Microorganism

Lett Appl Microbiol, 1997 Oct, 25(4), 261 - 4
Production of a cell wall-specific monoclonal antibody to Fibrobacter succinogenes; Brigmon RL et al.; A monoclonal antibody (mAb) was raised against Fibrobacter succinogenes and produced after fusion as ascites in BALB/c mice . An ELISA was used to test for specificity and sensitivity of the mAb to detect F . succinogenes . The mAb BD1 was tested for sensitivity and cross-reactivity in detecting F . succinogenes with ELISA . The lower limits for F . succinogenes detection in pure and mixed culture-using mAb BD1 with ELISA was 10(5) cells ml-1 . Twenty-six other species of bacteria, including 12 cellulolytic species, were tested for cross-reactivity with the ELISA but none was detected . Electron micrographs of F . succinogenes cells with immunogold labelling showed that the mAb BD1 reacted exclusively with cell wall epitopes but not intracellular material, as confirmed by ELISA.

J Clin Microbiol, 1997 Nov, 35(11), 2931 - 6
Evaluation of performances of three DNA enzyme immunoassays for detection of Helicobacter pylori PCR products from biopsy specimens; Monteiro L et al.; PCR is recognized as a promising method for the detection of Helicobacter pylori in gastric biopsy specimens . However, detection of PCR products by gel electrophoresis is difficult to implement in routine clinical laboratories . The aim of this study was to compare three new DNA enzyme immunoassays with the standard method in their ability to detect PCR products . The three assays were based on the amplification of a fragment of the ureC gene of H . pylori and a colorimetric hybridization assay . The first assay (GEN-ETI-K DNA enzyme immunoassay; Sorin, Sallugia, Italy) was based on the hybridization of amplified DNA with a probe bound in microtiter wells and detected with labelled anti-DNA antibody . The second assay (Pylori-prob; Biocode, Sclessin, Belgium) comprised a solid-phase sandwich hybridization system with a specific biotinylated probe being used for detection . Finally, the third assay (PCR enzyme-linked immunosorbent assay; Boehringer, Mannheim, Germany) was based on the hybridization of amplified DNA labelled with digoxigenin as a probe (used as a coating in microtiter wells) and detected with antidigoxigenin-peroxidase as conjugate . The sensitivity of the colorimetric assay was evaluated by using amplification products from PCR assays performed on several 10-fold dilutions of DNA from H . pylori CIP 101260, and the specificity was assessed with different urease-positive bacteria . Biopsy specimens from 199 patients were tested; 106 were classified as H . pylori positive, and 93 were classified as H . pylori negative by culture and/or histological examination as the "gold standard." The receiving operating characteristic curve was used to determine the best cutoff point for each assay . The detection of PCR products by colorimetric hybridization increases the sensitivity up to 100-fold compared to that with gel electrophoresis . The results are rapid (4 h) and easy to interpret and can be automated.

Biochem Mol Biol Int, 1997 Oct, 43(2), 399 - 408
The role of reactive oxygen species (ROS) in the effector mechanisms of human antimycobacterial immunity; Fazal N; The aim of this study was to determine the role of reactive oxygen species (ROS) in checking the growth of intracellular mycobacteria within human phagocytes . Peripheral blood-derived neutrophils and monocyte-derived macrophages were isolated from Chronic Granulomatous Disease (CGD) patients and normal healthy human volunteers . CGD patients are known to have a defect in the NADPH oxidase pathway, resulting in their neutrophils and monocyte-derived macrophages being unable to generate oxygen radicals which are required to kill intracellular bacteria . The cells were then infected with Bacille Calmette Guerin (BCG) or Mycobacterium avium, and the bacterial growth in each cell type determined by Colony Forming Units (CFU) estimate . The results obtained indicate that there was no demonstrable inhibition in the intracellular mycobacterial growth within neutrophils or macrophages derived from either Chronic Granulomatous Disease (CGD:deficient in NADPH oxidase pathway) or normal healthy volunteers . Macrophage treatment with either IFN-gamma or TNF-alpha had no effect.

APMIS, 1997 Sep, 105(9), 680 - 8
Characterization of the lactoferrin-dependent inhibition of the adhesion of Actinobacillus actinomycetemcomitans, Prevotella intermedia and Prevotella nigrescens to fibroblasts and to a reconstituted basement membrane; Alugupalli KR et al.; Lactoferrin was previously shown to inhibit the adhesion of A . actinomycetemcomitans, P . intermedia and P . nigrescens to human cells . Lactoferrin was also shown to competitively inhibit the binding of these bacteria to the basement membrane protein laminin . The present study aimed to determine the type of interactions inhibited by lactoferrin . Lactoferrin binds to fibroblast monolayers and Matrigel, a reconstituted basement membrane, through ionic interactions . The adhesion of A . actinomycetemcomitans to these substrata was mainly dependent on the ionic strength of the environment . P . intermedia and P . nigrescens also adhere to fibroblasts mainly by ionic interactions, while their adhesion to Matrigel seems to be mediated by specific mechanisms . Lectin-type interactions were not found to be involved in the binding of these bacteria to the substrata . Treatment of either A . actinomycetemcomitans or fibroblasts with lactoferrin decreased the adhesion in a dose-dependent manner, while lactoferrin treatment of Matrigel alone had no adhesion-counteracting effect . Adhesion of P . intermedia and P . nigrescens to Matrigel was not significantly affected by the ionic strength, but the presence of lactoferrin inhibited the adhesion . Lactoferrin bound to Matrigel, P . intermedia and P . nigrescens was rapidly released, while lactoferrin bound to A . actinomycetemcomitans and fibroblasts was retained . These findings indicate that lactoferrin-dependent inhibition of the adhesion of A . actinomycetemcomitans, P . intermedia and P . nigrescens to fibroblasts and Matrigel can involve binding of lactoferrin to both the bacteria and substrata . The decreased adhesion may be due to blocking of both specific adhesin-ligand as well as non-specific charge-dependent interactions.

Acta Otolaryngol, 1997 Sep, 117(5), 724 - 7
Nasal congestion in relation to low air exchange rate in schools . Evaluation by acoustic rhinometry; Walinder R et al.; Upper airway symptoms are common, but there is little information available on clinical findings in relation to indoor air pollution . This pilot study was conducted to test whether increased levels of indoor air pollutants in schools may correlate to a swelling of the nasal mucosa . The assumption was made that the degree of swelling could be related to the degree of decongestive effect of xylometazoline, and measured by acoustic rhinometry . The study was performed among 15 subjects in a school with low air exchange rate (0.6 air changes/h) and 12 subjects in a school with high air exchange rate (5.2 air changes/h) . Hygienic measurements were performed in both schools . Acoustic rhinometry was performed for each individual under standardized forms . Cross-sectional areas and volumes of the nasal cavity were measured before and after decongestion with xylometazoline hydrochloride . Absolute values of the minimal cross-sectional area were lower in the school with poor ventilation . The decongestive effect of xylometazoline was significantly higher in the school with low air exchange, when correction for the influence of age was made . A diminished decongestive effect was seen with increasing age . The exposure measurements showed that indoor concentrations of volatile organic compounds, bacteria and moulds were higher in the school with low ventilation . In conclusion, raised levels of indoor air pollutants due to inadequate ventilation in schools may affect the upper airways and cause a swelling of the nasal mucosa, and acoustic rhinometry could be a useful objective method to measure human nasal reactions to the indoor environment.

J Vet Diagn Invest, 1997 Oct, 9(4), 363 - 7
Experimental use of a dot-blot assay to measure serologic responses of cattle vaccinated with Brucella abortus strain RB51; Olsen SC et al.; Brucella abortus strain RB51 was recently approved as an official brucellosis calfhood vaccine for cattle by the Animal and Plant Health Inspection Service branch of the United States Department of Agriculture . Currently available serologic surveillance tests for B . abortus do not detect seroconversion following SRB51 vaccination . The purpose of this study was to evaluate a dot-blot assay using gamma-irradiated strain RB51 bacteria for its specificity and sensitivity to detect antibody responses of cattle vaccinated with strain RB51 . Dot-blot titers of sera at a recommended dosage (10(10) colony-forming units) were similar to those of sera from cattle vaccinated with similar numbers of B . abortus strain 19 and greater (P < 0.05) than titers of nonvaccinated cattle . In the first 12 weeks after vaccination with 10(10) colony-forming units of strain RB51, the RB51 dot-blot assay had 100% specificity for titers of 80 or less and a 53% sensitivity for titers of 160 or greater . Sensitivity of the RB51 dot-blot assay peaked at 4 weeks after vaccination with 10(10) colony-forming units of strain RB51 . Dot-blot responses of sera from cattle vaccinated with a reduced dosage of strain RB51 (10(9) colony-forming units) did not differ (P > 0.05) from titers of sera from nonvaccinated cattle . Following intraconjunctival challenge with B . abortus strain 2308, titers on the RB51 dot-blot assay did not differ (P > 0.05) between nonvaccinated cattle and cattle vaccinated at calfhood with strain 19 or strain RB51.

Mikrobiol Z, 1997 May-Jun, 59(3), 46 - 53
Specific identification of Mycobacterium bovis by monoclonal antibody-based enzyme immunoassay; Liashchenko KP et al.; To improve identification of M . bovis, rabbit immune sera and mouse monoclonal antibodies against M . bovis-secreted protein antigens were used in the enzyme-linked assay (ELISA) . In western blot analysis, M . bovis-specific epitopes within culture filtrate proteins were demonstrated to be mostly concentrated on the immunodominant 35-38 kDa antigen . Polyclonal and cross-reactive monoclonal antibodies were shown to be able to bind to all mycobacterial strains tested in ELISA (M . tuberculosis, M . bovis, M . kansasii, M . marinum, M . avium, M . smegmatis), but not to bacteria of the other genera . Among the monoclonal antibodies against M . bovis specific antigen, 2-6B was found to be the only one which could evidently react with whole cells of M . bovis in ELISA, but not with those of the other mycobacteria including M . tuberculosis.

J Biol Chem, 1997 Oct 31, 272(44), 27823 - 9
Characterization of a putative helix-loop-helix motif in nucleotide excision repair endonuclease, XPG; Park MS et al.; Complementation group G of xeroderma pigmentosum (XPG) is one of the most rare and pathophysiologically heterogeneous forms of this inherited disease . XPG patients exhibit varying phenotypes, from having a very mild defect in DNA repair to being severely affected, and a few cases are also associated with the neurological degeneracy and growth retardation of Cockayne's syndrome . The XPG gene encodes a 134-kDa nuclear protein that is essential for the incision steps of nucleotide excision repair . XPG protein contains a putative helix-loop-helix (HLH) motif in the region that is most conserved among the members of structure-specific endonuclease family . To establish the functional significance of the HLH motif, we used several approaches, including theoretical modeling, functional complementation assay, structure-specific endonuclease assay, and DNA binding assay . A secondary structure of the motif was predicted by energy minimization and the Monte Carlo simulation and empirically proven using the circular dichroism to contain a high content of alpha-helix . When an XPG mutant lacking the HLH was overexpressed in UV135 cells, which have defects in the hamster homolog of XPG, the mutant gene failed to confer to the hamster cells the resistance to UV light . A recombinant XPG protein lacking the HLH motif was purified from insect cells and tested for a structure-specific endonuclease activity . The mutant protein failed to cleave the flap strand . A recombinant peptide containing the HLH (amino acids 758-871) was expressed in and purified from bacteria, tested for DNA binding activity, and found to bind to a DNA substrate with the flap structure . These results suggest that the HLH motif is required for the catalytic and DNA binding activities of XPG.

Eur J Biochem, 1997 Sep 1, 248(2), 592 - 601
Lipopolysaccharides of Helicobacter pylori serogroups O:3 and O:6--structures of a class of lipopolysaccharides with reference to the location of oligomeric units of D-glycero-alpha-D-manno-heptose residues; Aspinall GO et al.; Lipopolysaccharides (LPS) from antigenically different strains assigned to serogroups O:3 and O:6 of Helicobacter pylori were isolated as water-soluble material of high Mr and as water-insoluble gels of low Mr . Chemical and spectroscopic analyses of the soluble LPS and oligosaccharides liberated from the water-insoluble gels led to proposed structures with Lewis (Le) antigen determinants terminating regular repeating units of different types, linked in turn to inner core regions of invariable structure . The O:6 LPS has two populations of related molecules with chains of 3-linked D-glycero-alpha-D-manno-heptose residues similar to those in the MO19 strain, one with and the other without a single terminal Lewis (Le(y)) epitope . In contrast, in the O:3 LPS, Lewis (Le(x) and Le(y)) epitopes terminate a partially fucosylated N-acetyllactosaminoglycan, but a heptan chain similar to that in the O:6 LPS was shown to connect the outer chains to the inner core . These LPS provide examples of the molecular mimicry of cell-surface glycoconjugates . Structural variations of LPS between strains, and differences in some aspects of structure within strains, between high Mr and low Mr LPS indicate a class of LPS whose mechanisms of biosynthesis lead to overall architectures different from those characteristic of most LPS from enteric bacteria.

Curr Opin Struct Biol, 1997 Oct, 7(5), 738 - 48
Femtosecond spectroscopy of photosynthetic light-harvesting systems; Fleming GR et al.; Observing the elementary steps of light-harvesting in real time is now possible using femtosecond spectroscopy . This, combined with new structural data, has allowed a fairly complete description of light-harvesting in purple bacteria and substantial insights into higher plant antenna systems.

Mult Scler, 1995 Jun, 1(2), 88 - 94
Is LM7 a new human retrovirus or a mycoplasma virion?
Froussard P.
A putative retrovirus called LM7 was recently isolated from a patient with MS . This retrovirus was detected in LM7 and LM711 cultured human leptomeningeal cells . In the present work, nucleic acids from LM711 cell culture supernatants were purified and subjected to avian myeloblastosis viral (AMV) reverse transcriptase and to random polymerase chain reaction (rPCR) in order to characterize the genomic material of LM7 virions . Analysis of reverse transcription products allowed the detection of an approximately 14 kb ribonucleic acid in all LM711 cell culture supernatants . However, sequencing of rPCR-amplified molecules as well as RNA blotting data showed essentially that all tested cells producing LM7 particles were infected with mycoplasma . Moreover, purification of LM7 particles onto a linear sucrose density gradient established that the 14 kb nucleic acid was always associated with the 1.19-1.21 g ml-1 sucrose fractions, which are known to correspond to the buoyant density of mycoplasma . In addition, no viral genomic RNA was detected in the 1.17 g ml-1 sucrose fraction containing the low reverse transcription activity . These results strongly suggest that microscopic images and serological data could be related to mycoplasma and/or to a virion associated with the bacteria . The LM7 particle might be a new and additional enveloped virus able to infect Mycoplasma hyorhinis hosts . Thus, for instance, it would be presumptuous to assert, with our current understanding, that the LM7 virion is one of the causal agents of MS in humans.

Biochem Biophys Res Commun, 1997 Oct 9, 239(1), 291 - 7
Chaperonin genes of the Synechocystis PCC 6803 are differentially regulated under light-dark transition during heat stress; Glatz A et al.; Transcriptional startpoints of the two heat inducible chaperonin genes of Synechocystis PCC 6803 were mapped within the conservative CIRCE element and proved to be identical irrespective of the temperature treatment . Finding of an ORF encoding for a potential CIRCE binding repressor (HrcA) further suggests that both groEL-analogs are regulated in a CIRCE-dependent manner . In contrast to the expectations, the chaperonin twins are differentially expressed under light-dark transition during heat stress . Not the light per se, but rather the photosynthetic electron transport appears to be accountable for the regulatory differences . Our findings support the hypothesis that multiple chaperonins play different physiological roles under stress conditions.

Exp Cell Res, 1997 Oct 10, 236(1), 351 - 4
G1-phase arrest is not a prerequisite for encystment in Physarum; Anderson RW et al.; Amoebae of the Myxomycete Physarum polycephalum form resistant, walled cysts when the food bacteria in a culture have been consumed . No G1 phase has been detected in the vegetative amoebal cell cycle, most of which comprises the G2 phase . Mature cysts are also in G2, but it has been reported that a G1 phase of roughly 24 h, followed by an S phase, is obligatory prior to encystment . We used flow cytometry to determine the distribution of DNA contents in amoebal cultures at intervals during vegetative growth and encystment . In all cultures, the cells were predominantly in G2 phase, and the percentage of cells with G1 DNA content remained very low . We conclude that an extended G1 phase of 24 h did not occur in our cultures and cannot be a prerequisite for encystment.

Nat Genet, 1997 Oct, 17(2), 198 - 200
Mutation of the gene encoding cellular retinaldehyde-binding protein in autosomal recessive retinitis pigmentosa; Maw MA et al.; Inadequate levels of all-trans-retinol in the blood cause retinal dysfunction; hence, genes implicated in retinal vitamin-A metabolism represent candidates for inherited retinal degenerations . In the current study, molecular genetic analysis of a consanguineous pedigree segregating for non-syndromic autosomal recessive retinitis pigmentosa (arRP) indicated that the affected siblings were homozygous by descent for a G4763A nucleotide substitution in RLBP1, the gene encoding cellular retinaldehyde-binding protein (CRALBP) . This substitution is predicted to replace an arginine with glutamine at residue 150 . CRALBP is not expressed in photoreceptors but is abundant in the retinal pigment epithelium (RPE) and Muller cells of the neuroretina, where it carries 11-cis-retinol and 11-cis-retinaldehyde . When expressed in bacteria, recombinant CRALBP (rCRALBP) containing the R150Q substitution was less soluble than wild-type rCRALBP . Mutant rCRALBP was purified from the soluble cell lysate and the protein structure was verified by mass spectrometry . The mutant protein lacked the ability to bind 11-cis-retinaldehyde . These findings suggest that arRP in the current pedigree results from a lack of functional CRALBP, presumably leading to disruption of retinal vitamin-A metabolism.

J Neurochem, 1997 Oct, 69(4), 1738 - 45
Amino-terminal analysis of tryptophan hydroxylase: protein kinase phosphorylation occurs at serine-58; Kumer SC et al.; Tryptophan hydroxylase (TPH) catalyzes the rate-limiting and committed step in serotonin biosynthesis . Within this enzyme, two distinct domains have been hypothesized to exist, an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain . In the present experiments, the functional boundary between the putative domains was defined using deletion mutagenesis . A full-length cDNA clone for rabbit TPH was engineered for expression in bacteria . Five amino-terminal deletions were constructed using PCR, i.e., Ndelta50, Ndelta60, Ndelta90, Ndelta106, and Ndelta116 (referring to the number of amino acids deleted from the amino terminus) . Enzymatic activity was determined for each mutant after expression in bacteria . Whereas deletion of 116 amino acids (Ndelta116) abolished enzyme activity, all of the other amino-terminal deletions exhibited increased specific activity relative to the recombinant wild-type TPH . The ability of the cyclic AMP-dependent protein kinase (PKA) to phosphorylate members of the deletion series was also examined . Deletion of the first 60 amino-terminal residues abolished the ability of the enzyme to serve as a substrate for PKA, yet the native and Ndelta50 enzymes were phosphorylated . Moreover, a serine-58 point mutant (S58A) was not phosphorylated by PKA . In conclusion, the first 106 amino acids comprise a regulatory domain that is phosphorylated by PKA at serine-58 . In addition, the boundary between regulatory and catalytic domains is analogous to the domain structure observed for the related enzyme tyrosine hydroxylase.

J Biol Chem, 1997 Oct 10, 272(41), 25899 - 906
Molecular and functional evidence for multiple Ca2+-binding domains in the type 1 inositol 1,4,5-trisphosphate receptor; Sienaert I et al.; Structural and functional analyses were used to investigate the regulation of the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) by Ca2+ . To define the structural determinants for Ca2+ binding, cDNAs encoding GST fusion proteins that covered the complete linear cytosolic sequence of the InsP3R-1 were expressed in bacteria . The fusion proteins were screened for Ca2+ and ruthenium red binding through the use of 45Ca2+ and ruthenium red overlay procedures . Six new cytosolic Ca2+-binding regions were detected on the InsP3R in addition to the one described earlier (Sienaert, I., De Smedt, H., Parys, J . B., Missiaen, L., Vanlingen, S., Sipma, H., and Casteels, R . (1996) J . Biol . Chem . 271, 27005-27012) . Strong 45Ca2+ and ruthenium red binding domains were localized in the N-terminal region of the InsP3R as follows: two Ca2+-binding domains were located within the InsP3-binding domain, and three Ca2+ binding stretches were localized in a 500-amino acid region just downstream of the InsP3-binding domain . A sixth Ca2+-binding stretch was detected in the proximity of the calmodulin-binding domain . Evidence for the involvement of multiple Ca2+-binding sites in the regulation of the InsP3R was obtained from functional studies on permeabilized A7r5 cells, in which we characterized the effects of Ca2+ and Sr2+ on the EC50 and cooperativity of the InsP3-induced Ca2+ release . The activation by cytosolic Ca2+ was due to a shift in EC50 toward lower InsP3 concentrations, and this effect was mimicked by Sr2+ . The inhibition by cytosolic Ca2+ was caused by a decrease in cooperativity and by a shift in EC50 toward higher InsP3 concentrations . The effect on the cooperativity occurred at lower Ca2+ concentrations than the inhibitory effect on the EC50 . In addition, Sr2+ mimicked the effect of Ca2+ on the cooperativity but not the inhibitory effect on the EC50 . The different {Ca2+} and {Sr2+} dependencies suggest that three different cytosolic interaction sites were involved . Luminal Ca2+ stimulated the release without affecting the Hill coefficient or the EC50, excluding the involvement of one of the cytosolic Ca2+-binding sites . We conclude that multiple Ca2+-binding sites are localized on the InsP3R-1 and that at least four different Ca2+-interaction sites may be involved in the complex feedback regulation of the release by Ca2+.

Anal Biochem, 1997 Oct 1, 252(1), 96 - 105
Construction of an epitope-tagged calmodulin useful for the analysis of calmodulin-binding proteins: addition of a hemagglutinin epitope does not affect calmodulin-dependent activation of smooth muscle myosin light chain kinase; Szymanska G et al.; An epitope-tagged calmodulin (CaM), capable of interacting with CaM-binding proteins in cellular extracts, would be a valuable tool for identifying proteins in signal transduction pathways involving calcium . A bacterial overexpression vector for epitope-tagged CaM has been constructed by inserting the coding sequence for a nine amino acid portion of the influenza virus hemagglutinin (HA) protein into the initiation site of an overexpression vector for chicken CaM . The HA-CaM fusion produced in bacteria was compared to native CaM for its ability to activate smooth muscle myosin light chain kinase (MLCK), one of the best understood CaM-dependent enzymes . MLCK activity was tested in both a purified system and a CaM-depleted "native actomyosin" preparation maintaining many of the regulatory properties of the intact smooth muscle . HA-CaM behaves identically to unmodified CaM in both systems, indicating that the HA epitope does not adversely affect CaM function . The recombinant HA-CaM was used to sensitively detect CaM interactions with smooth muscle proteins in a modified gel overlay assay, using a monoclonal antibody against the HA epitope as the secondary reagent . Enzymatically active complexes of HA-CaM and MLCK could be immunoprecipitated from actomyosin preparations using the same monoclonal antibody and protein G-Sepharose beads.

Anal Biochem, 1997 Oct 1, 252(1), 1 - 9
Measurement of phospholipase D activity; Morris AJ et al.; Phosphodiesteric cleavage of phosphatidylcholine by members of a growing family of phospholipases D produces choline and phosphatidic acid . These enzymes can also catalyse a transphosphatidylation reaction in which the aliphatic chain of a primary alcohol is transferred to the phosphatidyl moiety of the phosphatidic acid product . PLD enzymes are found in a variety of organisms including bacteria, yeast, plants, and vertebrates . In mammalian systems, biochemical and cell biological approaches have identified phosphatidic acid as a mediator (or progenitor of mediators) that play important roles in the transduction of extracellular signals . Phosphatidic acid or its metabolites may be regulators of key cellular processes such as the control of intracellular protein trafficking, secretion, and alterations in cell morphology and motility . This review discusses methods for the determination of PLD activity both in vitro and in intact cells.

Dtsch Tierarztl Wochenschr, 1997 Aug, 104(8), 306 - 12
{Prophylactic measures against infection in dairy cattle--necessity of hygiene management}; Fehlings K et al.; Prophylactic measures against infection are really essential to the preservation of the udder-health and the quality of the milk . When curing a dairy cattle population suffering from mastitis strict hygienic measures for the completion and maintenance of the therapy must be taken absolutely consequently . On the occasion of a study made in Bavaria for the period of one year 9948 dairy farmers were asked if hygienic measures against infection were taken . In detail it was evaluated if the secret was controlled during pre-milking, if the cleaning of the teats before milking and the post-milking teat end disinfection (teat dipping) was done, if dry cow therapy was practiced, if the milking machine was serviced and ist functioning checked regularly.

Immunity, 1997 Sep, 7(3), 433 - 44
ATP-induced killing of mycobacteria by human macrophages is mediated by purinergic P2Z(P2X7) receptors; Lammas DA et al.; The death of BCG-infected human macrophages induced in vitro by ligation of surface CD95 (Fas), CD69, or complement-mediated lysis was shown not to result in the death of intracellular mycobacteria, whereas exposure to extracellular ATP initiated both macrophage death and killed the intracellular bacteria . ATP acted via P2Z receptors because these effects were mimicked by benzoylbenzoic ATP (a known agonist of P2Z receptors) and blocked by oxidized ATP, DIDS, suramin, amiloride, and KN62 (known inhibitors of P2Z-mediated responses) . ATP-mediated bacterial killing was independent of reactive nitrogen and oxygen intermediates and of actinomycin D or cycloheximide inhibition . ATP-induced macrophage cell death, BCG killing, and lucifer yellow dye incorporation were minimal in 2 out of 19 healthy donors . The results suggest possible genetic heterogeneity of this mechanism of mycobacterial killing associated with P2Z-mediated pore formation.

Scand J Prim Health Care, 1997 Sep, 15(3), 156 - 60
Acute bronchitis in adults . How close do we come to its aetiology in general practice?
Jonsson JS, Sigurdsson JA, Kristinsson KG, Guthnadottir M, Magnusson S.
OBJECTIVE: To investigate how close we can come to the aetiology of acute bronchitis in adults in a primary care setting . DESIGN: Prospective study . SETTING: General practice population in Gardabaer district, south-western Iceland . SUBJECTS: 140 patients > or = 16 years old who were diagnosed as having acute bronchitis during a two-year period (1992-1993) . MAIN OUTCOME MEASURES: Laboratory investigations (twice with a minimum four-week interval), used in general practice to analyse respiratory tract infections . They included serology for Chlamydia pneumoniae, Mycoplasma pneumoniae, respiratory tract viruses, and the level of C-reactive protein . RESULTS: Of a total of 140 patients, two blood samples were taken on scheduled time in 113 patients . Serology confirmed recent infection in 18 (16%) of these patients . Only two (2%) had a bacterial infection (one C . pneumoniae, one M . pneumoniae) . The others (84%) did not have a significant increase in antibody titres . Only four (4%) had C-reactive protein levels higher than 48 mg/l . CONCLUSIONS: The study indicates that it is difficult to come close to a precise aetiology with respect to infectious agents of acute bronchitis in general practice . We conclude that the disease is rarely caused by atypical bacteria such as C . pneumoniae and M . pneumoniae, and rarely caused by bacterial infections severe enough significantly to increase the level of C-reactive protein.

J Lipid Res, 1997 Sep, 38(9), 1923 - 7
Preparation of a naturally occurring D-erythro-(2S, 3R)-sphingosylphosphocholine using Shewanella alga NS-589; Sueyoshi N et al.; Sphingosylphosphocholine, an N-deacylated derivative of sphingomyelin, has been found to be involved in many cellular events . This paper describes a new method for preparation of a D-erythro-sphingosylphosphocholine, which is naturally occurring but difficult to prepare by chemical methods, using marine bacteria as a biocatalyst . When cultured with Shewanella alga NS-589 in synthetic medium, sphingomyelin was found to be efficiently converted by sphingomyelin deacylase to sphingosylphosphocholine . Sphingosylphosphocholine was purified with a high yield from the culture supernatant and identified to be a D-erythro-(2S,3R)-isomer containing d18:1 sphingenine as a long-chain base by fast atom bombardment mass spectrometry and NMR analyses.

J Extra Corpor Technol, 1997 Dec, 29(4), 181 - 4
Extracorporeal circuit sterility after 168 hours; Young WV et al.; One of the most important tasks of the perfusionist is the proper assembly of the extracorporeal circuit (ECC) prior to the initiation of cardiopulmonary bypass (CPB) . The ECC is usually assembled, primed and debubbled 30 minutes to one hour prior to the patient entering the operating room . But there are occasions when the ECC may have been set up and the previously scheduled procedure cancelled . Perfusionists in this situation have found themselves in a quandary; dispose of the ECC because of required nursing compliance and the sterility question, or keep it and use it later because of the economic impact on the "bottom line" . Some hospitals may have satisfactorily answered the question of ECC sterility after 24 hours without observation, but the few reported papers regarding this issue, and our desire to save these circuits, inspired us to find out if they were in fact sterile after having been open for a long period of time . The purpose of this study was to evaluate ECC sterility using an open reservoir oxygenator, over a time period of seven days . After obtaining 792 bacterial cultures from three sites within the ECC, the study was terminated . There were no positive bacterial cultures during the study period . Assuming there is no deliberate contamination, pump circuits assembled in an unused operating room can be maintained sterile for a period of seven days.

Am J Infect Control, 1997 Oct, 25(5), 424 - 5
Who washes hands after using the bathroom?
Guinan ME, McGuckin-Guinan M, Sevareid A.
Handwashing is one of the most important control measures for preventing the spread of bacteria . Although young children are taught the procedure through different types of behavior modification, its effect has not been measured in older children . We have documentation that adults and health care workers have a compliance rate of only 50% with this basic control measure . This article reports on the compliance rate, duration, and handwashing techniques used by middle and high school students after using the bathroom.

J Mol Biol, 1997 Oct 3, 272(4), 493 - 508
Sensing homology at the strand-swapping step in lambda excisive recombination; Nunes-Duby SE et al.; lambda Site-specific recombination requires a short stretch of sequence homology that might be sensed during strand swapping, during ligation and/or during isomerization of the obligate Holliday junction intermediate . Here, we use half-att site suicide substrates to study single and double top-strand-transfers, isolated from the subsequent steps of the reaction . The double-strand-transfer is analogous to a top-strand exchange and consists of one normal top-strand and one "contrary" bottom-strand to top-strand ligation between the half-att site substrate and its full-site partner . The resulting covalent three-way DNA junctions are poor substrates for resolution in the forward or reverse direction . We show that both the rate and the efficiency of Y-junction formation are homology dependent . Pairing of three nucleotides (either in the forward or in the contrary alignment) provides maximal stability to strand swapping . Complementary base-pairing next to one top-strand site (with or without ligation) stimulates strand-transfer at the other mismatched site . The data suggest that homology can be sensed at the strand-swapping step before ligation . However, homology also stimulates ligation and stabilizes the products, as is evident from the different rates of closed Y-junction formation in the presence or absence of homology . Furthermore, under recombination conditions, single top-strand-transfers are subject to reversal even in the presence of sequence homology; stability depends on a double-strand-transfer, i.e . dissociation of covalent Int .

Proc Natl Acad Sci U S A, 1997 Oct 28, 94(22), 11911 - 6
A structural census of the current population of protein sequences; Gerstein M et al.; We examine the occurrence of the approximately 300 known protein folds in different groups of organisms . To do this, we characterize a large fraction of the currently known protein sequences ( approximately 140,000) in structural terms, by matching them to known structures via sequence comparison (or by secondary-structure class prediction for those without structural homologues) . Overall, we find that an appreciable fraction of the known folds are present in each of the major groups of organisms (e.g., bacteria and eukaryotes share 156 of 275 folds), and most of the common folds are associated with many families of nonhomologous sequences (i.e., >10 sequence families for each common fold) . However, different groups of organisms have characteristically distinct distributions of folds . So, for instance, some of the most common folds in vertebrates, such as globins or zinc fingers, are rare or absent in bacteria . Many of these differences in fold usage are biologically reasonable, such as the folds of metabolic enzymes being common in bacteria and those associated with extracellular transport and communication being common in animals . They also have important implications for database-based methods for fold recognition, suggesting that an unknown sequence from a plant is more likely to have a certain fold (e.g., a TIM barrel) than an unknown sequence from an animal.

Proc Natl Acad Sci U S A, 1997 Oct 28, 94(22), 11881 - 6
Conformation of coenzyme pyrroloquinoline quinone and role of Ca2+ in the catalytic mechanism of quinoprotein methanol dehydrogenase; Zheng YJ et al.; The ab initio structures of 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), semiquinone (PQQH), and dihydroquinone (PQQH2) have been determined and compared with ab initio structures of the (PQQ)Ca2+, (PQQH)Ca2+, and (PQQH2)Ca2+ complexes as well as the x-ray structure of (PQQ)Ca2+ bound at the active site of the methanol dehydrogenase (MDH) of methyltropic bacteria . Plausible mechanisms for the MDH oxidation of methanol involving the (PQQ)Ca2+ complex are explored via ab initio computations and discussed . Considering the reaction of methanol with PQQ in the absence of Ca2+, nucleophilic addition of methanol to the PQQ C-5 carbonyl followed by a retro-ene elimination is deemed unlikely due to large energy barrier . A much more favorable disposition of the methanol C-5 adduct to provide formaldehyde involves proton ionization of the intermediate followed by elimination of methoxide concerted with hydride transfer to the oxygen of the C-4 carbonyl . Much the same transition state is reached if one searches for the transition state beginning with Asp-303-CO2-general-base removal of the methanol proton of the (PQQ)Ca2+O(H)CH3 complex concerted with hydride transfer to the oxygen at C-4 . For such a mechanism the role of the Ca2+ moiety would be to (i) contribute to the formation of the ES complex (ii) provide a modest decrease in the pKa of methanol substrate,; and (iii) polarize the oxygen at C-5.

Eur J Biochem, 1997 Sep 15, 248(3), 889 - 96
Purification and characterization of an alcohol:N,N-dimethyl-4-nitrosoaniline oxidoreductase from the methanogen Methanosarcina barkeri DSM 804 strain Fusaro; Daussmann T et al.; Cell-free extracts of Methanosarcina barkeri DSM 804 showed alcohol dehydrogenase activity under aerobic conditions when N,N-dimethyl-4-nitrosoaniline (NDMA) was used as an artificial electron acceptor . The NDMA-dependent alcohol dehydrogenase (NDMA-ADH) was purified to approximate homogeneity by column chromatography . It is most probably a homodimeric enzyme consisting of subunits of 45 kDa, the native molecular mass estimated by gel filtration being about 87 kDa . The purified protein had an isoelectric point of 4.3 . It possesses a tightly but noncovalently bound NADP(H) cofactor . Each subunit contains 1 mol NADP(H)/mol, about 2 mol Zn2+/mol and significant amounts of magnesium . The purified enzyme preferably oxidized primary alcohols (including benzyl alcohol) . NDMA-ADH from M . barkeri also catalyzed the stoichiometric dismutation of aldehydes, especially higher aliphatic aldehydes, to form equimolar amounts of the corresponding alcohol and acid without addition of an electron carrier . The enzyme did not catalyze the dehydrogenation of methanol or the disproportionation of formaldehyde and therefore is not directly involved in methanogenesis . An alignment of the N-terminal amino acid sequence of the enzyme with the sequences of other alcohol dehydrogenases from methanogenic and nonmethanogenic bacteria indicated no significant identity . Nevertheless there was a quite interesting sequence similarity in the first 30 N-terminal amino acids to plant cinnamyl alcohol dehydrogenase . NDMA-ADH from M . barkeri is a novel type of alcohol dehydrogenase in methanogenic bacteria.

Mol Gen Genet, 1997 Sep, 256(1), 45 - 53
Overexpression of the protein kinase Pak1 suppresses yeast DNA polymerase mutations; Hovland PG et al.; This article presents the identification and characterization of the PAK1 gene of Saccharomyces cerevisiae, and the biochemical characterization of the protein kinase activity that it encodes . Overexpression of the PAK1 gene product suppresses temperature-sensitive mutations of the poll (cdc 17) gene, which encodes DNA polymerase alpha . Overexpression and suppression can be achieved either by expressing PAK1 from a high-copy-number plasmid, or by GAL1-induced transcription of PAK1 . Gene disruption of PAK1 indicates that it is not an essential gene . The PAK1 gene encodes a protein with a kinase consensus domain . By deletion analysis and site-directed mutagenesis, we demonstrate that the complete and active kinase consensus domain is required for suppression . A glutathione-S-transferase (GST)-Pak1 fusion protein, overproduced in bacteria, can be purified in an active form with glutathione affinity beads or by immunoprecipitation . Thus, other protein subunits of Pak1 are not required for its activity . In vitro protein kinase assays show that GST-Pak1 can autophosphorylate, and can phosphorylate casein as an exogenous substrate . The phenotype of the suppressed cdc17-1 cells indicates that Pak1 suppression is inefficient and does not restore the wild-type phenotype . Pak1 suppression requires Rad9 function, but Pak1 does not affect Rad9 function . Overexpression of PAK1 does not enhance the expression of the POL1 gene . Pak1 may function by modifying and partially stabilizing thermolabile DNA polymerases, perhaps during DNA repair, because pak1 mutant cells are caffeine sensitive.

Hosp Pract (Off Ed), 1997 Oct 15, 32(10), 23 - 4, 26
Massive hemoptysis in a woman with seizures; Dizon MN et al.; A 53-year-old woman presented with a productive cough, fever, chills, and night sweats of one month's duration . She reported having had lightly blood-streaked sputum initially but then experiencing massive hemoptysis (> 200 mL/2 hr) . Since the onset of symptoms, she had had malaise, body aches, and a 27-lb weight loss . For the last two weeks, she had also had increasing shortness of breath and pleuritic chest pain.

Anthropol Anz, 1997 Jun, 55(2), 131 - 41
{Biogenic decomposition of bone collagens}; Turban-Just S; Soil bacteria, which are an always present component of all burial conditions, substantially contribute to the decomposition of bone collagen, i.e . even modifications of preserved bone collagen . Archaeometric data, which were derived from collagen remnants, therefore may always contain diagenetic components which disturb the biological signals and must subsequently be eliminated . To adequately assess probable diagenetic influences one should refer to our model of the biogenic steps of decomposition . The taphonomic experiments and their verifications of archaeological human bones, which were gathered from different origins and of different historical times, were done with histological, biochemical and biophysical methods . Experimentally, as well as through archaeological bone analysis, we were able to identify a "high" molecular product of decomposition, which contains (1) changes in the amino acid composition caused by specific soil conditions and (2) significant deviations of stable nitrogen and carbon isotopes . Due to decomposition procedures, isotope analysis of isolated amino acids indicate rearrangements of collagen fragments.

Ugeskr Laeger, 1997 Sep 22, 159(39), 5800 - 4
{Occupational respiratory tract allergy in trout processing workers}; Tougard AB et al.; Out of 16 workers in a trout processing industry, ten experienced work-related cough, dyspnoea, and nasal secretion . A clinical examination was performed including specific IgE, precipitating antibodies IgG for trout and processing water, skin prick testing and peak flow monitoring . A total of four workers showed a positive allergic reaction . Processing water contained endotoxin and bacteria in high amounts . It is concluded, that work-related respiratory symptoms should be investigated and the cause at the workplace identified, so that preventive measures can be introduced.

