Schriftenr Ver Wasser Boden Lufthyg, 1999, 104, 551 - 9 {Risk to employees in garbage collection and transport from pathogen exposure}; Becker G et al.; In a current study funded by the Federal Institute of Occupational Safety and Hygiene the microbial exposure during waste collection and the health of waste collectors are being examined . The investigation of the exposure is carried out in standard experiments and in field studies . It should give information about the extent of emissions of bacteria and fungi spores und confounding factors . Medical examinations of waste collectors will show if there are special health risks attributable to waste collection. Schriftenr Ver Wasser Boden Lufthyg, 1999, 104, 533 - 45 {Microbial exposure in collection of residential garbage--results of field studies}; Neumann HD et al.; Since 1995 the communal accident insurance carrier of the county Wetfalen-Lippe conducts investigations into the exposure to biological agents related to refuse collection . Total fungal exposure during refuse collection turned out to range from 10,000 up to 750,000 colony forming units per cubic meter . Most of the measurement values exceeded the limit of 50,000 . During hot periods in the summertime, the concentration of Aspergillus fumigatus increased up to 90,000 cfu/m3 . The mean values of the bacterial concentrations ranged from 15,000 up to 50,000 cfu/m3, the endotoxin concentration from 12 up to 59 EU/m3 . In the driver's cabin fungal exposure sometimes exceeded 10,000 cfu/m3 especially in autumn and winter . Maximum values were 5,000 cfu/m3 for bacteria and 15 EU/m3 for endotoxins . High values were measured irrespective of the kind of refuse. Schriftenr Ver Wasser Boden Lufthyg, 1999, 104, 403 - 81 {Effect of microbial aerosols on the human}; Herr C et al.; Based on numerous publications dealing with the effects of microbial emissions on workers in waste processing plants and on few papers concerning the assessment of the environmental health relevance of microbial aerosols on people living in the vicinity of composting facilities, the current state of the art in this field is presented . With regard to occupational exposure the possible health effects like infections, toxicity and allergies are specified . Since to date only few studies have been made of populations exposed to microbial aerosols in ambient air, the environmental health aspects are reviewed in the context of a study of three compost plants in Hesse, where ambient air measurements as well as epidemiological investigations were carried out . Final recommendations are given and the research needs regarding the environmental health significance of microbial aerosols are formulated. Schriftenr Ver Wasser Boden Lufthyg, 1999, 104, 211 - 43 {"Microbial air pollutants--methods for detection of endotoxins, mycotoxins and MVOC"}; Tilkes F et al.; In the context of microbial emissions from composting facilities the methods for the detection and identification of the groups of substances released, i.e . endotoxins, mycotoxins and Microbial Volatile Compounds (MVOC) are discussed . With the aid of an overview of the different methods employed for the investigation of the single groups of compounds the current state of the art in this field is presented . In conclusion the enormous research needs, especially with regard to the mycotoxins and MVOC, are pointed out. Schriftenr Ver Wasser Boden Lufthyg, 1999, 104, 183 - 92 {MVOC of fungi--use as an indicator for exposure level}; Fischer G et al.; The production of microbial volatile organic compounds (MVOC) by fungi derived from biowaste was studied on synthetic agar (YES), compost-extract media (CEA VAR 1-3) and compost . Exposure assessment was carried out in composting facilities and the occurrence of VOC/MVOC was related to the presence of airborne fungi . A number of non-specific MVOC was found at all sampling sites or in each season, some of which showed preferences for certain sampling sites (3-methyl-1-butanol, 3-octanone) . They seemed to be correlated with a certain species composition or with microbial activity, and may thus be used to describe a certain state of decomposition . Correlations between single volatiles and certain fungi were found in situ, but these findings did not match with the species-specific volatiles obtained from pure cultures. Schriftenr Ver Wasser Boden Lufthyg, 1999, 104, 149 - 62 {Mycotoxins as exposure parameters in bioaerosols of composting sites}; Fischer G et al.; The potential to produce mycotoxins was investigated for freshly isolated strains of airborne fungi . The spectra of metabolites in conidial extracts and culture extracts were compared for some relevant species . Furthermore, their potential to produce mycotoxins on semi-natural media (compost extract agar) supplemented with sucrose, yeast extract, and carboxymethylcellulose in different combinations was investigated . In native bioaerosols in a compost facility (plant 2), tryptoquivaline, a compound with tremorgenic properties, and trypacidin, for which no toxic properties are described, were found . The highly toxic metabolites gliotoxin and verruculogen were not found in the bioaerosols, although they were produced by some strains in pure culture . An inventory of microbial metabolites in addition of fungal propagules has led to a more detailed identification of potential health hazards at the working place . In addition to the pathogenic and allergological relevance, airborne fungi are thus of toxicological concern. Biochim Biophys Acta, 2000 May 6, 1485(1), 56 - 62 Egg yolk lipoproteins as substrates for lipases; Abousalham A et al.; Egg yolk emulsions containing phospholipids (about 31%, w/w) are classically used as substrates for measuring phospholipase A2 activity using the pH-stat method . Here we investigated the susceptibility of egg yolk lipoproteins to lipolysis by various highly purified lipases of animal or microbial origin . Egg yolk lipoproteins, which contain up to 65% triacylglycerols, were found to be effective substrates for all the lipases tested . The specific activities measured on egg yolk lipoproteins using the pH-stat technique were found to be 8000, 1000, 1250 and 1700 U/mg in the case of human pancreatic lipase, horse pancreatic lipase, porcine pancreatic lipase and Humicola lanuginosa lipase, respectively . No activity was detected in the absence of colipase with any of the pancreatic lipases tested . Consequently, the classical egg yolk assay cannot be considered as a specific phospholipase A2 assay. Proc Natl Acad Sci U S A, 2000 May 23, 97(11), 6097 - 101 Specific phenotypic restoration of an attenuated virus by knockout of a host resistance gene; Leib DA et al.; To produce disease, viruses must enter the host, multiply locally in host tissues, spread from the site of entry, and overcome or evade host immune responses . At each stage in this infectious process, specific microbial and host genes determine the ultimate virulence of the virus . Genetic approaches have identified many viral genes that play critical roles in virulence and are presumed to target specific components of the host innate and acquired immune response . However, formal proof that a virulence gene targets a specific protein in a host pathway in vivo has not been obtained . Based on cell culture studies, it has been proposed that the herpes simplex virus type 1 gene ICP34.5 (ICP, infected cell protein) enhances neurovirulence by negating antiviral functions of the IFN-inducible double-stranded RNA-dependent protein kinase R or PKR {Chou, J., Chen, J.J., Gross, M . & Roizman, B . (1995) Proc . Natl . Acad . Sci . USA 92, 10516-10520} . Herein, we show that a virus that has been attenuated by deletion of ICP34.5 exhibits wild-type replication and virulence in a host from which the PKR gene has been deleted . We show that restoration of virulence is specific to ICP34.5 and PKR by using additional host and viral mutants . The use of recombinant viruses to infect animals with null mutations in host defense genes provides a formal genetic test for identifying in vivo mechanisms and targets of microbial virulence genes. Structure Fold Des, 2000 Apr 15, 8(4), 407 - 18 Solution structure of PCP, a prototype for the peptidyl carrier domains of modular peptide synthetases; Weber T et al.; BACKGROUND: Nonribosomal peptide synthetases (NRPSs) are large modular enzymes responsible for the synthesis of a variety of microbial bioactive peptides . They consist of modules that each recognise and incorporate one specific amino acid into the peptide product . A module comprises several domains, which carry out the individual reaction steps . After activation by the adenylation domain, the amino acid substrate is covalently tethered to a 4'-phosphopantetheinyl cofactor of a peptidyl carrier domain (PCP) that passes the substrate to the reaction centres of the consecutive domains . RESULTS: The solution structure of PCP, a distinct peptidyl carrier protein derived from the equivalent domain of an NRPS, was solved using NMR techniques . PCP is a distorted four-helix bundle with an extended loop between the first two helices . Its overall fold resembles the topology of acyl carrier proteins (ACPs) from Escherichia coli fatty acid synthase and actinorhodin polyketide synthase from Streptomyces coelicolor; however, the surface polarity and the length and relative alignment of the helices are different . The conserved serine, which is the cofactor-binding site, has the same location as in the ACPs and is situated within a stretch of seven flexible residues . CONCLUSIONS: The structure of PCP reflects its character as a protein domain . The fold is well defined between residues 8 and 82 and the structural core of the PCP domain can now be defined as a region spanning 37 amino acids in both directions from the conserved serine . The flexibility of the post-translationally modified site might have implications for interactions with the cooperating proteins or NRPS domains. J Mol Biol, 2000 May 19, 298(5), 859 - 73 Crystal structure of a plant ribonuclease, RNase LE; Tanaka N et al.; Ribonuclease LE (RNase LE) from cultured tomato (Lycopersicon esculentum) cells is a member of the RNase T(2) family showing broad base specificity . The crystal structure of RNase LE has been determined at 1.65 A resolution . The structure consists of seven alpha-helices and seven beta-strands, belonging to an alpha+beta type structure . Comparison of the structure of RNase LE with that of RNase Rh, a microbial RNase belonging to the RNase T(2) family, reveals that while the overall folding topologies are similar to each other, major insertions and deletions are found at the N-terminal regions . The structural comparison, an amino acid sequence alignment of the RNase T(2) enzymes, and comparison of the disulfide-bonding pattern of these enzymes show that the structure of RNase LE shown here is the basic framework of the animal/plant subfamily of RNase T(2) enzymes (including a self-incompatibility protein called S-RNase), and the structure of RNase Rh is that of the fungal subfamily of RNase T(2) enzymes (including RNase T(2)) . Subsequently, we superposed the active-site of the RNase LE with that of RNase Rh and found that (1) His39, Trp42, His92, Glu93, Lys96, and His97 of RNase LE coincided exactly with His46, Trp49, His104, Glu105, Lys108, and His109, respectively, of RNase Rh, and (2) two conserved water molecules were found at the putative P(1) sites of both enzymes . These facts suggest that plant RNase LE has a very similar hydrolysis mechanism to that of fungal RNase Rh, and almost all the RNase T(2) enzymes widely distributed in various species share a common catalytic mechanism . A cluster of hydrophobic residues was found on the active-site face of the RNase LE molecule and two large hydrophobic pockets exist . These hydrophobic pockets appear to be base binding sites mainly by hydrophobic interactions and are responsible for the base non-specificity of RNase LE . Copyright 12000 Academic Press. Biotechnol Bioeng, 2000 Jun 20, 68(6), 594 - 601 Power consumption in shaking flasks on rotary shaking machines: II . Nondimensional description of specific power consumption and flow regimes in unbaffled flasks at elevated liquid viscosity; Buchs J et al.; This article is the second part of a series presenting and modeling the hydrodynamics and specific power consumption in shaking flasks on rotary (orbital) shaking machines . In part I, a new method was introduced that enables the accurate determination of the specific power consumption in shaking flasks . The method was first applied to investigate unbaffled flasks with a nominal volume of < or =1 L at low viscosity . In part II, the results for the specific power consumption of unbaffled shaking flasks at elevated viscosities are investigated after varying shaking frequency, flask size, filling volume, and shaking diameter . The theory introduced in part I is extended to liquids of elevated viscosities using nondimensional equations . With these results, the specific power consumption in unbaffled shaking flasks can now be fully described . For the first time, the phenomenon of the liquid being "out of phase" is observed and described . This occurs at certain operating conditions and is characterized by an increasing amount of liquid not following the movement of the shaking table, thus reducing the specific power consumption . This, of course, has much relevance for practical work with microbial cultures . The phenomenon of being "out-of-phase" is described in the form of a newly defined nondimensional phase number (Ph) in analogy to a partially filled, rotating horizontal drum . The Ph can be used to determine reasonable operating conditions for shaking flask experiments when using viscous media, avoiding unfavorable "out-of-phase" operation . Clin Diagn Lab Immunol, 2000 