Microorganisms

Afr Health Sci, 2003 Dec, 3(3), 131 - 135
Imaging features of brain tuberculoma in Tanzania: case report and literature review; Lwakatare FA et al.; Brain tubercluomas are a rare manifestation of mycobacterium tuberculosis infection usually resulting from hematogenous spread of the bacteria from a primary focus elsewhere in the body . A 29-year-old female with no history of pulmonary tuberculosis or signs of pulmonary infection presented with signs and symptoms of raised intracranial pressure . She underwent CT and MR imaging where multiple enhancing lesions were revealed in the brain parenchyma . The features of tuberculoma on CT and MR imaging may mimic the appearance of several other brain lesions . Histological diagnosis of tuberculoma was obtained . In areas where tuberculosis is endemic, the imaging features of brain tuberculoma have to be readily recognized by attending doctors.

Curr Opin Struct Biol, 2003 Dec, 13(6), 665 - 73
Molecular shredders: how proteasomes fulfill their role; Groll M et al.; The 20S proteasome is a large, cylinder-shaped protease that is found in all domains of life and plays a crucial role in cellular protein turnover . It has multiple catalytic centers located within the hollow cavity of a molecular cage . This architecture prevents unwanted degradation of endogenous proteins and promotes processive degradation of substrates by restricting the dissociation of partially digested polypeptides . Although this kind of self-compartmentalization is generally conserved, the proteasomes of bacteria, archaea and eukaryotes show many differences in architecture, subunit composition and regulation . The structure of the 20S proteasome and its inherent role in the regulation of proteasome function are gradually being elucidated.

Stat Med, 2003 Dec 30, 22(24), 3713 - 24
Estimating the incidence of subclinical infections with Legionella Pneumonia using data augmentation: analysis of an outbreak in The Netherlands; Nagelkerke NJ et al.; Infections with Legionella bacteria can cause a potentially lethal form of pneumonia known as legionnaires' disease . In 1999 a major outbreak, causing 31 deaths, occurred among visitors and exhibitors of a consumer fair in The Netherlands . The epidemiology of subclinical infections is largely unknown, as there is no reliable method to diagnose such infections . To explore the incidence of subclinical infections, IgG and IgM antibody levels among exhibitors were compared to those among a representative sample of the Dutch population . As exhibitors were assumed to comprise both infected and uninfected individuals, their antibody levels were modelled as a mixture distribution . As infected individuals are expected to cluster around a point source, the spatial aspect of the spread of infections was taken into account . To estimate the distribution of antibody levels among infected individuals and to impute infection status among exhibitors, data augmentation was used . Subclinical infection appeared to be very common and its frequency declined with the distance from the putative source of the outbreak .

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Dec, 35(12), 1061 - 5
Evolution of phenylalanyl-tRNA synthetase by domain losing; Lin J et al.; The gene duplication, fusion and horizontal transfer are the frequent events during evolution of many proteins, including the aminoacyl-tRNA synthetases (AARSs) . However, in this work, it was shown that the main event during evolution of phenylalanyl-tRNA synthetase (PheRS) is a domain loss, and the function/activity of PheRS is not affected by domain losing . Generally, the size of genome and number of genes are increased during evolution from bacteria to eukaryote, but the interesting thing is that the type and number of PheRS domains in eukaryote are obviously less than those in bacteria . The evolution of PheRS by domain losing seems to be related to the functional evolution of some AARSs from the multiple specificities to the single specificity.

J Neurosci, 2003 Dec 10, 23(36), 11479 - 88
Effects of neurotoxic and neuroprotective agents on peripheral nerve regeneration assayed by time-lapse imaging in vivo; Pan YA et al.; A direct histological assay of axonal regeneration would have many advantages over currently available behavioral, electrophysiological, and radiometric assays . We show that peripheral sensory axons marked with the yellow fluorescent protein in transgenic mice can be viewed transcutaneously in superficial nerves . Degenerating and regenerating axons can be followed in live animals with a dissecting microscope and then, after fixation, studied at high resolution by confocal microscopy . Using this approach, we document differences in regenerative ability after nerve transection, crush injury, and crush injury after a previous "conditioning" lesion . We also show that the chemotherapeutic drug vincristine rapidly but transiently blocks regeneration and that the immunosuppressive drug FK506 modestly enhances regeneration . Moreover, FK506 nearly restores normal regenerative ability in animals treated with submaximal doses of vincristine . Because neuropathy is the major dose-limiting side effect of vincristine, we propose that its efficacy could be enhanced by coadministration of FK506 analogs that are neuroactive but not immunosuppressive.

Vet Res Commun, 2003 Dec, 27(8), 643 - 52
Influence of dexamethasone on some cellular aspects of the immune system in cats; Hoffmann-Jagielska M et al.; In view of the frequent use of glucocorticoids in the treatment of cats, we studied the effect of dexamethasone on their immunological system . The phagocytic activity and oxidative burst of neutrophils and monocytes were evaluated by cytometric analysis using commercial kits and the subpopulations of lymphocytes were assessed . Neutrophilia and monocytosis reduced phagocytic activity, as shown from the number of phagocytized bacteria, and variations in the intensity of the oxidative burst in activated neutrophils and monocytes were observed . Dexamethasone also caused an increase in the number of B lymphocytes.

Inflamm Bowel Dis, 2003 Nov, 9(6), 363 - 71
The intestinal anti-inflammatory activity of UR-12746S on reactivated experimental colitis is mediated through downregulation of cytokine production; Galvez J et al.; BACKGROUND: UR-12746S (dersalazine sodium) is cleaved by colonic bacteria delivering the PAF antagonist UR-12715 and 5-ASA . This study describes the anti-inflammatory activity of UR-12746S in an experimental model of reactivated colitis and its effects on cytokine production . METHODS: Rats were initially rendered colitic by a colonic instillation of 10 mg of trinitrobenzenesulphonic acid (TNBS) dissolved in 0.25 ml of 50 % ethanol, and colitis was reactivated two weeks after by a second administration of the same dose of TNBS . Two groups of colitic rats received UR-12746S (25 and 50 mg/kg daily, p.o.) and colonic damage was evaluated every week for 4 weeks . Different biochemical markers of colonic inflammation were assayed: MPO activity and cytokine (IL-1beta and TNFalpha) levels . Also, the in vitro effects of UR-12715 and 5-ASA on cytokine production were assayed . RESULTS: UR-12746S showed anti-inflammatory effect in reactivated colitis in rats, as evidenced by a significant reduction in MPO activity . Both doses of UR-12746S decreased IL-1beta production, while only the highest dose assayed inhibited TNFalpha production . In vitro studies revealed that UR-12715 or 5-ASA (from 10(-6) to 10(-4) M) inhibited IL-8 production (30-40%) in HT-29 cells when incubated with LPS . This inhibitory effect was enhanced when both compounds were administered simultaneously at 10(-4) M . In addition, UR-12715 inhibited IL-1beta or TNFalpha production in THP-1 or U937 cells, respectively, when these cells were stimulated by PMA and LPS; whereas 5-ASA only showed a weak effect in inhibiting IL-1beta production . CONCLUSION: UR-12746S was able to prevent relapse in experimental colitis and inhibition of proinflammatory cytokine production participates in the intestinal anti-inflammatory activity exerted by this compound.

J Virol, 2004 Jan, 78(1), 389 - 98
Spatial and temporal organization of adeno-associated virus DNA replication in live cells; Fraefel C et al.; Upon cell entry, the genomes of herpes simplex virus type 1 (HSV-1) and adenovirus (Ad) associate with distinct nuclear structures termed ND10 or promyelocytic leukemia (PML) nuclear bodies (NBs) . PML NB morphology is altered or disrupted by specific viral proteins as replication proceeds . We examined whether adeno-associated virus (AAV) replication compartments also associate with PML NBs, and whether modification or disruption of these by HSV-1 or Ad, both of which are helper viruses for AAV, is necessary at all . Furthermore, to add a fourth dimension to our present view of AAV replication, we established an assay that allows visualization of AAV replication in live cells . A recombinant AAV containing 40 lac repressor binding sites between the AAV inverted terminal repeats was constructed . AAV Rep protein and helper virus-mediated replication of this recombinant AAV genome was visualized by binding of enhanced yellow fluorescent protein-lac repressor fusion protein to double-stranded AAV replication intermediates . We demonstrate in live cells that AAV DNA replication occurs in compartments which colocalize with AAV Rep . Early after infection, the replication compartments were small and varied in numbers from 2 to more than 40 per cell nucleus . Within 4 to 8 h, individual small replication compartments expanded and fused to larger structures which filled out much of the cell nucleus . We also show that AAV replication compartments can associate with modified PML NBs in Ad-infected cells . In wild-type HSV-1-infected cells, AAV replication compartments and PML NBs did not coexist, presumably because PML was completely disrupted by the HSV-1 ICP0 protein . However, alteration or disruption of PML appears not to be a prerequisite for AAV replication, as the formation of replication compartments was normal when the ICP0 mutants HSV-1 dl1403 and HSV-1 FXE, which do not affect PML NBs, were used as the helper viruses; under these conditions, AAV replication compartments did not associate with PML NBs.

Plant Physiol, 2004 Jan, 134(1), 286 - 95 Epub 2003 Dec 11.
RHM2 is involved in mucilage pectin synthesis and is required for the development of the seed coat in Arabidopsis; Usadel B et al.; Pectins are major components of primary plant cell walls and the seed mucilage of Arabidopsis . Despite progress in the structural elucidation of pectins, only very few enzymes participating in or regulating their synthesis have been identified . A first candidate gene involved in the synthesis of pectinaceous rhamnogalacturonan I is RHM2, a putative plant ortholog to NDP-rhamnose biosynthetic enzymes in bacteria . Expression studies with a promoter beta-glucuronidase construct and reverse transcription PCR data show that RHM2 is expressed ubiquitously . Rhm2 T-DNA insertion mutant lines were identified using a reverse genetics approach . Analysis of the rhm2 seeds by various staining methods and chemical analysis of the mucilage revealed a strong reduction of rhamnogalacturonan I in the mucilage and a decrease of its molecular weight . In addition, scanning electron microscopy of the seed surface indicated a distorted testa morphology, illustrating not only a structural but also a developmental role for RGI or rhamnose metabolism in proper testa formation.

Biochim Biophys Acta, 2003 Dec 8, 1607(2-3), 131 - 40
Dissecting a cyanobacterial proteolytic system: efficiency in inducing degradation of the D1 protein of photosystem II in cyanobacteria and plants; Kanervo E et al.; A chromatography fraction, prepared from isolated thylakoids of a fatty acid desaturation mutant (Fad6/desA Colon, two colons Km(r)) of the cyanobacterium Synechocystis 6803, could induce an initial cleavage of the D1 protein in Photosystem II (PSII) particles of Synechocystis 6803 mutant and Synechococcus 7002 wild type as well as in supercomplexes of PSII-light harvesting complex II of spinach . Proteolysis was demonstrated both in darkness and in light as a reduction in the amount of full-length D1 protein or as a production of C-terminal initial degradation fragments . In the Synechocystis mutant, the main degradation fragment was a 10-kDa C-terminal one, indicating an initial cleavage occurring in the cytoplasmic DE-loop of the D1 protein . A protein component of 70-90 kDa isolated from the chromatographic fraction was found to be involved in the production of this 10-kDa fragment . In spinach, only traces of the corresponding fragment were detected, whereas a 24-kDa C-terminal fragment accumulated, indicating an initial cleavage in the lumenal AB-loop of the D1 protein . Also in Synechocystis the 24-kDa fragment was detected as a faint band . An antibody raised against the Arabidopsis DegP2 protease recognized a 35-kDa band in the proteolytically active chromatographic fraction, suggesting the existence of a lumenal protease that may be the homologue DegP of Synechocystis . The identity of the other protease cleaving the D1 protein in the DE-loop exposed on the stromal (cytoplasmic) side of the membrane is discussed.

Biochim Biophys Acta, 2003 Dec 8, 1607(2-3), 121 - 30
Kinetic analyses of state transitions of the cyanobacterium Synechococcus sp . PCC 7002 and its mutant strains impaired in electron transport; Huang C et al.; The state transitions of the cyanobacterium Synechococcus sp . PCC 7002 and of three mutant strains, which were impaired in PsaE-dependent cyclic electron transport (psaE(-)), respiratory electron transport (ndhF(-)) and both activities (psaE(-)ndhF(-)), were analyzed . Dark incubation of the wild type and psaE(-) cells led to a transition to state 2, while the ndhF(-) strains remained in state 1 after dark incubation . The ndhF(-) cells adapted to state 2 when the cells were incubated under anaerobic conditions or in the presence of potassium cyanide; these results suggest that the ndhF(-) cells were inefficient in performing state 1 to state 2 transitions in the dark unless cytochrome oxidase activity was inhibited . In the state 2 to state 1 transition of wild-type cells induced by light in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), there was still a significant reduction of the interphotosystem electron carriers by both respiration and cyclic electron flow around PSI . Kinetic analysis of the state 2 to state 1 transition shows that, in the absence of PSII activity, the relative contribution to the reduced state of the interphotosystem electron carriers by respiratory and cyclic electron transfer is about 72% and 28%, respectively . The state 2 to state 1 transition was prevented by the cytochrome b(6)f inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) . On the other hand, the state 1 to state 2 transition was induced by DBMIB with half times of approximately 8 s in all strains . The externally added electron acceptor 2,5-dimethyl-benzoquinone (DMBQ) induced a state 2 to state 1 transition in the dark and this transition could be prevented by DBMIB . The light-induced oxidation of P700 showed that approximately 50% of PSI could be excited by 630-nm light absorbed by phycobilisomes (PBS) under state 2 conditions . P700 oxidation measurements with light absorbed by PBS also showed that the dark-induced state 1 to state 2 transition occurred in wild-type cells but not in the ndhF(-) cells . The possible mechanism for sensing an imbalanced light regime in cyanobacterial state transitions is discussed.

Tuberculosis (Edinb), 2004, 84(1-2), 110 - 30
Iron, mycobacteria and tuberculosis; Ratledge C; The role of iron in the growth and metabolism of M . tuberculosis and other mycobacteria is discussed in relation to the acquisiton of iron from host sources, such as transferrin, lactoferrin and ferritin, and its subsequent assimilation and utilization by the bacteria . Key components involved in the acquisition of iron (as ferric ion) and its initial transport into the mycobacterial cell are extracellular iron binding agents (siderophores) which, in pathogenic mycobacteria, are the carboxymycobactins and, in saprophytic mycobacteria, are the exochelins . In both cases, iron may be transferred to an intra-envelope, short-term storage molecule, mycobactin . For transport across the cell membrane, a reductase is used which converts FeIII-mycobactin to the FeII form . The ferrous ion, possibly complexed with salicylic acid, is then shuttled across the membrane either for direct incorporation into various porphyrins and apoproteins or, for storage of iron within the bacterial cytoplasm, bacterioferritin . The overall process of iron acquisition and its utilization is under very genetic tight control . The importance of iron in the virulence of mycobacteria is discussed in relationship to the development of tuberculosis . The management of dietary iron can therefore be influential in aiding the outcome of this disease . The role of the old anti-TB compound, p-aminosalicylate (PAS), is discussed in its action as an inhibitor of iron assimilation, together with the prospects of being able to synthesize further selective inhibitors of iron metabolism that may be useful as future chemotherapeutic agents.

Biol Chem, 2003 Oct-Nov, 384(10-11), 1411 - 9
Ribosomal tolerance and peptide bond formation; Yonath A; In the ribosome, the decoding and peptide bond formation sites are composed entirely of ribosomal RNA, thus confirming that the ribosome is a ribozyme . Precise alignment of the aminoacylated and peptidyl tRNA 3'-ends, which is the major enzymatic contribution of the ribosome, is dominated by remote interactions of the tRNA double helical acceptor stem with the distant rims of the peptidyl transferase center . An elaborate architecture and a sizable symmetry-related region within the otherwise asymmetric ribosome guide the A --> P passage of the tRNA 3'-end by a spiral rotatory motion, and ensures its outcome: stereochemistry suitable for peptide bond formation and geometry facilitating the entrance of newly formed proteins into their exit tunnel.

Lipids, 2003 Oct, 38(10), 1085 - 92
On the origin of cis-vaccenic acid photodegradation products in the marine environment; Rontani JF et al.; The origin of 11-hydroxyoctadec-trans-12-enoic and 12-hydroxyoctadec-trans-10-enoic acids (photodegradation products of cis-vaccenic acid) in the marine environment was investigated . cis-Vaccenic acid is commonly used as a bacterial biomarker; however, in heterotrophic bacteria the observed rates of cis-vaccenic acid photodegradation are negligible . Here, two hypotheses explaining the source of the photoproducts were tested . According to the first hypothesis, the photoproducts originate from aerobic anoxygenic bacteria, i.e., photoheterotrophic organisms using bacteriochlorophyll-containing reaction centers . Alternatively, the photoproducts come from a heterotrophic bacterial community closely associated with senescent phytoplanktonic cells . cis-Vaccenic acid photodegradation was detected in both experimental setups . However, a detailed comparison of the cis-vaccenic acid photodegradation patterns with those observed in particulate matter samples of the DYFAMED station (Mediterranean Sea) suggests that photodegradation of heterotrophic bacteria attached to senescent phytoplanktonic cells constitutes the more likely source of cis-vaccenic acid oxidation products detected in situ.

Environ Technol, 2003 Oct, 24(10), 1315 - 21
Effects of temperature on post-methanation of digested dairy cow manure in a farm-scale biogas production system; Kaparaju PL et al.; A post-methanation process that could be adopted at farm-scale, operating at temperatures prevailing in farm manure digester post-storage tanks, was evaluated . Digested manure samples from a farm digester (35 degrees C) and post-storage tank (5-10 degrees C) were incubated in parallel batches at 5-20 degrees C and as reference at 35 and 55 degrees C . Specific methane yields (kg(-1) volatile solids (VS)(added waste)) were 0.20-0.26 m3 at 35-55 degrees C and 0.085-0.09 m3 at 10-20 degrees C for digester material (345 days of incubation) and 0.16-0.21 m3 at 35-55 degrees C, 0.053-0.087 kg(-1) VS(added waste) m3 at 15-20 degrees C and 0.026 m3 at 10 degrees C for post-storage tank material (250 days) . Both materials produced less than 0.005 m3 at 5 degrees C . However, an increase in temperature to 35 degrees C (40 days) improved methane production in assays pre-incubated at 5-20 degrees C (9 months) . These results suggest that the untapped methane potential of the digested manure cannot effectively be recovered at temperatures prevailing in farm digested manure storage tanks during the winter in Northern latitudes . Nevertheless, as ambient temperatures increase during the late spring, an increase in methanogenesis can be expected.

Arch Microbiol, 2004 Jan, 181(1), 1 - 7 Epub 2003 Dec 11.
Molecular analysis of a subcellular compartment: the magnetosome membrane in Magnetospirillum gryphiswaldense; Schuler D; The ability of magnetotactic bacteria (MTB) to orient and migrate along magnetic field lines is based on magnetosomes, which are membrane-enclosed intracellular crystals of a magnetic iron mineral . Magnetosome biomineralization is achieved by a process involving control over the accumulation of iron and deposition of the magnetic particle, which has a specific morphology, within a vesicle provided by the magnetosome membrane . In Magnetospirillum gryphiswaldense, the magnetosome membrane has a distinct biochemical composition and comprises a complex and specific subset of magnetosome membrane proteins (MMPs) . Classes of MMPs include those with presumed function in magnetosome-directed uptake and binding of iron, nucleation of crystal growth, and the assembly of magnetosome membrane multiprotein complexes . Other MMPs comprise protein families of so far unknown function, which apparently are conserved between all other MTB . The mam and mms genes encode most of the MMPs and are clustered within several operons, which are part of a large, unstable genomic region constituting a putative magnetosome island . Current research is directed towards the biochemical and genetic analysis of MMP functions in magnetite biomineralization as well as their expression and localization during growth.

J Biol Chem, 2004 Mar 5, 279(10), 8694 - 700 Epub 2003 Dec 10.
Structural requirements of synthetic muropeptides to synergize with lipopolysaccharide in cytokine induction; Traub S et al.; Muropeptides contribute to the recognition of bacteria by modulating immune responses: the structural requirements for adjuvant activity were described in the seventies . During the last years, our knowledge of bacterial pattern recognition has increased dramatically and the importance of the absence of contaminations in both muropeptide preparations and other bacterial stimuli has become clear . We investigated a panel of 15 synthetic Limulus-negative muropeptides, four of them synthesized for the first time, as to their potency to synergize with lipopolysaccharide (LPS) in cytokine induction in human whole blood . No muropeptide was capable of stimulating cytokine release from human blood . However, as little as 20 nM of the muropeptides N-acetyl-muramyl-l-alanyl-d-isoglutamine (muramyl dipeptide, M(AdiQ)), N-acetyl-glucosamine-muramyl dipeptide GM(AdiQ), or C(18)M(AdiQ), which carries a non-natural additional fatty acid, sufficed to induce an up to 3 log-order shift in tumor necrosis factor alpha-release in response to 100 pg/ml LPS . The release of interleukin-1beta, interleukin-6, and interleukin-10 was also significantly enhanced although to a lesser extent . The synergistic effect was stereoselective with M(AdiQ) being the minimal active principle . Synergy was also observed on the transcriptional level by means of real-time PCR . Smaller molecules like N-acetylmuramic acid (M), aM, carrying a naturally occurring 1,6-anhydro-bound in M or M(A), containing only the amino acid l-alanine neither synergized with LPS nor influenced the synergy of other muropeptides with LPS . In conclusion, these data show that nanomolar quantities of muropeptides dramatically potentiate LPS-induced monocyte activation . This has implications for pyrogenicity testing and endotoxemia in patients.

Food Chem Toxicol, 2004 Feb, 42(2), 221 - 35
Oral (gavage), in utero and post-natal exposure of Sprague-Dawley rats to low doses of tributyltin chloride . Part II: effects on the immune system; Tryphonas H et al.; The immunotoxic effects of tributyltin chloride (TBTC) were examined in the offspring of Sprague-Dawley rats exposed in utero from day 8 of gestation, through lactation and post-weaning until pups reached the age of 30 days (male and female), 60 days (female) and 90 days (male) . Daily oral (gavage) doses of 0.025, 0.25 and 2.5 mg/kg body weight/day were administered in olive oil 7 days/week . Immunologic endpoints were investigated at the termination of each study . Statistically significant results (P<0.05) included the following: At 30 days, the mean percent and absolute natural killer (NK) cell numbers were increased in male and female rats treated with the high TBTC dose . At 60 days, female rats had increased mean serum IgM levels at the low and high TBTC doses, increased mean percentage CD4(+)8(+) (immature) T lymphocytes at the middle and high doses, a non-linear dose-response increase in NK cell activity at the 50:1 and 100:1 effector:target cell ratios (pairwise comparisons significant at the low dose compared with control), and increased mean numbers of L . monocytogenes colony-forming bacteria on Day 2 post-infection (significant for trend) and Day 3 post infection (pairwise comparisons significant only in the middle dose) . The 90-day male rats had decreased mean serum IgA levels at the middle dose group; increased IgM levels at the high dose group, increased IgG levels at the middle and high doses; decreased IgG2(a) in the high dose compared to the control; a dose-related increase in the mean percentage NK cell numbers (pairwise comparisons significant at the high dose compared with the control) and increased mean NK cell activity (pairwise comparisons significant at all dose groups compared with the control) . The delayed-type hypersensitivity response to oxazolone was increased in the low and middle doses and decreased in the high dose . Thymus atrophy was observed in the high TBTC dose across all ages . Thus, in utero and post-natal treatment of F1 rats with low levels of TBTC affected some aspects of humoral and cell mediated immunity as well as the number and function of cells which are involved in the host's immunosurveillance mechanisms against tumours and viral infections.

Proc R Soc Lond B Biol Sci, 2003 Nov 7, 270 Suppl 2, S202 - 5
Characterization of methanogenic and methanotrophic assemblages in landfill samples; Uz I et al.; A greater understanding of the tightly linked trophic groups of anaerobic and aerobic bacteria residing in municipal solid waste landfills will increase our ability to control methane emissions and pollutant fate in these environments . To this end, we characterized the composition of methanogenic and methanotrophic bacteria in samples taken from two regions of a municipal solid waste landfill that varied in age . A method combining polymerase chain reaction amplification, restriction fragment length polymorphism analysis and phylogenetic analysis was used for this purpose . 16S rDNA sequence analysis revealed a rich assemblage of methanogens in both samples, including acetoclasts, H2/CO2-users and formate-users in the newer samples and H2/CO2-users and formate-users in the older samples, with closely related genera including Methanoculleus, Methanofollis, Methanosaeta and Methanosarcina . Fewer phylotypes of type 1 methanotrophs were observed relative to type 2 methanotrophs . Most type 1 sequences clustered within a clade related to Methylobacter, whereas type 2 sequences were broadly distributed among clades associated with Methylocystis and Methylosinus species . This genetic characterization tool promises rapid screening of landfill samples for genotypes and, therefore, degradation potentials.

Analyst, 2003 Oct, 128(10), 1286 - 90 Epub 2003 Jul 28.
Gas phase electrochemical detection of single latex particles; Caruana DJ et al.; In this study we describe zero current potentiometric measurements in a gaseous flame electrolyte, for the detection of single latex particles . Combustion of polystyrene latex particles when added to a premixed hydrogen/oxygen/nitrogen flame, results in an increase in charged species relative to the surrounding hydrogen flame . As a consequence of this increase in ionic concentration over background, short-lived potential difference transients were measured between two platinum indicator electrodes placed in a two-compartment flame electrochemical cell (described in Electrochem . Commun., 2001, 3, 675-681) . The frequency of the transient events was dependent on the number density of latex particles in solution . It is proposed that each short-lived transient event corresponds to the combustion of single latex particles in a flame . A potential difference maximum of 0.56 V when 3.0 microm diameter particles were added to the flame was measured . Also it was shown that it is possible to detect 0.3 microm diameter latex particles using the same technique . It is postulated that the physical basis of the potential difference is due to the establishment of diffusion/junction potential due to the increase in ionisation from polystyrene combustion at the surface of one indicator electrode . This methodology may be applied to the detection of particulates composed of ionisable species (organic or inorganic) in gaseous environment such as bacteria, viruses, pollen grains and dust.

J Vet Diagn Invest, 2003 Nov, 15(6), 570 - 4
Molecular and antigenic characterization of Bordetella bronchiseptica isolated from a wild southern sea otter (Enhydra lutris nereis) with severe suppurative bronchopneumonia; Staveley CM et al.; Bordetella bronchiseptica was isolated in pure culture from the lung, abdomen, and intestine of a wild free-ranging southern sea otter (Enhydra lutris nereis) with severe, suppurative bronchopneumonia . Immunohistochemistry, using antiserum raised to B . bronchiseptica, revealed strong positive staining of bacteria attached to bronchial ciliated epithelia as well as scattered positive staining in affected alveoli . Western blot analysis demonstrated that virulence factors, filamentous hemagglutinin, pertactin, and adenylate cyclase toxin are produced by the sea otter B . bronchiseptica isolate . Ribotype analysis using Pvu II restriction digests indicated that this isolate is most similar to strains commonly obtained in domestic dogs and cats.

Arch Inst Pasteur Tunis, 1999 Jan-Apr, 76(1-4), 19 - 22
{Oral-dental health status in patients with infectious endocarditis}; Ayadi R et al.; Mouth and dental examination is a systematic act in the etiologic follow up of an infectious endocarditis . Our study concerned 42 patients collected during 5 years period, starting july 1995 until July 2000, in the dental medical service of Hedi Chaker university hospital of Sfax oriented by the cardiovascular diseases service looking for a buccodental entry of bacteria . Our patients group is divided into 25 men and 17 women of 36.1 years mean age . Dental or parodontal lesions were observed in 25 patients . Panoramic X-rays showed periapical lesions of granuloma or cystic type in 15 patients . Among the etiological factors encountered we noticed dental acts accomplished without antibioprophylaxis and dental bacterial collection medically treated without extraction . Buccodental lesions were a frequent cause of the infectious endocarditis in 59.5% of the patients studied in this report, and were due more to defectious hygienic conditions than to a disabled dental act.

Genome, 2003 Dec, 46(6), 1059 - 69
SNP-based codominant markers for a recessive gene conferring resistance to corky root rot (Rhizomonas suberifaciens) in lettuce (Lactuca sativa); Moreno-Vazquez S et al.; The analysis of F2 progeny and derived F3 families of Lactuca sativa segregating for resistance to corky root rot caused by Rhizomonas suberifaciens permitted the identification of restriction fragment length polymorphism (RFLP) and single nucleotide polymorphism (SNP) markers linked to the recessive resistance gene cor . PCR-based markers were identified by bulked segregant analysis (BSA) . Allele-specific primers were generally designed with the 3 terminal base coinciding with an SNP, matching one of the alleles and mismatching the other, and with an additional subterminal 3 base mismatching both alleles . Codominant, robust, and inexpensive molecular markers were obtained that used standardized PCR conditions . Some of the markers could be analyzed in multiple Lactuca mapping populations that did not segregate for disease resistance allowing the cor locus to be located on several maps . The consistent low density of markers around cor in these maps suggests that cor may be in an area with an elevated rate of recombination . Evaluation of these markers in a large sample of cultivars and landraces identified pairs of flanking polymorphic markers that can be used for marker-assisted selection of corky root resistance.

Can J Microbiol, 2003 Oct, 49(10), 625 - 32
A metalloproteinase extracellularly released by Crithidia deanei; d'Avila-Levy CM et al.; Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4) . The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa . One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs . The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . The incorporation of gelatin into SDS-PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa . The enzyme was optimally active at 37 degrees C and pH 6.0 and showed 25% of residual activity at 28 degrees C for 30 min . The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class . A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C . deanei protease . After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases . The detection of an extracellular metalloproteinase produced by C . deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.

Can J Microbiol, 2003 Oct, 49(10), 602 - 12
Methanotrophic diversity in high arctic wetlands on the islands of Svalbard (Norway)--denaturing gradient gel electrophoresis analysis of soil DNA and enrichment cultures; Wartiainen I et al.; The methanotrophic community in arctic soil from the islands of Svalbard, Norway (78 degrees N) was analysed by combining group-specific PCR with PCR of the highly variable V3 region of the 16S rRNA gene and then by denaturing gradient gel electrophoresis (DGGE) . Selected bands were sequenced for identification . The analyses were performed with DNA extracted directly from soil and from enrichment cultures at 10 and 20 degrees C . The two genera Methylobacter and Methylosinus were found in all localities studied . The DGGE band patterns were simple, and DNA fragments with single base differences were separated . The arctic tundra is a potential source of extensive methane emission due to climatic warming because of its large reservoirs of stored organic carbon . Higher temperatures due to climatic warming can cause increased methane production, and the abundance and activity of methane-oxidizing bacteria in the arctic soil may be important regulators for methane emission to the atmosphere.

J Clin Microbiol, 2003 Dec, 41(12), 5434 - 41
Culture and phenotypic characterization of a Wolbachia pipientis isolate; Fenollar F et al.; The recent isolation of Wolbachia pipientis in the continuous cell line Aa23, established from eggs of a strain of the Asian tiger mosquito Aedes albopictus, allowed us to perform extensive characterization of the isolate . Bacterial growth could be obtained in C6/36, another A . albopictus cell line, at 28 degrees C and in a human embryonic lung fibroblast monolayer at 28 and 37 degrees C, confirming that its host cell range is broader than was initially thought . The bacteria were best visualized by Diff-Quik and May-Grunwald-Giemsa staining . Proteins from 213 to 18 kDa with two major protein bands of 65 and 25 kDa were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . By Western blotting with specific polyclonal mouse and rabbit antisera, dominant immunoreactive antigens were found at approximately 100, 80, and 30 kDa . The genome size was calculated to be 1,790 +/- 17 kb by pulsed-field gel electrophoresis . The sequence of the citrate synthase gene (gltA) of W . pipientis was determined by gene walking . Its position in the phylogenetic tree constructed with gltA confirmed that found in a phylogenetic tree constructed with 16S rRNA genes and that it belongs in the alpha subgroup of the class Proteobacteria and that it is closely related to but independent from the genera Ehrlichia, Anaplasma, and Neorickettsia.

Curr Opin Biotechnol, 2003 Dec, 14(6), 627 - 33
Novel biologically active natural and unnatural products; Myles DC; Natural products have been used as medicinal agents for many years . In addition, these compounds have served as the starting points for semisynthetic analogs with improved properties . This review highlights work on several classes of natural products and their derivatives, including both well established and emerging structural classes that are in, or nearing, clinical use for a variety of important indications.

Curr Opin Biotechnol, 2003 Dec, 14(6), 577 - 82
New substrates for reliable enzymes: enzymatic modification of polymers; Gubitz GM et al.; Recent studies clearly indicate that the modification of synthetic and natural polymers with enzymes is an environmentally friendly alternative to chemical methods using harsh conditions . New processes using lipases, proteases, nitrilases and glycosidases have been developed for the specific non-destructive functionalization of polymer surfaces . The specificity of enzymes has also been exploited in polymer synthesis; for example, lipases have been used for the production of optically active polyesters . Oxidoreductases have been used for the cross-linking and grafting of lignaceous materials and for the production of polymers from phenolics . Recent successes in this area are mainly attributable to advances in the design of reaction systems (e.g . biphasic systems and micellar solutions), while the enzymes are mainly from commercial sources.

Comp Biochem Physiol B Biochem Mol Biol, 2003 Dec, 136(4), 943 - 55
Cloning of bovine peroxiredoxins-gene expression in bovine tissues and amino acid sequence comparison with rat, mouse and primate peroxiredoxins; Leyens G et al.; The peroxiredoxin (PRDX) family is a recently identified family of peroxidases found in organisms ranging from bacteria to mammals . In mammals, six PRDX isoforms have been characterized in human (Homo sapiens), rat (Rattus norvegicus) and mouse (Mus musculus) . PRDXs are cytosolic, secreted or targeted to organelles such as peroxisomes, mitochondria and the nucleus . Some PRDXs are synthesized as larger precursor proteins with a presequence that is cleaved to produce the mature form . To study the expression of the six PRDXs in bovine (Bos taurus), we first cloned cDNAs coding for PRDX1, PRDX2, PRDX4 and PRDX5 . PRDX3 and PRDX6 had previously been cloned and characterized in bovine . The comparison of bovine PRDXs with their rat, mouse and primate orthologues reveals a minimum of 95% similarity of mature proteins . Even though mitochondrial or export signal presequences are normally less conserved, the unprocessed proteins still present a minimum of 84% similarity . Nevertheless, a major divergence lies at the N-terminus of bovine PRDX2, where a Cys-Val-Cys motif was identified . The expression of the six PRDXs in 22 bovine tissues has been studied by RT-PCR . Our results point out the ubiquity of the different PRDX transcripts in bovine tissues . The important conservation of the different PRDXs, the multiple processes they have been associated with, as well as the ubiquity of all the members of the family analyzed in this study for the first time altogether, suggest that they play a major role in the basal metabolism of mammalian cells.

Comp Biochem Physiol B Biochem Mol Biol, 2003 Dec, 136(4), 723 - 30
Molecular diversity of skin mucus lectins in fish; Suzuki Y et al.; Among lectins in the skin mucus of fish, primary structures of four different types of lectin have been determined . Congerin from the conger eel Conger myriaster and AJL-1 from the Japanese eel Anguilla japonica were identified as galectin, characterized by its specific binding to beta-galactoside . Eel has additionally a unique lectin, AJL-2, which has a highly conserved sequence of C-type lectins but displays Ca(2+)-independent activity . This is rational because the lectin exerts its function on the cutaneous surface, which is exposed to a Ca(2+) scarce environment when the eel is in fresh water . The third type lectin is pufflectin, a mannose specific lectin in the skin mucus of pufferfish Takifugu rubripes . This lectin showed no sequence similarity with any known animal lectins but, surprisingly, shares sequence homology with mannose-binding lectins of monocotyledonous plants . The fourth lectin was found in the ponyfish Leiognathus nuchalis and exhibits homology with rhamnose-binding lectins known in eggs of some fish species . These lectins, except ponyfish lectin, showed agglutination of certain bacteria . In addition, pufflectin was found to bind to a parasitic trematode, Heterobothrium okamotoi . Taken together, these results demonstrate that skin mucus lectins in fish have wide molecular diversity.

Biochemistry, 2003 Dec 16, 42(49), 14501 - 6
Lack of negative charge in the E46Q mutant of photoactive yellow protein prevents partial unfolding of the blue-shifted intermediate; Derix NM et al.; The long-lived light-induced intermediate (pB) of the E46Q mutant (glutamic acid is replaced by glutamine at position 46) of photoactive yellow protein (PYP) has been investigated by NMR spectroscopy . The ground state of this mutant is very similar to that of wild-type PYP (WT), whereas the pB state, formed upon illumination, appears to be much more structured in E46Q than in WT . The differences are most striking in the N-terminal domain of the protein . In WT, the side-chain carboxylic group of E46 is known to donate its proton to the chromophore upon illumination . The absence of the carboxylic group near the chromophore in the E46Q mutant prohibits the formation of a negative charge at this position upon formation of pB . This prevents the partial unfolding of the mutant, as evidenced from NMR chemical shift comparison and proton/deuterium (H/D) exchange studies.

J Biomater Sci Polym Ed, 2003, 14(10), 1029 - 42
Production of purified polyhydroxyalkanoates (PHAs) for applications in contact with blood; Sevastianov VI et al.; Samples of olyhydroxyalkanoates (PHAs), polyhydroxybutyrate (PHB) and copolymers poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) with 4 and 18 mol% hydroxyvalerate, synthesized by the bacteria Ralstonia eutropha B5786, were investigated . PHA films in contact with blood did not activate the hemostasis system at the level of cell response, but they did activate the coagulation system and the complement reaction . To detect biologically-active components in the PHAs, a detailed analysis of the composition of the polymers was conducted . Gas chromatography-mass spectrometry revealed long-chain fatty acids (FAs) in the tested PHAs . Their total concentration in the polymer ranged from tenths of mol% to 2-3 mol%, depending on the purification method . C16:0 constituted the largest proportion, up to 70% . Of the long-chain hydroxy acids, only beta-OH-C14:0 was detected and it did not exceed 0.06 mol% . The analysis of the hemocompatibility properties of the PHAs purified by a specialized procedure, including the quantitative and morphological estimation of platelets adherent to the surface of polymer films, the plasma recalcification time and complement activation studies, indicated that PHB and PHBV can be used in contact with blood . It has been found out that the lipopolysaccharides of bacteria producing PHAs, which contain mostly long-chain hydroxy acids, can be the factor activating the hemostasis systems . Thus, the technology of PHA purification must satisfy rather stringent specific requirements.

Shanghai Kou Qiang Yi Xue, 2003 Apr, 12(2), 109 - 11
{Clinical studies of calcium hydroxide disinfecting infectious root canal of deciduous tooth}; Zhao J et al.; OBJECTIVE: To search for a more ideal way to disinfect infectious root canal of deciduous tooth . METHODS: According to bacterial occurrence and clinical effect, 40 cases of infectious root canal of deciduous teeth disinfected with Ca(OH)2 were investigated, the other 40 cases with traditional way of using FC and CP respectively, were used as the control . RESULTS: Fist of all, the bacteria of deciduous tooth infectious root canal were same as the bacteria of permanent tooth infectious root canal . Secondly, Ca(OH)2 was stronger in disinfecting deciduous tooth infectious root canal . Thirdly, so far as sterilization was concerned, Ca(OH)2 had the same disinfectant as FC, and was better than CP, but FC had some irritation and poisonous side-effects to periodical tissue . So Ca(OH)2 was safe and effective for deciduous tooth infectious root canal . CONCLUSION: It was a more ideal way to disinfect deciduous tooth infectious root canal by Ca(OH)2.

