Microb Ecol, 2004 Jan, 47(1), 18 - 29 Quantitative importance, composition, and seasonal dynamics of protozoan communities in polyhaline versus freshwater intertidal sediments; Hamels I et al.; The quantitative importance and composition of protozoan communities was investigated in sandy and silty intertidal sediments of a polyhaline and a freshwater site in the Schelde estuary . Total biomass of the protozoans studied, integrated over the upper 4 cm of the sediment, ranged from 41 to 597 mg C m(-2) and was in the same order of magnitude at the polyhaline and the freshwater intertidal site . Nanoheterotrophs were the dominant protozoans, in terms of both abundance and biomass . Ciliate abundances appeared to be largely determined by physical constraints, namely, the amount of interstitial space and hydrodynamic disturbances . It remains unclear which factors control nanoheterotrophic abundances and biomasses, which showed comparatively little seasonal and between-site fluctuations . Salinity differences were clearly reflected in the protozoan community composition . The dominant role of sessile ciliates is a unique feature of sediments in the freshwater tidal reaches, which can be attributed to the dynamic nature of sedimentation and resuspension processes associated with the maximum turbidity zone . Based on biomass ratios and estimated weight-specific metabolic rates, protozoa possibly accounted for approximately 29 to 96% of the estimated combined metabolic rate of protozoan and metazoan consumers at our sampling stations in late spring/early autumn . The contribution of protozoa to this combined metabolic rate was higher at the sandy than at the silty stations and was mainly accounted for by the nanoheterotrophs . These data emphasize the potential importance of small protozoa in sediments and suggest that protozoa are important components of benthic food webs. J Med Virol, 2004 Sep, 74(1), 180 - 90 Differential expression of immunoregulatory genes in male and female Norway rats following infection with Seoul virus; Klein SL et al.; Males of many species are more susceptible than females to infections caused by parasites, bacteria, fungi, and viruses . Following inoculation with Seoul virus, male rats have more virus present in target organs and shed virus longer than females . The goal of this study was to test the hypothesis that variation in the expression of genes associated with immune function mediates sex differences in hantavirus infection . Using DNA microarrays, we examined changes in gene expression in lung tissue during the early (when animals are viremic and shedding virus; Day 15 post-inoculation (p.i.)) and late (animals have low levels of infectious virus, but high antibody titers; Day 40 p.i.) phases of infection in adult male and female rats . After normalizing the gene expression levels from infected animals to the gene expression levels from same-sex uninfected controls, our data revealed that 1,813 genes were differentially expressed between the sexes during infection . The expression of key transcriptional factors (e.g., eIF-2 alpha, NF-kappa B, IRF-1, NF-IL-6, and STAT6) and genes that encode for proinflammatory (e.g., TNF alpha R, IL-1R, and IL-1RAcP), antiviral (e.g., IFN gamma R and Mx proteins), T cell (e.g., CD3 and TCR), and Ig superfamily (e.g., IgM, IgG, and MHC class I and II) proteins was higher in females than males . Conversely, males had higher expression of heat shock protein genes (e.g., hsp70) suggesting that cellular stress is elevated in males . These data provide candidate genes and cellular pathways that may underlie sex differences in responses to Seoul virus and possibly other hemorrhagic fever viruses. Nat Struct Mol Biol, 2004 Aug, 11(8), 763 - 9 Epub 2004 Jul 18. Nucleosomes facilitate their own invasion; Li G et al.; DNA wrapped in nucleosomes is sterically occluded, creating obstacles for polymerase, regulatory, remodeling, repair and recombination complexes, which require access to the wrapped DNA . How such complexes recognize and gain access to their DNA target sites is not known . Here we report the direct detection of a dynamic equilibrium conformational transition in nucleosomes that greatly increases the distance between the end of the nucleosomal DNA and the histone core . We quantified the equilibrium constant for this transition under physiological conditions . As predicted by these findings, addition of LexA protein to nucleosomes containing the LexA target site drives this conformational equilibrium toward the unwrapped, accessible state, simultaneously allowing stable LexA binding . This inherent property of nucleosomes allows any protein, whether an energy-dependent machine or a passive binder, to gain access even to buried stretches of nucleosomal DNA. Nucleic Acids Res . 2004 Jul 16;32(12):e106. Design and in vivo characterization of self-inactivating human and non-human lentiviral expression vectors engineered for streptogramin-adjustable transgene expression; Mitta B et al.; Adjustable transgene expression is considered key for next-generation molecular interventions in gene therapy scenarios, therapeutic reprogramming of clinical cell phenotypes for tissue engineering and sophisticated gene-function analyses in the post-genomic era . We have designed a portfolio of latest generation self-inactivating human (HIV-derived) and non-human (EIAV-based) lentiviral expression vectors engineered for streptogramin-adjustable expression of reporter (AmyS(DeltaS), EYFP, SAMY, SEAP), differentiation-modulating (human C/EBP-alpha) and therapeutic (human VEGF) transgenes in a variety of rodent (CHO-K1, C2C12) and human cell lines (HT-1080, K-562), human and mouse primary cells (NHDF, PBMC, CD4+) as well as chicken embryos . Lentiviral design concepts include (i) binary systems harboring constitutive streptogramin-dependent transactivator (PIT) and PIT-responsive transgene expression units on separate lentivectors; (ii) streptogramin-responsive promoters (P(PIR8)) placed 5' of desired transgenes; (iii) within modified enhancer-free 3'-long terminal repeats; and (iv) bidirectional autoregulated configurations providing streptogramin-responsive transgene expression in a lentiviral one-vector format . Rigorous quantitative analysis revealed HIV-based direct P(PIR)-transgene configurations to provide optimal regulation performance for (i) adjustable expression of intracellular and secreted product proteins, (ii) regulated differential differentiation of muscle precursor cell lines into adipocytes or osteoblasts and (iii) conditional vascularization fine-tuning in chicken embryos . Similar performance could be achieved by engineering streptogramin-responsive transgene expression into an autoregulated one-vector format . Powerful transduction systems equipped with adjustable transcription modulation options are expected to greatly advance sophisticated molecular interventions in clinically and/or biotechnologically relevant primary cells and cell lines. Microbiology, 2004 Jul, 150(Pt 7), 2391 - 400 Adherence of Actinobacillus pleuropneumoniae to swine-lung collagen; Enriquez-Verdugo I et al.; Actinobacillus pleuropneumoniae serotype 1 adhered to immobilized swine-lung collagen . Bacteria bound to collagen type I, III, IV and V . At 5 min incubation, 30 % of bacteria adhered to collagen, reaching saturation in around 90 min . Treatment of bacteria with divalent-metal chelators diminished their attachment to collagen, and Ca(2+) but not Mg(2+) increased it, suggesting Ca(2+) dependence for adherence . Proteolytic enzymes drastically reduced bacterial adherence to collagen, showing that binding involved bacterial surface proteins . Porcine fibrinogen, haemoglobin and gelatin partially reduced collagen adhesion . A 60 kDa outer-membrane protein of A . pleuropneumoniae recognized the swine collagens by overlay . This membrane protein was apparently involved in adhesion to collagen and fibrinogen, but not to fibronectin and laminin . Antibodies against the 60 kDa protein inhibited the adhesion to collagen by 70 %, whereas pig convalescent-phase antibodies inhibited it by only 40 % . Serotypes 1 and 7 were the most adherent to pig collagen (taken as 100 %); serotypes 6 and 11 were the lowest (approximately 50 %), and neither showed the 60 kDa adhesin to biotinylated collagens . By negative staining, cells were observed initially to associate with collagen fibres in a polar manner, and the adhesin was detected on the bacterial surface . The results suggest that swine-lung collagen is an important target for A . pleuropneumoniae colonization and spreading, and that the attachment to this protein could play a relevant role in pathogenesis. Microbiology, 2004 Jul, 150(Pt 7), 2335 - 45 FixJ-regulated genes evolved through promoter duplication in Sinorhizobium meliloti; Ferrieres L et al.; The FixLJ two-component system of Sinorhizobium meliloti is a global regulator, turning on nitrogen-fixation genes in microaerobiosis . Up to now, nifA and fixK were the only genes known to be directly regulated by FixJ . We used a genomic SELEX approach in order to isolate new FixJ targets in the genome . This led to the identification of 22 FixJ binding sites, including the known sites in the fixK1 and fixK2 promoters . FixJ binding sites are unevenly distributed among the three replicons constituting the S . meliloti genome: a majority are carried either by pSymA or by a short chromosomal region of non-chromosomal origin . Thus FixJ binding sites appear to be preferentially associated with the pSymA replicon, which carries the fixJ gene . Functional analysis of FixJ targets led to the discovery of two new FixJ-regulated genes, smc03253 and proB2 . This FixJ-dependent regulation appears to be mediated by a duplication of the whole fixK promoter region, including the beginning of the fixK gene . Similar duplications were previously reported for the nifH promoter . By systematic comparison of all promoter regions we found 17 such duplications throughout the genome, indicating that promoter duplication is a common mechanism for the evolution of regulatory pathways in S . meliloti. Microbiology, 2004 Jul, 150(Pt 7), 2023 - 8 Communications blackout? Do N-acylhomoserine-lactone-degrading enzymes have any role in quorum sensing? Roche DM, Byers JT, Smith DS, Glansdorp FG, Spring DR, Welch M. A number of bacteria, including some significant pathogens, utilize N-acylhomoserine lactones (AHLs) as quorum sensing signals . There is considerable interest in the therapeutic potential of disrupting quorum sensing . Recently, a number of bacteria have been identified which are capable of enzymic inactivation of AHLs . These enzymes show considerable promise as 'quenchers' of quorum sensing . However, the assumption that the natural function of these enzymes is to disrupt or modulate quorum sensing has yet to be established . This review surveys the progress made to date in this field and examines what implications these findings have for our understanding of the role played by these enzymes in vivo. Plant J, 2004 Aug, 39(3), 354 - 65 The chloroplastic protein translocation channel Toc75 and its paralog OEP80 represent two distinct protein families and are targeted to the chloroplastic outer envelope by different mechanisms; Inoue K et al.; Toc75 is postulated to form the protein translocation channel in the chloroplastic outer envelope membrane . Proteins homologous to Toc75 are present in a wide range of organisms, with the closest homologs occurring in cyanobacteria . Therefore, an endosymbiotic origin of Toc75 has been postulated . Recently, a gene encoding a paralog to Toc75 was identified in Arabidopsis and its product was named atToc75-V . In the present study, we characterized this new Toc75 paralog, and investigated extensively the relationships among Toc75 homologs from higher plants and bacteria in order to gain insights into the evolutionary origin of the chloroplastic protein translocation channel . First, we found that the native molecular weight of atToc75-V is 80 kDa and renamed it (AtOEP80) Arabidopsis thalianaouter envelope protein of 80 kDa . Second, we found that AtOEP80 and Toc75 utilize different mechanisms for their targeting to the chloroplastic envelope . Toc75 is directed with a cleavable bipartite transit peptide partly via the general import pathway, whereas AtOEP80 contains the targeting information within its mature sequence, and its targeting is independent of the general pathway . Third, we undertook phylogenetic analyses of Toc75 homologs from various organisms, and found that Toc75 and OEP80 represent two independent gene families that are most likely derived from cyanobacterial sequences . Our results suggest that Toc75 and OEP80 diverged early in the evolution of plastids from their common ancestor with modern cyanobacteria. J Oral Implantol, 2004, 30(3), 134 - 43 AICRG, Part II: Crestal bone loss associated with the Ankylos implant: loading to 36 months; Chou CT et al.; PROBLEM: The Ankylos endosseous dental implant is a new implant design that will be available in the United States in early 2004 . It features an internal tapered abutment connection, a smooth polished collar without threads at the coronal part of the implant body, and a roughened surface with variable threads on the body of the implant fixture . A precise, tapered, conical abutment connection eliminates the microgap often found in 2-stage implant systems . This microgap may allow the accumulation of food debris and bacteria, as well as micromovement between the parts during clinical function, both of which can lead to a localized inflammation and crestal bone loss . PURPOSE: The purpose of this section of the study was to assess any crestal bone loss associated with this new implant . METHOD: The clinical performance of this new implant design was studied under well-controlled clinical conditions . Over 1500 implants were placed and restored . The vertical crestal bone loss was measured "directly" between the time of implant placement and uncovering, using a periodontal probe . Serial dental radiographs were taken between loading, and the 12-, 24-, and 36-month follow-up visits to