May, 7(3), 352 - 9 Preservation of lymphocyte immunophenotype and proliferative responses in cryopreserved peripheral blood mononuclear cells from human immunodeficiency virus type 1-infected donors: implications for multicenter clinical trials . The ACTG Immunology Advanced Technology Laboratories; Reimann KA et al.; Human immunodeficiency virus type 1 (HIV-1) infection results in impaired immune function that can be measured by changes in immunophenotypically defined lymphocyte subsets and other in vitro functional assays . These in vitro assays may also serve as early indicators of efficacy when new therapeutic strategies for HIV-1 infection are being evaluated . However, the use of in vitro assays of immune function in multicenter clinical trials has been hindered by their need to be performed on fresh specimens . We assessed the feasibility of using cryopreserved peripheral blood mononuclear cells (PBMC) for lymphocyte immunophenotyping and for lymphocyte proliferation at nine laboratories . In HIV-1-infected patients with moderate CD4(+) lymphocyte loss, the procedures of density gradient isolation, cryopreservation, and thawing of PBMC resulted in significant loss of CD19(+) B cells but no measurable loss of total T cells or CD4(+) or CD8(+) T cells . No significant changes were seen in CD28(-) CD95(+) lymphocytes after cell isolation and cryopreservation . However, small decreases in HLA-DR(+) CD38(+) lymphocytes and of CD45RA(+) CD62L(+) were observed within both the CD4(+) and CD8(+) subsets . Fewer than 10% of those specimens that showed positive PBMC proliferative responses to mitogens or microbial antigens lost their responsiveness after cryopreservation . These results support the feasibility of cryopreserving PBMC for immunophenotyping and functional testing in multicenter AIDS clinical trials . However, small changes in selected lymphocyte subsets that may occur after PBMC isolation and cryopreservation will need to be assessed and considered in the design of each clinical trial. Naturwissenschaften, 2000 Mar, 87(3), 137 - 41 Ice shelf microbial ecosystems in the high arctic and implications for life on snowball earth; Vincent WF et al.; The Ward Hunt Ice Shelf (83 degrees N, 74 degrees W) is the largest remaining section of thick (> 10 m) land-fast sea ice along the northern coastline of Ellesmere Island, Canada . Extensive meltwater lakes and streams occur on the surface of the ice and are colonized by photosynthetic microbial mat communities . This High Arctic cryo-ecosystem is similar in several of its physical, biological and geochemical features to the McMurdo Ice Shelf in Antarctica . The ice-mats in both polar regions are dominated by filamentous cyanobacteria but also contain diatoms, chlorophytes, flagellates, ciliates, nematodes, tardigrades and rotifers . The luxuriant Ward Hunt consortia also contain high concentrations (10(7)-10(8) cm-2) of viruses and heterotrophic bacteria . During periods of extensive ice cover, such as glaciations during the Proterozoic, cryotolerant mats of the type now found in these polar ice shelf ecosystems would have provided refugia for the survival, growth and evolution of a variety of organisms, including multicellular eukaryotes. Immunity, 2000 Apr, 12(4), 391 - 8 Control of antigen presentation by a single protease cleavage site; Antoniou AN et al.; Protein antigens require limited proteolytic processing to generate peptides for binding to class II MHC molecules, but the proteases and processing sites involved are largely unknown . Here we analyze the effect of eliminating the three major asparagine endopeptidase (AEP)-processing sites in the microbial antigen tetanus toxin C fragment . The mutant antigen is highly resistant to proteolysis by AEP and crude lysosomal extracts and is dramatically impaired in its ability to be processed and presented to T cells . Remarkably, processing at a single asparagine residue (1219) is obligatory for optimal presentation of many T cell epitopes in this antigen . These studies demonstrate that cleavage at a single processing site can be crucial for effective antigen presentation. Lett Appl Microbiol, 2000 May, 30(5), 396 - 401 Microbial degradation and detoxification of high molecular weight polycyclic aromatic hydrocarbons by Stenotrophomonas maltophilia strain VUN 10,003; Juhasz AL et al.; The ability of Stenotrophomonas maltophilia strain VUN 10,003 to degrade and detoxify high molecular weight polycyclic aromatic hydrocarbons (PAHs) was evaluated in a basal liquid medium . Using high cell density inocula of strain VUN 10,003, the concentration of pyrene, fluoranthene, benz{a}anthracene, benzo{a}pyrene, dibenz{a, h}anthracene and coronene decreased by 98, 45, 26, 22, 22 and 55% over periods ranging from 5 to 42 d . When a PAH mixture containing three- to seven-ring compounds was used, degradation of both low and high molecular weight compounds occurred concurrently . Mutagenicity assays (Ames Test) demonstrated a decrease in the mutagenic potential of dichloromethane culture extracts from all cultures containing single PAH over the incubation period, corresponding to the decrease in the concentration of the PAH . These observations indicate that strain VUN 10,003 could be used for the detoxification of PAH-contaminated wastes. Lett Appl Microbiol, 2000 Apr, 30(4), 272 - 6 Influence of the growth rate calculation on the relationship between growth rate and temperature; Demetz D et al.; Three calculations of the growth rate (e.g . slope of a plot of the log10 of cfu ml-1 vs time, mum of the Gompertz equation and the reciprocal of time to obtain 108 cfu ml-1) were compared for Escherichia coli TG1 growing in tryptone soy broth medium at temperatures ranging from 14 to 39 degrees C . Up to now, the influence of using such different definitions on the relationship between microbial growth rate and temperature has never been investigated . In order to compare these calculation procedures, a dimensionless analysis based on the following normalized variables, mudim = mu/muopt and Tdim = {T-Tmin}/{Topt-Tmin}, was used (Dantigny 1998) . The influence of suboptimal temperatures on the growth rate was represented by means of a Belehradek-type model based on a power function law: {mudim} = {Tdim}alpha . The influence of the different growth rate calculations on the model constants was assessed . Despite the great dependence of the raw growth rate values on the calculation procedure, the dimensionless analysis demonstrated that the alpha-value is independent of the growth rate definition . This result suggests that any definition for the growth rate can be utilized in studies aimed at determining the influence of temperature on microbial growth and highlights the interest of using dimensionless variables to overcome differences in the order of magnitude of the growth rate data and to avoid confusion between definitions. J Appl Microbiol, 2000 May, 88(5), 809 - 16 Rapid fluorescence assessment of intracellular pH as a viability indicator of Clavibacter michiganensis subsp . michiganensis; Chitarra LG et al.; The viability of Clavibacter michiganensis subsp . michiganensis (Cmm) was determined by measuring the intracellular pH (pHin) as a viability parameter . This was based on the observation that growth of Cmm was inhibited at pH 5.5 and below . Therefore, viable cells should maintain their pHin above this pH value . The pHin of Cmm was determined using the fluorescent probe 5(and 6-)-carboxyfluorescein succinimidyl ester (cFSE) . The pHin of Cmm cells exposed to acid treatments was determined using fluorescence spectrofluorometry, and for cells exposed to elevated temperatures, the pHin was determined using fluorescence spectrofluorometry and flow cytometry (FCM) . A good correlation was found between the presence of a pH gradient and the number of colony-forming units (cfu) observed in plate counts . However, with the spectrofluorometry technique, the analysis is based on the whole cell population and the detection sensitivity of this technique is rather low, i.e., cell numbers of at least 107 cfu ml-1 are needed for the analysis . Using FCM, heat-treated and non-treated Cmm cells could be distinguished based on the absence and presence of a pH gradient, respectively . The major advantage of FCM is its high sensitivity, allowing analysis of microbial populations even at low numbers, i.e., 102-103 cfu ml-1. Anaesthesia, 2000 May, 55(5), 466 - 8 Microbial contamination of gum elastic bougies; Cupitt JM; The gum elastic bougie is a simple device that is used to assist in the management of the difficult intubation . It is not uncommon for a bougie to be re-used many times . This study investigated the incidence of microbial contamination of the bougies in one hospital . Potentially pathogenic organisms were identified both on the bougies and in their storage containers . This has implications for their cleaning and maintenance, and raises the question as to whether we should replace them with single-use, disposable devices. Int J Food Microbiol, 2000 Apr 10, 55(1-3), 93 - 8 Quantifying the hurdle concept by modelling the bacterial growth/no growth interface; McMeekin TA et al.; The hurdle concept described eloquently over many years by Professor Leistner and his colleagues draws attention to the interaction of factors that affect microbial behaviour in foods . Under some circumstances these effects are additive . Under others the implication is that synergistic interactions lead to a combined effect of greater magnitude than the sum of constraints applied individually . Predictive modelling studies on the combined effects of temperature and water activity and temperature and pH suggest that the effect of these combinations on growth rate is independent . Where the effect of the two factors is interactive rather than independent is at the point where growth ceases--the growth/no growth interface . An interesting and consistent observation is that a very sharp cut off occurs between conditions permitting growth and those preventing growth, allowing those combinations of factors to be defined precisely and modelled . Growth/no growth interface models quantify the effects of various hurdles on the probability of growth and define combinations at which the growth rate is zero or the lag time infinite . Increasing the stringency of one or more hurdles at the interface by only a small amount will significantly decrease the probability of an organism growing . Understanding physiological processes occurring near the growth/no growth interface and changes induced by moving from one side of the interface to the other may well provide insights that can be exploited in a new generation of food preservation techniques with minimal impact on product quality. Bioelectrochemistry, 2000 Feb, 51(1), 53 - 65 Correction of the influence of baseline artefacts and electrode polarisation on dielectric spectra; Yardley JE et al.; The deconvolution of biological dielectric spectra can be difficult enough with artefact-free spectra but is more problematic when machine baseline artefacts and electrode polarisation are present as well . In addition, these two sources of anomalies can be responsible for significant interference with dielectric biomass measurements made using one- or two-spot frequencies . The aim of this paper is to develop mathematical models of baseline artefacts and electrode polarisation which can be used to remove these anomalies from dielectric spectra in a way that can be easily implemented on-line and in real-time on the Biomass Monitor (BM) . We show that both artefacts can be successfully removed in solutions of organic and inorganic ions; in animal cell and microbial culture media; and in yeast suspensions of varying biomass . The high quality of the compensations achieved were independent of whether gold and platinum electrodes were used; the electrode geometry; electrode fouling; current density; the type of BM; and of whether electrolytic cleaning pulses had been applied . In addition, the calibration experiments required could be done off-line using a simple aqueous KCl dilution series with the calibration constants being automatically calculated by a computer without the need for user intervention . The calibration values remained valid for a minimum of 3 months for the baseline model and indefinitely for the electrode polarisation one . Importantly, application of baseline correction prior to polarisation correction allowed the latter's application to the whole conductance range of the BM . These techniques are therefore exceptionally convenient to use under practical conditions. Yeast, 2000 Apr, 16(6), 553 - 60 A counterselection for the tryptophan pathway in yeast: 5-fluoroanthranilic acid resistance; Toyn JH et al.; The ability to counterselect, as well as to select for, a genetic marker has numerous applications in microbial genetics . Described here is the use of 5-fluoroanthranilic acid for the counterselection of TRP1, a commonly used genetic marker in the yeast Saccharomyces cerevisiae . Counterselection using 5-fluoroanthranilic acid involves antimetabolism by the enzymes of the tryptophan biosynthetic pathway, such that trp1, trp3, trp4 or trp5 strains, which lack enzymes required for the conversion of anthranilic acid to tryptophan, are resistant to 5-fluoroanthranilic acid . Commonly used genetic procedures, such as selection for loss of a chromosomally integrated plasmid, and a replica-plating method to rapidly assess genetic linkage in self-replicating shuttle vectors, can now be carried out using the TRP1 marker gene . In addition, novel tryptophan auxotrophs can be selected using 5-fluoroanthranilic acid . J Exp Med, 2000 May 1, 191(9), 1605 - 10 Primary role for Gi protein signaling in the regulation of interleukin 12 production and the induction of T helper cell type 1 responses; He J et al.; We explored