Mol Microbiol, 2002 Oct, 46(2), 395 - 409 Cell cycle-dependent expression of an essential SMC-like protein and dynamic chromosome localization in the archaeon Halobacterium salinarum; Herrmann U et al.; The genome of Halobacterium salinarum encodes four proteins of the structural maintenance of chromosomes (SMC) protein superfamily . Two proteins form a novel subfamily and are named 'SMC-like proteins of H . salinarum' (Sph1 and Sph2) . Northern blot analyses revealed that sph1 and hp24, the adjacent gene, are solely transcribed in exponentially growing, but not in stationary phase, cells . A synchronization procedure was developed, which makes use of the DNA polymerase inhibitor aphidicolin and leads to highly synchronous cultures . It allowed us for the first time to study cell cycle-dependent transcription in an archaeon . The sph1 transcript was found to be highly cell cycle regulated, with its maximal accumulation around the time of septum formation . The Sph1 protein level was also elevated at that time, but a basal protein level was found throughout the cell cycle . The hp24 transcript was sharply upregulated about 1 h before sph1 and had already declined at the time of sph1 induction . These and additional transcript patterns revealed that precisely controlled transcriptional regulation is involved in haloarchaeal cell cycle progression . A DNA staining protocol was developed, which opened the possibility of following the dynamic intracellular localization of haloarchaeal nucleoids using synchronized cultures . After an initial dispersed localization, the nucleoid is condensed at mid-cell . Subsequently, DNA is rapidly transported to the 1/4 and 3/4 positions . All staining patterns were also observed in untreated exponentially growing cells, excluding synchronization artifacts . The Sph1 concentration is elevated when segregation of the new chromosomes is nearly complete; therefore, it is proposed to play a role in a late step of replication, e.g . DNA repair, similar to eukaryotic Rad18 proteins. J Agric Food Chem, 2002 Nov 6, 50(23), 6923 - 8 Antimutagenic effect of various honeys and sugars against Trp-p-1; Wang XH et al.; Honey has been used since ancient times as a flavorful sweetener and for its therapeutic and medicinal effects . Consumers' demand for natural, healthy products has driven renewed interest in honey's health benefits . The commonly encountered food mutagen, Trp-p-1, has been demonstrated to be mutagenic in bacteria and carcinogenic in animals . Chemically, honey is quite complex . Honey is comprised primarily of sugars; however, it contains many other potentially biologically active components, such as antioxidants . Sugars have been reported to display both mutagenic and antimutagenic effects in different systems; antioxidants often display antimutagenic activity . Little information exists about potential antimutagenic effects of honey . Antimutagenicity of honeys from seven different floral sources against Trp-p-1 was tested via the Ames assay and compared to that of a sugar analogue and to individually tested simple sugars . All honeys exhibited significant inhibition of Trp-p-1 mutagenicity; most demonstrated a linear correlation between percentage inhibition and log transformed honey concentration from 10 microg/mL to 20 mg/mL . Each displayed significant degrees of inhibition of mutagenicity above concentrations of 1 mg/mL, with individual variations in degree of effectiveness . Buckwheat honey displayed the greatest inhibition at 1 mg/mL, with slightly less effectiveness at higher concentrations . A sugar analogue demonstrated a pattern of inhibition similar to that of the honeys, with enhanced antimutagenicity at concentrations greater than 1 mg/mL . Glucose and fructose were also similar to honeys and were more antimutagenic than maltose and sucrose. JPEN J Parenter Enteral Nutr, 2002 Nov-Dec, 26(6), 372 - 6 Effects of prebiotics on the immune response to vaccination in the elderly; Bunout D et al.; BACKGROUND: Prebiotics stimulate the growth of bifidogenic bacteria in the gut . The aim of this work was to assess the effects of a prebiotic mixture on the immune response in healthy elderly people . METHODS: Healthy free-living elderly people (age, > or = 70 years), receiving a nutritional supplement that provided 1.6 MJ, 15 g of protein, and 50% of vitamin daily reference values per day, were randomly assigned to receive a prebiotic mixture (6 g/d of a 70% raftilose and 30% raftiline mixture) or placebo (6 g of maltodextrin powder) for 28 weeks . At week 2 of the study, all subjects were vaccinated with influenza and pneumococcal vaccines . At weeks 0, 2, and 8 of the study, serum total proteins, albumin, immunoglobulins, saliva secretory immunoglobulin A (IgA), and serum titers of influenza A and B and pneumococcal antibodies were measured . At week 8, cultured peripheral monocyte cell secretion of interleukin-4, interferon-gamma, and lymphocyte proliferation, stimulated with phytohemagglutinin and influenza antigen, were measured . RESULTS: Sixty-six subjects were considered eligible for the study, and 43 (20 receiving prebiotics and 23 receiving placebo) were considered for final analyses on a per protocol basis . No changes in serum proteins, albumin, immunoglobulins, and secretory IgA were observed . Antibodies against influenza B and pneumococcus increased significantly from weeks 0 to 8, with no significant differences between groups . Antibodies against influenza A did not increase . No effects of prebiotics on interleukin-4 and interferon-gamma secretion by cultured monocytes were observed . CONCLUSIONS: No immunological effects of prebiotics were observed in this study. Comp Med, 2002 Oct, 52(5), 403 - 13 The mouse as a model for investigation of human granulocytic ehrlichiosis: current knowledge and future directions; Borjesson DL et al.; The use of laboratory mice to investigate correlates of infectious disease, including infection kinetics, cellular alterations, cytokine profiles, and immune response in the context of an intact host has expanded exponentially in the last decade . A marked increase in the availability of transgenic mice and research tools developed specifically for the mouse parallels and enhances this research . Human granulocytic ehrlichiosis (HGE) is an emerging, zoonotic disease caused by tick-borne bacteria . The HGE agent (Anaplasma phagocytophila) is one of two recognized pathogens to cause human granulocytic ehrlichiosis (HGE) . The mouse model of HGE complements in vitro tissue culture studies, limited in vivo large animal studies, and ex vivo studies of human and ruminant neutrophils, and promises new avenues to approach mechanisms of disease . In the overview reported here, we focus principally on current research into HGE pathogenesis using the mouse model . Included is a discussion of current changes in ehrlichial classification and nomenclature, a review of ehrlichial biology and ecology, and highlights of clinical disease in animals and people. JPEN J Parenter Enteral Nutr, 2002 Nov-Dec, 26(6 Suppl), S19 - 24; discussion S24-5 Aspiration pneumonia: incidence, mortality, and at-risk populations; DeLegge MH; Pulmonary aspiration in the hospitalized patient can be devastating . Most aspiration events occur in patients with a swallowing disorder . Aspiration can be divided into 3 separate etiologies: oropharyngeal bacteria, particulate matter, and acidified gastric contents . Reported prevalence data are extremely variable, ranging from 10% to 70% . Mortality is related to the volume and content of the aspirate and is reported to be as high as 70% . Neurologic dysfunction, decreased consciousness, advancing age, gastroesophageal reflux, and tube feeding are all potential risk factors for the development of aspiration. Water Res, 2002 Sep, 36(16), 4160 - 4 Degradation of the (R)- and (S)-enantiomers of the herbicides MCPP and dichlorprop in a continuous field-injection experiment; Rugge K et al.; An aerobic field-injection experiment was performed to study the degradation and migration of different herbicides at trace levels in an aerobic aquifer at Vejen, Denmark . Mecoprop (MCPP) and dichlorprop monitored in a dense network of multilevel samplers were both degraded within a distance of 1 m after a period of 120 days . The study showed that no preferential degradation of the (R)- and (S)-enantiomers of MCPP and of dichlorprop took place as the enantiomeric forms of the phenoxy acids were degraded simultaneously in the aquifer. Water Res, 2002 Sep, 36(16), 4095 - 105 Multi-metallic modelling for biosorption of binary systems; Pagnanelli F et al.; In this paper a specially propagated biomass of Sphaerotilus natans was tested as adsorbent for binary solutions of Cu-Cd, Cu-Pb and Cu--Zn at different equilibrium pH . The experimental results outline the buffering effect of H+ at low pH . which masks the competition among metals . In each binary system the biomass affinity follows the acidic properties of the heavy metals probably due to an ionic exchange mechanism operating among active sites and metals in solution . The experimental results were fitted according to an empirical approach with growing complexity that outlines the inadequacy of the predictive models and the non-ideal interactions among metals. Photochem Photobiol, 2002 Oct, 76(4), 373 - 80 Bacteriochlorophyll e monomers, but not aggregates, sensitize singlet oxygen: implications for a self-photoprotection mechanism in chlorosomes; Arellano JB et al.; Sensitization of singlet delta oxygen (O2(1delta(g))) by bacteriochlorophyll e (BChle) has been investigated to gain a better understanding of the photoprotection mechanism(s) operating in chlorosomes of green photosynthetic bacteria . The sensitization process has been studied in media where BChle forms monomers (acetone and aqueous solutions containing 0.5% Triton X-100 {TX}) and in systems where BChle aggregates, namely, aqueous solutions containing 0.003% monogalactosyl diglyceride (MGDG) and chlorosomes(control as well as hexanol perturbed) from Chlorobium phaeobacteroides strain CL1401 . In Ar-purged acetone, BChle triplets (BChle triplets) have a lifetime of a few tens of microseconds; however, in air-saturated acetone, quenching of BChle triplets by ground-state oxygen (O2(3sigma(-)g)) and formation of O2(1delta(g)) take place . The O2(1delta(g)) so formed is susceptible to quenching by BChle0, a ground-state BChle molecule . A Stern-Volmer analysis reveals a linear fit between the decay rate of O2(1delta(g)) and the BChle concentration . The rate constants for the quenching of O2(1delta(g)) by BChle0 and for the deactivation of O2(1delta(g)) by the solvent come out to be kq = (1.4 +/- 0.1) x 10(9) M(-1) s(-1) and k0 = (18.5 +/- 0.7) x 10(3) s(-1), respectively . The absolute quantum yield of O2(1delta(g)) sensitization by BChle monomers is 0.65 +/- 0.15 in air-saturated acetone . In aqueous phase, the triplet lifetime of BChle aggregates in native or hexanol-perturbed chlorosomes shortens by more than two orders of magnitude when compared with the triplet lifetime of BChle monomers in 0.5% TX solution (a few hundreds of microseconds) . Quenching by carotenoids (Car) makes only a minor contribution to the decay of BChle triplets in aggregates . Because O2(1delta(g)) sensitization by BChle triplets could be detected neither in MGDG aggregates nor in chlorosomes (control as well as hexanol perturbed), it is concluded that (1) this process is highly likely when BChle is present as a monomer but not when it is tightly packed in artificial aggregates or in chlorosomes; and (2) Car, though vital for the baseplate BChla, are dispensable for BChle. Bull Soc Pathol Exot, 2002 Aug, 95(3), 194 - 6 {Rapid immunotitration of individual toxins from Androctonus australis venom}; Aubrey N et al.; Passive immunotherapy against scorpion envenomations is facilitated by the preliminary titration of circulating toxins in envenomed patients . Currently, routinely used ELISA tests allow only the titration of the whole venom, without reference to the toxins which compose the venom and spread variably within the tissue . Taking as a model one of the three toxins responsible for the lethal effects of Androctonus australis hector (Aahl) venom, we developed an ELISA sandwich test based on a fragment of recombining antibody (scFv) consisting of the variable chains of the monoclonal IgG 9C2 coupled to a decapeptide showing high affinity for streptavidine . Conjugate scFvlStrep-tag was prepared by genetic engineering . It was produced in the periplasm of recombining bacteria, in a reproducible way, in a soluble form, at low cost and with an output, after purification, of 0.8 mg/L of bacterial culture . The recombinant protein, of small size (28 kDa), is bifunctional . It preserves a very high affinity for the toxin Aah I (Kd of 2.3 10(-10) M, very close to that of IgG 9C2), yet recognises streptavidine and its conjugate (streptavidine-peroxidase) . The titration of the Aahl toxin used an ELISA sandwich test in which the toxin was captured in a specific way by a monoclonal antibody; the immunocomplexes were then detected by recombinant immunoconjugate, thus conferring a high specificity on titration . The test is quick (90 mn), reproducible and sensitive, with a limit of detection of 0.6 toxin (ng.ml-1) . This method could be extended to two other lethal toxins of the venom of the scorpion Androctonus australis hector and to those of other species . New perspectives are thus possible for the diagnosis of the envenomations. J Cell Biol, 2002 Oct 28, 159(2), 279 - 90 Epub 2002 Oct 28. Myosin Va binding to neurofilaments is essential for correct myosin Va distribution and transport and neurofilament density; Rao MV et al.; The identification of molecular motors that modulate the neuronal cytoskeleton has been elusive . Here, we show that a molecular motor protein, myosin Va, is present in high proportions in the cytoskeleton of mouse CNS and peripheral nerves . Immunoelectron microscopy, coimmunoprecipitation, and blot overlay analyses demonstrate that myosin Va in axons associates with neurofilaments, and that the NF-L subunit is its