J Proteome Res, 2003 Jul-Aug, 2(4), 441 - 3 Living nanovesicles--chemical and physical survival strategies of primordial biosystems; Sommer AP et al.; Life on Earth and Mars could have started with self-assembled nanovesicles similar to the present nanobacteria (NB) . To resist extreme environmental stress situations and periods of nutritional deprivation, nanovesicles would have had a chemical composition protected by a closed mineralized compartment, facilitating their development in a primordial soup, or other early wet environment . Their survivability would have been enhanced if they had mechanisms for metabolic communication, and an ability to collect primordially available energy forms . Here, we establish an irreducible model system for life formation starting with NB. Parasitol Res, 2003 Jun, 90 Suppl 2, S55 - 62 Epub 2003 Feb 20. Natural products as antiparasitic drugs; Kayser O et al.; Natural products are not only the basis for traditional or ethnic medicine . Only recently, they have provided highly successful new drugs such as Artemisinin . Furthermore, screening natural products found in all sorts of environments such as the deep sea, rain forests and hot springs, and produced by all sorts of organisms ranging from bacteria, fungi and plants to protozoa, sponges and invertebrates, is a highly competitive field where all of the major pharmaceutical companies are encountered . Already, many new natural product groups have revealed antiparasitic properties of surprising efficacy and selectivity, as will be shown in this review for plant-derived alkaloids, terpenes and phenolics . Many novel lead structures, however, have severe chemico-physical drawbacks such as poor solubility . Here, innovative drug formulations and carrier systems might help, as discussed by the authors in another article of this series. J Contam Hydrol, 2003 Sep, 65(3-4), 269 - 91 Natural attenuation of xenobiotic organic compounds in a landfill leachate plume (Vejen, Denmark); Baun A et al.; Demonstration of natural attenuation of xenobiotic organic compounds (XOCs) in landfill leachate plumes is a difficult task and still an emerging discipline within groundwater remediation . One of the early studies was made at the Vejen Landfill in Denmark in the late 1980s, which suggested that natural attenuation of XOCs took place under strongly anaerobic conditions within the first 150 m of the leachate plume . This paper reports on a revisit to the same plume 10 years later . Within the strongly anaerobic part of the plume, 49 groundwater samples were characterized with respect to redox-sensitive species and XOCs . The analytical procedures have been developed further and more compounds and lower detection limits were observed this time . In addition, the samples were screened for degradation intermediates and for toxicity . The plume showed fairly stationary features over the 10-year period except that the XOC level as well as the level of chloride and nonvolatile organic carbon (NVOC) in the plume had decreased somewhat . Most of the compounds studied were subject to degradation in addition to dilution . Exceptions were benzene, the herbicide Mecoprop (MCPP), and NVOC . In the early study, NVOC seemed to degrade in the first part of the plume, but this was no longer the case . Benzyl succinic acid (BSA) was for the first time identified in a leachate plume as a direct indicator, and as the only intermediate of toluene degradation . Toxicity measurements on solid phase-extracted (SPE) samples revealed that toxic compounds not analytically identified were still present in the plume, suggesting that toxicity measurements could be helpful in assessing natural attenuation in leachate plumes. Leg Med (Tokyo), 2002 Sep, 4(3), 139 - 55 The evolution and formation of RH genes; Okuda H et al.; The Rh system clinically is one of the important blood groups . The major Rh antigens, which are constituted by over 40 types, are RhD, RhC/c, and RhE/e . Furthermore, Rh blood group system is characterized by the existence of many variants.It was considered that Rh blood group system was encoded on two genes termed the RHCE and RHD, which are composed of ten exons, respectively . It is inferred that the RHD gene encodes the RhD antigen and that the RHCE gene encodes the Rh C/c and RhE/e antigens . There are RHce, RHCe, RHcE and RHCE alleles as polymorphisms of RHCE gene . In 2000, the entire nucleotide sequences in all introns of both the RHD and RHCE genes were determined . Due to the new findings on RH genes, it is thought that multiple recombination (and/or gene conversion), nucleotide substitutions, small nucleotide gaps, replication slippage of microsatellite, large nucleotide gaps (due to Alu sequence) and the high level of the homology (%) between both RH genes are the important factors in the formation and evolution of both RH genes and Rh variants . Based on the advance of human genome project, the new interpretations on the evolution and formation of RH genes and Rh variants will be performed.Human Rh family (superfamily) and its counterparts in primates, mammals, fish, amphibians, bacteria, lower eukaryotes, archaea and plants have been identified . A lot of findings have been accumulated in their evolution and function . As gene conversions or recombination events confuse the phylogenetic tree of human RH genes and their counterparts, careful attention is necessary for researchers to calculate the time of gene duplication and to discuss the evolution of Rh family and its counterparts.Rh genotyping methods will never be perfect and both the clinicians and researchers have to recognize the limitation of Rh genotyping, especially RhD genotyping, because new Rh variants must have formed continually . In applying the Rh genotyping to clinical medicine, especially transfusion medicine, it is necessary to compare and examine the serological (phenotypic) data in Rh blood group system with caution. Hunan Yi Ke Da Xue Xue Bao, 2003 Apr, 28(2), 174 - 6 {Hela cells secrete interleukin-8 and interleukin-10 response to Chlamydia trachomatis entry}; Yu JL et al.; OBJECTIVE: To investigate the effect of Chlamydia trachomatis serotype K infection on productions of IL-8 and IL-10 in supernatant of cultured Hela cells . METHODS: The levels of IL-8 and IL-10 productions were detected by cell culture method and ELISA assay . RESULTS: The amount of IL-8 and IL-10 in the cell culture supernatant rose with the increasing doses of Chlamydia trachomatis . The concentrations of IL-8 and IL-10 began to increase obviously at 24 h, and reached their highest level at 72 h, and then decreased . Heat-inactivated Chlamydia trachomatis induced lower levels of IL-8 and IL-10 production than live bacteria in vitro . CONCLUSION: The Chlamydia trachomatis can induce the secretion of IL-8 and IL-10 of host cells in a dose and time dependent manner and the intracellar replication of the bacteria is required for enhancing IL-8 and IL-10 productions by the infected cells. Infect Immun, 2003 Sep, 71(9), 5418 - 21 Signaling via Toll-like receptor 5 can initiate inflammatory mediator production by murine osteoblasts; Madrazo DR et al.; Murine osteoblasts express Toll-like receptor 5 (TLR5), and this expression is upregulated following exposure to bacteria or to the TLR5 agonist, flagellin . Importantly, flagellin activates transcriptional regulators and elicits proinflammatory cytokine production, suggesting TLR5 functionality . TLR5 may represent an important mechanism underlying the recognition of bacterial pathogens by osteoblasts during bone infections. Infect Immun, 2003 Sep, 71(9), 5012 - 20 Regulation of expression of the paralogous Mlp family in Borrelia burgdorferi; Yang XF et al.; The Mlp (multicopy lipoproteins) family is one of many paralogous protein families in Borrelia burgdorferi . To examine the extent to which the 10 members of the Mlp family in B . burgdorferi strain 297 might be differentially regulated, antibodies specific for each of the Mlps were developed and used to analyze the protein expression profiles of individual Mlps when B . burgdorferi replicated under various cultivation conditions . All of the Mlps were upregulated coordinately when B . burgdorferi was cultivated at either elevated temperature, reduced culture pH, or increased spirochete cell density . Inasmuch as the expression of OspC is influenced by a novel RpoN-RpoS regulatory pathway, similar induction patterns for OspC and the Mlp paralogs prompted an assessment of whether the RpoN-RpoS pathway also was involved in Mlp expression . In contrast to wild-type B . burgdorferi, both RpoN- and RpoS-deficient mutants were unable to upregulate OspC or the Mlp paralogs when grown at lower pH (6.8), indicating that pH-mediated regulation of OspC and Mlp paralogs is dependent on the RpoN-RpoS pathway . However, when RpoN- or RpoS-deficient mutants were shifted from 23 degrees C to 37 degrees C or were cultivated to higher spirochete densities, some induction of the Mlps still occurred, whereas OspC expression was abolished . The combined findings suggest that the Mlp paralogs are coordinately regulated as a family and have an expression profile similar, but not identical, to that of OspC . Although Mlp expression as a family is influenced by the RpoN-RpoS regulatory pathway, there exists at least one additional layer of gene regulation, yet to be elucidated, contributing to Mlp expression in B . burgdorferi. Infect Immun, 2003 Sep, 71(9), 4936 - 42 Bordetella pertussis acquires resistance to complement-mediated killing in vivo; Pishko EJ et al.; In order to initially colonize a host, bacteria must avoid various components of the innate immune system, one of which is complement . The genus Bordetella includes three closely related species that differ in their ability to resist complement-mediated killing . Bordetella parapertussis and Bordetella bronchiseptica resist killing in naive serum, a characteristic that may aid in efficient respiratory tract colonization and has been attributed to expression of O antigen . Bordetella pertussis lacks O antigen and is sensitive to naive serum in vitro, yet it also efficiently colonizes the respiratory tract . Based on these observations, we hypothesized that B . pertussis may have an alternate mechanism to resist complement in vivo . While a number of reports on serum sensitivity of the bordetellae have been published, we show here that serum concentration and growth conditions can greatly alter the observed level of sensitivity to complement and that all but one strain of B . pertussis observed were sensitive to some level of naive serum in vitro, particularly when there was excess complement . However, B . pertussis rapidly acquires increased resistance in vivo to naive serum that is specific to the alternative pathway . Resistance is not efficiently acquired by B . parapertussis and B . bronchiseptica mutants lacking O antigen . This B . pertussis-specific mechanism of complement resistance does not appear to be dependent on either brkA or other genes expressed specifically in the Bvg(+) phase . This in vivo acquisition of alternative pathway resistance suggests that there is a novel O antigen-independent method by which B . pertussis evades complement-mediated killing. Endocrinology, 2003 Sep, 144(9), 4070 - 9 Yellow fluorescent protein-tagged and cyan fluorescent protein-tagged imaging analysis of glucocorticoid receptor and importins in single living cells; Tanaka M et al.; Glucocorticoid receptor (GR) acts as a ligand-dependent transcription factor after nuclear transport from the cytoplasm in the liganded state . Importins are docking proteins for karyopherin-mediated binding of substrate in a nuclear import pathway . To investigate the spatial and temporal relation between GR and importins, we analyzed the subcellular distribution of GR and importins in response to ligand in single living cells using fusion proteins labeled with different spectral variants of green fluorescent protein . Upon activation with ligand treatment, fluorescent protein-tagged (FP-) GR was translocated from the cytoplasm to the nucleus, showing a similar time course as FP-importin-alpha in the coexpressed cells with the fusion proteins . In contrast to FP-importin-alpha, the distribution of FP-importin-beta was little changed upon ligand treatment in the coexpressed cells with FP-GR and FP-importin-beta . Analysis using fluorescence resonance energy transfer proved that GR directly interacted with importin-alpha in the whole area of the cytoplasm upon ligand treatment and detached importin-alpha shortly after nuclear import . However, direct interaction between GR and importin-beta was not detected . These studies showed visual evidence of the nuclear importing of GR in association with importin-alpha in single living cells. Arthritis Res Ther, 2003, 5(5), R277 - 84 Epub 2003 Jul 07. An association between the acute phase response and patterns of antigen induced T cell proliferation in juvenile idiopathic arthritis; Black AP et al.; The aim of this research was to determine whether all memory T cells have the same propensity to migrate to the joint in patients with juvenile idiopathic arthritis . Paired synovial fluid and peripheral blood mononuclear cell proliferative responses to a panel of antigens were measured and the results correlated with a detailed set of laboratory and clinical data from 39 patients with juvenile idiopathic arthritis . Two distinct patterns of proliferative response were found in the majority of patients: a diverse pattern, in which synovial fluid responses were greater than peripheral blood responses for all antigens tested; and a restricted pattern, in which peripheral blood responses to some antigens were more vigorous than those in the synovial fluid compartment . The diverse pattern was generally found in patients with a high acute phase response, whereas patients without elevated acute phase proteins were more likely to demonstrate a restricted pattern . We propose that an association between the synovial fluid T cell repertoire and the acute phase response suggests that proinflammatory cytokines may influence