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| Appl Environ Microbiol, 2004 Nov, 70(11), 6473 - 80 Stages of infection during the tripartite interaction between Xenorhabdus nematophila, its nematode vector, and insect hosts; Sicard M et al.; Bacteria of the genus Xenorhabdus are mutually associated with entomopathogenic nematodes of the genus Steinernema and are pathogenic to a broad spectrum of insects . The nematodes act as vectors, transmitting the bacteria to insect larvae, which die within a few days of infection . We characterized the early stages of bacterial infection in the insects by constructing a constitutive green fluorescent protein (GFP)-labeled Xenorhabdus nematophila strain . We injected the GFP-labeled bacteria into insects and monitored infection . We found that the bacteria had an extracellular life cycle in the hemolymph and rapidly colonized the anterior midgut region in Spodoptera littoralis larvae . Electron microscopy showed that the bacteria occupied the extracellular matrix of connective tissues within the muscle layers of the Spodoptera midgut . We confirmed the existence of such a specific infection site in the natural route of infection by infesting Spodoptera littoralis larvae with nematodes harboring GFP-labeled Xenorhabdus . When the infective juvenile (IJ) nematodes reached the insect gut, the bacterial cells were rapidly released from the intestinal vesicle into the nematode intestine . Xenorhabdus began to escape from the anus of the nematodes when IJs were wedged in the insect intestinal wall toward the insect hemolymph . Following their release into the insect hemocoel, GFP-labeled bacteria were found only in the anterior midgut region and hemolymph of Spodoptera larvae . Comparative infection assays conducted with another insect, Locusta migratoria, also showed early bacterial colonization of connective tissues . This work shows that the extracellular matrix acts as a particular colonization site for X . nematophila within insects. Science, 2004 Nov 5, 306(5698), 1042 - 6 Tracking SNARE complex formation in live endocrine cells; An SJ et al.; Syntaxin, synaptosome-associated protein of 25 kD (SNAP25), and vesicle-associated membrane protein/synaptobrevin are collectively called SNAP receptor (SNARE) proteins, and they catalyze neuronal exocytosis by forming a "core complex." The steps in core complex formation are unknown . Here, we monitored SNARE complex formation in vivo with the use of a fluorescent version of SNAP25 . In PC12 cells, we found evidence for a syntaxin-SNAP25 complex that formed with high affinity, required only the amino-terminal SNARE motif of SNAP25, tolerated a mutation that blocks formation of other syntaxin-SNAP25 complexes, and assembled reversibly when Ca2+ entered cells during depolarization . The complex may represent a precursor to the core complex formed during a Ca2+-dependent priming step of exocytosis. FEBS Lett, 2004 Nov 5, 577(1-2), 170 - 4 Structure of the mitochondrial beta-ketoacyl-{acyl carrier protein} synthase from Arabidopsis and its role in fatty acid synthesis; Olsen JG et al.; Mitochondrial fatty acid synthesis is catalyzed by a dissociated fatty acid synthase similar to those of plant plastids and bacteria . The crystal structure of a mitochondrial beta-ketoacyl-{acyl carrier protein} synthase (mtKAS), namely that from Arabidopsis thaliana, has been determined for the first time . This enzyme accomplishes the vital condensation steps in constructing fatty acid carbon skeletons . The product profile of mtKAS is unusual in that C8 and C(14-16) fatty acyl chains predominate . An enzyme architecture that likely is the basis for the observed bimodal profile of mtKAS products can be derived from the shape of the acyl binding pocket. FEBS Lett, 2004 Nov 5, 577(1-2), 141 - 6 Identification of a novel shrimp protein phosphatase and its association with latency-related ORF427 of white spot syndrome virus; Lu L et al.; To characterize the role of latency-associated ORF427 of white spot syndrome virus (WSSV), a shrimp cDNA library was constructed to screen interacting proteins of ORF427 . Employing the yeast two-hybrid system, a novel shrimp protein phosphatase (named PPs), sharing 93% homology with human protein phosphatase 1, has been identified able to bind ORF427 in yeast . Through co-immunoprecipitation assays, the interaction between PPs and ORF427 was further confirmed both in vitro and in vivo . Interestingly, the novel shrimp protein phosphatase consists of only 199 aa and contains almost all the functional catalytic domains of human protein phosphatase, while it lacks the corresponding C-terminal non-catalytic sequence . Transcription and translation products of the identified cDNA can be detected in both normal and WSSV-infected shrimps; and PPs was found to localize mainly in the lysosome of shrimp cells . To characterize its function, the PPs cDNA was highly expressed in bacteria and the purified protein showed phosphatase activity when tested against pNPP in a standard phosphatase assay . Our results suggest that the identified protein phosphatase, PPs, may represent a novel member of protein phosphatase family and might be involved in the regulation of WSSV's life cycle through interaction with latency-related ORF427 of WSSV. Curr Gastroenterol Rep, 2004 Dec, 6(6), 441 - 6 The spectrum of gastric disease associated with Helicobacter pylori and other infectious gastritides; Guarner J; Helicobacter pylori is the most frequent infection of the stomach worldwide . Some of the people infected with H . pylori develop symptoms of dyspepsia that correlate with pathologic evidence of gastritis and peptic ulcers . In addition, H . pylori has been associated with preneoplastic lesions and with two neoplasias: intestinal-type gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma . Rarely, gastric pathology can also be caused by other infectious agents, including fungi, other bacteria, parasites, and viruses . This review describes H . pylori-associated pathology and pathologies related to other infectious agents. Curr Gastroenterol Rep, 2004 Dec, 6(6), 429 - 35 Helicobacter pylori infection and gastric cancer: host, bug, environment, or all three? Menaker RJ, Sharaf AA, Jones NL. Helicobacter pylori is a common bacterial pathogen that colonizes the gastric mucosa of over 50% of the world's population . All infected individuals exhibit chronic gastric inflammation, and approximately 1% of patients develop gastric cancers, including adenocarcinomas and mucosal-associated lymphoid tissue lymphomas . In 1994, the World Health Organization International Agency for Research on Cancer classified H . pylori as a type I, or definite carcinogen . Because the prevalence of gastric cancers among H . pylori-infected patients varies between individuals, countries, and geographic areas, H . pylori disease-related outcomes are believed to be determined by an interplay between host factors, bacterial factors, and their interaction with the environment . This review highlights recent advances in our knowledge on H . pylori disease pathogenesis, focusing on the role of the host, bacteria, and environment in the development of gastric carcinoma. Inflammopharmacology, 2004, 12(3), 247 - 60 Effect of Mycobacterium leprae lipids on BCG- and carrageenan-induced cellular recruitment in mouse pleurisy; Moura AC et al.; Pathogenic mycobacteria survive inside macrophages and deactivate these cells, using a mechanism that is still poorly understood . Mycobacterial cell wall lipids constitute the first contact with the host cell . Although Mycobaterium leprae and M . bovis BCG share common antigens, they induce opposite inflammatory responses . Apolar M . leprae lipids have been shown to be anti-inflammatory by down-regulating macrophage activation and T-cell functions . We wonder if these lipids would influence cellular migration to BCG or to other inflammatory agent . We investigated the effect of M . leprae, its lipids or delipidated bacteria on acute and chronic BCG- or carrageenan-induced pleurisy . Previous injection of intact or delipidated M . leprae did not alter either the BCG- or carrageenan-induced pleural inflammatory reaction . However, M . leprae lipids enhanced carrageenan-induced acute cellular migration without impairing BCG inflow; moreover, they reduced BCG chronic response . Together these data suggest distinct mechanisms for intracellular deactivation and pleural cell recruitment exerted by mycobacterial structures. Biochemistry (Mosc), 2004 Oct, 69(10), 1055 - 66 Apoptosis in unicellular organisms: mechanisms and evolution; Gordeeva AV et al.; Data about the programmed death (apoptosis) in unicellular organisms, from bacteria to ciliates, are discussed . Firstly apoptosis appeared in lower eukaryotes, but its mechanisms in these organisms are different from the classical apoptosis . During evolution, the apoptotic process has been improving gradually, with reactive oxygen species and Ca2+ playing an essential role in triggering apoptosis . All eukaryotic organisms have apoptosis inhibitors, which might be introduced by viruses . In the course of evolution, caspases and apoptosis-inducing factor appeared before other apoptotic proteins, with so-called death receptors being the last among them . The functional analogs of eukaryotic apoptotic proteins take parts in the programmed death of bacteria. Indian J Environ Health, 2003 Oct, 45(4), 275 - 80 Influence of Adyar river in the coastal waters of Chennai (Madras), Bay of Bengal; Subramanian B et al.; The impact of the river Adyar which is influenced by industrial wastes and domestic sewage from the city of Chennai (Madras), on the hydrobiology of coastal water in the Bay of Bengal was evaluated . Reduction in pH, dissolved oxygen (DO), nitrite, nitrate, primary productivity and increase of ammonia, phosphate, silicate, heavy metals, biological oxygen demand (BOD), chlorophyll pigments, particulate organic carbon (POC), respiration, suspended solids, ATP, and bacteria in the mixing site during low tide indicated that the Adyar water was heavily polluted and ultimately affect the quality of coastal water near the vicinity of mixing zone . The effects are diluted or nullified as the distance increased from the mixing site due to heavy mixing and littoral current . During high tide, the flow was completely stopped and little or no effect was recorded . The light, water current and wave action are the important factors which minimize the pollution impacts in the receiving coastal water. J Anim Sci, 1999, 77 Suppl 2, 101 - 10 Inflammation in the bovine female reproductive tract; Bondurant RH; Inflammation of the reproductive tract of a cow occurs when the physical and functional barriers to contamination are breached or specific infection occurs . Commonly, contamination occurs at parturition and to a lesser extent at estrus . Uterine contamination following calving is common, but most healthy cows are able to clear the uterus of bacteria in the first 2 to 3 wk after calving . Persistent infections are more likely to be caused by Actinomyces pyogenes . Specific venereal infections tend to be more host-adapted and produce a lower grade inflammation . Nonspecific bacterial contamination of the endometrium generally induces a neutrophilic influx into the stratum compactum and uterine lumen . Neutrophils phagocytize bacteria with the aid of opsonins in the uterine fluid . Mast cells and eosinophils may also contribute to the inflammatory reaction, which may damage the surface epithelium and release vasoactive substances that allow leakage of serum antibodies into the uterine secretions . Specific antibodies of immunoglobulin (Ig) isotype A, M, G1, and G2 in uterine secretions have been described . In model species, the immune capability of the uterus is influenced by steroid hormones, especially estradiol, which increases secretory component and both IgA and IgG content in uterine secretions and increases the activity of antigen-presenting cells in the uterus . Similar cyclic fluctuations in immune components have been described for cows, including changes in the population of subsurface cytotoxic and helper T cells and changes in the expression of major histocompatibility II antigen on surface cells. Nippon Hoigaku Zasshi, 2004 Sep, 58(2), 130 - 40 {Induction mechanism of shock: applying the etiology in judgment of the cause of death in forensic practice}; Tanaka N; In the field of forensic medicine, shock has been identified as a cause of death owing to various kinds of exogenous insults . The etiology and pathogenesis of shock cannot be explained well by the usual gross appearance in medicolegal autopsies, because it is now generally established that the shock is a functional reaction of the vascular system to bodily injury, and that several organs are secondarily impaired during shock . Thus it seemed to forensic pathologists that these morphological changes in several organs after shock did not reveal any significant differences among the causes of death . We approached to the induction mechanism of shock, and we investigated what etiology induced these morphological changes after shock in order to identify shock as the cause of death . It is now generally accepted that the kidney is a target organ of shock, so we mainly investigated the cause of kidney disorder in a case of burn shock and hemorrhagic shock . 1 . Consequences of bacterial translocation (BT) in the shock . The concept of BT indicates that the beginning of shock is induced by the loss of gut barrier function and consequent translocation of bacteria . In general, impaired gut barrier function can be caused either during the shock period by decreased intestinal blood flow and reduced oxygen delivery, resulting during reperfusion in a stage of increased intestinal blood flow, or at a later stage again by reduced flow . A variety of physiological stresses, such as trauma, hemorrhage, thermal injury, surgical operation, various kinds of drags and mental stress, have been shown to cause failure of the gut mucosal barrier, with translocation of bacteria/endotoxin from the gastrointestinal into the mesenteric lymph nodes, and translocation into remote organs and systemic circulation . 2 . Burn shock . We designed to evaluate the BT in a burn shock rat model (following 20% full-thickness scald injury) . The p38 MAPK pathway is an important stress-responsive signal molecule pathway, and it is responsible for the production and signal transduction of cytokines . This pathway is activated by the bacterial LPS or ischemia, so we examined the effects of FR167653, a specific inhibitor of p38 MAPK, on the development of renal failure after the burn-induced intestinal barrier damage . Our study demonstrated that viable bacteria reached the remote organs after burn by quantitative bacterial culture data and FR167653 blocked the burn-induced intestinal barrier damage, and the immunohistochemical data showed that FR167653 prevented the accumulation of polymorphonuclear leukocytes (PMNs) in the glomerular capillaries after burn, and blockaded the burn-induced renal failure by serum UN assay . FR167653 especially decreased the phosphorylation levels of p38 MAPK in the infant kidney after burn, and TNF-alpha and IL-1beta mRNA decreased through the p38 MAPK pathway . The above-mentioned facts do provide additional support for the hypothesis that postburn renal failure is mediated by endotoxin associated with the bacterial translocation, and we identified the pathophysiologic role of p38 MAPK pathway in the development of renal failure after the burn-induced intestinal barrier damage . 3 . Hemorrhagic shock . We evaluated the role of endogenous TNF-alpha in the renal failure and gut bacterial translocation induced by mild hemorrhagic shock (16.7% bleeding of total body blood via a common carotid catheter without fluid resuscitation) . FR167653, a potent inhibitor of TNF-alpha up regulation through p38 MAPK pathway, significantly inhibited these increases of TNF-alpha . Adding to this, our study demonstrated that FR167653 prevented renal failure, such as the infiltration of inflammatory cells and tubular cell necrosis after hemorrhage, and the intestinal barrier damage was also dramatically improved by FR167653 treatment . These results show that derived endogenous TNF-alpha plays a key role in renal failure through p38 MAPK activation during the early phase of mild hemorrhagic shock, including the possible participation of BT . According to these results, we hypothesized that the invading leukocytes induced these organs failures after hemorrhagic shock, so we examined the appearances of leukocytes by the immunohistochemical myeloperoxidase (MPO) staining (marker staining for PMNs) . The incidences of PMNs in these organs after mild hemorrhagic shock increased significantly, and FR157653 prevented the appearance of PMNs . These results showed the possible effective role of the PMNs on the occurrence of organ failure caused by mild hemorrhagic shock . 4 . Forensic practice . Six hundred and seven forensic autopsy cases in our department of forensic medicine during the past 11 years between 1992 and 2002 were analyzed with regard to the cause of death . Shock cases accounted for 18% of all forensic autopsy cases, and among them 65% of cases identified hemorrhagic shock as the cause of death . So we investigated what good grounds to clearly identify the cause of death induced by hemorrhagic shock . Our experimental hemorrhagic shock data showed PMNs activation and priming during hemorrhagic shock, and it might be closely related to BT and remote organ failure . Consequently, we used the MPO staining method, and we immunohistochemically investigated several organs of our practical autopsy cases to detect the appearance of PMNs as a marker of shock induction . We compared the hemorrhagic shock with other causes of death, such as blood loss, asphyxia, drawing and head injury (intracranial hemorrhage) . In every organ, a significant appearance of PMNs was observed in the hemorrhagic shock compared to the other causes of death . Especially, the appearance of PMNs in the heart was clear than that of the other organs in the hemorrhagic shock cases . Therefore, detecting the appearance of PMNs as a marker of shock induction is a very useful and significant method forjudging the cause of death in forensic practice. AORN J, 2004 Oct, 80(4), 652 - 60, 663-5 From handmaiden to right hand--World War I--the mud and the blood; Holder VL; By the beginning of World War I, modern innovations and advances in the field of medicine were common . Physicians knew about bacteria, how disease spread, and the importance of antisepsis . To prepare for the war, Great Britain developed elite corps of army nurses, fully-equipped military hospitals, and trained personnel who established field hospitals and base hospitals . Alice Fitzgerald, a nurse from Baltimore, was drawn into the conflict when she was asked to serve as the Edith Cavell Memorial Nurse with the British Army. Med Arh, 2004, 58(4), 218 - 9 {Acute polyradiculoneuritis--clinical course and outcome during the war and postwar period}; Loncarevic N et al.; The acute polyradiculoneuritis represent acute inflamatory polyneuropathy with unknown cause, frequently induced by prior virus and sometimes bacteria infection . The purpose of this paper is to establish particularities of the clinical course and results of the disease during the war and the post-war time . Within the retrospective study we performed analysis of all patients suffering from the acute polyradiculoneuritis between January 1992 and December 2003 year who during the mentioned period where treated at the Department of Neurology in Sarajevo . In addition to the anamnysis and the clinical examination, diagnostic criteria where laboratory parameters, EKG, cardiological finding, cerebrospinal liquor (LP) and the EMG finding. Phys Rev E Stat Nonlin Soft Matter Phys . 2004 Sep;70(3 Pt 1):031909 . Epub 2004 Sep 23. Topological generalizations of network motifs; Kashtan N et al.; Biological and technological networks contain patterns, termed network motifs, which occur far more often than in randomized networks . Network motifs were suggested to be elementary building blocks that carry out key functions in the network . It is of interest to understand how network motifs combine to form larger structures . To address this, we present a systematic approach to define "motif generalizations": families of motifs of different sizes that share a common architectural theme . To define motif generalizations, we first define "roles" in a subgraph according to structural equivalence . For example, the feedforward loop triad--a motif in transcription, neuronal, and some electronic networks--has three roles: an input node, an output node, and an internal node . The roles are used to define possible generalizations of the motif . The feedforward loop can have three simple generalizations, based on replicating each of the three roles and their connections . We present algorithms for efficiently detecting motif generalizations . We find that the transcription networks of bacteria and yeast display only one of the three generalizations, the multi-output feedforward generalization . In contrast, the neuronal network of C . elegans mainly displays the multi-input generalization . Forward-logic electronic circuits display a multi-input, multi-output hybrid . Thus, networks which share a common motif can have very different generalizations of that motif . Using mathematical modeling, we describe the information processing functions of the different motif generalizations in transcription, neuronal, and electronic networks. Biodegradation, 2004 Oct, 15(5), 327 - 36 Anaerobic degradability of alcohol ethoxylates and related non-ionic surfactants; Mosche M; The anaerobic degradability of alcohol ethoxylates with various degrees of branching and several related substances was studied . Different inocula were employed in order to increase the probability of obtaining capable bacteria, and the degradation assays were fed with several small doses of the test substances in order to avoid inhibition by too high initial concentrations . Mineralization was quantified by monitoring the biogas production and inorganic carbon concentration in the liquid phase . Almost complete mineralization was achieved in the assays with linear alcohol ethoxylate, poly(ethylene glycol), dodecanol, 2-ethyl-hexanoic acid and 3-methyl-valeric acid . No significant degradation was detected in the assays with highly branched alcohol ethoxylate, 2-butyl-branched alcohol ethoxylate, alcohol alkoxylate, poly(propylene glycol) and iso-tridecanol . A 2-ethyl-branched alcohol ethoxylate was transformed to (2-ethyl-hexyloxy)-acetate, which was not further degraded . Apparently already the first step of anaerobic degradation of alcohol ethoxylates, the ethoxylate chain shortening, is sterically hindered by the alkyl branching . Alkyl branching in alcohol ethoxylates and the inclusion of propylene oxide units in alcohol alkoxylates seem to have a clearly more detrimental effect on anaerobic degradability than on aerobic degradability. Gastroenterol Clin Biol, 2004 Oct, 28(10 Pt 1), 913 - 6 {Tropical sprue: two cases in the Paris area}; Macaigne G et al.; Tropical sprue (TS) is a postinfective tropical malabsorption that occurs in tropical countries . TS is associated with a persisting colonization of the small-intestine lumen by enterotoxinogenic bacteria that cause subsequent enterocyte damage affecting all or part of the small-intestine . We report two cases of TS that occurred in inhabitants of Paris area returned from endemic areas . The first observation concerned a 76-year old woman admitted for anorexia, loss of 20 kg and anemia . The second observation concerned a 53-year old man referred for chronic diarrhea and loss of 40 kg within 4 years . In both cases, duodenal lesions consisted of subtotal and total villous atrophy with prominent infiltration of the damaged surface epithelium with lymphocytes and infiltrate of lymphocytes and plasma cells of the lamina propria . The two patients recovered under antibiotics, confirming the diagnosis of TS. J Biol Chem, 2005 Jan 7, 280(1), 326 - 33 Epub 2004 Nov 02. Identification of the namH Gene, Encoding the Hydroxylase Responsible for the N-Glycolylation of the Mycobacterial Peptidoglycan; Raymond JB et al.; The peptidoglycan of most bacteria consists of a repeating disaccharide unit of beta-1,4-linked N-acetylmuramic acid and N-acetylglucosamine . However, the muramic acid moieties of the mycobacterial peptidoglycan are N-glycolylated, not N-acetylated . This is a rare modification seen only in the peptidoglycan of mycobacteria and five other closely related genera of bacteria . The N-glycolylation of sialic acids is a unique carbohydrate modification that has been studied extensively in eukaryotes . However, the significance of the N-glycolylation of bacterial peptidoglycan is unknown . The goal of this project was to identify the gene encoding the hydroxylase responsible for the N-glycolylation of the mycobacterial peptidoglycan . We developed a novel assay for the mycobacterial UDP-N-acetylmuramic acid hydroxylation reaction and demonstrated that Mycobacterium smegmatis has an enzyme activity that can convert UDP-N-acetylmuramic acid to UDP-N-glycolylmuramic acid . We identified the gene namH encoding the mycobacterial UDP-N-acetylmuramic acid hydroxylase by computer data base searching and motif comparisons with the eukaryotic enzymes responsible for the N-glycolyation of sialic acids . The namH gene is not essential for in vitro growth as we were successful in deleting the gene in M . smegmatis . The M . smegmatis mutant is devoid of UDP-N-acetylmuramic acid hydroxylase activity and synthesizes only N-acetylated muropeptide precursors . Furthermore, the mutant exhibits increased susceptibility to beta-lactam antibiotics and lysozyme . Our studies suggest that the N-glycolylation of mycobacterial peptidoglycan may play a role in lysozyme resistance or may contribute to the structural stability of the cell wall architecture. Trends Genet, 2004 Dec, 20(12), 625 - 30 An expanding genetic code; Cropp TA et al.; More than 30 novel amino acids have been genetically encoded in response to unique triplet and quadruplet codons including fluorescent, photoreactive and redox active amino acids, glycosylated and heavy atom derived amino acids in addition to those with keto, azido and acetylenic chains . In this article, we describe recent advances that make it possible to add new building blocks systematically to the genetic codes of bacteria, yeast and mammalian cells . Taken together these tools will enable the detailed investigation of protein structure and function, which is not possible with conventional mutagenesis . Moreover, by lifting the constraints of the existing 20-amino-acid code, it should be possible to generate proteins and perhaps entire organisms with new or enhanced properties. J Mol Biol, 2004 Nov 19, 344(2), 455 - 69 Conformational states of cytochrome P450cam revealed by trapping of synthetic molecular wires; Hays AM et al.; Members of the ubiquitous cytochrome P450 family catalyze a vast range of biologically significant reactions in mammals, plants, fungi, and bacteria . Some P450s display a remarkable promiscuity in substrate recognition, while others are very specific with respect to substrate binding or regio and stereo-selective catalysis . Recent results have suggested that conformational flexibility in the substrate access channel of many P450s may play an important role in controlling these effects . Here, we report the X-ray crystal structures at 1.8A and 1.5A of cytochrome P450cam complexed with two synthetic molecular wires, D-4-Ad and D-8-Ad, consisting of a dansyl fluorophore linked to an adamantyl substrate analog via an alpha,omega-diaminoalkane chain of varying length . Both wires bind with the adamantyl moiety in similar positions at the camphor-binding site . However, each wire induces a distinct conformational response in the protein that differs from the camphor-bound structure . The changes involve significant movements of the F, G, and I helices, allowing the substrate access channel to adapt to the variable length of the probe . Wire-induced opening of the substrate channel also alters the I helix bulge and Thr252 at the active site with binding of water that has been proposed to assist in peroxy bond cleavage . The structures suggest that the coupling of substrate-induced conformational changes to active-site residues may be different in P450cam and recently described mammalian P450 structures . The wire-induced changes may be representative of the conformational intermediates that must exist transiently during substrate entry and product egress, providing a view of how substrates enter the deeply buried active site . They also support observed examples of conformational plasticity that are believed be responsible for the promiscuity of drug metabolizing P450s . Observation of such large changes in P450cam suggests that substrate channel plasticity is a general property inherent to all P450 structures. J Mol Biol, 2004 Nov 19, 344(2), 395 - 407 RNA tertiary structure and cooperative assembly of a large ribonucleoprotein complex; Recht MI et al.; The mechanisms that govern the ordered assembly of multiprotein ribonucleoprotein complexes are not well understood . The in vitro reconstitution of the small subunit of the bacterial ribosome provides a tractable system for the detailed study of ordered assembly . We present a quantitative thermodynamic description of the hierarchical binding of ribosomal proteins to 16S rRNA during assembly of the platform of the 30S ribosomal subunit . The binding of S8, S11, S15, and the S6:S18 heterodimer to the central domain of 16S rRNA has been measured both individually and in combination using isothermal titration calorimetry and gel mobility shift assays . Both enthalpy and free energy measurements demonstrate the cooperative binding of S15 and the S6:S18 heterodimer, but no cooperativity is observed for either S8 or S11 . The results define a thermodynamic framework that describes cooperative platform assembly. Salud Publica Mex, 2004 Sep-Oct, 46(5), 451 - 63 {The role of epidemiology in mental disorder research}; Borges G et al.; Mental disorders, including substance abuse, are part of the Mexican epidemiologic scenario and will remain so during several decades . They may even become more prominent as causes of disease, disability, and death in our country . It is thus imperative to frame appropriate management strategies to curb these problems without delay . This paper aims at outlining epidemiology of mental diseases as a field of study, and to identify its limitations . Emphasis is made on common elements shared with other more traditional fields of epidemiology, as well as on the specific contributions made by this particular field to epidemiology and to psychiatry in general . This paper describes the main study designs and problems in this field of epidemiology, its usefulness in prevention actions, and future challenges . A unique characteristic of mental disorder epidemiology is that its target diseases manifest in two levels: behaviorally (for example, compulsive hand-washing) and as an element of the individual's mental life (e.g., obsession with bacteria being a constant, omnipresent health threat) . It follows that much of the knowledge currently available on the phenomena of mental disorders in general is based on the self-reported insight of individuals . Trained clinicians have collected such reports by interview or with standardized questionnaires . This field of epidemiology is characterized by having two-sides: a mental disorder is a problem in and of itself, causing suffering and prompting the search for specialized care, as it has peculiar clinical manifestations . On the other hand, mental disorder epidemiology also focuses on determining factors (drug use, abuse, or addiction) and on the way these independent variables result in certain processes and outcomes (such as accidents, homicide, suicide, liver cirrhosis, etc.) . Finally, the epidemiology of mental disorders has also been set apart by its focus in series of processes that are not suitably classified as syndromes, but which are germane to public health, for example, violence . The epidemiology of mental disorders faces great challenges in the new millennium, including a complex, changing epidemiologic scenario . Several important issues will influence the future development of mental disorder epidemiology: measurement of mental disorders and risk factors, more efficient sampling design and methods, the relationships among biological research, genetics, social studies, and epidemiology, and the interface between epidemiology and the evaluation of therapies and health services. Nippon Geka Gakkai Zasshi, 2004 Oct, 105(10), 664 - 8 {Hepatic failure following resection of cholestatic liver}; Arai T et al.; Hepatectomy for biliary cancer with obstructive jaundice is often followed by postoperative septic complications associated with hyperbilirubinemia, both of which could lead to cholestatic liver failure by affecting each other . Such septic complications seem to develop from contamination of bile, reduction of intestinal integrity, or impairment of host resistance to bacteria, each resulting from biliary obstruction . Hyperbilirubinemia after hepatectomy is demonstrated to develop due to hepatic mitochondrial dysfunction or impaired expression of bile efflux pumps on the canalicular membrane of hepatocytes . Since no therapeutic strategy is established for liver failure following hepatectomy, it is important to take all possible measures before surgery to enhance the functions of the liver, intestine, and host immunity and to prevent postoperative septic complications. Indian J Med Res, 2004 Oct, 120(4), 305 - 15 Abdominal tuberculosis; Sharma MP et al.; Tuberculosis can involve any part of the gastrointestinal tract and is the sixth most frequent site of extrapulmonary involvement . Both the incidence and severity of abdominal tuberculosis are expected to increase with increasing incidence of HIV infection . Tuberculosis bacteria reach the gastrointestinal tract via haematogenous spread, ingestion of infected sputum, or direct spread from infected contiguous lymph nodes and fallopian tubes . The gross pathology is characterized by transverse ulcers, fibrosis, thickening and stricturing of the bowel wall, enlarged and matted mesenteric lymph nodes, omental thickening, and peritoneal tubercles . Peritoneal tuberculosis occurs in three forms : wet type with ascitis, dry type with adhesions, and fibrotic type with omental thickening and loculated ascites . The most common site of involvement of the gastrointestinal tuberculosis is the ileocaecal region . Ileocaecal and small bowel tuberculosis presents with a palpable mass in the right lower quadrant and/or complications of obstruction, perforation or malabsorption especially in the presence of stricture . Rare clinical presentations include dysphagia, odynophagia and a mid oesophageal ulcer due to oesophageal tuberculosis, dyspepsia and gastric outlet obstruction due to gastroduodenal tuberculosis, lower abdominal pain and haematochezia due to colonic tuberculosis, and annular rectal stricture and multiple perianal fistulae due to rectal and anal involvement . Chest X-rays show evidence of concomitant pulmonary lesions in less than 25 per cent of cases . Useful modalities for investigating a suspected case include small bowel barium meal, barium enema, ultrasonography, computed tomographic scan and colonoscopy . Ascitic fluid examination reveals straw coloured fluid with high protein, serum ascitis albumin gradient less than 1.1 g/dl, predominantly lymphocytic cells, and adenosine deaminase levels above 36 U/l . Laparoscopy is a very useful investigation in doubtful cases . Management is with conventional antitubercular therapy for at least 6 months . The recommended surgical procedures today are conservative and a period of preoperative drug therapy is controversial. Nucleic Acids Res, 2004 Nov 01, 32(19), 5841 - 50 Print 2004. Alleviation of restriction by DNA condensation and non-specific DNA binding ligands; Keatch SA et al.; During conditions of cell stress, the type I restriction and modification enzymes of bacteria show reduced, but not zero, levels of restriction of unmethylated foreign DNA . In such conditions, chemically identical unmethylated recognition sequences also occur on the chromosome of the host but restriction alleviation prevents the enzymes from destroying the host DNA . How is this distinction between chemically identical DNA molecules achieved? For some, but not all, type I restriction enzymes, alleviation is partially due to proteolytic degradation of a subunit of the enzyme . We identify that the additional alleviation factor is attributable to the structural difference between foreign DNA entering the cell as a random coil and host DNA, which exists in a condensed nucleoid structure coated with many non-specific ligands . The type I restriction enzyme is able to destroy the 'naked' DNA using a complex reaction linked to DNA translocation, but this essential translocation process is inhibited by DNA condensation and the presence of non-specific ligands bound along the DNA. Proc Natl Acad Sci U S A, 2004 Nov 9, 101(45), 16040 - 5 Epub 2004 Nov 01. Different functions of HetR, a master regulator of heterocyst differentiation in Anabaena sp . PCC 7120, can be separated by mutation; Khudyakov IY et al.; The HetR protein has long been recognized as a key player in the regulation of heterocyst development . HetR is known to possess autoproteolytic and DNA-binding activities . During a search for mutants of Anabaena sp . PCC 7120 that can overcome heterocyst suppression caused by overexpression of the patS gene, which encodes a negative regulator of differentiation, a bypass mutant strain, S2-45, was isolated that produced a defective pattern (Pat phenotype) of irregularly spaced single and multiple contiguous heterocysts (Mch phenotype) in combined nitrogen-free medium . Analysis of the S2-45 mutant revealed a R223W mutation in HetR, and reconstruction in the wild-type background showed that this mutation was responsible for the Mch phenotype and resistance not only to overexpressed patS, but also to overexpressed hetN, another negative regulator of differentiation . Ectopic overexpression of the hetRR223W allele in the hetRR223W background resulted in a conditionally lethal (complete differentiation) phenotype . Analysis of the heterocyst pattern in the hetRR223W mutant revealed that heterocysts differentiate essentially randomly along filaments, indicating that this mutation results in an active protein that is insensitive to the major signals governing heterocyst pattern formation . These data provide genetic evidence that, apart from being an essential activator of differentiation, HetR plays a central role in the signaling pathway that controls the heterocyst pattern. Hepatol Res, 2004 Oct, 30(2), 57 - 62 Transmission of hepatitis C virus quasispecies between human adults; Saito T et al.; To elucidate how hepatitis C virus (HCV) with multiple variants (quasispecies) is transmitted and adapts to the host during infection, we compared nucleotide and deduced amino acid sequences from hypervariable region1 (HVR1) of the E2 gene of HCV between a donor and a recipient who developed hepatitis after a needlestick accident . Thirty clones from each subject were sequenced after PCR amplification, cloning, and purification of plasmid DNA from single colonies of transformed bacteria . Genetic analysis revealed that the recipient's viral sequences were much less diverse than the donor's . We found a single predominant HCV HVR1 clone of the recipient in 22/30 isolates with the same amino acid sequence, and mimic clones in 8/30 isolates with only one amino acid substitution . These were all absent in the donor, who had 21 highly diverse sequences . Phylogenetic analysis of virus E1/E2 gene sequences showed that the recipient's unique sequences were related to the population of variants from the donor, in whom one isolate had 96% similarity to the recipient's predominant amino acid sequence . These results suggest that a minor subset of the donor's HCV variants is selectively transmitted to the recipient, and that the selection determines the predominant variant in the new host. J Am Soc Mass Spectrom, 2004 Nov, 15(11), 1665 - 74 Strategies and data analysis techniques for lipid and phospholipid chemistry elucidation by intact cell MALDI-FTMS; Jones JJ et al.; Ions attributed to lipids and phospholipids are directly observed by desorption from whole bacteria using intact cell (IC) matrix-assisted laser desorption-ionization (MALDI) Fourier transform mass spectrometry (FTMS) . Saccharomyces cerevisiae are grown in rich media broth, concentrated, and applied directly to the MALDI surface without lysis or chemical treatment . FTMS of MALDI ions gives excellent signal to noise ratios with typical resolving powers of 90,000 and mass precision better than 0.002 Da . Use of accurate mass measurements and a simple set of rules allow assignment of major peaks into one of twelve expected lipid classes . Subsequently, fractional mass versus whole number mass plots are employed to enhance visual interpretation of the high-resolution data and to facilitate detection of related ions such as those representing homologous series or different degrees of unsaturation . This approach, coupled with rules based on bacterial biochemistry, is used to classify ions with m/z up to about 1000 . Major spectral peaks in the range m/z 200-1000 are assigned as lipids and phospholipids . In this study, it is assumed that biologically-derived ions with m/z values lower than 1000 are lipids . This is not unreasonable in view of the facts that molecular weights of lipids are almost always less than 1000 Da, that the copy numbers for lipids in a cell are higher than those for any single protein or other component, and that lipids are generally collections of distinct homologous partners, unlike proteins or other cell components . This paper presents a new rapid lipid-profiling method based on IC MALDI-FTMS. Biochemistry, 2004 Nov 9, 43(44), 13981 - 95 Divergence of function in the thioredoxin fold suprafamily: evidence for evolution of peroxiredoxins from a thioredoxin-like ancestor; Copley SD et al.; The thioredoxin fold is found in proteins that serve a wide variety of functions . Among these are peroxiredoxins, which catalyze the reduction of hydrogen peroxide and alkyl peroxides . Although the common structural fold shared by thioredoxins and peroxiredoxins suggests the possibility that they have evolved from a common progenitor, it has been difficult to examine this hypothesis in depth because pairwise sequence identities between proteins in these two superfamilies are statistically insignificant . Using the Shotgun program, we have found that sequences of reductases involved in maturation of cytochromes in certain bacteria bridge the sequences of thioredoxins and peroxiredoxins . Analysis of motifs found in a divergent set of thioredoxins, cytochrome maturation proteins, and peroxiredoxins provides further support for an evolutionary relationship between these proteins . Within the conserved motifs are specific residues that are characteristic of individual protein classes, and therefore are likely to be involved in the specific functions of those classes . We have used this information, in combination with existing structural and functional information, to gain new insight into the structure-function relationships in these proteins and to construct a model for the emergence of peroxiredoxins from a thioredoxin-like ancestor. Biochemistry, 2004 Nov 9, 43(44), 13963 - 71 Biochemical and biophysical characterization of serotonin 5-HT2C receptor homodimers on the plasma membrane of living cells; Herrick-Davis K et al.; While many studies have provided evidence of homodimerization and heterodimerization of G-protein-coupled receptors (GPCRs), few studies have used fluorescence resonance energy transfer (FRET) combined with confocal microscopy to visualize receptor dimerization on the plasma membrane, and there have been no reports demonstrating the expression of serotonin receptor dimers/oligomers