Bacteria

J Periodontol, 2000 Apr, 71(4), 664 - 78
Diabetes and periodontal diseases . Committee on Research, Science and Therapy . American Academy of Periodontology; The effect of interleukin-11 on the progression of ligature-induced periodontal disease in the beagle dog; Department of Periodontology, Harvard School of Dental Medicine, Boston, MA 02115, USABACKGROUND: The rate of progression of periodontal disease is dependent on the complex regulatory interactions between bacteria and the immune modulators of the host response . The purpose of this investigation was to determine if recombinant human interleukin-11 (rhIL-11), known to downregulate several inflammatory modulators, has the ability in subcutaneous administration to reduce the rate and/or extent of periodontal attachment loss and radiographic bone loss in a ligature-induced periodontal disease beagle dog model . METHODS: Twenty 18-month-old female beagle dogs were brought to optimal periodontal health over a 2-week period . Periodontal disease was induced by placing 2.0 silk ligatures around the mandibular first molar and premolar teeth . The dogs were divided into 3 treatment groups and one control group . The 3 treatment groups received subcutaneous injections of either 15, 30, or 80 microg/kg of rhIL-11 in saline buffer twice a week . The placebo group received buffer only subcutaneously twice a week . The gingival health of each animal was measured by recording the presence or absence of gingival inflammation, plaque, and bleeding upon probing . Attachment levels and bone height were also measured . Treatment administration and clinical and radiographic evaluations were performed in a masked fashion . RESULTS: At week 8, the placebo group had 3.89 mm of attachment loss and 73.8% radiographic bone remaining . The 15 microg/kg group had 1.99 mm attachment loss and 89.5% bone remaining; the 30 microg/kg group had 0.84 mm attachment loss and 92.5% bone remaining; and the 80 microg/kg group had 1.05 mm attachment loss and 85.5% bone remaining . All 3 treatment groups lost significantly less attachment and retained significantly more bone than did the placebo group . CONCLUSIONS: The study indicates that subcutaneous injections of rhIL-11 were able to slow the progression of attachment and radiographic alveolar bone loss in a ligature-induced beagle dog model.

J Periodontol, 2000 Apr, 71(4), 521 - 32
Treatment with subantimicrobial dose doxycycline improves the efficacy of scaling and root planing in patients with adult periodontitis; Caton JG et al.; BACKGROUND: In a previous study, subantimicrobial dose doxycycline (SDD) significantly improved clinical parameters associated with periodontal health in patients with adult periodontitis (AP) when used as an adjunct to a maintenance schedule of supragingival scaling and dental prophylaxis . In this double-blind, placebo-controlled, parallel-group, multicenter study, the efficacy and safety of SDD were evaluated in conjunction with scaling and root planing (SRP) in patients with AP . METHODS: Patients (n = 190) received SRP at the baseline visit and were randomized to receive either SDD 20 mg bid or placebo bid for 9 months . Efficacy parameters included the per-patient mean changes in clinical attachment level (CAL) and probing depth (PD) from baseline, the per-patient percentages of tooth sites with attachment loss (AL) > or = 2 mm and > or = 3 mm from baseline, and the per-patient percentage of tooth sites with bleeding on probing . Prior to analysis, tooth sites were stratified by the degree of disease severity evident at baseline RESULTS: In tooth sites with mild to moderate disease and severe disease (n = 183, intent-to-treat population), improvements in CAL and PD were significantly greater with adjunctive SDD than with adjunctive placebo at 3, 6, and 9 months (all P <0.05) . In tooth sites with severe disease, the per-patient percentage of sites with AL > or = 2 mm from baseline to month 9 was significantly lower with adjunctive SDD than with adjunctive placebo (P<0.05) . Improvements in clinical outcomes occurred without detrimental shifts in the normal periodontal flora or the acquisition of doxycycline resistance or multiantibiotic resistance . SDD was well tolerated, with a low incidence of discontinuations due to adverse events . CONCLUSIONS: The adjunctive use of SDD with SRP is more effective than SRP alone and may represent a new approach in the long-term management of AP.

J Cell Sci, 2000 Jun, 113 ( Pt 11), 1913 - 21
Expression of autofluorescent proteins reveals a novel protein permeable pathway between cells in the lens core; Shestopalov VI et al.; The lens of the eye is composed of concentric layers of tightly packed fiber cells . The oldest fibers, those in the lens core, lose their nuclei and other organelles during terminal differentiation . This is thought to ensure the clarity of the lens . The anucleated core fibers are sustained by gap junction-mediated communication with metabolically active cells near the lens surface . In this study, we expressed autofluorescent proteins and microinjected fluorescent markers to probe cell-to-cell communication in different regions of the developing lens . Our data indicate that a novel cell-cell diffusion pathway becomes patent in the lens core during development . This pathway is remarkable in that it is permeable to proteins and other large molecules and is thus distinct from gap junctions . Diffusion of large molecules probably occurs through regions of membrane fusion observed between neighboring cells in the lens core . Further direct evidence for a continuous plasma membrane system was provided by the observation that exogenous membrane proteins expressed in one core fiber cell were able to diffuse laterally into the membranes of adjacent fibers . Thus, the lens core appears to represent a true syncytium within which both membrane proteins and cytoplasmic proteins freely diffuse . Significantly, the outermost edge of the core syncytium encompasses a shell of nucleated, transcriptionally-competent, fiber cells . This arrangement could facilitate the delivery of newly synthesized protein components to the aged and metabolically quiescent cells in the center of the lens.

Cytometry, 2000 Jun 1, 40(2), 126 - 34
Simultaneous flow cytometric analyses of enhanced green and yellow fluorescent proteins and cell surface antigens in doubly transduced immature hematopoietic cell populations; Stull RA et al.; BACKGROUND: Cell transduction with multiple genes offers opportunities to investigate specific gene interactions on cell function . Detection of multiple transduced genes in hematopoietic cells requires strategies to combine measurements of gene expression with phenotypic cell discriminants . We describe simultaneous flow cytometric detection of two green fluorescent protein (GFP) variants in immunophenotypically defined human hematopoietic subpopulations using only a minor physical adjustment to a standard FACSCalibur . METHODS: The accuracy and sensitivity of enhanced GFP (EGFP) and enhanced yellow fluorescent protein (EYFP) detection in mixtures of transduced and nontransduced PG13 packaging cells were evaluated by flow cytometry . Retroviral vectors encoding EGFP or EYFP were used to transduce CD34(+) hematopoietic cells derived from umbilical cord blood . The transduction efficiency into subpopulations of hematopoietic cells was measured using multivariate flow cytometry . RESULTS: A bicistronic retroviral vector containing the EGFP and puromycin N-acetyltransferase (pac) genes afforded brighter EGFP signals in transduced cells than a retroviral vector encoding a pac-EGFP fusion protein . The sensitivity of detecting EGFP and EYFP-expressing cells among a background of nonexpressing cells was 0.01% and 0.05%, respectively . EGFP or EYFP was expressed in up to 95% of CD34(+) DR(-) or CD34(+) 38(-) subpopulations in cord blood 48 h posttransduction . Simultaneous transduction with EGFP and EYFP viral supernatants (1:1 mixture) led to coexpression of both GFP variants in 15% of CD34(+) DR(-) and 20% of CD34(+) 38(-) cells . CONCLUSIONS: These results demonstrate simultaneous detection of EGFP and EYFP in immunophenotypically discriminated human hematopoietic cells . This technique will be useful to quantify transduction of multiple retroviral constructs in discriminated subpopulations .

Proc Natl Acad Sci U S A, 2000 May 9, 97(10), 5351 - 6
LexA chimeras reveal the function of Drosophila Fos as a context-dependent transcriptional activator; Szuts D et al.; The transcriptional activation potential of proteins can be assayed in chimeras containing a heterologous DNA-binding domain that mediates their recruitment to reporter genes . This approach has been widely used in yeast and in transient mammalian cell assays . Here, we applied it to assay the transactivation potential of proteins in transgenic Drosophila embryos . We found that a chimera between the DNA-binding bacterial LexA protein and the transactivation domain from yeast GAL4 behaved as a potent synthetic activator in all embryonic tissues . In contrast, a LexA chimera containing Drosophila Fos (Dfos) required an unexpected degree of context to function as a transcriptional activator . We provide evidence to suggest that this context is provided by Djun and Mad (a Drosophila Smad), and that these partner factors need to be activated by signaling from Jun N-terminal kinase and decapentaplegic, respectively . Because Dfos behaves as an autonomous transcriptional activator in more artificial assays systems, our data suggest that context-dependence of transcription factors may be more prevalent than previously thought.

Proc Natl Acad Sci U S A, 2000 May 9, 97(10), 5095 - 100
DNA polymerase active site is highly mutable: evolutionary consequences; Patel PH et al.; DNA polymerases contain active sites that are structurally superimposable and highly conserved in sequence . To assess the significance of this preservation and to determine the mutational burden that active sites can tolerate, we randomly mutated a stretch of 13 amino acids within the polymerase catalytic site (motif A) of Thermus aquaticus DNA polymerase I . After selection, by using genetic complementation, we obtained a library of approximately 8, 000 active mutant DNA polymerases, of which 350 were sequenced and analyzed . This is the largest collection of physiologically active polymerase mutants . We find that all residues of motif A, except one (Asp-610), are mutable while preserving wild-type activity . A wide variety of amino acid substitutions were obtained at sites that are evolutionarily maintained, and conservative substitutions predominate at regions that stabilize tertiary structures . Several mutants exhibit unique properties, including DNA polymerase activity higher than the wild-type enzyme or the ability to incorporate ribonucleotide analogs . Bacteria dependent on these mutated polymerases for survival are fit to replicate repetitively . The high mutability of the polymerase active site in vivo and the ability to evolve altered enzymes may be required for survival in environments that demand increased mutagenesis . The inherent substitutability of the polymerase active site must be addressed relative to the constancy of nucleotide sequence found in nature.

Alcohol Clin Exp Res, 2000 Apr, 24(4 Suppl), 55S - 58S
Alcohol enhances lipopolysaccharide-induced increases in nitric oxide production by Kupffer cells via mechanisms dependent on endotoxin; Enomoto N et al.; BACKGROUND: Ethanol causes both tolerance and sensitization of Kupffer cells . Accordingly, this study examines the effect of acute ethanol consumption on nitric oxide (NO) production from Kupffer cells with or without lipopolysaccharide (LPS) treatment . METHODS: Rats were given ethanol (4 g/kg body weight) intragastrically, and Kupffer cells were isolated 2 and 24 hr later . Some rats were treated for 4 days with 150 mg/kg/day of polymyxin B and 450 mg/kg/day of neomycin to prevent growth of intestinal bacteria, the primary source of endotoxin in the gastrointestinal tract . After addition of LPS, NO was measured by the Griess reaction . RESULTS: Two hours after ethanol administration, LPS-induced NO production by Kupffer cells was diminished by 50% but was enhanced 2-fold at 24 hr . Sterilization of the gut with antibiotics blocked this enhancement . CONCLUSIONS: Kupffer cells isolated from rats early after ethanol exhibited tolerance to LPS, whereas sensitization was observed later . It is likely that sensitization to Kupffer cell is caused by gut-derived endotoxin.

Sb Lek, 1998, 99(4), 529 - 38
{High pressure technology and examples of its use in the biological sciences}; Kamarad J et al.; A short description of basic types of high pressure apparatuses is presented in view of their application in a research in the biosciences . General tendencies in an evolution of characteristic inter-atomic bonds and their changes under high pressure are shortly reviewed . A complex behaviour of organic macromolecular compounds under pressure is demonstrated by the effects of pressure on proteins (including p--T diagram of proteins denaturation) . In consequence, an effect of pressure on the simplest micro-organisms is mentioned and relevant critical pressures of sterilization are presented . Trends in a future application of results of the pressure research in biosciences are discussed.

Toxicol Lett, 2000 May 19, 115(2), 165 - 72
Cytotoxicity and keratinocyte microsome-mediated mutagenic activation of carcinogens in cultured epidermal cells; Chun HS et al.; Four model carcinogens (aflatoxin B(1), 6-nitrochrysene, 3-amino-1-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido{4,3-b}indole (Trp-P-2)) were examined for their ability to inhibit the growth of cultured human and rat epidermal cells . To find a basis for observed differences in growth inhibition, aflatoxin B(1), Trp-P-1 and Trp-P-2 were tested for activation by microsomes isolated from these cells in a bacterial mutagenesis assay . Treated rat cultures exhibited sensitivity to Trp-P-1 and Trp-P-2 and especially aflatoxin toxicity (growth inhibition) despite their microsomes being unable to induce bacterial mutagenicity . In treated human cultures, the toxicities of Trp-P-1, Trp-P-2 and AFB(1) were stimulated by 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), consistent with their dependence on the biotransformation reactions this agent induces; however, the toxicity correlated poorly with observed bacterial mutagenicity mediated by their isolated microsomes . 6-Nitrochrysene, a known direct-acting mutagen in bacteria, was highly toxic to the rat but not to the human cells . Since toxic effects can modify carcinogenic outcomes, these findings are compatible with a complex relationship between toxicity, mutagenicity and carcinogenicity and indicate the utility of keratinocytes for clarifying this relationship.

FEMS Microbiol Lett, 2000 May 15, 186(2), 177 - 80
Isolation of RNA from mycobacteria grown under in vitro and in vivo conditions; Dietrich G et al.; Isolation of RNA from mycobacteria is very difficult to perform, and the yields are generally very low . We describe an approach to isolate RNA from mycobacterial species which combines the disruption of mycobacterial cells by a silica/ceramic matrix in a reciprocal shaker with the ease and efficiency of subsequent RNA purification on spin columns with silica gel-based membranes . This method is rapid, easy to perform and yields high amounts of pure, intact total RNA . Due to its safety, this method is applicable even to group 3 biological hazard organisms like Mycobacterium tuberculosis . By combining a method for the isolation of phagosomal bacteria from infected primary macrophages with the novel RNA isolation technique, we are able to monitor gene expression during infection even in bacteria which are rather resistant to genetic manipulation, like Mycobacterium bovis.

J Microbiol Methods, 2000 May, 40(3), 213 - 20
BASIC program for reduction of data from community-level physiological profiling using biolog microplates: rationale and critical interpretation of data; O'Connell S et al.; A BASIC program is offered that reduces data resulting from mixed-species inoculations into Biolog microplates . The procedures of the program are supported by a critical review of the literature relating to Biolog data reduction . The availability of standardized, accelerated data reduction protocols will facilitate study comparisons and allow efficient evaluation of new data reduction approaches.

J Nutr, 2000 May, 130(5S Suppl), 1424S - 31S
Therapeutic application of zinc in human immunodeficiency virus against opportunistic infections; Mocchegiani E et al.; The relevance of zinc in resistance to infections by virus, fungi and bacteria is recognized because of its pivotal role in the efficiency of the entire immune system, in particular in conferring biological activity to a thymic hormone called thymulin, which has differentiation properties on T-cell lines . In infection with human immunodeficiency virus (HIV), the zinc-bound form of thymulin (active thymulin, ZnFTS) is strongly reduced in stage IV of the disease (Centers for Disease Control and Prevention classification) with concomitant decrements in CD4(+) cell count and zincemia values . The zinc-unbound form of thymulin (inactive thymulin, FTS) is, in contrast, very high . The in vitro addition of zinc to plasma samples induces a recovery of the thymulin active form, suggesting low zinc bioavailability as the cause of impaired thymic functions with consequent CD4(+) depletion . An analysis of risk factors for the incidence of recidivism opportunistic infections shows CD4(+) depletion and zinc deficiency to have significant scores . Supplementation with zinc for 1 mo (45 mg Zn(2+)/d) associated with zidovudine (AZT) therapy in stage IV induces recovery of active zinc-bound thymulin, of zincemia, of CD4(+) cells with concomitant reduction (50%) of recidivism opportunistic infections compared with the AZT-treated group . Complete disappearance of recidivism by Candida aesophagea or Pneumocystis carinii is observed after supplementation with zinc . The relative risk factors (CD4(+) depletion and zinc-deficiency) have lower scores in the HIV-positive zinc-treated group, confirming, as such, the relevance of zinc in opportunistic infections that involve extracellular matrix . Such an assumption is indirectly confirmed with new HAART, where no opportunistic infections occur . Indeed, HIV RNA is inversely correlated with both CD4(+) and zincemia values (r = -0.73, P<0.01) in HAART-treated subjects . Lower scores for the same relative factors for the appearance of opportunistic infections are present in HAART-treated subjects compared with those treated with AZT . These findings, on the one hand, show the poor efficacy of AZT therapy compared with HAART therapy for the progression of HIV, but on the other hand, they suggest that the lack of occurrence of opportunistic infections by HAART may also result from major zinc bioavailability . This further supports the key role played by zinc against opportunistic infections in HIV with a possible independent effect by either HIV or the pathogens involved.

J Nutr, 2000 May, 130(5S Suppl), 1388S - 92S
Zinc deficiency, malnutrition and the gastrointestinal tract; Wapnir RA; Recent clinical and experimental findings have reinforced the link among zinc deficiency, malnutrition and diarrheal disease . Because there is a strong association between protein and zinc content in virtually all types of foods, insufficient protein intake may often be the cause of zinc deficiency . Compensatory mechanisms operating in monogastric species during malnutrition are less effective for the absorption of transition divalent elements such as zinc, which remain bound to ligands of dietary or endogenous origin . Both protein and zinc deficiencies are strong negative determinants for normal cellular immunity . In zinc deficiency, the organism is more susceptible to toxin-producing bacteria or enteroviral pathogens that activate guanylate and adenylate cyclases, stimulating chloride secretion, producing diarrhea and diminishing absorption of nutrients, thus exacerbating an already compromised mineral status . In addition, zinc deficiency may impair the absorption of water and electrolytes, delaying the termination of normally self-limiting gastrointestinal disease episodes . The gastrointestinal tract may be one of the first target areas where zinc insufficiency may be manifested . A prolonged low zinc intake deprives the organism of the local potential beneficial effects of zinc, including interactions with oxidative free radicals and nitric oxide metabolism . Nitric oxide is a second messenger that plays an important part in the triggering of diarrheal disease . The possible interrelationship among infection, inflammation, free radical damage and its quenching by potential scavengers, such as zinc, in the intestinal lumen or within the enterocyte should be more extensively studied.

J Neurochem, 2000 May, 74(5), 2201 - 8
Characterization of arginine decarboxylase in rat brain and liver: distinction from ornithine decarboxylase; Regunathan S et al.; We compared the properties of mammalian arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) in rat liver and brain . Mammalian ADC is thermally unstable and associated with mitochondrial membranes . ADC decarboxylates both arginine (Km = 0.75 mM) and ornithine (Km = 0.25 mM), a reaction not inhibited by the specific ODC inhibitor, difluoromethylomithine . ADC activity is inhibited by Ca2+, Co2+, and polyamines, is present in many organs being highest in aorta and lowest in testis, and is not recognized by a specific monoclonal antibody to ODC . In contrast, ODC is thermally stable, cytosolic, and mitochondrial and is expressed at low levels in most organs except testis . Although ADC and ODC are expressed in cultured rat C6 glioma cells, the patterns of expression during growth and confluence are very different . We conclude that mammalian ADC differs from ADC isoforms expressed in plants, bacteria, or Caenorhabditis elegans and is distinct from ODC . ADC serves to synthesize agmatine in proximity to mitochondria, an organelle also harboring agmatine's degradative enzyme, agmatinase, and a class of imidazoline receptor (I2) to which agmatine binds with high affinity.

Przegl Epidemiol, 1999, 53(3-4), 231 - 43
{Chlamydia pneumoniae: an etiologic of coronary heart disease?}; Podsiadly E et al.; The hypothesis put forward in 1988 that Chlamydia pneumoniae is the aetiological agent in coronary disease and myocardial infraction has aroused an interest in these bacteria . The epidemiology of Ch . pneumoniae infections and researches on the role of it in the development of coronary artery lesions are reviewed, including animal models of this infection which could provide additional on the mechanism of atherosclerosis development.

J Immunol, 2000 May 15, 164(10), 4991 - 5
Cutting edge: inflammatory responses can be triggered by TREM-1, a novel receptor expressed on neutrophils and monocytes; Bouchon A et al.; We have identified new activating receptors of the Ig superfamily expressed on human myeloid cells, called TREM (triggering receptor expressed on myeloid cells) . TREM-1 is selectively expressed on blood neutrophils and a subset of monocytes and is up-regulated by bacterial LPS . Engagement of TREM-1 triggers secretion of IL-8, monocyte chemotactic protein-1, and TNF-alpha and induces neutrophil degranulation . Intracellularly, TREM-1 induces Ca2+ mobilization and tyrosine phosphorylation of extracellular signal-related kinase 1 (ERK1), ERK2 and phospholipase C-gamma . To mediate activation, TREM-1 associates with the transmembrane adapter molecule DAP12 . Thus, TREM-1 mediates activation of neutrophil and monocytes, and may have a predominant role in inflammatory responses.

Mol Cell Probes, 2000 Apr, 14(2), 101 - 8
Improved diagnosis of porcine proliferative enteropathy caused by Lawsonia intracellularis using polymerase chain reaction-enzyme-linked oligosorbent assay (PCR-ELOSA); Zhang P et al.; Proliferative enteropathy (PE) caused by Lawsonia intracellularis is a major diarrheal disease affecting swine worldwide . Routine laboratory diagnosis of PE is done by amplification of L . intracellularis -specific DNA sequences by PCR followed by agarose gel electrophoresis and staining of PCR products with ethidium bromide . We report the development of an enzyme-linked oligosorbent assay (ELOSA) for specific identification of chromosomal L . intracellularis 328-bp PCR amplified products . The ELOSA involved determination of optical density value at 450 nm (OD(450)) after hybridization of biotin-labelled PCR products with an amine-modified internal oligonucleotide capture probe immobilized in microwell plates, and avidin-biotin-peroxidase complex . A positive ELOSA cut-off value of > or =0.375 was established using the mean OD(450)of negative control specimens plus three times the standard deviation . Using this value, the detection limit of PCR amplified L . intracellularis -specific products by ethidium bromide-stained agarose gel electrophoresis, Southern blot, and ELOSA were estimated to be 6.1 ng, between 0.8 and 3.0 ng, and 0.8 ng of DNA, respectively . Comparison of ethidium bromide-stained agarose gel analysis with ELOSA for detection of L . intracellularis -specific PCR products from 315 clinical specimens revealed 78% sensitivity, 100% specificity and 94% accuracy . The ELOSA produced a spectrophotometric signal that confirmed the authenticity of PCR products without subjective interpretation of ethidium bromide-stained PCR products after agarose gel electrophoresis .

J Appl Toxicol, 2000 May-Jun, 20(3), 165 - 74
DNA adducts produced by oils, oil fractions and polycyclic aromatic hydrocarbons in relation to repair processes and skin carcinogenesis; Ingram AJ et al.; Ten polycyclic aromatic hydrocarbons (PAHs) mainly with three or four aromatic rings were tested for their ability to induce DNA adduct formation in mouse skin . Four of these were selected to investigate adduct formation and loss over a period of 8 days . Three mineral oils were also examined for their adduct forming ability and one was selected for adduct formation and loss over a period of 8 days . In addition, fractions derived from the same oil containing 2-3- and 4-6-ring aromatic compounds were applied to mouse skin in a non-carcinogenic oil vehicle and adduct levels were observed over an 8-day period . It was found that PAHs that had no mutagenic, initiating or carcinogenic activity and those that had mutagenic activity in bacteria but no initiating activity in mouse skin failed to produce DNA adducts in mouse skin . Two of the three PAHs with initiating activity and both complete carcinogens produced clear evidence of adduct formation, the adduct levels produced by complete carcinogens being 100-1000 times greater than those produced by initiators . Examination of adduct formation and loss with the carcinogenic PAHs benzo{a}pyrene and 5-methylchrysene over an 8-day period showed a peak at 24 h and an apparent two-phase process of adduct loss . It is suggested that the first steep loss was due to DNA repair and that the more gradual subsequent loss was probably due to epidermal hyperplasia and desquamation . With the initiator 1, 4-dimethylphenanthrene (three rings) a peak of adduct formation was seen at 2 days and adduct levels were not reduced much by 8 days . This suggested that, with initiators, adduct formation and repair may be spread over a longer period than with complete carcinogens . With the whole oils, clear evidence of adduct formation was seen with both a carcinogenic non-solvent-refined oil and with a non-carcinogenic residual oil . The level of adduct formation with the residual oil, however, was much lower than with the carcinogenic oil . When adduct formation by the carcinogenic oil was examined over 8 days, the pattern of adduct formation and loss was similar to that of a tumour initiator rather than a complete carcinogen . Peak adduct levels on the diagonal of the thin-layer chromatography (TLC) plates seemed to occur at 1 and 4 days after treatment, with no clear reduction after 8 days . From examination of adducts formed by the 2-3-ring and 4-6-ring aromatic fractions, it appeared that the main adduct spots produced by the carcinogenic oil were due to the 2-3-ring aromatic components of the oil . Adduct spots near the vertical axis of the TLC plates were also seen with the 2-3-ring and 4-6-ring fractions . The relevance of these spots is uncertain, but if they truly represent adducts, the findings suggest that they are due mainly to 4-ring PAHs . The studies suggest that the activity of carcinogenic oils is largely due to substituted 3- and 4-ring polycyclic aromatic compounds and that more attention should be paid to substituted 3-ring compounds in predicting the carcinogenic potential of oils from analytical data .

Cochrane Database Syst Rev . 2000;(2):CD001176.
Pharmacotherapy for inducing and maintaining remission in pouchitis; Sandborn W et al.; OBJECTIVES: To determine the effectiveness of medical therapy (including metronidazole, bismuth carbomer enemas, oral probiotic bacteria, butyrate suppositories, and glutamine suppositories) for inducing a response or maintaining remission in pouchitis . SEARCH STRATEGY: Studies were selected using the MEDLINE data base (1966 - December 1997), abstracts from major gastrointestinal meetings and references from published articles and reviews . The Cochrane Controlled Trials Register and the Inflammatory Bowel Disease Review Group Trials Register were also searched . SELECTION CRITERIA: Four randomized controlled trials of medical therapy in adult patients with pouchitis were identified: two placebo controlled trials in active chronic pouchitis; one maintenance of remission trial comparing two active agents in chronic pouchitis; and one placebo-controlled maintenance of remission trial for chronic pouchitis . A single patient "n-of-1" trial for active chronic pouchitis was excluded . DATA COLLECTION AND ANALYSIS: Data were extracted by three independent observers based on the intention to treat principle . Each study was given a quality score based on predetermined criteria . Extracted data were converted to 2X2 tables (response versus no response and medical therapy versus placebo or medical therapy versus medical therapy) and then synthesized in to a summary statistic using the pooled odds ratio and 95% confidence intervals as described by Cochran and Mantel and Haenszel (the "odds ratio" in MetaView) . MAIN RESULTS: The odds ratios of inducing a response using oral metronidazole or bismuth carbomer foam enemas compared with placebo in active chronic pouchitis were 26.67 (95% CI 2.31-308.01) and 1.00 (95% CI 0.29-3.48), respectively . The numbers needed to treat with these therapies to prevent an additional relapse was 2 for oral metronidazole, and undefined for bismuth carbomer foam enemas (due to equal efficacy between the enema and placebo) . The odds ratio of maintaining remission in chronic pouchitis for oral probiotic bacteria (VSL-3) compared with placebo was 205.00 (95% CI 9.89-4247.71), while the number needed to treat to prevent one additional relapse was 2 . After discontinuation of suppressive medical therapy for chronic pouchitis, there was no difference in the odds ratio of maintaining symptomatic remission with glutamine suppositories compared to butyrate suppositories, 3 . 00 (95% CI 0.46-19.59) . The numbers needed to treat with glutamine suppositories to prevent an additional relapse was 4 . REVIEWER'S CONCLUSIONS: The results presented in this review must be interpreted with extreme caution given the small numbers of trials and patients evaluated for any one comparison . Metronidazole appears to be an effective therapy for active chronic pouchitis . Bismuth carbomer foam enemas may not be an effective therapy for chronic active pouchitis . Oral probiotic therapy with VSL-3 appears to be an effective therapy for maintaining remission in patients with chronic pouchitis in remission . There is no evidence of a difference in the maintenance of symptomatic remission in patients with chronic pouchitis treated with glutamine versus butyrate suppositories, and it is unknown whether glutamine and butyrate are equally effective or ineffective . Additional randomized, double-blind, placebo-controlled, dose-ranging clinical trials are needed to determine the efficacy of empiric medical therapies currently being used in patients with pouchitis.

Arch Microbiol, 2000 Feb, 173(2), 154 - 63
Unusual ultrastructural features in three strains of Cyanothece (cyanobacteria); Porta D et al.; Three unicellular cyanobacterial strains (PCC 7425, PCC 8303, PCC 9308) assigned to the genus Cyanothece Komarek 1976, which showed an unusually high content of light refractile inclusions when viewed by phase-contrast microscopy, were characterized by confocal laser scanning microscopy and transmission electron microscopy . All strains had concentric cortical thylakoids and a compact central nucleoid . Frequently, the two innermost thylakoid membranes protruded to form circular enclosures containing cytoplasm or electron-transparent granules, or both . The largest granules were partially immersed in the nucleoid region, but they remained attached to the inner cortical thylakoids by a single narrow connection . The pattern of binary cell division in strain PCC 7425 was different than that in strains PCC 8303 and PCC 9308 . In the former, all cell wall layers invaginated simultaneously, whereas in the latter the invagination of the outer membrane was delayed compared to that of the cytoplasmic membrane and the peptidoglycan layer . Thus, prior to completion of cell division, the new daughter cells of strains PCC 8303 and PCC 9308 were transiently connected by a thick septum, which was not observed in strain PCC 7425 . Nucleoid partitioning coincided with initiation of cell division in all three strains and was unlike that reported in other bacteria and in archaea, in which separation of the nucleoids precedes cell division . Based on the common morphological and ultrastructural features, the three strains of Cyanothece examined constitute a distinct cluster, which might deserve independent generic status.

Int J Oral Maxillofac Implants, 2000 Mar-Apr, 15(2), 247 - 51
Improvement of epidermal adhesion by surface modification of craniofacial abutments; Klein M et al.; Craniofacial implants may present peri-implant inflammation because there is no close adhesion of the epithelium to abutments and because of bacteria infiltrating the subcutaneous tissue through the gap . Therefore an attempt was made to improve adhesion of epithelium to abutments . In an in vitro model, adhesion of epithelial cells (HaCat cells) to nonmodified and 3 modified Branemark System abutment surfaces was quantified . It was found that more cells were adherent in sequence at silicone-coated surfaces, sandblasted surfaces, and collagen-coated (Types I and IV) surfaces than on nonmodified abutments . It was concluded that it is possible to improve epidermal adhesion to abutments through modification of abutment surfaces.

Aquat Toxicol, 2000 Apr 1, 48(4), 529 - 547
Cytochrome P450 enzymes in aquatic invertebrates: recent advances and future directions; Snyder MJ; A variety of enzymes and other proteins are produced by organisms in response to xenobiotic exposures . Cytochrome P450s (CYP) are one of the major phase I-type classes of detoxification enzymes found in terrestrial and aquatic organisms ranging from bacteria to vertebrates . These enzymes metabolize a wide variety of substrates including endogenous molecules (e.g . fatty acids, eicosenoids, steroids) and xenobiotics (e.g . hydrocarbons, pesticides, drugs) . Aquatic invertebrates, especially those in marine habitats, occupy every aspect of the environment, from above the surface (intertidal) to below the sediments . In turn, they have extremely diverse physiologies and are exposed to a vast array of potential toxicants . Aspects of aquatic invertebrate cytochrome P450 enzymes have been studied for the last 25 years . In a few phyla, P450 activities have been measured and are responsive to xenobiotic exposures . Until the last several years, little progress had occurred in the identification of P450 gene diversity in aquatic invertebrates . Molecular biology tools have greatly aided this search, and are likely to identify as much diversity for this protein superfamily as is present in higher marine and terrestrial organisms . Recent work has expanded our knowledge of the CYP superfamily, and new developments will rapidly advance the usefulness of these genes into such fields as biomarker research . Advances of the last decade are reviewed and insights are presented from related insect studies.

Neurotoxicology, 2000 Feb-Apr, 21(1-2), 165 - 73
Brain hypoplasia caused by exposure to trichlorfon and dichlorvos during development can be ascribed to DNA alkylation damage and inhibition of DNA alkyltransferase repair; Mehl A et al.; Treatment of pregnant guinea pigs with trichlorfon causes cerebellar hypoplasia in offspring . The most sensitive period for treatment is days 42-47 of gestation, which coincides with the rapid brain growth spurt and with the development of cerebellar granule cells . When rat granule cells were exposed in vitro to trichlorfon and dichlorvos for 24 hours they died, whereas trichloroethanol had no effect . When the cells were exposed to trichlorfon and dichlorvos for 3 hours, only dichlorvos was lethal indicating that the metabolite dichlorvos was more potent than trichlorfon itself . Cultured cerebellar granule cells were also found to be quite sensitive to other DNA-alkylating agents such as methylazoxymethanol and methylmethane sulphonate and to O6-benzylguanine; a potent and specific inhibitor of the DNA alkyltransferase involved in the repair of DNA alkylation damage . The organophosphorous compounds were also found to cause inhibition of the alkyltransferase and the lethal effects of the tested compounds on granule cell culture correlated well with the potency of inhibition . In a bacterial test system for monitoring alkylation effects on the DNA, dichlorvos was demonstrated to have a strong DNA alkylation effect . These results suggest that alkylation of DNA and inhibition of its repair can contribute to the brain hypoplasia observed after exposure to trichlorfon and dichlorvos during brain development.

Dev Biol Stand, 2000, 102, 149 - 55
Photochemical decontamination of red blood cell concentrates with the silicon phthalocyanine PC 4 and red light; Ben-Hur E et al.; Various approaches are being developed for virus inactivation of red blood cell concentrates (RBCC) in order to increase the safety of the blood supply . We have been studying the silicon phthalocyanine Pc 4 for this purpose, a photosensitizer activated with red light . Pc 4 targets the envelope of pathogenic viruses such as HIV . To protect RBC during the process two main approaches are used: (i) inclusion of quenchers of reactive oxygen species produced during the treatment . Tocopherol succinate was found to be most effective for this purpose; (ii) formulation of Pc 4, a lipophilic compound, in liposomes that reduce its binding to RBC but not to viruses . As a light source we used a light emitting diode array emitting at 670-680 nm . An efficient mixing device ensures homogenous light exposure during treatment of intact RBCC . Treatment of 50 ml RBCC with 5 microM Pc 4 and 18 J/cm(2) light results in the inactivation of > or = 5.5 log(10) HIV, > or = 6.3 log(10), VSV and > or = 5 log(10) of PRV and BVDV . The relative sensitivities of these viruses based on the slope of virus kill versus light dose are 1.0, 1.25, 1.5 and 1.9 for HIV, VSV, PRV and BVDV, respectively . To achieve the same level of virus inactivation in 350 ml RBCC, the light dose needed is 40 J/cm(2) . HIV actively replicating in CEM cells is as sensitive as cell-free and HIV in latently infected cells is 3-4 times more sensitive . Parasites that can be transmitted by blood transfusion (P . falciparum and T . cruzi) are even more sensitive than viruses . Following treatment, RBCC can be stored for 28 days at 4 degrees C with haemolysis below 1% . Previous studies under less favourable conditions showed that baboon RBC circulated with an acceptable 24 hr recovery and half-life . Genetic toxicological studies of Pc 4 with or without light exposure (mutagenicity in bacteria, mammalian cells in vitro and clastogenicity in vivo) were negative . We conclude that a process using Pc 4 and red light can potentially reduce the risk of transmitting pathogens in RBCC.

IUBMB Life, 1999 Jul, 48(1), 13 - 8
Macromolecular mimicry of nucleic acid and protein; Pedersen GN et al.; Although proteins and nucleic acids consist of different chemical components, proteins can mimic structures and possibly also functions of nucleic acids . Recently, structural mimicry was observed between two elongation factors in bacterial protein biosynthesis leading to the introduction of the concept of macromolecular mimicry . Macromolecular mimicry has further been proposed among initiation and release factors, thereby adding a new element to the description of protein synthesis in bacteria . Such mimicry has also been observed in other biological processes such as autoimmunity, DNA repair, and gene regulation, at both transcriptional and translational levels.

J Periodontal Res, 2000 Feb, 35(1), 3 - 16
Herpesviruses in human periodontal disease; Contreras A et al.; Recent studies have identified various herpesviruses in human periodontal disease . Epstein-Barr virus type 1 (EBV-1) infects periodontal B-lymphocytes and human cytomegalovirus (HCMV) infects periodontal monocytes/ macrophages and T-lymphocytes . EBV-1, HCMV and other herpesviruses are present more frequently in periodontitis lesions and acute necrotizing ulcerative gingivitis-lesions than in gingivitis or periodontally healthy sites . Reactivation of HCMV in periodontitis lesions tends to be associated with progressing periodontal disease . Herpesvirus-associated periodontitis lesions harbor elevated levels of periodontopathic bacteria, including Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Bacteriodes forsythus, Prevotella intermedia, Prevotella nigrescens and Treponema denticola . It may be that active periodontal herpesvirus infection impairs periodontal defenses, thereby permitting subgingival overgrowth of periodontopathic bacteria . Alteration between latent and active herpesvirus infection in the periodontium might lead to transient local immunosuppression and explain in part the episodic progressive nature of human periodontitis . Tissue tropism of herpesvirus infections might help explain the localized pattern of tissue destruction in periodontitis . Absence of herpesvirus infection or viral reactivation might explain why some individuals carry periodontopathic bacteria while still maintaining periodontal health . Further studies are warranted to delineate whether the proposed herpesvirus-periodontopathic bacteria model might account for some of the pathogenic features of human periodontal disease.

J Electron Microsc (Tokyo), 2000, 49(1), 123 - 34
The discovery of the division apparatus of plastids and mitochondria; Kuroiwa T; Mitochondria and plastids contain distinct genomes and multiply by binary division of existing organelles . Mitochondrial and plastid division can be clearly separated into two main events: division of the organelle nuclei (nucleoids), and subsequent division of the rest of the organelles, the process of organellokinesis . Organellokinesis makes use of organelle dividing apparatuses such as plastid-dividing ring (PD ring) and mitochondrion-dividing ring (MD ring) . The plastid-dividing apparatus (PD apparatus) is composed of three electron-dense rings (the outer, middle and inner), while the mitochondrion-dividing apparatus (MD apparatus) is a pair of electron-dense rings in cytoplasm and inner ring in the mitochondrial matrix . The behaviour of both the PD and MD apparatuses throughout organelle division in Cyanidioschyzon merolae has been studied in detail by electron microscopy . When cells enter mitosis, the inner PD ring forms first, followed by the outer and middle rings and finally the MD rings . The PD rings begin to contract before the MD rings . However, the MD rings start to contract at about 4 times the speed of the PD rings and catch up to the PD rings . The cross-sectional areas of both the outer PD and MD rings increase as contraction in the plane of division progress . This suggests that the outer rings of organelle dividing apparatuses (OD apparatus) provide the motive force for contraction . FtsZ protein is located on the bacterial contractile ring at the equator of dividing bacteria, and controls bacterial division . Since FtsZ contains a tubulin motif, and host eukaryotic organisms and chloroplasts evolved from bacteria, there is debate whether that tubulins found in the cytoskeleton and the inner or outer PD ring evolved from FtsZ protein during eukaryogenesis.