Dtsch Tierarztl Wochenschr, 1997 Mar, 104(3), 120 - 2
{Case report: decreased laying performance as a contributing problem}; Zentek J et al.; In 2 laying hen flocks (housed in aviary systems) depressed performance, increased mortality and cannibalism were observed . Specific infectious diseases were excluded . The hygienic quality of the diet, however, based on self produced feed ingredients, was impaired . High concentrations of bacteria, moulds, and yeasts were found in the complete diet and also in the ingredients . These problems were eliminated by providing the birds with good quality feed ingredients . In a diagnostic feeding trial with a change in housing conditions hens showed a good performance . The poor feed quality, as demonstrated in this report, is regarded to be one of several factors, which have contributed to the problems observed in these flocks . Other negative environmental factors such as housing have to be eliminated.

Chirurg, 1997 Jul, 68(7), 744 - 8
{Percutaneous ultrasound controlled drainage of large splenic abscesses}; Schaberle W et al.; Because of the rare incidence of splenic abscesses and bleeding, only a few cases are described in the literature on percutaneous drainage of splenic abscesses . We report on eight cases (three male, five female, average age 74.1 +/- 10.76 years) of percutaneous, sonographically controlled catheter drainage of large splenic abscesses . Results: Percutaneous, sonographically controlled drainage of splenic abscesses was feasible in all eight cases in the last 5 years (trocar technique, drain: 12-16 F, abscess contents 70-750 ml) . In seven of eight cases, therapy with percutaneous, sonographically controlled drainage of the splenic abscess and rinsing of the abscess cavity over several days was successful . In two cases, however, a recurrent abscess had to be drained repeatedly with sonographic control, and this was successful . In one case a colitis-related fistula prevented successful drainage of an infected subcapsular splenic hematoma . The following splenectomy, however, proved the infected hematoma to be completely drained . In this study there were no complications like bleeding, injury of pleura or colon . The advantages of percutaneous drainage are: the spleen is not removed; conservative therapy is beneficial, particularly in multimorbid patients with a high surgical risk; there is no transmission of bacteria; the method is safe and effective.

Strahlenther Onkol, 1997 Sep, 173(9), 457 - 61
Molecular processes and radiosensitivity; Zdzienicka MZ; BACKGROUND: DNA double-strand breaks (DSB) are the most genotoxic lesions induced by ionizing radiation . At least 2 different pathways for DSB repair have been identified, homologous and non-homologous recombination . METHODS: Studies on X-ray-sensitive mutants have led to the identification of several genes involved in processing of DSB in bacteria, yeast and mammalian cells . RESULTS AND CONCLUSION: In mammalian cells non-homologous recombination is the main pathway for DSB repair, while the role of homologous recombination in DSB repair awaits clarification . It is known that, in addition to DNA repair, other safeguards control the human cellular response to ionizing radiation, such as cell cycle regulation and mechanisms involved in scavenging of free radicals produced by ionizing radiation.

J Bacteriol, 1997 Oct, 179(19), 6205 - 7
Characterization of a porin from Mycobacterium smegmatis; Mukhopadhyay S et al.; A pore-forming protein with an Mr of 40,000 has been extracted from the cell wall of Mycobacterium smegmatis with buffer containing the detergent Zwittergent 3-12 and 0.5 M NaCl and purified on an anion-exchange column . Although the pore diameter was large (2 nm), the specific activity was much lower than those of nonspecific porin channels of enteric bacteria . The channel allowed the permeation of small hydrophilic molecules such as sugars and amino acids . Its N-terminal sequence did not show any similarity to those of other porins sequenced so far.

J Bacteriol, 1997 Oct, 179(19), 6154 - 62
Characterization of novel long-chain 1,2-diols in Thermus species and demonstration that Thermus strains contain both glycerol-linked and diol-linked glycolipids; Wait R et al.; In this study, we purified and characterized tetra- and triglycosyl glycolipids (GL-1 and GL-2, respectively) from two different colonial forms of Thermus scotoductus X-1, from T . filiformis Tok4 A2, and from T . oshimai SPS-11 . Acid hydrolysis of the purified glycolipids liberated, in addition to the expected long-chain fatty acids, two components which were identified by gas chromatography-mass spectrometry as 16-methylheptadecane-1,2-diol and 15-methylheptadecane-1,2-diol . Fast atom bombardment mass spectrometry of the intact glycolipids indicated that a major proportion consisted of components with glycan head groups linked to long-chain 1,2-diols rather than to glycerol, although in all cases glycerol-linked compounds containing similar glycan head groups were also present . As in other Thermus strains, the polar head group of GL-1 from T . filiformis Tok4 A2 and from T . scotoductus X-1 colony type t2 was a glucosylgalactosyl-(N-acyl)glucosaminylglucosyl moiety . However, GL-2 from T . scotoductus X-1 colony type t1 and from T . oshimai SPS-11 was a truncated analog which lacked the nonreducing terminal glucose . Long-chain 1,2-diols have been previously reported in the polar lipids of Thermomicrobium roseum and (possibly) Chloroflexus aurantiacus, but to our knowledge, this is the first report of their detection in other bacteria and the first account of the structural determination of long-chain diol-linked glycolipids.

Cancer Res, 1997 Sep 15, 57(18), 3972 - 8
Mice carrying a truncated Apc gene have diminished gastric epithelial proliferation, gastric inflammation, and humoral immunity in response to Helicobacter felis infection; Fox JG et al.; Helicobacter pylori infection and adenomatous polyposis coli (Apc) gene mutations have been linked to gastric cancer in humans, but possible synergistic interaction(s) between these risk factors have not been examined . Fourteen C57BL/6 wild-type and 14 Apc1638 heterozygous mice were inoculated with Helicobacter felis at 6 weeks of age and compared at various time points with a similar number of uninfected control mice of the same genotype . Both infected and uninfected Apc1638 mice had a limited incidence of atypical proliferation foci in the mucosa of the antrum and pyloric junction at 4.5 and 6 months of age, whereas polyps of the antrum and pylorus were present in all mice, regardless of infection status, at 7.5 months . In contrast, no altered gastric mucosal foci were observed in control or infected C57BL/6 mice at any time point . Interestingly, the infected Apc1638 mice had less epithelial proliferation and inflammation in the body of the stomach, lower anti-H . felis serum IgG antibody responses (although both the wild-type and Apc mutant mice had a Th1-like immune response, based on a predominantly IgG2a immunoglobulin response), and higher bacteria and urease scores than did infected wild-type C57BL/6 mice . In conclusion, the Apc1638 truncating mutation leads to gastric dysplasia and polyposis of the antrum and pyloric junction, but H . felis infection of the Apc mutant mouse does not lead to an increased rate of gastric neoplasia . In addition, our data suggest this Apc mutation may actually lead to decreased immune, inflammatory, and gastric hyperplastic responses to Helicobacter infection, suggesting the possibility of a novel role for this tumor suppressor gene in the immune and local tissue responses to gastric bacterial infection.

Science, 1997 Oct 24, 278(5338), 631 - 7
A genomic perspective on protein families; Tatusov RL et al.; In order to extract the maximum amount of information from the rapidly accumulating genome sequences, all conserved genes need to be classified according to their homologous relationships . Comparison of proteins encoded in seven complete genomes from five major phylogenetic lineages and elucidation of consistent patterns of sequence similarities allowed the delineation of 720 clusters of orthologous groups (COGs) . Each COG consists of individual orthologous proteins or orthologous sets of paralogs from at least three lineages . Orthologs typically have the same function, allowing transfer of functional information from one member to an entire COG . This relation automatically yields a number of functional predictions for poorly characterized genomes . The COGs comprise a framework for functional and evolutionary genome analysis.

J Cell Sci, 1997 Sep, 110 ( Pt 18), 2239 - 48
Dictyostelium lysosomal proteins with different sugar modifications sort to functionally distinct compartments; Souza GM et al.; Many Dictyostelium lysosomal enzymes contain mannose-6-phosphate (Man-6-P) in their N-linked oligosaccharide chains . We have now characterized a new group of lysosomal proteins that contain N-acetylglucosamine-1-phosphate (GlcNAc-1-P) linked to serine residues . GlcNAc-1-P-containing proteins, which include papain-like cysteine proteinases, cofractionate with the lysosomal markers and are in functional vesicles of the endosomal/lysosomal pathway . Immunoblots probed with reagents specific for each carbohydrate modification indicate that the lysosomal proteins are modified either by Man-6-P or GlcNAc-1-P, but not by both . Confocal microscopy shows that the two sets of proteins reside in physically and functionally distinct compartments . Vesicles with GlcNAc-1-P fuse with nascent bacteria-loaded phagosomes less than 3 minutes after ingestion, while those with Man-6-P do not participate in bacterial digestion until about 15 minutes after phagocytosis . Even though both types of vesicles fuse with phagosomes, GlcNAc-1-P- and Man-6-P-bearing proteins rarely colocalize . Since both lysosomal enzymes and their bound carbohydrate modifications are stable in lysosomes, a targeting or retrieval mechanism based on these carbohydrate modifications probably establishes and/or maintains segregation.

Blood, 1997 Oct 15, 90(8), 2911 - 5
Interleukin-1beta secretion is impaired by inhibitors of the Atp binding cassette transporter, ABC1; Hamon Y et al.; The production of interleukin-1beta (IL-1beta), a powerful mediator of inflammation, is tightly regulated at several levels . However, in some pathologic conditions, a pharmacologic treatment is required to control the toxicity of excessive extracellular IL-1beta . Because of the heavy side effects of most therapies used in IL-1beta-mediated pathologies, a goal of pharmacologic research is the development of selective anti-IL-1beta drugs . We show here that the sulfonylurea glyburide, currently used in the oral therapy of noninsulin dependent diabetes, is an inhibitor of IL-1beta secretion from human monocytes and mouse macrophages . Glyburide reduces dramatically the recovery of extracellular 17-kD IL-1beta in the absence of toxic effects on the cells and without affecting the synthesis or processing of the IL-1beta precursor . IL-1beta belongs to the family of leaderless secretory proteins released from the cell by a nonclassical secretory route . In bacteria and yeast Atp binding cassette (ABC) transporters are involved in the secretion of leaderless secretory proteins . Interestingly, glyburide blocks the anion exchanger function of ABC1, a mammalian member of the family of ABC transporters . We thus investigated the involvement of ABC1 in IL-1beta secretion, through the analysis of the effects of drugs known to inhibit IL-1beta secretion, on the activity of ABC1 and in turn the ability of known inhibitors of ABC1 of blocking IL-1beta secretion . Our data show that IL-1beta secretion and the function of ABC1 as an anion exchanger are sensitive to the same drugs, therefore suggesting an involvement of the ABC1 transporter in the secretion of leaderless proteins in mammals.

Biosci Rep, 1997 Jun, 17(3), 347 - 66
Membrane-linked systems preventing superoxide formation; Skulachev VP; New facts and ideas concerning the membrane-linked mechanisms preventing superoxide formation are summarised here . It is assumed that aerobic cells possess several lines of anti-ROS defence, including optimisation of the intracellular oxygen concentration, decrease in the concentration and life-time of one-electron O2 reductants such as CoQH; and mitochondrial and cell selections, i.e . elimination of mitochondria and cells producing ROS at high rate . It is postulated that ROS-dependent pore opening and ROS-dependent apoptosis are involved in mitochondrial and cell selections.

J Physiol Pharmacol, 1997 Sep, 48(3), 383 - 91
Helicobacter pylori and gastric adaptation to repeated aspirin administration in humans; Konturek JW et al.; The gastric irritant properties of nonsteroidal anti-inflammatory drugs (NSAID) are well established but the pathogenic mechanisms by which these agents damage the mucosa or delay its repair are poorly understood . The phenomenon of gastric adaptation after repeated exposures to ASA is well documented but the involvement of Helicobacter pylori (H . pylori) in NSAID-induced gastropathy and adaptation has not been elucidated . The aim of this study was 1) to compare the gastric damage in response to repeated exposures to ASA in the same subjects before and after eradication of H . pylori and 2) to examine the morphological and functional changes of gastric mucosa during the 14 day treatment with ASA in H . pylori-infected subjects before and after eradication of this bacteria: Eight healthy volunteers (age 19-28) with H . pylori infection were given ASA 1g bd during 14 days before and after H . pylori eradication . Mucosal damage was evaluated by endoscopy before and at 3, 7 and 14 days of ASA administration using modified Lanza score . During endoscopy mucosal biopsies were obtained for determination of DNA synthesis, by measuring 3H-thymidine incorporation into DNA . Prior to each endoscopy gastric microbleeding was determined in three consecutive gastric washings . Three months after successful eradication of H . pylori confirmed by 13C-urea breath test and mucosal rapid urease test, the same subjects received again 14 day treatment with ASA and underwent the same examinations as prior to the therapy . In all subjects, ASA administration induced acute gastric damage with endoscopic Lanza score reaching maximum at 3rd day . In H . pylori-positive subjects, this damage was maintained at similar level up to day 14th, whereas in H . pylori-eradicated subjects, this damage was lessened at day 14th by about 60-75% . Gastric microbleeding also reached its maximum at 3rd day of ASA treatment being significantly higher in H . pylori-eradicated subjects than in those with H . pylori infection . This microbleeding decreased to almost normal values by the end of the study in all H . pylori-negative subjects but remained significantly elevated in H . pylori-infected subjects . DNA synthesis before and following ASA administration was significantly higher in subjects after H . pylori eradication than in those with H . pylori infection . Moreover, this DNA synthesis showed significant increase at day 7 of ASA administration only in H . pylori-eradicated subjects . We conclude that: 1) gastric adaptation to ASA is impaired in H . pylori-positive subjects but eradication of H . pylori restores this adaptation, 2) the DNA synthesis and possibly also mucosal cell turnover in response to ASA are suppressed in H . pylori infection and this can be reversed by eradication of H . pylori.

Int Rev Cytol, 1998, 177, 181 - 253
Signaling in unicellular eukaryotes; Christensen ST et al.; Aspects of intercellular and intracellular signaling systems in cell survival, proliferation, differentiation, chemosensory behavior, and programmed cell death in free-living unicellular eukaryotes have been reviewed . Comparisons have been made with both bacteria and metazoa . The central organisms were flagellates (Trypanosoma, Leishmania, and Crithidia), slime molds (Dictyostelium), yeast cells (Saccharomyces cerevisiae), and ciliates (Paramecium, Euplotes, and Tetrahymena) . There are two novel aspects in this review . First, cellular responses are viewed in an evolutionary perspective, rather than from the more prevailing one, in which the unicellular eukaryotes are seen by the mammalian organisms . Second, results obtained with cell cultures in minimal, chemically defined nutrient media at low cell densities where intercellular signaling is strongly reduced are discussed . These results shed light on control mechanisms and their cooperation inside the living cell . Intracellular systems have many common features in unicellular and multicellular organisms.

Crit Care Med, 1997 Oct, 25(10), 1707 - 12
Clinical utility of hygroscopic heat and moisture exchangers in intensive care patients; Boots RJ et al.; OBJECTIVE: To compare the degree of bacterial circuit colonization, frequency of ventilator-associated pneumonia (VAP), character of respiratory secretions, rewarming of hypothermic patients, disposable costs, and air flow resistance in intensive care patients ventilated using either a heat and moisture exchanger (HME) or hot water (HW) humidifier circuit . DESIGN: A prospective, randomized blinded trial of patients in the intensive care unit undergoing mechanical ventilation . SETTING: A metropolitan teaching hospital . PATIENTS: One hundred sixteen patients undergoing mechanical ventilation for a minimum period of 48 hrs were enrolled . INTERVENTIONS: Patients were randomized to three ventilation groups using a) an HW circuit with a 2-day circuit change (n = 41); or b) a bacterial-viral filtering HME in the circuit, with either a 2-day (n = 42); or c) a 4-day circuit change (n = 33) . MEASUREMENTS AND MAIN RESULTS: Circuit colonization was assessed using quantitative culture of washings taken from the circuit tubing and semiquantitative culture of swabs from the Y connectors . Sixty-seven percent of HW circuits became contaminated compared with 12% in the two HME groups (p < .0001) . Median colony counts were lower in the HME groups (p < .0001) . If circuits at first circuit change were contaminated in the HW group, 89% of subsequent circuit changes became contaminated compared with 0% and 25% for the 2- and 4-day HME groups, respectively . The frequency of VAP, the time to resolution of admission hypothermia, and the volume and fluidity of secretions were similar for all groups . The resistance of the HME after 24 hrs of use was < 0.025 cm H2O/L at gas flows of 40 L/min . HME use resulted in a cost reduction of $1.48 (Australian)/day . CONCLUSIONS: Circuits with a bacterial-viral filtering HME are less readily colonized by bacteria . Contamination is a random event . Humidification technique has no influence on the frequency rate of VAP, the effectiveness of rewarming, nor the character of the respiratory secretions . Breathing resistance is generally low and disposable costs are reduced when an HME is used.

J Physiol Pharmacol, 1997 Sep, 48(3), 393 - 404
Attachment of Helicobacter pylori strains to human epithelial cells; Chmiela M et al.; The aim of the study was to characterize several clinical isolates of H . pylori as regards the activity and specificity of their haemagglutinins and the involvement of surface sialic acid-specific and heparin-binding compounds in the adhesin of the bacteria to human epithelial cell lines . Although H . pylori strains caused haemagglutination (HA) of sheep erythrocytes, they differed markedly by activity and specificity . On the basis of haemagglutination inhibition study three types of H . pylori strains could be distinguished . The HA of Type I strains was inhibited with fetuin/mucin but not asialofetuin/asialomucin . The HA activity of Type II strains was inhibited with fetuin/mucin and asialofetuin/asialomucin . The HA of Type III strains was not influenced by any of these inhibitors . In vitro, H . pylori strains bound to the cells of human epithelial lines: HeLa, Kato-3, Ags . However, various compounds mediated the binding of H . pylori types distinguished by HA, to epithelial cells . The interaction of some of H . pylori strains with epithelial cells was mediated by bacterial sialic acid-binding compounds . The majority of H . pylori strains used heparin-binding surface compounds to attach to epithelial cells . Clinical H . pylori strains differ by the compounds used in adhesin to epithelial cell lines, however, this process also depends on the expression of appropriate receptors on the host cells.

Int J Syst Bacteriol, 1997 Oct, 47(4), 1255 - 7
Reassessment of the taxonomic position of Rickettsiella grylli; Roux V et al.; We determined the 16S rRNA gene sequence of Rickettsiella grylli, an intracellular parasite of Gryllus bimaculatus and related species of crickets . Phylogenetic inferences made from alignment of this sequence with the sequences of other bacteria demonstrated that R . grylli is most closely related to Coxiella burnetii and Legionella species in the gamma subclass of the phylum Proteobacteria . R . grylli was previously thought to be related to members of the order Rickettsiales, but the representatives of this order have been shown to be members of the alpha 1 subclass of the Proteobacteria . Our results indicate that R . grylli should be removed from the order Rickettsiales.

Int J Syst Bacteriol, 1997 Oct, 47(4), 1236 - 45
A novel pathogenic taxon of the Mycobacterium tuberculosis complex, Canetti: characterization of an exceptional isolate from Africa; van Soolingen D et al.; In an attempt to characterize an unusual mycobacterial strain isolated from a 2-year-old Somali patient with lymphadenitis, we applied various molecular methods not previously used for the taxonomic classification of mycobacteria . This isolate, designated So93, did not differ from Mycobacterium tuberculosis in the biochemical tests and in its 16S rRNA sequence, but produced smooth and glossy colonies, which is highly exceptional for this species . This smooth phenotype was unstable and switched nonreversibly to a rough colony morphology with a low frequency . The two colony types were equally virulent for the guinea pig, exhibiting characteristic tuberculous disease . Both morphotypes had shorter generation times than the M . tuberculosis reference laboratory strain H37Rv and clinical isolates of M . tuberculosis and Mycobacterium bovis . Furthermore, the So93 isolate differed from all M . tuberculosis complex strains described thus far by having only a single copy of insertion sequence IS1081, an unusual composition of the direct repeat cluster, and a characteristic phenolic glycolipid and lipooligosaccharide . This glycolipid had previously been observed only in a smooth isolate of M . tuberculosis obtained in 1969 by Canetti in France . Analysis of the Canetti strain showed that it shared virtually all genetic properties characteristic of So93, distinguishing these two strains from the known M . tuberculosis complex taxa, M . tuberculosis, Mycobacterium africanum, M . bovis, and Mycobacterium microti . The natural reservoir, host range, and mode of transmission of the group of bacteria described in this paper are presently unknown . This study, partly based on not previously used molecular criteria, supports the idea that the established members within the M . tuberculosis complex and the newly described Canetti grouping should be regarded as a single species, which likely will be designated "M . tuberculosis".

Int J Syst Bacteriol, 1997 Oct, 47(4), 1172 - 8
Reorganization of the genus Erythromicrobium: description of "Erythromicrobium sibiricum" as Sandaracinobacter sibiricus gen . nov., sp . nov., and of "Erythromicrobium ursincola" as Erythromonas ursincola gen . nov., sp . nov; Yurkov V et al.; The results of investigations on the morphology, physiology, pigment composition, light-harvesting antenna and reaction center organization, and electron carriers of five Erythromicrobium representatives, and on phylogenetic relations among them, are summarized . On the basis of clear phenotypic differences and distinct phylogenetic positions shown by 16S ribosomal DNA analysis, the tentative species "Erythromicrobium sibiricum" and "Erythromicrobium ursincola" are formally described as the type species of two new genera: Sandaracinobacter sibiricus gen . nov., sp . nov., and Erythromonas ursincola gen . nov., sp . nov., respectively . The genus Erythromicrobium is at present composed of the type species, E . ramosum, and two species, "E . hydrolyticum" and "E . ezovicum," whose nomenclature is yet to be validated . All species studied group within the alpha-4 subclass of Proteobacteria.

Int J Syst Bacteriol, 1997 Oct, 47(4), 948 - 51
Phylogeny of Photorhabdus and Xenorhabdus species and strains as determined by comparison of partial 16S rRNA gene sequences; Liu J et al.; Partial 16S rRNA gene sequences of 16 strains of the genera Photorhabdus and Xenorhabdus were determined by direct sequencing of PCR products . Aligned sequences were subjected to phylogenetic analysis by maximum-likelihood and maximum-parsimony methods . Distance matrix and phylogenetic analysis did not separate the genera unambiguously . Taxonomic grouping of the bacteria closely paralleled taxonomic grouping of their nematode associates and their geographic origins . We found at least two well-supported taxonomic groups in Photorhabdus species, which suggests that the genus Photorhabdus is coevolving with the nematodes and may be polyspecific.

Clin Microbiol Rev, 1997 Oct, 10(4), 694 - 719
Rickettsioses as paradigms of new or emerging infectious diseases; Raoult D et al.; Rickettsioses are caused by species of Rickettsia, a genus comprising organisms characterized by their strictly intracellular location and their association with arthropods . Rickettsia species are difficult to cultivate in vitro and exhibit strong serological cross-reactions with each other . These technical difficulties long prohibited a detailed study of the rickettsiae, and it is only following the recent introduction of novel laboratory methods that progress in this field has been possible . In this review, we discuss the impact that these practical innovations have had on the study of rickettsiae . Prior to 1986, only eight rickettsioses were clinically recognized; however, in the last 10 years, an additional six have been discovered . We describe the different steps that resulted in the description of each new rickettsiosis and discuss the influence of factors as diverse as physicians' curiosity and the adoption of molecular biology-based identification in helping to recognize these new infections . We also assess the pathogenic potential of rickettsial strains that to date have been associated only with arthropods, and we discuss diseases of unknown etiology that may be rickettsioses.

Nucleic Acids Res, 1997 Nov 1, 25(21), 4250 - 6
An Abf1p C-terminal region lacking transcriptional activation potential stimulates a yeast origin of replication; Wiltshire S et al.; Although it has been demonstrated that eukaryotic cellular origins of DNA replication may harbor stimulatory elements that bind transcription factors, how these factors stimulate origin function is unknown . In Saccharomyces cerevisiae , the transcription factor Abf1p stimulates origin function of ARS121 and ARS1 . In the results presented here, an analysis of Abf1p function has been carried out utilizing LexA(BD)-Abf1p fusion proteins and an ARS 121 derivative harboring LexA DNA-binding sites . A minimal region which stimulates origin function mapped to 50 amino acids within the C-terminus of Abf1p . When tested for transcriptional activation of a LacZ reporter gene, the same LexA(BD)-Abf1p fusion protein had negligible transcriptional activation potential . Therefore, stimulation of ARS 121 may occur independently of a transcriptional activation domain . It has been previously observed that the Gal4p, Rap1p DNA-binding sites and the LexA-Gal4p fusion protein can replace the role of Abf1p in stimulating ARS 1 . Here we show that the stimulatory function of Abf1p at ARS 121 cannot be replaced by these alternative DNA-binding sites and the potent chimeric transcriptional activator LexA(BD)-Gal4(AD)p . Hence, these results strongly suggest that the Abf1p stimulation of replication may differ for ARS 121 and ARS 1 , and imply specificity in the Abf1p/ARS 121 relationship.

Rev Fac Cien Med Univ Nac Cordoba, 1996, 54(1-2), 19 - 25
{Immune complex detection in nasal mucosa of patients with chronic and permanent nasal obstruction}; Sandez E et al.; Thirty patients with rhinologic problems and in which the main symptom was "Permanent Nasal Blockade" were studied . We found that they presented a "Permanent and Total Chronic Nasal Blockade"-an entity defined by us as a permanent chronic obstruction of the nasal cavities that would be the clinical and anatomopathologic expression and the immunological terminal phase of "Chronic Rhinitis", leading to Chronic Respiratory Nasal Insufficiency . The main symptoms of these "buccal breathers" were: throat dryness, night snores, nasal resonance of the voice, mechanic and secondary disturbances derived from mouth breathing, such as pharingitis, laryngitis, bronchitis, bronchial asthma and a tendency towards emphysema; dystrophia of the facial bones, disminution of the intellectual capacity, irritability, lack of concentration; disturbances of the circulatory function (alterations of the cardiac rhythm and of the blood pressure) . The patients were adolescents and young adults in which allergic and infectious causes dominated the etiology, showed by family allergic and personal atopic antecedents, positive intradermic tests for aeroallergenes and bacteria and corroborated by increase of immunoglobulin E and of blood eosinophiles . The methods of diagnosis used to verify the "Permanent and Total Chronic Nasal Blockade" were: X-rays, lineal tomography; rhinomanometry and rhinofibrolaringoscopy . The anatomopathologic results obtained by nasal biopsy showed a lymphomonocitary infiltrate around arterioles and venules: signs of vasculitis . All this led us to search for immunocomplexes . The presence of positive immunocomplexes surrounding the vessels contributed to a more precise focusing of the physiopathology, diagnosis and treatment of "Permanent and Total Chronic Nasal Blockade".

An Acad Bras Cienc, 1995, 67 Suppl 3, 329 - 45
Secondary metabolites and their role in evolution; Jarvis BB; The role of secondary metabolites in evolution will be examined with the view that they are chemicals released within a system by one component which have evolved to affect other component(s) within the system . Secondary metabolites are a natural outgrowth and consequence of an increase in complexity, and they are a critical part of the chemical "glue" that holds a system together . An analysis of secondary metabolites from a broad perspective (e.g . genetics, ecology, evolution, etc.) suggests that the nature of secondary metabolism can be viewed as a critical component of an emergent system (ecological) arising from a host of interlocking cycles and feedback processes.

J Bacteriol, 1997 Oct, 179(20), 6426 - 31
Identification of an essential Caulobacter crescentus gene encoding a member of the Obg family of GTP-binding proteins; Maddock J et al.; We have identified an essential Caulobacter crescentus gene (cgtA) that encodes a member of a recently identified subfamily of GTPases (the Obg family) conserved from Bacteria to Archaea to humans . This evolutionary conservation between distantly related species suggests that this family of GTP-binding proteins possesses a fundamental, yet unknown, cellular role . In this report, we describe the isolation and sequence of the cgtA gene . The predicted CgtA protein displays striking similarity to the Obg family of small, monomeric GTP-binding proteins, both in the conserved guanine nucleotide-binding domains and throughout the N-terminal glycine-rich domain that is found in many members of the Obg family . Disruption of the cgtA gene was lethal, demonstrating that this gene is essential for cell growth . Immunoblot analysis revealed that CgtA protein levels remained constant throughout the C . crescentus cell cycle.

J Bacteriol, 1997 Oct, 179(20), 6311 - 7
Characterization of the oriC region of Mycobacterium smegmatis; Qin MH et al.; A 3.5-kb DNA fragment containing the dnaA region of Mycobacterium smegmatis has been hypothesized to be the chromosomal origin of replication or oriC (M . Rajagopalan et al., J . Bacteriol . 177:6527-6535, 1995) . This region included the rpmH gene, the dnaA gene, and a major portion of the dnaN gene as well as the rpmH-dnaA and dnaA-dnaN intergenic regions . Deletion analyses of this region revealed that a 531-bp DNA fragment from the dnaA-dnaN intergenic region was sufficient to exhibit oriC activity, while a 495-bp fragment from the same region failed to exhibit oriC activity . The oriC activities of plasmids containing the 531-bp sequence was less than the activities of those containing the entire dnaA region, suggesting that the regions flanking the 531-bp sequence stimulated oriC activity . The 531-bp region contained several putative nine-nucleotide DnaA-protein recognition sequences {TT(G/C)TCCACA} and a single 11-nucleotide AT-rich cluster . Replacement of adenine with guanine at position 9 in five of the putative DnaA boxes decreased oriC activity . Mutations at other positions in two of the DnaA boxes also decreased oriC activity . Deletion of the 11-nucleotide AT-rich cluster completely abolished oriC activity . These data indicate that the designated DnaA boxes and the AT-rich cluster of the M . smegmatis dnaA-dnaN intergenic region are essential for oriC activity . We suggest that M . smegmatis oriC replication could involve interactions of the DnaA protein with the putative DnaA boxes as well as with the AT-rich cluster.

J Biomol NMR, 1997 Jul, 10(1), 21 - 7
Alterations in chemical shifts and exchange broadening upon peptide boronic acid inhibitor binding to alpha-lytic protease; Davis JH et al.; alpha-Lytic protease, a bacterial serine protease of 198 amino acids (19 800 Da), has been used as a model system for studies of catalytic mechanism, structure-function relationships, and more recently for studies of pro region-assisted protein folding . We have assigned the backbones of the enzyme alone, and of its complex with the tetrahedral transition state mimic N-tert-butyloxycarbonyl-Ala-Pro-boro Val, using double- and triple-resonance 3D NMR spectroscopy on uniformly 15N- and 13C/15N-labeled protein . Changes in backbone chemical shifts between the uncomplexed and inhibited form of the protein are correlated with distance from the inhibitor, the displacement of backbone nitrogens, and change in hydrogen bond strength upon inhibitor binding (derived from previously solved crystal structures) . A comparison of the solution secondary structure of the uninhibited enzyme with that of the X-ray structure reveals no significant differences . Significant line broadening, indicating intermediate chemical exchange, was observed in many of the active site amides (including three broadened to invisibility), and in a majority of cases the broadening was reversed upon addition of the inhibitor . Implications and possible mechanisms of this line broadening are discussed.

J Biol Chem, 1997 Oct 17, 272(42), 26627 - 33
N2 fixation by Streptomyces thermoautotrophicus involves a molybdenum-dinitrogenase and a manganese-superoxide oxidoreductase that couple N2 reduction to the oxidation of superoxide produced from O2 by a molybdenum-CO dehydrogenase; Ribbe M et al.; N2 fixation by Streptomyces thermoautotrophicus follows the equation N2 + 4-12MgATP + 8H+ + 8e- --> 2NH3 + H2 + 4-12MgADP + 4-12Pi and exhibits features which are not obvious in the diazotrophic bacteria studied so far . The reaction is coupled to the oxidation of carbon monoxide (CO) by a molybdenum-containing CO dehydrogenase that transfers the electrons derived from CO oxidation to O2, thereby producing superoxide anion radicals (O-2) . A manganese-containing superoxide oxidoreductase reoxidizes the O-2 anions to O2 and transfers the electrons to a MoFeS-dinitrogenase for the reduction of N2 to ammonium . Among the most striking properties of the S . thermoautotrophicus nitrogenase system are the dependence on O2 and O-2, the complete insensitivity of all components involved toward O2 and H2O2, the inability to reduce ethine or ethene, and a low MgATP requirement . In addition, the subunit structure of the S . thermoautotrophicus nitrogenase components and the polypeptides involved seem to be dissimilar from the known nitrogenases.

J Biol Chem, 1997 Oct 17, 272(42), 26132 - 8
Identification, cloning, and mutational analysis of the casein kinase 1 cDNA of the malaria parasite, Plasmodium falciparum . Stage-specific expression of the gene; Barik S et al.; The cDNA for casein kinase 1 (CK1) of Plasmodium falciparum was cloned, sequenced, and expressed in bacteria . The single major open reading frame of the 1.2-kilobase pair cDNA coded for a 324-amino acid polypeptide of approximately 37 kDa, the predicted sequence of which showed strong identity with known CK1 isoforms . The purified recombinant enzyme exhibited properties characteristic of CK1, such as inhibition by CK1-7, the ability to phosphorylate a highly specific peptide substrate, and a strong preference for ATP over GTP . A casein kinase activity, partially purified from soluble extracts of P . falciparum by affinity chromatography through CK1-7 columns displayed identical properties . The activity showed a stage-specific expression in the parasite, in the order trophozoite > ring >> schizont . Northern analysis indicated the existence of two major CK1 mRNAs, 2.4 and 3.2 kilobase pairs long, the levels of which were in the order ring > schizont > trophozoite . Mutagenesis of recombinant CK1 defined important amino acid residues and their potential role in the conformation of the enzyme . The malarial CK1 appeared to be the one of the smallest and perhaps the most primitive CK1 enzymes known, containing little sequence information beyond the minimal catalytic domain.

Gene, 1997 Sep 15, 197(1-2), 325 - 36
The human mitochondrial elongation factor tu (EF-Tu) gene: cDNA sequence, genomic localization, genomic structure, and identification of a pseudogene; Ling M et al.; The human mitochondrial elongation factor Tu (EF-Tu) is nuclear-encoded and functions in the translational apparatus of mitochondria . The complete human EF-Tu cDNA sequence of 1677 base pairs (bp) with a 101 bp 5'-untranslated region, a 1368 bp coding region, and a 207 bp 3'-untranslated region, has been determined and updated . The predicted protein from this cDNA sequence is approximately 49.8 kDa in size and is composed of 455 amino acids (aa) with a putative N-terminal mitochondrial leader sequence of approximately 50 aa residues . The predicted amino acid sequence shows high similarity to other EF-Tu protein sequences from ox, yeast, and bacteria, and also shows limited similarity to human cystolic elongation factor 1 alpha . The complete size of this cDNA (1677 bp) obtained by cloning and sequencing was confirmed by Northern blot analysis, which showed a single transcript (mRNA) of approximately 1.7 kb in human liver . The genomic structure of this EF-Tu gene has been determined for the first time . This gene contains nine introns with a predicted size of approximately 3.6 kilobases (kb) and has been mapped to chromosome 16p11.2 . In addition, an intronless pseudogene of approximately 1.7 kb with 92.6% nucleotide sequence similarity to the EF-Tu gene has also been identified and mapped to chromosome 17q11.2.

Structure, 1997 Sep 15, 5(9), 1129 - 34
Helicase structures: a new twist on DNA unwinding; Marians KJ; The crystal structures of two members of the SF1 family of helicases, Rep and PcrA, and one member of the SF2 family of helicases, the HCV RNA helicase, have recently been solved . These structures illuminate the roles of the conserved helicase motifs in catalytic function and offer clues as to how these proteins can translocate along DNA.

Dig Dis Sci, 1997 Sep, 42(9), 1835 - 40
Clinical application of gastric histology to monitor treatment of dual therapy in H . pylori eradication; Yang HB et al.; This preliminary study attempted to test whether pretreatment gastric histology of H . pylori infection may affect the success of dual therapy and to identify which parameter of gastric histology could be improved after dual therapy . One hundred forty-five dyspeptic patients with H . pylori infection received a two-week course of dual therapy (Amoxicillin 500 mg every 6 hr plus omeprazole 20 mg twice a day) . In each patient, three pairs of gastric biopsies, sampled from the antrum, lower body, and upper body near the cardia, were collected before treatment and four weeks after completion of dual therapy . The density of H . pylori (score 1-5) and parameters of the modified Sydney system were applied to test the severity of H . pylori-related gastric histology in each specimen . The total bacterial load (score 1-15) was a sum of the density of H . pylori sampled from three biopsies . The overall rate of H . pylori eradication rate by dual therapy is 73.1% (106/145) . Univariate analysis of parameters in pretreatment histology disclosed that the presence of mucosal atrophy (P < 0.01), lymphoid follicles (P < 0.005), and higher-density H . pylori (P < 0.001) predisposed to dual therapy failure . Multivariate analysis by stepwise logistic regression further confirmed that both the density of bacteria and the presence of lymphoid follicles are the two major factors related to the outcome of dual therapy (P < 0.001) . Four weeks after dual therapy was completed, only patients with successful eradication significantly improved in these gastric histology parameters: acute activity, chronic inflammation, eosinophil infiltration, and mucosal atrophy . However, the lymphoid follicle and intestinal metaplasia were not significantly improved during the study period . The eradication rates among three subgroups with different total bacterial loads (group I: 1-5; II: 6-10; III: 11-15) disclosed a downward trend (I: 89.1%; II: 73%; III: 52.7%) . It is concluded that dual therapy could improve gastric histology especially among patients with successful eradication of H . pylori . Evaluating pretreatment histologic parameters, including the density of H . pylori and the presence of lymphoid follicles, is valuable in predicting the success of dual therapy.

DNA Res, 1997 Jun 30, 4(3), 199 - 204
Synthesis of RepK of rolling circle plasmid pKYM is regulated by countertranscript and HU protein; Nakahama K et al.; Replication of rolling circle plasmid pKYM was regulated by RepK, a plasmid-encoded initiator protein, with HU protein and antisense RNA (copRNA) that block the expression of RepK . HU protein bound to the repK promoter in the presence of RepK protein and inhibited the transcription of repK mRNA . The copRNA would hybridize to repK mRNA and induce a stem-loop structure in which the repK Shine-Dalgarno sequence is sequestered by base pairing . Sequence substitution experiments demonstrated that this stem-loop not only inhibits translation but induces premature termination.

Ann R Coll Surg Engl, 1997 Sep, 79(5), 368 - 71
Hunterian peptic ulcers and Helicobacter pylori; Walker MM et al.; Gastric spiral organisms were first described in man in 1939 and identified as Helicobacter pylori causing peptic ulcers in the early 1980s . Surgical specimens of gastric resections from 1939 showed H . pylori to be present . Full-thickness sections of gastric mucosa from gastric specimens from the eighteenth-century Hunterian Collection at The Royal College of Surgeons of England were examined by histology for the presence of H . pylori . Four gastric ulcers and a section from an oesophageal varix showed remarkable preservation of the overall architecture, but surface autolysis did not allow identification of the bacteria . However, the presence of lymphoid aggregates in the Hunterian specimens suggests that H . pylori may have been present before autolysis.

