Proc Natl Acad Sci U S A, 2000 Aug 15, 97(17), 9665 - 70 In vivo transposon mutagenesis of the methanogenic archaeon Methanosarcina acetivorans C2A using a modified version of the insect mariner-family transposable element Himar1; Zhang JK et al.; We present here a method for in vivo transposon mutagenesis of a methanogenic archaeon, Methanosarcina acetivorans C2A, which because of its independence from host-specific factors may have broad application among many microorganisms . Because there are no known Methanosarcina transposons we modified the mariner transposable element Himar1, originally found in the insect Hematobia irritans, to allow its use in this organism . This element was chosen because, like other mariner elements, its transposition is independent of host factors, requiring only its cognate transposase . Modified mini-Himar1 elements were constructed that carry selectable markers that are functional in Methanosarcina species and that express the Himar1 transposase from known Methanosarcina promoters . These mini-mariner elements transpose at high frequency in M . acetivorans to random sites in the genome . The presence of an Escherichia coli selectable marker and plasmid origin of replication within the mini-mariner elements allows facile cloning of these transposon insertions to identify the mutated gene . In preliminary experiments, we have isolated numerous mini-mariner-induced M . acetivorans mutants, including ones with insertions that confer resistance to toxic analogs and in genes that encode proteins involved in heat shock, nitrogen fixation, and cell-wall structures. J Med Entomol, 2000 Jul, 37(4), 528 - 33 Tagging bloodmeals with phagemids allows feeding of multiple-sample arrays to single cages of mosquitoes (Diptera: Culicidae) and the recovery of single recombinant antibody fragment genes from individual insects; Killeen GF et al.; A recombinant single-chain variable-region human antibody fragment (scFv) was expressed in Escherichia coli, extracted in hypertonic sucrose, mixed directly with blood and fed to Anopheles gambiae Giles mosquitoes . When E . coli containing the phagemids that encode these scFv were included in bloodmeals, phagemids could be recovered from the mosquito midgut for up to 3 d after feeding . Furthermore, large arrays of such gene-tagged scFv-containing bloodmeals could be fed to cages of mosquitoes using microtiter plates . Arrays of phagemids with and without an antibody insert were fed to single cages of mosquitoes to test whether individual mosquitoes fed from single wells of such arrays . Phagemids were recovered from 95% of blood-fed females and > 80% of these phagemids were monoclonal . Therefore, it is possible to feed multiple sample arrays of recombinant proteins to single cages of mosquitoes and to recover the genetic material that encodes for only one of the array elements from individual mosquitoes . This demonstration indicates that multiple-sample feeding and recovery strategies are feasible and may represent a viable strategy for future rapid screening of biologically active genes, gene products or microorganisms in live arthropods. Virology, 2000 Aug 1, 273(2), 228 - 40 Chemokines and viruses: the dearest enemies; Lusso P; The relation between viruses and the chemokine system is characterized by a complex blend of enmity and attraction . Chemokines are key regulators of innate and adaptive immune responses against invading microorganisms, including viruses . They act not only as immune system "traffic officers," controlling leukocyte migration under both physiological and pathological conditions, but also as fine orchestrators that modulate the induction, amplification, and cytokine-secretion pattern of antiviral responses . However, viruses have succeeded in turning the chemokine system into an ally . During the course of a long parallel evolution, viruses have captured from their hosts the genetic information for encoding chemokines and chemokine receptors and have reprogrammed it for evading the control of the immune system . Moreover, selected viral agents, most notably primate immunodeficiency retroviruses, have adopted chemokine receptors as essential gateways for entry into their target cells . The endogenous secretion of chemokines is thus emerging as an important in vivo mechanism of viral control, which is potentially inducible by effective vaccines . The deepening knowledge of the interactions between viruses and chemokines may lead to novel therapeutic and preventive strategies for the control of viral and inflammatory diseases . J Periodontol, 2000 Jun, 71(6), 898 - 903 Metabolic diseases and their possible link to risk indicators of periodontitis; Noack B et al.; BACKGROUND: During the last few years, risk assessment has become one of the main topics of periodontal research . Therefore, the aim of this study was to determine whether a predisposition to metabolic disorders such as diabetes mellitus (in the absence of diagnosed diabetic disease) or hyperlipidemia may be risk indicators for periodontitis . METHODS: One hundred patients ranging in age from 40 to 70 years were examined . The patients were classified as having impaired glucose tolerance (IGT) but no manifest diabetes (56 patients), hyperlipidemia (17 patients, HL), or normal metabolic status (27 control patients) . Probing depth (PD), attachment level (AL), plaque index (PI), and gingival bleeding on probing (BOP) were recorded . Serum antibody titers (SAT) to A . actinomycetemcomitans (A.a.), P . intermedia (P.i.), and P . gingivalis (P.g.) were determined by enzyme-linked immunosorbent assay (ELISA) . Pooled subgingival plaque samples were analyzed using indirect immunofluorescence to detect the same organisms . In addition, respiratory burst activity of peripheral polymorphonuclear leukocytes (PMN) was evaluated by chemiluminescence (CL) . RESULTS: No significant differences were observed between the IGT group and normal controls in the following parameters: 1) percentage of sites exhibiting BOP; 2) mean PI; 3) mean PD and AL; 4) percentage of periodontal microorganisms; and 5) increased SAT . The IGT probands exhibited a significantly higher mean serum level of triglycerides, as well as higher formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated PMN chemiluminescence than the control group . Patients with hyperlipidemia (HL) showed a significantly higher number of sextants with increased PD (73.4%) than the control group (50.6%) . Similar results were obtained when comparing the percentage of all sites with increased PD (HL = 16.7%, control 12.3%) . The mean FMLP-stimulated CL in patients with hyperlipidemia was significantly higher than the control group . When looking at all patients, there was a small but statistically significant correlation between PD and lipid levels . In addition, a significant correlation was observed between lipid serum levels and the FMLP-stimulated chemiluminescence . CONCLUSIONS: These findings suggest that abnormal glucose tolerance, which is a predisposing factor for diabetes mellitus, does not appear to be a risk indicator for periodontal disease . On the other hand, impaired lipid metabolism does seem to be a risk indicator for periodontitis. Harefuah, 1999 Feb 15, 136(4), 257 - 61, 340 {Legionellosis in Israel--a view of the situation}; Boldur I et al.; Infection with Legionella remains an important cause of disease and death . We analyzed our laboratory data from 1993 through 1997, augmented by our 20 years of experience . The incidence of Legionella as a cause of pneumonia varied in our study from 5%-9%, with a slight increase during the winter . Isolation of these microorganisms from different water sources was higher during the summer and ranged from 7%-70% . Special laboratory tests are necessary to diagnose the disease and monitor these bacteria in water samples . The serologic method--indirect immunofluorescent assay--for 41 serogroups of Legionella was the main diagnostic method used . Legionella sg . 1 was the most frequent cause of the disease, with an incidence of 52% in 1993, decreasing to 15% in 1997 . An increase in the incidence of seropositivity to "other Legionellae" is characteristic for our country . No correlation was found between the incidence of isolation of a specific strain and exposure . However, it is well known that the disease is overtreated but underdiagnosed, which requires reversal . Larger studies of Legionella colonization in water supplies and in air are needed in order to establish the risk of infection . Water sources are presently under-studied, as are respiratory devices in hospitals, or they are not studied at all in Israel, such as in mist machines in supermarkets, in dental clinics, and in ships and airplanes. Arch Immunol Ther Exp (Warsz), 2000, 48(3), 189 - 93 Immune phenomena in echinoderms; Glinski Z et al.; Advances in biochemistry and molecular biology have made it possible to identify a number of mechanisms active in the immune phenomena of echinoderms . It is obvious that echinoderms have the ability to distinguish between different foreign objects (pathologically changed tissues, microorganisms, parasites, grafts) and to express variable effector mechanisms which are elicited specifically and repeatably after a variety of non-self challenges . The molecular and biochemical basis for the expression of these variable defense mechanisms and the specific signals which elicit one type of effector mechanism are not, however, yet well known . The high capacity of coelomocytes to phagocytose, entrap and encapsulate invading microorganisms is a valid immune cell-mediated mechanism of echinoderms . The entrapped bacteria, discharged cellular materials and disintegrating granular cells are compacted and provoke the cellular encapsulation reaction . Moreover, humoral-based reactions form an integral part of the echinoderm defense system against microbial invaders . Factors such as lysozyme, perforins (hemolysins) vitellogenin and lectins are normal constituents of hemolymph, while cytokines are synthesized by echinoderms in response to infection. Przegl Lek, 2000, 57(1), 32 - 5 {Health-toxicologic aspects of some fungi}; Ochmanski W et al.; Recent findings of fungi in food products of such renomed companies as Coca-Cola and Danone resulted in society-wide alert in Poland . Humans have contact with fungi everywhere . Every food product covered with mould or having marks of it should be discarded . We should mention that cutting of or skimming the mould is totally ineffective and dangerous, because of the fact that rest of the product will contain products of fungal metabolism such as mikotoxins, which are, of course, invisible . Modern food producing technologies effected in microorganism-free products, but sporadically we can find dead fungi debris due to improper washing procedures of multi-use bottles, like it was observed in Coca-Cola products . As for mould-covered cottage cheese type products of Danone, most probably reason was improper handling of ready, sealed products during transport and storage . Even minimal physical injuries to air-tight containers resulted in sporae penetration to milk products and finally contamination with mikotoxins. Biotechniques, 2000 Jul, 29(1), 108 - 12, 114-6 Fingerprinting method for phylogenetic classification and identification of microorganisms based on variation in 16S rRNA gene sequences; Raghava GP et al.; The paper describes a method for the classification and identification of microorganisms based on variations in 16S rRNA sequences . The 16S rRNA is one of the most conserved molecules within a cell . The nature of the variable and spacer regions has been found to be specific to a given organism . Thus, the method presented here can be very useful for the classification and identification of microorganisms for which very little information is available . To automate the method, a comprehensive computer program called FPMAP has been developed for the analysis of restriction fragment pattern data . The method involves the restriction digestion of genomic DNA, preferably using four-cutters that may recognize 6-9 sites within the 16S rDNA . The fragments are separated on a polyacrylamide gel along with a suitable marker, then transferred into a nylon membrane and hybridized with a radiolabeled 16S rDNA probe . After autoradiography, the fragment sizes are calculated, and the data are analyzed using the FPMAP software . We demonstrate that the method can be used for identification of strains of Streptomyces and mycobacteria . The software is available from our ftp site ftp: inverted question markimtech.chd.nic.in/pub/com/fpmap/unix/. Emerg Infect Dis, 2000 Jul-Aug, 6(4), 329 - 36 Male-killing bacteria in insects: mechanisms, incidence, and implications; Hurst GD et al.; Bacteria that are vertically transmitted through female hosts and kill male hosts that inherit them were first recorded in insects during the 1950s . Recent studies have shown these "male-killers" to be diverse and have led to a reappraisal of the biology of many groups of bacteria . Rickettsia, for instance, have been regarded as human pathogens transmitted by arthropods . The finding of a male-killing Rickettsia obligately associated with an insect suggests that the genus' members may be primarily associated with arthropods and are only sometimes pathogens of vertebrates . We examined both how killing of male hosts affects the dynamics of inherited bacteria and how male-killing bacteria affect their host populations . Finally, we assessed the potential use of these microorganisms in the control of insect populations. Minerva Ginecol, 2000 Mar, 52(3), 49 - 58 {Ureaplasma urealyticum vaginosis and premature rupture of membranes . What is its role?