Med Hypotheses, 2000 Feb, 54(2), 275 - 7
Helicobacter infection and cirrhosis in hepatitis C virus carriage: is it an innocent bystander or a troublemaker?
Ponzetto A, Pellicano R, Leone N, Cutufia MA, Turrini F, Grigioni WF, D'Errico A, Mortimer P, Rizzetto M, Silengo L.
Since it has been shown that Helicobacter hepaticus causes both chronic hepatitis and hepatocellular carcinoma (HCC) in mice, it is suggested that differences in the progression of chronic hepatitis C may be due to a cofactor stemming from co-infection by bacteria, especially Helicobacter pylori, and/or other Helicobacter species . An assessment was made of the prevalence of H . pylori infection in HCV-positive cirrhotic patients . The presence of Helicobacter species (spp) . was evaluated in resected liver tissue from HCC patients . Serum anti-H . pylori IgG antibodies were determined in 70 males with a clinical and/or histological diagnosis of cirrhosis and HCV infection and in 310 age-matched male blood donors . The prevalences of H . pylori antibody were 77% (54/70) and 59% (183/310) (P 0.004) . Primers identifying 26 Helicobacter species were used to determine the presence of the genomic 16S rRNA of this genus in liver tissue resected from 25 cirrhotic HCC patients . Genomic sequences corresponding to H . pylori and H . pullorum were identified in 23 of these 25 livers . Together, these findings support the proposal that H . pylori is implicated in the pathogenesis and progression of cirrhosis, particularly in HCV-infected individuals . Involvement of Helicobacter spp . in HCC also seems highly possible .

Microb Ecol, 2000 Jan, 39(1), 49 - 55
Natural Endophytic Occurrence of Acetobacter diazotrophicus in Pineapple Plants; Tapia-Hernandez A et al.; The presence of endophytic Acetobacter diazotrophicus was tested for pineapple plants (Ananas comosus {L.} Merr.) grown in the field . Diazotrophic bacteria were isolated from the inner tissues of surface sterilized roots, stems, and leaves of pineapple plants . Phenotypic tests permitted the selection of presumptive nitrogen-fixing A . diazotrophicus isolates . Restriction fragment length polymorphisms (RFLPs) of small subunit (SSU) rDNA using total DNA digested with endonuclease SphI and with endonuclease NcoI, hybridizations of RNA with an A . diazotrophicus large subunit (LSU) rRNA specific probe, as well as patterns in denaturing protein electrophoresis (SDS-PAGE) and multilocus enzyme tests allowed the identification of A . diazotrophicus isolates . High frequencies of isolation were obtained from propagative buds that had not been nitrogen-fertilized, and lower frequencies from 3-month-old plants that had been nitrogen-fertilized . No isolates were recovered from 5- to 7-month-old nitrogen-fertilized plants . All the A . diazotrophicus isolates recovered from pineapple plants belonged to the multilocus genotype which shows the most extensive distribution among all host species previously analyzed . </hea

Microsurgery, 2000, 20(3), 116 - 20
Establishment of a parabiotic rat model by anastomosis of common carotid artery; Rikihisa N et al.; A cross circulation model was established by replacing mutual blood between two rats by anastomoses of each common carotid artery . In this model, parabiotic rats were shown to replace their mutual blood completely without any artificial materials or instruments, such as silicon tubes or pumps . The rate of blood exchange was measured by intravenous injection of Evans blue dye without the use of the radioactive materials that were so far commonly used . The partial alteration of blood circulation through microvascular surgery may reveal the mechanisms of organ tropism of bacteria, protozoa, and parasites . Microvascular surgery is available for reconstruction and analysis of organ functions as well as for clinical use in organplasty .

J Clin Microbiol, 2000 May, 38(5), 1909 - 14
Distribution and molecular characterization of Porphyromonas gingivalis carrying a new type of fimA gene; Nakagawa I et al.; Fimbriae of Porphyromonas gingivalis are filamentous appendages on the cell surface and are thought to be one of the virulence factors . The fimA gene encoding the subunit protein of fimbriae, fimbrillin (FimA), was classified into four typeable variants (types I to IV) . We previously examined the distribution of P . gingivalis in terms of fimA genotypes in periodontitis patients using a fimA type-specific PCR assay . However, some patients harbored P . gingivalis with untypeable fimA . In this study, we have cloned a new type (type V) of fimA from dental plaque samples . P . gingivalis with type V fimA was isolated from dental plaque of a periodontitis patient, and the isolate was named HNA-99 . The deduced amino acid sequences were compared with those of type I P . gingivalis ATCC 33277, type II strain HW24D1, type III strain 6/26, and type IV strain HG564, and the homologies were found to be 45, 44, 43, and 55%, respectively . Southern blot analysis showed that the clinical isolate HNA-99 possessed P . gingivalis-specific genes sod and kgp . However, in terms of serological specificities, type V FimA showed a difference from other types of FimA . In addition, type V P . gingivalis bacteria were detected in 16.4% (12 of 73) of the P . gingivalis-positive patients with periodontitis by PCR assay using specific primers . Thus, a new type of fimA gene is now established, and the fimA genotyping could be useful in determining the disease-associated genotypes of P . gingivalis involved in the development of adult periodontitis.

Surg Laparosc Endosc Percutan Tech, 2000 Apr, 10(2), 59 - 62; discussion 62-5
Is it possible to resterilize disposable laparoscopy trocars in a hospital setting?
Ulualp KM, Hamzaoglu I, Ulgen SK, Sahin DA, Saribas S, Ozturk R, Cebeci H.
Nosocomial infections associated with interventional procedures have been attributed to improper decontamination of instruments . Disinfection of solid laparoscopic instruments, such as telescopes, by 2% glutaraldehyde and ethylene oxide was shown to be effective in preventing infection transmission . However, instrument design in more complex surgical instruments may hamper the quality of disinfection . The aim of this study is to investigate the safety of hospital disinfection of disposable laparoscopic instruments with a relatively more complex design . A total of 40 laparoscopic trocars were divided into two equal groups: group 1 was contaminated with bacteria and yeast, and group 2 was contaminated with the hepatitis B virus . Each group was then divided to two equal subgroups . After disinfecting subgroup A with 2% glutaraldehyde and B with ethylene oxide, samples were obtained for bacterial cultures and for virus detection using polymerase chain reaction (PCR) . Bacterial and yeast cultures were positive in three instruments in group 1A and in two instruments in group 1B . Tests results for the hepatitis B virus were negative in group 2A, but positive in group 2B . Results of this study indicate that disinfection for multiple use of disposable laparoscopic instruments with a relatively complex structure is not effective and may result in nosocomial disease transmission by bacteria, fungi, and viruses.

Anaesthesist, 2000 Mar, 49(3), 196 - 201
{The effect of different volume expanders on neutrophil granulocyte function in vitro}; Welters ID et al.; INTRODUCTION: The influence of kolloids on the immune system is not well documented . In this study we investigated the effects of gelatine, hydroxyethylstarch (HES), human albumine, and dextrane on neutrophil function and receptor expression by flow cytometry . METHODS: Whole blood of healthy volunteers was incubated for 30 minutes with either gelatine, HES (6% and 10%), dextrane 40 and 60, or human albumin 20% . Phagocytic capacity was determined by uptake of fluorescein-isothiocyanate labeled bacteria, the conversion of dihydrorhodamine 123 into fluorescent rhodamine 123 was used for oxidative burst measurements . Expression of complement receptors CD 11b and CD35 was investigated using fluorescein-isothiocyanate labeled antibodies . RESULTS: Incubation with gelatine significantly increased expression of complement receptors and oxidative burst . Dextranes and HES had no influence on neutrophil function . Human albumin reduced the oxidative burst, whereas CD 35 expression was increased . CONCLUSION: The physiological significance of these changes in a range of 10% has to be clarified in further investigations.

Curr Pharm Des, 2000 Apr, 6(6), 665 - 80
Cytokine therapeutics for infectious diseases; Rodriguez FH et al.; Cytokines are potent molecules, which function as growth factors and orchestrate both innate and adaptive immune responses . Over the last two decades the number of molecules in this class have greatly expanded, and as the biology of these factors is better understood, several of these factors have entered the clinical arena to support or augment components of the immune response . Recently the use of cytokines/growth factors has been studied in patients without a defective immune system but either have significant infection or infection with drug resistant organisms . The use of cytokines as adjuvants in the treatment of infectious diseases is reviewed both in the context of protein and gene-based therapies.

J Biol Chem, 2000 May 5, 275(18), 13759 - 70
Human homologue of the Drosophila discs large tumor suppressor protein forms an oligomer in solution . Identification of the self-association site; Marfatia SM et al.; The human homologue of the Drosophila discs large tumor suppressor protein (hDlg), a member of the membrane-associated guanylate kinase (MAGUK) superfamily, interacts with K(+) channels, N-methyl-d-aspartate receptors, calcium ATPase, adenomatous polyposis coli, and PTEN tumor suppressor proteins, and several viral oncoproteins through its PDZ domains . MAGUKs play pivotal roles in the clustering and aggregation of receptors, ion channels, and cell adhesion molecules at the synapses . To investigate the physiological basis of hDlg interactions, we examined the self-association state of full-length hDlg as well as defined segments of hDlg expressed as recombinant proteins in bacteria and insect Sf9 cells . Gel permeation chromatography of full-length hDlg revealed that the purified protein migrates as a large particle of size >440 kDa . Similar measurements of defined domains of hDlg indicated that the anomalous mobility of hDlg originated from its amino-terminal domain . Ultrastructural analysis of hDlg by low angle rotary shadow electron microscopy revealed that the full-length hDlg protein as well as its amino-terminal domain exhibits a highly flexible irregular shape . Further evaluation of the self-association state of hDlg using sedimentation equilibrium centrifugation, matrix-assisted laser desorption/ionization mass spectrometry, and chemical cross-linking techniques confirmed that the oligomerization site of hDlg is contained within its amino-terminal domain . This unique amino-terminal domain mediates multimerization of hDlg into dimeric and tetrameric species in solution . Sedimentation velocity experiments demonstrated that the oligomerization domain exists as an elongated tetramer in solution . In vitro mutagenesis was used to demonstrate that a single cysteine residue present in the oligomerization domain of hDlg is not required for its self-association . Understanding the oligomerization status of hDlg may help to explicate the mechanism of hDlg association with multimeric K(+) channels and dimeric adenomatous polyposis coli tumor suppressor protein . Our findings, therefore, begin to rationalize the role of hDlg in the clustering of membrane channels and formation of multiprotein complexes necessary for signaling and cell proliferation pathways.

Electrophoresis, 2000 Apr, 21(6), 1054 - 70
Membrane proteins and proteomics: un amour impossible?
Santoni V, Molloy M, Rabilloud T.
Proteome analysis implies the ability to separate proteins as a first step prior to characterization . Thus, the overall performance of the analysis strongly depends on the performance of the separation tool, usually two-dimensional electrophoresis . This review shows how two-dimensional electrophoresis performs with membrane proteins from bacteria or animal or vegetable cells and tissues, the recent progress in this field, and it examines future prospects in this area.

Curr Opin Hematol, 2000 May, 7(3), 161 - 7
Expression of hematopoietic growth factor receptors on early hematopoietic precursors: detection and regulation; Greenberger JS; Since the original isolation of colony-stimulating factors from human serum, conditioned medium of murine or human cell lines, or freshly isolated human mononuclear cells, a revolutionary explosion of ideas has occurred in our understanding of molecular controls of the hematopoietic stem cell self-renewal and differentiation . With the availability of techniques of molecular cloning in the early 1 980s, the first hematopoietically activated cytokines led to molecular clones expressed in bacteria, yeast, or mammalian cellular systems . There then followed a development of techniques leading to the molecular cloning and expression of many hematopoietic growth factors and their receptors, as well as the primary, secondary, and tertiary molecules in signal transduction into activation of specific genes for differentiation or self-renewal . The clinical use of these factors in the diagnosis, treatment, and incorporation into new cell therapies for a variety of diseases is a subject of current interest.

Trends Microbiol, 2000 May, 8(5), 238 - 44
The eukaryotic-like Ser/Thr protein kinases of Mycobacterium tuberculosis; Av-Gay Y et al.; In bacteria, extracellular signals are generally transduced into cellular responses via a two-component system . However, genome sequence data have now revealed the presence of 'eukaryotic-like' protein kinases and phosphatases . Mycobacterium tuberculosis appears to be unique among bacteria in that its genome contains 11 members of a newly identified protein kinase family . These M . tuberculosis eukaryotic-like protein kinases could be key regulators of metabolic processes, including transcription, cell development and interactions with host cells.

Trends Microbiol, 2000 May, 8(5), 226 - 31
Common molecular mechanisms of symbiosis and pathogenesis; Hentschel U et al.; Traditionally, symbiotic and pathogenic interactions were considered different manifestations of the bacteria-host interaction . However, the molecular mechanisms that mediate communication between and cellular modulation of the involved partners are quite similar . With this review we aim to contribute to a reduction of the traditional gap between symbiosis and pathogenesis research.

Trends Microbiol, 2000 May, 8(5), 221 - 5
Regulation of expression of methane monooxygenases by copper ions; Murrell JC et al.; Many methanotrophs contain both a soluble and a particulate methane monooxygenase . A unique metabolic switch, mediated by copper ions, regulates the expression of these enzymes . When the copper-to-biomass ratio of the cell is low, the soluble enzyme is expressed, and when the copper-to-biomass ratio is high, the particulate enzyme is expressed . A model for the mechanism of this switch is proposed.

J Dent, 2000 Jul, 28(5), 327 - 32
Human pulp reaction to dentine bonded amalgam restorations: a histologic study; Subay RK et al.; OBJECTIVE: The aim of this study was to investigate the human pulp response to Scotchbond Multi Purpose Plus (SMPP) bonding agent in non-exposed Class V cavities . METHODS: SMPP was placed in 24 of 40 cavites according to manufacturer's instructions and the cavities were restored with amalgam . The remaining 16 cavities were capped with a calcium hydroxide formulation (Dycal) sealed with zinc-oxide eugenol, and restored with the amalgam . After extraction at 10 and 35 days, the teeth were fixed, sectioned and stained for light microscopy . RESULTS: All Dycal-capped teeth, at both 10 and 35 days, exhibited no pulp inflammation and no demonstrable bacteria . Six cases sealed with SMPP at 10 days showed no pulp inflammation or stained bacterial profiles . The remaining six teeth demonstrated mild to moderate inflammatory pulpal responses and five out of these six cases exhibited stained bacterial profiles . Nine out of 12 teeth showed no inflammatory pulp responses at 35 days, the remaining three cases exhibited mild to moderate pulp inflammation without stained bacteria . CONCLUSIONS: None of the teeth sealed with SMPP presented severe inflammatory pulpal reactions histologically . SMPP did not exhibit significant deleterious effects on the human pulp tissue during the test periods.

Curr Opin Pulm Med, 2000 May, 6(3), 240 - 5
Human immunodeficiency virus and respiratory infection; Ashley EA et al.; Pulmonary disease remains a major problem for the 33 million individuals who are thought to be infected with human immunodeficiency virus (HIV) worldwide . Respiratory infections are responsible for a large number of the 2 million deaths that occur each year in association with HIV disease . In countries where the majority of the population can access highly active antiretroviral therapy, morbidity and mortality rates have been cut by up to 80% . This has allowed the withdrawal of specific opportunistic infection prophylaxis when immune restoration is deemed to be adequate . Recommendations have been published concerning Pneumocystis carinii prophylaxis . This year has also seen further reports of drug-resistant isolates of Pneumocystis carinii . The clinical relevance of this is still debated . Tuberculosis remains a global problem . The complexity of the interactions between specific anti-HIV and anti-tuberculous treatment have been highlighted . In the developing world, the importance of immunization and prophylaxis (against bacteria and mycobacteria) have recently been further defined in a number of studies.

Trends Biochem Sci, 2000 May, 25(5), 247 - 51
AAA proteases: cellular machines for degrading membrane proteins; Langer T; AAA proteases are a conserved class of ATP-dependent proteases that mediate the degradation of membrane proteins in bacteria, mitochondria and chloroplasts . They combine proteolytic and chaperone-like activities and thus form a membrane-integrated quality-control system . Inactivation of AAA proteases causes severe defects in various organisms, including neurodegeneration in humans . Proteolysis by AAA proteases is modulated by another membrane-protein complex that is composed of prohibitins in eukaryotic cells and related proteins in bacteria.

Vaccine, 2000 Jun 1, 18(24), 2677 - 85
Further development of the Helicobacter pylori mouse vaccination model; Sutton P et al.; Immunisation against Helicobacter infection in mouse models has thus far produced neither complete protection against the bacteria, nor a complete prevention of the associated gastritis . This study aimed firstly to compare the sensitivities of the various methods used to assess H . pylori infection in the mouse model, and secondly to develop the experimental design to induce a more effective immunity, aimed at further reducing bacterial burden in the gastric tissue . Various mouse strains were prophylactically immunised with whole bacterial sonicate and cholera toxin before challenge with H . pylori-SS1 . The relative sensitivities of the urease assay, histological assessment and the colony forming assay to detect levels of H . pylori colonisation were compared . Comparisons of different antigen doses and different timecourses of immunisation were performed . The colony forming assay was found to be far more sensitive than either the urease assay or histological assessment for determining the protective efficacies of immunisation . Mice which had 10(5) H . pylori per gram of stomach by colony assay were negative by histology and urease . Lower doses of whole cell sonicate were more protective than high doses and more effective immunisation was achieved by leaving at least 3 weeks between immunisation instead of weekly immunisations . In conclusion, for assessment of H . pylori colonisation in the mouse model, the colony forming assay should be used . The experimental protocol for immunisation has been altered to produce a significant improvement in protection . However, full protection has still not yet been achieved and more work is still required.

FEBS Lett, 2000 Apr 21, 472(1), 122 - 8
Molecular characterization of an additional shrimp hyperglycemic hormone: cDNA cloning, gene organization, expression and biological assay of recombinant proteins; Gu PL et al.; The crustacean eyestalk CHH/MIH/GIH neurohormone gene family represents a unique group of neuropeptides identified mainly in crustaceans . In this study, we report the cloning and characterization of the cDNA and the gene encoding the hyperglycemic hormone (MeCHH-B) of the shrimp Metapenaeus ensis . The amino acid sequence of MeCHH-B shows 85% identity to that of MeCHH-A (formerly MeCHH-like neuropeptide) . Two separate but identical MeCHH-B genes were identified in the genome of shrimp by library screening and they are located on different CHH gene clusters . The organization of the MeCHH-B gene is identical to other members of the CHH/MIH/GIH neurohormone family . MeCHH-B is expressed at a constant level in the eyestalks of juveniles and mature females . Unlike the MeCHH-A gene, a low level of MeCHH-B transcripts can also be detected in the central nervous system . Interestingly, the expression pattern of MeCHH-B in the eyestalk of vitellogenic females is reversed to that of the MeCHH-A gene . At the middle stage of gonad maturation, a minimum level of MeCHH-B transcript was recorded and a maximum level of MeCHH-A transcript was detected . Recombinant proteins for MeCHH-A and MeCHH-B were produced by a bacterial expression system . The hemolymph glucose level of bilaterally eyestalk-ablated shrimp increased two-fold 1 h after the rCHH injection and then returned to normal after 2 h . The hyperglycemic effect of these fusion proteins is comparable to that of de-stalked shrimp injected with crude extract from a single sinus gland.

Vet Microbiol, 2000 May 11, 73(4), 261 - 7
Detection and differentiation of Leptospira spp . serovars in bovine semen by polymerase chain reaction and restriction fragment length polymorphism; Heinemann MB et al.; In view of the importance of venereal transmission of bovine leptospirosis, the objective of the present study was to apply the polymerase chain reaction (PCR) to 26 serovars of Leptospira interrogans, L . borgpetersenii, L . santarosai, L . noguchii and L . biflexa, to determine the detection threshold in semen samples and to evaluate the possibility of differentiation among serovars using 19 restriction endonucleases . The results showed that all serovars were amplified and the detection threshold in semen samples of a bull was 100 bacteria/ml . Using endonucleases we could classify the 26 serovars into eight groups . The present results show that PCR is a method of great potential for the detection of Leptospira spp . at bovine artificial insemination centers.

J Bacteriol, 2000 May, 182(10), 2973 - 7
A study of the CopF repressor of plasmid pAMbeta1 by phage display; d'Alencon E et al.; We studied DNA binding of a transcriptional repressor, CopF, displayed on a filamentous phage . Mutagenesis of a putative helix-turn-helix motif of CopF and of certain bases of the operator abolished the protein-DNA interaction, establishing the elements involved in CopF function and showing that phage display can be used to study repressor proteins.

J Bacteriol, 2000 May, 182(10), 2909 - 18
Altered stationary-phase response in a Borrelia burgdorferi rpoS mutant; Elias AF et al.; The homolog of the chromosomally encoded stationary-phase sigma factor RpoS in Borrelia burgdorferi was inactivated using gyrB(r) as a selectable marker . Two-dimensional nonequilibrium pH gradient electrophoresis of stationary-phase cell lysates identified at least 11 differences between the protein profiles of the rpoS mutant and wild-type organisms . Wild-type B . burgdorferi had a growth phase-dependent resistance to 1 N NaCl, similar to the stationary-phase response reported for other bacteria . The B . burgdorferi rpoS mutant strain was less resistant to osmotic stress in stationary phase than the isogenic rpoS wild-type organism . The results indicate that the B . burgdorferi rpoS homolog influences protein composition and participates in stationary-phase-dependent osmotic resistance . This rpoS mutant will be useful for studying regulation of gene expression in response to changing environmental conditions.

Proc Natl Acad Sci U S A, 2000 Apr 25, 97(9), 4991 - 6
Cadmium and iron transport by members of a plant metal transporter family in Arabidopsis with homology to Nramp genes; Thomine S et al.; Metal cation homeostasis is essential for plant nutrition and resistance to toxic heavy metals . Many plant metal transporters remain to be identified at the molecular level . In the present study, we have isolated AtNramp cDNAs from Arabidopsis and show that these genes complement the phenotype of a metal uptake deficient yeast strain, smf1 . AtNramps show homology to the Nramp gene family in bacteria, yeast, plants, and animals . Expression of AtNramp cDNAs increases Cd(2+) sensitivity and Cd(2+) accumulation in yeast . Furthermore, AtNramp3 and AtNramp4 complement an iron uptake mutant in yeast . This suggests possible roles in iron transport in plants and reveals heterogeneity in the functional properties of Nramp transporters . In Arabidopsis, AtNramps are expressed in both roots and aerial parts under metal replete conditions . Interestingly, AtNramp3 and AtNramp4 are induced by iron starvation . Disruption of the AtNramp3 gene leads to slightly enhanced cadmium resistance of root growth . Furthermore, overexpression of AtNramp3 results in cadmium hypersensitivity of Arabidopsis root growth and increased accumulation of Fe, on Cd(2+) treatment . Our results show that Nramp genes in plants encode metal transporters and that AtNramps transport both the metal nutrient Fe and the toxic metal cadmium.

Br J Pharmacol, 2000 Apr, 129(8), 1553 - 60
Mechanisms of suppression of inducible nitric oxide synthase (iNOS) expression in RAW 264.7 cells by andrographolide; Chiou WF et al.; Andrographolide, an active component found in leaves of Andrographis paniculata, has been reported to exhibit nitric oxide (NO) inhibitory property in endotoxin-stimulated macrophages, however, the detailed mechanisms remain unclear . In the present study we investigated the effect of andrographolide on the expression of inducible NO synthase (iNOS) mRNA, protein, and enzyme activity in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) . RAW 264.7 cells stimulated with LPS/IFN-gamma activated NO production; in this condition andrographolide (1-100 microM) inhibited NO production in a dose-dependent manner with an IC(50) value of 17.4+/-1.1 microM . Andrographolide also reduces the expression of iNOS protein level but without a significant effect on iNOS mRNA . The reduction of iNOS activity is thought to be caused by decreased expression of iNOS protein . In a protein stability assay, andrographolide moderately but significantly reduced the amount of iNOS protein as suggested by accelerating degradation . Furthermore, andrographolide also inhibited total protein de novo synthesis as demonstrated by {(35)S}-methionine incorporation . As a whole, these data suggest that andrographolide inhibits NO synthesis in RAW 264.7 cells by reducing the expression of iNOS protein and the reduction could occur through two additional mechanisms: prevention of the de novo protein synthesis and decreasing the protein stability via a post-transcriptional mechanism . It is also possible that inhibition of iNOS protein expression and NO production under immune stimulation and/or bacteria infection may explain, in part, the beneficial effects of andrographolide as an anti-inflammatory agent.

Yale J Biol Med, 1999 Mar-Jun, 72(2-3), 195 - 202
Helicobacter pylori modulation of gastric acid; Calam J; Helicobacter pylori plays major causative roles in peptic ulcer disease and gastric cancer . Elevated acid secretion in patients with duodenal ulcers (DUs) contributes to duodenal injury, and diminished acid secretion in patients with gastric cancer allows carcinogen-producing bacteria to colonize the stomach . Eradication of H . pylori normalizes acid secretion both in hyper-secreting DU patients and hypo-secreting relatives of gastric cancer patients . Therefore, we and others have asked how H . pylori causes these disparate changes in acid secretion . H . pylori gastritis more or less restricted to the gastric antrum in DU patients is associated with increased acid secretion . This is probably because gastritis increases release of the antral acid-stimulating hormone gastrin and diminished mucosal expression of the inhibitory peptide somatostatin . Bacterial products and inflammatory cytokines including TNFalpha may cause these changes in endocrine function . Gastritis involving the gastric corpus tends to diminish acid secretion, probably because bacterial products and cytokines including IL-1 inhibit parietal cells . Pharmacological inhibition of acid secretion increases corpus gastritis in H . pylori-infected subjects, so it is envisaged that gastric hypo-secretion of any cause might become self-perpetuating . H . pylori-associated mucosal atrophy will also contribute to acid hypo-secretion and is more likely in when the diet is high in salt or lacking in antioxidant vitamins . Data on gastric acid secretion in patients with esophagitis are limited but suggest that acid secretion is normal or slightly diminished . Nevertheless, H . pylori infection may be relevant to the management of esophagitis because: (i) H . pylori infection increases the pH-elevating effect of acid inhibiting drugs; (ii) proton pump inhibitors may increase the tendency of H . pylori to cause atrophic gastritis; and (iii) successful eradication of H . pylori is reported to increase the likelihood of esophagitis developing in patients who had DU disease . Points (ii) and (iii) remain controversial and more work is clearly required to elucidate the relationship between H . pylori, acid secretion, gastric mucosa atrophy and esophagitis.

Yale J Biol Med, 1999 Mar-Jun, 72(2-3), 169 - 72
Drugs, bugs, and esophageal pH profiles; Robinson M; Until relatively recently, gastroesophageal reflux disease (GERD) was thought to be a relatively trivial problem, and pharmaceutical companies initially had remarkably little interest in clinical trials for GERD . Over the last ten years, GERD therapy has become the subject of intense interest, since reflux disease is now recognized as a major market for antisecretory and prokinetic drugs . Even low-technology antacids are now known to effectively neutralize esophageal acid prevent acid reflux for up to 90 minutes . Esophageal pH profiling is known to be an excellent surrogate for clinical efficacy of GERD drugs, particularly in erosive esophagitis . Years ago, famotidine normalized esophageal mucosal exposure to pH < 4.0 only when administered in doses of 40 mg twice a day . Subsequent studies confirmed that multiple daily dosing of histamine-2 receptor antagonists (H2RAs) was mandatory for GERD treatment, with clear dose-response relationships for each agent . Proton pump inhibitors (PPIs) have each been carefully assessed in terms esophageal and gastric pH profiles . Omeprazole has a particularly flat dose response curve, making it difficult to differentiate pH or clinical effects of 20 vs . 40 mg doses . Improved rapidity of onset and/or enhanced potency is demonstrable in pH data obtained with lansoprazole, rabeprazole and pantoprazole . Such differences will translate to improved clinical efficacy, based on the meta-analyses of Richard Hunt and his group in Canada that correlate pH effects and symptom relief/healing . PPI's have dependably surpassed H2RAs and prokinetic drugs in management of the more severe grades of esophagitis . Helicobacter pylori has a peculiar relationship to GERD . There has been some concern that PPIs given to patients with H . pylori might accelerate development of severe atrophic gastritis . It is also now known that eradication of H . pylori may increase symptomatic GERD (possibly as a result of increased gastric acid secretion once the bacteria have been eliminated) . New data confirm nocturnal breakthrough of acid secretion and esophageal acid exposure in three-fourths of patients on omeprazole 20 mg twice daily . This nocturnal acidity can be controlled more effectively with a nighttime dose of an H2RA than with a third dose of omeprazole . Control of acid secretion and improved gastric and esophageal pH profiles are goals of modern GERD therapy, and the product that most cost effectively normalizes esophageal acid exposure will have a substantial advantage in the ever-growing GERD marketplace.

Prikl Biokhim Mikrobiol, 2000 Mar-Apr, 36(2), 195 - 8
{Effect of gas phase composition on formation of hydrocarbons by Desulfovibrio desulfuricans}; Bagaeva TV; Changes in the synthesis of extracellular metabolic products generated by sulfate-reducing bacteria Desulfovibrio desulfuricans grown on a lactate-containing mineral medium in the presence of H2 and CO2 at various volume ratios in the gaseous phase were studied . An increase in the amount of extracellular products synthesized by the bacteria was observed at an H2/CO2 ratio of 3:1 . High concentrations of molecular hydrogen (80-95%) in the presence of 5-20% CO2 facilitated the synthesis of hydrocarbons (alkanes) whose highest concentrations were produced at an H2/CO2 ratio of 9:1 . An increase in the initial CO2 concentration in the gaseous phase above 20% increased the amount of oxygenated compounds in the culture.

FEMS Microbiol Lett, 2000 May 1, 186(1), 133 - 8
A consensus Porphyromonas gingivalis promoter sequence; Jackson CA et al.; We have determined the transcription start points (tsp) for recently identified Porphyromonas gingivalis W50 genes, kgp, rgpA, rgpB (formerly designated prtK, prtR, and prtRII respectively), fetB and the mcmAB operon . Alignment of the DNA upstream of these tsp and those from the literature has enabled us to identify consensus sequences that may represent a P . gingivalis promoter . There is a potential -10 hexamer sequence, 5'-TATATT-3' centred on average at -10/11 nt which is repeated at -19/20 nt and an upstream consensus, 5'-CAGAT(A/G)-3' which is centred at -39/40 nt.

Biochem Biophys Res Commun, 2000 Apr 29, 271(1), 36 - 41
Rat basophilic leukemia cells express syntaxin-3 and VAMP-7 in granule membranes; Hibi T et al.; In neuronal cells, it is generally agreed that SNARE proteins underlie the release of neurotransmitter . It is controversial, however, whether they also work functionally in the degranulation of RBL-2H3 cells because the expression of SNARE proteins has not been confirmed and the degranulation is not inhibited by tetanus toxin which cleaves one of SNARE proteins, VAMP-2 . We investigated the expression and the localization of SNARE proteins including VAMP-7 which is insensitive to tetanus toxin . RT-PCR analysis showed the existence of SNARE proteins, including syntaxin-2, -3, -4, SNAP-23, VAMP-2, and VAMP-7 . Experiments using GFP-conjugated proteins revealed that VAMP-7 was localized only in granule membranes, whereas syntaxin-3 was in both the plasma and granule membranes . Upon antigen stimulation, these proteins in granule membranes moved to the cell surface due to the fusion of granules with the plasma membrane . The results suggest the involvement of SNARE proteins in the degranulation of RBL-2H3 cells .

J Biol Chem, 2000 Apr 28, 275(17), 12374 - 80
TFIIA has activator-dependent and core promoter functions in vivo; Stargell LA et al.; The physiological role of TFIIA was investigated by analyzing transcription in a yeast strain that contains a TATA-binding protein (TBP) mutant (N2-1) defective for interacting with TFIIA . In cells containing N2-1, transcription from a set of artificial his3 promoters dependent on different activators is generally reduced by a similar extent, indicating that TFIIA function is largely nonselective for activators . In addition, TATA element utilization, a core promoter function, is altered at his3 promoters dependent on weak activators . Genomic expression analysis reveals that 3% of the genes are preferentially affected by a factor of 4 or more . Chimeras of affected promoters indicate that the sensitivity to the TFIIA-TBP interaction can map either to the upstream or core promoter region . Unlike wild-type TBP or TFIIA, the N2-1 derivative does not activate transcription when artificially recruited to the promoter via a heterologous DNA binding domain, indicating that TFIIA is important for transcription even in the absence of an activation domain . Taken together, these results suggest that TFIIA plays an important role in both activator-dependent and core promoter functions in vivo . Further, they suggest that TFIIA function may not be strictly related to the recruitment of TBP to promoters but may also involve a step after TBP recruitment.

Neuroimaging Clin N Am, 2000 May, 10(2), 333 - 53
Encephalitis, cerebritis, and brain abscess: pathophysiology and imaging findings; Falcone S et al.; This article discusses the imaging findings of encephalitis, cerebritis, and brain abscess in immunocompetent patients . MR imaging is the procedure of choice in evaluating suspected intracranial infections because of its inherent contrast resolution, multiplanar capability, improved sensitivity in the posterior fossa, sensitivity to the presence of subacute, and chronic hemorrhage, and its sensitivity to the detection of meningeal disease on postcontrast images . Discussion of pathologic conditions and imaging features of encephalitis are based on the most common causative agents of each type of disease . Imaging features and pathologic conditions of cerebritis and brain abscesses also are reviewed with emphasis on pyogenic bacteria.

Nippon Rinsho, 2000 Apr, 58(4), 933 - 8
{DNA vaccines against alphaherpesvirus infections}; Maeda K; DNA vaccines offer a number of unique and favorable features that distinguish them from conventional live attenuated, killed whole, or subunit vaccines . DNA vaccine has been used to elicit humoral and cellular immune responses against viruses, bacteria, parasites and tumors in various animals . In particular, DNA vaccine can induce cytotoxic T lymphocytes, which play an important role in protection against alphaherpesvirus infections . Therefore DNA vaccine is likely to be a new and better approach to protect human and animals from alphaherpesvirus infections . In this paper, current knowledge on DNA vaccines against alphaherpesvirus infections is summarized.

Gene, 2000 Apr 18, 247(1-2), 209 - 14
Expression of the transcripts of the sigma factors and putative sigma factor regulators of Chlamydia trachomatis L2; Douglas AL et al.; The steady state levels of the transcripts of the beta' subunit of RNA polymerase gene (rpoC), three sigma factor genes (rpoD, rpoN, and rpsD), and four putative sigma factor regulatory genes (rsbW, rsbV1, rsbV2, and rsbU) of Chlamydia trachomatis L2 were examined during the chlamydial developmental cycle by reverse transcription-polymerase chain reaction (RT-PCR) analysis . rpoC and the major sigma factor rpoD transcripts were detected at all times post-infection, consistent with their expected function in the expression of housekeeping genes . Transcripts of the alternative sigma factors and the putative regulatory genes (with the exception of those of rsbV2, which were present at near constant levels at all times) were present at low or undetectable levels at the time of elementary body (EB) to reticulate body conversion early in the cycle, but were easily detected during the logarithmic growth phase of RBs, indicating that these genes are not expressed in a cascade fashion and that it is unlikely that their major role is to recognize the promoters of stage-specific genes.

Biochim Biophys Acta, 2000 Apr 21, 1457(3), 129 - 44
Salt shock-inducible photosystem I cyclic electron transfer in Synechocystis PCC6803 relies on binding of ferredoxin:NADP(+) reductase to the thylakoid membranes via its CpcD phycobilisome-linker homologous N-terminal domain; van Thor JJ et al.; Relative to ferredoxin:NADP(+) reductase (FNR) from chloroplasts, the comparable enzyme in cyanobacteria contains an additional 9 kDa domain at its amino-terminus . The domain is homologous to the phycocyanin associated linker polypeptide CpcD of the light harvesting phycobilisome antennae . The phenotypic consequences of the genetic removal of this domain from the petH gene, which encodes FNR, have been studied in Synechocystis PCC 6803 . The in frame deletion of 75 residues at the amino-terminus, rendered chloroplast length FNR enzyme with normal functionality in linear photosynthetic electron transfer . Salt shock correlated with increased abundance of petH mRNA in the wild-type and mutant alike . The truncation stopped salt stress-inducible increase of Photosystem I-dependent cyclic electron flow . Both photoacoustic determination of the storage of energy from Photosystem I specific far-red light, and the re-reduction kinetics of P700(+), suggest lack of function of the truncated FNR in the plastoquinone-cytochrome b(6)f complex reductase step of the PS I-dependent cyclic electron transfer chain . Independent gold-immunodecoration studies and analysis of FNR distribution through activity staining after native polyacrylamide gelelectrophoresis showed that association of FNR with the thylakoid membranes of Synechocystis PCC 6803 requires the presence of the extended amino-terminal domain of the enzyme . The truncated DeltapetH gene was also transformed into a NAD(P)H dehydrogenase (NDH1) deficient mutant of Synechocystis PCC 6803 (strain M55) (T . Ogawa, Proc . Natl . Acad . Sci . USA 88 (1991) 4275-4279) . Phenotypic characterisation of the double mutant supported our conclusion that both the NAD(P)H dehydrogenase complex and FNR contribute independently to the quinone cytochrome b(6)f reductase step in PS I-dependent cyclic electron transfer . The distribution, binding properties and function of FNR in the model cyanobacterium Synechocystis PCC 6803 will be discussed.

J Mol Biol, 2000 May 5, 298(3), 477 - 91
Crystal structure of cancer chemopreventive Bowman-Birk inhibitor in ternary complex with bovine trypsin at 2.3 A resolution . Structural basis of Janus-faced serine protease inhibitor specificity; Koepke J et al.; Understanding molecular recognition on a structural basis is an objective with broad academic and applied significance . In the complexes of serine proteases and their proteinaceous inhibitors, recognition is governed mainly by residue P1 in accord with primary serine protease specificity . The bifunctional soybean Bowman-Birk inhibitor (sBBI) should, therefore, interact at LysI16 (subdomain 1) with trypsin and at LeuI43 (subdomain 2) with chymotrypsin . In contrast with this prediction, a 2:1 assembly with trypsin was observed in solution and in the crystal structure of sBBI in complex with trypsin, determined at 2.3 A resolution by molecular replacement . Strikingly, P1LeuI43 of sBBI was fully embedded into the S(1) pocket of trypsin in contrast to primary specificity . The triple-stranded beta-hairpin unique to the BBI-family and the surface loops surrounding the active site of the enzyme formed a protein-protein-interface far extended beyond the primary contact region . Polar residues, hydrophilic bridges and weak hydrophobic contacts were predominant in subdomain 1, interacting specifically with trypsin . However, close hydrophobic contacts across the interface were characteristic of subdomain 2 reacting with both trypsin and chymotrypsin . A Met27Ile replacement shifted the ratio with trypsin to the predicted 1:1 ratio . Thus, the buried salt-bridge responsible for trypsin specificity was stabilised in a polar, and destabilized in a hydrophobic, environment . This may be used for adjusting the specificity of protease inhibitors for applications such as insecticides and cancer chemopreventive agents .

J Mol Biol, 2000 May 5, 298(3), 351 - 64
Architecture of the Streptomyces lividans DnaA protein-replication origin complexes; Jakimowicz D et al.; The Streptomyces oriC region contains two clusters of 19 DnaA boxes separated by a spacer (134 bp) . The Streptomyces DnaA protein consists, like all other DnaA proteins, of four domains: domain III and the carboxyterminal part (domain IV) are responsible for binding of ATP and DNA, respectively . Binding of the DnaA protein to the entire oriC region analysed by electron microscopy showed that the DnaA protein forms separate complexes at each of the clusters of DnaA boxes, but not at the spacer separating them . In vivo mutational analysis revealed that the number of DnaA boxes and the presence of the spacer linking both groups of DnaA boxes seem to be important for a functional Streptomyces origin . We suggest that the arrangement of DnaA boxes allows the DNA-bound DnaA protein to induce bending and looping of the oriC region . As it was shown by electrophoretic mobility shift assay and "one hybrid system", two domains, I and III, facilitate interactions between DnaA molecules . We postulate that domain I and domain III could be involved in cooperativity at distant and at closely spaced DnaA boxes, respectively . The long domain II extends the range over which N termini (domain I) of DNA-bound DnaA protein can form dimers . Thus, interactions between DnaA molecules may bring two clusters of DnaA boxes separated by the spacer into functional contact by loop formation . Removal of the spacer region or deletion of domains I and II resulted, respectively, in nucleoprotein complexes which are not fully developed, or huge nucleoprotein aggregates .