on the plasma membrane of living cells . In the study presented here, biochemical and biophysical techniques were used to determine if 5-HT(2C) receptors exist as homodimers on the plasma membrane of living cells . Immunoprecipitation followed by Western blotting revealed the presence of immunoreactive bands the predicted size of 5-HT(2C) receptor monomers and homodimers that were detergent and cross-linker sensitive . Bioluminescence resonance energy transfer (BRET) was assessed in HEK293 cells expressing 5-HT(2C) receptors labeled with Renilla luciferase and yellow fluorescent protein . BRET levels were not altered by pretreatment with serotonin . Confocal microscopy provided direct visualization of FRET on the plasma membrane of live cells expressing 5-HT(2C) receptors labeled with cyan (donor) and yellow (acceptor) fluorescent proteins . FRET, assessed by acceptor photobleaching, was dependent on the donor/acceptor ratio and independent of acceptor expression levels, indicating that FRET resulted from receptor clustering and not from overexpression of randomly distributed receptors, providing evidence for GPCR dimers/oligomers in a clustered distribution on the plasma membrane . The results of this study suggest that 5-HT(2C) receptors exist as constitutive homodimers on the plasma membrane of living cells . In addition, a confocal-based FRET method for monitoring receptor dimerization directly on the plasma membrane of living cells is described. Pol Merkuriusz Lek, 2004 May, 16(95), 430 - 3 {Formation of the selected biochemical markers in the patients with atherosclerotic arterial obliteration and the coexistent infection with Chlamydia psittaci}; Niedziela P et al.; In the arterial wall which is atheromatously changed both basic EB and reticular RB bodies of Chlamydia bacteria may be present . The role of these micro-organisms in the pathogenesis of arteriosclerosis is not thoroughly recognized yet . It is unknown whether Chlamydia initiate or lead to the inflammation of the already existent disease process . That is why it is expedient to carry out the research determining the correlation between the presence of Chlamydia bacteria in the operated arteries and the formation of the selected biochemical indicators . During the operation the imprint specimen was taken from the arterial wall, which was subsequently painted using stamp's method . EB bodies were marked in the immunofluorescence test with the usage of specific antibodies . The concentration of C-reactive protein was marked with the help of the rocket immunoelectrophoresis . The examination of blood lipids and fibrinogens was conducted with the usage of a colorimetric method with the help of ready-made kits . On the basis of the obtained data two groups of patients were distinguished . The first group comprised 16 patients (26%); here elementary EB bodies were detected both in the imprint specimens and the blood serum . In all the patients assigned to the first group the presence of anti-Chlamydia antibodies in the blood serum was observed . The second group consisted of the remaining 47 patients (74%); here no currently lasting Chlamydia infection was stated . On the basis of the conducted research and statistic calculations it is justified to point the correlation between the concentration of C-reactive protein and HDL cholesterol in the group of patients with the detected Chlamydia bacteria in comparison to the group of patients with no infection . However no statistically significant relationship between the concentration of fibrinogen, total cholesterol, LDL cholesterol and triglycerides was observed in the relation to the analyzed groups. Nat Rev Immunol, 2004 Nov, 4(11), 841 - 55 Regulatory T cells: friend or foe in immunity to infection? Mills KH. Homeostasis in the immune system depends on a balance between the responses that control infection and tumour growth and the reciprocal responses that prevent inflammation and autoimmune diseases . It is now recognized that regulatory T cells have a crucial role in suppressing immune responses to self-antigens and in preventing autoimmune diseases . Evidence is also emerging that regulatory T cells control immune responses to bacteria, viruses, parasites and fungi . This article explores the possibility that regulatory T cells can be both beneficial to the host, through limiting the immunopathology associated with anti-pathogen immune responses, and beneficial to the pathogen, through subversion of the protective immune responses of the host. Am J Trop Med Hyg, 2004 Oct, 71(4), 393 - 9 Effect of blocking the CXCL9/10-CXCR3 chemokine system in the outcome of endothelial-target rickettsial infections; Valbuena G et al.; Rickettsiae cause systemic infections such as Rocky Mountain spotted fever and boutonneuse fever . The main cellular target of these obligately intracellular bacteria is the endothelium . T lymphocytes are the most important effectors of immunity, and the CXCR3 ligands CXCL9 and CXCL10 may play an important role in the T cell-mediated clearance of rickettsiae from the infected vasculature as suggested by recent expression studies . Here we showed that antibody-mediated neutralization of CXCL9 and CXCL10, and CXCR3 gene knockout, had no effect on survival or bacterial loads of mice infected with rickettsiae . We also demonstrated that rickettsiae triggered the endothelial expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 in vivo . These findings suggested that antigenic presentation by endothelial cells together with an endothelial inflammatory phenotype induced by the rickettsial infection may be sufficient to arrest T cells and trigger their anti-rickettsial effector mechanisms without the need for chemokines. Huan Jing Ke Xue, 2004 Jul, 25(4), 109 - 12 {Photocatalytic functional ceramic and its speciality of photodecomposition}; Liu P et al.; Photocatalytic ceramic was prepared by coating photocatalytic membrane on ceramic matrix . The photocatalytic behavior of the TiO2 coated ceramic for degradation of oleic acid, ethylene, SO2, NOx and sterilization was studied by using XRD, chromatogram, in-situ IR and spectrophotometer . The results showed that the photocatalytic ceramic prepared by special conditions have the function of environmental conservation such as the photodegradating organic contaminants, removing inorganic baleful gas and killing bacteria . Degradation ratio of ethylene, oleic acid, SO2 and NOx reached 95%-100% respectively for the photocatalytic functional ceramic. Lin Chuang Er Bi Yan Hou Ke Za Zhi, 2002 Oct, 16(10), 550 - 2 {The influence of medicine to the expression of cell adhesion molecule in experimental otitis media with effusion}; Wang S et al.; OBJECTIVE: To study the influence of Cefaclor and Prednisonum to the expression of cell adhesion molecule in experimental otitis media with effusion (OME) . METHOD: SD rats models of OME were made using formalin killed nontypeable Hemophilus influenzae and the expression of adhesion molecules Mac-1 and L-Selectin was analysed by flow cytometry using white blood cell obtained by cardiac puncture in control, bacterial and medicine groups . The pathology of middle ear mucosa was investigated to understand the relationship between the expression of adhesion molecules and the pathology . RESULT: It was found that the expression of adhesion molecules Mac-1 and L-Selection in bacteria group increased obviously with pathological change of the middle ear mucosa . Cefaclor and Prednisonum could decrease the expression of adhesion molecules and inhibited the leukocyte accumulation in the middle ear cavity though there was no significant differences except for the L-Selectin in the Cefaclor group between the bacteria group and the medicine group . CONCLUSION: Dead bacteria in the middle ear may act as an antigen to induce the inflammatory response and effusion of the middle ear . The expression of adhesion molecules Mac-1 and L-Selectin is highly related to the inflammatory response . Cefaclor and Prednisonum may improve otitis media with effusion, which may related to the decreasing expression of Mac-1 and L-Selectin. J Periodontol, 2004 Sep, 75(9), 1209 - 15 Relative proportions of T-cell subpopulations and cytokines that mediate and regulate the adaptive immune response in patients with aggressive periodontitis; Suarez LJ et al.; BACKGROUND: Both the virulence factors of periodontopathic bacteria and the immune response against them have been involved in tissue destruction observed in periodontal disease . Considering the regulatory role of cytokines produced by T cells, the purpose of this study was to compare the CD3+, CD4+, and CD8+ subpopulations of T cells, and to characterize the mRNA of cytokines involved in the adaptive immune response in a group of healthy/gingivitis 1 (HI/G1) individuals and aggressive periodontitis (AgP) patients . METHODS: The percentages of T-cell subpopulations were analyzed in 10 gingival samples of HI/G1 individuals and 10 gingival samples of AgP patients by immunohistochemistry . The presence of interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-5, IL-10, IL- 13, and transforming growth factor (TGF)-beta was measured by reverse transcription polymerase chain reaction (RT-PCR) of mRNA extracted from complete gingival biopsies . RESULTS: Significant differences were found in CD3+ and CD4+ cell counts between both groups . The parameters were lower in the gingival biopsies from AgP patients while CD8+ counts were similar in both groups . The cytokine mRNA analysis showed constant expression of IL-2 and IFN-gamma in all cases . The mRNA of IL-5 and IL-10 was present in the majority of HI/G1 (N = 10, N = 9, respectively) but was not in the AgP group (N = 2, N = 1) . IL-13 and TGF-beta were only detected in HI/G1 (N = 2, N = 3) and IL-4 was not detected in any of the individuals . CONCLUSIONS: These results indicate that the role of the CD8+ subpopulation in aggressive periodontitis lesions is limited . On the other hand, cytokines IL-2 and IFN-gamma may not be relevant in the progression of aggressive periodontitis. Int J Toxicol, 2004, 23(5), 335 - 44 Contamination is a frequent confounding factor in toxicology studies with anthraquinone and related compounds; Butterworth BE et al.; Anthraquinone (AQ) (9,10-anthracenedione) is an important compound in commerce . Many structurally related AQ derivatives are medicinal natural plant products . Examples include 1-hydroxyanthraquinone (1-OH-AQ) and 2-hydroxyanthraquinone (2-OH-AQ), which are also metabolites of AQ . Some commercial AQ is produced by the oxidation of anthracene (AQ-OX) . In the recent past, the anthracene used was distilled from coal tar and different lots of derived AQ often contained polycyclic aromatic hydrocarbon contaminants, particularly 9-nitroanthracene (9-NA) . Many toxicology studies on AQ used contaminated anthracene-derived AQ-OX, including a National Toxicology Program (NTP) 2-year cancer bioassay that reported a weak to modest increase in tumors in the kidney and bladder of male and female F344/N rats and in the livers of male and female B6C3F1 mice . The AQ-OX used in that bioassay was mutagenic and contained 9-NA and other contaminants . In contrast, purified AQ is not genotoxic . The purpose of this paper is to provide additional information to help iterpret the NTP cancer bioassay . This paper describes a quantitative analytical study of the NTP anthracene-derived AQ-OX test material, and presents the results of mutagenicity studies with the 1-OH-AQ and 2-OH-AQ metabolites and the primary contaminant 9-NA . Purified 1-OH-AQ and 2-OH-AQ exhibited only weak mutagenic activity in selected strains of tester bacteria and required S9 . Literature reports of potent mutagenic activity for 1-OH-AQ and 2-OH-AQ in bacteria minus S9 are, once again, very likely the result of the presence of contaminants in the test samples . Weak activity and limited production of the 1-OH-AQ and 2-OH-AQ metabolites are possible reasons that AQ fails to exhibit activity in numerous genotoxicity assays . 9-NA was mutagenic in tester strains TA98 and TA100 minus S9 . This pattern of activity is consistent with that seen with the contaminated AQ-OX used in the NTP bioassay . Analysis of all the mutagenicity and analytical data, however, indicates that the mutagenic contamination in the NTP bioassay probably resides with compounds in addition to 9-NA . 9-NA exhibited potent mutagenic activity in the L5178Y mammalian cell mutagenicity assay in the presence of S9 . The positive response was primarily associated with an increase in small colony mutants suggesting a predominance of a clastogenic mechanism . Quantitative mutagenicity and carcinogenicity potency estimates indicate that it is plausible that the contaminants alone in the NTP AQ-OX bioassay could have been responsible for all of the observed carcinogenic activity . Although AQ-OX is no longer commercially used in the United States, many of the reported genotoxicity and carcinogenicity results in the literature for AQ and AQ derivative compounds must be viewed with caution. Semin Ophthalmol, 2002 Mar, 17(1), 11 - 7 Infectious optic neuropathy; Golnik KC; A wide variety of infectious agents are known to cause optic neuropathy . This article will consider the bacteria, spirochetes, fungi, and viruses that most commonly affect the optic nerve . Clinical presentation is variable, but some pathogens often produce a characteristic funduscopic pattern . Diagnosis is usually made on the basis of clinical suspicion and serologic testing . Polymerase chain reaction is also increasingly utilized . Most infectious agents can be effectively treated but visual recovery is highly variable. Biochim Biophys Acta, 2004 Nov 4, 1659(1), 19 - 31 Molecular interference of Cd(2+) with Photosystem II; Sigfridsson KG et al.; Many heavy metals inhibit electron transfer reactions in Photosystem II (PSII) . Cd(2+) is known to exchange, with high affinity in a slow reaction, for the Ca(2+) cofactor in the Ca/Mn cluster that constitutes the oxygen-evolving center . This results in inhibition of photosynthetic oxygen evolution . There are also indications that Cd(2+) binds to other sites in PSII, potentially to proton channels in analogy to heavy metal binding in photosynthetic reaction centers from purple bacteria . In search for the effects of Cd(2+)-binding to those sites, we have studied how Cd(2+) affects electron transfer reactions in PSII after short incubation times and in sites, which interact with Cd(2+) with low affinity . Overall electron transfer and partial electron transfer were studied by a combination of EPR spectroscopy of individual redox components, flash-induced variable fluorescence and steady state oxygen evolution measurements . Several effects of Cd(2+) were observed: (i) the amplitude of the flash-induced variable fluorescence was lost indicating that electron transfer from Y(Z) to P(680)(+) was inhibited; (ii) Q(A)(-) to Q(B) electron transfer was slowed down; (iii) the S(2) state multiline EPR signal was not observable; (iv) steady state oxygen evolution was inhibited in both a high-affinity and a low-affinity site; (v) the spectral shape of the EPR signal from Q(A)(-)Fe(2+) was modified but its amplitude was not sensitive to the presence of Cd(2+) . In addition, the presence of both Ca(2+) and DCMU abolished Cd(2+)-induced effects partially and in different sites . The number of sites for Cd(2+) binding and the possible nature of these sites are discussed. BMC Infect Dis . 2004 Oct 29;4(1):45. Gastro-enteritis outbreak among Nordic patients with psoriasis in a health centre in Gran Canaria, Spain: a cohort study; Eriksen HM et al.; BACKGROUND: Between November 2 and 10, 2002 several patients with psoriasis and personnel staying in the health centre in Gran Canaria, Spain fell ill with diarrhoea, vomiting or both . Patient original came from Norway, Sweden and Finland . The patient group was scheduled to stay until 8 November . A new group of patients were due to arrive from 7 November . METHODS: A retrospective cohort study was conducted to assess the extent of the outbreak, to identify the source and mode of transmission and to prevent similar problems in the following group . RESULTS: Altogether 41% (48/116) of persons staying at the centre fell ill . Norovirus infection was suspected based on clinical presentations and the fact that no bacteria were identified . Kaplan criteria were met . Five persons in this outbreak were hospitalised and the mean duration of diarrhoea was 3 days . The consequences of the illness were more severe compared to many other norovirus outbreaks, possibly because many of the cases suffered from chronic diseases and were treated with drugs reported to affect the immunity (methotrexate or steroids).During the two first days of the outbreak, the attack rate was higher in residents who had consumed dried fruit (adjusted RR = 3.1; 95% CI: 1.4-7.1) and strawberry jam (adjusted RR = 1.9; 95% CI: 0.9-4.1) than those who did not . In the following days, no association was found . The investigation suggests two modes of transmission: a common source for those who fell ill during the two first days of the outbreak and thereafter mainly person to person transmission . This is supported by a lower risk associated with the two food items at the end of the outbreak . CONCLUSIONS: We believe that the food items were contaminated by foodhandlers who reported sick before the outbreak started . Control measures were successfully implemented; food buffets were banned, strict hygiene measures were implemented and sick personnel stayed at home >48 hours after last symptoms. APMIS, 2004 Jun, 112(6), 336 - 41 Identification of Bartonella species in rodents, shrews and cats in Denmark: detection of two B . henselae variants, one in cats and the other in the long-tailed field mouse; Engbaek K et al.; Small mammals and stray cats were trapped in two areas of North Zealand, Denmark, and their blood cultured for hemotrophic bacteria . Bacterial isolates were recovered in pure culture and subjected to 16S rDNA gene sequencing . Bartonella species were isolated from five mammalian species: B . grahamii from Microtus agrestis (field vole) and Apodemus flavicollis (yellow-necked field mouse); B . taylorii from M . agrestis, A . flavicollis and A . sylvaticus (long-tailed field mouse); B . tribocorum from A . flavicollis; B . vinsonii subsp . vinsonii from M . agrestis and A . sylvaticus; and B . birtlesii from Sorex vulgaris (common shrew) . In addition, two variant types of B . henselae were identified: variant I was recovered from three specimens of A . sylvaticus, and B . henselae variant II from 11 cats; in each case this was the only B . henselae variant found . No Bartonella species was isolated from Clethrionomys glareolus (bank vole) or Micromys minutus (harvest mouse) . These results suggest that B . henselae occurs in two animal reservoirs in this region, one of variant I in A . sylvaticus, which may be transmitted between mice by the tick Ixodes ricinus, and another of variant II in cats, which may be transmitted by the cat flea (Ctenocephalides felis) . To our knowledge, this is the first report of the occurrence of B . henselae and B . tribocorum in Apodemus mice. PLoS Biol . 2004 Nov;2(11):e367 . Epub 2004 Nov. Tuberculous granuloma formation is enhanced by a mycobacterium virulence determinant; Volkman HE et al.; Granulomas are organized host immune structures composed of tightly interposed macrophages and other cells that form in response to a variety of persistent stimuli, both infectious and noninfectious . The tuberculous granuloma is essential for host containment of mycobacterial infection, although it does not always eradicate it . Therefore, it is considered a host-beneficial, if incompletely efficacious, immune response . The Mycobacterium RD1 locus encodes a specialized secretion system that promotes mycobacterial virulence by an unknown mechanism . Using transparent zebrafish embryos to monitor the infection process in real time, we found that RD1-deficient bacteria fail to elicit efficient granuloma formation despite their ability to grow inside of infected macrophages . We showed that macrophages infected with virulent mycobacteria produce an RD1-dependent signal that directs macrophages to aggregate into granulomas . This Mycobacterium-induced macrophage aggregation in turn is tightly linked to intercellular bacterial dissemination and increased bacterial numbers . Thus, mycobacteria co-opt host granulomas for their virulence. J Biol Chem, 2005 Jan 14, 280(2), 1327 - 35 Epub 2004 Oct 26. Extended Neck Regions Stabilize Tetramers of the Receptors DC-SIGN and DC-SIGNR; Feinberg H et al.; The human cell surface receptors DC-SIGN (dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin) and DC-SIGNR (DC-SIGN-related) bind to oligosaccharide ligands found on human tissues as well as on pathogens including viruses, bacteria, and parasites . The extracellular portion of each receptor contains a membrane-distal carbohydrate-recognition domain (CRD) and forms tetramers stabilized by an extended neck region consisting of 23 amino acid repeats . Cross-linking analysis of full-length receptors expressed in fibroblasts confirms the tetrameric state of the intact receptors . Hydrodynamic studies on truncated receptors demonstrate that the portion of the neck of each protein adjacent to the CRD is sufficient to mediate the formation of dimers, whereas regions near the N terminus are needed to stabilize the tetramers . Some of the intervening repeats are missing from polymorphic forms of DC-SIGNR . Two different crystal forms of truncated DC-SIGNR comprising two neck repeats and the CRD reveal that the CRDs are flexibly linked to the neck, which contains alpha-helical segments interspersed with non-helical regions . Differential scanning calorimetry measurements indicate that the neck and CRDs are independently folded domains . Based on the crystal structures and hydrodynamic data, models for the full extracellular domains of the receptors have been generated . The observed flexibility of the CRDs in the tetramer, combined with previous data on the specificity of these receptors, suggests an important role for oligomerization in the recognition of endogenous glycans, in particular those present on the surfaces of enveloped viruses recognized by these proteins. Nihon Hansenbyo Gakkai Zasshi, 2004 Sep, 73(3), 253 - 61 {Functional changes of macrophages in Hansen's disease}; Fukutomi Y; As an obligate intracellular pathogen, the principal host cells for Mycobacterium leprae are mononuclear phagocytes or macrophages . The macrophage is a primitive cell type being found in both early and advanced life forms, and possesses a variety of functions, such as phagocytosis of invaded bacteria, production of cytokines, antigen presentation and tumor killing . Hansen's disease is a chronic infectious disease characterized by specific host immune responses against M . leprae . In this article the macrophage is focused to dissect its functions in the disease. Methods Mol Biol, 2004, 292, 387 - 400 Construction of a Gene Inactivation Library for Bovine herpesvirus 1 Using Infectious Clone Technology; Mahony TJ et al.; The application of infectious clone technology to herpesvirus biology has revolutionized the study of these viruses . Previously the ability to manipulate these large DNA viruses was limited to methods dependent on homologous recombination in mammalian cells . However, the construction of herpesvirus infectious clones using bacterial artificial chromosome vectors has permitted the application of powerful bacterial genetics for the manipulation of these viruses . A method is described for the construction and characterization of a gene inactivation library of Bovine herpesvirus 1 using an infectious clone . The method utilizes transposon-mediated gene inactivation, which permits gene inactivation without any prior knowledge of the viral genomic sequence . Furthermore, as the genetic manipulation is performed in bacteria the inactivation of those viral genes that are essential for viral replication is also possible . The method described here can be readily applied to any herpesvirus clone and provides the tools for precise characterization of all the genes contained within a herpesvirus genome. JAMA, 2004 Oct 27, 292(16), 1955 - 60 Risk of community-acquired pneumonia and use of gastric acid-suppressive drugs; Laheij RJ et al.; CONTEXT: Reduction of gastric acid secretion by acid-suppressive therapy allows pathogen colonization from the upper gastrointestinal tract . The bacteria and viruses in the contaminated stomach have been identified as species from the oral cavity . OBJECTIVE: To examine the association between the use of acid-suppressive drugs and occurrence of community-acquired pneumonia . DESIGN, SETTING, AND PARTICIPANTS: Incident acid-suppressive drug users with at least 1 year of valid database history were identified from the Integrated Primary Care Information database between January 1, 1995, and December 31, 2002 . Incidence rates for pneumonia were calculated for unexposed and exposed individuals . To reduce confounding by indication, a case-control analysis was conducted nested in a cohort of incident users of acid-suppressive drugs . Cases were all individuals with incident pneumonia during or after stopping use of acid-suppressive drugs . Up to 10 controls were matched to each case for practice, year of birth, sex, and index date . Conditional logistic regression was used to compare the risk of community-acquired pneumonia between use of proton pump inhibitors (PPIs) and H2-receptor antagonists . MAIN OUTCOME MEASURE: Community-acquired pneumonia defined as certain (proven by radiography or sputum culture) or probable (clinical symptoms consistent with pneumonia) . RESULTS: The study population comprised 364,683 individuals who developed 5551 first occurrences of pneumonia during follow-up . The incidence rates of pneumonia in non-acid-suppressive drug users and acid-suppressive drug users were 0.6 and 2.45 per 100 person-years, respectively . The adjusted relative risk for pneumonia among persons currently using PPIs compared with those who stopped using PPIs was 1.89 (95% confidence interval, 1.36-2.62) . Current users of H2-receptor antagonists had a 1.63-fold increased risk of pneumonia (95% confidence interval, 1.07-2.48) compared with those who stopped use . For current PPI users, a significant positive dose-response relationship was observed . For H2-receptor antagonist users, the variation in dose was restricted . CONCLUSION: Current use of gastric acid-suppressive therapy was associated with an increased risk of community-acquired pneumonia. Biochem Soc Trans, 2004 Dec, 32(Pt 6), 904 - 9 Structure and chemistry of the Sir2 family of NAD+-dependent histone/protein deactylases; Marmorstein R; The yeast Sir2 (silent information regulator-2) protein functions as an NAD(+)-dependent histone deacetylase to silence gene expression from the mating-type locus, tolomeres and rDNA and also promotes longevity and genome stability in response to calorie restriction . Homologues of yeast Sir2 have been identified in the three domains of bacteria, archaea and eukaryotes; in mammalian cells, Sir2 proteins also deacetylate non-histone proteins such as the p53 tumour suppressor protein, alpha-tubulin and forkhead transcription factors to mediate diverse biological processes including metabolism, cell motility and cancer . We have determined the X-ray crystal structure of a Sir2 homologue from yeast Hst2 (yHst2), in various liganded forms, including the yHst2/acetyl-Lys-16 histone H4/NAD(+) ternary complex; we have also performed related biochemical studies to address the conserved mode of catalysis by these enzymes as well as the distinguishing features that allow different members of the family to target their respective cognate substrates . These studies have implications for the structure-based design of Sir2-specific small molecule compounds, which might modulate Sir2 function for therapeutic application. Ying Yong Sheng Tai Xue Bao, 2004 Jul, 15(7), 1276 - 80 {Significance of senescence study on tree roots and its advances}; Wu C et al.; Root system is one of the important components of trees, and has some important physiological functions such as nutrient and water absorption, transport and storage, anchoring, and supporting . After tree root systems formed, roots often suffer from nutrient- and water-deficient stress, and thus, their absorption of nutrients and water appears more important . Soil nutrient and water have a great spatiotemporal heterogeneity . As the heterogeneity occurs, trees regulate carbon partitioning to roots, resulting in the senescence or death of some roots of the whole root system . In forestry, the senescence of tree roots is closely related to tree productivity, because there is a close relationship between the senescence of tree roots and the absorption of soil nutrient and water . At ecosystem and global scale, the senescence of tree roots influences the cycling of carbon and nutrients, because roots exhaust a great deal of carbon fixed by source leaves through photosynthesis, and there are great amounts of nutrients in tree roots . The senescence of tree roots is influenced by many environmental factors, biotic (e.g., fungi, bacteria, viruses, small edaphic animals) and abiotic (e.g., water, temperature, soil nutrients, heavy metals) . These factors affect the senescence of tree roots by different mechanisms . Although we have much knowledge on the senescence of tree roots and some hypotheses have been proposed, some problems still remain to be resolved, and further experiments are needed to test these hypotheses . Interdisciplinary studies integrating cytology, biochemistry, soil science, and genetics are the prerequisite for rapid advances in understanding the essence of tree root senescence. Med Hypotheses, 2004, 63(6), 925 - 32 Infant mouthing behavior: the immunocalibration hypothesis; Fessler DM et al.; Avid mouthing, the propensity of infants to suck objects and put them in their mouths, is a pattern characteristic of the first 2-3 years of life, with its most intensive manifestation occurring during the first year . Although traditional accounts explain infant mouthing as a source of sensual gratification and/or environmental exploration, these proximate hypotheses are inconsistent with the high costs of mouthing, including choking, poisoning, and exposure to pathogens . We propose that mouthing serves to proactively expose the naive gastrointestinal tract to environmental antigens and commensal bacteria while under the sheltering umbrella of breastfeeding . Mouthing functions to accurately calibrate the developing immune system, including antibody production and mucosal immunity, to the local disease ecology . The critical exposure period is not open-ended, as failure to expose the gut to an adequate number of antigens early in life is associated with an increased risk of allergies, asthma, and atopy . Weaning initiates a number of immune changes that may program the neonatal immune system into certain life-long responses. Biochemistry, 2004 Nov 2, 43(43), 13867 - 74 Micromechanical analysis of the binding of DNA-bending proteins HMGB1, NHP6A, and HU reveals their ability to form highly stable DNA-protein complexes; Skoko D et al.; The mechanical response generated by binding of the nonspecific DNA-bending proteins HMGB1, NHP6A, and HU to single tethered 48.5 kb lambda-DNA molecules is investigated using DNA micromanipulation . As protein concentration is increased, the force needed to extend the DNA molecule increases, due to its compaction by protein-generated bending . Most significantly, we find that for each of HMGB1, NHP6A, and HU there is a well-defined protein concentration, not far above the binding threshold, above which the proteins do not spontaneously dissociate . In this regime, the amount of protein bound to the DNA, as assayed by the degree to which the DNA is compacted, is unperturbed either by replacing the surrounding protein solution with protein-free buffer or by straightening of the molecule by applied force . Thus, the stability of the protein-DNA complexes formed is dependent on the protein concentration during the binding . HU is distinguished by a switch to a DNA-stiffening function at the protein concentration where the formation of highly stable complexes occurs . Finally, introduction of competitor DNA fragments into the surrounding solution disassembles the stable DNA complexes with HMGB1, NHP6A, and HU within seconds . Since spontaneous dissociation of protein does not occur on a time scale of hours, we conclude that this rapid protein exchange in the presence of competitor DNA must occur only via "direct" DNA-DNA contact . We therefore observe that protein transport along DNA by direct transfers occurs even for proteins such as NHP6A and HU that have only one DNA-binding domain. Proc R Soc Lond B Biol Sci, 2004 Aug 7, 271 Suppl 5, S277 - 80 Direct evidence for predation on trilobites in the Cambrian; Zhu MY et al.; A fossil arthropod similar to Fuxianhuia and displaying an exceptionally well-preserved alimentary canal with in situ gut contents is reported from the lower Middle Cambrian (ca . 510 Myr ago) of South China . Three-dimensionally preserved, paired phosphatic nodules, arranged in series along both sides of the gut and containing spherical bacteria, probably represent serial digestive glands . The gut is filled with fragments of the eodiscoid trilobite, Pagetia . The well-developed digestive glands and the fragmentary trilobite remains suggest (i) that the arthropod was a durophagous, possibly selective predator, and (ii) that small trilobites such as eodiscoids were a major food source in Cambrian marine ecosystems . This fossil association augments the small number of previously described associations providing more or less direct evidence of predator-prey relationships in Cambrian epibenthic communities. Refuat Hapeh Vehashinayim, 2004 Jul, 21(3), 67 - 74, 95 {Smoking--a proven risk factor for periodontal disease?}; Klinger A; While bacterial dental plaque is widely considered as the etiological risk factor for the development of periodontal diseases, numerous publications from recent years pointed out the role of environmental factors like smoking as aggravating the severity of disease expression . The present paper reviews the updated literature linking epidemiologically between the presence of smoking habit and disease prevalence in the same population, as well as studies reporting on cell toxicity of nicotine and its by-products on gingival cells, negative effect on humoral and cell-mediated immune system, enhancement of bacterial plaque pathogeneity, and disruption of the wound healing processes . Finally, a negative effect is apparent on the supportive stage of the periodontal treatment, indicating lasting effect of smoking for years to come following the termination of active periodontal treatment. Zhongguo Zhong Yao Za Zhi, 2004 Jul, 29(7), 663 - 6 {Experimental study on anti-pyretic effect of gegen qin lian decoction and its compounds}; Yu LZ et al.; OBJECTIVE: To investigate composition principles of Gegen Qin Lian decoction through anti-pyretic experiment . METHOD: Pharmacological effects of different compounds of Gegen Qin Lian decoction according to six hours temperature response index (TRI6) and average top temperature response height (deltaT) after the decoction was given to feverish animal model by inactived bacteria suspension . RESULT: As for reducing six hour temperature response index, Scutellaria baicalensis root was the main effective drug . Pueraria lobata root could enforce the effect while Coptis chinensis rhizome and Glycyrrhiza uralensis root counteracted it . As for reducing average top temperature response height, the Effects of four herbal drugs were the same as for TRI6 . CONCLUSION: Of the compounds of Gegen Qin Lian decoction, as to the pharmcological anti-pyretic effects, the best one is the compound of Scutellaria baicalensis and Pueraria lobata roots. Zhongguo Zhong Yao Za Zhi, 2004 Jul, 29(7), 624 - 7 {Development of effervescent tablet of Chinese traditional medicine and its technology study}; Tian XF et al.; Effervescent tablets which contain an effervescent mixture of a suitable organic acid and an alkali metal bicarbonate and/or carbonate can give out carbon dioxide when they meet water . The effervescent tablets for oral solution can be dissolved in cool water about 17-20 degrees C, therefore it is convenient to carry and use . It also has a good taste for patient with deodorizing agent added . The foam produced by external effervescent tablets is usually helpful in killing the local bacteria . The review displayed the main supplementary material, preparative technique and the study development of effervescent tablets of Chinese traditional medicine . Effervescent tablets that have been used to clinic were enumerated. J Nanosci Nanotechnol, 2004 May, 4(5), 532 - 8 Initial salivary pellicle formation on solid substrates studied by AFM; Hannig M et al.; Organic layers of salivary biopolymers adsorbed on soft and hard oral tissues, referred to also as salivary pellicle, play a critical role with respect to all surface phenomena taking place in the oral cavity . The initial stages of pellicle formation are of great interest since they determine the ensuing processes of salivary biopolymer adsorption and subsequent adherence of bacteria . In spite of the important physiological role of the pellicle in protecting the enamel surface against short-term acidic attacks, the composition and ultrastructure of the pellicle layer are not yet understood and resolved in detail . The present study utilized atomic force microscopy (AFM), for the first time, to elucidate the morphogenesis and ultrastructural pattern of initial salivary pellicle formation taking place in situ on solid substrates of mica, silicon wafer and graphite . Using tapping mode AFM, salivary pellicles were found in all intraorally exposed specimens and revealed a globular surface morphology of the adsorbed protein layer . The average diameter and height of the adsorbed salivary proteins were determined to be 15 +/- 3 nm and 2.0 +/- 0.5 nm, respectively . It was also found that the surface energy of the substrates affects the rate of pellicle formation, while the overall size of the adsorbed salivary proteins appears to be identical on all studied substrates. Mol Genet Genomics, 2004 Nov, 272(4), 460 - 9 Epub 2004 Nov. Mismatch repair ensures fidelity of replication and recombination in the radioresistant organism Deinococcus radiodurans; Mennecier S et al.; We have characterized the mismatch repair system (MMR) of the highly radiation-resistant type strain of Deinococcus radiodurans, ATCC 13939 . We show that the MMR system is functional in this organism, where it participates in ensuring the fidelity of DNA replication and recombination . The system relies on the activity of two key proteins, MutS1 and MutL, which constitute a conserved core involved in mismatch recognition . Inactivation of MutS1 or MutL resulted in a seven-fold increase in the frequency of spontaneous RifR mutagenesis and a ten-fold increase in the efficiency of integration of a donor point-mutation marker during bacterial transformation . Inactivation of the mismatch repair-associated UvrD helicase increased the level of spontaneous mutagenesis, but had no effect on marker integration--suggesting that binding of MutS1 and MutL proteins to a mismatched heteroduplex suffices to inhibit recombination between non identical (homeologous) DNAs . In contrast, inactivation of MutS2, encoded by the second mutS -related gene present in D . radiodurans, had no effect on mutagenesis or recombination . Cells devoid of MutS1 or MutL proteins were as resistant to gamma-rays, mitomycin C and UV-irradiation as wild-type bacteria, suggesting that the mismatch repair system is not essential for the reconstitution of a functional genome after DNA damage. Pediatr Infect Dis J, 2004 Oct, 23(10 Suppl), S173 - 8 Natural rotavirus infection is not associated to intussusception in Mexican children; Velazquez FR et al.; AIMS: To determine whether natural rotavirus infection or infection by another enteropathogen is associated to intussusception (IS); and to describe the seasonality of IS compared with severe diarrhea (SD) and rotavirus SD in Mexican children . METHODS: A prospective, observational, multicenter and case-control study was conducted in Mexico City from December 1999 to February 2001 . Cases were children younger than 1 year old hospitalized for IS; diagnosis was made by clinical features, radiologic and/or surgery findings . Controls were children younger than 1 year old hospitalized for another disease than a gastrointestinal illness (NGI) . Cases and controls were paired by age and date of admission (+/-3 months; for both), in a 1:2 ratio . A surveillance of IS cases, SD and rotavirus SD episodes was conducted during the study period . Stool samples collected soon after IS resolution or at admission were tested for rotavirus, adenovirus, astrovirus, bacteria and parasites . RESULTS: Thirty cases of IS and 60 controls with NGI were studied . Rotavirus was not detected in any case of IS . Adenovirus (17%) was the only enteropathogen detected in IS . Rotavirus (8%), adenovirus (2%), astrovirus (2%) and bacteria (2%) were detected in NGI . Rotavirus infection was not associated with IS (odds ratio, 0; 95% confidence interval, 0-2.9), whereas adenovirus infection was strongly associated as risk factor for IS (odds ratio undefined; P = 0.003), compared with NGI . Seasonal variation in admissions for IS was small, whereas admissions for SD and rotavirus SD showed a marked seasonal increase during fall-winter . CONCLUSIONS: In Mexican children, rotavirus infection was not associated to IS; whereas a significant association was observed between adenovirus and IS . Also there was no increase in IS cases during the sharply defined fall-winter rotavirus outbreak . Observations from this controlled study suggest that natural rotavirus infection is not a risk factor for IS . This information may have implications for development of a safer and effective rotavirus vaccine. Acta Crystallogr D Biol Crystallogr, 2004 Nov, 60(Pt 11), 2003 - 5 Epub 2004 Oct 20. Crystallization and preliminary X-ray crystallographic analysis of chorismate synthase from Mycobacterium tuberculosis; Dias MV et al.; The enzymes of the shikimate pathway are potential targets for the development of new therapies because they are essential for bacteria but absent from mammals . The last step in this pathway is performed by chorismate synthase (CS), which catalyzes the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate . Optimization of crystallization trials allowed the crystallization of homogeneous recombinant CS from Mycobacterium tuberculosis (MtCS) . The crystals of MtCS belong to space group P6(4)22 (or P6(2)22) and diffract to 2.8 A resolution, with unit-cell parameters a = b = 129.7, c = 156.8 A . There are two molecules in the asymmetric unit . Molecular-replacement trials were not successful . Heavy-atom derivative screening is in progress. Methods Mol Biol, 2004, 291, 337 - 50 An immunoassay for measuring repair of ultraviolet photoproducts; McCready S; A method is described that makes use of a polyclonal antiserum to measure repair of the principal photoproducts induced in DNA by short-wave ultraviolet light (UVC)-pyrimidine-pyrimidone 6-4 photoproducts ({6-4}PPs) and cyclobutane pyrimidine dimers (CPDs) . DNA extracted from irradiated cells is applied to a nitrocellulose dot blot and quantitated using an enzyme-conjugated secondary antibody and a color assay . Although the polyclonal antiserum contains antibodies to both {6-4}PPs and CPDs, repair of these can be measured separately by differential destruction or repair of one or other photoproduct . The method is useful for measuring repair in total genomic DNA and is sufficiently sensitive to measure repair of damage induced by doses of 10 J/m2 of UVC and less . The method is very versatile and has been used to measure repair in human cells, yeasts, plants, archaea, bacteria, and filamentous fungi. Nucleic Acids Res, 2004 Oct 22, 32(19), 5703 - 11 Print 2004. Unidirectional translocation from recognition site and a necessary interaction with DNA end for cleavage by Type III