Appl Environ Microbiol, 2003 Dec, 69(12), 7430 - 4
Real-time PCR assay for detection and enumeration of Dekkera bruxellensis in wine; Phister TG et al.; Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments . To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D . bruxellensis in wine . Specific PCR primers to D . bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified . The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine . The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay . This method will be helpful to identify possible routes of D . bruxellensis infection in winery environments . Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D . bruxellensis.

Res Vet Sci, 2004 Feb, 76(1), 1 - 18
Molecular differentiation of Mycobacterium bovis isolates . Review of main techniques and applications; Haddad N et al.; Until recently, none of the Mycobacterium bovis typing techniques permitted a satisfactory differentiation of isolates . During the last 10 years, the genome of pathogenic mycobacteria has been extensively studied, and phylogenetic analyses have shown that all (except Mycobacterium avium) belong to a single genetic species: the Mycobacterium tuberculosis complex . This increase in knowledge about the genome of these bacteria has lead to the discovery of molecular markers that allow us to differentiate isolates . Because of the phylogenetic proximity of the strains, even if most of these markers have been discovered in M . tuberculosis, they could be successfully adapted to the other bacteria of the M . tuberculosis complex, especially M . bovis . The most common markers in use today are the IS6110 insertion sequence, the direct repeat (DR) region, the poly(GC) rich (PGRS) sequences and the variable number tandem repeats (VNTR) sequences . The corresponding typing techniques are briefly described, and current knowledge of polymorphism and marker stability is detailed . If molecular markers are to offer wide perspectives for field studies, these two characteristics (polymorphism and stability) must be taken into account when choosing the marker(s) used in a study . In this context, examples of the application of molecular typing techniques for M . bovis are reviewed, on the one hand with epidemiological studies for which the major problem is the comparison between isolates and, on the other, with more general studies about the population genetics of M . bovis in a given country, and about its history and its phylogeny.

Proc Natl Acad Sci U S A, 2003 Dec 9, 100(25), 15271 - 5 Epub 2003 Dec 01.
The population structure of Mycobacterium bovis in Great Britain: clonal expansion; Smith NH et al.; We have analyzed 11,500 isolates of Mycobacterium bovis (the cause of tuberculosis in cattle and other mammals) isolated in Great Britain (England, Wales and Scotland)} and characterized by spoligotype . Genetic exchange between cells is rare or absent in strains of the Mycobacterium tuberculosis complex so that, by using spoligotypes, it is possible to recognize "clones" with a recent common ancestor . The distribution of variable numbers of tandem repeats types in the most common clone in the data set is incompatible with random mutation and drift . The most plausible explanation is a series of "clonal expansions," and this interpretation is supported by the geographical distribution of different genotypes . We suggest that the clonal expansion of a genotype is caused either by the spread of a favorable mutation, together with all other genes present in the ancestral cell in which the mutation occurred, or by the invasion of a novel geographical region by a limited number of genotypes . A similar pattern is observed in M . tuberculosis (the main cause of tuberculosis in humans) . The significance of clonal expansion in other bacteria that have recombination is discussed.

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 1833 - 42
Molecular signatures in protein sequences that are characteristic of cyanobacteria and plastid homologues; Gupta RS et al.; Fourteen conserved indels (i.e . inserts or deletions) have been identified in 10 widely distributed proteins that appear to be characteristic of cyanobacterial species and are not found in any other group of bacteria . These signatures include three inserts of 6, 7 and 28 aa in the DNA helicase II (UvrD) protein, an 18-21 aa insert in DNA polymerase I, a 14 aa insert in the enzyme ADP-glucose pyrophosphorylase, a 3 aa insert in the FtsH protein, an 11-13 aa insert in phytoene synthase, a 5 aa insert in elongation factor-Tu, two deletions of 2 and 7 aa in ribosomal S1 protein, a 2 aa insert in the SecA protein, a 1 aa deletion and a 6 aa insert in the enzyme inosine-5'-monophosphate dehydrogenase and a 1 aa deletion in the major sigma factor . These signatures, which are flanked by conserved regions, provide molecular markers for distinguishing cyanobacterial taxa from all other bacteria and they should prove helpful in the identification of cyanobacterial species, simply on the basis of the presence or absence of these markers in the corresponding proteins . The signatures in six of these proteins (SecA, elongation factor-Tu, ADP-glucose pyrophosphorylase, phytoene synthase, FtsH and ribosomal S1 protein) are also commonly present in plastid homologues from plants and algae (chlorophytes, chromophytes and rhodophytes), indicating their specific relationship to cyanobacteria and supporting their endosymbiotic origin from these bacteria . In phylogenetic trees based on a number of these proteins (SecA, UvrD, DNA polymerase I, elongation factor-Tu) that were investigated, the available cyanobacterial homologues grouped together with high affinity (>95 % bootstrap value), supporting the view that the cyanobacterial phylum is monophyletic and that the identified signatures were introduced in a common ancestor of this group.

Environ Sci Technol, 2003 Nov 15, 37(22), 5175 - 80
Virus inactivation in aluminum and polyaluminum coagulation; Matsui Y et al.; Inorganic aluminum salts, such as aluminum sulfate, are coagulants that cause small particles, such as bacteria and viruses as well as inorganic particles, to destabilize and combine into larger aggregates . In this investigation, batch coagulation treatments of water samples spiked with Qbeta, MS2, T4, and P1 viruses were conducted with four different aluminum coagulants . The total infectious virus concentration in the suspension of floc particles that eventually formed by dosing with coagulant was measured after the floc particles were dissolved by raising the pH with an alkaline beef extract solution . The virus concentrations were extremely reduced after the water samples were dosed with aluminum coagulants . Viruses mixed with and adsorbed onto preformed aluminum hydroxide floc were, however, completely recovered after the floc dissolution . These results indicated that the aluminum coagulation process inactivates viruses . Virucidal activity was most prominent with the prehydrolyzed aluminum salt coagulant, polyaluminum chloride (PACl) . Virucidal activity was lower in river water than in ultrapure water--natural organic matter in the river water depressed the virucidal activity . Mechanisms and kinetics of the virus inactivation were discussed . Our results suggest that intermediate polymers formed during hydrolysis of the aluminum coagulants sorbed strongly to viruses, either rendering them inactive or preventing infectivity.

Berl Munch Tierarztl Wochenschr, 2003 Nov-Dec, 116(11-12), 447 - 53
Molecular typing of pathogens; Wassenaar TM; Molecular typing and fingerprinting of bacterial pathogens has become a major part of epidemiology, disease monitoring, intervention and food safety research . This treatise will briefly address the aims of molecular typing and the criteria that are important to choose the optimal method of typing . However, since the method of choice will depend on a number of factors, including available facilities and research goals, no particular methods are recommended; instead, theoretical considerations are presented as a guide line for the best decision . Since molecular typing is examining the nature of the population genetics of a particular organism, it is important to understand how the degree of clonality of that particular organism will influence outcome and interpretation of results . The mechanisms that lead to non-clonality are briefly outlined . The problems with typing of clonal, non-clonal, and weakly clonal populations are briefly treated . The merits and myths of multilocus sequence typing (MLST) are discussed.

Turk J Gastroenterol, 2003 Sep, 14(3), 168 - 72
Expression of class I and II MHC receptors in Helicobacter pylori-positive patients with active gastritis and duodenal ulcer; Suleymanov Z; BACKGROUND/AIMS: Helicobacter pylori colonizes on the epithelial surface below the mucous membrane in the gastric antrum, causing chronic active gastritis and duodenal ulcer . However, we know little about the response of the immune system, which is responsible for protecting the organism, to this bacteria and the duodenal tissue damage . The aims of our study were to examine the expression of class I and II MHC receptors in the immune cells in the peripheral venous blood of patients with chronic active gastritis and duodenal ulcer . METHODS: The study included a total of 124 cases (Helicobacter pylori-positive patients without prior treatment, 47 of whom had small-sized and 56 medium-sized duodenal ulcer, and 21 healthy individuals) . Immune cells carrying receptors CD3, CD4, CD3/4, CD8, CD3/8, CD14, CD45, CD14/45, HLA-DR, CD3/HLA-DR, CD16/56, and CD3/16/56 by applying DAKO Dual Color Reagent, and non-specific indicators of reactivity, were investigated in the peripheral venous blood samples . RESULTS: In both study groups, nonspecific indicators of reactivity in peripheral venous blood were not different from those in the healthy group irrespective of the ulcer size . It was detected that NK cells which express only CD3/16/56 receptor are more aggressive than class I MHC carriers, which were increased in both patient groups more markedly than in the control group (p<0.05) . The number of CD14 cells in patient groups with small- and medium-sized duodenal ulcers was found to be lower than in the control group . This difference was more marked in the small-sized ulcer group than in the medium-sized ulcer group . While there was a similarity in the number of CD45 cells in all three groups, the number of CD14/45 cells was reduced in the study group . This reduction was found statistically insignificant in the medium-sized ulcer group, but significant in the small-sized ulcer group (p<0.05) . As compared to the control group, the number of HLA-DR cells was found to be increased in both study groups, and CD3/HLA-DR was found to be increased in the medium-sized ulcer group (p<0.05) . CONCLUSIONS: The reduced number of CD14 cells may have been among the factors predisposing to the development of Helicobacter pylori infection . Such a deficit in cellular defense may be compensated through the upregulation of cells with HLA-DR and CD3/HLA-DR markers by the organism . The increase in the number of cells expressing CD3/16/56 in chronic antral gastritis and duodenal ulcer patients positive for Helicobacter pylori suggests a role for autoimmune events in these diseases.

Mol Genet Metab, 2003 Dec, 80(4), 389 - 97
PCR methods in clinical investigations of human ureaplasmas: a minireview; Colaizy TT et al.; Human ureaplasmas are small, cell-wall-deficient organisms that are implicated in many human infections . Due to their small size and fastidious growth requirements, ureaplasmal infections are hard to diagnose clinically, and ureaplasmal disease is difficult to study in clinical samples . Standard culture methods for ureaplasmas are technically challenging, and 3 to 5 days are required to identify this pathogen . PCR methods have been increasingly used in the diagnosis of these infections and in the study of this pathogen in human specimens from multiple sites . These methods have theoretical advantages over traditional culture methods . Organism identification can occur in the presence of low numbers of bacteria, and viability is not necessary . Rapid identification of organisms within 24 h is also possible . In addition, subtyping of isolates can be performed faster with PCR methods than with culture methods . The use of PCR methods in translational research in multiple human ureaplasmal diseases will be reviewed in this paper, and their usefulness will be compared to culture methods.

Eur J Biochem, 2003 Dec, 270(24), 4909 - 20
The astacin protein family in Caenorhabditis elegans; Mohrlen F et al.; In the nematode Caenorhabditis elegans, 40 genes code for astacin-like proteins (nematode astacins, NAS) . The astacins are metalloproteases present in bacteria, invertebrates and vertebrates and serve a variety of physiological functions like digestion, hatching, peptide processing, morphogenesis and pattern formation . With the exception of one distorted pseudogene, all the other C . elegans astacins are expressed and are evidently functional . For 13 genes we found splicing patterns differing from the Genefinder predictions in WormBase, sometimes markedly . The GFP expression pattern for NAS-4 shows a specific localization in anterior pharynx cells and in the whole digestive tract (as the secreted form) . In contrast, NAS-7 is found in the head of adult hermaphrodites, but not in pharynx cells or in the lumen of the digestive tract . In embryos, NAS-7 fluorescence becomes detectable just before hatching . In C . elegans astacins, three basic structural and functional moieties can be discerned: a prepro portion, the central catalytic chain and long C-terminal extensions with presumably regulatory functions . Within the regulatory moiety, EFG-like, CUB, SXC, and TSP-1 domains can be distinguished . Based on structural differences of the regulatory unit we established six NAS subgroups, which seemingly represented different functional and evolutionary clusters . This pattern deduced exclusively from the domain arrangement in the regulatory moiety is perfectly reflected in an evolutionary tree constructed solely from amino acid sequence information of the catalytic chain . Related catalytic chains tend to have related regulatory extensions . The notable gene, NAS-39 shows a striking resemblance to human BMP-1 and the tolloids.

Respir Care, 2003 Dec, 48(12), 1204 - 13; discussion 1213-5
Exacerbations of chronic obstructive pulmonary disease; Wedzicha JA et al.; Exacerbations of chronic obstructive pulmonary disease (COPD) cause morbidity, hospital admissions, and mortality, and strongly influence health-related quality of life . Some patients are prone to frequent exacerbations, which are associated with considerable physiologic deterioration and increased airway inflammation . About half of COPD exacerbations are caused or triggered primarily by bacterial and viral infections (colds, especially from rhinovirus), but air pollution can contribute to the beginning of an exacerbation . Type 1 exacerbations involve increased dyspnea, sputum volume, and sputum purulence; Type 2 exacerbations involve any two of the latter symptoms, and Type 3 exacerbations involve one of those symptoms combined with cough, wheeze, or symptoms of an upper respiratory tract infection . Exacerbations are more common than previously believed (2.5-3 exacerbations per year); many exacerbations are treated in the community and not associated with hospital admission . We found that about half of exacerbations were unreported by the patients, despite considerable encouragement to do so, and, instead, were only diagnosed from patients' diary cards . COPD patients are accustomed to frequent symptom changes, and this may explain their tendency to underreport exacerbations . COPD patients tend to be anxious and depressed about the disease and some might not seek treatment . At the beginning of an exacerbation physiologic changes such as decreases in peak flow and forced expiratory volume in the first second (FEV(1)) are usually small and therefore are not useful in predicting exacerbations, but larger decreases in peak flow are associated with dyspnea and the presence of symptomatic upper-respiratory viral infection . More pronounced physiologic changes during exacerbation are related to longer exacerbation recovery time . Dyspnea, common colds, sore throat, and cough increase significantly during prodrome, indicating that respiratory viruses are important exacerbation triggers . However, the prodrome is relatively short and not useful in predicting onset . As colds are associated with longer and more severe exacerbations, a COPD patient who develops a cold should be considered for early therapy . Physiologic recovery after an exacerbation is often incomplete, which decreases health-related quality of life and resistance to future exacerbations, so it is important to identify COPD patients who suffer frequent exacerbations and to convince them to take precautions to minimize the risk of colds and other exacerbation triggers . Exacerbation frequency may vary with the severity of the COPD . Exacerbation frequency may or may not increase with the severity of the COPD . As the COPD progresses, exacerbations tend to have more symptoms and take longer to recover from . Twenty-five to fifty percent of COPD patients suffer lower airway bacteria colonization, which is related to the severity of COPD and cigarette smoking and which begins a cycle of epithelial cell damage, impaired mucociliary clearance, mucus hypersecretion, increased submucosal vascular leakage, and inflammatory cell infiltration . Elevated sputum interleukin-8 levels are associated with higher bacterial load and faster FEV(1) decline; the bacteria increase airway inflammation in the stable patient, which may accelerate disease progression . A 2-week course of oral corticosteroids is as beneficial as an 8-week course, with fewer adverse effects, and might extend the time until the next exacerbation . Antibiotics have some efficacy in treating exacerbations . Exacerbation frequency increases with progressive airflow obstruction; so patients with chronic respiratory failure are particularly susceptible to exacerbation.

Reprod Biol Endocrinol . 2003 Dec 02;1(1):122.
Immunology of term and preterm labor; Peltier MR; During pregnancy there is an alteration in maternal immunity within the uterus where innate, proinflammatory immune responses are tightly regulated to prevent immunological rejection of the fetal allograft . Disruption of the delicate balance of cytokines by bacteria or other factors increases the production of proinflammatory cytokines at the maternal-fetal interface and activates the parturition mechanism prematurely . Despite years of searching, there is still no broadly effective strategy for preventing preterm labor and most therapies are directed at inhibiting myometrial contractions and improving neonatal outcome . Recent studies with progestins and interleukin-10 (IL-10), however, are showing promise in randomized clinical trials and animal studies . Furthermore, the identification of the Toll-like receptors as upstream mediators of inflammation may offer alternative therapeutic targets for preventing this common pregnancy complication.

J Endod, 2003 Nov, 29(11), 729 - 34
Direct capping with four different materials in humans: histological analysis of odontoblast activity; Scarano A et al.; Pulp inflammation in restored teeth is mainly due to the presence of bacteria or bacterial products introduced by microleakage around the restoration or to the material toxicity . Recent knowledge has permitted a precise identification of the risks for pulpal irritation associated with adhesive materials and procedures . The purpose of this work was to evaluate the cellular events that occur in direct pulp exposure capped using different materials . Twenty-four vital teeth without caries, scheduled for extraction for orthodontic reasons, were selected . After a control of the hemostasis, each pulp was directly capped with a different material . The samples were randomly divided into four groups of six specimens each: group I: dental-bonding agent (Solist) followed by resin composite (Ecusit); group II: dental adhesive (Prompt) and resin composite (Pertac II); group III: traditional calcium hydroxide (Dycal) plus resin composite (Ecusit); group IV: light-curing calcium hydroxide (Ultrablend Plus) and amalgam (Dentsply) . After 15 days the teeth were extracted, immediately fixed in 10% buffered formalin, embedded in resin (7200 Technovit), and prepared for thin ground sections with Precise 1 System . In the specimens of all groups, there were active odontoblasts near the composite resins and no newly formed dentin . Small quantities of inflammatory cells were present . A 1- to 3-microm layer zone of necrosis was present . In conclusion, all materials tested in this study induced similar tissue responses.

Chang Gung Med J, 2003 Sep, 26(9), 621 - 36
Possible application of milk oligosaccharides for drug development; Kobata A; By applying a finger-printing method to the analysis of human milk oligosaccharides, several oligosacchartides were found to be deleted in the milk of non-secretor or Lewis negative individual . This finding afforded a clue to elucidate the enzymatic basis of blood types in humans . Furthermore, disappearance of some major oligosaccharides led to the finding of five novel minor oligosaccharides, which were hidden under the major oligosaccharides . Later on, structures of more than seventy oligosaccharides were elucidated . These oligosaccharides are derived from eleven core oligosaccharides by sialylation and/or fucosylation . All these oligosaccharides contain lactose at their reducing termini . This evidence, together with the deletion phenomena found in the milk of two blood type individuals, suggested that the oligosaccharides are formed by the concerted action of glycosyltransferases, which are responsible for formation of the sugar chains of glycoproteins on the surface of epithelial cells constructing the mucous membrane . The elongation may start by the action of iGnT . This enzyme is responsible for the addition of a beta-N-acetylglucosamine residue to the C-3 position of the galactose moiety constructing the N-acetyllactosamine group of the sugar chains of glycoconjugates . Therefore, oligosaccharides in human milk may include many structures, starting from the N-acetyllactosamine residues in the sugar chains of various glycoproteins . Many evidences, which indicate that virulent enteric bacteria and viruses start their infection by binding to particular sugar chains of glycoconjugates on the surface of their target cells, were presented recently . Therefore, milk oligosaccharides are expected to be useful to inhibit the infection of these bacteria and viruses.

Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2065 - 71 Epub 2003 Nov 27.
Structure of the hybrid cluster protein (HCP) from Desulfovibrio desulfuricans ATCC 27774 containing molecules in the oxidized and reduced states; Macedo S et al.; The hybrid cluster protein (HCP) from the sulfate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774 has been isolated and crystallized anaerobically . The protein sample used in the crystallization studies was several months old, having been stored at 193 K, and initial crystal structure studies were unable to fully resolve details of the hybrid cluster despite the use of high-resolution data to 1.25 A collected at the ESRF, Grenoble, France . Full elucidation of the structure has only become possible with the complete knowledge of the as-isolated and fully reduced crystal structures . The analysis clarifies the significant movements in the position of the Fe atom linked to the persulfide moiety in the oxidized as-isolated protein and the S atom of the persulfide itself as the protein is reduced . The structures of the as-isolated and reduced states are discussed in terms of the putative function of the HCP proteins.

Biol Trace Elem Res, 2003 Nov, 95(2), 97 - 106
Serum levels of vitamins A, C, and E, beta-carotene, selenium, and zinc in patients with Behçet's disease: a controlled study; Erel A et al.; Behcet's disease is a multisystemic disease characterized by activation and remission periods . The etiopathogenesis is not exactly known; a genetic defect in the immunoregulatory system induced by infectious agents, like viruses and bacteria, is thought to cause the disease . In this study, we examine the serum levels of vitamins A, C, and E, beta-carotene, selenium, and zinc in Behcet's disease patients and investigate the relationship between these serum levels and the activation of the disease . We conclude that adding vitamin E to the treatment of Behcet's disease patients and its effects on the prognosis of the disease need to be further investigated by controlled studies.

Biochim Biophys Acta, 2003 Dec 3, 1618(1), 59 - 66
Characterisation of subunit III and its oligomer from spinach chloroplast ATP synthase; Poetsch A et al.; Proton ATP synthases carry out energy conversion in mitochondria, chloroplasts, and bacteria . A key element of the membrane integral motor CFO in chloroplasts is the oligomer of subunit III: it converts the energy of a transmembrane electrochemical proton gradient into rotational movement . To enlighten prominent features of the structure-function relationship of subunit III from spinach chloroplasts, new isolation methods were established to obtain highly pure monomeric and oligomeric subunit III in milligram quantities . By Fourier-transform infrared (FTIR) and CD spectroscopy, the predominantly alpha-helical secondary structure of subunit III was demonstrated . For monomeric subunit III, a conformational change was observed when diluting the SDS-solubilized protein . Under the same conditions the conformation of the oligomer III did not change . A mass of 8003 Da for the monomeric subunit III was determined by MALDI mass spectrometry (MALDI-MS), showing that no posttranslational modifications occurred . By ionisation during MALDI-MS, the noncovalent homooligomer III14 disaggregated into its III monomers.

J Theor Biol, 2004 Feb 7, 226(3), 341 - 8
Chaos game representation of protein sequences based on the detailed HP model and their multifractal and correlation analyses; Yu ZG et al.; Similar to the chaos game representation (CGR) of DNA sequences proposed by Jeffrey (Nucleic Acid Res . 18 (1990) 2163), a new CGR of protein sequences based on the detailed HP model is proposed . Multifractal and correlation analyses of the measures based on the CGR of protein sequences from complete genomes are performed . The Dq spectra of all organisms studied are multifractal-like and sufficiently smooth for the Cq curves to be meaningful . The Cq curves of bacteria resemble a classical phase transition at a critical point . The correlation distance of the difference between the measure based on the CGR of protein sequences and its fractal background is also proposed to construct a more precise phylogenetic tree of bacteria.

Mutat Res, 2003 Dec 10, 533(1-2), 211 - 26
Signaling events for metallothionein induction; Haq F et al.; Metallothioneins (MTs) are low molecular weight, cysteine-rich metal-binding proteins found in a wide variety of organisms including bacteria, fungi and all eukaryotic plant and animal species . They are the single most abundant group of intracellular zinc-binding proteins in eukaryotic cells {Prog . Food Nutr . Sci . 14 (1990) 193}: 5-10% of zinc in human hepatocytes is bound to MTs {Methods Enzymol . 205 (1991) 613; FEBS Lett . 39 (1974) 229} . A critical physiological role for MTs has been suggested to encompass control of zinc availability to proteins requiring zinc for activity, but has not been definitively described . Nevertheless, a hallmark of MT gene regulation is their pattern of expression in different cell types and tissues, and their inducibility by a host of chemical and physical agents acting directly or indirectly on multiple cis-acting motifs in the regulatory regions of MT genes {Prog . Nucleic Acid . Res . Mol . Biol., 66 (2001) 357} . The pattern of physical and chemical events regulating basal MT gene transcription, and induction or repression of MT gene activity, provides valuable insight into the potential functions of MTs . In this review, we describe the transcriptional regulatory processes involved in MT gene expression.

Neurobiol Aging, 2003 Dec, 24(8), 1071 - 7
Use of YFP to study amyloid-beta associated neurite alterations in live brain slices; Brendza RP et al.; Neuritic plaques are one of the defining neuropathological features of Alzheimer's disease (AD) . These structures are composed of a buildup of fibrils of the amyloid-beta (Abeta) peptide (amyloid) surrounded by activated glial cells and degenerating nerve processes (dystrophic neurites) . To study neuritic plaques and possible abnormalities associated with dendrites, axons, and synaptic structures, we have developed an acute slice preparation model using PDAPP, yellow fluorescent protein (YFP) double transgenic mice (a mouse model with AD-like pathology that stably expresses YFP in a subset of neurons in the brain) . With laser scanning confocal microscopy, we have imaged living brain slices from PDAPP, YFP double transgenic mice as old as 20 months and have been able to visualize axons, dendrites, dendritic spines, and dystrophic neurites for many hours . Our initial studies suggest that dystrophic axons and dendrites within neuritic plaques are fairly stable structures in the absence of exogenous perturbations . This acute slice preparation model should prove to be a useful tool to explore the pathophysiology of Abeta-related axonal, dendritic, and synaptic dysfunction.

J Struct Biol, 2003 Oct-Nov, 144(1-2), 172 - 83
Windex: a toolset for indexing helices; Ward A et al.; We describe here a set of procedures and algorithms that may be used as an aid in determining the indexing rule of a helical specimen . Crystallizing macromolecules into helical arrays has the potential to speed up and simplify the process of three-dimensional reconstruction of the macromolecular structure . The process of helical reconstruction has been largely automated except for the critical first step of indexing the helical diffraction pattern . This is quite often the rate-limiting step in the overall process, particularly in the case of large helical tubes, which have complicated helical diffraction patterns that may vary from tube to tube . We have developed a set of procedures, supported by a graphical user interface, that provide a straightforward and semi-automated approach to indexing a helical structure . The new procedures have been tested using a number of helical specimens, including TMV, acto-myosin, decorated microtubules, and a variety of helical tubes of a bacterial membrane protein.

J Struct Biol, 2003 Oct-Nov, 144(1-2), 144 - 51
Low-resolution structure refinement in electron microscopy; Chen JZ et al.; A real-space structure refinement method, originally developed for macromolecular X-ray crystallography, has been applied to protein structure analysis by electron microscopy (EM) . This method simultaneously optimizes the fit of an atomic model to a density map and the stereo-chemical properties of the model by minimizing an energy function . The performance of this method is characterized at different resolution and signal-to-noise ratio conditions typical for EM electron density maps . A multi-resolution scheme is devised to improve the convergence of the refinement on the global energy minimum . Applications of the method to various model systems are demonstrated here . The first case is the arrangement of FlgE molecules in the helical filament of flagellar hook, in which refinement with segmented rigid bodies improves the density correlation and reduces severe van der Waals contacts among the symmetry-related subunits . The second case is a conformational analysis of the NSF AAA ATPase in which a multi-conformer model is used in the refinement to investigate the arrangement of the two ATPase domains in the molecule . The third case is a docking simulation in which the crystal structure of actin and the NOE data from NMR experiments on the dematin headpiece are combined with a low-resolution EM density map to generate an atomic model of the F-actin-dematin headpiece structure.

Br J Nutr, 2003 Dec, 90(6), 1071 - 80
The effect of feeding structured triacylglycerols enriched in eicosapentaenoic or docosahexaenoic acids on murine splenocyte fatty acid composition and leucocyte phagocytosis; Kew S et al.; The effects of altering the type of n-3 polyunsaturated fatty acid (PUFA) in the mouse diet on the ability of monocytes and neutrophils to perform phagocytosis were investigated . Male weanling mice were fed for 7 d on one of nine diets which contained 178 g lipid/kg and which differed in the type of n-3 PUFA and in the position of these in dietary triacylglycerol (TAG) . The control diet contained 4.4 g alpha-linolenic acid/100 g total fatty acids . In the other diets, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) replaced a proportion (50 or 100 %) of the alpha-linolenic acid, and were in the sn-2 or the sn-1(3) position of dietary TAG . There were significant increases in the content of n-3 PUFA in spleen-cell phospholipids when EPA or DHA was fed . These increases were largely independent of the position of EPA or DHA in dietary TAG except when EPA was fed at the highest level, when the incorporation was greater when it was fed in the sn-2 than in the sn-1(3) position . There was no significant effect of dietary DHA on monocyte or neutrophil phagocytic activity . Dietary EPA dose-dependently decreased the number of monocytes and neutrophils performing phagocytosis . However, when EPA was fed in the sn-2 position, the ability of active monocytes or neutrophils to engulf bacteria was increased in a dose-dependent fashion . This did not occur when EPA was fed in the sn-1(3) position . Thus, there appears to be an influence of the position of EPA, but not of DHA, in dietary TAG on its incorporation into cell phospholipids and on the activity of phagocytic cells.

J Oral Rehabil, 2003 Dec, 30(12), 1177 - 82
In vitro comparison of the efficacy of Carisolv and conventional rotary instrument in caries removal; Yazici AR et al.; The purpose of this in vitro study was to compare the efficacy of a new chemomechanical caries removal agent, Carisolv (MediTeam AB, Savedalen, Sweden), with conventional slow-speed rotary instrument (bur) . Fourteen extracted human molar teeth with deep dentine caries and no enamel coverage were selected for the study . Their laser fluorescence values were over 30 (DIAGNOdent; KaVo, Biberach, Germany) . After the teeth were sectioned through the centre of the carious lesion, one half was removed with conventional drilling (bur); the other half was removed with Carisolv gel . Removal of carious dentine was continued until the lesion was deemed caries-free by visual and tactile criteria . The preparation time for each caries removal technique was also noted . The two halves of each tooth were fixed in 10% buffered-formaldehyde for 1 week . They were then decalcified, dehydrated and embedded in paraffin blocks for histological studies . After taking serial sections of 5 microm thickness, sections were mounted on glass slides, deparaffinized, dehydrated and stained with toluidine blue for observation under a light microscope . Each section was examined for the presence of bacteria . Complete removal of caries was achieved in 13 (93%) of 14 conventionally prepared teeth, and 5 (36%) of 14 chemomechanically prepared teeth (P < 0.05) . Mean (+/-SD) time for caries removal was 272 s (+/-53.3) with Carisolv, and 116 s (+/-49.4) with drilling . The results of this study suggest that conventional rotary instrument (bur) was more effective than Carisolv in removal of carious tissue and also takes shorter time.

Environ Microbiol, 2003 Dec, 5(12), 1226 - 41
Integrated regulation in response to aromatic compounds: from signal sensing to attractive behaviour; Shingler V; Deciphering the complex interconnecting bacterial responses to the presence of aromatic compounds is required to gain an integrated understanding of how aromatic catabolic processes function in relation to their genome and environmental context . In addition to the properties of the catabolic enzymes themselves, regulatory responses on at least three different levels are important . At a primary level, aromatic compounds control the activity of specific members of many families of transcriptional regulators to direct the expression of the specialized enzymes for their own catabolism . At a second level, dominant global regulation in response to environmental and physiological cues is incorporated to subvert and couple transcription levels to the energy status of the bacteria . Mediators of these global regulatory responses include the alarmone (p)ppGpp, the DNA-bending protein IHF and less well-defined systems that probably sense the energy status through the activity of the electron transport chain . At a third level, aromatic compounds can also impact on catabolic performance by provoking behavioural responses that allow the bacteria to seek out aromatic growth substrates in their environment.

Cell Microbiol, 2003 Dec, 5(12), 861 - 73
Genome sequencing and comparative genomics of tropical disease pathogens; Carlton JM; The sequencing of eukaryotic genomes has lagged behind sequencing of organisms in the other domains of life, archae and bacteria, primarily due to their greater size and complexity . With recent advances in high-throughput technologies such as robotics and improved computational resources, the number of eukaryotic genome sequencing projects has increased significantly . Among these are a number of sequencing projects of tropical pathogens of medical and veterinary importance, many of which are responsible for causing widespread morbidity and mortality in peoples of developing countries . Uncovering the complete gene complement of these organisms is proving to be of immense value in the development of novel methods of parasite control, such as antiparasitic drugs and vaccines, as well as the development of new diagnostic tools . Combining pathogen genome sequences with the host and vector genome sequences is promising to be a robust method for the identification of host-pathogen interactions . Finally, comparative sequencing of related species, especially of organisms used as model systems in the study of the disease, is beginning to realize its potential in the identification of genes, and the evolutionary forces that shape the genes, that are involved in evasion of the host immune response.

Biofouling, 2003 Oct, 19(5), 327 - 34
A new antifouling bioassay monitoring brown algal spore swimming behaviour in the presence of echinoderm extracts; Iken K et al.; Epibiosis, the colonization of biogenic surfaces by epibiotic organisms such as bacteria, filamentous algae, and sessile invertebrates, poses a major threat to the fitness and survival of macroorganisms which could potentially be fouled . Fouling of artificial submerged structures can also cause severe economic problems, making the need for refined bioassays to determine the efficacy of potential antifouling compounds even more important . The aim of this study was to use the distinct swimming behaviour of zoospores of the fouling brown alga Hincksia irregularis to develop a new laboratory antifouling bioassay to test the effect of marine natural products . Spores were exposed to different concentrations of aqueous and organic extracts from body walls of sympatric echinoderms (Asteroidea: Luidia clathrata, Astropecten articulatus; Ophiuroidea: Astrocyclus caecilia) . Computer-assisted motion analysis was used to distinguish between the straight and fast swimming movements of undisturbed spores (controls) and the helical and erratic swimming patterns of chemically irritated spores, using the quantitative parameters rate of direction change (RCD) and swimming speed (SPD) . The ratio RCD/SPD of spore swimming paths at extract treatments compared to controls can be used to quantify the detrimental effect of echinoderm extracts . Echinoderm extracts had significant effects on spore swimming behaviour at concentrations three orders of magnitude lower than that present naturally in the echinoderm body walls (mg extract/dry weight echinoderm body wall) . Comparative studies on spore settlement and germination under similar treatment conditions show that changes in spore swimming behaviour reflect decreased fitness and survivourship of algal spores . It is suggested that this bioassay can be used to screen potential antifouling extracts and compounds at very low concentrations, making this assay particularly suitable for detection of concentration dependent effects and for bioassay-guided fractionation of extracts to identify active compounds.

Zhong Yao Cai, 2003 Jul, 26(7), 471 - 4
{Diseases and its control on Dendrobium}; Zeng S et al.; The diseases on the Dendrobium plants and their occurrence and damage as well as control methods have been investigated and studied . 11 kinds of fungi, 4 kinds of bacteria, 3 kinds of virus and one root-knot nematode were recorded . To control them effectively, cultural methods such as selecting cultivable field, keeping the field sanitation, enhancing cultural operations are mostly recommended . The application of chemical germicides is also stressed.

Ground Water, 2003 Nov-Dec, 41(6), 816 - 27
A modular injection system, multilevel sampler, and manifold for tracer tests; Mailloux BJ et al.; Ground water injection and sampling systems were developed for bacterial transport experiments in both homogenous and heterogeneous unconsolidated, surficial aquifers . Two types of injection systems, a large single tank and a dynamic mixing tank, were designed to deliver more than 800 L of amended ground water to the aquifer over 12 hours, without altering the ground water temperature, pH, Eh, or dissolved gas composition . Two types of multilevel samplers (MLSs) were designed and installed . Permanent MLSs performed well for the homogenous surficial aquifer, but their installation procedure promoted vertical mixing, which could obfuscate experimental data obtained from vertically stratified, heterogeneous aquifers . A novel, removable MLS was designed to fit in 2- and 4-inch wells . Expandable O-rings between each sampling port hydraulically isolated each port for sample collection when a nut was tightened at the land surface . A low-cost vacuum manifold system designed to work with both MLS designs used 50 mL centrifuge tubes to efficiently sample 12 MLS ports with one peristaltic pump head . The integrated system was developed and used during four field campaigns over a period of three years . During each campaign, more than 3000 ground water samples were collected in less than one week . This system should prove particularly useful for ground water tracer, injection, and push-pull experiments that require high-frequency and/or high-density sampling.

Rapid Commun Mass Spectrom, 2003, 17(23), 2655 - 60
Analysis of ketones by selected ion flow tube mass spectrometry; Smith D et al.; A selected ion flow tube mass spectrometry (SIFT-MS) study of the reactions of H3O+, NO+ and O2+* ions with the ketones (M) 2-heptanone, 2-octanone, 2-nonanone, 2-undecanone and 2-aminoacetophenone has been conducted in preparation for studies of volatile emissions from bacteria . The H3O+ reactions all proceed rapidly via exothermic proton transfer, producing only MH+ ions that form their monohydrates when water vapour is present in the helium carrier gas . The O2+* reactions proceed rapidly via dissociative charge transfer producing parent cations M+* and some fragment ions . The NO+ reactions form the NO+M adduct ions at rates which are dependent on the pressure of the helium carrier gas . Combining the present NO+ kinetic data with those available from previous SIFT studies, the phenomenon of charge transfer complexing is clearly demonstrated . This results in adduct formation in these NO+/ketone reactions at or near the collisional rate . SIFT-MS spectra are presented to illustrate the simplicity of SIFT-MS analysis of ketones using both H3O+ and NO+ precursor ions .

J Pathol, 2003 Dec, 201(4), 596 - 602
Long-term infection with Helicobacter felis and inactivation of the tumour suppressor gene p53 cumulatively enhance the gastric mutation frequency in Big Blue transgenic mice; Jenks PJ et al.; The aims of this study were to determine whether colonization with Helicobacter felis resulted in the accumulation of mutations within murine gastric tissue and whether the degree of genetic damage was increased by p53 deficiency . Female C57BL/6 mice carrying either the lambda/lacI transgene (Big Blue transgenic mice) or the lambda/lacI transgene and deficient in one allele of the p53 tumour suppressor gene (TSG-p53/Big Blue) were inoculated with H felis . Seven months after inoculation, mutations in the target lacI gene were assessed using the Big Blue transgenic mutagenesis assay system in these animals and in controls . There was an approximately two-fold increase in lacI mutations in gastric mucosa harvested from mice infected with H felis and also from non-infected mice heterozygous for the p53 allele relative to wild-type mice . The mutation frequency in mice infected with H felis and deficient in one allele of p53 was increased approximately three-fold . Active gastric inflammation was significantly greater in H felis-infected p53 hemizygous mice compared with H felis p53 wild-type mice . Gastric epithelial proliferation was similarly increased with infection in both of these latter groups of mice . In infected mice, there was a significant correlation between the mutation frequency and the degree of active gastric inflammation . These data suggest a synergistic action between infection with H felis and p53 deficiency in the accumulation of mutations within gastric tissue . Active neutrophil infiltration in gastric Helicobacter infection may contribute to the increased levels of mutation observed .

