|
|
|
|
|
| J Immunol, 2004 Sep 15, 173(6), 4164 - 70 Cigarette smoke inhibits lipopolysaccharide-induced production of inflammatory cytokines by suppressing the activation of activator protein-1 in bronchial epithelial cells; Laan M et al.; Chronic smoking is characterized by immunosuppressive changes in the airways, leading to chronic colonization with bacteria, which in turn may contribute to the chronic obstructive pulmonary disease . The mechanisms causing this immunosuppression, however, are poorly characterized . This study evaluated whether cigarette smoke can inhibit endotoxin (LPS)-induced inflammatory cytokine production in bronchial epithelial cells and, if so, what the mechanisms are behind this effect . Pretreatment with cigarette smoke extract (CSE) concentration dependently inhibited the LPS-induced GM-CSF and IL-8 protein release, which was accompanied by decreased expression of mRNA in human bronchial epithelial cells (Beas-2B) . The increase of neutrophil chemotaxis induced by conditioned medium from LPS-treated Beas-2B cells was also suppressed by CSE . In addition, the activity of LPS-induced transcription factor AP-1, but not NF-kappaB, was down-regulated by CSE . Notably, at the concentrations used, CSE had no effect on number or viability of Beas-2B cells . These data indicate that cigarette smoke possesses immunosuppressive properties by down-regulating the bacterial pathogen-induced neutrophil-mobilizing cytokine production via suppression of AP-1 activation in the airways . Hence, this study suggests a novel mechanism by which cigarette smoke may contribute to chronic colonization and chronic obstructive pulmonary disease in smokers . J Immunol, 2004 Sep 15, 173(6), 3647 - 52 Immunological synapse formation licenses CD40-CD40L accumulations at T-APC contact sites; Boisvert J et al.; The maintenance of tolerance is likely to rely on the ability of a T cell to polarize surface molecules providing "help" to only specific APCs . The formation of a mature immunological synapse leads to concentration of the TCR at the APC interface . In this study, we show that the CD40-CD154 receptor-ligand pair is also highly concentrated into a central region of the synapse on mouse lymphocytes only after the formation of the TCR/CD3 c-SMAC . Concentration of this ligand was strictly dependent on TCR recognition, the binding of ICAM-1 to T cell integrins and the presence of an intact cytoskeleton in the T cells . This may provide a novel explanation for the specificity of T cell help directing the help signal to the site of Ag receptor signal . It may also serve as a site for these molecular aggregates to coassociate and/or internalize alongside other signaling receptors . J Biol Chem, 2004 Nov 26, 279(48), 50514 - 23 Epub 2004 Nov 26. Crystal structure of T-protein of the glycine cleavage system . Cofactor binding, insights into H-protein recognition, and molecular basis for understanding nonketotic hyperglycinemia; Lee HH et al.; The glycine cleavage system catalyzes the oxidative decarboxylation of glycine in bacteria and in mitochondria of animals and plants . Its deficiency in human causes nonketotic hyperglycinemia, an inborn error of glycine metabolism . T-protein, one of the four components of the glycine cleavage system,is a tetrahydrofolate dependent aminomethyltransferase . It catalyzes the transfer of the methylene carbon unit to tetrahydrofolate from the methylamine group covalently attached to the lipoamide arm of H-protein . To gain insight into the T-protein function at the molecular level, we have determined the first crystal structure of T-protein from Thermotoga maritima by the multiwavelength anomalous diffraction method of x-ray crystallography and refined four structures: the apoform; the tetrahydrofolate complex; the folinic acid complex; and the lipoic acid complex . The overall fold of T-protein is similar to that of the C-terminal tetrahydrofolate-binding region (residues 421-830) of Arthrobacter globiformis dimethylglycine oxidase . Tetrahydrofolate (or folinic acid) is bound near the center of the tripartite T-protein . Lipoic acid is bound adjacent to the tetrahydrofolate binding pocket, thus defining the interaction surface for H-protein binding . A homology model of the human T-protein provides the structural framework for understanding the molecular mechanisms underlying the development of nonketotic hyperglycinemia due to missense mutations of the human T-protein. Lett Appl Microbiol, 2004, 39(4), 376 - 82 A real-time polymerase chain reaction-based method for rapid and specific detection of spoilage Alicyclobacillus spp . in apple juice; Luo H et al.; AIMS: To develop a real-time PCR-based rapid detection method for spoilage Alicyclobacillus spp . in juice products . METHODS AND RESULTS: The squalene-hopene cyclase-encoding gene was targeted for primer-and-probe development . Gene fragments from representative strains were cloned, and PCR primers and probe were designed by DNA sequence comparison . Selected bacteria were examined for cross-reactivity by the new method . Cells were serially diluted in apple juice and saline, and examined by the new method to establish detection sensitivity . Using the newly developed Taqman real-time PCR-based method, strains of Alicyclobacillus acidocaldarius and A . acidoterrestris were detected without cross reactivity with other common food-borne micro-organisms . Detection of <10 cells per PCR reaction from juice samples was accomplished within 3-5 h . CONCLUSION: This is the first reported real-time PCR-based detection method for Alicyclobacillus spp . and its application in juice products is demonstrated . SIGNIFICANCE AND IMPACT OF THE STUDY: As a favourable alternative for the laborious and time-consuming culture- or biochemical characterization-based techniques, the system has great potential for industrial applications from raw material screening to final product quality control. Clin Microbiol Infect, 2004 Sep, 10(9), 820 - 5 Chlamydia pneumoniae in HIV-infected patients and controls assessed by a novel whole blood interferon-gamma assay, serology and PCR; Woolley IJ et al.; Chlamydia pneumoniae seropositivity is associated with cardiovascular disease and HIV infection . Cell-mediated immune responses are important for control of C . pneumoniae, and such responses may be impaired in HIV-infected patients . An assay for detection of interferon (IFN)-gamma in whole blood stimulated with C . pneumoniae antigen was developed and studied in HIV-infected patients and uninfected controls . Among 34 HIV-infected patients, none had an IFN-gamma response to C . pneumoniae antigen, compared with five of 32 healthy controls (p < 0.001) . Fewer HIV-infected individuals elicited a serum IgG response when tested with a commercial enzyme immunoassay (p 0.009), but this was not so for serum IgA (p 0.12) . Additionally, the IFN-gamma and antibody assays showed a trend towards a bivariate response in normal controls . This indicates that cellular and antibody responses against C . pneumoniae may be mutually exclusive, with potential implications for the role of this organism in the genesis of cardiovascular disease in both immunocompetent and HIV-infected populations. MMW Fortschr Med, 2004 Jun 17, 146(25), 23 - 4, 26-8 {Humanopathogenic parasites--an unwelcome import}; Mehlhorn H; Humanopathogenic parasites represent a health risk for travelers to tropical countries . They can be picked up by direct contact, via contaminated food and drink, the bites of blood-sucking insects and leeches, as also via direct penetration through the skin . Not only the bite and sting wounds themselves are unpleasant . The true danger to the victim is represented, in particular, by the bacteria, viruses or parasites that are transmitted in this way . They can give rise to protracted diseases such as hepatitis B, cutaneous or intestinal disorders . The most effective preventive measure is exposure prophylaxis. Curr Opin Infect Dis, 2004 Oct, 17(5), 461 - 9 A review of viral gastroenteritis; Clark B et al.; PURPOSE OF REVIEW: Since Kapakian first identified a virus in the stool of a patient with diarrhoea in 1972, many viruses have been described that cause diarrhoea directly or indirectly . It is now appreciated that viruses are the most common cause of diarrhoeal illness worldwide . Although bacteria and other pathogens cause significant numbers of gastroenteritis, it is the viruses that are dealt with in this review . The viruses responsible will be discussed individually . RECENT FINDINGS: Rotavirus remains the leading cause of diarrhoeal disease overall, with the newly designated calicivirus family causing the most outbreaks in the industrialized nations . As diagnostic techniques improve, however, the importance of astrovirus and other previously under-reported pathogens is becoming more apparent and the number of viruses associated with gastroenteritis continues to increase . The emergence of severe acute respiratory syndrome coronavirus, arguably the most important emerging infection of recent years and a cause of significant gastrointestinal disease, is also discussed . SUMMARY: No effective treatments have been developed for viral gastroenteritis . Current efforts are targeted at the development of suitable vaccines and the implementation of infection control measures. J Vet Med Sci, 2004 Aug, 66(8), 985 - 7 Fibrinonecrotic rhinitis caused by a concurrent infection of Fusobacterium necrophorum and Arcanobacterium pyogenes in a cow; Seimiya YM et al.; An 8 year-old cow showing severe dyspnea and nasal mucosal necrosis immediately after parturition was subjected to pathological examination . The principal lesions were fibrinonecrotic rhinitis, necrotic bronchopneumonia and renal infarction . Fusobacterium necrophorum biotype A and Arcanobacterium pyogenes antigens were detected in the nasal and pulmonary lesions . These results suggest that the lesions were caused by a concurrent infection of the detected bacteria and that the pulmonary lesions were caused by the aspiration of infectious materials from the nasal ones . Mucosal coagulative necroses observed as the initial lesions in rhinitis were frequently associated with multiple thrombosis . The findings might suggest that thrombosis played an important role in the development of the nasal lesions. Microbiol Mol Biol Rev, 2004 Sep, 68(3), 453 - 73, table of contents CO-Sensing Mechanisms; Roberts GP et al.; Carbon monoxide (CO) has long been known to have dramatic physiological effects on organisms ranging from bacteria to humans, but recently there have a number of suggestions that organisms might have specific sensors for CO . This article reviews the current evidence for a variety of proteins with demonstrated or potential CO-sensing ability . Particular emphasis is placed on the molecular description of CooA, a heme-containing CO sensor from Rhodospirillum rubrum, since its biological role as a CO sensor is clear and we have substantial insight into the basis of its sensing ability. Front Biosci, 2004 Sep 01, 9, 1999 - 2019 Osmoadaptation and osmoregulation in archaea: update 2004; Roberts MF; The response of archaea to changes in external NaCl is reviewed and compared to what is known about osmoadaptation and osmoregulation in bacteria and eukaryotes . Cells placed in altered external NaCl exhibit short term and long term responses . The earliest events are likely to be water movement through aquaporin-like channels (efflux if external NaCl has been increased, influx into the cell if the external NaCl has been decreased) and ion movement (e.g., K+ moving in the direction opposite to water flow) through channels sensitive to osmotic pressure . A brief discussion of recent structures of homologues of these membrane proteins is presented . Accumulation of organic solutes, either by uptake from the medium or de novo synthesis, is triggered after these initial changes . Archaea have some unique organic solutes (osmolytes) that are not used by other organisms . These as well as other more common solutes have a role in stabilizing macromolecules from denaturation . Many osmolytes are distinguished by their stability in the cell and their lack of strong interactions with cellular components . A cell may respond by accumulating one or more temporary osmolytes, then over time readjust the intracellular solute distribution to what is optimal for cell growth under the new conditions . Coupled with the movement and accumulation of solutes is the induction of stress proteins (e.g., chaperonins) and, in some cases, transcriptional regulation of key enzymes . The response to NaCl stress of Methanococcus thermolithotrophicus is presented as an example of how one particular archaeon responds and adapts to altered osmotic pressure . The detailed response of many other archaea to osmotic stress will be needed in order to identify features (aside from some of the organic osmolytes) unique to the organisms in this kingdom. Aliment Pharmacol Ther, 2004 Oct, 20 Suppl 4, 79 - 83 Review article: the role of nutrition in the treatment of inflammatory bowel disease; Gassull MA; Nutrients may be involved in the modulation of the immune response through at least three different mechanisms . First, the intestinal ecosystem plays a pivotal role in the pathogenesis of inflammatory bowel disease, triggering the uncontrolled inflammatory response in genetically predisposed individuals . Nutrients, together with bacteria, are major components of, and can therefore influence, the intestinal environment . Second, as components of cell membranes, nutrients can mediate the expression of proteins involved in the immune response, such as cytokines, adhesion molecules and nitric oxide synthase . The composition of lipids in the cell membrane is modified by dietary changes and can influence cellular responses . Indeed, various epidemiological, experimental and clinical data suggest that the immune response may be sensitive to changes in dietary composition . Finally, suboptimal levels of micronutrients are often found in both children and adults with inflammatory bowel disease, although, with the exception of iron and folate, it is unusual to discover symptoms attributable to these deficits . However, subclinical deficits may have a pathophysiological significance, as they may favour the self-perpetuation of the disease (due to defects in the mechanisms of tissue repair), cause defective defence against damage produced by oxygen free radicals and facilitate lipid peroxidation . These events can occur even in clinically inactive or mildly active disease, as well as in the development of dysplasia in the intestinal mucosa . Some dietary manipulations have been attempted as primary treatment for rheumatoid arthritis, and specially formulated diets for enteral nutrition have proved to be an effective treatment for Crohn's disease . Most trials, although lacking sufficient patient numbers, have demonstrated a role for dietary manipulation as primary therapy for inflammatory disease . Dietary lipids are one of the most active nutritional substrates modulating the immune response . Recently, it has been demonstrated that lipids may be a key factor explaining the therapeutic effect of clinical nutrition in Crohn's disease. Biotechnol Bioeng, 2004 Sep 5, 87(5), 584 - 92 Estimating the potential refolding yield of recombinant proteins expressed as inclusion bodies; Ho JG et al.; Recombinant protein production in bacteria is efficient except that insoluble inclusion bodies form when some gene sequences are expressed . Such proteins must undergo renaturation, which is an inefficient process due to protein aggregation on dilution from concentrated denaturant . In this study, the protein-protein interactions of eight distinct inclusion-body proteins are quantified, in different solution conditions, by measurement of protein second virial coefficients (SVCs) . Protein solubility is shown to decrease as the SVC is reduced (i.e., as protein interactions become more attractive) . Plots of SVC versus denaturant concentration demonstrate two clear groupings of proteins: a more aggregative group and a group having higher SVC and better solubility . A correlation of the measured SVC with protein molecular weight and hydropathicity, that is able to predict which group each of the eight proteins falls into, is presented . The inclusion of additives known to inhibit aggregation during renaturation improves solubility and increases the SVC of both protein groups . Furthermore, an estimate of maximum refolding yield (or solubility) using high-performance liquid chromatography was obtained for each protein tested, under different environmental conditions, enabling a relationship between "yield" and SVC to be demonstrated . Combined, the results enable an approximate estimation of the maximum refolding yield that is attainable for each of the eight proteins examined, under a selected chemical environment . Although the correlations must be tested with a far larger set of protein sequences, this work represents a significant move beyond empirical approaches for optimizing renaturation conditions . The approach moves toward the ideal of predicting maximum refolding yield using simple bioinformatic metrics that can be estimated from the gene sequence . Such a capability could potentially "screen," in silico, those sequences suitable for expression in bacteria from those that must be expressed in more complex hosts. Int J Cancer, 2004 Nov 1, 112(2), 225 - 30 The tumor-suppressive reagent taurolidine is an inhibitor of protein biosynthesis; Braumann C et al.; Taurolidine has been successfully used as a disinfectant and to prevent the spreading and growth of tumor cells after surgical excision . However, the underlying mechanisms regarding its effects remain obscure . Here, we show that taurolidine treatment reduces endogenous levels of IkappaBalpha, p105, c-Jun, p53 and p27 in a dose-dependent manner in colon adenocarcinoma cells, which can be in part due to massive cell death . Because expression of tested proteins was affected by taurolidine, its influence on protein expression was studied . In the coupled transcription/translation system, taurolidine inhibited c-Jun expression with an IC50 value of 1.4 mM . There was no or little effect on transcription . In contrast, translation of c-Jun or p53 mRNA was completely inhibited by taurolidine . To determine which step of translation was affected, prominent complexes occurring in the course of translation were analyzed by density gradient centrifugation . In the presence of taurolidine, no preinitiation translation complex was assembled . Taurolidine also suppressed protein expression in bacteria . Based on our data, we conclude that taurolidine blocks a fundamental early phase of translation, which might explain its effects as a disinfectant and inhibitor of tumor growth . J Mol Biol, 2004 Sep 24, 342(4), 1325 - 35 Structure of the constitutively active double mutant CheYD13K Y106W alone and in complex with a FliM peptide; Dyer CM et al.; CheY is a member of the response regulator protein superfamily that controls the chemotactic swimming response of motile bacteria . The CheY double mutant D13K Y106W (CheY**) is resistant to phosphorylation, yet is a highly effective mimic of phosphorylated CheY in vivo and in vitro . The conformational attributes of this protein that enable it to signal in a phosphorylation-independent manner are unknown . We have solved the crystal structure of selenomethionine-substituted CheY** in the presence of its target, a peptide (FliM16) derived from the flagellar motor switch, FliM, to 1.5A resolution with an R-factor of 19.6% . The asymmetric unit contains four CheY** molecules, two with FliM16 bound, and two without . The two CheY** molecules in the asymmetric unit that are bound to FliM16 adopt a conformation similar to BeF3- -activated wild-type CheY, and also bind FliM16 in a nearly identical manner . The CheY** molecules that do not bind FliM16 are found in a conformation similar to unphosphorylated wild-type CheY, suggesting that the active phenotype of this mutant is enabled by a facile interconversion between the active and inactive conformations . Finally, we propose a ligand-binding model for CheY and CheY**, in which Ile95 changes conformation in a Tyr/Trp106-dependent manner to accommodate FliM. J Mol Biol, 2004 Sep 24, 342(4), 1237 - 48 Crystal structure of human triggering receptor expressed on myeloid cells 1 (TREM-1) at 1.47 A; Kelker MS et al.; The triggering receptor expressed on myeloid cells (TREM) family of single extracellular immunoglobulin receptors includes both activating and inhibitory isoforms whose ligands are unknown . TREM-1 activation amplifies the Toll-like receptor initiated responses to invading pathogens allowing the secretion of pro-inflammatory chemokines and cytokines . Hence, TREM-1 amplifies the inflammation induced by both bacteria and fungi, and thus represents a potential therapeutic target . We report the crystal structure of the human TREM-1 extracellular domain at 1.47 A resolution . The overall fold places it within the V-type immunoglobulin domain family and reveals close homology with Ig domains from antibodies, T-cell receptors and other activating receptors, such as NKp44 . With the additional use of analytical ultracentrifugation and 1H NMR spectroscopy of both human and mouse TREM-1, we have conclusively demonstrated the monomeric state of this extracellular ectodomain in solution and, presumably, of the TREM family in general. Anal Biochem, 2004 Oct 1, 333(1), 14 - 8 A coupled spectrophotometric enzyme assay for the determination of pectin methylesterase activity and its inhibition by proteinaceous inhibitors; Grsic-Rausch S et al.; Pectin methylesterase (PME; EC 3.1.1.11) activities are widespread in bacteria, fungi, and plants . PME-mediated changes in cell wall pectin structure play important roles in plant development . Genome sequencing projects have revealed the existence of large PME multigene families in higher plants . Additional complexity for PME regulation arises from the presence of specific PME inhibitor proteins (PMEI) in plant cells . Several assay procedures for the determination of PME activity have been reported . However, previous protocols suffered from various limitations . Here we report a protocol for a coupled enzyme assay based on methanol oxidation via alcohol oxidase (AO; EC 1.1.3.13) and subsequent oxidation of formaldehyde by formaldehyde dehydrogenase (FDH; EC 1.2.1.3) . This simple and robust assay allows the continuous monitoring of PME activity in the neutral pH range . Furthermore, as plant PMEIs do not interfer with AO and FDH activities, this assay is suitable for the characterization of the inhibition kinetics of PMEI. Spectrochim Acta A Mol Biomol Spectrosc, 2004 Oct, 60(12), 2767 - 74 Spectroscopic characterization of tetradentate macrocyclic ligand: it's transition metal complexes; Chandra S et al.; Manganese(II), cobalt(II), nickel(II) and copper(II) complexes are synthesized with a novel tetradentate ligand viz . 1,3,9,11-tetraaza-4,8,12,16-tetraoxo-2,6,10,14-tetrathiacyclohexadecane (L) and characterized by the elemental analysis, molar conductance measurements, magnetic susceptibility measurements, electron impact mass, 1H NMR, IR, electronic and EPR spectral studies . The molar conductance measurements of the complexes in DMSO correspond to be nonelectrolytic nature for Mn(II), Co(II) and Cu(II) while 1:2 electrolytes for Ni(II) complexes . Thus these complexes may be formulated as {M(L)X2} and {Ni(L)}X2 (where M: Mn(II), Co(II), and Cu(II) and X = Cl- and NO3-) . On the basis of IR, electronic and EPR spectral studies an octahedral geometry has been assigned for Mn(II) and Co(II) complexes, square-planar for Ni(II) whereas tetragonal for Cu(II) complexes . The ligand and its complexes were also evaluated against the growth of bacteria and pathogenic fungi in vitro. Microbiology, 2004 Sep, 150(Pt 9), 2843 - 55 Ciliostasis is a key early event during colonization of canine tracheal tissue by Bordetella bronchiseptica; Anderton TL et al.; The primary site of infection for Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis is the ciliated respiratory epithelium . Previous studies have implicated adherence of bacteria to cilia, induction of mucus production, induction of ciliostasis and damage to the ciliated epithelium in Bordetella pathogenesis . This paper describes the use of an air-interface organ culture system using canine tracheal tissue infected with B . bronchiseptica to assess the temporal relationship between these pathologies . Ciliostasis occurs very early during the host tissue-pathogen interaction, before mucus production and obvious signs of epithelial damage occur . A B . bronchiseptica bvg mutant does not colonize the organ culture model, induce ciliostasis or cause damage to the epithelial cell layer, but it does induce similar amounts of mucus release as does infection by wild-type bacteria . The authors propose that ciliostasis is a key early event during the B . bronchiseptica-host tissue interaction that abrogates the muco-ciliary defences of the host tissue, renders it susceptible to colonization by the bacteria and allows subsequent damage to the epithelium . The organ culture model described offers a physiologically relevant tool with which to characterize the molecular basis for interactions between Bordetella and its primary site of infection, the ciliated respiratory epithelium. Proc R Soc Lond B Biol Sci, 2004 Sep 22, 271(1551), 1941 - 6 A cost of Wolbachia-induced sex reversal and female-biased sex ratios: decrease in female fertility after sperm depletion in a terrestrial isopod; Rigaud T et al.; A number of parasites are vertically transmitted to new host generations via female eggs . In such cases, host reproduction is an intimate component of parasite fitness and no cost of the infection on host reproduction is expected to evolve . A number of these parasites distort host sex ratios towards females, thereby increasing either parasite fitness or the proportion of the host that transmit the parasite . In terrestrial isopods (woodlice), Wolbachia bacteria are responsible for sex reversion and female-biased sex ratios, changing genetic males into functional neo-females . Although sex ratio distortion is a powerful means for parasites to increase in frequency in host populations, it also has potential consequences on host biology, which may, in turn, have consequences for parasite prevalence . We used the woodlouse Armadillidium vulgare to test whether the interaction between Wolbachia infection and the resulting excess of females would limit female fertility through the reduction in sperm number that they receive from males . We showed that multiple male mating induces sperm depletion, and that this sperm depletion affects fertility only in infected females . This decrease in fertility, associated with male mate choice, may limit the spread of Wolbachia infections in host populations. Zhonghua Fu Chan Ke Za Zhi, 2004 Jul, 39(7), 478 - 81 {Killing effect of adenovirus mediated fusion gene cytosine and deaminase uracil phosphoribosyl transferase directed by glutathione S-transferase P1 promoter on cisplatin-resistant ovarian cancer cells in vitro}; Cai LQ et al.; OBJECTIVE: To construct an adenoviral vector in which the fusion gene cytosine and uracil phosphoribosyl (UPP) transferase was directed by glutathione S-transferase P1 (GSTP1) promoter, and to investigate specific killing effect of the suicide gene system on cisplatin-resistant ovarian cancer cells . METHODS: Recombinant adenovirus was generated through homologous recombination in bacteria . A2780 and A2780/DDP cells were infected with Ad and then received flucytosine (5-FC) administration . The relative survival of these cells was tested . And a bystander effect was observed by mixing gene-transferred and gene-untransferred A2780/DDP cells with 5-FC . RESULTS: In vitro, when MOI was 100 and 5-FC was 250 micro g/ml, relative survival rate of A2780/DDP cells was only (3.6 +/- 1.0)%; that of A2780 cells was (76.5 +/- 2.8)% . Significant bystander effect was caused by CD-UPP gene and 20% gene-transferred A2780/DDP cells induced 80.3% of total cells to death . CONCLUSION: Recombinant adenovirus carrying CD-UPP gene driven by GSTP1 promoter has a specific killing effect on cisplatin-resistant ovarian cancer cells. Acta Crystallogr C, 2004 Sep, 60(Pt 9), o662 - 4 Epub 2004 Aug 11. 6-(4-Methoxybenzylamino)purin-3-ium chloride; Travnicek Z et al.; The title compound, C13H14N5O+.Cl-, belongs to the group of aromatic cytokinins . These compounds affect a variety of important physiological processes in plants and animals as well as in bacteria, including cell division, differentiation and senescence . The structure consists of a 6-(4-methoxybenzylamino)purinium cation and a Cl- anion . The cation moiety exists as the N3-protonated N7 tautomer . The cation contains nearly planar benzene and purine ring systems, with a dihedral angle of 77.46 (5) degrees . The crystal structure is stabilized by N(amino)-H...N(purine) hydrogen bonds connecting two adjacent molecules, thus forming centrosymmetric dimers. Appl Environ Microbiol, 2004 Sep, 70(9), 5701 - 3 PCR-based identification of hyperthermophilic archaea of the family Thermococcaceae; Slobodkina GB et al.; A method for rapid detection and identification of hyperthermophilic archaea of the family Thermococcaceae based on PCR amplification of 16S rRNA gene fragments with primers TcPc 173F (5'-TCCCCCATAGGYCTGRGGTACTGGAAGGTC-3') and TcPc 589R (5'-GCCGTGRGATTTCGCCAGGGACTTACGGGC-3') was developed and used for identification of new isolates. Appl Environ Microbiol, 2004 Sep, 70(9), 5522 - 7 Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones; Ginolhac A et al.; The metagenomic approach provides direct access to diverse unexplored genomes, especially from uncultivated bacteria in a given environment . This diversity can conceal many new biosynthetic pathways . Type I polyketide synthases (PKSI) are modular enzymes involved in the biosynthesis of many natural products of industrial interest . Among the PKSI domains, the ketosynthase domain (KS) was used to screen a large soil metagenomic library containing more than 100,000 clones to detect those containing PKS genes . Over 60,000 clones were screened, and 139 clones containing KS domains were detected . A 700-bp fragment of the KS domain was sequenced for 40 of 139 randomly chosen clones . None of the 40 protein sequences were identical to those found in public databases, and nucleic sequences were not redundant . Phylogenetic analyses were performed on the protein sequences of three metagenomic clones to select the clones which one can predict to produce new compounds . Two PKS-positive clones do not belong to any of the 23 published PKSI included in the analysis, encouraging further analyses on these two clones identified by the selection process. Appl Environ Microbiol, 2004 Sep, 70(9), 5415 - 25 Shewanella oneidensis MR-1 restores menaquinone synthesis to a menaquinone-negative mutant; Myers CR et al.; The mechanisms underlying the use of insoluble electron acceptors by metal-reducing bacteria, such as Shewanella oneidensis MR-1, are currently under intensive study . Current models for shuttling electrons across the outer membrane (OM) of MR-1 include roles for OM cytochromes and the possible excretion of a redox shuttle . While MR-1 is able to release a substance that restores the ability of a menaquinone (MK)-negative mutant, CMA-1, to reduce the humic acid analog anthraquinone-2,6-disulfonate (AQDS), cross-feeding experiments conducted here showed that the substance released by MR-1 restores the growth of CMA-1 on several soluble electron acceptors . Various strains derived from MR-1 also release this substance; these include mutants lacking the OM cytochromes OmcA and OmcB and the OM protein MtrB . Even though strains lacking OmcB and MtrB cannot reduce Fe(III) or AQDS, they still release a substance that restores the ability of CMA-1 to use MK-dependent electron acceptors, including AQDS and Fe(III) . Quinone analysis showed that this released substance restores MK synthesis in CMA-1 . This ability to restore MK synthesis in CMA-1 explains the cross-feeding results and challenges the previous hypothesis that this substance represents a redox shuttle that facilitates metal respiration. Appl Environ Microbiol, 2004 Sep, 70(9), 5366 - 72 Heads or tails: host-parasite interactions in the Drosophila-Wolbachia system; Veneti Z et al.; Wolbachia strains are endosymbiotic bacteria typically found in the reproductive tracts of arthropods . These bacteria manipulate host reproduction to ensure maternal transmission . They are usually transmitted vertically, so it has been predicted that they have evolved a mechanism to target the host's germ cells during development . Through cytological analysis we found that Wolbachia strains display various affinities for the germ line of Drosophila . Different Wolbachia strains show posterior, anterior, or cortical localization in Drosophila embryos, and this localization is congruent with the classification of the organisms based on the wsp (Wolbachia surface protein) gene sequence . This embryonic distribution pattern is established during early oogenesis and does not change until late stages of embryogenesis . The posterior and anterior localization of Wolbachia resembles that of oskar and bicoid mRNAs, respectively, which define the anterior-posterior axis in the Drosophila oocyte . By comparing the properties of a single Wolbachia strain in different host backgrounds and the properties of different Wolbachia strains in the same host background, we concluded that bacterial factors determine distribution, while bacterial density seems to be limited by the host . Possible implications concerning cytoplasmic incompatibility and evolution of strains are discussed. Crit Care Med, 2004 Sep, 32(9), 1899 - 903 Mild preseptic hypothermia is detrimental in rats; Torossian A et al.; OBJECTIVE: We evaluated the effects of mild hypothermia (32 degrees C), established before experimental intra-abdominal sepsis, on outcome, cytokine pattern, and muscle tissue oxygenation . DESIGN: Clinic modeling randomized laboratory trial . SETTING: University laboratory . SUBJECTS: Ninety-six male rats . INTERVENTIONS: In a group-sequential design, using 42 rats per group, we compared mild hypothermia with normothermia before peritonitis . Peritoneal inoculation with human stool bacteria was performed to simulate clinical trial conditions . Additionally, 12 rats underwent preoperative mild hypothermia without infection . MEASUREMENTS AND MAIN RESULTS: Primary end point was mortality at 120 hrs . Secondary end points were systemic cytokine concentrations, granulocyte counts, and muscle oxygen partial pressure . Survival rate was 40% (16 of 42) after preseptic hypothermia and 62% (26 of 42) after preseptic normothermia (p =.048) . All hypothermic rats without infection survived . Interleukin-10 concentrations were 1843 +/- 96 pg/mL after preseptic hypothermia, 945 +/- 225 pg/mL with preseptic normothermia, and 520 +/- 121 pg/mL after hypothermia without infection (p<.001) . Macrophage inflammatory protein-2 was comparable in the treatment groups . Interleukin-6 concentrations were 106 +/- 24 pg/mL after preseptic hypothermia and 276 +/- 76 pg/mL with preseptic normothermia (p<.05) . Postinfection granulocyte count was 1.7 x 10(9)/L after hypothermia and 2.4 x 10(9)/L after normothermia (p =.2) . After infection, muscle oxygen partial pressure was 47 +/- 10 mm Hg with preseptic hypothermia, 85 +/- 12 mm Hg in preseptic normothermia, and 49 +/- 9 mm Hg after hypothermia without infection (p =.7) . CONCLUSIONS: In this rat model of intra-abdominal sepsis, mild preseptic hypothermia (32 degrees C) reduced survival, impaired granulocyte recruitment, and changed cytokine balance, suggesting immunosuppression. J Biol Chem, 2004 Nov 12, 279(46), 47564 - 71 Epub 2004 Sep 01. Coupling DNA supercoiling to transcription in defined protein systems; Leng F et al.; Transcription of closed circular DNA templates in the presence of DNA gyrase is known to stimulate negative DNA supercoiling both in vivo and in vitro . It has proven elusive, however, to establish a general system in vitro that supports transcription-coupled DNA supercoiling (TCDS) by the "twin-domain" mechanism (Liu, L . F . and Wang, J . C . (1987) Proc . Natl . Acad . Sci . USA 84, 7024-7027) that operates in bacteria . In this report, we examine the properties of TCDS in defined protein systems that minimally contained T7 RNA polymerase and DNA gyrase . Specifically designed plasmid DNA templates permitted us to control the location and length of RNA transcripts . We demonstrate that TCDS takes place by two separate, and apparently independent, mechanistic pathways in vitro . The first supercoiling pathway, which is not likely to be significant in vivo, was found to be dependent on R-loop formation and could be suppressed by the presence of RNase H or bacterial HU protein . The second pathway for TCDS was much more potent, but became predominant in vitro only when sequence-specific DNA-bending proteins were present during transcription, and RNA transcript lengths exceeded 3 kb . This major supercoiling route was shown to be resistant to RNase H and had functional properties consistent with those predicted for the twin-domain mechanism . For example, DNA supercoiling activity was proportional to RNA transcript length and was greatly stimulated by macromolecular crowding agents . Under optimal conditions, the twin domain pathway of TCDS rapidly and efficiently generated superhelicity levels more than twice that typically found in vivo. Genes Dev, 2004 Sep 1, 18(17), 2086 - 94 Regulatory circuit design and evolution using phage lambda; Atsumi S et al.; Bistable gene regulatory circuits can adopt more than one stable epigenetic state . To understand how natural circuits have this and other systems properties, several groups have designed regulatory circuits de novo . Here we describe an alternative approach . We have modified an existing bistable circuit, that of phage lambda . With this approach, we used powerful genetic selections to identify functional circuits and selected for variants with altered behavior . The lambda circuit involves two antagonistic repressors, CI and Cro . We replaced lambda Cro with a module that included Lac repressor and several lac operators . Using a combinatorial approach, we isolated variants with different types of regulatory behavior . Several resembled wild-type lambda--they could grow lytically, could form highly stable lysogens, and carried out prophage induction . Another variant could form stable lysogens in the presence of a ligand for Lac repressor but switched to the lytic state when the ligand was removed . Several isolates evolved toward a desired behavior under selective pressure . These results strongly support the idea that complex circuits can arise during the course of evolution by a combination of simpler regulatory modules . They also underscore the advantages of modifying a natural circuit as an approach to understanding circuit design, systems behavior, and circuit evolution. J Mol Biol, 2004 Sep 17, 342(3), 861 - 75 Structure and function of a regulated archaeal triosephosphate isomerase adapted to high temperature; Walden H et al.; Triosephophate isomerase (TIM) is a dimeric enzyme in eucarya, bacteria and mesophilic archaea . In hyperthermophilic archaea, however, TIM exists as a tetramer composed of monomers that are about 10% shorter than other eucaryal and bacterial TIM monomers . We report here the crystal structure of TIM from Thermoproteus tenax, a hyperthermophilic archaeon that has an optimum growth temperature of 86 degrees C . The structure was determined from both a hexagonal and an orthorhombic crystal form to resolutions of 2.5A and 2.3A, and refined to R-factors of 19.7% and 21.5%, respectively . In both crystal forms, T.tenax TIM exists as a tetramer of the familiar (betaalpha)(8)-barrel . In solution, however, and unlike other hyperthermophilic TIMs, the T.tenax enzyme exhibits an equilibrium between inactive dimers and active tetramers, which is shifted to the tetramer state through a specific interaction with glycerol-1-phosphate dehydrogenase of T.tenax . This observation is interpreted in physiological terms as a need to reduce the build-up of thermolabile metabolic intermediates that would be susceptible to destruction by heat . A detailed structural comparison with TIMs from organisms with growth optima ranging from 15 degrees C to 100 degrees C emphasizes the importance in hyperthermophilic proteins of the specific location of ionic interactions for thermal stability rather than their numbers, and shows a clear correlation between the reduction of heat-labile, surface-exposed Asn and Gln residues with thermoadaptation . The comparison confirms the increase in charged surface-exposed residues at the expense of polar residues. Structure (Camb), 2004 Sep, 12(9), 1729 - 40 Structure of superoxide reductase bound to ferrocyanide and active site expansion upon X-ray-induced photo-reduction; Adam V et al.; Some sulfate-reducing and microaerophilic bacteria rely on the enzyme superoxide reductase (SOR) to eliminate the toxic superoxide anion radical (O2*-) . SOR catalyses the one-electron reduction of O2*- to hydrogen peroxide at a nonheme ferrous iron center . The structures of Desulfoarculus baarsii SOR (mutant E47A) alone and in complex with ferrocyanide were solved to 1.15 and 1.7 A resolution, respectively . The latter structure, the first ever reported of a complex between ferrocyanide and a protein, reveals that this organo-metallic compound entirely plugs the SOR active site, coordinating the active iron through a bent cyano bridge . The subtle structural differences between the mixed-valence and the fully reduced SOR-ferrocyanide adducts were investigated by taking advantage of the photoelectrons induced by X-rays . The results reveal that photo-reduction from Fe(III) to Fe(II) of the iron center, a very rapid process under a powerful synchrotron beam, induces an expansion of the SOR active site. Structure (Camb), 2004 Sep, 12(9), 1595 - 605 The crystal and solution structure of a putative transcriptional antiterminator from Mycobacterium tuberculosis; Morth JP et al.; We describe the crystal structure of Rv1626 from Mycobacterium tuberculosis at 1.48 A resolution and the corresponding solution structure determined from small angle X-ray scattering . The N-terminal domain shows structural homology to the receiver domains found in bacterial two-component systems . The C-terminal domain has high structural homology to a recently discovered RNA binding domain involved in transcriptional antitermination . The molecule in solution was found to be monomeric as it is in the crystal, but in solution it undergoes a conformational change that is triggered by changes in ionic strength . This is the first structure that links the phosphorylation cascade of the two-component systems with the antitermination event in the transcriptional machinery . Rv1626 belongs to a family of proteins, which we propose calling phosphorylation-dependent transcriptional antitermination regulators, so far only found in bacteria, and includes NasT, a protein from the assimilatory nitrate/nitrite reductase operon of Azetobacter vinelandii. Mol Microbiol, 2004 Sep, 53(6), 1709 - 19 Newly secreted adenylate cyclase toxin is responsible for intoxication of target cells by Bordetella pertussis; Gray MC et al.; Adenylate cyclase (AC) toxin is present on the surface of Bordetella pertussis organisms and their addition to eukaryotic cells results in increases in intracellular cAMP . To test the hypothesis that surface-bound toxin is the source for intoxication of cells when incubated with B . pertussis, we characterized the requirements of intoxication from intact bacteria and found that this process is calcium-dependent and blocked by monoclonal antibody to AC toxin or antibody against CD11b, a surface glycoprotein receptor for the toxin . Increases in intracellular cAMP correlate with the number of adherent bacteria, not the total number present in the medium, suggesting that interaction of bacteria with target cells is important for efficient delivery of AC toxin . A filamentous haemagglutinin-deficient mutant (BP353) and a clinical isolate (GMT1), both of which have a marked reduction in AC toxin on their surface, and wild-type B . pertussis (BP338) from which surface AC toxin has been removed by trypsin, were fully competent for intoxicating target cells, demonstrating that surface-bound AC toxin is not responsible for intoxication . B . pertussis killed by gentamicin or gamma irradiation were unable to intoxicate, illustrating that toxin delivery requires viable bacteria . Furthermore, CCCP, a protonophore that disrupts the proton gradient necessary for the secretion of related RTX toxins, blocked intoxication by whole bacteria . These data establish that delivery of this toxin by intact B . pertussis is not dependent on the surface-associated AC toxin, but requires close association of live bacteria with target cells and the active secretion of AC toxin. Mol Microbiol, 2004 Sep, 53(6), 1559 - 62 Traffic spotting: poles apart; Pugsley AP et al.; Finding out where specific functions are carried out within a bacterial cell has now become technically feasible . Here we consider recent experiments aimed at determining where bacteria translocate proteins across the cytoplasmic membrane using the Sec machinery. Evolution Int J Org Evolution, 2004 Jul, 58(7), 1511 - 20 Parasites and sexual reproduction in psychid moths; Kumpulainen T et al.; Persistence of sexual reproduction among coexisting asexual competitors has been a major paradox in evolutionary biology . The number of empirical studies is still very limited, as few systems with coexisting sexual and strictly asexual lineages have been found . We studied the ecological mechanisms behind the simultaneous coexistence of a sexually and an asexually reproducing closely related species of psychid moth in Central Finland between 1999 and 2001 . The two species compete for the same resources and are often infected by the same hymenopteran parasitoids . They are extremely morphologically and behaviorally similar and can be separated only by their reproductive strategy (sexual vs . asexual) or by genetic markers . We compared the life-history traits of these species in two locations where they coexist to test predictions of the cost-of-sex hypothesis . We did not find any difference in female size, number of larvae, or offspring survival between the sexuals and asexuals, indicating that sexuals are subject to cost of sex . We also used genetic markers to check and exclude the possibility of Wolbachia bacteria infection inducing parthenogenesis . None of the samples was infected by Wolbachia and, thus, it is unlikely that these bacteria could affect our results . We sampled 38 locations to study the prevalence of parasitoids and the moths' reproductive strategy . We found a strong positive correlation between prevalence of sexual reproduction and prevalence of parasitoids . In locations where parasitoids are rare asexuals exist in high densities, whereas in locations with a high parasitoid load the sexual species was dominant . Spatial distribution alone does not explain the results . We suggest that the parasite hypothesis for sex may offer an explanation for the persistence of sexual moths in this system. