Phylogeography of a subterranean amphipod reveals cryptic diversity and dynamic evolution in extreme environments.

Extreme conditions in subsurface are suspected to be responsible for morphological convergences, and so to bias biodiversity assessment. Subterranean organisms are also considered as having poor dispersal abilities that in turn generate a large number of endemic species when habitat is fragmented. Here we test these general hypotheses using the subterranean amphipod Niphargus virei. All our phylogenetic analyses (Bayesian, maximum likelihood and distance), based on two independent genes (28S and COI), revealed the same tripartite structure. N. virei populations from Benelux, Jura region and the rest of France appeared as independent evolutionary units. Molecular rates estimated via global or Bayesian relaxed clock suggest that this split is at least 13 million years old and accredit the cryptic diversity hypothesis. Moreover, the geographical distribution of these lineages showed some evidence of recent dispersal through apparent vicariant barrier. In consequence, we argue that future analyses of evolution and biogeography in subsurface, or more generally in extreme environments, should consider dispersal ability as an evolving trait and morphology as a potentially biased marker.

Analysis of pFQ31, a 8551-bp cryptic plasmid from the symbiotic nitrogen-fixing actinomycete Frankia.

The actinomycete Frankia has never been transformed genetically. To favour the development of Frankia cloning vectors, we have fully sequenced the Frankia alni pFQ31 cryptic plasmid and performed analyses to characterise its coding and non-coding regions. This plasmid is 8551 bp-long and contains 72% G+C. Computer-assisted analyses identified 18 open reading frames (ORFs). These ORFs show a synonymous codon usage different from the one of Frankia chromosomal genes, suggesting an evolutionary bias linked to the nature of the replicon or a horizontal transfer. Three ORFs were found to encode genes likely to be involved in plasmid replication and stability: parFA (partition protein), ptrFA (transcriptional repressor of the GntR family) and repFA (initiation of replication). DNA signatures of a replication origin were identified in the ptrFA-repFA intergenic region. These structural motifs are similar to those observed among origins of iteron-containing plasmids replicating via a θ mode.

Phylogenetic analysis of the Saccharomyces cerevisiae group based on polymorphisms of rDNA spacer sequences.

The phylogenetic relationships between species of yeasts assigned to the Saccharomyces sensu stricto group, which includes Saccharomyces cerevisiae and Saccharomyces bayanus, were studied together with Saccharomyces pastorianus and Saccharomyces paradoxus. The experimental approaches used were RFLP analysis of the PCR-amplified rDNA internal transcribed spacer (ITS) and intergenic spacer, and total ITS sequence analysis. Both RFLP and sequence analyses gave fairly similar results. The gene trees generated with either of the two data sets showed the distribution of the yeasts into two major, well-separated, phylogenetic clusters called 'cerevisiae' and 'bayanus'. The 'cerevisiae' cluster included the S. cerevisiae type strain, together with most of the species (16 out of 23), whereas the 'bayanus' cluster included the remaining seven type strains. Therefore, analysis of rDNA sequences confirmed S. cerevisiae and S. bayanus as two well-defined taxa. However, S. pastorianus and S. paradoxus, the two other usually accepted taxa of the now-defined Saccharomyces sensu stricto complex, could not be clearly separated from S. bayanus and S. cerevisiae, respectively. However, in both PCR-RFLP and ITS sequence analyses, S. paradoxus had the outermost position in the 'cerevisiae' cluster. PCR-RFLP analysis of the ribosomal spacer sequences was also carried out on 26 Saccharomyces strains isolated in various wine-growing regions of France in an attempt to clarify their positions in the Saccharomyces phylogenetic tree. Compared to the diversity of the Saccharomyces type strains, less genetic diversity was detected among these yeasts and several of them exhibited identical RFLP patterns. Most of the wine yeast strains (16 out of 26) were closely related to each other and were found within the 'cerevisiae' cluster. The remaining 10 wine yeast strains branched within the 'bayanus' cluster. PCR-RFLP analysis of ribosomal spacer sequences thus appears to be a useful and appropriate method for the correct characterization of Saccharomyces yeast strains used in food processing.

Molecular phylogeny of Cyprinidae inferred from cytochrome b DNA sequences.

