compared with those from Wistar rats . The dysfunction of DC from BB-DP rats is likely to be caused by their relative immaturity as indicated by their low class II and costimulatory molecule expression and relatively high phagocytic activity.

J Bacteriol, 1999 Feb, 181(4), 1360 - 3
Chromosomal changes during experimental evolution in laboratory populations of Escherichia coli; Bergthorsson U et al.; Short-term rates of chromosome evolution were analyzed in experimental populations of Escherichia coli B that had been propagated for 2,000 generations under four thermal regimens . Chromosome alterations were monitored in 24 independent populations by pulsed-field gel electrophoresis of DNA treated with five rare-cutting restriction enzymes . A total of 11 changes, 8 affecting chromosome size and 3 altering restriction sites, were observed in these populations, with none occurring in strains cultured at 37 degreesC . Considering the changes detected in these experimental populations, the rate of chromosome alteration of E . coli is estimated to be half of that observed in experimental populations of yeast.

J Bacteriol, 1999 Feb, 181(4), 1334 - 7
Specific effects of a recB mutation on the HfrH strain of Escherichia coli; Dermic D et al.; The recB268::Tn10 mutation was introduced into the HfrH strain of Escherichia coli . Compared with recB F- and recB F+ cells, the viability of this mutant strain was much lower . Compared with wild-type HfrH, the recB derivative donated much shorter fragments of its chromosome to the recipient . It is suggested that the recB gene product (i.e., RecBCD enzyme) participates in Hfr transfer.

J Bacteriol, 1999 Feb, 181(4), 1269 - 80
Functional determinants of the Escherichia coli fis promoter: roles of -35, -10, and transcription initiation regions in the response to stringent control and growth phase-dependent regulation; Walker KA et al.; Escherichia coli Fis is a small DNA binding and bending protein that has been implicated in a variety of biological processes . A minimal promoter sequence consisting of 43 bp is sufficient to generate its characteristic growth phase-dependent expression pattern and is also subject to negative regulation by stringent control . However, information about the precise identification of nucleotides contributing to basal promoter activity and its regulation has been scant . In this work, 72 independent mutations were generated in the fis promoter (fis P) region from -108 to +78 using both random and site-directed PCR mutagenesis . beta-Galactosidase activities from mutant promoters fused to the (trp-lac)W200 fusion on a plasmid were used to conclusively identify the sequences TTTCAT and TAATAT as the -35 and -10 regions, respectively, which are optimally separated by 17 bp . We found that four consecutive substitutions within the GC-rich sequence just upstream of +1 and mutations in the -35 region, but not in the -10 region, significantly reduced the response to stringent control . Analysis of the effects of mutations on growth phase-dependent regulation showed that replacing the predominant transcription initiation nucleotide +1C with a preferred nucleotide (A or G) profoundly altered expression such that high levels of fis P mRNA were detected during late logarithmic and early stationary phases . A less dramatic effect was seen with improvements in the -10 and -35 consensus sequences . These results suggest that the acute growth phase-dependent regulation pattern observed with this promoter requires an inefficient transcription initiation process that is achieved with promoter sequences deviating from the -10 and -35 consensus sequences and, more importantly, a dependence upon the availability of the least favored transcription initiation nucleotide, CTP.

J Bacteriol, 1999 Feb, 181(4), 1249 - 55
Inhibition of homologous recombination by the plasmid MucA'B complex; Venderbure C et al.; By its functional interaction with a RecA polymer, the mutagenic UmuD'C complex possesses an antirecombination activity . We show here that MucA'B, a functional homolog of the UmuD'C complex, inhibits homologous recombination as well . In F- recipients expressing MucA'B from a Ptac promoter, Hfr x F- recombination decreased with increasing MucA'B concentrations down to 50-fold . In damage-induced pKM101-containing cells expressing MucA'B from the native promoter, recombination between a UV-damaged F lac plasmid and homologous chromosomal DNA decreased 10-fold . Overexpression of MucA'B together with UmuD'C resulted in a synergistic inhibition of recombination . RecA{UmuR} proteins, which are resistant to UmuD'C inhibition of recombination, are inhibited by MucA'B while promoting MucA'B-promoted mutagenesis efficiently . The data suggest that MucA'B and UmuD'C contact a RecA polymer at distinct sites . The MucA'B complex was more active than UmuD'C in promoting UV mutagenesis, yet it did not inhibit recombination more than UmuD'C does . The enhanced mutagenic potential of MucA'B may result from its inherent superior capacity to assist DNA polymerase in trans-lesion synthesis . In the course of this work, we found that the natural plasmid pKM101 expresses around 45,000 MucA and 13,000 MucB molecules per lexA(Def) cell devoid of LexA . These molecular Muc concentrations are far above those of the chromosomally encoded Umu counterparts . Plasmid pKM101 belongs to a family of broad-host-range conjugative plasmids . The elevated levels of the Muc proteins might be required for successful installation of pKM101-like plasmids into a variety of host cells.

J Bacteriol, 1999 Feb, 181(4), 1149 - 55
Spectra of spontaneous growth-dependent and adaptive mutations at ebgR; Hall BG; A comparison of the spectra of spontaneous growth-dependent and adaptive mutations in ebgR shows that both spectra are dominated by insertion sequence (IS)-mediated mutations . The difference between growth-dependent mutations (61% IS mediated) and adaptive mutations (80% IS mediated) is highly significant (P < 0.0001) . In contrast, the spectra of growth-dependent and adaptive non-IS-mediated mutations do not differ from each other and therefore do not provide support for the hypothesis that adaptive and growth-dependent mutations arise by substantially different mechanisms.

J Bacteriol, 1999 Feb, 181(4), 1141 - 8
Expression and study of recombinant ExoM, a beta1-4 glucosyltransferase involved in succinoglycan biosynthesis in Sinorhizobium meliloti; Lellouch AC et al.; Here we report on the overexpression and in vitro characterization of a recombinant form of ExoM, a putative beta1-4 glucosyltransferase involved in the assembly of the octasaccharide repeating subunit of succinoglycan from Sinorhizobium meliloti . The open reading frame exoM was isolated by PCR and subcloned into the expression vector pET29b, allowing inducible expression under the control of the T7 promoter . Escherichia coli BL21(DE3)/pLysS containing exoM expressed a novel 38-kDa protein corresponding to ExoM in N-terminal fusion with the S-tag peptide . Cell fractionation studies showed that the protein is expressed in E . coli as a membrane-bound protein in agreement with the presence of a predicted C-terminal transmembrane region . E . coli membrane preparations containing ExoM were shown to be capable of transferring glucose from UDP-glucose to glycolipid extracts from an S . meliloti mutant strain which accumulates the ExoM substrate (Glcbeta1-4Glcbeta1-3Gal-pyrophosphate-polyprenol) . Thin-layer chromatography of the glycosidic portion of the ExoM product showed that the oligosaccharide formed comigrates with an authentic standard . The oligosaccharide produced by the recombinant ExoM, but not the starting substrate, was sensitive to cleavage with a specific cellobiohydrolase, consistent with the formation of a beta1-4 glucosidic linkage . No evidence for the transfer of multiple glucose residues to the glycolipid substrate was observed . It was also found that ExoM does not transfer glucose to an acceptor substrate that has been hydrolyzed from the polyprenol anchor . Furthermore, neither glucose, cellobiose, nor the trisaccharide Glcbeta1-4Glcbeta1-3Glc inhibited the transferase activity, suggesting that some feature of the lipid anchor is necessary for activity.