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2003 Sep, 17(3), 251 - 3 {Investigation of an outbreak of acute diarrhea caused by human calicivirus}; Li WZ et al.; BACKGROUND: To survey a diarrhea outbreak in Guangan city and analyze the cause of the disease . METHODS: The population enrolled in the surveillance came from four different settings and was randomly sampled . Stool specimens collected from diarrhea patients were tested ordinarily for enteric bacteria and further examined for viral pathogens with PAGE, ELISA and RT-PCR . RESULTS: In total, 4,567 persons were surveyed, among them 942 had acute diarrhea (prevalence 20.63%) . The incidence was higher in rural area (28.6%) than in urban area (19.6%) (chi-square =22.29, P less than 0.005) with a peak in May 10 through 25 four human caliciviruses were detected from stool specimens by ELISA and RT-PCR in specimens from 4 and 1 patients, respectively . CONCLUSION: Human calicivirus probably was the cause of this diarrhea outbreak in Guangan city. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2003 Sep, 17(3), 225 - 8 {Characterization of internal genes of two strains of influenza A (H9N2) virus isolated from men}; Guo YJ et al.; BACKGROUND: To understand the characteristics of internal genes of two strains of influenza A(H9N2) virus isolated from men and on the basis of these to reveal the origin of these two strains of influenza A(H9N2)virus . METHODS: The target gene was amplified by RT-PCR,the PCR product was ligated with P GEM-T Vector (Promega Company, USA) at 4 degrees, the recombined plasmid was transferred into dH5a bacteria, and the positive colonies were selected and identified with restriction enzyme . Afterwards, they were sent to Liu He Tong Company in Beijing for nucleotide sequencing . Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (version 1.03)and Editseq (Version 3.69) softwares . RESULTS: Internal genes of the two strains of H9N2 virus were G9 lineage . There was a slight difference in nucleotide sequence in PA gene between the two strains, whereas another five gene segments were identical to each other . CONCLUSION: The genomes of the two strains of influenza A(H9N2)virus were G9 lineage.They were transmitted to men separately from different avian sources with different characteristics of gene of influenza A(H9N2) virus, respectively. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2003 Dec, 17(4), 315 - 8 {Origin of hemagglutinin and neuraminidase gene of swine influenza A H1N1 viruses}; Guo YJ et al.; OBJECTIVE: To understand the origin of hemagglutinin (HA), and neuraminidase (NA) gene of swine influenza A (H1N1) viruses isolated in pigs in mainland China in 2002 and reveal the reason of pathogenesis of them in pigs . METHODS: The target gene amplified by PCR,PCR product was linked with PGEM-T Easy Vector(Promega company, USA) at 4? degrees C, the recombined plasmid was transferred into DH 10B bacteria, positive colonies were selected and identified them with restriction enzyme . Afterwards, they were sent to Liu He Tong company in Beijing for testing nucleotide sequence . Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (Version 1.03)and Editseq (Version 3.69) softwares . RESULTS: The HA and NA genes of three strains of swine influenza A (H1N1) viruses isolated from pigs in China were closely related to those of swine influenza A (H1N1) virus, but different from those of avian or human influenza A (H1N1) virus . The swine strain of influenza A (H1N1) virus isolated in 2002 was derived from swine influenza A (H1N1) virus circulated in pigs in China in 1991 . Since the antigenic drifts of HA and NA proteins of the new isolates occurred, their activity in pigs is increasing and they can cause disease in pigs . CONCLUSION: The HA and NA genes of three strains of influenza A (H1N1) virus tested were identified to be derived from those of swine influenza A (H1N1) virus . The increased activity and pathogenesis of them in pigs were most likely due to antigenic drifts of HA and NA proteins of the new isolates. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2004 Mar, 18(1), 35 - 8 {Distribution and efficiency of recombinant adenovirus mediated hVEGF165 gene transfer in bone marrow transplanted mice}; Zhong ZD et al.; OBJECTIVE: To investigate the rapid construction of enhanced green fluorescent protein (EGFP) labeled recombinant adenovirus containing hVEGF165 and its distribution and efficiency in bone marrow transplanted mice . METHODS: The recombinant adenovirus Ad-EGFP/hVEGF165 was rapidly constructed by using AdEasy system based on the homologous recombination in bacteria, and its property was studied in vitro . Then 3x10(8) PFU adenovirus was injected into BALB/c mice via the tail vein accepted syngeneic bone marrow transplantation . The in vivo distribution of adenovirus and plasma levels of VEGF were measured at different phases . RESULTS: The adenovirus Ad-EGFP/hVEGF165 was quickly constructed by homologous recombination in bacteria using AdEasy system . The purified particles were homogenous hexagon with titers between 10(10) PFU/ml and 10(11) PFU/ml . The Hela cells infected with Ad-EGFP/hVEGF165 did not show cytopathic effects after several times passages . Under the fluorescent microscope, EGFP was revealed in the heart, lung, liver, spleen, kidney and intestine of mice at different phases . RT-PCR and immunohistochemistry showed hVEGF165 expressed significantly . No obvious damages were observed in different organs by HE staining . The plasma level of hVEGF165 was up to 866.67+/-97.13 pg/ml . CONCLUSION: These results suggested that the construction of adenovirus vector by homologous recombination in bacteria was an efficient and time-saving method, and high titer adenovirus could successfully mediate the safe and stable expression of hVEGF165 in post bone marrow transplanted mice . All these would make further gene therapy in bone marrow transplantation possible. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2004 Mar, 18(1), 7 - 11 {Characterization of HA and NA genes of swine influenza A (H9N2) viruses}; Guo YJ et al.; OBJECTIVE: To understand the origin of HA and NA genes of swine influenza A (H9N2) viruses isolated from pigs in the mainland of China and on basis of these to reveal the pathogenecity of them in pigs . METHODS: The target gene was amplified by PCR, the PCR product was ligated with PGEM-T Easy Vector (Promega company, USA) at 4 degrees, the recombined plasmid was transferred into DH-10-beta bacteria; positive colonies were selected and identified then digested with restriction enzyme . Afterwards,the nucleotide sequence was determined . Finally,phylogenetic analysis of the sequencing data was performed with MegAlign (Version 1.03) and Editseg (Version 3.69) softwares . RESULTS: Two strains of swine influenza A(H9N2) virus isolated in the mainland had an amino acid residue, leucine (L) at position 226 (H3 numbering) on HA protein molecule found in H9N2 viruses isolated either in pigs or humans previously; the amino acid sequence at HA connecting peptide of isolates possessed R-L-S-R, whereas the other H9N2 viruses with virulence in poultry had R-S-S-R at HA connecting peptide . The two pig H9N2 isolates shared the same three-amino-acids deletion in the NA stalk at 62.64 position found in A/Shaoguan/408/98 and A/Swine/Hong Kong/9/98, as well as A/Duck/Hong Kong /Y280/97(H9N2) viruses . The analysis of the phylogenetic tree indicated that the HA and NA genes of new isolates were closely related to those of A/Chicken/Hong Kong/G23/97 and A/Chickon/Hong Kong/G9/97 and A/Shaoguan/408/98 viruses, respectively . CONCLUSION: The HA and NA genes of swine influenza A(H9N2) viruses isolated in the mainland of China probably were derived from those of avian influenza A(H9N2) virus . The occurrence of substitution of amino acid sequence at HA connecting peptide, could result in the H9N2 virus from non pathogenic to pathogenic in pigs . However, avian influenza A(H9N2) virus had deletion in the stalk of the NA that resulted in host range transmission.Therefore they could infect pigs directly. PLoS Biol . 2004 Sep;2(9):E257 . Epub 2004 Aug 31. Monomethyl branched-chain fatty acids play an essential role in Caenorhabditis elegans development; Kniazeva M et al.; Monomethyl branched-chain fatty acids (mmBCFAs) are commonly found in many organisms from bacteria to mammals . In humans, they have been detected in skin, brain, blood, and cancer cells . Despite a broad distribution, mmBCFAs remain exotic in eukaryotes, where their origin and physiological roles are not understood . Here we report our study of the function and regulation of mmBCFAs in Caenorhabditis elegans, combining genetics, gas chromatography, and DNA microarray analysis . We show that C . elegans synthesizes mmBCFAs de novo and utilizes the long-chain fatty acid elongation enzymes ELO-5 and ELO-6 to produce two mmBCFAs, C15ISO and C17ISO . These mmBCFAs are essential for C . elegans growth and development, as suppression of their biosynthesis results in a growth arrest at the first larval stage . The arrest is reversible and can be overcome by feeding the arrested animals with mmBCFA supplements . We show not only that the levels of C15ISO and C17ISO affect the expression of several genes, but also that the activities of some of these genes affect biosynthesis of mmBCFAs, suggesting a potential feedback regulation . One of the genes, lpd-1, encodes a homolog of a mammalian sterol regulatory element-binding protein (SREBP 1c) . We present results suggesting that elo-5 and elo-6 may be transcriptional targets of LPD-1 . This study exposes unexpected and crucial physiological functions of C15ISO and C17ISO in C . elegans and suggests a potentially important role for mmBCFAs in other eukaryotes. Nat Biotechnol, 2004 Sep, 22(9), 1110 - 4 Monitoring and modeling horizontal gene transfer; Nielsen KM et al.; Monitoring efforts have failed to identify horizontal gene transfer (HGT) events occurring from transgenic plants into bacterial communities in soil or intestinal environments . The lack of such observations is frequently cited in biosafety literature and by regulatory risk assessment . Our analysis of the sensitivity of current monitoring efforts shows that studies to date have examined potential HGT events occurring in less than 2 g of sample material, when combined . Moreover, a population genetic model predicts that rare bacterial transformants acquiring transgenes require years of growth to out-compete wild-type bacteria . Time of sampling is there-fore crucial to the useful implementation of monitoring . A population genetic approach is advocated for elucidating the necessary sample sizes and times of sampling for monitoring HGT into large bacterial populations . Major changes in current monitoring approaches are needed, including explicit consideration of the population size of exposed bacteria, the bacterial generation time, the strength of selection acting on the transgene-carrying bacteria, and the sample size necessary to verify or falsify the HGT hypotheses tested. Proc Natl Acad Sci U S A, 2004 Sep 14, 101(37), 13642 - 7 Epub 2004 Aug 31. Isolation of Mycobacterium tuberculosis mutants defective in the arrest of phagosome maturation; Pethe K et al.; Mycobacterium tuberculosis resides within the phagocytes of its host . It ensures its continued survival through arresting the normal maturation of its phagosome, which is retained within the early endosomal system of the macrophage . Although individual bacterial components have been shown to modulate phagosome biogenesis, the mechanism(s) active in live, intact bacteria remain elusive . We have developed a genetic screen that facilitates the isolation of mutants defective in arresting the maturation of their phagosomes . Macrophages were incubated with iron-dextran that was chased into lysosomes . The cells were subsequently infected with M . tuberculosis from a library of transposon-mutagenized bacteria . After four rounds of enrichment, the majority of mutants isolated were unable to prevent acidification of their phagosomes and were attenuated for intracellular survival . The genes affected range in function from those with no known homologues to putative transporters and lipid synthesis enzymes . Further characterization of these bacteria is needed . In addition to clarifying the processes active in modulation of phagosome biogenesis by M . tuberculosis, this screen may be applicable to other pathogens that restrict the maturation of their phagosome. Zhong Xi Yi Jie He Xue Bao, 2004 Jan, 2(1), 42 - 5 {Effect of recombinant adenoviruses with CD/TK fusion suicide gene on human hepatocellular carcinoma cells}; Wang CJ et al.; OBJECTIVE: To investigate gene-therapy for human hepatocellular carcinoma with adenovirus vectors by double suicide gene CD/TK . METHODS: Double suicide gene CD/TK was liberated from eukaryotic vectors pCEA-CD/TK and subcloned into shuttle vectors, and the transfer plasmid pAdtrack-CMV-CD/TK was formed after linearizing with Pac 1 . It was recombinated with pAdeasy-1 in bacteria BJ5183 . The identified adenovirus plasmid was digested by Pac1 and was transfected into 293 cells to pack the adenoviruses . After PCR determination, its titre was measured, and the infection rate and efficacy were tested in human hepatocellular carcinoma cells . RESULTS: pAdtrack-CMV-CD/TK and pAd-CD/TK were tested by endonuclease digestion . Ad-CD/TK was produced in 293 cells, and the human hepatocellular carcinoma cells (SMMC7721) infected by Ad-CD/TK were killed after 5-FC was used, and bystander effects were observed . CONCLUSION: Recombinant adenoviruses with CD/TK fusion suicide gene have a high infection rate and efficacy for human hepatocellular carcinoma cells. Photochem Photobiol, 2004 Jul-Aug, 80, 78 - 83 Effects of UV-B irradiation on a marine microecosystem; Marangoni R et al.; Purpose of this work was to study the effect of UV irradiation on a microecosystem consisting of several interacting species . The system chosen was of a hypersaline type, where all the species present live at high salt concentration; it comprises different bacteria; a producer, the photosynthetic green alga Dunaliella salina; and a consumer, the ciliated protozoan Fabrea salina, which form a complete food chain . We were able to establish the initial conditions that give rise to a self-sustaining microecosystem, stable for at least 3 weeks . We then determined the effect of UV irradiation on this microecosystem under laboratory-controlled conditions, in particular by measuring the critical UV exposure for the two main components of the microecosystem (algae and protozoa) under UV-B irradiances comparable to those of solar irradiation . In our experiments, we varied irradiance, total dose and spectral composition of the actinic light . The critical doses at irradiances of the order of 56 kJ/m(2) (typical average daily irradiance in a sunny summer day in Pisa), measured for each main component of the microecosystem (algae and ciliates), turned out to be around 70 kJ/m(2) for ciliates and 50 kJ/m(2) for D . salina . By exposing microecosystems to daily UV-B irradiances of the order of 8 kJ/m(2) (typical average daily irradiance in a sunny winter day in Pisa), we found no effect at total doses of the order of the critical doses at high irradiances, showing that the reciprocity law does not hold . We have also measured a preliminary spectral-sensitive curve of the UV effects, which shows an exponential decay with wavelength. Chromosoma, 2004 Sep, 113(3), 103 - 12 Epub 2004 Aug 03. The mammalian circadian timing system: from gene expression to physiology; Gachon F et al.; Many physiological processes in organisms from bacteria to man are rhythmic, and some of these are controlled by self-sustained oscillators that persist in the absence of external time cues . Circadian clocks are perhaps the best characterized biological oscillators and they exist in virtually all light-sensitive organisms . In mammals, they influence nearly all aspects of physiology and behavior, including sleep-wake cycles, cardiovascular activity, endocrinology, body temperature, renal activity, physiology of the gastro-intestinal tract, and hepatic metabolism . The master pacemaker is located in the suprachiasmatic nuclei, two small groups of neurons in the ventral part of the hypothalamus . However, most peripheral body cells contain self-sustained circadian oscillators with a molecular makeup similar to that of SCN (suprachiasmatic nucleus) neurons . This organization implies that the SCN must synchronize countless subsidiary oscillators in peripheral tissues, in order to coordinate cyclic physiology . In this review, we will discuss some recent studies on the structure and putative functions of the mammalian circadian timing system, but we will also point out some apparent inconsistencies in the currently publicized model for rhythm generation. J Steroid Biochem Mol Biol, 2004 Aug, 91(4-5), 191 - 6 Functional analyses of an LXXLL motif in nuclear receptor corepressor (N-CoR); Loinder K et al.; Transcriptional repression is a major regulatory mechanism in cell differentiation, organogenesis, and oncogenesis . Two repressors of ligand-dependent transcription factors, nuclear receptor corepressor (N-CoR) and the related protein SMRT were identified as a silencing mediator for thyroid hormone receptor beta and as a silencing mediator for retinoic acid and thyroid hormone receptors, respectively . Nuclear receptor coactivators such as steroid receptor coactivator-1 (SRC-1) contain multiple LXXLL motifs, which are essential and sufficient for its ligand-dependent interaction with nuclear receptors . N-CoR also has an LXXLL motif, located between repressor domains 1 and 2, and conserved between mouse and man . In contrast, SMRT lacks this motif . This paper describes functional implications of the LXXLL motif in N-CoR . A 57-amino acid portion of N-CoR containing the LDNLL sequence (N-CoR(LDNLL)) fused to GST interacted with retinoic acid receptor alpha (RARalpha) and thyroid hormone receptor beta (TRbeta) in vitro . Similarly, {(35)S-methionine}N-CoR(LDNLL) interacted with a RARalpha fusion protein . N-CoR(LDNLL) also bound to RARalpha in vivo as determined in mammalian one-hybrid system in transfected CV-1 cells and by two-hybrid assays in bacteria . The interaction with RARalpha in vitro and in vivo was specific as determined by mutation of the sequence LDNLL to LDNAA . Our data suggest that the LDNLL motif in N-CoR has functional significance because it mediates interaction with nuclear receptors such as RARalpha and TRbeta. DNA Repair (Amst), 2004 Oct 5, 3(10), 1323 - 34 Attempted base excision repair of ionizing radiation damage in human lymphoblastoid cells produces lethal and mutagenic double strand breaks; Yang N et al.; A significant proportion of cellular DNA damages induced by ionizing radiation are produced in clusters, also called multiply damaged sites . It has been demonstrated by in vitro studies and in bacteria that clustered damage sites can be converted to lethal double strand breaks by oxidative DNA glycosylases during attempted base excision repair . To determine whether DNA glycosylases could produce double strand breaks at radiation-induced clustered damages in human cells, stably transformed human lymphoblastoid TK6 cells that inducibly overexpress the oxidative DNA glycosylases/AP lyases, hNTH1 and hOGG1, were assessed for their radiation responses, including survival, mutation induction and the enzymatic production of double strand breaks post-irradiation . We found that additional double strand breaks were generated during post-irradiation incubation in uninduced TK6 control cells . Moreover, overproduction of either DNA glycosylase resulted in significantly increased double strand break formation, which correlated with an elevated sensitivity to the cytotoxic and mutagenic effects of ionizing radiation . These data show that attempted repair of radiation damage, presumably at clustered damage sites, by the oxidative DNA glycosylases can lead to the formation of potentially lethal and mutagenic double strand breaks in human cells. Biochem Biophys Res Commun, 2004 Sep 24, 322(3), 766 - 71 The solubility and stability of recombinant proteins are increased by their fusion to NusA; De Marco V et al.; The new bacterial vector pETM60 enables the expression of His-tagged recombinant proteins fused to the C-terminus of NusA through a TEV protease recognition sequence . Three sequences coding for two protein domains (Xklp3A and Tep3Ag) and one membrane-bound viral protein (E8R) could not be expressed in a soluble form in bacteria . Their GST-fusions were mostly soluble but quickly degraded during purification . The same sequences cloned in pETM60 were efficiently purified by metal affinity and recovered soluble after the removal of the fusion partner . The NusA-fused constructs enabled to yield 13-20mg of fusion protein per litre of culture and 2.5-5mg of pure protein per litre of culture . Structural analysis indicated that the purified proteins were monodispersed and correctly folded . NusA has been used to raise antibodies that have been successfully used for Western blot and immunoprecipitation of NusA fusion proteins. Pathol Biol (Paris), 2004 Sep, 52(7), 407 - 14 {Macrophage activation syndrome, hemophagocytic syndrome}; Pradalier A et al.; Macrophage activation syndrome MAS describes the clinical, biological and histological symptoms related to a probably T lymphocytes/NK cell driven stimulation of macrophages with the consequence of a hemophagocytosis involving numerous organs, preferentially bone marrow, explaining the other term of "hemophagocytic syndrome" . Clinical symptoms include cytopenia, multiple organ dysfunction, fever unresponsive to antibiotics, fatigue and rash . Infections (bacteria, virus or parasites), lymphoproliferative disorders, cancers, systemic diseases are the most prevalent triggers or etiologies of M.A.S . Evidence of haemaphagocytosis is obtained in the majority of cases with bone marrow specimens . In some cases haemophagocytosis can spare the bone marrow with involvement confined to other tissues such as liver and spleen . Very high levels of ferritine seem to correlate well with the presence of haemophagocytosis and is a possible marker for an early diagnosis . Early treatment initiation is mandatory . Corticosteroids, cytostatic drugs such as etoposide, cyclosporine A, plasmapherese, intravenous immunoglobulins and anti TNFalpha are proposed but no randomized trials were published. FEMS Microbiol Lett, 2004 Sep 1, 238(1), 57 - 63 Identification of a promoter motif regulating the major DNA damage response mechanism of Mycobacterium tuberculosis; Gamulin V et al.; The principal response of many bacteria to DNA damage is mediated by a mechanism dependent on the LexA and RecA proteins . However, Mycobacterium tuberculosis was recently reported to regulate a majority of DNA repair genes independently of RecA and LexA, suggesting that an unknown RecA/LexA-independent mechanism controls the major DNA damage response pathway in this organism . Here we have identified a motif tTGTCRgtg-8nt-TAnnnT that defines a novel RecA/LexA-independent promoter (RecA-NDp) of M . tuberculosis . Furthermore, we show that the RecA-NDp type of promoter precedes DNA repair genes in other Actinomycetales. J Clin Forensic Med, 1996 Dec, 3(4), 157 - 60 Is the nasopharynx warmer in children than in adults? Molony NC, Kerr AI, Blackwell CC, Busuttil A. Recent studies on the aetiology of the Sudden Infant Death Syndrome (SIDS) have suggested that some of these deaths are the consequence of an overwhelming inflammatory response to the production of pyrogenic toxins from bacteria colonizing the upper respiratory tract, particularly the nasopharynx . The pyrogenic toxins of Staphlococcus aureus, one of the likelier bacterial candidates, are only produced in temperatures of over 37 degrees C . This study examined nasopharyngeal temperatures in children . It is a preliminary study to develop an accurate means to measure how close to 37 degrees C the nasopharyngeal temperature lies in infants at the age when SIDS deaths occur . Following a pilot study and power calculation, measurements of nasopharyngeal temperature were made on 30 apyrexial children aged 4-10 years and 30 adults with no nasal pathology, undergoing surgery under general anaesthesia, using an accurately sited thermocouple probe . The mean temperature in children (35.64 degrees C) was significantly higher than in adults (34.01 degrees C) . Comparable measurements attempted with the same subjects awake gave similar results. Br J Nutr, 2004 Aug, 92(2), 247 - 55 Reduction of allergic airway eosinophilia by dietary raffinose in Brown Norway rats; Watanabe H et al.; Oral administration of raffinose, a naturally occurring indigestible oligosaccharide, has reportedly ameliorated atopic dermatitis in human subjects although the mechanism is unknown . The present study investigated the effect of dietary raffinose on allergen-induced airway eosinophilia in ovalbumin-sensitised Brown Norway rats as an atopic disease model . Brown Norway rats were immunised by subcutaneous injection with ovalbumin on day 0 and fed either a control diet or the diet supplemented with raffinose (50 g/kg diet) . The rats were exposed to aerosolised ovalbumin on day 20, and broncho-alveolar lavage fluid was obtained on the next day . The number of eosinophils in the fluid was significantly lower in the rats fed the raffinose diet than in those fed the control diet . Dietary raffinose significantly reduced IL-4 and IL-5 mRNA levels in lung tissue and tended to lower ovalbumin-specific Ig E levels . Suppression of eosinophilia by dietary raffinose was still observed in caecectomised and neomycin-administered rats, suggesting little contribution by the colonic bacteria to the effect of raffinose . Intraperitoneal administration of raffinose also suppressed eosinophilia . Significant concentrations of raffinose were detected in portal venous and abdominal arterial plasma after the intragastric administration of raffinose . Overall, the findings suggest that dietary raffinose ameliorates allergic airway eosinophilia at least partly via post-absorptive mechanisms in Brown Norway rats. J Environ Sci Health A Tox Hazard Subst Environ Eng, 2004, 39(8), 2195 - 204 Anaerobic fluidized bed reactor for the treatment of landfill leachates; Gulsen H et al.; Treatability of the sanitary young landfill leachate in a pilot-scale anaerobic fluidized bed reactor during reactor startup and steady-state phases was investigated . All runs were carried out at 35 degrees C due to environmental conditions in the mesophilic anaerobic fluidized bed reactor (AFBR) while organic loading rate (OLR) was increased from 2.5 to 37gCOD/L-day during the 220 days of operation . The AFBR process attained steady-state conditions about on day 80 and a good and stable COD removal were achieved at about 90% . Biogas production in the bed continuously increased during the process . The mean specific biogas production was found 0.52 L biogas/gCODrem while the methane content was about 75% . The attached biomass concentration, measured as volatile solids rapidly increased as containing about 90% of the total biomass concentration . Furthermore, an increase in the suspended solid concentration was found as an evidence of biomass detachment from the media. Eur Respir J, 2004 Aug, 24(2), 247 - 50 Triggering receptor expressed on myeloid cells: role in the diagnosis of lung infections; Richeldi L et al.; The triggering receptor expressed on myeloid cells (TREM)-1 is a recently described molecule, which plays an important role in myeloid cell-activated inflammatory responses . TREM-1 is expressed on blood neutrophils and monocytes, and also on alveolar macrophages, thus suggesting a potential role in lung inflammatory responses against infections . To investigate the differential expression of TREM-1 in lung infections, its levels were assessed in bronchoalveolar lavage specimens from patients with community-acquired pneumonia or tuberculosis . TREM-1 was also investigated in patients with interstitial lung diseases, as a model of noninfectious inflammatory disease of the lung . TREM-1 expression was significantly increased in lung neutrophils and in lung macrophages of patients with pneumonia (n=7; 387.9+/-61.4 and 660.5+/-18.3, respectively) compared with patients with pulmonary tuberculosis (n=7; 59.2+/-13.1 and 80.6+/-291.2) and patients with interstitial lung diseases (n=10; 91.8+/-23.3 and 123.9+/-22.8) . In contrast, TREM-1 expression on peripheral blood neutrophils was no different among the three groups . In conclusion, these data suggest that triggering receptor expressed on myeloid cells-1 is selectively expressed in the lungs of patients with pneumonia caused by extracellular bacteria and not in patients with tuberculosis, providing a potential marker for differential diagnosis. J Biomol Tech, 2004 Sep, 15(3), 208 - 12 Characterization of a noncovalent lipocalin complex by liquid chromatography/electrospray ionization mass spectrometry; Doneanu CE et al.; Nanoscale liquid chromatography coupled to electrospray ionization mass spectrometry was used to identify the nature of the ligand that binds noncovalently to siderocalin (lipocalin 2) . The folded state siderocalin-ligand complex was separated from free, unfolded siderocalin using reversed phase chromatography, and the molecular weight of the siderocalin ligand was then determined from the deconvoluted molecular weights of the complex and of the free protein . The ligand was identified as dihydroxybenzoyl-serine, a breakdown product of enterobactin, an iron-chelating compound ("siderophore") synthesized in bacteria . These results demonstrate that, in some cases, electrostatic noncovalent protein complexes can survive the denaturing conditions of reversed phase liquid chromatography and the gas phase transfer occurring during electrospray ionization . Nature, 2004 Aug 26, 430(7003), 1027 - 32 High rates of N2 fixation by unicellular diazotrophs in the oligotrophic Pacific Ocean; Montoya JP et al.; The availability of nitrogen is important in regulating biological productivity in marine environments . Deepwater nitrate has long been considered the major source of new nitrogen supporting primary production in oligotrophic regions of the open ocean, but recent studies have showed that biological N2 fixation has a critical role in supporting oceanic new production . Large colonial cyanobacteria in the genus Trichodesmium and the heterocystous endosymbiont Richelia have traditionally been considered the dominant marine N2 fixers, but unicellular diazotrophic cyanobacteria and bacterioplankton have recently been found in the picoplankton and nanoplankton community of the North Pacific central gyre, and a variety of molecular and isotopic evidence suggests that these unicells could make a major contribution to the oceanic N budget . Here we report rates of N2 fixation by these small, previously overlooked diazotrophs that, although spatially variable, can equal or exceed the rate of N2 fixation reported for larger, more obvious organisms . Direct measurements of 15N2 fixation by small diazotrophs in various parts of the Pacific Ocean, including the waters off Hawaii where the unicellular diazotrophs were first characterized, show that N2 fixation by unicellular diazotrophs can support a significant fraction of total new production in oligotrophic waters. Heredity, 2004 Dec, 93(6), 592 - 6 Cytogenetic mechanism and genetic consequences of thelytoky in the wasp Trichogramma cacoeciae; Vavre F et al.; In Hymenoptera, complete parthenogenesis, that is thelytoky, is a common phenomenon where virgin females produce only daughters . Thelytoky is often induced by bacteria of the genus Wolbachia, but can also be genetically determined by the insect itself, as in the genus Trichogramma where both forms exist . In order to compare these two forms of thelytoky, chromosome behaviour analysis in young eggs and genetic analysis of microsatellite markers were carried out in the wasp Trichogramma cacoeciae, where thelytoky is genetically determined . Microscopic studies revealed that during female gamete formation meiotic cells undergo only a single equational division followed by the expulsion of a single polar body . This absence of meiotic recombination and reduction corresponds well with the high levels of heterozygosity observed in females collected from the field and a nonsegregation pattern in the offspring of heterozygous females . We therefore concluded that diploidy in T . cacoeciae is maintained through an apomictic cloning mechanism and that the incidence of thelytoky under genetic control of the wasp differs entirely from the mechanism induced by Wolbachia infection, where thelytoky is restored through gamete duplication. Nucleic Acids Res . 2004 Aug 25;32(15):e121. Genomic representations using concatenates of Type IIB restriction endonuclease digestion fragments; Tengs T et al.; We have developed a method for genomic representation using Type IIB restriction endonucleases . Representation by concatenation of restriction digests, or RECORD, is an approach to sample the fragments generated by cleavage with these enzymes . Here, we show that the RECORD libraries may be used for digital karyotyping and for pathogen identification by computational subtraction. Indian J Dent Res, 2003 Oct-Dec, 14(4), 279 - 83 DNA probe analysis in smoker and non smoker rapidly progressive periodontitis patients--a pilot study; Vandana KL et al.; Smoking is one of the most significant risk factors in the development and further advancement of inflammatory periodontal disease . The bacteria A . actinomycetemcomitans, P . gingivalis and P . intermedius as indicated as the potential pathogens associated with periodontal disease . Since the bacteria mentioned as well as smoking are factors associated with periodontitis it is of importance to elucidate the interrelationship between these factors . The purpose of this study was to investigate the prevalence of A . actinomycetemcomitans, P . gingivalis and P . intermedius in subgingival plaque samples obtained form healthy and diseased sites of patients with rapidly progressive periodontitis who were smokers and non smokers along with other clinical parameters. J Mol Biol, 2004 Aug 20, 341(4), 999 - 1013 Structural and enzymatic properties of 1-aminocyclopropane-1-carboxylate deaminase homologue from Pyrococcus horikoshii; Fujino A et al.; 1-Aminocyclopropane-l-carboxylate deaminase (ACCD) is a pyridoxal 5/-phosphate dependent enzyme that shows deaminase activity toward ACC, a precursor of plant hormone ethylene . ACCD from some soil bacteria has been reported to be able to break the cyclopropane ring of ACC to yield a-ketobutyrate and ammonia . We reported the crystal structure of ACCD from the yeast Hansenula saturnus in the absence/presence of substrate ACC, and proposed its ingenious reaction mechanisms . In order to study the enzyme further, we overexpressed the ACCD homologue protein (phAHP) from the fully decoded hyperthermophilic archearon, Pyrococcus horikoshii OT3 . However, phAHP does not show ACCD activity at high temperature as well as at room temperature, though it has significant sequence similarity . Instead of ACCD activity, the GC-MS analysis and enzymatic method show that phAHP has deaminase activity toward L and D-serine . Here, we present the crystal structures of the native and ACC-complexed phAHP . The overall topology of the phAHP structure is very similar to that of ACCD; however, critical differences were observed around the active site . Here, the differences of enzymatic activity between phAHP and ACCD are discussed based on the structural differences of these two proteins . We suggest that the catalytic disagreement between these two enzymes comes from the difference of the residues near the pyridine ring of pyridoxal 5'-phosphate (PLP), not the difference of the catalytic residues themselves . We also propose a condition necessary in the primary sequence to have ACCD activity . Nucleic Acids Res, 2004 Aug 24, 32(15), 4563 - 75 Print 2004. Thioredoxin can influence gene expression by affecting gyrase activity; Li K et al.; The expression of many genes of facultatively photosynthetic bacteria of the genus Rhodobacter is controlled by the oxygen tension . Among these are the genes of the puf and puc operons, which encode proteins of the photosynthetic apparatus . Previous results revealed that thioredoxins are involved in the regulated expression of these operons, but it remained unsolved as to the mechanisms by which thioredoxins affect puf and puc expression . Here we show that reduced TrxA of Rhodobacter capsulatus and Rhodobacter sphaeroides and oxidized TrxC of R.capsulatus interact with DNA gyrase and alter its DNA supercoiling activity . While TrxA enhances supercoiling, TrxC exerts a negative effect on this activity . Furthermore, inhibition of gyrase activity strongly reduces puf and puc expression . Our results reveal a new signaling pathway by which oxygen can affect the expression of bacterial genes. J Biol Chem, 2004 Nov 5, 279(45), 47233 - 41 Epub 2004 Aug 24. The minimal transactivation domain of the basic motif-leucine zipper transcription factor NRL interacts with TATA-binding protein; Friedman JS et al.; The basic motif-leucine zipper (bZIP) transcription factor NRL controls the expression of rhodopsin and other phototransduction genes and is a key mediator of photoreceptor differentiation . To delineate the molecular mechanisms underlying transcriptional initiation of rod-specific genes, we characterized different regions of the NRL protein using yeast-based autoactivation assays . We identified 35 amino acid residues in the proline- and serine-rich N-terminal region (called minimal transactivation domain, MTD), which, when combined with LexA or Gal4 DNA binding domains, exhibited activation of target promoters . Because this domain is conserved in all proteins of the large Maf family, we hypothesized that NRL-MTD played an important role in assembling the transcription initiation complex . Our studies showed that the NRL protein, including the MTD, interacted with full-length or the C-terminal domain of TATA-binding protein (TBP) in vitro . NRL and TBP could be co-immunoprecipitated from bovine retinal nuclear extract . TBP was also part of c-Maf and MafA (two other large Maf proteins)-containing complex(es) in vivo . Our data suggest that the function of NRL-MTD is to activate transcription by recruiting or stabilizing TBP (and consequently other components of the general transcription complex) at the promoter of target genes, and a similar function may be attributed to other bZIP proteins of the large Maf family. J Dairy Sci, 2004 Aug, 87(8), 2571 - 7 Effects of zinc and sodium monensin on ruminal degradation of lysine-HCl and liquid 2-hydroxy-4-methylthiobutanoic acid; Bateman HG 2nd et al.; Four nonlactating, mature, Holstein cows were fitted with ruminal cannula and used in a 4 x 4 Latin square-designed experiment to evaluate the impact of supplemental Zn and monensin on ruminal degradation of Lys and liquid 2-hydroxy-4-methylthiobutanoic acid (HMB) . Cows were fed 4.54 kg (as fed) of alfalfa hay top-dressed with 4.54 kg (as fed) concentrate once daily . Concentrates were formulated to provide 0 or 500 mg/kg of Zn as ZnSO4 and 0 or 40 mg/kg of monensin in the total diet . Zinc supplementation provided approximately 22-fold greater dietary Zn than estimated by NRC requirements . On d 14 of each period, cows were dosed via the rumen cannula with 50 g of HMB and 100 g of Lys-HCl, and the concentrations of Lys and HMB were monitored every 0.5 h for 8 h . Supplemental Zn tended to decrease the proportion of acetate in ruminal fluid postfeeding and increased the proportion of propionate in ruminal fluid postfeeding . Supplemental Zn increased mean fluid passage rate from the rumen . Monensin decreased the proportion of acetate and increased the mean proportion of propionate in ruminal fluid, resulting in a decrease in the ratio of acetate to propionate . Monensin also increased the mean fluid passage rate from the rumen . Neither Zn nor monensin affected the apparent rate of ruminal disappearance of HMB or Lys . However, Zn and monensin interacted to alter the ruminal degradability of free Lys but not HMB . These data indicate that Zn and monensin may interact to alter ruminal degradability of free amino acids. Antimicrob Agents Chemother, 2004 Sep, 48(9), 3530 - 5 Terbinafine resistance mediated by salicylate 1-monooxygenase in Aspergillus nidulans; Graminha MA et al.; Resistance to antifungal agents is a recurring and growing problem among patients with systemic fungal infections . UV-induced Aspergillus nidulans mutants resistant to terbinafine have been identified, and we report here the characterization of one such gene . A sib-selected, 6.6-kb genomic DNA fragment encodes a salicylate 1-monooxygenase (salA), and a fatty acid synthase subunit (fasC) confers terbinafine resistance upon transformation of a sensitive strain . Subfragments carrying salA but not fasC confer terbinafine resistance . salA is present as a single-copy gene on chromosome VI and encodes a protein of 473 amino acids that is homologous to salicylate 1-monooxygenase, a well-characterized naphthalene-degrading enzyme in bacteria . salA transcript accumulation analysis showed terbinafine-dependent induction in the wild type and the UV-induced mutant Terb7, as well as overexpression in a strain containing the salA subgenomic DNA fragment, probably due to the multicopy effect caused by the transformation event . Additional naphthalene degradation enzyme-coding genes are present in fungal genomes, suggesting that resistance could follow degradation of the naphthalene ring contained in terbinafine. Huan Jing Ke Xue, 2004 May, 25(3), 44 - 7 {Influential factors on the toxicity of pentachlorophenol sodium with MICROTOX system in water}; Shi W et al.; The biological toxicity of pentachlorophenol sodium (Na-PCP), a typical kind of aquatic organic pollutants, was tested with the MICROTOX system using luminescent bacteria with the influence of several factors taken into consideration . The EC50 of Na-PCP increases with the increasing of the pH and hardness of the sample . The EC50 (15 min) of Na-PCP is approximately equal to its EC50 (20 min) . Compared with some common organic pollutants, which exhibit little influence on the toxicity of Na-PCP when mixed, Na-PCP is more toxic . A reduction of 14% in relative luminescent rate was observed in the experiment with a natural sample . The natural sample exhibits similar influence on the toxicity of Na-PCP similarly compared with unionized water. J Biol Chem, 2004 Oct 29, 279(44), 46082 - 95 Epub 2004 Aug 23. Influence of the unusual covalent adduct on the kinetics and formation of radical intermediates in synechocystis catalase peroxidase: a stopped-flow and EPR characterization of the MET275, TYR249, and ARG439 variants; Jakopitsch C et al.; Catalase-peroxidases (KatGs) are heme peroxidases with a catalatic activity comparable to monofunctional catalases . They contain an unusual covalent distal side adduct with the side chains of Trp(122), Tyr(249), and Met(275) (Synechocysis KatG numbering) . The known crystal structures suggest that Tyr(249) and Met(275) could be within hydrogen-bonding distance to Arg(439) . To investigate the role of this peculiar adduct, the variants Y249F, M275I, R439A, and R439N were investigated by electronic absorption, steady-state and transient-state kinetic techniques and EPR spectroscopy combined with deuterium labeling . Exchange of these conserved residues exhibited dramatic consequences on the bifunctional activity of this peroxidase . The turnover numbers of catalase activity of M275I, Y249F, R439A, and R439N are 0.6, 0.17, 4.9, and 3.14% of wild-type activity, respectively . By contrast, the peroxidase activity was unaffected or even enhanced, in particular for the M275I variant . As shown by mass spectrometry and EPR spectra, the KatG typical adduct is intact in both Arg(439) variants, as is the case of the wild-type enzyme, whereas in the M275I variant the covalent link exists only between Tyr(249) and Trp(122) . In the Y249F variant, the link is absent . EPR studies showed that the radical species formed upon reaction of the Y249F and R439A/N variants with peroxoacetic acid are the oxoferryl-porphyrin radical, the tryptophanyl and the tyrosyl radicals, as in the wild-type enzyme . The dramatic loss in catalase activity of the Y249F variant allowed the comparison of the radical species formed with hydrogen peroxide and peroxoacetic acid . The EPR data strongly suggest that the sequence of intermediates formed in the absence of a one electron donor substrate, is por(.