recruitment of memory T cells to an inflammatory site, independent of their antigen specificity . Additionally, increased responses to enteric bacteria and the presence of alphaEbeta7 T cells in synovial fluid may reflect accumulation of gut associated T cells in the synovial compartment, even in the absence of an elevated acute phase response . This is the first report of an association between the acute phase response and the T cell population recruited to an inflammatory site. J Environ Qual, 2003 Jul-Aug, 32(4), 1523 - 33 Advanced solid-state carbon-13 nuclear magnetic resonance spectroscopic studies of sewage sludge organic matter: detection of organic "domains"; Smernik RJ et al.; Two novel solid-state 13C nuclear magnetic resonance (NMR) spectroscopic techniques, PSRE (proton spin relaxation editing) and RESTORE {Restoration of Spectra via T(CH) and T(1rho)H (T One Rho H) Editing}, were used to provide detailed chemical characterization of the organic matter from six Australian sewage sludges . These methods were used to probe the submicrometer heterogeneity of sludge organic matter, and identify and quantify spatially distinct components . Analysis of the T1H relaxation behavior of the sludges indicated that each sludge contained two types of organic domains . Carbon-13 PSRE NMR subspectra were generated to determine the chemical nature of these domains . The rapidly relaxing component of each sludge was rich in protein and alkyl carbon, and was identified as dead bacterial material . The slowly relaxing component of each sludge was rich in carbohydrate and lignin structures, and was identified as partly degraded plant material . The bacterial domains were shown, using the RESTORE technique, to also have characteristically rapid T(1rho)H relaxation rates . This rapid T(1rho)H relaxation was identified as the main cause of underrepresentation of these domains in standard 13C cross polarization (CP) NMR spectra of sludges . The heterogeneous nature of sewage sludge organic matter has implications for land application of sewage sludge, since the two components are likely to have different capacities for sorbing organic and inorganic toxicants present in sewage sludge, and will decompose at different rates. Vnitr Lek, 2003 Jul, 49(7), 555 - 8 {Occurrence of chlamydia infection in relation to lipidemia indicators in the etiology of unstable angina pectoris}; Zeman K et al.; The presence of Chlamydia pneumoniae infection was examined in 66 patients with unstable angina pectoris (UAP), 155 patients with acute myocardial infarction (AIM) and 112 controls without signs of a heart disease . Besides evaluation of anamnestic data, ECG and coronarographic examination, serologic examination of C . pneumoniae by the microfluorescent method anti-MOMP and ELISA of anti-LPS of globulin IgA and IgG serum classes in every patient was performed . Moreover, in patients with UAP, routine biochemical methods for the detection of total cholesterol levels and its lipoprotein fractions LDL, HDL and triacylglycerols were used . The levels of anti-MOMP C . pneumoniae antibodies and anti-LPS of the IgA class in sera of patients with UAP were statistically highly significantly increased (chi 2 = 19.54; chi 2 = 12.92; p < 0.01) and anti-LPS of the IgG class significantly increased (chi 2 = 6.15; p < 0.05) in comparison with controls . It can be assumed that the participation of C . pneumoniae is aetiologically possible . Total cholesterol levels, LDL, HDL and triacylgylcerols were increased above the normal range in 34.8%, 48.5%, 39.4% and 28.8% of patients, respectively . The anti-LPS C . pneumoniae ELISA test of globulin class IgA in patients with UAP seems to be the most suitable method for the determination of infections with C . pneumoniae. Surg Technol Int, 2003, 11, 23 - 31 Clinical efficacy and mechanism of bilayered living human skin equivalent (HSE) in treatment of diabetic foot ulcers; Brem H et al.; Bilayered living human skin equivalent (HSE) consists of cultured keratinocytes residing on the surface of a fibroblast-populated collagen lattice . Although HSE is FDA-approved for treatment of diabetic foot and venous stasis ulcers, its clinical efficacy remains limited, because the molecular mechanisms underlying its therapeutic effect are not fully understood . It is, therefore, often applied mistakenly as a skin graft . In this report, we delineate a mechanism of HSE biological effect and consequent optimal clinical use in accelerating closure of diabetic foot ulcers . Experimental: HSE was grafted onto nude mice and the release of various growth factors was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and immunochemistry . Clinical: HSE was grafted onto 11 consecutive patients with diabetes who had 13 non-ischemic foot ulcers and healing was measured as time to 100% closure (e.g., no drainage and 100% epithelialized) . Experimental: HSE cellular components were determined to express 15 different growth factors/cytokine genes known to promote wound healing . Histological evidence from the nude mice showed that the collagen component of HSE underwent remodeling within the first seven days of grafting . Clinical: All diabetic foot ulcers healed in 31.8 12.4 days . Local release of a unique combination of 15 growth factors expressed by HSE keratinocyte and fibroblast components generates closure of diabetic foot ulcers . HSE should be applied with the same surgical conditions for a skin graft (i.e., no cellulitis, no drainage, and negligible bacteria) . We hypothesize that bilayered HSE generates its effect by way of the local synthesis and release of multiple growth factors in specific combination and concentration, which improves the impaired reparative process of chronic wounds. Protein Sci, 2003 Sep, 12(9), 1833 - 43 Archaeal signal peptides--a comparative survey at the genome level; Bardy SL et al.; The correct delivery of noncytoplasmic proteins to locations both within and outside the cell depends on the appropriate targeting signals . Protein translocation across the bacterial plasma membrane and the eukaryal endoplasmic reticulum membrane relies on cleavable N-terminal signal peptides . Although the signal peptides of secreted proteins in Bacteria and Eukarya have been extensively studied at the sequence, structure, and functional levels, little is known of the nature of archaeal signal peptides . In this report, genome-based analysis was performed in an attempt to define the amino acid composition, length, and cleavage sites of various signal peptide classes in a wide range of archaeal species . The results serve to present a picture of the archaeal signal peptide, revealing the incorporation of bacterial, eukaryal, and archaeal traits. Nucleic Acids Res, 2003 Sep 1, 31(17), 5064 - 73 Nuclear factories for signalling and repairing DNA double strand breaks in living fission yeast; Meister P et al.; In mammalian and budding yeast cells treated with genotoxic agents, different proteins implicated in detecting, signalling or repairing DNA lesions form nuclear foci . We studied foci formed by proteins involved in these processes in living fission yeast cells, which is amenable to genetic and molecular analysis . Using fluorescent tags, we analysed subnuclear localisations of the DNA damage checkpoint protein Rad9, of the homologous recombination protein Rad22 and of PCNA, which are implicated in many aspects of DNA metabolism . After inducing double strand breaks (DSBs) with ionising radiations, Rad22, Rad9 and PCNA form a low number of nuclear foci . Rad9 recruitment to foci depends on the presence of Rad1, Hus1 and Rad17, but is independent of downstream checkpoint effectors and of homologous recombination proteins . Likewise, Rad22 and PCNA form foci despite inactive homologous recombination repair and impaired DNA damage checkpoint . Rad22 and Rad9 foci co-localise completely, whereas PCNA co-localises with Rad22 and Rad9 only partially . Foci do not disassemble in cells unable to repair DNA by homologous recombination . Thus, in fission yeast, DSBs are detected by the DNA damage checkpoint and are repaired by homologous recombination at a few spatially confined subnuclear compartments where Rad22, Rad9 and PCNA concentrate independently. Exp Dermatol, 2003 Aug, 12(4), 346 - 55 In vitro reconstructed mucosa-integrating Langerhans' cells; Sivard P et al.; All three-dimensional in vitro mucosal models constructed, thus far, have only been reconstituted by epithelial cells . We have developed a reconstructed oral and vaginal epithelium that integrates Langerhans' cells (LC), the dendritic cells (DC) of malpighian epithelia . The epithelium was composed of gingival or vaginal keratinocytes seeded on a de-epidermized dermis (DED) and grown in submerged culture for 2 weeks . LC precursors, obtained after differentiation of cord blood-derived CD34+ hematopoietic progenitor cells (CD34+HPC) by granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta) and Flt3-ligand (Flt3-L), were introduced after 6-8 days of culture into the reconstituted epithelium . The in vitro reconstituted mucosal epithelium formed a multilayered, well-differentiated epithelial structure, confirmed by the immunohistochemical expression of cytokeratins 4, 6, 10, 13, 14, 16 and involucrin . LC were identified in the basal and suprabasal epithelial layers by CD1a antigen, S100 protein and Langerin/CD207 expression, and by transmission electron microscopy . Type IV collagen was expressed at the chorio-epithelial junction, and most ultrastructural features of this junction were visualized by electron microscopy . This in vitro reconstructed gingiva or vagina integrating LC represents interesting models very similar to native tissues . Because LC play an important role in the mucosal immune system, our models could be useful for conducting studies on interactions with pathogenic agents (viruses, bacteria etc.), as well as in pharmacological, toxicological and clinical research. J Oral Sci, 2003 Jun, 45(2), 85 - 91 Hyaluronan (hyaluronic acid) and its regulation in human saliva by hyaluronidase and its inhibitors; Pogrel MA et al.; The presence of hyaluronan (HA), hyaluronidase and hyaluronidase inhibitors has been assayed in pure resting and stimulated parotid saliva and also in resting and stimulated mixed saliva utilizing an ELISA-type assay and its modifications . Results confirmed the presence of hyaluronan in all saliva specimens which generally decreased upon stimulation . Hyaluronan in parotid saliva was of high molecular weight (> 200,000 kDa) whilst that in whole saliva in the floor of the mouth had a molecular weight between 20,000 kDa and 200,000 kDa, presumably because of cleavage by bacterial hyaluronidases . Hyaluronidase detection was variable in saliva, being present in some specimens of unstimulated parotid saliva, but in fewer specimens of stimulated saliva . Hyaluronidase was detected in parotid and whole saliva, both in the resting and stimulated state, at pH 3.7 . Unstimulated whole saliva also showed hyaluronidase activity at pH 6.8, suggesting a different origin for this hyaluronidase . Hyaluronidase inhibitors were identified in both parotid and mixed whole saliva . There was an inverse relationship between the presence of hyaluranidase and the presence of hyluronidase inhibitors, suggesting a feedback mechanism . The possible significance, interactions and function of hyaluronan, hyaluronidase and its regulation by hyaluronidase inhibitors in saliva is discussed, particularly in relation to intra-oral wound healing and periodontal disease. Genet Res, 2003 Jun, 81(3), 221 - 8 Non-parametric interval mapping in half-sib designs: use of midranks to account for ties; Tilquin P et al.; In QTL analysis of non-normally distributed phenotypes, non-parametric approaches have been proposed as an alternative to the use of parametric tests on mathematically transformed data . The non-parametric interval mapping test uses random ranking to deal with ties . Another approach is to assign to each tied individual the average of the tied ranks (midranks) . This approach is implemented and compared to the random ranking approach in terms of statistical power and accuracy of the QTL position . Non-normal phenotypes such as bacteria counts showing high numbers of zeros are simulated (0-80% zeros) . We show that, for low proportions of zeros, the power estimates are similar but, for high proportions of zeros, the midrank approach is superior to the random ranking approach . For example, with a QTL accounting for 8% of the total phenotypic variance, a gain from 8% to 11% of power can be obtained . Furthermore, the accuracy of the estimated QTL location is increased when using midranks . Therefore, if non-parametric interval mapping is chosen, the midrank approach should be preferred . This test might be especially relevant for the analysis of disease resistance phenotypes such as those observed when mapping QTLs for resistance to infectious diseases. Org Biomol Chem, 2003 Jan 21, 1(2), 338 - 49 A stereodivergent, two-directional synthesis of stereoisomeric C-linked disaccharide mimetics; Harding M et al.; Dipyranones, such as 1,2-bis{(2R,3S,6S)-3-hydroxy-6-methoxy-3-oxo-6H-pyran-2-yl}ethane, were exploited as templates for the synthesis of some novel C-linked disaccharide analogues . Efficient methods, such as stereoselective reduction and dihydroxylation, were developed for two-directional functionalisation of these templates . Peracetylated derivatives of ten stereoisomeric disaccharide analogues {acetic acid 4,5-diacetoxy-6-methoxy-{(3',4',5'-triacetoxy-6'-methoxytetrahydropyran- 2'-yl)ethyl}tetrahydropyran-3-yl esters} were synthesised from a virtual library of 136 compounds; furthermore, an additional eight stereoisomers could have been synthesised simply by using the enantiomeric ligand in the enantioselective step . The ability of (2S,3S,4R,5R,6R)-6-methoxy-2-{2'-((2'R,3'R,4'S, 5'R,6'S)-3',4',5'-trihydroxy-6'-methoxytetrahydropyran-2'-yl) ethyl}tetrahydropyran-3,4,5-triol to bind to the repressor protein, LacI, was estimated to be similar to that of isopropyl-beta-thiogalactoside . The