restriction enzyme; Raghavendra NK et al.; Type III restriction enzymes have been demonstrated to require two unmethylated asymmetric recognition sites oriented head-to-head to elicit double-strand break 25-27 bp downstream of one of the two sites . The proposed DNA cleavage mechanism involves ATP-dependent DNA translocation . The sequence context of the recognition site was suggested to influence the site of DNA cleavage by the enzyme . In this investigation, we demonstrate that the cleavage site of the R.EcoP15I restriction enzyme does not depend on the sequence context of the recognition site . Strikingly, this study demonstrates that the enzyme can cleave linear DNA having either recognition sites in the same orientation or a single recognition site . Cleavage occurs predominantly at a site proximal to the DNA end in the case of multiple site substrates . Such cleavage can be abolished by the binding of Lac repressor downstream (3' side) but not upstream (5' side) of the recognition site . Binding of HU protein has also been observed to interfere with R.EcoP15I cleavage activity . In accordance with a mechanism requiring two enzyme molecules cooperating to elicit double-strand break on DNA, our results convincingly demonstrate that the enzyme translocates on DNA in a 5' to 3' direction from its recognition site and indicate a switch in the direction of enzyme motion at the DNA ends . This study demonstrates a new facet in the mode of action of these restriction enzymes. J Biol Chem, 2005 Jan 7, 280(1), 469 - 475 Epub 2004 Oct 22. Crystal Structure of the Human Cytosolic Sialidase Neu2: EVIDENCE FOR THE DYNAMIC NATURE OF SUBSTRATE RECOGNITION; Chavas LM et al.; Gangliosides play key roles in cell differentiation, cell-cell interactions, and transmembrane signaling . Sialidases hydrolyze sialic acids to produce asialo compounds, which is the first step of degradation processes of glycoproteins and gangliosides . Sialidase involvement has been implicated in some lysosomal storage disorders such as sialidosis and galactosialidosis . Neu2 is a recently identified human cytosolic sialidase . Here we report the first high resolution x-ray structures of mammalian sialidase, human Neu2, in its apo form and in complex with an inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA) . The structure shows the canonical six-blade beta-propeller observed in viral and bacterial sialidases with its active site in a shallow crevice . In the complex structure, the inhibitor lies in the catalytic crevice surrounded by ten amino acids . In particular, the arginine triad, conserved among sialidases, aids in the proper positioning of the carboxylate group of DANA within the active site region . The tyrosine residue, Tyr(334), conserved among mammalian and bacterial sialidases as well as in viral neuraminidases, facilitates the enzymatic reaction by stabilizing a putative carbonium ion in the transition state . The loops containing Glu(111) and the catalytic aspartate Asp(46) are disordered in the apo form but upon binding of DANA become ordered to adopt two short alpha-helices to cover the inhibitor, illustrating the dynamic nature of substrate recognition . The N-acetyl and glycerol moieties of DANA are recognized by Neu2 residues not shared by bacterial sialidases and viral neuraminidases, which can be regarded as a key structural difference for potential drug design against bacteria, influenza, and other viruses. Infect Immun, 2004 Nov, 72(11), 6676 - 9 Mucosal administration of flagellin induces innate immunity in the mouse lung; Honko AN et al.; Nonsurgical intratracheal instillation of 1 microg of purified, recombinant flagellin in several strains of mice stimulated a transient innate immune response in the lung characterized by the infiltration of neutrophils and the rapid production of tumor necrosis factor alpha, interleukin 6, granulocyte colony-stimulating factor, and the chemokines keratinocyte-derived chemokine, MIP1alpha, and MIP-2. Infect Immun, 2004 Nov, 72(11), 6615 - 21 Differences in cell activation by Chlamydophila pneumoniae and Chlamydia trachomatis infection in human endothelial cells; Krull M et al.; Seroepidemiological studies and demonstration of viable bacteria in atherosclerotic plaques have linked Chlamydophila pneumoniae infection to the development of chronic vascular lesions and coronary heart disease . In this study, we characterized C . pneumoniae-mediated effects on human endothelial cells and demonstrated enhanced phosphorylation and activation of the endothelial mitogen-activated protein kinase (MAPK) family members extracellular receptor kinase (ERK1/2), p38-MAPK, and c-Jun-NH2 kinase (JNK) . Subsequent interleukin-8 (IL-8) expression was dependent on p38-MAPK and ERK1/2 activation as demonstrated by preincubation of endothelial cells with specific inhibitors for the p38-MAPK (SB202190) or ERK (U0126) pathway . Inhibition of either MAPK had almost no effect on intercellular cell adhesion molecule 1 (ICAM-1) expression . While Chlamydia trachomatis was also able to infect endothelial cells, it did not induce the expression of endothelial IL-8 or ICAM-1 . These effects were specific for a direct stimulation with viable C . pneumoniae and independent of paracrine release of endothelial cell-derived mediators like platelet-activating factor, NO, prostaglandins, or leukotrienes . Thus, C . pneumoniae triggers an early signal transduction cascade in target cells that could lead to endothelial cell activation, inflammation, and thrombosis, which in turn may result in or promote atherosclerosis. Infect Immun, 2004 Nov, 72(11), 6324 - 9 RNA encoding the MPT83 antigen induces protective immune responses against Mycobacterium tuberculosis infection; Xue T et al.; We have previously demonstrated that vaccination of mice with plasmid DNA vectors expressing immunodominant mycobacterial genes induced cellular immune responses and significant protection against challenge with Mycobacterium tuberculosis . We demonstrate here, using in vitro-synthesized RNA, that vaccination with DNA or RNA constructs expressing the M . tuberculosis MPT83 antigen are capable of inducing specific humoral and T-cell immune responses and confer modest but significant protection against M . tuberculosis challenge in mice . This is the first report of protective immunity conferred against intracellular bacteria by an RNA vaccine . This novel approach avoids some of the drawbacks of DNA vaccines and illustrates the potential for developing new antimycobacterial immunization strategies. Ultrason Sonochem, 2005 Mar, 12(4), 301 - 6 Bioleaching with ultrasound; Swamy KM et al.; Application of bioleaching of metals in a large-scale operation is reviewed briefly . Continued technical innovation is vital for the wider utilisation of this extraction process within the mineral industries . Therefore, the use of power ultrasound in combination with bioleaching (referred to as sonobioleaching) has been studied and is shown to be beneficial in the recovery of nickel from lean grade ores . The role of ultrasound in improving the benefits and lowering the drawbacks of bioleaching to an acceptable level are described . A possible mechanism for improving and intensifying this process is hypothesised. Peptides, 2004 Nov, 25(11), 1899 - 907 The crustacean hyperglycemic hormone precursors a and b of the Norway lobster differ in the preprohormone but not in the mature peptide; Mettulio R et al.; The neuro-endocrine X-organ sinus-gland complex of crustaceans produces and releases the neuropeptides of the crustacean hyperglycemic hormone (cHH)/molt-inhibiting hormone (MIH)/gonad-inhibiting hormone (GIH) family that regulate important physiological processes, such as growth, reproduction and molting . We cloned two full-length cDNAs encoding the preprocHH-A and preprocHH-B of the Norway lobster Nephrops norvegicus of 132 and 131 amino acid residues . The two cHHs differ in the preprohormone but not in the mature peptide sequence . The mature cHH was expressed in bacteria as GST fusion protein that, in bioassay, shows a hyperglycemic activity similar to that of native cHH present in an eyestalk extract. J Theor Biol, 2004 Dec 7, 231(3), 357 - 76 Identifying control mechanisms of granuloma formation during M . tuberculosis infection using an agent-based model; Segovia-Juarez JL et al.; Infection with Mycobacterium tuberculosis is a major world health problem . An estimated 2 billion people are presently infected and the disease causes approximately 3 million deaths per year . After bacteria are inhaled into the lung, a complex immune response is triggered leading to the formation of multicellular structures termed granulomas . It is believed that the collection of host granulomas either contain bacteria resulting in a latent infection or are unable to do so, leading to active disease . Thus, understanding granuloma formation and function is essential for improving both diagnosis and treatment of tuberculosis . Granuloma formation is a complex spatio-temporal system involving interactions of bacteria, specific immune cells, including macrophages, CD4+ and CD8+ T cells, as well as immune effectors such as chemokine and cytokines . To study this complex dynamical system we have developed an agent-based model of granuloma formation in the lung . This model combines continuous representations of chemokines with discrete agent representations of macrophages and T cells in a cellular automata-like environment . Our results indicate that key host elements involved in granuloma formation are chemokine diffusion, prevention of macrophage overcrowding within the granuloma, arrival time, location and number of T cells within the granuloma, and an overall host ability to activate macrophages . Interestingly, a key bacterial factor is its intracellular growth rate, whereby slow growth actually facilitates survival. J Hosp Infect, 2004 Nov, 58(3), 233 - 7 In situ endoluminal brushing: a safe technique for the diagnosis of catheter-related bloodstream infection; Dobbins BM et al.; We assessed the safety of the endoluminal brush technique for the in situ diagnosis of central-venous-catheter (CVC)-related bloodstream infection (CRBSI) . The endoluminal brush was used to investigate patients with suspected CRBSI by sampling the CVC lumen to within 3-5 cm of the catheter tip (the brush was kinked to mark the length required) . Quantitative peripheral blood cultures were taken 1 min pre-brushing and 1 min and 1h post-brushing . In addition, a 1 mL sample of through-catheter blood was aspirated immediately pre- and post-brushing, and aliquots were directly plated on to blood agar to determine the load of CVC-associated bacteria . After CVC removal, catheter tips were sent for culture using the Maki roll and modified Cleri flush techniques . Of 139 suspected cases, 61 (45%) had confirmed episodes of CRBSI . No patients experienced fever or rigors, or had evidence of embolic phenomena after CVC endoluminal brushing . There was no difference overall in pre-brushing and 1-min post-brushing peripheral blood bacterial counts (P < 0.702) . Peripheral and CVC blood bacterial counts were significantly reduced 1h post-brushing (P < 0.03 and P < 0.001, respectively), possibly due to removal of endoluminal biomass . The endoluminal brush technique can be safely used to diagnose CRBSI without the need for CVC removal, provided that the brush is not allowed to protrude beyond the tip of the catheter. Trends Plant Sci, 2004 Nov, 9(11), 523 - 8 Plant fructan exohydrolases: a role in signaling and defense? Van den Ende W, De Coninck B, Van Laere A. Fructans are fructose oligomers and polymers synthesized by a small number of plant and bacterial species and mainly function as reserve carbohydrates . The terminal fructosyl-fructose linkages can be degraded by fructan exohydrolases (FEHs), occurring in bacteria, fungi and fructan plants . Unexpectedly, it was found that FEHs also occur in non-fructan plants such as Beta vulgaris and Arabidopsis thaliana that apparently lack endogenous fructan substrates . FEHs might have defense-related roles acting on bacterial fructan-containing slimes or might act on minute (up to now undetected) amounts of fructans acting as signals in plants. Mol Biochem Parasitol, 2004 Nov, 138(1), 29 - 36 Malaria parasite-inhibitory antibody epitopes on Plasmodium falciparum merozoite surface protein-1(19) mapped by TROSY NMR; Morgan WD et al.; Plasmodium falciparum merozoite surface protein 1 (MSP1)(19), the C-terminal fragment of merozoite surface protein 1, is a leading candidate antigen for development of a vaccine against the blood stages of the malaria parasite . Many human and animal studies have indicated the importance of MSP1(19)-specific immune responses . Anti-MSP1(19) antibodies can prevent invasion of red blood cells by P . falciparum parasites in vitro . However, the fine specificity of anti-MSP1(19) antibodies is also important, as only a fraction of monoclonal antibodies (mAbs) have parasite-inhibitory activity in vitro . Human sera from malaria-endemic locations show strong MSP1(19) reactivity, but individual serum samples vary greatly in inhibitory activity . NMR is an excellent method for studying protein-protein interactions, and has been used widely to study binding of peptides representing known epitopes (as well as non-protein antigens) to antibodies and antibody fragments . The recent development of transverse relaxation optimized spectroscopy (TROSY) and related methods has significantly extended the maximum size limit of molecules that can be studied by NMR . TROSY NMR experiments produce high quality spectra of Fab complexes that allow the mapping of epitopes by the chemical shift perturbation technique on a complete, folded protein antigen such as MSP1(19) . We studied the complexes of P . falciparum MSP1(19) with Fab fragments from three monoclonal antibodies . Two of these antibodies have parasite-inhibitory activity in vitro, while the third is non-inhibitory . NMR epitope mapping showed a close relationship between binding sites for the two inhibitory antibodies, distinct from the location of the non-inhibitory antibody . Together with a previously published crystal structure of the P . falciparum MSP1(19) complex with the Fab fragment of another non-inhibitory antibody, these results revealed a surface on MSP1(19) where inhibitory antibodies bind . This information will be useful in evaluating the anti-MSP1(19) immune response in natural populations from endemic areas, as well as in vaccine trials . It will also be valuable for optimizing the MSP1(19) antigen by rational vaccine design . This work also shows that TROSY NMR techniques are very effective for mapping conformational epitopes at the level of individual residues on small- to medium-sized proteins, provided that the antigen can be expressed in a system amenable to stable isotope labelling, such as bacteria or yeast. Adv Protein Chem, 2004, 68, 313 - 49 Ionotropic glutamate receptor recognition and activation; Oswald RE; Ionotropic glutamate receptors are the major excitatory neurotransmitters in mammalian brain but are found throughout the animal kingdom as well as in plants and bacteria . A great deal of progress in understanding the structure of these essential neurotransmitter receptors has been made since the first examples were cloned and sequenced in 1989 . The atomic structure of the ligand-binding domain of several ionotropic glutamate receptors has been determined, and a great deal of progress has been made in relating the structural properties of the binding site to the function of the intact receptor . In addition, the identification of glutamate receptors from a wide variety of organisms ranging from several types of bacteria to Arabidopsis to a range of animal species has made glutamate receptors a molecular laboratory for studying the evolution of proteins . The fact that glutamate receptors are a particularly ancient intercellular signaling molecule suggests a potential role in the transition from single celled to multicellular organisms . This review focuses on the structure and dynamics of ionotropic glutamate receptors and their relation to the function and evolution of these proteins. Biochem J . 2004 Oct 25; {Epub ahead of print} Characterization of phylogenetically distant members of the adenylyl cyclase family from mycobacteria: Rv1647 from M . tuberculosis and its ortholog ML1399 from M . leprae; Shenoy AR et al.; Analysis of the genome sequence of Mycobacterium tuberculosis H37Rv has identified 16 genes that are similar to the mammalian adenylyl and guanylyl cyclases . Rv1647 was predicted to be an active adenylyl cyclase but its position in a phylogenetically distant branch from the other enzymes characterized so far from M . tuberculosis, makes it an interestingly divergent nucleotide cyclase to study . In agreement with its divergence at the sequence level from other nucleotide cyclases, cloning, expression and purification of Rv1647 revealed differences in its biochemical properties from the earlier characterized Rv1625c adenylyl cyclase . Adenylyl cyclase activity of Rv1647 was activated by detergents but was resistant to high concentrations of salt . Mutations of substrate specifying residues to those present in guanylyl cyclases failed to convert the enzyme to a guanylyl cyclase, but did not alter its oligomeric status . Orthologs of Rv1647 could be found in M . leprae, M . avium and M . smegmatis . The ortholog from M . leprae (ML1399) was cloned, protein expressed, purified and shown biochemically to be an adenylyl cyclase, thus representing the first adenylyl cyclase to be described from M . leprae . Importantly, Western blot analysis of subcellular fractions from M . tuberculosis and M . leprae revealed that Rv1647 and ML1399 gene products were expressed in these bacteria respectively . Additionally, M . tuberculosis was also found to express the Rv1625c adenylyl cyclase, suggesting that multiple adenylyl cyclase proteins may be expressed simultaneously in this organism . These results suggest that Class III cyclase-like gene products are likely to have an important role to play in the physiology and perhaps the pathology of these medically important bacteria. Lin Chuang Er Bi Yan Hou Ke Za Zhi, 2004 Jul, 18(7), 411 - 3 {The expression of RANTES interleukin 1beta and endotoxin in the effusion of secretory otitis media}; Liang G et al.; OBJECTIVE: To study the protein expression of endotoxin, interleukin 1beta (IL-1beta) and regulated upon activation, normal T expressed and secreted (RANTES) in effusions of secretory otitis media and their roles in the pathogenesis of secretory otitis media . METHOD: Seventy-two effusion samples were taken from 53 patients with secretory otitis media by tympanocentesis . After swabs they were taken for bacteria culture . Limulus amebocyte lysate (LAL) test was used to quantify the content of endotoxin, radioimmunoassay to analyze the level of IL-1beta and enzyme-linked immunosorbent assay (ELISA) to detect the concentration of RANTES . RESULT: Endotoxin, IL-1beta and RANTES were detectable in 80.9%, 77.8% and 70.8% of middle ear effusion, with mean levels of (35.2 +/- 51.6) EU/ml, (1.10 +/- 0.84) microg/L and (0.52 +/- 0.43) microg/L respectively . All of them showed higher concentration in the mucoid-type effusions than those in the serous-type effusions (P<0.05) . Higher levels of the endotoxin and RANTES (P<0.05) were found in longer course patients . The effusions with positive bacteria culture displayed more abundant endotoxin, IL-1beta and RANTES protein than those with negative culture . Significant correlations were found between endotoxin and IL-1beta (r = 0.74, P<0.01), also between IL-1beta and RANTES (r = 0.48, P<0.01) . CONCLUSION: Endotoxin, IL-1beta and RANTES may take part in maintaining the inflammatory response in the tympanic cavity and stimulating mucin secretion . It indicates that they play an important role in the immune pathogenesis of secretory otitis media. Cutis, 2004 Sep, 74(3 Suppl), 9 - 12, 32-4 Rosacea and the pilosebaceous follicle; Powell FC; The pathophysiology of rosacea remains unknown . A leading theory suggests a vascular basis; however, clinical observations and histopathologic studies suggest that inflammation of the pilosebaceous follicle may be central to the pathogenesis of rosacea . Demodex folliculorum is a frequently seen commensal in the follicles of facial skin . According to evidence from biopsies of the skin surface, individuals with rosacea have a higher density of this parasite . This increased mite density may play a role in the pathophysiology of rosacea by triggering inflammatory or specific immune reactions, mechanically blocking the follicles, or acting as a vector for bacteria . Ongoing research has shown that bacteria from patients with rosacea may behave differently at the higher skin temperature that may be present in patients with rosacea . Another group has isolated bacteria from the Demodex mites; these bacteria may play a pathogenic role in papulopustular rosacea by facilitating follicular-based inflammatory changes. Am J Physiol Heart Circ Physiol . 2004 Oct 21; {Epub ahead of print} Human neutrophils promote angiogenesis by a paracrine feedforward mechanism involving endothelial interleukin-8; Schruefer R et al.; Neovascularization by sprouting angiogenesis is critical for inflammation-mediated tissue remodeling and wound healing . We report here that human polymorphonuclear neutrophils (PMN) stimulated for 1 h with 100 nM N-formyl-Met-Leu-Phe (fMLP) released a proangiogenic entity which induced sprouting of capillary-like structures in an in vitro angiogenesis assay . The effect was comparable to the response obtained upon stimulation with 100 ng/ml basic fibroblast growth factor . The PMN-mediated response was inhibited by neutralizing antibodies against VEGF or IL-8 . As measured by ELISA technique, we found that fMLPactivated PMN (5 x 10(6)/ml) released 78 pg/ml IL-8 and 39 pg/ml VEGF within 1 h after stimulation . The IL-8 release was blocked by actinomycin D or cycloheximide, but the inhibitors had no effect on the VEGF release suggesting that the IL-8 secretion required de novo synthesis whereas VEGF was secreted from preformed stores . Accordingly, RT-PCR analysis revealed that the IL-8 mRNA was upregulated upon PMN stimulation whereas the expression of the VEGF mRNA was not affected . Moreover, the supernatant derived from activated PMN induced an upregulation of the endothelial IL-8 mRNA expression suggesting that the release of VEGF and IL-8 from activated PMN may activate a paracrine feedforward mechanism involving endothelial IL-8 . Moreover, the VEGF-induced upregulation of endothelial IL-8 expression as well as the sprouting of capillary-like structures was inhibited by a neutralizing anti-IL-8 antibody . These findings suggest that bacteria-derived tripeptides stimulate human PMN to release VEGF and IL-8 which activate endothelial cells and induce angiogenesis by a paracrine feedforward mechanism involving endothelial IL-8 upregulation. Mol Plant Microbe Interact, 2004 Oct, 17(10), 1086 - 94 Enhancer trapping identifies TRI, an Arabidopsis gene up-regulated by pathogen infection; Fridborg I et al.; Enhancer trap Arabidopsis thaliana plants were screened for genes up-regulated by virus infection . The plants carried T-DNA insertions comprising a minimal -60-bp Cauliflower mosaic virus 35S promoter fused to the beta-glucuronidase (GUS) reporter gene . Approximately 12,000 plants were assayed for GUS activity before and after rub-inoculation with Tobacco rattle virus (TRV) tagged with the green fluorescent protein (GFP) . One plant and its progeny consistently showed upregulation of GUS activity in response to TRV-GFP infection, indicating that a virus-responsive enhancer element was "tagged" by the T-DNA in this line . Other viruses, bacteria, and oomycetes, but not wounding, up-regulated GUS activity in the enhancer trap line, indicating that the response was not specific to TRV-GFP infection . A pathogen-inducible, alternatively spliced gene was identified, which we have termed TRI for TRV-induced gene . A pathogen-responsive element was localized to a 1.1-kb region upstream of the T-DNA insertion, and two different cis-acting elements, both implicated in defense responses, were found in the sequence upstream of TRI . Sequence analyses revealed that TRI is similar to ACRE169, a gene that is up-regulated in Cf-9-expressing tobacco when treated with Avr-9, the Cladosporium fulvum elicitor of the Cf-9 resistance response. Appl Biochem Biotechnol, 2004 Oct, 119(1), 31 - 40 Decolorization and partial degradation of selected azo dyes by methanogenic sludge; Yemashova N et al.; The toxicity potential and decolorization of three acid azo dyes (Acid Orange 6, Acid Orange 7, and Acid Orange 52) by methanogenic granular sludge from an anaerobic expanded granular sludge bed reactor was assayed . Complete bioreduction was found for all three azo dyes . Sulfanilic acid and 4-aminoresorcinol were detected from the decolorization of Acid Orange 6, sulfanilic acid and 1-amino-2-naphtol were detected from the reduction of Acid Orange 7, and sulfanilic acid and N,N-dimethyl-1,4-phenylenediamine (DMP) were found to be intermediates of Acid Orange 52 degradation . Sulfanilic acid and 1-amino-2-naphtol were persistent in the anaerobic conditions, whereas 4-aminoresorcinol was completely mineralized by anaerobic sludge and DMP was transformed into 1,4-phenylenediamine . Enrichment cultures obtained via consecutive passages on basal medium with only azo dye as a carbon and an energy source seemed to be morphologically heterogeneous . Baculiform and coccus cells were found when viewed under a light microscope . Cocci were joined in chains . Because anaerobic sludge contains sulfate-reducing bacteria and therefore may generate sulfide, azo dyes were tested for chemical decolorization by sulfide to compare rates of chemical and biologic reduction. Biochem Soc Trans, 2004 Nov, 32(Pt 5), 733 - 7 Mechanisms of elongation on the ribosome: dynamics of a macromolecular machine; Wintermeyer W et al.; Protein synthesis in the cell is performed on ribosomes, large ribonucleoprotein particles, which in bacteria consist of three RNA molecules and over 50 proteins . This review summarizes recent progress in understanding the mechanisms of the elongation phase of protein synthesis . Results from rapid kinetic analysis of elongation reactions are discussed in the light of recent structural data. Biochem Soc Trans, 2004 Nov, 32(Pt 5), 647 - 54 Chemical insights from structural studies of enzymes; Naismith JH; The rapid progress in structural and molecular biology over the past fifteen years has allowed chemists to access the structures of enzymes, of their complexes and of mutants . This wealth of structural information has led to a surge in the interest in enzymes as elegant chemical catalysts . Enzymology is a distinguished field and has been making vital contributions to medicine and basic science long before structural biology . This review for the Colworth Medal Lecture discusses work from the author's laboratory . This work has been carried out in collaboration with many other laboratories . The work has mapped out the chemical mechanisms and structures of interesting novel enzymes . The review tries to highlight the interesting chemical aspects of the mechanisms involved and how structural analysis has provided a detailed insight . The review focuses on carbohydrate-processing pathways in bacteria, and includes some recent data on an integral membrane protein. Int J Med Microbiol, 2004 Sep, 294(2-3), 149 - 55 Preference, adaptation and survival of Mycoplasma pneumoniae subtypes in an animal model; Dumke R et al.; The interaction between Mycoplasma pneumoniae and its natural host, humans, cannot be studied directly for obvious reasons . Therefore, we used guinea pigs instead, which had been recently introduced as an acceptable alternative host organism . The following experimental approaches were taken to study the pathogen-host relationship: characterization and subtyping of M . pneumoniae strains isolated from human patients, infection of guinea pigs with selected M . pneumoniae strains, and analysis of adaptation, preference and survival of individual strains in guinea pigs under competitive conditions . The results of our study indicated that the species M . pneumoniae is genetically very homogeneous . From 115 independently isolated strains two subtypes and one variant were found . The subtypes differed significantly in the amino acid composition of the P1 protein, the main adhesin of M . pneumoniae, while the variant showed only minor amino acid exchanges . Infection of guinea pigs indicated differences between the subtypes and the variant in their ability to colonize and survive in the animal . Preinfection of the host with a certain subtype or variant caused a subtype-specific immunity and had a strong influence on the type of surviving bacteria in superinfection experiments . The results of these studies explain the shift of subtypes frequently observed in epidemic outbreaks of M . pneumoniae infection appearing in intervals of 3-7 years. Med Pr, 2004, 55(3), 289 - 92 {Allergy to cosmetics . II . Preservatives}; Kiec-Swierczynska M et al.; Disinfectants are essential components of body care preparations, household goods and industrial products . They inhibit growth of bacteria and fungi . Esters of parahydroxybenzoate acid and products that release small amounts of formaldehyde (Germal 115, Germal II, Dovicil 200, Bronopol, DMDM hydantoine) were most frequently used in the past . In the 1980s, Katon CG (5-chloro-2-methyl-4-isothiazolin-3-one + 2-methyl-4-isothiazolin-3-one) evoked the epidemics of contact dermatitis in Sweden, Finland, Germany, Italy and The Netherlands . In the next years, allergy to another preservative, Euxyl K 400 was dramatically growing . Studies carried out in 11 European countries showed that hypersensitivity increased from 0.7% in 1991 to 3.5% in 2000 . It was revealed that not only cosmetics left on the skin sensitize, but also those washable . Apart from preservatives, allergic reactions are induced by emulgators, antioxidants, moisteners, lubricants, stabilizers and stickers. Retina, 2004 Oct, 24(5), 699 - 705 Minimizing the risk of endophthalmitis following intravitreous injections; Ta CN; PURPOSE: To review techniques that are important in preventing endophthalmitis following intravitreous injections based on the available evidence . METHODS: A review of the literature regarding existing evidence that is relevant to post-injection endophthalmitis prophylaxis . RESULTS: The available evidence regarding endophthalmitis prophylaxis is largely based on intraocular surgery, particularly cataract surgery . Despite the controversy and the paucity of evidence regarding endophthalmitis prophylaxis for intravitreous injections, it is recommended that povidone-iodine should be used prior to intravitreous injections to minimize or eliminate the presence of ocular surface bacteria . Administration of topical antibiotics may also be considered before and after the intravitreous injections . Strict adherence to aseptic techniques, including the use of an eyelide speculum is also important . CONCLUSION: Endophthalmitic can occur following intravitreous injections . Prophylaxis with topical povidone-iodine, and possibly antibiotics, as well as adherence to aseptic technique may minimize the risk of post-injection endophthalmitis. Methods Mol Med, 2004, 107, 313 - 24 Primary culture of human antral endocrine and epithelial cells; Curtis SB et al.; The mucosal endocrine cells in the antrum are found as individual elements interspersed among the surrounding epithelial cells, the majority of these being the gastric mucous cells . To establish the factors regulating either endocrine or mucous cell function, the cells have to be separated not only from nonepithelial cells, but also from circulating and neuronal elements within the stomach.A major problem in obtaining cultures of gastric endocrine cells is their diffuse distribution in the stomach and the nonsterile nature of the gastric lumen . To overcome these problems, we have used a combination of collagenase digestion of the mucosal layer with centrifugal elutriation to remove small particles, such as bacteria and fungi, and provide an enriched preparation of endocrine cells . The technique represents a modification of the methodology originally developed to isolate endocrine cells from the canine stomach.Unfortunately, none of the techniques developed so far produce a 100% pure culture of an individual endocrine cell type, with the antral gastrin cell cultures ranging from 20 to 45% purity . However, by modifying the single-cell fractionation protocol cultures containing 95% gastric mucous cells can be obtained.These epithelial cell cultures have been used for a number of different experimental techniques: release studies examining regulation of hormonal secretion, intracellular ion flux in response to stimulation (e.g., Fura-2 measurement of intracellular calcium levels) , molecular studies of gene expression patterns using array technology, or the interaction between antral epithelial cells and Helicobacter pylori. Proc Natl Acad Sci U S A, 2004 Oct 26, 101(43), 15380 - 5 Epub 2004 Oct 18. A class of eukaryotic GTPase with a punctate distribution suggesting multiple functional replacements of translation elongation factor 1alpha; Keeling PJ et al.; Translation elongation factor 1alpha (EF-1alpha, or EF-Tu in bacteria) is a highly conserved core component of the translation machinery that is shared by all cellular life . It is part of a large superfamily of GTPases that are involved in translation initiation, elongation, and termination, as well as several other cellular functions . Eukaryotic EF-1alpha (eEF-1alpha) is well studied and widely sampled and has been used extensively for phylogenetic analyses . It is generally thought that such highly conserved and functionally integrated proteins are unlikely to be involved in events such as lateral gene transfer or ancient duplication and gene sorting, which would undermine phylogenetic reconstruction . Here we describe a GTPase called EF-like (EFL), which is very similar to, but also distinct from, canonical eEF-1alpha . EFL is found in a wide variety of eukaryotes (dinoflagellates, haptophytes, cercozoa, green algae, choanoflagellates, and fungi), but its distribution is punctate: organisms that possess EFL are not closely related to one another, and EFL appears to be absent from the closest relatives of organisms that do possess it . Moreover, in most genomes where EFL is present, canonical eEF-1alpha appears to be absent . Analysis of functional divergence suggests that, whereas EFL is divergent in general, putative functional binding sites involved in translation are not significantly divergent as a whole . Altogether, it appears that EFL has replaced eEF-1alpha several times independently . This finding could be an indication of an ancient paralogy or, more likely, eukaryote-to-eukaryote lateral gene transfer. J Control Release, 2004 Nov 5, 100(1), 111 - 9 Prolonged circulation of large polymeric nanoparticles by non-covalent adsorption on erythrocytes; Chambers E et al.; Polymeric nanoparticles have been extensively studied for use as intravascular drug delivery vehicles; however, their applications are limited by rapid clearance from circulation by the reticuloendothelial system (RES) . Previous attempts to improve vascular circulation have focused on surface modification using polymers such as poloxamines, poloxamers, and polyethylene glycol, to prevent opsononization . We report on a novel method of prolonging intravascular particle circulation by anchoring the nanoparticles to the surface of red blood cells (RBCs) . We hypothesize that particles adhered to RBCs can escape RES clearance due to the ability of RBCs to do so . This method is motivated by the strategy adopted by certain bacteria, for example, hemobartonella, that adhere to RBCs and remain in circulation for several weeks . Prolonged circulation of nanoparticles as large as 450 nm was observed after adsorption on RBCs . Although particles were eventually eliminated from circulation, RBCs were not cleared . RBC-anchored nanoparticles offer a novel approach for intravascular drug delivery and blood pool imaging. Mol Microbiol, 2004 Nov, 54(3), 808 - 22 Integration host factor is involved in transcriptional regulation of the Brucella abortus virB operon; Sieira R et al.; Type IV secretion systems (T4SSs) are multicomponent machineries that play an essential role in pathogenicity of many facultative intracellular bacteria . The virB operon of Brucella abortus codes for a T4SS essential for virulence and intracellular multiplication . Here, virB expression analyses carried out using lacZ transcriptional fusions showed that virB promoter (PvirB) is temporally activated within J774 cells . Primer extension experiments revealed that virB transcription starts at 27 bp upstream of the first gene of the virB operon . Structural analyses showed that PvirB and regulatory sequences involved in intracellular regulation span 430 bp upstream of the transcription start site . A protein able to bind PvirB was isolated and identified . This protein, homologue to integration host factor (IHF), specifically interacts with PvirB and induces a DNA bending with an angle of 50.36 degrees . DNAse I footprinting experiments showed that IHF protects a 51 bp region that contains two overlapped IHF binding consensus motifs . VirB expression experiments carried out with PvirB-lacZ fusions showed that in B . abortus IHF participates in the regulation of PvirB activity during the intracellular and vegetative growth in different media . A mutant strain with a 20 bp IHF binding site replacement failed to turn on the virB operon during the initial stages of macrophage infection and displayed severe intracellular multiplication defects . These data indicate that IHF plays a key role during intracellular virB operon expression being required for the biogenesis of the endoplasmic reticulum-derived replicative vacuole. Mol Microbiol, 2004 Nov, 54(3), 715 - 30 Regulation of FlbD activity by flagellum assembly is accomplished through direct interaction with the trans-acting factor, FliX; Muir RE et al.; The temporal and spatial transcription of late flagellar genes in Caulobacter crescentus is regulated by the sigma54 transcriptional activator, FlbD . One requirement for FlbD activity is the assembly of a structure encoded by early, class II flagellar genes . In this report, we show that the trans-acting factor FliX predominantly functions as a negative regulator of FlbD activity in the absence of the class II-encoded flagellar structure . In contrast, a mutant FliX that bypasses the transcriptional requirement for early flagellar assembly is incapable of repressing FlbD in a class II flagellar mutant . Expression of this mutant allele, fliX1, does not alter the temporal pattern of FlbD-dependent transcription . Remarkably, this mutation confers the correct cell cycle timing of hook operon transcription in a strain that cannot assemble the flagellum, indicating that the progression of flagellar assembly is a minor influence on temporal gene expression . Using a two-hybrid assay, we present evidence that FliX regulates FlbD through a direct interaction, a novel mechanism for this class of sigma54 transcriptional activator . Furthermore, increasing the cellular levels of FliX results in an increase in the concentration of FlbD, and a corresponding increase in FlbD-activated transcription, suggesting that FliX and FlbD form a stable complex in Caulobacter . FliX and FlbD homologues are present in several polar-flagellated bacteria, indicating that these proteins constitute an evolutionarily conserved regulatory pair in organisms where flagellar biogenesis is likely to be under control of the cell division cycle. J Periodontal Res, 2004 Dec, 39(6), 432 - 41 The interleukin-10 knockout mouse is highly susceptible to Porphyromonas gingivalis-induced alveolar bone loss; Sasaki H et al.; OBJECTIVE: Interleukin-10 is an anti-inflammatory cytokine that reduces periapical bone loss, but its role in periodontal bone loss is unclear . In the present study, we tested the hypothesis that endogenous interleukin-10 is a potent suppressor of Porphyromonas gingivalis-induced alveolar bone loss in vivo . METHODS: Interleukin-10 knockout (-/-) and wild-type mice were inoculated intraorally with P . gingivalis . Non-infected animals served as negative controls . Alveolar bone loss, gingival cytokine levels, and gingival gene expression were assessed using morphometric analysis, enzyme-linked immunosorbent assay (ELISA), and semiquantitative reverse transcription polymerase chain reaction (RT-PCR), respectively . RESULTS: P . gingivalis-infected interleukin-10(-/-) mice exhibited severe alveolar bone loss compared to non-infected interleukin-10(-/-) and wild-type mice by day 42 . Surprisingly, bone resorptive cytokines interleukin-1alpha and tumor necrosis factor alpha (TNF-alpha) were not up-regulated in gingival tissues by P . gingivalis-infection . Although interleukin-1beta was marginally increased, blockade of both interleukin-1 isoforms or interleukin-1 receptor type I with neutralizing antisera failed to reduce alveolar bone loss in interleukin-10(-/-) mice, indicating the operation of an interleukin-1-independent mechanism . No strong correlations between bone loss and other cytokines was observed, although interferon gamma (IFNgamma), interleukin-6, interleukin-4, and prostaglandin E2 were modestly up-regulated in infected interleukin-10(-/-) mice . P . gingivalis infection reduced the expression of cell markers in gingival tissue on days 7 and 14 in both interleukin-10(-/-) and wild-type animals, suggestive of bacteria-induced cytotoxicity or apoptosis . This was followed by up-regulated expression of receptor activator of nuclear factor kappa B ligand (RANKL) and CD40 ligand (CD40L on days 28 and 70 in infected interleukin-10(-/-) mice only . CONCLUSION: The interleukin-10(-/-) mouse is highly susceptible to bone loss induced by the periodontal pathogen P . gingivalis, which is mediated via an interleukin-1-independent pathway . (c)Blackwell Munksgaard 2004 Biochemistry, 2004 Oct 26, 43(42), 13467 - 77 NiFe hydrogenase active site biosynthesis: identification of Hyp protein complexes in Ralstonia eutropha; Jones AK et al.; Biosynthesis of the NiFe hydrogenase active site is a complex process involving the action of the Hyp proteins: HypA-HypF . Here we investigate the mechanism of NiFe site biosynthesis in Ralstonia eutropha by examining the interactions between HypC, HypD, HypE, and HypF1 . Using an affinity purification procedure based on the Strep-tag II, we purified HypC and HypE from different genetic backgrounds as complexes with other hydrogenase-related proteins and characterized them using immunological analysis . Copurification of HypC and HoxH, the active site-containing subunit of the soluble hydrogenase in R . eutropha, from several different genetic backgrounds suggests that this complex forms early in the maturation process . With respect to the Hyp proteins, it is shown that HypE and HypF1 formed a stable complex both in vivo and in vitro . Furthermore, HypC and HypD functioned as a unit . Together, they were able to interact with HypE to form a range of complexes probably varying in stoichiometry . The HypC/HypD/HypE complexes did not involve HypF1 but appeared to be more stable when HypF1 was also present in the cells . We hypothesize that HypF1 is able to modify some component of the HypC/HypD/HypE complex . Since we have also seen that HypF1 and HypE form a complex, it is likely that HypF1 modifies HypE . On the basis of these results, we propose a complete catalytic cycle for HypE . First, it is modified by HypF1, and then it can form a complex with HypC/HypD . This activated HypE/HypC/HypD complex could then decompose by donating active site components to the immature hydrogenase and regenerate unmodified HypE. Mikrobiyol Bul, 2004 Jul, 38(3), 295 - 303 {Immunopathogenesis of Legionnaires' disease}; Alim A; Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen of alveolar macrophages, alveolar epithelial cells and, blood monocytes . Infection begins with the inhalation of the L.pneumophila . Once the bacteria reach to alveolar spaces, they come in contact with the alveolar macrophages, phagocyted and the macrophage takes the bacteria into a phagosome . Inside the cell, Legionella inhibits the lysosome-phagosome fusion, resulting in multiplication and lysis of the cell . The newly formed bacteria infect the other cells . In-vitro studies have shown that the presence of antibody and complement do not enhance intracellular killing . Cellular immunity is more important than humoral immunity for resolution of Legionella infections . In this review article, immunopathogenesis of Legionella infections have been discussed under the light of literature. Syst Appl Microbiol, 2004 Sep, 27(5), 573 - 80 Comparative sequence analysis and oligonucleotide probe design based on 23S rRNA genes of Alphaproteobacteria from North Sea bacterioplankton; Peplies J et al.; Almost complete 23S rRNA gene sequences were obtained from 11 Alphaproteobacteria isolated from marine surface water of the German Bight . Five of the strains belong to the "marine alpha" group, a phylogenetic cluster which encompasses members of the genus Roseobacter and closely related bacteria . Phylogenetic sequence analysis based on 52 published as well as unpublished complete 23S rDNA sequences from Alphaproteobacteria including the newly obtained was in general consistent with the 16S rRNA gene sequence-derived phylogeny . 