Escherichia coli

Med Hypotheses, 1997 Apr, 48(4), 317 - 24
Sudden infant death syndrome and placental disorders: the thyroid-selenium link; Reid GM et al.; Placental insufficiency, inducing hypoxia-ischaemia, is considered a major cause of neuronal injury and impaired post natal development . Placental insufficiency alters the metabolism of arachidonic acid and its oxidation products . Premature labour and low-birth-weight infants are associated with reduced intrauterine blood-flow and infections of the reproductive tract . Thyroidal activity is depressed in undernutrition (placental insufficiency) . Premature infants require extra vitamin C for normal tyrosine metabolism (tyrosine is the thyroxine precursor) . Among the symptoms indicating infantile cretinism, which appear during 3-5 months of age are: delayed union of skull bones, torpid behaviour, slow feeding, cyanosis during feeding, excessive sleepiness, enlarged tongue, umbilical herniation, flabby musculature, short stature and delayed development . These symptoms have all been described in low-birth-weight infants and sudden infant death syndrome victims by various authors . Bacteria utilize selenium (at the expense of host tissue) . Escherichia coli is among the bacteria invading the reproductive tract . E . coli produce thiouracil and are goitrogenic . Some strains of E . coli produce phospholipase A2 which releases arachidonic acid from phospholipids for prostaglandin synthesis . Phospholipase A2 is more active against peroxidized than non-peroxidized lipids . Bacterial competition for intrauterine selenium and goitrogenic bacterial infections of the reproductive tract during pregnancy, depress thyroid function in the fetus but not in the mother.

Mol Microbiol, 1997 Apr, 24(2), 373 - 85
The sigmaE-mediated response to extracytoplasmic stress in Escherichia coli is transduced by RseA and RseB, two negative regulators of sigmaE; De Las Penas A et al.; The extracytoplasmic stress response in Escherichia coli is controlled by the alternative sigma factor, sigma(E) . sigma(E) activity is uniquely induced by the accumulation of outer membrane protein precursors in the periplasmic space, and leads to the increased production of several proteins, including the periplasmic protease DegP, that are thought to be required for maintaining cellular integrity under stress conditions . Genetic and biochemical experiments show that sigma(E) activity is under the control of three genes, rseABC (for regulator of sigma E), encoded immediately downstream of the sigma factor . Deletion of rseA leads to a 25-fold induction of sigma(E) activity . RseA is predicted to be an inner membrane protein, and the purified cytoplasmic domain binds to and inhibits sigma(E)-directed transcription in vitro, indicating that RseA acts as an anti-sigma factor . Deletion of rseB leads to a slight induction of sigma(E), indicating that RseB is also a negative regulator of sigma(E) . RseB is a periplasmic protein and was found to co-purify with the periplasmic domain of RseA, indicating that RseB probably exerts negative activity on sigma(E) through RseA . Deletion of rseC, in contrast, has no effect on sigma(E) activity under steady-state conditions . Under induction conditions, strains lacking RseB and/or C show wild-type induction of sigma(E) activity, indicating either the presence of multiple pathways regulating sigma(E) activity, or the ability of RseA alone to both sense and transmit information to sigma(E).

Mol Microbiol, 1997 Apr, 24(2), 355 - 71
Modulation of the Escherichia coli sigmaE (RpoE) heat-shock transcription-factor activity by the RseA, RseB and RseC proteins; Missiakas D et al.; The sigma(E) (RpoE) transcription factor of Escherichia coli regulates the expression of genes whose products are devoted to extracytoplasmic activities . The sigma(E) regulon is induced upon misfolding of proteins in the periplasm or the outer membrane . Similar to other alternative sigma factors, the activity of sigma(E) is tightly regulated in E . coli . We have previously shown that sigma(E) is positively autoregulated at the transcriptional level . DNA sequencing, coupled with transcriptional analyses, have shown that sigma(E) is encoded by the first gene of a four-gene operon . The second gene of this operon, rseA, encodes an anti-sigma(E) activity . This was demonstrated at both the genetic and biochemical levels . For example, mutations in rseA constitutively increase sigma(E) activity . Consistent with this, overproduction of RseA leads to an inhibitory effect on sigma(E) activity . Topological analysis of RseA suggests the existence of one transmembrane domain, with the N-terminal part localized in the cytoplasm . Overproduction of this N-terminal domain alone was shown to inhibit sigma(E) activity . These observations were confirmed in vitro, because either purified RseA or only its purified N-terminal domain inhibited transcription from Esigma(E)-dependent promoters . Furthermore, RseA and sigma(E) co-purify, and can be co-immunoprecipitated, and chemically cross-linked . The sigma(E) activity is further modulated by the products of the remaining genes in this operon, rseB and rseC . RseB is a periplasmic protein, which negatively regulates sigma(E) activity and specifically interacts with the C-terminal periplasmic domain of RseA . In contrast, RseC is an inner membrane protein that positively modulates sigma(E) activity . Most of these protein-protein interactions were verified in vivo using the yeast two-hybrid system.

Mol Microbiol, 1997 Apr, 24(2), 255 - 61
The Escherichia coli phage-shock-protein (psp) operon; Model P et al.; The phage-shock-protein (psp) operon helps to ensure survival of Escherichia coli in late stationary phase at alkaline pH, and protects the cell against dissipation of its proton-motive force against challenge . It is strongly induced by filamentous phage pIV and its bacterial homologues, and by mutant porins that don't localize properly, as well as by a number of other stresses . Transcription of the operon is dependent on sigma54 and a constitutively active, autogenously controlled activator . psp-operon expression is controlled by one negatively and several positively acting regulators, none of which is a DNA-binding protein . The major product of the operon, PspA, may also serve as a negative regulator of an unusual porin, OmpG.

Bioorg Med Chem, 1997 Apr, 5(4), 707 - 14
Synthesis of N-glyoxylyl peptides and their in vitro evaluation as HIV-1 protease inhibitors; Qasmi D et al.; A series of novel synthetic peptides containing an N-terminal glyoxylyl function (CHOCO-) have been tested as inhibitors of HIV-1 protease . The N-glyoxylyl peptide CHOCO-Pro-Ile-Val-NH2, which fulfills the specificity requirements of the MA/CA protease cleavage site together with the criteria of transition state analogue of the catalyzed reaction, was found to be a moderate competitive inhibitor although favorable interactions were visualized between its hydrated form and the catalytic aspartates using molecular modeling . Increasing the length of the peptide sequence led to compounds acting only as substrates.

J Clin Microbiol, 1997 Apr, 35(4), 867 - 72
Detection and characterization of the coli surface antigen 6 of enterotoxigenic Escherichia coli strains by using monoclonal antibodies; Helander A et al.; We describe, for the first time, the production of monoclonal antibodies (MAbs) against coli surface antigen 6 (CS6) of enterotoxigenic Escherichia coli (ETEC) and their use for characterization and diagnosis of CS6 . Two MAbs, MAbs CS6-20:11:9 and CS6-2A:14, were produced by immunizing mice with purified CS6 or CS6-containing bacterial extracts . The MAb specificity was demonstrated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and immunoelectron microscopy, which showed that the MAbs bound to CS6-expressing bacteria as well as to purified CS6 and CS6 structural subunits but not to CS6-negative bacteria or other purified ETEC colonization factors . By using bacterial recombinants, i.e., strains with a complete CS6 operon or parts thereof, it was found that both MAbs were specific for CssB, one of the two structural subunits of CS6 . Although the MAbs bound specifically to the entire surface of CS6-expressing bacteria, no structure of CS6 could be identified . The usefulness of the MAbs for the detection of CS6 was evaluated in an inhibition ELISA and in a dot blot test . Ninety-two ETEC strains with known colonization factors were analyzed, and all CS6-positive strains were identified by either assay with MAb CS6-2A:14, whereas MAb CS6-20:11:9 failed to identify two CS6-positive strains; in no instance was any CS6-negative strain identified by either of the MAbs . Parallel analyses of 48 strains with a gene probe specific for the other structural subunit of CS6, i.e., CssA, and the MAb-based assays gave identical results, suggesting the simultaneous presence of both subunits.

J Protein Chem, 1997 Apr, 16(3), 213 - 25
Changes in conformation and stability upon formation of complexes of erythropoietin (EPO) and soluble EPO receptor; Narhi LO et al.; Erythropoietin (EPO) is a glycoprotein hormone which belongs to the four-helical-bundle cytokine family and regulates the level of circulating red blood cells . The EPO receptor (EPOR) belongs to the cytokine-receptor family of proteins . While many of the downstream events following receptor/ligand interaction have been defined, both ligand-induced receptor dimerization and conformational changes induced by binding have been implicated as the initial step in signal transduction . In a recent paper {Philo et al . (1996), Biochemistry 38, 1681-1691} we described the formation of both 1:1 and 2:1 EPOR/EPO complexes . In this paper, we examine changes in protein conformation and stability resulting from the formation of both 1:1 and 2:1 complexes of the soluble extracellular domain of EPOR and the recombinant EPO derived from either Chinese hamster ovary cells or from Escherichia coli cells . Occupation of the first binding site results in a slight conformational change that is apparent in both the far- and near-UV circular dichroism spectra . Formation of the 2:1 complex results in an even greater change in conformation which involves the local environment of one or more aromatic amino acids, accompanied perhaps by a small increase in helical content of the complex . This change in local conformation could occur in the EPO molecule, in the EPOR, in both EPOR molecules due to dimerization, or in all molecules in the trimer . The 1:1 complex exhibits increased stability to thermal-induced denaturation relative to the individual protein component; indeed, the E . coli-derived (nonglycosylated) EPO stays folded in the complex at temperatures where the EPO alone would have unfolded and precipitated . Glycosylation of the receptor increases the reversibility of thermal denaturation, but does not affect the temperature at which this unfolding reaction occurs.

Plant Mol Biol, 1997 Apr, 33(6), 1105 - 10
Characterization of a structurally and functionally diverged acyl-acyl carrier protein desaturase from milkweed seed; Cahoon EB et al.; A cDNA for a structurally variant acyl-acyl carrier protein (ACP) desaturase was isolated from milkweed (Asclepias syriaca) seed, a tissue enriched in palmitoleic (16:1delta9)* and cis-vaccenic (18:1delta11) acids . Extracts of Escherichia coli that express the milkweed cDNA catalyzed delta9 desaturation of acyl-ACP substrates, and the recombinant enzyme exhibited seven- to ten-fold greater specificity for palmitoyl (16:0)-ACP and 30-fold greater specificity for myristoyl (14:0)-ACP than did known delta9-stearoyl (18:0)-ACP desaturases . Like other variant acyl-ACP desaturases reported to date, the milkweed enzyme contains fewer amino acids near its N-terminus compared to previously characterized delta9-18:0-ACP desaturases . Based on the activity of an N-terminal deletion mutant of a delta9-18:0-ACP desaturase, this structural feature likely does not account for differences in substrate specificities.

Plant Mol Biol, 1997 Apr, 33(6), 1073 - 84
Isolation and transcript analysis of gibberellin 20-oxidase genes in pea and bean in relation to fruit development; Garcia-Martinez JL et al.; PCR was used with degenerate primers based on conserved amino acid sequences in gibberellin (GA) 20-oxidases to isolate cDNA clones for these enzymes from young seeds of pea (Pisum sativum) and developing embryos of French bean (Phaseolus vulgaris) . One GA 20-oxidase cDNA (Ps27-12) was obtained from pea and three (Pv 15-11, Pv73-1 and Pv85-26) from bean . Their identities were confirmed by demonstrating that fusion proteins expressed in Escherichia coli exhibited GA 20-oxidase activity, converting {14C}GA12 to {14C}GA9 . The intermediates in this three-step reaction, GA15 and GA24, were also identified as products . The expression proteins from three of the clones (Ps27-12, Pv15-11 and Pv73-1) were also shown to convert GA53 to GA20, as effectively as they did GA12 . On the basis of transcript levels measured by northern blot analysis, the pea GA 20-oxidase gene is most highly expressed in young leaves, fully expanded internodes, very young seeds (until 4 days after anthesis) and expanding pods (from 3 days after anthesis at least until day 6) . Expression in pods from 3-day-old unpollinated ovaries is higher than in those from pollinated ovaries . Treatment of unpollinated ovaries with GA3 to induce parthenocarpic fruit-set severely reduced the amount of GA 20-oxidase mRNA, whereas treatment with 2,4-D, although inducing fruit-set, did not reduce the levels of these transcripts . Plant decapitation above an unpollinated ovary resulted in very high levels of GA 20-oxidase mRNA in the pod . The three GA 20-oxidase genes from French bean showed very different patterns of expression: Pv 15-1 was expressed in the roots, young leaves, and developing seeds, but most highly in immature cotyledons, while Pv73-1 has a similar expression pattern to Ps27-12, with transcripts found only in young seeds and young leaves, where it was particularly abundant . Transcripts corresponding to Pv85-26 were detected in developing seeds, and just traces in the young leaves . Southern blot analysis indicated that the bean GA 20-oxidases are each encoded by single-copy genes, whereas one more gene, homologous to Ps27-12, could also exist in pea.

Plant Mol Biol, 1997 Apr, 33(6), 1037 - 50
High content, size and distribution of single-stranded DNA in the mitochondria of Chenopodium album (L.); Backert S et al.; Mitochondrial (mt) DNA of higher plants is unique in its large size and complexity . We report here a hitherto unknown feature, the presence of large quantities of single-stranded (ss) DNA . About 2.0-8.5% of the chromosomal mtDNA from a suspension culture (depending on the growth stage) and 6.5% of the chromosomal mtDNA from whole plants of Chenopodium album were found to be in ss form by dot-blot hybridization after neutral transfer . Similar amounts of ss mtDNA were observed by binding of the single-strand binding (SSB) protein of Escherichia coli under the electron microscope . Significantly less ssDNA was found in plastids of C . album and in E . coli cells . We observed ss regions between 100 and 22,800 bases distributed in the mt genome spaced from 0.5-100 kb apart . After pulsed-field gel electrophoresis (PFGE), the well-bound fraction of mtDNA (found to consist of circular, sigma-shaped and rosette-like molecules), contained the major part of ssDNA as opposed to the migrating linear molecules . Digestion of mtDNA by ss-specific nucleases followed by PFGE mobilized all well-bound DNA and correspondingly increased the quantity of migrating linear DNA molecules . The implications of ssDNA for the structural organization on plant mt genomes are discussed.

Fukuoka Igaku Zasshi, 1997 Apr, 88(4), 128 - 31
{An old female case of vero-cytotoxin-producing Escherichia coli O-157: H7 infection}; Takaki Y et al.; It is well known that verocytotoxin-producing Escherichia coli O-157 : H7 infection can be severe in elderly persons . We report an 82-year-old female case of verocytotoxin-producing Escherichia coli O-157 : H7 infection . She was admitted to our hospital because of bloody diarrhea . The hemolytic-uremic syndrome (HUS) was not occurred, but systemic edema, ascites, pleural effusion and pulmonary edema with hypoxyemia gradually appeared . Treatment with human immunoglobulin in addition to fluid therapy, antibiotics, diuretics and human serum albumin resulted in dramatic improvement . Management of complications such as sepsis and electrolyte disturbance caused by this infection was considered to be important in elderly patients.

J Comp Physiol {B}, 1997 Apr, 167(3), 193 - 6
Endotoxin-challenges precocial neonates and iron changes; Tegowska E et al.; In adult mammals fever is associated with the reduction of blood plasma iron level . Immature mammals, however, show either a decrease (precocial animals such as guinea pig neonates) or a lack of reduction (altricial animals such as human neonates) of plasma iron in response to endotoxin . In order to determine whether this difference is connected with maturity just after delivery, plasma iron concentration, hematocrit, body temperature and body mass were measured in rat pups injected with E . coli endotoxin in doses of 50 or 200 micrograms kg-1 . Rat pups, like human neonates, are altricial animals . In 7-day-old rats injection of LPS led to a dose-dependent decrease in plasma iron level . The fall in plasma iron was accompanied by changes in body temperature and body mass . The results showed that plasma iron response to endotoxin in altricial rat neonates is similar to that observed in precocial guinea pig pups.

Glycobiology, 1997 Apr, 7(3), 367 - 72
Cloning and expression of a Xenopus laevis oocyte lectin and characterization of its mRNA levels during early development; Lee JK et al.; cDNA clones encoding a soluble, calcium-dependent, melibiose-binding lectin from Xenopus laevis oocytes have been isolated, characterized, and expressed in bacteria . This lectin has been shown by others to be localized in oocyte cortical granules where it ultimately is released and participates in the formation of the fertilization envelope . A lectin with similar specificity has been purified by others from blastula and immunolocalized to specific locations in developing embryos, which suggests it may also function after fertilization in regulating cell adhesion and migration . We have used melibiose affinity chromatography to isolate the oocyte lectin (monomer molecular masses of about 45 and 43 kDa) and shown that after exhaustive treatment with N-glycanase, only one major protein band at 35 kDa was observed, suggesting that a single polypeptide with variable N-linked glycosylation is expressed in the oocyte . After obtaining internal peptide sequences, a PCR-based cloning approach allowed the isolation of full length cDNAs from an ovary lambda gt11 library encoding a protein of 313 amino acids with three potential N-linked oligosaccharide sites . Although this lectin, termed XL35, requires calcium ions for oligosaccharide binding, its sequence does not contain the sequence motif defined for "C-type" lectins . A 6-Histagged from of the lectin was expressed in E . coli and purified on a Ni(2+)-NTA column from bacterial extracts . The recombinant lectin was active using an agglutination assay, and this activity was inhibitable by EDTA and melibiose, properties exhibited by the native lectin . Southern blot analysis revealed a single hybridizing band, arguing against the existence of a multigene family . Northern blot analysis demonstrated that the lectin mRNA is expressed in oocytes and remains at relatively high levels through late gastrulae, continuing until tadpole stages . The persistence of the lectin mRNA, coupled with results from earlier studies, strongly suggests that XL35 is zygotically expressed and functions during morphogenesis.

Chromosome Res, 1997 Apr, 5(2), 132 - 41
Characterization of internal DNA-binding and C-terminal dimerization domains of human centromere/kinetochore autoantigen CENP-C in vitro: role of DNA-binding and self-associating activities in kinetochore organization; Sugimoto K et al.; Human centromere protein C (CENP-C), a chromosomal component of the inner plate of kinetochores, was originally identified as one of the centromere autoantigens . In a previous study, we showed that it possesses DNA-binding activity in vitro . Recently, centromere-binding activity was suggested at the C-terminal region in vivo . However, little is known about the role of CENP-C in kinetochore organization . Here, to characterize its biochemical properties, three separate antigenic regions of human CENP-C were expressed in Escherichia coli, affinity purified and used in South-western blotting and chemical cross-linking analyses . We found that the internal DNA-binding domain was composed of two kinds of elements: the 'core' and two flanking 'stabilizing' elements that support the activity . When cross-linked with disuccinimidyl suberate (DSS), the N-terminal region produced the ladder bands of dimer and tetramer: the C-terminal region exclusively produced the dimer band, whereas the internal region was not affected at all . Dimer formation at the C-terminus in the native state was also indicated by gel filtration and the presence of conformation-specific autoantibodies in the patient's sera . These results suggest that human CENP-C consists of three functional units required for 'kinetochore assembly': a putative N-terminal oligomerization domain, an internal DNA-binding domain and a C-terminal dimerization domain.

Biol Pharm Bull, 1997 Apr, 20(4), 332 - 7
Participation of leukotriene D4 and tumor necrosis factor on lipopolysaccharide-induced airway hyperresponsiveness in guinea pigs; Uno T et al.; In guinea pigs, a marked increase in airway responsiveness to acetylcholine (Ach) was observed at 2 h after lipopolysaccharide (LPS) inhalation . To examine the mediators responsible for the airway hyperresponsiveness, the changes of peptide-leukotrienes (LTs), tumor necrosis factor (TNF), interleukin-1 (IL-1), histamine and 5-hydroxytryptamine (5-HT) levels in bronchoalveolar lavage fluid (BALF) were measured . Airway responsiveness to Ach reached a peak 2 h after LPS inhalation . The influx of neutrophil into BALF increased gradually and reached a peak 24 h after LPS inhalation . After the inhalation of LPS, LTD4 and TNF contents in BALF increased within the first 2 h after LPS inhalation . However, other mediators were not detected or increased 6 h after LPS inhalation . Aeroinhalation of LTD4 and murine recombinant TNF-alpha caused airway hyperresponsiveness in guinea pigs . In addition, a LTD4 antagonist, BAYx7195, and an inhibitor of TNF, pentoxifylline, inhibited the LPS-induced airway hyperresponsiveness . These results suggest that LTs and/or TNF play an important role in the onset of airway hyperresponsiveness in guinea pigs.

Biol Pharm Bull, 1997 Apr, 20(4), 327 - 31
Decrease in expression of the master operon of flagellin synthesis in a dnaA46 mutant of Escherichia coli; Mizushima T et al.; We examined the expression of the fliC, fliA and fihD genes in the dnaA46 mutant . In Northern blot analysis, the amounts of fliC, fliA, and flhD mRNA decreased in the dnaA46 mutant growing at 37 degrees C but not at 28 degrees C . The promoter activity of each gene also decreased in this mutant at 37 degrees C . The decrease in expression of the flh D gene was not related to the cAMP-catabolite activator protein (CAP) pathway . We tentatively conclude that DnaA protein is involved in the expression of the flhD gene by a mechanism independent of the cAMP-CAP pathway.

Biol Pharm Bull, 1997 Apr, 20(4), 322 - 6
Delayed release of prostaglandins from arachidonic acid and kinetic changes in prostaglandin H synthase activity on the induction of prostaglandin H synthase-2 after lipopolysaccharide-treatment of RAW264.7 macrophage-like cells; Tanaka Y et al.; In a mouse macrophage-like cell line, RAW264.7, arachidonic acid was cleaved within 30 min of lipopolysaccharide (LPS)-treatment . However, prostaglandin (PG) synthesis did not accompany this cleavage, being delayed by 4 h, although significant PGH synthase (PGHS) activity was detected in the lysate of LPS-nontreated cells . The K(m) value of this basal PGHS activity toward arachidonic acid was more than one hundred-fold higher than that for the lysate of cells treated with LPS for 4 h . Changes in the sensitivity of the PGHS activity to nonsteroidal antinflammatory drugs after LPS-treatment also suggested induction of PGHS with different properties from that in the case of basal PGHS . The concomitant increase in PGH synthase (PGHS) activity with the induction of PGHS-2 protein after LPS-treatment suggested a contribution by PGHS-2 to the delayed synthesis of PGs in LPS-treated macrophage cells . As for PGHS in the control cells without LPS-treatment, a different K(m) value from that of PGHS-1 and the lack of cross-reactivity to anti-PGHS-1 antibodies suggested that this basal PGHS was different from the typical PGHS-1 . The lower affinity of this enzyme for arachidonic acid might be the reason for the failure to release PGs by the cells without LPS-treatment.

Crit Care Med, 1997 Apr, 25(4), 684 - 8
Nitric oxide does not mediate lipopolysaccharide-induced myocardial depression in guinea pigs; Toth I et al.; OBJECTIVE: To determine the role of nitric oxide as a mediator of lipopolysaccharide-induced myocardial depression . DESIGN: Prospective, controlled study . SETTING: University research laboratory . SUBJECTS: Male and female Hartley guinea pigs . INTERVENTIONS: Animals (n = 97) received intraperitoneal injections of either saline or Escherichia coli lipopolysaccharide (2 mg/kg) . Some (n = 5) animals received two injections of dexamethasone before lipopolysaccharide . Left atria were harvested 6 hrs (n = 20) or 16 hrs (n = 77) later and placed in a tissue bath with Krebs-Henseleit buffer . Contractile tension was measured in the presence or absence of two inhibitors of nitric oxide synthase (NG-nitroarginine {NNA} or aminoguanidine) . Atrial and serum nitrite/ nitrate and atrial cyclic guanosine monophosphate (cGMP) concentrations were assayed . MEASUREMENTS AND MAIN RESULTS: Lipopolysaccharide caused significant atrial contractile depression at 16 hrs but not 6 hrs compared with control animals . Neither NNA nor aminoguanidine reversed the depression in atrial function . In contrast, exposure of control atria to NNA worsened contractile function . There were no significant differences between control and lipopolysaccharide-treated animals in atrial and serum nitrite/nitrate and atrial cGMP concentrations . CONCLUSIONS: Nitric oxide does not mediate lipopolysaccharide-induced myocardial depression in this animal model . Basal concentrations of nitric oxide may be important since NNA worsened contractile function in control atria.

Microbiology, 1997 Apr, 143 ( Pt 4), 1369 - 79
Molecular characterization of the bet genes encoding glycine betaine synthesis in Sinorhizobium meliloti 102F34; Pocard JA et al.; As a first step towards the elucidation of the molecular mechanisms responsible for the utilization of choline and glycine betaine (betaine) either as carbon and nitrogen sources or as osmoprotectants in Sinorhizobium meliloti, we selected a Tn5 mutant, LTS23-1020, which failed to grow on choline but grew on betaine . The mutant was deficient in choline dehydrogenase (CDH) activity, failed to oxidize {methyl-14C}choline to {methyl-14C}betaine, and did not use choline, but still used betaine, as an osmoprotectant . The Tn5 mutation in LTS23-1020 was complemented by plasmid pCHO34, isolated from a genomic bank of S . meliloti 102F34 . Subcloning and DNA sequencing showed that pCHO34 harbours two ORFs which showed 60% and 57% identity with the Escherichia coli betB gene encoding betaine-aldehyde dehydrogenase (BADH) and betA gene encoding CDH, respectively . In addition to the homology with E . coli genes, the deduced sequence of the sinorhizobial BADH protein displays consensus sequences also found in plant BADHs . The deduced sequence of the sinorhizobial CDH protein shares only 21% identical residues with choline oxidase from Arthrobacter globiformis . The structural organization of the betBA genes in S . meliloti differs from that described in E . coli: (i) the two ORFs are separated by a 210 bp sequence containing inverted repeats resembling a putative rho-independent transcription terminator, and (ii) no sequence homologous to betT (high-affinity choline transport system) or betI (regulator) was found in the vicinity of the sinorhizobial betBA genes . Evidence is also presented that the S . meliloti betBA genes are not located on the megaplasmids.

Microbiology, 1997 Apr, 143 ( Pt 4), 1221 - 33
A carboxy-terminal processing protease gene is located immediately upstream of the invasion-associated locus from Bartonella bacilliformis; Mitchell SJ et al.; A gene with homology to those encoding an unusual class of C-terminal processing proteases that flanks the invasion-associated locus ialAB of Bartonella bacilliformis has been identified . The 1302 bp gene, termed ctpA, is located immediately upstream of the ialA gene and encodes a predicted nascent product of 434 amino acids, producing a mature protein of 411 amino acid residues . The Bartonella CtpA appears to undergo autolysis in vitro, producing multiple products of 43-46 kDa, and a second group of products of 36-37 kDa . Production of CtpA in vivo gives a single product of 41.8 kDa . In addition to a computer-predicted N-terminal secretory signal sequence, the molecular mass difference in vivo versus in vitro indicates that CtpA is likely to be secreted and post-translationally modified . The full-length CtpA protein shows 30% identify to the CtpA protein of Synechocystis sp . 6803 (69% overall sequence similarity) . The mature CtpA protein also has significant homology to the tail-specific protease (Tsp) of Escherichia coli, with 22% identify and 62% similarity to an internal region of the 660 amino acid Tsp . The CtpA protein does not appear to exhibit haemolysin, collagenase, or caseinase activity . The ctpA gene is conserved in all Bartonella species examined, as determined by hybridization analyses, but it was not found in Brucella abortus or E . coli . The ctpA gene does not directly affect the erythrocyte-invasion phenotype conferred by ialAB, but its homology to other stress-response processing proteases implies an important role in survival of this intracellular pathogen.

Microbiology, 1997 Apr, 143 ( Pt 4), 1191 - 202
Copper-inducible transcriptional regulation at two promoters in the Escherichia coli copper resistance determinant pco; Rouch DA et al.; The pco determinant of Escherichia coli plasmid pRI1004 encodes inducible resistance to the trace element copper . The identification of two copper-dependent transcriptional initiation regions within pco that each contain a similar upstream hyphenated dyad motif is described . Deletion constructs showed that this 'copper box' motif was essential for copper-inducible activity at both pco promoters, PpcoA and PpcoE . The placement of the motif differs in the two promoters, and PpcoA contains an extended -10 nonamer typical of promoters for which RNA polymerase does not bind specifically to -35 sequences . PpcoE does not contain this motif and is the more strongly expressed promoter . The transcript from PpcoA contains the pcoABCDRS genes, while PpcoE expresses only pcoE . The induction profiles for PpcoA- and PpcoE-IacZ fusions were flattened sigmoidal curves with a gradual response to increasing copper concentration . On high-copy-number plasmids, zinc was found also to induce transcription from both promoters in vivo . Both promoters showed inducible activity in the absence of pcoRS, the plasmid-borne two-component regulatory system, indicating that a second trans-acting regulatory system is present on the chromosome . The pcoR product showed repressor action in the absence of pcoS, while still allowing induction, suggesting the chromosome encoded a similar two-component system to pco . TnphoA insertion mutagenesis identified chromosomal genes which affected promoter expression, including ptsH, ptsI (sugar phosphotransferase system) and cya (adenylate cyclase) . The results support that idea that pco-encoded copper resistance is an auxiliary mechanism for handling copper, the regulation of which is integrated with the chromosomal regulation of cellular copper metabolism.

Microbiology, 1997 Apr, 143 ( Pt 4), 1181 - 9
Tellurite reductase activity of nitrate reductase is responsible for the basal resistance of Escherichia coli to tellurite; Avazeri C et al.; Tellurite and selenate reductase activities were identified in extracts of Escherichia coli . These activities were detected on non-denaturing polyacrylamide gels using an in situ methyl viologen activity-staining technique . The activity bands produced from membrane-protein extracts had the same RF values as those of nitrate reductases (NRs) A and Z . Tellurite and selenate reductase activities were absent from membranes obtained from mutants deleted in NRs A and Z . Further evidence of the tellurite and selenate reductase activities of NR was demonstrated using rocket immunoelectrophoresis analysis, where the tellurite and selenate reductase activities corresponded to the precipitation arc of NR . Additionally, hypersensitivity to potassium tellurite was observed under aerobic growth conditions in nar mutants . The tac promoter expression of NR A resulted in elevated tellurite resistance . The data obtained also imply that a minimal threshold level of NR A is required to increase resistance . Under anaerobic growth conditions additional tellurite reductase activity was identified in the soluble fraction on non-denaturing gels . Nitrate reductase mutants were not hypersensitive under anaerobic conditions, possibly due to the presence of this additional reductase activity.

Microbiology, 1997 Apr, 143 ( Pt 4), 1163 - 74
Isolation and characterization of the gene encoding single-stranded-DNA-binding protein (SSB) from four marine Shewanella strains that differ in their temperature and pressure optima for growth; Chilukuri LN et al.; The ssb gene, coding for single-stranded-DNA-binding protein (SSB), was cloned from four marine Shewanella strains that differed in their temperature and pressure optima and ranges of growth . All four Shewanella ssb genes complemented Escherichia coli ssb point and deletion mutants, with efficiencies that varied with temperature and ssb gene source . The Shewanella SSBs are the largest bacterial SSBs identified to date (24.9-26.3 kDa) and may be divided into conserved amino- and carboy-terminal regions and a highly variable central region . Greater amino acid sequence homology was observed between the Shewanella SSBs as a group (72-87%) than with other bacterial SSBs (52-69%) . Analysis of the amino acid composition of the Shewanella SSBs revealed several features that could correlate with pressure or temperature adaptation . SSBs from the three low-temperature-adapted Shewanella strains were an order of magnitude more hydrophilic than that from the mesophilic strain, and differences in the distribution of eight amino acids were identified which could contribute to either the temperature or pressure adaptation of the proteins . The SSBs from all four Shewanella strains were overproduced and partially purified based upon their ability to bind single-stranded DNA . The differences found among the Shewanella SSBs suggest that these proteins will provide a useful system for exploring the adaptation of protein-protein and protein-DNA interactions at low temperature and high pressure.

Mol Microbiol, 1997 Apr, 24(1), 181 - 9
HIP1 propagates in cyanobacterial DNA via nucleotide substitutions but promotes excision at similar frequencies in Escherichia coli and Synechococcus PCC 7942; Robinson PJ et al.; The sequence 5'-GCGATCGC-3', designated HIP1, for highly iterated palindrome, was first identified at the borders of a gene-deletion event and subsequently shown to constitute up to 2.5% of the DNA in some cyanobacteria . It is now reported that HIP1 is polyphyletic, occurring in several distinct cyanobacterial lineages and not defining a clade . HIP1 does not introduce gaps into sequence alignments . It aligns with partial HIP1 sites in related sequences showing that it propagates by nucleotide substitutions rather than insertion . Constructs have been created to determine the frequencies at which deletion events occur between palindromes located within the selectable marker neo . Deletion between HIP1 sites was more frequent in Synechococcus PCC 7942 than deletion between control palindromes, 5'-CCGATCGG-3', designated PAL0 . However, this is not due to a recombinase that recognises HIP1 and is peculiar to cyanobacteria because similar deletion frequencies were detected in Escherichia coli . Furthermore, the frequency of deletion of DNA flanked asymmetrically by one HIP1 site and one PAL0 site was less than the frequency of deletion of DNA flanked asymmetrically by identical copies of either palindrome . This is consistent with deletion by copy-choice.

Mol Microbiol, 1997 Apr, 24(1), 129 - 39
Analysis of ssb mutations in vivo implicates SSB protein in two distinct pathways of SOS induction and in recombinational DNA repair; Carlini LE et al.; Site-directed mutations in the Escherichia coli ssb gene were tested for the ability to complement a chromosomal ssb deletion for viability, and only the ssb W54-->G mutation failed to do so at the pSC101 copy level . Non-aromatic amino acid substitutions for SSB Trp-54 (ssb W54-->L and ssb W54-->S) produced the greatest effects on in vivo protein function including altered marker linkage subsequent to generalized transduction, extreme UV sensitivity, and a lack of ability to support SOS induction . Additionally, the ssb-113 (ssb P176-->S) mutation demonstrated the existence of both uvrA-dependent and uvrA-independent components of SOS induction . Although nucleotide excision repair appeared unaffected by alterations in the SSB protein, the mutational analysis suggests a direct role for SSB in recombinational repair.

Mol Microbiol, 1997 Apr, 24(1), 93 - 104
Three disparately regulated genes for sigma 32-like transcription factors in Bradyrhizobium japonicum; Narberhaus F et al.; Bradyrhizobium japonicum possesses a subclass of heat-shock genes whose members are transcribed from a sigma 32 consensus promoter . Having identified previously one gene (rpoH1) encoding a sigma 32-like RNA polymerase transcription factor, we report here the characterization of two additional rpoH-like genes (rpoH2 and rpoH3) . B . japonicum thus represents the first example of an organism possessing an rpoH multigene family . All three rpoH genes encode functional proteins that are able to initiate transcription from the Escherichia coli groE promoter . Each rpoH gene is apparently regulated by a different mechanism . Although both rpoH1 and rpoH2 are transcribed from sigma 70-type promoters, transcription of the rpoH1 operon was found to be heat inducible by an unknown mechanism, whereas the level of rpoH2 mRNA decreased after heat shock . At extreme temperatures (48 degrees C), rpoH2 was transcribed from a second promoter that resembled the E . coli sigma E-type promoter . The rpoH3 gene was found to be associated with two upstream genes, ragA and ragB, coding for a classical two-component regulatory system . Transcription initiated from a promoter that mapped in front of the putative response regulator gene ragA, suggesting that ragA, ragB and rpoH3 are organized in an operon . The ragA promoter was similar to a sigma 32 consensus promoter . The three B . japonicum rpoH genes also varied in their significance to support growth of the organism . While the rpoH2 gene could not be eliminated by mutation, knock-out mutants of rpoH1 and/ or rpoH3 were readily obtained and shown to be indistinguishable from the wild type under aerobic growth conditions or during root-nodule symbiosis . We conclude that rpoH2 is essential for the synthesis of cellular proteins under physiological growth conditions, whereas rpoH1, and probably also rpoH3, are involved in their synthesis during the stress response.

Mol Microbiol, 1997 Apr, 24(1), 41 - 51
Characterization of the chemotaxis protein CheW from Rhodobacter sphaeroides and its effect on the behaviour of Escherichia coli; Hamblin PA et al.; In contrast to the situation in enteric bacteria, chemotaxis in Rhodobacter sphaeroides requires transport and partial metabolism of chemoattractants . A chemotaxis operon has been identified containing homologues of the enteric cheA, cheW, cheR genes and two homologues of the cheY gene . However, mutations in these genes have only minor effects on chemotaxis . In enteric species, CheW transmits sensory information from the chemoreceptors to the histidine protein kinase, CheA . Expression of R . sphaeroides cheW in Escherichia coli showed concentration-dependent inhibition of wild-type behaviour, increasing counter-clockwise rotation and thus smooth swimming--a phenotype also seen when E . coli cheW is overexpressed in E . coli . In contrast, overexpression of R . sphaeroides cheW in wild-type R . sphaeroides inhibited motility completely, the equivalent of inducing tumbly motility in E . coli . Expression of R . sphaeroides cheW in an E . coli delta cheW chemotaxis mutant complemented this mutation, confirming that CheW is involved in chemosensory signal transduction . However, unlike E . coli delta cheW mutants, in-frame deletion of R . sphaeroides cheW did not affect either swimming behaviour or chemotaxis to weak organic acids, although the responses to sugars were enhanced . Therefore, although CheW may act as a signal-transduction protein in R . sphaeroides, it may have an unusual role in controlling the rotation of the flagellar motor . Furthermore, the ability of a delta cheW mutant to swim normally and show wild-type responses to weak acids supports the existence of additional chemosensory signal-transduction pathways.

Microb Pathog, 1997 Apr, 22(4), 241 - 6
Cloning and nucleotide sequence analysis of a Brucella abortus gene encoding an 18 kDa immunoreactive protein; Kovach ME et al.; A DNA fragment encoding an approximately 18 kDa protein from Brucella abortus strain 2308 was cloned and expressed in Escherichia coli . This recombinant protein, designated BA18K, reacted in Western blot analysis with sera obtained from experimentally and naturally infected animals including mice, goats, dogs and humans . Restriction enzyme analysis of the plasmid (pBA28) encoding BA18K revealed the presence of an approximately 8.7 kbp Sau3A genomic DNA fragment within the vector and subsequent subcloning and Western blot analysis limited the region encoding BA18K to an approximately 3.0 kbp Pst 1 DNA fragment . DNA sequence analysis of this region identified an open reading frame capable of encoding a protein of 177 amino acids with a predicted relative molecular mass of 17529 . Comparison of the deduced amino acid sequence of BA18K with those in the protein sequence databases yielded no homology with previously described proteins from other bacterial genera . These searches did, however, indicate that BA18K is identical to the previously described outer membrane protein (OMP) from B . abortus strain 544 designated Omp 19.

Am J Physiol, 1997 Apr, 272(4 Pt 2), R1204 - 9
Leptin increases energy expenditure and selectively promotes fat metabolism in ob/ob mice; Hwa JJ et al.; Obesity occurs whenever energy intake exceeds energy expenditure . The ob gene product leptin is a potent anorectic agent when administered to ob/ob mice, but its effects on energy expenditure have not been investigated in detail . The present study was designed to analyze the acute metabolic effects of leptin in vivo . Analysis of oxygen consumption in ob/ob mice demonstrated a reduction in energy expenditure compared with lean controls; this reduction showed a diurnal fluctuation and was most evident during the light cycle . A single intraperitoneal dose of leptin increased oxygen consumption during the light cycle in ob/ob mice, ablating the circadian fluctuation in this parameter . In addition, leptin had a profound effect on fuel selection: the respiratory quotient was markedly reduced, indicating a reduction in carbohydrate oxidation and an increase in fat oxidation . These acute effects of leptin on metabolic parameters are consistent with the selective loss of body fat observed on chronic leptin treatment and suggest that increased energy utilization plays an important role in the anti-obese actions of leptin.

Biochem Mol Biol Int, 1997 Apr, 41(5), 1035 - 44
The large ribosomal protein gene cluster of a cryptomonad plastid: gene organization, sequence and evolutionary implications; Wang SL et al.; The complete sequence of the major ribosomal protein gene cluster of the plastid genome of the cryptomonad alga Guillardia theta (formerly Cryptomonas phi) is presented . The ribosomal protein genes (corresponding to the S10, spc, alpha and L13/S9 operons of E . coli) are found upstream of the previously reported plastid str operon, and transcribed in the same orientation . The genes are very tightly packed with as little as two nucleotides between the rpl14 and rpl24 genes . The gene arrangement is very similar to that reported for the rhodophyte alga, Porphyra purpurea, and the chromophyte diatom, Odontella sinensis, indicating a close evolutionary relationship between these groups of algae . Northern analysis indicates that the 29 genes are arranged as one operon and are transcribed as a single mRNA that is subsequently processed into smaller transcripts.

