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