}; Calleri LF et al.; BACKGROUND: The aim of this study was to evaluate a correlation between PROM and genital infections . METHODS: A total of 308 vaginal swabs were made in randomized study group composed by 184 pregnant women aged between 26 and 32 years with an extreme age of 19 (one) and 40 years (one) . Three vaginal swabs and one cervical swab (searching for Chlamydia) were made for every patient . Sixteen patients were excluded during this study, because they decided to have their babies in other hospitals . Therefore, the patients totally included in the study were 166: 109 at the first pregnancy, 33 at the second pregnancy and 5 at the third pregnancy . No one of them had any spontaneous abortion in the past . All possible other factor which ca be considered responsible and/or inductive of premature ruptures, such as cervical incontinence, cigarette smoke and coitus were excluded . RESULTS: 280 vaginal swabs were made in this study: 134 were positive, with a global positivity percentage of 47.85% . Premature rupture of membranes (PROM) was observed in 38 cases with an incidence of 23.03%: 26 were PROM and 12 were pPROM . The extreme pPROMs occurred, respectively, at the 21st and at 34th gestational week; 29 of the 38 cases of PROM, were associated with positive cultures . The results obtained show an evident correlation between PROM and Ureaplasma urealyticum vaginosis: this fact is improved by the high incidence percentage of this mycobacter in pregnant women and also by an absolute predomination of Ureplasma urealyticum in PROM cases (72.41%) . CONCLUSIONS: These data obtained confirm the importance of this microorganism in PROM genesis, according to some recent studies . It is suggested that Ureaplasma urealyticum infection can contribute to a premature start of labour. Mem Inst Oswaldo Cruz, 2000 Jul-Aug, 95(4), 491 - 4 Flow cytometry as a tool to identify Mycobacterium tuberculosis interaction with the immune system and drug susceptibility; Bonecini-Almeida MG; Flow cytometric analysis is a useful and widely employed tool to identify immunological alterations caused by different microorganisms, including Mycobacterium tuberculosis . However, this tool can be used for several others analysis . We will discuss some applications for flow cytometry to the study of M . tuberculosis, mainly on cell surface antigens, mycobacterial secreted proteins, their interaction with the immune system using inflammatory cells recovered from peripheral blood, alveolar and pleura spaces and the influence of M . tuberculosis on apoptosis, and finally the rapid determination of drug susceptibility . All of these examples highlight the usefulness of flow cytometry in the study of M . tuberculosis infection. J Immunol, 2000 Aug 1, 165(3), 1403 - 9 Impaired secretion of rat mannose-binding protein resulting from mutations in the collagen-like domain; Heise CT et al.; Serum mannose-binding protein (MBP) or mannose-binding lectin initiates the lectin branch of the innate immune response by binding to the surface of potentially pathogenic microorganisms and initiating complement fixation through an N-terminal collagen-like domain . Mutations in this region of human MBP are associated with immunodeficiency resulting from a reduction in the ability of the mutant MBPs to fix complement as well as from reduced serum concentrations . Inefficient secretion of the mutant proteins, which is one possible cause of the reduced serum levels, has been investigated using a mammalian expression system in which each of the naturally occurring human mutations has been recreated in rat serum MBP . The mutations Gly25-->Asp and Gly28-->Glu disrupt the disulfide-bonding arrangement of the protein and cause at least a 5-fold increase in the half-time of secretion of MBP compared with wild-type rat serum MBP . A similar phenotype, including a 3-fold increase in the half-time of secretion, disruption of the disulfide bonding arrangement, and inefficient complement fixation, is observed when nearby glucosylgalactosyl hydroxylysine residues at positions 27 and 30 are replaced with arginine residues . The results suggest that defective secretion resulting from structural changes in the collagen-like domain is likely to be a contributory factor for MBP immunodeficiency. Ann Surg, 2000 Aug, 232(2), 208 - 15 Bactericidal/permeability-increasing protein preserves leukocyte functions after major liver resection; Wiezer MJ et al.; OBJECTIVE: To analyze postoperative leukocyte functions in patients undergoing hemihepatectomy, and to assess the effect of treatment with the endotoxin-neutralizing agent bactericidal/permeability-increasing protein (rBPI21) . SUMMARY BACKGROUND DATA: Extensive liver resection is associated with a high incidence of infectious complications . Because elimination of pathogenic microorganisms occurs mainly by leukocytes, this increased rate of infections is most likely due to an impaired function of these cells . Endotoxin, translocated from the gut into the systemic circulation as a result of increased gut permeability and reduced hepatic clearance function after major liver resection, may play an important role in the impairment of posthepatectomy leukocyte function . METHODS: To investigate whether hemihepatectomy results in impaired leukocyte functions and to determine the role of endotoxin in this process, leukocyte oxidative burst and leukocyte antigen expression were studied in three groups of patients: patients undergoing a hemihepatectomy and receiving rBPI21 treatment, patients undergoing hemihepatectomy and receiving placebo, and as an extra control group patients undergoing other major abdominal surgeries . Blood samples were collected before surgery, 2 hours after surgery, and at days 1, 2, 5, and 7 . Phorbol myristate acetate-stimulated oxidative burst was measured using dihydrorhodamine, and leukocyte surface expression of the antigens CD11b, CD16, and CD14 was investigated by indirect immunofluorescence . Both oxidative burst and membrane surface expression were quantified by flow cytometry . An indication of the antiendotoxin effect of rBPI21 treatment was provided by assessment of plasma lipopolysaccharide binding protein (LBP) levels by enzyme-linked immunosorbent assay . RESULTS: The oxidative burst in the hemihepatectomized patients receiving placebo and the controls increased 2 hours after surgery, whereas it decreased in the rBPI21-treated patients, resulting in significant differences between the groups . On day 1, neutrophil CD11b expression and monocyte CD14 expression in the rBPI21-treated patients and controls were significantly lower than in the placebo group . At 2 hours, CD16 expression in the placebo-treated patients was significantly higher than in the rBPI21-treated patients and controls . On day 5 and day 7, plasma LBP levels were significantly higher in the placebo-treated patients compared with the rBPI21-treated patients . CONCLUSIONS: The results of this study show that patients undergoing major liver resection have an increased activation of leukocytes compared with those undergoing other major abdominal surgery . This enhanced activation may contribute to the increased risk of infection in these patients . Administration of the endotoxin-neutralizing agent rBPI21 to hemihepatectomy patients was shown to reduce plasma LBP levels, to preserve leukocyte functions partially, and to reduce leukocyte activation to the level of other, nonhepatic abdominal surgery. J Mol Evol, 2000 Jul, 51(1), 64 - 75 Comparative genomics of mitochondrial DNA in Drosophila simulans; Ballard JW; The current study compares the nucleotide variation among 22 complete mitochondrial genomes of the three distinct Drosophila simulans haplotypes with intron 1 of the alcohol dehydrogenase-related locus . This is the first study to investigate the sequence variation of multiple complete mitochondrial genomes within distinct mitochondrial haplotypes of a single species . Patterns of variation suggest distinct forces are influencing the evolution of mitochondrial DNA (mtDNA) and autosomal DNA in D . simulans . First, there is little variation within each mtDNA haplotype but strong differentiation among them . In contrast, there is no support for differentiation of the mitochondrial haplotypes at the autosomal locus . Second, there is a significant deficiency of mitochondrial variation in each haplotype relative to the autosomal locus . Third, the ratio of nonsynonymous to synonymous substitutions is not equal in all branches of the well-resolved phylogeny . There is an excess of nonsynonymous substitutions relative to synonymous substitutions within each D . simulans haplotype . This result is similar to that previously observed within the mtDNA of distinct species . A single evolutionary force may be causally linked to the observed patterns of mtDNA variation-a rickettsia-like microorganism, Wolbachia pipientis, which is known to directly influence mitochondrial evolution but have a less direct influence on autosomal loci. J Vet Med B Infect Dis Vet Public Health, 2000 Jun, 47(5), 343 - 9 Dynamics of the development of Chlamydophila psittaci inclusions in epithelial and fibroblast host cells; Escalante-Ochoa C et al.; The development of Chlamydophila psittaci (formerly Chlamydia psittaci, avian strains) inclusions in fibroblast L-929 and epithelial BGM cell lines was studied along the bacterial growth cycle using a BGM cell-adapted strain in the presence or absence of cycloheximide and cycloheximide + polyethylene glycol . Evolution of the inclusions was determined in terms of their number and size at 24, 30, 36, 48 and 54 h after infection . Significant differences in the chlamydial growth were found between both host cells, throughout the study . Higher numbers of inclusions (P < 0.05) were observed in L cells while larger inclusions (P < 0.01) were found in BGM cells . In both fibroblast and epithelial cells, inclusions showed a significant (P < 0.001) increase in size at the later times studied . Free extracellular chlamydial particles were noticed at 48 and 54 h post-infection (p.i.) in infected L cells, and at 54 h p.i . in BGM cells . Addition of cycloheximide or cycloheximide + polyethylene glycol had no significant effect on the number of inclusions or their size . The results suggest that host cell characteristics and innate compatibility between Chlamydophila strain and host cell are more important than host cell adaptation for the development of the microorganism. Chemosphere, 1999 Feb, 38(3), 655 - 62 Tolerance to trichlorophenols in microorganisms from a polluted and a pristine site of a river Godoy F, Zenteno P, Cerda F, Gonzalez B, Martinez M. The effect of 2,4,5- and 2,4,6-trichlorophenol on the microbiota from a polluted and a pristine site of a river was studied . Bacterial metabolic activity measurements by epifluorescence microscopy showed that the polluted site contained more metabolically active cells than the pristine site . Total culturable bacterial counts and tolerant bacterial counts from both sites were not affected by incubation (for up to 5 days) with 200 ppm of chlorophenols . However, the incubation with 500 ppm of 2,4,5-trichlorophenol prevented detection of total and tolerant bacterial counts in the pristine site, and inhibited tolerants in the polluted site . None of 250 bacterial colonies directly isolated from these samples was able to grow on chlorophenols . However, bacteria able to grow on 2,4,6-trichlorophenol, were obtained by enrichment of water and sediments samples. Infect Immun, 2000 Aug, 68(8), 4831 - 3 Role of Nramp1 deletion in Chlamydia infection in mice; Pal S et al.; Elicited macrophages from 129sv mice with a functional deletion of the natural-resistance-associated macrophage protein 1 gene (Nramp1) were shown to be as susceptible as wild-type mice to infection with the Chlamydia trachomatis mouse pneumonitis and L3 serovars and to Chlamydia pneumoniae . Furthermore, the two groups of mice were shown to be similarly susceptible to an intranasal infection with these microorganisms . In conclusion, the Nramp1 gene does not appear to play a major role in the regulation of the susceptibility of mice to a chlamydial infection. Infect Immun, 2000 Aug, 68(8), 4736 - 45 Nonopsonic phagocytosis of zymosan and Mycobacterium kansasii by CR3 (CD11b/CD18) involves distinct molecular determinants and is or is not coupled with NADPH oxidase activation; Le Cabec V et al.; Complement receptor type 3 (CR3) was initially described as an opsonic receptor . Subsequently, CR3-mediated lectin-sugar recognition mechanisms have been shown to play a major role in the nonopsonic phagocytosis of several pathogens, among them Mycobacterium tuberculosis . Little is known about the binding and signal transduction mechanisms operating during nonopsonic ingestion through CR3 of different microorganisms . In the present study, we used CHO cells stably transfected with CR3 to show that CR3 was able to mediate internalization of zymosan and pathogenic mycobacteria (Mycobacterium kansasii and Mycobacterium avium) but not that of nonpathogenic species (Mycobacterium smegmatis and Mycobacterium phlei) . A combination of mannan and beta-glucan inhibited the phagocytosis of zymosan but had no effect on M . kansasii ingestion . Among six monoclonal antibodies (MAbs) directed against the CD11b subunit of CR3 that decreased zymosan ingestion, only three inhibited M . kansasii phagocytosis . In particular, MAbs known to block the CR3 lectin site affected only internalization of zymosan . Using U937 macrophages, we observed that zymosan ingestion through CR3 induced superoxide production measured by cytochrome c reduction and by translocation of the NADPH oxidase cytosolic component p47phox to the phagosomal membrane, whereas phagocytosis of viable or heat-killed M . kansasii did not . Furthermore, lack of superoxide anion production during phagocytosis of M . kansasii was not due to inhibition