-)(+) --> Trp(.-) (or Trp(.-)(+)) --> Tyr(.-) . The M275I variant did not form the Trp(.-) species because of the dramatic changes on the heme distal side, most probably induced by the repositioning of the remaining Trp(122)-Tyr(249) adduct . The results are discussed with respect to the bifunctional activity of catalase-peroxidases. Biophys J, 2004 Oct, 87(4), 2905 - 11 Epub 2004 Aug 23. Sizing DNA using a nanometer-diameter pore; Heng JB et al.; Each species from bacteria to human has a distinct genetic fingerprint . Therefore, a mechanism that detects a single molecule of DNA represents the ultimate analytical tool . As a first step in the development of such a tool, we have explored using a nanometer-diameter pore, sputtered in a nanometer-thick inorganic membrane with a tightly focused electron beam, as a transducer that detects single molecules of DNA and produces an electrical signature of the structure . When an electric field is applied across the membrane, a DNA molecule immersed in electrolyte is attracted to the pore, blocks the current through it, and eventually translocates across the membrane as verified unequivocally by gel electrophoresis . The relationship between DNA translocation and blocking current has been established through molecular dynamics simulations . By measuring the duration and magnitude of the blocking current transient, we can discriminate single-stranded from double-stranded DNA and resolve the length of the polymer . Theriogenology, 2004 Oct 1, 62(7), 1353 - 64 Blood cells in rainbow trout Oncorhynchus mykiss milt: relation to milt collection method and sampling period; Ciereszko A et al.; The presence of blood cells in milt of rainbow trout (Oncorhynchus mykiss) collected every week between the middle at the end of the spawning season, either by stripping or by catheterization was investigated . Basic sperm biological and biochemical characteristics were also evaluated . Because milt often becomes contaminated with blood during collection, we also studied the influence of experimental blood contamination on sperm motility and biochemical parameters of seminal plasma . We demonstrated the presence of blood cells (erythrocytes, lymphoid, and phagocytes) in rainbow trout milt collected by both methods . Both sampling period and collection method influenced sperm characteristics, however the relationship between these characteristics and blood cells are not clear at present . A high number of blood cells in milt was found in some samples, possibly due to inflammation, because at the same time we observed bacteria and elevated levels of protein and antiproteinase activity in contaminated samples . Experimental contamination of milt with blood did not influence sperm motility, protein concentration and LDH activity of the 5-day-stored semen . Our study demonstrated that blood cells were present in rainbow trout milt . Blood cells may also appear in milt as a result of bleeding and their elevated levels are present during inflammation. FEMS Immunol Med Microbiol, 2004 Sep 1, 42(1), 21 - 33 Development of mucosal immunity in the first year of life and relationship to sudden infant death syndrome; Gleeson M et al.; The common mucosal immune system (CMIS) is an interconnecting network of immune structures that provides effective immunity to mucosal surfaces . The structures of the mucosal immune system are fully developed in utero by 28 weeks gestation, but in the absence of intrauterine infection, activation does not occur until after birth . Mucosal immune responses occur rapidly in the first weeks of life in response to extensive antigenic exposure . Maturation of the mucosal immune system and establishment of protective immunity varies between individuals but is usually fully developed in the first year of life, irrespective of gestational age at birth . In addition to exposure to pathogenic and commensal bacteria, the major modifier of the developmental patterns in the neonatal period is infant feeding practices . A period of heightened immune responses occurs during the maturation process, particularly between 1 and 6 months, which coincides with the age range during which most cases of sudden infant death syndrome (SIDS) occur . A hyper-immune mucosal response has been a common finding in infants whose death is classified as SIDS, particularly if in association with a prior upper respiratory infection . Inappropriate mucosal immune responses to an otherwise innocuous common antigen and the resulting inflammatory processes have been proposed as factors contributing to SIDS. Comp Biochem Physiol B Biochem Mol Biol, 2004 Aug, 138(4), 331 - 7 Catalase from the white shrimp Penaeus (Litopenaeus) vannamei: molecular cloning and protein detection; Tavares-Sanchez OL et al.; Catalase is an antioxidant enzyme that plays a very important role in the protection against oxidative damage by breaking down hydrogen peroxide . It is a very highly conserved enzyme that has been identified from numerous species including bacteria, fungi, plants and animals, but the information about catalase in crustaceans is very limited . A cDNA containing the complete coding sequence for catalase from the shrimp Penaeus (Litopenaeus) vannamei was sequenced and the mRNA was detected by RT-PCR in selected tissues . Catalase was detected in hepatopancreas crude extracts by Western blot analysis with anti-human catalase polyclonal antibodies . The nucleotide sequence is 1692 bp long, including a 72-bp 5'-UTR, a coding sequence of 1515 bp and a 104-bp 3'-UTR . The deduced amino acid sequence corresponds to 505 amino acids with high identity to invertebrate, vertebrate and even bacterial catalases and contains the catalytic residues His71, Asn144, and Tyr354 . The predicted protein has a calculated molecular mass of 57 kDa; which coincides with the size of the subunit (approximately 55 kDa) and the tetrameric protein (approximately 230 kDa) detected in hepatopancreas extracts under native conditions . Catalase mRNA level was higher in hepatopancreas, followed by gills and was not detected in muscle. Clin Lab Med, 2004 Sep, 24(3), 797 - 823, viii Emerging infections in transfusion medicine; Fiebig EW et al.; The risk of transfusion-transmitted infectious diseases (TTIDs) has declined dramatically in high-income nations over the past 2 decades, primarily because of extraordinary success in preventing HIV and other established transfusion-transmitted viruses from entering the blood supply . Despite this achievement, TTIDs remain a public health concern, and attention is refocusing on new and emerging pathogens, such as West Nile virus, infectious proteins (the presumed cause of variant Creutzfeldt-Jakob disease), and other transmissible organisms such as bacteria and parasites . In this article the authors concentrate on this heterogeneous group of infectious agents, describe individual pathogens and the risks they pose to transfusion recipients, and comment on existing and evolving procedures that are designed to protect the blood supply from this threat. Biochemistry, 2004 Aug 31, 43(34), 10886 - 95 Using networks to identify fine structural differences between functionally distinct protein states; Swint-Kruse L; The vast increase in available data from the "-omics" revolution has enabled the fields of structural proteomics and structure prediction to make great progress in assigning realistic three-dimensional structures to each protein molecule . The challenge now lies in determining the fine structural details that endow unique functions to sequences that assume a common fold . Similar problems are encountered in understanding how distinct conformations contribute to different phases of a single protein's dynamic function . However, efforts are hampered by the complexity of these large, three-dimensional molecules . To overcome this limitation, structural data have been recast as two-dimensional networks . This analysis greatly reduces visual complexity but retains information about individual residues . Such diagrams are very useful for comparing multiple structures, including (1) homologous proteins, (2) time points throughout a dynamics simulation, and (3) functionally different conformations of a given protein . Enhanced structural examination results in new functional hypotheses to test experimentally . Here, network representations were key to discerning a difference between unliganded and inducer-bound lactose repressor protein (LacI), which were previously presumed to be identical structures . Further, the interface of unliganded LacI was surprisingly similar to that of the K84L variant and various structures generated by molecular dynamics simulations . Apo-LacI appears to be poised to adopt the conformation of either the DNA- or inducer-bound structures, and the K84L mutation appears to freeze the structure partway through the conformational transition . Additional examination of the effector binding pocket results in specific hypotheses about how inducer, anti-inducer, and neutral sugars exert their effects on repressor function. Planta . 2004 Aug 18; {Epub ahead of print} Physcomitrella patens is highly tolerant against drought, salt and osmotic stress; Frank W et al.; In order to determine the degree of tolerance of the moss Physcomitrella patens to different abiotic stress conditions, we examined its tolerance against salt, osmotic and dehydration stress . Compared to other plants like Arabidopsis thaliana, P . patens exhibits a high degree of abiotic stress tolerance, making it a valuable source for the identification of genes effecting the stress adaptation . Plants that had been treated with NaCl tolerated concentrations up to 350 mM . Treatments with sorbitol revealed that plants are able to survive concentrations up to 500 mM . Furthermore, plants that had lost 92% water on a fresh-weight basis were able to recover successfully . For molecular analyses, a P . patens expressed sequence tag (EST) database was searched for cDNA sequences showing homology to stress-associated genes of seed plants and bacteria . 45 novel P . patens genes were identified and subjected to cDNA macroarray analyses to define their expression pattern in response to water deficit . Among the selected cDNAs, we were able to identify a set of genes that is specifically up-regulated upon dehydration . These genes encode proteins exerting their function in maintaining the integrity of the plant cell as well as proteins that are known to be members of signaling networks . The identified genes will serve as molecular markers and potential targets for future functional analyses. Proc Natl Acad Sci U S A, 2004 Oct 5, 101 Suppl 2, 14622 - 6 Epub 2004 Aug 20. Therapeutic vaccination using CD4+CD25+ antigen-specific regulatory T cells; Bluestone JA et al.; Autoimmune disease results from the dysregulation of basic tolerogenic processes designed to control self/non-self-discrimination . Approaches to treat autoimmunity have focused historically on potent immunosuppressives that block the activation and expansion of antigen-specific T cells before they differentiate into pathogenic T cell responses . These therapies are very efficient in reducing clonal expansion and altering early signaling pathways . However, once the pathogenic responses are established (i.e., autoimmunity), the interventions are less effective on activated and differentiated T cell subsets (including memory T cells) or acting in the presence of an inflammatory milieu to abort immune responses at the target tissue and systemically . Moreover, the current immunotherapies require continuous use because they do not redirect the immune system to a state of tolerance . The continuous treatment leads to long-term toxicities and can profoundly suppress protective immune responses targeted at viruses, bacteria, and other pathogens . Over the past decade, there have been tremendous advances in our understanding of the basic processes that control immune tolerance . Among the most exciting has been the identification of a professional regulatory T cell subset that has shown enormous potential in suppressing pathologic immune responses in autoimmune diseases, transplantation, and graft vs . host disease . In this review, we summarize current efforts to induce and maintain tolerance in the autoimmune diabetes setting by using therapeutic vaccination with CD4(+)CD25(+) regulatory T cells . Emphasis will be placed on approaches to exploit regulatory T cells either directly or through the use of anti-CD3 immunotherapy. Infect Immun, 2004 Sep, 72(9), 5506 - 10 Growth phase regulation of flaA expression in Helicobacter pylori is luxS dependent; Loh JT et al.; LuxS plays a role in the synthesis of an extracellular signaling molecule, autoinducer 2 (AI-2) . To analyze a possible role of AI-2 in regulating Helicobacter pylori gene expression, we constructed a panel of transcriptional reporter strains . We show that the expression of H . pylori flaA is growth phase dependent and that flaA transcription increases in association with increased culture density . Mutating the luxS gene eliminates growth-phase-dependent control of flaA, and this growth phase dependence is restored when the luxS mutant strain is complemented with the wild-type luxS gene. Infect Immun, 2004 Sep, 72(9), 5143 - 9 Differential requirements for VirB1 and VirB2 during Brucella abortus infection; den Hartigh AB et al.; The Brucella abortus virB operon, encoding a type IV secretion system (T4SS), is required for intracellular replication and persistent infection in the mouse model . The products of the first two genes of the virB operon, virB1 and virB2, are predicted to be localized at the bacterial surface, where they could potentially interact with host cells . Studies to date have focused on characterization of transposon mutations in these genes, which are expected to exert polar effects on downstream genes in the operon . In order to determine whether VirB1 and VirB2 are required for the function of the T4SS apparatus, we constructed and characterized nonpolar deletion mutations of virB1 and virB2 . Both mutants were shown to be nonpolar, as demonstrated by their ability to express the downstream gene virB5 during stationary phase of growth in vitro . Both VirB1 and VirB2 were essential for intracellular replication in J774 macrophages . The nonpolar virB2 mutant was unable to cause persistent infection in the mouse model, demonstrating the essential role of VirB2 in the function of the T4SS apparatus during infection . In contrast, the nonpolar virB1 mutant persisted at wild-type levels, showing that the function of VirB1 is dispensable in the mouse model of persistent infection. Nature, 2004 Aug 19, 430(7002), 877 - 81 Variable ageing and storage of dissolved organic components in the open ocean; Loh AN et al.; Seawater dissolved organic matter (DOM) is the largest reservoir of exchangeable organic carbon in the ocean, comparable in quantity to atmospheric carbon dioxide . The composition, turnover times and fate of all but a few planktonic constituents of this material are, however, largely unknown . Models of ocean carbon cycling are thus limited by the need for information on temporal scales of carbon storage in DOM subcomponents, produced via the 'biological pump', relative to their recycling by bacteria . Here we show that carbohydrate- and protein-like substances in the open Atlantic and Pacific oceans, though often significantly aged, comprise younger fractions of the DOM, whereas dissolved lipophilic material exhibits up to approximately 90 per cent fossil character . In contrast to the millennial mean ages of DOM observed throughout the water column, weighted mean turnover times of DOM in the surface ocean are only decadal in magnitude . An observed size-age continuum further demonstrates that small dissolved molecules are the most highly aged forms of organic matter, cycling much more slowly than larger, younger dissolved and particulate precursors, and directly links oceanic organic matter age and size with reactivity. Proc Natl Acad Sci U S A, 2004 Aug 31, 101(35), 12848 - 53 Epub 2004 Aug 18. Identification and characterization of phosphoseryl-tRNA{Ser}Sec kinase; Carlson BA et al.; In 1970, a kinase activity that phosphorylated a minor species of seryl-tRNA to form phosphoseryl-tRNA was found in rooster liver {Maenpaa, P . H . & Bernfield, M . R . (1970) Proc . Natl . Acad . Sci . USA 67, 688-695}, and a minor seryl-tRNA that decoded the nonsense UGA was detected in bovine liver . The phosphoseryl-tRNA and the minor UGA-decoding seryl-tRNA were subsequently identified as selenocysteine (Sec) tRNA{Ser}Sec, but the kinase activity remained elusive . Herein, by using a comparative genomics approach that searched completely sequenced archaeal genomes for a kinase-like protein with a pattern of occurrence similar to that of components of Sec insertion machinery, we detected a candidate gene for mammalian phosphoseryl-tRNA{Ser}Sec kinase (pstk) . Mouse pstk was cloned, and the gene product (PSTK) was expressed and characterized . PSTK specifically phosphorylated the seryl moiety on seryl-tRNA{Ser}Sec and, in addition, had a requirement for ATP and Mg2+ . Proteins with homology to mammalian PSTK occur in Drosophila, Caenorhabditis elegans, Methanopyrus kandleri, and Methanococcus jannaschii, suggesting a conservation of its function across archaea and eukaryotes that synthesize selenoproteins and the absence of this function in bacteria, plants, and yeast . The fact that PSTK has been highly conserved in evolution suggests that it plays an important role in selenoprotein biosynthesis and/or regulation . J Bacteriol, 2004 Sep, 186(17), 5799 - 807 Genetic evidence identifying the true gluconeogenic fructose-1,6-bisphosphatase in Thermococcus kodakaraensis and other hyperthermophiles; Sato T et al.; Fructose-1,6-bisphosphatase (FBPase) is one of the key enzymes in gluconeogenesis . Although FBPase activity has been detected in several hyperthermophiles, no orthologs corresponding to the classical FBPases from bacteria and eukaryotes have been identified in their genomes . An inositol monophosphatase (IMPase) from Methanococcus jannaschii which displayed both FBPase and IMPase activities and a structurally novel FBPase (FbpTk) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 have been proposed as the "missing" FBPase . For this study, using T . kodakaraensis, we took a genetic approach to elucidate which candidate is the major gluconeogenic enzyme in vivo . The IMPase/FBPase ortholog in T . kodakaraensis, ImpTk, was confirmed to possess high FBPase activity along with IMPase activity, as in the case of other orthologs . We therefore constructed Deltafbp and Deltaimp strains by applying a gene disruption system recently developed for T . kodakaraensis and investigated their phenotypes . The Deltafbp strain could not grow under gluconeogenic conditions while glycolytic growth was unimpaired, and the disruption resulted in the complete abolishment of intracellular FBPase activity . Evidently, fbpTk is an indispensable gene for gluconeogenesis and is responsible for almost all intracellular FBPase activity . In contrast, the endogenous impTk gene could not complement the defect of the fbp deletion, and its disruption did not lead to any detectable phenotypic changes under the conditions examined . These facts indicated that impTk is irrelevant to gluconeogenesis, despite the high FBPase activity of its protein product, probably due to insufficient transcription . Our results provide strong evidence that the true FBPase for gluconeogenesis in T . kodakaraensis is the FbpTk ortholog, not the IMPase/FBPase ortholog. J Bacteriol, 2004 Sep, 186(17), 5782 - 9 A novel Acetivibrio cellulolyticus anchoring scaffoldin that bears divergent cohesins; Xu Q et al.; Sequencing of a cellulosome-integrating gene cluster in Acetivibrio cellulolyticus was completed . The cluster contains four tandem scaffoldin genes (scaA, scaB, scaC, and scaD) bounded upstream and downstream, respectively, by a presumed cellobiose phosphorylase and a nucleotide methylase . The sequences and properties of scaA, scaB, and scaC were reported previously, and those of scaD are reported here . The scaD gene encodes an 852-residue polypeptide that includes a signal peptide, three cohesins, and a C-terminal S-layer homology (SLH) module . The calculated molecular weight of the mature ScaD is 88,960; a 67-residue linker segment separates cohesins 1 and 2, and two approximately 30-residue linkers separate cohesin 2 from 3 and cohesin 3 from the SLH module . The presence of an SLH module in ScaD indicates its role as an anchoring protein . The first two ScaD cohesins can be classified as type II, similar to the four cohesins of ScaB . Surprisingly, the third ScaD cohesin belongs to the type I cohesins, like the seven ScaA cohesins . ScaD is the first scaffoldin to be described that contains divergent types of cohesins as integral parts of the polypeptide chain . The recognition properties among selected recombinant cohesins and dockerins from the different scaffoldins of the gene cluster were investigated by affinity blotting . The results indicated that the divergent types of ScaD cohesins also differ in their preference of dockerins . ScaD thus plays a dual role, both as a primary scaffoldin, capable of direct incorporation of a single dockerin-borne enzyme, and as a secondary scaffoldin that anchors the major primary scaffoldin, ScaA and its complement of enzymes to the cell surface. J Bacteriol, 2004 Sep, 186(17), 5603 - 13 Identification and transcriptional control of Caulobacter crescentus genes encoding proteins containing a cold shock domain; Lang EA et al.; The cold shock proteins are small peptides that share a conserved domain, called the cold shock domain (CSD), that is important for nucleic acid binding . The Caulobacter crescentus genome has four csp genes that encode proteins containing CSDs . Three of these (cspA, cspB, and cspC) encode peptides of about 7 kDa and are very similar to the cold shock proteins of other bacteria . Analysis by reverse transcription-PCR of the fourth gene (cspD), which was previously annotated as encoding a 7-kDa protein, revealed that the mRNA is larger and probably encodes a putative 21-kDa protein, containing two CSDs . A search in protein sequences databases revealed that this new domain arrangement has thus far only been found among deduced peptides of alpha-proteobacteria . Expression of each Caulobacter csp gene was studied both in response to cold shock and to growth phase, and we have found that only cspA and cspB are induced by cold shock, whereas cspC and cspD are induced at stationary phase, with different induction rates . The transcription start sites were determined for each gene, and a deletion mapping of the cspD promoter region defined a sequence required for maximal levels of expression, indicating that regulation of this gene occurs at the transcriptional level . Deletion of cspA, but not cspD, caused a reduction in viability when cells were incubated at 10 degrees C for prolonged times, suggesting that cspA is important for adaptation to a low temperature. Respiration, 2004 Jul-Aug, 71(4), 402 - 9 Exposure of differentiated airway epithelial cells to volatile smoke in vitro; Beisswenger C et al.; BACKGROUND: Cigarette smoke (CS) is the predominant pathogenetic factor in the development of chronic bronchitis and chronic obstructive pulmonary disease . The knowledge about the cellular and molecular mechanisms underlying the smoke-induced inflammation in epithelial cells is limited . OBJECTIVES: The aim of this study was to develop an in vitro model to monitor the effects of volatile CS on differentiated airway epithelial cells . METHODS: The airway epithelial cell line MM-39 and primary human bronchial epithelial cells were cultivated as air-liquid interface cultures and exposed directly to volatile CS . We used two types of exposure models, one using ambient air, the other using humidified and warm air . Cytokine levels were measured by quantitative PCR and ELISA . Phosphorylation of p38 MAP kinase was assessed by Western blot analysis . To reduce the smoke-induced inflammation, antisense oligonucleotides directed against the p65 subunit of NF-kappaB were applied . RESULTS: Exposure of epithelia to cold and dry air resulted in a significant inflammatory response . In contrast, exposure to humidified warm air did not elicit a cellular response . Stimulation with CS resulted in upregulation of mRNA for IL-6 and IL-8 and protein release . Exposure to CS combined with heat-inactivated bacteria synergistically increased levels of the cytokines . Reactions of differentiated epithelial cells to smoke are mediated by the MAP kinase p38 and the transcription factor NF-kappaB . CONCLUSIONS: We developed an exposure model to examine the consequences of direct exposure of differentiated airway epithelial cells to volatile CS . The model enables to measure the cellular reactions to smoke exposure and to determine the outcome of therapeutic interventions . J Biol Chem, 2004 Nov 5, 279(45), 46896 - 906 Epub 2004 Aug 16. Conservation of the biochemical properties of IncA from Chlamydia trachomatis and Chlamydia caviae: oligomerization of IncA mediates interaction between facing membranes; Delevoye C et al.; The developmental cycle of Chlamydiaceae occurs in a membrane compartment called an inclusion . IncA is a member of a family of proteins synthesized and secreted onto the inclusion membrane by bacteria . IncA proteins from different species of Chlamydiaceae show little sequence similarity . We report that the biochemical properties of Chlamydia trachomatis and Chlamydia caviae are conserved . Both proteins self-associate to form multimers . When artificially expressed by the host cell, they localize to the endoplasmic reticulum . Strikingly, heterologous expression of IncA in the endoplasmic reticulum completely inhibits concomitant inclusion development . Using truncated forms of IncA from C . caviae, we show that expression of the C-terminal cytoplasmic domain of the protein at the surface of the endoplasmic reticulum is sufficient to disrupt the bacterial developmental cycle . On the other hand, development of a C . trachomatis strain that does not express IncA is not inhibited by artificial IncA expression, showing that the disruptive effect observed with the wild-type strain requires direct interactions between IncA molecules at the inclusion and on the endoplasmic reticulum . Finally, we modeled IncA tetramers in parallel four helix bundles based on the structure of the SNARE complex, a conserved structure involved in membrane fusion in eukaryotic cells . Both C . trachomatis and C . caviae IncA tetramers were highly stable in this model . In conclusion, we show that the property of IncA proteins to assemble into multimeric structures is conserved between chlamydial species, and we propose that these proteins may have co-evolved with the SNARE machinery for a role in membrane fusion. J Neuroinflammation . 2004 Aug 17;1(1):16. Immunopathogenesis of brain abscess; Kielian T; Brain abscess represents a significant medical problem despite recent advances made in detection and therapy . Due to the emergence of multi-drug resistant strains and the ubiquitous nature of bacteria, the occurrence of brain abscess is likely to persist . Our laboratory has developed a mouse experimental brain abscess model allowing for the identification of key mediators in the CNS anti-bacterial immune response through the use of cytokine and chemokine knockout mice . Studies of primary microglia and astrocytes from neonatal mice have revealed that S . aureus, one of the main etiologic agents of brain abscess in humans, is a potent stimulus for proinflammatory mediator production . Recent evidence from our laboratory indicates that Toll-like receptor 2 plays a pivotal role in the recognition of S . aureus and its cell wall product peptidoglycan by glia, although other receptors also participate in the recognition event . This review will summarize the consequences of S . aureus on CNS glial activation and the resultant neuroinflammatory response in the experimental brain abscess model. Oral Dis, 2004 Sep, 10(5), 249 - 57 Solubility properties of human tooth mineral and pathogenesis of dental caries; Aoba T; Dental research over the last century has advanced our understanding of the etiology and pathogenesis of caries lesions . Increasing knowledge of the dynamic demineralization/remineralization processes has led to the current consensus that bacteria-mediated tooth destruction can be arrested or even to some degree reversed by adopting fluoride and other preventive measures without using restorative materials . Our experimental approach provided new insight into the stoichiometries and solubility properties of human enamel and dentin mineral . The determination of the solubility product constant on the basis of the stoichiometric model (Ca)5.x(Mg)q(Na)u(HPO4)v(CO3)w(PO4)3.y(OH,F)1.z, verifies the difference in their solubility properties, supporting the phase transformation between tooth mineral and calcium phosphates in a wide range of fluid compositions as found in the oral environment . Further refinement of the stoichiometry and solubility parameters is essential to assess quantitatively the driving force for de- and remineralization of enamel and dentin in the oral fluid environment . Prediction of the effects of a combination of inhibitors and accelerator(s) on remineralization kinetics is also required . In order to develop devices efficient for optimizing remineralization in the lesion body, it is a critical question how, and to what extent, fluoride can compensate for the activity of any inhibitors in the mineralizing media. J Microsc, 2004 Aug, 215(Pt 2), 213 - 8 Five-colour in vivo imaging of neurons in Caenorhabditis elegans; Hutter H; In the last few years variants of the 'green fluorescent protein' (GFP) with different spectral properties have been generated . This has greatly increased the number of possible applications for these fluorochromes in cell biology . The significant overlap of the excitation and emission spectra of the different GFP variants imposes constraints on the number of variants that can be used simultaneously in a single sample . In particular, the two brightest variants, GFP and YFP, are difficult to separate spectrally . This study shows that GFP and YFP can be readily separated with little spectral overlap (cross-talk) with the use of a confocal microscope equipped with an acusto-optical beam splitter and freely adjustable emission windows . Under optimal recording conditions cross-talk is less than 10% . Together with two other fluorescent proteins and the lipophilic dye DiD a total of five different colours can now be used simultaneously to label in vivo distinct anatomical structures such as neurons and their processes . Spatial resolution of the confocal microscope is sufficient to resolve the relative position of labelled axons within a single axon bundle . The use of five distinct marker dyes allows the in vivo analysis of the Caenorhabditis elegans nervous system at unprecedented resolution and richness in detail at the light microscopic level. J Microsc, 2004 Aug, 215(Pt 2), 162 - 73 Quantitative fluorescence resonance energy transfer (FRET) measurement with acceptor photobleaching and spectral unmixing; Gu Y et al.; Fluorescence resonance energy transfer (FRET) by acceptor photobleaching is a simple but effective tool for measurements of protein-protein interactions . Until recently, it has been restricted to qualitative or relative assessments owing to the spectral bleed-through contamination resulting from fluorescence overlap between the donor and the acceptor . In this paper, we report a quantitative algorithm that combines the spectral unmixing technique with FRET by acceptor photobleaching . By spectrally unmixing the emissions before and after photobleaching, it is possible to resolve the spectral bleed-through and retrieve the FRET efficiency/interaction distance quantitatively . Using a human keratinocyte cell line transfected with cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-tagged Cx26 connexins as an example, FRET information at homotypic gap junctions is measured and compared with well-established methods . Results indicate that the new approach is sensitive, flexible, instrument independent and solely FRET dependent . It can achieve FRET estimations similar to that from a sensitized emission FRET method . This approach has a great advantage in providing the relative concentrations of the donor and the acceptor; this is, for example, very important in the comparative study of cell populations with variable expression levels. Mikrobiologiia, 2004 May-Jun, 73(3), 350 - 7 {Investigation of lipopolysaccharides from Sinorhizobium meliloti SKHM1-188 and two its mutants with decreased nodulation competitiveness}; Kosenko LV et al.; A comparative study of the lipopolysaccharides (LPS) isolated from Sinorhizobium meliloti SKHM 1-188 and two its LPS-mutants (Th29 and Ts22) with sharply decreased nodulation competitiveness was conducted . Polyacrylamide gel electrophoresis with sodium dodecyl sulfate revealed two forms of LPS in all the three strains: a higher molecular-weight LPS1, containing O-polysaccharide (O-PS), and a and lower molecular-weight LPS2 without O-PS . However, the LPS1 content in mutants was significantly smaller than in the parent strain . The LPS of the strains studied contained glucose, galactose, mannose, xylose, three nonidentified sugars--X1 (TGlc 0.53), X2 (TGlc 0.47), and X3 (TGlc 0.43), glucosamine, and ethanolamine, while the LPS of S . meliloti SKHM1-188 additionally contained galactosamine, glucuronic and galacturonic acids, and 2-keto-3-deoxyoctulosonic acid (KDO), as well as fatty acids, such as 3-OH C14:0, 3-OH C15:0, 3-OH C16:0, 3-OH C18:0, nonidentified hydroxy X (T3-OH C14:0 1.33), C18:0, and unsaturated C18:1 fatty acids . The LPS of both mutants were similar in the component composition but differed from the LPS of the parent strain by a lower X2, X3, and 3-OH C 14:0 content and a higher KDO, C18:0, and hydroxy X content . The LPS of all the strains were subjected to mild hydrolysis with 1% acetic acid and fractionated on a column with Sephadex G-25 . The higher molecular weight fractions (2500-4000 Da) contained a set of sugars typical of intact LPS and, supposedly, corresponded to the LPS polysaccharide portion (PS1) . In the lower molecular weight fractions (600-770 Da, PS2), glucose and uronic acids were the major components; galactose, mannose, and X1 were present in smaller amounts . The PS1/PS2 ratio for the two mutants was significantly lower than for strain SKHM1-188 . The data obtained show that the amount of O-PS-containing molecules (LPS1) in the heterogeneous lipopolysaccharide complex of the mutants was smaller than in the SKHM1-188 LPS; this increases the hydrophobicity of the cell surface of the mutant bacteria . This supposedly contributes to their nonspecific adhesion on the roots of the host plant, thus decreasing their nodulation competitiveness. Z Gastroenterol, 2004 Aug, 42(8), 735 - 8 Emphysematous gastritis -- case report and review of the literature; Ocepek A et al.; Emphysematous gastritis is a rare form of phlegmonous gastritis caused by invasion of the stomach wall by gas-forming bacteria . This clinical entity was first described by Fraenkel in 1889 . The authors report the case of a 58-year-old patient with end-stage renal failure under treatment with hemodialysis for the past year . He was admitted on account of pain in the lower abdomen . During the diagnostic procedure, emphysematous gastritis and acute cholecystitis were confirmed . An acute abdomen developed and an emergency laparotomy was performed . During the procedure, the necrotic gallbladder was removed . The patient died on the following day due to multiorgan failure and septic shock . According to the literature, emphysematous gastritis is generally a fatal disease . Air in the wall of the stomach is a rare finding with a broad differential diagnosis. J Med Microbiol, 2004 Sep, 53(Pt 9), 927 - 33 One-tube cell lysis and DNA extraction procedure for PCR-based detection of Mycobacterium ulcerans in aquatic insects, molluscs and fish; Kotlowski R et al.; The purpose of this study was to develop a simple procedure for cell lysis and DNA extraction for direct detection of Mycobacterium ulcerans in aquatic insects, gills and intestinal contents of fish, molluscs and human tissue samples using a nested PCR method specific for the insertion sequence IS2404 . The simultaneous action of sodium N-lauroyl sarcosine, guanidinium isothiocyanate, chloroform and Tris-saturated phenol on mycobacteria, followed by a DNA purification method using mini-columns fitted with silica-cellulose membranes was successfully employed to extract DNA from cultured bacteria, environmental and human tissue samples . All specimens were collected from Buruli ulcer endemic regions . M . ulcerans DNA was detected in 11 of 57 aquatic insects, one of six molluscs and three of 15 fish, supporting the hypothesis that the fauna of major Buruli ulcer endemic foci in swampy terrain of tropical and subtropical regions can be a source of M . ulcerans infection. Mol Cell Biol, 2004 Sep, 24(17), 7370 - 9 RNA-mediated programming of developmental genome rearrangements in Paramecium tetraurelia; Garnier O et al.; The germ line genome of ciliates is extensively rearranged during development of the somatic macronucleus . Numerous sequences are eliminated, while others are amplified to a high ploidy level . In the Paramecium aurelia group of species, transformation of the maternal macronucleus with transgenes at high copy numbers can induce the deletion of homologous genes in sexual progeny, when a new macronucleus develops from the wild-type germ line . We show that this trans-nuclear effect correlates with homology-dependent silencing of maternal genes before autogamy and with the accumulation of approximately 22- to 23-nucleotide (nt) RNA molecules . The same effects are induced by feeding cells before meiosis with bacteria containing double-stranded RNA, suggesting that small interfering RNA-like molecules can target deletions . Furthermore, experimentally induced macronuclear deletions are spontaneously reproduced in subsequent sexual generations, and reintroduction of the missing gene into the variant macronucleus restores developmental amplification in sexual progeny . We discuss the possible roles of the approximately 22- to 23-nt RNAs in the targeting of deletions and the implications for the RNA-mediated genome-scanning process that is thought to determine developmentally regulated rearrangements in ciliates . Cancer Res, 2004 Aug 15, 64(16), 5850 - 60 Synergistic activation of innate immunity by double-stranded RNA and CpG DNA promotes enhanced antitumor activity; Whitmore MM et al.; Double-stranded RNA (dsRNA) and unmethylated CpG sequences in DNA are pathogen-associated molecular patterns of viruses and bacteria that activate innate immunity . To examine whether dsRNA and CpG DNA could combine to provide enhanced stimulation of innate immune cells, murine macrophages were stimulated with poly-rI:rC (pIC), a dsRNA analog, and CpG-containing oligodeoxynucleotides (CpG-ODN) . Combined treatments demonstrated synergy in nitric oxide, interleukin (IL)-12, tumor necrosis factor alpha, and IL-6 production . Studies using neutralizing antibodies for type I interferons (IFNs), IFN-alpha and IFN-beta, indicated that nitric oxide synthase synergism is mediated by paracrine/autocrine effects of IFN-beta . In contrast, enhanced cytokine production occurred independent of type I IFN and was maintained in macrophages from IFN-alpha/beta receptor knockout mice . Cotransfection of human Toll-like receptors 3 and 9 (receptors for dsRNA and CpG DNA, respectively) into 293T cells supported synergistic activation of an IL-8 promoter reporter construct by pIC, indicating interaction of the signaling pathways in driving the synergy response . In vivo stimulation of mice with pIC and CpG-ODN demonstrated synergy for serum IL-6 and IL-12p40 levels that correlated with an enhanced antitumor effect against established B16-F10 experimental pulmonary metastases . Treatment of tumor-bearing mice with pIC and CpG-ODN in combination resulted in enhanced nitric oxide synthase expression in lung tissue and enhanced up-regulation of class I major histocompatibility complex on splenic dendritic cells relative to treatments with either agent alone . In conclusion, the combined detection of viral pathogen-associated molecular patterns, i.e., dsRNA and CpG DNA, may mimic definitive viral recognition, resulting in an enhanced innate immune response that could be used for tumor vaccination or immunotherapy. Hepatobiliary Pancreat Dis Int, 2004 Aug, 3(3), 444 - 7 Expression of vascular endothelial growth factor gene in primary cultured rat hepatocytes; Wang JL et al.; BACKGROUND: It is the key point for vascular endothelial growth factor (VEGF121) gene related therapy as to how to transfect and express the gene safely, effectively and repeatedly . This study was designed to investigate the VEGF121 transfection and expression in primary cultured rat hepatocyte . METHODS: After construction of vector internal ribosome entry site-enhanced yellow fluorescent protein (pIRES-EYFP)/VEGF121, the transfection and expression of the exogenous VEGF121 gene in primary cultured rat hepatocytes were observed through RT-PCR, Western blot and fluorescent microscopy . RESULTS: pIRES-EYFP/VEGF121 plasmid was constructed and transfected successfully into primary cultured rat hepatocytes, the transfection and expression of gene in primary cultured rat hepatocytes were examined by RT-PCR and Western blot, and yellow-green fluorescence was observed through a fluorescent microscope . CONCLUSION: The successful transfection and expression of plasmid pIRES-EYFP/VEGF121 in primary cultured rat hepatocytes provides a foundation for hepatocyte transplantation and gene therapy after modification of hepatocytes by the gene. Curr Opin Struct Biol, 2004 Aug, 14(4), 495 - 504 Periplasmic binding proteins: a versatile superfamily for protein engineering; Dwyer MA et al.; The diversity of biological function, ligand binding, conformational changes and structural adaptability of the periplasmic binding protein superfamily have been exploited to engineer biosensors, allosteric control elements, biologically active receptors and enzymes using a combination of techniques, including computational design . Extensively redesigned periplasmic binding proteins have been re-introduced into bacteria to function in synthetic signal transduction pathways that respond to extracellular ligands and as biologically active enzymes. Curr Opin Struct Biol, 2004 Aug, 14(4), 397 - 404 The machinery of membrane protein assembly; White SH et al.; The SecY (bacteria) and Sec61 (eukaryotes) translocon complexes, or protein-conducting channels, work in concert with bound ribosomes to insert proteins into membranes during the first step of membrane protein assembly . The crystallographic structure of an archaeal SecY translocon provides dramatic new insights into the mechanism of translocon function . This structure suggests an explanation for how the translocon can aid in establishing membrane protein topology via the positive-inside rule . The folding of membrane proteins may begin in the ribosome exit tunnel, before entering the translocon, according to cryo-electron microscopy and biophysical studies. Curr Opin Struct Biol, 2004 Aug, 14(4), 390 - 6 Structural insight into the protein translocation channel; Clemons WM Jr et al.; A structurally conserved protein translocation channel is formed by the heterotrimeric Sec61 complex in eukaryotes, and SecY complex in archaea and bacteria . Electron microscopy studies suggest that the channel may function as an oligomeric assembly of Sec61 or SecY complexes . Remarkably, the recently determined X-ray structure of an archaeal SecY complex indicates that the pore is located at the center of a single molecule of the complex . This structure suggests how the pore opens perpendicular to the plane of the membrane to