Exp Cell Res, 2000 May 1, 256(2), 392 - 9
Tetracycline-regulated gene expression switch in Xenopus laevis; Ridgway P et al.; Xenopus is a well-characterized model system for the investigation of biological processes at the molecular, cellular, and developmental level . The successful application of a rapid and reliable method for transgenic approaches in Xenopus has led to renewed interest in this system . We have explored the applicability of tetracycline-regulated gene expression, first described by Gossen and Bujard in 1992, to the Xenopus system . By optimizing conditions, tetracycline repressor induced expression of a luciferase reporter gene was readily and reproducibly achieved in both the Xenopus oocyte and developing embryo . This high level of expression was effectively abrogated by addition of low levels of tetracycline . The significance of this newly defined system for studies of chromatin dynamics and developmental processes is discussed .

J Food Prot, 2000 Apr, 63(4), 495 - 501
A method of assessing the efficacy of hand sanitizers: use of real soil encountered in the food service industry; Charbonneau DL et al.; In many outbreaks of foodborne illness, the food worker has been implicated as the source of the infection . To decrease the likelihood of cross-contamination, food workers must clean and disinfect their hands frequently . To ensure their effectiveness, hand disinfectants should be tested using rigorous conditions that mimic normal use . Currently, several different methods are used to assess the efficacy of hand disinfectants . However, most of these methods were designed with the health care worker in mind and do not model the specific contamination situations encountered by the food worker . To fill this void, we developed a model that uses soil from fresh meat and a means of quantifying bacteria that is encountered and transferred during food preparation activities . Results of studies using various doses of para-chloro-meta-xylenol and triclosan confirm that the method is reproducible and predictable in measuring the efficacy of sanitizers . Consistent, dose-dependent results were obtained with relatively few subjects . Other studies showed that washing hands with a mild soap and water for 20 s was more effective than applying a 70% alcohol hand sanitizer.

JPEN J Parenter Enteral Nutr, 2000 Mar-Apr, 24(2), 107 - 12
With medium-chain triglycerides, higher and faster oxygen radical production by stimulated polymorphonuclear leukocytes occurs; Kruimel JW et al.; BACKGROUND: Parenteral lipid emulsions are suspected of suppressing the immune function . However, study results are contradictory and mainly concern the conventional long-chain triglyceride emulsions . METHODS: Polymorphonuclear leukocytes were preincubated with parenteral lipid emulsions . The influence of the lipid emulsions on the production of oxygen radicals by these stimulated leukocytes was studied by measuring chemiluminescence . Three different parenteral lipid emulsions were tested: long-chain triglycerides, a physical mixture of medium- and long-chain triglycerides, and structured triglycerides . Structured triglycerides consist of triglycerides where the medium- and long-chain fatty acids are attached to the same glycerol molecule . RESULTS: Stimulated polymorphonuclear leukocytes preincubated with the physical mixture of medium- and long-chain triglycerides showed higher levels of oxygen radicals (p < .005) and faster production of oxygen radicals (p < .005) compared with polymorphonuclear leukocytes preincubated with long-chain triglycerides or structured triglycerides . Additional studies indicated that differences in results of various lipid emulsions were not caused by differences in emulsifier . The overall production of oxygen radicals was significantly lower after preincubation with the three lipid emulsions compared with controls without lipid emulsion . CONCLUSIONS: A physical mixture of medium- and long-chain triglycerides induced faster production of oxygen radicals, resulting in higher levels of oxygen radicals, compared with long-chain triglycerides or structured triglycerides . This can be detrimental in cases where oxygen radicals play either a pathogenic role or a beneficial one, such as when rapid phagocytosis and killing of bacteria is needed . The observed lower production of oxygen radicals by polymorphonuclear leukocytes in the presence of parenteral lipid emulsions may result in immunosuppression by these lipids.

Redox Rep, 1999, 4(6), 301 - 6
Sunscreens, oxidative stress and antioxidant functions in marine organisms of the Great Barrier Reef; Dunlap WC et al.; An overview of the biochemical photophysiology of tropical, reef-building corals is presented with a discussion on the biosynthetic relationship between natural UV-absorbing sunscreens and certain antioxidant functions in marine organisms . Our studies reveal that marine organisms, including 'UV-extremophilic' bacteria, are a rich source of novel antioxidants having potential for the development of commercial and biomedical applications . Novel sunscreening agents derived from tropical marine organisms of the Great Barrier Reef are in development . New marine-derived antioxidants are being isolated for testing as chemopreventatives in a variety of oxidatively degenerative diseases.

Indian J Pediatr, 1997 Mar-Apr, 64(2), 237 - 42
Recognition and management of ARI--a KAP study on private medical practitioners; Kumar D; We interviewed 113 private medical practitioners (PMPs) of all system of medicine in Ambedkar Nagar area of South Delhi to determine as to how they recognise and treat Acute Respiratory tract Infections (ARI) in children, in particular, pneumonia . Allopathic PMPs reported viruses and bacteria as causes of ARI as compared to PMPs of other system of medicine who often reported exposure to cold, change in weather and dietary habits as a cause of ARI . Sixty-eight PMPs out of 113 did not count the respiratory rate (RR) in children with ARI and among those who counted, only 19.5% PMPs could correctly tell the normal RR in children aged less than two months . In children aged 2-12 months, the percentage of PMPs responding correctly was 15.0% . Relatively greater proportion of PMPs (31.8%) could correctly tell the normal respiratory rate in children aged 1-5 years . X-ray to diagnose pneumonia was suggested by 102 (90.3%) PMPs . Majority of PMPs prescribed some form of medication including antibiotics for the treatment of cough and cold . Eighty-seven (77%) PMPs prescribed antibiotics, 53 (46.9%) antihistaminics and 49 (43.4%) prescribed allopathic cough syrups to treat cough and cold . For pneumonia, 108 (96.4%) PMPs prescribed antibiotics and 31 (27.7%) PMPs prescribed steroids among other things.

Acta Crystallogr D Biol Crystallogr, 2000 May, 56 ( Pt 5), 625 - 33
The use of wavelet transforms in low-resolution phase extension; Wilson J et al.; A method to extend low-resolution phases has been developed using histogram matching not only of the electron density itself but also of histograms obtained from the different levels of detail provided by the wavelet transform of the electron density . It is shown that the method can extend phases from 10 A to around 6-7 A on a wide range of trial structures differing in size, space group and solvent content . This level of phase extension can improve the electron-density map from little more than a molecular envelope to one in which secondary structure can often be identified.

Biochem J, 2000 May 1, 347 Pt 3, 845 - 55
A novel 50 kDa protein forms complexes with protein phosphatase 4 and is located at centrosomal microtubule organizing centres; Hastie CJ et al.; Protein phosphatase 4 (PPP4) is a protein serine/threonine phosphatase that has been implicated in microtubule organization at centrosomes . Complexes of PPP4 with high apparent molecular masses (450 and 600 kDa) were purified from mammalian skeletal muscle and testis to near homogeneity . Amino acid sequences derived from a protein component present in both complexes were utilized to identify a human cDNA . The encoded putative PPP4 regulatory subunit (termed PPP4R2), comprising 453 amino acids, had a molecular mass of 50.4 kDa . The interaction of PPP4R2 with PPP4 catalytic subunit (PPP4c) was confirmed by co-sedimentation of PPP4c with PPP4R2 expressed in bacteria and human cells . PPP4c formed a complex of 450 kDa with baculovirus expressed His(6)-tagged PPP4R2 . Immunocytological detection of PPP4R2 at centrosomes suggests that it may target PPP4c to this location . Native 450 kDa and 600 kDa PPP4 complexes are inactive, but can be activated by basic proteins, suggesting that PPP4R2 may also regulate the activity of PPP4c at centrosomal microtubule organising centres.

Infect Immun, 2000 May, 68(5), 2939 - 47
Intracellular growth of Legionella pneumophila in Dictyostelium discoideum, a system for genetic analysis of host-pathogen interactions; Solomon JM et al.; Conditions were established in which Legionella pneumophila, an intracellular bacterial pathogen, could replicate within the unicellular organism Dictyostelium discoideum . By several criteria, L . pneumophila grew by the same mechanism within D . discoideum as it does in amoebae and macrophages . Bacteria grew within membrane-bound vesicles associated with rough endoplasmic reticulum, and L . pneumophila dot/icm mutants, blocked for growth in macrophages and amoebae, also did not grow in D . discoideum . Internalized L . pneumophila avoided degradation by D . discoideum and showed evidence of reduced fusion with endocytic compartments . The ability of L . pneumophila to grow within D . discoideum depended on the growth state of the cells . D . discoideum grown as adherent monolayers was susceptible to L . pneumophila infection and to contact-dependent cytotoxicity during high-multiplicity infections, whereas D . discoideum grown in suspension was relatively resistant to cytotoxicity and did not support intracellular growth . Some known D . discoideum mutants were examined for their effect on growth of L . pneumophila . The coronin mutant and the myoA/B double myosin I mutant were more permissive than wild-type strains for intracellular growth . Growth of L . pneumophila in a G(beta) mutant was slightly reduced compared to the parent strain . This work demonstrates the usefulness of the L . pneumophila-D . discoideum system for genetic analysis of host-pathogen interactions.

Infect Immun, 2000 May, 68(5), 2888 - 98
Attenuation of and protection induced by a leucine auxotroph of Mycobacterium tuberculosis; Hondalus MK et al.; Attenuated mutants of Mycobacterium tuberculosis represent potential vaccine candidates for the prevention of tuberculosis . It is known that auxotrophs of a variety of bacteria are attenuated in vivo and yet provide protection against challenge with wild-type organisms . A leucine auxotroph of M . tuberculosis was created by allelic exchange, replacing wild-type leuD (Rv2987c), encoding isopropyl malate isomerase, with a mutant copy of the gene in which 359 bp had been deleted, creating a strain requiring exogenous leucine supplementation for growth in vitro . The frequency of reversion to prototrophy was <10(-11) . In contrast to wild-type M . tuberculosis, the DeltaleuD mutant was unable to replicate in macrophages in vitro . Its attenuation in vivo and safety as a vaccine were established by the fact that it caused no deaths in immunodeficient SCID mice . Complementation of the mutant with wild-type leuD abolished the requirement for leucine supplementation and restored the ability of the strain to grow both in macrophages and in SCID mice, thus confirming that the attenuated phenotype was due to the DeltaleuD mutation . As a test of the vaccine potential of the leucine auxotroph, immunocompetent BALB/c mice, susceptible to fatal infection with wild-type M . tuberculosis, were immunized with the DeltaleuD mutant and subsequently challenged with virulent M . tuberculosis by both the intravenous and aerosol routes . A comparison group of mice was immunized with conventional Mycobacterium bovis BCG vaccine . Whereas all unvaccinated mice succumbed to intravenous infection within 15 weeks, mice immunized with either BCG or the DeltaleuD mutant of M . tuberculosis exhibited enhanced and statistically equivalent survival curves . However, the leuD auxotroph was less effective than live BCG in reducing organ burdens and tissue pathology of mice challenged by either route . We conclude that attenuation and protection against M . tuberculosis challenge can be achieved with a leucine auxotroph and suggest that to induce optimal protection, attenuated strains of M . tuberculosis should persist long enough and be sufficiently metabolically active to synthesize relevant antigens for an extended period of time.

Antonie Van Leeuwenhoek, 2000 Feb, 77(2), 173 - 7
Density gradient separation of active and non-active cells from natural environments; Whiteley AS et al.; We present a method for the selective, physical separation of active and non-active bacterial cells from natural communities . The method exploits the reduction of tetrazolium salts to form insoluble formazan crystals intracellularly in response to the addition of different oxidisable substrates . The intracellular deposition of formazan alters the bouyant density of active cells enabling them to be separated by density gradient centrifugation . The method has been successfully applied to the fractionation and collection of large whole cell sub-populations of active and non-active cells from sea-water samples . Removal of the bands from the density gradient, followed by PCR amplification and DGGE analyses showed distinct differences in the PCR amplicon diversity associated with the active and non-active cell fractions; an indication of changes in bacterial community structure in response to the addition of oxidisable substrate . Thus, based on their in situ respiration potential, the approach enables the cytochemical enrichment and molecular characterisation of mixed bacterial populations in natural environments.

Pediatr Med Chir, 2000 Jul-Aug, 21(4), 181 - 4
{Reflux nephropathy in absence of obvious vesicoureteral reflux}; Vino L et al.; Although the majority of patients with vesicoureteric reflux presents DMSA scan alterations, parenchimal renal scars are found also in children without vesicoureteric reflux . Two clinical cases of reflux nephropathy without evidence of reflux are presented . Several explanations could be advocated to justify this picture, including haematogenous source of infection, inadequate timing and/or procedure of cystouretrography, intermittency of reflux, ascending bacteria, previous presence of reflux, and appearance of controlateral reflux during the natural history of a monolateral documented reflux . Tailored diagnostic and therapeutic strategy should discussed for each patient.

FEMS Immunol Med Microbiol, 2000 May, 28(1), 71 - 7
Membrane surface of Mycobacterium microti-infected macrophages antigenically differs from that of uninfected macrophages; Majumdar S et al.; Identification of the antigenic changes in mycobacteria-infected macrophage may be important in understanding the mechanisms responsible for the intracellular survival of the bacteria . In the present study, Mycobacterium microti-infected macrophages were utilized to investigate the possibility of differentiating the infected cells from normal cells, based on the antigenic changes occurring in the membranes . Antisera were generated against bacterial extract, heat-killed bacteria and crude preparation of M . microti-infected homologous macrophage membrane . The reactivity of these antisera, towards in vitro infected macrophages, was compared by flow cytometry . Unlike anti-bacterial extract antiserum or anti-heat-killed bacterial antiserum, anti-infected macrophage membrane antiserum reacted with infected macrophage surface . This reactivity increased with the increase in post-infection time . However, it was not observed with uninfected macrophages, PMA- or lipopolysaccharide-activated macrophages and those harboring Mycobacterium tuberculosis H37Ra, heat-killed M . microti and Leishmania donovani . Interestingly, anti-infected macrophage membrane antiserum identified a 63-kDa antigen in M . microti-infected macrophage membranes which was not present in the membranes of normal macrophages, activated macrophages and of those infected with M . tuberculosis H37Ra, heat-killed M . microti and L . donovani . Thus, membranes of M . microti-infected macrophages differ antigenically from those of the normal macrophages and infected homologous macrophage membrane antiserum provides a useful tool in studying such changes.

J Colloid Interface Sci, 2000 May 1, 225(1), 54 - 61
An Aqueous Polymer Two-Phase System as Carrier in the Spray-Drying of Biological Material; Millqvist-Fureby A et al.; This investigation describes a novel concept in the formulation of carrier systems for the spray-drying of biological materials . As carrier material a system composed of poly(vinyl pyrrolidone) (PVP) and dextran was used . This system yields an aqueous two-phase system in which each phase is enriched in one of the polymers . By varying the composition of the system, the effective structure of a "stirred" system can be varied, covering the entire range from dextran continuous to PVP continuous . This facilitates encapsulation of either of these polymers in a spray-drying operation . In an attempt to investigate the spray-drying from such a system, the surface composition of the spray-dried powder obtained from various compositions of the two-phase system was analyzed by electron spectroscopy for chemical analysis (ESCA), providing information on the distribution of the polymers in the powder and thus also in the spray droplets . The two-phase system was applied for the spray-drying of live bacteria . The survival rate of the bacteria depended on the composition of the two-phase system . The storage stability of the bacteria in these formulations was investigated after storage at room temperature under dry conditions for 4 weeks, and it was found that the survival rate was 10-45% . The results therefore show that this type of formulation holds promise for future applications for micro-organisms as well as other sensitive biological materials such as proteins .

J Neurocytol, 1999 Jun, 28(6), 439 - 53
Microglial motility in the rat facial nucleus following peripheral axotomy; Schiefer J et al.; Microglial motility was studied in living mammalian brain tissue using infrared gradient contrast microscopy in combination with video contrast enhancement and time lapse video recording . The infrared gradient contrast allows the visualization of living cells up to a depth of 60 microm in brain slices, in regions where cell bodies remain largely uninjured by the tissue preparation and are visible in their natural environment . In contrast to other techniques, including confocal microscopy, this procedure does not require any staining or labeling of cell membranes and thus guarantees the investigation of tissue which has not been altered, apart from during preparation . Microglial cells are activated and increase in number in the facial nucleus following peripheral axotomy . Thus we established the preparation of longitudinal rat brainstem slices containing the axotomized facial nucleus as a source of activated microglial cells . During prolonged video time lapse recordings, two different types of microglial cell motility could be observed . Microglial cells which had accumulated at the surface of the slice remained stationary but showed activity of the cell soma, developing pseudopods of different shape and size which undulated and which were used for phagocytosis of cell debris . Microglial phagocytosis of bacteria could be documented for the first time in situ . In contrast, ameboid microglia which did not display pseudopods but showed migratory capacity, could be observed exclusively in the depth of the tissue . Some of these cells maintained a close contact to neurons and appeared to move along their dendrites, a finding that may be relevant to the role of microglia in "synaptic stripping", the displacement of synapses following axotomy . This approach provides a valuable opportunity to investigate the interactions between activated microglial cells and the surrounding cellular and extracellular structures in the absence of staining or labeling, thus opening a wide field for the analysis of the cellular mechanisms involved in numerous pathologies of the CNS.

J Biol Chem, 2000 Jun 30, 275(26), 20197 - 203
Phosphorylation of osteopontin is required for inhibition of vascular smooth muscle cell calcification; Jono S et al.; Osteopontin (OPN) is a non-collagenous, glycosylated phosphoprotein associated with biomineralization in osseous tissues, as well as ectopic calcification . We previously reported that osteopontin was co-localized with calcified deposits in atherosclerotic lesions, and that osteopontin potently inhibits calcium deposition in a human smooth muscle cell (HSMC) culture model of vascular calcification . In this report, the role of phosphorylation in osteopontin's mineralization inhibitory function was examined . The ability of OPN to inhibit calcification completely depended on post-translational modifications, since bacteria-derived recombinant OPN did not inhibit HSMC mineralization . Following casein kinase II treatment, phosphorylated OPN (P-OPN) dose-dependently inhibited calcification of HSMC cultured in vitro about as effectively as native OPN . The inhibitory effect of osteopontin depended on the extent of phosphorylation . To determine the specific structural domains of OPN important for inhibition of calcification, we compared OPN fragments (N-terminal, C-terminal, and full-length), and compared the inhibitory effect of both phosphorylated and non-phosphorylated fragments . While none of the non-phosphorylated OPN fragments effected calcification, P-OPN caused dose dependent inhibition of HSMC calcification . P-OPN was treated with alkaline phosphatase to create dephosphorylated OPN . Dephosphorylated OPN did not have an inhibitory effect on calcification . The expression of OPN mRNA and P-OPN secretion by HSMC were decreased in a time-dependent manner during culture calcification . These results indicate that phosphorylation is required for the inhibitory effect of OPN on HSMC calcification, and that regulation of OPN phosphorylation represents one way in which mineralization may be controlled by cells.

Cell Mol Life Sci, 2000 Feb, 57(2), 250 - 64
Biomolecular stability and life at high temperatures; Daniel RM et al.; It is not clear what the upper temperature limit for life is, or what specific factors will set this limit, but it is generally assumed that the limit will be dictated by molecular instability . In this review, we examine the thermal stability of two key groups of biological molecules: the intracellular small molecules/metabolites and the major classes of macromolecules . Certain small molecules/metabolites are unstable in vitro at the growth temperatures of the hyperthermophiles in which they are found . This instability appears to be dealt with in vivo by a range of mechanisms including rapid turnover, metabolic channelling and local stabilisation . Evidence to date suggests that proteins have the potential to be stable at substantially higher temperatures than those known to support life, but evidence concerning degradative reactions above 100 degrees C is slight . DNA duplex stability is apparently achieved at high temperature by elevated salt concentrations, polyamines, cationic proteins, and supercoiling rather than manipulation of C-G ratios . RNA stability seems dependent upon covalent modification, although secondary structure is probably also critical . The diether-linked lipids, which make up the monolayer membrane of most organisms growing above 85 degrees C are chemically very stable and seem potentially capable of maintaining membrane integrity at much higher temperatures . However, the in vivo implications of the in vitro instability of biomolecules are difficult to assess, and in vivo data are rare.

Vestn Ross Akad Med Nauk, 2000, (3), 7 - 10
{Determination of Ehrlichia genome size by pulse gel electrophoresis}; Rydkina EB et al.; Ehrlichia infections are more and more common in the USA and Europe . The genetics and genome organization of Ehrlichia are little studied due to great difficulties in cultivating these bacteria . Pulse gel electrophoresis was first used to determine the sizes of a genome of 3 representatives of the genus Ehrlichia . The sizes of a genome was established for E . sennetsu (881 kb), for E . risticii (867 kb), E . chaffeensis (1,236 kb).

Vestn Ross Akad Med Nauk, 2000, (3), 3 - 7
{Evolutionary relationship of Rickettsia and eukaryotic mitochondria}; Emel'ianov VV; To clarify the evolutionary relationship of rickettsiae and mitochondria, the conserved flat heat-shock protein Hsp60 was phylogenetically studied in detail by using PHYLIP and PROTML packages . The ample data set (50 species) included as many as possible representatives from the Rickettsiaceae family, mitochondrial-type homologs from Archezoa and mitochondrial homologs from Protozoa . Rickettsia prowazekii (that is the genus Rickettsia) was shown to be the least diverging member within Rickettsia--a sister group to the monophyletic clade of mitochondria . These findings were also evidenced by the phylogenetic analysis of 16S rRNA . Rickettsia-like endosymbionts (the parasites Paramecium caudatum and the etiological agent of hepatopancreatitis in shrimps) included within the order Rickettsiales appear to have diverged prior to the Rickettsiaceae/mitochondria cluster . Thus, the Rickettsiales does not seem to be a monophyletic group . An idea concerning the nature of obligate intracellular parasitism of rickettsiae is proposed in the paper from the suggested profound similarity of Rickettsiae genus bacteria and mitochondria which could have a common evolutionary history.

Pol Merkuriusz Lek, 2000 Jan, 7(43), 23 - 6
{Serological screening examinations of atypical pathogens (Mycoplasma pneumoniae, Chlamydia pneumoniae) in respiratory tract infection}; Dudko S et al.; Lower respiratory tract infections are a heterogeneous group of disorders induced by plenty of pathogens . Atypical bacteria play an important role in the respiratory tract pathology . In our study 90 patients with acute infection of the respiratory tract were examined in serological screening test for Mycoplasma pneumonia and Chlamydia pneumoniae . It was confirmed that in 21 patients with community acquired pneumonia Mycoplasma pneumonia antibodies were detected in 38% and Chlamydia pneumoniae in 10% . In our opinion this screening serological tests are useful for early diagnosis of atypical bacterial infections of the respiratory tract.

J Biol Chem, 2000 Jun 23, 275(25), 18759 - 66
Mass spectrometry unravels disulfide bond formation as the mechanism that activates a molecular chaperone; Barbirz S et al.; The heat shock protein Hsp33 is a very potent molecular chaperone with a distinctive mode of functional regulation; its activity is redox-regulated . In its reduced form all six cysteinyl residues of Hsp33 are present as thiols, and Hsp33 displays no folding helper activity . Exposure of Hsp33 to oxidizing conditions like H(2)O(2), however, rapidly converts Hsp33 into an efficient molecular chaperone . Activated Hsp33 binds tightly to refolding intermediates of chemically denatured luciferase and suppresses efficiently their aggregation in vitro . Matrix-assisted laser desorption/ionization-mass spectrometry peptide mapping in combination with in vitro and on target protein chemical modification showed that this activation process of Hsp33 is accompanied by the formation of two intramolecular disulfide bonds within Hsp33: Cys(232)-S-S-Cys(234) and Cys(265)-S-S-Cys(268) . Cys(141), although not involved in disulfide bond formation, was found highly reactive toward chemical modifications . In contrast, Cys(239) is readily accessible under reducing conditions but becomes poorly accessible though still reduced when Hsp33 is in its active state . This indicates a significant conformational change during the activation process of Hsp33 . Mass spectrometry, thus, unraveled a novel molecular mechanism by which alteration of the disulfide bond structure, as a result of changes in the cellular redox potential, results in the activation of a molecular chaperone.

Microb Pathog, 2000 Apr, 28(4), 203 - 9
Attachment of Moraxella catarrhalis occurs to the positively charged domains of pharyngeal epithelial cells; Ahmed K et al.; Attachment of bacteria to host cells is the initial step in the pathogenesis of infection . Several factors, such as hydrophobicity, surface electric charge, and van der Waals force, are considered to be responsible for the attachment step . However, it is not clear why bacteria and epithelial cells, both of which possess a negative surface charge, do not repel one another . In the present study, we used Moraxella catarrhalis and pharyngeal epithelial cells to study the surface charges of structures involved in the attachment . By atomic force microscopy (AFM) equipped with surface potential spectroscopy, it was found that the cell surface microplicae have a positive charge of 30.1+/-3.6 mV (mean+/-SE) . The depressions between the microplicae have a negative surface charge of 43.5+/-4.0 mV . Using cationic ferritin and electron microscopy (EM) we confirmed that the depressions between the microplicae have a negative charge . By AFM and by using cationic ferritin with EM, it was found that the net surface charge of the bacterial cells is negative . By both AFM and EM, it was found that the bacterial cells attach to the microplicae of the pharyngeal epithelial cell . Our work confirmed the general belief that both kinds of cells do have a net negative charge . We conclude that there are positively and negatively charged domains on the surface of human pharyngeal epithelial cells . M . catarrhalis evidently attaches to the positively charged domain (i.e . microplicae) of pharyngeal epithelial cells .

J Gastroenterol Hepatol, 2000 Mar, 15(3), 263 - 70
Water extract of Helicobacter pylori stimulates interleukin-8 secretion by a human gastric epithelial cell line (JR-St) through protein tyrosine phosphorylation; Yakabi K et al.; BACKGROUND: Infection by Helicobacter pylori induces cytokine production in gastric mucosal cells . Production of interleukin-8 (IL-8) is known to be markedly increased and is believed to play an important role in gastric mucosal inflammation . The aim of this study was to elucidate the effects of soluble factors of H . pylori on IL-8 production in a gastric epithelial cell line, JR-St . METHODS: JR-St cells were cocultured with a H . pylori water extract, live H . pylori or culture medium supernatant for 24 h, then the IL-8 secreted into the culture medium was assayed . The effects of three different inhibitors; (i) an inhibitor of protein kinase C (PKC); (ii) an inhibitor of PKC and protein kinase A (PKA); and (iii) an inhibitor of protein tyrosine kinase (PTK) were also compared . Specific induction of IL-8 mRNA was also examined . RESULTS: Water extract of H . pylori increased IL-8 secretion 7.72-fold, more than the control . The increase was concentration dependent . Live bacteria, supernatant and water extract significantly stimulated IL-8 secretion . Addition of live bacteria increased IL-8 secretion most strongly, while the effect of water extract was small (22% that of live bacteria) . Secretion was not inhibited by the PKC inhibitor staurosporine or the inhibitors of PKA and PKC H7 . However, secretion was significantly reduced by the PTK inhibitor herbimycin in a dose-dependent manner . Furthermore, 24 h exposure to water extract increased IL-8 mRNA expression, suggesting water extract increased production of IL-8 . CONCLUSIONS: Some soluble factors of H . pylori can stimulate IL-8 production by JR-St cells . Stimulation was not dependent on PKA or PKC but was, at least partially, dependent on protein tyrosine phosphorylation . This suggests that soluble factors of H . pylori can play an important role in mediating the inflammatory response of H . pylori gastritis.

Arch Microbiol, 2000 Mar, 173(3), 200 - 5
Purification and properties of a novel azide-sensitive ATPase of Exiguobacterium aurantiacum; Suga S et al.; Exiguobacterium aurantiacum BL77/1 possesses at least two distinct membrane-bound ATPases . One of them was solubilized with decanoyl N-methylglucamide, a non-ionic detergent, and purified by successive chromatography on DEAE-Sepharose and hydroxyapatite . The purified ATPase appears to consist of a single polypeptide component with an apparent molecular mass of 54 kDa . Among the triphosphates of various nucleosides tested, ATP was the best substrate . The enzyme exhibited a Km of 0.5 mM for ATP and a Vmax of 109 micromol ATP (mg protein)(-1) min(-1); the optimum pH for activity was near 6.5 . The enzyme was sensitive to azide and inactivated by N,N'-dicyclohexylcarbodiimide . Analysis of the inhibition kinetics by N,N'-dicyclohexylcarbodiimide suggested that binding of the drug to a single carboxyl group per ATPase molecule is sufficient for inactivation.

Arch Microbiol, 2000 Mar, 173(3), 193 - 9
Induction of carbon monoxide dehydrogenase to facilitate redox balancing in a ribulose bisphosphate carboxylase/oxygenase-deficient mutant strain of Rhodospirillum rubrum; Joshi HM et al.; A ribulose-1,5-bisphosphate carboxylase/oxygenase-deficient mutant strain (strain I-19) of Rhodospirillum rubrum was capable of growth under photoheterotrophic conditions in the absence of exogenous electron acceptors . These results suggested that alternative means of removing reducing equivalents have been acquired that allow this strain to remove reducing equivalents in the absence of a functional Calvin-Benson-Bassham reductive pentose phosphate pathway . Previously, the proton-reducing activity of the dinitrogenase complex was implicated in helping to maintain redox balance . However, since considerable amounts of CO2 were still fixed in this strain, the complete profile of enzymes involved in alternative CO2 fixation schemes was assessed . A specific and substantial induction of carbon monoxide dehydrogenase (CO dehydrogenase) synthesis was found in the mutant strain; although none of the other CO2 fixation pathways or enzyme activities were altered . These results suggested that CO dehydrogenase contributes to the photoheterotrophic success of strain I-19 . Furthermore, the data implicate interacting and complex regulatory processes required to maintain the proper redox balance of this organism and other nonsulfur purple bacteria.

Int J Mol Med, 2000 May, 5(5), 511 - 3
Free DNA induces modification on the protein synthesis profile of human peripheral blood mononuclear cells of healthy donors; Martins GA et al.; Understanding how free DNA might act as a signal between cells is important for knowing how DNA orchestrates immune responses and for optimizing the therapeutic of cancer, infection and immunologic diseases . This communication demonstrates that DNAs from different origins (bacteria, T . cruzi, HeLa cells) and synthetic oligonucleotide containing an unmethylated CpG motif are capable of inducing alterations in the protein profile of normal human leukocytes . As far as we know there have been no similar studies regarding the comparative effects of different free DNAs on early protein synthesis of human peripheral blood mononuclear cells.

Immunol Cell Biol, 2000 Apr, 78(2), 118 - 23
Haemopoiesis in mice genetically lacking granulocyte-macrophage colony stimulating factor during chronic infection with Mycobacterium avium; Zhan Y et al.; In order to test the role of granulocyte-macrophage colony stimulating factor (GM-CSF) in haemopoiesis during chronic infection, mice with a targeted disruption of the gene for GM-CSF were infected intraperitoneally with the facultative intracellular pathogen, Mycobacterium avium . The bacteria spread to lungs, liver and spleen and persisted for more than 10 weeks at levels between 105 and 106 CFU . Bacterial numbers did not differ significantly between infected GM-CSF-/- and wild-type mice, making this an excellent model in which to study the effects of GM-CSF deficiency on haemopoietic cells without complications of interpretation relating to differences in bacterial load . Haemopoietic colony forming cells (CFC) in the bone marrow of GM-CSF-/- mice before infection were not different from wild-type . However, whereas CFC in wild-type mice increased 1.5-fold with infection, GM-CSF-/- mice were unable to increase their CFC and numbers were significantly lower than in infected wild-type mice . Cells attracted to the peritoneal cavity of the GM-CSF-/- mice following i.p . injection of bacteria were notably lacking in the large, granular macrophages of activated appearance, which were a feature in wild-type mice . Nitric oxide production by peritoneal cells from GM-CSF-/- mice was deficient . Thus, GM-CSF is not critical for haemopoiesis during chronic infection, but in its absence the mice are unable to increase their output of haemopoietic cells and there are deficiencies in macrophage activation.

Heredity, 2000 Feb, 84 ( Pt 2), 152 - 60
The evolutionary dynamics of male-killers and their hosts; Randerson JP et al.; Male-killing bacteria are cytoplasmic sex-ratio distorters that are transmitted vertically through females of their insect hosts . The killing of male hosts by their bacteria is thought to be an adaptive bacterial trait because it augments the fitness of female hosts carrying clonal relatives of those bacteria . Here we attempt to explain observations of multiple male-killers in natural host populations . First we show that such male-killer polymorphism cannot be explained by a classical model of male-killing . We then show that more complicated models incorporating the evolution of resistance in hosts can explain male-killer polymorphism . However, this is only likely if resistance genes are very costly . We also consider the long-term evolutionary dynamics of male-killers, and show that evolution towards progressively more 'efficient' male-killers can be thwarted by the appearance of host resistance . The presence of a resistance gene can allow a less efficient male-killer to outcompete its rival and hence reverse the trend towards more efficient transmission and reduced metabolic load on the host.

J Bacteriol, 2000 May, 182(9), 2597 - 603
ADP-Ribosylation of variants of Azotobacter vinelandii dinitrogenase reductase by Rhodospirillum rubrum dinitrogenase reductase ADP-ribosyltransferase; Grunwald SK et al.; In a number of nitrogen-fixing bacteria, nitrogenase is posttranslationally regulated by reversible ADP-ribosylation of dinitrogenase reductase . The structure of the dinitrogenase reductase from Azotobacter vinelandii is known . In this study, mutant forms of dinitrogenase reductase from A . vinelandii that are affected in various protein activities were tested for their ability to be ADP-ribosylated or to form a complex with dinitrogenase reductase ADP-ribosyltransferase (DRAT) from Rhodospirillum rubrum . R140Q dinitrogenase reductase could not be ADP-ribosylated by DRAT, although it still formed a cross-linkable complex with DRAT . Thus, the Arg 140 residue of dinitrogenase reductase plays a critical role in the ADP-ribosylation reaction . Conformational changes in dinitrogenase reductase induced by an F135Y substitution or by removal of the Fe(4)S(4) cluster resulted in dinitrogenase reductase not being a substrate for ADP-ribosylation . Through cross-linking studies it was also shown that these changes decreased the ability of dinitrogenase reductase to form a cross-linkable complex with DRAT . Substitution of D129E or deletion of Leu 127, which result in altered nucleotide binding regions of these dinitrogenase reductases, did not significantly change the interaction between dinitrogenase reductase and DRAT . Previous results showed that changing Lys 143 to Gln decreased the binding between dinitrogenase reductase and dinitrogenase (L . C . Seefeldt, Protein Sci . 3:2073-2081, 1994); however, this change did not have a substantial effect on the interaction between dinitrogenase reductase and DRAT.

J Interferon Cytokine Res, 2000 Mar, 20(3), 259 - 72
Viral double-stranded RNA, cytokines, and the flu; Majde JA; The symptoms of the flu, such as fever, drowsiness, and malaise, are the sole means by which this common clinical syndrome is defined . The syndrome is usually the first clinical manifestation of both acute bacterial and viral infections . In the case of acute bacterial infections, several proinflammatory cytokines induced by bacterial products have been implicated as the causative agents of the flu syndrome . Viruses induce similar cytokines to bacteria, plus substantial amounts of interferon-alpha (IFN-alpha), although the direct association of these cytokines with the viral flu syndrome is less clear . Furthermore, the viral inducer(s) of cytokines has not been defined . The best candidate cytokine inducer associated with a majority of viral infections is virus-associated double-stranded RNA (dsRNA) . This review examines the essential physical properties of toxic dsRNA, the cytokines induced by it, its viral and cellular sources, evidence for its presence in infected cells, its quantities in normal and infected cells, its cytotoxic mechanisms, and its cell-penetration properties . Toxic effects of viruses and dsRNA are compared . Energetics and extraction artifact issues are also discussed . Whereas most research on dsRNA toxicity has employed synthetic dsRNA, studies with virus-associated dsRNA are featured when available . Finally, a model for how viral dsRNA might initiate systemic disease is presented.

Annu Rev Entomol, 2000, 45, 287 - 306
Control of insect pests with entomopathogenic nematodes: the impact of molecular biology and phylogenetic reconstruction; Liu J et al.; Entomopathogenic nematodes are excellent biological control agents . Utilization of these nematodes is developing rapidly with almost a doubling of newly described species in the past five years . Advances in molecular biology and phylogenetic reconstruction have revolutionized understanding of population structure, identification, genetic improvement, systematics, and the symbiosis between entomopathogenic nematodes and their bacteria . Population structure provides the most fundamental information for reliable identification of species and unique genetic variants . Such information could be further assessed for nematode potential as biological control agents . Phylogenetic reconstruction is an important approach for understanding multitrophic interactions among entomopathogenic nematodes, symbiotic bacteria, and their insect hosts . Phylogenetic reconstruction is also important for the development of a natural and stable type of systematics, which can provide guidelines for selecting appropriate entomopathogenic nematode species for particular biological control programs.

Biochim Biophys Acta, 2000 Apr 25, 1491(1-3), 248 - 52
Molecular cloning and sequencing of the sodB gene from a heterocystous cyanobacterium Anabaena sp . PCC 7120; Liu Y et al.; Superoxide dismutase (Sod) plays an important role in all aerobic organisms . The sodB gene of a heterocystous cyanobacterium Anabaena sp . PCC 7120 was cloned and sequenced . The Sod protein is predicted to have 199 amino acids and a molecular mass of 22.5 kDa . Sequence comparison among SodB from cyanobacteria and chloroplasts revealed that the sodB gene indeed encodes an iron-Sod . Northern blot analysis showed that the sodB gene of Anabaena sp . PCC 7120 is transcribed as a single gene and its expression was up-regulated when the cells were subjected to a shift from a nitrogen repletion condition to a nitrogen depletion condition.

Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 4392 - 7
Mutants of Arabidopsis thaliana defective in the acquisition of tolerance to high temperature stress; Hong SW et al.; The ability of organisms to acquire thermotolerance to normally lethal high temperatures is an ancient and conserved adaptive response . However, knowledge of cellular factors essential to this response is limited . Acquisition of thermotolerance is likely to be of particular importance to plants that experience daily temperature fluctuations and are unable to escape to more favorable environments . We developed a screen, based on hypocotyl elongation, for mutants of Arabidopsis thaliana that are unable to acquire thermotolerance to high-temperature stress and have defined four separate genetic loci, hot1-4, required for this process . hot1 was found to have a mutation in the heat shock protein 101 (Hsp101) gene, converting a conserved Glu residue in the second ATP-binding domain to a Lys residue, a mutation that is predicted to compromise Hsp101 ATPase activity . In addition to exhibiting a thermotolerance defect as assayed by hypocotyl elongation, 10-day-old hot1 seedlings were also unable to acquire thermotolerance, and hot1 seeds had greatly reduced basal thermotolerance . Complementation of hot1 plants by transformation with wild-type Hsp101 genomic DNA restored hot1 plants to the wild-type phenotype . The hot mutants are the first mutants defective in thermotolerance that have been isolated in a higher eukaryote, and hot1 represents the first mutation in an Hsp in any higher plant . The phenotype of hot1 also provides direct evidence that Hsp101, which is required for thermotolerance in bacteria and yeast, is also essential for thermotolerance in a complex eukaryote.