of NADPH oxidase per se or superoxide anion scavenging . Together, our results indicate that (i) nonopsonic phagocytosis of zymosan and M . kansasii by CR3 implicates different molecular mechanisms involving multiple and distinct epitopes of CD11b and (ii) CR3 may transduce different cellular responses depending on the sites mediating nonopsonic phagocytosis. Enzyme Microb Technol, 2000 Aug 1, 27(3-5), 212 - 218 Characteristics of levan fructotransferase from Arthrobacter ureafaciens K2032 and difructose anhydride IV formation from levan; Song K et al.; A microorganism producing levan fructotransferase was isolated from sugar-disclosed soil and it was identified as Arthrobacter ureafaciens . The major product from levan by enzyme reaction was identified as di-D-fructofuranose 2,6':6,2' dianhydride by mass spectrometry, nuclear magnetic resonance, and chemical analyses . Small amounts of several oligosaccharides and free fructose were also formed by enzyme reaction . An extracellular enzyme that produces di-D-fructofuranose 2,6':6,2' dianhydride from levan was purified from the culture broth of A . ureafaciens K2032 . The enzyme had optimum activity around pH 5.8 and 45 degrees C and had a dimeric form in solution . The N-terminal amino acid residues of the purified enzyme were SAPGSLRAVYHMTPPSGXLXDPQ . The enzyme has narrow substrate range and converts the levan to di-D-fructofuranose 2,6':6,2' dianhydride with around 62.5% conversion yield. Am J Physiol Gastrointest Liver Physiol, 2000 Jul, 279(1), G20 - 7 Functional and molecular biological evidence of SGLT-1 in the ruminal epithelium of sheep; Aschenbach JR et al.; Because of the effective catabolism of D-glucose to short-chain fatty acids by intraruminal microorganisms, the absorption of D-glucose from the rumen was thought to be of minor importance . However, clinical studies suggested that significant quantities of D-glucose are transported from the ruminal contents to the blood . We therefore tested the ruminal epithelium of sheep for the presence of Na(+)-glucose cotransporter 1 (SGLT-1) on both the functional and mRNA levels . In the absence of an electrochemical gradient, 3-O-methylglucose (3-OMG) was net absorbed across isolated ruminal epithelia mounted in Ussing chambers . The net transport of 3-OMG followed Michaelis-Menten kinetics and was sensitive to phlorizin or decreasing Na(+) concentrations . The mucosal addition of 10 mM D-glucose induced an immediate, phlorizin-sensitive increase in short-circuit current (I(sc)) . I(sc) could also be increased by serosal addition of D-glucose or D-mannose, but electrogenic uptake of D-glucose or 3-OMG added on the mucosal side was still detectable after serosal stimulation of I(sc) . RT-PCR using primers specific for the ovine intestinal SGLT-1 with subsequent TA cloning and sequencing revealed 100% identity between the cloned cDNA and mRNA fragment 187-621 of ovine intestinal SGLT-1 . In conclusion, the ruminal epithelium has a high-affinity SGLT-1, which indicates that it maintains the capacity for D-glucose absorption. An R Acad Nac Med (Madr), 1999, 116(4), 793 - 811; discussion 811-3 {Gastroesophageal reflux and Helicobacter pylori}; Herrerias Gutierrez JM; The relationship between Helicobacter pylori (H . pylori) infection and gastroesophageal reflux disease (GERD) is not well known yet, and has some controversial issues . There is indirect epidemiological evident, not yet proven, that H . pylori may have a protective role against GERD . Hypochlorhydria caused by gastritis located mainly at corpus would be the principal physiopathological protector mechanism against GERD . Even knowing that more studies are needed, the risk of developing GERD after the H . pylori eradication seems to be increased in some groups of patients . In the presence of the microorganism, the efficacy of acid suppression therapy may decrease, and its long-term use could favour the progression to corpus atrophic gastritis . Specialized intestinal metaplasia in an endoscopically normal-appearing cardia may precede adenocarcinoma, and its prevention would vary, depending on its controversial origin, secondary to carditis, H . pylori or GERD. Exp Cell Res, 2000 Aug 1, 258(2), 298 - 309 CBP1 associates with the Dictyostelium cytoskeleton and is important for normal cell aggregation under certain developmental conditions; Dharamsi A et al.; In cells of the eukaryotic microorganism Dictyostelium discoideum, at least eight small, four-EF-hand Ca(2+)-binding proteins of unknown function are expressed at specific times during development . One of these proteins, calcium-binding protein 1 (CBP1), first appears just prior to cell aggregation and then is present at relatively constant levels throughout development . To determine a role for CBP1 during development, the protein was used as bait in a yeast two-hybrid screen to reveal putative CBP1-interacting proteins . Two proteins identified in this screen were the actin-binding proteins, protovillin and EF-1alpha . Using an in vitro binding assay, both of these proteins were found to interact with CBP1 in the absence of Ca(2+), but the interaction of CBP1 with EF-1alpha was increased substantially by Ca(2+) . CBP1 was also shown by fluorescence microscopy and by binding assays to associate with the actin cytoskeleton of Dictyostelium cells during development, and these interactions were partially Ca(2+)-dependent . cbpA-null cells grew normally, but under certain developmental conditions, cell aggregation was prolonged and irregular . This defect in aggregation appeared to be related to a general reduction in cell motility rather than to a decrease in the ability of the cells to respond to the chemoattractant cAMP . Together, these results suggest that CBP1 might function to help regulate the reorganization of the Dictyostelium actin cytoskeleton during cell aggregation . Rev Panam Salud Publica, 2000 May, 7(5), 325 - 31 {Epidemiology of leptospirosis in a livestock production area of the Andes}; Ochoa JE et al.; A cross-sectional study was conducted to estimate the prevalence of Leptospira infection in populations of workers, cattle, and pigs in livestock operations and to explore some environmental and production system variables associated with seropositivity . The research was done between November 1997 and February 1998 in the municipality of Don Matias, in the northern part of the department of Antioquia, Colombia . The area has a cold climate, and there is a "pigs-grasses-milk" production system that utilizes pig dung to fertilize grazing pastures . A total of 23 farms were studied, and blood samples were obtained from 67 dairy and pig-raising workers, 174 dairy cows, 68 pigs for fattening, and 214 pigs for breeding . The microagglutination test (MAT) was used for six Leptospira serotypes . The seropositivity prevalence was 22.4% among the workers (95% confidence interval: 13.1% to 34.2%), 60.9% among the dairy cows (95% CI: 53.2% to 68.2%), 10.3% in the fattening pigs, and 25.7% in the breeding pigs . Four logistic regression models were constructed to identify the variables that predicted infection in the workers and in the dairy cows . A high prevalence of infection with Leptospira (serotypes pomona, bratislava, and hardjo) was found in this production system, where conditions are favorable for transmitting this microorganism to the different animal species and to humans. Am J Ind Med, 2000 Aug, 38(2), 164 - 218 Occupational airway sensitizers: an overview on the respective literature; van Kampen V et al.; BACKGROUND: Worldwide, there is rigorous scientific activity concerning the further development of work safety regulations involving airway-sensitizing substances . Technical directives on hazardous substances are enforced in several countries and are being continuously updated . The European Union has established a code for several occupational substances, now labeled R 42 ("may cause sensitization by inhalation") . METHODS: We present an overview of the literature dealing with allergic occupational asthma . The literature was selected according to criteria of study design and diagnostic test methods . Approximately 300 publications were reviewed including both epidemiological studies and individual case reports . RESULTS: Airway sensitizers are systematically arranged and separately listed according to chemicals and their origin from animals, plants, and microorganisms . The clinical data as well as threshold limit values (TLV) and R 42 labeling of 250 airway-sensitizing substances are presented . CONCLUSIONS: The most common sensitizing substances causing occupational asthma were dust of cereal flours, enzymes, natural rubber latex, laboratory animals as well as low molecular substances such as isocyanates and acid anhydrides . FEMS Immunol Med Microbiol, 2000 Aug, 28(4), 343 - 6 Sensitivity of bacterial coaggregation to chelating agents; Taweechaisupapong S et al.; Coaggregation between pairs of microorganisms was found to be inhibited by chelating agents, such as acetylacetone, citrate, EDTA and carboxymethylcellulose . Assays were conducted on eight pairs of periodontopathogens and one pair consisting of Escherichia coli and Saccharomyces cerevisiae . The inhibitory effects of the chelating agents were reversible except for Actinomyces naeslundii 12104, the adhesin of which was irreversibly inactivated . Even though the bacteria possessed different kinds of adhesins, their sensitivity to chelating agents appears to be a common property . Non-toxic chelating agents, such as carboxymethylcellulose and citrate, may prove to be useful anti-adhesins. J Chromatogr A, 2000 Jun 2, 880(1-2), 3 - 33 Sample preparation for the analysis of flavors and off-flavors in foods; Wilkes JG et al.; Off-flavors in foods may originate from environmental pollutants, the growth of microorganisms, oxidation of lipids, or endogenous enzymatic decomposition in the foods . The chromatographic analysis of flavors and off-flavors in foods usually requires that the samples first be processed to remove as many interfering compounds as possible . For analysis of foods by gas chromatography (GC), sample preparation may include mincing, homogenation, centrifugation, distillation, simple solvent extraction, supercritical fluid extraction, pressurized-fluid extraction, microwave-assisted extraction, Soxhlet extraction, or methylation . For high-performance liquid chromatography of amines in fish, cheese, sausage and olive oil or aldehydes in fruit juice, sample preparation may include solvent extraction and derivatization . Headspace GC analysis of orange juice, fish, dehydrated potatoes, and milk requires almost no sample preparation . Purge-and-trap GC analysis of dairy products, seafoods, and garlic may require heating, microwave-mediated distillation, purging the sample with inert gases and trapping the analytes with Tenax or C18, thermal desorption, cryofocusing, or elution with ethyl acetate . Solid-phase microextraction GC analysis of spices, milk and fish can involve microwave-mediated distillation, and usually requires adsorption on poly(dimethyl)siloxane or electrodeposition on fibers followed by thermal desorption . For short-path thermal desorption GC analysis of spices, herbs, coffee, peanuts, candy, mushrooms, beverages, olive oil, honey, and milk, samples are placed in a glass-lined stainless steel thermal desorption tube, which is purged with helium and then heated gradually to desorb the volatiles for analysis . Few of the methods that are available for analysis of food flavors and off-flavors can be described simultaneously as cheap, easy and good. Br Poult Sci, 2000 May, 41(2), 168 - 73 The effect of changing the excreta moisture of caged laying hens on the excreta and microbial contamination of their egg shells; Smith A et al.; 1 . An experiment that included 1440 caged laying hens in 24 experimental units was conducted to determine the effect of differences in excreta moisture on the proportion of dirty eggs and the microbial contamination of eggs that were ostensibly uncontaminated by excreta . Excreta moisture contents were changed by giving the hens diets that contained 4 different concentrations of sodium . 2 . Diets containing 1.6, 5, 10 or 15 g/kg dietary sodium were fed ad libitum to 1140 laying hens for a 12-week feeding period . A sample of excreta was collected from each experimental unit each week and its moisture content determined . All eggs produced were classified as clean or dirty according to the European Community Egg Marketing Regulations . A sample of eggs were collected from each experimental unit on 4 separate occasions in the last 4 weeks of the feeding period and the total bacterial numbers on ostensibly clean egg shells were determined . 