J Cardiovasc Surg (Torino), 1998 Dec, 39(6), 783 - 9
The stimulation of neoangiogenesis in the ischemic human heart by the growth factor FGF: first clinical results; Schumacher B et al.; BACKGROUND: This paper is a report of our clinical experience with the human growth factor FGF as applied to the ischemic human myocardium . METHODS: After the completion of extensive preliminary animal experiments, the growth factor FGF, obtained from genetically manipulated E . coli bacteria and highly purified, was introduced into aortocoronary bypass surgery as an additional therapeutic agent . A double blind study was carried out on 40 patients with CHD, separated into "growth factor" and control groups, each containing 20 members . All the patients were treated for threefold vascular disease, in each case with an IMA bypass for the LAD and single venous bypasses for the RCX and/or RCA . In order to bridge over additional peripheral stenoses in the LAD or one of its branches, human growth factor FGF was injected into the myocardium of those in the growth factor group . Twelve weeks later, the IMA bypasses were selectively demonstrated by intraarterial DSA . These angiographs were then quantitatively evaluated . RESULTS: In all patients of the growth factor group, the formation of new vessels could be demonstrated in the region where FGF had been administered, in a manner strictly reminiscent of our experimental results . A capillary net sprouting from the coronary artery and making further connection with this vessel could be demonstrated, and the computer-supported evaluation of the angiographs showed a significant increase in the blood supply of the region of the myocardium injected . CONCLUSIONS: It is therefore our opinion that employment of the human growth factor FGF represents a useful extension to bypass surgery, particularly for patients with an additional peripheral stenosis that cannot be operatively revascularized.

Tokai J Exp Clin Med, 1998 Mar, 23(1), 39 - 44
Use of a glycoprotein gB promoter for expression of genes inserted into the human cytomegalovirus genome; Takekoshi M et al.; We attempted to utilize the human cytomegalovirus (HCMV) as an expression vector by replacing the dispensable genes of the viral genome with foreign genes . The selection of a promoter to be fused to the foreign gene is important to achieve a high expression rate in the recombinant virus . We selected the glycoprotein B (gB) promoter of HCMV as a target of analysis because gB is one of the most abundantly synthesized components in cell culture . The gB promoter, fused to the E . coli lacZ gene, was introduced into the HCMV HindIII-O fragment region by homologous recombination . It was confirmed that the gB promoter-lacZ construct was inserted in the targeted site of HCMV . The expression of the lacZ gene in the recombinant virus infection was initiated 24 h after infection and increased until 120 h post infection . The lacZ gene expression was inhibited by the presence of cytosine arabinoside . These observations indicate that the expression of the lacZ gene is under the control of the late promoter of gB.

Biosci Biotechnol Biochem, 1998 Dec, 62(12), 2408 - 14
Cloning of the gene for inorganic pyrophosphatase from a thermoacidophilic archaeon, Sulfolobus sp . strain 7, and overproduction of the enzyme by coexpression of tRNA for arginine rare codon; Wakagi T et al.; The gene encoding an extremely stable inorganic pyrophosphatase from Sulfolobus sp . strain 7, a thermoacidophilic archaeon, was cloned and sequenced . An open reading frame consisted of 516 base pairs coding for a protein of 172-amino acid residues . The deduced sequence was supported by partial amino acid sequence analyses . All the catalytically important residues were conserved . A unique 17-base-pair sequence motif was found to be repeated four times in frame in the gene, encoding a cluster of acidic amino acids essential for the function . Although the codon usage of the gene was quite different from that of Escherichia coli, the gene was effectively expressed in E . coli . Coexpression of tRNA(Arg), cognate for the rare codon AGA in E . coli, however, further improved the production of the enzyme, which occupied more than 85% of the soluble proteins obtained after removal of heat denatured E . coli proteins.

Biosci Biotechnol Biochem, 1998 Dec, 62(12), 2346 - 50
Transformation of the edible basidiomycete Lentinus edodes by restriction enzyme-mediated integration of plasmid DNA; Sato T et al.; We have used the restriction enzyme-mediated DNA integration (REMI) method to establish a transformation system in Lentinus edodes using the recombinant plasmid pLC1-hph, which contains the L . edodes transcriptional signals and an Escherichia coli hygromycin B phosphotransferase gene . Protoplasts of L . edodes were treated by the PEG transformation mixture containing 50 units of SalI, which cleaves pLC1-hph at a single site, yielding about 15 transformants per 2.5 micrograms of DNA . The conventional PEG transformation without SalI, however, yielded only 1.5 transformants per 25 micrograms of DNA . The optimal amount of SalI for increased transformation was 50 units . In the case of transformation with SphI, which cleaves the plasmid at one site, the optimal amount of the enzyme was 2.5 units . Southern blot analysis of the SphI-derived transformants suggested that 50% of the plasmid integrations were REMI events.

Biosci Biotechnol Biochem, 1998 Nov, 62(11), 2251 - 3
Isolation and properties of glucose-1-phosphatase from mycelia of Pholiota nameko; Joh T et al.; An acid phosphatase with a very high substrate specificity for glucose-1-phosphate was isolated for the first time from mycelia of Pholiota nameko . The molecular weight of the enzyme was estimated to be 31,000 on gel filtration and 35,000 on SDS-PAGE . The activity was inhibited by Cu2+, Hg2+, molybdate, and tartaric acid . The sequence of N-terminal 20 amino acid residues was analyzed.

Biosci Biotechnol Biochem, 1998 Nov, 62(11), 2236 - 8
Mutational analysis of the histidine-containing phosphotransfer (HPt) signaling domain of the ArcB sensor in Escherichia coli; Matsushika A et al.; The Escherichia coli ArcB sensor is involved in anaerobic phosphotransfer signal transduction . ArcB is a hybrid sensor that contains three types of phosphotransfer signaling domains in its primary amino acid sequence, namely, transmitter (or His-Kinase), receiver, and histidine-containing phosphotransfer (HPt) domains . However, examination of the function of the newly-discovered HPt domain (named ArcBc) is still at a very early stage . To gain a general insight into the structure and function of the widespread HPt domains, on the basis of its three-dimensional crystal structure, in this study we constructed a certain set of mutants each having a single amino acid substitution in the HPt domain of ArcB . These ArcBc mutants were characterized and evaluated, based on the in vivo ability to signal the OmpR receiver via trans-phosphorylation.

Biosci Biotechnol Biochem, 1998 Nov, 62(11), 2155 - 60
Cloning, sequencing, and heterologous expression of rat methionine synthase cDNA; Yamada K et al.; Methionine synthase catalyzes cobalamin-dependent methyl transfer reaction from 5-methyltetrahydrofolate to homocysteine, forming methionine . Rat methonine synthase cDNA was cloned and analyzed by RT-PCR, 3'- and 5'-RACE techniques . The cDNA consists of a 0.3-kb upstream untranslated region, a 3.8-kb coding region, and a 0.4-kb downstream untranslated region . The open reading frame encoded a polypeptide of 1,253 amino acid residues with a calculated molecular weight of 139,162 . This molecular weight was in good agreement with the observed one (143,000) of the purified rat liver enzyme . The deduced amino acid sequence was 53, 92, and 64% identical with those of the Escherichia coli, human, and presumptive Caenorhabditis elegans enzymes, respectively . All the fingerprint sequences, forming parts of the cobalamin- and S-adenosylmethionine-binding sites, were completely conserved in the rat methionine synthase . A high-level expression of catalytically active enzyme in insect cells was done by infection with a baculovirus containing the rat methionine synthase cDNA.