Biochem Mol Biol Int, 1997 Apr, 41(5), 995 - 1003
Isolation of a gene encoding nodulin-like intrinsic protein of Escherichia coli; Fushimi K et al.; Members of the membrane intrinsic protein (MIP) family are expressed in various organisms including plants, insects, and vertebrates . E . coli is known to have a MIP member gene, glycerol facilitator (G1pF) . Here we report the isolation of E . coli gene encoding BniP, bacterial nodulin-like intrinsic protein . BniP encodes a 231 amino acid, 24 kDa protein with 42% amino acid identity to Nod26, 38% amino acid identity to AQP1, and 29% amino acid identity to G1pF . Analysis of deduced amino acid sequence predicted a hydrophobic protein with six membrane-spanning domains . Expression of BniP in Xenopus oocytes induced slight increase in osmotic water permeability, but not glycerol or ion permeability . Our results showed that BniP is a new member of the MIP channel-forming proteins of E . coli.

Biochem Mol Biol Int, 1997 Apr, 41(5), 887 - 94
Mutation of Arg154 to Gly154 in urokinase augments its fibrin-specificity; Peng G et al.; Rscu-PA and its mutant constructed by in vitro site specific mutagenesis of Arg154 in rscu-PA to Gly154 (mscu-PA) were both expressed in Escherichia coli . After in vitro denaturation and renaturation, the rscu-PA and mscu-PA were purified to homogeneity by Zn2+ selective precipitation, anti-u-PA IgG-sepharose CL 4B affinity chromatography . After activation by plasmin, the kinetic constants for the resultant mtcu-PA against synthetic substrate S2444 hydrolysis were found to be essentially identical to rtcu-PA, suggesting that no impairment had been exerted on the catalytic active site of mtcu-PA . However, both 125I-fibrin plasma-clot lysis and fibrinogenolysis showed that mtcu-PA possessed a higher fibrinolytic activity but hardly any degradation of fibrinogen in plasma compared to rtcu-PA and rscu-PA . It was concluded that the substitution of Arg154 by Gly154 in tcu-PA promoted the fibrin-specificity of urokinase.

Cell Mol Life Sci, 1997 Apr, 53(4), 294 - 302
Dietary vitamin E does not protect from endotoxin-induced hepatic microvascular dysfunction; Rucker M et al.; Starting from the concept that lipopolysaccharide (LPS)-associated hepatotoxicity involves the action of reactive oxygen species, the present study was conducted to test whether vitamin E, a lipophilic antioxidant, prevents LPS-induced hepatic microvascular dysfunction and liver injury . Fifty-two rats were divided into three groups and fed diets containing 0 (n = 16), 75 (n = 18) or 8000 mg (n = 18) alpha-tocopherol acetate/kg food for four weeks . At 1 h and 6 h after intravenous LPS-exposure (10 mg/kg E . coli LPS) hepatic microvascular response and liver injury were assessed by the analysis of Kupffer cell phagocytic activity, leukocyte-endothelial cell interaction and nutritive sinusoidal perfusion (intravital fluorescence epi- illumination technique) as well as bile flow, serum liver enzyme activities and tissue histomorphology . In animals fed with 75 mg vitamin E/kg (standard diet), LPS caused hepatic Kupffer cell activation (increased phagocytic activity) and hepatic microvascular leukocyte activation, with stasis in sinusoids and adherence in postsinusoidal venules (1 h) followed by leukocytic infiltration into tissue (6 h) and progredient sinusoidal perfusion failure (6 h) . Hepatic microvascular injury was accompanied by reduced bile flow and enhanced liver enzyme release . Vitamin E-enriched diet (8000 mg/kg) and even vitamin E-deficient diet did not significantly affect LPS-induced hepatic microvascular cell activation and perfusion failure . Thus, we conclude, that vitamin E is not effective to protect from endotoxin-induced hepatic microvascular dysfunction.

Lett Appl Microbiol, 1997 Apr, 24(4), 291 - 5
Incidence of toxigenic Escherichia coli in soft cheese made with raw or pasteurized milk; Quinto EJ et al.; Soft cheeses made with raw (221 samples) or pasteurized (75) cow's milk were collected . Enterotoxigenic, verotoxigenic and necrotoxigenic Escherichia coli strains were studied . Three raw milk cheeses were positive for toxigenic E . coli (1.4%): the first with toxin CNF2 and serogroup O5 (40% of colonies studied with typical E . coli morphology); the second with VT and O2 (10%); and the third with LT and O51 (10%) . Toxigenic E . coli of bovine origin can pass to the milk destined to make cheese, and survive . Soft cheese should be considered as a possible vehicle of infection in future investigations.

Lett Appl Microbiol, 1997 Apr, 24(4), 286 - 90
An evaluation of the use of 4-methylumbelliferyl-beta-D-glucuronide (MUG) in different solid media for the detection and enumeration of Escherichia coli in foods; Villari P et al.; The use of 4-methylumbelliferyl-beta-D-glucuronide (MUG) in different solid media for the detection and enumeration of Escherichia coli in foods was evaluated by testing the effects of different substrate concentrations (50 or 100 micrograms ml-1), incubation temperatures (37 or 41.5 degrees C) and incubation times (8, 12, 24 and 48 h) . Different kinds of foods, both naturally and artificially contaminated, were analysed . The use of selective media without differential substances and an incubation time of 24 h seem to be worthy of recommendation . In this case an incubation temperature of 37 degrees C would be preferred and the MUG concentration could be reduced to 50 micrograms ml-1 . Incubation times shorter than 24 h, which may cause a loss of sensitivity, require higher incubation temperatures (41.5 degrees C) and MUG concentration (100 micrograms ml-1).

Thromb Haemost, 1997 Apr, 77(4), 755 - 9
Construction and expression of mouse-human chimeric antibody SZ-51 specific for activated platelet P-selectin; Gu J et al.; A murine monoclonal (mAb) SZ-51 specific for human P-selectin may be used for in vivo thrombus imaging and for the targeting of fibrinolytic agents to thrombi . In order to reduce the immunogenicity of the murine mAb SZ-51 in humans, we cloned and sequenced the cDNAs encoding the variable region of mAb SZ-51 in order to develop mouse/human chimeric reagents . The E . coli expression vector pHEN1-SZ51Fab/Hu was constructed by fusing the variable regions of mAb SZ-51 with human IgG gamma 1CH1 and C kappa genes . The constructs were introduced into E . coli HB2151 for expression of soluble chimeric Fab fragment . We also constructed two fusion products by joining the variable regions of mouse antibody to the appropriate constant regions of human Ig gamma 1 and kappa . These chimeras were cloned into two eukaryotic selectable expression vectors separately, which were then contransfected into a non-Ig secreting murine myeloma line SP2/0 with lipofectin reagent . Six cell lines remained positive for Ig secretion . The highest producing cell line, which showed stable integration and expression at 5 mg/l of culture, was selected for the large scale production of chimeric antibody . Immunoblotting analysis demonstrated that both of the chimeric antibodies (SZ51Fab/Hu, SZ51/Hu) in the culture supernatants, like the native mAb SZ-51, bind P-selectin . In addition, the whole chimeric antibody can compete for binding to activated platelets with murine SZ-51 . Therefore, the SZ-51 chimeric antibody may be a potential agent for diagnosis and treatment of thrombotic diseases in the future.

Thromb Haemost, 1997 Apr, 77(4), 710 - 7
The role of the low-density lipoprotein receptor-related protein (LRP) in the plasma clearance and liver uptake of recombinant single-chain urokinase-type plasminogen activator in rats; van der Kaaden ME et al.; Urokinase-type plasminogen activator (u-PA) is used as a thrombolytic agent in the treatment of acute myocardial infarction . In vitro, recombinant single-chain u-PA (rscu-PA) expressed in E.coli is recognized by the Low-Density Lipoprotein Receptor-related Protein (LRP) on rat parenchymal liver cells . In this study we investigated the role of LRP in the liver uptake and plasma clearance of rscu-PA in rats . A preinjection of the LRP inhibitor GST-RAP reduced the maximal liver uptake of 125I-rscu-PA at 5 min after injection from 50 to 30% of the injected dose and decreased the clearance of rscu-PA from 2.37 ml/min to 1.58 ml/min . Parenchymal, Kupffer and endothelial cells were responsible for 40, 50 and 10% of the liver uptake, respectively . The reduction in liver uptake of rscu-PA by the preinjection of GST-RAP was caused by a 91% and 62% reduction in the uptake by parenchymal and Kupffer cells, respectively . In order to investigate the part of rscu-PA that accounted for the interaction with LRP, experiments were performed with a mutant of rscu-PA lacking residues 11-135 (= delta 125-rscu-PA) . Deletion of residues 11-135 resulted in a 80% reduction in liver uptake and a 2.4 times slower clearance (0.97 ml/min) . The parenchymal, Kupffer and endothelial cells were responsible for respectively 60, 33 and 7% of the liver uptake of 125I-delta 125-rscu-PA . Preinjection of GST-RAP completely reduced the liver uptake of delta 125-rscu-PA and reduced its clearance to 0.79 ml/min . Treatment of isolated Kupffer cells with PI-PLC reduced the binding of rscu-PA by 40%, suggesting the involvement of the urokinase-type Plasminogen Activator Receptor (u-PAR) in the recognition of rscu-PA . Our results demonstrate that in vivo LRP is responsible for more than 90% of the parenchymal liver cell mediated uptake of rscu-PA and for 60% of the Kupffer cell interaction . It is also suggested that u-PAR is involved in the Kupffer cell recognition of rscu-PA.

Auris Nasus Larynx, 1997 Apr, 24(2), 185 - 91
Demonstration of antibodies against human papillomavirus type-11 E6 and L2 proteins in patients with recurrent respiratory papillomatosis; Sameshima A et al.; Recurrent respiratory papillomatosis (RRP) is highly prevalent in Thailand, with the human papillomavirus (HPV) type-11 being the most widespread . In this study, we isolated the HPV type-11 (HPV-11) genome from subjects with RRP and subcloned the E6 and L2 open reading frames (ORFs) with the expression vectors pEX1 and pEX3 . The recombinant E6/beta-gal and L2/beta-gal fusion proteins were expressed in E . coli . Using the recombinant proteins, we demonstrated the presence of antibodies against HPV-11 E6 and L2 in RRP patients by Western blot analysis . The prevalence of seropositivity for HPV-11 E6 and L2 were 5% (1/20) and 10% (2/20), respectively . Although RRP is caused by infection on the mucosal surface, it appears that an immune response occurs against viral proteins expressed in the epithelial lesions.

EMBO J, 1997 Apr 1, 16(7), 1742 - 50
The distal GATA sequences of the sid1 promoter of Ustilago maydis mediate iron repression of siderophore production and interact directly with Urbs1, a GATA family transcription factor; An Z et al.; The sid1 and urbs1 genes encode L-ornithine N5-oxygenase and a GATA family transcription regulator, respectively, for siderophore biosynthesis in Ustilago maydis . The basic promoter and iron-regulatory sequences of the U . maydis sid1 gene were defined by fusing restriction and Bal31 nuclease-generated deletion fragments of the promoter region with the Escherichia coli beta-glucuronidase (GUS) reporter gene . Sequences required for basal expression of sid1 mapped within 1043 bp upstream of the translation start site and include the first untranslated exon and first intron . Sequences needed for iron-regulated expression of sid1 were localized to a 306 bp region mapping 2.3 and 2.6 kb upstream of the ATG . The 306 bp region contains two G/TGATAA sequences, consensus DNA binding sites of GATA family transcription factors . Deletion or site-directed mutation of either or both GATA sequences resulted in deregulated expression of sid1 . In vitro DNA binding studies showed that Urbs1 binds to the 3'-GATA site in the 306 bp iron-responsive region . However, deletion of 1.1 kb between the distal GATA sites and the basal promoter region led to deregulated expression of GUS, indicating that these GATA sequences are by themselves insufficient to regulate sid1 . In vitro DNA binding and in vivo reporter gene analysis revealed that siderophores are not co-repressors of Urbs1.

EMBO J, 1997 Apr 1, 16(7), 1519 - 30
A novel ubiquitin-specific protease is dynamically associated with the PML nuclear domain and binds to a herpesvirus regulatory protein; Everett RD et al.; Herpes simplex virus type 1 immediate-early protein Vmw110 is a non-specific activator of gene expression and is required for efficient initiation of the viral lytic cycle . Since Vmw110-deficient viruses reactivate inefficiently in mouse latency models it has been suggested that Vmw110 plays a role in the balance between the latent and lytic states of the virus . The mechanisms by which Vmw110 achieves these functions are poorly understood . Vmw110 migrates to discrete nuclear structures (ND10) which contain the cellular PML protein, and in consequence PML and other constituent proteins are dispersed . In addition, Vmw110 binds to a cellular protein of approximately 135 kDa, and its interactions with the 135 kDa protein and ND10 contribute to its ability to stimulate gene expression and viral lytic growth . In this report we identify the 135 kDa protein as a novel member of the ubiquitin-specific protease family . The protease is distributed in the nucleus in a micropunctate pattern with a limited number of larger discrete foci, some of which co-localize with PML in ND10 . At early times of virus infection, the presence of Vmw110 increases the proportion of ND10 which contain the ubiquitin-specific protease . These results identify a novel, transitory component of ND10 and implicate a previously uncharacterized ubiquitin-dependent pathway in the control of viral gene expression.

EMBO J, 1997 Apr 1, 16(7), 1501 - 7
Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries; Rudiger S et al.; Hsp70 chaperones assist protein folding by ATP-dependent association with linear peptide segments of a large variety of folding intermediates . The molecular basis for this ability to differentiate between native and non-native conformers was investigated for the DnaK homolog of Escherichia coli . We identified binding sites and the recognition motif in substrates by screening 4360 cellulose-bound peptides scanning the sequences of 37 biologically relevant proteins . DnaK binding sites in protein sequences occurred statistically every 36 residues . In the folded proteins these sites are mostly buried and in the majority found in beta-sheet elements . The binding motif consists of a hydrophobic core of four to five residues enriched particularly in Leu, but also in Ile, Val, Phe and Tyr, and two flanking regions enriched in basic residues . Acidic residues are excluded from the core and disfavored in flanking regions . The energetic contribution of all 20 amino acids for DnaK binding was determined . On the basis of these data an algorithm was established that predicts DnaK binding sites in protein sequences with high accuracy.

J Biomol Struct Dyn, 1997 Apr, 14(5), 629 - 39
G and T nucleotide contents show specie-invariant negative correlation for all three codon positions; Frank GK et al.; The nucleotide contents of the three codon positions show a number of statistical pairwise correlations, some of which are universal for all analysed genomes . Among the most prominent of these correlations are negative correlations between G and T contents found in genes of all species analysed . The pair A/C, which is complementary to G/T shows similar negative correlation in genes of most species . In the genes of several species including all mammalian genes studied, positive correlations between A and T contents, and G and C contents are found . Since these regularities are observed in all three codon positions they are connected with amino-acid content of proteins . Such correlations may origin from features of the mutation process or/and translation reading frame check . The well-known bias of the preference for G in the first codon position and its deficiency in the second is accompanied by opposite bias in T content . In the third codon position there is no general nucleotide preference, but its content is often biased with regard to GC content of the gene . G and T contents in this case are always shifted in the opposite directions Several ideas are drawn to explain this preference.

Eur J Biochem, 1997 Apr 1, 245(1), 103 - 15
Sequence of a gene cluster from Malonomonas rubra encoding components of the malonate decarboxylase Na+ pump and evidence for their function; Berg M et al.; Malonate decarboxylation in Malonomonas rubra involves the formation of malonyl-S-{acyl-carrier protein} from acetyl-S-{acyl-carrier protein} and malonate, carboxyltransfer to a biotin protein and its decarboxylation that is coupled to delta mu Na+ generation . The genes encoding components of the malonate decarboxylase enzyme system have been cloned and sequenced . These are located within a gene cluster of approximately 11 kb comprising 14 genes that have been termed madYZGBAECDHKFLMN in the given order . Upstream of madY an open reading frame pointing into the opposite direction of the mad genes was found with structural similarities to insertion-sequence elements . The upstream region also contains DNA regions which are typical for an Escherichia coli sigma 70 promoter . Within 950 bp downstream of madN no other open reading frame was found . This region contains a putative terminator sequence . The intergenic regions within the mad gene cluster are short (usually < 70 bp, maximum 302 bp) and ribosome binding sites were defined before all 14 genes . Thus, this DNA region could form a transcriptional unit and all 14 genes could be translated into proteins . The genes madABCDEF encode the structural proteins of the malonate decarboxylase as yet identified . By comparing protein and DNA sequences and by data bank searches for related proteins with known function the following assignments could be made: MadA represents the acyl-carrier-protein-transferase component . MadB is the integral membrane-bound carboxybiotin protein decarboxylase, MadC and MadD are the two subunits of the carboxyltransferase, MadE is the acyl carrier protein and MadF is the biotin protein . Sequence comparison further indicates that MadH could be involved in the acetylation of the phosphoribosyl-dephospho-CoA prosthetic group and MadG could be involved in its biosynthesis . MadL and MadM are membrane proteins that could function as malonate carrier . The function of the madY,Z,K and N gene products is as yet unknown.

Eur J Biochem, 1997 Apr 1, 245(1), 32 - 9
Room temperature phosphorescence study of phosphate binding in Escherichia coli alkaline phosphatase; Sun L et al.; The phosphorescence spectrum and decay of Trp109 in Escherichia coli alkaline phosphatase was measured for the enzyme in 10 mM Tris/HCl, pH 7.4, at 21 degrees C . Changes in the spectrum and decay from the steady-state in response to non-covalent phosphate binding suggested a phosphate-induced alteration in the local environment surrounding Trp109 which lies buried below the active site . The seemingly inflexible structure in the region of Trp109, as judged by its very long phosphorescence lifetime, appeared unaltered when the enzyme was symmetrically bound with phosphate . However, the protein with phosphate bound to only one site displayed a marked increase in flexibility that extended over both subunits . For ratios of phosphate/enzyme (mol/mol) between 1.0 and 2.0, the observation of exponential phosphorescence decays with lifetimes that are a function of dilution provided evidence for the rapid exchange between phosphate half-saturated and fully-saturated enzymes consistent with observed enzyme turnover rates . The lifetimes under these conditions result in the calculation of a Kd for the dissociation of phosphate from the doubly occupied enzyme of 1.1 +/- 0.1 microM . The non-exponential decays at P/Ed (phosphate/dimeric enzyme) ratios less than 1.0 revealed that the exchange of phosphate between phosphate-free and half-saturated enzymes was not occurring on the timescale of the phosphorescence decay times, which implied that the half-saturated molecule cannot be contributing significantly to catalysis under steady-state conditions . The observation that the phosphorescence decay at a P/Ed ratio of 1.0 is exponential with a lifetime characteristic of the half-saturated species indicates that the binding of the first phosphate is significantly greater than the second, or that the binding exhibits negative cooperativity.

Am J Gastroenterol, 1997 Apr, 92(4), 700 - 2
Perforated appendicitis within an inguinal hernia: case report and review of the literature; Lyass S et al.; The finding of the vermiform appendix within an inguinal hernia sac is not uncommon . However, it is rare to find a perforated appendix within an inguinal hernia . An unusual case of an incarcerated and perforated appendix within an inguinal hernia complicated by an intra-abdominal abscess is reported herein . Perforated appendix as a cause of abscess was revealed during abdominal exploration . Clinicians are encouraged to be aware of this unusual entity, which is rarely recognized before exploration.

Eur J Cell Biol, 1997 Apr, 72(4), 287 - 96
Unusual distribution of gamma-tubulin in the giant fresh water amoeba Reticulomyxa filosa; Kube-Granderath E et al.; We have isolated a cDNA encoding gamma-tubulin of Reticulomyxa . The deduced amino acid sequence revealed a high homology to known gamma-tubulin sequences from other organisms, underlining the hypothesis of conserved functions for gamma-tubulin in the cell . After introduction of restriction sites by site-directed mutagenesis and polymerase chain reaction (PCR), we cloned the cDNA into a bacterial expression vector . Recombinant gamma-tubulin was expressed in considerable amounts . Polyclonal antibodies raised against the recombinant material recognized in Western blots the bacterially expressed gamma-tubulin and in the amoeba crude extract a single band representing most likely the amoebal gamma-tubulin . In immunofluorescence studies the antibody showed a diffuse staining in the cytoplasm and no enrichment at centrosome-like structures . Because the assembly of centrosome-independent microtubules can be observed in the far extensions of the amoeba, the data suggest that in this amoeba gamma-tubulin might be involved in the centrosome-independent assembly of microtubules.

Biotechnol Appl Biochem, 1997 Apr, 25 ( Pt 2), 109 - 15
Purification of (His)6EcoRV {recombinant restriction endonuclease EcoRV fused to a (His)6 affinity domain} by metal-chelate affinity chromatography; Oswald T et al.; The chromatographic purification of (His)6EcoRV, a fusion protein consisting of a hexahistidine affinity domain and restriction endonuclease EcoRV produced from recombinant Escherichia coli, led to high product concentrations (> or = 1 mg/ml) in the preparative mode . Increasing the amount of applied crude cell homogenate caused competition with host-specific proteins was achieved by pre-adsorption on to a DEAE anion-exchange sorbent . This, in combination with 0.5-1 M NaCl in the adsorption buffer, assured a purity > 95% and a total protein recovery of approximately 34% in the preparative mode . Contamination of the product with about 2 mol of Ni(11)/mol of (His)6EcoRV was found due to metal-ion transfer to the N-terminal high-affinity binding site at (His)6 . Tris(carboxymethyl)ethylenediamine (TED)-Sepharose was employed as an Ni(11) adsorber . One passage of Ni(11)-contaminated protein solutions through the TED-Sepharose column resulted in a decrease in the Ni(11) content in the (His)6EcoRV fractions below the detection limit (approximately 0.02 mg/l) of the atomic-adsorption spectrophotometer.

Protein Expr Purif, 1997 Apr, 9(3), 379 - 87
Human cysteine-rich intestinal protein: cDNA cloning and expression of recombinant protein and identification in human peripheral blood mononuclear cells; Khoo C et al.; Cysteine-rich intestinal protein (CRIP) is a small, 8.5-kDa protein with one double zinc-finger motif called a LIM domain . It is very abundant in intestine and some immune cells in rodents, and expression is influenced by development and the immune response . We have cloned a human CRIP cDNA from human small intestine poly(A)+ RNA by RT-PCR . Through sequencing, we found that the human intestinal CRIP protein (hCRIP) differed from the previously cloned rat CRIP by two amino acids (residues 8 and 58) . hCRIP was expressed with the pET vector/bacterial system and isolated by gel filtration and ion-exchange chromatography . The protein was purified to homogeneity as confirmed by PAGE, Western blotting, and immunodetection . Recombinant hCRIP has a molecular mass of 8390 Da based on mass spectrum analysis . Southern analysis suggests that there are three copies of the CRIP gene in the human genome . hCRIP mRNA was detected by RT-PCR in human monocytes purified from peripheral blood and THP-1 cells, a human monocytic cell line . Incubation of THP-1 cells with 65Zn and chromatography of the cytosol show that a significant amount of the radioactivity is associated with CRIP as was shown previously for rat intestine . The results are consistent with a functional role for CRIP in proliferation/differentiation of specific cell types, particularly those associated with host defense.

Protein Expr Purif, 1997 Apr, 9(3), 372 - 8
Application of a single-plasmid vector for mutagenesis and high-level expression of thioredoxin reductase and its use to examine flavin cofactor incorporation; Mulrooney SB; Thioredoxin reductase from Escherichia coli is a dimeric enzyme containing one FAD and one redox-active disulfide per monomer and catalyzes the transfer of electrons from NADPH to thioredoxin, which subsequently performs several important cellular functions . To overcome problems with site-directed mutagenesis and low expression, the thioredoxin reductase gene was adapted for use in the plasmid vector pSL350 (Brosius, J., Methods Enzymol . 216, 469-483, 1992), which is designed both for protein expression and for production of single-stranded template DNA for mutagenesis, and examined expression of wild-type thioredoxin reductase under different growth conditions . In the absence of IPTG inducer, expression of thioredoxin reductase in saturated cultures accounts for 19% of the soluble protein, and with 1 mM IPTG expression increases to 61% . Some of the thioredoxin reductase is expressed as apoenzyme with the amount of apoenzyme increasing at higher IPTG concentrations, accounting for as high as 68% of the total thioredoxin reductase expressed . The apoenzyme in cell extracts is activated rapidly by addition of FAD, indicating correct folding of the enzyme in the absence of cofactor . Purification of wild-type thioredoxin reductase from the new system yielded 189 mg of enzyme from a 300-ml uninduced culture . The new plasmid was also used to generate an N155Y mutant which is purified and partially characterized.

Protein Expr Purif, 1997 Apr, 9(3), 363 - 71
Purification, characterization, and in vitro phosphorylation of the neuron-specific membrane-associated protein SCG10; Antonsson B et al.; SCG10 is a neuron-specific, developmentally regulated protein which is highly enriched in growth cones . Sequence homology indicates that it is related to the phosphoprotein stathmin or Op18, an in vitro and in vivo substrate for several serine/threonine kinases which are involved in a variety of signaling pathways . As a first step to examine the biochemical properties of SCG10, the protein was expressed in Escherichia coli and purified to apparent homogeneity . The purified protein was used in in vitro phosphorylation assays . SCG10 was phosphorylated by MAP kinase, cAMP-dependent protein kinase, cGMP-dependent protein kinase, p34cdc2 kinase, DNA-dependent protein kinase, Ca2+/calmodulin kinase II, and casein kinase II . The protein was not a substrate for casein kinase I and protein kinase C . SCG10 was phosphorylated by src tyrosine kinase, which demonstrates that the protein can be phosphorylated in vitro on a tyrosine residue . Our data suggest that SCG10 is a phosphoprotein which might be involved in signal transduction in neurons.

Protein Expr Purif, 1997 Apr, 9(3), 337 - 45
Construction and overexpression of a synthetic gene for human DNA methylguanine methyltransferase: renaturation and rapid purification of the protein; Brown LR et al.; A synthetic gene was constructed that encodes human DNA methylguanine methyltransferase (hMGMT) . The synthetic gene was designed with a number of unique restriction sites to facilitate cassette mutagenesis and to reflect the preferences found among genes in Escherichia coli . Both the full-length gene and a gene for a functional variant (hMGMT delta C) that lacks the C-terminal 28 codons were constructed, and the genes were overexpressed using a T7 RNA polymerase promoter . The proteins are made in the form of insoluble aggregates but the truncated form of the protein (hMGMT delta C) has been successfully denatured, renatured, and purified to near homogeneity by ion exchange . Methyltransferase activity assays of hMGMT delta C demonstrate that the reconstituted protein has substantial DNA repair activity, though somewhat less than full-length hMGMT that had been expressed and purified in a soluble form . Mass spectrometry of a mixture of proteolytic fragments confirmed the protein sequence and indicated no detectable oxidation of the active site cysteine . The protein was determined to be monomeric by gel filtration chromatography, and circular dichroism spectra for renatured hMGMT delta C and fully soluble hMGMT are consistent with the renatured protein preparation being fully folded . Refolded hMGMT delta C had a curious propensity to form large aggregates in a time-dependent manner when injected into a dynamic light scattering instrument; this aggregation behavior was not observed for hMGMT purified in a soluble form . Differences in susceptibility to aggregation may account for differences in methyltransfer activity . Yields of purified protein were approximately 5 mg/liter of culture.

Protein Expr Purif, 1997 Apr, 9(3), 319 - 30
One-step immunoaffinity purification of recombinant human retinoic acid receptor gamma; Repa JJ et al.; Retinoic acid receptors (RAR) are members of the steroid/thyroid hormone receptor superfamily and serve as ligand-activated transcription factors . In order to facilitate studies of receptor protein, we have generated a monoclonal antibody to the human RAR gamma, and have developed a procedure to purify the full-length receptor expressed in insect cells . The monoclonal antibody (A10) was developed using as antigen a carboxy-terminal fragment of the human RAR gamma expressed as a bacterial fusion protein . The A10 monoclonal antibody binds to both native and denatured forms of the human RAR gamma . This antibody was immobilized on a resin and used to purify full-length, baculovirus-expressed human RAR gamma to near homogeneity . The immunoaffinity-purified receptor is > 90-95% pure as revealed by silver-stained gels . The identity of the single protein band as RAR gamma was verified by immunoblotting using a polyclonal antibody to an epitope distinct from that recognized by the A10 antibody . The pure human RAR gamma is functional with respect to both ligand and DNA binding . Scatchard analysis of 3H-labeled all-trans retinoic acid binding to purified human RAR gamma revealed a single, high-affinity binding site with a Kd of approximately 2 nM . Binding of the pure RAR gamma to a DR5-type retinoic acid response element was also studied . Response element binding by RAR gamma required the presence of the retinoid X receptor, but did not require the presence of additional proteins . Human RAR gamma protein purified in this fashion will be useful in future structural and functional studies.

Protein Expr Purif, 1997 Apr, 9(3), 315 - 8
Method for quantitative refolding of the link module from human TSG-6; Kahmann JD et al.; We have developed a procedure for the quantitative refolding of the Link module from human tumor necrosis factor-stimulated gene 6 . This significantly simplifies the previously described method of production of this protein domain (Day et al., Protein Expression Purif . 8, 1-16, 1996) . The refolding is carried out under nondenaturing conditions at pH 6.0 in the presence of a 100-fold molar excess of beta-mercaptoethanol . After 2 days the starting material, which consists of three species that differ only with respect to their disulfide bond organization, has rearranged to give a single homogeneous species with the correct disulfide bridges . This method allows the production of about 20 mg of folded protein per liter of Escherichia coli culture.

Arch Biochem Biophys, 1997 Apr 1, 340(1), 36 - 42
Escherichia coli F1-ATPase subunit interactions: beta and gamma subunit peptides inhibit in vitro reconstitution of the active alpha beta gamma complex; Shin Y et al.; For biochemical analysis of subunit interactions in the proton-translocating ATPase, a new approach with in vitro reconstitution of the Escherichia coli alpha beta gamma complex and the peptides derived from the subunits was established . Various portions of the beta or gamma subunits were used for in vitro reconstitution of the alpha beta gamma complex from the purified subunits . For the beta subunits, peptides corresponding to residues 226-459, 254-459, and 226-365 inhibited reconstitution, while those corresponding to residues 1-105, 1-146, and 295-459 did not . For the gamma subunits, peptides corresponding to residues 1-192 and 74-286 exhibited inhibitory effect on reconstitution, but the peptide containing residues 191-286 did not . Only inhibitory peptides blocked the assembly of the alpha beta gamma complex which was detected by nondenaturing polyacrylamide gel electrophoresis . These inhibitory peptides bound to the alpha or beta subunit on the filter, but the noninhibitory peptides did not . These results suggested that regions beta 254-294 and gamma 74-190 have sequences important for subunit interactions which interfered with those in the reconstitution mixtures . Based on comparison between X-ray crystallographic data of bovine alpha beta gamma complex and the present results, we discussed here the significance of the biochemical approach adopted in this study.

Dig Dis Sci, 1997 Apr, 42(4), 731 - 7
Pathophysiology of adynamic ileus; Cullen JJ et al.; We hypothesized that the inhibitory neurotransmitters nitric oxide (NO) and vasoactive intestinal peptide (VIP) may play a role in the disrupted gastrointestinal motility of endotoxemia . Strain gauge transducers on the stomach and small intestine of dogs determined interdigestive gastrointestinal motility . Tissue levels of NO synthase and VIP and serum levels of nitrite/nitrate (NO(2)-/NO(3)-) and VIP were measured . Following completion of the baseline studies, dogs were given a single dose of E . coli lipopolysaccharide, 200 microg/kg intravenously, and the studies were repeated for the next three days . Following endotoxin bolus, the migrating motor complex (MMC) was delayed for two days while serum VIP was increased on postendotoxin day 1 and serum NO(2)-/NO(3)- was increased on postendotoxin day 2 . There were no changes in gut smooth muscle levels of NO synthase or VIP . We conclude that a single, sublethal dose of endotoxin results in prolongation of the MMC with distinct but independent increases in serum levels of VIP and NO(2)-/NO(3)-.

Mol Cell Biol, 1997 Apr, 17(4), 2291 - 300
The Drosophila suppressor of sable protein binds to RNA and associates with a subset of polytene chromosome bands; Murray MV et al.; Mutations of the Drosophila melanogaster suppressor of sable {su(s)} gene, which encodes a 150-kDa nuclear protein {Su(s)}, increase the accumulation of specific transcripts in a manner that is not well understood but that appears to involve pre-mRNA processing . Here, we report biochemical analysis of purified, recombinant Su(s) {rSu(s)} expressed in baculovirus and in Escherichia coli as maltose binding protein (MBP) fusions and immunocytochemical analysis of endogenous Su(s) . This work has shown that purified, baculovirus-expressed rSu(s) binds to RNA in vitro with a high affinity and limited specificity . Systematic evolution of ligands by exponential enrichment was used to identify preferred RNA targets of rSu(s), and a large proportion of RNAs isolated contain a full or partial match to the consensus sequence UCAGUAGUCU, which was confirmed to be a high-affinity rSu(s) binding site . An MBP-Su(s) fusion protein containing the N-terminal third of Su(s) binds RNAs containing this sequence with a higher specificity than full-length, baculovirus-expressed rSu(s) . The consensus sequence resembles both a cryptic 5' splice site and a sequence that is found near the 5' end of some Drosophila transcripts . Immunolocalization studies showed that endogenous Su(s) is distributed in a reticulated pattern in Drosophila embryo and salivary gland nuclei . In salivary gland cells, Su(s) is found both in the nucleoplasm and in association with a subset of polytene chromosome bands . Considering these and previous results, we propose two models to explain how su(s) mutations affect nuclear pre-mRNA processing.

Mol Cell Biol, 1997 Apr, 17(4), 2257 - 65
PRH75, a new nucleus-localized member of the DEAD-box protein family from higher plants; Lorkovic ZJ et al.; The putative RNA helicases of the DEAD-box protein family are involved in pre-mRNA splicing, rRNA maturation, ribosome assembly, and translation . Members of this protein family have been identified in organisms from Escherichia coli to humans, but except for the translation initiation factor 4A, there have been no reports on the characterization of other DEAD-box proteins from plants . Here we report on a novel member of the DEAD-box protein family, the plant RNA helicase 75 (PRH75) . PRH75 is localized in the nucleus and contains two domains for RNA binding . One is located at the C terminus and is similar to RGG RNA-binding domains of nucleus-localized RNA-binding proteins . The other one is located between amino acids 308 and 622, a region containing the conserved motif VI characteristic of DEAD-box proteins and known as the RNA-binding site of eIF-4A . The N-terminal 81 amino acids are sufficient for nuclear targeting of the protein . Northern and Western blot analyses show that PRH75 is mainly expressed in young and rapidly developing tissues . The purified recombinant PRH75 has a weak ATPase activity which is barely stimulated by RNA ligands . The fractionation of spinach whole-cell extracts by glycerol gradient centrifugation and gel filtration on a Superdex 200 column shows that the protein exists in a complex of about 500 kDa . Possible biological functions of PRH75 as well as structure-function relationships in the context of its modular primary structure are discussed.

J Immunol, 1997 Apr 1, 158(7), 3259 - 69
Construction and characterization of human CD7-specific single-chain Fv immunotoxins; Pauza ME et al.; To develop novel therapeutic agents for treatment of human T cell malignancies, we constructed two single-chain Fv (sFv) immunotoxins specific for the T cell-associated Ag CD7 . The sFv fragments were derived from the murine hybridomas 3A1e and 3A1f and were expressed as soluble proteins in Escherichia coli . Surface plasmon resonance analyses demonstrated that the purified 3A1e and 3A1f sFv fragments specifically bound CD7 with high affinity, 8.1 and 1.8 nM, respectively . The difference in affinity is chiefly due to a slower dissociation rate for the 3A1f sFv fragment . Despite this difference, both monovalent sFv fragments were comparably internalized by CD7+ human T leukemic cells within 30 min . These data support findings of previous studies suggesting that CD7 internalization does not require cross-linking . The sFv immunotoxins were assembled by linking ricin toxin A chain to the C termini of the sFv fragments via disulfide bonds . Both sFv immunotoxins were comparably potent in their ability to inhibit protein synthesis in vitro in CD7+ Jurkat cells (50% inhibiting concentration = 15 pM) . Further preclinical studies on the use of the 3A1e and 3A1f sFv immunotoxins to treat human T cell diseases therefore appear warranted.

J Clin Invest, 1997 Apr 1, 99(7), 1673 - 81
Conversion of the major birch pollen allergen, Bet v 1, into two nonanaphylactic T cell epitope-containing fragments: candidates for a novel form of specific immunotherapy; Vrtala S et al.; A novel approach to reduce the anaphylactic activity of allergens is suggested . The strategy makes use of the presence of conformational immunoglobulin E (IgE) epitopes on one of the most common allergens . The three dimensional structure of the major birch pollen allergen, Bet v 1, was disrupted by expressing two parts of the Bet v 1 cDNA representing amino acids 1-74 and 75-160 in Escherichia coli . In contrast to the complete recombinant Bet v 1, the fragments showed almost no allergenicity and exhibited random coil conformation as analyzed by circular dichroism . Both nonanaphylactic fragments induced proliferation of human Bet v 1-specific T cell clones, indicating that they harbored all dominant T cell epitopes and therefore may be considered as a basis for the development of a safe and specific T cell immunotherapy.

J Clin Invest, 1997 Apr 1, 99(7), 1662 - 72
Development of experimental model of chronic pyelonephritis with Escherichia coli O75:K5:H-bearing Dr fimbriae: mutation in the dra region prevented tubulointerstitial nephritis; Goluszko P et al.; Escherichia coli that express Dr fimbriae and related adhesins recognize the common receptor decay accelerating factor . E . coli strains that express adhesins of the Dr family were postulated to be associated with cystitis (30-50%), pregnancy-associated pyelonephritis (30%), and chronic diarrhea (50%) . In this study, we investigated the hypothesis that E . coli renal interstitial binding mediated by the Dr adhesin may be important for the development of chronic pyelonephritis . An insertional dra mutant, E . coli DR14, of the clinical E . coli isolate IH11128 bearing Dr fimbriae, was constructed and used to characterize persistence of infection and interstitial tropism in an experimental model of ascending pyelonephritis . Quantitative cultures of kidney homogenates indicated that Dr hemagglutinin positive (Dr+) E . coli IH11128 established a 1-yr colonization of renal tissue . In the Dr hemagglutinin negative (Dr-) group, 50% of animals cleared infection within 20 wk and 100% between 32 to 52 wk . Dr+ E . coli colonized the renal interstitium . Significant histological changes corresponding to tubulointerstitial nephritis including interstitial inflammation, fibrosis, and tubular atrophy were found in the kidney tissue of the Dr+ but not the Dr- group . A substantial amount of fimbrial antigen was detected in the parenchymal regions affected by interstitial inflammation and fibrosis . The obtained results are consistent with the hypothesis that mutation within the dra region, affecting E . coli binding to tubular basement membranes, prevented renal interstitial tropism and the development of the changes characteristically seen in tubulointerstitial nephritis.

Infect Immun, 1997 Apr, 65(4), 1408 - 13
Effect of carbon source on localized adherence of enteropathogenic Escherichia coli; Vanmaele RP et al.; Enteropathogenic Escherichia coli (EPEC) strains attach to epithelial cells as discrete clusters of bacteria which are localized at a few sites on the cell surface . Previously, it was shown that this localized-adherence (LA) phenotype is induced by specific growth conditions . We found that wild-type EPEC attached to HEp-2 cells in an LA pattern when the bacteria were grown in Dulbecco's modified Eagle medium (DMEM) containing glucose as the carbon source . In contrast, bacteria incubated in DMEM containing galactose did not adhere to epithelial cells . The latter results were similar to those observed when JPN15, an LA-negative strain, was grown under conditions which promoted bacterial binding . The differences in attachment of wild-type EPEC were independent of the stage of log-phase growth of the cultures and of the number of CFU incubated with the HEp-2 monolayers . Expression of the adherence phenotype by organisms grown in glucose was associated with increased expression of intimin and bundle-forming pilin . In contrast, bacteria grown in medium containing galactose expressed these proteins at levels similar to those observed when JPN15 was grown in medium containing glucose.

Infect Immun, 1997 Apr, 65(4), 1299 - 306
Glucose up-regulates expression of the differentiation-associated brush border binding site for enterotoxigenic Escherichia coli colonization factor antigen I in cultured human enterocyte-like cells; Bernet-Camard MF et al.; The association of enterotoxigenic Escherichia coli expressing colonization factor antigen I (CFA/I) with the cultured human colon adenocarcinoma cell, a model of the mature enterocyte of the small intestine, is dependent on the binding of CFA/I to a brush border-associated component . Binding of the purified radiolabeled {125I}CFA/I- and 14C-labeled CFA/I-positive bacteria could be displaced by an increasing concentration of unlabeled CFA/I . Moreover, we showed that expression of the specific CFA/I binding developed as a function of cell differentiation in Caco-2 cells, whereas expression of the nonspecific binding did not . Expression of the brush border differentiation-associated component acting as a binding site for CFA/I was up-regulated by glucose . Indeed, the enterocyte-like HT-29 glc- cell subpopulation not expressing the CFA/I binding site when cultured in dialyzed serum and hexose-free medium regained the ability to bind CFA/I when the cells were returned to culture medium containing glucose . Furthermore, expression of the brush border-associated CFA/I binding site in the enterocyte-like Caco-2 cells was repressed when the cells were cultured in hexose-free conditions.