3 . Increasing dietary sodium concentration gave linear (P<0.01) increases in excreta moisture . Each 100 g/kg increase in excreta moisture increased (P<0.001) dirty egg numbers by 0.52% of the total eggs produced . Increasing excreta moisture gave a linear increase (P<0.001) in the (log-transformed) numbers of microorganisms that contaminated ostensibly clean egg shells. J Biochem Biophys Methods, 2000 Jul 10, 44(1-2), 1 - 30 Fractionation of cells and subcellular particles with Percoll; Pertoft H; At present, centrifugation is the most common method for separation and isolation of cells and subcellular particles . The technique can be used for a wide range of applications . During latter years it has become obvious what a powerful method density gradient centrifugation is, especially when used in conjunction with sensitive assays or clinical treatments . The most active areas for use of density gradient centrifugation include purification for in vitro fertilization of sperm of both human and bovine origin, isolation of cells for cell therapy of patients receiving chemo- and radiation therapy and basic research both on cellular and subcellular levels . These treatments and investigations require homogeneous populations of cells and cell organelles, which are undamaged after the separation procedure . Percoll, once introduced to reduce convection during centrifugation, has proved to be the density gradient medium of choice since it fulfills almost all criteria of an ideal density gradient medium . Recently good results have also been obtained after silanization of colloidal silica particles, e.g . BactXtractor . The latter medium has proved to be useful in recovery of microorganisms from food samples free of inhibitors to the Polymer Chain Reaction (PCR) . The separation procedures described for Percoll in this review seem to be applicable to any cells or organelles in suspension for which differences in size or bouyant density exist . Furthermore, since Percoll media are inert, they are well suited for the separation of fragile elements like enveloped viruses. Gastroenterology, 2000 Jul, 119(1), 23 - 31 Identification of a novel bacterial sequence associated with Crohn's disease; Sutton CL et al.; BACKGROUND & AIMS: Enteric microorganisms are implicated in the pathogenesis of Crohn's disease (CD), but no clear bacterial or viral species has been identified . In this study, representational difference analysis (RDA) was used to isolate DNA segments preferentially abundant in lamina propria mononuclear cells of lesional mucosa vs . adjacent uninvolved mucosa . METHODS: Two RDA-derived microbial sequences were isolated (I1 and I2) and identified as novel homologues of the ptxR and tetR bacterial transcription-factor families . RESULTS: Quantitative competitive polymerase chain reaction of paraffin-embedded intestinal specimens from 212 patients showed that I2 DNA was present in many CD colonic lesions (43%), but was infrequent in other colonic specimens (9% of ulcerative colitis lesions and 5% of non-inflammatory bowel disease diseases; P<0.0001) . I2 was prevalent in ileal specimens, regardless of disease status (43%-54%) . Enzyme-linked immunosorbent assay analysis of 150 individuals with an I2 glutathione-S-transferase fusion protein showed frequent immunoglobulin A seroreactivity in CD (54% of patients), but infrequent seroreactivity in patients with ulcerative colitis, other inflammatory enteric diseases, or normals (10%, 19%, and 4%, respectively; P<0.001 to 0.00001) . CONCLUSIONS: These findings relate CD to a novel lesion-localized and immunologically associated bacterial sequence, suggesting that the microorganism expressing the I2 gene product may be related to CD pathogenesis. Nat Biotechnol, 2000 Jul, 18(7), 750 - 3 Molecular breeding of carotenoid biosynthetic pathways; Schmidt-Dannert C et al.; The burgeoning demand for complex, biologically active molecules for medicine, materials science, consumer products, and agrochemicals is driving efforts to engineer new biosynthetic pathways into microorganisms and plants . We have applied principles of breeding, including mixing genes and modifying catalytic functions by in vitro evolution, to create new metabolic pathways for biosynthesis of natural products in Escherichia coli . We expressed shuffled phytoene desaturases in the context of a carotenoid biosynthetic pathway assembled from different bacterial species and screened the resulting library for novel carotenoids . One desaturase chimera efficiently introduced six rather than four double bonds into phytoene, to favor production of the fully conjugated carotenoid, 3, 4,3',4'-tetradehydrolycopene . This new pathway was extended with a second library of shuffled lycopene cyclases to produce a variety of colored products . One of the new pathways generates the cyclic carotenoid torulene, for the first time, in E . coli . This combined approach of rational pathway assembly and molecular breeding may allow the discovery and production, in simple laboratory organisms, of new compounds that are essentially inaccessible from natural sources or by synthetic chemistry. J Agric Food Chem, 2000 Jun, 48(6), 2387 - 90 Occurrence of 12-methyltridecanal in microorganisms and physiological samples isolated from beef; Kerscher R et al.; 12-Methyltridecanal (MT) smelling tallowy, beef-like was formed from plasmalogens when beef was boiled . To clarify the origin of MT, its concentration was determined by a stable isotope dilution assay in bacteria and protozoa isolated from the rumen of bovine animals as well as in the plasma, erythrocytes, and other physiological samples . The highest amounts of MT were found in bacteria followed by protozoa . The MT content of the erythrocytes was small . The results support the hypothesis that microorganisms are the main source of MT of which a small amount is resorbed by the animal and transported to the muscular tissue where MT is incorporated into plasmalogens. Rev Cubana Med Trop, 1999 Jan-Apr, 51(1), 20 - 5 {Specific humoral response in Balb/c mice inoculated with a genome library of expression of Trypanosoma cruzi}; Amador EA et al.; A genomic expression library of Trypanosoma cruzi (T . cruzi) was made using plasmid pcDNA3 as a vector, with which male mice from the Balb/c isogenic line were intramuscullary inoculated . It was used a positive control group that was administered soluble antigens of T . cruzi . Other 2 groups received genomic and plasmid DNA, respectively . One group was not immunized . Weekly blood samples were obtained from all the animals until the fourth week and 2 weeks after reimmunization to study the response of specific antibodies against the microorganism antigens by an indirect immunoenzymatic assay (ELISA) . It was observed a significant increase of specific antibodies in the animals reimmunized with 50 micrograms of the library, as well as in the group immunized with soluble antigens of T . cruzi. Sao Paulo Med J, 2000 Jul 6, 118(4), 105 - 8 Incidence of Gardnerella vaginalis, Candida sp and human papilloma virus in cytological smears; Murta EF et al.; CONTEXT: In spite of the wide-ranging literature on the microbiology of normal and abnormal flora of the vagina, there are few studies on the relationship between human papilloma virus (HPV) and other vaginal microorganisms . OBJECTIVE: To analyze the frequency of infection by human papilloma virus (HPV) and other agents like Candida sp., Gardnerella vaginalis and Trichomonas vaginalis in cytological smears . DESIGN STUDY: Retrospective study SETTING: A public tertiary referral center . SAMPLE: An analysis of 17,391 cytologies from outpatients seen between January 1997 and August 1998 . The control group was made up of patients in the same age group and same period with no cytological evidence of HPV infection . Patients with a diagnosis of cervical intraepithelial neoplasia (CIN) II or III were excluded from this analysis . MAIN MEASUREMENTS: The diagnosis of HPV infection was made in accordance with the criteria of Schneider et al . and the diagnosis of Gardnerella vaginalis was made with a finding of clue cells . RESULTS: 390 (2 . 24%) had alterations consistent with infection by HPV, sometimes associated with CIN I . The results showed that Gardnerella vaginalis was the most frequent agent in women with HPV infection (23.6% versus 17.4%; P <0.05), while in the control group the most frequent agent was Candida sp . (23.9% versus 13.8%; p <0.001) . CONCLUSION: In spite of this study being based solely on cytological criteria, in which specific HPV and Gardnerella diagnostic tests were not used, the cytological smear is widely used in clinical practice and the data presented in this investigation show that there is an association between Gardnerella vaginalis and HPV infection . It remains to be established whether the microorganisms favor each other. Pediatr Dermatol, 2000 May-Jun, 17(3), 169 - 73 Asymmetric periflexural exanthem of childhood: microbiologic case-control study; Coustou D et al.; Our objective was to study possible etiologic factors of asymmetric periflexural exanthem of childhood (APEC) among a large panel of microbiologic agents not yet investigated . To do so, we designed a prospective case-control study using throat, stool, blood, and skin samples, and enlisted 37 children with APEC and 37 age-matched controls without eruption seen consecutively from February 1995 to April 1996 from a mixed referral center and community-based population . No interventions were done . Used as the main outcome measure was the differences in the two groups for microbiologic investigations . No significant statistical differences between cases and controls for virus and bacteria investigated were found . No microorganism was identified as a possible etiologic agent in any of the APEC patients . APEC is not a nonspecific cutaneous eruptive pattern to several common microbiologic agents . More sophisticated molecular approaches are needed to address its etiology. J Nutr Sci Vitaminol (Tokyo), 2000 Apr, 46(2), 97 - 100 Forms of vitamin B12 compounds containing sulfitoB12 in corbiculas; Mizoguchi K et al.; Forms of vitamin B12 (B12) compounds in young and aged corbiculas were examined by bioautography using B12-requiring microorganisms combined with cellulose acetate membrane electrophoresis or high-performane liquid chromatography (HPLC) . Both corbiculas (per 100 g) contained ca . 30 microg of cobalamin, a complete type of B12 . Five known B12 compounds, adenosylB12 (AdoB12), methylB12, hydroxoB12, sulfitoB12 and cyanoB12, were identified by bioautography . Young corbicula contained more B12 compounds, assumed as methylB12, AdoB12 and sulfitoB12, as compared to aged corbicula . All of the B12 compounds detected around the location corresponding to those of methylB12, AdoB12 and sulfitoB12 in the bioautography were converted into compounds that behaved like hydroxoB12 after photolysis . Young corbicula was found to contain an unidentified B12 compound using bioautography combined with HPLC . A large portion of the B12 compound that moved like hydroxoB12 during cellulose acetate membrane electrophoresis using 0.5 N acetic acid might be identical with the unidentified B12 compound detected in the HPLC-bioautography. Microbes Infect, 2000 May, 2(6), 575 - 80 Mycobacterium genavense infection in normal and immunodeficient mice; Matsiota-Bernard P et al.; Mycobacterium genavense is a recently described microorganism causing disseminated infections in AIDS patients . In this study, we investigate its pathogenicity in mice and some mechanisms of the host response to this bacterium . Following an intravenous challenge of 10(6) organisms, M . genavense grew progressively in the spleens and livers of BALB/c and CBA mice over at least an 8-month period . Granulomas were present in the spleens, livers and lungs of the animals . The numbers of bacteria recovered from the spleens and livers were higher in BALB/c (Bcg(s)) than in CBA (Bcg(r)) mice from day 30 . The role of the Bcg gene, in the early phase of infection, was supported by the fact that the bacterial load, on day 15, was higher in BALB/c than in the congenic C.D2 (Bcg(r)) mice . The role of T cells in the host response was suggested by the high susceptibility of nude mice to M . genavense infection . In vivo depletion experiments in CBA mice indicated that gamma interferon and both CD4(+) and CD8(+) T cells participate in the containment of the bacterial load. Infection, 1999, 27(4-5), 283 - 5 Duke criteria-negative endocarditis caused by Bartonella quintana; Simon-Vermont I et al.; Bartonella spp . were recently recognized as causative agents of culture-negative endocarditis . Molecular techniques, such as broad-range PCR, are now available to detect fastidious or slow-growing microorganisms, or those which cannot be cultured . Such microorganisms may be responsible for culture-negative endocarditis . Alcoholism and homelessness are known risk factors for Bartonella quintana endocarditis, even in patients without previous valvular disease . We report a case of B . quintana endocarditis diagnosed by PCR amplification on the excised heart valve, in a young non-alcoholic and immunocompetent patient with few clinical signs of infection who did not fulfill the Duke criteria. Infection, 1999, 27(4-5), 280 - 2 Diagnosis by polymerase chain reaction of pneumonia caused by Legionella pneumophila in an immunocompetent child; Aebischer CC et al.; A 10-year-old, previously healthy child with pneumonia caused by Legionella pneumophila diagnosed by polymerase chain reaction (PCR) of serum is presented . Diagnostic methods were PCR of serum using two different primer sets, and the detection of specific antibody in paired sera using an indirect immunofluorescence assay . Legionella DNA was amplified from serum obtained before and on day 6, but not after completion of a 14-day course of oral clarithromycin.The etiologic role of L . pneumophila was confirmed by seroconversion.The report illustrates that L . pneumophila PCR of serum may contribute to the identification of this microorganism as a cause of severe pneumonia in immunocompetent children. J Theor Biol, 2000 Aug 7, 205(3), 377 - 408 Planktonic contact rates in homogeneous isotropic turbulence: theoretical predictions and kinematic simulations; Lewis DM et al.; The key role played by turbulence in the environment of plankton and larval fish populations has become appreciated in recent years . In particular, the turbulent enhancement of encounter rates between different species of microorganisms, either swimming or passively advected by the flow, is well established . However, most of the current modelling approaches are rather ad hoc, giving rise to ambiguities in the specification of certain key parameters . In this paper, the encounter problem in a turbulent flow of large Reynolds number is re-examined from first principles and a number of new formulae will be established