allow the passage of newly synthesized secretory proteins across the membrane and opens laterally to allow transmembrane segments of nascent membrane proteins to enter the lipid bilayer . The electron microscopy and X-ray results together suggest that only one copy of the SecY or Sec61 complex within an oligomer translocates a polypeptide chain at any given time. Clin Chim Acta, 2004 Sep, 347(1-2), 41 - 8 The proteomic approach to analysis of human milk fat globule membrane; Cavaletto M et al.; Lactating mammary epithelial cells do not adhere to the classical rule of lipid secretion since lipids are secreted enveloped by the milk fat globule membrane (MFGM) via a budding process . Historically, biochemical studies of MFGM proteins focused on the analysis of single molecules . Today, thanks to proteomic technologies, it is now possible to comprehensively analyse the protein component of MFGM . Questions concerning: (1) protein organization within the MFGM structure; (2) correlation to the intracellular secretion pathway; and (3) display of multiple specialized functions, can now be addressed . Proteomics allows for rapid identification of the total MFGM proteins thereby providing a global functional screen of the lactating cell . In addition to their nutritional value, human MFGM proteins have important protective functions against bacteria, viruses and protozoa . Identifying the complex nature of protein-protein interactions would further enhance our present knowledge of MFGM function . A speculative molecular architecture of the human MFGM is proposed. Microb Pathog, 2004 Aug, 37(2), 95 - 105 N-terminal-capturing screening system for the isolation of Brucella abortus genes encoding surface exposed and secreted proteins; Marchesini MI et al.; Secreted as well as surface exposed proteins are assumed to play major roles in bacterial virulence . In this report we describe the construction of an N-terminal protein-capturing system and its use for the isolation of Brucella abortus S2308 genes coding for putative surface exposed or secreted proteins . For this purpose, a cloning vector that generates gene fusions to a ribosome binding site and start codon deficient Chloramphenicol Acetyl Transferase (CAT) reporter gene was constructed and the resulting library introduced into B . abortus S2308 and virB mutant strains . Secreted translational fusions were identified by determining CAT activity in culture supernatants . Secretion was confirmed by Western Blot using a polyclonal anti-CAT antibody . A total of 864 clones were screened and 10 genes encoding putative secreted/surface exposed proteins were identified . Seven are Brucella proteins with an assigned function, whereas three are hypothetical proteins . The number of amino acid residues that promotes CAT secretion varies from 5 to 386 and no conserved motifs were detected . Secretion in a virB mutant background of some of the isolated fusion proteins was also determined . Interestingly, some hybrid proteins seemed to require a full VirB system for their secretion . J Mol Biol, 2004 Jul 30, 341(1), 199 - 214 SecA folding kinetics: a large dimeric protein rapidly forms multiple native states; Doyle SM et al.; SecA, a 202 kDa dimeric protein, is the ATPase for the Sec-dependent translocase of precursor proteins in vivo . SecA must undergo conformational changes, which may involve dissociation into a monomer, as it translocates the precursor protein across the inner membrane . To better understand the dynamics of SecA in vivo, protein folding studies to probe the native, intermediate, and unfolded species of SecA in vitro have been done . SecA folds through a stable dimeric intermediate and dimerizes in the dead-time of a manual-mixing kinetic experiment ( approximately 5-7 seconds) . Here, stopped-flow fluorescence and CD, as well as ultra-rapid continuous flow fluorescence techniques, were used to further probe the rapid folding kinetics of SecA . In the absence of urea, rapid, near diffusion-limited ( approximately 10(9)M(-1)s(-1)) SecA dimerization occurs following a rate-limiting unimolecular rearrangement of a rapidly formed intermediate . Multiple kinetic folding and unfolding phases were observed and SecA was shown to have multiple native and unfolded states . Using sequential-mixing stopped-flow experiments, SecA was determined to fold via parallel channels with sequential intermediates . These results confirm that SecA is a highly dynamic protein, consistent with the rapid, major conformational changes it must undergo in vivo. Cancer Lett, 2004 Sep 15, 213(1), 39 - 48 Spontaneous transformation of an immortalized hepatocyte cell line: potential role of a nuclear protease; Drubin DA et al.; In this study, we utilized an in vitro model of spontaneous transformation/progression, an SV40 large T antigen-immortalized rat hepatocyte cell line (designated CWSV14) that is very weakly tumorigenic at low-passage, but acquires a transformed phenotype upon extended passage in cell culture . Here we show that this mid-passage transformation is accompanied by development of aneuploidy and disorganization of the actin cytoskeleton, concomitant with a large increase in a chymotrypsin-like nuclear protease activity which we have previously implicated in chemical transformation of fibroblasts and ras-transformation of hepatocytes . Passage of the CWSV14 cells with AAPF(cmk), a relatively selective inhibitor of the nuclear protease activity, abrogates the acquisition of the transformed phenotype and prevents the changes in the actin cytoskeleton . We hypothesize that the nuclear protease may play a role in initiating development of genomic instability, paralleling the archetypical role of proteases in paradigms such as the SOS-type responses in bacteria and yeast. Fish Shellfish Immunol, 2004 Oct, 17(4), 315 - 23 Oligomerisation and carbohydrate binding in an Atlantic salmon serum C-type lectin consistent with non-self recognition; Stratton L et al.; A C-type lectin was previously isolated from the blood of healthy Atlantic salmon (Salmo salar) and this salmon serum lectin (SSL) was found to opsonise bacteria . Selective binding to bacteria in vivo requires that the lectin be able to recognise a carbohydrate pattern on the bacterial surface distinguishable from that of the host . In order to investigate this selectivity in the lectin, a phage-display antibody was prepared and then used for detection of lectin by Western blotting . A carbohydrate binding-inhibition assay with Western blot detection of the lectin showed mannose to be the primary ligand and related sugars including glucose, N-acetylglucosamine and methyl alpha-D-mannopyranoside to be additional ligands of this lectin . The SSL in serum detected by Western blotting was shown to form a complex oligomer . These results show that the salmon serum lectin is oligomeric in blood and that it recognizes a broad spectrum of carbohydrates with optimal binding to mannose . The lectin might therefore be an ideal opsonin for multiple salmon pathogens with carbohydrate arrays on their surfaces . No similar lectins were identified in the sera of other fish by Western blotting using the phage-display antibody . Molecular analysis will be required in order to determine whether homologous lectins are expressed in related fish species . It is anticipated that similar lectins might have related pathogen recognition roles in divergent fish species. Lik Sprava, 2001 Jan-Feb, (1), 109 - 11 {Claritromycin in the modern schemes of eradication of helicobacter infection in patients with ulcer disease}; Vdovychenko BI et al.; Out of the fifty patients with duodenal peptic ulcer having received conventional dosages of ranitidin (phamotidin) combined with Denol, metronidazole, and/or tetracycline over two weeks the relapse of the illness within one year occured in 22 percent . Of these, 44 patients had a culture isolated the sensitivity of which to antibiotics was studied with the aid of standard disks or end-point dilutions . 38.7 percent of strains of bacteria were found out to be insensitive to tetracycline, 6.8 percent--to metronidazole, 2.3 percent--to amoxicylline, and no strain was sensitive to claritromycin . The repeated treatment of patients with recurrences of the illness with a combination of omeprazole (20 mg two times daily), amoxicylline (1000 mg twice per day), and claritromycin (500 mg twice per day) for up to 7 days showed no infection in the patients. J Med Entomol, 2004 Jul, 41(4), 677 - 83 Molecular (sub) grouping of endosymbiont Wolbachia infection among mosquitoes of Taiwan; Tsai KH et al.; Wolbachia are maternally inherited bacteria that infect a wide range of arthropods as well as filarial worms . The infection usually results in reproductive distortions of the host, primarily cytoplasmic incompatibility, parthenogenesis, and feminization . This study showed that Wolbachia infection (15/29; 51.72%) was prevalent among field-caught mosquitoes in Taiwan . Three mosquito species were identified as having Wolbachia A infection, eight species as having Wolbachia B, and four other species were dually infected by both groups . Each Wolbachia isolate from different mosquitoes was further divided into a specific subgroup . However, there were still some isolates that did not belong to any known subgroup, suggesting that more subgroups remain to be identified . Investigation of tissue tropism in either Aedes albopictus (Skuse) or Armigeres subalbatus (Coquillett) revealed that Wolbachia were extensively distributed within the host, although the ovary was most susceptible to infection . This report provides preliminary features of molecular relationships among Wolbachia groups of mosquitoes from Taiwan. Pesqui Odontol Bras, 2004 Apr-Jun, 18(2), 168 - 73 Epub 2004 Aug 05. Toothbrushing with vegetable oil: a clinical and laboratorial analysis; Aguiar AA et al.; The dentifrices currently available in the marketplace contain many anticariogenic substances, fluoride and abrasives aimed to better clean the dental surface, remove dental plaque, improve salivary flow and its buffer capacity and reduce colonies of bacteria such as S . mutans, the causative agent of dental caries . The objective of this study was to evaluate the possibility of adequately removing dental plaque using an experimental almond oil dentifrice (Titoil) with no abrasives or antiplaque agents . This study was carried out with 80 volunteers, all of them 18-year-old recruits from the military training school of Aracatuba -- SP . Saliva sampling and dental plaque disclosing were undertaken both before and after 28 days of toothbrushing with a low abrasive dentifrice (Group 1: 40 volunteers) or with Titoil (Group 2: 40 volunteers) . Statistical analysis of the results revealed that the experimental dentifrice (Titoil) did not interfere with salivary flow and reduced dental plaque more than the low abrasive dentifrice, improved the salivary buffer capacity and decreased salivary S . mutans (Caritest-SM) as much as regular dentifrices . It was concluded that if the dental industry replaces abrasive by vegetable oil in dentifrices, these will be more effective in maintaining oral health and will cause less dental abrasion. Biochemistry (Mosc), 2004 Jul, 69(7), 813 - 7 Structural specificity of photosynthetic reaction centers provides high efficiency of excitation trapping and conversion; Borisov AY; The atomic structures of photosynthetic reaction centers of two species of purple bacteria and two photosystems 2 of cyanobacteria were resolved in the late last century . In this work I put forward the idea that of the huge body of data available thus far, only three structural factors are responsible for the unique function of conversion of physical energy of electronic excitation into electrochemical energy of separated opposite charges in reaction centers at least in purple bacteria and, perhaps, in other photosynthetic organisms. Dig Dis Sci, 2004 Jun, 49(6), 977 - 81 The activity of class I, II, III, and IV alcohol dehydrogenase isoenzymes and aldehyde dehydrogenase in colorectal cancer; Jelski W et al.; Chronic ethanol consumption is associated with an increased risk for cancer of the colorectum . The highly toxic and carcinogenic compound is acetaldehyde, the product of ethanol metabolism . Ethanol is metabolized to acetaldehyde by alcohol dehydrogenase (ADH) in colorectal mucosa and bacteria . The enzyme responsible for oxidation of acetaldehyde is aldehyde dehydrogenase . The aim of this study was to compare ADH isoenzymes and ALDH activity in colorectal cancer with the activity in normal colonic mucosa . Total ADH activity was measured by a photometric method with p-nitrosodimethylaniline (NDMA) as substrate, and ALDH activity by a fluorometric method with 6-methoxy-2-naphthaldehyde as a substrate . For measurement of the activity of class I and II isoenzymes we employed fluorometric methods, with class-specific fluorogenic substrates . The activity of class III ADH was measured by the photometric method with n-octanol as substrate, and class IV with m-nitrobenzaldehyde as substrate . Samples were taken surgically during routine operations of colorectal carcinomas from 32 patients . The activities of total ADH and, the most important in colon mucosa, class I ADH were significantly higher in cancer than in healthy tissues . The other tested classes of ADH had a tendency to higher-level activity in cancer cells than in healthy mucosa . ALDH activity was not significantly lower in the cancer cells . The activities of all tested enzymes and isoenzymes were not significantly higher in drinkers than in nondrinkers both in colorectal cancer and in normal mucosa . The differences in activities of total ADH and class I isoenzyme between cancer tissues and normal colon mucosa might be a factor for metabolic changes and disturbances in low-mature cancer cells and, additionally, might be a reason for the higher level of acetaldehyde, which can intensify carcinogenesis. J Antimicrob Chemother, 2004 Sep, 54(3), 579 - 81 Epub 2004 Aug 12. Glycodendritic structures: promising new antiviral drugs; Rojo J et al.; DC-SIGN, a C-type lectin expressed by dendritic cells, is able to recognize high mannosylated glycoproteins at the surface of a broad range of pathogens including viruses, bacteria, fungi and parasites . For at least some of these agents this interaction appears to be an important part of the infection process . Therefore, this lectin might be considered in the design of new antiviral drugs . In this manner, multivalent carbohydrate systems based on dendrimers and dendritic polymers are promising candidates as antiviral drugs . Boltorn hyperbranched dendritic polymers functionalized with mannose have been used to inhibit DC-SIGN-mediated infection in an Ebola-pseudotyped viral model . Their physiological solubility, lack of toxicity and especially their low price suggest the application of these glycodendritic polymers for possible formulation as microbicides. Vaccine, 2004 Sep 3, 22(25-26), 3386 - 94 DNA immunization followed by a viral vector booster in a Chlamydia pneumoniae mouse model; Penttila T et al.; Vaccination against Chlamydia pneumoniae would be a beneficial strategy for either preventing or controlling infection by this human respiratory pathogen that also causes persistent infections . In the present study, we used recombinant Semliki Forest virus (rSFV) particles for delivering C . pneumoniae antigens major outer membrane protein (MOMP) or outer membrane protein 2 (Omp2) to the mice or applied the prime-boost technique, where mice were first primed with naked DNA and then boosted with the viral vector coding for the same proteins . Partial protection suggested by the reduced number of cultivable bacteria from the lungs of the challenged mice was seen in mice immunized by either method with MOMP expressing constructs . A significant protection was also achieved after DNA/rSFV immunization with Omp2 . DNA priming followed by rSFV boosting induced a more prominent IFN-gamma production after challenge at the site of the infection in pulmonary and mediastinal cells. Zhonghua Liu Xing Bing Xue Za Zhi, 2004 Jul, 25(7), 602 - 6 {Study on Bartonella infection using molecular biological diagnostic techniques from China}; Li DM et al.; OBJECTIVE: To establish polymerase chain reaction (PCR) technique for the detection of specific genes related to species of genus Bartonella, and for diagnosing clinically suspected cat-scratch disease (CSD) case complicated with pneumonia on both lungs . The appearance of Bartonella infectious diseases calls for genus and species detection and tools for identification in order to make clinical diagnosis and carry on epidemiological studies . METHODS: One pair of primer TIle.455p-TAla.885n was designed based on the fact that tRNA(Ile)-tRNA(Ala) intergenic spacer region in 16S-23S rRNA intergenic spacer (ITS) of genus Bartonella were high variable sequences flanked by completely conserved tRNA-encoding genes . 16S-23S rRNA was longer than that which had been described in other bacteria . Two published pairs of primers were used to directly detect the specific gene fragments of Bartonella species DNA extracts from human blood, followed by PCR product Sequencing and nucleotide base sequence analysis . RESULTS: Amplification products of the three pairs of primers had the same predicted size of those in Bartonella spp . According to the different length of electrophoresis bank, the sample was identified as a species of genus Bartonella other than the positive control . Sequence analysis showed that the nuleotide sequence from the PCR product of primer TIle.455p-TAla.885n was identical to the Bartonella isolated from Yunnan in China . CONCLUSIONS: PCR-based assay provided a simple and rapid means to detect pathogenic Bartonella species in humans and mammalian hosts as well as in arthropod vecters . This study suggested that this pathogenic Bartonella species existed in patients in northern and southern parts of China. Scand J Infect Dis, 2004, 36(6-7), 516 - 9 Reactive hemophagocytosis associated with the initiation of highly active antiretroviral therapy (HAART) in a patient with AIDS; Huang DB et al.; Reactive hemophagocytosis has been associated with neoplasia, bacteria, parasitic, and viral infections including human immunodeficiency virus (HIV) . Here we present a case of reactive hemophagocytosis associated with the initiation of highly active antiretroviral therapy (HAART), probably representing a syndrome termed the immune reconstitution inflammatory syndrome (IRIS) or immune reconstitution syndrome. Scand J Infect Dis, 2004, 36(6-7), 410 - 6 Value of preoperative investigations in diagnosing prosthetic joint infection: retrospective cohort study and literature review; Bernard L et al.; The diagnosis of a prosthetic joint infection is difficult, but crucial for appropriate treatment . Scintigraphy with specific markers for infection (labelled white cells or immunoglobulin-G) has been reported as a more reliable diagnostic tool than clinical assessment (fever, fistula), laboratory studies (polynuclear neutrophil count, erythrocyte rate sedimentation, and C-reactive protein), and preoperative aspiration . In the first part of this study, we retrospectively reviewed 230 patients admitted with a suspected prosthetic joint infection, and examined the validity of the different diagnostic tools for the group as a whole and for subgroups according to the Coventry classification . In the second part, we reviewed 35 articles about preoperative evaluation of infection in prosthetic joints and compared them to our findings . Our study indicates that C-reactive protein and joint aspiration are the most useful tools to diagnose prosthetic joint infection even in situations of chronic infection (Coventry type II). Eur J Immunol, 2004 Sep, 34(9), 2579 - 88 Depletion of NK cells results in disseminating lethal infection with Bordetella pertussis associated with a reduction of antigen-specific Th1 and enhancement of Th2, but not Tr1 cells; Byrne P et al.; IFN-gamma plays a critical role in protection against Bordetella pertussis, but Th1 cells are only detectable after the infection has started to resolve, suggesting a protective role for innate IFN-gamma early in infection . Here, we demonstrate significant recruitment of NK cells and NKT cells into the lungs following respiratory challenge with B . pertussis . Furthermore, NK cells are the primary source of IFN-gamma in the lungs during the acute stage of infection . Stimulation of IFN-gamma production by NK cells was indirect through B . pertussis-activated IL-12 or IL-23 production by dendritic cells . Depletion of NK cells with anti-asialo ganglio-N-tetraosylceramide antibody resulted in a lethal infection, with enhancement of bacterial load in the lungs and dissemination of the bacteria to the liver via the blood . NK cell-depleted mice had significantly reduced B . pertussis-specific IFN-gamma and enhanced IgG1 and IL-5, but not IL-10 production, suggesting that regulatory T cells are induced simultaneously with Th1 cells, but the absence of NK cells resulted in enhancement of Th2-type responses . These findings suggest that NK cells confer resistance to B . pertussis by activating IL-12-mediated production of IFN-gamma, which enhances the anti-bacterial activity of macrophages, but also promotes the differentiation of Th1 cells . Philos Trans R Soc Lond B Biol Sci, 2004 Jun 29, 359(1446), 919 - 27 The archaeal Sec-dependent protein translocation pathway; Bolhuis A; Over the past three decades, transport of proteins across cellular membranes has been studied extensively in various model systems . One of the major transport routes, the so-called Sec pathway, is conserved in all domains of life . Very little is known about this pathway in the third domain of life, archaea . The core components of the archaeal, bacterial and eucaryal Sec machinery are similar, although the archaeal components appear more closely related to their eucaryal counterparts . Interestingly, the accessory factors of the translocation machinery are similar to bacterial components, which indicates a unique hybrid nature of the archaeal translocase complex . The mechanism of protein translocation in archaea is completely unknown . Based on genomic sequencing data, the most likely system for archaeal protein translocation is similar to the eucaryal co-translational translocation pathway for protein import into the endoplasmic reticulum, in which a protein is pushed across the translocation channel by the ribosome . However, other models can also be envisaged, such as a bacterial-like system in which a protein is translocated post-translationally with the aid of a motor protein analogous to the bacterial ATPase SecA . This review discusses the different models . Furthermore, an overview is given of some of the other components that may be involved in the protein translocation process, such as those required for protein targeting, folding and post-translational modification. Brain Res Mol Brain Res, 2004 Aug 23, 127(1-2), 68 - 78 Rapid dendritic transport of TGN38, a putative cargo receptor; McNamara JO 2nd et al.; Protein transport to and from the postsynaptic plasma membrane is thought to be of central importance for synaptic plasticity . However, the molecular details of such processes are poorly understood . One mechanism by which membrane and secretory proteins may be transported to and from postsynaptic membranes is via cargo receptors . We studied the dendritic transport of TGN38, a putative cargo receptor thought to mediate protein transport between the trans-Golgi network (TGN), endosomes, and the plasma membrane . With fluorescence time-lapse imaging of neurons expressing a TGN38-green fluorescent protein fusion protein (GFP-TGN38), we observed rapid bidirectional dynamics of the protein in dendritic shafts . In addition, the protein was present on the surface and on intracellular membranes of dendrites and dendritic spines . Finally, GFP-TGN38 was found to cycle rapidly between the plasma membrane and intracellular membranes within dendrites, including those of spines . Together, our results suggest a role for TGN38 in facilitating rapid changes in the protein composition of postsynaptic membranes. Heredity, 2004 Oct, 93(4), 379 - 89 Variable fitness effects of Wolbachia infection in Drosophila melanogaster; Fry AJ et al.; Maternally inherited Wolbachia bacteria are extremely widespread among insects and their presence is usually associated with parasitic modifications of host fitness . Wolbachia pipientis infects Drosophila melanogaster populations from all continents, but their persistence in this species occurs despite any strong parasitic effects . Here, we have investigated the symbiosis between Wolbachia and D . melanogaster and found that Wolbachia infection can have significant survival and fecundity effects . Relative to uninfected flies, infected females from three fly strains showed enhanced survival or fecundity associated with Wolbachia infection, one strain showed both and one strain responded positively to Wolbachia removal . We found no difference in egg hatch rates (cytoplasmic incompatibility) for crosses between infected males and uninfected females, although there were fecundity differences . Females from this cross consistently produced fewer eggs than infected females and these fecundity differences could promote the spread of infection just like cytoplasmic incompatibility . More surprising, we found that infected females often had the greatest fecundity when mated to uninfected males . This could also promote the spread of Wolbachia infection, though here the fitness benefits would also help to spread infection when Wolbachia are rare . We suggest that variable fitness effects, in both sexes, and which interact strongly with the genetic background of the host, could increase cytoplasmic drive rates in some genotypes and help explain the widespread persistence of Wolbachia bacteria in D . melanogaster populations . These interactions may further explain why many D . melanogaster populations are polymorphic for Wolbachia infection . We discuss our results in the context of host-symbiont co-evolution. Biol Pharm Bull, 2004 Aug, 27(8), 1158 - 64 Secretory phospholipase A2; Murakami M et al.; Secretory phospholipase A2 (sPLA2) is a growing family of structurally related, disulfide-rich, low molecular weight, lipolytic enzymes with a His-Asp catalytic dyad . sPLA2s are distributed in a wide variety of vertebrate and invertebrate animals, plants, bacteria, and viruses, and there are 10 catalytically active sPLA2 isozymes in mammals . Although the structural bases for mammalian sPLA2s have been well documented, their physiological functions are still subject to debate . Individual mammalian sPLA2s have distinct enzymatic properties and display distinct tissue expression patterns, suggesting that each enzyme acts on distinct phospholipid membrane moieties in vivo . In this article, we briefly review our latest understanding of the possible physiological functions of sPLA2s, in keeping with their diverse actions on mammalian and nonmammalian cell membranes. Appl Biochem Biotechnol, 2004 Jul-Sep, 118(1-3), 155 - 70 Lipases and their industrial applications: an overview; Houde A et al.; Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) are part of the family of hydrolases that act on carboxylic ester bonds . The physiologic role of lipases is to hydrolyze triglycerides into diglycerides, monoglycerides, fatty acids, and glycerol . These enzymes are widely found throughout the animal and plant kingdoms, as well as in molds and bacteria . Of all known enzymes, lipases have attracted the most scientific attention . In addition to their natural function of hydrolyzing carboxylic ester bonds, lipases can catalyze esterification, interesterification, and transesterification reactions in nonaqueous media . This versatility makes lipases the enzymes of choice for potential applications in the food, detergent, pharmaceutical, leather, textile, cosmetic, and paper industries . The most significant industrial applications of lipases have been mainly found in the food, detergent, and pharmaceutical sectors . Limitations of the industrial use of these enzymes have mainly been owing to their high production costs, which may be overcome by molecular technologies, enabling the production of these enzymes at high levels and in a virtually purified form. J Biol Chem, 2004 Oct 22, 279(43), 44407 - 16 Epub 2004 Aug 10. Light and redox control of photosynthesis gene expression in Bradyrhizobium: dual roles of two PpsR; Jaubert M et al.; The two closely related bacteria Bradyrhizobium and Rhodopseudomonas palustris show an unusual mechanism of regulation of photosystem formation by light thanks to a bacteriophytochrome that antirepresses the regulator PpsR . In these two bacteria, we found out, unexpectedly, that two ppsR genes are present . We show that the two Bradyrhizobium PpsR proteins exert antagonistic effects in the regulation of photosystem formation with a classical repressor role for PpsR2 and an unexpected activator role for PpsR1 . DNase I footprint analysis show that both PpsR bind to the same DNA TGTN12ACA motif that is present in tandem in the bchC promoter and the crtED intergenic region . Interestingly, the cycA and aerR promoter regions that contain only one conserved palindrome are recognized by PpsR2, but not PpsR1 . Further biochemical analyses indicate that PpsR1 only is redox sensitive through the formation of an intermolecular disulfide bond, which changes its oligomerization state from a tetramer to an octamer under oxidizing conditions . Moreover, PpsR1 presents a higher DNA affinity under its reduced form in contrast to what has been previously found for PpsR or its homolog CrtJ from the Rhodobacter species . These results suggest that regulation of photosystem synthesis in Bradyrhizobium involves two PpsR competing for the binding to the same photosynthesis genes and this competition might be modulated by two factors: light via the antagonistic action of a bacteriophytochrome on PpsR2 and redox potential via the switch of PpsR1 oligomerization state. FEBS Lett, 2004 Aug 13, 572(1-3), 201 - 5 A family of glycosyl hydrolase family 45 cellulases from the pine wood nematode Bursaphelenchus xylophilus; Kikuchi T et al.; We have characterized a family of GHF45 cellulases from the pine wood nematode Bursaphelenchus xylophilus . The absence of such genes from other nematodes and their similarity to fungal genes suggests that they may have been acquired by horizontal gene transfer (HGT) from fungi . The cell wall degrading enzymes of other plant parasitic nematodes may have been acquired by HGT from bacteria . B . xylophilus is not directly related to other plant parasites and our data therefore suggest that horizontal transfer of cell wall degrading enzymes has played a key role in evolution of plant parasitism by nematodes on more than one occasion. J Invest Dermatol, 2004 Sep, 123(3), 522 - 9 Differential gene induction of human beta-defensins (hBD-1, -2, -3, and -4) in keratinocytes is inhibited by retinoic acid; Harder J et al.; Human skin is able to mount a fast response against invading harmful bacteria through the rapid production of inducible peptide antibiotics such as the human beta-defensins (hBD) . To gain more insight into the role and regulation of inducible beta-defensins in the innate immunity of human skin, we investigated whether gene induction of the human beta-defensins hBD-1, -2, -3, and -4 in keratinocytes is regulated in a similar manner . Therefore, we performed a comparative study of gene expression of these four hBD in primary cultured keratinocytes using real-time PCR . A basal mRNA expression was observed for all four hBD in primary keratinocytes, which strongly increased for hBD-2, -3, and -4 during Ca(2+)-induced differentiation of the keratinocytes . This effect was completely abolished when the keratinocytes were pre-treated with all-trans-retinoic acid (RA) . Furthermore, the differential induction of hBD-2, -3, and -4 gene expression in keratinocytes by proinflammatory cytokines, phorbol-myristate-acetate (PMA), and bacteria was inhibited by more than 90% when the keratinocytes were pre-incubated with RA . Inhibition of IL-1beta-mediated hBD-2 induction through RA was further confirmed by gene reporter assays and western-blot analysis . We conclude that RA is a potent inhibitor of beta-defensin induction in keratinocytes and might downregulate the inducible innate chemical defense system of human skin. Water Sci Technol, 2004, 49(11-12), 35 - 40 The effect of upflow air velocity on the structure of aerobic granules cultivated in a sequencing batch reactor; Tay JH et al.; The effect of upflow air velocity on the formation and structure of aerobic granules was studied in three column sequencing batch reactors . Upflow aeration would be the major cause of hydrodynamic shear force in the column reactor . Results showed that high upflow air velocity resulted in more compact, denser, rounder, stronger and smaller aerobic granules, while high biomass retention in the reactor was achieved . It was found that high upflow air velocity could induce granular sludge to secrete more cell polysaccharides which in turn contributed to the compact and strong structure . It appears from this study that the structure of aerobic granules could be controlled by manipulating the upflow air velocity. J Chem Ecol, 2004 Jun, 30(6), 1165 - 81 Variation of brominated indoles and terpenoids within single and different colonies of the marine bryozoan Flustra foliacea; Peters L et al.; The variation of the brominated indole and diterpenoid content within single and different colonies of the bryozoan Flustra foliacea was investigated . The secondary metabolite profile and concentrations of individual components of F . foliacea samples were established using GC-MS . Samples from 17 different collecting sites were analyzed . The alkaloid and diterpene composition of F . foliacea varied greatly depending upon the site of collection . Investigation of F . foliacea samples from a single site (Helgoland, North Sea) over a period of time showed that the alkaloid and diterpenoid profile remained constant, however concentrations of individual components varied significantly . The alkaloid and diterpenoid composition of different segments of a single colony was found to be constant . Only small differences could be detected in the essential oil composition of different colonies and segments of single colonies of F . foliacea . Two of the F . foliacea alkaloids were found in the gastropods Hydrobia ulva and Gibbula cinerea, and one alkaloid in the common starfish Asteria rubens, all collected from the surface of the bryozoan. Eukaryot Cell, 2004 Aug, 3(4), 855 - 61 Multiple triclosan targets in Trypanosoma brucei; Paul KS et al.; Trypanosoma brucei genes encoding putative fatty acid synthesis enzymes are homologous to those encoding type II enzymes found in bacteria and organelles such as chloroplasts and mitochondria . It was therefore not surprising that triclosan, an inhibitor of type II enoyl-acyl carrier protein (enoyl-ACP) reductase, killed both procyclic forms and bloodstream forms of T . brucei in culture with 50% effective concentrations (EC(50)s) of 10 and 13 microM, respectively . Triclosan also inhibited cell-free fatty acid synthesis, though much higher concentrations were required (EC(50)s of 100 to 200 microM) . Unexpectedly, 100 microM triclosan did not affect the elongation of {(3)H}laurate (C(12:0)) to myristate (C(14:0)) in cultured bloodstream form parasites, suggesting that triclosan killing of trypanosomes may not be through specific inhibition of enoyl-ACP reductase but through some other mechanism . Interestingly, 100 microM triclosan did reduce the level of incorporation of {(3)H}myristate into glycosyl phosphatidylinositol species (GPIs) . Furthermore, we found that triclosan inhibited fatty acid remodeling in a cell-free assay in the same concentration range required for killing T . brucei in culture . In addition, we found that a similar concentration of triclosan also inhibited the myristate exchange pathway, which resides in a distinct subcellular compartment . However, GPI myristoylation and myristate exchange are specific to the bloodstream form parasite, yet triclosan kills both the bloodstream and procyclic forms . Therefore, triclosan killing may be due to a nonspecific perturbation of subcellular membrane structure leading to dysfunction in sensitive membrane-resident biochemical pathways. J Theor Biol, 2004 Sep 21, 230(2), 251 - 60 Fractal properties of the human genome; Garte S; The fractal dimension of the human chromosomes and four other genomes were determined using the box counting method . Human chromosomes exhibited a fractal dimension (D) of about 0.8, while values for a bacteria, yeast, worm and plant were higher . Analysis of three human chromosomes over five orders of magnitude of scale (from 10(8) to 10(4) bp), showed D to be non-constant at the smaller scales, when introns were included as gaps . The relationship between D and gene density fit an empirical equation related to that expected from theory, and allowed for the calculation of the fractal initiator or self-similarity ratio . This value (0.57) was constant at all scales for human chromosomes, and was similar for other species, except for Arabidopsis. Toxicon, 2004 Sep 15, 44(4), 385 - 403 Antinutritional properties of plant lectins; Vasconcelos IM et al.; Lectins are carbohydrate binding (glyco)proteins which are ubiquitous in nature . In plants, they are distributed in various families and hence ingested daily in appreciable amounts by both humans and animals . One of the most nutritionally important features of plant lectins is their ability to survive digestion by the gastrointestinal tract of consumers . This allows the lectins to bind to membrane glycosyl groups of the cells lining the digestive tract . As a result of this interaction a series of harmful local and systemic reactions are triggered placing this class of molecules as antinutritive and/or toxic substances . Locally, they can affect the turnover and loss of gut epithelial cells, damage the luminal membranes of the epithelium, interfere with nutrient digestion and absorption, stimulate shifts in the bacterial flora and modulate the immune state of the digestive tract . Systemically, they can disrupt lipid, carbohydrate and protein metabolism, promote enlargement and/or atrophy of key internal organs and tissues and alter the hormonal and immunological status . At high intakes, lectins can seriously threaten the growth and health of consuming animals . They are also detrimental to numerous insect pests of crop plants although less is presently known about their insecticidal mechanisms of action . This current review surveys the recent knowledge on the antinutritional/toxic effects of plant lectins on higher animals and insects. Acta Crystallogr D Biol Crystallogr, 1997 May, 53(Pt 3), 302 - 10 Structure of ferric soybean leghemoglobin a nicotinate at 2.3 a resolution; Ellis PJ; Soybean leghemoglobin a is a small (16 kDa) protein facilitating the transport of O(2) to respiring N(2)-fixing bacteria at low free-O(2) tension . The crystal structure of soybean ferric leghemoglobin a nicotinate has been refined at 2.3 A resolution . The final R factor is 15.8% for 6877 reflections between 6.0 and 2.3 A . The structure of soybean leghemoglobin a (143 residues) is closely similar to that of lupin leghemoglobin II (153 residues), the proteins having 82 identical residues when the sequences are aligned . The new structure provides support for the conclusion that the unique properties of leghemoglobin arise principally from a heme pocket considerably larger and more flexible than that of myoglobin, a strongly ruffled heme group, and a proximal histidine orientation more favourable to ligand binding. Acta Crystallogr D Biol Crystallogr, 1995 Nov, 51(Pt 6), 962 - 78 Crystal structures of three complexes between chito-oligosaccharides and lysozyme from the rainbow trout . How distorted is the NAG sugar in site D? Karlsen S. Like all c-type lysozymes, those from rainbow trout act as 1,4-beta-acetyl-muramidases to destroy bacteria by cleaving the polysaccharide chains of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) units in the cell walls . Lysozymes also hydrolyse chitin, the analogous N-acetylglucosamine polymer . The rainbow trout enzymes have been shown to be particularly effective in bacterial defence . We have determined the crystal structures of three complexes between rainbow trout lysozyme (RBTL) and the chito-oligosaccharides (NAG)(2), (NAG)(3) and (NAG)(4) to resolutions of 1.8, 2.0 and 1.6 A, respectively . Crystals of these complexes were obtained by co-crystallization, and intensity data were collected on a FAST area detector system . Refinement and model building gave final R values of 16.6, 15.9 and 16.5% for the di-, tri- and tetrasaccharide complexes, respectively . The results show that the chito-oligosaccharides bind to sites A, B and C as previously observed for complexes between the hen egg-white lysozyme (HEWL) and a variety of saccharides . The NAG ring in site D is not bound so deeply and is only slightly distorted towards a half-chair conformation as observed for the equivalent NAM residue in HEWL . From our results, there is reason to question the position and the degree of strain of the D saccharide and the mode of binding and importance of two saccharides in sites E and F for correct orientation of sugar D and effective hydrolysis of a productive substrate-lysozyme complex . Simple model building study from our structures implies a 'left-sided' binding mode of (NAG)(6) in the lower part of the active site of RBTL. Acta Crystallogr D Biol Crystallogr, 1996 May, 52(Pt 3), 435 - 46 Structure of human salivary alpha-amylase at 1.6 a resolution: implications for its role in the oral cavity; Ramasubbu N; Salivary alpha-amylase, a major component of human saliva, plays a role in the initial digestion of starch and may be involved in the colonization of bacteria involved in early dental plaque formation . The three-dimensional atomic structure of salivary amylase has been determined to understand the structure-function relationships of this enzyme . This structure was refined to an R value of 18.4% with 496 amino-acid residues, one calcium ion, one chloride ion and 170 water molecules . Salivary amylase folds into a multidomain structure consisting of three domains, A, B and C . Domain A has a (beta/alpha)(8-) barrel structure, domain B has no definite topology and domain C has a Greek-key barrel structure . The Ca(2+) ion is bound to Asnl00, Arg158, Asp167, His201 and three water molecules . The Cl(-) ion is bound to Arg195, Asn298 and Arg337 and one water molecule . The highly mobile glycine-rich loop 304-310 may act as a gateway for substrate binding and be involved in a 'trap-release' mechanism in the hydrolysis of substrates . Strategic placement of calcium and chloride ions, as well as histidine and tryptophan residues may play a role in differentiating between the glycone and aglycone ends of the polysaccharide substrates . Salivary amylase also possesses a suitable site for binding to enamel surfaces and provides potential sites for the binding of bacterial adhesins. Acta Crystallogr D Biol Crystallogr, 1996 Nov, 52(Pt 6), 1202 - 8 Preliminary crystallographic analysis and further characterization of a dodecaheme cytochrome c from Desulfovibrio desulfuricans ATCC 27774; Coelho AV; Dodecaheme cytochrome c has been purified from Desulfovibrio (D.) desulfuricans ATCC 27774 cells grown under both nitrate and sulfate-respiring conditions . Therefore, it is likely to play a role in the electron-transfer system of both respiratory chains . Its molecular mass (37768 kDa) was determined by electrospray mass spectrometry . Its first 39 amino acids were sequenced and a motif was found between amino acids 32 and 37 that seems to exist in all the cytochromes of the c(3) type from sulfate-reducing bacteria sequenced at present . The midpoint redox potentials of this cytochrome were estimated to be -68, -120, -248 and -310 mV . Electron paramagnetic resonance spectroscopy of the oxidized cytochrome shows several low-spin components with a g(max) spreading from 3.254 to 2.983 . Two crystalline forms were obtained by vapour diffusion from a solution containing 2% PEG 6000 and 0.25-0.75 M acetate buffer pH = 5.5 . Both crystals belong to monoclinic space groups: one is P2(1), with a = 61.00, b = 106.19, c = 82.05 A, beta = 103.61 degrees, and the other is C2 with a = 152.17, b = 98.45, c = 89.24 A, beta = 119.18 degrees . Density measurements of the P2(1) crystals suggest that there are two independent molecules in the asymmetric unit . Self-rotation function calculations indicate, in both crystal forms, the presence of a non-crystallographic axis perpendicular to the crystallographic twofold axis . This result and the calculated values for the volume per unit molecular weight of the C2 crystals suggest the presence of two or four molecules in the asymmetric unit. Acta Crystallogr D Biol Crystallogr, 1994 Jul, 50(Pt 4), 596 - 602 Crystallization and X-ray structure determination of cytochrome c(2) from Rhodobacter sphaeroides in three crystal forms; Axelrod HL; Cytochrome c(2) serves as the secondary electron donor that reduces the photo-oxidized bacteriochlorophyll dimer in photosynthetic bacteria . Cytochrome c(2) from Rhodobacter sphaeroides