major ligand . A physiological association is indicated by observations that the level of myosin Va is reduced in axons of NF-L-null mice lacking neurofilaments and increased in mice overexpressing NF-L, but unchanged in NF-H-null mice . In vivo pulse-labeled myosin Va advances along axons at slow transport rates overlapping with those of neurofilament proteins and actin, both of which coimmunoprecipitate with myosin Va . Eliminating neurofilaments from mice selectively accelerates myosin Va translocation and redistributes myosin Va to the actin-rich subaxolemma and membranous organelles . Finally, peripheral axons of dilute-lethal mice, lacking functional myosin Va, display selectively increased neurofilament number and levels of neurofilament proteins without altering axon caliber . These results identify myosin Va as a neurofilament-associated protein, and show that this association is essential to establish the normal distribution, axonal transport, and content of myosin Va, and the proper numbers of neurofilaments in axons. J Microbiol Methods, 2003 Jan, 52(1), 85 - 91 A TaqMan-PCR protocol for quantification and differentiation of the phytopathogenic Clavibacter michiganensis subspecies; Bach HJ et al.; Real-time TaqMan-PCR assays were developed for detection, differentiation and absolute quantification of the pathogenic subspecies of Clavibacter michiganensis (Cm) in one single PCR run . The designed primer pair, targeting intergenic sequences of the rRNA operon (ITS) common in all subspecies, was suitable for the amplification of the expected 223-nt DNA fragments of all subspecies . Closely related bacteria were completely discriminated, except of Rathayibacter iranicus, from which weak PCR product bands appeared on agarose gel after 35 PCR cycles . Sufficient specificity of PCR detection was reached by introduction of the additional subspecies specific probes used in TaqMan-PCR . Only Cm species were detected and there was clear differentiation among the subspecies C . michiganensis sepedonicus (Cms), C . michiganensis michiganensis (Cmm), C . michiganensis nebraskensis (Cmn), C . michiganensis insidiosus (Cmi) and C . michiganensis tessellarius (Cmt) . The TaqMan assays were optimized to enable a simultaneous quantification of each subspecies . Validity is shown by comparison with cell counts. FEBS Lett, 2002 Oct 30, 531(1), 74 - 80 Current thoughts on the phosphatidylinositol transfer protein family; Allen-Baume V et al.; Monomeric transport of lipids is carried out by a class of proteins that can shield a lipid from the aqueous environment by binding the lipid in a hydrophobic cavity . One such group of proteins is the phosphatidylinositol transfer proteins (PITP) that can bind phosphatidylinositol and phosphatidylcholine and transfer them from one membrane compartment to another . PITPs are found in both unicellular and multicellular organisms but not bacteria . In mice and humans, the PITP domain responsible for lipid transfer is found in five proteins, which can be classified into two classes based on sequence . Class I PITPs comprises two family members, alpha and beta, small 35 kDa proteins with a single PITP domain which are ubiquitously expressed . Class IIA PITPs (RdgBalphaI and II) are larger proteins possessing additional domains that target the protein to membranes and are only able to bind lipids but not mediate transfer . Finally, Class IIB PITP (RdgBbeta) is similar to Class I in size (38 kDa) and is also ubiquitously expressed . Class III PITPs, exemplified by the Sec14p family, are found in yeast and plants but are unrelated in sequence and structure to Class I and Class II PITPs . In this review we discuss whether PITP proteins are passive transporters or are regulated proteins that are able to couple their transport and binding properties to specific biological functions including inositol lipid signalling and membrane turnover. J Radiol Prot, 2002 Sep, 22(3A), A159 - 61 Dose-effect modifying factors in radiation protection--a 1967 National Commission on Radiation Protection document revisited; Waligorski MP; In 1967 Subcommittee M-4 of the National Commission on Radiation Protection proposed a system for evaluating, summing and reporting occupational exposures . It appears that some 30 years later these concepts could be implemented in a system of radiation protection based on Katz's cellular track structure model, which is able to quantify and predict the response of systems relevant to radiation protection, such as survival or oncogenic transformations in cell cultures, enzymes, bacteria or whole organisms . As a consequence of this approach, an m-power-law (with m ranging between 2 and 3.5) dependence of effect, or radiation risk, after doses of standard Co-60 radiation, would follow. Biosci Biotechnol Biochem, 2002 Sep, 66(9), 1976 - 80 Gene encoding a trehalose phosphorylase from Thermoanaerobacter brockii ATCC 35047; Maruta K et al.; A gene encoding a trehalose phosphorylase was cloned from Thermoanaerobacter brockii ATCC 35047 . The gene encodes a polypeptide of 774 amino acid residues . The deduced amino acid sequence was homologous to bacterial maltose phosphorylases and a trehalose 6-phosphate phosphorylase catalyzing anomer-inverting reactions . On the other hand, no homology was found between the T . brockii enzyme and an anomer-retaining trehalose phosphorylase from Grifola frondosa. Izv Akad Nauk Ser Biol, 2002 Sep-Oct, (5), 534 - 40 {Functional characteristics of bacterioplankton with reference to its aggregation in water bodies of different types}; Shchur LA et al.; The functional characteristics of bacterioplankton have been studied with reference to its aggregation in water bodies of different types . Several methods were used for calculation of the total numbers of bacteria and proportion of aggregated cells . Analysis of the experimental data has shown a relationship between the functional characteristics of bacterioplankton, such as bacterial production, destruction of organic matter, and energy coefficient, and the proportion of aggregated bacteria . It has been shown that as this proportion increases, the production per cell and energy coefficients also increase. Biopolymers, 2003, 72(1), 10 - 20 Hydration of polysaccharide hyaluronan observed by IR spectrometry . I . Preliminary experiments and band assignments; Haxaire K et al.; This article is the first one in a series dedicated to the study of hyaluronan as observed by IR spectrometry . The goal is to determine its hydration mechanism and the structural changes this mechanism implies . Hyaluronan is a natural polysaccharide that is widely used in biomedical applications and cosmetics . Its macroscopic properties are significantly dependent on its degree of hydration . In this article we record the IR spectrum of a several micron thick dried film and deduce that four or five residual H(2)O molecules remain around each disaccharide repeat unit in the dried film . We then compare the spectra of sodium hyaluronan and its acid form to assign vibrational bands linked to the carboxylate group . We proceed with a qualitative analysis of the spectral changes induced by changes of temperature and hygroscopicity, two independent parameters that act by modifying the hydrogen bond network of the sample . This enables us to assign most of the vibrational bands of the hydrophilic groups and to distinguish the bands that are due to these hydrophilic groups when they are or are not hydrogen bonded . It constitutes a prerequisite for the quantitative analysis of hydration spectra that will be described in the following articles of this series . J Biol Chem, 2002 Dec 20, 277(51), 49727 - 34 Epub 2002 Oct 23. Intraphagosomal Mycobacterium tuberculosis acquires iron from both extracellular transferrin and intracellular iron pools . Impact of interferon-gamma and hemochromatosis; Olakanmi O et al.; Mycobacterium tuberculosis multiplies within the macrophage phagosome and requires iron for growth . We examined the route(s) by which intracellular M . tuberculosis acquires iron . During intracellular growth of the virulent Erdman M . tuberculosis strain in human monocyte-derived macrophages (MDM), M . tuberculosis acquisition of (59)Fe from transferrin (TF) provided extracellularly (exogenous source) was compared with acquisition when MDM were loaded with (59)Fe from TF prior to M . tuberculosis infection (endogenous sources) . M . tuberculosis (59)Fe acquisition required viable bacteria and was similar from exogenous and endogenous sources at 24 h and greater from exogenous iron at 48 h . Interferon-gamma treatment of MDM reduced (59)Fe uptake from TF 51% and TF receptor expression by 34% . Despite this, intraphagosomal M . tuberculosis iron acquisition in IFN-gamma-treated cells was decreased by only 30% . Macrophages from hereditary hemochromatosis patients have altered iron metabolism . Intracellular M . tuberculosis acquired markedly less iron in MDM from these individuals than in MDM from healthy donors, regardless of the iron source (exogenous and endogenous): 36 +/- 3.8% and 17 +/- 9.6% of control, respectively . Thus, intraphagosomal M . tuberculosis can acquire iron from both extracellular TF and endogenous macrophage sources . Acquisition of iron from macrophage cytoplasmic iron pools may be critical for the intracellular growth of M . tuberculosis . This acquisition is altered by IFN-gamma treatment to a small extent, but is markedly reduced in macrophages from hemochromatosis patients. Trends Plant Sci, 2002 Oct, 7(10), 451 - 6 Cutting edge of chloroplast proteolysis; Adam Z et al.; Chloroplasts have a dynamic protein environment and, although proteases are presumably major contributors, the identities of these crucial regulatory proteins have only recently been revealed . There are defined proteases within each of the major chloroplast compartments: the ATP-dependent Clp and FtsH proteases in the stroma and stroma-exposed thylakoid membranes, respectively, the ATP-independent DegP proteases within the thylakoid lumen and on both sides of thylakoid membranes, and the SppA protease on the stromal side of the thylakoid . All four types are homologous to proteases characterized in bacteria, but most have many isomers in higher plants . With such diversity, the challenge is to link the mode of action of each protease to the chloroplast enzymes and regulatory proteins that it targets. J Zoo Wildl Med, 2002 Jun, 33(2), 166 - 71 Systemic mycosis caused by Scedosporium apiospermum in a stranded northern elephant seal (Mirounga angustirostris) undergoing rehabilitation; Haulena M et al.; A recently weaned, stranded, male northern elephant seal (Mirounga angustirostris) pup that had been undergoing rehabilitation was found severely obtunded with hyponatremia, hypokalemia, hypochloremia, and hypophosphatemia after a history of intermittent regurgitation . The animal was euthanatized, and gross postmortem findings included multifocal abscessation affecting brain, spleen, kidney, muscle, and subcutaneous tissue . Scedosporium apiospermum and mixed bacteria were cultured from brain, kidney, and subcutaneous tissue . Histopathologic examination revealed multiple fungal granulomas of variable size in the kidneys, brain, liver, and skeletal muscle . This is the first report of S . apiospermum infection associated with lesions in a marine mammal. Int J Med Microbiol, 2002 Sep, 292(3-4), 257 - 66 CagA tyrosine phosphorylation and interleukin-8 induction by Helicobacter pylori are independent from alpAB, HopZ and bab group outer membrane proteins; Odenbreit S et al.; In several studies Helicobacter pylori type I strains (cag-positive strains) have been described to translocate their CagA protein into epithelial cells, where it is tyrosine-phosphorylated . The intimate contact allows a Cag-dependent bacteria-to-cell signaling inducing the secretion of the chemokine interleukin-8 . Although a contact between the bacterial and the eukaryotic cell is known to be necessary for these signal transduction events the bacterial adhesin and the cellular receptor are unknown, so far . In this study, we investigated the influence of several outer membrane proteins associated with adherence on CagA translocation and IL-8 induction . The quantitative assessment of a cag deletion mutant strain binding to epithelial cells revealed that the Cag secretion apparatus is not primarily necessary for attachment . In contrast, the knockout mutation of the adherence-associated alpAB locus significantly reduced the binding capacity in two independent strains . Despite this partial adherence defect, the alpAB mutation did not affect CagA translocation and IL-8 induction . The mutagenesis of the bab group genes hp317, hp896 and hp1243 in H . pylori 26695 did not influence the Cag-dependent signaling either . No causative linkage could be found between the production of the outer membrane proteins HopZ, OipA or seven additional outer membrane proteins and CagA translocation or IL-8 induction. Proc Natl Acad Sci U S A, 2002 Nov 12, 99(23), 14893 - 6 Epub 2002 Oct 23. Origin of plant glycerol transporters by horizontal gene transfer and functional recruitment; Zardoya R et al.; Gene-family evolution mostly relies on gene duplication coupled with functional diversification of gene products . However, other evolutionary mechanisms may also be important in generating protein diversity . The ubiquitous membrane intrinsic protein (MIP) gene family is an excellent model system to search for such alternative evolutionary mechanisms . MIPs are proteins that transport water, glycerol, and small solutes across cell membranes in all living organisms . We reconstructed the molecular phylogeny of MIPs based on amino acid sequence data by using neighbor-joining, maximum-likelihood, and Bayesian methods of phylogenetic inference . The recovered trees show an early and distinct separation of water and glycerol transporters, i.e., aquaporins (AQPs), and aquaglyceroporins . The latter are absent from plants . As expected, gene duplication and functional diversification account for most of the diversity of animal and plant members of the family . However, in contrast to