for different swimming strategies . The key innovation is the proposal of a model form for the conditional joint probability density function of predator and prey velocities when the organisms are separated by their given contact radius, R . Particular attention will be paid to the case when a microorganism follows a random trajectory, due to a combination of its own swimming and the action of the flow . The theoretical predictions are subsequently tested against corresponding quantities derived from a series of kinematic simulations of a turbulent-like flow field . Good agreement is demonstrated between the predictions and simulations . Clin Infect Dis, 2000 Jun, 30(6), 982 - 3 Need for diagnostic screening of Herpes simplex virus in patients with nongonococcal urethritis; Madeb R et al.; The prevalence of various microorganisms known to cause nongonococcal urethritis, including herpes simplex virus (HSV), was evaluated . The findings suggest that HSV can be a significant etiological agent in nongonococcal urethritis (NGU) and that the necessary laboratory investigations should be performed for all patients with clinical symptoms of NGU. Immunobiology, 2000 May, 202(1), 42 - 50 Human MO subsets as defined by expression of CD64 and CD16 differ in phagocytic activity and generation of oxygen intermediates; Grage-Griebenow E et al.; Phagocytosis and killing of microorganisms by reactive oxygen radicals are important defence mechanisms of the immune system and it was shown that human monocytes (MO) are heterogeneous in exerting these functions . Previously, we described that human peripheral blood MO consist of a major subset of Fc gamma-receptor-I (CD64)-positive cells exhibiting low accessory cell capacity but high phagocytic activity, and a minor subset of CD64-negative cells with dendritic cell (DC)-like high T cell accessory cell capacity but low phagocytic capacity . Recently, we could show that each subset itself further differs in the expression of the Fc gamma-receptor-III (CD16) and T cell accessory activities resulting in four different subsets: two CD16+ subsets (CD64+ or CD64-) with high T cell stimulation capacity and two CD16- subsets (CD64+ or CD64-) with low accessory activities . In the present study we demonstrate that these subsets also differ in their ability to phagocytose opsonized bacteria (S . aureus and E . coli) and in the generation of reactive oxygen species . Both CD64+ subsets (CD16+ or CD16-) exhibit high phagocytic activity accompanied by intracellular superoxide induction . Luminol-dependent (mainly myeloperoxidase (MPO)-mediated) chemiluminescence (CL) response to latex and FMLP (formylmethionylleucylphenylalanine) was also high in these cell populations . Phagocytic activity and modest CL response was shown in CD64-/CD16+ but not in CD64-/CD16- cells, indicating that each subset except for CD64-/CD16- cells may engulf bacteria and exhibit MPO activity . Taken together, these data demonstrate further heterogeneity of peripheral blood MO in both, phagocytic activity and generation of reactive oxygen species indicating differences between the four subsets in this kind of defence mechanisms against pathogens. Cytometry, 2000 Jul 1, 40(3), 214 - 8 Two and three-color fluorescence flow cytometric analysis of immunoidentified viable bacteria; Barbesti S et al.; BACKGROUND: Traditional culture methods well established in the past and still in use are not able to detect the environmental microorganisms that exist in a viable but not culturable state . A number of different fluorescence-based assays have been developed over the past decade to detect and identify viable bacteria in the environment . METHODS: We have developed a simple and rapid method for measuring the number and viability of immunolabeled bacteria by means of a two/three color fluorescence flow cytometric analysis . After washing, cultured bacteria in suspension were labeled with a rabbit polyclonal antibody recognizing the wall lipopolysaccharide complex . A secondary biotinylated anti-rabbit polyclonal antibody was added allowing the cells to be labeled with the streptavidin R-phycoerythrin-Cyanine 5 (RPE-Cy5) fluorochrome . Before flow cytometric analysis, bacterial suspensions were stained with SYBR Green I and propidium iodide which stain all of the cells and the non viable ones, respectively . RESULTS AND CONCLUSIONS: With the appropriate filter sets of both Bryte-HS (Bio-Rad, Hercules, CA) and FACScan (Becton Dickinson, San Jose, CA) flow cytometers, the measurement of separated green (SYBR Green I), orange-red (propidium iodide), and far red (RPE-Cy5) fluorescence was possible, allowing the enumeration of viable immunodetected bacteria . The entire protocol is completed in less than 3 h, offering numerous possibilities for rapid and precise analyses in sanitary, industrial, and environmental microbiology . Neuroendocrinology, 2000 Jun, 71(6), 375 - 85 Diurnal variations in lipopolysaccharide-induced sleep, sickness behavior and changes in corticosterone levels in the rat; Mathias S et al.; Inoculation of rats with microorganisms or microbial constituents that activate host defense promotes non-rapid eye movement sleep (non-REMS) and suppresses REMS . In this study, we evaluated circadian influences on the effects of lipopolysaccharide (LPS) on sleep, sickness behavior and plasma corticosterone levels in the rat . Three sets of experiments were performed . In each, the animals were intraperitoneally injected with vehicle for LPS (30 microg/kg) during 2 consecutive days, at the beginning of either the circadian rest or the activity phase . In experiment 1, sleep-wake behavior and brain temperature were recorded, and in experiment 2, core body temperature, locomotor activity as well as food and water intake . In experiment 3, corticosterone blood levels were measured . The results show that LPS-evoked changes in temperature, sleep and other behavioral parameters depend markedly on the time of day LPS is administered . However, a direct comparison of the LPS data demonstrates that, except for sleep parameters, the absolute time course of the assessed parameters was rather similar between the rest and activity phases . These findings suggest that LPS evokes a state characterized by high temperature and low vigilance, which is reached independently of the circadian phase . J Clin Microbiol, 2000 Jul, 38(7), 2768 - 71 Fluorescent whole-cell hybridization with 16S rRNA-targeted oligonucleotide probes to identify Brucella spp . by flow cytometry; Fernandez-Lago L et al.; A whole-cell hybridization assay with fluorescent oligonucleotide probes derived from the 16S rRNA sequence of Brucella abortus in combination with flow cytometry has been developed . With the three fluorescent probes selected, a positive signal was observed with all the representative strains of the species and biovars of Brucella and with a total of nine different Brucella clinical isolates . Using the B9 probe in the hybridization assay, it was possible to discriminate between Brucella suis biovars 2, 3, 4, and 5 and almost all the other Brucella spp . On the basis of differences in fluorescence intensities, no discrimination was established between Brucella spp . and other phylogenetically related microorganisms . No positive fluorescence signals were detected with any of the bacteria showing serological cross-reactions with Brucella spp . and with a total of 17 clinical isolates not belonging to the genus Brucella . These results suggest that the 16S rRNA whole-cell hybridization technique could be a valuable diagnostic tool for the detection and identification of Brucella spp. Appl Environ Microbiol, 2000 Jul, 66(7), 3088 - 92 Naphthalene and donor cell density influence field conjugation of naphthalene catabolism plasmids; Hohnstock AM et al.; We examined transfer of naphthalene-catabolic genes from donor microorganisms native to a contaminated site to site-derived, rifampin-resistant recipient bacteria unable to grow on naphthalene . Horizontal gene transfer (HGT) was demonstrated in filter matings using groundwater microorganisms as donors . Two distinct but similar plasmid types, closely related to pDTG1, were retrieved . In laboratory-incubated sediment matings, the addition of naphthalene stimulated HGT . However, recipient bacteria deployed in recoverable vessels in the field site (in situ) did not retrieve plasmids from native donors . Only when plasmid-containing donor cells and naphthalene were added to the in situ mating experiments did HGT occur. J Dairy Sci, 2000 Jun, 83(6), 1310 - 21 Synergy between ruminal fibrolytic enzymes and enzymes from Trichoderma longibrachiatum; Morgavi DP et al.; The mechanism by which enzyme additives improve feed digestion in ruminants is not fully understood . Direct hydrolysis of feed in the rumen is a potential mode of action, but the importance of this mode needs to be quantified because of the relatively low exogenous hydrolase activity added compared with the total activity present in the rumen . We examined the interactions between ruminal and exogenous enzymes on fiber degradation using a completely randomized experimental design, with an 11 (enzyme preparations and their combinations) x 5 (assay pH) arrangement of treatments . Ruminal enzymes were extracted from cattle receiving high fiber or high concentrate diets and exogenous enzymes were Trichoderma longibrachiatum preparations containing different proportions of xylanase and cellulase activities . Ruminal and exogenous enzyme preparations and their combinations were tested for the ability to degrade soluble cellulose, xylan, and corn silage over a range of pH from 4.5 to 6.5 at 39 degrees C . T . longibrachiatum enzymes acted synergistically with enzymes from mixed rumen microorganisms in degrading soluble cellulose, xylan, and corn silage . Hydrolysis increased by up to 35, 100, and 40% for soluble cellulose, xylan, and corn silage, respectively, and was most evident at a pH range between 5.0 and 6.0 . The synergistic effect between ruminal and exogenous enzymes increases the hydrolytic potential within the rumen environment and is likely a significant mechanism by which enzyme additives improve feed digestion. J Eur Acad Dermatol Venereol, 2000 Jan, 14(1), 61 - 3 Extensive and deep dermatophytosis caused by Trichophyton mentagrophytes var . interdigitalis in an HIV-1 positive patient; Munoz-Perez MA et al.; BACKGROUND: Cutaneous infections are common in HIV-1 positive patients and are usually severe, recurrent, and caused by microorganisms that are unusual in immunocompetent patients . OBJECTIVE: We report a case of an HIV-1-positive 23-year-old male, with a history of intravenous drug use, in stage C-II (CDC '86), with a CD4 lymphocyte count of 335 cells/mm3 . He had multiple, large erythematous, circinate and pustular plaques on his abdomen, back, arms and legs . RESULTS: We isolated Trichophyton mentagrophytes var . interdigitalis from the lesions . The biopsy showed suppurative deep dermatophytosis and folliculitis . The patient satisfactorily responded to itraconazole (100 mg/d for 14 days) . CONCLUSION: This is the first reported case of deep dermatophytosis caused by T . mentagrophytes in an HIV-positive patient. Zh Mikrobiol Epidemiol Immunobiol, 2000 Jan-Feb, (1), 103 - 6 {The ecological determinacy of the intraspecies diversity of pathogenic bacteria}; Bukharin OV et al.; The review deals with some theoretical and applied problems of the intraspecific diversity of pathogenic microorganisms . Special attention is given to the role of ecological factors in the formation of the phenotypic polymorphism of bacteria . The possibility of using the methods of mathematical analysis for the evaluation of the influence of the environment (ecotopes) on individual phenotypic characteristics of bacteria and their complex (bioprofile) is shown . The proposition on the bioprofile of microorganisms as a criterion of their ecological (ecotopic) marking is substantiated . The examples of using the data of the ecological marking of pathogenic bacteria in clinical epidemiology and ecological practice are presented. Zh Mikrobiol Epidemiol Immunobiol, 2000 Jan-Feb, (1), 60 - 3 {The effect of immunotherapy with the VP-4 multicomponent vaccine in children with frequent acute respiratory diseases and obstructive bronchitis}; Obol'skaia NM; The results of prolonged observations on children with frequent acute respiratory diseases (ARD), subject to immunoprophylaxis with the use of polycomponent vaccine (VP-4), prepared from the antigens of opportunistic microorganisms, are presented . The vaccine was introduced to 30 children in 3 intranasal administrations and 6-8 oral administrations . The morbidity rate of the children was registered and their clinical status was evaluated for a year after the introduction of vaccine VP-4 . As revealed in these observations, the frequency of ARD cases among the immunized children decreased 3 times or more in comparison with that among the same children, registered during a year prior to the introduction of the vaccine . In addition to a decrease in the frequency of ARD cases, a decrease in their duration and in the number of antibiotic administrations, as well as in the necessity of hospitalization, were also registered. Mol Gen Mikrobiol Virusol, 2000, (2), 37 - 40 {Mobilization of a cryptic plasmid from the melioidosis pathogen in heterologous species of microorganisms}; Merinova LK et al.; Cryptic plasmids with different molecular weights have been detected in B . pseudomallei strains . Mobilization of B . pseudomallei plasmid DNA in heterologous B . mallei species was performed by the conjugate plasmid RP1::Tn10 . Possibility of detecting phenotypical characteristics of plasmids and behavior of B . pseudomallei non-chromosomal replicons in B . mallei have been determined. ANNA J, 1999 