disaccharide mimetics were concluded to be a new and interesting class of C-linked disaccharide mimetics with promising, though largely unstudied, biological activity. Theor Appl Genet, 2003 Oct, 107(6), 1094 - 101 Epub 2003 Aug 20. Barley putative hypersensitive induced reaction genes: genetic mapping, sequence analyses and differential expression in disease lesion mimic mutants; Rostoks N et al.; The hypersensitive response (HR) is one of the most-efficient forms of plant defense against biotrophic pathogens, and results in localized cell death and the formation of necrotic lesions; however, the molecular components of pathways leading to HR remain largely unknown . Barley ( Hordeum vulgare ssp . vulgare L.) cDNAs for putative hypersensitive-induced reaction ( HIR) genes were isolated based on DNA and amino-acid homologies to maize HIR genes . Analyses of the cDNA and genomic sequences and genetic mapping found four distinct barley HIR genes, Hv-hir1, Hv-hir2, Hv-hir3 and Hv-hir4, on chromosomes 4(4H) bin10, 7(5H) bin04, 7(5H) bin07 and 1(7H) bin03, respectively . Hv-hir1, Hv-hir2 and Hv-hir3 genes were highly homologous at both DNA and the deduced amino-acid level, but the Hv-hir4 gene was similar to the other genes only at the amino-acid sequence level . Amino-acid sequence analyses of the barley HIR proteins indicated the presence of the SPFH protein-domain characteristic for the prohibitins and stomatins which are involved in control of the cell cycle and ion channels, as well as in other membrane-associated proteins from bacteria, plants and animals . HIR genes were expressed in all organs and developement stages analyzed, indicating a vital and non-redundant function . Barley fast-neutron mutants exhibiting spontaneous HR (disease lesion mimic mutants) showed up to a 35-fold increase in Hv-hir3 expression, implicating HIR genes in the induction of HR. J Immunol, 2003 Sep 1, 171(5), 2563 - 70 Francisella tularensis selectively induces proinflammatory changes in endothelial cells; Forestal CA et al.; Naturally acquired infections with Francisella tularensis, the bacterial agent of tularemia, occur infrequently in humans . However, the high infectivity and lethality of the organism in humans raise concerns that it might be exploited as a weapon of bioterrorism . Despite this potential for illicit use, the pathogenesis of tularemia is not well understood . To examine how F . tularensis interacts with cells of its mammalian hosts, we tested the ability of a live vaccine strain (LVS) to induce proinflammatory changes in cultured HUVEC . Living F . tularensis LVS induced HUVEC to express the adhesion molecules VCAM-1 and ICAM-1, but not E-selectin, and to secrete the chemokine CXCL8, but not CCL2 . Stimulation of HUVEC by the living bacteria was partially suppressed by polymyxin B, an inhibitor of LPS, but did not require serum, suggesting that F . tularensis LVS does not stimulate endothelium through the serum-dependent pathway that is typically used by LPS from enteric bacteria . In contrast to the living organisms, suspensions of killed F . tularensis LVS acquired the ability to increase endothelial expression of both E-selectin and CCL2 . Up-regulation of E-selectin and CCL2 by the killed bacteria was not inhibited by polymyxin B . Exposure of HUVEC to either live or killed F . tularensis LVS for 24 h promoted the transendothelial migration of subsequently added neutrophils . These data indicate that multiple components of F . tularensis LVS induce proinflammatory changes in endothelial cells in an atypical manner that may contribute to the exceptional infectivity and virulence of this pathogen. Trends Plant Sci, 2003 Aug, 8(8), 387 - 93 Plants, humans and hemoglobins; Kundu S et al.; New developments have forced a re-evaluation of our understanding of the structure and function of hemoglobins . Leghemoglobins regulate oxygen affinity through a mechanism different from that of myoglobin using a novel combination of heme pocket amino acids that lower the oxygen affinity . The hexacoordinate hemoglobins are characterized by intramolecular coordination of the ligand binding site at the heme iron, and were first identified in plants as the 'non-symbiotic plant hemoglobins' . They are now known to be present in animals and bacteria . Many of these proteins are upregulated in both plants and animals during hypoxia or similar stresses . Therefore, there might be a common physiological function for hexacoordinate hemoglobins in plants and animals. Trends Plant Sci, 2003 Aug, 8(8), 355 - 7 Building stress tolerance through over-producing trehalose in transgenic plants; Penna S; Trehalose is a rare sugar with unique abilities to protect biomolecules from environmental stresses and is present in many bacteria, fungi and some desiccation-tolerant higher plants . Increasing trehalose accumulation in crop plants could improve drought and salinity tolerance . Transgenic plants have been developed with trehalose biosynthetic genes--a recent study on the stress-inducible overexpression of the bifunctional TPSP fusion gene in transgenic rice could offer novel strategies for improving abiotic stress tolerance in crop plants. Anal Biochem, 2003 Sep 15, 320(2), 223 - 33 Capillary electrophoresis-based profiling and quantitation of total salicylic acid and related phenolics for analysis of early signaling in Arabidopsis disease resistance; Shapiro AD et al.; A capillary electrophoresis-based method for quantitation of total salicylic acid levels in Arabidopsis leaves was developed . Direct comparison to previous high-performance liquid chromatography (HPLC)-based measurements showed similar levels of salicylic acid . Simultaneous quantitation of trans-cinnamic acid, benzoic acid, sinapic acid, and an internal recovery standard was achieved . A rapid, streamlined protocol with requirements for plant tissue reduced relative to those of HPLC-based protocols is presented . Complicated, multiparameter experiments were thus possible despite the labor-intensive nature of inoculating plants with bacterial pathogens . As an example of this sort of experiment, detailed time course studies of total salicylic acid accumulation by wild-type Arabidopsis and two lines with mutations affecting salicylic acid accumulation in response to either of two avirulent bacterial strains were performed . Accumulation in the first 12h was biphasic . The first phase was partially SID2 and NDR1 dependent with both bacterial strains . The second phase was largely independent of both genes with bacteria carrying avrB, but dependent upon both genes with bacteria carrying avrRpt2 . Virulent bacteria did not elicit salicylic acid accumulation at these time points . Application of this method to various Arabidopsis pathosystems and the wealth of available disease resistance signaling mutants will refine knowledge of disease resistance and associated signal transduction. Biochem Biophys Res Commun, 2003 Sep 5, 308(4), 820 - 5 Protein PknE, a novel transmembrane eukaryotic-like serine/threonine kinase from Mycobacterium tuberculosis; Molle V et al.; Protein PknE from Mycobacterium tuberculosis has been overproduced and purified, and its biochemical properties have been analyzed . This protein is shown to be a eukaryotic-like (Hanks'-type) protein kinase with a structural organization similar to that of membrane-bound eukaryotic sensor serine/threonine kinases . It consists of a N-terminal catalytic domain located in the cytoplasm, linked via a single transmembrane-spanning region to an extracellular C-terminal domain . The full-length enzyme, as well as the cytosolic domain alone, can autophosphorylate on serine and threonine residues . Such autokinase activity requires the presence of a lysine residue at position 45 in subdomain II, which is known to be essential also for eukaryotic kinase activity . Involvement of PknE in the transduction of external signals into the cytosol of bacteria is proposed. Biochem Biophys Res Commun, 2003 Sep 5, 308(4), 684 - 8 Preparation and X-ray crystallographic analysis of rubredoxin crystals from Desulfovibrio gigas to beyond ultra-high 0.68 A resolution; Chen CJ et al.; Rubredoxin (D.g . Rd), a small non-heme iron-sulfur protein shown to function as a redox coupling protein from the sulfate reducing bacteria Desulfovibrio gigas, has been crystallized using the hanging-drop vapor diffusion method and macroseeding method . Rubredoxin crystals diffract to an ultra-high resolution 0.68 A using synchrotron radiation X-ray, and belong to the space group P2(1) with unit-cell parameters a=19.44 A, b=41.24 A, c=24.10 A, and beta=108.46 degrees . The data set of single-wavelength anomalous dispersion signal of iron in the native crystal was also collected for ab initio structure re-determination . Preliminary analysis indicates that there is one monomer with a {Fe-4S} cluster in each asymmetric unit . The crystal structure at this ultra-high resolution will reveal the details of its biological function . The crystal character and data collection strategy for ultra-high resolution will also be discussed. Int J Food Microbiol, 2003 Oct 15, 87(1-2), 181 - 5 Estimating the precision of serial dilutions and colony counts: contribution of laboratory re-calibration of pipettes; Hedges AJ; The basic (inherent) precision of serial dilutions and of colony counts made from them may be reliably estimated by reference solely to the pipette-manufacturer's specifications . Such estimates do not include external sources of variation and may be regarded as minima . The quality of estimation can be improved by using information gained by laboratory ('in-house') re-calibration of the pipettes . The degree of improvement was assessed by comparison with similar series made without re-calibration . It was found that improvement was minimal for colony counts but worthwhile for homogeneous solutions. Neuroscience, 2003, 120(4), 1005 - 17 The fragile X mental retardation protein binds and regulates a novel class of mRNAs containing U rich target sequences; Chen L et al.; Fragile X syndrome is a common form of inherited mental retardation caused by the absence of the fragile X mental retardation protein (FMRP) . It has been hypothesized that FMRP is involved in the processing and/or translation of mRNAs . Human and mouse target-mRNAs, containing purine quartets, have previously been identified . By using cDNA-SELEX (systematic evolution of ligands by exponential enrichment), we identified another class of human target-mRNAs which contain U rich sequences . This technique, in contrast to oligonucleotide-based SELEX, allows the identification of FMRP targets directly from mRNA pools . Many of the proteins encoded by the identified FMRP targets have been implicated in neuroplasticity . Steady state levels of target-mRNAs were unchanged in the brain of fragile X mice . However, levels of two target-encoded proteins, an L-type calcium channel subunit and MAP1B, were downregulated in specific brain regions suggesting a defect in the expression of target-encoded proteins in fragile X syndrome. Am J Vet Res, 2003 Aug, 64(8), 969 - 75 Evaluation of treatment of colostrum-deprived kittens with equine IgG; Crawford PC et al.; OBJECTIVE: To evaluate equine IgG as a treatment for kittens with failure of passive transfer of immunity (FPT) . ANIMALS: 13 specific pathogen-free queens and their 77 kittens . PROCEDURE: Kittens were randomized at birth into 9 treatment groups . One group contained colostrum-fed (nursing) kittens; the other groups contained colostrum-deprived kittens that were administered supplemental feline or equine IgG PO or SC during the first 12 hours after birth . Blood samples were collected at serial time points from birth to 56 days of age for determination of serum IgG concentrations . The capacity of equine IgG to opsonize bacteria for phagocytosis by feline neutrophils was determined via flow cytometry . RESULTS: Kittens that received feline or equine IgG SC had significantly higher serum IgG concentrations than those of kittens that received the supplements PO . In kittens that were administered supplemental IgG SC, serum IgG concentrations were considered adequate for protection against infection . The half-life of IgG in kittens treated with equine IgG was shorter than that in kittens treated with feline IgG . Feline IgG significantly enhanced the phagocytosis of bacteria by feline neutrophils, but equine IgG did not . CONCLUSIONS AND CLINICAL RELEVANCE: Serum concentrations of equine IgG that are considered protective against infection are easily attained in kittens, but the failure of these antibodies to promote bacterial phagocytosis in vitro suggests that equine IgG may be an inappropriate treatment for FPT in kittens. Przegl Epidemiol, 2003, 57(1), 211 - 9 {Usefulness of plasma procalcitonin (PCT) estimation to diagnose patients in departments of infectious diseases}; Wrodycki W; PCT is a new highly sensitive and specific marker of bacterial and fungi infection--to be used in differential diagnosis at Infectious Diseases Departments . Author in this paper presents structure and mechanism of stimulation of PCT as a factor of "early infection's fase" for many infectious agents: bacteria, fungi, viruses and parasites . PCT may be found useful in diagnosing diseases; for ex.: sepsis, meningitis, inflammation of respiratory system, spontaneous bacterial peritonitis (SPB) and other local inflammatory foci (otitis media, endocarditis) . PCT level is low in systemic inflammatory response syndrome (SIRS) and multiorgan dysfunction syndrome (MODS) of non-infectious origin (< 0.5 ng/ml), medium in case of localized infections (1.0-2.0 ng/ml) and in severe cases of disseminated infections (sepsis-->SIRS-->MODS) high (approximately 20 ng/ml). Mar Biotechnol (NY), 2003 Jan-Feb, 5(1), 92 - 101 Expression and characterization of European sea bass (Dicentrarchus labrax) somatolactin: assessment of in vivo metabolic effects; Vega-Rubin de Celis S et al.; The complementary DNA coding for European sea bass somatolactin was expressed in the pET-3a bacteria expression vector . The recombinant somatolactin (rbSL) was purified by