Water Sci Technol, 2003, 48(6), 263 - 9
Effect of natural and modified zeolite addition on anaerobic digestion of piggery waste; Milan Z et al.; The effect of natural and modified zeolites on the anaerobic degradation of acetate and methanol was evaluated by the determination of specific methane productivity (SMP) in batch minidigesters of 50 mL at doses of 0.01, 0.05 and 0.1 g of zeolite/g of VSS . The effects of the different zeolite concentrations were determined by the results of the SMP . A kinetic characterization with data of accumulated methane gas volume was also carried out . In the second phase of the study, the effects of natural and nickel zeolite concentrations were tested with piggery waste in laboratory scale digesters of 2.5 L operating at semi continuous mode, by increasing the organic load applied from 0.2 to 22.0 g TCOD/d corresponding to organic loading rates (OLR) of 0.1-8.8 g TCOD/l x d . A greater effect of modified zeolite on SMP was observed, with an increase of 8.5 times with magnesium zeolite, 4.4 times with cobalt zeolite and 2.8 times with nickel zeolite . Two phases were defined in the kinetic study and an increase of more than 2 times the apparent constant of digesters with modified zeolites was observed in the second phase when compared to unmodified natural zeolite . Modified natural zeolite addition to digesters can allow an increase in the potential biodegradability of piggery waste solid fraction and/or a considerable reduction of digestion volume.

Water Sci Technol, 2003, 48(6), 249 - 54
The influence of pH in the hydrolytic stage of anaerobic digestion of the organic fraction of urban solid waste; Dinamarca S et al.; The influence of the pH in the first stage, the hydrolytic stage, of the anaerobic digestion of the organic fraction of urban solid waste in a two phase anaerobic reactor was studied . The reactor was fed with a solution of the organic fraction of urban solid residues containing 5 to 7% solids . Four reactors with a working volume of 3 L were used, the experiments were done at three controlled pHs; 6, 7, and 8, and one with free pH, the temperature was keep at 37 degrees C in all the experiments . The higher degradation of TSS and VSS was obtained in the reactors operated at pH 7 and 8; 75% degradation of TSS and 85% degradation of VSS . The volatile fatty acids were determined at the different pH conditions, no significant differences were found, and as was expected, the acetic acid was found at the higher value among them (from 25 to 29 g/L) . According to the results obtained it is possible to conclude that in the case of the hydrolytic stage of the anaerobic digestion of the organic fraction of urban solid waste it is not necessary to control the pH, the pH is kept stable by the buffer effect of the protein residues and other macromolecules present in the residue.

Water Sci Technol, 2003, 48(6), 227 - 34
Performance evaluation of an UASB reactor used for combined treatment of domestic sewage and excess aerobic sludge from a trickling filter; Pontes PP et al.; This work aimed at evaluating the influence of the excess sludge produced in a trickling filter (TF) on the performance of a UASB reactor used for the combined treatment of domestic sewage and aerobic sludge . During phase 1 of the research, the UASB reactor/TF system was fed with domestic sewage pumped directly from the sewer collector of Arrudas stream, in Belo Horizonte-Brazil . During phase 2, besides feeding the reactor with domestic sewage, the UASB reactor was also fed with the aerobic sludge from the trickling filter . The UASB reactor, with a volume of 420 litres, was operated at a mean hydraulic detention time of 5.6 hours in both operational phases . After 133 days of continuous monitoring, no detrimental effect was noticed on the performance of the UASB reactor regarding the return of the aerobic sludge produced in the TF . On the contrary, the COD results indicated a higher percentage of compliance with the discharge standards set forth by the Brazilian environmental legislation . During phase 2 of the research, when the UASB reactor was used for combined treatment of domestic sewage and excess aerobic sludge from the TF, the anaerobic effluent presented mean concentrations of 108 mgCOD x L(-1), 57 mgBOD x L(-1) and 18 mgTSS x L(-1).

Water Sci Technol, 2003, 48(6), 219 - 26
Decentralised treatment of concentrated sewage at low temperature in a two-step anaerobic system: two upflow-hybrid septic tanks; Elmitwalli TA et al.; The decentralised treatment of concentrated sewage (about 3,600 mgCOD/l) at low temperature was investigated in a two-step anaerobic system: two-anaerobic hybrid (AH) septic tanks (each 0.575 m3) . The two reactors were placed in a temperature controlled-room and the HRT was 2.5 days for each reactor . The system was fed with concentrated domestic sewage, mainly black water from about 40 toilets flushed with only 4 litre of water and a limited amount of grey water . The system showed high removal efficiency for the different COD fractions . Mean removal efficiencies in the two-step AH-septic tank at 5 days HRT and 13 degrees C were 94, 98, 74 and 78% for total COD, suspended COD, colloidal COD and dissolved COD respectively . The results of short run experiments indicated that the presence of reticulated polyurethane foam (RPF) media in the AH-septic tank improved the removal of suspended COD by 22% . The first AH-septic tank was full of sludge after 4 months of operation due to the high removal of particulate COD and the limited hydrolysis at low temperature conditions . Therefore, a simple mathematical model was developed based on ADM1 (the IWA model in 2002) . Based on the experimental results and the mathematical model, only a one-step AH septic tank is required . An HRT of 5.5-7.5 days is needed for that one-step AH septic tank to treat concentrated sewage at a low temperature of 13 degrees C . Such a system can provide a total COD removal as high as 87% and will be full of sludge after a period of more than a year.

Water Sci Technol, 2003, 48(6), 179 - 86
Operating feasibility of anaerobic whey treatment in a stirred sequencing batch reactor containing immobilized biomass; Ratusznei SM et al.; The scope of this work was to evaluate the operating feasibility of anaerobic whey treatment in a stirred sequencing batch reactor (ASBR) containing biomass immobilized on inert support . Assays were performed using 8-hour cycles and agitation rate of 200 rpm at 30 +/- 1 degrees C, for treating cheese whey containing 500 to 4,000 mgCOD/L, which corresponded to a volumetric organic load (VOL) of 0.81 to 5.7 gCOD/L x d . Stability and high organic matter removal of about 96% were achieved at effluent concentration below 160 mgCOD/L for non filtered samples . Operating stability of the reactor was shown to be strongly dependent on the alkalinity supplementing strategy during the assay, especially during the startup period, where NaHCO3 supplementation was approximately 20-30% of the chemical oxygen demand (mgNaHCO3/mgCOD) . After startup, alkalinity supplementation could be reduced down to 10% maintaining efficiency and stability . Moreover, proper homogenization of the system through mechanical agitation was also shown to be indispensable, especially with increasing organic load.

Water Sci Technol, 2003, 48(6), 141 - 7
Anaerobic degradation kinetics of a cholesteryl ester; Gutierrez S et al.; The most important components of wool scouring effluent grease are esters of sterols . Cholesteryl palmitate (CP) is the main ester in this grease . In this paper, the influence of the ester concentration in the anaerobic digestion and the relative rate of the different degradation steps, are studied . The experiment was carried out to measure methane production in the anaerobic degradation of acetate, palmitic acid (PA) and CP . A first-order kinetic model was assumed for hydrolysis and Monod models were assumed for both the methanogenic and acetogenic steps . Maximum hydrolysis rate was found to be around 20 times faster than the maximum methanogenic reaction rate during the experience . The lanolin emulsion drop size effect was also evaluated employing fine and coarse stock lanolin emulsions and no adapted sludge . Concentrations of 13.7 to 4.6 gCOD x l(-1) were employed . In a previous study, the effect of palmitic acid emulsion size was found important when similar sludge was tested . When esters are degraded, a significant effect of drop size on the degradation rate was not found . The difference between CP and PA emulsions behavior could be due to the fact that cholesterol produced during the ester degradation has a protective effect on the sludge.

Water Sci Technol, 2003, 48(6), 33 - 40
Accumulation of long chain fatty acids onto anaerobic sludge under steady state and shock loading conditions: effect on acetogenic and methanogenic activity; Pereira MA et al.; Accumulation of substrate onto the biomass was quantified under steady-state and shock conditions in a fixed bed reactor fed with an oleic acid-based synthetic effluent . The accumulation of substrate onto the sludge was more dependent on oleic acid concentration than on oleic acid loading rate and decreased the acetogenic, acetoclastic and hydrogenophilic activity . However, even when the methanogenic activity measurements indicate a severe inhibition, the anaerobic sludge was able to methanise efficiently the accumulated substrate that was mainly adsorbed LCFA . Methanogenic activity measurements for a sludge loaded with 2,861 mg COD/gVSS as LCFA, revealed that only hydrogenophilic activity was detected, whereas the methanogenic activities with acetate, propionate and butyrate as substrates were null . However the methanogenic activity of the same sludge after allowing the depletion of the adsorbed LCFA were significantly enhanced in the presence of all substrates, except in propionate . A discussion about the relative importance of metabolic inhibition and transport limitations for the anaerobic degradation of LCFA is launched.

Acta Med Croatica, 2003, 57(4), 295 - 301
{Detection of human papillomaviruses and other agents causing sexually transmitted diseases with molecular diagnosis methods}; Grce M et al.; Causative agents of sexually transmitted diseases (STD) are different types of bacteria, viruses, fungi and protozoa . The last two decades of the twentieth century were marked with a sudden rise in the number of cases of STDs . Human immunodeficiency virus (HIV), which emerged in the 1980s, is the most prominent STD agent because of its fast spread and severity of the disease it causes, acquired immunodeficiency syndrome (AIDS) . Beside HIV, human papillomaviruses (HPVs), herpes simplex viruses (HSVs) and Chlamydia trachomatis are nowadays among most health-threatening STD pathogens . In order to stop the spread of infection, apart from education about precautions, early detection of the disease is essential . Although most STD pathogens can be detected by classical methods of cultivation, biochemical and/or serologic methods, molecular diagnosis of infectious diseases has largely simplified and accelerated their detection . For instance, HPVs that cause benign and malignant tumors of genital skin and mucosa cannot be routinely detected on cell culture, whereas serologic analysis is not sensitive and informative enough . Moreover, cytologic (Pap smear) and histologic analyses can indicate changes associated with HPV infection, but neither of these methods can prove the presence of HPV . That is why the molecular methods are essential to demonstrate the presence of the infection and, even more important, to determine the type of the virus, which is associated either with low-grade or high-grade genital lesions . There are numerous methods based on hybridization with DNA or RNA probes, some of them are suitable for detecting wide range of types and screening of large collection of samples . However, the most sensitive and informative methods are based on polymerase chain reaction (PCR), and they have the advantage of being able to determine the type of the virus and distinguishing between multiple infections . Herein, we present when and why molecular analysis is useful and necessary for the detection of STD agents.

Acta Med Croatica, 2003, 57(4), 255 - 9
{MHC tetramers: tracking specific immunity}; Kosor E et al.; In an adaptive immune response, antigen is recognized by two distinct sets of highly variable receptor molecules: (1) immunoglobulins, that serve as antigen receptors on B cells and (2) the antigen-specific receptors on T cells . T cells play important role in the control of infection and in the development of protective immunity . These cells can also mediate anti-tumor effects and, in case of autoimmune syndromes, contribute to the development and pathology of disease . The specificity of T cells is determined by T cell receptors (TCR) . Understanding of the success of immune responses requires the direct measurement of antigen-specific T lymphocytes . Cell with major histocompatibility complex (MHC) class I molecules are able to present antigens to antigen-specific CD8+ cytotoxic T lymphocytes . MHC class I molecules present small peptides (epitopes) processed from intracellular antigens such as viruses and intracellular bacteria . MHC class I molecules in humans are designated as human leukocyte antigen (HLA) class I and divided into HLA-A, -B and -C . CD8+ T cells recognize MHC class I molecules and after activation produce proteins that destroy infected cells . MHC class II molecules receive their peptides mainly from extracellular and soluble antigens and present them to the CD4+ T helper cells . A recently described technique that can be used in flow cytometry enables us to quantify ex vivo antigen-specific T cells by binding of soluble tetramer MHC-peptide complexes attached to fluorochrome . Quantitative analyses of antigen-specific T cell populations provide important information on the natural course of immune responses . The interaction of T cell receptors on T lymphocytes with tetrameric MHC-peptide complexes mimics the situation on the cell surface, and allows for reliable binding . Tetramers consist of four biotinylated HLA-peptide epitope complexes bound to streptavidin conjugated with fluorescent dye . Tetramer technology has sensitivity of detection as little as 0.02% of total cytotoxic T cell pool or T helper cell pool (i.e . approximately 1 in 50.000 lymphocytes) . The combination of this technology with intracellular cytokine staining methods opens up significantly better ways of studying these cells than previously possible, allowing immunologists to look at their life cycle (activation and proliferation), manner of death (aging and apoptosis) and effector function (cytotoxic potential and cytokine production) . MHC tetramers class I have yielded useful insights into in vivo dynamic and function of antigen-specific CD8+ T cells in viral infections, parasitic infections, cancer, autoimmune disease and transplantation . This knowledge is of special interest for immunotherapy, diagnostic monitoring of T cell mediated immunity, and the development of new vaccination strategies . There is some possibility for cell therapy with antigen-specific CD8+ T cells for various diseases including cancer and viral infections . Targeted immunotherapy of selective deletion of auto--or alloreactive T cells with MHC tetramers may be important for the treatment of autoimmune disease, or to prevent the rejection of transplanted organs . The utility of this technique for the immunotherapy in vivo needs to be confirmed and modified in further research . Understanding how antigen-specific cells develop and function in different circumstances and pathologies will be the key to unravelling the secrets of cellular immune system.

J Am Acad Dermatol, 2003 Dec, 49(6), 979 - 1000; quiz 1000-2
Tropical dermatology: viral tropical diseases; Lupi O et al.; Viruses are important pathogens in tropical areas; most of them, especially the tropical hemorrhagic fevers, produce mucocutaneous manifestations . More than any other kind of pathogen, viruses have the possibility for being widespread, since they have a greater probability of mutation than do bacteria, can cross species barriers easily, and infect both human beings and animals in habitats with a great biodiversity . Tropical habitats also have been subject to major ecologic changes in the last few decades, exposing humans to direct contact with these viruses and allowing hemorrhagic fevers due to new emergent viruses such as flaviviruses, filoviruses, arenaviruses, and hantaviruses to become major threats to public health . The collapse of eradication programs in many countries, as well as population increases and ecologic modifications, have led to the spread of dengue and yellow fever to large portions of the world owing to the dissemination of vectors, especially mosquitoes, with broad ecologic ranges . Viruses previously restricted to some geographic areas, such as Rift Valley fever, Crimean-Congo hemorrhagic fever, West Nile fever, and monkeypox are now affecting new countries and populations . Other viruses such as herpes B infection often affect travelers and animal handlers in most parts of the world . Dermatologic lesions occur in all these diseases and can facilitate a rapid diagnosis, leading to control of the virus and helping prevent possible outbreaks.

J Cell Biol, 2003 Nov 24, 163(4), 715 - 21
Dynamics and calcium sensitivity of the Ca2+/myristoyl switch protein hippocalcin in living cells; O'Callaghan DW et al.; Hippocalcin is a neuronal calcium sensor protein that possesses a Ca2+/myristoyl switch allowing it to translocate to membranes . Translocation of hippocalcin in response to increased cytosolic {Ca2+} was examined in HeLa cells expressing hippocalcin-enhanced yellow fluorescent protein (EYFP) to determine the dynamics and Ca2+ affinity of the Ca2+/myristoyl switch in living cells . Ca2+-free hippocalcin was freely diffusible, as shown by photobleaching and use of a photoactivable GFP construct . The translocation was dependent on binding of Ca2+ by EF-hands 2 and 3 . Using photolysis of NP-EGTA, the maximal kinetics of translocation was determined (t1/2 = 0.9 s), and this was consistent with a diffusion driven process . Low intensity photolysis of NP-EGTA produced a slow {Ca2+} ramp and revealed that translocation of hippocalcin-EYFP initiated at around 180 nM and was half maximal at 290 nM . Histamine induced a reversible translocation of hippocalcin-EYFP . The data show that hippocalcin is a sensitive Ca2+ sensor capable of responding to increases in intracellular Ca2+ concentration over the narrow dynamic range of 200-800 nM free Ca2+.

Infect Immun, 2003 Dec, 71(12), 7219 - 22
Abortive potency of Chlamydophila abortus in pregnant mice is not directly correlated with placental and fetal colonization levels; Bouakane A et al.; Abortion, placental and fetal colonization, and levels of gamma interferon were analyzed for four Chlamydophila abortus strains presenting antigenic variations in a mouse model . Expression of virulence of these strains varied and indicated that abortion was not directly related to the number of bacteria in the placenta, and thus, other factors may have an important role in activating the abortion process.

Infect Immun, 2003 Dec, 71(12), 7208 - 10
Functional BvgAS virulence control system in Bordetella bronchiseptica is necessary for induction of Ca2+ transients in ciliated tracheal epithelial cells; Groathouse NA et al.; To study initial Bordetella bronchiseptica-tracheal epithelial cell interactions, we coincubated B . bronchiseptica with rabbit tracheal explant cultures and assayed bacterial adherence and host cell Ca(2+) signaling . Wild-type B . bronchiseptica (RB50) preferentially adhered to cilia and induced ciliated host cell Ca(2+) transients within 2 min of coincubation, whereas coincubation with an avirulent strain (RB57) resulted in limited binding and Ca(2+) signaling . The described cell system allows for assessment of initial B . bronchiseptica-host cell interactions that can contribute to pathogenicity or to host cell defense.

Infect Immun, 2003 Dec, 71(12), 7140 - 8
Role for complement in development of Helicobacter-induced gastritis in interleukin-10-deficient mice; Ismail HF et al.; The mechanisms by which the immune response can eradicate gastric Helicobacter infection are unknown . We hypothesized that Helicobacter-induced activation of the complement system could promote both inflammation and eradication of Helicobacter from the stomach . In vitro studies demonstrated that Helicobacter felis activates complement in normal mouse serum but not in serum from Rag2(-/-) mice, indicating that H . felis activates complement through the classical pathway . Next, we infected complement-depleted wild-type control and interleukin-10-deficient (IL-10(-/-)) mice with H . felis . Helicobacter infection of wild-type mice elicited a mild, focal gastritis and did not alter serum complement levels . Infection of IL-10(-/-) mice with H . felis elicited severe gastritis . After the initial colonization, the IL-10(-/-) mice completely cleared Helicobacter from the stomach by day 8 . In contrast to wild-type mice, H . felis-infected IL-10(-/-) mice had a marked increase in serum complement levels . Complement depletion of wild-type mice did not affect the intensity of gastric inflammation or the extent of Helicobacter colonization compared to that for the wild-type control mice . In contrast, complement depletion of Helicobacter-infected IL-10(-/-) mice decreased the severity of gastritis, decreased the Helicobacter-induced infiltration of neutrophils into the stomach, and delayed the clearance of bacteria . In vitro studies of stimulated splenocytes and neutrophils from IL-10(-/-) mice produced a twofold increase in complement production compared to that for wild-type mice . Pretreatment with IL-10 inhibited this increase . These studies identify a role for complement in the local immune response to gastric Helicobacter in IL-10(-/-) mice and suggest a role for IL-10 in the regulation of complement production.

Infect Immun, 2003 Dec, 71(12), 6986 - 94
Removal of Wolbachia from Brugia pahangi is closely linked to worm death and fecundity but does not result in altered lymphatic lesion formation in Mongolian gerbils (Meriones unguiculatus); Chirgwin SR et al.; Approximately 30 years ago, researchers reported intracellular bacteria in filarial nematodes . These bacteria are relatives of the arthropod symbiont Wolbachia and occur in many filarial nematodes, including Brugia pahangi and Brugia malayi . Wolbachia bacteria have been implicated in a variety of roles, including filaria development and fecundity and the pathogenesis of lymphatic lesions associated with filarial infections . However, the role of the bacteria in worm biology or filarial disease is still not clear . The present experiments support previous data showing that tetracycline eliminates or reduces Wolbachia bacteria in B . pahangi in vivo . The elimination of Wolbachia was closely linked to a reduction in female fecundity and the viability of both sexes, suggesting that the killing of Wolbachia is detrimental to B . pahangi . The gerbils treated with tetracycline showed reduced levels of interleukin-4 (IL-4) and IL-5 mRNA in renal lymph nodes and spleens compared with the levels in B . pahangi-infected gerbils not treated with tetracycline . However, similar findings were noted in B . pahangi-infected gerbils treated with ivermectin, suggesting that the loss of circulating microfilariae, not the reduction of Wolbachia bacteria, was associated with the altered cytokine profile . Despite the change in T-cell cytokines, there was no difference in the sizes of renal lymph nodes isolated from gerbils in each treatment group . Furthermore, the numbers, sizes, or cellular compositions of granulomas examined in the lymphatics or renal lymph nodes did not differ with treatment . These data suggest that Wolbachia may not play a primary role in the formation of lymphatic lesions in gerbils chronically infected with B . pahangi.

Infect Immun, 2003 Dec, 71(12), 6943 - 52
Temporal analysis of Borrelia burgdorferi Erp protein expression throughout the mammal-tick infectious cycle; Miller JC et al.; Previous immunological studies indicated that the Lyme disease spirochete, Borrelia burgdorferi, expresses Erp outer surface proteins during mammalian infection . We conducted analyses of Erp expression throughout the entire tick-mammal infectious cycle, which revealed that the bacteria regulate Erp production in vivo . Bacteria within unfed nymphal ticks expressed little to no Erp proteins . However, as infected ticks fed on mice, B . burgdorferi increased production of Erp proteins, with essentially all transmitted bacteria expressing these proteins . Mice infected with B . burgdorferi mounted rapid IgM responses to all tested Erp proteins, followed by strong immunoglobulin G responses that generally increased in intensity throughout 11 months of infection, suggesting continued exposure of Erp proteins to the host immune system throughout chronic infection . As naive tick larvae acquired B . burgdorferi by feeding on infected mice, essentially all transmitted bacteria produced Erp proteins, also suggestive of continual Erp expression during mammalian infection . Shortly after the larvae acquired bacteria, Erp production was drastically downregulated . The expression of Erp proteins on B . burgdorferi throughout mammalian infection is consistent with their hypothesized function as factor H-binding proteins that protect the bacteria from host innate immune responses.

Infect Immun, 2003 Dec, 71(12), 6728 - 33
Divergent interactions of Ehrlichia chaffeensis- and Anaplasma phagocytophilum-infected leukocytes with endothelial cell barriers; Park J et al.; Human anaplasmosis (formerly human granulocytic ehrlichiosis) and human monocytic ehrlichiosis (HME) are emerging tick-borne infections caused by obligate intracellular bacteria in the family Anaplasmataceae . Clinical findings include fever, headache, myalgia, leukopenia, thrombocytopenia, and hepatic inflammatory injury . Whereas Ehrlichia chaffeensis (HME) often causes meningoencephalitis, this is rare with Anaplasma phagocytophilum infection . The abilities of infected primary host monocytes and neutrophils and of infected HL-60 cells to cross human umbilical vein endothelial cell-derived EA.hy926 cell barriers and human brain microvascular cells (BMEC), a human blood-brain barrier model, were studied . Uninfected monocyte/macrophages crossed endothelial cell barriers six times more efficiently than neutrophils . More E . chaffeensis-infected monocytes transmigrated than uninfected monocytes, whereas A . phagocytophilum suppressed neutrophil transmigration . Differences were not due to barrier dysfunction, as transendothelial cell resistivities were the same for uninfected cell controls . Similar results were obtained for HL-60 cells used as hosts for E . chaffeensis and A . phagocytophilum . Differential transmigration of E . chaffeensis- and A . phagocytophilum-infected leukocytes and HL-60 cells confirmed a role for the pathogen in modifying cell migratory capacity . These results support the hypothesis that Anaplasmataceae intracellular infections lead to unique pathogen-specific host cell functional alterations that are likely important for pathogen survival, pathogenesis, and disease induction.

Eur J Pharmacol, 2003 Nov 14, 481(1), 15 - 23
Agonist-induced desensitization and endocytosis of heterodimeric GABAB receptors in CHO-K1 cells; Gonzalez-Maeso J et al.; gamma-Aminobutyric acid B (GABA(B)) receptor is the first discovered G protein-coupled receptor that requires two subunits, GB1 and GB2, to form a functional receptor . Whereas the molecular and functional characteristics of GABA(B) receptors have been recently extensively studied, the mechanisms underlying receptor desensitization and endocytosis are still poorly understood . We have investigated the effect of continuous agonist exposure on the human GABA(B) receptor functional response and redistribution when expressed in Chinese hamster ovary (CHO-K1) cells . The wild-type GABA(B) receptor-mediated inhibition of the adenylate cyclase activity appeared desensitized after 2 h in the presence of GABA (100 microM) . Fusion proteins were generated by attachment of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) to GB1 and GB2, respectively, and confocal microscopy experiments in intact living cells semi-stably expressing the constructs were performed . Incubation of co-expressing CFP-GB1 and YFP-GB2 cells in the presence of GABA (100 microM) for 2 h induced a profound receptor internalization, and CFP-GB1 and YFP-GB2 appeared co-localized in the endosome (labelled with Cy3-transferrin) . The internalization was blocked by a selective GABA(B) receptor antagonist . These results represent the first clear visualization of agonist-induced internalization of the unique heterodimeric GABA(B) receptor.

Scand J Immunol, 2003 Dec, 58(6), 628 - 41
Interferon-gamma tempers the expression of carcinoembryonic antigen family molecules in human colon cells: a possible role in innate mucosal defence; Fahlgren A et al.; Four carcinoembryonic antigen-related cell adhesion molecule (CEACAM)s, i.e . CEA, CEACAM1, CEACAM6 and CEACAM7, are localized to the apical glycocalyx of normal colonic epithelium and have been suggested to play a role in innate immunity . The expression of these molecules in colon carcinoma cells was studied at the mRNA and protein levels after treatment with interferon-gamma (IFN-gamma), interleukin-1beta, live bacteria or lipopolysaccharide . The colon carcinoma cell lines LS174T and HT-29 were studied in detail using real-time quantitative reverse transcriptase-polymerase chain reaction, immunoflow cytometry and immunoelectron microscopy . IFN-gamma, but not the other agents, modified expression of CEA, CEACAM1 and CEACAM6 . None of the agents upregulated CEACAM7 expression . Two expression patterns were seen . HT-29 cells, which initially showed low quantities of mRNAs and proteins, displayed marked upregulation of both mRNAs and proteins . LS174T cells transcribed stable high levels of mRNA before and after treatment . Additionally, IFN-gamma induced increased cell surface expression of CEA, CEACAM1 and CECAM6 . IFN-gamma has two important effects on the expression levels of the CEA family molecules in colon epithelial cells: direct upregulation of CEACAM1 and promotion of cell differentiation resulting in increased expression of CEA and CEACAM6 and decreased expression of CEACAM7.

Immunol Cell Biol, 2003 Dec, 81(6), 415 - 23
Depletion of cellular cholesterol interferes with intracellular trafficking of liposome-encapsulated ovalbumin; Rao M et al.; Cholesterol is a major constituent of plasma cell membranes and influences the functions of proteins residing in the membrane . To assess the role of cholesterol in phagocytosis and intracellular trafficking of liposomal antigen, macrophages were treated with inhibitors of cholesterol biosynthesis for various time periods and levels of cholesterol depletion were assessed by thin layer chromatography . In control macrophages, cholesterol was present in the plasma membrane and in intracellular stores, as visualised by staining with the cholesterol-binding compound filipin, whereas macrophages treated with cholesterol inhibitors failed to stain with filipin . However, these macrophages were still capable of phagocytosis as evidenced by their internalisation of fluorescent-labelled bacteria and liposome-encapsulated Texas red labelled-ovalbumin, L(TR-OVA) . While fluorescent ovalbumin (OVA) was consistently transported to the Golgi in macrophages incubated with L(TR-OVA), in cells treated with cholesterol inhibitors, OVA remained spread diffusely throughout the cytoplasm . Even though the mean fluorescence intensity of MHC class I molecules on cholesterol inhibitor-treated macrophages was equivalent to that of the control macrophages, the amount of MHC class I-liposomal OVA-peptide complex detected on the cell surface of cholesterol inhibitor-treated macrophages, was only 45.6 +/- 7.4% (n = 4, mean +/- SEM) of control levels after intracellular processing of L(OVA) . We conclude that cholesterol depletion does not eliminate phagocytosis or MHC class I surface expression, but does affect the trafficking and consequently the MHC class I antigen-processing pathway.

Biochemistry, 2003 Dec 2, 42(47), 13893 - 900
Role of an N-terminal loop in the secondary structural change of photoactive yellow protein; Harigai M et al.; Photoactive yellow protein (PYP) is photoconverted to its putative active form (PYP(M)) with global conformational change(s) . The changes in the secondary structure were studied by far-UV circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy using PYP, which lacks N-terminal 6, 15, or 23 amino acid residues (T6, T15, and T23, respectively) . Irradiation of truncated PYPs induced the loss of the CD signal, where the maximal difference was located at 222 nm . The reduction of the CD signal was significantly larger than the calculated CD of the N-terminal helices, indicating that it is mainly accounted for by the unfolding and/or structural change of the helices located outside the N-terminal region . The difference FTIR spectra between dark and photosteady states recorded using the solution samples demonstrated that large absorbance changes in the amide mode of the beta-sheet were reduced and downshifted by truncation . The structural change of the beta-sheet is therefore closely correlated with the N-terminal loop . NaCl decelerates the decay of intact PYP(M) and T6(M) at low concentrations (<500 mM) but accelerates decay at high concentrations (>1000 mM) . For T15(M) and T23(M), NaCl accelerates their decay at >100 mM but never decelerates their decay, suggesting that the electrostatic interaction, which plays an important role for the recovery of PYP from PYP(M), is lost by removing positions 7-15 . The electrostatic interaction between this region and the beta-scaffold is likely to promote the conformational change of PYP(M) for recovery of PYP.

Parasitology, 2003 Oct, 127(Pt 4), 337 - 47
Interaction of the proteasome S5a/Rpn10 multiubiquitin-binding protein and the 8 kDa calcium-binding protein of Schistosoma mansoni; Ram D et al.; A distinct 8 kDa calcium-binding protein (CaBP) is preferentially expressed at the cercarial stage during the life-cycle of the schistosome . Available data indicate that this CaBP may be associated with tissue/organ remodelling (involving protein degradation and synthesis of new proteins) during transformation of the cercariae from free-living form in water to parasitic life in the vertebrate host . Many CaBP molecules (e.g . calmodulin) show Ca(++)-dependent interaction with target proteins and thus modulate their activity . Accordingly, the parasite 8 kDa CaBP was used as a probe to clone and identify putative target protein(s) directly by binding interaction . Screening of schistosome lambdagt11 expression library with radio-iodinated CaBP yielded several overlapping clones showing Ca(++)-dependent binding of the CaBP . Sequence analyses revealed that these clones encode the S5a/Rpn10 multiubiquitin-binding protein which is a component of the regulatory 19S subunit of the 26S proteasome . The schistosome molecule, designated SmS5a, is 420 amino acids long . The nearly full length molecule (Gln3-Ser420) as well as the amino terminal (N-S5a, Gln3-Gly200) and carboxyl-terminal (C-S5a, Asp225-Ser420) portions were synthesized in bacteria, purified, and antibodies to the parasite SmS5a were prepared . Interaction between SmS5a and the 8 kDa CaBP in a Ca(++)-dependent manner was found under various experimental conditions: CaBP-Sepharose bound soluble SmS5a, immobilized SmS5a bound soluble CaBP, and complex formation was found when both molecules were in solution . Furthermore, it was shown that the C-terminal portion of SmS5a, but not the N-terminal portion of the molecule, reacted with the CaBP . SmS5a synthesized in a cell-free system and Western blots revealed 2 species, conceivably corresponding to the naked molecule (approximately 50 kDa) and the molecule subjected to post-translational modification (approximately 70 kDa) . The present studies suggest that proteasome activity may be modulated by calcium, and this modulation is mediated via CaBP molecule(s).

Exp Appl Acarol, 2003, 29(3-4), 253 - 64
Sorting out the effects of Wolbachia, genotype and inbreeding on life-history traits of a spider mite; Vala F et al.; Wolbachia bacteria manipulate host reproduction by inducing cytoplasmic incompatibility (CI) and sex ratio distortion . Wolbachia are transmitted from mother to offspring through the cytoplasm of the egg . Therefore, reproduction of Wolbachia is tightly coupled to reproduction of its host . Mathematical analysis predicts that in the course of evolution, traits that reduce the physiological costs of the infection will be selectively favored . For a Wolbachia-host system to evolve, traits under selection must have some genetic component and variation must be present in the population . We have previously established that highly inbred isofemale lines of the two-spotted spider mite Tetranychus urticae may differ regarding the effects of infection by Wolbachia, and that at least some of the traits affected had a genetic component . However, the effects measured could have been affected by the fact that the lines were severely inbred prior to the experiments . In this paper we attempt to distinguish between the effects of Wolbachia, isofemale line, and inbreeding . We show that Wolbachia did not affect longevity but infected females produced smaller clutch sizes, more daughter-biased sex ratios and had decreased F1 mortality; between-line variation was found for clutch size, F1 mortality and sex ratio; finally, inbreeding resulted in an overall reduction of clutch sizes, and a change in survival curves and mean longevity.

Bioessays, 2003 Dec, 25(12), 1178 - 91
Building and breaking bridges between sister chromatids; Haering CH et al.; Eukaryotic chromosomes undergo dramatic changes and movements during mitosis . These include the individualization and compaction of the two copies of replicated chromosomes (the sister chromatids) and their subsequent segregation to the daughter cells . Two multisubunit protein complexes termed 'cohesin' and 'condensin', both composed of SMC (Structural Maintenance of Chromosomes) and kleisin subunits, have emerged as crucial players in these processes . Cohesin is required for holding sister chromatids together whereas condensin, together with topoisomerase II, has an important role in organizing individual axes of sister chromatids prior to their segregation during anaphase . SMC and kleisin complexes also regulate the compaction and segregation of bacterial nucleoids . New research suggests that these ancient regulators of chromosome structure might function as topological devices that trap chromosomal DNA between 50 nm long coiled coils .

J Neurosci Res, 2003 Dec 1, 74(5), 794 - 800
Adenoviral-mediated expression of functional Na+ channel beta1 subunits tagged with a yellow fluorescent protein; Fry M et al.; Voltage-gated sodium (Na(+)) channels typically contain a pore-forming alpha subunit and one or two auxiliary beta subunits . Although initial characterization of known alpha and beta subunits has been facilitated by expression in heterologous cells, to understand fully the differences between individual subunits and the functional consequences of selective subunit expression, there is a need to acutely manipulate expression in cells that endogenously express Na(+) channels . To this end, we have constructed a recombinant adenovirus containing a cDNA for a mouse Na(+) channel beta1 subunit with a yellow fluorescent protein fused to its C-terminus (Ad-beta1-EYFP), and with fluorescence microscopy detected beta1-EYFP expression in primary cerebellar neurons and Chinese hamster ovary (CHO) cells upon transduction with this adenovirus, including expression in the plasma membrane . Consistent with this, patch clamp recordings confirmed that Na(+) currents in CHO cells expressing mouse Na(v)1.4 alpha subunits were appropriately modified by the viral-mediated expression of beta1-EYFP subunits . The results demonstrate that adenoviral-mediated gene delivery can be used effectively to express epitope-tagged Na(+) channel subunits with properties similar to wild-type subunits, and suggest that Ad-beta1-EYFP will be a useful reagent for investigating Na(+) channels in a variety of excitable cell types, including neurons .

J Neurosci Res, 2003 Dec 1, 74(5), 782 - 93
An antisense construct reduces N-methyl-D-aspartate receptor 2A expression and receptor-mediated excitotoxicity as determined by a novel flow cytometric approach; Mattar PA et al.; The N-methyl-D-aspartate receptor (NMDAR) is a major neurotransmitter receptor in the central nervous system (CNS), with functional roles in learning, memory, and sensation . Several mechanisms potentiate NMDARs, and NMDAR hyperexcitability plays pathophysiological roles in many conditions, such as neurodegenerative disease, ischemia, and chronic conditions arising from spinal cord injury . Previous research suggests that the NR2A subunit of the receptor contributes to NMDAR excitotoxicity in heterologous cells and in neurons in vivo . To investigate the role of NR2A in NMDAR excitotoxicity, we have developed a system based on flow cytometry that allows rapid evaluation of the effect of antisense constructs on protein expression and channel function . The enhanced yellow fluorescent protein (EYFP) was fused to obligatory NMDAR subunits, allowing expression to be monitored in living cultured cells . An NR2A antisense construct, asNR2A, specifically and effectively reduced NR2A-EYFP expression . NR1 and NR2A fusion proteins formed functional, excitotoxic channels upon co-expression . The asNR2A RNA significantly reduced NMDAR excitotoxicity when NR2A levels were limiting for channel formation . Using our assay system, further optimization can be achieved rapidly . The asNR2A construct and the assays developed for this study can be used to provide insights into NMDAR biology and disease .

Nat Cell Biol, 2003 Dec, 5(12), 1104 - 10 Epub 2003 Nov 23.
Rac-MEKK3-MKK3 scaffolding for p38 MAPK activation during hyperosmotic shock; Uhlik MT et al.; Sensing the osmolarity of the environment is a critical response for all organisms . Whereas bacteria will migrate away from high osmotic conditions, most eukaryotic cells are not motile and use adaptive metabolic responses for survival . The p38 MAPK pathway is a crucial mediator of survival during cellular stress . We have discovered a novel scaffold protein that binds to actin, the GTPase Rac, and the upstream kinases MEKK3 and MKK3 in the p38 MAPK phospho-relay module . RNA interference (RNAi) demonstrates that MEKK3 and the scaffold protein are required for p38 activation in response to sorbitol-induced hyperosmolarity . FRET identifies a cytoplasmic complex of the MEKK3 scaffold protein that is recruited to dynamic actin structures in response to sorbitol treatment . Through its ability to bind actin, relocalize to Rac-containing membrane ruffles and its obligate requirement for p38 activation in response to sorbitol, we have termed this protein osmosensing scaffold for MEKK3 (OSM) . The Rac-OSM-MEKK3-MKK3 complex is the mammalian counterpart of the CDC42-STE50-STE11-Pbs2 complex in Saccharomyces cerevisiae that is required for the regulation of p38 activity.

Diabetes, 2003 Dec, 52(12), 2943 - 50
On-line monitoring of apoptosis in insulin-secreting cells; Kohler M et al.; Apoptosis was monitored in intact insulin-producing cells both with microfluorometry and with two-photon laser scanning microscopy (TPLSM), using a fluorescent protein based on fluorescence resonance energy transfer (FRET) . TPLSM offers three-dimensional spatial information that can be obtained relatively deep in tissues . This provides a potential for future in vivo studies of apoptosis . The cells expressed a fluorescent protein (C-DEVD-Y) consisting of two fluorophores, enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), linked by the amino acid sequence DEVD selectively cleaved by caspase-3-like proteases . FRET between ECFP and EYFP in C-DEVD-Y could therefore be monitored on-line as a sensor of caspase-3 activation . The relevance of using caspase-3 activation to indicate beta-cell apoptosis was demonstrated by inhibiting caspase-3-like proteases with Z-DEVD-fmk and thereby showing that caspase-3 activation was needed for high-glucose-and cytokine-induced apoptosis in the beta-cell and for staurosporine-induced apoptosis in RINm5F cells . In intact RINm5F cells expressing C-DEVD-Y and in MIN6 cells expressing the variant C-DEVD-Y2, FRET was lost at 155 +/- 23 min (n = 9) and 257 +/- 59 min (n = 4; mean +/- SE) after activation of apoptosis with staurosporine (6 micromol/l), showing that this method worked in insulin-producing cells.

