Mol Diagn, 2003, 7(3-4), 155 - 62 Improvement in the laboratory recognition of lyme borreliosis with the combination of culture and PCR methods; Chmielewski T et al.; BACKGROUND: Lyme disease is a multisystem, multistage infection caused by three genospecies of the Borrelia burgdorferi sensu lato species . The diagnosis of Lyme disease is based on a history of tick-bite, physical examination, and serological tests . In the seronegative patients with Lyme borreliosis symptoms, additional testing should be introduced . METHODS: The study group was composed of 240 hospitalized patients presented with various clinical symptoms suggesting Lyme borreliosis: 221 of the patients with neurological abnormalities and 19 with oligoarticular arthritis . Citrated blood and serum samples were collected from the patients for culture and serological examination, respectively . Moreover, 173 cerebrospinal and 6 synovial fluid samples were tested . New oligonucleotide primers based on B . burgdorferi sensu lato 16SrRNA gene sequences were designed for the detection of the bacteria in blood, cerebrospinal, and synovial fluid specimens with PCR . Levels of specific antibodies were measured in serum, cerebrospinal fluid and synovial fluid samples using ELISA and Western blot . B . burgdorferi spirochetes from blood, cerebrospinal fluid, and synovial fluid samples were cultured in cell line . Extracted and purified B . burgdorferi DNA was identified by PCR with new oligonucleotide primers . Then three genospecies were identified by PCR amplification with other primer sets specific for 16S rDNA and/or by the restriction fragment length polymorphism of 23S(rrl)-5S(rrf) . RESULTS: Bacterial DNA were found in samples from 32 patients, including 28 patients with neuroborreliosis and 4 with Lyme arthritis . B . burgdorferi-specific IgM and/or IgG serum antibodies were detected in 14 of these patients . Fourteen strains of Borrelia garini, 4 strains of Borrelia afzelii and 1 strain of B . burgdorferi sensu stricto were identified by PCR . Genospecies were not recognized in 13 specimens . CONCLUSIONS: The procedure can be a rapid and sensitive diagnostic method for the detection of etiological agents in clinical materials derived from patients with the clinical symptoms of Lyme borreliosis . It can be utilized for both basic research as well as routine laboratory diagnosis. Adv Drug Deliv Rev, 2004 Apr 23, 56(7), 987 - 97 pH-sensitive toxins: interactions with membrane bilayers and application to drug delivery; Cabiaux V; pH-sensitive toxins are secreted by bacteria and reach the cytosol of eukaryotic target cells by complex mechanisms involving receptor binding, membrane interaction and translocation across a cell lipid membrane . Membrane interaction and ability to reach the cytoplasm have been used respectively to present proteins at the cell surface and to transport foreign peptides or DNA into the cytoplasm . The first approach is used in anticancer vaccination and the second in inducing a major histocompatibility (MHC) class I presentation of exogenous peptides or proteins . A brief overview of the use of toxins themselves for targeting cancer cells is also presented . Altogether, the data suggest that pH sensitive toxins have a huge potential for surface presentation or cytosol transport of biomacromolecules and that many ways could still be explored to develop new strategies in vaccination or therapeutic methods. Vet Microbiol, 2004 Apr 19, 99(3-4), 295 - 9 Efficacy of live Chlamydophila abortus vaccine 1B in protecting mice placentas and foetuses against strains of Chlamydophila pecorum isolated from cases of abortion; Rekiki A et al.; The efficacy of Chlamydophila abortus vaccine strain 1B in protecting against two selected Chlamydophila pecorum strains, isolated from an aborted goat (M14) in Morocco and a ewe (AB10) in France, was investigated in a mouse model, by comparing the reduction in number of bacteria in the placentas of vaccinated mice challenged intraperitoneally at 11 days of pregnancy with the reference C . abortus (AB7) and C . pecorum (M14, or AB10) strains, to those of unvaccinated mice . Vaccine 1B was shown to provide effective protection against the field strains of C . pecorum, since it significantly reduced the placental Chlamydophila colonisation . The two C . pecorum strains were not sufficiently abortifacient in mice to use reduction in abortion as a criterion of protection. Vet Microbiol, 2004 Apr 19, 99(3-4), 215 - 25 Analysis of differential protein expression in Actinobacillus pleuropneumoniae by Surface Enhanced Laser Desorption Ionisation--ProteinChip (SELDI) technology; Hodgetts A et al.; Actinobacillus pleuropneumoniae (APP) is the aetiological agent of porcine pleuropneumonia . An increased understanding of its molecular basis of pathogenicity and vaccine development will be facilitated by the availability of sequence data from a complete genome which, by analogy to other bacteria, is predicted to encode many proteins in the molecular mass range 3-20kDa . However, conventional techniques to study bacterial protein expression, such as SDS-PAGE and 2-dimensional electrophoresis, typically focus on the 15-200kDa range . In this study we have evaluated Surface Enhanced Laser Desorption Ionisation-ProteinChip (SELDI) technology for the analysis of protein expression, in particular those of <20kDa, of APP grown under different environmental conditions . Cytoplasmic/periplasmic and outer membrane protein fractions were obtained from the APP wildtype serotype 1 strain 4074 grown in Brain Heart Infusion (BHI) broth (+different concentrations of NAD), BHI containing pig serum or defined medium . Optimum conditions for SELDI profiles included a sample size of 1 microg and the use of sinapinic acid as the energy absorbing matrix . In the <20kDa range, the SELDI profiles obtained from wild-type bacteria grown in rich medium plus 33-66% pig serum were most similar to those grown in defined medium . The SELDI profiles of extracts of the wild-type and of an rpoE mutant were similar although there were clear differences . The results suggest that SELDI is a useful complementary approach to conventional proteomic analytical methods with APP, and presumably other bacterial pathogens, being particularly suited for analysis of proteins in the <20kDa mass range. Dev Growth Differ, 2004 Apr, 46(2), 195 - 9 Defect in peroxisomal multifunctional enzyme MFE1 affects cAMP relay in Dictyostelium; Matsuoka S et al.; We have previously reported that cells of Dictyostelium discoideum lacking the fatty acid oxidation enzyme MFE1 accumulate excess cyclopropane fatty acids from ingested bacteria . Cells in which mfeA(-) is disrupted fail to develop when grown in association with bacteria but form normal fruiting bodies when grown in axenic media . Bacterially grown mfeA(-) cells express the genes for the cyclic AMP (cAMP) receptor (carA) and adenylyl cyclase (acaA) but fail to respond to a cAMP pulse by synthesis of additional cAMP which normally relays the signal . Moreover, they do not accumulate the adhesion protein, gp80, which is encoded by the cAMP-induced gene, csaA . As a consequence, they do not acquire developmentally regulated EDTA-resistant cell-cell adhesion . When mutant cells are mixed with wild-type cells and allowed to develop together, they co-aggregate and differentiate into both spores and stalk cells . Thus, most of the developmental consequences of excess cyclopropane fatty acids appear to result from impaired cAMP relay. Mol Microbiol, 2004 Apr, 52(2), 515 - 27 Origins and evolution of isoprenoid lipid biosynthesis in archaea; Boucher Y et al.; A characteristic feature of the domain archaea are the lipids forming the hydrophobic core of their cell membrane . These unique lipids are composed of isoprenoid side-chains stereospecifically ether linked to sn-glycerol-1-phosphate . Recently, considerable progress has been made in characterizing the enzymes responsible for the synthesis of archaeal lipids . However, little is known about their evolution . To better understand how this unique biosynthetic apparatus came to be, large-scale database surveys and phylogenetic analyses were performed . All characterized enzymes involved in the biosynthesis of isoprenoid side-chains and the glycerol phosphate backbone along with their assembly in ether lipids were included in these analyses . The sequence data available in public databases was complemented by an in-depth sampling of isoprenoid lipid biosynthesis genes from multiple genera of the archaeal order Halobacteriales, allowing us to look at the evolution of these enzymes on a smaller phylogenetic scale . This investigation of the isoprenoid biosynthesis apparatus of archaea on small and large phylogenetic scales reveals that it evolved through a combination of evolutionary processes, including the co-option of ancestral enzymes, modification of enzymatic specificity, orthologous and non-orthologous gene displacement, integration of components from eukaryotes and bacteria and lateral gene transfer within and between archaeal orders. Mol Microbiol, 2004 Apr, 52(2), 485 - 500 Structural characterization of extracellular polysaccharides of Azorhizobium caulinodans and importance for nodule initiation on Sesbania rostrata; D'Haeze W et al.; During lateral root base nodulation, the microsymbiont Azorhizobium caulinodans enters its host plant, Sesbania rostrata, via the formation of outer cortical infection pockets, a process that is characterized by a massive production of H(2)O(2) . Infection threads guide bacteria from infection pockets towards nodule primordia . Previously, two mutants were constructed that produce lipopolysaccharides (LPSs) similar to one another but different from the wild-type LPS, and that are affected in extracellular polysaccharide (EPS) production . Mutant ORS571-X15 was blocked at the infection pocket stage and unable to produce EPS . The other mutant, ORS571-oac2, was impaired in the release from infection threads and was surrounded by a thin layer of EPS in comparison to the wild-type strain that produced massive amounts of EPS . Structural characterization revealed that EPS purified from cultured and nodule bacteria was a linear homopolysaccharide of alpha-1,3-linked 4,6-O-(1-carboxyethylidene)-D-galactosyl residues . In situ H(2)O(2) localization demonstrated that increased EPS production during early stages of invasion prevented the incorporation of H(2)O(2) inside the bacteria, suggesting a role for EPS in protecting the microsymbiont against H(2)O(2) . In addition, ex planta assays confirmed a positive correlation between increased EPS production and enhanced protection against H(2)O(2). Biochem J, 2004 Jul 1, 381(Pt 1), 307 - 12 Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer; Karasawa S et al.; GFP (green fluorescent protein)-based FRET (fluorescence resonance energy transfer) technology has facilitated the exploration of the spatio-temporal patterns of cellular signalling . While most studies have used cyan- and yellow-emitting FPs (fluorescent proteins) as FRET donors and acceptors respectively, this pair of proteins suffers from problems of pH-sensitivity and bleeding between channels . In the present paper, we demonstrate the use of an alternative additional donor/acceptor pair . We have cloned two genes encoding FPs from stony corals . We isolated a cyan-emitting FP from Acropara sp., whose tentacles exhibit cyan coloration . Similar to GFP from Renilla reniformis, the cyan FP forms a tight dimeric complex . We also discovered an orange-emitting FP from Fungia concinna . As the orange FP exists in a complex oligomeric structure, we converted this protein into a monomeric form through the introduction of three amino acid substitutions, recently reported to be effective for converting DsRed into a monomer (Clontech) . We used the cyan FP and monomeric orange FP as a donor/acceptor pair to monitor the activity of caspase 3 during apoptosis . Due to the close spectral overlap of the donor emission and acceptor absorption (a large Forster distance), substantial pH-resistance of the donor fluorescence quantum yield and the acceptor absorbance, as well as good separation of the donor and acceptor signals, the new pair can be used for more effective quantitative FRET imaging. Biochemistry, 2004 Apr 13, 43(14), 4394 - 9 The nuclear protein p34SEI-1 regulates the kinase activity of cyclin-dependent kinase 4 in a concentration-dependent manner; Li J et al.; Previous studies have shown that p34(SEI-1), also known as TRIP-Br1, is involved in cell cycle regulations by interacting with a number of important proteins including CDK4 . However, the detailed mechanism and structural basis of the interaction remains to be determined . We report the use of in vitro studies to address these problems . First, it was shown that p34(SEI-1) binds to CDK4 directly, and the binding does not compete directly with p16 . In the presence of p16, a quaternary complex is formed between p34(SEI-1), CDK4, cyclin D2, and p16 . Second, it was found that p34(SEI-1) activates the kinase activity of CDK4 at lower concentrations (reaching the maximum at 500 nM) but inhibits the same activity at higher concentrations, implying that p34(SEI-1)-mediated CDK4 activation is dose-dependent . Again, the effects of p34(SEI-1) and p16 are independent of each other . Third, it was shown that p34(SEI-1) possesses a LexA-mediated transactivation activity . Finally, a set of truncation mutants were used to dissect the structural elements responsible for the different functions of p34(SEI-1) . The results indicate that the fragment 30-160 can bind, activate, and inhibit CDK4; the fragment 30-132 can bind and activate but does not inhibit CDK4, while the fragment 30-88 cannot bind, activate, or inhibit but retains the LexA-mediated transactivation activity. J Struct Biol, 2004 Jan-Feb, 145(1-2), 100 - 10 SLEUTH--a fast computer program for automatically detecting particles in electron microscope images; Short JM; A method has been developed to locate biological complexes in a digitized electron micrograph by matching small windows to a set of reference images using a