Bacteria

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 bacteria . Active recombinant Cgamma was optimally expressed and purified to homogeneity from Sf9 cells with a 273-fold increase in specific activity and a 21% recovery after sequential CM-Sepharose and Sephacryl S-300 chromatography . The specific activity of pure Cgamma was 0.31 and 0.81 U/mg with kemptide and histone as substrates, respectively . Physical characterization showed Cgamma had a lower apparent molecular weight and Stokes radii than Calpha, suggesting differences in tertiary structures . Steady-state kinetics demonstrated that like Calpha and Cbeta, Cgamma phosphorylates substrates requiring basic amino acids at P-3 and P-2 . However, Cgamma generally exhibited a lower Km and Vmax than Calpha for peptide substrates tested . Cgamma also exhibited a distinct pseudosubstrate specificity showing inhibition by homogeneous preparations of RIalpha and RIIalpha-subunits, but not by pure recombinant protein kinase inhibitors PKIalpha and PKIbeta, PKA-specific inhibitors . These studies suggest that Cgamma and Calpha exhibit differences in structure and function in vitro, supporting the hypothesis that functionally different C-subunit isozymes could diversify and/or fine-tune cAMP signal transduction downstream of PKA activation.

Dis Aquat Organ, 2004 Jan 28, 58(1), 1 - 8
Phenotypic characterization and description of two major O-serotypes in Tenacibaculum maritimum strains from marine fishes; Avendano-Herrera R et al.; Tenacibaculum maritimum is the etiological agent of marine flexibacteriosis disease, with the potential to cause severe mortalities in various cultured marine fishes . The development of effective preventive measures (i.e . vaccination) requires biochemical, serological and genetic knowledge of the pathogen . With this aim, the biochemical and antigenic characteristics of T . maritimum strains isolated from sole, turbot and gilthead sea bream were analysed . Rabbit antisera were prepared against sole and turbot strains to examine the antigenic relationships between the 29 isolates and 3 reference strains . The results of the slide agglutination test, dot-blot assay and immunoblotting of lipopolysaccharides (LPS) and membrane proteins were evaluated . All bacteria studied were biochemically identical to the T . maritimum reference strains . The slide agglutination assays using O-antigens revealed cross-reaction for all strains regardless of the host species and serum employed . However, when the dot-blot assays were performed, the existence of antigenic heterogeneity was demonstrated . This heterogeneity was supported by immunoblot analysis of the LPS, which clearly revealed 2 major serological groups that were distinguishable without the use of absorbed antiserum: Serotypes O1 and O2 . These 2 serotypes seem to be host-specfic . In addition, 2 sole isolates and the Japanese reference strains displayed cross-reaction with both sera in all serological assays, and are considered to constitute a minor serotype, O1/O2 . Analysis of total and outer membrane proteins revealed that all strains share a considerable number of common bands that are antigenically related.

Nippon Rinsho, 2004 Mar, 62(3), 470 - 6
{Immunological rapid urease test}; Kato M et al.; Immunological rapid urease test for detection of Helicobacter pylori infection, composed of a solid-phase tip coated with monoclonal antibody against H . pylori's urease and ion-sensitive field transistor (ISFT)-based pH sensor system, was developed . The monoclonal antibody against H . pylori's urease was useful to avoid the contamination of urease activity in other bacteria . Because ISFTT had high ability to detect pH change, the sensitivity and specificity of immunological rapid urease test was significantly improved comparing with that of conventional rapid urease test . The utility of immunological rapid urease test was evaluated in some clinical studies.

Microb Ecol, 2004 May, 47(4), 305 - 15
Strong indirect effects of a submersed aquatic macrophyte, Vallisneria americana, on bacterioplankton densities in a mesotrophic lake; Huss AA et al.; Phytoplankton and allochthonous matter are important sources of dissolved organic carbon (DOC) for planktonic bacteria in aquatic ecosystems . But in small temperate lakes, aquatic macrophytes may also be an important source of DOC, as well as a source or sink for inorganic nutrients . We conducted micro- and mesocosm studies to investigate the possible effects of an actively growing macrophyte, Vallisneria americana, on bacterial growth and water chemistry in mesotrophic Calder Lake . A first microcosm (1 L) study conducted under high ambient NH4+ levels (NH4+ > or = 10 microM) demonstrated that macrophytes had a positive effect on bacterial densities through release of DOC and P . A second microcosm experiment, conducted under NH4+-depleted conditions (NH4+ < 10 microM), examined inter- active effects of macrophytes and their sediments on bacterial growth and water chemistry . Non-rooted macrophytes had negative effects on bacterial numbers, while rooted macrophytes had no significant effects, despite significant increases in DOC and P . A 70-L mesocosm experiment manipulated macrophytes, as well as N and P supply under surplus NH4-+conditions (NH4+ > or = 10 gmicro), and measured effects on bacterial growth, Chl a concentrations, and water chemistry . Bacterial growth and Chl a concentrations declined with macrophyte additions, while bacterial densities increased with P addition (with or without N) . Results suggest that the submersed macrophyte Vallisneria exerts a strong but indirect effect on bacteria by modifying nutrient conditions and/or suppressing phytoplankton . Effects of living macrophytes differed with ambient nutrient conditions: under NH4+-surplus conditions, submersed macrophytes stimulated bacterioplankton through release of DOC or P, but in NH4-+depleted conditions, the influence of Vallisneria was negative or neutral . Effects of living macrophytes on planktonic bacteria were apparently mediated by the macrophytes use and/or release of nutrients, as well as through possible effects on phytoplankton production.

Nucleic Acids Res, 2004 Mar 22, 32(5), 1836 - 47 Print 2004.
The CtBP2 co-repressor is regulated by NADH-dependent dimerization and possesses a novel N-terminal repression domain; Thio SS et al.; The C-terminal binding protein 2 (CtBP2) is a 48 kDa phosphoprotein reported to function as a co- repressor for a growing list of transcriptional repressors . It was recently demonstrated that CtBP is a dimeric NAD+-regulated d-isomer-specific 2-hydroxy acid dehydrogenase . However, the specific substrate(s) of CtBP enzymatic activity and the relationship of this activity to its co-repression function remain unknown . The ability of a human CtBP to bind and serve as a co-repressor of E1A has been shown to be regulated by nuclear NADH levels . Here we extend the functional characterization of CtBP by demonstrating that amino acid substitutions at Gly189 in the conserved NAD+-binding fold both abrogate the ability of CtBP2 to homodimerize and are associated with a dramatic loss of co-repressor activity . Consistent with the known enzymatic activity of CtBP2, mutations at Arg272 in the substrate-binding domain and at His321 in the catalytic domain result in significant loss of CtBP2 transcriptional co-repressor activity . High resolution serial C-terminal deletion analysis of CtBP2 also revealed a novel N-terminal repression domain that is distinct from its dehydrogenase domain . Our results suggest a model in which CtBP2 co-repressor function is regulated, at least in part, through the effect of NADH on CtBP2 homodimerization.

Mol Cancer Res, 2004 Mar, 2(3), 170 - 82
The interaction of specific peptide aptamers with the DNA binding domain and the dimerization domain of the transcription factor Stat3 inhibits transactivation and induces apoptosis in tumor cells; Nagel-Wolfrum K et al.; The transcription factor signal transducer and activator of transcription (Stat) 3 is activated through the interleukin-6 family of cytokines and by binding of growth factors to the epidermal growth factor (EGF) receptor . It plays an essential role in embryonic development and assumes specialized tasks in many differentiated tissues . Constitutively activated Stat3 has been found in tumor cell lines and primary tumors and plays a crucial role in tumor cell survival and proliferation . To inhibit the oncogenic action of Stat3 in tumor cells, we have selected short peptides, so-called peptide aptamers, which specifically interact with defined functional domains of this transcription factor . The peptide aptamers were selected from a peptide library of high complexity by an adaptation of the yeast two-hybrid procedure . Peptide aptamers specifically interacting with the Stat3 dimerization domain caused inhibition of DNA binding activity and suppression of transactivation by Stat3 in EGF-responsive cells . Similarly, a peptide aptamer selected for its ability to recognize the Stat3 DNA binding domain inhibited DNA binding and transactivation by Stat3 following EGF stimulation of cells . Peptide aptamers were expressed in bacteria as fusion proteins with a protein transduction domain and introduced into human myeloma cells . This resulted in dose-dependent growth inhibition, down-regulation of Bcl-x(L) expression, and induction of apoptosis . The inhibition of Stat3 functions through the interaction with peptide aptamers counteracts the transformed phenotype and could become useful in targeted tumor therapy.

J Biol Chem, 2004 May 28, 279(22), 23250 - 4 Epub 2004 Mar 22.
XRCC3 ATPase activity is required for normal XRCC3-Rad51C complex dynamics and homologous recombination; Yamada NA et al.; Homologous recombinational repair preserves chromosomal integrity by removing double-strand breaks, cross-links, and other DNA damage . In eukaryotic cells, the Rad51 paralogs (XRCC2/3, Rad51B/C/D) are involved in this process, although their exact functions are largely undetermined . All five paralogs contain ATPase motifs, and XRCC3 exists in a single complex with Rad51C . To examine the function of this Rad51C-XRCC3 complex, we generated mammalian expression vectors that produce human wild-type XRCC3 or mutant XRCC3 with either a nonconservative mutation (K113A) or a conservative mutation (K113R) in the GKT Walker A box of the ATPase motif . The three vectors were independently transfected into Xrcc3-deficient irs1SF Chinese hamster ovary cells . Wild-type XRCC3 complemented irs1SF cells, albeit to varying degrees, whereas ATPase mutants had no complementing activity, even when the mutant protein was expressed at comparable levels to that in wild-type-complemented clones . Because of dysfunction of the mutants, we propose that ATP binding and hydrolyzing activities of XRCC3 are essential . We tested in vitro complex formation by wild-type and mutant XRCC3 with His6-tagged Rad51C upon co-expression in bacteria, nickel-affinity purification, and Western blotting . Wild-type and K113A mutant XRCC3 formed stable complexes with Rad51C and co-purified with Rad51C, whereas the K113R mutant did not and was predominantly insoluble . The addition of 5 mm ATP but not ADP also abolished complex formation by the wild-type proteins . These results suggest that XRCC3 probably regulates the dissociation and formation of Rad51C-XRCC3 complex through ATP binding and hydrolysis with both processes being essential for the ability of the complex to participate in homologous recombinational repair.

J Insect Physiol, 2004 Jan, 50(1), 51 - 9
The MRJP/YELLOW protein family of Apis mellifera: identification of new members in the EST library; Albert S et al.; Major royal jelly proteins (named MRJP1-5) of honeybee (Apis mellifera), yellow proteins of Drosophila, together with putative proteins found in several bacteria, form a protein family termed the MRJP/yellow family . Members of the family exert diverse physiological functions and amongst eukaryotes appear to be restricted to the order Insecta . MRJPs constitute about 90% of total protein of royal jelly, which is secreted by nurse bees to feed the queen and growing larvae . We looked for mrjp and yellow homologues in a honeybee brain expressed sequence tags (EST) library . In addition to the five mrjp cDNAs previously characterized, we found three additional cDNAs encoding novel MRJPs and importantly, two cDNAs coding for orthologues of Drosophila yellow proteins . One yellow cDNA and all three cDNAs coding for the novel MRJPs were assembled completely, the sequence of the other yellow homologue was partially assembled . The data we present here supports the view that repeated duplications and functional divergence occurred during the evolution of MRJPs in honeybees, with even closely related MRJPs appearing to perform diverse physiological functions . Conversely, yellow protein orthologues appear to be conserved and thus candidates for maintaining the former function(s) of yellow proteins.

Mech Ageing Dev, 2004 Feb, 125(2), 129 - 31
Protection of the elderly from pneumococcal pneumonia with a protein-based vaccine?
Briles DE.
Vaccines exist to protect children and adults from pneumococcal infection . The adult vaccine contains capsular polysaccharides from those pneumococci causing the vast majority of pneumococcal infection around the world . This vaccine is, however, poorly immunogenic and not as protective as would be desired . The vaccine for children is a seven-valent conjugate vaccine, which is highly protective against invasive infection and offers some protection against otitis media and pneumococcal carriage . The capsular types in the vaccine are not all appropriate for the developing world and the vaccine is too expensive for use in the developing world . As a result of these problems there have been extensive efforts to develop pneumococcal vaccines for adults and children based on cross-reactive protein antigens . The molecules used are in general virulence factors and the antibodies to them neutralize their function, thus reducing the virulence of the infecting bacteria . Studies in humans have revealed that the proteins studied are invariably immunogenic in humans, as at least low levels of antibody are seen following colonization or infection . Studies in mice have demonstrated that vaccines containing more than one of these virulence proteins are generally more protective than those involving just one . Proteins that have been studied the most in mice are pneumococcal surface protein A (PspA), PspC, PsaA, and pneumolysin . PspA has been used in human safety trials and was shown to elicit antibodies that can protect mice from otherwise fatal pneumococcal infections.

Endeavour, 2004 Mar, 28(1), 14 - 9
Resisting insects: shifting strategies in chemical control; Ceccatti JS; Throughout the 20th century, scientists developed a variety of chemical compounds to kill insects and other menaces of agriculture and public health . Yet, in many cases, the target insects outmaneuvered the scientists by developing resistance to insecticides--in much the same way as some bacteria can tolerate antibiotics . Insecticide resistance research has involved scientists from a range of disciplines and a variety of institutional contexts that have often guided research strategy . For example, entomologists working at agricultural stations and universities concentrated on insect physiology and evolutionary genetics, while industrial chemists continued the development of novel compounds capable of killing resistant pests . Collaboration between the two groups beginning in the 1940s did not provide a solution to the resistance, but did lead to a strategic shift from pest control to pest management that continues to the present.

Toxicology, 2004 Mar 1, 196(1-2), 1 - 19
Failure of the standard battery of short-term tests in detecting some rodent and human genotoxic carcinogens; Brambilla G et al.; Theoretical reasons and experimental evidence indicate that a no-effect level generally cannot be expected for genotoxic carcinogens; as a consequence, in quantitative risk assessment the capability of distinguishing genotoxic from non-genotoxic carcinogens is of fundamental importance in order to identify relevant levels of human exposure . According to generally accepted guidelines, the standard three-test battery for the detection of genotoxic compounds consists of: (i) an in vitro test for gene mutation in bacteria; (ii) an in vitro test in mammalian cells with cytogenetic evaluation of chromosomal damage and/or a test that detects gene mutations; (iii) an in vivo test for chromosomal damage using rodent hematopoietic cells . This test battery is designed to avoid the risk of false negative results for compounds with genotoxic potential, but it cannot be taken for granted that the risk is completely eliminated . As a matter of fact there are some chemicals, classified by the International Agency for Research on Cancer (IARC) as probably or possibly carcinogenic to humans, which gave consistent negative results in this test battery, and in contrast provided positive results in other not routinely employed genotoxicity assays . The failure of the standard test battery in detecting some genotoxic carcinogens is attributable to several causes, but the principal of them are the following ones: in vitro, the artificial metabolic activity of the liver S9-mix, and the different biotransformation of chemicals in cells of different type and from different animal species; in vivo, the pharmacokinetic behaviour of the test compound, and its possible species-, sex- and tissue-specificity.

Cancer Lett, 2004 Mar 8, 205(1), 23 - 9
20(S)-Protopanaxatriol, one of ginsenoside metabolites, inhibits inducible nitric oxide synthase and cyclooxygenase-2 expressions through inactivation of nuclear factor-kappaB in RAW 264.7 macrophages stimulated with lipopolysaccharide; Oh GS et al.; Ginsenosides from Panax ginseng are metabolized by human intestinal bacteria after oral administration of ginseng extract . 20(S)-Protopanaxatriol (PPT) is one of the major metabolites of ginsenosides . Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are important enzymes that mediate inflammatory processes . Improper up-regulation of iNOS and/or COX-2 has been associated with the pathogenesis of inflammatory diseases and certain types of human cancers . Here, we investigated whether PPT could modulate iNOS and COX-2 expressions in RAW 264.7 macrophages stimulated with the endotoxin lipopolysaccharide (LPS) . We found that PPT blocked the increase in LPS-induced iNOS and COX-2 expressions through inactivation of nuclear factor-kappaB by preventing I-kappaBalpha phosphorylation and degradation . Thus, it may be possible to develop PPT as a useful agent for chemoprevention of cancer or inflammatory diseases.

Vet Microbiol, 2004 Mar 5, 98(3-4), 235 - 41
Molecular characterisation of chlamydial isolates from birds; Sudler C et al.; Fifty-one chlamydial isolates from birds collected in Switzerland were classified by amplification and restriction analysis of the 16S-23S rRNA intergenic spacer region as Chlamydophila psittaci . The aim was to characterise a broad panel of chlamydial strains from birds and to apply and verify the methods of classification and differentiation described for chlamydial organisms . Two of the six known avian chlamydial serovars (A and B) were found by serotyping with monoclonal antibodies . One isolate was not typable . Digestion of ompA-PCR amplicons by AluI generated four distinct restriction patterns (genotypes A, B, F and G) . Genotypes A and B correlated in most cases to serovars A and B, respectively . One serovar A isolate was verified as genotype B instead of A and one serovar B isolate belonged to genotype A . The non-serotypable isolate was of genotype F and one serovar A generated genotype G . OmpA sequences of one strain of each genotype were determined and compared to data bank entries . Amino acid sequences of genotype A and B strains corresponded well, showing more than 98.0% homology . The homologies of genotypes F and G sequences to genotype A strain were 82.0 and 83.0% respectively.

Vet Microbiol, 2004 Mar 5, 98(3-4), 229 - 34
Contagious bovine pleuropneumonia (CBPP) caused by vaccine strain T1/44 of Mycoplasma mycoides subsp . mycoides SC; Mbulu RS et al.; A study was carried out on four adult cattle to assess the pathogenicity of Mycoplasma mycoides subsp . mycoides SC strain T1/44, currently used as a vaccine for the control of contagious bovine pleuropneumonia (CBPP) in Namibia . Post mortem examination 9 weeks after endobronchial inoculation of the vaccine strain to three of the four animals revealed unilateral pleuropneumonic lesions, pleuritis and well-developed sequesters in two of the three inoculated animals and several small sequesters surrounded by pleuropneumonic lesions in the diaphragmatic and apical lobes in one animal . The fourth animal, which was not directly inoculated but was in close contact with the inoculated animals, revealed only an adhesion area of the lung to the ribcage . Serological examination carried out using the complement fixation test (CFT) detected positive titres in all three intubated animals and the indirect CBPP-LppQ-ELISA was positive for two of the three inoculated animals . The contact animal showed no seroconversion . M . mycoides subsp . mycoides SC was isolated from the sequesters of two of the inoculated animals . Isolation of mycoplasmas was not possible from the third inoculated animal due to heavy contamination of the samples by other bacteria, but the presence of M . mycoides subsp . mycoides SC could be evidenced by PCR from clinical samples . The identity of the T1/44 vaccine strain isolated from the sequesters of two animals was confirmed by T1/44-specific PCR analysis and by IS1296 typing using Southern blot . These results clearly show that inoculation of T1/44 vaccine via the endobronchial route can lead to CBPP.

J Ethnopharmacol, 2004 Mar, 91(1), 31 - 6
In vivo topical anti-inflammatory and in vitro antioxidant activities of two extracts of Thymus satureioides leaves; Ismaili H et al.; Four extracts at increasing polarity were prepared from the leaves of Thymus satureioides Coss . (Labiatae) and assayed for the in vivo topical anti-inflammatory effect using the croton oil ear test in mice, and for in vitro both antioxidant (DPPH degrees test) and anti-bacterial (broth microdilution method) activities . The chloroform extract showed a topical anti-inflammatory activity (ID50=282 microg cm(-2)), only three times lower than that of the reference drug indomethacin (ID50=93 microg cm(-2)) and its active components were identified as ursolic and oleanolic acids . The methanol extract, showing a significant radical-scavenging effect (SC50=14.54 microg), was characterized by the isolation and identification of some flavonoids . On the contrary, the extracts did not show any anti-bacterial effect against four standard aerobial bacteria strains.

Semin Immunol, 2004 Apr, 16(2), 107 - 17
Antigen-specific regulatory T cells--their induction and role in infection; Mills KH et al.; In addition to the well-established role of natural CD4(+)CD25(+) regulatory T (Tr) cells in the maintenance of tolerance to self-antigens, there is accumulating evidence for distinct populations of Tr cells induced in the periphery after encounter with pathogens and foreign antigens . These antigen-specific T cells, termed Tr1 or Th3 cells, secrete IL-10 and or TGF-beta, but no IL-4 and little or no IFN-gamma, and are induced by semi-mature dendritic cells under the influence of regulatory cytokines, including IL-10, TGF-beta and IL-4 . Tr1 or Th3 cells are capable of suppressing Th1 and Th2 responses and function in infection to limit pathogen-induced immunopathology, but can also be exploited in therapies for immune-mediated diseases.

Inflammopharmacology, 2003, 11(4), 415 - 22
Nitric oxide and the gut injury induced by non-steroidal anti-inflammatory drugs; Whittle BJ; Nitric oxide (NO) can protect the gastrointestinal tract from injury, including that provoked by non-steroidal anti-inflammatory drugs (NSAIDs) . This protective profile of NO, which predominantly reflects actions on the microcirculation, is mimicked by NO donors . Moreover, the NO-donating agents know as the NO-NSAIDs or CINODs (cyclo-oxygenase-inhibiting nitric oxide-donating drugs) exhibit reduced gut injury in experimental models, which is considered to reflect these local beneficial actions of NO . NSAIDs cause chronic inflammatory lesions in the small intestine in experimental models . This injury results from initial COX inhibition and other local events, with translocation of indigenous luminal bacteria, leading to induction of NO synthase isoform, iNOS, and subsequent production of the cytotoxic moiety, peroxynitrite from NO and superoxide . Agents that inhibit iNOS or superoxide production can attenuate such intestinal injury . In the absence of reactive oxygen moieties, NO may play a beneficial role in the resolution of inflammatory damage to the gut, thus reconciling the potential opposing properties of NO in tissue inflammation and injury.

Nat Rev Microbiol, 2003 Nov, 1(2), 127 - 36
Comparative genomics, minimal gene-sets and the last universal common ancestor; Koonin EV; Comparative genomics, using computational and experimental methods, enables the identification of a minimal set of genes that is necessary and sufficient for sustaining a functional cell . For most essential cellular functions, two or more unrelated or distantly related proteins have evolved; only about 60 proteins, primarily those involved in translation, are common to all cellular life . The reconstruction of ancestral life-forms is based on the principle of evolutionary parsimony, but the size and composition of the reconstructed ancestral gene-repertoires depend on relative rates of gene loss and horizontal gene-transfer . The present estimate suggests a simple last universal common ancestor with only 500-600 genes.

J Immunol, 2004 Apr 1, 172(7), 4470 - 9
CD14 is an acute-phase protein; Bas S et al.; The origin of soluble CD14 (sCD14) in the circulation is uncertain . To examine whether CD14 could be an acute-phase protein (APP), the levels of sCD14, IL-6, and C-reactive protein were determined by ELISA in serum and synovial fluid (SF) of patients with various arthropathies, and the regulation of CD14 synthesis was examined in liver cells . In patients with crystal-mediated or immunologically mediated arthritis (rheumatoid arthritis), serum levels of sCD14 were higher than or similar to those found in infection-mediated arthritis (reactive arthritis), precluding a relation with bacteria exposure . Levels of sCD14 were similar in SF and serum, and did not correlate with the number of SF leukocytes, excluding an important source from leukocyte membrane-bound CD14, by protease-mediated shedding . In contrast, serum levels of sCD14 in patients correlated with those of C-reactive protein, a classical APP, and IL-6, a cytokine known to regulate the synthesis of APP in the liver . Serum levels of sCD14 also correlated with disease activity in rheumatoid arthritis and reactive arthritis patients . IL-6 stimulated the production of CD14 by HepG2 hepatoma cells . By real-time PCR, the inducibility of CD14 by IL-6 was also observed at the mRNA level both in HepG2 cells and human primary hepatocytes . These in vitro results were confirmed by in vivo studies in IL-6(-/-) mice injected with turpentine, an experimental model of acute-phase response . Liver levels of CD14 mRNA increased in IL-6(+/+), but not in IL-6(-/-) mice . These results indicate that sCD14 can be considered as a type 2 APP.

J Mol Biol, 2004 Apr 2, 337(4), 893 - 903
Crystal structure of HEL4, a soluble, refoldable human V(H) single domain with a germ-line scaffold; Jespers L et al.; The antigen binding site of antibodies usually comprises associated heavy (V(H)) and light (V(L)) chain variable domains, but in camels and llamas, the binding site frequently comprises the heavy chain variable domain only (referred to as V(HH)) . In contrast to reported human V(H) domains, V(HH) domains are well expressed from bacteria and yeast, are readily purified in soluble form and refold reversibly after heat-denaturation . These desirable properties have been attributed to highly conserved substitutions of the hydrophobic residues of V(H) domains, which normally interact with complementary V(L) domains . Here, we describe the discovery and characterisation of an isolated human V(H) domain (HEL4) with properties similar to those of V(HH) domains . HEL4 is highly soluble at concentrations of > or =3 mM, essentially monomeric and resistant to aggregation upon thermodenaturation at concentrations as high as 56 microM . However, in contrast to V(HH) domains, the hydrophobic framework residues of the V(H):V(L) interface are maintained and the only sequence changes from the corresponding human germ-line segment (V3-23/DP-47) are located in the loops comprising the complementarity determining regions (CDRs) . The crystallographic structure of HEL4 reveals an unusual feature; the side-chain of a framework residue (Trp47) is flipped into a cavity formed by Gly35 of CDR1, thereby increasing the hydrophilicity of the V(H):V(L) interface . To evaluate the specific contribution of Gly35 to domain properties, Gly35 was introduced into a V(H) domain with poor solution properties . This greatly enhanced the recovery of the mutant from a gel filtration matrix, but had little effect on its ability to refold reversibly after heat denaturation . Our results confirm the importance of a hydrophilic V(H):V(L) interface for purification of isolated V(H) domains, and constitute a step towards the design of isolated human V(H) domains with practical properties for immunotherapy.

Physiol Plant, 2004 Mar, 120(3), 358 - 369
The respiratory chain of blue-green algae (cyanobacteria); Peschek GA et al.; Electron transport components on the way from reduced substrates to the terminal respiratory oxidase(s) are discussed in relation to analogous and/or homologous enzymes and electron carriers in the generally much better known bacteria, mitochondria and chloroplasts . The kinetic behaviour of the components, their localization within the cell and their evolutionary position are given special attention . Pertinent results from molecular genetics are also mentioned . The unprecedented role of cyanobacteria for our biosphere and our whole planet earth appears to deserve a more extended introductory chapter.

Curr Drug Targets Inflamm Allergy, 2004 Mar, 3(1), 87 - 96
The possibilities and pitfalls for anti-complement therapies in inflammatory diseases; Mizuno M et al.; The complement system is a key component of innate immunity, acting to protect the host from micro-organisms such as bacteria and other "foreign" threats, including tumor cells . However, excessive activation of complement can injure the host and can even be life threatening . These toxic effects are caused primarily by the excessive production of the anaphylatoxins C3a and C5a during complement activation and excessive formation of membrane attack complex on the host cell membrane . Many inflammatory diseases, including rheumatoid arthritis and glomerulonephritis, are thought to involve excessive activation of complement, both for their development and perpetuation . Uncontrolled complement activation is also implicated in post-ischemic inflammation and tissue damage and in sepsis . Therefore, it is important to regulate the complement system to treat disease . There are still no broadly applicable agents for the therapeutic regulation of excessive complement activation . However, there are now some agents in the development that might provide useful anti-complement therapies in the near future . Current strategies include the use of neutralizing antibodies, small synthetic antagonists, soluble recombinant forms of the natural complement regulators, and gene therapies to control excessive complement activation . Here we describe these new agents, their strengths and weaknesses and progress in testing the agents in relevant animal models.