Phylogenetic relationships between European cyprinids (Teleostei:Cypriniformes:Cyprinidae) were investigated by comparing cytochrome b gene sequences from 29 species, among which 20 were newly sequenced. Results were in general agreement with previous morphologically-based studies, but new interesting relationships were found. The classical barbelled/lacking barbels split is dubious. Genus Leuciscus appears paraphyletic. The phylogenetic location of some American cyprinid species was recovered; at least two distinct invasions of the New World are likely. Finally, the problem of intergeneric cyprinid hybrids is addressed. The genus rank for these interbreeding entities is supported and hybrids are seen as the consequence of a high genetic flexibility. This is the first molecularly based study of cyprinid diversity. It sheds light on the evolution and taxonomy of this major freshwater fish family.

Molecular phylogeny of rodents, with special emphasis on murids: evidence from nuclear gene LCAT.

Phylogenetic relationships among 19 extant species of rodents, with special emphasis on rats, mice, and allied Muroidea, were studied using sequences of the nuclear protein-coding gene LCAT (lecithin:cholesterol acyltransferase), an enzyme of cholesterol metabolism. Analysis of 705 base pairs from the exonic regions of LCAT confirmed known groupings in and around Muroidea. Strong support was found for the families Sciuridae (squirrel and marmot) and Gliridae (dormice) and for suprafamilial taxa Muroidea and Caviomorpha (guinea pig and allies). Within Muroidea, the first branching leads to the fossorial mole rats Spalacinae and bamboo rats Rhizomyinae. The other Muroidea appear as a polytomy from which are issued Gerbillinae (gerbils), Murinae (rats and mice), Sigmodontinae (New World cricetids), Cricetinae (hamsters), and Arvicolinae (voles). Evidence from LCAT sequences agrees with that from a number of previous molecular and morphological studies, both concerning branching orders inside Muroidea and the bush-like radiation of rodent suprafamilial taxa (caviomorphs, sciurids, glirids, muroids), thus suggesting that this nuclear gene is an appropriate candidate for addressing questions of rodents relationships.

Phylogenetic relationships within genus Leuciscus (Pisces, Cyprinidae) in Portuguese fresh waters, based on mitochondrial DNA cytochrome b sequences.

To investigate phylogenetic relationships among Leuciscus species occurring in Portuguese inland waters, the cytochrome b gene was sequenced from representatives of the main rivers. This study supports the recognition of the species level for L. pyrenaicus, including populations from the southern Portuguese drainages (Tejo, Sado, and Guadiana drainages), and for L. carolitertii, including populations from the northern Portuguese drainages. The existence of two new species occurring in the extreme southwestern drainages of Mira and Arade is also suggested. The present results support the monophyly of the Mira and the Arade populations, as well as an early divergence of these two lineages. The present-day distribution of Leuciscus species is seen as a consequence of Pliocene and Pleistocene events, such as river disjunctions and posterior confluence in epicontinental seas and river captures. A mixture of haplotypes was observed in the Mondego and the Tejo drainages, which could be a consequence of ancient river captures, with a possible mitochondrial DNA introgression in the Tejo drainage and a recent introduction by man in the Mondego drainage. The pattern of differentiation among mtDNA haplotypes and their geographic distribution is discussed in terms of evolutionary aspects.

Intracellular reactive oxygen species as apparent modulators of heat-shock protein 27 (hsp27) structural organization and phosphorylation in basal and tumour necrosis factor alpha-treated T47D human carcinoma cells.

The small stress protein heat-shock protein 27 (hsp27) is an oligomeric phosphoprotein, constitutively expressed in most human cells, which enhances cellular resistance to tumour necrosis factor alpha (TNF alpha). This phenomenon correlates with dramatic changes in hsp27 cellular location, structural organization and phosphorylation. To gain a better understanding of the molecular mechanisms regulating these properties of hsp27, we investigated whether they were a consequence of the intracellular production of reactive oxygen species (ROS) generated by TNF alpha. Here, we report that, in T47D carcinoma cell lines, the rapid burst of intracellular ROS production and changes in hsp27 locale, structural organization and phosphoisoform composition induced by TNF alpha were abolished by the overexpression of the antioxidant enzyme seleno-glutathione peroxidase (GSHPx). These effects were greatly diminished when GSHPx-expressing cells were grown in the absence of selenium, a cofactor that is essential for seleno-GSHPx activity, indicating that they are directly linked to the increased GSHPx activity. Moreover, in growing T47D cells, GSHPx expression induced intracellular redistribution of hsp27 and decreased the phosphorylation of this protein without altering its pattern of oligomerization. In contrast, the heat-mediated phosphorylation of hsp27 was not altered by decreased intracellular ROS levels. Hence, in growing and TNF-treated cells, several hsp27 properties appear to be modulated by fluctuations in intracellular ROS levels.

