Ultrason Sonochem, 2004 Sep, 11(6), 379 - 84 The direct effect of ultrasound on the extraction of date syrup and its micro-organisms; Entezari MH et al.; In the date syrup industry, date fruits are mixed with a suitable amount of water at a temperature greater than 50 degrees C for about 1 h . This condition is not sufficient for killing the micro-organisms present in the fruit . In addition, Overheating for a long time can damages nutritious materials and also changes the final product's color . Ultrasound was applied for improving the quantity and quality of the extraction, and also to overcome the health problem . In this research, the following variables were examined: date fruit/water ratio, ultrasonic intensity and temperature . The results showed that sonication under the proper conditions can lead to a higher extraction in a shorter time with a better physical quality of the product . Most importantly, the sonication significantly decreased the microbial count in comparison to the classical method . This study also confirmed the presence of anti-microbial substances in date fruit, and that ultrasonic waves can accelerate their effects. Anal Biochem, 2004 Sep 1, 332(1), 109 - 15 Enzyme-coupled assay of acetylxylan esterases on monoacetylated 4-nitrophenyl beta-D-xylopyranosides; Biely P et al.; Three different monoacetates of 4-nitrophenyl beta-D-xylopyranoside were tested as substrates for beta-xylosidase and for microbial carbohydrate esterases and a series of non-hemicellulolytic esterases . The acetyl group in 2-O-acetyl, 3-O-acetyl, and 4-O-acetyl 4-nitrophenyl beta-D-xylopyranoside makes the glycoside resistant to the action of beta-xylosidase (EC 3.2.1.37) . This fact was explored to introduce a new enzyme-coupled assay of acetylxylan esterases (EC 3.1.1.72) and other carbohydrate-deacetylating enzymes . The deacetylation converts the monoacetates into the substrate of beta-xylosidase, the auxiliary enzyme . The effect of the acetyl group migration along the xylopyranoid ring in aqueous media can be avoided by shortening the assay duration . The assay enables an easy examination of the positional specificity of the enzymes, which is important for classification of acetylxylan esterases and for elucidation of the structure-function relationship among carbohydrate esterases in general . Non-hemicellulolytic esterases showed different positional specificity of deacetylation than did acetylxylan esterases. Clin Exp Rheumatol, 2004 Jul-Aug, 22(4), 433 - 40 Low density of CD1+ cells in the synovial tissue of patients with rheumatoid arthritis; Weidler C et al.; OBJECTIVE: CD1 molecules present microbial and self glycolipid antigens to a defined T cell subset with features of natural killer cells . CD1 molecules are up-regulated by inflammatory stimuli such as GM-CSF, and we would expect to find increased CD1 expression in the synovium of patients with rheumatoid arthritis (RA) as compared to osteoarthritis (OA) . This study was initiated to compare the density of CD1a+, CD1b+, and CD1c+ synovial cells in RA and OA patients . METHODS: Expression of CD1a+, CD1b+, and CD1c+ molecules in synovial tissue was assessed by semiquantitative immunohistochemistry . For comparison, serological, functional, and typical immunohistochemical markers of inflammation were detected . RESULTS: Although patients with RA as compared to OA had highly significantly increased signs of inflammation, the density of CD1a+, CD1b+, and CD1c+ synovial cells was similar This was also true for the density of CD1+ cells in relation to that of activated CD163+ macrophages . There was a high correlation between the densities of CD1a,b,c positive cells, which suggests the existence of similar regulatory pathways . In a combined analysis of RA and OA patients, there existed a negative association between prior NSAID therapy and the density of CD1a+, CD1b+, and CD1c+ synoviocytes in relation to CD163+ macrophages . This is interesting because a similar immunosuppressive aspect of NSAID has never been shown before and this might represent a hitherto unrecognized immunosuppressive aspect of NSAID . CONCLUSION: Considering the high synovial inflammation in patients with RA, the densities of CD1a+, CD1b+, and CD1c+ synovial cells were low compared to patients with OA . Further studies in RA patients are needed to clarify whether a defect in CD1 regulation may exist . Such a defect may lead to an insufficient immune response against microbial glycolipids, which would support smoldering or repeated inadequately responded infection. Surgery, 2004 Aug, 136(2), 384 - 9 Beta-glucan affects leukocyte navigation in a complex chemotactic gradient; Tsikitis VL et al.; BACKGROUND: Polymorphonuclear leukocytes (PMNs) must traverse endogenous chemotactic gradients (interleukin 8 {IL-8}) before reaching target chemoattractants (fMLP {N-formylmethionine-leucine-phenylalanine}, C5a) produced at a site of bacterial infection . Complement receptor 3 (CR3; CD11b/CD18) contains 2 distinct binding sites, one that mediates adhesion and a lectin-like domain (LLD) that binds polysaccharides of microbial origin . This laboratory previously reported an increase in the chemotactic capacity of PMNs toward fMLP upon ligation of the CR3 LLD with beta-glucan, a CR3 agonist . Current studies sought to determine the effect of beta-glucan on PMN navigation toward other chemoattractants alone and in a competing chemotactic environment . METHODS: Migration was assessed by serum agarose overlay with the use of chambered slides containing or not, beta-glucan . Migration of human PMNs at 37 degrees C for 2 hours was evaluated toward C5a or IL-8 alone and in competing gradients . Selected groups were treated with anti-CR3-blocking antibodies . The number of chemotactic cells was quantified by microscopy . RESULTS: beta-glucan significantly enhanced chemotaxis toward C5a and suppressed that toward IL-8 in a CR3-dependent fashion . In the competing chemotactic gradient assays (C5a vs IL-8), beta-glucan further enhanced migration toward C5a while not affecting that toward IL-8 . CONCLUSIONS: beta-glucan selectively upregulates PMN chemotaxis toward C5a while suppressing chemotaxis toward IL-8 . Environ Sci Technol, 2004 Jul 15, 38(14), 3877 - 84 Role of speciation in organotin toxicity to the yeast Candida maltosa; White JS et al.; Assessment of organometal pollution requires an understanding of the various processes that influence the bioavailability and toxicity of the contaminant . Organotins may exist as both cationic species and neutral hydroxides in aqueous solution, with the formation of chloride species in the presence of Cl- . Although these species have different chemical properties, there is very little information on the influence of speciation on organotin and microbial cell interactions . Tributyltin (TBT) and triphenyltin (TPT) interactions with the yeast Candida maltosa were investigated between pH 3.5 and 7.5 and in up to 0.5 M NaCl at pH 5.5 . Toxicity increased with both pH and NaCl concentration and the mechanisms of interaction depended on the species present in solution . TBT and TPT interacted by different mechanisms, as evidenced by action on membrane fluidity . Furthermore, there was a strong correlation between toxicity and overall octanol-water distribution ratio (D(OW)) of organotin compounds . Triorganotin cations are less toxic than triorganotin hydroxides, which are in turn less toxic than triorganotin chlorides . These findings underline the importance of speciation effects on organotin interactions in the environment. J Vasc Surg, 2004 Aug, 40(2), 359 - 66 Genomic, serologic, and clinical case-control study of Chlamydia pneumoniae and peripheral artery occlusive disease; Linares-Palomino JP et al.; OBJECTIVES: Chlamydia pneumoniae has been related to atherosclerotic disease in both seroepidemiologic and genomic studies . We performed a case-control study to determine seropositivity and DNA detection in arteries of patients with peripheral artery occlusive disease and of healthy subjects . METHODS: The study included 64 patients with peripheral artery occlusive disease, and 50 control subjects who underwent varicose vein surgery, matched to the patient group for age, sex, and tobacco use . The fibrinogen level in all study subjects was measured as a marker of inflammation . Blood samples were taken from all subjects for determination of immunoglobulin (Ig) G elementary bodies (EB) against C pneumoniae with microimmunofluorescence (MIF) and enzyme-linked immunosorbent assay (ELISA), and of IgA EB with ELISA . The cutoff titers were 1:32 for MIF and 1.1 for ELISA . Biopsy specimens of arterial atheromatous plaque were obtained from patients, and of pudendal artery and saphenous vein from control subjects, and were studied with hemi-nested polymerase chain reaction . RESULTS: There were no differences in fibrinogen level between patients and controls . The prevalence of IgG anti-EB with MIF was 78% in patients and 24% in control subjects (P =.0001; odds ratio {OR}, 11.3; 95% confidence interval {CI}, 4.7-27.2) . Prevalence of IgG anti-EB with ELISA was 75% in patients and 16% in control subjects (P =.0001; OR, 15.7; 95% CI, 6.1-40) . There were no differences in IgA anti-EB titers . Bacterial DNA was detected in 67% of atheromatous plaques versus 12% of pudendal arteries (P =.0001) and 4% of saphenous veins . A weak correlation was found between seropositivity and the presence of intravascular DNA . CONCLUSIONS: Our results support the hypothesis that C pneumoniae is related to the pathogenesis of atherosclerotic peripheral artery occlusive disease . CLINICAL RELEVANCE: This study explored the infectious hypothesis in the context of the pathogenesis of atherosclerosis . This hypothesis has been supported by findings that certain infectious agents can cause or accelerate the course of diseases in which the possibility of a microbial cause was not previously proposed, as in the case of peptic ulcer and spongiform encephalopathy . The present study demonstrated the presence of Chlamydia pneumoniae and seropositivity in atheromatous plaques in patients with peripheral artery occlusive disease . These results contribute to a body of research that is opening up the possibility of treating atherosclerotic disease with antibiotic agents, and preventing it with immunization. J Environ Radioact, 2004, 77(1), 1 - 27 Uranium mill tailings: nuclear waste and natural laboratory for geochemical and radioecological investigations; Landa ER; Uranium mill tailings (UMT) are a high volume, low specific activity radioactive waste typically disposed in surface impoundments . This review focuses on research on UMT and related earth materials during the past decade relevant to the assessment of: (1) mineral hosts of radionuclides; (2) the use of soil analogs in predicting long-term fate of radionuclides; (3) microbial and diagenetic processes that may alter radionuclide mobility in the surficial environment; (4) waste-management technologies to limit radionuclide migration; and (5) the impact of UMT on biota. Dev Cell, 2004 Aug, 7(2), 217 - 28 EspFU is a translocated EHEC effector that interacts with Tir and N-WASP and promotes Nck-independent actin assembly; Campellone KG et al.; Several microbial pathogens including enteropathogenic E . coli (EPEC) exploit mammalian tyrosine-kinase signaling cascades to recruit Nck adaptor proteins and activate N-WASP-Arp2/3-mediated actin assembly . To promote localized actin "pedestal formation," EPEC translocates the bacterial effector protein Tir into the plasma membrane, where it is tyrosine-phosphorylated and binds Nck . Enterohemorrhagic E . coli (EHEC) also generates Tir-dependent pedestals, but in the absence of phosphotyrosines and Nck recruitment . To identify additional EHEC effectors that stimulate phosphotyrosine-independent actin assembly, we systematically generated EHEC mutants containing specific deletions in putative pathogenicity-islands . Among 0.33 Mb of deleted sequences, only one ORF was critical for pedestal formation . It lies within prophage-U, and encodes a protein similar to the known effector EspF . This proline-rich protein, EspFU, is the only EHEC effector of actin assembly absent from EPEC . Whereas EHEC Tir cannot efficiently recruit N-WASP or trigger actin polymerization, EspFU associates with Tir, binds N-WASP, and potently stimulates Nck-independent actin assembly. Environ Sci Technol, 2004 Jul 1, 38(13), 3623 - 31 Deriving soil critical limits for Cu, Zn, Cd, and Pb: a method based on free ion concentrations; Lofts S et al.; We present a method to calculate critical limits of cationic heavy metals accounting for variations in soil chemistry . We assume the free metal ion concentration (Mfree) to be the most appropriate indicator of toxicity, combined with a protective effect of soil cations (e.g., H+, Ca2+) . Because soil metal cations tend to covary with pH, the concentration of Mfree exerting a given level of toxic effect (Mfree,toxic) can be expressed as a function of pH alone . We use linear regression equations to derive Mfree,toxic in toxicity experiments from soil pH, organic matter content, and endpoint soil metal . Chronic toxicity data from the literature, for plants, invertebrates, microbial processes, and fungi are interpreted in terms of an average log Mfree,toxic together with distributions of species sensitivity . This leads to critical limit functions to protect 95% of species, of the form log Mfree,CRIT = (pH + gamma . Appreciable effects of soil pH upon log Mfree,CRIT are found, with alpha = -1.21 (Cu), -0.34 (Zn), -0.43 (Cd), and -0.83 (Pb) . Critical limit functions in terms of the geochemically active soil metal (Msoil,CRIT), that pool of metal which controls the free ion concentration, have also been derived, with soil pH and organic matter content as variables . The pH effect on Msoil,CRIT is relatively