FEBS Lett, 1997 Sep 15, 414(3), 485 - 91
ATP synthase: a tentative structural model; Engelbrecht S et al.; Adenosine triphosphate (ATP) synthase produces ATP from ADP and inorganic phosphate at the expense of proton- or sodium-motive force across the respective coupling membrane in Archaea, Bacteria and Eucarya . Cation flow through the intrinsic membrane portion of this enzyme (Fo, subunits ab2c9-12) and substrate turnover in the headpiece (F1, subunits alpha3beta3 gammadeltaepsilon) are mechanically coupled by the rotation of subunit gamma in the center of the catalytic hexagon of subunits (alphabeta)3 in F1 . ATP synthase is the smallest rotatory engine in nature . With respect to the headpiece alone, it probably operates with three steps . Partial structures of six out of its at least eight different subunits have been published and a 3-dimensional structure is available for the assembly (alphabeta)3gamma . In this article, we review the available structural data and build a tentative topological model of the holoenzyme . The rotor portion is proposed to consist of a wheel of at least nine copies of subunits c, epsilon and a portion of gamma as a spoke, and another portion of gamma as a crankshaft . The stator is made up from a, the transmembrane portion of b2, delta and the catalytic hexagon of (alphabeta)3 . As an educated guess, the model may be of heuristic value for ongoing studies on this fascinating electrochemical-to-mechanical-to-chemical transducer.

Gastroenterology, 1997 Oct, 113(4), 1246 - 57
Inhibition of inducible nitric oxide synthase ameliorates endotoxin-induced gut mucosal barrier dysfunction in rats; Unno N et al.; BACKGROUND & AIMS: The permeability of intestinal epithelial monolayers increases after exposure to nitric oxide . The aim of this study was to investigate the role of excessive NO production on intestinal barrier function in rats injected with lipopolysaccharide (LPS) . METHODS: Rats were injected with saline or LPS (5 mg/ kg) . Bacterial translocation to mesenteric lymph nodes, liver, and spleen was assessed 24 hours after LPS injection . Mucosal permeability was determined by loading fluorescein-labeled dextran (mol wt, 4000 daltons) into an intestinal segment and measuring its appearance in plasma . Intestinal mucosal mitochondrial respiration was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide . RESULTS: Intestinal tissue from LPS-challenged rats showed upregulation of inducible NO synthase (iNOS) messenger RNA expression and subsequent up-regulation of iNOS enzymatic activity . Plasma concentrations of nitrite plus nitrate (NO2-/NO3-) were increased for at least 24 hours after injection of LPS . Treatment with the selective iNOS inhibitor, aminoguanidine, inhibited iNOS enzymatic activity and overproduction of NO2-/NO3- . LPS-induced bacterial translocation was reduced by aminoguanidine . LPS-induced intestinal hyperpermeability was ameliorated by both aminoguanidine and another selective iNOS inhibitor, S-methylisothiourea . LPS depressed intestinal mucosal mitochondrial function, and this effect was ameliorated by aminoguanidine . CONCLUSIONS: Overproduction of NO may contribute to intestinal barrier dysfunction in LPS challenged rats, possibly by interfering with mitochondrial oxidative metabolism.

Enzyme Microb Technol, 1997 Oct, 21(5), 335 - 40
Purification and characterization of alkaline phosphatase from Thermotoga neapolitana; Dong G et al.; A hyperthermophilic alkaline phosphatase was purified from Thermotoga neapolitana by heat treatment at 100 degrees C in the presence of Co2+ followed by ion-exchange and affinity chromatographies . The enzyme was purified 2,880-fold with 44% yield . The purified enzyme showed a single protein band of M(r) 45,000 on SDS-PAGE and an apparent molecular weight of 87,000 estimated by gel filtration chromatography . This suggested a homogenous dimer structure . The optimal pH and temperature for enzyme activity were 9.9 and 85 degrees C, respectively . Under optimal conditions, T . neapolitana alkaline phosphatase displayed 30% higher activity than calf intestine alkaline phosphatase did with p-nitrophenyl-phosphate as substrate . The hyperthermostable enzyme had a half-life of 238 min at 90 degrees C and K(m) and Vmax values of 183 microM and 1,352 U mg-1, respectively . Co2+ enhanced the enzyme activity, thermostability, and ligand affinity during column chromatography . The alkaline phosphatase was twice as active with Co2+ than with either Zn2+ or Mn2+ as the metal cofactor.

Hum Gene Ther, 1997 Sep 1, 8(13), 1545 - 54
In situ histochemical detection of beta-galactosidase activity in lung: assessment of X-Gal reagent in distinguishing lacZ gene expression and endogenous beta-galactosidase activity; Weiss DJ et al.; Bacterial lacZ is one of the most commonly used reporter genes for assessing gene transfer to lung . However, lung contains endogenous beta-galactosidase (beta-Gal), which can confound estimation of exogenous lacZ expression by histochemical techniques (i.e., X-Gal) for in situ demonstration of enzyme activity . We investigated several parameters of the X-Gal reaction, including time and temperature of X-Gal exposure as well as lung tissue processing and fixation techniques, and found that none of these could be used to distinguish between endogenous and exogenous beta-Gal activities . The mammalian and bacterial beta-Gal enzymes, however, have pH optima in the acidic and neutral ranges, respectively . Exposing whole lung, lung minces, or mounted frozen sections of lung to X-Gal at mildly alkaline pH (pH 8.0-8.5), minimized detection of endogenous activity in lungs from a variety of species while preserving that resulting from bacterial enzyme activity in a transgenic mouse expressing lacZ . This technique was also useful in distinguishing endogenous activity from that resulting from adenovirus-mediated lacZ gene transfer to diploid lung fibroblasts in primary culture . An appropriate buffer that maintains the desired pH throughout the duration of X-Gal exposure must be used.

Infect Immun, 1997 Oct, 65(10), 4350 - 4
Endobronchial inoculation with Apx toxins of Actinobacillus pleuropneumoniae leads to pleuropneumonia in pigs; Kamp EM et al.; To establish the role of the Apx toxins in the pathogenesis of porcine pleuropneumonia, specific-pathogen-free pigs were inoculated deeply endobronchially with either culture filtrates of Actinobacillus pleuropneumoniae serotype 8 or 9, culture filtrates depleted of the Apx toxins by affinity chromatography, depleted culture filtrate supplemented with purified recombinant Apx toxins (rApx), or purified rApx toxins alone . Results of these experiments indicate that ApxI, ApxIII, and, to a lesser extent, ApxII are the bacterial factors that trigger the development of clinical symptoms and lung lesions typical for porcine pleuropneumonia.

Infect Immun, 1997 Oct, 65(10), 4258 - 66
Nonopsonic binding of Mycobacterium tuberculosis to complement receptor type 3 is mediated by capsular polysaccharides and is strain dependent; Cywes C et al.; The choice of host cell receptor and the mechanism of binding (opsonic versus nonopsonic) may influence the intracellular fate of Mycobacterium tuberculosis . We have identified two substrains of M . tuberculosis H37Rv, designated H37Rv-CC and -HH, that differed in their modes of binding to complement receptor type 3 (CR3) expressed in transfected Chinese hamster ovary (CHO-Mac-1) cells: H37Rv-CC bound nonopsonically, whereas H37Rv-HH bound only after opsonization in fresh serum . H37Rv-CC also bound nonopsonically to untransfected CHO cells, whereas H37Rv-HH binding was enhanced by serum and was mediated by the 1D1 antigen, a bacterial adhesin previously identified as a polar phosphatidylinositol mannoside . H37Rv-CC and -HH had identical IS6110 DNA fingerprint patterns . Of five M . tuberculosis clinical isolates examined, four displayed the same binding phenotype as H37Rv-CC, as did the Erdman strain, whereas one isolate, as well as Mycobacterium smegmatis, behaved like H37Rv-HH . Nonopsonic binding of H37Rv-CC to CHO cell-expressed CR3 was apparently to the beta-glucan lectin site, as it was cation independent and inhibited by laminarin (seaweed beta-glucan) and N-acetylglucosamine; laminarin also inhibited the binding of H37Rv-CC to monocyte-derived macrophages . Further, binding of H37Rv-CC to CHO-Mac-1 cells was inhibited by prior agitation of bacteria with glass beads (which strips outer capsular polysaccharides) and by preincubation with amyloglucosidase, as well as by the presence of capsular D-glucan and D-mannan from M . tuberculosis Erdman, but not by Erdman D-arabino-D-mannan, yeast mannan, or capsular components from H37Rv-HH . Analysis of capsular carbohydrates revealed that H37Rv-CC expressed 5-fold more glucose and 2.5-fold more arabinose and mannose than H37Rv-HH . Flow cytometric detection of surface epitopes indicated that H37Rv-CC displayed twofold less surface-exposed phosphatidylinositol mannoside and bound complement C3 less efficiently than H37Rv-HH; these differences were eliminated after treatment of H37Rv-CC with glass beads . Thus, outer capsular polysaccharides mediate the binding of H37Rv-CC to CR3, likely to the beta-glucan site . Moreover, there are strain-dependent differences in the thickness or composition of capsular polysaccharides that determine the mode of binding of M . tuberculosis to mammalian cells.

Infect Immun, 1997 Oct, 65(10), 4222 - 8
Role of the Bordetella pertussis minor fimbrial subunit, FimD, in colonization of the mouse respiratory tract; Geuijen CA et al.; Bordetella pertussis fimbriae are composed of a major subunit, Fim2 or Fim3, and the minor subunit FimD . Using immunoelectron microscopy, we provide evidence that FimD is located at the fimbrial tip . The role of FimD in colonization of the mouse respiratory tract was studied by using two fimbrial mutants: a mutant completely devoid of fimbriae (designated FimD-) and a mutant devoid of the major fimbrial subunits but still producing the minor subunit (designated FimD+) . The ability of the two fimbrial mutants to colonize the nasopharynx, trachea, and lungs was compared with those of the wild type parental strain and a filamentous hemagglutinin (FHA) mutant . Of the three mutants studied, the FimD- mutant showed the greatest defect, colonizing less well in the nasopharynx, trachea, and lungs . The most pronounced defect in colonizing ability of the three mutants was observed in the trachea . However, the colonizing defect of the FHA and FimD+ mutants in the trachea was observed only during the first 3 days of infection . After 10 days, the colonization level was nearly restored to wild-type levels . The FHA and FimD+ mutants showed a slight colonization defect in the nasopharynx but no defect in the lungs . A maltose binding protein-FimD fusion protein and a peptide derived from FimD were able to bind to heparin, a member of a class of sulfated sugars which are ubiquitous in the respiratory tract . Recently it was shown (W . L . W . Hazenbos, C . A . W . Geuijen, B . M . van den Berg, F . R . Mooi, and R . van Furth, J . Infect . Dis . 171:924-929, 1995) that FimD also binds to the integrin VLA-5, and our results suggest that the binding of B . pertussis to these two molecules plays an important role in colonization of the respiratory tract of the mouse.

J Clin Microbiol, 1997 Oct, 35(10), 2649 - 50
Histoplasma capsulatum sinusitis; Butt AA et al.; Sinusitis is commonly reported in patients with AIDS . In addition to the usual bacterial pathogens isolated from immunocompetent patients, sinusitis in patients with AIDS may be caused by a variety of unusual bacteria, viruses, fungi, parasites, and mycobacteria . Histoplasma capsulatum has not typically been associated with sinusitis in either group of patients . We report a case of sinusitis caused by H . capsulatum in a patient with AIDS.

Trop Anim Health Prod, 1997 Aug, 29(3), 158 - 64
Hydropericardium syndrome (HPS) in India: a preliminary study on the causative agent and control of the disease by inactivated autogenous vaccine; Kumar R et al.; Hydropericardium syndrome (HPS) in broiler birds of 3 to 6 weeks of age was recorded for the first time in the Haldwani area of Nainital district (UP) in India in November, 1994 . The overall mortality in 6 poultry farms was 61.62 per cent . The disease was experimentally transmitted by bacteria free infected liver homogenate extract passed through membrane filters of 0.22 and 0.1 mu APD . The aetiological agent was inactivated by heat treatment at 56 degrees C for one hour and 80 degrees C for 10 min . A precipitin band was demonstrated in agar gel immunodiffusion and counter immunoelectrophoresis using infected liver homogenate extract as antigen and homologous antisera raised in the laboratory . The disease was effectively controlled by formalinised and heat inactivated autogenous vaccine prepared from the infected livers of birds which died of natural infection.

J Theor Biol, 1997 Oct 7, 188(3), 289 - 99
Dynamics of the emergence of genetic resistance to biocides among asexual and sexual organisms; Jaffe K et al.; A stochastic, agent based, evolutionary algorithm, modeling mating, reproduction, genetic variation, phenotypic expression and selection was used to study the dynamic interactions affecting a multiple-gene system . The results suggest that strong irreversible constraints affect the evolution of resistance to biocides . Resistant genes evolve differently in asexual organisms compared with sexual ones in response to various patterns of biocide applications . Asexual populations (viruses and bacteria) are less likely to develop genetic resistance in response to multiple pesticides or if pesticides are used at low doses, whereas sexual populations (insects for example) are more likely to become resistant to pesticides if susceptibility to the pesticide relates to mate selection . The adaptation of genes not related to the emergence of resistance will affect the dynamics of the evolution of resistance . Increasing the number of pesticides reduces the probability of developing resistance to any of them in asexual organisms but much less so in sexual organisms . Sequential applications of toxins, were slightly less efficient in slowing emergence of resistance compared with simultaneous application of a mix in both sexual and asexual organisms . Targeting only one sex of the pest speeds the development of resistance . The findings are consistent to most of the published analytical models but are closer to known experimental results, showing that nonlinear, agent based simulation models are more powerful in explaining complex processes .

Biochemistry, 1997 Oct 7, 36(40), 12329 - 36
Fourier transform infrared study on the primary donor P798 of Heliobacterium modesticaldum: cysteine S-H coupled to P798 and molecular interactions of carbonyl groups; Noguchi T et al.; Light-induced Fourier transform infrared (FTIR) difference spectra of the primary donor P798 upon its cation formation (P798(+)/P789) were measured using the membranes and purified RC complex of Heliobacterium modesticaldum . A differential signal at 2550/2560 cm-1 was observed in the difference spectra and assigned to the S-H stretching mode of cysteine by an isotopic shift to 1854/1861 cm-1 upon deuteration . The observed frequencies indicate that this S-H forms a strong hydrogen bond and that the bond is further strengthened upon P798(+) formation . Polarized FTIR difference spectra showed that this S-H group is oriented at <40 degrees with respect to the membrane normal . It was proposed that the cysteine S-H is coupled to P798 through a hydrogen-bond network or by direct hydrogen bonding to either a P798 carbonyl or a ligand histidine . In the carbonyl stretching region, differential signals were observed at 1741/1737, 1725/1718, 1702/1693, and 1687/1666 cm-1 . In a dry membrane film, the signal at 1687/1666 cm-1 was mostly lost and hence was assigned to the amide I bands arising from the protein conformational change, which was suppressed upon dehydration of the membranes . The 1702/1693 cm-1 signal was assigned to the 13(1)-keto C&dbd;O of P798, which was free from hydrogen bonding and had a nearly parallel orientation to the membrane plane . The upshift by 9 cm-1 upon P798 oxidation, which is much smaller than upshifts of monomeric (bacterio)chlorophylls {(B)Chls} in organic solution, indicates that the positive charge on P798(+) is significantly delocalized in a BChlg dimer . The signals at 1741/1737 and 1725/1718 cm-1 were assigned to a free and a hydrogen-bonded ester C=O group, respectively . The dichroism measurement showed that the C=O of 1741/1737 cm-1 was oriented nearly parallel to the membrane plane while that of 1725/1718 cm-1 was considerably tilted by <31 degrees to the membrane normal . It was proposed that one of the two ester signals arose from the 13(2)-carbomethoxy C=O of P798 while the other arose either from the 17(2)-ester C=O of P798 or from an ester C&dbd;O of adjacent BChlg or 8(1)-OH-Chla that was electrostatically influenced by oxidation of P798.

Biochemistry, 1997 Oct 7, 36(40), 12303 - 16
Torsional constraints on the formation of open promoter complexes on DNA minicircles carrying sigma 54-dependent promoters; Qureshi M et al.; A topoisomer gel retardation assay has been used to examine the topological requirements for the formation of open promoter complexes on DNA minicircles carrying sigma 54-dependent promoters . In the absence of intercalators, individual topoisomers carrying both the nifL and nifF promoters could be resolved as discrete species by electrophoresis, but exhibited anomalous electrophoretic behavior at relatively high negative superhelical density, indicative of a structural transition . In the presence of phosphorylated activator protein NTRC, ATP, and sigma 54 RNA polymerase holoenzyme, discrete topoisomer shifts were detected associated with the formation of open promoter complexes . At the nifL promoter open complexes could be formed on all negatively supercoiled topoisomers examined as well as on nicked circular DNA, but not on the DeltaLk = 0 topoisomer or positively supercoiled DNA . Minicircles carrying the sigma 54-dependent glnAp2 promoter could not be resolved in the electrophoresis system, but using a combination of potassium permanganate footprinting and topoisomerase I relaxation assays, we found in contrast to the nifL promoter, that open complexes were formed not only on negatively supercoiled topoisomers but also on relaxed minicircles and the Delta Lk = +1 topoisomer . These results indicate there is a thermodynamic barrier to the formation of open complexes on DNA minicircles carrying the nifL promoter which is not evident at glnAp2.

Indian J Exp Biol, 1997 Feb, 35(2), 103 - 10
Phytolectins: natural molecules with immense biotechnological potential; Sengupta S et al.; Lectins are structurally diverse, carbohydrate binding proteins that bind reversibly to specific mono- or oligosaccharides . Their abundance in the plant kingdom suggest that they have diverse roles to perform . They serve as recognition factor between symbiotic nitrogen fixing bacteria and host plants, as a deterrent to phytopathogens like fungi, insects, and animals, as storage protein and as an aid in sexual reproduction in Chlamydomonas, amongst others . The possible application of lectins as a factor in increasing soil fertility and as a biopesticide by genetically engineered organisms is yet to be fully explored by the biotechnologists . However, they are being used by the biomedical scientists and biochemists in blood typing and stimulation of cells for chromosome analysis and gene mapping, in cell separation, identification of complex glycoproteins and typing of bacteria . Cell targeting by lectins in cancer therapy is still in its infancy . This review gives an insight into the potential of these wonder biomolecules in agriculture, biochemistry, cell biology and medicine for the benefit of mankind.

Proc Natl Acad Sci U S A, 1997 Sep 30, 94(20), 10955 - 60
Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis; Pelicic V et al.; A better understanding of Mycobacterium tuberculosis virulence mechanisms is highly dependent on the design of efficient mutagenesis systems . A system enabling the positive selection of insertional mutants having lost the delivery vector was developed . It uses ts-sacB vectors, which combine the counterselective properties of the sacB gene and a mycobacterial thermosensitive origin of replication and can therefore be efficiently counterselected on sucrose at 39 degrees C . This methodology allowed the construction of M . tuberculosis transposition mutant libraries . Greater than 10(6) mutants were obtained, far exceeding the number theoretically required to obtain at least one insertion in every nonessential gene . This system is also efficient for gene exchange mutagenesis as demonstrated with the purC gene: 100% of the selected clones were allelic exchange mutants . Therefore, a single, simple methodology has enabled us to develop powerful mutagenesis systems, the lack of which was a major obstacle to the genetic characterization of M . tuberculosis.

Folia Med Cracov, 1996, 37(1-2), 49 - 59
{The importance of nitric oxide (NO) in the development of Helicobacter pylori infection}; Kopanski Z et al.; In this work we discussed selected questions about the Helicobacter pylori pathogenesis linked with the components of cell-walls as well as with toxins and enzymes externally secreted by this bacteria . We also paid attention to some theories which try to imperf the damaging effects of the bacteria on the mucosa of the upper part of the alimentary canal, specifically underlining the possible link between the Helicobacter pylori infection and the increase of the concentration of cytotoxically acting nitric oxide (NO).

J Biol Chem, 1997 Oct 3, 272(40), 24800 - 4
pH dependence of structural and functional properties of oxidized cytochrome c" from Methylophilus methylotrophus; Coletta M et al.; Cytochrome c" from Methylophilus methylotrophus is an unusual monoheme protein that undergoes a major redox-linked change in the heme arrangement: one of the two axial histidines bound to the iron in the oxidized form is detached upon reduction and a proton is taken up . The kinetics of reduction by sodium dithionite and the spectroscopic properties of the oxidized cytochrome c" have been investigated over the pH range between 1.4 and 10.0 . The rate of reduction displays proton-linked transitions of pKa congruent with 5.5 and 2.4, and a spectroscopic transition with a pKa congruent with 2.4 is also observed . The protein displays a complete reversibility after exposure to low pH, and both electronic absorption and resonance Raman spectroscopic properties suggest that the transition at lower pH brings about a drastic change in the heme coordination geometry . Circular dichroism spectra indicate that over the same proton-linked transition, the protein undergoes a marked decrease (approximately 60%) of the alpha-helical content toward a random coil arrangement, which is recovered upon increasing the ionic strength . The structural change at low pH is linked to a concerted two-proton transition, suggesting the detachment and protonation of axial histidine(s) . Such kinetic and spectroscopic features along with the remarkable capacity of this protein to recover its native structure after exposure to extremely low pH values makes it a promising model for studying folding processes and stability in heme proteins.

EMBO J, 1997 Sep 1, 16(17), 5188 - 97
Direct interaction of aminopeptidase A with recombination site DNA in Xer site-specific recombination; Alen C et al.; Xer site-specific recombination at ColE1 cer converts plasmid multimers into monomers, thus ensuring the heritable stability of ColE1 . Two related recombinase proteins, XerC and XerD, catalyse the strand exchange reaction at a 30 bp recombination core site . In addition, two accessory proteins, PepA and ArgR, are required for recombination at cer . These two accessory proteins are thought to act at 180 bp of accessory sequences adjacent to the cer recombination core to ensure that recombination only occurs between directly repeated sites on the same molecule . Here, we demonstrate that PepA and ArgR interact directly with cer, forming a complex in which the accessory sequences of two cer sites are interwrapped approximately three times in a right-handed fashion . We present a model for this synaptic complex, and propose that strand exchange can only occur after the formation of this complex.

Clin Infect Dis, 1997 Sep, 25 Suppl 2, S291 - 4
Bilophila wadsworthia clinical isolates compared by polymerase chain reaction fingerprinting; Hunt Gerardo S et al.; Bilophila wadsworthia isolates recovered from a right-ear cholesteatoma and brain abscess of the same patient were analyzed by means of polymerase chain reaction (PCR) fingerprinting with single primers (T3B and M13 core) to ascertain if they originated from the same clone . Their PCR fingerprint profiles were compared with those of three additional B . wadsworthia clinical isolates and the type strain (ATCC 49260) . The two isolates from the same patient produced PCR fingerprint profiles identical to each other, regardless of which primer was used . All isolates' PCR fingerprint profiles, with use of either the T3B or M13 core primer, shared some major and minor bands . However, differences in additional major and minor bands distinguished each of the additional isolates, suggesting that there are different subgroups of B . wadsworthia.

J Pept Res, 1997 Sep, 50(3), 159 - 70
Beta-turn induction by C60-based fulleroproline: synthesis and conformational characterization of Fpr/Pro small peptides; Bianco A et al.; We have recently described the preparation of Fpr (C60-based fulleroproline) . In this paper the synthesis and a conformational characterization of heterochiral di- and tripeptides containing this new alpha-amino acid are reported . A folded structure, induced by the -L-Fpr-D-Ala-sequence in chloroform solution and detected by Fourier transform infrared absorption and 1H nuclear magnetic resonance, has been compared with the known propensity of the cognate -L-Pro-D-Ala-sequence to adopt a beta II-turn conformation, which has also been confirmed in this work . The beta II-turn structure is retained in the crystal state by the Pro-peptides, as shown by the X-ray diffraction structures of Ibu-L-Pro-D-Ala-NHtBu and Z-L-Pro-D-Ala-L-Ala-OtBu.

Cancer Detect Prev, 1997, 21(5), 406 - 11
Human cancer and DNA repair-deficient diseases; Sarasin A et al.; Cancer development requires the accumulation of numerous genetic changes which are usually believed to occur through the presence of unrepaired DNA lesions . Exogenous or endogenous DNA-damaging agents can lead to mutations in the absence of efficient error-free repair, via replication of DNA damage . Several DNA repair pathways are present in living cells and well conserved from bacteria to human cells . Apart from mismatch repair, photolyases, base excision, and postreplication repair, the nucleotide excision repair (NER), the most versatile of these DNA repair systems, recognizes and eliminates a wide variety of DNA lesions and particularly those induced by ultraviolet (UV) light . The phenotypic consequences of an NER defect in humans are apparent in rare but dramatic diseases characterized by hypersensitivity to UV and a striking clinical and genetic heterogeneity . The xeroderma pigmentosum syndrome (XP), the Cockayne's syndrome (CS), and the photosensitive form of trichothiodystrophy (TTD) are three of these clinically distinct human disorders inherited as an autosomal recessive trait . Persistence of unrepaired DNA damage produced by exposure to UV light is associated, in the XP syndrome, with an extremely high level of skin tumors in sun-exposed sites . But the direct link of defective DNA repair to cancer seems to be complex, since, in contrast to patients with XP, those with TTD or CS do not have an increased frequency of skin cancers . The understanding of the absence of skin tumors in TTD and CS patients may offer a way to better protect normal individuals from the most rapidly increasing cancer: skin cancer.

Hosp Pract (Off Ed), 1997 Sep 15, 32(9), 37 - 8, 41-4, 52-3 passim
Polysaccharide vaccines: determinants of clinical efficacy; Musher DM; Many common bacteria cause disease because their polysaccharide capsules prevent phagocytosis . Antibody directed against these capsules is the basis for some of the most effective bacterial vaccines . Nevertheless, many diseases caused by such bacteria continue to cause serious morbidity . Recent findings indicate that several previously unrecognized factors (most notably, genetics) determine responsiveness to capsular polysaccharides.

Curr Microbiol, 1997 Oct, 35(4), 237 - 9
Molecular characterization of genes encoding a novel ABC transporter in Thermoanaerobacterium thermosulfurigenes EM1; Matuschek M et al.; The nucleotide sequence of two open reading frames (ORFs) from Thermoanaerobacterium thermosulfurigenes EM1 was determined that encode proteins with similarity to components of ATP-binding cassette (ABC) transport systems . Sequence analysis suggests that the deduced proteins AbcA and AbcB consist of an NH2-terminal membrane-spanning domain and a COOH-terminal ATP-binding domain . The deduced proteins AbcA and AbcB showed highest similarity to proteins of the MsbA subfamily of ABC transporters . AbcA and AbcB probably function as a heterodimer . An ORF predicted to encode the primary sigma factor SigA was identified downstream of abcB.

Gene, 1997 Aug 22, 195(2), 141 - 9
CROC-1 encodes a protein which mediates transcriptional activation of the human FOS promoter; Rothofsky ML et al.; The cloning of signal transducing molecules capable of activating the human FOS proto-oncogene promoter was achieved by co-transfecting a modified human FOS promoter-driven polyomavirus large T antigen gene (P(f)LAG-8) with a human brain cDNA library, incorporated into a replication-competent mammalian retroviral expression vector whose replication occurs in the presence of T antigen . In murine cells, transcriptional activation of the P(f)LAG-8 promoter by a biologically active, cDNA-encoded signalling molecule resulted in plasmid replication . Replicated plasmids, following selective cleavage of unreplicated plasmids by Dpn1, were recovered by transformation into competent bacteria . Successive P(f)LAG-8/cDNA library co-transfections, using library plasmids resulting from prior transfections, ultimately resulted in the identification of individual plasmids capable of transcriptionally activating the FOS promoter . DNA sequencing revealed the first plasmid, denoted CROC-1, to contain a 1.8-kb cDNA encoding a 16.5-kDa nuclear protein possessing a bipartite structure comprised an amino-terminal acidic domain and a carboxy-terminal basic domain, and displaying partial homology to the HMG domain of the TAF(II)250 transcription cofactor . Co-transfection of CROC-1 with various FOS/CAT reporter genes revealed that the human FOS promoter region spanning -56 to - 105, encompassing two identical 8-bp DR enhancer sequences, was necessary for CROC-1-mediated transcriptional activation . Results suggest that CROC-1 participates in intracellular signalling pathways involved in induction of the human FOS promoter.

EMBO J, 1997 Aug 15, 16(16), 5019 - 29
From genetic to structural characterization of a new class of RNA-binding domain within the SacY/BglG family of antiterminator proteins; Manival X et al.; SacY is the prototype of a family of regulatory proteins able to prevent transcription termination . It interacts with a 29 nucleotide RNA sequence able to fold into a stem-loop structure and partially overlapping with a terminator sequence located in the 5' leader mRNA region of the gene it controls . We show here that the N-terminal fragment of SacY, SacY(1-55), and the corresponding fragments of other members of the family have antiterminator activities with efficiency and specificity identical to those of the full-length proteins . In vitro, this activity correlates with the specific affinity of SacY(1-55) for its RNA target . UV melting experiments demonstrate that SacY(1-55) binding stabilizes the RNA target structure . The NMR solution structure of SacY(1-55) is very similar to that obtained in the crystal (van Tilbeurgh et al., 1997): the peptide is folded as a symmetrical dimer without any structural homology with other RNA-binding domains yet characterized . According to a preliminary NMR analysis of the SacY(1-55)-RNA complex, the protein dimer is not disrupted upon RNA binding and several residues implicated in RNA recognition are located at the edge of the dimer interface . This suggests a new mode of protein-RNA interaction.

Ophthalmic Surg Lasers, 1997 Sep, 28(9), 758 - 61
An orbital abscess secondary to acute dacryocystitis; Ntountas I et al.; An orbital abscess is an ophthalmic surgical emergency that is typically caused by the spread of bacteria from adjacent structures, such as the sinuses, eyelids, or teeth . Although acute dacryocystitis is commonly associated with preseptal cellulitis, it rarely causes orbital infection . Infection of the lacrimal sac will typically localize in the preseptal space because the lacrimal sac lies anterior to the orbital septum . To the authors' knowledge, this is the first report of an intraconal abscess secondary to acute dacryocystitis . The key points in the surgical management of this entity are discussed.

Zh Mikrobiol Epidemiol Immunobiol, 1997 May-Jun, (3), 84 - 8
{The immunotherapy of bronchial asthma with a multicomponent vaccine administered intranasally and orally}; Chuchalin AK et al.; Multicomponent vaccine prepared from opportunistic bacteria, when administered intranasally and orally to patients with bronchial asthma and chronic bronchitis, was well tolerated by the patients . Side effects induced by the vaccine in a few patients lasted for a short time and were mainly local . Immunotherapy with the vaccine led to a decrease in the severity of the course of the disease, as well as in the frequency and severity of exacerbations . In 26 out of 35 adult patients (74.3%) good and excellent effect was noted; during the observation of 20 sick children aged up to 3 years for a period of 1 year similar effect was noted in 60% of cases . Vaccinal therapy, in addition to its main effect, also contributed to a considerable drop in the morbidity rate of acute respiratory diseases among susceptible children.

FEMS Microbiol Lett, 1997 Aug 15, 153(2), 479 - 84
Evidence for the role of 2-hydroxychromene-2-carboxylate isomerase in the degradation of anthracene by Sphingomonas yanoikuyae B1; Kim E et al.; Sphingomonas yanoikuyae B1 is extremely versatile in its catabolic ability . An insertional mutant strain, S . yamoikuyae EK504, which is unable to grow on naphthalene due to the loss of 2-hydroxychromene-2-carboxylate isomerase activity, was utilized to investigate the role of this enzyme in the degradation of anthracene by S . yanoikuyae B1 . Although EK504 is unable to grow on anthracene, this strain could transform anthracene to some extent . A metabolite in the degradation of anthracene by EK504 was isolated by high-pressure liquid chromatography (HPLC) and was identified as 6,7-benzocoumarin by UV-visible, gas-chromatographic, HPLC/mass-spectrometric, and 1H nuclear magnetic resonance spectral techniques . The identification of 6,7-benzocoumarin provides direct chemical and genetic evidence for the involvement of nahD in the degradation of anthracene by S . yanoikuyae B1.

Rev Esp Enferm Dig, 1997 Jun, 89(6), 479 - 80
{Gas gangrene secondary to carcinoma of the colon}; Ruiz de Adana JC et al.; Systemic infections due to enteric bacteria can develop in patients with occult intestinal tumours . A patient with a sigmoid adenocarcinoma that developed crepitation and necrosis of soft tissues in gluteous region and thigh of left lower limb is presented . No pus or free fluid was observed in the peritoneal cavity; a sigmoid tumor infiltrating and perforating the retroperitoneum with necrosis of the psoas muscle was found . The infection spread subsequently through the inguinal canals and sciatic foramen to the lower limb . Necrotizing infections of soft tissues without previous trauma or ischemic accident leads to the suspicion of an occult digestive disease.

FEBS Lett, 1997 Aug 25, 413(3), 473 - 6
Electrospray ionization mass spectrometry analysis of the apo- and metal-substituted forms of the Fur protein; Michaud-Soret I et al.; Fur has been purified and reconstituted with Co2+ and Mn2+ . The ESI-MS spectra of the apoprotein as well as Mn-Fur and Co-Fur under acidic denaturating conditions showed the existence of two species of molecular mass 16,660 +/- 3 and 16,792 +/- 3 Da, which correspond, respectively, to the N-terminal methionine 'excised' or 'non-excised' forms of the monomer . This result proves the absence of any other post-translational modification or modification due to metal incorporation . On the other hand, under soft conditions, ESI spectra provided for the first time direct evidence for dimeric metal-containing forms in solution.

Mem Inst Oswaldo Cruz, 1997 Jan-Feb, 92(1), 123 - 8
Establishment and characterization of a cell line from the mosquito Anopheles albimanus (Diptera: Culicidae); Bello FJ et al.; A new cell line designated LSB-AA695BB, was established from embryos of the mosquito Anopheles albimanus . The primary culture was initiated in April, 1995, and the first passage was made 48 days later . Serial subcultures of the cells have been carried through 90 passage from Abril 1995 to February 1996 . The cells were grown at 28 degrees C in MK/VP12 medium, supplemented with 20% fetal bovine serum: the pH tolerance ranged between 6.8 to 7.0 . The cells have also been adapted to MM/VP12 medium under the same pH, temperature and serum concentration . The majority of the cells were a fibroblast-type . Isozyme characterization showed a pattern similar to that of An . albimanus pupae and adults but distinct from Ae . taeniorhynchus and Ae . albopictus (C6/36) mosquito cell lines . The culture was shown to be free of mycoplasma, bacteria and fungi . Microsporidia contamination of transovarial transmission was controlled with 6.0 micrograms/ml of albendazole.

Biol Chem, 1997 Aug, 378(8), 827 - 36
A unique cascade of oxidoreductases catalyses trypanothione-mediated peroxide metabolism in Crithidia fasciculata; Nogoceke E et al.; Parasitic trypanosomatids comprise causative agents of debilitating or life-threatening tropical diseases . The limited capacity of these parasites to cope with oxidative stress has been discussed as a target area for therapeutic approaches but success has been hampered by a lack of comprehension of their peculiar oxidant defense system depending on the unique redox metabolite trypanothione . Here we report that trypanothione-dependent hydroperoxide metabolism in Crithidia fasciculata is catalysed by two distinct proteins working in concert . One is Cf16, a unique protein which, apart from a WCPPC sequence that resembles the thioredoxin-type WCG(A)PC motif, only shows low similarity to thioredoxin-like proteins of bacteria and invertebrates . The second component is Cf21, which can be classified as a member of the peroxiredoxin family of proteins . The two proteins have been purified to homogeneity and shown to be essential for the trypanothione-dependent removal of hydroperoxides . By means of selective derivatisation of the substrate-reduced proteins the flux of reduction equivalents from trypanothione to Cf16, Cf21 and finally to the hydroperoxide was elucidated . Cf21 proved to be a moderately efficient peroxidase with broad specificity . The rate constants for the reaction of the reduced protein with H2O2, t-butyl hydroperoxide, linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide were 1.0 x 10(5), 1.2 x 10(5), 1.0 x 10(5) and 0.4 x 10(5) M-1S-1, respectively . The apparent rate constant for the regeneration of reduced Cf21 by Cf16 was in the range of 1.5-3.5 x 10(6) M-1S-1 . This newly discovered metabolic pathway adds two further candidates to the list of potential targets for trypanocidal drugs.

J Immunol, 1997 Sep 15, 159(6), 2840 - 8
CD28 expression by mouse mast cells is modulated by lipopolysaccharide and outer surface protein A lipoprotein from Borrelia burgdorferi; Marietta EV et al.; The concept of costimulation has been best defined in T cells and B cells . However, other cells that respond in an Ag-specific fashion, such as the mast cell, may be regulated by similar mechanisms . We have found that murine mast cells express one such costimulatory molecule, CD28, which was previously defined as a T and NK cell-specific protein . While CD28 transcription appeared to be constitutive in murine mast cells, its cell surface expression was not . CD28 cell surface expression by mast cells derived from bone marrow with stem cell factor (SCF) was dependent upon activation with agents such as LPS, the Borrelia burgdorferi lipoprotein outer surface protein A, and PMA . Peak cell surface expression of CD28 by such cells occurred 24 h after LPS stimulation, 18 h after outer surface protein A stimulation, and 3 h after PMA stimulation . In contrast, mast cells derived from bone marrow with IL-3 did not demonstrate induction-specific cell surface expression of CD28 . Instead, maturation of such cells in vitro allowed for the increased cell surface expression of CD28 . Peritoneal mast cells cultured in SCF also expressed CD28 . Mast cell CD28 was functional, in that cross-linking of CD28 on the surface of the IL-3-derived cells resulted in an increased level of c-jun transcripts . Additionally, cross-linking of CD28 simultaneously with PMA treatment of SCF-derived mast cells resulted in an increased level of IL-13 transcripts . These data suggest that mast cell CD28 has functions similar to those of T cell CD28.

Virology, 1997 Sep 15, 236(1), 30 - 6
Mutational analysis of transcriptional activation by the bovine papillomavirus type 1 E6; Chen JJ et al.; While the bovine papillomavirus type 1 (BPV-1) E6 induces tumorigenic transformation of murine C127 cells, it does not bind or promote the degradation of p53 . We recently showed the cellular protein ERC-55/E6BP binds BPV-1 E6 as well as the cancer-related human papillomavirus (HPV) E6 proteins . BPV-1 E6 also binds E6-AP, a ubiquitin ligase necessary for HPV E6-induced p53 degradation . We previously reported that the transforming activity of a set of BPV-1 E6 mutants correlated with their E6BP-binding ability . Another function of BPV-1 E6 is stimulation of transcription when targeted to a promoter, although cellular promoters responsive to BPV-1 E6 have not been identified . To examine whether its transcriptional function is required for oncogenic activity, or is related to its interactions with E6-AP or E6BP, a series of BPV-1 E6 mutants were analyzed as fusions to a sequence-specific DNA binding domain for activity in yeast and in mammalian cells . We show that some transformation defective mutants retained substantial levels of transcriptional activation activity . These mutants also distinguish transcriptional activation from E6-AP and E6BP binding . These results suggest the transcriptional activation function of BPV-1 E6 is not sufficient for cell transformation .