has been crystallized in three different forms . At high ionic strength, crystals of a hexagonal space group (P6(1)22) were obtained, while at low ionic strength, triclinic (P1) and tetragonal (P4(1)2(1)2) crystals were formed . The three-dimensional structures of the cytochrome in all three crystal forms have been determined by X-ray diffraction at resolutions of 2.20 A (hexagonal), 1.95 A, (triclinic) and 1.53 A (tetragonal) . The most significant difference observed was the binding of an imidazole molecule to the iron atom of the heme group in the hexagonal structure . This binding displaces the sulfur atom of Met l00, which forms the axial ligand in the triclinic and tetragonal structures. J Exp Biol, 2004 Aug, 207(Pt 18), 3125 - 9 Role of trehalose phosphate synthase and trehalose during hypoxia: from flies to mammals; Chen Q et al.; Trehalose is a nonreducing disaccharide in which the two glucose units are linked in an alpha,alpha-1,1-glycosidic linkage . The best known and most widely distributed pathway of trehalose synthesis involves the transfer of glucose from UDP-glucose to glucose 6-phosphate to form trehalose-6-phosphate and UDP via the trehalose-6-phosphate synthase (TPS1) . Trehalose-6-phosphate phosphatase (TPS2) then converts trehalose-6-phosphate to free trehalose . This sugar is present in a wide variety of organisms, including bacteria, yeast, fungi, insects, invertebrates and plants, and because of its particular physical features, trehalose is able to protect the integrity of cells against a variety of environmental stresses such as desiccation, dehydration, heat, cold and oxidation . Our current studies described here indicate that trehalose protects Drosophila and mammalian cells from hypoxic and anoxic injury . The mechanism of this protection is probably related to a decrease in protein denaturation through protein-trehalose interactions. J Med Food, 2004 Summer, 7(2), 252 - 5 Antitumor activity of beta-D-glucan from Libyan dates; Ishurd O et al.; Beta-glucan with antitumor activities was isolated from Libyan dates, and the structure of the purified glucan was characterized using methods such as methylation, periodate oxidation, and acetolysis . Glucans were found to exhibit potent antitumor activity; this activity could be correlated to their (1-->3)-beta-D-glucan linkages . Such antitumor glucans have also been obtained from a number of other sources, such as yeast, fungi, bacteria, and plants . This is the first study to report antitumor activity for date glucan. Aliment Pharmacol Ther, 2004 Jul, 20 Suppl 1, 165 - 70 Histological aspects and role of mast cells in Helicobacter pylori-infected gastritis; Nakajima S et al.; BACKGROUND: Mast cells are one of the main pro-inflammatory cells, while their knowledge in Helicobacter pylori infection has not been summarized . METHODS: We reviewed studies on mast cells in H . pylori infection, and summarized the histological aspects and roles . RESULTS: The density of mast cells is greater in H . pylori-infected than in non-infected subjects . Increased mast cell density in infected gastritis significantly decreases after eradication . On electron microscopy, mast cells in infected gastric mucosa show degranulation . Some experimental studies demonstrate that mast cells are degranulated with H . pylori-derived products . CONCLUSIONS: Mast cells are actively involved in the pathogenesis of H . pylori-infected gastritis . The possible roles are to initiate and promote the formation of oedema through degranulated and secreted mediators, and to release multiple chemotactic factors, which induce inflammatory cells to infiltrate to the site of oedema, showing acute inflammatory changes . Mast cells also stimulate the degradation of pericellular matrices and the growth of cells in their vicinity, and thereby promote tissue turnover . In chronic H . pylori infection, these reactions continue until the bacteria are eradicated . Mast cells may act both to maintain gastritis and to repair tissue damage in H . pylori-infected chronic gastritis. J Clin Microbiol, 2004 Aug, 42(8), 3505 - 12 Species of the family Helicobacteraceae detected in an Australian sea lion (Neophoca cinerea) with chronic gastritis; Oxley AP et al.; We describe the first case of gastritis in a male Australian sea lion (Neophoca cinerea) in which members of the family Helicobacteraceae, particularly the genus Wolinella, were detected . The sea lion exhibited clinical signs of gastrointestinal disease, including abdominal pain, lack of appetite, and lethargy . Examination of one ileal and five gastric biopsy specimens collected over a 10-year period revealed persistent fibrosis and/or superficial focal erosion and ulceration of the lamina propria . Spiral-shaped organisms 5 to 12 microm long were observed in two of the gut biopsy specimens . While Helicobacter species were detected by PCR in one of the gastric biopsy specimens, Wolinella species were detected in four of the five gastric specimens, including those in which spiral-shaped organisms were observed . Comparisons of biopsy specimen ribosomal DNA sequences with those obtained from the feces of this animal, the gastric tissue of a clinically healthy individual, and the feces of several other cohoused sea lions and fur seals revealed a separate and possibly novel gastric Helicobacter species . A possibly novel Wolinella species, along with Wolinella succinogenes, was also identified . These findings highlight the pathogenic potential of other members of this family in the etiopathogenesis of gastric disease in these animals. J Biol Chem, 2004 Oct 15, 279(42), 44101 - 12 Epub 2004 Aug 05. Live cell imaging of Gs and the beta2-adrenergic receptor demonstrates that both alphas and beta1gamma7 internalize upon stimulation and exhibit similar trafficking patterns that differ from that of the beta2-adrenergic receptor; Hynes TR et al.; To visualize and investigate the regulation of the localization patterns of Gs and an associated receptor during cell signaling, we produced functional fluorescent fusion proteins and imaged them in HEK-293 cells . alphas-CFP, with cyan fluorescent protein (CFP) inserted into an internal loop of alphas, localized to the plasma membrane and exhibited similar receptor-mediated activity to that of alphas . Functional fluorescent beta1gamma7 dimers were produced by fusing an amino-terminal yellow fluorescent protein (YFP) fragment to beta1 (YFP-N-beta1) and a carboxyl-terminal YFP fragment to gamma7 (YFP-C-gamma7) . When expressed together, YFP-N-beta1 and YFP-C-gamma7 produced fluorescent signals in the plasma membrane that were not seen when the subunits were expressed separately . Isoproterenol stimulation of cells co-expressing alphas-CFP, YFP-N-beta1/YFP-C-gamma7, and the beta2-adrenergic receptor (beta2AR) resulted in internalization of both fluorescent signals from the plasma membrane . Initially, alphas-CFP and YFP-N-beta1/YFP-C-gamma7 stained the cytoplasm diffusely, and subsequently they co-localized on vesicles that exhibited minimal overlap with beta2AR-labeled vesicles . Moreover, internalization of beta2AR-GFP, but not alphas-CFP or YFP-N-beta1/YFP-C-gamma7, was inhibited by a fluorescent dominant negative dynamin 1 mutant, Dyn1(K44A)-mRFP, indicating that the Gs subunits and beta2AR utilize different internalization mechanisms . Subsequent trafficking of the Gs subunits and beta2AR also differed in that vesicles labeled with the Gs subunits exhibited less overlap with RhoB-labeled endosomes and greater overlap with Rab11-labeled endosomes . Because Rab11 regulates traffic through recycling endosomes, co-localization of alphas and beta1gamma7 on these endosomes may indicate a means of recycling specific alphasbetagamma combinations to the plasma membrane. Bioelectrochemistry, 2004 Sep, 64(2), 125 - 31 Electrochemical investigation of the dynamics of Mycobacterium smegmatis cells' transformation to dormant, nonculturable form; Kuznetsov BA et al.; Dynamics of transformation of Mycobacterium smegmatis cells by cultivation under nonoptimal conditions (partial starvation) to dormant, nonculturable form has been studied . For this aim, an electrochemical method was developed to detect both viable and 'viable but nonculturable' (VBNC) cells . The current produced by bacteria placed at the electrode surface was measured in the presence of 2,6-dichlorophenol indophenol (DCIP) at the applied potential of 350 mV . It has been established that electrochemical activity changes parallel with the growth of biomass . The transition of M . smegmatis to a dormant, nonculturable state goes along with the decrease of the detection current up to 20% of the maximum level . This means that nonculturable cells have rather high rest metabolic activity . The course of the CFU values has a complicated character during bacterial growth . The placement of the bacterial culture on the solid medium appears to cause a new stress that stops proliferation and stimulates aggregation . Both processes distort CFU measurement results . The quantitative estimation of the viable but nonculturable cells by counting colonies, measuring optical density and current produced by bacteria has been discussed. Pharmazie, 2004 Jul, 59(7), 513 - 5 Absolute configuration of chloro-bisabolene sesquiterpene; Zhu Y et al.; The crystal structure of a cholor-bisabolene sesquiterpene has been determined for the absolute configuration . Its structure was elucidated as (-)-(1R,2R,3R,4R,6S,8S,10S)-chloro-2,10-diacetoxy-1,8-diangeloyloxy-3-hydroxy-11-methoxy-bisabol-7(14)-ene . The chlorine atom at C4 is axial in the cyclohexane ring . The molecule shows a stable chair conformation, and crystallizes in the monoclinic space group P2(1) with one molecule in the asymmetric unit. Photochem Photobiol Sci, 2004 Aug, 3(8), 823 - 9 Epub 2004 May 26. Early molecular events in the photoactive yellow protein: role of the chromophore photophysics; Changenet-Barret P et al.; We report a comparative study of the isomerization reaction in native and denatured photoactive yellow protein (PYP) and in various chromophore analogues in their trans deprotonated form . The excited-state relaxation dynamics was followed by subpicosecond transient absorption and gain spectroscopy . The free p-hydroxycinnamate (pCA(2-)) and its amide analogue (pCM(-)) are found to display a quite different transient spectroscopy from that of PYP . The excited-state deactivation leads to the formation of the ground-state cis isomer without any detectable intermediate with a mechanism comparable to trans-stilbene photoisomerization . On the contrary, the early stage of the excited-state deactivation of the free thiophenyl-p-hydroxycinnamate (pCT(-)) and of the denatured PYP is similar to that of the native protein . It involves the formation of an intermediate absorbing in the spectral region located between the bleaching and gain bands in less than 2 ps . However, in these two cases, the formation of the cis isomer has not been proved yet . This difference with pCA(-) and pCM(-) might result from the fact that, in the thioester substituted chromophore, simultaneous population of two quasi-degenerate excited states occurs upon excitation . This comparative study highlights the determining role of the chromophore structure and of its intrinsic properties in the primary molecular events in native PYP. Nature, 2004 Aug 5, 430(7000), 700 - 4 Structural basis for template-independent RNA polymerization; Tomita K et al.; The 3'-terminal CCA nucleotide sequence (positions 74-76) of transfer RNA is essential for amino acid attachment and interaction with the ribosome during protein synthesis . The CCA sequence is synthesized de novo and/or repaired by a template-independent RNA polymerase, 'CCA-adding enzyme', using CTP and ATP as substrates . Despite structural and biochemical studies, the mechanism by which the CCA-adding enzyme synthesizes the defined sequence without a nucleic acid template remains elusive . Here we present the crystal structure of Aquifex aeolicus CCA-adding enzyme, bound to a primer tRNA lacking the terminal adenosine and an incoming ATP analogue, at 2.8 A resolution . The enzyme enfolds the acceptor T helix of the tRNA molecule . In the catalytic pocket, C75 is adjacent to ATP, and their base moieties are stacked . The complementary pocket for recognizing C74-C75 of tRNA forms a 'protein template' for the penultimate two nucleotides, mimicking the nucleotide template used by template-dependent polymerases . These results are supported by systematic analyses of mutants . Our structure represents the 'pre-insertion' stage of selecting the incoming nucleotide and provides the structural basis for the mechanism underlying template-independent RNA polymerization. Plant Cell Physiol, 2004 Jul, 45(7), 855 - 63 Capturing in vivo dynamics of the actin cytoskeleton stimulated by auxin or light; Holweg C et al.; We present here a transient expression system that allows the response of actin microfilaments to physiological stimuli (changes in auxin content, light) to be observed in single cells in vivo . Etiolated, intact rice seedlings are attached to glass slides, transfected biolistically with talin fused to yellow-fluorescent protein to visualize actin microfilaments, and either treated with auxin or irradiated . The talin marker labels distinct populations of actin that are differentially expressed depending on the physiological state of the coleoptile (active elongation versus ceased elongation) . Whereas longitudinal transvacuolar bundles prevail in cells that have ceased to elongate, fine cortical strands are characteristic for elongating cells . The visualized actin structures remain dynamic and responsive to signals . Exogenous auxin triggers a loosening of the bundles and an extension of the cortical strands, whereas irradiation reorientates cortical strands into longitudinal arrays . These responses correspond in quality and timing to the signal responses inferred previously from fixed specimens and biochemical studies . In big advantage over those methods it is now possible to observe them directly at the single cell level . Thus, the rice coleoptile system can be used as a convenient model to study actin dynamics in vivo, in response to physiologically relevant stimuli. J Immunol, 2004 Aug 15, 173(4), 2231 - 5 Cutting edge: expression of chemokine receptor CXCR1 on human effector CD8+ T cells; Takata H et al.; IL-8 is a potent inflammatory cytokine that induces chemotaxis of neutrophils expressing CXCR1 and CXCR2, thus indicating its involvement in the migration of these cells to inflammatory sites where bacteria proliferate . Presently, we showed that CXCR1(+) cells were predominantly found among CD8(+) T cells having effector phenotype, and that the expression of CXCR1 was positively correlated with that of perforin, suggesting that CXCR1 is expressed on effector CD8(+) T cells . Indeed, human CMV-specific CD8(+) T cells from healthy individuals, which mostly express the effector phenotype and have cytolytic function, expressed CXCR1, whereas EBV-specific CD8(+) T cells, which mostly express the memory phenotype and have no cytolytic function, did not express this receptor . The results of a chemotaxis assay showed that the migration of CXCR1(+)CD8(+) T cells was induced by IL-8 . These results suggest that the IL-8-CXCR1 pathway plays an important role in the homing of effector CD8(+) T cells. J Biol Chem, 2004 Oct 29, 279(44), 45844 - 54 Epub 2004 Aug 04. The C-terminal tail of presenilin regulates Omi/HtrA2 protease activity; Gupta S et al.; Presenilin mutations are responsible for most cases of autosomal dominant inherited forms of early onset Alzheimer disease . Presenilins play an important role in amyloid beta-precursor processing, NOTCH receptor signaling, and apoptosis . However, the molecular mechanisms by which presenilins regulate apoptosis are not fully understood . Here, we report that presenilin-1 (PS1) regulates the proteolytic activity of the serine protease Omi/HtrA2 through direct interaction with its regulatory PDZ domain . We show that a peptide corresponding to the cytoplasmic C-terminal tail of PS1 dramatically increases the proteolytic activity of Omi/HtrA2 toward the inhibitor of apoptosis proteins and beta-casein and induces cell death in an Omi/HtrA2-dependent manner . Consistent with these results, ectopic expression of full-length PS1, but not PS1 lacking the C-terminal PDZ binding motif, potentiated Omi/HtrA2-induced cell death . Our results suggest that the C terminus of PS1 is an activation peptide ligand for the PDZ domain of Omi/HtrA2 and may regulate the protease activity of Omi/HtrA2 after its release from the mitochondria during apoptosis . This mechanism of Omi/HtrA2 activation is similar to the mechanism of activation of the related bacterial DegS protease by the outer-membrane porins. Appl Environ Microbiol, 2004 Aug, 70(8), 4950 - 60 Correspondence between community structure and function during succession in phenol- and phenol-plus-trichloroethene-fed sequencing batch reactors; Ayala-Del-Rio HL et al.; The effects of more than 2 years of trichloroethene (TCE) application on community succession and function were studied in two aerobic sequencing batch reactors . One reactor was fed phenol, and the second reactor was fed both phenol and TCE in sequence twice per day . After initiation of TCE loading in the second reactor, the TCE transformation rates initially decreased, but they stabilized with an average second-order rate coefficient of 0.044 liter mg(-1) day(-1) for 2 years . In contrast, the phenol-fed reactor showed higher and unstable TCE transformation rates, with an average rate coefficient of 0.093 liter mg(-1) day(-1) . Community analysis by terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes showed that the phenol-plus-TCE-fed reactor had marked changes in community structure during the first 100 days and remained relatively stable afterwards, corresponding to the period of stable function . In contrast, the community structure of the phenol-fed reactor changed periodically, and the changes coincided with the periodicity observed in the TCE transformation rates . Correspondence analysis of each reactor community showed that different community structures corresponded with function (TCE degradation rate) . Furthermore, the phenol hydroxylase genotypes, as determined by restriction fragment length polymorphism analysis, corresponded to community structure patterns identified by T-RFLP analysis and to periods when the TCE transformation rates were high . Long-term TCE stress appeared to select for a different and stable community structure, with lower but stable TCE degradation rates . In contrast, the community under no stress exhibited a dynamic structure and dynamic function. Appl Environ Microbiol, 2004 Aug, 70(8), 4906 - 10 Investigation of spa pools associated with lung disorders caused by Mycobacterium avium complex in immunocompetent adults; Lumb R et al.; Three cases of Mycobacterium avium complex-related lung disorders were associated with two poorly maintained spa pools by genotypic investigations . Inadequate disinfection of the two spas had reduced the load of environmental bacteria to less than 1 CFU/ml but allowed levels of M . avium complex of 4.3 x 10(4) and 4.5 x 10(3) CFU/ml . Persistence of the disease-associated genotype was demonstrated in one spa pool for over 5 months until repeated treatments with greater than 10 mg of chlorine per liter for 1-h intervals eliminated M . avium complex from the spa pool . A fourth case of Mycobacterium avium complex-related lung disease was associated epidemiologically but not genotypically with another spa pool that had had no maintenance undertaken . This spa pool contained low numbers of mycobacteria by smear and was culture positive for M . avium complex, and the nonmycobacterial organism count was 5.2 x 10(6) CFU/ml . Public awareness about the proper maintenance of private (residential) spa pools must be promoted by health departments in partnership with spa pool retailers. Appl Environ Microbiol, 2004 Aug, 70(8), 4821 - 30 Biogeography, evolution, and diversity of epibionts in phototrophic consortia; Glaeser J et al.; Motile phototrophic consortia are highly regular associations in which numerous cells of green sulfur bacteria surround a flagellated colorless beta-proteobacterium in the center . To date, seven different morphological types of such consortia have been described . In addition, two immotile associations involving green sulfur bacteria are known . By employing a culture-independent approach, different types of phototrophic consortia were mechanically isolated by micromanipulation from 14 freshwater environments, and partial 16S rRNA gene sequences of the green sulfur bacterial epibionts were determined . In the majority of the lakes investigated, different types of phototrophic consortia were found to co-occur . In all cases, phototrophic consortia with the same morphology from the same habitat contained only a single epibiont phylotype . However, morphologically indistinguishable phototrophic consortia collected from different lakes contained different epibionts . Overall, 19 different types of epibionts were detected in the present study . Whereas the epibionts within one geographic region were very similar (Dice coefficient, 0.582), only two types of epibionts were found to occur on both the European and North American continents (Dice coefficient, 0.190) . None of the epibiont 16S rRNA gene sequences have been detected so far in free-living green sulfur bacteria, suggesting that the interaction between epibionts and chemotrophic bacteria in the phototrophic consortia is an obligate interaction . Based on our phylogenetic analysis, the epibiont sequences are not monophyletic . Thus, the ability to form symbiotic associations either arose independently from different ancestors or was present in a common ancestor prior to the radiation of green sulfur bacteria and the transition to the free-living state in independent lineages . The present study thus demonstrates that there is great diversity and nonrandom geographical distribution of phototrophic consortia in the natural environment. Appl Environ Microbiol, 2004 Aug, 70(8), 4692 - 701 Chemotaxis of Silicibacter sp . strain TM1040 toward dinoflagellate products; Miller TR et al.; The alpha-proteobacteria phylogenetically related to the Roseobacter clade are predominantly responsible for the degradation of organosulfur compounds, including the algal osmolyte dimethylsulfoniopropionate (DMSP) . Silicibacter sp . strain TM1040, isolated from a DMSP-producing Pfiesteria piscicida dinoflagellate culture, degrades DMSP, producing 3-methylmercaptopropionate . TM1040 possesses three lophotrichous flagella and is highly motile, leading to a hypothesis that TM1040 interacts with P . piscicida through a chemotactic response to compounds produced by its dinoflagellate host . A combination of a rapid chemotaxis screening assay and a quantitative capillary assay were used to measure chemotaxis of TM1040 . These bacteria are highly attracted to dinoflagellate homogenates; however, the response decreases when homogenates are preheated to 80 degrees C . To help identify the essential attractant molecules within the homogenates, a series of pure compounds were tested for their ability to serve as attractants . The results show that TM1040 is strongly attracted to amino acids and DMSP metabolites, while being only mildly responsive to sugars and the tricarboxylic acid cycle intermediates . Adding pure DMSP, methionine, or valine to the chemotaxis buffer resulted in a decreased response to the homogenates, indicating that exogenous addition of these chemicals blocks chemotaxis and suggesting that DMSP and amino acids are essential attractant molecules in the dinoflagellate homogenates . The implication of Silicibacter sp . strain TM1040 chemotaxis in establishing and maintaining its interaction with P . piscicida is discussed. Chemosphere, 2004 Oct, 57(2), 135 - 45 Monitoring of toxicity during degradation of selected pesticides using ionizing radiation; Drzewicz P et al.; The optimization of experimental conditions for radiolytic removal of organic pollutants from water and waste with the use of ionizing radiation via controlling the concentration of target compound(s) requires also monitoring the toxicity changes during the process . Commonly used herbicides 2,4-D and dicamba were shown to increase toxicity measured with the Microtox test at low irradiation doses resulting from formation of more toxic transient products, which can be decomposed at larger doses . The changes of toxicity were examined with respect to dose magnitude and the presence of commonly occurring scavengers of radiation. Proc R Soc Lond B Biol Sci, 2004 May 22, 271(1543), 1065 - 72 Evolution of the human ABO polymorphism by two complementary selective pressures; Seymour RM et al.; The best-known example of terminal-glycan variation is the ABO histo-blood group polymorphism in humans . We model two selective forces acting on histo-blood group antigens that may account for this polymorphism . The first is generated by the invasion of opportunistic bacterial or other pathogens that interact with the epithelial-mucosal surfaces . The bacteria adapt to the microenvironments of common host phenotypes and so create frequency-dependent selection for rarer host alleles . The second is generated by intracellular viruses, and accounts for the observed differentials between the ABO-phenotype frequencies . It is thought that viruses acquire histo-blood group structures as part of their envelope from their previous host . The presence of host antigens on the viral envelope causes differential transmission of the virus between host types owing to the asymmetric action of ABO natural antibodies . Our model simulations show that these two forces acting together can account for the major features of the ABO polymorphism in humans. Int J Med Microbiol, 2004 Jul, 294(1), 53 - 7 Growth factor production in human endothelial cells after Chlamydia pneumoniae infection; Prochnau D et al.; Seroepidemiological and histopathological studies have suggested a link of atherosclerosis with chronic Chlamydia pneumoniae infection . The present study was designed to examine the effect of C . pneumoniae on expression of basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) in human endothelial cells . Using reverse transcription-polymerase chain reaction, immunoblotting and enzyme-linked immunosorbent assay we found stimulation of bFGF expression depending on the number of infecting bacteria and the time of infection as well . This stimulatory effect was diminished by heat and UV light treatment of the chlamydial inoculum, suggesting that viable bacteria but not bacterial LPS may be essential for eliciting this growth factor . In contrast, the expression of both PDGF-A and PDGF-B was not increased following C . pneumoniae infection . This study demonstrates that C . pneumoniae activates endothelial cells to produce bFGF, a growth factor which is linked to the development of atherosclerotic plaques. J Gastroenterol, 2004 Jul, 39(7), 621 - 8 Comparison between a new 13C-urea breath test, using a film-coated tablet, and the conventional 13C-urea breath test for the detection of Helicobacter pylori infection; Ohara S et al.; BACKGROUND: In Japan, urea breath-testing includes mouth rinsing with water immediately after the ingestion of (13)C-urea solution, to prevent false-positive results that are caused by oral bacteria with urease activity . Our objective was to evaluate the diagnostic performance of a urea breath test using a film-coated (13)C-urea tablet and omitting mouth rinsing . METHODS: The study was a multicenter trial comparing the solution- and tablet-based urea breath tests (UBTs) . Helicobacter pylori status was determined by histology, culture, and rapid urease testing . RESULTS: Of the 255 subjects who completed the study, evaluation of the tablet-based UBT was possible in 254, and comparison of the tablet-based UBT and the solution-based UBT was possible in 250 patients . When the assessment achieved by a combination of biopsy-based methods was used as a reference standard, the sensitivity, specificity, and accuracy of the tablet-based method were determined to be 97.7%, 98.4%, and 98.0%, respectively . When the results of the solution-based UBT were used as a reference standard, the sensitivity, specificity, and accuracy of the tablet-based UBT were determined to be 96.9%, 97.6%, and 97.2%, respectively . CONCLUSIONS: The (13)C-urea tablet-based method proved to be a simple and accurate test for the diagnosis of H . Pylori infection . Mouth rinsing was not required. J Bacteriol, 2004 Aug, 186(16), 5473 - 9 Comparative whole-genome analysis of virulent and avirulent strains of Porphyromonas gingivalis; Chen T et al.; We used Porphyromonas gingivalis gene microarrays to compare the total gene contents of the virulent strain W83 and the avirulent type strain, ATCC 33277 . Signal ratios and scatter plots indicated that the chromosomes were very similar, with approximately 93% of the predicted genes in common, while at least 7% of them showed very low or no signals in ATCC 33277 . Verification of the array results by PCR indicated that several of the disparate genes were either absent from or variant in ATCC 33277 . Divergent features included already reported insertion sequences and ragB, as well as additional hypothetical and functionally assigned genes . Several of the latter were organized in a putative operon in W83 and encoded enzymes involved in capsular polysaccharide synthesis . Another cluster was associated with two paralogous regions of the chromosome with a low G+C content, at 41%, compared to that of the whole genome, at 48% . These regions also contained conserved and species-specific hypothetical genes, transposons, insertion sequences, and integrases and were located adjacent to tRNA genes; thus, they had several characteristics of pathogenicity islands . While this global comparative analysis showed the close relationship between W83 and ATCC 33277, the clustering of genes that are present in W83 but divergent in or absent from ATCC 33277 is suggestive of chromosomal islands that may have been acquired by lateral gene transfer. J Bacteriol, 2004 Aug, 186(16), 5460 - 72 The LuxR homolog ExpR, in combination with the Sin quorum sensing system, plays a central role in Sinorhizobium meliloti gene expression; Hoang HH et al.; Quorum sensing, a population density-dependent mechanism for bacterial communication and gene regulation, plays a crucial role in the symbiosis between alfalfa and its symbiont Sinorhizobium meliloti . The Sin system, one of three quorum sensing systems present in S . meliloti, controls the production of the symbiotically active exopolysaccharide EPS II . Based on DNA microarray data, the Sin system also seems to regulate a multitude of S . meliloti genes, including genes that participate in low-molecular-weight succinoglycan production, motility, and chemotaxis, as well as other cellular processes . Most of the regulation by the Sin system is dependent on the presence of the ExpR regulator, a LuxR homolog . Gene expression profiling data indicate that ExpR participates in additional cellular processes that include nitrogen fixation, metabolism, and metal transport . Based on our microarray analysis we propose a model for the regulation of gene expression by the Sin/ExpR quorum sensing system and another possible quorum sensing system(s) in S . meliloti. Photochem Photobiol, 2004 Jun, 79(6), 551 - 5 Involvement of cyanobacterial phytochromes in growth under different light qualities and quantities; Fiedler B et al.; Inactivation of the genes for the cyanobacterial phytochromes cph1 and cph2 in the unicellular cyanobacterium Synechocystis sp . strain PCC 6803 affected the growth of the cells under certain light conditions . Differences in growth were detected by recording growth curves and in competition experiments . Mutation of cph1 and cph2 resulted in different effects . The cph1-mutant strains exhibited a reduced growth rate under far-red light (FRL), whereas the growth of the cph2-mutant strains was inhibited by red light (RL) . The growth rate of a cph1- / cph2- double mutant was reduced under both RL and FRL . Furthermore, cph1-, cph2- as well as double-mutant strains showed impaired growth under high-light (HL) conditions . Acclimation of the photosynthetic apparatus of the mutants to RL, FRL and HL, as determined by pigment analysis, was similar to that of the wild type. Int J Artif Organs, 2004 Jun, 27(6), 480 - 7 Endotoxin removal from whole blood by a novel adsorption resin: efficiency and hemocompatibility; Amoureux MC et al.; The structural component of Gram- bacteria, endotoxin (ET), induces the release of endogenous mediators of sepsis . Attempts to remove these downstream molecules in vivo, have not improved survival . However, extracorporeal strategies such as continuous renal replacement therapy or therapeutic plasmapheresis have shown benefit . We are presenting an affinity-based extracorporeal technology for the removal of ET from whole blood . The small-scale device contains an adsorbent that removed 75% of ET present in whole blood . This affinity resin displayed good hemocompatibility regarding the coagulation pathway . Minimal platelet, neutrophil and complement activation were observed . There was also no evidence of consumption of coagulation factors or cell loss . In as much as ET participates in both the inflammatory and coagulation abnormalities in sepsis, this method represents an efficient and hemocompatible way to remove ET from whole blood, which, in an extracorporeal setting, may improve the outcome of sepsis. Emerg Radiol, 2002 Nov, 9(5), 292 - 5 Epub 2002 Nov 05. "Cecal gangrene": a rare cause of right-sided inferior abdominal quadrant pain, fever, and leukocytosis; Wiesner W et al.; We report on a 58-year-old man with known diabetes, congestive heart failure, and need for chronic hemodialysis presenting with right lower abdominal quadrant pain, fever, and leukocytosis . Although initial clinical findings were highly suggestive of acute appendicitis, CT revealed marked circumferential wall thickening of the cecum, which was interpreted as cecal infarction by the radiologist . Intraoperatively, cecal necrosis was confirmed, but the ileocecal valve and, especially, the appendix showed no ischemia . No vascular occlusions were found . Histopathologic analysis of the resected cecum demonstrated isolated transmural cecal necrosis with marked infiltration of the cecal wall by numerous bacteria and neutrophils . We present the CT features and histopathologic findings of isolated cecal gangrene, review the pathogenesis of occlusive and nonocclusive cecal ischemia or infarction, and discuss the role of bacterial superinfection as a potential cofactor in the pathogenesis of isolated cecal necrosis which should be included in the differential diagnosis of right-sided inferior abdominal quadrant pain. Chromosoma . 2004 Aug 3; {Epub ahead of print} The mammalian circadian timing system: from gene expression to physiology; Gachon F et al.; Many physiological processes in organisms from bacteria to man are rhythmic, and some of these are controlled by self-sustained oscillators that persist in the absence of external time cues . Circadian clocks are perhaps the best characterized biological oscillators and they exist in virtually all light-sensitive organisms . In mammals, they influence nearly all aspects of physiology and behavior, including sleep-wake cycles, cardiovascular activity, endocrinology, body temperature, renal activity, physiology of the gastro-intestinal tract, and hepatic metabolism . The master pacemaker is located in the suprachiasmatic nuclei, two small groups of neurons in the ventral part of the hypothalamus . However, most peripheral body cells contain self-sustained circadian oscillators with a molecular makeup similar to that of SCN (suprachiasmatic nucleus) neurons . This organization implies that the SCN must synchronize countless subsidiary oscillators in peripheral tissues, in order to coordinate cyclic physiology . In this review, we will discuss some recent studies on the structure and putative functions of the mammalian circadian timing system, but we will also point out some apparent inconsistencies in the currently publicized model for rhythm generation. Int J Mol Med, 2004 Sep, 14(3), 343 - 51 Regularity of distribution of immunoreactive pulmonary surfactant protein A in rat tissues; Luo JM et al.; Existing data has shown that SP-A-like protein or mRNA is widely distributed in lamellar bodies such as tissues and mucosal surfaces . Using immunohistochemistry method with a polyclonal antibody against human SP-A, in this study we investigated distribution of immunoreactive pulmonary surfactant protein A (IR-SP-A) in a number of rat tissues . The SP-A-like immunoreactivity was found in alveolar, parenchyma, pleura of lung; myelin sheath of brain; epithelia of Bowman's capsule, glomerulus and renal tubules of kidney; epithelia of colon, stomach, duct of salivary gland, pharynx; and blood vessel wall and connective tissue of extracellular matrix . The positive signal was blocked by pre-absorbed SP-A antigen from recombinant or bronchoalveolar lavage (BAL) . SP-A has long been considered as an important frontier host defense molecule which participates in immune and inflammatory regulation of lung . With every inhalation, small particles, viruses, bacteria, and antigens from environment are continuously deposited onto the vast pulmonary epithelial surface . While a proper host defense is required to protect the lung, an over-exuberant response can disrupt the appropriate balance between pro- and anti-inflammatory . Traditional Chinese medicine believes that body is an open system relevant to the external environment . The physical, chemical and biological environmental factors constantly affect the open system, and the body properly reacts to maintain homeostasis of body machinery . The Chinese traditional medicine scholars have thus hypothesized that 'Qi' (meaning air) is the communication way between the body and external environment . What is 'Qi'? The results from our study suggest that IR-SP-A is a candidate of 'Qi' . It is compatible with the sites, theoretically containing collagenous and lectin domain molecules, also compatible with the primary injury sites of some autoimmune diseases . SP-A may be as one of 'Qi' molecules mentioned in traditional Chinese medicine that trigger some of autoimmune diseases. Vet Microbiol, 2004 Aug 19, 102(1-2), 55 - 65 Development of two real-time PCR assays for the detection of Mycoplasma hyopneumoniae in clinical samples; Dubosson CR et al.; In order to improve the diagnosis of enzootic pneumonia (EP) in pigs two real-time polymerase chain reaction (rtPCR) assays for the detection of Mycoplasma hyopneumoniae in bronchial swabs from lung necropsies were established and validated in parallel . As a gold standard, the current "mosaic diagnosis" was taken, including epidemiological tracing, clinical signs, macro- and histopathological lesions of the lungs and immunofluorescence . One rtPCR is targeting a repeated DNA element of the M . hyopneumoniae genome (REP assay), the other a putative ABC transporter gene (ABC assay) . Both assays were shown to be specific for M . hyopneumoniae and did not cross react with other bacteria and mollicutes from pig . With material from pigs of defined EP-negative farms the two assays showed to be 100% specific . When testing lungs from pig farms with EP, the REP assay detected 50% and the ABC assay 90% of the farms as positive . Both tests together detected all positive farms . Within a positive herd the two assays tested similarly with on average over 90% of the lung samples analysed from a single farm showing positive scores . A series of samples with suspicion of EP and samples from pigs with diseases other than respiratory taken from current routine diagnostic was assayed . None of the assays showed false positive results . The sensitivities in this sample group were 50% for the REP and 70% for the ABC assays and for both assays together 85% . The two assays run in parallel are therefore a valuable tool for the improvement of the current diagnosis of EP. Biochemistry, 2004 Aug 10, 43(31), 10166 - 72 Catalytic mechanism of S-ribosylhomocysteinase (LuxS): stereochemical course and kinetic isotope effect of proton transfer reactions; Zhu J et al.; S-ribosylhomocysteinase (LuxS) catalyzes the cleavage of the thioether bond in S-ribosylhomocysteine (SRH) to produce homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of type II bacterial quorum sensing molecule . The proposed mechanism involves a series of proton-transfer reactions, which are catalyzed by an Fe2+ ion and two general acids/bases in the LuxS active site, resulting in the migration of the ribose carbonyl group from its C1 to C3 position . Subsequent beta-elimination at C4 and C5 positions completes the catalytic cycle . In this work, the regiochemistry and stereochemical course of the proton transfer reactions were determined by carrying out the reactions using various specifically deuterium-labeled SRH as substrate and analyzing the reaction products by 1H NMR spectroscopy and mass spectrometry . Our data indicate a suprafacial transfer of the ribose C2 proton to its C1 position and the C3 proton to the C2 position during catalysis, whereas the ribose C4 proton is completely washed into solvent . The primary deuterium kinetic isotope effect suggests that the conversion of 2-keto intermediate to 3-keto intermediate is partially rate limiting . However, mutation of Glu-57, the putative second general acid/base in catalysis, to an aspartic acid renders the final beta-elimination step rate limiting. Kansenshogaku Zasshi, 2004 Jun, 78(6), 496 - 502 {Mycoplasma pneumoniae infection in hospitalized children with acute pneumonia under the Mycoplasma epidemic}; Nariai A; Since October 2000, Mycoplasma pneumonia has been a recurring epidemic in Japan . To become clear the importance of Mycoplasma pneumoniae infection in children, we investigated cross-sectionally M . pneumoniae infection by serology in the hospitalized children age under seven years with acute pneumonia retrospectively reviewing pediatric patients of the four studies about lower respiratory tract infection which we had been treated during 2001 to 2003 . Firstly, we found M . pneumoniae infection in 75 patients (33.8%) among a total of 222 patients with asthma exacerbation and acute pneumonia in 2001 . Second, we had evaluated a total of 46 hospitalized children with acute pneumonia for M . pneumoniae infection in November 2002 and 18 patients (39.1%) were found . Thirdly, we found M . pneumoniae infection in 8 patients (34.8%) among 23 patients with respiratory syncitial virus and acute pneumonia age under two years during October 2002 to April 2003 . Fourthly, we found M . pneumoniae infection in 19 patients (35.8%) among 53 patients with asthma exacerbation and acute pneumonia during January from June in 2003 . Even only among the patients age under two years M . pneumoniae infection was found to be 24.3% (16/70), 27.8% (5/8), 34.8% (8/23) and 33.3% (7/21), respectively . These findings demonstrate that M . pneumoniae is common pathogen of acute pneumonia even in infants and young children under Mycoplasma epidemic . Not only typical bacteria and but also M . pneumoniae should be considered as important pathogens in the treatment of acute pneumonia in infants and young children under Mycoplasma epidemic. Bone Marrow Transplant, 2004 Sep, 34(6), 497 - 504 Infectious complications and outcomes after allogeneic hematopoietic stem cell transplantation in Korea; Yoo JH et al.; We reviewed 242 allogeneic hematopoietic stem cell transplantation (HSCT) recipients retrospectively over a 2-year period (January 1998-December 1999) in order to analyze the characteristics and assess the outcomes of infectious complications in patients after HSCT in Korea . Bacteria were the major pathogens before engraftment, and viral and fungal infections predominated during the post-engraftment period . Varicella zoster virus was the most common viral pathogen after engraftment . Cytomegalovirus disease occurred mainly in the late-recovery phase . The frequency of mold infection was higher than that of yeast . There was a relatively high incidence of tuberculosis (3.0%) and Pneumocystis carinii pneumonia (6.5%) . One case of death by measles confirmed by autopsy was also noted . Overall, cumulative mortality was 43% (104/242), and 59.6% of these deaths (62/104) were infection-related . Allogeneic HSCT recipients from unrelated donors were prone to infectious complication and higher mortality than those from matched sibling (17/39 (43.6%) vs 45/203 (22.2%), respectively; P<0.01; odd ratio 2.5; 95% confidence interval 1.2-5.1) . As infection was the main post-HSCT complication in our data, more attention should be given to the management of infections in HSCT recipients. Crit Care Med, 2004 Aug, 32(8), 1759 - 63 Role of Toll-like receptor 4 on pancreatic and pulmonary injury in a mice model of acute pancreatitis associated with endotoxemia; Pastor CM et al.; OBJECTIVE: Infection of pancreatic necrosis is a severe complication of acute pancreatitis . Because Toll-like receptor 4 (TLR4) has been identified as a receptor necessary to transduct the signal of bacteria-derived lipopolysaccharide into cells, we investigated the role of TLR4 on pancreatic and pulmonary injury in acute pancreatitis and acute pancreatitis associated with endotoxemia in wild-type and TLR4-deficient mice . DESIGN: Laboratory investigation . SETTING: University laboratory . SUBJECTS: Heterozygous TLR4 mice . INTERVENTIONS: Mice were injected intraperitoneally with a supramaximal dose of cerulein each hour for 10 hrs . To mimic infection, additional groups of mice were injected with lipopolysaccharide in the presence or absence of cerulein injections . MEASUREMENTS AND MAIN RESULTS: The severity of acute pancreatitis was assessed by serum amylase activity, pancreatic edema, acinar cell necrosis, and pancreas myeloperoxidase activity . Lung injury was quantitated by lung microvascular permeability and lung myeloperoxidase activity . Injections of cerulein induced an edematous pancreatitis that was of similar severity in wild-type and TLR4-deficient mice . Lipopolysaccharide alone had no toxic effect on pancreas and lungs and did not worsen the pancreatic injury induced by cerulein in wild-type and TRL4-deficient mice . In contrast, lipopolysaccharide worsened pancreatitis-associated lung injury, and the deficiency in TLR4 fully prevented this aggravation . CONCLUSIONS: TLR4 may not play a role in the pancreatitis-associated lung injury but participates in the pulmonary injury mediated by endotoxemia. Mol Biol (Mosk), 2004 May-Jun, 38(3), 524 - 31 {Survival strategy of photosynthetic organisms . 