Annu Rev Immunol, 2004, 22, 217 - 46
Starting at the beginning: new perspectives on the biology of mucosal T cells; Cheroutre H; The gastrointestinal tract is the central organ for uptake of fluids and nutrients, and at the same time it forms the main protective barrier between the sterile environment of the body and the outside world . In mammals, the intestine has further evolved to harbor a vast load of commensal bacteria that have important functions for the host . Discrimination by the host defense system of nonself from self can prevent invasion of pathogens, but equivalent responses to dietary or colonizing bacteria can lead to devastating consequences for the organism . This dilemma imposed by the gut environment has probably contributed significantly to the evolutionary drive that has led to sophisticated mechanisms and diversification of the immune system to allow for protection while maintaining the integrity of the mucosal barrier . The immense expansion and specialization of the immune system is particularly mirrored in the phylogeny, ontogeny, organization, and regulation of the adaptive intraepithelial lymphocytes, or IEL, which are key players in the unique intestinal defense mechanisms that have evolved in mammals.

Antonie Van Leeuwenhoek, 2004 May, 85(4), 335 - 41
Magnetosome chain arrangement and stability in magnetotactic cocci; Lins U et al.; We have studied the disposition of chains of magnetosomes inside magnetotactic cocci with light and electron microscopy . Light microscopy of isolated cocci indicated that the chains of magnetosomes are disposed on opposite sides of the cell . Electron spectroscopic imaging of whole unprocessed bacteria, showed the magnetosome chains in the cells . Freeze-etching of the cell surface allowed the observation of the close association of the chain with the cell surface . During the replication process of the freeze-etching, the magnetosome chains remained attached to the replicas, which indicates that chains were very close to the cell surface before freezing . We provide evidence that the large area of the contact faces between magnetosomes in a chain may provide an extra mechanical stability that helps keep the magnetosomes in chains even after isolation from the bacteria . Comparison with pointed magnetosomes from different cocci present in the same samples showed that the maintenance of linear chains is more difficult to be achieved because of the geometry of the crystals.

Respiration, 2004 Mar-Apr, 71(2), 174 - 7
Bronchoscopy with the Vision Sciences BF100 disposable-sheath device: French experience after 328 procedures; Margery J et al.; BACKGROUND: In spite of adhesion to recommended disinfection procedures, the transmission of infections by bronchoscopes is a permanent problem . OBJECTIVE: The new device may prevent nosocomial infections because it consists of two parts: a specific bronchoscope Vision Sciences BF100 and a single-use protective sheath for each procedure . The aim of this paper is to report our practice and the difficulties encountered when using this system . METHODS: We report our experience from 1997 to 2002 after 328 elective and emergency endoscopic procedures with the BF100 device . In a retrospective study, we describe the population and the incidents during procedure . We discuss the impact of the use of BF100 on the cost of bronchoscopies . RESULTS: The major constraint is the care required in assembling the optical device and disposable sheath . The intrinsic qualities of the optics are confirmed; any sample may be taken although image quality and suction capacity are inferior to videoscopes . Maneuverability is inferior to videoscopes, but improves with a short experience . In addition, this device is expensive . CONCLUSIONS: The technical performances of the BF100 device are inferior to those of videoscopes but the concept of sterile single-use sheaths is able to prevent the nosocomial infections related to bronchoscopes . Because of the cost, examination with the BF100 should be reserved to patients with proved or suspected infection (multiresistant bacteria, tuberculosis, hepatitis C and B virus, HIV, prions) and immunosuppression (hematologic diseases) .

Science, 2004 Mar 19, 303(5665), 1862 - 6
Stathmin-tubulin interaction gradients in motile and mitotic cells; Niethammer P et al.; The spatial organization of the microtubule cytoskeleton is thought to be directed by steady-state activity gradients of diffusible regulatory molecules . We visualized such intracellular gradients by monitoring the interaction between tubulin and a regulator of microtubule dynamics, stathmin, using a fluorescence resonance energy transfer (FRET) biosensor . These gradients were observed both during interphase in motile membrane protrusions and during mitosis around chromosomes, which suggests that a similar mechanism may contribute to the creation of polarized microtubule structures . These interaction patterns are likely to reflect phosphorylation of stathmin in these areas.

J Agric Food Chem, 2004 Mar 24, 52(6), 1594 - 600
Selenate reduction in river water by Citerobacter freundii isolated from a selenium-contaminated sediment; Zhang Y et al.; Bacterial reduction of selenate {Se(VI)} to insoluble elemental Se {Se(0)} is an important remedial technology to remove selenium (Se) from Se-impacted water . Citerobacter freundii, a Se(VI) reducer, isolated from a Se-contaminated sediment was assessed for its ability to reduce Se(VI) in a mineral culture medium and natural river water in a series of laboratory batch experiments . The results showed that a combination of yeast extract and glucose used in the culture medium was more effective than yeast extract alone, yeast extract plus sodium acetate, and yeast extract plus sodium lactate for reduction of Se(VI) to Se(0) by C . freundii . About 89-96% of the added Se(VI) (500-4500 microg/L) was reduced to Se(0) in the culture medium amended with 500 mg/L each of yeast extract and glucose . C . freundii can also survive in natural river water and reduce Se(VI) . During an 8-day experiment in both sterile and nonsterile river water, 63-70 and 21-22% of the added Se(VI) was reduced to Se(0) and Se(-II), respectively . These results suggest that C . freundii has great potential for Se(VI) reduction and may be used for remediating Se-impacted water.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2004, 39(1), 103 - 12
Evaluation of bioassays for analyzing biodegradable dissolved organic carbon in drinking water; Park SK et al.; Three different bioassays for analysis of biodegradable dissolved organic carbon (BDOC) were evaluated to identify which method is most applicable to analysis of drinking water . The determination of BDOC is primarily based on the differences between initial and final DOC levels during a certain incubation period using indigenous bacterial consortium as an inoculum . The assay procedures basically differ in the preparation method of inoculum . Inoculum was added in the form of suspended bacteria in one assay . In the other two assays, bacterial inoculum attached to either sand or inert media was used in a continuous reactor column . Standard solutions containing sodium acetate, sodium oxalate, or glucose at 1 mg C/L, and tap water were tested . The bioassay using bacteria attached to sand was shown to be superior to the two other methods for BDOC determination in terms of its incubation period, recovery, and reproducibility . Tap water samples, when analyzed by this assay, could not be guaranteed for biological stability due to their high BDOC concentrations (0.17 to 0.23 mg/L) that corresponded to 26-36% of the initial DOC level.

DNA Res, 2003 Dec 31, 10(6), 239 - 47
Comprehensive analysis of NAC family genes in Oryza sativa and Arabidopsis thaliana; Ooka H et al.; The NAC domain was originally characterized from consensus sequences from petunia NAM and from Arabidopsis ATAF1, ATAF2, and CUC2 . Genes containing the NAC domain (NAC family genes) are plant-specific transcriptional regulators and are expressed in various developmental stages and tissues . We performed a comprehensive analysis of NAC family genes in Oryza sativa (a monocot) and Arabidopsis thaliana (a dicot) . We found 75 predicted NAC proteins in full-length cDNA data sets of O . sativa (28,469 clones) and 105 in putative genes (28,581 sequences) from the A . thaliana genome . NAC domains from both predicted and known NAC family proteins were classified into two groups and 18 subgroups by sequence similarity . There were a few differences in amino acid sequences in the NAC domains between O . sativa and A . thaliana . In addition, we found 13 common sequence motifs from transcriptional activation regions in the C-terminal regions of predicted NAC proteins . These motifs probably diverged having correlations with NAC domain structures . We discuss the relationship between the structure and function of the NAC family proteins in light of our results and the published data . Our results will aid further functional analysis of NAC family genes.

Przegl Epidemiol, 2003, 57(4), 593 - 8
Plague in Kazakhstan at the present time; Aikimbajev A et al.; Since 1990 to 2002, 19 human plague cases and 2 bacteria-carriers cases were registered in natural plague foci on the territory of the republic . For last 12 years plague in forms of bubonic and bubonic-septic was diagnosed in Kazakhstan . Out of 19 patients (14 men--73.6% and 5 women--26.3%), 13 persons recovered (8.4%), 6 persons died (31.6%) . Employment of the treatment in the new scheme sharply lowered lethality from plague . Live attenuated Y . pestis EV vaccine available for human and camel use is applied as a basic preventive measure in plague natural foci . Epidemiological importance of ill camels remains high . At the analyzed period, in 5 cases infection was a result of forced camel slaughter . 11 people (57.8%) were infected through flea bites . Central Asian desert plague focus is still the most active and its potential to start epidemic increases in connection with rising of anthropogenic influence . All these changes occur on the background of epidemiological survey decreasing in natural plague foci.

Prikl Biokhim Mikrobiol, 2004 Jan-Feb, 40(1), 66 - 9
{Features of transformation of phosphates in Rhodobacter sphaeroides chromatophores}; Goncharova NV et al.; We have found the ATP production in the Rhodobacter sphaeroides chromatophores illuminated by single short light flash, that is under conditions when the proton gradient formed as a result of electron transport after the second flash, is absent . The ATP synthesis was accompanied by the H2O2 formation . Simultaneous formation of H2O2 is indicative of the oxidative activation of phosphate during the ATP synthesis, as in the model systems with isolated chlorophyll . These data provide a theoretical background to the fitting of illumination parameters in both laboratory and industrial photobioreactors with photosynthetic bacteria used in biotechnological processes.

Prikl Biokhim Mikrobiol, 2004 Jan-Feb, 40(1), 24 - 7
{Effect of deuteration on methanol dehydrogenase activity in Methylophilus sp . B-7741}; Pshenichnikova AB et al.; We studied the effect of deuterium oxide present in the medium on the activity of methanol dehydrogenase (EC 1.1.99.8) from methylotrophic bacteria Methylophilus sp . B-7741 . Methanol dehydrogenase activity in extracts of the biomass obtained in a highly deuterated medium (2H-enzyme) was 34-47% of enzyme activity in the control biomass, which depended on reaction conditions . The isotopic effects of substrate deuterium (methanol) for 1H-enzyme and 2H-enzyme were 1.37 +/- 0.05 and 1.38 +/- 0.01, respectively . We revealed for the first time the reverse isotopic effect of solvent deuterium in the reaction catalyzed by methanol dehydrogenase (0.80 +/- 0.02 and 0.60 +/- 0.01 for 1H-enzyme and 2H-enzyme, respectively).

Ann N Y Acad Sci, 2003 Dec, 1009, 30 - 3
Vertebrate agmatinases: what role do they play in agmatine catabolism?
Morris SM Jr.
Whereas agmatine in vertebrates may be derived from multiple sources such as the diet, endogenous synthesis via arginine decarboxylase, and possibly also from enteric bacteria, agmatinase is the only enzyme specific for agmatine catabolism . As it hydrolyzes a guanidino group within agmatine and also contains signature amino acid residues that act as ligand binding sites for the Mn(++) cofactor, agmatinase is classified as a member of the arginase superfamily . Very little information is available regarding how much agmatine in vertebrate species is catabolized by agmatinase versus other enzymes such as diamine and amine oxidases . Moreover, comparisons of primary sequences of several vertebrate agmatinases demonstrate that several residues essential for catalytic activity are not conserved in the mouse . This leads to the prediction that the agmatinase protein in mouse has little or no catalytic activity, not only raising questions about the physiologic routes of agmatine disposal in this organism, but also suggesting the existence of species-specific differences in mechanisms for regulating agmatine levels.

Curr Biol, 2004 Mar 9, 14(5), R207 - 9
Chaperones: inserting beta barrels into membranes; Ryan MT; How beta-barrel proteins are inserted into cellular membranes is poorly understood . New work has identified a sorting and assembly machinery that chaperones beta-barrels into the mitochondrial outer membrane and is evolutionarily conserved from bacteria to man.

Environ Technol, 2004 Jan, 25(1), 69 - 77
Precipitation of heavy metals from landfill leachates by microbially-produced sulphide; Moller A et al.; Four leachates from two landfills in Sweden were treated for the removal of heavy metals with the aid of sulphate-reducing bacteria (SRB) . Both continuous and batch experiments were performed . A packed-bed process was used for sulphide production . The metals studied were As, Cd, Cr, Cu, Hg, Ni, Pb, and Zn . The continuous experiments showed that Cd and Cu were most efficiently removed and that Cr was the most difficult to precipitate . In a continuous experiment with one of the leachates, the removal of Cd, Cu and Zn depended upon the retention time in the system . In the batch experiments, precipitation of the metals was a relatively fast process . No significant differences in metal concentrations were found between experiments terminated after a day and those terminated after a week . In a batch experiment involving one of the leachates, the precipitation of Cd and Cu was shown to be dependent upon the metal:sulphide ratio . Removal of the metals increased with an increase in the sulphide:metal ratio up to 45:1 . The process with SRB showed an interesting potential for removal of heavy metals from leachates . One of the two leachates for which the highest metal removals were obtained came from a landfill for hazardous waste.

Presse Med, 2004 Jan 31, 33(2), 119 - 22
{Bioterrorism with brucellosis}; Guihot A et al.; A CONTAMINATING SPRAY: Brucellosis is a zoonosis due to a bacteria of the Brucella . B . melitensis species and is the principle agent of human brucellosis . The interest of Brucella as a biological weapon resides in the fact that transmission through a spray is possible, as has been reported with human contamination during abortion of infected animals or bacterial spraying in laboratories . The bacteria is highly contagious . It is suggested that 10 to 100 bacteria would be sufficient to produce a contaminating spray for humans . THE MENACE: In 1954, B . suis was the first infectious agent used as a biological weapon in the United States . Several other countries have been suspected of studying the agent as a biological weapon, but to date, no use of Brucella in a bioterrorist attack has been reported . THE EXTENT OF RISK: Brucella, and notably B . melitensis and B . suis, are considered as agents unlikely to be used as biological weapons: their incubation is long, the majority of infections are asymptomatic and mortality is low . However, the morbidity of this agent should not be underrated since it leads to chronic and disabling pathologies.

Water Res, 2004 Apr, 38(7), 1838 - 52
Decolorization and toxicity of reactive anthraquinone textile dyes under methanogenic conditions; Lee YH et al.; Reductive decolorization of two anthraquinone reactive dyes (Reactive Blue 4, RB4; Reactive Blue 19, RB19) under methanogenic conditions was performed using a mixed, methanogenic culture . Decolorization of the two anthraquinone dyes was investigated to evaluate the rate and extent of color removal as well as to assess possible toxic effects of the dyes and their decolorization product(s) on the methanogenic culture as a function of initial dye concentration ranging from 50 to 300 mg x L(-1) . A dextrin/peptone mixture was used as the carbon and electron source . A high rate and extent of color removal was achieved ranging from 4.3 to 29.9 mg x L(-1)h(-1) and 73-91% for RB4, and 13.0-74.4 mg x L(-1)h(-1) and 90-95% for RB19 . Initial RB4 concentrations up to 100 mg x L(-1) did not result in any significant inhibition . Both the 200 and 300 mg x L(-1) RB4-amended cultures, and all RB19-amended cultures resulted in severe inhibition of both acidogenesis and methanogenesis . Sequential dye addition at 300 mg x L(-1) for both RB4 and RB19 resulted in accumulation of volatile fatty acids (VFAs) and a very low methane production at the end of the first dye addition after 44 days of incubation . However, at the end of the second dye addition, after a relatively long incubation (384 days), recovery of methanogens in the RB4-amended culture was observed in contrast to the complete inhibition of methanogenesis in the RB19-amended culture . Therefore, RB19 resulted in a higher degree of inhibition of both acidogenesis and methanogenesis than RB4 . Addition of dextrin/peptone to dye-inhibited cultures resulted in acidogenesis and a gradual recovery of methanogenesis (mainly aceticlastic methanogenesis) in the RB4-inhibited culture, and a slow recovery of acidogenesis but no recovery of methanogenesis in the RB19-inhibited culture . In contrast, addition of 80% H(2)-20% CO(2) gas to dye-inhibited cultures resulted in recovery of hydrogenotrophic methanogenesis in both the RB4- and RB19-inhibited cultures . In spite of the relatively severe inhibition of the two anthraquinone dyes on the mixed, methanogenic culture, a high extent of color removal was achieved.

Mol Cell Endocrinol, 2004 Feb 27, 215(1-2), 149 - 59
Modulation of aldosterone and cortisol synthesis on the molecular level; Lisurek M et al.; CYP11B1 and the closely related CYP11B2 are involved in the production of adrenal steroid hormones . Although in human their primary structure is 93% identical they are involved in the biosynthesis of functionally diverse products, such as glucocorticoids and mineralocorticoids, respectively . In contrast, bovine CYP11B1 combines both activities in one single enzyme . The CYP11B family belongs to class I cytochromes P450 that have been described in bacteria and mitochondria and receive their electrons from a low molecular weight iron sulphur protein which is reduced by a NADPH-dependent FAD-containing reductase . In this review, we summarise the current knowledge on the modulation of aldosterone and cortisol synthesis by transcriptional regulation, on the molecular level as consequence of mutations found in patients suffering from steroid hormone-related diseases as well as introduced by site-directed mutagenesis and as consequence of protein-protein interaction with both CYP11A1 and the natural redox partner adrenodoxin.

J Endotoxin Res, 2004, 10(1), 15 - 23
Molecular cloning and characterization of mouse LITAF cDNA: role in the regulation of tumor necrosis factor-alpha (TNF-alpha) gene expression; Bolcato-Bellemin AL et al.; The inflammatory response to bacteria and bacterial products, such as lipopolysaccharides (LPSs), is mediated by a variety of secreted factors, but cytotoxic effects of LPS have been ascribed to the tumor necrosis factor alpha (TNF-alpha) activity . TNF-alpha is probably the most pleiotropic cytokine and, given the deleterious effects to the host of this factor, it has been postulated that its expression must be tightly regulated . Our laboratory has recently isolated, cloned and characterized a novel human transcription factor named LITAF or LPS-induced TNF-alpha factor . The present study reports the isolation, cloning and characterization of the mouse LITAF cDNA . Chromosomal localization revealed that mouse LITAF mapped to mouse chromosome 16, in a region highly homologous with the area on which human LITAF was previously located . Northern blot analysis shows that mouse LITAF is already expressed at embryonic day 7 of development, and is highly expressed in adult liver, heart and kidney . Moreover, upon LPS stimulation, we show that: (i) LITAF expression is increased in a mouse monocyte/macrophage cell line; and (ii) TNF-alpha expression is reduced in ES cell-derived macrophages lacking one copy of LITAF gene . Taken together, these results highlight the important role of LITAF in the regulation of TNF-alpha gene expression and suggest a potential role of LITAF in mouse organogenesis.

J Periodontol, 2004 Jan, 75(1), 146 - 53
Locally produced anti-phosphorylcholine and anti-oxidized low-density lipoprotein antibodies in gingival crevicular fluid from aggressive periodontitis patients; Schenkein HA et al.; BACKGROUND: Sera from patients with periodontal attachment loss contain higher concentrations of IgG anti-phosphorylcholine (anti-PC) than sera from healthy subjects . Furthermore, a large proportion of plaque bacteria bear PC-containing surface antigens, implicating the oral flora as a source of immunogen for anti-PC . Additionally, anti-PC is cross-reactive with a variety of oral bacterial antigens and human antigens such as oxidized low-density lipoprotein (oxLDL) . We hypothesized that, if the oral flora is a source of PC antigens, then we should be able to detect local anti-PC and anti-oxLDL production in gingival crevicular fluid (GCF) . METHODS: To test this, we collected 66 GCF samples from 15 patients with aggressive periodontitis and examined both the GCF samples and serum samples for their content of IgG anti-PC, IgG anti-LDL, and IgG anti-oxLDL by enzyme-linked immunosorbent assay . We also determined levels of anti-tetanus toxoid (anti-TT) as a non-oral antigen control . Serum and GCF concentrations of serum albumin (HSA) were also determined for use as a dilution marker . A conservative GCF:serum antibody ratio of greater than 1.5 was considered to be evidence of local antibody production . RESULTS: For the non-oral antigen TT, only one out of 62 samples contained locally produced antibody . Eight out of 64 samples (7 from a single subject) demonstrated local production of anti-LDL . In contrast, 28 out of 66 samples demonstrated local production of anti-PC, and 47 out of 66 samples contained locally produced anti-oxLDL . It was observed that A . actinomycetemcomitans strains containing or devoid of PC could absorb anti-oxLDL from human sera . Although there was a correlation between the ratios of anti-PC and anti-oxLDL (Spearman's rho = 0.35, P = 0.0037), local production of both antibodies was found in only 17 out of 65 samples, indicating that these antibodies are not always reflective of reactivity to the same antigens . CONCLUSION: The local production of anti-PC and anti-oxLDL further implicates the oral flora as a source of antigen that may mediate immune reactions of relevance to cardiovascular and other systemic diseases.

Biochemistry, 2004 Mar 23, 43(11), 3204 - 13
X-ray crystal structure of Desulfovibrio vulgaris rubrerythrin with zinc substituted into the {Fe(SCys)4} site and alternative diiron site structures; Jin S et al.; The X-ray crystal structure of recombinant Desulfovibrio vulgaris rubrerythrin (Rbr) that was subjected to metal constitution first with zinc and then iron, yielding ZnS(4)Rbr, is reported . A {Zn(SCys)(4)} site with no iron and a diiron site with no appreciable zinc in ZnS(4)Rbr were confirmed by analysis of the anomalous scattering data . Partial reduction of the diiron site occurred during the synchrotron X-ray irradiation at 95 K, resulting in two different diiron site structures in the ZnS(4)Rbr crystal . These two structures can be classified as containing mixed-valent Fe1(III)(mu-OH(-))(mu-GluCO(2)(-))(2)Fe2(II) and Fe1(II)(mu-GluCO(2)(-))(2)Fe2(III)-OH(-) cores . The data do not show any evidence for alternative positions of the protein or solvent ligands . The iron and ligand positions of the solvent-bridged site are close to those of the diferric site in all-iron Rbr . The diiron site with only the two carboxylato bridges differs by an approximately 2 A shift in the position of Fe1, which changes from six- to four-coordination . The Fe1- - -Fe2 distance (3.6 A) in this latter site is significantly longer than that of the site with the additional solvent bridge (3.4 A) but significantly shorter than that previously reported for the diferrous site (4.0 A) in all-iron Rbr . The apparent redox-induced movement of Fe1 at 95 K in the ZnS(4)Rbr crystal implies an extremely low activation barrier, which is consistent with the rapid (approximately 30 s(-1)) room temperature turnover of the all-iron Rbr during its catalysis of two-electron reduction of hydrogen peroxide . ZnS(4)Rbr does not show peroxidase activity, presumably because the {Zn(SCys)(4)} site, unlike the {Fe(SCys)(4)} site, cannot mediate electron transfer to the diiron site . One or both of the diiron site structures in the cryoreduced ZnS(4)Rbr crystal are likely to represent that (those) of transient mixed-valent diiron site(s) that must occur upon return of the diferric to the diferrous oxidation level during peroxidase turnover.

Mol Phylogenet Evol, 2004 Jan, 30(1), 24 - 37
Molecular systematics of aphids (Homoptera: Aphididae): new insights from the long-wavelength opsin gene; Ortiz-Rivas B et al.; Viviparous aphids (Aphididae) constitute a monophyletic group within the Homoptera with more than 4000 extant species worldwide but higher diversity in temperate regions . Several aspects of their biology account for attention paid to this group of insects . Their plant-sap-sucking way of feeding with many species transmitting viruses to crop plants has important implications on crop management strategies . Cyclical parthenogenesis associated in many groups to host alternation and elaborate polyphenisms is of special interests for evolutionists . Finally, the ancient association of most aphid species with intracellular endosymbiotic bacteria (Buchnera sp.) has also received much attention from evolutionists interested in mechanisms involved in the symbiotic process . Knowing the phylogenetic relationships among major aphid taxa is of special interest to evolutionists interested in the above issues . However, until recently, molecular approaches to aphid phylogeny were absent and discussions on the evolution of aphid life-cycles and on evolutionary aspects of their symbiotic association with Buchnera were framed by morphology-based phylogenies . Recently, two reports using molecular approaches attempted to address the yet unresolved phylogeny of Aphididae with limited although somehow different conclusions . In the present report we study the utility of the long-wave opsin gene in resolving phylogenetic relationships among seven subfamilies within the Aphididae . Our results corroborate some previously proposed relationships and suggest a revision of some others . In particular, our data support grouping the analysed aphid species into three main clades, being the subfamily Lachninae one of them, which contradicts its generally accepted sistership relationship with the subfamily Aphidinae . Moreover, our data also suggest a basal position of Lachninae which has implications on current discussions about the ancestrality of conifer-feeding in modern aphids.

Methods Mol Biol, 2004, 260, 129 - 44
The use of double-stranded RNA to knock down specific gene activity; Montgomery MK; In many eukaryotes, the introduction of double-stranded RNA (dsRNA) into cells triggers the degradation of cognate mRNAs through a posttranscriptional gene silencing mechanism . This phenomenon has been called RNA interference or RNAi . Several methods for delivering dsRNA into the model organism C . elegans are described; these methods include (1) microinjecting dsRNA synthesized in vitro into the body cavity of the worm, (2) soaking worms in a solution of dsRNA, (3) feeding worms dsRNA-expressing bacteria, and (4) engineering transgenic worm strains to express dsRNA in vivo . Variations of these methods may be used to perform RNAi in other species as well . The choice of which delivery method to use, along with other options (region to target, length of dsRNA) are also discussed.

Methods Mol Biol, 2004, 264, 183 - 9
SH3 domain protein-binding arrays; Chamnongpol S et al.; First identified as part of the Rous sarcoma oncogene product Src, SH3 (Src Homology 3) domains play an important role in intercellular communication and intracellular signal transduction . A high-throughput assay for ligand binding to SH3 domains--SH3 domain proteins immobilized on a membrane--allows rapid visualization of numerous SH3 domain protein-protein interactions with no expensive equipment or radioactivity required . Once the array is constructed or obtained commercially, the procedure is straightforward: The protein of interest is cloned into a fusion-tagged expression vector and expressed in bacteria, the prepared bacterial extract is incubated with the array membrane, and the signal is measured using a chemiluminescence detection system.