small, with slopes of 0.05 (Cu), 0.19 (Zn), 0.16 (Cd), and 0.20 (Pb), since the effect of pH on Mfree,CRIT is countered by the variation of Mfree with pH. J Infect Dis, 2004 Sep 1, 190(5), 946 - 56 Epub 2004 Jul 30. Transcription profile of Helicobacter pylori in the human stomach reflects its physiology in vivo; Boonjakuakul JK et al.; BACKGROUND: Little is known about levels of expression of Helicobacter pylori genes in the human host . We therefore developed a quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) assay to measure transcript profiles of H . pylori in the human stomach . METHODS: In vivo expression of 16 genes on the cag pathogenicity island and of 18 putative virulence genes was quantitated by isolation of total RNA directly from infected human gastric mucosa . The results were compared with in vitro expression determined from H . pylori cells grown in culture . RESULTS: The highest levels of expression were found for cag1 and cag25 and for genes, such as urease and catalase, that may be important for bacterial homeostasis in the relatively hostile environment of the gastric mucosa . Transcript abundance, relative to 16S rRNA, was lower in vivo than in vitro, which suggests that H . pylori cells are in stationary phase in the gastric environment . This was particularly apparent for cagA . Since CagA is arguably of unique importance, in terms of interaction with the host, tight control of its in vivo expression might be particularly important . CONCLUSIONS: qRT-PCR is a powerful tool to measure gene expression in human or animal tissue that contains minute amounts of microbial mRNA, and the results reflect on the physiology of the pathogen in its natural host. J Immunol, 2004 Aug 15, 173(4), 2725 - 35 The induction of acute ileitis by a single microbial antigen of Toxoplasma gondii; Rachinel N et al.; The role of specific microbial Ags in the induction of experimental inflammatory bowel disease is poorly understood . Oral infection of susceptible C57BL/6 mice with Toxoplasma gondii results in a lethal ileitis within 7-9 days postinfection . An immunodominant Ag of T . gondii (surface Ag 1 (SAG1)) that induces a robust B and T cell-specific response has been identified and a SAG1-deficient parasite (Deltasag1) engineered . We investigated the ability of Deltasag1 parasite to induce a lethal intestinal inflammatory response in susceptible mice . C57BL/6 mice orally infected with Deltasag1 parasites failed to develop ileitis . In vitro, the mutant parasites replicate in both enterocytes and dendritic cells . In vivo, infection with the mutant parasites was associated with a decrease in the chemokine and cytokine production within several compartments of the gut-associated cell population . RAG-deficient (RAG1(-/-)) mice are resistant to the development of the ileitis after T . gondii infection . Adoptive transfer of Ag-specific CD4(+) effector T lymphocytes isolated from C57BL/6-infected mice into RAG(-/-) mice conferred susceptibility to the development of the intestinal disease . In contrast, CD4(+) effector T lymphocytes from mice infected with the mutant Deltasag1 strain failed to transfer the pathology . In addition, resistant mice (BALB/c) that fail to develop ileitis following oral infection with T . gondii were rendered susceptible following intranasal presensitization with the SAG1 protein . This process was associated with a shift toward a Th1 response . These findings demonstrate that a single Ag (SAG1) of T . gondii can elicit a lethal inflammatory process in this experimental model of pathogen-driven ileitis. J Immunol, 2004 Aug 15, 173(4), 2211 - 5 Regulatory cells and infectious agents: detentes cordiale and contraire; Rouse BT et al.; This brief review describes the types of interactions that occur between CD4(+)CD25(+) regulatory T cells (Treg) and microbial pathogens . These interactions range from one of mutual benefit (detente cordiale) such as occurs in Leishmania major infection of resistant mouse strains, to instances where the Treg response appears to mainly favor the pathogen and be detrimental to the host (detente contraire) . Examples of the latter include chronic persistent infections with retroviruses, perhaps including HIV, and hepatitis C virus . The Treg response also hampers the effectiveness of immunity against some acute virus infections such as HSV . Evidence is also discussed showing that Treg can play a benevolent role to limit the severity of bystander tissue damage in circumstances where the immune response to pathogens is immunopathological . Finally, emerging approaches are discussed that either blunt or activate Treg and that could be used practically to manage host-pathogen interaction. Appl Environ Microbiol, 2004 Aug, 70(8), 4930 - 40 Dominant microbial composition and its vertical distribution in saline meromictic Lake Kaiike (Japan) as revealed by quantitative oligonucleotide probe membrane hybridization; Koizumi Y et al.; Vertical distributions of dominant bacterial populations in saline meromictic Lake Kaiike were investigated throughout the water column and sediment by quantitative oligonucleotide probe membrane hybridization . Three oligonucleotide probes specific for the small-subunit (SSU) rRNA of three groups of Chlorobiaceae were newly designed . In addition, three general domain (Bacteria, Archaea, and Eukarya)-specific probes, two delta-Proteobacteria-specific probes, a Chlorobiaceae-specific probe, and a Chloroflexi-specific probe were used after optimization of their washing conditions . The abundance of the sum of SSU rRNAs hybridizing with probes specific for three groups of Chlorobiaceae relative to total SSU rRNA peaked in the chemocline, accounting for up to 68% . The abundance of the delta-proteobacterial SSU rRNA relative to total SSU rRNA rapidly increased just below the chemocline up to 29% in anoxic water and peaked at the 2- to 3-cm sediment depth at ca . 34% . The abundance of SSU rRNAs hybridizing with the probe specific for the phylum Chloroflexi relative to total SSU rRNA was highest (31 to 54%) in the top of the sediment but then steeply declined with depth and became stable at 11 to 19%, indicating the robust coexistence of sulfate-reducing bacteria and Chloroflexi in the top of the sediment . Any SSU rRNA of Chloroflexi in the water column was under the detection limit . The summation of the signals of group-specific probes used in this study accounted for up to 89% of total SSU rRNA, suggesting that the DGGE-oligonucleotide probe hybridization approach, in contrast to conventional culture-dependent approaches, was very effective in covering dominant populations. Appl Environ Microbiol, 2004 Aug, 70(8), 4800 - 6 Comparisons of different hypervariable regions of rrs genes for use in fingerprinting of microbial communities by PCR-denaturing gradient gel electrophoresis; Yu Z et al.; Denaturing gradient gel electrophoresis (DGGE) has become a widely used tool to examine microbial diversity and community structure, but no systematic comparison has been made of the DGGE profiles obtained when different hypervariable (V) regions are amplified from the same community DNA samples . We report here a study to make such comparisons and establish a preferred choice of V region(s) to examine by DGGE, when community DNA extracted from samples of digesta is used . When the members of the phylogenetically representative set of 218 rrs genes archived in the RDP II database were compared, the V1 region was found to be the most variable, followed by the V9 and V3 regions . The temperature of the lowest-melting-temperature (T(m(L))) domain for each V region was also calculated for these rrs genes, and the V1 to V4 region was found to be most heterogeneous with respect to T(m(L)) . The average T(m(L)) values and their standard deviations for each V region were then used to devise the denaturing gradients suitable for separating 95% of all the sequences, and the PCR-DGGE profiles produced from the same community DNA samples with these conditions were compared . The resulting DGGE profiles were substantially different in terms of the number, resolution, and relative intensity of the amplification products . The DGGE profiles of the V3 region were best, and the V3 to V5 and V6 to V8 regions produced better DGGE profiles than did other multiple V-region amplicons . Introduction of degenerate bases in the primers used to amplify the V1 or V3 region alone did not improve DGGE banding profiles . Our results show that DGGE analysis of gastrointestinal microbiomes is best accomplished by the amplification of either the V3 or V1 region of rrs genes, but if a longer amplification product is desired, then the V3 to V5 or V6 to V8 region should be targeted. Appl Environ Microbiol, 2004 Aug, 70(8), 4756 - 65 Determining rates of change and evaluating group-level resiliency differences in hyporheic microbial communities in response to fluvial heavy-metal deposition; Feris KP et al.; Prior field studies by our group have demonstrated a relationship between fluvial deposition of heavy metals and hyporheic-zone microbial community structure . Here, we determined the rates of change in hyporheic microbial communities in response to heavy-metal contamination and assessed group-level differences in resiliency in response to heavy metals . A controlled laboratory study was performed using 20 flowthrough river mesocosms and a repeated-measurement factorial design . A single hyporheic microbial community was exposed to five different levels of an environmentally relevant metal treatment (0, 4, 8, 16, and 30% sterilized contaminated sediments) . Community-level responses were monitored at 1, 2, 4, 8, and 12 weeks via denaturing gradient gel electrophoresis and quantitative PCR using group-specific primer sets for indigenous populations most closely related to the alpha-, beta-, and gamma-proteobacteria . There was a consistent, strong curvilinear relationship between community composition and heavy-metal contamination (R(2) = 0.83; P < 0.001), which was evident after only 7 days of metal exposure (i.e., short-term response) . The abundance of each phylogenetic group was negatively affected by the heavy-metal treatments; however, each group recovered from the metal treatments to a different extent and at a unique rate during the course of the experiment . The structure of hyporheic microbial communities responded rapidly and at contamination levels an order of magnitude lower than those shown to elicit a response in aquatic macroinvertebrate assemblages . These studies indicate that hyporheic microbial communities are a sensitive and useful indicator of heavy-metal contamination in streams. Asia Pac J Clin Nutr . 2004;13(Suppl):S162. Watermelon juice concentrate; Ghazizadeh M et al.; Production of watermelon is in great amount in Iran during summer time . Consumption of this fruit is recommended by health and nutrition authorities . Findings from USDA scientists indicate that watermelon contains high levels of lycopene an antioxidant that may help the body fight against cancer and other chronic diseases . Thus it can be considered as a functional food - one that may help prevent certain diseases . However, production of this fruit in some parts of country is in excess of its consumption, so large amounts are spoiled and wasted . Therefore, in this study we tried to process two kinds of watermelons and to formulate their juice and concentrate which preserves its flavor, appearance and nutrient contents . Seventy two Charleston Gray and local variety of Khorasan watermelon samples were analyzed . Water content of local watermelon and Charleston Gray were 55.9 % +/- 3.1 and 58 % +/- 1.12 respectively, rind 34.3 % +/- 0.7 and 31.8 % +/- 1.56 (N.S), seeds 4.8% +/- 1.29 and 2.3 %+/- 0.32 (P<002) and pulps were 4.9% +/- 1/39 and 7.9 % +/- 0.45 (P<0.001) . Sixty different formulae from a mixture of watermelon Juice and food additives (30 formulae of each variety) were prepared and their organoleptic characteristics underwent preliminary investigation by category scale . Three formulae from each variety were selected (A, B and C) and their organoleptic characteristics were evaluated . The suitable formula for each variety was nominated by test panel . The formula A of Charleston Gray variety including 100 ml Juice, 4gr sugar and 0.1 gr citric acid with 68.3% acceptability and the formula B of local variety including 100ml juice, 5 gr Sugar and 0.2 gr citric acid with 30.4 % acceptability were selected as preferred samples . The selected formula of Charleston Gray variety was concentrated (up to Brix 54) and packed . Changes of chemical, microbial and organoleptic characteristics in different condition (room, refrigerator and freezer) during 0, 15, 30, 45, . 90 days storage were determined . Ninety days storage in different temperatures did not cause notable changes of chemical, microbial and organoleptic characteristics. Asia Pac J Clin Nutr . 2004 Aug;13(Suppl):S91. The efficacy of phytase in corn soybean meal based broiler diets; Mulyantini NG et al.; Background - To overcome the inability of poultry to utilise phosphorus bound to phytate in plant feed ingredients, phytase of microbial origin is added to poultry diets . This has the added advantage of reducing phosphorus build up in manure . Objective - To examine the efficacy of two commercial phytase sources on the performance of broiler chickens fed corn soybean meal based diets . Design - Day-old, male broiler chicks (Ross) were allocated to 30 pens in a completely randomised experimental design with each diet offered to 6 pens of 50 chicks per pen . The 6 experimental mash diets were formulated as: 1) standard Phosphorus (P), 2) low-P, 3) low-P + phytase A at 500 U/kg feed, and 4) low-P + phytase A at 1000 U/kg feed, 5) low-P + phytase B at 500 U and 6) low-P + phytase B at 1000 U/kg feed . A starter diet was fed from day old to day 22 and a finisher diet from day 23 to day 42 . The standard-P starter and finisher diets contained 0.4% and 0.32% available phosphorous respectfully compared to 0.28% and 0.20% in the low-P diets . Outcomes - At the completion of the study body weight of the birds fed the standard-P diet were significantly (P<0.05) heavier than those fed the unsupplemented low-P diets . There was no significant difference in body weight between the groups fed the standard-P diet and the phytase supplemented diets . Feed conversion ratio was not significantly affected by phytase supplementation . Conclusion - The performance of broilers fed low phosphorus corn soybean meal based diets can be significantly improved by phytase supplementation . Both sources of phytase gave similar results, and can replace approximately 50% dicalcium phosphate in diets, without affecting broiler performance. Parasitol Res . 