Dec, 26(6), 591 - 6 Does iron cause bacterial infections in patients with end stage renal disease? Cieri E. Anemia management using erythropoietin and intravenous iron supplementation has improved the lives of many patients with end stage renal disease (ESRD) . However, because iron is an essential nutrient for microorganisms, it is plausible that iron supplementation may promote infection . This review examines the literature on the connection between iron and infection, with a focus on the relevance of these data to hemodialysis patients treated according to the National Kidney Foundation--Dialysis Outcomes Quality Initiative (NKF-DOQI) Guidelines for Anemia Management . The current evidence does not show a cause-and-effect relationship between intravenous iron administration and an increased susceptibility to infection in hemodialysis patients . Therefore, the author does not recommend changing current iron management practices in ESRD patients because of concern about infectious risk. Microb Ecol, 2000 May, 39(4), 290 - 300 Horizontal Distribution Patterns of Testate Amoebae (Protozoa) in a Sphagnum magellanicum Carpet; Mitchell EA et al.; The distribution of soil microorganisms is generally believed to be patchy and to reflect habitat heterogeneity . Despite this general rule, the amount of existing data on species distribution patterns is scarce . Testate amoebae (Protozoa; Rhizopoda) are an important component of soil microbial communities and are increasingly used in ecological and paleoecological studies of Sphagnum-dominated peatlands, but data on the spatial structure of communities are completely lacking . This is an important aspect since quantitative models used for paleoecological reconstruction and monitoring are based on species assemblages . We explored the distribution patterns of testate amoebae distribution in a macroscopically homogeneous Sphagnum carpet, down to a scale of several centimeters . Distributions maps of the species and spatially constrained sample groups were produced . Multivariate and individual spatial autocorrelations were calculated . The importance of spatial structure was quantified by canonical correspondence analysis . Our ultimate goal is to find the finest resolution of environmental monitoring using testate amoebae . The distribution patterns differed among species, resulting in a complex spatial structure of the species assemblage in a whole . Spatial structure accounted for 36% of the total variation of species abundance in a canonical correspondence analysis constrained by spatial variables . This structure was partly correlated to altitude (microtopography) at a very fine scale . These results confirmed the existence of significant broad- and fine-scale spatial structures within testate amoebae communities that could in part be interpreted as effects of ecological gradients . This shows that, on a surface area of 0.25 m(2), ecological conditions which look uniform from a macroscopic point of view are not perceived as such by Sphagnum-inhabiting organisms . Therefore, testate amoebae could prove very useful to monitor fine-scale ecological processes or disturbances . Studies of the species' spatial distribution patterns in combination with autoecological studies are needed and should be included in the toolbox of biomonitoring itself. Poult Sci, 2000 Jun, 79(6), 838 - 42 Heat lability of five strains of infectious bursal disease virus; Mandeville WF et al.; An extensive world trade environment has created a need for many nations to protect their food animal industries against the importation of pathogenic microorganisms . We initiated studies to determine how cooking conditions would affect the viability of different infectious bursal disease virus (IBDV) strains . Five different viral strains were tested . The strains included two serotype 1 variant viruses, Del-A and MD; two serotype 1 classic viruses, STC and D78; and a serotype 2 virus, OH . A 100-microL aliquot of each viral stain in Dulbecco's Modified Eagle Medium with 2% fetal calf serum was heated to 37,65, 71, 74,77, 82, or 100 C for 1 min . Following heat treatment, the virus titers were determined in a BGM-70 cell culture . Virus titers declined following each incubation, and there were no appreciable differences among the five viral strains tested . The greatest decline in titer occurred at temperatures above 65 C . After 1 min at 65, 71, and 100 C, reduction of viable viruses were > or =90, > or =99, and >99.9%, respectively, compared to nonheated controls . A viral reduction curve similar to the first heating trial was observed when viruses were heated at 71 or 74 C for 6 min . Drumsticks and boneless chicken patties were seeded with the virus at a mean value of 10(5.5) TCID50/25 microL of IBDV and were cooked to internal temperatures of 71 and 74 C, respectively . All samples were quickly cooled after achieving target temperatures . Viable virus was recovered from both products following cooking. Ann Fr Anesth Reanim, 2000 May, 19(5), 395 - 402 {Consequences of antibiotic therapy to the intestinal ecosystem}; Andremont A; Ecological impact of antibiotherapy results from the interaction between microorganisms in the ecosystems and antibiotics at which they are exposed . The amount of antibiotics use in the world is continuously increasing . The fraction devoted to human care is only about half the total amount . There are multiple other fields of usage, in agriculture, breeding and veterinary medicine . Bacterial ecosystems exposed at antibiotherapy in man are mainly the skin and the gastrointestinal and respiratory tracts . The gastrointestinal system is quantitatively predominant and the consequences of the bacterial imbalance induced by antibiotics are potentially severe . It is the reason why it is the most extensively studied, in the literature and in the present review . The origin of resistant bacteria will be briefly discussed. Methods, 2000 Jul, 21(3), 241 - 7 Alveolar macrophage-environmental particle interaction: analysis by flow cytometry; Palecanda A et al.; Inhaled particulates such as pollutant particles, allergens, and microorganisms are rapidly cleared by alveolar macrophages (AMs) . Methods for analysis of AM-particle interaction have been hindered by the lack of a convenient assay . Flow cytometry offers rapid, sensitive, and reproducible measurements of single cells in suspension . Multiple parameters can be measured in real time . Here we will review the application of flow cytometry to the study and characterization of AM receptors for unopsonized environmental particles . We will discuss the role of this technique in identifying a key AM receptor system involved in lung defense . Multiparametric flow cytometry to analyze intracellular functional parameters, though a powerful and unique tool, needs to be interpreted with caution . We will also discuss the advantages and limitations of flow cytometry in analysis of AM-particle interaction . Int J STD AIDS, 2000 Jun, 11(6), 356 - 60 The association of Mycoplasma hominis, Ureaplasma urealyticum and Mycoplasma genitalium with bacterial vaginosis: observations on heterosexual women and their male partners; Keane FE et al.; The prevalence of 3 mycoplasmas (Mycoplasma hominis, Ureaplasma urealyticum and Mycoplasma genitalium) was determined in a cohort of women with or without bacterial vaginosis (BV) and in their respective male partners . Heterosexual women with or without BV and their male partners were recruited and genital sampling for these microorganisms was performed . Seventeen women with BV and 21 women with normal flora, and their respective male partners, were recruited . M . hominis was present in 9 (53%) of 17 women with BV compared with none of 21 women without BV (P=0.0001) . Of the 17 male partners of women with BV, 8 (47%) had M . hominis compared to 5 (24%) of 21 male partners of women without BV (not significant {n/s}) . U . urealyticum was detected in 11 (65%) of 17 women with BV in comparison with 10 (48%) of 21 women without BV (n/s) . U . urealyticum was present in 4 (24%) of 17 male partners of women with BV compared to 6 (29%) of 21 male partners of women without BV (n/s) . M . genitalium was not detected in any of 15 women with BV and in only 2 (12%) of 17 women without BV (n/s) . M . genitalium was present in 4 (25%) male partners of 16 women with BV in comparison with 3 (16%) male partners of 19 women without BV (n/s) . Thus, M . hominis was the only mycoplasma detected significantly more often in women with, rather than in those without, BV . None of the mycoplasmas was found significantly more often in male partners of women with, rather than those without, BV . Overall, M . genitalium behaved somewhat similar to Chlamydia trachomatis . It was the least commonly occurring mycoplasma, a reflection perhaps of the relatively low incidence of partner change in this study population. Cell Mol Biol (Noisy-le-grand), 2000 May, 46(3), 637 - 47 Iron, metalloenzymes and cytotoxic reactions; Nappi AJ et al.; There is considerable evidence implicating iron and other redox-active transition metals as progenitors of reactive intermediates of oxygen (ROI), molecules which lead to oxidative stress and contribute to various neurodegenerative processes . An important aspect of such metal-mediated damage to biomolecules is the site-specific nature of such pathological activity . Iron sequestering molecules, such as ferritin, transferrin, lactotransferrin, melanotransferrin, hemosiderin and heme can serve as cytoprotectants against metal-mediated oxidant damage . Metalloenzymes also constitute an important group of iron sequestering molecules . Metalloenzyme-catalyzed reactions in which metal ions at the enzyme active site undergo redox-cycling in association with O2 are site-specific in nature, and may represent a potential source of ROI-mediated damage to biomolecules . Dysregulation of brain iron and alterations in the levels of metalloenzymes involved in reactions with O2 derived molecules can contribute to neuronal damage . Iron may increase the cytotoxicity of neuronal dopamine by increasing its rate of oxidation to quinones and semiquinones, thereby reducing the level of this neurotransmitter . Interestingly, dopamine also may play an important role in the maintenance of transition-metal homeostasis as an iron chelator, since it can form both catecholate and hydroxamate groups, molecules employed by many microorganisms to sequester iron. Mikrobiol Z, 2000 Mar-Apr, 62(2), 51 - 68 {Associative nitrogen-fixing microorganisms}; Volkogon VV; State of the study of associative nitrogen-fixing microorganisms has been evaluated . The paper also deals with the problem of spatial and functional interrelations of diasotrophs with the higher plants, formation peculiarities of nitrogen-fixing associations under inoculation of annual and perennial plants. Can J Microbiol, 2000 May, 46(5), 425 - 32 The potential of soil microorganisms to mineralize atrazine as predicted by MCH-PCR followed by nested PCR; Shapir N et al.; The potential of soil microorganisms to mineralize atrazine was studied in soil samples collected from fields with various histories of atrazine application . In contrast to many previous studies, which showed no atrazine mineralization activity, all the tested soils mineralized atrazine regardless of their atrazine application history . However, the delay before mineralization and the variation in the subsequent mineralization rate were in agreement with the initial copy number of the atrazine dechlorinaze gene, and the proliferation rate of the degraders . Soils from corn fields, which had up to 100 copies of the atzA gene per gram of soil, had a lag period of 4-5 days before atrazine mineralization started, and final mineralization percentages ranged from 40% to 54% . However, soils from fields that were never amended with atrazine had much longer lag periods (more than 17 days), which decreased after enrichment of the degrader population with high concentrations of atrazine for 15 days . Generally the mineralization rate and the atzA gene copy number increased after the enrichment period . The atrazine mineralization potential was measured by PCR of genes from the atrazine mineralization pathway . Magnetic capture hybridization was the most efficient of the two tested methods for purifying target DNA of PCR inhibitors, without reducing the copy number of the required fragment . Nested PCR proved to be the most effective method for predicting the exact potential of the soil to mineralize the pollutant even without enrichment of a small population with the target genes . This method can complement microcosm studies and eliminate futile efforts when the potential to mineralize the pollutant does not exist in the soil. Trends Plant Sci, 2000 Jul, 5(7), 304 - 8 Are microorganisms more effective than plants at competing for nitrogen? Hodge A, Robinson D, Fitter A. Plant scientists have long debated whether plants or microorganisms are the superior competitor for nitrogen in terrestrial ecosystems . Microorganisms have traditionally been viewed as the victors but recent evidence that plants can take up organic nitrogen compounds intact and can successfully acquire N from organic patches in soil raises the question anew . We argue that the key determinants of 'success' in nitrogen competition are spatial differences in nitrogen availability and in root and microbial distributions, together with temporal differences in microbial and root turnover . Consequently, it is not possible to discuss plant-microorganism competition without taking into account this spatiotemporal context. Aust N Z J Obstet Gynaecol, 2000 Feb, 40(1), 44 - 7 Single dose oral azithromycin versus seven day doxycycline in the treatment of non-gonococcal mucopurulent endocervicitis; Sendag F et al.; The aim of this study