Sox18 expression in blood vessels and feather buds during chicken embryogenesis

Sox18 encodes a transcription factor known to be important for the development of blood vessels and hair follicles in mice. In order to study the functional conservation of this gene through evolution, we have isolated and characterized Sox18 in chickens. cSox18 shows a high degree of sequence homology to both the mouse and human orthologues, particularly in the high mobility group DNA-binding domain and to a lesser extent in the transcriptional activation domain. A region of unusually high sequence conservation at the C-terminus may represent a further, previously unrecognized functional domain. Both the chicken and human proteins appear to be truncated at the N-terminus relative to mouse SOX18. In situ hybridization analyses showed expression in the developing vasculature and feather follicles, consistent with reported expression in the mouse embryo. In addition, cSox18 mRNA was observed in the retina and claw beds. (C) 2001 Elsevier Science B.V. All rights reserved.

Genetic dependence of the specific T-cell cytokine response to Porphyromonas gingivalis in mice

Background: Susceptibility to periodontal infections may, in part, be genetically determined. Porphyromonas gingivalis is a major periodontopathogen, and the immune response to this organism requires T-cell help. The aim of the present study was to examine the specific T-cell cytokine responses to P gingivalis outer membrane antigens in a mouse model and their relationship with H-2 haplotype. Methods: BALB/c and DBA/2J (H-2(d)), CBACaH (H-2(k)), and C57BL6 (H-2(b)) mice were immunized with P gingivalis outer membrane antigens weekly for 3 weeks. One week after the final injection, the spleens were removed, and 6 T-cell lines specific for P gingivalis were established for each mouse strain. The percentage of CD4 and CD8 cells in the P gingivalis-specific T-cell lines staining positive for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma, and IL-10 was determined by 2-color flow cytometry. Results: The cytokine profiles of T-cell lines from BALB/c and DBA/2J mice showed no significant differences. Significantly fewer IL4+, IFN-gamma+, and IL-10+ CD4 cells than IL-4+, IFN-gamma+, and IL-10+ CD8 cells, respectively, were demonstrated for both strains. P gingivalis-specific T-cell lines generated from CBACaH mice were similar to those generated from BALB/c and DBA/2J mice; however, the mean percentage of IL4+ CD4 cells in CBACaH mice was lower than the percentage of IFN-gamma+ CD4 cells. Also, the mean percentage of IFN-gamma+ CD4 cells in CBACaH mice was significantly increased compared to DBA/2J mice. Unlike the other 3 strains, T-cell lines established from C57BL6 mice contained similar percentages of cytokine-positive cells, although the percentage of IL-4+ CD4 cells was reduced in comparison to the percentage of CD8 cells. However, comparisons with the other 3 strains demonstrated a higher percentage of IL-4+ CD4 cells than in lines established from the spleens of DBA/2J mice, IFN-gamma+ CD4 cells than in lines established from BALB/c and CBACaH mice, and IL-10+ CD4 cells than in lines established from all 3 other strains. No significant differences in the percentage of positive CD8 cells were demonstrated between lines in the 4 strains of mice. Conclusion: The specific T-cell response to P gingivalis in mice may, in the case of the CD4 response, depend on MHC genes. These findings are consistent with the concept that patient susceptibility is important to the outcome of periodontal infection and may, in part, be genetically determined.

Enrichment of autotrophic anaerobic ammonium-oxidizing consortia from various wastewaters

The option for biological nitrogen removal has recently been broadened with the description of simultaneous nitrification/denitrification, anaerobic ammonium oxidation (ANAMMOX) and the concept of CANON (completely autotrophic nitrogen removal over nitrite). An autotrophic anaerobic ammonium oxidation (AAAO) consortium was successfully selected and enriched from municipal treatment plant sludges in Sydney, Australia, but not from industrial coke-oven wastewater sludges. Chemolithoautotrophic basic salt (CLABS) medium was used in the selection of AAAO organisms and chloramphenicol was added to the initial stage of selection to eliminate denitrifiers. Two different temperatures, 37degreesC and 55degreesC, were used in the selection of mesophilic and thermophilic consortia, respectively. Thermophilic AAAO organisms were not selected at 55degreesC. Mesophilic AAAO activities, however, were evident in both batch and continuous cultures, whereby ammonium was consumed concurrently with a decrease of nitrite, giving a ratio of 1:1-1:1.3 in ammonium removal rate over nitrite consumption rate. A continuous-mode mesophilic fixed-bed reactor was established to enrich the AAAO consortium. After 1 year, biofilms, pinkish in color, had developed on the support media and side wall of the feed-line tubing. Ammonium and nitrite consumption increased from similar to15 mg to 60 mg d(-1) L-1 over a period of 243 days. Later, transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH) techniques revealed that the dominant cell type in the AAAO consortium had a similar morphology and 16S rDNA sequence homology to that of the recently described ANAMMOX organism, Brocadia anammoxidans.