Development, 1997 Apr, 124(7), 1343 - 54
Concentration-dependent patterning by an ectopic expression domain of the Drosophila gap gene knirps; Kosman D et al.; The asymmetric distribution of the gap gene knirps (kni) in discrete expression domains is critical for striped patterns of pair-rule gene expression in the Drosophila embryo . To test whether these domains function as sources of morphogenetic activity, the stripe 2 enhancer of the pair-rule gene even-skipped (eve) was used to express kni in an ectopic position . Manipulating the stripe 2-kni expression constructs and examining transgenic lines with different insertion sites led to the establishment of a series of independent lines that displayed consistently different levels and developmental profiles of expression . Individual lines showed specific disruptions in pair-rule patterning that were correlated with the level and timing of ectopic expression . These results suggest that the ectopic domain acts as a source for morphogenetic activity that specifies regions in the embryo where pair-rule genes can be activated or repressed . Evidence is presented that the level and timing of expression, as well as protein diffusion, are important for determining the specific responses of target genes.

Can J Microbiol, 1997 Apr, 43(4), 395 - 9
Restriction enzyme and DNA hybridization analysis of cellulolytic Streptomyces isolates of different origin; Marri L et al.; Streptomyces rochei A2 endoglucanase (eglS) and beta-glucosidase (bgs1) genes were used as probes to survey their distribution among 16 Streptomyces strains isolated from different sources and characterized for their cellulolytic activities . The eglS probe hybridized to the genomic DNA of 12 strains with a restriction pattern different from that of S . rochei A2 . The DNA from all strains, except one, hybridized with the bgs1 probe and one strain showed the same restriction pattern as seen in S . rochei A2 . The sequence localized by the eglS probe in S . thermoviolaceus and the one localized by the bgs1 probe in strain EC1 were cloned and expressed in E . coli in plasmids pTAE and pCSF203, respectively . The restriction maps showed that the cloned genes were identical to eglS and bgs1 . The restriction enzyme analysis and genomic DNA from all the strains identified nine different groups, each characterized by a distinctive pattern and in agreement with the results of the hybridization experiments.

Chem Res Toxicol, 1997 Apr, 10(4), 369 - 77
Sequence specific mutagenesis of the major (+)-anti-benzo{a}pyrene diol epoxide-DNA adduct at a mutational hot spot in vitro and in Escherichia coli cells; Hanrahan CJ et al.; In the supF gene, most (+)-anti-benzo{a}pyrene diol epoxide ((+)-anti-B{a}PDE) mutagenesis hot spots in Escherichia coli are in 5'-GG sequences {Rodriguez and Loechler (1993) Carcinogenesis 14, 373-383} . A major hot spot was detected at G1 in the sequence 5'-GCG1G2-CCAAAG, whereas G2 yielded very few mutants . In order to investigate the details of such sequence context effects of (+)-anti-B{a}PDE mutagenesis, we have constructed 25-mer oligonucleotides and single-stranded M13 genomes containing the above decamer sequence, in which the trans-N2-dG adduct induced by (+)-anti-B{a}PDE {(+)-trans-anti-B{a}P-N2-dG} at G1 or G2 was introduced . In vitro DNA synthesis on the adducted 25-mers was strongly blocked at each site, although the 3'-->5' exonuclease-deficient Klenow fragment could incorporate a nucleotide opposite the adduct in the presence of Mn2+ . For both sites purine nucleotides were preferred . The ratio Vmax/K(m) indicated that the efficiency of incorporation of dGTP opposite these sites was very similar, but dATP incorporation opposite the adduct at G1 was five-fold more efficient than that at G2 . For each site, further extension beyond the adducted nucleotide was investigated by annealing four different primers, in which only the nucleotide opposite the adducted deoxyguanosine was altered . Significant extension was only observed when deoxyadenosine was located opposite adducted G1 . When the M13 genomes containing the (+)-trans-anti-B{a}P-N2-dG were replicated in E . coli, survival of each adducted genome was less than 1% as compared to the unadducted genome . Upon induction of SOS, viability increased 2-6-fold . DNA sequencing showed no base substitutions in the progeny from SOS-uninduced cells, although small deletions in a quasipalindromic sequence occurred with the adduct being located at either site . However, following SOS induction, up to 40% targeted base substitutions were detected when the adduct was located at G1, while approximately 12% of the progeny were mutants with the adduct at G2 . Most base substitutions were targeted G-->T transversions . We conclude that (+)-trans-anti-B{a}P-N2-dG is a highly mutagenic and replication blocking lesion . In addition, the biological consequence of this adduct depends on whether it is located at G1 or G2, suggesting that sequence context plays a major role in the mutagenic processing of this adduct.

Photochem Photobiol, 1997 Apr, 65(4), 660 - 5
Irradiation of DNA with 193 nm light yields formamidopyrimidine-DNA glycosylase(Fpg) protein-sensitive lesions; Melvin T et al.; Irradiation of aqueous solutions of plasmid DNA (pUC18) at pH 7.6 with 193 nm laser light results in low yields of prompt single strand breakage (air-saturated sample phi ssh = {1.5 +/- 0.1} x 10(4), argon-saturated sample phi ssh = {0.9 +/- 0.1} x 10(4) . Treatment of the irradiated DNA samples with Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) protein results in an approximate 20-fold increase in the yield of single strand break-age (air-saturated sample phi fpg = {33.1 +/- 3.1} x 10(-4), argon-saturated sample phi fpg = {23.8 +/- 2.6} x 10(-4) . This result indicates that 193 nm light induces other modification(s) (most likely of the purine moieties) that are 20 times more abundant than prompt strand breakage within the DNA matrix.

Plant Physiol, 1997 Apr, 113(4), 1427 - 35
Molecular cloning of mannose-6-phosphate reductase and its developmental expression in celery; Everard JD et al.; Compared with other primary photosynthetic products (e.g . sucrose and starch), little is known about sugar alcohol metabolism, its regulation, and the manner in which it is integrated with other pathways . Mannose-6-phosphate reductase (M6PR) is a key enzyme that is involved in mannitol biosynthesis in celery (Apium graveolens L.) . The M6PR gene was cloned from a leaf cDNA library, and clonal authenticity was established by assays of M6PR activity, western blots, and comparisons of the deduced amino acid sequence with a celery M6PR tryptic digestion product . Recombinant M6PR, purified from Escherichia coli, had specific activity, molecular mass, and kinetic characteristics indistinguishable from those of authentic celery M6PR . Sequence analyses showed M6PR to be a member of the aldo-keto reductase superfamily, which includes both animal and plant enzymes . The greatest sequence similarity was with aldose-6-phosphate reductase (EC 1.1.1.200), a key enzyme in sorbitol synthesis in Rosaceae . Developmental studies showed M6PR to be limited to green tissues and to be under tight transcriptional regulation during leaf initiation, expansion, and maturation . These data confirmed a close relationship between the development of photosynthetic capacity, mannitol synthesis, and M6PR activity.

Plant Physiol, 1997 Apr, 113(4), 1369 - 77
Gibberellin biosynthesis from gibberellin A12-aldehyde in endosperm and embryos of Marah macrocarpus; MacMillan J et al.; Soluble enzyme preparations from embryos and endosperm of Marah macrocarpus (previously Echinocystis macrocarpa) were incubated with {14C4}gibberellin(GA)12-aldehyde, {14C4}GA12, {14C1} GA9, 2,3-didehydro{14C1}GA9, {14C1}GA20, and {17-13C, 3H}GA5 . Embryo preparations converted GA12-aldehyde, GA12, and GA9 to GA4 and GA7; 2,3-didehydroGA9 to GA7; GA5 to GA3; and GA20 (incompletely) to GA1 and GA60, but not to GA3 . Endosperm preparations converted GA12-aldehyde and GA12 to GA15, GA24, and GA9, but, unlike embryo preparations, not to GA4 or GA7 . However, GA4 and GA7 were formed from GA9 and GA7 was formed from 2,3-didehydroGA9 . Metabolism of GA5 to GA3 and GA20 to GA1 was low . 2,3-DidehydroGA9 accumulated when GA9 was incubated with a desalted endosperm preparation . A cDNA clone (M3-8), selected from an embryo-derived cDNA library using a DNA fragment generated by reverse transcriptase polymerase chain reaction, was expressed in Escherichia coli . The fusion protein converted GA12 to GA9 (major) and GA25 (minor); GA53 was metabolized less effectively and only to GA44 . Thus, the M3-8 protein is functionally similar to GA 20-oxidases from Arabidopsis thaliana, Spinacia oleracea, and Pisum sativum, but different from that from Cucurbita maxima seeds, to which its amino acid sequence is most closely related . mRNA hybridizing to M3-8 accumulated in embryos and endosperm of M . macrocarpus, but was absent in vegetative tissues.

Plant Physiol, 1997 Apr, 113(4), 1177 - 83
Increased resistance to oxidative stress in transgenic plants by targeting mannitol biosynthesis to chloroplasts; Shen B et al.; To investigate the potential role of a polyol, mannitol, in oxidative stress protection, a bacterial mannitol-1-phosphate dehydrogenase gene was targeted to chloroplasts by the addition of an amino-terminal transit peptide . Transgenic tobacco (Nicotiana tabacum) lines accumulate mannitol at concentrations ranging from 2.5 to 7 mumol/g fresh weight . Line BS1-31 accumulated approximately 100 mM mannitol in chloroplasts and was identical to the wild type in phenotype and photosynthetic performance . The presence of mannitol in chloroplasts resulted in an increased resistance to methyl viologen (MV)-induced oxidative stress, documented by the increased retention of chlorophyll in transgenic leaf tissue following MV treatment . In the presence of MV, isolated mesophyll cells of BS1-31 exhibited higher CO2 fixation than the wild type . When the hydroxyl radical probe dimethyl sulfoxide was introduced into cells, the initial formation rate of methane sulfinic acid was significantly lower in cells containing mannitol in the chloroplast compartment than in wild-type cells, indicating an increased hydroxyl radical-scavenging capacity in BS1-31 tobacco . We suggest that the chloroplast location of mannitol can supplement endogenous radical-scavenging mechanisms and reduce oxidative damage of cells by hydroxyl radicals.

Plant Physiol, 1997 Apr, 113(4), 1081 - 90
Novel, highly expressed late nodulin gene (LjNOD16) from Lotus japonicus; Kapranov P et al.; We have isolated a Lotus japonicus cDNA corresponding to a highly abundant, late nodule-specific RNA species that encodes a polypeptide with a predicted molecular mass of 15.6 kD . The protein and its corresponding gene were designated Nlj16 and LjNOD16, respectively . LjNOD16 was found to be expressed only in the infected cells of L . japonicus nodules . Related DNA sequences could be identified in the genomes of both Glycine max and Medicago sativa . In the latter, a homologous mRNA species was detected in the nodules . Unlike LjNOD16, its alfalfa homologs appear to represent low-abundance mRNA species . However, the proteins corresponding to the LjNOD16 and its alfalfa homolog could be detected at similar levels in nodules but not in roots of both legume species . The predicted amino acid sequence analysis of nodulin Nlj16 revealed the presence of a long alpha-helical region and a positively charged C terminus . The former domain has a very high propensity to form a coiled-coil type structure, indicating that nodulin Nlj16 may interact with an as-yet-unidentified protein target(s) in the nodule-infected cells . Homology searches revealed no significant similarities to any known sequences in the databases, with the exception of two related, anonymous Arabidopsis expressed sequence tags.

Biochem Mol Biol Int, 1997 Apr, 41(4), 657 - 63
Molecular cloning of the leuB genes from Mycobacterium bovis BCG and Mycobacterium tuberculosis; Han MY et al.; A gene responsible for the biosynthesis of leucine has been cloned by the complementation of the Escherichia coli leuB6 auxotroph mutant after transformation with the Mycobacterium bovis BCG genomic DNA library, which was constructed by ligating the partially digested BCG DNA with Sau3A1 into the pUC19 digested with BamHI . Sequencing of the leuB gene of BCG revealed an ORF (open reading frame) of 1.011 bp encoding isopropylmalate dehydrogenase with a calculated molecular weight of 42 kDa . The leuB gene of Mycobacterium tuberculosis isolated from Korean tuberculosis patient is shown to be identical to that of BCG except one bp.

Carcinogenesis, 1997 Apr, 18(4), 851 - 4
Metabolism of carcinogenic heterocyclic and aromatic amines by recombinant human cytochrome P450 enzymes; Hammons GJ et al.; The N-hydroxylation of carcinogenic arylamines represents an initial step in their metabolic activation . Animal studies have shown that this reaction is catalyzed by the cytochrome P450 (P450) enzymes P450 1A1 and P450 1A2 . In this study, utilizing enzymes expressed in Escherichia coli (and purified) or in human B-lymphoblastoid cells, the catalytic activities of recombinant human P450 1A1, P450 1A2, and P450 3A4 for N-hydroxylation of several carcinogenic arylamines were determined . P450 1A2 from both expression systems catalyzed the N-hydroxylation of 4-aminobiphenyl and the heterocyclic amines, 2-amino-3-methylimidazo{4,5-f/quinoline (IQ), 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) . Rates were similar, with values of 1.1-7.8 nmol/min/nmol P450 . In contrast, P450 1A1 catalyzed N-hydroxylation of only PhIP, and no activity was observed with P450 3A4 . Further kinetic analysis with purified P450 1A2 showed similar Km and Vmax values for N-hydroxylation of the arylamines . Furafylline and fluvoxamine, inhibitors of P450 1A2 activity in human liver microsomes, were found to be inhibitory of the recombinant P450 1A2 N-hydroxylation activity . Results from this study are supportive of a major role for human P450 1A2 in the metabolic activation of arylamines.

Carcinogenesis, 1997 Apr, 18(4), 745 - 8
Agreement of mutational characteristics of heterocyclic amines in lacI of the Big Blue mouse with those in tumor related genes in rodents; Okonogi H et al.; The mutational spectra of carcinogenic heterocyclic amines (HCAs), 2-amino-3,4-dimethylimidazo{4,5-b}quinoline (MeIQ), 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) and 2-amino-9H-pyrido{2,3-b}indole (A alphaC) were studied in the colon of Big Blue mice . In 90, 115 and 105 lacI mutants from mice fed 300 p.p.m . MeIQ, 400 p.p.m . PhIP and 800 p.p.m . A alphaC, respectively, 92, 115 and 105 mutations were identified . G:C-->T:A transversions predominated with these HCAs . Mutational hot spots for base-substitution mutations caused by MeIQ, PhIP and A alphaC were in distinct sequence contexts; at 5'-GC-3', in runs of guanine and in 5'-CGT-3', respectively . Further, 30 of 115 (26%) PhIP-induced mutations were G:C base pair deletions, and eight of these deletions were in 5'-GGGA-3' . The mutational characteristics of MeIQ in the lacI gene coincided well with those in the Ha-ras gene of MeIQ-induced mouse forestomach tumors and rat Zymbal gland tumors . The characteristic single-base deletion induced by PhIP in the lacI gene also coincided well with those in the Apc gene of PhIP-induced rat colon tumors . These results suggest that the mutational characteristics of each chemical are conserved across different genes in different species.

Shock, 1997 Apr, 7(4), 254 - 62
Induction of heat shock protein 70 by zinc-bis-(DL-hydrogenaspartate) reduces cytokine liberation, apoptosis, and mortality rate in a rat model of LD100 endotoxemia; Klosterhalfen B et al.; A prospective, randomized model of LD100/24 h endotoxemia was performed in male Wistar rats (n = 26; 250-300 g) . The animals were divided into four groups: Group I (n = 5; saline treatment only), Group II (n = 5; Zn2+ treatment only), Group III (n = 8; saline pretreatment, lipopolysaccharide (LPS) treatment), and Group IV (n = 8; Zn2+ pretreatment, LPS treatment) . Zn2+ pretreatment was carried out by intraperitoneal injection of 50 mg/kg zinc-bis-(DL-hydrogenaspartate) (10 mg/kg Zn2+) . LD100/24 h endotoxemia was induced by intraperitoneal administration of 20 mg/kg LPS of the Escherichia coli strain WO111:B4 . Tumor necrosis factor alpha, interleukin-1 beta, and interleukin-6 were detected by enzyme-linked immunosorbent assay (ELISA) . HSP70 expression in the lungs, the liver, and the kidneys was determined by immunohistochemistry, Western blotting, and an HSP70 ELISA . Apoptosis was also detected by an in situ apoptosis detection kit (TUNEL) and a cell death detection ELISA, respectively . This rat model of endotoxemia proves the close relationship between HSP70 expression, cytokine liberation, and development of apoptosis . The data demonstrate that: 1) Zn2+ is a potent inducer of HSP70 expression; 2) the application of Zn2+ leads to slightly increased cytokine plasma levels; and 3) the manipulation of the heat shock response by Zn2+ significantly increases the survival rate after LD100 endotoxemia . Enhanced survival rate in animals pretreated with Zn2+ may be explained by increased tissue levels of HSP70, a subsequent significantly decreased liberation of the proinflammatory cytokines after LPS challenge, and a significantly decreased rate of apoptosis.

FEBS Lett, 1997 Apr 1, 405(3), 267 - 72
Post-translational modification of heterologously expressed Streptomyces type II polyketide synthase acyl carrier proteins; Cox RJ et al.; Expression in Escherichia coli of Streptomyces acyl carrier proteins (ACPs) associated with polyketide biosynthesis using the pT7-7 expression system of Tabor and Richardson led to the production predominantly of inactive apo-proteins lacking the 4'-phosphopantetheinyl prosthetic group essential for polyketide synthase activity . Modification of growth conditions led to an increase of production of active holo-protein for the actinorhodin (act) ACP, but this technique was ineffective for oxytetracycline (otc) and griseusin (gris) ACPs . Labelling experiments revealed that a low level of otc ACP expressed prior to induction was produced mainly as active holo-protein, while post-induction 15N-labelled protein was almost exclusively in the apo-ACP form . Limiting endogenous holo-acyl carrier protein synthase (ACPS) concentration was implicated as responsible for low apo-ACP to holo-ACP conversion, rather than limiting substrate (coenzyme A) and cofactor (Mg2+) concentrations . Co-expression of act and gris ACPs with ACPS in E . coli led to high levels of production of active holo-ACPs and ACPS . We have also made the significant observation that ACPS is able to transfer acylated CoA moieties to act apo-ACP.

FEBS Lett, 1997 Apr 1, 405(3), 260 - 2
Ligands regulate GroEL thermostability; Surin AK et al.; Escherichia coli heat-shock proteins GroEL and GroES stimulate (in an ATP-dependent manner) the folding of various proteins . In this study scanning microcalorimetry was applied to investigate GroEL thermostability in the presence of its ligands . Mg2+ and K+ ions stabilize while ADP destabilizes the GroEL molecule against the action of temperature . Furthermore, ADP essentially increases the number of binding sites for the hydrophobic probe (ANS) and the number of GroEL SH-groups accessible to Ellman's reagent as well as the accessibility of the protein to the action of trypsin . The interaction of GroEL with GroES in the presence of Mg2+-ADP eliminates the destabilizing effect of ADP on the GroEL molecule against the action of temperature and Ellman's reagent but does not change its hydrophobicity and accessibility to trypsin.

Curr Genet, 1997 Apr, 31(4), 302 - 7
Oxa1p, which is required for cytochrome c oxidase and ATP synthase complex formation, is embedded in the mitochondrial inner membrane; Kermorgant M et al.; We have previously isolated the yeast nuclear gene OXA1 and showed that Oxa1p is required for the formation of the cytochrome c oxidase and ATP synthase complexes . We have expressed Oxa1p in E . coli and shown that it is toxic and rapidly degraded . Nevertheless, a truncated protein was successfully expressed and antibodies have been raised against this truncated protein . These antibodies recognise a protein in mitochondrially enriched fractions . In vitro mitochondrial import experiments demonstrate that the import of Oxa1p is accompanied by the cleavage of a long pre-sequence . Osmotic swelling and alkaline carbonate extraction show that Oxa1p is an integral membrane protein located in the inner membrane of mitochondria . The relationships between the sub-mitochondrial location and the function of Oxa1p are discussed.

Immunol Cell Biol, 1997 Apr, 75(2), 217 - 21
Comparison of strategies for the construction of libraries of artificial antibodies; Iba Y et al.; Construction of libraries of artificial antibodies has been reported by several groups of investigators . Various forms of antibody fused to surface protein, cpII, are expressed on the surface of filamentous phage . Since phages that encode desired antibodies can be easily grown in Escherichia coli after selection with target antigens, the phage-display antibody system appears to be very useful for various biological purposes . In this brief review, recent progress in research into the production of artificial antibodies is summarized and the strategies used for the construction of libraries of artificial antibodies are compared.

Parasitology, 1997 Apr, 114 ( Pt 4), 395 - 406
Production of a monoclonal antibody specific for ovine immunoglobulin E and its application to monitor serum IgE responses to Haemonchus contortus infection; Kooyman FN et al.; Part of the C epsilon 3-C epsilon 4 region of the ovine immunoglobulin E (IgE) gene (nucleotides 1111-1575) was amplified by PCR . The recombinant protein (recIgE1-2) was expressed in E . coli and both monoclonal and polyclonal antibodies were produced . These antibodies recognized recIgE1-2 and native IgE on Western blots and in ELISA . The polyclonal serum showed cross-reactivity with other sheep immunoglobulin classes . The monoclonal antibody was specific for ovine IgE and goat IgE . Infection of sheep with the abomasal nematode Haemonchus contortus resulted in elevated IgE levels in serum 2-4 weeks after infection, as measured by sandwich ELISA using the rabbit polyclonal as capture antibody and the monoclonal antibody against ovine IgE as second antibody . A negative correlation between worm counts and total serum IgE levels at the end of the experiment was found in repeatedly infected sheep . Significant increased levels of excretory-secretory antigens specific IgE levels were found after H . contortus infection . In contrast, no significant changes in 3rd-stage larvae (L3) antigen-specific IgE titre in sera could be detected after infection.

Mol Pharmacol, 1997 Apr, 51(4), 576 - 82
Molecular cloning and expression of a 2-arylpropionyl-coenzyme A epimerase: a key enzyme in the inversion metabolism of ibuprofen; Reichel C et al.; The 2-arylpropionic acid derivatives, including ibuprofen, are the most widely used anti-inflammatory analgesic cyclooxygenase inhibitors . The (-)-R-enantiomer, which is inactive in terms of cyclooxygenase inhibition, is epimerized in vivo via the 2-arylpropionyl-coenzyme A (CoA) epimerase to the cyclooxygenase-inhibiting (+)-S-enantiomer . The molecular biology of the epimerization pathway is largely unknown . To clarify this mechanism, the sequence of the 2-arylpropionyl-CoA epimerase was identified, and the enzyme cloned and expressed . A cDNA clone encoding the 2-arylpropionyl-CoA epimerase was isolated from a rat liver cDNA library . The nucleotide and the deduced amino acid sequence of this enzyme was determined . Significant amino acid sequence similarity was found between the rat epimerase and carnitine dehydratases from Caenorhabditis elegans (41%) and Escherichia coli (27%) . A bacterial expression system (E . coli strain M15{pREP4}) was used to express the epimerase protein, representing up to 20-30% of the total cellular E . coli protein . The expression of the epimerase was confirmed with Western blots using specific anti-epimerase antibodies and by measuring the rate of inversion of (R)-ibuprofenoyl-CoA . Northern blot analysis revealed a prominent 1.9-kb mRNA transcript in different rat tissues . In addition to its obvious importance in drug metabolism, the homology of the epimerase with carnitine dehydratases from several species suggests that this protein, which up to now has only been characterized as having a role in drug transformation, has a function in lipid metabolism.

Br J Pharmacol, 1997 Apr, 120(7), 1249 - 54
Regulation of guanosine 3':5'-cyclic monophosphate in ovine tracheal epithelial cells; Range SP et al.; 1 . Guanosine 3':5'-cyclic monophosphate (cyclic GMP) is an important second messenger mediating the effects of nitric oxide (NO) and natriuretic peptides . Cyclic GMP pathways regulate several aspects of lung pathophysiology in a number of airway cells . The regulation of this system has not been extensively studied in pulmonary epithelial tissue . 2 . We have studied the production of cyclic GMP by suspensions of ovine tracheal epithelial cells in response to activators of soluble guanylyl cyclase (sodium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP) and particulate guanylyl cyclase (atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP) and E . coli heat stable enterotoxin (STa)) . 3 . Both 10(-7)-10(-3) M and 10(-7)-10(-3) M SNAP generated a concentration-dependent marked elevation in cyclic GMP production when incubated with 10(-3) M 3-isobutyl-l -methylxanthine (IBMX) (both greater than 25 x baseline values with highest drug concentration) . 4 . The increase in production of cyclic GMP in response to 10(-6) M SNP and 10(-5) M SNAP was markedly inhibited by both 5 x 10(-5) M haemoglobin (102% and 92% inhibition) and 5 x 10(-5) M methylene blue (82% and 84% inhibition) . 5 . The increase in cyclic GMP in response to 10(-3) M SNP was measured following co-incubation with the phosphodiesterase inhibitors 10(-7)-10(-3) M IBMX, 10(-7)-10(-4) M milrinone and 10(-7)-10(-4) M SKF 96231 . Only 10(-4)-10(-3) M IBMX significantly increased cyclic GMP levels . 6 . Cyclic GMP production was also significantly elevated from baseline by 10(-5) M ANP, 10(-5) M BNP, 10(-5) M CNP and 200 iu ml-3 of E . coli STa toxin in the presence of 10(-3) M IBMX . Increases with these natriuretic peptides and STa toxin were smaller in magnitude (2-4 fold) than those seen with SNP and SNAP . CNP was the most potent of the natriuretic peptides studied suggesting type B membrane bound guanylate cyclase is the predominant form expressed . 7 . These results suggest that ovine tracheal epithelial cells contain active guanylyl cyclases . The more marked response to SNP and SNAP than to natriuretic peptides suggests that soluble guanylyl cyclase predominates.

Biotechniques, 1997 Apr, 22(4), 744 - 51
Heat-mediated activation of affinity-immobilized Taq DNA polymerase; Nilsson J et al.; A novel strategy for heat-mediated activation of recombinant Taq DNA polymerase is described . A serum albumin binding protein tag is used to affinity-immobilize an E . coli-expressed Taq DNA polymerase fusion protein onto a solid support coated with human serum albumin (HSA) . Analysis of heat-mediated elution showed that elevated temperatures (> 70 degrees C) were required to significantly release the fusion protein from the solid support . A primer-extension assay showed that immobilization of the fusion protein resulted in little or no extension product . In contrast, fusion protein released from the HSA ligand by heat showed high polymerase activity . Thus, a heat-mediated release and reactivation of the Taq DNA polymerase fusion protein from the solid support can be obtained to allow for hot-start PCR with improved amplification performance.

J Parasitol, 1997 Apr, 83(2), 224 - 9
Immunological detection and subcellular localization of Hsp70 and Hsp60 homologs in Trichomonas vaginalis; Bozner P; Heat shock protein (Hsp) homologs of Trichomonas vaginalis belonging to Hsp70 and Hsp60 families with Mr 72 and 58 kDa, respectively, were detected by cross-reacting polyclonal antibody to DnaK of Escherichia coli . polyclonal antibody to Hsp60 of Heliothis virescens, and polyclonal antibody to GroEL/Hsp60 of cyanobacteria . A diffuse and finely granular intracellular staining of T . vaginalis was obtained with anti-DnaK on frozen 4-micron sections of the parasite as revealed by light microscopy . This antibody also recognized T . vaginalis antigen(s) present mainly in the Golgi apparatus, endoplasmic reticulum, and hydrogenosomes, as demonstrated by immunoelectron microscopy . Both antibodies against Hsp60 variants localized a homolog antigen in light microscopic granules, corresponding to hydrogenosomes of T . vaginalis.

Int Arch Allergy Immunol, 1997 Apr, 112(4), 348 - 55
Isolation and characterization of two cDNA clones coding for isoforms of the Parietaria judaica major allergen Par j 1.0101; Duro G et al.; Two cDNA clones named P9* and P1* of 794 and 631 bp, respectively, were isolated from a lambda ZAP cDNA expression library using Parietaria judaica (Pj) pollen-specific IgE antibodies from a pool of sera (n = 23) of patients allergic to Pj . Sequence analysis showed open reading frames of 176 and 138 amino acids . Both clones contain a putative signal peptide giving two mature processed proteins named Par j 1.0102 of 14,726 D and Par j 1.0201 of 10,677 D . These proteins represent isoallergenic forms of the major Pj allergen Par j 1.0101 (clone P5) previously reported . The Par j 1.0102 shared 98% amino acid sequence homology with the P5, while the Par j 1.0201 shared 89% homology . Since P1, P5 and P9 clones were expressed in Escherichia coli, and since the three allergenic proteins shared a very high degree of sequence identity and comparable binding to the Pj-specific IgE, we decided to analyze in more detail the immunological properties of only one allergen, the recombinant Par j 1.0101 . The allergenic activity determined by the histamine release assay ranged between 9 and 56%, depending on the allergic patient analyzed, while it blocked approximately 40% of all the Pj-specific IgE antibodies, as detected after ELISA and cross-absorption analysis.

FEMS Microbiol Lett, 1997 Apr 1, 149(1), 115 - 20
Antigen 43, a phase-variable bipartite outer membrane protein, determines colony morphology and autoaggregation in Escherichia coli K-12; Henderson IR et al.; The product controlling colony form variation and autoaggregation in Escherichia coli K-12 (the flu gene product) has been identified as the phase-variable, bipartite, outer membrane protein, termed antigen 43 (Ag43) . Identification is based: (i) on complete correlation in authentic flu variants between colony morphology/autoaggregation and Ag43 expression as determined by colony and Western immunoblotting and immunofluorescence microscopy; and (ii) on the use of a specific probe to map the gene encoding Ag43 to a position (min 43) on the E . coli chromosome previously established for flu.

FEMS Microbiol Lett, 1997 Apr 1, 149(1), 107 - 13
Growth of Escherichia coli in acetate as a sole carbon source is inhibited by ankyrin-like repeats present in the 2',5'-linked oligoadenylate-dependent human RNase L enzyme; Diaz-Guerra M et al.; Expression of low levels of the 2',5'-linked oligoadenylate-dependent human RNase L, an enzyme induced by interferons, is highly toxic in Escherichia coli . This protein contains an ankyrin domain responsible for RNase L toxicity . The only known ORF in E . coli containing ankyrin repeats is yjaC in the acetate metabolic cluster . We have investigated if expression of mutant forms of RNase L interfere with metabolism of acetate in E . coli . Our findings demonstrate that E . coli expressing RNase L ankyrin repeats is unable to grow in medium containing acetate as the sole carbon source, while it can grow when expressing other domains of the protein . This defect correlates with a severe decrease in the levels of induction of enzymes in the glyoxylate bypass.

FEMS Microbiol Lett, 1997 Apr 1, 149(1), 99 - 105
Virulence patterns from septicemic Escherichia coli O78 strains; Babai R et al.; Several septicemic Escherichia coli O78 strains, isolated from different sources, were characterized phenotypically and genotypically . Two avian isolates, one of which is known to carry the AC/I fimbriae, hybridized with the sfa determinant in colony dot-blot assay . Southern hybridizations with specific sfa probes, following pulsed-field gel electrophoresis (PFGE), showed positive hybridization to the same fragment in each of these strains . Determination of the N-terminal amino acid sequence of the AC/I major subunit gene revealed high similarity to the sequence of the SfaA-II protein . These data suggest that the adhesin gene cluster, coding for AC/I fimbriae, belongs to the S-fimbrial adhesin family.

Am J Vet Res, 1997 Apr, 58(4), 364 - 9
Effects of thermal environment on response to acute peripheral lipopolysaccharide challenge exposure in neonatal pigs; Klir JJ et al.; OBJECTIVE: To evaluate effects of thermal environment on response to acute peripheral lipopolysaccharide (LPS) challenge exposure in neonatal pigs . ANIMALS: 26 neonatal pigs . PROCEDURE: Pigs were assigned to the following treatment groups: 1 warm environment/LPS; 2 warm environment/saline solution; 3 cool environment/LPS; and 4 cool environment/saline solution . For each pig given LPS, 1 littermate of the same sex was given saline solution . Sows with baby pigs were housed in a warm (32 C) or cool (21 C) thermal environment . At 28 days of age, pigs were given 150 micrograms/kg of body weight of Escherichia coli LPS or saline solution intraperitonealy as a control . Rectal temperature and signs of sickness were monitored for 3 hours after LPS administration, when pigs were euthanatized and blood samples were collected to determine serum concentrations of tumor necrosis factor (TNF) alpha and cortisol . To determine in vitro production of TNF alpha, alveolar macrophages were collected by tracheal lavage and incubated for 24 hours at 37 or 41 C, with or without LPS (10 micrograms/ml) . RESULTS: Thermal environment had a significant (P = 0.0004) effect on rectal temperature; LPS administration induced a febrile response (P = 0.0007) only in pigs in the warm environment . All LPS-injected pigs developed signs of endotoxemia; serum TNF alpha and cortisol concentrations were significantly increased (TNF alpha, P = 0.003; cortisol, P = 0.0001); there was no significant in vivo thermal effect on serum TNF alpha and cortisol concentrations . LPS-stimulated alveolar macrophages produced significantly less (P = 0.0086) TNF alpha when incubated at 41 C . CONCLUSIONS: Thermal environment can have a significant impact on the response of neonatal pigs exposed to bacterial endotoxins.

Protein Sci, 1997 Apr, 6(4), 916 - 8
Production, crystallization, and preliminary X-ray analysis of rabbit skeletal muscle troponin complex consisting of troponin C and fragment (1-47) of troponin I; Saijo Y et al.; Troponin is a ternary protein complex consisting of subunits TnC . TnI, and TnT, and plays a key role in calcium regulation of the skeletal and cardiac muscle contraction . In the present study, a partial complex (CI47) was prepared from Escherichia coli-expressed rabbit skeletal muscle TnC and fragment 1-47 of TnI, which is obtained by chemical cleavage of an E . coli-expressed mutant of rabbit skeletal muscle TnI . Within the ternary troponin complex, CI47 is thought to form a core that is resistant to proteolytic digestion, and the interaction within CI47 likely maintains the integrity of the troponin complex . Complex CI47 was crystallized in the presence of sodium citrate . The addition of trehalose improved the diffraction pattern of the crystals substantially . The crystal lattice belongs to the space group P3(1)(2)21, with unit cell dimensions a = b = 48.2 A, c = 162 A . The asymmetric unit presumably contains one CI47 complex . Soaking with p-chloromercuribenzenesulfonate (PCMBS) resulted in loss of isomorphism, but enhanced the quality of the crystals . The crystals diffracted up to 2.3 A resolution, with completeness of 91% and R(merge) = 6.4% . The crystals of PCMBS-derivative should be suitable for X-ray studies using the multiple-wavelength anomalous diffraction technique . This is the first step for elucidating the structure of the full troponin complex.

Protein Sci, 1997 Apr, 6(4), 834 - 42
Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site; Weaver T et al.; Two mutant forms of fumarase C from E . coli have been made using PCR and recombinant DNA . The recombinant form of the protein included a histidine arm on the C-terminal facilitating purification . Based on earlier studies, two different carboxylic acid binding sites, labeled A- and B-, were observed in crystal structures of the wild type and inhibited forms of the enzyme . A histidine at each of the sites was mutated to an asparagine . H188N at the A-site resulted in a large decrease in specific activity, while the H129N mutation at the B-site had essentially no effect . From the results, we conclude that the A-site is indeed the active site, and a dual role for H188 as a potential catalytic base is proposed . Crystal structures of the two mutant proteins produced some unexpected results . Both mutations reduced the affinity for the carboxylic acids at their respective sites . The H129N mutant should be particularly useful in future kinetic studies because it sterically blocks the B-site with the carboxyamide of asparagine assuming the position of the ligand's carboxylate . In the H188N mutation at the active site, the new asparagine side chain still interacts with an active site water that appears to have moved slightly as a result of the mutation.

Protein Sci, 1997 Apr, 6(4), 781 - 8
Remodeling domain interfaces to enhance heterodimer formation; Zhu Z et al.; An anti-p185HER2/anti-CD3 humanized bispecific diabody was previously constructed from two cross-over single-chain Fv in which YH and VL domains of the parent antibodies are present on different polypeptides . Here this diabody is used to evaluate domain interface engineering strategies for enhancing the formation of functional heterodimers over inactive homodimers . A disulfide-stabilized diabody was obtained by introducing two cysteine mutations, VL L46C and VH D101C, at the anti-p185HER2.VL/VH interface . The fraction of recovered diabody that was functional following expression in Escherichia coli was improved for the disulfide-stabilized compared to the parent diabody (> 96% versus 72%), whereas the overall yield was > 60-fold lower . Eleven "knob-into-hole" diabodies were designed by molecular modeling of sterically complementary mutations at the two VL/VH interfaces . Replacements at either interface are sufficient to improve the fraction of functional heterodimer, while maintaining overall recoverable yields and affinity for both antigens close to that of the parent diabody . For example, diabody variant v5 containing the mutations VL Y87A:F98M and VH V37F:L45W at the anti-p185HER2 VL/VH interface was recovered as 92% functional heterodimer while maintaining overall recovered yield within twofold of the parent diabody . The binding affinity of v5 for p185HER2 extracellular domain and T cells is eightfold weaker and twofold stronger than for the parent diabody, respectively . Domain interface remodeling based upon either sterically complementary mutations or interchain disulfide bonds can facilitate the production of a functional diabody heterodimer . This study expands the scope of domain interface engineering by demonstrating the enhanced assembly of proteins interacting via two domain interfaces.

J Bacteriol, 1997 Apr, 179(8), 2769 - 71
Insertional inactivation of dsbA produces sensitivity to cadmium and zinc in Escherichia coli; Rensing C et al.; In a search for genes that produce hypersensitivity to cadmium salts in Escherichia coli, random transposon mutagenesis with TnphoA was used . One of the mutant strains obtained was sensitive to Cd2+ and Zn2+ . Sequence analysis showed that the TnphoA insertion was located in the dsbA gene coding for a periplasmic protein required for disulfide bond formation.

J Bacteriol, 1997 Apr, 179(8), 2678 - 89
Transcription of glutamine synthetase genes (glnA and glnN) from the cyanobacterium Synechocystis sp . strain PCC 6803 is differently regulated in response to nitrogen availability; Reyes JC et al.; In the cyanobacterium Synechocystis sp . strain PCC 6803 we have previously reported the presence of two different proteins with glutamine synthetase activity: GSI, encoded by the glnA gene, and GSIII, encoded by the glnN gene . In this work we show that expression of both the glnA and glnN genes is subjected to transcriptional regulation in response to changes in nitrogen availability . Northern blot experiments and transcriptional fusions demonstrated that the glnA gene is highly transcribed in nitrate- or ammonium-grown cells and exhibits two- to fourfold-higher expression in nitrogen-starved cells . In contrast, the glnN gene is highly expressed only under nitrogen deficiency . Half-lives of both mRNAs, calculated after addition of rifampin or ammonium to nitrogen-starved cells, were not significantly different (2.5 or 3.4 min, respectively, for glnA mRNA; 1.9 or 1.4 min, respectively, for glnN mRNA), suggesting that changes in transcript stability are not involved in the regulation of the expression of both genes . Deletions of the glnA and glnN upstream regions were used to delimit the promoter and the regulatory sequences of both genes . Primer extension analysis showed that structure of the glnA gene promoter resembles those of the NtcA-regulated promoters . In addition, mobility shift assays demonstrated that purified, Escherichia coli-expressed Synechocystis NtcA protein binds to the promoter of the glnA gene . Primer extension also revealed the existence of a sequence related to the NtcA binding site upstream from the glnN promoter . However, E . coli-expressed NtcA failed to bind to this site . These findings suggest that an additional modification of NtcA or an additional factor is required for the regulation of glnN gene expression.