Helicobacter, 2003 Dec, 8(6), 590 - 3
Development of Barrett's esophagus after 'spontaneous' healing of atrophic corpus gastritis; Kokkola A et al.; We report here the case of a 58-year-old asymptomatic male smoker who had a gastroscopy performed in 1990 because of a low serum pepsinogen I level (16 microg/l) . The patient had severe atrophic corpus gastritis and elevated Helicobacter pylori antibody titers, but no histologic evidence of the bacteria . Additionally, a hiatal hernia without esophagitis was seen . He was followed up endoscopically because of the atypical changes (indefinite for dysplasia) in addition to atrophic gastritis in some of the gastric biopsy samples . During the follow-up period, H . pylori antibody titers declined to normal levels without eradication therapy, and atrophic gastritis healed . The patient developed first erosive esophagitis, and, 5 years later, a 3-cm-long Barrett's esophagus was detected.

Curr Issues Mol Biol, 2004 Jan, 6(1), 1 - 15
Signal transduction in T helper cells: CD4 coreceptors exert complex regulatory effects on T cell activation and function; Konig R et al.; The immune system provides a highly sophisticated surveillance mechanism to detect diverse antigens and to protect the host organism from invading pathogens and altered cells (e.g., virus-infected and tumor cells) . Adaptive immune responses depend on the recognition of antigen by specific antigen receptors that are expressed on the surface of T and B lymphocytes . Helper T cells provide regulatory functions and direct the adaptive immune system to respond appropriately to a particular antigen (i.e., cytotoxic T cell responses against viral infections and tumor cells, humoral responses against extracellular bacteria and parasitic worms) . Helper T cells express CD4 coreceptors, which recognize conserved domains on proteins expressed by the class II major histocompatibility complex, the same proteins that present antigen to the T cell receptor . Recent progress in T cell biology has identified multiple regulatory functions of CD4 during thymocyte development and antigen stimulation of mature T helper cells . Signaling pathways induced by engagement of CD4 independently of T cell receptor signaling mediate these regulatory functions . In this review, we discuss the regulation of T cell signaling and emphasize the functional consequences of proper and improper CD4 coreceptor signaling.

Bull Exp Biol Med, 2003 Aug, 136(2), 209 - 11
Oxidative stress monitoring in biological samples by bioluminescent method; Remmel' NN et al.; The integral bioluminescent biotest with lyophilized fluorescent bacteria was used for monitoring of LPO processes in tissue extracts and serum of rats exposed to stress . A relationship between the content of MDA (LPO indicator) and fluorescence of bacteria was observed in all biological samples.

Can J Gastroenterol, 2003 Nov, 17(11), 667 - 72
How can we battle the scourge of diarrhea? 2003 McKenna Memorial Lecture; Barrett KE; Diarrheal diseases exact a considerable toll of morbidity and mortality worldwide, including in developed countries where the incidence of foodborne illness, in particular, may be increasing . This article summarizes the current understanding of the basis of diarrheal illness, focusing particularly on intracellular signaling mechanisms that limit the extent of intestinal epithelial chloride secretion, which may offer new targets for antidiarrheal therapies . Recent information regarding the mechanisms whereby invasive bacteria cause diarrhea is also reviewed along with effects of beneficial bacteria (so-called probiotics) in limiting dysfunction associated with enteric infections . Finally, the author provides some speculations as to the possible benefits to the host of mounting a diarrheal response to an offending pathogen, and possible consequences of the failure of this primitive host defense mechanism.

J Biol Chem, 2004 Feb 13, 279(7), 5053 - 4 Epub 2003 Nov 20.
Light-induced 3-O-sulfotransferase expression alters pineal heparan sulfate fine structure . A surprising link to circadian rhythm; Kuberan B et al.; Proteoglycans are dominant glycoconjugates located on the cell surface and in extracellular spaces and consist of a core protein with one or more glycosaminoglycan side chains linked covalently . Heparan sulfate (HS) belongs to the family of glycosaminoglycans . HS has been assigned a variety of physiological and pathological functions, such as cell-cell adhesion, cell-matrix adhesion, cell proliferation, motility and differentiation, lipoprotein metabolism, blood coagulation, inflammation, tissue regeneration, tumor progression and invasion, pathogenic infection by bacteria, protozoa, and viruses, through specific interaction with a wide array of proteins, ligands, receptors, and pathogens (Bernfield, M., Gotte, M., Park, P . W., Reizes, O., Fitzgerald, M . L., Lincecum, J., and Zako, M . (1999) Annu . Rev . Biochem . 68, 729-777) . We have shown here for the first time that light induces changes in pineal HS fine structure and that occurrence of the rare 3-O sulfation catalyzed by HS 3-O-sulfotransferase (3-OST2) is predominantly restricted to daytime pineal glands.

Vet Parasitol, 2003 Nov 14, 117(3), 195 - 211
Ultrastructural study of a tetratrichomonad species isolated from prepucial smegma of virgin bulls; Cobo ER et al.; We present observations on an unusual tetratrichomonad species isolated from preputial smegma of virgin bulls . Ultrastructural studies were performed using scanning and electron microscopy techniques . This protozoan presents four anterior flagella of unequal length and a recurrent one forming the undulating membrane . It shows one anterior nucleus, a Golgi complex, an axostyle, and a costa . The hydrogenosomes are rather elongated, seen in groups, and presenting different electron densities . Vacuoles of different sizes containing bacteria and material in process of digestion were frequently found . PCR was also used in order to compare the species herein described with other trichomonad species . The amplification products were seen only with primers TFR1 and TFR2 (specific to trichomonads), but not with TFR3 and TFR4 (specific to Tritrichomonas foetus), suggesting that although collected from the genital tract of the bull, this protist was not T . foetus . We propose that the appearance of these tetratrichomonads were probably due to the sodomy practiced among bulls . Concomitant contamination of preputial cavity with feces could explain the presence of the opportunistic organism . The observations presented here show the importance of the correct diagnostic when investigating samples obtained from the urogenital tract of cattle . We also suggest that this flagellate belongs to the species Tetratrichomonas buttreyi.

Food Chem Toxicol, 2004 Jan, 42(1), 51 - 63
Intragastric formation and modulation of N-nitrosodimethylamine in a dynamic in vitro gastrointestinal model under human physiological conditions; Krul CA et al.; Human exposure to carcinogenic N-alkylnitrosamines can occur exogenously via food consumption or endogenously by formation of these compounds through nitrosation of amine precursors . Information on the intragastric formation of NDMA from complex mixtures of precursors and inhibitors in humans is not available . In this study the formation of N-nitrosodimethylamine (NDMA) has been quantitatively analysed in a dynamic in vitro gastrointestinal model, in which gastric conditions can be modulated and closely simulates the physiological situation in humans . Substantial amounts of NDMA were produced when nitrite and dimethylamine or codfish were simultaneously introduced into the model . However, humans are gradually exposed to nitrite by the intake of nitrate-containing food . Nitrate secreted in saliva is converted to nitrite by oral bacteria . To mimic the human exposure to nitrite in a realistic way, nitrite was gradually added into the gastric compartment, simulating the swallowing of nitrite containing oral fluid after the intake of nitrate at the level of 0.1-10 times the ADI . Under these conditions, the cumulative amounts of NDMA formed were 2.3-422 microg NDMA and 1.8-42.7 microg NDMA at a rapid and slow gastric pH decrease, respectively . Beside codfish, various fish species and batches in combination with nitrite, simulating the intake of for times the ADI of nitrate, were investigated . Herring, pollack and plaice were also able to induce NDMA formation . Mackerel, salmon and pike perch did not result in increased NDMA formation . Furthermore, the effect of nitrosation modulators on NDMA formation was investigated . Thiocyanate (2 mM) increased NDMA formation, but the increase was not statistically significant . In contrast, orange jus and tea effectively, but not totally, reduced the amount of NDMA formed in the gastric compartment . These experiments show that (1) . the dynamic in vitro gastrointestinal model is an appropriate tool for mechanistic studies on the intragastric formation of nitrosamines, and (2) . that the results obtained with this model are helpful in evaluating human cancer risk for the combined intake of codfish-like fish species and nitrate-containing vegetables.

J Mol Evol, 2003 Sep, 57(3), 309 - 24
Intra- and interbacterial genetic exchange of Lyme disease spirochete erp genes generates sequence identity amidst diversity; Stevenson B et al.; All isolates of the spirochete Borrelia burgdorferi contain multiple, different plasmids of the cp32 family, each of which contains a locus encoding Erp surface proteins . Many of these proteins are known to bind host complement regulatory factor H, enabling the bacteria to avoid killing by the alternative complement pathway during vertebrate infection . In the present study, we characterized the erp loci and cp32 plasmids of strains N40, Sh-2-82, and 297 and compared them to the previously determined cp32 sequences of type strain B31 . Bacteria of strain N40 contain 6 different cp32s, those of Sh-2-82 contain 10, and 297 bacteria contain 9 cp32s . Significant conservation between all strains was noted for the cp32 loci responsible for plasmid maintenance, indicating close relationships that appear to correspond with incompatibility groups . In contrast, considerable diversity was found between erp gene sequences, both within individual bacteria and between different strains . However, examples of identities among erp loci were found, with strains Sh-2-82, 297, and B31 each containing three identical loci that likely arose through intrabacterial genetic rearrangements . These studies also found the first evidence of large-scale genetic exchanges between Lyme disease spirochetes in nature, including the apparent transfer of an entire cp32 plasmid between two different bacteria.

J Mol Evol, 2003 Sep, 57(3), 255 - 60
Structure-based phylogenies of the serine beta-lactamases; Hall BG et al.; The serine beta-lactamases present a special problem for phylogenetics because they have diverged so much that they fall into three classes that share no detectable sequence homology among themselves . Here we offer a solution to the problem in the form of two phylogenies that are based on a protein structure alignment . In the first, structural alignments were used as a guide for aligning amino acid sequences and in the second, the average root mean square distances between the alpha carbons of the proteins were used to create a pairwise distance matrix from which a neighbor-joining phylogeny was created . From those phylogenies, we show that the Class A and Class D beta-lactamases are sister taxa and that the divergence of the Class C beta-lactamases pre-dated the divergence of the Class A and Class D beta-lactamases.

J Mol Evol, 2003 Sep, 57(3), 249 - 54
The metallo-beta-lactamases fall into two distinct phylogenetic groups; Hall BG et al.; The Ambler Class B metallo-beta-lactamases fall into two distinct phylogenetic groups based on the observation that there is no significant sequence homology between the sequences of members of different groups . Structural alignments confirm that those groups are no more closely related to each other than are the three classes of serine beta-lactamases, Classes A, C, and D . We present phylogenies of these two groups and suggest a new classification scheme for the beta-lactamases.

Curr Microbiol, 2003 Oct, 47(4), 341 - 6
The heat shock gene, htpG, and thermotolerance in the cyanobacterium, Synechocystis sp . PCC 6803; Fang F et al.; The htpG null mutant was obtained by inserting a chloramphenicol resistance cassette (Cm(r)) in the htpG coding sequence . The htpG null mutant (delta htpG), delta hsp16.6, and the double mutant, delta htpG::hsp16.6 cells showed little growth disadvantage at 30 degrees C and 37 degrees C, but not at 40 degrees C . This suggests that HtpG and HSP16.6 proteins do not have an essential role during growth at normal and mildly elevated temperatures . Cell growth, cell survival rate, and oxygen electrode measurements demonstrated that delta htpG, delta hsp16.6, and delta htpG::hsp16.6 cells were sensitive to heat stress . Decreased basal and acquired thermotolerance was observed when mutants were heat shocked, with delta htpG::hsp16.6 being the most sensitive . A comparison of mutants showed that delta hsp16.6 was more sensitive to heat shock than delta htpG.

Curr Microbiol, 2003 Oct, 47(4), 314 - 8
Molecular evidence of Wolbachia infection in natural populations of tropical odonates; Thipaksorn A et al.; Wolbachia are endosymbiotic bacteria that cause reproductive alterations in numerous arthropod species . Using a PCR-based method, we found that, out of 33 odonate species, four species were infected with Wolbachia . This finding represents the first record of Wolbachia infection in tropical odonates . Identical wsp gene sequences were found in the Wolbachia-infected common odonate species, Agriocnemis f . femina, collected from different locations in Thailand . The infection frequencies in several natural populations suggest that replacement of uninfected populations by Wolbachia-infected ones has recently occurred in this damselfly species.

J Vet Med B Infect Dis Vet Public Health, 2003 Aug, 50(6), 289 - 93
Effect of endobronchial challenge with Actinobacillus pleuropneumoniae serotype 10 of pigs vaccinated with bacterins consisting of A . pleuropneumoniae serotype 10 grown under NAD-rich and NAD-restricted conditions; van Overbeke I et al.; The efficacy of two bacterins containing an Actinobacillus pleuropneumoniae serotype 10 strain was evaluated . The bacterial cells constituting bacterin 1 and 2 were grown under nicotinamide adenine dinucleotide (NAD)-rich (low-adherence capacity to alveolar epithelial cell cultures) and NAD-restricted (high-adherence capacity to alveolar epithelial cell cultures) conditions, respectively . Ten pigs were vaccinated twice with the bacterin 1 and nine pigs with the bacterin 2 . Ten control animals were injected twice with a saline solution . Three weeks after the second vaccination, all pigs were endobronchially inoculated with 106.5 colony-forming units (CFU) of an A . pleuropneumoniae serotype 10 strain . In the bacterin 1 and 2 group, three and two pigs died after inoculation, respectively . Only two pigs of the control group survived challenge . Surviving pigs were killed at 7 days after challenge . The percentage of pigs with severe lung lesions (> 10% of the lung affected) was 100% in the control group, 70% in the bacterin 1 group and 22% in the bacterin 2 group . Actinobacillus pleuropneumoniae was isolated from the lungs of all animals . The mean bacterial titres of the caudal lung lobes were 7.0 x 10(6) CFU/g in the control group, 6.3 x 10(5) CFU/g in the bacterin 1 group and 1.3 x 10(6) CFU/g in the bacterin 2 group . It was concluded that both bacterins induced partial protection against severe challenge . Furthermore, there are indications that the bacterin 2, containing A . pleuropneumoniae bacteria grown under conditions resulting in high in vitro adhesin, induced better protection than the bacterin 1.

Nucleic Acids Res, 2003 Dec 1, 31(23), 6778 - 87
Solution structure and DNA binding of the effector domain from the global regulator PrrA (RegA) from Rhodobacter sphaeroides: insights into DNA binding specificity; Laguri C et al.; Prr/RegA response regulator is a global transcription regulator in purple bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus, and is essential in controlling the metabolic changes between aerobic and anaerobic environments . We report here the structure determination by NMR of the C-terminal effector domain of PrrA, PrrAC . It forms a three-helix bundle containing a helix-turn-helix DNA binding motif . The fold is similar to FIS protein, but the domain architecture is different from previously characterised response regulator effector domains, as it is shorter than any characterised so far . Alignment of Prr/RegA DNA targets permitted a refinement of the consensus sequence, which contains two GCGNC inverted repeats with variable half-site spacings . NMR titrations of PrrAC with specific and non-specific DNA show which surfaces are involved in DNA binding and suggest residues important for binding specificity . A model of the PrrAC/DNA complex was constructed in which two PrrAC molecules are bound to DNA in a symmetrical manner.

Ned Tijdschr Geneeskd, 2003 Nov 1, 147(44), 2153 - 6
{Gene therapy with interleukin 10 in Crohn's disease: too early yet}; Dijkstra G et al.; An improved understanding of the pathogenesis of inflammatory bowel disease (IBD) has led to the development of new drugs such as infliximab and insights into the modes of action of commonly used drugs such as azathioprine and methotrexate . These drugs act, at least in part, by inducing apoptosis of activated T-cells, which are an important phenomenon in the aberrant mucosal immune response in Crohn's disease . Gene therapy directed towards delivering anti-inflammatory proteins such as interleukin 10 (IL-10) to the inflamed mucosa is another new means of correcting the balance between the proinflammatory and anti-inflammatory cytokines in IBD . Gene-therapeutic manipulation of T-cells or bacteria to make them selectively deliver IL-10 to the gut mucosa has been successfully described in experimental models of IBD . Although interleukin-10-based gene therapy for Crohn's disease is an attractive option, safety aspects concerning the gene transfection method and questions about the efficacy of interleukin-10 in Crohn's disease in particular, prevent its application in the near future.

Arch Immunol Ther Exp (Warsz), 2003, 51(5), 289 - 94
The immunology of Chlamydia trachomatis; Zdrodowska-Stefanow B et al.; Chlamydia trachomatis (C . trachomatis) is one of the most common sexually transmitted bacterial agents . What distinguishes it from other organisms is its intracellular reproductive cycle . Up to now, four antigens have been identified in the Chlamydia genus: genus-specific antigen as well as species-specific, type-specific and subspecies-specific . C . trachomatis is a powerful immunogen which stimulates the host's immunological processes . The intracellular parasitism of the bacteria is the basis for both symptomatic or asymptomatic infection as well as for chronic ones . The primary infection leads to a local inflammatory reaction due to penetration and reproduction of the bacteria in the epithelial cells and to IgA secretory antibody production . In most cases the host's reaction to the primary infection is transient and does not cause tissue damage . In the course of chronic infection or reinfection, the most important processes are those of delayed hypersensitivity, which lead to a fast and intense immunological reaction of specifically sensitized Th1 lymphocytes . This reaction leads to progressive damage of the epithelial cells and to cicatrization and fibrosis, which means irreversible complications . Interferon gamma is of special importance in the process of C . trachomatis infection . High concentrations of it inhibit the bacteria's reproductive cycle, while lower concentrations promote the development of atypical, non-contagious forms of Chlamydia of diminished metabolic activity and altered antigenicity . The chlamydial heat shock proteins are considered to be of great importance lately . Their molecular weights of 60 and 10 kDa are a powerful stimulant of immunological reactions and show significant homology (40-90%) to human and other bacterial heat shock proteins.

Nurs Times, 2003 Oct 28-Nov 3, 99(43), 22 - 4
Examining the literature on using tap water in wound cleansing; Patel S et al.; Practitioners working in the community setting and whose caseload predominantly consists of treating chronic wounds often use tap water as a wound cleanser with the rationale that chronic wounds are already colonised with bacteria . However, there is some controversy, as discussed within this review, of the suitability of tap water as a cleansing agent for acute wounds.

Proteomics, 2003 Oct, 3(10), 2044 - 51
Functional proteomics of neurokinin B in the placenta indicates a novel role in regulating cytotrophoblast antioxidant defences; Sawicki G et al.; Neurokinin B (NKB) has recently been demonstrated to be secreted from the placenta in abnormally high amounts in preeclampsia (PE) and to cause hypertension in rats, suggesting it may be a mediator of some pathophysiological features of PE . It is also known that NKB receptors exist in the placenta . To determine the effect of high levels of NKB on the placenta, we have performed proteomics on five separate preparations of cultured purified human term cytotrophoblast cells . The results showed a statistically significant decrease in 20 proteins, of which five were unknown proteins . Proteins important in antioxidant defenses that decreased were thioredoxin, cyclophilin A, cytokeratin 1, and peroxiredoxin 5 . Two proteins that inhibit intravascular anticoagulation, cytokeratin 1 and annexin 11 were also decreased . Pathways involving pro-inflammatory cytokine activation of NF-kappa B are opposed by Raf kinase inhibitor protein, which was also decreased . Cofilin 1, a protein involved in defense against bacteria, was also decreased . Among other proteins that were suppressed by NKB were proteasome proteins, desmoplakin, and calgizzarin . Western blots confirmed the decrease in cytokeratin 1 and cyclophilin A protein after NKB exposure . In PE, there is reduced antioxidant activity and increased intravascular coagulation . The findings that high levels of NKB, similar to those observed in PE, can impair these two classes of activity support the hypothesis that high NKB levels may contribute to the pathogenesis of PE.

J Biol Inorg Chem, 2004 Jan, 9(1), 49 - 58 Epub 2003 Nov 18.
Biophysical characterization of the MerP-like amino-terminal extension of the mercuric reductase from Ralstonia metallidurans CH34; Rossy E et al.; The purified native mercuric reductase (MerA) from Ralstonia metallidurans CH34 contains an N-terminal sequence of 68 amino acids predicted to be homologous to MerP, the periplasmic mercury-binding protein . This MerP-like protein has now been expressed independently . The protein was named MerAa by homology with Ccc2a, the first soluble domain of the copper-transporting ATPase from yeast . Deltaa has been characterized using a set of biophysical techniques . The binding of mercury was followed using circular dichroism spectroscopy and electrospray mass spectrometry . The two cysteine residues contained in the consensus sequence GMTC XXC are involved in the binding of one mercury atom, with an apparent affinity comparable to that of MerP for the same metal . The metal-binding site is confirmed by NMR chemical shift changes observed between apo- and metal-bound MerAa in solution . NMR shift and NOE data also indicate that only minor structural changes occur upon metal binding . Further NMR investigation of the fold of MerAa using long-range methyl-methyl NOE and backbone residual dipolar coupling data confirm the expected close structural homology with MerP . (15)N relaxation data show that MerAa is a globally rigid molecule . An increased backbone mobility was observed for the loop region connecting the first beta-strand and the first alpha-helix and comprising the metal-binding domain . Although significantly reduced, this loop region keeps some conformational flexibility upon metal binding . Altogether, our data suggest a role of MerAa in mercury trafficking.

Proc Natl Acad Sci U S A, 2003 Nov 25, 100(24), 14321 - 6 Epub 2003 Nov 17.
Differential expression of iron-, carbon-, and oxygen-responsive mycobacterial genes in the lungs of chronically infected mice and tuberculosis patients; Timm J et al.; Pathogenetic processes that facilitate the entry, replication, and persistence of Mycobacterium tuberculosis (MTB) in the mammalian host likely include the regulated expression of specific sets of genes at different stages of infection . Identification of genes that are differentially expressed in vivo would provide insights into host-pathogen interactions in tuberculosis (TB); this approach might be particularly valuable for the study of human TB, where experimental opportunities are limited . In this study, the levels of selected MTB mRNAs were quantified in vitro in axenic culture, in vivo in the lungs of mice, and in lung specimens obtained from TB patients with active disease . We report the differential expression of MTB mRNAs associated with iron limitation, alternative carbon metabolism, and cellular hypoxia, conditions that are thought to exist within the granulomatous lesions of TB, in the lungs of wild-type C57BL/6 mice as compared with bacteria grown in vitro . Analysis of the same set of mRNAs in lung specimens obtained from TB patients revealed differences in MTB gene expression in humans as compared with mice.

Tuberculosis (Edinb), 2003, 83(6), 351 - 9
Catalase-peroxidase activity has no influence on virulence in a murine model of tuberculosis; Cardona PJ et al.; The capacity to generate a chronic and persistent infection in the experimental murine model of tuberculosis induced aerogenically by a low-dose inoculum was determined in eight isoniazid-resistant clinical strains of Mycobacterium tuberculosis showing different catalase-peroxidase (C-P) activities . Determination of bacillary concentration in lung and spleen and the percentage of pulmonary parenchyma occupied by granulomas were monitored . Data showed no relation between the lack of C-P activity and the ability to develop a persistent infection, highlighting the potential of C-P negative strains to spread through the community.

FEBS Lett, 2003 Nov 20, 554(3), 394 - 8
A microscope-based screening platform for large-scale functional protein analysis in intact cells; Liebel U et al.; A modular microscope-based screening platform, with applications in large-scale analysis of protein function in intact cells is described . It includes automated sample preparation, image acquisition, data management and analysis, and the genome-wide automated retrieval of bioinformatic information . The modular nature of the system ensures that it is rapidly adaptable to new biological questions or sets of proteins . Two automated functional assays addressing protein secretion and the integrity of the Golgi complex were developed and tested . This shows the potential of the system in large-scale, cell-based functional proteomic projects.

FEBS Lett, 2003 Nov 20, 554(3), 323 - 9
Selection and characterisation of binders based on homodimerisation of immunoglobulin V(H) domains; Jin H et al.; The antigen-binding surface of antibodies is formed by the heterodimerisation of the two variable domains of the light (V(L)) and heavy (V(H)) chains . We have previously described the spontaneous formation of V(H) dimers (VHD) in both bacteria and mammalian cells . The self-association of a single domain produces a homo-VHD, in which the two identical V(H) domains generate a unique symmetric surface for antigen binding that is never found in the normal V(L)/V(H) antibody binding site . We developed a phagemid vector for the construction of phage display libraries in which a cysteine residue, introduced at the C-terminus of the only V(H) cloned, allowed display of homo-VHDs . Panning of the library on different proteins yielded antigen specific binders against lysozyme, glutathione S-transferase and streptavidin . A lysozyme specific homo-VHD was further characterised with an apparent affinity determined to be 216+/-6.6 nM . Importantly, the results showed that its binding activity was fully dependent on the dimerisation of both identical V(H) domains.

Microbes Infect, 2003 Nov, 5(13), 1165 - 9
Inducible nitric oxide synthase promoter polymorphism in human brucellosis; Orozco G et al.; Nitric oxide (NO), produced by the inducible nitric oxide synthase (NOS2) protein, is a defence mechanism against various pathogens, including bacteria of the genus Brucella . The aim of this study was to investigate the possible association between the NOS2 gene promoter polymorphism, TAAA(n) and CCTTT(n) microsatellites, and the predisposition to human brucellosis . We performed a case-control study in 85 patients with brucellosis and 100 healthy individuals, matched for age and sex, living in the same geographic area, in whom the NOS2 promoter was genotyped by using a polymerase chain reaction (PCR)-based method combined with fluorescent technology . No statistically significant differences were observed in the distribution of TAAA(n) alleles between the groups under study . When the overall NOS2 CCTTT(n) allele distribution of the brucellosis patients was compared with that of the control subjects, a significant skewing was observed (P = 0.04, by chi(2) test from 2 x 9 contingency table) . Interestingly, we observed a trend towards Brucella infection protection with the 9 repeat (181 bp) allele (1.8% patients vs . 7.5% controls; P = 0.01, odds ratios = 0.22, 95% confidence interval = 0.05-0.83), which turned out to be non-significant after applying multiple testing . We concluded that the NOS2 microsatellite polymorphism might not have a major effect on brucellosis; nevertheless, the fact that a non-significant trend towards protection was detected in the CCTTT(n) alleles may be an indication for a follow-up study.

Mol Microbiol, 2003 Nov, 50(4), 1329 - 38
An anti-repression Fur operator upstream of the promoter is required for iron-mediated transcriptional autoregulation in Helicobacter pylori; Delany I et al.; The Fur protein acts as a regulator of iron-dependent gene transcription in bacteria . In Helicobacter pylori, Fur regulates iron-activated and iron-repressed promoters . It also acts as an autoregulatory rheostat of transcription to fine-tune its own expression in response to iron by binding to three operators at its own promoter Pfur . Using biochemical and genetic analyses, here we show that the distal upstream operator III (centred at -110) is essential for iron regulation of Pfur and functions as an anti-repression site that is bound by the iron-free form of Fur to induce transcription . Furthermore, operator I (centred at -50) may have a dual role both as a high-affinity binding site for Fur and as an UP element . We propose that its role is ensuring that Fur expression is not repressed below a minimum threshold level . Our data supports a novel promoter architecture and mechanism of regulation by Fur.

J Pak Med Assoc, 2003 Sep, 53(9), 413 - 7
Management of superficial neck abscesses; Samad L et al.; OBJECTIVE: To evaluate clinical features, management options and outcomes in children with neck abscesses, with a view to correlating this data with the different causative micro-organisms, specifically mycobacteria . PATIENTS AND METHODS: A retrospective chart review of fifty-one consecutive children who were admitted with suspected superficial neck abscesses to the Unit between January 1994 and June 1999 was performed . RESULTS: The causative organisms were identified in 21 cases--bacteria in 13 patients, atypical mycobacteria in 6 cases, and mycobacterium tuberculosis in 2 instances . Children with atypical mycobacterial infection had a significantly longer duration of symptoms at the time of admission (mean = 87 days) versus those with bacterial infection (mean = 6 days) . Ultrasonography was performed in 20 patients in this series, and was seen to be of value in demonstrating a collection in situations where there was doubt on clinical basis alone . The outcome was seen to be complicated in all children with mycobacterial infections--either atypical or tuberculous . CONCLUSIONS: Neck abscesses are commonly encountered in paediatric practice . In most instances the diagnosis and treatment is straightforward, with an uncomplicated outcome . However, it is important to bear in mind that there exists a subset of abscesses caused by atypical mycobacteria, with greater risk of complications and recurrence, that needs special attention at the time of diagnosis, intervention and follow-up.

Nippon Rinsho, 2003 Nov, 61(11), 1936 - 44
{Clinical features of pneumonia associated with influenza virus infection}; Fujita J; The history of influenza pandemics was reviewed and clinical manifestations of pneumonia associated with influenza virus infections are described . Several types of pneumonia associated with the influenza virus infection have been reported: 1) influenza complicated by secondary bacterial pneumonia, 2) primary influenza virus pneumonia, 3) combined influenza virus and bacterial pneumonia . Secondary bacterial pneumonia often produces a syndrome that is clinically distinguishable from that of primary viral pneumonia . In primary influenza virus pneumonia, chest roentgenography revealed bilateral infiltrations but no consolidation . Histologically, diffuse alveolar damage and hemorrhagic bronchiolitis were frequently observed in primary influenza virus pneumonia, in which case the prognosis was the worst . Although rare, the possibility of bronchiolitis obliterans organizing pneumonia associated with influenza virus infection should be recognized . H . influenzae, S . pneumoniae, or S . aureus were frequently associated with influenza viral infection, and treatment against these bacteria should be considered.

Mikrobiol Z, 2003 Jul-Aug, 65(4), 3 - 10
{Survival of Bradyrhizobium japonicum strain 646a introduced into soil of the soybean, common bean and maize rhizosphere}; Tolkachev NZ; The number dynamics of Bradyrhizobium japonicum strain 646a introduced into soil in the soybean, common bean and maize rhizosphere and its dependence on inoculum doses have been studied in greenhouse experiments . It was shown that B . japonicum number in the maize, common bean and soybean rhizosphere depends on the inoculum doses (r = 0.79-0.95) . It was less than 0.1% of total saprophytic bacteria number and it had no essential effect on this plant productivity . The soybean symbiosis with nodule bacteria enhanced its yield by 28.9-42.6% . In nodulating soybean rhizosphere B . japonicum reproduced most intensively and reached the maximum number 52-225 thousand bacteria/g of soil in the period of soybean maturing and nodule destruction . This indicated to important role of soybean nodules not only in symbiotic nitrogen fixation, but also in formation of the rhizosphere and soil B . japonicum population.

Am Nat, 2003 Nov, 162(5), 597 - 614 Epub 2003 Nov 06.
Comparative tests of parasite species richness in primates; Nunn CL et al.; Some hosts harbor diverse parasite communities, whereas others are relatively parasite free . Many factors have been proposed to account for patterns of parasite species richness, but few studies have investigated competing hypotheses among multiple parasite communities in the same host clade . We used a comparative data set of 941 host-parasite combinations, representing 101 anthropoid primate species and 231 parasite taxa, to test the relative importance of four sets of variables that have been proposed as determinants of parasite community diversity in primates: host body mass and life history, social contact and population density, diet, and habitat diversity . We defined parasites broadly to include not only parasitic helminths and arthropods but also viruses, bacteria, fungi, and protozoa, and we controlled for effects of uneven sampling effort on per-host measures of parasite diversity . In nonphylogenetic tests, body mass was correlated with total parasite diversity and the diversity of helminths and viruses . When phylogeny was taken into account, however, body mass became nonsignificant . Host population density, a key determinant of parasite spread in many epidemiological models, was associated consistently with total parasite species richness and the diversity of helminths, protozoa, and viruses tested separately . Geographic range size and day range length explained significant variation in the diversity of viruses.

J Bacteriol, 2003 Dec, 185(23), 6902 - 12
Analysis of bvgR expression in Bordetella pertussis; Merkel TJ et al.; Bordetella pertussis, the causative agent of whooping cough, produces a wide array of factors that are associated with its ability to cause disease . The expression and regulation of these virulence factors are dependent upon the bvg locus, which encodes three proteins: BvgA, a 23-kDa cytoplasmic protein; BvgS, a 135-kDa transmembrane protein; and BvgR, a 32-kDa protein . It is hypothesized that BvgS responds to environmental signals and interacts with BvgA, a transcriptional regulator, which upon modification by BvgS binds to specific promoters and activates transcription . An additional class of genes is repressed by the products of the bvg locus . The repression of these genes is dependent upon the third gene, bvgR . Expression of bvgR is dependent upon the function of BvgA and BvgS . This led to the hypothesis that the binding of phosphorylated BvgA to the bvgR promoter activates the expression of bvgR . We undertook an analysis of the transcriptional activation of bvgR expression . We identified the bvgR transcript by Northern blot analysis and identified the start site of transcription by primer extension . We determined that transcriptional activation of the bvgR promoter in an in vitro transcription system requires the addition of phosphorylated BvgA . Additionally, we have identified cis-acting regions that are required for BvgA activation of the bvgR promoter by in vitro footprinting and in vivo deletion and linker scanning analyses . A model of BvgA binding to the bvgR promoter is presented.

J Bacteriol, 2003 Dec, 185(23), 6860 - 9
Two paralogous families of a two-gene subtilisin operon are widely distributed in oral treponemes; Correia FF et al.; Certain oral treponemes express a highly proteolytic phenotype and have been associated with periodontal diseases . The periodontal pathogen Treponema denticola produces dentilisin, a serine protease of the subtilisin family . The two-gene operon prcA-prtP is required for expression of active dentilisin (PrtP), a putative lipoprotein attached to the treponeme's outer membrane or sheath . The purpose of this study was to examine the diversity and structure of treponemal subtilisin-like proteases in order to better understand their distribution and function . The complete sequences of five prcA-prtP operons were determined for Treponema lecithinolyticum, "Treponema vincentii," and two canine species . Partial operon sequences were obtained for T . socranskii subsp . 04 as well as 450- to 1,000-base fragments of prtP genes from four additional treponeme strains . Phylogenetic analysis demonstrated that the sequences fall into two paralogous families . The first family includes the sequence from T . denticola . Treponemes possessing this operon family express chymotrypsin-like protease activity and can cleave the substrate N-succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide (SAAPFNA) . Treponemes possessing the second paralog family do not possess chymotrypsin-like activity or cleave SAAPFNA . Despite examination of a range of protein and peptide substrates, the specificity of the second protease family remains unknown . Each of the fully sequenced prcA and prtP genes contains a 5' hydrophobic leader sequence with a treponeme lipobox . The two paralogous families of treponeme subtilisins represent a new subgroup within the subtilisin family of proteases and are the only subtilisin lipoprotein family . The present study demonstrated that the subtilisin paralogs comprising a two-gene operon are widely distributed among treponemes.

BMC Infect Dis . 2003 Nov 14;3(1):27.
Water dispersible microbicidal cellulose acetate phthalate film; Neurath AR et al.; BACKGROUND: Cellulose acetate phthalate (CAP) has been used for several decades in the pharmaceutical industry for enteric film coating of oral tablets and capsules . Micronized CAP, available commercially as "Aquateric" and containing additional ingredients required for micronization, used for tablet coating from water dispersions, was shown to adsorb and inactivate the human immunodeficiency virus (HIV-1), herpesviruses (HSV) and other sexually transmitted disease (STD) pathogens . Earlier studies indicate that a gel formulation of micronized CAP has a potential as a topical microbicide for prevention of STDs including the acquired immunodeficiency syndrome (AIDS) . The objective of endeavors described here was to develop a water dispersible CAP film amenable to inexpensive industrial mass production . METHODS: CAP and hydroxypropyl cellulose (HPC) were dissolved in different organic solvent mixtures, poured into dishes, and the solvents evaporated . Graded quantities of a resulting selected film were mixed for 5 min at 37 degrees C with HIV-1, HSV and other STD pathogens, respectively . Residual infectivity of the treated viruses and bacteria was determined . RESULTS: The prerequisites for producing CAP films which are soft, flexible and dispersible in water, resulting in smooth gels, are combining CAP with HPC (other cellulose derivatives are unsuitable), and casting from organic solvent mixtures containing approximately equal to 50 to approximately equal to 65% ethanol (EtOH) . The films are approximately equal to 100 micron thick and have a textured surface with alternating protrusions and depressions revealed by scanning electron microscopy . The films, before complete conversion into a gel, rapidly inactivated HIV-1 and HSV and reduced the infectivity of non-viral STD pathogens >1,000-fold . CONCLUSIONS: Soft pliable CAP-HPC composite films can be generated by casting from organic solvent mixtures containing EtOH . The films rapidly reduce the infectivity of several STD pathogens, including HIV-1 . They are converted into gels and thus do not have to be removed following application and use . In addition to their potential as topical microbicides, the films have promise for mucosal delivery of pharmaceuticals other than CAP.

Traffic, 2003 Nov, 4(11), 724 - 38
Lipid rafts and caveolae as portals for endocytosis: new insights and common mechanisms; Parton RG et al.; Clathrin-coated pits and caveolae are two of the most recognizable features of the plasma membrane of mammalian cells . While our understanding of the machinery regulating and driving clathrin-coated pit-mediated endocytosis has progressed dramatically, including the elucidation of the structure of individual components and partial in vitro reconstitution, the role of caveolae as alternative endocytic carriers still remains elusive 50 years after their discovery . However, recent work has started to provide new insights into endocytosis by caveolae and into apparently related pathways involving lipid raft domains . These pathways, distinguished by their exquisite sensitivity to cholesterol-sequestering agents, can involve caveolae but also exist in cells devoid of caveolins and caveolae . This review examines the current evidence for the involvement of rafts and caveolae in endocytosis and the molecular players involved in their regulation.

Transfus Med, 2003 Oct, 13(5), 303 - 10
Autologous red cells derived from cord blood: collection, preparation, storage and quality controls with optimal additive storage medium (Sag-mannitol); Garritsen HS et al.; To investigate whether packed red cells (PRCs) prepared from autologous cord blood-packed red cells (AC-PRCs) could be used as an alternative for homologous-packed red cells (H-PRCs), we developed a system to collect and prepare AC-PRCs and determined standard storage parameters during 35 days of storage in extended storage medium (Sag-mannitol) . We collected and fractionated cord blood from 390 newborns . The amount and quality of the AC-PRCs were analysed . The bacterial contamination rate was 1.84% . Twelve AC-PRCs were stored for 35 days, and standard laboratory parameters were measured at day 1 and day 35 . The initial laboratory parameters of the AC-PRCs were similar to the parameters of the H-PRCs . After 35 days, the AC-PRCs displayed an increased haemolysis rate compared to H-PRCs (1.1 versus 0.2%) and also a significant decreased adenosine triphosphate value (1.2 versus 2.3 micromol L(-1)) . Haemoglobin, haematocrit and pH were comparable in both groups . AC-PRCs meet the quality criteria for H-PRCs after 35 days . Utilizing a closed collection system for cord blood and an extended storage medium will increase safety and quality and facilitate the routine transfusion of autologous red cells derived from cord blood.