Biochem Biophys Res Commun, 1997 Sep 18, 238(2), 452 - 6
Identification of a novel determinant of glutathione affinity in dichloromethane dehalogenases/glutathione S-transferases; Vuilleumier S et al.; Bacterial dichloromethane dehalogenases catalyze the glutathione-dependent hydrolysis of dichloromethane to formaldehyde and are members of the enzyme superfamily of glutathione S-transferases involved in the detoxification of electrophilic compounds . Numerous protein engineering studies have addressed questions pertaining to the substrate specificity, the reaction mechanism, and the kinetic pathway of glutathione S-transferases . In contrast, the molecular determinants for binding of the glutathione cofactor have been less well investigated . Dichloromethane dehalogenases from Hyphomicrobium sp . DM2 and Methylobacterium sp . DM4 displayed significantly different affinities for glutathione, but not for the dichloromethane substrate . The sequence of dcmA, the dichloromethane dehalogenase gene from strain DM2, was determined and featured a single base difference from the previously determined sequence of dcmA from strain DM4 . This base change resulted in a single amino acid difference in the corresponding proteins at sequence position 27 . Site-directed variants of the homologous dichloromethane dehalogenase from Methylophilus sp . DM11 (56% amino acid identity) at the corresponding residue in the protein sequence provided further evidence that this residue selectively modulated the dependence of dichloromethane dehalogenase activity on glutathione .

J Theor Biol, 1997 Sep 7, 188(1), 69 - 78
Sexual communication; Bernstein C et al.; Sexual communication refers to the use of signals to promote or modulate sexual interaction . We suggest that these signals operate at three levels: (a) a primary level at which signals are used to increase the likelihood of sexual interaction between two individuals; (b) a secondary level at which signals inhibit inbreeding or facilitate outbreeding; and (c) a tertiary level at which signals allow selection among potential mates . Evidence is cited that the selective advantage of the primary, secondary and tertiary levels are, respectively, repair of DNA damage, masking of mutation, and selection for fitness in the mating partner . We illustrate how these advantageous processes are facilitated by sexual communication in bacteria, fungi, protozoa, insects, plants and vertebrates .

J Theor Biol, 1997 Aug 21, 187(4), 529 - 40
Modular evolution of the respiratory NADH:ubiquinone oxidoreductase and the origin of its modules; Friedrich T et al.; The proton-pumping NADH:ubiquinone oxidoreductase is the foremost of the respiratory chain complexes providing the proton motive force required for the synthesis of ATP . The complex is found in purple bacteria and in the mitochondria of most eukaryotes . The bacterial complex consists of 14 different subunits while the mitochondrial complex contains at least 28 accessory proteins which do not directly participate in the electron and proton transport function . A homologous complex which has 11 subunits in common with the respiratory complex I exists in cyanobacteria and chloroplasts . This complex might probably work as a NADPH:plastoquinone oxidoreductase being possibly involved in a cyclic photosynthetic electron transport . Homologues of the functional modules of the complex are also found in other bacterial electron transfer and ion transport proteins . The modular evolution of the complex and the possible origin of its modules are discussed in this paper.

Gynecol Oncol, 1997 Sep, 66(3), 464 - 74
Establishment and characterization of cell lines derived from uterine malignant mixed Müllerian tumor; Yuan Y et al.; OBJECTIVE: We report the establishment and characterization of three new cell lines derived from uterine malignant mixed mullerian tumor (MMMT) . METHODS: Three uterine MMMT cell lines from primary tumors of Korean patients were cultured and the involved cell morphology, growth properties, DNA profiles, immunohistochemical properties, tumor-associated antigen secretion, and genetic alterations of related oncogenes and tumor suppressor genes were studied as well . RESULTS: Three MMMT cell lines were successfully established including one homologous tumor SNU-539 and two heterologous tumors SNU-685 and SNU-1077 . All lines showed substrate adherence and high viability and were proven by DNA fingerprinting analysis to be unique . Contamination by mycoplasma and bacteria was excluded . SNU-539 and SNU-1077 cells stained positively for both epithelial and mesenchymal antigens, while SNU-685 cells only stained positively for mesenchymal antigens . The level of secretion of tumor-associated antigens, CA125 and CEA, was shown to be undetectable in all three lines . One missense mutation from AAC to GAC at codon 239 of exon 7 in the p53 gene was identified in SNU-539 . CONCLUSIONS: These newly established and characterized permanent uterine MMMT cell lines might be regarded as valuable resources for a multitude of in vitro investigations, which should be used for clarifying the obscure histogenetic origin and understanding the biological behavior of this aggressive tumor .

Gynecol Oncol, 1997 Sep, 66(3), 378 - 87
Establishment and characterization of human ovarian carcinoma cell lines; Yuan Y et al.; Five human ovarian carcinoma cell lines cultured from primary and metastatic tumors of Korean patients were characterized . These lines were isolated from two papillary serous cystadenocarcinomas, two endometrioid carcinomas, and one malignant Brenner tumor . It was shown that the growth of these cell lines was stable when cultured after at least 20 passages . Population doubling times varied from 40 to 67 hr . All lines showed high viability and were proven by DNA fingerprinting analysis to be unique . Contamination by mycoplasma or bacteria was excluded . In two lines, SNU-8 and SNU-840, an elevated level of CA125 antigen secretion could be detected, whereas CEA was undetectable in all five lines . Four different mutations in functional and highly conserved regions of the p53 gene were identified in three of our five lines (60%), namely in SNU-119, SNU-251, and SNU-563 . Included were two missense mutations, one in-frame 3-base-pair deletion, and one out-of-frame 1-base-pair deletion . It is interesting to note that one of these three lines, SNU-251, presented an additional simultaneous nonsense mutation of the BRCA1 gene and missense mutation of the hMLH1 gene . In its lacking both wild-type alleles of the BRCA1 gene, SNU-251 might serve as an unusual and important in vitro model for studies related to ovarian carcinoma and the BRCA1 gene . It is thus likely that the establishment and characterization of these permanent human ovarian carcinoma cell lines in continuous cultures can provide useful tools for in vitro studies related to human ovarian carcinomas .

Electrophoresis, 1997 Aug, 18(8), 1491 - 7
Identification and characterization of Brucella ovis immunogenic proteins using two-dimensional electrophoresis and immunoblotting; Teixeira-Gomes AP et al.; In a previous report, proteins from Brucella melitensis were characterized by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and N-terminal microsequencing . In the present report, we have extended this study to the second etiologic agent in ovine brucellosis, B . ovis, responsible for ram epididymitis and infertility . The combination of 2-D gel electrophoresis and protein microsequencing facilitated the location and identification of the major proteins of B . ovis on the 2-D pattern . These proteins comprised cytoplasmic, periplasmic, and some membrane proteins except the major outer membrane proteins . By comparing 2-D gel profiles of B . ovis with that of B . melitensis described previously, a few proteins with different expression levels were readily identified . Serum from a ram naturally infected with B . ovis was used in immunoblotting studies to identify immunogenic proteins recognized during the course of infection . This serum showed antibody reactivity against approximately 82 protein spots . Twenty-one of these proteins were identified either by use of monoclonal antibodies or by N-terminal microsequencing . Several proteins previously described in earlier Brucella works were identified: the 89 kDa outer membrane protein, DnaK, GroEL, BP26, and Cu-Zn superoxide dismutase . Eight proteins had amino acid sequences homologous to those of various proteins from other bacteria found in protein databases; NikA, dihydrolipoamide succinyltransferase, a hypothetical 31 kDa protein, malate dehydrogenase, succinyl-CoA synthetase alpha subunit, an amino acid ABC type transporter, Leu/Ile/Val-binding protein precursor, and ClpP . The remaining eight proteins had N-terminal sequences lacking similarity to existing databases entries . Thus, the 2-D PAGE analysis provided a convenient first approach in the characterization of immunogenic proteins.

Electrophoresis, 1997 Aug, 18(8), 1373 - 83
Proteomic 'contigs' of Ochrobactrum anthropi, application of extensive pH gradients; Wasinger VC et al.; The most extensive linear pH gradients yet employed in combination with two-dimensional gel electrophoresis are described, along with their application in proteome analysis . A significant proportion of the protein compliment of bacterial species is believed to be accessible using an extended linear pH gradient of 2.3 to 11.0 . Protein standards with predicted isoelectric points (pI) ranging from 3.24 to 9.56 were used to confirm focusing positions with respect to the immobilised pH gradients (IPG) prior to mapping studies of Ochrobactrum anthropi . Multiple gel images were used to construct contiguous windows of protein expression ('proteomic contigs') within 18 cm pH gradients 2.3-5, 4-7, and 6-11 in conjunction with 15% T and 7.5% T acrylamide gels, the latter being used to resolve higher molecular weight (M(r)) proteins . Each IPG had a 5 cm region of similar pH gradient overlap at pH 4-5 and pH 6-7 that was used to construct an image of protein expression characteristic of whole cell lysates . This is reminiscent of genomic sequencing initiatives whereby portions are combined to form a contiguous picture of the whole . The protein maps obtained demonstrated a means of resolving the many tens of thousands of cellular proteins likely to occur in eukaryotic systems, but also highlighted the need to further optimise protein extraction, equilibration buffers, and separation conditions of higher M(r) proteins occurring at extreme pI . Theoretical 2-D protein maps were constructed for five organisms for which the total DNA sequence is now available . In all cases, higher M(r) acidic and basic proteins were shown to be common.

Acta Odontol Scand, 1997 Aug, 55(4), 261 - 4
The effect of some metal ions on volatile sulfur-containing compounds originating from the oral cavity; Waler SM; Halitosis originates mainly from the oral cavity, and the volatile sulfur-containing compounds (VSC) are the major contributors of the unpleasant odor . Anaerobic G- bacteria use sulfur-containing amino acids in their production of VSC . Zinc has been shown to inhibit production of odiferous VSC, and the mechanism proposed has been that zinc, with its affinity for sulfur, oxidizes thiol groups and thereby inhibits the precursors of VSC . The aim of the study was to investigate whether, and to what extent, other metal ions with affinity for sulfur exert the same effect and whether a correlation exists between the sulfur affinity and VSC-inhibiting activity of these metals . VSC levels were measured on the 'morning breath' of 10 test subjects, using a portable sulfide monitor . The mouthrinses tested were aqueous solutions of zinc chloride, zinc citrate, stannous fluoride, cuprous gluconate, ferrous gluconate, and silver acetate, and they contained equimolar amounts of metals (1.47 mmol/I) . The results showed that the ranking of Zn++ and Sn++ differed in the clinical test compared with sulfur affinity, and likewise with Ag+ and Fe++ . It may therefore be concluded that there is no positive correlation between the inhibiting effect of metal ions on VSC and their affinity for sulfur.

Bioessays, 1997 Sep, 19(9), 827 - 32
Origin of eukaryotic programmed cell death: a consequence of aerobic metabolism?
Frade JM, Michaelidis TM.
A marked feature of eukaryotic programmed cell death is an early drop in mitochondrial transmembrane potential . This results from the opening of permeability transition pores, which are composed of adenine nucleotide translocators and mitochondrial porins . The latter share striking similarities with bacterial porins, including down-regulation of their pore size by purine nucleotides), suggesting a common origin . The porins of some invasive bacteria play a crucial role during their accommodation inside the host cell and this coexistence resembles the endosymbiotic origin of mitochondria . The above observations suggest that early in eukaryotic evolution, former invaders may have used porin-type channels to enter their host and to induce its death when the levels of its cytoplasmic purine nucleotides were dropped . The appearance of adenosine nucleotide translocators in the primitive eukaryotes, which permitted usage of the oxidative metabolism of the invaders, provided the basis for the permeability transition phenomena, now linked to the apoptotic process . Bcl-2-type molecules, being able to modulate the permeability transition pores by interaction with adenosine nucleotide translocators, may have played an essential role in conferring a means of controlling apoptosis.

FEMS Microbiol Lett, 1997 Sep 1, 154(1), 151 - 7
Development and use of a conditional antisense mutagenesis system in mycobacteria; Parish T et al.; Expression from a 2.3 kb region upstream of the inducible acetamidase gene from Mycobacterium smegmatis was shown to be upregulated by acetamide . A DNA fragment containing the start of the M . smegmatis hisD gene was cloned in front of the promoter, such that the antisense message was produced . When this construct was induced in vivo, the bacteria became phenotypically histidine auxotrophs; this auxotrophy was restored by histidine supplementation . Auxotrophy was not observed under non-induced conditions . Antisense mutagenesis may be useful for observing the phenotypic inactivation of specific mycobacterial genes, and an inducible system such as that described would allow the study of essential genes.

Hum Immunol, 1997 May, 54(2), 194 - 201
MHC class II-dependent peptide antigen versus superantigen presentation to T cells; Shoukry NH et al.; T lymphocytes expressing the CD4 coreceptor can be activated by two classes of major histocompatibility complex (MHC) class II-bound ligands . The elaboration of a conventional T-cell mediated immune response involves recognition of an antigenic peptide bound to the MHC class II molecules by a T-cell receptor (TCR) specific to that particular antigen . Conversely, superantigens (SAgs) also bind to MHC class II molecules and activate T cells, leading to a completely different functional outcome; indeed, SAg-responsive T cells die through apoptosis following stimulation . Superantigens are proteins that are secreted by various bacteria . They interact with the TCR using molecular determinants that are distinct from the residues involved in the recognition of nominal antigenic peptides . Despite the similarities between the recognition of the two classes of ligands by the TCR, considerable structural difference is observed . Here, we discuss the current knowledge on the presentation of SAgs to T cells and compare the different aspects of the SAg response with the recognition of antigenic peptide/MHC complexes.

Arch Microbiol, 1997 Oct, 168(4), 277 - 81
The major carotenoid in all known species of heliobacteria is the C30 carotenoid 4,4'-diaponeurosporene, not neurosporene; Takaichi S et al.; The carotenoids of five species of heliobacteria (Heliobacillus mobilis, Heliophilum fasciatum, Heliobacterium chlorum, Heliobacterium modesticaldum, and Heliobacterium gestii) were examined by spectroscopic methods, and the C30 carotene 4,4'-diaponeurosporene was found to be the dominant pigment; heliobacteria were previously thought to contain the C40 carotenoid neurosporene . In addition, trace amounts of the C30 diapocarotenes diapolycopene, diapo-zeta-carotene, diapophytofluene, and diapophytoene were also found . Up to now, diapocarotenes have been found in only three species of chemoorganotrophic bacteria, but not in phototropic organisms . Furthermore, the esterifying alcohol of bacteriochlorophyll g from all known species of heliobacteria was determined to be farnesol (C15) instead of the usual phytol (C20) . Heliobacteria may be unable to produce geranylgeranyol (C20).

Biochim Biophys Acta, 1997 Aug 21, 1358(1), 39 - 45
2',3'-Dideoxycytidine cytotoxicity in human macrophages; Antonelli A et al.; Human macrophages when cultured for several weeks in the presence of therapeutically relevant 2',3'-dideoxycytidine (ddC) concentrations show a time-dependent decay in mitochondrial DNA content . This decay is associated with a reduction of Rhodamine 123 fluorescence, a marker for mitochondrial membrane potential suggesting that impairment of mitochondrial functions occurs . Mitochondrial metabolic impairment was confirmed by direct evaluation of lactate production, which is markedly increased in cells treated with ddC . The activity of protein kinase C and intracellular free Ca2+ upon addition of phorbol 12-myristate 13-acetate (PMA) were lower in the drug-treated cells compared to controls . A 50% reduction in O2-release was also found upon PMA stimulation . Fluorescent latex beads, yeast and bacteria phagocytosis were normal, but intracellular bacteria killing was markedly impaired in ddC-exposed macrophages . Thus, ddC exerts a delayed mitochondrial toxicity also on differentiated macrophages with impairment of several metabolic properties and O2 production causing a reduced ability of these phagocytic cells to kill phagocytosed bacteria.

Zhonghua Hu Li Za Zhi, 1996 Jul, 31(7), 381 - 3
{Study on drawing aseptic gas in diluting drugs}; Fan ZC et al.; In this study, the gas was drawn from sealed aseptic bottles, the blue flame of an alcohol lamp, and the air of the same treatment room . And the gas was put into aseptic solutions of 10% glucose separately and dripped . Then the samples were taken for bacteriaculture at appointed time-points . Meanwhile, the gases were drawn and put into aseptic solutions of 10% glucose separately . Then deactived penicillines were diluted with the solutions separately . Finally, the penicillines were mixed with 10% glucose and dripped . The samples were taken for bacteria-culture in the same way . The results showed that there was no colony existed in the gas from the sealed aseptic bottles and the flame of the alcohol lamp . However, colonies existed in the samples from the air of the treatment room . It is suggested that sealed aseptic gas should be drawn and kept for use in diluting drugs.

J Biol Chem, 1997 Sep 19, 272(38), 23690 - 5
Activation of the mitogen-activated protein kinase ERK2 by the chemoattractant folic acid in Dictyostelium; Maeda M et al.; The Dictyostelium MAP kinase ERK2 is activated by extracellular cAMP in aggregation-competent cells and is required for receptor activation of adenylyl cyclase (Maeda, M., Aubry, L., Insall, R., Gaskins, C., Devreotes, P . N., and Firtel, R . A . (1996) J . Biol . Chem . 271, 3351-3354; Segall, J., Kuspa, A., Shaulsky, G., Ecke, M., Maeda, M., Gaskins, C., Firtel, R., and Loomis, W . (1995) J . Cell Biol . 128, 405-413) . This cAMP-dependent activation of ERK2 is mediated by the serpentine, G protein-coupled cAMP receptors . However, ERK2 activation by cAMP is at least partially heterotrimeric G protein-independent, with a level of activation in cells lacking the sole Gbeta subunit or the G protein-coupled cAMP receptors-coupled Galpha2 subunit that is approximately 50% that of wild-type cells (Maeda, M., Aubry, L., Insall, R., Gaskins, C., Devreotes, P . N., and Firtel, R . A . (1996) J . Biol . Chem . 271, 3351-3354; Segall, J., Kuspa, A., Shaulsky, G., Ecke, M., Maeda, M., Gaskins, C., Firtel, R., and Loomis, W . (1995) J . Cell Biol . 128, 405-413) . Folic acid, a chemoattractant in the vegetative cells that enables amoebae to find bacteria in the wild, also triggers the activation of adenylyl cyclase, which is impaired in the vegetative cells lacking the Galpha protein subunit Galpha4 (Hadwiger, J., Lee, S., and Firtel, R . (1994) Proc . Natl . Acad . Sci . U . S . A . 91, 10566-10570) . In this study, we show that folic acid activates ERK2 in developmentally regulated manner and is required for ERK2 stimulation of adenylyl cyclase activity . Maximum levels of folate-stimulated ERK2 activity occur in cells from very early in development, prior to aggregation, and again at the tipped aggregate stages, corresponding to the stages in which folate receptors and the coupled Galpha subunit Galpha4 are maximally expressed . During the activation by folic acid, ERK2 is phosphorylated on tyrosine residue(s) and contemporaneously shows a mobility shift on SDS-PAGE . Interestingly, this activation is not elicited in the absence of Gbeta subunits, in contrast to the response to cAMP . This response also requires the Galpha4 subunit known to be required for other folic acid-mediated responses (Hadwiger, J., Lee, S., and Firtel, R . (1994) Proc . Natl . Acad . Sci . U . S . A . 91, 10566-10570) . Furthermore, we show that the activation of ERK2 by cAMP is independent of the Galpha4 subunit, while the activation of ERK2 by folate is independent of Galpha2 . Taken together, these data indicate that there are at least two pathways of ERK2 activation, heterotrimeric G protein-dependent and -independent pathways.

Pharmacogenetics, 1997 Aug, 7(4), 255 - 69
The UDP glycosyltransferase gene superfamily: recommended nomenclature update based on evolutionary divergence; Mackenzie PI et al.; This review represents an update of the nomenclature system for the UDP glucuronosyltransferase gene superfamily, which is based on divergent evolution . Since the previous review in 1991, sequences of many related UDP glycosyltransferases from lower organisms have appeared in the database, which expand our database considerably . At latest count, in animals, yeast, plants and bacteria there are 110 distinct cDNAs/genes whose protein products all contain a characteristic 'signature sequence' and, thus, are regarded as members of the same superfamily . Comparison of a relatedness tree of proteins leads to the definition of 33 families . It should be emphasized that at least six cloned UDP-GlcNAc N-acetylglucosaminyltransferases are not sufficiently homologous to be included as members of this superfamily and may represent an example of convergent evolution . For naming each gene, it is recommended that the root symbol UGT for human (Ugt for mouse and Drosophila), denoting 'UDP glycosyltransferase,' be followed by an Arabic number representing the family, a letter designating the subfamily, and an Arabic numeral denoting the individual gene within the family or subfamily, e.g . 'human UGT2B4' and 'mouse Ugt2b5' . We recommend the name 'UDP glycosyltransferase' because many of the proteins do not preferentially use UDP glucuronic acid, or their nucleotide sugar preference is unknown . Whereas the gene is italicized, the corresponding cDNA, transcript, protein and enzyme activity should be written with upper-case letters and without italics, e.g . 'human or mouse UGT1A1.' The UGT1 gene (spanning > 500 kb) contains at least 12 promoters/first exons, which can be spliced and joined with common exons 2 through 5, leading to different N-terminal halves but identical C-terminal halves of the gene products; in this scheme each first exon is regarded as a distinct gene (e.g . UGT1A1, UGT1A2, .. . UGT1A12) . When an orthologous gene between species cannot be identified with certainty, as occurs in the UGT2B subfamily, sequential naming of the genes is being carried out chronologically as they become characterized . We suggest that the Human Gene Nomenclature Guidelines be used for all species other than the mouse and Drosophila . Thirty published human UGT1A1 mutant alleles responsible for clinical hyperbilirubinemias are listed herein, and given numbers following an asterisk (e.g . UGT1A1*30) consistent with the Human Gene Nomenclature Guidelines . It is anticipated that this UGT gene nomenclature system will require updating on a regular basis.

Eur J Immunol, 1997 Aug, 27(8), 2118 - 21
B cell-deficient muMT mice as an experimental model for Mycoplasma infections in X-linked agammaglobulinemia; Berglof A et al.; B cell-deficient muMT mice were investigated as an experimental model for human X-linked agammaglobulinemia (XLA) . Mice were intranasally infected with Mycoplasma pulmonis and in 16 out of 17 muMT mice, dissemination of the bacteria from the airways was observed . More than 50% of these mice developed arthritis and/or changes in periarticular tissues . Mycoplasmal infection in muMT mice thus resembles the disease seen in XLA patients implying the usefulness of the model.

Trends Microbiol, 1997 Sep, 5(9), 355 - 9
Selfish operons and speciation by gene transfer; Lawrence JG; Bacterial genes providing for single metabolic functions are found in operons, possibly because this organization allows efficient horizontal transfer among organisms . Transferred genes can confer novel metabolic phenotypes on their new hosts and allow rapid, effective exploitation of new environmental niches . Moreover, the mobility of selfish operons may facilitate bacterial speciation.

Clin Cardiol, 1997 Sep, 20(9), 813 - 5
Transesophageal echocardiographic evaluation of aortitis; Harris KM et al.; Aortitis is an uncommon infection which may occur in patients with preexisting atherosclerotic disease of the aorta . The clinical features in two patients presenting with fever and nonspecific symptoms are reviewed . In these patients, transesophageal echocardiographic features of wall thickening and false aneurysm formation were suggestive of the diagnosis of aortitis . Both patients were taken for surgical excision of the descending aorta and subsequently improved.

J Bacteriol, 1997 Sep, 179(18), 5951 - 5
Testosterone-regulated expression of enzymes involved in steroid and aromatic hydrocarbon catabolism in Comamonas testosteroni; Mobus E et al.; The effect of testosterone as the sole carbon source on protein expression was analyzed in Comamonas testosteroni . Testosterone simultaneously induced the expression of steroid- and aromatic hydrocarbon-catabolizing enzymes and repressed one amino acid-degrading enzyme . It is suggested that steroids play a regulative role in catabolic enzyme synthesis during adaptive growth of C . testosteroni.

J Bacteriol, 1997 Sep, 179(18), 5827 - 34
A Tn10 derivative (T-POP) for isolation of insertions with conditional (tetracycline-dependent) phenotypes; Rappleye CA et al.; A new Tn10-based transposon has been constructed and used to isolate insertion mutations with tetracycline-conditional phenotypes . Classes of mutants include conditional lethal mutations, conditional auxotrophs, and conditional mutants of the eut (ethanolamine utilization) operon . The described mutations were made with a new derivative of Tn10dTet that we have called Tn10d(T-POP) . Others have noted that transposon Tn10dTet directs weak tetracycline-inducible transcripts out of both ends of the element into adjacent sequences . We have increased this level of outward transcription from Tn10dTet by selecting deletion mutations within the element that presumably remove transcription-termination signals . Insertion of the Tn10d(T-POP) element within an operon disrupts the target gene and makes expression of distal genes dependent on induction of outward transcription by tetracycline . Insertion mutations made with Tn10d(T-POP) can cause tetracycline-correctable conditional phenotypes based on expression of distal genes.

Proc Natl Acad Sci U S A, 1997 Sep 16, 94(19), 10279 - 84
Macrophage inflammatory protein-2: chromosomal regulation in rat small intestinal epithelial cells; Ohno Y et al.; Nonpathogenic, resident bacteria participate in the pathogenesis of inflammation in the small intestine, but the molecular messages produced by such bacteria are unknown . Inflammatory responses involve the recruitment of specific leukocyte subsets . We, therefore, hypothesized that butyrate, a normal bacterial metabolite, may modulate chemokine secretion by epithelial cells, by amplifying their response to proinflammatory signals . We studied the expression of the chemokine, macrophage inflammatory protein-2 (MIP-2) by the rat small intestinal epithelial cell line, IEC-6 . Cells were stimulated with lipopolysaccharide or with interleukin 1beta (IL-1beta) and incubated with sodium butyrate . Acetylation of histones was examined in Triton X acetic acid-urea gels by PAGE . Unstimulated IEC-6 cells did not secrete MIP-2 . However, lipopolysaccharide and IL-1beta induced MIP-2 expression . Butyrate enhanced MIP-2 secretion both in lipopolysaccharide-stimulated and IL-1beta-stimulated enterocytes; but butyrate alone did not induce MIP-2 expression . Butyrate increased the acetylation of histones extracted from the nuclei of IEC-6 cells . Furthermore, acetylation of histones (induced by trichostatin A, a specific inhibitor of histone deacetylase) enhanced MIP-2 expression by cells stimulated with IL-1beta . In conclusion, trichostatin A reproduced the effects of butyrate on MIP-2 secretion . Butyrate, therefore, increases MIP-2 secretion in stimulated cells by increasing histone acetylation . We speculate that butyrate carries information from bacteria to epithelial cells . Epithelial cells transduce this signal through histone deacetylase, modulating the secretion of chemokines.

J Clin Invest, 1997 Sep 15, 100(6), 1557 - 65
Lipopolysaccharide-stimulated osteoclastogenesis is mediated by tumor necrosis factor via its P55 receptor; Abu-Amer Y et al.; Chronic bone infection, as attends periodontitis, is often complicated by severe osteolysis . While LPS is believed to be central to the pathogenesis of the osteolytic lesion, the mechanisms by which this bacteria-derived molecule promotes bone resorption are unknown . We find that LPS induces bone marrow macrophages (BMMs) to express c-src, a protooncogene product that we demonstrate is a specific marker of commitment to the osteoclast phenotype . We next turned to possible soluble mediators of LPS-induced c-src . Of a number of osteoclastogenic cytokines tested, only TNF-alpha mirrors the c-src-enhancing effect of LPS . Suggesting that LPS augmentation of c-src is TNF-mediated, endotoxin sequentially induces BMM expression of TNF, followed by c-src . TNF and c-src expression, by cultured BMMs derived from LPS-injected mice, reflects duration of exposure to circulating endotoxin, intimating that endotoxin's effect in vivo is also mediated by TNF . Consistent with these findings, thalidomide (which antagonizes TNF action) attenuates c-src induction by LPS . An anti-TNF antibody blocks LPS enhancement of c-src mRNA, validating the cytokine's modulating role in vitro . Using BMMs of TNF receptor-deleted mice, we demonstrate that TNF induction of c-src is transmitted through the cytokine's p55, but not p75, receptor . Most importantly, LPS administered to wild-type mice prompts osteoclast precursor differentiation, manifest by profound osteoclastogenesis in marrow cultured ex vivo, and by a profusion of marrow-residing cells expressing the osteoclast marker tartrate resistant acid phosphatase, in vivo . In contrast, LPS does not substantially enhance osteoclast proliferation in mice lacking the p55TNF receptor, confirming that LPS-induced osteoclastogenesis is mediated by TNF in vivo via this receptor . Thus, therapy targeting TNF and/or its p55 receptor presents itself as a means of preventing the osteolysis of chronic bacterial infection.

Biochim Biophys Acta, 1997 Aug 7, 1353(2), 103 - 6
Cloning and sequencing of the hup gene encoding the histone-like protein HSl of Streptomyces lividans; Yokoyama E et al.; The hup gene encoding the histone-like HU-type protein HSl of Streptomyces lividans TK24 was cloned and sequenced . The deduced N-terminal amino acid sequence, molecular mass (9851 Da) and pI (9.95) are in good agreement with characteristics of the HSI protein . The hup transcript of about 500 nucleotides was detected . The 2.3-kb HincII fragment containing the hup gene hybridized with the AseI fragment C in the 9-10 o'clock region of the chromosome of S . lividans ZX7.

Exp Dermatol, 1997 Aug, 6(4), 161 - 6
Superantigens but not mitogens are capable of inducing upregulation of E-selectin ligands on human T lymphocytes; Zollner TM et al.; Bacterial infections can exacerbate immune mediated dermatoses, possibly via superantigens produced by these bacteria . Therefore, we asked whether superantigens induce the expression of adhesion molecules which may then facilitate invasion of highly activated T cells into different organs . The influence of exfoliative toxin (ET) and toxic shock syndrome toxin-1 (TSST-1) stimulation on the expression of a broad panel of adhesion and costimulatory molecules was investigated by flow cytometry . We found that only the E-selectin ligands cutaneous lymphocyte-associated antigen (CLA) and sialylated Lewis(x) (CD15s) are significantly upregulated by these superantigens but not by mitogen stimulation . In contrast, the mucosal lymphocyte-associated antigen (MLA) recognized by the monoclonal antibody Ber-Act8 was not differentially induced by mitogen or superantigen stimulation . Therefore, T lymphocyte stimulation by bacterial superantigens might directly influence their skin homing capacity . Furthermore, the superantigen-driven induction of CD15s-an adhesion molecule which is absent or only weakly expressed by resting or mitogen stimulated T cells-may indicate a role of this antigen for T cell skin homing . An additional adhesion pathway via E-selectin may thus be available to lymphocytes, comparable to granulocytes which constitutively express CD15s.

Br J Clin Pract, 1997 Apr-May, 51(3), 169 - 72
Reactive arthritis: is it a useful concept?
Beutler AM, Schumacher HR Jr.
Reactive arthritis (ReA) is one of the most common arthritides affecting young adults . In most cases it follows urogenital or enteric bacterial infection, but its pathogenesis is not fully understood . It is generally considered a sterile arthritis which appears to involve immune response to bacterial organisms and genetic host susceptibility associated with the presence of HLA-B27 antigen . New findings suggest that in some ReA cases, viable bacteria are present inside the joints, and these organisms may cause the disease and trigger the inflammatory response . ReA manifests clinically as a rheumatoid factor negative oligoarthritis associated with enthesopathy and certain mucosal and skin lesions . Laboratory findings in ReA are non-specific . Although concepts of its pathogenesis are still evolving, so-called ReA remains an important condition to be distinguished from rheumatoid arthritis . Prognosis is generally better . Treatments with known effects in some cases include non-steroidal anti-inflammatory drugs, intra-articular corticosteroids, oral tetracyclines and sulphasalazine . The occasional chronic and severe ReA may be very difficult to treat.

Trends Biotechnol, 1997 Sep, 15(9), 359 - 64
Cold-adapted enzymes; Marshall CJ; It is an article of faith among biochemists and molecular biologists that precious enzymes must be stored on ice . The usual reason given is that, at temperatures around freezing, enzyme activity is minimized and protein stability maximized . There is considerable evidence supporting this, but is it true for all enzymes? What about enzymes from organisms that spend part or all of their lives at temperatures around freezing? How do they manage to maintain normal enzymatic function at low temperatures? Can we learn something from cold-adapted proteins that would allow us better to understand how proteins function?

Appl Environ Microbiol, 1997 Sep, 63(9), 3676 - 83
Coffea arabica L., a new host plant for Acetobacter diazotrophicus, and isolation of other nitrogen-fixing acetobacteria; Jimenez-Salgado T et al.; Acetobacter diazotrophicus was isolated from coffee plant tissues and from rhizosphere soils . Isolation frequencies ranged from 15 to 40% and were dependent on soil pH . Attempts to isolate this bacterial species from coffee fruit, from inside vesicular-arbuscular mycorrhizal fungi spores, or from mealybugs (Planococcus citri) associated with coffee plants were not successful . Other acid-producing diazotrophic bacteria were recovered with frequencies of 20% from the coffee rhizosphere . These N2-fixing isolates had some features in common with the genus Acetobacter but should not be assigned to the species Acetobacter diazotrophicus because they differed from A . diazotrophicus in morphological and biochemical traits and were largely divergent in electrophoretic mobility patterns of metabolic enzymes at coefficients of genetic distance as high as 0.950 . In addition, these N2-fixing acetobacteria differed in the small-subunit rRNA restriction fragment length polymorphism patterns obtained with EcoRI, and they exhibited very low DNA-DNA homology levels, ranging from 11 to 15% with the A . diazotrophicus reference strain PAI 5T . Thus, some of the diazotrophic acetobacteria recovered from the rhizosphere of coffee plants may be regarded as N2-fixing species of the genus Acetobacter other than A . diazotrophicus . Endophytic diazotrophic bacteria may be more prevalent than previously thought, and perhaps there are many more potentially beneficial N2-fixing bacteria which can be isolated from other agronomically important crops.

J Formos Med Assoc, 1997 Aug, 96(8), 621 - 7
Nontuberculous mycobacteria isolates: clinical significance and disease spectrum; Shih JY et al.; The incidence of diseases caused by nontuberculous mycobacteria (NTM) is increasing worldwide . There has been no previous report regarding the clinical significance and disease spectrum of these bacteria in Taiwan . From January 1992 to June 1996, 201 isolates of NTM were recovered from clinical specimens from 143 patients at National Taiwan University Hospital . We retrospectively studied the clinical records and radiographs of these patients . A total of 86 isolates of NTM were considered clinically significant; they were cultured from 39 patients with soft-tissue infections and/or osteomyelitis (16 patients), isolated pulmonary infections (10), keratitis (6), disseminated infections (4), peritonitis, enteritis, and conjunctivitis . The most common organisms involved in these patients were Mycobacterium fortuitum complex, followed by Mycobacterium avium-intracellulare . Positive cultures of NTM were derived from respiratory sources (sputum, bronchial washing, and pleural effusion) from 111 patients; in 11 the isolates were associated with clinically significant disease, in two they were persistent colonizers, in 79 the isolates were considered to be contaminants, and for the remainder there were insufficient cultures to classify . The organisms involved in pulmonary diseases were M . avium-intracellular (4 patients), Mycobacterium chelonae (1), Mycobacterium abscessus (1), M . fortuitum (2), Mycobacterium gordonae (1), and unidentified scotochromogens (2), M . fortuitum complex (55%) was the most common pathogen of keratitis and soft-tissue infection . Three of the four cases of disseminated disease were caused by M . avium-intracellulare . The only isolate of Mycobacterium kansasii found in this study was a contaminant . The strains of clinically significant NTM isolates found in our hospital and their disease spectrum differ from those reported in other regions of the world.

Nutrition, 1997 Sep, 13(9 Suppl), 58S - 63S
The route of nutritional support in the critically ill: physiological and economical considerations; Frost P et al.; Although it generally is accepted that early enteral nutrition is of benefit to critically ill patients, there is little evidence to support this assertion . Nevertheless, enteral nutrition has many advantages over total parenteral nutrition (TPN), the latter being associated with several complications . Animal studies have shown that injury and infection can lead to gut atrophy and increased mucosal permeability . Translocation of bacteria and endotoxin in these animal models may initiate a systemic inflammatory response and cause multiple organ failure (MOF) . Again, there is little direct evidence to suggest that similar mechanisms operate in humans . As a cause of MOF, simple splanchnic ischemia and reperfusion may be sufficient with no absolute requirement for translocation . In this setting, enteral nutrition may preserve splanchnic blood flow and prevent mucosal breakdown . Unfortunately there is a widespread misconception that gastric stasis, the absence of bowel sounds, and recent abdominal surgery preclude enteral feeding . There are few absolute contraindications to early enteral feeding and with motivated staff, the use of prokinetics, and the availability of jejunal feeding tubes, the majority of intensive care patients can be fed enterally . Enteral feeding is more cost effective than TPN, but TPN remains a common therapeutic intervention in the intensive care unit and represents a significant burden on health care budgets . Nutrition support teams have led to savings, particularly by identifying patients who have been inappropriately prescribed TPN and also by preventing excessive enteral feeding.

Biochemistry, 1997 Sep 16, 36(37), 11282 - 91
The role of betaArg-10 in the B800 bacteriochlorophyll and carotenoid pigment environment within the light-harvesting LH2 complex of Rhodobacter sphaeroides; Fowler GJ et al.; Previous work has suggested that the betaArg-10 residue forms part of the binding site for the B800 bacteriochlorophyll in the LH2 complex of Rhodobactersphaeroides {Crielaard, W., Visschers, R . W., Fowler, G . J . S., van Grondelle, R., Hellingwerf, K . J., Hunter, C . N . (1994) Biochim . Biophys . Acta1183, 473-482}, and this is consistent with the X-ray crystallographic data that have been subsequently obtained for the related LH2 complex from Rhodopseudomonas acidophila {McDermott, G., Prince, S . M., Freer, A . A., Hawthornthwaite-Lawless, A . M., Papiz, M . Z., Cogdell, R . J., Isaacs, N . W . (1995) Nature 374, 517-521} . Therefore, in order obtain more information about the B800 binding site and its effect on the B800 absorption band, betaArg-10 was replaced by residues Met, His, Asn, Leu, and Lys (in addition to the Glu mutant described in our previous work); these residues were thought to represent a suitable range of amino acid shape, charge, and hydrogen-bonding ability . This new series of betaArg-10 mutants, in the form of LH2 complexes in the native membrane, has been characterized using a variety of biochemical and spectroscopic techniques in order to determine the ways in which the mutants differ from wild-type (WT) LH2 . For example, most of the mutant LH2 complexes were found to have blue-shifted B800 absorption bands ranging from 794 to 783 nm at 77 K; the exception to this trend is the betaArg-10 to Met mutant, which absorbs maximally at 798 nm . These blue shifts decrease the spectral overlap between the "B800" and B850 pigments, which allowed us to examine the nature of the B800 to B850 transfer step for the betaArg-10 mutant LH2 complexes by carrying out a series of room temperature subpicosecond energy transfer measurements . The results of these measurements demonstrated that the reduced overlap leads to a slower B800 to B850 transfer, although the alterations at betaArg-10 were found to have little effect on the efficiency of internal energy transfer within LH2 . Similarly, carotenoid to bacteriochlorophyll energy transfer was largely unaffected, although shifts in the excitation spectra in the carotenoid region were noted . These betaArg-10 mutant complexes provide an opportunity to investigate the structural requirements for the binding of monomeric bacteriochlorophyll and to examine the basis of the red shift seen for bacteriochlorophyll in photosynthetic complexes, in addition to providing new information about the environment of the carotenoid pigments in this complex.