series of simple criteria . From the reference images, the program calculates parameters such as the radius of gyration, the density sum and variance . It compares them with corresponding values from a moving square window of densities extracted from the micrograph and records the coordinates of successfully matched candidate squares . Since the same particle is detected in a series of overlapping windows, candidates found to be within close proximity are grouped and the best-fitting one is selected from each cluster . The user is required only to select a small stack of boxed reference images and provide a few parameters, such as the particle radius and the minimum acceptable distance between particle centres . Micrograph labels and other areas that do not contain appropriate specimens are automatically ignored in order to minimize false positives . The program has been tested successfully on a variety of different biological structures, from both negatively stained and ice-embedded specimens. Plant Physiol, 2004 Apr, 134(4), 1775 - 83 Epub 2004 Apr 02. Adenylate gradients and Ar:O2 effects on legume nodules . II . Changes in the subcellular adenylate pools; Wei H et al.; Central infected zone tissue of soybean (Glycine max L . Merr.) nodules was fractionated into separate subcellular compartments using density gradient centrifugation in nonaqueous solvents to better understand how exposure to Ar:O(2) (80:20%, v/v) atmosphere affects C and N metabolism, and to explore a potential role for adenylates in regulating O(2) diffusion . When nodules were switched from air to Ar:O(2), adenylate energy charge (AEC) in the plant cytosol rose from 0.63 +/- 0.02 to 0.73 +/- 0.02 within 7 min and to 0.80 +/- 0.01 by 60 min . In contrast, AEC of the mitochondrial compartment of this central zone tissue remained high (0.80 +/- 0.02 to 0.81 +/- 0.02) following Ar treatment while that of the bacteroid compartment was unchanged, at 0.73 +/- 0.02, after 7 min, but declined to 0.57 +/- 0.03 after 60 min . These results were consistent with a simulation model that predicted Ar:O(2) exposure would first reduce ATP demand for ammonia assimilation and rapidly increase cytosolic AEC, before the Ar:O(2)-induced decline mediated by a decrease in nodule O(2) permeability reduces bacteroid AEC . The possibility that adenylates play a key, integrating role in regulating nodule permeability to oxygen diffusion is discussed. J Theor Biol, 2004 May 7, 228(1), 55 - 80 A model for the study of Helicobacter pylori interaction with human gastric acid secretion; Joseph IM et al.; We present a comprehensive mathematical model describing Helicobacter pylori interaction with the human gastric acid secretion system . We use the model to explore host and bacterial conditions that allow persistent infection to develop and be maintained . Our results show that upon colonization, there is a transient period (day 1-20 post-infection) prior to the establishment of persistence . During this period, changes to host gastric physiology occur including elevations in positive effectors of acid secretion (such as gastrin and histamine) . This is promoted by reduced somatostatin levels, an inhibitor of acid release . We suggest that these changes comprise compensatory mechanisms aimed at restoring acid to pre-infection levels . We also show that ammonia produced by bacteria sufficiently buffers acid promoting bacteria survival and growth. Curr Opin Microbiol, 2004 Apr, 7(2), 126 - 31 Regulation of gene expression by effectors that bind to RNA; Grundy FJ et al.; Recent studies have revealed several genetic systems in bacteria that use complex RNA structural elements to monitor regulatory signals and control expression of downstream genes . These include RNA thermosensors, in which an inhibitory structure melts at high temperature, and systems where binding of small RNAs or cellular metabolites modulates the structure of the RNA . The remarkable feature of these systems is the ability of the regulatory RNA elements to specifically sense the regulatory signal, without accessory components, and convey that information to the gene expression machinery via a structural change in the nascent RNA. Curr Opin Microbiol, 2004 Apr, 7(2), 115 - 9 A link between transcription and intermediary metabolism: a role for Sir2 in the control of acetyl-coenzyme A synthetase; Starai VJ et al.; The silent information regulator protein (Sir2) and its homologs (collectively known as sirtuins) are NAD+-dependent deacetylase enzymes involved in chromosome stability, gene silencing and cell aging in eukaryotes and archaea . The discovery that sirtuin-dependent protein deacetylation is a NAD+-consuming reaction established a link with the energy generation systems of the cell . This link to metabolism was recently extended to the post-translational control of the activity of short-chain fatty acyl-coenzyme A (adenosine monophosphate-forming) synthetases in bacteria and yeast . The crystal structure of the Sir protein complexed with a peptide of a protein substrate provided insights into how sirtuins interact with their protein substrates. FEMS Microbiol Lett, 2004 Apr 15, 233(2), 333 - 9 Increased transcription of a potential sigma factor regulatory gene Rv1364c in Mycobacterium bovis BCG while residing in macrophages indicates use of alternative promoters; Li MS et al.; Alternative sigma factors are key global regulators that coordinate bacterial responses to environmental changes necessary for adaptation and survival . In turn these sigma factors are controlled by regulators such as anti-sigma and anti-anti-sigma factors . In this report, using a cDNA-total RNA subtractive hybridisation strategy that we have developed previously, we identified increased transcription of a potential sigma factor regulatory gene, Rv1364c, in Mycobacterium bovis BCG upon phagocytosis by macrophages and this was confirmed by Northern blot analysis . Primer extension analysis revealed the use of alternative promotors, P1 and P2, and that the increased expression inside macrophages coincided with promoter switching from P2 to P1 . Rv1364c (653 amino acids), originally annotated as RsbU, contains structural domains homologous to the PAS redox sensor, the protein phosphatases anti-anti-sigma factor RsbU/SpoIIE, the protein kinase anti-sigma factor RsbW/SpoIIAB and the anti-anti-sigma factor RsbV/SpoIIAA found in other bacteria . These findings have important implications for understanding coordination of the expression of sigma factors under intra-macrophage conditions . Other potentially differentially expressed genes, including genes for fatty acid metabolism, membrane transportors, heat shock proteins, potential sigma factors and energy metabolic pathways are also listed and their biological significance discussed. Biotechnol Adv, 2004 May, 22(5), 363 - 82 Plants as models for the study of human pathogenesis; Guttman DS; There are many common disease mechanisms used by bacterial pathogens of plants and humans . They use common means of attachment, secretion and genetic regulation . They share many virulence factors, such as extracellular polysaccharides and some type III secreted effectors . Plant and human innate immune systems also share many similarities . Many of these shared bacterial virulence mechanisms are homologous, but even more appear to have independently converged on a common function . This combination of homologous and analogous systems reveals conserved and critical steps in the disease process . Given these similarities, and the many experimental advantages of plant biology, including ease of replication, stringent genetic and reproductive control, and high throughput with low cost, it is proposed that plants would make excellent models for the study of human pathogenesis. J Microbiol Methods, 2004 May, 57(2), 241 - 9 Recovery of Mycobacterium avium subspecies paratuberculosis from the natural host for the extraction and analysis in vivo-derived RNA; Granger K et al.; RNA has been extracted and analysed from in vivo-derived Mycobacterium avium subspecies paratuberculosis recovered from the natural host . The bacteria were selectively extracted from the intestinal tissue of two goats exhibiting clinical signs of Johne's disease . Small intestine was rapidly removed, luminal contents washed away and the mucosa and submucosa harvested . Mycobacteria in this material were released from the macrophages by isotonic lysis and differential centrifugation . RNA was extracted and compared with RNA extracted from bacteria grown in vitro . Real-time polymerase chain reaction was used to analyse the katG gene from the bacterial messenger RNA . The katG mRNA encoding the putative catalase/peroxidase showed differential expression in the in vivo and in vitro-derived samples . We hypothesize that the increase in katG expression for in vivo-derived M . paratuberculosis may represent a response to the oxidative stress encountered within the intra-macrophage environment. Curr Opin Chem Biol, 2004 Apr, 8(2), 120 - 6 Recent advances in the biocatalytic reduction of ketones and oxidation of sec-alcohols; Kroutil W et al.; To improve the efficiency and applicability of biocatalytic redox-reactions for asymmetric ketone-reduction and enantioselective alcohol-oxidation catalyzed by nicotinamide-dependent dehydrogenases/reductases, several achievements for cofactor-recycling have been made during the last two years . First, the use of hydrogenases for NADPH recycling in a two enzyme system . Second, preparative transformations with alcohol dehydrogenases coupled with NADH oxidases for NAD+/NADP+ recycling . Third, an exceptional chemo-stable alcohol dehydrogenase can efficiently use i-propanol and acetone as cosubstrates for reduction and oxidation, respectively, in a single-enzyme system . Novel carbonyl reductases and dehydrogenases derived from plant cells are particularly suited for sterically demanding substrates. Curr Opin Chem Biol, 2004 Apr, 8(2), 114 - 9 Enzyme catalysed deracemisation and dynamic kinetic resolution reactions; Turner NJ; New catalysts and reaction conditions have been developed for the dynamic kinetic resolution or deracemisation of racemic mixtures of chiral compounds . Specific functional groups that lend themselves particularly well to this approach include chiral secondary alcohols, alpha-amino acids, amines and carboxylic acids . A general theme of these processes is the combination of an enantioselective enzyme with a chemical reagent, the latter being used either to racemise the unreactive enantiomer or alternatively recycle an intermediate in the deracemisation process . In some examples of dynamic kinetic resolution, a second enzyme (racemase) is used to interconvert the enantiomers of the starting material. Curr Biol, 2004 Apr 6, 14(7), 625 - 9 Isolation and characterization of hemolymph clotting factors in Drosophila melanogaster by a pullout method; Scherfer C et al.; Clotting is critical in limiting loss of hemolymph and initiating wound healing in insects as well as in vertebrates . Clotting is also an important immune defense, quickly forming a secondary barrier to infection, thereby immobilizing, and possibly killing bacteria directly . Here, we describe methods to assess clotting and to extract the clot from Drosophila larval hemolymph by using aggregation of paramagnetic beads . The validity of the assay was demonstrated by characterization of mutants . We show that clotting occurs in the absence of phenoloxidase and that the Drosophila clot binds bacteria . We also describe a pullout assay to purify the clot as a whole, free from entrapped hemocytes and cellular debris . Proteins subsequently identified by mass spectrometry include both predicted and novel clot proteins . Immune induction has been shown for three of the latter, namely Tiggrin and two unknown proteins (GC15825 and CG15293) that we now propose function in hemolymph clotting . The most abundant clot protein is Hemolectin, and we confirm that hemolectin mutant larvae show clotting defects. Structure (Camb), 2004 Apr, 12(4), 677 - 87 Aspergillus niger protein EstA defines a new class of fungal esterases within the alpha/beta hydrolase fold superfamily of proteins; Bourne Y et al.; From the fungus Aspergillus niger, we identified a new gene encoding protein EstA, a member of the alpha/beta-hydrolase fold superfamily but of unknown substrate specificity . EstA was overexpressed and its crystal structure was solved by molecular replacement using a lipase-acetylcholinesterase chimera template . The 2.1 A resolution structure of EstA reveals a canonical Ser/Glu/His catalytic triad located in a small pocket at the bottom of a large solvent-accessible, bowl-shaped cavity . Potential substrates selected by manual docking procedures were assayed for EstA activity . Consistent with the pocket geometry, preference for hydrolysis of short acyl/propyl chain substrates was found . Identification of close homologs from the genome of other fungi, of which some are broad host-range pathogens, defines EstA as the first member of a novel class of fungal esterases within the superfamily . Hence the structure of EstA constitutes a lead template in the design of new antifungal agents directed toward its pathogenic homologs. Structure (Camb), 2004 Apr, 12(4), 669 - 76 A docking approach to the study of copper trafficking proteins; interaction between metallochaperones and soluble domains of copper ATPases; Arnesano F et al.; A structural model of the transient complex between the yeast copper chaperone Atx1 and the first soluble domain of the copper transporting ATPase Ccc2 was obtained with HADDOCK, combining NMR chemical shift mapping information with in silico docking . These two proteins are involved in copper trafficking in yeast cells . Calculations were performed starting with the copper ion either bound to Atx1 or to Ccc2 and using the experimental structures of the copper-loaded and apo forms of each protein . The copper binding motifs of the two proteins are found in close proximity . Copper tends to move from Atx1 to Ccc2, consistent with the physiological direction of transfer, with concomitant structural