J Mol Biol, 2004 Mar 26, 337(3), 743 - 59
Highly discriminating protein-protein interaction specificities in the context of a conserved binding energy hotspot; Li W et al.; We explore the thermodynamic basis for high affinity binding and specificity in conserved protein complexes using colicin endonuclease-immunity protein complexes as our model system . We investigated the ability of each colicin-specific immunity protein (Im2, Im7, Im8 and Im9) to bind the endonuclease (DNase) domains of colicins E2, E7 and E8 in vitro and compared these to the previously studied colicin E9 . We find that high affinity binding (Kd < or = 10(-14) M) is a common feature of cognate colicin DNase-Im protein complexes as are non-cognate protein-protein associations, which are generally 10(6)-10(8)-fold weaker . Comparative alanine scanning of Im2 and Im9 residues involved in binding the E2 DNase revealed similar behaviour to that of the two proteins binding the E9 DNase; helix III forms a conserved binding energy hotspot with specificity residues from helix II only contributing favourably in a cognate interaction, a combination we have termed as "dual recognition" . Significant differences are seen, however, in the number and side-chain chemistries of specificity sites that contribute to cognate binding . In Im2, Asp33 from helix II dominates colicin E2 specificity, whereas in Im9 several hydrophobic residues, including position 33 (leucine), help define its colicin specificity . A similar distribution of specificity sites was seen using phage display where, with Im2 as the template, a library of randomised sequences was generated in helix II and the library panned against either the E2 or E9 DNase . Position 33 was the dominant specificity site recovered in all E2 DNase-selected clones, whereas a number of Im9 specificity sites were recovered in E9 DNase-selected clones, including position 33 . In order to probe the relationship between biological specificity and in vitro binding affinity we compared the degree of protection afforded to bacteria against colicin E9 toxicity by a set of immunity proteins whose affinities for the E9 DNase differed by up to ten orders of magnitude . This analysis indicated that the Kd required for complete biological protection is <10(-10)M and that the "affinity window" over which the selection of novel immunity protein specificities likely evolves is 10(-6)-10(-10)M . This comprehensive survey of colicin DNase-immunity protein complexes illustrates how high affinity protein-protein interactions can be very discriminating even though binding is dominated by a conserved hotspot, with single or multiple specificity sites modulating the overall binding free energy . We discuss these results in the context of other conserved protein complexes and suggest that they point to a generic specificity mechanism in divergently evolved protein-protein interactions.

Mol Phylogenet Evol, 2004 Apr, 31(1), 204 - 13
Phylogenetic comparison of metabolic capacities of organisms at genome level; Ma HW et al.; Horizontal gene transfer (HGT) has been shown to widely spread in organisms by comparative genomic studies . However, its effect on the phylogenetic relationship of organisms, especially at a system level of different cellular functions, is still not well understood . In this work, we have constructed phylogenetic trees based on the enzyme, reaction, and gene contents of metabolic networks reconstructed from annotated genome information of 82 sequenced organisms . Results from different phylogenetic distance definitions and based on three different functional subsystems (i.e., metabolism, cellular processes, information storage and processing) were compared . Results based on the three different functional subsystems give different pictures on the phylogenetic relationship of organisms, reflecting the different extents of HGT in the different functional systems . In general, horizontal transfer is prevailing in genes for metabolism, but less in genes for information processing . Nevertheless, the major results of metabolic network-based phylogenetic trees are in good agreement with the tree based on 16S rRNA and genome trees, confirming the three domain classification and the close relationship between eukaryotes and archaea at the level of metabolic networks . These results strongly support the hypothesis that although HGT is widely distributed, it is nevertheless constrained by certain pre-existing metabolic organization principle(s) during the evolution . Further research is needed to identify the organization principle and constraints of metabolic network on HGT which have large impacts on understanding the evolution of life and in purposefully manipulating cellular metabolism.

Mol Phylogenet Evol, 2004 Apr, 31(1), 123 - 30
The origin of polynucleotide phosphorylase domains; Leszczyniecka M et al.; In this report, we document the presence of polynucleotide phosphorylase (PNPase) in the animal eukaryotes . These proteins contain several domains, including 2 RNase PH domains (PNPase 1 and PNPase 2) which are closely related functionally and in sequence similarity to ribonuclease PH (RPH) protein . Phylogenetic analysis of the gene genealogy of these three domains suggests that PNPase was formed via a duplication event that also produced the RNase PH protein . Given the current distribution of these domains in the tree of life, these duplication events most likely occurred in the common ancestor of the three organismal superkingdoms, Archaea, Eukarya, and Bacteria . In particular, PNPase 2 and RPH are more closely related to each other than either one is to PNPase 1, suggesting a deeper differentiation of PNPase 1 in the common organismal ancestor . In addition, while PNPase 1 and PNPase 2 appear to have the same evolutionary signal as determined by the incongruence length difference (ILD) test, RPH appears to have an incongruent signal with both of the PNPase domains . This result suggests that RPH experienced different evolutionary divergence patterns than the PNPase domains, consistent with the linked nature of the two PNPase domains.

Immunol Lett, 2004 Feb 15, 91(2-3), 247 - 53
A phagemid system enabling easy estimation of the combinatorial antibody library size; Hong SH et al.; We developed a new phagemid system for the generation of combinatorial antibody libraries . This system allows the recombination efficiency to be estimated easily, which aids the accurate measurement of the antibody library size . Two phagemids were constructed . pRTCB bears the structural tetracycline-resistant gene (tetR) and pRTCA bears its promoter region (Ptet) . pRTCA and pRTCB were constructed so that recombinase-mediated chain exchange (RMCE) in Cre-expressing bacteria results in the simultaneous acquisition of Tet resistance and newly recombined single chain variable fragment (scFv) genes . PCR and restriction enzyme analysis of randomly selected tetR phagemids showed that all Tet-resistant phagemids have an active tetR gene and a recombined scFv gene . RMCE efficiency was measured by the ratio of the tetR colonies to the total colonies . The scFv proteins expressed from the recombined phagemid vectors were functional . Thus, our phagemid vector system may be useful for making combinatorial antibody libraries.

Virus Res, 2004 Mar 15, 100(2), 179 - 89
Characterization and phylogenetic analysis of the chitinase gene from the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus; Wang H et al.; A putative chitinase gene was identified within the fragment EcoRI-K of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HearNPV, also called HaSNPV) genome . The open reading frame (ORF) contains 1713 nucleotides (nt) and encodes a protein of 570 amino acids (aa) with a predicted molecular weight of 63.6 kDa . Transcription started at about 18 h post infection (p.i.) and the protein was first detected at 20 h p.i . The times of transcription and expression are characteristic of a late baculovirus gene . 5' and 3' RACE indicated that transcription was initiated from the adenine residue located at -246 nt upstream from the ATG start site and the poly (A) tail was added at 267 nt downstream from the stop codon . This is the first report on the molecular characterization of a chitinase from a single nucleocapsid NPV . The phylogeny of baculoviral chitinase genes were extensively examined in comparison with chitinases derived from bacteria, fungi, nematode, actinomycetes, viruses, insects and mammals . Neighbor-joining and most parsimony analyses showed that the baculoviral chitinases were clustered exclusively within gamma-proteobacteria . Our results strongly suggest that baculoviruses acquired their chitinase genes from bacteria.

Burns, 2004 Mar, 30(2), 140 - 7
In vitro cytotoxity of silver: implication for clinical wound care; Poon VK et al.; In this study, we look at the cytotoxic effects of silver on keratinocytes and fibroblasts . We have assessed the viability of monolayer cultures using the MTT and BrdU assays . The composition of the culture medium and also the culture technique were modified to assess the effects of culture 'environment' on the susceptibility of the cells to the toxic action of silver . Further in vitro, experiments were performed using tissue culture models to allow cellular behavior in three dimensional planes which more closely simulated in vivo behavior . The silver source was both silver released from silver nitrate solution but also nanocrystalline silver released from a commercially available dressing . The results show that silver is highly toxic to both keratinocytes and fibroblasts in monolayer culture . When using optimized and individualized culture the fibroblasts appear to be more sensitive to silver than keratinocytes . However, when both cell types were grown in the same medium their viability was the same . Using tissue culture models again indicated an 'environmental effect' with decreased sensitivity of the cells to the cytotoxic effects of the silver . Nevertheless in these studies the toxic dose of skin cells ranging from 7 x 10(-4) to 55 x 10(-4)% was similar to that of bacteria . These results suggest that consideration of the cytotoxic effects of silver and silver-based products should be taken when deciding on dressings for specific wound care strategies . This is important when using keratinocyte culture, in situ, which is playing an increasing role in contemporary wound and burn care.

Vet Microbiol, 2004 Mar 26, 99(1), 59 - 66
Immunoblotting, ELISA and culture evidence for Chlamydiaceae in sows on 258 Belgian farms; Vanrompay D et al.; The prevalence of Chlamydiaceae infections on 258 closed pig breeding farms in Belgium was examined . For this purpose, 258 farms were randomly selected in the provinces West-Vlaanderen (44%), Oost-Vlaanderen (20%), Antwerpen (10%) and Vlaams-Brabant (6%) . Of all farms examined, 96.5% were positive for Chlamydia-specific antibodies in ELISA and most were moderately to strongly positive . ELISA results revealed only 9 (3.5%) sero-negative farms . None of the ELISA negative sera reacted in immunoblotting . Only 212 of 249 ELISA positive sera reacted positive in immunoblotting . Additionally, 23 autopsy samples were examined by isolation in Vero cells . The major outer membrane sequence of the one isolate obtained showed 98.6% amino acid homology to the one of Chlamydophila psittaci strain CP3, formerly isolated from a pigeon . Present observations indicate that chlamydial infections are nearly endemic in the Belgian pig population and that Belgian pigs can become infected with C . psittaci . Nevertheless, the role and significance of Chlamydiaceae as pathogens in pigs remain unsolved and require further investigation.

Vector Borne Zoonotic Dis, 2004 Spring, 4(1), 23 - 32
Density of questing Ixodes ricinus nymphs and adults infected by Borrelia burgdorferi sensu lato in Switzerland: spatio-temporal pattern at a regional scale; Jouda F et al.; Lyme borreliosis, the most important vector-borne disease in the Northern hemisphere, causes health problem for populations in endemic areas . In the present study, the density of questing Ixodes ricinus ticks and their infection with Borrelia burgdorferi sensu lato (sl) was examined in 11 areas located on the Swiss Plateau and in an alpine valley . From 1999 to 2001, free-living I . ricinus ticks were collected on a monthly basis by flagging vegetation in these areas . Each tick was examined for the presence of B . burgdorferi sl using direct fluorescent antibody assay, and for isolation of the bacteria . Borreliae were characterized by PCR followed by RFLP . Density of questing ticks varied greatly between studied areas . Borreliae were observed in ticks collected in all investigated sites . However, the prevalence of infection differed significantly among areas . Infection prevalence varied from 9% to 40% in nymphs and from 22% to 47% in adults . Adult ticks were significantly more infected (129/366, 35%) than nymphs (109/552, 20%) . There was no correlation between nymphal density and infection prevalence as well as between adult density and infection prevalence, but there was a correlation between density of ticks and density of infected ticks . During the spring peak of questing tick density, a range of 2-30.3 infected ticks per 100 m(2) was observed . B . burgdorferi sl isolates (n = 129) were obtained from ticks collected in 10/11 areas . Five Borrelia species were identified: B . garinii, B . burgdorferi sensu stricto, B . afzelii, B . valaisiana, B . lusitaniae, and six mixed infections were also obtained . Borrelia species were heterogeneously distributed in the different areas.

BMC Microbiol . 2004 Feb 05;4(1):7.
Immune response to Mycoplasma pneumoniae P1 and P116 in patients with atypical pneumonia analyzed by ELISA; Drasbek M et al.; BACKGROUND: Serology is often used for the diagnosis of Mycoplasma pneumoniae . It is important to identify specific antigens that can distinguish between the presence or absence of antibodies against M . pneumoniae . The two proteins, P116 and P1, are found to be immunogenic . By using these in ELISA it is possible to identify an immune response against M . pneumoniae in serum samples . RESULTS: A recombinant protein derived from the P116 protein and one from the P1 protein were used in two ELISA tests, rP116-ELISA and rP1-ELISA . Human serum samples from patients with atypical pneumonia were tested and compared to the results of the complement fixation test . There was a good agreement between the two tests but the rP1-ELISA showed the best discrimination between positive and negative samples . CONCLUSION: Two ELISA tests based on recombinant proteins have been analysed and compared to the complement fixation test results . The two ELISA tests were found suitable for use in serodiagnostics of M . pneumoniae infections . The use of specific antigens eliminates the risk of cross reaction to an immune response against other bacteria.

Hua Xi Kou Qiang Yi Xue Za Zhi, 2004 Feb, 22(1), 52 - 3
{Study on endotoxin levels of acute and chronic periapical periodontitis}; Hu T et al.; OBJECTIVE: To compare the endotoxin levels between acute and chronic periapical periodontitis with different clinical symptoms . METHODS: 10 cases of acute periapical priodontitis(Group 1), 10 cases of chronic periapical periodontitis (group 2, the diameter of apical radiolucency area was less than 2 mm), 10 cases of chronic periapical periodontitis with sinus(group 3, the diameter of apical radiolucency area was greater than 2 mm), 10 cases of chronic periapical periodontitis without sinus (group 4, the diameter of apical radiolucency area was greater than 2 mm), were included in the study . Chromogenic substrate method of limulus amebocyte lysate(LAL) test was used to measure the endotoxin level . RESULTS: Endotoxin concentrations in group 2 were significantly lower than those in group 1, group 3 and group 4(P < 0.01) . CONCLUSION: Endotoxin plays a very important role in the initiation and development of periapical periodotitis and is closely associated with clinical symptoms and apical radiolucency degree.

J Med Microbiol, 2004 Apr, 53(Pt 4), 341 - 3
Demonstration of Brachyspira aalborgi lineages 2 and 3 in human colonic biopsies with intestinal spirochaetosis by specific fluorescent in situ hybridization; Jensen TK et al.; Sequences of known 16S rRNA genes, derived from sequence analysis of cloned 16S rDNA, were used to design a specific oligonucleotide probe targeting spirochaetes of Brachyspira aalborgi lineages 2 and 3 . The probe was used with fluorescent in situ hybridization to study the involvement of these organisms in human intestinal spirochaetosis . Seventeen human colonic biopsies from Norway and Denmark with intestinal spirochaetosis caused by Brachyspira-like organisms different from the type strain of B . aalborgi (lineage 1) were examined . Application of the probe gave a positive signal in two Norwegian biopsies, whereas the 15 other biopsies were hybridization-negative . The positive reaction visualized the spirochaetes as a fluorescent, 3-5 microm-high fringe on the surface epithelium, extending into the crypts . The study verified the presence of B . aalborgi lineages 2 and 3 and identified the bacteria as an aetiological agent of human intestinal spirochaetosis.

J Med Microbiol, 2004 Apr, 53(Pt 4), 293 - 300
Brachyspira hyodysenteriae and other strongly beta-haemolytic and indole-positive spirochaetes isolated from mallards (Anas platyrhynchos); Jansson DS et al.; The aims of the current study were to collect intestinal spirochaetes (genus Brachyspira) from farmed and wild mallards (Anas platyrhynchos) and to identify and classify those isolates that phenotypically resembled Brachyspira hyodysenteriae, an enteric pathogen of pigs . The isolation rate of Brachyspira spp . was high from both farmed (93 %) and wild mallards (78 %) . In wild mallards, it appeared that Brachyspira spp . were more likely to be found in migratory birds (multivariate analysis: RR = 1.8, 95 % CI 1.1-3.1) than in mallards sampled in a public park . Pure cultures of putative B . hyodysenteriae were obtained from 22 birds . All five isolates from farmed mallards and ten randomly selected isolates with this phenotype were used for further studies . All isolates from farmed mallards and two of the isolates from wild mallards were PCR-positive for the tlyA gene of B . hyodysenteriae . Two isolates from farmed mallards were selected for pulsed field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) analysis . These isolates clustered with the type and reference strains of B . hyodysenteriae . 16S rDNA sequence analysis performed on 11 of the strains showed that they were all closely related to each other and to the B . hyodysenteriae-Brachyspira intermedia cluster . Three of the mallard isolates had 16S rDNA sequences that were identical to those of B . hyodysenteriae strains R1 and NIV-1 previously isolated from common rheas (Rhea americana) . To conclude, the isolates from farmed mallards and two isolates from wild mallards were classified as B . hyodysenteriae based on the fact that they could not be differentiated by any of the applied methods from type, reference and field strains of B . hyodysenteriae . The remaining isolates could not be assigned irrefutably to any of the presently recognized Brachyspira species . These results point to a broader host spectrum of B . hyodysenteriae than is generally recognized, and to the presence in mallards of strongly beta-haemolytic and indole-producing spirochaetes that possess many, but not all, of the currently recognized characteristics of B . hyodysenteriae.

J Clin Periodontol, 2004 Feb, 31(2), 85 - 90
Impact of mouthrinses on morning bad breath in healthy subjects; Carvalho MD et al.; BACKGROUND: During sleep, a proliferation of oral bacteria is responsible for the release of offending gases in morning breath even in healthy people . Thus, the aim of this study was to evaluate the bad breath-inhibiting effect of four commercially available mouthrinses (0.03% triclosan, 0.12% chlorhexidine gluconate, 0.05% cetylpyridinium chloride and essential oils) on morning breath when compared with a positive and a negative control . METHOD: A six-step double-blind, crossover, randomised study was conducted in 12 dental students with healthy periodontium, who refrained from mechanical plaque control during a 4-day period . The subjects were instructed to rinse twice daily with the assigned product during each period . Fifteen-day washout intervals were used . Before professional plaque and tongue coating removal (baseline), the morning breath was scored through volatile sulphur compounds (VSCs) level measured by a sulphide monitor . After 4 days, VSCs and plaque index (PI) were recorded . RESULTS: Even in the absence of mechanical plaque control, there was a decrease in VSC level with the use of all mouthrinses, with the exception of an increase with the use of the negative control . The VSC formation was inhibited in descending order, by positive control (0.2% chlorhexidine), 0.12% chlorhexidine, triclosan and essential oils and cetylpyridinium chloride . Plaque formation was inhibited by chlorhexidine mouthrinses and essential oils . CONCLUSIONS: These findings suggest that mouthrinses can reduce morning bad breath, and that such a reduction is not attributable only to the reduction of supragingival plaque formation . Copyright Blackwell Munksgaard, 2004.

Vet Pathol, 2004 Mar, 41(2), 122 - 30
Granulomatous uveitis associated with vaccination in the atlantic salmon; Koppang EO et al.; This study addressed histologic and immunopathologic changes in ocular tissues and investigated the distribution of major histocompatibility class II (MHC class II)-positive cells in Atlantic salmon (Salmo salar) suffering from severe postvaccination disease . Twenty-nine fish with generalized inflammation, probably a result of vaccination, were investigated . One individual that had escaped vaccination was included in the study . Material was investigated by cultivation methods for fungi and bacteria . Histology using conventional staining procedures and immunohistochemistry with antisera against MHC class II beta chain were performed . No growth was observed from the cultivation investigations . Histology revealed occlusion of the lumen in the larger choroid vessels and in the choriocapillaris, inflammatory infiltrations and loss of structure in the choroid rete, and, in some cases, aggregations of multinucleated giant cells (MGC) and Splendore-Hoeppli material . Immunohistochemistry demonstrated massive MHC class II+ cellular infiltrations in the uveal tract . Such infiltrations were also seen in the ventral ciliary cleft, a condition that is associated with glaucoma . Immunoreactive cells included dendritelike cells, epithelioid cells, and MGCs . The endothelia of smaller vessels were frequently MHC class II+, and immunoreactive infiltrations were seen in the optic nerve in several individuals . No pathologic changes were detected in the unvaccinated individual . In conclusion, generalized inflammatory reactions in fish may lead to severe ocular inflammation, occlusion of uveal vessels, and perivascular changes with MHC class II+ upregulation in cells in the uveal tract and optic nerve.

J Biol Chem, 2004 May 14, 279(20), 21520 - 5 Epub 2004 Mar 11.
Heme-ligand tunneling in group I truncated hemoglobins; Milani M et al.; Truncated hemoglobins (trHbs) are small hemoproteins forming a separate cluster within the hemoglobin superfamily; their functional roles in bacteria, plants, and unicellular eukaryotes are marginally understood . Crystallographic investigations have shown that the trHb fold (a two-on-two alpha-helical sandwich related to the globin fold) hosts a protein matrix tunnel system offering a potential path for ligand diffusion to the heme distal site . The tunnel topology is conserved in group I trHbs, although with modulation of its size/structure . Here, we present a crystallographic investigation on trHbs from Mycobacterium tuberculosis, Chlamydomonas eugametos, and Paramecium caudatum, showing that treatment of trHb crystals under xenon pressure leads to binding of xenon atoms at specific (conserved) sites along the protein matrix tunnel . The crystallographic results are in keeping with data from molecular dynamics simulations, where a dioxygen molecule is left free to diffuse within the protein matrix . Modulation of xenon binding over four main sites is related to the structural properties of the tunnel system in the three trHbs and may be connected to their functional roles . In a parallel crystallographic investigation on M . tuberculosis trHbN, we show that butyl isocyanide also binds within the apolar tunnel, in excellent agreement with concepts derived from the xenon binding experiments . These results, together with recent data on atypical CO rebinding kinetics to group I trHbs, underline the potential role of the tunnel system in supporting diffusion, but also accumulation in multiple copies, of low polarity ligands/molecules within group I trHbs.

Virology, 2004 Mar 15, 320(2), 291 - 300
Bovine adenovirus type 3 containing heterologous protein in the C-terminus of minor capsid protein IX; Zakhartchouk A et al.; Earlier, we detected pIX of BAdV-3 as a 14-kDa protein in purified virions . Analysis of BAdV-3 pIX using different region antibodies revealed that the N-terminus and central domain of the pIX contain immunogenic sites and are not exposed on the surface of BAdV-3 virion . This suggested that the C-terminus of BAdV-3 pIX (125 amino acid) may be exposed on the virion and may be used as a site for incorporation of heterologous peptides or proteins . We constructed recombinant BAV950 containing a small peptide (21 amino acid), including the RGD motif or recombinant BAV951 containing enhanced yellow-green fluorescent protein (EYFP) fused to the C-terminus of pIX . Western blot analysis demonstrated that the chimeric pIX-RGD was incorporated into virion capsids . Incorporation of the RGD motif into the pIX resulted in significant augmentation of BAdV-3 fiber knob-independent infection of the integrin-positive cells, suggesting that RGD motifs are displayed on the surface of virion capsids and are accessible for binding to integrins . Analysis of BAV951 revealed that the chimeric pIX is incorporated into virion capsids and EYFP containing the C-terminus of pIX is exposed on the surface of the virion . Moreover, insertion of chimeric pIXs was maintained without change through successive rounds of viral replication . These results suggested that in contrast to major capsid proteins (hexon, penton, fiber), the minor capsid protein IX can be use for the incorporation of targeting ligands based on either small peptides or longer polypeptides.

Oecologia, 2004 May, 139(4), 560 - 7 Epub 2004 Mar 10.
Upward cascading effects of nutrients: shifts in a benthic microalgal community and a negative herbivore response; Armitage AR et al.; We evaluated the effects of nutrient addition on interactions between the benthic microalgal community and a dominant herbivorous gastropod, Cerithidea californica (California horn snail), on tidal flats in Mugu Lagoon, southern California, USA . We crossed snail and nutrient (N and P) addition treatments in enclosures on two tidal flats varying from 71 to 92% sand content in a temporally replicated experiment (summer 2000, fall 2000, spring 2001) . Diatom biomass increased slightly (approximately 30%) in response to nutrient treatments but was not affected by snails . Blooms of cyanobacteria (up to 200%) and purple sulfur bacteria (up to 400%) occurred in response to nutrient enrichment, particularly in the sandier site, but only cyanobacterial biomass decreased in response to snail grazing . Snail mortality was 2-5 times higher in response to nutrient addition, especially in the sandier site, corresponding to a relative increase in cyanobacterial biomass . Nutrient-related snail mortality occurred only in the spring and summer, when the snails were most actively feeding on the microalgal community . Inactive snails in the fall showed no response to nutrient-induced cyanobacterial growths . This study demonstrated strongly negative upward cascading effects of nutrient enrichment through the food chain . The strength of this upward cascade was closely linked to sediment type and microalgal community composition .

Mol Biol Evol, 2004 Jul, 21(7), 1234 - 41 Epub 2004 Mar 10.
The enigmatic planctomycetes may hold a key to the origins of methanogenesis and methylotrophy; Chistoserdova L et al.; Methanogenesis and methane oxidation are the major biological processes affecting the global cycling of the powerful greenhouse gas methane . To carry out the two alternative bioconversions, Nature has cleverly recycled key reactions for the C1 transfers between the oxidation levels of formaldehyde and formate, and these involve analogous enzyme systems and common specialized cofactors, methanopterin and methanofuran . Until recently, the distribution of these functions has been limited to methanogenic archaea and methylotrophic proteobacteria, and their evolutionary history remained obscure . Single interdomain lateral transfer of the respective genes has been suggested to play a role . Here we show that genes for C1 transfer reactions linked to methanopterin and methanofuran are also present in diverse representatives of the enigmatic bacterial clade, the Planctomycetes . Phylogenetic analysis places the planctomycete sequences as distantly from their archaeal counterparts as from their proteobacterial counterparts, suggesting novel scenarios for the evolution of the C1 transfer functions in both methanogens and methylotrophs . This finding suggests a possible role for Planctomycetes in the evolution of the methane cycle on Earth.

Clin Diagn Lab Immunol, 2004 Mar, 11(2), 423 - 5
Prevalence of Bartonella clarridgeiae and Bartonella henselae in domestic cats from France and detection of the organisms in erythrocytes by immunofluorescence; Rolain JM et al.; The prevalence of Bartonella infection in a pet cat population from France was found to be 8.1% (8 of 99 cats) . The intraerythrocytic location of Bartonella clarridgeiae is shown for the first time, and we show that immunofluorescence detection of the organism in erythrocytes correlates with the number of bacteria in blood.

Chemosphere, 2004 May, 55(5), 653 - 9
The dechlorination of cyclodiene pesticides by methanogenic granular sludge; Baczynski TP et al.; Cyclodiene pesticides: aldrin, isodrin, dieldrin and endrin were dechlorinated by methanogenic granular sludge in spiked batch tests . Initial pesticides concentration was 7 or 9 mgl(-1) . Two monodechlorinated analogues were formed during the conversions of aldrin and isodrin . Dieldrin was transformed into two monodechlorinated products as well as into aldrin and two monodechlorinated derivatives of aldrin . In respect of endrin three monodechlorinated and three didechlorinated products were found . The dechlorination of endrin was the most rapid, and was almost complete within 28 days . The dechlorinations of aldrin, isodrin and dieldrin were much slower: over a period of 3 months only 59%, 70% and 88% was transformed, respectively . The amounts of released chloride corresponded 0.54 +/- 0.23, 1.05 +/- 0.25, 1.10 +/- 0.12 of theoretical value for suggested reactions, for aldrin, isodrin and dieldrin respectively . For endrin it was much higher . These transformations did not occur in control samples containing autoclaved granules or in control blank samples without sludge . However, in aldrin spiked blanks, a conversion into a six-chlorinated analogue was found.

Biochem Biophys Res Commun, 2004 Feb 27, 315(1), 107 - 12
NF-kappaB-dependent induction of osteoprotegerin by Porphyromonas gingivalis in endothelial cells; Kobayashi-Sakamoto M et al.; Porphyromonas gingivalis is a major etiological pathogen of adult periodontitis characterized by alveolar bone resorption . Vascular endothelial cells supply many inflammatory cytokines into periodontal tissue . However, whether the cells contribute to bone metabolism in periodontitis remains unknown . In this study, we investigated the effect of P . gingivalis on osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) production, both of which are key regulators of bone metabolism, in human microvascular endothelial cells (HMVECs) . We showed that P . gingivalis upregulated expression of OPG but not RANKL mRNA in HMVEC . P . gingivalis induced NF-kappaB activation, and the induction of OPG in HMVEC by the pathogen was blocked by the inhibitors of NF-kappaB . In addition, incubation of OPG with P . gingivalis supernatant resulted in loss of the protein . These results indicate that P . gingivalis-stimulated HMVEC secrete OPG via a NF-kappaB-dependent pathway, while the OPG is partly degraded by the bacteria . Thus, microvascular endothelial cells can act as a source of OPG and thereby may play an important role in regulating bone metabolism in periodontitis.

