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