J Virol, 1999 Mar, 73(3), 2365 - 75
Identification of an active reverse transcriptase enzyme encoded by a human endogenous HERV-K retrovirus; Berkhout B et al.; Of the numerous endogenous retroviral elements that are present in the human genome, the abundant HERV-K family is distinct because several members are transcriptionally active and coding for biologically active proteins . A detailed phylogeny of the HERV-K family based on the partial sequence of the reverse transcriptase (RT) gene revealed a high incidence of an intact RT open reading frame within the HML-2 subgroup of HERV-K elements . In this study, we report the cloning of six full-length HML-2 RT genes, of which five contain an uninterrupted open reading frame . The RT enzymes were expressed as glutathione S-transferase fusion proteins in Escherichia coli, and several HERV-K RT enzymes demonstrated polymerase as well as RNase H activity . Several biochemical properties of the RT polymerase were analyzed, including the template requirements and optimal reaction conditions (temperature, type of divalent cation) . Inspection of the nucleotide sequence of the HERV-K RT genes demonstrated a mosaic structure, suggesting that a high level of genetic recombination has occurred in this virus family, which is a hallmark of replication by means of reverse transcription . The selective pressure to maintain the RT coding potential is illustrated by the sequence of a particular HERV-K isolate that contains three 1-nucleotide deletions within a small RT segment, thus maintaining the open reading frame . These combined results may suggest that these endogenous RT enzymes still have a biological function . It is possible that the RT activity was involved in the spread of this major class of retroelements by retrotransposition, and in fact it cannot be excluded that this retrovirus group is still mobile . The endogenous RT activity may also have been involved in the shaping of the human genome, e.g., by formation of pseudogenes.

J Virol, 1999 Mar, 73(3), 2270 - 9
In vitro assembly properties of human immunodeficiency virus type 1 Gag protein lacking the p6 domain; Campbell S et al.; Human immunodeficiency virus type 1 (HIV-1) normally assembles into particles of 100 to 120 nm in diameter by budding through the plasma membrane of the cell . The Gag polyprotein is the only viral protein that is required for the formation of these particles . We have used an in vitro assembly system to examine the assembly properties of purified, recombinant HIV-1 Gag protein and of Gag missing the C-terminal p6 domain (Gag Deltap6) . This system was used previously to show that the CA-NC fragment of HIV-1 Gag assembled into cylindrical particles . We now report that both HIV-1 Gag and Gag Deltap6 assemble into small, 25- to 30-nm-diameter spherical particles in vitro . The multimerization of Gag Deltap6 into units larger than dimers and the formation of spherical particles required nucleic acid . Removal of the nucleic acid with NaCl or nucleases resulted in the disruption of the multimerized complexes . We conclude from these results that (i) N-terminal extension of HIV-1 CA-NC to include the MA domain results in the formation of spherical, rather than cylindrical, particles; (ii) nucleic acid is required for the assembly and maintenance of HIV-1 Gag Deltap6 virus-like particles in vitro and possibly in vivo; (iii) a wide variety of RNAs or even short DNA oligonucleotides will support assembly; (iv) protein-protein interactions within the particle must be relatively weak; and (v) recombinant HIV-1 Gag Deltap6 and nucleic acid are not sufficient for the formation of normal-sized particles.

J Virol, 1999 Mar, 73(3), 2153 - 60
Extensive mutagenesis of the hepatitis B virus core gene and mapping of mutations that allow capsid formation; Koschel M et al.; We generated a large number of mutations in the hepatitis B virus (HBV) core gene inserted into a bacterial expression vector . The new mutagenesis procedure generated deletions and insertions (as sequence repeats) of various lengths at random positions between M1 and E145 but not substitutions . The R-rich 30-amino-acid C-terminal domain was not analyzed . A total of 50,000 colonies were tested with a polyclonal human serum for the expression of hepatitis B core or e antigen . A total of 110 mutants randomly chosen from 1,500 positive colonies were genotyped . Deletions and insertions were clustered in four regions: D2 to E14, corresponding to the N-terminal loop in a model for the core protein fold (B . Bottcher, S . A . Wynne, and R . A . Crowther, Nature 386:88-91, 1997); V27 to P50 (second loop); L60 to V86 (upper half of the alpha helix forming the N-terminal part of the spike and the tip of the spike); and V124 to L140 (C-terminal part of the C-terminal helix and downstream loop) . Deletions or insertions in the remaining parts of the molecule forming the compact center of the fold seemed to destabilize the protein . Of the 110 mutations, 38 allowed capsid formation in Escherichia coli . They mapped exclusively to nonhelical regions of the proposed fold . The mutations form a basis for subsequent analysis of further functions of the HBV core protein in the viral life cycle.

J Virol, 1999 Mar, 73(3), 2084 - 93
Analysis of the effects of charge cluster mutations in adeno-associated virus Rep68 protein in vitro; Davis MD et al.; The Rep78 and Rep68 proteins of adeno-associated virus type 2 (AAV) are multifunctional proteins which are required for viral replication, regulation of AAV promoters, and preferential integration of the AAV genome into a region of human chromosome 19 . These proteins bind the hairpin structures formed by the AAV inverted terminal repeat (ITR) origins of replication, make site- and strand-specific endonuclease cuts within the AAV ITRs, and display nucleoside triphosphate-dependent helicase activities . Additionally, several mutant Rep proteins display negative dominance in helicase and/or endonuclease assays when they are mixed with wild-type Rep78 or Rep68, suggesting that multimerization may be required for the helicase and endonuclease functions . Using overlap extension PCR mutagenesis, we introduced mutations within clusters of charged residues throughout the Rep68 moiety of a maltose binding protein-Rep68 fusion protein (MBP-Rep68Delta) expressed in Escherichia coli cells . Several mutations disrupted the endonuclease and helicase activities; however, only one amino-terminal-charge cluster mutant protein (D40A-D42A-D44A) completely lost AAV hairpin DNA binding activity . Charge cluster mutations within two other regions abolished both endonuclease and helicase activities . One region contains a predicted alpha-helical structure (amino acids 371 to 393), and the other contains a putative 3,4 heptad repeat (coiled-coil) structure (amino acids 441 to 483) . The defects displayed by these mutant proteins correlated with a weaker association with wild-type Rep68 protein, as measured in coimmunoprecipitation assays . These experiments suggest that these regions of the Rep molecule are involved in Rep oligomerization events critical for both helicase and endonuclease activities.

Biochem Pharmacol, 1999 Mar 1, 57(5), 531 - 8
Removal by human apurinic/apyrimidinic endonuclease 1 (Ape 1) and Escherichia coli exonuclease III of 3'-phosphoglycolates from DNA treated with neocarzinostatin, calicheamicin, and gamma-radiation; Chaudhry MA et al.; DNA strand breaks with terminal 3'-phosphoglycolate groups are produced by agents that can abstract the hydrogen atom from the 4'-carbon of DNA deoxyribose groups . Included among these agents are gamma-radiation (via the OH radical) and enediyne compounds, such as neocarzinostatin and calicheamicin . However, while the majority of radiation-induced phosphoglycolates are found at single-strand breaks, most of the phosphoglycolates generated by these two enediynes are found at bistranded lesions, including double-strand breaks . Using a 32P-post-labelling assay, we have compared the enzyme-catalyzed removal of phosphoglycolates induced by each of these agents . Both human apurinic/apyrimidinic endonuclease 1 (Ape 1) and its Escherichia coli homolog exonuclease III rapidly removed over 80% of phosphoglycolates from gamma-irradiated DNA, although there appeared to be a small resistant subpopulation . The neocarzinostatin-induced phosphoglycolates were removed more slowly, though not to completion, while the calicheamicin-induced phosphoglycolates were extremely refractory to both enzymes . These data suggest that unless other enzymes are capable of acting upon the phosphoglycolate termini at enediyne-induced double-strand breaks, such termini will be resistant to end rejoining repair pathways.

