Population genetics of the endangered Canarian endemic Atractylis arbuscula (Asteraceae): implications for taxonomy and conservation

We use the information provided by 36 RAPD loci and 15 morphological traits to describe and construe the population differentiation in A. arbuscula, a Canarian endemic Asteraceae threatened with extinction that exhibits a disjunct distribution in the islands of Gran Canaria (var. schizogynophylla) and Lanzarote (var. arbuscula). Our evaluation of morphological characters and the extent of RAPD differentiation found sets the stage for a taxonomic restructuring to hoist both var. arbuscula and var. schizogynophylla to subspecies category. Our genetic results suggest that fragmentation and generally low population sizes are jeopardising the survival of this species through a predominance of stochastic processes in microevolutionary dynamics, especially in Gran Canaria, where subpopulations exhibit the lowest levels of genetic variation and gene flow. If, as most Asteraceae, these plants have a sporophytic self-incompatibility system, the scarce reproductive turnover observed in the much smaller subpopulations from Gran Canaria (ssp. schizogynophylla) is possibly more influenced by a deficiency of S-alleles (that would be provoking the unavailability of compatible mating types, and a cascade of deleterious effects associated with the Allee effect) than by a reduced pollinator visitation frequency. Based on the depauperated values of genetic parameters for this subspecies, urgent mixed subpopulation reinforcements and reintroductions (with specimens belonging to the same subspecies) seem advisable on the grounds that they might allow the isolated habitat remnants to retrieve from inbreeding through the introduction of a new stock of S-alleles and the subsequent production of genotypes that may have a higher selective value.     

Formation of the gap junction nexus: binding partners for connexins.

Gap junctions are the morphological correlates of direct cell-cell communication and are formed of hexameric assemblies of gap junction proteins (connexins) into hemichannels (or connexons) provided by each coupled cell. Gap junction channels formed by each of the connexin subtypes (of which there are as many as 20) display different properties, which have been attributed to differences in amino acid sequences of gating domains of the connexins. Recent studies additionally indicate that connexin proteins interact with other cellular components to form a protein complex termed the Nexus. This review summarizes current knowledge regarding the protein-protein interactions involving of connexin proteins and proposes hypothesized functions for these interactions.     

Water flux through human aquaporin 1: inhibition by intracellular furosemide and maximal response with high osmotic gradients

This work studies water permeability properties of human aquaporin 1 (hAQP1) expressed in Xenopus laevis oocyte membranes, applying a technique where cellular content is replaced with a known medium, with the possibility of measuring intracellular pressure. Consequences on water transport—produced by well-known anisotonic gradients and by the intracellular effect of probable aquaporin inhibitors—were tested. In this way, the specific intracellular inhibition of hAQP1 by the diuretic drug furosemide was demonstrated. In addition, experiments imposing anisotonic mannitol gradients with a constant ionic strength showed that the relationship between water flux and the applied mannitol gradient deflects from a perfect osmometer response when the gradient is higher than 150 mosmol kg_W⁻¹. These results would indicate that the passage of water molecules through hAQP1 may have a maximum rate. As a whole, this work demonstrates the technical advantage of controlling both intracellular pressure and medium composition in order to study biophysical properties of hAQP1, and contributes information on water channel behavior under osmotic challenges and the discovery of new inhibitors.     

Functional evaluation of domain-domain interactions and human protein interaction networks

MOTIVATION: Large amounts of protein and domain interaction data are being produced by experimental high-throughput techniques and computational approaches. To gain insight into the value of the provided data, we used our new similarity measure based on the Gene Ontology (GO) to evaluate the molecular functions and biological processes of interacting proteins or domains. The applied measure particularly addresses the frequent annotation of proteins or domains with multiple GO terms. RESULTS: Using our similarity measure, we compare predicted domain-domain and human protein-protein interactions with experimentally derived interactions. The results show that our similarity measure is of significant benefit in quality assessment and confidence ranking of domain and protein networks. We also derive useful confidence score thresholds for dividing domain interaction predictions into subsets of low and high confidence. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Systemic increased immune response to Nocardia brasiliensis co-exists with local immunosuppressive microenvironment