rearrangements, in agreement with experimental observations . The interaction is mainly of an electrostatic nature with hydrogen bonds stabilizing the complex . The structural data are relevant for a number of proteins homologous to Atx1 and Ccc2 and conserved from bacteria to humans. Plant Physiol Biochem, 2004 Jan, 42(1), 27 - 33 Ferroxidase activity in a laccase-like multicopper oxidase from Liriodendron tulipifera; Hoopes JT et al.; Ferroxidase activity was detected in a laccase-like multicopper oxidase (LMCO) produced in transgenic tobacco cells expressing an LMCO cDNA (Ltlacc2.2) cloned from yellow-poplar (Liriodendron tulipifera) . This marks the first report of ferroxidase activity associated with a plant laccase and suggests that some members of this plant enzyme family may have physiological functions based on activities other than their more widely recognized phenoloxidase activity . Recent work with LMCOs from bacteria, yeast and mammals has shown that metal oxidase activities in these enzymes can be important for their primary physiological functions, With respect to ferroxidase activity in certain plant LMCOs, it is proposed that the high levels of LMCO expression in plant vascular tissues may reflect the need for high-efficiency iron uptake pumps in tissues that undergo lignification during normal development . Such iron uptake pumps would function to minimize levels of free iron so that reactive oxygen species do not reach toxic levels when H2O2 is generated for peroxidase-mediated monolignol coupling during lignin deposition. Genome, 2004 Apr, 47(2), 404 - 13 Soybean FGAM synthase promoters direct ectopic nematode feeding site activity; Vaghchhipawala ZE et al.; Soybean cyst nematode (SCN) resistance in soybean is a complex oligogenic trait . One of the most important nematode resistance genes, rhg1, has been mapped to a distal region of molecular linkage group G in soybean . A simplified genetic system to identify soybean genes with modified expression in response to SCN led to the identification of several genes within the nematode feeding sites . The genes were mapped to reveal their linkage relationship to known QTLs associated with soybean cyst nematode (SCN) resistance . One candidate, a phosphoribosyl formyl glycinamidine (FGAM) synthase (EC 6.3.5.3) gene, mapped to the same genomic interval as the major SCN resistance gene rhg1 within linkage group G . Isolation of FGAM synthase from a soybean bacterial artificial chromosome (BAC) library revealed two highly homologous paralogs . The genes appeared to be well conserved between bacteria and humans . Promoter analysis of the two soybean homologs was carried out with the Arabidopsis thaliana - Heterodera schachtii system to investigate gene response to nematode feeding . The two promoters and their derived deletion constructions effected green fluorescent protein (GFP) expression within nematode feeding sites . The 1.0-kb promoter sequence immediately adjacent to the translation start site was sufficient to direct expression of GFP within syncytia . A wound-inducible element and a floral organ expression sequence were also identified within these promoters . Although a nematode-responsive element could not be identified, the observed expression of GFP within feeding sites supports the hypothesis that plant gene expression is redirected within feeding sites to benefit the parasite. Genome, 2004 Apr, 47(2), 299 - 303 Cytology of Wolbachia-induced parthenogenesis in Leptopilina clavipes (Hymenoptera: Figitidae); Pannebakker BA et al.; Parthenogenesis induced by cytoplasmatically inherited Wolbachia bacteria has been found in a number of arthropod species, mainly Hymenoptera . Previously, two different forms of diploidy restoration have been reported to underlie parthenogenesis induction in Hymenoptera by Wolbachia . Both are a form of gamete duplication, but each differs in their timing . We investigated the cytology of the early embryonic development of a Wolbachia-infected strain of the parasitoid wasp Leptopilina clavipes and compared it with that of an uninfected sexual strain . Both strains have a similar meiosis . In the infected parthenogenetic strain, diploidy is restored by anaphase restitution during the first somatic mitosis, similar to Trichogramma, but not to Muscidifurax . Our results confirm the occurrence of different cytological mechanisms of diploidy restoration associated with parthenogenesis-inducing Wolbachia in the order Hymenoptera. J Bacteriol, 2004 Apr, 186(8), 2457 - 65 Population-based genetic and evolutionary analysis of Chlamydia trachomatis urogenital strain variation in the United States; Millman K et al.; Chlamydia trachomatis is a major cause of ocular and sexually transmitted diseases worldwide . While much of our knowledge about its genetic diversity comes from serotyping or ompA genotyping, no quantitative assessment of genetic diversity within serotypes has been performed . To accomplish this, 507 urogenital samples from a multicenter U.S . study were analyzed by phylogenetic and statistical modeling . No B, Da, or I serotypes were represented . Based on our analyses, all but one previous urogenital B serotype was identified as Ba . This, coupled with the lack of B serotypes in our population, suggests that B has specific tropism for ocular mucosa . We identified a Ba/D recombinant (putative crossover nucleotide 477; P < 0.0001) similar to a B/D mosaic we described previously from an African trachoma patient . Computational analyses of the Ba/D recombinant indicated that upstream changes were less important for tissue tropism than downstream incorporation of the D sequence . Since most serotypes had nonsynonymous/synonymous ratios of <1.0, the major outer membrane protein, encoded by ompA, has many functional constraints and is under purifying selection . Surprisingly, all serotype groups except for J had a unimodal population structure indicating rapid clonal expansion . Of the groups with a unimodal structure, E and Ia and, to a lesser extent, G and K were prevalent, had infrequent incorporation of mutations, and, compared to other groups, had a relatively greater degree of diversifying selection, consistent with a selective sweep of mutations within these groups . Collectively, these data suggest a diverse evolutionary strategy for different serogroups of the organism. J Bacteriol, 2004 Apr, 186(8), 2253 - 65 The Helicobacter pylori flaA1 and wbpB genes control lipopolysaccharide and flagellum synthesis and function; Merkx-Jacques A et al.; flaA1 and wbpB are conserved genes with unknown biological function in Helicobacter pylori . Since both genes are predicted to be involved in lipopolysaccharide (LPS) biosynthesis, flagellum assembly, or protein glycosylation, they could play an important role in the pathogenesis of H . pylori . To determine their biological role, both genes were disrupted in strain NCTC 11637 . Both mutants exhibited altered LPS, with loss of most O-antigen and core modification, and increased sensitivity to sodium dodecyl sulfate compared to wild-type bacteria . These defects could be complemented in a gene-specific manner . Also, flaA1 could complement these defects in the wbpB mutant, suggesting a potential redundancy of the reductase activity encoded by both genes . Both mutants were nonmotile, although the wbpB mutant still produced flagella . The defect in the flagellum functionality of this mutant was not due to a defect in flagellin glycosylation since flagellins from wild-type strain NCTC 11637 were shown not to be glycosylated . The flaA1 mutant produced flagellins but no flagellum . Overall, the similar phenotypes observed for both mutants and the complementation of the wbpB mutant by flaA1 suggest that both genes belong to the same biosynthesis pathway . The data also suggest that flaA1 and wbpB are at the interface between several pathways that govern the expression of different virulence factors . We propose that FlaA1 and WbpB synthesize sugar derivatives dedicated to the glycosylation of proteins which are involved in LPS and flagellum production and that glycosylation regulates the activity of these proteins. Res Microbiol, 2004 Apr, 155(3), 192 - 200 The Mycobacterium tuberculosis Rv2358-furB operon is induced by zinc; Milano A et al.; The Mycobacterium tuberculosis genome encodes two ferric uptake regulator homologues, furA and furB, the function of which is under investigation . Using Mycobacterium smegmatis as a model system, we investigated the transcriptional pattern of Rv(Ms)2358-furB genes . Transcripts covering the two genes could be identified by northern blotting and by reverse transcriptase PCR . The transcriptional start point was mapped at one base upstream of the Ms2358 start codon by the RACE technique . By cloning M . smegmatis and M . tuberculosis DNA regions upstream of a reporter gene, we demonstrated the presence of one promoter, located immediately upstream of the Rv(Ms)2358 gene . Promoter induction was tested on several cultures grown under different conditions of pH and temperature, and in the presence of different concentrations of metallic ions . The promoter was found to be specifically induced by zinc . The recombinant M . tuberculosis FurB protein typically contained two zinc ions per protein monomer . Complete removal of zinc could not be obtained, even with strong denaturation treatment . Our data are in favour of the hypothesis that Rv2358 and FurB are transcriptional regulators involved in zinc homeostasis. Res Microbiol, 2004 Apr, 155(3), 154 - 61 Repeatability and reproducibility of ribotyping and its computer interpretation; Lefresne G et al.; Many molecular typing methods are difficult to interpret because their repeatability (within-laboratory variance) and reproducibility (between-laboratory variance) have not been thoroughly studied . In the present work, ribotyping of coryneform bacteria was the basis of a study involving within-gel and between-gel repeatability and between-laboratory reproducibility (two laboratories involved) . The effect of different technical protocols, different algorithms, and different software for fragment size determination was studied . Analysis of variance (ANOVA) showed, within a laboratory, that there was no significant added variance between gels . However, between-laboratory variance was significantly higher than within-laboratory variance . This may be due to the use of different protocols . An experimental function was calculated to transform the data and make them compatible (i.e., erase the between-laboratory variance) . The use of different interpolation algorithms (spline, Schaffer and Sederoff) was a significant source of variation in one laboratory only . The use of either Taxotron (Institut Pasteur) or GelCompar (Applied Maths) was not a significant source of added variation when the same algorithm (spline) was used . However, the use of Bio-Gene (Vilber Lourmat) dramatically increased the error (within laboratory, within gel) in one laboratory, while decreasing the error in the other laboratory; this might be due to automatic normalization attempts . These results were taken into account for building a database and performing automatic pattern identification using Taxotron . Conversion of the data considerably improved the identification of patterns irrespective of the laboratory in which the data were obtained. Zhonghua Shao Shang Za Zhi, 2004 Feb, 20(1), 23 - 5 {Study of biosensor technology on the detection of endotoxin-neutralizing materials}; Lu GF et al.; OBJECTIVE: To explore the application of biosensor technology in the determination of endotoxin-neutralizing materials . METHODS: After mixing polymyxin B (PMB) with endotoxin in certain concentration, the neutralizing ratio of PMB to endotoxin was assessed by biosensor technique and limulus amebocyte lysate test respectively . The results from the two methods were compared . RESULTS: The neutralizing ratio of PMB to endotoxin as assessed by biosensor technology was 0.35 microg to 1 ng, while that by dynamic turbidimetric and chromogenic limulus amebocyte lysate (LAL) technique was 0.5 mg to 1 ng and 1 mg to 1 ng, respectively . The results obtained by biotechnology were similar to that by biosensor technique . CONCLUSION: Biosensor technology was an accurate, convenient and rapid method for the determination of potency of endotoxin-neutralizing materials. Zhonghua Shao Shang Za Zhi, 2004 Feb, 20(1), 20 - 2 {Prospective study on the gastro-pulmonary infection route of ventilator-associated pneumonia}; Zhang QL et al.; OBJECTIVE: To explore the role of gastro-pulmonary infection route in the development of ventilator-associated pneumonia (VAP), so as to improve the management of VAP . METHODS: Forty-three patients who received mechanical ventilation (MV) were enrolled in the study . Intra-gastric contents were labeled with (99)mTc-DTPA . Randomized two-period crossover trial was employed to determine the radioactive level in the oropharyngeal and bronchial secretion when patients were in supine or semi-reclining position . Gastric juice, oropharyngeal secretion and tracheal lavage fluid were collected for bacterial culture every other day . Bronchoalveolar lavage fluid (BALF) was harvested from those suspected of VAP for quantitative bacterial culture . Infrequent-restriction site amplification (IRS-PCR) was employed in the identification of the identity of the bacteria from intra-gastric colonization with those causing VAP . The sIgA content in the BALF was determined . RESULTS: The gastroesophageal regurgitation rate was higher (89.7%) with lower aspiration rate (28.5%) in patients receiving MV . Moreover, the aspiration rate and the radioactivity of deep tracheal aspirates in patients in supine position were significantly higher than those in semi-reclining position (P < 0.01) . There was high homology of the bacteria isolated from intra-gastric colonization with that causing VAP (55.8%) . The sIgA content in BALF in VAP patients was evidently lower than that in non-VAP patients (P < 0.01) . CONCLUSION: Regurgitation and aspiration of stomach contents are very common in patients receiving MV . Intra-gastric colonized bacteria