was to compare single dose oral azithromycin versus seven-day doxycycline in the treatment of non-gonococcal mucopurulent cervicitis (MPC) . One hundred and thirty-one women with non-gonococcal MPC were enrolled in a prospective-randomised study to compare the efficacy and safety of a single oral dose of 1 g azithromycin and a seven-day course of 100 mg doxycycline twice daily . Clinical examination and culture samples for Chlamydia trachomatis and other microorganisms were performed before and approximately 14 days after starting the treatment . Of the 131 women recruited (67 in the azithromycin group and 64 in the doxycycline group), Ureaplasma urealyticum was isolated from 21 (16%); Chlamydia trachomatis from 15 (11.5%); and Mycoplasma hominis from 3 (2.3%) of the patients at the initial examination . The eradication rate of baseline culture-positive cases at the follow-up visit in the azithromycin group was 71.4%, and 77.3% in the doxycycline group . There was no statistically significant difference in efficacy between the single dose azithromycin and seven-day course of doxycycline in the treatment of culture-positive cases . Azithromycin 1 g appears to be an effective and safe alternative to doxycycline for the treatment of non-gonococcal MPC. Res Microbiol, 2000 Apr, 151(3), 179 - 82 Do bacterial cryptic genes really exist? Tamburini E, Mastromei G. Cryptic genes have been defined as phenotypically silent DNA sequences, usually not expressed during the life cycle of a microorganism, but capable of expression in a few members of a large population by mutation, recombination, insertion processes, or other genetic mechanisms . Recently, the crypticity of several genetic systems has been questioned . It appears that in many cases cryptic genes are silent only under the experimental conditions analysed and that their expression can be induced in the natural environment . Therefore, we propose that cryptic genes might not be a peculiar class of uniquely regulated genes, but rather genes encoding unusual functions. Med Dosw Mikrobiol, 1999, 51(1-2), 37 - 46 {Evaluation of interactions between strains of S . aureus isolated from different clinical specimens with peripheral blood phagocytic cells}; Baran J et al.; The aim of this study was to investigate the interactions occurring between peripheral blood phagocytes and strains of S . aureus isolated from different clinical specimens (blood, respiratory tract, pus) . To evaluate the sensitivity of microorganisms to bactericidal activity of phagocytes, monocytes and granulocytes separated from peripheral blood by standard density gradient and by counter-current centrifugal elutriation were incubated with suspensions of opsonized bacteria . In parallel, the viability of phagocytes was examined by flow cytometry, and the ability of bacteria to trigger reactive oxygen intermediates (ROI) production was evaluated by chemiluminescence measurement . To investigate efficiency of phagocytosis, bacteria were labelled with fluorescein isothiocyanate (FITC) and the percentage of cells containing FITC-labelled bacteria was analysed by flow cytometry . The data obtained show that strains of S . aureus originated from different clinical specimens, differ in their sensitivity to bactericidal activity of phagocytes--strains isolated from the blood show the highest, but strains isolated from respiratory tract show the lowest sensitivity for killing . These strains differ too in their ability to trigger monocyte CL response . Contrary, there was no difference in toxicity of bacteria against phagocytes . Strains isolated from peripheral blood showed significant negative correlation between the ability to trigger CL response and toxicity against phagocytes. Science, 2000 Jun 23, 288(5474), 2222 - 6 A primitive T cell-independent mechanism of intestinal mucosal IgA responses to commensal bacteria; Macpherson AJ et al.; The immunoglobulin A (IgA) is produced to defend mucosal surfaces from environmental organisms, but host defenses against the very heavy load of intestinal commensal microorganisms are poorly understood . The IgA against intestinal commensal bacterial antigens was analyzed; it was not simply "natural antibody" but was specifically induced and responded to antigenic changes within an established gut flora . In contrast to IgA responses against exotoxins, a significant proportion of this specific anti-commensal IgA induction was through a pathway that was independent of T cell help and of follicular lymphoid tissue organization, which may reflect an evolutionarily primitive form of specific immune defense. J Indian Soc Pedod Prev Dent, 1999 Sep, 17(3), 73 - 89 Relationship between the existing caries status, plaque S . mutans and Cariostat caries activity test in children; Munshi AK et al.; An attempt was made in this study to find out the sensitivity and specificity of a caries activity test, CARIOSTAT and its relationship to the existing caries status and the plaque S . mutans level . The test proved to be highly sensitive and specific with significant relationship to the S.mutans count in the dental plaque . There also was a significant relationship between both the cultured microorganisms on MSB agar and the plaque in the Cariostat medium. Ann Periodontol, 1999 Dec, 4(1), 74 - 8 Necrotizing ulcerative periodontitis; Novak MJ; In patients with no known systemic disease or immune dysfunction, necrotizing periodontitis (NUP) appears to share many of the clinical and etiologic characteristics of necrotizing ulcerative gingivitis (NUG) except that patients with NUP demonstrate loss of clinical attachment and alveolar bone at affected sites . In these patients, NUP may be a sequela of a single or multiple episodes of NUG or may be the result of the occurrence of necrotizing disease at a previously periodontitis-affected site . The existence of immune dysfunction may predispose patients to NUG and NUP, especially when associated with an infection of microorganisms frequently associated with periodontal disease such as Treponema and Selenomonas species, Fuscobacterium nucleatum, Prevotella intermedia, and Porphyromonas gingivalis . The role of immune dysfunction is exemplified by the occasionally aggressive nature of necrotic forms of periodontal disease seen in patients with HIV infection or malnutrition, both of which may impact host defenses . Clinical studies of HIV-infected patients have shown that patients with NUP are 20.8 times more likely to have CD4+ cell counts below 200 cells/mm3 . However, these same studies have demonstrated that most patients with CD4+ cell counts below 200 cells/mm do not have NUP, suggesting that other factors, in addition to immunocompromisation, are involved . Further studies are needed to define the complex interactions between the microbial, or viral, etiology of necrotic lesions and the immunocompromised host . It is, therefore, recommended that NUG and NUP be classified together under the grouping of necrotizing periodontal diseases based on their clinical characteristics. Enzyme Microb Technol, 2000 Jul 1, 27(1-2), 114 - 121 A bifunctional beta-xylosidase-xylose isomerase from Streptomyces sp . EC 10; Belfaquih N et al.; beta-Xylosidase (1,4-beta-D-xylan xylohydrolase EC 3.2.1.37) and xylose isomerase (D-xylose ketol-isomerase EC 5.3.1.5) produced by Streptomyces sp . strain EC 10, were cell-bound enzymes induced by xylan, straw, and xylose . Enzyme production was subjected to a form of carbon catabolite repression by glycerol . beta-Xylosidase and xylose isomerase copurified strictly, and the preparation was found homogeneous by gel electrophoresis after successive chromatography on DEAE-Sephacel and gel filtration on Biogel A . Streptomyces sp . produced apparently a bifunctional beta-xylosidase-xylose isomerase enzyme . The molecular weight of the enzyme was measured to be 163,000 by gel filtration and 42,000 by SDS-PAGE, indicating that the enzyme behaved as a tetramer of identical subunits . The Streptomyces sp . beta-xylosidase was a typical glycosidase acting as an exoenzyme on xylooligosaccharides, and working optimally at pH 7.5 and 45 degrees C . The xylose isomerase optimal temperature was 70 degrees C and maximal activity was observed in a broad range pH (5-8) . Enhanced saccharification of arabinoxylan caused by the addition of the enzyme to endoxylanase suggested a cooperative enzyme action . The first 35 amino acids of the N-terminal sequence of the enzyme showed strong analogies with N-terminal sequences of xylose isomerase produced by other microorganisms but not with other published N-terminal sequences of beta-xylosidases. J Mol Biol, 2000 Jun 2, 299(2), 463 - 76 The 1.5 A resolution crystal structure of the carbamate kinase-like carbamoyl phosphate synthetase from the hyperthermophilic Archaeon pyrococcus furiosus, bound to ADP, confirms that this thermostable enzyme is a carbamate kinase, and provides insight into substrate binding and stability in carbamate kinases; Ramon-Maiques S et al.; Carbamoyl phosphate (CP), an essential precursor of arginine and the pyrimidine bases, is synthesized by CP synthetase (CPS) in three steps . The last step, the phosphorylation of carbamate, is also catalyzed by carbamate kinase (CK), an enzyme used by microorganisms to produce ATP from ADP and CP . Although the recently determined structures of CPS and CK show no obvious mutual similarities, a CK-like CPS reported in hyperthermophilic archaea was postulated to be a missing link in the evolution of CP biosynthesis . The 1.5 A resolution structure of this enzyme from Pyrococcus furiosus shows both a subunit topology and a homodimeric molecular organization, with a 16-stranded open beta-sheet core surrounded by alpha-helices, similar to those in CK . However, the pyrococcal enzyme exhibits many solvent-accessible ion-pairs, an extensive, strongly hydrophobic, intersubunit surface, and presents a bound ADP molecule, which does not dissociate at 22 degrees C from the enzyme . The ADP nucleotide is sequestered in a ridge formed over the C-edge of the core sheet, at the bottom of a large cavity, with the purine ring enclosed in a pocket specific for adenine . Overall, the enzyme structure is ill-suited for catalyzing the characteristic three-step reaction of CPS and supports the view that the CK-like CPS is in fact a highly thermostable and very slow (at 37 degrees C) CK that, in the extreme environment of P . furiosus, may have the new function of making, rather than using, CP . The thermostability of the enzyme may result from the extension of the hydrophobic intersubunit contacts and from the large number of exposed ion-pairs, some of which form ion-pair networks across several secondary structure elements in each enzyme subunit . The structure provides the first information on substrate binding and catalysis in CKs, and suggests that the slow rate at 37 degrees C is possibly a consequence of slow product dissociation . J Can Dent Assoc . 2000 May;66(5):262. An evaluation of sampling and laboratory procedures for determination of heterotrophic plate counts in dental unit waterlines; Noce L et al.; BACKGROUND: The high numbers of heterotrophic microorganisms that have been cultured from dental unit waterlines (DUWs) have raised concern that this water may exceed suggested limits for heterotrophic plate counts (HPCs) . The main purpose of this investigation was to examine HPC variability in DUWs and to examine in detail the effect of laboratory processing of water samples on HPC values . METHODS: Water samples were collected from dental offices either at the beginning of or during the clinic day and were transported to the laboratory, where they were analyzed . RESULTS: Measuring HPC levels within an office would involve testing all units, because significant differences were found between units connected to the same municipal water supply . Within a unit, the average microbial count from high-speed lines was approximately twice the average count from air/water lines . The laboratory processing of water samples significantly affected the numbers of heterotrophic microorganisms that were recovered . Incubation temperature, time and media, as well as neutralization of residual chlorine, all had significant effects on the HPC values . However, no significant differences in microbial counts were found between samples plated with the spread plate method on R2A agar and those plated with the pour plate method with Plate Count Agar . CONCLUSIONS: Dental organizations have suggested target limits in terms of numbers of heterotrophic microorganisms recovered in water from dental units, but standards for laboratory handling must be established as well . A protocol for sample collection and laboratory handling is proposed. Q Rev Biol, 2000 Jun, 75(2), 113 - 48 Morning sickness: a mechanism for protecting mother and embryo; Flaxman SM et al.; Approximately two-thirds of women experience nausea or vomiting during the first trimester of pregnancy . These symptoms are commonly known as morning sickness . Hook (1976) and Profet (1988) hypothesized that morning sickness protects the embryo by causing pregnant women to physically expel and subsequently avoid foods that contain teratogenic and abortifacient chemicals, especially toxic chemicals in strong-tasting vegetables, caffeinated beverages and alcohol . We examined this hypothesis by comprehensively reviewing the relevant medical, psychological and anthropological literature . In its support, (i) symptoms peak when embryonic organogenesis is most susceptible to chemical disruption (weeks 6-18), (ii) women who experience morning sickness are significantly less likely to miscarry than women who do not (9 of 9 studies), (iii) women who vomit suffer fewer miscarriages than those who experience nausea alone, and (iv) many pregnant women have aversions to alcoholic and