Constitutive expression of human hsp27, Drosophila hsp27, or human alpha B-crystallin confers resistance to TNF- and oxidative stress-induced cytotoxicity in stably transfected murine L929 fibroblasts.

Hyperthermia and other forms of stress that induce and/or stimulate heat shock or stress protein (hsp) expression enhance the cellular resistance to TNF-alpha. One of the stress proteins, hsp70, has been shown to participate in the molecular mechanisms that regulate this phenomenon. Here we have tested the capability of small hsps from different species to protect against this cytokine in the TNF-sensitive L929 fibrosarcoma cells. The genes that encode human hsp27, Drosophila hsp27, and human alpha B-crystallin were placed under the control of the constitutive SV40 early promoter and were stably introduced into L929 cells. We observed that all clones that constitutively expressed the exogenous small hsps exhibited a strong protection against TNF-mediated killing, which was proportional to the level of the expressed proteins. This phenomenon did not correlate with altered binding of TNF-alpha to its receptors, and no protection was observed as a consequence of the transfection or selection procedures. In addition, the overexpression of the exogenous small hsps did not modify the level of the endogenous stress proteins in the transfected clones. Remarkably, the small hsps also induced a protection against oxidative stresses generated by either hydrogen peroxide or menadione. In L929 cells, the killing induced by TNF-alpha and oxidative stress is thought to occur through the accumulation of intracellular reactive oxygen intermediates. Hence, our data suggest that the small hsps from different species share the property to protect L929 cells against the deleterious effects of reactive oxygen intermediates generated by either TNF-alpha or oxidative stress.

IL-4 inhibits growth factor-stimulated rheumatoid synoviocyte proliferation by blocking the early phases of the cell cycle.

A major feature of rheumatoid arthritis is an uncontrolled proliferation of synoviocytes. This is consistent with the active production of factors such as platelet-derived growth factor (PDGF) and IL-1 by the synovitis, which act in vivo as well as in vitro as potent synoviocyte growth factors. We have previously shown that IL-4 is able to inhibit growth factor production in an ex vivo model of synovitis. Herein, we show that IL-4 strongly inhibited PDGF and IL-1 beta stimulated rheumatoid arthritis synoviocyte proliferation in a dose-dependent manner and through its 130 kDa receptor. This antiproliferative effect of IL-4 directly correlated with a blockade of the synoviocyte cell cycle at the G0 + G1 phases. We also observed that IL-4 induced striking morphologic changes in IL-1 beta or PDGF-stimulated synoviocytes, including increased volume and granulosity. These changes led to major perturbations of the cell monolayer, associated with a marked decrease of synoviocyte viability. Taken together, these data indicate that IL-4 inhibits growth factor-induced proliferation of synoviocytes by interfering with the cell cycle, and by decreasing cell survival.

Analysis of the resistance to heat and hydrogen peroxide stresses in COS cells transiently expressing wild type or deletion mutants of the Drosophila 27-kDa heat-shock protein.