Mol Microbiol, 2003 Nov, 50(3), 1031 - 42
The majority of inducible DNA repair genes in Mycobacterium tuberculosis are induced independently of RecA; Rand L et al.; In many species of bacteria most inducible DNA repair genes are regulated by LexA homologues and are dependent on RecA for induction . We have shown previously by analysing the induction of recA that two mechanisms for the induction of gene expression following DNA damage exist in Mycobacterium tuberculosis . Whereas one of these depends on RecA and LexA in the classical way, the other mechanism is independent of both of these proteins and induction occurs in the absence of RecA . Here we investigate the generality of each of these mechanisms by analysing the global response to DNA damage in both wild-type M . tuberculosis and a recA deletion strain of M . tuberculosis using microarrays . This revealed that the majority of the genes that were induced remained inducible in the recA mutant stain . Of particular note most of the inducible genes with known or predicted functions in DNA repair did not depend on recA for induction . Amongst these are genes involved in nucleotide excision repair, base excision repair, damage reversal and recombination . Thus, it appears that this novel mechanism of gene regulation is important for DNA repair in M . tuberculosis.

Mol Microbiol, 2003 Nov, 50(3), 949 - 59
RshA, an anti-sigma factor that regulates the activity of the mycobacterial stress response sigma factor SigH; Song T et al.; SigH, an alternative sigma factor of Mycobacterium tuberculosis, is a central regulator of the response to oxidative and heat stress . Exposure to these stresses results in increased expression of sigH itself, and of genes encoding additional regulators and effectors of the bacterial response to these stresses . In this work we show that RshA, a protein encoded by a gene in the sigH operon, is an anti-sigma factor of SigH . We demonstrate that RshA binds to SigH in vitro and in vivo . This protein-protein interaction, as well as the ability of RshA to inhibit SigH-dependent transcription, is redox-dependent, with RshA functioning as a negative regulator of SigH activity only under reducing conditions . The interaction of SigH and RshA is also disrupted in vitro by elevated temperature . RshA, a protein of 101 amino acids, contains five conserved cysteine residues of which two appear to be essential for RshA to inhibit SigH activity, suggesting that these cysteines may be important for the redox state dependence of RshA function . Our results indicate that RshA is a sensor that responds to oxidative stress, and also to heat stress, resulting in activation of SigH and expression of the SigH-dependent genes that allow M . tuberculosis to adapt to these stresses.

APMIS, 2003 Oct, 111(10), 951 - 4
Nanobacteria promote crystallization of psammoma bodies in ovarian cancer; Sedivy R et al.; Serous papillary adenocarcinomas of the ovary are often associated with microcalcifications, namely psammoma bodies . Archebacteria such as nanobacteria are, for example, involved in kidney stone formation . Nanobacteria deserve close scrutiny as their cytotoxicity and ability to cross the placenta present a potential clinical risk . In this study we investigated whether nanobacteria are associated with psammoma bodies in ovarian cancer . We identified in all seven carcinomas with multiple psammoma body nanobacterial antigens in histological specimens and in the ascitic fluid . Control cases of adenocarcinomas without such calcifications did not present nanobacterial antigens . This finding indicates that apart from non-malignant conditions, nanobacteria could also be found in cancer . Hence, nanobacterial infection may be of clinical importance as these bacteria are related to microcalifications in ovarian cancer.

Annu Rev Genet, 2003, 37, 513 - 43
A cyanobacterial circadian timing mechanism; Ditty JL et al.; Cyanobacteria such as Synechococcus elongatus PCC 7942 exhibit 24-h rhythms of gene expression that are controlled by an endogenous circadian clock that is mechanistically distinct from those described for diverse eukaryotes . Genetic and biochemical experiments over the past decade have identified key components of the circadian oscillator, input pathways that synchronize the clock with the daily environment, and output pathways that relay temporal information to downstream genes . The mechanism of the cyanobacterial circadian clock that is emerging is based principally on the assembly and disassembly of a large complex at whose heart are the proteins KaiA, KaiB, and KaiC . Signal transduction pathways that feed into and out of the clock employ protein domains that are similar to those in two-component regulatory systems of bacteria.

Proc Natl Acad Sci U S A, 2003 Nov 25, 100(24), 13863 - 8 Epub 2003 Nov 13.
Coevolution of an aminoacyl-tRNA synthetase with its tRNA substrates; Salazar JC et al.; Glutamyl-tRNA synthetases (GluRSs) occur in two types, the discriminating and the nondiscriminating enzymes . They differ in their choice of substrates and use either tRNAGlu or both tRNAGlu and tRNAGln . Although most organisms encode only one GluRS, a number of bacteria encode two different GluRS proteins; yet, the tRNA specificity of these enzymes and the reason for such gene duplications are unknown . A database search revealed duplicated GluRS genes in >20 bacterial species, suggesting that this phenomenon is not unusual in the bacterial domain . To determine the tRNA preferences of GluRS, we chose the duplicated enzyme sets from Helicobacter pylori and Acidithiobacillus ferrooxidans . H . pylori contains one tRNAGlu and one tRNAGln species, whereas A . ferrooxidans possesses two of each . We show that the duplicated GluRS proteins are enzyme pairs with complementary tRNA specificities . The H . pylori GluRS1 acylated only tRNAGlu, whereas GluRS2 was specific solely for tRNAGln . The A . ferrooxidans GluRS2 preferentially charged tRNA(UUG)(Gln) . Conversely, A . ferrooxidans GluRS1 glutamylated both tRNAGlu isoacceptors and the tRNA(CUG)(Gln) species . These three tRNA species have two structural elements in common, the augmented D-helix and a deletion of nucleotide 47 . It appears that the discriminating or nondiscriminating natures of different GluRS enzymes have been derived by the coevolution of protein and tRNA structure . The coexistence of the two GluRS enzymes in one organism may lay the groundwork for the acquisition of the canonical glutaminyl-tRNA synthetase by lateral gene transfer from eukaryotes.

Immunity, 2003 Nov, 19(5), 689 - 99
Hematopoietic stem cells expressing the myeloid lysozyme gene retain long-term, multilineage repopulation potential; Ye M et al.; Single cell PCR studies showed that hematopoietic stem cells (HSCs) express a variety of lineage-affiliated genes . However, it remains unclear whether these cells exhibiting "lineage priming" represent bona fide stem cells or a subpopulation earmarked for differentiation . Here we have used a Cre-Lox approach to follow the fate of cells expressing a lineage-affiliated marker . We crossed lysozyme Cre mice with yellow fluorescent protein (EYFP) reporter mice and found EYFP gene expression not only in myelomonocytic cells but also in a fraction of HSCs as well as B cells and T cells . Transplantation of EYFP+ HSCs into primary and secondary recipients generated mice in which all hematopoietic cells were EYFP+ . In contrast, crosses between CD19 Cre and lck Cre mice with reporter mice showed no EYFP expression in HSCs or intermediate progenitors . Our results demonstrate that lysozyme expression does not mark myeloid commitment and that long-term repopulation potential is maintained in primed HSCs.

Oncogene, 2003 Nov 13, 22(51), 8337 - 42
Effects of Helicobacter pylori CagA protein on the growth and survival of B lymphocytes, the origin of MALT lymphoma; Umehara S et al.; Helicobacter pylori (H . pylori) is a causative agent of gastrointestinal diseases such as atrophic gastritis and gastroduodenal ulcer . Infection of cagA-positive H . pylori is also associated with gastric carcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma . The cagA gene product CagA is directly injected into the bacteria-attached host cells via the bacterial type IV secretion system . The translocated CagA deregulates intracellular signaling pathways and thereby initiates pathogenesis . In this work, we examined the biological effects of CagA on B cells, from which MALT lymphoma arises . Ectopic expression of CagA in interleukin 3-dependent B cells inhibited cell proliferation by suppressing the JAK-STAT signaling . CagA was also capable of preventing hydroxyurea-induced B-cell apoptosis through inhibiting p53 accumulation . In contrast to the effects of CagA in gastric epithelial cells, the observed CagA activities in B cells were independent of its tyrosine phosphorylation . Our results indicate that CagA possesses both phosphorylation-dependent and -independent activities in mammalian cells and that biological impacts of CagA depend on cell-type context . As a result of B-cell growth inhibition, CagA may diminish anti-H . pylori immune responses . Furthermore, CagA may play a role in the development of MALT lymphoma by impairing p53-dependent apoptosis.

Mutagenesis, 2003 Nov, 18(6), 491 - 6
Mechanisms of induction of chromosomal aberrations by hydroquinone in V79 cells; do Ceu Silva M et al.; Hydroquinone occurs naturally in bacteria and plants and it is also manufactured for commercial use . Human exposure to this compound can occur by environmental, occupational, dietary and cigarette smoke exposure and from exposure to benzene, which can be metabolized to this compound . However, the main source of exposure to this compound is dietary, since hydroquinone is a naturally occurring compound in many foods . Hydroquinone can be metabolized to benzoquinones, which are potent haematotoxic, genotoxic and carcinogenic compounds that can also induce the formation of radical species, predisposing cells to oxidative damage . In order to clarify the involvement of radical species in the genotoxicity of hydroquinone, the induction of chromosomal aberrations in V79 cells was studied along with the assessment of the production of hydroxyl radicals at different pH values (6.0, 7.4 and 8.0), as well as the effect of antioxidant enzymes {catalase and superoxide dismutase (SOD)} on the clastogenic effect of hydroquinone . The results obtained indicate that the clastogenic activity of hydroquinone is dependent on the pH, suggesting that deprotonation is a fundamental step leading to DNA lesions under the experimental conditions used . The addition of S9 mix, SOD or SOD and catalase significantly decreased the clastogenic activity, suggesting the involvement of superoxide anion and hydrogen peroxide in the genotoxicity of hydroquinone . However, other species generated in the auto-oxidation process of hydroquinone, such as the semiquinone radical or the quinone, also seem to play a role in its genotoxicity, since the addition of antioxidant enzymes (catalase and SOD) or S9 mix do not lead to a complete abolition of the observed genotoxic activity . These results suggest the existence of at least two mechanisms associated with the genotoxic activity of this compound.

Clin Cancer Res, 2003 Nov 1, 9(14), 5423 - 8
Peptides inhibitory for the transcriptional regulatory function of human papillomavirus E2; Fujii T et al.; PURPOSE: Human papillomavirus (HPV) infections are associated with cervical neoplasia . Cellular and viral proteins are known to interact with the papillomavirus E2 protein to initiate transcription and DNA replication in the HPV life cycle . Our aim was to identify peptides that bind to the HPV16 E2 protein and thereby inhibit its ability to alter the transcriptional activity of other genes . EXPERIMENTAL DESIGN: The HPV16 E2 protein was expressed and purified to near homogeneity in bacteria . We screened a phage display library of random peptides for ones that bound to HPV16 E2 protein . Among the isolated phage clones, we found that tryptophan-rich peptide sequences appeared repetitively in successive cycles of phage library panning . Replacement of the tryptophan amino acids in these dodecapeptides reduced the degree to which these peptides bound to the E2 protein . These E2-binding peptides were tested for their ability to inhibit the transcriptional regulatory function of E2 in a test cell line, which contained an E2 gene and a luciferase reporter gene driven by an E2-dependent transcriptional promoter . RESULTS: Delivery of four of the E2 binding peptides into the intracellular compartment of the test cell line resulted in suppression of the E2-dependent luciferase expression . Deletion of the tryptophan residues from these peptides reduced their E2 binding and their ability to suppress E2-dependent luciferase expression in the test cell line . CONCLUSIONS: These results suggest a strategy for the development of chemical inhibitors of E2-dependent transcription of viral genes in HPV-infected cells as an approach to the therapy of chronic HPV infections.

Mar Biotechnol (NY), 2004 Jan-Feb, 6(1), 67 - 82 Epub 2003 Nov 14.
Seasonal variation of antifouling activities of marine algae from the Brittany coast (France); Hellio C et al.; The antifouling activity of extracts (aqueous, ethanol, and dichloromethane) of 9 marine macroalgae against bacteria, fungi, diatoms, macroalgal spores, mussel phenoloxidase activity, and barnacle cypris larvae has been investigated in relation to season in bimonthly samples from the Bay of Concarneau (France) . Of the extracts tested, 48.2% were active against at least one of the fouling organisms, and of these extracts, 31.2% were seasonally active with a peak of activity in summer corresponding to maximal values for water temperature, light intensity, and fouling pressure, and 17% were active throughout the year . This seasonal activity may be adaptive as it coincides with maximal fouling pressure in the Bay of Concarneau . Dichloromethane extracts of Rhodophyceae were the most active in the antifouling assays.

J Biol Chem, 2004 Feb 13, 279(7), 5739 - 51 Epub 2003 Nov 11.
Alterations in global patterns of gene expression in Synechocystis sp . PCC 6803 in response to inorganic carbon limitation and the inactivation of ndhR, a LysR family regulator; Wang HL et al.; The cyanobacterium Synechocystis sp . PCC 6803 possesses multiple inorganic carbon (Ci) uptake systems that are regulated by Ci availability . The control mechanisms of these systems and their integration with other cell functions remain to be clarified . An analysis of the changes in global gene expression in response to Ci downshift and the inactivation of ndhR (sll1594), a LysR family regulator of Ci uptake is presented in this report . Mild Ci limitation (3% CO2 (v/v) in air to air alone) induced a dramatic up-regulation of genes encoding both inducible CO2 and HCO3- uptake systems . An induction of ndhD5/ndhD6 and other genes in a probable transcriptional unit was observed, suggesting a function in inducible Ci uptake . The expression of slr1513 and sll1735, physically clustered with sbtA and ndhF3/ndhD3/cupA, respectively, were also coordinated with upstream genes encoding the essential components for HCO3- and CO2 uptake . Ci limitation induced the regulatory genes slr1214, sll1292, slr1594, sigD, sigG, and sigH, among which slr1214, a two-component response regulator, showed the earliest induction, implying a role for the early response to Ci limitation . Opposite regulation of genes encoding the assimilation of carbon and nitrogen demonstrated a striking coordination of expression to balance C- and N-fluxes . The analyses revealed that ndhR inactivation up-regulated the expression of sbtA/sbtB, ndhF3/ndhD3/cupA/sll1735, and slr2006-13 including ndhD5 and ndhD6, indicating a vital role of this regulatory gene in both CO2 and HCO3- acquisition of the cyanobacterium . We therefore suggest that ndhR be renamed ccmR to better represent its broader regulatory characteristics.

Phys Rev Lett . 2003 Oct 10;91(15):158104 . Epub 2003 Oct 10.
Nonlocal interaction effects on pattern formation in population dynamics; Fuentes MA et al.; We consider a model for population dynamics such as for the evolution of bacterial colonies which is of the Fisher type but where the competitive interaction among individuals is nonlocal, and show that spatial structures with interesting features emerge . These features depend on the nature of the competitive interaction as well as on its range, specifically on the presence or absence of tails in, and the central curvature of, the influence function of the interaction.

J Exp Biol, 2003 Dec, 206(Pt 24), 4533 - 8
Heterotrophy on ultraplankton communities is an important source of nitrogen for a sponge-rhodophyte symbiosis; Pile AJ et al.; Grazing on ultraplankton by the sponge partner of an invertebrate/algal symbiotic association can provide enough particulate organic nitrogen to support the nitrogen needs of both partners . The previously unknown natural diet of the sponge in the Haliclona-Ceratodictyon association consists of bacteria and protozoans, which are rich sources of nitrogen . Retention of ultraplankton varied with season and time of day . During the winter there was an order of magnitude more nitrogen taken up than in summer . Time of day during each season also affected the amount of ultraplankton retained . In summer retention was higher at night whereas the opposite was true during winter . Overall, the Haliclona-Ceratodictyon association is able to meet its metabolic nitrogen demands through grazing on the naturally occurring water column community.

Novartis Found Symp, 2003, 252, 291 - 302; discussion 302-5
The role of dendritic cells in regulating mucosal immunity and tolerance; Mowat AM et al.; The intestinal immune system discriminates between invasive pathogens and antigens that are harmless, such as food proteins and commensal bacteria . The latter groups of antigens normally induce tolerance and a breakdown in this homeostatic process can lead to diseases such as coeliac disease or Crohn's disease . The nature ofthe intestinal immune response depends on how antigen is presented to CD4+ T cells by dendritic cells (DCs) . Both oral tolerance and priming are influenced by the numbers and activation status of DCs in the gut and its draining lymphoid tissues, and our current work indicates that dietary proteins are taken up preferentially by DCs in the lamina propria of the small intestine . These then migrate to interact with antigen-specific CD4+ T cells in the mesenteric lymph node . In vivo and in vitro studies using purified lamina propria DCs suggest these may play a unique role in the regulation of intestinal immune responses . We propose that local DCs are the gatekeepers of the mucosal immune system, inducing tolerance under physiological conditions, but being sufficiently responsive to inflammatory stimuli to allow T cell priming and protective immunity when necessary . In addition, we will discuss evidence that adjuvant vectors such as ISCOMS may be effective mucosal vaccines due to an ability to activate intestinal DCs.

Novartis Found Symp, 2003, 252, 92 - 8; discussion 98-105, 106-14
Control of immune pathology by regulatory T cells; Powrie F et al.; CD4+CD25+ T(reg) cells inhibit colitis in the severe combined immune deficient (SCID) T cell adoptive transfer model . Cells with this function are present in the thymus suggesting that T(reg) cells capable of inhibiting bacteria-induced immune pathology are similar to those that inhibit organ-specific autoimmunity . CD4+CD25+ T(reg) cells inhibit both T cell-dependent and T cell-independent intestinal inflammation . The latter point illustrates that in addition to direct effects on other T cells, T(reg) cells can alsoprevent immune pathology in vivo by inhibiting the actions of innate immune cells . T(reg) cells suppress intestinal inflammation through mechanisms that involve interleukin 10 and transforming growth factor beta and blockade of the negative regulator of T cell activation CTLA4 abrogates T(reg) cell function in vivo . Importantly adoptive transfer of CD4+CD25+ T(reg) cells to mice with established colitis reverses inflammation and restores normal intestinal architecture suggesting that CD4+CD25+ T(reg) cells may be utilized for cellular therapy of inflammatory diseases.

IUBMB Life, 2003 Aug, 55(8), 451 - 8
The role of DNA recombination in herpes simplex virus DNA replication; Wilkinson DE et al.; In many organisms the processes of DNA replication and recombination are closely linked . For instance, in bacterial and eukaryotic systems, replication forks can become stalled or damaged, in many cases leading to the formation of double stranded breaks . Replication restart is an essential mechanism in which the recombination and repair machinery can be used to continue replication after such a catastrophic event . DNA viruses of bacteria such as lambda and T4 also rely heavily on DNA recombination to replicate their genomes and both viruses encode specialized gene products which are required for recombination-dependent replication . In this review, we examine the linkage between replication and recombination in the eukaryotic pathogen, Herpes Simplex Virus Type 1 (HSV-1) . The evidence that recombination plays an intrinsic role in HSV-1 DNA replication and the infection process will be reviewed . We have recently demonstrated that HSV-1 encodes two proteins which may be analogous to the lambda phage recombination system, Red(alpha) and beta . The HSV-1 alkaline nuclease, a 5' to 3' exonuclease, and ICP8, a single stranded DNA binding protein, can carry out strand annealing reactions similar to those carried out by the lambda Red system . In addition, evidence suggesting that host recombination proteins may also be important for HSV-1 replication will be reviewed . In summary, it is likely that HSV-1 infection will require both viral and cellular proteins which participate in various pathways of recombination and that recombination-dependent replication is essential for the efficient replication of viral genomes.

IUBMB Life, 2003 Aug, 55(8), 435 - 41
Biosynthesis and control of mosquito gut proteases; Borovsky D; Earlier investigations of the proteases that digest the blood meal in mosquitoes identified the midgut epithelial cells as the source of the main blood digesting protease, trypsin . Surgical manipulations and cytoimmunochemical studies indicated that trypsin in the mosquito is not stored in the epithelial cells as an inactive enzyme, but is synthesized, activated and released into the gut lumen after the blood meal . Using molecular biology techniques the major trypsin genes have been sequenced and some information on the transcription factors that regulate the induction of the genes have been reported . Although all the hormone(s) and the factors that are responsible for the induction of trypsin biosynthesis are not known, the termination of trypsin biosynthesis is controlled by a decapeptide hormone; trypsin modulating oostatic factor (TMOF) that is secreted by the mosquito ovary . Genetic engineering and expression of TMOF in bacteria, yeast and algae is emerging as a potential future larval control of mosquitoes in the field.

Lab Anim, 2003 Oct, 37(4), 341 - 51
Comparison of the sensitivity of in vivo antibody production tests with in vitro PCR-based methods to detect infectious contamination of biological materials; Bootz F et al.; Bacteria and viruses may be transmitted to laboratory rodents by contaminated biological materials such as transplantable tumours, cell lines, sera or other biological materials . Biological materials are currently being screened using the mouse or rat antibody production (MAP/RAP) test (serological testing) . We decided to test and validate an alternative assay using polymerase chain reaction (PCR/realtime PCR) technology to detect viral contamination directly in biological material . The aim of this study therefore is the validation of our new PCR assays and the comparison of PCR and the MAP test . For 8/14 viruses, conventional PCR was more sensitive and more specific than the MAP test in detecting murine viruses . For 12/14 viruses, the realtime PCR was more sensitive than the MAP test . In 2/14 cases, all three detection methods had the same sensitivity . Furthermore, PCR screening clearly conforms to the principles of the 3Rs as a replacement technique because it eliminates the need for using animals to screen for murine viruses in biological material.

J Immunol, 2003 Nov 15, 171(10), 5345 - 52
Mast cell degranulation requires N-ethylmaleimide-sensitive factor-mediated SNARE disassembly; Puri N et al.; Mast cells possess specialized granules that, upon stimulation of surface FcR with IgE, fuse with the plasma membrane, thereby releasing inflammatory mediators . A family of membrane fusion proteins called SNAREs, which are present on both the granule and the plasma membrane, plays a role in the fusion of these granules with the plasma membrane of mast cells . In addition to the SNAREs themselves, it is likely that the SNARE accessory protein, N-ethylmaleimide-sensitive factor (NSF), affects the composition and structure of the SNARE complex . NSF is a cytoplasmic ATPase that disassembles the SNARE complexes . To investigate the role of NSF in mast cell degranulation, we developed an assay to measure secretion from transiently transfected RBL (rat basophilic leukemia)-2H3 mast cells (a tumor analog of mucosal mast cells) . RBL-2H3 cells were cotransfected with a plasmid encoding a human growth hormone secretion reporter along with either wild-type NSF or an NSF mutant that lacks ATPase activity . Human growth hormone was targeted to and released from secretory granules in RBL-2H3 cells, and coexpression with mutant NSF dramatically inhibited regulated exocytosis from the transfected cells . Biochemical analysis of SNARE complexes in these cells revealed that overexpression of the NSF mutant decreased disassembly and resulted in an accumulation of SNARE complexes . These data reveal a role for NSF in mast cell exocytosis and highlight the importance of SNARE disassembly, or priming, in regulated exocytosis from mast cells.

J Biol Chem, 2004 Feb 6, 279(6), 4849 - 61 Epub 2003 Nov 07.
Substrate discrimination by formamidopyrimidine-DNA glycosylase: a mutational analysis; Zaika EI et al.; Formamidopyrimidine-DNA glycosylase (Fpg) is a primary participant in the repair of 8-oxoguanine, an abundant oxidative DNA lesion . Although the structure of Fpg has been established, amino acid residues that define damage recognition have not been identified . We have combined molecular dynamics and bioinformatics approaches to address this issue . Site-specific mutagenesis coupled with enzyme kinetics was used to test our predictions . On the basis of molecular dynamics simulations, Lys-217 was predicted to interact with the O8 of extrahelical 8-oxoguanine accommodated in the binding pocket . Consistent with our computational studies, mutation of Lys-217 selectively reduced the ability of Fpg to excise 8-oxoguanine from DNA . Dihydrouracil, also a substrate for Fpg, served as a nonspecific control . Other residues involved in damage recognition (His-89, Arg-108, and Arg-109) were identified by combined conservation/structure analysis . Arg-108, which forms two hydrogen bonds with cytosine in Fpg-DNA, is a major determinant of opposite-base specificity . Mutation of this residue reduced excision of 8-oxoguanine from thermally unstable mispairs with guanine or thymine, while excision from the stable cytosine and adenine base pairs was less affected . Mutation of His-89 selectively diminished the rate of excision of 8-oxoguanine, whereas mutation of Arg-109 nearly abolished binding of Fpg to damaged DNA . Taken together, these results suggest that His-89 and Arg-109 form part of a reading head, a structural feature used by the enzyme to scan DNA for damage . His-89 and Lys-217 help determine the specificity of Fpg in recognizing the oxidatively damaged base, while Arg-108 provides specificity for bases positioned opposite the lesion.

Hepatobiliary Pancreat Dis Int, 2003 Feb, 2(1), 147 - 51
Killing effects of cytosine deaminase gene mediated by adenovirus vector on human pancreatic cancer cell lines in vitro; Li ZS et al.; OBJECTIVE: To evaluate the killing effects of the cytosine deaminase (CD) gene mediated by adenovirus vector on human pancreatic cancer cell lines in vitro . METHODS: The CD gene was cloned into pAdTrack-CMV-CD, and pAdTrack-CMV-CD and pAdEasy-1 were recombinated in bacteria . The newly recombinated Ad-CD containing green fluorescent protein (GFP) was propagated in 293 cells and purified by cesium chloride gradient centrifugation . Human pancreatic cancer cell lines Patu8988 and SW1990 were infected with this virus, then 5-FC was added . XTT assay was used to estimate relative numbers of viable cells . RESULTS: The positive clones were selected by using endonuclease to digest the combinatants and the concentration of viral liquids containing the CD gene was 2X10(11) pfu/ml . It was found that significant cytotoxic activities were possessed by 5-FC for the CD gene transduced pancreatic cell lines, but little effects exerted on the nontransduced pancreatic carcinoma cells . CONCLUSIONS: The CD gene mediated by adenovirus with a high infectivity is efficient for gene therapy of pancreatic carcinoma cell lines . These data demonstrate the therapeutic efficacy of an enzyme prodrug strategy in experimental pancreatic cancer.

J Microbiol Methods, 2003 Dec, 55(3), 841 - 50
Flow cytometry assessment of bacterioplankton in tropical marine environments; Andrade L et al.; Flow cytometry was used to characterize bacterioplankton from two tropical environments in Brazil: the eutrophic Guanabara Bay and the oligotrophic southwest Atlantic Ocean . Bacterial abundance was evaluated by flow cytometry, and cells were stained with SYTO 13, allowing demonstration of differences in nucleic acid content . Bacterial production was also evaluated by means of 3H-leucine incorporation . Bacterial numbers were different for both sites . In Atlantic Ocean samples, we found a maximum of 5.50 x 10(5) cells ml(-1), and low nucleic acid content organisms predominated . In Guanabara Bay, bacterial numbers were one order of magnitude higher than in the ocean, and they varied from outer bay (1.01 x 10(6) cells ml(-1)) to inner bay (6.90 x 10(6) cells ml(-1)) . Bacterial activity in ocean samples varied from 4.6 to 126 ng C l(-1) h(-1), while in the bay, mean values ranged from 1.95 microg C l(-1) h(-1) (outer bay) to 7.35 microg C l(-1) h(-1) (inner bay) . Values found for both parameters are characteristic of different trophic situations . These results illustrate the utility of cytometric analyses of bacterioplankton populations in characterizing their large spatial and temporal scales of distribution in aquatic ecosystems.

J Microbiol Methods, 2003 Dec, 55(3), 787 - 90
Selective adhesion of extremophiles for scanning electron microscopy; Bizzoco RL et al.; Polyethyleneimine (PEI)-coated slides were used for attachment of unknown extremophiles and their cultures grown at high temperature and low pH . Selective adhesion of cells in natural samples and cultural isolates was compared to polylysine (PL) and several other coatings . PEI is superior to PL for adhesion of extremophiles.

Biomacromolecules, 2003 Nov-Dec, 4(6), 1703 - 12
In vitro assessment of the enzymatic degradation of several starch based biomaterials; Azevedo HS et al.; The susceptibility of starch-based biomaterials to enzymatic degradation by amylolytic enzymes (glucoamylase and alpha-amylase) was investigated by means of incubating the materials with a buffer solution, containing enzymes at different concentrations and combinations, at 37 degrees C for 6 weeks . Two polymeric blends of corn starch with poly(ethylene-vinyl alcohol) copolymer and poly(epsilon-caprolactone), designated by SEVA-C and SPCL, respectively, were studied . The material degradation was characterized by gravimetry measurements, tensile mechanical testing, scanning electron microscopy (SEM), and Fourrier transform infrared-attenuated total reflectance (FTIR-ATR) . The degradation liquors were analyzed for determination of reducing sugars, as a result of enzyme activity, and high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was used to identify the degradation products . All of the analysis performed showed that starch polymeric blends are susceptible to enzymatic degradation, as detected by increased weight loss and reducing sugars in solution . alpha-Amylase caused significant changes on the overall mechanical properties of the materials, with a decrease of about 65% and 58% being observed in the moduli for SEVA-C and SPCL, respectively, when compared with the control (samples incubated in buffer only) . SEM analysis detected the presence of fractures and pores at the material's surface as a result of starch degradation by amylolytic enzymes . FTIR spectra confirmed a decrease on the band corresponding to glycosidic linkage (-C-O-C-) of starch after incubation of the materials with alpha-amylase . In contrast, the incubation of the polymers in buffer only, did not cause significant changes on the material's properties and morphology . Comparing the two materials, SEVA-C exhibited a higher degradability, which is related to the physicochemical structure of the materials and also to the fact that the starch concentration is higher in SEVA-C . The identification of the degradation products by HPAEC-PAD revealed that glucose was the major product of the enzymatic degradation of starch-based polymers . alpha-Amylase, as expected, is the key enzyme involved in the starch degradation, contributing to major changes on the physicochemical properties of the materials . Nevertheless, it was also found that starch-based polymers can also be degraded by other amylolytic enzymes but in a smaller extent.

World J Gastroenterol, 2003 Nov, 9(11), 2419 - 23
Construction and analysis of SSH cDNA library of human vascular endothelial cells related to gastrocarcinoma; Liu YB et al.; AIM: To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database . METHODS: Human VECs related to gastric adenocarcinoma and corresponding normal tissue were separated by magnetic beads coupled with antibody CD31 (Dynabeads CD31) . A few amount of total RNA were synthesized and amplified by SMART PCR cDNA Synthesis Kit . Then, using SSH and T/A cloning techniques, cDNA fragments of differentially expressed genes in human VECs of gastric adenocarcinoma were inserted into JM109 bacteria . One hundred positive bacteria clones were randomly picked and identified by colony PCR method . To analyze cDNA libraries of gastrocarcinoma and VECs in CGAP database, the tools of Library Finder, cDNA xProfiler, Digital GENE Expression Displayer (DGED), and Digital Differential Display (DDD) were used . RESULTS: Forward and reverse subtraction cDNA libraries of human VECs related to gastrocarcinoma were constructed successfully with SSH and T/A cloning techniques . Analysis of CGAP database indicated that no appropriate library of VECs related to carcinoma was constructed . CONCLUSION: Construction of subtraction cDNA libraries of human VECs related to gastrocarcinoma was successful and necessary, which laid a foundation for screening and cloning new and specific genes of VECs related to gastrocarcinoma.

Arch Microbiol, 2003 Dec, 180(6), 484 - 9 Epub 2003 Nov 07.
Expression of the trxA gene for thioredoxin 1 in Rhodobacter sphaeroides during oxidative stress; Li K et al.; Expression of the thioredoxin ( trxA) gene of Rhodobacter sphaeroides is regulated by oxidative stress at the transcriptional and post-transcriptional levels . All oxidative stress agents tested resulted in a moderate or strong increase of trxA mRNA levels, which was not due to increased mRNA stability . While the kinetics of increased trxA mRNA and of sodB mRNA, encoding superoxide dismutase, were similar after addition of tert-butyl hydroperoxide ( t-BOOH) or hydrogen peroxide (H(2)O(2)), different kinetics were observed after addition of diamide or paraquat, indicating the involvement of different stress responses in the regulation of these genes . The level of TrxA did not increase to the same extent as trxA mRNA levels . Furthermore, the addition of H(2)O(2) or t-BOOH led to increased turnover of the protein . Apparently, increased txA transcription compensated, at least in part, for the reduced stability of the protein . A strain expressing lower levels of thioredoxin 1 showed decreased resistance to diamide and H(2)O(2) but increased resistance to paraquat and t-BOOH compared to the wild-type . These data implicate the involvement of various systems in the response to different types of oxidative stress and the participation of thioredoxin 1 in the defense against oxidative stress caused by diamide or H(2)O(2).

Science, 2003 Dec 19, 302(5653), 2124 - 6 Epub 2003 Nov 06.
Yeast life-span extension by calorie restriction is independent of NAD fluctuation; Anderson RM et al.; Calorie restriction (CR) slows aging in numerous species . In the yeast Saccharomyces cerevisiae, this effect requires Sir2, a conserved NAD+-dependent deacetylase . We report that CR reduces nuclear NAD+ levels in vivo . Moreover, the activity of Sir2 and its human homologue SIRT1 are not affected by physiological alterations in the NAD+:NADH ratio . These data implicate alternate mechanisms of Sir2 regulation by CR.

Vaccine, 2003 Dec 8, 22(1), 70 - 6
Oral vaccination with Mycobacterium bovis BCG in a lipid formulation induces resistance to pulmonary tuberculosis in brushtail possums; Aldwell FE et al.; A method was developed for formulating Mycobacterium bovis bacille Calmette-Guerin (BCG) for oral vaccination against tuberculosis . Selected lipid-based formulations of BCG were tested in the brushtail possum for their ability to elicit immune responses and protection against bovine tuberculosis . Formulation of BCG in lipid matrices maintained bacteria in a dormant but viable state . Oral delivery of 2 x 10(8) colony forming units of formulated BCG to possums induced strong lymphocyte proliferation responses to bovine purified protein derivative (PPD) in peripheral blood lymphocytes . Oral vaccination of possums also reduced the severity of disease following aerosol challenge with virulent M . bovis compared with animals vaccinated with non-formulated BCG . In a second experiment, levels of protection with lipid-formulated oral BCG were similar to those seen with subcutaneous BCG vaccination . Our data shows that formulated oral BCG is an efficient means of inducing protection against bovine tuberculosis in possums and should be a practical means of vaccinating wildlife against tuberculosis.

Vaccine, 2003 Dec 8, 22(1), 49 - 56
Efficacy of DNA-hsp65 vaccination for tuberculosis varies with method of DNA introduction in vivo; Lima KM et al.; A DNA vaccine codifying the mycobacterial hsp65 can prevent infection with Mycobacterium tuberculosis in a prophylactic setting and also therapeutically reduce the number of bacteria in infected mice . The protective mechanism is thought to be related to Th1-mediated events that result in bacterial killing . To determine the best method of hsp65 introduction for vaccination efficacy against tuberculosis (TB), we evaluated the immunogenicity and protection of DNA-hsp65 administered by gene gun bombardment or intramuscular (i.m.) injection of naked DNA . Immunization by gene gun induced immune response with plasmid doses 100-fold lower than those required for intramuscular immunization . However, in contrast to intramuscular immunization, which was protective in these studies, gene gun immunization did not protect BALB/c mice against challenge infection.

Orig Life Evol Biosph, 2003 Oct, 33(4-5), 405 - 32
Origin of sex revisited; Santos M et al.; Why did sex ever arise in the first place? Why it does not disappear in view of the greater efficiency of asexuals? These are clearly two different questions, and we suggest here that the solution for the origin of sex does not necessarily come from theoretical considerations based on currently existing genetic systems . Thus, while we agree with a number of authors in that the emergence of sex (understood as the exchange of genetic material between genomes) is deeply rooted in the origin of life and happened during the very early stages in the transition from individual genes ('replicators') to bacteria-like cells ('reproducers'), we challenge the idea that recombinational repair was the major selective force for the emergence of sex . Taking the stochastic corrector model as a starting point, we provide arguments that question the putative costs of redundancy in primitive protocells . In addition, if genes that cause intragenomic conflict (i.e., parasites) are taken into account, it is certainly wrong to suggest that cellular fusion would be beneficial at the population level (although this strong claim needs some qualifications) . However, when a continuous input of deleterious mutations that impair the fitness of the protocell as a whole is considered in the model (in the realistic range in which stable mutant distributions of quasi-species within compartments are established), there are circumstances when sex could be beneficial as a side effect of the dynamic equilibrium between cellular fusion-mutation-selection . The scenario we have explored numerically is fully consistent with the idea that the universal ancestor was not a discrete entity but an ensemble of proto-organisms that exchanged much genetic information.

Nature, 2003 Nov 6, 426(6962), 65 - 7
Sulphide mining by the superextensile foot of symbiotic thyasirid bivalves; Dufour SC et al.; In a symbiotic association between an invertebrate host and chemoautotrophic bacteria, each partner has different metabolic requirements, and the host typically supplies the bacteria with necessary reduced chemicals (sulphide or methane) . Some combination of anatomical, physiological and behavioural adaptations in the host often facilitates uptake and transport of reduced chemicals to the symbionts . We have studied five species of bivalve molluscs of the family Thyasiridae (that is, thyasirids) three of which harbour chemoautotrophic bacteria . Here we show that the symbiotic bivalves extend their feet to form elongated and ramifying burrows in the sediment, most probably to gain access to reduced sulphur . Closely related bivalves (including some thyasirid species) without bacterial symbionts show no comparable foot extension behaviour . The length and number of burrows formed by chemosymbiotic thyasirids are related to the concentration of hydrogen sulphide in the sediment . The burrows are formed by the foot of each bivalve, which can extend up to 30 times the length of the shell, and may be the most extreme case of animal structure elongation documented to date.

Nucleic Acids Res, 2003 Nov 15, 31(22), 6610 - 8
Host specificity of mollicutes oriC plasmids: functional analysis of replication origin; Lartigue C et al.; Recently, artificial oriC plasmids containing the chromosomal dnaA gene and surrounding DnaA box sequences were obtained for the mollicutes Spiroplasma citri and Mycoplasma pulmonis . In order to study the specificity of these plasmids among mollicutes, a set of similar oriC plasmids was developed for three mycoplasmas belonging to the mycoides cluster, Mycoplasma mycoides subsp . mycoides LC (MmmLC), M.mycoides subsp . mycoides SC (MmmSC) and Mycoplasma capricolum subsp . capricolum . Mycoplasmas from the mycoides cluster, S.citri and M.pulmonis were used as recipients for transformation experiments by homologous and heterologous oriC plasmids . All five mollicutes were successfully transformed by homologous plasmids, suggesting that the dnaA gene region represents the functional replication origin of the mollicute chromosomes . However, the ability of mollicutes to replicate heterologous oriC plasmids was found to vary noticeably with the species . For example, the oriC plasmid from M.capricolum did not replicate in the closely related species MmmSC and MmmLC . In contrast, plasmids harbouring the oriC from MmmSC, MmmLC and the more distant species S.citri were all found to replicate in M.capricolum . Our results suggest that the cis-elements present in oriC sequences are not the only determinants of this host specificity.