2 . Experimental proof of the size variability of the unit building block of light-harvesting oligomeric antenna}; Iakovlev AG et al.; The present series of papers is part of an integrated research program to understand the effective functional strategy of native light-harvesting molecular antennae in photosynthetic organisms . This work tackles the problem of the structural optimization of light-harvesting antennae of variable size . In vivo, the size responds to the illumination intensity, thus implying more sophisticated optimization strategies, since larger antenna size demands finer structural tuning . Earlier modeling experiments showed that the aggregation of the antenna pigments, apart from being itself a universal structural factor of functional antenna optimization with any (!) spatial lattice of light-harvesting molecules, determines the antenna performance provided that the degree of aggregation varies: the larger the unit building block, the higher the efficacy of the whole structure . It means that altering the degree of pigment aggregation in response to the antenna size is biologically expedient . In the case of the oligomeric chlorosomal antenna of green bacteria, the strategy of variable antenna structural optimization in response to the illumination intensity was demonstrated to take place in vivo and facilitate high antenna performance regardless of its size, thus allowing bacteria to survive in diverse illumination conditions. Arh Hig Rada Toksikol, 2004 Jun, 55(2-3), 213 - 20 Organic aerosols and the development of allergic disorders; Kanceljak-Macan B et al.; The aim of this study was to investigate skin reactivity to organic dust extracts and total serum IgE and their relation to the prevalence of respiratory symptoms and ventilatory capacity in workers occupationally exposed to organic aerosols . It included workers employed in processing coffee, tea, dried fruits, spices, animal food, soy, hemp, cotton, swine farmers, and control groups of workers non-exposed to organic dust . All underwent a skin prick test (SPT) with water extracts of organic dust 1:10 w/v, Dermatophagoides pteronyssinus, mixed moulds, bacteria, histamin solution (1 mg/ml) and buffer solution . SPT was considered positive if the diameter of the observed wheal was 3 mm greater than that of buffer solution . The total IgE was measured by the PRIST method (Pharmacia Diagnostics AB, Upsala) and the values > 125 kU/L were considered increased . Data on respiratory symptoms were collected by standardized questionnaire . Ventilatory capacity was measured by recording MEFV curve . Airborne industrial dust were measured as total and respirable fraction . The exposed workers had a greater prevalence of positive SPT to organic dust extracts, except in soy processing . Increased IgE was found in workers processing coffee, tea, hemp, cotton and animal food, compared to non-exposed workers (P<0.05) . Workers with positive SPT had a significantly higher total IgE . As there was no correlation between acute and chronic changes in ventilatory function, positive SPT, and level of total IgE, our findings could not predict objective respiratory impairment. Sheng Li Ke Xue Jin Zhan, 2004 Apr, 35(2), 139 - 42 {The role of myeloid differential protein-2 in innate immunity}; Xu FL et al.; As typical PRRs (pattern-recognition receptors), TLRs (Toll-like receptors) play an important role during innate immunity recognition . MD-2 (myeloid differential protein-2) may contain distinct functional domains that can separately and simultaneously bind TLRs (TLR4 or TLR2) and TLR ligands, such as lipopolysaccharide (LPS) . The special structure of MD-2 may result in its three main functions: (1) An association with TLR4 that amplifies TLR4 responsiveness to ligands, especially LPS . (2) Enabling TLR2-mediated responses to LPS and enhancing TLR2-mediated responses to bacteria and their cell wall components . (3) Increasing the expression of TLR2 and TLR4 and possibly influencing the correct intracellular distribution of TLR4 . Importantly, while MD-2 regulation of TLR expression and distribution is well established, determining whether the interaction is direct or not will require further study . Thus, MD-2 is not only an assistant molecule of TLR4 but is also a key regulatory molecule in innate immunity, and may play important roles during infection, inflammation, immune responses and many other pathologic and physiologic processes. Environ Toxicol Chem, 2004 Mar, 23(3), 606 - 12 Derivation of a chronic site-specific water quality standard for selenium in the Great Salt Lake, Utah, USA; Brix KV et al.; The purpose of this study was to develop a site-specific water quality standard for selenium in the Great Salt Lake, Utah, USA . The study examined the bioavailability and toxicity of selenium, as selenate, to biota resident to the Great Salt Lake and the potential for dietary selenium exposure to aquatic dependent birds that might consume resident biota . Because of its high salinity, the lake has limited biological diversity with bacteria, algae, diatoms, brine shrimp, and brine flies being the only organisms present in the main (hypersaline) portions of the lake . To evaluate their sensitivity to selenium, a series of acute and chronic toxicity studies were conducted on brine shrimp (Artemia franiciscana), brine fly (Ephydra cinerea), and a hypersaline alga (Dunaliella viridis) . The resulting acute and chronic toxicity data indicated that resident species are more selenium tolerant than many freshwater species . Because sulfate is known to reduce selenate bioavailability, this selenium tolerance is thought to result in part from the lake's high ambient sulfate concentrations (>5,800 mg/L) . The acute and chronic test results were compared to selenium concentrations expected to occur in a mining effluent discharge located at the south end of the lake . Based on these comparisons, no appreciable risks to resident aquatic biota were projected . Field and laboratory data collected on selenium bioaccumulation in brine shrimp demonstrated a linear relationship between water and tissue selenium concentrations . Applying a dietary selenium threshold of 5 mg/kg dry weight for aquatic birds to this relationship resulted in an estimate of 27 microg/L Se in water as a safe concentration for this exposure pathway and an appropriate chronic site-specific water quality standard . Consequently, protection of aquatic birds represents the driving factor in determining a site-specific water quality standard for selenium. World J Gastroenterol, 2004 Aug 15, 10(16), 2455 - 6 A case of leptospirosis simulating colon cancer with liver metastases; Granito A et al.; We report a case of a 61-year-old man who presented with fatigue, abdominal pain and hepatomegaly . Computed tomography (CT) of the abdomen showed hepatomegaly and multiple hepatic lesions highly suggestive of metastatic diseases . Due to the endoscopic finding of colon ulcer, colon cancer with liver metastases was suspected . Biochemically a slight increase of transaminases, alkaline phosphatase and gammaglutamyl transpeptidase were present; alpha-fetoprotein, carcinoembryogenic antigen and carbohydrate 19-9 antigen serum levels were normal . Laboratory and instrumental investigations, including colon and liver biopsies revealed no signs of malignancy . In the light of spontaneous improvement of symptoms and CT findings, his personal history was reevaluated revealing direct contact with pigs and their tissues . Diagnosis of leptospirosis was considered and confirmed by detection of an elevated titer of antibodies to leptospira . After two mo, biochemical data, CT and colonoscopy were totally normal. World J Gastroenterol, 2004 Aug 15, 10(16), 2334 - 9 Helicobacter pylori enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in human gastric epithelial cells; Wu YY et al.; AIM: To investigate the relations between tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Helicobacter pylori (H pylori) infection in apoptosis of gastric epithelial cells and to assess the expression of TRAIL on the surface of infiltrating T-cells in H pylori-infected gastric mucosa . METHODS: Human gastric epithelial cell lines and primary gastric epithelial cells were co-cultured with H pylori in vitro, then recombinant TRAIL proteins were added to the culture . Apoptosis of gastric epithelial cells was determined by a specific ELISA for cell death . Infiltrating lymphocytes were isolated from H pylori-infected gastric mucosa, and expression of TRAIL in T cells was analyzed by flow cytometry . RESULTS: The apoptosis of gastric epithelial cell lines and primary human gastric epithelial cells was mildly increased by interaction with either TRAIL or H pylori alone . Interestingly, the apoptotic indices were markedly elevated when gastric epithelial cells were incubated with both TRAIL and H pylori (Control vs TRAIL and H pylori: 0.51+/-0.06 vs 2.29+/-0.27, P = 0.018) . A soluble TRAIL receptor (DR4-Fc) could specifically block the TRAIL-mediated apoptosis . Further studies demonstrated that infiltrating T-cells in gastric mucosa expressed TRAIL on their surfaces, and the induction of TRAIL sensitivity by H pylori was dependent upon direct cell contact of viable bacteria, but not CagA and VacA of H pylori . CONCLUSION: H pylori can sensitize human gastric epithelial cells and enhance susceptibility to TRAIL-mediated apoptosis . Modulation of host cell sensitivity to apoptosis by bacterial interaction adds a new dimension to the immunopathogenesis of H pylori infection. Can J Microbiol, 2004 Jun, 50(6), 383 - 96 {The genome of alpha-proteobacteria : complexity, reduction, diversity and fluidity.}; Teyssier C et al.; The alpha-proteobacteria displayed diverse and often unconventional life-styles . In particular, they keep close relationships with the eucaryotic cell . Their genomic organization is often atypical . Indeed, complex genomes, with two or more chromosomes that could be linear and sometimes associated with plasmids larger than one megabase, have been described . Moreover, polymorphism in genome size and topology as well as in replicon number was observed among very related bacteria, even in a same species . Alpha-proteobacteria provide a good model to study the reductive evolution, the role and origin of multiple chromosomes, and the genomic fluidity . The amount of new data harvested in the last decade should lead us to better understand emergence of bacterial life-styles and to build the conceptual basis to improve the definition of the bacterial species. Ann Ital Chir, 2004 Jan-Feb, 75(1), 11 - 6 {Risk factors of surgical wound infection}; Fabiano G et al.; Surgical Site Infection (SSI) continues to be a major source of morbidity following operative procedures . The aging of the population means that not only will the number of operations likely increase, but the National Nosocomial Infections Surveillance (NNIS) Risk Index, which standardizes the risk of SSI for an aging population, will be greater . The NNIS report for 1986-1996 described an SSI rate of 2.6% for all operations at the reporting hospitals . It seems likely that overall SSI rates are likely to be greater than reported . All surgical wounds are contaminated by bacteria, but only a minority actually demonstrate clinical infection . The SSI are the biological summation of several factors: the inoculum of bacteria introduced into the wound during the procedure, the unique virulence of contaminants, the microenvironment of each wound, and the integrity of the patients host defense mechanisms . Risk factors were studied in single and multivariate analyses . Although an SSI rate of zero may not be achievable, continued progress in understanding the biology of infection at the surgical site and consistent applications of proven methods of prevention will allow us to further reduce the frequency, cost, and morbidity associated with SSI. Indian J Ophthalmol, 2004 Jun, 52(2), 127 - 31 Sterile endophthalmitis in vitrectomised eyes due to suspected heat resistant endotoxins in the infusion fluid; Patnaik B et al.; PURPOSE: To report on to the possibility of development of severe postoperative sterile endophthalmitis due to heat-resistant bacterial endotoxins in commercially available infusion fluids METHODS: A case study of 4 eyes that had previously undergone vitreoretinal surgery, which developed clinical endophthalmitis within 18 hours of surgery and two eyes that had vitreous surgery with intraocular gas and did not develop clinical endophthalmitis following intraocular surgery on three consecutive operative days RESULTS: The vitreous samples were sterile, both for bacteria and fungi . The only common supply in all cases was a new batch of Ringer's lactate infusion fluid . Though the Ringer's lactate solutions in the same batch were also sterile, the infusion fluids contained abnormally high levels of bacterial endotoxins detected by gel clot method . CONCLUSION: Commercially available infusion fluid may be sterile, yet contain endotoxins from killed bacteria . This could cause postoperative sterile endophthalmitis. J Mass Spectrom, 2004 Jul, 39(7), 752 - 61 Collision-induced dissociation tandem mass spectrometry of desferrioxamine siderophore complexes from electrospray ionization of UO2(2+), Fe3+ and Ca2+ solutions; Groenewold GS et al.; Desferrioxamine (DEF) is a trihydroxamate siderophore typical of those produced by bacteria and fungi for the purpose of scavenging Fe(3+) from environments where the element is in short supply . Since this class of molecules has excellent chelating properties, reaction with metal contaminants such as actinide species can also occur . The complexes that are formed can be mobile in the environment . Because the natural environment is extremely diverse, strategies are needed for the identification of metal complexes in aqueous matrices having a high degree of chemical heterogeneity, and electrospray ionization mass spectrometry (ESI-MS) has been highly effective for the characterization of siderophore-metal complexes . In this study, ESI-MS of solutions containing DEF and either UO(2)(2+), Fe(3+) or Ca(2+) resulted in generation of abundant singly charged ions corresponding to {UO(2)(DEF - H)}(+), {Fe(DEF - 2H)}(+) and {Ca(DEF - H)}(+) . In addition, less abundant doubly charged ions were produced . Mass spectrometry/mass spectrometry (MS/MS) studies of collision-induced dissociation (CID) reactions of protonated DEF and metal-DEF complexes were contrasted and rationalized in terms of ligand structure . In all cases, the most abundant fragmentation reactions involved cleavage of the hydroxamate moieties, consistent with the idea that they are most actively involved with metal complexation . Singly charged complexes tended to be dominated by cleavage of a single hydroxamate, while competitive fragmentation between two hydroxamate moieties increased when the doubly charged complexes were considered . Rupture of amide bonds was also observed, but these were in general less significant than the hydroxamate fragmentations . Several lower abundance fragmentations were unique to the metal examined: abundant loss of H(2)O occurred only for the singly charged UO(2)(2+) complex . Further, NH(3) was eliminated only from the singly charged Fe(3+) complex; this and fragmentation of C-C and C-N bonds derived from neither the hydroxamate nor the amide groups suggested that Fe(3+) insertion reactions were competing with ligand complexation . In no experiments were coordinating solvent molecules observed, attached either to the intact complexes or to the fragment ions, which indicated that both intact DEF and its fragments were occupying all of the coordination sites around the metal centers . This conclusion was based on previous experiments that showed that undercoordinated UO(2)(2+) and Fe(3+) readily added H(2)O and methanol in the ESI quadrupole ion trap mass spectrometer that was used in this study. J Antimicrob Chemother, 2004 Sep, 54(3), 609 - 16 Epub 2004 Jul 28. In vitro effects of nitazoxanide on Echinococcus granulosus protoscoleces and metacestodes; Walker M et al.; OBJECTIVES: Infection of humans and domestic ruminants with the larval stage (metacestode) of Echinococcus granulosus results in cystic echinococcosis (CE) . The metacestode causes a space-occupying lesion in visceral organs, most commonly in the liver . Benzimidazole carbamate derivatives, such as mebendazole and albendazole, are currently used for chemotherapeutic treatment of CE . In human patients, benzimidazoles have to be applied in high doses for extended periods of time, and adverse side effects are frequently observed . In order to evaluate alternative treatment options, the in vitro efficacy of nitazoxanide, a broad-spectrum drug used against intestinal parasites and bacteria, was investigated . METHODS: Freshly isolated E . granulosus protoscoleces were subjected to nitazoxanide treatment (1, 5 and 10 microg/mL), and the effects on parasite viability were monitored by Trypan Blue staining and scanning electron microscopy . Protoscolex cultures were maintained further, until metacestode development took place . Metacestodes were then subjected to nitazoxanide treatment (10 microg/mL), and corresponding effects were visualized by scanning and transmission electron microscopy . RESULTS: Dose-dependent protoscolex death within a few days of nitazoxanide treatment was observed . Subsequent in vitro culture of drug-treated protoscoleces confirmed the non-viability of parasites, while further cultivation of non-treated protoscoleces for a period of at least 3 months resulted in stage conversion and the formation of small metacestodes 3-4 mm in diameter . Nitazoxanide had a deleterious effect on these metacestodes, which was comparable to that of albendazole . CONCLUSIONS: Our study indicates a potential for nitazoxanide as an alternative treatment option against CE. Am J Physiol Cell Physiol, 2004 Dec, 287(6), C1547 - 59 Epub 2004 Jul 28. Detection of intracellular iron by its regulatory effect; Li JY et al.; Intracellular iron regulates gene expression by inhibiting the interaction of iron regulatory proteins (IRPs) with RNA motifs called iron-responsive elements (IREs) . To assay this interaction in living cells we have developed two fluorescent IRE-based reporters that rapidly, reversibly, and specifically respond to changes in cellular iron status as well as signaling that modifies IRP activity . The reporters were also sufficiently sensitive to distinguish apo- from holotransferrin in the medium, to detect the effect of modifiers of the transferrin pathway such as HFE, and to detect the donation or chelation of iron by siderophores bound to the lipocalin neutrophil gelatinase-associated lipocalin (Ngal) . In addition, alternative configurations of the IRE motif either enhanced or repressed fluorescence, permitting a ratio analysis of the iron-dependent response . These characteristics make it possible to visualize iron-IRP-IRE interactions in vivo. Biochim Biophys Acta, 2004 Jul 23, 1658(1-2), 95 - 105 Protonmotive cooperativity in cytochrome c oxidase; Papa S et al.; Cooperative linkage of solute binding at separate binding sites in allosteric proteins is an important functional attribute of soluble and membrane bound hemoproteins . Analysis of proton/electron coupling at the four redox centers, i.e . Cu(A), heme a, heme a(3) and Cu(B), in the purified bovine cytochrome c oxidase in the unliganded, CO-liganded and CN-liganded states is presented . These studies are based on direct measurement of scalar proton translocation associated with oxido-reduction of the metal centers and pH dependence of the midpoint potential of the redox centers . Heme a (and Cu(A)) exhibits a cooperative proton/electron linkage (Bohr effect) . Bohr effect seems also to be associated with the oxygen-reduction chemistry at the heme a(3)-Cu(B) binuclear center . Data on electron transfer in cytochrome c oxidase are also presented, which, together with structural data, provide evidence showing the occurrence of direct electron transfer from Cu(A) to the binuclear center in addition to electron transfer via heme a . A survey of structural and functional data showing the essential role of cooperative proton/electron linkage at heme a in the proton pump of cytochrome c oxidase is presented . On the basis of this and related functional and structural information, variants for cooperative mechanisms in the proton pump of the oxidase are examined. J Environ Pathol Toxicol Oncol, 1999, 18(4), 323 - 34 Transmission electron microscopy findings in the respiratory epithelium of guinea pigs exposed to the polluted air of southwest Mexico City; Villegas-Castrejon H et al.; We evaluated the nature and the extent of the damage to the respiratory epithelium of guinea pigs after a 4-month exposure to the mixture of air pollutants in southwest Mexico City . Guinea pigs were placed outdoors on the roof of our facility, 8 hours daily, from February to May 1995 . At the same time, control guinea pigs were kept indoors breathing filtered air . Air pollutants, temperature, and humidity data were obtained from the nearest station of the Environmental Monitoring Net . The airways and lung parenchyma were analyzed after 120 days using transmission electron microscopy (TEM) . During the 4-month exposure period, ozone (O3) exceeded the norm during 511 hours, and suspended particles less than 10 microm in diameter (PM10) during 52 hours . Both pollutants reached peak levels of more than twice the norm . TEM revealed no important abnormalities in the control group . In the exposed group, there was loss of cilia, detachment of epithelial cells, and eosinophil and macrophage migration toward alveolar spaces through type I pneumocytes with destruction of their basal membranes . In six guinea pigs in the exposed group, we noted bacteria along the airways, with associated inflammatory response . We explain the colonization of the respiratory epithelium by bacteria as the result of the impairment on the defense mechanism caused by the exposure to environmental O3 and PM10. Proteins, 2004 Sep 1, 56(4), 795 - 807 The SHS2 module is a common structural theme in functionally diverse protein groups, like Rpb7p, FtsA, GyrI, and MTH1598/TM1083 superfamilies; Anantharaman V et al.; Using structural comparisons, we identified a novel domain with a simple fold in the bacterial cell division ATPase FtsA, the archaeo-eukaryotic RNA polymerase subunit Rpb7p, the GyrI superfamily, and the uncharacterized MTH1598/Tm1083-like proteins . The fold contains a core of 3 strands, forming a curved sheet, and a single helix in a strand-helix-strand-strand (SHS2) configuration . The SHS2 domain may exist either in single or duplicate copies within the same polypeptide . The single-copy versions of the domain in FtsA and Rbp7p are most closely related, and appear to mediate protein-protein interactions by means of strand 1, and the loop between strand 2 and strand 3 of the domain . We predict that the interactions between FtsA and its functional partners in bacterial cell division are likely to be similar to the interactions of Rbp7p in the archaeo-eukaryotic RNA polymerase complex . The dimeric versions typified by the GyrI superfamily appear to have been adapted for small-molecule binding . Sequence profiles searches helped us to identify several new versions of the GyrI superfamily, including a family of secreted forms that is found only in animals and the bacterial pathogen Leptospira . Through sequence-structure comparisons, we predict the positions that are likely to be important for ligand specificity in the GyrI superfamily . In the MTH1598/Tm1083-like proteins, a SHS2 domain is inserted into the loop between strand 1 and helix 1 of another SHS2 domain . This has resulted in a structure that has convergent similarities with the Hsp33 and green fluorescent protein folds . The sequence conservation pattern and its phyletic profile suggest that it might function as an enzyme in some conserved aspect of nucleic acid metabolism . Thus, the SHS2 domain is an example of a simple module that has been adapted to perform an entire spectrum of functions ranging from protein-protein interactions to small-molecule recognition and catalysis . J Clin Pathol, 2004 Aug, 57(8), 822 - 8 Topographical localisation of cagA positive and cagA negative Helicobacter pylori strains in the gastric mucosa; an in situ hybridisation study; Camorlinga-Ponce M et al.; BACKGROUND: The cagA gene is a marker for the presence of the cag pathogenicity island, and the presence of cagA positive strains of Helicobacter pylori can identify individuals with a higher risk of developing gastrointestinal diseases . AIMS: To study the interaction between H . pylori cagA(+) and cagA(-) strains and the gastric mucosa . METHODS: Patients with H . pylori associated gastritis and peptic ulcers were studied . Biopsies were obtained from the antrum, corpus, fundus, and incisura for H pylori culture, and for in situ hybridisation studies . From each biopsy, multiple single H . pylori colonies were isolated and propagated for DNA isolation, and cagA was detected by the polymerase chain reaction (PCR) . For in situ detection of H . pylori an oligonucleotide specific for an H . pylori common antigen and an oligonucleotide specific for cagA were used as probes . Biotinylated probes were incubated with biopsy sections, developed with streptavidin-horseradish peroxidase, and amplified with the tyramide system . RESULTS: PCR results for cagA in isolated colonies confirmed the in situ hydridisation studies . In situ hybridisation identified cagA(+) bacteria in patients with cagA(+) isolates; cagA(-) bacteria in patients with cagA(-) isolates, and cagA(+) and cagA (-) bacteria in patients with both cagA(+) and cagA(-) isolates . CagA(-) bacteria usually colonised the mucous gel or the apical epithelial surface, whereas cagA(+) bacteria colonised the immediate vicinity of epithelial cells or the intercellular spaces . CONCLUSIONS: These results document a different in vivo interaction between H . pylori cagA(+) or cagA(-) strains and the gastric mucosa. J Biol Chem, 2004 Oct 15, 279(42), 44046 - 56 Epub 2004 Jul 27. Mapping sites of potential proximity between the dihydropyridine receptor and RyR1 in muscle using a cyan fluorescent protein-yellow fluorescent protein tandem as a fluorescence resonance energy transfer probe; Papadopoulos S et al.; Excitation-contraction coupling in skeletal muscle involves conformational coupling between the dihydropyridine receptor (DHPR) and the type 1 ryanodine receptor (RyR1) at junctions between the plasma membrane and sarcoplasmic reticulum . In an attempt to find which regions of these proteins are in close proximity to one another, we have constructed a tandem of cyan and yellow fluorescent proteins (CFP and YFP, respectively) linked by a 23-residue spacer, and measured the fluorescence resonance energy transfer (FRET) of the tandem either in free solution or after attachment to sites of the alpha1S and beta1a subunits of the DHPR . For all of the sites examined, attachment of the CFP-YFP tandem did not impair function of the DHPR as a Ca2+ channel or voltage sensor for excitation-contraction coupling . The free tandem displayed a 27.5% FRET efficiency, which decreased significantly after attachment to the DHPR subunits . At several sites examined for both alpha1S (N-terminal, proximal II-III loop of a two fragment construct) and beta1a (C-terminal), the FRET efficiency was similar after expression in either dysgenic (alpha1S-null) or dyspedic (RyR1-null) myotubes . However, compared with dysgenic myotubes, the FRET efficiency in dyspedic myotubes increased from 9.9 to 16.7% for CFP-YFP attached to the N-terminal of beta1a, and from 9.5 to 16.8% for CFP-YFP at the C-terminal of alpha1S . Thus, the tandem reporter suggests that the C terminus of alpha1S and the N terminus of beta1a may be in close proximity to the ryanodine receptor. J Biol Chem, 2004 Oct 15, 279(42), 44057 - 64 Epub 2004 Jul 27. Metabolic biotinylation as a probe of supramolecular structure of the triad junction in skeletal muscle; Lorenzon NM et al.; Excitation-contraction coupling in skeletal muscle involves conformational coupling between dihydropyridine receptors (DHPRs) in the plasma membrane and ryanodine receptors (RyRs) in the sarcoplasmic reticulum . However, it remains uncertain what regions, if any, of the two proteins interact with one another . Toward this end, it would be valuable to know the spatial interrelationships of DHPRs and RyRs within plasma membrane/sarcoplasmic reticulum junctions . Here we describe a new approach based on metabolic incorporation of biotin into targeted sites of the DHPR . To accomplish this, cDNAs were constructed with a biotin acceptor domain (BAD) fused to selected sites of the DHPR, with fluorescent protein (XFP) attached at a second site . All of the BAD-tagged constructs properly targeted to junctions (as indicted by small puncta of XFP) and were functional for excitation-contraction coupling . To determine whether the introduced BAD was biotinylated and accessible to avidin (approximately 60 kDa), myotubes were fixed, permeablized, and exposed to fluorescently labeled avidin . Upon expression in beta1-null or dysgenic (alpha1S-null) myotubes, punctate avidin fluorescence co-localized with the XFP puncta for BAD attached to the beta1a N- or C-terminals, or the alpha1S N-terminal or II-III loop . However, BAD fused to the alpha1S C-terminal was inaccessible to avidin in dysgenic myotubes (containing RyR1) . In contrast, this site was accessible to avidin when the identical construct was expressed in dyspedic myotubes lacking RyR1 . These results indicate that avidin has access to a number of sites of the DHPR within fully assembled (RyR1-containing) junctions, but not to the alpha1S C-terminal, which appears to be occluded by the presence of RyR1. Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1333 - 44 Comparative analysis of ribonuclease P RNA of the planctomycetes; Butler MK et al.; The planctomycetes, order Planctomycetales, are a distinct phylum of domain Bacteria . Genes encoding the RNA portion of ribonuclease P (RNase P) of some planctomycete members were sequenced and compared with existing database planctomycete sequences . rnpB gene sequences encoding RNase P RNA were generated by a conserved primer PCR strategy for Planctomyces brasiliensis, Planctomyces limnophilus, Pirellula marina, Pirellula staleyi strain ATCC 35122, Isosphaera pallida, one other Isosphaera strain, Gemmata obscuriglobus and three other strains of the Gemmata group . These sequences were aligned against reference bacterial sequences and secondary structures of corresponding RNase P RNAs deduced by a comparative approach . P12 helices were found to be highly variable in length, as were helices P16.1 and P19, when present . RNase P RNA secondary structures of Gemmata isolates were found to have unusual features relative to other planctomycetes, including a long P9 helix and an insert in the P13 helix not found in any other member of domain Bacteria . These unique features are consistent with other unusual properties of this genus, distinguishing it from other bacteria . Phylogenetic analyses indicate that relationships between planctomycetes derived from RNase P RNA are consistent with 16S rRNA-based analyses. Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1203 - 7 Hydrocarboniphaga effusa gen . nov., sp . nov., a novel member of the gamma-Proteobacteria active in alkane and aromatic hydrocarbon degradation; Palleroni NJ et al.; Novel alkane-degrading strains of bacteria were isolated from soil contaminated with fuel oil from a leaking underground tank in New Jersey, USA . Two phenotypically similar strains (designated AP102 and AP103T) possessed 16S rRNA sequences unique among the majority of known hydrocarbon-degrading bacteria . The 16S rRNA sequences showed a moderate but distant relationship to the genus Nevskia and a substantial similarity to strains that had previously been isolated for growth on phenol (in Japan) and on toluene (in Canada) by other researchers . The hydrocarbon-degrading strains from Japan, Canada and New Jersey showed no resemblance to the typical morphology of Nevskia but did share a striking similarity among themselves in cell morphology, in the unusual appearance of colonies on various solid media and in various physiological properties . A full taxonomic analysis was performed, including DNA-DNA hybridization and nutritional screening with 117 organic compounds as sole sources of carbon and energy . The strains are active in the degradation of important environmental pollutants, and their phenotypic, physiological, metabolic and genomic properties suggest that they are members of a novel taxon in the gamma-Proteobacteria, for which the name Hydrocarboniphaga gen . nov . is proposed, with the single species Hydrocarboniphaga effusa sp . nov . The type strain is AP103T (=ATCC BAA-332T=DSM 16095T). Development, 2004 Sep, 131(17), 4225 - 37 Epub 2004 Jul 27. Real-time lineage analysis reveals oriented cell divisions associated with morphogenesis at the shoot apex of Arabidopsis thaliana; Reddy GV et al.; Precise knowledge of spatial and temporal patterns of cell division, including number and orientation of divisions, and knowledge of cell expansion, is central to understanding morphogenesis . Our current knowledge of cell division patterns during plant and animal morphogenesis is largely deduced from analysis of clonal shapes and sizes . But such an analysis can reveal only the number, not the orientation or exact rate, of cell divisions . In this study, we have analyzed growth in real time by monitoring individual cell divisions in the shoot apical meristems (SAMs) of Arabidopsis thaliana . The live imaging technique has led to the development of a spatial and temporal map of cell division patterns . We have integrated cell behavior over time to visualize growth . Our analysis reveals temporal variation in mitotic activity and the cell division is coordinated across clonally distinct layers of cells . Temporal variation in mitotic activity is not correlated to the estimated plastochron length and diurnal rhythms . Cell division rates vary across the SAM surface . Cells in the peripheral zone (PZ) divide at a faster rate than in the central zone (CZ) . Cell division rates in the CZ are relatively heterogeneous when compared with PZ cells . We have analyzed the cell behavior associated with flower primordium development starting from a stage at which the future flower comprises four cells in the L1 epidermal layer . Primordium development is a sequential process linked to distinct cellular behavior . Oriented cell divisions, in primordial progenitors and in cells located proximal to them, are associated with initial primordial outgrowth . The oriented cell divisions are followed by a rapid burst of cell expansion and cell division, which transforms a flower primordium into a three-dimensional flower bud . Distinct lack of cell expansion is seen in a narrow band of cells, which forms the boundary region between developing flower bud and the SAM . We discuss these results in the context of SAM morphogenesis. FEBS Lett, 2004 Jul 30, 571(1-3), 212 - 6 Nucleoside diphosphate kinase of Mycobacterium tuberculosis acts as GTPase-activating protein for Rho-GTPases; Chopra P et al.; Several bacterial pathogens secrete proteins into the host cells that act as GTPase-activating proteins (GAPs) for Rho-GTPases and convert GTP-bound active form to GDP-bound inactive form . However, no such effector molecule has been identified in Mycobacterium tuberculosis . In this study, we show that culture supernatant of M . tuberculosis H(37)Rv harbors a protein that stimulates the conversion of GTP-bound Rho-GTPases to the GDP-bound form . Nucleoside diphosphate kinase (Ndk) was identified as this culture supernatant protein that stimulated in vitro GTP hydrolysis by members of Rho-GTPases . The histidine-117 mutant of Ndk, which is impaired for autophosphorylation and nucleotide-binding activities, shows GAP activity . These results suggest that Ndk of M . tuberculosis functions as a Rho-GAP to downregulate Rho-GTPases, and this activity may aid in pathogenesis of the bacteria. FEBS Lett, 2004 Jul 30, 571(1-3), 61 - 6 NO-degradation by alfalfa class 1 hemoglobin (Mhb1): a possible link to PR-1a gene expression in Mhb1-overproducing tobacco plants; Seregelyes C et al.; Tobacco plants overproducing alfalfa class 1 hemoglobin (HOT plants) have been shown to have reduced necrotic symptom development . Here, we show that this altered pathogenic response is linked to a significant increase in the nitric oxide (NO)-affected pathogenesis-related (PR-1a) transcript accumulation in the transgenic plants . Homogenates of HOT transgenic seedlings were also found to have higher NO-scavenging activity than non-transformed ones . The NO-scavenging properties of recombinant alfalfa class1 hemoglobin have been examined . Recombinant Mhb1 (rMhb1) was produced in bacteria and purified using polyethylene glycol (10-25%) fractionation, chromatography on DEAE-Sephacel, and Phenyl Superose columns . After the final purification step, the obtained preparations were near homogeneous and had a molecular weight of 44 kDa determined by size-exclusion chromatography and 23 kDa by SDS-PAGE, indicating that rMhb1 is a dimer . The protein participated in NO-degradation activity with NAD(P)H as a cofactor . After ion-exchange columns, addition of FAD was necessary for exhibiting maximal NO-degradation activity . The NAD(P)H-dependent NO-scavenging activity of rMhb1, which is similar to that of barley hemoglobin, supports a conclusion that both monocot and dicot class 1 hemoglobins can affect cellular NO levels by scavenging NO formed during hypoxia, pathogen attack and other stresses. J Microbiol Methods, 2004 Sep, 58(3), 361 - 6 Chemotactic responses of the fish-parasitic scuticociliate Philasterides dicentrarchi to blood and blood components of the turbot Scophthalmus maximus, evaluated using a new microplate multiassay; Parama A et al.; This study describes a new capillary-type microplate multiassay for characterization of protozoal chemotactic responses, allowing up to 32 assays to be run simultaneously . We used the new multiassay to evaluate the chemoattractant activity of turbot blood components and turbot cells for the facultative parasite Philasterides dicentrarchi, which is responsible for significant losses in turbot farming . Preliminary tests indicated that the assay requires 3-4 h for detection of chemoattractant activity, that it can be performed effectively using the ciliate axenic culture medium, and that it distinguishes clearly between different concentrations of chemoattractant . Application of the assay indicated that whole blood and serum from normal turbot, and especially infected turbot, have strong chemoattractant activity for P . dicentrarchi trophozoites, whereas neither turbot blood cells nor other turbot cells nor bacteria were significant chemoattractants . These results raise the possibility that turbot serum components are involved in host detection and host invasion by P . dicentrarchi, in line with previous findings indicating that turbot with skin lesions show increased susceptibility to P . dicentrarchi infection. J Med Assoc Thai, 2004 Jun, 87(6), 581 - 8 Severity of measles: a study at the Queen Sirikit National Institute of Child Health; Ariyasriwatana C et al.; INTRODUCTION: Thousands of measles cases are reported annually in Thailand even though measles vaccine has been introduced in the expanded program of immunization for every 9-month-old infant for nearly 20 years . Severe cases are admitted to the hospital, usually with complications, some cases lead to death . OBJECTIVES: To study the clinical presentations of severe cases of measles and its complications and find the correlations of severity of pneumonia with age, nutritional status and history of vaccination . MATERIAL AND METHOD: The hospital charts of measles patients admitted to the Queen Sirikit National Institute of Child Health (QSNICH) during 1998-2002 were retrospectively reviewed . Demographic data, history including history of measles vaccination, physical examinations, laboratory investigations, treatment and hospital course which were relevant were recorded . Paired t-test and Pearson's correlation were used for data analysis . RESULTS: There were 156 cases of measles admitted to the QSNICH . There were 95 boys and 61 girls and the male to female ratio was 1.56:1 . The age range was 2 months to 14.8 years, median = 1.5 years, mode 8 months . Fifty-nine percent of the cases were under 2 years of age; 40% under one year and 23.9% were under 9 months . About 44% of the cases had one dose of previous measles vaccination, no history of measles vaccination in 91.4% of cases whose age was under 1 year in contrast to 80% of cases over 5 years that had a history of measles vaccination . Sixty-six percent of the cases had normal nutritional status while 12.4%, 4.8% and 2.1% had mild, moderate and severe protein calorie malnutrition . Fourteen cases (9%) had underlying diseases . At least 3 of the classical signs and symptoms of measles (rash, cough and coryza) were found in 92.3% of the cases . The mean duration of fever at the time of admission was 5.3 days . The common complications in admitted measles cases were pneumonia (62.2%) and diarrhea (38.1%) . The likely causes of pneumonitis were measles viruses (52.6%) and bacteria (47.4%) . There was one dead case with severe pneumonia, with ARDS and respiratory failure . Young infants had a higher incidence of diarrhea with dehydration (p = 0.000) but severity of pneumonia was not different from older children (p = 0.512) . The severity of pneumonia was not correlated with the age (r = 0.087), nutritional status (r = 0122) or the history of receiving measles vaccine (r = 0.116) . CONCLUSION: Measles is one of the important diseases of in-patients admitted to the QSNICH, because of the severity of the diseases due to pneumonia and diarrhea . One severe case died because of severe pneumonia that lead to ARDS and respiratory failure . Young infants had a higher incidence of diarrhea and dehydration, while there was no correlation between severe pneumonia with age, nutritional status and history of vaccination. Lasers Med Sci, 2004, 19(1), 52 - 6 Epub 2004 Jul 15. Nitrogen laser irradiation (337 nm) causes temporary inactivation of clinical isolates of Mycobacterium tuberculosis; Dube A et al.; We have investigated the effect of nitrogen laser irradiation (337 nm) on viability of clinical isolates of Mycobacterium tuberculosis . Bacteria were exposed to a nitrogen laser (average power 2.0 mW) in vitro at power density of 70 +/- 0.7 W/m2 for 0-30 min, and the cell viability was determined by luciferase reporter phage (LRP) assay . Immediately after laser exposure, all the clinical isolates investigated showed a dose-dependent decrease in cell viability . However, when the laser-exposed isolates were incubated in broth medium for 3 days, most of these showed significant recovery from laser-induced damage . Addition of 5.0 microg/ml acriflavine (a DNA repair inhibitor) in the incubation medium had no significant effect on recovery . This suggests that DNA damage may not be involved in the cell inactivation . Electron paramagnetic resonance (EPR) studies using 5-doxyl strearic acid (5-DS) as a probe suggest alterations in lipid regions of the cell wall . Implications of these results for understanding therapeutic effect of nitrogen laser on drug-resistant tuberculosis are discussed. Minerva Stomatol, 2004 Jul-Aug, 53(7-8), 389 - 402 The challenge of severe acute respiratory syndrome (SARS) in dentistry; Testarelli L et al.; Severe Acute Respiratory Syndrome (SARS) is caused by a newly identified coronavirus, called SARS-associated coronavirus (SARS-CoV) that appears to be transmitted primarily through droplets of saliva . This is the reason why the most important international organizations recommend that the dentists adopt a unique preventive approach to the problem: SARS patients should not be treated in the dental office . This is possible only if a suspected case of SARS is correctly and promptly identified . But a correct identification is made difficult by several factors, such as the incubation period, a possibly asymptomatic onset of the illness, the still low specificity and sensitivity of laboratory and instrumental tests . A case or suspected case of SARS may thus unwillingly be treated at the dental office . It is therefore necessary to adopt protective measures for the dental personnel and to implement and enforce infection control measures in order to eliminate the risk of viral contamination . Nonetheless, these procedures do not ensure a complete elimination of SARS-CoV contamination risk since a major risk is represented by blood-borne infection, which is originated by the mouth of patients, and the contamination of dental units water lines (DUWLs) is most difficult to control . Blood-borne contamination may be achieved only by adopting a high level, between-patients disinfection protocol of the DUWLs based on the use of chemical agents with biocidal activity against spores, viruses, bacteria and fungi (Autosteril method) . In conclusion a fully effective control of the cross-infection risk will be obtained only by adopting a correct, integrated use of different infection control procedures. J Exp Biol, 2004 Aug, 207(Pt 17), 2935 - 46 Cytotoxicity of diatom-derived oxylipins in organisms belonging to different phyla; Adolph S et al.; The cytotoxicity of several saturated and unsaturated marine diatom-derived aldehydes and an oxo-acid have been screened in vitro and in vivo against different organisms, such as bacteria, algae, fungi, echinoderms, molluscs and crustaceans . Conjugated unsaturated aldehydes like 2E,4E-decadienal, 2E,4E-octadienal, 5E,7E-9-oxo-nonadienoic acid and 2E-decenal were active against bacteria and fungi and showed weak algicidal activity . By contrast, the saturated aldehyde decanal and the non-conjugated aldehyde 4Z-decenal had either low or no significant biological activity . In assays with oyster haemocytes, 2E,4E-decadienal exhibited a dose-dependent inhibition of cytoskeleton organisation, rate of phagocytosis and oxidative burst and a dose-dependent promotion of apoptosis . A maternal diatom diet that was rich in unsaturated aldehydes induced arrest of cell division and apoptotic cell degradation in copepod embryos and larvae, respectively . This wide spectrum of physiological pathologies reflects the potent cell toxicity of diatom-derived oxylipins, in relation to their non-specific chemical reactivity towards nucleophilic biomolecules . The cytotoxic activity is conserved across six phyla, from bacteria to crustaceans . Deregulation of cell homeostasis is supposed to induce the elimination of damaged cells through apoptosis . However, efficient protection mechanisms possibly exist in unicellular organisms . Experiments with a genetically modified yeast species exhibiting elevated membrane and/or cell wall permeability suggest that this protection can be related to the inability of the oxylipin compounds to enter the cell. Arch Androl, 2004 Jul-Aug, 50(4), 239 - 46 Effects of semen characteristics on IUI combined with mild ovarian stimulation; Yalti S et al.; To determine the influence of sperm parameters inseminated on the outcome of intrauterine insemination (IUI) in patients undergoing ovarian stimulation with clomiphen citrat (CC) or human menopausal gonadotropin (HMG) therapy, a retrospective review was performed for 2 years on data from the IUI program . 