size exclusion chromatography, and 95% of the protein remained in the oxidized form with negligible aggregation over prolonged cold storage . The identity of the recombinant protein was demonstrated by Western blotting with a rabbit polyclonal antibody against gilthead sea bream somatolactin . The same antibody was utilized in a radioimmunoassay procedure, using rbSL as standard and radioiodinated tracer . Curve displacements of pituitary and plasma samples paralleled the rbSL standard, and the midrange of the assay (8 ng/ml) was low enough to measure in a consistent manner the circulating SL concentration . To assess biological activity a single dose of rbSL (0.1 microg/g of body mass) was administered to juvenile gilthead sea bream by intraperitioneal injection . In comparison with saline-treated fish, rbSL did not modify the circulating amount of insulin-like growth factor I, whereas a 50% increase was found with the same dose of recombinant trout growth hormone (rtGH) . Hormone treatment did not modify nitrogen-ammonia excretion, but both rbSL and rtGH increased carbon dioxide output and oxygen uptake, which in turn decreased the respiratory quotient (CO2 output per O2 uptake) . This pattern of gas exchange suggests the enhancement of lipid catabolism, which is consistent with the observation that both hormones were able to inhibit the hepatic activity of acetyl-coenzyme A carboxylase . These new insights provide direct evidence for the involvement of fish somatolactin in energy homeostasis, which may serve to maintain the lipolytic tonus in different physiologic states. Mar Biotechnol (NY), 2003 Jan-Feb, 5(1), 1 - 12 Polyketide synthase genes from marine dinoflagellates; Snyder RV et al.; Rapidly developing techniques for manipulating the pathways of polyketide biosynthesis at the genomic level have created the demand for new pathways with novel biosynthetic capability . Polyketides derived from dinoflagellates are among the most complex and unique structures identified thus far, yet no studies of the biosynthesis of dinoflagellate-derived polyketides at the genomic level have been reported . Nine strains representing 7 different species of dinoflagellates were screened for the presence of type I and type II polyketide synthases (PKSs) by polymerase chain reaction (PCR) and reverse transcriptase PCR . Seven of the 9 strains yielded products that were homologous with known and putative type I PKSs . Unexpectedly, a PKS gene was amplified from cultures of the dinoflagellate Gymnodinium catenatum, a saxitoxin producer, which is not known to produce a polyketide . In each case the presence of a PKS gene was correlated with the presence of bacteria in the cultures as identified by amplification of the bacterial 16S ribosomal RNA gene . However, amplification from polyadenylated RNA, the lack of PKS expression in light-deprived cultures, residual phylogenetic signals, resistance to methylation-sensitive restriction enzymes, and the lack of hybridization to bacterial isolates support a dinoflagellate origin for most of these genes. Med Microbiol Immunol (Berl), 2004 Nov, 193(4), 155 - 62 Epub 2004 Nov. The functional interaction between CD98 and CD147 in regulation of virus-induced cell fusion and osteoclast formation; Mori K et al.; Membrane fusion is an important event in the functioning of a living organism . Life starts as a sperm fuses with the membrane of an egg, leading to its fertilization . Membrane fusion is also required for myogenesis, osteogenesis and placenta formation . Multinucleated giant cells originating from monocytes-macrophages are associated with granulomatous lesions formed in response to foreign bodies, viruses, and bacteria . The CD4 molecule acts as a receptor for HIV . The major virus envelope glycoprotein, gp120, attaches to CD4 molecules expressed on the host cell surface . After binding to CD4 on the target cells, HIV is internalized via direct, pH-independent fusion of the viral and cell membranes . However, attachment of HIV to CD4 on the target cells is not sufficient for fusion . Interaction of gp160-expressing cells with neighboring cells bearing surface CD4 molecules is also required for syncytium formation . Syncytium formation and subsequent generalized cell fusion have been reported as potentially important mechanisms of virus-induced cytotoxic effects . Some antibodies against CD98/FRP-1 suppressed virus-induced cell fusion and CD98-mediated cell fusion of monocytes, indicating that CD98/FRP-1 molecules are able to regulate cell fusion . In this study, the functional interaction between CD98 and CD147 was investigated . Three kinds (Ab1, 2, and 3) of anti-CD147 and three kinds of anti-CD98 antibodies were used . Ab1 suppressed CD98-mediated cell fusion, but showed no effect on cell aggregation of Cd(+)U2ME-7 cells, U937-2 cells expressing HIV gp160 . On the other hand, Ab2 enhanced the CD98-mediated cell fusion . Ab1 showed suppressive effect at early stage and Ab2 showed enhancing effect at later stage . Ab2 and 3 suppressed the spontaneous cell agglutination and cell fusion of Cd(+)JME-2 cells, Jurkat cells expressing HIV gp160 . Ab2 suppressed CD98-mediated cell fusion, but showed no effect on cell aggregation of Cd(+)JME-2 cells . Ab2 cancelled suppression of cell fusion induced by suppressive antibody against CD98 . Ab2 and 3 also suppressed CD98-mediated cell fusion of monocytes . This study indicates the functional interaction between CD98 and CD147 in the regulation of cell fusion. Nat Neurosci, 2003 Sep, 6(9), 956 - 60 Synaptic dynamism measured over minutes to months: age-dependent decline in an autonomic ganglion; Gan WB et al.; Naturally occurring rearrangements of synaptic terminals are common in the nervous systems of young mammals, but little is known about their incidence in adults . Using transgenic mice that express yellow fluorescent protein (YFP) in axons, we repeatedly imaged nerve terminals in the parasympathetic submandibular ganglion . We found that the pattern of synaptic branches underwent significant rearrangements over several weeks in young adult mice . In older mice, rearrangements were less common, and synaptic patterns on individual neurons were recognizable for many months to years . Axonal branches frequently retracted or extended on a time scale of minutes in young adult mice, but seldom in mature animals . These results provide direct evidence for a decrease in plasticity of interneuronal connections as animals make the transition from young adulthood to middle age . The long-term stability of synaptic patterns could provide a structural basis for the persistence of memory in the adult nervous system. Acta Crystallogr D Biol Crystallogr, 2003 Sep, 59(Pt 9), 1670 - 3 Epub 2003 Aug 19. Preliminary X-ray characterization and phasing of a type II cohesin domain from the cellulosome of Acetivibrio cellulolyticus; Noach I et al.; The N-terminal type II cohesin from the cellulosomal ScaB subunit of Acetivibrio cellulolyticus was crystallized in two different crystal systems: orthorhombic (space group P2(1)2(1)2(1)), with unit-cell parameters a = 37.455, b = 55.780, c = 87.912 A, and trigonal (space group P3(1)21), with unit-cell parameters a = 55.088, b = 55.088, c = 112.553 A . The two crystals diffracted to 1.2 and 1.9 A, respectively . A selenomethionine derivative was also crystallized and exhibited trigonal symmetry (space group P3(1)21), with unit-cell parameters a = 55.281, b = 55.281, c = 112.449 A and a diffraction limit of 1.97 A . Initial phasing of the trigonal crystals was successfully performed by the SIRAS method using Cu Kalpha radiation with the selenomethionine derivative as a heavy-atom derivative . The structure of the orthorhombic crystal form was solved by molecular replacement using the coordinates of the trigonal form. Acta Crystallogr D Biol Crystallogr, 2003 Sep, 59(Pt 9), 1545 - 50 Epub 2003 Aug 19. Auracyanin B structure in space group P6(5); Lee M et al.; The structure of auracyanin B, a 'blue' copper protein produced by Chloroflexus aurantiacus, has previously been solved and refined in the hexagonal space group P6(4)22 with a single molecule in the asymmetric unit . The protein has now been crystallized in space group P6(5), with unit-cell parameters a = b = 115.9, c = 108.2 A . In the new crystal form, the asymmetric unit contains four protein molecules . The structure has been solved by molecular replacement and refined at 1.9 A resolution . The final residuals are R = 19.2% and R(free) = 21.9% . In relation to the earlier crystal structure, the doubling of the unit-cell volume and the lower symmetry are explained by small rotations of the molecules with respect to one another. J Exp Med, 2003 Aug 18, 198(4), 545 - 56 Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum; Celli J et al.; The intracellular pathogen Brucella is the causative agent of brucellosis, a worldwide zoonosis that affects mammals, including humans . Essential to Brucella virulence is its ability to survive and replicate inside host macrophages, yet the underlying mechanisms and the nature of the replicative compartment remain unclear . Here we show in a model of Brucella abortus infection of murine bone marrow-derived macrophages that a fraction of the bacteria that survive an initial macrophage killing proceed to replicate in a compartment segregated from the endocytic pathway . The maturation of the Brucella-containing vacuole involves sustained interactions and fusion with the endoplasmic reticulum (ER), which creates a replicative compartment with ER-like properties . The acquisition of ER membranes by replicating Brucella is independent of ER-Golgi COPI-dependent vesicular transport . A mutant of the VirB type IV secretion system, which is necessary for intracellular survival, was unable to sustain interactions and fuse with the ER, and was killed via eventual fusion with lysosomes . Thus, we demonstrate that live intracellular Brucella evade macrophage killing through VirB-dependent sustained interactions with the ER . Moreover, we assign an intracellular function to the VirB system, as being required for late maturation events necessary for the biogenesis of an ER-derived replicative organelle. Aliment Pharmacol Ther, 2003 Jul, 18 Suppl 1, 82 - 9 Review article: inflammation-related promotion of gastrointestinal carcinogenesis--a perigenetic pathway; Tsuji S et al.; Chronic inflammation has been reported to accelerate neoplasmas in gastrointestinal tract . Certain bacteria including Helicobacter pylori directly interact with host cells, induce proinflammatory cytokines and stimulate production of free radicals . Free radicals cause mutations in target cells so that neoplastic clones are established . Accumulation of such genetic alterations may cause malignant transformation of some established clones . In addition, inflammatory alterations may promote growth, expansion and invasion of gastrointestinal epithelial cells . The latter changes caused by inflammation may occur even without further genetic mutations or epigenetic alterations, and therefore may be categorized as 'perigenetic alterations' of neoplastic cells . For an example, tumour necrosis factor alpha (TNF-alpha) plays pivotal roles not only in the reduction but also in the growth, invasion and metastases of certain neoplasmas . Our studies show that TNF-alpha increases intracellular radical production, degradates E-cadherin / beta-catenin complex and promotes dispersion and migration in epithelial cells transformed with an activated src oncogene (v-src) . These data indicate that an inflammatory cytokine induces the malignant potential of src-activated neoplastic cells . Interestingly, TNF-alpha also induced these phenotypic changes in nonmutated cells whose c-Src was activated by TGF-alpha, suggesting that the invasive properties of the cell were not necessarily related to gene mutation . Furthermore, certain radical scavengers suppressed the invasive phenotype of the cells . These results indicate that perigenetic alterations are an important target of pharmacological intervention of carcinogenesis. Aliment Pharmacol Ther, 2003 Jul, 18 Suppl 1, 39 - 44 The effects of cure of Helicobacter pylori infection on the signal transduction of gastric epithelial cells; Azuma T et al.; BACKGROUND: The CagA protein of Helicobacter pylori is directly injected from the bacteria into cells via the bacterial type IV secretion system and undergoes tyrosine phosphorylation in the gastric epithelial cells . Translocated CagA forms a physical complex with the SRC homology 2 domain (SH2)-containing tyrosine phosphatase SHP-2, which plays an important role in mitogenic signal transduction in the host cells . AIM: We examined the effect of eradication therapy on the signal transduction pathway of gastric epithelial cells induced by the CagA protein of H . pylori . METHODS: Gastric biopsy samples were obtained from 20 H . pylori-positive atrophic gastritis patients before, and 3 months after, H . pylori infection eradication therapy, and subjected to immunoblot analysis to detect tyrosine phosphorylated CagA protein and CagA co-immunoprecipitated endogenous SHP-2 . RESULTS: Tyrosine phosphorylated CagA protein and CagA co-immunoprecipitated endogenous SHP-2 were detected in the gastric mucosa from H . pylori-positive atrophic gastritis patients . All H . pylori strains from these patients were cagA-positive type I strains . After curing H . pylori infection, the tyrosine phosphorylated CagA protein and CagA co-immunoprecipitated endogenous SHP-2 disappeared from the gastric mucosa . CONCLUSION: The cure of infection reduces the stimulated signal transduction of gastric epithelial cells by the translocated CagA protein of H . pylori, and may confer a beneficial effect on the reduction of cancer risk. Cell Microbiol, 2003 Sep, 5(9), 649 - 60 Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells; Danelishvili L et al.; Mycobacterium tuberculosis interacts with macrophages and epithelial cells in the alveolar space of the lung, where it is able to invade and replicate in both cell types . M . tuberculosis-associated cytotoxicity to these cells