Appl Environ Microbiol, 2003 Nov, 69(11), 6740 - 9
Usefulness of rpoB gene sequencing for identification of Afipia and Bosea species, including a strategy for choosing discriminative partial sequences; Khamis A et al.; Bacteria belonging to the genera Afipia and Bosea are amoeba-resisting bacteria that have been recently reported to colonize hospital water supplies and are suspected of being responsible for intensive care unit-acquired pneumonia . Identification of these bacteria is now based on determination of the 16S ribosomal DNA sequence . However, the 16S rRNA gene is not polymorphic enough to ensure discrimination of species defined by DNA-DNA relatedness . The complete rpoB sequences of 20 strains were first determined by both PCR and genome walking methods . The percentage of homology between different species ranged from 83 to 97% and was in all cases lower than that observed with the 16S rRNA gene; this was true even for species that differed in only one position . The taxonomy of Bosea and Afipia is discussed in light of these results . For strain identification that does not require the complete rpoB sequence (4,113 to 4,137 bp), we propose a simple computerized method that allows determination of nucleotide positions of high variability in the sequence that are bordered by conserved sequences and that could be useful for design of universal primers . A fragment of 740 to 752 bp that contained the most highly variable area (positions 408 to 420) was amplified and sequenced with these universal primers for 47 strains . The variability of this sequence allowed identification of all strains and correlated well with results of DNA-DNA relatedness . In the future, this method could be also used for the determination of variability "hot spots" in sets of housekeeping genes, not only for identification purposes but also for increasing the discriminatory power of sequence typing techniques such as multilocus sequence typing.

Transgenic Res, 2003 Oct, 12(5), 529 - 40
Chemically regulated expression systems and their applications in transgenic plants; Wang R et al.; In the past 20 years, several systems have been developed to control transgene expression in plants using chemicals . The components used to construct these systems are derived from regulatory sequences mostly from non-plant organisms such as bacteria, fungi, insects and mammals . These constructs allowed transgene expression to be controlled temporally, spatially and quantitatively with the help of exogenous chemicals, without disturbing endogenous plant gene expression . Various chemically regulated transgene expression systems, their advantages/disadvantages and their potential for large-scale field application are reviewed.

Ann Acad Med Stetin, 2002, 48, 395 - 405
{Occurrence of genomic species of Borrelia burgdorferi sensu lato in populations of Ixodes ricinus ticks from West Pomerania}; Bukowska K; The aim of this study was to determine the incidence of Borrelia burgdorferi sensu lato infections in Ixodes ricinus ticks from recreational areas of the province of West Pomerania . Ticks (nymphs and adults) were collected between April and October 2000 and 2001 from 10 sampling sites . The presence of the spirochete, Borrelia burgdorferi sensu lato, was confirmed with an indirect fluorescence assay (IFA) and the polymerase chain reaction (PCR) . In addition, a nested PCR technique was used to detect one of three genomic species of the bacteria (B . burgdorferi sensu stricto, B . garinii, B . afzelii) . A total of 2380 ticks were studied, including 2195 nymphs, 89 females and 96 males . Peak collection was in June of 2000 (330 ticks) and in August of 2001 (156 ticks) (Table 4) . Collection was lowest in October of 2000 (93 ticks) and 2001 (127 ticks) (Tables 2 and 4) . The mean infection frequency of Ixodes ricinus as determined with PCR was 12.8% in 2000 and 10.9% in 2001 . The frequency determined with IFA was 11.6% (2000) and 9.6% (2001) (Tables 1 and 3, Figs . 1 and 2) . The nested PCR technique revealed that the dominant genospecies was B . burgdorferi sensu stricto (83.5% of ticks in 2000 and 87.7% in 2001), followed by B . garinii (12% and 10.2% in 2000 and 2001, respectively) . B . afzelii was detected in 4.4% (2000) and 2% (2001) of ticks . Co-infection with B . burgdorferi sensu stricto and B . garinii occurred in 3.4% (2000) and 1.8% of ticks (2001) . Determination of the genomic species was not possible in approximately 7% of ticks (Tables 5 and 6) . The present results show that there is a substantial risk of contacting borreliosis in recreational areas in the vicinity of Szczecin.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2003 Nov, 96(5), 578 - 81
The effect of chlorhexidine as an endodontic disinfectant; Zamany A et al.; OBJECTIVE: The purpose of this study was to establish whether addition of a 2% chlorhexidine rinse to a conventional treatment protocol enhances the rate of the successful disinfection of the root canal system in vivo . STUDY DESIGN: Twenty-four teeth with infected necrotic pulps and resorbing apical periodontitis were treated with a conventional technique in which 1% NaOCl as irrigant was used . Half of the cases received an additional rinse with 2% chlorhexidine . Prereduced thioglycollate medium was used to take cultures that were incubated for 4 weeks . RESULTS: Cultivable bacteria were retrieved at the conclusion of the first visit in 1 out of 12 chlorhexidine cases whereas in the control group 7 out of 12 cases showed growth . This difference was significant (P < .05) . CONCLUSION: The findings are clinically important.

Biol Pharm Bull, 2003 Nov, 26(11), 1581 - 4
Antiallergic activity of ginsenoside Rh2; Park EK et al.; The antiallergic activities of ginsenosides, which were isolated from acid-treated ginseng (Panax ginseng, Araliaceae), and their metabolites by human intestinal bacteria were measured . Ginsenoside Rh2, which is a main metabolite, had the most potent inhibitory activity on beta-hexosaminidase release from RBL-2H3 cells and in the passive cutaneous anaphylaxis reaction . The inhibitory activity of ginsenoside Rh2 was more potent than that of disodium cromoglycate, a commercial antiallergic drug . This compound showed membrane stabilizing action upon differential scanning calorimetry and inhibited nitric oxide (NO) and prostaglandin E2 (PGE2) in lipopolysaccharide-stimulated RAW cells . However, this ginsenoside Rh2 did not inhibit the activation of hyaluronidase and did not scavenge active oxygen . These results suggest that ginsenoside Rh2 can exhibit antiallergic activity originating from cell membrane-stabilizing activity and antiinflammatory activity by the inhibition of NO and PGE2 production.

Fungal Genet Biol, 2003 Dec, 40(3), 187 - 96
Septation and cytokinesis in fungi; Walther A et al.; Cytokinesis is the ultimate step of a cell cycle resulting in the generation of two progeny . Failure of correct cell division may be lethal for both, mother and daughter cells, and thus such a process must be tightly regulated with other events of the cell cycle . Differing solutions to the same problem have been developed in bacteria and plants while cytokinesis in animal and fungal cells is highly similar and requires a contractile ring containing actomyosin . Cytokinesis in fungi can be viewed as a three-stage process: (i) selection of a division site, (ii) orderly assembly of protein complexes, and finally (iii) dynamic events that lead to a constriction of the contractile ring and septum construction . Elaborate mechanisms known as the Mitotic Exit Network (MEN) and the Septation Initiation Network (SIN) have evolved to link these events, particularly the final steps of cytokinesis, with nuclear division . The purpose of this review was to discuss the latest developments in the fungal field and to describe the central known players required for key steps on the road to cell division . Differences in the cytokinesis of yeast-like fungi that result in complete cell separation in contrast to septation which leads to the compartmentalization of fungal hyphae are highlighted.

J Biomol Screen, 2002 Dec, 7(6), 526 - 30
High-throughput fluorescence polarization method for identification of FKBP12 ligands; Bollini S et al.; FKBP12 is best known as the target of the widely used immunosuppressive drug FK506 but may also play a role in neuronal survival . Nonimmunosuppressive ligands of FKBP12 have been shown to have neuroprotective and neuroregenerative activity both in vitro and in vivo, stimulating interest in the development of high-throughput screens to rapidly identify novel ligands . FKBP12 was expressed as a His(6)-fusion in bacteria and purified by metal ion affinity and gel filtration chromatography . A high-throughput fluorescence polarization assay was developed to identify novel ligands of FKBP12 . Dissociation constant values of known FKBP12 ligands measured by the new method agreed closely with K(i) values obtained by assaying inhibition of the rotamase activity of the enzyme . The fluorescence polarization assay is rapid, robust, and inexpensive and does not generate radioactive waste . It is very well suited for high-throughput screening efforts.

Environ Technol, 2003 Sep, 24(9), 1079 - 86
The methane production of poultry slaughtering residues and effects of pre-treatments on the methane production of poultry feather; Salminen E et al.; The biological methane production rate and yield of different poultry slaughtering residues were studied . Poultry offal, blood, and bonemeal were rich in proteins and lipids and showed high methane yields, 0.7-0.9, 0.5, and 0.6-0.7 m3 kg(-1) volatile solids(added), respectively (270-340, 100, and 150-170 m3 ton(-1) wet weight) . Blood and bonemeal produced methane rapidly, whereas the methane production of offal was more delayed probably due to long-chain fatty acid inhibition . The length of delay depended on the source and concentration of inoculum and incubation temperature, sewage sludge at 35 degrees C having the shortest delay of a few days, while granular sludge did not produce methane within 94 days of incubation . Feather showed a somewhat lower methane yield, 0.21 m3 kg(-1) volatile solids(added) (50 m3 ton(-1) wet weight) . Combined thermal (120 degrees C, 5 min) and enzymatic (commercial alkaline endopeptidase, 2-10 g l(-1)) pre-treatments increased its methane yield by 37 to 51% . Thermal (70-120 degrees C, 5-60 min), chemical (NaOH 2-10 g l(-1), 2-24 h), and enzymatic pre-treatments were less effective, with methane yield increasing by 5 to 32% . Based on the present results, anaerobic digestion of the studied poultry slaughtering residues appears a promising possibility because of the high methane yield and nitrogen content of these residues (8 to 14% N of total solids), whereas pre-treatments were shown to improve the methane production of feather.

Environ Technol, 2003 Sep, 24(9), 1061 - 7
A simple spectrophotometric method based on pH-indicators for monitoring partial and total alkalinity in anaerobic processes; Jantsch TG et al.; Partial alkalinity, as a measure of bicarbonate concentration, and total alkalinity, as a measure of the concentration of bicarbonate and volatile fatty acids, are useful parameters for monitoring anaerobic digestion processes . This paper reports a new method based on pH-indicators and spectrophotometric measurements for the monitoring of partial and total alkalinity . The method was used in an off-line procedure for monitoring of an anaerobic process and displayed less than 5% deviation from the traditional method of measuring partial and total alkalinity, as well as being rapid and reproducible . A flow injection analysis system based on the method was used on-line for monitoring overload in a UASB reactor, which demonstrated changes in alkalinity not easily registered using traditional methods.

Probl Tuberk Bolezn Legk, 2003, (9), 3 - 5
{Organization of tuberculosis-controlling work and its efficiency in the penitentiaries of the Ivanovo Region}; Pavlov IuA et al.; The incidence rate of tuberculosis is high in the penitentiaries of the Ivanovo Region and in 2000 it was 32 times greater than that in the Region (3122 versus 96.7 per 100,000) . Fluorography remains to be the main method of detection of pulmonary tuberculosis . It is performed on day 3 of admission of jail, then every 6 months . Fluorography allows one to identify 85-90% of the patients with pulmonary tuberculosis . In the fluorgraphic interval, the sputum from patients with symptoms of suspected tuberculosis is tested by the Ziehl-Neelsen procedure, which makes it possible to detect more than 15% of the tuberculosis patients who isolate bacteria . The patients are treated with the routine regimens including 4 first-line drugs . In 1997-2000, the rates of bacterial isolation session and decay cavity closure were in the range of 82.1-93 and 66.7-87.5%, respectively . Integration with civil tuberculosis service, the social security administration of the region, and the regional branch of the red-cross society has proved to be highly effective . The proportion of patients with tuberculosis who continued their treatment in tuberculosis facilities after release increased from 51.1% in 2000 to 80% in 2001.

J Hepatobiliary Pancreat Surg, 2003, 10(4), 292 - 4
Anterior approach for right hepatectomy for hilar cholangiocarcinoma; Liu CL et al.; BACKGROUND/PURPOSE: Right hepatectomy is indicated for hilar cholangiocarcinoma, but mobilization of the right lobe could be difficult when perihepatic adhesion develops in response to repeated cholangitis and forceful mobilization may disseminate bacteria if the obstructed biliary tract contains pus . We encountered a patient who died from postoperative sepsis and multiorgan failure . METHODS: To circumvent such a difficulty, we employed the anterior approach right hepatectomy in a second patient with hilar cholangiocarcinoma . In this patient, liver transection and division of the hepatic vein were performed before mobilization of the right lobe . RESULTS: The second patient recovered uneventfully . CONCLUSION: The anterior approach (utilizing the "no-touch" technique) may be a preferred procedure for right hepatectomy for hilar cholangiocarcinoma.

Gene, 2003 Nov 27, 320, 97 - 108
Early divergence of the FtsZ1 and FtsZ2 plastid division gene families in photosynthetic eukaryotes; Stokes KD et al.; Homologues of the bacterial cell division protein FtsZ are found in higher plants where they function as key components of the chloroplast division complex . In contrast to most bacteria that encode a single FtsZ protein, plants encode multiple proteins that group into two families, FtsZ1 and FtsZ2 . Using new sequence data from a broad range photosynthetic organisms, we performed a series of analyses to better understand the evolutionary history of the plant FtsZ families . Multiple phylogenetic analyses strongly support the grouping of the plant FtsZ genes and proteins into distinct FtsZ1 and FtsZ2 clades . Protein features representing potentially significant functional differences between FtsZ1 and FtsZ2 are identified . Genomic structure comparisons show that exon length and intron position are conserved within each clade, but differ between the clades except at one position . Our data indicate that the divergence of the FtsZ1 and FtsZ2 families occurred long before the evolution of land plants, preceding the emergence of the green algae . The results are consistent with proposals that the two FtsZ families evolved distinct functions during evolution of the chloroplast division apparatus, and indicate that genetic and functional differentiation occurred much earlier than previously hypothesized.

Exp Neurol, 2003 Nov, 184 Suppl 1, S97 - 105
Metabotropic glutamate receptor regulation of neuronal cell death; Spillson AB et al.; The metabotropic glutamate receptors (mGluRs) are a family of glutamate-sensitive receptors that regulate neuronal function separately from the ionotropic glutamate receptors . By coupling to guanosine nucleotide-binding proteins (G proteins), mGluRs are able to regulate neuronal injury and survival, likely through a series of downstream protein kinase and cysteine protease signaling pathways that affect mitochondrial regulated programmed cell death (PCD) . The physiological relevance of this system is supported by evidence that mGluRs are associated with cell survival in several central nervous system neurodegenerative diseases . Evidence is presented that mGluRs are also able to prevent PCD in the peripheral nervous system, and that this may provide a novel mechanism for treatment of diabetic neuropathy . In dorsal root ganglion (DRG) neurons, a high glucose load increases generation of reactive oxygen species (ROS), destabilizes the inner mitochondrial membrane potential (Deltapsi(M)), induces cytochrome c release from the mitochondrial intermembrane space, and induces downstream activation of caspases . In high-glucose conditions, the group II metabotropic glutamate agonist N-acetylaspartylglutamate (NAAG) blocks caspase activation and is completely reversed by the mGluR3 antagonist (S)-alpha-ethylglutamic acid (EGLU) . Furthermore, the direct mGluR3 agonist (2R,4R)-4-aminopyrrolidine-2, 4-dicarboxylate (APDC) prevents induction of ROS . Together these findings are consistent with an emerging concept that mGluRs may protect against cellular injury by regulating oxidative stress in the neuron . More complete understanding of the complex PCD regulatory pathways mediated by mGluRs will provide new therapeutic approaches for the treatment of a wide variety of neurodegenerative diseases.

Vet Parasitol, 2003 Nov 3, 117(1-2), 73 - 83
Immunological role of the endosymbionts of Dirofilaria immitis: the Wolbachia surface protein activates canine neutrophils with production of IL-8; Bazzocchi C et al.; Filarial nematodes, including Dirofilaria immitis and D . repens, harbour intracellular bacteria belonging to the genus Wolbachia . These bacteria have been implicated in the pathogenesis of filarial diseases, possibly through their endotoxins . Recent studies have shown that a major surface protein of Wolbachia (WSP) induces a specific IgG response in hosts infected by D . immitis . WSP from the Wolbachia of D . immitis was produced in recombinant form . The purified protein was used in stimulation assays on canine neutrophils . The assays performed using a modified Boyden chamber showed that WSP stimulates neutrophil chemokinesis . In addition, RT-PCR revealed increased production of chemokine IL-8 by cells incubated with this protein . Neutrophils have been shown to play a major role in the pathogenesis of river blindness, and to accumulate in the nodules of onchocerciasis patients . In dogs infected by D . immitis, neutrophils accumulate in kidneys and in the wall of pulmonary arteries . As shown by our studies, Wolbachia could contribute to these inflammatory phenomena through its surface protein WSP, independently from its endotoxin component.

J Pediatr Adolesc Gynecol, 2003 Oct, 16(5), 269 - 83
Common pediatric and adolescent skin conditions; Sanfilippo AM et al.; Skin lesions are encountered in all areas of medicine, and it is therefore important for physicians to understand the fundamentals of explaining and diagnosing common skin conditions . This article begins with a discussion of description and documentation of skin lesions based on color, size, morphology, and distribution . Pigmentation disorders such as vitiligo are depicted.Cutaneous growths that are found in the pediatric and adolescent population include acrochordons, dermatofibromas, keloids, milia, neurofibromas, and pyogenic granulomas . Treatment of these growths usually involves observation or curettage with electrodessication.Psoriasis, atopic dermatitis, poison ivy, and eczema are comprised of scaling patches and plaques; poison ivy and atopic dermatitis may also present with bullous and vesicular changes . Therapy typically consists of topical emollients and corticosteroids; phototherapy is reserved for refractory cases.Acne vulgaris is the most common skin disease of the pediatric and adolescent population . This condition can be psychologically debilitating and, therefore, proper treatment is of paramount importance . Therapeutic options include topical as well as oral antibiotics and retinoids . Extreme caution must be used when prescribing retinoids to post-pubescent females, as these agents are teratogenic.Vascular anomalies are most commonly exemplified as port wine stains and hemangiomas . Port wine stains may be treated with pulsed dye laser or may be observed if they are not of concern to the patient or physician . Hemangiomas typically spontaneously regress by age ten; however, there has been recent concern that certain cases may need to be treated.Dermal rashes may be localized or generalized . Treatment of generalized drug eruptions involves elimination of the inciting agent, topical antipruritics, and systemic corticosteroids for severe reactions.Infectious etiologic agents of skin disease include bacteria, fungi, and viruses . Many sexually transmitted diseases are bacterial or viral in origin and present as a rash or ulcer . Impetigo is a bacterial infection which may present as a bullous eruption or as an erosion with a honey colored crust . Other bacterial infections include erythema chronicum migrans, folliculitis, and cellulitis . Fungal infections include the various forms of tinea and are usually treated with topical antifungals; if the infection is located in a hair-bearing area, systemic antifungals are necessary . Viral infections include warts, varicella, molluscum contagiosum, and herpes . Treatment varies from observation or antivirals for varicella to cryosurgery and topical imiquimod for warts . Finally, scabies and lice are infectious agents that can be treated with permethrin and pyrethrin solutions.

Plasmid, 2003 Nov, 50(3), 236 - 41
pHP489, a Helicobacter pylori small cryptic plasmid, harbors a novel gene coding for a replication initiation protein; Song JY et al.; We have analyzed a Helicobacter pylori plasmid, pHP489 . The 1222-bp nucleotide sequence had one open reading frame, a DnaA-binding site, one direct repeat, and three inverted repeats . The (G+C) content of pHP489 was 33.3% . Although the nucleic acid sequence and deduced amino acid sequence were homologous to those of other bacterial plasmid Rep proteins, the degree of similarity was very low . A deletion analysis showed that the Rep protein was not required for the replication of pHP489 in its H . pylori host, but the host replication machinery was needed.

Diagn Microbiol Infect Dis, 2003 Nov, 47(3), 503 - 9
Evaluation of sputum decontamination methods for Mycobacterium tuberculosis using viable colony counts and flow cytometry; Burdz TV et al.; Continuous monitoring systems for the detection of Mycobacterium tuberculosis are reported to have higher contamination rates than traditional radiometric technologies . Multiple decontamination methods have recently been reported in an attempt to optimize contamination rates for these systems . In this study, several decontamination methods for sputum were evaluated using viable colony count and flow cytometry . The decontamination protocols evaluated include N-Acetyl-L-Cysteine-Sodium Hydroxide (NALC-NaOH), modified Petroffs's method, and the Yamane procedure . Several parameters of the NALC-NaOH method were analyzed including final NaOH concentrations of 0.5-3%, NaOH exposure times of 0-30 min, and variations in resuspension media for the resultant pellet . All decontamination methods were performed on pooled and sterilized sputum seeded separately with either a mixture of common contaminating bacteria or M . tuberculosis H37Ra . Viability of organisms following decontamination was assessed by both colony counts and flow cytometric analysis . Flow cytometry viability assays utilized a combination of viability dyes and reference beads to determine viable organism concentrations . The results indicated that no decontamination method was clearly superior, however a concentration of 1-2% NaOH and an increase in the time of NaOH exposure to 30 min will effectively kill contaminating bacteria without significantly affecting the viability of M . tuberculosis H37Ra . While flow cytometry viability analysis did not directly correspond to viable colony counts, it was a useful tool for rapid viability analysis M . tuberculosis.

Brain Res Bull, 2003 Nov 15, 62(1), 15 - 28
The role of metals in neurodegenerative processes: aluminum, manganese, and zinc; Zatta P et al.; Until the last decade, little attention was given by the neuroscience community to the neurometabolism of metals . However, the neurobiology of heavy metals is now receiving growing interest, since it has been linked to major neurodegenerative diseases . In the present review some metals that could possibly be involved in neurodegeneration are discussed . Two of them, manganese and zinc, are essential metals while aluminum is non-essential . Aluminum has long been known as a neurotoxic agent . It is an etiopathogenic factor in diseases related to long-term dialysis treatment, and it has been controversially invoked as an aggravating factor or cofactor in Alzheimer's disease as well as in other neurodegenerative diseases . Manganese exposure can play an important role in causing Parkinsonian disturbances, possibly enhancing physiological aging of the brain in conjunction with genetic predisposition . An increased environmental burden of manganese may have deleterious effects on more sensitive subgroups of the population, with sub-threshold neurodegeneration in the basal ganglia, generating a pre-Parkinsonian condition . In the case of zinc, there has as yet been no evidence that it is involved in the etiology of neurodegenerative diseases in humans . Zinc is redox-inactive and, as a result of efficient homeostatic control, does not accumulate in excess . However, adverse symptoms in humans are observed on inhalation of zinc fumes, or accidental ingestion of unusually large amounts of zinc . Also, high concentrations of zinc have been found to kill bacteria, viruses, and cultured cells . Some of the possible mechanisms for cell death are reviewed.

Arch Oral Biol, 2003 Dec, 48(12), 797 - 804
Hepatocyte growth factor/scatter factor in development, inflammation and carcinogenesis: its expression and role in oral tissues; Ohnishi T et al.; Hepatocyte growth factor (HGF) was discovered as a potent mitogen for adult hepatocytes from the plasma of patients with fulminant hepatic failure . It is now known to be a broad-spectrum, multi-functional mitogen, motogen and morphogen . The activities of HGF are mediated through the signalling pathway of its receptor, c-Met . During tooth development, HGF is expressed in the dental papilla and c-Met is expressed in the inner enamel epithelium . The expression of HGF and c-Met indicates that HGF is involved in morphogenesis of the tooth by mediating epithelial-mesenchymal interactions . In the mature tooth, HGF expression by fibroblasts is enhanced in pulpitis and mediated through the induction of prostaglandin (PG) E(2); it is induced not only by inflammatory cytokines, but also by components of oral bacteria . Consequently, concentrations of HGF in gingival crevicular fluid (GCF) increase in periodontitis . The mitogenic and other biological activities, such as angiogenesis, of HGF contribute towards wound healing . Both HGF and c-Met are expressed in the developing tongue, and the signalling pathway of the latter is shown to be essential for myogenesis . Dysregulation of c-Met signalling is observed in carcinogenesis, but HGF also has cytotoxic activity to certain tumour cells . The reason for the discrepancy between these observations is not clear at present.

Anal Biochem, 2003 Nov 15, 322(2), 179 - 84
Microscale synthesis of 2-tritiated isopropanol and 4R-tritiated reduced nicotinamide adenine dinucleotide phosphate; Agrawal N et al.; An improved microscale synthesis of 2-tritiated isopropanol ({2-3H}iPrOH) and R-tritiated reduced beta-nicotinamide adenine dinucleotide 2(')-phosphate (R-{4-3H}NADPH) is presented . The current procedure offers high yield, high purity, and small-quantity synthesis and is shorter than previous procedures . {2-3H}iPrOH was prepared by reduction of acetone with {3H}NaBH(4) under reflux conditions . {2-3H}iPrOH was used to reduce NADP(+) to R-{4-3H}NADPH with alcohol dehydrogenase from Thermoanaerobium brockii at 40 degrees C . This equilibrium reaction was drawn toward products by trapping the acetone with an excess of semicarbazide . This improvement enables a better control of the reaction time, as the enzymatic reduction became rate determining . Greater than 75% of the product was identified as reduced cofactor by reverse-phase (RP) HPLC . Longer reaction led to decomposition of the product and lower yield . Purification was carried out by semipreparative RP HPLC followed by lyophilization and yielded a compound of high purity that was preserved at -80 degrees C . Applications of the new procedure to a wide variety of specific radioactivities ranging from carrier-free to a few Ci/mmol are discussed . The intriguing formation of radioactive NADP(+) by-product (the major product in some reported procedures), was also studied and minimized in the procedure described below . Finally, the usage of the labeled cofactor is demonstrated with the enzymes dihydrofolate reductase and thymidylate synthase.

Biochemistry, 2003 Nov 11, 42(44), 13019 - 26
Membrane protein stability: high pressure effects on the structure and chromophore-binding properties of the light-harvesting complex LH2; Gall A et al.; Using the bacteriochlorophyll a (Bchl) cofactors as intrinsic probes to monitor changes in membrane protein structure, we investigate the response to high-pressure of the LH2 complexes from the photosynthetic bacteria Rhodobacter sphaeroides 2.4.1 and Rhodopseudomonas acidophila 10050 . By FT-Raman spectroscopy, we demonstrate that high pressure does not induce significant distortion of the protein-bound 850 nm-absorbing bacteriochlorophyll molecules, or break of the hydrogen bond they are involved in . This indicates in particular that the oligomerization of the polypeptides is not perturbed up to 0.6 GPa . The pressure-induced changes in the Bchl absorption spectra are attributed to pigment-pigment interactions . In contrast, the loss of 800 nm-absorbing bacteriochlorophyll reflects pressure-induced alterations to the tertiary structure of the protein in proximity to the membrane/cytosol interface . This suggests that the LH2 protein does have two independent structural domains . The first domain is pressure independent and comprises mostly the C-terminal domain . The second domain located on the N-terminal side exhibits sensitivity to pressure and pH reminiscent of soluble proteins . The LH2 thus constitutes a suitable model system for studying in detail the stability of membrane-embedded hydrophobic helices and helices located at or close to the solvent/membrane interface.

J Med Entomol, 2003 Sep, 40(5), 723 - 31
Detection and identification of pathogens and host DNA in unfed host-seeking Ixodes ricinus L . (Acari: Ixodidae); Pichon B et al.; In this study, we have developed molecular methods for the identification of reservoir hosts of sylvatic tick-borne zoonoses . The methods are based on the analysis of the blood meal remnant in the tick gut and include detection of pathogens and identification of the host origin of the blood meal . For host identification, a universal primer pair was used to amplify part of the vertebrate 18S rRNA gene followed by reverse line blot hybridization using subgroup-specific probes . Analyses of DNA from whole blood of vertebrates identified the correct subgroup of a broad range of vertebrate species (e.g., Ruminantia, Leporidea, Canidae, Murinae, Arvicolinae, Insectivora, Galliformes, Passeriformes) using probes based on the 18S rDNA sequences . Host DNA in the remnants of larval blood meals was detected in the gut of Ixodes ricinus nymphs maintained under natural conditions up to 9 mo after molting . For pathogen identification, a multiplex polymerase chain reaction was used that targeted parts of the 18S rRNA gene of piroplasm protozoa, the 16S rRNA gene of bacteria, and the intergenic spacer of the Borrelia burgdorferi genospecies complex . The utility of both methods was demonstrated under laboratory conditions by detecting Babesia microti (Franca) and gerbil DNA in 3-mo-old I . ricinus nymphs that had fed on B . microti-infected gerbils as larvae, and under field conditions by analyzing unfed ticks that were collected in a forest . The field study showed that the majority of ticks had fed on ruminants or birds and few on rodents, which is in accord with our knowledge of the fauna in this forest . Few pathogens were detected but the discovery of Borrelia valaisiana and B . burgdorferi s.s . in ticks that had fed on deer and Borrelia afzelii in a tick that had fed on a bird raises questions about the mode of transmission of these spirochetes and possibly about their host specificity.

Mem Inst Oswaldo Cruz, 2003 Sep, 98(6), 817 - 21 Epub 2003 Oct 29.
Prevalence of cagA and vacA genes in isolates from patients with Helicobacter pylori-associated gastroduodenal diseases in Recife, Pernambuco, Brazil; Brito CA et al.; Geographical differences in the prevalence of Helicobacter pylori genes and their association with disease severity have been identified . This study analyzes the prevalences of the cagA gene and alleles of the vacA gene in H . pylori-associated gastroduodenal diseases in isolates from Recife, PE, Brazil . Gastric biopsy of 61 H . pylori-positive patients were submitted to DNA extraction and gene amplification by polymerase chain reaction . Among the 61 patients, 21 suffered from duodenal ulcer (DU) and 40 from gastritis (GT) . The prevalence of H . pylori strains harbouring the cagA gene was higher in the DU group (90.5%) than in the GT group (60%) (p=0.02) . The vacA gene was amplified in 56 out of 61 biopsies, of which 43 (76.8%) contained bacteria carrying the s1 allele and 13 (23.2%) the s2 . However, the prevalence of the vacA s1 genotyping was the same in either DU or GT group . The majority of the s1-typed strains, 39 (90.7%) out of 43, were subtype s1b . In resume there was a strong association between the H . pylori cagA+ gene and DU . However, there were no differences between the DU and GT groups in relation to the vacA s1 and s2 alleles distribution, albeit the subtype s1b was predominant.

J Bacteriol, 2003 Nov, 185(22), 6548 - 55
Identification and characterization of a unique adenosine kinase from Mycobacterium tuberculosis; Long MC et al.; Adenosine kinase (AK) is a purine salvage enzyme that catalyzes the phosphorylation of adenosine to AMP . In Mycobacterium tuberculosis, AK can also catalyze the phosphorylation of the adenosine analog 2-methyladenosine (methyl-Ado), the first step in the metabolism of this compound to an active form . Purification of AK from M . tuberculosis yielded a 35-kDa protein that existed as a dimer in its native form . Adenosine (Ado) was preferred as a substrate at least 30-fold (Km = 0.8 +/- 0.08 microM) over other natural nucleosides, and substrate inhibition was observed when Ado concentrations exceeded 5 micro M . M . tuberculosis and human AKs exhibited different affinities for methyl-Ado, with Km values of 79 and 960 microM, respectively, indicating that differences exist between the substrate binding sites of these enzymes . ATP was a good phosphate donor (Km = 1100 +/- 140 microM); however, the activity levels observed with dGTP and GTP were 4.7 and 2.5 times the levels observed with ATP, respectively . M . tuberculosis AK activity was dependent on Mg2+, and activity was stimulated by potassium, as reflected by a decrease in the Km and an increase in Vmax for both Ado and methyl-Ado . The N-terminal amino acid sequence of the purified enzyme revealed complete identity with Rv2202c, a protein currently classified as a hypothetical sugar kinase . When an AK-deficient strain of M . tuberculosis (SRICK1) was transformed with this gene, it exhibited a 5,000-fold increase in AK activity compared to extracts from the original mutants . These results verified that the protein that we identified as AK was coded for by Rv2202c . AK is not commonly found in bacteria, and to the best of our knowledge, M . tuberculosis AK is the first bacterial AK to be characterized . The enzyme shows greater sequence homology with ribokinase and fructokinase than it does with other AKs . The multiple differences that exist between M . tuberculosis and human AKs may provide the molecular basis for the development of nucleoside analog compounds with selective activity against M . tuberculosis.

J Biol Chem, 2004 Jan 16, 279(3), 1787 - 93 Epub 2003 Oct 31.
A new function of the Desulfovibrio vulgaris Hildenborough {Fe} hydrogenase in the protection against oxidative stress; Fournier M et al.; Sulfate-reducing bacteria, like Desulfovibrio vulgaris Hildenborough, have developed a set of reactions allowing them to survive in oxic environments and even to reduce molecular oxygen to water . D . vulgaris contains a cytoplasmic superoxide reductase (SOR) and a periplasmic superoxide dismutase (SOD) involved in the elimination of superoxide anions . To assign the function of SOD, the periplasmic {Fe} hydrogenase activity was followed in both wild-type and sod deletant strains . This activity was lower in the strain lacking the SOD than in the wild-type when the cells were exposed to oxygen for a short time . The periplasmic SOD is thus involved in the protection of sensitive iron-sulfur-containing enzyme against superoxide-induced damages . Surprisingly, production of the periplasmic {Fe} hydrogenase was higher in the cells exposed to oxygen than in those kept in anaerobic conditions . A similar increase in the amount of {Fe} hydrogenase was observed when an increase in the redox potential was induced by addition of chromate . Viability of the strain lacking the gene encoding {Fe} hydrogenase after exposure to oxygen for 1 h was lower than that of the wild-type . These data reveal for the first time that production of the periplasmic {Fe} hydrogenase is up-regulated in response to an oxidative stress . A new function of the periplasmic {Fe} hydrogenase in the protective mechanisms of D . vulgaris Hildenborough toward an oxidative stress is proposed.

Mikrobiyol Bul, 2003 Apr-Jun, 37(2-3), 131 - 6
{Comparison of auramine-rhodamine and Erlich-Ziehl-Neelsen staining methods for the diagnosis of tuberculosis}; Tarhan G et al.; The aim of this study was to compare auramin-rhodamine (A-R) and Erlich-Ziehl-Neelsen (EZN) staining techniques for the diagnosis of tuberculosis . Of 311 sputum samples collected from active pulmonary tuberculosis patients and tuberculosis-suspected patients, 103 (33%) were found culture positive . In the direct microscopic examination of EZN stained smears, 86.4% (89/103) of culture positive samples, and 3.8% (8/208) of culture negative samples yielded asido-resistant bacteria, while these rates were 74.8% (77/103) and 11.5% (24/208) for A-R staining method . When culture was accepted as reference method, the specificity and sensitivity of the staining techniques were found as 88.5% and 74.8% for A-R, and 96.2% and 86.4% for EZN, respectively . As a result, it was concluded that, the use of A-R staining alone, could not be an alternative method to EZN staining.

J Infect Dis, 2003 Nov 1, 188(9), 1332 - 5 Epub 2003 Oct 15.
Nosocomial Mycobacterium bovis-bacille Calmette-Guérin infections due to contamination of chemotherapeutics: case finding and route of transmission; Vos MC et al.; We studied nosocomial infections due to Mycobacterium bovis bacille Calmette-Guerin (BCG) Onco-TICE bacteria, transmitted by contamination of medication prepared in BCG Onco-TICE-contaminated hoods in the pharmacy, in 5 immunocompromised patients at 3 hospitals . The BCG strains cultured from the patients had the same DNA profile as the BCG Onco-TICE strain used for bladder instillation . To prevent these infections, a change from open to closed preparation was made; strictly separated preparation in time of BCG Onco-TICE instillation and chemotherapy was enforced, the biological safety cabinet was disinfected between preparations, and gloves were changed between preparations.

FEMS Microbiol Lett, 2003 Oct 24, 227(2), 255 - 62
Repression by Fur is not the main mechanism controlling the iron-inducible isiAB operon in the cyanobacterium Synechocystis sp . PCC 6803; Kunert A et al.; The iron deficiency-dependent regulation of isiAB transcription in Synechocystis sp . PCC 6803 was analyzed by fusion of modified isiAB promoter fragments to gfp and in vivo quantification of Gfp fluorescence . For the putative Fur box only a slight repressing impact on promoter activity could be shown . In a heteroallelic fur mutant a corresponding incomplete repression of isiAB transcription under iron-replete conditions confirmed the role of Fur in isiAB regulation . However, a 90 bp region upstream of the putative -35 box of the isiAB promoter was essential for full promoter activity under iron-deplete conditions . This pattern indicates a dual promoter regulation by both a repressing mechanism exhibited via the Fur system and an unknown activating mechanism.

FEMS Microbiol Lett, 2003 Oct 24, 227(2), 197 - 202
The hydE gene is essential for the formation of Wolinella succinogenes NiFe-hydrogenase; Gross R et al.; Wolinella succinogenes grows by anaerobic respiration using hydrogen gas as electron donor . The hydE gene is located on the genome downstream of the structural genes encoding the membrane-bound NiFe-hydrogenase complex (HydABC) and a putative protease (HydD) possibly involved in hydrogenase maturation . Homologs of hydE are found in the vicinity of NiFe-hydrogenase-encoding genes on the genomes of several other proteobacteria . A hydE deletion mutant of W . succinogenes does not catalyze hydrogen oxidation with various electron acceptors . The hydrogenase iron-sulfur subunit HydA is absent in mutant cells whereas the apparently processed NiFe subunit (HydB) is located exclusively in the soluble cell fraction . It is suggested that HydE is involved in the maturation and/or stability of HydA or the HydAB complex in some, but not all bacteria containing NiFe-hydrogenases.

Bioorg Med Chem Lett, 2003 Nov 17, 13(22), 3897 - 900
Chemical synthesis of S-ribosyl-L-homocysteine and activity assay as a LuxS substrate; Zhao G et al.; Bacterial quorum sensing is mediated by autoinducers, small signaling molecules generated by bacteria . It has been proposed that the LuxS enzyme converts S-ribosyl-L-homocysteine to 4,5-dihydroxy-2,3-pentanedione, the precursor of autoinducer 2 (AI-2) . We report here a chemical synthesis of S-ribosyl-L-homocysteine and its analogue using Mitsunobu coupling . Chemically synthesized ribosylhomocysteine has been confirmed as a substrate for LuxS in both an enzyme assay and a whole cell quorum sensing assay . The chemical entities of products from the LuxS reaction were also established . Several ribosylhomocysteine analogues have been tested as LuxS inhibitors.

Environ Toxicol Chem, 2003 Nov, 22(11), 2554 - 60
Effects of ammonia on juvenile unionid mussels (Lampsilis cardium) in laboratory sediment toxicity tests; Newton TJ et al.; Ammonia is a relatively toxic compound generated in water and sediments by heterotrophic bacteria and accumulates in sediments and pore water . Recent data suggest that unionid mussels are sensitive to un-ionized ammonia (NH3) relative to other organisms . Existing sediment exposure systems are not suitable for ammonia toxicity studies with juvenile unionids; thus, we modified a system to expose juveniles to ammonia that was continuously infused into sediments . This system maintained consistent concentrations of ammonia in pore water up to 10 d . Juvenile Lampsilis cardium mussels were exposed to NH3 in pore water in replicate 96-h and 10-d sediment toxicity tests . The 96-h median lethal concentrations (LC50s) were 127 and 165 microg NH3-N/L, and the 10-d LC50s were 93 and 140 microg NH3-N/L . The median effective concentrations (EC50s) (based on the proportion affected, including dead and inactive mussels) were 73 and 119 microg NH3-N/L in the 96-h tests and 71 and 99 microg NH3-N/L in the 10-d tests . Growth rate was substantially reduced at concentrations between 31 and 76 microg NH3-N/L . The lethality results (when expressed as total ammonia) are about one-half the acute national water quality criteria for total ammonia, suggesting that existing criteria may not protect juvenile unionids.