this model, we find that the sister group of plant glycerol transporters are bacterial AQPs . This relationship suggests first that plant glycerol transporters may resulted from a single event of horizontal gene transfer from bacteria, which we have estimated to have occurred approximately 1,200 million years ago, at the origin of plants, and second that bacterial AQPs were likely recruited to transport glycerol in plants because of their absence of aquaglyceroporins . This striking example of adaptive evolution at the molecular level was demonstrated further by finding convergent or parallel replacements at particular amino acid positions related to water- and glycerol-transporting specificity. Avian Pathol, 2002 Jun, 31(3), 267 - 70 A potential new serotype of Riemerella anatipestifer isolated from ducks in Thailand; Pathanasophon P et al.; Eighty isolates of Riemerella anatipestifer representing 71 outbreaks of riemerellosis in Thailand between 1994 and 1999 were serotyped using the gel diffusion precipitin test . Based on the precipitation patterns, 25 serological profiles containing one to three antigenic determinants were recognized . Heat-stable antigens of the organism reacted with antisera raised against 16 known serotypes and an untypable strain 698/95 . The most prevalent serotype appeared to be serotype 7, followed by serotypes 5, 10, 21 and 1 . Further study demonstrated that the untypable strain probably represents a new serotype . Analysis of the polymerase chain reaction-amplified rrs genes for restriction fragment length polymorphisms verified the inclusion of strain 698/95 within the species R . anatipestifer and supported earlier work excluding strain 670/89, which had originally been designated the reference strain of serotype 20 . Therefore, it is suggested that the strain 698/95 could be adopted as a replacement for the reference strain of serotype 20 . Attention should be paid to strains with multiple antigenic factors as they may be useful for the preparation of vaccines. Ultrastruct Pathol, 2002 Sep-Oct, 26(5), 323 - 9 Copper accumulation in actinomyces druses during endometritis after long-term use of an intrauterine contraceptive device; Jonas L et al.; A 32-year-old woman carried a copper intrauterine contraceptive device (IUCD) or intrauterine pessar (IAP) for more than 5 years . She had acyclic menstrual bleedings and underwent a corpus abrasio after explantation of IUCD . The histological study of paraffin sections showed an actinomycotic endometritis with brown to black deposits in or around typical actinomyces druses, but there was no carcinoma . The electron microscopic study of these accumulations by electron energy loss spectroscopy (EELS) in TEM demonstrated copper deposits in the shell and matrix of these druses as well as inside the bacteria . With scanning electron microscopy (SEM) and Energy Dispersive X-Ray Analysis (EDX), the electron-dense accumulations revealed high signals for copper and sulfur, but also of phosphorus and oxygen in a lower extent . This copper accumulation is discussed as an active uptake and concentration by these actinomyces bacteria. Immunobiology, 2002 Sep, 205(4-5), 476 - 89 Structural and functional aspects of the collectin SP-A; Haagsman HP; Surfactant protein A (SP-A), member of the collectin family, is implicated in innate host defense of the lung . SP-A is a "pattern recognition molecule" and interacts with glycoconjugates on the surface of micro-organisms . It protects the lung by interacting with a wide variety of potential pathogens, including viruses, bacteria and fungi . This may result in enhanced killing and clearance by phagocytes . SP-A is a link between the innate and adaptive immune system because it directly affects lymphocyte proliferation and function . Although most extensively studied in the lung, SP-A is found in a number of other sites in the body . The presence of SP-A at these mucosal surfaces, which are in close contact with numerous potentially harmful micro-organisms, supports a more general role for this collectin in mucosal defense. Environ Pollut, 2002, 120(2), 183 - 90 Spatial and seasonal variation in heavy metals in interstitial water of salt marsh soils; Otero XL et al.; The composition of interstitial water collected from a salt marsh in NW Spain showed clear seasonal and spatial variations associated with redox cycles of Fe and S . In the summer, salinity increased in all soils as a consequence of the increase in evapotranspiration . The pH and concentrations of heavy metals also differed with season, but not all environments showed the same variations . Soils not colonized by plants had the highest pH and lowest heavy metal concentrations in the summer . These results support the idea that higher temperatures lead to an increase in the activity of sulfate-reducing bacteria, which in turn leads to an increase in alkalinity and concentration of sulfides in the water . Trace metals tend to precipitate with sulfides under these conditions and are removed from the interstitial water . In contrast, in the soils colonized by Spartina maritima, the oxidation of metal sulfides during the summer led to a decrease in pH and an increase in the metal concentrations in the interstitial water . The results obtained concur with those found for seasonal variations in metal sulfides in soils from the same salt marsh. Radiats Biol Radioecol, 2002 Jul-Aug, 42(4), 433 - 9 {Prognostic radioecological mathematical model of the Yenisei river}; Degermendzhi AG et al.; A one-dimensional mathematical model of the Yenisei river ecosystem including hydrological, ecosystem and radioecological blocks has been developed . The model was used to evaluate contribution of different processes (transfer by water masses, dilution, radioactive decay, bioaccumulation) into self-purification of the river water from a radiation pollution and calculate pollution density of ecosystem components (bacteria, phyto-, zooplankton, phyto-, zoobenthos, detritus) with 137Cs and 32P. J Clin Gastroenterol, 2002 Nov-Dec, 35(5), 375 - 8 A new semiquantitative method of quantifying Helicobacter pylori in antigen stools; Ierardi E et al.; Stool antigen test for Helicobacter pylori, a noninvasive assay, is emerging as a strong competitor to urea breath test (UBT) . Nevertheless, although the UBT delta value is a semiquantitative indicator of H . pylori intragastric load, until now the H . pylori stool antigen test has been used only as a qualitative investigation . We report here the results of a study performed with the aim of obtaining a semiquantitative measurement of bacterial amount in stools . We studied 15 patients with dyspepsia using H . pylori positivity at histology, the rapid urease test, UBT, and the H . pylori stool antigen test . The result of this last test was expressed by a numerical value we obtained by applying the principle of "standard points" to the absorbance units at spectrophotometric reading . This measurement was previously validated by testing probe sampling of H . pylori stool antigen with known pure and stool-mixed bacterial amounts . A numerical result for H . pylori stool antigen was correlated to UBT delta for each patient using Pearson's r test . Finally, a Student t test was performed to investigate possible differences in UBT and H . pylori stool antigen test values between anti-CagA-positive and -negative patients . We obtained a curve of saturation with both known amount of pure and stool-mixed bacteria . Pearson's r test showed a significant correlation between UBT delta value and H . pylori stool antigen measurement (r = 0.77; p < 0.001) . Urea breath test delta and H . pylori stool antigen test values were significantly higher in anti-CagA-positive patients . Our data suggest that a numerical estimation of H . pylori stool antigen may be feasible . This evaluation, similarly to UBT delta, may represent a semiquantitative determination of bacterial intragastric load. Blood, 2002 Dec 15, 100(13), 4410 - 9 Epub 2002 Aug 15. Transgenic targeting with regulatory elements of the human CD34 gene; Radomska HS et al.; The human CD34 gene is expressed on early progenitor and stem cells in the bone marrow . Here we report the isolation of the human CD34 locus from a human P1 artificial chromosome (PAC) library and the characterization and evaluation of this genomic fragment for expression of reporter genes in stable cell lines and transgenic mice . We show that a 160-kb fragment spanning 110 kb of the 5' flanking region and 26 kb of the 3' flanking region of the CD34 gene directs expression of the human CD34 gene in the bone marrow of transgenic mice . The expression of human CD34 transgenic RNA in tissues was found to be similar to that of the endogenous murine CD34 gene . Colony-forming cell assays showed that bone marrow cells staining positive for human CD34 consist of early progenitor cells in which expression of CD34 decreased with cell maturation . In order to test the construct for its ability to express heterologous genes in vivo, we used homologous recombination in bacteria to insert the tetracycline-responsive transactivator protein tTA . Analysis of transgenic human CD34-tTA mice by cross breeding with a strain carrying Cre recombinase under control of a tetracycline-responsive element demonstrated induction of Cre expression in mice in a pattern consistent with the expression of the human CD34 transgene. J Immunol, 2002 Nov 1, 169(9), 4797 - 804 T cell requirement for development of chronic ulcerative dermatitis in E- and P-selectin-deficient mice; Forlow SB et al.; C57BL/6 mice deficient in E- and P-selectin (E(-/-)P(-/-)) kept under specific pathogen-free barrier conditions have high circulating neutrophil counts and develop hypercellular cervical lymph nodes with substantial plasma cell infiltrates, severe ulcerative dermatitis, conjunctivitis, and lung pathology, which eventually lead to premature death . To test the hypothesis that the pathology in E(-/-)P(-/-) mice may be caused by dysfunctional lymphocyte activity, we crossed E(-/-)P(-/-) mice with recombination activation gene (Rag)-1(-/-) mice to generate E(-/-)P(-/-)Rag-1(-/-) mice lacking mature T and B lymphocytes . E(-/-)P(-/-)Rag-1(-/-) mice had circulating neutrophil counts and plasma G-CSF levels similar to E(-/-)P(-/-) mice . Remarkably, none of the E(-/-)P(-/-)Rag-1(-/-) mice developed conjunctivitis or ulcerative dermatitis typical of E(-/-)P(-/-) mice . These mice were overall healthier in appearance than E(-/-)P(-/-) mice, and histopathologic changes in the lung were reduced . Cervical lymph nodes in E(-/-)P(-/-)Rag-1(-/-) mice were much smaller than those of E(-/-)P(-/-) mice, containing few mononuclear cells and no plasma cells . These data show that the severe disease phenotype of E(-/-)P(-/-) mice depends on lymphocyte function . We conclude that a dysregulated immune response in E(-/-)P(-/-) mice causes disease development, but is not necessary for elevated neutrophil counts. J Cell Biol, 2002 Oct 28, 159(2), 291 - 302 Epub 2002 Oct 21. Ca2+-controlled competitive diacylglycerol binding of protein kinase C isoenzymes in living cells; Lenz JC et al.; The cellular decoding of receptor-induced signaling is based in part on the spatiotemporal activation pattern of PKC isoforms . Because classical and novel PKC isoforms contain diacylglycerol (DAG)-binding C1 domains, they may compete for DAG binding . We reasoned that a Ca2+-induced membrane association of classical PKCs may accelerate the DAG binding and thereby prevent translocation of novel PKCs . Simultaneous imaging of fluorescent PKC fusion proteins revealed that during receptor stimulation, PKC alpha accumulated in the plasma membrane with a diffusion-limited kinetic, whereas translocation of PKC epsilon was delayed and attenuated . In BAPTA-loaded cells, however, a selective translocation of PKC epsilon, but not of coexpressed PKC alpha, was evident . A membrane-permeable DAG analogue displayed a higher binding affinity for PKC epsilon than for PKC alpha . Subsequent photolysis of caged Ca2+ immediately recruited PKC alpha to the membrane, and DAG-bound PKC epsilon was displaced . At low expression levels of PKC epsilon, PKC alpha concentration dependently prevented the PKC epsilon translocation with half-maximal effects at equimolar coexpression . Furthermore, translocation of endogenous PKCs in vascular smooth muscle cells corroborated the model that a competition between PKC isoforms for DAG binding occurs at native expression levels . We conclude that Ca2+-controlled competitive DAG binding contributes to the selective recruitment of PKC isoforms after receptor activation. J Exp Med, 2002 Oct 21, 196(8), 1099 - 104 The CD8alpha(+) dendritic cell is responsible for inducing peripheral self-tolerance to tissue-associated antigens; Belz GT et al.; We previously described a mechanism for the maintenance of peripheral self-tolerance . This involves the cross-presentation of tissue-associated antigens by a bone marrow-derived cell type that stimulates the proliferation and ultimate deletion of self-reactive CD8 T cells . This process has been referred to as cross-tolerance . Here, we characterize the elusive cell type responsible for inducing cross-tolerance as a CD8alpha(+) dendritic cell (DC) . To achieve this aim, transgenic mice were generated expressing yellow fluorescent protein (YFP) linked to CTL epitopes for ovalbumin and glycoprotein B (gB) of herpes simplex virus under the rat insulin promoter (RIP) . Although tracking of YFP was inconclusive, the use of a highly sensitive gB-specific hybridoma that produced beta-galactosidase on encounter with antigen, enabled detection of antigen presentation by cells isolated from the pancreatic lymph node . This showed that a CD11c(+)CD8alpha(+) cell was responsible for cross-tolerance, the same DC subset as previously implicated in cross-priming . These data indicate that CD8alpha(+) DCs play a critical role in both tolerance and immunity to cell-associated antigens, providing a potential mechanism by which cytotoxic T lymphocyte can be immunized to viral antigens while maintaining tolerance to self. Biol Reprod, 2002 Nov, 67(5), 