16S and 23S rRNA based phylogenies both showed a distinct cluster for strains associated with the "marine alpha" group . The suitability of both markers for the design of oligonucleotide probes targeting selected groups of Alphaproteobacteria was systematically evaluated and compared in silico . Six clusters of sequences covering different phylogenetic levels as well as two strains were selected in a case study . To compensate for the quantitative difference in the two data sets, the 16S rRNA dataset was truncated to sequences with an equivalent in the 23S rRNA data set . Our results show, that the overall number of phylogenetically redundant probes available could be more than doubled by extending probe design to the 23S rRNA . For small clusters of high sequence similarity and single strains, up to 8 times more discriminating binding sites were provided by the 23S rRNA. Anasthesiol Intensivmed Notfallmed Schmerzther, 2004 Sep, 39 Suppl 1, S19 - 27 {How did the earth's oxygen atmosphere originate?}; Schafer G; The planet earth did not carry an oxygen atmosphere from the beginning . Though oxygen could arise from radiation mediated water splitting, these processes were not efficient enough to create a global gas atmosphere . Oxygen in the latter is a product of the photosynthetic activity of early green organisms . Only after biological mass-formation of oxygen the UV-protective ozone layer could develop, then enabeling life to move from water onto land . This took billions of years . The basics of the processes of biological oxygen liberation and utilization are described in the following as well as the importance of their steady state equilibrium . Also a hint is given to oxygen as a toxic compound though being a chemical prerequisite for aerobic life on earth. Shock, 2004 Nov, 22(5), 446 - 52 Butyrate suppresses hypoxia-inducible factor-1 activity in intestinal epithelial cells under hypoxic conditions; Miki K et al.; Interaction between the products of intestinal bacteria and the intestinal epithelial cells is a key event in understanding the biological, physiological, and pathological functions of the intestinal epithelium . Here, we examined the effect of butyrate, one of the major intestinal bacterial products, on hypoxia-inducible factor-1 (HIF-1) activity under hypoxic conditions in intestinal epithelial cells . HIF-1 activity was assessed by luciferase assay using cytoplasmic extracts of intestinal epithelial cells, Caco-2, and IEC-6 cells . These cells were transiently transfected with hypoxia response element (HRE)-luciferase reporter plasmids and cultured under hypoxic conditions in the presence or absence of sodium butyrate (NaB) . The effect of NaB on HRE DNA binding activity in Caco-2 cells under hypoxic conditions was assessed by electrophoretic mobility shift assay . Expression of a hypoxia-responsive gene encoding intestinal trefoil factor (ITF) in Caco-2 cells after NaB treatment was assessed using reverse-transcription PCR . The barrier function of Caco-2 cells under hypoxic conditions was also evaluated by transepithelial electrical resistance measurement . NaB suppressed up-regulation of HIF-1 transcriptional activity under hypoxic conditions in Caco-2 and IEC-6 cells . In parallel, NaB reduced HRE DNA binding activity under the same conditions . Furthermore, NaB down-regulated enhanced transcription of ITF gene . Addition of NaB under hypoxic conditions delayed recovery of transepithelial electrical resistance of the monolayers after hypoxia-reoxygenation treatment . These findings indicate that NaB suppresses HIF-1 transcriptional activity on hypoxia-responsive genes by reducing the HRE DNA binding activity under hypoxic conditions in intestinal epithelial cells. J Bacteriol, 2004 Nov, 186(21), 7302 - 11 The BfeR regulator mediates enterobactin-inducible expression of Bordetella enterobactin utilization genes; Anderson MT et al.; Utilization of the enterobactin siderophore by the respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica is dependent on the BfeA outer membrane receptor . This study determined that production of BfeA was increased significantly in iron-starved bacteria upon supplementation of cultures with enterobactin . A 1.01-kb open reading frame, designated bfeR, encoding a predicted positive transcriptional regulator of the AraC family was identified upstream and divergently oriented from bfeA . In iron-depleted cultures containing enterobactin, a Bordetella bfeR mutant exhibited markedly decreased BfeA receptor production compared to that of the wild-type strain . Additionally, B . pertussis and B . bronchiseptica bfeR mutants exhibited impaired growth with ferric enterobactin as the sole source of iron, demonstrating that effective enterobactin utilization is bfeR dependent . Transcriptional analysis using bfeA-lacZ reporter fusions in wild-type strains demonstrated that bfeA transcription was stimulated in iron-depleted conditions in the presence of enterobactin, compared to modest expression levels in cultures lacking enterobactin . In contrast, bfeA transcription in B . pertussis and B . bronchiseptica bfeR mutants was completely unresponsive to the enterobactin inducer . bfeA transcriptional analyses of a bfeA mutant demonstrated that induction by enterobactin did not require BfeA receptor-mediated uptake of the siderophore . These studies establish that bfeR encodes an enterobactin-dependent positive regulator of bfeA transcription in these Bordetella species. J Bacteriol, 2004 Nov, 186(21), 7243 - 53 The "intracellular" poly(3-hydroxybutyrate) (PHB) depolymerase of Rhodospirillum rubrum is a periplasm-located protein with specificity for native PHB and with structural similarity to extracellular PHB depolymerases; Handrick R et al.; Rhodospirillum rubrum possesses a putative intracellular poly(3-hydroxybutyrate) (PHB) depolymerase system consisting of a soluble PHB depolymerase, a heat-stable activator, and a 3-hydroxybutyrate dimer hydrolase (J . M . Merrick and M . Doudoroff, J . Bacteriol . 88:60-71, 1964) . In this study we reinvestigated the soluble R . rubrum PHB depolymerase (PhaZ1) . It turned out that PhaZ1 is a novel type of PHB depolymerase with unique properties . Purified PhaZ1 was specific for amorphous short-chain-length polyhydroxyalkanoates (PHA) such as native PHB, artificial PHB, and oligomer esters of (R)-3-hydroxybutyrate with 3 or more 3-hydroxybutyrate units . Atactic PHB, (S)-3-hydroxybutyrate oligomers, medium-chain-length PHA, and lipase substrates (triolein, tributyrin) were not hydrolyzed . The PHB depolymerase structural gene (phaZ1) was cloned . Its deduced amino acid sequence (37,704 Da) had no significant similarity to those of intracellular PHB depolymerases of Wautersia eutropha or of other PHB-accumulating bacteria . PhaZ1 was found to have strong amino acid homology with type-II catalytic domains of extracellular PHB depolymerases, and Ser(42), Asp(138), and His(178) were identified as catalytic-triad amino acids, with Ser(42) as the putative active site . Surprisingly, the first 23 amino acids of the PHB depolymerase previously assumed to be intracellular revealed features of classical signal peptides, and Edman sequencing of purified PhaZ1 confirmed the functionality of the predicted cleavage site . Extracellular PHB depolymerase activity was absent, and analysis of cell fractions unequivocally showed that PhaZ1 is a periplasm-located enzyme . The previously assumed intracellular activator/depolymerase system is unlikely to have a physiological function in PHB mobilization in vivo . A second gene, encoding the putative true intracellular PHB depolymerase (PhaZ2), was identified in the genome sequence of R . rubrum. Clin Microbiol Rev, 2004 Oct, 17(4), 942 - 64, table of contents Role of microglia in central nervous system infections; Rock RB et al.; The nature of microglia fascinated many prominent researchers in the 19th and early 20th centuries, and in a classic treatise in 1932, Pio del Rio-Hortega formulated a number of concepts regarding the function of these resident macrophages of the brain parenchyma that remain relevant to this day . However, a renaissance of interest in microglia occurred toward the end of the 20th century, fueled by the recognition of their role in neuropathogenesis of infectious agents, such as human immunodeficiency virus type 1, and by what appears to be their participation in other neurodegenerative and neuroinflammatory disorders . During the same period, insights into the physiological and pathological properties of microglia were gained from in vivo and in vitro studies of neurotropic viruses, bacteria, fungi, parasites, and prions, which are reviewed in this article . New concepts that have emerged from these studies include the importance of cytokines and chemokines produced by activated microglia in neurodegenerative and neuroprotective processes and the elegant but astonishingly complex interactions between microglia, astrocytes, lymphocytes, and neurons that underlie these processes . It is proposed that an enhanced understanding of microglia will yield improved therapies of central nervous system infections, since such therapies are, by and large, sorely needed. Genome Res, 2004 Oct, 14(10B), 2201 - 6 Generation of the Brucella melitensis ORFeome version 1.1; Dricot A et al.; The bacteria of the Brucella genus are responsible for a worldwide zoonosis called brucellosis . They belong to the alpha-proteobacteria group, as many other bacteria that live in close association with a eukaryotic host . Importantly, the Brucellae are mainly intracellular pathogens, and the molecular mechanisms of their virulence are still poorly understood . Using the complete genome sequence of Brucella melitensis, we generated a database of protein-coding open reading frames (ORFs) and constructed an ORFeome library of 3091 Gateway Entry clones, each containing a defined ORF . This first version of the Brucella ORFeome (v1.1) provides the coding sequences in a user-friendly format amenable to high-throughput functional genomic and proteomic experiments, as the ORFs are conveniently transferable from the Entry clones to various Expression vectors by recombinational cloning . The cloning of the Brucella ORFeome v1.1 should help to provide a better understanding of the molecular mechanisms of virulence, including the identification of bacterial protein-protein interactions, but also interactions between bacterial effectors and their host's targets. Chem Biol, 2004 Oct, 11(10), 1373 - 81 The biosynthesis of the thiazole phosphate moiety of thiamin: the sulfur transfer mediated by the sulfur carrier protein ThiS; Dorrestein PC et al.; Thiamin-pyrophosphate is an essential cofactor in all living systems . The biosynthesis of both the thiazole and the pyrimidine moieties of this cofactor involves new biosynthetic chemistry . Thiazole-phosphate synthase (ThiG) catalyses the formation of the thiazole moiety of thiamin-pyrophosphate from 1-deoxy-D-xylulose-5-phosphate (DXP), dehydroglycine and the sulfur carrier protein (ThiS), modified on its carboxy terminus as a thiocarboxylate (ThiS-thiocarboxylate) . Thiazole biosynthesis is initiated by the formation of a ThiG/DXP imine, which then tautomerizes to an amino-ketone . In this paper we study the sulfur transfer from ThiS-thiocarboxylate to this amino-ketone and trap a new thioenolate intermediate . Surprisingly, thiazole formation results in the replacement of the ThiS-thiocarboxylate sulfur with an oxygen from DXP and not from the buffer, as shown by electrospray ionization Fourier transform mass spectrometry (ESI-FTMS) using (18)O labeling of the 13C-, 15N-depleted protein . These observations further clarify the mechanism of the complex thiazole biosynthesis in bacteria. Mol Cell Probes, 2004 Dec, 18(6), 429 - 35 Differential detection of B . pertussis from B . parapertussis using a polymerase chain reaction (PCR) in presence of SYBR green1 and amplicon melting analysis; Poddar SK; Nucleic acid (DNA) from control stock strains of B . pertussis and B . parapertussis (B . pertussis strain # 9797 and B . parapertussis strain # 15234 from ATCC) was amplified by polymerase chain reaction (PCR) targeting pertussis toxin (PT) promotor region, in presence of SYBR green1 a dye that fluoresces on binding specifically to double stranded DNA; and fluorescent melting profile of amplicon (amplified DNA) was studied . Amplicon of B . pertussis and B . parapertussis generated distinctly different melting bands with melting temperature (Tm) at 89.8 and 91.7 degrees C, respectively . Melting profile and Tm of each randomly selected isolates of B . pertussis and B . parapertussis was identical to that of respective control strains . Distinct difference in Tm between B . pertussis and B . parapertussis specific amplicons allowed differential detection of the two Bordetella species based on a single PCR product . The amplified product of serial diluted control stock of bacteria was analyzed by both agarose gel electrophoresis and melting profile analysis . The analytical sensitivity of detection (1-10 CFU equivalent in the tested volume) by melting profile and Tm analysis was in agreement with that obtained by agarose gel analysis. Annu Rev Microbiol, 2004, 58, 555 - 85 Biophysical analyses of designed and selected mutants of protocatechuate 3,4-dioxygenase1; Brown CK et al.; The catechol dioxygenases allow a wide variety of bacteria to use aromatic compounds as carbon sources by catalyzing the key ring-opening step . These enzymes use specifically either catechol or protocatechuate (2,3-dihydroxybenozate) as their substrates; they use a bare metal ion as the sole cofactor . To learn how this family of metalloenzymes functions, a structural analysis of designed and selected mutants of these enzymes has been undertaken . Here we review the results of this analysis on the nonheme ferric iron intradiol dioxygenase protocatechuate 3,4-dioxygenase. Annu Rev Microbiol, 2004, 58, 521 - 54 The cellulosomes: multienzyme machines for degradation of plant cell wall polysaccharides; Bayer EA et al.; The discrete multicomponent, multienzyme cellulosome complex of anaerobic cellulolytic bacteria provides enhanced synergistic activity among the different resident enzymes to efficiently hydrolyze intractable cellulosic and hemicellulosic substrates of the plant cell wall . A pivotal noncatalytic subunit called scaffoldin secures the various enzymatic subunits into the complex via the cohesin-dockerin interaction . The specificity characteristics and tenacious binding between the scaffoldin-based cohesin modules and the enzyme-borne dockerin domains dictate the supramolecular architecture of the cellulosome . The diversity in cellulosome architecture among the known cellulosome-producing bacteria is manifest in the arrangement of their genes in either multiple-scaffoldin or enzyme-linked clusters on the genome . The recently described three-dimensional crystal structure of the cohesin-dockerin heterodimer sheds light on the critical amino acids that contribute to this high-affinity protein-protein interaction . In addition, new information regarding the regulation of cellulosome-related genes, budding genetic tools, and emerging genomics of cellulosome-producing bacteria promises new insight into the assembly and consequences of the multienzyme complex. Annu Rev Microbiol, 2004, 58, 453 - 88 Biosynthesis of nonribosomal peptides1; Finking R et al.; Bacteria and fungi use large multifunctional enzymes, the so-called nonribosomal peptide synthetases (NRPSs), to produce peptides of broad structural and biological activity . Biochemical studies have contributed substantially to the understanding of the key principles of these modular enzymes that can draw on a much larger number of catalytic tools for the incorporation of unusual features compared with the ribosomal system . Several crystal structures of NRPS-domains have yielded deep insight into the catalytic mechanisms involved and have led to a better prediction of the products assembled and to the construction of hybrid enzymes . In addition to the structure-function relationship of the core- and tailoring-domains of NRPSs, which is the main focus of this review, different biosynthetic strategies and essential enzymes for posttranslational modification and editing are discussed. Photochem Photobiol . 2004 Jul 1; {Epub ahead of print} Effect of Carotenoids and Monogalactosyl Diglyceride on Bacteriochlorophyll c Aggregates in Aqueous Buffer: Implications for the Self-Assembly of Chlorosomes; Klinger P et al.; Aggregation of bacteriochlorophyll (BChl) c from chlorosomes, the main light harvesting complex of green bacteria, has been studied in aqueous buffer . Unlike other chlorophyll-like molecules, BChl c is rather soluble in aqueous buffer, forming dimers . When BChl c is mixed with carotenoid (Car), the BChl c Qy transition is further red-shifted, in respect to that of monomers and dimers . The results suggest that Car are incorporated in the aggregates and induce further aggregation of BChl c . The red shift of the BChl c Qy band is proportional to the Car concentration . In contrast, the mixture of bacteriochlorophyllide (BChlide) c, which lacks the non-polar esterifying alcohol, does not form aggregates with Car in aqueous buffer or non-polar solvents . Instead, the position of the BChlide c Qy transition remains unshifted in respect to that of the monomeric molecule, and Car precipitates with the course of time in aqueous buffer . Similar effects on both BChl c and BChlide c are also observed when monogalactosyl diglyceride (MGDG), which forms the monolayer envelope of chlorosomes, is used instead of (or together with) Car . The results show that the hydrophobic interactions of the BChl c esterifying alcohols with themselves and the non-polar carbon skeleton of Car, or the fatty acid tails of MGDG, are essential driving forces for BChl aggregation in chlorosomes. Environ Sci Technol, 2004 Sep 15, 38(18), 4745 - 52 pH dependence of carbon tetrachloride reductive dechlorination by magnetite; Danielsen KM et al.; Magnetite is precipitated by dissimilatory iron-reducing bacteria or forms through corrosion of zero-valent iron (ZVI) in permeable reactive barriers . Reduction of carbon tetrachloride (CCl4) by synthetic magnetite was examined in batch reactors to evaluate the pH dependence of the reaction rates and product distributions . This work presents the first data where magnetite promotes CCl4 dechlorination independent of added sorbed Fe(II) or coexisting minerals that maintained Fe2+ above the magnetite solubility limit . In this system, reaction rate constants increase with increasing pH values between 6 and 10 . The pH dependence is explained by acid-base equilibrium between two surface sites, where the more deprotonated exhibits greater dechlorination reactivity . The distribution of reaction products was also found to depend on pH . The primary reaction product is carbon monoxide (CO) followed by chloroform (CHCl3) . CHCl3 production is at a minimum at pH 6 but increases through pH 10 . Formation rate constants for both products increase with increasing pH, but the values for CHCl3 increase at a much faster rate . A hypothesis is proposed that relates the CHCl3 rate enhancement to the reduced capacity of deprotonated surface sites to stabilize the trichlorocarbanion transition-state complex . These data form a basis to assess the natural attenuation capacity of magnetite formed under iron reducing conditions . Application of this information to permeable barrier technology suggests that, in the long term, oxidation of ZVI to magnetite may be accompanied by a shift toward more benign reaction products as well as a 2 order of magnitude decrease in reaction rate constants. J Gen Appl Microbiol, 2004 Jun, 50(3), 129 - 35 Phylogenetic analyses of the nitrogen-fixing genus Derxia; Xie CH et al.; Phylogenetic analyses of the 16S rRNA gene sequence indicate that the genus Derxia forms a distinct lineage in the beta-Proteobacteria . On the NJ tree Derxia has a low bootstrap value (30.9%) with Alcaligeneceae, and on the ML tree it shows an independent cluster separated from other families . Moreover, there is below 93.4% 16S rDNA sequence similarity between genus Derxia and the genera of the beta-Proteobacteria . These facts reveal that Derxia is not grouped with any known family of beta-Proteobacteria and should be placed as a separate genus of beta-Proteobacteria . The data on high G+C content (71 mol%), the cellular fatty acid composition, and the physiological characteristics of facultative hydrogen autotrophy and nitrogen fixation are unique for Derxia . The nifH gene sequence was found in this genus and phylogenetically compared among nitrogen-fixing bacteria to indicate that Derxia is clustered with the diazotrophs of beta-Proteobacteria. Neuroreport, 2004 Sep 15, 15(13), 2039 - 43 Imaging of gene expression during long-term potentiation; Behnisch T et al.; Long term potentiation (LTP) at hippocampal Schaffer collateral-CA1 synapses involves an early and a late phase, where only the latter is sensitive to protein synthesis inhibitors . Here we characterized the dynamics of protein synthesis associated with the induction of L-LTP using a transgenic mouse model in which a cAMP responsive element (CRE)-regulated promoter drives production of an enhanced yellow fluorescent protein (eYFP) . We found that eYFP fluorescence increased after less than 30 min following L-LTP induction . Application of transcription and translation suppressors and the NMDA receptor antagonist D-AP5 inhibited the L-LTP and prevented the rise in eYFP levels . The early-phase of LTP was not affected by inhibiting protein synthesis. Science . 2004 Oct 15;306(5695):455. arrA is a reliable marker for As(V) respiration; Malasarn D et al.; Arsenate {As(V)}-respiring bacteria affect the speciation and mobilization of arsenic in the environment . This can lead to arsenic contamination of drinking water supplies and deleterious consequences for human health . Using molecular genetics, we show that the functional gene for As(V) respiration, arrA, is highly conserved; that it is required for As(V) reduction to arsenite when arsenic is sorbed onto iron minerals; and that it can be used to identify the presence and activity of As(V)-respiring bacteria in arsenic-contaminated iron-rich sediments . The expression of arrA thus can be used to monitor sites in which As(V)-respiring bacteria may be controlling arsenic geochemistry. Water Sci Technol, 2004, 50(4), 257 - 65 The development of experimental procedures for the evaluation of additives to attenuate manure odour, and the impact of these additives on workers, animals and the environment; Masse I et al.; The objective of this project was to develop a laboratory research protocol to evaluate the effect of additives on manure odour and physico-chemical characteristics, and establish conditions that are representative of those found in farm storage structures (temperature, solids content, pH, ventilation above the manure surface, storage period) . The results suggested that system configuration might have an impact on additive effect . An open system should be used when it is recommended that additives be applied in the animal diet or the gutters . Additionally, the surface/depth ratio of the gutter should be respected, since it will impact on the relative importance of the aerobic layer and on ammonia volatilization . On the other hand, a closed system should be used when the additive is applied to the manure storage tank, especially if the tank has a cover . Odour analysis still requires fundamental research to establish reliable procedures and protocols, especially in the area sample collection and dilution levels required to decrease H2S concentration to safe levels for the panellists . Odour analysis should also be conducted in triplicate, because of the possible large experimental error due to dilution, the human factors, and also instrumental error. Br Poult Sci, 2004 Aug, 45(4), 471 - 5 Hatching success and embryonic mortality on lesser rhea (Pterocnemia pennata) farms in northern Patagonia, Argentina; Reissig EC et al.; 1 . The hatching success and embryonic mortality of 724 lesser rhea eggs from 12 farms in northern Patagonia, Argentina were monitored during the 2000 breeding season . 2 . Average infertility was 24.5%, embryonic mortality was 33.5%, hatchability of fertile eggs was 66.5% and the neonatal mortality was 57.0% . There were large variations between different farms . 3 . Bacterial contamination was high and egg hatchability was also affected by the occurrence of oedematous and malformed embryos . 4 . No significant relationships were found between embryonic loss, duration of egg storage, initial egg weight or weight loss of hatched and failed eggs . 5 . The low productivity on lesser rhea farms is caused not only by embryonic mortality but also by a high incidence of infertile eggs and neonatal mortality . 6 . Bacterial infection may not have been the most important cause of incubation failure, indicating that nutritional deficiencies and inbreeding may play an important role in the productivity of these farms. Br Poult Sci, 2004 Aug, 45(4), 453 - 62 Individual production differences do not explain cannibalistic behaviour in laying hens; Yngvesson J et al.; 1 . Sixty-three laying hens were collected from 5 commercial farms experiencing outbreaks of cannibalism . It is known that cannibalistic behaviour varies strongly between hybrids and between individuals of the same hybrid . The aim of this study was to test specific hypotheses about the causation of cannibalism by comparing matched individuals varying in their cannibalistic behaviour . 2 . To investigate feed efficiency, egg production and feed consumption, data were collected over 30 d . Nutrient deficiencies have been associated with outbreaks of cannibalism and we hypothesised that cannibals are more feed efficient than their flock mates and, therefore, more susceptible to transient nutrient deficiencies or imbalances that trigger cannibalistic behaviour . 3 . Video recordings were made of victims of cloacal cannibalism and their matched controls during oviposition . Victims were expected to have longer oviposition durations and a higher incidence of short-term cloacal prolapses than controls, because the starting point for cannibalism is often suggested to be pecking at the red mucosa of the victim during, or just after, oviposition . 4 . Oviduct swab samples were taken from surviving victims of cloacal cannibalism and their matched controls 4 weeks after the cannibalistic attack . If peck wounds provide a route for subsequent bacterial invasion of the reproductive tract, victims would be expected to have greater bacterial colonisation of the oviduct . 5 . Mortality within each matched trio was recorded over a 23-month period after the cannibalistic attack . Victims that survived a cannibalistic attack were expected to have reduced longevity compared to controls and cannibals . This could occur if they developed secondary infections as a result of the cannibalistic attack, or if they had poorer health even before the attack . 6 . No significant differences were found between cannibals, victims and controls in feed efficiency or any of the production variables measured . Victims of cloacal cannibalism did not expose their cloacal mucosa more, or for longer, than matched control hens during oviposition, and had no more bacteria in their oviduct . Victims tended to die sooner than their matched cannibals or cannibals during a 23-month period after a cannibalistic attack. World J Gastroenterol, 2004 Nov 15, 10(22), 3289 - 91 Pathogenicty and immune prophylaxis of cag pathogenicity island gene knockout homogenic mutants; Lin HJ et al.; AIM: To clarify the role of cag pathogenicity island (cagPAI) of Helicobacter pylori (H pylori ) in the pathogenicity and immune prophylaxis of H pylori infection . METHODS: Three pairs of H pylori including 3 strains of cagPAI positive wildtype bacteria and their cagPAI knockout homogenic mutants were utilized . H pylori binding to the gastric epithelial cells was analyzed by flow cytometry assays . Apoptosis of gastric epithelial cells induced by H pylori was determined by ELISA assay . Prophylaxis effect of the wildtype and mutant strains was compared by immunization with the sonicate of the bacteria into mice model . RESULTS: No difference was found in the apoptasis between cagPAI positive and knockout H pylori strains in respective of the ability in the binding to gastric epithelial cells as well as the induction of apoptosis . Both types of the bacteria were able to protect the mice from the infection of H pylori after immunization, with no difference between them regarding to the protection rate as well as the stimulation of the proliferation of splenocytes of the mice . CONCLUSION: The role of cagPAI in the pathogenicity and prophylaxis of H pylori infection remains to be cleared. J Gen Virol, 2004 Nov, 85(Pt 11), 3421 - 30 Maintenance of coat protein N-terminal net charge and not primary sequence is essential for zucchini yellow mosaic virus systemic infectivity; Kimalov B et al.; Zucchini yellow mosaic virus (ZYMV) surface exposed coat protein (CP) N-terminal domain (Nt) is 43 aa long and contains an equal number of positively and negatively charged amino acid residues (CP-Nt net charge = 0) . A ZYMV-AGII truncation mutant lacking the first 20 aa of its CP-Nt (AGII-CP Delta 20; CP-Nt net charge = +2) was found to be systemically non-infectious even though AGII mutants harbouring larger CP-Nt deletions were previously demonstrated to be fully infectious . Nevertheless, AGII-CP Delta 20 infectivity was restored by fusion to its CP-Nt two Asp residues or a negatively charged Myc peptide, both predicted to neutralize CP-Nt net positive charge . To evaluate further the significance of CP-Nt net charge for AGII infectivity, a series of CP-Nt net charge mutants was generated and analysed for systemic infectivity of squash plants . AGII-CP(KKK) harbouring a CP-Nt amino fusion of three Lys residues (CP-Nt net charge = +3) was not systemically infectious . Addition of up to four Asp residues to CP-Nt did not abolish virus infectivity, although certain mutants were genetically unstable and had delayed infectivity . Addition of five negatively charged residues abolished infectivity (AGII-CP(DDDDD); CP-Nt net charge = -5) even though a recombinant CP(DDDDD) could assemble into potyviral-like particle in bacteria . Neutralization of CP-Nt net charge by fusing Asp or Lys residues recovered infectivity of AGII-CP(KKK) and AGII-CP(DDDDD) . GFP-tagging of these mutants has demonstrated that both viruses have defective cell-to-cell movement . Together, these findings suggest that maintenance of CP-Nt net charge and not primary sequence is essential for ZYMV infectivity. J Invest Dermatol, 2004 Nov, 123(5), 888 - 91 Non-invasive visualization of epidermal responses to injury using a fluorescent transgenic reporter; Pan YA et al.; We describe transgenic mice in which expression of a reporter, the yellow fluorescent protein (YFP), is locally activated in epidermal cells at sites of injury . YFP is detectable a day after injury; its levels peak within 3 d, and then decline over the subsequent week . Expression is also activated by a chemical irritant, and is suppressed by topical administration of hydrocortisone . These mice permit non-invasive time-lapse monitoring of responses to injury in vivo . They can be used to detect epidermal activation and to test agents that may provoke or attenuate epidermal responses. Crit Rev Immunol, 2004, 24(3), 179 - 92 From pattern recognition receptor to regulator of homeostasis: the double-faced macrophage mannose receptor; Allavena P et al.; The mannose receptor (MR) is an endocytic and phagocytic receptor belonging to the C-type lectin superfamily . A number of functions have been ascribed to this receptor, which is involved in innate and adaptive immune responses . The MR binds carbohydrate moieties on several pathogens, such as bacteria, fungi, parasites, and viruses, and, therefore, is considered a pattern recognition receptor (PRR) . In addition, MR binds endogenous molecules and was originally described as a membrane-associated component binding lysosomal glycosidases in alveolar macrophages . Since its identification more than 25 years ago, many other endogenous ligands were described, including hormones, enzymes, cell membranes, extracellular matrix components, and normal as well as tumoral mucins . The MR is preferentially expressed on immune cells of myeloid lineage, especially subsets of dendritic cells (DC) and tissue macrophages . In addition to immune cells, specialized endothelial cells are also MR-positive . Increasing evidence shows that the MR is involved in the silent clearance of inflammatory molecules . In this review, we discuss current knowledge about the receptor and show that endogenous ligands activate an anti-inflammatory and tolerogenic program in DC and macrophages, thus reinforcing the hypothesis that the MR has an important role in the maintenance of homeostasis. Expert Rev Anti Infect Ther, 2004 Feb, 2(1), 43 - 9 Nitazoxanide: a new broad spectrum antiparasitic agent; White CA Jr; Nitazoxanide (Alinia, Romark Laboratories) was synthesized based on the structure of niclosamide . In vitro studies have demonstrated activity against a broad range of parasites as well as some bacteria . Three controlled trials demonstrated efficacy in cryptosporidiosis, however, the efficacy in advanced AIDS patients (CD4 cell counts = 50) at approved doses was limited . Trials have also demonstrated efficacy comparable to metronidazole (Flagyl, GD Searle and Co.) in giardiasis with fewer side effects . Nitazoxanide is also effective versus intestinal helminths and tapeworms as well as in chronic fascioliasis . Side effects in clinical trials have been similar to placebo . Nitazoxanide is the first agent proven to be effective in cryptosporidiosis . It has also proven efficacy in giardiasis . Nitazoxanide is efficacious again intestinal helminths . Additional indications may be developed in the future. Expert Rev Anti Infect Ther, 2003 Dec, 1(4), 571 - 7 Chemotherapy for lymphatic filariasis: progress but not perfection; Melrose WD; Currently, only three drugs are used to control and treat the mosquito-borne parasitic disease lymphatic filariasis: diethylcarbamazine, ivermectin (Mectizan) and albendazole (Zentel) . All interrupt transmission by eliminating microfilaria, the parasite stage that is responsible for transmission between hosts, but do not reliably kill the adult worms that are responsible for much of the pathology seen in the disease . There is an urgent need to develop drugs that will reliably kill adult worms and several compounds are under-going in vitro and animal testing . An alternative strategy - that of targeting symbiont bacteria within the parasite - has also shown promising results. Expert Rev Anti Infect Ther, 2003 Aug, 1(2), 319 - 35 Natural products and synthetic compounds as immunomodulators; Kayser O et al.; Research on immunomodulation by natural products or synthetic derivatives is of key interest for anti-infective therapy for a number of reasons . Many plant remedies well-known in traditional medicine or refined natural products in clinical use exert their anti-infective effects not only (if at all) by directly affecting the pathogen . At least part of their effect is indirect, by stimulating natural and adaptive defense mechanisms of the host . These findings have now given many empirical therapies a rationale, scientific basis and thereby a means for 'intelligent' improvement . In discovering the molecular mechanisms by which known remedies exert their effects, chosen elements further down the 'chain of command' might be synthesized and applied directly for more rapid and selective cure, omitting unwanted side effects . The direct use of recombinant cytokines, often in combination with antibiotics, is one consequence of this rationale. Anal Bioanal Chem . 2004 Oct 7; {Epub ahead of print} Efficient and rapid method for extraction of intact phospholipids from sediments combined with molecular structure elucidation using LC-ESI-MS-MS analysis; Zink KG et al.; This paper presents the application of an efficient method for extraction and fractionation of intact phospholipids (PLs) from complex sediment matrices and elucidation of their molecular structure by normal-phase HPLC-ESI-MS-MS . Flow-blending extraction was tested with different solvent mixtures and the best recovery of all PLs classes from the sediment matrix was achieved by using methanol-dichloromethane-buffer, 2:1:0.8 . The applied LC-ESI-MS system has linearity of R(2)=0.98 and a detection limit of 0.5 ng/PL, sufficient for reliable identification of complex mixtures of PLs . MS-MS analyses using a triple-quadrupole mass spectrometer enables detection of individual PL side-chain composition and, hence, characterization of the living organisms contributing to the sedimentary organic material . Parallel GC-MS analysis of the hydrolysed phospholipid fatty acids supports the characterized fatty acid patterns determined from intact PLs . The PL inventory of different investigated lacustrine surface sediments shows predominantly high abundance of phosphatidylglycerols and phosphatidylethanolamines and phosphatidyl-mono- and dimethyl-ethanolamines with fatty acyl side-chains typically known from bacteria . In a sample from Lake Baikal intense signals of bacterial 14:0-acyl-PGs were also identified, for the first time in sediments as far as we are aware. Genome Res, 2004 Nov, 14(11), 2279 - 86 Epub 2004 Oct 12. Codon usage bias from tRNA's point of view: redundancy, specialization, and efficient decoding for translation optimization; Rocha EP; The selection-mutation-drift theory of codon usage plays a major role in the theory of molecular evolution by explaining the co-evolution of codon usage bias and tRNA content in the framework of translation optimization . Because most studies have focused only on codon usage, we analyzed the tRNA gene pool of 102 bacterial species . We show that as minimal generation times get shorter, the genomes contain more tRNA genes, but fewer anticodon species . Surprisingly, despite the wide G+C variation of bacterial genomes these anticodons are the same in most genomes . This suggests an optimization of the translation machinery to use a small subset of optimal codons and anticodons in fast-growing bacteria and in highly expressed genes . As a result, the overrepresented codons in highly expressed genes tend to be the same in very different genomes to match the same most-frequent anticodons . This is particularly important in fast-growing bacteria, which have higher codon usage bias in these genes . Three models were tested to understand the choice of codons recognized by the same anticodons, all providing significant fit, but under different classes of genes and genomes . Thus, co-evolution of tRNA gene composition and codon usage bias in genomes seen from tRNA's point of view agrees with the selection-mutation-drift theory . However, it suggests a much more universal trend in the evolution of anticodon and codon choice than previously thought . It also provides new evidence that a selective force for the optimization of the translation machinery is the maximization of growth. J Am Chem Soc, 2004 Oct 20, 126(41), 13424 - 31 Catalytic nanomotors: autonomous movement of striped nanorods; Paxton WF et al.; Rod-shaped particles, 370 nm in diameter and consisting of 1 microm long Pt and Au segments, move autonomously in aqueous hydrogen peroxide solutions by catalyzing the formation of oxygen at the Pt end . In 2-3% hydrogen peroxide solution, these rods move predominantly along their axis in the direction of the Pt end at speeds of up to 10 body lengths per second . The dimensions of the rods and their speeds are similar to those of multiflagellar bacteria . The force along the rod axis, which is on the order of 10(-14) N, is generated by the oxygen concentration gradient, which in turn produces an interfacial tension force that balances the drag force at steady state . By solving the convection-diffusion equation in the frame of the moving rod, it was found that the interfacial tension force scales approximately as SR(2)gamma/muDL, where S is the area-normalized oxygen evolution rate, gamma is the liquid-vapor interfacial tension, R is the rod radius, mu is the viscosity, D is the diffusion coefficient of oxygen, and L is the length of the rod . Experiments in ethanol-water solutions confirmed that the velocity depends linearly with the product Sgamma, and scaling experiments showed a strong dependence of the velocity on R and L . The direction of motion implies that the gold surface is hydrophobic under the conditions of the experiment . Tapping-mode AFM images of rods in air-saturated water show soft features that are not apparent in images acquired in air . These features are postulated to be nanobubbles, which if present in hydrogen peroxide solutions, would account for the observed direction of motion. Mol Biochem Parasitol, 2004 Aug, 136(2), 249 - 55 RNA interference of Trypanosoma brucei topoisomerase IB: both subunits are essential; Bakshi RP et al.; Type IB topoisomerases are enzymes essential for the orderly synthesis of nucleic acids and are the molecular target for antitumor camptothecins . In dozens of organisms, including eukaryotes, bacteria, and viruses, this enzyme is monomeric . However, we previously found that topoisomerase IB in trypanosomes is a heteromultimer, comprised of two distinct subunits encoded by separate genes . A large 90 kDa subunit contains the DNA binding domain and a small 36 kDa subunit contains the catalytic domain . In this study we use RNA interference to silence each of the subunits separately . For each subunit, tetracycline-induced expression of double-stranded RNA results in drastic reduction of cognate mRNA and protein . For the large subunit, nucleic acid biosynthesis (as monitored by the incorporation of radiolabeled precursors into DNA and RNA) is halved by 39 h, and cell growth halts by 72 h, after induction . The steady state level of both nuclear and mitochondrial mRNAs is reduced . Virtually identical results are obtained by silencing the small subunit . Interestingly, although interference is specific at the level of mRNA, silencing of one subunit leads to a profound reduction in the level of protein for both subunits, suggesting that survival, or perhaps synthesis, of each subunit depends upon the presence of the other . These findings underscore the essential nature of type IB topoisomerase activity in Trypanosoma brucei and its suitability as a target for rational drug design. Kansenshogaku Zasshi, 2004 Aug, 78(8), 710 - 6 {Distribution of Legionella spp . in hot spring baths in Japan}; Furuhata K et al.; We investigated the inhabitation of Legionella spp . in hot spring water in various regions in Japan . The following results were obtained . 