2004 Jul 31; {Epub ahead of print} The involvement of an integrin-like protein and protein kinase C in amoebic adhesion to fibronectin and amoebic cytotoxicity; Han KL et al.; Adherence of a pathogen to the host cell is one of the critical steps in microbial infections . Naegleria fowleri, a causative agent of primary amoebic meningoencephalitis in humans, is expected to interact with extracellular components of the host, such as fibronectin, in a receptor-mediated mode . In this study, we investigated the interaction between N . fowleri and fibronectin to understand its cytopathology . In binding assays using immobilized fibronectin, the number of amoebae bound to fibronectin was increased compared to the controls, and was dependent on the amount of coated fibronectin present . A fibronectin binding protein of 60 kDa was found in extracts of N . fowleri . Western blot and immunolocalization assays using integrin alpha(5)/FnR antibodies showed that a 60 kDa protein reacted with the antibodies in extracts of N . fowleri, which was localized on the surface of N . fowleri . Preincubation of N . fowleri with the integrin antibodies significantly inhibited amoebic binding to fibronectin and cytotoxicity to the CHO cells . Additionally, protein kinase C activity was detected in the extract of N . fowleri . When N . fowleri was pretreated with protein kinase C activator or inhibitor, the abilities of amoebic adhesion to fibronectin and cytotoxicity to the host cells were markedly affected compared to untreated amoebae . These results suggest that an amoebic integrin-like receptor and protein kinase C play important roles in amoebic cellular processes in response to fibronectin. Genome Res, 2004 Aug, 14(8), 1669 - 75 cpnDB: a chaperonin sequence database; Hill JE et al.; Type I chaperonins are molecular chaperones present in virtually all bacteria, some archaea and the plastids and mitochondria of eukaryotes . Sequences of cpn60 genes, encoding 60-kDa chaperonin protein subunits (CPN60, also known as GroEL or HSP60), are useful for phylogenetic studies and as targets for detection and identification of organisms . Conveniently, a 549-567-bp segment of the cpn60 coding region can be amplified with universal PCR primers . Here, we introduce cpnDB, a curated collection of cpn60 sequence data collected from public databases or generated by a network of collaborators exploiting the cpn60 target in clinical, phylogenetic, and microbial ecology studies . The growing database currently contains approximately 2000 records covering over 240 genera of bacteria, eukaryotes, and archaea . The database also contains over 60 sequences for the archaeal Type II chaperonin (thermosome, a homolog of eukaryotic cytoplasmic chaperonin) from 19 archaeal genera . As the largest curated collection of sequences available for a protein-encoding gene, cpnDB provides a resource for researchers interested in exploiting the power of cpn60 as a diagnostic or as a target for phylogenetic or microbial ecology studies, as well as those interested in broader subjects such as lateral gene transfer and codon usage . We built cpnDB from open source tools and it is available at Cancer Res, 2004 Aug 1, 64(15), 5518 - 24 Modern criteria to establish human cancer etiology; Carbone M et al.; The Cancer Etiology Branch of the National Cancer Institute hosted a workshop, "Validation of a causal relationship: criteria to establish etiology," to determine whether recent technological advances now make it possible to delineate improved or novel criteria for the rapid establishment for cancer causation . The workshop was held in Washington, D.C., December 11-12, 2003, and participants were among the international leaders in the fields of epidemiology, chemistry, biochemistry, microbiology, virology, environmental and chemical carcinogenesis, immunology, pathology, molecular pathology, genetics, oncology, and surgical oncology . There was a general consensus that the rapid identification of human carcinogens and their removal (when possible) or the establishment of specific preventive and therapeutic measures was the most desirable and effective way to have a rapid and positive impact in the fight against cancer . From a clinical perspective, it may be as important to target initiators, cocarcinogens and promoters, if by removing any one of them tumor growth can be prevented . Future studies should focus on interactions among and between different biological, chemical, and physical agents . Analyses of single agents can at times miss their carcinogenic potential when such agents are carcinogenic only in subgroups of individuals because of their genetic background, diet, exposure to other carcinogens, or microbial infection . Epidemiology, molecular pathology (including chemistry, biochemistry, molecular biology, molecular virology, molecular genetics, epigenetics, genomics, proteomics, and other molecular-based approaches), and animal and tissue culture experiments should all be seen as important integrating evidence in the determination of human carcinogenicity . Concerning the respective roles of epidemiology and molecular pathology, it was noted that epidemiology allows the determination of the overall effect of a given carcinogen in the human population (e.g., hepatitis B virus and hepatocellular carcinoma) but cannot prove causality in the individual tumor patient . Molecular pathology cannot determine the overall impact of a carcinogen in the population but can at times prove causality in the individual tumor patient {such as the detection of high-risk human papillomavirus (HPV) in a cervical carcinoma biopsy} . This is possible when molecular techniques have shown that the agent is required for transformation or malignant growth of human cells (such as antisense HPV strategies showing the requirement for the expression of HPV proteins for tumor cell growth) and when there is supportive experimental animal evidence . Ideally, epidemiology and molecular pathology information together with experimental evidence in animals should be available for the most reliable identification of human carcinogens . All sets of data are not always available, and a rapid identification of human carcinogens is in the best public health interest . Swift validation of a causal relationship when followed by a rapid deployment of preventive and therapeutic approaches should lead to a favorable public health impact (such as hepatitis B virus vaccination to prevent hepatocellular carcinoma). Mutat Res, 2004 Aug 18, 552(1-2), 101 - 17 Emerging contaminants--pesticides, PPCPs, microbial degradation products and natural substances as inhibitors of multixenobiotic defense in aquatic organisms; Smital T et al.; The environmental presence of chemosensitizers or inhibitors of the multixenobiotic resistance (MXR) defense system in aquatic organisms could cause increase in intracellular accumulation and toxic effects of other xenobiotics normally effluxed by MXR transport proteins (P-glycoprotein (P-gps), MRPs) . MXR inhibition with concomitant detrimental effects has been shown in several studies with aquatic organisms exposed to both model MXR inhibitors and environmental pollutants . The presence of MXR inhibitors has been demonstrated in environmental samples from polluted locations at concentrations that could abolish P-gp transport activity . However, it is not clear whether the inhibition observed after exposure to environmental samples is a result of saturation of MXR transport proteins by numerous substrates present in polluted waters or results from the presence of powerful MXR inhibitors . And are potent environmental MXR inhibitors natural or man-made chemicals? As a consequence of these uncertainties, no official action has been taken to monitor and control the release and presence of MXR inhibitors into aquatic environments . In this paper we present our new results addressing these critical questions . Ecotoxicological significance of MXR inhibition was supported in in vivo studies that demonstrated an increase in the production of mutagenic metabolites by mussels and an increase in the number of sea urchin embryos with apoptotic cells after exposure to model MXR inhibitors . We also demonstrated that MXR inhibitors are present among both conventional and emerging man-made pollutants: some pesticides and synthetic musk fragrances show extremely high MXR inhibitory potential at environmentally relevant concentrations . In addition, we emphasized the biological transformation of crude oil hydrocarbons into MXR inhibitors by oil-degrading bacteria, and the risk potentially caused by powerful natural MXR inhibitors produced by invasive species. Waste Manag, 2004, 24(7), 673 - 9 Biodegradable organic matter in municipal solid waste incineration bottom ash; Zhang S et al.; For investigation of the behavior of municipal solid waste incineration bottom ash in landfill, we have analysed bottom ash samples taken after the quench tank as well as after five months of storage in the laboratory for elements and organic constituents . Water extractable organic carbon, particulate organic carbon, amino acids, hexosamines and carbohydrates considerably decreased during the five months of storage and their spectra revealed microbial reworking . This shows that the organic matter present in the bottom ash after incineration can provide a substrate for microbial activity . The resulting changes of the physico-chemical environment may effect the short-term behavior of the bottom ash in landfill . Bioresour Technol, 2004 Dec, 95(3), 287 - 91 Single cell oil (SCO) production by Mortierella isabellina grown on high-sugar content media; Papanikolaou S et al.; Mortierella isabellina cultivated in nitrogen-limited media presented remarkable cell growth (up to 35.9 g/l) and high glucose uptake even with high initial sugar concentrations (e.g . 100 g/l) in media . After nitrogen depletion, significant fat quantities were accumulated inside the fungal mycelia (50-55%, wt/wt oil in dry biomass), resulting in a notable single cell oil production of 18.1 g/l of culture medium . Total dry biomass and lipid yields presented greatly increased values (0.34 and 0.17 g respectively per gram of glucose consumed) . The microbial lipid produced contained gamma-linolenic acid (GLA) at a concentration of 3.5+/-1.0%, wt/wt, which corresponded to 16-19 mg GLA per gram of dry microbial mass and a maximum concentration of 0.801 g GLA per liter of culture medium. Autoimmun Rev, 2004 Jul, 3(5), 394 - 400 Inflammatory bowel disease: the role of environmental factors; Danese S et al.; Environmental factors are essential components of the pathogenesis of inflammatory bowel disease (IBD) and primarily responsible for its growing incidence around the globe . Epidemiological, clinical and experimental evidence support an association between IBD and a large number of seemingly unrelated environmental factors, which include smoking, diet, drugs, geographical and social status, stress, microbial agents, intestinal permeability and appendectomy . Data supporting the involvement of each of these factors in predisposing to, triggering, or modulating the course or outcome of IBD vary from strong to tenuous . Smoking and the enteric bacterial flora are the ones for which the most solid evidence is currently available . Smoking increases the risk of Crohn's disease (CD) and worsens its clinical course, but has a protective effect in ulcerative colitis (UC) . Presence of enteric bacteria is indispensable to develop gut inflammation in most animal models of IBD, and modulation of the quantity or quality of the flora can be beneficial in patients with IBD . Surprisingly, evidence for a major role of the diet in inducing or modifying IBD is limited, while that for nonsteroidal anti-inflammatory drugs is more convincing than for oral contraceptives . Northern geographic location, and a high social, economical, educational or occupational status increase the risk of IBD, an observation fitting the hygiene hypothesis for allergic and autoimmune diseases . Stress is also associated with IBD, but more as a modifier than an inducing factor, and its contribution is more obvious in IBD animal models than human IBD . Finally, an increased intestinal permeability may increase the risk for developing CD, whereas an appendectomy lowers the risk of developing UC. Microb Cell Fact . 