the role of Gi protein signaling in the regulation of interleukin (IL)-12 production and T helper cell type 1 (Th1) T cell differentiation . In initial studies, we showed that treatment of normal mice with pertussis toxin (PT), which inhibits Gi protein signaling, enhanced the capacity of splenocytes to produce IL-12 in response to both microbial and nonmicrobial stimuli . In addition, PT treatment increased the production of tumor necrosis factor (TNF)-alpha and IL-10 by stimulated cells . These findings were corroborated by the fact that untreated Gi2alpha(2/-) mice exhibited enhanced production of IL-12 and TNF-alpha by splenocytes, and of IL-12 p40 by purified spleen CD8alpha(+) lymphoid dendritic cells . Finally, we showed that while normal BALB/c mice infected with Leishmania major exhibited a nonhealing phenotype, those treated with PT when infection was initiated exhibited a healing phenotype along with an enhancement of leishmania-specific Th1 responses in draining lymph nodes . Further, healing was prevented by coadministration of anti-IL-12 and PT . These data demonstrate that endogenous Gi protein signaling has a primary role in the regulation of IL-12 production and the induction of Th1 responses in vivo. Microb Ecol, 2000 Jan, 39(1), 80 - 91 Phosphatase Activities of Endolithic Communities in Rocks of the Antarctic Dry Valleys; Banerjee M et al.; Phosphorus is scarce in Beacon Sandstone of the McMurdo Dry Valleys, Antarctica, and any input from precipitation is minimal . In endolithic microbial communities recycling of P by the action of phosphatases may therefore be important . The phosphatase activities of three different types of endolithic communities in the McMurdo Dry Valley, Antarctica, were studied in the laboratory . The dominant phototrophs were Chroococcidiopsis, mixed Gloeocapsa and Trebouxia, and Trebouxia . Bacteria were also visually conspicuous in the latter two communities, and the Trebouxia in both cases formed a lichenized association with fungal hyphae . In each case marked phosphomonoesterase (PMEase) activity was found in assays with 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate as substrate, and phosphodiesterase activity with bis-p-nitrophenyl phosphate as substrate . The pH optimum of PMEase (assayed at 0.5 pH intervals) of the Chroococcidiopsis, Gloeocapsa-Trebouxia, and Trebouxia communities was 9.5, 5.5, and 8.0, respectively . These values are similar for aqueous extracts of the respective rocks (pH 9.2, 6.2, 7.5) . All three communities showed significantly higher PMEase activity at 5 degrees than 1 degrees C, and the first two also showed much higher activity at 5 degrees than 10 degrees C . All three communities also showed slightly lower activity in the light (7 micromol photon m(-2) s(-1)) than the dark; this was found with all substrates and substrate concentrations . Prior exposure of a moistened sample to light for 2 h led to a reduction in activity even when the subsequent assay was done in the dark . The rate of PMEase activity (using 100 microM MUP) in the Gloeocapsa-Trebouxia and Trebouxia communities was approximately linear with time up to 24 h, whereas the Chroococcidiopsis community showed a marked decrease after 6 h . At least part of this was due to retention of the 4-methylumbelliferone (MU) hydrolysis product . In spite of the assays being conducted on a whole community, the activity-substrate relationship in each case quite closely resembled a typical Michaelis-Menten relationship . Estimates were made of the apparent half-saturation value and the concentration of MUP required to support half-maximal rates . The apparent K (m) values were: Chroococcidiopis, 230 microM; Gloeocapsa-Trebouxia 169 microM; Trebouxia, 135 microM . The respective values for apparent V (max) were 0.053, 0.55, and 0.35 micromol MU g(-1) h(-1) . In view of the greater dependence of these communities on the rock for their sole supply of P than for C and probably N, it is suggested that the cycling of P within the communities is a key factor influencing their overall metabolic activity when moisture permits their activation . </hea Microb Ecol, 2000 Jan, 39(1), 72 - 79 Effects of Agricultural Chemicals on DNA Sequence Diversity of Soil Microbial Community: A Study with RAPD Marker; Yang Y et al.; The DNA sequence diversities for microbial communities in four soils affected by agricultural chemicals (mainly triadimefon and ammonium bicarbonate and their intermediates) were evaluated by Random Amplified Polymorphic DNA (RAPD) analysis . Fourteen random primers were used to amplify RAPDs from four soil microbial community DNAs . The products of 12 primers were separated in gel and generated 155 reliable fragments, of which 134 were polymorphic . The richness, modified richness, Shannon-Weaver index, and a similarity coefficient of DNA were calculated to quantify the diversity to access DNA sequence diversities for four soil microbial communities . The results showed that agricultural chemicals affected soil microbial community diversity at the DNA level . The four soil microbial communities were distinguishable in terms of DNA sequence richness, modified richness, Shannon-Weaver index, and coefficient of DNA similarity . Analysis also showed that the amounts of organic C and microbial biomass C were low in the soil polluted by pesticide (mainly triadimefon and its intermediates), but high in the soil polluted by chemical fertilizer (mainly ammonium bicarbonate and its intermediates) . The above results combined may indicate that pesticide pollution caused a decrease in the soil microbial biomass but kept high diversity at DNA level, compared with the control without chemical pollution . In contrast, chemical fertilizer pollution caused an increase in the soil biomass but decrease in the DNA diversity . The RAPD marker technique combined with analysis of soil microbial biomass appears to be an effective approach for studying the diversity of soil microbial communities, although the effects of PCR bias on community composition, such as dominating and rare populations in soils, on the diversity needed to be addressed further . </hea Microb Ecol, 2000 Jan, 39(1), 1 - 11 Plant Species and Plant Incubation Conditions Influence Variability in Epiphytic Bacterial Population Size; Kinkel LL et al.; The influences of plant species and plant incubation conditions on the variability in bacterial population sizes among leaves were investigated in field and growth chamber studies . Pseudomonas syringae strains TLP2 and Cit7 were inoculated onto plants and population sizes were measured at intervals after inoculation . Total bacterial population sizes were also assessed in field studies . Levels of leaf-to-leaf variability in both P . syringae population size and bacterial community size differed significantly among plant species . For all plant species, variability among leaves in population sizes of inoculated bacteria was consistently greater than the leaf-to-leaf variability in numbers of total bacteria . Considering levels of variability in population size immediately prior to and following incubation under either wet or dry physical conditions, leaf-to-leaf variability in the population sizes of inoculated P . syringae strains increased significantly following incubation under dry, but not under wet, conditions . Measurements of leaf-to-leaf variability immediately prior to and following incubation were positively correlated regardless of whether the incubation was under wet or dry conditions, though the correlation was greater following dry incubation . These data provide insight into the biological and physical factors that may be important in generating variability in bacterial population sizes among leaves, and they have important implications for the design of appropriate strategies for sampling leaf surface microbial populations . </hea Curr Pharm Des, 2000 Apr, 6(6), 633 - 49 Anti-inflammatory cytokines and cytokine antagonists; Standiford TJ; Cytokines are critical mediators of protective host responses, including defense against microbial invasion and tumorigenesis . However, the production of specific proinflammatory cytokines must be tightly regulated and compartmentalized to prevent the overzealous expression of these molecules that can culminate in unabated inflammation and tissue injury . Cytokine production and/or biologic effects can be inhibited by a variety of endogenous molecules, including anti-inflammatory cytokines, soluble cytokine receptors, and receptor antagonist proteins . Additionally, synthetic molecules have been constructed to selectively block the synthesis, post-translation processing, or receptor binding of proinflammatory cytokines . Relevant anti-inflammatory cytokines and cytokine inhibitors (both endogenous and synthetic) will be the subject of this review, with a particular emphasis on those anti-inflammatory cytokines and cytokine inhibitors that have been used experimentally or clinically in the treatment of diseases that are believed to be mediated by excessive proinflammatory cytokine responses. Toxicol Sci, 2000 May, 55(1), 36 - 43 Ethane sulfonate metabolite of alachlor: assessment of oncogenic potential based on metabolic and mechanistic considerations; Heydens WF et al.; Chronic administration of alachlor has been shown to produce neoplastic responses in the nasal turbinate mucosa, glandular stomach mucosa, and thyroid follicular epithelium of rats . Subsequent studies have shown that specific metabolic activation of alachlor is required for nasal tumor formation, and that non-genotoxic, threshold-sensitive processes produce all three tumors . The herbicide alachlor is degraded in the soil by microbial action to the tertiary ethane sulfonate metabolite (ESA) . The acute and subchronic toxicity of ESA is very low, and the metabolite did not produce developmental toxicity or genotoxicity . The studies described here were conducted to determine whether ESA shares a common mechanism of oncogenicity with alachlor in rats . Specifically, we studied ESA's pharmacokinetics and ability to produce changes that are causally associated with the oncogenicity of alachlor . These studies demonstrated that ESA was poorly absorbed and underwent minor metabolism, which contrasted with the significant absorption and substantial metabolism observed with alachlor . ESA was also excreted more quickly than alachlor and showed no evidence of accumulation in the nasal turbinates, a site of oncogenicity for alachlor in the rat . In addition, ESA did not elicit the characteristic preneoplastic changes observed in the development of alachlor-induced nasal, stomach, and thyroid tumors . The results of these studies support the conclusion that ESA does not share a common oncogenic mechanism with alachlor and would not be expected to produce the same oncogenic responses observed following chronic alachlor exposure in rats. Biol Bull, 2000 Apr, 198(2), 168 - 87 Chemical signaling processes in the marine environment; Zimmer RK et al.; Understanding the mechanisms by which environmental chemical signals, chemical defenses, and other chemical agents mediate various life-history processes can lead to important insights about the forces driving the ecology and evolution of marine systems . For chemical signals released into the environment, establishing the principles that mediate chemical production and transport is critical for interpreting biological responses to these stimuli within appropriate natural, historical contexts . Recent technological advancements provide outstanding opportunities for new discoveries, thus allowing quantification of interactions between hydrodynamic, chemical, and biological factors at numerous spatial and temporal scales . Past work on chemically mediated processes involving organisms and their environment have emphasized habitat colonization by larvae and trophic relationships . Future research priorities should include these topics as well as courtship and mating, fertilization, competition, symbiosis, and microbial chemical ecology . There are now vast new opportunities for determining how organisms respond to chemical signals and employ chemical defenses under environmentally realistic conditions . Integrating these findings within a larger ecological and evolutionary framework should lead to improved understanding of natural physicochemical phenomena that constrain biological responses at the individual, population, and community levels of organization. Electrophoresis, 2000 Apr, 21(6), 1094 - 103 Subproteomics based upon protein cellular location and relative solubilities in conjunction with composite two-dimensional electrophoresis gels; Cordwell SJ et al.; Progress in the field of proteomics is dependent upon an ability to visualise close to an entire protein complement via a given array technology . These efforts have previously centred upon two-dimensional gel electrophoresis in association with immobilised pH gradients in the first dimension . However, limitations in this technology, including the inability to separate hydrophobic, basic, and low copy number proteins have hindered the analysis of complete proteomes . The challenge is now to overcome these limitations through access to new technology and improvements in existing methodologies . Proteomics can no longer be equated with a single two-dimensional electrophoresis gel . Greater information can be obtained using targeted biological approaches based upon sample prefractionation into specific cellular compartments to determine protein location, while novel immobilised pH gradients spanning single pH units can be used to display poorly abundant proteins due to their increased resolving power and loading capacity . In this study, we show the effectiveness of a combined use of two differential subproteomes (as defined by relative solubilities, cellular location and