1) Of 710 hot spring water samples nationwide, Legionella spp . was isolated from 204 samples (28.7%), covering all 47 prefectures . By region, the isolation rate was the highest at 31.0% in the Chugoku district, while the isolation rates in Hokkaido, Kinki, and Kyushu were low, ranging from 25.0 to 26.2% . The rate in Tohoku, Kanto, Chubu, and Shikoku districts was 28.6-30.7% . Regarding the isolation rate by pH of hot spring water, the isolation rate was 4.9% at pH 3 or lower, but 34.8% at pH 3.1-7.5 . When pH was 7.6 or higher, the isolation rate was 24.8% . 2) Most frequently, the number of bacteria detected was below 10(2) CFU/100ml (98 samples, 48.0%) . The count was between 10(2) and 10(3) CFU/100ml in 71 samples (34.8%), and between 10(3) and 10(4) CFU/100ml in 29 samples (14.2%) . In 6 samples (2.9%), the count was higher than 10(4) CFU/100 ml . 3) Among the isolates identified, L . pneumophila was the predominant species, and particularly, serogroups 1 and 5 were frequently isolated . The above findings clarified that although the number of the bacteria is low, Legionella spp . inhabits hot spring water throughout Japan. Spectrochim Acta A Mol Biomol Spectrosc, 2004 Nov, 60(13), 3079 - 85 Mass, IR, electronic and EPR spectral studies on transition metal complexes with a new tetradentate 12-membered new macrocyclic ligand; Chandra S et al.; Complexes of Cr(III), Co(II), Ni(II) and Cu(II) containing a novel macrocyclic tetradentate nitrogen donor (N4) ligand prepared via reaction of 2,3-hexanedione and ethylenediamine has been prepared and characterized . The newly synthesized ligand (L) and its complexes have been characterized on the basis of elemental analysis, molar conductance, magnetic moment susceptibility, EI-Mass, IR, Electronic and EPR spectral studies . The complexes are of high-spin type and four coordinated tetrahedral, five coordinated square pyramidal and six coordinated octahedral/tetragonal geometries . The ligand (L) and its soluble transition metal complexes have also been screened against different bacteria and plant pathogenic fungi in vitro. Protein Expr Purif, 2004 Nov, 38(1), 84 - 98 Purification of the recombinant human serotonin N-acetyltransferase (EC 2.3.1.87): further characterization of and comparison with AANAT from other species; Ferry G et al.; Melatonin is synthesized by a series of enzymes, the penultimate one, serotonin N-acetyltransferase, catalyzing the limiting reaction . In the present study, we compared the recombinant serotonin N-acetyltransferases from rat, ovine, and human . The human protein is particularly difficult to purify because it interacts strongly with a putative chaperone protein from bacteria whereas the rat and sheep enzymes, which interact less strongly with this protein, have been purified close to homogeneity . We identified the contaminating protein as GroEL, the bacterial equivalent of Hsp60 . We present numerous catalytic activities (substrate and cosubstrate specificities as well as inhibitor specificities) measured on the three species enzymes from which we deduced that the presence of the chaperone might partly explain the differences between the various species enzyme characteristics, beside the inter-species ones resulting from sequence differences . Despite several trials reported in the literature, a purification to homogeneity of the human (recombinant) enzyme has never been described . We present a new purification method, by using an original denaturation/renaturation process in which the enzyme is immobilized on an affinity chromatography column . The enzyme is then eluted in an active and pure form (i.e., absence of chaperone) . The up-scaled system permitted us to perform the necessary experiments for the measurement of more accurate affinities of human serotonin N-acetyltransferase towards its main natural substrates, showing that only the activity of the enzyme towards phenylethylamine was modified. Free Radic Biol Med, 2004 Nov 15, 37(10), 1564 - 77 Reevaluating gel-forming mucins' roles in cystic fibrosis lung disease; Perez-Vilar J et al.; The existence of mucus plugs, containing mucins, bacteria, and neutrophils, blocking the lower airways in the lung of cystic fibrosis (CF) patients has raised the possibility that production of "abnormal" mucins is a critical characteristic of this disease . The molecular nature, if any, of this abnormality is unknown . Recent studies suggest that CF lung disease progression is characterized by an early phase in which airway surface liquid (ASL) increased dehydration is accompanied by altered pH and levels of reduced glutathione (GSH) . In a later phase, bacterial infection and neutrophil invasion lead to increased ASL of concentrations myeloperoxidase and hypochlorous acid (HOCl) . Independent studies indicate that gel-forming mucins, the key components of airway mucus, form disulfide-linked polymers through a pH-dependent, likely self-catalyzed mechanism . In this article, we present the hypothesis that increased mucus concentration (dehydration) and altered pH, and levels of GSH, myeloperoxidase, and/or HOCl result in the extracellular formation of additional interchain bonds among airway mucins . These novel interactions would create an atypical mucin network with abnormal viscoelastic and adhesive properties. J Mol Biol, 2004 Oct 29, 343(4), 833 - 49 Mechanistic insights from the structures of HincII bound to cognate DNA cleaved from addition of Mg2+ and Mn2+; Etzkorn C et al.; The three-dimensional X-ray crystal structures of HincII bound to cognate DNA containing GTCGAC and Mn(2+) or Mg(2+), at 2.50A and 2.95A resolution, respectively, are presented . In both structures, the DNA is found cleaved, and the positions of the active-site groups, cleaved phosphate group, and 3' oxygen atom of the leaving group are in very similar positions . Two highly occupied Mn(2+) positions are found in each active site of the four crystallographically independent subunit copies in the HincII/DNA/Mn(2+) structure . The manganese ion closest to the previously identified single Ca(2+) position of HincII is shifted 1.7A and has lost direct ligation to the active-site aspartate residue, Asp127 . A Mn(2+)-ligated water molecule in a position analogous to that seen in the HincII/DNA/Ca(2+) structure, and proposed to be the attacking nucleophile, is beyond hydrogen bonding distance from the active-site lysine residue, Lys129, but remains within hydrogen bonding distance from the proRp oxygen atom of the phosphate group 3' to the scissile phosphate group . In addition, the position of the cleaved phosphate group is on the opposite side of the axis connecting the two metal ions relative to that found in the BamHI/product DNA/Mn(2+) structure . Mechanistic implications are discussed, and a model for the two-metal-ion mechanism of DNA cleavage by HincII is proposed. Cell Motil Cytoskeleton, 2004 Dec, 59(4), 215 - 26 Dynamics of endocytic traffic of Entamoeba histolytica revealed by confocal microscopy and flow cytometry; Meza I et al.; Entamoeba histolytica, the protozoan parasite of humans, manifests constitutive endocytosis to obtain nutrients and, when induced to express invasive behavior, as a means of ingesting and processing host cells and tissue debris . E . histolytica trophozoites were grown in liquid axenic medium that contained fluorescently labeled fluid-phase markers, so that the kinetics of uptake, the transit of loaded endosomes through the cytoplasm, and the time of release of the markers could be monitored by flow cytometry . Confocal microscopy of live trophozoites revealed uptake of fluid by avid macropinocytosis and the occurrence of fusion between young and older endosomes, as well as between pinosomes and phagosomes containing bacteria . Endosomes were rapidly acidified, then gradually neutralized; finally, indigestible material was released . Transit of endosomes containing fluid-phase markers required about 2 h . Uptake and release of fluid-phase markers were impaired by drugs that inhibited actin dynamics and actin-myosin interaction; uptake was also impaired by inhibition of PI 3-kinase . A striking feature of the trophozoites was the great heterogeneity of their endocytic behavior . 2004 Wiley-Liss, Inc. Inflamm Bowel Dis, 2004 Jul, 10(4), 462 - 78 Mechanisms of natural tolerance in the intestine: implications for inflammatory bowel disease; Jump RL et al.; Tolerance, the regulated inability to respond to a specific immunologic stimulant, is a physiological event important to normal immune function . Just as loss of tolerance to self-proteins results in autoimmune diseases, we assert that loss of tolerance to commensal flora in the intestinal lumen leads to inflammatory bowel disease (IBD) . Mechanisms through which the mucosal immune system establishes and remains hyporesponsive toward the presence of food proteins and commensal flora, which we define as natural tolerance, are discussed . In addition to the contributions by commensal flora, the innate host defense and the adaptive immune systems promote natural tolerance to sustain normal mucosal homeostasis . Understanding the molecular and cellular events that mediate natural tolerance will lead to more advanced insights into IBD pathogenesis and improved therapeutic options. Proc R Soc Lond B Biol Sci, 2004 Oct 22, 271(1553), 2099 - 107 Mortality in cultures of the dinoflagellate Amphidinium carterae during culture senescence and darkness; Franklin DJ et al.; The study of cell death in higher plants and animals has revealed the existence of an active ('programmed') process in most types of cell, and similarities in cell death between plants, animals, yeast and bacteria suggest an evolutionarily ancient origin of programmed cell death (PCD) . Despite their global importance in primary production, information on algal cell death is limited . Algal cell death could have similarities with metazoan cell death . One morphotype of metazoan PCD, apoptosis, can be induced by light deprivation in the unicellular chlorophyte Dunaliella tertiolecta . The situation in other algal taxa is less clear . We used a model dinoflagellate (Amphidinium carterae) to test whether mortality during darkness and culture senescence showed apoptotic characteristics . Using transmission electron microscopy, fluorescent biomarkers, chlorophyll fluorescence and particulate carbon analysis we analysed the process of cell mortality and found that light deprivation caused mass mortality . By contrast, fewer dead cells (5-20% of the population) were found in late-phase cultures, while a similar degenerate cell morphology (shrunken, chlorotic) was observed . On morphological grounds, our observations suggest that the apoptotic cell death described in D . tertiolecta does not occur in A . carterae . Greater similarity was found with paraptosis, a recently proposed alternative morphotype of PCD . A paraptotic conclusion is supported by inconclusive DNA fragmentation results . We emphasize the care that must be taken in transferring fundamental paradigms between phylogenetically diverse cell types and we argue for a greater consistency in the burden of proof needed to assign causality to cell death processes. Exp Cell Res, 2004 Nov 1, 300(2), 308 - 19 Differential inside-out activation of beta2-integrins by leukotriene B4 and fMLP in human neutrophils; Patcha V et al.; We have investigated how LTB4, an endogenous chemoattractant encountered early in the inflammatory process, and fMLP, a bacteria-derived chemotactic peptide emanating from the site of infection, mediate inside-out regulation of the beta2-integrin . The role of the two chemoattractants on beta2-integrin avidity was investigated by measuring their effect on beta2-integrin clustering and surface mobility, whereas their effect on beta2-integrin affinity was measured by the expression of a high affinity epitope, a ligand-binding domain on beta2-integrins, and by integrin binding to s-ICAM . We find that the two chemoattractants modulate the beta2-integrin differently . LTB4 induces an increase in integrin clustering and surface mobility, but only a modest increase in integrin affinity . fMLP evokes a large increase in beta2-integrin affinity as well as in clustering and mobility . Lipoxin, which acts as a stop signal for the functions mediated by pro-inflammatory agents, was used as a tool for further examining the inside-out mechanisms . While LTB4-induced integrin clustering and mobility were inhibited by lipoxin, only a minor inhibition of fMLP-induced beta2-integrin avidity and no inhibition of integrin affinity were detected . The different modes of the inside-out regulation of beta2-integrins suggest that distinct mechanisms are involved in the beta2-integrin modulation induced by various chemoattractants. FEBS Lett, 2004 Oct 8, 576(1-2), 141 - 4 Functional and molecular characterization of the frataxin homolog from Arabidopsis thaliana; Busi MV et al.; Frataxin is a highly conserved protein from bacteria to mammals that has been proposed to participate in iron-sulfur cluster assembly and mitochondrial iron homeostasis . In higher organisms, the frataxin gene is nuclear-encoded and the protein is required for maintenance of normal mitochondrial iron levels and respiration . We describe here AtFH, a plant gene with significant homology to other members of the frataxin family . Plant frataxin has five segments of beta regions and two alpha helices, which are characteristics of human frataxin, as well as a potential N-terminal targeting peptide for the mitochondrial localization . Transcription analysis showed that AtFH is ubiquitously expressed with high levels in flowers . Complementation of a Saccharomyces cerevisiae mutant (Deltayfh) lacking the frataxin gene proved that AtFH is a functional protein, because it restored normal rates of respiration, growth and sensitivity to H2O2 of the null mutant . Our results support the involvement of AtFH in mitochondrial respiration and survival during oxidative stress in plants . This is the first report of a functional frataxin gene in plants . Yi Chuan Xue Bao, 2004 Feb, 31(2), 166 - 70 {Transcriptional differences between a heterokaryon and its segregants of Fusarium oxysporum f . sp . vasinfectum}; Yang H et al.; In order to elucidate the mechanism of fungal heterokayosis, a wild type strain of Fusarium oxysporum f . sp . vasinfectum was isolated from the cotton field in Anyang, Henan Province . Through single hyphal-tip isolation, a heterokaryon, Ag149, was obtained, and its two different phenotypic segregants, Ag149-I and Ag149-III, were separated from the mutated sectors on colony of the heterokaryon . They have remarkable differences on color of colony, morphology of hypha and pathogenicity . After analyzing by RAPD on their nuclear DNA with 100 random primers, no polymorphic difference was found among them . On going to find the different expressed gene, the mRNA differential display method was performed . Two kinds of reverse transcriptase AMV and MMLV, and a kit which consists of three kinds of 3' terminal anchor primers and eight kinds of 5' terminal arbitrary primers were used in differential display PCR (DD-PCR) . Total RNA as template was reverse transcribed into corresponding cDNA by 3' terminal anchor primers, and the cDNA were amplified by polymerase chain reaction with a set of one same 3' anchor primer and one 5' arbitrary primer . The PCR products were then resolved on denaturing polyacylamimide gel, and the cDNA bands were visualized by silver staining . Among the 144 PCR products, 19 differentially expressed cDNA fragments ranged from 300 bp to 700 bp were purified . All of them were ligated to pGEM-T vector respectively for sequencing and Rev-Northern blotting . Two cDNA fragments (G5 and C6) were observed to be positive after Rev-Northern blotting . The C6 was highly expressed in the heterokaryon Ag149 and its segregant Ag149-I . It is 564 bp in length and can be predicted 77 amino acids from the beginning of the 3rd to 233th base, and then searched from GenBank . The amino acid sequence of C6 shared homologies with the 6th subunit of NADH dehydrogenase found in some bacteria, plants and animals at 30% - 70% level . While the G5 was highly expressed in Ag149 and its segregant Ag149-III . It is 432 bp in length and can be predicted 101 amino acids from the beginning of the 2nd to 304th base, and the amino acid sequence is 35% homologies comparing with the tetracycline efflux protein (OtrB) of Streptomyces rimosus . We also checked these two kinds of the pGEM-T vector harboring cDNA fragments (C6 and G5) by Southern blotting with their nuclear DNA and mitochondrial DNA (mtDNA) separately . The positive identification signals only appeared from nuclear DNA,and it addressed that C6 and G5 locate on their nuclear genome . The result indicated that the difference between the heterokaryon and its segregants is distinct on gene transcriptional level . Thus a molecular evidence for the formation of heterokaryon in filamentous fungi was provided. Inflamm Bowel Dis, 2004 Sep, 10(5), 666 - 76 Regulatory T cells: peace keepers in the gut; Allez M et al.; The mucosa-associated lymphoid tissue (MALT) has the task of protecting the host from pathogens while maintaining the integrity of the gut . Immune responses are tightly regulated such that there is tolerance of nonpathogenic bacteria as well as dietary antigens present in the intestinal lumen . The failure to control these responses leads to a disruption in tolerance, which has been proposed as one mechanism involved in the development of inflammatory bowel disease (IBD) . Different mechanisms are involved in the control of immune responses in the intestinal tract, including active suppression by regulatory T cells . Distinct subsets of regulatory T cells coexist in the intestinal mucosa, which is a fertile environment for their growth . Most of these are defined by their phenotype and/or their ability to produce regulatory cytokines such as interleukin-10 and transforming growth factor-beta A lack of activation and/or expansion of regulatory cells could play a role in the uncontrolled inflammation seen in IBD . Regulatory T cells may be activated by cytokines, and their inductive phase may be antigen-driven . There are limited data relating to the true surface interactions regulating the activation of these cells . Most of the CD4 regulatory T cells (Tr1, Th3, and CD4 CD25+) are thought to interact with dendritic cells . Subsets of regulatory T cells (such as CD8 TrE cells) may recognize antigens presented by intestinal epithelial cells . A better understanding of the mechanisms by which these regulatory T cells are expanded and/or activated in the intestinal mucosa may provide clues as how to use them as a novel therapeutic tool in the treatment of patients with IBD . Microbiology, 2004 Oct, 150(Pt 10), 3451 - 62 Detoxification of hydrogen peroxide and expression of catalase genes in Rhodobacter; Zeller T et al.; The two related facultatively photosynthetic bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus show different sensitivities against peroxide stress . R . sphaeroides is able to tolerate higher concentrations of H2O2 and exhibits higher catalase activity than R . capsulatus . The katE gene of R . sphaeroides and the katG gene of R . capsulatus are strongly induced by H2O2 . This induction depends on the presence of the OxyR protein, which is able to bind to the promoter regions of these genes . In addition to katE R . sphaeroides harbours the katC gene, which shows no significant response to H2O2 but is induced in stationary phase. Photochem Photobiol . 2004 Jul 1; {Epub ahead of print} Self-Aggregation of Synthetic Protobacteriochlorophyll-d Derivatives; Tamiaki H et al.; 3(1)-Racemically pure zinc 3(1)-hydroxy-13(1)-oxo-porphyrins (zinc methyl 17,18-dehydro- bacteriopheophorbides-d) as well as their 3(1)- demethyl form were prepared by modifying chlorophyll-a through oxidation by 2,3-dichloro-5,6-dicyano-benzoquinone . From visible, circular dichroism and infrared spectral analyses, these synthetic pigments self-aggregated in 1%(v/v) tetrahydrofuran and cyclohexane to give large oligomers by an intermolecular bonding of 13-C=O...H-O(3(1))...Zn(central) and pi-pi interaction of the porphyrin chromophores . The supramolecular structures are similar to those of the corresponding chlorins and a core part of extramembranous light-harvesting antennas of photosynthetic green bacteria . The 17,18-dehydrogenation of a chlorin to porphyrin moiety did not disturb its self-aggregation and the synthetic zinc porphyrins are good models for naturally occurring self-aggregative bacteriochlorophylls. Proteins, 2005 Jan 1, 58(1), 14 - 21 Effects of mutations in tyrosine hydroxylase associated with progressive dystonia on the activity and stability of the protein; Royo M et al.; Tyrosine hydroxylase (TyrH) catalyzes the conversion of tyrosine to dihydroxyphenylalanine (DOPA), the rate-limiting step in the biosynthesis of dopamine . Four mutations in the TyrH gene have recently been described in cases of autosomal recessive DOPA-responsive dystonia (Swaans et al., Ann Hum Genet 2000;64:25-31) . All four are predicted to result in changes in single amino acid residues in the catalytic domain of the protein: T245P, T283M, R306H, and T463M . To determine the effects of these mutations on the molecular properties of the enzyme, mutant proteins containing the individual single amino acid changes have been expressed in bacteria and purified . Only the T283M mutation results in a decrease in the enzyme k(cat) value, while the T245P enzyme has a slightly higher value than the wild-type enzyme . The only case in which a K(m) value for either tyrosine or tetrahydrobiopterin is perturbed is the T245P enzyme, for which the K(m) value for tyrosine has increased about 50% . In contrast to the minor effects of the mutations on enzyme activity, the stability is decreased significantly by the mutations . The R306H and T283M enzymes are the least stable, losing activity 30- and 50-fold more rapidly than the wild-type enzyme . The apparent T(m) value for unfolding was decreased by 3.9, 8.2, and 7.2 degrees for the T245P, R306H, and T463M enzymes, while the T283M enzyme was too unstable for measurement of a T(m) value . The results establish that the physiological effects of the mutations are primarily due to the decreased stability of the mutant proteins rather than decreases in their intrinsic activities . (c) 2004 Wiley-Liss, Inc. Arch Immunol Ther Exp (Warsz), 2004 Jul-Aug, 52(4), 225 - 39 Rethinking oxidized low-density lipoprotein, its role in atherogenesis and the immune responses associated with it; Shaw PX; Atherosclerosis is a chronic inflammatory disease, resulting from hyperlipidemia and a complex interplay of many environmental, metabolic, and genetic risk factors . The unregulated macrophage uptake of cholesterol and lipids through modified forms of low-density lipoprotein (LDL), such as "OxLDL", transforms macrophages into "foam cells" to form the initial morphological lesion (the fatty streak) . The modification of LDL not only enhances its uptake by macrophages, but also changes the natural structures of these otherwise ubiquitous molecules to generate a variety of modified lipids and proteins that represent highly immunogenic neo-determinants . For example, in ApoE-/- mice, autoantibody titers to epitopes on OxLDL are correlated with the extent of atherosclerosis . Similarly, oxidative stress on cellular membranes could also give rise to "oxidation-specific" epitopes and common autoantibodies . However, OxLDL is not uniform, but rather contains complex structures, ranging from a small conformational change in surface lipids to the breakdown of the peptide chain . Therefore, the immune responses to the variety of OxLDL and their association to atherosclerosis progression are very different . For example, phosphorylcholine (PC) is a natural component of phospholipids and exists in LDL and plasma membranes . "Natural" antibodies against PC can distinctively react to PC on bacteria, OxLDL and apoptotic cells, but not to those on unoxidized phospholipids, native LDL and viable cells, which suggests the broader role of such autoantibodies in maintaining the homeostasis of the host . While malondialdehyde-modified structures resemble more the exogenous changes and associate with advanced stage of lesion, they are more likely to associate with adaptive immunity. Proc Natl Acad Sci U S A, 2004 Oct 12, 101(41), 14736 - 41 Epub 2004 Oct 04. Dynamical transition and proteinquake in photoactive yellow protein; Itoh K et al.; Conformational dynamics in protein functioning covers a wide range of time scales from nanosecond fluctuations around a conformation to the large-amplitude conformational changes of milliseconds or longer . We illustrate a picture of cooperative coupling among such motions of different time scales in a model protein, photoactive yellow protein, by proposing a model that can consistently explain the experimental results on the photocycle of photoactive yellow protein . The model provides a scenario in which the global collective motion induced by the unfolding of the N-terminal domain promotes the loosening of the atomistic packing around the chromophore, which produces the favorable molecular environment for the photoexcited chromophore, thereby stabilizing the partially unfolded intermediate in the photocycle . The proteinquake, the large conformational change triggered by the local structural disturbance, plays a decisive role in controlling the kinetics of functioning. Proc Natl Acad Sci U S A, 2004 Oct 12, 101(41), 14949 - 54 Epub 2004 Oct 04. Sigma-1 receptors at galactosylceramide-enriched lipid microdomains regulate oligodendrocyte differentiation; Hayashi T et al.; In the brain, myelin is important in regulating nerve conduction and neurotransmitter release by providing insulation at axons . Myelin is a specialized yet continuous sheet structure of differentiated oligodendrocytes (OLs) that is enriched in lipids, specifically galactosylceramides (GalCer) originated at the endoplasmic reticulum (ER) . GalCer are known to affect OL differentiation . However, the mechanism whereby GalCer affect OL differentiation is not well understood . Sigma-1 receptors (Sig-1Rs), shown by us to exist in detergent-insoluble lipid microdomains at lipid-enriched loci of ER in NG108 cells, are important in the compartmentalization/transport of ER-synthesized lipids and in cellular differentiation . In this study, we used rat primary hippocampal cultures and found that Sig-1Rs form GalCer-enriched lipid rafts at ER lipid droplet-like structures in the entire myelin sheet of mature OLs . In rat OL progenitors (CG-4 cells), levels of lipid raft-residing Sig-1Rs and GalCer increase as cells differentiate . Sig-1Rs also increase in OLs and myelin of developing rat brains . Sig-1R, GalCer, and cholesterol are colocalized and are resistant to the Triton X-100 solubilization . Treating cells with a Sig-1R agonist or targeting Sig-1Rs at lipid rafts by overexpression of Sig-1Rs in CG-4 cells enhances differentiation, whereas reducing Sig-1Rs at lipid rafts by transfection of functionally dominant-negative Sig-1Rs attenuates differentiation . Furthermore, Sig-1R siRNA inhibits differentiation . Our findings indicate that, in the brain, Sig-1Rs targeting GalCer-containing lipid microdomains are important for OL differentiation and that Sig-1Rs may play an important role in the pathogenesis of certain demyelinating diseases. Appl Environ Microbiol, 2004 Oct, 70(10), 6320 - 3 Repressor mutant forms of the Azospirillum brasilense NtrC protein; Huergo LF et al.; The Azospirillum brasilense mutant strains FP8 and FP9, after treatment with nitrosoguanidine, showed a null Nif phenotype and were unable to use nitrate as their sole nitrogen source . Sequencing of the ntrC genes revealed single nucleotide mutations in the NtrC nucleotide-binding site . The phenotypes of these strains are discussed in relation to their genotypes. Appl Environ Microbiol, 2004 Oct, 70(10), 6166 - 72 "Candidatus hepatoplasma crinochetorum," a new, stalk-forming lineage of Mollicutes colonizing the midgut glands of a terrestrial isopod; Wang Y et al.; Uncultivated bacteria that densely colonize the midgut glands (hepatopancreas) of the terrestrial isopod Porcellio scaber (Crustacea: Isopoda) were identified by cloning and sequencing of their 16S rRNA genes . Phylogenetic analysis revealed that these symbionts represent a novel lineage of the Mollicutes and are only distantly related (<82% sequence identity) to members of the Mycoplasmatales and Entomoplasmatales . Fluorescence in situ hybridization with a specific oligonucleotide probe confirmed that the amplified 16S rRNA gene sequences indeed originated from a homogeneous population of symbionts intimately associated with the epithelial surface of the hepatopancreas . The same probe also detected morphotypically identical symbionts in other crinochete isopods . Scanning and transmission electron microscopy revealed uniform spherical bacterial cells without a cell wall, sometimes interacting with the microvilli of the brush border by means of stalk-like cytoplasmic appendages, which also appeared to be involved in cell division through budding . Based on the isolated phylogenetic position and unique cytological properties, the provisional name "Candidatus Hepatoplasma crinochetorum" is proposed for this new taxon of Mollicutes colonizing the hepatopancreas of P . scaber. J Bacteriol, 2004 Oct, 186(20), 7019 - 22 Treponema pallidum fibronectin-binding proteins; Cameron CE et al.; Putative adhesins were predicted by computer analysis of the Treponema pallidum genome . Two treponemal proteins, Tp0155 and Tp0483, demonstrated specific attachment to fibronectin, blocked bacterial adherence to fibronectin-coated slides, and supported attachment of fibronectin-producing mammalian cells . These results suggest Tp0155 and Tp0483 are fibronectin-binding proteins mediating T . pallidum-host interactions. J Bacteriol, 2004 Oct, 186(20), 6759 - 67 Functional and structural analysis of HrcA repressor protein from Caulobacter crescentus; Susin MF et al.; A large number of bacteria regulate chaperone gene expression during heat shock by the HrcA-CIRCE system, in which the DNA element called CIRCE serves as binding site for the repressor protein HrcA under nonstress conditions . In Caulobacter crescentus, the groESL operon presents a dual type of control . Heat shock induction is controlled by a sigma32-dependent promoter and the HrcA-CIRCE system plays a role in regulation of groESL expression under physiological temperatures . To study the activity of HrcA in vitro, we purified a histidine-tagged version of the protein, and specific binding to the CIRCE element was obtained by gel shift assays . The amount of retarded DNA increased significantly in the presence of GroES/GroEL, suggesting that the GroE chaperonin machine modulates HrcA activity . Further evidence of this modulation was obtained using lacZ transcription fusions with the groESL regulatory region in C . crescentus cells, producing different amounts of GroES/GroEL . In addition, we identified the putative DNA-binding domain of HrcA through extensive protein sequence comparison and constructed various HrcA mutant proteins containing single amino acid substitutions in or near this region . In vitro and in vivo experiments with these mutated proteins indicated several amino acids important for repressor activity. J Nutr, 2004 Oct, 134(10), 2634 - 9 Alpha-eleostearic acid (9Z11E13E-18:3) is quickly converted to conjugated linoleic acid (9Z11E-18:2) in rats; Tsuzuki T et al.; We previously showed that alpha-eleostearic acid (alpha-ESA; 9Z11E13E-18:3) is converted to conjugated linoleic acid (CLA; 9,11-18:2) in the liver and plasma of rats that were given diets including 1% alpha-ESA for 4 wk . In this study, we investigated this phenomenon in detail . First, the chemical structure of CLA produced by alpha-ESA administration was determined . After alpha-ESA was orally administered to rats, CLA in rat liver was isolated by HPLC . The positional and geometric isomerism was determined using GC-EI/MS and (13)C-NMR, respectively, and the CLA generated in rats after alpha-ESA feeding was confirmed to be 9Z11E-CLA . Next, the concentrations of alpha-ESA and CLA were determined 0, 3, 6, and 24 h after oral administration of alpha-ESA to rats . Moreover, we also investigated whether enteric bacteria are involved in the conversion of alpha-ESA to CLA using germ-free rats . alpha-ESA was orally administered to germ-free and normal rats and alpha-ESA and CLA were detected in the organs of both groups . In addition, to confirm that this reaction was enzyme-mediated, alpha-ESA was reacted with tissue homogenates (liver, kidney, and small intestine mucous) and coenzymes (NADH, NAD(+), NADPH, and NADP(+)), and the enzyme activities were estimated from the amount of CLA produced . CLA was detected when alpha-ESA was reacted with liver, kidney, and small intestine mucous homogenates and a coenzyme (NADPH) . These results indicated that alpha-ESA is converted to 9Z11E-CLA in rats by a Delta13-saturation reaction carried out by an NADPH-dependent enzyme. J Wildl Dis, 2004 Jul, 40(3), 548 - 54 Occurrence of Helicobacter Infection in the gastric mucosa of free-living red foxes (Vulpes vulpes); Erginsoy SD et al.; We studied gastric Helicobacter spp . in five red foxes (Vulpes vulpes) . Samples of stomach from the cardia, corpus, pyloric antrum, and duodenum were subjected to histopathologic, immunohistochemical, and transmission electron microscopy (TEM) examination for the presence of Helicobacter and gastritis . All foxes had gastric Helicobacter-like organisms (GHLOs) on examination by light microscopy and TEM . Gastric Helicobacter-like organisms were present in all areas of the stomachs . Chronic mild or moderate gastric inflammation was associated with infection by GHLOs in one or more regions of the stomach, but there was no correlation between inflammation and infection . It is not clear whether the organisms were causing the minimal histologic lesions observed, but the gastric mucosa of free-living foxes appears to be commonly colonized with GHLOs . The frequent colonization of free-living foxes with distinct GHLOs possibly reflects their special characteristic in feeding and/or social behavior or the potential commensal nature of the bacteria in free-ranging foxes. J Mol Biol, 2004 Oct 22, 343(3), 649 - 57 The structures of inhibitor complexes of Pyrococcus furiosus phosphoglucose isomerase provide insights into substrate binding and catalysis; Berrisford JM et al.; Pyrococcus furiosus phosphoglucose isomerase (PfPGI) is a metal-containing enzyme that catalyses the interconversion of glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P) . The recent structure of PfPGI has confirmed the hypothesis that the enzyme belongs to the cupin superfamily and identified the position of the active site . This fold is distinct from the alphabetaalpha sandwich fold commonly seen in phosphoglucose isomerases (PGIs) that are found in bacteria, eukaryotes and some archaea . Whilst the mechanism of the latter family is thought to proceed through a cis-enediol intermediate, analysis of the structure of PfPGI in the presence of inhibitors has led to the suggestion that the mechanism of this enzyme involves the metal-dependent direct transfer of a hydride between C1 and C2 atoms of the substrate . To gain further insight in the reaction mechanism of PfPGI, the structures of the free enzyme and the complexes with the inhibitor, 5-phospho-d-arabinonate (5PAA) in the presence and absence of metal have been determined . Comparison of these structures with those of equivalent complexes of the eukaryotic PGIs reveals similarities at the active site in the disposition of possible catalytic residues . These include the presence of a glutamic acid residue, Glu97 in PfPGI, which occupies the same position relative to the inhibitor as that of the glutamate that is thought to function as the catalytic base in the eukaryal-type PGIs . These similarities suggest that aspects of the catalytic mechanisms of these two structurally unrelated PGIs may be similar and based on an enediol intermediate. Dent Clin North Am, 2004 Oct, 48(4), 1017 - 59, viii Erbium lasers in dentistry; van As G; Erbium hard tissue lasers have the capability to prepare enamel, dentin, caries, cementum, and bone in addition to cutting soft tissue . The ability of hard tissue lasers to reduce or eliminate vibrations, the audible whine of drills, microfractures, and some of the discomfort that many patients fear and commonly associate with high-speed handpieces is impressive . In addition, these lasers can be used with a reduced amount of local anesthetic for many procedures . Today, these instruments have evolved from their initial use for all classes of cavity preparations to their ability for removing soft tissue, their usefulness in the disinfection of bacteria within endodontic canals, and most recently, as an alternative to the high speed handpiece for the removal of bone in oral and maxillofacial surgery . In addition, recent research has centered on the value of the erbium family of laser wavelengths in periodontics, including the removal of calculus. Dent Clin North Am, 2004 Oct, 48(4), 999 - 1015, viii Lasers in dental implantology; Martin E; The parallels in the expansion of implant dentistry and laser dentistry in clinical practice are apparent . As advocates for laser dentistry continue to seek new ways to use the technology and as more practitioners become involved in implant dentistry, it is logical to see the concurrent use of both technologies in clinical practice . This article provides data that clearly support the value of dental lasers in the practice of implant dentistry . The challenge for the practitioner is the same as for any other area of dentistry: knowing when, where, and what armamentarium to use in any given situation . Not all dental laser wavelengths are necessarily useful in every dental implant situation . After clinicians know the characteristics of the wavelengths available to them, the application of the technology to the specific situation certainly is warranted. Dent Clin North Am, 2004 Oct, 48(4), 923 - 36, vii Lasers for initial periodontal therapy; Raffetto N; With a greater understanding of the components that initiate the host into the downward spiral of periodontal disease, the clinician must look for better treatment and therapy options . Clinical observations and studies show good results, making incorporation of the laser into the first phase of nonsurgical periodontal therapy an excellent choice. Parasitol Today, 1993, 9(9), 329 - 33 Pharmacokinetic disposition of benzimidazole drugs in the ruminant gastrointestinal tract; Hennessy DR; Orally administered benzimidazole (BZ) drugs are ideally deposited in the rumen where they associate extensively with particulate digesta material, the residence time of this drug-digesta complex being a major influence on the subsequent rate and duration of BZ availability . This duration is shortened if the dose should bypass the rumen due to oesophageal groove closure . Benzimidazole metabolites flow from the rumen primarily in association with particulate digesta . In the abomasum, the majority of soluble metabolites result from gastric secretions . These metabolites flow into the small intestine where they are absorbed into the systemic circulation . Depending on the chemical structure a significant portion are secreted in bile either in a free (ie . unconjugated) or conjugated form . Free biliary metabolites are absorbed from the upper small intestine whereas bacteria in the large intestine hydrolyse the conjugated biliary metabolites to promote further absorption . Biliary derived metabolites are enterohepatically recycled but contribute little to the peripheral plasma metabolite pool . In this review, Des Hennessy discusses these issues in relation to the pharmacology of BZ drugs in the gastrointestinal tract of ruminants. Parasitol Today, 1993 Jun, 9(6), 218 - 22 Trypanosoma cruzi trans-sialidase and cell invasion; Schenkman S et al.; Trypanosoma cruzi does not synthesize sialic acid but does contain a trans-sialidase, an enzyme capable of transferring sialic acid between host glycoconjugates and the parasite . Sialic acids are negatively charged carbohydrates attached to the terminal non-reducing end of glycoproteins and glycolipids, and their presence can dramatically influence many cell-surface recognition processes . Since sialic acids have been implicated in several ligand-receptor interactions, including the interaction of pathogenic viruses, bacteria and protozoans with their hosts, the expression of trans-sialidase and the acquisition of sialic acid by T . cruzi may be relevant to the interaction of the parasite with the host, and consequently may influence the pathobiology of Chagas disease . In this review, Sergio Schenkman and Daniel Eichinger discuss recent data about the structure and function of T . cruzi trans-sialidase. Parasitol Today, 1993 Oct, 9(10), 361 - 4 Naturally occurring antibodies that react with protozoan parasites; Konishi E; In this paper, Eiji Konishi reviews general features of naturally occurring (natural) antibodies that react with protozoan parasites . Several functions of natural antibodies have been identified in relation to their multireactivity, but reports on protozoan infection have dealt mainly with the role of natural antibodies in the innate immunity of the host, These antibodies lyse cells in the presence of complement and have opsonizing activity, in vitro . Studies of their origin have shown the possibilities of (1) continuous polyclonal stimulation by gastrointestinal bacteria, and (2) there being multireactive antibodies secreted by CD5(+) B cells . The protective functions of natural antibodies are important in the interpretation of the host range, the mode of infection, and the course of the disease of certain protozoan parasites. Parasitol Today, 1991, 7(7), 163 - 72 The interaction between intestinal mucus glycoproteins and enteric infections; Tse SK et al.; Adherence of pathogenic enteric organisms to specific receptors on mucosal surfaces is widely recognized as an important first step in the initiation of infectious diseases . The specific interactions whereby parasites and bacteria exploit mucus substrates for colonization, and the host uses them as a nonimmunological defense mechanism, is only now being unravelled . In this review, Sil-King Tse and Kris Chadee discuss various hypothetical models for interaction, including the role of the immune system in the regulation of mucus secretion. Parasitol Today, 1991, 7(12), 335 - 40 Phagolysosomal escape by intracellular pathogens; Andrews NW et al.; It has often been suggested that intracellular parasites invade cells in order to evade the host's immune response . Whether or not this view is correct, have successfully avoided excessive scrutiny by biomedical investigators . Published descriptions of the intracellular compartments occupied by parasites often contradict each other, reflecting the fact that the early events following host cell invasion remained, until recently, poorly understood . In this review, Norma Andrews and Paul Webster focus on what is now known about a dramatic transition that some parasites undergo after invading cells: escape from a membrane-bound vacuole into the cytosol . They discuss the information available on strategies for phagolysosomal escape of pathogens ranging from bacteria to protozoa, with emphasis on the cases in which the molecular mechanisms controlling this event have been investigated. Parasitol Today, 1990 May, 6(5), 163 - 5 A role for pore-forming proteins in the pathogenesis by parasites? Ojcius DM, Ding-E Young J. Over the past decade or so, pore-forming proteins (PFPs) have been isolated from various immune cells and nonpathogenic bacteria . It is now becoming apparent that PFPs may also be produced by a number of parasites . Although far from definitive, the evidence currently available for the role of PFPs in the survival and pathogenesis by parasites in briefly presented by David Ojcius and John Ding-E Young. Parasitol Today, 1989 May, 5(5), 156 - 60 Xenobiotic metabolism in helminths; Precious WY et al.; During its life cycle a parasite, like mammals and other organisms, comes into contact with a variety of toxic molecules . In mammals the main line of defence against such compounds is a group of oxidative enzymes that occur predominantly in the liver . Many of these oxidations are dependent on the haemoprotein cytochrome P-450 which serves as a terminal oxidase accepting electrons from NADPH and cytochrome {Formula: see text} . This review by Wendy Precious and John Barrett illustrates that in contrast to mammals, xenobiotic metabolism in both parasitic and free-living platyhelminths and nematodes is predominantly reductive and hydrolytic as the cytochrome P-450 system is absent . This is surprising since it is present in many groups including bacteria, fungi and protozoa, which suggests an ancient origin . The absence of the cytochrome P-450 system not only severely limits the ability of helminths to detoxify compounds but also limits their ability to activate prodrugs. Parasitol Today, 1988 Sep, 4(9), 262 - 6 Differential diagnosis of eosinophilic meningitis; Jaroonvesama N; Meningitis - infection o f the meninges - can be attributed to a variety o f agents, including bacteria, protozoa and some helminths . In helminth infections, but rarely in protozoal infections, eosinophilia is a common sign . Thus eosinophilic meningitis (EOM) is typically associated with certain helminth infections in which nervous system involvement occurs . Among the most important of these are ongiostrongyliosis, gnathostomiasis, porogonimiasis and cysticercosis (see Fig . 