J Appl Microbiol, 2004, 96(4), 871 - 7
Intraspecific diversity of the marine fish pathogen Tenacibaculum maritimum as determined by randomly amplified polymorphic DNA-PCR; Avendano-Herrera R et al.; AIM: The aim of the present study was to evaluate the intraspecific genetic variability within Tenacibaculum maritimum strains isolated from different species of marine fish . METHODS AND RESULTS: Twenty-nine strains isolated from five different fish species and three reference strains were characterized by randomly amplified polymorphic DNA (RAPD) method . Cluster analysis of RAPD-PCR profiles showed that the strains, regardless of the oligonucleotide primer employed (P2 and P6), were separated into two main groups that strongly correlated with the host species and/or O-serotypes described for this pathogen . One group composed all strains isolated from sole (Solea senegalensis and S . solea) and gilthead seabream (Sparus aurata), and the other compiled the T . maritimum isolates from yellowtail (Seriola quinqueradiata), Atlantic salmon (Salmo salar) and turbot (Scophthalmus maximus) . An important exception was observed in the RAPD patterns of the reference strains, which were included in different genetic groups depending on the primer employed . CONCLUSIONS: The results obtained demonstrated genetic variability within the T . maritimum isolated from different marine fish . Such genetic variability proved to be strongly associated with the host and/or serogroups described for this pathogen . SIGNIFICANCE AND IMPACT OF THE STUDY: The RAPD analysis constitutes a valuable molecular technique for epidemiological studies of T . maritimum . Interestingly, this is the first report of intraspecific differentiation and characterization of T . maritimum strains isolated from cultured fish.

Annu Rev Plant Physiol Plant Mol Biol, 1998 Jun, 49, 151 - 171
LESSONS FROM SEQUENCING OF THE GENOME OF A UNICELLULAR CYANOBACTERIUM, SYNECHOCYSTIS SP . PCC6803; Kotani H et al.; The nucleotide sequence of the entire genome of the unicellular cyanobacterium, Synechocystis sp . PCC6803, has been determined . The length of the circular genome was 3,573,480 bp, and a total of 3168 protein-coding genes were assigned to the genome by a computer-assisted analysis . The functions of approximately 45% of the genes were deduced based on sequence similarity to known genes . Here are distinctive features of genetic information carried by the cyanobacteria, which have a phylogenetic relationship to both bacteria and plants.

Annu Rev Plant Physiol Plant Mol Biol, 1998 Jun, 49, 53 - 75
BIOSYNTHESIS AND FUNCTION OF THE SULFOLIPID SULFOQUINOVOSYL DIACYLGLYCEROL; Benning C; The sulfolipid sulfoquinovosyl diacylglycerol is an abundant sulfur-containing nonphosphorous glycerolipid that is specifically associated with photosynthetic membranes of higher plants, mosses, ferns, algae, and most photosynthetic bacteria . The characteristic structural feature of sulfoquinovosyl diacylglycerol is the unique head group constituent sulfoquinovose, a derivative of glucose in which the 6-hydroxyl is replaced by a sulfonate group . While there is growing evidence for the final assembly of the sulfolipid by the transfer of the sulfoquinovosyl moiety from UDP-sulfoquinovose to the sn-3 position of diacylglycerol, very little is known about the biosynthesis of the precursor UDP-sulfoquinovose . Recently, a number of mutants deficient in sulfolipid biosynthesis and the corresponding sqd genes have become available from different organisms . These provide novel tools to analyze sulfolipid biosynthesis by a combination of molecular and biochemical approaches . Furthermore, the analysis of sulfolipid-deficient mutants has provided novel insights into the function of sulfoquinovosyl diacylglycerol in photosynthetic membranes.

Annu Rev Plant Physiol Plant Mol Biol, 1999 Jun, 50, 505 - 537
ASYMMETRIC CELL DIVISION IN PLANTS; Scheres B et al.; Asymmetric cell divisions generate cells with different fates . In plants, where cells do not move relative to another cell, the specification and orientation of these divisions is an important mechanism to generate the overall cellular pattern during development . This review summarizes our knowledge of selected cases of asymmetric cell division in plants, in the context of recent insights into mechanisms underlying this process in bacteria, algae, yeast, and animals.

Curr Mol Med, 2004 Feb, 4(1), 31 - 40
Animal models of HLA-B27-associated diseases; Breban M et al.; The HLA-B27 molecule is strongly associated with the spondyloarthropathies (SpA), a group of inflammatory conditions affecting the skeleton, the skin and several mucosae . The mechanism of this association remains unknown, largely because the HLA-B27 molecule displays normal function . A disease that closely mimicks SpA arises spontaneously in HLA-B27 transgenic rats . This disease is dependent on the presence of a normal bacterial flora and implicates the immune system . The presence of both CD4+ T cells and antigen presenting cells (APCs) expressing high levels of HLA-B27, seems of critical importance in its pathogenesis, whereas CD8+ T cells are dispensable . The T cell stimulatory function of APCs is disturbed by the HLA-B27 molecule . This disease could result from a failure of tolerance, related in part to high level of B27 expression in professional APCs and to the immune response to gut bacteria . In contrast, HLA-B27 transgenic mice have usually remained healthy . However, two types of inflammatory conditions affecting the skeleton, which arise in mice of susceptible background after exposure to a conventional bacterial flora, are increased by an HLA-B27 transgene . The first is ANKENT, a spontaneous ankylosing enthesitis that affects ankle and/or tarsal joints of ageing mice; the second is a spontaneous arthritis of hindpaws developing in mice lacking endogenous mbeta2m . As in rats, the absence of CD8+ T cells in the latter model, argues against the "arthritogenic peptide" hypothesis . In these mbeta2m0 mice, B27 free heavy chain could be implicated in the pathogenesis of arthritis by presenting extracellular peptides to CD4+ T cells.

Hepatogastroenterology, 2004 Jan-Feb, 51(55), 282 - 4
Systemic humoral anti-Helicobacter pylori immune response in patients with gastric malignancies and benign gastroduodenal disease; Manojlovic N et al.; BACKGROUND/AIMS: Helicobacter pylori elicits a strong local and systemic humoral immune response, but it is not able to eliminate bacteria . Immune response may be important for the course of infection that may lead to different gastroduodenal disease . In order to investigate differences in systemic humoral immune response between patients with different gastroduodenal diseases, we conducted clinical and serological studies . METHODOLOGY: From 1999 to 2001 we enrolled 80 patients with dyspeptic symptoms: 26 with gastritis, 12 with duodenal ulcer, 29 with gastric cancer and 13 with gastric lymphoma . In all patients during diagnostic work-up we performed ELISA test with Helicobacter pylori-specific IgG and IgA . We investigated difference in stimulation of different immunoglobulin classes in patients with different gastroduodenal diseases, particularly benign and malignant . We estimated significance of differences with Mann-Whitney and Fisher exact probability test . RESULTS: All patients enrolled in the study were seropositive . Patients with gastritis had statistically significant higher level of IgG than patients with gastric cancer (p=0.0001), and gastric lymphoma (p=0.006) . Patients with duodenal ulcer had statistically significant higher level of IgG than patients with gastric cancer (p=0.02), and gastric lymphoma (p=0.046) . IgA level was significantly higher in patients with gastritis than in patients with gastric cancer (p=0.03) . IgA>IgG ratio was significantly more frequent in patients with gastric cancer and gastric lymphoma than in patients with gastritis and duodenal ulcer (p=0.0002) . CONCLUSIONS: Result of our study suggested that Helicobacter pylori elicits different systemic humoral immune response in patients with gastritis and duodenal ulcer than in patients with gastric cancer and gastric lymphoma at least in intensity of stimulation of different immunoglobulin classes.

Neuropsychopharmacology, 2004 May, 29(5), 860 - 8
Abeta(25-35)-induced memory impairment, axonal atrophy, and synaptic loss are ameliorated by M1, A metabolite of protopanaxadiol-type saponins; Tohda C et al.; We previously screened neurite outgrowth activities of several Ginseng drugs in human neuroblastoma, and demonstrated that protopanaxadiol (ppd)-type saponins were active constituents . Since ppd-type saponins are known to be completely metabolized to 20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol (M1) by intestinal bacteria when taken orally, M1 and ginsenoside Rb1, as a representative of ppd-type saponins, were examined for cognitive disorder . In a mouse model of Alzheimer's disease (AD) by Abeta(25-35) i.c.v . injection, impaired spatial memory was recovered by p.o . administration of ginsenoside Rb1 or M1 . Although the expression levels of phosphorylated NF-H and synaptophysin were reduced in the cerebral cortex and the hippocampus of Abeta(25-35)-injected mice, their levels in ginsenoside Rb1- and M1-treated mice were almost completely recovered up to control levels . Potencies of the effects were not different between ginsenoside Rb1 and M1 when given orally, suggesting that most of the ginsenoside Rb1 may be metabolized to M1, and M1 is an active principal of ppd-type saponins for the memory improvement . In cultured rat cortical neurons, M1 showed extension activity of axons, but not dendrites . The axon-specific outgrowth was seen even when neuritic atrophy had already progressed in response to administration of Abeta(25-35) as well as in the normal condition . These results suggest that M1 has axonal extension activity in degenerated neurons, and improve memory disorder and synaptic loss induced by Abeta(25-35) . M1 was shown to be effective in vitro and in vivo, indicating that Ginseng drugs containing ppd-type saponins may reactivate neuronal function in AD by p.o . administration.

Mol Microbiol, 2004 Mar, 51(6), 1677 - 89
The H2-sensing complex of Ralstonia eutropha: interaction between a regulatory {NiFe} hydrogenase and a histidine protein kinase; Buhrke T et al.; Two {NiFe} hydrogenases enable the proteobacterium Ralstonia eutropha H16 to grow on molecular hydrogen as the sole energy source . A third {NiFe} hydrogenase (RH) acts as an H2 sensor in a multiple component signal transduction chain that controls hydrogenase gene transcription . The RH forms a dimeric heterodimer (HoxBC)2 in which HoxC contains the H2-sensing active site and HoxB the electron-transferring components including an organic, not yet identified redox cofactor . This oligomer forms a tight complex with the histidine protein kinase HoxJ . Both the sensor and the kinase were analysed by mutagenesis for functional domains that are instrumental in H2 signal transmission . A mutant deleted for a C-terminal peptide of 55 amino acids in HoxB lost its H2-sensing ability but still catalysed H2 oxidation . The mutant protein failed to form the dimeric heterodimer and a complex with HoxJ . The organic redox cofactor was no longer detectable in the truncated sensor . H2 sensing was also abolished by deletion of the PAS domain of HoxJ, indicating that this domain is involved in signal transduction . A truncated version of HoxJ consisting of only the input domain of the kinase was still capable of forming a complex with the RH . Mass determination of the purified HoxJ protein revealed that the kinase forms a homotetramer . The unique oligomeric structure of the H2-sensing complex with respect to its regulatory function is discussed.

Mol Microbiol, 2004 Mar, 51(6), 1551 - 62
The principal sigma factor sigA mediates enhanced growth of Mycobacterium tuberculosis in vivo; Wu S et al.; The ability of Mycobacterium tuberculosis to grow in macrophages is central to its pathogenicity . We found previously that the widespread 210 strain of M . tuberculosis grew more rapidly than other strains in human macrophages . Because principal sigma factors influence virulence in some bacteria, we analysed mRNA expression of the principal sigma factor, sigA, in M . tuberculosis isolates during growth in human macrophages . Isolates of the 210 strain had higher sigA mRNA levels and higher intracellular growth rates, compared with other clinical strains and the laboratory strain H37Rv . SigA was also upregulated in the 210 isolate TB294 during growth in macrophages, compared with growth in broth . In contrast, H37Rv sigA mRNA levels did not change under these conditions . Overexpression of sigA enhanced growth of recombinant M . tuberculosis in macrophages and in lungs of mice after aerosol infection, whereas recombinant strains expressing antisense transcripts to sigA showed decreased growth in both models . In the presence of superoxide, sense sigA transformants showed greater resistance than vector controls, and the antisense sigA transformant did not grow . We conclude that M . tuberculosis sigA modulates the expression of genes that contribute to virulence, enhancing growth in human macrophages and during the early phases of pulmonary infection in vivo . This effect may be mediated in part by increased resistance to reactive oxygen intermediates.

J Periodontal Res, 2004 Apr, 39(2), 129 - 35
Effect of low dose Actinobacillus actinomycetemcomitans lipopolysaccharide pretreatment on cytokine production by human whole blood; Nakamura T et al.; BACKGROUND AND OBJECTIVE: Periodontal disease is known to influence the systemic condition in various ways, and the bacteria and their products, such as lipopolysaccharides (LPS), may spread from periodontal lesions via the systemic circulation to affect distant organs . The level of LPS in plasma from such patients is reported to be very low, and this low level of LPS is suspected to have priming or desensitizing effect . Thus, we investigated the effects of low dose LPS pretreatment on LPS-dependent cytokine production by whole blood cells ex vivo . METHODS: Blood samples obtained from seven systemically and periodontally healthy individuals were pretreated with or without 5 pg/ml Actinobacillus actinomycetemcomitans LPS, followed by further stimulation with 1 ng/ml A . actinomycetemcomitans LPS . The concentrations of interleukin-1 beta (IL-1beta), IL-6, IL-10 and tumor necrosis factor-alpha (TNF-alpha) in the culture supernatants were then determined using enzyme-linked immunosorbent assay (ELISA) . In addition, intracytoplasmic cytokine staining of whole blood cells was performed for flow cytometry . RESULTS: Pretreatment with 5 pg/ml A . actinomycetemcomitans LPS significantly enhanced the production of IL-1beta and IL-6 from whole blood when further induced by 1 ng/ml LPS (1.72 times higher for IL-1beta, 2.18 times higher for IL-6 than without pretreatment) . The pretreatment did not enhance the production of either TNF-alpha or IL-10 . Intracytoplasmic staining showed that the monocyte fraction was primarily involved in producing IL-1beta and IL-6 . Flow cytometric analysis revealed that pretreatment increased the number of IL-1beta and IL-6 producing cells as well as mean fluorescence intensity of the stained cells . CONCLUSION: A low dose of bloodstream LPS found in periodontitis patients appears to be sufficient to prime monocytes, and may be capable of affecting the systemic responses of immune and inflammatory cells.

J Thromb Haemost, 2004 Mar, 2(3), 485 - 91
ADAMTS-13 activity in plasma is rapidly measured by a new ELISA method that uses recombinant VWF-A2 domain as substrate; Whitelock JL et al.; The metalloprotease ADAMTS-13 cleaves von Willebrand factor (VWF) at the Y842/M843 peptide bond located in the A2 domain . Measurement of ADAMTS-13 activity is a clinical utility for thrombotic diseases, but the current assays used for diagnostic and clinical research are non-physiological and time consuming . We have expressed in bacteria a recombinant VWF-A2 peptide (aa 718-905) that contains both a 6xHis tag at the N-terminal end and a Tag-100 epitope at the C-terminal end . Diluted plasma was mixed with the VWF-A2 peptide and digestion was allowed to proceed in a Ni2+-coated microtiter well plate for 2 h . The immobilized Ni2+ captures the VWF-A2 peptide by its 6xHis tag and cleavage of the A2 peptide is measured by the removal of the C-terminus fragment of the A2 peptide that contains the Tag-100 . The cleavage activity for this assay was defined by the low detection of A2 peptide containing the Tag-100 epitope by the antiTag-100 monoclonal antibody . The assay was completed in <5 h . We then used the assay to analyze ADAMTS-13 activity in plasma from 39 healthy donors and 16 samples from patients diagnosed as thrombotic thrombocytopenic purpura . The average of enzyme activity +/- SEM for normal plasmas diluted 1 : 50 was 40 +/- 4.2% while the value obtained for the patients was 2.4 +/- 0.7% . These results were validated by a traditional long incubation assay (24 h) . Our assay provides significant advantages over currently used assays because it is quicker, reproducible, cost effective and measures ADAMTS-13 activity under physiological and non-denaturing conditions . This assay is clinically useful and significant in measuring ADAMTS-13 activity in plasma.

Appl Environ Microbiol, 2004 Mar, 70(3), 1688 - 97
Possible association of GroES and antigen 85 proteins with heat resistance of Mycobacterium paratuberculosis; Sung N et al.; Conflicting reports on the heat resistance of Mycobacterium paratuberculosis prompted an examination of the effect of culture medium on this property of the organism . M . paratuberculosis was cultured in three types of media (fatty acid-containing medium 7H9-OADC (oleic acid-albumin-dextrose-catalase supplement) and glycerol-containing media WR-GD and 7H9-GD {glycerol-dextrose supplement}) at pH 6.0 . M . paratuberculosis grown under these three culture conditions was then tested for heat resistance in distilled water at 65 degrees C . Soluble proteins and mycolic acids of M . paratuberculosis were evaluated by two-dimensional electrophoresis (2-DE) and thin-layer chromatography (TLC), respectively . The type of culture medium used significantly affected the heat resistance of M . paratuberculosis . The decimal reduction times at 65 degrees C (D(65 degrees C) values; times required to reduce the concentration of bacteria by a factor of 10 at 65 degrees C) for M . paratuberculosis strains grown in 7H9-OADC were significantly higher than those for the organisms grown in WR-GD medium (P < 0.01) . When the glycerol-dextrose supplement of WR was substituted for the fatty acid supplement (OADC) in 7H9 medium (resulting in 7H9-GD), the D(65 degrees C) value was significantly lower than that for the organism grown in 7H9-OADC medium (P = 0.022) but higher than that when it was cultured in WR-GD medium (P = 0.005) . Proteomic analysis by 2-DE of soluble proteins extracted from M . paratuberculosis grown without heat stress in the three media (7H9-OADC, 7H9-GD, and WR-GD) revealed that seven proteins were more highly expressed in 7H9-OADC medium than in the other two media . When the seven proteins were subjected to matrix-assisted laser desorption ionization-mass spectrometric analysis, four of the seven protein spots were unidentifiable . The other three proteins were identified as GroES heat shock protein, alpha antigen, and antigen 85 complex B (Ag85B; fibronectin-binding protein) . These proteins may be associated with the heat resistance of M . paratuberculosis . Alpha antigen and Ag85B are both trehalose mycolyltransferases involved in mycobacterial cell wall assembly . TLC revealed that 7H9-OADC medium supported production of more trehalose dimycolates and cell wall-bound mycolic acids than did WR-GD medium . The present study shows that in vitro culture conditions significantly affect heat resistance, cell wall synthesis, and protein expression of M . paratuberculosis and emphasize the importance of culture conditions on in vitro and ex vivo studies to estimate heat resistance.

Appl Environ Microbiol, 2004 Mar, 70(3), 1651 - 7
Rapid detection and enumeration of Legionella pneumophila in hot water systems by solid-phase cytometry; Aurell H et al.; A new method for the rapid and sensitive detection of Legionella pneumophila in hot water systems has been developed . The method is based on an IF assay combined with detection by solid-phase cytometry . This method allowed the enumeration of L . pneumophila serogroup 1 and L . pneumophila serogroups 2 to 6, 8 to 10, and 12 to 15 in tap water samples within 3 to 4 h . The sensitivity of the method was between 10 and 100 bacteria per liter and was principally limited by the filtration capacity of membranes . The specificity of the antibody was evaluated against 15 non-Legionella strains, and no cross-reactivity was observed . When the method was applied to natural waters, direct counts of L . pneumophila were compared with the number of CFU obtained by the standard culture method . Direct counts were always higher than culturable counts, and the ratio between the two methods ranged from 1.4 to 325 . Solid-phase cytometry offers a fast and sensitive alternative to the culture method for L . pneumophila screening in hot water systems.

Appl Environ Microbiol, 2004 Mar, 70(3), 1455 - 65
Development and testing of a DNA macroarray to assess nitrogenase (nifH) gene diversity; Steward GF et al.; A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene . Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes . Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence . A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%) . When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity . Results implied a detection limit of roughly 13 pg of target ml(-1), a half-saturation of signal at 0.26 ng ml(-1), and a dynamic range of about 2 orders of magnitude . The threshold for cross-hybridization varied between 78 and 88% sequence identity . Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (r = 0.98, P < 0.0001, n = 88) . A mixed nifH target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes . The natural community results were well simulated (r = 0.941, P < 0.0001, n = 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes . Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.

Expert Opin Biol Ther, 2004 Mar, 4(3), 397 - 406
Exploiting immune surveillance mechanisms in mucosal vaccine development; Lo D; Historically, immune responsiveness was regarded by many as an ability to discriminate self from non-self, but this definition has recently been revised to be a distinction between threatening infectious organisms versus innocuous molecules from autologous tissues . Such distinctions can be made in the context of adjuvant effects from triggering of 'pattern recognition receptors' by pathogen-associated molecules . Mucosal sites such as airway and intestinal passages present a particularly interesting challenge to this system, as distinctions must be effectively made between innocuous non-self molecules associated with food and commensal bacteria versus pathogenic viruses and bacteria . Given the simultaneous presence of all these molecular types at mucosal lymphoid sites, immunological discrimination mechanisms must be especially precise, as immune responses must be directed only at pathogen-associated targets . Ongoing research is identifying genes that may be critical to triggering mucosal immunity; an understanding of their role in discrimination may lead to the development of new vaccines.

Bull Math Biol, 2004 May, 66(3), 559 - 81
A metabolic force for gene clustering; Svetic RE et al.; Bacterial chromosomes frequently contain arrays of contiguous genes that group according to related metabolic roles . We propose that clustering of genes for metabolically related functions confers thermodynamic advantage to the organism based upon our protein immobility model (PIM) of intracellular diffusion . This thermodynamic effect provides the selection force argument that is missing from previous models of gene clustering . The PIM posits that clustered genes produce local clusters of enzymes in bacteria owing to the co-linearity of transcription and translation, and to the relative immobility of large proteins released into the cytosol . We maintain that the resulting physical proximity of enzymes for related pathway steps minimizes the steady state level of reaction step intermediates and thus conserves the energy and material required for rapid growth and maintenance . Support for this idea comes from in silico experiments using the PIM applied to a model metabolic pathway A --> B --> C . The metabolites A, B, and C are small molecules that diffuse freely in a cytosol crowded with macromolecules, whereas the large enzyme molecules, E1 and E2, tend to remain in the vicinity of their point of release . Modeling E1 as a source of B from A, and E2 as a sink for B, numerical experiments suggest that the steady state concentration of B in the cytosol increases approximately in proportion to the square of the distance of the E1 and E2 separation . A further model prediction is that the steady state concentration of B is influenced by the geometric effects of the spatial location and orientation of E1 relative to E2 . These results suggest that: (i) gene clustering reduces the energy and material costs of enzyme reactions linked by metabolic intermediates; (ii) gene clusters near ori, the origin of replication, utilize the geometric effect to conserve free energy by further reducing the steady state concentration of the intermediate; (iii) gene organization on a chromosome influences the organism's capacity to accelerate into steady state growth and is, in turn, influenced by the abundance and frequency of access to nutrients.

Cytogenet Genome Res, 2003, 103(1-2), 111 - 21
Cytogenetics, conserved synteny and evolution of chicken fucosyltransferase genes compared to human; Coullin P et al.; Fucosyltransferases appeared early in evolution, since they are present from bacteria to primates and the genes are well conserved . The aim of this work was to study these genes in the bird group, which is particularly attractive for the comprehension of the evolution of the vertebrate genome . Twelve fucosyltransferase genes have been identified in man . The orthologues of theses genes were looked for in the chicken genome and cytogenetically localized by FISH . Three families of fucosyltransferases: alpha6-fucosyltransferases, alpha3/4-fucosyltransferases, and protein-O-fucosyltransferases, were identified in the chicken with their associated genes . The alpha2-fucosyltransferase family, although present in some invertebrates and amphibians was not found in birds . This absence, also observed in Drosophila, may correspond to a loss of these genes by negative selection . Of the eight chicken genes assigned, six fell on chromosome segments where conservation of synteny between human and chicken was already described . For the two remaining loci, FUT9 and FUT3/5/6, the location may correspond to a new small syntenic area or to an insertion . FUT4 and FUT3/5/6 were found on the same chicken chromosome . These results suggest a duplication of an ancestral gene, initially present on the same chromosome before separation during evolution . By extension, the results are in favour of a common ancestor for the alpha3-fucosyltransferase and the alpha4-fucosyltransferase activities . These observations suggest a general mechanism for the evolution of fucosyltransferase genes in vertebrates by duplication followed by divergent evolution .

Proc Natl Acad Sci U S A, 2004 Mar 16, 101(11), 3839 - 44 Epub 2004 Mar 02.
Magnetosome vesicles are present before magnetite formation, and MamA is required for their activation; Komeili A et al.; Bacterial magnetosomes are intracellular compartments that house highly ordered magnetite crystals . By using Magnetospirillum sp . AMB-1 as a model system, we show that magnetosome vesicles exist in the absence of magnetite, biomineralization of magnetite proceeds simultaneously in multiple vesicles, and biomineralization proceeds from the same location in each vesicle . The magnetosome-associated protein, MamA, is required for the formation of functional magnetosome vesicles and displays a dynamic subcellular localization throughout the growth cycle of magnetotactic bacteria . Together, these results suggest that the magnetosome precisely coordinates magnetite biomineralization and can serve as a model system for the study of organelle biogenesis in noneukaryotic cells.

J Immunol, 2004 Mar 15, 172(6), 3678 - 85
A disintegrin and metalloproteinase 10-mediated cleavage and shedding regulates the cell surface expression of CXC chemokine ligand 16; Gough PJ et al.; CXC chemokine ligand (CXCL)16 and scavenger receptor for phosphatidylserine and oxidized low-density lipoprotein were independently identified as a chemokine and a scavenger receptor, respectively, but have since been shown to be identical . CXCL16 is synthesized as a transmembrane protein with its chemokine domain at the end of a mucin-rich stalk . When expressed at the cell surface, CXCL16 functions as a scavenger receptor, binding and internalizing oxidized low-density lipoprotein and bacteria . As a soluble form, CXCL16 is a chemoattractant for activated CD4+ and CD8+ T cells through binding its receptor, CXCR6 . In this study, we examined the mechanisms that regulate the conversion between these two functionally distinct forms of CXCL16 . We demonstrate that murine CXCL16 is synthesized as an intracellular precursor that is rapidly transported to the cell surface where it undergoes metalloproteinase-dependent cleavage, causing the release of a fragment that constitutes the majority of the CXCL16 extracellular domain . Using a novel retroviral system for the generation of short interfering RNAs, we show that knockdown of a disintegrin and metalloproteinase (ADAM) family protease ADAM10 decreases this constitutive shedding of CXCL16 . Furthermore, we show that overexpression of ADAM10 increases CXCL16 shedding, whereas overexpression of a dominant-negative form of ADAM10 lowers shedding of CXCL16 in a similar manner to short interfering RNAs . Through the modulation of ADAM10 function, we demonstrate that ADAM10-mediated constitutive shedding is a key regulator of CXCL16 cell surface expression . The identification of ADAM10 as a major protease responsible for the conversion of CXCL16 from a membrane-bound scavenger receptor to a soluble chemoattractant will provide new information for understanding the physiological function of this molecule.

