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 whic