190 couples underwent a total of 268 IUI cycles in which CC or HMG was used for ovulation induction . The initial sperm concentration (mil/ml), motility (percent), preprocessing total motile sperm (TMS) count (million), fast motile sperm (percent) and postprocessing sperm concentration (mil/ml), motility (percent), TMS count, fast motile sperm (percent), sperm morphology, hypoosmotic swelling (HOS) scores, semen leuocytes, and bacteria were analyzed . 268 inseminations were followed by a pregnancy rate of 12% and couple pregnancy rate of 17% . On multivariable logistic regression analysis, total motile sperm (TMS) count, percent motility, and percent of fast motile sperm were independent prognostic factors of fertility . The impact of the preprocessing and postprocessing sperm parameters on pregnancy outcome after IUI was evaluated . There was a trend toward an increasing percent of conception with increasing TMS count, motility, and percent of fast motile sperm . The TMS count, motility and percent of fast motile sperm independently predict success with IUI . Patients with original sperm motility > or = 30% had a higher cumulative pregnancy rate (74%) than patient with motility < 30% (p < 0.005) . Pregnancy rate increased 4 times with motility of > or = 30%. J Mol Biol, 2004 Aug 6, 341(2), 419 - 28 Modulation of DNA conformations through the formation of alternative high-order HU-DNA complexes; Sagi D et al.; HU is an abundant, highly conserved protein associated with the bacterial chromosome . It belongs to a small class of proteins that includes the eukaryotic proteins TBP, SRY, HMG-I and LEF-I, which bind to DNA non-specifically at the minor groove . HU plays important roles as an accessory architectural factor in a variety of bacterial cellular processes such as DNA compaction, replication, transposition, recombination and gene regulation . In an attempt to unravel the role this protein plays in shaping nucleoid structure, we have carried out fluorescence resonance energy transfer measurements of HU-DNA oligonucleotide complexes, both at the ensemble and single-pair levels . Our results provide direct experimental evidence for concerted DNA bending by HU, and the abrogation of this effect at HU to DNA ratios above about one HU dimer per 10-12 bp . These findings support a model in which a number of HU molecules form an ordered helical scaffold with DNA lying in the periphery . The abrogation of these nucleosome-like structures for high HU to DNA ratios suggests a unique role for HU in the dynamic modulation of bacterial nucleoid structure. Res Vet Sci, 2004 Dec, 77(3), 197 - 202 Prevalence of exposure and infection of Lawsonia intracellularis among slaughter-age pigs; van der Heijden HM et al.; The extent of clinical or subclinical infection associated with Lawsonia intracellularis within Dutch pig herds was uncertain . A case-control study of slaughter age pigs was used to study natural infection within Dutch herds and to compare diagnostic methods . From six case herds where clinical disease had been identified recently, and six disease-free herds, 40 pigs of slaughter-age were examined postmortem . The diagnostic methods used were: serology, gross examination, Haematoxylin and Eosin stain (HE), Warthin-Starry silver stain, Lawsonia-specific indirect immunoperoxidase of the ileum, and PCR of ileum mucosa and colon contents . There were 59% seropositive pigs in case herds and 26% seropositive pigs in control herds . Using immunohistochemistry, 57% of case herds and 46% of control herds were bacteria positive in the ileum mucosa . It was concluded that a majority of Dutch herds contain L . intracellularis infected finisher pigs . In some herds this is associated with clinical outbreaks of acute haemorrhagic enteropathy but in other herds no clinical disease is apparent . Many seropositive pigs in herds without clinical disease had evidence of Lawsonia antigen in sites other than the apical cytoplasm of proliferating epithelial cells, particularly the supranuclear region . It was uncertain whether to classify these pigs as having "recovered" from an infection or whether they have a sub-clinical or chronic form of the disease . We concluded that PCR examination of faeces and serology probably provide more specific results than gross examinations at slaughter, and that a monoclonal antibody-based examination of ileum mucosa should be the accepted screening method for this infection. Trends Microbiol, 2004 Aug, 12(8), 373 - 7 Multilocus sequence typing--what is resolved? Cooper JE, Feil EJ. Nucleotide sequence-based methods for bacterial typing (multilocus sequence typing; MLST) allow rapid and global comparisons between results from different laboratories . Combining this advantage with the reduced cost of high throughput sequencing, increasing automation and the amenability of sequence data for evolutionary analysis, it seems inevitable that sequence-based typing will eventually predominate over gel-based methods such as pulsed-field gel electrophoresis (PFGE) for most bacterial species . The increasing availability of multiple genome sequences for single pathogenic species, and the recent development of many new MLST schemes, means that a re-examination of the utility of multilocus sequencing, and in particular the choice of gene loci, is now appropriate. Ann Chir Plast Esthet, 2004 Jun, 49(3), 302 - 5 {GESNOMA (Geneva Study Group on Noma): state-of-the-art medical research for humanitarian purposes}; Baratti-Mayer D et al.; Noma is a devastating gangrenous disease that leads to severe tissue destructions in the face . It is seen almost exclusively in children living in less developed countries . The exact prevalence of the disease is unknown and the cause remains unknown too . Risk factors are: malnutrition, a compromised immune system, poor oral hygiene and a lesion of the gingival mucosal barrier, as well as an unidentified bacterial factor . Herpes viruses might also contribute . Studies of the buccal flora in acute phases of noma and comparison with control children do not exist . Our study takes place in Niger . For each child (cases and controls) we take samples of gingival fluid, saliva, blood and mouth mucosal swabs . The samples are analysed in Geneva in different laboratories . We control the serologies for Herpes viruses and measles . We also perform a nutritional assessment and the mucosal swabs are cultivated for the presence of viruses . The gingival flora is investigated by microarrays . These microarrays are instrumental to test for the presence of thousands of different bacteria in each clinical sample . This method allows a qualitative and quantitative description of the oral flora in noma-children and control cases . This is the first large scale study on the etiology of noma which uses new technical approaches for humanitarian purposes. Trends Biochem Sci, 2004 Jun, 29(6), 289 - 92 Vitamin K epoxide reductase: homology, active site and catalytic mechanism; Goodstadt L et al.; Vitamin K epoxide reductase (VKOR) recycles reduced vitamin K, which is used subsequently as a co-factor in the gamma-carboxylation of glutamic acid residues in blood coagulation enzymes . VKORC1, a subunit of the VKOR complex, has recently been shown to possess this activity . Here, we show that VKORC1 is a member of a large family of predicted enzymes that are present in vertebrates, Drosophila, plants, bacteria and archaea . Four cysteine residues and one residue, which is either serine or threonine, are identified as likely active-site residues . In some plant and bacterial homologues the VKORC1 homologous domain is fused with domains of the thioredoxin family of oxidoreductases . These might reduce disulfide bonds of VKORC1-like enzymes as a prerequisite for their catalytic activities. Parasitol Today, 1994, 10(3), 98 - 102 Variability and its implications for host-parasite interactions; Schmid-Hempel P et al.; Variability in host-parasite interactions has considerable impact on the ecology and evolution of parasites and on the epidemiology of disease . The nature of the impact depends largely on the level of ecological organization where variability occurs: variability of parasites within their individual hosts, variability of host individuals within populations, or variability of hosts and parasites among populations . In this review, Paul Schmid-Hempel and Jacob Koella give some examples of variability at each of these levels, with particular emphasis on microparasites (defined broadly as viruses, bacteria and protozoa), consider the maintenance of the variability, and describe the implications of variability for the epidemiology of disease and the ecology of host parasite associations . In particular, they describe how variability at each level of ecological organization can affect the perception of AIDS and the evolution of virulence. Parasitol Today, 1994, 10(7), 258 - 62 Transgenic mice and the study of cytokine function in infection; Taverne J; Most information about the involvement of the different cytokines in immunity to infection has been obtained by the administration to infected animals of recombinant molecules or of antibodies against them . Now another approach to the study of cytokine function, in vivo, is available in the form of transgenic mice that express a transgene encoding a particular cytokine, and of 'knockout' animals, in which a cytokine gene, or a gene for its receptor (which usually comes to the same thing), have been rendered inactive by targeted disruption . While a few of these lines of mice have been analysed for their response to infection by protozoan parasites or worms, more have been tested for their ability to withstand intracellular infections by bacteria or viruses . In this review, Janice Taverne outlines those described to date in which the immune (or immunopathological mechanisms concerned may be relevant to parasitic diseases. Parasitol Today, 1996 Oct, 12(10), 388 - 96 Tick salivary prostaglandins: Presence, origin and significance; Bowman AS et al.; Prostaglandins (PGs) are oxygenated metabolites of polyunsaturated fatty acids, most notably arachidonic acid, that act as 'local hormones', regulating a plethora of physiological processes in mammals and other vertebrates . For a long time, PGs were reported only in higher vertebrates, but more recently they have been reported in lower organisms such as bacteria, yeasts and protozoa, and much information is now available on PGs in insects . Prostaglandins are increasingly reported to exist at the host-parasite interface and are thought to aid the parasite by modulating the inflammatory and immune response . Ticks secrete saliva containing extremely high concentrations of PGs into the host, and in this article Alan Bowman, Jack Dillwith and John Sauer provide a synopsis of the information, to date, on the presence, synthesis and proposed roles for these tick salivary PGs. Parasitol Today, 1996 Dec, 12(12), 472 - 9 Co-feeding ticks: Epidemiological significance for tick-borne pathogen transmission; Randolph SE et al.; Until recently, the transmission of tick-borne pathogens via vertebrates was thought to depend on the development of a systemic infection in the vertebrate hosts . Pathogen transmission has now been shown to occur between infected and uninfected ticks co-feeding in time or space in the absence of a systemic infection, originally for viruses, but now also for bacteria . The epidemiological consequences of this new non-systemic transmission pathway necessitate a major reassessment of the components and dynamics of tick-borne pathogen enzootic cycles . Here Sarah Randolph, Lise Gern and Pat Nuttall show that a much wider range of natural hosts than was previously recognized may contribute significantly to the transmission of tick-borne diseases, and compare quantitatively the relative contributions made by the systemic and non-systemic transmission pathways. Parasitol Today, 1997 Dec, 13(12), 468 - 71 Studies with artificial extrachromosomal elements in trypanosomatids: Could specificity in the initiation of DNA replication be linked to that in transcription? Patnaik PK. Historically, artificial replicons have served as useful models for the definition of regulatory elements involved in chromosomal replication and transmission in yeast and DNA replication in bacteria . Here, Pradeep Patnaik examines what we have learnt so far from the replicative behaviour of various artificial extrachromosomal elements available for trypanosomatids . He highlights the involvement of transcription regulatory elements in virtually every eukaryotic origin of replication analysed in detail and, by drawing upon the extensive literature supporting a close association between DNA replication and transcription, he speculates that the nature and organization of origins of replication on a chromosome also may hold clues to the manner by which an organism regulates gene expression. Structure (Camb), 2004 Jun, 12(6), 1039 - 45 Chromophore conformation and the evolution of tertiary structural changes in photoactive yellow protein; Anderson S et al.; We use time-resolved crystallography to observe the structural progression of a bacterial blue light photoreceptor throughout its photocycle . Data were collected from 10 ns to 100 ms after photoactivation of the E46Q mutant of photoactive yellow protein . Refinement of transient chromophore conformations shows that the spectroscopically distinct intermediates are formed via progressive disruption of the hydrogen bond network to the chromophore . Although structural change occurs within a few nanoseconds on and around the chromophore, it takes milliseconds for a distinct pattern of tertiary structural change to fully progress through the entire molecule, thus generating the putative signaling state . Remarkably, the coupling between the chromophore conformation and the tertiary structure of this small protein is not tight: there are leads and lags between changes in the conformation of the chromophore and the protein tertiary structure. J Clin Apheresis, 2004, 19(2), 98 - 102 L-carnitine improves pH and decreases surface phosphatidylserine expression in extended stored apheresis platelets; Sweeney JD et al.; Extension of the storage period of apheresis platelets to seven or ten days may be possible with the implementation of screening for bacteria . This, however, may impair platelet quality, and additive compounds that improve storage parameters would be desirable . Apheresis platelets were harvested using the Cobe LRS device . Part of the product was aliquoted into two CLX bags, 60 ml into each, on day 0 . L-carnitine (LC) to a final concentration of 5 mM was added to one container and saline to the other . pH, morphology score, and surface expression of phosphatidylserine were measured on day 1, and, in addition, hypotonic shock response (HSR) and the extent of shape change (ESC) on days 5, 10, and 13 . Differences between test and controls were analyzed using paired t-tests . The addition of LC improved pH by day 5, but was more evident by days 10 and 13 . By day 10, significant differences (<0.01) were observed in pH (6.54 +/- 0.3 vs . 6.75 +/- 0.3), lactate (176 +/- 31 vs . 150 +/- 24 mg %), morphology score (213 +/- 27 vs . 229 +/- 35) and ESC (7 +/- 6 vs . 11 +/- 6) . Percent surface phosphatidylserine expression was less in the LC treated platelets (16 +/- 7 vs . 12 +/- 4, P<0.03) . Much of the benefit observed was attributable to improved parameters in some donors . LC improves the quality of extended stored apheresis platelets. Bull Exp Biol Med, 2004 Feb, 137(2), 150 - 1 Absorption and digestion of phagocytized objects by mononuclear phagocytes during rheumatoid arthritis; Arleevskaya MI et al.; Radioisotope study of mononuclear phagocytes from patients with rheumatoid arthritis showed impaired ingestion of bacteria in the presence of pronounced digestive activity . Excessive accumulation methylumbelliferyl phosphate beta-glucuronide (product of hydrolysis catalyzed by glucuronidase released from cells) into the incubation medium was observed . This was probably related to the predominance of extracellular digestion. Plant J, 2004 Aug, 39(4), 477 - 86 Metabolic engineering of ketocarotenoid formation in higher plants; Ralley L et al.; Although higher plants synthesize carotenoids, they do not possess the ability to form ketocarotenoids . In order to generate higher plants capable of synthesizing combinations of ketolated and hydroxylated carotenoids the genes responsible for the carotene 4,4' oxygenase and 3,3' hydroxylase have been transformed into tomato and tobacco . The gene products were produced as a polyprotein . Subsequent cleavage of the polyprotein, targeting of the two enzymes to the plastid and enzyme activities have been shown for both gene products . Metabolite profiling has shown the formation of ketolated carotenoids from beta-carotene and its hydroxylated intermediates in tobacco and tomato leaf . In the nectary tissues of tobacco flowers a quantitative increase (10-fold) as well as compositional changes were evident, including the presence of astaxanthin, canthaxanthin and 4-ketozeaxanthin . Interestingly, in this tissue the newly formed carotenoids resided predominantly as esters . These data are discussed in terms of metabolic engineering of carotenoids and their sequestration in higher plant tissues. J Biol Chem, 2004 Sep 10, 279(37), 38095 - 8 Epub 2004 Jul 22. Fluorescent biosensor for quantitative real-time measurements of inositol 1,4,5-trisphosphate in single living cells; Tanimura A et al.; The second messenger inositol 1,4,5-trisphosphate (IP(3)) plays a central role in the generation of a variety of spatiotemporally complex intracellular Ca(2+) signals involved in the regulation of many essential physiological processes . Here we describe the development of "LIBRA", a novel ratiometric fluorescent IP(3) biosensor that allows for the quantitative monitoring of intracellular IP(3) concentrations in single living cells in real time . LIBRA consists of the IP(3)-binding domain of the rat type 3 IP(3) receptor fused between the fluorescence resonance energy transfer pair cyan fluorescent protein and yellow fluorescent protein and preceded by a membrane-targeting signal . We show that the LIBRA fluorescent signal is highly selective for IP(3) and unaffected by concentrations of Ca(2+) and ATP in the physiological range . In addition, LIBRA can be calibrated in situ . We demonstrate the utility of LIBRA by monitoring the temporal relationship between the responses intracellular IP(3) and Ca(2+) concentrations in SH-SY5Y cells following acetylcholine stimulation. J Biol Chem, 2004 Oct 8, 279(41), 42916 - 23 Epub 2004 Jul 22. Lipid utilization, gluconeogenesis, and seedling growth in Arabidopsis mutants lacking the glyoxylate cycle enzyme malate synthase; Cornah JE et al.; The aim of this research was to test the role of the glyoxylate cycle enzyme malate synthase (MLS) in lipid utilization, gluconeogenesis, and seedling growth in Arabidopsis . We hypothesized that in the absence of MLS, succinate produced by isocitrate lyase (ICL) could still feed into the tricarboxylic acid cycle, whereas glyoxylate could be converted to sugars using enzymes of the photorespiratory pathway . To test this hypothesis we isolated knock-out mls mutants and studied their growth and metabolism in comparison to wild type and icl mutant seedlings . In contrast to icl seedlings, which grow slowly and are unable to convert lipid into sugars (Eastmond, P . J., Germain, V., Lange, P . R., Bryce, J . H., Smith, S . M . & Graham, I . A . (2000) Proc . Natl . Acad . Sci . U . S . A . 97, 5669-5674), mls seedlings grow faster, use their lipid more rapidly, and are better able to establish as plantlets . Transcriptome and metabolome analyses show that icl seedlings exhibit many features characteristic of carbohydrate starvation, whereas mls seedlings differ relatively little from wild type . In the light mls seedlings generate more sugars than icl seedlings, and when fed with {14C}acetate, 14C-labeling of sugars is three times greater than in icl seedlings and more than half that in wild type seedlings . The mls seedlings also accumulate more glycine and serine than icl or wild type seedlings, consistent with a diversion of glyoxylate into these intermediates of the photorespiratory pathway . We conclude that, in contrast to bacteria and fungi in which MLS is essential for gluconeogenesis from acetate or fatty acids, MLS is partially dispensable for lipid utilization and gluconeogenesis in Arabidopsis seedlings. Infect Immun, 2004 Aug, 72(8), 4864 - 7 The luxS gene is not required for Borrelia burgdorferi tick colonization, transmission to a mammalian host, or induction of disease; Blevins JS et al.; luxS mutants of Borrelia burgdorferi strain 297 naturally colonized their arthropod (Ixodes scapularis) vector, were maintained in ticks throughout the molting process (larvae to nymphs), were tick transmitted to uninfected mice, and elicited histopathology in mice indistinguishable from that induced by wild-type B . burgdorferi. Infect Immun, 2004 Aug, 72(8), 4503 - 11 A specific genomic location within the icm/dot pathogenesis region of different Legionella species encodes functionally similar but nonhomologous virulence proteins; Feldman M et al.; Legionella pneumophila, the major causative agent of Legionnaires' disease, is a facultative intracellular pathogen that grows within human macrophages and amoebae . Intracellular growth involves the formation of a replicative phagosome that requires the Icm/Dot type IV secretion system . Part of the icm/dot region in L . pneumophila contains the icmTSRQPO genes . The proteins encoded by the icmR and icmQ genes were shown to exhibit a chaperone-substrate relationship . Analysis of this region from other pathogenic Legionella species, i.e., L . micdadei and L . longbeachae, indicated that the overall organization of this region is highly conserved, but it was found to contain a favorable site for gene variation . In the place where the icmR gene was expected to be located, other open reading frames that are nonhomologous to each other or to any entry in the GenBank database were found (migAB in L . micdadei and ligB in L . longbeachae) . Examination of these unique genes revealed an outstanding phenomenon; by use of interspecies complementation, the icmR, migB, and ligB gene products were found to be functionally similar . In addition, the function of these proteins was usually dependent on the presence of the corresponding IcmQ proteins . Moreover, each of these proteins (IcmR, LigB, and MigB) was found to interact with the corresponding IcmQ proteins, and the genes encoding these proteins were found to be regulated by CpxR . This study reveals new evidence of gene variation occurring in the same genomic location within the icm/dot locus in various Legionella species . The genes found at this site were shown to be similarly regulated and to encode species-specific, nonhomologous, but functionally similar proteins. Infect Immun, 2004 Aug, 72(8), 4480 - 5 Helicobacter pylori induces apoptosis of human monocytes but not monocyte-derived dendritic cells: role of the cag pathogenicity island; Galgani M et al.; Monocytes are circulating precursors of the dendritic cell subset, professional antigen-presenting cells with a unique ability to initiate the innate and adaptive immune response . In this study, we have investigated the effects of wild-type Helicobacter pylori strains and their isogenic mutants with mutations in known bacterial virulence factors on monocytes and monocyte-derived dendritic cells . We show that H . pylori strains induce apoptosis of human monocytes by a mechanism that is dependent on the expression of a functional cag pathogenicity island . This effect requires an intact injection organelle for direct contact between monocytes and the bacteria but also requires a still-unidentified effector that is different from VacA or CagA . The exposure of in vitro-generated monocyte-derived dendritic cells to H . pylori stimulates the release of inflammatory cytokines by a similar mechanism . Of note is that dendritic cells are resistant to H . pylori-induced apoptosis . These phenomena may play a critical role in the evasion of the immune response by H . pylori, contributing to the persistence of the infection. Am J Respir Crit Care Med, 2004 Nov 1, 170(9), 1000 - 5 Epub 2004 Jul 21. Pulmonary surfactant, lung function, and endobronchial inflammation in cystic fibrosis; Griese M et al.; Cystic fibrosis (CF) lung disease is primarily a disease of the small airways . We hypothesized that even in patients with normal lung function, a reduced surfactant function would be present and favor small airway obstruction . Bronchoalveolar lavages from 76 patients with CF (5-31 years, median 11) with well-conserved lung function (FEV1 94% predicted, range 78-121) and from 10 healthy control subjects were investigated . The deviation of the biophysical surfactant performance from normal, assessed in a bubble surfactometer, was small; however, the ability of the surfactant to maintain the patency of a narrow airway (% open) was significantly reduced . Surfactant protein (SP)-C level was increased, SP-B and SP-D were unchanged, whereas SP-A was decreased . Among the patients with CF, neutrophilic inflammation was modestly related to a poorer surfactant activity, but not to lung function . SP-D was reduced in proportion to the degree of inflammation and in the presence of bacteria . These findings in a large cohort of patients with CF with normal lung function show that the endobronchial airway inflammation is linked to early perturbations of the biophysical properties and immunologic components of pulmonary surfactant and opens fields for novel therapeutic interventions. Insect Mol Biol, 2004 Aug, 13(4), 387 - 98 Identification and molecular characterization of two immune-responsive chitinase-like proteins from Anopheles gambiae; Shi L et al.; Two haemolymph proteins that are processed rapidly and specifically in response to exposure to bacteria have been identified from Anopheles gambiae . Both proteins, Anopheles gambiae bacteria-responsive 1 (AgBR1) and AgBR2, are similar to chitinases but belong to a family of proteins that have lost chitinolytic activity . AgBR1 and AgBR2 are converted to smaller forms in vivo or in vitro on exposure to bacteria, and AgBR2 also can be processed on exposure to peptidoglycan alone . AgBR1 and AgBR2 do not bind to bacteria or chitin beads . The AgBR1 and AgBR2 genes are expressed in all developmental stages . In adults, AgBR1 expression is restricted to the fat body, whereas AgBR2 is expressed in many tissues. Insect Mol Biol, 2004 Aug, 13(4), 359 - 64 Distribution and prevalence of Wolbachia in Japanese populations of Lepidoptera; Tagami Y et al.; Wolbachia are cytoplasmically inherited bacteria that are reported to infect at least 18-30% of all insect species . Our survey of Lepidoptera indicated that 44.9% of forty-nine species and 77.8% of nine families tested positive for Wolbachia using PCR with wsp primers . Nineteen species had not been described previously as infected . In particular, although Pieris rapae, which is a common species in Japan, is infected by Wolbachia, the prevalence was very low (3.4%) and there were some localities where Wolbachia could not be detected . The probability of detection of Wolbachia depends on the number of screened individuals of P . rapae . The results indicate that the actual number of species that are positive for Wolbachia may be higher than previously reported. Helicobacter, 2004 Aug, 9(4), 313 - 23 Comparative chemical and biological characterization of the lipopolysaccharides of gastric and enterohepatic helicobacters; Hynes SO et al.; BACKGROUND: The lipopolysaccharide of Helicobacter pylori plays an important role in colonization and pathogenicity . The present study sought to compare structural and biological features of lipopolysaccharides from gastric and enterohepatic Helicobacter spp . not previously characterized . MATERIALS AND METHODS: Purified lipopolysaccharides from four gastric Helicobacter spp . (H . pylori, Helicobacter felis, Helicobacter bizzozeronii and Helicobacter mustelae) and four enterohepatic Helicobacter spp . (Helicobacter hepaticus, Helicobacter bilis, 'Helicobacter sp . flexispira' and Helicobacter pullorum) were structurally characterized using electrophoretic, serological and chemical methods . RESULTS: Structural insights into all three moieties of the lipopolysaccharides, i.e . lipid A, core and O-polysaccharide chains, were gained . All species expressed lipopolysaccharides bearing an O-polysaccharide chain, but H . mustelae and H . hepaticus produced truncated semirough lipopolysaccharides . However, in contrast to lipopolysaccharides of H . pylori and H . mustelae, no blood group mimicry was detected in the other Helicobacter spp . examined . Intra-species, but not interspecies, fatty acid profiles of lipopolysaccharides were identical within the genus . Although shared lipopolysaccharide-core epitopes with H . pylori occurred, differing structural characteristics were noted in this lipopolysaccharide region of some Helicobacter spp . The lipopolysaccharides of the gastric helicobacters, H . bizzozeronii and H . mustelae, had relative Limulus amoebocyte lysate activities which clustered around that of H . pylori lipopolysaccharide, whereas H . bilis, 'Helicobacter sp . flexispira' and H . hepaticus formed a cluster with approximately 1000-10,000-fold lower activities . H . pullorum lipopolysaccharide had the highest relative Limulus amoebocyte lysate activity of all the helicobacter lipopolysaccharides (10-fold higher than that of H . pylori lipopolysaccharide), and all the lipopolysaccharides of enterohepatic Helicobacter spp . were capable of inducing nuclear factor-Kappa B(NF-kappaB) activation . CONCLUSIONS: The collective results demonstrate the structural heterogeneity and pathogenic potential of lipopolysaccharides of the Helicobacter genus as a group and these differences in lipopolysaccharides may be indicative of adaptation of the bacteria to different ecological niches. Biochem Soc Trans, 2004 Aug, 32(Pt 4), 601 - 5 Regulation of chloroplast translation: interactions of RNA elements, RNA-binding proteins and the plastid ribosome; Manuell A et al.; Chloroplast gene expression is primarily controlled during the translation of plastid mRNAs into proteins, and genetic studies have identified cis-acting RNA elements and trans-acting protein factors required for chloroplast translation . Biochemical analysis has identified both general and specific mRNA-binding proteins as components of the regulation of chloroplast translation, and has revealed that chloroplast translation is related to bacterial translation but is more complex . Utilizing proteomic and bioinformatic analyses, we have identified the proteins that function in chloroplast translation, including a complete set of chloroplast ribosomal proteins, and homologues of the 70 S initiation, elongation and termination factors . These analyses show that the translational apparatus of chloroplasts is related to that of bacteria, but has adopted a number of eukaryotic mechanisms to facilitate and regulate chloroplast translation. Parassitologia, 2003 Mar, 45(1), 13 - 8 dnaA gene sequences from Wolbachia pipientis support subdivision into supergroups and provide no evidence for recombination in the lineages infecting nematodes; Casiraghi M et al.; Wolbachia pipientis is an intracellular bacterial endosymbiont of arthropods and filarial nematodes . Six main supergroups of W . pipientis have been described: supergroups A, B, E, and F encompass arthropod wolbachiae; supergroups C and D encompass nematode wolbachiae . The description of these six supergroups has been based on the analysis of only two genes (ftsZ and 16S rDNA) and before decisions are taken on the taxonomic status of the six supergroups, analysis of further genes is required . In addition, the branching order of the six supergroups is still unresolved . Sequence information from other genes is also needed to allow phylogenesis to be addressed through the analysis of a higher number of characters . Here we report sequences from a portion of the gene coding for the DNAA protein of W pipientis, generated from the endosymbionts of 22 host species . Phylogenies based on dnaA gene sequences are congruent with the existence of at least six supergroups of W pipientis . In addition, subtrees generated for nematode wolbachiae in supergroups C and D were compared to the trees based on the already available gene sequences (ftsZ, 16S rDNA and wsp) . The congruence observed among the trees based on the different genes agrees with the hypothesis that recombination does not occur in nematode wolbachiae. Cell Stress Chaperones, 2004 Mar, 9(1), 38 - 48 The mitochondrial 60-kDa heat shock protein in marine invertebrates: biochemical purification and molecular characterization; Choresh O et al.; Sessile marine invertebrates undergo constant direct exposure to the surrounding environmental conditions, including local and global environmental fluctuations that may lead to fatal protein damage . Induction of heat shock proteins (Hsps) constitutes an important defense mechanism that protects these organisms from deleterious stress conditions . In a previous study, we reported the immunological detection of a 60-kDa Hsp (Hsp60) in the sea anemone Anemonia viridis (formerly called Anemonia sulcata) and studied its expression under a variety of stress conditions . In the present study, we show that the sponge Tetilla sp . from tidal habitats with a highly variable temperature regime is characterized by an increased level of Hsp60 . Moreover, we show the expression of Hsp60 in various species among Porifera and Cnidaria, suggesting a general importance of this protein among marine invertebrates . We further cloned the hsp60 gene from A viridis, using a combination of conventional protein isolation methods and screening of a complementary deoxyribonucleic acid library by polymerase chain reaction . The cloned sequence (1764 bp) encodes for a protein of 62.8 kDa (588 amino acids) . The 62.8-kDa protein, which contains an amino terminal extension that may serve as a mitochondrial targeting signal, shares a significant identity with mitochondrial Hsp60s from several animals but less identity with Hsp60s from either bacteria or plants. Environ Toxicol, 2004 Aug, 19(4), 421 - 4 Impact of crude oil on bacteriocenosis of the digestive tract of mollusks; Syvokiene J et al.; In this study the effect of crude oil on intestinal bacterial populations of the mollusk Viviparus contactus was investigated . The addition of crude oil into an environment of mollusks induced no clear changes in the saprophytic, amylolytic, and total coliform bacterial counts in the digestive tract of the mollusk . After 10 days of contamination, the saprophytic, amylolytic, and total coliform bacterial numbers were of the same order of magnitude as the initial numbers . Significant numbers of indigenous hydrocarbon-degrading bacteria were observed in the intestinal tracts of mollusks before contamination with crude oil . Introduction of crude oil into the mollusk environment resulted in an increase of 2 orders of magnitude in the number of hydrocarbon-degrading bacteria in the digestive tract . Therefore, measuring the hydrocarbon-degrading bacterial populations in the digestive tracts of hydrobionts can be considered an important component of contaminated-site assessment studies . Lab Chip, 2004 Aug, 4(4), 372 - 7 Epub 2004 Apr 20. Measurements of scattered light on a microchip flow cytometer with integrated polymer based optical elements; Wang Z et al.; Flow cytometry is widely used for analyzing microparticles, such as cells and bacteria . In this paper, we report an innovative microsystem, in which several different optical elements (waveguides, lens and fiber-to-waveguide couplers) are integrated with microfluidic channels to form a complete microchip flow cytometer . All the optical elements, the microfluidic system, and the fiber-to-waveguide couplers were defined in one layer of polymer (SU-8, negative photoresist) by standard photolithography . With only a single mask procedure required, all the fabrication and packaging processes can be finished in one day . Polystyrene beads were measured in the microchip flow cytometer, and three signals (forward scattering, large angle scattering and extinction) were measured simultaneously for each bead . To our knowledge this is the first time forward scattered light and incident light extinction were measured in a microsystem using integrated optics . The microsystem can be applied for analyzing different kinds of particles and cells, and can easily be integrated with other microfluidic components. J Biol Chem, 2004 Sep 17, 279(38), 40122 - 9 Epub 2004 Jul 21. Coenzyme specificity of Sir2 protein deacetylases: implications for physiological regulation; Schmidt MT et al.; Sir2 (silent information regulator 2) enzymes catalyze a unique protein deacetylation reaction that requires the coenzyme NAD(+) and produces nicotinamide and a newly discovered metabolite, O-acetyl-ADP-ribose (OAADPr) . Conserved from bacteria to humans, these proteins are implicated in the control of gene silencing, metabolism, apoptosis, and aging . Here we examine the role of NAD(+) metabolites/derivatives and salvage pathway intermediates as activators, inhibitors, or coenzyme substrates of Sir2 enzymes in vitro . Also, we probe the coenzyme binding site using inhibitor binding studies and alternative coenzyme derivatives as substrates . Sir2 enzymes showed an exquisite selectivity for the nicotinamide base coenzyme, with the most dramatic losses in binding affinity/reactivity resulting from relatively minor changes in the nicotinamide ring, either by reduction, as in NADH, or by converting the amide to its acid analogue . Both ends of the dinucleotide NAD(+) are shown to be critical for high selectivity and high affinity . Among the NAD(+) metabolites tested none were able to allosterically activate, although all led to various extents of inhibition, consistent with competition at the coenzyme binding site . Nicotinamide was the most potent inhibitor examined, suggesting that cellular nicotinamide levels would provide an effective small molecule regulator of protein deacetylation and generation of OAADPr . The presented findings also suggest that changes in the physiological NAD(+):NADH ratio, without a change in NAD(+), would yield little alteration in Sir2 activity . That is, NADH is an extremely ineffective inhibitor of Sir2 enzymes (average IC(50) of 17 mm) . We propose that changes in both free nicotinamide and free NAD(+) afford the greatest contribution to cellular activity of Sir2 enzymes but with nicotinamide having a more dramatic effect during smaller fluctuations in concentration. FEMS Microbiol Lett, 2004 Aug 1, 237(1), 163 - 70 Brefeldin A enhances Helicobacter pylori vacuolating cytotoxin-induced vacuolation of epithelial cells; Argent RH et al.; Intracellular VacA localises to the vacuolar (late endosome/lysosome) membrane, but little is known about the trafficking of the toxin beyond this region . We show that the Golgi-disturbing agent brefeldin A (BFA) enhances VacA-induced vacuolation of epithelial cells by Helicobacter pylori co-culture and, importantly, BFA treatment induces vacuolation by less toxic forms of VacA . The effect is BFA dose-dependent and occurs within 2.5 h . These data suggest that VacA may be routed deeper within the cell than the vacuole, and that vacuolation is minimised when this occurs efficiently . This may explain why some forms of VacA do not cause vacuolation and why vacuolation is minimal at the low bacteria:cell ratios observed in vivo. Curr Biol, 2004 Jul 27, 14(14), R575 - 7 Comparative genomics: prediction of the ciliary and basal body proteome; Pazour GJ; Defects in mammalian cilia lead to a range of diseases, but our understanding of the composition of these organelles and of the basal bodies from which they arise is limited . Two recent studies used comparative genomics to predict the ciliary and basal body proteomes, providing datasets that are rich sources of human disease gene candidates. Curr Mol Med, 2004 Aug, 4(5), 539 - 47 Therapeutic antibodies; Groner B et al.; Monoclonal antibodies had the lure of drugs very much since their first description . The ability to bind to a predetermined chemical structure stimulated the imagination of drug discoverers and developers . Nevertheless it took many years before a drug was registered which started to make good on the promise . The complexity of the molecule, made up of four polypeptide chains, its large molecular weight, its multiple and versatile functional domains and its mouse origin initially were obstacles for the production and the utilisation . Also the selection of appropriate target structures on the surface of cells turned out be difficult . Many of these difficulties have been overcome . The replacement of most of the murine sequences with equivalent human sequences and the concomittant decrease in immunogenicity, and the identification of cell surface components which are causative and limiting in cellular transformation have made monoclonal antibodies valuable weapons in the fight against cancer . Multiple mechanisms of monoclonal antibody action are being exploited for this purpose . Antibodies can sequester growth factors and prevent the activation of crucial growth factor receptors . A monoclonal antibody directed against the vascular endothelial growth factor (VEGF) has been shown to be a potent neo-vascularisation inhibitor (bevacizumab) . An antibody against the extracellular domain of the EGF receptor prevents the binding of the ligand to the receptor and thereby its activation (cetuximab) . EGFR activity, however, is absolutely required for the survival and proliferation of certain human tumour cells . An antibody which interferes with the dimerisation of the ErbB2 and the ErbB3 members of the EGF receptor family prevents the association of a most potent signaling module (pertuxumab) . The signals emenating from this dimer determine many phenotypic properties of e.g . human breast cancer cells . A monoclonal antibody also directed against ErbB2 has been most successful, clinically and commercially (trastuzumab) . This antibody interferes with signals generated by the receptor and causes the arrest of the cell cycle in tumour cells . In addition, it recruits immune effector cells as cytotoxic agents . Finally, monoclonal antibody derivatives, single chain Fv fragments, have been used as a basis for the construction of recombinant tumour toxins . These molecules harness the exquisite binding specificity of the antibodies and combine them with the toxic principles of bacteria. Acta Otolaryngol, 2004 Jun, 124(5), 621 - 7 Actinobacillus actinomycetemcomitans suppresses rat natural killer cell activity in vivo; Ghoneum M et al.; OBJECTIVE: To examine the immune suppressive effect of Actinobacillus actinomycetemcomitans (Aa) on rat natural killer (NK) cell activity in vivo . MATERIAL AND METHODS: Sprague Dawley rats were given Aa in 2 different manners: (i) by mixing Aa with food at a dose of 10(8) cells/rat/day for 3 months; or (ii) by a single i.m . injection of live Aa at doses of 10(6) and 10(7) cells/rat/day . NK cell activity was measured by means of a 51Cr-release assay using YAC-1 tumor cells as targets . RESULTS: Rats that had been infected by Aa mixed with food experienced significant suppression of their NK cell activity; this reached approximately =50% of control values at 2 and 3 months post-Aa infection . The suppression in NK cell activity was related to decreases in the extent of conjugate formation between effectors and YAC-1 target tumor cells (51.7%) and the extent of lysis of target cells (75.7%) . The results also showed that addition of an admixture of Aa-treated NK cells to the control NK cells caused 75% and 53% decreases in activity at effector:target ratios of 25:1 and 50:1, respectively . In addition, a significant increase in the extent of T-suppressor cells (154.8% of control) was detected at 3 months post-Aa infection . In contrast, the single injection of live bacteria resulted in a remarkable, dose-dependent inhibition of NK cell activity (55% and 71% at doses of 10(6) and 10(7) cells/rat/day, respectively) as early as 2 days post-treatment . This also reflected significant suppression in the effector:target conjugate formation ratio (52% of control) . The data also revealed a 150-188% increase in the number of splenic lymphocytes post-Aa injection . These effects were transient and normal levels were re-established by the fifth day CONCLUSION: Aa treatment causes suppression of NK cell activity and the mode of action may be due to induction of T-suppressor cells or dilution of NK cells with other lymphoid cell populations . The degree of suppression is affected by the way in which Aa is introduced to the host . These results may contribute to the understanding of how Aa evades host defense. Cochrane Database Syst Rev . 