Analysis of the microbial community structure and function of a laboratory scale enhanced biological phosphorus removal reactor

A laboratory scale sequencing batch reactor (SBR) operating for enhanced biological phosphorus removal (EBPR) and fed with a mixture of volatile fatty acids (VFAs) showed stable and efficient EBPR capacity over a four-year-period. Phosphorus (P), poly-beta-hydroxyalkanoate (PHA) and glycogen cycling consistent with classical anaerobic/aerobic EBPR were demonstrated with the order of anaerobic VFA uptake being propionate, acetate then butyrate. The SBR was operated without pH control and 63.67+/-13.86 mg P l(-1) was released anaerobically. The P% of the sludge fluctuated between 6% and 10% over the operating period (average of 8.04+/-1.31%). Four main morphological types of floc-forming bacteria were observed in the sludge during one year of in-tensive microscopic observation. Two of them were mainly responsible for anaerobic/aerobic P and PHA transformations. Fluorescence in situ hybridization (FISH) and post-FISH chemical staining for intracellular polyphosphate and PHA were used to determine that 'Candidatus Accumulibacter phosphatis' was the most abundant polyphosphate accumulating organism (PAO), forming large clusters of coccobacilli (1.0-1.5 mum) and comprising 53% of the sludge bacteria. Also by these methods, large coccobacillus-shaped gammaproteobacteria (2.5-3.5 mum) from a recently described novel cluster were glycogen-accumulating organisms (GAOs) comprising 13% of the bacteria. Tetrad-forming organisms (TFOs) consistent with the 'G bacterium' morphotype were alphaproteobacteria , but not Amaricoccus spp., and comprised 25% of all bacteria. According to chemical staining, TFOs were occasionally able to store PHA anaerobically and utilize it aerobically.

A real-time PCR assay for the detection of Neisseria gonorrhoeae by LightCycler

The detection of Neisseria gonorrhoeae by the polymerase chain reaction (PCR) is now recognized as a sensitive and specific method of diagnosing infection by the organism. In this Study 152 urine specimens were examined for N. gonorrhoeae by a real-time PCR method using the LightCycler platform and results were compared to an in-house PCR assay using an ELISA-based detection method. N. gonorrhoeae DNA was detected in 29 (19%) specimens by LightCycler PCR (LC-PCR) and in 31 (20%) specimens by the in house PCR method. The LightCycler assay proved to be specific and 94% sensitive when compared to the in house PCR method. These features combined with the rapid turn-around time for results makes the LC-PCR particularly suitable for the detection of N. gonorrhoeae in a routine clinical laboratory. (C) 2002 Elsevier Science Inc. All rights reserved.

Cytokine regulation of syndecan-1 and-2 gene expression in human periodontal fibroblasts and osteoblasts