2004 Aug 2;3(1):10. Use and improvement of microbial redox enzymes for environmental purposes; Garcia-Arellano H et al.; Industrial development may result in the increase of environmental risks . The enzymatic transformation of polluting compounds to less toxic or even innocuous products is an alternative to their complete removal . In this regard, a number of different redox enzymes are able to transform a wide variety of toxic pollutants, such as polynuclear aromatic hydrocarbons, phenols, azo dyes, heavy metals, etc . Here, novel information on chromate reductases, enzymes that carry out the reduction of highly toxic Cr(VI) to the less toxic insoluble Cr(III), is discussed . In addition, the properties and application of bacterial and eukaryotic proteins (lignin-modifying enzymes, peroxidases and cytochromes) useful in environmental enzymology is also discussed. Lett Appl Microbiol, 2004, 39(3), 306 - 9 Kinetics and effects of trace elements and electron complexes on 2-keto-4-methylthiobutyric acid-dependent biosynthesis of ethylene in soil; Arshad M et al.; AIMS: 2-Keto-4-methylthiobutyric acid (KMBA) is an established intermediate in microbial biosynthesis of ethylene from methionine . This study demonstrates the kinetics and effects of trace elements and electron complexes on substrate (KMBA)-derived C2H4 biosynthesis in soil . METHODS AND RESULTS: We have previously reported KMBA-dependent C2H4 production in soil . We studied the kinetics and effects of various trace elements and electron complexes on KMBA-derived C2H4 biosynthesis in soil by gas chromatography . Kinetic analysis revealed that ethylene forming enzyme (EFE) reaction was linear (R2 = 0.9448) when velocity of reaction (V) was plotted against substrate {S} over the range from 2.5 to 10 mmol l(-1) and thus followed a first order reaction . Application of three linear transformations of the Michaelis-Menten equation indicated high affinity of EFE for the substrate because Km values ranged between 5.4 and 6.67 mmol l(-1) and Vmax of reaction was between 22.4 and 35.7 nmol kg(-1) soil 120 cm(-1) . Most of the trace elements exhibited positive effects on KMBA-dependent C2H4 production in soil . Maximum stimulatory effect on C2H4 biosynthesis was observed in response to Co(II) application, while Fe(III) inhibited the biotransformation of KMBA into C2H4 . Contrarily, most of the tested electron complexes inhibited KMBA-derived C2H4 biosynthesis in the soil . However, lower concentrations (1.0 mmol l(-1)) of mannitol and hydroquinone were stimulatory to C2H4 production in soil compared with controls (substrate only) . Conclusions: The results revealed that both kind and concentration of trace elements and electron complexes affected the substrate-dependent production of C2H4 in soil with different degrees of efficacy . SIGNIFICANCE AND IMPACT OF THE STUDY: The C2H4 in the root environment could be physiologically active even at low concentrations, so knowledge regarding various factors which regulate C2H4 biosynthesis in soil could be of significance for plant growth and development. J AOAC Int, 2004 May-Jun, 87(3), 587 - 91 Rapid fluorescence screening assay for tetracyclines in chicken muscle; Schneider MJ et al.; A simple, rapid fluorescence assay was developed for screening tetracyclines in chicken muscle at the U.S . tolerance level (2 mg/kg) . The method requires only a homogenization of the tissue in acetonitrile-ammonium hydroxide, centrifugation, addition of Mg+2, and another centrifugation before fluorescence of the supernatant is measured at 505 nm (excitation at 385 nm) . Comparison of the fluorescence of control chicken muscle extracts with extracts from muscle fortified with either 2 mg/kg tetracycline, oxytetracycline, or chlortetracycline showed no overlap . A threshold level set at the average fluorescence for a series of fortified 2 mg/kg samples minus 3sigma minimized false-negative responses to provide a successful screening method . The method was tested with blinded samples as controls or samples fortified with tetracycline, oxytetracycline, or chlortetracycline in order to demonstrate its utility . This approach can provide an alternative to microbial screening assays. Int J Oral Maxillofac Surg, 2004 Apr, 33(3), 221 - 34 Recurrent aphthous ulcers today: a review of the growing knowledge; Natah SS et al.; Recurrent aphthous ulcers represent a very common but poorly understood mucosal disorder . They occur in men and women of all ages, races and geographic regions . It is estimated that at least 1 in 5 individuals has at least once been afflicted with aphthous ulcers . The condition is classified as minor, major, and herpetiform on the basis of ulcer size and number . Attacks may be precipitated by local trauma, stress, food intake, drugs, hormonal changes and vitamin and trace element deficiencies . Local and systemic conditions, and genetic, immunological and microbial factors all may play a role in the pathogenesis of recurrent aphthous ulceration (RAU) . However, to date, no principal cause has been discovered . Since the aetiology is unknown, diagnosis is entirely based on history and clinical criteria and no laboratory procedures exist to confirm the diagnosis . Although RAU may be a marker of an underlying systemic illness such as coeliac disease, or may present as one of the features of Behcet's disease, in most cases no additional body systems are affected, and patients remain otherwise fit and well . Different aetiologies and mechanisms might be operative in the aetiopathogenesis of aphthous ulceration, but pain, recurrence, self-limitation of the condition, and destruction of the epithelium seem to be the ultimate outcomes . There is no curative therapy to prevent the recurrence of ulcers, and all available treatment modalities can only reduce the frequency or severity of the lesions. Biotechnol Bioeng, 2004 Aug 20, 87(4), 516 - 24 Metabolic engineering and protein directed evolution increase the yield of L-phenylalanine synthesized from glucose in Escherichia coli; Baez-Viveros JL et al.; L-phenylalanine (L-Phe) is an aromatic amino acid with diverse commercial applications . Technologies for industrial microbial synthesis of L-Phe using glucose as a starting raw material currently achieve a relatively low conversion yield (Y(Phe/Glc)) . The purpose of this work was to study the effect of PTS (phosphotransferase transport system) inactivation and overexpression of different versions of feedback inhibition resistant chorismate mutase-prephenate dehydratase (CM-PDT) on the yield (Y(Phe/Glc)) and productivity of L-Phe synthesized from glucose . The E . coli JM101 strain and its mutant derivative PB12 (PTS(-)Glc(+) phenotype) were used as hosts . PB12 has an inactive PTS, but is capable of transporting and phosphorylating glucose by using an alternative system constituted by galactose permease (GalP) and glucokinase activities (Glk) . JM101 and PB12 were transformed with three plasmids, harboring genes that encode for a feedback inhibition resistant DAHP synthase (aroG(fbr)), transketolase (tktA) and either a truncated CM-PDT (pheA(fbr)) or its derived evolved genes (pheA(ev1) or pheA(ev2)) . Resting-cells experiments with these engineered strains showed that JM101 and PB12 strains expressing either pheA(ev1) or pheA(ev2) genes produced l-Phe from glucose with Y(Phe/Glc) of 0.21 and 0.33 g/g, corresponding to 38 and 60% of the maximum theoretical yield (0.55 g/g), respectively . In addition, in both engineered strains the reached q(Phe) high levels of 40 mg/g-dcw.h . The metabolic engineering strategy followed in this work, including a strain with an inactive PTS, resulted in a positive impact over the Y(Phe/Glc), enhancing it nearly 57% compared with its PTS(+) counterpart . This is the first report wherein PTS inactivation was a successful strategy to improve the Y(Phe/Glc). Rev Hosp Clin Fac Med Sao Paulo, 2004 May-Jun, 59(3), 138 - 44 Epub 2004 Jul 28. Involvement of C4 allotypes in the pathogenesis of human diseases; Samano ES et al.; The complement system is an important humoral defense mechanism that plays a relevant role against microbial agents, inflammatory response control, and immunocomplex clearance . Classical complement pathway activation is antibody-dependent . The C4 component participates in the initial step of activation, and C4 expression is determined by 2 pairs of allotypes: C4A and C4B . Deficiencies in C4 allotypes have been associated with several diseases . The aim of the present review is evaluate the reported data in the literature regarding specific C4A and C4B deficiencies and characterize their clinical relevance . We searched the MEDLINE and LILACS databases . Papers referring to total C4 deficiency without allotype evaluation and case reports of primary C4 deficiency were not included . Deficiencies in C4 allotypes have been associated with Mycobacterium leprae infection, erythema nodosum, systemic sclerosis with anti-topoisomerase I antibodies, intermediate congenital adrenal hyperplasia with DR5 genotype, diabetes mellitus type 1 with DR3,4 genotype, and diabetes mellitus with antibodies against islet cells . C4 allotype deficiency is also related to C4B deficiency and autoimmune-associated diseases, such as systemic lupus erythematosus, or diseases with an autoimmune component, such as autism . Some reports associate C4A with thyroiditis after delivery as well as limited and systemic sclerosis without anti-topoisomerase I antibodies . However, the studies with C4A and C4B have been concentrated in isolated populations, and some of the studies could not be reproduced by other authors. Rev Inst Med Trop Sao Paulo, 2004 May-Jun, 46(3), 119 - 26 Epub 2004 Jul 20. The influence of the human genome on chronic viral hepatitis outcome; de Andrade DR Jr et al.; The mechanisms that determine viral clearance or viral persistence in chronic viral hepatitis have yet to be identified . Recent advances in molecular genetics have permitted the detection of variations in immune response, often associated with polymorphism in the human genome . Differences in host susceptibility to infectious disease and disease severity cannot be attributed solely to the virulence of microbial agents . Several recent advances concerning the influence of human genes in chronic viral hepatitis B and C are discussed in this article: a) the associations between human leukocyte antigen polymorphism and viral hepatic disease susceptibility or resistance; b) protective alleles influencing hepatitis B virus (HBV) and hepatitis C virus (HCV) evolution; c) prejudicial alleles influencing HBV and HCV; d) candidate genes associated with HBV and HCV evolution; d) other genetic factors that may contribute to chronic hepatitis C evolution (genes influencing hepatic stellate cells, TGF-beta 1 and TNF-alpha production, hepatic iron deposits and angiotensin II production, among others) . Recent discoveries regarding genetic associations with chronic viral hepatitis may provide clues to understanding the development of end-stage complications such as cirrhosis or hepatocellular carcinoma . In the near future, analysis of the human genome will allow the elucidation of both the natural course of viral hepatitis and its response to therapy. J Cell Sci, 2004 Aug 1, 117(Pt 17), 4007 - 14 Lateral diffusion of Toll-like receptors reveals that they are transiently confined within lipid rafts on the plasma membrane; Triantafilou M et al.; The innate immune system utilises pattern recognition receptors in order to recognise microbial conserved molecular patterns . The family of Toll-like receptors (TLRs) has been shown to act as the main pattern recognition receptors for the innate immune system . Using biochemical as well as fluorescence imaging techniques, TLR2 and TLR4 were found to be recruited within microdomains upon stimulation by bacterial products . Furthermore their lateral diffusion in the cell membrane as determined by fluorescence recovery after photobleaching revealed that upon stimulation by bacterial products TLRs encounter barriers to their lateral movement, thus supporting the notion that specialised domains on the plasma membrane facilitate the innate recognition. Environ Toxicol Chem, 2004 Mar, 23(3), 613 - 20 Glycosidation of chlorophenols by Lemna minor; Day JA et al.; Metabolic fate of xenobiotics in plant tissues has an important role in the ultimate fate of these compounds in natural and engineered systems . Chlorophenols are an important class of xenobiotics used in a variety of biocides and have been shown to be resistant to microbial degradation . Three chlorophenyl glycosides were extracted from tissues of Lemna minor exposed to 2,4-dichlorophenol (DCP) . The products were identified as 2,4-dichlorophenyl-beta-D-glucopyranoside (DCPG), 2,4-dichlorophenyl-beta-D-(6-O-malonyl)-glucopyranoside (DCPMG) and 2,4-dichlorophenyl-beta-D-glucopyranosyl-(6 --> 1)-beta-D-apiofuranoside (DCPAG) . Identification was based on reverse phase retention (C18), electrospray mass spectra collected in negative and positive mode (ESI-NEG and ESI-POS, respectively), and nuclear magnetic resonance (NMR) spectra comparisons to reference materials synthesized in the laboratory . Liquid chromatography-mass spectrometry (LC-MS) analysis of plants exposed to 2,4,5-trichlorophenol (TCP) formed analogous compounds: 2,4,5-trichlorophenyl-beta-D-glucopyranoside (TCPG), 2,4,5-trichlorophenyl-beta-D-(6-O-malonyl)-glucopyranoside (TCPMG) and 2,4,5-trichlorophenyl-beta-D-glucopyranosyl-(6 --> 1)-beta-D-apiofuranoside (TCPAG) . Enzyme catalyzed hydrolysis with beta-glucosidase was ineffective in releasing the beta-glucosides with chemical modifications at C6 . Presence of these glucoconjugates confirmed that L . minor was capable of xenobiotic uptake and transformation . Identification of these products suggested that chlorophenols were incorporated into vacuoles and cell walls of L . minor. Nahrung, 2004 Jun, 48(3), 165 - 8 Casein gelation under simultaneous action of transglutaminase and glucono-delta-lactone; Menendez O et al.; Casein solutions (5% w/v) were treated with microbial transglutaminase (MTG) and glucono-delta-lactone (GDL) under varying conditions in order to obtain gels . Storage modulus (G') and gelation time of the gels were measured by oscillation rheometry, while protein cross-linking was determined by gel permeation chromatography . The addition of only GDL to milk resulted in very weak gels, while MTG on its own was not able to create gel networks . Simultaneous action of both ingredients