narrow-range immobilised pH gradients) to increase the resolution of proteins contained on two-dimensional gels . We also present new results confirming that this method is capable of displaying up to a further 45% of a given microbial proteome . Subproteomics, utilising up to 40 two-dimensional gels per sample will become a powerful tool for near-to-total proteome analysis in the postgenome era . Furthermore, this new approach can direct biological focus towards molecules of specific interest within complex cells and thus simplify efforts in discovery-based proteome research. Nature, 2000 Apr 20, 404(6780), 858 - 61 Evidence that decomposition rates of organic carbon in mineral soil do not vary with temperature; Giardina CP et al.; It has been suggested that increases in temperature can accelerate the decomposition of organic carbon contained in forest mineral soil (Cs), and, therefore, that global warming should increase the release of soil organic carbon to the atmosphere . These predictions assume, however, that decay constants can be accurately derived from short-term laboratory incubations of soil or that in situ incubations of fresh litter accurately represent the temperature sensitivity of Cs decomposition . But our limited understanding of the biophysical factors that control Cs decomposition rates, and observations of only minor increases in Cs decomposition rate with temperature in longer-term forest soil heating experiments and in latitudinal comparisons of Cs decomposition rates bring these predictions into question . Here we have compiled Cs decomposition data from 82 sites on five continents . We found that Cs decomposition rates were remarkably constant across a global-scale gradient in mean annual temperature . These data suggest that Cs decomposition rates for forest soils are not controlled by temperature limitations to microbial activity, and that increased temperature alone will not stimulate the decomposition of forest-derived carbon in mineral soil. Curr Opin Hematol, 2000 May, 7(3), 174 - 7 Interleukin-15 and its impact on neutrophil function; Cassatella MA et al.; Interleukin-15 is a recently discovered cytokine produced by several cell types (including fibroblasts, keratinocytes, endothelial cells, and macrophages) in response to endotoxin or microbial infection . In turn, interleukin-15 has been shown to act on various cells of the immune system, including T and B lymphocytes, natural killer cells, monocytes, eosinophils, and circulating neutrophils . In the latter instance, interleukin-15 was initially observed to induce cytoskeletal rearrangements, to enhance phagocytosis, to increase the synthesis of several cellular proteins, and to delay apoptosis . Recently, interleukin-15 has been found to elicit other functional responses in neutrophils, such as chemokine production . This review recapitulates advances made in the area of interleukin-15/neutrophil interactions. J Magn Reson, 2000 May, 144(1), 175 - 85 An iterative fitting procedure for the determination of longitudinal NMR cross-correlation rates; Wang L et al.; We present a method to measure (15)N-(1)H dipolar/(15)N CSA longitudinal cross-correlation rates in protonated proteins . The method depends on the measurement of four observables: the cumulative proton-proton cross relaxation rates, the (15)N R(1) relaxation rate, the multiexponential decay of 2N(Z)H(N)(Z) spin-order, and multiexponential buildup of 2N(Z)H(N)(Z) spin-order . The (15)N-(1)H dipolar/(15)N CSA longitudinal cross-correlation rate is extracted from these measurements by an iterative fitting procedure to the solution of differential equations describing the coupled relaxation dynamics of the z-magnetization of the (15)N nucleus, the two-spin-order 2N(Z)H(N)(Z), and a two-spin-order term 2N(Z)H(Q)(Z) describing the interaction with remote protons . The method is applied to the microbial ribonuclease binase . The method can also extract longitudinal cross-correlation rates for those amide protons that are involved in rapid solvent exchange . The experiment that serves for extracting proton-proton cross-relaxation rates is a modification of 3D (15)N-resolved NOESY-HSQC . The experiment restores the solvent magnetization to its equilibrium state during data detection for all phase cycling steps and all values of NOE mixing times and is recommended for use in standard applications as well . Science, 2000 Apr 28, 288(5466), 658 - 61 The Archean sulfur cycle and the early history of atmospheric oxygen; Canfield DE et al.; The isotope record of sedimentary sulfides can help resolve the history of oxygen accumulation into the atmosphere . We measured sulfur isotopic fractionation during microbial sulfate reduction up to 88 degrees C and show how sulfate reduction rate influences the preservation of biological fractionations in sediments . The sedimentary sulfur isotope record suggests low concentrations of seawater sulfate and atmospheric oxygen in the early Archean (3.4 to 2.8 billion years ago) . The accumulation of oxygen and sulfate began later, in the early Proterozoic (2.5 to 0.54 billion years ago). Chem Pharm Bull (Tokyo), 2000 Apr, 48(4), 563 - 5 Efficient synthesis of a key intermediate of DV-7751 via optical resolution or microbial reduction; Miyadera A et al.; Two efficient and practical methods of synthesis of the C-10 substituent of DV-7751 (1), a novel quinolone carboxylic acid, were established . The first method utilizes an optical resolution of racemic 8-amino-6-benzyl-6-azaspiro{3.4}octane (13), while the second employs an enantioselective microbial reduction of 6-benzyl-5,8-dioxo-6-azaspiro{3.4}octane (8b) . The enantiomeric excess of (S)-8-amino-6-benzyl-6-azaspiro{3.4}octane (11) with each method of synthesis is greater than 96%. Hum Reprod Update, 2000 Mar-Apr, 6(2), 149 - 59 The role of heat shock proteins in reproduction; Neuer A et al.; Heat shock proteins (HSP) were first identified in cells after exposure to elevated temperature . Subsequently HSP have been identified as a critical component of a very complex and highly conserved cellular defence mechanism to preserve cell survival under adverse environmental conditions . HSP are preferentially expressed in response to an array of insults, including hyperthermia, free oxygen radicals, heavy metals, ethanol, amino acid analogues, inflammation and infection . HSP interact with intracellular polypeptides and prevent their denaturation or incorrect assembly . In addition HSP are also involved in several processes essential for cellular function under physiological conditions . HSP production is enhanced during in-vitro embryo culture and they are among the first proteins produced during mammalian embryo growth . The spontaneous expression of HSP as an essential part of embryo development is well documented and the presence or absence of HSP influences various aspects of reproduction in many species . Finally, HSP are immunodominant antigens of numerous microbial pathogens, e.g . Chlamydia trachomatis, which have been recognized as the main cause of tubal infertility . Many couples with fertility problems have had a previous genital tract infection, have become sensitized to microbial HSP, and a prolonged and asymptomatic infection may trigger immunity to microbial HSP epitopes that are also expressed in man . Antibodies to both bacterial and human HSP are present at high titres in sera and hydrosalpinx fluid of many patients undergoing in-vitro fertilization (IVF) . In a mouse in-vitro embryo culture model, these antibodies impaired the mouse embryo development at unique developmental stages . Recent studies indicate an association between a previous infection, immunity to HSP and reproductive failure. Phytomedicine, 2000 Mar, 7(1), 1 - 6 A comparative multi-centre study of the efficacy of propolis, acyclovir and placebo in the treatment of genital herpes (HSV); Vynograd N et al.; Ninety men and women with recurrent genital HSV type 2 participated in a randomized, single-blind, masked investigator, controlled multi-centre study comparing the efficacy of ointment of Canadian propolis containing natural flavonoids with ointments of acyclovir and placebo (vehicle) on healing ability and capacity to remedy symptoms . Thirty individuals were randomized to each group . Treatment was intended to start in the blister phase . All participants had HSV type 2 isolated, confirmed by serum immunoglobulin levels . The participants were examined on the 3rd, 7th and 10th days of treatment by gynaecologists, dermatovenerologists or urologists at seven different medical centres . Apart from clinical symptoms the number and size of the herpetic lesions were noted . At each examination the lesions were classified into four stages: vesicular, ulcerated, crusted and healed . The study ointments were applied to affected areas four times daily . In women with vaginal or cervical lesions a tampon with the appropriate ointment was inserted four times daily for 10 days . Endpoint variables were healing time and time until loss of symptoms . RESULTS: On Day 10, 24 out of 30 individuals in the propolis group had healed . In the acyclovir group 14 out of 30 and in the placebo group 12 out of 30 had healed . (p = 0.0015) . The healing process appeared to be faster in the propolis group . In the propolis group 15 individuals had crusted lesions on Day 3 compared to 8 individuals in the acyclovir group and none in the placebo group (p = 0.0006) . On Day 7, 10 participants in the propolis group, 4 in the acyclovir group and 3 in the placebo group had healed . At the initial examination all patients had local symptoms and 28% general symptoms . At Day 3, 3 patients in the propolis group had local symptoms compared to 8 and 9 in the acyclovir and placebo groups respectively . Of the women, 66% had vaginal superinfections of microbial pathogens at the initial examination . In the acyclovir and placebo groups no change in the vaginal flora was found following treatment whereas in the propolis group the incidence of superinfection was reduced by 55% . (p = 0.10 n.s.) . CONCLUSION: An ointment containing flavonoids appeared to be more effective than both acyclovir and placebo ointments in healing genital herpetic lesions, and in reducing local symptoms. Carbohydr Res, 2000 Jan 12, 323(1-4), 57 - 62 A new bioactive steroidal saponin, furcreastatin, from the plant Furcraea foetida; Itabashi M et al.; Microbial and plant secondary metabolites were screened for compounds that are selectively cytotoxic to mutant p53-expressing mouse fibroblasts . As a result, furcreastatin, a novel steroidal saponin, was isolated from an EtOH extract of the leaves of Furcraea foetida . Furcreastatin consisted of hecogenin as the aglycone and a hexasaccharide containing D-galactose, L-rhamnose and four D-glucose residues . The structure was determined to be (3 beta,5 alpha,25R)- 3-hydroxyspirostan-12-one 3-O-{alpha-L-Rhap-(1-->4)-beta-D-Glcp-(1-->3)- inverted question markbeta-D-Glcp-(1-->3) -beta-D- Glcp-(1-->2) inverted question mark-beta-D-Glcp-(1-->4)-beta-D-Galp} by extensive NMR spectroscopic studies . Furcreastatin decreased the viability of mutant p53-over-expressing cells with an ED50 of 4.0 micrograms/mL, and decreased that of the parental cell-line with an ED50 of 9.6 micrograms/mL. Nucleic Acids Symp Ser, 1999, (42), 215 - 6 Specific RNA protease inhibitors from in vitro selection; Yamamoto S et al.; RNA aptamers that bind to and inhibit the proteolytic activity of subtilisin BPN' are selected in vitro from pools of random RNA . The RNAs in vitro transcribed from the isolated clones show highly specific inhibition toward the microbial serine proteases . From the sequences of the isolated clones, a C/A-rich sequence was obtained . The kinetic features of the common C/A-rich sequence will be discussed. Prikl Biokhim Mikrobiol, 2000 Mar-Apr, 36(2), 204 - 8 {Effect of conditions of culturing Pseudomonas rathonis T on characteristics of a biosensor for determining anionic surface-active agents}; Taranova LA et al.; The dependence of the sensitivity of a microbial biosensor of anionic surfactants (AS) on the growth phase of Pseudomonas rathonis T, a strain capable of degrading surfactants, was studied . Correlations were found between the optimum values of temperature and pH of microbial growth, substrate utilization, and functional performance of the microbial biosensor . These results allow the process of AS detection to be optimized. Prikl Biokhim Mikrobiol, 2000 Mar-Apr, 36(2), 189 - 94 {Study of microbial degradation of nonionic surface-active agents in designing technologies for purifying waste water}; Panchenko LV et al.; Studies of degradation of non-ionic surfactants (NISA) in a model purification plant of an original design demonstrated an high rate and depth of degradation processes compared with periodic cultivation of free or immobilized degrading strains . A virtually complete primary degradation (99-99.5%), with destruction of the oxyethyl moiety of the molecule, was observed . In addition, NISA molecules were degraded to a greater extent, including considerable degradation of the hydrocarbon radical, partial degradation of aromatic structures in Neonol, and utilization of biologically "unyielding" fractions of commercial NISA preparations: polyethylene glycol (PEG) and long-chain fractions of polymer homologues. J Immunol, 2000 May 1, 164(9), 4790 - 6 Evidence for human CD4+ T cells in the CD1-restricted repertoire: derivation of mycobacteria-reactive T cells from leprosy lesions; Sieling PA et al.; Both the CD4-CD8- (double negative) and CD4-CD8+ T cell lineages have been shown to contain T cells which recognize microbial lipid and glycolipid Ags in the context of human CD1 molecules . To determine whether T cells expressing the CD4 coreceptor could recognize Ag in the context of CD1, we derived CD4+ T cell lines from the lesions of