determine "indirect" crestal bone loss within a specific period . RESULTS: Bone loss varied among the participating centers from less than 0.5 mm to 2.0 mm . The largest amount of bone loss occurred between the time of placement and uncovering . Following loading, the mean bone loss for all implants for a period of 3 years was about 0.2 mm/y . CONCLUSIONS: The extent of the crestal bone loss after loading was minimal for patients regardless of age, gender, prosthetic applications, bone density, and remote or crestal incisions, as well as for smokers or nonsmokers . Bone loss per year is well within the guidelines of 0.2 mm/y proposed by others. Planta Med, 2004 Jul, 70(7), 620 - 6 High molecular weight polysaccharides from black currant seeds inhibit adhesion of Helicobacter pylori to human gastric mucosa; Lengsfeld C et al.; Several crude and purified polysaccharides from black currant seeds (Ribes nigrum L.) have been isolated, analysed and examined on their effects against Helicobacter pylori in in situ adhesion studies on sections of human gastric mucosa . After pre-treatment of Helicobacter pylori with 0.01 to 0.1 % solutions of the isolated raw polysaccharide (RPS), the epithelial binding of the bacteria was considerably reduced in a concentration-dependent manner, as compared with a non-treated control suspension . Preincubation of the mucosal sections with 0.1 % solutions did not result in a reduced binding of non-treated bacteria . An anion-exchange fraction of RPS eluted with 0.1 M phosphate buffer exhibited a comparable, concentration-dependent reduction of adhesion, whereas the water-eluted fraction was ineffective at the respective concentrations . Both subfractions consisted of similar 1,3-linked galactans, decorated with side chains possessing 1,4-galacturonic acid, galactose and arabinose residues . Molecular weight profiling by GPC revealed that the antiadhesive activity of the buffer eluate correlated with high molecular weight components ranging from about 1000 Da to 340 kDa, whereas the ones of the inactive water eluate had molecular weights of about 100 and 25 kDa, respectively . None of the active fractions revealed inhibitory effects on bacterial growth in vitro . We conclude that acidic, high molecular weight galactans are responsible for the antiadhesive qualities of black currant seed extracts and that these polymers are able to block Helicobacter surface receptors, thus inhibiting their interaction with specific binding factors located on human gastric epithelia. J Environ Qual, 2004 Jul-Aug, 33(4), 1256 - 70 Effect of cobalt sorption on metal fractionation in anaerobic granular sludge; Osuna MB et al.; A sequential extraction procedure was applied to two anaerobic methanogenic sludges (Eerbeek and Nedalco) to examine the speciation of micro- and macronutrients in the sludges after cobalt sorption by exposing the sludge to a 1 mM Co solution for 4 d at pH 7 and 30 degrees C . The effect of different physicochemical conditions on cobalt sorption was studied as well: effect of pH (6-8), effect of competition by a second trace element (Ni or Fe), modification of the granular matrix by glutaraldehyde or heat treatment, and EDTA (ethylenediaminetetraacetic acid) addition . Sorbed Co was found to distribute between the carbonates, organic matter + sulfides, and residual fractions . Cobalt adsorption resulted in an antagonistic interaction with other metals present in the granular matrix, evidenced by the solubilization of other trace elements (e.g., Ni, Cu, and Zn) as well as macronutrients (especially Ca and Fe) . Modification of the sludge matrix by glutaraldehyde or heat treatment, or exposure to EDTA, led to serious modifications of the Co sorption capacity and strong interactions with multivalent cations (i.e., Ca(2+) and Fe(2+)). J Proteome Res, 2004 May-Jun, 3(3), 670 - 2 Could reduced bone mineral densities in HIV be caused by nanobacteria? Sommer AP. From the observations of different research groups reporting on reduced bone mineral density (BMD) and on a pronounced tendency for kidney stone formation, both in HIV-infected patients, and from results achieved in the treatment of severest peripheral neuropathy with lasers, it is concluded that nanobacteria (NB) could actively contribute to the reduction of BMD . A reduced BMD could primarily stem from NB, extracting calcium and phosphate from blood, affecting the calcium and phosphate homeostasis in humans. Acta Biochim Biophys Sin (Shanghai), 2004 Apr, 36(4), 243 - 9 Dissecting and exploiting nonribosomal peptide synthetases; Shen QT et al.; A large number of therapeutically useful cyclic and linear peptides of bacteria or fungal origin are synthesized via a template-directed, nucleic-acid-independent nonribosomal mechanism . This process is carried out by mega-enzymes called nonribosomal peptide synthetases (NRPSs) . NRPSs contain repeated coordinated groups of active sites called modules, and each module is composed of several domains with different catalytic activities . The familiarity to these domains lays base for the future genetic engineering of NRPSs to generate entirely "unnature" products . The details about NRPSs domain structures and the exploitation of NRPSs are described in this review. Glia, 2004 Aug 15, 47(3), 275 - 83 D-Serine as a glial modulator of nerve cells; Miller RF; Until the last decade, it was widely accepted that D-amino acids had no functional role in higher organisms, but that they were restricted to lower organisms, such as bacteria, where they are integrated into the proteoglycans of the cell wall . However, D-serine proved to be an effective coagonist at the "glycine-binding" site of the N-methyl-D-aspartate (NMDA) glutamate receptors, and this observation led to chemical analyses that have now revealed the presence of high levels of D-serine in the central nervous system, including many regions of the brain and retina . D-Serine has been localized to astrocytes and can be released by glutamate through stimulation of AMPA receptors . A new enzyme, serine racemase has been localized to glial cells and converts L-serine to D-serine . Degradation of D-serine takes place through D-amino acid oxidase, an enzyme once thought to metabolize D-amino acids from external sources . Although the "glycine-binding" site of NMDA receptors was initially regarded as a saturated site, evidence in many brain regions has established that this site is not saturated and is therefore modulated by interactions between glial cells and neurons . In some, but not all, studies, D-serine enhances NMDA-mediated currents; a light-evoked enhancement to NMDA currents has been reported in the retina . D-serine also plays a role in synaptic and cellular development, particularly in the cerebellum, where the normal developmental sequences underlying synapse formation onto Purkinje cells and the migration of granule cells are dependent on NMDA receptors during a time when high levels of D-serine are expressed in the Bergmann glia and other cerebellar astrocytes . D-serine must be added to the list of agents through which glial cells participate in controlling the excitability of neurons . J Biol Chem, 2004 Sep 17, 279(38), 39745 - 9 Epub 2004 Jul 13. The cofactor function of the N-terminal domain of tissue factor; Kittur FS et al.; Tissue factor (TF) is an integral membrane protein cofactor for factor VIIa (fVIIa) that initiates the blood coagulation cascade during vascular injury . TF has two fibrinonectin type III-like domains, both of which make extensive interactions with both the light and heavy chains of fVIIa . In addition to interaction with fVIIa, the membrane proximal C-terminal domain of TF is also known to bind the natural substrates factors IX and X, thereby facilitating their assembly and recognition by fVIIa in the activation complex . Both fVIIa and TF are elongated proteins, and their complex appears to be positioned nearly perpendicular to the membrane surface . It is possible that, similar to fVIIa, the N-terminal domain of TF also contacts the natural substrates . To investigate this possibility, we substituted all 23 basic and acidic residues of the N-terminal domain of TF with Ala or Asn and expressed the mutants as soluble TF(2-219) in a novel expression/purification vector system in the periplasmic space of bacteria . Following purification to homogeneity, the cofactor properties of mutants in promoting the amidolytic and proteolytic activity of fVIIa were analyzed in appropriate kinetic assays . The amidolytic activity assays indicated that several charged residues spatially clustered at the junction of the N- and C-terminal domains of TF are required for high affinity interaction with fVIIa . On the other hand, the proteolytic activity assays revealed that none of the residues under study may be an interactive site for either factor IX or factor X . However, it was discovered the Arg(74) mutant of TF was defective in enhancing both the amidolytic and proteolytic activity of fVIIa, suggesting that this residue may be required for the allosteric activation of the protease. J Immunol Methods, 2004 Jun, 289(1-2), 211 - 23 Recombinant HMGB1 with cytokine-stimulating activity; Li J et al.; We describe methods for the isolation, purification, and characterization of full-length high-mobility group box 1 (HMGB1) and truncated mutants expressed in bacteria and in mammalian Chinese Hamster Ovary (CHO) cells . HMGB1 is an abundant nuclear and cytoplasmic protein, highly conserved across species and widely distributed in eukaryotic cells from yeast to man . As a ubiquitous nuclear DNA binding protein, HMGB1 binds DNA, facilitates gene transcription, and stabilizes nucleosome structure . In addition to these intracellular roles, HMGB1 can be released into the extracellular milieu by activated innate immune cells (i.e., macrophages, monocytes) and functions as a mediator of lethal endotoxemia and sepsis . The proinflammatory cytokine activity of HMGB1 has become an intense area of research and recombinant protein can be a useful tool to probe HMGB1 functions . Due to its dipolar charged properties, HMGB1 isolated by some methods can be contaminated with bacterial products (such as CpG DNA or lipopolysaccharide {LPS}) that may interfere with immunological analyses . Here we report our newly developed methods for the isolation and purification of biologically active HMGB1 from bacteria or mammalian CHO cells that is essentially free of contaminants . This strategy provides an important advance in methodology to facilitate future HMGB1 studies. J Immunol Methods, 2004 Jun, 289(1-2), 191 - 9 CrELISA: a fast and robust enzyme-linked immunosorbent assay bypassing the need for purification of recombinant protein; Tureci O et al.; A multitude of antigens has been recently identified by screening of cDNA expression libraries derived from human tumors with autologous sera . Using a phage autoantibody assay and small panels of sera derived from cancer patients or controls it has been shown that some of these antigens display cancer-associated autoantibody responses . The diagnostic and prognostic significance of these potentially cancer-related autoantibodies remains unclear until large-scale assays are developed and serological data are available for hundreds of cancer patients and controls . The major bottleneck for the development of large-scale assays are the cloning, expression and the purification of each of the respective antigens . Due to these limitations and despite the potential clinical relevance large-scale autoantibody tests are established for only a few of these tumor antigens . Here we describe an enzyme-linked immunosorbent assay, Crude lysate ELISA (CrELISA), suitable for antigens identified by expression screening based on crude lysates of antigen-expressing bacteria . This assay permits sensitive and specific autoantibody seroscreening without the need of laborious and time-consuming cloning, expression and purification of recombinant proteins . CrELISA is robust and provides a versatile high throughput procedure for the rapid evaluation of multiple antigens in large-scale serology. FEMS Microbiol Lett, 2004 Jul 15, 236(2), 333 - 40 Novel protein domains and motifs in the marine planctomycete Rhodopirellula baltica; Studholme DJ et al.; The planctomycetes are a phylum of bacteria that have a unique cell compartmentalisation and yeast-like budding cell division and peptidoglycan-less proteinaceous cell walls . We wished to further our understanding of these unique organisms at the molecular level by searching for conserved amino acid sequence motifs and domains in the proteins encoded by Rhodopirellula baltica . Using BLAST and single-linkage clustering, we have discovered several new protein domains and sequence motifs in this planctomycete . R . baltica has multiple members of the newly discovered GEFGR protein family and the ASPIC C-terminal domain family, whilst most other organisms for which whole genome sequence is available have no more than one . Many of the domains and motifs appear to be restricted to the planctomycetes . It is possible that these protein domains and motifs may have been lost or replaced in other phyla, or they may have undergone multiple duplication events in the planctomycete lineage . One of the novel motifs probably represents a novel N-terminal export signal peptide . With their unique cell biology, it may be that the planctomycete cell compartmentalisation plan in particular needs special membrane transport mechanisms . The discovery of these new domains and motifs, many of which are associated with secretion and cell-surface functions, will help to stimulate experimental work and thus enhance further understanding of this fascinating group of organisms. FEMS Microbiol Lett, 2004 Jul 15, 236(2), 227 - 34 Aggregation of mycobacteria caused by disruption of fibronectin-attachment protein-encoding gene; Miyamoto Y et al.; The fibronectin-attachment protein (FAP) is conserved among several species of mycobacteria . Although this protein is associated with attachment and internalization of bacteria to host cells via fibronectin, the physiological role of the protein still remains unclear . To investigate this point, we generated FAP gene disruptant in Mycobacterium smegmatis . The gene disruption, verified by Southern blot and PCR analysis, induced changes on the bacteria, which are associated with strong aggregation and alteration of cell surface properties . Increased hydrophobicity and Congo red accumulation was observed in the FAP gene disruptant . In addition, the complementation experiment demonstrated that the corresponding gene restored wild type morphology in the disruptant . These results indicate that the FAP affects the cell surface properties, and its deletion lead to enhanced aggregation of the M . smegmatis. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2004 Jun, 21(3), 460 - 3 {Development of a freezing drier for lyophilization of biomaterials}; Wu Z et al.; To observe and assess the performance and effect of our self-made FD-1 freezing drier on biomaterials . R502 compressor and R502 refrigerating agent were adopted . In the experiment, FD-1 lyophilized collagen sponge, strain and defibrinogenase . The evaporating-condenser temperature reached -45 degrees C and the small icebox temperature reached -30 degrees C under the loading or free-loading circumstances in the lyophilizing box . The lyophilized collagen sponge had many pores in the structure, and the strain and the defibrinogenase were lyophilized and maintained satisfactorily . This freezing drier is suitable for lyophilizing some biomaterial samples in small or medium batches. Chest, 2004 Jul, 126(1), 220 - 37 Inhaled combination therapy with long-acting beta 2-agonists and corticosteroids in stable COPD; Cazzola M et al.; Long-acting beta(2)-agonists (LABAs) have been shown to be effective first-line bronchodilators in the treatment of COPD patients, and inhaled corticosteroids (ICSs) have been shown to reduce the frequency and/or severity of exacerbations in COPD patients . The concomitant use of a LABA and an ICS can influence both airway obstruction (ie, smooth muscle contraction, increased cholinergic tone, and loss of elastic recoil), and airway inflammation (ie, increased numbers of neutrophils, macrophages, and CD8+ lymphocytes, elevated interleukin-8 and tumor necrosis factor-alpha levels, and protease/antiprotease imbalance) . They are also able to reduce the total number of bacteria adhering to the respiratory mucosa in a concentration-dependent manner without altering the bacterial tropism for mucosa, and to preserve ciliated cells . Several clinical trials support the concept of inhaled combination therapy with LABAs and corticosteroids in stable COPD patients . This type of therapy not only improves airflow obstruction but also provides clinical benefits, as manifested by sustained reduction in overall symptoms, improvements in health-related quality of life, and reductions in exacerbations . All of these effects are very important because, despite recent advances in our understanding of COPD and its treatment, therapy remains suboptimal for a considerable number of patients. Spectrochim Acta A Mol Biomol Spectrosc, 2004 Aug, 60(10), 2411 - 7 Spectroscopic studies on Mn(II), Co(II), Ni(II), and Cu(II) complexes with N-donor tetradentate (N4) macrocyclic ligand derived from ethylcinnamate moiety; Chandra S et al.; Manganese(II), cobalt(II), nickel(II), and copper(II) complexes are synthesized with a novel tetradentate ligand, viz . 1,5,9,13-tetraaza-6,14-dioxo-8,16-diphenylcyclohexadecane (L) and characterized by the elemental analysis, molar conductance measurements, magnetic susceptibility measurements, mass, 1H NMR, IR, electronic, and EPR spectral studies . The molar conductance measurements of the complexes in DMSO correspond to be nonelectrolyte nature for Mn(II), Co(II), and Cu(II) whereas 1:2 electrolytes for Ni(II) complexes . Thus, these complexes may be formulated as {M(L)X(2)} and {Ni(L)}X(2), respectively (where M = Mn(II), Co(II), and Cu(II) and X = Cl- and NO(3-)) . On the basis of IR, electronic, and EPR spectral studies an octahedral geometry has been assigned for Mn(II) and Co(II) complexes, square-planar for Ni(II) whereas tetragonal for Cu(II) complexes . The ligand and its complexes were also evaluated against the growth of bacteria and pathogenic fungi in vitro. Spectrochim Acta A Mol Biomol Spectrosc, 2004 Jul, 60(8-9), 1751 - 61 EPR, IR and electronic spectral studies on Mn(II), Co(II), Ni(II) and Cu(II) complexes with a new 22-membered azamacrocyclic {N4} ligand; Chandra S et al.; The complexes of transition metal ions with an azamacrocyclic tetradentate nitrogen donor {N4} ligand viz . 2,6,12,16,21,22-hexaaza;3,5,13,15-tetramethyltricyclo{15.3.1.1(7-11)} docosa;1(21),2,5,7,9,11(22),12,15,17,19-decaene (L) have been synthesized . All the complexes were found to have general composition M(L)X2 {where M = manganese(II), cobalt(II), nickel(II) and copper(II) and X = Cl- & NO3-} . All the complexes are characterized by the elemental analysis, molar conductance measurements, magnetic susceptibility measurements, mass, 1H NMR, IR, electronic, EPR spectral and cyclic voltammetric studies . An octahedral geometry was assigned for Mn(II), Co(II) and Ni(II) complexes and tetragonal for Cu(II) complexes . The biological actions of the ligand and complexes have been screened in vitro against many bacteria and pathogenic fungi to study their comparative capacity to inhibit the growth. Biochemistry, 2004 Jul 20, 43(28), 9185 - 94 Site-directed mutagenesis of human soluble calcium-activated nucleotidase 1 (hSCAN-1): identification of residues essential for enzyme activity and the Ca(2+)-induced conformational change; Yang M et al.; Human soluble calcium-activated nucleotidase 1 (hSCAN-1) is the human homologue of soluble apyrases found in blood-sucking insects . This family of nucleotidases is unrelated in sequence to more well-studied nucleotidases, and very little is known about the enzymatic mechanism . By multiple sequence alignment, eight regions that are highly conserved in the hSCAN-1 family were identified and named . To identify amino acids important for catalytic activity and enzyme specificity, seven point mutations were constructed, expressed in bacteria, refolded, purified, and characterized . Substitution of glutamic acid 130 with tyrosine resulted in dramatically increased nucleotidase activities, while mutagenesis of aspartic acid 151 to alanine and aspartic acid 84 to alanine completely abolished activity . Mutagenesis of arginine 133 and arginine 271 resulted in enzymes with very little nucleotidase activity . Mutagenesis of aspartic acid 175 to alanine and glycine 122 to glutamic acid had smaller negative effects on enzyme activities . Previously, our laboratory showed that calcium triggers a conformational change in hSCAN-1 necessary for nucleotidase activity . Here we show that several mutants (D84A, R133A, and D151A) that lost most of their activity were unable to undergo the conformational change induced by Ca(2+), as shown by Cibacron blue binding, limited proteolysis, and tryptophan fluorescence . We conclude that aspartic acid residues 84 and 151, as well as arginine residue 133, are essential for the Ca(2+)-induced conformational change that is necessary for enzyme activity . Aspartic acid 175 and glutamic acid 130 are important for determining substrate specificity . In addition, we show that Sr(2+), unlike Mg(2+) and other divalent cations, can substitute for Ca(2+) to induce the conformational change necessary for enzyme activity . However, Sr(2+) cannot substitute for Ca(2+) to support nucleotide hydrolysis, presumably because Sr(2+) cannot substitute for Ca(2+) in its second role as a nucleotide cosubstrate . The ramifications of our results on the interpretation of a recently published crystal structure are discussed . This information will facilitate future engineering of this enzyme designed to enhance its ability to hydrolyze ADP and thus increase its potential for therapeutic use in the treatment of pathological ischemic events triggered via activation of platelets by ADP. Biochemistry, 2004 Jul 20, 43(28), 9029 - 35 CO-induced structural rearrangement of the C cluster in Carboxydothermus hydrogenoformans CO dehydrogenase-evidence from Ni K-edge X-ray absorption spectroscopy; Gu W et al.; We have examined the C cluster in type II CO dehydrogenase (CODH) from Carboxydothermus hydrogenformans using Ni K-edge X-ray absorption near edge spectroscopy and extended X-ray absorption fine structure (EXAFS) spectroscopy . The enzyme was studied under three conditions: "as-isolated" and after treatment with CO or Ti(III) . The shape of the Ni K-edge changes slightly between the different conditions, but no significant edge shift is seen, suggesting that the C cluster contains Ni(II) in both forms . The Ni EXAFS of as-isolated CODH can be simulated with 4 Ni-S interactions at 2.20 A with a large spread in distances . A light atom (C, N, O) is not required to fit the spectrum . After CO treatment, significant changes are observed in the EXAFS . A new feature appears at approximately 2.7 A; this component is consistent with a Ni-Fe interaction . The average Ni-S distance also expands to approximately 2.25 A . The changes between the two forms suggest that the active site (C cluster) undergoes structural rearrangement after CO treatment, and the observed changes help reconcile the two different crystal structures . The implications of the structural change for the enzyme activation and mechanism are discussed. Biochemistry, 2004 Jul 20, 43(28), 8894 - 900 Structural characterization of the stringent response related exopolyphosphatase/guanosine pentaphosphate phosphohydrolase protein family; Kristensen O et al.; Exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GPPA) enzymes play central roles in the bacterial stringent response induced by starvation . The high-resolution crystal structure of the putative Aquifex aeolicus PPX/GPPA phosphatase from the actin-like ATPase domain superfamily has been determined, providing the first insights to features of the common catalytic core of the PPX/GPPA family . The protein has a two-domain structure with an active site located in the interdomain cleft . Two crystal forms were investigated (type I and II) at resolutions of 1.53 and 2.15 A, respectively . This revealed a structural flexibility that has previously been described as a "butterfly-like" cleft opening around the active site in other actin-like superfamily proteins . A calcium ion is observed at the center of this region in type I crystals, substantiating that PPX/GPPA enzymes use metal ions for catalysis . Structural analysis suggests that nucleotides bind at a similar position to that seen in other members of the superfamily. Cell Biochem Funct, 2004 Jul-Aug, 22(4), 207 - 11 Tumour necrosis factor alpha production by polymorphonuclear neutrophils treated with mouse anti-human CD19 monoclonal antibody; Nishimura M; Polymorphonuclear neutrophils (PMNs) play pivotal roles as phagocytic cells in immune defence against bacteria and parasites, exerting their effects by production of reactive oxygen species, several cytokines, chemokines and by phagocytotic reaction . In our investigation of properties of activated PMNs, we discovered that one of the two kinds of mouse anti-human CD19 monoclonal antibodies (mAbs) clone SJ25-C1, weakly binds to freshly prepared PMNs . Moreover, the treatment of freshly prepared PMNs with anti-CD19 mAb (clone SJ25-C1) at 37 degrees C for 6 h induces the production and the secretion of tumour necrosis factor alpha (TNF alpha) by PMNs in vitro which was detectable in culture supernatants by bioassay using mouse cell line L929 cells . The concentration of TNF alpha secreted into the culture supernatant of PMNs cultured in the presence of anti-CD19 mAb (clone SJ25-C1) was higher than those of PMNs treated at 37 degrees C for 6 h with various PMN activators, such as anti-CD24 mAb, granulocytes-macrophage colony stimulation factor (GM-CSF) or interferon gamma (IFN gamma) . In contrast, another clone of anti-CD19 mAb, HD37, did not bind to freshly prepared PMNs and failed to produce TNF alpha . To confirm that anti-CD19 mAb (clone SJ25-C1)-treated PMNs definitely produce TNF alpha, we measured the levels of intracellular expression of TNF alpha in PMNs permeabilized by saponin . These cells were treated with fluorescence-conjugated mouse anti-human TNF alpha mAb for detection of intracellular TNF alpha expression . Consequently, large amounts of intracellular TNF alpha were detected in PMNs treated with anti-CD19 mAb (clone SJ25-C1) but not in those treated with anti-CD19 mAb (clone HD37) . Appl Microbiol Biotechnol, 2004 Aug, 65(3), 243 - 9 Epub 2004 Jul 10. Current studies on biological tagatose production using L-arabinose isomerase: a review and future perspective; Kim P; D-Tagatose is a hexoketose monosaccharide sweetener, which is an isomer of D-galactose and is rarely found in nature . Recently, there has been industrial interest in D-tagatose as a low-calorie sugar-substituting sweetener . This article describes the properties and metabolism of tagatose as well as its commercial importance . The comparison between the biological tagatose production and the chemical production was reviewed based on the example of the glucose isomerization into fructose . The industrial problems facing its commercial application is described and evolving potential solutions are suggested. Pediatr Infect Dis J, 2004 Jul, 23(7), 688 - 9 Facial nerve palsy after human herpesvirus 6 infection; Pitkaranta A et al.; Facial nerve palsy has long been considered to have an infectious etiology, either viruses, mainly herpesviruses, or bacteria, such as Borrelia burgdorferi . We report for the first time the association of human herpesvirus 6 and facial palsy in a previously healthy 1-year 9-month-old boy who developed left facial nerve palsy 7 days after exanthema subitum caused by human herpesvirus 6. Proc Natl Acad Sci U S A, 2004 Jul 20, 101(29), 10554 - 9 Epub 2004 Jul 09. Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins; Nagai T et al.; Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for various cellular functions . Although most indicators have cyan- and yellow-emitting fluorescent proteins (CFP and YFP) as FRET donor and acceptor, their poor dynamic range often prevents detection of subtle but significant signals . Here, we optimized the relative orientation of the two chromophores in the Ca(2+) indicator, yellow cameleon (YC), by fusing YFP at different angles . We generated circularly permuted YFPs (cpYFPs) that showed efficient maturation and acid stability . One of the cpYFPs incorporated in YC absorbs a great amount of excited energy from CFP in its Ca(2+)-saturated form, thereby increasing the Ca(2+)-dependent change in the ratio of YFP/CFP by nearly 600% . Both in cultured cells and in the nervous system of transgenic mice, the new YC enables visualization of subcellular Ca(2+) dynamics with better spatial and temporal resolution than before . Our study provides an important guide for the development and improvement of indicators using GFP-based FRET. J Biol Chem, 2004 Sep 17, 279(38), 40174 - 84 Epub 2004 Jul 09. The transcriptional responses of Mycobacterium tuberculosis to inhibitors of metabolism: novel insights into drug mechanisms of action; Boshoff HI et al.; The differential transcriptional response of Mycobacterium tuberculosis to drugs and growth-inhibitory conditions was monitored to generate a data set of 430 microarray profiles . Unbiased grouping of these profiles independently clustered agents of known mechanism of action accurately and was successful at predicting the mechanism of action of several unknown agents . These predictions were validated biochemically for two agents of previously uncategorized mechanism, pyridoacridones and phenothiazines . Analysis of this data set further revealed 150 underlying clusters of coordinately regulated genes offering the first glimpse at the full metabolic potential of this organism . A signature subset of these gene clusters was sufficient to classify all known agents as to mechanism of action . Transcriptional profiling of both crude and purified natural products can provide critical information on both mechanism and detoxification prior to purification that can be used to guide the drug discovery process . Thus, the transcriptional profile generated by a crude marine natural product recapitulated the mechanistic prediction from the pure active component . The underlying gene clusters further provide fundamental insights into the metabolic response of bacteria to drug-induced stress and provide a rational basis for the selection of critical metabolic targets for screening for new agents with improved activity against this important human pathogen. J Biol Chem, 2004 Sep 10, 279(37), 39051 - 7 Epub 2004 Jul 09. Purification and crystallization of the cystic fibrosis transmembrane conductance regulator (CFTR); Rosenberg MF et al.; The cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane protein that is mutated in patients suffering from cystic fibrosis . Here we report the purification and first crystallization of wild-type human CFTR . Functional characterization of the material showed it to be highly active . Electron crystallography of negatively stained two-dimensional crystals of CFTR has revealed the overall architecture of this channel for two different conformational states . These show a strong structural homology to two conformational states of another eukaryotic ATP-binding cassette transporter, P-glycoprotein . In contrast to P-glycoprotein, however, both conformational states can be observed in the presence of a nucleotide, which may be related to the role of CFTR as an ion channel rather than a transporter . The hypothesis that the two conformations could represent the "open" and "closed" states of the channel is considered. J Biol Chem, 2004 Sep 10, 279(37), 39026 - 34 Epub 2004 Jul 07. The H,K-ATPase beta subunit as a model to study the role of N-glycosylation in membrane trafficking and apical sorting; Vagin O et al.; The role of N-glycosylation in trafficking of an apical membrane protein, the gastric H,K-ATPase beta subunit linked to yellow fluorescent protein, was analyzed in polarized LLC-PK1 cells by confocal microscopy and surface-specific biotinylation . Deletion of the N-glycosylation sites at N1, N3, N5, and N7 but not at N2, N4, and N6 significantly slowed endoplasmic reticulum-to-Golgi trafficking, impaired apical sorting, and enhanced endocytosis from the apical membrane, resulting in decreased apical expression . Golgi mannosidase inhibition to prevent carbohydrate chain branching and elongation resulted in faster internalization and degradation of the beta subunit, indicating that terminal glycosylation is important for stabilization of the protein in the apical membrane and protection of internalized protein from targeting to the degradation pathway . The decrease in the apical content of the beta subunit was less with mannosidase inhibition compared with that found in the N1, N3, N5, and N7 site mutants, suggesting that the core region sugars are more important than the terminal sugars for apical sorting. Ann N Y Acad Sci, 2004 Jun, 1019, 370 - 4 Alternative pathways might mediate toxicity of high concentrations of superoxide dismutase; Kowald A et al.; One of the most important antioxidant enzymes is superoxide dismutase (SOD), which catalyzes the dismutation of superoxide radicals to peroxide . The gene for CuZnSOD lies in humans on chromosome 21, and its activity is increased in patients with Down syndrome . However, instead of being beneficial, increased lipid peroxidation is associated with this increased expression, and also studies on bacteria and transgenic animals show that high levels of SOD actually lead to increased lipid peroxidation and hypersensitivity to oxidative stress . Using mathematical models, we investigated the question of how overexpression of SOD can lead to increased oxidative stress, although it is an antioxidant enzyme . We considered several possibilities that have been proposed in the literature, such as CuZnSOD-catalyzed hydroxyl radical formation, superoxide-mediated inhibition of membrane peroxidation, and short-circuiting of the Cu(I)ZnSOD/Cu(II)ZnSOD redox cycle . We found that one of the proposed mechanisms under certain circumstances is able to explain the increased oxidative stress caused by SOD . Furthermore, we identified an additional mechanism that agrees well with experimental observations . We call it the "alternative pathway" mechanism, because it depends on superoxide radicals having alternative pathways besides their reaction with SOD . The alternative pathway mechanism is a very general explanation for SOD-associated oxidative stress, because it does not depend on the specific type of SOD, nor on the redox status of the cell . We therefore think that it might be the common mechanism for the detrimental effects seen in cells and organisms with increased levels of the different forms of superoxide dismutase. Vaccine, 2004 Jul 29, 22(21-22), 2873 - 80 Identification of adjuvants that enhance the therapeutic antibody response to host IgE; Johansson J et al.; In the development of a novel vaccine against atopic allergies, we have screened for adjuvants that enhance the therapeutic antibody response against self immunoglobulin E (IgE) . The response against self IgE is induced by administration of a vaccine antigen, which contains both self and non-self IgE regions, together with an adjuvant . We evaluated five commonly used adjuvants; Freund's, aluminium hydroxide, ISCOMs, Montanide ISA 51 and Montanide ISA 720, and found that the mineral oil-based adjuvants; Montanide ISA 51 and Freund's induced at least 5-10-fold higher anti-self IgE titers than any of the other candidates . However, with one exception, Alum, the immune responses against the carrier, i.e . the non-self regions, were similar for all adjuvants, indicating that the ability to induce responses against self and non-self antigens differ among adjuvants . The responses against non-self IgE were more than 50-fold higher than antibody responses against self IgE in both the Freund's and Montanide 51-administered animals, indicating that the response against self molecules is markedly inhibited by tolerance-inducing mechanisms . Co-administration of Montanide ISA 51 with immuno-stimulatory substances from bacteria; muramyldipeptide (MDP), monophosphoryl-lipid A (MPL) or a formyl-methionine-containing tripeptide (fMLP), did not elevate the anti-self IgE response . Hence, adjuvants based on pure mineral oil without additional immuno-stimulatory substances appear to be the best adjuvant candidates in therapeutic vaccines aimed at regulating the in vivo levels of self-proteins. Peptides, 2004 Jul, 25(7), 1215 - 22 Antifungal proteins and peptides of leguminous and non-leguminous origins; Ng TB; Antifungal proteins and peptides, as their names imply, serve a protective function against fungal invasion . They are produced by a multitude of organisms including leguminous flowering plants, non-leguminous flowering plants, gymnosperms, fungi, bacteria, insects and mammals . The intent of the present review is to focus on the structural and functional characteristics of leguminous, as well as non-leguminous, antifungal proteins and peptides . A spectacular diversity of amino acid sequences has been reported . Some of the antifungal proteins and peptides are classified, based on their structures and/or functions, into groups including chitinases, glucanases, thaumatin-like proteins, thionins, and cyclophilin-like proteins . Some of the well-known proteins such as lectins, ribosome inactivating proteins, ribonucleases, deoxyribonucleases, peroxidases, and protease inhibitors exhibit antifungal activity . Different antifungal proteins may demonstrate different fungal specificities . The mechanisms of antifungal action of only some antifungal proteins including thaumatin-like proteins and chitinases have been elucidated. Mol Ecol, 2004 Aug, 13(8), 2405 - 20 Reproductive effects and geographical distributions of two Wolbachia strains infecting the Neotropical beetle, Chelymorpha alternans Boh . (Chrysomelidae, Cassidinae); Keller GP et al.; Wolbachia are maternally inherited endocellular bacteria known to alter insect host reproduction to facilitate their own transmission . Multiple Wolbachia infections are more common in tropical than temperate insects but few studies have investigated their dynamics in field populations . The beetle, Chelymorpha alternans, found throughout the Isthmus of Panama, is infected with two strains of Wolbachia, wCalt1 (99.2% of beetles) and wCalt2 (53%) . Populations infected solely by the wCalt1 strain were limited to western Pacific Panama, whereas populations outside this region were either polymorphic for single (wCalt1) and double infections (wCalt1 + wCalt2) or consisted entirely of double infections . The wCalt2 strain was not found as a single infection in the wild . Both strains caused cytoplasmic incompatibility (CI) . The wCalt1 strain caused weak CI (approximately 20%) and the double infection induced moderate CI (approximately 70-90%) in crosses with uninfected beetles . The wCalt1 strain rescued about 75% of eggs fertilized by sperm from wCalt2 males . Based on the relationships of beetle mtDNA and infection status, maternal transmission, and repeated population sampling we determined that the double infection invaded C . alternans populations about 100,000 years ago and that the wCalt2 strain appears to be declining in some populations, possibly due to environmental factors . This may be the first study to demonstrate an association between widespread strain loss and environmental factors in the field . J Clin Microbiol, 2004 Jul, 42(7), 3256 - 61 Purification of Enterocytozoon bieneusi spores from stool specimens by gradient and cell sorting techniques; Kucerova Z et al.; A three-step method for the purification of Enterocytozoon bieneusi spores from stool specimens was developed . The primary process of purification of the spores from bacterial contaminants involved Percoll gradient centrifugation followed by additional separation using cesium chloride density gradient centrifugation . The cesium chloride-isolated spores were further purified using a flow cytometer with cell sorting capabilities . Sorting was performed without the use of antibodies, fluorochromes, or dyes, leaving the sorted spores in their native state, which appears to be less destructive for spores . When quantified by flow cytometry using tubes with known numbers of highly fluorescent polystyrene beads, the sorted material showed a slight decrease in light scatter characteristics compared with the slightly larger Encephalitozoon species spores . Although the overall recovery of the E . bieneusi spores was low, calcofluor and Gram chromotrope staining, indirect immunofluorescence assay, and transmission electron microscopy revealed that the sorted material was highly purified and contained large numbers of E . bieneusi spores and relatively few bacteria and other debris . The sorted material appeared to be sufficiently pure and could be used for in vitro culture and for the development of a variety of diagnostic reagents as well as in studying the genome of E . bieneusi and host-parasite interactions. Clin Diagn Lab Immunol, 2004 Jul, 11(4), 720 - 8 Delineation of the role of platelet-activating factor in the immunoglobulin G2 antibody response; Al-Darmaki S et al.; Localized aggressive periodontitis (LAgP) is a chronic inflammatory disease characterized by severe destruction of periodontal tissues surrounding the first molars and incisors . LAgP subjects produce large amounts of immunoglobulin G2 (IgG2) antibody against oral pathogens, and this response is inversely correlated with the severity of disease . We previously demonstrated that platelet-activating factor (PAF) is required for optimal IgG2 responses . The present investigation was designed to determine the mechanism of IgG2 induction by PAF . Exogenous PAF acetylhydrolase suppressed approximately 80% of pokeweed mitogen-stimulated IgG2 production, confirming that PAF is essential for optimal responses . PAF-activated leukocytes produced gamma