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