J Bacteriol, 1997 Apr, 179(8), 2616 - 22
Suppressor analysis of mutations in the loop 2-3 motif of lactose permease: evidence that glycine-64 is an important residue for conformational changes; Jessen-Marshall AE et al.; A superfamily of transport proteins, which includes the lactose permease of Escherichia coli, contains a highly conserved motif, G-X-X-X-D/E-R/K-X-G-R/K-R/K, in the loops that connect transmembrane segments 2 and 3 and transmembrane segments 8 and 9 . Previous analysis of this motif in the lactose permease (A . E . Jessen-Marshall, N . J . Paul, and R . J . Brooker, J . Biol . Chem . 270:16251-16257, 1995) has shown that the conserved glycine residue found at the first position in the motif (i.e., Gly-64) is important for transport function . Every substitution at this site, with the exception of alanine, greatly diminished lactose transport activity . In this study, three mutants in which glycine-64 was changed to cysteine, serine, and valine were used as parental strains to isolate 64 independent suppressor mutations that restored transport function . Of these 64 isolates, 39 were first-site revertants to glycine or alanine, while 25 were second-site mutations that restored transport activity yet retained a cysteine, serine, or valine at position 64 . The second-site mutations were found to be located at several sites within the lactose permease (Pro-28 --> Ser, Leu, or Thr; Phe-29 --> Ser; Ala-50 --> Thr, Cys-154 --> Gly; Cys-234 --> Phe; Gln-241 --> Leu; Phe-261 --> Val; Thr-266 --> Iso; Val-367 --> Glu; and Ala-369 --> Pro) . A kinetic analysis was conducted which compared lactose uptake in the three parental strains and several suppressor strains . The apparent Km values of the Cys-64, Ser-64, and Val-64 parental strains were 0.8 mM, 0.7 mM, and 4.6 mM, respectively, which was similar to the apparent Km of the wild-type permease (1.4 mM) . In contrast, the Vmax values of the Cys-64, Ser-64, and Val-64 strains were sharply reduced (3.9, 10.1, and 13.2 nmol of lactose/min x mg of protein, respectively) compared with the wild-type strain (676 nmol of lactose/min x mg of protein) . The primary effect of the second-site suppressor mutations was to restore the maximal rate of lactose transport to levels that were similar to the wild-type strains . Taken together, these results support the notion that Gly-64 in the wild-type permease is at a site in the protein which is important in facilitating conformational changes that are necessary for lactose translocation across the membrane . According to our tertiary model, this site is at an interface between the two halves of the protein.

J Bacteriol, 1997 Apr, 179(8), 2608 - 15
Open reading frame 3, which is adjacent to the mycocerosic acid synthase gene, is expressed as an acyl coenzyme A synthase in Mycobacterium bovis BCG; Fitzmaurice AM et al.; The aim of this study was to test for expression of a 900-bp open reading frame (ORF), ORF3, located at the 5' end of the mycocerosic acid synthase gene in Mycobacterium bovis BCG and to determine the nature of the ORF3 protein . ORF3 was expressed as a 61-kDa C-terminal fusion protein with glutathione S-transferase in Escherichia coli . Polyclonal rabbit antiserum, prepared against this fusion protein, cross-reacted with a 65-kDa protein in M . bovis BCG crude extracts . Since this protein was larger than that predicted from the nucleotide sequence (32 kDa), ORF3 was resequenced, revealing an ORF of 1,749 bp that encodes a 64.8-kDa protein containing 583 amino acids . Reverse transcription-PCR revealed that ORF3 is expressed in M . bovis BCG . The ORF3 product has a high degree of similarity to the acyladenylate family of enzymes . Immunoaffinity absorption chromatography was used to isolate the 65-kDa cross-reacting protein from M . bovis BCG . This purified protein catalyzed coenzyme A (CoA) ester synthesis of n-C10 to n-C18 fatty acids but not mycocerosic acids . ORF3 antibodies severely inhibited acyl-CoA synthase activities of the purified protein and extracts of M . bovis BCG, Mycobacterium smegmatis, and E . coli . They also showed immunological cross-reactivity with proteins in these extracts . Both the ORF3 protein and the acyl-CoA synthase activity were located in the cell cytosol or were loosely associated with the cell membrane . These results indicate that ORF3 encodes an acyl-CoA synthase-like protein.

J Bacteriol, 1997 Apr, 179(8), 2567 - 72
DNA binding by the Xis protein of the conjugative transposon Tn916; Rudy CK et al.; We purified the Xis protein of the conjugative transposon Tn916 and showed by nuclease protection experiments that Xis bound specifically to sites close to each end of Tn916 . These specific binding sites are close to, and in the same relative orientation to, binding sites for the N-terminal domain of Tn916 integrase protein . These results suggest that Xis is involved in the formation of nucleoprotein structures at the ends of Tn916 that help to correctly align the ends so that excision can occur.

J Bacteriol, 1997 Apr, 179(8), 2486 - 93
Mutations in the rpmBG operon of Escherichia coli that affect ribosome assembly; Maguire BA et al.; The rpmBG operon of Escherichia coli codes for ribosomal proteins L28 and L33 . Two strains with mutations in the operon are AM81, whose ribosomes lack protein L28, and AM90, whose ribosomes are without protein L33 . Neither strain showed major defects in ribosome assembly . However, when the mutations were transferred to other strains of E . coli, ribosome synthesis was greatly perturbed and precursor ribonucleoproteins accumulated . In the new backgrounds, the mutation in rpmB was complemented by synthesis of protein L28 from a plasmid; the rpmG mutation was not complemented by protein L33 because synthesis of protein L28 from the upstream rpmB gene was also greatly reduced . The results suggest that protein L33, in contrast to protein L28, has at best a minor role in ribosome assembly and function.

Clin Exp Immunol, 1997 Apr, 108(1), 128 - 37
Induction of oral tolerance with effects on numbers of IgE-carrying mast cells and on bystander suppression in young rats; Dahlman-Hoglund A et al.; The presence of IgE+ mast cells in the small intestine, bystander suppression of DTH and antibody responses to human serum albumin (HSA) were studied in young rats, made tolerant to ovalbumin (OA) by feeding an OA-containing diet for 1-4 weeks starting from weaning, and in sensitized control rats . One week after finishing the OA diet, both groups of rats were immunized with a mixture of OA and HSA in Freund's complete adjuvant (FCA) at one site on the back . The animals were then colonized for 5 days with a genetically manipulated Escherichia coli producing OA . Immunohistochemical staining of the small intestine of the rats fed the OA diet for 4 weeks showed significantly fewer IgE+ mast cells in the lamina propria, a lower level of MHC class antigen was found in the epithelial cells and in the lamina propria, and the villus crypt depth was also significantly less in tolerant compared with sensitized rats (P = 0.003, 0.007, 0.003, respectively) . Sensitized rats showed a mild diarrhoea during the colonization in contrast to tolerant rats . All rats fed OA showed a significantly reduced IgE anti-OA antibody and DTH response to OA before colonization compared with the sensitized rats . Bystander suppression of IgG and IgE anti-HSA antibody responses was also seen, but only in the rats fed OA for either 1 or 4 weeks . Rats fed the OA-containing diet for 1, 3, or 4 weeks showed bystander suppression of the DTH response to HSA . After colonization with E . coli producing OA, rats tolerant to OA after either 1 or 4 weeks on an OA diet maintained tolerance to OA and bystander suppression HSA . These results suggest that oral tolerance to OA down-regulates signs of local inflammatory response by IgE, IgG antibody and T cell responses to OA, but also provides bystander suppression to an unrelated antigen, HSA.

Proc Natl Acad Sci U S A, 1997 Apr 1, 94(7), 3122 - 7
Elevated levels of mutation in multiple tissues of mice deficient in the DNA mismatch repair gene Pms2; Narayanan L et al.; The Pms2 gene has been implicated in hereditary colon cancer and is one of several mammalian homologs of the Escherichia coli mutL DNA mismatch repair gene . To determine the effect of Pms2 inactivation on genomic integrity in vivo, hybrid transgenic mice were constructed that carry targeted disruptions at the Pms2 loci along with a chromosomally integrated mutation reporter gene . In the absence of any mutagenic treatment, mice nullizygous for Pms2 showed a 100-fold elevation in mutation frequency in all tissues examined compared with both wild-type and heterozygous litter mates . The mutation pattern in the nullizygotes was notable for frequent 1-bp deletions and insertions within mononucleotide repeat sequences, consistent with an essential role for PMS2 in the repair of replication slippage errors . Further, the results demonstrate that high rates of mutagenesis in multiple tissues are compatible with normal development and life and are not necessarily associated with accelerated aging . Also, the finding of genetic instability in all tissues tested contrasts with the limited tissue distribution of cancers in the animals, raising important questions regarding the role of mutagenesis in carcinogenesis.

Proc Natl Acad Sci U S A, 1997 Apr 1, 94(7), 2909 - 14
Minus-strand origin of filamentous phage versus transcriptional promoters in recognition of RNA polymerase; Higashitani A et al.; Replication of complementary-strand DNA in filamentous phages is initiated by a primer RNA that is synthesized at the minus-strand origin on the viral single-stranded DNA by Escherichia coli RNA polymerase holoenzyme containing the sigma70 subunit . We have demonstrated that the affinity of RNA polymerase in vitro to the origin is about 16-fold higher than that to the lacUV5 promoter . We have also shown that the temperature dependence of the primer RNA synthesis is much lower than that of lacUV5 transcription . The high affinity of RNA polymerase to the origin depends on the single strandedness of the "-10 region." A nucleotide sequence of the nontemplate strand in the -10 region was found to be important for the function, but that of the template strand was not . These observations suggest that sigma70 subunit directly interacts with the single-stranded nontemplate strand containing adenine residue(s) at the -10 region of promoter.

Proc Natl Acad Sci U S A, 1997 Apr 1, 94(7), 2891 - 6
Lucigenin luminescence as a measure of intracellular superoxide dismutase activity in Escherichia coli; Liochev SI et al.; Lucigenin and paraquat are similar in that each can be taken into Escherichia coli and can then mediate O2.- production by cycles of univalent reduction, to the corresponding monocation radical, followed by autoxidation . Thus, both compounds caused induction of enzymes that are regulated by the soxRS regulon . The lucigenin cation radical has the added property of reacting with O2.-, in a radical-radical addition, to yield an unstable dioxetane, whose decomposition yields light . Superoxide dismutases (SOD), by decreasing {O2.-}, inhibit light production and to the same degree inhibit other O2.(-)-dependent reactions in the cell . Lucigenin luminescence was used to show that the levels of SOD in the parental strain provide approximately 95% protection of all O2.(-)-sensitive targets in E . coli . This degree of protection was so close to the limit of 100% that halving the parental level of {SOD}, or increasing it 5-fold, had only marginal effects on the intensity of lucigenin-dependent luminescence.

Proc Natl Acad Sci U S A, 1997 Apr 1, 94(7), 2880 - 4
A novel arachidonate-preferring acyl-CoA synthetase is present in steroidogenic cells of the rat adrenal, ovary, and testis; Kang MJ et al.; We report herein the cDNA cloning of a novel rat acyl-CoA synthetase (ACS) that preferentially uses arachidonate and eicosapentaenoate . This newly identified ACS (designated ACS4) contains 670 amino acids and is 68% identical to rat ACS3, a previously characterized ACS that is highly expressed in brain . ACS4 was overproduced in Escherichia coli and the resulting enzyme was purified to homogeneity . The purified enzyme utilizes arachidonate and eicosapentaenoate most preferentially among C8-C22 saturated fatty acids and C14-C22 unsaturated fatty acids . Kinetic analyses revealed that the enzyme has a high affinity for arachidonate and eicosapentaenoate and low affinity for palmitate . ACS4 transcripts are detectable in a wide range of tissues, with the highest level in adrenal gland . Immunoreactivity to ACS4 was detected in the zona fasciculata and reticularis of adrenal gland, in the corpus luteum and stromal luteinized cells in ovary, and in the Leydig cells of testis.

Proc Natl Acad Sci U S A, 1997 Apr 1, 94(7), 2843 - 7
The structure of a CAP-DNA complex having two cAMP molecules bound to each monomer; Passner JM et al.; The 2.2 A resolution crystal structure of the Escherichia coli catabolite gene activator protein (CAP) complexed with cAMP and a 46-bp DNA fragment reveals a second cAMP molecule bound to each protein monomer . The second cAMP is in the syn conformation and is located on the DNA binding domain interacting with the helix-turn-helix, a beta-hairpin from the regulatory domain and the DNA (via water molecules) . The presence of this second cAMP site resolves the apparent discrepancy between the NMR and x-ray data on the conformation of cAMP, and explains the cAMP concentration-dependent behaviors of the protein . In addition, this site's close proximity to mutations affecting transcriptional activation and its water-mediated interactions with a DNA recognition residue (E181) and DNA raise the possibility that this site has biological relevance.

Proc Natl Acad Sci U S A, 1997 Apr 1, 94(7), 2828 - 32
Phosphorylation stimulates the cooperative DNA-binding properties of the transcription factor OmpR; Huang KJ et al.; The two-component regulatory proteins OmpR and EnvZ of Escherichia coli K-12 regulate expression of the major outer membrane porin protein, OmpF . OmpR is a DNA-binding protein that is involved in both the positive and negative control of ompF transcription . EnvZ is a histidine kinase that phosphorylates OmpR in response to environmental signals . We used DNA migration retardation analysis to examine the interactions of OmpR and the phosphorylated form of OmpR (OmpR-P) with the regulatory region immediately upstream of the ompF promoter . Our results indicate that the binding of OmpR to this regulatory region is cooperative and that phosphorylation significantly stimulates these cooperative interactions . Moreover, although phosphorylation increases the intrinsic binding of OmpR to a single OmpR-binding site, the primary role of phosphorylation in ompF regulation is to facilitate cooperative interactions between OmpR molecules bound at adjacent sites . Based on these results, we propose a model to explain how the phosphorylation of OmpR could stimulate the occupancy of specific sites in the ompF regulatory region, thereby resulting in the activation or repression of ompF transcription under the appropriate environmental conditions.

Nat Struct Biol, 1997 Apr, 4(4), 298 - 304
The kinetic folding intermediate of ribonuclease H resembles the acid molten globule and partially unfolded molecules detected under native conditions; Raschke TM et al.; Folding of ribonuclease HI from Escherichia coli populates a kinetic intermediate detectable by stopped-flow circular dichroism . Pulse labelling hydrogen exchange reveals that this intermediate consists of a structured core region of the protein, namely helices A and D and beta-strand 4 . This kinetic intermediate resembles both the acid molten globule of ribonuclease HI and rarely populated, partially unfolded forms detected under native conditions . These results indicate that the first portion of ribonuclease HI to fold is the most thermodynamically stable region of the native state, and that folding of this protein follows a hierarchical process.

Am J Pathol, 1997 Apr, 150(4), 1231 - 44
Epitope-mapped monoclonal antibodies as tools for functional and morphological analyses of the human urokinase receptor in tumor tissue; Luther T et al.; uPAR (CD87), the receptor for the urokinase-type plasminogen activator (uPA) facilitates tumor cell invasion and metastasis by focusing uPA proteolytic activity to the cell surface . As uPAR exists in various molecular forms, it is desirable to use well defined antibodies for analyses of uPAR antigen expression in human malignant tumors by immunological methods . Therefore, twelve monoclonal antibodies (MAbs) directed against uPAR were generated by using nonglycosylated, recombinant human uPAR (spanning amino acids 1 to 284), expressed in Escherichia coli, as the immunogen . The reaction pattern of these MAbs with the immunogen and a series of carboxyl-terminally truncated versions of uPAR demonstrated that at least six different epitopes of uPAR are recognized . All MAbs reacted under reducing conditions in immunoblot analyses with E . coli-expressed uPA and also with highly glycosylated, functionally intact, recombinant human uPAR expressed in Chinese hamster ovary (CHO) cells . Seven of the MAbs recognized CHO uPAR under nonreducing conditions as well . By flow cytofluorometric analyses, three of these MAbs were shown to bind to native human uPAR present on the cell surface of monocytoid U937 cells with MAb IIIF10 being the best . Saturation of uPAR with uPA on U937 cells completely blocked interaction of MAb IIIF10 with uPAR (mapped epitope, amino acids 52 to 60 of domain I of uPAR) . In turn, preincubation of U937 cells with MAb IIIF10 efficiently reduced binding of uPA to uPAR, indicating that the epitope detected by MAb IIIF10 is located within or closely to the uPA-binding site of uPAR, and thus, this site may be a target to influence uPA/uPAR-mediated proteolysis in tumors . Binding of MAbs IID7 or IIIB11 (mapped epitope, amino acids 125 to 132 of domain II of uPAR) to uPAR is not affected when uPAR is occupied by uPA . As these MAbs reacted strongly with cellular uPAR antigen in formalin-fixed paraffin-embedded tumor sections, the domain-II-specific antibodies IID7 and IIIB11 may be useful for immunohistochemical studies of uPAR expression in tissue remodeling processes in tumor invasion . In conclusion, we have devised well defined and epitope-mapped MAbs to uPAR that are highly specific tools for detection and targeting of uPAR in tumor tissue.

Curr Opin Struct Biol, 1997 Apr, 7(2), 266 - 72
The ribosome at higher resolution--the donut takes shape; Frank J; Major new results in the 3D cryoimaging of ribosomes have advanced our understanding of ribosomal structure and function . For the first time, 3D difference maps have been used to image tRNA molecules in situ . With this new technology, the stage is set for detailed ligand-binding experiments that explore the binding states of elongation factors and tRNA, and that pinpoint locations of proteins and RNA on the surface of the ribosome.

Genetics, 1997 Apr, 145(4), 877 - 89
Gene replacement with linear DNA fragments in wild-type Escherichia coli: enhancement by Chi sites; Dabert P et al.; During conjugation and transduction of Escherichia coli even numbers of recombinational exchanges are required for replacement of a gene on the circular chromosome . We studied gene replacement using a related method of gene transfer (transformation with 6.5-kb linear DNA fragments) as an experimental model for conjugation and transduction . Two properly situated Chi sites, 5' GCTGGTGG 3', stimulated gene replacement approximately 50-fold, more than the sum of the stimulation by the individual Chi sites . Gene replacement was dependent on RecA and RecB functions . Similar results were obtained with an alternative experimental model in which linear DNA fragments were generated from phage lambda by intracellular EcoRI restriction following infection . Dual Chi site-stimulation of these RecA-, RecB-dependent recombination events thus did not depend upon the mode of delivery of the linear DNA into the cells . A single DNA fragment with two Chi sites was sufficient for gene replacement . These results support a one Chi-one exchange hypothesis ("long chunk" gene replacement), stemming from studies with purified RecBCD enzyme, and argue against models in which Chi converts RecBCD enzyme to a state capable of promoting multiple exchanges on one DNA molecule . These results also provide a method for gene targeting in wild-type E . coli and suggest a method for gene targeting in other organisms.

Genetics, 1997 Apr, 145(4), 867 - 75
Isolation and characterization of suppressors of two Escherichia coli dnaG mutations, dnaG2903 and parB; Britton RA et al.; The dnaG gene of Escherichia coli encodes the primase protein, which synthesizes a short pRNA that is essential for the initiation of both leading and lagging strand DNA synthesis . Two temperature-sensitive mutations in the 3' end of the dnaG gene, dnaG2903 and parB, cause a defect in chromosome partitioning at the nonpermissive temperature 42 degrees . We have characterized 24 cold-sensitive suppressor mutations of these two dnaG alleles . By genetic mapping and complementation, five different classes of suppressors have been assigned; sdgC, sdgD, sdgE, sdgG and sdgH . The genes responsible for suppression in four of the five classes have been determined . Four of the sdgC suppressor alleles are complemented by the dnaE gene, which encodes the enzymatic subunit of DNA polymerase III . The sdgE class are mutations in era, an essential GTPase of unknown function . The sdgG suppressor is likely a mutation in one of three genes: ubiC, ubiA or yjbI . The sdgH class affects rpsF, which encodes the ribosomal protein S6 . Possible mechanisms of suppression by these different classes are discussed.

Radiat Res, 1997 Apr, 147(4), 401 - 8
Strand breaks after the decay of iodine-125 in proximity to plasmid pBR322 DNA; Sahu SK et al.; To elucidate the kinetics of DNA strand breaks caused by low-energy Auger electron emitters in proximity to DNA molecules, we synthesized (125)I-labeled 2-iodoacridine (2-(125)IA), which intercalates with DNA, and 4-iodoacridine (4-(125)IA), which does not . Supercoiled DNA from pBR322 plasmid, labeled with 3H, was purified and incubated with 2-(125)IA or 4-(125)IA in aqueous solution . Reaction mixtures were stored at 4 degrees C to accumulate radiation dose from the decay of (125)I, and DNA was resolved by gel electrophoresis into supercoiled (DNA-I), nicked-circular (DNA-II) and linear (DNA-III) forms, representing undamaged DNA, single-strand breaks (SSBs) and double-strand breaks (DSBs), respectively . Gamma irradiation from an external (137)Cs source led to an exponential decrease in DNA-I with a D0 value of 10.8 +/- 0.3 Gy . Under identical conditions, the D0 values for 2-(125)IA and 4-(125)IA were 22.4 +/- 0.6 x 10(11) disintegrations and 4.7 +/- 0.4 x 10(11) disintegrations, respectively . External gamma irradiation and 4-(125)IA produced SSB/DSB ratios of 26.5 +/- 2.1 and 15.9 +/- 2, respectively, while that for 2-(125)IA was 0.6 . The average number of DSBs from each decay of (125)I was 0.67 for 2-(125)IA and 0.27 for 4-(125)IA . The results indicate that the decay of (125)I bound to a DNA-intercalating compound produces DSBs 2.5-fold more efficiently than (125)I bound to a nonintercalating compound and support the theoretical expectations that predict a DSB yield that is highly dependent on the proximity of the Auger electron emitter to DNA.

Biochemistry, 1997 Apr 1, 36(13), 3981 - 90
Ability of single-site mutants of citrate synthase to catalyze proton transfer from the methyl group of dethiaacetyl-coenzyme A, a non-thioester substrate analog; Kurz LC et al.; The catalytic strategies of enzymes (such as citrate synthase) whose reactions require the abstraction of the alpha-proton of a carbon acid remain elusive . Citrate synthase readily catalyzes solvent proton exchange of the methyl protons of dethiaacetyl-coenzyme A, a sulfur-less, ketone analog of acetyl-coenzyme A, in its ternary complex with oxaloacetate . Because no further reaction occurs with this analog, it provides a uniquely simple probe of the roles of active site interactions on carbon acid proton transfer catalysis . In view of the high reactivity of the analog for proton transfer to the active site base, its failure to further condense with oxaloacetate to form a sulfur-less analog of citryl-coenzyme A was unexpected, although we offer several possible explanations . We have measured the rate constants for exchange, k(exch), at saturating concentrations of the analog for six citrate synthase mutants with single changes in active site residues . Comparisons between the values of k(exch) are straightforward in two limits . If the rate of exchange of the transferred proton with solvent protons is rapid, then k(exch) equals the forward rate constant for proton transfer, and k(exch) values for different mutants compare directly the rate constants for proton transfer . If the exchange of the transferred proton with protons in the bulk solution is the slow step and the equilibrium constant for proton transfer is unfavorable (as is likely), then k(exch) equals the product of the equilibrium constant for proton transfer and the rate constant for exchange of the transferred proton with bulk solvent . If that exchange rate with bulk solution remains constant for a series of mutant enzymes, then k(exch) values compare the equilibrium constants for proton transfer . The importance of the acetyl-CoA site residues, H274 and D375, is confirmed with D375 again implicated as the active site base . The results with the series of oxaloacetate site mutants, H320X, strongly suggest that activation of the first substrate, oxaloacetate, through carbonyl bond polarization, not just oxaloacetate binding in the active site, is required for the enzyme to efficiently catalyze proton transfer from the methyl group of the second substrate.

Biochemistry, 1997 Apr 1, 36(13), 3894 - 902
Inhibition of alpha-lytic protease by pro region C-terminal steric occlusion of the active site; Sohl JL et al.; alpha-Lytic protease, a chymotrypsin-like serine protease, is synthesized with an N-terminal 166 amino acid pro region which is absolutely required for folding of the protease . The pro region is also the most potent inhibitor of the protease known with a Ki of approximately 10(-10) M . Compared to its role in the folding reaction, relatively little is known about the mechanism by which the pro region inhibits the mature protease . While proteinaceous protease inhibitors generally function by occluding the active sites of their respective targets {Bode, W., & Huber, R . (1992) Eur . J . Biochem . 204, 433-451}, the pro region of alpha-lytic protease with its dual roles in folding and inhibition might be expected to show a novel mechanism of inhibition . However, experiments that probe both the structural and enzymatic consequences of pro region binding indicate that the pro region does not measurably distort the protease active site . Instead, the catalytic site is fully functional in the complex . Pro region inhibition of the protease is due to simple steric obstruction; the pro region C-terminus lies in the substrate binding sites of the protease . The implications of these results are discussed with regard to alpha-lytic protease maturation and folding . In addition, the proposed mechanism of alpha-lytic protease pro region inhibition is discussed with respect to data from other pro region families.

Biochemistry, 1997 Apr 1, 36(13), 3755 - 9
Hydrogen exchange/electrospray ionization mass spectrometry studies of substrate and inhibitor binding and conformational changes of Escherichia coli dihydrodipicolinate reductase; Wang F et al.; Escherichia coli dihydrodipicolinate reductase is one of seven enzymes in the succinylase pathway of bacterial L-lysine biosynthesis . The binding of NADH, a substrate, and 2,6-pyridinedicarboxylate, an inhibitor, to the recombinant, overexpressed enzyme has been analyzed using hydrogen/deuterium exchange and electrospray ionization/mass spectrometry . NADH binding reduces the extent of deuterium exchange, as does the subsequent binding of 2,6-pyridinedicarboxylate . Pepsin digestion of the deuterated enzyme and enzyme-inhibitor complex coupled with liquid chromatography/mass spectrometry has allowed the identification of four peptides whose deuterium exchange slows considerably upon the binding of the substrate or inhibitor . Two of these peptides represent regions known or thought to bind NADH and 2,6-pyridinedicarboxylate . Two additional peptides are located at the interdomain hinge region and are proposed to be exchangeable in the "open", catalytically inactive, conformation but are nonexchangeable in the "closed", catalytically active conformation formed after NADH and 2,6-pyridinedicarboxylate binding and domain closure . These studies provide a clear example of a catalytically essential domain movement in this enzyme.

Vet Res Commun, 1997 Apr, 21(3), 187 - 200
Cardiovascular responses to glibenclamide during endotoxaemia in the pig; Vanelli G et al.; The effects of blockading the ATP-sensitive potassium channel (K+ATP channels) on endotoxin-induced vascular derangements was studied . Escherichia coli endotoxin was infused (20 micrograms/kg per h) intravenously for 180 min into anaesthetized, mechanically ventilated, indomethacin-treated pigs . After 150 min of endotoxaemia, glibenclamide (a K+ ATP channel blocker) was infused intravenously at 2 mg/kg per min for 5 min . The cardiovascular parameters were recorded before (control), every 30 min up to 150 min during endotoxaemia, and then at 5, 15 and 30 min after administration of glibenclamide . Infusion of endotoxin reduced the systemic arterial pressure to 60.6% +/- 3.7% (p < 0.01) and increased the pulmonary arterial pressure by 75.9% +/- 11.0% (p < 0.01) of the control values . Within 5 min, infusion of glibenclamide transiently but significantly reversed the systemic hypotension by raising the systemic vascular resistance, whereas the increased pulmonary arterial pressure was further augmented . Glibenclamide infusion did not influence the cardiac output . Within 30 min, the cardiovascular parameters had returned to the values induced by endotoxin, except for the systemic vascular resistance . Infusion of glibenclamide into normal pigs did not change the systemic pressure or resistance, but the pulmonary pressure and resistance were augmented transiently . These data suggest that, in pigs, the ATP-sensitive K+ channels may be one factor playing a role in the vascular changes due to endotoxaemia, especially in the systemic circulation.

Enzyme Microb Technol, 1997 Apr, 20(5), 393 - 400
An efficient process for production of N-acetylneuraminic acid using N-acetylneuraminic acid aldolase; Mahmoudian M et al.; N-acetyl-D-neuraminic acid (Neu5Ac) aldolase (EC 4.1.3.3) has bee reported for synthesis of Neu5Ac,1-5 but there are no reports of processes which do not have significant drawbacks for large-scale operation . Here, Neu5Ac aldolase from an overexpressing recombinant strain of Escherichia coli has been used to develop an immobilized enzyme process for production of Neu5Ac . The enzyme was immobilized onto Eupergit-C and could be reused many times in the reaction . Base-catalyzed epimerization of N-acetyl-D-glucosamine (GlcNAc) yielded GlcNAc/N-acetyl-D-mannosamine (ManNAc) mixtures (c 4:1) which could be used directly in the aldolase reaction; however, inhibition of the enzyme by GlcNAc limited the concentration of ManNAc which could be used in the reaction by this approach . This necessitated the addition of a large molar excess of pyruvate (five- to seven-fold) to drive the equilibrium over to Neu5Ac; nevertheless, a method has been developed to remove the excess pyruvate effectively by complexation with bisulfite, thus allowing Neu5Ac to be recovered by absorption onto an anion-exchange resin . In a second approach, a method has been developed to enrich GlcNAc/ManNAc mixtures for ManNAc . ManNAc can be used at high concentrations in the reaction, thus obviating the need to use a large molar excess of pyruvate . Neu5Ac can be isolated from such reaction mixtures by a simple crystallization . This work shows the importance of integrated process solutions for the effective scale-up of biotransformation reactions.

Biophys J, 1997 Apr, 72(4), 1900 - 7
Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion; Swaminathan R et al.; The green fluorescent protein (GFP) was used as a noninvasive probe to quantify the rheological properties of cell cytoplasm . GFP mutant S65T was purified from recombinant bacteria for solution studies, and expressed in CHO cell cytoplasm . GFP-S65T was brightly fluorescent in solution (lambda ex 492 nm, lambda em 509 nm) with a lifetime of 2.9 ns and a rotational correlation time (tc) of 20 ns . Recovery of GFP fluorescence after photobleaching was complete with a half-time (t1/2) in aqueous saline of 30 +/- 2 ms (5-micron diameter spot), giving a diffusion coefficient of 8.7 x 10(-7) cm2/s . The t1/2 was proportional to solution viscosity and was dependent on spot diameter . In contrast to fluorescein . GFP photobleaching efficiency was not affected by solution O2 content, triplet state quenchers, singlet oxygen scavengers, and general radical quenchers . In solutions of higher viscosity, an additional, rapid GFP recovery process was detected and ascribed to reversible photobleaching . The t1/2 for reversible photobleaching was 1.5-5.5 ms (relative viscosity 5-250), was independent of spot diameter, and was unaffected by O2 or quenchers . In cell cytoplasm, time-resolved microfluorimetry indicated a GFP lifetime of 2.6 ns and a tc of 36 +/- 3 ns, giving a relative viscosity (cytoplasm versus water) of 1.5 . Photobleaching recovery of GFP in cytoplasm was 82 +/- 2% complete with a t1/2 of 83 +/- 6 ms, giving a relative viscosity of 3.2 . GFP translational diffusion increased 4.7-fold as cells swelled from a relative volume of 0.5 to 2 . Taken together with measurements of GFP translation and rotation in aqueous dextran solutions, the data in cytoplasm support the view that the primary barrier to GFP diffusion is collisional interactions between GFP and macromolecular solutes.

J Bacteriol, 1997 Apr, 179(7), 2446 - 8
Characterization of a temperature-sensitive Escherichia coli mutant and revertants with altered seryl-tRNA synthetase activity; Ferri ML et al.; A mutation in the structural gene coding for seryl-tRNA synthetase in temperature-sensitive Escherichia coli K28 has been reported to alter the level of enzyme expression at high temperature (R . J . Hill and W . Konigsberg, J . Bacteriol . 141:1163-1169, 1980) . We identified this mutation as a C-->T transition in the first base of codon 386, resulting in a replacement of histidine by tyrosine . The steady-state levels of serS mRNA in K28 and in the wild-type strains are very similar . Pulse-chase labeling experiments show a difference in protein stability, but not one important enough to account for the temperature sensitivity of K28 . The main reason for the temperature sensitivity of K28 appears to be the low level of specific activity of the mutant synthetase at nonpermissive temperature, not a decreased expression level . Spontaneous temperature-resistant revertants were selected which were found to have about a fivefold-higher level of SerRS than the K28 strain . We identified the mutation responsible for the reversion as being upstream from the -10 sequence in the promoter region . The steady-state levels of serS mRNA in the revertants are significantly higher than that in the parental strain.

J Bacteriol, 1997 Apr, 179(7), 2426 - 32
Purification and characterization of the Streptomyces lividans initiator protein DnaA; Majka J et al.; The Streptomyces lividans DnaA protein (73 kDa) consists, like the Escherichia coli DnaA protein (52 kDa), of four domains . The larger size of the S . lividans protein is due to an additional stretch of 120 predominantly acidic amino acids within domain II . The S . lividans protein was overproduced as a His-tagged fusion protein . The purified protein (isoelectric point, 5.7) has a weak ATPase activity . By DNase I footprinting studies, each of the 17 DnaA boxes (consensus sequence, TTGTCCACA) in the S . lividans oriC region was found to be protected by the DnaA fusion protein . Purified mutant proteins carrying a deletion of the C-terminally located helix-loop-helix (HLH) motif or with amino acid substitutions in helix A (L577G) or helix B (R595A) no longer interact with DnaA boxes . A substitution of basic amino acids in the loop of the HLH motif (R587A or R589A) entailed the formation of S . lividans mutant DnaA proteins with little or no capacity for binding to DnaA boxes . Thus, like in E . coli, the C-terminally located domain IV is absolutely necessary for the specific binding of DnaA . A mutant protein lacking a stretch of acidic amino acids corresponding to domain II is not affected in its DNA binding capacity . Whether the acidic domain II interacts with accessory proteins remains to be elucidated.

J Bacteriol, 1997 Apr, 179(7), 2300 - 4
Expression and control of an operon from an intracellular symbiont which is homologous to the groE operon; Sato S et al.; Members of the genus Buchnera are intracellular symbionts harbored by the aphid bacteriocyte which selectively synthesize symbionin, a homolog of the Escherichia coli GroEL protein, in vivo . Symbionin and SymS, a GroES homolog, are encoded in the symSL operon . Northern blotting and primer extension analyses revealed that the symSL operon invariably gives rise to a bicistronic mRNA under the control of a heat shock promoter, though the amount of the symSL mRNA in the isolated symbiont did not increase in response to heat shock . The sigma32 protein that recognizes the heat shock promoter in E . coli was scarcely detected in Buchnera cells even after heat shock . Although the functionally essential regions of the Buchnera sigma32 protein were well conserved, the Buchnera rpoH gene did not complement an E . coli delta rpoH mutant . On the one hand, the A-T evolutionary pressure imposed on the Buchnera genome may have not only decreased the activity of its sigma32 but also ruined the nucleotide sequences necessary for the expression of rpoH; on the other hand, it may have facilitated expression of the symSL operon without activation by sigma32.

J Bacteriol, 1997 Apr, 179(7), 2259 - 66
In vivo nickel insertion into the carbon monoxide dehydrogenase of Rhodospirillum rubrum: molecular and physiological characterization of cooCTJ; Kerby RL et al.; The products of cooCTJ are involved in normal in vivo Ni insertion into the carbon monoxide dehydrogenase (CODH) of Rhodospirillum rubrum . Located on a 1.5-kb DNA segment immediately downstream of the CODH structural gene (cooS), two of the genes encode proteins that bear motifs reminiscent of other (urease and hydrogenase) Ni-insertion systems: a nucleoside triphosphate-binding motif near the N terminus of CooC and a run of 15 histidine residues regularly spaced over the last 30 amino acids of the C terminus of CooJ . A Gm(r)omega-linker cassette was developed to create both polar and nonpolar (60 bp) insertions in the cooCTJ region, and these, along with several deletions, were introduced into R . rubrum by homologous recombination . Analysis of the exogenous Ni levels required to sustain CO-dependent growth of the R . rubrum mutants demonstrated different phenotypes: whereas the wild-type strain and a mutant bearing a partial cooJ deletion (of the region encoding the histidine-rich segment) grew at 0.5 microM Ni supplementation, strains bearing Gm(r)omega-linker cassettes in cooT and cooJ required approximately 50-fold-higher Ni levels and all cooC insertion strains, bearing polar or nonpolar insertions, grew optimally at 550 microM Ni.

J Bacteriol, 1997 Apr, 179(7), 2194 - 201
Cloning and characterization of an Mn-containing superoxide dismutase (SodA) of Bordetella pertussis; Graeff-Wohlleben H et al.; The Fur titration assay (FURTA) recently developed by I . Stojiljkovic and coworkers (J . Mol . Biol . 236:531-545, 1994) was applied to clone iron-regulated genes of Bordetella pertussis . After sequence analysis, one of the clones obtained by this selection procedure was shown to contain an open reading frame with significant sequence similarities to Mn-containing superoxide dismutases (SodA) . The open reading frame was preceded by a Fur consensus binding site, which according to primer extension analysis overlaps the -10 region of the sodA promoter . Southern blot analysis also revealed the presence of sodA homologous sequences in Bordetella bronchiseptica . On the transcriptional level, sodA expression is strictly iron regulated in both organisms and also in the heterologous host Escherichia coli harboring a plasmid with the sodA gene . Accordingly, SodA-mediated superoxide dismutase activity in Bordetella lysates was detected only after cultivation of the bacteria in iron-restricted media . A B . bronchiseptica fur mutant constitutively expressed SodA, thereby confirming the functional similarity of the iron regulatory systems in the two genera . Apart from iron regulation, sodA expression was affected by changes in DNA topology induced by coumermycin A but not by the global virulence regulatory Bvg system . B . pertussis and B . bronchiseptica sodA deletion mutants did not show significant changes in their growth properties . In contrast, mutation of the previously described Fe-containing SodB enzyme resulted in clones strongly impaired in viability . No direct involvement of SodA in bacterial virulence could be revealed because deletion of the sodA gene affected survival of Bordetella species neither in cultured macrophages nor in a mouse respiratory infection model.

J Bacteriol, 1997 Apr, 179(7), 2163 - 8
Role of the RepA1 protein in RepFIC plasmid replication; Maas R et al.; Using a sensitive primer extension technique, we have carried out studies to localize the start site of replication of the replicon RepFIC . In the course of these studies, we have found evidence that supports the hypothesis that transcription is an integral component of the initiation of replication . On the basis of our findings, we suggest that the transcript is processed to act as a primer, and therefore we propose that the transcript has a dual role as primer of replication and mRNA for the RepA1 protein . We present a model, based on our evidence, for the initiation of replication of the replicon RepFIC . This model provides as well an alternative explanation for what has been called the cis action of RepA1, and we show that RepA1 may act in trans as well as in cis.

J Bacteriol, 1997 Apr, 179(7), 2141 - 6
Effects of sigmaS and the transcriptional activator AppY on induction of the Escherichia coli hya and cbdAB-appA operons in response to carbon and phosphate starvation; Atlung T et al.; The transcriptional regulation of two energy metabolism operons, hya and cbdAB-appA, has been investigated during carbon and phosphate starvation . The hya operon encodes hydrogenase 1, and the cbdAB-appA operon encodes cytochrome bd-II oxidase and acid phosphatase, pH 2.5 . Both operons are targets for the transcriptional activator AppY . In exponential growth, expression of the hya and cbd operons was reduced in an rpoS mutant lacking the RNA polymerase sigmaS factor, and the induction of the two operons by entry into stationary phase in rich medium was strongly dependent on sigmaS . Both operons were induced by carbon starvation, but only induction of the hya operon was dependent on sigmaS, whereas that of the cbd promoter was dependent on AppY . The appY gene also showed sigmaS-dependent induction by carbon starvation . The cbd and hya operons were also found to exhibit a sigmaS-dependent transient twofold induction by osmotic upshift . Like the cbd operon, the hya operon was highly induced by phosphate starvation . For both operons the induction was strongly dependent on AppY . The induction ratio of the two operons was the same in rpoS+ and rpoS mutant strains, indicating that the phosphate starvation-induced increase in sigmaS concentration is not involved in the phosphate regulation of these operons.

J Bacteriol, 1997 Apr, 179(7), 2109 - 15
DNA polymerase I in constitutive stable DNA replication in Escherichia coli; Kogoma T et al.; We examined the effects of mutations in the polA (encoding DNA polymerase I) and polB (DNA polymerase II) genes on inducible and constitutive stable DNA replication (iSDR and cSDR, respectively), the two alternative DNA replication systems of Escherichia coli . The polA25::miniTn10spc mutation severely inactivated cSDR, whereas polA1 mutants exhibited a significant extent of cSDR . cSDR required both the polymerase and 5'-->3' exonuclease activities of DNA polymerase I . A similar requirement for both activities was found in replication of the pBR322 plasmid in vivo . DNA polymerase II was required neither for cSDR nor for iSDR . In addition, we found that the lethal combination of an rnhA (RNase HI) and a polA mutation could be suppressed by the lexA(Def) mutation.

Arch Microbiol, 1997 Apr, 167(4), 209 - 16
Effect of chelating agents and respiratory inhibitors on regulation of the cadA gene in Escherichia coli; Reams SG et al.; The cadA gene that encodes lysine decarboxylase in Escherichia coli is induced by low pH and - during anaerobic growth - by the substrate, lysine . We used operon fusions of cadA to lacZ to investigate the effects of aeration on cadA regulation . When an insertion mutation in osmZ (= hns) was introduced, a cadA-lacZ fusion was derepressed in the presence of air to approximately the same level as seen during anaerobic growth . However, the pH-dependent regulation of cadA was not affected by osmZ . Introduction of mutations in rpoS, fur, or fnr had no significant effect on cadA expression . However, defects in arcB or arcA largely abolished expression of cadA during anaerobic growth . Nonetheless, strains defective in both arcB and osmZ showed the same high cadA-lac expression in air as seen in the single osmZ derivatives . Blocking the respiratory chain with mutations or chemical inhibitors also caused derepression of a cadA-lacZ fusion in air, while agents affecting the proton gradient had no effect . Derepression of cadA in air was also mediated by several chelating agents, in particular by methoxyindole carboxylic acid . Addition of Fe2+ overcame this effect . Chelating agents also abolished the expression during aerobic growth of several genes known to be under arcAB control and which are normally repressed during anaerobic growth but induced in the presence of air . This implies that the effect of chelating agents on cadA expression is mediated via the arcAB regulatory system.