Plant Cell, 2000 Apr, 12(4), 519 - 33
A role for ectophosphatase in xenobiotic resistance; Thomas C et al.; Xenobiotic resistance in animals, plants, yeast, and bacteria is known to involve ATP binding cassette transporters that efflux invading toxins . We present data from yeast and a higher plant indicating that xenobiotic resistance also involves extracellular ATP degradation . Transgenic upregulation of ecto-ATPase alone confers resistance to organisms that have had no previous exposure to toxins . Similarly, cells that are deficient in extracellular ATPase activity are more sensitive to xenobiotics . On the basis of these and other supporting data, we hypothesize that the hydrolysis of extracellular ATP by phosphatases and ATPases may be necessary for the resistance conferred by P-glycoprotein.

Eur J Clin Invest, 2000 Apr, 30(4), 359 - 66
Antithrombin III and local serum application: adjuvant therapy in peritonitis; Schorr M et al.; BACKGROUND: Patients with diffuse peritonitis show an overall mortality of about 20%, probably caused by the breakdown of local defence mechanisms combined with a systemic outspread of bacteria and toxins, which often results in sepsis syndrome . DESIGN: In a prospective, randomized, controlled study 50 patients with diffuse secondary peritonitis were included . Patients in the therapy group were treated with an adjuvant medication consisting of a continuous intravenous infusion of antithrombin III and two intraperitoneal instillations of fresh frozen human donor serum . The aim of the study was the reduction of mortality and incidence of multiple organ failure . RESULTS: Mean antithrombin III plasma levels in the therapy group were raised above 140% for 4 days and were significantly higher than in the control group . With the intraperitoneal application of fresh frozen serum and antithrombin III opsonic capacity as well as thrombin, inhibitory activity in the exudate could be significantly elevated over 2 days . The 90-day-mortality rate was 6/26 (23%) in the control group and 6/24 (25%) in the therapy group . Although no improvement of mortality was achieved, a slight but not significant reduction of the severity of the multiple organ failure was seen . CONCLUSIONS: The chosen therapeutic approach was feasible and showed no side-effects . Yet, neither mortality nor multiple organ failure were significantly improved by the applied short-term adjuvant therapy . Thus, for future trials in severely-ill patients a longer treatment period and/or combinations of antithrombin III with other anti-inflammatory agents should be considered.

Eur J Biochem, 2000 Apr, 267(8), 2354 - 61
Morphine-like substance in leech ganglia . Evidence and immune modulation; Laurent V et al.; Binding experiments followed by measurement of nitric oxide release revealed an opiate alkaloid high affinity receptor with no affinity to opioids, representing a new mu-subtype receptor in the brain of the leech Theromyzon tessulatum . In addition, evidence of morphine-like substances was found in immunocytochemical studies and HPLC coupled to electrochemical detection (500 mV and 0.02 Hz) . Based on previous evidence of the involvement of morphine as an immune response inhibitor, we demonstrate that in leech ganglia injection of lipopolysaccharide (LPS; a potent immunostimulatory agent derived from bacteria) provoked an increase in the level of ganglionic morphine-like substances after a prolonged latency period of 24 h (from 2.4 +/- 1.1 pmol per ganglion to 78 +/- 12.3 pmol per ganglion; P < 0.005; LPS injected 1 microg x mL-1); this effect is both concentration- and time-dependent . Finally, we have demonstrated that morphine, after binding to its own receptor, inhibits leech immunocyte activation through adenylate cyclase inhibition and nitric oxide release . This report confirms that morphine is an evolutionarily stable potent immunomodulator.

Clin Exp Immunol, 2000 Apr, 120(1), 154 - 61
Selective expansion of T cells in gingival lesions of patients with chronic inflammatory periodontal disease; Yamazaki K et al.; Chronic inflammatory periodontal diseases are characterized by a cellular infiltrate and are similar in many respects to other chronic inflammatory diseases . While periodontopathic bacteria have been recognized as the principal causative agent and the immune response to these bacteria is thought to be responsible for the tissue destruction, the full aetiological spectrum is still incompletely understood . In addition to many cell types such as polymorphonuclear leucocytes and macrophages, T cells have been implicated in pathogenesis and are considered to have regulatory roles in progression of the disease . Based on our recent studies demonstrating biased expression of several Vbeta families in periodontitis tissues, the aim of this study was to characterize further the T cells relevant to the disease process by reverse transcription-polymerase chain reaction-single-strand conformation polymorphism (RT-PCR-SSCP) and subsequent nucleotide sequence analysis of complementarity-determining region 3 (CDR3) of the TCR beta-chain . In spite of the likely involvement of numerous bacteria, the present study has clearly shown the oligoclonality of infiltrating T cells in periodontitis lesions in contrast to low clonality of peripheral blood T cells as evidenced by the appearance of distinct bands in gingival tissue samples and smear pattern of peripheral blood on SSCP gels . These were confirmed by the DNA sequencing of the CDR3 of Vbeta16 of selected samples . The analysis of deduced amino acid sequences demonstrated amino acid motifs in the CDR3 region of the periodontitis lesion-derived sequences from each patient . The results indicate that gingival tissue-infiltrating T cells recognizing a limited number of antigens or epitopes are involved in the disease process.

Clin Exp Immunol, 2000 Apr, 120(1), 85 - 92
Cross-reactive epitopes and HLA-restriction elements in human T cell recognition of the Mycobacterium leprae 18-kD heat shock protein; Mustafa AS et al.; We have previously demonstrated that the Mycobacterium leprae 18-kD heat shock protein (HSP18) is represented among the antigenic targets of human T cell responses induced by M . leprae immunization and that the peptide 38-50 serves as an immunodominant epitope recognized by CD4+ T cell clones . By using peripheral blood mononuclear cells and T cell lines from the same donor group, we have in this study shown that the M . leprae HSP18 and peptide 38-50 were recognized by memory T cells 8 years after immunization with M . leprae . The finding that M . bovis BCG-induced T cell lines responded to M . leprae HSP18, but not to the peptide 38-50, suggested the existence of additional T cell epitopes of a cross-reactive nature . Consistent with this, testing of the T cell lines for proliferative responses to the complete HSP18 molecule, truncated HSP18 (amino acid (aa) residues 38-148) and overlapping synthetic peptides, made it possible to identify two cross-reactive epitope regions defined by aa residues 1-38 and 41-55 . While peptide 38-50-reactive T cell clones showed limited cross-reactivity by responding to M . leprae, M . avium and M . scrofulaceum, the T cell lines specific to the epitopes 1-38 and 41-55 were broadly cross-reactive, as demonstrated by their response to M . leprae, M . tuberculosis complex, M . avium and other mycobacteria . MHC restriction analysis of the HSP18-responding T cell lines showed that the epitopes 1-38 and 38-50 were presented by one of the two HLA-DR molecules expressed from self HLA-DRB1 genes, whereas the epitope 41-55 was recognized in the presence of autologous as well as HLA-DR and HLA-DQ mismatched allogeneic antigen-presenting cells . The results obtained in this study made it possible to identify cross-reactive T cell epitopes of the M . leprae HSP18, and provide an explanation for T cell recognition of this antigen in individuals infected with species of the M . tuberculosis complex or environmental mycobacteria.

Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 3901 - 6
A sequence resembling a peroxisomal targeting sequence directs the interaction between the tetratricopeptide repeats of Ssn6 and the homeodomain of alpha 2; Smith RL et al.; The tetratricopeptide repeat (TPR) is a 34-aa sequence motif, typically found in tandem clusters, that occurs in proteins of bacteria, archea, and eukaryotes . TPRs interact with other proteins, although few details on TPR-protein interactions are known . In this paper we show that a portion of a loop in the homeodomain of the DNA-binding protein alpha2 is required for its recognition by the TPRs of the corepressor Ssn6 . The amino acid sequence of this loop is similar to the sequences recognized by the TPRs of an entirely different protein, Pex5, which directs peroxisomal import . We further show that alpha2 can be made to bind specifically in vitro to the TPRs of Pex5 and that a point mutation that disrupts the alpha2-Ssn6 interaction also disrupts the alpha2-Pex5 interaction . These results demonstrate that two different TPR proteins recognize their target by a similar mechanism, raising the possibility that other TPR-target interactions could occur through the same means.

Eur Respir J, 2000 Mar, 15(3), 560 - 5
Farming practices and the respiratory health risks of swine confinement buildings; Cormier Y et al.; This study investigated whether clean swine confinement buildings (SCB) are less harmful to the respiratory system than older and dirtier facilities . Eight healthy volunteers were exposed for 4 h, at 1 week intervals, to eight SCB representing the widest possible range of cleanliness . Each volunteer and a technician rated the SCB for cleanliness from 1-10, 1 being the cleanest possible . Airborne dust, bacteria, endotoxin levels, molds, and ammonia were measured . For each volunteer measured, before and after each exposure, forced expiratory flows (forced expiratory volume in one second (FEV1), and forced vital capacity), white cells in nasal wash and venous blood, and nasal lavage levels of interleukin (IL)-8 and serum levels of IL-6 . A methacholine challenge was obtained at baseline and following each exposure . Cleanliness scores ranged 1.5-8.25 . Mean airborne levels were: dust 3.54 mg x m(-3) bacteria 4.25 x 10(5) CFU x m(-3); endotoxins 404 EU x m(-3); molds 883 CFU x m(-3); ammonia 20.7 parts per million (ppm) . Expiratory flows decreased after exposure (FEV1 from 4.8+/-0.7 to 4.4+/-0.7, p<0.001), neutrophils in the nasal wash and white blood cells increased (28.5+/-37 to 424+/-207 x 10(3), 5.4+/-1.0 to 7.4+/-1.7 x 10(9) cells x mL(-1) respectively), IL-8 increased from 158+/-311 to 2679+/-639 pg x mL(-1), IL-6 from 0.15+/-0.26 to 2.34+/-0.92 pg x mL(-1), (p<0.001) . All SCB were similarly harmful . In conclusion, modern farming has not succeeded in making swine confinement buildings inoffensive to exposed subjects.

Prog Neurobiol, 2000 May, 61(1), 61 - 74
Neural development and neurodegeneration: two faces of neuropathy target esterase; Glynn P; Neuropathy target esterase (NTE) is an integral membrane protein in vertebrate neurons . Recent evidence suggests that NTE plays an important role in neural development, possibly via involvement in a signalling pathway between neurons and glial cells . NTE is a member of a novel protein family, represented in organisms from bacteria to man . NTE comprises an N-terminal regulatory domain (with some sequence similarity to cyclic nucleotide-binding proteins) and a C-terminal catalytic domain: the latter has three predicted transmembrane segments and requires membrane-association for activity . In vitro, NTE potently catalyses hydrolysis of phenyl valerate: however, its physiological substrate is likely to be a metabolite of a much longer chain carboxylic acid, possibly associated with cell membranes . NTE was discovered originally as the primary target for those organophosphorus esters (OPs) which cause a delayed neuropathy with degeneration of long axons in peripheral nerves and spinal cord . Paradoxically, NTE's catalytic activity appears redundant in adult vertebrates . Neuropathic OPs react covalently with NTE in a rapid two-step process which not only inhibits catalytic activity but also leaves a negatively-charged OP group attached to the active site serine . The latter event is proposed to induce a toxic gain of function in NTE . OP-modified NTE somehow engenders a "chemical transection of the axon" . In turn, this leads to calcium entry, elevation of axonal calpain activity and Wallerian-type degeneration . The net damage to peripheral nerve axons is a balance between ongoing degenerative and repair processes: the latter involve serine hydrolases which can be inhibited by the same OPs used to modify NTE.

Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 649 - 59
Reassessment of the taxonomic structure of the diazotrophic genus Azoarcus sensu lato and description of three new genera and new species, Azovibrio restrictus gen . nov., sp . nov., Azospira oryzae gen . nov., sp . nov . and Azonexus fungiphilus gen . nov., sp . nov; Reinhold-Hurek B et al.; The taxonomic structure of members of the genus Azoarcus sensu lato was reassessed in a polyphasic approach . Two species, Azoarcus communis and Azoarcus indigens, three unnamed species containing diazotrophs associated with Kallar grass roots (groups C, D) and a group of strains (E) isolated from fungi were analysed . They were compared by PAGE analyses of cellular proteins, genomic fingerprints, morphological and nutritional features to new isolates from rice roots . All strains within groups C, D and E containing 5-12 isolates showed group-specific cell and colony morphology and carbon source utilization patterns, with exception of the obligately microaerobic strain BS20-3, a member of group C . All strains, with this exception, also had almost indistinguishable electrophoretic protein patterns and genomic fingerprints generated with tDNA-directed primers, suggesting they belong to the same species . Phylogenetic analyses of almost complete 16S rDNA sequences carried out with three different algorithms (neighbour-joining, maximum-likelihood, parsimony) revealed that Azoarcus sensu lato is not monophyletic . Groups C, D and E formed three distinct lineages located between the Azoarcus/Thauera and the Rhodocyclus clusters . Phylogenetic distances between groups C, D and E were as large as between other genera (93-94% sequence similarity) . This suggested they have the rank of three different genera . Since it was possible to differentiate them from each other and other related bacteria by phenotypic features, three new genera with one type species each are proposed: Azovibrio restrictus gen . nov., sp . nov., Azospira oryzae gen . nov., sp . nov . and Azonexus fungiphilus gen . nov., sp . nov.

Plant J, 2000 Mar, 21(6), 571 - 8
Arbuscular mycorrhizal fungi induce the non-mevalonate methylerythritol phosphate pathway of isoprenoid biosynthesis correlated with accumulation of the 'yellow pigment' and other apocarotenoids; Walter MH et al.; Plants and certain bacteria use a non-mevalonate alternative route for the biosynthesis of many isoprenoids, including carotenoids . This route has been discovered only recently and has been designated the deoxyxylulose phosphate pathway or methylerythritol phosphate (MEP) pathway . We report here that colonisation of roots from wheat, maize, rice and barley by the arbuscular mycorrhizal fungal symbiont Glomus intraradices involves strong induction of transcript levels of two of the pivotal enzymes of the MEP pathway, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) . This induction is temporarily and spatially correlated with specific and concomitant accumulation of two classes of apocarotenoids, namely glycosylated C13 cyclohexenone derivatives and mycorradicin (C14) conjugates, the latter being a major component of the long-known 'yellow pigment' . A total of six cyclohexenone derivatives were characterised from mycorrhizal wheat and maize roots . Furthermore, the acyclic structure of mycorradicin described previously only from maize has been identified from mycorrhizal wheat roots after alkaline treatment of an 'apocarotenoid complex' of yellow root constituents . We propose a hypothetical scheme for biogenesis of both types of apocarotenoids from a common oxocarotenoid (xanthophyll) precursor . This is the first report demonstrating (i) that the plastidic MEP pathway is active in plant roots and (ii) that it can be induced by a fungus.

Plant J, 2000 Feb, 21(4), 317 - 27
Maize high chlorophyll fluorescent 60 mutation is caused by an Ac disruption of the gene encoding the chloroplast ribosomal small subunit protein 17; Schultes NP et al.; The maize mutation high chlorophyll fluorescence 60-muTable 1 (hcf60-m1), generated through Activator (Ac) tagging, has insufficient photosynthetic electron transport . Here we show that the Hcf60 gene encodes a protein with substantial amino acid similarity to plant plastid and bacterial ribosomal small subunit protein 17 (RPS17) proteins . The lack of detectable HCF60 transcripts in mutant leaves, and insertion of the transposed Ac element 17 bp upstream of the start of translation in the mutated locus, suggest that little if any RPS17 is produced . The mutant phenotype is consistent with reduced plastid translation . Seedling lethal hcf60-m1 plants display temperature and light-dependent chlorophyll deficiencies, a depletion of plastid rRNA pools, and few high-molecular-weight polysomal complexes . Growth under moderate light conditions (27 degrees C, 100 microE m-2 sec-1) allows for substantial chlorophyll accumulation in mutant leaves, yet the number of functional photosystem II complexes appears low . Nevertheless, the presence of a limited but intact C4 system indicates that some plastid translation occurs.

Microbes Infect, 2000 Mar, 2(3), 313 - 6
The macrophage receptor MARCO; Kraal G et al.; MARCO (macrophage receptor with collagenous structure) belongs to the class A scavenger receptor molecules . The structure and function of the molecule is described . Although it is expressed on subsets of macrophages, it can be upregulated on other macrophages after bacterial infection . The strategic position of MARCO-expressing cells in lymphoid organs suggests an important role for this bacteria-binding molecule in removal of pathogens.

Microbes Infect, 2000 Mar, 2(3), 273 - 8
Collectins and innate immunity in the lung; Clark HW et al.; Evidence from both in vitro and in vivo studies suggests that the collectins are important elements in host innate immune defences against infectious agents . Study of the collectins in specific disease settings now raises the prospects of developing therapies exploiting these mechanisms of innate immunity.

Rev Med Chir Soc Med Nat Iasi, 1999 Jul-Dec, 103(3-4), 35 - 43
{Chaperone proteins--essential proteins for cellular activity}; Sandovici I et al.; Molecular chaperones are an ubiquitous, abundant and highly conserved group of proteins which bind and stabilize proteins at intermediate stages of folding, assembly, translocation across membranes and degradation . They first came to attention because of their specific induction during the cellular response of all organisms to heat shock, but are now known to be constitutively and abundantly expressed in the absence of any stress . Despite the obvious importance of stress responses, only recently has scrutiny focused on the role of heat shock proteins in the control of disease pathology . Knowledge about Hsp functions in bacteria is much further advanced than in eukaryotes, but already some hints of Hsp involvement in mammalian diseases have emerged.

Rev Med Chir Soc Med Nat Iasi, 1999 Jan-Jun, 103(1-2), 57 - 62
{Multiparticulate systems in the controlled release of active substances in the gastrointestinal tract}; Dumistracel I et al.; The design of multiparticulate systems as carriers for molecules that have to be delivered at specific sites of the gastrointestinal tract must take into account the common events that govern this region . The intrusion of the multiparticulate systems has to face, by meanings of synthesis methods, or only by lack of aggressiveness the host system defences . Thus, non-specific and specific defence mechanisms are deployed to deal with the intruding material . Several strategies can be mentioned, as (a) prevent the contact and remove if contacted (b) kill if not remove, by the micro-fold cells that engulf bacteria and the gastrointestinal-associated lymphoid tissue . A physical barrier that adheres, prevents the contact, en-traps and removes the foreign matter is the mucus produced by the goblet cells.

Acta Microbiol Pol, 1999, 48(3), 277 - 81
Anti-Lewis X IgM and IgG in H . pylori infections in children and adults; Chmiela M et al.; A role of autoimmune processes in the pathology of Helicobacter pylori infections has been suggested . The Lewis determinants present in LPS molecule of H . pylori bacteria have been indicated as the cause of antigenic mimicry . In this study, the prevalence of IgM and IgG antibodies to Lewis X antigen in the sera from children and adults, with or without dyspepsia, infected or not infected with H . pylori, seropositive and seronegative for anti-H . pylori IgG were determined immuno-enzymatically (ELISA) . Our results revealed that humans may produce anti-Lewis X antibodies, particularly of IgM class, in the absence of H . pylori infection or H . pylori independent dyspepsia . The production of such antibodies, by healthy children who had never been infected with H . pylori suggested that anti-Lewis X antibodies may occur naturally.

Acta Microbiol Pol, 1999, 48(3), 261 - 75
Genotypes of Helicobacter pylori in Polish population; Gzyl A et al.; Here we have studied the genetic diversity of Helicobacter pylori strains recovered from 64 individual patients, 5 family members and 13 unsuccessfully treated patients . The recovered bacteria were finger-printed by the PCR-RFLP and RAPD methods and virulence associated loci (cagPAI, vacA) were PCR studied . Unique differentiation of every independently isolated strain from not-related persons was possible by RAPD technique . In PCR-RFLP technique several profile groups (7 and 15) for particular endonuclease tested were found . Eleven patients carried strains of the same gene profile (PCR-RFLP) and the same overall genotype (RAPD) before and after therapy . In the family studies, essentially the same strain was found in different relatives in three cases, and different strains were found in the other two cases . Island of cagPAI was present in 79% of all strains tested, half and one-fifth of all strains tested presented, s1am2 and s1m1 alleles of vacA gene, respectively . Independently from identity or diversity of pre- and post-treatment strains and strains recovered from the family members we have been observed identical cagPAI/vacA genotypes . These results suggest that H . pylori infections in Poland can be mixed, although just one strain may often predominate, and that inter-family transmission may be significant even in this high risk society . The genetic feature of virulence-associated loci are similar to those seen elsewhere in Europe, although strains that carry the cagPAI and the potentially more toxigenic alleles of the vacA gene are more common . RAPD technique is proven as most differentiating, however PCR-RFLP allows for easy recognition of mixed infection with two or more different strains . Molecular typing study in case of children therapy may allow reduce rate of relapses by reduction of possible transmission from family source.

J Mol Biol, 2000 Apr 21, 298(1), 83 - 94
The solution structure of Rhodobacter sphaeroides LH1beta reveals two helical domains separated by a more flexible region: structural consequences for the LH1 complex; Conroy MJ et al.; Here, the solution structure of the Rhodobacter sphaeroides core light-harvesting complex beta polypeptide solubilised in chloroform:methanol is presented . The structure, determined by homonuclear NMR spectroscopy and distance geometry, comprises two alpha helical regions (residue -34 to -15 and -11 to +6, using the numbering system in which the conserved histidine residue is numbered zero) joined by a more flexible four amino acid residue linker . The C-terminal helix forms the membrane spanning region in the intact LH1 complex, whilst the N-terminal helix must lie in the lipid head groups or in the cytoplasm, and form the basis of interaction with the alpha polypeptide . The structure of a mutant beta polypeptide W(+9)F was also determined . This mutant, which is deficient in a hydrogen bond donor to the bacteriochlorophyll, showed an identical structure to the wild-type, implying that observed differences in interaction with other LH1 polypeptides must arise from cofactor binding . Using these structures we propose a modification to existing models of the intact LH1 complex by replacing the continuous helix of the beta polypeptide with two helices, one of which lies at an acute angle to the membrane plane . We suggest that a key difference between LH1 and LH2 is that the beta subunit is more bent in LH1 . This modification puts the N terminus of LH1beta close to the reaction centre H subunit, and provides a rationale for the different ring sizes of LH1 and LH2 complexes .

Genomics, 2000 Mar 15, 64(3), 252 - 63
A new gene family including DSCR1 (Down Syndrome Candidate Region 1) and ZAKI-4: characterization from yeast to human and identification of DSCR1-like 2, a novel human member (DSCR1L2); Strippoli P et al.; A new gene family has been identified on the basis of in-depth bioinformatics analysis of the Down syndrome candidate region 1 (DSCR1) gene, located on 21q22.1 . We have determined the complete coding sequences of similar genes in Saccharomyces cerevisiae and Caenorhabditis elegans, as well as that of a novel human gene, named DSCR1L2 (DSCR1-like 2) . Peripheral blood leukocyte cDNA sequencing predicts as its product a 241-amino-acid protein highly similar to products of the human genes DSCR1 and ZAKI-4 (HGMW-approved symbol DSCR1L1) . The highest level of expression of DSCR1L2 mRNA was found by Northern blot analysis in heart and skeletal muscles, liver, kidney, and peripheral blood leukocytes (three transcripts of 3.2, 5 . 2, and 7.5 kb) . The gene consists of four exons and spans about 22 kb on chromosome 1 (1p33-p35.3) (Human Chromosome 1, Sanger Centre) . Exon/intron organization is highly conserved between DSCR1 and DSCR1L2 . Two alternative DSCR1L2 mRNA splicing forms have been recognized, with one lacking 10 amino acids in the middle of the protein . Analysis of expressed sequence tags (ESTs) shows DSCR1L2 expression in fetal tissues (heart, liver, and spleen) and in adenocarcinomas . ESTs related to the murine DSCR1L2 orthologue are found in the 2-cell stage mouse embryo, in developing brain stem and spinal cord, and in thymus and T cells . The most prominent feature identified in the protein family is a central short, unique serine-proline motif (including an ISPPXSPP box), which is strongly conserved from yeast to human but is absent in bacteria . Moreover, homology with the RNA-binding domain was weakly but consistently detected in a stretch of 80 amino acids at the amino-terminus by fine sequence analysis based on tools utilizing both hidden Markov models and BLAST . The identification of this new gene family should allow a better understanding of the functions of the genes belonging to it .

Nucleic Acids Res, 2000 May 1, 28(9), 1871 - 8
Functional analysis of the homeodomain protein SIX5; Harris SE et al.; SIX5 (previously known as myotonic dystrophy associated homeodomain protein - DMAHP ) is a member of the SIX { sine oculis homeobox (Drosophila ) homologue } gene family which encodes proteins containing a SIX domain adjacent to a homeo-domain . To investigate the DNA binding specificities of these two domains in SIX5, they were expressed as GST fusion proteins, both separately and together . Affinity purified recombinant proteins and cell lysates from bacteria expressing the recombinant proteins were used in gel retardation assays with double stranded oligonucleotides representing putative DNA binding sites . The putative sites included two in the promoter region of DMPK (dystrophia myotonica protein kinase ) and the previously characterised murine Six4 DNA binding site in the Na(+)/K(+) ATPase alpha 1 subunit gene ( ATP1A1 ) regulatory element (ARE) . None of the recombinant proteins showed any affinity for the two putative sites in DMPK . However, the two recombinant proteins containing the homeodomain both formed at least one specific complex with the ARE . The recombinant protein containing both domains formed a second specific complex with the ARE, assumed to be a dimer complex . Finally, a whole genome PCR-based screen was used to identify genomic DNA sequences to which SIX5 binds, as an initial stage in the identification of genes regulated by SIX5.

J Chromatogr B Biomed Sci Appl, 2000 Mar 10, 739(2), 337 - 44
Analysis of N(alpha)-methylhistamine by gas chromatography-mass spectrometry; Murray S et al.; A gas chromatography-electron capture mass spectrometry assay has been developed for the histamine H3 receptor agonist, N(alpha)-methylhistamine (N(alpha)-MH) . The assay is linear from 50 pg-10 ng, with a limit of detection of 50 pg/ml for gastric juice and plasma, and 50 pg/sample for bacteria (10(7)-10(8) CFU) and gastric tissue (5-10 mg wet weight) . The limits of quantification are 100 pg/ml for gastric juice (%RSD=1.4) and plasma (%RSD=9.4), and 100 pg/sample for bacteria (%RSD=3.9) and tissue (%RSD=5.8) . N(alpha)-MH was not present in human plasma, but low levels (1.4 ng/ml and 0.4 ng/ml) were detected in two samples of human gastric juice obtained from patients infected with Helicobacter pylori.

Carcinogenesis, 2000 Apr, 21(4), 533 - 41
Threshold mechanisms and site specificity in chromium(VI) carcinogenesis; De Flora S; Ten years have elapsed since the International Agency for Research on Cancer (IARC) evaluated the carcinogenicity of chromium and chromium compounds . Further studies performed during the last decade have provided further epidemiological, experimental and mechanistic data which support the IARC conclusions . A wealth of results indicate that, at variance with chromium(0) and chromium(III), chromium(VI) can induce a variety of genetic and related effects in vitro . The lack of carcinogenicity of chromium(0) and chromium(III) compounds in experimental animals is well established, and only a minority of animal carcinogenicity data with chromium(VI) compounds were positive (30 out of 70, i.e . 42.9%) . Moreover, most positive studies used administration routes which do not mimic any human exposure and by-pass physiological defense mechanisms . Typically, positive results were only obtained at implantation sites and at the highest dose tested . Exposure to chromium(VI) has been known for more than a century to be associated with induction of cancer in humans . Carcinogenicity requires massive exposures, as is only encountered in well defined occupational settings, and is site specific, being specifically targeted to the lung and, in some cases, to the sinonasal cavity . Increased death rates for cancers at other sites, which were occasionally reported in some epidemiological studies, were almost invariably not statistically significant, and inconsistent (being counterbalanced by other studies which apparently showed decreased rates for the same cancers) . As we recently quantified in human body compartments, chromium(VI) can be reduced in body fluids and non-target cells, which results in its detoxification, due to the poor ability of chromium(III) to cross cell membranes . In target cells, chromium(VI) tends to be metabolized by a network of mechanisms leading to generation of reduced chromium species and reactive oxygen species, which will result either in activation or in detoxification depending on the site of the intracellular reduction and its proximity to DNA . When introduced by the oral route, chromium(VI) is efficiently detoxified upon reduction by saliva and gastric juice, and sequestration by intestinal bacteria . If some chromium(VI) is absorbed by the intestine, it is massively reduced in the blood of the portal system and then in the liver . These mechanisms explain the lack of genotoxicity, carcinogenicity, and induction of other long-term health effects of chromium (VI) by the oral route . Within the respiratory tract, chromium(VI) is reduced in the epithelial-lining fluid, pulmonary alveolar macrophages, bronchial tree and peripheral lung parenchyma cells . Hence, lung cancer can only be induced when chromium(VI) doses overwhelm these defense mechanisms . The efficient uptake and reduction of chromium(VI) in red blood cells explains its lack of carcinogenicity at a distance from the portal of entry into the body . All experimental and epidemiological data, and the underlying mechanisms, point to the occurrence of thresholds in chromium(VI) carcinogenesis.

Am J Pathol, 2000 Apr, 156(4), 1177 - 82
Microvascular effects of oral interleukin-6 on ischemia/reperfusion in the murine small intestine; Rollwagen FM et al.; Oral administration of interleukin-6 (IL-6) has been shown to reduce hemorrhage-induced bacterial translocation from the gut in mice and rats . To examine the intestinal microvasculature, mice were given the electron-dense tracer horseradish peroxidase (HRP) after hemorrhage and IL-6 or vehicle administration . In normal mice and in those hemorrhaged and given IL-6, the electron-dense marker, administered intravenously, could be found in intestinal capillaries and between mucosal epithelial cells, suggesting that the microvasculature was patent . In mice given saline after shock, however, no marker was present in the gut, suggesting that the intestinal microvasculature was unable to deliver the marker to the epithelia . When mice were given HRP intralumenally (il) the tracer was able to penetrate between intestinal epithelial cells only in mice given vehicle after hemorrhage . This finding suggests that hemorrhaged mice were susceptible to sepsis and endotoxic shock from the leaky gut . In normal and IL-6-treated mice, the tracer was unable to pass from the lumen between mucosal epithelial cells, because the presence of an intact zonula occludens prevented passage . Functional studies supported the electron microscopy findings . Bacteria were cultured from the livers of mice fed vehicle after hemorrhage, but not from those fed IL-6 . These data support the conclusions that parts of the intestinal microvasculature remain diminished after hemorrhage and resuscitation and that oral IL-6 restores this circulation.

Curr Opin Oncol, 2000 Mar, 12(2), 163 - 73
Melanoma vaccines; Brinckerhoff LH et al.; Remarkable advances in tumor vaccination have been made since Coley first deliberately infected cancer patients with both live and heat-killed bacteria . Melanoma is the most immunogenic solid tumor and, as such, has served as the major model for tumor vaccine investigation in both the laboratory and the clinic . Many advances in the field of melanoma vaccination have been based on an improved understanding of the cellular interaction required to induce a specific antitumor immune response . As a result of this new knowledge, many clinical trials of melanoma vaccines are now under way, and vaccines for metastatic melanoma have shown evidence of clinical effectiveness . This paper outlines the current status of melanoma vaccination.

Mol Biol Cell, 2000 Apr, 11(4), 1213 - 24
Axonal membrane proteins are transported in distinct carriers: a two-color video microscopy study in cultured hippocampal neurons; Kaether C et al.; Neurons transport newly synthesized membrane proteins along axons by microtubule-mediated fast axonal transport . Membrane proteins destined for different axonal subdomains are thought to be transported in different transport carriers . To analyze this differential transport in living neurons, we tagged the amyloid precursor protein (APP) and synaptophysin (p38) with green fluorescent protein (GFP) variants . The resulting fusion proteins, APP-yellow fluorescent protein (YFP), p38-enhanced GFP, and p38-enhanced cyan fluorescent protein, were expressed in hippocampal neurons, and the cells were imaged by video microscopy . APP-YFP was transported in elongated tubules that moved extremely fast (on average 4.5 micrometer/s) and over long distances . In contrast, p38-enhanced GFP-transporting structures were more vesicular and moved four times slower (0.9 micrometer/s) and over shorter distances only . Two-color video microscopy showed that the two proteins were sorted to different carriers that moved with different characteristics along axons of doubly transfected neurons . Antisense treatment using oligonucleotides against the kinesin heavy chain slowed down the long, continuous movement of APP-YFP tubules and increased frequency of directional changes . These results demonstrate for the first time directly the sorting and transport of two axonal membrane proteins into different carriers . Moreover, the extremely fast-moving tubules represent a previously unidentified type of axonal carrier.

J Pediatr Gastroenterol Nutr, 2000 Mar, 30(3), 276 - 82
A prospective trial of lansoprazole triple therapy for pediatric Helicobacter pylori infection; Shashidhar H et al.; BACKGROUND: Triple therapy with a proton-pump inhibitor and two antibiotics is widely used in the treatment of Helicobacter pylori infection in adults . Experience with such therapy in the pediatric population is limited . This was a prospective, nonrandomized, open-label trial to evaluate safety and efficacy of a combination of lansoprazole, clarithromycin, and amoxicillin in symptomatic children with H . pylori infection . METHODS: Children with H . pylori gastritis diagnosed by endoscopy performed for persistent nausea, vomiting, recurrent abdominal pain, and diarrhea with consistent histology were treated with the regimen of 0.45 mg/kg per day lansoprazole divided into two doses (maximum dose, 15 mg twice daily), amoxicillin 40 mg/kg per day in two doses (maximum dose, 1.0 g twice daily), and 250 mg clarithromycin twice daily (<10 years old) or 500 mg twice daily (>10 years old) for 2 weeks . Pre- and posttreatment endoscopic biopsy specimens were graded for the severity of gastritis and H . pylori density by a blinded pathologist . A questionnaire for assessing the severity of symptoms at the time of initial and second endoscopy were completed by patient and/or parent . RESULTS: Thirty-two children (age range, 1-25 years; mean age, 11 years; 19 females, 13 males) were treated with this regimen during an 18-month period . H . pylori organisms with varying grades of gastritis were present in tissue specimens of all patients . Only 28 children had follow-up endoscopy, which showed eradication of H . pylori in 15 (54%) children . Histologic symptoms of gastritis improved after therapy in the whole group . Overall, symptoms of vomiting, abdominal pain, diarrhea, anorexia, and halitosis significantly improved (P < 0.05) . Minor adverse effects of therapy occurred in 25% of patients . CONCLUSIONS: Symptoms, histologic, and endoscopic findings improved after triple therapy in children with H . pylori gastritis; however, eradication of bacteria was achieved in only 56% of children.

Baillieres Best Pract Res Clin Gastroenterol, 2000 Feb, 14(1), 1 - 12
Conceivable mechanisms by which Helicobacter pylori provokes duodenal ulcer disease; Olbe L et al.; A conceivable concept for the development of duodenal ulcers in Helicobacter pylori (H . pylori) infected subjects is presented in this chapter . The concept includes an explanation of the fact that only a minority of all H . pylori-infected subjects will develop a duodenal ulcer . Helicobacter pylori infection of the antrum induces a hypersecretion of gastric acid secretion, giving rise to gastric metaplasia in the duodenal bulb . This gastric metaplasia is a prerequisite for H . pylori colonization of the bulb . These events are common to all H . pylori-infected subjects . However, a much higher density of H . pylori bacteria and colonization with virulent organisms has been found in the bulb of duodenal ulcer patients, resulting in a much stronger inflammatory reaction with active duodenitis and an impaired bicarbonate secretion . These characteristics, together with acid hypersecretion, seem to be the important factors in evoking a duodenal ulcer.

Med Pregl, 1999 Nov-Dec, 52(11-12), 469 - 74
{Helicobacter pylori infection and a decrease in the pH of gastric juice as a possible cause of bleeding in erosive gastritis}; Stevanovic D et al.; INTRODUCTION: Bleeding erosive gastritis and ulcer disease are the most common causes of bleeding from the upper parts of the alimentary tract . Over the period of the last 10 years there has been an increased number of patients where Helicobacter pylori is mentioned as the possible cause of infection . At the same time in this group of patients pH values were decreased . During our research we wanted to examine the influence of local pH value and Helicobacter pylori infection on the occurrence of bleeding erosive gastritis . MATERIAL AND METHODS: During this research we examined two groups of patients . The following parameters were examined within these two groups of patients: 1 . presence of Helicobacter pylori infection; 2 . histopathologic changes in mucus; 3 . there were 57 patients with a bleeding form of gastritis of unknown etiology, whereas the second group consisted of 351 patients with non-bleeding form of gastritis . We examined the localization: 4 . local pH . RESULTS: The presence of Helicobacter pylori infection is statistically significantly higher in the group of patients with bleeding gastritis, when compared to the non-bleeding group (72%:57%) . Within the Helicobacter positive patients the most common change of mucose (over 90%) is active chronic gastritis . The presence of erosions and histopathological changes characteristic for Helicobacter pylori infection coincides with colonization of Helicobacter in the antrum . The pathogenetic action of bacteria in antrum decreases the local pH . Therefore the lowest value of local pH occurred in the group of bleeding Helicobacter pylori positive gastritis (1.72) . DISCUSSION: Results of this research helped us in treatment of helicobacter pylori positive patients making a risk group for development of recurrent bleeding . We accomplished therapeutical effect in 80% of patients by using a three component eradication therapeutic protocol for helicobacter pylori . Within the group of patients who have been cured, regression of histopathological changes occurred and increase of local pH took place . In the group with persistent infection we have come across with high percentage of recurrent bleeding (37.5%) . CONCLUSION: In our opinion helicobaceter pylori infection and decrease of local pH, which take place during the reaction of mucose on the presence of infection, are very aggressive factors which can cause bleeding erosions.

Nat Biotechnol, 2000 Apr, 18(4), 433 - 7
A genetic system for detection of protein nuclear import and export; Rhee Y et al.; We have developed a simple genetic assay to detect active nuclear localization (NLS) and export signals (NES) on the basis of their function within yeast cells . The bacterial LexA protein was modified (mLexA) to abolish its intrinsic NLS and fused to the activation domain of the yeast Gal4p (Gal4AD) with or without the SV40 large T-antigen NLS . In the import assay, if a tested protein fused to mLexA-Gal4AD contains a functional NLS, it will enter the cell nucleus and activate the reporter gene expression . In the export assay, if a tested protein fused to mLexA-SV40 NLS-Gal4AD contains a functional NES, it will exit into the cytoplasm, decreasing the reporter gene expression . We tested this system with known NLS and NES and then used it to demonstrate a NES activity of the capsid protein of a plant geminivirus . This approach may help to identify, analyze, and select for proteins containing functional NLS and NES.

Biochim Biophys Acta, 2000 May 1, 1465(1-2), 324 - 42
The role of aquaporins in cellular and whole plant water balance; Johansson I et al.; Aquaporins are water channel proteins belonging to the major intrinsic protein (MIP) superfamily of membrane proteins . More than 150 MIPs have been identified in organisms ranging from bacteria to animals and plants . In plants, aquaporins are present in the plasma membrane and in the vacuolar membrane where they are abundant constituents . Functional studies of aquaporins have hitherto mainly been performed by heterologous expression in Xenopus oocytes . A main issue is now to understand their role in the plant, where they are likely to be important both at the cellular and at the whole plant level . Plants contain a large number of aquaporin isoforms with distinct cell type- and tissue-specific expression patterns . Some of these are constitutively expressed, whereas the expression of others is regulated in response to environmental factors, such as drought and salinity . At the protein level, regulation of water transport activity by phosphorylation has been reported for some aquaporins.