led to gels, the firmness of which was linearly related to the added amount of MTG, but passed through a maximum with rising GDL concentrations . Using chromatographical analysis, increasing G' values were interrelated with the formation of MTG-induced oligomers . The gelation time was directly proportional to the GDL concentration but not influenced by the addition of MTG within the studied range of concentration. Annu Rev Phytopathol, 2004, 42, 35 - 59 Assessment and management of soil microbial community structure for disease suppression; Mazzola M; Identification of the biological properties contributing to the function of suppressive soils is a necessary first step to the management of such systems for use in the control of soilborne diseases . The development and application of molecular methods for the characterization and monitoring of soil microbial properties will enable a more rapid and detailed assessment of the biological nature of soil suppressiveness . Although suppressive soils have provided a wealth of microbial resources that have subsequently been applied for the biological control of soilborne plant pathogens, the full functional capabilities of the phenomena have not been realized in production agricultural ecosystems . Cultural practices, such as the application of soil amendments, have the capacity to enhance disease suppression, though the biological modes of action may vary from that initially resident to the soil . Plants have a distinct impact on characteristics and activity of resident soil microbial communities, and therefore play an important role in determining the development of the disease-suppressive state . Likewise, plant genotype will modulate these same biological communities, and should be considered when developing strategies to exploit the potential of such a natural disease control system . Implementation of consistently effective practices to manage this resource in an economically and environmentally feasible manner will require more detailed investigation of these biologically complex systems and refinement of currently available methodologies. Anal Chem, 2004 Aug 1, 76(15), 4452 - 8 Raman microscopic analysis of single microbial cells; Huang WE et al.; We demonstrate the utility of the Raman confocal microscope to generate a spectral profile from a single microbial cell and the use of this approach to differentiate bacterial species . In general, profiles from different bacterial taxa shared similar peaks, but the relative abundances of these components varied between different species . The use of multivariate methods subsequently allowed taxa discrimination . Further investigations revealed that the single-cell spectra could be used to differentiate between growth phases of a single species, but these differences did not obscure the overall interspecies discrimination . Finally, we tested the efficacy of the method as a means to identify cells responsible for the uptake of a specific substrate . A single strain was grown in media containing incrementally varying ratios of (13)C(6) to (12)C(6) glucose, and it was found that (13)C incorporation shifted characteristic peaks to lower wavenumbers . These findings suggest that Raman microscopy has significant potential for studies requiring the taxonomic identity and functioning of single microbial cells to be determined. Am J Rhinol, 2004 May-Jun, 18(3), 151 - 6 Multi-use Venturi nasal atomizer contamination in a clinical rhinologic practice; Dubin MG et al.; INTRODUCTION: Cross-contamination of Venturi atomizers has been reported . METHODS: In phase I, 16 atomizers were sterilized and refilled with either 1% lidocaine or 0.1% Tyzine (day 0) . During phase II, atomizers were wiped with isopropyl alcohol wipes between uses . In both phases, on days 7, 14, 21, and 28, the contents of the atomizer were sprayed onto two culture media . If a culture was found positive, cultures from the nozzle, lumen, and solution were taken and cultured in depth . RESULTS: Twelve lidocaine bottles and three Tyzine bottles were initially positive . There were more positive cultures from lidocaine bottles than Tyzine bottles at 2 weeks (p = 0.02) . After wiping with isopropyl alcohol, contamination was significantly reduced in the lidocaine bottles at 2 weeks (p = 0.02) . CONCLUSION: Microbial contamination of questionable clinical significance may occur with nasal atomizers . Regardless of this significance, wiping the devices with isopropyl alcohol can eliminate microbial growth for a 2-week interval. Prikl Biokhim Mikrobiol, 2004 May-Jun, 40(3), 366 - 9 {Effects of treatment with a composite preparation (2-chloroethylphosphonic acid and methacide) or butylated hydroxyanisole on ethylene release in apples}; Chernykh AS et al.; We studied the effect of a Russian composite preparation (2-chloroethylphosphonic acid and methacide) and butylated hydroxyanisole on ethylene release in whole fruit and peel disks of two apple cultivars, Antonovka obyknovennaya (Antonovka) and Simirenko's rennet (Simirenko) . Treatment with the composite preparation was followed by an increase in ethylene release from whole apples and peel disks . The development of microbial infection (fruit rot) in whole apples became less pronounced after the treatment . Treatment of whole apples with the antioxidant butylated hydroxyanisole (BHA) increased the intensity of ethylene release during the first subsequent days; thereafter, ethylene release decreased and was 10-15% lower than in the control on days 10-12 . In model experiments, BHA decreased ethylene release from apple peel disks below control levels as early as on day 1 after the treatment . Antonovka apples gave quick responses to the treatment . In the late-ripening Simirenko apples, the response persisted for a longer period . Our results suggest that treatment with physiologically active preparations affects ethylene release, ripening, and preservation of apples in storage. Int J Food Microbiol, 2004 Sep 1, 95(2), 169 - 75 Predictive modelling of growth and measurement of enzymatic synthesis and activity by a cocktail of Brochothrix thermosphacta; Braun P et al.; The possibility was examined of developing a predictive model that combined microbial growth (increase in cellular number) and extracellular enzyme activity of a cocktail of three strains of Brochothrix thermosphacta . Estimations of growth and enzyme activity were made within a three-dimensional matrix of conditions: temperature 2-20 degrees C, pH value 4.0-7.5 and water activity (a(w)) 0.95-0.995 . A model which predicted growth based on increases in cell number was constructed . No extracellular lipases were detected, but slight proteolytic reactions were observed . Although it was not possible to model protease activity, the growth model and information relating to enzyme activity will be made freely available in a database on the Internet. Clin Experiment Ophthalmol, 2004 Aug, 32(4), 349 - 53 Evaluation of surveillance methods for an epidemiological study of contact lens related microbial keratitis; Keay L et al.; PURPOSE: To evaluate surveillance methods in a pilot epidemiological study of contact lens related microbial keratitis (MK) cases identified by ophthalmic practitioners in Australia and New Zealand between May and August 2003 inclusive . METHODS: Twelve ophthalmologists and 55 optometrists from rural and metropolitan locations were sent a study information pack with postal reporting forms . After 2 months, practitioners were emailed a link to a website for Internet reporting . After 4 months, practitioners were prompted by email and then by telephone if a response was not received . Passive response rates were the rate of returns after posting information and emailing the website link . Active response rates included personalized email and telephone follow-up . RESULTS: Ten cases of MK were identified by optometrists and five by ophthalmologists . The passive response rates were 79% and 58% for the first and second reporting periods, respectively . There was a lower response rate in the second reporting period compared to the first (P = 0.02) . With active surveillance the response rate increased to 97% and 96% . A large proportion of optometrists (62%) and ophthalmologists (55%) used the website for at least one reporting period . Internet reporting was used by all New Zealand practitioners (5/5) . CONCLUSIONS: A surveillance study to estimate the incidence of contact lens related MK in Australia and New Zealand is feasible and acceptable . Internet-based reporting offers a reliable, rapid and cost-effective means of running a large scale, international surveillance study . Active surveillance methods are necessary to enhance reporting rates. J Appl Microbiol, 2004, 97(3), 546 - 56 Assessment of the rind microbial diversity in a farmhouse-produced vs a pasteurized industrially produced soft red-smear cheese using both cultivation and rDNA-based methods; Feurer C et al.; AIMS: The diversity of the surface flora of two French red-smear soft cheeses was examined by cultivation-dependent and cultivation-independent methods to assess their composition and to evaluate the accuracy of both approaches . METHODS AND RESULTS: Culture-independent methods used involved 16S ribosomal DNA gene cloning and sequencing and single-strand conformation polymorphism analysis (SSCP) . The culture-dependent method used involved direct culture and macroscopic observation, polymerase chain reaction of the 16S rRNA gene from DNA extracted from single colonies followed by complete sequencing of the gene . Only few species were recovered by both approaches either in the pasteurized and the farmer cheese . A large diversity of isolates or 16S rDNA sequences related to marine bacteria was identified at the surface of both cheeses . CONCLUSIONS: The results indicated that all three techniques were informative and complementary to allow a more accurate representativeness of the cheese surface biodiversity . SIGNIFICANCE AND IMPACT OF THE STUDY: Cultivation and molecular methods have to be combined in order to obtain an extended view of the bacterial populations of complex ecosystems . J Exp Med, 2004 Aug 2, 200(3), 273 - 6 Epub 2004 Jul 26. Suppressor T cells in human diseases; Baecher-Allan C et al.; Although central and peripheral tolerance are important for the regulation of human immune responses to self- and microbial antigens, an important role of suppressor CD4(+) CD25(+) T cells is suggested from the recent investigations of human autoimmune diseases and HIV . These new data provide increasing evidence that altered function of CD4(+) CD25(+) T cells may be an important factor in a wide range of human inflammatory and infectious diseases. J Microbiol Methods, 2004 Sep, 58(3), 297 - 302 First use of two-dimensional polyacrylamide gel electrophoresis to determine phylogenetic relationships; Dopson M et al.; Methods for microbial classification are not always capable of distinguishing between isolates at the species level . We have previously characterised four Ferroplasma isolates that were >98.9% similar at the 16S rDNA level, the isolates showed marked phenotypic differences, and one isolate was borderline on the 70% species boundary from DNA-DNA similarity data . In this study we have used statistical comparisons of two-dimensional polyacylamide gel electrophoresis gels for classification of closely related isolates . From the protein profile similarities an un-rooted tree was constructed that was congruent with a tree derived from DNA-DNA similarities. J Med Assoc Thai, 2004 Jun, 87(6), 697 - 703 Microbial air quality in mass transport buses and work-related illness among bus drivers of Bangkok Mass Transit Authority; Luksamijarulkul P et al.; The air quality in mass transport buses, especially air-conditioned buses may affect bus drivers who work full time . Bus numbers 16, 63, 67 and 166 of the Seventh Bus Zone of Bangkok Mass Transit Authority were randomly selected to investigate for microbial air quality . Nine air-conditioned buses and 2-4 open-air buses for each number of the bus (36 air-conditioned buses and 12 open-air buses) were included . Five points of in-bus air samples in each studied bus were collected by using the Millipore A ir Tester Totally, 180 and 60 air samples collected from air-conditioned buses and open-air buses were cultured for bacterial and fungal counts . The bus drivers who drove the studied buses were interviewed towards histories of work-related illness while working . The results revealed that the mean +/- SD of bacterial counts in the studied open-air buses ranged from 358.50 +/- 146.66 CFU/m3 to 506 +/- 137.62 CFU/m3; bus number 16 had the highest level . As well as the mean +/- SD of fungal counts which ranged from 93.33 +/- 44.83 CFU/m3 to 302 +/- 294.65 CFU/m3; bus number 166 had the highest level . Whereas, the mean +/- SD of bacterial counts in the studied air-conditioned buses ranged from 115.24 +/- 136.01 CFU/m3 to 244.69 +/- 234.85 CFU/m3; bus numbers 16 and 67 had the highest level . As well as the mean +/- SD of fungal counts which rangedfrom 18.84 +/- 39.42 CFU/m3 to 96.13 +/- 234.76 CFU/m3; bus number 166 had the highest level . When 180 and 60 studied air samples were analyzed in detail, it was found that 33.33% of the air samples from open-air buses and 6.11% of air samples from air-conditioned buses had a high level of bacterial counts (> 500 CFU/m3) while 6.67% of air samples from open-air buses and 2.78% of air samples from air-conditioned buses had a high level of fungal counts (> 500 CFU/m3) . Data from the history of work-related illnesses among the studied bus drivers showed that 91.67% of open-air bus drivers and 57.28% of air-conditioned bus drivers had symptoms of work-related illnesses, p = 0.0185. Appl Microbiol Biotechnol . 