Cell-surface proteoglycans participate in several biological functions including interactions with a variety of growth factors and cytokines. Regulation of syndecan-1 and -2 gene expression was investigated in human periodontal ligament fibroblasts (PDLF), osteoblasts (OB) and gingival fibroblasts (GF), in response to platelet-derived growth factor (PDGF-BB), transforming growth factor (TGF-beta(1)), and interleukin (IL-1beta) by Northern blot analyses. We also compared the effect of PDGF-BB and TGF-beta(1), separately and in combination, in the prolonged presence of IL-1beta on the expression of both syndecan genes. The results demonstrated that the three cell lines regulated the expression of syndecan-1 and -2 in response to growth factors and cytokines in different manners. These cell lines increased syndecan-1 mRNA levels in response to either PDGF-BB or TGF-beta(1) and decreased levels in response to IL-1beta. The effect of IL-1beta on syndecan-1 mRNA synthesis was partially reversed after adding PDGF-BB and TGF-beta(1), separately or in combination, in the presence of IL-1beta. In contrast, syndecan-2 mRNA level was markedly upregulated in response to either TGF-beta(1) or IL-1beta in OB when compared with the other two cell lines. However, the stimulatory effect of TGF-beta(1) on syndecan-2 mRNA production in OB was abolished in the prolonged presence of IL-1beta. These findings lend support to the notion that syndecan-1 and syndecan-2 have distinct functions which correlate with their source and functions within the periodontium.

Evolution and developmental expression of nuclear receptor genes in the ascidian Herdmania

Nuclear receptors are a superfamily of metazoan transcription factors that have been shown to be involved in a wide range of developmental and physiological processes. A PCR-based survey of genomic DNA and developmental cDNAs from the ascidian Herdmania identifies eight members of this multigene family. Sequence comparisons and phylogenetic analyses reveal that these ascidian nuclear receptors are representative of five of the six previously defined nuclear receptor subfamilies and are apparent homologues of retinoic acid [NR1B], retinoid X [NR2B], peroxisome proliferator-activated [NR1C], estrogen related [NR3B], neuron-derived orphan (NOR) [NR4A3], nuclear orphan [NR4A], TR2 orphan [NR2C1] and COUP orphan [NR2F3] receptors. Phylogenetic analyses that include the ascidian genes produce topologically distinct trees that suggest a redefinition of some nuclear receptor subfamilies. These trees also suggest that extensive gene duplication occurred after the vertebrates split from invertebrate chordates. These ascidian nuclear receptor genes are expressed differentially during embryogenesis and metamorphosis.

Testing the beneficial acclimation hypothesis

Recent developments in evolutionary physiology have seen many of the long-held assumptions within comparative physiology receive rigorous experimental analysis. Studies of the adaptive significance of physiological acclimation exemplify this new evolutionary approach. The beneficial acclimation hypothesis (BAH) was proposed to describe the assumption that all acclimation changes enhance the physiological performance or fitness of an individual organism. To the surprise of most physiologists, all empirical examinations of the BAH have rejected its generality. However, we suggest that these examinations are neither direct nor complete tests of the functional benefit of acclimation. We consider them to be elegant analyses of the adaptive significance of developmental plasticity, a type of phenotypic plasticity that is very different from the traditional concept of acclimation that is used by comparative physiologists.

Selective loss of expression of glutamate GluR2/R3 receptor subunits in cerebellar tissue from a patient with olivopontocerebellar atrophy

Expression of the mRNAs encoding the astrocytic (EAAT1, EAAT2) and neuronal (EAAT3, EAAT4) excitatory amino acid transporters and the AMPA-type glutamate receptor subunits GluR2 and GluR3 was investigated in postmortem cerebellar extracts from a patient with olivopontocerebellar atrophy (OPCA) and in material from three age-matched controls. Decreased expression in the steady state level of EAAT4 mRNA in the OPCA sample was correlated with the selective loss of Purkinje cells. Neuropathological evaluation revealed reactive gliosis and concomitantly increased expression of the mRNA encoding astrocytic glial fibrillary acidic protein (GFAP). Expression of the mRNAs encoding the AMPA receptor subunits GluR2 and GluR3 subunits was found to be decreased in OPCA suggesting that excitotoxic mechanism could play a role in the pathogenesis of the selective neuronal cell death in this disorder.