Clin Immunol Immunopathol, 1997 Apr, 83(1), 12 - 4
Safety evaluation of recombinant human interleukin-4 . II . Clinical studies; Leach MW et al.; The safety and tolerability of Escherichia coli-derived recombinant human interleukin-4 (rhuIL-4) have been evaluated in phase I and phase II studies in human patients with a variety of malignancies . Clinical trials have demonstrated that subcutaneous administration of rhuIL-4 is safe and well tolerated at doses as high as 5 micrograms/kg/day and as high as 10 micrograms/kg when administered 3 times/week . Although preclinical safety studies in cynomolgus monkeys demonstrated a number of adverse effects following repeated daily dosing with rhuIL-4, similar effects have generally not been observed in human patients.

Clin Immunol Immunopathol, 1997 Apr, 83(1), 8 - 11
Safety evaluation of recombinant human interleukin-4 . I . Preclinical studies; Leach MW et al.; Recombinant human IL-4 (rhuIL-4) has been evaluated in a series of preclinical studies . These studies have demonstrated that rhuIL-4 is a very potent cytokine with a wide range of pharmacologic and toxicologic effects . Target systems/organs included the cardiovascular system, liver, spleen, and bone marrow . The incidence and severity of effects correlated strongly with both the dose level and the duration of rhuIL-4 administration . The major dose-limiting toxicities identified included death, cardiac inflammation and necrosis, hepatitis, and hepatic necrosis and occurred at sc doses > or = 25 micrograms/kg/day, while a sc dose of 5 micrograms/kg/day was the highest tested that did not result in major dose-limiting toxicity . Clinical trials in humans have demonstrated that sc administration of Escherichia coli-derived rhuIL-4 is safe and well tolerated at doses up to and including 5 micrograms/kg/day and up to 10 micrograms/kg when administered 3 times/week.

J Virol, 1997 Apr, 71(4), 3312 - 8
Expression of a murine leukemia virus Gag-Escherichia coli RNase HI fusion polyprotein significantly inhibits virus spread; VanBrocklin M et al.; The antiviral strategy of capsid-targeted viral inactivation (CTVI) was designed to disable newly produced virions by fusing a Gag or Gag-Pol polyprotein to a degradative enzyme (e.g., a nuclease or protease) that would cause the degradative enzyme to be inserted into virions during assembly . Several new experimental approaches have been developed that increase the antiviral effect of the CTVI strategy on retroviral replication in vitro . A Moloney murine leukemia virus (Mo-MLV) Gag-Escherichia coli RNase HI fusion has a strong antiviral effect when used prophylactically, inhibiting the spread of Mo-MLV and reducing virus titers 1,500- to 2,500-fold . A significant (approximately 100-fold) overall improvement of the CTVI prophylactic antiviral effect was produced by a modification in the culture conditions which presumably increases the efficiency of delivery and expression of the Mo-MLV Gag fusion polyproteins . The therapeutic effect of Mo-MLV Gag-RNase HI polyproteins is to reduce the production of infectious Mo-MLV up to 18-fold . An Mo-MLV Gag-degradative enzyme fusion junction was designed that can be cleaved by the Mo-MLV protease to release the degradative enzyme.

J Virol, 1997 Apr, 71(4), 3069 - 76
Identification, expression, and immunogenicity of Kaposi's sarcoma-associated herpesvirus-encoded small viral capsid antigen; Lin SF et al.; We describe a recombinant antigen for use in serologic tests for antibodies to Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) . The cDNA for a small viral capsid antigen (sVCA) was identified by immunoscreening of a library prepared from the BC-1 body cavity lymphoma cell line induced into KSHV lytic gene expression by sodium butyrate . The cDNA specified a 170-amino-acid peptide with homology to small viral capsid proteins encoded by the BFRF3 gene of Epstein-Barr virus and the ORF65 gene of herpesvirus saimiri . KSHV sVCA was expressed from a 0.85-kb mRNA present late in lytic KSHV replication in BC-1 cells . This transcript was sensitive to phosphonoacetic acid and phosphonoformic acid, inhibitors of herpesvirus DNA replication . KSHV sVCA expressed in mammalian cells or Escherichia coli or translated in vitro was recognized as an antigen by antisera from KS patients . Rabbit antisera raised to KSHV sVCA expressed in E . coli detected a 22-kDa protein in KSHV-infected human B cells . Overexpressed KSHV sVCA purified from E . coli and used as an antigen in immunoblot screening assay did not cross-react with EBV BFRF3 . Antibodies to sVCA were present in 89% of 47 human immunodeficiency virus (HIV)-positive patients with KS, in 20% of 54 HIV-positive patients without KS, but in none of 122 other patients including children born to HIV-seropositive mothers and patients with hemophilia, autoimmune disease, or nasopharyngeal carcinoma . Low-titer antibody was detected in three sera from 28 healthy subjects . Antibodies to recombinant sVCA correlate with KS in high-risk populations . Recombinant sVCA can be used to examine the seroepidemiology of infection with KSHV in the general population.

J Virol, 1997 Apr, 71(4), 2988 - 95
Expression of the human papillomavirus type 11 L1 capsid protein in Escherichia coli: characterization of protein domains involved in DNA binding and capsid assembly; Li M et al.; The L1 major capsid protein of human papillomavirus type 11 (HPV-11) was expressed in Escherichia coli, and the soluble recombinant protein was purified to near homogeneity . The recombinant L1 protein bound DNA as determined by the Southwestern assay method, and recombinant mutant L1 proteins localized the DNA-binding domain to the carboxy-terminal 11 amino acids of L1 . Trypsin digestion of the full-length L1 protein yielded a discrete 42-kDa product (trpL1), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, resulting from cleavage at R415, 86 amino acids from the L1 carboxy terminus . Sucrose gradient sedimentation analysis demonstrated that trpL1 sedimented at 11S, while L1 proteins with amino-terminal deletions of 29 and 61 residues sedimented at 4S . Electron microscopy showed that the full-length L1 protein appeared as pentameric capsomeres which self-assembled into capsid-like particles . The trpL1 protein also had a pentameric morphology but was unable to assemble further . In an enzyme-linked immunosorbent assay, the trpL1 and L1 capsids reacted indistinguishably from virus-like particles purified after expression of HPV-11 L1 in insect cells . The carboxy terminus of L1 therefore constitutes the interpentamer linker arm responsible for HPV-11 capsid formation, much like the carboxy-terminal domain of the polyomavirus VP1 protein . The trypsin susceptibility of HPV-11 L1 capsids suggests a possible mechanism for virion disassembly.

J Virol, 1997 Apr, 71(4), 2722 - 30
Mutational analysis of the adeno-associated virus Rep68 protein: identification of critical residues necessary for site-specific endonuclease activity; Walker SL et al.; The Rep68 and Rep78 proteins of adeno-associated virus type 2 (AAV) are multifunctional proteins which contain overlapping amino acid sequences . They are required for viral replication and preferential integration of the AAV genome into a region of human chromosome 19 . During the terminal resolution process of AAV DNA replication, these proteins make a site-specific and strand-specific endonuclease cut within the AAV inverted terminal repeat DNA . The Rep68 and Rep78 proteins also have helicase and DNA-binding activities . The endonuclease activity is believed to involve the covalent attachment of Rep68 or Rep78 at the cut site via a phosphotyrosine linkage . In an attempt to identify the active-site tyrosine residue of Rep78 and Rep68, tyrosine residues were site specifically mutated to phenylalanines by overlap extension PCR, and the resulting PCR fragments were cloned into a maltose binding protein-Rep68 fusion (MBP-Rep68delta) expression vector . The mutant MBP-Rep68delta proteins were expressed in Escherichia coli cells, purified with amylose resin, and assayed in vitro for Rep68-specific activities . Although several of the mutations disrupted the endonuclease activity, only the mutation of tyrosine 152 abrogated the endonuclease activity with no discernible effect on the helicase or DNA-binding activities . Our data therefore suggest that there are distinct active sites for the helicase and endonuclease activities.

J Virol, 1997 Apr, 71(4), 2666 - 73
The U(L)15 gene of herpes simplex virus type 1 contains within its second exon a novel open reading frame that is translated in frame with the U(L)15 gene product; Baines JD et al.; The U(L)15 gene of herpes simplex virus type 1 is composed of two exons . A mutation previously shown to preclude viral DNA cleavage and packaging at the nonpermissive temperature was identified as a change from a highly conserved serine to proline at codon 653 . Separate viral mutants that contained stop codons inserted into exon I of U(L)15 (designated S648) or an insertion of the Escherichia coli lacZ gene into a truncated U(L)15 exon II {designated HSV-1(delta U(L)15ExII)} were constructed . Recombinant viruses derived from S648 and HSV-1(delta U(L)15ExII) and containing restored U(L)15 genes were constructed and designated S648R and HSV-1(delta U(L)15ExIIR), respectively . Unlike HSV-1(delta U(L)15ExIIR) and S648R, the viruses containing mutant U(L)15 genes failed to cleave and package viral DNA when propagated on noncomplementing cells . As revealed by electron microscopy, large numbers of enveloped capsids lacking viral DNA accumulated within the cytoplasm of cells infected with either S648 or HSV-1(delta U(L)15ExII) but not in cells infected with HSV-1(delta U(L)15ExIIR) or S648R . Thus, one function of the U(L)15 gene is to effectively prevent immature particles lacking DNA from exiting the nucleus by envelopment at the inner lamella of the nuclear membrane . Cells infected with HSV-1(delta U(L)15ExII) did not express the 75,000- or 35,000-apparent-Mr proteins previously shown to be products of the U(L)15 open reading frame, whereas the 35,000-apparent-Mr protein was readily detectable in cells infected with S648 . We conclude that at least the 75,000-Mr protein is required for viral DNA cleavage and packaging and hypothesize that the 35,000-Mr protein is derived from translation of a novel mRNA located partially or completely within the second exon of U(L)15.

J Virol, 1997 Apr, 71(4), 2628 - 35
Specific repression of Tax trans-activation by TAR RNA-binding protein TRBP; Donzeau M et al.; The human T-cell leukemia virus type 1 (HTLV-1)-encoded Tax protein activates transcription from the long terminal repetition via association with host cellular factors . In this study, we searched for cellular proteins that interact with Tax and modulate its activity by using the yeast two-hybrid system . One of the strongest interactors was found to be identical with TRBP, which was previously shown to bind to the RNA encoded by the Tat response element of human immunodeficiency virus type 1 . Interactions are demonstrated with Escherichia coli-expressed proteins in vitro and in mammalian cells, using one- and two-hybrid systems, and with antibodies that coprecipitate Tax and TRBP at physiological TRBP concentrations . Moreover, TRBP, when directed into the cytoplasm, is capable of preventing transport of Tax into the nucleus . A 60-amino-acid polypeptide suffices for binding to Tax . TRBP inhibits activation of transcription by both Tax and GAL4-Tax fusion proteins . Inhibition is specific for Tax and is not seen with the other activators tested . Our data are consistent with the interpretation that TRBP inhibits the interplay of Tax with the transcription machinery or accessory factors.

Nucleic Acids Res, 1997 Apr 1, 25(7), 1405 - 12
RNase YI* and RNA structure studies; Cannistraro VJ et al.; The enzymology of RNase YI*, a recently discovered endoribonuclease from yeast, was studied and compared to other endonucleases for detection of single-strand regions and base pair mismatches in RNA . Its value for RNA structure analyses was assessed with Escherichia coli 5S rRNA as a model substrate . The generally accepted structure of the 5S rRNA is based on thermodynamic energy considerations as well as structures conserved in regions of the molecule during evolution . S1 and mung bean nucleases gave similar results with very marked preference only for the longest single-stranded region in the model . RNase YI* was much more discriminating for detecting unpaired nucleotides as well as short single-strand regions and predicted the generally accepted 5S rRNA structure . Preliminary experiments also indicated that RNase YI* was more sensitive than RNase I for detecting single or multiple base pair mismatches in an RNA-DNA hybrid.

Nucleic Acids Res, 1997 Apr 1, 25(7), 1397 - 404
Codon bias in Escherichia coli: the influence of codon context on mutation and selection; Berg OG et al.; The codon bias in Escherichia coli for all two-fold degenerate amino acids was studied as dependent on the context from the six bases in the nearest surrounding codons . By comparing the results in genes at different expression levels, effects that are due to differences in mutation rates can be distinguished from those that are due to selection . Selective effects on the codon bias is found mostly from the first neighbouring base in the 3'direction, while neighbouring bases further away influence mostly the mutational bias . In some cases it is also possible to identify specific molecular processes, repair or avoidance of frame shift, that lead to the context dependence of the bias.

Nucleic Acids Res, 1997 Apr 1, 25(7), 1390 - 6
Preparation and characterization of a uniformly 2 H/ 15 N-labeled RNA oligonucleotide for NMR studies; Nikonowicz EP et al.; An RNA oligonucleotide that contains the binding site for Escherichia coli ribosomal protein S8 was prepared with uniform 15N isotopic enrichment and uniform deuterium enrichment at all non-exchangeable sites using enzymatic methods . The RNA binding site, which contains 44 nt, forms a hairpin in solution and requires Mg2+for proper folding . The longitudinal magnetization recovery rates of the exchangeable protons were compared for the {2H,15N}-enriched RNA molecule and for the corresponding fully {2H,15N}-enriched RNA hairpin . It was found that 1H-1H dipolar relaxation significantly contributes to the recovery of exchangeable proton longitudinal magnetization . The exchangeable proton resonance line widths were less affected by deuteration, indicating that chemical exchange with H2O remains the dominant mechanism of transverse magnetization relaxation . Nevertheless, deuteration of this RNA hairpin was found to enhance the sensitivity of NOE-based experiments relative to the fully protonated hairpin and to simplify 2D NMR spectra . The increased signal-to-noise ratio facilitated the assignment of the cytidine amino resonances and several of the purine nucleotide amino resonances and permitted the identification of NOE crosspeaks that could not be observed in spectra of the fully protonated RNA hairpin.

Curr Microbiol, 1997 Apr, 34(4), 205 - 11
Serine319 and 321 are functional in isocitrate lyase from Escherichia coli; Rehman A et al.; With site-directed mutagenesis, Ser319 and Ser321 in conserved stretch 3 of tetrameric isocitrate lyase from Escherichiacoli were each substituted with alanine, cysteine, asparagine, or threonine in addition to simultaneous alanine/alanine substitutions . Besides their absolute conservation in all aligned isocitrate lyase sequences, the location of these serine residues, which flank a completely conserved proline, had been suggested in the active site in previous research by studies of photoinactivation of the enzyme by vanadate {Ko et al . (1992) J Biol Chem 267:91} . All substitutions for Ser321 and 319 except by threonine appreciably reduced the kcat of E . coli isocitrate lyase relative to that for wild-type (100) as follows: S319A, 0.4; S319C, 0.05; S319N, 0.01; S319T, 32.3; S321A, 2.9; S321C, 0.3; S321N, 0.1; S321T, 0.3; and S319A/S321A, 0, with little or no effect on the Km for the substrate Mg2+-Ds-isocitrate . The most active variant S319T exhibited threefold less activity than the wild-type enzyme; all variants assembled into tetramers . The S319T mutant isocitrate lyase was 100-fold more active than the S321T variant . This observation suggests that the requirement for a beta-hydroxymethyl group of serine in catalysis is less important at position 319 than at position 321 . Although most singly substituted variants had very low isocitrate lyase activity, all variants harboring mutant isocitrate lyase of very low activity did grow on acetate asa sole carbon source albeit with longer doubling times and lag phases.Substitution of Pro320 by Ala, Asp, Gly, or His was highly detrimental to activity and increased the Km for substrate 3.5- to 8-fold; this suggests that Pro fixes the location of adjacent Ser OH groups and facilitates substrate binding and catalysis . From these collective results, it is proposed that Ser319 and Ser321 are involved in E.coli isocitrate lyase catalysis, perhaps by stabilizing the postulated reaction intermediate succinate trianion in the aci-carboxylate form and the related transition state via hydrogen bonding.

Cell Tissue Res, 1997 Apr, 288(1), 177 - 84
Cellular distribution, levels, and function of the diazepam-binding inhibitor/acyl-CoA-binding protein in last instar Manduca sexta midgut; Snyder MJ et al.; The diazepam-binding inhibitor (DBI) was originally isolated as an endogenous competitor of diazepam for mammalian central nervous system binding sites . Later DBI was found to be identical with an intracellular medium-long chain acyl-CoA-ester-binding protein (ACBP) . Its phylogenetic distribution was also extended outside vertebrates to insects and fungi . We studied DBI/ACBP biochemistry and ultrastructural distribution to learn more about the potential role(s) of the insect protein in lipid transport in the tobacco hornworm, Manduca sexta . We expressed and purfied the M . sexta DBI/ACBP from E . coli for the production of specific polyclonal antisera . We also showed specific binding of {14C}-oleoyl-CoA to the purified protein, supporting its evolutionarily conserved role as an acyl-CoA-binding protein . With the use of an enzyme-linked immunosorbant assay (ELISA) and Northern analysis, it was determined that the M . sexta ACBP is expressed highest during times of active feeding and lipid transport by the larval midgut . Study of the ultrastructural distribution, by immunocytochemistry and electron microscopy, of ACBP in larval midgut showed that acyl-CoA transport is localized throughout the M . sexta columnar cell.

Virology, 1997 Mar 31, 230(1), 1 - 10
Characterization of feline immunodeficiency virus integrase and analysis of functional domains; Shibagaki Y et al.; The wild-type and mutant derivatives of the integrase protein of feline immunodeficiency virus (FIV) were cloned and expressed in Escherichia coli . The purified proteins were examined using various model DNA substrates for their catalytic activities: 3'-end processing, 3'-end joining, and disintegration . The reactions required the presence of either Mn2+ or Mg2+ as a divalent cation . The N-terminal and C-subterminal domains (residues 1-52 and 189-235, respectively) were necessary for 3'-end processing and joining reactions but not for disintegration . Substitution of asparagine for the highly conserved aspartic acid at position 118 resulted in a complete loss of all three activities, confirming that the catalytic domain resides in the central core region (residues 53-188) of the protein . Deletion of the C-terminus (residues 236-281) resulted in a FIV integrase mutant that had efficient 3'-end processing and disintegration activities but weak 3'-end joining activity, a finding that has not been reported previously with other retroviral integrases . The result suggests that the C-terminus is the primary binding site for target DNA . Attachment of a histidine-tag at the N-terminus of the wild-type and deletion derivatives increased the binding affinity to the DNA substrate, resulting in altered levels of catalytic activities and selection of integration sites . Similar to other retroviral integrases, certain pairs of mutant derivatives of FIV integrase could complement each other to restitute 3'-end processing and joining activities, suggesting that formation of functional multimers is a general feature of proteins in the integrase family.

J Chromatogr A, 1997 Mar 28, 765(2), 201 - 6
Purification of monoclonal antibodies by simulated moving-bed chromatography; Gottschlich N et al.; A simulated moving bed (SMB) system has been developed for the biospecific purification of monoclonal antibodies . Adsorption and desorption of the desired product is performed under different conditions . To increase the purity and yield of the antibodies, two purge steps have to be introduced . The steady-state performance of the SMB system was modelled by solving the governing differential equations using a linear driving force approximation . The model parameters were determined independently in batch experiments . They were used to determine the operating conditions of the SMB system for the purification of monoclonal antibodies from cell culture supernatant . The antibodies could be isolated with a yield of > or = 90 . SDS gel electrophoresis of the feed and product stream showed that more than 99% of the contaminating proteins were removed in a single step by SMB chromatography.

Biochim Biophys Acta, 1997 Mar 28, 1319(1), 19 - 58
Catalytic mechanism of F1-ATPase; Weber J et al.; The structure of the core catalytic unit of ATP synthase, alpha 3 beta 3 gamma, has been determined by X-ray crystallography, revealing a roughly symmetrical arrangement of alternating alpha and beta subunits around a central cavity in which helical portions of gamma are found . A low-resolution structural model of F0, based on electron spectroscopic imaging, locates subunit a and the two copies of subunit b outside of a subunit c oligomer . The structures of individual subunits epsilon and c (largely) have been solved by NMR spectroscopy, but the oligomeric structure of c is still unknown . The structures of subunits a and delta remain undefined, that of b has not yet been defined but biochemical evidence indicates a credible model . Subunits gamma, epsilon, b, and delta are at the interface between F1 and F0; gamma epsilon complex forms one element of the stalk, interacting with c at the base and alpha and beta at the top . The locations of b and delta are less clear . Elucidation of the structure F0, of the stalk, and of the entire F1F0 remains a challenging goal.

J Mol Biol, 1997 Mar 28, 267(2), 250 - 63
A simple screen for permissive sites in proteins: analysis of Escherichia coli lac permease; Manoil C et al.; Proteins can be remarkably tolerant of major mutational changes . Sites that accomodate large insertions without loss of function ("permissive" sites) appear generally to correspond to surface regions at which the added sequences do not disrupt overall folding . The identification of such sites can aid in the engineering of functional derivatives of a protein with novel properties . To screen for permissive sites, we developed a simple two-step procedure for generating 31-codon insertions in cloned genes . In a first step, a beta-galactosidase or alkaline phosphatase gene fusion is generated by insertion of a transposon derivative into the target gene . Requiring beta-galactosidase or alkaline phosphatase activity fixes the translational reading frame of the transposon relative to the target gene . In a second step, most of the transposon sequences are excised in vitro, leaving the in-frame insertion . Insertions may be targeted either to cytoplasmic or exported protein sequences, and the inserted sequence acts as an epitope in a variety of proteins . As a test case, a set of 31-codon insertions in the Escherichia coli lac permease gene was generated . The lactose transport activities of the mutant proteins followed a simple pattern: most of the proteins (10/12) with insertions in sequences thought to face the cytoplasm or periplasm were at least partially active, whereas all proteins (9/9) with insertions in membrane-spanning sequences were inactive . The only exceptions were two inactive proteins with insertions in the third cytoplasmic region . Most of the inactive proteins were detected at reduced levels in cells, presumably due to proteolytic breakdown . These studies thus illustrate the use of the new method to identify permissive sites and help document the remarkable sequence flexibility of many of the hydrophilic loops in lac permease . In addition to screening for permissive sites, 31-codon insertion mutagenesis may be useful in epitope-tagging proteins at multiple internal positions, in analyzing membrane protein topology, and in dissecting structure-function relationships in proteins.

MMWR Morb Mortal Wkly Rep, 1997 Mar 28, 46(12), 258 - 61
Foodborne Diseases Active Surveillance Network, 1996; Incorporation of dinitrophenyl protein L23 into totally reconstituted Escherichia coli 50 S ribosomal subunits and its localization at two sites by immune electron microscopy; Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, California 90095-1737, USAEscherichia coli ribosomal protein L23 was derivatized with {3H}2, 4-dinitrofluorobenzene both at the N terminus and at internal lysines . Dinitrophenyl-L23 (DNP-L23) was taken up into 50 S subunits from a reconstitution mixture containing rRNA and total 50 S protein depleted in L23 . Unmodified L23 competed with DNP-L23 for uptake, indicating that each protein form bound in an identical or similar position within the subunit . Modified L23, incorporated at a level of 0.7 or 0.4 DNP groups per 50 S, was localized by electron microscopy of subunits complexed with antibodies to dinitrophenol . Antibodies were seen at two major sites with almost equal frequency . One site is beside the central protuberance, in a region previously identified as the peptidyltransferase center . The second location is at the base of the subunit, in the area of the exit site from which the growing peptide leaves the ribosome . Models derived from image reconstruction show hollows or canyons in the subunit and a tunnel that links the transferase and exit sites . Our results indicate that L23 is at the subunit interior, with separate elements of the protein at the subunit surface at or near both ends of this tunnel.

J Biol Chem, 1997 Mar 28, 272(13), 8686 - 94
Tetramerization and single-stranded DNA binding properties of native and mutated forms of murine mitochondrial single-stranded DNA-binding proteins; Li K et al.; We examined previously unexplored aspects of the tetramerization and single-stranded DNA (ssDNA) binding properties of native, precursor, and mutated forms of mitochondrial ssDNA-binding protein (mtSSB) from a mammalian organism (mouse) . Tetramic forms of mtSSB reassemble spontaneously after thermal denaturation and undergo subunit exchange . Binding of mtSSB to ssDNA as a function of protein concentration is nonlinear, suggesting a concentration-dependent transition in intrinsic binding affinity and in the topology of the DNA-protein complex . The cleavable presequence at the amino terminus of the precursor form of mtSSB does not disrupt tetramer formation but has a specific inhibitory effect on DNA binding that is not seen in a fusion protein that substitutes a bulkier peptide moiety in this position . Mutated forms of mtSSB bearing amino acid substitutions at highly conserved amino acid positions exhibit subtle or severe defects in ssDNA binding activity and/or tetramerization, even when assembled into heterotetramers in combination with wild-type mtSSB monomers . These experiments provide new insights into structural and functional properties of mammalian mtSSB and have implications for the pathogenesis of human diseases resulting from defects in mtDNA replication.

J Biol Chem, 1997 Mar 28, 272(13), 8644 - 52
Nuclease activity of T7 RNA polymerase and the heterogeneity of transcription elongation complexes; Sastry SS et al.; We have discovered that T7 RNA polymerase, purified to apparent homogeneity from overexpressing Escherichia coli cells, possesses a DNase and an RNase activity . Mutations in the active center of T7 RNA polymerase abolished or greatly decreased the nuclease activity . This nuclease activity is specific for single-stranded DNA and RNA oligonucleotides and does not manifest on double-stranded DNAs . Under the conditions of promoter-driven transcription on double-stranded DNA, no nuclease activity was observed . The nuclease attacks DNA oligonucleotides in mono- or dinucleotide steps . The nuclease is a 3' to 5' exonuclease leaving a 3'-OH end, and it degrades DNA oligonucleotides to a minimum size of 3 to 5 nucleotides . It is completely dependent on Mg2+ . The T7 RNA polymerase-nuclease is inhibited by T7 lysozyme and heparin, although not completely . In the presence of rNTPs, the nuclease activity is suppressed but an unusual 3'-end-initiated polymerase activity is unmasked . RNA from isolated pre-elongation and elongation complexes arrested by a psoralen roadblock or naturally paused at the 3'-end of an oligonucleotide template exhibited evidence of nuclease activity . The nuclease activity of T7 RNA polymerase is unrelated to pyrophosphorolysis . We propose that the nuclease of T7 RNA polymerase acts only in arrested or paused elongation complexes, and that in combination with the unusual 3'-end polymerizing activity, causes heterogeneity in elongation complexes . Additionally, during normal transcription elongation, the kinetic balance between nuclease and polymerase is shifted in favor of polymerase.

J Biol Chem, 1997 Mar 28, 272(13), 8611 - 7
The Escherichia coli polB locus is identical to dinA, the structural gene for DNA polymerase II . Characterization of Pol II purified from a polB mutant; Qiu Z et al.; Escherichia coli DNA polymerase II (Pol II) is a member of the group B, "alpha-like" family of DNA polymerases . Pol II is encoded by the damage-inducible dinA gene and exhibits SOS induction under the control of Lex A repressor . The polB gene was originally designated as the structural gene for Pol II based on the absence of detectable Pol II activity in cell lysates prepared from a strain containing the mutant polB100 allele . Because polB and dinA mapped at different chromosomal locations, it remained an open question whether polB, in addition to lexA, might be involved in regulating the expression of Pol II . We have cloned and sequenced the polB100 mutant allele, including adjacent surrounding sequences, and have expressed the mutant dinA gene from Pol B100 on a high copy number plasmid . Our sequence data reveal that polB and dinA represent the same gene and that the original transduction mapping of polB was inaccurate . We purified the mutant Pol B100 polymerase and show that it retains 5 to 10% of the wild-type level of polymerase activity . The Pol B100 mutation, Gly401 --> Asp401, is not located within any of the five conserved domains that define group B polymerases . Pol B100 retains a wild-type level of 3' --> 5' exonuclease activity . We suggest that the normal level of exonucleolytic proofreading associated with the mutant Pol B100 enzyme may explain the repeated failures, over the past two decades, to detect phenotypes in polB mutant strains.

J Biol Chem, 1997 Mar 28, 272(13), 8454 - 8
Cloning of the cDNA and chromosome localization of the gene for human thymidine kinase 2; Johansson M et al.; Human thymidine kinase 2 (TK2) is a deoxyribonucleoside kinase that phosphorylates thymidine, deoxycytidine, and deoxyuridine . The enzyme also phosphorylates anti-viral and anti-cancer nucleoside analogs . We have identified an expressed sequence tag cDNA that encoded a 27.5-kDa protein approximately 30% similar to the human deoxycytidine kinase and deoxyguanosine kinase . The protein was expressed in Escherichia coli and shown to have similar substrate specificity as reported for purified native human TK2 . The recombinant TK2 was shown to phosphorylate the anti-cancer nucleoside analog 2',2'-difluorodeoxycytidine . Northern blot analysis showed two mRNA species at 2.4 and 4.0 kilobases predominantly expressed in liver, pancreas, muscle, and brain . We identified a sequence-tagged site designed from the 3' region of the TK2 cDNA . The sequence-tagged site has been mapped to 81-84 centimorgans from the top linkage group of chromosome 16, which corresponds to the 16q22 region . Our data show that deoxycytidine kinase, deoxyguanosine kinase, and TK2 belong to a family of closely related enzymes . At the time of this report all four of the known human deoxyribonucleoside kinases have been cloned . This provides the opportunity to characterize their individual contribution to therapeutic and toxic effects of nucleoside analogs.

J Biol Chem, 1997 Mar 28, 272(13), 8332 - 9
Repair of O6-benzylguanine by the Escherichia coli Ada and Ogt and the human O6-alkylguanine-DNA alkyltransferases; Goodtzova K et al.; O6-Methylguanine is removed from DNA via the transfer of the methyl group to a cysteine acceptor site present in the DNA repair protein O6-alkylguanine-DNA alkyltransferase . The human alkyltransferase is inactivated by the free base O6-benzylguanine, raising the possibility that substantially larger alkyl groups could also be accepted as substrates . However, the Escherichia coli alkyltransferase, Ada-C, is not inactivated by O6-benzylguanine . The Ada-C protein was rendered capable of reaction by the incorporation of two site-directed mutations converting Ala316 to a proline (A316P) and Trp336 to alanine (W336A) or glycine (W336G) . These changes increase the space at the active site of the protein where Cys321 is buried and thus permit access of the O6-benzylguanine inhibitor . Reaction of the mutant A316P/W336A-Ada-C with O6-benzylguanine was greatly stimulated by the presence of DNA, providing strong support for the concept that binding of DNA to the Ada-C protein activates the protein . The Ada-C protein was able to repair O6-benzylguanine in a 16-mer oligodeoxyribonucleotide . However, the rate of repair was very slow, whereas the E . coli Ogt, the human alkyltransferase, and the mutant A316P/W336A-Ada-C alkyltransferases reacted very rapidly with this 16-mer substrate and preferentially repaired it when incubated with a mixture of the methylated and benzylated 16-mers . These results show that benzyl groups are better substrates than methyl groups for alkyltransferases provided that steric factors do not prevent binding of the substrate in the correct orientation for alkyl group transfer.

J Biol Chem, 1997 Mar 28, 272(13), 8165 - 71
Functional analysis of ribosomal protein L2 in yeast mitochondria; Pan C et al.; The yeast nuclear gene RML2, identified through genomic sequencing of Saccharomyces cerevisiae chromosome V, was shown to encode a mitochondrial homologue of the bacterial ribosomal protein L2 . Immunoblot analysis showed that the mature Rml2p is a 37-kDa polypeptide component of the mitochondrial 54 S large ribosomal subunit . Null mutants of RML2 are respiration-deficient and convert to {rho-} or {rho degrees } cytoplasmic petites, indicating that Rml2p is essential for mitochondrial translation . RML2 is regulated transcriptionally in response to carbon source and the accumulation of Rml2p is dependent on the presence of the 21 S large rRNA . Site-directed mutagenesis showed that a highly conserved 7-amino acid sequence (Val336 to Asp342) of Rml2p is essential for function . Substitution of Gln for His-343, the most highly conserved histidine in the L2 protein family, caused cold-sensitive respiratory growth but did not affect the assembly of 54 S ribosomal subunits . Mitochondrial protein synthesis was normal in the His343 to Gln (H343Q) mutant grown at the permissive temperature (30 degrees C) and was severely impaired after growth at the nonpermissive temperature (18 degrees C) . His343 corresponds to His229 in Escherichia coli L2, which has been implicated in a direct involvement in peptidyl transferase activity . The conditional phenotype of the H343Q mutant indicates that His343 is not essential for peptidyl transferase activity in yeast mitochondria.

Oncogene, 1997 Mar 27, 14(12), 1407 - 17
The tumor suppressor p53 is subject to both nuclear import and export, and both are fast, energy-dependent and lectin-inhibited; Middeler G et al.; Human p53 was expressed in E . coli, purified, labeled with fluorescein iodoacetamide (IAF) and characterized for sequence-specific DNA binding and epitope disposition . Injected into the cytoplasm or nuclei of 3T3 cells IAF-p53 was imported into or exported from nuclei within minutes . Import was inhibited by coinjection of the lectin wheat germ agglutinine (WGA) . In contrast, the peptide-protein conjugate NLS-HSA carrying the nuclear localization sequence (NLS) of the SV40 T antigen was only imported but not exported . 3T3 polykaryons were injected with IAF-p53 and photo-bleached by Scanning Microphotolysis in such a manner that only a single nucleus per polykaryon remained non-bleached . IAF-p53 was found to migrate rapidly (halftime 10 min) from non-bleached into bleached nuclei, while NLS-HSA did not . In digitonin permeabilized cells IAF-p53 was imported into nuclei . When removed from the medium after nuclear accumulation IAF-p53 was exported from the nuclei . Nuclear import and export of IAF-p53 both were rapid (halftimes of a few minutes, 22 C) and strongly inhibited by WGA or incubation on ice . NLS-HSA was only imported but not exported . We conclude that the nucleocytoplasmic transport of p53, in contrast to that of NLS-HSA, is bidirectional and that transport in both directions is carrier mediated and energy dependent . These results suggest that p53 contains nuclear export signals (NES) in addition to import signals (NLS) and thus open new views on the potential regulation of p53 cellular fractions.

Biochem Biophys Res Commun, 1997 Mar 27, 232(3), 682 - 6
Norepinephrine-induced expression of the K99 pilus adhesin of enterotoxigenic Escherichia coli; Lyte M et al.; This study examined whether the provision of norepinephrine, as would be encountered within the highly innervated gastrointestinal system, affected the growth rate of enterotoxigenic Escherichia coli (ETEC) and the expression of the K99 pilus adhesin virulence-related factor . The addition of norepinephrine to serum-containing medium resulted in a 3- to 7-fold increase in the growth rate of the K99+ ETEC strain B44 as compared to growth in vehicle supplemented medium or medium supplemented with normetanephrine, a norepinephrine metabolite that contains one more methyl group than norepinephrine . ELISA analysis revealed that K99 pilus adhesin expression was increased in norepinephrine supplemented culture as compared to normetanephrine and vehicle supplemented controls . This increase occurred from 9 to 15 hours of incubation which represented the exponential growth phase for the norepinephrine supplemented culture . These results indicate that addition of norepinephrine affects both ETEC growth and expression of a specific virulence factor.

Biochem Biophys Res Commun, 1997 Mar 27, 232(3), 678 - 81
Characterization of a cDNA encoding Arabidopsis thaliana inositol 1,3,4-trisphosphate 5/6-kinase; Wilson MP et al.; We have sequenced and recombinantly expressed as a fusion protein an expressed sequence tag clone (GB Z25963) from Arabidopsis thaliana that represents the plant homologue of human inositol 1,3,4 trisphosphate 5/6-kinase . The 1365 base pair clone has an open reading frame of 960 base pairs that predicts a protein product of 36.2 kDa, with a pI of 6.1 . There is no polyadenylation signal or poly (A) tail, suggesting that additional 3' sequence remains to be identified . The amino acid sequence is 30% identical to the human protein . There are several short regions with particularly high degrees of identity between the human and Arabidopsis protein sequences, and these may be useful in identifying the active site of the enzyme . The expressed sequence tag was expressed as a fusion protein in Escherichia coli, with a carboxyl terminal deletion removing one region of high identity between the two proteins . The protein product of this construct was found to have inositol 1,3,4-trisphosphate 5/6-kinase activity . The Arabidopsis enzyme produced both inositol 1,3,4,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate as products in a ratio of 1:3, in contrast with the human enzyme which gives a product ratio of 3:1.

Biochim Biophys Acta, 1997 Mar 27, 1356(1), 71 - 83
Ca2+-dependent membrane bound protein fraction from rabbit gastric mucosa contains a protein whose histidyl residue is phosphorylated; Urushidani T et al.; We found an autophosphorylated protein with a molecular weight of 40 kDa (p40) in the crude annexin fraction of rabbit gastric mucosa, i.e., the materials released by EGTA from the membrane fraction obtained in the presence of Ca2+ . This protein was enriched in chief cells in the gastric glands, and also found in the heart and the liver by Western blotting . The protein bound to phenyl-Sepharose in the presence of Ca2+ and showed extremely basic nature . The phosphorylation site of p40 was considered to be histidyl residue based on the stability to the various agents, the synthesizing activity of ATP from ADP, and the results of phosphoamino acid analysis . The autophosphorylation of p40 was augmented several tenth fold by GDP, Ras, myelin basic protein, or H1 histone at micromolar range . The phosphorylated form was rapidly dephosphorylated in the presence of cold ATP, succinate, and CoA, suggesting that p40 has succinyl-CoA synthetase activity . In fact, a peptide fragment from p40 showed a striking homology with the alpha subunits of succinyl-CoA synthetases from Escherichia coli, Dictyostelium discoideum, and rat liver . These results suggest that p40 is extramitochondrial alpha subunit of succinyl-CoA synthetase or its homologue.

Biochim Biophys Acta, 1997 Mar 27, 1356(1), 53 - 63
Analysis of expression of an alternative La (SS-B) cDNA and localization of the encoded N- and C-terminal peptides; Bachmann M et al.; A deletion of an (A)-residue was detected in a cDNA encoding for the nuclear autoantigen La/SS-B . The cDNA was recently isolated from a cDNA library made from peripheral blood lymphocytes of a patient with primary Sjogren's Syndrome . The region, where the deletion occurred, represents a hot spot region in the La gene(s) . It leads to a frame shift mutation and a premature stop codon eleven amino acids downstream of the deletion site within one of the protease sensitive regions of the La protein . In spite of the frame shift mutation expression of full length La protein occurred efficiently in E . coli . Full length La protein was also made in SF9 cells infected with recombinant baculoviruses, although the efficiency of full length protein production was less . Two major peptides with molecular weights of 29 kDa and 25 kDa were made . The size of these peptides was similar to the known proteolytic degradation products of La protein . The N-terminal 29 kDa fragment containing the RNP consensus sequence located in the cytoplasm . The 25 kDa C-terminal fragment containing the nuclear location signal entered in the nucleus and associated with nuclear speckles . In conclusion, the ability to (i) enter, (ii) remain in the nucleus and (iii) assemble with nuclear speckles resides in the C-terminal domain of La protein and does not depend on the N-terminal RNP-consensus motif.

Mol Gen Genet, 1997 Mar 26, 254(2), 207 - 17
Characterization and mutagenesis of the leucine biosynthetic genes of Azotobacter vinelandii: an analysis of the rarity of amino acid auxotrophs; Manna AC et al.; An 8.7 kb region of the Azotobacter vinelandii chromosome has been analyzed by genetic complementation and nucleotide sequencing . The sequence reveals that leuC and leuD comprise an operon- and leuB is adjacent to leuD . leuA was not detected . Experiments involving lac fusion constructs have confirmed the existence of separate promoters for leuC-leuD and for leuB . Primer extension studies have localized the transcription initiation sites of the leuC-leuD operon and also of the leuB operon . Five more open reading frames showing homology with the Escherichia coli genes yoh1, ibpB, cynR, asd and usg1 have also been found . Auxotrophic mutations are rare in A . vinelandii . We have been able to generate, for the first time, stable mutations in leuB, leuC and leuD by insertion of various gene blocks in vitro and integration by double crossover in vivo . Homogenotization of the mutation into all of the multiple chromosomes of A . vinelandii has been achieved . Evidence has been obtained suggesting the presence of a permease in A . vinelandii capable of leucine transport . Possible reasons for the dearth of auxotrophic mutations in A . vinelandii are discussed.