2004;(3):CD004507. Semen preparation techniques for intrauterine insemination; Boomsma CM et al.; BACKGROUND: Semen preparation techniques for assisted reproduction, including intrauterine insemination (IUI), were developed to separate the motile morphological normal spermatozoa . Leucocytes, bacteria and dead spermatozoa produce oxygen radicals that negatively influence the ability to fertilize the egg . The yield of as many motile, morphologically normal spermatozoa might influence treatment choices and therefore outcomes . OBJECTIVES: To compare the effectiveness of gradient, swim-up, or wash and centrifugation in subfertile couples undergoing intrauterine insemination (IUI) on clinical outcome as well as on semen parameters . SEARCH STRATEGY: We searched the Menstrual Disorders and Subfertility Group's trials register (30 June 2003), MEDLINE (1966 to July 2003), EMBASE (1980 to July 2003), Science Direct Database (1966 to July 2003), Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library Issue 3, 2003), National Research Register (2000 to Issue 2, 2003), Biological Abstracts (2000 to June 2003), CINAHL (1982 to July 2003) and reference lists of relevant articles . We also contacted experts and authors in the field . SELECTION CRITERIA: Parallel randomised controlled trials (RCTs), comparing the efficacy of semen preparation techniques used for subfertile couples undergoing IUI, were included . RCTs or split sample studies examining semen parameters after different semen preparation techniques were also included . DATA COLLECTION AND ANALYSIS: Two reviewers independently assessed trial quality and extracted data . Study authors were contacted for additional information . MAIN RESULTS: Two randomised controlled trials comparing clinical outcomes, including 81 participants in total, were included in the meta-analysis (Dodson 1998-I/ II; Xu 2000-I/ II) . Both studies compared swim-up technique versus gradient technique in 65 subfertile couples undergoing IUI . One study compared the effectiveness of both techniques with wash technique . No trials reported the primary outcome of live birth . There was no statistically significant difference between pregnancy rates (PR) for swim-up versus gradient / wash centrifuge (Peto OR 0.55, 95% CI 0.17 to 1.76; Peto OR 1.74, 95% CI 0.2 to 14.9; PR/ couple swim-up 20%, gradient 40%, wash 12.5%) or gradient versus wash centrifuge (Peto OR 4.01, 95% CI 0.82 to 19.56; PR/ couple swim-up 15%, gradient 20%) . There was no significant difference in the miscarriage rate (MR) per couple between either of the three treatment groups in the one trial reporting this outcome (MR/ couple swim-up 0%, gradient 10.3%, wash 0% . MR/ pregnancy gradient 30.3%) . There was no statistically significant difference in the multiple pregnancy rate (MPR) per couple between either of the three treatment groups in Dodson 1998-I/ II (MPR/ couple swim -up 0%, gradient 0%, wash 6.3%) . One triplet pregnancy was recorded . Fifteen studies comparing semen parameters after processing were included . Two studies were included in the meta-analysis, we were not able to pool results . REVIEWERS' CONCLUSIONS: There is insufficient evidence to recommend any specific preparation technique . Large high quality randomised controlled trials, comparing the effectiveness of a gradient and/ or a swim-up and/ or wash and centrifugation technique on clinical outcome are lacking . Further randomised trials are warranted . Results from studies comparing semen parameters may suggest a preference for gradient technique, but firm conclusions cannot be drawn and the limitations should be taken into consideration. Cochrane Database Syst Rev . 2004;(3):CD004153. Ozone therapy for the treatment of dental caries; Rickard GD et al.; BACKGROUND: Dental caries is a bacterially mediated disease characterised by demineralisation of the tooth surface, which may lead to cavitation, discomfort, pain and eventual tooth loss . Ozone is toxic to certain bacteria in vitro and it has been suggested that delivering ozone into a carious lesion might reduce the number of cariogenic bacteria . This possibly could arrest the progress of the lesion and may, in the presence of fluoride, perhaps allow remineralisation to occur . This may in turn delay or prevent the need for traditional dental conservation by 'drilling and filling' . OBJECTIVES: To assess whether ozone is effective in arresting or reversing the progression of dental caries . SEARCH STRATEGY: We searched the Cochrane Oral Health Group's Trials Register (to 7 November 2003); Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library Issue 3, 2003); MEDLINE and PREMEDLINE (OVID, 1966 to November 2003); EMBASE (OVID, 1980 to November 2003); CINAHL (OVID, 1982 to November 2003); AMED (OVID, 1985 to November 2003) . Quintessence was handsearched through 2002 and KaVo were contacted as manufacturers of the HealOzone apparatus for any additional published or unpublished trials . SELECTION CRITERIA: Inclusion was assessed independently by at least two reviewers . Trials were only included if they met the following criteria: randomisation in a controlled trial; single surface in vivo carious lesion accessible to ozone application; clear allocation concealment; ozone application to the lesions in the intervention group; no such application of ozone in the control group; outcomes measured after at least 6 months . DATA COLLECTION AND ANALYSIS: Reviewers independently extracted information in duplicate . A paucity of comparable data did not allow meta-analytic pooling of the included studies . MAIN RESULTS: Three trials were included, with a combined total of 432 randomised lesions (137 participants) . Forty-two conference papers, abstracts and posters were excluded (from an unknown number of studies) . The risk of bias in all studies appeared high . The analyses of all three studies were conducted at the level of the lesion, which is not independent of the person, for this reason pooling of data was not appropriate or attempted . Individual studies showed inconsistent effects of ozone on caries, across different measures of caries progression or regression . Few secondary outcomes were reported, but one trial reported an absence of adverse events . REVIEWERS' CONCLUSIONS: Given the high risk of bias in the available studies and lack of consistency between different outcome measures, there is no reliable evidence that application of ozone gas to the surface of decayed teeth stops or reverses the decay process . There is a fundamental need for more evidence of appropriate rigour and quality before the use of ozone can be accepted into mainstream primary dental care or can be considered a viable alternative to current methods for the management and treatment of dental caries. Cochrane Database Syst Rev . 2004;(3):CD003949. Preoperative skin antiseptics for preventing surgical wound infections after clean surgery; Edwards PS et al.; BACKGROUND: Approximately 15% of elective surgery patients and 30% of patients receiving contaminated or dirty surgery are estimated to develop post-operative wound infections . The costs of surgical wound infection can be considerable in financial as well as social terms . Preoperative skin antisepsis is performed to reduce the risk of post-operative wound infections by removing soil and transient organisms from the skin . Antiseptics are thought to be both toxic to bacteria and aid their mechanical removal . The effectiveness of preoperative skin preparation is thought to be dependent on both the antiseptic used and the method of application, however it is unclear whether preoperative skin antisepsis actually reduces post-operative wound infection and if so which antiseptic is most effective . OBJECTIVES: To determine whether preoperative skin antisepsis reduces post-operative surgical wound infection . SEARCH STRATEGY: We searched the Cochrane Wounds Group Specialised Trials Register and the Cochrane Central Register of Controlled Trials in April 2004 . In addition we handsearched journals, conference proceedings and bibliographies . SELECTION CRITERIA: Randomised controlled trials evaluating the use of preoperative skin antiseptics applied immediately prior to incision in clean surgery . There were no restrictions based on language, date or publication status . DATA COLLECTION AND ANALYSIS: Three reviewers independently undertook data extraction and assessment of study quality . Pooling was inappropriate and trials are discussed in a narrative review . MAIN RESULTS: We identified six eligible RCTs evaluating preoperative antiseptics . There was significant heterogeneity in the comparisons and the results could not be pooled . In one study, infection rates were significantly lower when skin was prepared using chlorhexidine compared with iodine . There was no evidence of a benefit in four trials associated with the use of iodophor impregnated drapes . REVIEWERS' CONCLUSIONS: There is insufficient research examining the effects of preoperative skin antiseptics to allow conclusions to be drawn regarding their effects on post-operative surgical wound infections . Further research is needed. Nephrol Dial Transplant, 2004 Sep, 19(9), 2296 - 301 Epub 2004 Jul 20. IGG and complement receptor expression on peripheral white blood cells in uraemic children; Bouts AH et al.; BACKGROUND: Phagocytosis of IgG- or complement-opsonized bacteria and antibody production by lymphocytes are regulated by cell surface receptors for IgG (FcgammaRI, FcgammaRII and FcgammaRIII) and complement (CR1 and CR3) . We measured the effect of uraemia and dialysis treatment on FcgammaR and CR expression on leukocytes in blood . METHODS: Blood samples were obtained from children: 40 treated with peritoneal dialysis (PD), 23 with haemodialysis (HD), 46 not yet dialysed (CRF) and 33 healthy (HC) . White blood cells, isolated from EDTA-blood by centrifugation after cell fixation with paraformaldehyde, were labelled with FITC-conjugated CD16 (FcgammaRIII), CD32 (FcgammaRII), CD64 (FcgammaRI), CD11b (CR3) and CD35 (CR1) monoclonal antibodies and analysed by flow cytometry . RESULTS: In PD, HD, CRF and HC, monocytes and neutrophils were all positive for FcgammaR and CR, except for CD16 on monocytes (20% positive) . Lymphocytes expressed CD16 and CD32 but not CD64 . PD, HD and CRF children had lower percentages of CD16(+) and CD32(+) lymphocytes compared with HC . The percentage of CD11b(+) lymphocytes was lower only in PD and the percentage of CD35(+) lymphocytes was lower in HD and CRF compared with HC . The median CD32 mean fluorescense intensity (MFI) on lymphocytes, monocytes and neutrophils was lower in PD, HD and CRF compared with HC . On the other hand, CD11b MFI on lymphocytes, monocytes and neutrophils was higher in PD, HD and CRF children compared with HC . CD16 and CD64 MFI were not different among the groups and CD35 MFI was only lower on lymphocytes from PD, HD and CRF compared with HC . CONCLUSIONS: In children with chronic renal failure, whether dialysed or not, FcgammaRII expression on lymphocytes, monocytes and neutrophils was reduced and CR3 expression was increased . Furthermore, CR1 expression on lymphocytes, important for the humoral response, was lower in children with renal failure . Age and uraemia are associated with these abnormalities and might contribute to impaired immune function in children with chronic renal failure. J Cell Sci, 2004 Aug 1, 117(Pt 17), 3923 - 33 Epub 2004 Jul 20. Analysis of Chlamydia caviae entry sites and involvement of Cdc42 and Rac activity; Subtil A et al.; In epithelial cells, endocytic activity is mostly dedicated to nutrient and macromolecule uptake . To invade these cells, Chlamydiaceae, like other pathogens, have evolved strategies that utilise the existing endocytic machineries and signalling pathways, but little is known about the host cell molecules involved . In this report, we show that within five minutes of infection of HeLa cells by Chlamydia caviae GPIC strain several events take place in the immediate vicinity of invasive bacteria: GM1-containing microdomains cluster, tyrosine-phosphorylated proteins accumulate, and intense actin polymerization occurs . We show that actin polymerization is controlled by the small GTPases Cdc42 and Rac, which become activated upon infection . Expression of dominant negative forms of these GTPases inhibits C . caviae entry and leads to abnormal actin polymerization . In contrast, the small GTPase Rho does not seem essential for bacterial entry . Finally, phosphatidylinositol 3-kinase activity is also required for internalization of C . caviae, probably downstream of the other molecular events reported here . We present the first scheme of the events occurring at the sites of invasion of epithelial cells by a member of the Chlamydiaceae family. J Immunol, 2004 Aug 1, 173(3), 1934 - 40 Bordetella type III secretion and adenylate cyclase toxin synergize to drive dendritic cells into a semimature state; Skinner JA et al.; Bordetella bronchiseptica establishes persistent infection of the murine respiratory tract . We hypothesize that long-term colonization is mediated in part by bacteria-driven modulation of dendritic cells (DCs) leading to altered adaptive immune responses . Bone marrow-derived DCs (BMDCs) from C57BL/6 mice infected with live B . bronchiseptica exhibited high surface expression of MHCII, CD86, and CD80 . However, B . bronchiseptica-infected BMDCs did not exhibit significant increases in CD40 surface expression and IL-12 secretion compared with BMDCs treated with heat-killed B . bronchiseptica . The B . bronchiseptica type III secretion system (TTSS) mediated the increase in MHCII, CD86, and CD80 surface expression, while the inhibition of CD40 and IL-12 expression was mediated by adenylate cyclase toxin (ACT) . IL-6 secretion was independent of the TTSS and ACT . These phenotypic changes may result from differential regulation of MAPK signaling in DCs . Wild-type B . bronchiseptica activated the ERK 1/2 signaling pathway in a TTSS-dependent manner . Additionally, ACT was found to inhibit p38 signaling . These data suggest that B . bronchiseptica drive DC into a semimature phenotype by altering MAPK signaling . These semimature DCs may induce tolerogenic immune responses that allow the persistent colonization of B . bronchiseptica in the host respiratory tract. Exp Cell Res, 2004 Aug 15, 298(2), 388 - 98 Interaction between p230 and MACF1 is associated with transport of a glycosyl phosphatidyl inositol-anchored protein from the Golgi to the cell periphery; Kakinuma T et al.; The molecular basis by which proteins are transported along cytoskeletal tracts from the trans-Golgi network (TGN) to the cell periphery remains poorly understood . Previously, using human autoimmune sera, we identified and characterized a TGN protein, p230/Golgin-245, an extensively coiled-coil protein with flexible amino- and carboxyl-terminal ends, that is anchored to TGN membranes and TGN-derived vesicles by its carboxyl-terminal GRIP domain . To identify molecules that interact with the flexible amino-terminal end of p230, we used this domain as bait to screen a human brain cDNA library in a yeast two-hybrid assay . We found that this domain interacts with the carboxyl-terminal domain of MACF1, a protein that cross-links microtubules to the actin cytoskeleton . The interaction was confirmed by co-immunoprecipitation, an in vitro binding assay, double immunofluorescence images demonstrating overlapped localization in HeLa cells, and co-localization of FLAG-tagged constructs containing the interacting domains of these two proteins with their endogenous partners . Expression in HeLa cells of FLAG-tagged constructs containing the interacting domains of p230 and MACF1 disrupted transport of the glycosyl phosphatidyl inositol-anchored marker protein conjugated with yellow fluorescent protein (YFP-SP-GPI), while trafficking of the transmembrane marker protein, vesicular stomatitis virus glycoprotein conjugated with YFP (VSVG3-GL-YFP), was unaffected . Our results suggest that p230, through its interaction with MACF1, provides the molecular link for transport of GPI-anchored proteins along the microtubule and actin cytoskeleton from the TGN to the cell periphery. J Am Chem Soc, 2004 Jul 28, 126(29), 9094 - 100 Effect of sodium sulfide on Ni-containing carbon monoxide dehydrogenases; Feng J et al.; The structure of the active-site C-cluster in CO dehydrogenase from Carboxydothermus hydrogenoformans includes a mu(2)-sulfide ion bridged to the Ni and unique Fe, whereas the same cluster in enzymes from Rhodospirillum rubrum (CODH(Rr)) and Moorella thermoacetica (CODH(Mt)) lack this ion . This difference was investigated by exploring the effects of sodium sulfide on activity and spectral properties . Sulfide partially inhibited the CO oxidation activity of CODH(Rr) and generated a lag prior to steady-state . CODH(Mt) was inhibited similarly but without a lag . Adding sulfide to CODH(Mt) in the C(red1) state caused the g(av) = 1.82 EPR signal to decline and new features to appear, including one with g = 1.95, 1.85 and (1.70 or 1.62) . Removing sulfide caused the g(av) = 1.82 signal to reappear and activity to recover . Sulfide did not affect the g(av) = 1.86 signal from the C(red2) state . A model was developed in which sulfide binds reversibly to C(red1), inhibiting catalysis . Reducing this adduct causes sulfide to dissociate, C(red2) to develop, and activity to recover . Using this model, apparent K(I) values are 40 +/- 10 nM for CODH(Rr) and 60 +/- 30 microM for CODH(Mt) . Effects of sulfide are analogous to those of other anions, including the substrate hydroxyl group, suggesting that these ions also bridge the Ni and unique Fe . This proposed arrangement raises the possibility that CO binding labilizes the bridging hydroxyl and increases its nucleophilic tendency toward attacking Ni-bound carbonyl. Rev Biol Trop, 2003 Jun, 51 Suppl 4, 1 - 6 The effect of elevated temperature on the toxicity of the laboratory cultured dinoflagellate Ostreopsis lenticularis (Dinophyceae); Ashton M et al.; Ostreopsis lenticularis Fukuyo 1981, is the major benthic dinoflagellate vector implicated in ciguatera fish poisoning in finfish on the southwest coast of Puerto Rico . Clonal laboratory cultures of O . lenticularis (clone 301) exposed to elevated temperatures (30-31 degrees C) for 33 and 54 days showed significant increases in the quantity of extractable toxin they produced as compared to their toxicities versus cells grown at temperatures of 25-26 degrees C . O lenticularis samples collected directly from the field following exposure to elevated temperatures for comparable periods of time also showed significant increases in extractable toxin . The increased toxicity of both field sampled and laboratory grown O . lenticularis exposed to elevated temperatures may result from the effects of elevated temperatures on their metabolism and/or the bacterial symbionts found associated with these microalgae . The number of bacteria associated with cultured O . lenticularis exposed to elevated temperatures was significantly reduced . Increased toxin recovery from O . lenticularis exposed to elevated temperatures may have resulted from the direct effect of temperature on toxin production and/or the reduction of Ostreopsis associated bacterial flora that consume toxin in the process of their growth . This reduction in the quantity of associated bacterial flora in temperature treated cultures may result in increased toxin recovery from O . lenticularis due to a reduction in the consumption of toxin by these symbiont bacteria. J Struct Funct Genomics, 2004, 5(1-2), 167 - 72 Towards higher-throughput membrane protein production for structural genomics initiatives; Laible PD et al.; Integral membrane proteins present unparalleled challenges for structural genomics programs . Samples from this class of proteins are not only difficult to produce in quantities sufficient for analysis by X-ray diffraction or NMR, but their hydrophobic properties add extra dimension to their purification and subsequent crystallization . New systems that seek to tackle the production problems are in development . In our laboratory, one such strategy exploits the unique physiology of the Rhodobacter species of photosynthetic bacteria where we have designed an overexpression system that coordinates the heterologous production of targeted hydrophobic proteins with nascent, unfilled membranes that can be used to harbor them . In this study, we describe the means by which purification of recombinant membrane proteins produced in such a fashion can be purified efficiently from Rhodobacter membranes using relatively higher-throughput, semi-automated methods . These protocols utilize a state-of-the-art FPLC system for affinity chromatography, followed by gel filtration or ion exchange chromatography to enhance purity for crystallization attempts . The Rhodobacter expression system coupled with the semi-automation of purification steps represents an advance towards the development of a strategy for obtaining structures for membrane proteins at a more rapid pace. J Mol Microbiol Biotechnol, 2004, 7(3), 115 - 32 Four promoters subject to regulation by ExoR and PhoB direct transcription of the Sinorhizobium melilotiexoYFQ operon involved in the biosynthesis of succinoglycan; Quester I et al.; Succinoglycan (EPS I), the main acidic exopolysaccharide of Sinorhizobium meliloti, is required for the initiation and elongation of infection threads during nodulation of the host plant alfalfa . The gene products of the exoYFQ operon are involved in the first step of succinoglycan biosynthesis as well as in the polymerisation of subunits to the high-molecular-mass form of this exopolysaccharide . One promoter region that directs transcription of exoX and two promoter regions that drive transcription of exoY were mapped in the exoX-exoY intergenic region . The distal exoY promoter region containing three putative -10 promoter elements was active under standard growth conditions and was subject to ExoR-dependent regulation . Although this promoter region was stimulated in a phoB mutant, no PHO box-like sequences were found, suggesting an indirect regulatory effect of PhoB . The proximal promoter contains a PHO box-like sequence in the putative -35 region and was affected by low and high phosphate concentrations dependent on PhoB . In the case of deleted upstream regions, this promoter was also controlled by ExoR . An additional promoter displaying activity in exoR, mucR and phoB mutants under standard conditions was identified upstream of exoF . The putative -35 promoter element of this promoter is covered by a second PHO box-like sequence . J Mol Microbiol Biotechnol, 2004, 7(3), 102 - 8 Sequence analyses of cyanobacterial bicarbonate transporters and their homologues; von Rozycki T et al.; The primary HCO3- uptake system in the cyanobacterium Synecocystis is the Na+-dependent transporter SbtA . SbtA and its homologues were identified and shown to display a common topology of ten transmembrane segments (TMSs) . These proved to have arisen by an intragenic duplication event from an ancestral gene encoding a five TMS protein product . A region of SbtA shows sufficient similarity to 10 TMS ABC-type integral membrane transport proteins to suggest a common origin . Phylogenetic analyses of the SbtA family revealed two clusters of cyanobacterial homologues with all non-cyanobacterial family members outside of these two clusters . The tree topology suggests that SbtA family members display multiple transport functions . J Biol Chem, 2004 Oct 8, 279(41), 42970 - 6 Epub 2004 Jul 15. The cytoplasmic membrane-proximal domain of the HtrII transducer interacts with the E-F loop of photoactivated Natronomonas pharaonis sensory rhodopsin II; Yang CS et al.; The structures of the cytoplasmic loops of the phototaxis receptor sensory rhodopsin II (SRII) and the membrane-proximal cytoplasmic domain of its bound transducer HtrII were examined in the dark and in the light-activated state by fluorescent probes and cysteine cross-linking . Light decreased the accessibility of E-F loop position 154 in the SRII-HtrII complex, but not in free SRII, consistent with HtrII proximity, which was confirmed by tryptophans placed within a 5-residue region identified in the HtrII membrane-proximal domain that exhibited Forster resonance energy transfer to a fluorescent probe at position 154 in SRII . The Forster resonance energy transfer was eliminated in the signaling deficient HtrII mutant G83F without loss of affinity for SRII . Finally, the presence of SRII and HtrII reciprocally inhibit homodimer disulfide cross-linking reactions in their membrane-proximal domains, showing that each interferes with the others self-interaction in this region . The results demonstrate close proximity between SRII-HtrII in the membrane-proximal domain, and in addition, light stimulation of the SRII inhibition of HtrII cross-linking was observed, indicating that the contact is enhanced in the photoactivated complex . A mechanism is proposed in which photoactivation alters the SRII-HtrII interaction in the membrane-proximal region during the signal relay process. J Bacteriol, 2004 Aug, 186(15), 5172 - 7 Biochemical study of multiple CheY response regulators of the chemotactic pathway of Rhodobacter sphaeroides; Ferre A et al.; The six copies of the response regulator CheY from Rhodobacter sphaeroides bind to the switch protein FliM . Phosphorylation by acetyl phosphate (AcP) was detected by tryptophan fluorescence quenching in three of the four CheYs that contain this residue . Autophosphorylation with Ac(32)P was observed in five CheY proteins . We also show that all of the cheY genes are expressed simultaneously; therefore, in vivo all of the CheY proteins could bind to FliM to control the chemotactic response . Consequently, we hypothesize that in this complex chemotactic system, the binding of some CheY proteins to FliM, does not necessarily imply switching of the flagellar motor. J Bacteriol, 2004 Aug, 186(15), 4960 - 71 Identification of the protease and the turnover signal responsible for cell cycle-dependent degradation of the Caulobacter FliF motor protein; Grunenfelder B et al.; Flagellar ejection is tightly coupled to the cell cycle in Caulobacter crescentus . The MS ring protein FliF, which anchors the flagellar structure in the inner membrane, is degraded coincident with flagellar release . Previous work showed that removal of 26 amino acids from the C terminus of FliF prevents degradation of the protein and interferes with flagellar assembly . To understand FliF degradation in more detail, we identified the protease responsible for FliF degradation and performed a high-resolution mutational analysis of the C-terminal degradation signal of FliF . Cell cycle-dependent turnover of FliF requires an intact clpA gene, suggesting that the ClpAP protease is required for removal of the MS ring protein . Deletion analysis of the entire C-terminal cytoplasmic portion of FliF C confirmed that the degradation signal was contained in the last 26 amino acids that were identified previously . However, only deletions longer than 20 amino acids led to a stable FliF protein, while shorter deletions dispersed over the entire 26 amino acids critical for turnover had little effect on stability . This indicated that the nature of the degradation signal is not based on a distinct primary amino acid sequence . The addition of charged amino acids to the C-terminal end abolished cell cycle-dependent FliF degradation, implying that a hydrophobic tail feature is important for the degradation of FliF . Consistent with this, ClpA-dependent degradation was restored when a short stretch of hydrophobic amino acids was added to the C terminus of stable FliF mutant forms. Bioinformatics, 2004 Aug 4, 20 Suppl 1, I297 - I302 Prediction of class I T-cell epitopes: evidence of presence of immunological hot spots inside antigens; Srinivasan KN et al.; MOTIVATION: Processing and presentation of major histocompatibility complex class I antigens to cytotoxic T-lymphocytes is crucial for immune surveillance against intracellular bacteria, parasites, viruses and tumors . Identification of antigenic regions on pathogen proteins will play a pivotal role in designer vaccine immunotherapy . We have developed a system that not only identifies high binding T-cell antigenic epitopes, but also class I T-cell antigenic clusters termed immunological hot spots . METHODS: MULTIPRED, a computational system for promiscuous prediction of HLA class I binders, uses artificial neural networks (ANN) and hidden Markov models (HMM) as predictive engines . The models were rigorously trained, tested and validated using experimentally identified HLA class I T-cell epitopes from human melanoma related proteins and human papillomavirus proteins E6 and E7 . We have developed a scoring scheme for identification of immunological hot spots for HLA class I molecules, which is the sum of the highest four predictions within a window of 30 amino acids . RESULTS: Our predictions against experimental data from four melanoma-related proteins showed that MULTIPRED ANN and HMM models could predict T-cell epitopes with high accuracy . The analysis of proteins E6 and E7 showed that ANN models appear to be more accurate for prediction of HLA-A3 hot spots and HMM models for HLA-A2 predictions . For illustration of its utility we applied MULTIPRED for prediction of promiscuous T-cell epitopes in all four SARS coronavirus structural proteins . MULTIPRED predicted HLA-A2 and HLA-A3 hot spots in each of these proteins. Pathol Biol (Paris), 2004 Jul, 52(6), 351 - 64 {Monoclonal antibodies and therapeutics}; Desgranges C; More than 25 years after their discovery, monoclonal antibodies are now the most rapid expanding pharmaceutical viable drugs in clinical trials . The emergence of these antibodies was made possible by the development of genetic recombinant techniques . It is now possible to obtain engineered antibodies: chimearic or humanized or fully human monoclonal antibodies via the use of phage display technology or of transgenic mice . These antibodies are tolerable to the human immune system and eleven have been approved for therapeutic by the US Food and Drug Administration (FDA), the majority of them in the past four years . At least an additional 400 monoclonal antibodies are in clinical trials to treat cancer, transplant rejection or to combat autoimmune or infectious diseases . Important advances have been made in the design of highly specific fragment antibodies, fused or not with drugs or radioisotopes, and in the large industrial scale production with different expression systems (bacteria, yeasts, mammalian cells and transgenic plants and animals) . In the next future new molecular promising strategies will enhance affinity, stability and expression levels and reduce the price of these engineering monoclonal to permit their use to treat a large number of diseases. J Chem Phys, 2004 Jul 8, 121(2), 946 - 59 Chiral exciton wave functions in cylindrical J aggregates; Didraga C et al.; We study the exciton wave functions and the optical properties of cylindrical molecular aggregates . The cylindrical symmetry allows for a decomposition of the exciton Hamiltonian into a set of effective one-dimensional Hamiltonians, characterized by a transverse wave number k2 . These effective Hamiltonians have interactions that are complex if the cylinder exhibits chirality . We propose analytical ansatze for the eigenfunctions of these one-dimensional problems that account for a finite cylinder length, and present a general study of their validity . A profound difference is found between the Hamiltonian for the transverse wave number k2=0 and those with k2 not equal 0 . The complex nature of the latter leads to chiral wave functions, which we characterize in detail . We apply our general formalism to the chlorosomes of green bacteria and compare the wave functions as well as linear optical spectra (absorption and dichroism) obtained through our ansatze with those obtained by numerical diagonalization as well as those obtained by imposing periodic boundary conditions in the cylinder's axis direction . It is found that our ansatze, in particular, capture the finite-length effect in the circular dichroism spectrum much better than the solution with periodic boundary conditions . Our ansatze also show that in finite-length cylinders seven superradiant states dominate the linear optical response . (c) 2004 American Institute of Physics. Biochemistry, 2004 Jul 27, 43(29), 9467 - 76 A look within LHCII: differential analysis of the Lhcb1-3 complexes building the major trimeric antenna complex of higher-plant photosynthesis; Caffarri S et al.; The major antenna complex of higher-plant photosynthesis, LHCII, is composed by the products of three genes, namely, Lhcb1-2-3 . In this paper, the biochemical and spectroscopic properties of each of the three gene products were investigated . The three complexes were obtained by overexpression of the apoproteins in bacteria and refolding in vitro with purified pigments, thus allowing detection of differences in the structure/function of the pigment-binding gene products . The analyses showed that Lhcb1 and Lhcb2 complexes have similar pigment binding properties, although not identical, while Lhcb3 is clearly different with respect to both pigment binding and spectral properties and cannot produce homotrimers in vitro . Heterotrimers containing Lhcb3 together with Lhcb1 and/or -2 proteins were obtained upon assembly with Lhcb proteins purified from thylakoids . The major functional characteristics of Lhcb3 with respect to Lhcb1 and -2 consisted in (i) a red-shift of one specific chlorophyll a chromophore, strongly affecting the red-most region of the absorption spectrum and (ii) a different specificity for xanthophylls binding to sites L2 and N1 . These properties make Lhcb3 a relative sink for excitation energy in isolated heterotrimers with Lhcb1 + Lhcb2, and potentially, a preferential site of regulation of the antenna function in excess light conditions. Environ Sci Technol, 2004 Jun 15, 38(12), 3418 - 24 Thermal wet oxidation improves anaerobic biodegradability of raw and digested biowaste; Lissens G et al.; Anaerobic digestion of solid biowaste generally results in relatively low methane yields of 50-60% of the theoretical maximum . Increased methane recovery from organic waste would lead to reduced handling of digested solids, lower methane emissions to the environment, and higher green energy profits . The objective of this research was to enhance the anaerobic biodegradability and methane yields from different biowastes (food waste, yard waste, and digested biowaste already treated in a full-scale biogas plant (DRANCO, Belgium)) by assessing thermal wet oxidation . The biodegradability of the waste was evaluated by using biochemical methane potential assays and continuous 3-L methane reactors . Wet oxidation temperature and oxygen pressure (T, 185-220 degrees C; O2 pressure, 0-12 bar; t, 15 min) were varied for their effect on total methane yield and digestion kinetics of digested biowaste . Measured methane yields for raw yard waste, wet oxidized yard waste, raw food waste, and wet oxidized food waste were 345, 685, 536, and 571 mL of CH/g of volatile suspended solids, respectively . Higher oxygen pressure during wet oxidation of digested biowaste considerably increased the total methane yield and digestion kinetics and permitted lignin utilization during a subsequent second digestion . The increase of the specific methane yield for the full-scale biogas plant by applying thermal wet oxidation was 35-40%, showing that there is still a considerable amount of methane that can be harvested from anaerobic digested biowaste. Water Sci Technol, 2004, 49(10), 209 - 16 Sludge thermal oxidation processes: mineral recycling, energy impact, and greenhouse effect gases release; Guibelin E; Different treatment routes have been studied for a mixed sludge: the conventional agricultural use is compared with the thermal oxidation processes, including incineration (in gaseous phase) and wet air oxidation (in liquid phase) . The interest of a sludge digestion prior to the final treatment has been also considered according to the two major criteria, which are the fossil energy utilisation and the greenhouse effect gases (CO2, CH4, N2O) release . Thermal energy has to be recovered on thermal processes to make these processes environmentally friendly, otherwise their main interest is to extract or destroy micropollutants and pathogens from the carbon cycle . In case of continuous energy recovery, incineration can produce more energy than it consumes . Digestion is especially interesting for agriculture: according to these two schemes, the energy final balance can also be in excess . As to wet air oxidation, it is probably one of the best ways to minimize greenhouse effect gases emission. Water Sci Technol, 2004, 49(10), 105 - 13 Process performance and change in sludge characteristics during anaerobic digestion of sewage sludge with ozonation; Goel R et al.; A new process configuration combining anaerobic digestion with ozonation, and operated at long SRT, was studied with the objective of on-site reduction in sludge quantity and improving biogas recovery . The process performance with respect to solid reduction efficiency and other important process parameters like accumulation of inorganic solids, changes in sludge viscosity and dewatering characteristics were evaluated from the data of long term pilot scale continuous experiments conducted using a mixture of primary and secondary municipal sewage sludge . Due to sludge ozonation and long SRT, high VSS degradation efficiency of approximately 80% was achieved at a reactor solid concentration of 6.5% . A high fraction of inorganic solid (>50%) consisting mainly of acid insoluble and iron compounds was found to accumulate in the reactor . The high inorganic content accumulated in the digested sludge did not, however, contribute to the observed increase in sludge viscosity at high solid concentration . The sludge viscosity was largely found to depend on the organic solid concentration rather than the total solid content . Moreover, higher inorganic content in the digested sludge resulted in better sludge dewaterability . For a quick assessment of the economic feasibility of the new process, an economic index based on the unit cost of digested sludge disposal to unit electric cost is proposed. Water Sci Technol, 2004, 49(10), 97 - 104 Investigation and assessment of sludge pre-treatment processes; Muller JA et al.; The pre-treatment of sludges by disintegration will result in a number of changes in sludge properties . Floc destruction as well as cell disintegration will occur . This leads to an increase of soluble substances and fine particles . Furthermore, biochemical reactions may appear during or immediately after disintegration . The influence of disintegration of excess sludge on anaerobic digestion was studied in full scale . A stirred ball mill, an ultrasound disintegrator, a lysate centrifuge and ozone treatment were used . The results of the degradation process were compared to a reference system without pre-treatment . An enhancement of the degree of degradation of 7.4% to 20% was observed . The pollution of sludge water as well as the dewatering properties of the digested sludge were investigated . COD and ammonia in the sludge water were increased and a higher polymer demand was observed while the solid content after dewatering stayed almost unchanged . Based on these results the cost effectiveness has been assessed taking into account different conditions (size of WWTP, cost for disposal, etc.) . Capital and energy costs are the main factors while the decrease in disposal costs due to the reduced amount of sludge is the main profit factor. Arch Environ Health, 2003 Aug, 58(8), 498 - 504 Atmospheric transport of mold spores in clouds of desert dust; Shinn EA et al.; Fungal spores can be transported globally in clouds of desert dust . Many species of fungi (commonly known as molds) and bacteria--including some that are human pathogens--have characteristics suited to long-range atmospheric transport . Dust from the African desert can affect air quality in Africa, Europe, the Middle East, and the Americas . Asian desert dust can affect air quality in Asia, the Arctic, North America, and Europe . Atmospheric exposure to mold-carrying desert dust may affect human health directly through allergic induction of respiratory stress . In addition, mold spores within these dust clouds may seed downwind ecosystems in both outdoor and indoor environments. Microb Ecol, 2004 Jan, 47(1), 80 - 6 Impact of clay minerals on sulfate-reducing activity in aquifers; Wong D et al.; Previous studies have shown that sulfate-reduction activity occurs in a heterogeneous manner throughout the terrestrial subsurface . Low-activity regions are often observed in the presence of clay minerals . Here we report that clays inhibit sulfate reduction activity in sediments and in a pure culture of Desulfovibrio vulgaris . Clay minerals including bentonite and kaolinite inhibited sulfate reduction by 70-90% in sediments . Intact clays and clay colloids or soluble components, capable of passing through a 0.2-microm filter, were also inhibitory to sulfate-reducing bacteria . Other adsorbent materials, including anion or cation exchangers and a zeolite, did not inhibit sulfate reduction in sediments, suggesting that the effect of clays was not due to their cation-exchange capacity . We observed a strong correlation between the Al2O3 content of clays and their relative ability to inhibit sulfate reduction in sediments (r2 = 0.82) . This suggested that inhibition might be a direct effect of Al3+ (aq) on the bacteria . We then tested pure aluminum oxide (Al2O3) and showed it to act in a similar manner to clay . As dissolved aluminum is known to be toxic to a variety of organisms at low concentrations, our results suggest that the effects of clay on sulfate-reducing bacteria may be directly due to aluminum . Thus, our experiments provide an explanation for the lack of sulfate-reduction activity in clay-rich regions and presents a mechanism for the effect.
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