Biotechnol Bioeng, 1999 Mar 20, 62(6), 722 - 9
Incorporating qualitative knowledge in enzyme kinetic models using fuzzy logic; Lee B et al.; Modeling of metabolic pathway dynamics requires detailed kinetic equations at the enzyme level . In particular, the kinetic equations must account for metabolite effectors that contribute significantly to the pathway regulation in vivo . Unfortunately, most kinetic rate laws available in the literature do not consider all the effectors simultaneously, and much kinetic information exists in a qualitative or semiquantitative form . In this article, we present a strategy to incorporate such information into the kinetic equation . This strategy uses fuzzy logic-based factors to modify algebraic rate laws that account for partial kinetic characteristics . The parameters introduced by the fuzzy factors are then optimized by use of a hybrid of simplex and genetic algorithms . The resulting model provides a flexible form that can simulate various kinetic behaviors . Such kinetic models are suitable for pathway modeling without complete enzyme mechanisms . Three enzymes in Escherichia coli central metabolism are used as examples: phosphoenolpyruvate carboxylase; phosphoenolpyruvate carboxykinase; and pyruvate kinase I . Results show that, with fuzzy logic-augmented models, the kinetic data can be much better described . In particular, complex behavior, such as allosteric inhibition, can be captured using fuzzy rules . The resulting models, even though they do not provide additional physical meaning in enzyme mechanisms, allow the model to incorporate semiquantitative information in metabolic pathway models.

Plant Physiol, 1999 Feb, 119(2), 713 - 24
Use of ubiquitin fusions to augment protein expression in transgenic plants; Hondred D et al.; A major goal of plant biotechnology is the production of genetically engineered crops that express natural or foreign proteins at high levels . To enhance protein accumulation in transgenic plants, we developed a set of vectors that express proteins and peptides as C-terminal translational fusions with ubiquitin (UBQ) . Studies of several proteins in tobacco (Nicotiana tabacum) showed that: (a) proteins can be readily expressed in plants as UBQ fusions; (b) by the action of endogenous UBQ-specific proteases (Ubps), these fusions are rapidly and precisely processed in vivo to release the fused protein moieties in free forms; (c) the synthesis of a protein as a UBQ fusion can significantly augment its accumulation; (d) proper processing and localization of a protein targeted to either the apoplast or the chloroplast is not affected by the N-terminal UBQ sequence; and (e) single amino acid substitutions surrounding the cleavage site can inhibit in vivo processing of the fusion by Ubps . Noncleavable UBQ fusions of beta-glucuronidase became extensively modified, with additional UBQs in planta . Because multiubiquitinated proteins are the preferred substrates of the 26S proteasome, noncleavable fusions may be useful for decreasing protein half-life . Based on their ability to augment protein accumulation and the sequence specificity of Ubps, UBQ fusions offer a versatile way to express plant proteins.

Plant Physiol, 1999 Feb, 119(2), 593 - 8
Expression of a soybean gene encoding the tetrapyrrole-synthesis enzyme glutamyl-tRNA reductase in symbiotic root nodules; Sangwan I et al.; Heme and chlorophyll accumulate to high levels in legume root nodules and in photosynthetic tissues, respectively, and they are both derived from the universal tetrapyrrole precursor delta-aminolevulinic acid (ALA) . The first committed step in ALA and tetrapyrrole synthesis is catalyzed by glutamyl-tRNA reductase (GTR) in plants . A soybean (Glycine max) root-nodule cDNA encoding GTR was isolated by complementation of an Escherichia coli GTR-defective mutant for restoration of ALA prototrophy . Gtr mRNA was very low in uninfected roots but accumulated to high levels in root nodules . The induction of Gtr mRNA in developing nodules was subsequent to that of the gene Enod2 (early nodule) and coincided with leghemoglobin mRNA accumulation . Genomic analysis revealed two Gtr genes, Gtr1 and a 3' portion of Gtr2, which were isolated from the soybean genome . RNase-protection analysis using probes specific to Gtr1 and Gtr2 showed that both genes were expressed, but Gtr1 mRNA accumulated to significantly higher levels . In addition, the qualitative patterns of expression of Gtr1 and Gtr2 were similar to each other and to total Gtr mRNA in leaves and nodules of mature plants and etiolated plantlets . The data indicate that Gtr1 is universal for tetrapyrrole synthesis and that a Gtr gene specific for a tissue or tetrapyrrole is unlikely . We suggest that ALA synthesis in specialized root nodules involves an altered spatial expression of genes that are otherwise induced strongly only in photosynthetic tissues of uninfected plants.

Plant Physiol, 1999 Feb, 119(2), 497 - 506
Organellar and cytosolic localization of four phosphoribosyl diphosphate synthase isozymes in spinach; Krath BN et al.; Four cDNAs encoding phosphoribosyl diphosphate (PRPP) synthase were isolated from a spinach (Spinacia oleracea) cDNA library by complementation of an Escherichia coli Deltaprs mutation . The four gene products produced PRPP in vitro from ATP and ribose-5-phosphate . Two of the enzymes (isozymes 1 and 2) required inorganic phosphate for activity, whereas the others were phosphate independent . PRPP synthase isozymes 2 and 3 contained 76 and 87 amino acid extensions, respectively, at their N-terminal ends in comparison with other PRPP synthases . Isozyme 2 was synthesized in vitro and shown to be imported and processed by pea (Pisum sativum) chloroplasts . Amino acid sequence analysis indicated that isozyme 3 may be transported to mitochondria and that isozyme 4 may be located in the cytosol . The deduced amino acid sequences of isozymes 1 and 2 and isozymes 3 and 4 were 88% and 75% identical, respectively . In contrast, the amino acid identities of PRPP synthase isozyme 1 or 2 with 3 or 4 was modest (22%-25%), but the sequence motifs for binding of PRPP and divalent cation-nucleotide were identified in all four sequences . The results indicate that PRPP synthase isozymes 3 and 4 belong to a new class of PRPP synthases that may be specific to plants.

Plant Physiol, 1999 Feb, 119(2), 481 - 8
Complementary DNA cloning and characterization of ferredoxin localized in bundle-sheath cells of maize leaves; Matsumura T et al.; In maize (Zea mays L.) two leaf-specific ferredoxin (Fd) isoproteins, Fd I and Fd II, are distributed differentially in mesophyll and bundle-sheath cells . A novel cDNA encoding the precursor of Fd II (pFD2) was isolated by heterologous hybridization using a cDNA for Fd I (pFD1) as a probe . The assignment of the cDNAs to the Fds was verified by capillary liquid-chromatography/electrospray ionization-mass spectrometry . RNA-blot analysis demonstrated that transcripts for Fd I and Fd II accumulated specifically in mesophyll and bundle-sheath cells, respectively . The mature regions of pFD1 and pFD2 were expressed in Escherichia coli as functional Fds . Fd I and Fd II had similar redox potentials of -423 and -406 mV, respectively, but the Km value of Fd-NADP+ reductase for Fd II was about 3-fold larger than that for Fd I . Asparagine at position 65 of Fd II is a unique residue compared with Fd I and other Fds from various plants, which have aspartic acid or glutamic acid at the corresponding position as an electrostatic interaction site with Fd-NADP+ reductase . Substitution of asparagine-65 with aspartic acid increased the affinity of Fd II with Fd-NADP+ reductase to a level comparable to that of Fd I . These structural and functional differences of Fd I and Fd II may be related to their cell-specific expression in the leaves of a C4 plant.