2004 Jul 20; {Epub ahead of print} Microbial population dynamics during fed-batch operation of commercially available garbage composters; Narihiro T et al.; Microbial populations in terms of quantity, quality, and activity were monitored during 2 months of start-up operation of commercially available composters for fed-batch treatment of household biowaste . All the reactors, operated at a waste-loading rate of 0.7 kg day(-1) (wet wt), showed a mass reduction efficiency of 88-93% . The core temperature in the reactors fluctuated between 31show $132# degrees show $132#C and 58show $132# degrees show $132#C due to self-heating . The pH declined during the early stage of operation and steadied at pH 7.4-9.3 during the fully acclimated stage . The moisture content was 48-63% early in the process and 30-40% at the steady state . Both direct total counts and plate counts of bacteria increased via two phases (designated phases I, II) and reached an order of magnitude of 10(11) cells g(-1) (dry wt) at the steady state . Microbial community changes during the start-up period were studied by culture-independent quinone profiling and denatured gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA . In all the reactors, ubiquinones predominated during phase I, whereas partially saturated menaquinones became predominant during phase II . This suggested that there was a drastic population shift from ubiquinone-containing Proteobacteria to Actinobacteria during the start-up period . The DGGE analysis of the bacterial community in one of the reactors also demonstrated a drastic population shift during phase I and the predominance of members of the phyla Proteobacteria and Bacteroidetes during the overall period . But this molecular analysis failed to detect actinobacterial clones from the reactor at any stage. Water Res, 2004 Aug-Sep, 38(14-15), 3449 - 57 Neural networks provide superior description of Giardia lamblia inactivation by free chlorine; Haas CN; Use of conventional models to describe data on microbial inactivation during disinfection suffers from limitations with respect to flexibility and direct quantitative incorporation of water quality variables . This paper develops an approach to analysis of such data using neural networks (NNs) . Using the data on free chlorine inactivation of Giardia lamblia previously reported, it was found that the use of an NN with a single hidden layer and four hidden neurons provided a superior (better) fit to the data with a reduced number of model parameters when compared to the fitting of this data using a conventional approach . Therefore, the use of NN models should be considered in the future assessment of microbial inactivation during disinfection . Incorporation of additional facets of the disinfection process, such a disinfectant decay, needs to be considered in subsequent development of this approach. Water Res, 2004 Aug-Sep, 38(14-15), 3442 - 8 Dynamics and influencing factors of heterotrophic bacterial utilization of acetate in constructed wetlands treating woodwaste leachate; Tao W et al.; 14C-acetate was used as a tracer of substrates for heterotrophic bacteria to investigate the dynamics and influencing factors of bacterial activity in surface flow wetlands treating woodwaste leachate . Epiphytic assimilation and mineralization of acetate increased considerably over three weeks of microbial colonization . No significant longitudinal variation in acetate uptake was found . The temporal variation of bacterial activity was neither correlated to water temperature nor acetic acid concentration . Mineralization percentage varied significantly with acetate uptake rate . Acetate uptake rates of bacterioplankton (408+/-258 microg L(-1) h(-1)), epiphyton (67.7+/-41.7 mg m(-2) h(-1)) and sedimentary bacteria (26.7+/-25.6 microg g(-1) h(-1)) were influenced by the concentrations of organic substrates and inorganic nutrients . Sedimentary bacteria contributed to the majority (55-73%) of the total heterotrophic acetate uptake, while epiphytic bacteria played only a minor role (1-3%) . The acetate mineralization percentage of sediment (16%) was much lower than that of water (55%) and epiphyton (64%) . Addition of ammonium nitrate fertilizer increased acetate uptake rates significantly, especially for sediment . No remarkable changes in mineralization percentage and the relative importance of water, sediment and epiphyton were found due to fertilization. Parasitol Today, 1994, 10(4), 154 - 7 Borrelia burgdorferi and the macrophage: Routine annihilation but occasional haven? Montgomery RR, Malawista SE. Borrelia burgdorferi, the agent for Lyme disease, has a typical pattern of bacterial interaction with phagocytes: attachment, stimulation o f release o f inflammatory mediators and, in most cases, ingestion and killing . Spirochetes are killed extracellulorly by antibody plus complement via the classical pathway, as well as by phagocytes through apparently nonoxidative means . Yet rare persistent spirochetes (mutants?) have been identified both in patients' tissues and in cells grown in vitro . Ruth Montgomery and Stephen Malawista here ask: are some Borrelia wolves in sheeps' clothing, evading macrophage anti-microbial action? Parasitol Today, 1994, 10(8), 324 - 7 MVR-PCR analysis of hypervariable DNA sequence variation; Arnot DE et al.; Techniques for accurate marking of infectious microbial agents circulating in populations would be very useful to epidemiologists . In this article, David Arnot, Cally Roper and Ali Sultan review recent progress in transferring MVR-PCR DNA finger-printing techniques from human forensic medicine to parasitology. BMC Immunol . 2004 Jul 26;5(1):15. Age-associated alterations in CXCL1 chemokine expression by murine B cells; Hu L et al.; BACKGROUND: The CXCL1 chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC), have been shown to play a role in a number of pathophysiological disease states including endotoxin-induced inflammation and bacterial meningitis . While the expression of these chemokines has been identified in a variety of cell types in the mouse, little is known about their expression with murine B-lymphocytes . RESULTS: Here, we demonstrate that highly purified murine splenic B cells are capable of expressing both MIP-2 and KC protein and mRNA upon activation with lipopolysaccharide (LPS) but not in response to anti-micro and anti-CD40 in combination with interleukin-4 (IL-4) stimulation . Moreover, these chemokines are expressed at higher levels in B cells derived from young (4 m) compared to old (24-29 m) mice . Upon fractionation into distinct B-cell subsets, we found that the expression of MIP-2 and KC by aged follicular (FO) B cells is significantly decreased when compared to the same cells from younger mice, while only MIP-2 production was found to be diminished in aged marginal zone (MZ) B cells . Interestingly, MIP-2 and KC production by newly formed (NF) B cells did not significantly differ with age . Moreover, the potential relevance of these findings is supported by the poor ability of LPS-activated aged B cells to specifically mediate CXCL1-dependent leukocyte recruitment when compared to younger B cells . CONCLUSION: Overall, the decreased expression of CXCL1 chemokines by aged B cells in response to LPS may have potential implications on the secondary recruitment of leukocytes to sites of microbial infections and inflammation possibly contributing to the increased susceptibility of older subjects to pathogen challenge. J Chem Ecol, 2004 May, 30(5), 1067 - 82 Allelochemicals of Polygonella myriophylla: chemistry and soil degradation; Weidenhamer JD et al.; Gallic acid and hydroquinone have been identified as the major allelochemicals of the known allelopathic plant Polygonella myriophylla . Both of these compounds occur in the foliage as glycosides . Quercetin and rhamnetin were identified as the major flavonoid constituents, but in much lower concentration . The behavior of gallic acid, hydroquinone, the hydroquinone glycoside arbutin, and benzoquinone in sterile and nonsterile soil from beneath Polygonella was investigated . Sterilization effectively stabilized arbutin, hydroquinone, and gallic acid . Concentrations of benzoquinone rapidly diminished in sterilized soil, and the compound was almost completely gone after 7 days . In nonsterile soils, all four compounds degraded rapidly . The order of persistence was hydroquinone > benzoquinone > gallic acid > arbutin . Persistence was rate-dependent . Arbutin degraded to hydroquinone, and benzoquinone formed as a degradation product of hydroquinone . Hydroquinone was also observed as a degradation product of benzoquinone . Benzoquinone degrades rapidly by nonmicrobial oxidative processes . These results support the hypothesis that microbial and nonmicrobial oxidative transformations of soil allelochemicals are crucial in mediating the allelopathic effects of Polygonella myriophylla. Protein Sci, 2004 Aug, 13(8), 2108 - 19 Biophysical and kinetic analysis of wild-type and site-directed mutants of the isolated and native dehydroquinate synthase domain of the AROM protein; Park A et al.; Dehydroquinate synthase (DHQS) is the N-terminal domain of the pentafunctional AROM protein that catalyses steps 2 to 7 in the shikimate pathway in microbial eukaryotes . DHQS converts 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) to dehydroquinate in a reaction that includes alcohol oxidation, phosphate beta-elimination, carbonyl reduction, ring opening, and intramolecular aldol condensation . Kinetic analysis of the isolated DHQS domains with the AROM protein showed that for the substrate DAHP the difference in Km is less than a factor of 3, that the turnover numbers differed by 24%, and that the Km for NAD+ differs by a factor of 3 . Isothermal titration calorimetry revealed that a second (inhibitory) site for divalent metal binding has an approximately 4000-fold increase in KD compared to the catalytic binding site . Inhibitor studies have suggested the enzyme could act as a simple oxidoreductase with several of the reactions occurring spontaneously, whereas structural studies have implied that DHQS participates in all steps of the reaction . Analysis of site-directed mutants experimentally test and support this latter hypothesis . Differential scanning calorimetry, circular dichroism spectroscopy, and molecular exclusion chromatography demonstrate that the mutant DHQS retain their secondary and quaternary structures and their ligand binding capacity . R130K has a 135-fold reduction in specific activity with DAHP and a greater than 1100-fold decrease in the kcat/Km ratio, whereas R130A is inactive. Swed Dent J, 2004, 28(2), 67 - 76 The microbial outcome observed with polymerase chain reaction in subjects with recurrent periodontal disease following local treatment with 25% metronidazole gel; Jansson H et al.; The aim of this study was to evaluate the microbial outcome in patients with recurrent periodontal disease following treatment with 25% metronidazole gel using the polymerase chain reaction (PCR) . Twenty subjects in a maintenance care program but with recurrent periodontal disease participated . Three months after scaling and root planing a total of 40 sites, 2 in each patient, with pocket probing depth of > or = 5 mm were selected . One site randomly selected was treated with 25% metronidazole gel (test) and the other site with a placebo gel (control) . A bacterial sample was collected on paperpoint from each test and control site at baseline and 12 weeks after treatment . The following pathogens were analysed and detected with PCR:Actinobacillus actinomycetemcomitans (A.a.), Porphyromonas gingivalis (P.g.) and Prevotella nigrescens (P.n.) . At baseline, A.a., P.g . and P.n . were detected in 30, 60 and 70% of all test sites and in 32, 58 and 21% of all control sites . There was a statistically significant difference between the test and control sites for P.n . at baseline . The major difference after treatment with 25% metronidazole gel was the increase of positive control sites for P.g . and P.n . However, there were no statistically significant differences in the occurrence rate of A.a., P.g . and P.n . at test and control sites after treatment . This study has shown that 25% metronidazole gel treatment did not seem to influence the microbial outcome, when PCR was used to analyse the presence/absence of A.a., P.g . and P.n . in this group of subjects with recurrent periodontal disease. EMBO Rep, 2004 Aug, 5(8), 825 - 30 Epub 2004 Jul 23. HMGB1 is an endogenous immune adjuvant released by necrotic cells; Rovere-Querini P et al.; Immune responses against pathogens require that microbial components promote the activation of antigen-presenting cells (APCs) . Autoimmune diseases and graft rejections occur in the absence of pathogens; in these conditions, endogenous molecules, the so-called 'innate adjuvants', activate APCs . Necrotic cells contain and release innate adjuvants; necrotic cells also release high-mobility group B1 protein (HMGB1), an abundant and conserved constituent of vertebrate nuclei . Here, we show that necrotic HMGB1(-/-) cells have a reduced ability to activate APCs, and HMGB1 blockade reduces the activation induced by necrotic wild-type cell supernatants . In vivo, HMGB1 enhances the primary antibody responses to soluble antigens and transforms poorly immunogenic apoptotic lymphoma cells into efficient vaccines. J Inorg Biochem, 2004 Aug, 98(8), 1331 - 7 Inhibition of alcohol dehydrogenase by bismuth; Jin L et al.; Bismuth compounds have been widely used for the treatment of ulcers and Helicobacter pylori infection, and enzyme inhibition was thought to be crucial for bismuth anti-microbial activity . We have investigated the interaction of colloidal bismuth subcitrate (CBS) with alcohol dehydrogenase and our results demonstrate that bismuth can effectively inhibit the enzyme . Kinetic analysis revealed that CBS acted as a non-competitive inhibitor of yeast alcohol dehydrogenase . Both UV-vis and fluorescence data show that interaction of CBS with the enzyme exhibits biphasic processes . Bismuth can replace only half of Zn(II) from the enzyme (i.e., about one Zn(II) per monomer) . Surprisingly, binding of CBS also induces the enzyme dissociation from its native form, tetramer into dimers . The inhibition of Bi(III) on the enzyme is probably due to its direct interference with the zinc sites . This study is likely to provide an insight into the mechanism of action of bismuth drugs. Clin Exp Immunol, 2004 Aug, 137(2), 253 - 62 DNA vaccination encoding glutamic acid decarboxylase can enhance insulitis and diabetes in correlation with a specific Th2/3 CD4 T cell response in non-obese diabetic mice; Gauvrit A et al.; DNA vaccination encoding beta cell autoantigens has been shown very recently to prevent type I diabetes in non-obese diabetic (NOD) mice . However, DNA vaccination encoding microbial or reporter antigens is known to induce specific long-lasting CD4 Th1 and strong cytolytic CD8 T cell responses . As this immune phenotype is associated strongly with beta cell destruction leading to diabetes, we have chosen to study the effects of plasmids encoding glutamic acid decarboxylase (GAD), a crucial beta cell autoantigen, in female NOD mice that developed a 'moderate' diabetes incidence . In the present study, 3-week-old female NOD mice were vaccinated twice in tibialis muscles with plasmid-DNA encoding 65-kDa GAD or betagalactosidase . In GAD-DNA immunized mice, diabetes cumulative incidence (P < 3.10(-3)) and insulitis (P < 7.10(-3)) increased significantly . Simultaneously, DNA immunization induced GAD-specific CD4 T cells secreting interleukin (IL)-4 (P < 0.05) and transforming growth factor (TGF)-beta (P = 0.03) . These cells were detected in spleen and in pancreatic lymph nodes . Furthermore, vaccination produced high amounts of Th2 cytokine-related IgG1 (P < 3.10(-3)) and TGF-beta-related IgG2b to GAD (P = 0.015) . Surprisingly, diabetes onset was correlated positively with Th2-related GAD-specific IgG1 (P < 10(-4)) and TGF-beta-related IgG2b (P < 3.10(-3)) . Moreover, pancreatic lesions resembled Th2-related allergic inflammation . These results indicate, for the first time, that GAD-DNA vaccination could increase insulitis and diabetes in NOD mice . In addition, our study suggests that Th2/3 cells may have potentiated beta cell injury. Immunology, 2004 Aug, 112(4), 651 - 60 Enteroaggregative Escherichia coli flagellin-induced interleukin-8 secretion requires Toll-like receptor 5-dependent p38 MAP kinase activation; Khan MA et al.; Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen that causes acute and chronic diarrhoea in a number of clinical settings . EAEC diarrhoea involves bacterial aggregation, adherence to intestinal epithelial cells and elaboration of several toxigenic bacterial mediators . Flagellin (FliC-EAEC), a major bacterial surface protein of EAEC, causes interleukin (IL)-8 release from several epithelial cell lines . The host response to flagellins from E . coli and several other bacteria is mediated by Toll-like receptor 5 (TLR5), which signals through nuclear factor kappa B (NF-kappaB) to induce transcription of pro-inflammatory cytokines . p38 mitogen-activating protein (MAP) kinase (MAPK) is a member of a family of stress-related kinases that influences a diverse range of cellular functions including host inflammatory responses to microbial products . We studied the role of p38 MAPK in FliC-EAEC-induced IL-8 secretion from Caco-2 human intestinal epithelial cells and THP-1 human monocytic cells . We found that IL-8 secretion from both cell types is dependent on p38 MAPK, which is phospho-activated in response to FliC-EAEC . The role of TLR5 in p38 MAPK-dependent IL-8 secretion was verified in HEp-2 cells transiently transfected with a TLR5 expression construct . Activation of interleukin-1 receptor-associated kinase (IRAK) was also observed in Caco-2 and TLR5-transfected HEp-2 cells after exposure to FliC-EAEC . Finally, we demonstrated that pharmacological inhibition of p38 MAPK reduced IL-8 transcription and mRNA levels, but did not affect NF-kappaB activation . Collectively, our results suggest that TLR5 mediates p38 MAPK-dependent IL-8 secretion from epithelial and monocytic cells incubated with FliC-EAEC, and that this effect requires IL-8 promoter activation independent of NF-kappaB nuclear migration. Biochem Soc Trans, 2004 Aug, 32(Pt 4), 629 - 32 Functional domains of HSP70 stimulate generation of cytokines and chemokines, maturation of dendritic cells and adjuvanticity; Lehner T et al.; Microbial HSP70 (heat-shock protein 70) consists of three functionally distinct domains: an N-terminal 44 kDa ATPase portion (amino acids 1-358), followed by an 18 kDa peptide-binding domain (amino acids 359-494) and a C-terminal 10 kDa fragment (amino acids 495-609) . Immunological functions of these three different domains in stimulating monocytes and dendritic cells have not been fully defined . However, the C-terminal portion (amino acids 359-610) stimulates the production of CC chemokines, IL-12 (interleukin-12), TNFalpha(tumour necrosis factor alpha), NO and maturation of dendritic cells and also functions as an adjuvant in the induction of immune responses . In contrast, the ATPase domain of microbial HSP70 mostly lacks these functions . Since the receptor for HSP70 is CD40, which with its CD40 ligand constitutes a major co-stimulatory pathway in the interaction between antigen-presenting cells and T-cells, HSP70 may function as an alternative ligand to CD40L . HSP70-CD40 interaction has been demonstrated in non-human primates to play a role in HIV infection, in protection against Mycobacterium tuberculosis and in conversion of tolerance to immunity. Biochem Soc Trans, 2004 Aug, 32(Pt 4), 622 - 5 Peptides complexed with the protein HSP70 generate efficient human cytolytic T-lymphocyte responses; Javid B et al.; Microbial HSPs (heat-shock proteins) are implicated in the induction of the innate and adaptive arms of the immune response . We set out to determine whether peptides complexed with HSP70 generate efficient CTL (cytolytic T-lymphocyte) responses . Human dendritic cells pulsed with peptide-loaded microbial HSP70 complexes generate potent antigen-specific CTL responses . Using fluorescence anisotropy, we have calculated the peptide-binding affinity of mycobacterial HSP70 (K(D)=14 microM) and show that 120 pM HSP70-bound peptide is sufficient to generate a peptide-specific CTL response that is four orders of magnitude more efficient than the peptide alone . Through the generation of mycobacterial HSP70 truncations, we find that the minimal 136 amino acid, mycobacterial HSP70 peptide-binding domain is sufficient to generate CTL responses . The design of an HSP70 mutant, in which the peptide-binding site of HSP70 is filled with a bulky hydrophobic residue, leads to a large decrease in the peptide-binding affinity . This mutant HSP70 retains stimulatory capacity but is unable to generate CTL and has separated antigen delivery from immunostimulation of dendritic cells. Thromb Haemost, 2004 Aug, 92(2), 288 - 97 Aberrant mucosal wound repair in the absence of secretory leukocyte protease inhibitor; Angelov N et al.; Secretory leukocyte protease inhibitor (SLPI) is a cationic serine protease inhibitor with anti-microbial and anti-inflammatory properties found in large quantities in mucosal fluids, including saliva . SLPI is expressed during cutaneous wound healing, however, its role in oral wound repair is unknown . We have used a novel approach involving a murine buccal mucosal acute wound model to investigate the role of SLPI in oral healing . In parallel to the observed cutaneous healing phenotype, an absence of SLPI results in markedly impaired oral wound healing associated with increased inflammation and raised elastase activity . Moreover, matrix deposition was decreased, while MMP activity was enhanced in the oral SLPI null wounds suggesting deregulated proteolysis . Intriguingly, regardless of genotype, reduced collagen deposition was observed in oral compared to dermal wounds, associated with reduced TGF-beta expression and decreased fibroblast collagen expression in vitro . We propose that SLPI is a pivotal endogenous factor necessary for optimal tissue repair including intra-oral wound healing . In addition, our model provides a unique opportunity to delineate the cellular and molecular mechanisms underlying the differences between dermal scarring and oral scar-free healing. Lab Chip, 2004 Aug, 4(4), 337 - 41 Epub 2004 Apr 05. Filter-based microfluidic device as a platform for immunofluorescent assay of microbial cells; Zhu L et al.; A filter-based microfluidic device was combined with immunofluorescent labeling as a platform to rapidly detect microbial cells . The coin-sized device consisted of micro-chambers, micro-channels and filter weirs (gap = 1-2 microm), and was demonstrated to effectively trap and concentrate microbial cells (i.e., Cryptosporidium parvum and Giardia lamblia), which were larger in size than the weir gap . After sample injection, a staining solution containing fluorescently-labeled antibodies was continuously provided into the device (flow rate = 20 microl min(-1)) to flush the microbial cells toward the weirs and to accelerate the fluorescent labeling reaction . Using a staining solution that was 10 to 100 times more dilute than the recommended concentration used in a conventional glass method, those target cells with a fluorescent signal-to-noise ratio of 12 could be microscopically observed at single-cell level within 2 to 5 min prior to secondary washing. J Biochem (Tokyo), 2004 Jul, 136(1), 81 - 7 Protective properties of neoechinulin A against SIN-1-induced neuronal cell death; Maruyama K et al.; Peroxynitrite (ONOO-) is thought to be involved in the neurodegenerative process . To screen for neuroprotective compounds against ONOO- -induced cell death, we developed 96-well based assay procedures for measuring surviving cell numbers under oxidative stress caused by 3-(4-morpholinyl) sydnonimine hydrochloride (SIN-1), a generator of ONOO-, and sodium N,N-dietyldithiocarbamate trihydrate (DDC), an inhibitor of Cu/Zn superoxide (O2-) dismutase . Using these procedures, we obtained a microbial metabolite that rescued primary neuronal cells from SIN-1-induced damage, but not from DDC-induced damage . By NMR analysis, the compound was identified as neoechinulin A, an antioxidant compound that suppresses lipid oxidation . We found that the compound rescues neuronal cells such as primary neuronal cells and differentiated PC12 cells from damage induced by extracellular ONOO- . However, non-neuronal cells, undifferentiated PC12 cells and cells of the fibroblast cell line 3Y1 were not rescued . Neoechinulin A has scavenging, neurotrophic factor-like and anti-apoptotic activities . This compound specifically scavenges ONOO-, but not O2- or nitric oxide (NO) . Similar to known neuroprotective substances such as nerve growth factor and extracts of Gingko biloba leaves, neoechinulin A inhibits the SIN-1-induced activation of caspase-3-like proteases and increases NADH-dehydrogenase activity . These results suggest that neoechinulin A might be useful for protecting against neuronal cell death in neurodegenerative diseases. Chemosphere, 2004 Sep, 56(10), 999 - 1009 Abiotic reductive dechlorination of chlorinated ethylenes by iron-bearing phyllosilicates; Lee W et al.; Abiotic reductive dechlorination of chlorinated ethylenes (tetrachloroethylene (PCE), trichloroethylene (TCE), cis-dichloroethylene (c-DCE), and vinylchloride (VC)) by iron-bearing phyllosilicates (biotite, vermiculite, and montmorillonite) was characterized to obtain better understanding of the behavior of these contaminants in systems undergoing remediation by natural attenuation and redox manipulation . Batch experiments were conducted to evaluate dechlorination kinetics and some experiments were conducted with addition of Fe(II) to simulate impact of microbial iron reduction . A modified Langmuir-Hinshelwood kinetic model adequately described reductive dechlorination kinetics of target organics by the iron-bearing phyllosilicates . The rate constants stayed between 0.08 (+/-10.4%) and 0.401 (+/-8.1%) day(-1) and the specific initial reductive capacity of iron-bearing phyllosilicates for chlorinated ethylenes stayed between 0.177 (+/-6.1%) and 1.06 (+/-7.1%) microM g(-1) . The rate constants for the reductive dechlorination of TCE at reactive biotite surface increased as pH (5.5-8.5) and concentration of sorbed Fe(II) (0-0.15 mM g(-1)) increased . The appropriateness of the model is supported by the fact that the rate constants were independent of solid concentration (0.0085-0.17 g g(-1)) and initial TCE concentration (0.15-0.60 mM) . Biotite had the greatest rate constant among the phyllosilicates both with and without Fe(II) addition . The rate constants were increased by a factor of 1.4-2.5 by Fe(II) addition . Between 1.8% and 36% of chlorinated ethylenes removed were partitioned to the phyllosilicates . Chloride was produced as a product of degradation and no chlorinated intermediates were observed throughout the experiment. Cloning Stem Cells, 2004, 6(2), 172 - 7 Nutritional value of milk and meat products derived from cloning; Tome D et al.; The development and use of milk and meat products derived from cloning depends on their safety and on the nutritional advantages they can confer to the products as perceived by consumers . The development of such products thus implies (i) to demonstrate their safety and security, (ii) to show that their nutritional value is equivalent to the traditional products, and (iii) to identify the conditions under which cloning could allow additional nutritional and health benefit in comparison to traditional products for the consumers . Both milk and meat