has been well documented, but the mechanisms of host cell death are not well understood . We examined the induction of apoptosis and necrosis of human macrophages (U937) and type II alveolar epithelial cells (A549) by virulent (H37Rv) and attenuated (H37Ra) M . tuberculosis strains . Apoptosis was determined by both enzyme-linked immunosorbent assay (ELISA) and TdT-mediated dUTP nick end labelling (TUNEL) assay, whereas necrosis was evaluated by the release of lactate dehydrogenase (LDH) . Both virulent and attenuated M . tuberculosis induced apoptosis in macrophages; however, the attenuated strain resulted in significantly more apoptosis than the virulent strain after 5 days of infection . In contrast, cytotoxicity of alveolar cells was the result of necrosis, but not apoptosis . Although infection with M . tuberculosis strains resulted in apoptosis of 14% of the cells on the monolayer, cell death associated with necrosis was observed in 59% of alveolar epithelial cells after 5 days of infection . Infection with M . tuberculosis suppressed apoptosis of alveolar epithelial cells induced by the kinase inhibitor, staurosporine . Because our findings suggest that M . tuberculosis can modulate the apoptotic response of macrophages and epithelial cells, we carried out an apoptosis pathway-specific cDNA microarray analysis of human macrophages and alveolar epithelial cells . Whereas the inhibitors of apoptosis, bcl-2 and Rb, were upregulated over 2.5-fold in infected (48 h) alveolar epithelial cells, the proapoptotic genes, bad and bax, were downregulated . The opposite was observed when U937 macrophages were infected with M . tuberculosis . Upon infection of alveolar epithelial cells with M . tuberculosis, the generation of apoptosis, as determined by the expression of caspase-1, caspase-3 and caspase-10, was inhibited . Inhibition of replication of intracellular bacteria resulted in an increase in apoptosis in both cell types . Our results showed that the differential induction of apoptosis between macrophages and alveolar epithelial cells represents specific strategies of M . tuberculosis for survival in the host. Biochemistry, 2003 Aug 26, 42(33), 9829 - 40 How cytochromes with different folds control heme redox potentials; Mao J et al.; The electrochemical midpoint potentials (E(m)'s) of 13 cytochromes, in globin (c, c(2), c(551), c(553)), four-helix bundle (c', b(562)), alpha beta roll (b(5)), and beta sandwich (f) motifs, with E(m)'s spanning 450 mV were calculated with multiconformation continuum electrostatics (MCCE) . MCCE calculates changes in oxidation free energy when a heme-axial ligand complex is moved from water into protein . Calculated and experimental E(m)'s are in good agreement for cytochromes with His-Met and bis-His ligated hemes, where microperoxidases provide reference E(m)'s . In all cytochromes, E(m)'s are raised by 130-260 mV relative to solvated hemes by the loss of reaction field (solvation) energy . However, there is no correlation between E(m) and heme surface exposure . Backbone amide dipoles in loops or helix termini near the axial ligands raise E(m)'s, but amides in helix bundles contribute little . Heme propionates lower E(m)'s . If the propionic acids are partially protonated in the reduced cytochrome, protons are released on heme oxidation, contributing to the pH dependence of the E(m) . In all cytochromes studied except b(5)'s and low potential globins, buried side chains raise E(m)'s . MCCE samples ionizable group protonation states, heme redox states, and side chain rotamers simultaneously . Globins show the largest structural changes on heme oxidation and four-helix bundles the least . Given the calculated protein-induced E(m) shift and measured cytochrome E(m) the five-coordinate, His heme in c' is predicted to have a solution E(m) between that of isolated bis-His and His-Met hemes, while the reference E(m) for His-Ntr ligands in cytochrome f should be near that of His-Met hemes. Eur J Cell Biol, 2003 Jul, 82(7), 333 - 42 The interaction between human PEX3 and PEX19 characterized by fluorescence resonance energy transfer (FRET) analysis; Muntau AC et al.; The process of peroxisome biogenesis involves several PEX genes that encode the machinery required to assemble the organelle . Among the corresponding peroxins the interaction between PEX3 and PEX19 is essential for early peroxisome biogenesis . However, the intracellular site of this protein interaction is still unclear . To address this question by fluorescence resonance energy transfer (FRET) analysis, we engineered the enhanced yellow fluorescent protein (EYFP) to the C-terminus of PEX3 and the enhanced cyan fluorescent protein (ECFP) to the N-terminus of PEX19 . Functionality of the fusion proteins was shown by transfection of human PEX3- and PEX19-deficient fibroblasts from Zellweger patients with tagged versions of PEX3 and PEX19 . This led to reformation of import-competent peroxisomes in both cell lines previously lacking detectable peroxisomal membrane structures . The interaction of PEX3-EYFP with ECFP-PEX19 in a PEX3-deficient cell line during peroxisome biogenesis was visualized by FRET imaging . Although PEX19 was predominantly localized to the cytoplasma, the peroxisome was identified to be the main intracellular site of the PEX3-PEX19 interaction . Results were confirmed and quantified by donor fluorescence photobleaching experiments . PEX3 deletion proteins lacking the N-terminal peroxisomal targeting sequence (PEX3 34-373-EYFP) or the PEX19-binding domain located in the C-terminal half of the protein (PEX3 1-140-EYFP) did not show the characteristic peroxisomal localization of PEX3, but were mislocalized to the cytoplasm (PEX3 34-373-EYFP) or to the mitochondria (PEX3 1-140-EYFP) and did not interact with ECFP-PEX19 . We suggest that FRET is a suitable tool to gain quantitative spatial information about the interaction of peroxins during the process of peroxisome biogenesis in single cells . These findings complement and extend data from conventional in vitro protein interaction assays and support the hypothesis of PEX3 being an anchor for PEX19 at the peroxisomal membrane. Shock, 2003 Sep, 20(3), 245 - 50 Increased plasma D-lactate is associated with the severity of hemorrhagic/traumatic shock in rats; Szalay L et al.; D-lactate is produced by indigenous bacteria in the gastrointestinal tract . Mammals do not have the enzyme systems to metabolize D-lactate rapidly . The present study was designed to determine the kinetics of circulating D-lactate levels and to examine whether the severity of shock affects circulating D-lactate levels in rats subjected to hemorrhagic/traumatic shock . Anesthetized rats underwent midline laparotomy (duration 30 min) and were bled to 30-35 mmHg mean arterial pressure (MAP) . After the onset of decompensation, MAP was either increased to 40-45 mmHg immediately by administration of Ringer's solution (moderate shock) or after 40% of shed blood volume had been re-infused as Ringer's solution (severe shock) . MAP was then maintained at 40-45 mmHg for 40 min by further administration of Ringer's solution (inadequate resuscitation) . Subsequently, adequate resuscitation was performed for 60 min with shed blood and additional Ringer's solution . Metabolic acidosis was significantly more pronounced in severe than in moderate hemorrhagic/traumatic shock . Plasma D-lactate levels were already significantly increased at the end of severe hemorrhagic/traumatic shock and remained high during inadequate resuscitation . D-lactate levels were significantly higher after severe than after moderate shock . Endotoxin levels did not correlate with shock severity . Damage to the intestinal mucosa was more profound in severe shock than in moderate shock . Our data suggest that hemorrhagic/traumatic shock is associated with mucosal damage and increased plasma D-lactate levels . The severity of shock affects D-lactate concentrations in plasma . Plasma D-lactate may be a useful marker of intestinal injury after hemorrhagic/traumatic shock. J Bacteriol, 2003 Sep, 185(17), 5029 - 36 Proteomic analysis of wild-type Sinorhizobium meliloti responses to N-acyl homoserine lactone quorum-sensing signals and the transition to stationary phase; Chen H et al.; Proteome analysis revealed that two long-chain N-acyl homoserine lactones (AHLs) produced by Sinorhizobium meliloti 1021 induced significant differences in the accumulation of more than 100 polypeptides in early-log-phase cultures of the wild type . Fifty-six of the corresponding proteins have been identified by peptide mass fingerprinting . The proteins affected by addition of these two AHLs had diverse functions in carbon and nitrogen metabolism, energy cycles, metabolite transport, DNA synthesis, and protein turnover . Two hours of exposure to 3-oxo-C(16:1)-homoserine lactone (3-oxo-C(16:1)-HL) affected the accumulation of 40 of the 56 identified proteins, whereas comparable exposure to C(14)-HL affected 13 of the 56 proteins . Levels of four proteins were affected by both AHLs . Exposure to 3-oxo-C(16:1)-HL for 8 h affected the accumulation of 17 proteins, 12 of which had reduced accumulation . Of the 80 proteins identified as differing in accumulation between early-log- and early-stationary-phase cultures, only 13 were affected by exposure to 3-oxo-C(16:1)-HL or C(14)-HL . These results provide a foundation for future studies of the functions regulated by AHL quorum sensing in S . meliloti and help to establish proteomic analysis as a powerful global approach to the identification of quorum-sensing regulatory patterns in wild-type bacteria. Arch Biochem Biophys, 2003 Sep 1, 417(1), 123 - 7 Glycolaldehyde induces apoptosis in a human breast cancer cell line; Al-Maghrebi MA et al.; Activated phagocytes employ myeloperoxidase to generate glycolaldehyde, 2-hydroxypropanal, and acrolein . Because alpha-hydroxy and alpha,beta-unsaturated aldehydes are highly reactive, phagocyte-mediated formation of these products may play a role in killing bacteria and tumor cells . Using breast cancer cells, we demonstrate that glycolaldehyde inactivates glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and Cu,Zn superoxide dismutase, suppresses cell growth, and induces apoptosis . These results suggest that glycolaldehyde might be an important mediator of neutrophil anti-tumor activity. J Comp Pathol, 2003 Aug-Oct, 129(2-3), 179 - 85 Comparison of diagnostic techniques for porcine proliferative enteropathy (Lawsonia intracellularis infection); Huerta B et al.; In studying the post-mortem diagnosis of porcine proliferative enteropathy (PPE) a "double-blind" study was performed on 77 apparently healthy "finisher" pigs at the time of slaughter, to compare the results of a polymerase chain reaction (PCR) technique with those of (1) an indirect immunofluorescence assay (IFA), (2) examination for gross proliferative lesions at slaughter, (3) histopathological study of sections stained with haematoxylin and eosin (HE), and (4) Warthin-Starry (WS) silver staining for intracellular bacteria . The IFA, with a sensitivity of 89% and a specificity of 97% (positive "likelihood ratio"=30) and an agreement of 82% with PCR, is suggested as an alternative to the PCR in the post-mortem diagnosis of PPE . Histopathological examination was shown to be of little use as a principal diagnostic method, although it appeared to be effective in diagnosing infection by Lawsonia intracellularis in cases with proliferative-type lesions (positive likelihood ratio=29.6) . Finally, the values obtained from an examination of gross lesions (k=0.075; positive likelihood ratio of 1.3) and WS staining (k=0.42; positive likelihood ratio of 5.3) demonstrated the lack of validity of these tests for the diagnosis of L . intracellularis infection. Zhonghua Shao Shang Za Zhi, 2003 Jun, 19(3), 141 - 4 {An experimental study on the apoptosis of rabbit small intestinal cells during early postburn stage}; Wang H et al.; OBJECTIVE: To explore the significance of apoptosis of rabbit small intestinal mucosal epithelial cells and lymphocytes, and lymphocytes of lumbrical process at early postburn stage . METHODS: Twenty-five Japanese white rabbits were randomly divided into 5 groups with 5 in each group, i.e . normal control (N), 3-postburn-hour group (3 PBH), 6 PBH, 12 PBH and 24 PBH groups . The rabbits in all PBH groups were inflicted with 30% TBSA III degree of flame burn on the back . The intestinal tissue samples were harvested from 5 anatomical sites for HE staining, electron microscopic examination and the detection of apoptosis in situ by TUNEL method at all the postburn time points . The results of TUNEL slides were analyzed statistically . RESULTS: HE staining revealed that there were relatively abundant apoptotic cells scattering solitarily in the lymph nodules and diffuse lymphatic tissue in the mucosal epithelial and mucosal lamina propria (and partially extended into the submucosal layer) of the intestine and lumbrical process in all burn groups . There were some disruption of intestinal mucosa in 24 PBH group . But no obvious inflammatory reaction and signs of necrosis were observed in all the slides . Apoptotic body formation could be identified by EM . Large number of blue-black positive cellular nuclei were revealed by TUNEL method with their distribution as similar to that found by HE staining . When comparing with those in control group, the apoptotic cells in small intestine and lumbrical process were increased obviously (P < 0.01) in 3 PBH group and reached the top level in 6 and 12 PBH groups (P < 0.01), declining thereafter to near value of 3 PBH in 24 PBH group, though it was still higher than control (P < 0.05) . The number of apoptotic epithelial cells in middle distal portions of small intestinal mucosa in burn groups was much higher than that in proximal intestine (P < 0.05) . CONCLUSION: There was a large number of apoptotic cells in rabbit small intestinal mucosal epithelium, gut associated lymphoid tissue and lymphocytes in the lumbrical process, especially in the middle and distal portions of the intestine . These change might be the cellular