Mol Gen Genet, 1997 Mar 26, 254(2), 195 - 206
The dnaKJ operon belongs to the sigma32-dependent class of heat shock genes in Bradyrhizobium japonicum; Minder AC et al.; The dnaKJ genes of Bradyrhizobium japonicum were cloned and sequenced . They map adjacent to each other, as in other proteobacteria of the alpha and gamma subgroups . Primer extension experiments identified two strongly heat-inducible transcripts starting 99 bp (T1) and 204 bp (T2) upstream of dnaK . Synthesis of the shorter transcript T1 in Escherichia coli required the presence of a recently characterized sigma32 homologue (RpoH1) from B . japonicum . The -35 and -10 regions of the promoters associated with the transcription start sites T1 and T2 displayed nucleotide sequence motifs that are characteristic for sigma32-dependent promoters in E . coli and alpha-proteobacteria . Heat shock regulation of dnaK expression was confirmed by immunoblot analysis of DnaK protein . All of these results put dnaK into the sigma32-dependent class, not the CIRCE-dependent class, of heat shock genes in B . japonicum . At normal growth temperature dnaK was expressed at a significant basal level . All attempts to eliminate dnaK function by insertion or deletion mutagenesis failed . By contrast, dnaJ null mutants and insertions in the dnaKJ intergenic region were easily obtained . The growth rate of dnaJ mutants was reduced but the final cell density reached in rich medium and their symbiotic properties were indistinguishable from the wild type.

Biochemistry, 1997 Mar 25, 36(12), 3680 - 6
Site-specific recognition by an isolated DNA-binding domain of the sine oculis protein; Hazbun TR et al.; The sine oculis (so) gene is required for the development of the Drosophila visual system . The 416 amino acid SO protein contains a 40 amino acid region homologous to the helix-turn-helix (HtH) region of the homeodomain . Three HtH-containing peptides ranging in size from 63 to 93 amino acids (SO(218-279), SO(204-279), and SO(188-279)) were expressed in Escherichia coli and characterized in vitro . These fragments show circular dichroism spectra characteristic of helical proteins and cooperative unfolding transitions . Derivatization of these three peptides with the chemical nuclease 1,10-phenanthroline:copper (OP-Cu) allowed the identification of specific DNA-binding sites within the 3.1 kb pUC119 plasmid . Similar cleavage patterns with similar relative affinities were obtained for all three peptides . Nucleotide resolution mapping of the predominant cleavage area identified two primary cleavage sites with a similar core sequence . The DNA cleavage sites were confirmed by DNase I footprinting with both native and OP-Cu-conjugated SO HtH peptides . This study identifies a 63 amino acid peptide as sufficient for specific DNA binding.

Biochemistry, 1997 Mar 25, 36(12), 3543 - 53
Physical studies of conformational plasticity in a recombinant prion protein; Zhang H et al.; PrP(Sc) is known to be the major, if not the only, component of the infectious prion . Limited proteolysis of PrP(Sc) produces an N-terminally truncated polypeptide of about 142 residues, designated PrP 27-30 . Recently, a recombinant protein (rPrP) of 142 residues corresponding to the Syrian hamster PrP 27-30 was expressed in Escherichia coli and purified (Mehlhorn et al., 1996) . rPrP has been refolded into both alpha-helical and beta-sheet structures as well as various intermediates in aqueous buffers . The beta-sheet state and two pH-dependent alpha-helical states were characterized by CD and NMR . The alpha-helical conformation occurred only after the formation of an intramolecular disulfide bond, whereas the beta-sheet form was accessible either with or without the disulfide . Of the different alpha-helical forms studied, only those refolded in the pH range 5-8 were substantially soluble at physiological pH, exhibiting similar conformations and monomeric analytical sedimentation profiles throughout the above pH range . Furthermore, refolded alpha-rPrP showed NMR chemical shift dispersion typical of proteins with native conformations, although 2D NMR indicated large segments of conformational flexibility . It displayed a cooperative thermal denaturation transition; at elevated temperatures, it converted rapidly and irreversibly to the thermodynamically more stable beta-sheet form . Unfolding of alpha-rPrP by GdnHCl revealed a two-phase transition with a relatively stable folding intermediate at 2 M GdnHCl . The deltaG values were estimated to be 1.9 +/- 0.4 kcal/mol for the first phase and 6.5 +/- 1.2 kcal/mol for the second, consistent with a folding core surrounded by significant segments of flexible conformation . By NMR, alpha-rPrP(acid) isolated at pH 2 without refolding exhibited heterogeneous line widths, consistent with an acid-denatured molten globular state . We conclude that to the extent that rPrP constitutes a relevant folding domain of PrP(C), the various conformations exhibited by rPrP suggest that the PrP sequence may be intrinsically plastic in its conformations; indeed, portions of PrP(C) may possess a relatively open conformation which makes it susceptible to conversion into PrP(Sc) under appropriate conditions.

Biochemistry, 1997 Mar 25, 36(12), 3506 - 13
Evidence for a recombination-independent pathway for the repair of DNA interstrand cross-links based on a site-specific study with nitrogen mustard; Berardini M et al.; DNA-DNA interstrand cross-links are thought to be important for the cytotoxicity of many chemotherapeutic agents . To study this more definitively, adduct site-specific methods are used to construct a plasmid with a single nitrogen mustard interstrand cross-link (inter-HN2-pTZSV28) . Replication efficiency (RE = {colonies from (inter-HN2-pTZSV28)/(control with no cross-link)}) is approximately 0.3 following transformation into Escherichia coli, implying that the cross-link is repaired . The commonly accepted pathway for cross-link repair, which involves both nucleotide excision repair (NER) and recombination, is ruled out since RE is approximately 0.3 in a delta recA strain . Non-RecA-directed recombination such as copy-choice is also unlikely . However, NER is involved since RE was approximately 0.02 in strains deficient in NER . Base excision repair is not important since RE is approximately 0.3 in strains deficient in 3-methyladenine DNA glycosylases I and II, FAPY DNA glycosylase, both known apurinic/apyrimidinic endonucleases, or DNA deoxyribophosphodiesterase . Another hypothetical repair pathway hinging on a 5' --> 3' exonuclease activity is unlikely since RE is approximately 0.3 in cells deficient in either the 5' --> 3' exonuclease activities of DNA polymerase I, exonuclease VII, or RecJ . Thus, aside from NER, it is unclear what else participates in this recombination-independent repair pathway, although a pathway involing NER followed by replicative bypass of the lesion is the current working hypothesis . Psoralen interstrand cross-links appear not to be repairable by this second pathway, which may have implications for the relative cytotoxicity of interstrand cross-links from different agents.

Biochemistry, 1997 Mar 25, 36(12), 3473 - 82
Design of an active fragment of a class II aminoacyl-tRNA synthetase and its significance for synthetase evolution; Augustine J et al.; Primordial aminoacyl-tRNA synthetases (aaRSs) based on the Rossman nucleotide binding fold of class I enzymes or the seven-stranded antiparallel beta-sheet fold of class II enzymes have been proposed to predate the contemporary aaRS . As part of an inquiry into class II aaRS evolution, the individual domains of the homodimeric Escherichia coli histidyl-tRNA synthetase (HisRS) were separately expressed and purified to determine their individual contributions to catalysis . A 320-residue fragment (Ncat HisRS) truncated immediately following motif 3 catalyzes both the specific aminoacylation of tRNA and pyrophosphate exchange, albeit less efficiently than the full-length enzyme . Ncat HisRS showed no mischarging of noncognate tRNAs but exhibited reduced selectivity for the C73 discriminator base, a principal aminoacylation determinant for histidine tRNAs . Size exclusion chromatography showed that Ncat HisRS is monomeric, indicating that the C-terminal domain is essential for maintaining the dimeric structure of the enzyme . The stably folded C-terminal domain (Cter HisRS) was inactive for both reactions and did not enhance the activity of Ncat HisRS when added in trans . The fusion of one or more accessory domains to a primordial catalytic domain may therefore have been a critical evolutionary step by which aminoacyl-tRNA synthetases acquired increased catalytic efficiency and substrate specificity.

Biochemistry, 1997 Mar 25, 36(12), 3417 - 22
GrpE accelerates nucleotide exchange of the molecular chaperone DnaK with an associative displacement mechanism; Packschies L et al.; The ATP hydrolysis and protein-binding and release cycle of the molecular chaperone DnaK is regulated by the accessory proteins GrpE and DnaJ . Here we describe a study of the formation of complexes between the molecular chaperone DnaK, its nucleotide exchange factor GrpE, and the fluorescent ADP analog N8-{4-{(N'-methylanthraniloyl)amino}butyl}-8-aminoadenosine 5'-diphosphate (MABA-ADP) by equilibrium and stopped flow kinetic experiments . The catalytic cycle of the GrpE-stimulated nucleotide exchange involves a ternary DnaK x GrpE x ADP complex as well as the binary DnaK x GrpE and DnaK x ADP complexes . The equilibrium data of the interaction of GrpE with DnaK x ADP and the nucleotide-free DnaK can be described by a simple equilibrium system where GrpE reduces the affinity of ADP for DnaK 200-fold . However, transient kinetic studies revealed that the functional cycle of GrpE in addition includes at least two distinct ternary DnaK x GrpE x ADP complexes . Our data indicate that the initial weak binding of GrpE to DnaK x ADP is followed by an isomerization of the ternary complex which leads to weakening of nucleotide binding and finally to its rapid dissociation . The maximal stimulation for nucleotide exchange brought about by GrpE was found to be 5000-fold . We propose that this kinetically observed isomerization represents a structural change (opening) of the nucleotide binding pocket of DnaK that allows for fast nucleotide exchange.

Gene, 1997 Mar 25, 188(1), 119 - 22
Functional characterization of a human DNase-like protein encoded by a gene positioned in Xq28; Appierto V et al.; Xib, a gene recently reported to reside on the q28 region of the human X chromosome {Pergolizzi et al . (1996) Gene 168, 267-270}, contains an open reading frame homologous to those of the DNase I family enzymes . The full open reading frame of this gene has been fused to the E . coli gene of the maltose binding protein and expressed in bacteria as a chimeric protein . The partially purified chimeric protein is enzymatically active . It introduces single and double stranded breaks into supercoiled DNA, at 30 degrees C in the absence of divalent cations and at a pH optimum of 5.2 . To our knowledge this enzyme represents the first cloned human endonuclease with characteristics similar to those of acidic DNase II.

Gene, 1997 Mar 25, 188(1), 109 - 13
Cloning and expression of the gene for a protein disulfide oxidoreductase from Azotobacter vinelandii: complementation of an Escherichia coli dsbA mutant strain; Ng TC et al.; The gene for a disulfide oxidoreductase was cloned and sequenced from Azotobacter vinelandii and termed the dsbA locus . The deduced amino acid sequence contains 214 residues with a potential 17-residue signaling sequence on the N-terminal end . This gives the mature protein a calculated molecular mass of 21 799 Da . The A . vinelandii DsbA protein contains the well-conserved motif of C-P-H-C, which is found in the catalytic site of other bacterial DsbA enzymes . The A . vinelandii dsbA gene was expressed in Escherichia coli and was found to be able to complement an E . coli dsbA mutant strain by restoring flagellar and alkaline phosphatase activities . A . vinelandii dsbA mutant strains were impossible to characterize because of the extreme deleterious effect of the mutation . Therefore, the in vivo role of A . vinelandii DsbA is unknown, but it may function to form disulfide bonds and/or be involved in cytochrome biogenesis.

Gene, 1997 Mar 25, 188(1), 101 - 7
The Xenopus Sox3 gene expressed in oocytes of early stages; Koyano S et al.; We have isolated an SRY-type HMG box (Sox) cDNA, XSox3, from a Xenopus immature ovary cDNA library . The XSox3 cDNA contains an open reading frame (ORF) of 309 amino-acid residues, showing 62.7 and 77.0% homology with human and chicken Sox3 proteins, respectively . We also showed that the XSox3 gene is composed of a single exon by sequence analysis of the genomic clone and the determination of the transcription start site of the XSox3 . The XSox3 mRNA was detected only in ovary and was at a higher level in immature ovary than in mature ovary . During oocyte maturation, the XSox3 mRNA was most abundant in stage I oocytes, and the XSox3 protein was detected in stage I and II oocytes . Recombinant XSox3 protein produced in Escherichia coli bound specifically to sequences containing the binding motif for the HMG box of SRY or SOX proteins, AACAAT or AACAAAG, demonstrating its sequence-specific DNA binding property . Taken together, these results indicate that the XSox3 protein may participate in early oogenesis of Xenopus as a transcription factor.

Gene, 1997 Mar 25, 188(1), 41 - 4
Cloning and expression of the cDNA for rat NAD+-dependent 15-hydroxyprostaglandin dehydrogenase; Zhang H et al.; The cDNA for rat NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) was cloned from an intestinal cDNA library . The sequence of this cDNA was found to be identical to that of the reverse transcription-polymerase chain reaction (RT-PCR) product obtained using rat lung RNA as a template . The cDNA contains a 798-bp open reading frame that codes for a protein of 266 amino acids (M(r) 28,775) which shares 88.7% identity with the human 15-PGDH and 92.1% identity with the mouse 15-PGDH protein . The regions that are believed to be the NAD+ binding domain and the active site are conserved in the enzymes from the three different species . However, the sequence of the C-terminal 9 amino acids appears to be significantly different . The authenticity of the rat cDNA was confirmed by the expression of an enzymatically active 15-PGDH in E . coli.

Gene, 1997 Mar 25, 188(1), 23 - 8
Structure of the rice glutaredoxin (thioltransferase) gene; Sha S et al.; We have isolated the gene encoding a glutaredoxin in rice (Oryza sativa L.) and determined the nucleotide (nt) sequence of about a 4.2 kb long . The cloned gene (gRASC8) was found to contain four exons interrupted by three introns . The first exon begins the ATG translation start codon and the four exons code for a protein composed of 112 amino acids . The tetrapeptide -Cys-Pro-Phe-Cys- {-Cys-Pro-Phe(Tyr)-Cys-} which constitutes an active site of Escherichia coli and mammalian glutaredoxins, was conserved . The nt sequence contained consensus TATA and CAAT boxes, and two polyadenylation signals . Southern blot analysis of rice genomic DNA suggests that there are two copies of the glutaredoxin genes in rice.

FEBS Lett, 1997 Mar 24, 405(2), 224 - 8
Production of a soluble and active MBP-scFv fusion: favorable effect of the leaky tolR strain; Chames P et al.; The 6D6 anti-cortisol scFv was prepared as fusion protein with maltose-binding protein (MBP) to increase the amount of soluble product . This fusion was almost completely insoluble when produced in a wild-type strain of Escherichia coli . However, when MBP-scFv fusion was produced in a tolR leaky strain, it was secreted into the culture medium as an active, soluble protein . Production of recombinant proteins in the tolR strain greatly enhances the recovery of active protein and may be a useful system to produce MBP fusion proteins that would normally aggregate when produced in wild-type bacterial strains.

FEBS Lett, 1997 Mar 24, 405(2), 195 - 9
Symmetric GroEL-GroES complexes can contain substrate simultaneously in both GroEL rings; Llorca O et al.; Incubation of rhodanese with hche aperonins GroEL and GroES (1:2 GroEL14:GroES7 molar ratio) under functional and steady state conditions for ATP leads to the formation of a high proportion of rhodanese-bound symmetric complexes (GroEL14(GroES7)2), as revealed by native electrophoresis . Aliquots of such samples were observed under the electron microscope, and the symmetric particles were classified using neuronal networks and multivariate statistical analysis . Three different populations of symmetric particles were obtained which contained substrate in none, one or both GroEL cavities, respectively . The presence of substrate in the symmetric complexes under functional conditions supports their role as active intermediates in the protein folding cycle . These results also suggest that symmetric GroEL-GroES complexes can use both rings simultaneously for folding, probably increasing the efficiency of the reaction.

J Theor Biol, 1997 Mar 21, 185(2), 165 - 71
Allelopathy in Spatially Distributed Populations
Durrett R, Levin S.
In a homogeneously mixing population of E . coli, colicin-producing and colicin-sensitive strategies both may be evolutionarily stable for certain parameter ranges, with the outcome of competition determined by initial conditions . In contrast, in a spatially-structured population, there is a unique ESS for any given set of parameters; the outcome is determined by how effective allelopathy is in relation to its costs . Furthermore, in a spatially-structured environment, a dynamic equilibrium may be sustained among a colicin-sensitive type, a high colicin-producing type, and a "cheater" that expends less on colicin production but is resistant .

Mutat Res, 1997 Mar 21, 374(2), 277 - 85
Evaluation of MutS as a tool for direct measurement of point mutations in genomic DNA; Parsons BL et al.; The MutEx assay is a technique that was developed to detect and map mutations . This assay takes advantage of the Escherichia coli mismatch binding protein MutS, which binds and protects mismatched, heteroduplex DNA from subsequent exonuclease digestion . The plausibility of using the MutEx assay as part of a genotypic selection scheme was investigated . Heteroduplexes were formed between mouse H-ras gene PCR products or restriction fragments that contained wild-type sequence and sequence with a single base change at codon 61 (wild-type, CAA and mutant, AAA) . The heteroduplexes were incubated with MutS and then treated with the exonuclease activity of T7 DNA polymerase . MutS-protected DNA sequences were amplified by PCR . When this method was linked to single nucleotide primer extension (SNuPE) for mutant base identification, original mutant fractions of 1 in 50000 and above were detected . Using comparable DNA template mixtures, the sensitivity of SNuPE alone was 1 in 5 or 1 in 50, depending on the direction of SNuPE priming and the particular base being incorporated . We conclude that the MutEx assay was able to enrich the mutant sequence approximately 1000-fold and, therefore, has considerable potential as a tool for mutation detection.

J Mol Biol, 1997 Mar 21, 267(1), 142 - 9
The thermosome: alternating alpha and beta-subunits within the chaperonin of the archaeon Thermoplasma acidophilum; Nitsch M et al.; The thermosome of the archaeon Thermoplasma acidophilum is composed of two subunits, alpha and beta, which are arranged in two stacked, eight-membered rings . Electron cryo-microscopy in conjunction with image analysis revealed a 4-fold symmetry in the heterooligomeric alpha + beta thermosome isolated from Thermoplasma, but not in the homooligomeric alpha-only thermosome expressed in Escherichia coli . This indicates that alpha and beta-subunits alternate within the rings of the Thermoplasma thermosome rather than forming two different homooligomeric rings . In addition, a small subpopulation of 9-fold symmetric complexes was found among the recombinant alpha-only thermosomes, and a central mass most likely representing bound substrate molecules was observed in about half of the native and recombinant thermosome particles.

Pediatr Surg Int, 1997 Mar 21, 12(2/3), 198 - 9
Cholelithiasis in early infancy
Wilcox DT, Casson D, Bowen J, Thomas A, Bruce J.
This report describes three children, age range 7 weeks to 5 months, who presented with obstructive jaundice secondary to gallstones . Previous Escherichia coli septicaemia and frusemide therapy were predisposing risk factors in two of the patients . All three were successfully treated with cholecystectomy and exploration of the common bile duct.

Biochim Biophys Acta, 1997 Mar 20, 1351(1-2), 239 - 47
Cloning and expression of the rubredoxin gene from Desulfovibrio vulgaris (Miyazaki F)--comparison of the primary structure of desulfoferrodoxin; Kitamura M et al.; A gene encoding rubredoxin from Desulfovibrio vulgaris (Miyazaki F) was cloned and overexpressed in Escherichia coli . A 1.1-kilobase pair DNA fragment, isolated from D . vulgaris (Miyazaki F) by double digestion with SmaI and SalI, contained two genes, the rubredoxin gene (rub) and the desulfoferrodoxin gene (rbo) which was situated upstream of rub . The deduced amino acid sequence of desulfoferrodoxin was homologous to those from other strains and Cys residues that are responsible to bind irons were also conserved . The expression system for rub was constructed under the control of the T7 promoter in E . coli . The purified protein was soluble and had a characteristic visible absorption spectrum . Inductively coupled plasma-atomic emission analysis and electron paramagnetic resonance analysis of the recombinant rubredoxin revealed the presence of an iron ion in a distorted tetrahedral geometry that was the same as native D . vulgaris rubredoxin . In vitro NADH reduction analysis indicated that recombinant rubredoxin was active, and its redox potential was determined as -5 mV.

Biochim Biophys Acta, 1997 Mar 20, 1351(1-2), 231 - 8
Cloning, characterization and expression of a cDNA clone encoding rabbit ubiquitin-conjugating enzyme, E2(32k); Sun B et al.; A cDNA clone encoding rabbit E2(32k) was obtained by library screening and PCR . The cDNA contains an open reading frame coding for 238 amino acids which shows an overall identity of 81% to human CDC34, the cell cycle-related ubiquitin-conjugating enzyme . A 50% homology to yeast CDC34 within the conserved core domain was also observed . Northern blot analysis indicated that three transcripts existed in all six rabbit tissues examined but their expression levels varied over a wide range . The putative cDNA coding region was highly expressed in Escherichia coli as a his-tagged protein which was purified to homogeneity . The ability of this expressed protein to form a thiolester bond with ubiquitin showed that it was functionally active . The ability of this protein to catalyze the conjugation of ubiquitin to histone H2A and H2B was also examined.

Biochim Biophys Acta, 1997 Mar 20, 1351(1-2), 223 - 30
Nucleotide sequence and developmental expression of Acanthamoeba S-adenosylmethionine synthetase gene; Ahn KS et al.; We have isolated and characterized a cDNA (cDNA1) from an Acanthamoeba cDNA library encoding the enzyme S-adenosylmethionine (SAM) synthetase (ATP: L-methionine S-adenosyltransferase; EC 2.5.1.6) . The nucleotide sequence exhibits about 61-73% overall similarity to the corresponding gene of other organisms . The cDNA displays extreme codon bias with a preference for C or G in the third position . A putative initiation site and an ATP-binding site are identified . An amino acid content of 388 and a molecular mass of about 44,000 Daltons are deduced for the enzyme . Putative phosphorylation sites which might be involved in regulation of the enzyme are revealed . The cDNA was expressed in Escherichia coli BL21(DE3), and the identity of the protein product confirmed by Western blotting analysis . Northern analyses of the expression of the Acanthamoeba SAM synthetase gene during development revealed a pronounced reduction in the level of transcripts as amoebae converted to cysts.

Biochim Biophys Acta, 1997 Mar 20, 1351(1-2), 203 - 12
Repair analysis of promutagenic (+)-anti-BPDE DNA adduct in transcriptionally active sequences of plasmid DNA in Escherichia coli; Musarrat J et al.; The extent of formation and repair of promutagenic (+)-anti-BPDE-N2-dG in transcriptionally active thymidine kinase (tk) gene insert and vector DNA fragments was assessed in the (+)-anti-BPDE treated plasmid p220-tk within the Escherichia coli hosts of varying repair potential . Polyclonal antibody (BP1), specific for (+)-anti-BPDE DNA adduct, was utilized for quantitative estimation of this bulky lesion in nanograms amounts of membrane transblotted DNA fragments . A carcinogen dose-dependent quantitative antibody binding response, due to selective recognition of the major (+)-anti-BPDE adduct, was seen with various DNA fragments separated by gel electrophoresis . The sensitivity of the immunodetection at 0.2 fmol (+)-anti-BPDE DNA adduct, allowed a linear detection in the range of modification level of 0.64 x 10(-7) to 86 x 10(-7) adducts per nucleotide in plasmid DNA . Based on this sensitivity, detection of 0.07 and 0.46 (+)-anti-BPDE DNA adducts in respective tk and vector DNA fragments was achieved upon immunoanalysis of the in vitro modified DNA . Adduct concentration dependent antibody binding was independent of size of the vector or insert fragments . Antibody binding response, to DNA modified in vivo, was dependent upon the dose of (+/-)-anti-BPDE to plasmid DNA replicating within bacterial hosts . The repair of (+)-anti-BPDE DNA adducts was determined as the loss of antibody binding sites in the specific fragments of plasmid DNA within host E . coli . About 50% of the initial DNA damage was repaired from the individual fragments during 15 min post-incubation in the repair-proficient (wild-type) E . coli cells . Complete adduct removal occurred in approx . 60 min of post-incubation period . A significant (91%) decrease in the survival of mutant (uvrA- recA-) cells was observed at 4 microM (+/-)-anti-BPDE treatment without any reduction in the colony forming units in the wild-type cells . On the contrary, no repair was seen in the excision repair-deficient (uvrA-) E . coli cells . The results indicate (1) the selectivity of the immunological method and the unique ability of the (+)-anti-BPDE specific antibodies to monitor the direct loss of this promutagenic base lesion from the in vivo modified DNA (2) the role of host excision repair pathway in efficient removal of adducts from bacterial genome determines the survival of the bacterial cells and (3) the repair of (+)-anti-BPDE DNA adducts in episomally replicating, transcriptionally active sequences occur at a rapid rate presumably due to the ease of accessibility of repair enzymes to lesions within DNA.

Biochim Biophys Acta, 1997 Mar 20, 1351(1-2), 192 - 202
Cloning, sequencing, expression and characterization of three anti-estradiol-17beta Fab fragments; Pajunen M et al.; In order provide data for a basic understanding of the mechanisms of antibody specificity and for the design of antibodies with desired properties, we have sequence-analysed three high affinity anti-estradiol-17beta monoclonal antibodies . All three monoclonal antibodies to estradiol-17beta had been raised by conjugation of the 6-carboxymethyloxime derivative to protein carrier . The genes encoding heavy (Fd) and light (L) chains of these three antibodies were cloned and sequenced . The sequenced antibody chains were found to be from 46.0 to 89.7% sequence identical to a monoclonal antibody (DB3) binding a related steroid, progesterone . The Fd and L chains were paired with all possible Fd-L combinations and the corresponding proteins were expressed in Escherichia coli and characterized for their binding (immunoreactivity) to estradiol-17beta . Under the lac promoter and using the pelB signal sequences the production levels of the soluble (total) heavy and light chain Fab fragment combinations in periplasm and in supernatant varied from 115 to 2207 microg/l, while the immunoreactivity percentages (IR%) varied from < 1 to 45% . The production levels and IR% were dependent on the first constant domain subclasses of the heavy chain as well as the Fd-L chain combination expressed.

J Neurol Sci, 1997 Mar 20, 147(1), 13 - 20
2.1 kb 5'-flanking region of the brain type dystrophin gene directs the expression of lacZ in the cerebral cortex, but not in the hippocampus; Kimura S et al.; Duchenne muscular dystrophy is a muscle-wasting disease accompanied by a variable, but often significant degree of mental retardation, possibly due to the absence of dystrophin . However, the function of brain type dystrophin remains insufficiently clear . With this background, in order to study the cell-specific regulation of brain type dystrophin expression in mice, we generated transgenic mice carrying the 2.1 kb 5'-fragment of the mouse brain type dystrophin gene, fused to the coding region of the bacterial lacZ gene . Three transgenic mice lines showed lacZ expression in the cerebral cortex . However, lacZ expression was not detected in the CA region of the hippocampus . These results suggest that the 2.1 kb 5'-fragment of the mouse brain type dystrophin gene contains the regulatory element required for its expression in the cerebral cortex, but not in the hippocampus.

Nature, 1997 Mar 20, 386(6622), 299 - 302
Direct observation of the rotation of F1-ATPase; Noji H et al.; Cells employ a variety of linear motors, such as myosin, kinesin and RNA polymerase, which move along and exert force on a filamentous structure . But only one rotary motor has been investigated in detail, the bacterial flagellum (a complex of about 100 protein molecules) . We now show that a single molecule of F1-ATPase acts as a rotary motor, the smallest known, by direct observation of its motion . A central rotor of radius approximately 1 nm, formed by its gamma-subunit, turns in a stator barrel of radius approximately 5nm formed by three alpha- and three beta-subunits . F1-ATPase, together with the membrane-embedded proton-conducting unit F0, forms the H+-ATP synthase that reversibly couples transmembrane proton flow to ATP synthesis/hydrolysis in respiring and photosynthetic cells . It has been suggested that the gamma-subunit of F1-ATPase rotates within the alphabeta-hexamer, a conjecture supported by structural, biochemical and spectroscopic studies . We attached a fluorescent actin filament to the gamma-subunit as a marker, which enabled us to observe this motion directly . In the presence of ATP, the filament rotated for more than 100 revolutions in an anticlockwise direction when viewed from the 'membrane' side . The rotary torque produced reached more than 40 pN nm(-1) under high load.

Nature, 1997 Mar 20, 386(6622), 239 - 46
Expression cloning of GABA(B) receptors uncovers similarity to metabotropic glutamate receptors; Kaupmann K et al.; GABA (gamma-amino-butyric acid), the principal inhibitory neurotransmitter in the brain, signals through ionotropic (GABA(A)/ GABA(c)) and metabotropic (GABA(B)) receptor systems . Here we report the cloning of GABA(B) receptors . Photoaffinity labelling experiments suggest that the cloned receptors correspond to two highly conserved GABA(B) receptor forms present in the vertebrate nervous system . The cloned receptors negatively couple to adenylyl cyclase and show sequence similarity to the metabotropic receptors for the excitatory neurotransmitter L-glutamate.

Proc Natl Acad Sci U S A, 1997 Mar 18, 94(6), 2705 - 10
Regulation of intestinal uroguanylin/guanylin receptor-mediated responses by mucosal acidity; Hamra FK et al.; Guanylin and uroguanylin are intestinal peptides that stimulate chloride secretion by activating a common set of receptor-guanylate cyclase signaling molecules located on the mucosal surface of enterocytes . High mucosal acidity, similar to the pH occurring within the fluid microclimate domain at the mucosal surface of the intestine, markedly enhances the cGMP accumulation responses of T84 human intestinal cells to uroguanylin . In contrast, a mucosal acidity of pH 5.0 renders guanylin essentially inactive . T84 cells were used as a model epithelium to further explore the concept that mucosal acidity imposes agonist selectivity for activation of the intestinal receptors for uroguanylin and guanylin, thus providing a rationale for the evolution of these related peptides . At an acidic mucosal pH of 5.0, uroguanylin is 100-fold more potent than guanylin, but at an alkaline pH of 8.0 guanylin is more potent than uroguanylin in stimulating intracellular cGMP accumulation and transepithelial chloride secretion . The relative affinities of uroguanylin and guanylin for binding to receptors on the mucosal surface of T84 cells is influenced dramatically by mucosal acidity, which explains the strong pH dependency of the cGMP and chloride secretion responses to these peptides . The guanylin-binding affinities for peptide-receptor interaction were reduced by 100-fold at pH 5 versus pH 8, whereas the affinities of uroguanylin for these receptors were increased 10-fold by acidic pH conditions . Deletion of the N-terminal acidic amino acids in uroguanylin demonstrated that these residues are responsible for the increase in binding affinities that are observed for uroguanylin at acidic pH . We conclude that guanylin and uroguanylin evolved distinctly different structures, which enables both peptides to regulate, in a pH-dependent fashion, the activity of receptors that control intestinal salt and water transport via cGMP.

Proc Natl Acad Sci U S A, 1997 Mar 18, 94(6), 2681 - 6
Hyperresponsive febrile reactions to interleukin (IL) 1alpha and IL-1beta, and altered brain cytokine mRNA and serum cytokine levels, in IL-1beta-deficient mice; Alheim K et al.; IL-1beta is an endogenous pyrogen that is induced during systemic lipopolysaccharide (LPS)- or IL-1-induced fever . We have examined the fever and cytokine responses following i.p . injection of IL-1 agonists, IL-1alpha and IL-1beta, and compared these with response to LPS (i.p.) in wild-type and IL-1beta-deficient mice . The IL-1beta deficient mice appear to have elevated body temperature but exhibit a normal circadian temperature cycle . Exogenously injected IL-1beta, IL-1alpha, or LPS induced hyperresponsive fevers in the IL-1beta-deficient mice . We also observed phenotypic differences between wild-type and IL-1beta-deficient mice in hypothalamic basal mRNA levels for IL-1alpha and IL-6, but not for IL-1beta-converting enzyme or IL-1 receptor type I or type II . The IL-1alpha mRNA levels were down-regulated, whereas the IL-6 mRNA levels were up-regulated in the hypothalamus of IL-1beta-deficient mice as compared with wild-type mice . The IL-1beta-deficient mice also responded to LPS challenge with significantly higher serum corticosterone and with lower serum tumor necrosis factor type alpha levels than the wild-type mice . The data suggest that, in the redundant cascade of proinflammatory cytokines, IL-1beta plays an important but not obligatory role in fever induction by LPS or IL-1alpha, as well as in the induction of serum tumor necrosis factor type alpha and corticosterone responses either by LPS or by IL-1alpha or IL-1beta.

Proc Natl Acad Sci U S A, 1997 Mar 18, 94(6), 2626 - 31
A genetic system for Archaea of the genus Methanosarcina: liposome-mediated transformation and construction of shuttle vectors; Metcalf WW et al.; New methods that allow, for the first time, genetic analysis in Archaea of the genus Methanosarcina are presented . First, several autonomously replicating plasmid shuttle vectors have been constructed based on the naturally occurring plasmid pC2A from Methanosarcina acetivorans . These vectors replicate in 9 of 11 Methanosarcina strains tested and in Escherichia coli . Second, a highly efficient transformation system based upon introduction of DNA by liposomes has been developed . This method allows transformation frequencies of as high as 2 x 10(8) transformants per microgram of DNA per 10(9) cells or approximately 20% of the recipient population . During the course of this work, the complete 5467-bp DNA sequence of pC2A was determined . The implications of these findings for the future of methanoarchaeal research are also discussed.

Proc Natl Acad Sci U S A, 1997 Mar 18, 94(6), 2557 - 62
Obesity increases sensitivity to endotoxin liver injury: implications for the pathogenesis of steatohepatitis; Yang SQ et al.; Genetically obese fatty/fatty rats and obese/obese mice exhibit increased sensitivity to endotoxin hepatotoxicity, quickly developing steatohepatitis after exposure to low doses of lipopolysaccharide (LPS) . Among obese animals, females are more sensitive to endotoxin liver injury than males . LPS induction of tumor necrosis factor alpha (TNF alpha), the proven affecter of endotoxin liver injury, is no greater in the livers, white adipose tissues, or sera of obese animals than in those of lean controls . Indeed, the lowest serum concentrations of TNF occur in female obese rodents, which exhibit the most endotoxin-induced liver injury . Several cytokines that modulate the biological activity of TNF are regulated abnormally in the livers of obese animals . After exposure to LPS, mRNA of interferon gamma, which sensitizes hepatocytes to TNF toxicity, is overexpressed, and mRNA levels of interleukin 10, a TNF inhibitor, are decreased . The phagocytic activity of liver macrophages and the hepatic expression of a gene encoding a macrophage-specific receptor are also decreased in obesity . This new animal model of obesity-associated liver disease demonstrates that hepatic macrophage dysfunction occurs in obesity and suggests that this might promote steatohepatitis by sensitizing hepatocytes to endotoxin.

Proc Natl Acad Sci U S A, 1997 Mar 18, 94(6), 2278 - 83
Coexpression of nuclear receptor partners increases their solubility and biological activities; Li C et al.; The biological activities of the retinoids are mediated by two nuclear hormone receptors: the retinoic acid receptor (RAR) and the retinoid-X receptor (RXR) . RXR (and its insect homologue ultraspiracle) is a common heterodimeric partner for many other nuclear receptors, including the insect ecdysone receptor . As part of a continuing analysis of nuclear receptor function, we noticed that, whereas RXR can be readily expressed in Escherichia coli to produce soluble protein, many of its heterodimeric partners cannot . For example, overexpression of RAR results mostly in inclusion bodies with the residual soluble component unable to interact with RXR or ligand efficiently . Similar results are seen with other RXR/ultraspiracle partners . To overcome these problems, we designed a novel double cistronic vector to coexpress RXR and its partner ligand-binding domains in the same bacterial cell . This resulted in a dramatic increase in production of soluble and apparently stable heterodimer . Hormone-binding studies using the purified RXR-RAR heterodimer reveal increased ligand-binding capacity of both components of 5- to 10-fold, resulting in virtually complete functionality . Based on these studies we find that bacterially expressed receptors can exist in one of three distinct states: insoluble, soluble but unable to bind ligand, or soluble with full ligand-binding capacity . These results suggest that coexpression may represent a general strategy for biophysical and structural analysis of receptor complexes.

Proc Natl Acad Sci U S A, 1997 Mar 18, 94(6), 2192 - 7
Evidence for a lipochaperonin: association of active protein-folding GroESL oligomers with lipids can stabilize membranes under heat shock conditions; Torok Z et al.; During heat shock, structural changes in proteins and membranes may lead to cell death . While GroE and other chaperone proteins are involved in the prevention of stress-induced protein aggregation and in the recovery of protein structures, a mechanism for short-term membrane stabilization during stress remains to be established . We found that GroEL chaperonin can associate with model lipid membranes . Binding was apparently governed by the composition and the physical state of the host bilayer . Limited proteolysis of GroEL oligomers by proteinase K, which removes selectively the conserved glycine- and methionine-rich C terminus, leaving the chaperonin oligomer intact, prevented chaperonin association with lipid membranes . GroEL increased the lipid order in the liquid crystalline state, yet remained functional as a protein-folding chaperonin . This suggests that, during stress, chaperonins can assume the functions of assisting the folding of both soluble and membrane-associated proteins while concomitantly stabilizing lipid membranes.

Proc Natl Acad Sci U S A, 1997 Mar 18, 94(6), 2139 - 44
Structural features of the binding site for ribosomal protein S8 in Escherichia coli 16S rRNA defined using NMR spectroscopy; Kalurachchi K et al.; Ribosomal protein S8 of Escherichia coli plays a key role in 30S ribosomal subunit assembly through its interaction with 16S rRNA . S8 also participates in the translational regulation of ribosomal protein expression through its interaction with spc operon mRNA . The binding site for protein S8 within the 16S rRNA encompasses nucleotides G588 to G604 and C634 to C651 and is composed of two base paired helical regions that flank a phylogenetically conserved core element containing nine residues . We have investigated the structure of the rRNA binding site for S8 both in the free state and in the presence of protein using NMR spectroscopy . The integrity of the two helical segments has been verified, and the presence of G597 x C643 and A596 x U644 base pairs within the conserved core, predicted from comparative analysis, have been confirmed . In addition, we have identified a base triple within the core that is composed of residues A595 x (A596 x U644) . The NMR data suggest that S8-RNA interaction is accomplished without significant changes in the RNA . Nonetheless, S8 binding promotes formation of the U598 x A640 base pair and appears to stabilize the G597 x C643 and A596 x U644 base pairs.

Proc Natl Acad Sci U S A, 1997 Mar 18, 94(6), 2128 - 32
Generation of cDNA expression libraries enriched for in-frame sequences; Davis CA et al.; Bacterial cDNA expression libraries are made to reproduce protein sequences present in the mRNA source tissue . However, there is no control over which frame of the cDNA is translated, because translation of the cDNA must be initiated on vector sequence . In a library of nondirectionally cloned cDNAs, only some 8% of the protein sequences produced are expected to be correct . Directional cloning can increase this by a factor of two, but it does not solve the frame problem . We have therefore developed and tested a library construction methodology using a novel vector, pKE-1, with which translation in the correct reading frame confers kanamycin resistance on the host . Following kanamycin selection, the cDNA libraries contained 60-80% open, in-frame clones . These, compared with unselected libraries, showed a 10-fold increase in the number of matches between the cDNA-encoded proteins made by the bacteria and database protein sequences . cDNA sequencing programs will benefit from the enrichment for correct coding sequences, and screening methods requiring protein expression will benefit from the enrichment for authentic translation products.

Biochemistry, 1997 Mar 18, 36(11), 3270 - 7
Altering the reaction coordinate of the ATP sulfurylase-GTPase reaction; Yang M et al.; ATP sulfurylase, isolated from Escherichia coli K-12, catalyzes and couples two reactions: the hydrolysis of GTP and the synthesis of APS (adenosine 5'-phosphosulfate) . Its GTPase activity is regulated in response to ligand binding at the APS-forming active site . In particular, AMP mimics an intermediate-like form of the enzyme that increases the k(cat) for GTP hydrolysis 180-fold . Using equilibrium and pre-steady-state methods, we have determined the relative Gibbs energies for many of the ground and transition states in the GTPase catalytic cycle, in the presence and absence of AMP . GTP and AMP energetically interact throughout the substrate branch of the reaction coordinate; however, once bond breaking occurs, communication between nucleotides ceases . Stopped-flow experiments, using the fluorescent nucleotides 2'-deoxy-mant-GTP and -GDP, indicate that the binding of AMP fosters a conformation of the enzyme that hinders the addition of 2'-deoxy-mant-GTP into the active site without affecting its escaping tendency . These results explain the effects of AMP on the equilibrium binding of the 2'-deoxy-mant-GTP . The second-order rate constants for the binding of 2'-deoxy-mant-GTP or -GDP, approximately 1 x 10(-6) M(-1) s(-1), are 2-3 orders of magnitude less than expected for simple diffusion models, and the binding progress curves appear biphasic . These findings suggest the presence of an intermediate(s) in the binding reactions . The Gibbs energy changes that occur in the reaction coordinate upon binding of AMP clearly show that the catalytic effect of AMP is due primarily to its -3.1 kcal/mol stabilization of the rate-limiting transition state.