Plasmid, 2004 Mar, 51(2), 127 - 47
Comparative sequence analysis of the icm/dot genes in Legionella; Morozova I et al.; The icm/dot genes in Legionella pneumophila are essential for the ability of the bacteria to survive within macrophages in lung infections such as Legionnaires' disease, or amoebae in nature . The 22 genes of the complex, thought to encode a transport apparatus for transfer of effector molecules into the host cell cytoplasm, are located in two chromosomal loci . We demonstrate that these genes are present in all the L . pneumophila strains examined herein, but display a wide range of sequence variation among the different strains, none of which are clearly associated with virulence potential . The strains fall within seven phylogenetic groups, but discrepancies among the gene trees indicate a complicated evolutionary history for the icm/dot loci, with perhaps two independent gene acquisition events and subsequent genomic rearrangements . Significant findings include a probable t-SNARE domain in IcmG that may indicate a direct role for this putative inner membrane protein in altering the host's membrane fusion machinery, a potential functional domain in the central hydrophobic portion of IcmK that may allow it to participate in forming the pore of the secretion complex, and strict conservation of the amino acid physicochemical characteristics in the IcmP region corresponding to the trbA domain that could play a role in molecular transfer.

Methods, 2004 Apr, 32(4), 451 - 5
Imaging protein phosphorylation by fluorescence in single living cells; Sato M et al.; Protein phosphorylation by intracellular kinases plays one of the most pivotal roles in signaling pathways within cells . To reveal the biological issues related to the kinase proteins, electrophoresis, immunocytochemistry, and in vitro kinase assay have been used . However, these conventional methods do not provide enough information about spatial and temporal dynamics of the signal transduction based on protein phosphorylation and dephosphorylation in living cells . To overcome the limitation for investigating the kinase signaling, we developed genetically encoded fluorescent indicators for visualizing the protein phosphorylation in living cells . Using these indicators, we visualized under a fluorescence microscope when, where, and how the protein kinases are activated in single living cells.

Methods, 2004 Apr, 32(4), 371 - 80
A two-step two-hybrid system to identify functionally significant protein-protein interactions; Poustovoitov M et al.; The two-step two-hybrid approach described here is an adaptation of the classic two-hybrid system . Its purpose is to identify proteins that interact with a relatively small, defined, functionally significant domain of a protein of interest . In this method, a first round of screening is performed to identify proteins that interact with bait comprised of the wild type protein . Next, each of the prey identified in this first round is tested for its ability to interact with functionally impaired, mutant bait . Any proteins that interact with the wild type bait, but not the mutant bait, are candidate effectors or regulators of the protein of interest.

Biochem Biophys Res Commun, 2004 Mar 26, 316(1), 203 - 10
Actinohivin, a novel anti-human immunodeficiency virus protein from an actinomycete, inhibits viral entry to cells by binding high-mannose type sugar chains of gp120; Chiba H et al.; We searched human immunodeficiency virus (HIV) entry inhibitors and found a novel anti-HIV protein, actinohivin (AH), in a culture filtrate of the newly discovered genus actinomycete Longispora albida gen . nov., sp . nov . This paper deals with the mechanism of action of the anti-HIV activity of AH . AH exhibited potent anti-HIV activities against various strains of HIV-1 and HIV-2 . AH bound to the glycoprotein gp120 of various strains of HIV-1 and gp130 of simian immunodeficiency virus (SIV), but did not bind to non-glycosylated gp120 nor to cells having CD4 and coreceptors, suggesting that AH inhibits viral entry to cells by binding to the envelope glycoprotein . The investigation of the effects of various sugars on AH-gp120 binding by ELISA revealed that yeast mannan alone strongly inhibited the binding (IC50 = 3.0 microg/ml) . Experiments investigating the binding of AH to other glycoproteins revealed that AH binds to ribonuclease B and thyroglobulin that have a high-mannose type saccharide chain, but not to other glycoproteins having a N-glycoside type saccharide chain . The above results indicate that high-mannose type saccharide chains of gp120 are molecular targets of AH in its anti-HIV activity.

Postepy Hig Med Dosw, 2003, 57(6), 727 - 37
{Role of the adaptins, dynamin like GTP-ases and Rab proteins in metabolic disorders and various infections}; Kierczak M et al.; Numerous metabolic and genetic diseases are due to mutations in adaptins, dynamin-like GTP-ases or disorders in trafficking machinery mediated by Rab proteins . A great number of pathogenes including viruses (HIV, SIV), bacteria and protozoa use various elements of endocytic/trafficking machinery to get into the host cells and to make their infection successful . Their different strategies are discussed.

J Pediatr, 2004 Mar, 144(3), 316 - 20
Correlation of in situ detection of infectious agents in the placenta with neonatal outcome; Genen L et al.; OBJECTIVES: To determine if infections involving the placenta are associated with unexplained systemic illness in the newborn infant and subsequent poor neonatal outcome (death or significant neurodevelopmental abnormalities) . STUDY DESIGN: Placental tissue from 33 newborn infants with systemic illness and poor neonatal outcome were tested by in situ hybridization or reverse transcriptase-polymerase chain reaction for infectious pathogens . Control placentas came from mothers delivering infants with poor neonatal outcome of known cause (ie, cord prolapse, uterine rupture), mothers with known infections, and normal births (n=21) . RESULTS: There were 5 deaths among the newborn infants, and all survivors had poor neonatal outcome . Placentas from 24 of 33 cases (73%) had positive test results for Coxsackie virus (46%), bacteria (38%), herpes (8%), and parvovirus (4%) and picornavirus (4%) . At autopsy, multiple organs from the newborn infant had positive test results for the same organism isolated from the placenta . No infectious agents were detected in the control infants, except those from mothers with known infections . CONCLUSIONS: In utero infection of the placenta is associated with systemic illness in the newborn infant and poor neonatal outcome . These results emphasize the importance of pathologic and molecular examination of the placenta in critically ill newborn infants.

J Dent, 2004 Mar, 32(3), 173 - 96
Resin bonding to cervical sclerotic dentin: a review; Tay FR et al.; Several reports have indicated that resin bond strengths to noncarious sclerotic cervical dentine are lower than bonds made to normal dentine . This is thought to be due to tubule occlusion by mineral salts, preventing resin tag formation . The purpose of this review was to critically examine what is known about the structure of this type of dentine . Recent transmission electron microscopy revealed that in addition to occlusion of the tubules by mineral crystals, many parts of wedge-shaped cervical lesions contain a hypermineralised surface that resists the etching action of both self-etching primers and phosphoric acid . This layer prevents hybridisation of the underlying sclerotic dentine . In addition, bacteria are often detected on top of the hypermineralised layer . Sometimes the bacteria were embedded in a partially mineralised matrix . Acidic conditioners and resins penetrate variable distances into these multilayered structures . Examination of both sides of the failed bonds revealed a wide variation in fracture patterns that involved all of these structures . Microtensile bond strengths to the occlusal, gingival and deepest portions of these wedge-shaped lesions were significantly lower than similar areas artificially prepared in normal teeth . When resin bonds to sclerotic dentine are extended to include peripheral sound dentine, their bond strengths are probably high enough to permit retention of class V restorations by adhesion, without additional retention.

Microb Pathog, 2004 Apr, 36(4), 219 - 25
Role of Syndecan-4 in the cellular invasion of Orientia tsutsugamushi; Kim HR et al.; Cell surface heparan sulfate proteoglycans (HSPGs) play a critical role in the cellular invasion of intracellular bacteria and are presumed to have a role in the infection of host cells by Orientia tsutsugamushi . Previously, we showed that O . tsutsugamushi infection decreased markedly after treating host cells with heparinase, which suggests that HSPGs play an important role in oriential infection . We tested oriential infection in REF-Syn4 cells over-expressing syndecan-4, and in REF-Syn4AS cells in which the expression of syndecan-4 was down regulated by transfecting with anti-sense syndecan-4 cDNA . Oriential infection was found to be dependent on the expression level of syndecan-4 on the cell surface . Furthermore, the infectivity of O . tsutsugamushi was specifically reduced by treating O . tsutsugamushi with the purified recombinant core protein of syndecan-4 (Syn4E) . These results suggest that the core protein of syndecan-4 and the heparin/heparan sulfate chain of syndecan play an important role in oriential infection by O . tsutsugamushi.

Microb Pathog, 2004 Apr, 36(4), 197 - 203
Growth phase mediated regulation of the Actinobacillus pleuropneumoniae ApxI and ApxII toxins; Jarma E et al.; Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, a highly contagious and often fatal respiratory tract disease of pigs . The Apx toxins are primary virulence factors of this pathogen, with ApxI and ApxII being produced by all highly virulent strains in North America . Further characterization of these hemolytic toxins is needed to fully understand their role in disease and elucidate the environmental signals and genes that affect their production during infection . Many bacteria regulate genes in response to growth phase, and in this report we examined the effect of growth phase on ApxI and ApxII gene expression . Batch cultures of ApxI- and ApxII-producing strains were grown in heart infusion broth supplemented with beta-NAD, and samples were prepared throughout the growth curve . Maximal gene expression occurred in late exponential or early stationary phase, as indicated by a peak in apx mRNA concentration in Northern blot analysis . The amount of accumulated Apx protein and Apx hemolytic activity confirmed this increase in gene expression . These findings suggest a novel transcriptional regulatory mechanism that enhances Apx gene expression under in vitro conditions of high cell density and/or slow growth rate.

Semin Neonatol, 2003 Dec, 8(6), 449 - 59
Treatment and prevention of necrotizing enterocolitis; Lee JS et al.; Necrotizing enterocolitis (NEC) is the most common serious, acquired gastrointestinal disorder in the newborn infant . Although many variables are associated with development of NEC, only prematurity has been consistently identified in case-controlled studies . Traditionally, the diving seal reflex has been invoked as the mechanism responsible for ischaemic injury and necrosis . Intestinal ischaemia is likely to be the final common pathway in NEC; however, it is due to the release of vasoconstricting substances, such as platelet activating factor, rather than perinatal asphyxia . Bacteria and/or bacterial toxins are likely to have a key role in the pathogenesis of NEC by fostering production of inflammatory mediators . The role of feeding practices in the pathogenesis of NEC remains controversial . Treatment of infants with NEC generally includes a regimen of bowel rest, gastric decompression, systemic antibiotics and parenteral nutrition . Infants with perforation are generally operated upon; however, there has been recent interest in primary peritoneal drainage as an alternative . Prevention of NEC still remains elusive . Avoidance of preterm birth, use of antenatal steroids and breast-milk feeding are practices that offer the greatest potential benefits . Use of any other strategy should await further trials.

J Evol Biol, 2004 Jan, 17(1), 48 - 54
Molecular evolution of plant haemoglobin: two haemoglobin genes in Nymphaeaceae Euryale ferox; Guldner E et al.; We isolated and sequenced two haemoglobin genes from the early-branching angiosperm Euryale ferox (Nymphaeaceae) . The two genes belong to the two known classes of plant haemoglobin . Their existence in Nymphaeaceae supports the theory that class 1 haemoglobin was ancestrally present in all angiosperms, and is evidence for class 2 haemoglobin being widely distributed . These sequences allowed us to unambiguously root the angiosperm haemoglobin phylogeny, and to corroborate the hypothesis that the class 1/class 2 duplication event occurred before the divergence between monocots and eudicots . We addressed the molecular evolution of plant haemoglobin by comparing the synonymous and nonsynonymous substitution rates in various groups of genes . Class 2 haemoglobin genes of legumes (functionally involved in a symbiosis with nitrogen-fixing bacteria) show a higher nonsynonymous substitution rate than class 1 (nonsymbiotic) haemoglobin genes . This suggests that a change in the selective forces applying to plant haemoglobins has occurred during the evolutionary history of this gene family, potentially in relation with the evolution of symbiosis.

Mol Plant Microbe Interact, 2004 Mar, 17(3), 292 - 303
Global changes in gene expression in Sinorhizobium meliloti 1021 under microoxic and symbiotic conditions; Becker A et al.; Sinorhizobium meliloti is an alpha-proteobacterium that alternates between a free-living phase in bulk soil or in the rhizosphere of plants and a symbiotic phase within the host plant cells, where the bacteria ultimately differentiate into nitrogen-fixing organelle-like cells, called bacteroids . As a step toward understanding the physiology of S . meliloti in its free-living and symbiotic forms and the transition between the two, gene expression profiles were determined under two sets of biological conditions: growth under oxic versus microoxic conditions, and in free-living versus symbiotic state . Data acquisition was based on both macro- and microarrays . Transcriptome profiles highlighted a profound modification of gene expression during bacteroid differentiation, with 16% of genes being altered . The data are consistent with an overall slow down of bacteroid metabolism during adaptation to symbiotic life and acquisition of nitrogen fixation capability . A large number of genes of unknown function, including potential regulators, that may play a role in symbiosis were identified . Transcriptome profiling in response to oxygen limitation indicated that up to 5% of the genes were oxygen regulated . However, the microoxic and bacteroid transcriptomes only partially overlap, implying that oxygen contributes to a limited extent to the control of symbiotic gene expression.

Chemphyschem, 2004 Jan 23, 5(1), 57 - 67
Excitonic coupling strength and coherence length in the singlet and triplet excited states of meso-meso directly linked Zn(II)porphyrin arrays; Ha JH et al.; Excitation-energy transport (EET) phenomena in meso-meso directly linked Zn(II)porphyrin arrays in the singlet and triplet excited states were investigated with a view to electronic coupling strength and coherence length by steady-state and time-resolved spectroscopic measurements . To investigate energy transfer in the triplet states, we modified the Zn(II)porphyrin arrays with bromo substituents at both ends . The coupling strength of the Soret bands of the arrays was estimated to be about 2200 cm-1, and that of the Q bands is about 570 cm-1 . The coherence length in the S1 state of the Zn(II)porphyrin arrays was determined to be 4-5 porphyrin units, which is comparable to that of the well-ordered two-dimensional circular structure B850 in the peripheral light-harvesting antenna (LH2) in photosynthetic purple bacteria . This indicates that the Zn(II)porphyrin arrays are well suited for mimicking natural light-harvesting antenna complexes . On the other hand, the rate of energy transfer in the triplet state is estimated to be on the order of 100 microseconds-1, and the very weak coupling between the triplet states (ca . 0.003 cm-1), indicates that the triplet excitation energy is essentially localized on a single porphyrin moiety.

Adv Space Res, 2003, 32(11), 2373 - 8
The "SCORPION" experiment onboard the International Space Station . Preliminary results; Borisov V et al.; The "SCORPION" program onboard the Russian Segment (RS) of the International Space Station (ISS) is designed to carry out complex research of the effects of the nar-Earth space parameters on the conditions under which various experiments and operations are being conducted . Special attention in this program was paid to the biological objects onboard the orbital station, e.g . it was found that variation in the number of colony forming units (micromicets and bacteria) correlates with the solar activity and the absorbed dose . The "SCORPION" experiment onboard the RS ISS started in January 2002 . It was designed to measure the following parameters inside the space absorbed doses in different places inside the RS ISS, the fluxes of energetic charged particles, neutrons and gamma-quanta; the vectors of the magnetic field and low-frequency electromagnetic waves . At the same time the growth of micromicets on the samples of various materials was studied . The description of the "SCORPION" experiment and the preliminary results obtained onboard the RS ISS in 2002 are presented . c2003 COSPAR . Published by Elsevier Ltd . All rights reserved.

Clin Infect Dis, 2004 Mar 15, 38(6), 805 - 11 Epub 2004 Mar 01.
Rickettsia parkeri: a newly recognized cause of spotted fever rickettsiosis in the United States; Paddock CD et al.; Ticks, including many that bite humans, are hosts to several obligate intracellular bacteria in the spotted fever group (SFG) of the genus Rickettsia . Only Rickettsia rickettsii, the agent of Rocky Mountain spotted fever, has been definitively associated with disease in humans in the United States . Herein we describe disease in a human caused by Rickettsia parkeri, an SFG rickettsia first identified >60 years ago in Gulf Coast ticks (Amblyomma maculatum) collected from the southern United States . Confirmation of the infection was accomplished using serological testing, immunohistochemical staining, cell culture isolation, and molecular methods . Application of specific laboratory assays to clinical specimens obtained from patients with febrile, eschar-associated illnesses following a tick bite may identify additional cases of R . parkeri rickettsiosis and possibly other novel SFG rickettsioses in the United States.

Microbes Infect, 2004 Feb, 6(2), 141 - 9
Wolbachia endosymbionts of Onchocerca volvulus express a putative periplasmic HtrA-type serine protease; Jolodar A et al.; Wolbachia are intracellular bacteria of many filarial nematodes . A mutualistic interaction between the endobacteria and the filarial host is likely, because the clearance of Wolbachia by tetracycline leads to the obstruction of embryogenesis and larval development . Databases were searched for exported molecules to identify candidates involved in this mutualism . Fragments of a Wolbachia serine protease from the human filarial parasite Onchocerca volvulus were obtained (Wol-Ov-HtrA) by the use of a PCR technique and primers based on the Rickettsia prowazekii genome . The deduced amino acid sequence exhibited 87% and 81% identity to the homologous Wolbachia proteases identified from Brugia malayi and Drosophila melanogaster, respectively . The full-length cDNA encodes 494 amino acids with a calculated mass of 54 kDa . Three characteristic features, (i) a catalytic triad of serine proteases, (ii) two PDZ domains and (iii) a putative signal peptide, classify the endobacterial protein as a member of the periplasmic HtrA family of proteases known to express chaperone and regulator activity of apoptosis . Using a rabbit antiserum raised against a recombinantly expressed 33-kDa fragment of Wol-Ov-HtrA, strong labelling of the antigen was found associated with endobacteria in hypodermis, oocytes, zygotes, all embryonic stages and microfilariae of O . volvulus . Staining of hypodermal cytoplasm surrounding the endobacteria indicated a possible release of the protein from the Wolbachia . The demonstration of Wol-Ov-HtrA-reactive IgG1 antibodies in sera of O . volvulus-infected persons indicated the exposure to the protein and its recognition by the human immune system . Wol-Ov-HtrA is a candidate for an exported Wolbachia protein that may interact with the filarial host metabolism.

Cancer Cell, 2004 Feb, 5(2), 137 - 49
Prune cAMP phosphodiesterase binds nm23-H1 and promotes cancer metastasis; D'Angelo A et al.; We identify a new enzymatic activity underlying metastasis in breast cancer and describe its susceptibility to therapeutic inhibition . We show that human prune (h-prune), a phosphoesterase DHH family appertaining protein, has a hitherto unrecognized cyclic nucleotide phosphodiesterase activity effectively suppressed by dipyridamole, a phosphodiesterase inhibitor . h-prune physically interacts with nm23-H1, a metastasis suppressor gene . The h-prune PDE activity, suppressed by dipyridamole and enhanced by the interaction with nm23-H1, stimulates cellular motility and metastasis processes . Out of 59 metastatic breast cancer cases analyzed, 22 (37%) were found to overexpress h-prune, evidence that this novel enzymatic activity is involved in promoting cancer metastasis.

Environ Sci Technol, 2004 Feb 15, 38(4), 1045 - 53
Carbon tetrachloride transformation on the surface of nanoscale biogenic magnetite particles; McCormick ML et al.; Iron-reducing conditions in subsurface environments promote dechlorination reactions via both biotic and abiotic pathways, the latter often mediated via biologically activated minerals formed by dissimilatory iron-reducing bacteria (DIRB) . Here we report the major products and pathways associated with the abiotic transformation of carbon tetrachloride (CT) by nanoscale biogenic magnetite/maghemite particles produced by the DIRB Geobacter metallireducens . Product formation and free radical/carbene trapping studies indicate that CT transformation occurs via three parallel pathways . The first pathway (hydrogenolysis) results in the formation of chloroform (45-50%) via a trichloromethyl free radical (*CCl3) and possibly a trichloromethyl carbanion (**CCl3-) . The second and third pathways involve a dichlorocarbene intermediate (**CCl2), which either hydrolyzes to form CO (approximately 38%) (carbene hydrolysis), or undergoes further reduction to yield methane (8-10%) (carbene reduction) . The mechanism of methane formation from **CCl2 is not known, but is speculated to involve a sequence of surface coordinated carbenoid and free radical complexes . The large fraction of relatively benign products formed by the carbene-mediated pathways suggests that magnetite/maghemite particles may have a beneficial application in the remediation of CT contaminated environments.

Ying Yong Sheng Tai Xue Bao, 2003 Nov, 14(11), 2079 - 80
{Respiration and production of bacterioplankton in shrimp cultural enclosure ecosystems}; Liu G et al.; The study on the respiration and production of bacterioplankton in five shrimp cultural enclosure ecosystems showed that the production and respiration fluctuated from 90 to 909 and 248 to 1785 micrograms C.L-1.d-1, respectively . There existed a significant positive relationship between production and respiration . The daily P/B ratio of bacterioplancton averaged 0.93 d-1, and its production efficiency averaged 0.34.

Med Sci (Paris), 2004 Feb, 20(2), 248 - 53
{Genetics: still a discipline?}; Gayon J; In the institutional sense of the term "discipline" (laboratories, societies, congresses, curricula, etc.), genetics remains a discipline . In the intellectual sense of the term (consensus on a definite array of concepts, methods and theoretical purposes), it is doubtful that genetics is still a discipline . At first, molecular biology seemed to have introduced an unequivocal structural (or molecular) definition of the gene: a definite sequence of nucleotides that code for a protein . In fact, as it appears in retrospect, this was not the case . Even in 1961, when Jacob and Monod proposed their first model of genetic regulation in bacteria, there was no possibility of constructing a non equivocal concept of the gene . More recent developments in molecular genetics have made this situation worse . There is no possible definition of the gene as a general category . The reasons why biologists keep the word are pragmatic rather than theoretical: communication among scientists, economic interests and ideology.

Can J Gastroenterol, 2004 Feb, 18(2), 83 - 6
Failure to improve parameters of lactose maldigestion using the multiprobiotic product VSL3 in lactose maldigesters: a pilot study; Yesovitch R et al.; Lactose maldigestion is a common genetic trait in up to 70% of the world's population . In these subjects, the ingestion of lactose may lead to prebiotic effects which can be confirmed by measurement of breath hydrogen . After a period of continuous lactose ingestion, colonic bacterial adaptation is measurable as improved parameters of lactose digestion . There may be inherent benefits in this process of adaptation which may protect against some diseases . We attempt to link therapeutically beneficial probiotics (VSL3, Seaford Pharmaceuticals Inc, Ontario) with improvement in parameters of lactose maldigestion . Two groups of five subjects with maldigestion were fed one or four packets of VSL3 (one packet containing 450 x 10(9) live bacteria) before testing and then 17 days later . A 50 g lactose challenge was carried out before and after feeding . While there was a trend toward increasing rather than reducing of summed breath hydrogen, no statistically significant changes were observed between results from before testing and those from testing 17 days later . The authors conclude that direct consumption of the probiotic VSL3 may not improve parameters of lactose maldigestion without metabolic activation . In its present format, therefore, the test for colonic adaptation cannot be used to demonstrate direct bacterial embedding with VSL3.

J Biol Chem, 2004 May 7, 279(19), 19739 - 46 Epub 2004 Mar 02.
Purification and characterization of NafY (apodinitrogenase gamma subunit) from Azotobacter vinelandii; Rubio LM et al.; The formation of an active dinitrogenase requires the synthesis and the insertion of the iron-molybdenum cofactor (FeMo-co) into a presynthesized apodinitrogenase . In Azotobacter vinelandii, NafY (also known as gamma protein) has been proposed to be a FeMo-co insertase because of its ability to bind FeMo-co and apodinitrogenase . Here we report the purification and biochemical characterization of NafY and reach the following conclusions . First, NafY is a 26-kDa monomeric protein that binds one molecule of FeMo-co with very high affinity (K(d) approximately equal to 60 nm); second, the NafY-FeMo-co complex exhibits a S = 3/2 EPR signal with features similar to the signals for extracted FeMo-co and the M center of dinitrogenase; third, site-directed mutagenesis of nafY indicates that the His(121) residue of NafY is involved in cofactor binding; and fourth, NafY binding to apodinitrogenase or to FeMo-co does not require the presence of any additional protein . In addition, we have obtained evidence that suggests the ability of NafY to bind NifB-co, an FeS cluster of unknown structure that is a biosynthetic precursor to FeMo-co.

J Bacteriol, 2004 Mar, 186(6), 1658 - 66
The Legionella pneumophila PilT homologue DotB exhibits ATPase activity that is critical for intracellular growth; Sexton JA et al.; The ability of Legionella pneumophila to grow and cause disease in the host is completely dependent on a type IV secretion system known as the Dot/Icm complex . This membrane-spanning apparatus translocates effector molecules into host cells in a process that is poorly understood but that is known to require the putative ATPase DotB . One possible role for DotB is suggested by its similarity to the PilT family of proteins, which mediate pilus retraction . To better understand the molecular behavior of DotB, we have purified the protein and shown that it forms stable homohexameric rings and hydrolyzes ATP with a specific activity of 6.4 nmol of ATP/min/mg of protein . ATPase activity is critical to the function of DotB, as alteration of the conserved Walker box lysine residue resulted in a mutant protein, DotB K162Q, which failed to bind or hydrolyze ATP and which could not complement a DeltadotB strain for intracellular growth in macrophages . Consistent with the ability of DotB to interact with itself, the dotBK162Q allele exhibited transdominance over wild-type dotB, providing the first example of such a mutation in L . pneumophila . Finally, the DotB K162Q mutant protein had a significantly enhanced membrane localization in L . pneumophila compared to wild-type DotB, suggesting a relationship between nucleotide binding and membrane association . These results are consistent with a model in which DotB cycles between the cytoplasm and the Dot/Icm complex at the membrane, where it hydrolyzes nucleotides to provide energy to the complex.

Plant J, 2004 Mar, 37(6), 853 - 63
A cytosolic glucosyltransferase is required for conversion of starch to sucrose in Arabidopsis leaves at night; Chia T et al.; Maltose is exported from the Arabidopsis chloroplast as the main product of starch degradation at night . To investigate its fate in the cytosol, we characterised plants with mutations in a gene encoding a putative glucanotransferase (disproportionating enzyme; DPE2), a protein similar to the maltase Q (MalQ) gene product involved in maltose metabolism in bacteria . Use of a DPE2 antiserum revealed that the DPE2 protein is cytosolic . Four independent mutant lines lacked this protein and displayed a decreased capacity for both starch synthesis and starch degradation in leaves . They contained exceptionally high levels of maltose, and elevated levels of glucose, fructose and other malto-oligosaccharides . Sucrose levels were lower than those in wild-type plants, especially at the start of the dark period . A glucosyltransferase activity, capable of transferring one of the glucosyl units of maltose to glycogen or amylopectin and releasing the other, was identified in leaves of wild-type plants . Its activity was sufficient to account for the rate of starch degradation . This activity was absent from dpe2 mutant plants . Based on these results, we suggest that DPE2 is an essential component of the pathway from starch to sucrose and cellular metabolism in leaves at night . Its role is probably to metabolise maltose exported from the chloroplast . We propose a pathway for the conversion of starch to sucrose in an Arabidopsis leaf.