Biochim Biophys Acta, 2000 May 1, 1465(1-2), 127 - 39
Molecular insights into the structure and function of plant K(+) transport mechanisms; Schachtman DP; Our understanding of plant potassium transport has increased in the past decade through the application of molecular biological techniques . In this review, recent work on inward and outward rectifying K(+) channels as well as high affinity K(+) transporters is described . Through the work on inward rectifying K(+) channels, we now have precise details on how the structure of these proteins determines functional characteristics such as ion conduction, pH sensitivity, selectivity and voltage sensing . The physiological function of inward rectifying K(+) channels in plants has been clarified through the analysis of expression patterns and mutational analysis . Two classes of outward rectifying K(+) channels have now been cloned from plants and their initial characterisation is reviewed . The physiological role of one class of outward rectifying K(+) channel has been demonstrated to be involved in long distance transport of K(+) from roots to shoots . The molecular structure and function of two classes of energised K(+) transporters are also reviewed . The first class is energised by Na(+) and shares structural similarities with K(+) transport mechanisms in bacteria and fungi . Structure-function studies suggest that it should be possible to increase the K(+) and Na(+) selectivity of these transporters, which will enhance the salt tolerance of higher plants . The second class of K(+) transporter is comprised of a large gene family and appears to have a dual affinity for K(+) . A suite of molecular techniques, including gene cloning, oocyte expression, RNA localisation and gene inactivation, is now being used to fully characterise the biophysical and physiological function of plants K(+) transport mechanisms.

Biochim Biophys Acta, 2000 May 1, 1465(1-2), 37 - 51
Vacuolar H(+)-pyrophosphatase; Maeshima M; The H(+)-translocating inorganic pyrophosphatase (H(+)-PPase) is a unique, electrogenic proton pump distributed among most land plants, but only some alga, protozoa, bacteria, and archaebacteria . This enzyme is a fine model for research on the coupling mechanism between the pyrophosphate hydrolysis and the active proton transport, since the enzyme consists of a single polypeptide with a calculated molecular mass of 71-80 kDa and its substrate is also simple . Cloning of the H(+)-PPase genes from several organisms has revealed the conserved regions that may be the catalytic site and/or participate in the enzymatic function . The primary sequences are reviewed with reference to biochemical properties of the enzyme, such as the requirement of Mg(2)(+) and K(+) . In plant cells, H(+)-PPase coexists with H(+)-ATPase in a single vacuolar membrane . The physiological significance and the regulation of the gene expression of H(+)-PPase are also reviewed.

J Biol Chem, 2000 Jul 28, 275(30), 23204 - 10
Structural model of the Fe-hydrogenase/cytochrome c553 complex combining transverse relaxation-optimized spectroscopy experiments and soft docking calculations; Morelli X et al.; Fe-hydrogenase is a 54-kDa iron-sulfur enzyme essential for hydrogen cycling in sulfate-reducing bacteria . The x-ray structure of Desulfovibrio desulfuricans Fe-hydrogenase has recently been solved, but structural information on the recognition of its redox partners is essential to understand the structure-function relationships of the enzyme . In the present work, we have obtained a structural model of the complex of Fe-hydrogenase with its redox partner, the cytochrome c(553), combining docking calculations and NMR experiments . The putative models of the complex demonstrate that the small subunit of the hydrogenase has an important role in the complex formation with the redox partner; 50% of the interacting site on the hydrogenase involves the small subunit . The closest contact between the redox centers is observed between Cys-38, a ligand of the distal cluster of the hydrogenase and Cys-10, a ligand of the heme in the cytochrome . The electron pathway from the distal cluster of the Fe-hydrogenase to the heme of cytochrome c(553) was investigated using the software Greenpath and indicates that the observed cysteine/cysteine contact has an essential role . The spatial arrangement of the residues on the interface of the complex is very similar to that already described in the ferredoxin-cytochrome c(553) complex, which therefore, is a very good model for the interacting domain of the Fe-hydrogenase-cytochrome c(553).

J Biol Chem, 2000 Jul 21, 275(29), 22255 - 67
DNA recognition, strand selectivity, and cleavage mode during integrase family site-specific recombination; Tribble G et al.; We have probed the association of Flp recombinase with its DNA target using protein footprinting assays . The results are consistent with the domain organization of the Flp protein and with the general features of the protein-DNA interactions revealed by the crystal structures of the recombination intermediates formed by Cre, the Flp-related recombinase . The similarity in the organization of the Flp and Cre target sites and in their recognition by the respective recombinases implies that the overall DNA-protein geometry during strand cleavage in the two systems must also be similar . Within the functional recombinase dimer, it is the interaction between two recombinase monomers bound on either side of the strand exchange region (or spacer) that provides the allosteric activation of a single active site . Whereas Cre utilizes the cleavage nucleophile (the active site tyrosine) in cis, Flp utilizes it in trans (one monomer donating the tyrosine to its partner) . By using synthetic Cre and Flp DNA substrates that are geometrically restricted in similar ways, we have mapped the positioning of the active and inactive tyrosine residues during cis and trans cleavage events . We find that, for a fixed substrate geometry, Flp and Cre cleave the labile phosphodiester bond at the same spacer end, not at opposite ends . Our results provide a model that accommodates local heterogeneities in peptide orientations in the two systems while preserving the global functional architecture of the reaction complex.

J Biol Chem, 2000 Jun 23, 275(25), 19224 - 30
Interaction of CbbR and RegA* transcription regulators with the Rhodobacter sphaeroides cbbIPromoter-operator region; Dubbs JM et al.; The form I (cbb(I)) Calvin-Benson-Bassham (CBB) reductive pentose phosphate cycle operon of Rhodobacter sphaeroides is regulated by both the transcriptional activator CbbR and the RegA/PrrA (RegB/PrrB) two-component signal transduction system . DNase I footprint analyses indicated that R . sphaeroides CbbR binds to the cbb(I) promoter between -10 and -70 base pairs (bp) relative to the cbb(I) transcription start . A cosmid carrying the R . capsulatus reg locus was capable of complementing an R . sphaeroides regA-deficient mutant to phototrophic growth with restored regulated synthesis of both photopigments and ribulose-bisphosphate carboxylase/oxygenase (Rubisco) . DNase I footprint analyses, using R . capsulatus RegA*, a constitutively active mutant version of RegA, detected four RegA* binding sites within the cbb(I) promoter . Two sites were found within a previously identified cbb(I) promoter proximal regulatory region from -61 to -110 bp . One of these proximal RegA* binding sites overlapped that of CbbR . Two sites were within a previously identified promoter distal positive regulatory region between -301 and -415 bp . Expression from promoter insertion mutants showed that the function of the promoter distal regulatory region was helical phase-dependent . These results indicated that RegA exerts its regulatory affect on cbb(I) expression through direct interaction with the cbb(I) promoter.

J Biol Chem, 2000 Jun 23, 275(25), 18692 - 7
Cysteine-scanning mutagenesis around transmembrane segments 1 and 11 and their flanking loop regions of Tn10-encoded metal-Tetracycline/H+ antiporter; Kimura-Someya T et al.; Putative transmembrane helices (TM) 1 and 11 in the metal-tetracycline/H(+) antiporter are predicted to be close to each other on the basis of disulfide cross-linking experiments of the double-cysteine mutants in the periplasmic loop regions (Kubo, Y., Konishi, S., Kawabe, T., Nada, S., and Yamaguchi, A . (2000) J . Biol . Chem . 275, 5270-5274) . In this study, each amino acid from Asn-2 to Gly-44 in the putative TM1 and loop1-2 regions or that from Ser-328 to Gly-366 in TM11 and its flanking regions was individually replaced with cysteine . With respect to the TM1 region, 10 mutants, from T5C to L14C, were all not reactive with N-ethylmaleimide (NEM), and from D15C to I22C, NEM-reactive and non-reactive mutations periodically appeared every two residues . Three mutants, M23C to V25C, were all NEM-reactive, but the degree of the latter two mutants was very low . Seven mutants, from L26C to E32C, were all highly reactive with NEM . Therefore, the region of TM1 is composed of the 21 amino acid residues from Thr-5 to Val-25 . It is a partially amphiphilic helix, that is, the N-terminal (cytoplasmic) half is embedded in the hydrophobic interior, and the C-terminal (periplasmic) half faces a water-filled channel . With respect to TM11, nine mutants, from S328C to G336C, and six mutants, from L361C to G366C, were all reactive with NEM . On the other hand, out of the 24 mutants, from L337C to S360C, 17 were not reactive with NEM, and the 7 NEM-reactive mutants were scattered, indicating that this region is a transmembrane segment . The 7 residues from Val-347 to Phe-353 including Pro-350 formed a central hydrophobic core, and the 7 NEM-reactive mutations were periodically distributed in its flanking regions, indicating that both ends of TM11 face a water-filled channel . Ala-354 is located at about 1/3 of the length from the periplasmic end of TM11 . Disulfide cross-linking experiments on double-cysteine mutants having the combination of A354C and a cysteine-scanning mutation in the loop1-2 region indicated that loop1-2 is very flexible and close to the periplasmic end of TM11 . Tetracycline prevented the cross-linking formation between the periplasmic ends of TM1 and TM11; however, it did not affect the cross-linking between loop1-2 and TM11, indicating that the substrate-induced conformational change involves a shift in the relative locations of TM1 and TM11.

J Clin Microbiol, 2000 Apr, 38(4), 1569 - 74
Borrelia burgdorferi B31 Erp proteins that are dominant immunoblot antigens of animals infected with isolate B31 are recognized by only a subset of human lyme disease patient sera; Miller JC et al.; Sera from animals infected with Borrelia burgdorferi isolates yield intense immunoblot signals from the B31 ErpA/I/N and ErpB/J/O proteins, which have apparent molecular masses of 19 and 60 kDa, respectively . Since B . burgdorferi proteins with those molecular masses are of immunodiagnostic importance, Lyme disease patient sera were used in studies of B31 lysates and recombinant B31 ErpA/I/N and ErpB/J/O proteins . Immunoblot analyses indicated that only a minority of the patients produced antibodies that recognized the tested B31 Erp proteins . Southern blot analyses of Lyme disease spirochetes cultured from 16 of the patients indicated that all these bacteria contain genes related to the B31 erpA/I/N and erpB/J/O genes, although signal strengths indicated only weak similarities in many cases, suggestive of genetic variability of erp genes among these bacteria . These data indicate that Erp proteins are generally not the 19- and 60-kDa antigens observed on serodiagnostic immunoblots.

J Clin Microbiol, 2000 Apr, 38(4), 1498 - 501
New agar medium for testing susceptibility of Mycobacterium tuberculosis to pyrazinamide; Heifets L et al.; A new agar medium to perform pyrazinamide (PZA) susceptibility testing with Mycobacterium tuberculosis has been developed . This medium has an acidic pH of 6.0 instead of the usual for agar media, pH 6.8, to provide optimal conditions for PZA activity, and it also differs from conventional Middlebrook 7H10/7H11 agar in that animal serum (fetal or calf bovine or fetal equine serum) is used instead of oleic acid-albumin-dextrose-catalase to support good growth of M . tuberculosis at the low pH of 6.0 . A critical concentration of 900 or 1,200 microg of PZA/ml in this medium made it possible to differentiate between PZA-susceptible and PZA-resistant clinical isolates . This agar medium has the following advantages compared to a liquid medium: it allows determination of the actual proportion of PZA-resistant bacteria in the isolate and it is simple and inexpensive . In addition, it has the potential of being used for a direct susceptibility test with PZA, but this approach will require further confirmation . Further studies to develop critical concentrations of other drugs for this low-pH medium, as well as to investigate the possibility of cultivation in regular (non-CO(2)) incubators, are in progress.

J Clin Microbiol, 2000 Apr, 38(4), 1390 - 6
Diagnosis and differentiation of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis infections in pigs by PCR amplification of the p36 and p46 genes; Caron J et al.; The genome of Mycoplasma hyopneumoniae encodes several immunodominant proteins, including a cytosolic protein (p36), three membranous proteins (p46, p65, and p74), and an adhesin (p97) . Cross-reactions with M . flocculare and M . hyorhinis reduce the specificity of conventional serological detection methods . However, certain antigenic determinants of the p36 and p46 proteins have been shown to be specific for M . hyopneumoniae . In the present study, pairs of oligonucleotide primers were designed to permit PCR amplification of entire p36 and p46 genes and of internal fragments of these genes . Specific amplicons could be obtained with as low as 0.5 to 50 pg of extracted chromosomal DNA . No amplification product was obtained when testing p36 and p46 primer pairs with genomic DNA or RNA from other mycoplasma species, bacteria, and viruses commonly associated with respiratory diseases in pigs . By using the single p36-PCR method, a positive reaction was demonstrated in 100% (30 of 30) of lungs from pigs that developed typical lesions associated with an M . hyopneumoniae infection, and no false-positive results were detected when 62 apparently normal lungs were tested . On the other hand, with the single p46-PCR method a sensitivity of 86.6% (26 of 30) and a specificity of 96.7% (60 of 62) were obtained in comparison with the necropsy findings . A mixed infection with M . hyorhinis was diagnosed in 13.3% (4 of 30) of the cases by using species-specific primers for the heterologous p37 gene . The sensitivity of the single p36-PCR method for the detection of M . hyopneumoniae, when tested on tracheobronchial swabs, was 100% (20 positive samples), with a specificity of 93.3% (14 of 15 negative samples), compared to the necropsy findings . Both expected amplicons were obtained with 86.6% (26 of 30) positive lungs when p36 and p46 primers were used simultaneously (multiplex PCR) to further increase the specificity of the PCR assay.

J Clin Microbiol, 2000 Apr, 38(4), 1364 - 9
Comparison of the nucleotide sequences of 16S rRNA, 444 Ep-ank, and groESL heat shock operon genes in naturally occurring Ehrlichia equi and human granulocytic ehrlichiosis agent isolates from Northern California; Chae JS et al.; We examined 11 naturally occurring isolates of Ehrlichia equi in horses and two human granulocytic ehrlichiosis agent isolates in California for sequence diversity in three genes . Ehrlichia equi isolates were from Sierra (n = 6), Mendocino (n = 3), Sonoma (n = 1), and Marin (n = 1) counties, and human granulocytic ehrlichiosis (HGE) agent isolates were obtained from Humboldt county . PCR with specific primers for 16S rRNA, 444 Ep-ank and groESL heat shock operon genes successfully produced amplicons for all 13 clinical samples . The 444 Ep-ank gene of the HGE agent and E . equi isolates from northern California is different from the eastern U.S . isolates BDS and USG3 . The translated amino acid sequence of the groESL heat shock operon gene fragment is identical among E . equi, the HGE agent, and E . phagocytophila, with the exception of the northern Californian equine CASOLJ isolate . Microheterogeneity was observed in the 16S rRNA gene sequences of HGE agent and E . equi isolates from northern California . These results suggest that E . equi and the HGE agent found in California are similar or identical but may differ from the isolates of equine and human origin found in the eastern United States.

J Cell Biol, 2000 Apr 3, 149(1), 33 - 40
Fusion of constitutive membrane traffic with the cell surface observed by evanescent wave microscopy; Toomre D et al.; Monitoring the fusion of constitutive traffic with the plasma membrane has remained largely elusive . Ideally, fusion would be monitored with high spatial and temporal resolution . Recently, total internal reflection (TIR) microscopy was used to study regulated exocytosis of fluorescently labeled chromaffin granules . In this technique, only the bottom cellular surface is illuminated by an exponentially decaying evanescent wave of light . We have used a prism type TIR setup with a penetration depth of approximately 50 nm to monitor constitutive fusion of vesicular stomatitis virus glycoprotein tagged with the yellow fluorescent protein . Fusion of single transport containers (TCs) was clearly observed and gave a distinct analytical signature . TCs approached the membrane, appeared to dock, and later rapidly fuse, releasing a bright fluorescent cloud into the membrane . Observation and analysis provided insight about their dynamics, kinetics, and position before and during fusion . Combining TIR and wide-field microscopy allowed us to follow constitutive cargo from the Golgi complex to the cell surface . Our observations include the following: (1) local restrained movement of TCs near the membrane before fusion; (2) apparent anchoring near the cell surface; (3) heterogeneously sized TCs fused either completely; or (4) occasionally larger tubular-vesicular TCs partially fused at their tips.

Microbiology, 2000 Mar, 146 ( Pt 3), 669 - 76
Fur-independent regulation of iron metabolism by Irr in Bradyrhizobium japonicum; Hamza I et al.; Bradyrhizobium japonicum expresses both Fur and Irr, proteins that mediate iron-dependent regulation of gene expression . Control of irr mRNA accumulation by iron was aberrant in a fur mutant strain, and Fur repressed an irr::lacZ promoter fusion in the presence of iron . Furthermore, metal-dependent binding of Fur to an irr gene promoter was demonstrated in a region with no significant similarity to the Fur-binding consensus DNA element . These data suggest that the modest control of irr transcription by iron is mediated by Fur . However, Irr protein levels were regulated normally by iron in the fur strain, indicating that Fur is not required for post-transcriptional control of the irr gene . Accordingly, regulation of hemB, a haem biosynthesis gene regulated by Irr, was controlled normally by iron in a fur strain . In addition, the hemA gene was shown to be controlled by Fur, but not by Irr . It was concluded that Fur cannot be the only protein by which B . japonicum cells sense and respond to iron, and that Irr may be involved in Fur-independent signal transduction . Furthermore, iron-dependent regulation of haem biosynthesis involves both Irr and Fur.

Zhonghua Yi Xue Za Zhi (Taipei), 2000 Mar, 63(3), 196 - 204
Splanchnic endotoxin levels in cirrhotic rats induced by carbon tetrachloride; Chu CJ et al.; BACKGROUND: Bacterial translocation (passage of intestinal bacteria to mesenteric lymph nodes) observed in cirrhosis may be a source of endotoxin that can stimulate nitric oxide production and participate in the pathogenesis of hyperdynamic circulation . Currently, there are no published data concerning splanchnic endotoxin levels in cirrhotic rats . This study was designed to determine systemic and portal hemodynamics and to detect endotoxins in the portal and systemic circulation . METHODS: Liver cirrhosis was induced by carbon tetrachloride intragastric gavage . Systemic and splanchnic endotoxin levels in control rats and cirrhotic rats with or without ascites were measured using a chromogenic Limulus assay . In addition, systemic and portal hemodynamic data were obtained using a thermodilution technique and catheterization . RESULTS: Cirrhotic rats with ascites had the lowest systemic vascular resistance (2.6 +/- 0.1 mmHg.ml-1.min.100 g body weight, BW) compared with control rats (6.3 +/- 0.3 mmHg.ml-1.min.100 g BW; p < 0.05) and cirrhotic rats without ascites (3.7 +/- 0.3 mmHg.ml-1.min.100 g BW; p < 0.05) . Cirrhotic rats with ascites displayed the highest splanchnic levels of endotoxin (10.6 +/- 3.1 pg/ml) compared with cirrhotic rats without ascites (2.0 +/- 0.7 pg/ml; p < 0.05) and control rats (2.0 +/- 0.4 pg/ml; p < 0.05) . There was no difference in the splanchnic endotoxin levels between control rats and cirrhotic rats without ascites (p > 0.05) . Similar results were observed with systemic endotoxin values (cirrhotic rats with ascites, 10.8 +/- 2.8 pg/ml; cirrhotic rats without ascites, 2.7 +/- 0.6 pg/ml; control rats, 2.5 +/- 0.4 pg/ml; p < 0.05) . A significant correlation existed between portal and systemic endotoxin values in cirrhotic rats with or without ascites (r = 0.96, p < 0.001 and r = 0.9, p < 0.05, respectively), whereas this correlation did not exist in control rats (r = 0.5, p > 0.05) . CONCLUSIONS: Cirrhotic rats with ascites had the lowest systemic vascular resistance and the highest splanchnic endotoxin levels when compared with cirrhotic rats without ascites and control rats . These results suggest that splanchnic endotoxemia may be involved in the development and/or maintenance of hyperdynamic circulation.

Hum Exp Toxicol, 2000 Jan, 19(1), 41 - 75
Radiation hormesis: its historical foundations as a biological hypothesis; Calabrese EJ et al.; This paper represents the first systematic effort to describe the historical foundations of radiation hormesis . Spanning the years from 1898 to the early 1940's the paper constructs and assesses the early history of such research and evaluates how advances in related scientific fields affected the course of hormetic related research . The present effort was designed to not only address this gap in current knowledge, but to offer a toxicological basis for how the concept of hormetic dose-response relationships may affect the nature of the bioassay and its role in the risk assessment process.

FEBS Lett, 2000 Mar 31, 470(3), 300 - 4
A gene fusion event in the evolution of aminoacyl-tRNA synthetases; Berthonneau E et al.; The genes of glutamyl- and prolyl-tRNA synthetases (GluRS and ProRS) are organized differently in the three kingdoms of the tree of life . In bacteria and archaea, distinct genes encode the two proteins . In several organisms from the eukaryotic phylum of coelomate metazoans, the two polypeptides are carried by a single polypeptide chain to form a bifunctional protein . The linker region is made of imperfectly repeated units also recovered as singular or plural elements connected as N-terminal or C-terminal polypeptide extensions in various eukaryotic aminoacyl-tRNA synthetases . Phylogenetic analysis points to the monophyletic origin of this polypeptide motif appended to six different members of the synthetase family, belonging to either of the two classes of aminoacyl-tRNA synthetases . In particular, the monospecific GluRS and ProRS from Caenorhabditis elegans, an acoelomate metazoan, exhibit this recurrent motif as a C-terminal or N-terminal appendage, respectively . Our analysis of the extant motifs suggests a possible series of events responsible for a gene fusion that gave rise to the bifunctional glutamyl-prolyl-tRNA synthetase through recombination between genomic sequences encoding the repeated units.

Curr Opin Microbiol, 2000 Apr, 3(2), 165 - 70
Two-component and phosphorelay signal transduction; Hoch JA; Two-component and phosphorelay signal transduction systems are the major means by which bacteria recognize and respond to a variety of environmental stimuli . Recent results have implicated these systems in the regulation of a variety of essential processes including cell-cycle progression, pathogenicity, and developmental pathways . Elucidation of the structures of the interacting domains is leading to an understanding of the mechanisms of molecular recognition and phosphotransfer in these systems.

J Chromatogr B Biomed Sci Appl, 2000 Feb 28, 739(1), 101 - 7
Determination of pyrroloquinoline quinone by capillary zone electrophoresis; Glatz Z et al.; A new method for the determination of pyrroloquinoline quinone by capillary zone electrophoresis has been developed . Separation conditions have been optimised with the respect to different parameters including pH and ionic strength of the background electrolyte, separation voltage and temperature of the capillary . A buffer consisting of 50 mM beta-alanine-HCl pH 3.0 was found to be the most suitable electrolyte for this separation . An applied voltage of 25 kV (negative polarity) and a temperature of 25 degrees C gave the best analysis of pyrroloquinoline quinone . The linear detection range for concentration versus peak area for the assay is from 5 to 500 microM (correlation coefficient 0.9998) with a detection limit of 0.1-0.2 microM . The inter-day reproducibility of the peak area was 2.5% and the inter-day reproducibility of the migration time was below 0.18%.

RNA, 2000 Mar, 6(3), 311 - 24
An unusual structure formed by antisense-target RNA binding involves an extended kissing complex with a four-way junction and a side-by-side helical alignment; Kolb FA et al.; The antisense RNA CopA binds to the leader region of the repA mRNA (target: CopT) . Previous studies on CopA-CopT pairing in vitro showed that the dominant product of antisense RNA-mRNA binding is not a full RNA duplex . We have studied here the structure of CopA-CopT complex, combining chemical and enzymatic probing and computer graphic modeling . CopI, a truncated derivative of CopA unable to bind CopT stably, was also analyzed . We show here that after initial loop-loop interaction (kissing), helix propagation resulted in an extended kissing complex that involves the formation of two intermolecular helices . By introducing mutations (base-pair inversions) into the upper stem regions of CopA and CopT, the boundaries of the two newly formed intermolecular helices were delimited . The resulting extended kissing complex represents a new type of four-way junction structure that adopts an asymmetrical X-shaped conformation formed by two helical domains, each one generated by coaxial stacking of two helices . This structure motif induces a side-by-side alignment of two long intramolecular helices that, in turn, facilitates the formation of an additional intermolecular helix that greatly stabilizes the inhibitory CopA-CopT RNA complex . This stabilizer helix cannot form in CopI-CopT complexes due to absence of the sequences involved . The functional significance of the three-dimensional models of the extended kissing complex (CopI-CopT) and the stable complex (CopA-CopT) are discussed.

Plant J, 2000 Jan, 21(2), 199 - 213
Allene oxide synthases of barley (Hordeum vulgare cv . Salome): tissue specific regulation in seedling development; Maucher H et al.; Allene oxide synthase (AOS) is the first enzyme in the lipoxygenase (LOX) pathway which leads to formation of jasmonic acid (JA) . Two full-length cDNAs of AOS designated as AOS1 and AOS2, respectively, were isolated from barley (H . vulgare cv . Salome) leaves, which represent the first AOS clones from a monocotyledonous species . For AOS1, the open reading frame encompasses 1461 bp encoding a polypeptide of 487 amino acids with calculated molecular mass of 53.4 kDa and an isoelectric point of 9.3, whereas the corresponding data of AOS2 are 1443 bp, 480 amino acids, 52.7 kDa and 7.9 . Southern blot analysis revealed at least two genes . Despite the lack of a putative chloroplast signal peptide in both sequences, the protein co-purified with chloroplasts and was localized within chloroplasts by immunocytochemical analysis . The barley AOSs, expressed in bacteria as active enzymes, catalyze the dehydration of LOX-derived 9- as well as 13-hydroperoxides of polyenoic fatty acids to the unstable allene oxides . In leaves, AOS mRNA accumulated upon treatment with jasmonates, octadecanoids and metabolizable carbohydrates, but not upon floating on abscisic acid, NaCl, Na-salicylate or infection with powdery mildew . In developing seedlings, AOS mRNA strongly accumulated in the scutellar nodule, but less in the leaf base . Both tissues exhibited elevated JA levels . In situ hybridizations revealed the preferential occurrence of AOS mRNA in parenchymatic cells surrounding the vascular bundles of the scutellar nodule and in the young convoluted leaves as well as within the first internode . The properties of both barley AOSs, their up-regulation of their mRNAs and their tissue specific expression suggest a role during seedling development and jasmonate biosynthesis.

Zhonghua Er Bi Yan Hou Ke Za Zhi, 1997 Feb, 32(1), 15 - 7
{Clinical and pathological observation of recurrent nasal polyp formation}; Chen W et al.; To investigate the mechanism of nasal polyp recurrence, 19 patients with recurrent nasal polyp were retrospectively analysed . In 4 patients, nasal polyp specimens had been sent for pathological examination more than three times, and in 6 patients, bacteria culture was performed . There were two types of recurrent nasal polyp: water-cyst type (fast recurrence) and edematous granulation type (slow recurrence) . Both types were the result of inflammatory reaction, perhaps related to trauma of operation . Water-cyst formation indicated stronger inflammatory reaction . During follow-up, 10 patients were cured and 9 recurred . Preventive measures against nasal polyp recurrence were discussed.

Zhonghua Er Bi Yan Hou Ke Za Zhi, 1997 Dec, 32(6), 348 - 9
{Accumulation sites of kanamycin in the organ of Corti by microautoradiography}; Ding D et al.; The accumulation sites of the 3H-labelled kanamycin in the organ of Corti were observed with electron microscope . The results showed that the accumulation of kanamycin was mainly in the mitochondria, cell membrane and stereocilia of the hair cells . The possible mechanism of the kanamycin accumulation in the mitochondria is their similarity with bacteria in the protein synthesis, i.e . the way that kanamycin might inhibit the protein synthesis in the mitochondria is similar to that of bacteria . The kanamycin accumulation in the cell membrane may indicate the entrance of kanamycin into the hair cells . The kanamycin deposition in the stereocilia was most likely related to its high chemical affinity with glycocalyx.

Appl Environ Microbiol, 2000 Apr, 66(4), 1692 - 7
Natural assemblages of marine proteobacteria and members of the Cytophaga-Flavobacter cluster consuming low- and high-molecular-weight dissolved organic matter; Cottrell MT et al.; We used a method that combines microautoradiography with hybridization of fluorescent rRNA-targeted oligonucleotide probes to whole cells (MICRO-FISH) to test the hypothesis that the relative contributions of various phylogenetic groups to the utilization of dissolved organic matter (DOM) depend solely on their relative abundance in the bacterial community . We found that utilization of even simple low-molecular-weight DOM components by bacteria differed across the major phylogenetic groups and often did not correlate with the relative abundance of these bacterial groups in estuarine and coastal environments . The Cytophaga-Flavobacter cluster was overrepresented in the portion of the assemblage consuming chitin, N-acetylglucosamine, and protein but was generally underrepresented in the assemblage consuming amino acids . The amino acid-consuming assemblage was usually dominated by the alpha subclass of the class Proteobacteria, although the representation of alpha-proteobacteria in the protein-consuming assemblages was about that expected from their relative abundance in the entire bacterial community . In our experiments, no phylogenetic group dominated the consumption of all DOM, suggesting that the participation of a diverse assemblage of bacteria is essential for the complete degradation of complex DOM in the oceans . These results also suggest that the role of aerobic heterotrophic bacteria in carbon cycling would be more accurately described by using three groups instead of the single bacterial compartment currently used in biogeochemical models.

Appl Environ Microbiol, 2000 Apr, 66(4), 1532 - 7
Sequencing and expression of additional xylanase genes from the hyperthermophile Thermotoga maritima FjSS3B.1; Reeves RA et al.; Two genes, xynB and xynC, coding for xylanases were isolated from Thermotoga maritima FjSS3B.1 by a genomic-walking-PCR technique . Sequencing of the genes showed that they encode multidomain family 10 xylanases . Only XynB exhibited activity against xylan substrates . The temperature optimum (87 degrees C) and pH optimum (pH 6.5) of XynB are different from the previously reported xylanase, XynA (also a family 10 enzyme), from this organism . The catalytic domain expressed without other domains has a lower temperature optimum, is less thermostable, and has optimal activity at pH 6.5 . Despite having a high level of sequence similarity to xynB, xynC appears to be nonfunctional since its encoded protein did not show significant activity on xylan substrates.

Appl Environ Microbiol, 2000 Apr, 66(4), 1369 - 74
16S rRNA gene-based detection of tetrachloroethene-dechlorinating Desulfuromonas and Dehalococcoides species; Loffler FE et al.; Members of the genera Desulfuromonas and Dehalococcoides reductively dechlorinate tetrachloroethene (PCE) and trichloroethene . Two primer pairs specific to hypervariable regions of the 16S rRNA genes of the Dehalococcoides group (comprising Dehalococcoides ethenogenes and Dehalococcoides sp . strain FL2) and the acetate-oxidizing, PCE-dechlorinating Desulfuromonas group (comprising Desulfuromonas sp . strain BB1 and Desulfuromonas chloroethenica) were designed . The detection threshold of a nested PCR approach using universal bacterial primers followed by a second PCR with the Desulfuromonas dechlorinator-targeted primer pair was 1 x 10(3) BB1 cells added per gram (wet weight) of sandy aquifer material . Total community DNA isolated from sediments of three Michigan rivers and six different chloroethene-contaminated aquifer samples was used as template in nested PCR . All river sediment samples yielded positive signals with the BB1- and the Dehalococcoides-targeted primers . One chloroethene-contaminated aquifer tested positive with the Dehalococcoides-targeted primers, and another contaminated aquifer tested positive with the Desulfuromonas dechlorinator-targeted primer pair . Restriction fragment analysis of the amplicons could discriminate strain BB1 from other known Desulfuromonas species . Microcosm studies confirmed the presence of PCE-dechlorinating, acetate-oxidizing Desulfuromonas and hydrogenotrophic Dehalococcoides species in samples yielding positive PCR signals with the specific primers.

Appl Environ Microbiol, 2000 Apr, 66(4), 1334 - 9
Effect of three factors in cheese production (pH, salt, and heat) on Mycobacterium avium subsp . paratuberculosis viability; Sung N et al.; Low pH and salt are two factors contributing to the inactivation of bacterial pathogens during a 60-day curing period for cheese . The kinetics of inactivation for Mycobacterium avium subsp . paratuberculosis strains ATCC 19698 and Dominic were measured at 20 degrees C under different pH and NaCl conditions commonly used in processing cheese . The corresponding D values (decimal reduction times; the time required to kill 1 log(10) concentration of bacteria) were measured . Also measured were the D values for heat-treated and nonheated M . avium subsp . paratuberculosis in 50 mM acetate buffer (pH 5.0, 2% {wt/vol} NaCl) and a soft white Hispanic-style cheese (pH 6.0, 2% {wt/vol} NaCl) . Samples were removed at various intervals until no viable cells were detected using the radiometric culture method (BACTEC) for enumeration of M . avium subsp . paratuberculosis . NaCl had little or no effect on the inactivation of M . avium subsp . paratuberculosis, and increasing NaCl concentrations were not associated with decreasing D values (faster killing) in the acetate buffer . Lower pHs, however, were significantly correlated with decreasing D values of M . avium subsp . paratuberculosis in the acetate buffer . The D values for heat-treated M . avium subsp . paratuberculosis ATCC 19698 in the cheese were higher than those predicted by studies done in acetate buffer . The heat-treated M . avium subsp . paratuberculosis strains had lower D values than the nonheated cells (faster killing) both in the acetate buffer (pH 5, 2% {wt/vol} NaCl) and in the soft white cheese . The D value for heat-treated M . avium subsp . paratuberculosis ATCC 19698 in the cheese (36.5 days) suggests that heat treatment of raw milk coupled with a 60-day curing period will inactivate about 10(3) cells of M . avium subsp . paratuberculosis per ml.

Mol Biol Evol, 2000 Apr, 17(4), 584 - 600
Molecular evolution and phylogenetic utility of Wolbachia ftsZ and wsp gene sequences with special reference to the origin of male-killing; Schulenburg JH et al.; A detailed assessment of the evolution and phylogenetic utility of two genes, ftsZ and wsp, was used to investigate the origin of male-killing Wolbachia, previously isolated from the ladybird Adalia bipunctata and the butterfly Acraea encedon . The analysis included almost all available sequences of B-group Wolbachia and two outgroup taxa and showed that (1) the two gene regions differ in phylogenetic utility, (2) sequence variation is here correlated with phylogenetic information content, (3) both genes show significant rate heterogeneity between lineages, (4) increased substitution rates are associated with homoplasy in the data, (5) wsp sequences of some taxa appear to be subject to positive selection, and (6) only a limited number of clades can be inferred with confidence due to either lack of phylogenetic information or the presence of homoplasy . With respect to the evolution of male-killing, the two genes nevertheless seemed to provide unbiased information . However, they consistently produce contradictory results . Current data therefore do not permit clarification of the origin of this behavior . In addition, A . bipunctata was found to be a host to two recently diverged strains of male-killing Wolbachia that showed increased substitution rates for both genes . Moreover, the wsp gene, which codes for an outer membrane protein, was found to be subject to positive selection in these taxa . These findings were postulated to be the product of high selection pressures due to antagonistic host-symbiont interactions in this ladybird species . In conclusion, our study demonstrates that the results of a detailed phylogenetic analysis, including characterization of the limitations of such an approach, can serve as a valuable basis for an understanding of the evolution of Wolbachia bacteria . Moreover, particular features of gene evolution, such as elevated substitution rates or the presence of positive selection, may provide information about the dynamics of Wolbachia-host associations.

Mol Biol Evol, 2000 Apr, 17(4), 576 - 83
A nucleomorph-encoded CbbX and the phylogeny of RuBisCo regulators; Maier UG et al.; Chloroplasts contain proteins that are encoded by different genetic systems, the plastid genome and the nuclear chromosomes . By comparing the gene content of plastid genomes of different taxa, some predictions about nuclear-encoded genes for plastid proteins are possible . However, early in evolution, many genes were transferred from the plastid to the cell nucleus and are therefore missing from all known plastid genomes and escape such predictions . By sequencing the miniaturized chromosomes of the nucleomorph of the cryptophyte Guillardia theta, as well as the plastid genome, we uncovered two genes encoding CbbX which are predicted to be involved in plastid function . Our findings suggest that (1) red-type plastid rbcLS genes evolved together with cbbX, which is related to cbbX genes of purple bacteria; (2) early in rhodoplast evolution, the cbbX gene was duplicated and transferred into the nucleus; (3) the plastid-encoded LysR transcriptional activator gene, rbcR, is homologous to rbcR and cbbR transcriptional activator genes of purple bacteria and cyanobacteria; and (4) the ancestral plastid probably harbored both types of form I RuBisCo.

Eur J Gastroenterol Hepatol, 2000 Feb, 12(2), 165 - 73
The effect of intra-gastric acidity and flora on the concentration of N-nitroso compounds in the stomach; Viani F et al.; BACKGROUND: Correa's hypothesis proposes that gastric carcinogenesis is due to atrophic gastritis and hypochlorhydria which permit gastric bacterial colonization, the reduction of dietary nitrates to nitrites and the formation of potentially carcinogenic N-nitroso compounds (NOCs) . OBJECTIVE: To test the hypothesis that omeprazole-induced hypochlorhydria is associated with increased intra-gastric concentrations of nitrate-reducing bacteria (NRB), nitrites and NOCs . DESIGN: Single-blind study in healthy volunteers . PARTICIPANTS: Fourteen healthy subjects (seven female, mean age 24 years), free of Helicobacter pylori infection, received a one-week course of placebo followed by a two-week course of omeprazole, 20 mg daily . METHODS: Fasted gastric samples, aspirated using a sterile double-lumen nasogastric tube at the end of the 1 st week (placebo) and the 2nd and 3rd weeks (omeprazole), were cultured aerobically and anaerobically; gastric pH and intra-gastric concentrations of nitrates, nitrites and NOCs were also determined . RESULTS: After weeks 1, 2 and 3, the intra-gastric concentrations of nitrate-reducing bacteria exceeded 10(5) colony-forming units (c.f.u.)/ml in 3, 7 and 9 subjects, respectively (P > 0.05) . A gastric pH greater than 4.0 was associated with increased NRB (P < 0.05); however, neither increased gastric pH nor increased NRB, alone or in combination, was associated with increased intra-gastric concentrations of nitrites or NOCs (P > 0.05) . CONCLUSIONS: A two-week increase in gastric pH in healthy, H . pylori-negative subjects was associated with increased intra-gastric concentrations of nitrate-reducing bacteria but not of nitrites or N-nitroso compounds . These data suggest that reduced gastric acid secretion is not a necessary precursor to the formation of carcinogenic N-nitroso compounds and that other mechanisms should be invoked to explain gastric carcinogenesis.

Biol Blood Marrow Transplant, 2000, 6(2), 100 - 8
Humoral immune response to proteins of human cytomegalovirus latency-associated transcripts; Landini MP et al.; Latent human cytomegalovirus (CMV) infection of hematopoietic progenitor cells is associated with the presence of latency-associated transcripts that may express 6 proteins larger than 44 amino acids in size (open reading frame {ORF} 55, ORF45, ORF94, ORF59, ORF154, ORF152/UL124) . The serologic response to these proteins was evaluated in healthy seropositive individuals as well as in individuals undergoing active CMV infection . Individual recombinant GST-fusion proteins, prepared from bacteria, were found by enzyme-linked immunosorbent assay to be recognized by between 8% and 44% long-term healthy seropositive individuals, with ORF94 and ORF55 being the most broadly and significantly recognized . Although nearly all of serum samples (85%) recognized at least 1 of these proteins, none reacted with all 6 . Patterns of antibody prevalence to these proteins in long-term seropositive individuals were similar to many antigens expressed during productive replication (IE1, ppUL57, ppUL83/pp65), but none were broadly detected by a majority of individuals, a characteristic of only a few productive-phase antigens, including ppUL44/ICP36 and ppUL32/pp150 . Consistent with prevalence in long-term seropositive individuals, commercial preparations of pooled human gamma globulin were also found to recognize latency-associated proteins . Serologic reactivity to latency-associated proteins was slow to develop following primary infection, in a pattern distinct from any of the characterized replication-phase proteins tested here, and was boosted late after secondary infection or reactivation in solid-organ transplant recipients without showing a correlation with viremia or disease . These results provide evidence that proteins expressed from the latent region during natural infection exhibit immunogenicity comparable with most other characterized viral antigens, although the narrow response to individual latency-associated proteins likely precludes their use in serologic assays to investigate clinical correlates or outcome in transplant recipients.