Biochemistry, 1997 Mar 18, 36(11), 3262 - 9
A 1H NMR study of the paramagnetic active site of the CuA variant of amicyanin; Dennison C et al.; The dinuclear paramagnetic center of the CuA variant of the cupredoxin amicyanin has been investigated using 1H NMR . The hyperfine-shifted resonances have been assigned using a combination of 1D NOE difference and 2D WEFT-NOESY spectroscopy . The shifts experienced by the assigned resonances have been used to calculate hyperfine coupling constants for these protons from which the spin density distribution on the ligands at the CuA center is obtained . A comparison with published data for the paramagnetic form of wild type amicyanin highlights a number of similarities and differences between these evolutionary related sites . In both cases 50-60% of the unpaired spin density is distributed on the ligands, which in the case of the CuA center involves two cysteine and two histidine ligands . The two weak axial interactions at the CuA center carry less than 1% spin density.

Biochemistry, 1997 Mar 18, 36(11), 3193 - 8
Reciprocal size-effect relationship of the key residues in determining regio- and stereospecificities of DHEA hydroxylase activity in P450 2a5; Uno T et al.; Collectively, the P450 2a4/2a5 system hyrdoxylates DHEA in at least three positions (7alpha, 7beta, and 2alpha) . An individual P450, however, exhibits high specificity to one of these products . Using site-directed mutagenesis of mP450 2a5 from the wild mouse Mus minutoides and bacterial expression, we have associated the function of residues 117, 209, and 481 with the respective specificity observed in each P450 . Ala at position 117 determines the 7beta-hydroxylase activity, whereas Val at this position defines the 2alpha-hydroxylase activity . Leu at position 209 is essential for high DHEA 7alpha-hydroxylase activity . The substitutions of residue 481 with various hydrophobic amino acids elicited a profound alteration of the specific hydroxylation rates, but did not influence the regio- and stereospecificities at either of the three positions of DHEA . The alterations caused by residue 481 also depended on the residue identity at position 117 or 209 . The results indicate that the sizes of several key residues obey a concerted reciprocal relationship whereby the substrate pocket of the P450s adjusts to accommodate DHEA . A limited molecular modeling study successfully correlates DHEA binding to experimental DHEA hydroxylase activities for a series of mutants at key positions.

Biochemistry, 1997 Mar 18, 36(11), 3179 - 85
Inhibition of the ribonuclease H and DNA polymerase activities of HIV-1 reverse transcriptase by N-(4-tert-butylbenzoyl)-2-hydroxy-1-naphthaldehyde hydrazone; Borkow G et al.; HIV-1 reverse transcriptase (RT) is multifunctional, with RNA-dependent DNA polymerase (RDDP), DNA-dependent DNA polymerase (DDDP), and ribonuclease H (RNase H) activities . N-(4-tert-Butylbenzoyl)-2-hydroxy-1-naphthaldehyde hydrazone (BBNH) inhibited both the polymerase and the RNase H activities of HIV-1 RT in vitro . IC50 values for inhibition of RDDP were 0.8-3.4 microM, depending on the template/primer (T/P) used in the assay . The IC50 for DDDP inhibition was about 12 microM, while that for inhibition of RNase H was 3.5 microM . EC50 for inhibition of HIV-1 replication in cord blood mononuclear cells was 1.5 microM . BBNH inhibition of RNase H in vitro was time-dependent, whereas inhibition of RT polymerase activities was immediate . BBNH was a linear mixed-type inhibitor of RT RDDP activity with respect to both T/P and to dNTP, whereas BBNH inhibition of RT RNase H activity was linear competitive . Protection experiments using an azidonevirapine photolabel showed that BBNH binds to the non-nucleoside RT inhibitor (NNRTI) binding pocket . Importantly, the compound inhibited recombinant RT containing mutations associated with high-level resistance to other NNRTI . While BBNH did not inhibit the DNA polymerase activities of other retroviral reverse transcriptases and DNA polymerases, the compound inhibited Escherichia coli RNase HI and the RNase H activity of murine leukemia virus RT . BBNH also inhibited HIV-1 RT RNase H in the presence of high concentrations of other non-nucleoside inhibitors with higher affinities for the NNRTI binding pocket, and of RT in which the NNRTI binding pocket had been irreversibly blocked by the azidonevirapine photolabel . We conclude that BBNH may therefore bind to two sites on HIV-1 RT . One site is the polymerase non-nucleoside inhibitor binding site and the second may be located in the RNase H domain . BBNH is therefore a promising lead compound for the development of multisite inhibitors of HIV-1 RT.

Biochemistry, 1997 Mar 18, 36(11), 3115 - 25
A two-site mechanism for ATP hydrolysis by the asymmetric Rep dimer P2S as revealed by site-specific inhibition with ADP-A1F4; Wong I et al.; The Escherichia coli Rep helicase is a dimeric motor protein that catalyzes the transient unwinding of duplex DNA to form single-stranded (ss) DNA using energy derived from the binding and hydrolysis of ATP . In an effort to understand this mechanism of energy transduction, we have used pre-steady-state methods to study the kinetics of ATP binding and hydrolysis by an important intermediate in the DNA unwinding reaction--the asymmetric Rep dimer state, P2S, where ss DNA {dT(pT)15} is bound to only one subunit of the Rep dimer . To differentiate between the two potential ATPase active sites inherent in the dimer, we constructed dimers with one subunit covalently cross-linked to ss DNA and where one or the other of the ATPase sites was selectively complexed to the tightly bound transition state analog ADP-A1F4 . We found that when ADP-A1F4 is bound to the Rep subunit in trans from the subunit bound to ss DNA, steady-state ATPase activity of 18 s(-1) per dimer (equivalent to wild-type P2S) was recovered . However, when the ADP-A1F4 and ss DNA are both bound to the same subunit (cis), then a titratable burst of ATP hydrolysis is observed corresponding to a single turnover of ATP . Rapid chemical quenched-flow techniques were used to resolve the following minimal mechanism for ATP hydrolysis by the unligated Rep subunit of the cis dimer: E + ATP <==> E-ATP <==> E'-ATP <==> E'-ADP-Pi <==> E-ADP-Pi <==> E-ADP + Pi <==> E + ADP + Pi, with K1 = (2.0 +/- 0.85) x 10(5) M(-1), k2 = 22 +/- 3.5 s(-1), k(-2) < 0.12 s(-1), K3 = 4.0 +/- 0.4 (k3 > 200 s(-1)), k4 = 1.2 +/- 0.14 s(-1), k(-4) << 1.2 s(-1), K5 = 1.0 +/- 0.2 mM, and K6 = 80 +/- 8 microM . A salient feature of this mechanism is the presence of a kinetically trapped long-lived tight nucleotide binding state, E'-ADP-Pi . In the context of our "subunit switching" model for Rep dimer translocation during processive DNA unwinding {Bjornson, K . B., Wong, I., & Lohman, T . M . (1996) J . Mol . Biol . 263, 411-422}, this state may serve an energy storage function, allowing the energy from the binding and hydrolysis of ATP to be harnessed and held in reserve for DNA unwinding.

Biochemistry, 1997 Mar 18, 36(11), 3095 - 103
Molecular, biological, and preliminary structural analysis of recombinant bryodin 1, a ribosome-inactivating protein from the plant Bryonia dioica; Gawlak SL et al.; Bryonia dioica (Cucurbitaceae family) produces at least two type I ribosome-inactivating proteins, bryodin 1 (BD1) and bryodin 2 (BD2) . A cDNA sequence encoding BD1 was isolated from B . dioica leaf mRNA using degenerative oligonucleotides and codes for a 22 amino acid signal peptide followed by a protein of 267 residues . Expression of two recombinant BD1 (rBD1) forms in Escherichia coli yielded proteins of 267 (to the natural stop codon) and 247 amino acids (to the putative cleavage site yielding the mature protein) that had identical protein synthesis inhibition activity as compared to native BD1 . The substitution of Lys for Glu at position 189 near the active site reduced the ability of rBD1 to inhibit protein synthesis by 10-fold . Toxicologic analysis showed that rBD1 was well tolerated in rodents with LD50 values of 40 mg/kg in mice and >25 mg/kg in rats . A crystal of mature rBD1 protein was used to collect X-ray diffraction data to 2.1 A resolution . The rBD1 crystal structure was solved and showed extensive homology with other type I RIPs and A chains of type II RIPs . The studies described here demonstrate that rBD1 retains full biologic activity and serve as a guide for using this potent, yet nontoxic, RIP in the construction of single-chain immunotoxin fusion proteins.

Biochemistry, 1997 Mar 18, 36(11), 3068 - 75
Identification of the active site nucleophile in the thermostable beta-glycosidase from the archaeon Sulfolobus solfataricus expressed in Escherichia coli; Febbraio F et al.; Sulfolobus solfataricus beta-glycosidase expressed in Escherichia coli was fully inactivated at 65 degrees C, according to pseudo-first-order kinetics, by {3H}conduritol B epoxide (DL-1,2 anhydro-myo-inositol) synthesized as the active site directed inhibitor by a slight modification of Legler's procedure {Legler, G . (1977) Methods Enzymol . 46, 368-381} . The determination of kinetic constants for the inactivation showed that the process took place through the formation of a stabilized inhibitor-enzyme intermediate . Inactivation and reactivation studies suggested that the inhibitor-enzyme intermediate complex was formed more rapidly and hydrolyzed at a lower rate than it was for other glycosidases . Moreover, the stoichiometry of the binding, determined by electrospray mass spectrometric analysis, revealed that one molecule of the inhibitor was covalently bound to each enzyme subunit . The binding site for {3H}conduritol B epoxide was identified by the isolation and partial sequence analysis of the radioactive peptide obtained by cyanogen bromide and pepsin digests . Electrospray tandem mass analysis of the labeled peptide showed that the inhibitor was covalently bound to E387 . This result, in agreement with data obtained from sequence alignments of S . solfataricus beta-glycosidase with other gluco- and galactosidases of the glycosyl hydrolase family 1 {Henrissat, B . (1991) Biochem . J . 280, 309-316}, indicates that the conserved E387 is the nucleophilic amino acid residue in the active site of the enzyme.

Mol Gen Genet, 1997 Mar 18, 254(1), 98 - 103
Treatment with DNA-damaging agents increases expression of polA'-'lacZ gene fusions in Escherichia coli K-12; Wandt G et al.; The polA gene of Escherichia coli encodes the DNA polymerase I that is involved in DNA replication and repair . In contrast to the extensive body of data on the structure and function of polymerase I, there is little information available concerning the mechanisms that govern polA expression . Here, we studied the expression of the polA gene using translational fusions to lacZ . We found that treatment with the DNA-damaging agents 4-nitroquinoline-N-oxide (4-NQO), UV light mitomycin C (MC) and methyl methanesulfonate (MMS) leads to enhanced expression of polA'-'lacZ fusions . The increase in expression of polA reflects stimulation of transcription from a single promoter, as determined by S1 nuclease analyses . This was not observed in mutants that are blocked in induction of the SOS regulon . However, mutants with defective excision repair were more susceptible to polA stimulation . These results support the hypothesis that increased polA expression may be important for the ability to repair bulky DNA adducts that interfere with replication.

Mol Gen Genet, 1997 Mar 18, 254(1), 37 - 42
Mutations occurring at the human minisatellite MS1 integrated in haploid yeast are similar to MS1 mutations in humans; Maleki S et al.; MS1 is one of the most variable minisatellites so far isolated from the human genome . We have previously reported an MS1 length-mutant frequency of 29.6% in overnight cultures of haploid yeast cells carrying a 1.35 kb MS1 allele . Here we present data on the instability of alleles with lengths ranging from 0.15 kb to 2.05 kb, which revealed a threshold of 0.75 kb, at and below which MS1 alleles were entirely stable . Larger alleles exhibited a length-related increase in mutation frequency . Chromosomal integration of various MS1 alleles, isolated from bacterial transformants, in haploid yeast cells also revealed a threshold for the onset of instability and a higher degree of mutability for longer alleles . DNA sequencing of alleles showed that the length changes were due to mutational events involving repeat units in the central region of MS1 which is composed of two variant repeat units only . The similarity between MS1 mutations in yeast and humans argues that yeast represents a suitable model organism for mechanistic studies on mutations occurring in human minisatellites.

Mol Gen Genet, 1997 Mar 18, 254(1), 13 - 20
Control of Escherichia coli type 1 fimbrial gene expression in stationary phase: a negative role for RpoS; Dove SL et al.; Expression of type 1 fimbriae in Escherichia coli is subject to phase-dependent control, with many regulatory inputs from proteins which organise or rearrange the structure of DNA . Inversion of a DNA segment carrying the promoter for expression of fimA, the gene encoding the fimbrial subunit protein, makes a key contribution to the switching of cells between fimbriate and afimbriate states . We have discovered that transcription of fimA is repressed as cells enter stationary phase . This repression is not seen in isogenic strains deficient in rpoS, the gene coding for the stationary phase-specific sigma factor RpoS . RpoS-deficient strains are also altered in the frequency of inversion of the fimA promoter segment . In the strains used in this study, inversion is catalysed by an integrase-like site-specific recombinase encoded by the fimB gene . We report that fimB transcription is repressed strongly as wild-type cells enter stationary phase but that this repression is alleviated in cells deficient in RpoS . These data suggest that RpoS has a negative regulatory role which may be indirect, at both the fimA and the fimB promoters.

Gene, 1997 Mar 18, 187(2), 289 - 94
Inducible high-level expression vector for mammalian cells, pEF-LAC carrying human elongation factor 1alpha promoter and lac operator; Edamatsu H et al.; We have constructed an inducible high-level expression vector, pEF-LAC . pEF-LAC has a modified human polypeptide chain elongation factor 1alpha (EF-1alpha) promoter containing three lactose operator sequences . Using the cat reporter gene, we characterized the transcriptional activity of pEF-LAC . In the transient transfection of NIH3T3 and BaF3 cells, the transcriptional activity of pEF-LAC was higher than that of the original human elongation factor 1alpha promoter, simian virus 40 (SV40) promoter, and Rous sarcoma virus (RSV) long terminal repeat (LTR) . Cotransfection of the lactose repressor expression plasmid effectively suppressed the promoter activity of pEF-LAC, and the activity was fully recovered by addition of isopropyl beta-D-thiogalactopyranoside (IPTG) . Even in the stable transfection of Rat-1 cells, the promoter activity of the integrated pEF-LAC was much higher than that of the RSV-LTR and regulated in an IPTG-dependent manner . These results suggest that pEF-LAC is a useful vector for the inducible high-level expression of the cloned gene in a variety of mammalian cells.

EMBO J, 1997 Mar 17, 16(6), 1464 - 72
The RuvC protein dimer resolves Holliday junctions by a dual incision mechanism that involves base-specific contacts; Shah R et al.; The Escherichia coli RuvC protein resolves DNA intermediates produced during genetic recombination . In vitro, RuvC binds specifically to Holliday junctions and resolves them by the introduction of nicks into two strands of like polarity . In contrast to junction recognition, which occurs without regard for DNA sequence, resolution occurs preferentially at sequences that exhibit the consensus 5'-(A/T)TT/(G/C)-3' (where / indicates the site of incision) . Synthetic Holliday junctions containing modified cleavage sequences have been used to investigate the mechanism of cleavage . The results indicate that specific DNA sequences are required for the correct docking of DNA into the two active sites of the RuvC dimer . In addition, using chemically modified oligonucleotides to introduce a hydrolysis-resistant 3'-S-phosphorothiolate linkage at the cleavage site, it was found that, as long as the sequence requirements are fulfilled, the two incisions could be uncoupled from each other . These results indicate that RuvC protein resolves Holliday junctions by a mechanism similar to that exhibited by certain restriction enzymes.

EMBO J, 1997 Mar 17, 16(6), 1444 - 54
A novel role of ImmE7 in the autoregulatory expression of the ColE7 operon and identification of possible RNase active sites in the crystal structure of dimeric ImmE7; Hsieh SY et al.; Site-specific cleavage of mRNA has been identified in vivo for the polycistronic colicin E7 operon (ColE7), which occurs between G and A nucleotides located at the Asp52 codon (GAT) of the immunity gene (ceiE7) . In vitro, this specific cleavage occurs only in the presence of the ceiE7 gene product (ImmE7) . The crystal structure of dimeric ImmE7 has been determined at 1.8 A resolution by X-ray crystallographic analysis . We found that several residues located at the interface of dimeric ImmE7 bear surprising resemblance to the active sites of some RNases . These results suggest that dimeric ImmE7 may possess a novel RNase activity that cleaves its own mRNA at a specific site and thus autoregulates translational expression of the downstream celE7 gene as well as degradation of the upstream ceaE7 mRNA.

EMBO J, 1997 Mar 17, 16(6), 1181 - 8
Projection structure of the cytochrome bo ubiquinol oxidase from Escherichia coli at 6 A resolution; Gohlke U et al.; The haem-copper cytochrome oxidases are terminal catalysts of the respiratory chains in aerobic organisms . These integral membrane protein complexes catalyse the reduction of molecular oxygen to water and utilize the free energy of this reaction to generate a transmembrane proton gradient . Quinol oxidase complexes such as the Escherichia coli cytochrome bo belong to this superfamily . To elucidate the similarities as well as differences between ubiquinol and cytochrome c oxidases, we have analysed two-dimensional crystals of cytochrome bo by cryo-electron microscopy . The crystals diffract beyond 5 A . A projection map was calculated to a resolution of 6 A . All four subunits can be identified and single alpha-helices are resolved within the density for the protein complex . The comparison with the three-dimensional structure of cytochrome c oxidase shows the clear structural similarity within the common functional core surrounding the metal-binding sites in subunit I . It also indicates subtle differences which are due to the distinct subunit composition . This study can be extended to a three-dimensional structure analysis of the quinol oxidase complex by electron image processing of tilted crystals.

Biochem Biophys Res Commun, 1997 Mar 17, 232(2), 555 - 8
Expression in Escherichia coli, phosphorylation with cAMP-dependent protein kinase and proteolysis by calpain of a 71-kDa domain of human endothelial actin binding protein; Jay D et al.; A middle region of human endothelial actin-binding protein (ABP) was subcloned and expressed in the pT7-7/E . coli BL21 (DE3) system . As predicted by the amino acid sequence this 71 kD truncated protein (residues 1717-2360) contained a calpain cleavage site and two of the three presumptive cAMP-dependent protein kinase phosphorylation sites . This peptide fragment comprised all the elements needed to confer stability against calpain proteolysis to ABP after PKA phosphorylation.

Biochem Biophys Res Commun, 1997 Mar 17, 232(2), 388 - 93
Characterization of cysteine residues involved in the reductive activation and the structural stability of rapeseed (Brassica napus) chloroplast fructose-1,6-bisphosphatase; Rodriguez-Suarez RJ et al.; In higher plants, light enhances the activity of chloroplast fructose-1,6-bisphosphatase via a cascade of thiol/disulfide exchanges . We have examined the structural and functional role of seven conserved cysteine residues in the rapeseed (Brassica napus) enzyme by site-directed mutagenesis . After lysis of Escherichia coli cells, C53S and C191S variants partitioned mainly in the insoluble fraction whereas C96S, C157S, C174S, C179S, and C307S mutants were soluble . Homogeneous preparations of the latter hydrolyzed fructose 1,6-bisphosphate at similar rates in the presence of 10 mM Mg2+ but only C157S, C174S and C179S mutants were both efficient catalysts at 1 mM Mg2+ and nearly insensitive to dithiothreitol . These results demonstrate the contribution of Cys53 and Cys191 to the stability of the enzyme and the participation of Cys157, Cys174 and Cys179 in the reductive process responsive of the light-dependent regulation . Given that mutations at Cys96 and Cys307 neither destabilize the enzyme nor affect the reductive modulation, their function remains unknown.

Mutat Res, 1997 Mar 17, 389(2-3), 237 - 42
Intracellular generation of superoxide by copper sulphate in Escherichia coli; Kimura T et al.; Oxidative stress exerted by superoxide-generating agents such as paraquat triggers induction of the soxRS regulon of E . coli . In the system, SoxR protein is the superoxide-sensitive activator of the soxS gene . We found that copper sulphate (CuSO4) is a powerful inducer of soxS in a manner tightly dependent on both a functional soxR gene and the presence of molecular oxygen . E . coli strain lacking either superoxide dismutases or oxidative DNA repair enzymes was hypersensitive to killing by CuSO4 . These results suggest that superoxide may be generated intracellularly by CuSO4 treatment, and that the generation may be involved in the Cu cytotoxicity.

Mutat Res, 1997 Mar 17, 389(2-3), 191 - 7
Genotoxicity of selected metal compounds in the SOS chromotest; Lantzsch H et al.; Knowledge concerning the genotoxicity of inorganic metal compounds in the SOS chromotest is limited . Up to now, only Cr(VI), Sn(II) and the platinum antitumor compound cisplatin(II) were shown to be genotoxic in this test system . However, for Cr(VI) and Sn(II), a positive reaction could only be achieved in cytotoxic dose ranges . The aim of the present study was to provide additional data concerning metal salt genotoxicity in the SOS chromotest . Therefore, 14 metal/metalloid salt compounds of platinum, palladium, rhodium, arsenic, antimony and chromium were tested . Four platinum salts, K2PtCl4, cis-Pt(NH3)2Cl2 (cisplatin), trans-Pt(NH3)2Cl2 (transplatin) and PtCl4 as well as two rhodium compounds tested, K2RhCl5 and (NH4)3RhCl6, could be shown to be genotoxic in the chromotest using the tester strain Escherichia coli PQ37 . A moderate genotoxicity was shown by the two Cr(VI) compounds K2CrO4 and K2Cr2O7 . All palladium compounds and all the other metal salts tested were unable to induce a significant SOS response.

J Exp Med, 1997 Mar 17, 185(6), 1143 - 8
Epinephrine exerts anticoagulant effects during human endotoxemia; van der Poll T et al.; To determine the effect of a physiologically relevant elevation in the plasma concentrations of epinephrine on the activation of the hemostatic mechanism during endotoxemia, 17 healthy men were studied after intravenous injection of lipopolysaccharide (LPS, 2 ng/kg), while receiving a continuous infusion of epinephrine (30 ng/kg/min) started either 3 h (n = 5) or 24 h (n = 6) before LPS injection, or an infusion of normal saline (n = 6) . Activation of the coagulation system (plasma concentrations of thrombin-antithrombin III complexes and prothrombin fragment F1+2) was significantly attenuated in the groups treated with epinephrine when compared with subjects injected with LPS only (P <0.05) . Epinephrine enhanced LPS-induced activation of fibrinolysis (plasma levels of tissue-type plasminogen activator and plasmin-alpha2-antiplasmin complexes; P <0.05), but did not influence inhibition of fibrinolysis (plasminogen activator inhibitor type I) . In subjects infused with epinephrine, the ratio of maximal activation of coagulation and maximal activation of fibrinolysis was reduced by >50% . Hence, epinephrine exerts antithrombotic effects during endotoxemia by concurrent inhibition of coagulation, and stimulation of fibrinolysis . Epinephrine, whether endogenously produced or administered as a component of treatment, may limit the development of disseminated intravascular coagulation during systemic infection.

Biochem J, 1997 Mar 15, 322 ( Pt 3), 853 - 8
Activation of the ATPase activity of heat-shock proteins Hsc70/Hsp70 by cysteine-string protein; Chamberlain LH et al.; DnaJ proteins are characterized by a 'J' domain which is homologous to a region of the Escherichia coli protein DnaJ . DnaJ has been shown to interact with the chaperone protein DnaK, and a number of eukaryotic DnaJ-like proteins have been found to interact with the 70 kDa heat-shock protein/70 kDa heat-shock cognate protein (Hsp70/Hsc70), the eukaryotic homologues of DnaK . Cysteine-string proteins (Csps) are believed to function in calcium-stimulated exocytosis and in this paper we describe a specific ATP-dependent interaction between a Csp (Csp1) and Hsc70/Hsp70 . We also show that Csp1 can stimulate the ATPase activity of both Hsc70 and Hsp70 several-fold . Furthermore, we demonstrate that Csp2, a Csp variant found in adrenal chromaffin cells, can enhance the ATPase activity of Hsc70 to a similar extent as Csp1, whereas Csp(137-198), a truncated protein lacking the 'J' domain of Csp1 is unable to stimulate the ATPase activity of Hsc70 . This suggests that the functions of Csp1 and Csp2 must differ in some aspect other than interaction with Hsc70 . This study is also important from a general view of DnaJ/Hsc70 interactions, as Csps lack a G/F-rich region which has been suggested to be essential for activation of the ATPase activity of DnaK by DnaJ . Thus, this work would imply that a G/F-rich region is not an essential feature of DnaJ proteins for stimulation of the ATPase activity of Hsp70 proteins.

Biochem J, 1997 Mar 15, 322 ( Pt 3), 815 - 22
Heterologous expression and characterization of wild-type and mutant forms of a 26 kDa endochitinase from barley (Hordeum vulgare L.); Andersen MD et al.; To investigate structure-function relationships in plant chitinases, we have developed a heterologous expression system for the 26 kDa endochitinase from Hordeum vulgare L . (barley) . Escherichia coli cells harbouring the gene in a T7 RNA polymerase-based expression vector synthesized completely insoluble recombinant protein under standard induction conditions at 37 degrees C . However, a concentration of soluble recombinant protein of approx . 15 mg/l was achieved by inducing bacteria at low temperature (15 degrees C) . Recombinant endochitinase was purified to homogeneity and shown to be structurally and functionally identical to the seed protein . An average of three disulphide bonds are present in the recombinant enzyme, consistent with the number found in the natural form . The seed and recombinant proteins showed the same specific activity towards a high-molecular-mass substrate and exhibited similar anti-fungal activity towards Tricoderma reesei . Site-directed mutagenesis was used to replace residues that are likely to be involved in the catalytic event, based on structural similarities with lysozyme and on sequence alignments with related chitinases . The Glu67-->Gln mutation resulted in a protein with undetectable activity, while the Glu89-->Gln mutation yielded an enzyme with 0 . 25% of wild-type specific activity . This suggests that two acidic residues are essential for catalytic activity, similar to the situation with many other glycosyl hydrolases . Examination of conserved residues stretching into the proposed substrate binding cleft suggests that Asn124 also plays an important functional role.

Biochem J, 1997 Mar 15, 322 ( Pt 3), 771 - 6
Recombinant expression and isolation of human L-arginine:glycine amidinotransferase and identification of its active-site cysteine residue; Humm A et al.; Creatine and its phosphorylated form play a central role in the energy metabolism of muscle and nerve tissues . l-Arginine:glycine amidinotransferase (AT) catalyses the committed step in the formation of creatine . The mitochondrial and cytosolic forms of the enzyme are believed to derive from the same gene by alternative splicing . We have expressed recombinant human AT in Escherichia coli with two different N-termini, resembling the longest two forms of the enzyme that we had isolated recently from porcine kidney mitochondria as a mixture . The enzymes were expressed with N-terminal histidine tags followed by factor Xa-cleavage sites . We established a new method for the removal of N-terminal fusion peptides by means of an immobilized snake venom prothrombin activator . We identified cysteine-407 as the active-site residue of AT by radioactive labelling and isolation of labelled peptides, and by site-directed mutagenesis of the protein.

Biochem J, 1997 Mar 15, 322 ( Pt 3), 719 - 27
Characterization of a saporin isoform with lower ribosome-inhibiting activity; Fabbrini MS et al.; We have expressed in Escherichia coli five isoforms of saporin, a single-chain ribosome-inactivating protein (RIP) . Translation inhibition activities of the purified recombinant polypeptides in vitro were compared with those of recombinant dianthin 30, a less potent and closely related RIP, and of ricin A chain . Dianthin 30, and a saporin isoform encoded by a cDNA from leaf tissue (SAP-C), both had about one order of magnitude lower activity in translation inhibition assays than all other isoforms of saporin tested . We recently demonstrated that saporin extracted from seeds of Saponaria officinalis binds to alpha2-macroglobulin receptor (alpha2MR; also termed low density lipoprotein-receptor-related-protein), indicating a general mechanism of interaction of plant RIPs with the alpha2MR system {Cavallaro, Nykjaer, Nielsen and Soria (1995) Eur . J . Biochem . 232, 165-171} . Here we report that SAP-C bound to alpha2MR equally well as native saporin . However, the same isoform had about ten times lower cytotoxicity than the other saporin isoforms towards different cell lines . This indicates that the lower cell-killing ability of the SAP-C isoform is presumably due to its altered interaction with the protein synthesis machinery of target cells . Since saporin binding to the alpha2MR is competed by heparin, we also tested in cell-killing experiments Chinese hamster ovary cell lines defective for expression of either heparan sulphates or proteoglycans . No differences were observed in cytotoxicity using native saporin or the recombinant isoforms . Therefore saporin binding to the cell surface should not be mediated by interaction with proteoglycans, as is the case for other alpha2MR ligands.

Eur J Biochem, 1997 Mar 15, 244(3), 931 - 9
Identification of the regions on the M110 subunit of protein phosphatase 1M that interact with the M21 subunit and with myosin; Johnson D et al.; We have previously isolated a form of protein phosphatase-1 (PP1M) from avian smooth muscle myofibrils that is composed of the catalytic subunit of PP1 (PP1C) bound to an M-complex consisting of 110-kDa (M110) and 21-kDa (M21) subunits . The interaction of PP1C with an N-terminal region of the M110 subunit enhances the dephosphorylation of myosin and suppresses the dephosphorylation of other substrates {Alessi, D . R., MacDougall, L . K., Sola, M . M., Ikebe, M . & Cohen, P . (1992) Eur . J . Biochem . 210, 1023-1035; Chen, Y . H., Chen, M . X., Alessi, D . R., Campbell, D . G., Shanahan, C., Cohen, P . & Cohen, P . T . W . (1994) FEBS Lett . 356, 51-56; Johnson, D . F., Moorhead, G., Caudwell, F . B., Cohen, P., Chen, Y . H., Chen, M . X . & Cohen, P . T . W . (1996) Eur . J . Biochem . 239, 317-325} . In this paper, we establish that PP1M accounts for nearly all the myosin phosphatase activity in myofibrils, that the M110 and M21 subunits are present at similar concentrations in the myofibrillar fraction, and that these subunits are entirely bound to PP1 . We demonstrate that the M21 subunit does not interact with PP1C, but with the C-terminal 72 residues of the M110 subunit, a region which is 43% identical to residues 87-161 of the M21 subunit . A fragment of the M21 subunit, M21-(M1-L146), which lacks the C-terminal leucine zipper, also bound to the M110 subunit, but two other fragments M21-(M1-E110) and M21-(E110-K186) did not . The M110 and M21 subunits were both found to be myosin-binding proteins . The C-terminal 291 residues of the M110 subunit, but not the C-terminal 72 residues, bound to myosin, but the N-terminal fragments M110-(M1-E309) and M110-(M1-S477) did not . Thus, the region of the M110 subunit that stimulates the dephosphorylation of myosin by PP1C is distinct from the region that targets PP1M to myosin . Remarkably, each myosin dimer was capable of binding about 20 mol M21 subunit and many of the M21-binding sites were located in the myosin rod domain . The potential significance of this observation is discussed.

Eur J Biochem, 1997 Mar 15, 244(3), 904 - 12
Altered methylation substrate kinetics and calcium binding of a calmodulin with a Val136-->Thr substitution; Han CH et al.; Calmodulin is trimethylated on Lys115 by a specific calmodulin methyltransferase . Previously, it was shown that the cam2 mutant (Ile136-->Thr) of Paramecium has a decreased level of methylated Lys115 {Lukas, T . J., Friedman, M . W., Kung, C . & Watterson, D . M . (1989) Proc . Natl Acad . Sci . USA 86, 7331-7335} . To investigate how this substitution affects calmodulin structure, function and recognition by the calmodulin methyltransferase, a calmodulin with a Thr136 substitution ({Thr136}calmodulin) was expressed in Escherichia coli in an unmethylated form for in vitro enzyme activator, calcium binding and methylation kinetic analyses . {Thr136}calmodulin was indistinguishable from wild-type calmodulin in saturating (1 mM) calcium in its ability to activate calmodulin-dependent enzymes and in its steady-state kinetic properties with isolated calmodulin methyltransferase . However, {Thr136}calmodulin did show two defects: a complete inability to be methylated in the absence of calcium; and defective calcium binding . As a result, an approximate 10-fold shift in the K0.5 values for calcium dependence of enzyme activation (shifted from 1.1 microM to 9.1 microM of Ca2+ for NAD kinase) and methylation (from 0.71 microM to 7.2 microM of Ca2+ in 0.15 M K+, 2 mM Mg2+) were observed . Non-denaturing electrophoresis and Tyr138 spectroscopic measurements suggest a difference in the conformation of the calcium-depleted structures of normal calmodulin and {Thr136}calmodulin . Overall, the results suggest that the mutation in this conserved position in the COOH-terminal hydrophobic core lowers calcium-binding affinity and alters the calcium-depleted structure leading to decreased methylation at physiological Ca2+ concentrations.

Eur J Biochem, 1997 Mar 15, 244(3), 869 - 75
Phosphoprotein PII from cyanobacteria--analysis of functional conservation with the PII signal-transduction protein from Escherichia coli; Forchhammer K et al.; The signal transduction protein PII from Escherichia coli is modified by uridylylation, whereas its counterpart from the cyanobacterium Synechococcus PCC 7942 is phosphorylated at a seryl residue . To elucidate functional conservations between these proteins, we compared the Synechococcus PII protein with the known properties of the E . coli PII protein . Similar to the E . coli protein, Synechococcus PII binds the metabolites 2-oxoglutarate and ATP in a mutually dependent manner . The synergism of ligand binding was analyzed in detail . The ATP-binding site of Synechococcus PII could be labelled with 5'-p-fluorosulfonylbenzoyladenosine . By heterologous expression of the cyanobacterial glnB gene in E . coli we showed that Synechococcus PII can be modified by the E . coli PII uridylyltransferase . The presence of Synechococcus PII prevents signal transduction of E . coli PII to NtrB, presumably by non-functional competition . We therefore propose that the primary function of Synechococcus PII is to sense 2-oxoglutarate, the carbon skeleton required for nitrogen assimilation.

Eur J Biochem, 1997 Mar 15, 244(3), 706 - 12
Molecular cloning and characterization of beta-1,4-galactosyltransferase expressed in mouse testis; Uehara K et al.; Using an affinity-purified antibody against beta-1,4-galactosyltransferase purified from F9 embryonal carcinoma cells, we screened a lambda gt11 expression library constructed from the same cell line . The cDNA clone obtained encoded a 46-kDa protein . Among adult mouse organs, the testis was found to express large amounts of the corresponding mRNA . An antibody against the 46-kDa protein was raised in a rabbit by immunization with a maltose-binding-protein fusion protein produced in Escherichia coli . The affinity-purified antibody against the cloned 46-kDa protein reacted with a 59-kDa protein in sperm extract on western blotting . Among the proteins in the F9 beta-1,4-galactosyltransferase preparation, which were mostly 68-kDa and 59-kDa species, 59-kDa and 50-kDa bands reacted with the antibody against the cloned 46-kDa protein . The cloned 46-kDa protein had type-II transmembrane topology and some blocks of sequence similarity with the known beta-1,4-galactosyltransferase . Furthermore, predicted secondary structures were similar over large portions of the two proteins . The histidine-tagged 46-kDa protein produced in E . coli was partially adsorbed onto a N-acetylglucosamine-Affi-Gel column and eluted by 20 mM N-acetylglucosamine . The histidine-tagged 46-kDa protein, which was purified to homogeneity, had a small, but significant amount of beta-1,4-galactosyltransferase activity . These results strongly suggest that the 46-kDa protein is a beta-1,4-galactosyltransferase.

Eur J Biochem, 1997 Mar 15, 244(3), 700 - 5
Cloning, expression, purification and characterization of triosephosphate isomerase from Trypanosoma cruzi; Ostoa-Saloma P et al.; The gene that encodes for triosephosphate isomerase from Trypanosoma cruzi was cloned and sequenced . In T . cruzi, there is only one gene for triosephosphate isomerase . The enzyme has an identity of 72% and 68% with triosephosphate isomerase from Trypanosoma brucei and Leishmania mexicana, respectively . The active site residues are conserved: out of the 32 residues that conform the interface of dimeric triosephosphate isomerase from T . brucei, 29 are conserved in the T . cruzi enzyme . The enzyme was expressed in Escherichia coli and purified to homogeneity . Data from electrophoretic analysis under denaturing techniques and filtration techniques showed that triosephosphate isomerase from T . cruzi is a homodimer . Some of its structural and kinetic features were determined and compared to those of the purified enzymes from T . brucei and L . mexicana . Its circular dichroism spectrum was almost identical to that of triosephosphate isomerase from T . brucei . Its kinetic properties and pH optima were similar to those of T . brucei and L . mexicana, although the latter exhibited a higher Vmax with glyceraldehyde 3-phosphate as substrate . The sensitivity of the three enzymes to the sulfhydryl reagent methylmethane thiosulfonate (MeSO2-SMe) was determined; the sensitivity of the T . cruzi enzyme was about 40 times and 200 times higher than that of the enzymes from T . brucei and L . mexicana, respectively . Triosephosphate isomerase from T . cruzi and L . mexicana have the three cysteine residues that exist in the T . brucei enzyme (positions 14, 39, 126, using the numbering of the T . brucei enzyme); however, they also have an additional residue (position 117) . These data suggest that regardless of the high identity of the three trypanosomatid enzymes, there are structural differences in the disposition of their cysteine residues that account for their different sensitivity to the sulfhydryl reagent . The disposition of the cysteine in triosephosphate isomerase from T . cruzi appears to make it unique for inhibition by modification of its cysteine.

Biochim Biophys Acta, 1997 Mar 15, 1334(2-3), 261 - 72
Catalytic domain comparisons of human fibroblast-type collagenase, stromelysin-1, and matrilysin; Windsor LJ et al.; The propeptide plus the catalytic domain of human fibroblast-type collagenase, stromelysin-1, and matrilysin were expressed in Escherichia coli to directly compare the properties of all three catalytic domains utilizing the same assays . Truncated fibroblast-type collagenase (mini-CL), truncated stromelysin-1 (mini-SL-1), and matrilysin, like their native counterparts, could be activated by organomercurials, trypsin, or SDS . The mini-CL and mini-SL-1 displayed catalytic properties similar to their native counterparts, except that the mini-CL could not cleave native type I collagen . The k(cat)/Km for matrilysin (355 microM(-1) h(-1)) on the synthetic Mca-peptide was much higher than that for mini-CL (69 microM(-1) h(-1)) or mini-SL-1 (23.6 microM(-1) h(-1)) . Mini-SL-1 and matrilysin, but not mini-CL, were capable of superactivating collagenase thus increasing the rate of collagen cleavage . Mini-CL and mini-SL-1, but not matrilysin, were able to form SDS-stable complexes with TIMP-1 when co-incubated with an organomercurial and TIMP-1 . The second-order rate constant (k(on)) for TIMP-1 inhibition of mini-CL and mini-SL-1 were similar, 0.635 x 10(5) M(-1) s(-1) and 1.52 x 10(5) M(-1) s(-1), respectively . The k(on) for TIMP-1 inhibition of matrilysin was lower (0.130 x 10(5) M(-1) s(-1)) supporting the observation that no SDS stable complexes were detected . This study demonstrates that these catalytic domains are distinct and play a major role in the specificity of these enzymes in regard to rate of catalysis, TIMP-1 binding, and superactivation of collagenase.

Ann N Y Acad Sci, 1997 Mar 15, 813, 338 - 43
Salivary glands, their hormones, and thermoregulation; Mathison RD et al.; The effects of the removal of the submandibular glands (sialadenectomy) on the fever induced by bacterial lipopolysaccharide (LPS) were examined . Thermally sensitive radiotransmitters were implanted into the abdomens of adult male Sprague-Dawley rats that experienced at this time either a sham operation or a sialadenectomy, and one week later body temperatures were recorded by telemetry in these rats when conscious . The initial fever (up to 180 min following LPS) response, following the intraperitoneal injection of 150 micrograms/kg E . coli LPS, was similar in the two groups of rats, but the second phase of the fever (240 to 420 min post-LPS) was modestly, but significantly higher (mean = 0.26 degree C) in sialadenectomized rats . A submandibular gland peptide (compound T; 100 micrograms/kg), given one-half hour before the LPS, did not affect the early fever, but suppressed the late-phase fever by 0.37 degree C (mean) . The submandibular glands, which form an integral part of the neuroendocrine mechanisms responsible for attenuating the responses of the immune system to inflammatory stimuli, also appear to modulate thermogenic responses to these stimuli.

Nucleic Acids Res, 1997 Mar 15, 25(6), 1315 - 6
Simple and rapid preparation of plasmid template by a filtration method using microtiter filter plates; Itoh M et al.; We developed a new simple high-throughput plasmid DNA extraction procedure, based on a modified alkaline lysis method, using only one 96-well microtiter glassfilter plate . In this method, cell harvesting, lysis by alkaline and plasmid purification are performed on only one microtiter glassfilter plate . After washing out RNAs or other contaminants, plasmid DNA is eluted by low-ion strength solution, although precipitated chromosomal DNA is not eluted . The plasmid prepared by this method can be applied to sequencing reactions or restriction enzyme cleavage.