Plant Physiol, 1999 Feb, 119(2), 471 - 80
Cloning of nicotianamine synthase genes, novel genes involved in the biosynthesis of phytosiderophores; Higuchi K et al.; Nicotianamine synthase (NAS), the key enzyme in the biosynthetic pathway for the mugineic acid family of phytosiderophores, catalyzes the trimerization of S-adenosylmethionine to form one molecule of nicotianamine . We purified NAS protein and isolated the genes nas1, nas2, nas3, nas4, nas5-1, nas5-2, and nas6, which encode NAS and NAS-like proteins from Fe-deficient barley (Hordeum vulgare L . cv Ehimehadaka no . 1) roots . Escherichia coli expressing nas1 showed NAS activity, confirming that this gene encodes a functional NAS . Expression of nas genes as determined by northern-blot analysis was induced by Fe deficiency and was root specific . The NAS genes form a multigene family in the barley and rice genomes.

Plant Physiol, 1999 Feb, 119(2), 385 - 97
Structure, properties, and tissue localization of apoplastic alpha-glucosidase in crucifers; Monroe JD et al.; Apoplastic alpha-glucosidases occur widely in plants but their function is unknown because appropriate substrates in the apoplast have not been identified . Arabidopsis contains at least three alpha-glucosidase genes; Aglu-1 and Aglu-3 are sequenced and Aglu-2 is known from six expressed sequence tags . Antibodies raised to a portion of Aglu-1 expressed in Escherichia coli recognize two proteins of 96 and 81 kD, respectively, in vegetative tissues of Arabidopsis, broccoli (Brassica oleracea L.), and mustard (Brassica napus L.) . The acidic alpha-glucosidase activity from broccoli flower buds was purified using concanavalin A and ion-exchange chromatography . Two active fractions were resolved and both contained a 96-kD immunoreactive polypeptide . The N-terminal sequence from the 96-kD broccoli alpha-glucosidase indicated that it corresponds to the Arabidopsis Aglu-2 gene and that approximately 15 kD of the predicted N terminus was cleaved . The 81-kD protein was more abundant than the 96-kD protein, but it was not active with 4-methylumbelliferyl-alpha-D-glucopyranoside as the substrate and it did not bind to concanavalin A . In situ activity staining using 5-bromo-4-chloro-3-indolyl-alpha-D-glucopyranoside revealed that the acidic alpha-glucosidase activity is predominantly located in the outer cortex of broccoli stems and in vascular tissue, especially in leaf traces.

Hepatogastroenterology, 1998 Nov-Dec, 45(24), 2042 - 3
Urgent bedside endoscopic nasobiliary drainage without fluoroscopic monitoring; Sreenivas DV et al.; Emergency endoscopic nasobiliary drainage was performed successfully in 2 patients with severe cholangitis and septic shock without the help of fluoroscopy because of technical problems in the fluoroscopy units . Definitive procedure was performed after the recovery . Nasobiliary drainage can be performed without fluoroscopic guidance, but should be attempted in exceptional situations.

Planta, 1998 Dec, 207(2), 286 - 95
Clp protease complexes and their diversity in chloroplasts; Sokolenko A et al.; The Clp proteases represent a large, ancient ATP-dependent protease family which in higher plants is known to be located in chloroplasts . The soluble, presumably multisubunit, enzyme of the organelle stroma is of dual genetic origin . It consists of a nuclear-encoded, regulatory subunit ClpC, which is an ATPase, and a plastid-encoded proteolytic subunit ClpP, which is a serine protease . An additional, nuclear-encoded proteolytic subunit resembling ClpP has been recently reported from tomato (Schaller and Ryan, 1995 plant gene Register 95-00) . We demonstrate that in both tomato Lycopersicon esculentum Mill . and Arabidopsis thaliana, (L.) Heynh . the nuclear-encoded ClpP (nClpP) is made as a precursor molecule that can be imported into isolated intact chloroplasts of spinach (Spinacia oleracea L.) and processed in two or three steps, respectively, to the size of the authentic protein . Furthermore, both gel electrophoresis under non-denaturing conditions and size-exclusion chromatography verified that the three proteins can form distinct heteromeric supramolecular complexes of approximately 860, 1380 and 1700 kDa (probably also of 600 kDa) molecular mass . The size ranges of the former two are reminiscent of those of Clp complexes described from Escherichia coli . In addition, various complexes between 160 and 560 kDa are detectable with the individual components . Both the processing "intermediates" and the mature nClpP are found in assembled form.

Mol Biotechnol, 1998 Dec, 10(3), 269 - 72
Efficient identification of point mutations by automated DNA sequencing of artificial heterozygote samples; Staedtler F et al.; DNA sequencing templates of individual point mutants of the lacI target gene were amplified by polymerase chain reaction (PCR) . By mixing the PCR fragments from two individual mutants in a defined ratio, samples of artificial heterozygous composition were prepared . These samples were then submitted to automated DNA sequencing . The simultaneous, visual comparison of the mixed mutant traces using a graphics program efficiently revealed all heterozygous positions . Based on the individual intensities of the heterozygous base signals the identified point mutations could be assigned to the corresponding mutants . This efficient approach doubles the sample throughput for both the sequencing reactions and the gel electrophoresis using an automated DNA sequencing system.

Mol Biotechnol, 1998 Dec, 10(3), 217 - 22
Cloning and nucleotide sequence of the thermostable beta-galactosidase gene from Pyrococcus woesei in Escherichia coli and some properties of the isolated enzyme; Dabrowski S et al.; Pyrococcus woesei (DSM 3773) beta-galactosidase gene amplified by polymerase chain reaction was cloned into KpnI and HindIII binding sites of pET-30LIC expression plasmid . The obtained pGal2 (6785 bp) transcription vector was then transferred to Escherichia coli B121 (DE3) cells . High identity (99.9%) of DNA sequences suggests that beta-galactosidases from P . woesei and Pyrococcus furiosus are closely related . This enzyme from E . coli transformant is a unique thermostable protein in the cells and can be successfully separated by thermal precipitation of other bacterial proteins at 85 degrees C . The crude beta-galactosidase remaining in the solution comprises about 21% of the total amount of proteins extracted from E . coli cells and has maximal activity at pH 5.4 and temperature of 93 degrees C . Isolated enzyme is active at temperatures up to 110 degrees C and the activity loss after 4 h of incubation at 85 and 93 degrees C did not exceed 11 and 15% of the initial value respectively.

Mol Biotechnol, 1998 Dec, 10(3), 199 - 208
High-level expression of murine terminal deoxynucleotidyl transferase in Escherichia coli grown at low temperature and overexpressing argU tRNA; Boule JB et al.; Terminal deoxynucleotidyl transferase (TdT) is a highly conserved vertebrate enzyme that possesses the unique ability to catalyze the random addition of deoxynucleoside 5'-triphosphates onto the 3'-hydroxyl group of a single-stranded DNA . It plays an important role in the generation of immunoglobin and T-cell receptor diversity . TdT is usually obtained from animal thymus gland or produced in a baculovirus system, but both procedures are rather tedious, and proteolysis occurs during purification . Attempts to overexpress TdT in bacteria have been unsuccessful or have yielded an enzyme with a lower specific activity . A dearth of TdT has thus hampered detailed structural and functional studies . In the present study, we report that by lowering growth temperature and overexpressing a rare arginyl tRNA, it is possible to boost the production in Escherichia coli of murine TdT with minimal proteolysis and high specific activity.