might be one of the important origins causing VAP . The lowering of sIgA in BALF in patients with MV could be a risk factor for VAP. Clin Microbiol Infect, 2004 Apr, 10(4), 272 - 88 Resistance integrons and super-integrons; Fluit AC et al.; Integrons are genetic elements composed of a gene encoding an integrase, gene cassettes and an integration site for the gene cassettes (att) . The integrase excises and integrates the gene cassettes from and into the integron, but integrons themselves are not mobile . Two groups of integrons are known: resistance integrons and super-integrons . Nearly all known gene cassettes from resistance integrons encode resistance to antibiotics or disinfectants . These integrons are found on transposons, plasmids and the bacterial chromosome . Gene cassettes in super-integrons encode a variety of different functions . Super-integrons are located on the bacterial chromosome . More than 100 gene cassettes may be present, in contrast to resistance integrons where less than ten cassettes are present . Many species harbour super-integrons, which are species-specific, whereas particular resistance integrons can be found in a variety of species . The gene cassettes in resistance integrons probably originated from super-integrons . In the last few years, a variety of new gene cassettes have been described . Many of these encode resistance against newer antibiotics such as cephalosporins and carbapenems . Resistance integrons have been found in isolates from a wide variety of sources, including food. Hepatology, 2004 Apr, 39(4), 970 - 7 Variation of hepatic glucuronidation: Novel functional polymorphisms of the UDP-glucuronosyltransferase UGT1A4; Ehmer U et al.; UDP-glucuronosyltransferases are a family of drug metabolizing enzymes contributing to hepatic drug metabolism and protection against environmental toxins . The aim of this study was to identify polymorphisms at the human UGT1A gene locus and to characterize their function and potential association with hepatocellular carcinoma (HCC) . Genomic DNA from the blood of 363 subjects (128 patients with HCC, 235 blood donors) was analyzed for polymorphisms of the UGT1A3, UGT1A4, UGT1A8, UGT1A9, UGT1A10 genes using polymerase chain reaction, sequencing analysis . Recombinant variant UGT protein was analyzed by activity assays . In the UGT1A8 gene an A173G variant and a conserved G to A exchange at position 765 were detected in 25% and 15% . UGT1A9 exhibited two variants C3Y and M33T in 1% and 3% . UGT1A10 exhibited conserved nucleotide exchanges (128 G-->A and 696 C-->T) in 2% and 13% . In the UGT1A3 gene a W11R, a V47A variant, and a conserved G to A exchange at position 81 with an incidence of 65%, 58%, and 65%, respectively, were identified . UGT1A4 exhibited a P24T and an L48V variant in 8% and 9% . UGT1A SNPs were not associated with HCC . UGT1A4 P24T and L48V exhibited reduced glucuronidation activities: beta-naphthylamine 30% and 50%, and dihydrotestosterone 50% and 0%, respectively . In conclusion, the high prevalence of SNPs throughout the human UGT1A gene locus illustrates a genetic basis of interindividual variations of hepatic metabolism . Two polymorphisms of the hepatic UGT1A4 protein show a differential metabolic activity toward mutagenic amines and endogenous steroids, altering hepatic metabolism and detoxification. IDrugs, 2004 Apr, 7(4), 359 - 73 Betulinic acid: a promising anticancer candidate; Eiznhamer DA et al.; Betulinic acid is a naturally occurring pentacyclic triterpenoid which has demonstrated selective cytotoxicity against a number of specific tumor types, a variety of infectious agents such as HIV, malaria and bacteria, and the inflammatory process in general . Biological activity was first demonstrated in melanoma cell lines and was confirmed in mice bearing human melanoma xenografts . These in vivo studies also established a favorable safety margin for betulinic acid, as systemic side effects were not observed at any dose . Recently, considerable in vitro evidence has demonstrated that betulinic acid is effective against small- and non-small-cell lung, ovarian, cervical, and head and neck carcinomas . Published data suggest that betulinic acid induces apoptosis in sensitive cells in a p53- and CD95-independent fashion . While the precise molecular target and mechanism of action remain elusive and are the focus of a number of ongoing research programs, accumulated experimental evidence indicates that betulinic acid functions through a mitochondrial-mediated pathway . Supplemental reports suggest that the generation of reactive oxygen species, inhibition of topoisomerase I, activation of the MAP kinase cascade, inhibition of angiogenesis, and modulation of pro-growth transcriptional activators and aminopeptidase N activity may play a role in betulinic acid-induced apoptosis . These potential mechanisms of action may enable betulinic acid to be effective in cells resistant to other chemotherapeutic agents . Arguments supporting the role of this agent in the treatment of cancers and other infectious conditions will be reviewed. Biol Pharm Bull, 2004 Apr, 27(4), 480 - 5 Human DNA glycosylases involved in the repair of oxidatively damaged DNA; Ide H et al.; Reactive oxygen species from endogenous and environmental sources induce oxidative damage to DNA, and hence pose an enormous threat to the genetic integrity of cells . Such oxidative DNA damage is restored by the base excision repair (BER) pathway that is conserved from bacteria to humans and is initiated by DNA glycosylases, which simply remove the aberrant base from the DNA backbone by hydrolyzing the N-glycosidic bond (monofunctional DNA glycosylase), or further catalyze the incision of a resulting abasic site (bifunctional DNA glycosylase) . In human cells, oxidative pyrimidine lesions are generally removed by hNTH1, hNEIL1, or hNEIL2, whereas oxidative purine lesions are removed by hOGG1 . hSMUG1 excises a subset of oxidative base damage that is poorly recognized by the above enzymes . Unlike these enzymes, hMYH removes intact A misincorporated opposite template 8-oxoguanine during DNA replication . Although hNTH1, hOGG1, and hMYH account for major cellular glycosylase activity for inherent substrate lesions, mouse models deficient in the enzymes exhibit no overt phenotypes such as the development of cancer, implying backup mechanisms . Contrary to the mouse model, hMYH mutations have been shown to lead to a multiple colorectal adenoma syndrome and high colorectal cancer risk . For cleavage of the N-glycosidic bond, bifunctional DNA glycosylases (hNTH1, hNEIL1, hNEIL2, and hOGG1) use Lys or Pro for direct attack on sugar C1', whereas monofunctional DNA glycosylases (hSMUG1 and hMYH) use an activated water molecule . DNA glycosylases for oxidative damage, if not all, are covalently trapped by DNA containing 2-deoxyribonolactone or oxanine . Thus, the depletion of functional DNA glycosylases using covalent trapping may reduce the BER capacity of cancer cells, hence potentiating the efficacy of anticancer drugs or radiation therapy. Curr Allergy Asthma Rep, 2004 May, 4(3), 200 - 7 Refractory chronic rhinosinusitis: pathophysiology and management of chronic rhinosinusitis persisting after endoscopic sinus surgery; Desrosiers M; Refractory chronic rhinosinusitis (RCRS) is defined as persistence of signs and symptoms of chronic rhinosinusitis, despite technically adequate endoscopic sinus surgery . Rather than a simple, prolonged bout of acute sinusitis, it instead appears to be secondary to an interaction of a susceptible host with the outside environment . Inflammatory responses to colonizing bacteria appear to be responsible for a significant portion of the pathophysiology . Reduction of bacterial load and inflammation of the mucosa play an important role in controlling the disease . Novel treatment strategies, with an emphasis on topical therapies, seem to offer optimal management . In this review, current concepts on the pathophysiology and current therapies available for RCRS are outlined . A practical management strategy based on the author's personal experience draws upon these concepts, and is detailed in this review of an unusual topic. Cell Microbiol, 2004 May, 6(5), 435 - 45 Adherence of Brucella to human epithelial cells and macrophages is mediated by sialic acid residues; Castaneda-Roldan EI et al.; The basis for the interaction of Brucella species with the surface of epithelial cells before migration in the host within polymorphonuclear leucocytes is largely unknown . Here, we studied the ability of Brucella abortus and Brucella melitensis to adhere to cultured epithelial (HeLa and HEp-2) cells and THP-1-derived macrophages, and to bind extracellular matrix proteins (ECM) . The brucellae adhered to epithelial cells forming localized bacterial microcolonies on the cell surface, and this process was inhibited significantly by pretreatment of epithelial cells with neuraminidase and sodium periodate and by preincubation of the bacteria with heparan sulphate and N-acetylneuraminic acid . Trypsinization of epithelial cells yielded increased adherence, suggesting unmasking of target sites on host cells . Notably, the brucellae also adhered to cultured THP-1 cells, and this event was greatly reduced upon removal of sialic acid residues from these cells with neuraminidase . B . abortus bound in a dose-dependent manner to immobilized fibronectin and vitronectin and, to a lesser extent, to chondroitin sulphate, collagen and laminin . In sum, our data strongly suggest that the adherence mechanism of brucellae to epithelial cells and macrophages is mediated by cellular receptors containing sialic acid and sulphated residues . The recognition of ECM (fibronectin and vitronectin) by the brucellae may represent a mechanism for spread within the host tissues . These are novel findings that offer new insights into understanding the interplay between Brucella and host cells. Allergy Asthma Proc, 2004 Jan-Feb, 25(1), 27 - 30 The biology of the mast cell; Boyce JA; Mast cells are ancient, versatile immune effector cells . On the one hand, they are endowed with unique effector capabilities and activation responses that initiate innate immunity to bacteria and are essential to host defense against helminthic parasites . On the other hand, they are the major effector of type I hypersensitivity and an important participant in a number of disease processes . This review focuses on the mechanisms of mast cell development, the cytokine control of this process, and the amplification of mast cell effector systems as an important determinant of disease. J Endod, 2004 Mar, 30(3), 167 - 72 Evaluation of ultrasonically placed MTA and fracture resistance with intracanal composite resin in a model of apexification; Lawley GR et al.; The purpose of this study was to evaluate whether intracoronal delivery of an apical barrier of mineral trioxide aggregate (MTA) placed ultrasonically, non-ultrasonically, or ultrasonically with the addition of an intracanal composite resin provided a better seal against bacterial leakage . A second purpose was to determine whether intracanal composite resin or gutta-percha and sealer placed against an apical barrier of MTA provided greater resistance to root fracture . In a standardized in vitro open apex model, MTA was placed as an apical barrier at a thickness of 4 mm, with and without ultrasonic vibration . The barriers were challenged with bacteria exposure within a leakage model, and fracture resistance was assessed with increasing forces applied via an Instron machine . After 45 days, the addition of ultrasonics significantly improved the MTA seal, compared with the non-ultrasonics treatment (Kruskal Wallis nonparametric ANOVA with Dunn multiple comparison test p < 0.05) . Bacterial leakage occurred in 6 (33%) of 18 in the non-ultrasonic MTA group, 2 (11%) of 18 in the ultrasonic MTA group, and 1 (6%) of 18 in the ultrasonic MTA-composite group . There were no significant differences at 90 days . A 4-mm thickness of MTA followed with an intracanal composite resin demonstrated a significantly greater resistance to root fracture than MTA followed with gutta-percha and sealer (one-way ANOVA with Newman-Keuls multiple comparison test, p < 0.01) . The MTA-gutta-percha group was not significantly different than the MTA unrestored positive control. Cell Biochem Biophys, 2004, 40(2), 149 - 65 Fluorescence spectroscopy of single photosynthetic light-harvesting supramolecular systems; Saga Y et al.; Recent spectroscopic studies of photosynthetic light-harvesting supramolecular complexes at the single supramolecule level are reviewed . This report describes the "single-molecule" investigation on light-harvesting complex 2 (LH2) of purple photosynthetic bacteria, phycobiliproteins of cyanobacteria and red algae, light-harvesting complex 2 (LHC2) of higher plants, and chlorosomes of green photosynthetic bacteria . Unique behaviors and spectral features of single light-harvesting apparatus have been unraveled that were hidden by the ensemble averaging of many of the complexes . The information obtained will be useful for understanding the electronic structures and energy-transfer mechanism of photosynthetic light-harvesting supramolecular systems. Mol Cell, 2004 Mar 26, 13(6), 843 - 51 Positional recognition of a tRNA determinant dependent on a peptide insertion; Lovato MA et al.; The crystal structure of a catalytic fragment of Aquifex aeolicus AlaRS and additional data suggest how the critical G3:U70 identity element of its cognate tRNA acceptor stem is recognized . Though this identity element is conserved from bacteria to the cytoplasm of eukaryotes, Drosophila melanogaster mitochondrial (Dm mt) tRNA(Ala) contains a G:U base pair that has been translocated to the adjacent 2:71 position . This G2:U71 is the major determinant for identity of Dm mt tRNA(Ala) . Sequence alignments showed that Dm mt AlaRS is differentiated from G3:U70-recognizing AlaRSs by an insertion of 27 amino acids in the region of the protein that contacts the acceptor stem . Precise deletion of this insertion from Dm mt AlaRS gave preferential recognition to a G3:U70-containing