Human diseases produced by pathogenic actinomycetes are increasing because they may be present as opportunistic infections. Some of these microbes cause systemic infections associated with immunosuppressive conditions, such as chemotherapy for cancer, immunosuppressive therapy for transplant, autoimmune conditions, and AIDS; while others usually cause localized infection in immunocompetent individuals. Other factors related to this increase in incidence are: antibiotic resistance, not well defined taxonomy, and a delay in isolation and identification of the offending microbe. Examples of these infections are systemic disease and brain abscesses produced by Nocardia asteroides or the located disease by Nocardia brasiliensis, named actinomycetoma. During the Pathogenic Actinomycetes Symposium of the 16th International Symposium on Biology of Actinomycetes (ISBA), held in Puerto Vallarta, Mexico, several authors presented recent research on the mechanisms by which N. brasiliensis modulates the immune system to survive in the host and advances in medical treatment of human actinomycetoma. Antibiotics and antimicrobials that are effective against severe actinomycetoma infections with an excellent therapeutic outcome and experimental studies of drugs that show promising bacterial inhibition in vivo and in vitro were presented. Here we demonstrate a systemic strong acquired immune response in humans and experimental mice at the same time of a local dominance of anti inflammatory cytokines environment. The pathogenic mechanisms of some actinomycetes include generation of an immunosuppressive micro environment to evade the protective immune response. This information will be helpful in understanding pathogenesis and to design new drugs for treatment of actinomycetoma.     

Impact of DNA methyltransferases on the epigenetic regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor expression in malignant melanoma

Aberrant promoter methylation and resultant silencing of TRAIL decoy receptors were reported in a variety of cancers, but to date little is known about the relevance of this epigenetic modification in melanoma. In this study, we examined the methylation and the expression status of TRAIL receptor genes in cutaneous and uveal melanoma cell lines and specimens and their interaction with DNA methyltransferases (DNMTs) DNMT1, DNMT3a, and DNMT3b. DR4 and DR5 methylation was not frequent in cutaneous melanoma but on the contrary it was very frequent in uveal melanoma. No correlation between methylation status of DR4 and DR5 and gene expression was found. DcR1 and DcR2 were hypermethylated with very high frequency in both cutaneous and uveal melanoma. The concordance between methylation and loss of gene expression ranged from 91% to 97%. Here we showed that DNMT1 was crucial for DcR2 hypermethylation and that DNMT1 and DNMT3a coregulate the methylation status of DcR1. Our work also revealed the critical relevance of DcR1 and DcR2 expression in cell growth and apoptosis either in cutaneous or uveal melanoma. In conclusion, the results presented here claim for a relevant impact of aberrant methylation of decoy receptors in melanoma and allow to understand how the silencing of DcR1 and DcR2 is related to melanomagenesis.     

Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets.

BACKGROUND: Natural killer (NK) and NK T (NKT) cells are important in innate immune defense. Their unequivocal identification requires at least four antigens. Based on the expression of additional antigens, they can be further divided into functional subsets. For more accurate immunophenotyping and to describe multiple expression patterns of leukocyte subsets, an increased number of measurable colors is necessary. To take advantage of the technologic features offered by slide-based cytometry, repeated analysis was combined with sequential optical-filter changing. METHODS: Human peripheral blood leukocytes from healthy adult volunteers were labeled with antibodies by direct or indirect staining. Tandem dyes of Cy7 (phycoerythrin [PE]-/allophycocyanin [APC]-Cy7), Cy5.5 (PE-/APC-Cy5.5), and PE-Cy5 and the fluorochromes fluorescein isothiocyanate (FITC), PE, and APC were tested alone and in combinations. Optical filters of the laser scanning cytometer were 555 DRLP/BP 530/30 nm for photomultiplier tube (PMT) 1/FITC, 605 DRLP/BP 580/30 nm for PMT 2/PE, 740 DCXR/BP 670/20 nm for PMT 3/Cy5/APC, and BP 810/90 nm for PMT 4/Cy7. Filter PMT 3 was replaced for detection of PE/Cy5.5 and APC/Cy5.5 by 740 LP/BP 710/20 nm and the sample was remeasured. Both data files were merged into one to combine the different information on a single-cell basis. The combination of eight antibodies against CD3, CD4, CD8, CD14, CD16, CD19, CD45, and CD56 was used to characterize NK and NKT cells and their subsets. RESULTS: In this way Cy5.5 is measurable at 488-nm and 633-nm excitation. Further, with the two different filters it is possible to distinguish Cy5 from Cy5.5 in the same detection channel (PMT 3). With this method we identified NK and NKT cells, subsets of NK (CD3-16+56+, CD3-16+56-, CD3-16-56+) and NKT (CD3+16+56+, CD3+16-56+) and their CD4+8-, CD4-8+, CD4-8- and CD4+8+ subsets. CONCLUSION: With our adaptations it is possible to discriminate tandem conjugates of Cy5, Cy5.5, and Cy7 for eight-color immunophenotyping. Using this method, novel rare subsets of NK and NKT cells that are CD4/CD8 double positive are reported for the first time.     