Med Oncol, 1998 Dec, 15(4), 229 - 33
Glycosylated and non-glycosylated recombinant human granulocyte colony-stimulating factor (rhG-CSF)--what is the difference?
Hoglund M.
Two forms of recombinant human G-CSF (rhG-CSF) are available for clinical use: filgrastim is expressed in E coli and non-glycosylated, whereas lenograstim is derived from Chinese hamster ovary (CHO) cells and glycosylated . The function of the sugar chain, accounting for approximately 4% of the molecular weight of lenograstim (and native G-CSF), is not known . Glycosylation of the G-CSF molecule does not prolong its circulation half life . Lenograstim is more active than filgrastim (and research-use deglycosylated G-CSF) on a weight-by-weight basis in in vitro colony-forming and cell line assays . An international potency standard assigns a specific activity of 100,000 IU/microgram to filgrastim and 127,760 IU/microgram to lenograstim . Correspondingly, two randomised crossover studies in normal subjects, comparing mass equivalent doses of the two rhG-CSFs, have demonstrated a 25-30% higher concentration of blood stem cells (CD34+, CFU-GM) during lenograstim administration . No difference in side effects was observed . Results from a prospective, randomised, non-crossover trial in breast cancer patients suggest that bioequivalent doses of filgrastim and lenograstim have a similar effect on mobilisation of CD34+ cells and immature CD34+ cell subsets, respectively . Although comparisons outside the setting of stem cell mobilisation are lacking, the clinical relevance of the greater specific activity of lenograstim may thus be limited . The difference in potency between microgram identical doses of the two rhG-CSFs makes dosing in biological units (IU) rather than mass units (microgram) more appropriate.

Parasitol Res, 1999 Jan, 85(1), 41 - 6
Characterization of a Plasmodium chabaudi gene encoding a protein with glutamate-rich tandem repeats; Giraldo LE et al.; Several highly antigenic proteins containing tandem repeats rich in glutamic acid residues have been described in Plasmodium falciparum . However, relatively little information is available about analogous genes in rodent parasites . This report describes a 4.2-kb genomic DNA fragment from P . chabaudi with a deduced amino acid sequence that is predominantly glutamate-rich tandem repeats . Several different monoclonal antibodies raised against a 93-kDa P . chabaudi protein, which does not correspond to the cloned DNA fragment, recognize a recombinant protein expressed from the 4.2-kb DNA fragment . The only sequence similarities between these two genes are tandem repeats with a predominance of glutamate pairs followed by a hydrophobic residue . This repetitious-sequence motif may be the basis for the observed cross-reactivity . A similar motif has been demonstrated to be the basis for antibody cross-reactivity between glutamate-rich proteins of P . falciparum . The expression of multiple glutamate-rich proteins with cross-reacting epitopes may be a general phenomenon in Plasmodium species.

Proc Natl Sci Counc Repub China B, 1999 Jan, 23(1), 38 - 44
Identification of a novel family of human Rab-like small GTP-binding proteins; Peng HL et al.; The presence of a novel family of Rab-like proteins (Rlp) in the human genome is reported . The gene encoding the Rlp-2 was isolated from a human lymphocyte genomic library . The Rlp-2 gene is intronless and was mapped to chromosome Xq21.3 using fluorescence in situ hybridization . Several cDNA clones encoding the Rlp-1 were identified in a human hippocampus lambda library . Northern analysis revealed a 2.1-kb transcript of Rlp-1 expressed predominantly in brain, heart and skeletal muscle . The transcript was also observed in all examined regions of the human brain at a similar level . An additional gene, termed Rlp-3, which is highly related to Rlp-1 and Rlp-2, was found in the GenBank Data Base . The predicted molecular mass for Rlp is approximately 31 kDa and is consistent with that of Rlp-1 synthesized in Escherichia coli.

J Biomed Sci, 1999 Jan, 6(1), 64 - 70
Expression of foreign antigens on the surface of Escherichia coli by fusion to the outer membrane protein traT; Chang HJ et al.; The traT gene is one of the F factor transfer genes and encodes an outer membrane protein which is involved in interactions between an Escherichia coli and its surroundings . This protein was altered so as to permit the expression of foreign proteins on the outer membrane of E . coli in this study . A 729-bp DNA fragment, including the leader and entire structural gene sequence of traT, was amplified and obtained by PCR . This sequence was then subcloned downstream of the tac promoter of pDR540, resulting in a TraT expression vector, pT2 . Here, we report that the expression of TraT protein, fused either with a partial pre-S antigen of hepatitis B virus (60 and 98 amino acids, respectively) or with the snake venom rhodostomin (72 amino acids), was successfully achieved on the outer membrane of E . coli, using the pT2 plasmid . This result was demonstrated using dot blot and immunofluorescence analysis . This finding supports the notion that the pT2 plasmid can be used as an E . coli display system . This system can detect a foreign peptide of about 100 amino acid residues in length on the bacterial surface.

J Biol Chem, 1999 Feb 12, 274(7), 3923 - 6
The in vitro ligation of bacterially expressed proteins using an intein from Methanobacterium thermoautotrophicum; Evans TC Jr et al.; The smallest known intein, found in the ribonucleoside diphosphate reductase gene of Methanobacterium thermoautotrophicum (Mth RIR1 intein), was found to splice poorly in Escherichia coli with the naturally occurring proline residue adjacent to the N-terminal cysteine of the intein . Splicing proficiency increased when this proline was replaced with an alanine residue . However, constructs that displayed efficient N- and C-terminal cleavage were created by replacing either the C-terminal asparagine or N-terminal cysteine of the intein, respectively, with an alanine . Furthermore, these constructs were used to specifically generate complementary reactive groups on protein sequences for use in ligation reactions . Reaction between an intein-generated C-terminal thioester on E . coli maltose-binding protein (43 kDa) and an intein-generated cysteine at the N terminus of either T4 DNA ligase (56 kDa) or thioredoxin (12 kDa) resulted in the ligation of the proteins through a native peptide bond . Thus the smallest of the known inteins is capable of splicing and its unique properties extend the utility of intein-mediated protein ligation to include the in vitro fusion of large, bacterially expressed proteins.

Diagn Microbiol Infect Dis, 1998 Dec, 32(4), 255 - 8
The use of an IpaC-specific ELISA to identify enteroinvasive Escherichia coli strains of unusual serogroups; Pal T et al.; Twenty five Escherichia coli isolates expressing O antigens different from the conventionally recognized enteroinvasive E . coli were tested in the Sereny test, with an invasion plasmid-specific DNA probe, and by an enzyme-linked immunosorbent assay recognizing the secreted IpaC antigen . These results indicate that the IpaC enzyme-linked immunosorbent assay is a sensitive method to recognize enteroinvasive E . coli, irrespective of their serogroups.

Bioorg Med Chem Lett, 1998 Dec 15, 8(24), 3511 - 4
Methionine analogues as inhibitors of methionyl-tRNA synthetase; Lee J et al.; A series of methionine analogues have been synthesized as inhibitors of methionyl-tRNA synthetase and evaluated for their inhibitory activities of E . coli methionyl-tRNA synthetase and bacterial growth . Among them, L-methionine hydroxamate 20 has proved to be the best inhibitor of the enzyme with Ki = 19 microM and showed a growth inhibition against E.coli JM 109, P . vulganis 6059 and C . freundii 8090.