substrate . Larger or smaller deletions were ineffective . The crystal structure of the orthologous A . aeolicus protein places this insertion on the surface, where it can act as a hinge that provides positional switching of G:U recognition. J Am Chem Soc, 2004 Apr 7, 126(13), 4228 - 33 Photoactivation of the photoactive yellow protein: why photon absorption triggers a trans-to-cis Isomerization of the chromophore in the protein; Groenhof G et al.; Atomistic QM/MM simulations have been carried out on the complete photocycle of Photoactive Yellow Protein, a bacterial photoreceptor, in which blue light triggers isomerization of a covalently bound chromophore . The "chemical role" of the protein cavity in the control of the photoisomerization step has been elucidated . Isomerization is facilitated due to preferential electrostatic stabilization of the chromophore's excited state by the guanidium group of Arg52, located just above the negatively charged chromophore ring . In vacuo isomerization does not occur . Isomerization of the double bond is enhanced relative to isomerization of a single bond due to the steric interactions between the phenyl ring of the chromophore and the side chains of Arg52 and Phe62 . In the isomerized configuration (ground-state cis), a proton transfer from Glu46 to the chromophore is far more probable than in the initial configuration (ground-state trans) . It is this proton transfer that initiates the conformational changes within the protein, which are believed to lead to signaling. J Agric Food Chem, 2004 Apr 7, 52(7), 2079 - 83 Dehydrotomatine and alpha-tomatine content in tomato fruits and vegetative plant tissues; Kozukue N et al.; Tomato plants (Lycopersicon esculentum) synthesize the glycoalkaloids dehydrotomatine and alpha-tomatine, possibly as a defense against bacteria, fungi, viruses, and insects . We used a high-performance liquid chromatography method with UV detection at 208 nm for the analysis of these compounds in various tissues . An Inertsil ODS-2 column with a mobile phase of acetonitrile/20 mM KH2PO4 (24/76, v/v) afforded good separation of the two glycoalkaloids in mini-tomato extracts, fruit harvested at different stages of maturity, and calyxes, flowers, leaves, roots, and stems . The two peaks appeared at approximately 17 and approximately 21 min . Recoveries from tomato fruit extracts spiked with dehydrotomatine and alpha-tomatine were 87.7 +/- 6.8 and 89.8 +/- 3.4% (n = 5), respectively . The detection limit is estimated to be 0.39 microg for dehydrotomatine and 0.94 microg for alpha-tomatine . The dehydrotomatine and alpha-tomatine content of tomatoes varied from 42 to 1498 and 521 to 16 285 microg/g of fresh weight, respectively . The ratio of alpha-tomatine to dehydrotomatine ranged from 10.9 to 12.5 in tomatoes and from 2.3 to 7.8 in the other plant tissues . These results suggest that the biosynthesis of the glycoalkaloids is under separate genetic control in each plant part . Degradation of both glycoalkaloids occurred at approximately the same rate during maturation of the tomatoes on the vine . An Inertsil NH2 column, with acetonitrile/1 mM KH2PO4 (96/4, v/v) as the eluent, enabled the fractionation of commercial tomatidine into tomatidenol and tomatidine, the aglycons of dehydrotomatine and alpha-tomatine, respectively . The information should be useful for evaluating tomatoes and vegetative tissues for dehydrotomatine/alpha-tomatine content during fruit development and their respective roles in host-plant resistance and the diet. World J Gastroenterol, 2004 Apr 1, 10(7), 930 - 3 Intestinal failure: pathophysiological elements and clinical diseases; Ding LA et al.; There are two main functions of gastrointestinal tract, digestion and absorption, and barrier function . The latter has an important defensive effect, which keeps the body away from the invading and damaging of bacteria and endotoxin . It maintains the systemic homeostasis . Intestinal dysfunction would happen when body suffers from diseases or harmful stimulations . The lesser dysfunction of GI tract manifests only disorder of digestion and absorption, whereas the more serious intestinal disorders would harm the intestinal protective mechanism, or intestinal barrier function, and bacterial/endotoxin translocation, of intestinal failure (IF) would ensue . This review discussed the theory of the intestinal failure, aiming at attracting recognition and valuable comments by clinicians. Biotechnol Bioeng, 2004 Apr 20, 86(2), 125 - 35 Bulking sludge in biological nutrient removal systems; Martins AM et al.; Bulking sludge problems are commonly reported in biological nutrient removal (BNR) systems . This has led to the general belief that intrinsic BNR conditions favor the growth of undesirable and excessive filamentous bacteria . The present study shows that other factors have a major role in bulking, and not the BNR conditions . These factors have been verified in well-controlled, strictly anoxic-aerobic and strictly anaerobic-aerobic sequencing batch reactor systems . The experimental results show that conditions known to be responsible for bulking sludge in aerobic systems (i.e., low concentration of electron donor and/or electron acceptor) did not lead to bulking . Even when acetate was present at very low concentrations in the aerobic stage of an anaerobic-aerobic bio-P system, the sludge settleability remained very good . This clearly demonstrates that good bio-P activity can stabilize and improve sludge settleability . The presence of microaerophilic conditions in the anoxic stage of the anoxic-aerobic system was the only factor leading to worsening sludge settling characteristics . The results are discussed in light of our previous hypothesis about the importance of diffusion-limited substrate uptake for the development of filamentous structures in biological flocs . The hypothesis is extended to anaerobic-aerobic and anoxic-aerobic conditions, typical of BNR-activated sludge systems . Taking into account the effect of feeding patterns on biochemical rates and on the development of filamentous bacterial structures, we recommend the adoption of plug-flow selector configurations, with strictly anaerobic and/or strictly anoxic conditions, wherein microaerophilic conditions are excluded, in order to maintain reliable and robust BNR performance . Gastric Cancer, 2004, 7(1), 9 - 16 The future of gastric cancer prevention; Correa P et al.; Despite advances in surgical treatment and chemotherapy, gastric cancer remains a major global health burden . The most recent estimates show that it is the fourth most common cancer and the second most common cause of cancer deaths worldwide . Various etiologic factors have been linked with the disease . It is widely accepted that Helicobacter pylori infection and high salt intake are positively associated with this neoplastic process . Controversial associations have been found with smoking or drinking habits . In contrast, there is convincing evidence that the adequate consumption of fresh fruits and vegetables reduces the risk of gastric cancer . Prevention intervention trials involving antioxidant supplements and anti- H . pylori treatment have shown beneficial effects in preventing the progression of pathologic changes in the gastric mucosa . On the other hand, recent advances related to differences in the genotypes of the bacteria and in human cytokine polymorphisms would allow the design and implementation of large-scale screening programs to identify subjects at the highest risk of gastric cancer . Curing the infection in such subjects and supplying adequate amounts of antioxidants should prevent a neoplastic outcome, and this intervention should be monitored by endoscopic surveillance. Biochem Cell Biol, 2004 Feb, 82(1), 225 - 53 Phospholipase D; McDermott M et al.; Phospholipase D catalyses the hydrolysis of the phosphodiester bond of glycerophospholipids to generate phosphatidic acid and a free headgroup . Phospholipase D activities have been detected in simple to complex organisms from viruses and bacteria to yeast, plants, and mammals . Although enzymes with broader selectivity are found in some of the lower organisms, the plant, yeast, and mammalian enzymes are selective for phosphatidylcholine . The two mammalian phospholipase D isoforms are regulated by protein kinases and GTP binding proteins of the ADP-ribosylation and Rho families . Mammalian and yeast phospholipases D are also potently stimulated by phosphatidylinositol 4,5-bisphosphate . This review discusses the identification, characterization, structure, and regulation of phospholipase D . Genetic and pharmacological approaches implicate phospholipase D in a diverse range of cellular processes that include receptor signaling, control of intracellular membrane transport, and reorganization of the actin cytoskeleton . Most ideas about phospholipase D function consider that the phosphatidic acid product is an intracellular lipid messenger . Candidate targets for phospholipase-D-generated phosphatidic acid include phosphatidylinositol 4-phosphate 5-kinases and the raf protein kinase . Phosphatidic acid can also be converted to two other lipid mediators, diacylglycerol and lyso phosphatidic acid . Coordinated activation of these phospholipase-D-dependent pathways likely accounts for the pleitropic roles for these enzymes in many aspects of cell regulation. Proc Natl Acad Sci U S A, 2004 Apr 6, 101(14), 4848 - 53 Epub 2004 Mar 29. HetR homodimer is a DNA-binding protein required for heterocyst differentiation, and the DNA-binding activity is inhibited by PatS; Huang X et al.; HetR plays a key role in regulation of heterocyst differentiation . When the Cys-48 residue of the HetR from Anabaena sp . PCC 7120 was replaced with an Ala residue, the mutant HetR (HetR(C48A)) could not dimerize, indicating that HetR forms a homodimer through a disulfide bond . The Anabaena strain C48, containing the hetRc48a gene, could not produce HetR homodimer and failed to form heterocyst . We show that HetR is a DNA-binding protein and that its homodimerization is required for the DNA binding . HetR binds the promoter regions of hetR, hepA, and patS, suggesting a direct control of the expression of these genes by HetR . We present evidence that shows that the up-regulation of patS and hetR depends on DNA binding by HetR dimer . The pentapeptide RGSGR, which is present at the C terminus of PatS and blocks heterocyst formation, inhibits the DNA binding of HetR and prevents hetR up-regulation. Environ Int, 2004 Jul, 30(5), 741 - 59 Fate and transport of pathogens in lakes and reservoirs; Brookes JD et al.; Outbreaks of water-borne disease via public water supplies continue to be reported in developed countries even though there is increased awareness of, and treatment for, pathogen contamination . Pathogen episodes in lakes and reservoirs are often associated with rain events, and the riverine inflow is considered to be major source of pathogens . Consequently, the behaviour of these inflows is of particular importance in determining pathogen transport and distribution . Inflows are controlled by their density relative to that of the lake, such that warm inflows will flow over the surface of the lake as a buoyant surface flow and cold, dense inflows will sink beneath the lake water where they will flow along the bathymetry towards the deepest point . The fate of pathogens is determined by loss processes including settling and inactivation by temperature, UV and grazing . The general trend is for the insertion timescale to be shortest, followed by sedimentation losses and temperature inactivity . The fate of Cryptosporidium due to UV light inactivation can occur at opposite ends of the scale, depending on the location of the oocysts in the water column and the extinction coefficient for UV light . For this reason, the extinction coefficient for UV light appears to be a vitally important parameter for determining the risk of Cryptosporidium contamination . For risk assessment of pathogens in supply reservoirs, it is important to understand the role of hydrodynamics in determining the timescale of transport to the off-take relative to the timescale of inactivation . The characteristics of the riverine intrusion must also be considered when designing a sampling program for pathogens . A risk management framework is presented that accounts for pathogen fate and transport for reservoirs. Bioresour Technol, 2004 Jun, 93(2), 155 - 67 Mesophilic anaerobic treatment of sludge from saline fish farm effluents with biogas production; Gebauer R; The mesophilic anaerobic treatment of sludge from saline fish farm effluents (total solids (TS): 8.2-10.2 wt%, chemical oxygen demand (COD): 60-74 g/l, sodium (Na): 10-10.5 g/l) was carried out in continuously stirred tank reactors (CSTRs) at 35 degrees C . COD stabilization between 36% and 55% and methane yields between 0.114 and 0.184 l/g COD added were achieved . However, the process was strongly inhibited, presumably by sodium, and unstable, with propionic acid being the main compound of the volatile fatty acids (VFA) . When diluting the sludge 1:1 with tap water (Na: 5.3 g/l), the inhibition could be overcome and a stable process with low VFA concentrations was achieved . The results of the study are used to make recommendations for the configuration of full-scale treatment plants for the collected sludge from one salmon farming licence and to estimate the energy production from these plants . Microbes Infect, 2004 Apr, 6(4), 369 - 76 Protection against Chlamydia trachomatis infection in vitro and modulation of inflammatory response in vivo by membrane-bound glycosaminoglycans; Darville T et al.; Glycosaminoglycans (GAG) efficiently inhibit adherence of several strains of Chlamydia trachomatis to cell lines in vitro, but none of the GAG have been able to inhibit infections in vivo . One possible cause for failure of GAG inhibition in vivo is the inability to deliver a sustained concentration of GAG at the mucosal surface . We tested the possibility of enhancing cell protection by increasing the cell-surface concentration of GAG using membrane-anchored GAG (MAG), composed of