Exp Cell Res, 2000 Apr 10, 256(1), 150 - 7
Dispersive initiation of replication in the Chinese hamster rhodopsin locus; Dijkwel PA et al.; Several higher eukaryotic replication origins appear to be composed of broad zones of potential nascent strand start sites, while others are more circumscribed, resembling those of yeast, bacteria, and viruses . The most delocalized origin identified so far is approximately 55 kb in length and lies between the convergently transcribed dihydrofolate reductase (DHFR) and the 2BE2121 genes on chromosome 2 in the Chinese hamster genome . In some of our studies, we have utilized the rhodopsin origin as an early replicating internal standard for assessing the effects of deleting various parts of the DHFR locus on DHFR origin activity . However, it had not been previously established that the rhodopsin locus was located at a site far enough away to be immune to such deletions, nor had the mechanism of initiation at this origin been characterized . In the present study, we have localized the rhodopsin domain to a pair of small metacentric chromosomes and have used neutral/neutral 2-D gel replicon mapping to show that initiation in this origin is also highly delocalized, encompassing a region more than 50 kb in length that includes the nontranscribed rhodopsin gene itself . The initiation zone is flanked at least on one end by an actively transcribed gene that does not support initiation . Thus, the DHFR and rhodopsin origins belong to a class of complex, polydisperse origins that appears to be unique to higher eukaryotic cells .

J Investig Med, 2000 Mar, 48(2), 93 - 101
Pathogen analysis and genetic predisposition testing using microelectronic arrays and isothermal amplification; Edman CF et al.; BACKGROUND: A simple yet powerful tool for providing for rapid gene identification in the clinic would be the combination of isothermal gene amplification with electronic microchip analysis . This is a first report of such a union of these technologies . METHODS: The first assay demonstrates discrimination between four bacterial pathogens . For this, one portion of the bacterial 16S rRNA gene encompassing a microheterogeneous region was isothermally amplified using Strand Displacement Amplification (SDA) . Type identification was then made by "sandwich" assay format either using selective electronic hybridization of amplicons to sequence-specific capture oligonucleotides and a universal, fluorescently labeled reporter oligonucleotide, or, alternatively, sequence-specific reporters and a universal capture oligonucleotide . The second assay tested for the presence or absence of the Factor V Leiden point mutation using DNA obtained from 18 patients in a blind assay . For this, allele-specific SDA was developed . Following amplification using a sense-biotinylated primer and either the corresponding antisense wild type or mutant primer, multiple patient amplicons were targeted to specified locations on the microarray and visualized using a fluorescently labeled reporter oligonucleotide . Positive signals were scored as greater than or equal to two times the background . RESULTS: Bacterial type-specific signals were between 3- to 10-fold greater than nonspecific in both assay formats . Using allele-specific SDA, 100% agreement was observed between PAGE analysis, microarray results, and clinical diagnosis in Factor V mutation analysis . CONCLUSIONS: We demonstrated two model clinical assays combining amplified materials and microelectronic arrays, one potentially suitable for pathogen screening and the other for a deleterious genetic mutation.

Dev Comp Immunol, 2000 Jun, 24(4), 417 - 32
Interaction between turkey monocytes and avian Chlamydia psittaci in the presence of Mycoplasma sp.: the importance of nitric oxide; Van Nerom A et al.; The interaction between Chlamydia psittaci and turkey monocytes was studied in vitro . Purified monocytes were inoculated with C . psittaci, in the presence or absence of Mycoplasma hyorhinis . Whereas turkey monocytes produced high amounts of nitric oxide (NO) following the inoculation with M . hyorhinis, inoculation with C . psittaci did not induce NO production in these phagocytes . The monocytes strongly supported chlamydial growth, as demonstrated by the presence of inclusion forming units, the positive direct immunofluorescence staining and transmission electron microscopy . In contrast, upon co-inoculation of the monocytes with C . psittaci and M . hyorhinis, a reduced replication rate of C . psittaci was observed . N(G)-monomethyl-L-Arginine, a competitive inhibitor for the enzyme NO-synthase, inhibited the NO production and reversed the antichlamydial activity of the M . hyorhinis co-inoculated turkey monocytes . These results imply two considerations . First, as chlamydiae are obligate intracellular bacteria, special care should be taken to guard chlamydial cultures from mycoplasmal contamination, in order to prevent false results when investigating the response of immunomodulating cells to chlamydial infection . Secondly, as a mycoplasmal co-infection in vitro has the capacity of inducing antichlamydial activity in turkey monocytes, through the action of NO, it could be suggested that a similar interaction might take place in vivo . Moreover, it was shown that avian M . gallisepticum strains were also able to induce NO in turkey monocytes . Considering the high prevalence of both C . psittaci and Mycoplasma sp . in turkeys, this interaction, through the pivotal role of NO, might influence the outcome of respiratory diseases in turkeys.

Scand J Immunol, 2000 Apr, 51(4), 345 - 53
Protective DNA immunization against Chlamydia pneumoniae; Svanholm C et al.; We have investigated the efficacy of the DNA vaccination using the heat shock protein 60 (HSP-60) gene of C . pneumoniae, for protection of mice against infection with the bacteria . C57Bl/6 mice had a 5-20-fold reduction of C . pneumoniae numbers in lungs when immunized intranasally (i.n.) with plasmids (p) encoding pHSP-60 . The reduction of the bacterial load coincided with a decreased severity of disease . No specific antibodies were detected after protective i . n . immunization . In contrast, mice immunized intradermally (i.d.) were not protected against challenge with C . pneumoniae, although specific humoral Immunoglobulin (Ig)G responses were generated . Co-inoculation i.n . of pHSP-60 with pIL-12 but not with pGM-CSF further increased protection of mice against infection with C . pneumoniae . Lungs from pHSP-60 i.n . immunized and infected mice showed higher levels of interferon (IFN)-gamma mRNA, and spleen cells from these mice co-cultured with r-HSP-60 released higher levels of IFN-gamma and displayed higher proliferative responses than nonimmunized and infected controls . pHSP-60 immunized IFN-gamma receptor (R)-/- mice were not protected against infection with C . pneumoniae . Likewise, i.n . administration of pIFN-gamma alone induced significant protection . DNA vaccine-induced protection was CD4+ and CD8+ T-cell dependent, as shown by DNA-vaccination of MHC class II-/-, CD4-/-, CD8-/- and CD4-/-CD8-/-mice . Interestingly, DNA vaccine induced CD4+ T cells, in the absence of CD8+ T cells, were involved in worsening the outcome of infection . This worsening was linked with a shift towards a Th2 cytokine pattern.

J Appl Microbiol, 2000 Feb, 88(2), 358 - 63
Influence of physiological factors on the lysis effect of Cytophaga on the red microalga Rhodella reticulata; Toncheva-Panova T et al.; The influence of different factors on the lysis of the red microalga, Rhodella reticulata, by Cytophaga sp . LR2 was studied . The pathogenic bacterial strain was more resistant than the alga to the physiological parameters studied, which assured long-term survival of bacteria in algal cultures . Cytophaga sp . LR2 infected R . reticulata at temperatures between 15 and 30 degrees C, in the illuminated as well as the non-illuminated cultures, at pH values between 5.0 and 9.0, and in the presence of NaCl and CaCl2 in the culture medium . SEM showed a different morphology of the bacteria in algal cultures from those of axenic cultures of Cytophaga . Observations of specific associations between algal and bacterial cells revealed that the role of the slime extrusions on the bacterial surface was attachment of Cytophaga to algal cells, and that their clumping leads to rapid lysis.

J Bacteriol, 2000 Apr, 182(8), 2068 - 76
Molecular characterization of two-component systems of Helicobacter pylori; Beier D et al.; Two-component systems are frequently involved in the adaptation of bacteria to changing environmental conditions at the level of transcriptional regulation . Here we report the characterization of members of the two-component systems of the gastric pathogen Helicobacter pylori deduced from the genome sequence of strain 26695 . We demonstrate that the response regulators HP166, HP1043, and HP1021 have essential functions, as disruption of the corresponding genes is lethal for the bacteria, irrespective of the fact that HP1043 and HP1021 have nonconserved substitutions in crucial amino acids of their receiver domains . An analysis of the in vitro phosphorylation properties of the two-component proteins demonstrates that HP244-HP703 and HP165-HP166 are cognate histidine kinase-response regulator pairs . Furthermore, we provide evidence that the variability of the histidine kinase HP165 caused by a poly(C) tract of variable length close to the 3' end of open reading frame 165/164 does not interfere with the kinase activity of the transmitter domain of HP165.

J Appl Microbiol, 2000 Jan, 88(1), 98 - 106
Chromium(VI) reductase activity is associated with the cytoplasmic membrane of anaerobically grown Shewanella putrefaciens MR-1; Myers CR et al.; Shewanella putrefaciens MR-1 can reduce a diverse array of compounds under anaerobic conditions, including manganese and iron oxides, fumarate, nitrate, and many other compounds . These reductive processes are apparently linked to a complex electron transport system . Chromium (Cr) is a toxic and mutagenic metal and bacteria could potentially be utilized to immobilize Cr by reducing the soluble and bioavailable state, Cr(VI), to the insoluble and less bioavailable state, Cr(III) . Formate-dependent Cr(VI) reductase activity was detected in anaerobically grown cells of S . putrefaciens MR-1, with highest specific activity in the cytoplasmic membrane . Both formate and NADH served as electron donors for Cr(VI) reductase, whereas L-lactate or NADPH did not support any activity . The addition of 10 micromol l(-1) FMN markedly stimulated formate-dependent Cr(VI) reductase, and the activity was almost completely inhibited by diphenyliodonium chloride, an inhibitor of flavoproteins . Cr(VI) reductase activity was also inhibited by p-chloromercuriphenylsulphonate, azide, 2-heptyl-4-hydroxyquinolone-N-oxide, and antimycin A, suggesting involvement of a multi-component electron transport chain which could include cytochromes and quinones . Cr(V) was detected by electron paramagnetic resonance (EPR) spectroscopy, suggesting a one-electron reduction as the first step.

Ann Plast Surg, 2000 Mar, 44(3), 330 - 3
A prosthetic breast implant infected with Mycobacterium fortuitum; Heistein JB et al.; Augmentation mammaplasty is a common operation performed in the United States . Postoperative wound infections are rare, but can be devastating . Most often, bacteria from the normal skin flora cause these infections, but more atypical organisms can lead to similar situations . The authors present a case of a prosthetic breast implant infected with Mycobacterium fortuitum after augmentation mammaplasty . The patient, diagnosis, and treatment are discussed so that others may recognize and treat this entity successfully before encountering major complications . Although it is an infrequent occurrence, plastic surgeons, infectious disease specialists, and primary care doctors who may see postoperative wound infections should be aware of this potential pathogen . It is important in any postimplant infection and especially crucial in cases of unresolving or recurrent infections with unusual or even clear drainage . With proper identification through acid-fast smear and culture, multiagent therapy can be initiated early . Additional complications, including implant removal, may thus be avoided.

Gastroenterology, 2000 Apr, 118(4), 749 - 59
Induction and maintenance of immune effector cells in the gastric tissue of mice orally immunized to Helicobacter pylori requires salivary glands; Shirai Y et al.; BACKGROUND & AIMS: Helicobactor pylori mostly colonizes the gastric mucus that contains salivary antibodies . We studied the role of saliva in the induction and maintenance of gastric immunity conferred by oral vaccination against H . pylori . METHODS: C57BL/6 mice underwent a sialoadenectomy before and after intragastric immunization using whole-cell sonicates of H . pylori and cholera toxin as an adjuvant . At 1 and 6 months after oral inoculation, we assessed the density of the H . pylori colonizing the stomach, specific antibodies in gastric secretion and sera, and the constituents of cellular infiltrates in the tissue . RESULTS: A sialoadenectomy before, but not after, immunization abrogated protection by the vaccination at 1 month after inoculation . Protected mice had more neutrophils, plasma cells, and lymphocytes, but fewer eosinophils, in the gastric tissue than nonprotected mice . Protected mice had a greater increase of immunoglobulin (Ig) G1 specific to H . pylori than IgG2a in sera . At 6 months after inoculation, oral immunization was less effective in mice who had a sialoadenectomy than in control immunized mice . The antibody titers in both gastric secretion and in sera did not correlate with the density of bacteria colonizing the stomach . CONCLUSIONS: It is suggested that, in intragastric immunization against H . pylori, saliva is necessary for both the induction and maintenance of optimal immunity in the stomach . Effective immunity was associated with an increased number of neutrophils and lymphocytes in gastric tissue.

Biophys J, 2000 Apr, 78(4), 2127 - 37
Global analysis of fluorescence lifetime imaging microscopy data; Verveer PJ et al.; Global analysis techniques are described for frequency domain fluorescence lifetime imaging microscopy (FLIM) data . These algorithms exploit the prior knowledge that only a limited number of fluorescent molecule species whose lifetimes do not vary spatially are present in the sample . Two approaches to implementing the lifetime invariance constraint are described . In the lifetime invariant fit method, each image in the lifetime image sequence is spatially averaged to obtain an improved signal-to-noise ratio . The lifetime estimations from these averaged data are used to recover the fractional contribution to the steady-state fluorescence on a pixel-by-pixel basis for each species . The second, superior, approach uses a global analysis technique that simultaneously fits the fractional contributions in all pixels and the spatially invariant lifetimes . In frequency domain FLIM the maximum number of lifetimes that can be fit with the global analysis method is twice the number of lifetimes that can be fit with conventional approaches . As a result, it is possible to discern two lifetimes with a single-frequency FLIM setup . The algorithms were tested on simulated data and then applied to separate the cellular distributions of coexpressed green fluorescent proteins in living cells.

Photochem Photobiol, 2000 Mar, 71(3), 263 - 72
DNA damage induced by 4,6,8,9-tetramethyl-2H-furo{2,3-h}quinolin-2-one, a new furocoumarin analog: biological consequences; Marzano C et al.; 4,6,8,9-Tetramethyl-2H-furo{2,3-h}quinolin-2-one (HFQ) and its isomer FQ (1,4,6,8-tetramethyl-2H-furo{2,3-h}quinolin-2-one) showed very strong antiproliferative activity in mammalian cells, about two times greater than 8-methoxypsoralen (8-MOP) . Both compounds induced DNA-protein cross-links (DPC) but not interstrand cross-links . The FQ generated DPC in a biphotonic process, yielding a new kind of diadduct, whereas HFQ induced DPC by a monophotonic one, probably without its physical participation in the covalent bridge . These lesions gave different toxic responses . Sensitization of FQ led to extensive DNA fragmentation and to a number of chromosomal aberrations . Conversely, HFQ seemed to be completely inactive and 8-MOP gave intermediate results . A strict relationship between DPC formation and induction of chromosomal aberrations was observed . The HFQ did not induce light skin erythemas, whereas FQ was more phototoxic than 8-MOP, thus suggesting that FQ lesions, DPC in particular, may be implicated in skin phototoxicity . Ehrlich ascites cells, a transplantable mouse tumor, inactivated by furoquinolinone sensitization and injected into healthy mice, protected them from a successive challenge by viable tumor cells . This response appeared to be based on an immune mechanism . Comparable amounts of base substitution revertants were scored when testing furoquinolinones and 8-MOP in bacteria but no DPC were detected . This suggests that classic mutagenesis tests on bacteria are insufficient to give adequate information on furocoumarin genotoxicity . Given its features, HFQ can be regarded as an interesting new agent for psoralen plus UVA photochemotherapy and photopheresis.

Vet Microbiol, 2000 Apr 4, 73(1), 1 - 12
Effect of temperature modulation and bvg mutation of Bordetella bronchiseptica on adhesion, intracellular survival and cytotoxicity for swine alveolar macrophages; Brockmeier SL et al.; Bordetella bronchiseptica causes respiratory disease in swine, yet there are no studies examining the interaction of B . bronchiseptica with swine alveolar macrophages . A swine isolate of B . bronchiseptica was able to adhere to, and survive intracellularly in, swine alveolar macrophages, but the relative ability of the bacteria to accomplish these functions was dependent on its phenotypic phase and culture conditions . More bacteria were observed extracellularly as well as intracellularly by immunofluorescent staining when B . bronchiseptica was cultured at 23 degrees C as compared to 37 degrees C . However, more bacteria cultured at 37 degrees C were found surviving intracellularly after the macrophages were cultured with polymyxin B to kill extracellular bacteria . Similar results were seen in experiments performed with an isogenic Bvg(-) phase-locked mutant of B . bronchiseptica cultured at 37 or 23 degrees C, indicating that another temperature dependent mechanism in addition to bvg may play a role in adhesion and intracellular survival . B . bronchiseptica was cytotoxic for swine alveolar macrophages in the Bvg(+) phase only . The cytotoxicity of B . bronchiseptica for alveolar macrophages, and its ability to survive phagocytosis, are no doubt important to escape from immune clearance mechanisms and establish infection, and could leave the host susceptible to secondary respiratory pathogens.

FEMS Microbiol Lett, 2000 Apr 1, 185(1), 17 - 22
Identification of a 36-kDa fibronectin-binding protein expressed by a virulent variant of Leptospira interrogans serovar icterohaemorrhagiae; Merien F et al.; We investigated the ability of a virulent strain of Leptospira interrogans serovar icterohaemorrhagiae, its isogenic avirulent variant and a saprophytic strain to bind fibronectin using alkaline phosphatase-labelled fibronectin . A single 36-kDa fibronectin-binding protein was expressed only by the virulent strain and was located in the outer sheath according to proteinase K treatment results . The interaction of this protein with fibronectin was specific and the region of fibronectin bound to this potential adhesin overlapped the gelatin-binding domain . The inability of a RGDS synthetic peptide to inhibit the binding of fibronectin indicated that the cell-binding domain was not involved in this interaction . Considering the wide distribution of fibronectin within a host and the diversity of mammals involved in the epidemiology of leptospirosis, its implication in the cell attachment process of virulent leptospires is coherent with the multiplicity of target cells.

Int J Parasitol, 2000 Apr 10, 30(4), 411 - 9
The filarial genome project: analysis of the nuclear, mitochondrial and endosymbiont genomes of Brugia malayi; Williams SA et al.; The Filarial Genome Project (FGP) was initiated in 1994 under the auspices of the World Health Organisation . Brugia malayi was chosen as the model organism due to the availability of all life cycle stages for the construction of cDNA libraries . To date, over 20000 cDNA clones have been partially sequenced and submitted to the EST database (dbEST) . These ESTs define approximately 7000 new Brugia genes . Analysis of the EST dataset provides useful information on the expression pattern of the most abundantly expressed Brugia genes . Some highly expressed genes have been identified that are expressed in all stages of the parasite's life cycle, while other highly expressed genes appear to be stage-specific . To elucidate the structure of the Brugia genome and to provide a basis for comparison to the Caenorhabditis elegans genome, the FGP is also constructing a physical map of the Brugia chromosomes and is sequencing genomic BAC clones . In addition to the nuclear genome, B . malayi possesses two other genomes: the mitochondrial genome and the genome of a bacterial endosymbiont . Eighty percent of the mitochondrial genome of B . malayi has been sequenced and is being compared to mitochondrial sequences of other nematodes . The bacterial endosymbiont genome found in B . malayi is closely related to the Wolbachia group of rickettsia-like bacteria that infects many insect species . A set of overlapping BAC clones is being assembled to cover the entire bacterial genome . Currently, half of the bacterial genome has been assembled into four contigs . A consortium has been established to sequence the entire genome of the Brugia endosymbiont . The sequence and mapping data provided by the FGP is being utilised by the nematode research community to develop a better understanding of the biology of filarial parasites and to identify new vaccine candidates and drug targets to aid the elimination of human filariasis.

J Trop Pediatr, 2000 Feb, 46(1), 21 - 4
Viral isolates during febrile neutropaenia in children with cancer; Uys R et al.; We prospectively studied South African children with cancer for viral isolates during episodes of febrile neutropaenia . Viruses were found in seven (31.8 per cent) and bacteria in five (22.7 per cent) of 22 episodes . The most common isolate was the herpes simplex virus and the most common source was from nasopharyngeal aspirates . There was no dual detection of viral and bacterial isolates . This study emphasizes the important contribution of viruses to febrile neutropaenia.

Cells Tissues Organs, 2000, 166(2), 233 - 46
Early embryonic coats: morphology, function, practical applications . An overview; Herrler A et al.; Mammalian egg and embryo coats are primarily represented by the zona pellucida which, however, undergoes biochemical and structural changes during fertilization and embryo development . It serves several functions, from ovulation until shortly before implantation . Initially the zona pellucida induces sperm-oocyte interaction, acrosome reaction and prevents polyspermy . Later, it prevents disaggregation of the noncompacted blastomeres and the premature attachment to the oviductal and endometrial surface . Additionally, it protects the embryo against toxins and xenobiotics, as well as bacteria, viruses and phagocytes . As the embryo is covered by the zona pellucida until immediately before implantation, all signals of embryo-maternal signalling have to pass the zona and are detectable within it . Logically we may define the zona pellucida as a mailbox of the embryo-maternal signalling, especial for investigating these messages . Oviductal, uterine and embryonic proteins are incorporated into the zona pellucida as embryonic development goes on and change the zona's morphological and biochemical properties . Whether a protein is able to penetrate the zona, whether it accumulates within the zona or whether it is rejected by the zona depends on its biochemical properties . Three specific proteins have been detected within the embryonic coats (IGFBP3, HB-EGF, P19) . New insights into the physiology of the zona pellucida might present new achievements in the in vitro culture of embryos, and present new aspects as to how to prevent zona hardening . Furthermore, knowledge of the zona proteins enables the development of immunocontraceptive vaccines . Consequently, the zona pellucida is not only significant with regard to fertilization, but also during early embryonic development . Investigations of the zona pellucida will yield new insights into early embryo-maternal signalling which in turn may lead to improvements in classic IVF and new approaches to in vitro culture.

J Virol, 2000 Apr, 74(8), 3642 - 9
Identification of amino acid residues in CD81 critical for interaction with hepatitis C virus envelope glycoprotein E2; Higginbottom A et al.; Human CD81 has been previously identified as the putative receptor for the hepatitis C virus envelope glycoprotein E2 . The large extracellular loop (LEL) of human CD81 differs in four amino acid residues from that of the African green monkey (AGM), which does not bind E2 . We mutated each of the four positions in human CD81 to the corresponding AGM residues and expressed them as soluble fusion LEL proteins in bacteria or as complete membrane proteins in mammalian cells . We found human amino acid 186 to be critical for the interaction with the viral envelope glycoprotein . This residue was also important for binding of certain anti-CD81 monoclonal antibodies . Mutating residues 188 and 196 did not affect E2 or antibody binding . Interestingly, mutation of residue 163 increased both E2 and antibody binding, suggesting that this amino acid contributes to the tertiary structure of CD81 and its ligand-binding ability . These observations have implications for the design of soluble high-affinity molecules that could target the CD81-E2 interaction site(s).

Lett Appl Microbiol, 2000 Jan, 30(1), 42 - 6
Triton X-114 phase partitioning for the isolation of a pediocin-like bacteriocin from Carnobacterium divergens; Metivier A et al.; A new procedure combining Triton X-114 phase partitioning and cation exchange chromatography was developed to purify a bacteriocin from a complex culture medium . This pediocin-like bacteriocin, secreted by Carnobacterium divergens and named divercin V41, was entirely recovered in the lower detergent-rich phase whereas all other substances (compounds from culture medium, bacterial metabolites) remained in the upper detergent-poor phase . Subsequent cation-exchange chromatography of the TX-114-rich phase allowed recovery of the pure active bacteriocin and also detergent removing . This new purification method is versatile, fast (only two steps) and can be carried out on whole broth.

Eur J Biochem, 2000 Apr, 267(7), 2079 - 87
Requirements for the mitochondrial import and localization of dihydroorotate dehydrogenase; Rawls J et al.; In animals, dihydroorotate dehydrogenase (DHODH) is a mitochondrial protein that carries out the fourth step in de novo pyrimidine biosynthesis . Because this is the only enzyme of this pathway that is localized to mitochondria and because the enzyme is cytosolic in some bacteria and fungi, we carried out studies to understand the mode of targeting of animal DHODH and its submitochondrial localization . Analysis of fractionated rat liver mitochondria revealed that DHODH is an integral membrane protein exposed to the intermembrane space . In vitro-synthesized Drosophila, rat and human DHODH proteins were efficiently imported into the intermembrane space of isolated yeast mitochondria . Import did not alter the size of the in vitro synthesized protein, nor was there a detectable size difference when compared to the DHODH protein found in vivo . Thus, there is no apparent proteolytic processing of the protein during import either in vitro or in vivo . Import of rat DHODH into isolated yeast mitochondria required inner membrane potential and was at least partially dependent upon matrix ATP, indicating that its localization uses the well described import machinery of the mitochondrial inner membrane . The DHODH proteins of animals differ from the cytosolic proteins found in some bacteria and fungi by the presence of an N-terminal segment that resembles mitochondrial-targeting presequences . Deletion of the cationic portion of this N-terminal sequence from the rat DHODH protein blocked its import into isolated yeast mitochondria, whereas deletion of the adjacent hydrophobic segment resulted in import of the protein into the matrix . Thus, the N-terminus of the DHODH protein contains a bipartite signal that governs import and correct insertion into the mitochondrial inner membrane.

Biochem J, 2000 Apr 1, 347 Pt 1, 285 - 9
Protein kinase C-beta contributes to NADPH oxidase activation in neutrophils; Dekker LV et al.; We have analysed the involvement of the beta isotype of the protein kinase C (PKC) family in the activation of NADPH oxidase in primary neutrophils . Using immunofluorescence and cell fractionation, PKC-beta is shown to be recruited to the plasma membrane upon stimulation with phorbol ester and to the phagosomal membrane upon phagocytosis of IgG-coated particles (Fcgamma-receptor stimulus) . The time course of recruitment is similar to that of NADPH oxidase activation by these stimuli . The PKC-beta specific inhibitor 379196 inhibits the response to PMA as well as to IgG-coated bacteria . Partial inhibition occurs between 10 and 100 nM of inhibitor, the concentration at which PKC-beta, but not other PKC isotypes, is targeted . Neutrophils isolated from a mouse that lacks PKC-beta also showed an inhibition of NADPH oxidase activation by PMA and IgG-coated particles . The level of inhibition is comparable to that achieved with 379196 in human neutrophils . Thus the PKC-beta isotype mediates activation of NADPH oxidase by PMA and by stimulation of Fcgamma receptors in neutrophils.

J Dent Res, 2000 Feb, 79(2), 770 - 7
The pH of dental plaque in its relation to early enamel caries and dental plaque flora in humans; Lingstrom P et al.; Dental caries appears to result from the action of multiple, interrelated factors . A companion study dealt with the plaque-flora/caries relationship (van Ruyven et al., 2000) . The plaque-pH/caries relationship is the subject of this study . Since both studies involve the same subjects, plaques, and tooth surfaces, data on the examined factors have also been integrated . In vivo plaque pH determinations (microelectrode) were done on buccal sound (s) and "white-spot" (ws) caries surfaces in a selected dentition area in a low-caries (no ws) and higher-caries subject group . The pH response to sugar was evaluated before and after a sugar rinse, a local sugar application, or sucking on a sugary lozenge . pH profiles with sugar rinsing and normal or limited salivary flow conditions, showed progressively decreasing plaque pH values at various time points in the order of: low-caries subjects (s sites), higher-caries subjects (s sites), higher-caries subjects (s + ws sites), and higher-caries subjects (ws sites) . The minimum pH values showed the same trend . Analyses of all data indicated only a statistical difference for minimum values for s sites in low-caries subjects vs . ws sites in higher-caries subjects, and for s and ws sites in the latter . Local sugar application and sucking on a sugary lozenge induced smaller pH drops than sugar rinsing; such suboptimal sugar exposure caused a disappearance of the difference between the minimum pH values for s and ws sites observed with sugar rinsing in the higher-caries subjects . Initial plaque pH values were similar regardless of subject or tooth caries status . The values were also not correlated with the plaque levels of strongly iodophilic polysaccharide-storing bacteria . Collectively, both studies indicate that increasing subject caries status is characterized by increasing plaque levels of highly-acid-tolerant, acidogenic bacteria and an increasing plaque-pH-lowering potential and support the dynamic relationship between these parameters.

Biochemistry, 2000 Mar 28, 39(12), 3297 - 303
Pathways of energy transformation in antenna reaction center complexes of Heliobacillus mobilis; Neerken S et al.; The conversion of excitation energy in the antenna reaction center complex of Heliobacillus mobilis was investigated at 10 K as well as at 275 K by means of time-resolved absorbance difference spectroscopy of isolated membranes in the (sub)picosecond time range . Selective excitation of the primary electron acceptor, chlorophyll (Chl) a 670, and of the different spectral pools of bacteriochlorophyll (BChl) g (BChl g 778, BChl g 793, and BChl g 808) was applied . At 10 K, excitation at 770 or 793 nm resulted on the one hand in rapid energy transfer to BChl g 808 and on the other hand in fast charge separation from excited BChl g 793 ( approximately 1 ps) . Once the excitations were on BChl g 808, the bleaching band shifted gradually to the red, from 806 to 813 nm, and charge separation from excited BChl g 808 occurred by a very slow process ( approximately 500 ps) . The main purpose of our experiments was to answer the question whether an "alternative" pathway for charge separation exists upon excitation of Chl a 670 . Our measurements showed that the amount of oxidized primary donor (P798(+)) relative to that of excited BChl g produced by excitation of Chl a 670 was considerably larger than upon direct excitation of BChl g . This indicates the existence of an alternative pathway for charge separation that does not involve excited antenna BChl g . This effect occurred at 10 K as well as at 275 K . The mechanism for this process is discussed in relation to different trapping models; it is concluded that charge separation occurs directly from excited Chl a 670.

Antisense Nucleic Acid Drug Dev, 2000 Feb, 10(1), 1 - 9
Infection of the macrophage cell line NR8383 with Mycobacterium tuberculosis (H37Ra) leads to an increase in oligodeoxynucleotide accumulation; Rosenblatt MN et al.; Mycobacterium tuberculosis infection continues to be a daunting clinical challenge . Although it may well be one of the most studied bacteria in history, several aspects of its pathology remain a mystery . The resurgence of drug-resistant M . tuberculosis strains and with its unusual pathology have promoted a renewed basic and clinical research interest in developing new therapies to combat this pathogen . The primary localization site for M . tuberculosis is within alveolar macrophages . Drug delivery strategies and novel therapeutic agents designed to target alveolar macrophages may lead to efficient destruction of M . tuberculosis . Oligodeoxynucleotides (ODN) are short segments of nucleic acids that can interfere with transcription and translation processes . In this report, a monocyte-macrophage cell line was characterized in regard to ODN transport in the presence or absence of M . tuberculosis infection . The cells accumulated ODN in a time-dependent and concentration-dependent manner, regardless of the presence of serum . After 4 hours of incubation with M . tuberculosis (multiplicity of infection {MOI} 10:1), infected NR8383 cells demonstrated 1.5-7-fold increase in fluorescein isothiocyanate (FITC)-labeled phosphorothioate ODN accumulation as measured by flow cytometry . The increase in uptake was associated only with fluorescent-labeled ODN and not labeled markers of fluid phase endocytosis (e.g., tetramethylrhodamine isothiocyanate {TRITC}, FITC-labeled dextran) . NR8383 cells activated by phytohemagglutinin (PHA) did not demonstrate a significant increase in the uptake of either FITC-labeled dextran or FITC-labeled ODN . These studies demonstrate that NR8383 cells that have been infected with M . tuberculosis can specifically accumulate ODN, and this route of accumulation may lead to a means of drug targeting to mycobacteria-containing cells.

Berl Munch Tierarztl Wochenschr, 2000 Feb, 113(2), 65 - 6
Exclusion of strain 670/89 as type strain for serovar 20 of Riemerella anatipestifer; Ryll M et al.; Classical phenotypic characterisation and numeric analysis of whole-cell fatty acid patterns of twenty-one type strains of hitherto reported Riemerella anatipestifer serovars revealed that the type strain of serovar 20 does not belong to the species Riemerella anatipestifer sensu stricto and, therefore, it has to be excluded as a representative Riemerella anatipestifer serotype strain.

Nihon Kokyuki Gakkai Zasshi, 2000 Jan, 38(1), 67 - 72
{Nontuberculous mycobacterial infection followed for 12 years}; Kodama Y et al.; A 67-year-old woman presented in September 1985 with productive cough, bloody sputum, and dyspnea on exertion . Productive cough and bloody sputum had developed when the patient was 55 years old . Sputum culture and radiologic findings yielded a diagnosis of nontuberculous mycobacteriosis (NTM) . Antituberculous therapy with INH, RFP, and EB was initiated in November 1987 because of the development of a cavity in the right upper lobe, and led to resolution of the lesion and clinical symptoms . Despite progression of bronchiectatic changes in both lungs and a relapse of her clinical symptoms during the following 10 years, the patient retained enough pulmonary function to be able to maintain an active daily life until she died of advanced gastric cancer at the age of 79 . Autopsy revealed cystic bronchiectasis accompanied by bronchial wall thickening in both lungs, with some granuloma and acid-fast-bacteria observed in lung tissue . In this report, we concluded that patients with NTM usually experience a gradual progression of symptoms and radiographic changes during their clinical course, and that their pulmonary function may be conserved well enough to maintain an active daily life.

Mol Biol Evol, 2000 Mar, 17(3), 352 - 61
Horizontal gene transfer of glycosyl hydrolases of the rumen fungi; Garcia-Vallve S et al.; By combining analyses of G + C content and patterns of codon usage and constructing phylogenetic trees, we describe the gene transfer of an endoglucanase (celA) from the rumen bacteria Fibrobacter succinogenes to the rumen fungi Orpinomyces joyonii . The strong similarity between different glycosyl hydrolases of rumen fungi and bacteria suggests that most, if not all, of the glycosyl hydrolases of rumen fungi that play an important role in the degradation of cellulose and other plant polysaccharides were acquired by horizontal gene transfer events . This acquisition allows fungi to establish a habitat within a new environmental niche: the rumen of the herbivorous mammals for which cellulose and plant hemicellulose constitute the main raw nutritive substrate.

Int Rev Immunol, 2000, 19(1), 123 - 38
Spontaneous chronic colitis in TCR alpha-mutant mice; an experimental model of human ulcerative colitis; Bhan AK et al.; Mice with targeted disruption of the T cell receptor alpha gene (TCR alpha-/-) spontaneously develop chronic colitis . Colonic inflammation begins at 6-8 weeks of age and chronic colitis is established in about 60% of mice by 16-20 weeks of age . The disease is also associated with autoantibodies (anti-tropomyosin antibodies, anti-neutrophil cytoplasmic antibodies) and an oligoclonal immune response to luminal bacterial antigens . Although T cells, but not B cells or autoantibodies, are essential for the development of colitis, B cells and/or autoantibodies may have a regulatory role in the pathogenesis of this colitis because the colitis is more severe in B cell deficient TCR alpha-/- mice . Cytokines, specifically IL-4 and IL-1, also play an important role in the development of colitis in TCR alpha-/- mice . Enteric bacteria located in the large intestine are an important factor in the pathogenesis of this colitis because germ-free TCR alpha-/- mice do not develop colitis and appendectomy at an early age delays the onset of this colitis . The colitis in TCR alpha-/- mice resembles human ulcerative colitis and provides a useful model to study the pathogenesis of human inflammatory bowel disease.

J Dent, 2000 May, 28(4), 241 - 7
A new electrical method for detecting marginal leakage of in vitro resin restorations; Iwami Y et al.; OBJECTIVES: Ingress of bacteria at sites of marginal leakage has been suggested to cause pulpal inflammation . The purpose of this in vitro study was to evaluate the validity of a new electrical method to detect marginal leakage of restoratives by comparing the results obtained with those of a dye penetration test . METHODS: After cavities were prepared on the buccal coronal surfaces and root surfaces of 16 extracted non-carious human molar teeth, eight specimens were treated with a dentin bonding system (bonding group) and the other eight specimens were not treated (non-bonding group) . Resin composites were filled in the cavities of all specimens, and physiological saline was applied to the margin of the restorative . Any excess saline was wiped off, leaving only the electrolyte, which had penetrated into the marginal gap . The change in conductance was measured continuously across the margin of each specimen during this process . The marginal leakage of specimens was confirmed using the dye penetration test, and the results were evaluated by the microleakage score . RESULTS: In both coronal and root surface cavities, the changes in conductance in the non-bonding group after filling were significantly larger than those of the bonding group (p<0.05) . The change in conductance of each specimen correlated with the microleakage score (p<0.05) . CONCLUSIONS: It was concluded that the relative electrical method could detect marginal leakage in both coronal and root surface cavities.

FEBS Lett, 2000 Mar 17, 470(1), 7 - 10
The symbiotically essential cbb(3)-type oxidase of Bradyrhizobium japonicum is a proton pump; Arslan E et al.; Purified cbb(3)-type oxidase of Bradyrhizobium japonicum was reconstituted into phospholipid vesicles . Tight vesicles were obtained as shown by the disturbance of deltapH with CCCP and the membrane potential with valinomycin, which led to a six-fold increase in cytochrome c oxidase activity . The vesicles were thus suitable for proton translocation experiments . In the presence of valinomycin, a pulse with reduced cytochrome c caused an acidification with a subsequent alkalinization, whereas the same pulse caused only an alkalinization in the presence of valinomycin plus CCCP . We conclude that the cbb(3)-type oxidase of B . japonicum is a proton pump.

Infect Immun, 2000 Apr, 68(4), 2024 - 33
Role of Bordetella bronchiseptica fimbriae in tracheal colonization and development of a humoral immune response; Mattoo S et al.; Fimbriae are filamentous, cell surface structures which have been proposed to mediate attachment of Bordetella species to respiratory epithelium . Bordetella bronchiseptica has four known fimbrial genes: fim2, fim3, fimX, and fimA . While these genes are unlinked on the chromosome, their protein products are assembled and secreted by a single apparatus encoded by the fimBCD locus . The fimBCD locus is embedded within the fha operon, whose genes encode another putative adhesin, filamentous hemagglutinin (FHA) . We have constructed a Fim(-) B . bronchiseptica strain, RB63, by introducing an in-frame deletion extending from fimB through fimD . Western blot analysis showed that RB63 is unable to synthesize fimbriae but is unaffected for FHA expression . Using this mutant, we assessed the role of fimbriae in pathogenesis in vitro and in vivo in natural animal hosts . Although RB63 was not significantly defective in its ability to adhere to various tissue culture cell lines, including human laryngeal HEp-2 cells, it was considerably altered in its ability to cause respiratory tract infections in rats . The number of DeltafimBCD bacteria recovered from the rat trachea at 10 days postinoculation was significantly decreased compared to that of wild-type B . bronchiseptica and was below the limit of detection at 30 and 60 days postinoculation . The number of bacteria recovered from the nasal cavity and larynx was not significantly different between RB63 and the wild-type strain at any time point . The ability of fimbriae to mediate initial attachment to tracheal tissue was tested in an intratracheal inoculation assay . Significantly fewer RB63 than wild-type bacteria were recovered from the tracheas at 24 h after intratracheal inoculation . These results demonstrate that fimbriae are involved in enhancing the ability of B . bronchiseptica to establish tracheal colonization and are essential for persistent colonization at this site . Interestingly, anti-Bordetella serum immunoglobulin M (IgM) levels were significantly lower in animals infected with RB63 than in animals infected with wild-type B . bronchiseptica at 10 days postinoculation . Even at 30 days postinoculation, RB63-infected animals had lower serum anti-Bordetella antibody titers in general . This disparity in antibody profiles suggests that fimbriae are also important for the induction of a humoral immune response.