1439 - 49 Differential expression and estrogen response of lactoferrin gene in the female reproductive tract of mouse, rat, and hamster; Teng CT et al.; Lactoferrin, an iron-binding glycoprotein, kills bacteria and modulates inflammatory and immune responses . Presence of lactoferrin in the female reproductive tract suggests that the protein may be part of the mucosal immune system and act as the first line of defense against pathogenic organisms . We have discovered that lactoferrin is a major estrogen-inducible protein in the uterus of immature mice and is up-regulated by physiological levels of estrogen during proestrous in mature mice . In the present study, we examined lactoferrin gene expression and its response to estrogen stimulation in the female reproductive tract of several strains of immature mouse, rat, and hamster . The lactoferrin expression in the cycling adult female rat was also evaluated . Lactoferrin gene polymorphism exists among the different mouse strains . In the three inbred mouse strains studied, lactoferrin gene expression is stimulated by estrogen in the immature uterus, although it is less robust than in the outbred CD-1 mouse . We found that the lactoferrin gene is constitutively expressed in the epithelium of the vagina and the isthmus oviduct; however, it is estrogen inducible in the uterus of immature mice and rats . Furthermore, lactoferrin is elevated in the uterine epithelium of the mature rat during the proestrous and estrous stages of the estrous cycle . Estrogen stimulation of lactoferrin gene expression in the reproductive tract of an immature hamster is limited to the vaginal epithelium . The present study demonstrates differential expression and estrogen responsiveness of the lactoferrin gene in different regions of the female rodent reproductive tract and variation among the rodent species studied. J Clin Periodontol, 2002 Aug, 29(8), 663 - 71 Pathologic interactions in pulpal and periodontal tissues; Zehnder M et al.; Both endodontic and periodontal disease are caused by a mixed anaerobic infection . The pathways for the spread of bacteria between pulpal and periodontal tissues have been discussed with controversy . This article is an attempt to provide a rational approach to the perio-endo/endo-perio question based on a review of the relevant literature . In the light of evidence, clinical concepts for the diagnosis and treatment of lesions involving both periodontal and pulpal tissues are discussed. Lett Appl Microbiol, 2002, 35(5), 414 - 8 Zirconium hydroxide effectively immobilizes and concentrates human enteric viruses; D'Souza DH et al.; BACKGROUND: Detection of human enteric viruses in foods and environmental samples requires concentration of viruses from complex matrices before application of molecular or cultural methods . Previous studies have described the use of zirconium hydroxide to concentrate bacteria from clinical, environmental, and food samples . AIMS: Our study describes the application of zirconium hydroxide to concentrate human enteric viruses . METHODS: Poliovirus type 1, hepatitis A virus (HAV) strain HM-175, and Norwalk virus (NV) were used as models . Virus recovery was evaluated both as loss to discarded supernatants and as recovery in the precipitated pellets . RESULTS: Poliovirus type 1, based on the plaque assay recoveries, ranged from 16 to 59% with minimal loss to the supernatant (1-5%) . For both HAV and NV, RT-PCR amplicons of appropriate sizes were detected and confirmed in the pellet fraction with no visible amplicons from the supernatant . SIGNIFICANCE AND IMPACT OF THE STUDY: This rapid and inexpensive method shows promise as an alternative means to concentrate enteric viruses. Br J Surg, 2002 Nov, 89(11), 1457 - 64 Granulocyte colony-stimulating factor but not peritoneal lavage increases survival rate after experimental abdominal contamination and infection; Bauhofer A et al.; BACKGROUND: The value of peritoneal lavage for intra-abdominal contamination and infection has never been proven scientifically . In contrast, the stimulation of host defence mechanisms with cytokines such as granulocyte colony-stimulating factor (G-CSF) has appeared promising in recent clinical trials . METHODS: Clinic modelling randomized trials (CMRTs), which model the complexity of the clinical reality, were used in rats in which peritoneal contamination and infection (PCI) was produced with human stool bacteria . The following groups were compared: trial 1, intraoperative peritoneal lavage with saline versus taurolin (18 rats per group); trial 2, no lavage versus saline lavage versus saline lavage plus subcutaneous administration of G-CSF (18 rats per group); trial 3, lavage with saline versus no lavage (30 rats per group) . The primary endpoint was mortality at 120 h . Secondary endpoints were the phagocytic activity of granulocytes, and systemic and peritoneal cytokine levels . RESULTS: In trial 1 lavage with taurolin was not superior to that with saline (five of 18 versus eight of 18 animals survived; P = 0.32) . In trial 2, six of 18 animals having no lavage and three of 18 receiving saline lavage survived . The combination of lavage and G-CSF increased the number of animals surviving to 11 of 18 (P < 0.05) . Lavage combined with G-CSF stimulated granulocyte phagocytic activity (P < 0.01) and reduced the levels of interleukin (IL) 6 (P < 0.01) and tumour necrosis factor alpha (P < 0.05) in peritoneal fluid, as well as plasma levels of IL-6 (P < 0.05) and IL-10 (P < 0.01) . In trial 3, survival was not significantly different in animals having lavage (14 of 30) and no lavage (19 of 30) (P = 0.14) . CONCLUSION: In these CMRTs of intra-abdominal contamination and infection, peritoneal lavage was not beneficial, but when lavage was combined with subcutaneous administration of G-CSF mortality was reduced and the local and systemic cytokine response was downgraded . Results from these CMRTs were used directly to define the trial conditions of a randomized clinical trial with G-CSF . Peritoneal lavage is not recommended. Clin Microbiol Infect, 2002 Oct, 8(10), 654 - 61 Does infection with Chlamydia pneumoniae and/or Helicobacter pylori increase the expression of endothelial cell adhesion molecules in humans? Schumacher A, Seljeflot I, Lerkerod AB, Sommervoll L, Otterstad JE, Arnesen H. OBJECTIVE: To investigate if Chlamydia pneumoniae and/or Helicobacter pylori seropositivity is associated with elevated levels of soluble endothelial cell adhesion molecules (sCAMs) as markers of atherosclerotic activity . METHODS: Immunoglobulin A (IgA) and IgG antibodies to the two bacteria, soluble intercellular cell adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1) and E-selectin were measured in coronary heart disease (CHD) patients (n = 193) and age- and sex-matched controls (n = 193) . Two different serological methods were used for the detection of Chlamydia antibodies: Labsystems microimmunofluorescence to detect species-specific C . pneumoniae antibodies and Medac's recombinant enzyme-linked immunosorbent assay to detect genus-specific lipopolysaccharide antibodies . RESULTS: The concentrations of sICAM-1 and E-selectin were higher in CHD patients with positive vs . negative Chlamydia lipopolysaccharide IgA (P = 0.044 for both) . H . pylori antibodies alone did not predict raised levels of sCAMs, but in CHD patients sICAM-1 was increased with IgA seropositivity to both bacteria compared to double seronegativity (P = 0.034) . Concentrations of sVCAM-1 were elevated in CHD patients with double IgA seropositivity compared to those with Chlamydia lipopolysaccharide IgA seropositivity alone (P = 0.018) . CONCLUSION: Our results may indicate that C . pneumoniae contributes to increased inflammation in CHD, and that this contribution is even more pronounced when present in combination with H . pylori IgA antibodies. Biochemistry, 2002 Oct 29, 41(43), 12952 - 8 Structure-based mechanism of O2 sensing and ligand discrimination by the FixL heme domain of Bradyrhizobium japonicum; Hao B et al.; Structures of the Bradyrhizobium japonicum FixL heme domain have been determined in the absence and presence of specific ligands to elucidate the detailed features of its O2 sensing mechanism . The putative roles of spin-state and steric hindrance were evaluated by the structure determination of ferrous CO-bound BjFixLH and correlating its features with other ligand-bound structures . As found for NO-BjFixLH, no protein conformational change was observed in CO-BjFixLH, suggesting a more complicated mechanism than solely spin state or ligand sterics . To evaluate the role of oxidation state, the structure of the ferrous deoxy-BjFixLH was determined . The structure of deoxy-BjFixLH was found to be virtually identical to the structure of the ferric met-BjFixLH . The role of hydrogen bonding of substrates to a heme-pocket water was evaluated by determining the structure of BjFixLH bound to 1-methyl-imidazole that cannot form a hydrogen bond with this water . In this case, the heme-mediated conformational change was observed, limiting the potential importance of this interaction . Finally, the structure of cyanomet-BjFixLH was revisited to rule out concerns regarding the partial occupancy of the cyanide ligand in a previous structure . In the revised structure, Arg 220 was found to move into the heme pocket to form a hydrogen bond to the bound cyanide ligand . The implications of these results on FixL's sensing mechanism are discussed. Protist, 2002 Sep, 153(3), 239 - 50 Vertical distribution and abundance of gymnamoebae (Rhizopoda) in bottom sediments of the brackish water Nivå Bay (Baltic Sea, The Sound); Smirnov AV; The sandy sediments of Niva Bay (Baltic Sea, The Sound, Denmark) are often covered with the mats of sulphur bacteria and are temporarily anoxic . The vertical distribution and abundance of naked amoebae species in three sediment cores from this bay were studied . Amoebae were most abundant and diverse in the upper 1 cm of sediment, and their number and diversity decreased with increasing depth into the sediment . Amoebae were recovered from both upper oxygenated and deep anoxic layers of sediments . The species composition and abundance of amoebae was very heterogeneous, even at spatial scales of several centimeters, suggesting the existence of microhabitats selectively occupied by particular species . All species found were recorded from aerobic cultures and some of these amoebae occur in both the aerobic and anaerobic layers of the sediment . Minimal possible number of amoebae in the sediments, estimated for the first time as areal abundance integrated for depth was: core 1 -597 cm(-2); core 2 -1,110 cm(-2); core 3 -1,430 cm(-2) . These abundances are probably best regarded as "potential" abundances of amoebae hidden in the sediments, as the question of the ratio between active and resting amoebae remains open. Acta Histochem, 2002, 104(3), 285 - 7 Silver staining combined with alcian blue and hematoxylin-eosin for the detection of Lawsonia intracelullaris in swine proliferative enteropathy; Driemeier D et al.; Fragments of ileum from 663 pigs were collected in abattoirs, prepared with the use of standard histological methods and stained with a novel sensitive histochemical method for the detection of porcine proliferative enteropathy . The method is a combination of the following 3 well-known methods, the Warthin-Starry method, alcian blue and hematoxylin-eosin . In 11 cases, mucus-producing cells were completely absent, severe adenomatous proliferation was observed and intracellular bacteria were found in enterocytes . Disappearance of goblet cells and the presence of adenomatous proliferation without any detectable intracellular bacteria were observed in 16 cases . In the remaining 636 cases, histological changes and intracellular bacteria were not found . When comparing the conventional Warthin-Starry method with the modified staining method presented here, the same 16 cases were found . However, the method presented here enables examination of large numbers of sections in a relatively short period of time. Am J Physiol Gastrointest Liver Physiol, 2003 Jan, 284(1), G96 - G106 Epub 2002 Sep 04. Interactions among the seven Helicobacter pylori proteins encoded by the urease gene cluster; Voland P et al.; Survival of Helicobacter pylori in acid depends on intrabacterial urease . This urease is a Ni(2+)-containing oligomeric heterodimer . Regulation of its activity and assembly is important for gastric habitation by this neutralophile . The gene complex encodes catalytic subunits (ureA/B), an acid-gated urea channel (ureI), and accessory assembly proteins (ureE-H) . With the use of yeast two-hybrid analysis for determining protein-protein interactions, UreF as bait identified four interacting sequences encoding UreH, whereas UreG as bait detected five UreE sequences . These results were confirmed by coimmunoprecipitation and beta-galactosidase assays . Native PAGE immunoblotting of H . pylori inner membranes showed interaction of UreA/B with UreI, whereas UreI deletion mutants lacked this protein interaction . Deletion of ureE-H did not affect this interaction with UreI . Hence, the accessory proteins UreE/G and UreF/H form dimeric complexes and UreA/B form a membrane complex with UreI, perhaps enabling assembly of the urease apoenzyme at the membrane surface and immediate urea access to intrabacterial urease to allow rapid periplasmic neutralization. FEBS Lett, 2002 Oct 23, 530(1-3), 117 - 23 Insights into the evolution of the antenna domains of Type-I and Type-II photosynthetic reaction centres through homology modelling; Fyfe PK et al.; The (bacterio)chlorophylls of photosynthetic antenna and reaction centre complexes are bound to the protein via a fifth, axial ligand to the central magnesium atom . A number of the amino acids identified as providing such ligands are conserved between the large antenna of the cyanobacterial Type-I reaction centre and smaller antennas of the Type-I reaction centres of green sulphur bacteria and heliobacteria, and these numbers match closely the estimated number of antenna bacteriochlorophylls in the latter . The possible organisation of the antenna in the latter reaction centres is discussed, as is the mechanism by which the more pigment-rich antenna of the cyanobacterial reaction centre evolved . The homology modelling approach is also extended to the six-helix antenna proteins CP47 and CP43 associated with the Photosystem II reaction centre. Food Chem Toxicol, 2002 Oct, 40(10), 1463 - 7 Induction of micronuclei in V79 cells after combined treatments with heterocyclic aromatic amines; Perez C et al.; Heterocyclic aromatic amines (HAs) appear in foods rich in proteins when subjected to different cooking processes . These amines have been demonstrated to be mutagenic in bacteria; in eucaryotic cells, controversial results have been referred . The objective of this study is to evaluate the clastogenic and/or aneugenic capacity of three HAs--2-amino-3-methylimidazo{4,5-f}quinoline (IQ), 2-amino-3-methylimidazo{4,5-f}quinoxaline (IQx), and 2-amino-3-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP)--in isolated as well as in combined treatments . The micronucleus test in vitro was used on V79 cells in the presence and absence of metabolic activation . The duration of the treatment was 2 h, and cytochalasin B was added for 21 h to stop cytokinesis; then, micronuclei (MN) were counted in binucleated cells . In the presence of metabolic activation, the three amines showed a significant increase in the number of MN with respect to the negative control . The PhIP amine presented the highest values and it also resulted slightly active in the absence of metabolic activation, although these differences have not been considered to be significant . The combined treatments of these amines have shown that the effects attributed to them when administered together are those that are expected for a possible additive effect; the effect attributed to each HA separately is not potentiated nor inhibited. Rinsho Byori, 2002 Sep, 50(9), 847 - 52 {Present trends of drug-resistant tuberculosis and how to manage it by mycobacterial laboratories}; Suzuki K et al.; Global, domestic, and local trends of drug-resistant tuberculosis and how to manage increase in the resistance by mycobacterial laboratories were discussed based on literatures and our own data . At first, how to make drug-resistant tuberculosis was explained . Genetic drug-resistant bacteria were emerged spontaneously by mutation of the genome and were selected by inadequate treatment(mono-therapy or functional mono-therapy): acquired drug resistance(single, and then multi-drug resistance) . In a mean time, some people were infected with the drug-resistant bacteria from the beginning and a part of them developed active disease: primary drug resistance (single or multi-drug resistance) . Estonia, Latvia, Iran, and some part of Russia, China, and India were reported to be the most endemic region of drug resistant and multi-drug resistant tuberculosis in the world . The rate of primary resistance in Japan was as high as the median of the world, but the rate of acquired resistance was almost twice of the median . Since delays in reporting of the drug resistance from the laboratory seemed one of reasons for the inadequate treatment, drug susceptibility testing should be more rapid than usual, by using liquid media such as BACTEC MGIT 960 system, or gene analysis. Adv Health Sci Educ Theory Pract, 1997, 2(2), 115 - 130 Basic Science Reasoning and Clinical Reasoning Intertwined: Epistemological Analysis and Consequences for Medical Education; Magnani L; The aim of this paper is to emphasize the distinction between basic medical science (and reasoning) and clinical science (and reasoning) in order to illuminate some basic philosophical and cognitive issues in medical education . The Kunhian concept of exemplar refers to the field of growth of scientific knowledge and in this sense is related to the "anti-theoretical" emphasis on problem solving performance . In cognitive science this (and similar) types of postpositivistic objections to the formalistic excess of the neopositivistic tradition are exploited to stress the relevance of the distinction between theories and their domains of application . This objection is exploited to stress the difference between established bodies of scientific knowledge and their processes of discovery and/or application and, in medical knowledge, between clinical reasoning (situated, concerned with attributes of people) and basic science reasoning (unsituated, concerned with attributes of entities such as organs, bacteria, viruses) . Exploiting the theoretical consequences of the previous analysis I will try to answer some questions: What is the role of problem solving in teaching and learning, as different from conventional basic science-centred education? Is it relevant, in medical education, an epistemological and logical awareness of the main methodological topics? Finally, the analysis of the significance of abduction in a unified epistemological model of medical reasoning is exploited to individuate the proper ontological level dealing with the entities and relationships belonging to the dynamism of the underlying domain knowledge (for instance biomedical physics) and the consequences for medical education. Drug Metab Dispos, 2002 Nov, 30(11), 1186 - 93 Down-regulation of alpha class glutathione S-transferase by interleukin-1beta in human intestinal epithelial cells (Caco-2) in culture; Romero L et al.; The influence of pro-inflammatory cytokines on alpha class glutathione S-transferase A1 and A2 (GSTA1/A2) expression was examined in human colonic epithelial cells (Caco-2) in culture . Dose-dependent reductions in GSTA1/A2 mRNA, protein, and activity levels occurred in Caco-2 cells cultured in conditioned medium (CM) from lipopolysaccharide-stimulated murine monocyte-macrophage cells (RAW 264.7) . Neutralizing anti-interleukin-1beta (IL-1beta) antibodies attenuated this repression of GSTA1/A2 expression by CM . Moreover, recombinant human IL-1beta reduced GSTalpha expression at the mRNA, protein, and activity levels in a dose-related fashion . Reduction of GSTA1/A2 mRNA levels by IL-1beta was attenuated by pretreatment with IL-1 receptor antagonist . GSTA1/A2 mRNA half-lives were similar in control and IL-1beta-treated cells, indicating that IL-1beta has no effect on mRNA stability . In reporter gene studies, IL-1beta caused a dose-related reduction of luciferase activity in Caco-2 cells transfected with the full-length GSTA1 promoter-luciferase construct . Using truncated constructs, IL-1beta responsiveness was mapped to a region 286 base pairs upstream to the coding region . Deletion of a hepatic nuclear factor 1 (HNF-1) site in this region abrogated the IL-1beta-mediated repression of GSTA1 promoter activity . These results demonstrate that IL-1beta down-regulates GSTA1/A2 expression in cultured human enterocytes by a transcriptional mechanism involving an HNF-1 site. Bioinformatics, 2002 Oct, 18 Suppl 2, S219 - S230 BioMiner-modeling, analyzing, and visualizing biochemical pathways and networks; Sirava M et al.; Motivation: Understanding the biochemistry of a newly sequenced organism is an essential task for post-genomic analysis . Since, however, genome and array data grow much faster than biochemical information, it is necessary to infer reactions by comparative analysis . No integrated and easy to use software tool for this purpose exists as yet . Results: We present a new software system-BioMiner-for analyzing and visualizing biochemical pathways and networks . BioMiner is based on a new comprehensive, extensible and reusable data model-BioCore-which can be used to model biochemical pathways and networks . As a first application we present PathFinder, a new tool predicting biochemical pathways by comparing groups of related organisms based on sequence similarity . We successfully tested PathFinder with a number of experiments, e.g . the well studied glycolysis in bacteria . Additionally, an application called PathViewer for the visualization of metabolic networks is presented . PathViewer is the first application we are aware of which supports the graphical comparison of metabolic networks of different organisms . Availability: Contact: schaefer@bioinf.uni-sb.de Supplementary Information: Additional information on experimental results can be found on our web site . Keywords: Biochemical data model, metabolic and regulatory pathways, visualization, Java, XML. Eur J Immunol, 2002 Nov, 32(11), 3050 - 8 Mycobacterium tuberculosis subverts the differentiation of human monocytes into dendritic cells; Mariotti S et al.; Intracellular pathogens have developed strategies for evading elimination by the defenses of the host immune system . Here we describe an escape mechanism utilized by Mycobacterium tuberculosis that involves the interference with the generation of fully competent DC from monocytes . We show that monocytes infected with live M . tuberculosis differentiated into mature, CD83+ and CCR7+ DC (Mt-MoDC), but were characterized by a selective failure in the expression of the family of CD1 molecules . These cells also showed levels of MHC class II and CD80 (B7.1) that were reduced in comparison with LPS-matured DC . In addition, Mt-MoDC produced TNF-alpha and IL-10, but were unable to secrete IL-12 . The generation of Mt-MoDC required the infection of monocytes with live M . tuberculosis, since infection with heat-killed bacteria partially abrogated the effects on monocyte differentiation . Interestingly, Mt-MoDC revealed an impaired antigen-presentation function as assessed by the reduced capability to induce proliferation of cord blood T lymphocytes . Further, naive T lymphocytes expanded by Mt-MoDC were unable to secrete cytokines, in particular IL-4 and IFN-gamma, suggesting that they could be ineffective in helping the macrophage-mediated killing of intracellular mycobacteria . Our results suggest that the interference with monocyte differentiation into fully competent DC is an evasion mechanism of M . tuberculosis that could contribute to its intracellular persistence by avoiding immune recognition. Adv Drug Deliv Rev, 2002 Oct 18, 54(8), 1131 - 43 Genetic engineering of fibrous proteins: spider dragline silk and collagen; Wong Po Foo C et al.; Various strategies have been employed to genetically engineer fibrous proteins . Two examples, the subject of this review, include spider dragline silk from Nephila clavipes and collagen . These proteins are highlighted because of their unique mechanical and biological properties related to controlled release, biomaterials and tissue engineering . Cloning and expression of native genes and synthetic artificial variants of the consensus sequence repeats from the native genes has been accomplished . Expression of recombinant silk and collagen proteins has been reported in a variety of host systems, including bacteria, yeast, insect cells, plants and mammalian cells . Future utility for these proteins for biomedical materials is expected to increase as needs expand for designer materials with tailored mechanical properties and biological interactions to elicit specific responses in vitro and in vivo. Gene, 2002 Sep 4, 297(1-2), 189 - 96 Molecular cloning and characterization of the mouse thymocyte protein gene; Miyaji H et al.; Chicken thymocyte protein (cThy28) has recently been identified and implicated to be involved in apoptosis of avian lymphocytes, while its functional role remains undefined . To elucidate the role of Thy28, we have molecularly cloned the mouse Thy28 (mThy28) cDNA and clarified its genomic organization in the present study . The mThy28 cDNA encodes a 226 amino acid protein, whose sequence is highly conserved among bacteria, yeast and plants as well as vertebrates . Northern blot analysis revealed abundant expression of approximately 1 kb mRNA in testis, liver, brain and kidney with lower levels of the expression in thymus, spleen, heart and stomach . The mThy28 gene spans at least 7 kb of genomic DNA, and contains eight exons and seven introns . The 5'-flanking region of the mThy28 gene does not contain a typical TATA box, but contains a putative CCAAT box and presumably multiple transcription initiation sites . Potential Sp1, GATA-1, -2, -3, AP-1, and p300 binding sites were found in the 5'-flanking region of the mThy28 gene . The gene was mapped to mouse chromosome 9. Mol Phylogenet Evol, 2002 Oct, 25(1), 101 - 11 Proteome-wide analysis of protein function composition reveals the clustering and phylogenetic properties of organisms; Ling L et al.; A 17-dimensional vector named the proteome vector is defined to represent an organism . The components of the vector reflect the relative contents of protein-encoding genes of the 17 cluster of orthologous groups of proteins (COGs) classes in the whole genome of the relevant organism . Based on the definition of this proteome vector, the fuzzy clustering of 36 completely sequenced organisms (8 archaea, 24 bacteria, and 4 eukarya) was performed and a proteome tree was constructed . Our results show that (1) the 36 organisms can be 100% correctly classified into three clusters corresponding to the three primary kingdoms, (2) our proteome tree is remarkably similar to that derived from 16S rRNA, and (3) the chromosomes and/or plasmids belonging to the same organism have very similar gene composition . Based on these results, we argue that the 17-dimensional proteome vector could be a good criterion for clustering approaches and to a large extent reveals the phylogenetic properties of organisms; the Three Primary Kingdoms Hypothesis is trustworthy although the existence of lateral gene transfer (LGT) brings controversy to the construction of the "universal tree of life." Gene, 2002 Aug 21, 296(1-2), 139 - 50 Cytoplasmic SIR2 homologue overexpression promotes survival of Leishmania parasites by preventing programmed cell death; Vergnes B et al.; The Silent Information Regulator (SIR2) family of genes have been cloned from a variety of species ranging from bacteria to man . In previous studies, we reported the characterization of a Leishmania major gene encoding a protein with extensive homology to yeast SIR2p and