interferon (IFN-gamma), a Th1 cytokine that has been associated with IgG2 responses in previous studies . The monocyte-derived cytokines interleukin-12 (IL-12) and IL-18 are upstream of IFN-gamma production, and IgG2 production was suppressed by neutralizing antibodies against these proteins . In addition, PAF induced monocyte-derived dendritic cells (DC) but not macrophages (MPhi) to secrete IL-12 and IL-18 . This observation was interesting because monocyte differentiation in LAgP subjects is skewed to the DC phenotype . Although other investigators have implicated IFN-gamma in IgG2 production, its precise role in this response is controversial . Our studies suggest that IFN-gamma induces isotype switching to IgG2 but only in concert with the Th2 cytokine IL-4 . Thus, it appears that the unique PAF metabolism of LAgP monocytes or DC promotes Th1 responses that are essential for optimal IgG2 antibody production . As IgG2 antibodies opsonize oral bacteria and promote their clearance and destruction, these alterations in PAF metabolism may be essential for limiting disease severity in LAgP patients. Insect Biochem Mol Biol, 2004 Jul, 34(7), 723 - 9 Wolbachia and cytoplasmic incompatibility in mosquitoes; Sinkins SP; Wolbachia are maternally inherited bacteria that induce cytoplasmic incompatibility in mosquitoes, and are able to use these patterns of sterility to spread themselves through populations . For this reason they have been proposed as a gene drive system for mosquito genetic replacement, as well as for the reduction of population size or for modulating population age structure in order to reduce disease transmission . Here, recent progress in the study of mosquito Wolbachia is reviewed . We now have much more comprehensive estimates of the parameters that can affect the spread of Wolbachia through natural populations from low starting frequencies, and for waves of spread to be maintained in the face of partial barriers to gene flow . In Aedes albopictus these dynamics are extremely favourable, with very high maternal transmission fidelity and levels of incompatibility recorded . Correspondence between measurements taken in the lab and field is much better than in the Drosophila simulans model system . Important research goals are also discussed, including Wolbachia transformation, interspecific transfer and the elucidation of the mechanisms of incompatibility and rescue; all will be aided by a wealth of new Wolbachia genome information. J Gastroenterol Hepatol, 2004 Aug, 19(8), 897 - 903 Efficacy of electrolyzed acid water in reprocessing patient-used flexible upper endoscopes: Comparison with 2% alkaline glutaraldehyde; Lee JH et al.; BACKGROUND AND AIM: Two percent glutaraldehyde, the most widely used liquid chemical germicide (LCG), may be hazardous to patients and medical personnel . Alternatives to glutaraldehyde, such as electrolyzed acid water (EAW), are being developed, but data from well-controlled studies with patient-used endoscopes are rare . The purpose of the present paper was to evaluate the high-level disinfection capability of EAW and compare it with glutaraldehyde . METHODS: A random sample of 125 endoscopes was collected immediately after upper endoscopic examination . After careful manual cleaning, endoscopes were divided into a glutaraldehyde and EAW group . After the disinfection procedure, samples from working channel (S-1), insertion tube (S-2), umbilical cord (S-3), and angulation knob (S-4) were taken and cultured . Another twenty endoscopes were experimentally contaminated with hepatitis B virus (HBV) and samples were collected after contamination (T-1), after manual cleaning (T-2), and after final disinfection (T-3) . Polymerase chain reaction (PCR) for HBV-DNA was performed . RESULTS: In the EAW group, culture-positive rates were 3.2% in S-1, 9.5% in S-2, 3.2% in S-3, and 27.0% in the S-4 samples . There was no significant difference between the EAW and glutaraldehyde groups for all sampling sites . However, in both groups, disinfection of the angulation knobs (S-4) was less efficient than the others . For the T-1 site, HBV-DNA was detected from all of them, and in 95% (19/20) of T-2 . However, HBV-DNA was not detected from T-3 samples . CONCLUSIONS: Electrolyzed acid water is as efficient as glutaraldehyde in eliminating bacteria from patient-used endoscopes . After disinfection procedures using both methods, HBV-DNA was not detected from any endoscopes experimentally contaminated with HBV-positive mixed sera . However, some bacteria may remain on the surface of the endoscopes . Therefore, more careful precleaning of the endoscopes may help achieve high-level disinfection in the clinical setting. J Gastroenterol Hepatol, 2004 Aug, 19(8), 891 - 6 Heterotopic gastric mucosa in the cervical esophagus (inlet patch): endoscopic prevalence, histological and clinical characteristics; Akbayir N et al.; BACKGROUND AND AIM: Heterotopic gastric mucosal patch, which has a 0.1-10% frequency, is encountered when the cervical esophagus is examined carefully during endoscopy . In this study, we aimed to determine the prevalence of the patch in the cervical esophagus, to identify its macroscopic and histological characteristics and to evaluate demographic and clinical features . METHODS: Six hundred and sixty patients (317 male, 343 female; mean age 50.28 years, range 14-90) with upper gastrointestinal symptoms had elective esophagogastroduodenoscopy and the cervical esophagus was examined for the patch during withdrawal of the endoscope . Biopsies were obtained from the antrum and the patch . Helicobacter pylori was assessed using an immunohistochemical method . RESULTS: The patch was found in 11 patients of 660, with a prevalence of 1.67% . Patch size ranged between 5 and 30 mm, appeared as a single patch in nine patients and as twin patches in two patients . Mean age and male : female ratio were not significantly different from the patient population without patches, but the female sex was predominant (three men, eight women; mean age 43.6 years, range 32-64) . In five of 11 patients, the upper esophageal and laryngopharyngeal symptoms were remarkable . Eight patients in whom histological confirmation was carried out showed three fundic and five antral-type mucosa . Two of five patients with antral H . pylori also had the bacteria in the patch . H . pylori prevalence in the patch was 25% . CONCLUSION: Heterotopic gastric mucosal patches in the proximal esophagus should not be overlooked during endoscopy because they may lead to important complications in relation to their acid secretion, which may vary according to their parietal cell mass. Indian J Exp Biol, 2003 Sep, 41(9), 967 - 71 Biorecovery of gold; Eisler R; Recovery of ionic and metallic gold (Au) from a wide variety of solutions by selected species of bacteria, yeasts, fungi, algae, and higher plants is documented . Gold accumulations were up to 7.0 g/kg dry weight (DW) in various species of bacteria, 25.0 g/kg DW in freshwater algae, 84.0 g/kg DW in peat, and 100.0 g/kg DW in dried fungus mixed with keratinous material . Mechanisms of accumulation include oxidation, dissolution, reduction, leaching, and sorption . Uptake patterns are significantly modified by the physicochemical milieu . Crab exoskeletons accumulate up to 4.9 g Au/kg DW; however, gold accumulations in various tissues of living teleosts, decapod crustaceans, and bivalve molluscs are negligible. Environ Technol, 2004 May, 25(5), 613 - 9 Stimulation of methanogenesis in a laboratory scale UASB reactor treating domestic sewage by Fe(0) application; Xu H et al.; The effects of application of zero valence Fe (Fe(0)) on the anaerobic digestion of sewage was investigated using two laboratory scale UASB reactors . One reactor had Fe(0) addition in a container found midway along the recycling loop . The other one was a control reactor . In a test run period of 76 days, the Fe(0) application significantly increased the CH, yield by 8.7% and decreased the effluent COD concentration by 21.0% relative to the control reactor . A decrease of the H, concentration of biogas and the CODs/CODt ratio in effluent by Fe(0) application were observed . The obtained results imply that the methanogenesis and COD removal efficiency of the UASB reactor were stimulated by Fe(0) application . The higher performance of the reactor with Fe(0) application arises from the integrated functions of Fe(0) or its ionic state as donor of H2, macronutrient, and flocculant . This study showed that the supply of Fe(0) to a UASB can improve the methanogenesis and the overall COD removal of a UASB reactor treating low-strength domestic waste water. J Gene Med, 2004 Jul, 6(7), 769 - 76 A model for non-viral gene delivery: through syndecan adhesion molecules and powered by actin; Kopatz I et al.; BACKGROUND: Cell transfection requires cationic DNA complexes and heparan sulfate proteoglycans (HSPGs) at the cell surface . Syndecans are transmembrane HSPGs that are ubiquitously expressed on adherent cells . Their polyanionic heparan sulfate moieties are bound at the distal end of their ectodomain, thus facilitating interaction with large cationic particles . METHODS: We propose a model for cell entry involving syndecans as receptors for the DNA complexes by comparing transfection with bacteria uptake and using drug inhibition experiments along with confocal microscopy . RESULTS: When combined with results from the literature, our data suggest the following sequence of events: after initial particle binding, gradual electrostatic zippering of the plasma membrane onto the particle is sustained by lateral diffusion of syndecan molecules that cluster into cholesterol-rich rafts . Clustering in turn triggers PKC activity and linker protein-mediated actin binding to the cytoplasmic tail of the syndecans . Resulting tension fibers and a growing network of cortical actin may then pull the particle into the cell . CONCLUSIONS: Diversion of integrin- and syndecan-mediated cell adhesion processes for particle engulfment appears to be widely exploited by animals (chylomicrons), by pathogens (bacteria, viruses) and, as suggested here, by non-viral vectors . Eur J Nucl Med Mol Imaging, 2004 Nov, 31(11), 1505 - 11 Epub 2004 Jul 06. Clinical feasibility of two-step streptavidin/111In-biotin scintigraphy in patients with suspected vertebral osteomyelitis; Lazzeri E et al.; PURPOSE: Streptavidin accumulates at sites of inflammation and infection as a result of increased capillary permeability . In addition to being utilised by bacteria for their own growth, biotin forms a stable, high-affinity non-covalent complex with avidin . The objective of this investigation was to determine the diagnostic performance of two-step streptavidin/111In-biotin imaging for evaluating patients with suspected vertebral osteomyelitis . METHODS: We evaluated 55 consecutive patients with suspected vertebral osteomyelitis (34 women and 21 men aged 27-86 years), within 2 weeks after the onset of clinical symptoms . Thirty-two of the patients underwent magnetic resonance imaging (MRI) and 24, computed tomography (CT) . DTPA-conjugated biotin was radiolabelled by incubating 500 microg of DTPA-biotin with 111 MBq of 111In-chloride . Two-step scintigraphy was performed by first infusing 3 mg streptavidin intravenously, followed 4 h later by 111In-biotin . Imaging was begun 60 min later . RESULTS . Streptavidin/111In-biotin scintigraphy was positive in 32/34 patients with spinal infection (94.12% sensitivity) . The study was negative in 19/21 patients without infection (95.24% specificity) . The corresponding results for MRI and CT were 54.17% and 35.29% (sensitivity), and 75% and 57.14% (specificity), respectively . All statistical parameters of diagnostic performance (Youden's J index, kappa measure of agreement with correct classification, accuracy, sensitivity, specificity, positive likelihood and negative likelihood) were clearly better for streptavidin/111In-biotin scintigraphy than for either MRI or CT . CONCLUSION . Streptavidin/111In-biotin scintigraphy is highly sensitive and specific for detecting vertebral osteomyelitis in the first 2 weeks after the onset of clinical symptoms, and is potentially very useful for guiding clinical decisions on instituting appropriate therapy. J Allergy Clin Immunol, 2004 Jul, 114(1), 174 - 82 Activation of mast cells by double-stranded RNA: evidence for activation through Toll-like receptor 3; Kulka M et al.; BACKGROUND: Although mast cells (MCs) have been clearly implicated in innate immune responses involving bacteria, their ability to respond to viral infection is less clear . OBJECTIVE: Given that MCs increase at sites of inflammation and are located at surfaces where exposure to invading viruses may occur, we explored the ability of MCs to produce cytokines including type I IFNs after exposure to viruses and to polyinosine-polycytidylic acid (polyI:C), a synthetic mimic of viral double-stranded RNA, and characterized the receptors involved, if any . METHODS: Human peripheral blood-derived cultured MCs and 2 MC lines, Laboratory of Allergic Disease MC line and human MC line 1, were stimulated with viruses and polyI:C, and cytokine production, degranulation, and signaling pathway activation were examined . Because polyI:C is a ligand for Toll-like receptor (TLR)-3, human MCs were also analyzed for TLR expression . RESULTS: Viruses and polyI:C induced IFN-alpha and IFN-beta production . PolyI:C did not induce TNF, IL-1beta, IL-5, or GM-CSF production, in contrast with other TLR ligands (LPS, peptidoglycan, CpG-A, or flagellin) . IFN-alpha production involved nuclear factor-kappaB, p38, and C-Jun NH2-terminal kinase and mitogen-activated protein kinase . RT-PCR and Western blot analysis confirmed expression of TLR-3 by all MCs . Human cultured MCs also expressed TLR-1, TLR-2, TLR-4, TLR-5, TLR-6, TLR-7 and TLR-9 . Antibodies to TLR-3 significantly decreased IFN-alpha production . Bone marrow-derived MCs from TLR-3 knockout mice showed an ablated response to polyI:C . CONCLUSIONS: Murine and human MCs produce type I IFNs after exposure to double-stranded RNA and/or virus, the former via specific