Infect Immun, 2000 Apr, 68(4), 1934 - 41
Role of adhesins and toxins in invasion of human tracheal epithelial cells by Bordetella pertussis; Bassinet L et al.; Bordetella pertussis, the agent of whooping cough, can invade and survive in several types of eukaryotic cell, including CHO, HeLa 229, and HEp-2 cells and macrophages . In this study, we analyzed bacterial invasiveness in nonrespiratory human HeLa epithelial cells and human HTE and HAE0 tracheal epithelial cells . Invasion assays and transmission electron microscopy analysis showed that B . pertussis strains invaded and survived, without multiplying, in HTE or HAE0 cells . This phenomenon was bvg regulated, but invasive properties differed between B . pertussis strains and isolates and the B . pertussis reference strain . Studies with B . pertussis mutant strains demonstrated that filamentous hemagglutinin, the major adhesin, was involved in the invasion of human tracheal epithelial cells by bacteria but not in that of HeLa cells . Fimbriae and pertussis toxin were not found to be involved . However, we found that the production of adenylate cyclase-hemolysin prevents the invasion of HeLa and HTE cells by B . pertussis because an adenylate cyclase-hemolysin-deficient mutant was found to be more invasive than the parental strain . The effect of adenylate cyclase-hemolysin was mediated by an increase in the cyclic AMP concentration in the cells . Pertactin (PRN), an adhesin, significantly inhibited the invasion of HTE cells by bacteria, probably via its interaction with adenylate cyclase-hemolysin . Isolates producing different PRNs were taken up similarly, indicating that the differences in the sequences of the PRNs produced by these isolates do not affect invasion . We concluded that filamentous hemagglutinin production favored invasion of human tracheal cells but that adenylate cyclase-hemolysin and PRN production significantly inhibited this process.

Infect Immun, 2000 Apr, 68(4), 1855 - 63
Secreted enzymatic activities of wild-type and pilD-deficient Legionella pneumophila; Aragon V et al.; Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular pathogen of protozoa and macrophages . Previously, we had determined that the Legionella pilD gene is involved in type IV pilus biogenesis, type II protein secretion, intracellular infection, and virulence . Since the loss of pili and a protease do not account for the infection defect exhibited by a pilD-deficient strain, we sought to define other secreted proteins absent in the mutant . Based upon the release of p-nitrophenol (pNP) from p-nitrophenyl phosphate, acid phosphatase activity was detected in wild-type but not in pilD mutant supernatants . Mutant supernatants also did not release either pNP from p-nitrophenyl caprylate and palmitate or free fatty acid from 1-monopalmitoylglycerol, suggesting that they lack a lipase-like activity . However, since wild-type samples failed to release free fatty acids from 1,2-dipalmitoylglycerol or to cleave a triglyceride derivative, this secreted activity should be viewed as an esterase-monoacylglycerol lipase . The mutant supernatants were defective for both release of free fatty acids from phosphatidylcholine and degradation of RNA, indicating that PilD-negative bacteria lack a secreted phospholipase A (PLA) and nuclease . Finally, wild-type but not mutant supernatants liberated pNP from p-nitrophenylphosphorylcholine (pNPPC) . Characterization of a new set of mutants defective for pNPPC-hydrolysis indicated that this wild-type activity is due to a novel enzyme, as opposed to a PLC or another known enzyme . Some, but not all, of these mutants were greatly impaired for intracellular infection, suggesting that a second regulator or processor of the pNPPC hydrolase is critical for L . pneumophila virulence.

APMIS Suppl, 2000, 97, 1 - 45
Molecular dissection of Mycoplasma hominis; Ladefoged SA; M . hominis is commonly found as part of the normal flora in the female genital tract, but several studies have shown that it may be involved in a variety of urogenital infections . The basis for clinical manifestations in some patients has varyingly been attributed to host and M . hominis factors . The host factors involved in the infection process are largely unknown . M . hominis have no cell wall and outer membranes, and at present it seems plausible that M . hominis possesses genetic systems allowing the bacteria in vivo to alter its antigenic structure on the membrane surface and consequently circumvent the host immune system . The studies of M . hominis have shown that the antigenic variation is pronounced between surface exposed membrane proteins from different isolates . The genetic background for this variation has been investigated for three surface exposed membrane proteins: P120, Lmp, and Vaa . P120 and P120' are similar proteins in M . hominis without any homology to other known proteins . A hypervariable region in the otherwise conserved P120 protein seems to be very antigenic in patients with immunologically verified M . hominis infection . The remaining part of P120 as well as the entire P120' protein do not seem to elicit significant antibody formation . Two genes in M . hominis, lmp1 and lmp3, contain numerous highly similar 0.5 kb tandem repeats at their 3'-end . The proteins, Lmp1 and Lmp3, are synthesized from the lmp1 and lmp3 genes, respectively . Lmp1 shows size variation among M . hominis isolates . M . hominis isolates investigated in detail show that the size variation of Lmp1 corresponds to the variation in number of 0.5 kb repeats contained within the lmp1 gene . Lmp3 appears to have a lesser tendency to size variation . M . hominis isolates were found with deletions involving the lmp1 stop codon leading to translation of the downstream gene lmp2 and expression of a chimeric Lmp1-Lmp2 protein . The number of repeated elements in the lmp1 gene of a M . hominis isolate correlates with the extent of anti-Lmp antibody induced agglutination between the bacteria . Vaa is a protein involved in cell adherence . vaa is a single copy gene containing tandem repeated elements like the lmp gene family . The number of repeats in the Vaa protein differs between M . hominis isolates leading to size variation . It has been suggested that the number of repeated elements is of importance in the bacteria-host adhesion process . Beside the size variation Vaa demonstrates phase variation due to frequent frame shift mutation in a specific region near the 5'-end of the structural gene . Based on the investigations of M . hominis and other mycoplasmas several genetic mechanisms seem to be responsible for the antigenic variation of surface exposed membrane proteins in mycoplasmas: 1) variation in protein size due to insertions or deletion of repeated elements in the structural gene, 2) presence of multi-gene families, and 3) phase variation due to mutations in the promotor region or the coding region . The influence of specific antibodies on antigenic variation of membrane proteins has not been studied in greater detail in mycoplasmas . In M . hominis it was investigated whether the presence in the culture medium of monoclonal antibodies directed against the repeated elements in the M . hominis Lmp proteins would affect gene structure and consequently protein expression . The presence of anti-Lmp antibodies resulted in overgrowth of bacteria with specific deletions in the repeated elements of lmp1 leaving the lmp3 gene unchanged . The precise mechanism leading to the dominance of M . hominis isolates with fewer 0 . (ABSTRACT TRUNCATED)

Ann Ist Super Sanita, 1999, 35(3), 401 - 4
Phytoplankton extracellular production and leakage with considerations on the polysaccharide accumulation; Myklestad SM; It is well known that many diatoms release and accumulate copious amounts of extracellular polysaccharides . Dramatic differences between diatom species exist . Examples from Prymnesiophyceae and Prasinophyceae show that algae from these classes as well may produce large amounts of extracellular polysaccharides . Soluble cell content may be released as extracellular components as a result of leaky membranes caused by unfavourable light conditions, pH, salinity, temperature, nutrient status and attack by bacteria or virus . Cellular soluble compounds may differ markedly from extracellular compounds produced under normal photosynthetic conditions . Production of polysaccharides in the Adriatic Sea may be promoted by heavy phytoplankton growth over long time under nutrient limitation . Sinking and flotation seem important processes in making aggregates and massive gels.

Ann Ist Super Sanita, 1999, 35(3), 365 - 72
General features of the Adriatic Sea: the key role of carbon cycling; Pagnotta R et al.; Major inputs into the northern Adriatic Sea and critical problems affecting this basin are revised strengthening the need for a thorough knowledge of organic carbon cycling . Results gathered in the context of the PRISMA 2 project by our research group give information on the distribution and composition of organic matter in terms of major biochemical components, molecular weight sizes and biodegradability characteristics . Seasonal accumulation of dissolved organic matter in summer along with an increased role of carbohydrate and colloidal matter (being on average 15-20 and 60-65% of DOC, respectively) and restricted bacterial activity due to P deficiency are all priority subjects to be further investigated.

Adv Exp Med Biol, 1999, 467, 73 - 8
Fructose malabsorption is associated with decreased plasma tryptophan; Ledochowski M et al.; Fructose malabsorption is characterized by the inability to absorb fructose efficiently . Consequently fructose reaches the colon and is broken down by bacteria to short-fatty-acids, CO2 and H2 . Recently we found that fructose malabsorption was associated with signs of depression . It was therefore of interest to find out whether fructose malabsorption is associated with abnormal tryptophan metabolism . Breath hydrogen concentrations were measured in 50 after an oral dose of 50 g fructose allowing to classify them as normals (n = 15) or fructose malabsorbers (n = 35) . Blood samples were taken for tryptophan and kynurenine measurements . Fructose malabsorbers showed significantly lower plasma tryptophan concentrations and significantly higher depression scores compared to normals . Fructose malabsorption is associated with lower tryptophan levels which may play a role in the development of depressive disorders.

Toxicol Lett, 2000 Mar 15, 112-113, 325 - 31
Biomarkers of immunotoxicity in fish: from the lab to the ocean; Zelikoff JT et al.; Historically, host immunocompetence has been monitored using a battery of immune parameters . Recently, many of these same assays have been employed as biomarkers for predicting chemical-induced immunotoxicity in wildlife species . In this laboratory, assays measuring immunopathology, immune cell function, and host resistance against bacteria have been used successfully to assess immunotoxicity in laboratory-reared Japanese medaka (Oryzias latipes) and in feral fish populations . As an example of the latter, smallmouth bass collected from a PCB-contaminated site demonstrated significantly reduced phagocyte function and antioxidant activity compared to reference site fish . Taken together, these studies along with those from other investigators demonstrate the usefulness of immune assays as indicators to predict the toxicological risk associated with 'real-world' polluted aquatic environments.

Microbios, 2000, 101(398), 23 - 36
Flow cytometric analysis of a marine LAS-degrading consortia; Lopez-Amoros R et al.; The specific nucleic acid fluorochrome SYTO-13 was used in flow cytometric analysis to assess changes in the density and heterogeneity of marine bacterial populations which biodegrade linear alkylbenzene sulphonate (LAS) . Seawater samples with LAS and incubated in the laboratory (20 degrees C, 100 rpm, 30 days) were used to monitor LAS-degrading consortia . Flow cytometric studies and culture methods were used to characterize the LAS degrading bacterioplankton consortia . Fluorescence and scatter signals enabled us to define three regions (R1, R2 and R3) in the dual parameter cytograms . The distribution of the bacterial counts in these regions allowed us to monitor the formation and evolution of the consortia.

Immunol Rev, 2000 Feb, 173, 66 - 78
The roles of surfactant proteins A and D in innate immunity; Lawson PR et al.; Research over the last decade on the surfactant proteins SP-A and SP-D suggests roles beyond surfactant lipid homeostasis, involving their participation in innate immune defence . SP-A and SP-D bind and agglutinate an impressive array of non-self structures, ranging from bacteria and fungi to allergens and environmental inorganic substrates . Complementing binding . SP-A and SP-D initiate and enhance immune cell ingestion and killing of targets . Recently, some exciting developments have extended and clarified their contributions to innate immunity . Knockout mice for SP-A and SP-D have been developed . The SP-A knockout confirms that SP-A plays a key role in defence against lung pathogens and reveals the underlying defense mechanisms that require SP-A . These surfactant proteins have also been shown to have important roles in modulating the immune response, instructing, yet quenching, the immune reactions in the lung . The crystal structure of SP-D plus functional studies with recombinantly altered forms of SP-A and SP-D has begun to characterise the structural motifs responsible for mediating their immune functions . Linkage and polymorphism analysis is explaining the role these genes may play in lung diseases and infection.

FEMS Microbiol Ecol, 2000 Mar 1, 31(3), 231 - 239
Archaeaplankton in the Columbia River, its estuary and the adjacent coastal ocean, USA; Crump BC et al.; PCR-amplified 16S rRNA genes from particle-attached and free-living Archaea in the Columbia River estuary, particle-attached Archaea in the river, and Archaea in the adjacent coastal ocean were cloned, and 43 partial sequences were determined . There was a high diversity of Archaea in the estuary, especially among the particle-attached Archaea, with representatives from four major phylogenetic clusters . Eighteen of 21 estuarine clones were closely related to clones from the river and the coastal ocean or to clusters of marine and soil clones identified in other studies . This contrasts with a similar study of the estuarine bacterial community that found 62% of bacterial 16S rRNA clones to be unique to the estuary . Archaea in the estuary were primarily allochthonous, and therefore, unlike the bacteria, probably do not form a native estuarine community.

J Fam Pract, 2000 Feb, 49(2), 153 - 6
Patient beliefs about the characteristics, causes, and care of the common cold: an update; Braun BL et al.; BACKGROUND: Many people seek medical care for cold symptoms . The cold-related knowledge and beliefs of adults seeking medical care for themselves or their children may not correspond with current medical opinion . METHODS: A total of 249 parents of symptomatic children and 257 symptomatic adults who sought medical advice in the spring of 1997 from 1 of 3 primary care clinics in the Minneapolis-St . Paul, Minnesota, area were surveyed by telephone 48 to 96 hours after contact with the medical system . RESULTS: Of the adults seeking care for a child or themselves, 44% believed viruses alone cause the common cold: an additional 42% believed both viruses and bacteria play a role . Most thought rest (97%) and nonprescription medications (63%) were helpful for colds, which was consistent with published reports . Contrary to medical reports, however, most felt vitamin C (67%) and the inhalation of steam (70%) reduced cold symptoms, and 44% believed antibiotics help colds (chi2=19.57; P=.0002) . But 85% believed colds could resolve on their own . CONCLUSIONS: Those adults seeking medical care for uncomplicated colds are misinformed about the primary cause of the common cold, the use of prescription medications for treating cold symptoms, and the effectiveness of some palliative care techniques . Care providers should address these perceptions rather than enabling overuse of antibiotics.

Shock, 2000 Mar, 13(3), 236 - 43
Endotoxin inducible transcription is repressed in endotoxin tolerant cells; Yoza BK et al.; Stimulation of the human promonocytic cell line, THP-1, with endotoxin results in a rapid and transient increase in interleukin 1beta expression . Endotoxin pretreatment of THP-1 cells results in tolerance, characterized by decreased levels of endotoxin-induced interleukin 1beta expression due to decreased transcription of the interleukin 1beta gene . We hypothesized that tolerant cells could not activate transcription factors necessary to express the interleukin 1beta gene . This hypothesis was tested in tolerant THP-1 cells by using stable and transiently transfected reporter genes containing the interleukin 1beta promoter . We found decreased endotoxin-induced transcription of all reporter genes tested; however, individual transcription factors, such as NFkappaB, retain normal, CD14-dependent, nuclear translocation and DNA binding . Tolerance is specific for endotoxin, because phorbol ester is still able to activate transcription of the endogenous interleukin 1beta gene and transfected reporter genes . A constitutively active reporter gene that is not inducible by endotoxin is unaffected . We further show that nuclear extracts of tolerant cells show transcription inhibitor activity that is specific for promoter sequences of the interleukin 1beta gene . These results support a mechanism of endotoxin tolerance that is independent of transcription factor DNA binding and appears to be associated with the inability of DNA-bound transcription factors to activate transcription, perhaps through the activity of a repressor.

Am Nat, 2000 Mar, 155(3), 335 - 345
Effect of Predator-Prey Phylogenetic Similarity on the Fitness Consequences of Predation: A Trade-off between Nutrition and Disease?
Pfennig DW.
A largely neglected aspect of foraging behavior is whether the costs and benefits of predation vary as a function of phylogenetic (i.e., genetic) similarity between predator and prey . Prey of varying phylogenetic similarities to predators might differ in value because both the risk of pathogen transmission and the nutritional quality of prey typically decline with decreasing phylogenetic similarity between predator and prey . I experimentally evaluated this hypothesis by feeding omnivorous spadefoot toad tadpoles (Spea bombifrons, Spea multiplicata, and Scaphiopus couchii) either conspecific tadpoles or an equal mass of three different species of heterospecific prey, all of which contained naturally occurring bacteria . I also examined which prey species Spea tadpoles preferred . I found that all three species of tadpoles performed best on, and preferred to eat, prey that were of intermediate phylogenetic similarity to the predators . Prey of intermediate phylogenetic similarity may provide the greatest fitness benefits to predators because such prey balance the nutritional benefits of closely related prey with the cost of parasite transmission between closely related individuals.

Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3314 - 8
Reciprocal domain evolution within a transactivator in a restricted sequence space; Juarez K et al.; offhough the concept of domain merging and shuffling as a major force in protein evolution is well established, it has been difficult to demonstrate how domains coadapt . Here we show evidence of coevolution of the Sinorhizobium meliloti NifA (SmNifA) domains . We found that, because of the lack of a conserved glycine in its DNA-binding domain, this transactivator protein interacts weakly with the enhancers . This defect, however, was compensated by evolving a highly efficient activation domain that, contrasting to Bradyrhizobium japonicum NifA (BjNifA), can activate in trans . To explore paths that lead to this enhanced activity, we mutagenized BjNifA . After three cycles of mutagenesis and selection, a highly active derivative was obtained . Strikingly, all mutations changed to amino acids already present in SmNifA . Our artificial process thus recreated the natural evolution followed by this protein and suggests that NifA is trapped in a restricted sequence space with very limited solutions for higher activity by point mutation.

Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3304 - 8
An archaeal genomic signature; Graham DE et al.; Comparisons of complete genome sequences allow the most objective and comprehensive descriptions possible of a lineage's evolution . This communication uses the completed genomes from four major euryarchaeal taxa to define a genomic signature for the Euryarchaeota and, by extension, the Archaea as a whole . The signature is defined in terms of the set of protein-encoding genes found in at least two diverse members of the euryarchaeal taxa that function uniquely within the Archaea; most signature proteins have no recognizable bacterial or eukaryal homologs . By this definition, 351 clusters of signature proteins have been identified . Functions of most proteins in this signature set are currently unknown . At least 70% of the clusters that contain proteins from all the euryarchaeal genomes also have crenarchaeal homologs . This conservative set, which appears refractory to horizontal gene transfer to the Bacteria or the Eukarya, would seem to reflect the significant innovations that were unique and fundamental to the archaeal "design fabric." Genomic protein signature analysis methods may be extended to characterize the evolution of any phylogenetically defined lineage . The complete set of protein clusters for the archaeal genomic signature is presented as supplementary material (see the PNAS web site, www.pnas.org).

Protein Sci, 2000 Feb, 9(2), 290 - 301
Microscopic stability of cold shock protein A examined by NMR native state hydrogen exchange as a function of urea and trimethylamine N-oxide; Jaravine VA et al.; Native state hydrogen exchange of cold shock protein A (CspA) has been characterized as a function of the denaturant urea and of the stabilizing agent trimethylamine N-oxide (TMAO) . The structure of CspA has five strands of beta-sheet . Strands beta1-beta4 have strongly protected amide protons that, based on experiments as a function of urea, exchange through a simple all-or-none global unfolding mechanism . By contrast, the protection of amide protons from strand beta5 is too weak to measure in water . Strand beta5 is hydrogen bonded to strands beta3 and beta4, both of which afford strong protection from solvent exchange . Gaussian network model (GNM) simulations, which assume that the degree of protection depends on tertiary contact density in the native structure, accurately predict the strong protection observed in strands beta1-beta4 but fail to account for the weak protection in strand beta5 . The most conspicuous feature of strand beta5 is its low sequence hydrophobicity . In the presence of TMAO, there is an increase in the protection of strands beta1-beta4, and protection extends to amide protons in more hydrophilic segments of the protein, including strand beta5 and the loops connecting the beta-strands . TMAO stabilizes proteins by raising the free energy of the denatured state, due to highly unfavorable interactions between TMAO and the exposed peptide backbone . As such, the stabilizing effects of TMAO are expected to be relatively independent of sequence hydrophobicity . The present results suggest that the magnitude of solvent exchange protection depends more on solvent accessibility in the ensemble of exchange susceptible conformations than on the strength of hydrogen-bonding interactions in the native structure.

Int J Cardiol, 2000 Feb 15, 72(3), 209 - 13
Inflammatory mediators in heart failure; Niebauer J; Cytokines have been identified as a major player in the pathogenesis and the functional status of patients with heart failure . Herein I review studies which are under way to assess the effects of anti-cytokine therapy, where soon we may see treatments directed against bacteria in the bowel, the translocation process, and endotoxin itself, the binding sites of bacterial endotoxin on immune competent cells, or both.

Arch Oral Biol, 2000 Feb, 45(2), 179 - 83
Suppression of interleukin-10 release from human periodontal ligament cells by interleukin-1beta in vitro; Deschner J et al.; Periodontitis is characterized by an inflammatory process induced by periodontopathogenic bacteria in the subgingival plaque . Periodontal inflammation can be enhanced by both an increase of inflammatory stimulators, e.g . interleukin (IL)-6, and a decrease of inflammatory inhibitors, e.g . IL-10 . The amount of IL-1beta is known to be increased in gingival tissues and in the gingival crevicular fluid from inflamed sites compared to healthy sites . This in vitro study sought to clarity whether IL-1beta (1 ng/ml) has a regulatory effect on the release of these two cytokines from human periodontal ligament (PDL) cells . PDL cells derived from healthy premolars were grown in the presence and absence (control) of IL-1beta . The concentration of IL-6 and IL-10 in the supernatants was assessed by enzyme-linked immunosorbent assay after 48 h of culture . PDL cells incubated with IL-1beta released significantly (p < 0.05) higher amounts of IL-6 and significantly (p < 0.01) smaller amounts of IL-10 compared to control . These results give further support to the observation that IL-1beta can increase the IL-6 secretion from PDL cells . Moreover, they provide original evidence that PDL cells secrete IL-10, which can be suppressed by IL-1beta . It is concluded that PDL cells can function as accessory immunoinflammatory cells amplifying the inflammatory process in periodontitis and, thereby, contributing to periodontal breakdown.

Protist, 1999 Dec, 150(4), 383 - 98
Symbiomonas scintillans gen . et sp . nov . and Picophagus flagellatus gen . et sp . nov . (Heterokonta): two new heterotrophic flagellates of picoplanktonic size; Guillou L et al.; Two new oceanic free-living heterotrophic Heterokonta species with picoplanktonic size (< 2 microm) are described . Symbiomonas scintillans Guillou et Chretiennot-Dinet gen . et sp . nov . was isolated from samples collected both in the equatorial Pacific Ocean and the Mediterranean Sea . This new species possesses ultrastructural features of the bicosoecids, such as the absence of a helix in the flagellar transitional region (found in Cafeteria roenbergensis and in a few bicosoecids), and a flagellar root system very similar to that of C . roenbergensis, Acronema sippewissettensis, and Bicosoeca maris . This new species is characterized by a single flagellum with mastigonemes, the presence of endosymbiotic bacteria located close to the nucleus, the absence of a lorica and a R3 root composed of a 6+3+x microtubular structure . Phylogenetical analyses of nuclear-encoded SSU rDNA gene sequences indicate that this species is close to the bicosoecids C . roenbergensis and Siluania monomastiga . Picophagus flagellatus Guillou et Chretiennot-Dinet gen . et sp . nov . was collected in the equatorial Pacific Ocean . Cells are naked and possess two flagella . This species is characterized by the lack of a transitional helix and lateral filaments on the flagellar tubular hairs, the absence of siliceous scales, two unequal flagella, R1 + R3 roots, and the absence of a rhizoplast . SSU rDNA analyses place this strain at the base of the Chrysophyceae/Synurophyceae lineages.

Protist, 1999 Dec, 150(4), 375 - 82
The effect of electrostatic charge of food particles on capture efficiency by Oxyrrhis marina Dujardin (dinoflagellate); Hammer A et al.; Laboratory experiments were carried out to investigate the effect of food quality, measured as surface charge of the particles, on capture efficiency and ingestion rate by the heterotrophic dinoflagellate Oxyrrhis marina . Fluorescent particles in two size classes of around 1 and 4 microm and of 7 different qualities were offered to the flagellate: carbohydrate and albumin particles, the algae Synechocystis spec . and Chlorella spec., carboxylated microspheres, silicate particles and bacteria . Rates of particle uptake showed significant differences depending on particle size and quality, and ranged from 0 to 4 particles cell(-1) h(-1) . Ingestion rates were up to 4 times higher for 4 pm particles than for 1 microm particles, which indicates strong size-selective feeding . Our main result is that the surface charge or zeta potential, of artificial particles, i.e . carboxylated microspheres (> or = -107 mV) and silicate particles, strongly differ from more natural and natural food (< or = -17 mV) . For both size classes Oxyrrhis had ingestion rates up to 4 times higher for particles with less negative charge, such as albumin particles or algae . Thus, the zeta potential of the model food should be considered in experimental design . Particles with a zeta potential similar to that of natural food, e.g . albumin, seem to be the preferred model food.

FEMS Microbiol Lett, 2000 Mar 15, 184(2), 261 - 4
A simple method to evaluate the concentration of pentachlorophenol degraders in contaminated soils; Becaert V et al.; A new most probable number (MPN) method for the determination of pentachlorophenol (PCP) degraders in soil using the change in pH due to PCP degradation is compared with a well documented MPN method using radiolabeled PCP . The results of all MPN counts were similar within a 95% confidence limit . The results obtained in MPN per gram of dry soil using pH measurements were 1.8 (+3.1, -1.03) x10 (4) compared to 0.64 (+1.34, -0.42) x 10(4) when using production of {(14)C}CO(2).

FEMS Microbiol Lett, 2000 Mar 15, 184(2), 247 - 51
Binding and utilization of myoglobin by Porphyromonas gingivalis; Fujimura S et al.; Myoglobin was found to bind reversibly to the envelope of Porphyromonas gingivalis in a pH-dependent manner; the binding took place below neutral pHs of the incubation mixtures and myoglobin bound released from the envelope at high pHs . The amounts of myoglobin bound to 1 mg of the envelope at pH 5.0 per min under the presence of sufficient myoglobin were 1.4 microg . K(d) for the reaction at pH 5.0 was 2.2 x 10(-10) M . From the dot blot assay, myoglobin obviously bound to hemoglobin-binding protein (HbBP) of P . gingivalis, however, the amounts of myoglobin that bound to HbBP were half those of hemoglobin . One of the fractions, separated by gel filtration, of the digested materials of myoglobin by the detergent-solubilized envelope containing proteinases was found to support the growth of P . gingivalis in the iron source-depleted medium.

FEMS Microbiol Lett, 2000 Mar 15, 184(2), 215 - 8
Detection of sequence variation in PCR-amplified fragments of omp2 gene from three species of the family Chlamydiaceae using agarose gel electrophoresis containing bisbenzimide-PEG; Demkin VV et al.; A simple technique providing a means for rapid genetic differentiation of chlamydial strains is described . The technique is based on a single-step sequence-specific separation of PCR-amplified DNA fragments by electrophoresis in an agarose gel containing a DNA ligand - bisbenzimide-PEG . A hypervariable region at the 5' end of the omp2 gene of Chlamydiaceae species encoding the 60-kDa cysteine-rich outer membrane protein was selected as a target for PCR . The appropriate fragments were amplified from strains of Chlamydia trachomatis, Chlamydophila pneumoniae, and Chlamydophila psittaci, and the PCR products originating from different species were electrophoretically separated in the presence of the DNA ligand . We therefore demonstrated that PCR with a single pair of primers followed by simple agarose gel electrophoresis with bisbenzimide-PEG can be applied to the differentiation of three members of the family Chlamydiaceae which are commonly recognized as human pathogens.

Curr Opin Plant Biol, 2000 Apr, 3(2), 147 - 52
Novel genes for disease-resistance breeding; Melchers LS et al.; Plant disease control is entering an exciting period during which transgenic plants showing improved resistance to pathogenic viruses, bacteria, fungi and insects are being developed . This review summarizes the first successful attempts to engineer fungal resistance in crops, and highlights two promising approaches . Biotechnology provides the promise of new integrated disease management strategies that combine modern fungicides and transgenic crops to provide effective disease control for modern agriculture.

Chem Biol, 2000 Mar, 7(3), 211 - 24
Predictive, structure-based model of amino acid recognition by nonribosomal peptide synthetase adenylation domains; Challis GL et al.; BACKGROUND: Nonribosomal peptide synthetases (NRPSs) are large modular proteins that selectively bind, activate and condense amino acids in an ordered manner . Substrate recognition and activation occurs by reaction with ATP within the adenylation (A) domain of each module . Recently, the crystal structure of the A domain from the gramicidin synthetase (GrsA) with L-phenylalanine and adenosine monophosphate bound has been determined . RESULTS: Critical residues in all known NRPS A domains have been identified that align with eight binding-pocket residues in the GrsA A domain and define sets of remarkably conserved recognition templates . Phylogenetic relationships among these sets and the likely specificity determinants for polar and nonpolar amino acids were determined in light of extensive published biochemical data for these enzymes . The binding specificity of greater than 80% of the known NRPS A domains has been correlated with more than 30 amino acid substrates . CONCLUSIONS: The analysis presented allows the specificity of A domains of unknown function (e.g . from polymerase chain reaction amplification or genome sequencing) to be predicted . Furthermore, it provides a rational framework for altering of A domain specificity by site-directed mutagenesis, which has significant potential for engineering the biosynthesis of novel natural products.

Enzyme Microb Technol, 2000 Mar 1, 26(5-6), 451 - 458
Critical evaluation of p-nitrophenylphosphorylcholine (p-NPPC) as artificial substrate for the detection of phospholipase C*
Fliegera A, Gong S, Faigle M, Neumeister B.
Phospholipase C (PLC) activity secreted by bacteria as a virulence factor is commonly detected by use of the artificial substrate p-nitrophenylphosphorylcholine (p-NPPC) . We examined several commercially available enzymes (phosphodiesterases, phosphomonoesterases, phospholipase A, lipase, protease) for their hydrolytic activity towards p-NPPC and compared these results with those of PLC tests using phospholipid substrates . Our data indicate that, in addition to PLC, several other enzymes which can affect phosphate esters are able to hydrolyze p-NPPC . We therefore suggest to use lipid substrates for correct characterization of bacterial PLCs, especially when whole bacteria or crude enzyme preparations are investigated.

Mol Microbiol, 2000 Mar, 35(5), 1220 - 34
Identification of a candidate glycosaminoglycan-binding adhesin of the Lyme disease spirochete Borrelia burgdorferi; Parveen N et al.; Binding of glycosaminoglycans (GAGs) by Borrelia burgdorferi, the Lyme disease spirochete, has the potential to promote the colonization of diverse tissues . GAG binding by B . burgdorferi is associated with haemagglutination and we have identified a 26 kDa protein, which we have termed Bgp (Borrelia GAG-binding protein), on the basis of its ability to bind to heparin and erythrocytes . Bgp was found in outer membrane fractions of B . burgdorferi and on the surface of intact bacteria, as assayed by labelling with a membrane-impermeable biotinylating agent or anti-Bgp antibodies . Purified recombinant Bgp agglutinated erythrocytes, binds to the same spectrum of GAGs as the B . burgdorferi strain from which the cloned bgp sequence was obtained, and inhibited B . burgdorferi binding to purified GAGs and to cultured mammalian cells . Thus, Bgp is a strong candidate for a GAG-binding adhesin of B . burgdorferi.

Mol Microbiol, 2000 Mar, 35(5), 991 - 1004
Modulation of host immune responses, induction of apoptosis and inhibition of NF-kappaB activation by the Bordetella type III secretion system; Yuk MH et al.; Bordetella bronchiseptica establishes respiratory tract infections in laboratory animals with high efficiency . Colonization persists for the life of the animal and infection is usually asymptomatic in immunocompetent hosts . We hypothesize that this reflects a balance between immunostimulatory events associated with infection and immunomodulatory events mediated by the bacteria . We have identified 15 loci that are part of a type III secretion apparatus in B . bronchiseptica and three secreted proteins . The functions of the type III secretion system were investigated by comparing the phenotypes of wild-type bacteria with two strains that are defective in type III secretion using in vivo and in vitro infection models . Type III secretion mutants were defective in long-term colonization of the trachea in immunocompetent mice . The mutants also elicited higher titres of anti-Bordetella antibodies upon infection compared with wild-type bacteria . Type III secretion mutants also showed increased lethal virulence in immunodeficient SCID-beige mice . These observations suggest that type III-secreted products of B . bronchiseptica interact with components of both innate and adaptive immune systems of the host . B . bronchiseptica induced apoptosis in macrophages in vitro and inflammatory cells in vivo and type III secretion was required for this process . Infection of an epithelial cell line with high numbers of wild type, but not type III deficient B . bronchiseptica resulted in rapid aggregation of NF-kappaB into large complexes in the cytoplasm . NF-kappaB aggregation was dependent on type III secretion and aggregated NF-kappaB did not respond to TNFalpha activation, suggesting B . bronchiseptica may modulate host immunity by inactivating NF-kappaB . Based on these in vivo and in vitro results, we hypothesize that the Bordetella type III secretion system functions to modulate host immune responses during infection.

Plant Physiol, 2000 Mar, 122(3), 907 - 14
Molecular cloning and characterization of ATP-phosphoribosyl transferase from Arabidopsis, a key enzyme in the histidine biosynthetic pathway; Ohta D et al.; We have characterized two isoforms of ATP-phosphoribosyl transferase (ATP-PRT) from Arabidopsis (AtATP-PRT1 {accession no . AB025251} and AtATP-PRT2), catalyzing the first step of the pathway of hisidine (His) biosynthesis . The primary structures deduced from AtATP-PRT1 and AtATP-PRT2 cDNAs share an overall amino acid identity of 74.6% and contain N-terminal chloroplast transit peptide sequences . DNA-blot analyses indicated that the ATP-PRTs in Arabidopsis are encoded by two separate genes with a closely similar gene structural organization . Both gene transcripts were detected throughout development, and protein-blot analysis revealed predominant accumulation of the AtATP-PRT proteins in Arabidopsis leaves . The His auxotrophy of a his1 mutant of Saccharomyces cerevisiae was suppressed by the transformation with AtATP-PRT1 and AtATP-PRT2 cDNAs, indicating that both isoforms are functionally active ATP-PRT enzymes . The K(m) values for ATP and phosphoribosyl pyrophosphate of the recombinant AtATP-PRT proteins were comparable to those of the native ATP-PRTs from higher plants and bacteria . It was demonstrated that the recombinant AtATP-PRTs were inhibited by L-His (50% inhibition of initial activity = 40-320 microM), suggesting that His biosynthesis was regulated in plants through feedback inhibition by L-His.

Plant Physiol, 2000 Mar, 122(3), 747 - 56
Genetic engineering of glycinebetaine production toward enhancing stress tolerance in plants: metabolic limitations; Huang J et al.; Glycinebetaine (betaine) affords osmoprotection in bacteria, plants and animals, and protects cell components against harsh conditions in vitro . This and a compelling body of other evidence have encouraged the engineering of betaine production in plants lacking it . We have installed the metabolic step for oxidation of choline, a ubiquitous substance, to betaine in three diverse species, Arabidopsis, Brassica napus, and tobacco (Nicotiana tabacum), by constitutive expression of a bacterial choline oxidase gene . The highest levels of betaine in independent transgenics were 18.6, 12.8, and 13 micromol g(-1) dry weight, respectively, values 10- to 20-fold lower than the levels found in natural betaine producers . However, choline-fed transgenic plants synthesized substantially more betaine . Increasing the choline supplementation further enhanced betaine synthesis, up to 613 micromol g(-1) dry weight in Arabidopsis, 250 micromol g(-1) dry weight in B . napus, and 80 micromol g(-1) dry weight in tobacco . These studies demonstrate the need to enhance the endogenous choline supply to support accumulation of physiologically relevant amounts of betaine . A moderate stress tolerance was noted in some but not all betaine-producing transgenic lines based on relative shoot growth . Furthermore, the responses to stresses such as salinity, drought, and freezing were variable among the three species.

Am J Respir Crit Care Med, 2000 Mar, 161(3 Pt 1), 723 - 9
The utility of open lung biopsy in patients with hematologic malignancies; White DA et al.; The yield and impact of open lung biopsies in patients with hematologic malignancies and unexplained pulmonary processes were assessed and analyzed to determine factors that affected the yield . Records of 63 patients with hematologic malignancy, who underwent 67 open lung biopsies for diagnosis of an unknown pulmonary process from 1996 to 1998 at Memorial Sloan-Kettering Cancer Center, were retrospectively reviewed . A specific diagnosis was found in 41 (62%) of the biopsies . Changes in therapy were made in 37 (57%) of patients after biopsy results, but in 69% of those with a specific diagnosis . Survival at 30 and 90 d was increased in those with specific rather than a nonspecific pulmonary diagnosis . The factor most predictive of finding a specific diagnosis was the presence of a focal rather than a diffuse radiographic abnormality (79% versus 36%, p = 0.003) . Neutropenic patients or those on mechanical ventilation had a low chance of finding a specific diagnosis . Having received pulmonary toxic chemotherapy in the 6 mo before the biopsy was associated with finding a nonspecific lung injury . Specific pulmonary diagnoses found were inflammatory diseases in 23% of cases, infections in 21%, and malignancy in 18% . Bronchiolitis obliterans with organizing pneumonia (BOOP) was the most common inflammatory disorder and fungi and bacteria were the most frequent infectious pathogens . Complications occurred in 13% of the biopsies, including five patients who required mechanical ventilation post-procedure; one death was associated with the biopsy . The risk was increased in those with less than 50,000 platelets . Complications were similar with video-assisted thoracoscopy (VATS) compared with thoracotomy . We conclude that open lung biopsy in patients with hematologic malignancy has a significant yield and impact on management of patients with hematologic malignancy.

Microbiol Immunol, 2000, 44(1), 29 - 39
Therapeutic oral vaccination induces mucosal immune response sufficient to eliminate long-term Helicobacter pylori infection; Ikewaki J et al.; We examined the efficacy of therapeutic oral vaccination using Helicobacter pylori-whole cell sonicate and cholera toxin (CT) in mice persistently infected with H . pylori . Efficacy was determined by bacterial culture and microscopic examination of gastric tissues for the persistence of bacteria at 6 weeks after the last vaccination . Vaccination of H . pylori-whole cell sonicate combined with CT eradicated bacteria in 10/16 mice (62.5%) . Interestingly, oral vaccination with CT alone also eliminated the bacteria in 8/17 mice (47.1%) . However, a therapeutic intraperitoneally administered vaccine failed to eradicate H . pylori from the stomach (1/17 mice, 5.9%) . Identification of the type of immunity involved in the eradication process showed that oral vaccination enhanced the antigen-specific IgA in the feces and saliva . The efficacy of eradication of H . pylori correlated well with increases in IgA secretion in mucosal tissue and a higher labeling index of IgA-positive lumina of pyloric glands . Moreover, the expression of IL-4 mRNA in the stomach of mice with eradicated bacteria was higher than in the uneradicated group . Our results suggest that the efficacy of vaccination depends on the mucosal IgA response in the gastrointestinal tract against H . pylori via Th2 cell activation and that therapeutic oral vaccination induces a mucosal immune response sufficient to eradicate long-term infection with H . pylori.