expressed by different Leishmania species and parasite developmental stages and thus termed LmSIR2 . Unlike the yeast SIR2p, LmSIR2p is mainly localized within the cytoplasm . In the present study, sequencing of a homologue encoding gene in another Leishmania species, Leishmania infantum, revealed 93% overall amino acid identity with L . major SIR2 gene . Further, using L . infantum as a recipient for a plasmid vector (pTEX) which allows overexpression of LmSIR2p led to the accumulation of the protein in the parasite cytoplasm of both promastigote and amastigote forms and a striking increase in the survival of amastigotes, the vertebrate stage of the parasite, when maintained under normal axenic culture conditions . This phenotype was also observed when L . infantum parasites were transfected with a cosmid vector (CLHyg), isolated from a L . infantum cosmid library, carrying the L . infantum SIR2 gene (CLHyg-LiSIR2) . In contrast, no effect was observed on survival of the promastigote forms (insect stage) under similar culture conditions . However, when the glucose was used as a unique source of energy under starvation conditions, the viability of promastigotes was significantly enhanced . Moreover, we showed that amastigote forms in the stationary phase of culture died with a feature of apoptosis as revealed by the appearance of YOPRO-1 positive cells and that expression of LmSIR2 protein substantially delays this phenomenon . Taken together, these results demonstrate the existence of SIR2-related proteins encoding genes in different Leishmania species and suggest that LmSIR2p could participate among other factors in the control of cell death. Gene, 2002 Aug 21, 296(1-2), 111 - 9 Isolation and characterization of cold-shock domain protein genes, Oryzias latipes Y-box protein 2 (OlaYP2) and Fugu rubripes Y-box protein 1 (FruYP1), in medakafish and pufferfish; Zend-Ajusch E et al.; The Y-box protein (YP) family shares a nucleic acid binding domain, called cold-shock domain, that has been evolutionarily highly conserved from bacteria to human . The different YPs identified so far in vertebrates are thought to function as transcriptional activators, transcriptional repressors and/or translational repressors . Medakafish and pufferfish are very suitable vertebrate models for the study of developmental genetics and comparative genomics, respectively . Here we report the isolation of two teleost YP genes, medakafish Oryzias latipes (Ola)YP2 and Fugu rubripes (Fru)YP1, which are expressed in multiple tissues . Phylogenetic analysis demonstrated that OlaYP2 and FruYP1 belong to different subclasses of the cold-shock domain protein genes . Future studies in suitable model systems, like the medaka for developmental biology and Fugu for evolutionary genomics, are expected to contribute to our understanding of YPs. Plant J, 2002 Oct, 32(2), 205 - 19 Phototropin LOV domains exhibit distinct roles in regulating photoreceptor function; Christie JM et al.; Phototropins (phot1 and phot2) are autophosphorylating serine/threonine kinases that function as photoreceptors for phototropism, light-induced chloroplast movement, and stomatal opening in Arabidopsis . The N-terminal region of phot1 and phot2 contains two specialized PAS domains, designated LOV1 and LOV2, which function as binding sites for the chromophore flavin mononucleotide (FMN) . Both LOV1 and LOV2 undergo a self-contained photocycle, which involves the formation of a covalent adduct between the FMN chromophore and a conserved active-site cysteine residue (Cys39) . Replacement of Cys39 with alanine abolishes the light-induced photochemical reaction of LOV1 and LOV2 . Here we have used the Cys39Ala mutation to investigate the role of LOV1 and LOV2 in regulating phototropin function . Photochemical analysis of a bacterially expressed LOV1 + LOV2 fusion protein indicates that LOV2 functions as the predominant light-sensing domain for phot1 . LOV2 also plays a major role in mediating light-dependent autophosphorylation of full-length phot1 expressed in insect cells and transgenic Arabidopsis . Moreover, photochemically active LOV2 alone in full-length phot1 is sufficient to elicit hypocotyl phototropism in transgenic Arabidopsis, whereas photochemically active LOV1 alone is not . Further photochemical and biochemical analyses also indicate that the LOV1 and LOV2 domains of phot2 exhibit distinct roles . The significance for the different roles of the phototropin LOV domains is discussed. Extremophiles, 2002 Oct, 6(5), 377 - 83 Epub 2002 May 30. Comparison of high pressure-induced dissociation of single-stranded DNA-binding protein (SSB) from high pressure-sensitive and high pressure-adapted marine Shewanella species; Chilukuri LN et al.; The effects of hydrostatic pressure on protein quaternary structure were compared for recombinant single-stranded DNA-binding protein (SSB) derived from piezosensitive, piezotolerant, and obligately piezophilic ("pressure-loving") marine Shewanella strains . The pressure-induced dissociation of the oligomeric SSB proteins was investigated using fluorescence anisotropy . The SSBs all exhibited striking similarity in the pressure-dependent behavior of the fluorescence intensity and emission spectrum as well as in their dissociation constants at atmospheric pressure . The free energies of subunit association into tetramers for all SSBs were between -27 and -30 kcal mol(-1) . However, SSB from the piezosensitive Shewanella strain S . hanedai was more sensitive to pressure than that of the SSB proteins from the piezotolerant or piezophilic bacteria . The volume change of association obtained from the pressure dependence of dissociation at a fixed protein concentration (Delta V(p)) for SSB from S . hanedai was 394-402 ml mol(-1) . The Delta V(p) values for SSB from the deeper-living Shewanellas were smaller and ranged from 253 to 307 ml mol(-1) . Differences between the primary structures of the SSB proteins that could correlate with differences in sensitivity to pressure-induced dissociation were examined. Protein Sci, 2002 Nov, 11(11), 2622 - 30 Molecular dynamics of the FixJ receiver domain: movement of the beta4-alpha4 loop correlates with the in and out flip of Phe101; Roche P et al.; FixJ is a two-domain response regulator involved in nitrogen fixation in Sinorhizobium meliloti . Recent X-ray characterization of both the native (unphosphorylated) and the active (phosphorylated) states of the protein identify conformational changes of the beta4-alpha4 loop and the conserved residue Phe101 as the key switches in activation . These structures also allowed investigation of the transition between conformations of this two-component regulatory receiver domain by molecular dynamics simulations . The path for the conformational change was studied with a distance constraint directing the system from one state to the other . The simulations provide evidence for a correlation between the conformation of the beta4-alpha4 loop and the orientation of the residue Phe101 . A model presenting the sequence of events during the activation/deactivation process is discussed. Proc Natl Acad Sci U S A, 2002 Oct 29, 99(22), 14268 - 73 Epub 2002 Oct 14. Solving the freeloaders paradox: Genetic associations and frequency-dependent selection in the evolution of cooperation among nonrelatives; Aviles L; One of the enduring problems in the study of social evolution has been to understand how cooperation can be maintained in the presence of freeloaders, individuals that take advantage of the more cooperative members of groups they are eager to join . The freeloader problem has been particularly troublesome when groups consist of nonrelatives, and no inclusive fitness benefits accrue to individuals that contribute more heavily to communal activities . These theoretical difficulties, however, are not mirrored by the numerous examples of cooperative or even altruistic behaviors exhibited by groups of nonrelatives in nature (e.g., many human groups, communally nesting bees, multiple queen-founding ants, cellular slime molds, and social bacteria) . Using a model in which cooperation and grouping tendencies are modeled as coevolving dynamical variables, I show that the freeloader problem can be addressed when group-size effects on fitness are considered explicitly . I show that freeloaders, whose presence is reflected in the development of linkage disequilibrium between grouping and cooperation, increase in frequency when rare, but are selected against when common due to the reduced productivity of the groups they overburden with their presence . Freeloader frequencies thus periodically rise and fall around an equilibrium shown here to be dynamic . These results highlight the importance of group-level effects in the origin and maintenance of sociality, illustrate the dynamic nature of equilibria when multiple levels of selection are involved, and provide a solution to the freeloaders paradox. Cell Immunol, 2002 Mar-Apr, 216(1-2), 65 - 72 CD4 is required for the development of a protective granulomatous response to pulmonary tuberculosis; Saunders BM et al.; To confirm the primary role of CD4 T cells in pulmonary tuberculosis, mice with a disruption of their CD4 gene (CD4 KO) were exposed to an aerosol of Mycobacterium tuberculosis and survival, cellular responses in the lung and granuloma development followed . CD8 and NK cells from the lungs of infected CD4 KO mice expressed IFN-gamma and were recruited in numbers similar to those seen in the C57BL/6 mice; recruitment correlated with initial control of bacteria . The major defect in mice lacking CD4 was the significant reduction in total cellular recruitment into the lungs . CD4 KO mice did not generate the typical mononuclear granulomatous lesions, instead the cellular influx was macrophage in character and was localized as perivascular cuffing . Early control of M . tuberculosis growth is therefore independent of CD4+ cells but such cells are required to ensure recruitment of mononuclear cells to the lung and thus ensure long-term survival. J Mol Biol, 2002 Oct 25, 323(3), 585 - 98 A method for prediction of the locations of linker regions within large multifunctional proteins, and application to a type I polyketide synthase; Udwary DW et al.; Multifunctional proteins often appear to result from fusion of smaller proteins and in such cases typically can be separated into their ancestral components simply by cleaving the linker regions that separate the domains . Though possibly guided by sequence alignment, structural evidence, or light proteolysis, determination of the locations of linker regions remains empirical . We have developed an algorithm, named UMA, to predict the locations of linker regions in multifunctional proteins by quantification of the conservation of several properties within protein families, and the results agree well with structurally characterized proteins . This technique has been applied to a family of fungal type I iterative polyketide synthases (PKS), allowing prediction of the locations of all of the standard PKS domains, as well as two previously unidentified domains . Using these predictions, we report the cloning of the first fragment from the PKS norsolorinic acid synthase, responsible for biosynthesis of the first isolatable intermediate in aflatoxin production . The expression, light proteolysis and catalytic abilities of this acyl carrier protein-thioesterase didomain are discussed. Eur J Epidemiol, 2001, 17(11), 1029 - 32 A seroepidemiological study of the risks of Q fever infection in Japanese veterinarians; Abe T et al.; The causative agent of Q fever, a widespread zoonotic disease, is the bacteria Coxiella burnetii . Although cases of Q fever have been documented in countries throughout the world, the prevalence of the disease in Japan is not yet known . Q fever is a demonstrated occupational hazard to those employed in zoological professions, but the risk to Japanese veterinarians has not yet been quantified . In order to evaluate the risk to Japanese veterinarians, we performed a serological survey using serum samples from 267 veterinarians . Two control groups consisting of 352 medical workers and 2003 healthy blood donors were also evaluated . The antibody titers of the serum samples were measured by indirect immunofluorescence assay (IFA) using phase II C . burnetii Nine Mile strain as the antigen . The positive rate of IgG antibody was 13.5% in the veterinarians, which was higher than in the blood donors (3.6%, p < 0.001) and medical workers (5.1 %,p < 0.001) . These findings suggest that Japanese veterinarians have a higher risk of infection by C . burnetii than other members of the Japanese population . An interesting finding of this study was that positive rates of IgG and IgM antibodies in the blood donor group were higher in younger individuals . The IgM antibody positive rate was the highest in females under 30 years old. Rheum Dis Clin North Am, 2002 Aug, 28(3), 531 - 60, vi The juvenile-onset spondyloarthritides; Burgos-Vargas R; The juvenile-onset spondyloarthritides comprise a group of HLA-B27-associated disorders, which are mainly characterized by enthesitis and arthritis affecting the lower extremities, and in a variable proportion of cases, the sacroiliac and spinal joints . Additional features include a variety of extra-articular manifestations, and in some cases, bacterial infections as triggers . Except for the prevalence of some clinical features at onset and severity throughout the course of the disease, juvenile-onset SpA resemble their adult counterpart in most clinical aspects, strength of HLA-B27 association, and the role of arthritogenic bacteria in their pathogenesis . Not surprisingly, several aspects, from nomenclature to classification, and diagnostic criteria reflect to some extent those developed in the adult onset populations. Stomatologiia (Mosk), 2002, 81(4), 24 - 8 {Efficiency of combined action of a chemical disinfectant and plasma sterilization on stomatological instruments and casts}; Tsarev VN et al.; The efficiency of combined treatment of dentures by argon plasma and chemical disinfectant Ocadez is compared using anaerobic