The orosomucoid 1 protein (alpha1 acid glycoprotein) is overexpressed in odontogenic myxoma

ABSTRACT: BACKGROUND: Odontogenic myxoma (OM) is a benign, but locally invasive, neoplasm occurring in the jaws. However, the molecules implicated in its development are unknown. OM as well as Dental Follicle (DF), an odontogenic tissue surrounding the enamel organ, is derived from ectomesenchymal/mesencyhmal elements. To identify some protein that could participate in the development of this neoplasm, total proteins from OM were separated by two-dimensional electrophoresis and the profiles were compared with those obtained from DF, used as a control. RESULTS: We identified eight proteins with differential expression; two of them were downregulated and six upregulated in OM. A spot consistently overexpressed in odontogenic myxoma, with a molecular weight of 44-kDa and a pI of 3.5 was identified as the orosomucoid 1 protein. Western blot experiments confirmed the overexpression of this protein in odontogenic myxoma and immunohistochemical assays showed that this protein was mainly located in the cytoplasm of stellate and spindle-shaped cells of this neoplasm. CONCLUSION: Orosomucoid 1, which belongs to a group of acute-phase proteins, may play a role in the modulation of the immune system and possibly it influences the development of OM.     

A constituent of green tea, epigallocatechin-3-gallate, activates endothelial nitric oxide synthase by a phosphatidylinositol-3-OH-kinase-, cAMP-dependent protein kinase-, and Akt-dependent pathway and leads to endothelial-dependent vasorelaxation

Epidemiological studies suggest that tea catechins may reduce the risk of cardiovascular disease, but the mechanisms of benefit have not been determined. The objective of the present study was to investigate the effects of epigallocatechin-3-gallate (EGCG), the major constituent of green tea, on vasorelaxation and on eNOS expression and activity in endothelial cells. EGCG (1-50 microm) induced dose-dependent vasodilation in rat aortic rings. Vasodilation was abolished by pretreatment with Ng-nitro L-arginine methyl ester. In bovine aortic endothelial cells, EGCG increased endothelial nitric oxide (eNOS) activity dose-dependently after 15 min. Treatment with EGCG induced a sustained activation of Akt, ERK1/2, and eNOS Ser1179 phosphorylation. Inhibition of extracellular signal-regulated kinase (ERK)1/2 had no influence on eNOS activity or Ser1179 phosphorylation. Simultaneous treatment of cells with selective inhibitors for cAMP-dependent protein kinase (PKA) and Akt completely prevented the increase in eNOS activity by EGCG after 15 min, indicating that both kinases act in concert. Specific phosphatidylinositol-3-OH-kinase inhibitors yielded identical results. Akt inhibition prevented eNOS Ser1179 phosphorylation, whereas inhibition of PKA did not influence Akt and eNOS Ser1179 phosphorylation. Pretreatment of endothelial cells with EGCG for 4 h markedly enhanced the increase in eNOS activity stimulated by Ca-ionomycin, suggesting that Akt accounts for prolonged eNOS activation. Treatment of cells for 72 h with EGCG did not change eNOS protein levels. Our results indicate that EGCG-induced endothelium-dependent vasodilation is primarily based on rapid activation of eNOS by a phosphatidylinositol 3-kinase-, PKA-, and Akt-dependent increase in eNOS activity, independently of an altered eNOS protein content.