J Clin Pathol, 1999 Dec, 52(12), 914 - 6
A reliable method for the simultaneous identification of H pylori and gastric metaplasia in the duodenum; el-Zimaity HM et al.; While an association of Helicobacter pylori infection with duodenal mucosa gastric metaplasia has been described, the details and extent of the interaction are lacking . One of the limiting factors has been the lack of a staining technique that allows simultaneous visualisation of the bacteria and gastric metaplasia in the duodenum . This report describes a new stain that allows the simultaneous visualisation of duodenal gastric metaplasia and H pylori and compares the new stain with the component stains.

Br J Nurs, 1999 Aug 12-Sep 8, 8(15), 1027 - 31
Improving the nutrition of patients using Entera Fibre Plus; Kemp S; This article examines the role of fibre in the diet with a special focus on Entera Fibre Plus, a new high energy, fibre-containing sip feed . Early enteral feeds were all fibre-free or 'low residue' . In the last 10 years fibre-containing feeds have become available, mostly designed for tube feeding and containing a single fibre source . Entera Fibre Plus contains a mixed fibre source, providing both soluble and insoluble fibre types, including inulin . This helps provide the full range of fibre benefits, which include reduction of constipation and diarrhoea, maintaining the health of the gut and encouraging beneficial gut bacteria to assist the body's defence systems . Inulin has a specific role in the latter . Entera Fibre Plus provides 300 kcal and 5 g of fibre per 200 ml pack and is available in six flavours . It is a convenient and palatable way of improving the nutritional status of patients who are malnourished and may be of particular benefit to elderly people, those on long-term supplements and those who suffer from chronic constipation or diarrhoea . Entera Fibre Plus is approved by the Advisory Committee on Borderline Substances (ACBS) and is available at retall pharmacles or on prescription.

Genetica, 1999, 106(1-2), 75 - 84
Modelling DNA stretching for physics and biology; Lavery R et al.; We have used internal coordinate molecular mechanics calculations to study how the DNA double helix deforms upon stretching . Results obtained for polymeric DNA under helical symmetry constraints suggest that two distinct forms, an unwound ribbon and a narrow fibre, can be formed as a function of which ends of the duplex are pulled . Similar results are also obtained with DNA oligomers . These experiments lead to force curves which exhibit a plateau as the conformational transition occurs . This behaviour is confirmed by applying an increasing force to DNA and observing a sudden length increase at a critical force value . It is finally shown some DNA binding proteins can also stretch DNA locally, to conformations related to those created by nanomanipulation.

Genetica, 1999, 106(1-2), 3 - 13
The skeletal function of non-genic nuclear DNA: new evidence from ancient cell chimaeras; Cavalier-Smith T et al.; DNA can be divided functionally into three categories: (1) genes--which code for proteins or specify non-messenger RNAs; (2) semons--short specific sequences involved in the replication, segregation, recombination or specific attachments of chromosomes, or chromosome regions (e.g . loops or domains) or selfish genetic elements; (3) secondary DNA--which does not function by means of specific sequences . Probably more than 90% of DNA in the biosphere is secondary DNA present in the nuclei of plants and phytoplankton . The amount of genic DNA is related to the complexity of the organism, whereas the amount of secondary DNA increases proportionally with cell volume, and not with complexity . This correlation is most simply explained by the skeletal DNA hypothesis, according to which nuclear DNA functions as the basic framework for the assembly of the nucleus and the total genomic DNA content functions (together with relatively invariant folding rules) in determining nuclear volumes . Balanced growth during the cell cycle requires the cytonuclear ratio to be basically constant, irrespective of cell volume; thus nuclear volumes, and therefore the overall genome size, have to be evolutionarily adjusted to changing cell volumes for optimal function . Bacteria, mitochondria, chloroplasts and viruses have no nuclear envelope; and the skeletal DNA hypothesis simply explains why secondary DNA is essentially absent from them but present in large cell nuclei . Hitherto it has been difficult to refute the alternative hypothesis that nuclear secondary DNA (whether 'junk' or selfish DNA) accumulates merely by mutation pressure, and that selection for economy is not strong enough to eliminate it, whereas accumulation in mitochondria and plastids is prevented by intracellular replicative competition between their multiple genomes . New data that discriminate clearly between these explanations for secondary DNA come from cryptomonads and chlorarachneans, two groups of algae that originated independently by secondary symbiogenesis (i.e., the merger of two radically different eukaryote cells) several hundred million years ago . In both groups the nucleus and plasma membrane of the former algal symbiont persist as the nucleomorphs and periplastid membrane, respectively . The fact that nucleomorphs have undergone a 200- to 1000-fold reduction in genome size and have virtually no secondary DNA shows that selection against non-functional nuclear DNA is strong enough to eliminate it very efficiently; therefore, the large amounts of secondary DNA in the former host nuclei of these chimaeras, and in nuclei generally, must be being maintained by positive selection . The divergent selection for secondary DNA in the nucleus and against it in nucleomorphs is readily explicable by the skeletal DNA hypothesis, given the different spectrum of gene functions that it encodes.

Mol Cell, 2000 Jan, 5(1), 181 - 7
VASA mediates translation through interaction with a Drosophila yIF2 homolog; Carrera P et al.; The Drosophila gene vasa (vas) encodes an RNA-binding protein required for embryonic patterning and germ cell specification . In vas mutants, translation of several germline mRNAs is reduced . Here we show that VAS interacts directly with the Drosophila homolog of yeast translation initiation factor 2, encoded by a novel gene, dIF2 . Embryos produced by vas/+; dIF2/+ females have pattern defects and fewer germline progenitor cells, indicating a functional interaction between endogenous vas and dIF2 activities . Mutations in other translation initiation factors do not enhance the vas phenotype, suggesting that dIF2 has a particular role in germ plasm function . We conclude that VAS regulates translation of germline mRNAs by specific interaction with dIF2, an essential factor conserved from bacteria to humans.

Mol Cell, 2000 Jan, 5(1), 133 - 40
Mapping interactions between nuclear transport factors in living cells reveals pathways through the nuclear pore complex; Damelin M et al.; The interactions between transport receptors and proteins of the nuclear pore complex (NPC) are fundamental to understanding nucleocytoplasmic transport . In order to delineate the path that a particular transport receptor takes through the NPC, we have employed fluorescence resonance energy transfer (FRET) between enhanced cyan and yellow fluorescent proteins (ECFP, EYFP) in living cells . A panel of yeast strains expressing functional receptor--ECFP and nucleoporin--EYFP fusions has been analyzed with a FRET assay . With this approach, we define points of contact in the NPC for the related importin Pse1/Kap121 and exportin Msn5 . These data demonstrate the utility of FRET in mapping dynamic protein interactions in a genetic system . Furthermore, the data indicate that an importin and exportin have overlapping pathways through the NPC.

Mol Cell, 2000 Jan, 5(1), 109 - 19
The eIF1A solution structure reveals a large RNA-binding surface important for scanning function; Battiste JL et al.; The translation initiation factor eIF1A is necessary for directing the 43S preinitiation complex from the 5' end of the mRNA to the initiation codon in a process termed scanning . We have determined the solution structure of human eIF1A, which reveals an oligonucleotide-binding (OB) fold and an additional domain . NMR titration experiments showed that eIF1A binds single-stranded RNA oligonucleotides in a site-specific, but non-sequence-specific manner, hinting at an mRNA interaction rather than specific rRNA or tRNA binding . The RNA binding surface extends over a large area covering the canonical OB fold binding site as well as a groove leading to the second domain . Site-directed mutations at multiple positions along the RNA-binding surface were defective in the ability to properly assemble preinitiation complexes at the AUG codon in vitro.

Mol Biol Evol, 2000 Feb, 17(2), 213 - 23
Compartment-specific isoforms of TPI and GAPDH are imported into diatom mitochondria as a fusion protein: evidence in favor of a mitochondrial origin of the eukaryotic glycolytic pathway; Liaud MF et al.; Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI) are essential to glycolysis, the major route of carbohydrate breakdown in eukaryotes . In animals and other heterotrophic eukaryotes, both enzymes are localized in the cytosol; in photosynthetic eukaryotes, GAPDH and TPI exist as isoenzymes that function in the glycolytic pathway of the cytosol and in the Calvin cycle of chloroplasts . Here, we show that diatoms--photosynthetic protists that acquired their plastids through secondary symbiotic engulfment of a eukaryotic rhodophyte--possess an additional isoenzyme each of both GAPDH and TPI . Surprisingly, these new forms are expressed as an TPI-GAPDH fusion protein which is imported into mitochondria prior to its assembly into a tetrameric bifunctional enzyme complex . Homologs of this translational fusion are shown to be conserved and expressed also in nonphotosynthetic, heterokont-flagellated oomycetes . Phylogenetic analyses show that mitochondrial GAPDH and its N-terminal TPI fusion branch deeply within their respective eukaryotic protein phylogenies, suggesting that diatom mitochondria may have retained an ancestral state of glycolytic compartmentation that existed at the onset of mitochondrial symbiosis . These findings strongly support the view that nuclear genes for enzymes of glycolysis in eukaryotes were acquired from mitochondrial genomes and provide new insights into the evolutionary history (host-symbiont relationships) of diatoms and other heterokont-flagellated protists.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2000 Mar, 89(3), 323 - 32
Necrotizing ulcerative stomatitis in human immunodeficiency virus-seropositive individuals: a review of the histopathologic, immunohistochemical, and virologic characteristics of 18 cases; Jones AC et al.; OBJECTIVE: The purpose of this retrospective study was to delineate the histopathologic, immunohistochemical, and virologic characteristics of 18 cases of necrotizing ulcerative stomatitis . STUDY DESIGN: Eighteen examples or oral ulcerations in human immunodeficiency virus-seropositive individuals were identified that displayed unique histopathologic features . Immunohistochemic staining for CD1a, CD3, CD23, CD68, HLA-DR, p24, cytomegalovirus, HSV-1, and HSV-2 was performed, along with in situ hybridization for Epstein-Barr virus RNA and special staining for bacteria and fungi . RESULTS: The lesions demonstrated ulceration, extensive necrosis, leukocytoclasia, histiocytic vasculitis with luminal fibrin clots, and a prominent infiltrate of large atypical cells with amphophilic cytoplasm, vesicular nuclei, and prominent nucleoli, interspersed with crescentic histiocytes, a histologic picture resembling extranodal Kikuchi's disease . Immunohistochemical findings suggested that the large atypical cells were histiocytes . Fifty-six percent (10/18) of the cases were immunoreactive for human immunodeficiency virus p24 within focal histiocytes, whereas Epstein-Barr virus RNA was identified in 1 (6%) of 17 cases . CONCLUSIONS: Necrotizing ulcerative stomatitis is an inflammatory disease characterized by specific, reproducible microscopic features . We postulate that the histopathologic resemblance of necrotizing ulcerative stomatitis to extranodal Kikuchi's disease reflects a similar immune response to differing pathogens.

Int J Periodontics Restorative Dent, 1999 Aug, 19(4), 373 - 7
Skin-prick test for severe marginal periodontitis; Lindskog S et al.; The present study tested the hypothesis that treatment-resistant periodontitis patients present with a more intense inflammatory response to marginal bacterial plaque as a sign of an inflammatory overreaction . Patients with severe marginal periodontitis (Gingival Index > 20%) who had not responded to treatment showed almost no positive response to lipid A in a skin-prick test, which was significantly different from the results from patients with severe marginal periodontitis who had responded to treatment and from healthy control individuals without marginal periodontitis . This finding can be interpreted as an impaired inflammatory reactivity to periodontitis pathogens in treatment-resistant patients, rejecting the hypothesis.

Biochim Biophys Acta, 2000 Mar 7, 1477(1-2), 122 - 45
Structure-function relationships of glutamine synthetases; Eisenberg D et al.; As a highly regulated enzyme at the core of nitrogen metabolism, glutamine synthetase has been studied intensively . We review structural and functional studies of both bacterial and eukaryotic glutamine synthetases, with emphasis on enzymatic inhibitors.

Arch Oral Biol, 2000 Apr, 45(4), 277 - 91
In situ studies of pellicle formation on hydroxyapatite discs; Vacca Smith AM et al.; The formation of acquired enamel pellicle on hydroxyapatite (HA) discs of known surface area carried in the mouth was studied; discs were carried in the mouth for 30 s, 1, 5, 10 and 20 min . Similar amounts of protein were found on the discs at each time-point, as determined by ninhydrin analyses . The amounts of amylase and lysozyme detected remained stable after 5 min of exposure of the discs to the mouth . Assay of the discs for fructosyl- and glucosyltransferase activities revealed that fructosyltransferase activity increased up to 1 min of exposure to the mouth and decreased when kept in the mouth for longer periods; glucosyltransferase activity, in contrast, increased the longer the discs were kept in the mouth . This in situ model provides insight into the activities of various enzymes during the first 20 min of pellicle formation . The effects of rinsing with sucrose and sugar alcohols on pellicle formation on the discs were also explored . The discs were placed in the mouth for 30 s, 1, 5, 10 and 20 min, preceded by rinsing with either distilled deionized water, sucrose, sorbitol, xylitol or phosphate-buffered saline . Western blot analyses of disc eluates with antiserum/antibody preparations to various salivary components revealed distinct patterns of deposition of bacterial and salivary components depending on the composition of the rinse . These studies confirm that salivary molecules and bacteria are deposited on apatitic surfaces in a selective manner and reveal that pellicle formation may be influenced by composition of diet . It is apparent that this in situ model could be used in screening potential antiplaque agents.

Trends Microbiol, 2000 Mar, 8(3), 120 - 8
Phages will out: strategies of host cell lysis; Young I et al.; Most phages accomplish host lysis using a muralytic enzyme, or endolysin, and a holin, which permeabilizes the membrane at a programmed time and thus controls the length of the vegetative cycle . By contrast, lytic single-stranded RNA and DNA phages accomplish lysis by producing a single lysis protein without muralytic activity.

Am J Ind Med, 2000 Apr, 37(4), 438 - 42
Two year follow-up of a garbage collector with allergic bronchopulmonary aspergillosis (ABPA); Allmers H et al.; BACKGROUND: Separate collection of biodegradable garbage and recyclable waste is expected to become mandatory in some western countries . A growing number of persons engaged in garbage collection and separation might become endangered by high loads of bacteria and fungi . Case history and examination A 29 year old garbage collector involved in emptying so-called biological garbage complained of dyspnea, fever, and flu-like symptoms during work beginning in the summer of 1992 . Chest x-ray showed streaky shadows near both hili reaching into the upper regions . IgE- and IgG-antibodies (CAP, Pharmacia, Sweden) were strongly positive for Aspergillus fumigatus with 90.5 kU/L and 186%, respectively . Total-IgE was also strongly elevated with 5430 kU/L . Bronchial challenge testing with commercially available Aspergillus fumigatus extract resulted in an immediate-type asthmatic reaction . Two years later he was still symptomatic and antibodies persisted at lower levels . CONCLUSIONS: Our diagnosis was allergic bronchopulmonary aspergillosis (ABPA) including asthmatic responses as well as hypersensitivity pneumonitis (extrinsic allergic alveolitis) due to exposure to moldy household waste . A growing number of persons engaged in garbage collection and handling are exposed and at risk to develop sensitization to fungi due to exposure to dust of biodegradable waste . Further studies are necessary to show if separate collection of biodegradable waste increases the health risks due to exposure to bacteria and fungi in comparison to waste collection without separation .

J Immunol, 2000 Mar 15, 164(6), 3236 - 45
Superfibronectin, a multimeric form of fibronectin, increases HIV infection of primary CD4+ T lymphocytes; Tellier MC et al.; The ability of viruses and bacteria to interact with the extracellular matrix plays an important role in their infectivity and pathogenicity . Fibronectin is a major component of the extracellular matrix in lymph node tissue, the main site of HIV deposition and replication during the chronic phase of infection . Therefore, we asked whether matrix fibronectin (FN) could affect the ability of HIV to infect lymphocytes . To study the role of matrix FN on HIV infection, we used superfibronectin (sFN), a multimeric form of FN that closely resembles in vivo matrix FN . In this study we show that HIV-1IIIB efficiently binds to multimeric fibronectin (sFN) and that HIV infection of primary CD4+ lymphocytes is enhanced by >1 order of magnitude in the presence of sFN . This increase appears to be due to increased adhesion of viral particles to the cell surface in the presence of sFN, followed by internalization of virus . Enzymatic removal of cell surface proteoglycans inhibited the adhesion of HIV-1IIIB/sFN complexes to lymphocytes . In contrast, Abs to integrins had no effect on binding of HIV-1IIIB/sFN complexes to lymphocytes . The III1-C peptide alone also bound HIV-1IIIB efficiently and enhanced HIV infection, although not as effectively as sFN . HIV-1IIIB gp120 envelope protein binds to the III1-C region of sFN and may be important in the interaction of virus with matrix FN . We conclude that HIV-1IIIB specifically interacts with the III1-C region within matrix FN, and that this interaction may play a role in facilitating HIV infection in vivo, particularly in lymph node tissue.

Planta Med, 2000 Feb, 66(1), 40 - 3
Beta-glucuronidase inhibitory activity and hepatoprotective effect of 18 beta-glycyrrhetinic acid from the rhizomes of Glycyrrhiza uralensis; Shim SB et al.; An inhibitor of beta-glucuronidase from the rhizomes of Glycyrrhiza uralensis was isolated and its hepatoprotective activity on CCI4-induced hepatotoxicity of rats was investigated . From the water-soluble extract of G . uralensis, glycyrrhizin was isolated as a potent inhibitor of beta-glucuronidase . When glycyrrhizin was orally administered, it had a hepatoprotective activity . However, when glycyrrhizin was intraperitoneally administered, it did not have a hepatoprotective activity . 18 beta-Glycyrrhetinic acid, which is a major metabolite of glycyrrhizin by human intestinal bacteria, was also a potent inhibitor of beta-glucuronidase . When 18 beta-glycyrrhetinic acid was intraperitoneally administered, it also had some hepatoprotective activity . These results suggest that glycyrrhizin may be a natural prodrug for the observed hepatoprotective effect in rats and that serum beta-glucuronidase levels have implications for the liver injury, as reductions of its activity by administration of inhibitors such as G . uralensis or its derived products and silymarin correlate with reductions in biochemical indices of liver injury.

Biosci Biotechnol Biochem, 2000 Jan, 64(1), 52 - 60
Molecular modeling study of highly branching (1-->3)-alpha-D-glucan, a model polysaccharide for cariogenic glucan, using the N-H mapping method; Yui T et al.; A systematic search for possible regular helical structures of a highly branching (1-->3)-alpha-D-glucan was done using the n-h mapping technique, combined with MM3-generated relaxed-residue energy map calculations with respect to the conformations of the backbone glycosidic linkages . The alpha-D-glucan, consisting of a (1-->3)-alpha-linked backbone with alpha-D-glucose side residues attaching to an O6 atom of every second backbone residue, was considered as a model polysaccharide of a branching part of the glucan produced by oral bacteria, which was known to be related to dental plaque formation and to contribute to dental caries . The potential energy surfaces of the trisaccharide repeating unit of the branching alpha-D-glucan indicated that (1-->6)-alpha-linked side residues did not appear to interfere significantly with the backbone stereochemistry, probably due to a further separation of the three-bond-linked side residue compared with an ordinary two-bond-linked residue . Based on the n-h maps of the branching alpha-D-glucan, the side residues, when involved in a complete helix, mostly contributed additional stabilizations to particular helical structures . It was found by checking the typical helix models that formation of hydrogen bonds involving side residues was probably a major cause of the stabilization . This hydrogen bonding was expected to increase insolubility for the glucan chain--a typical, physical property observed for the bacterial alpha-D-glucan--by introducing its backbone stereochemistry as an additional stiff feature.

Nutrition, 2000 Mar, 16(3), 165 - 7
Effect of high ambient temperature on contamination and physical stability of one-liter ready-to-hang enteral delivery systems; Hsu TC et al.; The effect of high ambient temperature on the physical stability and bacterial contamination of 1-L, prefilled, closed enteral feeding systems was examined under simulated clinical conditions . One hundred Jevity Ready-to-Hang enteral feeding systems (Abbott Park, IL, USA) were placed in a 37 degrees C incubator for 24 h . The Ready-to-Hang formula containers were visually inspected at 0 and 24 h . Formula samples were collected from the containers at 24 h and plated on trypticase soy agar . Two samples had insignificant bacterial growth of one colony-forming unit per milliliter that was not demonstrated in repeat culture . No growth was observed for any other sample . Additional samples collected from the two apparently contaminated delivery sets showed no growth . No set showed signs of formula instability, such as coagulation, clumping, or curdling . These findings suggest that, even at a high ambient temperature of 37 degrees C, the risk of bacterial contamination or compromised physical integrity is very low with the use of 1-L, prefilled, closed enteral feeding systems.

Microbiol Mol Biol Rev, 2000 Mar, 64(1), 202 - 36
Aminoacyl-tRNA synthetases, the genetic code, and the evolutionary process; Woese CR et al.; The aminoacyl-tRNA synthetases (AARSs) and their relationship to the genetic code are examined from the evolutionary perspective . Despite a loose correlation between codon assignments and AARS evolutionary relationships, the code is far too highly structured to have been ordered merely through the evolutionary wanderings of these enzymes . Nevertheless, the AARSs are very informative about the evolutionary process . Examination of the phylogenetic trees for each of the AARSs reveals the following . (i) Their evolutionary relationships mostly conform to established organismal phylogeny: a strong distinction exists between bacterial- and archaeal-type AARSs . (ii) Although the evolutionary profiles of the individual AARSs might be expected to be similar in general respects, they are not . It is argued that these differences in profiles reflect the stages in the evolutionary process when the taxonomic distributions of the individual AARSs became fixed, not the nature of the individual enzymes . (iii) Horizontal transfer of AARS genes between Bacteria and Archaea is asymmetric: transfer of archaeal AARSs to the Bacteria is more prevalent than the reverse, which is seen only for the "gemini group . " (iv) The most far-ranging transfers of AARS genes have tended to occur in the distant evolutionary past, before or during formation of the primary organismal domains . These findings are also used to refine the theory that at the evolutionary stage represented by the root of the universal phylogenetic tree, cells were far more primitive than their modern counterparts and thus exchanged genetic material in far less restricted ways, in effect evolving in a communal sense.

Microbiol Mol Biol Rev, 2000 Mar, 64(1), 13 - 33
Membrane topology and insertion of membrane proteins: search for topogenic signals; van Geest M et al.; Integral membrane proteins are found in all cellular membranes and carry out many of the functions that are essential to life . The membrane-embedded domains of integral membrane proteins are structurally quite simple, allowing the use of various prediction methods and biochemical methods to obtain structural information about membrane proteins . A critical step in the biosynthetic pathway leading to the folded protein in the membrane is its insertion into the lipid bilayer . Understanding of the fundamentals of the insertion and folding processes will significantly improve the methods used to predict the three-dimensional membrane protein structure from the amino acid sequence . In the first part of this review, biochemical approaches to elucidate membrane protein topology are reviewed and evaluated, and in the second part, the use of similar techniques to study membrane protein insertion is discussed . The latter studies search for signals in the polypeptide chain that direct the insertion process . Knowledge of the topogenic signals in the nascent chain of a membrane protein is essential for the evaluation of membrane topology studies.

J Cell Biol, 2000 Mar 6, 148(5), 997 - 1008
Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells; Pralle A et al.; To probe the dynamics and size of lipid rafts in the membrane of living cells, the local diffusion of single membrane proteins was measured . A laser trap was used to confine the motion of a bead bound to a raft protein to a small area (diam < or = 100 nm) and to measure its local diffusion by high resolution single particle tracking . Using protein constructs with identical ectodomains and different membrane regions and vice versa, we demonstrate that this method provides the viscous damping of the membrane domain in the lipid bilayer . When glycosylphosphatidylinositol (GPI) -anchored and transmembrane proteins are raft-associated, their diffusion becomes independent of the type of membrane anchor and is significantly reduced compared with that of nonraft transmembrane proteins . Cholesterol depletion accelerates the diffusion of raft-associated proteins for transmembrane raft proteins to the level of transmembrane nonraft proteins and for GPI-anchored proteins even further . Raft-associated GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 min . The measurements agree with lipid rafts being cholesterol-stabilized complexes of 26 +/- 13 nm in size diffusing as one entity for minutes.

J Mol Biol, 2000 Mar 17, 297(1), 49 - 65
Roles of the ccoGHIS gene products in the biogenesis of the cbb(3)-type cytochrome c oxidase; Koch HG et al.; In many bacteria the ccoGHIS cluster, located immediately downstream of the structural genes (ccoNOQP) of cytochrome cbb(3) oxidase, is required for the biogenesis of this enzyme . Genetic analysis of ccoGHIS in Rhodobacter capsulatus demonstrated that ccoG, ccoH, ccoI and ccoS are expressed independently of each other, and do not form a simple operon . Absence of CcoG, which has putative (4Fe-4S) cluster binding motifs, does not significantly affect cytochrome cbb(3) oxidase activity . However, CcoH and CcoI are required for normal steady-state amounts of the enzyme . CcoI is highly homologous to ATP-dependent metal ion transporters, and appears to be involved in the acquisition of copper for cytochrome cbb(3) oxidase, since a CcoI-minus phenotype could be mimicked by copper ion starvation of a wild-type strain . Remarkably, the small protein CcoS, with a putative single transmembrane span, is essential for the incorporation of the redox-active prosthetic groups (heme b, heme b(3 )and Cu) into the cytochrome cbb(3) oxidase . Thus, the ccoGHIS products are involved in several steps during the maturation of the cytochrome cbb(3) oxidase .

Biochemistry, 2000 Mar 14, 39(10), 2530 - 7
Heteronuclear NMR and soft docking: an experimental approach for a structural model of the cytochrome c553-ferredoxin complex; Morelli X et al.; The combination of docking algorithms with NMR data has been developed extensively for the studies of protein-ligand interactions . However, to extend this development for the studies of protein-protein interactions, the intermolecular NOE constraints, which are needed, are more difficult to access . In the present work, we describe a new approach that combines an ab initio docking calculation and the mapping of an interaction site using chemical shift variation analysis . The cytochrome c553-ferredoxin complex is used as a model of numerous electron-transfer complexes . The 15N-labeling of both molecules has been obtained, and the mapping of the interacting site on each partner, respectively, has been done using HSQC experiments . 1H and 15N chemical shift analysis defines the area of both molecules involved in the recognition interface . Models of the complex were generated by an ab initio docking software, the BiGGER program (bimolecular complex generation with global evaluation and ranking) . This program generates a population of protein-protein docked geometries ranked by a scoring function, combining relevant stabilization parameters such as geometric complementarity surfaces, electrostatic interactions, desolvation energy, and pairwise affinities of amino acid side chains . We have implemented a new module that includes experimental input (here, NMR mapping of the interacting site) as a filter to select the accurate models . Final structures were energy minimized using the X-PLOR software and then analyzed . The best solution has an interface area (1037.4 A2) falling close to the range of generally observed recognition interfaces, with a distance of 10.0 A between the redox centers.

Br J Anaesth, 1999 Dec, 83(6), 845 - 9
Gram stain of bronchoalveolar lavage fluid in the early diagnosis of ventilator-associated pneumonia; Allaouchiche B et al.; To assess the usefulness of the Gram stain in the early diagnosis of ventilator-associated pneumonia (VAP), we performed 146 protected specimen brushings (PSB) and bronchoalveolar lavages (BAL) in 118 patients suspected of having nosocomial pneumonia . Gram stain and counts of infected cells were performed in all samples from BAL fluid . A final diagnosis of pneumonia was established in 51 patients and there was no infection in 95 cases . A threshold of 2% of infected cells was used to distinguish between VAP and the group without VAP (sensitivity 86.3%, specificity 78.9%, positive predictive value 68.7% and negative predictive value 91.4%); there was good agreement with the final diagnosis (kappa statistic 0.616; concordance 81.5%) . Regarding detection of bacteria using the Gram stain, we found a sensitivity of 90.2%, specificity 73.7%, positive predictive value 64.8% and negative predictive value 93.3%; there was moderate agreement with the final diagnosis (kappa statistic 0.586; concordance 79.4%) . In the VAP group, we analysed the degree of qualitative agreement between Gram stain and PSB quantitative cultures: the correlation was complete in 51% (26 of 51 VAP), partial in 39.2% (20 of 51 VAP) and there was no correlation in 9.8% (five of 51 VAP) . We conclude that the Gram stain is useful for rapid diagnosis of VAP but unreliable for early adaptation of empiric therapy.

Rapid Commun Mass Spectrom, 2000, 14(5), 307 - 10
Characterization of fungal spores by laser desorption/ionization time-of-flight mass spectrometry; Welham KJ et al.; A considerable volume of research has now been completed on the application of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to the analysis of bacteria; however, to date no definitive studies have been made using this technique on fungi . Preliminary studies on the application of the MALDI-MS methodology, previously developed for the analysis of bacteria, to the analysis of intact fungal spores are described here . MALDI-MS and electrospray mass spectrometry enable the high molecular weight analysis of proteins, glycoproteins, oligosaccharides and oligonucleotides . Using MALDI-MS with bacteria has demonstrated the ability to produce 'fingerprints' of the intact cells with the ions observed being associated with the proteinaceous components of the cell wall . This paper reports the adaptation of this technique to the direct analysis of fungal cells . The high percentage of carbohydrate in the fungal cell wall indicates that the ions observed in the mass spectrometric experiments may be of carbohydrate origin . Penicillium spp., Scytalidium dimidiatum and Trichophyton rubrum have been studied in this preliminary investigation and all show individually distinctive spectra which would appear to provide a profile of the cellular material with discrete peaks being observed over the mass range 2 to 13 kDa . The spectra obtained are reproducible within the method used but, as shown in our previous studies on bacteria, washing may selectively release components from the fungal cell wall .

Pharmacol Rev, 2000 Mar, 52(1), 91 - 112
C1-Esterase inhibitor: an anti-inflammatory agent and its potential use in the treatment of diseases other than hereditary angioedema; Caliezi C et al.; C1-esterase inhibitor (C1-Inh) therapy was introduced in clinical medicine about 25 years ago as a replacement therapy for patients with hereditary angioedema caused by a deficiency of C1-Inh . There is now accumulating evidence, obtained from studies in animals and observations in patients, that administration of C1-Inh may have a beneficial effect as well in other clinical conditions such as sepsis, cytokine-induced vascular leak syndrome, acute myocardial infarction, or other diseases . Activation of the complement system, the contact activation system, and the coagulation system has been observed in these diseases . A typical feature of the contact and complement system is that on activation they give rise to vasoactive peptides such as bradykinin or the anaphylatoxins, which in part explains the proinflammatory effects of either system . C1-Inh, belonging to the superfamily of serine proteinase inhibitors (serpins), is a major inhibitor of the classical complement pathway, the contact activation system, and the intrinsic pathway of coagulation, respectively . It is, therefore, endowed with anti-inflammatory properties . However, inactivation of C1-Inh occurs locally in inflamed tissues by proteolytic enzymes (e.g., elastase) released from activated neutrophils or bacteria thereby leading to increased local activation of the various host defense systems . Here we will give an overview on the biochemistry and biology of C1-Inh . We will discuss studies addressing therapeutic administration of C1-Inh in experimental and clinical conditions . Finally, we will provide an explanation for the therapeutic benefit of C1-Inh in so many different diseases.

EMBO J, 2000 Mar 1, 19(5), 800 - 6
The importance of aquaporin water channel protein structures; Engel A et al.; The history of the water channel and recent structural and functional analyses of aquaporins are reviewed . These ubiquitous channels are important for bacteria, plants and animals, exhibit a pronounced sequence homology and share functional as well as structural similarities . Aquaporins allow water or small specific solutes to pass unhindered, but block the passage of ions to prevent dissipation of the transmembrane potential . Besides advances in structure determination, recent experiments suggest that many of these channels are regulated by pH variations, phosphorylation and binding of auxiliary proteins.

Appl Environ Microbiol, 2000 Mar, 66(3), 914 - 9
Influence of Acanthamoeba castellanii on intracellular growth of different Legionella species in human monocytes; Neumeister B et al.; Previous studies using a murine model of coinhalation of Legionella pneumophila and Hartmannella vermiformis have shown a significantly enhanced intrapulmonary growth of L . pneumophila in comparison to inhalation of legionellae alone (J . Brieland, M . McClain, L . Heath, C . Chrisp, G . Huffnagle, M . LeGendre, M . Hurley, J . Fantone, and C . Engleberg, Infect . Immun . 64:2449-2456, 1996) . In this study, we introduce an in vitro coculture model of legionellae, Mono Mac 6 cells (MM6) and Acanthamoeba castellanii, using a cell culture chamber system which separates both cell types by a microporous polycarbonate membrane impervious to bacteria, amoebae, and human cells . Whereas L . pneumophila has shown a maximal 4-log-unit multiplication within MM6, which could not be further increased by coculture with Acanthamoeba castellanii, significantly enhanced replication of L . gormanii, L . micdadei, L . steigerwaltii, L . longbeachae, and L . dumoffii was seen after coculture with amoebae . This effect was seen only with uninfected amoebae, not with Legionella-infected amoebae . The supporting effect for intracellular multiplication in MM6 could be reproduced in part by addition of a cell-free coculture supernatant obtained from a coincubation experiment with uninfected A . castellanii and Legionella-infected MM6, suggesting that amoeba-derived effector molecules are involved in this phenomenon . This coculture model allows investigations of molecular and biochemical mechanisms which are responsible for the enhancement of intracellular multiplication of legionellae in monocytic cells after interaction with amoebae.

Mol Immunol, 1999 Sep-Oct, 36(13-14), 905 - 14
Complement activation and inhibition in experimental models of arthritis; Linton SM et al.; Complement activation has been implicated as a pathological process in a number of inflammatory and autoimmune disorders including chronic rheumatoid arthritis (RA) . Animal models of experimental arthritis have been widely used to investigate the pathogenesis of RA and also in the development of novel therapies . Many of these models are complement-dependent and both incidence and progression of disease can be influenced by complement inhibition . In certain situations, local inhibition is of greater therapeutic benefit than systemic decomplementation . An increasing awareness and availability of a wide range of naturally occurring complement regulatory proteins can now offer a more targeted approach to complement inhibition while the availability of novel engineering strategies has also improved the efficiency of this process . The success of complement inhibition in the experimental models described should offer a novel therapeutic approach to the treatment of human inflammatory arthritis.

Acta Biochim Pol, 1999, 46(3), 673 - 7
A comparative CD and fluorescence study of a series of model calcium-binding peptides; Goch G et al.; Lanthanide-saturated peptides analogous to calcium-binding loops of EF-hand proteins can be used to stabilize the alpha-helical structure of peptide or protein segments attached to their C-termini . To study conformational properties of such loop-containing hybrids it is necessary to produce them in bacteria . In peptides obtained in this way the helix will be destabilized by the negatively charged C-terminal alpha-carboxyl groups . We propose to block them by the homoserine lactone . The results presented in this paper indicate that the presence of the lactone even at the C-terminus of the loop does not have any negative effect on the loop helix-nucleation ability . On the other hand, the presence of the alpha-NH3+ at the loop N-terminus leads to a drop of metal-binding constant and loss of the rigid structure of the alpha-helical segment of the loop . The alpha-amino group separated by one glycine residue from the loop N-terminus should also be avoided because it perturbs the conformation of the N-terminal part of the loop and may reduce the loop affinity to lanthanide ions.

Am J Dermatopathol, 2000 Feb, 22(1), 75 - 8
Tumorlike eosinophilic granuloma of the skin; Gerbig AW et al.; In 1952, Kuske reported on a patient with a peculiar tumor on the dorsum of the right hand; histological analysis revealed a dense dermal infiltrate with numerous eosinophils . Not aware of any similar case report in the literature, he coined the descriptive term "tumor-like eosinophilic granuloma of the skin." In 1995, a 55-year-old white man with cancer of the prostate presented with a 4-month history of two reddish-brown, solid skin tumors on his left forearm and on the right side of his abdomen, respectively . Histologic examination revealed a dense, superficial and deep, tumorlike dermal inflammatory infiltrate consisting mainly of eosinophils as well as neutrophils and in part epithelioid, in part foamy histiocytes . Flame figures were absent . Immunohistochemical analysis was negative for S-100 protein, whereas sporadic cells in the infiltrate were CD1a positive and many mononuclear-histiocytic cells reacted with MAC 387 . Stains as well as cultures for bacteria, mycobacteria, and fungi were negative . The descriptive diagnosis of tumorlike eosinophilic granuloma of the skin was made . Seven weeks after prostatectomy, both tumors resolved spontaneously and so far has not recurred . In our opinion, this is the second report of Kuske's tumorlike eosinophilic granuloma of the skin . Perhaps tumorlike eosinophilic granuloma of the skin, eosinophilic ulcer of the mucosa, and transient eosinophilic nodulomatosis should be considered a mucocutaneous reaction pattern as is seen in cats . In humans, hypersensitivity reactions or atopy could emerge as an etiological link.

J Endod, 1975 Aug, 1(8), 276 - 8
Povidone-iodine and isopropyl alcohol as disinfectants in preparation for endodontics; Baumgartner JC et al.; The clinical effectiveness of povidone-iodine as a disinfectant in teeth isolated by a rubber dam was compared to the effectiveness of isopropyl alcohol . The results showed no statistically significant difference between povidone-iodine or isopropyl alcohol for disinfecting intact enamel surfaces . When a scrub-type application of povidone-iodine was used, preliminary removal of plaque with pumice did not increase its effectiveness . Both agents were significantly less effective at 15 minutes after application than they were at 90 seconds after application.

J Endod, 1975 Jun, 1(6), 193 - 202
Methodology and criteria in the evaluation of dental implants; Neuman G et al.; This study was designed to develop an inexpensive, reproducible method for studying the reaction of the tissues of the oral cavity to the endosseous implant . Thirty-six guinea pigs, three materials (Teflon, Vitallium, and Titanium 6Al-4V), two observation periods (two and 12 weeks), and two implant designs (one with exposure to the oral cavity and one without exposure to the oral cavity) were used . The inflammatory response was significantly greater in the exposed implants than in the unexposed implants . In the implants that were exposed 12 weeks, there was a strong interrelationship between severe inflammation, bacteria, and epithelial invagination . These factors are significant causes for failures of implants.

Endoscopy, 2000 Feb, 32(2), 131 - 7
Inflammatory bowel disease; Marteau P; This article reviews important papers on inflammatory bowel disease published between May 1998 and June 1999 . It does not review every aspect of treatment, but focuses on the effects of anti-tumor necrosis factor antibodies on the inflammatory lesions . The new information summarized includes: the role of bacteria and the modulating effects of probiotics; the frequency of appendiceal orifice inflammation in ulcerative colitis; progress in imaging based on endoscopic ultrasonography, magnetic resonance imaging, and leukocyte scintigraphy; frequency and treatment of massive hemorrhage, viral superinfection, and persistent perineal sinus; and the pathogenesis, detection, and treatment of dysplasia and cancer.

What Is Biofilter?, What Is Anthrax?, What Is Environmental Microbiology?, What Is Molecular Microbiology?, What Is Salmonella?, a, Bacteria, s, Microbiology, a, Microorganisms, s, Microbe, e, Microorganism, s, Antimicrobial, c, Enterobacters, n, Escherichia coli, o, Ps. putida, o, Escherichia coli, e, Enterobacters, e, Escherichia coli, n, Enterobacteriacea, s, Campylobacter, i, Streptococci, a, Acinetobacter, c, Penicillin, n, Meningococcus, c, Staphylococcus aureus, c, Bacillus subtilis, a, Escherichia coli, n, Escherichia coli, e, Escherichia coli, o, Multidrug resistant, s, Bacteriological, r, Salmonella typhimurium

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