products are a source of high quality protein as determined from their protein content and essential amino acid profile . Milk is a source of calcium, phosphorus, zinc, magnesium and vitamin B2 and B12 . Meat is a source of iron, zinc and vitamin B12 . An important issue regarding the nutritional quality of meat and milk is the level and quality of fat which usually present a high content in saturated fat and some modification of the fat fraction could improve the nutritional quality of the products . The role of the dietary proteins as potential allergens has to be taken into account and an important aspect regarding this question is to evaluate whether the cloning does not produce the appearance of novel allergenic structures . The presence of bio-activities associated to specific components of milk (lactoferrin, immunoglobulins, growth factors, anti-microbial components) also represents a promising development . Preliminary results obtained in rats fed cow's milk or meat-based diets prepared from control animals or from animals derived from cloning did not show any difference between control and cloning-derived products. Am Nat, 2004 Jun, 163(6), E126 - 35 Epub 2004 May 06. Evolution of cross-feeding in microbial populations; Pfeiffer T; Although limited by a single resource, microbial populations that grow for long periods in continuous culture (chemostat) frequently evolve stable polymorphisms . These polymorphisms may be maintained by cross-feeding, where one strain partially degrades the primary energy resource and excretes an intermediate that is used as an energy resource by a second strain . It is unclear what selective advantage cross-feeding strains have over a single competitor that completely degrades the primary resource . Here we show that cross-feeding may evolve in microbial populations as a consequence of the following optimization principles: the rate of ATP production is maximized, the concentration of enzymes of the pathway is minimized, and the concentration of intermediates of the pathway is minimized. J Immunol, 2004 Aug 1, 173(3), 2060 - 7 Platelet factor 4/CXCL4 induces phagocytosis and the generation of reactive oxygen metabolites in mononuclear phagocytes independently of Gi protein activation or intracellular calcium transients; Pervushina O et al.; Platelet factor 4 (PF-4), a platelet-derived CXC chemokine, is known to prevent human monocytes from apoptosis and to promote differentiation of these cells into HLA-DR(-) macrophages . In this study, we investigated the role of PF-4 in the control of acute monocyte proinflammatory responses involved in the direct combat of microbial invaders . We show that PF-4 increases monocyte phagocytosis and provokes a strong formation of oxygen radicals but lacks a chemotactic activity in these cells . Compared with FMLP, PF-4-induced oxidative burst was later in its onset but was remarkably longer in its duration (lasting for up to 60 min) . Furthermore, in PF-4-prestimulated cells, FMLP- as well as RANTES-induced burst responses became synergistically enhanced . As we could show, PF-4-mediated oxidative burst in monocytes does not involve Gi proteins, elevation of intracellular free calcium concentrations, or binding to CXCR3B, a novel PF-4 receptor recently discovered on endothelial cells . Moreover, we found that PF-4 acts on macrophages in a dual manner . On the one hand, very similar to GM-CSF or M-CSF, PF-4 treatment of monocytes generates macrophages with a high capacity for unspecific phagocytosis . On the other hand, short term priming of GM-CSF-induced human macrophages with PF-4 substantially increases their capability for particle ingestion and oxidative burst . A comparable effect was also observed in murine bone marrow-derived macrophages, indicating cross-reactivity of human PF-4 between both species . Taken together, PF-4 may play a crucial role in the induction and maintenance of an unspecific immune response. J Immunol, 2004 Aug 1, 173(3), 1966 - 77 Characterization of a Mycobacterium tuberculosis peptide that is recognized by human CD4+ and CD8+ T cells in the context of multiple HLA alleles; Shams H et al.; The secreted Mycobacterium tuberculosis 10-kDa culture filtrate protein (CFP)10 is a potent T cell Ag that is recognized by a high percentage of persons infected with M . tuberculosis . We determined the molecular basis for this widespread recognition by identifying and characterizing a 15-mer peptide, CFP10(71-85), that elicited IFN-gamma production and CTL activity by both CD4(+) and CD8(+) T cells from persons expressing multiple MHC class II and class I molecules, respectively . CFP10(71-85) contained at least two epitopes, one of 10 aa (peptide T1) and another of 9 aa (peptide T6) . T1 was recognized by CD4(+) cells in the context of DRB1*04, DR5*0101, and DQB1*03, and by CD8(+) cells of A2(+) donors . T6 elicited responses by CD4(+) cells in the context of DRB1*04 and DQB1*03, and by CD8(+) cells of B35(+) donors . Deleting a single amino acid from the amino or carboxy terminus of either peptide markedly reduced IFN-gamma production, suggesting that they are minimal epitopes for both CD4(+) and CD8(+) cells . As far as we are aware, these are the shortest microbial peptides that have been found to elicit responses by both T cell subpopulations . The capacity of CFP10(71-85) to stimulate IFN-gamma production and CTL activity by CD4(+) and CD8(+) cells from persons expressing a spectrum of MHC molecules suggests that this peptide is an excellent candidate for inclusion in a subunit antituberculosis vaccine. J Neuroimmunol, 2004 Aug, 153(1-2), 7 - 15 Innate immunity in the retina: Toll-like receptor (TLR) signaling in human retinal pigment epithelial cells; Kumar MV et al.; Toll-like receptors (TLRs) are crucial components of innate immunity that participate in host defense against microbial pathogens . We evaluated the expression and function of TLRs in human retinal pigment epithelial (RPE) cells . Real time PCR analysis revealed gene expression for TLRs 1-7, 9, and 10 in RPE cells . TLRs 1 and 3 were the most highly expressed TLRs . Protein expression for TLRs 2, 3, and 4 was observed on RPE cells and this expression was augmented by treatment with poly I:C or interferon-gamma (IFN-gamma) . TLR 3 is the receptor for dsRNA, an intermediate of virus replication . Because RPE cells express TLR 3 and are frequently the site of virus replication within the retina, we evaluated TLR 3 signaling . RPE cells treated with poly I:C produced IFN-beta but not IFN-alpha, and this was inhibited by the treatment of RPE cells with anti-TLR 3 antibody . Human recombinant IFN-beta was shown to be biologically active on RPE cells by inhibiting viral replication . Poly I:C treatment of RPE resulted in an increase in the production of IL-6, IL-8, MCP-1, and sICAM-1 . The presence of TLRs on RPE cells and the resultant TLR signaling in RPE cells suggest that these molecules may play an important role in innate and adaptive immune responses within the retina. Brain Behav Immun, 2004 Sep, 18(5), 458 - 67 Acute stress effects on local Il-1beta responses to pathogens in a human in vivo model; Deinzer R et al.; The combined effects of stress and antigen on interleukin-1beta (Il-1beta) have rarely been studied locally at the site of microbial challenges in vivo, so far . We here propose a model for the analysis of such effects in humans and examine its utility for acute stress trials . Twelve students (6 male, 6 female) refrained from oral hygiene in two antagonistic quadrants for 28 days to allow for increasing bacterial stimulation of the respective gingival sites due to accumulation of microbial plaque . Good oral hygiene was maintained in the remaining quadrants . At day 27 and 28 students were subjected to either stress ('public speech') or a control condition, in a cross-over design . Samples of gingival crevicular fluid (GCF) which emerges between the tooth surface and the gingival epithelium as transudate of healthy and exudate of inflamed gingival tissue, were taken immediately after stress and 60 min later for Il-1beta analysis . Salivary cortisol was assessed to prove the validity of the stress protocol . Stress induced a profound increase of salivary cortisol (p=.001) . Repeated measures (stress x time x hygiene) ANOVA with gender as between factor revealed significant stress (p=.014) and hygiene (p=.038) effects on GCF-Il-1beta concentrations and tentatively significant hygiene x time (p = .097) and stress x time x hygiene x gender (p=.107) interactions . Stress induced an increase of Il-1beta as did plaque accumulation . The merits of the proposed model are discussed . It is concluded that it is well suited for the assessment of the effects of stress on inflammatory responses in vivo in humans. J Struct Funct Genomics, 2004, 5(1-2), 111 - 8 Automation of protein purification for structural genomics; Kim Y et al.; A critical issue in structural genomics, and in structural biology in general, is the availability of high-quality samples . The additional challenge in structural genomics is the need to produce high numbers of proteins with low sequence similarities and poorly characterized or unknown properties . 'Structural-biology-grade' proteins must be generated in a quantity and quality suitable for structure determination experiments using X-ray crystallography or nuclear magnetic resonance (NMR) . The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples . The purification procedure must yield a homogeneous protein and must be highly reproducible in order to supply milligram quantities of protein and/or its derivative containing marker atom(s) . At the Midwest Center for Structural Genomics we have developed protocols for high-throughput protein purification . These protocols have been implemented on AKTA EXPLORER 3D and AKTA FPLC 3D workstations capable of performing multidimensional chromatography . The automated chromatography has been successfully applied to many soluble proteins of microbial origin . Various MCSG purification strategies, their implementation, and their success rates are discussed in this paper. Eur J Obstet Gynecol Reprod Biol, 2004 Aug 10, 115(2), 159 - 60 Infectious and inflammatory mechanisms in preterm birth and cerebral palsy; Jacobsson B; OBJECTIVE: In a thesis examine infectious and inflammatory mechanisms involved in preterm birth and cerebral palsy . STUDY DESIGN: Four cross-sectional studies and a case control study . RESULTS: Microbial invasion of the amniotic cavity and inflammation in this population of Swedish women in preterm labor and preterm prelabor rupture of membranes was similar to that reported in data from populations with a higher incidence of preterm birth . Our data support an association between antenatal infection/inflammation and cerebral palsy . CONCLUSIONS: Infectious and inflammatory mechanisms are involved in preterm birth and cerebral palsy in a population with low incidence of preterm birth. J Chromatogr B Analyt Technol Biomed Life Sci, 2004 Sep 5, 808(2), 249 - 54 Characterization of spirochetal isolates from arthropods collected in South Moravia, Czech Republic, using fatty acid methyl esters analysis; Cechova L et al.; Aim of this study was to evaluate cellular fatty acid analysis for characterization of spirochetes . Strains were isolated from arthropods collected in South Moravia, Czech Republic . Fatty acid methyl esters (FAME) profile was determined for five Borrelia burgdorferi sensu lato (s.l.) strains isolated from Ixodes ricinus ticks, one "Spironema culicis" strain recovered from mosquito Culex pipiens and seven spirochetal strains (not identified yet) isolated from mosquitoes and blackflies . Analysis was performed using a gas chromatography column in conjunction with Microbial Identification System Sherlock (MIDI Inc., Newark, DE, USA) . Results obtained on the basis of cluster analysis of FAME profiles showed, that the B . burgdorferi sensu lato isolates could be well separated from other spirochetal isolates . We recommended method used in this study as a useful tool for preliminary identification of spirochetes isolated from ticks and dipterans. Rheum Dis Clin North Am, 2004 Aug, 30(3), 559 - 74, ix The stimulation of Toll-like receptors by nuclear antigens: a link between apoptosis and autoimmunity; Marshak-Rothstein A et al.; As immunologists have long understood, effective responses to foreign antigens require adjuvants . It is now apparent that the initiation of autoimmune disease is comparably facilitated by adjuvant activity . In the case of antinuclear antibodies, it seems that DNA itself can serve as an endogenous adjuvant . Similar to many of the microbial adjuvants, mammalian DNA mediates its effect through a Toll-like receptor--in this case, TLR9. J Steroid Biochem Mol Biol, 2004 Jun, 91(1-2), 87 - 98 Novel cytotoxic bufadienolides derived from bufalin by microbial hydroxylation and their structure-activity relationships; Ye M et al.; Microbial transformation was used to prepare novel cytotoxic bufadienolides . Twelve products (3-14) were obtained from bufalin (1) by the fungus Mucor spinosus . Their structures were elucidated by high-resolution mass spectroscopy (HR-MS) and extensive NMR techniques, including 1H NMR, 13C NMR, DEPT, 1H-1H correlation spectroscopy (COSY), two dimensional nuclear Overhauser effect correlation spectroscopy (NOESY), heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond coherence (HMBC) . Compounds 3, 4, 9 and 11-14 are new mono- or dihydroxylated derivatives of bufalin with novel oxyfunctionalities at C-1beta, C-7beta, C-11beta, C-12beta and C-16alpha positions . The in vitro cytotoxic activities against human cancer cell lines of 3-14, together with 16 biotransformed products derived from cinobufagin (15-30) were determined by the MTT method, and their structure-activity relationships (SAR) were discussed. Biochemistry, 2004 Ju