The identification of the transcriptional regulator CRP in Aeromonas hydrophila JMP636 and its involvement in amylase production and the 'acidic toxicity' effect

Aims: The physiological examination of amylase production by Aeromonas hydrophila JMP636 and identification of the mechanism of regulation. Methods and Results: Aeromonas hydrophila JMP636 was grown with single, then dual carbon sources; the growth cycle was followed and amylase activity throughout was monitored. The levels of cAMP, a known secondary messenger for the regulatory gene crp, were also examined. Amylase activity was regulated by catabolite repression. Physiological studies revealed that JMP636 exhibited both diauxic growth, with two carbon sources, and the 'acid toxicity' effect on glucose. The crp gene was cloned, expressed and inactivated from the JMP636 chromosome. Catabolite repression of amylase production and the 'acid toxicity' effect both require crp and were linked to cAMP levels. Conclusions: Regulation of amylase production was predicted to follow the model CRP-mediated cAMP-dependent Escherichia coli catabolite regulation system. Significance and Impact of the Study: This work provides an understanding of the physiology of the opportunistic pathogen Aer. hydrophila through identification of the mechanism of catabolite repression of amylase production and the existence of crp within this cell. It also provides a broader knowledge of global gene regulation and suggests regulatory mechanisms of other Aer. hydrophila gene/s.

Characterization of a chemsensory protein (ASP3c) from honeybee (Apis mellifera L.) as a brood pheromone carrier

Chemosensory proteins (CSPs) are ubiquitous soluble small proteins isolated from sensory organs of a wide range of insect species, which are believed to be involved in chemical communication. We report the cloning of a honeybee CSP gene called ASP3c, as well as the structural and functional characterization of the encoded protein. The protein was heterologously secreted by the yeast Pichia pastoris using the native signal peptide. ASP3c disulfide bonds were assigned after trypsinolysis followed by chromatography and mass spectrometry combined with microsequencing. The pairing (Cys(I)-Cys(II), Cys(III)-Cys(IV)) was found to be identical to that of Schistocerca gregaria CSPs, suggesting that this pattern occurs commonly throughout the insect CSPs. CD measurements revealed that ASP3c mainly consists of alpha-helices, like other insect CSPs. Gel filtration analysis showed that ASP3c is monomeric at neutral pH. Using ASA, a fluorescent fatty acid anthroyloxy analogue as a probe, ASP3c was shown to bind specifically to large fatty acids and ester derivatives, which are brood pheromone components, in the micromolar range. It was unable to bind tested general odorants and other tested pheromones (sexual and nonsexual). This is the first report on a natural pheromonal ligand bound by a recombinant CSP with a measured affinity constant.

Cytochrome P450(cin) (CYP176A), isolation, expression, and characterization

Cytochromes P450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiologic and xenobiotic compounds in eukaryotes and prokaryotes. Studies on bacterial P450s, particularly those involved in monoterpene oxidation, have provided an integral contribution to our understanding of these proteins, away from the problems encountered with eukaryotic forms. We report here a novel cytochrome P450 (P450(cin), CYP176A1) purified from a strain of Citrobacter braakii that is capable of using cineole 1 as its sole source of carbon and energy. This enzyme has been purified to homogeneity and the amino acid sequences of three tryptic peptides determined. By using this information, a PCR-based cloning strategy was developed that allowed the isolation of a 4-kb DNA fragment containing the cytochrome P450(cin) gene (cinA). Sequencing revealed three open reading frames that were identified on the basis of sequence homology as a cytochrome P450, an NADPH-dependent flavodoxin/ferrodoxin reductase, and a flavodoxin. This arrangement suggests that P450(cin) may be the first isolated P450 to use a flavodoxin as its natural redox partner. Sequencing also identified the unprecedented substitution of a highly conserved, catalytically, important active site threonine with an asparagine residue. The P450 gene was subcloned and heterologously expressed in Escherichia coli at similar to2000 nmol/liter of original culture, and purification was achieved by standard protocols. Postulating the native E. coli flavodoxin/flavodoxin reductase system might mimic the natural redox partners of P450,in, it was expressed in E. coli in the presence of cineole 1. A product was formed in vivo that was tentatively identified by gas chromatography-mass spectrometry as 2-hydroxycineole 2. Examination of P450(cin) by UV-visible spectroscopy revealed typical spectra characteristic of P450s, a high affinity for cineole 1 (K-D = 0.7 mum), and a large spin state change of the heme iron associated with binding of cineole 1. These facts support the hypothesis that cineole 1 is the natural substrate for this enzyme and that P450(cin) catalyzes the initial monooxygenation of cineole 1 biodegradation. This constitutes the first characterization of an enzyme involved in this pathway.