leprosy patients . We identified three CD4+ Mycobacterium leprae-reactive, CD1-restricted T cell lines: two CD1b restricted and one CD1c restricted . These T cell lines recognize mycobacterial Ags, one of which has not been previously described for CD1-restricted T cells . The response of CD4+ CD1-restricted T cells, unlike MHC class II-restricted T cells, was not inhibited by anti-CD4 mAb, suggesting that the CD4 coreceptor does not impact positive or negative selection of CD1-restricted T cells . The CD4+ CD1-restricted T cell lines produced IFN-gamma and GM-CSF, the Th1 pattern of cytokines required for cell-mediated immunity against intracellular pathogens, but no detectable IL-4 . The existence of CD4+ CD1-restricted T cells that produce a Th1 cytokine pattern suggests a contributory role in immunity to mycobacterial infection. Genome Res, 2000 Apr, 10(4), 502 - 10 MAGPIE/EGRET annotation of the 2.9-Mb Drosophila melanogaster Adh region; Gaasterland T et al.; Our challenge in annotating the 2.91-Mb Adh region of the Drosophila melanogaster genome was to identify genetic and genomic features automatically, completely, and precisely within a 6-week period . To do so, we augmented the MAGPIE microbial genome annotation system to handle eukaryotic genomic sequence data . The new configuration required the integration of eukaryotic gene-finding tools and DNA repeat tools into the automatic data collection module . It also required us to define in MAGPIE new strategies to combine data about eukaryotic exon predictions with functional data to refine the exon predictions . At the heart of the resulting new eukaryotic genome annotation system is a reverse comparison of public protein and complementary DNA sequences against the input genome to identify missing exons and to refine exon boundaries . The software modules that add eukaryotic genome annotation capability to MAGPIE are available as EGRET (Eukaryotic Genome Rapid Evaluation Tool). Clin Immunol, 2000 May, 95(2), 93 - 8 The microbial product lipopolysaccharide confers diabetogenic potential on the T cell repertoire of BDC2.5/NOD mice: implications for the etiology of autoimmune diabetes; Balasa B et al.; Both genetic predisposition and environmental factors participate in the etiology of Type-1 diabetes . To test the role of the microbial product lipopolysaccharide (LPS) as an environmental trigger of autoimmune diabetes, we employed transgenic (tg) BDC2.5/NOD mice that bear an islet-specific CD4(+) T cell repertoire (>95%), but do not develop the spontaneous diabetes that typifies the NOD (nonobese diabetic) strain . LPS administration provoked diabetes in BDC2.5/NOD mice by their 16th week of age . However, LPS administration in NOD mice did not accelerate their diabetes . This finding indicates that the frequency of islet-specific T cells influences LPS-mediated diabetes . Furthermore, in vitro LPS-cultured splenocytes from BDC2 . 5/NOD and BDC2.5-microMT (B-cell-deficient) mice effectively transferred diabetes into immunodeficient NOD-scid/scid mice but not immunosufficient NOD mice . Therefore, B lymphocytes are not required for LPS-provoked autoimmune diabetes . Flow cytometric analysis then revealed that LPS-stimulation in vitro induced the expression of an IL-2 receptor (CD25) on CD4 T cells; this indicates that the activation of islet-specific T cells is a prerequisite to eliciting diabetes in this situation . Overall, these results point to microbial LPS as an etiopathogenic agent of autoimmune diabetes . Biochim Biophys Acta, 2000 May 1, 1474(3), 360 - 4 Inverting character of alpha-glucuronidase A from Aspergillus tubingensis; Biely P et al.; Alpha-glucuronidase A from Aspergillus tubingensis was found to be capable of liberating 4-O-methyl-D-glucuronic acid (MeGlcA) only from those beechwood glucuronoxylan fragments in which the acid is attached to the non-reducing terminal xylopyranosyl residue . Reduced aldotetrauronic acid, 4-O-methyl-D-glucuronosyl-alpha-1,2-D-xylopyranosyl-beta-1,4-xylopyranosyl-beta-1,4-xylitol, was found to be a suitable substrate to follow the stereochemical course of the hydrolytic reaction catalyzed by the purified enzyme . The configuration of the liberated MeGlcA was followed in a D(2)O reaction mixture by (1)H-NMR spectroscopy . It was unambiguously established that MeGlcA was released from the substrate as its beta-anomer from which the alpha-anomer was formed on mutarotation . This result represents the first experimental evidence for the inverting character of a microbial alpha-glucuronidase, a member of glycosyl hydrolase family 67 (EC 3.1.1.139). FEMS Microbiol Ecol, 2000 Apr 1, 32(1), 25 - 34 Comparison of pure cultures and natural assemblages of planktonic photosynthetic sulfur bacteria by low molecular mass RNA fingerprinting; Casamayor EO et al.; Pure cultures of phototrophic sulfur bacteria were compared to natural populations that bloom in karstic lakes by electrophoretic analysis of low molecular mass RNA molecules (lmwRNA) and microscopy . Similarities between dominant community members, field isolates and reference strains were established by comparing the lmwRNA band patterns through dendrograms produced with Euclidean distances and the average linkage clustering method, by a single, quick, one-step method . The fingerprinting analysis had three objectives: (i) to compare microbial assemblages from different geographical locations, (ii) to compare those organisms which grow in pure culture to those forming planktonic blooms and (iii) to give a preliminary view of the identity of the predominant community members . The lmwRNA analysis yielded a number of clusters consistent with the microscopic observations and allowed rapid comparison of the microbial communities without the need to isolate their individual components . The fingerprints of natural communities were different from most of the laboratory strains tested . Purple sulfur bacteria responsible for the blooms analyzed in karstic lakes were more closely related to the Thiocystis group than to the classical strains extensively studied in the laboratory. J Biol Chem, 2000 Apr 28, 275(17), 12430 - 7 Steady-state kinetic characterization and crystallization of a polychlorinated biphenyl-transforming dioxygenase; Imbeault NY et al.; The oxygenase component of biphenyl dioxygenase (BPDO) from Comamonas testosteroni B-356 dihydroxylates biphenyl and some polychlorinated biphenyls (PCBs), thereby initiating their degradation . Overexpressed, anaerobically purified BPDO had a specific activity of 4.9 units/mg, and its oxygenase component appeared to contain a full complement of Fe(2)S(2) center and catalytic iron . Oxygenase crystals in space group R3 were obtained under anaerobic conditions using polyethylene glycol as the precipitant . X-ray diffraction was measured to 1.6 A . Steady-state kinetics assays demonstrated that BPDO had an apparent k(cat)/K(m) for biphenyl of (1.2 +/- 0.1) x 10(6) M(-1) s(-1) in air-saturated buffer . Moreover, BPDO transformed dichlorobiphenyls (diClBs) in the following order of apparent specificities: 3,3'- > 2,2'- > 4, 4'-diClB . Strikingly, the ability of BPDO to utilize O(2) depended strongly on the biphenyl substrate: k(cat)/K(m(O(2))) = (3.6 +/- 0 . 3), (0.06 +/- 0.02), and (0.4 +/- 0.07) x 10(5) M(-1) s(-1) in the presence of biphenyl and 2,2'- and 3,3'-diClBs, respectively . Moreover, biphenyl/O(2) consumed was 0.97, 0.44, 0.63, and 0.48 in the presence of biphenyl and 2,2'-, 3,3'-, and 4,4'-diClBs, respectively . Within experimental error, the balance of consumed O(2) was detected as H(2)O(2) . Thus, PCB congeners such as 2, 2'-diClB exact a high energetic cost, produce a cytotoxic compound (H(2)O(2)), and can inhibit degradation of other congeners . Each of these effects would be predicted to inhibit the aerobic microbial catabolism of PCBs. Shock, 2000, 13(4), 314 - 9 Sodium arsenite induces the stress response in the gut and decreases bacterial translocation in a burned mouse model with gut-derived sepsis; Eaves-Pyles T et al.; Bacteria translocation from the bowel to systemic organs after burn injury may contribute to or be a cause of sepsis and multiple organ failure . The stress response confers protection under stressful conditions that would otherwise lead to cell damage or death . We investigated whether prior induction of the stress response by sodium arsenite could affect bacterial translocation after thermal injury . HSP-70, a highly stress-inducible protein, was used as a marker for induction of the stress response . Balb/c mice were intravenously injected with 4 mg/kg of sodium arsenite and killed at selected times post-treatment . Other treated mice were then gavaged with 10(10) E . coil or 10(10) 111In-labeled E . coil followed by a 20% burn . Survival was observed for 10 days . Mice gavaged with radiolabeled E . coil were killed 4 h post-burn to determine the effect of HSP-70 induction on microbial translocation in mesenteric lymph nodes (MLN), liver, and spleen . Sodium arsenite-injected mice showed HSP-70 induction in the ileum that increased in a time-dependent manner with peak expression 12 h post-injection . Treated mice showed a significantly higher survival rate (93%) than controls (46%; P < 0.05), and detection of 111In-labeled E . coli was significantly less in the liver and spleen (P < 0.05) . These data show that sodium arsenite induced HSP-70 expression in the small intestine . The stress response was associated with significantly increased survival and significant decrease in detection of 111In-labeled E . coil in the liver and spleen in a burned mouse model with gut-derived sepsis. Gene, 2000 Apr 18, 247(1-2), 255 - 64 A family of removable cassettes designed to obtain antibiotic-resistance-free genomic modifications of Escherichia coli and other bacteria; Palmeros B et al.; Modifications of microbial genomes often require the use of the antibiotic-resistance (Anb(R))-encoding genes and other easily selectable markers . We have developed a set of such selectable markers (Cm(R), Km(R) and Gm(R)), which could easily be inserted into the genome and subsequently removed by using the Cre/loxP site-specific recombination system of bacteriophage P1 . In this manner the same marker could be used more than once in the same background, while the resulting strain could or would remain Anb(R) marker-free.Three plasmids were constructed, each containing a cassette consisting of the Cm(R), Km(R), or Gm(R) gene flanked by two parallel loxP sites and two polylinkers (MCS) . To test insertion and excision, cassettes were inserted into the lacZ or galE genes carried on an origamma/pir-dependent suicide plasmid, which contained a dominant Sm(R) gene . The cassettes were crossed into the E . coli genome by homologous recombination (allelic exchange), in a manner analogous to that described by Posfai et al . {Nucl . Acids Res . 22 (1994) 2392-2398}, selecting for the Cm(R), Km(R), or Gm(R), for the LacZ(-) or GalE(-) and for the Sm(S) phenotypes (the latter to assure allelic exchange rather than insertion of the entire plasmid) . When required, after selecting the strain with the desired modification, the Cm(R), Km(R), or Gm(R) marker was excised by supplying the Cre function . Cre was provided by the thermosensitive plasmid pJW168, which was transformed into the Anb(R) host at 30 degrees C, and was subsequently eliminated at 42 degrees C . Thus the Anb(R) marker was removed, whereas the lacZ or galE gene remained interrupted by the retained loxP site. Acta Odontol Scand, 1999 Dec, 57(6), 325 - 9 Current concepts on the theories of the mechanism of action of fluoride; ten Cate JM; The caries-preventive effect of fluoride is mainly attributed to the effects on demineralization/remineralization at the tooth oral fluids interface . Sub ppm levels of fluoride in saliva are effective in shifting the balance from demineralization, leading to caries, to remineralization . This is attributed to the fluoride-enhanced precipitation of calcium phosphates, and the formation of fluorhydroxyapatite in the dental tissues . Low fluoride levels are found in saliva after toothbrushing with fluoride containing dentifrices . Similar concentrations are ineffective in interfering with processes of growth and metabolism of bacteria, and also do not result in a significantly reduced dissolution of tooth mineral as a result of (firmly bound) fluoride incorporation . Comparative studies of fluoride efficacy have shown that higher concentrations in solution are needed in pH-cycling studies of dentine than in enamel to maintain the mineral balance or to induce remineralization . This is attributed to the greater solubility of the dentine and the smaller size of the dentine crystallites compared to enamel . Fluoride slow-release devices, in the form of fluoride-releasing restorative materials, may serve to increase the fluoride levels in saliva and plaque to levels at which caries can be prevented, also in high-risk patients . Research questions for the next millennium and future perspectives for fluoride applications should be found in the retention and slow release of fluoride after various combinations of fluoride treatment, the combination of fluoride and anti-microbial treatment, the individualization of caries prevention, and the combination of preventive schemes with new developments in caries diagnosis. Microbiol Res, 2000 Mar, 154(4), 285 - 8 Characterization of a novel organophosphorus hydrolase from Nocardiodes simplex NRRL B-24074; Mulbry W; We characterized a novel organophosphorus hydrolase (OPH) activity expressed by Nocardiodes simplex NRRL B-24074, a member of a coumaphos-degrading microbial consortium from cattle dip waste . Like the previously characterized OPH from Nocardia sp . strain B- (NRRL B- 16944), OPH activity in N . simplex is located in the cytoplasm and is expressed constitutively . The purified