basis of postburn intestinal translocation of bacteria and endotoxin. Curr Issues Mol Biol, 2003 Oct, 5(4), 123 - 7 An historical perspective on genomic technologies; Galas DJ et al.; Genomic technologies are best defined as technologies used to manipulate and analyze genomic information . The evolution of this collective power began in earnest with the invention of DNA cloning in the 1970's and most of the technology derives from the last quarter of the 20th century . The historical impact of these technologies is clearly immense . With the genome sequence becoming available for many organisms, including humans, another new view of biology has recently emerged . This review examines the shape and texture of this recent evolution, with a particular emphasis on new technology: DNA cloning, macromolecular structure analysis (X-ray crystallography and NMR), DNA sequencing, DNA synthesis, amplification by the polymerase chain reaction, and transgenic animals (bacteria through mammals). J Biol Chem, 2003 Oct 24, 278(43), 42717 - 27 Epub 2003 Aug 14. Identification and characterization of two isoenzymes of methionine gamma-lyase from Entamoeba histolytica: a key enzyme of sulfur-amino acid degradation in an anaerobic parasitic protist that lacks forward and reverse trans-sulfuration pathways; Tokoro M et al.; To better understand the metabolism of sulfur-containing amino acids, which likely plays a key role in a variety of cell functions, in Entamoeba histolytica, we searched the genome data base for genes encoding putative orthologs of enzymes known to be involved in the metabolism . The search revealed that E . histolytica possesses only incomplete cysteine-methionine conversion pathways in both directions . Instead, this parasite possesses genes encoding two isoenzymes of methionine gamma-lyase (EC 4.4.1.11, EhMGL1/2), which has been implicated in the degradation of sulfur-containing amino acids . The two amebic MGL isoenzymes, showing 69% identity to each other, encode 389- and 392-amino acid polypeptides with predicted molecular masses of 42.3 and 42.7 kDa and pIs of 6.01 and 6.63, respectively . Amino acid comparison and phylogenetic analysis suggested that these amebic MGLs are likely to have been horizontally transferred from the Archaea, whereas an MGL from another anaerobic protist Trichomonas vaginalis has MGL isotypes that share a common ancestor with bacteria . Enzymological and immunoblot analyses of the partially purified native amebic MGL confirmed that both of the MGL isotypes are expressed in a comparable amount predominantly in the cytosol and form a homotetramer . Recombinant EhMGL1 and 2 proteins catalyzed degradation of L-methionine, DL-homocysteine, L-cysteine, and O-acetyl-L-serine to form alpha-keto acid, ammonia, and hydrogen sulfide or methanethiol, whereas activity toward cystathionine was negligible . These two isoenzymes showed notable differences in substrate specificity and pH optimum . In addition, we showed that EhMGL is an ideal target for the development of new chemotherapeutic agents against amebiasis by demonstrating an amebicidal effect of the methionine analog trifluoromethionine on trophozoites in culture (IC50 18 mum) and that this effect of trifluoromethionine was completely abolished by the addition of the MGL-specific inhibitor DL-propargylglycine. Blood, 2004 Feb 1, 103(3), 890 - 6 Epub 2003 Aug 14. Does Helicobater pylori initiate or perpetuate immune thrombocytopenic purpura? Michel M, Cooper N, Jean C, Frissora C, Bussel JB. To determine the prevalence of Helicobacter pylori (H pylori) infection in North American patients with immune thrombocytopenic purpura (ITP) and the effect of H pylori eradication on the platelet count, a prospective study was performed . Seventy-four patients aged 10 years and older (mean age of 41 years) with chronic ITP and a platelet count below 60 x 10(9)/L were enrolled . H pylori infection was found in 22% of patients by means of a breath test and could not be predicted by gastrointestinal symptoms . H pylori-positive patients (52.5 years of age) were older than H pylori-negative patients (38.5 years of age; P =.0035) . Fifteen of the 16 H pylori-positive patients were treated and the bacteria was eradicated in 14 (93%) . After 3 months, a significant response (platelet count > 50 x 10(9)/L and doubling the initial count) was observed in only one patient . After a median follow-up of 11.5 months, none of the 14 patients had responded . Ten H pylori-negative patients treated with the same regimen also did not increase their platelet counts . In conclusion, unlike several previous reports, this study does not implicate H pylori in the pathogenesis of ITP since the prevalence of H pylori infection was low and eradication of H pylori did not positively influence the course of the ITP. Biomol Eng, 2003 Jul, 20(4-6), 389 - 99 Qualitative and quantitative aspects of 2-5A synthesizing capacity of different marine sponges; Reintamm T et al.; 2-5A synthetase is an important component of the mammalian antiviral 2-5A system . At present, the existence of 2-5A synthetase in the lowest animals, the marine sponges, has been demonstrated, although this enzyme has not been found in bacteria, yeast or plants . Here, we studied the 2-5A synthesizing capacity and the product profile of a variety of marine sponges belonging to Demospongia subclasses Tetractinomorpha and Ceractinomorpha . The 2-5A synthetase activity varied largely, in the range of four orders of magnitude, depending on the sponge species . Compared with the enzymes of the mammalian 2-5A synthetase family, the most active sponge species exhibited a surprisingly high 2-5A synthetase specific activity . Unlike the mammalian 2-5A synthetases that produce 2-5A oligomers in the presence of a double-stranded RNA activator, the 2-5A synthetase(s) from sponges were active without the addition of dsRNA . The sponge species differed in their product profiles . A novel product pool formed by Chondrosia reniformis was identified as a series of long 2-5A oligomers (up to 17-mers) with the prevalence of heptamers and octamers . The large variability of qualitative and quantitative characteristics of sponge 2-5A synthetases may refer to the occurrence of a variety of 2-5A synthetase isozymes in sponges. Biomol Eng, 2003 Jul, 20(4-6), 325 - 31 Piezotolerance as a metabolic engineering tool for the biosynthesis of natural products; Wright PC et al.; Thermodynamically, high-pressure (>10's of MPa) has a potentially vastly superior effect on reactions and their rates within metabolic processes than temperature . Thus, it might be expected that changes in the pressure experienced by living organisms would have effects on the products of their metabolism . To examine the potential for modification of metabolic pathways based on thermodynamic principles we have performed simple molecular dynamics simulations, in vacuo and in aquo on the metabolites synthesized by recombinant polyketide synthases (PKS) . We were able to determine, in this in silico study, the volume changes associated with each reaction step along the parallel PKS pathways . Results indicate the importance of explicitly including the solvent in the simulations . Furthermore, the addition of solvent and high pressure reveals that high pressure may have a beneficial effect on certain pathways over others . Thus, the future looks bright for pressure driven novel secondary metabolite discoveries, and their sustained and efficient production via metabolic engineering. Mol Ecol, 2003 Sep, 12(9), 2447 - 55 Genetic markers for analysing symbiotic relationships and lateral gene transfer in Neotropical bradyrhizobia; Parker MA; Assays with seven sets of lineage-specific polymerase chain reaction (PCR) primers in the ribosomal RNA region were performed on 96 isolates of the Bradyrhizobium sp . nodule bacteria from Barro Colorado Island, Panama . The isolates were derived from 10 legume host species in six genera (Centrosema, Desmodium, Dioclea, Inga, Machaerium and Vigna) . The PCR assays differentiated 13 composite genotypes, and sequencing of a 5' 23S rRNA region indicated that all but one had a unique sequence . The most common genotype (seen in 44% of the isolates) was associated with all six legume host genera, and had a marker profile and 5' 23S rRNA sequence identical to a Bradyrhizobium lineage associated with several other legume genera in Panama and Costa Rica . Another 46% of the isolates had genotypes found to be associated with two to three legume genera . Bradyrhizobium strains with low host specificity thus appear to be prevalent in this tropical forest . Based on 16S rRNA and 5' 23S rRNA markers, most of the isolates had clear affinities to either B . japonicum or B . elkanii . However, one strain (Cp5-3) with a B . elkanii-type 16S rRNA marker had a 5' 23S rRNA region resembling B . japonicum . A partition homogeneity test indicated that relationships of strain Cp5-3 were significantly discordant for 16S rRNA vs . 23S rRNA sequences, and a runs test detected significant mosaic structure across the rRNA region . Lateral gene transfer events have therefore played a role in the evolution of symbiotic bacteria in this environment. Environ Microbiol, 2003 Sep, 5(9), 804 - 9 Acidovorax-like symbionts in the nephridia of earthworms; Schramm A et al.; Dense accumulations of bacteria in the excretory organs, nephridia, were first described more than 75 years ago in members of the annelid family Lumbricidae (earthworms) . These nephridial symbionts were assumed to play a role in the degradation of proteins in the excretory fluid for nitrogen recycling . In the present study, the phylogenetic affiliation of the nephridial bacteria of the earthworms Lumbricus terrestris, Aporrectodea tuberculata, Octolasion lacteum and Eisenia foetida was resolved . The 16S rRNA gene sequences of the symbionts formed a monophyletic cluster within the genus Acidovorax . Similarity between symbiont sequences from different host species was 95.5-97.6%, whereas similarity was> 99% between symbiont sequences from individuals of the same species . Densely packed bacteria were detected in the ampulla of the nephridia by fluorescence in situ hybridization (FISH) using Acidovorax-specific oligonucleotide probes . No other bacterial cells could be found by FISH, although a few sequences other than Acidovorax had been found by PCR and cloning . These results suggest that the Acidovorax-earthworm symbiosis is a stable, host-specific association that has evolved from a common bacterial ancestor . Given the close phylogenetic relationship of the symbionts to proteolytic, free-living Acidovorax species, they may indeed play a role in protein degradation during nitrogen excretion by earthworms. J Vet Diagn Invest, 2003 Jul, 15(4), 374 - 8 Evaluation of the Brucella abortus species-specific polymerase chain reaction assay, an improved version of the Brucella AMOS polymerase chain reaction assay for cattle; Bricker BJ et al.; In a blind test, 344 samples representing 80 bacterial isolates were analyzed by the Brucella abortus species-specific polymerase chain reaction (BaSS PCR) assay for the identification and discrimination of B . abortus field strains (wild-type biovars 1, 2, and 4) from 1) B . abortus vaccine strains, 2) other Brucella species, and 3) non-Brucella bacteria . Identical samples were tested in 2 laboratories . Half the samples were fully viable, and half were bacteria that had been killed by methanol fixation . The results in 1 laboratory correctly identified 100% of the samples, resulting in a predictive value of 100% for all categories and 100% sensitivity and specificity under the prescribed conditions . The second laboratory misidentified 31 samples, resulting in a range of 66.7-100% sensitivity, 93.2-99.7% specificity, and 77.3-98.2% predictive values depending on the category . There was no significant difference in viable versus fixed bacteria for either laboratory . Subsequent review of the protocol indicated that contamination was the likely cause of 26 of the 31 erroneous identifications . The results show that the BaSS PCR assay has the potential to be a very reliable screening tool for B . abortus identification . However, the data also provide a cautionary reminder of the importance of preventing contamination in diagnostic PCR. East Afr Med J, 2003 Apr, 80(4), 218 - 22 Arcobacter butzleri strains from poultry abattoir effluent in Nigeria {corrected}; Amisu KO et al.; OBJECTIVE: To investigate the prevalence, species distribution and genetic diversity of zoonotic Arcobacter species . DESIGN: Prospective study . SETTING: Drainage system of a cosmopolitan chicken abattoir in Lagos, Nigeria . METHODS: One hundred and fifty drainage water samples were enriched in a minimal antibiotics-containing medium at room temperature and bacteria then isolated by use of a membrane filtration method . RESULTS: Twenty six (14%) of samples were positive for Arcobacter spp . Of these, 20 were examined by a comprehensive probabilistic identification scheme for Epsilobacteria and all strains identified as A . butzleri . AFLP analysis of these strains revealed considerable genetic diversity among the strains, with 12 genotypes defined at the 90% similarity level . CONCLUSION: The prevalence of A . butzleri in Nigerian poultry abattoir effluent indicates this species may constitute a public health problem in this country . AFLP profiling could be a useful tool for molecular epidemiological and population genetic studies of this organism . This is the first known report of A . butzleri in Nigeria, and first application of AFLP analysis for genotyping the species. J Biomed Mater Res A, 2003 Sep 1, 66(3), 596 - 604 In vitro biocompatibility assessment of sulfonated polyrotaxane-immobilized polyurethane surfaces; Park HD et al.; Sulfonated polyrotaxanes (PRx-SO(3)'s), in which sulfonated alpha-cyclodextrins (alpha-CDs) were threaded onto the poly(ethylene glycol) (PEG) segments in a PEG-b-poly(propylene glycol) (PPG)-b-PEG triblock copolymer (Pluronic) capped with benzyloxycarbonyl (Z)-L-phenylalanine (Z-L-Phe), were prepared as a novel