Br J Dermatol, 2004 Feb, 150(2), 312 - 6
Dermatoses associated with travel to Burkina Faso and diagnosed by means of teledermatology; Caumes E et al.; BACKGROUND: The pattern of dermatoses occurring in travellers to tropical areas is poorly documented . OBJECTIVES: To diagnose skin diseases in travellers to Burkina Faso by means of teledermatology; to assess the educational value of teledermatology for the local general practitioner (GP) . METHODS: Patients (Westerners and Burkinabese nationals) were included in the study if they presented with a cutaneous disease to the GP based in Ouagadougou, Burkina Faso . Images of the skin lesions were acquired with a point-and-shoot digital camera and sent via the Internet, together with the clinical history . Diagnostic concordance between dermatologists in France and the GP in Ouagadougou was analysed as a simple proportion of agreement and 95% confidence interval . RESULTS: One hundred and twenty-four patients (M/F ratio 1.17; 80.6% Westerners) were included in the study . One hundred and thirty dermatoses were identified: 73 (56%) were of infectious origin, and 19 (15%) were related to eczematous dermatitis . The skin infections were mainly due to bacteria (18%), fungi (14%) or arthropods (13%) . Parasitic dermatoses were observed only in Burkinabese nationals . Among Westerners, fungal dermatoses were observed only in long-term residents . The diagnostic agreement between the local GP and the remote dermatologists was 49% overall (95% confidence interval 41-58) . Agreement between the GP and the dermatologists on the dermatological category improved significantly over time (P<0.05) . CONCLUSIONS: Telemedecine can improve the management of cutaneous diseases among Western travellers . Most dermatoses observed in Western travellers to Burkina Faso are of infectious origin . Teledermatology has educational value for local GPs.

Microb Ecol, 2004 Apr, 47(3), 243 - 51 Epub 2004 Mar 04.
Identification of planctomycetes with order-, genus-, and strain-specific 16S rRNA-targeted probes; Gade D et al.; A specific 16S rRNA-targeted oligonucleotide probe (PIR1223) for the genus Pirellula and a species-specific probe (RB454) for Pirellula sp . strain SH1 have been designed and optimized . Together with the already existing order-specific probe PLA886, the two newly designed probes were used to detect and identify planctomycetes, pirellulae, and close relatives of Pirellula sp . strain SH1 in different habitats . With the help of these probes for detection and identification, bacteria of the genus Pirellula were detected and cultivated from tissue of the Mediterranean sponge Aplysina aerophoba and from the water column of the Kiel Fjord . An unexpected result was the close phylogenetic relationship of the isolate from the sponge and the brackish water habitat Kiel Fjord as revealed by DNA/DNA hybridization.

Microbiology, 2004 Mar, 150(Pt 3), 707 - 13
Formaldehyde dehydrogenase preparations from Methylococcus capsulatus (Bath) comprise methanol dehydrogenase and methylene tetrahydromethanopterin dehydrogenase; Adeosun EK et al.; In methylotrophic bacteria, formaldehyde is an important but potentially toxic metabolic intermediate that can be assimilated into biomass or oxidized to yield energy . Previously reported was the purification of an NAD(P)(+)-dependent formaldehyde dehydrogenase (FDH) from the obligate methane-oxidizing methylotroph Methylococcus capsulatus (Bath), presumably important in formaldehyde oxidation, which required a heat-stable factor (known as the modifin) for FDH activity . Here, the major protein component of this FDH preparation was shown by biophysical techniques to comprise subunits of 64 and 8 kDa in an alpha(2)beta(2) arrangement . N-terminal sequencing of the subunits of FDH, together with enzymological characterization, showed that the alpha(2)beta(2) tetramer was a quinoprotein methanol dehydrogenase of the type found in other methylotrophs . The FDH preparations were shown to contain a highly active NAD(P)(+)-dependent methylene tetrahydromethanopterin dehydrogenase that was the probable source of the NAD(P)(+)-dependent formaldehyde oxidation activity . These results support previous findings that methylotrophs possess multiple pathways for formaldehyde dissimilation.

J Cell Biol, 2004 Mar 1, 164(5), 701 - 15
Septin collar formation in budding yeast requires GTP binding and direct phosphorylation by the PAK, Cla4; Versele M et al.; Assembly at the mother-bud neck of a filamentous collar containing five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) is necessary for proper morphogenesis and cytokinesis . We show that Cdc10 and Cdc12 possess GTPase activity and appropriate mutations in conserved nucleotide-binding residues abrogate GTP binding and/or hydrolysis in vitro . In vivo, mutants unable to bind GTP prevent septin collar formation, whereas mutants that block GTP hydrolysis do not . GTP binding-defective Cdc10 and Cdc12 form soluble heteromeric complexes with other septins both in yeast and in bacteria; yet, unlike wild-type, mutant complexes do not bind GTP and do not assemble into filaments in vitro . Absence of a p21-activated protein kinase (Cla4) perturbs septin collar formation . This defect is greatly exacerbated when combined with GTP binding-defective septins; conversely, the septin collar assembly defect of such mutants is suppressed efficiently by CLA4 overexpression . Cla4 interacts directly with and phosphorylates certain septins in vitro and in vivo . Thus, septin collar formation may correspond to septin filament assembly, and requires both GTP binding and Cla4-mediated phosphorylation of septins.

J Biol Chem, 2004 May 7, 279(19), 19705 - 11 Epub 2004 Mar 01.
Iron-sulfur cluster assembly: NifU-directed activation of the nitrogenase Fe protein; Dos Santos PC et al.; The NifU protein is a homodimer that is proposed to provide a molecular scaffold for the assembly of {Fe-S} clusters uniquely destined for the maturation of the nitrogenase catalytic components . There are three domains contained within NifU, with the N-terminal domain exhibiting a high degree of primary sequence similarity to a related family of {Fe-S} cluster biosynthetic scaffolds designated IscU . The C-terminal domain of NifU exhibits sequence similarity to a second family of proposed {Fe-S} cluster biosynthetic scaffolds designated Nfu . Genetic experiments described here involving amino acid substitutions within the N-terminal and C-terminal domains of NifU indicate that both domains can separately participate in nitrogenase-specific {Fe-S} cluster formation, although the N-terminal domain appears to have the dominant function . These in vivo experiments were supported by in vitro {Fe-S} cluster assembly and transfer experiments involving the activation of an apo-form of the nitrogenase Fe protein.

J Biol Chem, 2004 May 14, 279(20), 21217 - 22 Epub 2004 Mar 01.
Intracellular localization of homopolymeric amino acid-containing proteins expressed in mammalian cells; Oma Y et al.; Many human proteins have homopolymeric amino acid (HPAA) tracts, which are involved in protein-protein interactions and also have intrinsic polymerization properties . Polyglutamine or polyalanine expansions cause several neurodegenerative diseases . To examine the properties of HPAAs, we expressed 20 kinds of 30-residue HPAA fused to the C terminus of yellow fluorescent protein in mammalian cells . Specific localization was observed depending on the HPAA . Polyarginine and polylysine aggregated in the nucleus . Polyalanine, polyhistidine, polyisoleucine, polyleucine, polymethionine, polyphenylalanine, polythreonine, polytryptophan, and polyvaline localized in the cytoplasm, and some of these HPAAs formed aggregate(s) . Hydrophobic HPAAs such as polyisoleucine, polyleucine, polyphenylalanine, and polyvaline were found as one major aggregate or cumulus in the perinuclear region . Western blot analysis indicated that hydrophobic HPAA tracts appear to oligomerize and form high molecular weight complexes . These results indicate that hydrophobicity itself may trigger the oligomerization and aggregation of proteins when overexpressed in cells . Our experiments provide novel insights into the nature of the HPAAs that are often seen in human and other organisms.

Br J Pharmacol, 2004 Mar, 141(6), 951 - 60 Epub 2004 Mar 01.
Anti-inflammatory effect of diosmectite in hapten-induced colitis in the rat; Gonzalez R et al.; 1 . Diosmectite is a natural silicate effectively used in the treatment of infectious diarrhoea . Its antidiarrhoeal properties involve adsorption of toxins and bacteria and modifications of the rheological characteristics of gastrointestinal mucus . Hence, the aim of this study was to test the intestinal anti-inflammatory activity of diosmectite . 2 . Diosmectite (500 mg x kg(-1) day(-1), p.o.) was administered as a post-treatment to rats with chronic trinitrobenzene sulphonic acid colitis . Colonic status was checked 1 and 2 weeks after colitis induction by macroscopic, histological and biochemical examination . 3 . Diosmectite post-treatment resulted in amelioration of the morphological signs (intestinal weight, macroscopic damage, necrosed area, histology) and biochemical markers (myeloperoxidase activity, glutathione levels, MUC2 expression, inducible nitric oxide synthase and interleukin-1beta (IL-1beta) and leukotriene B(4) synthesis), as well as in the reduction of the severity of diarrhoea . The effect of the clay was comparable to that of sulphasalazine (50 mg x kg(-1) day(-1)) . 4 . 5 . Diosmectite exhibited a dose-dependent capacity to adsorb proteins in vitro as well as a dose-dependent inhibitory effect on the basolateral secretion of IL-8 by lipopolysaccharide (LPS)-stimulated HT29 cells . Diosmectite had a dose-dependent inhibitory effect on IL-1beta production by LPS-stimulated THP-1 cells . 6 . The effect of diosmectite on MUC2 was post-transcriptional, since mRNA levels were unaffected . However, diosmectite is able to upregulate MUC2 mRNA levels in HT29-MTX cells . 7 . Diosmectite has anti-inflammatory activity administered as a post-treatment . Possible mechanisms include adsorption of luminal antigens, increase of colonic mucin levels and possibly a direct modulatory action of cytokine production by mucosal cells.

World J Gastroenterol, 2004 Mar 1, 10(5), 620 - 5
IL-10 and its related cytokines for treatment of inflammatory bowel disease; Li MC et al.; Inflammatory bowel diseases (IBDs), including Crohn's disease and ulcerative colitis are chronic inflammatory disorders of gastrointestinal tract . Although the etiology is incompletely understood, initiation and aggravation of the inflammatory process seem to be due to a massive local mucosal immune response . Interleukin-10 (IL-10) is a regulatory cytokine which inhibits both antigen presentation and subsequent pro-inflammatory cytokine release, and it is proposed as a potent anti-inflammatory biological therapy in chronic IBD . Many methods of IL-10 as a treatment for IBD have been published . The new strategies of IL-10 treatment, including recombinant IL-10, the use of genetically modified bacteria, gelatine microsphere containing IL-10, adenoviral vectors encoding IL-10 and combining regulatory T cells are discussed in this review . The advantages and disadvantages of these IL-10 therapies are summarized . Although most results of recombinant IL-10 therapies are disappointing in clinical testing because of lacking efficacy or side effects, therapeutic strategies utilizing gene therapy may enhance mucosal delivery and increase therapeutic response . Novel IL-10-related cytokines, including IL-19, IL-20, IL-22, IL-24, IL-26, IL-28 and IL-29, are involved in regulation of inflammatory and immune responses . The use of IL-10 and IL-10-related cytokines will provide new insights into cell-based and gene-based treatment against IBD in near future.

Nat Biotechnol, 2004 Apr, 22(4), 445 - 9 Epub 2004 Feb 29.
An improved cyan fluorescent protein variant useful for FRET; Rizzo MA et al.; Many genetically encoded biosensors use Forster resonance energy transfer (FRET) between fluorescent proteins to report biochemical phenomena in living cells . Most commonly, the enhanced cyan fluorescent protein (ECFP) is used as the donor fluorophore, coupled with one of several yellow fluorescent protein (YFP) variants as the acceptor . ECFP is used despite several spectroscopic disadvantages, namely a low quantum yield, a low extinction coefficient and a fluorescence lifetime that is best fit by a double exponential . To improve the characteristics of ECFP for FRET measurements, we used a site-directed mutagenesis approach to overcome these disadvantages . The resulting variant, which we named Cerulean (ECFP/S72A/Y145A/H148D), has a greatly improved quantum yield, a higher extinction coefficient and a fluorescence lifetime that is best fit by a single exponential . Cerulean is 2.5-fold brighter than ECFP and replacement of ECFP with Cerulean substantially improves the signal-to-noise ratio of a FRET-based sensor for glucokinase activation.

Med Oral, 2004 Mar-Apr, 9(2), 131 - 7, 125-31
Periodontitis as a risk factor in patients with ischemic heart disease; Luis-Delgado O et al.; Cardiovascular disease, and particularly ischemic heart disease (IHD), constitutes one of the principal causes of mortality in the western world . Interest has recently increased in the relationship between IHD and different infectious processes as triggering factors of the former, such as Chlamydia pneumoniae and Helicobacter pylori infection . Periodontitis has also been related to an increased risk of coronary disease, since both disorders share common characteristics such as patient age and sex, and a smoking habit, among other aspects . There are many similarities between vascular pathology induced by bacteria and the natural history of atherogenesis . The principal mechanism of action underlying periodontitis and IHD centers on the effect of bacteria and their endotoxins upon inflammatory reaction, hemostasia and lipid metabolic alterations . However, some authors are of the opinion that periodontitis constitutes an epiphenomenon, and that further studies are needed to clarify the cause-effect relation between these two multifactor pathologies.

Free Radic Biol Med, 2004 Mar 15, 36(6), 745 - 56
Role of intestine in postsurgical complications: involvement of free radicals; Thomas S et al.; Surgery at any location in the body leads to surgical stress response and alterations in normal body homeostasis . The intestine is extremely sensitive to surgical stress even at remote locations and the gastrointestinal tract plays an important role in the development of postsurgical complications such as sepsis, the systemic immune response syndrome (SIRS), and multiple organ failure syndrome (MOFS) . The generation of free radicals and subsequent biochemical alterations at the cellular and subcellular level in the intestine has been suggested to play an important role in this process . These oxidative stress-induced events in the mucosa might act as an initiator of distant organ damage and also facilitate bacterial adherence onto the epithelium and translocation into the systemic circulation . This review attempts to highlight the important role of intestine and oxygen free radicals in initiating post-surgical complications.

Bioorg Chem, 2004 Apr, 32(2), 82 - 91
Structures of two new "minimalist" modified nucleosides from archaeal tRNA; Zhou S et al.; The wyeosine (or wye) family of tricyclic ribonucleosides from archaeal and eukaryal tRNA(Phe) constitutes one of the most complex and interesting series of posttranscriptional RNA modifications, and has been the object of numerous studies of their chemical and biological synthesis and distribution . We report the structures of two minimally elaborated wye derivatives from archaea, raising the known number of wye nucleosides to eight: 3,4-dihydro-6-methyl-3-beta-d-ribofuranosyl-9H-imidazo{1,2-a}purine-9-one (symbol imG-14), and 3,4-dihydro-6,7-dimethyl-3-beta-d-ribofuranosyl-9H-imidazo{1,2-a}purine-9-one (symbol imG2) . Structures were determined primarily by mass spectrometry, and confirmed by comparison of physicochemical properties with those of chemically synthesized nucleosides . The nucleosides contain no amino acid side chains at C-7 (1H-imidazo{1,2-a}purine nomenclature) and are the only wye derivatives not methylated at N-4 . These features suggest a minimal role for wye methyl groups and side chains in maintenance of anticodon stem-loop structures, and support the concept that archaeal tRNA nucleoside modification motifs are generally simpler than those of their counterparts in eukarya and bacteria.

Res Microbiol, 2004 Mar, 155(2), 98 - 104
Stringent control in the archaeal genus Sulfolobus; Cellini A et al.; Six Archaea belonging to the phylum Euryarchaeota were previously analyzed with respect to stringent control . Only one of the strains studied was shown to possess Bacteria-like stringent control over stable RNA accumulation; ppGpp and pppGpp production was totally lacking in all Archaea analyzed . To broaden our knowledge of stringent control in the Archaea, we examined here the accumulation of stable RNA and the production of ppGpp and pppGpp under amino acid starvation in three species of the genus Sulfolobus belonging to the Crenarchaeota, an archaeal phylum distant from the Euryarchaeota . In these species the accumulation of sRNA was arrested when aminoacylation of tRNA was inhibited by pseudomonic acid . Furthermore, stringent control of stable RNA accumulation was relaxed by some protein synthesis inhibitors that do not interfere with aminoacylation of tRNA, a feature typical of bacterial stringent control . Neither ppGpp nor pppGpp could be detected during growth or under amino acid starvation, and the intracellular GTP levels did not decrease in the course of the stringent response . These results show that: (1) stringency is widespread in wild-type thermoacidophilic archaea; (2) in the crenarchaeal species analyzed here SC depends on the deaminoacylation of tRNA; (3) in the strains analyzed ppGpp is not produced during normal growth nor during the stringent reaction; it is therefore not an effector either of SC over sRNA synthesis or of growth control . (p)ppGpp appears to be completely absent from the Archaea and thus constitutes an additional feature that distinguishes the Bacteria from the Archaea.

Res Microbiol, 2004 Mar, 155(2), 53 - 60
The autotransporter secretion system; Desvaux M et al.; The type V secretion system includes the autotransporter family, the two-partner system and the Oca family . The autotransporter secretion process involving first the translocation of the precursor through the inner membrane and then its translocation through the outer membrane via a pore formed by a beta-barrel is reviewed.

Biotechniques, 2004 Feb, 36(2), 240 - 7
Computer-assisted image analysis protocol that quantitatively measures subnuclear protein organization in cell populations; Voss TC et al.; Many nuclear proteins, including the nuclear receptor co-repressor (NCoR) protein are localized to specific regions of the cell nucleus, and this subnuclear positioning is preserved when NCoR is expressed in cells as a fusion to a fluorescent protein (FP) . To determine how specific factors may influence the subnuclear organization of NCoR requires an unbiased approach to the selection of cells for image analysis . Here, we use the co-expression of the monomeric red FP (mRFP) to select cells that also express NCoR labeled with yellow FP (YFP) . The transfected cells are selected for imaging based on the diffuse cellular mRFP signal without prior knowledge of the subnuclear organization of the co-expressed YFP-NCoR . The images acquired of the expressed FPs are then analyzed using an automated image analysis protocol that identifies regions of interest (ROIs) using a set of empirically determined rules . The relative expression levels of both fluorescent proteins are estimated, and YFP-NCoR subnuclear organization is quantified based on the mean focal body size and relative intensity . The selected ROIs are tagged with an identifier and annotated with the acquired data . This integrated image analysis protocol is an unbiased method for the precise and consistent measurement of thousands of ROIs from hundreds of individual cells in the population.

Gastroenterology, 2004 Mar, 126(3), 765 - 73
Urea movement across mouse colonic plasma membranes is mediated by UT-A urea transporters; Stewart GS et al.; BACKGROUND & AIMS: Urea is a major nitrogen source for commensal bacteria that inhabit the large intestine . UT-A urea transporters mediate urea movement across plasma membranes . The aim of this study was to determine whether UT-A proteins are expressed in the mouse colon and, if so, whether they have a functional role in transcellular urea transport . METHODS: Mouse colonic UT-A transporters were investigated with Northern blot analysis, immunoblotting, immunolocalization, and refractive light flux experiments . RESULTS: Northern blot analysis showed that 4 UT-A transcripts were present in mouse colon . Two peptide-targeted polyclonal antibodies showed the presence of UT-A immunoreactive proteins in mouse colon . Antiserum ML446 targeted to the N-terminus of mouse UT-A1 detected proteins of 34 and 48 kilodaltons . Antiserum ML194 targeted to the C-terminus of mouse UT-A1 detected proteins of 48, 75, and 100 kilodaltons . Immunolocalization studies using ML446 showed the presence of UT-A proteins in cells throughout the colonic crypts . ML194 specifically stained cells located in the proliferative and stem regions of the lower portion of colonic crypts . Differential centrifugation and immunoblotting of colonic epithelia showed that UT-A proteins were present in plasma membrane-enriched fractions . Refractive light flux experiments using colonic plasma membrane vesicles showed a significant urea flux, which was completely inhibited by the UT-A inhibitor phloretin . CONCLUSIONS: Functional UT-A transporters are expressed in the plasma membranes of mouse colon, indicating that these proteins may play a key role in host/bacterial interaction.

Physiol Biochem Zool, 2003 Nov-Dec, 76(6), 836 - 42
Physiology of immunity in the water flea Daphnia magna: environmental and genetic aspects of phenoloxidase activity; Mucklow PT et al.; In an attempt to understand the ecological correlates of immunocompetence in Daphnia magna (Crustacea, Cladocera), we tested for variation in immune function in relation to feeding conditions, host conditions, and host genotype . We investigated both phenotypic (environmental dependent and condition dependent) as well as genotypic aspects of the prophenoloxidase activating system (Pro-POAS), which has been described as a key factor in invertebrate immunity . Daphnia magna is an ideal study system to disentangle phenotypic and genetic variation because females can reproduce clonally . Well-fed Daphnia showed higher phenoloxidase (PO) activity than Daphnia kept at a low food level . Wounding provoked a higher level of PO activity, indicating that the Pro-POAS was condition dependent . Further, we found clonal variation in PO activity among four clones of D . magna isolated from four different populations . The same four clones were tested for their resistance to the bacterial pathogen Pasteuria ramosa . High resistance corresponded to high PO activity . Our results suggest adaptive variation in PO activity and suggest that its expression is costly . These costs may influence the evolution of the PO activity level and the maintenance of its genotypic variation.

Chemosphere, 2004 Apr, 55(3), 291 - 317
Environmental VOSCs--formation and degradation of dimethyl sulfide, methanethiol and related materials; Bentley R et al.; Volatile organic sulfur compounds (VOSCs) play a major role in the global sulfur cycle . Two components, dimethyl sulfide (DMS) and methanethiol (MT) are formed in large amounts by living systems (e.g . algae, bacteria, plants), particularly in marine environments . A major route to DMS is by action of a lyase enzyme on dimethylsulfoniopropionate (DMSP) . DMSP has other roles, for instance as an osmoprotectant and cryoprotectant . Demethiolation of DMSP and other materials leads to MT . A major transport process is release of DMS from the oceans to the atmosphere . Oxidation of DMS in the atmosphere by hydroxyl and nitrate radicals produces many degradation products including CO2, COS, dimethyl sulfoxide, dimethyl sulfone, organic oxyacids of sulfur, and sulfate . These materials also have roles in biotic processes and there are complex metabolic interrelationships between some of them . This review emphasizes the chemical reactions of the organic sulfur cycle . For biotic reactions, details of relevant enzymes are provided when possible.

Enferm Infecc Microbiol Clin, 2004 Mar, 22(3), 160 - 76
Prevention of opportunistic infections in adult and adolescent patients with HIV infection . GESIDA/National AIDS Plan guidelines, 2004 {correction}; Berenguer J et al.; OBJECTIVE: To provide an update of guidelines from the Spanish AIDS Study Group (GESIDA) and the National AIDS Plan (PNS) committee on the prevention of opportunistic infections in adult and adolescent HIV-infected patients . METHODS: These consensus recommendations have been produced by a group of experts from GESIDA and/or the PNS after reviewing the earlier document and the scientific advances in this field in the last years . The system used by the Infectious Diseases Society of America and the United States Public Health Service has been used to classify the strength and quality of the data . RESULTS: This document provides a detailed review of the measures for the prevention of infections caused by viruses, bacteria, fungi and parasites in the context of HIV infection . Recommendations are given for preventing exposure and for primary and secondary prophylaxis for each group of pathogens . In addition, criteria are established for the withdrawal of prophylaxis in patients who respond well to highly active antiretroviral therapy (HAART) . CONCLUSIONS: HAART is the best strategy for the prevention of opportunistic infections in HIV-positive patients . Nevertheless, prophylaxis is still necessary in countries with limited economic resources, in highly immunodepressed patients until HAART achieves beneficial effects, in patients who refuse to take or who cannot take HAART, in those in whom HAART is not effective, and in the small group of infected patients with inadequate recovery of CD4+ T lymphocyte counts despite good inhibition of HIV replication.

Ying Yong Sheng Tai Xue Bao, 2003 Oct, 14(10), 1795 - 8
{Global consequences and control strategies of biological invasion}; Xie Z et al.; Biological invasion is a worldwide ecological phenomenon, but its mechanism is still not very clear . Invasive species give impacts on native species and ecosystems through competitions, predations, changing habitats, and dispersing diseases . They pose an increasing threat to the composition and structure of natural communities across the globe . Biological invasion has been greatly damaging the ecological and evolutionary integrity of natural ecosystems, which will weaken the functions of the ecosystems and frequently cause natural disasters . A better understanding of the causes, patterns, predictability, consequences, and management options associated with this threat to biodiversity is necessary to guide managers, policy makers, researchers, and general publics . Biological invasion also causes huge economic losses, and 137 billion dollar losses per year from biological invasion were estimated in USA . Invasive diseases impair human health and kill thousands and thousands of people, and invasive bacteria lead to so serious social panic and turbulence that people could feel uneasy even when eating and sleeping . Biological invasion largely decreases global biodiversity, which will threaten the survival and development of our descendants . Three steps are used in prevention and control of biological invasions . Comprehensive quarantine is the most effective way to prevent exotic invasion by accident . Ecological evaluation and monitoring is helpful to avoid disasters from species introduction . Physical methods, chemical approaches and biological controls are used to eradicate and control the spread of invaded species . Before biological controls are chosen, risk analysis of controlling organism is needed . Ideally, there should be both pre-eradication assessment to tailor removal to avoid unwanted ecological effects and post-removal assessment of eradication effects on both the target organism and the invaded ecosystem.

Ying Yong Sheng Tai Xue Bao, 2003 Oct, 14(10), 1775 - 9
{Effects of different arbuscular mycorrhizal fungi on Tagetes erecta growth and diesel degradation}; Geng C et al.; The effects of G . mosseae, G . geospora, G . constrictum and bacteria on diesel tolerance of Tagetes erecta were investigated under greenhouse conditions . The results showed that AM fungi could still develop mycorrhizal assosiations with mum when the diesel concentration was 5,000 mg.kg-1 . White mum was better than yellow mum in diesel tolerance, with 63.1% total biomass increased . The colonization rate of inoculating AM fungi treatment was 3.5%-29.9% higher than the control . G . mosseae and G . geospora were better strains, their biomass increasing 9.0% and 42.7% than the control, respectively, while the effect of inoculating mixed AM fungi was not obvious . Bacteria inhibited the colonization of arbuscular mycorrhizal fungi on white mum, but promoted the vegetative and reproductive growth of mycorrhizal mum . Among 5 inoculation treatments, treatments of inoculating G . geospora and inoculating mixed AM fungi and bacteria were better, with 16.51% and 14.05% more diesel degradation rate than that of the control, respectively.

J Biol Chem, 2004 May 7, 279(19), 20356 - 62 Epub 2004 Feb 24.
The structural mechanism for transcription activation by MerR family member multidrug transporter activation, N terminus; Newberry KJ et al.; Transcription regulators of the MerR family respond to myriad stress signals to activate sigma70/sigmaA-targeted genes, which contain suboptimal 19-bp spacers between their -35 and -10 promoter elements . The crystal structure of a BmrR-TPP(+)-DNA complex provided initial insight into the transcription activation mechanism of the MerR family, which involves base pair distortion, DNA undertwisting and shortening of the spacer, and realignment of the -35 and -10 boxes . Here, we describe the crystal structure of MerR family member MtaN bound to the mta promoter . Although the global DNA binding modes of MtaN and BmrR differ somewhat, homologous protein-DNA interactions are maintained . Moreover, despite their different sequences, the mta promoter conformation is essentially identical to that of the BmrR-TPP(+)-bound bmr promoter, indicating that this DNA distortion mechanism is common to the entire MerR family . Interestingly, DNA binding experiments reveal that the identity of the two central bases of the mta and bmr promoters, which are conserved as either a thymidine or an adenine in nearly all MerR promoters, is not important for DNA affinity . Comparison of the free and DNA-bound MtaN structures reveals that a conformational hinge, centered at residues N-terminal to the ubiquitous coiled coil, is key for mta promoter binding . Analysis of the structures of BmrR, CueR, and ZntR indicates that this hinge may be common to all MerR family members.