enzyme is monomeric, has a native molecular size of 45,000 Da and has a specific activity toward ethyl parathion of 33 micromole/min x mg protein . Km constants for the enzyme with the structurally related organophosphate pesticides ethyl parathion and EPN were 100 microM and 345 microM, respectively . Although OPH activity in extracts did not require the addition of divalent cations, the purified enzyme lost activity during dialysis against phosphate buffer and this activity could be restored after incubation in buffer containing either CoSO4 or CuSO4 . Our results suggest that OPH activity in N . simplex is distinct from other known OPHs and that the responsible gene is unrelated to known genes. J Exp Med, 2000 Apr 17, 191(8), 1381 - 94 Localization of distinct Peyer's patch dendritic cell subsets and their recruitment by chemokines macrophage inflammatory protein (MIP)-3alpha, MIP-3beta, and secondary lymphoid organ chemokine; Iwasaki A et al.; We describe the anatomical localization of three distinct dendritic cell (DC) subsets in the murine Peyer's patch (PP) and explore the role of chemokines in their recruitment . By two-color in situ immunofluorescence, CD11b(+) myeloid DCs were determined to be present in the subepithelial dome (SED) region, whereas CD8alpha(+) lymphoid DCs are present in the T cell-rich interfollicular region (IFR) . DCs that lack expression of CD8alpha or CD11b (double negative) are present in both the SED and IFR . By in situ hybridization, macrophage inflammatory protein (MIP)-3alpha mRNA was dramatically expressed only by the follicle-associated epithelium overlying the SED, while its receptor, CCR6, was concentrated in the SED . In contrast, CCR7 was expressed predominantly in the IFR . Consistent with these findings, reverse transcriptase polymerase chain reaction analysis and in vitro chemotaxis assays using freshly isolated DCs revealed that CCR6 was functionally expressed only by DC subsets present in the SED, while all subsets expressed functional CCR7 . Moreover, none of the splenic DC subsets migrated toward MIP-3alpha . These data support a distinct role for MIP-3alpha/CCR6 in recruitment of CD11b(+) DCs toward the mucosal surfaces and for MIP-3beta/CCR7 in attraction of CD8alpha(+) DCs to the T cell regions . Finally, we demonstrated that all DC subsets expressed an immature phenotype when freshly isolated and maintained expression of subset markers upon maturation in vitro . In contrast, CCR7 expression by myeloid PP DCs was enhanced with maturation in vitro . In addition, this subset disappeared from the SED and appeared in the IFR after microbial stimulation in vivo, suggesting that immature myeloid SED DCs capture antigens and migrate to IFR to initiate T cell responses after mucosal microbial infections. Biochemistry, 2000 Apr 25, 39(16), 4761 - 8 Lipoxin A(4) analogues inhibit leukocyte recruitment to Porphyromonas gingivalis: a role for cyclooxygenase-2 and lipoxins in periodontal disease; Pouliot M et al.; The potential involvement of the inducible cyclooxygenase isoform (COX-2) and the role of novel lipid mediators were investigated in the pathogenesis of periodontal disease . Crevicular fluids from localized juvenile periodontitis (LJP) patients contained prostaglandin (PG)E(2) and 5-lipoxygenase-derived products, leukotriene B(4), and the biosynthesis interaction product, lipoxin (LX)A(4) . Neutrophils from peripheral blood of LJP patients, but not from asymptomatic donors, also generated LXA(4), suggesting a role for this immunomodulatory molecule in periodontal disease . To characterize host responses of interest to periodontal pathogens, Porphyromonas gingivalis was introduced within murine dorsal air pouches . In the air pouch cavity, P . gingivalis elicited leukocyte infiltration, concomitant with elevated PGE(2) levels in the cellular exudates, and upregulated COX-2 expression in infiltrated leukocytes . In addition, human neutrophils exposed to P . gingivalis also upregulated COX-2 expression . Blood borne P . gingivalis gave significant increases in the murine tissue levels of COX-2 mRNA associated with both heart and lungs, supporting a potential role for this oral pathogen in the evolution of systemic events . The administration of metabolically stable analogues of LX and of aspirin-triggered LX potently blocked neutrophil traffic into the dorsal pouch cavity and lowered PGE(2) levels within exudates . Together, these results identify PMN as an additional and potentially important source of PGE(2) in periodontal tissues . Moreover, they provide evidence for a novel protective role for LX in periodontitis, limiting further PMN recruitment and PMN-mediated tissue injury that can lead to loss of inflammatory barriers that prevent systemic tissue invasion of oral microbial pathogens. J Dent Educ, 2000 Apr, 64(4), 276 - 82 Management of patients needing antibiotic prophylaxis in a dental education setting; Johnson TE et al.; The management of antibiotic prophylaxed (ABX) patients at an educational institution was evaluated to identify areas for improvement . Management criteria, reflecting guidelines to prevent oral-induced hematogenous microbial seeding, were pretested and applied to 1,225 record entries of eighty-five patients needing ABX for dental treatment between 1991 and 1996 . Seven hundred twenty-two of the visits had 857 management or documentation problems, including no documentation indicating whether or not patients premedicated (n = 281); incomplete, insufficient, or repeated treatment (n = 214); and preventive concerns (n = 172), among others . The proportion of providers' patient visits with one or more management problems differed significantly (p < 0.001) by provider type, as did the distribution of problem categories (documentation, treatment, preventive, and scheduling concerns p < 0.001; compliance issues p < 0.005) . Fifty-one percent of postgraduates' and 39 percent of faculty's record entries omitted patients' ABX status . Improved documentation, outcome measures, and patient, faculty and student education are indicated. J Am Vet Med Assoc, 2000 Apr 15, 216(8), 1282 - 7 Effects of pulmonary abscesses on racing performance of horses treated at referral veterinary medical teaching hospitals: 45 cases (1985-1997); Ainsworth DM et al.; OBJECTIVE: To determine whether results of physical or radiographic examination or biochemical analyses in adult racehorses with primary lung abscesses were associated with ability to race following treatment . DESIGN: Multiple-center retrospective study . ANIMALS: 25 Standardbreds and 20 Thoroughbreds . PROCEDURE: Medical records of horses with a primary lung abscess that were admitted to any of 4 veterinary teaching hospitals were reviewed . Results of physical examination, laboratory testing, and thoracic radiography were reviewed . Racing performance after treatment was compared with performance before illness and with performance of the general population of racehorses of similar age, sex, and breed . RESULTS: 23 of 25 Standardbreds and 13 of 20 Thoroughbreds raced after diagnosis and treatment of a lung abscess . Most horses had a solitary abscess in the dorsal to caudodorsal lung fields . Results of initial physical examination, biochemical analyses, and culture and identification of the microbial isolate were not associated with whether a horse returned to racing . For horses that had raced prior to the illness, race performance after treatment of the lung abscess was not significantly different from performance before the illness . CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of racing performance in those horses that resumed racing after treatment, long-term residual lung damage did not develop in horses with primary lung abscesses that were treated appropriately . It is not known whether horses that recovered would be more likely to bleed from the site of a prior infection when resuming strenuous exercise and whether lung abscesses contributed to a failure to resume racing. Eur J Obstet Gynecol Reprod Biol, 2000 May, 90(1), 13 - 25 Human umbilical cord blood banking and transplantation: a state of the art; Fasouliotis SJ et al.; Human umbilical cord blood has proven to be a feasible alternative source of hematopoietic stem cells for pediatric and some adult patients with major hematologic disorders . This has promoted the establishment of cord blood banks for use in unrelated transplants worldwide . The banking of umbilical cord blood offers many advantages: absence of donor risk, absence of donor attrition, immediate availability, and the ability to expand available donor pools in targeted ethnic and racial minorities currently underrepresented in all bone marrow registries . Preliminary clinical experience suggests that, due to the immunological immaturity of cord blood cells, graft versus host disease might be lower than when using bone marrow from adult donors and HLA restrictions might be less stringent . Techniques to improve the efficacy of blood banks are currently under investigation . Closed cord blood collection methods have proven to be superior to open in reducing the risk of microbial contamination . Efficient banking requires volume reduction of cord blood units without significant loss of progenitor cells, in order to decrease storage space and cost, and this may be achieved by using the separation techniques . Cryopreservation and thawing techniques have been established and do not seem to affect the viability and progenitor cell recovery or the feasibility of CD34(+) selection and ex vivo expansion . Nevertheless, many scientific, ethical, and social questions have arisen in connection with cord blood banking that need to be addressed. Arterioscler Thromb Vasc Biol, 2000 Apr, 20(4), 1061 - 7 Detection of Chlamydia pneumoniae-reactive T lymphocytes in human atherosclerotic plaques of carotid artery; Mosorin M et al.; Linkage between Chlamydia pneumoniae infection and atherosclerosis has been confirmed in several studies, but the precise role of this organism in the disease process is not known . We investigated the relation and reactivity of T lymphocytes of human carotid plaques to C pneumoniae antigens . Tissue specimens were obtained from 17 patients who underwent carotid endarterectomy . Immunohistological staining and/or in situ hybridization revealed the presence of C pneumoniae in 11 (64%) of the 17 of the cases . Inflammatory infiltration seen in the vessel walls consisted primarily of CD45RO+ T-memory lymphocytes (median 80%, range 50% to 90%), whereas CD20+ B cells and monocytes were in minor proportion . In vivo activated T lymphocytes were propagated from the specimens with interleukin-2, and the antigen specificity of the established T-cell lines (TLLs) was analyzed against C pneumoniae elementary body antigen . TLLs were established from all 17 carotid tissues but none from the control specimens of ascending aorta . C pneumoniae was recognized as a specific T-cell-stimulating antigen in 7 (41%) of 17 cases . Further analyses of the C pneumoniae-reactive TLLs showed that chlamydial 60-kDa heat-shock protein induced specific proliferation in 5 (71%) of 7 cases and revealed 2 haplotype (DRB1*1502 and DQB1*06) binding motifs in human 60-kDa heat-shock protein . C pneumoniae was identified as a specific microbial antigen recognized by 41% of TLLs propagated from in vivo activated plaque T cells . Our results suggests that cell-mediated immunity to C pneumoniae plays a role in the atherosclerotic process and that this response may involve autoimmunity. Int Rev Immunol, 2000, 19(2-3), 221 - 45 DNA vaccination and the immune responsiveness of neonates; Bot A; Neonates often respond poorly to conventional vaccines or microbial infections . Immaturity of the immune system has been considered to play a role in this regard . However, accumulating evidence shows that in certain conditions, neonatal inoculation of antigens leads to protective immunity . In the particular case of DNA vaccines administered to neonates, the rule is immunity rather than tolerance . Exceptions to the rule give opportunities to further understand the neonatal responsiveness and the mechanism of DNA vaccination . Due to the very nature of the vaccine vector, inhibition of neonatal DNA vaccination by maternal antibodies may be limited to the humoral immunity. J Infect Dis, 2000 Apr, 181(4), 1496 - 500 Epub 2000 Apr 13. Intimin-mediated tissue specificity in enteropathogenic Escherichia coli interaction with human intestinal organ cultures; Phillips AD et al.; The hallmark of enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E . coli (EPEC) adhesion to cultured human host cells is intimate attachment and the formation of attaching and effacing (A/E) lesions . Recently, EHEC O157:H7 was shown to induce A/E lesions on human intestinal explants . Unlike EPEC, which colonized the small intestine, EHEC adhesion was restricted to follicle-associated epithelium (FAE) of ileal Peyer's patches . This study tested the hypothesis that the bacterial adhesin intimin contributes to tissue specificity . Complementing the eae gene mutation in CVD206 (derived from EPEC strain E2348/69) with EPEC eaealpha (encoding intimin-alpha) restored the ability to colonize small intestinal mucosa like the parent strain . In contrast, complementing with EHEC eaegamma (encoding intimin-gamma) resulted in the strain adhering and inducing A/E lesion on Peyer's patches, similar to EHEC . An intimin-gamma-positive O55:H7 EPEC also targeted FAE . Thus, intimin contributes to the tissue specificity of A/E lesion-forming microbial pathogens. J Infect, 2000 Jan, 40(1), 3 - 15 Emerging and re-emerging infectious diseases; Desselberger U; Microbial pathogens discovered as aetiological agents of human disease over the last 25 years are reviewed . Strengthening of laboratory and public health surveillance is of paramount importance for early detection and management of emerging infectious diseases. Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 3832 - 7 Removal of oxygen free-radical-induced 5',8-purine cyclodeoxynucleosides from DNA by the nucleotide excision-repair pathway in human cells; Kuraoka I et al.; Exposure of cellular DNA to reactive oxygen species generates several classes of base lesions, many of which are removed by the base excision-repair pathway . However, the lesions include purine cyclodeoxynucleoside formation by intramolecular crosslinking between the C-8 position of adenine or guanine and the 5' position of 2-deoxyribose . This distorting form of DNA damage, in which the purine is attached by two covalent bonds to the sugar-phosphate backbone, occurs as distinct diastereoisomers . It was observed here that both diastereoisomers block primer extension by mammalian and microbial replicative DNA polymerases, using DNA with a site-specific purine cyclodeoxynucleoside residue as template, and consequently appear to be cytotoxic lesions . Plasmid DNA containing either the 5'R or 5'S form of 5',8-cyclo-2-deoxyadenosine was a substrate for the human nucleotide excision-repair enzyme complex . The R diastereoisomer was more efficiently repaired than the S isomer . No correction of the lesion by direct damage reversal or base excision repair was detected . Dual incision around the lesion depended on the core nucleotide excision-repair protein XPA . In contrast to several other types of oxidative DNA damage, purine cyclodeoxynucleosides are chemically stable and would be expected to accumulate at a slow rate over many years in the DNA of nonregenerating cells from xeroderma pigmentosum patients . High levels of this form of DNA damage might explain the progressive neurodegeneration seen in XPA individuals. Crit Rev Oral Biol Med, 1999, 10(2), 120 - 38 Molecular and genetic analyses of Actinomyces spp; Yeung MK; Members of the genus Actinomyces are predominant primary colonizers of the oral cavity and play an important role in initiating plaque development . These bacteria have evolved unique mechanisms that favor colonization and persistence in this micro-environment . The expression of cell-surface fimbriae is correlated with the ability of these bacteria to adhere to specific receptors on the tooth and mucosal surfaces, and to interact with other plaque bacteria . The elaboration of sialidase is thought to enhance fimbriae-mediated adherence by unmasking the fimbrial receptors on mammalian cells . The presence of certain cell-associated or extracellular enzymes, including those involved in sucrose or urea metabolism, may provide the means for these bacteria to thrive under conditions when other growth nutrients are not available . Moreover, these enzyme activities may influence the distribution of other plaque bacteria and promote selection for Actinomyces spp . in certain ecological niches . The recent development of a genetic transfer system for Actinomyces spp . has allowed for studies the results of which demonstrate the existence of multiple genes involved in fimbriae synthesis and function, and facilitated the construction of allelic replacement mutants at each gene locus . Analyses of these mutants have revealed a direct correlation between the synthesis of assembled fimbriae and the observed adherence properties . Further genetic analysis of the various enzyme activities detected from strains of Actinomyces should allow for an assessment of the role of these components in microbial ecology, and their contribution to the overall success of Actinomyces spp . as a primary colonizer and a key player in oral health and disease. Microb Ecol, 1999 Nov, 38(4), 307 - 320 Is H(2) the Universal Energy Source for Long-Term Survival? Morita RY. This review revisits anabiosis (cryptobiosis or latent life); but more specifically with the discrepancy (time factor) between the finding of viable bacteria in ancient material and the racemization of amino acids and depurination of DNA that would have contributed to their death . The omnipresence of H(2) in the biosphere since life began, its ability to penetrate the microbial cell, its low energy of activation, its ability to form protons and electrons in the presence of Fe(II), and its (including electrons and protons) role in many biochemical reactions make H(2) the best candidate as the energy of survival for microbial cells . Although the concentration of H(2) in most environments is below the threshold level for microbial growth, the surviving cells have a long period of time to carry out the necessary metabolism to offset the racemization and depurination processes . This paper explores a hypothesis that explains this discrepancy. Genetics, 2000 Mar, 154(3), 1231 - 8 Adaptive evolution of relish, a Drosophila NF-kappaB/IkappaB protein; Begun DJ et al.; NF-kappaB and IkappaB proteins have central roles in regulation of inflammation and innate immunity in mammals . Homologues of these proteins also play an important role in regulation of the Drosophila immune response . Here we present a molecular population genetic analysis of Relish, a Drosophila NF-kappaB/IkappaB protein, in Drosophila simulans and D . melanogaster . We find strong evidence for adaptive protein evolution in D . simulans, but not in D . melanogaster . The adaptive evolution appears to be restricted to the IkappaB domain . A possible explanation for these results is that Relish is a site of evolutionary conflict between flies and their microbial pathogens. J Allergy Clin Immunol, 2000 Apr, 105(4), 760 - 8 Identification of cytokine-regulated genes in human leukocytes in vivo; Pals CM et al.; BACKGROUND: Human polymorphic nuclear granulocytes (PMNs) such as neutrophils and eosinophils play a critical role in mediating inflammatory responses to microbial and parasitic infections . Exposure of these leukocytes to cytokines leads to an amplification of granulocyte effector functions by a mechanism termed "priming." Although many studies have investigated the effects of granulocyte priming, little is known concerning the molecular mechanisms that lead to this phenomenon . OBJECTIVE: The purpose of this study was to identify potential markers for granulocyte priming and thus also to gain further insight into the pathogenesis of inflammatory responses . METHODS: We used a modified differential display technique, random arbitrary primed-PCR to identify genes regulated during the priming of human polymorphic nuclear granulocytes by GM-CSF in vitro . Genes identified were validated by Northern blot analysis of in vitro and in vivo primed leukocytes . RESULTS: Several genes were identified and their expression characterized in vitro . One of these genes, 5-lipoxygenase-activating protein, was also found to be up-regulated in leukocytes isolated after allergen challenge of allergic asthmatic patients . CONCLUSION: The use of differential display technology is a rapid and effective means of identifying genes whose expression is regulated by priming in vitro and in vivo . Further analysis will lead to a better understanding of the priming phenotype and may provide further insight into the pathologic mechanisms of inflammatory processes. Prikl Biokhim Mikrobiol, 2000 Jan-Feb, 36(1), 80 - 4 {Effect of various methods of immobilization on stability of a microbial biosensor based on Pseudomonas rathonis T during detection of surfactants}; Semenchuk IN et al.; The operating and storage stability of a receptor element of an amperometric biosensor based on the Pseudomonas rathonis strain T capable of degrading surfactants was tested . Microbial cells were immobilized by incorporation in gels (agar, agarose, and calcium-alginate), polyvinyl alcohol membrane, adhesion to the chromatographic paper GF/A, or by the cross-linking induced by glutaric aldehyde . Incorporation of microbial cells in agar gel provides the long-standing conservation of their activity and viability during measurements of high concentrations of surfactants and allows the receptory element of the biosensor to be rapidly recovered after the measurements. J Infect Dis, 2000 Apr, 181(4), 1264 - 72 Epub 2000 Apr 05. Strong human immunodeficiency virus (HIV)-specific CD4+ T cell responses in a cohort of chronically infected patients are associated with interruptions in anti-HIV chemotherapy; Haslett PA et al.; Virus-specific CD4+ T-helper cell function is important in controlling human immunodeficiency virus (HIV) infection but is impaired in patients with progressive HIV disease . It has been reported that after highly active antiretroviral therapy (HAART), HIV-specific lymphoproliferative responses remain absent, whereas responses to non-HIV microbial antigens are restored . However, in analyzing immune responses in a cohort of chronically infected adults on HAART, we observed strong HIV-specific CD4+ T cell responses of Th-1 phenotype in 11 of 22 patients . The magnitude and frequency of HIV-specific lymphoproliferative responses was strongly associated with previous interruptions in HAART (P=.001) . In contrast, the magnitude of CD8+ T cell responses to HIV Gag, Pol, Env, and Nef was similar in patients who had and those who had not interrupted HAART . We conclude that (1) a significant proportion of chronically HIV-infected patients on HAART can generate strong HIV-specific CD4+ and CD8+ T cell immunity and (2) transient interruptions in antiviral treatment may prime or boost HIV-specific CD4+ T-helper responses. J Eukaryot Microbiol, 2000 Mar-Apr, 47(2), 148 - 55 Abundance of terrestrial gymnamoebae at a northeastern U . S . site: a four-year study, including the El Niño winter of 1997-1998; Anderson OR; The abundance, sizes, and when appropriate, diversity of gymnamoebae were documented at approximately monthly intervals for four years (1995-1998) at a grassy, terrestrial site slightly upslope from a freshwater pond . Soil samples were analyzed for viable gymnamoebae using a standard laboratory culturing protocol . The mean density of gymnamoebae based on the total data set was ca . 1,600/g (s.e.+/-190) . Minimum densities of gymnamoebae (156/g) occurred in January 1995, and a maximum for the sampling period (5,838/g) occurred in July 1997, when a rainy period followed an extended period of drought . Among the environmental variables monitored (precipitation, soil moisture, organic content, and temperature) only precipitation correlated significantly with abundance of gymnamoebae (r = 0.34, p = 0.02) . During the mild, moist El Nino winter of 1997-1998, a larger than usual number of gymnamoebae was recorded at the site (approximately 3,800/g) compared to a mean density of approximately 900/g for comparable periods in preceding years . The mean sizes were also larger . Since gymnamoebae are increasingly recognized as major members of soil microbial communities enhancing soil fertility through nutrient mineralization, it is important to document environmental variables that influence their abundance and activity in terrestrial ecosystems. Biochim Biophys Acta, 2000 May 1, 1465(1-2), 79 - 103 Plant ABC transporters; Theodoulou FL; The ATP binding cassette (ABC) superfamily is a large, ubiquitous and diverse group of proteins, most of which mediate transport across biological membranes . ABC transporters have been shown to function not only as ATP-dependent pumps, but also as ion channels and channel regulators . Whilst members of this gene family have been extensively characterised in mammalian and microbial systems, the study of plant ABC transporters is a relatively new field of investigation . Sequences of over 20 plant ABC proteins have been published and include homologues of P-glycoprotein, MRP, PDR5 and organellar transporters . At present, functions have been assigned to a small proportion of these genes and only the MRP subclass has been extensively characterised . This review aims to summarise literature relevant to the study of plant ABC transporters, to review methods of cloning, to discuss the utility of yeast and mammalian systems as models and to speculate on possible roles of uncharacterised ABC transporters in plants. J Exp Med, 2000 Apr 3, 191(7), 1117 - 26 The cysteine-rich domain of the macrophage mannose receptor is a multispecific lectin that recognizes chondroitin sulfates A and B and sulfated oligosaccharides of blood group Lewis(a) and Lewis(x) types in addition to the sulfated N-glycans of lutropin; Leteux C et al.; The mannose receptor (MR) is an endocytic protein on macrophages and dendritic cells, as well as on hepatic endothelial, kidney mesangial, tracheal smooth muscle, and retinal pigment epithelial cells . The extracellular portion contains two types of carbohydrate-recognition domain (CRD): eight membrane-proximal C-type CRDs and a membrane-distal cysteine-rich domain (Cys-MR) . The former bind mannose-, N-acetylglucosamine-, and fucose-terminating oligosaccharides, and may be important in innate immunity towards microbial pathogens, and in antigen trapping for processing and presentation in adaptive immunity . Cys-MR binds to the sulfated carbohydrate chains of pituitary hormones and may have a role in hormonal clearance . A second feature of Cys-MR is binding to macrophages in marginal zones of the spleen, and to B cell areas in germinal centers which may help direct MR-bearing cells toward germinal centers during the immune response . Here we describe two novel classes of carbohydrate ligand for Cys-MR: chondroitin-4 sulfate chains of the type found on proteoglycans produced by cells of the immune system, and sulfated blood group chains . We further demonstrate that Cys-MR interacts with cells in the spleen via the binding site for sulfated carbohydrates . Our data suggest that the three classes of sulfated carbohydrate ligands may variously regulate the trafficking and function of MR-bearing cells. Lett Appl Microbiol, 2000 