culturing of oral bacteria and polymerase chain reaction with hepatitis B, C, and D virus primers . Different protocols of processing dental instruments and casts directly after dentist's manipulations with outpatients were tried and model experiments with hepatitis viruses were carried out . The optimal protocol of plasma sterilization is as follows: 10 min at 86-90 degrees C after treatment with Ocadez or 15 min without additional chemical treatment. Environ Sci Technol, 2002 Oct 1, 36(19), 4156 - 61 Determination of surfactants and some of their metabolites in untreated and anaerobically digested sewage sludge by subcritical water extraction followed by liquid chromatography-mass spectrometry; Bruno F et al.; Enormous amounts of sewage sludge are worldwide generated and released into the environment . Analysis of the most common and/or toxic chemicals in sludge should be mandatory before deciding its destination . Surfactants and some of their breakdown products are invariably the most common organic contaminants in domestic sewage sludge . For determining these compounds, we have developed a method based on extraction with subcritical water followed by liquid chromatography-mass spectrometry . On extracting surfactants and their metabolites from 50 mg of sludge, the efficiency of the water extraction device was evaluated in terms of pH of the extractant, temperature, and time of the static extraction . The best extraction conditions were obtained by using carbonate buffer (pH 9.4) at 200 degrees C as extractant, 10 min of static extraction at the pressure of 100 bar followed by 17 min of dynamic extraction . Analyte collection was performed by inserting a solid-phase extraction cartridge downstream the extraction cell . Compared to 16-h Soxhlet extraction with methanol, this procedure was remarkably more efficient in extracting anionic surfactants and acidic metabolites of nonylphenol ethoxylates (NPECs) . A short survey was conducted to estimate concentration changes of target compounds after 14-d sludge anaerobic digestion . Results showed that 54-74% of both neutral and weakly acidic ethoxylate species were removed after residence of the sludge in the digester . On the contrary, little, if any, removal of anionic surfactants was observed after the digestion treatment . As expected, the level of nonylphenol increased under anaerobic conditions. Infect Immun, 2002 Nov, 70(11), 6464 - 7 The N-terminal domain of RTX toxin ApxI of Actinobacillus pleuropneumoniae elicits protective immunity in mice; Seah JN et al.; We expressed three Actinobacillus pleuropneumoniae ApxI deletion derivatives to map the domain that could induce protective immunity . Antiserum to ApxI N-terminal covered by residues 40 to 380 was found to neutralize ApxI hemolytic activity but not ApxIII cytotoxicity . When used as a subunit vaccine in mice, this recombinant N-terminal fragment elicited protection against lethal infection with heterologous A . pleuropneumoniae serovars. Infect Immun, 2002 Nov, 70(11), 6383 - 8 Protection against experimental Helicobacter pylori infection after immunization with inactivated H . pylori whole-cell vaccines; Raghavan S et al.; The protective effect of therapeutic oral immunization with homologous and heterologous formalin-inactivated Helicobacter pylori cells given together with cholera toxin as an adjuvant was evaluated with C57BL/6 mice infected with H . pylori Sydney strain 1 (SS1) . The bacteria used for immunization were strains that were either homologous or heterologous with regard to the O antigen (i.e., the Lewis antigen {Le antigen}) expressed by the lipopolysaccharide of the infecting H . pylori SS1 strain . We found that repeated oral immunization with inactivated H . pylori SS1 cells can significantly inhibit an existing infection (P < 0.001) and that the protection induced by such therapeutic immunization extends to protection against reinfection (P < 0.001) . A similar level of protection was also achieved by immunization with another inactivated H . pylori strain having the same O antigen (Le antigen) as the infecting H . pylori SS1 strain . In contrast, immunization with inactivated strains expressing a heterologous O antigen, Le(x), provided less protection or no protection . Immunization with H . pylori lysate preparations, on the other hand, resulted in significant comparable protection whether the lysates were prepared from an Le(x) strain or an Le(y) strain . Postimmunization gastritis was seen in mice that were protected after vaccination but not in unimmunized or unprotected mice . In conclusion, therapeutic immunization with inactivated H . pylori whole-cell vaccines may provide strong protection both against experimental H . pylori infection and against later reinfection. Infect Immun, 2002 Nov, 70(11), 6346 - 54 Transcriptional analysis of Rickettsia prowazekii invasion gene homolog (invA) during host cell infection; Gaywee J et al.; An invasion gene homolog, invA, of Rickettsia prowazekii has recently been identified to encode a member of the Nudix hydrolase subfamily which acts specifically on dinucleoside oligophosphates (Np(n)N; n >/= 5), a group of cellular signaling molecules known as alarmones . InvA is thought to enhance intracellular survival by regulating stress-induced toxic nucleotide levels during rickettsial infection . To further characterize the physiological function of InvA, the gene expression pattern during various stages of rickettsial intracellular growth was investigated . Using semiquantitative reverse transcription-PCR (RT-PCR) and real-time fluorescent probe-based quantitative RT-PCR, a differential expression profile of invA during rickettsial host cell infection was examined . The invA transcript temporarily increased during the early period of infection . Expression of rickettsial groEL, a molecular indicator of cellular stresses, was also shown to be upregulated during the early period of infection . Furthermore, invA was cotranscribed in a polycistronic message with rrp, a gene encoding the response regulator protein homolog, which is a part of a two-component signal transduction system . These results support our earlier findings that under such stress conditions dinucleoside oligophosphate pyrophosphatase may function as a buffer, enhancing rickettsial survival within the cytoplasm of a eukaryotic cell . The expression of rickettsial dinucleoside oligophosphate pyrophosphatase may be regulated by a part of the two-component signal transduction system similar to that described for response regulators in other bacterial systems. Infect Immun, 2002 Nov, 70(11), 6223 - 30 Mycobacterium tuberculosis uptake by recipient host macrophages is influenced by environmental conditions in the granuloma of the infectious individual and is associated with impaired production of interleukin-12 and tumor necrosis factor alpha; Li YJ et al.; Transmission of Mycobacterium tuberculosis from one individual to another usually is associated with episodes of coughing . The bacteria leave the environment of the lung cavity of the infected person and travel in droplets to reach the recipient's respiratory tract . Therefore, at the time that the bacteria encounter alveolar cells (macrophages and epithelial cells) in the new host, they express virulence determinants that are regulated by the environmental conditions in the infected person . To determine if those environmental conditions encountered in the lung cavity (hyperosmolarity, acidic pH, and low oxygen tension, among others) would influence the uptake of M . tuberculosis by the recipient's alveolar macrophages, M . tuberculosis H37Rv was incubated under several conditions for different periods of time, washed at 4 degrees C, and used to infect human monocyte-derived macrophages . While increased osmolarity had no effect on M . tuberculosis uptake compared to the uptake of bacteria grown on 7H10 Middlebrook medium, both acidic pH and anaerobiosis increased the uptake of the H37Rv strain four- to sixfold . Using anti-CD11b receptor blocking antibodies or mannoside to inhibit the uptake of M . tuberculosis by macrophages, we determined that while uptake of M . tuberculosis cultured on 7H10 medium was inhibited 77% +/- 6% in the presence of anti-CD11b antibody, the antibody had no effect on the uptake of M . tuberculosis incubated at pH 6.0 and was associated with 27% inhibition of M . tuberculosis previously exposed to anaerobic conditions . The mannose receptor was also not involved with invasion after exposure to acidic conditions, and mannoside resulted in only 32% inhibition of uptake by macrophages of M . tuberculosis exposed to anaerobiosis . Uptake by macrophages also resulted in the secretion of significantly lower amounts of interleukin-12 and tumor necrosis factor alpha than that by macrophages infected with a strain cultured under laboratory conditions . M . tuberculosis cultured under the pH and oxygen concentration found in the granuloma expresses a large number of proteins that are different from the proteins expressed by bacteria grown under laboratory conditions . The results suggest that M . tuberculosis in vivo may be adapted to gain access to the intracellular environment in a very efficient fashion and may do so by using different receptors from the complement and mannose receptors. Infect Immun, 2002 Nov, 70(11), 6005 - 12 Expression of Anaplasma marginale major surface protein 2 operon-associated proteins during mammalian and arthropod infection; Lohr CV et al.; The antigenically variant major surface protein 2 (MSP2) of Anaplasma marginale is expressed from a 3.5-kb operon that contains, in a 5'-to-3' direction, four open reading frames, opag3, opag2, opag1, and msp2 . This operon structure was shown to be conserved among genotypically and phenotypically distinct A . marginale, A . ovis, and A . centrale strains . The individual OpAG amino acid sequences are highly conserved among A . marginale strains, with identities ranging from 95 to 99% . OpAG2 and OpAG3 were expressed by all examined A . marginale strains during the acute rickettsemia in the mammalian host and, like MSP2, localize to the bacterial surface . OpAG2 and OpAG3 were also expressed in an infected Ixodes scapularis tick cell line . In contrast, the same A . marginale strains expressed only OpAG2 in two different Dermacentor spp . during transmission feeding . OpAG1 expression was not detected in the infected mammalian host, the infected tick cell line, or within infected Dermacentor ticks . The differential expression of outer membrane proteins from within an operon is a novel finding in tick-transmitted bacteria, and the regulation of expression may be broadly applicable to understanding how the pathogen adapts to the mammalian host-tick vector transition. Brain Res Brain Res Protoc, 2002 Aug, 10(1), 12 - 5 An improved method of preparing microcarriers for biolistic transfection; O'Brien J et al.; The hand-held gene gun uses a pulse of helium to fire small gold particles coated with desiccated DNA (microcarriers) at target cells . This method of biolistic transfection is becoming increasingly popular as an effective means of rapid gene delivery into mammalian tissue . Current methods of microcarrier preparation, however, are slow (up to 2 days) and can result in variations in transfection efficiency due to a number of problems including shearing of DNA, agglomeration and adhesion of gold particles . Here we describe an improved, more rapid method of microcarrier preparation . To evaluate the new procedure we have used DNA encoding yellow fluorescent protein (EYFP), a modified version of the green fluorescent protein, which we have transfected into HEK293 cells . The data show that transfection by the new method results in high levels of transfection efficiency and low variability compared to an alternative method . Med Arh, 2002, 56(3), 131 - 3 Q-fever, human and animal morbidity in some regions of Bosnia and Herzegovina, in 2000; Zvizdic S et al.; Q-Fever is a worldwide zoonosis caused by Coxiella burnetti . C . burnetti is an obligate intracellular parasite . It lives in phagolysosome of the host cell . By its infection of the sensitive persons develops the acute noncharacteristic disease, which passes noncharacteristically, with the appearance of higher temperature, headache, fever, weakness of the organism or by the appearance of symptoms of the undifferentiated infection of the upper parties of the respiratory system . In the course of the infection is being developed the intersticial pneumonia, what is the reason of the infected hospitalization . Most often get sick the sheep, cows and goats, what showed also on our examined sample . In most animals the symptoms of this bacterial infection are not present, pass unobviously, and get turned out during their gravidity . The most important carriers of the causes of this disease on the domestic or wild animals are artropodes, in which within the kind is possible also the transvatial and transstadial transfer . The wild animals transfer the disease at the domestic ones, and people most often are infected by contact with these animals, their consuming of meat or milk or by contact with their secretions . Though, the most important way of getting infected of people is aerosol contaminated by the carrier as these bacteria for a long get kept in the contaminated dust, wool, animal skin, fur, straw and the excretions of the infected animals . In the illusorilly healthy and pregnant animals the bacteria are to be found in the fertile water, chorions, and placenta, that is C . burnetti becomes the cause of the premature birth or abortion in these animals . In this way comes to the bacterial contamination of the environment of the animal itself . The diagnosis of Q.