Schweiz Rundsch Med Prax, 2003 Oct 8, 92(41), 1740 - 3
{Liver abscess: a practical approach}; Baudat V et al.; The management of a liver abscess suspected on the basis of clinical and radiological findings is radically different depending on its amoebic or pyogenic etiology . Medical management is usually enough to treat amoebic abscess, the prognosis of which is excellent while percutaneous aspiration puncture, drainage and antibiotics is the rule in pyogenic abscess, the prognosis of which depends on the quickness of diagnosis and risk factors associated . This article first relates a case of liver abscess we had in our service and then propose, on the basis of a literature review, a synthesis of the different characteristics, diagnostic and therapeutic approaches and follow-up of amoebic and pyogenic liver abscesses.

Biochem Soc Symp, 2003, (70), 15 - 30
Papain-like lysosomal cysteine proteases and their inhibitors: drug discovery targets?
Turk D, Turk B, Turk V.
Papain-like lysosomal cysteine proteases are processive and digestive enzymes that are expressed in organisms from bacteria to humans . Increasing knowledge about the physiological and pathological roles of cysteine proteases is bringing them into the focus of drug discovery research . These proteases have rather short active-site clefts, comprising three well defined substrate-binding subsites (S2, S1 and S1') and additional broad binding areas (S4, S3, S2' and S3') . The geometry of the active site distinguishes cysteine proteases from other protease classes, such as serine and aspartic proteases, which have six and eight substrate-binding sites respectively . Exopeptidases (cathepsins B, C, H and X), in contrast with endopeptidases (such as cathepsins L, S, V and F), possess structural features that facilitate the binding of N- and C-terminal groups of substrates into the active-site cleft . Other than a clear preference for free chain termini in the case of exopeptidases, the substrate-binding sites exhibit no strict specificities . Instead, their subsite preferences arise more from the specific exclusion of substrate types . This presents a challenge for the design of inhibitors to target a specific cathepsin: only the cumulative effect of an assembly of inhibitor fragments will bring the desired result.

Expert Rev Mol Med, 2000 Apr 19, 2000, 1 - 24
Strategies for cancer therapy using carcinoembryonic antigen vaccines; Horig H et al.; Advances in molecular biology and immunology have renewed interest in the development of vaccines for the treatment or prevention of cancer . Research over the past 10 years has focused on the identification of suitable tumour antigens to use as targets for a variety of vaccine strategies . Carcinoembryonic antigen (CEA) was one of the first tumour antigens described, and is commonly expressed by a wide range of adenocarcinomas . Recent studies have identified several human-leukocyte-antigen-restricted epitopes (short peptides) within the CEA protein that can be recognised by human T lymphocytes (T cells) . Although CEA-expressing tumour cells are generally weakly recognised by the immune system, several new strategies have been used to enhance immune responses against CEA . This includes using antibodies directed against CEA; inserting the CEA gene into recombinant viruses and bacteria as viral and bacterial vaccines; pulsing the CEA protein, peptides, DNA or RNA onto dendritic cells (specialised antigen-presenting cells); and combining CEA vaccines with cytokines or co-stimulatory molecules to increase vaccine effectiveness . Other factors that might be important in establishing systemic immunity against CEA are the dose, route, timing, and choice of vector and adjuvants for vaccine administration . Further research in understanding the fundamental processes involved in tumour-cell recognition by the immune system, better animal models, and improved clinical trial designs will help to define the full potential of CEA as a target for cancer vaccine development.

J Am Vet Med Assoc, 2003 Oct 15, 223(8), 1142 - 50
Clinicopathologic and radiographic features and etiologic agents in cats with histologically confirmed infectious pneumonia: 39 cases (1991-2000); Macdonald ES et al.; OBJECTIVE: To determine clinicopathologic and radiographic features and etiologic agents in cats that died as a result of infectious pneumonia . DESIGN: Retrospective study . ANIMALS: 39 cats . PROCEDURE: Medical records of cats in which infectious pneumonia was confirmed by histologic examination of necropsy specimens were reviewed . Signalment, clinical signs, and results of a CBC, viral serologic tests, and thoracic radiography were evaluated . Infectious agents were classified as bacterial, viral, fungal, protozoal, or parasitic . Histologic features (severity, duration, anatomic location, and distribution) were analyzed . RESULTS: Clinical signs referable to the respiratory tract were not detected in 14 of 39 (36%) cats, and results of a CBC (4/18 cats) and radiography (3/13) were unremarkable . Sixteen of 39 (41%) cats lacked clinical signs of systemic illness . Etiologic agents identified included bacteria (n = 21), viruses (11), fungi (6), protozoa (2), and parasites (1) . Cats with clinical signs related to the respiratory tract (19/24 {79%} cats) were more likely to have severe histologic changes than cats without signs related to the respiratory system (6/14) . Twenty-nine of 38 (76%) cats had histologic evidence of systemic disease, whereas the remaining cats had lesions limited to the respiratory tract . CONCLUSIONS AND CLINICAL RELEVANCE: Infectious pneumonia is uncommon in cats . Cats with infectious pneumonia may lack clinical signs and have unremarkable results for a CBC and thoracic radiography, yet frequently have systemic infections . Therefore, clinicians should maintain an index of suspicion for pneumonia and evaluate the respiratory tract when infection is detected in other organ systems.

Microb Ecol . 2003 Oct 31; {Epub ahead of print}
Impact of Clay Minerals on Sulfate-Reducing Activity in Aquifers; Wong D et al.; Previous studies have shown that sulfate-reduction activity occurs in a heterogeneous manner throughout the terrestrial subsurface . Low-activity regions are often observed in the presence of clay minerals . Here we report that clays inhibit sulfate reduction activity in sediments and in a pure culture of Desulfovibrio vulgaris . Clay minerals including bentonite and kaolinite inhibited sulfate reduction by 70-90% in sediments . Intact clays and clay colloids or soluble components, capable of passing through a 0.2- micro m filter, were also inhibitory to sulfate-reducing bacteria . Other adsorbent materials, including anion or cation exchangers and a zeolite, did not inhibit sulfate reduction in sediments, suggesting that the effect of clays was not due to their cation-exchange capacity . We observed a strong correlation between the Al(2)O(3) content of clays and their relative ability to inhibit sulfate reduction in sediments ( r(2) = 0.82) . This suggested that inhibition might be a direct effect of Al(3+) (aq) on the bacteria . We then tested pure aluminum oxide (Al(2)O(3)) and showed it to act in a similar manner to clay . As dissolved aluminum is known to be toxic to a variety of organisms at low concentrations, our results suggest that the effects of clay on sulfate-reducing bacteria may be directly due to aluminum . Thus, our experiments provide an explanation for the lack of sulfate-reduction activity in clay-rich regions and presents a mechanism for the effect.

Viral Immunol, 2003, 16(3), 369 - 79
Measles virus infection of rhesus macaques affects neutrophil expression of IL-12 and IL-10; Hoffman SJ et al.; Cytokine production by phagocytic cells is an important component of the immune response to infection with a variety of pathogens . Neutrophils are phagocytic cells capable of expressing interleukin (IL)-12 and IL-10 in response to infection with parasites, fungi, and bacteria . These cytokines are postulated to play important roles in the immune response during measles . Neutrophils isolated from naive or measles virus (MV)-infected rhesus macaques expressed IL-12 and IL-10 following in vitro stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) . Stimulated neutrophils secreted proportionally more of the biologically active IL-12 heterodimer p70 and more IL-10 than similarly stimulated peripheral blood mononuclear cells (PBMCs) . During MV infection, the number of IL-12 and IL-10-producing neutrophils and monocytes initially decreased . Subsequently, IL-12 levels were restored, and IL-10 expression rose above baseline in both cell types . Increased IL-10 production by neutrophils and monocytes during MV infection may play a role in measles-induced immunosuppression.

Microb Ecol, 2003 Jul, 46(1), 33 - 42
Exploring bacteriplankton growth and protein synthesis to determine conversion factors across a gradient of dissolved organic matter; Pulido-Villena E et al.; The effect of bacterial specific growth rates of abundance (micro) and protein synthesis (b) on conversion factor (CF) variability was explored in order to provide an alternative approach to the controversial application of just one universal CF to field data . Nine regrowth cultures (RCs) were set up from very diverse aquatic ecosystems, controlling temperature and adding N and P to avoid mineral limitation and force organic carbon limitation . The values of micro varied one order of magnitude from 0.26 to 3.34 d(-1), whereas b values varied two orders of magnitude from 0.28 to 34.87 d(-1) . We found no relationships between micro or b values and the dissolved organic carbon (DOC) concentration or the dissolved organic matter (DOM) quality indexes assayed . Abundance and protein synthesis increased exponentially and synchronously in four RCs, leading to balanced growth (micro = b) . In contrast, abundance and protein synthesis increased logistically in the other five RCs and b values were significantly higher than g values, leading to unbalanced growth (micro not equal b) . CFs ranged from 0.0062 to 0.0576 x 10(18) cells mol leucine(-1) with an average of 0.0305 x 10(18) cells mol leucine(-1) . CFs obtained in RCs with balanced growth were generally higher than CFs obtained in RCs with unbalanced growth and were not alike, impeding the establishment of an upper limit for CFs . A positive and significant relationship (n = 8, p < 0.0 1, r2 = 0.71) was found between CFs and DOC concentration (CF (x10(18) cells mol leucine(-1)) = 0.0104 + 0.0094 DOC (mM)) when the value for the most productive system was excluded . This function permits the estimation of site-specific CFs based on DOC concentration instead of the controversial use of a single CF for different systems.

Ostomy Wound Manage, 2003 Sep, 49(9), 34 - 42
Preparing the wound for healing: the effect of activated polyacrylate dressing on debridement; Paustian C et al.; Activated polyacrylate dressings facilitate wound debridement by retaining moisture while attracting and retaining proteins and bacteria . A 55-patient retrospective study was conducted to quantify the effect of this dressing on debridement of chronic wounds in clinical practice . All patients attended one of four outpatient wound clinics between June 1, 2001 and February 10, 2002 and received treatment with the polyacrylate dressing for an average of 3.9 weeks (SD 4.1) . During that time, the rate of wound necrotic tissue debridement was 37.7% per week . Older patients (>80 years of age) had significantly lower rates of wound debridement (mean 18.1% per week) than those <51 years of age (mean 36% per week, P = 0.009) . Other variables (age, wound type, wound duration and diagnosis of diabetes) were not found to significantly affect the rate of wound debridement . Wound debridement rates of commonly available modalities are largely unknown . However, these results suggest that activated polyacrylate dressings are an effective, atraumatic, and easy-to-use method of debriding chronic wounds.

Biophys J, 2003 Nov, 85(5), 3173 - 86
Exciton theory for supramolecular chlorosomal aggregates: 1 . Aggregate size dependence of the linear spectra; Prokhorenko VI et al.; The interior of chlorosomes of green bacteria forms an unusual antenna system organized without proteins . The steady-spectra (absorption, circular dichroism, and linear dichroism) have been modeled using the Frenkel Hamiltonian for the large tubular aggregates of bacteriochlorophylls with geometries corresponding to those proposed for Chloroflexus aurantiacus and Chlorobium tepidum chlorosomes . For the Cf . aurantiacus aggregates we apply a structure used previously (V . I . Prokhorenko., D . B . Steensgaard, and A . R . Holzwarth, Biophys: J . 2000, 79:2105-2120), whereas for the Cb . tepidum aggregates a new extended model of double-tube aggregates, based on recently published solid-state nuclear magnetic resonance studies (B.-J . van Rossum, B . Y . van Duhl, D . B . Steensgaard, T . S . Balaban, A . R . Holzwarth, K . Schaffner, and H . J . M . de Groot, Biochemistry 2001, 40:1587-1595), is developed . We find that the circular dichroism spectra depend strongly on the aggregate length for both types of chlorosomes . Their shape changes from "type-II" (negative at short wavelengths to positive at long wavelengths) to the "mixed-type" (negative-positive-negative) in the nomenclature proposed in K . Griebenow, A . R . Holzwarth, F . van Mourik, and R . van Grondelle, Biochim: Biophys . Acta 1991, 1058:194-202, for an aggregate length of 30-40 bacteriochlorophyll molecules per stack . This "size effect" on the circular dichroism spectra is caused by appearance of macroscopic chirality due to circular distribution of the transition dipole moment of the monomers . We visualize these distributions, and also the corresponding Frenkel excitons, using a novel presentation technique . The observed size effects provide a key to explain many previously puzzling and seemingly contradictory experimental data in the literature on the circular and linear dichroism spectra of seemingly identical types of chlorosomes.

Chem Phys Lipids, 2003 Nov, 126(1), 49 - 53
Facile synthesis of (Z)-tetracos-5-enoic acid and racemic cis-4-(2-octadecylcyclopropane-1-yl)-butanoic acid; Coxon GD et al.; (Z)-tetracos-5-enoic acid and racemic cis-4-(2-octadecylcyclopropane-1-yl)-butanoic acid have been prepared from 1-eicosene by a new facile route . Periodic acid cleavage of the epoxide of 1-eicosene gave nonadecanal which was condensed with 4-carboxybutyltriphenylphosphonium bromide to give predominately (Z)-tetracos-5-enoic acid . Simmons-Smith type cyclopropanation of (Z)-tetracos-5-enoic acid gave a minor proportion of racemic cis-4-(2-octadecylcyclopropane-1-yl)-butanoic acid accompanied by major amounts of its methyl ester.

Curr Opin Biotechnol, 2003 Oct, 14(5), 484 - 90
Enabling inverse metabolic engineering through genomics; Gill RT; Inverse metabolic engineering (IME) is a powerful framework for engineering cellular phenotypes . Progress in this field has been limited by a lack of comprehensive methods for efficiently identifying the genetic basis of relevant phenotypes . Advances in genomics technologies, including DNA microarrays and gene sequencing, have dramatically improved our ability to relate changes in phenotype with associated changes in genotype . When applied in the context of IME, these tools should enable the integration of "evolutionary" and "direct" approaches to engineering cell physiology, which should improve our understanding of the complex interactions affecting the expression, evolution and engineering of traits in natural and industrial hosts.

Curr Opin Chem Biol, 2003 Oct, 7(5), 557 - 62
Mechanisms of protein fluorophore formation and engineering; Miyawaki A et al.; Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and GFP-like proteins from Anthozoa species contain light-absorbing chromophores within their protein sequences . Recent studies have made progress in obtaining bright variants of these proteins that develop chromophores quickly and efficiently, as well as novel fluorescent proteins that photoconvert (i.e . change color upon illumination at specific wavelengths) . Further molecular characterization of the structure and maturation of these proteins is in progress, aimed at providing information for rational design of variants with desired fluorescence properties.

Mol Cell, 2003 Oct, 12(4), 1003 - 13
RNA polymerase II/TFIIF structure and conserved organization of the initiation complex; Chung WH et al.; The structure of an RNA polymerase II/general transcription factor TFIIF complex was determined by cryo-electron microscopy and single particle analysis . Density due to TFIIF was not concentrated in one area but rather was widely distributed across the surface of the polymerase . The largest subunit of TFIIF interacted with the dissociable Rpb4/Rpb7 polymerase subunit complex and with the mobile "clamp." The distribution of the second largest subunit of TFIIF was very similar to that previously reported for the sigma subunit in the bacterial RNA polymerase holoenzyme, consisting of a series of globular domains extending along the polymerase active site cleft . This result indicates that the second TFIIF subunit is a true structural homolog of the bacterial sigma factor and reveals an important similarity of the transcription initiation mechanism between bacteria and eukaryotes . The structure of the RNAPII/TFIIF complex suggests a model for the organization of a minimal transcription initiation complex.

Exp Appl Acarol, 2003, 29(1-2), 1 - 12
No variation for Wolbachia-induced hybrid breakdown in two populations of a spider mite; Vala F et al.; Wolbachia are cytoplasmically transmitted bacteria that infect several species of mites . In the two-spotted spider mite Tetranychus urticae Koch this symbiont can induce reproductive incompatibility . Wolbachia-induced reproductive incompatibility is observed in crosses between Wolbachia-infected (W) males and uninfected (U) females . This incompatibility is expressed in F1 broods as male-biased sex ratios, an effect called cytoplasmic incompatibility (CI) . However, in the two-spotted spider mite, Wolbachia-induced reproductive incompatibility may extend to the F2: broods of virgin F1 females from U x W crosses sometimes suffer increased mortality rates . This F2 effect is called hybrid breakdown (HB) . Several isofemale lines derived from mites collected from rose and cucumber plants had been previously tested for CI . Here we report on the results obtained for HB.

Chromosoma, 2003 Oct, 112(3), 133 - 43 Epub 2003 Sep 17.
The ThioredoxinT and deadhead gene pair encode testis- and ovary-specific thioredoxins in Drosophila melanogaster; Svensson MJ et al.; So far, two thioredoxin proteins, DHD and Trx-2, have been biochemically characterized in Drosophila melanogaster . Here, with the cloning and characterization of TrxT we describe an additional thioredoxin with testis-specific expression . TrxT and dhd are arranged as a gene pair, transcribed in opposite directions and sharing a 471 bp regulatory region . We show that this regulatory region is sufficient for correct expression of the two genes . This gene pair makes a good model for unraveling how closely spaced promoters are differentially regulated by a short common control region . Both TrxT and DHD proteins are localized within the nuclei in testes and ovaries, respectively . Use of a transgenic construct expressing TrxT fused to Enhanced Yellow Fluorescent Protein reveals a clear association of TrxT with the Y chromosome lampbrush loops ks-1 and kl-5 in primary spermatocytes . The association is lost in the absence of the Y chromosome . Our results suggest that nuclear thioredoxins may have regulatory functions in the germline.

J Dent Res, 2003 Nov, 82(11), 870 - 6
Proteome analysis of oral pathogens; Macarthur DJ et al.; The oral environment contains diverse communities of micro-organisms including bacteria, fungi, protozoa, and viruses . Studies of oral ecology have led to an appreciation of the complexity of the interactions that oral micro-organisms have with the host in both health and disease . Despite this, diseases such as dental caries and periodontal diseases are still worldwide human ailments, resulting in a high level of morbidity and an economic burden to society . Proteomics offers a new approach to the understanding of holistic changes occurring as oral micro-organisms adapt to environmental change within their habitats in the mouth.

J Am Acad Dermatol, 2003 Nov, 49(5 Suppl), S240 - 3
Late-onset Papillon-Lefèvre syndrome without alteration of the cathepsin C gene; Pilger U et al.; Mutations in the cathepsin C gene have recently been detected in Papillon-Lefevre syndrome (PLS) . Until now, 5 cases with the late-onset variation of this disease have been reported in the literature . The genetic background of this type of PLS is still unknown . We describe a 46-year-old woman with late-onset transgredient palmar hyperkeratosis and a 10-year history of severe periodontal disease . Histology of skin biopsy specimens revealed a psoriasiform pattern . Dental examination showed severe gingival inflammation with loss of alveolar bone . Dental plaque investigated by a polymerase chain reaction method revealed DNA signals of 5 different dental bacteria . DNA from EDTA blood was investigated for mutations in the cathepsin C gene by polymerase chain reaction analysis and direct sequencing . A silent variation in the codon for proline-459 was detected but interpreted as a polymorphism of this gene . All genetic linkage and mutation studies for PLS performed so far have shown that PLS is genetically homogeneous . Our patient with late-onset variation of PLS, however, did not show a mutation in the cathepsin C gene . Thus, we suspect that there is another genetic cause for the late-onset forms of PLS.

Presse Med, 2003 Oct 18, 32(34), 1618 - 21
{Q fever, a potential biowarfare agent}; Bossi P et al.; SEVERAL POSSIBLE METHODS OF TRANSFUSION: Q fever is a zoonosis due to Coxiella burnetii . Its interest as a potential biowarfare agent is in its possible transmission by inhalation of sprayed particles . This form of transmission would probably be used: the inhalation of 1 to 10 bacteria could provoke the development of an infection in humans . Another possible method of transmission in the context of a terrorist act would be the intentional introduction of the bacteria into foodstuff . A DISABLING WEAPON: However, C . burnetii has never been used as a biological weapon . The probability that this germ could be used is very low: indeed, the incubation of Q fever is very long, and the majority of the infections is asymptomatic and mortality is low . In fact C . burnetii would more likely be used as a disabling weapon.

Cornea, 2003 Nov, 22(8), 740 - 5
Clinical appearance and microscopic analysis of mucin balls associated with contact lens wear; Millar TJ et al.; PURPOSE: The structure of mucin balls collected from silicone hydrogel contact lens wearers was examined to determine their nature . METHODS: Tears containing mucin balls were collected using a capillary tube . These were processed for light microscopic histochemistry, scanning electron microscopy, and electron microscopic elemental analysis . Mucin balls were also observed in vivo using confocal microscopy . RESULTS: Histology showed that the mucin balls were PAS positive, indicating that glycoproteins form a major component . Lipids and bacteria were not detected . Scanning electron microscopy did not show the surface to be smooth but revealed a variation in density across the surface . Elemental analysis was inconclusive . CONCLUSIONS: Mucin balls are likely to be made from collapsed mucin and are unlikely to have been formed as a result of pearling around a silicon, lipid, or bacterial kernel.

Lancet, 2003 Oct 18, 362(9392), 1285 - 6
Solar disinfection of infectious biomedical waste: a new approach for developing countries; Chitnis V et al.; Poor developing countries cannot afford expensive technologies such as incineration for management of infectious biomedical waste . We assessed solar heating as an alternative technology . We immersed simulated infectious waste with added challenge bacteria in water in a box-type solar cooker, which was left in the sun for 6 h . In 24 sets of observations, the amount of viable bacteria was reduced by about 7 log . We also tested infectious medical waste with a heavy load of bacteria (10(8)-10(9)/g) from our hospital's burn unit for solar heat disinfection in 20 experiments . Our results showed a similar 7 log reduction in the amount of viable bacteria . Solar heating thus seems to be a cheap method to disinfect infectious medical waste in less economically developed countries.

J Fish Dis, 2003 Sep, 26(9), 499 - 509
Melano-macrophage centres and their role in fish pathology; Agius C et al.; Melano-macrophage centres, also known as macrophage aggregates, are distinctive groupings of pigment-containing cells within the tissues of heterothermic vertebrates . In fish they are normally located in the stroma of the haemopoietic tissue of the spleen and the kidney, although in amphibians and reptiles, and some fish, they are also found in the liver . They may also develop in association with chronic inflammatory lesions elsewhere in the body and during ovarian atresia . In higher teleosts, they often exist as complex discrete centres, containing lymphocytes and macrophages, and may be primitive analogues of the germinal centres of lymph nodes . Melano-macrophage centres usually contain a variety of pigments, including melanins, and these increase in range and volume in older fish or in the presence of cachectic disease . Melano-macrophage centres act as focal depositories for resistant intracellular bacteria, from which chronic infections may develop . Iron capture and storage in haemolytic diseases appears to be a primary function, but antigen trapping and presentation to lymphocytes, sequestration of products of cellular degradation and potentially toxic tissue materials, such as melanins, free radicals and catabolic breakdown products are among other functions that have been ascribed . Recent work suggests that they are a site of primary melanogenesis rather than mere storage . Melano-macrophage centres increase in size or frequency in conditions of environmental stress and have been suggested as reliable biomarkers for water quality in terms of both deoxygenation and iatragenic chemical pollution.

Parasitol Res, 2003 Oct, 91(4), 265 - 6 Epub 2003 Aug 16.
Endoparasite KC5/2 encloses large areas of sol-like cytoplasm within Acanthamoebae . Normal behavior or aberration?
Michel R, Schmid EN, Hoffmann R, Muller KD.
In addition to the characteristic development of the rod-shaped or tube-like endocytobionts replicating within the sol-like cytoplasm of acanthamoebae which was published recently (Michel et al . 2003) we observed the strange behavior of the endoparasites described as follows . The material normally forming the characteristic cell walls of KC5/2 organisms was found to form a long array along the borderline between the granuloplasm and the central homogenous sol-like cytoplasm, so that about one third of the sol plasm had already been surrounded . We do not know whether this process belongs to the normal developmental repertoire of the endocytobiont that was interrupted by fixation for electron microscopy, or whether it is an aberrant behavior.

Protein Sci, 2003 Nov, 12(11), 2523 - 41
Allosteric transition pathways in the lactose repressor protein core domains: asymmetric motions in a homodimer; Flynn TC et al.; The crystal structures of lactose repressor protein (LacI) provide static endpoint views of the allosteric transition between DNA- and IPTG-bound states . To obtain an atom-by-atom description of the pathway between these two conformations, motions were simulated with targeted molecular dynamics (TMD) . Strikingly, this homodimer exhibited asymmetric dynamics . All asymmetries observed in this simulation are reproducible and can begin on either of the two monomers . Asymmetry in the simulation originates around D149 and was traced back to the pre-TMD equilibrations of both conformations . In particular, hydrogen bonds between D149 and S193 adopt a variety of configurations during repetitions of this process . Changes in this region propagate through the structure via noncovalent interactions of three interconnected pathways . The changes of pathway 1 occur first on one monomer . Alterations move from the inducer-binding pocket, through the N-subdomain beta-sheet, to a hydrophobic cluster at the top of this region and then to the same cluster on the second monomer . These motions result in changes at (1) side chains that form an interface with the DNA-binding domains and (2) K84 and K84', which participate in the monomer-monomer interface . Pathway 2 reflects consequent reorganization across this subunit interface, most notably formation of a H74-H74rsquo; pi-stacking intermediate . Pathway 3 extends from the rear of the inducer-binding pocket, across a hydrogen-bond network at the bottom of the pocket, and transverses the monomer-monomer interface via changes in H74 and H74rsquo; . In general, intermediates detected in this study are not apparent in the crystal structures . Observations from the simulations are in good agreement with biochemical data and provide a spatial and sequential framework for interpreting existing genetic data.

Infect Immun, 2003 Nov, 71(11), 6256 - 63
Identification of a gene that affects the efficiency of host cell infection by Legionella pneumophila in a temperature-dependent fashion; Ridenour DA et al.; The ability to infect host cells is critical for the survival and replication of intracellular pathogens in humans . We previously found that many genes involved in the ability of Legionella pneumophila to infect macrophages are not expressed efficiently under standard laboratory growth conditions . We have developed an approach using expression of L . pneumophila genes from an exogenous constitutive promoter on a low-copy-number vector that allows identification of genes involved in host cell infection . Through the use of this strategy, we found that expression of a gene, lvhB2, enhances the efficiency of L . pneumophila infection of mammalian cells . The putative protein encoded by lvhB2 has similarity to structural pilin subunits of type IV secretion systems . We confirmed that this gene plays a role in host cell infection by the construction of an in-frame deletion in the L . pneumophila lvhB2 gene and complementation of this mutant with the wild-type gene . The lvhB2 mutant does not display a very obvious defect in interactions with host cells when the bacteria are grown at 37 degrees C, but it has an approximately 100-fold effect on entry and intracellular replication when grown at 30 degrees C . These data suggest that lvhB2 plays an important role in the efficiency of host cell infection by L . pneumophila grown at lower temperatures.

J Biol Chem, 2004 Jan 2, 279(1), 245 - 50 Epub 2003 Oct 22.
Helicobacter pylori heat shock protein 60 mediates interleukin-6 production by macrophages via a toll-like receptor (TLR)-2-, TLR-4-, and myeloid differentiation factor 88-independent mechanism; Gobert AP et al.; Helicobacter pylori has been reported to induce interleukin-6 (IL-6) production in monocytes/macrophages and in chronically inflamed gastric tissues . The mechanism by which H . pylori induces IL-6 production in macrophages, however, has not been investigated . To identify the H . pylori factor responsible for this activity, we fractionated soluble proteins from H . pylori strain 26695 by ion exchange and size exclusion chromatography and screened the fractions for IL-6-inducing activity on RAW 264.7 macrophages . A single protein was purified and identified by mass spectrometry as H . pylori heat shock protein 60 (HSP60) . Consistent with the observed IL-6-inducing activity of H . pylori HSP60, soluble protein extracts of H . pylori 26695 and SS1 strains that were depleted of this protein by affinity chromatography had dramatically reduced IL-6-inducing activities . The immunopurified HSP60 stimulated IL-6 production in macrophages . When stimulated with H . pylori HSP60 or intact bacteria, peritoneal macrophages from mice deficient in Toll-like receptor (TLR)-2, TLR-4, TLR-2/TLR-4, and myeloid differentiation factor 88 produced the same amount of IL-6 than macrophages from wild-type mice, demonstrating the independence of H . pylori HSP60 responses from these signaling molecules . H . pylori HSP60-induced IL-6 mRNA expression, and NF-kappaB activation in RAW 264.7 cells was abrogated in the presence of MG-132, a proteasome inhibitor . In contrast, inhibitors of protein kinase A or C, mitogen-activated protein kinase kinase, and phosphoinositide 3-kinase had no effect on IL-6 mRNA levels . This study demonstrates the induction of innate immune responses by H . pylori HSP60, thereby implicating this highly conserved protein in the pathophysiology of chronic gastritis.

Life Sci, 2003 Nov 14, 73(26), 3313 - 21
Vitamin D receptor gene polymorphism is associated with chronic periodontitis; Tachi Y et al.; Chronic periodontitis (CP) is caused by enhanced resorption of the alveolar bone supporting the teeth and is associated with intraoral inflammation after infection with certain bacteria . The VDR gene polymorphism was reported recently to be deeply related to the occurrence of tuberculosis and infection of chronic hepatitis B virus . This may be interpreted to indicate a close relationship between VDR gene polymorphism and the immunological action, because vitamin D activates monocytes, stimulates cell-mediated immunity, and suppresses lymphocyte proliferation . The purpose of the present study was to clarify whether polymorphisms in VDR gene exons are associated with the incidence of CP . A case-controlled study was performed on a group of 168 unrelated Japanese subjects whose ages ranged from 35 to 65 years . The Taq I polymorphism in the VDR gene was found to be associated significantly with CP (X2=4.48, P=0.034) . We performed multiple logistic regression analyses on the TT genotype, which was found to be associated with CP, and on well-recognized risk factors, smoking and diabetes . The odds ratio (OR) for the genotype (TT/Tt) was 2.73 (95% CI 1.11-6.68, P=0.028), being larger than the unadjusted value . This indicates that the VDR gene polymorphism (TT genotype) is a risk factor for CP, independently of smoking and diabetes.

Am J Gastroenterol, 2003 Oct, 98(10), 2245 - 52
Lactulose/mannitol test has high efficacy for excluding organic causes of chronic diarrhea; Di Leo V et al.; OBJECTIVES: Diagnosis in chronic diarrhea in the absence of a distinctive clinical pattern is often challenging, as biochemical tests prescribed at the first evaluation do not show enough sensitivity and specificity to tailor further investigation . Intestinal permeability to sugars is an accurate test for detecting intestinal damage . The aim of this study was to evaluate the diagnostic value of the lactulose/mannitol (L/M) test in patients with chronic diarrhea . METHODS: We conducted a prospective cohort study to evaluate the diagnostic value of the L/M test in chronic diarrhea . The test was administered to 261 consecutive patients presenting with three or more bowel movements daily for at least 3 wk . Biochemical tests including complete blood cell count, acute phase reactive proteins, serum albumin and iron, and stool cultures for bacteria, ova, and parasites were assessed at the same time . Additional diagnostic investigations were directed by clinical features as well as first-line test results . RESULTS: Over 3 yr, 120 (46%) of our patients were found to have an organic cause for chronic diarrhea, whereas in 141 (54%) a functional condition was diagnosed . Multivariate logistic regression analysis revealed that the L/M test and C-reactive protein were independent predictors for the final diagnosis of organic cause of chronic diarrhea, with odds ratios of 1.5 (95% CI = 1.29-1.78) and 5.2 (95% CI = 1.90-14.12), respectively . The area under the receiver operating characteristic (ROC) curve of the adjusted model was 0.82, with positive predictive value of 80.4% and negative predictive value of 77.7% . CONCLUSIONS: The L/M test is a powerful tool for workup in patients with chronic diarrhea . Introducing the L/M test as first-level test effectively improves the selection of patients who need further evaluation.

Curr Opin Microbiol, 2003 Oct, 6(5), 472 - 7
Treatment of infectious diseases with immunostimulatory oligodeoxynucleotides containing CpG motifs; Dittmer U et al.; Bacterial DNA with CpG motifs can efficiently stimulate the vertebrate immune system . Thus, synthetic oligodeoxynucleotides that contain such CpG motifs (CpG-ODN) are currently used in preclinical and clinical studies to develop new allergy or cancer therapies and vaccine adjuvants . Recent animal studies indicate that CpG-ODN therapies can also be used for successful treatment of infections caused by bacteria, parasites or viruses . In these experiments, innate and adaptive immune responses against pathogens were augmented by CpG-ODN and subsequently induced resistance against infectious diseases . The stimulation of dendritic cells played a central role for the therapeutic effect of CpG-ODN . However, CpG-ODN can also have negative side effects, which accelerate disease progression in some viral infections . Clinical studies with CpG-ODN will determine their potential for the therapy of infectious diseases in humans.

J Food Prot, 2003 Oct, 66(10), 1924 - 6
High hydrostatic pressure treatment of finfish to inactivate Anisakis simplex; Dong FM et al.; High hydrostatic pressure has been demonstrated to be a useful technique for treating food to reduce the number of pathogenic organisms and to extend shelf life . Most research in this area has focused on bacteria . However, a concern in the sashimi (raw fish) industry is that nematode worms such as Anisakis simplex occur naturally in cold-water marine fish . The objectives of this research were to perform a pilot study to determine the effect of high hydrostatic pressure on the viability of Anisakis simplex larvae, commonly found in king salmon and arrowtooth flounder, and to evaluate the effects of high hydrostatic pressure on the color and texture of the fish fillets . Pieces of fish (ca . 100 g per bag) containing 13 to 118 larvae were exposed to pressures of up to 80,000 lb/in2 (552 MPa) for up to 180 s . The times and pressures required to kill 100% of the larvae were as follows: 30 to 60 s at 60,000 lb/in2 (414 MPa), 90 to 180 s at 40,000 lb/in2 (276 MPa), and 180 s at 30,000 lb/in2 (207 MPa) . For all salmon treatments that killed 100% of the larvae, a significant increase in the whiteness of the flesh was observed . Although high hydrostatic pressure was effective in killing A . simplex larvae in raw fish fillets, its significant effect on the color and overall appearance of the fillet may limit its application to the processing of fish for raw-fish markets.

Environ Sci Technol, 2003 Oct 1, 37(19), 4457 - 62
A temperature responsive biopolymer for mercury remediation; Kostal J et al.; Tunable biopolymers based on elastin-like polypeptides (ELP) were engineered for the selective removal of mercury . ELP undergoes a reversible thermal precipitation within a wide range of temperatures and was exploited to enable easy recovery of the sequestered mercury . A bacterial metalloregulatory protein, MerR, which binds mercury with an unusually high affinity and selectivity, was fused to the ELP to provide the highly selective nature of the biopolymers . Selective binding of mercury was demonstrated at an expected ratio of 0.5 mercury/biopolymer, and minimal binding of competing heavy metals (cadmium, nickel, and zinc), even at 100-fold excess, was observed . The sequestered mercury was extracted easily, enabling continuous reuse of the biopolymers . In repeating cycles, mercury concentration was reduced to ppb levels, satisfying even drinking water limits . Utility of the biopolymers with mercury-contaminated Lake Elsinore water was demonstrated with no decrease in efficiency . The nanoscale biopolymers reported here using metalloregulatory proteins represent a "green" technology for environmentally benign mercury removal . As nature offers a wide selection of specific metalloregulatory proteins, this technology offers promising solutions to remediation of other important pollutants such as arsenic or chromium.

Environ Sci Technol, 2003 Oct 1, 37(19), 4435 - 41
Rejection efficiency of water quality parameters by reverse osmosis and nanofiltration membranes; Peng W et al.; The objective of this study was to evaluate the effectiveness of reserve osmosis (RO) and nanofiltration (NF) membranes, under various solution chemistries, on water quality . The effects of organic carbon, divalent and monovalent cations, bacteria, and permeate drag on the rejection efficiencies of three different membranes were investigated through a series of laboratory bench-scale experiments . Quantitative models were successfully developed to predict the rejection of turbidity, divalent and monovalent cations, ultraviolet absorbance at 253.7 nm (UV254), and dissolved organic carbon (DOC) by membrane filtration . It was found that mechanical sieving (measured as molecular weight cutoff, MWCO) and electrostatic interactions were the most significant parameters since they were found to be important in nearly all models developed . For negatively charged membranes, under high ionic strength solution environments that repress electrostatic interaction between charged compounds and membranes, passage of compounds was mainly a function of size exclusion (i.e . MWCO) . Further, of the feedwater parameters tested, bacteria concentration was observed to be the most significant influence on UV254, divalent cation and monovalent cation rejections . The developed models revealed that interactions between feedwater composition and membrane properties impacted the rejection efficiency of membranes as significantly as water composition and membrane properties individually.

Environ Sci Technol, 2003 Oct 1, 37(19), 4374 - 81
Simultaneous release of metals and sulfide in lacustrine sediment; Motelica-Heino M et al.; A single DGT (diffusive gradient in thin films) probe that could measure metals and sulfide simultaneously and at the same location was deployed in the surface sediment of a productive lake (Esthwaite Water) . It contained a layer of AgI that binds sulfide overlying a layer of chelating resin that binds metals . Analysis for sulfide in two dimensions showed local sources of sulfide, 1-5 mm in diameter, at 8-11 cm depth within the sediment . A transect of trace metals measured at 100-microm intervals through the largest sulfide "hot spot" demonstrated concomitant release of Fe, Mn, Cu, Ni, and Co . Substantial supersaturation with respect to metal sulfides was observed for Fe and Co at the site of metal generation, but at a distance of less than 1 mm, solution concentrations were consistent with equilibration with amorphous FeS and CoS phases . Simple mass balance calculations were consistent with Fe being supplied from reductive dissolution of its oxides and with sulfide being supplied from reduction of sulfate . The observed concentrations of Cu, Ni, Co, and Mn could be accounted for by their release from iron oxides without invoking Mn reduction . The metals are removed rapidly (approximately 1 min) at the edge of the hot spot . These first observations of the simultaneous release of trace metals and sulfide are consistent with the known removal of metals by formation of their insoluble sulfides if the in situ kinetics of metal sulfide formation is on this time scale . The coproduction of reduced Fe and S suggests that iron- and sulfate-reducing bacteria may exist together in the same localized zone of actively decomposing organic matter.