J Cell Biochem, 1997 Sep 15, 66(4), 524 - 31
Evidence for inhibition of MyEF-2 binding to MBP promoter by MEF-1/Pur alpha; Muralidharan V et al.; Myelin basic protein (MBP) is a major component of the myelin sheath whose production is developmentally controlled during myelinogenesis . Earlier studies have indicated that programmed expression of the MBP gene is regulated at the level of transcription . Evidently, the MB1 regulatory motif located between nucleotides -14 to -50 plays an important role in transcription of the MBP promoter in both in vivo systems . The MB1 element contains binding sites for the activator protein MEF-1/Pur alpha and the repressor protein MyEF-2 . In this study we use bandshift assays with purified MEF-1/Pur alpha and MyEF-2 and demonstrate that binding of MyEF-2 to its target sequence is inhibited by MEF-1/Pur alpha . Under similar conditions, MyEF-2 enhances the association of MEF-1/Pur alpha with MB1 DNA . MEF-1/Pur alpha binds to MB1 in mono- and dimeric forms . Inclusion of MyEF-2 in the binding reaction increases the dimeric association of MEF-1/Pur alpha with the MB1 sequence . The use of MEF-1/Pur alpha variants in the bandshift assay suggests that two distinct regions of this protein may be involved in its binding to the MB1 sequences, and its ability to block MyEF-2 interaction with the MB1 sequence . Based on previous studies on the programmed expression of MEF-1/Pur alpha and MyEF-2 during myelination and the current findings on their interplay for binding to the MB1 motif, a model is proposed for their involvement in transcriptional regulation of the MBP gene during the course of brain development.

J Biol Chem, 1997 Sep 12, 272(37), 23328 - 33
Elevated homologous recombination activity in fanconi anemia fibroblasts; Thyagarajan B et al.; It is widely believed that Fanconi anemia cells possess a reduced ability to repair inter-strand DNA cross-links . While the mechanism through which inter-strand DNA cross-links are removed from mammalian chromosomes is unknown, these lesions are repaired via homologous recombination in lower eukaryotes and bacteria . Based on the hypothesis that a similar mechanism of DNA repair functions in mammalian somatic cells, we measured homologous recombination activity in diploid fibroblasts from healthy donors, and Fanconi anemia patients . Somewhat surprisingly, homologous recombination levels in nuclear protein extracts prepared from Fanconi anemia cells were nearly 100-fold higher than in extracts prepared from control cells . We observed a similar increase in the activity of a 100-kDa homologous DNA pairing protein in extracts from Fanconi anemia cells . Transfection studies confirmed that plasmid homologous recombination levels in intact Fanconi anemia cells were substantially elevated, compared with control cells . These results suggest that inappropriately elevated levels of homologous recombination activity may contribute to the genomic instability and cancer predisposition that characterize Fanconi anemia.

J Biol Chem, 1997 Sep 12, 272(37), 23151 - 6
KIS is a protein kinase with an RNA recognition motif; Maucuer A et al.; Protein phosphorylation is involved at multiple steps of RNA processing and in the regulation of protein expression . We present here the first identification of a serine/threonine kinase that possesses an RNP-type RNA recognition motif: KIS . We originally isolated KIS in a two-hybrid screen through its interaction with stathmin, a small phosphoprotein proposed to play a general role in the relay and integration of diverse intracellular signaling pathways . Determination of the primary sequence of KIS shows that it is formed by the juxtaposition of a kinase core with little homology to known kinases and a C-terminal domain that contains a characteristic RNA recognition motif with an intriguing homology to the C-terminal motif of the splicing factor U2AF . KIS produced in bacteria has an autophosphorylating activity and phosphorylates stathmin on serine residues . It also phosphorylates in vitro other classical substrates such as myelin basic protein and synapsin but not histones that inhibit its autophosphorylating activity . Immunofluorescence and biochemical analyses indicate that KIS overexpressed in HEK293 fibroblastic cells is partly targetted to the nucleus . Altogether, these results suggest the implication of KIS in the control of trafficking and/or splicing of RNAs probably through phosphorylation of associated factors.

J Bacteriol, 1997 Sep, 179(17), 5643 - 7
A flagellar sheath protein of Helicobacter pylori is identical to HpaA, a putative N-acetylneuraminyllactose-binding hemagglutinin, but is not an adhesin for AGS cells; Jones AC et al.; The gene encoding a 29-kDa flagellar sheath protein was cloned and found to be similar to hpaA, reported to encode an N-acetylneuraminyllactose-binding fibrillar hemagglutinin (D . G . Evans, T . K . Karjalainen, D . J . Evans, Jr., D . Y . Graham, and C . H . Lee, J . Bacteriol . 175:674-683, 1993) . The transcriptional start was mapped by primer extension from Helicobacter pylori mRNA, indicating an active consensus promoter at a location different from that suggested by Evans et al . Immunogold labelling of the flagellar sheath with a monoclonal antibody to HpaA was demonstrated in four strains, contrary to previous reports of a surface (D . G . Evans, T . K . Karjalainen, D . J . Evans, Jr., D . Y . Graham, and C . H . Lee, J . Bacteriol . 175:674-683, 1993) or a cytoplasmic (P . W . O'Toole, L . Janzon, P . Doig, J . Huang, M . Kostrzynska, and T . J . Trust, J . Bacteriol . 177:6049-6057, 1995) locale . Agglutination of erythrocytes and adherence to AGS cells by a delta hpaA mutant were no different from those of the parent strain, confirming a recent finding of O'Toole et al.

J Bacteriol, 1997 Sep, 179(17), 5602 - 4
Cell cycle regulation of flagellar genes; Pruss BM et al.; The expression of the flagellar master operon, flhDC, peaked in the middle of three consecutive cell cycles . The level of expression was lowest at the time of cell division . The expression of the second-level operon, flhB, peaked at cell division . The swimming speed of individual cells was also highest at the time of cell division.

J Bacteriol, 1997 Sep, 179(17), 5560 - 9
A succinate dehydrogenase with novel structure and properties from the hyperthermophilic archaeon Sulfolobus acidocaldarius: genetic and biophysical characterization; Janssen S et al.; The sdh operon of Sulfolobus acidocaldarius DSM 639 is composed of four genes coding for the 63.1-kDa flavoprotein (SdhA), the 36.5-kDa iron-sulfur protein (SdhB), and the 32.1-kDa SdhC and 14.1-kDa SdhD subunits . The four structural genes of the sdhABCD operon are transcribed into one polycistronic mRNA of 4.2 kb, and the transcription start was determined by the primer extension method to correspond with the first base of the ATG start codon of the sdhA gene . The S . acidocaldarius SdhA and SdhB subunits show characteristic sequence similarities to the succinate dehydrogenases and fumarate reductases of other organisms, while the SdhC and SdhD subunits, thought to form the membrane-anchoring domain, lack typical transmembrane alpha-helical regions present in all other succinate:quinone reductases (SQRs) and quinol:ifumarate reductases (QFRs) so far examined . Moreover, the SdhC subunit reveals remarkable 30% sequence similarity to the heterodisulfide reductase B subunit of Methanobacterium thermoautotrophicum and Methanococcus jannaschii, containing all 10 conserved cysteine residues . Electron paramagnetic resonance (EPR) spectroscopic studies of the purified enzyme as well as of membranes revealed the presence of typical S1 {2Fe2S} and S2 {4Fe4S} clusters, congruent with the deduced amino acid sequences . In contrast, EPR signals for a typical S3 {3Fe4S} cluster were not detected . However, EPR data together with sequence information implicate the existence of a second {4Fe4S} cluster in S . acidocaldarius rather than a typical {3Fe4S} cluster . These results and the fact that the S . acidocaldarius succinate dehydrogenase complex reveals only poor activity with caldariella quinone clearly suggest a unique structure for the SQR of S . acidocaldarius, possibly involving an electron transport pathway from the enzyme complex into the respiratory chain different from those for known SQRs and QFRs.

Infect Immun, 1997 Sep, 65(9), 3847 - 51
Elevated levels of macrophage inflammatory protein 2 in severe murine peritonitis increase neutrophil recruitment and mortality; Walley KR et al.; We hypothesized that chemokines may play important roles in a cecal ligation and puncture (CLP) model of septic peritonitis in CD-1 mice . Concentrations of C-X-C (macrophage inflammatory protein 2 {MIP-2} and ENA-78) and C-C (MIP-1alpha and JE) chemokines were measured (by enzyme-linked immunosorbent assay) in serum, peritoneal lavage fluid, lung, and liver at 4, 8, 24, 48, and 96 h after CLP . Significant elevations in all measured chemokines occurred in peritoneal fluid after CLP (P < 0.05) . MIP-2, in particular, increased dramatically (>400-fold, P < 0.001) in peritoneal fluid, serum, and to a lesser extent lung and liver (P < 0.05) . Increased MIP-2 was correlated with severity of sepsis (P < 0.001) . To determine the significance of this finding, mice were passively immunized prior to CLP with polyclonal antibody to MIP-2, which decreased mortality from 85 to 38% at 96 h (P < 0.01) . To further understand the mechanism of the effect of MIP-2, additional measurements demonstrated that anti-MIP-2 prior to CLP decreased the percent neutrophils in peritoneal fluid (55% +/- 12%, compared with 82% +/- 10% in controls), but no significant changes in tumor necrosis factor alpha, interleukin-6, or interleukin-10 occurred . MIP-2 contributes to the inflammatory response and overall mortality in this model of severe septic peritonitis, possibly by increasing recruitment of neutrophils, which clear bacteria but may also injure the host.

Infect Immun, 1997 Sep, 65(9), 3759 - 67
Interaction of Mycobacterium avium with environmental amoebae enhances virulence; Cirillo JD et al.; Environmental mycobacteria are a common cause of human infections . Recently, contaminated domestic water supplies have been suggested as a potential environmental source of several mycobacterial diseases . Since many of these mycobacterial species replicate best intracellularly, environmental hosts have been sought . In the present study, we examined the interaction of Mycobacterium avium with a potential protozoan host, the water-borne amoeba Acanthamoeba castellanii . We found that M . avium enters and replicates in A . castellanii . In addition, similar to that shown for mycobacteria within macrophages, M . avium inhibits lysosomal fusion and replicates in vacuoles that are tightly juxtaposed to the bacterial surfaces within amoebae . In order to determine whether growth of M . avium in amoebae plays a role in human infections, we tested the effects of this growth condition on virulence . We found that growth of M . avium in amoebae enhances both entry and intracellular replication compared to growth of bacteria in broth . Furthermore, amoeba-grown M . avium was also more virulent in the beige mouse model of infection . These data suggest a role for protozoa present in water environments as hosts for pathogenic mycobacteria, particularly M . avium.

Physiol Zool, 1997 Sep-Oct, 70(5), 578 - 88
Sulfide binding in the body fluids of hydrothermal vent alvinellid polychaetes; Martineu P et al.; Dissolved H2S is a major environmental factor in hydrothermal vent ecosystems . In a study of adaptations to sulfide by alvinellid polychaetes, the sulfide-binding capacity of body fluids was examined in Paralvinella palmiformis from northeast Pacific ridges and Alvinella species from the East Pacific Rise . Sulfide concentrations in vascular blood and coelomic fluid of freshly collected animals were notably variable . Separation of P . palmiformis body-fluid components revealed that most sulfide (ca . 77%) was accumulated in the dissolved fraction . In P . palmiformis, both vascular blood and coelomic fluid could reversibly bind sulfide in vitro with a low affinity, saturating only at high dialysate concentrations (ca . 2 mmol L-1) . No sulfide-binding activity was observed in the vascular blood from Alvinella species . A dissolved protein component of greater than 90 kDa appears to be involved in sulfide binding in Paralvinella, probably a vascular extracellular high-molecular-weight hemoglobin . Some sulfide may also adsorb onto a 15.38-kDa intracellular hemoglobin present in the coelomic erythrocyte fraction . In the absence of epibiotic bacteria, Paralvinella body fluids may function as a sulfide buffer to protect tissues from deleterious effects of sulfide exposure.

J Clin Microbiol, 1997 Sep, 35(9), 2393 - 7
DNA fingerprinting of Mycobacterium tuberculosis strains from patients with pulmonary tuberculosis in Honduras; Pineda-Garcia L et al.; Mycobacterium tuberculosis isolates from 84 patients with pulmonary tuberculosis in Honduras were characterized by restriction fragment length polymorphism analysis . Seventy-three different IS6110 patterns were found; 63 of these were unique and 10 were shared by two to three strains each . Thus, no ongoing spread of any specific clone of bacteria could be demonstratedPIP: The relationship among clinical isolates of Mycobacterium tuberculosis from patients with pulmonary tuberculosis in Honduras was investigated through use of DNA fingerprinting . Restriction fragment length polymorphism analysis of isolates from 84 patients revealed 73 different IS6110 patterns . 63 of these patterns were unique and 10 were shared by 2-3 strains each . Smear-positive samples were found for 64 (76%) of all patients, and an isolate derived from at least 1 smear-positive patient was represented in each cluster of strains with identical patterns . Identical or highly related strains--suggestive of ongoing transmission--were more common in men than women, in patients 15-42 years of age, and in those from the Tegucigalpa area . 11 of 76 tested patients were HIV-infected, and only 5 of 11 strains isolated from tuberculosis patients with AIDS had unique IS6110 banding patterns . The fact that no clonal spread of tuberculosis among AIDS patients or those with drug-resistant tuberculosis could be demonstrated indicates that drug-resistant tuberculosis in Honduras emerged in unrelated patients rather than from an ongoing spread of initially resistant M . tuberculosis strains .

J Clin Microbiol, 1997 Sep, 35(9), 2275 - 8
Rapid detection and typing of herpes simplex virus DNA in clinical specimens by the hybrid capture II signal amplification probe test; Cullen AP et al.; A second-generation signal amplification, nucleic acid-based test for the rapid detection and typing of herpes simplex virus (HSV) DNA was developed and evaluated with artificial and clinical specimens . The analytical sensitivity of the Hybrid Capture II (HC II) HSV DNA assay was determined by testing either cloned HSV DNA or total genomic HSV DNA titrations and resulted in detection thresholds of between 5 x 10(3) and 1 x 10(4) copies per assay . Specificity was assessed by testing a panel of bacteria and viruses commonly found in the female genital tract . Sensitivity was assessed by testing 112 ulcerative genital lesions by the HC II assay and comparing the results to those obtained by routine cell culture . Discrepant results were resolved by PCR testing . After resolution of the discrepant results, the sensitivity of the HC II assay compared to the consensus result (the results of two of three tests, the HC II assay, culture, and PCR, were in agreement) was 93.2% (41 of 44 specimens), and the specificity was 100% (60 of 60 specimens) . Culture gave a sensitivity of 84.1% (37 of 44 specimens) and a specificity of 100% (60 of 60 specimens) compared to the consensus result . The results of HSV typing by the HC II assay and culture agreed in all cases . The HC II assay is a rapid and accurate assay for detecting and typing HSV types 1 and 2, with a sensitivity comparable to that of culture and greater ease of use than culture.

Mol Biochem Parasitol, 1997 Sep, 88(1-2), 105 - 14
A cytidine deaminase expressed in the post-infective L3 stage of the filarial nematode, Brugia pahangi, has a novel RNA-binding activity; Anant S et al.; A number of genes have been identified that are highly expressed in the post-infective L3 stage of the filarial parasite, Brugia pahangi . Amongst these was a cDNA with homology to the cytidine deaminase (CDD) gene family . Phylogenetic analysis of the various cytosine nucleoside deaminases suggest that Brugia pahangi CDD evolved with significant divergence from the RNA editing family . In order to characterize its function, we have expressed Brugia pahangi CDD in bacteria as a chimera with maltose-binding protein (MBP) . Biochemical analysis demonstrates the MBP-CDD fusion protein functions as an authentic cytidine deaminase with an obligate requirement for zinc . In addition to cytidine deaminase activity, however, the fusion protein demonstrates RNA binding activity with specificity for AU-rich sequences and was found to bind an RNA template spanning the edited site of mammalian apolipoprotein B (apoB) mRNA . This RNA binding activity was not found in two different recombinant bacterial CDD proteins . In vitro RNA editing assays revealed that MBP-CDD failed to mediate cytidine deamination of a mammalian apoB RNA template . Furthermore, binding of MBP-CDD to the apoB RNA did not inhibit in vitro editing of this template by apobec-1 . The data suggest that the cytosine nucleoside deaminases and RNA editing deaminases have acquired different mechanisms of binding to an AU-rich RNA template, presumably with different functional implications.

Mol Biochem Parasitol, 1997 Sep, 88(1-2), 35 - 42
Polyamine biosynthesis in Cryptosporidium parvum and its implications for chemotherapy; Keithly JS et al.; This study demonstrates that polyamine biosynthesis in Cryptosporidium parvum occurs via a pathway chiefly found in plants and some bacteria . The lead enzyme of this pathway, arginine decarboxylase (ADC) was sensitive to the specific, irreversible inhibitor DL-alpha-difluoromethyl-arginine (IC50 30 microM), and intracellular growth of C . parvum was significantly reduced by inhibitors of ADC . No activity was detected using ornithine as substrate, and the irreversible inhibitor of ornithine decarboxylase, DL-alpha-difluoromethyl-ornithine, had no effect upon ADC activity or upon growth of the parasite . Back-conversion of spermine to spermidine and putrescine via spermidine:spermine-N1-acetyltransferase (SSAT) was also detected . Compounds such as his(ethyl)norspermine, which have been demonstrated to down-regulate SSAT activity in tumor cells, were synergistic in the inhibition of growth when used in combination with inhibitors of the forward pathway . Thus, C . parvum differs fundamentally in its polyamine metabolism from the majority of eukaryotes, including humans . Such differences indicate that polyamine metabolism may serve as a chemotherapeutic target in this organism.

J Virol, 1997 Sep, 71(9), 6749 - 56
Functional and structural analysis of the sialic acid-binding domain of rotaviruses; Isa P et al.; The infectivity of most animal rotaviruses is dependent on the interaction of the virus spike protein VP4 with a sialic acid (SA)-containing cell receptor, and the SA-binding domain of this protein has been mapped between amino acids 93 and 208 of its trypsin cleavage fragment VP8 . To identify which residues in this region are essential for the SA-binding activity, we performed alanine mutagenesis of the rotavirus RRV VP8 expressed in bacteria as a fusion polypeptide with glutathione S-transferase . Tyrosines were primarily targeted since tyrosine has been involved in the interaction of other viral hemagglutinins with SA . Of the 15 substitutions carried out, 10 abolished the SA-dependent hemagglutination activity of the protein, as well as its ability to bind to glycophorin A in a solid-phase assay . However, only alanine substitutions for tyrosines 155 and 188 and for serine 190 did not affect the overall conformation of the protein, as judged by their interaction with a panel of conformationally sensitive neutralizing VP8 monoclonal antibodies (MAbs) . These findings suggest that these three amino acids play an essential role in the SA-binding activity of the protein, presumably by interacting directly with the SA molecule . The predicted secondary structure of VP8 suggests that it is organized as 11 beta-strands separated by loops; in this model, Tyr-155 maps to loop 7 while Tyr-188 and Ser-190 map to loop 9 . The close proximity of these two loops is also supported by previous results from competition experiments with neutralizing MAbs directed at RRV VP8.

Nucleic Acids Res, 1997 Sep 1, 25(17), 3471 - 7
The RNase P RNA from cyanobacteria: short tandemly repeated repetitive (STRR) sequences are present within the RNase P RNA gene in heterocyst-forming cyanobacteria; Vioque A; The RNase P RNA gene (rnpB) from 10 cyanobacteria has been characterized . These new RNAs, together with the previously available ones, provide a comprehensive data set of RNase P RNA from diverse cyanobacterial lineages . All heterocystous cyanobacteria, but none of the non-heterocystous strains analyzed, contain short tandemly repeated repetitive (STRR) sequences that increase the length of helix P12 . Site-directed mutagenesis experiments indicate that the STRR sequences are not required for catalytic activity in vitro . STRR sequences seem to have recently and independently invaded the RNase P RNA genes in heterocyst-forming cyanobacteria because closely related strains contain unrelated STRR sequences . Most cyanobacteria RNase P RNAs lack the sequence GGU in the loop connecting helices P15 and P16 that has been established to interact with the 3'-end CCA in precursor tRNA substrates in other bacteria . This character is shared with plastid RNase P RNA . Helix P6 is longer than usual in most cyanobacteria as well as in plastid RNase P RNA.

J Neurosci, 1997 Sep 1, 17(17), 6657 - 68
Evidence that the homeodomain protein Gtx is involved in the regulation of oligodendrocyte myelination; Awatramani R et al.; We have investigated the patterns of postnatal brain expression and DNA binding of Gtx, a homeodomain transcription factor . Gtx mRNA accumulates in parallel with the RNAs encoding the major structural proteins of myelin, myelin basic protein (MBP), and proteolipid protein (PLP) during postnatal brain development; Gtx mRNA decreases in parallel with MBP and PLP mRNAs in the brains of myelin-deficient rats, which have a point mutation in the PLP gene . Gtx mRNA is expressed in differentiated, postmitotic oligodendrocytes but is not found in oligodendrocyte precursors or astrocytes . These data thus demonstrate that Gtx is expressed uniquely in differentiated oligodendrocytes in postnatal rodent brain and that its expression is regulated in parallel with the major myelin protein mRNAs, encoding MBP and PLP, under a variety of physiologically relevant circumstances . Using a Gtx fusion protein produced in bacteria, we have confirmed that Gtx is a sequence-specific DNA-binding protein, which binds DNA sequences containing a core AT-rich homeodomain binding site . Immunoprecipitation of labeled DNA fragments encoding either the MBP or PLP promoter regions with this fusion protein has identified several Gtx-binding fragments, and we have confirmed these data using an electrophoretic mobility shift assay . In this way we have identified four Gtx binding sites within the first 750 bp of the MBP promoter and four Gtx binding sites within the first 1 . 3 kb of the PLP promoter . In addition, inspection of the PLP promoter sequence demonstrates the presence of six additional Gtx binding sites . These data, taken together, strongly suggest that Gtx is important for the function of differentiated oligodendrocytes and may be involved in the regulation of myelin-specific gene expression.

J Biol Chem, 1997 Aug 29, 272(35), 21793 - 802
Topography of the photosystem I core proteins of the cyanobacterium Synechocystis sp . PCC 6803; Sun J et al.; PsaA and PsaB are homologous integral membrane proteins that form the heterodimeric core of photosystem I . Domain-specific antibodies were generated to examine the topography of PsaA and PsaB . The purified photosystem I complexes from the wild type strain of Synechocystis sp . PCC 6803 were treated with eight proteases to study the accessibility of cleavage sites in PsaA and PsaB . Proteolytic fragments were identified using the information from N-terminal amino acid sequencing, reactivity to antibodies, apparent mass, and specificity of proteases . The extramembrane loops of PsaA and PsaB differed in their accessibility to proteases, which indicated the folded structure of the loops or their shielding by the small subunits of photosystem I . NaI-treated and mutant photosystem I complexes were used to identify the extramembrane loops that were exposed in the absence of specific small subunits . The absence of PsaD exposed additional proteolytic sites in PsaB, whereas the absence of PsaE exposed sites in PsaA . These studies distinguish PsaA and PsaB in the structural model for photosystem I that has been proposed on the basis of x-ray diffraction studies (Krauss, N., Schubert, W.-D., Klukas, O., Fromme, P., Witt, H . T., and Saenger, W . (1996) Nat . Struct . Biol . 3, 965-973) . Using osmotically shocked cells for protease treatments, the N terminus of PsaA was determined to be on the n side of the photosynthetic membranes . Based on these data and available published information, we propose a topological model for PsaA and PsaB.

Biochemistry, 1997 Aug 26, 36(34), 10492 - 7
Three tRNA binding sites in rabbit liver ribosomes and role of the intrinsic ATPase in 80S ribosomes from higher eukaryotes; El'skaya AV et al.; Three tRNA binding sites have been found in organisms of all domains (former kingdoms) with only one exception: Four binding sites have been reported for cytoplasmic 80S ribosomes from rabbit liver . Therefore, the issue was reconsidered, and the data revealed that rabbit liver ribosomes contain three tRNA binding sites, underlining the universal character of this ribosomal feature . Furthermore, a first analysis of the role of the ribosome intrinsic ATPase was performed . This ATPase is found in ribosomes of higher eukarya but not in lower eukarya such as yeast or ribosomes of the domains archea and bacteria . The results suggest that the intrinsic ATPase fulfills the same function as the essential third elongation factor EF-3, an ATPase in higher fungi (yeast etc.), that facilitates the release of the deacylated tRNA from the E site.

Biochemistry, 1997 Aug 26, 36(34), 10482 - 91
Zinc binding properties of the DNA binding domain of the 1,25-dihydroxyvitamin D3 receptor; Craig TA et al.; To assess the zinc binding stoichiometry and the structural changes induced upon the binding of zinc to the human vitamin D receptor (VDR), we expressed the DNA binding domain (DBD) of the human VDR in bacteria as a soluble glutathione-S-transferase fusion protein at 20 degrees C, and examined the apo-protein and metal-liganded protein by mass spectrometry, and circular dichroism and nuclear magnetic resonance spectroscopy . Following final preparation with a zinc-free buffer, the VDR DBD bound 2 mol of zinc/mol of protein as measured by inductively coupled plasma-mass spectrometry and electrospray ionization-mass spectrometry . When protein preparation was carried out in a zinc containing buffer and zinc content of the protein was assesed by the same methods, VDR DBD bound 4 mol of zinc/mol of protein . Analysis of the protein using circular dichroism spectroscopy demonstrated that the EDTA-treated protein increased in alpha-helical content from 16 to 27% on the addition of zinc . Equilibrium ultracentrifugal analyses of the VDR DBD indicated that the protein was present in solution as a monomer . Gel mobility shift analyses of the VDR DBD with several vitamin D response elements (VDREs) in the absence of accessory proteins such as retinoic acid receptor, showed that VDR DBD was able to form a protein/VDRE DNA structural complex . In the presence of zinc, proton NMR NOESY spectra showed that the protein possessed elements of secondary structure . The addition of VDRE DNA, but not random DNA, caused changes in the proton NMR spectra of VDRE DNA indicating specific interaction between protein and DNA groups . We conclude that the DBD of the VDR binds zinc and DNA and undergoes conformational changes on binding to the metal and DNA.

J Mol Biol, 1997 Aug 22, 271(3), 456 - 71
Crystal structure of the bacteriochlorophyll a protein from Chlorobium tepidum; Li YF et al.; The bacteriochlorophyll (BChl) a protein from Chlorobium tepidum, which participates in energy transfer in green photosynthetic bacteria, has been crystallized using the sitting drop method of vapor diffusion . X-ray diffraction data collected from these crystals indicate that the crystals belong to the cubic space group P4132 with cell dimensions of a=b=c=169.5 A . A native X-ray diffraction data set has been collected to a resolution of 2.2 A . The initial solution was determined by using the molecular replacement method using the structure of the previously solved BChl a protein from Prosthecochloris aestuarii . A unique rotation and translation solution was obtained for two monomers in the asymmetric unit giving a pseudo-body centered packing . After rebuilding and refinement the model yields an R factor of 19.0%, a free R-factor of 28.3%, and good geometry with root-mean-square deviations of 0.013 A and 2.1 degrees for the bond lengths and angles, respectively . The structure of the BChl a protein from C . tepidum consists of three identical subunits related by a 3-fold axis of crystallographic symmetry . In each subunit the polypeptide backbone forms large beta-sheets and encloses a central core of seven BChl a molecules . The distances between neighboring bacteriochlorin systems within a subunit range between 4 A to 11 A and that between two bacteriochlorins from different subunits is more than 20 A . The overall structure is comparable with that of P . aestuarii but significant differences are observed for the individual bacteriochlorophyll structures . The surface of the trimer has a hydrophobic region that is modeled as the complex being a peripheral membrane protein partially embedded in the membrane . A general model is presented for the membrane organization with two of the bacteriochlorophyll structures in the membrane and transferring energy to the reaction center complex . In this model these two bacteriochlorophyll structures serve a similar role to the cofactors of integral membrane light-harvesting complexes although the protein structure surrounding the cofactors is significantly different for the BChl a protein compared with the integral membrane complexes.

Proc Natl Acad Sci U S A, 1997 Aug 19, 94(17), 9285 - 90
Recombinant human granzyme A binds to two putative HLA-associated proteins and cleaves one of them; Beresford PJ et al.; The release of cytotoxic granule contents by cytotoxic T lymphocytes triggers apoptotic target cell death . Cytotoxic granules contain a pore-forming protein, perforin, and a group of serine proteases called granzymes . We expressed human granzyme A in bacteria as a proenzyme capable of in vitro activation by enterokinase . The recombinant activated enzyme has catalytic activity against substrates with Arg, preferably, or Lys at the P1 position, comparable to trypsin . An enzymatically inactive recombinant granzyme A, with the active site Ser mutated to Ala, was produced and used with affinity chromatography to identify potential substrates . Two granzyme A-binding cytoplasmic proteins of molecular mass 33 and 44 kDa were isolated and identified by tryptic fragment sequencing as PHAP I and II, ubiquitous putative HLA-associated proteins, previously coisolated by binding to an HLA class II peptide . PHAP II forms an SDS-stable complex with recombinant mutant granzyme A and coprecipitates with it from cytoplasmic extracts . PHAP II, either purified or in cell lysates, is cleaved by the recombinant enzyme at nanomolar concentrations to a 25-kDa fragment . PHAP II begins to be degraded within minutes of initiation of cytotoxic T lymphocyte attack . PHAP I and II are candidate participants in the granzyme A pathway of cell-mediated cytotoxicity.

FEMS Microbiol Lett, 1997 Aug 15, 153(2), 371 - 7
Disruption of a sigma factor gene, sigF, affects an intermediate stage of spore pigment production in Streptomyces aureofaciens; Rezuchova B et al.; The Streptomyces aureofaciens sigF gene encodes a sigma factor . By integrative transformation, via double cross-over, a stable null mutant of sigF gene was obtained . This mutation appeared to have no obvious effect on vegetative growth, but affected the late stage of spore maturation . Microscopic examination showed that spores were deformed, and spore wall was thinner, compared with the wild-type spores . The spore pigment of sigF mutant was green, compared to wild-type grey-pink spore pigmentation . The plasmid-born wild-type sigF gene complemented the mutation after transformation of the mutant strain.

J Biol Chem, 1997 Aug 15, 272(33), 20901 - 6
The mitochondrial hsp70 chaperone system . Effect of adenine nucleotides, peptide substrate, and mGrpE on the oligomeric state of mhsp70; Azem A et al.; Mitochondrial hsp70 (mhsp70) is a key component in the import and folding of mitochondrial proteins . In both processes, mhsp70 cooperates with the mitochondrial nucleotide exchange factor mGrpE (also termed Mge1p) . In this work we have characterized the self-association of purified mhsp70, the interaction of mhsp70 with isolated mGrpE and protein substrate, and the effect of nucleotides on these interactions . mhsp70 can form oligomers that are dissociated by ATP or by a nonhydrolyzable ATP analog . A substrate peptide binds to mhsp70 in the absence of added nucleotides and is released by ATP but not by ADP . Binding of the peptide causes nucleotide-independent dissociation of the mhsp70 oligomers and enhances the mhsp70 ATPase . Purified mGrpE forms a homodimer . In the absence of added nucleotides, one mGrpE dimer binds to one molecule of mhsp70, forming a stable 122 kDa hetero-oligomer . This complex is weakened by ADP and completely dissociated by ATP.

JAMA, 1997 Aug 6, 278(5), 399 - 411
Clinical recognition and management of patients exposed to biological warfare agents; Franz DR et al.; Concern regarding the use of biological agents--bacteria, viruses, or toxins--as tools of warfare or terrorism has led to measures to deter their use or, failing that, to deal with the consequences . Unlike chemical agents, which typically lead to violent disease syndromes within minutes at the site of exposure, diseases resulting from biological agents have incubation periods of days . Therefore, rather than a paramedic, it will likely be a physician who is first faced with evidence of the results of a biological attack . We provide here a primer on 10 classic biological warfare agents to increase the likelihood of their being considered in a differential diagnosis . Although the resultant diseases are rarely seen in many countries today, accepted diagnostic and epidemiologic principles apply; if the cause is identified quickly, appropriate therapy can be initiated and the impact of a terrorist attack greatly reduced.

Transfus Sci, 1997 Sep, 18(3), 345 - 50
Platelet therapy: current opinions on laboratory and clinical aspects; Seghatchian J; The rapid development in blood component technologies has lead to production of various types of platelet concentrates which are highly heterogenous in terms of cellular content, subpopulation of platelet and leucocytes and in vitro characteristics and storage stability . This emphasizes the need for a stringent in-process validation and statistical process control to ensure that optimal process efficiency and platelet functional integrity are continuously attained and maintained throughout the 5 days shelf life . Several unresolved issues such as development of cytokines and microparticles, some with immunomodulatory effects; fears of bacterial contamination of leucodepleted platelets, in particular in storage media; lack of progress in reduction of some unquantified risk of all types of transfusion transmitted infection (TTI), still remain the focus of current interest . While some processing results recently succeeded in killing both viruses and bacteria through a recently developed viral inactivation and filtration process, nevertheless much still remains to be elucidated in respect to long term effects of such treatments . Apart from development of purer products to reduce transfusion reactions, some complimentory alternatives to platelet transfusion are becoming available, which in the near future, may influence the demand for platelet support.

Med Device Technol, 1997 Sep, 8(7), 20 - 1, 24-7
Anti-adhesive surfaces through hyaluronan coatings; Pavesio A et al.; The ability to resist adhesion of proteins, bacteria, cells and tissue is an important requirement for the surfaces of medical devices . This article describes a surface-modification process that imparts anti-adhesive properties to the surfaces of biomedical materials through the covalent binding of hyaluronan . It can be applied to a wide range of materials to yield anti-adhesive surfaces on plastics, metals and ceramics.

Health Care Cost Reengineering Rep, 1997 Sep, 2(9), 135 - 40
Hospital's aggressive screening efforts save money in long run; Non-enzymatic and enzymatic hydrolysis of alkyl halides: a haloalkane dehalogenation enzyme evolved to stabilize the gas-phase transition state of an SN2 displacement reaction; Department of Chemistry, University of California, Santa Barbara, CA 93106, USAThe semiempirical PM3 method, calibrated against ab initio HF/6-31+G(d) theory, has been used to elucidate the reaction of 1, 2-dichloroethane (DCE) with the carboxylate of Asp-124 at the active site of haloalkane dehalogenase of Xanthobacter autothropicus . Asp-124 and 13 other amino acid side chains that make up the active site cavity (Glu-56, Trp-125, Phe-128, Phe-172, Trp-175, Leu-179, Val-219, Phe-222, Pro-223, Val-226, Leu-262, Leu-263, and His-289) were included in the calculations . The three most significant observations of the present study are that: (i) the DCE substrate and Asp-124 carboxylate, in the reactive ES complex, are present as an ion-molecule complex with a structure similar to that seen in the gas-phase reaction of AcO- with DCE; (ii) the structures of the transition states in the gas-phase and enzymatic reaction are much the same where the structure formed at the active site is somewhat exploded; and (iii) the enthalpies in going from ground states to transition states in the enzymatic and gas-phase reactions differ by only a couple kcal/mol . The dehalogenase derives its catalytic power from: (i) bringing the electrophile and nucleophile together in a low-dielectric environment in an orientation that allows the reaction to occur without much structural reorganization; (ii) desolvation; and (iii) stabilizing the leaving chloride anion by Trp-125 and Trp-175 through hydrogen bonding.

Virology, 1997 Aug 4, 234(2), 203 - 14
Mutational analyses support a model for the HRV2 2A proteinase; Sommergruber W et al.; The proteinase 2A of human rhinovirus 2 is a cysteine proteinase which contains a tightly bound Zn ion thought to be required for structural integrity . A three-dimensional model for human rhinovirus type 2 proteinase 2A (HRV2 2A) was established using sequence alignments with small trypsin-like Ser-proteinases and, for certain regions, elastase . The model was tested by expressing selected proteinase 2A mutants in bacteria and examining the effect on both intramolecular ("cis") and intermolecular ("trans") activities . The HRV2 proteinase 2A is proposed to have a two domain structure, with the catalytic site and substrate binding region on one face of the molecule and a Zn-binding motif on the opposite face . Residues Gly 123, Gly 124, Thr 121, and Cys 101 are proposed to be involved in the architecture of the substrate binding pocket and to provide the correct environment for the catalytic triad of His 18, Asp 35, and Cys 106 . Residues Tyr 85 and Tyr 86 are thought to participate in substrate recognition . The presence of an extensive C-terminal helix, in which Asp 132, Arg 134, Phe 130, and Phe 136 play important roles, explains why mutations in this region are generally detrimental to proteinase activity . The proposed Zn-binding motif comprises Cys 52, Cys 54, Cys 112, and His 114 . Exchange of these residues inactivates the enzyme . Furthermore, as measured by atom emission spectroscopy, Zn was absent from purified preparations of proteinase 2A in which His 114 had been replaced by Asn . The absence of disulphide bridges was confirmed by subjecting highly purified HRV2 proteinase 2A to one- and two-step alkylation procedures.

Surgery, 1997 Aug, 122(2), 295 - 301; discussion 301-2
Evidence for an unknown component of pancreatic ascites that induces adult respiratory distress syndrome through an interleukin-1 and tumor necrosis factor-dependent mechanism; Denham W et al.; BACKGROUND: The development of acute respiratory distress syndrome (ARDS) during acute pancreatitis is associated with interleukin (IL)-1 and tumor necrosis factor (TNF) gene expression within the pulmonary parenchyma . Although activated pancreatic enzymes have been thought to mediate pancreatitis-induced ARDS, they are not capable of inducing cytokine production in vitro . We hypothesized that IL-1 and TNF production in the lungs is essential to the development of ARDS and is induced by a mediator released from the inflamed pancreas . METHODS: Pancreatic ascites was obtained from rats after induction of bile-salt pancreatitis, cultured, and assayed for IL-1, TNF, IL-6, IL-8, IL-10, interferon-gamma, and endotoxin . Sterile, cytokine-free ascites or saline (control) was subsequently administered intravenously (20 ml/kg) to healthy rats and to IL-1 R1 or TNF R1 knockout mice . RESULTS: Animals administered intravenous ascites had a 30-fold rise in pulmonary IL-1 and TNF mRNA, as well as increased alveolar leukocytes and protein . Knockout animals devoid of active IL-1 or TNF receptors failed to develop increased alveolar protein or leukocytes . CONCLUSIONS: A component of pancreatic ascites other than activated enzymes, bacteria, or inflammatory cytokines is capable of inducing ARDS in healthy animals . The mechanism appears to be directly attributable to the activity of pulmonary IL-1 and TNF.