Mar, 30(3), 188 - 91 Properties and stability of glycerophosphate oxidase isolated from a mutant strain of Aerococcus viridans; MacKova M et al.; The properties of microbial L-alpha-glycerophosphate oxidase (GPO) isolated from a mutant strain of Aerococcus viridans DBM 1509 were estimated . The stability at different temperatures and pH were detected . At 4 degrees C the enzyme lost activity during 15 d, at 20 degrees C and 30 degrees C GPO activity decreased during 30 and 25 h, respectively . The highest stability was measured at - 20 degrees C and pH 9 . At 4 degrees C the stability was enhanced by the addition of 0.1 M EDTA or by lyophilization in the presence of dextrin . These conditions allow the prolongation of the low stability of microbial GPO which limited its use, and give the opportunity to increase the stability of other enzymes J Appl Microbiol, 2000 Mar, 88(3), 442 - 8 Evaluation of plate count methods for determination of maximum specific growth rate in mixed microbial communities, and its possible application for diversity assessment; Salvesen I et al.; Two plate count methods are proposed for direct assessment of the maximum specific growth rate (microm) of bacteria in mixed communities . An estimate of microm of individual colonies is obtained by plating samples on an agar medium and determining either the time required to form macroscopically visible colonies, Tv (diameter < or =0.2 mm), or the linear radial growth rate, Kr, of single colonies . In accordance with theoretical models, a linear relationship was found between microm determined in liquid culture and 1/Tv, and between microm and Kr . Empirical relationships were established for these relationships . The time required to form a visible colony was 17 +/- 9 h longer for cells in the stationary growth phase, whereas the linear radial growth rate was not affected by the physiological state of the cells . The proposed plate count methods are simple and applicable for describing the community structure, and for estimating the frequency distribution of maximum specific growth rates in mixed communities . By using this frequency distribution it is possible to calculate diversity indexes and to assign a microbial community a position on an r/K-gradient. EMBO J, 2000 Apr 3, 19(7), 1476 - 85 Highly purified glycosylphosphatidylinositols from Trypanosoma cruzi are potent proinflammatory agents; Almeida IC et al.; Intracellular protozoan parasites are potent stimulators of cell-mediated immunity . The induction of macrophage proinflammatory cytokines by Trypanosoma cruzi is considered to be important in controlling the infection and the outcome of Chagas' disease . Here we show that the potent tumour necrosis factor-alpha-, interleukin-12- and nitric oxide-inducing activities of T.cruzi trypomastigote mucins were recovered quantitatively in a highly purified and characterized glycosylphosphatidylinositol (GPI) anchor fraction of this material . The bioactive trypomastigote GPI fraction was compared with a relatively inactive GPI fraction prepared from T . cruzi epimastigote mucins . The trypomastigote GPI structures were found to contain additional galactose residues and unsaturated, instead of saturated, fatty acids in the sn-2 position of the alkylacylglycerolipid component . The latter feature is essential for the extreme potency of the trypomastigote GPI fraction, which is at least as active as bacterial endotoxin and Mycoplasma lipopeptide and, therefore, one of the most potent microbial proinflammatory agents known. Curr Opin Microbiol, 2000 Apr, 3(2), 189 - 96 Microbial circadian oscillatory systems in Neurospora and Synechococcus: models for cellular clocks; Iwasaki H et al.; Common regulatory patterns have emerged among the feedback loops lying within circadian systems . Significant progress in dissecting the mechanism of clock resetting by temperature and the role of the WC proteins in the Neurospora light response has accompanied documentation of the importance of nuclear localization and phosphorylation-induced turnover of FRQ to this circadian cycle . The long-awaited molecular description of a transcription/translation loop in the Synechococcus circadian system represents a quantal step forward, followed by the identification of additional important proteins and interactions . Finally, the adaptive significance of rhythms in Synechococcus and by extension in all clocks nicely ties up an extraordinary year. Biotechnol Appl Biochem, 2000 Apr, 31 ( Pt 2), 135 - 52 Advances in microbial amylases; Pandey A et al.; This review makes a comprehensive survey of microbial amylases, i.e . alpha-amylase, beta-amylase and glucoamylase . Amylases are among the most important enzymes and are of great significance in present-day biotechnology . Although they can be derived from several sources, such as plants, animals and micro-organisms, the enzymes from microbial sources generally meet industrial demands . Microbial amylases could be potentially useful in the pharmaceutical and fine-chemical industries if enzymes with suitable properties could be prepared . With the advent of new frontiers in biotechnology, the spectrum of amylase application has widened in many other fields, such as clinical, medicinal and analytical chemistries, as well as their widespread application in starch saccharification and in the textile, food, brewing and distilling industries . In this review, after a brief description of the sources of amylases, we discuss the molecular biology of amylases, describing structures, cloning, sequences, and protoplast fusion and mutagenesis . This is followed by sections on their production and finally the properties of various amylases. J Rheumatol, 2000 Mar, 27(3), 708 - 13 T cell responses to 60/65 kDa heat shock protein derived peptides in Turkish patients with Behçet's disease; Direskeneli H et al.; OBJECTIVE: Sequence homology and cross reactivity between microbial and human heat shock proteins (HSP) led to the concept that HSP might be involved in the etiopathogenesis of Behcet's disease (BD) . We investigated T cell responses to 8 synthetic peptides derived from the mycobacterial 65 kDa and homologous human 60 kDa HSP in patients with BD . METHODS: T cell proliferative responses to synthetic peptides were studied in 49 patients with BD and 46 disease (DC) and 34 healthy controls (HC) with 3H-thymidine uptake test . RESULTS: Positive T cell responses to one or more of the mycobacterial peptides were observed in 52% (12/23) of patients with BD compared with 17% (3/18) of DC (p = 0.02) and to homologous human peptides in 57% (13/23) of BD and 11% (2/18) of DC (p < 0.01) . Responses to the mixtures of 4 mycobacterial peptides were also significantly higher in BD compared with controls (stimulation index in BD 4.7 +/- 3.5 vs DC 2.0 +/- 1.2, HC 1.6 +/- 0.4; BD vs DC and HC, p < 0.001) . Similar elevated responses to the mixture of 4 human peptides was also observed in patients with BD (BD 3.4 +/- 2.3; DC 1.9 +/- 0.8; HC 1.4 +/- 0.6; BD vs DC, p < 0.01; BD vs HC, p < 0.001) . CONCLUSION: These results suggest that cellular immunity against the 65 kDa mycobacterial and 60 kDa human HSP derived peptides is significantly increased in Turkish patients with BD compared to controls, as observed in the UK and Japan. Br J Nutr, 1999 Aug, 82(2), 139 - 47 Ileal amino acid digestibilities in pigs of barley-based diets with inclusion of lucerne (Medicago sativa), white clover (Trifolium repens), red clover (Trifolium pratense) or perennial ryegrass (Lolium perenne); Reverter M et al.; Two experiments were performed with post-valve T-cannulated growing pigs, using five animals in each experiment in a change-over design to evaluate the effect of inclusion of four different dried forage meals on ileal crude protein (CP) and amino acid (AA) digestibilities . The control diets (C1 and C2) were barley-based and the experimental diets were formulated by replacing the barley with 100 or 200 g/kg of either lucerne (Medicago sativa) or white clover (Trifolium repens) meal in Expt 1 and red clover (Trifolium pratense) or perennial ryegrass (Lolium perenne) meal in Expt 2 . A decrease (P < 0.05) in the apparent ileal digestibility of CP and most of the essential and nonessential AA was found with the inclusion of luceme, white clover and perennial ryegrass meal in the barley-based diets . When red clover meal was included, only the apparent ileal digestibilities of CP, leucine, phenylalanine, tyrosine and glutamic acid were found to decrease (P < 0.05) . The estimated apparent ileal digestibilities of most essential AA in the forage meals were lower than in the barley-based diets . The ileal flow of glucosamine and ornithine was found to increase (P < 0.05) with increasing proportion of fibre in the diet, suggesting an increase in endogenous N secretions and small-intestinal microbial activity . With the minor changes found for ileal essential AA digestibilities with forage meal inclusion in the diet the present data confirm the potential of forage meals as a source of protein in pig diets. J Control Release, 2000 May 15, 66(2-3), 199 - 214 Cutaneous vaccination: the skin as an immunologically active tissue and the challenge of antigen delivery; Babiuk S et al.; Vaccination is one of the major achievements of modern medicine . As a result of vaccination, diseases such as polio and measles have been controlled and small pox has been eradicated . However, despite these successes there are still many microbial diseases that cause tremendous suffering because there is no vaccine or the vaccines available are inadequate . In addition, even if vaccines were available for all infectious diseases there is no guarantee that people would use them routinely . One of the major impediments to ensuring vaccine efficacy and compliance is that of delivery . Presently most vaccines are given by intramuscular administration . Unfortunately this is often traumatic, especially in infants . Thus, if it was possible to replace intramuscular immunization by mucosal (oral/intranasal) or transdermal delivery it may be possible to both enhance mucosal immunity as well as improve overall compliance rates . The transdermal route has been used by the pharmaceutical industry for the delivery of various low molecular weight drugs . Some of the approaches used for smaller compounds may also have potential for delivery of either protein or polynucleotide vaccines . However, there is a greater challenge to delivering large molecular weight molecules through the skin due to size, charge and other physicochemical properties . This review will describe the recent advances that have been made in dermal and topical delivery as related to vaccines. Appl Environ Microbiol, 2000 Apr, 66(4), 1479 - 88 Sediment microbial community structure and mercury methylation in mercury-polluted Clear Lake, California; Macalady JL et al.; Spatial and temporal variations in sediment microbial community structure in a eutrophic lake polluted with inorganic mercury were identified using polar lipid fatty acid (PLFA) analysis . Microbial community structure was strongly related to mercury methylation potential, sediment organic carbon content, and lake location . Pore water sulfate, total mercury concentrations, and organic matter C/N ratios showed no relationships with microbial community structure . Seasonal changes and changes potentially attributable to temperature regulation of bacterial membranes were detectable but were less important influences on sediment PLFA composition than were differences due to lake sampling location . Analysis of biomarker PLFAs characteristic of Desulfobacter and Desulfovibrio groups of sulfate-reducing bacteria suggests that Desulfobacter-like organisms are important mercury methylators in the sediments, especially in the Lower Arm of Clear Lake. Immunol Res, 1999, 20(3), 187 - 94 Complement receptor type two (CR2,CR21): a target for influencing the humoral immune response and antigen-trapping; Prodinger WM; Cellular receptors for complement C3 fragments deposited on antigens are important bricks in the wall defending against microbial pathogens . The part of complement receptor type 2 (CR2; CD21) deals with enhancing humoral immune responses and with long-term trapping of C3d-coated antigen by follicular dendritic cells . CR2 is also pivotal for Epstein-Barr virus (EBV) infection . Here, the current understanding, how CR2 interacts with its ligands C3d, EBV, and CD23 is summarized . The potential to target CR2 for clinical therapy or immunization purposes are discussed. Extremophiles, 2000 Feb, 4(1), 61 - 7 Microbial diversity at 83 degrees C in Calcite Springs, Yellowstone National Park: another environment where the Aquificales and "Korarchaeota" coexist; Reysenbach AL et al.; The use of molecular phylogenetic approaches in microbial ecology has revolutionized our view of microbial diversity at high temperatures and led to the proposal of a new kingdom within the Archaea, namely, the "Korarchaeota." We report here the occurrence of another member of this archaeal group and a deeply rooted bacterial sequence from a thermal spring in Yellowstone National Park (USA) . The DNA of a mixed community growing at 83 degrees C, pH 7.6, was extracted and the small subunit ribosomal RNA gene (16S rDNA) sequences were obtained using the polymerase chain reaction . The products were cloned and five different phylogenetic types ("phylotypes") were identified: four archaeal phylotypes, designated pBA1, pBA2, pBA3, and pBA5, and only one bacterial phylotype, designated pBB . pBA5 is very closely related to the korarchaeotal phylotype, pJP27, from Obsidian Pool in Yellowstone National Park . The pBB phylotype is a lineage within the Aquificales and, based on 16S rRNA sequence, is different enough from the members of the Aquificales to constitute a different genus . In situ hybridization with