1) . Here, Nibha Jaroonvesoma discusses the key distinctions between such infections. Parasitol Today, 1988 Mar, 4(3), 82 - 3 Reducing agents and Entamoeba histolytica; Mehlotra RK et al.; Entamoeba histolytica remains an important but enigmatic parasite . It displays both non-pathogenic and invasive pathogenic types, which can be distinguished clinically and by isoenzyme markers . Yet as debated in Parasitology Today last year(1), the relationship between these two forms remains unclear . Bacterial associates and reducing agents are known to play on important role in the culture of E . histolytica, and possibly in its differentiation and invasive mechanisms . This article briefly reviews available information on the role o f reducing agents, and explores the possibility that bacteria may play a role in reduction o f toxic oxygen product - thereby promoting the virulence of E . histolytica . The review is not definitive, but should help to stimulate further research in this neglected area. Parasitol Today, 1988 Oct, 4(10), 289 - 90 Viruses of parasitic protozoa; Miles MA; Associations are increasingly recognized between parasitic protozoa and smaller organisms such as bacteria (see Parasitology Today 1, 143), and viruses . Their revelation, originally by ultrastructural studies, may now be indicated by observations of 'rogue bands' on gel electrophoresis of parasite nucleic acids . The origins and roles of parasite 'parasites' are still uncertain, but, as Michael Miles suggests here, they may prove of particular importance as agents to introduce genes for important immunogens into non-pathogenic protozoa for vaccine delivery. Parasitol Today, 1987 Apr, 3(4), 121 - 3 Summer mastitis: Vector transmission or not? Hillerton JE. Summer mastitis is on acute, suppurative infection of cattle with considerable importance to European agriculture . The cousotive bacteria are thought to be transmitted by the fly Hydrotaea irritans (L.) but evidence for this remains circumstantial . In this article, Eric Hillerton presents a working hypothesis to explain the transmission mechanism. Microbiol Res, 2004, 159(3), 233 - 43 Occurrence of Gluconacetobacter diazotrophicus in tropical and subtropical plants of Western Ghats, India; Madhaiyan M et al.; Endophytic bacteria were isolated from the tissues of surface sterilized roots, stems, and leaves of fifty different crop plants . Phenotypic, biochemical tests and species-specific PCR assay permitted identification of four isolates of Gluconacetobacter diazotrophicus from root tissues of carrot (Daucus carota L.), raddish (Raphanus sativus L.), beetroot (Beta vulgaris L.) and coffee (Coffea arabica L.) . Further the plant growth promoting traits such as nitrogenase activity, production of phytohormone indole acetic acid (IAA), phosphorus and zinc solubilization were assessed . Significant nitrogenase activity was recorded among the isolates and all the isolates produced IAA in the presence of tryptophan . Though all the four isolates efficiently solubilized phosphorus, the zinc solubilizing ability differed among the isolates. Microbiol Res, 2004, 159(3), 193 - 201 Butachlor inhibits production and oxidation of methane in tropical rice soils under flooded condition; Mohanty SR et al.; In laboratory incubation experiments, application of a commercial formulation of the herbicide butachlor (N-butoxymethyl-2-chloro-2',6'-diethyl acetanilide) to three tropical rice soils, widely differing in their physicochemical characteristics, under flooded condition inhibited methane (CH4) production . The inhibitory effect was concentration dependent and most remarkable in the alluvial soil . Thus, following application of butachlor at 5, 10, 50 and 100 microg g(-1) soil, respectively, cumulative CH4 production in the alluvial soil was inhibited by 15%, 31%, 91% and 98% over unamended control . Since CH4 production was less pronounced in the sandy loam and acid sulfate soil, the impact of amendment with butchalor, albeit inhibitory, was less extensive than the alluvial soil . Inhibition of CH4 production in butachlor-amended alluvial soil was related to the prevention in the drop in redox potential as well as low methanogenic bacterial population especially at high concentrations of butachlor . CH4 oxidation was also inhibited in butachlor-amended alluvial soil with the inhibitory effect being more prevalent under flooded condition . Inhibition in CH4 oxidation was related to a reduction in the population of soluble methane monooxygenase producing methanotrophs . Results demonstrate that butachlor, a commonly used herbicide in rice cultivation, even at very low concentrations can affect CH4 production and its oxidation, thereby influencing the biogeochemical cycle of CH4 in flooded rice soils. Bull Soc Pathol Exot, 2004 Aug, 97(3), 193 - 8 {Rickettsia africae, the agent of African tick-bite fever: an emerging pathogen in the West Indies and Reunion Island (Indian Ocean)}; Parola P et al.; Rickettsia africae is the agent of African tick bite fever, an emerging disease transmitted by Amblyomma ticks in sub-Saharan Africa . In 1998, we reported the first documented case of R . africae in the New World, in a patient who had returned from Guadeloupe . In order to confirm the presence of R . africae in the West Indies, entomologic surveys were conducted from 1999 to 2003 to collect Amblyomma, which are considered as potential vectors and reservoirs of the bacteria . Ticks were used as epidemiological tools to detect R . africae by molecular tools and/or cultivate the bacteria in shell-vial cell culture . This paper summarizes the results obtained in the West Indies . R . africae was detected and isolated for the first time in Guadeloupe, and then detected by molecular tools in Martinique and St-Kitts and Nevis . These last results confirm our first hypotheses--that is R . africae is prevalent on all the Caribbean islands where A . variegatum ticks have been introduced from Africa in the 18th and 19th centuries . We also present the results of a study conducted on the Reunion Island, a French island in the Indian Ocean . For the first time there, R . africae was detected in A . variegatum ticks, which were probably introduced from the African mainland or Madagascar with the human colonization during the 17th century Thus, clinicians should be aware that patient presenting in the West Indies or on Reunion Island (or after a trip over there) with fever, eschar (often multiple), regional lymphadenopathy and a rash, might be infected by R . africae. Int J Appl Philos, 2003 Fall, 17(2), 267 - 75 Patenting genes: a fast and furious primer; Askland A; Patents have been issued in the United States for genes and gene sequences since 1980 . Patent protection has provided incentives to aggressively probe the genome of humans and non-humans alike in search of profitable applications . Yet it is not clear that patent protection should have been afforded to genes and gene sequences and it is increasingly clear that patent protection, as currently formulated, is not an appropriate means to realize the full benefits of genetic research . As we stand on the threshold of a genetically enhanced future, in which we shall have the power to consciously steer our evolution as a species, we need to carefully consider how to shape the benefits of genetic research and how to recognize and contain its detriments. Biochemistry, 2004 Oct 12, 43(40), 12761 - 9 A novel antioxidant role for ligandin behavior of glutathione S-transferases: attenuation of the photodynamic effects of hypericin; Lu WD et al.; Hypericin (HYP) is a major constituent of the herbal antidepressant St . John's wort with potential utility as an antitumor photodynamic sensitizer and antiviral agent . Upon irradiation at 540-600 nm, HYP generates reactive oxygen species (ROS) and induces oxidative stress . Here, human glutathione S-transferase (GST) isoforms GSTP1-1 (P1-1) and GSTA1-1 (A1-1) are shown to bind with high affinity to HYP and to differentially quench its photodynamic properties . In steady-state turnover studies, HYP inhibits A1-1 and P1-1 with IC(50) values of 160 and 190 nM, respectively . Fluorescence titration experiments and fitting of the data to the Hill equation yield apparent K(D)s for binding to A1-1 and P1-1 of 0.65 and 0.51 microM, respectively . The recovered Hill coefficients are 1.8 for both GSTA1-1 and GSTP1-1, indicating that multiple HYPs bind to each isoform . This behavior is reminiscent of classic "ligandin" activity of GSTs, wherein nonsubstrate planar aromatic anions are sequestered on, and inhibit, the enzyme . However, HYP complexed with P1-1 is photodynamically attenuated, with minimal protein oxidation . In contrast, light-dependent, oxygen-dependent, oxidation of A1-1 was modest and oxidation of human albumin was extensive in the presence of HYP, as monitored by electrospray mass spectrometry (ESI-MS) . A peptide "trap" of diffusive ROS was oxidized extensively upon irradiation of HYP in the presence of albumin but very little in the presence of P1-1 or A1-1 . Solute quenching studies were used to probe the accessibility of the bound HYP in each of the protein complexes . The fluorescence of HYP complexed with albumin, A1-1, or P1-1 was quenched by I(-) with quenching rate constants (k(q)) of 1.1 x 10(9), 2.4 x 10(9) and 0.5 x10(9) M(-1) s(-1), respectively, indicating that small molecules such as O(2) have similar diffusional access to the complexed HYP in each of the proteins, eliminating the possibility of differential accessibility of oxygen as the source of a different yield of ROS . This is the first demonstration of a possible antioxidant role for the ligandin activity of GSTs and a striking example of protein-specific effects on hypericin photodynamic activity . Even highly homologous protein isoforms can differentially promote or inhibit photosensitizer activity. Dis Aquat Organ, 2004 Aug 9, 60(2), 89 - 96 Multiple pathogens found in growth-retarded black tiger shrimp Penaeus monodon cultivated in Thailand; Chayaburakul K et al.; In 2001-2002 throughout Thailand, black tiger shrimp Penaeus monodon farmers reported very unusual retarded growth . We have called this problem monodon slow growth syndrome (MSGS) . Based on decreased national production, estimated losses due to this phenomenon were in the range of 13 000 million baht (approximately 300 million US dollars) in 2002 . Since rearing practices had not changed, it was considered possible that the MSGS problem may have arisen from a new or existing pathogen . To examine this possibility, cultivated shrimp were sampled from 32 commercial rearing ponds that reported abnormally slow growth from eastern, central and southern regions of Thailand . Shrimp were randomly sampled from each pond and grouped into normal and small shrimp . Normal shrimp were defined as those with body weights (BW) of 24 g or more while small shrimp were defined as those that weighed 16.8 g or less . Pleopods were used for detection of monodon baculovirus (MBV), heptopancreatic parvovirus (HPV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) using specific polymerase chain reaction (PCR) assays . In addition, some shrimp were processed for normal histopathology and transmission electron microscopy (TEM) . Most of the shrimp specimens were infected by at least 1 of these viruses but many had dual or multiple infections . Prevalence of HPV and combined HPV/MBV infections in the small shrimp was significantly higher than in the normal shrimp . In addition to the viruses, a new microsporidian species, gregarines and bacteria were also observed but were not significantly associated with the MSGS problem . Some of the small shrimp gave negative results for all these pathogens by PCR and histology and no new and unique histopathology was recognized in any of the samples . The findings suggested that HPV infection was a contributing factor but not the overriding factor responsible for MSGS . It is possible that MSGS is caused by an unknown pathogen or by some other presently unknown, non-pathogenic factor. Clin Rheumatol, 2004 Oct, 23(5), 435 - 40 Epub 2004 Jul 28. An elevated level of IL-10- and TGFbeta-secreting T cells, B cells and macrophages in the synovial membrane of patients with reactive arthritis compared to rheumatoid arthritis; Appel H et al.; A relative high secretion level of IL-10 and a low secretion of TNF-alpha has been described in the synovial fluid and peripheral blood of patients with reactive arthritis (ReA), possibly contributing to the persistence of bacteria . The role of TGF-beta is less clear . We investigated these cytokines in the synovial membrane of patients with ReA and rheumatoid arthritis (RA) and tried to identify their cellular source . We used sections from the synovial membrane of 4 ReA and 4 RA patients which were double stained with immunofluorescence antibodies against cell surface markers for T cells (CD3), macrophages (CD68) and B cells (CD20) in combination with antibodies against intracellular cytokines TNF-alpha, IFN-gamma, TGF-beta, IL-4 and IL-10, and quantified these using a fluorescence microscope . A lower number of TNF-alpha-secreting cells were found in ReA compared to RA: CD3+: 1.78 +/- 0.54% versus 5.02% +/- 0.47% (p = 0.034) . CD68+: 2.86 +/- 0.52 versus 5.37 +/- 0.53% (p = 0.034), CD20+ : 3.02 +/- 0.42% versus 3.58 +/- 0.48% (p > 0.05) . A higher number of IL-10 positive cells were found in ReA compared to RA: CD3+: 3.27 +/- 1.5% versus 1.13 +/- 0.50% (p = 0.034), CD68+ 1.23 +/- 0.75% versus 0.83 +/- 0.35% (p > 0.05), CD20+: 3.70 +/- 1.6% versus 1.6 +/- 1.1% (p > 0.05) . A difference between ReA and RA was also found for TGF-beta+ T cells: CD3+ 7.86 + 1.5% versus 1.78 + 0.35% (p = 0.032); CD20+: 7.91 + 2.1% versus 2.1 + 2.8% (p > 0.05), CD68+: 7.81% + 1.24% versus 2.12 + 0.28% (p = 0.032) . In conclusion, we saw a different cytokine secretion pattern in the synovial membrane of ReA and RA . For T cells in ReA we found a cytokine secretion profile typical for T regulatory cells 1 (Tr1), with an elevated level of IL-10- and TGF-beta-secreting cells . Whether this is due to a more general difference in TNF-alpha, IL-10 or TGF-beta production which is genetically determined or regulated by T cells remains to be determined. J Microbiol, 2004 Sep, 42(3), 181 - 7 Comparative analysis of cyanobacterial communities from polluted reservoirs in Korea; Kim JB et al.; Cyanobacteria are the dominant phototrophic bacteria in water environments . Here, the diversity of cyanobacteria in seven Korean reservoir waters where different levels of algal blooms were observed during the summer of 2002, was examined by T-RFLP analysis . The number of T-RF bands in the HaeIII T-RFLP profiles analyzed from those water samples ranged from 20 to 44 . Of these, cyanobacteria accounted for 6.1 to 27.2% of the total bacteria . The water samples could be clustered into 2 groups according to the Dice coefficient of the T-RF profiles . The eutrophic Dunpo and oligotrophic Chungju reservoirs were selected, and several representative clones from both reservoir waters analyzed for the nucleotide sequences of their 16S rDNA . The major clones were found to belong to the Microcystis and Anabaena species in the waters from the Dunpo and Chungju reservoirs, respectively, which was in agreement with the T-RFLP result . That is, the Microcystis and Anabaena species were dominant in the eutrophic and polluted Dunpo and oligotrophic Chungju reservoir waters, respectively . These results indicated that there is a correlation between prevalence of cyanobacterial species and levels of pollution in reservoir waters . Microb Pathog, 2004 Oct, 37(4), 205 - 13 Identification of Francisella tularensis genes encoding exported membrane-associated proteins using TnphoA mutagenesis of a genomic library; Gilmore RD Jr et al.; Francisella tularensis, the causative agent of tularemia, is a highly infectious pathogen of humans and animals, yet little is known about the surface proteins of this organism that mediate mechanisms of pathogenicity . lambdaTnphoA was used to generate random alkaline phosphatase gene fusions in a F . tularensis subsp . tularensis (strain Schu S4) genomic library to identify genes encoding exported extracytoplasmic proteins . Eleven genes encoding membrane-associated proteins were identified by this method and their respective signal peptides were characterized . Three of the genes encoded conserved 'housekeeping' enzymes, while the other eight genes were unique to F . tularensis, encoding proteins with molecular masses ranging from 11 to 78kDa as deduced from the amino acid sequences . Two genes putatively encoded lipoproteins based on the presence of characteristic signal peptidase II cleavage sites . Four selected proteins were found associated with outer membranes from Schu S4 and LVS strains by Western blotting . Indirect immunofluorescence of strain Schu S4 cells also showed evidence of protein localization to the outer membrane . Protein database searches produced significant alignments with proteins from other bacteria involved in carbohydrate transport, lipid metabolism, and cell envelope biogenesis, thereby providing clues for putative functions . These findings demonstrated that TnphoA mutagenesis can be used in conjunction with F . tularensis genome sequence data to provide a foundation for studies to identify and define cellular surface protein virulence factors of this pathogen. Respir Care Clin N Am, 2004 Sep, 10(3), 309 - 15, v The "best" tidal volume for managing acute lung injury/acute respiratory distress syndrome; MacIntyre NR; An inappropriate tidal volume setting can overstretch and injure the lung . Maximal stretch, tidal stretch, frequency of stretch, and rate of stretch are all implicated in such injury . Moreover, the stretch injury produces systemic injury by liberating cytokines and translocating bacteria in the lung . Clinical trials have shown that limiting maximal and tidal stretch improves outcomes, even if gas exchange is partially compromised . Current strategies should focus on limiting tidal and maximal stretch as much as possible. Structure (Camb), 2004 Oct, 12(10), 1881 - 9 A novel structure of DNA repair protein RecO from Deinococcus radiodurans; Makharashvili N et al.; Recovery of arrested replication requires coordinated action of DNA repair, replication, and recombination machineries . Bacterial RecO protein is a member of RecF recombination repair pathway important for replication recovery . RecO possesses two distinct activities in vitro, closely resembling those of eukaryotic protein Rad52: DNA annealing and RecA-mediated DNA recombination . Here we present the crystal structure of the RecO protein from the extremely radiation resistant bacteria Deinococcus radiodurans (DrRecO) and characterize its DNA binding and strand annealing properties . The RecO structure is totally different from the Rad52 structure . DrRecO is comprised of three structural domains: an N-terminal domain which adopts an OB-fold, a novel alpha-helical domain, and an unusual zinc-binding domain . Sequence alignments suggest that the multidomain architecture is conserved between RecO proteins from other bacterial species and is suitable to elucidate sites of protein-protein and DNA-protein interactions necessary for RecO functions during the replication recovery and DNA repair . Development, 2004 Nov, 131(21), 5297 - 308 Epub 2004 Sep 29. Testis-specific TAF homologs collaborate to control a tissue-specific transcription program; Hiller M et al.; Alternate forms of the PolII transcription initiation machinery have been proposed to play a role in selective activation of cell-type-specific gene expression programs during cellular differentiation . The cannonball (can) gene of Drosophila encodes a homolog of a TBP-associated factor (dTAF5) protein expressed only in spermatocytes, where it is required for normal transcription of genes required for spermatid differentiation . We show that Drosophila primary spermatocytes also express four additional tissue-specific TAFs: nht (homolog of dTAF4), mia (homolog of dTAF6), sa (homolog of dTAF8) and rye (homolog of dTAF12) . Mutations in nht, mia and sa have similar effects in primary spermatocytes on transcription of several target genes involved in spermatid differentiation, and cause the same phenotypes as mutations in can, blocking both meiotic cell cycle progression and spermatid differentiation . The nht, mia, sa and rye proteins contain histone fold domain dimerization motifs . The nht and rye proteins interact structurally when co-expressed in bacteria, similarly to their generally expressed homologs TAF4 and TAF12, which heterodimerize . Strikingly, the structural interaction is tissue specific: nht did not interact with dTAF12 and dTAF4 did not interact with rye in a bacterial co-expression assay . We propose that the products of the five Drosophila genes encoding testis TAF homologs collaborate in an alternative TAF-containing protein complex to regulate a testis-specific gene expression program in primary spermatocytes required for terminal differentiation of male germ cells. Gene Ther, 2004 Oct, 11 Suppl 1, S33 - 42 DNA electrotransfer: its principles and an updated review of its therapeutic applications; Andre F et al.; The use of electric pulses to transfect all types of cells is well known and regularly used in vitro for bacteria and eukaryotic cells transformation . Electric pulses can also be delivered in vivo either transcutaneously or with electrodes in direct contact with the tissues . After injection of naked DNA in a tissue, appropriate local electric pulses can result in a very high expression of the transferred genes . This manuscript describes the evolution in the concepts and the various optimization steps that have led to the use of combinations of pulses that fit with the known roles of the electric pulses in DNA electrotransfer, namely cell electropermeabilization and DNA electrophoresis . A summary of the main applications published until now is also reported, restricted to the in vivo preclinical trials using therapeutic genes. Genetics, 2004 Sep, 168(1), 341 - 9 The genetic basis of male fertility in relation to haplodiploid reproduction in Leptopilina clavipes (Hymenoptera: Figitidae); Pannebakker BA et al.; Traits under relaxed selection are expected to become reduced or disappear completely, a process called vestigialization . In parthenogenetic populations, traits historically involved in sexual reproduction are no longer under selection and potentially subject to such reduction . In Leptopilina clavipes, thelytokous (parthenogenetic) populations are infected by Wolbachia bacteria . Arrhenotokous populations do not harbor Wolbachia . When antibiotics are applied to infected females, they are cured from their infection and males arise . Such males are capable of producing offspring with uninfected females, but with lower fertilization success than sexual males . This can be attributed to the lack of selection on male fertility in thelytokous lines . In this study we used this variation in L . clavipes male fertility to determine the genetic basis of this trait . Males from cured thelytokous populations were crossed to females from uninfected populations . Using AFLP markers, a genetic linkage map was generated, consisting of five linkage groups and spanning a total distance of 219.9 cM . A single QTL of large effect (explaining 46.5% of the phenotypic variance) was identified for male fertility, which we call male fertility factor (mff) . We discuss possible mechanisms underlying the effect of mff, as well as mechanisms involved in vestigialization of traits involved in sexual reproduction. Cell, 2004 Oct 1, 119(1), 137 - 44 Combinatorial marking of cells and organelles with reconstituted fluorescent proteins; Zhang S et al.; Expression of GFP and other fluorescent proteins depends on cis-regulatory elements . Because these elements rarely direct expression to specific cell types, GFP production cannot always be sufficiently limited . Here we show that reconstitution of GFP, YFP, and CFP previously split into two polypeptides yields fluorescent products when coexpressed in C . elegans . Because this reconstitution involves two components, it can confirm cellular coexpression and identify cells expressing a previously uncharacterized promoter . By choosing promoters whose expression patterns overlap for a single cell type, we can produce animals with fluorescence only in those cells . Furthermore, when one partial GFP polypeptide is fused with a subcellularly localized protein or peptide, this restricted expression leads to the fluorescent marking of cellular components in a subset of cells. Naturwissenschaften, 2004 Dec, 91(12), 575 - 8 Epub 2004 Dec. Pheromone-modulated behavioral suites influence colony growth in the honey bee ( Apis mellifera); Pankiw T et al.; The success of a species depends on its ability to assess its environment and to decide accordingly which behaviors are most appropriate . Many animal species, from bacteria to mammals, are able to communicate using interspecies chemicals called pheromones . In addition to exerting physiological effects on individuals, for social species, pheromones communicate group social structure . Communication of social structure is important to social insects for the allocation of its working members into coordinated suites of behaviors . We tested effects of long-term treatment with brood pheromone on suites of honey bee brood rearing and foraging behaviors . Pheromone-treated colonies reared significantly greater brood areas and more adults than controls, while amounts of stored pollen and honey remained statistically similar . Brood pheromone increased the number of pollen foragers and the pollen load weights they returned . It appeared that the pheromone-induced increase in pollen intake was directly canalized into more brood rearing . A two-way pheromone priming effect was observed, such that some workers from the same age cohorts showed an increased and extended capacity to rear larvae, while others were recruited at significantly younger ages into pollen-specific foraging . Brood pheromone affected suites of nursing and foraging behaviors allocating worker and pollen resources associated with an important fitness trait, colony growth. Bull Exp Biol Med, 2004 Apr, 137(4), 379 - 81 Modification of biological activity of lipopolysaccharide in the complex with chitosan; Ermak IM et al.; In the complex with chitosan, lipopolysaccharide partially lost its ability to induce lymphokines tumor necrosis factor and interleukin-8, but retained immunostimulating properties and increased phagocytic function of macrophages by improving digestion of bacteria. J Biol Chem, 2004 Dec 3, 279(49), 50904 - 14 Epub 2004 Dec 3. A cyclic peptide mimicking the third intracellular loop of the V2 vasopressin receptor inhibits signaling through its interaction with receptor dimer and G protein; Granier S et al.; In this study, we investigated the mechanism by which a peptide mimicking the third cytoplasmic loop of the vasopressin V2 receptor inhibits signaling . This loop was synthesized as a cyclic peptide (i3 cyc) that adopted defined secondary structure in solution . We found that i3 cyc inhibited the adenylyl cyclase activity induced by vasopressin or a nonhydrolyzable analog of GTP, guanosine 5'-O-(3-thio)triphosphate . This peptide also affected the specific binding of {3H}AVP by converting vasopressin binding sites from a high to a low affinity state without any effect on the global maximal binding capacity . The inhibitory actions of i3 cyc could also be observed in the presence of maximally uncoupling concentration of guanosine 5'-O-(3-thio)triphosphate, indicating a direct effect on the receptor itself and not exclusively on the interaction between the Gs protein and the V2 receptor (V2-R) . Bioluminescence resonance energy-transfer experiments confirmed this assumption, because i3 cyc induced a significant inhibition of the bioluminescence resonance energy-transfer signal between the Renilla reniformis luciferase and the enhanced yellow fluorescent protein fused V2-R . This suggests that the proper arrangement of the dimer could be an important prerequisite for triggering Gs protein activation . In addition to its effect on the receptor itself, the peptide exerted some of its actions at the G protein level, because it could also inhibit guanosine 5'-O-(3-thio)triphosphate-stimulated AC activity . Taken together, the data demonstrate that a peptide mimicking V2-R third intracellular loop affects both the dimeric structural organization of the receptor and has direct inhibitory action on Gs. Clin Immunol, 2004 Nov, 113(2), 130 - 6 Mycobacterial granulomas: keys to a long-lasting host-pathogen relationship; Co DO et al.; Chronic infection with mycobacteria is controlled by the formation of granulomas . The failure of granuloma maintenance results in reactivation of disease . Macrophages are the dominant cell type in granulomas, but CD4+ T cells are the master organizers of granuloma structure and function . Recent work points to an unrecognized role for nonspecific T cells in maintaining granuloma function in the chronic phase of infection . In addition, it has become clear that mycobacteria and host T cells collaborate in formation of granulomas . Further understanding of how nonspecific T cells contribute to granuloma formation, as well as how bacteria and T cells maintain a harmonious relationship over the life of the host, will facilitate the development of new strategies to treat mycobacterial disease. Dent Mater, 2004 Nov, 20(9), 814 - 22 Investigation of initial pellicle formation on modified titanium dioxide (TiO2) surfaces by reflectometric interference spectroscopy (RIfS) in a model system; Lange K et al.; OBJECTIVES: Plaque accumulation, leading to inflammatory processes and bone loss, is one of the main reasons for failure of dental implants . Pellicle formation plays a key role in bacterial adhesion and plaque accumulation . The influence of experimental bioactive implant surface coatings on the initial process of pellicle formation was investigated in a model system . METHODS: TiO2-films were modified by covalent binding of laminin and human epidermal growth factor (EGF) to promote adhesion of epithelial cells . Adsorption and dissociation behavior of bovine serum albumin (BSA) and salivary proteins on these surfaces were monitored by time-resolved reflectometric interference spectroscopy (RIfS) . RESULTS: The thickness of the irreversibly adsorbed salivary protein layer was reduced from 2.78 +/- 0.71 nm on unmodified TiO2 to 0.78 +/- 0.22 nm on laminin-coated surfaces and to 1.18 +/- 0.29 nm on EGF-coated surfaces . The percentage of initially adsorbed proteins remaining irreversibly bound was reduced from 51 +/- 8% on titanium to 23 +/- 5% by laminin coating and to 44 +/- 11% on EGF-coated surfaces . The highest reduction of protein adsorption (layer thickness lower than 0.05 nm) was achieved on DC-PEG-layers used as spacer for protein coupling . SIGNIFICANCE: Laminin and EGF were shown to be promising candidates for use as biological coatings on the transmucosal part of titanium dental implants where the objective is to enhance epithelial adhesion and inhibit adsorption of salivary proteins and bacteria. Antiviral Res, 2004 Oct, 64(1), 27 - 33 Inactivation of adenovirus types 5 and 6 by Virkon S; McCormick L et al.; Throughout the pharmaceutical industry, adenovirus-based products are being developed for human use as vaccine vectors and gene therapy delivery vehicles . The implementation of effective decontamination procedures is critical to the successful manufacture of these products to minimize the risk of personnel exposure and prevent product cross-contamination in the manufacturing facility . In this investigation, we have conducted small-scale decontamination studies to determine the efficacy of Virkon S on the inactivation of adenovirus types 5 and 6 in suspension . Virkon S is a commercially available oxidative disinfectant used against a variety of bacteria, spores, fungi, and viruses . A cytotoxicity-based endpoint dilution assay was used to quantify adenovirus potency before and after Virkon S treatment . We show that the level of organic content in the inactivation sample matrix has a significant impact on Virkon S activity . The potency of adenovirus types 5 and 6 was reduced by greater than six logs upon a five minute exposure to the appropriate concentration of Virkon S . Based on these results, we propose that Virkon S liquid decontamination procedures for adenovirus types 5 and 6 use 0.9% Virkon S for contact times greater than five minutes. Mutat Res, 2004 Oct 4, 554(1-2), 387 - 98 Comparison of the mutagenic activity of the benzene metabolites, hydroquinone and para-benzoquinone in the supF forward mutation assay: a role for minor DNA adducts formed from hydroquinone in benzene mutagenicity; Gaskell M et al.; Benzene, a ubiquitous environmental pollutant and occupational hazardous chemical, is a recognised human leukaemogen and rodent carcinogen . The mechanism by which benzene exerts its carcinogenic effects is to date unknown but it is considered that mutations induced by benzene-DNA adducts may play a role . The benzene metabolite, para-benzoquinone (p-BQ) following reaction in vitro with DNA, forms four major adducts, which include two adducts on 2'-deoxyguanosine 3'-monophosphate (dGp) . Reaction of DNA with the benzene metabolite hydroquinone (HQ) results in only one major DNA adduct, which corresponds to one of the dGp adducts formed following reaction with p-BQ . The mutagenicity of the adducts formed from these two benzene metabolites was investigated using the supF forward mutation assay . Metabolite-treated plasmid (pSP189) containing the supF gene was replicated in human Ad293 cells before being screened in indicator bacteria . Treatment with 5-20 mM p-BQ gave a 12 to 40-fold increase in mutation rate compared to 5-20 mM HQ treatment, a result reflected in the level of DNA modification observed (8 to 26-fold increase compared to HQ treatment) . Treatment with p-BQ gave equal numbers of GC --> TA transversions and GC --> AT transitions, whereas treatment with HQ gave predominantly GC-->AT transitions . The spectra of mutations achieved for the two individual treatments were shown to be significantly different (P = 0.004) . A combination of both treatments also resulted in a high level of GC --> AT transitions and a synergistic increase in the number of multiple mutations, which again predominated as GC --> AT transitions . Sites of mutational hotspots were observed for both individual treatments and one mutational hotspot was observed in the multiple mutations for the combined treatment . These results suggest that the dGp adducts formed from benzene metabolite treatment may play an important role in the mutagenicity and myelotoxicity of benzene. FEMS Microbiol Rev, 2004 Jun, 28(3), 291 - 318 Outer membrane proteins of pathogenic spirochetes; Cullen PA et al.; Pathogenic spirochetes are the causative agents of several important diseases including syphilis, Lyme disease, leptospirosis, swine dysentery, periodontal disease and some forms of relapsing fever . Spirochetal bacteria possess two membranes and the proteins present in the outer membrane are at the site of interaction with host tissue and the immune system . This review describes the current knowledge in the field of spirochetal outer membrane protein (OMP) biology . What is known concerning biogenesis and structure of OMPs, with particular regard to the atypical signal peptide cleavage sites observed amongst the spirochetes, is discussed . We examine the functions that have been determined for several spirochetal OMPs including those that have been demonstrated to function as adhesins, porins or to have roles in complement resistance . A detailed description of the role of spirochetal OMPs in immunity, including those that stimulate protective immunity or that are involved in antigenic variation, is given . A final section is included which covers experimental considerations in spirochetal outer membrane biology . This section covers contentious issues concerning cellular localization of putative OMPs, including determination of surface exposure . A more detailed knowledge of spirochetal OMP biology will hopefully lead to the design of new vaccines and a better understanding of spirochetal pathogenesis. J Biol Chem, 2004 Dec 3, 279(49), 51541 - 4 Epub 2004 Dec 3. Receptor-mediated reversible translocation of the G protein betagamma complex from the plasma membrane to the Golgi complex; Akgoz M et al.; Heterotrimeric G proteins have been thought to function on the plasma membrane after activation by transmembrane receptors . Here we show that, after activation by receptors, the G protein betagamma complex selectively translocates to the Golgi . Receptor inactivation results in Gbetagamma translocating back to the plasma membrane . Both translocation processes occur rapidly within seconds . The efficiency of translocation is influenced by the type of gamma subunit present in the G protein . Distinctly different receptor types are capable of inducing the translocation . Receptor-mediated translocation of Gbetagamma can spatially segregate G protein signaling activity. Acta Crystallogr D Biol Crystallogr, 2004 Oct, 60(Pt 10), 1874 - 6 Epub 2004 Sep 23. Cloning, expression, purification and crystallization of a transcriptional regulatory protein (Rv3291c) from Mycobacterium tuberculosis H37Rv; Shrivastava T et al.; Rv3291c, the translational product of the Mycobacterium tuberculosis Rv3291c gene, is an 18 kDa protein . It is a putative transcriptional regulatory protein belonging to the leucine-responsive regulatory protein/asparagine synthase C (Lrp/AsnC) family, which are proteins that have been identified in archaea and bacteria . Rv3291c probably plays a significant role during the persistent/latent phase of M . tuberculosis, as supported by its up-regulation several-fold during this stage . Orthorhombic crystals of recombinant Rv3291c have been grown from trisodium citrate dihydrate-buffered solutions containing monoammonium dihydrogen phosphate . Diffraction data extending to 2.7 A have been collected from a single crystal with unit-cell parameters a = 99.6, b = 100.7, c = 100.6 A . Assuming an octamer in the asymmetric unit results in a Matthews coefficient (VM) of 1.75 A3 Da(-1), corresponding to a solvent content of about 30%. Nucleic Acids Res, 2004 Sep 23, 32(17), 4969 - 78 Print 2004. Transcription-coupled and splicing-coupled strand asymmetries in eukaryotic genomes; Touchon M et al.; Under no-strand bias conditions, each genomic DNA strand should present equimolarities of A and T and of G and C . Deviations from these rules are attributed to asymmetric properties intrinsic to DNA mutation-repair processes . In bacteria, strand biases are associated with replication or transcription . In eukaryotes, recent studies demonstrate that human genes present transcription-coupled biases that might reflect transcription-coupled repair processes . Here, we study strand asymmetries in intron sequences of evolutionarily distant eukaryotes, and show that two superimposed intron biases can be distinguished . (i) Biases that are maximum at intron extremities and decrease over large distances to zero values in internal regions, possibly reflecting interactions between pre-mRNA and splicing machinery; these extend over approximately 0.5 kb in mammals and Arabidopsis thaliana, and over 1 kb in Caenorhabditis elegans and Drosophila melanogaster . (ii) Biases that are constant along introns, possibly associated with transcription . Strikingly, in C.elegans, these latter biases extend over intergenic regions that separate co-oriented genes . When appropriately examined, all genomes present transcription-coupled excess of T over A in the coding strand . On the opposite, GC skews are either positive (mammals, plants) or negative (invertebrates) . These results suggest that transcription-coupled asymmetries result from mutation-repair mechanisms that differ between vertebrates and invertebrates. Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1567 - 80 Taxonomic heterogeneity within the Planctomycetales as derived by DNA-DNA hybridization, description of Rhodopirellula baltica gen . nov., sp . nov., transfer of Pirellula marina to the genus Blastopirellula gen . nov . as Blastopirellula marina comb . nov . and emended description of the genus Pirellula; Schlesner H et al.; Ninety-seven strains of budding bacteria originating from various aquatic habitats and morphologically resembling planctomycetes were investigated taxonomically . Taxonomic differentiation was based on DNA-DNA hybridization, physiological properties and chemotaxonomic tests . Nineteen hybridization groups, containing 79 of the tested strains, were established . Eighteen strains, however, did not fit into any of these groups . Rhodopirellula baltica gen . nov., sp . nov . is described, with strain SH 1T (= IFAM 1310T = DSM 10527T = NCIMB 13988T) as the type strain . Pirellula marina is transferred to the genus Blastopirellula gen . nov . as Blastopirellula marina comb . nov., with strain SH 106T (= IFAM 1313T = DSM 3645T = ATCC 49069T) as the type strain . An emended description of the genus Pirellula is also provided . Differentiation between R . baltica, B . marina and Pirellula staleyi was achieved by the integration of morphological, physiological, chemotaxonomic and genetic characteristics. Biochimie, 2004 Aug, 86(8), 509 - 18 Characterization of a cytosolic malate dehydrogenase cDNA which encodes an isozyme toward oxaloacetate reduction in wheat; Ding Y et al.; Malate dehydrogenase (MDH), which is ubiquitous in nature, catalyzes the interconversion of oxaloacetate and malate . Higher plants contain multiple forms of MDH that differ in co-enzyme specificity, subcellular localization and physiological function . Cytosolic NAD-dependent MDH (cyMDH) is one class of MDH that has not been extensively characterized in plants . Here we report the cloning of a cDNA from wheat by RT-PCR and cDNA library screening, which is designated as TaMDH . Sequence analysis indicated that TaMDH exhibits a highly similarity to other plant cyMDHs . Immunological analysis confirmed that TaMDH encoded a cytosolic NAD-dependent MDH . The secondary and three-dimensional structures of TaMDH were analyzed by molecular modeling . DNA gel-blot analyses demonstrated that TaMDH gene exists as two copies in the wheat genome . RNA and protein gel-blot hybridization indicated that both TaMDH mRNA and protein were constitutively expressed in vegetative tissues of wheat, with slightly lower levels in roots than in leaves and stems . In silico analysis indicated that TaMDH was also expressed in various reproductive tissues and tissues under many different stress conditions . Kinetic analysis of bacterially expressed and purified protein confirmed that TaMDH catalyzed a reaction driven towards malate synthesis, which is consistent with other cyMDHs . Evolutionary analysis showed that this class of genes evolved from a very ancestral gene . The cyMDH represents an ancestral form of MDH, which is highly conserved in plants, animals and bacteria . This implies that cyMDHs are housekeeping genes and may have very essential functions in plant metabolism. Mol Microbiol, 2004 Sep, 53(5), 1501 - 13 Analysis of the Frz signal transduction system of Myxococcus xanthus shows the importance of the conserved C-terminal region of the cytoplasmic chemoreceptor FrzCD in sensing signals; Bustamante VH et al.; The Frz chemosensory system controls directed motility in Myxococcus xanthus by regulating cellular reversal frequency . M . xanthus requires the Frz system for vegetative swarming on rich media and for cellular aggregation during fruiting body formation on starvation media . The Frz signal transduction pathway is formed by proteins that share homology with chemotaxis proteins from enteric bacteria, which are encoded in the frzA-F putative operon and the divergently transcribed frzZ gene . FrzCD, the Frz system chemoreceptor, contains a conserved C-terminal module present in methyl-accepting chemotaxis proteins (MCPs); but, in contrast to most MCPs, FrzCD is localized in the cytoplasm and the N-terminal region of FrzCD does not contain transmembrane or sensing domains, or even a linker region . Previous work on the Frz system was limited by the unavailability of deletion strains . To understand better how the Frz system functions, we generated a series of in-frame deletions in each of the frz genes as well as regions encoding the N-terminal portion of FrzCD . Analysis of mutants containing these deletions showed that FrzCD (MCP), FrzA (CheW) and FrzE (CheA-CheY) control vegetative swarming, responses to repellents and directed movement during development, thus constituting the core components of the Frz pathway . FrzB (CheW), FrzF (CheR), FrzG (CheB) and FrzZ (CheY-CheY) are required for some but not all responses . Furthermore, deletion of approximately 25 amino acids from either end of the conserved C-terminal region of FrzCD results in a constitutive signalling state of FrzCD, which induces hyper-reversals with no net cell movement . Surprisingly, deletion of the N-terminal region of FrzCD shows only minor defects in swarming . Thus, signal input to the Frz system must be sensed by the conserved C-terminal module of FrzCD and not the usual N-terminal region . These results indicate an alternative mechanism for signal sensing with this cytoplasmic MCP . Curr Microbiol, 2004 Oct, 49(4), 288 - 94 Isolation and characterization of dibenzofuran-degrading Comamonas sp . strains isolated from white clover roots; Wang Y et al.; Three dibenzofuran (DF)-degrading strains were newly isolated from roots of white clover ( Trifolium repens L.) and poplar trees grown in DF-contaminated soil samples . These strains, designated KD2, KD7, and PD1, were characterized as Comamonas sp . on the basis of their 16S rDNA sequences and physiological characteristics . The metabolites produced when strain KD7 was incubated with DF were identified by gas chromatography-mass spectrometry (GC-MS) analysis . Interestingly, strain KD7 was found to have two pathways for DF degradation, beginning with angular dioxygenation at carbons 4 and 4a, and lateral dioxygenation at carbons 1 and 2, respectively . Furthermore, strains KD2 and KD7 not only achieved efficient root colonization in clover but also promoted clover growth . They are the first reported Comamonas sp . strains capable of utilizing DF as a sole carbon source . This provides additional information on the diversity of DF-degrading bacteria. Curr Microbiol, 2004 Sep, 49(3), 203 - 7 Siderophore activity among members of the Legionella genus; Starkenburg SR et al.; Members of the Legionella genus are ubiquitous aquatic bacteria and the etiologic agents of Legionnaires' disease, a potentially fatal form of pneumonia . Using the chrome azurol S (CAS) assay, we previously determined that Legionella pneumophila secretes a siderophore (legiobactin) when it is grown in a low-iron, chemically defined medium (CDM) . In the present study, we examined 29 other species of Legionella for their ability to produce CAS-reactive material when grown in deferrated CDM . Although some of the species did not grow in CDM, the majority replicated and secreted CAS reactivity, suggesting that siderophores are conserved among the legionellae. Nature, 2004 Sep 23, 431(7007), 471 - 6 Exogenous control of mammalian gene expression through modulation of RNA self-cleavage; Yen L et al.; Recent studies on the control of specific metabolic pathways in bacteria have documented the existence of entirely RNA-based mechanisms for controlling gene expression . These mechanisms involve the modulation of translation, transcription termination or RNA self-cleavage through the direct interaction of specific intracellular metabolites and RNA sequences . Here we show that an analogous RNA-based gene regulation system can effectively be designed for mammalian cells via the incorporation of sequences encoding self-cleaving RNA motifs into the transcriptional unit of a gene or vector . When correctly positioned, the sequences lead to potent inhibition of gene or vector expression, owing to the spontaneous cleavage of the RNA transcript . Administration of either oligonucleotides complementary to regions of the self-cleaving motif or a specific small molecule results in the efficient induction of gene expression, owing to inhibition of self-cleavage of the messenger RNA . Efficient regulation of transgene expression is shown in a variety of mammalian cell lines and live animals . In conjunction with other emerging technologies, this methodology may be particularly applicable to the development of gene regulation systems tailored to any small inducer molecule, and provide a novel means of biological sensing in vivo that may have an important application in the regulated delivery of protein therapeutics. Ned Tijdschr Tandheelkd, 2004 Aug, 111(8), 311 - 5 {Pain after cementation of cast restorations}; Gerritsen AE; Some patients experience pain after cementation of a cast restoration on a vital tooth . Limited data are available on the prevalence of this pain . In many cases the pain disappears within two weeks . The major cause of the pain might be attributed to more permeability of the dentine after preparation . Consequently, bacteria have free access towards the pulp . With respect to the cement, it is more likely that microleakage after cementation will be the cause of pulpal problems than toxicity of the cement . In order to prevent pain, the use of a desensitizer (a primer or a bonding) can be considered . However, use of a desensitizer in combination with some cements, can reduce the retention of a cast restoration . More important than the use of a desensitizer seem precautions during the procedures of preparing the restoration, like using sufficient cooling water and preventing a tooth from dehydration. J Ky Med Assoc, 2004 Aug, 102(8), 349 - 52 Delayed external ventriculoperitoneal shunt infection; Owen R et al.; Infection is a common complication of ventriculoperitoneal shunt placement . About 10% of shunt infections are found more than one year after operation . We recently treated two children with what appears to be a unique manifestation of late shunt infection: chronic, draining sinuses along the course of the shunt tubing . Neither child had a shunt malfunction or a CSF (cerebral spinal fluid) infection . The infections were external and responded to removal of the infected shunt system . These cases are described, and delayed and external shunt infections are discussed. PLoS Biol . 2004 Oct;2(10):e303 . Epub 2004 Sep 21. Genomic insights into methanotrophy: the complete genome sequence of Methylococcus capsulatus (Bath); Ward N et al.; Methanotrophs are ubiquitous bacteria that can use the greenhouse gas methane as a sole carbon and energy source for growth, thus playing major roles in global carbon cycles, and in particular, substantially reducing emissions of biologically generated methane to the atmosphere . Despite their importance, and in contrast to organisms that play roles in other major parts of the carbon cycle such as photosynthesis, no genome-level studies have been published on the biology of methanotrophs . We report the first complete genome sequence to our knowledge from an obligate methanotroph, Methylococcus capsulatus (Bath), obtained by the shotgun sequencing approach . Analysis revealed a 3.3-Mb genome highly specialized for a methanotrophic lifestyle, including redundant pathways predicted to be involved in methanotrophy and duplicated genes for essential enzymes such as the methane monooxygenases . We used phylogenomic analysis, gene order information, and comparative analysis with the partially sequenced methylotroph Methylobacterium extorquens to detect genes of unknown function likely to be involved in methanotrophy and methylotrophy . Genome analysis suggests the ability of M . capsulatus to scavenge copper (including a previously unreported nonribosomal peptide synthetase) and to use copper in regulation of methanotrophy, but the exact regulatory mechanisms remain unclear . One of the most surprising outcomes of the project is evidence suggesting the existence of previously unsuspected metabolic flexibility in M . capsulatus, including an ability to grow on sugars, oxidize chemolithotrophic hydrogen and sulfur, and live under reduced oxygen tension, all of which have implications for methanotroph ecology . The availability of the complete genome of M . capsulatus (Bath) deepens our understanding of methanotroph biology and its relationship to global carbon cycles . We have gained evidence for greater metabolic flexibility than was previously known, and for genetic components that may have biotechnological potential. PLoS Biol . 2004 Oct;2(10):e320 . Epub 2004 Sep 21. Mechanism of association and reciprocal activation of two GTPases; Shan SO et al.; The signal recognition particle (SRP) mediates the cotranslational targeting of nascent proteins to the eukaryotic endoplasmic reticulum membrane or the bacterial plasma membrane . During this process, two GTPases, one in SRP and one in the SRP receptor (named Ffh and FtsY in bacteria, respectively), form a complex in which both proteins reciprocally activate the GTPase reaction of one another . Here, we explore by site-directed mutagenesis the role of 45 conserved surface residues in the Ffh-FtsY interaction . Mutations of a large number of residues at the interface impair complex formation, supporting the importance of an extensive interaction surface . Surprisingly, even after a stable complex is formed, single mutations in FtsY can block the activation of GTP hydrolysis in both active sites . Thus, activation requires conformational changes across the interface that coordinate the positioning of catalytic residues in both GTPase sites . A distinct class of mutants exhibits half-site reactivity and thus allows us to further uncouple the activation of individual GTPases . Our dissection of the activation process suggests discrete conformational stages during formation of the active SRP*SRP receptor complex . Each stage provides a potential control point in the targeting reaction at which regulation by additional components can be exerted, thus ensuring the binding and release of cargo at the appropriate time. Blood, 2005 Jan 15, 105(2), 742 - 9 Epub 2004 Sep 21. Selective Rac1 inhibition in dendritic cells diminishes apoptotic cell uptake and cross-presentation in vivo; Kerksiek KM et al.; To better understand the influence of cytoskeletal regulation on dendritic cell (DC) function in vivo, the Rho guanosine triphosphatase (GTPase) Rac1 was selectively inhibited in DCs in transgenic (Tg) mice . Although transgene expression did not interfere with the migratory capacities of DC in vivo, a decreased uptake of fluorescent probes was observed . Interestingly, the absence of full Rac1 function most strongly affected the development and function of CD8(+) DCs . Apoptotic cell uptake was severely reduced in Tg mice, impairing subsequent DC-mediated cross-presentation and priming of bacteria-specific T-cell responses . These findings highlight a special role for Rac1 in the capacity of CD8(+) DCs to endocytose apoptotic cells and prime T cells via cross-presentation. IEEE Trans Nanobioscience, 2003 Mar, 2(1), 40 - 3 Preliminary evaluation of nanoscale biogenic magnetite-based ferromagnetic transduction mechanisms for mobile phone bioeffects; Cranfield C et al.; Ferromagnetic transduction models have been proposed as a potential mechanism for mobile phone bioeffects . These models are based on the coupling of RF and pulsed electromagnetic emissions to biogenic magnetite (Fe3O4) present in the human brain via either ferromagnetic resonance or mechanical activation of cellular ion channels . We have tested these models experimentally for the first time using a bacterial analogue (Magnetospirillum magnetotacticum) which produces intracellular biogenic magnetite similar to that present in the human brain . Experimental evaluation revealed that exposure to mobile phone emissions resulted in a consistent and significantly higher proportion of cell death in exposed cultures versus sham exposure (p = 0.037) . Though there appears to be a repeatable trend toward higher cell mortality in magnetite-producing bacteria exposed to mobile phone emissions, it is not yet clear that this would extrapolate to a deleterious health effect in humans. Am J Trop Med Hyg, 2004 Sep, 71(3), 363 - 6 Detection of Rickettsia africae in patients and ticks along the coastal region of Cameroon; Ndip LM et al.; Rickettsia africae was identified in seven (6%) of 118 patients with acute fevers of unknown etiology proven not to be malaria or typhoid fever from clinics along the coastal region of Cameroon by polymerase chain reaction (PCR) amplification and sequencing of the citrate synthase (gltA) and outer membrane protein A (ompA) genes of Rickettsia . The majority (71%) of the patients were female . Clinical manifestations included fever (100%), headache (71%), myalgia (71%), arthralgia (43%), pulmonary involvement (29%), and diffuse rash (14%) . Moreover, R . africae was detected by PCR amplification and sequence analysis of the gltA and ompA genes in 62 (75%) of 83 adult Amblyomma variegatum ticks collected from cattle in the same region . These results confirm the presence of a previously unrecognized infectious disease in the indigenous Cameroonian population, as well as extend the established range of R . africae. J Dent Res, 2004 Oct, 83(10), 786 - 90 Oral epithelial overexpression of IL-1alpha causes periodontal disease; Dayan S et al.; Periodontal disease is a bacterial infection that results in inflammatory destruction of tissues that support the teeth, including connective tissue and bone . In this study, we report that transgenic mice that overexpress the 17-kDa form of IL-1alpha in the basal layer of oral mucosal epithelium develop a syndrome that possesses all of the cardinal features of periodontal disease, including epithelial proliferation and apical migration, loss of attachment, and destruction of cementum and alveolar bone . In this model, bacterial colonization and infection were not required, since levels of periodontal bacteria were equivalent in transgenic and wild-type mice, and continuous treatment with antibiotics from birth did not ameliorate the disease . Our findings therefore indicate that elevated levels of IL-1alpha in the oral micro-environment can mediate all of the clinical features of periodontal disease. Vet Microbiol, 2004 Oct 5, 103(1-2), 121 - 6 Occurrence of Chlamydiaceae spp . in a wild boar (Sus scrofa L.) population in Thuringia (Germany); Hotzel H et al.; Tissue samples from lungs, pulmonary lymph nodes, large intestine, and uteri of 14 wild boar bagged at a seasonal hunt were examined for the presence of chlamydiae, mycobacteria and mycoplasmas . Nested PCR detected chlamydial DNA in 57.1% of the animals, predominantly in the lung . DNA sequencing identified Chlamydophila psittaci as the predominant species, but Chlamydophila abortus and Chlamydia suis were also encountered . Immunohistochemical staining of tissue sections confirmed the presence of typical chlamydial inclusions in lungs and uteri . While the role of Chlamydiaceae as pathogens in wild boar has yet to be established, the present findings revealed a possible wildlife reservoir of these bacteria. Water Res, 2004 Nov, 38(18), 3845 - 58 Xenobiotic organic compounds in leachates from ten Danish MSW landfills--chemical analysis and toxicity tests; Baun A et al.; A monitoring program comprising chemical analysis and biological toxicity testing of leachate samples from 10 Danish landfills (six engineered and four uncontrolled) revealed the presence of 55 different xenobiotic organic compounds (XOCs) and 10 degradation products of XOCs . The compounds belong to the following groups: BTEX, C3-benzenes, bicyclo compounds, napthalenes, chlorinated aliphatics, phenols (chloro-, methyl-, dimethyl, nonyl-), pesticides, and phthalates . Concentrations of single XOCs ranged from <0.1 to 2220 microg/L . A pesticide screening including 101 different compounds resulted in detection of 18 pesticides and three degradation products . The findings of degradation products of toluene, phenols, phthalates, pesticides, and nonylphenol ethoxylates show that degradation occurred inside the landfills . In biotests with bacteria and algae it was found that the non-volatile organic compounds were toxic as the samples only needed to be pre-concentrated from 1.3 to 9.4 times to give 50% inhibition of the test organisms . One of the ten samples proved to be genotoxic in the umuC test after 141 times pre-concentration . A major part of the organic chemicals causing toxicity remains unknown and it is recommended to combine chemical analyses and biotests in future monitoring programs. Trends Endocrinol Metab, 2004 Oct, 15(8), 393 - 7 Oxysteroids: a new class of steroids with autocrine and paracrine functions; Javitt NB; Oxysteroids are a new classification for sterol intermediates in cholesterol synthesis that undergo enzyme-catalyzed stereo-specific 25R,26-hydroxylation and thus bypass cholesterol as the expected end-product . Recently, they were identified in micromolar amounts in the plasma of patients with Smith-Lemli-Opitz syndrome (SLOS) . An additional three oxysteroids, the 25,26-hydroxy derivatives of lanosterol, zymosterol, and desmosterol, respectively, were generated in vitro by CYP27A1-transfected bacteria . As there are 19 steps between cholesterol and lanosterol, the first post-squalene sterol, a potentially large class of oxysteroids exists . Limited studies of 25r,26-7-dehydrocholesterol indicate a traditional role as a ligand for nuclear receptors, but complete evaluation of oxysteroids for novel biologic activities is lacking . Currently, the lack of authentic oxysteroid standards limits both their detection in biologic fluids and evaluation of their biologic effects. Microbes Infect, 2004 Oct, 6(12), 1110 - 6 Parasites and individual major histocompatibility complex diversity--an optimal choice? Wegner KM, Kalbe M, Schaschl H, Reusch TB. Overdominant selection or heterozygote advantage can partly explain the extraordinary polymorphism found at classical major histocompatibility complex (MHC) loci . However, several studies employing only single infectious agents often failed to detect it . Here, we review recent studies suggesting that due to the dominant nature of MHC-mediated resistance, a heterozygote advantage is most likely to be detected in multiple pathogen challenges. Hua Xi Kou Qiang Yi Xue Za Zhi, 2004 Aug, 22(4), 293 - 4, 297 {Comparison of preventive effects of two kinds of dental handpieces on viral contamination at different rotating times}; Hu T et al.; OBJECTIVE: It has been demonstrated that when a high-speed handpiece stops rotating, negative pressure will form . Thus, contaminating fluid in which there are many kinds of bacteria and viruses from the external environment will retract into various compartments of the handpiece and the dental unit . The purpose of the study is to compare the preventing effect of antisuction designed handpiece and conventional handpiece on viral contamination at different rotating times . METHODS: Twenty handpieces with or without antisuction device (10 of each) were used in the study . Each handpiece was submerged into 10(-6) microg/microl HBV particle solution rotating 5 and 10 times respectively (every time rotating for 10 seconds) . Samples were obtained from the water line and chip air line of the handpieces and examined by RT-PCR . RESULTS: At the same rotating times, there was statistical significance of the viral concentration between the two kinds of handpieces (P < 0.05) . However, there was no statistical significance of the viral concentration between different rotating times in each group (P > 0.05) . CONCLUSION: Contamination taking place in both water and air lines of dental handpiece was not enhanced by increasing the number of rotating times of the handpiece . The antisuction devices installed into the water line and chip air line were demonstrated to prevent viral contamination effectively. Orthop Nurs, 2004 Jul-Aug, 23(4), 267 - 73 Reactive arthritis . Immune-mediated synovitis or joint infection; Childs SG; Reactive arthritis is one form of the seronegative Spondyloarthropathies . Susceptibility to reactive arthritis is closely linked to individuals who have the genetic predisposition to the HLA-B27 allele (gene form) . Although there is a reactive-inflammatory joint reaction present, the synovium is not damaged by infectious agents (bacteria, fungi, or virus) . This article discusses the pathogenesis of reactive arthritis. Proteomics, 2004 Oct, 4(10), 2961 - 8 The Helicobacter pylori CagA protein induces tyrosine dephosphorylation of ezrin; Selbach M et al.; Helicobacter pylori is one of the most wide-spread bacterial pathogens and infects the human stomach to cause diseases, such as gastritis, gastric ulceration, and gastric cancer . A major virulence determinant is the H . pylori CagA protein (encoded by the cytotoxin-associated gene A) which is translocated from the bacteria into the cytoplasm of host cells by a type IV secretion system . In the host cell, CagA is phosphorylated on tyrosine residues and induces rearrangements of the actin cytoskeleton . We have previously shown that tyrosine-phosphorylated CagA inhibits the catalytic activity of Src family kinases and induces tyrosine dephosphorylation of several host cell proteins . Here, we identified one of these proteins as ezrin by a combination of preparative gel electrophoresis, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) . Specific pharmacological inhibition of Src family kinases also induces ezrin dephosphorylation . Therefore, ezrin dephosphorylation appears to be induced by CagA-mediated Src inactivation . Ezrin is the founding member of the ezrin-radixin-moesin (ERM) family of proteins which are signalling integrators at the cell cortex . Since ezrin is a component of microvilli and a linker protein between actin filaments and membrane proteins, this observation has important implications for H . pylori pathogenesis and might also help to explain the development of gastric cancer. J Basic Microbiol, 2004, 44(5), 360 - 73 Isolation of mak1 from Actinoplanes missouriensis and evidence that Pep2 from Streptomyces coelicolor is a maltokinase; Jarling M et al.; The gene mak1FN coding for maltokinase from Actinoplanes missouriensis is located in a cluster similar to glycogen metabolism clusters identified in Streptomyces coelicolor . Sequence comparisons demonstrate that mak1-related genes coding for homologous proteins are present in many bacterial genomes including taxonomic distantly related groups such as Rhodospirillales or green sulfur bacteria . More than 50% of the aligned sequences are longer than the mak1 gene from A . missouriensis, and the N-terminal portion of these putative maltokinases exhibit high sequence homologies with trehalose synthases . A more detailed sequence comparison indicates a relationship of maltokinases to aminoglycoside phospho-transferases and protein kinases . Transformation of S . lividans with plasmid vectors containing either the mak1 gene from A . missouriensis or the pep2 gene from S . coelicolor resulted in recombinant strains, which produced measurable amounts of maltokinase activity . The proteins Pep2 and Mak1 were over expressed with Streptomyces lividans 66 as a heterologous host and further characterized . The possible physiological function of maltokinases is discussed . Pediatr Radiol, 2004 Nov, 34(11), 876 - 85 Epub 2004 Sep 17. Definitive neuroradiological diagnostic features of tuberculous meningitis in children; Andronikou S et al.; BACKGROUND: Although CT scanning is used widely for making the diagnosis and detecting the complications of tuberculous meningitis (TBM) in children, the radiological features are considered non-specific . CT is particularly suggestive of the diagnosis when there is a combination of basal enhancement, hydrocephalus and infarction, and even then the diagnosis may be in doubt . In this paper we introduce a new CT feature for making the diagnosis of TBM, namely, hyperdensity in the basal cisterns on non-contrast scans, and we assess which of the recognized CT features is most sensitive and specific . OBJECTIVE: To determine the sensitivity and specificity of the presence of high-density exudates in the basal cisterns (on non-contrast CT) and basal enhancement (on contrast-enhanced CT) for the diagnosis of TBM in children, and to correlate these with the complications of infarction and hydrocephalus . MATERIALS AND METHODS: Retrospective review of CT scans with readers blinded to the diagnosis, which was based on a definitive culture of cerebrospinal fluid (CSF) for TBM or other bacteria . Computer-aided conversion of hard-copy film density to Hounsfield units was employed as well as a density threshold technique for determining abnormally high densities . Results: The most specific feature for TBM is hyperdensity in the basal cisterns prior to IV contrast medium administration (100%) . The most sensitive feature of TBM is basal enhancement (89%) . A combination of features (hydrocephalus, infarction and basal enhancement) is as specific as pre-contrast hyperdensity, but has a lower sensitivity (41%) . There were statistically significant differences in the presence of hydrocephalus (p=0.0016), infarcts (P=0.0014), basal enhancement (P<0.0001) and pre-contrast density (P<0.0001) between the negative and positive TBM patient groups . The presence of granulomas was not statistically significant between the two groups (P=0.44) . CONCLUSIONS: The presence of high density within the basal cisterns on non-contrast CT scans is a very specific sign for TBM in children . This will enhance diagnostic confidence, allow early institution of therapy and could reduce expenditure on contrast medium, scan time and radiation exposure . With the use of threshold techniques we believe that the pre-contrast hyperdensity may be detectable by a computer program that will facilitate diagnosis, and may also be modified to detect abnormal enhancement . Basal enhancement is a sensitive sign for the diagnosis of TBM and should be sought after contrast medium administration when no hyperdensity is seen in the basal cisterns or when this finding needs to be confirmed . The CT scan feature of hyperdense exudates on pre-contrast scans should be added to the inclusion criteria for the diagnosis of TBM in children. J Biol Chem, 2004 Nov 26, 279(48), 49787 - 94 Epub 2004 Nov 26. The N-terminal region of the CCAAT displacement protein (CDP)/Cux transcription factor functions as an autoinhibitory domain that modulates DNA binding; Truscott M et al.; The CCAAT displacement protein/Cut homeobox (CDP/Cux) transcription factor is expressed as multiple isoforms that may contain up to four DNA-binding domains: Cut repeats 1, 2, and 3 (CR1, CR2, CR3) and the Cut homeodomain (HD) . The full-length protein, which contains all four DNA-binding domains, is surprisingly less efficient than the shorter isoforms in DNA binding . Using a panel of recombinant proteins expressed in mammalian or bacterial cells, we have identified a domain at the extreme N terminus of the protein that can inhibit DNA binding . This domain was able to inhibit the activity of full-length CDP/Cux and of proteins containing various combinations of DNA-binding domains: CR1CR2, CR3HD, or CR2CR3HD . Since inhibition of DNA binding was also observed with purified proteins obtained from bacteria, we conclude that autoinhibition does not require post-translational modification or interaction with an interacting protein but instead functions through an intramolecular mechanism . Antibodies directed against the N-terminal region were able to partially relieve inhibition . In vivo, the transition between the inactive and active states for DNA binding is likely to be governed by posttranslational modifications and/or interaction with one or more protein partners . In addition, we show that the relief of autoinhibition can be accomplished via the proteolytic processing of CDP/Cux . Altogether, these results reveal a novel mode of regulation that serves to modulate the DNA binding activity of CDP/Cux. Rev Med Chil, 2004 Mar, 132(3), 381 - 7 {Spotted fever Rickettsiae . Their immunodominant proteins and immune response}; Zavala J et al.; The organisms of Rickettsia species are gram (-) bacteria that cause severe illnesses in humans and are an important health problem in several countries around the world, including Mexico . The presence of different antigens between species and also in different strains of the same specie is an obstacle for vaccine development and serological diagnosis . There are important advances in the knowledge of the rickettsial antigenic structure and the resulting immune response in infected hosts, including humans . This review covers these topics and provides an overview about the development of vaccines and accessible diagnostic methods for diseases caused by Rickettsia. Am J Ind Med, 2004 Oct, 46(4), 400 - 3 Metalworking fluid bioaerosols at selected workplaces in a steelworks; Gorny RL et al.; BACKGROUND: Exposure to metalworking fluid (MWF) aerosols has been shown to be associated with a variety of respiratory and skin diseases of workers . METHODS: Measurements of particulate aerosols, bacterial bioaerosol, and endotoxins and 3-hydroxy fatty acids (3-OH FAs) content in water-based and straight oil MWFs were done during an 8 hr work shift in the grindery and oil basement sections of a steelworks located in Upper Silesia, Poland . RESULTS: Particulate aerosol concentrations were below NIOSH recommended occupational exposure level . Differences in concentration and taxonomical composition of airborne bacteria between grindery and oil basement were found, due to the more aggressive creation of oil mist during machining operations and different MWF characteristics, which favor growth of different bacterial strains . CONCLUSIONS: The GC-MS analysis of 3-OH FAs as a marker of endotoxin contamination of the MWF and of the air seems to be a promising tool for evaluation of occupational exposure to bacterial bioaerosols. Histol Histopathol, 2004 Oct, 19(4), 1219 - 26 Helicobacter pylori (H . pylori) molecular signature in conjunctival mucosa-associated lymphoid tissue (MALT) lymphoma; Chan CC et al.; Conjunctival mucosa-associated lymphoid tissue (MALT) lymphoma is an extranodal marginal zone B-cell lymphoma that is characterized by an exaggerated clonal expansion of B cells, which implicate a pathological proliferative response to antigen(s) including bacteria . Helicobacter pylori (H . pylori) infection is recognized as one of the causative agents of gastric MALT lymphoma; however, it has not been reported in extra gastric MALT lymphoma . We studied 5 patients (4 adults and 1 child) with salmon-colored conjunctival lesions . One patient also had a history of abnormal bone marrow biopsy a year earlier with lymphoid aggregates involving 5% of the overall bone marrow . The conjunctival lesions of the 5 patients were biopsied . Histopathological diagnoses were consistent with conjunctival MALT lymphoma . Lymphoma and normal conjunctival cells were microdissected using laser capture microscopy or manual techniques . DNA was extracted and subjected to PCR amplification using H . pylori gene-specific primers from the urease B and vac/m2 gene . Cells from chronic conjunctivitis (normal lymphocytes), conjunctival human T-cell lymphotropic virus type-1/adult T-cell leukemia/lymphoma (HTLV-1/ATL), and orbital B-cell lymphoma were also microdissected, processed and analyzed . PCR amplification and Southern blot hybridization demonstrated H . pylori DNA in the conjunctival MALT lymphoma cells of 4/5 cases . The negative case was the one with a history of abnormal bone marrow . In contrast, H . pylori gene was not detected in normal conjunctival cells from the cases of MALT lymphoma or the lymphocytes, ATL and orbital B-lymphoma cells from the controls . These data suggest that H . pylori may play a role in conjunctival MALT lymphoma. Plant Biol (Stuttg), 2004 Sep, 6(5), 537 - 44 Origin and evolution of the light-dependent protochlorophyllide oxidoreductase (LPOR) genes; Yang J et al.; Light-dependent NADPH-protochlorophyllide oxidoreductase (LPOR) is a nuclear-encoded chloroplast protein in green algae and higher plants which catalyzes the light-dependent reduction of protochlorophyllide to chlorophyllide . Light-dependent chlorophyll biosynthesis occurs in all oxygenic photosynthetic organisms . With the exception of angiosperms, this pathway coexists with a separate light-independent chlorophyll biosynthetic pathway, which is catalyzed by light-independent protochlorophyllide reductase (DPOR) in the dark . In contrast, the light-dependent function of chlorophyll biosynthesis is absent from anoxygenic photosynthetic bacteria . Consequently, the question is whether cyanobacteria are the ancestors of all organisms that conduct light-dependent chlorophyll biosynthesis . If so, how did photosynthetic eukaryotes acquire the homologous genes of LPOR in their nuclear genomes? The large number of complete genome sequences now available allow us to detect the evolutionary history of LPOR genes by conducting a genome-wide sequence comparison and phylogenetic analysis . Here, we show the results of a detailed phylogenetic analysis of LPOR and other functionally related enzymes in the short chain dehydrogenase/reductase (SDR) family . We propose that the LPOR gene originated in the cyanobacterial genome before the divergence of eukaryotic photosynthetic organisms . We postulated that the photosynthetic eukaryotes obtained their LPOR homologues through endosymbiotic gene transfer. J Bacteriol, 2004 Oct, 186(19), 6626 - 33 Coexistence of Wolbachia with Buchnera aphidicola and a secondary symbiont in the aphid Cinara cedri; Gomez-Valero L et al.; Intracellular symbiosis is very common in the insect world . For the aphid Cinara cedri, we have identified by electron microscopy three symbiotic bacteria that can be characterized by their different sizes, morphologies, and electrodensities . PCR amplification and sequencing of the 16S ribosomal DNA (rDNA) genes showed that, in addition to harboring Buchnera aphidicola, the primary endosymbiont of aphids, C . cedri harbors a secondary symbiont (S symbiont) that was previously found to be associated with aphids (PASS, or R type) and an alpha-proteobacterium that belongs to the Wolbachia genus . Using in situ hybridization with specific bacterial probes designed for symbiont 16S rDNA sequences, we have shown that Wolbachia was represented by only a few minute bacteria surrounding the S symbionts . Moreover, the observed B . aphidicola and the S symbionts had similar sizes and were housed in separate specific bacterial cells, the bacteriocytes . Interestingly, in contrast to the case for all aphids examined thus far, the S symbionts were shown to occupy a similarly sized or even larger bacteriocyte space than B . aphidicola . These findings, along with the facts that C . cedri harbors the B . aphidicola strain with the smallest bacterial genome and that the S symbionts infect all Cinara spp . analyzed so far, suggest the possibility of bacterial replacement in these species. J Dairy Sci, 2004 Sep, 87(9), 3010 - 6 Effects of amide-protected and lipid-encapsulated conjugated linoleic acid supplements on milk fat synthesis; Perfield JW 2nd et al.; The trans-10, cis-12 isomer of conjugated linoleic acid (CLA) is a potent inhibitor of milk fat synthesis; its ability to reduce milk fat output in a controlled manner as a feed supplement, has potential management applications in the dairy industry . The effectiveness of dietary supplements of trans-10, cis-12 CLA is related to the extent to which their metabolism by rumen bacteria is minimized . A number of processes have been used to manufacture "rumen-protected" feed supplements, and their efficacy can be described by the extent of protection from rumen bacteria as well as postruminal bioavailability . The objective of this study was to investigate the effects of 2 rumen-protected CLA supplements on milk fat synthesis . Using the same initial batch of CLA, supplements were manufactured by the formation of fatty acyl amide bonds or by lipid encapsulation . Three rumen fistulated Holstein cows were randomly assigned in a 3 x 3 Latin square experiment . Treatments were 1) no supplement (control), 2) amide-protected CLA supplement, and 3) lipid-encapsulated CLA supplement . Supplements were fed to provide 10 g/d of the trans-10, cis-12 CLA isomer . Over the 7-d treatment period, 21 and 22% reductions in milk fat yield were observed for the amide-protected and lipid-encapsulated supplements, respectively . Transfer of trans-10, cis-12 CLA into milk fat was also similar for the amide-protected (7.1%) and lipid-encapsulated (7.9%) supplements . Overall, the amide-protected and lipid-encapsulated CLA supplements were equally effective at reducing milk fat synthesis and had no effect on milk yield or dry matter intake. Clin Exp Immunol, 2004 Oct, 138(1), 54 - 60 Effects of Chlamydia trachomatis infection on the expression of natural killer (NK) cell ligands and susceptibility to NK cell lysis; Hook CE et al.; Natural killer (NK) cells are an important component of the immediate immune response to infections, including infection by intracellular bacteria . We have investigated recognition of Chlamydia trachomatis (CT) by NK cells and show that these cells are activated to produce interferon (IFN)-gamma when peripheral blood mononuclear cells (PBMC) are stimulated with CT organisms . Furthermore, infection of epithelial cell lines with CT renders them susceptible to lysis by human NK cells . Susceptibility was observed 18-24 h following infection and required protein synthesis by the infecting chlamydiae, but not by the host cell; heat or UV inactivated chlamydiae did not induce susceptibility to NK cell lysis . CT infection was also shown to decrease the expression of classical and non-classical major histocompatibility complex (MHC) molecules on infected cells, thus allowing recognition by NK cells when combined with an activating signal . A candidate activating signal is MICA/B, which was shown to be expressed constitutively on epithelial cells. Clin Exp Immunol, 2004 Oct, 138(1), 30 - 8 A mycobacterial lipoarabinomannan specific monoclonal antibody and its F(ab') fragment prolong survival of mice infected with Mycobacterium tuberculosis; Hamasur B et al.; Lipoarabinomannan (LAM) is a major structural carbohydrate antigen of the outer surface of Mycobacterium tuberculosis . High antibody titres against LAM are often seen in active tuberculosis (TB) . The role of such LAM-specific antibodies in the immune response against TB is unknown . Here we have investigated a monoclonal antibody (MoAb) SMITB14 of IgG1 subclass and its corresponding F(ab')(2) fragment directed against LAM from M . tuberculosis strain H37Rv . MoAb SMITB14 was shown by immunofluorescence to bind to whole cells of the clinical isolate M . tuberculosis strain Harlingen as well as to M . tuberculosis H37Rv . The binding of MoAb SMITB14 to LAM was inhibited by arabinomannan (AM) and oligosaccharides (5.2 kDa) derived from LAM, showing that the MoAb binds specifically to the AM carbohydrate portion of LAM . In passive protection experiments BALB/c mice were infected intravenously with M . tuberculosis Harlingen . MoAb SMITB14 was added intravenously either prior to, or together with, the bacteria . The antibody proved to be protective against the M . tuberculosis infection in terms of a dose-dependent reduction in bacterial load in spleens and lungs, reduced weight loss and, most importantly, increased long-term survival. J Toxicol Environ Health A, 2004 Oct 22-Nov 26, 67(20-22), 1805 - 12 Guidelines for ultraviolet disinfection of drinking water: considerations for Ontario; Hofmann R et al.; The Ontario Ministry of the Environment is actively investigating protocols for approving the installation of ultraviolet (UV) disinfection systems for drinking water disinfection . This paper discusses issues that may be considered for selecting the appropriate UV dose, validating UV reactor performance, and monitoring the performance of the reactor once installed. Int Rev Immunol, 2004 Sep-Dec, 23(5-6), 465 - 86 Safety and ethical consideration of AIDS vaccine; Veljkovic V et al.; It has been demonstrated that HIV-1 gp120 resembles several important properties of immunoglobulins allowing it strong influence on the human immune system, especially through induction of the deceptive imprinting and deregulation of the immune network . On the other hand there are many unanswered questions concerning properties and control of the genetically modified viruses and bacteria used as vectors in AIDS vaccines . This situation opens a serious question about the safety of vectored AIDS vaccine and the ethics of their trials in humans. Cell, 2004 Sep 17, 118(6), 675 - 85 Robustness of cellular functions; Stelling J et al.; Robustness, the ability to maintain performance in the face of perturbations and uncertainty, is a long-recognized key property of living systems . Owing to intimate links to cellular complexity, however, its molecular and cellular basis has only recently begun to be understood . Theoretical approaches to complex engineered systems can provide guidelines for investigating cellular robustness because biology and engineering employ a common set of basic mechanisms in different combinations . Robustness may be a key to understanding cellular complexity, elucidating design principles, and fostering closer interactions between experimentation and theory. Cell, 2004 Sep 17, 118(6), 671 - 4 At the crossroads of inflammation and cancer; Clevers H; Chronic inflammation and cancer are closely associated in the intestine . Anti-inflammatory medication reduces intestinal neoplasia, while colorectal cancer incidence is increased in ulcerative colitis . Cyclooxygenases are key to both diseases, yet the molecular basis of the association remains incompletely understood . Two recent Cell (Greten et al., 2004; Rakoff-Nahoum et al., 2004) papers illuminate roles of Toll-like receptors and the NF-kappaB pathway in the control of epithelial homeostasis in health and disease. J Int Acad Periodontol, 2004 Jul, 6(3), 89 - 94 Maternal chronic infection as a risk factor in preterm low birth weight infants: the link with periodontal infection; Sanchez AR et al.; In the past decade, there has been mounting scientific evidence suggesting that periodontal disease may play an important role as a risk factor for adverse pregnancy outcomes . This article focuses on the definition, incidence, risk factors associated with preterm low birthweight infants (PLBW), the evidence linking chronic infections and PLBW, and the scientific evidence linking periodontal infections with adverse pregnancy outcomes . Additionally, this review summarizes the current epidemiological studies on the PLBW/infection relation and makes conclusions