Invest Ophthalmol Vis Sci, 1997 Aug, 38(9), 1719 - 25
Different responsiveness to nitric oxide-cyclic guanosine monophosphate pathway in cholinergic and tachykinergic contractions of the rabbit iris sphincter muscle; Chuman H et al.; PURPOSE: In the rabbit iris sphincter muscle, sodium nitroprusside (SNP), a nitric oxide (NO) donor, inhibits cholinergic contraction but does not affect tachykinergic contraction in vitro . The objectives of the current study were to clarify the mechanism for the different responsiveness to NO in cholinergic and tachykinergic muscular contractions, and to examine whether the mechanism for NO-induced inhibition of cholinergic muscular contraction is operative in vivo . METHODS: Iris sphincter muscle was dissected from the rabbit eye pretreated with or without endotoxin (lipopolysaccharide, LPS) in vivo . Cyclic guanosine monophosphate (cGMP) content in the iris sphincter muscle was determined by radioimmunoassay . The motor activity of the ring-shaped iris sphincter muscle was measured isometrically . Sodium nitroprusside, carboxy-2-phenyl-4,4,5,5,-tetramethyl-imidazoline-1-oxyl-3-oxide (C-PTIO, a scavenger of NO radicals), and 8-bromo cGMP (a permeable cGMP analogue) were administered between the first and second administrations of carbachol and neurokinin A, both of which had caused sustained contraction in the iris sphincter muscle . RESULTS: Sodium nitroprusside inhibited the contraction of the iris sphincter muscle caused by carbachol but had no effect on the contraction caused by neurokinin A . Application of C-PTIO significantly reduced SNP-induced cGMP accumulation in the muscle, as well as the SNP-induced inhibition of muscular contraction caused by carbachol . Neither carbachol nor neurokinin A influenced SNP-induced cGMP accumulation in the muscle . Induction of 8-bromo-cGMP significantly diminished the muscular contraction caused by carbachol but not that caused by neurokinin A . In vivo pretreatment of the eye with LPS increased, in a time-dependent manner, the cGMP accumulation in the iris sphincter muscle, which was significantly inhibited by pretreatment of NG-nitro-L-arginine methyl ester (an inhibitor of NO synthesis) in vivo . CONCLUSIONS: These results demonstrate that in rabbits the increase in cGMP accumulation induced by NO in the iris sphincter muscle is involved in the cholinergic contraction but not in the tachykinergic contraction, suggesting that different sensitivities to cGMP are essential for the different responsiveness to NO . Furthermore, the results of this study showed that the NO-cGMP pathway is operative in vivo and regulates iris sphincter muscle tone, at least when the eyes are infected with bacteria.

Nippon Rinsho, 1997 Aug, 55(8), 2131 - 9
{Structure and function of symporter and antiporter}; Tsuchiya T et al.; Structure and function of symporters and antiporters which utilize Na+ as the coupling cation are briefly reviewed . The SGLT is an Na+/glucose symporter present in animal cell membranes . So far, five isoforms of the SGLT (1 to 5) have been found in various tissues . The Me1B is an Na+/galactoside symporter present in membranes of enteric bacteria . Regions or amino acid residues of these symporters which are important for substrate recognition or cation recognition have been identified . Furthermore amino acid substitutions in the SGLT1 of glucose-galactose malabsortion families have been identified . Regions and amino acid residues which are important for the function of the Na+/H+ antiporters (or exchangers) which extrude H+ (in animal cells) or Na+ (in bacterial cells) have been also identified.

Scand J Gastroenterol, 1997 Aug, 32(8), 835 - 40
Inhibition of nitric oxide modulates the effect of oral arginine supplementation in acute liver injury; Adawi D et al.; BACKGROUND: Arginine possesses numerous unique and advantageous biochemical and pharmacologic properties . We have previously shown that arginine supplementation in an acute liver injury model reduces both the extent of the liver injury and bacterial translocation . We therefore studied the role of nitric oxide on the effects of oral arginine supplementation in acute liver injury, bacterial translocation, ileal and cecal mucosal nucleotides, and RNA and DNA, to investigate pathogenetic mechanisms . METHODS: Sprague-Dawley rats were divided into normal, liver injury control, N-nitro-L-arginine methyl ester (L-NAME), arginine, and L-NAME + arginine supplementation groups . Oral supplementation was performed daily through a nasogastric tube for 8 days . Acute liver injury was induced on the 8th day by intraperitoneal injection of D-galactosamine (1.1 g/kg body weight) . Twenty-four hours after the liver injury, liver function tests, bacterial translocation, and ileal and cecal mucosal nucleotides, RNA, and DNA were evaluated . RESULTS: Bilirubin and liver enzymes increased significantly in the L-NAME group compared with the arginine group, whereas the liver enzymes increased significantly compared with the liver injury control group . In the L-NAME group the number of bacteria translocated to the portal and arterial blood increased significantly compared with all groups . In the arginine group the bacteria translocated to the liver and mesenteric lymph nodes decreased significantly compared with the liver injury control and L-NAME groups . The ileal and cecal mucosal nucleotides, RNA, and DNA in the arginine group increased significantly compared with the normal, liver injury, and L-NAME groups . CONCLUSIONS: Nitric oxide plays a role in the beneficial effect of the arginine supplementation in acute liver injury . It significantly improves the liver damage indicated by the increase of liver enzymes when its production was inhibited by L-NAME . Nitric oxide has a role in bacterial translocation since the number of bacteria significantly increased in arterial and portal blood when L-NAME was used to inhibit its production . Furthermore, arginine supplementation improved mucosal nucleotides, RNA, and DNA in ileum and colon.

Lett Appl Microbiol, 1997 Aug, 25(2), 123 - 6
Detection of Xylella fastidiosa in potential insect vectors by immunomagnetic separation and nested polymerase chain reaction; Pooler MR et al.; A sensitive and specific assay for detecting Xylella fastidiosa in potential insect vectors was developed . This assay involves immunomagnetic separation of the bacteria from the insect, followed by a two-step, nested polymerase chain reaction (PCR) amplification using previously developed oligonucleotide primers specific to X . fastidiosa . A total of 347 leafhoppers representing 16 species were captured and sampled from American elm (Ulmus americana L.) trees growing in a nursery where bacterial leaf scorch caused by X . fastidiosa occurs . Two of these leafhopper species, Graphocephala coccinea and G . versuta, regularly tested positive for X . fastidiosa using this technique . These insects are therefore potential vectors of X . fastidiosa . Using immunocapture and nested PCR, it was possible to detect as few as five bacteria per sample.

Carcinogenesis, 1997 Aug, 18(8), 1535 - 9
Influence of diets containing high and low risk factors for colon cancer on early stages of carcinogenesis in human flora-associated (HFA) rats; Hambly RJ et al.; Germ-free rats colonised with a human intestinal flora were fed diets containing high risk (HR) or low risk (LR) factors for colorectal cancer, and putative biomarkers were evaluated in the colonic mucosa; (i) proliferation, (ii) 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci and (iii) DMH-induced DNA damage . The HR diet was high in fat (45% of calories) and low in calcium and fibre, reflecting levels characteristic of typical western diets . The LR diet was low in fat (<5% of calories), and high in calcium and fibre . The nutrient/energy ratio of the two diets were similar . Mucosal crypt cell proliferation, assessed after microdissection, was higher on the LR diet (mean number of mitoses per crypt was 2.65 on the LR diet, and 1.62 on the HR diet; P < 0.05) . Aberrant crypt foci (ACF) were assessed in the mucosa 12 weeks after DMH treatment . On the HR diet there were significantly more small ACF with 1 and 2 crypts per focus, but fewer ACF with 3, 5 and 7 or more crypts per focus . There was no significant difference in total ACF or the total number of crypts . The effect of diet on DNA damage in the colon was assessed in vivo by the comet assay . Animals were fed a HR or LR diet for 12 weeks before treatment with DMH or saline . For carcinogen-treated animals, DNA damage was significantly higher in colon cells from animals on the HR diet . On the LR diet both DNA damage and the induction of small ACF were reduced despite an increase in cell proliferation . The increase in large ACF on the LR diet may be attributable to elevated crypt cell proliferation possibly increasing crypt fission rates.

Chest, 1997 Aug, 112(2), 491 - 5
Gut decontamination reduces bowel ischemia-induced lung injury in rats; Sorkine P et al.; OBJECTIVE: To evaluate the effects of gut decontamination on endotoxin, tumor necrosis factor (TNF) levels, and the associated lung injury in a rat model of bowel ischemia . SUMMARY BACKGROUND DATA: Gut ischemia induces disruption of the intestinal mucosal barrier, allowing translocation of bacteria and endotoxin into the blood, which may trigger a systemic inflammatory response and lung injury . METHODS: Thirty anesthetized rats were randomized into three groups: (1) ischemia-reperfusion (I/R) alone (a 60-min superior mesenteric artery occlusion and 4 h of reperfusion, n=10); (2) rats that underwent gut decontamination prior to ischemia (I/R+GD, n=10); and (3) control rats (sham operated, n=10) . Serum levels of lipopolysaccharide (LPS) and TNF were measured at the end of the experiment . Lung permeability was measured using bovine serum albumin labeled with 125I, and organ injury was assessed histologically . RESULTS: One hour of bowel ischemia and 4 h of reperfusion (I/R) led to elevations of blood LPS and TNF levels of 0.33+/-0.005 EU/mL and 173+/-56 pg/mL, which were higher than the sham group (p<0.01) . Gut decontamination (I/R+GD) significantly attenuated the LPS and TNF generation, 0.09+/-0.005 and 56.2+/-6 pg/mL (p<0.01) . Lung injury as assessed by pulmonary permeability index was also reduced by gut decontamination, 2.1+/-0.42 vs 5.3+/-0.82 in the control group (p<0.03) . Surprisingly no difference was detected in the number of pulmonary neutrophils in sequestration between the groups . CONCLUSIONS: Our data suggest that gut decontamination can reduce the generation of LPS, TNF, and the severity of lung damage that often follows ischemia and reperfusion of the intestine in rats.

J Prosthet Dent, 1997 Aug, 78(2), 153 - 8
Lased and sandblasted denture base surface preparations affecting resilient liner bonding; Jacobsen NL et al.; STATEMENT OF PROBLEM: Adhesive failure between the liner and the denture base creates an environment for potential bacterial growth and accelerated breakdown of the soft liner resulting in a deteriorating prosthesis . PURPOSE: This study evaluated the effects of a specific sandblasted or lased preparation on the interfacial bonding of polymethyl methacrylate and silicone and polyethyl methacrylate resilient liners . MATERIAL AND METHODS: Polymethyl methacrylate test specimens were fabricated and received one of three surface treatments: untreated (control), sandblasted (250 microns aluminum oxide particles), and lased (carbon dioxide) . Polyethyl methacrylate and silicone resilient lining materials were applied to these surfaces and the peel strengths were determined with the American Society for Testing and Materials peelin-adhesion test . RESULTS: Altering the polymethyl methacrylate surface by sandblasting significantly reduced the peel strengths for the polymethyl methacrylate/polyethyl methacrylate and polymethyl methacrylate/silicone specimens . Altering the polymethyl methacrylate surface by delivering carbon dioxide laser energy to form a grid pattern produced lower peel strengths that were statistically significant from the controls for the polymethyl methacrylate/polyethyl methacrylate specimens, but not so for the polymethyl methacrylate/silicone specimens . Untreated polymethyl methacrylate/polyethyl methacrylate peel strengths were significantly higher than polymethyl methacrylate/silicone . CONCLUSIONS: Results of this study imply that mechanical surface preparation of denture bases before application of a resilient liner may not be warranted.

Curr Biol, 1997 Aug 1, 7(8), R487 - 8
Evolution: setting the mutation rate; Sniegowski P; A recent study of X-chromosome and autosome genes in mammals suggests that selective trade-offs are important in the long-term evolution of mutation rates; but recent studies with bacteria show that high mutation rates can nonetheless evolve in the short term in clonal populations.

FEMS Microbiol Lett, 1997 Aug 1, 153(1), 129 - 34
Identification and characterization of the gyrB gene from Treponema pallidum subsp . pallidum; Stamm LV et al.; The nucleotide sequence of a DNA gyrase B subunit gene (gyrB) from Treponema pallidum has been determined . Southern blot analysis of T . pallidum chromosomal DNA indicated that this gene is present as a single copy . The organization of genes flanking the gyrB gene is unique in comparison to that of other bacteria . The gyrB gene encodes a 637 amino acid protein whose deduced sequence has a high degree of homology with type-II topoisomerase ATPase subunits (GyrB and ParE) . Five type-II topoisomerase motifs, an ATP-binding site (Walker A), and amino acid residues that putatively interact with ATP, are highly conserved in the T . pallidum GyrB protein.

Int Arch Allergy Immunol, 1997 Aug, 113(4), 516 - 8
Lactose-intolerance may induce severe chronic eczema; Grimbacher B et al.; The primary acquired lactase deficiency of the adult is known to cause various disturbances in the gastrointestinal tract while extraintestinal symptoms are unusual . Here we report on a histologically proven chronic eczema requiring corticosteroid treatment for several months . It was obviously induced by a concomitant lactose intolerance since the introduction of a lactose-free diet led to a complete disappearance of the eczema and allowed the discontinuation of the corticosteroid treatment . As far as we know, this is the first case report of an eczema caused by a lactose intolerance.

Am J Clin Nutr, 1997 Aug, 66(2), 521S - 525S
Role of food in the stimulation of cytokine production; Solis-Pereyra B et al.; To study the role of food in the stimulation of cytokine production, the effects of lactic bacteria on production of interferon alpha, beta, and gamma; interleukin 1 beta; and tumor necrosis factor alpha were evaluated in mice and humans . Yogurt bacteria induced plasma interferon alpha and beta production in mice . Yogurt intake containing 10(11) bacteria led to increased 2'-5' A synthetase activity in human blood mononuclear cells . This result may suggest an interferon action in a peripheral way . This effect was also found when subjects consumed 10(8) yogurt bacteria/d for 15 d . In an in vitro model, blood mononuclear cells cultured in the presence of yogurt bacteria produced interleukin 1 beta, tumor necrosis factor, and interferon alpha and gamma . These results suggest the involvement of a certain type of food in cytokine production under healthy conditions.

J Am Coll Surg, 1997 Aug, 185(2), 156 - 62
Effect of glutamine or glucagon-insulin enriched total parenteral nutrition on liver and gut in 70% hepatectomized rats with colon stenosis; Yamaguchi T et al.; BACKGROUND: Malnutrition of enterocytes is believed to facilitate the breakdown of the intestinal mucosal barrier, furthering a translocation of enteric bacteria with subsequent severe infection, which has been described after extensive hepatectomy . Glutamine and glucagon insulin are said to attenuate the malnutrition of enterocytes . To determine whether this was true, the effects on the remnant liver and the gut of total parenteral nutrition supplemented by admixtures of glutamine and/or glucagon insulin were investigated in rats subjected to massive hepatectomy and transient intestinal stasis . STUDY DESIGN: Rats underwent a permanent cannulation of the superior caval vein without restraining their mobility, a 70% hepatectomy, and a 24 hour string-ligation stenosis of the colon . A standard total parenteral nutrition solution was infused without or with 2% glutamine and without or with glucagon-insulin supplementation, respectively . RESULTS: Glutamine and glucagon-insulin supplemented total parenteral nutrition increased ileal mucosal DNA concentrations during and after intestinal stasis . Glutamine or glucagon-insulin alone had less pronounced effects . In the liver, the combined supplementation resulted in reduced adenosine triphosphate concentrations and increased mitochondrial adenosine triphosphate synthesis as well as in an early increase in DNA concentrations . CONCLUSION: Glutamine and glucagon-insulin enriched total parenteral nutrition attenuates malnutrition of enterocytes after massive abdominal stress and promotes liver regeneration after extensive hepatectomy.

Immunopharmacol Immunotoxicol, 1997 Aug, 19(3), 393 - 404
Sulfide influence on polymorphonuclear functions: a possible role for Ca2+ involvement; Mariggio MA et al.; Polymorphonuclear cells (PMN) of gingival sulcus play an important role in host defense against periodontal tissue-invading bacteria, but their phagocytic activity is conditioned by several virulence factors released by oral pathogens . In this report we have studied the influence of sulfide, a toxic bacterial metabolite, on the main PMN functions: chemotaxis, degranulation and oxidative burst . PMN exposed to sodium sulfide (up to 2 mM) used as a source of H2S showed a depression of the calcium-dependent cytoskeleton activities such as chemotaxis and azurophilic granule release induced by FMLP . No effect was observed on the calcium-independent specific granule release obtained by PMA . These data were in agreement with the sulfide inhibition of cytosolic free Ca2+ concentration {Ca2+}i increase normally induced by ionomycin . On the other hand, hydrogen sulfide was able to prime PMN for a stronger oxidative response both to calcium-dependent or calcium-independent stimulation . This finding may account for a more efficient oxidative killing under reoxygenation of the anaerobic infectious areas.

J Mol Biol, 1997 Aug 1, 270(5), 751 - 62
Reduction in the amide hydrogen exchange rates of an anti-lysozyme Fv fragment due to formation of the Fv-lysozyme complex; Williams DC Jr et al.; The Fv fragment of the monoclonal antibody D1.3 was expressed in bacteria . Standard triple resonance techniques were used to obtain the NMR resonance assignments for 211 out of 215 backbone 15N/NH atoms for D1.3 Fv . Using these assignments, hydrogen exchange rates are measured for 82 amide hydrogen atoms in D1.3 Fv free and bound to hen egg-white lysozyme . Upon binding to antigen, exchange rates are decreased for residues throughout the Fv . Many of these residues are located remote from the site of interaction with the antigen . These changes are larger than previously observed for the antigen portion of the complex . Evidently, the beta-sheet structure of the Fv propagates the effects of binding more efficiently than the antigen . These effects are compared between the three different polypeptide chains that make up the complex . These data suggest that reduced dynamics are a general feature of antibody binding to antigen.

J Bacteriol, 1997 Aug, 179(15), 4778 - 88
The Tla protein of Porphyromonas gingivalis W50: a homolog of the RI protease precursor (PrpRI) is an outer membrane receptor required for growth on low levels of hemin; Aduse-Opoku J et al.; The prpR1 gene of Porphyromonas gingivalis W50 encodes the polyprotein precursor (PrpRI) of an extracellular arginine-specific protease . PrpRI is organized into four distinct domains (pro, alpha, beta, and gamma) and is processed to a heterodimeric protease (RI) which comprises the alpha and beta components in a noncovalent association . The alpha component contains the protease active site, whereas the beta component appears to have a role in adherence and hemagglutination processes . DNA sequences homologous to the coding region for the RI beta component are present at multiple loci on the P . gingivalis chromosome and may represent a family of related genes . In this report, we describe the cloning, sequence analysis, and characterization of one of these homologous loci isolated in plasmid pJM7 . The 6,041-bp P . gingivalis DNA fragment in pJM7 contains a major open reading frame of 3,291 bp with coding potential for a protein with an Mr 118,700 . An internal region of the deduced sequence (V304 to N768) shows 98% identity to the beta domain of PrpRI, and the recombinant product of pJM7 is immunoreactive with an antibody specific to the RI beta component . The N terminus of the deduced sequence has regional similarity to TonB-linked receptors which are frequently involved in periplasmic translocation of hemin, iron, colicins, or vitamin B12 in other bacteria . We have therefore designated this gene tla (TonB-linked adhesin) . In contrast to the parent strain, an isogenic mutant of P . gingivalis W50 in which the tla was insertionally inactivated was unable to grow in medium containing low concentrations of hemin (<2.5 mg liter(-1)), and hemin-depleted cells of this mutant failed to respond to hemin in an agar diffusion plate assay . These data suggest a role for this gene product in hemin acquisition and utilization . Furthermore, the mutant produced significantly less arginine- and lysine-specific protease activities than the parent strain, indicating that there may be a regulatory relationship between tla and other members of this gene family.

J Bacteriol, 1997 Aug, 179(15), 4676 - 83
Identification and characterization of an operon of Helicobacter pylori that is involved in motility and stress adaptation; Beier D et al.; We identified a novel stress-responsive operon (sro) of Helicobacter pylori that contains seven genes which are likely to be involved in cellular functions as diverse as chemotaxis, heat shock response, ion transport, and posttranslational protein modification . The products of three of these genes show amino acid homologies to known proteins, such as the flagellar motor switch protein CheY, a class of heat shock proteins, and the ribosomal protein L11 methyltransferase, and to a phosphatidyltransferase . In addition to containing an open reading frame of unknown function, the product of which is predicted to be membrane associated, the sro locus contains three open reading frames that have previously been described as constituting two separate loci, the ftsH gene and the copAP operon of H . pylori . Knockout mutants showed that CheY is essential for bacterial motility and that CopA, but not CopP, relieves copper toxicity . Transcriptional analyses indicated that this locus is regulated by a single promoter and that a positive effect on transcription is exerted by the addition of copper to the medium and by temperature upshift from 37 to 45 degrees C . The possible role of this locus in H . pylori virulence is discussed.

J Bacteriol, 1997 Aug, 179(15), 4671 - 5
Involvement of NtcB, a LysR family transcription factor, in nitrite activation of the nitrate assimilation operon in the cyanobacterium Synechococcus sp . strain PCC 7942; Aichi M et al.; Nitrite, either exogenously supplied or endogenously generated by nitrate reduction, activates transcription of the nitrate assimilation operon (nirA-nrtABCD-narB) in Synechococcus sp . strain PCC 7942 cells treated with L-methionine-DL-sulfoximine (an inhibitor of glutamine synthetase), in which there is no negative feedback resulting from fixation of the ammonium generated by nitrite reduction (Kikuchi et al., J . Bacteriol . 178:5822-5825, 1996) . Other transcription units related to nitrogen assimilation, i.e., the nirB-ntcB operon, glnA, and ntcA, were not activated by nitrite . Nitrite did not activate nirA operon transcription in a mutant with a deletion of ntcB, an ammonium-repressible gene encoding a LysR-type DNA-binding protein . Introduction of plasmid-borne ntcB into the ntcB deletion mutant restored the response of the cells to nitrite, indicating that NtcB activates the nirA operon in response to nitrite . Supplementation of nitrite or nitrate to nitrogen-starved cultures of the wild-type strain, but not of the ntcB deletion mutant, caused activation of the nirA operon without L-methionine-DL-sulfoximine treatment of the cells . The results suggested that the positive-regulation mechanism of nirA operon transcription plays a role in rapid adaptation of nitrogen-starved cells to changing availability of nitrate and nitrite.

J Biol Chem, 1997 Aug 1, 272(31), 19171 - 5
Defects in auxiliary redox proteins lead to functional methionine synthase deficiency; Gulati S et al.; Methionine synthase catalyzes a methyl transfer reaction from methyltetrahydrofolate to homocysteine to form methionine and tetrahydrofolate and is dependent on methylcobalamin, a derivative of vitamin B12, for activity . Due to the lability of the intermediate, cob(I)alamin, the activity of methionine synthase is additionally dependent on a redox activation system . In bacteria, two flavoproteins, NADPH-flavodoxin reductase and flavodoxin, shuttle electrons from NADPH to methionine synthase . Their mammalian counterparts are unknown, and a putative intrinsic thiol oxidase activity of the mammalian methionine synthase has been proposed to be involved . We demonstrate that the mammalian methionine synthase can be activated in an NADPH-dependent reaction and requires a minimum of two redox proteins . This model is consistent with our results from biochemical complementation studies between cblG and cblE cell lines and mutation detection analysis in cblG cell lines . These demonstrate that the cblG cell line has defects affecting methionine synthase directly, whereas the cblE cell line has defects in the redox proteins . We have also identified a P1173L mutation in the activation domain of methionine synthase in the cblG cell line WG1505.

Infect Immun, 1997 Aug, 65(8), 3469 - 73
Phase variation affects long-term survival of Bordetella bronchiseptica in professional phagocytes; Banemann A et al.; Several Bordetella bronchiseptica isolates were investigated for intracellular survival in macrophages . A significant number of viable bacteria of all strains could be recovered even after 96 h postinfection . In all cases bvg mutants of the B . bronchiseptica strains showed a significant survival advantage over the respective wild-type strains . The bacteria were already located in phagolysosomes early after uptake . Neither opsonization of the bacteria nor activation of the macrophages with gamma interferon or lipopolysaccharide prior to infection affected uptake and survival of the bacteria.

Infect Immun, 1997 Aug, 65(8), 3218 - 24
Mechanisms involved in Helicobacter pylori-induced interleukin-8 production by a gastric cancer cell line, MKN45; Aihara M et al.; Accumulating evidence suggests an important role of interleukin-8 (IL-8) in Helicobacter pylori infection-associated chronic atrophic gastritis and peptic ulcer . We observed in this study that a gastric cancer-derived cell line, MKN45, produced a massive amount of IL-8 upon coculture with live H . pylori but not with killed H . pylori, H . pylori culture supernatants, or live H . pylori separated by a permeable membrane, indicating that IL-8 production requires a direct contact between the cells and live bacteria . Moreover, the tyrosine kinase inhibitor herbimycin but neither a protein kinase C inhibitor (staurosporine) nor a protein kinase A inhibitor (H89) inhibited IL-8 production by MKN45 cells cocultured with live bacteria, suggesting the involvement of a tyrosine kinase(s) in H . pylori-induced IL-8 production . In addition, coculture of H . pylori induced IL-8 mRNA expression in MKN45 cells and an increase in luciferase activity in cells which were transfected with a luciferase expression vector linked with a 5'-flanking region of the IL-8 gene (bp -133 to +44), indicating that the induction of IL-8 production occurred at the transcriptional level . This region contain three cis elements important for induction of IL-8 gene expression: AP-1 (-126 to -120 bp), NF-IL6 (-94 to -81 bp), and NF-kappaB (-80 to -70 bp) binding sites . Mutation of the NF-kappaB binding site abrogated completely the induction of luciferase activity, whereas that of the AP-1 site partially reduced the induction . However, mutation of the NF-IL6 binding site resulted in no decrease in the induction of luciferase activity . Moreover, specific NF-kappaB complexes were detected in the nuclear proteins extracted from MKN45 cells which were infected with H . pylori . Collectively, these results suggest that H . pylori induced the activation of NF-kappaB as well as AP-1, leading to IL-8 gene transcription.

Mol Cell Biol, 1997 Aug, 17(8), 4852 - 8
A large protein complex containing the yeast Sin3p and Rpd3p transcriptional regulators; Kasten MM et al.; The SIN3 gene is required for the transcriptional repression of diverse genes in Saccharomyces cerevisiae . Sin3p does not bind directly to DNA but is thought to be targeted to promoters by interacting with sequence-specific DNA-binding proteins . We show here that Sin3p is present in a large multiprotein complex with an apparent molecular mass, estimated by gel filtration chromatography, of greater than 2 million Da . Genetic studies have shown that the yeast RPD3 gene has a function similar to that of SIN3 in transcriptional regulation, as SIN3 and RPD3 negatively regulate the same set of genes . The SIN3 and RPD3 genes are conserved from yeasts to mammals, and recent work suggests that RPD3 may encode a histone deacetylase . We show that Rpd3p is present in the Sin3p complex and that an rpd3 mutation eliminates SIN3-dependent repression . Thus, Sin3p may function as a bridge to recruit the Rpd3p histone deacetylase to specific promoters.

J Virol, 1997 Aug, 71(8), 5915 - 21
The 222- to 234-kilodalton latent nuclear protein (LNA) of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) is encoded by orf73 and is a component of the latency-associated nuclear antigen; Rainbow L et al.; Kaposi's sarcoma (KS)-associated herpesvirus or human herpesvirus 8 (KSHV/HHV8) is the likely cause of KS and primary effusion lymphomas or body cavity-based lymphomas (BCBLs) . A latency-associated nuclear immunofluorescence antigen (LANA) (D . H . Kedes, E . Operskalski, M . Busch, R . Kohn, J . Flood, and D . Ganem, Nat . Med . 2:918-924, 1996; S . J . Gao, L . Kingsley, M . Li, W . Zheng, C . Parravicini, J . Ziegler, R . Newton, C . R . Rinaldo, A . Saah, J . Phair, R . Detels, Y . Chang, and P . S . Moore, Nat . Med . 2:925-928, 1996) and a 222- to 234-kDa nuclear protein (LNA) (S . J . Gao, L . Kingsley, D . R . Hoover, T . J . Spira, C . R . Rinaldo, A . Saah, J . Phair, R . Detels, P . Parry, Y . Chang, and P . S . Moore, N . Engl . J . Med . 335:233-241, 1996) have previously been described in BCBL cell lines by immunofluorescence and Western blotting techniques, respectively . To identify the viral gene(s) encoding this antigen(s) we screened a cDNA library from HBL-6 cells, a B-cell lymphoma cell line persistently infected with KSHV/HHV8, with KS patient sera . One set of positive clones contained the 3' end of orf73, as well as the complete orf72 and orfK13, and another set contained the 5' end of orf73 . Comparison of cDNA sequences with the KSHV/HHV8 genomic sequence revealed a splice event, occurring upstream of orf73 . Immunoaffinity purified antibodies to a recombinant carboxy-terminal fragment of the orf73-encoded protein showed the characteristic speckled nuclear immunofluorescence pattern of LANA and reacted with the 222- to 234-kDa LNA on Western blots . Expression of full-length orf73 in bacteria and COS7 cells reproduced the LNA banding pattern . Immunohistochemistry on cases of nodular KS revealed that orf73/LNA is expressed in the nucleus of KS spindle cells . These findings demonstrate that orf73 encodes the 222- to 234-kDa LNA, is a component of LANA, and is expressed in KS tumor cells.

Curr Microbiol, 1997 Aug, 35(2), 122 - 3
Buchnera aphidicola (endosymbiont of aphids) contains nuoC(D) genes that encode subunits of NADH dehydrogenase; Clark MA et al.; A two-kilobase DNA fragment from Buchnera aphidicola, the endosymbiont of aphids, was cloned and sequenced . One open reading frame was detected, coding for a putative protein of 600 amino acids . The N-terminal portion of this protein corresponded to NuoC, while the C-terminal portion corresponded to NuoD . These proteins are constituents of the membrane-associated NADH dehydrogenase . Our results suggest that these two proteins are fused in Buchnera aphidicola, a result consistent with their previously postulated spatial association.

Vet Parasitol, 1997 Jul 31, 71(2-3), 155 - 75
Application of molecular biology in veterinary parasitology; Prichard R; The number of applications of molecular biology in veterinary parasitology is increasing rapidly . The techniques used with eukaryotic cells are generally applicable to the study of parasites and their hosts . The polymerase chain reaction is particularly important for identification and diagnosis of parasites, as well as for many other applications . With species and type specific probes or primers, sensitivities and specificities unheard of with conventional techniques can be achieved . The accumulation of more information on the DNA sequences of parasites will reveal many more unique sequences which can be used for identification, diagnosis, molecular epidemiology, vaccine development and for studying the evolutionary biology and the physiology of parasites and the host-parasite relationship . Similarly, the completion of genome projects on host organisms will greatly assist efforts to select for hosts that are genetically resistant to parasite infection . The study of the molecular biology of antiparasitic drug receptors, potential targets for chemotherapy, and the molecular genetics of drug resistance will allow molecular screens to be used with combinatorial chemistry in the search for new antiparasitic drugs, improvements to existing chemotherapeutic families and better diagnosis and monitoring of drug resistance . While there is a proliferation of molecular biology techniques, the availability of simple kits and of automated techniques and services for sequencing, library construction and oligonucleotide synthesis and other procedures is making it easier for non-specialists to apply many of the common techniques of molecular biology . Molecular biology and the benefits from its application are relevant for veterinary parasitologists in developing countries as well as developed countries and we should introduce aspects of molecular biology to the teaching and training of veterinary parasitologists.

Proc R Soc Lond B Biol Sci, 1997 Jul 22, 264(1384), 985 - 91
Optimal killing for obligate killers: the evolution of life histories and virulence of semelparous parasites; Ebert D et al.; Many viral, bacterial and protozoan parasites of invertebrates first propagate inside their host without releasing any transmission stages and then kill their host to release all transmission stages at once . Life history and the evolution of virulence of these obligately killing parasites are modelled, assuming that within-host growth is density dependent . We find that the parasite should kill the host when its per capita growth rate falls to the level of the host mortality rate . The parasite should kill its host later when the carrying capacity, K, is higher, but should kill it earlier when the parasite-independent host mortality increases or when the parasite has a higher birth rate . When K(t), for parasite growth, is not constant over the duration of an infection, but increases with time, the parasite should kill the host around the stage when the growth rate of the carrying capacity decelerates strongly . In case that K(t) relates to host body size, this deceleration in growth is around host maturation.

Proc Natl Acad Sci U S A, 1997 Jul 22, 94(15), 8110 - 5
Modified cytokeratins expressed on the surface of carcinoma cells undergo endocytosis upon binding of human monoclonal antibody and its recombinant Fab fragment; Ditzel HJ et al.; Previously, we have reported on successful imaging of colon, rectal, and pancreatic carcinomas in patients by using a radiolabeled all-human monoclonal antibody, COU-1, directed against modified cytokeratin . To further develop this antibody for use as an immunoconjugate, COU-1 was cloned by phage display selection and the human Fab fragment was expressed in bacteria . Analysis by confocal laser scanning microscopy demonstrated that COU-1 bound in a uniform punctate pattern to the surface of viable carcinoma cells stained at 4 degrees C, and binding increased significantly when cells were cultured on fibronectin, laminin, or collagen IV . In the case of fibronectin, COU-1 staining was particularly enhanced at intercellular junctions . When carcinoma cells were cultured with COU-1 at 37 degrees C for 6 hr, the antibody was found in large perinuclear vesicles and the punctate surface staining was significantly reduced . Similar results were obtained using intact IgM COU-1 and the recombinant Fab fragment . Immunohistological studies indicated that COU-1, in contrast to murine monoclonal antibodies against normal cytokeratin 8 and 18, could differentiate between malignant and normal colon epithelia, and between colon cancer metastasis in the liver and surrounding normal hepatocytes . Within biopsies of malignant tissue, COU-1 exhibited membrane-associated staining of proliferating cells, while resting cells had a filamentous pattern . Thus, modified cytokeratin at the surface of carcinoma cells may represent a new target for immunoconjugates and may explain the promising results of the phase I/II clinical study.

Proc Natl Acad Sci U S A, 1997 Jul 22, 94(15), 7982 - 6
An important developmental role for oligosaccharides during early embryogenesis of cyprinid fish; Bakkers J et al.; Derivatives of chitin oligosaccharides have been shown to play a role in plant organogenesis at nanomolar concentrations . Here we present data which indicate that chitin oligosaccharides are important for embryogenesis in vertebrates . We characterize chitin oligosaccharides synthesized in vitro by zebrafish and carp embryos in the late gastrulation stage by incorporation of radiolabeled N-acetyl-D-{U14C}glucosamine and by HPLC in combination with enzymatic conversion using the Bradyrhizobium NodZ alpha-1, 6-fucosyltransferase and chitinases . A rapid and sensitive bioassay for chitin oligosaccharides was also used employing suspension-cultured plant cells of Catharanthus roseus . We show that chitin oligosaccharide synthase activity is apparent only during late gastrulation and can be inhibited by antiserum raised against the Xenopus DG42 protein . The DG42 protein, a glycosyltransferase, is transiently expressed between midblastula and neurulation in Xenopus and zebrafish embryogenesis . Microinjection of the DG42 antiserum or the Bradyrhizobium NodZ enzyme in fertilized eggs of zebrafish led to severe defects in trunk and tail development.

Proc Natl Acad Sci U S A, 1997 Jul 22, 94(15), 7971 - 5
Roles of phospholipase C beta2 in chemoattractant-elicited responses; Jiang H et al.; The physiological roles of phospholipase C (PLC) beta2 in hematopoiesis, leukocyte function, and host defense against infection were investigated using a mouse line that lacks PLC beta2 . PLC beta2 deficiency did not affect hematopoiesis, but it blocked chemoattractant-induced Ca2+ release, superoxide production, and MAC-1 up-regulation in neutrophils . In view of these effects, it was surprising that the absence of PLC beta2 enhanced chemotaxis of different leukocyte populations and sensitized the in vivo response of the PLC beta2-deficient mice to bacteria, viruses, and immune complexes . These data raise questions about the roles that PLC beta2 may play in signal transduction induced by chemoattractants in leukocytes.

Proc Natl Acad Sci U S A, 1997 Jul 22, 94(15), 7837 - 40
Helical repeat of DNA in the region of homologous pairing; Kiianitsa K et al.; The process of DNA strand exchange during general genetic recombination is initiated within protein-stabilized synaptic filaments containing homologous regions of interacting DNA molecules . The RecA protein in bacteria and its analogs in eukaryotic organisms start this process by forming helical filamentous complexes on single-stranded or partially single-stranded DNA molecules . These complexes then progressively bind homologous double-stranded DNA molecules so that homologous regions of single- and double-stranded DNA molecules become aligned in register while presumably winding around common axis . The topological assay presented herein allows us to conclude that in synaptic complexes containing homologous single- and double-stranded DNA molecules, all three DNA strands have a helicity of approximately 19 nt per turn.

Biochim Biophys Acta, 1997 Jul 19, 1336(1), 51 - 8
The enantiomeric purity of alcohols formed by enzymatic reduction of ketones can be improved by optimisation of the temperature and by using a high co-substrate concentration; Yang H et al.; The stereoselective reduction of ketones by alcohol dehydrogenase from Thermoanaerobium brockii was studied in organic reaction media . 2-Propanol was used as co-substrate to regenerate the coenzyme NADPH . The enantiomeric excess of the alcohol formed from the ketone decreased during the course of the reaction (from 53 to 0% e.e . in the formation of (R)-2-butanol) . This was interpreted as being due to the reversibility of all the reactions involved . By using a large excess of 2-propanol this effect was suppressed . In the reduction of 2-butanone to (R)-2-butanol, the enantiomeric excess increased with increasing temperature, but in the reduction of 2-pentanone to (S)-2-pentanol the enantiomeric excess decreased with increasing temperature . The data were evaluated in terms of free energy of activation of the reaction pathways leading to the different possible products.

Gene, 1997 Jul 18, 194(1), 143 - 55
The majority of long non-stop reading frames on the antisense strand can be explained by biased codon usage; Silke J; In recent studies it has been suggested that long reading frames on the antisense strand of open reading frames (ORFs) are more frequent than expected . The vertebrate DNA database was searched for long (greater than 900 bp) antisense non-stop reading frames (aNRFs) that overlap known coding regions . The sequences obtained were predominantly positioned in DNA with a high usage of G or C in the third codon position of the sense ORF . The major class of sequences revealed by the search was that of the heat-shock protein 70 kDa (Hsp70) family . A long Hsp70 aNRF was found in many Hsp70 sequences and occurred in species as diverse as fish, flies, fungi and bacteria . The role of codon usage bias was analysed both in the specific case of the Hsp70 genes and in a general species-wide context . The data obtained showed that even the very long aNRFs present in the Hsp70 family could be explained by codon usage bias on the sense strand . Codon usage bias is determined by GC content at the third codon position of the sense ORF and, in some species, by a high expression level of the gene in question . Such an explanation for the occurrence of long aNRFs cannot exclude that some aNRFs are transcribed and translated.