interactions with TLR-3 . These data suggest that MCs contribute to innate immune responses to viral infection via the production of type I IFNs . J Vet Med Sci, 2004 Jun, 66(6), 619 - 25 The detection of bovine lactoferrin binding protein on Trypanosoma brucei; Tanaka T et al.; Trypanosoma brucei, the causative agent of sleeping sickness in humans, requires transferrin (TF) for growth . Therefore, T . brucei has a TF receptor that allows it to obtain iron from TF . Lactoferrin (LF), a member of the TF family protein, is an iron-binding protein that is found in most biological fluids of mammals . LF has been shown to interact with some bacteria species by specific receptor-ligand binding . We examined the ability of T . brucei to bind bovine LF (bLF) by using a fluorescence test, streptavidin-biotin (SAB) microplate analysis, and far Western blotting using a biotin-streptavidin system . We found that bLF bound to components of T . brucei, and that bLF hydrolysate disrupted the sites responsible for binding to parasite proteins . Furthermore, bLF, human LF, bovine TF, and ovotransferrin bound same proteins of T . brucei, which exhibited molecular masses of 40 and 43 kDa . The N-terminal amino acid sequence of the 40 kDa bLF binding protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Proc Natl Acad Sci U S A, 2004 Jul 13, 101(28), 10260 - 5 Epub 2004 Jul 06. A freestanding proofreading domain is required for protein synthesis quality control in Archaea; Korencic D et al.; Threonyl-tRNA synthetase (ThrRS) participates in protein synthesis quality control by selectively editing the misacylated species Ser-tRNA(Thr) . In bacteria and eukaryotes the editing function of ThrRS resides in a highly conserved N-terminal domain distant from the active site . Most archaeal ThrRS proteins are devoid of this editing domain, suggesting evolutionary divergence of quality-control mechanisms . Here we show that archaeal editing of Ser-tRNAThr is catalyzed by a domain unrelated to, and absent from, bacterial and eukaryotic ThrRSs . Despite the lack of sequence homology, the archaeal and bacterial editing domains are both reliant on a pair of essential histidine residues suggestive of a common catalytic mechanism . Whereas the archaeal editing module is most commonly part of full-length ThrRS, several crenarchaeal species contain individual genes encoding the catalytic (ThrRS-cat) and editing domains (ThrRS-ed) . Sulfolobus solfataricus ThrRS-cat was shown to synthesize both Thr-tRNAThr and Ser-tRNAThr and to lack editing activity against Ser-tRNAThr . In contrast, ThrRS-ed lacks aminoacylation activity but can act as an autonomous protein in trans to hydrolyze specifically Ser-tRNAThr, or it can be fused to ThrRS-cat to provide the same function in cis . Deletion analyses indicate that ThrRS-ed is dispensable for growth of S . solfataricus under standard conditions but is required for normal growth in media with elevated serine levels . The growth phenotype of the ThrRS-ed deletion strain suggests that retention of the discontinuous ThrRS quaternary structure relates to specific physiological requirements still evident in certain Archaea. J Immunol, 2004 Jul 15, 173(2), 1266 - 75 Type I IFN protects permissive macrophages from Legionella pneumophila infection through an IFN-gamma-independent pathway; Schiavoni G et al.; Legionella pneumophila is an intracellular pathogen whose replication in macrophages is mainly controlled by IFN-gamma . Freshly isolated peritoneal macrophages elicited in vivo with thioglycolate (TG) from A/J mice are highly permissive to L . pneumophila growth in vitro, while TG-elicited macrophages from CD1 mice are resistant . In this study, we show that when CD1 TG-macrophages are cultured for 7 days, they become permissive to Legionella infection . We demonstrate that treatment with type I IFN (IFN-alphabeta) totally inhibits the growth of L . pneumophila in both freshly isolated A/J and in vitro-aged CD1 TG-macrophages . IFN-alphabeta protective effect on permissive macrophages was comparable to that induced by IFN-gamma . Even low doses of either IFN-alpha or IFN-beta alone were effective in inhibiting L . pneumophila multiplication in macrophage cultures . Notably, treatment of resistant, freshly isolated CD1 TG-macrophages with Ab to mouse IFN-alphabeta significantly enhanced their susceptibility to Legionella infection in vitro, thus implying a role of endogenous IFN-alphabeta in mediating the natural resistance of macrophages to L . pneumophila infection . Finally, addition of anti-IFN-gamma-neutralizing Ab did not restore Legionella growth in IFN-alpha- or IFN-beta-treated A/J or CD1 permissive macrophages, indicating that IFN-alphabeta effect was not mediated by IFN-gamma . This observation was further confirmed by the finding that IFN-alphabeta was effective in inhibiting L . pneumophila replication in macrophages from IFN-gamma receptor-deficient mice . Taken together, our results provide the first evidence for a role of IFN-alphabeta in the control of L . pneumophila infection in mouse models of susceptible macrophages and suggest the existence of different pathways for the control of intracellular bacteria in macrophages. J Immunol, 2004 Jul 15, 173(2), 1033 - 42 Specific engagement of TLR4 or TLR3 does not lead to IFN-beta-mediated innate signal amplification and STAT1 phosphorylation in resident murine alveolar macrophages; Punturieri A et al.; The innate immune response must be mobilized promptly yet judiciously via TLRs to protect the lungs against pathogens . Stimulation of murine peritoneal macrophage (PMphi) TLR4 or TLR3 by pathogen-associated molecular patterns (PAMPs) typically induces type I IFN-beta, leading to autocrine activation of the transcription factor STAT1 . Because it is unknown whether STAT1 plays a similar role in the lungs, we studied the response of resident alveolar macrophages (AMphi) or control PMphi from normal C57BL/6 mice to stimulation by PAMPs derived from viruses (polyriboinosinic:polyribocytidylic acid, specific for TLR3) or bacteria (Pam(3)Cys, specific for TLR2, and repurified LPS, specific for TLR4) . AMphi did not activate STAT1 by tyrosine phosphorylation on Y701 following stimulation of any of these three TLRs, but readily did so in response to exogenous IFN-beta . This unique AMphi response was not due to altered TLR expression, or defective immediate-early gene response, as measured by expression of TNF-alpha and three beta chemokines . Instead, AMphi differed from PMphi in not producing bioactive IFN-beta, as confirmed by ELISA and by the failure of supernatants from TLR-stimulated AMphi to induce STAT1 phosphorylation in PMphi . Consequently, AMphi did not produce the microbicidal effector molecule NO following TLR4 or TLR3 stimulation unless exogenous IFN-beta was also added . Thus, murine AMphi respond to bacterial or viral PAMPs by producing inflammatory cytokines and chemokines, but because they lack the feed-forward amplification typically mediated by autocrine IFN-beta secretion and STAT1 activation, require exogenous IFN to mount a second phase of host defense. Appl Environ Microbiol, 2004 Jul, 70(7), 4384 - 6 Degradation and turnover of extracellular DNA in marine sediments: ecological and methodological considerations; Dell'Anno A et al.; Degradation rates of extracellular DNA determined in marine sediments were much higher than those in the water column . However, due to the high sediment DNA content, turnover times were much shorter in seawater . Results reported here provide new insights into the role of extracellular DNA in P cycling in marine ecosystems. Appl Environ Microbiol, 2004 Jul, 70(7), 4349 - 55 Fur is involved in manganese-dependent regulation of mntA (sitA) expression in Sinorhizobium meliloti; Platero R et al.; Fur is a transcriptional regulator involved in iron-dependent control of gene expression in many bacteria . In this work we analyzed the phenotype of a fur mutant in Sinorhizobium meliloti, an alpha-proteobacterium that fixes N(2) in association with host plants . We demonstrated that some functions involved in high-affinity iron transport, siderophore production, and iron-regulated outer membrane protein expression respond to iron in a Fur-independent manner . However, manganese-dependent expression of the MntABCD manganese transport system was lost in a fur strain as discerned by constitutive expression of a mntA::gfp fusion reporter gene in the mutant . Thus, Fur directly or indirectly regulates a manganese-dependent function . The data indicate a novel function for a bacterial Fur protein in mediating manganese-dependent regulation of gene expression. Appl Environ Microbiol, 2004 Jul, 70(7), 4144 - 50 Respiration strategies utilized by the gill endosymbiont from the host lucinid Codakia orbicularis (Bivalvia: Lucinidae); Duplessis MR et al.; The large tropical lucinid clam Codakia orbicularis has a symbiotic relationship with intracellular, sulfide-oxidizing chemoautotrophic bacteria . The respiration strategies utilized by the symbiont were explored using integrative techniques on mechanically purified symbionts and intact clam-symbiont associations along with habitat analysis . Previous work on a related symbiont species found in the host lucinid Lucinoma aequizonata showed that the symbionts obligately used nitrate as an electron acceptor, even under oxygenated conditions . In contrast, the symbionts of C . orbicularis use oxygen as the primary electron acceptor while evidence for nitrate respiration was lacking . Direct measurements obtained by using microelectrodes in purified symbiont suspensions showed that the symbionts consumed oxygen; this intracellular respiration was confirmed by using the redox dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride) . In the few intact chemosymbioses tested in previous studies, hydrogen sulfide production was shown to occur when the animal-symbiont association was exposed to anoxia and elemental sulfur stored in the thioautotrophic symbionts was proposed to serve as an electron sink in the absence of oxygen and nitrate . However, this is the first study to show by direct measurements using sulfide microelectrodes in enriched symbiont suspensions that the symbionts are the actual source of sulfide under anoxic conditions. Appl Environ Microbiol, 2004 Jul, 70(7), 4079 - 87 Light dependence of {3H}leucine incorporation in the oligotrophic North Pacific ocean; Church MJ et al.; The influence of irradiance on bacterial incorporation of {(3)H}leucine was evaluated at Station ALOHA in the oligotrophic North Pacific subtropical gyre . Six experiments were conducted on three cruises to Station ALOHA to examine how {(3)H}leucine incorporation varied as a function of irradiance . Two experiments were also conducted to assess the photoautotrophic response to irradiance (based on photosynthetic uptake of {(14)C}bicarbonate) in both the upper and lower photic zones . Rates of {(3)H}leucine incorporation responded to irradiance in a photosynthesis-like manner, increasing sharply at low light and then saturating and sometimes declining with increasing light intensity . The influence of irradiance on bacterial growth was evaluated in both the well-lit (5 to 25 m) and dimly lit regions of the upper ocean (75 to 100 m) to determine whether the bacterial response to irradiance differed along the depth-dependent light gradient of the photic zone . {(3)H}leucine incorporation rates were analyzed with a photosynthesis-irradiance model for a quantitative description of the relationships between {(3)H}leucine incorporation and irradiance . Maximum rates of {(3)H}leucine incorporation in the upper photic zone increased 48 to 92% relative to those of dark-incubated samples, with {(3)H}leucine incorporation saturating at light intensities between 58 and 363 micromol of quanta m(-2) s(-1) . Rates of {(3)H}leucine incorporation in the deep photic zone were photostimulated 53 to 114% and were susceptible to photoinhibition, with rates declining at light intensities of >100 micromol of quanta m(-2) s(-1) . The results of these experiments revealed that sunlight directly influences bacterial growth in this open-ocean ecosystem. Brief Funct Genomic Proteomic, 2003 Oct, 2(3), 254 - 65 PROCEED: A proteomic method for analysing plasma membrane proteins in living mammalian cells; Bledi Y et al.; Elucidating the profile of extracellular integral membrane proteins on live cells is vital for uncovering diagnostic disease biomarkers, therapeutic agents and drug receptor candidates . Exploring the realm of these proteins has proved to be an intricate task, mainly due to their hydrophobic nature and low abundance . Furthermore, the level of purity achieved by classical methods of purification and cell fractionation is insufficient . These restrictions pose major limitations for gel electrophoresis or chromatography-based separation techniques as the preferred methodologies for high-throughput analysis . Mass spectrometry has alleviated most of the difficulties in the identification of proteins in general; however, the Achilles' heel is still the isolation and separation of membrane proteins . In order to circumvent these limitations, a high-throughput platform has been devised, whereby proteases are applied to whole intact living cells . The resulting peptide fragments are then analysed by liquid chromatology followed by tandem MS (LC-MS/MS) technology to provide a detailed profile of proteins exposed on the surface of the plasma membrane . This kind of protein trimming offers the advantages that no prior manipulation or fractionation of the cell is required, contaminating proteins are remarkably reduced and the procedure is adequate for high-throughput purposes . This method, referred to as PROCEED (PROteome of Cell Exposed Extracellular Domains) is compatible with isotope labelling techniques which facilitate comparative protein expression studies . The methodology is extendable to all cell types including yeast and bacteria . Finally, the advantages and the limitations of PROCEED are discussed in view of other current technologies. Crit Rev Microbiol, 2004, 30(2), 123 - 43 The phylogeny and