surface-modifying biomaterial . Surface modification of the polyurethane (PU) was carried out by blending the PRx-SO(3)'s with a PU solution, followed by solution casting . The incorporated PRx-SO(3)'s led to the enhanced hydrophilicity by changing the surface properties of the PU matrix . Modified PUs showed the stable entrapment of the PRx-SO(3)'s with little extraction into water and enhanced mechanical properties after exposure to water compared to the PU control . The incorporated PRx-SO(3)'s repelled the proteins and kept them from closely approaching the surface areas, prevented platelet activation by thrombin, and effectively repelled bacteria . These results suggest that both the supramolecular structure of the polyrotaxanes and exposure of the sulfonated groups onto the surfaces contribute to these phenomena . Thus, surface modification with PRx-SO(3)'s is suggested to be useful for the fabrication of biocompatible medical devices . World J Surg, 2003 Oct, 27(10), 1161 - 4 Epub 2003 Aug 18. Obstructive jaundice alters proliferating cell nuclear antigen expression in rat small intestine; Sheen-Chen SM et al.; Translocation of bacteria and endotoxin has long been documented in obstructive jaundice, and altered intestinal barrier function is considered to be one of the important mechanisms for this phenomenon . Proliferating cell nuclear antigen (PCNA), also known as cyclin, is an auxiliary protein of DNA polymerase-delta, and the level of synthesis correlates directly with rates of cellular proliferation and DNA synthesis . This study was designed with the aim of evaluating the effect of obstructive jaundice on PCNA expression in small bowel epithelium . Male Sprague-Dawley rats were randomized to four groups . Group A (n = 10, control group) underwent a sham operation . Group B (n = 9, obstructive jaundice group for 1 week) underwent common bile duct ligation . Group C (n = 8, obstructive jaundice group for 2 weeks) underwent common bile duct ligation . Group D (n = 8, obstructive jaundice group for 2 weeks) underwent common bile duct ligation with oral glutamine intake . After periods of 7 days and 2 weeks, segments of small bowel were harvested from groups A & B and groups C & D, respectively . Nuclear immunohistochemical expression of PCNA in small bowel was evaluated . The PCNA-labeling index {(PCNA-positive cells/500 cells) x 100} was quantified . Comparisons among the four groups were performed . The PCNA-labeling index in small bowel of group B was significantly higher than that of group A (29.0% vs 21.2%, p = 0.001) . After 2 weeks of common bile duct ligation, the PCNA-labeling index in small bowel of group C was significantly lower than that of group A (19.4% vs 21.2%, p = 0.045) . With oral glutamine intake daily, the PCNA-labeling index in small bowel of Group D was restored and was significantly higher than that of group A (24.5% vs 21.2%, p = 0.002) . Obstructive jaundice for 1 week upgraded PCNA expression in rat small intestine . PCNA expression in rat small intestine later became depressed after obstructive jaundice for 2 weeks . Oral glutamine intake daily could effectively restore the PCNA expression in small bowel of rats subjected to obstructive jaundice for 2 weeks. Oncogene, 2003 Aug 14, 22(34), 5367 - 73 Specific inhibition of transcription factor NF-kappaB through intracellular protein delivery of I kappaBalpha by the Herpes virus protein VP22; Stroh C et al.; In many cancers, a high constitutive activation of transcription factor NF-kappaB has been implicated in tumor progression and apoptosis resistance, making NF-kappaB an attractive target for cancer therapy . Here, we describe the specific inhibition of NF-kappaB by the intracellular delivery of IkappaBalpha through VP22-mediated protein transduction . The Herpes virus protein VP22 has attracted great attention in gene therapy, because of its ability to migrate from an original expressing cell into surrounding recipient cells, resulting in high levels of protein transduction . To evaluate the use of VP22 as a vehicle for NF-kappaB inhibition, we expressed several versions of VP22-IkappaBalpha fusion proteins in baculovirus, bacteria, and mammalian cells . While we could not detect transcellular migration of different VP22-IkappaBalpha constructs, interestingly, baculovirally expressed VP22-IkappaBalpha was efficiently delivered into cells after exogenous administration . The purified and imported VP22-IkappaBalpha retained its function and efficiently inhibited both constitutive and inducible NF-kappaB activation . We further show that the 34 C-terminal amino acids of VP22 were sufficient for the import property, suggesting also that the ability of intercellular migration and cellular import are not linked to each other . Together, our results demonstrate that recombinant VP22 acts as an efficient vehicle for the exogenous delivery of IkappaBalpha and, moreover, might find applications to block NF-kappaB activation specifically. J Environ Sci Health A Tox Hazard Subst Environ Eng, 2003 Jul, 38(7), 1341 - 59 Process kinetics of pilot structured media anaerobic reaction tank for spentwash treatment; Kanhe NM et al.; This article presents the results obtained from pilot plant studies carried out on Structured Media Anaerobic Reaction Tank . Using various models, process kinetics such as pH, temperature, detention time, reactor volume, flow rate, organic loading rate, COD removal efficiency, substrate removal rate constant, specific growth rate, decay coefficient etc., are evaluated . These optimum process kinetics obtained may be used for the design and efficient operation of such type of full-scale anaerobic reactor for spentwash treatment. Pflugers Arch, 2004 Feb, 447(5), 594 - 602 Epub 2003 Aug 12. The SLC13 gene family of sodium sulphate/carboxylate cotransporters; Markovich D et al.; The SLC13 gene family consist of five sequence-related members that have been identified in a variety of animals, plants, yeast and bacteria . Proteins encoded by these genes are divided into two functionally unrelated groups: the Na(+)-sulphate (NaS) cotransporters and the Na(+)-carboxylate (NaC) cotransporters . Members of this family include the renal Na(+)-dependent inorganic sulphate transporter-1 (NaSi-1, SLC13A1), the Na(+)-dependent dicarboxylate transporters NaDC-1/SDCT1 (SLC13A2), NaDC-3/SDCT2 (SLC13A3), the sulphate transporter-1 (SUT-1, SLC13A4) and the Na(+)-coupled citrate transporter (NaCT, SLC13A5) . The general characteristics of the SLC13 proteins are that they encode multi-spanning proteins with 8-13 transmembrane domains, have a wide tissue distribution with most being expressed in the epithelial cells of the kidney and the gastrointestinal tract . They are Na(+)-coupled symporters, DIDS-insensitive, with strong cation preference for Na(+), with a Na(+):anion coupling ratio of around 3:1 and have a substrate preference for divalent anions, which include tetraoxyanions (for the NaS cotransporters) or Krebs cycle intermediates, including mono-, di-, and tri-carboxylates (for the NaC cotransporters) . The purpose of this review is to provide an update on the most recent advances and to summarize the biochemical, physiological and structural aspects of the vertebrate SLC13 gene family. Genome Res, 2003 Sep, 13(9), 2069 - 81 Epub 2003 Aug 12. A general approach for identifying distant regulatory elements applied to the Gdf6 gene; Mortlock DP et al.; Regulatory sequences in higher genomes can map large distances from gene coding regions, and cannot yet be identified by simple inspection of primary DNA sequence information . Here we describe an efficient method of surveying large genomic regions for gene regulatory information, and subdividing complex sets of distant regulatory elements into smaller intervals for detailed study . The mouse Gdf6 gene is expressed in a number of distinct embryonic locations that are involved in the patterning of skeletal and soft tissues . To identify sequences responsible for Gdf6 regulation, we first isolated a series of overlapping bacterial artificial chromosomes (BACs) that extend varying distances upstream and downstream of the gene . A LacZ reporter cassette was integrated into the Gdf6 transcription unit of each BAC using homologous recombination in bacteria . Each modified BAC was injected into fertilized mouse eggs, and founder transgenic embryos were analyzed for LacZ expression mid-gestation . The overlapping segments defined by the BAC clones revealed five separate regulatory regions that drive LacZ expression in 11 distinct anatomical locations . To further localize sequences that control expression in developing skeletal joints, we created a series of BAC constructs with precise deletions across a putative joint-control region . This approach further narrowed the critical control region to an area containing several stretches of sequence that are highly conserved between mice and humans . A distant 2.9-kilobase fragment containing the highly conserved regions is able to direct very specific expression of a minimal promoter/LacZ reporter in proximal limb joints . These results demonstrate that even distant, complex regulatory sequences can be identified using a combination of BAC scanning, BAC deletion, and comparative sequencing approaches. FEBS Lett, 2003 Aug 14, 549(1-3), 57 - 62 Targeted inactivation of the hrcA repressor gene in cyanobacteria; Nakamoto H et al.; To determine if the CIRCE/HrcA system operates in cyanobacteria, we have inactivated the hrcA repressor gene in Synechocystis sp . PCC 6803 by gene targeting . In the hrcA mutant, the groESL1 operon and the groEL2 gene, both of which have the CIRCE operator in their upstream regions, were derepressed at 30 degrees C without affecting expression of other major heat-shock genes . However, expression of these groE genes in the mutant was not fully derepressed . Their transcription increased further upon heat shock, and was initiated from the same sites as those used under normal conditions . This suggests that their expression is regulated by at least two different mechanisms, a negative one controlled by HrcA and an unknown positive one . The heat-induced expression of clpB1 and htpG was greatly repressed by the absence of HrcA . The hrcA mutant which constitutively overexpressed GroEL displayed improved cellular thermotolerance and also reduced photobleaching of phycocyanin under heat stress conditions. FEBS Lett, 2003 Aug 14, 549(1-3), 52 - 6 A photosystem 1 psaFJ-null mutant of the cyanobacterium Synechocystis PCC 6803 expresses the isiAB operon under iron replete conditions; Jeanjean R et al.; A psaFJ-null mutant of Synechocystis sp . strain PCC 6803 was characterised . As opposed to similar mutants in chloroplasts of green algae, electron transfer from plastocyanin to photosystem 1 was not affected . Instead, a restraint in full chain photosynthetic electron transfer was correlated to malfunction of photosystem 1 at its stromal side . Our hypothesis is that absence of PsaF causes oxidative stress, which triggers the induction of the 'iron stress inducible' operon isiAB . Products are the IsiA chlorophyll-binding protein (CP43') and the isiB gene product flavodoxin . Supporting evidence was obtained by similar isiAB induction in wild type cells artificially exposed to oxidative stress. Mol Immunol, 2003 Sep, 40(2-4), 159 - 70 Complement therapeutics; history and current progress; Morgan BP et al.; Complement (C) performs vital roles in immune surveillance, from killing of bacteria to generation of an optimal antibody response . However, the mediators responsible for this protective role can inappropriately target self tissues and cause pathology in many inflammatory diseases, in ischaemia-reperfusion injuries and also as a result of therapeutic intervention, such as in cardiopulmonary bypass . Here we review the history of anti-complement therapeutics and describe the plethora of reagents that have evolved to treat complement-mediated pathologies . These agents range from small compounds, including natural products isolated from plants and synthetic peptides designed to target and inhibit the complement cascade, to large, intricately engineered biological reagents . Recombinant, humanised antibody fragments which inhibit at specific points in the complement cascade have been generated and used successfully in man . Other reagents, mimicking the action of the natural complement regulatory proteins present on the surface of self cells, have also been developed and extensively tested . We discuss the pros and cons of these different reagents and describe recent advances in the field, such as specific targeting of drugs to sites of inflammation, which have opened the door to the use of anti-complement therapy in both acute and chronic inflammatory conditions. Genome Biol . 2003;4(8):115 . Epub 2003 Jul 16. Comparative genomics of Archaea: how much have we learned in six years, and what's next? Makarova KS, Koonin EV. Archaea comprise one of the three distinct domains of life (with bacteria and eukaryotes) . With 16 complete archaeal genomes sequenced to date, comparative genomics has revealed a conserved core of 313 genes that are represented in all sequenced archaeal genomes, plus a variable 'shell' that is prone to lineage-specific gene loss and horizontal gene exchange . The majority of archaeal genes have not been experimentally characterized, but novel functional pathways have been predicted. Kisaengchunghak Chapchi, 1973 Apr, 11(1), 1 - 11 {Fine Structures Of Trichomonas Tenax And Trichomonas Hominis}; Lee JM et al.; Trichomonas tenax(T . tenax) and Trichomonas hominis (T . hominis) were collected, cultured and sampled for comparative microscopical studies using electron microscope . 1 . Both flagellates were oval in shape and surrounded by a distinct outer membrane . Five recurrent flagella and one anterior flagellum had, each, 9 paris of peripheral and 1 pair of central fibrils, Undulating membrane was curved over the recurrent flagella, and bended in the middle at right angles with cell surface . Cytostome, engulfing bacteria, was observed in T . hominis . 