Phytochemistry, 1999 Jan, 50(2), 209 - 14
Expression of barley ADP-glucose pyrophosphorylase in Escherichia coli: processing and regulatory considerations; Luo C et al.; Full length cDNAs for barley ADP-glucose pyrophosphorylase (AGPase) coding for the large subunits of the endosperm and leaf homologues of the enzyme (AGPase-S1 and -S2, respectively) and for the small subunit protein from endosperm (AGPase-B1), have been expressed in Escherichia coli . The cDNAs for AGPase-S1 and -S2 required different induction conditions for their maximal expression and they encoded immunologically distinct proteins . The AGPase-S1 that was produced by E . coli had the same M(r) (58 kDa) as the corresponding protein in barley crude endosperm extracts, whereas the bacteria-produced AGPase-S2 (55 kDa) was larger than its counterpart from barley leaf preparations (53 kDa) . An enzymatically active AGPase expressed in E . coli from a double construct containing cDNAs for AGPase-S1 and -B1 subunits was insensitive to the activation by 3-phosphoglycerate and to inhibition by inorganic phosphate, similarly to the enzyme in barley endosperm . Neither AGPase-S1 nor -B1 were active when expressed alone in the bacteria . The data are discussed with respect to possible mechanisms of intracellular targeting of immature AGPase-S proteins in barley tissues and regarding previous data on effector regulation of the barley enzyme.

FEMS Microbiol Lett, 1999 Jan 15, 170(2), 389 - 98
The F420H2-dehydrogenase from Methanolobus tindarius: cloning of the ffd operon and expression of the genes in Escherichia coli; Westenberg DJ et al.; The membrane-bound F420H2-dehydrogenase from the methylotrophic methanogen Methanolobus tindarius oxidizes reduced coenzyme F420 and feeds the electrons into an energy-conserving electron transport chain . Based on the N-terminal amino acid sequence of the 40-kDa subunit of F420H2-dehydrogenase the corresponding gene ffdB was detected in chromosomal DNA of M . tindarius . Sequence analysis, primer extension, and RT-PCR experiments indicated that ffdB is part of an operon harboring three additional open reading frames (ffdA, ffdC, ffdD) . The corresponding mRNA transcript and transcription start sites were determined . All four genes could be heterologously expressed in Escherichia coli.

J Biol Chem, 1999 Feb 12, 274(7), 4281 - 92
Export of recombinant Mycobacterium tuberculosis superoxide dismutase is dependent upon both information in the protein and mycobacterial export machinery . A model for studying export of leaderless proteins by pathogenic mycobacteria; Harth G et al.; We have investigated the expression and extracellular release of enzymatically active superoxide dismutase, one of the 10 major extracellular proteins of Mycobacterium tuberculosis, both in its native host and in the heterologous host Mycobacterium smegmatis . We found that the M . tuberculosis superoxide dismutase gene, encoding a leaderless polypeptide of Mr approximately 23,000 representing one of the four identical subunits of the enzyme, is expressed constitutively under normal growth conditions and at a 5-fold increased level under conditions of hydrogen peroxide stress . The highly pathogenic mycobacterium M . tuberculosis expresses 93-fold more superoxide dismutase than the nonpathogenic mycobacterium M . smegmatis, and it exports a much higher proportion of expressed enzyme (76 versus 21%); taking both expression and export into consideration, M . tuberculosis exports approximately 350-fold more enzyme than M . smegmatis . In M . smegmatis, recombinant M . tuberculosis superoxide dismutase is expressed at 8.4 times the level of the endogenous enzyme and the proportion exported (66%) approaches that in the homologous host; hence M . smegmatis exports up to 26-fold more of the recombinant than endogenous enzyme . Interestingly, subunits of the M . tuberculosis and M . smegmatis enzymes readily and stoichiometrically exchange with each other, forming five different complexes of four subunits, both when the enzymes are expressed in the recombinant host and when the purified enzymes are incubated together; however, each subunit retains its characteristic metal ion, iron for M . tuberculosis and manganese for M . smegmatis . Compared with the cell-associated enzyme, the supernatant enzyme of recombinant M . smegmatis is enriched for M . tuberculosis enzyme subunits, consistent with preferential export of the M . tuberculosis enzyme . Recombinant M . tuberculosis superoxide dismutase transcomplements a superoxide dismutase-deficient Escherichia coli, resulting in a reduction of sensitivity of the strain to oxidative stress, but the enzyme is not exported from this nonmycobacterial host . Our findings indicate that the information for export of the M . tuberculosis superoxide dismutase is contained within the protein but that export additionally requires export machinery specific to mycobacteria.

J Biol Chem, 1999 Feb 12, 274(7), 4273 - 80
Cloning and expression of a potato cDNA encoding hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase; Schmidt A et al.; Hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110) catalyzes the transfer of hydroxycinnamic acids from the respective CoA esters to tyramine and other amines in the formation of N-(hydroxycinnamoyl)amines . Expression of THT is induced by Phytophthora infestans, the causative agent of late blight disease in potato . The amino acid sequences of nine endopeptidase LysC-liberated peptides from purified potato THT were determined . Using degenerate primers, a THT-specific fragment was obtained by reverse transcription-polymerase chain reaction, and THT cDNA clones were isolated from a library constructed from RNA of elicitor-treated potato cells . The open reading frame encoding a protein of 248 amino acids was expressed in Escherichia coli . Recombinant THT exhibited a broad substrate specificity, similar to that of native potato THT, accepting cinnamoyl-, 4-coumaroyl-, caffeoyl-, feruloyl- and sinapoyl-CoA as acyl donors and tyramine, octopamine, and noradrenalin as acceptors tested . Elicitor-induced THT transcript accumulation in cultured potato cells peaked 5 h after initiation of treatment, whereas enzyme activity was highest from 5 to 30 h after elicitation . In soil-grown potato plants, THT mRNA was most abundant in roots . Genomic Southern analyses indicate that, in potato, THT is encoded by a multigene family.

J Biol Chem, 1999 Feb 12, 274(7), 4202 - 6
Why superoxide imposes an aromatic amino acid auxotrophy on Escherichia coli . The transketolase connection; Benov L et al.; The lack of superoxide dismutase and the consequent elevation of {O2-} imposes, on Escherichia coli, auxotrophies for branched chain, sulfur-containing, and aromatic amino acids . The former two classes of auxotrophies have already been explained, whereas the third is explained herein . Thus O2- is shown to interfere with the production of erythrose-4-phosphate, which is essential for the first step of the aromatic biosynthetic pathway . It does so by oxidizing the 1, 2-dihydroxyethyl thiamine pyrophosphate intermediate of transketolase and inactivating this enzyme.

J Biol Chem, 1999 Feb 12, 274(7), 4166 - 73
The nuclear cap-binding complex is a novel target of growth factor receptor-coupled signal transduction; Wilson KF et al.; In an attempt to further understand how nuclear events (such as gene expression, nuclear import/export, and cell cycle checkpoint control) might be subject to regulation by extracellular stimuli, we sought to identify nuclear activities under growth factor control . Using a sensitive photoaffinity labeling assay that measured {alpha-32P}GTP incorporation into nuclear proteins, we identified the 20-kDa subunit of the nuclear cap-binding complex (CBC) as a protein whose binding activity is greatly enhanced by the extracellular stimulation of serum-arrested cells . The CBC represents a 20- and 80-kDa heterodimer (the subunits independently referred to as CBP20 and CBP80, respectively) that binds the 7-methylguanosine cap on RNAs transcribed by RNA polymerase II . This binding facilitates precursor messenger RNA splicing and export . We have demonstrated that the {alpha-32P}GTP incorporation into CBP20 was correlated with an increased ability of the CBC to bind capped RNA and have used the {alpha-32P}GTP photoaffinity assay to characterize the activation of the CBC in response to growth factors . We show that the CBC is activated by heregulin in HeLa cells and by nerve growth factor in PC12 cells as well as during the G1/S phase of the cell cycle and when cells are stressed with UV irradiation . Additionally, we show that cap-dependent splicing of precursor mRNA, a functional outcome of CBC activation, can be catalyzed by growth factor addition to serum-arrested cells . Taken together, these data identify the CBC as a nuclear target for growth factor-coupled signal transduction and suggest novel mechanisms by which growth factors can influence gene expression and cell growth.