Int J Cancer, 1997 Jul 17, 72(2), 313 - 20
Establishment and characterization of 12 uterine cervical-carcinoma cell lines: common sequence variation in the E7 gene of HPV-16-positive cell lines; Ku JL et al.; A total of 12 carcinoma cell lines of the human uterine cervix were established from 5 keratinizing and 5 nonkeratinizing squamous-cell carcinomas, and 2 small-cell carcinomas . Of these, 10 lines grew as adherent cells and 2 as floating aggregates . All lines showed (i) similarity in morphology to the primary tumor from which they were derived; (ii) high viability with relatively long doubling times (48-96 hr); (iii) absence of Mycoplasma and other bacteria, apart from one Mycoplasma-contaminated line; (iv) genetic heterogeneity by DNA-fingerprinting analysis; (v) absence of p53 mutation from exon 4 through 9; and (vi) the presence of HPV DNA sequence . Among the lines, 7 were infected by HPV-16, 3 by HPV-18, 1 by HPV-31, and 1 by HPV-33; the 2 cell lines derived from small-cell carcinomas contained HPV-18 . Interestingly, 6 of the 7 cell lines containing HPV-16-type DNA harbored the same alteration of E7 at nucleotide position 647 (amino acid 29, AAT --> AGT, Asn --> Ser), whereas the 3 HPV-18-positive lines did not; 3 cell lines proved to have intact E1/E2 of HPV, suggesting the presence of episomally replicating HPV DNA as well as the integrated form, whereas the other 9 lines were shown to have integrated HPV . Taken together, these cell lines would be very useful for studying the biology of uterine cervical carcinoma.

AIDS, 1997 Jul 15, 11(9), 1165 - 71
Disseminated Mycobacterium avium complex disease in the Swiss HIV Cohort Study: increasing incidence, unchanged prognosis; Low N et al.; OBJECTIVES: Disseminated disease due to Mycobacterium avium complex (MAC) bacteria is thought to occur less frequently in Europe than in the USA . This study investigated time trends in the occurrence of, and survival with, disseminated MAC disease in the Swiss HIV Cohort Study (SHCS) . DESIGN, SETTING AND PARTICIPANTS: The SHCS participants who were free of disseminated MAC disease at registration were stratified by calendar period (1987-1989, 1990-1992, 1993-1995) in which the first recorded CD4 count was 0-49, 50-99, or 100-199 x 10(6)/l . Kaplan-Meier estimates of the probability of developing and surviving disseminated MAC disease were calculated for these nine independent groups . Multivariate analyses were performed using Cox proportional hazards regression . RESULTS: The analysis was based on 6052 participants enrolled between January 1987 and December 1995 and 202 incident episodes of disseminated MAC disease recorded during a mean follow-up time of 3.5 years . The cumulative probability of MAC disease at 2 years in individuals with CD4 counts of 0-49 x 10(6)/l in 1987-1989 was 9.8% {95% confidence interval (CI) 4.4-15.2%}, increasing to 29.8% (95% CI, 20.8-38.8%) in 1993-1995 . Amongst those with CD4 counts from 50-99 x 10(6)/l these probabilities were 11.9% (95% CI, 5.9-17.8%), and 21.6% (95% CI, 13.9-29.2%), respectively . After adjusting for CD4 count the relative hazard of developing disseminated MAC disease in 1993-1995, compared with 1987-1989, was 1.37 (95% CI, 0.92-2.04) . Median survival following diagnosis was 7.9 months with no improvement over time . CONCLUSIONS: The incidence of disseminated MAC disease among SHCS participants has increased over time . More profound levels of immunosuppression amongst recent study entrants were found to explain this . When compared with US cohorts studied over the same calendar period the incidence of disseminated MAC disease in the SHCS appears to be lower . These findings are consistent with a secular effect of a more mature HIV epidemic in the US but direct comparison between the SHCS and a similar prospective cohort in the US should be undertaken to clarify this issue.

FEMS Microbiol Lett, 1997 Jul 15, 152(2), 205 - 11
A flagellar-specific ATPase (FliI) is necessary for flagellar export in Helicobacter pylori; Jenks PJ et al.; Although flagellar motility is essential for the colonisation of the stomach by Helicobacter pylori, little is known about the regulation of flagellar biosynthesis in this organism . We have identified a gene in H . pylori, designated fliI, whose deduced amino acid sequence revealed extensive homology with the FliI/LcrB/InvC family of proteins which energise the export of flagellar and other virulence factors in several bacterial species . An isogenic mutant of fliI was non-motile and synthesised reduced amounts of flagellin and hook protein subunits . The majority (> 99%) of mutant cells were completely aflagellate . These results suggest that FliI is a novel ATPase involved in flagellar export in H . pylori.

Biochem J, 1997 Jul 15, 325 ( Pt 2), 405 - 10
Multiple C-terminal serine phosphorylation accompanies both protein kinase C-dependent and -independent activation of cytosolic 85 kDa phospholipase A2 in macrophages; Wijkander J et al.; Exposure of mouse macrophages to either phorbol ester or certain bacteria was previously shown to cause increased phosphorylation of the cytosolic 85 kDa phospholipase A2 as well as a stable increase in its catalytic activity . We have now attempted to map the major phosphorylation sites on the enzyme in such cells . Phosphorylation occurred on serine residues without a detectable increase in either phosphothreonine or phosphotyrosine . After CNBr cleavage five fragments showed increased 32P labelling . Among those the most heavily labelled fragment was identified as the most C-terminal (residues 698-749), containing six serine residues . This was true whether phorbol ester or bacteria, causing protein kinase C-independent phospholipase A2 activation, was used as stimulus . The heavy phosphorylation of the most C-terminal fragment and an analysis of tryptic peptides derived from it suggested that more than one of the six serine residues became phosphorylated . Smaller increases also occurred in other CNBr-cleaved fragments from the C-terminal part of the protein, including that carrying Ser-505, a known target of the mitogen-activated protein kinase ERK-2 (extracellular-signal regulated kinase) . Dexamethasone treatment (1-100 nM for 20 h), which was earlier shown to dose-dependently down-regulate the 85 kDa phospholipase A2 and its activation by phorbol ester and zymosan, was here shown also to counteract the protein kinase C-independent activation and arachidonate release elicited by bacteria . It remains to be determined whether all phosphorylation sites are equally affected under those conditions.

J Immunol, 1997 Jul 15, 159(2), 599 - 605
Targeting of natural killer cells to mammary carcinoma via naturally occurring tumor cell-bound iC3b and beta-glucan-primed CR3 (CD11b/CD18); Vetvicka V et al.; Previous reports have suggested that malignant cells frequently generate a humoral immune response that is ineffective in tumor destruction . Despite coating tumors with IgM and IgG that activate the C system via the classical pathway, normal membrane regulators of C (e.g., membrane cofactor protein and CD59) prevent cytotoxicity . Moreover, C3 deposition on tumors does not result in cytotoxic recognition by phagocytes or NK cells bearing C3 receptors capable of mediating destruction of C3-opsonized bacteria or yeast . The current investigation showed that freshly excised mammary tumors bore IgM, IgG, and C3 detectable by flow cytometry . Normal sera contained natural IgM and IgG Abs reactive with breast tumor cell lines, and IgG Ab titers were increased in patients with breast cancer . Breast tumor cell lines incubated in normal serum from AB+ individuals activated the classical, but not the alternative, pathway of C and became coated with C3 . Despite exhibiting membrane-bound C3, serum-opsonized breast tumor cell lines were not killed by CR3 (CD11b/CD18)-bearing NK cells . Priming of NK cell CR3 with small soluble yeast beta-glucan polysaccharides enabled CR3-dependent killing of these same C3-bearing tumor cell lines . Tests of mammary carcinoma cells from freshly excised tumors demonstrated that they also bore sufficient amounts of opsonic C3 for cytotoxic recognition by NK cells bearing polysaccharide-primed CR3, whereas they were largely resistant to NK cells bearing unprimed CR3 . This study demonstrates the potential utility of using naturally occurring opsonic C3 on tumor cells for specific immunotherapeutic targeting by NK cells and phagocytes bearing polysaccharide-primed CR3.

Nucleic Acids Res, 1997 Jul 15, 25(14), 2723 - 9
Tetracycline-controlled transcription in eukaryotes: novel transactivators with graded transactivation potential; Baron U et al.; Several tetracycline-controlled transactivators (tTA) were generated which differ in their activation potential by >3 orders of magnitude . The transactivators are fusions between the Tet repressor and minimal transcriptional activation domains derived from Herpes simplex virus protein 16 (VP16) . By reducing the VP16 moiety of the previously described tTA to 12 amino acids, potential targets for interactions with various cellular transcription factors were eliminated, as were potential epitopes which may elicit a cellular immune response . When compared with the originally described tTA, these new transactivators are tolerated at higher intracellular concentrations . This will facilitate establishment of tet regulatory systems under a variety of conditions, but particularly when cell type-restricted tetracycline-controlled gene expression is to be achieved in transgenic organisms via homologous recombination.

FEBS Lett, 1997 Jul 14, 411(2-3), 359 - 64
Remarkably slow folding of a small protein; Aronsson G et al.; Equilibrium denaturation of the 72 amino acid alpha/beta-protein MerP, by acid, guanidine hydrochloride, or temperature, is fully reversible and follows a two-state model in which only the native and unfolded states are populated . A cis-trans equilibrium around a proline peptide bond causes a heterogeneity of the unfolded state and gives rise to a slow- and a fast folding population . With a rate constant of 1.2 s(-1) for the major fast folding population, which has none of the common intrinsically slow steps, MerP is the slowest folding protein of this small size yet reported.

J Biol Chem, 1997 Jul 11, 272(28), 17836 - 42
Interaction of arrestins with intracellular domains of muscarinic and alpha2-adrenergic receptors; Wu G et al.; The intracellular domains of G-protein-coupled receptors provide sites for interaction with key proteins involved in signal initiation and termination . As an initial approach to identify proteins interacting with these receptors and the receptor motifs required for such interactions, we used intracellular subdomains of G-protein-coupled receptors as probes to screen brain cytosol proteins . Peptides from the third intracellular loop (i3) of the M2-muscarinic receptor (MR) (His208-Arg387), M3-MR (Gly308-Leu497), or alpha2A/D-adrenergic receptor (AR) (Lys224-Phe374) were generated in bacteria as glutathione S-transferase (GST) fusion proteins, bound to glutathione-Sepharose and used as affinity matrices to detect interacting proteins in fractionated bovine brain cytosol . Bound proteins were identified by immunoblotting following SDS-polyacrylamide gel electrophoresis . Brain arrestins bound to the GST-M3 fusion protein, but not to the control GST peptide or i3 peptides derived from the alpha2A/D-AR and M2-MR . However, each of the receptor subdomains bound purified beta-arrestin and arrestin-3 . The interaction of the M3-MR and M2-MR i3 peptides with arrestins was further investigated . The M3-MR i3 peptide bound in vitro translated {3H}beta-arrestin and {3H}arrestin-3, but did not interact with in vitro translated or purified visual arrestin . The properties and specificity of the interaction of in vitro translated {3H}beta-arrestin, {3H}visual arrestin, and {3H}beta-arrestin/visual arrestin chimeras with the M2-MR i3 peptide were similar to those observed with the intact purified M2-MR that was phosphorylated and/or activated by agonist . Subsequent binding site localization studies indicated that the interaction of beta-arrestin with the M3-MR peptide required both the amino (Gly308-Leu368) and carboxyl portions (Lys425-Leu497) of the receptor subdomain . In contrast, the carboxyl region of the M3-MR i3 peptide was sufficient for its interaction with arrestin-3.

Gene, 1997 Jul 9, 193(2), 157 - 61
Human type II arginase: sequence analysis and tissue-specific expression; Morris SM Jr et al.; A full-length cDNA encoding type II arginase was isolated from a human kidney cDNA library and its sequence compared to those of vertebrate type I arginases as well as to arginases of bacteria, fungi and plants . The predicted sequence of human type II arginase is 58% identical to the sequence of human type I arginase but is 71% identical to the sequence of Xenopus type II arginase, suggesting that duplication of the arginase gene occurred before mammals and amphibians diverged . Seven residues known to be essential for activity were found to be conserved in all arginases . Type II arginase mRNA was detected in virtually all human and mouse RNA samples tested whereas type I arginase mRNA was found only in liver . At least five mRNA species hybridizing to type II arginase cDNA were found in the human RNA samples whereas only a single type II arginase mRNA species was found in the mouse . This raises the possibility that the multiple type II arginase mRNAs in humans arise from differential RNA processing or usage of alternative promoters.

Proc Natl Acad Sci U S A, 1997 Jul 8, 94(14), 7595 - 9
Induction of host signal transduction pathways by Helicobacter pylori; Segal ED et al.; Adherence of Helicobacter pylori to cultured gastric epithelial cells is associated with several cellular events, including the tyrosine phosphorylation of a 145-kDa host protein; the reorganization of the host cell actin and associated cellular proteins, like vasodilator-stimulated phosphoprotein, adjacent to the attached bacterial cell; and the subsequent release of the cytokine, interleukin 8 (IL-8) . H . pylori isolated from patients with ulcer disease and gastric cancer contain a DNA insertion, the cag pathogenicity island (PAI), that is not present in bacteria isolated from individuals with asymptomatic infection . Mutations in a number of PAI genes abolish tyrosine phosphorylation and IL-8 synthesis but not the cytoskeletal rearrangements . Kinase inhibition studies suggest there are two distinct pathways operative in stimulating IL-8 release from host cells and one of these H . pylori pathways is independent of the tyrosine phosphorylation step.

Virology, 1997 Jul 7, 233(2), 339 - 57
Bovine immunodeficiency virus tat gene: cloning of two distinct cDNAs and identification, characterization, and immunolocalization of the tat gene products; Fong SE et al.; cDNAs encoding the bovine immunodeficiency virus (BIV) transactivator gene (tat) were cloned from virally infected cells and characterized . BIV expresses two distinct tat mRNAs composed of three exons that are derived by alternative splicing . The BIV tat mRNA splice variants encode Tat proteins of 103 (Tat103) and 108 (Tat108) amino acids . The Tat103 coding region is specified only by exon 2, while that of Tat108 is specified by a truncated exon 2 and the first 30 nt of exon 3 . Thus, the first 98 amino acids of each Tat are identical, and have amino terminal, cysteine-rich, conserved core, basic, and carboxyl-terminal domains similar to Tats encoded by primate lentiviruses . BIV-infected bovine cells express a 14-kDa phosphorylated Tat protein identical in size to recombinant Tat expressed in bacteria . BIV Tat was shown to localize exclusively in the nucleoli of virally infected and Tat-expressing cells . Reporter gene assays indicated that Tat103 and Tat108 can strongly transactivate the BIV long terminal repeat (LTR) in virally permissive canine Cf2Th and nonpermissive HeLa and mouse NIH 3T3 cells, but not in permissive lapine EREp cells . However, an intact BIV tat gene is required for viral replication in both Cf2Th and EREp cells . Strong LTR activation by BIV Tat requires a TAR (transactivation responsive) element delimited by viral nt +1 to +31 and the Tat basic domain . BIV Tat strongly cross-transactivates the HIV-1 LTR in a TAR-dependent manner in Cf2Th, but not in EREp, HeLa, or NIH 3T3 cells . In contrast, strong, TAR-dependent cross-transactivation of the BIV LTR by HIV-1 Tat could not be demonstrated in any of these cell types . In Cf2Th cells Tat108 effects a moderately stronger transactivation of the BIV LTR than Tat103, indicative of a functional difference in BIV Tat proteins encoded by the mRNA splice variants . The present studies demonstrate that BIV Tat parallels the primate lentiviral Tats in structure and biochemistry but is not interchangeable with the latter.

J Biol Chem, 1997 Jul 4, 272(27), 17061 - 9
Structural organization of the major subunits in cyanobacterial photosystem 1 . Localization of subunits PsaC, -D, -E, -F, and -J; Kruip J et al.; Based on an improved isolation procedure using perfusion chromatography, trimeric Photosystem 1 (PS1) complexes have been isolated from various deletion mutants of the mesophilic cyanobacterium Synechocystis PCC 6803 . These mutants are only deficient in the deleted subunits, which was carefully checked by high resolution gel electrophoresis in combination with immunoblotting . These highly purified and well characterized PS1 particles were then examined by electron microscopy, followed by computer-aided image processing with single particle averaging techniques as described earlier (Kruip, J., Boekema, E . J., Bald, D., Boonstra, A . F., and Rogner, M . (1993) J . Biol . Chem . 268, 23353-23360) . This precise methodological approach allowed a confident localization of the PS1 subunits PsaC, -D, -E, -F, and -J; it also shows shape and size of these subunits once integrated in the PS1 complex . Subunits PsaC, -D, and -E form a ridge on the stromal site, with PsaE toward the edge of each monomer within the trimer and PsaD extending toward the trimeric center, leaving PsaC in between . PsaF (near PsaE) and PsaJ are close together on the outer edge of each monomer; their proximity is also supported by chemical cross-linking, using the zero-length cross-linker EDC . This localization of PsaF contradicts the position suggested by the published low resolution x-ray analysis and shows for the first time the existence of at least one transmembrane alpha-helix for PsaF . A topographic three-dimensional map has been drawn from this set of results showing the location of the major PS1 subunits (besides PsaA and PsaB) . These data also led to the assignment of electron density in the recent medium resolution x-ray structure for PS1 (Krauss, N., Schubert, W.-D., Klukas, O., Fromme, P., Witt, H . T., Saenger, W . (1996) Nat . Struct . Biol . 3, 965-973).

Mol Microbiol, 1997 Jul, 25(2), 399 - 409
Copper-dependent reciprocal transcriptional regulation of methane monooxygenase genes in Methylococcus capsulatus and Methylosinus trichosporium; Nielsen AK et al.; The methanotrophic bacteria Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b convert methane to methanol using the enzyme, methane monooxygenase (MMO) . These bacteria are able to express two distinct MMOs: a cytoplasmic or soluble form (sMMO) and a membrane-bound or particulate form (pMMO) . Differential expression of sMMO and pMMO is regulated by the amount of copper ions available to the cells; sMMO is expressed at low copper-biomass ratios, whereas pMMO is expressed at high copper-biomass ratios . In both methanotrophs, transcription of the sMMO gene cluster is negatively regulated by copper ions . Data suggest that transcription of the M . trichosporium OB3b sMMO gene cluster is directed from a sigma54-like and a sigma70-like promoter . The pMMO (pmo) genes of M . capsulatus (Bath) are transcribed into a polycistronic mRNA of 3.3 kb . The synthesis of this mRNA was activated by copper ions . Activation of pmo transcription by copper ions was concomitant with repression of sMMO gene transcription in both methanotrophs . This suggests that a common regulatory pathway may be involved in the transcriptional switch between sMMO and pMMO gene expression.

Mol Microbiol, 1997 Jul, 25(2), 361 - 73
The Borrelia burgdorferi circular plasmid cp26: conservation of plasmid structure and targeted inactivation of the ospC gene; Tilly K et al.; The 26 to 28kb circular plasmid of B . burgdorferi sensu lato (cp26) is ubiquitous among bacteria of this group and contains loci implicated in the mouse-tick transmission cycle . Restriction mapping and Southern hybridization indicated that the structure of cp26 is conserved among isolates from different origins and culture passage histories . The cp26 ospC gene encodes an outer surface protein whose synthesis within infected ticks increases when the ticks feed, and whose synthesis in culture increases after a temperature upshift . Previous studies of ospC coding sequences showed them to have stretches of sequence apparently derived from the ospC genes of distantly related isolates by homologous recombination after DNA transfer . We found conservation of the promoter regions of the ospC and guaA genes, which are divergently transcribed . We also demonstrated that the increase in OspC protein after a temperature upshift parallels increases in mRNA levels, as expected if regulatory regions adjoin the conserved sequences in the promoter regions . Finally, we used directed insertion to inactivate the ospC gene of a non-infectious isolate . This first example of directed gene inactivation in B . burgdorferi shows that the OspC protein is not required for stable maintenance of cp26 or growth in culture.

Mol Microbiol, 1997 Jul, 25(2), 351 - 9
Type III secretion genes identify a putative virulence locus of Chlamydia; Hsia RC et al.; Four genes of Chlamydia psittaci strain guinea pig inclusion conjunctivitis (GPIC), whose predicted products are highly homologous to structural and regulatory components of a contact-dependent or type III secretion apparatus, were isolated . Related to genes present in several animal and plant bacterial pathogens, these genes may represent a section of a previously undetected chromosomal virulence locus analogous to several recently described virulence-associated type III secretion loci . The existence of contact-dependent secretion in Chlamydia strongly suggests that these bacteria use pathogenic mechanisms that are similar to those of other intracellular bacterial pathogens . Unlike other intracellular bacteria, however, chlamydiae are metabolically inactive extracellularly and only become capable of global protein synthesis several hours after infection . This implies that chlamydial contact-dependent secretion is only active from within, uniquely after the bacteria have been internalized by eukaryotic cells . The possible role(s) of this pathway in chlamydial pathogenesis are discussed.

Mol Microbiol, 1997 Jul, 25(2), 219 - 28
Arginine boxes and the argR gene in Streptomyces clavuligerus: evidence for a clear regulation of the arginine pathway; Rodriguez-Garcia A et al.; The argR gene of Streptomyces clavuligerus has been located in the upstream region of argG . It encodes a protein of 160 amino acids with a deduced M(r) of 17117 for the monomer . Transformants containing the amplified argR gene showed lower activity (50%) of the biosynthetic ornithine carbamoyltransferase (OTC) activity and higher levels (380%) of the catabolic ornithine aminotransferase (OAT) activity than control strains . Amplification of an arginine (ARG) box-containing sequence results in a 2- to 2.5-fold derepression of ornithine acetyltransferase and OTC, suggesting that the repressor is titrated out . Footprinting experiments using the pure homologous arginine repressor (AhrC) of B . subtilis showed a protected 38 nt region (ARG box) in the coding strand upstream of argC . The protected region contained two tandemly repeated imperfect palindromic 18-nt ARG boxes . The repressor-operator interaction was confirmed by bandshift experiments of the DNA fragment containing the protected region . By computer analysis of the Streptomyces sequences available in the databases, a consensus ARG box has been deduced for the genus Streptomyces . This is the first example of a clear regulation of an amino acid biosynthetic pathway in Streptomyces species, challenging the belief that actinomycetes do not have a well-developed regulatory system of these pathways.

Parasite Immunol, 1997 Jul, 19(7), 291 - 300
Eosinophilic interleukin 5 (IL-5) transgenic mice: eosinophil activity and impaired clearance of Schistosoma mansoni; Dent LA et al.; Eosinophilia is a feature common to many invasive helminth infections and eosinophils are often considered to be effector cells in immunity to helminths . This study examined the possible influence of constitutive eosinophilia on the clearance of Schistosoma mansoni infections in mice . Eosinophils from interleukin-5 transgenic mice exhibit normal ultrastructure and function with regard to phagocytosis and killing of bacteria and responses to chemotactic stimuli . IL-5 transgenics and non-transgenic littermates were immunized once or four (hyperimmunization) times with irradiated cercariae of S . mansoni . Animals were challenged percutaneously with unirradiated cercariae one month after their last exposure to irradiated parasites . One month after challenge transgenic animals, whether unimmunized, vaccinated or hypervaccinated, carried significantly more liver-stage parasites than non-transgenic animals . These results suggest that although eosinophils from IL-5 transgenic mice are functional for a number of key parameters, large numbers of eosinophils and/or high levels of IL-5 may in some way impair clearance of S . mansoni . A re-evaluation of the roles of eosinophils and IL-5 in infections with this and other parasites may therefore be warranted.

J Intern Med, 1997 Jul, 242(1), 27 - 33
Hyaluronan: its nature, distribution, functions and turnover; Fraser JR et al.; Hyaluronan is a polysaccharide found in all tissues and body fluids of vertebrates as well as in some bacteria . It is a linear polymer of exceptional molecular weight, especially abundant in loose connective tissue . Hyaluronan is synthesized in the cellular plasma membrane . It exists as a pool associated with the cell surface, another bound to other matrix components, and a largely mobile pool . A number of proteins, the hyaladherins, specifically recognize the hyaluronan structure . Interactions of this kind bind hyaluronan with proteoglycans to stabilize the structure of the matrix, and with cell surfaces to modify cell behaviour . Because of the striking physicochemical properties of hyaluronan solutions, various physiological functions have been assigned to it, including lubrication, water homeostasis, filtering effects and regulation of plasma protein distribution . In animals and man, the half-life of hyaluronan in tissues ranges from less than 1 to several days . It is catabolized by receptor-mediated endocytosis and lysosomal degradation either locally or after transport by lymph to lymph nodes which degrade much of it . The remainder enters the general circulation and is removed from blood, with a half-life of 2-5 min, mainly by the endothelial cells of the liver sinuoids.

Skeletal Radiol, 1997 Jul, 26(7), 431 - 3
Sternal abscess due to Bartonella (Rochalimaea) henselae in a renal transplant patient; Bruckert F et al.; Bartonella henselae, previously called Rochalimaea henselae, is the causative agent of cat scratch disease (CSD) in immunocompetent subjects and bacillary angiomatosis in immunocompromised ones . Bone lesions are common in bacillary angiomatosis, but not in CSD . We present the case of a patient with a renal transplant treated by immunosuppressive therapy who developed a sternal abscess with a histological pattern of CSD . The CT pattern was that of a lytic bone lesion with adjacent fluid collection . The diagnosis was made on the basis of a polymerase chain reaction amplification performed on bone material . Bartonella henselae is a newly described bacteria that causes CSD in a normal host and bacillary angiomatosis in immunocompromised patients . We report a case of an osteolytic lesions of the sternum with adjacent fluid collection related to CSD, which occurred in a patient with a renal transplant.

J Clin Immunol, 1997 Jul, 17(4), 311 - 21
Specificity and function of "natural" antibodies in immunodeficient subjects: clues to B cell lineage and development; Parker W et al.; The origin of natural antibodies has long been a subject of controversy . Polyreactive natural antibodies recognize multiple ligands and are thought to arise from B1 B cells . Natural antibodies against carbohydrate antigens such as Gal alpha 1-3Gal or against blood groups A and B are thought to be "elicited" by gut bacteria, but their origin is uncertain . To explore the origin of naturally occurring anticarbohydrate antibodies, the specificity and function of the xenoreactive antibodies and isohemagglutinins were investigated in immunodeficient subjects . Subjects with defects in T cell-dependent antibody synthesis had normal levels of xenoreactive natural antibodies, most of which, like xenoreactive antibodies from normal individuals, were specific for Gal alpha 1-3Gal . On the other hand, some subjects with hyper-IgM syndrome who were able to synthesize abundant quantities of xenoreactive antibodies and polyreactive antibodies were devoid of anti-Gal alpha 1-3Gal antibodies . These results suggest that the lineages of B cells giving rise to anti-Gal alpha 1-3Gal antibodies and isohemagglutinins are distinct from B1 B cells or at least exist at a more "advanced" stage of development than those B1 B cells that give rise to polyreactive antibodies . The findings also suggest that B cells which synthesize anti-Gal alpha 1-3Gal antibodies and isohemagglutinins may be distinct from B2 B cells or exist at a more "primitive" stage of development than B2 B cells that synthesize elicited antibodies in normal individuals.

Am J Physiol, 1997 Jul, 273(1 Pt 1), G93 - 105
Signal transduction pathways via guanylin and uroguanylin in stomach and intestine; London RM et al.; Guanylin and uroguanylin are peptides that activate receptor guanylate cyclases (GCs) and elicit increased intestinal secretion . Bacteria that cause traveler's diarrhea produce heat-stable toxins (STs) that mimic this action . Investigation of the distribution and identity of receptor GCs in the gastrointestinal tract of rats revealed that receptors were localized to epithelial cells in stomach and intestine . Clusters of cells in gastric mucosa and enterocytes lining the intestine exhibited specific binding of 125I-labeled ST . Ligated loops of stomach and intestine treated with intraluminal ST had significant increases in guanosine 3',5'-cyclic monophosphate (cGMP), with duodenum exhibiting the greatest response . Expression of guanylate cyclase C (GCC) mRNA and a truncated, GCC-like mRNA was found in both stomach and intestine . Both mRNAs were isolated as cDNAs encoding the GC catalytic domain . The 0.9-kilobase (kb) cDNA is 99.8% identical to GCC, whereas the truncated, 0.75-kb GCC-like cDNA has a 159-nucleotide deletion and is 96.6% identical to GCC at the protein level . Uroguanylin and guanylin mRNAs were detected in stomach and intestine . Uroguanylin mRNA was most abundant in small intestine, whereas guanylin mRNA was highest in large intestine . Thus the stomach and intestine are targets for regulation of transport by guanylin and uroguanylin via cGMP.

Auris Nasus Larynx, 1997 Jul, 24(3), 233 - 8
Experimental otitis media induced by nonviable Moraxella catarrhalis in the guinea pig model; Sato K; Moraxella catarrhalis is a normal resident of the human nasopharyngeal flora, but it is also isolated from middle ear fluid of acute otitis media and otitis media with effusion patients . To determine whether M . catarrhalis has direct pathogenicity in the middle ear, heat-killed M . catarrhalis was inoculated into the middle ear bullae of guinea pigs, and the inflammatory response was investigated . Middle ear mucosal histopathology observed in M . catarrhalis-inoculated ears included subepithelial edema, capillary dilatation, thickening of lamina propria mucosa, inflammatory cell and erythrocyte infiltration into the lamina propria mucosa . Inflammatory cell numbers, lysozyme and myeloperoxidase concentrations in the middle ear washing suspensions of M . catarrhalis-inoculated ears were significantly higher than control ears throughout the experiment . Therefore, nonviable M . catarrhalis induced middle ear inflammation and mucoperiosteal histopathology, which might be caused by direct injury of the nonviable bacteria (e.g . lipooligosaccharide or outer membrane proteins) and metabolic products of inflammatory cells.

Acta Cytol, 1997 Jul-Aug, 41(4), 1085 - 90
Quality assurance in cytology . Rescreening of previously negative smears from high grade squamous intraepithelial lesions; Howell S et al.; OBJECTIVE: To determine the problem areas in the cytologic diagnosis of high grade squamous intraepithelial lesion (HSIL) (cervical intraepithelial neoplasia {CIN} 3) . STUDY DESIGN: Previously negative smears from cases with histologically proven CIN 3 in 1988 and 1992 were reviewed for any discrepancies between the original and reviewed diagnoses . Such features as the presence of excessive inflammation, blood or bacteria, and atypical and dysplastic cellular changes were assessed . Original and reviewed reports for both years were compared, and the false negative rate was calculated . RESULTS: In the 1992 study, small numbers of abnormal cells, abnormal cells masked by inflammation and pale-staining HSIL were the common patterns when the diagnosis of HSIL was initially overlooked . In the 1988 study, the abnormal cells were overlooked mainly because of suboptimal smears . CONCLUSION: Determining cytologic patterns that pose problems in the diagnosis of HSIL is a valuable quality assurance procedure used in our laboratory . In performing these studies, we are able to pinpoint problem areas and educate our staff so as to minimize the number of false negative smears.

Am J Physiol, 1997 Jul, 273(1 Pt 2), F1 - 8
Cellular and molecular mechanisms of renal peptide transport; Daniel H et al.; Renal epithelial cells express membrane transport proteins capable of cellular uptake of a large variety of di- and tripeptides . These transporters contribute to renal amino acid homeostasis and the efficiency of conservation of amino acid nitrogen . In addition, these transporters appear to play a role in the renal handling of xenobiotics that possess a peptide backbone . Peptide carriers specialized in transport of di- and tripeptides have been identified in bacteria, fungi, plants, and epithelial cells of mammalian intestine and kidney . They appear to represent an archaic transporter family conserved throughout evolution . As a unique feature, these peptide carriers utilize a transmembrane-electrochemical proton gradient as the driving force that enables them to transport peptides against a concentration gradient . Renal peptide transporters have been characterized in terms of mechanism of transport function and substrate specificity in a number of model systems . Within the last two years, kidney peptide transporters of a variety of species have been identified by cloning techniques . In this review we discuss the physiological importance of renal peptide carriers and the transport mechanisms at the cellular level . We also present the recent advancements in functional expression of the cloned proteins that provide first insights into their molecular architecture and mode of operation.

Gene, 1997 Jul 1, 193(1), 39 - 47
Asymmetrical progression of replication forks just after initiation on Mycoplasma capricolum chromosome revealed by two-dimensional gel electrophoresis; Miyata M et al.; Previously, we mapped the replication initiation site of the Mycoplasma capricolum chromosome into a region containing the dnaA gene {M . Miyata et al., 1993a . Nucleic Acids Res . 21, 4816-4823} . In this study, various regions including this functional domain were analyzed by two complementary two-dimensional (2D) gel electrophoretic methods . Sizes of nascent strands in a 10.7-kb and a 5.6-kb region were examined by a neutral/alkaline (N/A) method . The shortest nascent strand was detected in an 875-bp region composed of the 3' end of the dnaA gene and its downstream non-coding sequence . The shortest nascent strand detected became longer in an asymmetrical manner as position of the probe became further from the putative initiation site in both directions . The intermediate forms of eight regions restricted at different sites were examined by a neutral/neutral (N/N) method . Bubble arcs were observed in four regions including the 875-bp region . The region containing the 875-bp region at about its center showed an asymmetrical arc, although that containing the 875-bp region at its end showed a symmetrical arc . These results show that the replication forks develop in the 875-bp region and proceed bidirectionally in an asymmetrical manner around the initiation site . The results of N/A analysis of the 5.6-kb region showed a shift of intensity in the nascent strand signal, which suggests an upshift of fork progression velocity.

J Appl Microbiol, 1997 Jul, 83(1), 36 - 42
Effects of physico-chemical factors influencing tyramine production by Carnobacterium divergens; Masson F et al.; Tyramine production by a strain of Carnobacterium divergens was tested in relation to different conditions of pH, temperature, glucose, oxygen availability, potassium nitrate and sodium chloride content, using a combination of a Doehlert and Plackett-Burman experimental design . A second degree polynomial model was chosen to describe tyramine production which was quantified by high performance liquid chromatography . Maximal tyramine production occurred during the stationary phase in acidic conditions obtained by low initial pH (< 5) or addition of glucose (0.6%) to the medium . Production was slower at 5 degrees C than at 23 degrees C and 10% sodium chloride inhibited this production . However, the formation of tyramine was not affected by the presence of potassium nitrate or oxygen availability.

Microbiology, 1997 Jul, 143 ( Pt 7), 2167 - 77
Divergence and conservation of the partitioning and global regulation functions in the central control region of the IncP plasmids RK2 and R751; Macartney DP et al.; The central control region (Ctl) of IncP plasmids is associated with two phenotypes: the coordinate expression of replication and transfer genes; and the ability to increase the segregational stability of a low-copy number test plasmid . This region of the IncP beta plasmid R751 shows significant sequence divergence from the IncP alpha plasmid RK2 sequence, and two genes, korF and korG, present in the IncP alpha region are missing in the IncP beta Ctl . In other respects the organization of the Ctl is basically the same . Although the two key global regulatory genes korA and korB are highly conserved, studies on their ability to repress transcription from a variety of IncP alpha and IncP beta plasmid promoters suggest differences in operator recognition by KorA and synergy with other repressors . The products of kfrA, upf54.8 and upf54.4 genes are conserved; KfrA shows least conservation and, while retaining the ability to act as a transcriptional repressor, appears to have completely different DNA-binding specificity . The genes required for the plasmid segregational stabilization (partitioning) phenotype--incC, korB and the korB operator OB3--are conserved and contribute to a more efficient plasmid stabilization than the IncP alpha equivalents . This may indicate that the Ctl plays an especially important role in partitioning of IncP beta plasmids, since they lack the second stability region (parlmrs) found in IncP alpha plasmids.

Anat Embryol (Berl), 1997 Jul, 196(1), 1 - 11
Platelets and inflammation; Klinger MH; Besides their functions in the haemostatic process and in thrombus formation after an endothelial injury, blood platelets also take part in the processes of inflammation and tissue repair that follows . For this purpose, they closely collaborate with all types of leukocytes . Activated platelets secret chemotactic substances, they facilitate the binding of leukocytes to the endothelium and their subsequent extravasation, and they may influence the inflammatory responses of leukocytes in both stimulating and inhibiting ways . However, platelets themselves also contain an array of potent proinflammatory substances, and therefore they are regarded as mediator and effector cells in inflammation . Their capability to interact with bacteria, parasites, and other foreign materials is possibly a phylogenetic vestige and may explain the existence of IgE-dependent killing mechanisms of platelets . On the other hand, the connection between IgE and platelets, besides the platelet-induced eosinophil infiltration, offers a functional basis for the involvement of platelets in allergic processes, particular in the skin and the airways.

J Dairy Sci, 1997 Jul, 80(7), 1381 - 8
Zinc oxide and amino acids as sources of dietary zinc for calves: effects on uptake and immunity; Kincaid RL et al.; Calf starter diets were formulated to contain 60 ppm of Zn, 150 or 300 ppm of Zn in the form of Zn-Met and Zn-Lys, or 300 ppm of Zn in the form of ZnO to compare relative bioavailability and effects on immunity . Holstein heifer calves were weaned at wk 5 and fed experimental starter diets from wk 6 to 12 . Feed intake, body weight, Zn concentrations in liver and serum fractions, and mineral concentrations in serum were measured to determine the effects of treatment . In addition, peripheral blood lymphocyte blastogenesis, interleukin-2 production, cytotoxic activity, and the ability of blood neutrophils to phagocytose and kill bacteria were assessed at wk 0, 2, 4, and 6 of the trial . Feed intakes and body weight gains were similar among calves . Concentrations of Zn in serum were elevated in calves fed 300 ppm of Zn as Zn-Met and Zn-Lys but not in calves fed ZnO . Concentrations of Zn in liver were significantly elevated by 300 ppm of Zn in the form of Zn-Met and Zn-Lys (360 micrograms/g) but not by the other Zn treatments or by the control (245 micrograms/g) . No treatment had an effect on the concentrations of Lys and Met in serum; however, concentrations of Lys did decrease in serum as the age of the calves increased . There was no significant treatment effect on mitogen-induced lymphocyte blastogenesis, interleukin-2 production, lymphocyte cytotoxicity, or phagocytic and intracellular killing ability of blood neutrophils . These data indicated greater absorption and retention of Zn when administered in the form of Zn-Met and Zn-Lys than that when ZnO was administered to young calves . However, there was no advantage to the immune function of extra dietary Zn.

Photochem Photobiol, 1997 Jul, 66(1), 97 - 104
Triplet energy transfer between the primary donor and carotenoids in Rhodobacter sphaeroides R-26.1 reaction centers incorporated with spheroidene analogs having different extents of pi-electron conjugation; Farhoosh R et al.; Three carotenoids, spheroidene, 3,4-dihydrospheroidene and 3,4,5,6-tetrahydrospheroidene, having 8, 9 and 10 conjugated carbon-carbon double bonds, respectively, were incorporated into Rhodobacter (Rb.) sphaeroides R-26.1 reaction centers . The extents of binding were found to be 95 +/- 5% for spheroidene, 65 +/- 5% for 3,4-dihydrospheroidene and 60 +/- 10% for 3,4,5,6-tetrahydrospheroidene . The dynamics of the triplet states of the primary donor and carotenoid were measured at room temperature by flash absorption spectroscopy . The carotenoid, spheroidene, was observed to quench the primary donor triplet state . The triplet state of spheroidene that was formed subsequently decayed to the ground state with a lifetime of 7.0 +/- 0.5 microseconds . The primary donor triplet lifetime in the Rb . sphaeroides R-26.1 reaction centers lacking carotenoids was 60 +/- 5 microseconds . Quenching of the primary donor triplet state by the carotenoid was not observed in the Rb . sphaeroides R-26.1 reaction centers containing 3,4-dihydrospheroidene nor in the R-26.1 reaction centers containing 3,4,5,6-tetrahydrospheroidene . Triplet-state electron paramagnetic resonance was also carried out on the samples . The experiments revealed carotenoid triple-state signals in the Rb . sphaeroides R-26.1 reaction centers incorporated with spheroidene, indicating that the primary donor triplet is quenched by the carotenoid . No carotenoid signals were observed from Rb . sphaeroides R-26.1 reaction centers incorporating 3,4-dihydrospheroidene nor in reaction centers incorporating 3,4,5,6-tetrahydrospheroidene . Circular dichroism, steady-state absorbance band shifts accompanying the primary photochemistry in the reaction center and singlet energy transfer from the carotenoid to the primary donor confirm that the carotenoids are bound in the reaction centers and interacting with the primary donor . These studies provide a systematic approach to exploring the effects of carotenoid structure and excited-state energy on triplet transfer between the primary donor and carotenoids in reaction centers from photosynthetic bacteria.