The Drosophila melanogaster small heat-shock protein, hsp27 (Dhsp27) belongs to a family of polypeptides which shares a sequence related to alpha-crystallin and which protect cell against heat shock. Dhsp27 accumulates following heat shock and, in absence of stress, in the central nervous system, imaginal discs and the gonads of the developing fly. Two internal and adjacent deletion mutants in the conserved alpha-crystallin domain of Dhsp27 were constructed. Expression vectors containing either the coding sequence of Dhsp27 or that of the two deletion mutants linked to the Simian-Virus-40 late promoter were used to transfect monkey COS cells. The transient expression of Dhsp27 was found to decrease the sensitivity of COS cells to heat and hydrogen-peroxide stresses as judged by Trypan-blue staining and indirect immunofluorescence analysis. Using this rapid test, we observed that a deletion of 62 amino acids, which lies at the 5' end of the conserved alpha-crystallin domain and covers the first 41 amino acids of this region had only a weak effect on the protective activity of Dhsp27. This suggests that the N-terminal half of the conserved alpha-crystallin domain may not be essential for the protective activity of the small hsp. In contrast, Dhsp27 was no more active when the last 42 amino acids of the alpha-crystallin domain were deleted. Biochemical fractionation and indirect immunofluorescence analysis indicated that the protective function of Dhsp27 was localized at the level of the nucleus.

Increased production of soluble CD23 in rheumatoid arthritis, and its regulation by interleukin-4.

OBJECTIVE: To assess CD23 status in rheumatoid arthritis (RA) patients, as defined by the levels of CD23 expression on peripheral blood mononuclear cells (PBMC), the levels of soluble CD23 (sCD23) in sera, and the production of sCD23 by PBMC cultures and its regulation by interleukin-4 (IL-4). METHODS: CD23 expression as determined by double fluorescence-activated cell sorter analysis and sCD23 production as determined by immunoradiometric assay were investigated in 24 RA patients and 21 controls. Soluble CD23 was measured in sera and supernatants of PBMC, activated with polyclonal activators (pokeweed mitogen [PWM] or Staphylococcus aureus Cowan strain 1, [SAC]) used either alone or in combination with IL-2 or IL-4. RESULTS: The percentage of B cells expressing CD23 and serum levels of sCD23 were increased in patients with RA. IL-4 was a potent inducer of sCD23 production in supernatants, whereas IL-2 was inactive. Costimulation with SAC or PWM did not increase the effect obtained with IL-4 alone. When sCD23 levels in RA and control supernatants were compared, spontaneous production was found to be increased in RA PBMC: This difference from control values was even more pronounced when sCD23 levels in PBMC and purified B cells in response to IL-4, either alone or in combination with SAC or PWM, were tested. In the same supernatants, the increased secretion of sCD23 induced by IL-4 was associated with an inhibitory effect of IL-4 on Ig production, a phenomenon that was more pronounced in RA PBMC than in controls. CONCLUSION: CD23 status in RA is characterized by increased expression of CD23 on B cells, increased production of sCD23 in sera and supernatants, and increased sensitivity of RA PBMC and B cells to IL-4.

Proliferation and differentiation of human CD5+ and CD5- B cell subsets activated through their antigen receptors or CD40 antigens.

The pan-T cell antigen CD5 has been shown to delineate two different mouse B cell subsets, originating from distinct progenitors. In man, on average, 30% of the tonsillar B cell pool expresses this antigen. In the present report, a detailed comparison of the CD5+ and CD5- B cell response to cytokines, following activation via surface immunoglobulins (sIg) or CD40 antigen, was undertaken. CD5+ B cells were positively selected by panning or by sorting from tonsils. Two-color immunofluorescence analysis performed on tonsillar B cell populations showed that CD5+ B cells displayed most of the phenotypic features of mantle zone B cells. CD5+ B cells could be stimulated for DNA synthesis by mitogenic concentrations of Staphylococcus aureus, Cowan I strain (SAC), insolubilized anti-IgM antibodies, immobilized anti-CD40 antibodies and phorbol 12-myristate 13-acetate (PMA). The growth-response of small dense CD5- B cells to these T cell-independent mitogens was comparable to that of CD5+ B cells, whereas the low-density, in vivo-activated, CD5- B cells were only marginally stimulated by Ig-cross-linking agents and PMA. Following ligation of sIg, both B cell subsets proliferated essentially in response to interleukin (IL)-2 and IL-4. When used in co-stimulation with immobilized anti-CD40 antibodies, IL-4 promoted growth of CD5+ and CD5- B cells, whereas IL-2 displayed only moderate stimulatory effects. CD5+ and CD5- B cells differentiated into Ig-secreting cells when they were co-cultured with SAC or cross-linked anti-CD40 antibodies and IL-2. However, IgM constituted the major component of the Ig response of CD5+ B cells, whereas high levels of IgG were secreted by CD5- B cells.