J Small Anim Pract, 2004 Feb, 45(2), 117 - 21
Sclerosing encapsulating peritonitis in a dog with leishmaniasis; Adamama-Moraitou KK et al.; Canine sclerosing encapsulating peritonitis is a rarely reported condition . A 10-year-old male German shepherd dog cross was presented with a history of ascites, vomiting, soft faeces, anorexia and depression . Gathering of the intestinal loops in the middle portion of the abdomen was detected by radiography and ultrasonography . Cytological examination of Giemsa-stained smears from the popliteal lymph nodes revealed Leishmania species . The results of culture of serosanguineous fluid obtained by abdominocentesis were negative for bacteria and fungi . Laparotomy revealed a sac of fibrous tissue encasing most of the intestinal loops and numerous adhesions extending between them . Histologically, an uneven, diffusely thickened, visceral peritoneal membrane was found . A diagnosis of idiopathic sclerosing encapsulating peritonitis was made . The dog was euthanased because the intestinal wall was torn at many sites during dissection of the membrane.

Shanghai Kou Qiang Yi Xue, 2002 Jun, 11(2), 143 - 6
{Relationship between the gingival crevicular fluid occult blood test and periodontal inflammation}; Wang ZH et al.; OBJECTIVE: To seek a new non-traumative method applied to the diagnosis of gingival bleeding; Studies on the internal relationship between gingival bleeding and microbacteria . METHODS: 102 saliva samples were tested for salivary occult blood test(Sobt),1600 sites for gingival crevicular fluid occult blood test (GCFobt) by the test strips, clinical assessments including sulcus bleeding index(SBI) and probing depth(PD); 79,32 subgingival plaque samples for smearing and bacteria culture respectively . Studies on the relationship between GCFobt and clinical index and subgingival bacteria . RESULTS: The sensitivity of GCFobt as a predictor for gingival bleeding was 68.0% and the specificity was 80.5%, GCFobt could more correctly indicated the local gingival inflammation than Sobt . Significant correlation was found between GCFobt and SBI (P<0.001); The percentage of spirochetes, rods and cocci had significant differences between GCFobt negative and positive( P<0.001); Significant differences in the detection of black bacteria within the GCFobt 0 and 3( P<0.01), the same as fusobacteria within GCFobt 0 and 2,3(P<0.05) . CONCLUSION: GCFobt is a rapid,convenient susceptible and non-traumative method for assessing gingival bleeding, can be used as an objective index for clinical periodontal examination.

Shanghai Kou Qiang Yi Xue, 2002 Jun, 11(2), 129 - 30
{Comparison of effect on S.mutans producing acid ability between organic weak acid and fluoride}; Zhu CL et al.; OBJECTIVE: To figure out the effects of organic weak acids, benzoate, citric acid and malic acid on the main cariogenic bacteria S.mutans Ingbritt . To evaluate the possibility of above weak acids as anti-caries reagents . METHODS: S.mutans Ingbritt was inoculated into tryptic soy broth containing different concentrations of fluoride, benzoate, citric acid and malic acid, grew in anaerobic conditions at 37 degrees centigrade for 48h . The final pH values of suspension were measured . RESULTS: Fluoride had best inhibitory effect on S.mutans Ingbritt in low concentrations, whereas other 3 weak acids, benzoate, citric acid and malic acid could inhibit S.mutans Ingbritt in a higher concentrations . The order of effectiveness was fluoride>malic acid>benzoate>citric acid . CONCLUSION: Fluoride has the best inhibitory effect on S.mutans producing acid ability . Though weak acids benzoate, malic acid and citric acid in high concentration had different inhibitory effects on S.mutans Ingbritt, however high concentration of organic acids can make enamel demineralization.

Protein Eng, 2003 Dec, 16(12), 1107 - 13
Isolation from phage display libraries of lysine-deficient human epidermal growth factor variants for directional conjugation as targeting ligands; Bach M et al.; Ligand-targeted anticancer therapeutics represent an opportunity for the selective and efficient delivery of drugs to tumours . The chemical coupling of ligands to drugs or drug carrier systems is, however, often hampered by the presence of multiple reactive groups within the ligand, for example, epsilon-NH(2) groups in lysine side chains . In this paper, we describe the isolation by phage display of human epidermal growth factor (EGF) variants without lysine and a reduced number of arginine residues . The selection on A431 carcinoma cells also revealed that R41 is indispensable for EGF binding activity as all EGF variants contained an arginine residue at this position . One EGF variant (EGFm1) with K28Q, R45S, K48S and R53S mutations was expressed in bacteria and showed an identical binding activity as wild-type EGF . EGFm1 could be labelled with fluorescein isothiocyanate demonstrating the accessibility of the N-terminal amino group for coupling reagents . Furthermore, coupling of EGFm1 to PEGylated liposomes resulted in target cell-specific binding and internalization of the liposomes . These human EGF variants should be advantageous for the generation of anticancer therapeutics targeting the EGF receptor, which is overexpressed by a wide variety of different tumours.

J Am Chem Soc, 2004 Mar 3, 126(8), 2378 - 85
Conservation of mechanism in three chorismate-utilizing enzymes; He Z et al.; Chorismate is the end-product of the shikimate pathway for biosynthesis of carbocyclic aromatic compounds in plants, bacteria, fungi, and some parasites . Anthranilate synthase (AS), 4-amino-4-deoxychorismate synthase (ADCS), and isochorismate synthase (IS) are homologous enzymes that carry out the initial transformations on chorismate in the biosynthesis of tryptophan, p-aminobenzoate, and enterobactin, respectively, and are expected to share a common mechanism . Poor binding to ADCS of two potential transition state analogues for addition of a nucleophile to C6 of chorismate implies that it, like AS and IS, initiates reaction by addition of a nucleophile to C2 . Molecular modeling based on the X-ray structures of AS and ADCS suggests that the active site residue K274 is the nucleophile employed by ADCS to initiate the reaction, forming a covalent intermediate . The K274A and K274R mutants were shown to have 265- and 640-fold reduced k(cat) values when PabA (the cognate amidotransferase) + glutamine are used as the nitrogen source . Under conditions of saturating chorismate and NH(4)(+), ADCS and the K274A mutant have identical k(cat) values, suggesting the participation of NH(4)(+) as a rescue agent . Such participation was confirmed by the buildup of 2-amino-2-deoxyisochorismate in the reactions of the K274A mutant but not ADCS, when either NH(4)(+) or PabA + glutamine is used as the nitrogen source . Additionally, the inclusion of ethylamine in the reactions of K274A yields the N-ethyl derivative of 2-amino-2-deoxyisochorismate . A unifying mechanism for AS, ADCS, and IS entailing nucleophile addition to C2 of chorismate in an S(N)2' ' process is proposed.

Quintessence Int, 2003 Oct, 34(9), 678 - 85
New device for selective dentin caries removal; Boston DW; OBJECTIVES: To develop prototype rotary selective dentin caries excavators and to demonstrate their ability to remove only carious dentin in extracted teeth . METHOD AND MATERIALS: Milled polymer prototype and formed wire loop prototype burs were made . They were tested on normal dentin with standardized force application and compared to carbide burs for ability to cut by weighing three extracted teeth at pre- and postcutting for each prototype version . They were tested on carious dentin of three teeth for each prototype version . The resulting excavated surfaces were analyzed with dentin caries dye, the teeth were decalcified and examined histologically, and the used prototypes were examined in light and scanning electron microscopes . RESULTS: For both prototypes, noncarious teeth did not lose weight from prototype instrumentation, but each lost 9 or 10 mg after instrumentation with the control carbide bur . Both prototypes quickly removed carious dentin in each of the carious teeth until a definitive cutting endpoint was reached . The excavated surfaces from the milled polymer prototypes did not stain with caries dye, but those from the wire loop prototypes stained lightly in some areas . In the light microscope, all sections of excavated carious teeth were bacteria-free . Light and scanning electron microscope images of the prototypes revealed scratch patterns on the wire loop prototypes and deformation of blades on the polymer prototypes . CONCLUSION: Both prototypes removed carious dentin but did not remove normal dentin in the extracted teeth used in this study . Further development is indicated.

Arch Insect Biochem Physiol, 2004 Mar, 55(3), 153 - 68
Dietary phosphorus affects the growth of larval Manduca sexta; Perkins MC et al.; Although phosphorus has long been considered an important factor in the growth of diverse biota such as bacteria, algae, and zooplankton, insect nutrition has classically focused on dietary protein and energy content . However, research in elemental stoichiometry has suggested that primary producer biomass has similar N:P ratios in aquatic and terrestrial systems, and phosphorus-rich herbivores in freshwater systems frequently face phosphorus-limited nutritional conditions . Therefore, herbivorous insects should also be prone to phosphorus limitation . We tested this prediction by rearing Manduca sexta larvae on artificial and natural (Datura wrightii leaves) diets containing varying levels of phosphorus (approximately 0.20, 0.55, or 1.2% phosphorus by dry weight) . For both artificial and natural diets, increased dietary phosphorus significantly increased growth rates and body phosphorus contents, and shortened the time to the final instar molt . Caterpillars did not consistently exhibit compensatory feeding for phosphorus on either type of diet . The growth and body phosphorus responses were not explicable by changes in amounts of potassium or calcium, which co-varied with phosphorus in the diets . Concentrations of phosphorus in D . wrightii leaves collected in the field varied over a range in which leaf phosphorus is predicted to affect M . sexta's growth rates . These results suggest that natural variation in dietary phosphorus is likely to affect the growth rate and population dynamics of M . sexta, and perhaps larval insects more generally .

MMWR Morb Mortal Wkly Rep, 2004 Feb 20, 53(6), 131 - 2
Fatal case of unsuspected pertussis diagnosed from a blood culture--Minnesota, 2003; Centers for Disease Control and Prevention (CDC); Pertussis (i.e., whooping cough) is a prolonged cough illness caused by the bacteria Bordetella pertussis and associated typically with an inspiratory "whoop," paroxysmal cough, and posttussive vomiting . B . pertussis can cause severe illness or death, especially in infants who have not completed their pertussis vaccination series . Adolescents (i.e., persons aged 13-17 years), adults, and recently vaccinated persons often report atypical symptoms, resulting in delay of recognition and creation of infectious reservoirs for further transmission . In 2003, the Minnesota Department of Health (MDH) investigated a fatal case of unsuspected B . pertussis infection in an elderly adult . This report summarizes the case investigation, which documents the rare isolation of B . pertussis from blood and underscores the need for clinicians to consider pertussis infection in adolescents and adults who have a prolonged cough illness.

J Dent Res, 2004 Mar, 83(3), 216 - 21
Collagen degradation by host-derived enzymes during aging; Pashley DH et al.; Incompletely infiltrated collagen fibrils in acid-etched dentin are susceptible to degradation . We hypothesize that degradation can occur in the absence of bacteria . Partially demineralized collagen matrices (DCMs) prepared from human dentin were stored in artificial saliva . Control specimens were stored in artificial saliva containing proteolytic enzyme inhibitors, or pure mineral oil . We retrieved them at 24 hrs, 90 and 250 days to examine the extent of degradation of DCM . In the 24-hour experimental and 90- and 250-day control specimens, we observed 5- to 6-microm-thick layers of DCM containing banded collagen fibrils . DCMs were almost completely destroyed in the 250-day experimental specimens, but not when incubated with enzyme inhibitors or mineral oil . Functional enzyme analysis of dentin powder revealed low levels of collagenolytic activity that was inhibited by protease inhibitors or 0.2% chlorhexidine . We hypothesize that collagen degradation occurred over time, via host-derived matrix metalloproteinases that are released slowly over time.

J Exp Med, 2004 Mar 1, 199(5), 649 - 59 Epub 2004 Feb 23.
Diacylated sulfoglycolipids are novel mycobacterial antigens stimulating CD1-restricted T cells during infection with Mycobacterium tuberculosis; Gilleron M et al.; Mycobacterial lipids comprise a heterogeneous group of molecules capable of inducing T cell responses in humans . To identify novel antigenic lipids and increase our understanding of lipid-mediated immune responses, we established a panel of T cell clones with different lipid specificities . Using this approach we characterized a novel lipid antigen belonging to the group of diacylated sulfoglycolipids purified from Mycobacterium tuberculosis . The structure of this sulfoglycolipid was identified as 2-palmitoyl or 2-stearoyl-3-hydroxyphthioceranoyl-2'-sulfate-alpha-alpha'-D-trehalose (Ac2SGL) . Its immunogenicity is dependent on the presence of the sulfate group and of the two fatty acids . Ac2SGL is mainly presented by CD1b molecules after internalization in a cellular compartment with low pH . Ac2SGL-specific T cells release interferon gamma, efficiently recognize M . tuberculosis-infected cells, and kill intracellular bacteria . The presence of Ac2SGL-responsive T cells in vivo is strictly dependent on previous contact with M . tuberculosis, but independent from the development of clinically overt disease . These properties identify Ac2SGL as a promising candidate to be tested in novel vaccines against tuberculosis.

Paediatr Respir Rev, 2004, 5 Suppl A, S197 - 200
Viral pneumoniae in children: incidence and aetiology; Sinaniotis CA; Community-acquired pneumonia remains a common and serious illness, which affects children of all age groups . The spectrum of causative organisms is wide and it differs according to the age of the patients . With the advent of new and improved diagnostic techniques our understanding of the aetiology of the disease has been improved considerably . Viruses have been shown to cause up to 90% of pneumonias, especially in the first year of life, with the respiratory syncytial virus to be the most important pathogen and this percentage decreases to approximately 50% by school age . Viral pneumonias frequently are complicated by bacterial infections and mixed infections are identified in 30% of the cases . The precise role of viruses and bacteria in these cases, remains to be clarified.

Lab Anim, 2004 Jan, 38(1), 25 - 37
The bedding of laboratory animals as a source of airborne contaminants; Kaliste E et al.; In work environments with laboratory animals, the bedding of animals binds the excreta as well as other compounds originating from the animals and their environment . These may be generated into the ambient air when the personnel handle bedding in different procedures . This study compares the dustiness of different types of six clean and four soiled beddings from rat or mouse cages . The dust generation of clean bedding varied from <1 to 25 mg/m(3) . When used in the cages of rats or mice for 4 days, the dust concentration of the beddings decreased, increased or stayed the same, depending on the type of bedding and animal species . A decrease in dustiness was, however, more common . The levels in the soiled beddings varied from <1 to 8.6 mg/m(3) . In the case of the aspen chip bedding, the contents of bedding used in mouse, rat or rabbit cages were analysed for mesophilic bacteria and fungi, mycobacteria and endotoxins . All of these contaminants were variably found in the bedding samples, the maximal concentrations for bacteria were >6 500 000 colony-forming units (cfu)/g, for fungi 212 000 cfu/g, and for endotoxins 6500 ng/g (81 000 EU/g) . The results showed that the bedding of laboratory animals may contain biologically effective compounds, and that these may be distributed into the ambient air depending on the characteristics of the bedding material . The dustiness of different bedding types is an important factor affecting the amount and quality of the occupational exposure of the personnel to airborne contaminants.

Biochemistry, 2004 Mar 2, 43(8), 2279 - 87
Resonance Raman spectroscopy reveals the origin of an intermediate wavelength form in photoactive yellow protein; El-Mashtoly SF et al.; Photoactive yellow protein (PYP) is a bacterial blue light receptor containing a 4-hydroxycinnamyl chromophore, and its absorption maximum is 446 nm . In a dark state, the hydroxyl group of the chromophore is deprotonated and forms hydrogen bonds with Tyr42 and Glu46 . Either removal of a hydrogen bond with Tyr42 or addition of chaotropes such as thiocyanate produces a blue-shifted species called an intermediate wavelength form, in which absorption maximum ranges from 355 to 400 nm . To examine the structural origin of the intermediate wavelength form, we have performed resonance Raman investigations of wild-type PYP and some mutants (Tyr42 --> Ala, Tyr42 --> Phe, Glu46 --> Gln, and Thr50 --> Val) in the presence or absence of potassium thiocyanate . These studies show that the chromophore of the intermediate wavelength form is protonated, implying an increase in a pK(a) of the chromophore . Hence, the removal of the hydrogen bond between Tyr42 and chromophore or partial protein denaturation in the presence of thiocyanate results in a spectral blue-shift . Quantum chemical calculations based on density functional theory further support the idea that the pK(a) of the chromophore is increased by removing a hydrogen bond or by increasing the dielectric constant in the vicinity of the chromophore.

Biochemistry, 2004 Mar 2, 43(8), 2159 - 66
Cholesterol superlattice modulates the activity of cholesterol oxidase in lipid membranes; Wang MM et al.; Here, the interplay between membrane cholesterol lateral organization and the activity of membrane surface-acting enzymes was addressed using soil bacteria cholesterol oxidase (COD) as a model . Specifically, the effect of the membrane cholesterol mole fraction on the initial rate of cholesterol oxidation catalyzed by COD was investigated at 37 degrees C using cholesterol/1-palmitoyl-2-oleoyl-l-alpha-phosphatidylcholine (POPC) large unilamellar vesicles (LUVs, approximately 800 nm in diameter) . In the three concentration ranges examined (18.8-21.2, 23.6-26.3, and 32.2-34.5 mol % cholesterol), the initial activity of COD changed with cholesterol mole fraction in a biphasic manner, exhibiting a local maximum at 19.7, 25.0, and 33.4 mol % . Within the experimental errors, these mole fractions agree with the critical cholesterol mole fractions (C(r)) (20.0, 25.0, and 33.3) theoretically predicted for maximal superlattice formation . The activity variation with cholesterol content was correlated well with the area of regular distribution (A(reg)) in the plane of the membrane as determined by nystatin fluorescence . A similar biphasic change in COD activity was detected at the critical sterol mole fraction 20 mol % in dehydroergosterol (DHE)/POPC LUVs (approximately 168 nm in diameter) . These results indicate that the activity of COD is regulated by the extent of sterol superlattice for both sterols (DHE and cholesterol) and for a wide range of vesicle sizes (approximately 168-800 nm) . The present work on COD and the previous study on phospholipase A(2) (sPLA(2)) {Liu and Chong (1999) Biochemistry 38, 3867-3873} suggest that the activities of some surface-acting enzymes may be regulated by the extent of sterol superlattice in the membrane in a substrate-dependent manner . When the substrate is a sterol, as it is with COD, the enzyme activity reaches a local maximum at C(r) . When phospholipid is the substrate, the minimum activity is at C(r), as is the case with sPLA(2) . Both phenomena are in accordance with the sterol superlattice model and manifest the functional importance of membrane cholesterol content.

Eur Respir J, 2004 Feb, 23(2), 224 - 31
Genetic background affects susceptibility in nonfatal pneumococcal bronchopneumonia; Preston JA et al.; A nonfatal pneumococcal lung infection model was required to investigate immune responses during recovery, and the interaction of other diseases subsequent to infection . A murine model of nonfatal pneumococcal lung infection was developed and the effect of genetic background on susceptibility was determined in BALB/c and C57BL/6 mice . Bacteria colonised the lungs and mice developed mild clinical illness with pathophysiology similar to human bronchopneumonia . Recovery was associated with immune cell influx, which cleared bacteria but induced tissue damage characteristic of pneumococcal bronchopneumonia . After clearance, immune cell populations returned to normal and tissues appeared less inflamed . Although bacterial exposure and clearance were similar, the extent of immune cell influx and tissue damage differed significantly . Larger numbers of neutrophils and lymphocytes entered lung tissue and the affected area was greater in BALB/c compared with C57BL/6 mice . An inflammatory basis for differences was determined with greater levels of phagocytosis and oxidative burst observed in BALB/c mice . C57BL/6 mice cleared the low inoculum with a reduced immune response; however, C57BL/6 mice are more susceptible to larger inocula, which overwhelms the immune system . These different susceptibilities result from a greater inflammatory response in BALB/c compared with C57BL/6 mice.

Can J Vet Res, 2004 Jan, 68(1), 33 - 41
Adherence of actinobacillus pleuropneumoniae serotype 1 to swine buccal epithelial cells involves fibronectin; Hamer-Barrera R et al.; The swine pathogen Actinobacillus pleuropneumoniae serotype 1 was investigated for its ability to adhere to swine, rat, and human buccal epithelial cells (BEC) . The highest number of bacteria adhered was to swine BEC . This binding ability was affected by heating, extreme pH, treatment with sodium dodecyl sulfate, ethylenediamine tetra-acetate, or periodate, and proteolysis, suggesting that cell-surface glycoproteins participate in adherence and that adherence is based mostly on ionic interactions . Mannose and swine fibronectin may play a direct role in this interaction . Convalescent-phase serum from naturally infected pigs inhibited the adhesion . There was a correlation between bacterial pathogenicity as well as host specificity and the capacity for adherence to swine BEC . Adhesion to swine BEC provides a convenient method to study in vitro the adherence of A . pleuropneumoniae and other pathogens of the pig respiratory tract.

FEMS Microbiol Lett, 2004 Feb 9, 231(1), 1 - 12
Brucella pathogenesis, genes identified from random large-scale screens; Delrue RM et al.; Pathogenicity islands, specialized secretion systems, virulence plasmids, fimbriae, pili, adhesins, and toxins are all classical bacterial virulence factors . However, many of these factors, though widespread among bacterial pathogens, are not necessarily found among bacteria that colonize eukaryotic cells in a pathogenic/symbiotic relationship . Bacteria that form these relationships have developed other strategies to infect and grow in their hosts . This is particularly true for Brucella and other members of the class Proteobacteria . Thus far the identification of virulence factors for Brucella has been largely dependent on large-scale screens and testing in model systems . The genomes of the facultative intracellular pathogens Brucella melitensis and Brucella suis were sequenced recently . This has identified several more potential virulence factors for Brucella that were not found in large screens . Here, we present an overall view of Brucella virulence by compiling virulence data from the study of 184 attenuated mutants.

Mov Disord, 2004 Feb, 19(2), 220 - 2
Cerebrospinal fluid analysis for Whipple's disease in patients with progressive supranuclear palsy; Pezzella FR et al.; The clinical picture of neurological involvement in Whipple's disease (WD) may resemble progressive supranuclear palsy (PSP) . We looked for WD pathogen DNA in the cerebrospinal fluid of 9 patients with a clinical diagnosis of PSP . The analysis was negative for all samples, showing that WD is not commonly involved in the aetiopathogenesis of PSP.

Cardiovasc Drug Rev, 2004 Spring, 22(1), 7 - 16
Agmatine signaling: odds and threads; Berkels R et al.; Agmatine is a metabolite of L-arginine . It is formed by the decarboxylation of L-arginine via arginine decarboxylase in bacteria, plants and mammals . It is becoming clear that it has multiple physiological functions as a potential transmitter . Agmatine binds to alpha2-adrenoceptors and to imidazoline binding sites . It blocks NMDA receptors and other ligand-gated cation channels . It also inhibits nitric oxide synthase, induces release of peptide hormones and antizyme and plays a role during cell proliferation by interacting with the generation and transport of polyamines . Although the precise function of endogenously released agmatine is presently still unclear, this review will summarize several aspects concerning the biological function of agmatine.

Microbiol Immunol, 2004, 48(2), 125 - 30
Identification of the Coxiella sp . detected from Haemaphysalis longicornis ticks in Korea; Lee JH et al.; Two Haemaphysalis longicornis ticks were found positive in PCR assay of com-1 gene to detect Coxiella burnetii DNA from 100 ticks . The nucleotide sequences of com-1 and 16S rRNA gene were determined from 2 ticks and compared with those of other C . burnetii strains . The results suggest that H . longicornis harbor Coxiella sp . bacteria in Korea . Furthermore, icd, cbhE', and cbbE' genes are C . burnetii specific genes whereas com-1 gene is Coxiella genus specific gene . This study gives the first documentation to prove the existence of Coxiella sp . in tick collected in Korea.

J Biol Chem, 2004 Apr 30, 279(18), 18511 - 20 Epub 2004 Feb 21.
Single-stranded breaks in DNA but not oxidative DNA base damages block transcriptional elongation by RNA polymerase II in HeLa cell nuclear extracts; Kathe SD et al.; Transcription and repair of many DNA helix-distorting lesions such as cyclobutane pyrimidine dimers have been shown to be coupled in cells across phyla from bacteria to humans . The signal for transcription-coupled repair appears to be a stalled transcription complex at the lesion site . To determine whether oxidative DNA lesions can block correctly initiated human RNA polymerase II, we examined the effect of site-specifically introduced oxidative damages on transcription in HeLa cell nuclear extracts . We found that transcription was blocked by single-stranded breaks, common oxidative DNA lesions, when present in the transcribed strand of the transcription template . Cyclobutane pyrimidine dimers, which have been previously shown to block transcription both in vitro and in vivo, also blocked transcription in the HeLa cell nuclear transcription assay . In contrast, the oxidative DNA base lesions, 8-oxoguanine, 5-hydroxycytosine, and thymine glycol did not inhibit transcription, although pausing was observed with the thymine glycol lesion . Thus, DNA strand breaks but not oxidative DNA base damages blocked transcription by RNA polymerase II.

Infect Immun, 2004 Mar, 72(3), 1733 - 45
Attenuation of late-stage disease in mice infected by the Mycobacterium tuberculosis mutant lacking the SigF alternate sigma factor and identification of SigF-dependent genes by microarray analysis; Geiman DE et al.; The Mycobacterium tuberculosis alternate sigma factor, SigF, is expressed during stationary growth phase and under stress conditions in vitro . To better understand the function of SigF we studied the phenotype of the M . tuberculosis DeltasigF mutant in vivo during mouse infection, tested the mutant as a vaccine in rabbits, and evaluated the mutant's microarray expression profile in comparison with the wild type . In mice the growth rates of the DeltasigF mutant and wild-type strains were nearly identical during the first 8 weeks after infection . At 8 weeks, the DeltasigF mutant persisted in the lung, while the wild type continued growing through 20 weeks . Histopathological analysis showed that both wild-type and mutant strains had similar degrees of interstitial and granulomatous inflammation during the first 12 weeks of infection . However, from 12 to 20 weeks the mutant strain showed smaller and fewer lesions and less inflammation in the lungs and spleen . Intradermal vaccination of rabbits with the M . tuberculosis DeltasigF strain, followed by aerosol challenge, resulted in fewer tubercles than did intradermal M . bovis BCG vaccination . Complete genomic microarray analysis revealed that 187 genes were relatively underexpressed in the absence of SigF in early stationary phase, 277 in late stationary phase, and only 38 genes in exponential growth phase . Numerous regulatory genes and those involved in cell envelope synthesis were down-regulated in the absence of SigF; moreover, the DeltasigF mutant strain lacked neutral red staining, suggesting a reduction in the expression of envelope-associated sulfolipids . Examination of 5'-untranslated sequences among the downregulated genes revealed multiple instances of a putative SigF consensus recognition sequence: GGTTTCX(18)GGGTAT . These results indicate that in the mouse the M . tuberculosis DeltasigF mutant strain persists in the lung but at lower bacterial burdens than wild type and is attenuated by histopathologic assessment . Microarray analysis has identified SigF-dependent genes and a putative SigF consensus recognition site.