Hepatogastroenterology, 2003 Sep-Oct, 50(53), 1208 - 13
The clinical utility of string-PCR test in diagnosing Helicobacter pylori infection; Wang SW et al.; BACKGROUND/AIMS: Non-invasive string test has been reported as being convenient and capable of yielding bacteria by means of gastric juice sampling in the diagnosis of Helicobacter pylori infection . Molecular methods, such as polymerase chain reaction for the amplification of DNA, are desirable for the detection of minute quantities of H . pylori . We planned to evaluate the diagnostic efficiency of the combination of the string test and polymerase chain reaction and determine whether the string polymerase chain reaction test could obtain more information in conditions where the bacterial load is so low that other diagnostic tests fail to confirm the presence of H . pylori . METHODOLOGY: We enrolled 48 dyspeptic patients, including 29 males and 19 females, with a mean age of 52.5 years . Each patient received endoscopy and biopsy-based tests, including RUT (rapid urease test), cultures, and histology, followed by 13C-UBT (13Carbon urea breath test) . We used the string test, (Entero-Test H . pylori, HDC Corporation, CA, US), for gastric juice sampling . The specimen was further analyzed by polymerase chain reaction for the presence of H . pylori with the primer for cagA gene, which is highly prevalent in Taiwan . H . pylori infection was considered as positive when either culture yield was positive, or when two of the other three tests, including RUT, histology, and 13C-UBT, were positive . RESULTS: Of the total 48 patients, 34 patients were H . pylori-positive, and 14 were H . pylori-negative . A fragment of 349 bp of polymerase chain reaction products was detected by agarose gel electrophoresis in 32 out of 34 patients who was classified as H . pylori-positive . The sensitivity, specificity, positive predictive value, and negative predictive value of the string polymerase chain reaction test were 94.12%, 96.97%, 92.86%, and 86.67%, respectively . These results are comparable to 13C-UBT and RUT, and better than histology and culture . One subject, who tested as H . pylori-negative according to the diagnostic criteria, had positive 13C-UBT and string polymerase chain reaction test results . Further sequencing of the DNA obtained from the results of polymerase chain reaction product was performed and it showed 98% identities with the known sequence of cagA strain H . pylori (GenBank accession number: AF249275) . CONCLUSIONS: The string polymerase chain reaction test is non-invasive and provides direct bacterial yields . Its diagnostic efficiency is comparable with 13C-UBT and RUT in detecting H . pylori infection . Also, with the assistance of polymerase chain reaction and DNA sequencing, we can diagnose H . pylori infection even when the bacterial load is low . Further application of string polymerase chain reaction test in the genetic analysis of virulent and resistant strains seems promising.

J Biol Chem, 2003 Dec 5, 278(49), 48520 - 3 Epub 2003 Oct 21.
An essential role of Sam50 in the protein sorting and assembly machinery of the mitochondrial outer membrane; Kozjak V et al.; The preprotein translocase of the outer mitochondrial membrane (TOM complex) contains one essential subunit, the channel Tom40 . The assembly pathway of the precursor of Tom40 involves the TOM complex and the sorting and assembly machinery (SAM complex) with the non-essential subunit Mas37 . We have identified Sam50, the second essential protein of the mitochondrial outer membrane . Sam50 contains a beta-barrel domain conserved from bacteria to man and is a subunit of the SAM complex . Yeast mutants of Sam50 are defective in the assembly pathways of Tom40 and the abundant outer membrane protein porin, while the import of matrix proteins is not affected . Thus the protein sorting and assembly machinery of the mitochondrial outer membrane involves an essential, conserved protein.

J Biol Chem, 2004 Jan 23, 279(4), 3003 - 13 Epub 2003 Oct 21.
Epitope tagging of the yeast K(+) carrier Trk2p demonstrates folding that is consistent with a channel-like structure; Zeng GF et al.; TRK family proteins, which mediate the concentrative uptake of potassium by plant cells, fungi, and bacteria, resemble primitive potassium channels in sequence and have recently been proposed actually to fold like potassium channels in a 4-MPM motif (Durell, S . R., and Guy, H . R . (1999) Biophys . J . 77, 789 - 807), instead of like conventional substrate porters in the 12-TM motif (Gaber, R . F., Styles, C . A., and Fink, G . R . (1988) Mol . Cell . Biol . 8, 2848-2859) . The known fungal members of this family possess a very long hydrophilic loop, positioned intracellularly in the K(+)-channel model and extracellularly in the substrate porter model . This and two shorter hydrophilic segments have been tested as topological markers for the true folding pattern of TRK proteins using Saccharomyces cerevisiae Trk2p . Hemagglutinin epitope tags were inserted into all three segments, and the enhanced green fluorescent protein (EGFP) was fused to the C terminus of Trk2p . The gene constructs were expressed from a high copy plasmid, and sidedness of the tags was determined by native fluorescence (EGFP), indirect immunofluorescence, and immunoelectron microscopy . Both the long-loop tag and the C-terminal EGFP fusion allowed abundant protein to reach the plasma membrane and support normal yeast growth . In all determinations, the long-loop tag was localized to the inner surface of the yeast cell plasma membrane, thus strongly supporting the channel-like folding model . Additional observations showed (i) . membrane-associated Trk2p to lie in proteolipid rafts; (ii) . significant tagged protein, expressed from the plasmid, to be sequestered in cytoplasmic vesicular-tubular clusters; and (iii) . suppression of such clusters by yeast growth in 5-10% glycerol . This chaperone-like effect may assist other membrane proteins (overexpressed or heterologously expressed) to function within the yeast plasma membrane.

Yakushigaku Zasshi, 2003, 38(1), 82 - 92
{A study of the medical treatment for Ho-shun-in, Mrs . Toshiie Maeda, described in "Igaku-tensho-ki"}; Yoshizawa C et al.; The medical commentary "Igaku-tensho-ki," written by Gensaku Manase in the Edo era, Japan, describes the medical treatment of Ho-shun-in, Mrs . Toshiie Maeda, the first seignior of Kaga . The Igaku-tensho-ki is famous for its clinical descriptions of the diseases of many notables such as Emperors and Shoguns by their antonyms, and thus is very important for both medical and historical studies . Several variant versions are known to exist now . As to the description of Ho-shun-in, the details such as the date and place of the treatment, and the recipe of the Kampo medicine are not clear . In this study, to clarify these things, a comparative study of various descriptions in all of the variants was carried out . The results show that the medical treatment was conducted on June 7th of the lunar calendar, in the 11th year of the Keicho Era (1606) in Edo (present-day Tokyo) . The disease was thought to be diarrhea caused by the infection of bacteria and round worms . Gensaku , effectively prescribed Sho-ko-en, Kakko-shoki-san with Gorei-san, and Jinryo-byakujutu-san to Ho-shun-in over a 4-day period . All of these prescriptions were basically based on those in "Manbyo-kaishun", archaic medical text written in the Ming Dynasty in China, though they were derived from different origins.

Nat Rev Mol Cell Biol, 2003 Oct, 4(10), 814 - 9
Can transcription factors function as cell-cell signalling molecules?
Prochiantz A, Joliot A.
Recent data support the view that transcription factors - in particular, homeoproteins - can be transferred from cell to cell and have direct non-cell-autonomous (and therefore paracrine) activities . This intercellular transfer, based on atypical internalization and secretion, has important biotechnological consequences . But the real excitement stems from the physiological and developmental implications of this mode of signal transduction.

J Immunol, 2003 Nov 1, 171(9), 4801 - 8
In vivo impairment of neutrophil recruitment during lentivirus infection; Kubes P et al.; Evidence indicates that the lentivirus, HIV, infection affects neutrophil response to bacteria and bacterial products in vitro . We used a novel model of rapid onset immunosuppression following infection with a similar lentivirus, feline immunodeficiency virus (FIV), in cats to examine neutrophil function within the microvasculature in vivo and to determine the steps that are impaired in the neutrophil recruitment cascade . In uninfected cats and cats infected neonatally with FIV, the mesentery was exteriorized, but remained autoperfused during intravital microscopy for 4 h . When the tissue was superfused with 10 micro g/ml of LPS for 4 h, intravital microscopy displayed a profound increase in neutrophil rolling at both 8 and 12 wk of age in uninfected cats . At 12 wk of age, FIV-infected animals showed a profound decrease in the number of rolling neutrophils . In vitro studies revealed that neutrophils from infected and uninfected animals rolled equally well on surrogate selectin substrata . In addition, in vivo neutrophil adhesion and emigration out of the vasculature were severely reduced, and in vitro neutrophil chemotaxis from FIV-infected animals was significantly impaired in response to fMLP or IL-8 . However, FIV infection of neutrophils could not be detected . In summary, in vivo lentivirus infection with immunosuppression leads to a severe impairment in neutrophil rolling, adhesion, and emigration in response to bacterial stimulants potentially involving both endothelial and neutrophil dysfunction . These in vivo studies also indicate that neutrophil dysfunction should be taken into account when treating infections and tissue injury.

J Immunol, 2003 Nov 1, 171(9), 4742 - 9
An inclusion membrane protein from Chlamydia trachomatis enters the MHC class I pathway and stimulates a CD8+ T cell response; Starnbach MN et al.; During its developmental cycle, the intracellular bacterial pathogen Chlamydia trachomatis remains confined within a protective vacuole known as an inclusion . Nevertheless, CD8(+) T cells that recognize Chlamydia Ags in the context of MHC class I molecules are primed during infection . MHC class I-restricted presentation of these Ags suggests that these proteins or domains from them have access to the host cell cytoplasm . Chlamydia products with access to the host cell cytoplasm define a subset of molecules uniquely positioned to interface with the intracellular environment during the pathogen's developmental cycle . In addition to their use as candidate Ags for stimulating CD8(+) T cells, these proteins represent novel candidates for therapeutic intervention of infection . In this study, we use C . trachomatis-specific murine T cells and an expression-cloning strategy to show that CT442 from Chlamydia is targeted by CD8(+) T cells . CT442, also known as CrpA, is a 15-kDa protein of undefined function that has previously been shown to be associated with the Chlamydia inclusion membrane . We show that: 1) CD8(+) T cells specific for an H-2D(b)-restricted epitope from CrpA are elicited at a significant level (approximately 4% of splenic CD8(+) T cells) in mice in response to infection; 2) the response to this epitope correlates with clearance of the organism from infected mice; and 3) immunization with recombinant vaccinia virus expressing CrpA elicits partial protective immunity to subsequent i.v . challenge with C . trachomatis.

FEMS Microbiol Lett, 2003 Oct 10, 227(1), 73 - 9
Characterization of human humoral responses to the major outer membrane protein and OMP2 of Chlamydophila pneumoniae; Cunningham AF et al.; Chlamydophila pneumoniae infection is associated with a range of diseases including pneumonia, asthma and heart disease . Although an obligate intracellular pathogen, high levels of antigen-specific antibody are induced and serology is frequently used to diagnose these infections . Proteins targeted by the humoral response include the major outer membrane protein (MOMP) and outer membrane protein 2 (OMP2) . Using human anti-chlamydial sera we have defined the B cell epitopes recognized on MOMP and OMP2 . Peptides from MOMP, unlike OMP2, were not strongly recognized . Two of these epitopes when linked to an inert carrier reacted strongly with high-titer anti-C . pneumoniae sera.

FEMS Microbiol Lett, 2003 Oct 10, 227(1), 17 - 23
Filling a gap in the central metabolism of archaea: prediction of a novel aconitase by comparative-genomic analysis; Makarova KS et al.; Aconitase, an essential enzyme of the tricarboxylic acid cycle (TCA), so far has been identified only in a minority of archaeal genomes . This enzyme belongs to the aconitase A family, which is represented in most bacteria and eukaryotes . Using iterative sequence database search, we linked two previously uncharacterized protein families (COG1679 and COG1786), respectively, to the three Fe-S-cluster-associated aconitase domains and the swiveling domain, the four domains that are present in all known aconitase families . The respective genes are often found in one predicted operon and, moreover, are fused in several species, suggesting a functional and physical interaction . We predict that these proteins together comprise a previously undetected, distinct aconitase family, which we designated aconitase X . Aconitase X is encoded in the genomes of many archaea and some proteobacteria . Among archaea, the pattern of aconitase X occurrence complements that of aconitase A such that together the two enzymes account for aconitase activity in all archaea . Phylogenetic analysis indicates that aconitase X is likely to be the ancestral archaeal form, with non-orthologous displacement in some of the archaea apparently brought about by horizontal transfer of the gene for bacterial aconitase A . The prediction of aconitase X completes the TCA cycle for Methanothermobacter thermoautotrophicus and Archaeoglobus fulgidus and suggests that most archaea have a full TCA cycle.

Water Res, 2003 Nov, 37(19), 4711 - 8
Improvement of organics removal by bio-ceramic filtration of raw water with addition of phosphorus; Sang J et al.; The purpose of this study was to investigate the effect of phosphorus addition on biological pretreatment of raw water . Experiments were conducted in pilot-scale bio-ceramic filters with raw water from a reservoir located in Beijing, China . The results demonstrated that phosphorus was the limiting nutrient for bacterial growth in the raw water investigated in this study . The measured values of bacterial regrowth potential (BRP) and biodegradable dissolved organic carbon (BDOC) of the raw water increased by 50-65% and 30-40% with addition of 50 microg of PO4(3-)-PL(-1), respectively . Addition of 25 microg of PO4(3-)-PL(-1) to the influent of bio-ceramic filter enhanced the percent removal of organics by 4.6, 5.7 and 15 percentage points in terms of COD(Mn), TOC and BDOC, respectively . Biomass in terms of phospholipid content increased by 13-22% and oxygen uptake rate (OUR) increased by 35-45% . The ratio of C:P for bacteria growth was 100:1.6 for the raw water used in this study . Since change of phosphorus concentrations can influence the performance of biological pretreatment and the biological stability of drinking water, this study is of substantial significance for waterworks in China . The role of phosphorus in biological processes of drinking water should deserve more attention.

Water Res, 2003 Nov, 37(19), 4623 - 30
Partitioning variation of heavy metals in contaminated river sediment via bioleaching: effect of sulfur added to total solids ratio; Tsai LJ et al.; The aim of this study was to determine the effect of the ratio of sulfur added to total sediment solids (SA/TS) on the remobilization of heavy metals from contaminated river sediment by indigenous sulfur-oxidizing bacteria . Also, the difference in metal binding fractions before and after bioleaching was explored.It was found that sediment pH decreased at a significantly faster rate at higher SA/TS ratios (0.413 and 0.199) than at lower ones . Sulfate concentrations increased at a faster rate at these higher SA/TS ratios . At the lower SA/TS ratios, more acid must be produced and therefore it took more time for sulfur-oxidizing bacteria to lower the sediment pH . Remobilization efficiency of total extractable Pb and Cr was significantly higher at higher SA/TS ratios . After bioleaching, Mn-oxides became a stronger binding pool, and the percentage of Pb and Zn bound to Mn-oxides and Cr and Cu bound to organic matter increased with the decrease of SA/TS . Different heavy metals showed different binding behavior at the various SA/TS ratios.

J Wildl Dis, 2003 Jul, 39(3), 495 - 509
Patterns of mortality in southern sea otters (Enhydra lutris nereis) from 1998-2001; Kreuder C et al.; Detailed postmortem examination of southern sea otters (Enhydra lutris nereis) found along the California (USA) coast has provided an exceptional opportunity to understand factors influencing survival in this threatened marine mammal species . In order to evaluate recent trends in causes of mortality, the demographic and geographic distribution of causes of death in freshly deceased beachcast sea otters necropsied from 1998-2001 were evaluated . Protozoal encephalitis, acanthocephalan-related disease, shark attack, and cardiac disease were identified as common causes of death in sea otters examined . While infection with acanthocephalan parasites was more likely to cause death in juvenile otters, Toxoplasma gondii encephalitis, shark attack, and cardiac disease were more common in prime-aged adult otters . Cardiac disease is a newly recognized cause of mortality in sea otters and T . gondii encephalitis was significantly associated with this condition . Otters with fatal shark bites were over three times more likely to have pre-existing T . gondii encephalitis suggesting that shark attack, which is a long-recognized source of mortality in otters, may be coupled with a recently recognized disease in otters . Spatial clusters of cause-specific mortality were detected for T . gondii encephalitis (in Estero Bay), acanthocephalan peritonitis (in southern Monterey Bay), and shark attack (from Santa Cruz to Point Ano Nuevo) . Diseases caused by parasites, bacteria, or fungi and diseases without a specified etiology were the primary cause of death in 63.8% of otters examined . Parasitic disease alone caused death in 38.1% of otters examined . This pattern of mortality, observed predominantly in juvenile and prime-aged adult southern sea otters, has negative implications for the overall health and recovery of this population.

Bone Marrow Transplant, 2004 Jan, 33(1), 109 - 12
West Nile virus encephalitis causing fatal CNS toxicity after hematopoietic stem cell transplantation; Reddy P et al.; We describe here a patient who died of progressive CNS deterioration following allogeneic stem cell transplant with West Nile virus as the sole pathogen on the cerebrospinal fluid and brain tissue analysis . A 50-year-old male with Philadelphia chromosome-positive acute lymphocytic leukemia (ALL) underwent allogeneic PBSCT from his HLA identical sister . After engraftment, the patient developed fever with progressive and ultimately fatal neurological deterioration . Imaging studies of the brain including CT and MRI scans were remarkable for mild low attenuation lesions of the white matter . CSF analysis was negative for neoplastic cells, bacteria, AFB, CMV, HSV, fungal infections and leukemic relapse . However, serological analysis of both the serum and CSF was positive for West Nile virus-specific IgM antibodies . At autopsy, West Nile virus PCR and cultures were positive in the mid-brain tissue . Electron micrographs showed evidence of viral particles . Given the recent increase in the spread of West Nile virus infections and the increased susceptibility of BMT patients to infectious complications, West Nile virus encephalitis should be considered in patients undergoing transplantation.

Zh Mikrobiol Epidemiol Immunobiol, 2003 Sep-Oct, (5), 45 - 9
{Morbidity: terminology, mechanisms of formation and classification}; Kolpakov SL et al.; In the 70-ies of the last century the evolution of the epidemic process doctrine resulted in developing population approach to morbidity analysis, in addition to the traditional focal approach . On the one hand, this led to the discussion on the possibility of applying such notion to non-infectious diseases and, on the other hand, to the discussion on the mechanisms of the development and formation of different manifestations in infectious diseases . In both cases the discussion was centered on the phenomenon of morbidity . The present article deals with the nature of morbidity as a phenomenon and the acceptability of general scientific approaches in the interpretation of seasonal and cyclic character of morbidity from the viewpoint of the wave process in an open system . On the basis of the data presented in this work the terms used in epidemiology in considering manifestations of the epidemic process are discussed . The improved classification of morbidity by qualitative and quantitative signs for infectious diseases is proposed.

Mar Biotechnol (NY), 2003 Nov-Dec, 5(6), 519 - 27 Epub 2003 Oct 20.
In vitro culture of the tropical sponge Axinella corrugata (Demospongiae): effect of food cell concentration on growth, clearance rate, and biosynthesis of stevensine; Duckworth AR et al.; In vitro culture is one possible method for supplying sponge metabolites for pharmaceutical applications, but appropriate feeding regimens that maximize both growth and metabolite biosynthesis are largely unknown . According to the natural concentration (NC) of cells 1 to 50 micro m in size that are available to wild Axinella corrugata, we fed explants a multispecific diet of bacteria, microalgae, and yeast at 4 different concentrations: 1NC, 3NC, 5NC, and 5+1NC (the last consisted of 5 NC of bacteria and 1 NC of microalgae and yeast) . Explants fed a 3NC diet had the best culture response, growing on average from 8.5 g to 10.3 g in 8 weeks, and showing a 110% increase in concentration (milligrams per gram of dry weight) of the antitumor compound stevensine . Stevensine production in 3NC explants, representing the total milligrams of metabolite per explant, increased by 157% over the study . Explants fed at 1NC had relatively stable weights, indicating that the diet met metabolic costs only . Explants fed at the two highest concentrations lost weight after 4 weeks, possibly because long-term high cell concentration blocked their aquiferous system, reducing their ability to feed efficiently . Stevensine production in explants fed the 1NC, 5NC, or 5+1NC diets were similar, and varied little from the initial amount . A separate experiment showed that the clearance rate for A . corrugata is similar between the examined food types and cell concentrations over 5 hours, averaging 766 ml h(-1) g DW(-1).Overall, this study demonstrates that relatively small changes in food abundance can greatly affect both sponge growth and metabolite biosynthesis . The good growth and increased production of the target metabolite stevensine for A . corrugata explants fed a 3NC diet suggests that in vitro culture is a viable method of supplying some sponge metabolites.

Mol Endocrinol, 2004 Jan, 18(1), 26 - 42 Epub 2003 Oct 16.
Imaging analysis of subcellular correlation of androgen receptor and estrogen receptor alpha in single living cells using green fluorescent protein color variants; Ochiai I et al.; Androgen and estrogen act not only in a sex-specific manner but also interactively and synergistically . In the present study, to examine the possible interaction between androgen receptor (AR) and estrogen receptor-alpha (ERalpha), we investigated the subcellular dynamics of AR and ERalpha fused with green fluorescent protein color variants in single living cells using time-lapse microscopy and the technique of fluorescence recovery after photobleaching . AR and ERalpha showed punctate colocalization in the nucleus with estrogen, but not androgen . N-terminal AR deletion mutant did not form a nuclear punctate pattern with either androgen or estrogen . In the presence of AR, but not ERalpha, N-terminal AR deletion mutant formed a punctate nuclear pattern with androgen . AR had different mobility depending on the ligand and the presence of ERalpha . On the other hand, AR had little effect on the stability of ERalpha . ERalpha mutant that does not bind coactivators did not alter the mobility of AR . Taken together, using an imaging technique, we clarified that possible homo/hetero dimerization between AR and ERalpha could be attributed to androgen-estrogen interaction in living cells.

J Mol Evol, 2003 Aug, 57(2), 200 - 11
Phylogenetic analysis of the cytochrome P450 3 (CYP3) gene family; McArthur AG et al.; Cytochrome P450 genes (CYP) constitute a superfamily with members known from the Bacteria, Archaea, and Eukarya . The CYP3 gene family includes the CYP3A and CYP3B subfamilies . Members of the CYP3A subfamily represent the dominant CYP forms expressed in the digestive and respiratory tracts of vertebrates . The CYP3A enzymes metabolize a wide variety of chemically diverse lipophilic organic compounds . To understand vertebrate CYP3 diversity better, we determined the killifish (Fundulus heteroclitus) CYP3A30 and CYP3A56 and the ball python (Python regius) CYP3A42 sequences . We performed phylogenetic analyses of 45 vertebrate CYP3 amino acid sequences using a Bayesian approach . Our analyses indicate that teleost, diapsid, and mammalian CYP3A genes have undergone independent diversification and that the ancestral vertebrate genome contained a single CYP3A gene . Most CYP3A diversity is the product of recent gene duplication events . There is strong support for placement of the guinea pig CYP3A genes within the rodent CYP3A diversification . The rat, mouse, and hamster CYP3A genes are mixed among several rodent CYP3A subclades, indicative of a complex history involving speciation and gene duplication.

J Mol Evol, 2003 Aug, 57(2), 181 - 99
Amino acid sequences and distribution of high-potential iron-sulfur proteins that donate electrons to the photosynthetic reaction center in phototropic proteobacteria; Van Driessche G et al.; High-potential iron-sulfur protein (HiPIP) has recently been shown to function as a soluble mediator in photosynthetic electron transfer between the cytochrome bc1 complex and the reaction-center bacteriochlorophyll in some species of phototrophic proteobacteria, a role traditionally assigned to cytochrome c2 . For those species that produce more than one high-potential electron carrier, it is unclear which protein functions in cyclic electron transfer and what characteristics determine reactivity . To establish how widespread the phenomenon of multiple electron donors might be, we have studied the electron transfer protein composition of a number of phototrophic proteobacterial species . Based upon the distribution of electron transfer proteins alone, we found that HiPIP is likely to be the electron carrier of choice in the purple sulfur bacteria in the families Chromatiaceae and Ectothiorhodospiraceae, but the majority of purple nonsulfur bacteria are likely to utilize cytochrome c2 . We have identified several new species of phototrophic proteobacteria that may use HiPIP as electron donor and a few that may use cytochromes c other than c2 . We have determined the amino acid sequences of 14 new HiPIPs and have compared their structures . There is a minimum of three sequence categories of HiPIP based upon major insertions and deletions which approximate the three families of phototrophic proteobacteria and each of them can be further subdivided prior to construction of a phylogenetic tree . The comparison of relationships based upon HiPIP and RNA revealed several discrepancies.

J Biol Chem, 2004 Jan 2, 279(1), 385 - 93 Epub 2003 Oct 15.
Oxidative phosphorylation and rotenone-insensitive malate- and NADH-quinone oxidoreductases in Plasmodium yoelii yoelii mitochondria in situ; Uyemura SA et al.; Respiration, membrane potential, and oxidative phosphorylation of mitochondria of Plasmodium yoelii yoelii trophozoites were assayed in situ after permeabilization with digitonin . ADP induced an oligomycin-sensitive transition from resting to phosphorylating respiration in the presence of oxidizable substrates . A functional respiratory chain was demonstrated . In addition, the ability of the parasite to oxidize exogenous NADH, as well as the insensitivity of respiration to rotenone and its sensitivity to flavone, suggested the presence of an alternative NADH-quinone (NADH-Q) oxidoreductase . Rotenone-insensitive respiration and membrane potential generation in the presence of malate suggested the presence of a malate-quinone oxidoreductase . These results are in agreement with the presence of genes in P . yoelii encoding for proteins with homology to NADH-Q oxidoreductases of bacteria, plant, fungi, and protozoa and malate-quinone oxidoreductases of bacteria . The complete inhibition of respiration by antimycin A and cyanide excluded the presence of an alternative oxidase as described in other parasites . An uncoupling effect of fatty acids was partly reversed by bovine serum albumin and GTP but was unaffected by carboxyatractyloside . These results provide the first biochemical evidence of the presence of an alternative NADH-Q oxidoreductase and a malate-quinone oxidoreductase and confirm the operation of oxidative phosphorylation in malaria parasites.

J Biol Chem, 2004 Jan 2, 279(1), 812 - 9 Epub 2003 Oct 15.
Residues in internal repeats of the rice cation/H+ exchanger are involved in the transport and selection of cations; Kamiya T et al.; In plants, the cation/H+ exchanger (CAX) translocates Ca2+ and other metal ions into vacuoles using the H+ gradient formed by H+-ATPase and H+-pyrophosphatase . Such exchangers carrying 11 transmembrane domains (TMs) have been isolated from plants, yeast, and bacteria . In this study, multiple sequence alignment of several CAXs revealed the presence of highly conserved 36-residue regions between TM3 and TM4 and between TM8 and TM9 . These two repetitive motifs are designated repeats c-1 and c-2 . Using site-directed mutagenesis, we generated 31 mutations in the repeats of the Oryza sativa CAX, which translocates Ca2+ and Mn2+ . Mutant exchangers were expressed in a Saccharomyces cerevisiae strain that is sensitive to Ca2+ and Mn2+ because of the absence of vacuolar Ca2+-ATPase and the Ca2+/H+ exchanger . Mutant exchangers were classified into six classes according to their tolerance for Ca2+ and Mn2+ . For example, the class III mutants had no tolerance for either ion, and the class IV mutants had tolerance only for Ca2+ . The biochemical function of each residue was estimated . We investigated the membrane topology of the repeats using a method combining cysteine mutagenesis and sulfhydryl reagents . Our results suggest that repeat c-1 re-enters the membrane from the vacuolar luminal side and forms a solution-accessible region . Furthermore, several residues in repeats c-1 and c-2 were found to be conserved in animal Na+/Ca2+ exchangers . Finally, we suggest that these re-entrant repeats may form a vestibule or filter for cation selection.

J Clin Invest, 2003 Oct, 112(8), 1176 - 85
Inhibition of NF-kappaB activation in macrophages increases atherosclerosis in LDL receptor-deficient mice; Kanters E et al.; Atherosclerosis is now generally accepted as a chronic inflammatory condition . The transcription factor NF-kappaB is a key regulator of inflammation, immune responses, cell survival, and cell proliferation . To investigate the role of NF-kappaB activation in macrophages during atherogenesis, we used LDL receptor-deficient mice with a macrophage-restricted deletion of IkappaB kinase 2 (IKK2), which is essential for NF-kappaB activation by proinflammatory signals . These mice showed increased atherosclerosis as quantified by lesion area measurements . In addition, the lesions were more advanced and showed more necrosis and increased cell number in early lesions . Southern blotting revealed that deletion of IKK2 was approximately 65% in macrophages, coinciding with a reduction of 50% in NF-kappaB activation, as compared with controls . In both groups, the expression of differentiation markers, uptake of bacteria, and endocytosis of modified LDL was similar . Upon stimulation with LPS, production of TNF was reduced by approximately 50% in IKK2-deleted macrophages . Interestingly, we also found a major reduction in the anti-inflammatory cytokine IL-10 . Our data show that inhibition of the NF-kappaB pathway in macrophages leads to more severe atherosclerosis in mice, possibly by affecting the pro- and anti-inflammatory balance that controls the development of atherosclerosis.

Phytochemistry, 2003 Nov, 64(5), 997 - 1001
Isolation and anti-oomycete activity of nyasol from Anemarrhena asphodeloides rhizomes; Park HJ et al.; The methanol extract of Anemarrhena asphodeloides rhizomes exhibited strong antifungal activity against the plant pathogenic fungi Magnaphothe grisea, Rhizoctonia solani, and the plant pathogenic oomycete Phytophthora capsici . The antifungal substance isolated from the rhizomes of A . asphodeloides was identified to be nyasol, (Z)-1,3-bis(4-hydroxyphenyl)-1,4-pentadiene by NMR and mass spectral analysis . Nyasol effectively inhibited the mycelial growth of Colletotrichum orbiculare, P . capsici, Pythium ultimum, R . solani, and Cladosporium cucumerinum in a range of 1-50 mug/ml, but did not affect the growth of bacteria and yeast . In a greenhouse test, treatment with the antifungal compound nyasol was significantly effective in suppressing the Phytophthora blight on pepper plants.

Curr Hematol Rep, 2003 Nov, 2(6), 495 - 502
Pathogen reduction technology: methods, status of clinical trials, and future prospects; Corash L; Transfusion-transmitted infections caused by viruses, bacteria, and protozoa have been extensively reported for the past three decades . In the early 1980s, the HIV epidemic heightened the focus on transfusion safety and demonstrated the potential for new pathogens to enter the donor population and rapidly spread through the transfusion of blood components . The foundation for the prevention of transfusion-transmitted infections has been donor screening and testing, and significant advances have been made to reduce the risk of transfusion-associated infection . However, despite these efforts, transfusion-transmitted infections continue to be reported, and new infectious agents, such as the West Nile virus, continue to enter the donor population with transmission to recipients . For the past decade, several technologies to inactivate infectious pathogens that may contaminate donor blood have been developed and several of these methods have been introduced into clinical practice . These technologies offer the potential for a paradigm shift to further improve the safety of blood transfusion.

Eur J Epidemiol, 2003, 18(9), 907 - 12
Distribution and prevalence of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks of canton Ticino (Switzerland); Jouda F et al.; Free-living Ixodes ricinus ticks were collected from 12 different sites of canton Ticino, south of the Alps (Switzerland) . Each tick was examined for the presence of Borrelia burgdorferi sensu lato (sl), the etiologic agent of Lyme borreliosis using direct fluorescent antibody assay, and isolation of the bacteria . Borreliae were characterized by PCR followed by RFLP . The abundance and infection rates of I . ricinus ticks varied greatly between the areas . Two localities were found free of Borrelia . The prevalence of infected ticks ranged from 5 to 19% . Most ticks (96%) were found infected by < 50 spirochetes . Three B . burgdorferi sl species were successfully isolated: B . garinii dominated, followed by B . lusitaniae and B . valaisiana . Additionally, a mixed infection with B . garinii and B . valaisiana was observed . The distribution of the various Borrelia species in the different areas was heterogeneous . This is the first report of the presence of B . lusitaniae in I . ricinus in Switzerland.

Curr Top Microbiol Immunol, 2004, 279, 283 - 98
mTOR: a mediator of intracellular homeostasis; Jaeschke A et al.; Earlier studies have shown that mTOR plays a key role in ribosome biogenesis . In bacteria, amino acids and ATP levels independently control ribosome biogenesis . Here, we describe recent findings demonstrating that homeostatic levels of amino acids, most notably branched-chain amino acids, and ATP, independently regulate the activity of mTOR . Unlike the effects of amino acids, the effects of ATP appear to be direct . Based on these findings we propose a model by which tumor cells existing in the anaerobic environment may have an advantage in growth by exploiting the rapid, although less efficient, production of ATP to drive growth via the mTOR signaling pathway.

Lakartidningen, 2003 Sep 25, 100(39), 3055 - 9
{A national database could solve the issue of sudden sensorineural hearing loss}; Hultcrantz E et al.; A national database is being organised for the disease Sudden Sensorineural Hearing-loss, SSH (often called "Sudden deafness") . The cause of SSH is unknown . Many theories have given rise to many treatment regimens: the vascular theory, assuming a reduced blood flow to the inner ear, the infection theory, assuming bacteria or virus being the etiological agent, and the auto-immune theory, where also blood vessels may be involved . Still another theory concerns ruptured membranes, either towards the middle ear, leaking perilymph, or intra-cochlear ruptures, resulting in perilymph and endolymph mixing and giving rise to the hearing loss . Many cases recover spontaneously: up to one third completely and another one third significantly improved . The most common therapy today is high-dose corticosteroids . Collecting data on a large number of patients in a database will allow the assessment of different background factors and the possible effects of therapy . We hope to obtain data on 400 patients per year during a 4-year period in this multi-centre project . A controlled study is planned to assess the effect of cortico-steroid treatment.

Implant Dent, 2003, 12(3), 259 - 65
Histological analysis of the Ankylos peri-implant soft tissues in a dog model; Tenenbaum H et al.; PURPOSE: The importance of the soft tissue-implant interface is enhanced by the presence of a microgap between the implant and the abutment, which represents a contamination site for bacteria . The aim of this study was to investigate the interface between the Ankylos gap-free implant system and the surrounding soft tissues in a dog model . MATERIALS AND METHODS: Six Labrador dogs were included in the study and two Ankylos implants were inserted per dog . The dogs were killed 6 months after abutment placement without functional loading and without plaque control . The implants were analysed histologically by scanning electron microscopy, light microscopy, and histomorphometry . RESULTS: Some sections exhibited histologic signs of a mild inflammation . The connective tissue between the most apical epithelial cells of the junctional epithelium and the alveolar crest was characterized by collagen fibers running from the periosteum and the alveolar crest toward the oral epithelium and, in front of the cone-shaped abutment, by a narrow zone of extracellular matrix with a few collagen fibers . CONCLUSION: Compared with results obtained in other studies using different types of implant (Astra, Branemark, ITI), the Ankylos implant showed a higher length and a larger width of connective tissue contact as well as a shorter epithelial downgrowth . The absence of a microgap in the Ankylos system could explain the histologic mild inflammation in the connective tissue.

World J Surg, 2003 Sep, 27(9), 994 - 8 Epub 2003 Jul 24.
Effect of Helicobacter pylori infection in Barrett's esophagus and the genesis of esophageal adenocarcinoma; Clark GW; The relation between Helicobacter pylori and gastroesophageal reflux disease is unclear . Recent reports have suggested a possible protective role for H . pylori, particularly in preventing the complications of gastroesophageal reflux disease (GERD) . The purpose of this article is to present a brief overview of the recent literature regarding the role of H . pylori in the genesis of the complications of GERD, focusing on Barrett's esophagus and esophageal adenocarcinoma . The prevalence of H . pylori infection in the population of the West is around 40% and is not different in cohorts of patients with GERD . When the infection induces pangastritis or corpus-predominant gastritis, there may be concomitant reduced gastric acid secretion . Eradication of the bacteria in this subgroup of patients may enhance gastric acid secretion and provoke reflux symptoms . H . pylori organisms do not colonize the specialized intestinal metaplasia characteristic of Barrett's esophagus . H . pylori infection rates in gastric mucosa of patients with Barrett's esophagus occur at a similar or slightly lower frequency than is found in controls . Gastric infection with cagA-positive strains of H . pylori appears to be uncommon in patients with Barrett's esophagus . Furthermore, epidemiologic studies indicate that cagA-positive strains are protective against esophageal adenocarcinoma . Several investigators have proposed that the decreasing prevalence of H . pylori infection might be an important factor in the rising incidence of this tumor.

Genome Res, 2003 Nov, 13(11), 2444 - 9 Epub 2003 Oct 14.
Functionality of system components: conservation of protein function in protein feature space; Jensen LJ et al.; Many protein features useful for prediction of protein function can be predicted from sequence, including posttranslational modifications, subcellular localization, and physical/chemical properties . We show here that such protein features are more conserved among orthologs than paralogs, indicating they are crucial for protein function and thus subject to selective pressure . This means that a function prediction method based on sequence-derived features may be able to discriminate between proteins with different function even when they have highly similar structure . Also, such a method is likely to perform well on organisms other than the one on which it was trained . We evaluate the performance of such a method, ProtFun, which relies on protein features as its sole input, and show that the method gives similar performance for most eukaryotes and performs much better than anticipated on archaea and bacteria . From this analysis, we conclude that for the posttranslational modifications studied, both the cellular use and the sequence motifs are conserved within Eukarya.

Placenta, 2003 Oct, 24 Suppl B, S20 - 6
The impact of embryo transfer on implantation--a review; Levi Setti PE et al.; Embryo transfer has received little clinical attention and has been, until recently, the most inefficient step in in-vitro fertilization (IVF) . In this article, the authors review the literature and their personal experience regarding the process of intrauterine transfer of embryos, which remains the object of much discussion . Factors which appear to influence implantation rates are: contamination of the catheter tip with cervical bacteria, stimulation of uterine contractions during the procedure, the type of catheter, ultrasound guidance during the transfer, and the position of the embryos in the uterine cavity . Easy and atraumatic transfer is essential for successful implantation and the embryos need to be placed in the middle of the cavity, away from the fundus . Knowing, beforehand, the position and length of the uterus can provide better results and may reduce the rate of ectopic pregnancies . Evidence from randomized studies has supported this claim . Despite the number of available studies controlling certain variables, most authors, even using the same catheter, ultrasound guidance and/or a trial transfer use different protocols or similar instruments in different ways . Standardization of the transcervical intrauterine transfer of embryos in a large randomized study is needed before definitive conclusions can be drawn . The goal of improved implantation and pregnancy rates deserve these efforts.

J Agric Food Chem, 2003 Oct 22, 51(22), 6627 - 34
A combination of tea (Camellia senensis) catechins is required for optimal inhibition of induced CYP1A expression by green tea extract; Williams SN et al.; It was previously demonstrated that the commercial green tea extract Polyphenon 100 (P100), and to a lesser extent (-)-epigallocatechin-3-gallate (EGCG), partially antagonizes 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced transcription of human CYP1A1 (Williams, S . N.; Shih, H.; Guenette, D . K.; Brackney, W.; Denison, M . S.; Pickwell, G . V.; Quattrochi, L . C . Chem.-Biol . Interact . 2000, 128, 211-229) . Here, P100 is compared to a reconstituted mixture of the four major tea catechins (referred to as P100R) to determine whether inhibition was due to additional polyphenols in the extract or from synergistic interactions among the tea catechins . It was found that cotreatment of cells with TCDD and either P100 or P100R inhibited TCDD-induced CYP1A promoter-driven luciferase reporter activity (HepG2 cells) and CYP1A expression (HepG2 and primary human hepatocytes), similarly . These results indicate that modulation of human CYP1A expression by P100 can be attributed entirely to the combination of the four tea catechins . These findings may be important in the evaluation of future chemoprevention strategies using green tea and single catechin agents.

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