signature sequences characteristics of Fibrobacteres, Chlorobi, and Bacteroidetes; Gupta RS; Fibrobacteres, Chlorobi, and Bacteroidetes (FCB group) comprise three main bacterial phyla recognized on the basis of 16S rRNA trees . Presently, there are no distinctive biochemical or molecular characteristics known that can distinguish these bacteria from other bacterial phyla . The relationship of these bacteria to other phyla is also not known . This review describes many signatures, consisting of defined and conserved inserts in widely distributed proteins, that provide distinctive molecular markers for these groups of bacteria . These signatures serve to clarify the evolutionary relationship between members of the FCB group, and to other bacterial phyla . A 4 aa insert in DNA Gyrase B (GyrB) and a 45 aa insert in the SecA proteins are uniquely shared by various Bacteroidetes species . The insert in GyrB is present in all Bacteroidetes species (>100) covering different orders and families, indicating that it is a distinctive characteristic of the group . Three signatures consisting of an 18 aa insert in ATPase alpha-subunit, an 8-9 aa insert in the FtsK protein and a 1 aa insert in the UvrB protein are commonly shared only by the Bacteroidetes and Chlorobi homologs providing evidence that these two groups are specifically related to each other . Two additional inserts in the RNA polymerase beta'-subunit (5-7 aa) and Serine hydroxymethyl-transferase (14-16 aa), which are commonly present in various Bacteroidetes, Chlorobi, and Fibrobacteres homologs, but not any other bacteria, provide evidence that these groups shared a common ancestor exclusive of all other bacteria . The FCB groups of bacteria are indicated to have diverged from this common ancestor in the following order: Fibrobacteres --> Chlorobi --> Bacteriodetes . The inferences from signature sequences are strongly supported by phylogenetic analyses . These observations suggest that the FCB groups of bacteria should be placed in a single phylum rather than three distinct phyla . Signature sequences in a number of other proteins provide evidence that the FCB group of bacteria diverged at a similar time as the Chlamydiae group, and that the Spirochetes and Aquificales groups are its closest relatives. Genetics, 2004 Jun, 167(2), 827 - 34 Natural Wolbachia infections in the Drosophila yakuba species complex do not induce cytoplasmic incompatibility but fully rescue the wRi modification; Zabalou S et al.; In this study, we report data about the presence of Wolbachia in Drosophila yakuba, D . teissieri, and D . santomea . Wolbachia strains were characterized using their wsp gene sequence and cytoplasmic incompatibility assays . All three species were found infected with Wolbachia bacteria closely related to the wAu strain, found so far in D . simulans natural populations, and were unable to induce cytoplasmic incompatibility . We injected wRi, a CI-inducing strain naturally infecting D . simulans, into the three species and the established transinfected lines exhibited high levels of CI, suggesting that absence of CI expression is a property of the Wolbachia strain naturally present or that CI is specifically repressed by the host . We also tested the relationship between the natural infection and wRi and found that it fully rescues the wRi modification . This result was unexpected, considering the significant evolutionary divergence between the two Wolbachia strains. Scand J Immunol, 2004 Jul-Aug, 60(1-2), 9 - 13 On immunity against infections and vaccines: credo 2004; Zinkernagel RM et al.; Resistance of vertebrate hosts against infections comprises important natural or innate resistance combined with adaptive immune responses of T and B cells . Viruses, bacteria or classical parasites all probe the limit of immune responses and of immunity . They, therefore, offer an excellent opportunity to assess the biology, physiology and molecular aspects of immune responses and help in characterizing the three basic parameters of immunology-- specificity, tolerance and memory . Various experiments are summarized that indicate that the rules of antiviral, antitumour, antiorgan graft and of autoimmune responses are basically the same . The practical specificity repertoire of T and B cells is probably in the order of 10(4)-10(5) specificities expressed by T cells or by neutralizing antibodies . Tolerance is best defined by rules of reactivity to eliminate infections while avoiding destruction of normal cells by complete elimination of T cells that are specific for antigens persisting within the blood and lymphatic (lymphohaemopoietic) system . Induction of a T-cell response is the result of antigens newly entering lymph nodes or spleen, initially in a local fashion and exhibiting an optimal distribution kinetics within the lymphohaemopoietic system . Antigen staying outside lymphatic tissues are immunologically ignored (e.g . are non-events) . Thus immune reactivity is regulated by antigen dose, time and relative distribution kinetics . Memory is the fact that a host is resistant against disease caused by reinfection with the same agent . Memory correlates best with antigen-dependent maintenance of elevated antibody titres in serum and mucosal secretions, or with an antigen-driven activation of T cells, such that they are protective immediately against peripheral reinfections in solid tissues . While antibodies transferred from mother to offspring are a prerequisite for the survival of otherwise unprotected immuno-incompetent offsprings, activated memory T cells cannot be transmitted . Thus, attenuation of infections in newborns and babies by maternal antibodies is the physiological correlate of man-made vaccines . T cells not only play an essential role in maintaining T-help-dependent memory antibody titres, but also in controlling the many infections that persist in a host at rather low levels (such as tuberculosis, measles and HIV). Med Hypotheses, 2004, 63(2), 208 - 10 High infectious burden, low cancer incidence, and early malignancy in developing countries: a molecular hypothesis in term of the role of nitric oxide; Vijh AK; Nitric oxide (NO) is a neurotransmitter which plays a powerful role in the immune system: it kills bacteria, and, it also destroys the tumor cells . Specifically, immune system stimuli gamma interferon and lipopolysaccharide transmit signals to a macrophage nucleus causing the production of nitric oxide synthase, the enzyme that converts arginine to NO . The NO thus produced not only destroys bacteria but also attacks the tumor cells by inhibiting the energy-producing Krebs cycle, electron transport activity and DNA synthesis . People in developing countries who survive repeated childhood infections must be inferred to have robust microphage/NO systems and thus, also, a strong immunity against cancer--thence the low incidence of cancers in these countries . However, those unfortunate few in these countries who do develop cancer, despite a robust microphage/NO system, must be presumed to have a markedly virulent tumor development micro-environment (e.g., activation of tumor promotion genes, inactivation of tumor suppression genes, multiple mutations, etc.) that escapes even the particularly alert immune surveillance--thence the earlier (by about a decade) death by cancer in those countries . Thus the NO hypothesis put forward here simultaneously provides a mechanistic causation for (i) low cancer incidence in countries subjected to heavy infectious burdens, and (ii) the earlier occurrence (by about a decade) of major cancers in those countries when the immune surveillance, despite its robustness, fails to destroy the incipient formation of cancer cells. BMC Dev Biol . 2004 Jul 05;4(1):8. Gdt2 regulates the transition of Dictyostelium cells from growth to differentiation; Chibalina MV et al.; BACKGROUND: Dictyostelium life cycle consists of two distinct phases - growth and development . The control of growth-differentiation transition in Dictyostelium is not completely understood, and only few genes involved in this process are known . RESULTS: We have isolated a REMI (restriction enzyme-mediated integration) mutant, which prematurely initiates multicellular development . When grown on a bacterial lawn, these cells aggregate before the bacteria are completely cleared . In bacterial suspension, mutant cells express the developmental marker discoidin Igamma even at low cell densities and high concentrations of bacteria . In the absence of nutrients, mutant cells aggregate more rapidly than wild type, but the rest of development is unaffected and normal fruiting bodies are formed . The disrupted gene shows substantial homology to the recently described gdt1 gene, and therefore was named gdt2 . While GDT1 and GDT2 are similar in many ways, there are intriguing differences . GDT2 contains a well conserved protein kinase domain, unlike GDT1, whose kinase domain is probably non-functional . The gdt2 and gdt1 mRNAs are regulated differently, with gdt2 but not gdt1 expressed throughout development . The phenotypes of gdt2- and gdt1- mutants are related but not identical . While both initiate development early, gdt2- cells grow at a normal rate, unlike gdt1- mutants . Protein kinase A levels and activity are essentially normal in growing gdt2- mutants, implying that GDT2 regulates a pathway that acts separately from PKA . Gdt1 and gdt2 are the first identified members of a family containing at least eight closely related genes . CONCLUSIONS: We have isolated and characterised a new gene, gdt2, which acts to restrain development until conditions are appropriate . We also described a family of related genes in the Dictyostelium genome . We hypothesise that different family members might control similar cellular processes, but respond to different environmental cues. Biochemistry, 2004 Jul 13, 43(27), 8652 - 61 Electron transfer in acetohydroxy acid synthase as a side reaction of catalysis . Implications for the reactivity and partitioning of the carbanion/enamine form of (alpha-hydroxyethyl)thiamin diphosphate in a "nonredox" flavoenzyme; Tittmann K et al.; Acetohydroxy acid synthases (AHAS) are thiamin diphosphate- (ThDP-) and FAD-dependent enzymes that catalyze the first common step of branched-chain amino acid biosynthesis in plants, bacteria, and fungi . Although the flavin cofactor is not chemically involved in the physiological reaction of AHAS, it has been shown to be essential for the structural integrity and activity of the enzyme . Here, we report that the enzyme-bound FAD in AHAS is reduced in the course of catalysis in a side reaction . The reduction of the enzyme-bound flavin during turnover of different substrates under aerobic and anaerobic conditions was characterized by stopped-flow kinetics using the intrinsic FAD absorbance . Reduction of enzyme-bound FAD proceeds with a net rate constant of k' = 0.2 s(-1) in the presence of oxygen and approximately 1 s(-1) under anaerobic conditions . No transient flavin radicals are detectable during the reduction process while time-resolved absorbance spectra are recorded . Reconstitution of the binary enzyme-FAD complex with the chemically synthesized intermediate 2-(hydroxyethyl)-ThDP also results in a reduction of the flavin . These data provide evidence for the first time that the key catalytic intermediate 2-(hydroxyethyl)-ThDP in the carbanionic/enamine form is not only subject to covalent addition of 2-keto acids and an oxygenase side reaction but also transfers electrons to the adjacent FAD in an intramolecular redox reaction yielding 2-acetyl-ThDP and reduced FAD . The detection of the electron transfer supports the idea of a common ancestor of acetohydroxy acid synthase and pyruvate oxidase, a homologous ThDP- and FAD-dependent enzyme that, in contrast to AHASs, catalyzes a reaction that relies on intercofactor electron transfer. Ann Agric Environ Med, 2004, 11(1), 99 - 103 Human anaplasmosis in north-eastern Poland: seroprevalence in humans and prevalence in Ixodes ricinus ticks; Grzeszczuk A et al.; Sera of 500 inhabitants of north-eastern Poland, 450 suspected for Lyme borreliosis and 50 blood donors (control group), were analysed for the presence of IgG antibodies against Anaplasma phagocytophilum, the causative agent of human anaplasmosis (HA), known so far as human granulocytic ehrlichiosis (HGE) . Forty one (9.1 %) sera of the study group and one serum (2 %) of the control group were positive using indirect fluorescence assay (IFA) . The seropositivity tended to be more frequent among males (10.3 %) than females (7.6 %) and among the rural (10.3 %) than urban population (7.5 %); however, differences were of no statistical significance (p = 0.4) . No age difference was found between the seropositive and the seronegative individuals (p = 0.77) . The only factor increasing the risk of HA seropositivity found was forestry employment (p < 0.05) . Additionally, a total of 559 Ixodes ricinus ticks, collected in the same area as sera, were investigated for the presence of A . phagocytophilum by the polymerase chain reaction (PCR) and 41 (8.7 %) of them were found to be positive . The infection level ranged from 2.3-13.7 %, depending on the area studied . Bacteria were significantly less frequently detected in nymphs - 2.1 % (5/235) than in adult ticks - 13.6 % (44/324) and in males--4.2 % (74/165) than in females--23.3 % (37/159) (p < or = 0.05) . The obtained results confirm both the occurrence of HA foci in north-eastern Poland with I . ricinus as the principal vector of the A . phagocytophilum infection, and forestry workers as the main group at risk. Gastroenterology, 2004 Jul, 127(1), 224 - 38 Toll-like receptor 2 enhances ZO-1-associated intestinal epithelial barrier integrity via protein kinase C; Cario E et al.; BACKGROUND & AIMS: Protein kinase C (PKC) has been implicated in regulation of intestinal epithelial integrity in response to lumenal bacteria . Intestinal epithelial cells (IECs) constitutively express Toll-like receptor (TLR)2, which contains multiple potential PKC binding sites . The aim of this study was to determine whether TLR2 may activate PKC in response