phosphatidylethanolamine (PE)-linked GAG . These lipid conjugates were originally designed as extracellular phospholipase A2 (PLA2) inhibitors and exhibit a dual effect: the lipid moiety incorporates into the cell membrane, interfering with the action of PLA2 on cell membranes, and the anchored GAG protects the cell membrane from exogenous inflammatory mediators . We tested the ability of MAG to block chlamydia infection in vitro and in vivo . The MAG blocked infection of epithelial cells in vitro when added to the cells at the same time or before infection, but not if added after the bacteria had already invaded the host cells . One of the MAG led to the production of aberrant Chlamydia vacuoles, suggesting it may inhibit intracellular PLA2 associated with development of the vacuole . Although the MAG did not inhibit vaginal infection of mice, they decreased significantly the level of secretion of the inflammatory cytokines TNF-alpha and IFN-gamma but had no effect on secretion of the neutrophil chemokine, macrophage inflammatory protein-2 (MIP-2) . Acute and chronic inflammatory cell infiltrates were not altered by MAG treatment . These findings suggest that lipid conjugation of GAG could be used as a novel approach for increasing cell-surface concentrations of GAG . The inconclusive in vivo results might be due to the physical properties of the tested MAG or an insufficient application protocol, and their improvement might provide the desired inhibitory effects. Mol Microbiol, 2004 Apr, 52(1), 81 - 92 The VirB type IV secretion system of Bartonella henselae mediates invasion, proinflammatory activation and antiapoptotic protection of endothelial cells; Schmid MC et al.; Bartonella henselae is an arthropod-borne zoonotic pathogen causing intraerythrocytic bacteraemia in the feline reservoir host and a broad range of clinical manifestations in incidentally infected humans . Remarkably, B . henselae can specifically colonize the human vascular endothelium, resulting in inflammation and the formation of vasoproliferative lesions known as bacillary angiomatosis and bacillary peliosis . Cultured human endothelial cells provide an in vitro system to study this intimate interaction of B . henselae with the vascular endothelium . However, little is known about the bacterial virulence factors required for this pathogenic process . Recently, we identified the type IV secretion system (T4SS) VirB as an essential pathogenicity factor in Bartonella, required to establish intraerythrocytic infection in the mammalian reservoir . Here, we demonstrate that the VirB T4SS also mediates most of the virulence attributes associated with the interaction of B . henselae during the interaction with human endothelial cells . These include: (i) massive rearrangements of the actin cytoskeleton, resulting in the formation of bacterial aggregates and their internalization by the invasome structure; (ii) nuclear factor kappaB-dependent proinflammatory activation, leading to cell adhesion molecule expression and chemokine secretion, and (iii) inhibition of apoptotic cell death, resulting in enhanced endothelial cell survival . Moreover, we show that the VirB system mediates cytostatic and cytotoxic effects at high bacterial titres, which interfere with a potent VirB-independent mitogenic activity . We conclude that the VirB T4SS is a major virulence determinant of B . henselae, required for targeting multiple endothelial cell functions exploited by this vasculotropic pathogen. Mol Microbiol, 2004 Apr, 52(1), 25 - 38 Mycobacterium tuberculosis ECF sigma factor sigC is required for lethality in mice and for the conditional expression of a defined gene set; Sun R et al.; Bacterial alternative RNA polymerase sigma factors are key global adaptive response regulators with a likely role in Mycobacterium tuberculosis pathogenesis . We constructed a mutant lacking the sigma factor gene, sigC, by allelic exchange, in the virulent CDC1551 strain of M . tuberculosis and compared the resulting mutant with the isogenic wild-type strain and complemented mutant strain . In vitro, compared to the wild-type and complemented strains, the mutant was found to have similar ability to survive in both murine bone marrow-derived macrophages and activated J774 macrophages . In time-to-death experiments in the mouse model, the DeltasigC mutant was significantly attenuated, causing no death in infected mice whereas the wild-type and complemented strains caused 100% mortality within 235 days after aerosol infection with a median time to death of 170 days . Mouse organ bacterial burdens indicated that the mutant proliferated and persisted at the same level as the wild-type and complemented strains in lung tissue and was able to persist in mice without causing death for > 300 days . A complete genomic microarray study demonstrated that SigC modulates the expression of several key virulence-associated genes including hspX, senX3 and mtrA, encoding the alpha-crystallin homologue, a two-component sensor kinase and a two-component response regulator respectively . Altered expression of a subset of these genes was confirmed by quantitative RT-PCR analysis . Analysis of genes modulated by SigC also revealed a putative consensus DNA recognition sequence for SigC of SSSAAT-N(16-20)-CGTSSS (S = C or G) . Promoter recognition for one of these genes was confirmed by in vitro transcription analysis after purification of recombinant SigC and reconstitution of an Esigma(C) RNA polymerase holoenzyme . These data indicate that the M . tuberculosis transcription factor SigC governs expression of an important M . tuberculosis regulon and is essential for lethality in mice, but is not required for bacterial survival in this species . These observations place the DeltasigC mutant in a class of M . tuberculosis mutants which persist in tissues but are attenuated in their ability to elicit lethal immunopathology. Can J Microbiol, 2001 Jan, 47(1), 63 - 71 Characterization of the iron superoxide dismutase gene of Azotobacter vinelandii: sodB may be essential for viability; Qurollo BA et al.; Azotobacter vinelandii contains two superoxide dismutases (SODs), a cytoplasmic iron-containing enzyme (FeSOD), and a periplasmic copper/zinc-containing enzyme (CuZnSOD) . In this study, the FeSOD was found to be constitutive, while the activity of CuZnSOD increased as the culture entered the stationary phase . Total SOD (units/mg protein) in stationary phase cells grown under nitrogen-fixing conditions was not significantly different from those grown under non-nitrogen-fixing conditions . The gene encoding FeSOD (sodB) was isolated from an A . vinelandii cosmid library . A 1-kb fragment containing the coding region and 400 base pairs of upstream sequence was cloned and sequenced . The nucleotide sequence and the deduced amino acid sequence had a high degree of homology with other bacterial FeSODs, particularly with P . aeruginosa . Attempts to construct a sodB mutant by recombination of a sodB::kan insertion mutation into the multicopy chromosome of A . vinelandii were unsuccessful even in the presence of SOD mimics or nutritional supplements . These results suggest that FeSOD may be essential for the growth and survival of A . vinelandii, and that the periplasmic CuZnSOD cannot replace the function of FeSOD. Unfallchirurg . 2004 Mar 27; {Epub ahead of print} {The periprosthetic total hip infection}; Ruchholtz S et al.; Therapy of infected hip prosthesis should always be based on a structured treatment concept . When short-termed early infection is present or impending, with meticulous debridement of the soft tissue surroundings, the implant may be left in place . Chronic infection (>30 days) should lead to complete removal of implant and cement . A one-staged revision of the implant may be considered for patients without additional chronic disease, good vascularization of soft tissue and bones and bacteria susceptible to antibiotics . In most cases though two-staged revision is indicated . The interval between implant removal and re-implantation ranges between one and four months . Re-implantation should only be performed when laboratory parameters are normalized and the local wound site has turned to an unsuspicious condition . By insertion of a cement spacer during the interval period soft tissue shortening and local scar formation can be prevented . Parenteral antibiotics should be applied for four to six weeks . In patients with reduced general health state and extremely severe infection permanent resection of the hip or limp ablation may be indicated. Plant Physiol, 2004 Apr, 134(4), 1248 - 67 Epub 2004 Mar 26. Immunophilins and parvulins . Superfamily of peptidyl prolyl isomerases in Arabidopsis; He Z et al.; Immunophilins are defined as receptors for immunosuppressive drugs including cyclosporin A, FK506, and rapamycin . The cyclosporin A receptors are referred to as cyclophilins (CYPs) and FK506- and rapamycin-binding proteins are abbreviated as FKBPs . These two groups of proteins (collectively called immunophilins) share little sequence homology, but both have peptidyl prolyl cis/trans isomerase (PPIase) activity that is involved in protein folding processes . Studies have identified immunophilins in all organisms examined including bacteria, fungi, animals, and plants . Nevertheless, the physiological function of immunophilins is poorly understood in any organism . In this study, we have surveyed the genes encoding immunophilins in Arabidopsis genome . A total of 52 genes have been found to encode putative immunophilins, among which 23 are putative FKBPs and 29 are putative CYPs . This is by far the largest immunophilin family identified in any organism . Both FKBPs and CYPs can be classified into single domain and multiple domain members . The single domain members contain a basic catalytic domain and some of them have signal sequences for targeting to a specific organelle . The multiple domain members contain not only the catalytic domain but also defined modules that are involved in protein-protein interaction or other functions . A striking feature of immunophilins in Arabidopsis is that a large fraction of FKBPs and CYPs are localized in the chloroplast, a possible explanation for why plants have a larger immunophilin family than animals . Parvulins represent another family of PPIases that are unrelated to immunophilins in protein sequences and drug binding properties . Three parvulin genes were found in Arabidopsis genome . The expression of many immunophilin and parvulin genes is ubiquitous except for those encoding chloroplast members that are often detected only in the green tissues . The large number of genes and diversity of structure domains and cellular localization make PPIases a versatile superfamily of proteins that clearly function in many cellular processes in plants. Biosens Bioelectron, 2004 May 15, 19(10), 1203 - 8 Assessment of copper toxicity using an acoustic wave sensor; Yamasaki A et al.; A piezoelectric quartz crystal microbalance has been shown to be useful to monitor real time bacterial growth . Monitoring bacterial growth can give an insight into the ecosystem, as it is highly affected by the presence of toxic elements or nutrients . The frequency of an uncoated piezoelectric quartz crystal was monitored while in contact with bacteria, isolated from water sampled from a Portuguese lagoon, growing in two different media: a saline nutrient broth (NM) and the natural water . The sensor was used to evaluate the effect of copper on bacterial growth . Copper concentrations up to 18.8 microg l(-1) showed an increase in bacterial growth in NM, and a decrease beyond 25.0 microg l(-1) . Copper added to the natural water had negative effects on bacterial growth beyond 18.8 microg l(-1) . Copper concentrations in the natural water from the lagoon were determined using a similar quartz crystal to detect the mass deposited by anodic stripping voltammetry, and was found to be 3.38 +/- 0.09 microg l(-1). Neuron, 2004 Mar 25, 41(6), 859 - 65 A gating hinge in Na+ channels; a molecular switch for electrical signaling; Zhao Y et al.; Voltage-gated sodium channels are members of a large family with similar pore structures . The mechanism of opening and closing is unknown, but structural studies suggest gating via bending of the inner pore helix at a glycine hinge . Here we provide functional evidence for this gating model for the bacterial sodium channel NaChBac . Mutation of glycine 219 to proline, which would strongly favor bending of the alpha helix, greatly enhances activation by shifting its voltage dependence -51 mV and slowing deactivation by 2000-fold . The mutation also slows voltage-dependent inactivation by 1200-fold . The effects are specific because substitutions of proline at neighboring positions and substitutions of other amino acids at position 219 have much smaller functional effects . Our results fit a model in which concerted bending at glycine 219 in the S6 segments of NaChBac serves as a gating hinge . This gating motion may be conserved in most members of this large ion channel protein family. Biochem Soc Trans, 2004 Apr, 32(Pt 2), 310 - 3 Arginine regulation in Thermotoga neapolitana and Thermotoga maritima; Charlier D; Experimental data and in silico analyses of sequenced bacterial genomes indicate that arginine repressor (ArgR) proteins and their respective target sites are surprisingly well conserved in very diverse bacteria . Arginine regulation therefore constitutes an interesting model system from the study of evolutionary aspects of bacterial regulation . Moreover, arginine repressor molecules are multifunctional, they repress the arginine biosynthetic genes and are involved in the activation of the various arginine catabolic pathways . Studies on the arginine repressor from the hyperthermophiles Thermotoga neapolitana and Thermotoga maritima have reinforced the uniform view of the bacterial ArgR-operator interaction, but have also revealed that the Thermotoga repressor exhibits unique features . Thus, its DNA-binding activity is nearly arginine-independent and exhibits poor sequence specificity . ArgR(Tn) has a remarkable capacity to bind