2 . In the cytoplasm, there were fine dense glycogen particles, and vacuoles containing ingested materials . Dense pigment rods were also observed in both flagellates, but the rods were not distributed around the vacuoles in T . hominis . 3 . In T . tenax axostyle appeared as a cup-shaped structure comprising a single row of 41 fibrils, each about 120 a in diameter . It enclosed glycogen particles, and the open side was faced to the nucleus . 4 . Endoplasmic reticulum was observed around the nucleus, but it was less developed in T . hominis . 5 . Nucleus was ovoid having double nuclear membrane, which was clearly defined in T . hominis . 6 . Blepharoplast, parabasal body, Golgi appartus and mitochondrion was not observed in both flagellates. Eukaryot Cell, 2003 Aug, 2(4), 788 - 97 CF45-1, a secreted protein which participates in Dictyostelium group size regulation; Brock DA et al.; Developing Dictyostelium cells aggregate to form fruiting bodies containing typically 2 x 10(4) cells . To prevent the formation of an excessively large fruiting body, streams of aggregating cells break up into groups if there are too many cells . The breakup is regulated by a secreted complex of polypeptides called counting factor (CF) . Countin and CF50 are two of the components of CF . Disrupting the expression of either of these proteins results in cells secreting very little detectable CF activity, and as a result, aggregation streams remain intact and form large fruiting bodies, which invariably collapse . We find that disrupting the gene encoding a third protein present in crude CF, CF45-1, also results in the formation of large groups when cells are grown with bacteria on agar plates and then starve . However, unlike countin(-) and cf50(-) cells, cf45-1(-) cells sometimes form smaller groups than wild-type cells when the cells are starved on filter pads . The predicted amino acid sequence of CF45-1 has some similarity to that of lysozyme, but recombinant CF45-1 has no detectable lysozyme activity . In the exudates from starved cells, CF45-1 is present in a approximately 450-kDa fraction that also contains countin and CF50, suggesting that it is part of a complex . Recombinant CF45-1 decreases group size in colonies of cf45-1(-) cells with a 50% effective concentration (EC(50)) of approximately 8 ng/ml and in colonies of wild-type and cf50(-) cells with an EC(50) of approximately 40 ng/ml . Like countin(-) and cf50(-) cells, cf45-1(-) cells have high levels of cytosolic glucose, high cell-cell adhesion, and low cell motility . Together, the data suggest that CF45-1 participates in group size regulation in Dictyostelium. Eukaryot Cell, 2003 Aug, 2(4), 737 - 45 PAK paradox: Paramecium appears to have more K(+)-channel genes than humans; Haynes WJ et al.; K(+)-selective ion channels (K(+) channels) have been found in bacteria, archaea, eucarya, and viruses . In Paramecium and other ciliates, K(+) currents play an essential role in cilia-based motility . We have retrieved and sequenced seven closely related Paramecium K(+)-channel gene (PAK) sequences by using previously reported fragments . An additional eight unique K(+)-channel sequences were retrieved from an indexed library recently used in a pilot genome sequencing project . Alignments of these protein translations indicate that while these 15 genes have diverged at different times, they all maintain many characteristics associated with just one subclass of metazoan K(+) channels (CNG/ERG type) . Our results indicate that most of the genes are expressed, because all predicted frameshifts and several gaps in the homolog alignments contain Paramecium intron sequences deleted from reverse transcription-PCR products . Some of the variations in the 15 genomic nucleotide sequences involve an absence of introns, even between very closely related sequences, suggesting a potential occurrence of reverse transcription in the past . Extrapolation from the available genome sequence indicates that Paramecium harbors as many as several hundred of this one type of K(+)-channel gene . This quantity is far more numerous than those of K(+)-channel genes of all types known in any metazoan (e.g., approximately 80 in humans, approximately 30 in flies, and approximately 15 in Arabidopsis) . In an effort to understand this plurality, we discuss several possible reasons for their maintenance, including variations in expression levels in response to changes in the freshwater environment, like that seen with other major plasma membrane proteins in Paramecium. Vet Res, 2003 Jul-Aug, 34(4), 423 - 33 Experimental Coxiella burnetii infection in pregnant goats: excretion routes; Arricau Bouvery N et al.; Q fever is a widespread zoonosis caused by Coxiella burnetii . Infected animals, shedding bacteria by different routes, constitute contamination sources for humans and the environment . To study Coxiella excretion, pregnant goats were inoculated by the subcutaneous route in a site localized just in front of the shoulder at 90 days of gestation with 3 doses of bacteria (10(8), 10(6) or 10(4) i.d.) . All the goats aborted whatever the dose used . Coxiella were found by PCR and immunofluorescence tests in all placentas and in several organs of at least one fetus per goat . At abortion, all the goats excreted bacteria in vaginal discharges up to 14 days and in milk samples up to 52 days . A few goats excreted Coxiella in their feces before abortion, and all goats, excreted bacteria in their feces after abortion . Antibody titers against Coxiella increased from 21 days post inoculation to the end of the experiment . For a Q fever diagnostic, detection by PCR and immunofluorescence tests of Coxiella in parturition products and vaginal secretions at abortion should be preferred to serological tests. Clin Exp Allergy, 2003 Aug, 33(8), 1083 - 9 Mycobacterium vaccae administration during allergen sensitization or challenge suppresses asthmatic features; Smit JJ et al.; BACKGROUND AND OBJECTIVE: The hygiene hypothesis suggests that a lack of bacterial infections would favour the development of allergic disease . For this reason, bacteria or their components can be used as potential treatment for allergic asthma . We investigated whether heat-killed Mycobacterium vaccae is either able to suppress the induction of allergic asthma or able to suppress already established allergic asthma . METHODS: Mice were sensitized with ovalbumin (OVA)/alum on days 0 and 14 . Thereafter, mice were challenged on days 35, 39 and 42 by inhalation of either OVA or saline aerosols . M . vaccae-treated mice received an injection with 106, 107 or 108 CFU heat-killed M . vaccae on days 0 and 14 or 107 CFU on days 35 and 39 . On day 43, the airway responsiveness of the mice to increasing concentrations of methacholine was assessed, blood was withdrawn to measure serum parameters, and lung lavage was performed to detect cytokines and inflammatory cell number . RESULTS: Treatment of OVA-sensitized mice with 107 CFU M . vaccae either during sensitization or challenge suppresses airway hyper-responsiveness, airway eosinophilia and IL-5 production after OVA challenge . The increases in OVA-specific serum IgE and in IL-4 by respiratory challenges with OVA were only diminished after M . vaccae treatment (107 CFU) during sensitization . CONCLUSIONS: Heat-killed M . vaccae prevents allergic and asthmatic manifestations in a mouse model and, more importantly, M . vaccae treatment during challenge suppresses features of asthma, which opens up possibilities for new therapeutic interventions. J Appl Microbiol, 2003, 95(3), 528 - 35 Effect of some abiotic factors on the biological activity of Gluconacetobacter diazotrophicus; Tejera NA et al.; AIMS: The effect of some abiotic factors, dryness, heat and salinity on the growth and biological activity of Gluconacetobacter diazotrophicus, and the influence of a salt stress on some enzymes involved in carbon metabolism of these bacteria is studied under laboratory conditions . METHODS AND RESULTS: Strain PAL-5 of G . diazotrophicus was incubated under different conditions of drying, heat and salinity . Cells showed tolerance to heat treatments and salt concentrations, and sensitivity to drying conditions . Higher NaCl dosage of 150 and 200 mmol l -1 limited its growth and drastically affected the nitrogenase activity and the enzymes glucose dehydrogenase, alcohol dehydrogenase, fumarase, isocitrate dehydrogenase and malate dehydrogenase . CONCLUSIONS: Gluconacetobacter diazotrophicus, despite its endophytic nature, tolerated heat treatments and salinity stress, but its nitrogenase activity and carbon metabolism enzymes were affected by high NaCl dosage . SIGNIFICANCE AND IMPACT OF THE STUDY: The investigation of the biological activity of G . diazotrophicus in response to different abiotic factors led to more knowledge of this endophyte and may help to clarify pathways involved in its transmission into the host plant. J Appl Microbiol, 2003, 95(3), 492 - 9 Mediation of arsenic oxidation by Thiomonas sp . in acid-mine drainage (Carnoulès, France); Bruneel O et al.; AIMS: To isolate, identify, and characterize heterotrophic bacteria in acid-mine drainage that mediate oxidation of As(III) . METHODS AND RESULTS: Samples of acid-mine drainage were collected over a period of 14 months . Heterotrophic and non-obligatory acidophilic bacteria in the samples were cultured on a solid medium (pH 7.0-7.2), and three strains were isolated . The three different strains belong to the genus Thiomonas, and have more than 99% homology with the group Ynys1 . Culturing in mineral media demonstrated that the isolated strains used thiosulphate as an energy source, and oxidized iron in the presence of thiosulphate . However, none of the strains were able to oxidize arsenic in the presence of thiosulphate, nor could they use iron or arsenic alone as an energy source . In vitro experiments demonstrated that two of the Thiomonas strains were able to oxidize more than 90% of the As(III) present in the acid-mine drainage, whereas no abiotic oxidation of arsenic occurred . CONCLUSIONS: Two strains of newly identified Thiomonas sp . found in acid-mine drainage are capable of oxidizing arsenic . SIGNIFICANCE AND IMPACT OF STUDY: These results represent the first reported oxidation of arsenic by Thiomonas sp . Biologically mediated oxidation and subsequent immobilization of arsenic is of great interest for the remediation of contaminated mine sites. J Mol Evol, 2003 Jun, 56(6), 665 - 72 Comparative study of translation termination sites and release factors (RF1 and RF2) in procaryotes; Ozawa Y et al.; Translation termination is catalyzed by release factors that recognize stop codons . However, previous works have shown that in some bacteria, the termination process also involves bases around stop codons . Recently, Ito et al . analyzed release factors and identified the amino acids therein that recognize stop codons . However, the amino acids that recognize bases around stop codons remain unclear . To identify the candidate amino acids that recognize the bases around stop codons, we aligned the protein sequences of the release factors of various bacteria and searched for amino acids that were conserved specifically in the sequence of bacteria that seemed to regulate translation termination by bases around stop codons . As a result, species having several highly conserved residues in RF1 and RF2 showed positive correlations between their codon usage bias and conservation of the bases around the stop codons . In addition, some of the residues were located very close to the SPF motif, which deciphers stop codons . These results suggest that these conserved amino acids enable the release factors to recognize the bases around the stop codons. J Wildl Dis, 2003 Apr, 39(2), 407 - 11 Lawsonia intracellularis in wild mammals in the Slovak Carpathians; Tomanova K et al.; Feces of wild mammals were collected in the Bukovske Vrchy Hills (north-eastern Slovakia) in January and February 2002 . The feces were examined for Lawsonia intracellularis by means of nested polymerase chain reaction . A total of 194 samples of feces from red deer (Cervus elaphus), 46 samples from roe deer (Capreolus capreolus), 31 samples from red fox (Vulpes vulpes), 23 samples from gray wolf (Canis lupus), and 12 samples from brown hare (Lepus europaeus) were examined . Lawsonia intracellularis was found in two samples from wolves, in two samples from foxes, and one sample from red deer . This is the first description of L . intracellularis in these three species. J Wildl Dis, 2003 Apr, 39(2), 329 - 37 An outbreak of fungal dermatitis and stomatitis in a free-ranging population of pigmy rattlesnakes (Sistrurus miliarius barbouri) in Florida; Cheatwood JL et al.; Between September 1997 and March 1998, a severe skin, eye, and mouth disease was observed in a population of dusky pigmy rattlesnakes (Sistrurus miliarius barbouri), at the Lake Woodruff National Wildlife Refuge in Volusia County, Florida (USA) . Three affected pigmy rattlesnakes were submitted for necropsy . All snakes had severe necrotizing and predominantly granulomatous dermatitis, stomatitis, and ophthalmitis, with involvement of the subadjacent musculature and other soft tissues . Numerous fungal hyphae were seen throughout tissue sections stained with periodic acid Schiff and Gomori's methenamine silver . Samples of lesions were cultured for bacteria and fungi . Based on hyphae and spore characteristics, four species of fungi were identified from culture: Sporothrix schenckii, Pestalotia pezizoides, Geotrichum candidum (Galactomyces geotrichum), and Paecilomyces sp . While no additional severely affected pigmy rattlesnakes were seen at the study site, a garter snake (Thamnophis sirtalis) and a ribbon snake (Thamnophis sauritis) with similar lesions were found . In 1998 and 1999, 42 pigmy rattlesnakes with multifocal minimal to moderate subcutaneous masses were seen at the study site . Masses from six of these snakes were biopsied in the field . Hyphae morphologically similar to those seen in the severe cases were observed with fungal stains . Analysis of a database representing 10,727 captur