Inhibition of the production of proinflammatory cytokines and immunoglobulins by interleukin-4 in an ex vivo model of rheumatoid synovitis.

OBJECTIVE: To assess the spontaneous production of proinflammatory cytokines and immunoglobulins in rheumatoid arthritis (RA) synovitis and modulation by interleukin-4 (IL-4). METHODS: We developed an ex vivo model of RA synovitis using pieces of RA synovium, and have studied the regulation of the production of IL-1 beta, IL-6, tumor necrosis factor alpha (TNF alpha), IgM, and IgG. RESULTS: Spontaneous production of proinflammatory cytokines in vitro was active, with prolonged cytokine gene transcription and translation. IL-6 was produced at higher levels than either IL-1 beta or TNF alpha, and explants produced more IgG than IgM. In contrast, IL-4 and interferon-gamma were undetectable. When pieces of synovium were incubated in the presence of IL-4, reduction of spontaneous proinflammatory cytokine and Ig production was observed. CONCLUSION: These results extend the observations of the antiinflammatory properties of IL-4 to an ex vivo situation, and provide the rationale for the clinical use of IL-4 in RA.

Interleukin 4 inhibits polyclonal immunoglobulin secretion and cytokine production by peripheral blood mononuclear cells from rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is associated with T- and B-cell dysfunction. Immunoglobulin (Ig) production is under the control of T cells and their derived cytokines such as interleukin 2 (IL-2) and IL-4. Herein we studied the regulation of the production of immunoglobulins and cytokines by peripheral blood mononuclear cells from RA patients and controls. In the controls, IL-4 inhibited Ig production in response to Staphylococcus aureus and pokeweed mitogen stimulation. IL-2 induced maximal Ig production in association with Staphylococcus aureus, whereas it inhibited pokeweed mitogen-induced production. In patients, levels of Ig production in response to mitogens and cytokines were reduced, particularly for the response to IL 2. The inhibitory effect of IL-4 on mitogen-induced Ig production was observed in RA patients as in the controls. Spontaneous production of IL-6 was increased in RA patients. These levels were correlated with indicators of active disease such as sedimentation rate and Ritchie index. IL-4 inhibited the production of IL-6, IL-1 beta, and tumor necrosis factor alpha (TNF alpha) by both controls and rheumatoid patients. Thus as first described for the T-cell response, mononuclear cells from RA patients display a reduced response to mitogens and cytokines which induce their B-cell differentiation into Ig-screening cells. However, IL-4 was able to inhibit Ig and cytokine production, properties suggesting a potential antiinflammatory role for this cytokine.

Antinuclear antibodies detected by indirect immunofluorescence on HEp2 cells and by immunoblotting in patients with systemic sclerosis.

The HEp2 cell cultures appeared highly sensitive in detecting the antinuclear antibodies (ANAb) in systemic sclerosis, principally anticentromere antibodies of the CREST syndrome. The immunoblotting used with either complex cellular extracts from HeLa and rabbit thymus or purified nuclear components (high mobility group (HMG) proteins and histones) is able to identify precisely the ANAb targets and to contribute to diagnosis. With nuclear extracts of HeLa cells, the sera from 75.8% of CREST syndrome subjects stained 18 and 22 kD proteins. Corresponding antibodies were also detected in 72.7% of these patients, on HEp2 centromers by indirect immunofluorescence. With the same extracts, 33.3% of sera from diffuse sclerosis/acrosclerosis patients contain antibodies staining 86, 73, 32 and 30kD. These sera also stain 77, 66 and 63kD from thymus extracts. Corresponding antibodies will be the anti-SCL-70 antibodies defined by double immunodiffusion. The anti-HMG antibodies were infrequent in systemic sclerosis, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) and consequently without interest for diagnosis. The anti-whole histones antibodies which are less frequent in diffuse sclerosis/acrosclerosis (35.7%) than in SLE (41.3%) recognize especially H1 and H2A in the first diseases, H1 and H2B in SLE and H1 and H3 in RA.

Improvement of light sensitive staining of immunoblots for routine analysis.

We have demonstrated that alpha-naphthol is a peroxidase substrate that produces a stable staining of immunoblots, which may be kept for further examination and retrospective comparison.