Mechanistic aspects of HIV-1 reverse transcription initiation

During reverse transcription, the positive-strand HIV-1 RNA genome is converted into a double-stranded DNA copy which can be permanently integrated into the host cell genome. Recent analyses show that HIV-1 reverse transcription is a highly regulated process. The initiation reaction can be distinguished from a subsequent elongation reaction carried out by a reverse transcription complex composed of (at least) heterodimeric reverse transcriptase, cellular tRNA(lys3) and HIV-1 genomic RNA sequences. In addition, viral factors including Tat, Nef, Vif, Vpr, IN and NCp7, cellular proteins, and TAR RNA and other RNA stem-loop structures appear to influence this complex and contribute to the efficiency of the initiation reaction. As viral resistance to many antiretroviral compounds is a continuing problem, understanding the ways in which these factors influence the reverse transcription complex will likely lead to novel antiretroviral strategies. Copyright (C) 2001 John Wiley Sons, Ltd.

Differential regulation of twitching motility and elastase production by Vfr in Pseudomonas aeruginosa

Vfr, a homolog of Escherichia coli cyclic AMP (cAMP) receptor protein, has been shown to regulate quorum sensing, exotoxin A production, and regA transcription in Pseudomonas aeruginosa. We identified a twitching motility-defective mutant that carries a transposon insertion in vfr and confirmed that vfr is required for twitching motility by construction of an independent allelic deletion-replacement mutant of vfr that exhibited the same phenotype, as well as by the restoration of normal twitching motility by complementation of these mutants with wild-type vfr. Vfr-null mutants exhibited severely reduced twitching motility with barely detectable levels of type IV pili, as well as loss of elastase production and altered pyocyanin production. We also identified reduced-twitching variants of quorum-sensing mutants (PAK lasl::Tc) with a spontaneous deletion in vfr (S. A. Beatson, C. B. Whitchurch, A. B. T. Semmler, and J. S. Mattick, J. Bacteriol., 184:3598-3604,2002), the net result of which was the loss of five residues (EQERS) from the putative cAMP-binding pocket or Vfr. This allele (VfrDeltaEQERS) was capable of restoring elastase and pyocyanin production to wild-type levels in vfr-null mutants but not their defects in twitching motility. Furthermore, structural analysis of Vfr and VfrDeltaEQERS in relation to E. coli CRP suggests that Vfr is capable of binding both cAMP and cyclic GMP whereas VfrDeltaEQERS is only capable of responding to cAMP. We suggest that Vfr controls twitching motility and quorum sensing via independent pathways in response to these different signals, bound by the same cyclic nucleotide monophosphate-binding pocket.

Quorum sensing is not required for twitching motility in Pseudomonas aeruginosa

It has been reported that mutations in the quorum-sensing genes lasI and rhlI in Pseudomonas aeruginosa result in, among many other things, loss of twitching motility (A. Glessner, R. S. Smith, B. H. Iglewski, and J. B. Robinson, J. Bacteriol. 181:1623-1629, 1999). We constructed knockouts of lasI and rhlI and the corresponding regulatory genes lasR and rhlR and found no effect on twitching motility. However, twitching-defective variants accumulated during culturing of lasI and rhlI mutants. Further analysis showed that the stable twitching-defective variants of lasI and rhlI mutants had arisen as a consequence of secondary mutations in vfr and algR, respectively, both of which encode key regulators affecting a variety of phenotypes, including twitching motility. In addition, when grown in shaking broth culture, lasI and rhlI mutants, but not the wild-type parent, also accumulated unstable variants that lacked both twitching motility and swimming motility and appeared to be identical in phenotype to the S1 and S2 variants that were recently reported to occur at high frequencies in P. aeruginosa strains grown as a biofilm or in static broth culture (E. Deziel, Y. Comeau, and R. Villemur, J. Bacteriol. 183:1195-1204, 2001). These results indicate that mutations in one regulatory system may create distortions that select during subsequent culturing for compensatory mutations in other regulatory genes within the cellular network. This problem may have compromised some past studies of regulatory hierarchies controlled by quorum sensing and of bacterial regulatory systems in general.