based on these results . Data from a limited number of studies available support the hypothesis that periodontal disease may act as a risk factor for PLBW infants . The fetal exposure to different periodontal pathogens needs to be confirmed, the mechanisms associated with the potential passage of periodontal bacteria across the placental barrier, and the efficacy of different periodontal treatments in reducing the risk for PLBW need to be studied further. J Cell Biochem, 2004 Oct 1, 93(2), 409 - 17 Mutation of the highly conserved Arg165 and Glu168 residues of human Gsalpha disrupts the alphaD-alphaE loop and enhances basal GDP/GTP exchange rate; Hinrichs MV et al.; G protein signalling regulates a wide range of cellular processes such as motility, differentiation, secretion, neurotransmission, and cell division . G proteins consist of three subunits organized as a Galpha monomer associated with a Gbetagamma heterodimer . Structural studies have shown that Galpha subunits are constituted by two domains: a Ras-like domain, also called the GTPase domain (GTPaseD), and an helical domain (HD), which is unique to heterotrimeric G-proteins . The HD display significantly higher primary structure diversity than the GTPaseD . Regardless of this diversity, there are small regions of the HD which show high degree of identity with residues that are 100% conserved . One of such regions is the alpha helixD-alpha helixE loop (alphaD-alphaE) in the HD, which contains the consensus aminoacid sequence R*-{RSA}-{RSAN}-E*-{YF}-{QH}-L in all mammalian Galpha subunits . Interestingly, the highly conserved arginine (R*) and glutamic acid (E*) residues form a salt bridge that stabilizes the alphaD-alphaE loop, that is localized in the top of the cleft formed between the GTPaseD and HD . Because the guanine nucleotide binding site is deeply buried in this cleft and those interdomain interactions are playing an important role in regulating the basal GDP/GTP nucleotide exchange rate of Galpha subunits, we studied the role of these highly conserved R and E residues in Galpha function . In the present study, we mutated the human Gsalpha R165 and E168 residues to alanine (A), thus generating the R165--> A, E168--> A, and R165/E168--> A mutants . We expressed these human Gsalpha (hGsalpha) mutants in bacteria as histidine tagged proteins, purified them by niquel-agarose chromatography and studied their nucleotide exchange properties . We show that the double R165/E168--> A mutant exhibited a fivefold increased GTP binding kinetics, a higher GDP dissociation rate, and an augmented capacity to activate adenylyl cyclase . Structure analysis showed that disruption of the salt bridge between R165 and E168 by the introduced mutations, caused important structural changes in the HD at the alphaD-alphaE loop (residues 160-175) and in the GTPaseD at a region required for Gsalpha activation by the receptor (residues 308-315) . In addition, other two GTPaseD regions that surround the GTP binding site were also affected . Int Arch Occup Environ Health, 2004 Oct, 77(7), 451 - 60 Epub 2004 Oct. Occupational exposure to chemical agents in the paper industry; Korhonen K et al.; As part of an International Agency for Research on Cancer (IARC) international epidemiological study of workers in the pulp and paper industry, previously unpublished exposure measurements were assembled in a database . This article summarizes the results of 3,873 measurements carried out in the production departments of paper and paperboard mills and recycling plants in 12 countries . In the paper and paperboard mills, most of the agents were measured in the pulping and refining departments and in on-machine coating and winding of paper/paperboard . Exposures to asbestos, carbon monoxide, formaldehyde, fungal spores, bacteria, nitrogen dioxide, minerals dusts, paper dust, sulphuric acid and different solvents sometimes exceeded exposure limit values . In the re-pulping and de-inking departments of recycling plants high exposures to formaldehyde, fungal spores, bacteria and paper dust were observed . High exposures to asbestos, bioaerosols, carbon monoxide and paper dust were found in many departments; ammonia, formaldehyde, mineral and paper dust and solvents were found in coating machines; and diphenyl and polychlorobiphenyls (PCBs) were found in some special circumstances . Measurements in the newsprint and uncoated paper machine departments revealed only a few elevated exposures . In nearly all departments, measurements of epichlorohydrin, PCBs, sulphur dioxide, hydrogen sulphide and mercaptans tended to be low, often even below their detection limits . In spite of some uncertainties in the measurement data, the study provides new insights into the level and variation of occupational exposures of production workers in the paper and paperboard industry. J Virol, 2004 Oct, 78(19), 10390 - 8 RNAs are packaged into human cytomegalovirus virions in proportion to their intracellular concentration; Terhune SS et al.; The assembly of human cytomegalovirus (HCMV) virions is a complex process and involves the incorporation of viral transcripts . These RNAs are delivered to the newly infected cells and have the potential to be translated in the absence of HCMV gene expression . We have quantified the relative amount of RNAs in HCMV virions and infected cells with real-time reverse transcription-PCR and observed that viral and cellular RNAs are packaged in proportion to the amount of RNA within the cell at the time of assembly . To determine whether cis elements influenced RNA packaging, we constructed a recombinant HCMV mutant virus that expressed the yellow fluorescence protein (YFP) gene fused to the virion RNA UL21.5 . We also constructed a mutant virus in which the UL21.5 transcription unit was replaced with the YFP gene . YFP RNA was incorporated into both viruses, indicating that RNA is incorporated in the absence of a virus-specific signal motif . Furthermore, with in situ hybridization, packaged transcripts were observed throughout the cytoplasm of the infected cells, including the site of virus assembly . Several proteins that nonspecifically interact with RNA, including the tegument protein pp28, were found within HCMV virions . These studies demonstrate that both viral and cellular RNAs are nonspecifically incorporated into HCMV, potentially through interactions with several virion proteins. J Agric Food Chem, 2004 Sep 22, 52(19), 5987 - 94 Effects of alpha-amylases from different sources on the firming of concentrated wheat starch gels: relationship to bread staling; Palacios HR et al.; The firming and carbohydrate fractions of concentrated starch gels supplemented with four alpha-amylases from different sources were evaluated . Correlations were found between the firmness data and results for the carbohydrate fractions extracted from the gels . The thermostable (TBA) and intermediate temperature stability (ISBA) bacterial alpha-amylases were most effective in decreasing the rate of firming . The cereal alpha-amylase at the high level (CAH) was also effective . The CAH produced the largest quantity of dextrins at storage time zero and the thermostable bacterial alpha-amylase at the high level (TBAH) after storage for 5 days . None of the maltooligosaccharides appeared to be responsible for the decreased rate of firming of the gels . The results indicated that the TBA and ISBA most effectively inhibited firming because they degraded the external branches and the intercluster regions of amylopectin during storage . Consideration of previously reported differential scanning calorimetry and X-ray crystallography results leads to the conclusion that the antifirming action of the TBA and ISBA is due to their ability to degrade the amylopectin and amorphous regions of the gels during storage, which inhibits the formation of double helices and decreases the strength of the starch gel matrix . Gels supplemented with the TBA and ISBA were most crystalline but firmed to a lesser extent . These results are similar to those previously reported by other researchers for bread and strongly suggest that starch retrogradation plays a primary role in bread staling. J Agric Food Chem, 2004 Sep 22, 52(19), 5978 - 86 Effect of alpha-amylases from different sources on the retrogradation and recrystallization of concentrated wheat starch gels: relationship to bread staling; Palacios HR et al.; Concentrated starch gels were supplemented with four alpha-amylases from different sources . The retrogradation and recrystallization of the gels were evaluated using differential scanning calorimetry (DSC) and X-ray crystallography . Correlations between the retrogradation data and the carbohydrate fractions extracted from these gels were determined . The thermostable (TBA) and intermediate temperature stability (ISBA) bacterial alpha-amylases were most effective in decreasing the rate of retrogradation of the starch in the gels . The cereal alpha-amylase at the high level (CAH) was also effective . Supplementation with the alpha-amylases increased the crystallinity of the gels . Gels supplemented with TBA or ISBA were most crystalline and retrograded to a lesser extent . The results indicated that DSC gives not only a measure of recrystallized amylopectin but also a measure of total order (recrystallized amylopectin and double-helical content) . The maltooligosaccharides produced by the enzymes did not appear to be responsible for the reduced rates of retrogradation, but they appeared to be an expression of the degree of starch modification that was responsible for the inhibition of retrogradation . The crystallinity and retrogradation data were similar to results reported for bread and strongly suggest that bread staling is caused by the retrogradation of starch . The results also indicate that alpha-amylases decrease the rate and extent of retrogradation of starch gels by inhibiting the formation of double helices. Biol Rev Camb Philos Soc, 2004 Aug, 79(3), 557 - 81 Sexually transmitted diseases of insects: distribution, evolution, ecology and host behaviour; Knell RJ et al.; Sexually transmitted diseases (STDs) of insects are known from the mites, nematodes, fungi, protists and viruses . In total 73 species of parasite and pathogen from approximately 182 species of host have been reported . Whereas nearly all vertebrate STDs are viruses or bacteria, the majority of insect STDs are multicellular ectoparasites, protistans or fungi . Insect STDs display a range of transmission modes, with 'pure' sexual transmission only described from ectoparasites, all of which are mites, fungi or nematodes, whereas the microparasitic endo-parasites tend to show vertical as well as sexual transmission . The distribution of STDs within taxa of insect hosts appears to be related to the life histories of the hosts . In particular, STDs will not be able to persist if host adult generations do not overlap unless they are also transmitted by some alternative route . This explains the observation that the Coleoptera seem to suffer from more STDs than other insect orders, since they tend to diapause as adults and are therefore more likely to have overlapping generations of adults in temperate regions . STDs of insects are often highly pathogenic, and are frequently responsible for sterilizing their hosts, a feature which is also found in mammalian STDs . This, combined with high prevalences indicates that STDs can be important in the evolution and ecology of their hosts . Although attempts to demonstrate mate choice for uninfected partners have so far failed it is likely that STDs have other effects on host mating behaviour, and there is evidence from a few systems that they might manipulate their hosts to cause them to mate more frequently . STDs may also play a part in sexual conflict, with males in some systems possibly gaining a selective advantage from transmitting certain STDs to females . STDs may well be important factors in host population dynamics, and some have the potential to be useful biological control agents, but empirical studies on these subjects are lacking. J Biol Chem, 2004 Nov 26, 279(48), 50401 - 9 Epub 2004 Nov 26. Catalytic domain crystal structure of protein kinase C-theta (PKCtheta); Xu ZB et al.; A member of the novel protein kinase C (PKC) subfamily, PKC, is an essential component of the T cell synapse and is required for optimal T cell activation and interleukin-2 production . Selective involvement of PKC in TCR signaling makes this enzyme an attractive therapeutic target in T cell-mediated disease processes . In this report we describe the crystal structure of the catalytic domain of PKC at 2.0-A resolution . Human recombinant PKC kinase domain was expressed in bacteria as catalytically active phosphorylated enzyme and co-crystallized with its subnanomolar, ATP site inhibitor staurosporine . The structure follows the classic bilobal kinase fold and shows the enzyme in its active conformation and phosphorylated state . Inhibitory interactions between conserved features of staurosporine and the ATP-binding cleft are accompanied by closing of the glycine-rich loop, which also maintains an inhibitory arrangement by blocking the phosphate recognition subsite . The two major phosphorylation sites, Thr-538 in the activation loop and Ser-695 in the hydrophobic motif, are both occupied in the structure, playing key roles in stabilizing active conformation of the enzyme and indicative of PKC autocatalytic phosphorylation and activation during bacterial expression . The PKC-staurosporine complex represents the first kinase domain crystal structure of any PKC isotypes to be determined and as such should provide valuable insight into PKC specificity and into rational drug design strategies for PKC selective leads. Comp Biochem Physiol B Biochem Mol Biol, 2004 Sep, 139(1), 99 - 106 In vivo DNA double-strand breaks enhance gene targeting in cultured silkworm cells; Mon H et al.; Alteration of genomic information through homologous recombination (HR) is a powerful tool for reverse genetics in bacteria, yeast, and mice . The low frequency of HR is, however, a major obstacle to achieve efficient gene targeting . In this study, we have developed an assay system for investigating the frequency of gene targeting in cultured silkworm cells using a firefly luciferase gene as a reporter . The introduction of a DNA double-strand break (DSB) either in the chromosomal target locus or in the targeting construct drastically increased the frequency of gene targeting . Interestingly, the inhibition of poly(ADP-ribose) polymerase (PARP), a protein known to play an important role in overall suppression of the HR pathway, stimulated the targeting efficiency, whereas the overexpression of two silkworm RecA homologs, BmRad51 and BmDmc1, had no effect . The presently devised assay system may serve as a useful tool to improve the gene targeting efficiency in the silkworm (Bombyx mori). Comp Biochem Physiol B Biochem Mol Biol, 2004 Sep, 139(1), 27 - 33 Phospholipase A2 in salivary glands isolated from tobacco hornworms, Manduca sexta; Tunaz H et al.; We describe a phospholipase A2 (PLA2) associated with the salivary glands of tobacco hornworms, Manduca sexta . This enzyme is able to hydrolyze arachidonic acid from the sn-2 position of PLs . Addition of the calcium chelator, EGTA, or calcium, to the Tris reaction buffer impaired the PLA2 activity, from which we infer the enzyme requires very low concentrations of calcium or perhaps other ions for optimal activity . PLA2 activity was sensitive to protein concentration (highest activity at 25 microg protein per microl), reaction time (optimal at 30 min), buffer pH (optimal at pH 8-10), and reaction temperature (optimal range 18-38 degrees C) . The salivary gland PLA2 was sensitive to the site-specific inhibitor, oleyloxyethylphosphorylcholine and stable to freezing at -80 degrees C, but not -20 degrees C . The biological significance of this enzyme may relate to hydrolysis of fatty acid moieties from dietary PLs for absorption by midgut epithelia . This salivary gland enzyme may also be responsible for killing food-borne bacteria. Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2004 Aug, 12(4), 519 - 21 {Effects of preparation and quality control of cryopreserved platelets}; Li XJ et al.; The purpose of this study was to establish a set of techniques for cryopreservation of platelets with dimethylsulphoxide (DMSO) to insure high quality of cryopreserved platelet . The methods were as following: (1) DMSO was filtered in stead of being sterilized before infusion into the bag with platelets . (2) The whole blood was centrifuged immediately after blood collection and the attached tube was tied on the top of the bucket . (3) The related centrifugal force was 480 x g, the accelerating and braking grades of the centrifuge for acceleration and deceleration were 9 and 4 respectively . (4) The flow rate of platelet rich plasma (PRP) could not be too high, 80 - 100 ml PRP should be harvested at 1 minute or so . The infusion rate of DMSO into the PRP was 1 ml/min . After the infusion of DMSO, the PRP bag must be put into the -80 degrees C ultra low freezer at once to make the product to be freezed as soon as possible . The cryopreserved platelet should be thawed in the cycling warm water at the temperature of 38 - 40 degrees C . (5) After thawing of platelet, the platelet, red blood cell and white blood cell were counted, and the bacteria culturing, tests for HBsAg, anti-HCV, anti-HIV, TP and ALT were carried out . The results showed that altogether 14 800 units of cryopreserved platelets were prepared including 80 units collected with blood cell separator, of which quality control was accomplished in 300 units . The manually collected platelet mean count >/= 2.4 x 10(10)/unit, while the apheresis platelet count >/= 2.5 x 10(11)/unit . The yield was over 70% . The contaminated red and white blood cells were </= 1 x 10(9) and </= 1 x 10(7)/unit respectively . All the bacteria cultures were negative, while tests for HBsAg, anti-HCV, anti-HIV and TP were negative too . The ALT values were all in normal range . The transfusion of cryopreserved platelets showed obvious effect of haemostasis . In conclusion, the cryopreserved platelets prepared with this method were of high quality and efficaciousness in haemostasis clinically. Can J Public Health, 2004 Jul-Aug, 95(4), 309 - 13 From stool to statistics: reporting of acute gastrointestinal illnesses in Canada; Flint JA et al.; BACKGROUND: Limitations associated with the under-reporting of enteric illnesses have long been recognized but the extent and variation of this under-reporting in Canada has not been examined . Given the public health value of surveillance data, a closer examination of under-reporting of enteric illnesses in Canada was warranted . METHODS: Paper-based surveys were administered (a) to all laboratories in Canada licensed to process stool specimens and (b) to all local public health authorities in two provinces . RESULTS: Of the laboratories surveyed, 67% (n=274) conducted on-site testing of stool specimens for enteric bacteria, 31% (n=126) for parasites and 10% (n=42) for viruses . In the year 2000, these laboratories processed 459,982 stool specimens, of which 5%, 15%, 8% and 19% were positive for enteric bacteria (excluding C . difficile), C . difficile, parasites and viruses, respectively . Variations in laboratory testing and health authority reporting protocols and policies were identified . Of the laboratory-confirmed cases of AGI reported to local public health authorities, 5% (n=846) were not reported to provincial counterparts . CONCLUSION: A significant proportion of AGI cases submitting stool specimens are not captured in Canada's passive surveillance system due to unknown etiology . A much smaller proportion of laboratory-confirmed cases reported to local public health authorities are not captured at the provincial or national level . Given that the number of laboratory-confirmed AGI cases represents such a small fraction of all community cases, strategies to compensate for under-reporting and efforts directed at harmonizing laboratory and local public health authority policies and practices would be welcomed. Helicobacter, 2004 Oct, 9(5), 460 - 8 Detection of Helicobacter ganmani-like 16S rDNA in pediatric liver tissue; Tolia V et al.; BACKGROUND: To determine the presence of Helicobacter species in the liver biopsy specimens from children with various chronic liver diseases as data in adult literature suggests a possible role of these bacteria in their pathogenesis . MATERIALS AND METHODS: Paraffin sections of 61 liver biopsies of pediatric patients with miscellaneous diseases and autopsy liver tissue from 10 control subjects with no evidence of preexisting liver disease were examined for the presence of Helicobacter species by a genus-specific seminested polymerase chain reaction (PCR) assay . PCR-products of positive samples were further characterized by denaturing gradient gel electrophoresis (DGGE) and DNA-sequence analysis . Based on those results, a seminested PCR assay for H . ganmani was developed and applied to the samples . RESULTS: On analysis, 40/61 patient samples were positive in the genus-specific Helicobacter PCR and 4/10 from the control group . The nucleotide sequences of 16S rDNA fragments were 99-100% similar to mainly Helicobacter sp . 'liver' and H . ganmani . PCR-products similar to H . canis and H . bilis were also found . The 16S rDNAs of control specimens showed similarity to Helicobacter sp . 'liver' . In the H . ganmani-specific PCR analysis 19 patients, but none of the controls, were positive . CONCLUSIONS: Amplified Helicobacter 16S rDNAs were related to Helicobacter sp . 'liver' or H . ganmani in liver biopsy specimens of pediatric patients . The possible significance of Helicobacter species in pediatric liver diseases needs to be evaluated further in prospective studies. Helicobacter, 2004 Oct, 9(5), 408 - 16 Experimental Helicobacter felis infection in transgenic mice expressing human group IIA phospholipase A2; Huhtinen HT et al.; BACKGROUND: Both various virulence factors of Helicobacter pylori and host factors influence the clinical outcome of H . pylori infection . In animal experiments with Helicobacter felis, large variations in the severity of disease have been observed between different mouse strains infected with a single isolate of H . felis . C57BL/6 J mouse strain that lacks the expression of group IIA phospholipase A2 has been shown to develop more severe gastric inflammation than other mouse strains . Thus, group IIA phospholipase A2 has been suggested to play a role in regulating inflammation in gastric mucosa . The aim of this study was to examine the possible role of group IIA phospholipase A2 in experimental Helicobacter infection . MATERIALS AND METHODS: Transgenic mice expressing human group IIA phospholipase A2 and their group IIA phospholipase A2 deficient nontransgenic C57BL/6 J littermates were infected with H . felis . The mice were killed 3, 8, and 19 weeks after inoculation of bacteria to determine the histopathological changes in gastric mucosa . RESULTS: The infected mice developed chronic inflammation in gastric mucosa . We found no differences in the colonization of bacteria between transgenic and nontransgenic mice . At 3 and 8 weeks, no difference was found in the severity of inflammation between the two groups . Nineteen weeks after the administration of bacteria the inflammation was more marked in nontransgenic than transgenic mice . Group IIA phospholipase A2 was expressed by in situ hybridization in the neck cells of the glandular stomach in transgenic mice . CONCLUSIONS: The results of the present study suggest that the endogenous expression of group IIA phospholipase A2 diminishes chronic inflammation in gastric mucosa in experimental H . felis infection in mice. Biochem J, 2004 Dec 1, 384(Pt 2), 307 - 16 Structural diversity and specific distribution of O-glycans in normal human mucins along the intestinal tract; Robbe C et al.; Purified human mucins from different parts of the intestinal tract (ileum, cecum, transverse and sigmoid colon and rectum) were isolated from two individuals with blood group ALe(b) (A-Lewis(b)) . After alkaline borohydride treatment the released oligosaccharides were structurally characterized by nano-ESI Q-TOF MS/MS (electrospray ionization quadrupole time-of-flight tandem MS) without prior fractionation or derivatization . More than 100 different oligosaccharides, with up to ten monosaccharide residues, were identified using this technique . Oligosaccharides based on core 3 structures, GlcNAc(beta1-3)GalNAc (where GlcNAc is N-acetyl-D-glucosamine and GalNAc is N-acetylgalactosamine), were widely distributed in human intestinal mucins . Core 5 structures, GalNAc(alpha1-3)GalNAc, were also recovered in all fractions . Moreover, a comparison of the oligosaccharide repertoire, with respect to size, diversity and expression of glycans and terminal epitopes, showed a high level of mucin-specific glycosylation: highly fucosylated glycans, found specifically in the small intestine, were mainly based on core 4 structures, GlcNAc-(beta1-3){GlcNAc(beta1-6)}GalNAc, whereas the sulpho-Le(X) determinant carrying core 2 glycans, Gal(beta1-3){GlcNAc(beta1-6)}-GalNAc (where Gal is galactose), was recovered mainly in the distal colon . Blood group H and A antigenic determinants were present exclusively in the ileum and cecum, whereas blood group Sd(a)/Cad related epitopes, GalNAc(beta1-4){NeuAc(alpha2-3)}Gal (where NeuAc is N-acetylneuraminate), were found to increase along the length of the colon . Our findings suggest that mucins create an enormous repertoire of potential binding sites for micro-organisms that could explain the regio-specific colonization of bacteria in the human intestinal tract. Kansenshogaku Zasshi, 2004 Jul, 78(7), 545 - 53 {Physicochemical factors influencing distribution of Legionella species in Japanese hot springs}; Sasahara T et al.; We examined the relationship between the distribution of Legionella bacteria and various physicochemical characteristics of hot springs in Japan . Legionella bacteria were isolated from 52 (49.5%) out of 105 water samples, particularly from outdoor hot springs (67.3%) . The bacterial count in the water samples positive for Legionella (86.5%) ranged from 10(1) to < 10(3) cfu/100 mL . L . pneumophila serogroup (SG) 4 (27.8%) was predominant in the water samples, followed by SG 5 (12.2%) . The pulsefield gel electrophoresis (PFGE) patterns of chromosomal DNA for L . pneumophila SG 4 isolated from different parts of a hot spring resort were identical . Isolation of Legionella species from hot spring waters did not occur at pH 1.8-3.3, SO4(2-): > 780 mg/L, and H2SiO3: > 146 mg/L . The hot water-recirculating systems were applied to 18 out of 20 (90%) hot spring facilities which were found positive for Legionella . These results indicate that Legionella species are widespread in hot springs throughout Japan, except for waters with a low pH and non-recirculating waters, and that a single strain of L . pneumophila SG 4 is predominant in a particular hot spring resort. J Bioinform Comput Biol, 2004 Sep, 2(3), 569 - 87 Identifying patterns of DNA for tumor diagnosis using capillary electrophoresis-amplified fragment length polymorphism (CE-AFLP) screening; Wong KY et al.; Amplified Fragment Length Polymorphism (AFLP) screening is a genome-wide genotyping strategy that has been widely used in plants and bacteria, but little has been reported concerning its use in humans . We investigated if the AFLP procedure could be coupled with high-throughput capillary electrophoresis (CE) for use in tumor diagnosis and classification . Using CE-AFLP, a series of molecular 'fingerprints' were generated for a set of gastric tumor and normal genomic DNA samples . The CE-AFLP procedure was qualitatively and quantitatively robust, and a variety of clustering tools were used to identify a specific DNA marker 'pattern' of 20 features that classified the tumor and normal samples to reasonable degrees of accuracy (Sensitivity 95%, Specificity 80%) . The CE-AFLP-based approach also correctly classified 16 tumor samples, which in a previous study had exhibited no detectable genomic aberrations by comparative genome hybridization (CGH) . This is the first reported application of CE-AFLP screening in tumor diagnosis . As the procedure is relatively inexpensive and requires minimal prior sequence knowledge and biological material, we suggest that CE-AFLP-based protocols may represent a promising new approach for DNA-based cancer screening and diagnosis. Clin Diagn Lab Immunol, 2004 Sep, 11(5), 909 - 12 Comparison of oral fluid collectors for use in a rapid point-of-care diagnostic device; Holm-Hansen C et al.; Orally based diagnostic testing is emerging as an alternative, noninvasive method for analyzing a variety of analytes . These analytes include pathogens, antibodies, drugs, and nucleic acids . In the present study we developed a protocol for evaluation of collectors that could be used in orally based, point-of-care diagnostics . A performance comparison was carried out with a number of commercially available collectors, and their ability to deliver fluid, proteins, bacteria, and nucleic acid from pathogens compatible with PCR was assessed . The collectors were all capable of picking up and delivering test materials, albeit at various levels. Protein Expr Purif, 2004 Oct, 37(2), 493 - 8 Over-expression of the human MDM2 p53 binding domain by fusion to a p53 transactivation peptide; Liu Z et al.; MDM2 binds to the tumor suppressor protein p53 and regulates the level of p53 in cells . Although it is possible to prepare a small amount of the region of MDM2 that binds to p53, the expression level of this fragment of MDM2 is relatively low, limiting the studies involving this protein . Here, we describe a construct for the optimized bacterial expression and purification of the MDM2 p53 binding domain . We found that the expression level of the soluble MDM2 p53 binding domain in bacteria was increased dramatically by fusing it to its interaction partner, the p53 transactivation peptide . Attachment of the p53 transactivation peptide (residues 17-29) to the N-terminus of MDM2 resulted in a more than 200-fold increase of soluble protein expression of the p53 binding domain in bacteria . To obtain the final MDM2 p53 binding domain (residues 5-109) we inserted a tobacco etch virus protease recognition site between the P53 peptide and the MDM2 p53 binding domain . To weaken the protein/peptide interaction and facilitate the separation of the protein from the complex, we introduced a point mutation of one of the key interaction residues (F19A or W23A) in the p53 peptide . The advantages of our new construct are high yield and easy purification of the MDM2 protein. Risk Anal, 2004 Aug, 24(4), 847 - 58 Ecological risk assessment conceptual model formulation for nonindigenous species; Landis WG; This article addresses the application of ecological risk assessment at the regional scale to the prediction of impacts due to invasive or nonindigenous species (NIS) . The first section describes risk assessment, the decision-making process, and introduces regional risk assessment . A general conceptual model for the risk assessment of NIS is then presented based upon the regional risk assessment approach . Two diverse examples of the application of this approach are presented . The first example is based upon the dynamics of introduced plasmids into bacteria populations . The second example is the application risk assessment approach to the invasion of a coastal marine site of Cherry Point, Washington, USA by the European green crab . The lessons learned from the two examples demonstrate that assessment of the risks of invasion of NIS will have to incorporate not only the characteristics of the invasive species, but also the other stresses and impacts affecting the region of interest. J Microbiol, 2004 Jun, 42(2), 147 - 51 Identification of sigmaB-dependent promoters using consensus-directed search of Streptomyces coelicolor genome; Lee EJ et al.; SigmaB plays an important role in both osmoprotection and proper differentiation in Streptomyces coelicolor A3(2) . We searched for candidate members of the sigmaB regulon from the genome database, using the consensus promoter sequence (GNNTN14-16GGGTAC/T) . The list consists of 115 genes, and includes all the known sigmaB target genes and many other genes whose functions are related to stress protection and differentiation. J Microbiol, 2004 Mar, 42(1), 56 - 9 Transposition of IntAs into the conserved regions of IS3 family elements; Han CG; Together with the previous reports, my computer survey revealed that several bacteria contain six copies of the type group II intron IntA . The sequence analysis of IntAs showed the high level of homology in the nucleotide sequence (91.9-99.8%) . The consensus sequence, 2,270 base pair long, was derived from the nucleotide sequences of all IntA members . The size of the open reading frame intA was 502 amino acids long, that is homologous to reverse transcriptase-like proteins encoded within the group II introns . It was reported that EPEC.IntA and Sf.IntA were inserted into IS911 and IS629, respectively . The sequence of the flanking region IntA was analyzed here . The data show the insertion of EC.IntA into IS629, the insertion of EHEC.IntA into IS3, the insertion of Yp.IntA into IS904-like sequence, and the insertion of EK12.IntA into IS911 . Interestingly, these IS elements nested by IntAs were the members of IS3 family elements . The sequences of the IS3 members correspond to the OrfB with the DDE motif conserved in retroviral integrases . Alignment of the flanking sequences of IntAs revealed that the flanking regions -25 to +10 of insertion sites, that are generally believed to be required for the retrohoming, were not strongly conserved . The data presented here suggests that the retrohoming pathway of IntA seems to differ from those of other group II introns. Curr Mol Med, 2004 Sep, 4(6), 681 - 9 Macrophage specific drug delivery in experimental leishmaniasis; Basu MK et al.; Macrophage-specific delivery systems are the subject of much interest nowadays, because of the fact that macrophages act as host cells for many parasites and bacteria, which give rise to outbreak of so many deadly diseases(eg . leishmaniasis, tuberculosis etc.) in humans . To combat these deadly diseases initially macrophage specific liposomal delivery system were thought of and tested in vivo against experimental leishmaniasis in hamsters using a series of indigenous or synthetic antileishmanial compounds and the results were critically discussed . In vitro testing was also done against macrophages infected with Leishmania donovani, the causative agent for visceral leishmaniasis . The common problem of liposome therapy being their larger size, stability and storage, non-ionic surfactant vesicles, niosomes were prepared, for their different drug distribution and release characteristics compared to liposomes . When tested in vivo, the retention capacity of niosomes was found to be higher than that of liposomes due to the absence of lipid molecules and their smaller size . Thus the therapeutic efficacy of certain antileishmanial compounds was found to be better than that in the liposomal form . The niosomes, being cheaper, less toxic, biodegradable and non-immunogenic, were considered for sometime as suitable alternatives to liposomes as drug carriers . Besides the advent of other classical drugs carriers(e.g . neoglycoproteins), the biggest challenge came from polymeric delivery vehicles, specially the polymeric nanoparticles which were made of cost effective biodegradable polymers and different natural polymers . Because of very small size and highly stable nature, use of nanoparticles as effective drug carriers has been explored in experimental leishmaniasis using a series of antileishmanial compounds, both of indigenous and synthetic origin . The feasibility of application in vivo, when tested for biological as well as for other physicochemical parameters, the polymeric nanoparticles have turned out to be the best and thus may be projected for effective use in the clinics. J Parasitol, 2004 Aug, 90(4), 891 - 3 Cloning, characterization, and functional expression of Taenia solium calreticulin; Mendlovic F et al.; Calreticulin is an endoplasmic reticulum protein involved in the homeostasis of intracellular Ca++ and other physiological processes . A complementary DNA clone containing the complete coding sequence of Taenia solium calreticulin (TsCRT) was isolated and characterized . Recombinant TsCRT was expressed in bacteria as a 50-kDa protein that specifically bound calcium when tested in a radioassay . The deduced amino acid sequence has 47-50% identity with other reported calreticulins . Poor recognition of TsCRT by human and pig sera with confirmed cysticercosis discourages its use for diagnosis of the disease . However, further characterization and localization studies could provide insights into the role of TsCRT in T . solium physiology and host-parasite interactions. Clin Infect Dis, 2004 Sep 1, 39(5), 681 - 6 Epub 2004 Aug 13. Etiology of community-acquired pneumonia in hospitalized school-age children: evidence for high prevalence of viral infections; Tsolia MN et al.; BACKGROUND: Community-acquired pneumonia (CAP) in young children is most commonly associated with viral infections; however, the role of viruses in CAP of school-age children is still inconclusive . METHODS: Seventy-five school-age children hospitalized with CAP were prospectively evaluated for the presence of viral and bacterial pathogens . Nasopharyngeal washes were examined by polymerase chain reaction for viruses and atypical bacteria . Antibody assays to detect bacterial pathogens in acute-phase and convalescent-phase serum samples were also performed . RESULTS: A viral infection was identified in 65% of cases . Rhinovirus RNA was detected in 45% of patients; infection with another virus occurred in 31% . The most common bacterial pathogen was Mycoplasma pneumoniae, which was diagnosed in 35% of cases . Chlamydia pneumoniae DNA was not detected in any patient; results of serological tests were positive in only 2 patients (3%) . Mixed infections were documented in 35% of patients, and the majority were a viral-bacterial combination . CONCLUSIONS: The high prevalence of viral and mixed viral-bacterial infections supports the notion that the presence of a virus, acting either as a direct or an indirect pathogen, may be the rule rather than the exception in the development of CAP in school-age children requiring hospitalization. J Allergy Clin Immunol, 2004 Sep, 114(3), 650 - 6 Human nuclear factor kappa B essential modulator mutation can result in immunodeficiency without ectodermal dysplasia; Orange JS et al.; BACKGROUND: Many receptors rely on the appropriate activation of nuclear factor (NF) kappa B to induce cellular function . This process depends critically on the phosphorylation of the inhibitor of NF-kappa B (I kappa B) by the I kappa B kinase . This targets I kappa B for ubiquitination and degradation, allowing NF-kappa B to translocate to the nucleus, where it can direct transcription . Hypomorphic human mutations affecting one I kappa B kinase component, the NF-kappa B essential modulator (NEMO), result in impaired signaling from receptors required for ectodermal development and immune function . Male subjects with these mutant NEMO molecules have an X-linked syndrome known as ectodermal dysplasia with immunodeficiency, which is characterized by severe infections, with herpesviruses, bacteria, and mycobacterial susceptibility . OBJECTIVE: We sought to genetically and biochemically characterize a patient with a mutant NEMO molecule without ectodermal abnormalities . METHODS: We evaluated NEMO in a patient who had immunodeficiency and atypical mycobacterial infection but normal ectoderm . RESULTS: We identified a novel NEMO mutant causing immunodeficiency without ectodermal dysplasia . The mutation, which altered the exon 9 splice site, was present in cells of ectodermal and hematopoetic origin and resulted in a heterogeneous mixture of mutant and wild-type cDNA species . Immunologic function was variably impaired, with reduced CD40-induced B-cell proliferation, partially reduced NF-kappa B p65 nuclear translocation, and variable Toll-like receptor-induced TNF production . This variability might be explained by an inconsistent ratio of mutant to wild-type NEMO . The lack of any ectodermal phenotype, however, suggested a separation in the hematopoetic and ectodermal function of NEMO that leads to NF-kappa B activation . CONCLUSION: Mutation of the gene encoding NEMO can result in immunodeficiency without ectodermal dysplasia. Plant Mol Biol, 2004 Mar, 54(5), 623 - 39 The promoters of two isoflavone synthase genes respond differentially to nodulation and defense signals in transgenic soybean roots; Subramanian S et al.; Isoflavonoids are a group of secondary metabolites common to leguminous plants that play roles in nodulation and defense responses . Isoflavone synthase (IFS) catalyzes the key entry point step of isoflavone biosynthesis from the general phenylpropanoid pathway . We have cloned the 5' upstream regions of the genes encoding the two isoflavone synthase isoforms from soybean . We characterized the tissue-specific expression patterns of IFS1 and IFS2 genes of soybean by quantitative RT-PCR . Isoflavone synthase transcripts were detected primarily in the roots and seeds . In transgenic soybean plants in which the IFS1 promoter drove expression of the bacterial uidA (GUS) gene, the expression was localized to the root epidermis and root hairs . We detected differential tissue-specific expression of IFS1:GUS in response to nodulation and defense signals . Bradyrhizobium japonicum, a diazotropic symbiont of soybean, induced the expression of IFS1:GUS in root hairs and xylem poles in the young differentiation zone of the root . This observation suggests novel roles for isoflavonoids in nodulation . The defense related hormone salicylic acid induced the expression of IFS1:GUS in all cell types of the root . We also noticed corresponding alterations in the transcript levels of IFS1 and IFS2 in response to B . japonicum or salicylic acid as identified by quantitative RT-PCR . Using hairy root transformed soybean composite plants, we have identified regions of the IFS1 and IFS2 promoters mediating response to B . japonicum . We have also shown conserved root-specific expression of IFS1 in rice and Arabidopsis . The expression pattern of IFS1 in soybean is consistent with the physiological roles of isoflavonoids as defense compounds against pathogens and signal molecules to symbiotic bacteria in soybean. Mol Biol Cell, 2004 Nov, 15(11), 5064 - 74 Epub 2004 Sep 08. Differential regulation of the TRAIL death receptors DR4 and DR5 by the signal recognition particle; Ren YG et al.; TRAIL (TNF-related apoptosis-inducing ligand) death receptors DR4 and DR5 facilitate the selective elimination of malignant cells through the induction of apoptosis . From previous studies the regulation of the DR4 and DR5 cell-death pathways appeared similar; nevertheless in this study we screened a library of small interfering RNA (siRNA) for genes, which when silenced, differentially affect DR4- vs . DR5-mediated apoptosis . These experiments revealed that expression of the signal recognition particle (SRP) complex is essential for apoptosis mediated by DR4, but not DR5 . Selective diminution of SRP subunits by RNA interference resulted in a dramatic decrease in cell surface DR4 receptors that correlated with inhibition of DR4-dependent cell death . Conversely, SRP silencing had little influence on cell surface DR5 levels or DR5-mediated apoptosis . Although loss of SRP function in bacteria, yeast and protozoan parasites causes lethality or severe growth defects, we observed no overt phenotypes in the human cancer cells studied--even in stable cell lines with diminished expression of SRP components . The lack of severe phenotype after SRP depletion allowed us to delineate, for the first time, a mechanism for the differential regulation of the TRAIL death receptors DR4 and DR5--implicating the SRP complex as an essential component of the DR4 cell-death pathway.
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