heterologous arginine operators and half-site targets. Biochem Soc Trans, 2004 Apr, 32(Pt 2), 259 - 63 Structure, function and evolution of the Archaeal class I fructose-1,6-bisphosphate aldolase; Lorentzen E et al.; FBPA (fructose-1,6-bisphosphate aldolase) catalyses the reversible aldol condensation of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate to form fructose 1,6-bisphosphate . Two classes of FBPA, which rely on different reaction mechanisms, have so far been discovered, class I mainly found in Eucarya and class II mainly in Bacteria . Only recently were genes encoding proteins with FBPA activity identified in Archaea . Archaeal FBPAs do not share any significant overall sequence identity with members of the traditional classes of FBPAs, raising the interesting question of whether they have evolved independently by convergent evolution or diverged from a common ancestor . Biochemical characterization of FBPAs of the two hyperthermophilic Archaea Thermoproteus tenax and Pyrococcus furiosus showed that the enzymes use a Schiff-base mechanism and thus belong to the class I aldolases . The crystal structure of the archaeal FBPA from T . tenax revealed that the protein fold, as for the classical FBPA I and II, is that of a parallel (betaalpha)(8) barrel . A substrate-bound crystal structure allowed detailed active-site comparisons which showed the conservation of six important catalytic and substrate-binding residues between the archaeal and the classical FBPA I . This observation provides further evidence that the two sequence families of proteins share a common evolutionary origin . Furthermore, structure and sequence analysis indicate that the class I FBPA shares a common evolutionary origin with several other enzyme superfamilies of the (betaalpha)(8) barrel fold. Biochem Soc Trans, 2004 Apr, 32(Pt 2), 168 - 71 The hyperthermophilic origin of life revisited; Schwartzman DW et al.; We revisit the case for the hyperthermophilic scenario for the origin of life and the last common ancestor . Evidence includes studies of phylogenetic trees, rRNA, G and C content, hyperthermophilic proteins, correlations between maximal temperature tolerances and genetic distances, saline stabilization of DNA/RNA, and the inferred climatic temperatures of the early Earth . Although some doubts remain, the case for hot biogenesis and the last common ancestor has gotten stronger. J Mol Evol, 2004 Mar, 58(3), 291 - 303 Two types of FtsZ proteins in mitochondria and red-lineage chloroplasts: the duplication of FtsZ is implicated in endosymbiosis; Miyagishima SY et al.; The ancestors of plastids and mitochondria were once free-living bacteria that became organelles as a result of endosymbiosis . According to this theory, a key bacterial division protein, FtsZ, plays a role in plastid division in algae and plants as well as in mitochondrial division in lower eukaryotes . Recent studies have shown that organelle division is a process that combines features derived from the bacterial division system with features contributed by host eukaryotic cells . Two nonredundant versions of FtsZ, FtsZ1 and FtsZ2, have been identified in green-lineage plastids, whereas most bacteria have a single ftsZ gene . To examine whether there is also more than one type of FtsZ in red-lineage chloroplasts (red algal chloroplasts and chloroplasts that originated from the secondary endosymbiosis of red algae) and in mitochondria, we obtained FtsZ sequences from the complete sequence of the primitive red alga Cyanidioschyzon merolae and the draft sequence of the stramenopile (heterokont) Thalassiosira pseudonana . Phylogenetic analyses that included known FtsZ proteins identified two types of chloroplast FtsZ in red algae (FtsZA and FtsZB) and stramenopiles (FtsZA and FtsZC) . These analyses also showed that FtsZB emerged after the red and green lineages diverged, while FtsZC arose by the duplication of an ftsZA gene that in turn descended from a red alga engulfed by the ancestor of stramenopiles . A comparison of the predicted proteins showed that like bacterial FtsZ and green-lineage FtsZ2, FtsZA has a short conserved C-termmal sequence (the C-terminal core domain), whereas FtsZB and FtsZC, like the green-lineage FtsZ1, lack this sequence . In addition, the Cyanidioschyzon and Dictyostelium genomes encode two types of mitochondrial FtsZ proteins, one of which lacks the C-terminal variable domain . These results suggest that the acquisition of an additional FtsZ protein with a modified C terminus was common to the primary and secondary endosymbioses that produced plastids and that this also occurred during the establishment of mitochondria, presumably to regulate the multiplication of these organelles. Mod Pathol, 2004 Jun, 17(6), 684 - 9 Reliable detection of macrolide-resistant Helicobacter pylori via fluorescence in situ hybridization in formalin-fixed tissue; Juttner S et al.; Macrolide-resistant Helicobacter (H.) pylori represent an increasing therapeutic problem . Macrolide resistance is usually determined phenotypically in vitro with methods such as E-test or agar dilution test . A prerequisite for those tests, however, is bacterial culture that is not routinely set up in the course of gastroscopy . In contrast, formalin-fixed, paraffin-embedded biopsies are regularly available from patients who have undergone gastroscopy . In such biopsies macrolide-resistant H . pylori can be detected by the genotype-based technique of fluorescence in situ hybridization (FISH) . Experience gained by this new method, however, is still extremely limited, especially in formalin-fixed tissue . Therefore, we retrospectively investigated formalin-fixed, paraffin-embedded biopsy specimens by FISH in 104 patients suffering from therapy-resistant H . pylori gastritis . To test the accuracy of FISH, we initially examined specimens from 53 patients for whom results of the E-test were available . Next we analyzed biopsies from another 51 patients that had been selected since phenotypical resistance testing had failed despite documented culturing attempts . In all 104 patients, H . pylori was detected by FISH and could thus be investigated for macrolide resistance . Overall, macrolide-resistant bacteria were found in 71 patients (68.3%) . In 49 of 53 patients (92.4%), FISH and E-test returned identical results (no significant discordance according to McNemar's chi(2)-test) . Taken together, our study demonstrates that FISH is a highly sensitive and reliable method for detecting macrolide-resistant H . pylori in formalin-fixed, paraffin-embedded biopsy specimens, which represents the routine method of processing tissue obtained upon gastroscopy. Science, 2004 Mar 26, 303(5666), 2004 - 7 Oxygen isotope constraints on the sulfur cycle over the past 10 million years; Turchyn AV et al.; Oxygen isotopes in marine sulfate (delta18O(SO4)) measured in marine barite show variability over the past 10 million years, including a 5 per mil decrease during the Plio-Pleistocene, with near-constant values during the Miocene that are slightly enriched over the modern ocean . A numerical model suggests that sea level fluctuations during Plio-Pleistocene glacial cycles affected the sulfur cycle by reducing the area of continental shelves and increasing the oxidative weathering of pyrite . The data also require that sulfate concentrations were 10 to 20% lower in the late Miocene than today. Proc Natl Acad Sci U S A, 2004 Apr 6, 101(14), 5024 - 9 Epub 2004 Mar 25. The spatial orientation of Helicobacter pylori in the gastric mucus; Schreiber S et al.; The highly motile human pathogen Helicobacter pylori lives deep in the gastric mucus layer . To identify which chemical gradient guides the bacteria within the mucus layer, combinations of luminal perfusion, dialysis, and ventilation were used to modify or invert transmucus gradients in anaesthetized Helicobacter-infected mice and Mongolian gerbils . Neither changes in lumen or arterial pH nor inversion of bicarbonate/CO2 or urea/ammonium gradients disturbed Helicobacter orientation . However, elimination of the mucus pH gradient by simultaneous reduction of arterial pH and bicarbonate concentration perturbed orientation, causing the bacteria to spread over the entire mucus layer . H . pylori thus uses the gastric mucus pH gradient for chemotactic orientation. Physiol Rev, 2004 Apr, 84(2), 579 - 621 Protease-activated receptors: contribution to physiology and disease; Ossovskaya VS et al.; Proteases acting at the surface of cells generate and destroy receptor agonists and activate and inactivate receptors, thereby making a vitally important contribution to signal transduction . Certain serine proteases that derive from the circulation (e.g., coagulation factors), inflammatory cells (e.g., mast cell and neutrophil proteases), and from multiple other sources (e.g., epithelial cells, neurons, bacteria, fungi) can cleave protease-activated receptors (PARs), a family of four G protein-coupled receptors . Cleavage within the extracellular amino terminus exposes a tethered ligand domain, which binds to and activates the receptors to initiate multiple signaling cascades . Despite this irreversible mechanism of activation, signaling by PARs is efficiently terminated by receptor desensitization (receptor phosphorylation and uncoupling from G proteins) and downregulation (receptor degradation by cell-surface and lysosomal proteases) . Protease signaling in tissues depends on the generation and release of proteases, availability of cofactors, presence of protease inhibitors, and activation and inactivation of PARs . Many proteases that activate PARs are produced during tissue damage, and PARs make important contributions to tissue responses to injury, including hemostasis, repair, cell survival, inflammation, and pain . Drugs that mimic or interfere with these processes are attractive therapies: selective agonists of PARs may facilitate healing, repair, and protection, whereas protease inhibitors and PAR antagonists can impede exacerbated inflammation and pain . Major future challenges will be to understand the role of proteases and PARs in physiological control mechanisms and human diseases and to develop selective agonists and antagonists that can be used to probe function and treat disease. Mol Hum Reprod, 2004 Jun, 10(6), 433 - 44 Epub 2004 Mar 25. Expression analysis of the human testis-specific serine/threonine kinase (TSSK) homologues . A TSSK member is present in the equatorial segment of human sperm; Hao Z et al.; Two members of the human testis-specific serine/threonine (Ser/Thr) kinase family, TSSK 1 and TSSK 2, were cloned and sequenced from a human testis adaptor-ligated cDNA library using a PCR strategy . Within the cDNA, open reading frames (ORF) were defined encoding proteins of 367 and 358 amino acids respectively, as well as conserved kinase domains typical of the superfamily of Ser/Thr kinases . Both genes were intronless and mapped to chromosomes 5 and 22 respectively . The human and mouse homologues of TSSK 1 and TSSK 2, together with TSSK 3 and SSTK/FKSG82, constitute a kinase subfamily closely related to the calmodulin kinases and SNF/nim 1 kinase subfamilies . Similar to the mouse, tissue expression by northern and dot blot analysis revealed that human TSSK 1 and 2 messages are expressed exclusively in the testis . However, mRNA for these kinases can be detected in other tissues using real-time PCR . In addition, TSKS, the human homologue of a putative substrate of TSSK 1 and 2, was cloned . TSKS had an ORF of 592 amino acids and was also expressed exclusively in the testis as demonstrated by northern and dot blot analyses; however, lower levels of expression in other tissues were detected using real-time PCR . Human TSSK 2 and TSKS interacted in a yeast two-hybrid system and also co-immunoprecipitated after in vitro translation . TSSK 2 expressed in yeast and bacteria was able to autophosphorylate and also phosphorylated recombinant TSKS in vitro . Antibodies against recombinant TSSK 2 demonstrated that a member of the TSSK family was present in human testis and localized to the equatorial segment of ejaculated human sperm . In contrast, TSKS was only found in the testis . The finding of a TSSK family member in mature sperm suggests that this family of kinases might play a role in sperm function. J Pharmacol Exp Ther, 2004 Aug, 310(2), 821 - 7 Epub 2004 Mar 25. Induction of small intestinal damage in rats following combined treatment with cyclooxygenase-2 and nitric-oxide synthase inhibitors; Ohno R et al.; Nitric oxide (NO) produced by constitutively expressed NO synthase (cNOS) plays an important role in maintaining the mucosal integrity of the small intestine, in collaboration with prostaglandins produced by cyclooxygenase (COX)-1 . We examined whether intestinal damage is provoked in rats under inhibition of both cNOS and COX-2 . The animals were given L-NAME (N(G)-nitro-L-arginine methyl ester), aminoguanidine, or rofecoxib, either alone or in combination, and killed 24 h later . Neither L-NAME nor aminoguanidine alone provoked damage in the small intestinal mucosa within 24 h, yet L-NAME produced damage in a L-arginine-sensitive manner when administered together with rofecoxib . L-NAME up-regulated the expression of COX-2 mRNA, and the prostaglandin E(2) (PGE(2)) content following the L-NAME administration significantly increased 12 h later, in both a rofecoxib- and a L-arginine-inhibitable manner . L-NAME enhanced intestinal motility, decreased mucus secretion, and increased the number of bacteria in the mucosa . The up-regulation of COX-2 expression and PGE(2) production by L-NAME was inhibited by prior administration of atropine, at a dose that inhibited the intestinal hypermotility . The intestinal lesions induced by L-NAME plus rofecoxib were prevented by pretreatment with ampicillin and aminoguanidine as well as atropine, indicating the involvement of bacteria, inducible nitric oxide synthase, and hypermotility in the pathogenesis . These results suggest that inhibition of both cNOS and COX-2 provokes intestinal damage, similar to inhibition of both COX-1 and COX-2 . Inhibition of cNOS, similar to COX-1, up-regulates COX-2 expression, the process being associated with intestinal hypermotility and bacterial invasion, and this may be a key to the occurrence of intestinal damage associated with COX-2 inhibition. Nat Rev Microbiol, 2004 Feb, 2(2), 109 - 22 Virus entry: molecular mechanisms and biomedical applications; Dimitrov DS; Viruses have evolved to enter cells from all three domains of life--Bacteria, Archaea and Eukaryotes . Of more than 3,600 known viruses, hundreds can infect human cells and most of those are associated with disease . To gain access to the cell interior, animal viruses attach to host-cell receptors . Advances in our understanding of how viral entry proteins interact with their host-cell receptors and undergo conformational changes that lead to entry offer unprecedented opportunities for the development of novel therapeutics and vaccines. J Mol Evol, 2004 Feb, 58(2), 218 - 24 Pathway length and evolutionary constraint in amino acid biosynthesis; Rutter MT et al.; The evolutionary properties of a metabolic network may be determined by the topology of the network . One attribute of pathways that make up the network is the number of enzymatic steps between initial substrates and final products . To determine the effect of pathway length on evolutionary lability of pathway structure, we examined amino acid biosynthetic pathways across 48 sequenced organisms . We demonstrate that longer pathways exhibit lower rates of change in pathway structure than shorter pathways . This finding suggests that increasing complexity may increase constraint on evolutionary change. MMWR Morb Mortal Wkly Rep . 