J Biol Chem, 1999 Feb 12, 274(7), 4074 - 81
A K319N/E325Q double mutant of the lactose permease cotransports H+ with lactose . Implications for a proposed mechanism of H+/lactose symport; Johnson JL et al.; In this study, we have examined the transport characteristics of the wild-type lactose permease, single mutants in which Lys-319 was changed to asparagine or alanine or Glu-325 was changed to glutamine or alanine, and the corresponding double mutant strains . The wild-type and Asn-319 mutant showed high levels of lactose uptake, with Km values of 0.42 and 1.30 mM and Vmax values of 102.6 and 48.3 nmol of lactose/min/mg of protein, respectively . The Asn-319/Gln-325 strain had a normal Km of 0.36 mM and a moderate Vmax of 18.5 nmol of lactose/min/mg of protein . By comparison, the single E325Q strain had a normal Km of 0.27 mM but a very defective Vmax of 1.3 nmol of lactose/min/mg of protein . A similar trend was observed among the alanine substitutions at these positions, although the Vmax values were lower for the Ala-319 mutations . When comparing the Vmax values between the single position 325 mutants with those of the double mutants, these results indicate that neutral 319 mutations substantially alleviate a defect in Vmax caused by neutral 325 mutations . With regard to H+/lactose coupling, the wild-type permease is normally coupled and can transport lactose against a gradient . The position 325 single mutants showed no evidence of H+ transport with lactose or thiodigalactoside (TDG) and were unable to facilitate uphill lactose transport . The single Asn-319 mutant and double Asn-319/Gln-325 mutant were able to transport H+ upon the addition of lactose or TDG . In addition, both of these strains catalyzed a sugar-dependent H+ leak that inhibited cell growth in the presence of TDG . These two strains were also defective in uphill transport, which may be related to their sugar-dependent leak pathway . Based on these and other results in the literature, a model is presented that describes how the interactions among several ionizable residues within the lactose permease act in a concerted manner to control H+/lactose coupling . In this model, Lys-319 and Glu-325 play a central role in governing the ability of the lactose permease to couple the transport of H+ and lactose.

J Biol Chem, 1999 Feb 12, 274(7), 4009 - 16
Polyadenylation promotes degradation of 3'-structured RNA by the Escherichia coli mRNA degradosome in vitro; Blum E et al.; Polyadenylation contributes to the destabilization of bacterial mRNA . We have investigated the role of polyadenylation in the degradation of RNA by the purified Escherichia coli degradosome in vitro . RNA molecules with 3'-ends incorporated into a stable stem-loop structure could not readily be degraded by purified polynucleotide phosphorylase or by the degradosome, even though the degradosome contains active RhlB helicase which normally facilitates degradation of structured RNA . The exoribonucleolytic activity of the degradosome was due to polynucleotide phosphorylase, rather than the recently reported exonucleolytic activity exhibited by a purified fragment of RNase E (Huang, H., Liao, J., and Cohen, S . N . (1998) Nature 391, 99-102) . Addition of a 3'-poly(A) tail stimulated degradation by the degradosome . As few as 5 adenosine residues were sufficient to achieve this stimulation, and generic sequences were equally effective . The data show that the degradosome requires a single-stranded "toehold" 3' to a secondary structure to recognize and degrade the RNA molecule efficiently; polyadenylation can provide this single-stranded 3'-end . Significantly, oligo(G) and oligo(U) tails were unable to stimulate degradation; for oligo(G), at least, this is probably due to the formation of a G quartet structure which makes the 3'-end inaccessible . The inaccessibility of 3'-oligo(U) sequences is likely to have a role in stabilization of RNA molecules generated by Rho-independent terminators.

Biotechnol Prog, 1999 Jan-Feb, 15(1), 81 - 90
Modeling of overflow metabolism in batch and fed-batch cultures of Escherichia coli; Xu B et al.; A dynamic model of glucose overflow metabolism in batch and fed-batch cultivations of Escherichia coli W3110 under fully aerobic conditions is presented . Simulation based on the model describes cell growth, respiration, and acetate formation as well as acetate reconsumption during batch cultures, the transition of batch to fed-batch culture, and fed-batch cultures . E . coli excreted acetate only when specific glucose uptake exceeded a critical rate corresponding to a maximum respiration rate . In batch cultures where the glucose uptake was unlimited, the overflow acetate made up to 9 . 0 +/- 1.0% carbon/carbon of the glucose consumed . The applicability of the model to dynamic situations was tested by challenging the model with glucose and acetate pulses added during the fed-batch part of the cultures . In the presence of a glucose feed, E . coli utilized acetate 3 times faster than in the absence of glucose . The cells showed no significant difference in maximum specific uptake rate of endogenous acetate produced by glucose overflow and exogenous acetate added to the culture, the value being 0.12-0.18 g g-1 h-1 during the entire fed-batch culture period . Acetate inhibited the specific growth rate according to a noncompetitive model, with the inhibition constant (ki) being 9 g of acetate/L . This was due to the reduced rate of glucose uptake rather than the reduced yield of biomass.

Biotechnol Prog, 1999 Jan-Feb, 15(1), 58 - 64
Library of synthetic 5' secondary structures to manipulate mRNA stability in Escherichia coli; Carrier TA et al.; A DNA cassette system has been developed to allow for the convenient introduction of synthetic DNA oligonucleotides between the transcription and translation start sites of a gene in order to examine the effect of 5' hairpin structure and strength on mRNA stabilization . Rationally designed synthetic DNA cassettes were introduced into the 5' untranslated region of a modified lacZ gene to form hairpins at the 5' end of the mRNA . These DNA inserts influenced mRNA half-lives over an order-of-magnitude range, with some groups of predicted structures having half-lives that showed a strong correlation with hairpin strength while half-lives for another group of predicted structures exhibited little or no dependence on this property . These results indicate the importance of 5' hairpin structure and strength in determining stabilization of Escherichia coli mRNA . This synthetic library, as well others generated using the DNA cassette system described here, should prove useful in understanding the mechanisms of mRNA stabilization and in designing structures for recombinant gene expression control.

Curr Genet, 1999 Jan, 34(6), 472 - 7
Cloning and expression of the cDNA encoding an alternative oxidase gene from Aspergillus niger WU-2223L; Kirimura K et al.; A cDNA fragment encoding the mitochondrial alternative oxidase, the enzyme responsible for cyanide-insensitive and salicylhydroxamic acid (SHAM)-sensitive respiration, from the citric acid-producing fungus Aspergillus niger WU-2223L was cloned and expressed in Escherichia coli as a host strain . Synthetic primers were designed from the conserved nucleotide sequences of the alternative oxidase genes from higher plants and a yeast . The 210-bp DNA fragment was amplified by PCR with these primers using chromosomal DNA of WU-2223L as a template, and was employed to screen a cDNA library of A . niger . One full-length cDNA clone of 1.2 kb was obtained, and was sequenced to reveal that the clone contained an open reading frame (ORF-AOX1) encoding a polypeptide of 351 amino acids . The predicted amino-acid sequence exhibited 50%, 55%, and 52% homology to the alternative oxidases of Hansenula anomala, Neurospora crassa and Sauromatum guttatum, respectively . In the 5'-terminus region of the ORF-AOX1, a mitochondrial targeting motif was found . The whole open reading frame of ORF-AOX1 was ligated to plasmid pKK223-3 to construct the expression vector pKAOX1 . The E . coli transformant harboring pKAOX1 showed cyanide-insensitive and SHAM-sensitive respiration, and expression was increased approximately two-fold by the addition of IPTG . These results indicated that the ORF-AOX1 encodes an alternative oxidase of A . niger.

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