Infect Immun, 2004 Mar, 72(3), 1580 - 6
Dissolved oxygen levels alter gene expression and antigen profiles in Borrelia burgdorferi; Seshu J et al.; The Lyme disease spirochete, Borrelia burgdorferi, encounters many environmental signals as it cycles between the arthropod vector and mammalian hosts, including temperature, pH, and other host factors . To test the possibility that dissolved oxygen modulates gene expression in B . burgdorferi, spirochetes were exposed to differential levels of dissolved oxygen, and distinct alterations were observed at both the transcriptional and translational levels . Specifically NapA, a Dps/Dpr homologue involved in the oxidative stress response in other bacteria, was reduced when B . burgdorferi was grown under oxygen-limiting conditions . In contrast, several immunoreactive proteins were altered when tested with infection-derived sera from different hosts . Specifically, OspC, DbpA, and VlsE were synthesized at greater levels when cells were grown in limiting oxygen, whereas VraA was reduced . The levels of oxygen in the medium did not affect OspA production . Real-time reverse transcription-PCR analysis of RNA isolated from infectious isolates of strains B31 and cN40 indicated that the expression of ospC, dbpA, and vlsE increased while napA expression decreased under dissolved-oxygen-limiting conditions, whereas flaB was not affected . The reverse transcription-PCR results corroborated the immunoblot analyses and indicated that the increase in OspC, DbpA, and VlsE was due to regulation at the transcriptional level of the genes encoding these antigens . These results indicate that dissolved oxygen modulates gene expression in B . burgdorferi and imply that the redox environment may be an additional regulatory cue that spirochetes exploit to adapt to the disparate niches that they occupy in nature.

Infect Immun, 2004 Mar, 72(3), 1519 - 29
Helicobacter pylori and complex gangliosides; Roche N et al.; Recognition of sialic acid-containing glycoconjugates by the human gastric pathogen Helicobacter pylori has been repeatedly demonstrated . To investigate the structural requirements for H . pylori binding to complex gangliosides, a large number of gangliosides were isolated and characterized by mass spectrometry and proton nuclear magnetic resonance . Ganglioside binding of sialic acid-recognizing H . pylori strains (strains J99 and CCUG 17874) and knockout mutant strains with the sialic acid binding adhesin SabA or the NeuAcalpha3Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta-binding neutrophil-activating protein HPNAP deleted was investigated using the thin-layer chromatogram binding assay . The wild-type bacteria bound to N-acetyllactosamine-based gangliosides with terminal alpha3-linked NeuAc, while gangliosides with terminal NeuGcalpha3, NeuAcalpha6, or NeuAcalpha8NeuAcalpha3 were not recognized . The factors affecting binding affinity were identified as (i) the length of the N-acetyllactosamine carbohydrate chain, (ii) the branches of the carbohydrate chain, and (iii) fucose substitution of the N-acetyllactosamine core chain . While the J99/NAP(-) mutant strain displayed a ganglioside binding pattern identical to that of the parent J99 wild-type strain, no ganglioside binding was obtained with the J99/SabA(-) mutant strain, demonstrating that the SabA adhesin is the sole factor responsible for the binding of H . pylori bacterial cells to gangliosides.

Infect Immun, 2004 Mar, 72(3), 1463 - 9
Cross-species surface display of functional spirochetal lipoproteins by recombinant Borrelia burgdorferi; Zuckert WR et al.; Surface-exposed lipoproteins of relapsing fever (RF) and Lyme borreliosis Borrelia spirochetes mediate certain interactions of the bacteria with their arthropod and vertebrate hosts . RF spirochetes such as Borrelia hermsii serially evade the host's antibody response by multiphasic antigenic variation of Vsp and Vlp proteins . Furthermore, the expression of Vsp1 and Vsp2 by Borrelia turicatae is associated with neurotropism and higher blood densities, respectively . In contrast to RF Borrelia species, the Lyme borreliosis spirochete Borrelia burgdorferi is amenable to genetic manipulation . To facilitate structure-function analyses of RF surface lipoproteins, we used recombinant plasmids to introduce full-length vsp1 and vsp2 as well as two representative vlp genes into B . burgdorferi cells . Recombinant B . burgdorferi cells constitutively expressed the proteins under the control of the B . burgdorferi flaB promoter . Antibody and protease accessibility assays indicated proper surface exposure and folding . Expression of Vsp1 and Vsp2 conferred glycosaminoglycan binding to recombinant B . burgdorferi cells that was similar to that observed with purified recombinant proteins and B . turicatae expressing native Vsp . These data demonstrate that the lipoprotein modification and export mechanisms in the genus Borrelia are conserved . They also validate the use of recombinant B . burgdorferi in studies of surface lipoprotein structure-function and the biogenesis of spirochete membranes.

Front Biosci, 2004 May 01, 9, 1398 - 411
Metabolism of diadenosine tetraphosphate (Ap4A) and related nucleotides in plants; review with historical and general perspective; Guranowski A; This review presents our knowledge of potential biochemical conversions of minor mononucleotides, such as adenosine-5'-tetraphosphate (p4A) and adenosine-5'-pentaphosphate (p5A), and dinucleotides, such as diadenosine-5',5"'-P1,P3-triphosphate (Ap3A) and diadenosine-5',5"'-P1,P4-tetraphosphate (Ap4A), in plants . Although the occurrence of p4A, Ap3A and/or Ap4A has been demonstrated in various bacteria, fungi and animals, identification of these compounds in plants has not been reported as yet . However, the ubiquity of both the compounds and enzymes that can synthesize them (certain ligases and transferases), the demonstration that certain plant ligases can synthesize pnAs and ApnNs in vitro, and the existence in plants of specific and nonspecific degradative enzymes strongly suggest that these various pnNs and NpnN's do indeed occur and play a biological role in plant cells . In fact, some of the plant enzymes involved in the synthesis and degradation of these minor mono- and dinucleotides have been studied even more thoroughly than their counterparts from other organisms.

Annu Rev Physiol, 2004, 66, 385 - 417
Oral rehydration therapy: new explanations for an old remedy; Rao MC; Diarrheal diseases are among the most devastating illnesses globally, but the introduction of oral rehydration therapy has reduced mortality due to diarrhea from >5 million children, under the age of 5, in 1978 to 1.3 million in 2002 . Variations of this simple therapy of salts and sugars are prevalent in traditional remedies in cultures world-wide, but only in the past four decades have the scientific bases for these remedies begun to be elucidated . This review aims to provide a broad understanding of the cellular basis of oral rehydration therapy . The features integral to the success of oral rehydration therapy are active glucose transport in the small intestine, commensal bacteria, and short-chain fatty acid transport in the colon . The review examines these processes and their regulation and considers new approaches that might supplement oral rehydration therapy in controlling diarrheal diseases.

Methods Mol Biol, 2004, 263, 281 - 92
Flow cytometric analysis of fluorescence resonance energy transfer: a tool for high-throughput screening of molecular interactions in living cells; Chan FK et al.; The study of cellular processes has been facilitated by the use of methods to detect molecular associations both in vivo and in vitro . An invaluable tool to study molecular associations associated with dynamic processes in living cells utilizes the phenomenon of fluorescence resonance energy transfer (FRET), together with selected fluorophores that are attached to molecules of interest . Many reports have utilized fluorophores conjugated to antibodies for FRET pairs . However, these methods are restricted to extracellular molecules and dependent upon the availability of appropriate antibodies . The recent development of green fluorescent protein (GFP) variants suitable for FRET has expanded the utility of this methodology by permitting the study of intracellular as well as extracellular processes . Combining FRET with flow cytometric analysis results in a powerful high-throughput assay for molecular associations . This article details the use of green fluorescent protein (GFP) mutants cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) to measure the association of the signaling component TRAF2 with the TNFR-2 receptor to illustrate the versatility of this methodology.

Methods Mol Biol, 2004, 263, 219 - 38
Multiparameter flow cytometry of fluorescent protein reporters; Hawley TS et al.; Reporters based on the green fluorescent protein (GFP) from the jellyfish Aequorea victoria and GFP-like proteins from other marine organisms provide valuable tools to monitor gene transfer and expression noninvasively in living cells . Stable cell lines were generated from the Sp2/0-Ag14 hybridoma that express up to three spectral enhanced versions of GFP, the enhanced cyan fluorescent protein (ECFP), the enhanced green fluorescent protein (EGFP), and the enhanced yellow fluorescent protein (EYFP), and/or a variant of the Discosoma coral red fluorescent protein (DsRed) . The panel of lines was used to demonstrate a flow cytometric procedure for simultaneous analysis of all four fluorescent proteins that utilizes dual-laser excitation at 488 nm and 407 nm . Additional schemes for simultaneous detection of two, three or four of these fluorescent proteins are also presented.

J Biol Chem, 2004 May 14, 279(20), 21533 - 42 Epub 2004 Feb 19.
Regulation of immature protein dynamics in the endoplasmic reticulum; Kamada A et al.; The quality of nascent protein folding in vivo is influenced by the microdynamics of the proteins . Excessive collisions between proteins may lead to terminal misfolding, and the frequency of protein interactions with molecular chaperones determines their folding rates . However, it is unclear how immature protein dynamics are regulated . In this study, we analyzed the diffusion of immature tyrosinase in the endoplasmic reticulum (ER) of non-pigmented cells by taking advantage of the thermal sensitivity of the tyrosinase . The diffusion of tyrosinase tagged with yellow fluorescence protein (YFP) in living cells was directly measured using fluorescent correlation spectroscopy . The diffusion of folded tyrosinase in the ER of cells treated with brefeldin A, as measured by fluorescent correlation spectroscopy, was critically affected by the expression level of tyrosinase-YFP . Under defined conditions in which random diffusional motion of folded protein was allowed, we found that the millisecond-order diffusion rate observed for folded tyrosinase almost disappeared for the misfolded molecules synthesized at a nonpermissive high temperature . This was not because of enhanced aggregation at the high temperature, as terminally misfolded tyrosinase synthesized in the absence of calnexin interactions showed comparable, albeit slightly slower, diffusion . Yet, the thermally misfolded tyrosinase was not immobilized when measured by fluorescence recovery after photobleaching . In contrast, terminally misfolded tyrosinase synthesized in cells in which alpha-glucosidases were inhibited showed extensive immobilization . Hence, we suggest that the ER represses random fluctuations of immature tyrosinase molecules while preventing their immobilization.

FEMS Microbiol Rev, 2004 Feb, 28(1), 3 - 24
Translocation of proteins across archaeal cytoplasmic membranes; Pohlschroder M et al.; All cells need to transport proteins across hydrophobic membranes . Several mechanisms have evolved to facilitate this transport, including: (i) the universally-conserved Sec system, which transports proteins in an unfolded conformation and is thought to be the major translocation pathway in most organisms and (ii) the Tat system, which transports proteins that have already obtained some degree of tertiary structure . Here, we present the current understanding of these processes in the domain Archaea, and how they compare to the corresponding pathways in bacteria and eukaryotes.

Med Hypotheses, 2004, 62(3), 354 - 7
Hypothesis links emergence of chloroquine-resistant malaria and other intracellular pathogens and suggests a new strategy for treatment of diseases caused by intracellular parasites; Parris GE; Chloroquine and related anti-malarial drugs appear to promote apoptosis in T-cells by suppressing NF-kappa-B, which enhances the expression of anti-apoptotic proteins (e.g., Bcl-2) . Thus, chloroquine has found applications in autoimmune diseases where it apparently facilitates apoptosis of abnormally persistent T-cell clones . The mode of action of chloroquine in prevention of malaria is not known, but it may be to minimize replication of the parasite in the liver cells, which occurs before invasion of the erythrocytes, by facilitating premature apoptosis of the infected host cells . After introduction of chloroquine in the 1950s world-wide for prophylactic use, chloroquine-resistant malaria emerged . Here it is hypothesized that concurrent with emergence of chloroquine-resistant malaria (presumably with enhanced anti-apoptotic capabilities), other intracellular parasites have evolved to enhance their ability to prevent apoptosis in host cells . Two examples of viral diseases that have emerged from areas of high incidence of chloroquine-resistant malaria are AIDS from HIV and SARS from coronavirus . The hypothesis holds that prophylactic exposure to pro-apoptotic chloroquine drugs caused natural selection for strains of viruses and other parasites that have enhanced anti-apoptotic abilities . When transmitted to host organisms that are not under the influence of the pro-apoptotic drug, the new "anti-apoptotic" strains may cause unexpected diseases . In the case of SARS, the coronavirus appears to have accessed a new niche where it proves to be lethal to its host . In the case of AIDS, the HIV (which has had a long-term symbiotic relationship with primates) has run amuck because the infected cells are now substantially more tolerant to the toxins (i.e., resistant to apoptosis) that they secrete than the uninfected bystander cells, which are not unusually resistant to apoptosis . A corollary to the hypothesis is that if the level of resistance to apoptosis in the infected cells were no higher than the level of resistance in the bystander cells, then the infected cells would preferentially kill themselves through apoptosis . It appears that in the case of HIV, the increased resistance to apoptosis is provided by expression of Bcl-2 and suppression of p53 . Hence, drugs that suppresses Bcl-2 or restore p53 function might be effective in restoring the parity of resistance to apoptosis between infected and uninfected cells . Currently, an antisense drug targeting Bcl-2 (G3139/Genasense(TM), Genta, Inc.) is in late-stage cancer trials and may be on the market for those indications in months . It would be interesting to try these drugs against various intracellular parasites including HIV . This approach to prevent or eliminate active infections might be particularly attractive against a range of parasites (virus, bacteria, protozoa, fungus) when safe and effective vaccines are not available.

J Periodontol, 2003 Dec, 74(12), 1803 - 7
Alveolar bone response in an experimental model of renal failure and periodontal disease: a histomorphometric and histochemical study; Mandalunis PM et al.; BACKGROUND: Chronic destructive periodontal disease is characterized by gingival inflammation, periodontal pocket formation, and bacterial plaque that lead to alveolar bone destruction . Polymorphonuclear neutrophil leukocytes (PMNs) are the first line of defense against infection caused by dental plaque bacteria . Renal patients present functional abnormalities of PMN, including impaired chemotaxis, phagocytosis, and intracellular killing of bacteria . In view of the above, the aim of this work was to evaluate the effect of renal failure on bone damaged by periodontal disease using histomorphometric and histochemical parameters . METHODS: Twenty male Wistar rats weighing 250 g were assigned to one of the following four groups: 1) control (no treatment); 2) renal failure (RF); 3) periodontal disease (PD); and 4) renal failure plus periodontal disease (RF+PD) . All the animals were sacrificed 31 days after the onset of the experiment . Mesio-distally oriented sections of the first lower molar were obtained for histomorphometric and histochemical evaluation . RESULTS: Total erosion, active erosion, and total number of tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts were found to be increased in the RF+PD group compared with the PD group . CONCLUSION: Our results demonstrate increased bone resorption in animals with untreated renal failure and periodontal disease, and thus indicate that the release of different factors by inflammatory cells is magnified, accelerating the progression of the disease in this animal model.

Photochem Photobiol, 2004 Jan, 79(1), 55 - 61
Diastereoselective self-aggregation of synthetic 3-(1-hydroxyethyl)-bacteriopyrochlophyll-a as a novel photosynthetic antenna model absorbing near the infrared regions; Kunieda M et al.; 3-Deacetyl-3-(1-hydroxyethyl)bacteriopyrochlorophyll-a (1), 7,8-dihydrobacteriochlorophyll-d possessing 8-ethyl, 12-methyl and 17(4)-phytyl groups, was prepared by modifying naturally occurring bacteriochlorophyll-a . The synthetic 3(1)-epimers were separated by high-performance liquid chromotagraphy, and the absolute configuration at the 3(1)-position was determined by derivatization of 1 to a structurally determined chlorin . A dichloromethane solution of 3(1)R-1 or 3(1)S-1 was diluted with a 1000-fold volume of cyclohexane to give self-aggregation species absorbing light at a near-infrared (NIR) region (<910 nm) . The resulting Qy maximum in 3(1)R-1 was 860 nm and redshifted by 2170 cm(-1) from the monomeric one, whereas epimeric 3(1)S-1 showed a less redshifted peak at ca 800 nm, with a small dimeric band around 740 nm . Such visible spectra indicated that 3(1)R/S-1 formed different supramolecular structures in the self-aggregates . In contrast, self-aggregation of the 7,8-dehydro-compound 2, bacteriochlorophyll-dP, found in natural antennas of photosynthetic green bacteria showed much smaller diastereomeric control . The self-aggregates of 3(1)R-1 absorbing light in the NIR region would be models of intrinsic membraneous light-harvesting complexes 1 in photosynthetic purple bacteria as well as extramembranous antennas in green bacteria.

Photochem Photobiol, 2004 Jan, 79(1), 11 - 20
Fluorescence polarization studies of B-phycoerythrin oriented in polymer film; Frackowiak D et al.; Polarized steady-state fluorescence and fluorescence excitation spectra as well as time-resolved fluorescence for B-phycoerythrin (B-PE) from red algae, Porphyridium cruentum, embedded in polyvinyl stretched films were measured . The lifetimes of polarized fluorescence were analyzed using exponential components and fractal models . The interactions between various chromophores of the pigment-protein complexes investigated were discussed . The anisotropy of fluorescence excitation spectra differs from the anisotropy of absorption spectra and depends on the wavelength of observation . This shows that differently oriented chromophores take part in various paths of excitation energy transfer (ET) or change their excitation into heat with various efficiencies (or both) . Also, analysis of time-resolved fluorescence measured in various spectral regions gives different polarized components of emission . Fractal analysis of lifetimes, done under supposition of the Foerster resonance ET mechanism, suggests different arrangements of energy donors and acceptors for molecules absorbing in different spectral regions . It shows that several fractions of differently oriented "forms" of chromophores exhibiting different spectral properties occur in B-PE complexes . Small changes in the orientation of the chromophores can be followed by modification of the path of excitation energy migration . Based on the results obtained a new reorientational mechanism of the State 1 --> State 2 transition was proposed: Even small conformational modifications of biliproteins, which could be caused in vivo by the change in the conditions of preillumination of bacteria, are able to modify the path of excitation ET . Such a reorientation may be responsible for the change in the partition of biliprotein excitation energy between photosystem II (PSII) and PSI (State 1 --> State 2 transition) . The proposed mechanism needs further verification by the investigation of whole bacteria cells.

Cochrane Database Syst Rev . 2004;(1):CD001480.
Pneumococcal vaccines for preventing otitis media; Straetemans M et al.; BACKGROUND: Acute otitis media (AOM) is one of the most common diseases in early infancy and childhood . Long term effects of recurrent episodes of otitis media, rapid emergence of drug resistant bacteria associated with AOM worldwide and huge estimated direct and indirect annual costs associated with otitis media have emphasized the need for an effective vaccination program to prevent episodes of AOM . OBJECTIVES: The object of this review was to assess the effect of pneumococcal vaccination in preventing AOM in children up to 12 years of age . SEARCH STRATEGY: We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (issue 2, 2003) which contains the Cochrane Acute Respiratory Infection Group's specialised register (30th June 2003), MEDLINE (January 1966 to June 2003), EMBASE (January 1990 to June 2003) and reference lists of all studies and review articles retrieved . We also contacted two vaccine manufacturers and first or corresponding authors of some of the included studies . SELECTION CRITERIA: Randomised controlled clinical trials of pneumococcal vaccination with prevention of AOM as outcome in children aged 12 years or younger and a follow-up of at least six months after vaccination . DATA COLLECTION AND ANALYSIS: Five reviewers independently assessed trial quality and two reviewers extracted data . Two study authors were contacted . MAIN RESULTS: Eight trials on 8-to 14-valent pneumococcal polysaccharide vaccine (PPV) and four trials on 7-to 9-valent pneumococcal conjugate vaccine (PCV) were included . The highest efficacy of PPV was found in children aged 24 months and older: the rate ratio was 0.779 {95% CI: 0.625-0.970} . PPV has little effect on the prevention of AOM in children without documented prior episodes of AOM and only a moderate effect in the group of children with documented AOM episodes prior to vaccination . Pooled results of the four PCV trials in infants vaccinated as early as two months of age and toddlers attending daycare and toddlers with recurrent AOM showed only a small effect on prevention of AOM (rate ratio 0.921; 95% CI: 0.894-0.950) . REVIEWER'S CONCLUSIONS: Based on the currently available results of the effectiveness of pneumococcal vaccination for the prevention of AOM, a large scale use of pneumococcal polysaccharide and conjugate vaccination for this specific indication is not yet recommended . So far, pneumococcal conjugate vaccinations are not indicated in the management of recurrent AOM in toddlers and older children . The results of currently ongoing trials of 9- and 11-valent conjugate vaccines should provide more information as to whether pneumococcal vaccines are more effective in specific high-risk populations like infants and older children with recurrent AOM or immunodeficiency.

Plant Cell, 2004 Mar, 16(3), 672 - 93 Epub 2004 Feb 18.
Identification of multivesicular bodies as prevacuolar compartments in Nicotiana tabacum BY-2 cells; Tse YC et al.; Little is known about the dynamics and molecular components of plant prevacuolar compartments (PVCs) . We have demonstrated recently that vacuolar sorting receptor (VSR) proteins are concentrated on PVCs . In this study, we generated transgenic Nicotiana tabacum (tobacco) BY-2 cell lines expressing two yellow fluorescent protein (YFP)-fusion reporters that mark PVC and Golgi organelles . Both transgenic cell lines exhibited typical punctate YFP signals corresponding to distinct PVC and Golgi organelles because the PVC reporter colocalized with VSR proteins, whereas the Golgi marker colocalized with mannosidase I in confocal immunofluorescence . Brefeldin A induced the YFP-labeled Golgi stacks but not the YFP-marked PVCs to form typical enlarged structures . By contrast, wortmannin caused YFP-labeled PVCs but not YFP-labeled Golgi stacks to vacuolate . VSR antibodies labeled multivesicular bodies (MVBs) on thin sections prepared from high-pressure frozen/freeze substituted samples, and the enlarged PVCs also were indentified as MVBs . MVBs were further purified from BY-2 cells and found to contain VSR proteins via immunogold negative staining . Similar to YFP-labeled Golgi stacks, YFP-labeled PVCs are mobile organelles in BY-2 cells . Thus, we have unequivocally identified MVBs as PVCs in N . tabacum BY-2 cells . Uptake studies with the styryl dye FM4-64 strongly indicate that PVCs also lie on the endocytic pathway of BY-2 cells.

J Bacteriol, 2004 Mar, 186(5), 1565 - 70
Characterization of two methanopterin biosynthesis mutants of Methylobacterium extorquens AM1 by use of a tetrahydromethanopterin bioassay; Rasche ME et al.; An enzymatic assay was developed to measure tetrahydromethanopterin (H(4)MPT) levels in wild-type and mutant cells of Methylobacterium extorquens AM1 . H(4)MPT was detectable in wild-type cells but not in strains with a mutation of either the orf4 or the dmrA gene, suggesting a role for these two genes in H(4)MPT biosynthesis . The protein encoded by orf4 catalyzed the reaction of ribofuranosylaminobenzene 5'-phosphate synthase, the first committed step of H(4)MPT biosynthesis . These results provide the first biochemical evidence for H(4)MPT biosynthesis genes in bacteria.

J Bacteriol, 2004 Mar, 186(5), 1409 - 14
A dominant-negative fur mutation in Bradyrhizobium japonicum; Benson HP et al.; In many bacteria, the ferric uptake regulator (Fur) protein plays a central role in the regulation of iron uptake genes . Because iron figures prominently in the agriculturally important symbiosis between soybean and its nitrogen-fixing endosymbiont Bradyrhizobium japonicum, we wanted to assess the role of Fur in the interaction . We identified a fur mutant by selecting for manganese resistance . Manganese interacts with the Fur protein and represses iron uptake genes . In the presence of high levels of manganese, bacteria with a wild-type copy of the fur gene repress iron uptake systems and starve for iron, whereas fur mutants fail to repress iron uptake systems and survive . The B . japonicum fur mutant, as expected, fails to repress iron-regulated outer membrane proteins in the presence of iron . Unexpectedly, a wild-type copy of the fur gene cannot complement the fur mutant . Expression of the fur mutant allele in wild-type cells leads to a fur phenotype . Unlike a B . japonicum fur-null mutant, the strain carrying the dominant-negative fur mutation is unable to form functional, nitrogen-fixing nodules on soybean, mung bean, or cowpea, suggesting a role for a Fur-regulated protein or proteins in the symbiosis.

Brain Res, 2004 Mar 19, 1001(1-2), 37 - 50
Late onset Tay-Sachs disease in mice with targeted disruption of the Hexa gene: behavioral changes and pathology of the central nervous system; Miklyaeva EI et al.; Tay-Sachs disease is an autosomal recessive neurodegenerative disease resulting from a block in the hydrolysis of GM2 ganglioside, an intermediate in ganglioside catabolism . The mouse model of Tay-Sachs disease (Hexa -/-) has been described as behaviorally indistinguishable from wild type until at least 1 year of age due to a sialidase-mediated bypass of the metabolic defect that reduces the rate of GM2 ganglioside accumulation . In this study, we have followed our mouse model to over 2 years of age and have documented a significant disease phenotype that is reminiscent of the late onset, chronic form of human Tay-Sachs disease . Onset occurs at 11-12 months of age and progresses slowly, in parallel with increasing storage of GM2 ganglioside . The disease is characterized by hind limb spasticity, weight loss, tremors, abnormal posture with lordosis, possible visual impairment, and, late in the disease, muscle weakness, clasping of the limbs, and myoclonic twitches of the head . Immunodetection of GM2 ganglioside showed that storage varies widely in different regions, but is most intense in pyriform cortex, hippocampus (CA3 field, subiculum), amygdala, hypothalamus (paraventricular supraoptic, ventromedial and arcuate nuclei, and mammilary body), and the somatosensory cortex (layer V) in 1- to 2-year-old mutant mice . We suggest that the Tay-Sachs mouse model is a phenotypically valid model of disease and may provide for a reliable indicator of the impact of therapeutic strategies, in particular geared to the late onset, chronic form of human Tay-Sachs disease.

Mol Genet Metab, 2004 Mar, 81(3), 225 - 36
Human recombinant mutated forms of the mitochondrial COX assembly Sco2 protein differ from wild-type in physical state and copper binding capacity; Foltopoulou PF et al.; The human Sco2 protein is a cytochrome c oxidase assembly protein that participates in mitochondrial copper pathway, acting downstream of Cox17 protein . In a previous work, we detected mutations in the human SCO2 gene in three unrelated infants with fatal cardioencephalomyopathy and COX deficiency . In this study, full-length processed recombinant wild-type and two mutated forms of hSco2p (w/t-rhSco2p, E140K-rhSco2p, and S225F-rhSco2p) were produced in bacteria as soluble recombinant peptides for the first time and evaluated for differences in their physical state and ability to bind copper . Our data indicate the following: (a) w/t-rhSco2p and S225F-rhSco2p were found to be in a monomeric form in contrast to E140K-rhSco2p that was in a major non-reducible dimer and a minor monomer form; (b) wild-type and mutated rhSco2p exhibited clear differences in their physical conformational state, as shown by circular dichroism and thermal denaturation analyses; (c) copper binding studies showed that E140K-rhSco2p bound markedly less copper while S225F-rhSco2p more than expected as compared to amount of the copper bound with w/t-rhSco2p . rhCox17p served as positive control experiment . These data indicate that S225F and E140K mutations found in the SCO2 gene derived from patients alter the physical conformational state of encoded hSco2p that may disturb the normal copper transport pathway in mitochondria . These findings are valuable for understanding the molecular basis of fatal cardioencephalomyopathy and COX deficiency and for designing appropriate pharmacological interventions.

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