to specific ligands, thus potentially modulating barrier function in IECs . METHODS: TLR2 agonist (synthetic bacterial lipopeptide Pam(3)CysSK4, peptidoglycan)-induced activation of PKC-related signaling cascades were assessed by immunoprecipitation, Western blotting, immunofluorescence, and kinase assays-combined with functional transfection studies in the human model IEC lines HT-29 and Caco-2 . Transepithelial electrical resistance characterized intestinal epithelial barrier function . RESULTS: Stimulation with TLR2 ligands led to activation (phosphorylation, enzymatic activity, translocation) of specific PKC isoforms (PKCalpha and PKCdelta) . Phosphorylation of PKC by TLR2 ligands was blocked specifically by transfection with a TLR2 deletion mutant . Ligand-induced activation of TLR2 greatly enhanced transepithelial resistance in IECs, which was prevented by pretreatment with PKC-selective antagonists . This effect correlated with apical tightening and sealing of tight junction (TJ)-associated ZO-1, which was mediated via PKC in response to TLR2 ligands, whereas morphologic changes of occludin, claudin-1, or actin cytoskeleton were not evident . Downstream the endogenous PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS), but not transcriptional factor activator protein-1 (AP-1), was activated significantly on stimulation . CONCLUSIONS: The present study provides evidence that PKC is an essential component of the TLR2 signaling pathway with the physiologic consequence of directly enhancing intestinal epithelial integrity through translocation of ZO-1 on activation. Gastroenterology, 2004 Jul, 127(1), 26 - 40 Loss of detoxification in inflammatory bowel disease: dysregulation of pregnane X receptor target genes; Langmann T et al.; BACKGROUND & AIMS: Phase 1, phase 2, and cellular efflux transporters are critical components in intestinal barrier function against xenobiotics and bacteria . We therefore performed global gene expression profiling in patients with ulcerative colitis (UC) and Crohn's disease as well as control specimens, with a special emphasis on genes involved in detoxification and epithelial membrane integrity . METHODS: Mucosal biopsy specimens from nonaffected regions of the colon and the terminal ileum were subjected to DNA microarray analysis and pathway-related data mining . Real-time reverse-transcription polymerase chain reaction was used for verification of selected regulated candidate genes in larger inflammatory bowel disease sample numbers and intestinal cell lines . RESULTS: Several dysregulated genes were identified in both disease groups and tissues . A set of genes coordinately down-regulated in the colon of patients with UC was composed of cellular detoxification and defense genes, which are target genes for the transcription factor pregnane X receptor (PXR) . Messenger RNA expression of ABCB1 (MDR1) and PXR was significantly reduced in the colon of patients with UC but was unaffected in patients with Crohn's disease . In contrast to some of its target genes, the expression of PXR was not sensitive to tumor necrosis factor alpha stimulation of intestinal cell lines . CONCLUSIONS: A disease- and tissue-specific decrease in the expression of detoxification enzymes and ABC transporters was observed, which may be explained by a loss of PXR expression . Thus, dysregulation of xenobiotic metabolism and PXR activity in the gut is likely to contribute to the pathophysiology of UC. J Virol Methods, 2004 Sep 1, 120(1), 9 - 12 Conditional expression of vaccinia virus genes in mammalian cell lines expressing the tetracycline repressor; Hedengren-Olcott M et al.; A new system was developed for producing conditional-lethal vaccinia virus mutants using the tetracycline controlled gene expression system from bacteria . The tetracycline resistance operon (tetO) sequence was placed between the promoter and coding sequence of the target gene of the virus . To regulate the expression of the target gene, the tetO-containing virus was used to infect a tetracycline repressor (TetR) expressing cell line . This method allowed isolation of a tetO-containing virus in the absence of the TetR thereby eliminating the risk of selecting for an inactivated viral tetR gene caused by recombination of the virus genome and improving the stability of the engineered mutants. Ann Fr Anesth Reanim, 2004 Jun, 23(6), 597 - 600 {Spontaneous anaerobic osteomyelitis with necrotizing fasciitis of femur: two reasons for the use of hyperbaric oxygenotherapy}; Bilbault P et al.; This is a case report of a 50-year-old male patient who had septic shock with anaerobic bacterial septicaemia coming from a spontaneous left femoral osteomyelitis . The combined treatment with antibiotics, surgery and hyperbaric oxygenotherapy restored normal mobility of the lower limb . Two years later, there was no recurrence . Despite many efforts the aetiology of the disease is unknown . The authors, discuss the relevance of hyperbaric oxygenotherapy in such cases. Dtsch Tierarztl Wochenschr, 2004 May, 111(5), 193 - 5 Detection of pathogenic micro-organisms--a contribution to discussion; Glaeser H; When applying methods for the detection of pathogenic micro-organisms in foodstuffs, information on the distribution of the target organism in the foodstuff submitted to a test should be available . The sampling plan used should allow to detect the presence of low levels of the target organism with a high probability . The individual steps of a detection procedure (pre-enrichment, selective enrichment, isolation and confirmation) need careful examination . It is important that inoculation of low levels of the target organism leads to successful enrichment even in the presence of relatively large numbers of competing organisms . In cases where competing micro-organisms form suspect colonies, there is a risk that false-negative results are obtained, because colonies of the target organism may not be isolated . Collaborative trials have to be carried out to assess the performance of presence/absence tests . Meaningful results are obtained only, if the test samples contain low levels of the target organism and if the effect of competing micro-organisms is checked . While it can hardly be disputed that the determination of the sensitivity and specificity of the test method provides valuable information, this cannot be said for "accordance" and "concordance", two recently proposed parameters which correspond to repeatability and reproducibility in quantitative tests . A better alternative may be to specify the probability to obtain two positive results, when analysing samples containing the target organism under repeatability or reproducibility conditions . In an analogous way, the probability to obtain two negative results with samples containing competing micro-organisms, but not the target organism, could be specified. J Biol Chem, 2004 Sep 10, 279(37), 39000 - 9 Epub 2004 Jul 01. Enzyme-catalyzed mechanism of isoniazid activation in class I and class III peroxidases; Pierattelli R et al.; There is an urgent need to understand the mechanism of activation of the frontline anti-tuberculosis drug isoniazid by the Mycobacterium tuberculosis catalase-peroxidase . To address this, a combination of NMR spectroscopic, biochemical, and computational methods have been used to obtain a model of the frontline anti-tuberculosis drug isoniazid bound to the active site of the class III peroxidase, horseradish peroxidase C . This information has been used in combination with the new crystal structure of the M . tuberculosis catalase-peroxidase to predict the mode of INH binding across the class I heme peroxidase family . An enzyme-catalyzed mechanism for INH activation is proposed that brings together structural, functional, and spectroscopic data from a variety of sources . Collectively, the information not only provides a molecular basis for understanding INH activation by the M . tuberculosis catalase-peroxidase but also establishes a new conceptual framework for testing hypotheses regarding the enzyme-catalyzed turnover of this compound in a number of heme peroxidases. J Biol Chem, 2004 Sep 10, 279(37), 38991 - 9 Epub 2004 Jul 01. Crystal structure of Mycobacterium tuberculosis catalase-peroxidase; Bertrand T et al.; The Mycobacterium tuberculosis catalase-peroxidase is a multifunctional heme-dependent enzyme that activates the core anti-tuberculosis drug isoniazid . Numerous studies have been undertaken to elucidate the enzyme-dependent mechanism of isoniazid activation, and it is well documented that mutations that reduce activity or inactivate the catalase-peroxidase lead to increased levels of isoniazid resistance in M . tuberculosis . Interpretation of the catalytic activities and the effects of mutations upon the action of the enzyme to date have been limited due to the lack of a three-dimensional structure for this enzyme . In order to provide a more accurate model of the three-dimensional structure of the M . tuberculosis catalase-peroxidase, we have crystallized the enzyme and now report its crystal structure refined to 2.4-A resolution . The structure reveals new information about dimer assembly and provides information about the location of residues that may play a role in catalysis including candidates for protein-based radical formation . Modeling and computational studies suggest that the binding site for isoniazid is located near the delta-meso heme edge rather than in a surface loop structure as currently proposed . The availability of a crystal structure for the M . tuberculosis catalase-peroxidase also permits structural and functional effects of mutations implicated in causing elevated levels of isoniazid resistance in clinical isolates to be interpreted with improved confidence. J Bacteriol, 2004 Jul, 186(14), 4824 - 8 Whole-genome expression profiling of Thermotoga maritima in response to growth on sugars in a chemostat; Nguyen TN et al.; To provide data necessary to study catabolite-linked transcriptional networks in Thermotoga maritima, we used full-genome DNA microarray analysis of global transcriptional responses to growth on glucose, lactose, and maltose in a chemostat . A much larger number of genes changed expression in cells grown on lactose than on maltose, each relative to genes expressed in cells grown on glucose . Genes encoding putative oligopeptide transporters were often coregulated with adjacent glycosidase-encoding genes . Genes encoding enzymes catalyzing NADH oxidation were up-regulated on both lactose and maltose . Genes involved in iron and sulfur metabolism were differentially expressed in response to lactose . These data help define the sets of coregulated genes and suggest possible functions for their encoded products . J Bacteriol, 2004 Jul, 186(14), 4620 - 7 First characterization of an archaeal GTP-dependent phosphoenolpyruvate carboxykinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1; Fukuda W et al.; Phosphoenolpyruvate carboxykinase (PCK), which catalyzes the nucleotide-dependent, reversible decarboxylation of oxaloacetate to yield phosphoenolpyruvate and CO2, is one of the important enzymes in the interconversion between C3 and C4 metabolites . This study focused on the first characterization of the enzymatic properties and expression profile of an archaeal PCK from the hyperthermophilic archaeon Thermococcus kodakaraensis (PckTk) . PckTk showed 30 to 35% identities to GTP-dependent PCKs from mammals and bacteria but was located in a branch distinct from that of the classical enzymes in the phylogenetic tree, together with other archaeal homologs from Pyrococcus and Sulfolobus spp . Several catalytically important regions and residues, found in all known PCKs irrespective of their nucleotide specificities, were conserved in PckTk . However, the predicted GTP-binding region was unique compared to those in other GTP-dependent PCKs . The recombinant PckTk actually exhibited GTP-dependent activity and was suggested to possess dual cation-binding sites specific for Mn2+ and Mg2+ . The enzyme preferred phosphoenolpyruvate formation from oxaloacetate, since the Km value for oxaloacetate was much lower than that for phosphoenolpyruvate . The transcription and activity levels in T . kodakaraensis were higher under gluconeogenic conditions than under glycolytic conditions . These results agreed with the role of PckTk in providing phosphoenolpyruvate from oxaloacetate as the first step of gluconeogenesis in this hyperthermophilic archaeon . Additionally, under gluconeogenic conditions, we observed higher expression levels of PckTk on pyruvate than on amino acids, implying that it plays an additional role in the recycling of excess phosphoenolpyruvate produced from pyruvate, replacing the function of the anaplerotic phosphoenolpyruvate carboxylase that is missing from this archaeon . Zhonghua Liu Xing Bing Xue Za Zhi, 2004 Jun, 25(6), 492 - 4 {The detection of nanobacteria infection in serum of healthy Chinese people}; Wang XJ et al.; OBJECTIVE: Nanobacteria, a new kind of bacteria found by a Finnish scholar, is considered to relate to many human diseases like nephrolithiasis . However, there are no data available on nanobacteria infection in Chinese people . METHODS: Nanobacteria was detected in serum of 336 cases of healthy adults in Southern China by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry stain . The monoclonal antibody of nanobacterum was supplied by Kuipio University of Finland . RESULTS: Nanobacteria infection rates were 27 (8.0%), 19 (5.7%) in the healthy adults by ELISA and immunohistochemistry stain respectively . No difference was shown between the 2 methods and between male and female, statistically . Age and sex did not seem to be related to the infectious risk of nanobacteria . However, the infectious rate was lower in those below 30-year-old than that of people over 60-year-old (P < 0.05) . CONCLUSION: Nanobacteria was existed in the serum of Chinese healthy people with an infectious rate of 8.0%. BMC Microbiol . 2004 Jul 01;4(1):24. Chlamydial development is blocked in host cells transfected with Chlamydophila caviae incA; Alzhanov D et al.; BACKGROUND: Chlamydiae produce