Nucleic Acids Res, 1997 Mar 15, 25(6), 1254 - 64
Transfer RNA docking pair model in the ribosomal pre- and post-translocational states; Nagano K et al.; A consensus has been reached that the conformation of the anticodon-codon interactions of two adjacent tRNA molecules on the ribosome is a Sundaralingam-type (S-type) . Even if it is kept to the S-type, there are still various possibilities . Various experimental data have been supporting an idea that the conformation of A-site tRNA is different from that of P-site tRNA . Those data as well as the recent result of Brimacombe and co-workers that U20:1 of lupin tRNAmMetbound to the A-site was cross-linked to a region, 875-905, of 23S rRNA in combination with the other recent findings of Nierhaus and co-workers about the spin-contrast method of neutron diffraction of the ribosome and the better accessible nucleotide patterns of phosphorothioated tRNAs on the ribosome have led to a new tRNA docking pair model, in which the highly conserved G18 and G19 of D-loop in A-site tRNA and C56 and C61 of TpsiC-loop in P-site tRNA base pair along with the conventional base pairs of adjacent codon-anticodon interactions . This A-P tRNA pair model can be translocated to the P-E tRNA pair model without changing the conformation except the ACCA termini, keeping the position of the growing nascent polypeptide chain.

Nucleic Acids Res, 1997 Mar 15, 25(6), 1203 - 10
Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements; Lutz R et al.; Based on parameters governing promoter activity and using regulatory elements of the lac, ara and tet operon transcription control sequences were composed which permit the regulation in Escherichia coli of several gene activities independently and quantitatively . The novel promoter PLtetO-1 allows the regulation of gene expression over an up to 5000-fold range with anhydrotetracycline (aTc) whereas with IPTG and arabinose the activity of Plac/ara-1 may be controlled 1800-fold . Escherichia coli host strains which produce defined amounts of the regulatory proteins, Lac and Tet repressor as well as AraC from chromosomally located expression units provide highly reproducible in vivo conditions . Controlling the expression of the genes encoding luciferase, the low abundance E.coli protein DnaJ and restriction endonuclease Cfr9I not only demonstrates that high levels of expression can be achieved but also suggests that under conditions of optimal repression only around one mRNA every 3rd generation is produced . This potential of quantitative control will open up new approaches in the study of gene function in vivo, in particular with low abundance regulatory gene products . The system will also provide new opportunities for the controlled expression of heterologous genes.

Nucleic Acids Res, 1997 Mar 15, 25(6), 1185 - 93
Decoding fidelity at the ribosomal A and P sites: influence of mutations in three different regions of the decoding domain in 16S rRNA; O'Connor M et al.; The involvement of defined regions of Escherichia coli 16S rRNA in the fidelity of decoding has been examined by analyzing the effects of rRNA mutations on misreading errors at the ribosomal A and P sites . Mutations in the 1400-1500 region, the 530 loop and in the 1050/1200 region (helix 34) all caused readthrough of stop codons and frameshifting during elongation and stimulated initiation from non-AUG codons at the initiation of protein synthesis . These results indicate the involvement of all three regions of 16S rRNA in decoding functions at both the A and P sites . The functional similarity of all three mutant classes are consistent with close physical proximity of the 1400- 1500 region, the 530 loop and helix 34 and suggest that all three regions of rRNA comprise a decoding domain in the ribosome.

Nucleic Acids Res, 1997 Mar 15, 25(6), 1136 - 41
Isolation of human complexes proficient in nucleotide excision repair; He Z et al.; More than 20 polypeptides are required for the process of nucleotide excision repair (NER) in both human and yeast cells . This pathway of excision repair has most often been viewed as an ordered multi-step process involving steps of damage recognition, incision/excision and finally repair DNA synthesis . Here we present evidence for the existence of a complex of human NER proteins pre-assembled in the absence of damaged DNA . This multi-protein complex was initially isolated from HeLa cell extracts by affinity chromatography on a matrix containing the damage recognition protein XPA . Subsequent co-immunoprecipitation and gel filtration experiments demonstrated that a significant portion of the human NER proteins was present in the form of a high molecular weight complex and that these complexes, or repairosomes, were capable of performing all steps of NER in vitro . Consistent with studies indicating that DNA polymerasesdeltaandstraightepsiloncan both function in NER, these two polymerases are found in these repairosome complexes.

Genes Dev, 1997 Mar 15, 11(6), 738 - 47
Analysis of a cAMP-responsive activator reveals a two-component mechanism for transcriptional induction via signal-dependent factors; Nakajima T et al.; We have examined the mechanism by which the cAMP-responsive factor CREB stimulates target gene expression following its phosphorylation at Ser-133 . Using an in vitro transcription assay, we found that two signals were required for target gene activation: a phospho(Ser-133)-dependent interaction of CREB with RNA polymerase II via the coactivator CBP and a glutamine-rich domain interaction with TFIID via hTAF(II)130 . The adenovirus E1A oncoprotein was found to inhibit phospho(Ser-133) CREB activity by binding to CBP and specifically blocking recruitment of RNA Pol II to the promoter . Our results suggest that the recruitment of CBP-RNA Pol II complexes per se is not sufficient for transcriptional activation and that activator-mediated recruitment of TFIID is additionally required for induction of signal-dependent genes.

FEMS Microbiol Lett, 1997 Mar 15, 148(2), 255 - 60
Clonal relationships among Escherichia coli serogroup O6 isolates based on RAPD; Pacheco AB et al.; The genetic diversity in a group of Escherichia coli strains belonging to serogroup O6 but expressing different H antigens was investigated by random amplification of polymorphic DNA (RAPD) . Isolates of serotypes H16, H1, H31, and non-motile (NM) strains were typed using a set of 3 primers with different G + C contents . The amplified band arrays allowed the identification of 3 main clonal clusters corresponding to each O:H serotype analyzed . Based on their RAPD profiles NM strains could be assigned to either H1 or H31 serotypes . The results indicate that the flagellar antigen and the RAPD fingerprint represent reliable clonal markers in this E . coli group.

Structure, 1997 Mar 15, 5(3), 443 - 58
A mechanism for toxin insertion into membranes is suggested by the crystal structure of the channel-forming domain of colicin E1; Elkins P et al.; BACKGROUND: Channel-forming colicins, including colicin E1, are a sub-family of bacteriocins . The toxic action of colicin E1 is derived from its ability to form a voltage-gated channel, which causes depolarization of the cytoplasmic membrane of sensitive Escherichia coli cells . In this process, the toxin-like colicin E1 molecule must undergo a substantial structural transition from a soluble state, in which it binds the target cell, to a membrane-bound state . Details of the structural changes that accompany this conversion may be directly applicable to other channel-forming toxins, as well as to the mechanism by which proteins insert into or cross membranes . RESULTS: The structure of the 190-residue channel-forming domain of colicin E1 in its soluble form has been solved at 2.5 A resolution . This structure contains 10alpha helices arranged in three layers (A-C) with a central hydrophobic helical hairpin in layer B, which is proposed to anchor the membrane-bound form in the bilayer . The extended N-terminal helix I provides a connection to the rest of the colicin E1 molecule, and the loop I-II may act as a hinge for re-orientation of the domain for membrane binding . A set of conserved positively charged residues on layer C may provide the docking surface on the molecule for membrane attachment . A large internal cavity between layers B and C may allow these layers to disengage, suggesting a mechanism for unfolding the molecule on the membrane that involves the perturbation of the interhelical hydrophobic interactions in layer C . CONCLUSION: On the basis of the structure of the colicin E1 channel-forming domain, its comparison with the structure of the colicin A domain and the known requirement for initial electrostatic and subsequent hydrophobic interactions, molecular details of the docking, unfolding and insertion of the channel-forming domain into the membrane are proposed . The model for docking and initial interaction with the membrane positions the hydrophobic hairpin 'anchor' approximately parallel to the membrane surface . Hydrophobic interactions in the docking layer may then be displaced by interactions with the membrane, spreading the helices on the surface and exposing the hydrophobic hairpin for insertion into the membrane.

Structure, 1997 Mar 15, 5(3), 313 - 7
The long and short of colicin action: the molecular basis for the biological activity of channel-forming colicins; Gouaux E; Channel-forming colicins undergo a remarkable series of conformational gyrations during their voyage from the extracellular milieu to the periplasmic membrane . Crystal structures of the intact colicin la molecule and a channel-forming domain of colicin E1 illuminate relationships between the molecular structure and biological function of these voltage-dependent channel-forming toxins.

Transplantation, 1997 Mar 15, 63(5), 636 - 9
Effect of oral supplementation of ornithine-alpha-ketoglutarate on the intestinal barrier after orthotopic small bowel transplantation; de Oca J et al.; The aim of this study was to analyze the possible protective effects of a glutamine and arginine precursor (ornithine-alpha-ketoglutarate {OKG}) on the mucosa of a transplanted intestine when administered with either a defined formula oral diet (DFD) or a standard chow . Isogenic male Lewis rats (250 g) were submitted to a laparotomy (groups 1 and 2) or to an orthotopic small bowel transplantation (SBT; groups 3-6) . Groups 1, 3, and 5 received a DFD 14 days after surgery . Groups 2, 4, and 6 received standard chow . In addition, groups 5 and 6 received a daily oral supplementation of 1.4 g/kg of OKG . Weight changes and food intake were recorded daily . At the end of the study, bacterial translocation (BT) was measured in mesenteric lymph nodes, liver, and spleen . The protein/DNA index was also determined in intestinal mucosa . SBT induced BT in all transplanted groups, especially in those fed DFD . Addition of OKG (groups 5 and 6) significantly reduced BT in comparison with groups 3 and 4 and improved the protein/DNA index as well as weight gain . It is concluded that OKG supplementation protects the intestinal barrier after SBT, and that this effect is more marked when it is added to a standard chow.

J Membr Biol, 1997 Mar 15, 156(2), 105 - 15
Electrophysiological characteristics of the PhoE porin channel from Escherichia coli . Implications for the possible existence of a superfamily of ion channels; Berrier C et al.; Purified PhoE porins from Escherichia coli were reconstituted in giant proteoliposomes obtained by dehydration-rehydration, and studied by the patch-clamp technique . The following electrophysiological characteristics were observed . (i) The channels for which the probability of opening is maximum around 0 mV, closed at positive and negative potentials, at voltages higher than +/-120 mV . (ii) The channels behaved asymmetrically in response to positive and negative potentials . (iii) The channels exhibited two types of kinetics (fast and slow) on very different time scales . (iv) The channels had several closed states including a reversible inactivated state and a large number of substates . Similar characteristics have been described for channels other than bacterial porins, in particular mitochondrial porins and maxi-chloride channels of the plasma membrane of animal cells . These characteristics might constitute the electrophysiological fingerprint of a superfamily of ion channels for which the basic structure, rather than sequence, would have been conserved during evolution.

Anal Biochem, 1997 Mar 15, 246(2), 234 - 8
Resolution of recombinant human interleukin 10 from variants by recycling free flow focusing; Bondoc LL Jr et al.; Recombinant human interleukin 10 (rhIL-10) is a potential human therapeutic agent for treating inflammatory bowel diseases and rheumatoid arthritis . The rhIL-10 molecule derived from Escherichia coli including bodies consists of two identical subunits forming a noncovalent dimer . Since the ability to separate rhIL-10 from closely related impurities was highly desirable, recycling free flow focusing (RFFF) was utilized for the purification process development of rhIL-10 . Under nondenaturing conditions, RFFF was able to separate rhIL-10 from fractions enriched in rhIL-10 variants . Three major monomeric variants (A, B, and C) can be identified and quantitated by reversed phase HPLC . The isoelectric point (pI) of rhIL-10 was empirically determined to be 8.2 while that for the three variant populations were in the range 7.3-7.5 . Knowledge of these pI's would potentially facilitate the optimization process for ion-exchange chromatography . Furthermore, the technique provided a mild and fast preparation procedure for obtaining the recombinant protein and its variants for further characterization, as evidenced in the separation of rhIL-1- from variant C by successive RFFF treatments.

J Mol Biol, 1997 Mar 14, 266(5), 1032 - 42
Characterization of recombinant soybean leghemoglobin a and apolar distal histidine mutants; Hargrove MS et al.; The cDNA for soybean leghemoglobin a (Lba) was cloned from a root nodule cDNA library and expressed in Escherichia coli . The crystal structure of the ferric acetate complex of recombinant wild-type Lba was determined at a resolution of 2.2 A . Rate constants for O2, CO and NO binding to recombinant Lba are identical with those of native soybean Lba . Rate constants for hemin dissociation and auto-oxidation of wild-type Lba were compared with those of sperm whale myoglobin . At 37 degrees C and pH 7, soybean Lba is much less stable than sperm whale myoglobin due both to a fourfold higher rate of auto-oxidation and to a approximately 600-fold lower affinity for hemin . The role of His61(E7) in regulating oxygen binding was examined by site-directed mutagenesis . Replacement of His(E7) with Ala, Val or Leu causes little change in the equilibrium constant for O2 binding to soybean Lba, whereas the same mutations in sperm whale myoglobin cause 50 to 100-fold decreases in K(O2) . These results show that, at neutral pH, hydrogen bonding with His(E7) is much less important in regulating O2 binding to the soybean protein . The His(E7) to Phe mutation does cause a significant decrease in K(O2) for Lba, apparently due to steric hindrance of the bound ligand . The rate constants for O2 dissociation from wild-type and native Lba decrease significantly with decreasing pH . In contrast, the O2 dissociation rate constants for mutants with apolar E7 residues are independent of pH, suggesting that hydrogen bonding to the distal histidine residue in the native protein is enhanced under acid conditions . All of these results support the hypothesis that the high affinity of Lba for oxygen and other ligands is determined primarily by enhanced accessibility and reactivity of the heme group.

J Mol Biol, 1997 Mar 14, 266(5), 866 - 76
Design of CytR regulated, cAMP-CRP dependent class II promoters in Escherichia coli: RNA polymerase-promoter interactions modulate the efficiency of CytR repression; Kristensen HH et al.; In CytR regulated promoters in Escherichia coli, the cAMP-CRP complex acts as a transcriptional activator as well as a co-repressor for the CytR protein . Repression by CytR depends on the formation of nucleoprotein complexes in which CytR binds cooperatively to the DNA with one or two cAMP-CRP complexes . Here, we demonstrate that in order to establish CytR regulation in a cAMP-CRP dependent class II promoter with a single CRP site (CRP site centred around position -40.5) in which the CytR operator is located upstream of the CRP site, high affinity binding sites for both regulators are required . The efficiency of CytR regulation was observed to be modulated by RNA polymerase (RNAP)-promoter interactions . Specifically, in class II promoters with a single CRP site, the efficiency of CytR regulation was found to correlate inversely with cAMP-CRP independent promoter activity . These observations can be reconciled in a competition model for CytR regulation in which CytR and RNAP compete for cooperative binding with cAMP-CRP to the promoters in vivo . In this model, two mutually exclusive ternary complexes can be formed: a CytR/cAMP-CRP/promoter repression complex and an RNAP/cAMP-CRP/promoter activation complex . Thus, CytR regulation critically depends on formation of a repression complex that binds the promoter with sufficiently high affinity to exclude formation of the competing activation complex . We suggest that the transition from repression to activation involves a switch in the protein-protein interactions made by cAMP-CRP from CytR to RNAP . On the basis of the regulatory features of the promoters analysed here, we speculate about the advantages offered by the structural complexity of natural CytR/cAMP-CRP regulated promoters.

J Biol Chem, 1997 Mar 14, 272(11), 7494 - 500
cDNA sequence and catalytic properties of a chick embryo alcohol dehydrogenase that oxidizes retinol and 3beta,5alpha-hydroxysteroids; Kedishvili NY et al.; This study was undertaken to identify the cytosolic 40-kDa zinc-containing alcohol dehydrogenases that oxidize all-trans-retinol and steroid alcohols in fetal tissues . Degenerate oligonucleotide primers were used to amplify by polymerase chain reaction 500-base pair fragments of alcohol dehydrogenase cDNAs from chick embryo limb buds and heart . cDNA fragments that encode an unknown putative alcohol dehydrogenase as well as the class III alcohol dehydrogenase were identified . The new cDNA hybridized with two messages of approximately 2 and 3 kilobase pairs in the adult chicken liver but not in the adult heart, muscle, testis, or brain . The corresponding complete cDNA clones with a total length of 1390 base pairs were isolated from a chicken liver lambdagt11 cDNA library . The open reading frame encoded a 375-amino acid polypeptide that exhibited 67 and 68% sequence identity with chicken class I and III alcohol dehydrogenases, respectively, and had lower identity with mammalian class II (55-58%) and IV (62%) isozymes . Expression of the new cDNA in Escherichia coli yielded an active alcohol dehydrogenase (ADH-F) with subunit molecular mass of approximately 40 kDa . The specific activity of the recombinant enzyme, calculated from active site titration of NADH binding, was 3.4 min-1 for ethanol at pH 7.4 and 25 degrees C . ADH-F was stereospecific for the 3beta,5alpha- versus 3beta,5beta-hydroxysteroids . The Km value for ethanol at pH 7.4 was 17 mM compared with 56 microM for all-trans-retinol and 31 microM for epiandrosterone . Antiserum against ADH-F recognized corresponding protein in the chicken liver homogenate . We suggest that ADH-F represents a new class of alcohol dehydrogenase, class VII, based on its primary structure and catalytic properties.

J Biol Chem, 1997 Mar 14, 272(11), 7473 - 81
The hemopexin-like domain (C domain) of human gelatinase A (matrix metalloproteinase-2) requires Ca2+ for fibronectin and heparin binding . Binding properties of recombinant gelatinase A C domain to extracellular matrix and basement membrane components; Wallon UM et al.; The binding properties of the COOH-terminal hemopexin-like domain (C domain) of human gelatinase A (matrix metalloproteinase-2, 72-kDa gelatinase) were investigated to determine whether the C domain has binding affinity for extracellular matrix and basement membrane components . Recombinant C domain (rC domain) (Gly417-Cys631) was expressed in Escherichia coli, and the purified protein, identified using two antipeptide antibodies, was determined by electrospray mass spectrometry to have a mass of 25,925 Da, within 0.1 Da of that predicted . As assessed by microwell substrate binding assays and by column affinity chromatography, the matrix proteins laminin, denatured type I collagen, elastin, SPARC (secreted protein that is acidic and rich in cysteine), tenascin, and MatrigelTM were not bound by the rC domain . Unlike the hemopexin-like domains of collagenase and stromelysin, the rC domain also did not bind native type I collagen . Nor were native or denatured types II, IV, V, and X collagen, or the NC1 domain of type VII collagen bound . However, binding to heparin and fibronectin (Kd, 1.1 x 10(-6) M) could be disrupted by 0.58-0.76 and 0.3 M NaCl, respectively . Using nonoverlapping chymotrypsin-generated fragments of fibronectin, binding sites for the rC domain were found on both the 40-kDa heparin binding and the 120-kDa cell binding fibronectin domains (Kd values, approximately 4-6 x 10(-7) M) . The Ca2+ ion, but not the potential structural Zn2+ ion, were found to be essential for maintaining the binding properties of the protein . The apo-form of the rC domain did not bind heparin, and both ethylenediaminetetraacetic acid and the specific Ca2+ ion chelator 1, 2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, but not the Zn2+ ion chelator 1,10-phenanthroline, eluted the holo form of the rC domain from both heparin-Sepharose and fibronectin . Inductive coupled plasma mass spectrometry also did not detect a Zn2+ ion in the rC domain . In contrast, reduction with 65 mM dithiothreitol did not interfere with heparin binding, further emphasizing the crucial structural role played by the Ca2+ ion . Together, these data demonstrate for the first time that the hemopexin-like domain of gelatinase A has a binding site for fibronectin and heparin, and that Ca2+ ions are important in maintaining the structure and function of the domain.

J Biol Chem, 1997 Mar 14, 272(11), 7201 - 10
Domain organization of Escherichia coli transcript cleavage factors GreA and GreB; Koulich D et al.; The GreA and GreB proteins of Escherichia coli induce cleavage of the nascent transcript in ternary elongation complexes of RNA polymerase . Gre factors are presumed to have two biologically important and evolutionarily conserved functions: the suppression of elongation arrest and the enhancement of transcription fidelity . A three-dimensional structure of GreB was generated by homology modeling on the basis of the known crystal structure of GreA . Both factors display similar overall architecture and surface charge distribution, with characteristic C-terminal globular and N-terminal coiled-coil domains . One major difference between the two factors is the "basic patch" on the surface of the coiled-coil domain, which is much larger in GreB than in GreA . In both proteins, a site near the basic patch cross-links to the 3' terminus of RNA in the ternary transcription complex . GreA/GreB hybrid molecules were constructed by genetic engineering in which the N-terminal domain of one protein was fused to the C-terminal domain of the other . In the hybrid molecules, both the coiled-coil and the globular domains contribute to specific binding of Gre factors to RNA polymerase, whereas the antiarrest activity and the GreA or GreB specificity of transcript cleavage is determined by the N-terminal domain . These results implicate the basic patch of the N-terminal coiled-coil domain as an important functional element responsible for the interactions with nascent transcript and determining the size of the RNA fragment to be excised during the course of the cleavage reaction.

J Biol Chem, 1997 Mar 14, 272(11), 7173 - 81
Localization of structural elements of bee venom phospholipase A2 involved in N-type receptor binding and neurotoxicity; Nicolas JP et al.; We have shown previously that neurotoxic venom secretory phospholipases A2 (sPLA2s) have specific receptors in brain membranes called N-type receptors that are likely to play a role in the molecular events leading to neurotoxicity of these proteins . The sPLA2 found in honey bee venom is neurotoxic and binds to this receptor with high affinity . In this paper, we have used a number of mutants of bee venom sPLA2 produced in Escherichia coli to determine the structural elements of this protein that are involved in its binding to N-type receptors . Mutations in the interfacial binding surface, in the Ca2+-binding loop and in the hydrophobic channel lead to a dramatic decrease in binding to N-type receptors, whereas mutations of surface residues localized in other parts of the sPLA2 structure do not significantly modify the binding properties . Neurotoxicity experiments show that mutants with low affinity for N-type receptors are devoid of neurotoxic properties, even though some of them retain high enzymatic activity . These results provide further evidence for the involvement of N-type receptors in neurotoxic processes associated with venom sPLA2s and identify the surface region surrounding the hydrophobic channel of bee venom sPLA2 as the N-type receptor recognition domain.

J Biol Chem, 1997 Mar 14, 272(11), 7078 - 84
A study of Escherichia coli adenylosuccinate synthetase association states and the interface residues of the homodimer; Wang W et al.; The state of aggregation of adenylosuccinate synthetase from Escherichia coli is a point of controversy, with crystal structures indicating a dimer and some solution studies indicating a monomer . Crystal structures implicate Arg143 and Asp231 in stabilizing the dimer, with Arg143 interacting directly with bound IMP of the 2-fold related subunit . Residue Arg143 was changed to Lys and Leu, and residue Asp231 was changed to Ala . Matrix-assisted laser desorption ionization mass spectroscopy and analytical ultracentrifugation of the wild-type and the mutant enzymes indicate a mixture of monomers and dimers, with a majority of the enzyme in the monomeric state . In the presence of active site ligands, the wild-type enzyme exists almost exclusively as a dimer, whereas the mutant enzymes show only slightly decreased dissociation constants for the dimerization . Initial rate kinetic studies of the wild-type and mutant enzymes show similar kcat and Km values for aspartate . However, increases in the Km values of GTP and IMP are observed for the mutant . Changes in dissociation constants for IMP are comparable with changes in Km values . Our results suggest that IMP binding induces enzyme dimerization and that two residues in the interface region, Arg143 and Asp231, play significant roles in IMP and GTP binding.

J Biol Chem, 1997 Mar 14, 272(11), 7055 - 61
Molecular cloning and functional analysis of polyphosphoinositide-dependent phospholipase D, PLDbeta, from Arabidopsis; Pappan K et al.; A novel plant phospholipase D (PLD; EC 3.1.4.4) activity, which is dependent on phosphatidylinositol 4,5-bisphosphate (PIP2) and nanomolar concentrations of calcium, has been identified in Arabidopsis . This report describes the cloning, expression, and characterization of an Arabidopsis cDNA that encodes this PLD . We have designated names of PLDbeta for this PIP2-dependent PLD and PLDalpha for the previously characterized PIP2-independent PLD that requires millimolar Ca2+ for optimal activity . The PLDbeta cDNA contains an open reading frame of 2904 nucleotides coding for a 968-amino acid protein of 108,575 daltons . Expression of this PLDbeta cDNA clone in Escherichia coli results in the accumulation of a functional PLD having PLDbeta, but not PLDalpha, activity . The activity of the expressed PLDbeta is dependent on PIP2 and submicromolar amounts of Ca2+, inhibited by neomycin, and stimulated by a soluble factor from plant extracts . Sequence analysis reveals that PLDbeta is evolutionarily divergent from PLDalpha and that its N terminus contains a regulatory Ca2+-dependent phospholipid-binding (C2) domain that is found in a number of signal transducing and membrane trafficking proteins.

J Biol Chem, 1997 Mar 14, 272(11), 6994 - 7002
Comparative kinetic analysis and substrate specificity of the tandem catalytic domains of the receptor-like protein-tyrosine phosphatase alpha; Wu L et al.; The catalytic activity and substrate specificity of protein-tyrosine phosphatase alpha (PTPalpha) is primarily controlled by the membrane proximal catalytic domain (D1) . The membrane distal (D2) domain of PTPalpha by itself is a genuine PTPase, possessing catalytic activity comparable to that of D1 using aryl phosphates as substrates . Surprisingly, kcat and kcat/Km for the D2-catalyzed hydrolysis of phosphotyrosine-containing peptides are several orders of magnitude reduced in comparison with those of D1 . Substitution of the putative general acid/base Glu-690 in D2 by an Asp, which is invariably found in the WPD motifs in all cytoplasmic PTPases and all the D1 domains of receptor-like PTPases, only increases the kcat for D2 by 4-fold . Thus the much reduced D2 activity toward peptide substrates may be due to structural differences in the active sites other than the general acid/base . Alternatively, the D2 domain may have a functional active site with a highly stringent substrate specificity . PTPalpha display modest peptide substrate selectivity and are sensitive to charges adjacent to phosphotyrosine . In the sequence context of DADEpYLIPQQG (where pY stands for phosphotyrosine), the minimal sizes recognized by PTPalpha are either ADEpYLI or DADEpY-NH2.

J Biol Chem, 1997 Mar 14, 272(11), 6909 - 17
Heme oxygenase-1, intermediates in verdoheme formation and the requirement for reduction equivalents; Liu Y et al.; Conversion of heme to verdoheme by heme oxygenase-1 (HO-1) is thought to involve alpha-meso-hydroxylation and elimination of the meso-carbon as CO, a reaction supported by both H2O2 and NADPH-cytochrome P450 reductase/O2 . Anaerobic reaction of the heme-HO-1 complex with 1 eq of H2O2 produces an enzyme-bound intermediate identified by spectroscopic methods as alpha-meso-hydroxyheme . This is the first direct evidence for HO-1-catalyzed formation of alpha-meso-hydroxyheme . alpha-meso-Hydroxyheme exists as a mixture of Fe(III) phenolate, Fe(III) keto anion, and Fe(II) keto pi neutral radical resonance structures . EPR shows that complexation with CO enhances the Fe(II) pi neutral radical component . Reaction of the alpha-meso-hydroxyheme-HO-1 complex with O2 generates Fe(III) verdoheme, which can be reduced in the presence of CO to the Fe(II) verdoheme-CO complex . Thus, conversion of alpha-meso-hydroxyheme to Fe(III) verdoheme, in contrast to a previous report (Matera, K . M., Takahashi, S., Fujii, H., Zhou, H., Ishikawa, K., Yoshimura, T., Rousseau, D . L., Yoshida, T., and Ikeda-Saito, M . (1996) J . Biol . Chem . 271, 6618-6624), does not require a reducing equivalent . An electron is only required to reduce ferric to ferrous verdoheme in the first step of its conversion to biliverdin.

J Biol Chem, 1997 Mar 14, 272(11), 6846 - 9
Mapping of the novel protein kinase catalytic domain of Dictyostelium myosin II heavy chain kinase A; Cote GP et al.; Myosin heavy chain kinase A (MHCK A) in Dictyostelium was identified as a biochemical activity that phosphorylates threonine residues in the myosin II tail domain and regulates myosin filament assembly . The catalytic domain of MHCK A has now been mapped through the functional characterization of a series of MHCK A truncation mutants expressed in Escherichia coli . A recombinant protein comprising the central nonrepetitive domain of MHCK A (residues 552-841) was isolated in a soluble form and shown to phosphorylate Dictyostelium myosin II, myelin basic protein, and a synthetic peptide substrate . The functionally mapped catalytic domain of MHCK A shows no detectable sequence similarity to known classes of eukaryotic protein kinases but shares substantial sequence similarity with a transcribed Caenorhabditis elegans gene and with the mammalian elongation factor-2 kinase (calcium/calmodulin-dependent protein kinase III) . We suggest that MHCK A represents the prototype for a novel, widely occurring protein kinase family.

J Biol Chem, 1997 Mar 14, 272(11), 6842 - 5
ATP hydrolysis is critical for induction of conformational changes in GroEL that expose hydrophobic surfaces; Gorovits BM et al.; The degree of hydrophobic exposure in the molecular chaperone GroEL during its cycle of ATP hydrolysis was analyzed using 1,1'-bis(4-anilino)naphthalene-5,5'disulfonic acid (bisANS), a hydrophobic probe, whose fluorescence is highly sensitive to the environment . In the presence of 10 mM MgCl2 and 10 mM KCl the addition of ATP, but not ADP or AMP-PNP, resulted in a time-dependent, linear increase in the bisANS fluorescence . The rate of the increase in the bisANS fluorescence depended on the concentrations of both GroEL and the probe . The effect could be substantially inhibited by addition of excess ADP or by converting ATP to ADP using hexokinase, showing that the increase in the bisANS fluorescence was correlated with ATP hydrolysis . The rate of ATP hydrolysis catalyzed by GroEL was uncompetitively inhibited in the presence of bisANS . This uncompetitive inhibition suggests that the probe can interact with the GroEL-ATP complex . The inability of the nonhydrolyzable ATP analog, AMP-PNP, to cause a similar effect is explained by the interaction of bisANS with a transient conformational state of GroEL formed consequent to ATP hydrolysis . It is suggested that this short lived hydrophobic exposure reflects a conformational shift in GroEL that results from electrostatic repulsion between the bound products of ATP hydrolysis, and it plays an important role in the mechanism of the chaperonin cycle.

Biochim Biophys Acta, 1997 Mar 13, 1324(2), 263 - 72
A freeze-fracture study of the membrane morphology of phosphatidylethanolamine-deficient Escherichia coli cells; Rietveld AG et al.; Freeze-fracture electron microscopy was applied to study membrane morphology in a phosphatidylethanolamine-deficient E . coli strain . For growth, this strain requires millimolar concentrations of specific divalent cations like Mg2+ or Ca2+ . These cations bring the bilayer to nonbilayer phase transition temperature of the lipids back to wild type levels by shifting the phase preference of cardiolipin in the membrane towards the inverted hexagonal (H(II)) phase . Under growth conditions, these cells show a bilayer based membrane with an intramembrane particle distribution as in wild type cells . Upon lowering the temperature, smooth areas are observed corresponding to gel state lipid bilayer domains . Ca2+ was used to manipulate the phase behavior of the membrane lipids in situ . Exposing the cells to Ca2+ up to 100 mM at 42 degrees C did not result in the appearance of nonbilayer structures, despite the fact that in total lipid extracts under these conditions the hexagonal H(II) phase was observed . However, the addition of a Ca2+ ionophore, which leads to exposure to Ca2+ of both faces of the plasma membrane, gives rise to formation of H(II) phase, stacked bilayer domains and blebbing upon addition of 50 mM CaCl2 at 42 degrees C . We conclude that the asymmetrical localization of divalent cations in the periplasm of this strain allows them to be functionally effective while membrane stability is maintained.

Mutat Res, 1997 Mar 12, 383(2), 137 - 42
Hydrogen peroxide induces protection against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) effects in Escherichia coli; Asad LM et al.; Cross-adaptive response is defined as the capacity of cells to become resistant to a lethal agent when pretreated with a different lethal substance . In the present paper, the cross-adaptive response between hydrogen peroxide and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was studied in Escherichia coli repair mutants . Our results suggest that high doses of H2O2 induces protection against the lethal effects of MNNG in wild-type strain, ada, ogt, ada-ogt, aidB and alkA mutants . On the other hand, the MNNG induced mutagenesis is reduced by H2O2 pretreatment in wild-type and ogt mutant strains, but not in ada mutant . Furthermore, the protecting effect induced by H2O2 is time dependent: it decreases 15 min after the pretreatment and, after 30 min, is almost abolished . This reduction in the protecting effect is followed by an augmentation in the mutation frequency when MNNG is added 30 min after H2O2 pretreatment . This cross-adaptive response may be due to a modification of the MNNG alkylation pattern in the oxidized DNA.

Biochemistry, 1997 Mar 11, 36(10), 3037 - 46
Characterization of an unfolding intermediate and kinetic analysis of guanidine hydrochloride-induced denaturation of the colicin E1 channel peptide; Steer BA et al.; The equilibrium unfolding pathway of the colicin E1 channel peptide was shown in a previous study to involve an unfolding intermediate, stable in approximately 4 M guanidine hydrochloride, which comprised primarily the C-terminal hydrophobic alpha-helical hairpin segment of the peptide {Steer, B . A., & Merrill, A . R . (1995) Biochemistry 34, 7225-7233} . In this study, the structural nature of this unfolding intermediate was investigated further, and it was found that the intermediate primarily consists of a dimer species and is comprised of two partially denatured monomeric peptides, which appear to be associated by hydrophobic interactions . The dimerized structure was detected by size-exclusion high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, chemical cross-linking, and intermolecular fluorescence energy transfer . Using stopped-flow fluorescence spectroscopy, the kinetics of the denaturation and dimerization of the colicin E1 channel peptide in 4 M guanidine hydrochloride were examined . Denaturation kinetics were also investigated by wild-type peptide Trp fluorescence and 1-anilinonaphthalene-8-sulfonic acid binding . The kinetics of dimer formation were examined by monitoring the time dependence of intermolecular Trp to 5-{{2-{(iodoacetyl)amino}ethyl}amino}naphthalene-1-sulfonic acid fluorescence resonance energy transfer upon denaturation in 4 M guanidine hydrochloride . In addition, single Trp mutant peptides were employed as site-specific fluorescent probes of unfolding kinetics and reported diverse and characteristic unfolding kinetics . However, it was shown that following a rapid and major unfolding transition the peptide's core residues cluster slowly, by hydrophobic association, forming an intermediate species which is a prerequisite to dimerization . These equilibrium and kinetic unfolding data describe a unique unfolding mechanism where the channel peptide forms a partially unfolded dimerized structure in 4 M guanidine hydrochloride.

Biochemistry, 1997 Mar 11, 36(10), 2968 - 76
Recombinant expression of a type IV, cAMP-specific phosphodiesterase: characterization and structure-function studies of deletion mutants; Kovala T et al.; A potential role for cAMP in regulating the differentiation of myoblasts has led us to examine the components of the cAMP signaling system, including the type IV, cAMP-specific phosphodiesterases . The full coding sequence of the phosphodiesterase PDE4D1 was inserted in the bacterial expression vector pGEX-KG . N- and C-terminal truncations were also placed in the same vector, allowing the expression and purification of glutathione S-transferase (GST)-PDE fusion proteins using glutathione-Sepharose . The purified PDE was active {V(max) = 318 +/- 18 nmol min(-1)(mg of protein)(-1)} and inhibited by RO 20-1724, rolipram, and MIX (IC50 values of 2, 0.4, and 40 microM, respectively) . The requirement of PDE4D1 for a divalent cation was also examined . It was able to use Mg2+, Co2+, and Mn2+, but not Zn2+, suggesting that it is not a zinc hydrolase as has been proposed for other PDE types . Deletion of both C- and N-terminal regions affected the apparent native size of the enzyme . The C-terminal region was involved in dimer formation, whereas an N-terminal region was responsible for larger aggregates . Removal of the last 35 amino acids of an N-terminal 80-residue highly conserved region (UCR2) resulted in a 6-fold increase in PDE activity, providing evidence that this part of the molecule acts as an intramolecular inhibitor . The availability of a highly purified, enzymatically active protein in substantial quantities has allowed us to directly examine PDE4D1 for the first time.

Biochemistry, 1997 Mar 11, 36(10), 2932 - 8
Chemical modification and site-directed mutagenesis of the single cysteine in motif 3 of class II Escherichia coli prolyl-tRNA synthetase; Stehlin C et al.; Class II prolyl-tRNA synthetase (ProRS) from Escherichia coli contains all three of the conserved consensus motifs characteristic of class II aminoacyl-tRNA synthetases . In this study, chemical modification and site-directed mutagenesis of the single cysteine located at position 443 in motif 3 of Escherichia coli ProRS is carried out . We show that chemical modification of C443 blocks the ability of the enzyme to form the activated aminoacyl-adenylate, a prerequisite for tRNA(Pro) aminoacylation . Nearly complete protection from inactivation is achieved by preincubating the enzyme with ATP or ATP and proline, but not proline alone or tRNA(Pro) . Mutagenesis of C443 to amino acids Ala, Gly, and Ser resulted in significant decreases (16-225-fold) in k(cat)/K(M)(Pro) as measured by the ATP-PP(i) exchange reaction . The Ala and Gly mutations have a relatively small effect (4-7-fold) on the overall aminoacylation reaction, while the activity of the C443S mutant in this same assay is substantially reduced (80-fold) . A sequence comparison of the motif 3 region of class II synthetases shows that C443 aligns with residues that have been implicated in amino acid binding specificity . The results of our study suggest that while the thiol located at position 443 of Escherichia coli ProRS is not essential for catalysis, this residue is likely to be in a buried region that forms the prolyl-adenylate substrate binding pocket.

Biochemistry, 1997 Mar 11, 36(10), 2844 - 52
Study of structure and function of recombinant pea root plastid porin by biophysical methods; Popp B et al.; Pea root plastid porin (Fischer et al . (1994) J . Biol . Chem . 269, 25754-25760), which belongs to the family of mitochondrial (eukaryotic) porins, was expressed in Escherichia coli in high amounts using the pQE expression system . The recombinant protein was reconstituted into lipid bilayer membranes, and its characteristic properties were compared to those of the native porin isolated from pea root plastids . No significant difference was found between the native and the recombinant form when the protein was preincubated in detergent and sterol . The recombinant porin seems to be a valuable model system for the study of eukaryotic porins by spectroscopic methods, in which high amounts of protein are needed . CD spectroscopy was performed to determine the secondary structure of the porin under different conditions . It was found to have a high degree of beta-sheet structure in the nonionic detergent Genapol X-80 and in lipid vesicles . The more polar detergent sodium dodecyl sulfate (SDS) induced a large amount of alpha-helix structure in the protein . Addition of sterol to the porin in Genapol buffer did not influence its secondary structure to any measurable extent, whereas it had a strong influence on channel forming activity in black lipid bilayers . First refolding experiments performed in decreasing urea concentrations are discussed together with the results of the other measurements with regard to protein folding and channel formation.

FEBS Lett, 1997 Mar 10, 404(2-3), 314 - 8
Human mitochondrial import receptor, Tom20p . Use of glutathione to reveal specific interactions between Tom20-glutathione S-transferase and mitochondrial precursor proteins; Schleiff E et al.; The cytosolic domain of the human mitochondrial protein import receptor, hTom20, has been expressed as a fusion protein with glutathione S-transferase (GST) in bacteria and the purified protein immobilized on Sepharose beads . To discriminate between specific binding of precursor proteins with the receptor and non-specific binding, precursors were recovered as a complex with GST-hTom20 following competitive elution from the beads with reduced glutathione . Here, we describe the specificity of this assay and demonstrate that the cytosolic domain of hTom20 interacts directly with the transcription-translation product of precursor proteins that bear a diverse array of targeting signals . Such proteins include a matrix protein (pODHFR), a polytopic integral protein of the inner membrane (uncoupling protein), a beta-barrel protein of the outer membrane (VDAC/porin) as well as bitopic integral proteins which are inserted into the outer membrane by either an NH2-terminal or COOH-terminal signal anchor sequence (yTom70(1-29)DHFR and Bcl-2, respectively).

FEBS Lett, 1997 Mar 10, 404(2-3), 263 - 8
Cloning, sequencing and expression of VAT, a CDC48/p97 ATPase homologue from the archaeon Thermoplasma acidophilum; Pamnani V et al.; A member of the AAA family of Mg2(+)-ATPases from the archaeon Thermoplasma acidophilum has been cloned and expressed in Escherichia coli . The protein, VCP-like ATPase of Thermoplasma acidophilum (VAT), is a homologue of SAV from Sulfolobus acidocaldarius and CdcH of Halobacterium salinarium, and belongs to the CDC48/VCP/p97 subfamily . The deduced product of the vat gene is 745 residues long (Mr 83,000), which has an optimal Mg2(+)-ATPase activity at 70 degrees C . Electron microscopy shows the purified protein to form single and double homo-hexameric rings . Although the symmetry is different, the appearance of the complexes formed of two rings resembles the 20S proteasome and Hsp60/GroEL.

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