Curr Opin Rheumatol, 1997 Jul, 9(4), 317 - 20
Septic bone and joint infections; Hedstrom SA; During the past few years, new information on the pathogenesis of skeletal and joint infections and the host-parasite interaction has been presented . Although this is basic research, several recent case reports have shown that experimental laboratory findings are often relevant for the practical treatment of patients . Bacteria long known as causative agents in orthopedic infections, as well as new and rare ones, challenge our skills in diagnosis and encourage new thinking in host-parasite interaction and epidemiology . Another expanding problem is increased resistance against antibiotics, which is now being observed in pneumococci causing joint infections.

Curr Opin Rheumatol, 1997 Jul, 9(4), 302 - 7
Pathogenic role of gut inflammation in the spondyloarthropathies; Gaston JS; Some of today's most exciting immunologic research relevant to the pathogenesis of spondyloarthropathies concerns the relationship between the host immune system in the gut and in the intestinal bacteria . Through transgenic and gene knockout studies in rodents and the development of appropriate experimental models, it has become clear that this relationship depends on a fine balance in the production of different cytokines . Surprisingly, HLA-B27, acting at the level of T-cell recognition, disturbs this balance in ways that promote the development of arthritis . Exploring the mechanism of this disturbance and the role of genes other than B27 promises to illuminate the connection between gut inflammation and spondyloarthropathy--which has been carefully documented by clinicians over the past 30 years.

Ann Plast Surg, 1997 Jul, 39(1), 9 - 19
An outcome analysis of 100 women after explantation of silicone gel breast implants; Peters W et al.; A prospective outcome analysis was conducted on 100 consecutive women who requested explantation of their silicone gel breast implants from January 6, 1992 (the moratorium), through 1995 . Eighteen patients were referred by rheumatologists with a diagnosis of autoimmune or rheumatic disease . Six had autoimmune disease (systemic lupus, 2 patients; rheumatoid arthritis, 2 patients; multiple sclerosis, 1 patient; and Raynaud's disease, 1 patient) . Twelve had rheumatic disease (fibromyalgia, 10 patients; inflammatory arthritis, 2 patients) . All of these 18 patients had developed symptoms of their disease after they had received implants . All 100 patients were extensively evaluated pre- and postoperatively by interviews, clinical assessment, and by assay of the following laboratory tests: rheumatoid factor, ESR, ANA, and anti-Ro/SSA, -La/SSP, -Sm, -RNP, -double-stranded deoxyribonucleic acid, -Scl-70, -centromere, and -cardiolipin . Patients were also evaluated by a questionnaire that was sent at a mean time of 2.7 years postexplantation (range, 1-5 years), which had a 75% response rate . Reasons for implants were augmentation, 75%; lifting, 11%; reconstruction, 12%; and congenital aplasia, 2% . The mean age at first implant was 28.9 years (range, 13-55 years) and at explantation was 41.5 years (range, 25-65 years) . The mean duration of implantation was 12.0 years (range, 1-27 years) . Thirty-six percent of the patients had undergone at least one closed capsulotomy and 54% at least one open capsulotomy . The mean reasons for explantation were suspected silicone-related health problems, 76%; suspected rupture, 59%; breast firmness, 36%; breast pain, 36%; and musculoskeletal pain, 23% . Before explantation 75% of the questionnaire respondees had lost some sensitivity in their nipples following their breast augmentation . In 36% of those 75 patients, that loss was almost complete . Loss of sensitivity was related to capsular contracture and to pain (p < 0.05) . Following explantation there was significant improvement in nipple sensitivity in 38% of breasts in the 75 respondees . A total of 186 implants were removed . Fifty-seven percent had failed by rupturing or leaking . Only 3.2% demonstrated extravasation extracapsularly . Twenty-five percent of the capsules were calcified, demonstrating visible plaques of calcification on their inner surface . Forty-two percent were colonized by bacteria . The prevalence of class III-IV capsular contracture was 61% and it was related to implant location, duration in situ, and capsular calcification (p < 0.05), but not to capsular colonization or implant integrity (p > 0.05) . Only 43 of the 100 patients elected to have saline implants inserted . Of the others, 56% felt that the shell of the saline implant could be associated with medical problems . The others felt that breast size was of minor importance to them at this time . There were few complications from the explantation procedure . Two "masses" were discovered-one was an occult carcinoma, the other a galactocele . There was one wound infection, which responded to antibiotics . Three patients developed decreased sensitivity and 3 developed increased breast pain . From the patient questionnaires, in those women who did not have saline implants inserted, 15% felt that their breast appearance was improved after explantation, 36% were "pleased," 33% were disappointed, and 13% felt "mutilated" . In women who did have saline implants inserted, 18% felt that their breast appearance was now improved, 60% were "pleased," and 14% were disappointed, mainly because of wrinkling . At a mean time of 2.7 years (range, 1-5 years) after explantation, 45% of the 75 questionnaire respondees felt that their implants had caused permanent health problems and 56% felt that they had not been given adequate informed consent by their original surgeon (particularly regarding implant rupture and a possible relationship to medical disease) . (ABSTRACT TRUNCATED)

Nat Struct Biol, 1997 Jul, 4(7), 548 - 52
Protein alchemy: changing beta-sheet into alpha-helix; Dalal S et al.; For most proteins the amino acid sequence determines the tertiary structure . The relative importance of the individual amino acids in specifying the fold, however, remains unclear . To highlight this, Creamer and Rose put forth the 'Paracelsus challenge': Design a protein with 50% sequence identity to a protein with a different fold . We have met this challenge by designing a sequence which retains 50% identity to a predominantly beta-sheet protein, but which now adopts a four helix bundle conformation and possesses the attributes of a native protein . Our results emphasize that a subset of the amino acid sequence is sufficient to specify a fold, and have implications both for structure prediction and design.

FEMS Microbiol Lett, 1997 Jul 1, 152(1), 51 - 6
Functional interactions between homologous conditional killer systems of plasmid and chromosomal origin; Santos-Sierra S et al.; parD and chpA are homologous conditional killer systems of plasmid and chromosomal origin, respectively, encoding a killer protein (Kid and ChpAK) and an antidote (Kis and ChpAI) . Here it is shown that these systems can functionally interact . A multicopy chpA recombinant partially complements two mutations in the antidote of the parD system . These mutations affect either autoregulation or neutralization of the killer component . Following in vitro mutagenesis with hydroxylamine, chpA mutants that improve this complementation were isolated . Sequence analysis shows that these mutants are clustered in the 5' end of the chpAI gene and structure predictions suggest that they affect a putative loop in the secondary structure of the ChpAI antidote . It is proposed that this region is part of a protein-protein interface required for the functional interaction between the antidote and the killer components in the two homologous systems.

Int J Syst Bacteriol, 1997 Jul, 47(3), 693 - 7
rRNA sequences and evolutionary relationships among toxic and nontoxic cyanobacteria of the genus Microcystis; Neilan BA et al.; A primary-structure analysis of the 16S rRNA gene was performed with 10 strains representing five described and one unidentified species of the genus Microcystis . The phylogenies determined illustrate the evolutionary affiliations among Microcystis strains, other cyanobacteria, and related plastids and bacteria . A cluster of 10 strains that included hepatotoxic isolates identified as Microcystis aeruginosa formed a monophyletic group . However, the genus Microcystis appeared to be polyphyletic and contained two strains that clustered with unicellular cyanobacteria belonging to the genus Synechococcus . The clustering of related Microcystis strains, including strains involved in the production of the cyclic peptide toxin microcystin, was consistent with cell morphology, gas vacuolation, and the low G + C contents of the genomes . The Microcystis lineage was also distinct from the lineage containing the unicellular genus Synechocystis and the filamentous, heterocyst-forming genus Nostoc . The secondary structure of a Microcystis 16S rRNA molecule was determined, and genus-specific sequence signatures were used to design primers that permitted identification of the potentially toxic cyanobacteria belonging to the genus Microcystis via DNA amplification.

Int J Syst Bacteriol, 1997 Jul, 47(3), 607 - 14
Tsukamurella tyrosinosolvens sp . nov; Yassin AF et al.; Chemotaxonomic and 16S ribosomal DNA sequence analyses of four bacterial isolates from blood cultures from patients with cardiac pacemaker implants and sputa of patients with chronic lung infections clearly demonstrated that these bacteria belong to the genus Tsukamurella . DNA-DNA hybridization data, as well as the physiological characteristics of the isolates, indicate that they are closely related and belong to a single species that differs from previously described members of the genus Tsukamurella . The name Tsukamurella tyrosinosolvens sp . nov . is proposed for these isolates, and the new species is represented by strain IMMIB D-1397T (= DSM 44234T) . Strain IMMIB D-1397T exhibits 53.4, 53.5, and 54.7% DNA-DNA relatedness to Tsukamurella paurometabola DSM 20162T, Tsukamurella inchonensis DSM 44067T, and Tsukamurella pulmonis DSM 44142T, respectively.

J Eukaryot Microbiol, 1997 Jul-Aug, 44(4), 284 - 92
Phagosomal proteins of Dictyostelium discoideum; Rezabek BL et al.; In recognizing food particles . Dictyostelium cell-surface molecules initiate cytoskeletal rearrangements that result in phagosome formation . After feeding D . discoideum cells latex beads, early phagosomes were isolated on sucrose step gradients . Protein analyses of these vesicles showed that they contained glycoproteins and surface-labeled species corresponding to integral plasma membrane proteins . Cytoskeletal proteins also were associated with phagosomes, including myosin II, actin and a 30 kDa-actin bundling protein . As seen by the acridine orange fluorescence of vesicles containing bacteria, phagosomes were acidified rapidly by a vacuolar H(+)-ATPase that was detected by immunoblotting . Except for the loss of cytoskeletal proteins, few other changes over time were noted in the protein profiles of phagosomes, suggesting that phagosome maturation was incomplete . The indigestibility of the beads possibly inhibited further endocytic processing, which has been observed by others . Since nascent phagosomes contained molecules of both the cytoskeleton and plasma membrane, they will be useful in studies aimed at identifying specific protein associations occurring between membrane proteins and the cytoskeleton during phagocytosis.

Dev Biol, 1997 Jul 1, 187(1), 79 - 93
Characterization of the binding of recombinant mouse sperm fertilin beta subunit to mouse eggs: evidence for adhesive activity via an egg beta1 integrin-mediated interaction; Evans JP et al.; The sperm protein fertilin (also known as PH-30) is a candidate for mediating the interactions between sperm and egg plasma membranes . Fertilin is a heterodimer . The beta subunit, which has a region with homology to the family of integrin ligands known as disintegrins, has been hypothesized to be involved in the binding of sperm to the egg surface . To investigate this hypothesis and determine what role fertilin beta plays in fertilization, we have expressed the putative extracellular domain of mouse fertilin beta in bacteria as a fusion protein with maltose-binding protein (hereafter referred to as recombinant fertilin beta-EC) and used two assays to characterize its binding to mouse eggs . Immunocytochemistry was used to examine the localization of recombinant fertilin beta-EC binding . A luminometric assay was also developed to quantify levels of binding of recombinant fertilin beta-EC to single eggs . We find that recombinant fertilin beta-EC binds to the region of the plasma membrane of the egg to which sperm bind, thus providing the first direct evidence that fertilin beta has adhesive properties . Peptides corresponding to the disintegrin domain of fertilin beta reduce its binding to eggs, suggesting that this domain is at least partially involved in the recognition of fertilin beta by binding sites on the egg . Treatment of zona pellucida-free eggs with chymotrypsin reduces the ability of the eggs to support the binding of recombinant fertilin beta-EC, implicating an egg surface protein as a binding site for recombinant fertilin beta-EC . Binding of recombinant fertilin beta-EC to eggs is also reduced in the absence of divalent cations and is supported by 2.0 mM Ca2+, Mg2+, or Mn2+ . Furthermore, eggs incubated in recombinant fertilin beta-EC prior to in vitro fertilization show reduced levels of sperm binding . Finally, we have examined the possible role of integrins on eggs as receptors for fertilin beta, since an anti-alpha6 integrin subunit monoclonal antibody, GoH3, has been shown to inhibit sperm binding (E . A . C . Almeida et al . (1995) Cell 81, 1095-1104) . We find that: (a) an increased amount of GoH3 epitope on the egg surface does not correlate with an increased ability of the eggs to bind sperm or recombinant fertilin beta-EC; (b) the GoH3 antibody has virtually no inhibitory effect on recombinant fertilin beta-EC binding; and (c) recombinant fertilin beta-EC binding is reduced in the presence of anti-beta1 integrin antibodies . These results suggest that a beta1-containing integrin participates in the binding of recombinant fertilin beta-EC to mouse eggs.

Clin Diagn Lab Immunol, 1997 Jul, 4(4), 423 - 8
Expression, characterization, and immunoreactivities of a soluble hepatitis E virus putative capsid protein species expressed in insect cells; Zhang Y et al.; The hepatitis E virus (HEV) open reading frame-2 (ORF-2) is predicted to encode a 71-kDa putative capsid protein involved in virus particle formation . When insect Spodoptera frugiperda (Sf9) cells were infected with a recombinant baculovirus containing the entire ORF-2 sequence, two types of recombinant proteins were produced; an insoluble protein of 73 kDa and a soluble protein of 62 kDa . The 62-kDa species was shown to be a proteolytic cleavage product of the 73-kDa protein . N-terminal sequence analysis of the 62-kDa protein indicated that it lacked the first 111 amino acids that are present in the full-length 73-kDa protein . A soluble 62-kDa protein was produced without the proteolytic processing by inserting the coding sequence of amino acids 112 to 660 of ORF-2 in a baculovirus expression vector and using the corresponding virus to infect Sf9 cells . The two recombinant 62-kDa proteins made by different mechanisms displayed immunoreactivities very compatible to each other . The 62-kDa proteins obtained by both proteolytic processing and reengineering demonstrated much higher sensitivities in detecting anti-HEV antibodies in human sera than the antigens made from bacteria, as measured by enzyme-linked immunosorbent assay . The data suggest that the soluble 62-kDa protein made from insect cells contains additional epitopes not present in recombinant proteins made from bacteria . Therefore, the 62-kDa protein may be useful for HEV diagnostic improvement and vaccine development . The reengineered construct allows for the consistent large-scale production of the soluble 62-kDa protein without proteolytic processing.

Nat Biotechnol, 1997 Jul, 15(7), 632 - 6
Retargeting serum immunoglobulin with bispecific diabodies; Holliger P et al.; Monospecific antibody fragments produced in bacteria lack the Fc portion of antibodies, and are therefore unable to recruit natural effector functions . We describe the use of a bispecific antibody fragment (diabody) to recruit the whole spectrum of antibody effector functions by retargeting serum immunoglobulin (Ig) . One arm of the diabody was directed against the target antigen, and the other against the serum Ig . The bispecific diabodies were able to recruit complement, induce mononuclear phagocyte respiratory burst and phagocytosis, and promote synergistic cytotoxicity towards colon carcinoma cells in conjunction with CD8+ T-cells . Further, by virtue of binding to serum Ig their half-life (beta-phase) was increased fivefold compared to a control diabody of the same molecular weight . Such bispecific diabodies may provide an attractive alternative to monoclonal antibodies for serotherapy.

Nat Biotechnol, 1997 Jul, 15(7), 629 - 31
Complement recruitment using bispecific diabodies; Kontermann RE et al.; We describe the engineering of antibody fragments produced in bacteria for recruitment of complement effector functions . From a phage display repertoire we isolated human antibody fragments directed against complement C1q, and linked these to lysozyme-specific antibody fragments, creating bispecific antibodies (diabodies) . One diabody was able to recruit C1q, resulting in efficient lysis of lysozyme-coated sheep erythrocytes, and also induced rosette-formation of erythrocytes with human monocytes and phagocytosis after phorbol ester stimulation . These diabodies may have therapeutic applications requiring the activation of complement.

Brain Pathol, 1997 Jul, 7(3), 943 - 63
Trinucleotide repeat instability: genetic features and molecular mechanisms; La Spada AR; Trinucleotide repeat expansions are an important cause of inherited neurodegenerative disease . The expanded repeats are unstable, changing in size when transmitted from parents to offspring (intergenerational instability, "meiotic instability") and often showing size variation within the tissues of an affected individual (somatic mosaicism, "mitotic instability") . Repeat instability is a clinically important phenomenon, as increasing repeat lengths correlate with an earlier age of onset and a more severe disease phenotype . The tendency of expanded trinucleotide repeats to increase in length during their transmission from parent to offspring in these diseases provides a molecular explanation for anticipation (increasing disease severity in successive affected generations) . In this review, I explore the genetic and molecular basis of trinucleotide repeat instability . Studies of patients and families with trinucleotide repeat disorders have revealed a number of factors that determine the rate and magnitude of trinucleotide repeat change . Analysis of trinucleotide repeat instability in bacteria, yeast, and mice has yielded additional insights . Despite these advances, the pathways and mechanisms underlying trinucleotide repeat instability in humans remain largely unknown . There are many reasons to suspect that this uniquely human phenomenon will significantly impact upon our understanding of development, differentiation and neurobiology.

Laryngoscope, 1997 Jul, 107(7), 923 - 5
Adenovirus and respiratory syncytial virus in chronic sinusitis using polymerase chain reaction; Ramadan HH et al.; The aim of this study is to investigate the role of adenovirus and respiratory syncytial virus (RSV) in chronic sinusitis using the polymerase chain reaction (PCR) to assay for the viruses . PCR has proved to be more sensitive and specific than viral cultures and immunoassays in the detection of viruses . Adenovirus and RSV are among the most common viruses to cause upper respiratory tract infections . Sinus mucosa biopsies from 20 patients undergoing endoscopic sinus surgery were sterilely collected . Four specimens (20%) tested positive for RSV by PCR and none tested positive for adenovirus . Only one specimen tested positive for RSV and one for adenovirus by viral culture and immunofluorescence . Bacterial cultures tested positive in 40% of the 20 specimens . PCR can be used to detect RSV in patients with chronic sinusitis and PCR is more sensitive than viral culture and immunofluorescence techniques on sinus polyps and mucosa.

Radiat Res, 1997 Jul, 148(1), 2 - 21
Can low-level 50/60 Hz electric and magnetic fields cause biological effects?
Valberg PA, Kavet R, Rafferty CN.
Some epidemiological studies have suggested that exposure to ambient, low-level 50/60 Hz electric and magnetic fields (EMFs) increases risk of disease . Whether this association has a causal basis depends in part on whether the electrical, chemical and mechanical "signals" induced within living cells by ambient EMFs are detectable in the complex milieu of voltages, currents and forces present within the living organism . Magnetic responsiveness has been found in some animals and bacteria; aquatic animals (e.g . sharks and rays) can sense weak electric fields . We outline the physics of several mechanisms by which EMFs may interact: (1) Energy transfer by acceleration of ions and charged proteins modifies cell membranes and receptor proteins; however, EMF energies are far below those typical of biomolecules in the cell . (2) Electric fields induced inside the body exert force on electric charges and electric moments; however, these forces are considerably smaller than typical biological forces . (3) The magnetic moments of ferromagnetic particles and free radical molecules interact with magnetic fields, but magnetic-moment sensory cells have not been found in humans, and modification of radical recombination rates by EMFs in a biological system is highly problematic . (4) Resonant interactions involve EMFs driving vibrational or orbital transitions in ion-biomolecule complexes; these mechanisms conflict with accepted physics, and many experimental tests have not found the predicted effects . (5) Temporal averaging or spatial summation can improve the ratio of "signal" to "noise" in any system, but this "mechanism" requires biological structures and neural processes having the necessary capabilities of EMF detection and temporal averaging that have not been found in humans . In summary, biological effects in humans due to extremely low-frequency EMFs of the order of those found in residential environments {< or = 2 microT (< or = 20 mG)} are implausible based on current understanding of physics and biology . Biological effects in humans at higher fields {> 10 microT (> 100 mG)} might reach plausibility as a result of time-averaging in combination with a magnetic-moment transduction mechanism; but even here, neither specialized EMF transduction structures nor appropriate averaging networks have been demonstrated . The bypothesis that the epidemiological associations observed between 50/60 Hz EMFs and disease reflect a causal relationship is not supported by what is known about mechanisms.

J Infect Dis, 1997 Jul, 176(1), 292 - 5
Isolation of Chlamydia pneumoniae from a carotid endarterectomy specimen; Jackson LA et al.; Chlamydia pneumoniae has been associated with atherosclerotic cardiovascular disease by both seroepidemiologic studies and direct detection of the organism in atherosclerotic plaque by electron microscopy (EM), immunocytochemistry (ICC), and polymerase chain reaction (PCR) . Despite the frequent detection of the organism in atheromatous cardiovascular specimens by these methods, only 1 cardiovascular isolate of C . pneumoniae, obtained from a coronary artery, has been previously reported . This study reports the isolation of C . pneumoniae from a prospectively obtained carotid endarterectomy specimen . The organism appears to be identical to other C . pneumoniae isolates by EM morphology, reactivity to species-specific monoclonal antibodies, and Southern hybridization analysis of chromosomal digests using C . pneumoniae-specific DNA probes . C . pneumoniae was detected by PCR or ICC (or both) in 11 (69%) of 16 other endarterectomy specimens tested by both of these methods . These results provide further evidence for an association of C . pneumoniae and atherosclerosis by confirming the presence of viable bacteria within atherosclerotic plaque.

Mol Plant Microbe Interact, 1997 Jul, 10(5), 560 - 70
Specific flavonoids promote intercellular root colonization of Arabidopsis thaliana by Azorhizobium caulinodans ORS571; Gough C et al.; The ability of Azorhizobium caulinodans ORS571 and other diazotrophic bacteria to internally colonize roots of Arabidopsis thaliana has been studied . Strains tagged with lacZ or gusA reporter genes were used, and the principal colonization sites were found to be the points of emergence of lateral roots, lateral root cracks (LRCs) . High frequencies of colonization were found; 63 to 100% of plants were colonized by ORS571, and 100% of plants were colonized by Herbaspirillum seropedicae . After LRCs were colonized, bacteria moved into intercellular spaces between the cortical and endodermal cell layers . Specific flavonoids, naringenin and daidzein, at 5 x 10(-5) M, significantly promoted colonization by ORS571 . By using a nodC and a nodD mutant of ORS571, it was shown that neither Nod factors nor NodD are involved in colonization or flavonoid stimulation of colonization . Flavonoids did not appear to be stimulating LRC colonization by their activity as nutritional factors . LRC and intercellular colonization by H . seropedicae was stimulated by naringenin and daidzein at the same concentration that stimulated colonization by ORS571.

Biomaterials, 1997 Jul, 18(13), 903 - 6
alpha-Amylase and salivary albumin adsorption onto titanium, enamel and dentin: an in vivo study; Kohavi D et al.; In vivo adsorption of salivary albumin and alpha-amylase onto titanium, enamel and dentin was analysed following their exposure to the oral cavity for 2h . Oral appliances in six adults served as a platform for carrying 4-mm discs of the three materials . Adherent proteins were eluted from the discs and the amounts of salivary albumin and alpha-amylase were measured by an enzyme-linked immunosorbent assay . While significant difference between the adsorption of albumin and alpha-amylase onto enamel as compared with dentin was observed, adsorption onto titanium was significantly lower . A sample of whole saliva was also collected from each participant . The mean total amounts of albumin and alpha-amylase in the participants' whole saliva were 0.03 and 0.54 mg ml-1, respectively . Titanium adsorbed significantly less (4.43%) of the total albumin than did enamel (14.30%) or dentin (18.80%) . No significant difference was found in the relative amounts of alpha-amylase adsorbed by the three materials . This significantly selective adsorption of proteins may enable the attachment of specific bacteria and thus alter the composition of the dental plaque and its potential pathogenicity.

Infect Immun, 1997 Jul, 65(7), 2987 - 91
Lymphocyte proliferative responses of goats vaccinated with Brucella melitensis 16M or a delta purE201 strain; Olsen SC et al.; The response to a Brucella melitensis purEK deletion mutant, delta purE201 (referred to as strain 201), was compared with the response to its parental strain, 16M, in juvenile goats . Proliferative responses to gamma-irradiated bacteria were detected earlier in strain 201-infected goats . Lymphocytes from strain 16M- or 201-infected goats proliferated in response to one-dimensional polyacrylamide gel electrophoresis-separated proteins of similar mass isolated from strain 16M or Brucella abortus RB51 . Data from this study suggest that some antigens stimulating cell-mediated responses are conserved among Brucella species, as 201- and 16M-infected goats recognized similar proteins expressed by RB51 and 16M.

Infect Immun, 1997 Jul, 65(7), 2629 - 39
Synthesis and function of Actinomyces naeslundii T14V type 1 fimbriae require the expression of additional fimbria-associated genes; Yeung MK et al.; The nucleotide sequence of the chromosomal DNA flanking the Actinomyces naeslundii (formerly A . viscosus) T14V type 1 fimbrial structural subunit gene (fimP) was determined . Six open reading frames (ORFs), in the order 5' ORF3, ORF2, ORF1,fimP, ORF4, ORF5, ORF6 3', were identified . ORF1 encoded a protein of 408 amino acid residues (Mr = 39,270) and had significant sequence homology with the A . naeslundii T14V type 1 and A . naeslundii WVU45 type 2 fimbrial structural subunits . An in-frame fusion of ORF1 to the malE gene of the expression vector, pMAL-c2, yielded a protein that was immunostained with antibodies raised against the maltose binding protein and A . naeslundii T14V whole bacteria . Digestion of the fusion protein with factor Xa released a protein (apparent molecular mass of 34 kDa) that was immunostained only with the antibody directed against A . naeslundii T14V whole bacterial cells . Integration plasmids carrying a kanamycin resistance gene (kan) that was used to substitute for ORF1 or for DNA fragments internal to the coding region of the other five ORFs were used to transform A . naeslundii T14V . Neither type 1 fimbriae nor the 65-kDa fimbrial structural subunit was detected in mutants obtained by allelic replacement of ORF1 or ORF2 . Mutants obtained by allelic replacement of ORF3 or ORF4 expressed only the 65-kDa fimbrial structural subunit . These mutants did not bind, in vitro, to proline-rich proteins that serve as the receptors for Actinomyces type 1 fimbriae . In contrast, a mutant in which the integration plasmid DNA had been inserted at a site close to the carboxyl terminus of ORF6 expressed type 1 fimbriae and had adherence properties similar to those observed in the wild-type strain . These results demonstrate the existence of additional genes near fimP that are likely to be involved in the synthesis and function of cell surface fimbriae of A . naeslundii T14V.

Mol Cell Biol, 1997 Jul, 17(7), 3649 - 62
Intra- and intermolecular cooperative binding of high-mobility-group protein I(Y) to the beta-interferon promoter; Yie J et al.; The mammalian high-mobility-group protein I(Y) {HMG I(Y)}, while not a typical transcriptional activator, is required for the expression of many eukaryotic genes . HMG I(Y) appears to recruit and stabilize complexes of transcriptional activators through protein-DNA and protein-protein interactions . The protein binds to the minor groove of DNA via three short basic repeats, preferring tracts of adenines and thymines arranged on the same face of the DNA helix . However, the mode by which these three basic repeats function together to recognize HMG I(Y) binding sites has remained unclear . Here, using deletion mutants of HMG I(Y), DNase I footprinting, methylation interference, and in vivo transcriptional assays, we have characterized the binding of HMG I(Y) to the model beta-interferon enhancer . We show that two molecules of HMG I(Y) bind to the enhancer in a highly cooperative fashion, each molecule using a distinct pair of basic repeats to recognize the tandem AT-rich regions of the binding sites . We have also characterized the function of each basic repeat, showing that only the central repeat accounts for specific DNA binding and that the presence of a second repeat bound to an adjacent AT-rich region results in intramolecular cooperativity in binding . Surprisingly, the carboxyl-terminal acidic tail of HMG I(Y) is also important for specific binding in the context of the full-length protein . Our results present a detailed examination of HMG I(Y) binding in an important biological context, which can be extended not only to HMG I(Y) binding in other systems but also to the binding mode of many other proteins containing homologous basic repeats, which have been conserved from bacteria to humans.

Gen Pharmacol, 1997 Jul, 29(1), 39 - 47
Opiate signaling regulates microglia activities in the invertebrate nervous system; Sonetti D et al.; 1 . Evidence supporting the presence in the invertebrate nervous system of a class of glial cells resembling vertebrate microglia was obtained in the freshwater snail Planorbarius corneus . These cells are easily identified by their immunopositivity to anti-pro-opiomelanocortin (POMC)-derived peptide antibodies . 2 . Invertebrate microglia, as in vertebrates, exhibit macrophage-like activity in vivo and in cell cultures . These cells respond to the trauma of ganglionic excision and their organotypic culture by leaving their location around neurons and moving to the lesion site from which they migrate in the culture dish . 3 . In vitro, these microglia undergo conformational changes and show phagocytic properties in the presence of bacteria or lipopolysaccharide . The activated cells also express tumor necrosis factor-alpha-like material and an increase in nitric oxide synthase, as shown by immunocytochemistry . 4 . The inhibitory effect of morphine on the mobility and phagocytic activity of invertebrate microglia provide additional functional evidence for a possible role of opiate-like compounds in downregulating immunoregulatory processes, as also observed in the circulating immunocytes.

Comp Biochem Physiol A Physiol, 1997 Jul, 117(3), 279 - 90
"Compensatory" organic osmolytes in high osmolarity and dehydration stresses: history and perspectives; Gilles R; As stated in the conclusion, "life is a thing of macromolecular cohesion in salty water." This brief historical overview shows that "compensatory" organic osmolytes take an essential place in this cohesion . It reviews the major steps of the study of these compounds over more than 100 years, from the early beginnings of 1885 until now, showing some of its fascinating developments and ending on the idea that the most fascinating is still to come . This study can be taken as an example of the richness of the comparative approach.

Br Dent J, 1997 Jun 28, 182(12), 455 - 9
The future of dental amalgam: a review of the literature . Part 6: Possible harmful effects of mercury from dental amalgam; Eley BM; This is the sixth article in a series of seven on the future of dental amalgam . It considers the possible toxic and allergic effects which could occur as a result of exposure to mercury from dental amalgam . The main toxic effects covered are neurotoxicity, kidney dysfunction, reduced immunocompetence, effects on the oral and intestinal bacterial flora, fetal and birth effects and effects on general health . The relevant studies in all these areas are described and extensively discussed . In addition, the possible development of hypersensitivity to mercury from amalgam is described and the production of delayed hypersensitivity contact reactions on the skin and mucous membrane, including lichenoid lesions, are considered.

J Mol Biol, 1997 Jun 27, 269(5), 892 - 901
Importance of electrostatic interactions in the rapid binding of polypeptides to GroEL; Perrett S et al.; The question of how chaperones rapidly bind non-native proteins of very different sequence and function has been examined by determining the effect of ionic strength on the refolding of barnase on GroEL, and on the thermal denaturation of barnase in the presence of GroEL and SecB . Both chaperones bind the denatured state of barnase, so lowering the T(m) value . The refolding of barnase in the presence of GroEL is multiphasic, the slowest phase corresponding to the refolding of a singly bound molecule of barnase in the complex with GroEL . The fastest phase is related to the association of barnase and GroEL . At high ratios of GroEL to barnase and low ionic strength (less than 200 mM) this fast phase corresponds to the observed rate of binding . The rate of association of barnase and GroEL was found to be highly dependent on ionic strength, and at high ionic strength (greater than 600 mM) the majority of barnase molecules escaped binding and refolded free in solution . The data are consistent with an initial, transient, ionic interaction between barnase and GroEL, before hydrophobic binding occurs, allowing diffusion-controlled association and slow dissociation of unfolded polypeptide.

Proc Natl Acad Sci U S A, 1997 Jun 24, 94(13), 6927 - 32
RAB22 and RAB163/mouse BRCA2: proteins that specifically interact with the RAD51 protein; Mizuta R et al.; The human RAD51 protein is a homologue of the bacteria RecA and yeast RAD51 proteins that are involved in homologous recombination and DNA repair . RAD51 interacts with proteins involved in recombination and also with tumor suppressor proteins p53 and breast cancer susceptibility gene 1 (BRCA1) . We have used the yeast two-hybrid system to clone murine cDNA sequences that encode two RAD51-associated molecules, RAB22 and RAB163 . RAB163 encodes the C-terminal portion of mouse BRCA2, the homologue of the second breast cancer susceptibility gene protein in humans, demonstrating an in vitro association between RAD51 and BRCA2 . RAB22 is a novel gene product that also interacts with RAD51 in vitro . To detect RAD51 interactions in vivo, we developed a transient nuclear focus assay that was used to demonstrate a complete colocalization of RAB22 with RAD51 in large nuclear foci.

Proc Natl Acad Sci U S A, 1997 Jun 24, 94(13), 6820 - 5
Exon/intron structure of aldehyde dehydrogenase genes supports the "introns-late" theory; Rzhetsky A et al.; Whether or not nuclear introns predate the divergence of bacteria and eukaryotes is the central argument between the proponents of the "introns-early" and "introns-late" theories . In this study we compared the goodness-of-fit of each theory with a probabilistic model of exon/intron evolution and multiple nonallelic genes encoding human aldehyde dehydrogenases (ALDHs) . Using a reconstructed phylogenetic tree of ALDH genes, we computed the likelihood of obtaining the present-day ALDH sequences under the assumptions of each competing theory . Although on the grounds of its own assumptions each theory accounted for the ALDH data significantly better than its rival, the introns-early model required frequent intron slippage, and the estimated slippage rates were too high to be consistent with reported correlations between the boundaries of ancient protein modules and the ends of ancient exons . Because the molecular mechanisms proposed to explain intron slippage are incapable of providing such high rates and are incompatible with the observed distribution of introns in higher eukaryotes, the ALDH data support the introns-late theory.

J Immunol Methods, 1997 Jun 23, 205(1), 43 - 54
Importance of the linker in expression of single-chain Fv antibody fragments: optimisation of peptide sequence using phage display technology; Turner DJ et al.; We have investigated the potential for enhancing the production of functional single-chain Fv antibody fragments (sFv), by altering the sequence of the linker that joins the variable domains of the molecule . To identify new functionally improved linkers we have used a phage display library containing a random sequence of six amino acids in the linker . Multiple rounds of panning on the antigen led to the selection of six different linker sequences that enhanced the binding of phage to the antigen . Five of the linkers also improved the secretion of soluble sFv by approximately five-fold . Analysis of the predominant linker sequence isolated showed that this improvement is not due to an increased affinity for the antigen, nor to alterations in the toxicity to bacteria . However the linker did affect the denaturation of the sFv in urea . It is therefore possible that the novel sequence helps in the refolding or secretion of the molecule.

J Theor Biol, 1997 Jun 21, 186(4), 441 - 7
Escape from evolutionary stasis by transposon-mediated deleterious mutations; McFadden J et al.; Evolution within a rugged fitness landscape is limited by the tendency for organisms to become trapped on local optima resulting in evolutionary stasis . It is presently unclear how founder populations escape from an adaptive peak to found a new species . Insertion sequences, transposons and other mobile DNA elements are found in all species of eukaryotes, bacteria and archaebacteria, where they have been sought and are usually considered to be genomic parasites or selfish genes . However, many transposons and other mobile repetitive DNA are remarkably species or phyla-specific, indicating that infection with transposable elements coincides with speciation events and is involved in promoting evolutionary change . We propose here a model in which transposable elements are involved in speciation events by their ability to produce irreversible deleterious mutations that promote escape from evolutionary stasis . We have constructed a genetic algorithm designed to model both spontaneous and transposon-mediated mutations in populations of asexual digital organisms . We use this model to investigate the effect of transposon-mediated mutations on the rate of evolution of digital organisms as they compete for resources within an artificial adaptive landscape . In the absence of transposon mutations the seed organisms quickly evolve to occupy the nearest adaptive peak but thereafter evolutionary stasis ensues and adjacent empty peaks are left unoccupied . In the presence of transposon mutations, evolution is again dominated by stasis but is punctuated by bursts of rapid evolution in with consecutive unoccupied adaptive peaks are filled with organisms derived from single transposition events . Rapid evolutionary events leading to founding of new biological species, may be similarly initiated by irreversible deleterious mutations induced by transposition.

J Mol Biol, 1997 Jun 20, 269(4), 505 - 13
Partitioning of plasmid R1 . The ParM protein exhibits ATPase activity and interacts with the centromere-like ParR-parC complex; Jensen RB et al.; The parA system of plasmid R1 consists of two genes, parM and parR, and a cis-acting centromere-like site parC . The ParM protein exhibits similarity with a superfamily of ATPases that includes actin, hsp70 and hexokinase . ParM was purified to near-homogeneity and assayed for in vitro ATPase activity . The wild-type ParM protein was found to posses ATPase activity . Mutant ParM derivatives that exhibited decreased in vitro ATPase activity were non-functional in vivo, indicating that the ATP turnover by ParM is essential for correct plasmid partitioning . The mutant ParM proteins exhibited trans-dominance, suggesting that ParM participates as a structural component of the partitioning apparatus . The ATPase activity of ParM was activated slightly by the presence of ParR and activated to a much greater extent when ParR was bound to the centromere-like parC region . An analysis using the yeast two-hybrid system indicated that ParM and ParR interact, and demonstrated that ParR interacts with itself . Thus our results suggest a direct interaction of ParM and ParR at the natural partition site parC, and that the ATPase activity of ParM is specifically stimulated by this interaction.

J Biol Chem, 1997 Jun 20, 272(25), 15599 - 602
A point mutation of human nucleoside diphosphate kinase A found in aggressive neuroblastoma affects protein folding; Lascu I et al.; The point mutation serine 120 to glycine in the human nucleoside diphosphate kinase A has been identified in several aggressive neuroblastomas (Chang, C . L., Zhu, X . X., Thoraval, D . H., Ungar, D., Rawwas, J., Hora, N., Strahler, J . R., Hanash, S . M . & Radany, E . (1994) Nature 370, 335-336) . We expressed in bacteria and purified wild-type and S120G mutant nucleoside diphosphate kinase A . The mutant enzyme had enzymatic and structural properties similar to the wild-type enzyme, whereas its stability to denaturation by heat and urea was markedly reduced . More importantly, upon renaturation of the urea-denatured mutant protein, a folding intermediate accumulated, having the characteristics of a molten globule . It had no tertiary structure, as shown by near UV circular dichroism, whereas the secondary structure was substantially recovered . The hydrophobic probe 8-anilino-1-naphthalene sulfonate bound to the intermediate species with an increase in fluorescence intensity and a blue shift . The hydrodynamic size was between that expected for a folded and an unfolded monomer . Finally, electrophoresis in a transverse urea gradient displayed no renaturation curve, and the protein showed the tendency to aggregate at the lowest urea concentrations . The existence of a molten globule folding intermediates resulting from an altered folding in the mutated protein might be related to the aggressiveness of neuroblastomas.

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