2004 Mar 26;53(11):244. Kingella kingae infections in children--United States, June 2001-November 2002; Centers for Disease Control and Prevention (CDC); Kingella kingae is recognized increasingly as a cause of skeletal infections in children . Recent studies indicate that direct inoculation of clinical specimens into aerobic blood culture bottles (ABCBs), instead of direct plating of specimens on solid media, might improve recovery of the fastidious bacteria . Prompted by a report of a possible cluster of osteoarticular infections caused by K . kingae among children, the Infectious Diseases Society of America Emerging Infections Network (IDSA-EIN) surveyed pediatric infectious disease consultants (PIDCs) about their experiences in diagnosing K . kingae and other skeletal infections in children . This report summarizes the findings of that survey, which identified 23 K . kingae pediatric cases and indicated that 35% of responding PIDCs did not use ABCBs in diagnosing skeletal infections . Efforts to increase use of ABCBs among clinicians and laboratorians might lead to increased detection of K . kingae cases. Proc Natl Acad Sci U S A, 2004 Apr 6, 101(14), 4799 - 804 Epub 2004 Mar 23. Protein kinetics: structures of intermediates and reaction mechanism from time-resolved x-ray data; Schmidt M et al.; We determine the number of authentic reaction intermediates in the later stages of the photocycle of photoactive yellow protein at room temperature, their atomic structures, and a consistent set of chemical kinetic mechanisms, by analysis of a set of time-dependent difference electron density maps spanning the time range from 5 micros to 100 ms . The successful fit of exponentials to right singular vectors derived from a singular value decomposition of the difference maps demonstrates that a chemical kinetic mechanism holds and that structurally distinct intermediates exist . We identify two time-independent difference maps, from which we refine the structures of the corresponding intermediates . We thus demonstrate how structures associated with intermediate states can be extracted from the experimental, time-dependent crystallographic data . Stoichiometric and structural constraints allow the exclusion of one kinetic mechanism proposed for the photocycle but retain other plausible candidate kinetic mechanisms. Biophys J, 2004 Apr, 86(4), 2363 - 73 In vitro self-assembly of the light harvesting pigment-protein LH2 revealed by ultrafast spectroscopy and electron microscopy; Schubert A et al.; Controlled ensemble formation of protein-surfactant systems provides a fundamental concept for the realization of nanoscale devices with self-organizing capability . In this context, spectroscopic monitoring of pigment-containing proteins yields detailed structural information . Here we have studied the association behavior of the bacterial light-harvesting protein LH2 from Rhodobacter spheroides in an n,n-dimethyldodecylamine-n-oxide/water environment . Time-resolved studies of the excitation annihilation yielded information about aggregate sizes and packing of the protein complexes therein . The results are compared to transmission electron microscopy images of instantaneously frozen samples . Our data indicate the manifestation of different phases, which are discussed with respect to the thermodynamic equilibrium in ternary protein-surfactant-water systems . Accordingly, by varying the concentration the formation of different types of aggregates can be controlled . Conditions for the appearance of isolated LH2 complexes are defined. Vaccine, 2004 Feb 17, 22(7), 888 - 97 Clearance of Helicobacter pylori infection through immunization: the site of T cell activation contributes to vaccine efficacy; Blanchard TG et al.; Helicobacter pylori vaccine development has progressed rapidly in animal models . Both H . pylori-associated pathogenesis and protective immunity are CD4+ T cell dependent, with no discernable phenotypic difference to distinguish pathogenic T cells from protective T cells . Functionally however, protective T cells promote enhanced inflammation upon H . pylori challenge . Additionally, only mouse models such as phagocyte oxidase- or IL-10-deficient mice that respond to H . pylori infection with intense gastritis are capable of demonstrating spontaneous eradication of the bacteria . These data, combined with recent descriptions of down-regulatory T cells in infected humans and mice, support an emerging model of H . pylori pathogenesis in which H . pylori induces inflammation that is limited by regulatory T cells in the stomach . Immunization therefore may succeed by activating T cells in peripheral lymph nodes that are capable of promoting qualitatively or quantitatively different inflammation when recruited to the stomach . Evidence in support of this model will be discussed. Infect Immun, 2004 Apr, 72(4), 2358 - 68 Genes of Helicobacter pylori regulated by attachment to AGS cells; Kim N et al.; Reciprocal interactions between Helicobacter pylori and cells of the gastric epithelium to which it adheres may affect colonization . Changes in gene expression of H . pylori induced by adhesion to AGS gastric cancer cells by coculture were compared to changes in gene expression of H . pylori cultured without AGS cells by using cDNA filter macroarrays . Adhesion was quantitatively verified by confocal microscopy of green fluorescent protein-expressing bacteria . Four experiments showed that 22 and 21 H . pylori genes were consistently up- and down-regulated, respectively . The up-regulated genes included pathogenicity island, motility, outer membrane protein, and translational genes . The sigma(28) factor antagonist flgM, flgG, the stress response gene, flaA, omp11, and the superoxide dismutase gene (sodB) were down-regulated . The up-regulation of cag3, flgB, tonB, rho, and deaD was confirmed by quantitative PCR, and the up-regulation of lpxD, omp6, secG, fabH, HP1285, HP0222, and HP0836 was confirmed by reverse transcription (RT)-PCR . The down-regulation of flaA, sodB, and HP0874 was confirmed by quantitative PCR, and the down-regulation of omp11 was confirmed by RT-PCR . The alteration of gene expression in H . pylori after adhesion to gastric cells in vitro suggests that changes in motility, outer membrane composition, and stress responses, among other changes, may be involved in gastric colonization. Infect Immun, 2004 Apr, 72(4), 2303 - 11 Impairment of intramacrophagic Brucella suis multiplication by human natural killer cells through a contact-dependent mechanism; Dornand J et al.; Brucella spp . are facultative intracellular bacteria that can establish themselves and cause chronic disease in humans and animals . NK cells play a key role in host defense . They are implicated in an early immune response to a variety of pathogens . However, it was shown that they do not control Brucella infection in mice . On the other hand, NK cell activity is impaired in patients with acute brucellosis, and recently it was demonstrated that human NK cells mediate the killing of intramacrophagic Mycobacterium tuberculosis in in vitro infection . Therefore, we have analyzed the behavior of Brucella suis infecting isolated human macrophages in the presence of syngeneic NK cells . We show that (i) NK cells impair the intramacrophagic development of B . suis, a phenomenon enhanced by NK cell activators, such as interleukin-2; (ii) NK cells cultured in the presence of infected macrophages are highly activated and secrete gamma interferon and tumor necrosis factor alpha; (iii) impairment of bacterial multiplication inside infected cells is marginally associated with the cytokines produced during the early phase of macrophage-NK cell cocultures; (iv) direct cell-to-cell contact is required for NK cells to mediate the inhibition of B . suis development; and (v) inhibition of B . suis development results from an induction of NK cell cytotoxicity against infected macrophages . Altogether, these findings show that NK cells could participate early in controlling the intramacrophagic development of B . suis in humans . It seems thus reasonable to hypothesize a role for NK cells in the control of human brucellosis . However, by impairing the activity of these cells in the acute phase of the illness, the pathogen should avoid this control. Infect Immun, 2004 Apr, 72(4), 2111 - 22 Novel program of macrophage gene expression induced by phagocytosis of Leishmania chagasi; Rodriguez NE et al.; Leishmania spp . are protozoans that survive and replicate intracellularly in mammalian macrophages . Antileishmanial immunity requires gamma interferon (IFN-gamma)-mediated macrophage activation and generation of microbicidal effector molecules . The presence of intracellular Leishmania sp . impairs macrophage responses to IFN-gamma, which has led to the description of macrophages as deactivated . It has recently become apparent that in addition to classical activation, macrophages can be activated by distinct triggers to express noninflammatory or anti-inflammatory genes . These nonclassical activation programs have been called alternative or type II pathways . We hypothesized that during initial contact with a phagocyte, leishmaniae activate one of these nonclassical pathways, resulting in expression of genes whose products suppress microbicidal responses . Using DNA microarrays, we studied gene expression in RNAs from BALB/c bone marrow macrophages with and without Leishmania chagasi infection . Some changes were verified by an RNase protection assay, reverse transcription-PCR, immunoblotting, or a bioassay . The pattern of genes activated by leishmania phagocytosis differed from the pattern of genes activated by bacteria or lipopolysaccharide and IFN-gamma . Genes encoding some proinflammatory cytokines, receptors, and Th1-type immune response genes were down-modulated, and some genes associated with anti-inflammatory or Th2-like immune responses were up-regulated . Nonetheless, some markers of alternative (arginase) or type II activation (interleukin-10, tumor necrosis factor alpha) were unchanged . These data suggest that macrophages infected with L . chagasi exhibit a hybrid activation profile that is more characteristic of alternative or type II activation than of classical activation but does not strictly fall into either of these categories . We speculate that the pattern of genes upregulated by leishmania phagocytosis optimizes the chance of parasite survival in this hostile environment. Infect Immun, 2004 Apr, 72(4), 2057 - 66 Analysis of relative levels of production of pertussis toxin subunits and Ptl proteins in Bordetella pertussis; Cheung AM et al.; Pertussis toxin is transported across the outer membrane of Bordetella pertussis by the type IV secretion system known as the Ptl transporter, which is composed of nine different proteins . In order to determine the relative levels of production of pertussis toxin subunits and Ptl proteins in B . pertussis, we constructed translational fusions of the gene for alkaline phosphatase, phoA, with various ptx and ptl genes . Comparison of the alkaline phosphatase activity of strains containing ptx'- or ptl'-phoA fusions indicated that pertussis toxin subunits are produced at higher levels than Ptl proteins, which are encoded by genes located toward the 3' end of the ptx-ptl operon . We also engineered strains of B . pertussis by introducing multiple copies of the ptl genes or subsets of these genes and then examined the ability of each of these strains to secrete pertussis toxin . From these studies, we determined that certain Ptl proteins appear to be limiting in the secretion of pertussis toxin from the bacteria . These results represent an important first step in assessing the stoichiometric relationship of pertussis toxin and its transporter within the bacterial cell. Protein Expr Purif, 2004 May, 35(1), 156 - 69 Characterization of the cAMP-dependent protein kinase catalytic subunit Cgamma expressed and purified from sf9 cells; Zhang W et al.; The Cgamma and Calpha subunits of the cAMP-dependent protein kinase (PKA) contain 350 amino acids that are highly homologous (83% amino acid sequence), with 91% homology within the catalytic domain (a.a . 40-300) . Unlike Cgamma, the Calpha subunit has been readily purified and characterized as a recombinant protein in vitro, in intact cells, and in vivo . This report describes for the first time the expression, purification, and characterization of Cgamma . The expression of active Cgamma was eukaryote-specific, from mammalian and insect cells, but not