-Fever is complement fixation test, indirect immunofluorescence assay (IFT) and enzyme immunoassay (EIA). Gene Ther, 2002 Nov, 9(21), 1447 - 54 Establishment of an oriP/EBNA1-based episomal vector transcribing human genomic beta-globin in cultured murine fibroblasts; Black J et al.; A novel oriP/EBNA1-based episomal vector has been constructed that persists episomally in cultured murine fibroblasts . The vector, pBH148, is equipped with the entire 185-kb human beta-globin gene locus . After amplification in bacteria, column-purified episomal pBH148 was transfected into both cultured EBNA1-expressing human D98/Raji positive control fusion cells (DRpBH148) and cultured EBNA1-negative murine fibroblast cells (A9pBH148) . Cell cultures were maintained concurrently with and without hygromycin selection for a period of 3 months . We show long-term stable episome maintenance of the full-size 200-kb circular double-stranded pBH148 in both the DRpBH148 cultures and the A9pBH148 cultures, regardless of selective pressure by agarose gel electrophoresis and Southern blot . EBNA1 transgene was detected by PCR in all transfected cultures . In addition, we were able to detect correctly spliced human beta-globin mRNA by RT-PCR in all transfected late-passage DRpBH148 and A9pBH148 cell cultures . These findings illustrate that this oriP/EBNA1-based episomal vector is stable in a previously nonpermissive murine cell line and is a potential vector for human gene therapy. Structure (Camb), 2002 Oct, 10(10), 1337 - 47 Solution structure of CopC: a cupredoxin-like protein involved in copper homeostasis; Arnesano F et al.; The structure of the metal-free form of CopC, a protein involved in copper homeostasis, has been obtained . The fold is a Greek key beta barrel similar to that of functionally unrelated blue copper proteins but with important structural variations . The protein binds one equivalent of copper (II) with relatively high affinity and contains a cluster of conserved residues (His1, Glu27, Asp89, and His91) which could form a water-accessible metal binding site . The structure also reveals a loop containing the M(X)(n)M motif which is present in a number of proteins also involved in copper homeostasis . The present structure represents a link between copper-trafficking proteins and cupredoxins . Within a structural and genomic analysis, the role of CopC in copper trafficking is discussed. Arch Virol, 2002 Oct, 147(10), 1899 - 911 NSP5 phosphorylation regulates the fate of viral mRNA in rotavirus infected cells; Chnaiderman Xiao J et al.; Elucidation of the function of the non-structural rotavirus proteins during infection is difficult in the absence of a reverse genetic system . To study the role of NSP5, nonstructural phosphoprotein NSP5, we constructed a reassortant strain (SACC11) in the SA11 background that harbours a heterologous segment 11 encoding a variant protein (h-NSP5) . Cells infected by SACC11 produced viral polypeptides at earlier times than SA11 infected cells while showing less accumulation of genomic dsRNA . These changes suggested that NSP5 might direct viral messenger RNA to protein synthesis or genome replication . Distinct patterns of proteins were shown to form complexes with NSP5 in co-immunoprecipitation studies with SA11 and SACC11 infected cells . Recombinant h-NSP5 from either bacteria or eucaryotic cells migrated faster in PAGE suggesting that it was hypophosphorylated . Indeed, the kinase inhibitor H-7 enhanced translation of viral proteins in SA11 but not SACC11 infected cells suggesting that NSP5 function in the regulation of the fate of viral positive strand RNA is mediated by phosphorylation. Bioinformatics, 2002 Oct, 18(10), 1340 - 9 Selecting signature oligonucleotides to identify organisms using DNA arrays; Kaderali L et al.; MOTIVATION: DNA arrays are a very useful tool to quickly identify biological agents present in some given sample, e.g . to identify viruses causing disease, for quality control in the food industry, or to determine bacteria contaminating drinking water . The selection of specific oligos to attach to the array surface is a relevant problem in the experiment design process . Given a set S of genomic sequences (the target sequences), the task is to find at least one oligonucleotide, called probe, for each sequence in S . This probe will be attached to the array surface, and must be chosen in a way that it will not hybridize to any other sequence but the intended target . Furthermore, all probes on the array must hybridize to their intended targets under the same reaction conditions, most importantly at the temperature T at which the experiment is conducted . RESULTS: We present an efficient algorithm for the probe design problem . Melting temperatures are calculated for all possible probe-target interactions using an extended nearest-neighbor model, allowing for both non-Watson-Crick base-pairing and unpaired bases within a duplex . To compute temperatures efficiently, a combination of suffix trees and dynamic programming based alignment algorithms is introduced . Additional filtering steps during preprocessing increase the speed of the computation . The practicability of the algorithms is demonstrated by two case studies: The identification of HIV-1 subtypes, and of 28S rDNA sequences from >or=400 organisms. Am J Physiol Lung Cell Mol Physiol, 2002 Nov, 283(5), L1011 - 22 Surfactant protein A enhances the phagocytosis of C1q-coated particles by alveolar macrophages; Watford WT et al.; Surfactant protein-A (SP-A) plays multiple roles in pulmonary host defense, including stimulating bacterial phagocytosis by innate immune cells . Previously, SP-A was shown to interact with complement protein C1q . Our goal was to further characterize this interaction and elucidate its functional consequences . Radiolabeled SP-A bound solid-phase C1q but not other complement proteins tested . The lectin activity of SP-A was not required for binding to C1q . Because C1q is involved in bacterial clearance but alone does not efficiently enhance the phagocytosis of most bacteria, we hypothesize that SP-A enhances phagocytosis of C1q-coated antigens . SP-A enhanced by sixfold the percentage of rat alveolar macrophages in suspension that phagocytosed C1q-coated fluorescent beads . Furthermore, uptake of C1q-coated beads was enhanced when either beads or alveolar macrophages were preincubated with SP-A . In contrast, SP-A had no significant effect on the uptake of C1q-coated beads by alveolar macrophages adhered to plastic slides . We conclude that SP-A may serve a protective role in the lung by interacting with C1q to enhance the clearance of foreign particles. Diagn Microbiol Infect Dis, 2002 Sep, 44(1), 23 - 7 Candida peritonitis due to peptic ulcer perforation: incidence rate, risk factors, prognosis and susceptibility to fluconazole and amphotericin B; Lee SC et al.; Sixty-two cases of peritonitis due to peptic ulcer perforation were diagnosed between January 2000 and December 2000 . Of these 62 cases, 23 isolates of Candida in 23 cases (CP) were cultured from peritoneal fluid . Cultures of peritoneal fluid of 10 (BP) of the remaining 39 cases was positive for bacteria only . Cultures of peritoneal fluid of the remaining 29 cases was negative . Comparison of CP, BP and culture-negative cases did not reveal any significant risk factor . Of the 23 Candida isolates, the Candida species and 48-h MICs of fluconazole and amphotericin B (mean, range ug/ml) were C . albicans 18 (0.688, 0.125-1.0; 0.297, 0.031-0.5), C . glabrata 3 (0.542, 0.125-1.0; 0.25, 0.125-0.5), C . tropicalis 1 (0.25; 0.5), C . intermedia 1 (1.0; 0.125) respectively . Mortality rates of CP, BP and culture-negative peritonitis due to infection were 5/23(21.7%), 0/10 and 1/29(3.4%) respectively . Without effective antifungal therapy, the mortality rate of CP was not low. J Biol Chem, 2002 Dec 13, 277(50), 48456 - 62 Epub 2002 Oct 08. Regulation of WNK1 by an autoinhibitory domain and autophosphorylation; Xu BE et al.; WNK family protein kinases are large enzymes that contain the catalytic lysine in a unique position compared with all other protein kinases . These enzymes have been linked to a genetically defined form of hypertension . In this study we introduced mutations to test hypotheses about the position of the catalytic lysine, and we examined mechanisms involved in the regulation of WNK1 activity . Through the analysis of enzyme fragments and sequence alignments, we have identified an autoinhibitory domain of WNK1 . This isolated domain, conserved in all four WNKs, suppressed the activity of the WNK1 kinase domain . Mutation of two key residues in this autoinhibitory domain attenuated its ability to inhibit WNK kinase activity . Consistent with these results, the same mutations in a WNK1 fragment that contain the autoinhibitory domain increased its kinase activity . We also found that WNK1 expressed in bacteria is autophosphorylated; autophosphorylation on serine 382 in the activation loop is required for its activity. Eur J Clin Nutr, 2002 Oct, 56(10), 952 - 7 Phloem fortification in rye bread elevates serum enterolactone level; Vanharanta M et al.; OBJECTIVE: To analyse the lignan content of phloem powder enriched rye bread and to study the dose-response relationship of the effect of dietary plant lignans derived from phloem on intestinal production of enterolactone by measuring enterolactone concentration in serum . DESIGN: A randomized double-blind supplementation trial . SUBJECTS: Seventy-five non-smoking men recruited by newspaper advertisements . INTERVENTION: Subjects were randomized to three study groups receiving either rye bread high in phloem (HP, 14% of rye flour substituted with phloem powder), rye bread low in phloem (LP, 7% of rye flour substituted with phloem powder) or placebo rye bread . Participants consumed 70 g of study bread daily for 4 weeks and provided serum samples for enterolactone analysis at baseline and at the end of the intervention . RESULTS: There was a significant increase in serum enterolactone concentration in the LP and HP groups compared with the placebo group (P=0.009 and P=0.003, respectively) . Considerable interindividual differences were observed in the response to dietary lignans within the study groups . CONCLUSIONS: Our results indicate that plant lignans attached to insoluble fibre layer in phloem can be further metabolized and converted to enterolactone presumably by the bacteria present in the colon . Phloem powder is useful source of lignans for functional foods aimed to elevate serum enterolactone levels . SPONSORSHIP: Phloem powder and the study breads were provided by Finnpettu Oy and Linkosuo Oy, respectively . The clinical study work was sponsored in part by Oy Jurilab Ltd. Arch Dermatol Res, 2002 Oct, 294(7), 310 - 7 Epub 2002 Sep 05. U3 snoRNP associates with fibrillarin a component of the scleroderma clumpy nucleolar domain; Herrera-Esparza R et al.; Serum from patients with scleroderma recognizes the clumpy autoantigen . The present studies addressed the issue as to whether the clumpy nucleolar autoantigen recognized by scleroderma serum is fibrillarin-U3 snoRNP . Clones encoding for clumpy autoantigen were immunodetected from a lambdagt11 HeLa cell random-primed library with the serum from a patient with diffuse scleroderma and autoautoantibodies against clumpy autoantigen . Sequences from the recombinant phages were amplified by PCR and subcloned into a pCRII vector . The DNA was sequenced by a dideoxy termination reaction . Ten lambdagt11 clumpy clones were detected by immunoscreening . One containing the glycine-rich and RNP2 fibrillarin domains was expressed in lysogenic bacteria . The recombinant proteins were used to elicit antibodies in rabbits, and these exhibited clumpy nucleolar reactivity . The recombinant fibrillarin tested by ELISA was recognized by the clumpy scleroderma serum from the majority of patients . In situ hybridization assays showed that the fibrillarin tagged by the elicited antibodies was colocalized with U3 snoRNP in the nucleolus in a clumpy manner and coprecipitated the U3 snoRNP . In conclusion, the fibrillarin-U3 snoRNP complex is the major component of the clumpy subcellular domain . Therefore these molecules constitute an important target of scleroderma autoantibodies. Genome Biol . 2002 Sep 25;3(10):REVIEWS3013 . Epub 2002 Sep 25. Histidine protein kinases: key signal transducers outside the animal kingdom; Wolanin PM et al.; Histidine protein kinases (HPKs) are a large family of signal-transduction enzymes that autophosphorylate on a conserved histidine residue . HPKs form two-component signaling systems together with their downstream target proteins, the response regulators, which have a conserved aspartate in a so-called 'receiver domain' that is phosphorylated by the HPK . Two-component signal transduction is prevalent in bacteria and is also widely used by eukaryotes outside the animal kingdom . The typical HPK is a transmembrane receptor with an amino-terminal extracellular sensing domain and a carboxy-terminal cytosolic signaling domain; most, if not all, HPKs function as dimers . They show little similarity to protein kinases that phosphorylate serine, threonine or tyrosine residues, but may share a distant evolutionary relationship with these enzymes . In excess of a thousand known genes encode HPKs, which are important for multiple functions in bacteria, including chemotaxis and quorum sensing, and in eukaryotes, including hormone-dependent developmental processes . The proteins divide into at least 11 subfamilies, only one of which is present in eukaryotes, suggesting that lateral gene transfer gave rise to two-component signaling in these organisms. Genome Biol . 2002 Sep 19;3(10):research0054 . Epub 2002 Sep 19. Genomic analysis of membrane protein families: abundance and conserved motifs; Liu Y et al.; BACKGROUND: Polytopic membrane proteins can be related to each other on the basis of the number of transmembrane helices and sequence similarities . Building on the Pfam classification of protein domain families, and using transmembrane-helix prediction and sequence-similarity searching, we identified a total of 526 well-characterized membrane protein families in 26 recently sequenced genomes . To this we added a clustering of a number of predicted but unclassified membrane proteins, resulting in a total of 637 membrane protein families . RESULTS: Analysis of the occurrence and composition of these families revealed several interesting trends . 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