nonalcoholic (mostly caffeinated) beverages and strong-tasting vegetables, especially during the first trimester . Surprisingly, however, the greatest aversions are to meats, fish, poultry, and eggs . A cross-cultural analysis using the Human Relations Area Files revealed 20 traditional societies in which morning sickness has been observed and seven in which it has never been observed . The latter were significantly less likely to have animal products as dietary staples and significantly more likely to have only plants (primarily corn) as staples than the 20 societies in which morning sickness occurred . Animal products may be dangerous to pregnant women and their embryos because they often contain parasites and pathogens, especially when stored at room temperatures in warm climates . Avoiding foodborne microorganisms is particularly important to pregnant women because they are immunosuppressed, presumably to reduce the chances of rejecting tissues of their own offspring (Haig 1993) . As a result, pregnant women are more vulnerable to serious, often deadly infections . We hypothesize that morning sickness causes women to avoid foods that might be dangerous to themselves or their embryos, especially foods that, prior to widespread refrigeration, were likely to be heavily laden with microorganisms and their toxins . The alternative hypotheses that morning sickness is (i) an epiphenomenon of mother-offspring genetic conflict or hormones associated with viable pregnancies, or (ii) an indicator to potential sexual partners and kin that the woman is pregnant, resulting in reduced sexual behavior and increased nepotistic aid, were not well supported . Available data are most consistent with the hypothesis that morning sickness serves an adaptive, prophylactic function. Infect Immun, 2000 Jul, 68(7), 4255 - 63 Intracellular trafficking of Brucella abortus in J774 macrophages; Arenas GN et al.; Brucella abortus is a facultative intracellular bacterium capable of surviving inside professional and nonprofessional phagocytes . The microorganism remains in membrane-bound compartments that in several cell types resemble modified endoplasmic reticulum structures . To monitor the intracellular transport of B . abortus in macrophages, the kinetics of fusion of phagosomes with preformed lysosomes labeled with colloidal gold particles was observed by electron microscopy . The results indicated that phagosomes containing live B . abortus were reluctant to fuse with lysosomes . Furthermore, newly endocytosed material was not incorporated into these phagosomes . These observations indicate that the bacteria strongly affect the normal maturation process of macrophage phagosomes . However, after overnight incubation, a significant percentage of the microorganisms were found in large phagosomes containing gold particles, resembling phagolysosomes . Most of the Brucella bacteria present in phagolysosomes were not morphologically altered, suggesting that they can also resist the harsh conditions prevalent in this compartment . About 50% colocalization of B . abortus with LysoSensor, a weak base that accumulates in acidic compartments, was observed, indicating that the B . abortus bacteria do not prevent phagosome acidification . In contrast to what has been described for HeLa cells, only a minor percentage of the microorganisms were found in compartments labeled with monodansylcadaverine, a marker for autophagosomes, and with DiOC6 (3,3'-dihexyloxacarbocyanine iodide), a marker for the endoplasmic reticulum . These results indicate that B . abortus bacteria alter phagosome maturation in macrophages . However, acidification does occur in these phagosomes, and some of them can eventually mature to phagolysosomes. Infect Immun, 2000 Jul, 68(7), 3815 - 21 Coxiella burnetii exhibits morphological change and delays phagolysosomal fusion after internalization by J774A.1 cells; Howe D et al.; Coxiella burnetii, the etiological agent of Q fever, is an obligate intracellular bacterium proliferating within the harsh environment of the phagolysosome . Mechanisms controlling trafficking to, and survival of pathogens within, the phagolysosome are unknown . Two distinct morphological variants have been implicated as playing a role in C . burnetii survival . The dormant small-cell variant (SCV) is resistant to extracellular stresses and the more metabolically active large-cell variant (LCV) is sensitive to environmental stresses . To document changes in the ratio of SCVs to LCVs in response to environment, a protein specific to SCV, ScvA, was quantitated . During the first 2 h after internalization of C . burnetii by J774A.1 cells, the level of ScvA decreased, indicating a change from a population containing primarily SCVs to one containing primarily LCVs . In vitro experiments showed that 2 h of incubation at pH 5.5 caused a significant decrease in ScvA in contrast to incubation at pH 4.5 . Measuring in vitro internalization of {(35)S}methionine-{(35)S}cysteine in response to pH, we found the uptake to be optimal at pH 5.5 . To explore the possibility that after uptake C . burnetii was able to delay phagolysosomal fusion, we used thorium dioxide and acid phosphatase to label phagolysosomes during infection of J774A.1 cells . We determined that viable C . burnetii was able to delay phagolysosomal fusion . This is the first time that a delay in phagolysosomal fusion has been shown to be a part of the infection process of this pathogenic microorganism. Ophthalmology, 2000 Jun, 107(6), 1047 - 51; discussion 1051-2 Improved detection of microorganisms by polymerase chain reaction in delayed endophthalmitis after cataract surgery; Lohmann CP et al.; OBJECTIVE: To evaluate whether the use of polymerase chain reaction (PCR) improves the identification of the causative pathogen in eyes developing delayed endophthalmitis after cataract surgery . DESIGN: Prospective, noncomparative case series . PARTICIPANTS: Consecutive series of 25 eyes with the clinical diagnosis of delayed endophthalmitis after cataract . MAIN OUTCOME MEASURE: Presence of bacterial or fungal DNA in aqueous humor and vitreous samples . RESULTS: In the aqueous humor the causative pathogen was identified in 84% (n = 21) of the eyes by PCR compared with 0% by diagnostic culture and 0% by microscopy . In the vitreous samples the pathogen was identified in 92% (n = 23) of the eyes by PCR compared with 24% by diagnostic culture (n = 6) and 0% by microscopy . CONCLUSIONS: PCR is useful for the identification of the causative pathogen in delayed endophthalmitis and had a higher rate of positive identification of the causative organism than microscopy or diagnostic culture. Anal Chem, 2000 Jun 1, 72(11), 2475 - 81 Characterization of microorganisms and biomarker development from global ESI-MS/MS analyses of cell lysates; Xiang F et al.; The capability for sensitive and accurate identification of microorganisms has potential applications that include the monitoring of industrial bioprocessing operations, food safety analyses, disease diagnosis, and detection of potential biological hazards . Efforts based upon matrix-assisted laser desorption/ionization mass spectrometry to detect and identify specific microorganisms have been actively pursued for several years . We report a new method being developed to select useful biomarkers for the identification of microorganisms based upon electrospray ionization (ESI)-ion trap mass spectrometry . Crude cell lysates are processed using a recently developed dualmicrodialysis device and then directly infused into an ion trap MS . The low ESI flow rate and precursor ion accumulation capability of the ion trap MS enables high-sensitivity MS/MS analyses . Precursor ions are automatically selected and analyzed using tandem MS (MS/MS) to produce "global" MS/MS surveys and processed to yield two-dimensional MS/MS spectral displays . Such global MS/MS surveys are demonstrated for Escherichia coli lysates . The distinctive MS/MS spectral patterns can be used to identify mass spectrometric-detected species useful as biomarkers, which then provide a basis for confident microorganism identification . The results presented demonstrate the application of this method for the identification of microorganisms, as well as for detection of bacteriophage MS2 in the presence of a large excess of E . coli. Anal Chem, 2000 Jun 1, 72(11), 2414 - 7 Determination of motility forces of bovine sperm cells using an "optical funnel"; Kaneta T et al.; An optical funnel, a new technique for the evaluation of the force of a microorganism, was applied to the determination of the motility force of bovine sperm cells . In this approach, sperm cells, suspended in an aqueous solution, are introduced into a flow cell, to which radiation pressure is applied from the direction opposite to a medium flow . The sperm cell, which is moving in a stream, is captured by radiation pressure and forced to move to the position at which the force induced by the laser radiation is equal to the force induced by a medium flow . The sperm cell then escapes by its own power on the way to this equilibrium (entrapping) position . The radiation force increases with decreasing distance from the focal point, and as a result, the force of the sperm cell can be determined by measuring the position where the sperm cell escaped against the laser irradiation field . The motility force of the sperm cell was measured in aqueous solution at different pH values and potassium ion concentrations . It was possible to measure more than 250 sperm cells in 3 h . Thus, the optical funnel has potential for use as a rapid and repetitive means for the determination of the motility force of the sperm cell. Vaccine, 2000 Jul 15, 18(27), 3097 - 105 Induction of Th2-directed immune responses by IL-4-transduced dendritic cells in mice; Hayashi S et al.; Dendritic cell (DC)-based vaccines have been used to generate Th1-mediated, protective immunity against cancers and infectious microorganisms . As an attempt to develop a new vaccine protocol for the induction of Th2-directed responses, we introduced an IL-4 plasmid vector into the XS106 DC line (derived from A/J mice) . Although relatively small fractions of XS106 cells exhibited apparent intracellular deposition of IL-4, they secreted biologically relevant amounts of the cytokine . IL-4-transduced XS106 DC and control XS106 DC transfected with vector alone were pulsed with KLH and injected s.c . into A/J mice . The overall magnitude of KLH-specific cellular and humoral responses was comparable between the two animal groups . However, they differed in the isotype profile albeit only transiently, with the IL-4-transduced DC group showing higher IgE and lower IgG2a responses, and in the cytokine profile, with spleen cells isolated from the IL-4-transduced DC group producing higher IL-13 and lower IL-12 . Thus, delivery of IL-4 gene to relatively small numbers of DC is sufficient to modify the immunological outcome of DC-based vaccines. FEMS Microbiol Lett, 2000 Jun 15, 187(2), 139 - 44 Invasion of endothelial and epithelial cells by strains of Porphyromonas gingivalis; Dorn BR et al.; Porphyromonas gingivalis is a periodontal pathogen that may also be involved in the pathogenesis of coronary heart disease . This microorganism has the ability to invade several cell lines . In this study, 26 different strains of P . gingivalis were tested for invasion of human umbilical vein endothelial cells and KB cells, a human oral epidermoid cell line . Abilities to invade both cell lines by an individual strain were similar, and their invasion efficiencies could be assembled into four groups: high, moderate, low and non-invasive . Of the 26 strains, only P . gingivalis AJW4 was non-invasive . Since the fimbriae are implicated as having a key role in invasion by this species, the presence of fimbriae on strain AJW4 was investigated . Using polymerase chain reaction (PCR), strain AJW4 was found to contain the fimA gene . Sequence analysis revealed it to be type IV according to the typing scheme developed by Amano et al . Further, fimA is transcribed in this strain as demonstrated by reverse transcription PCR and is expressed on the cell surface as visualized by negative staining and electron microscopy . The adherence+invasion of strain AJW4 was 38.7% of the most invasive strain (strain 381) . However, the CFU ml(-1) of strain AJW4 recovered from within cells was 2.9% of strain 381 . Even though strains AJW4 and W50 have the same type IV fimbriae, strain AJW4 is 8.9-fold more adhesive yet is internalized 170-fold less . These data indicate that the invasion efficiency of P . gingivalis is variable among the different strains, and that the expression of FimA is not sufficient for invasion. Photochem Photobiol, 2000 Jun, 71(6), 691 - 9 Hydrogen peroxide formation and decay in iron-rich geothermal waters: the relative roles of abiotic and biotic mechanisms Wilson CL, Hinman NW, Sheridan RP. Hydrogen peroxide (H2O2) is widely distributed in surface waters where the primary photochemical formation pathway involves the interaction between dissolved organic carbon (DOC) and ultraviolet radiation (UVR) . In laboratory studies using iron-rich water from Yellow-stone's Chocolate Pots spring, H2O2 formation depended on sample treatment (unfiltered, < 0.2 micron filtered, autoclaved) prior to irradiation, suggesting several formation pathways . Similar H2O2 formation in filtered and unfiltered water indicates that it is primarily soluble material that is responsible for H2O2 formation . H2O2 formation with soluble material probably includes only photochemical reactions with DOC and/or metals . Greater H2O2 formation in unfiltered and filtered water than in autoclaved water suggests that the agent(s) involved in H2O2 formation is (are) not stable at high temperatures and pressures and degrade to n