Identification of an EcoRI restriction site for a rapid and precise determination of beta-asarone-free Acorus calamus cytotypes

Calamus (Acorus calamus L., Araceae) is an aromatic herb, indigenous to Central Asia and Eastern Europe. The fragrant oils obtained by alcoholic extraction of the rhizome are mainly used in the pharmaceutical and oenological industries. Nevertheless, the occurrence of beta-asarone [(Z)-1,2,4-trimethoxy-5-prop-1-enyl-benzene] limits the possibility of its use due to the carcinogenic properties of this compound. The aim of this work was to identify a diploid beta-asarone-free A. calamus by using chemical and molecular approaches. For these purposes alcoholic extracts of both diploid and triploid A. calamus were analyzed by gas chromatography-mass spectrometry (GC-MS) and comparison of the 700 bp sequence of the non-transcribed spacer (NTS) in the 5S-rRNA gene was also performed. Alcoholic extracts of the triploid A. calamus were characterized by a higher percentage of beta-asarone (11%), which was the main compound, followed by higher percentages of camphene (2.27%), E-beta-ocimene (3.28%), camphor (1.54%), calarene (1.42%), alpha-selinene (5.02%) and tau-cadinol (2.00%), when compared to the diploid A. calamus. The latter had higher percentages of iso-shyobunone (8.62%), beta-sesquiphellandrene (3.28%), preiso calamendiol (22.81%) and acorone (26.33%), and completely lacked of beta-asarone. The 5S-rRNA spacer region of both diploid and triploid A. calamus were amplified by PCR using a pair of primers located at the 3' and 5' ends of the coding sequence of 5S-rRNA gene. The resulting PCR products (about 700 bp) were gel purified, subcloned into pGEM-T Easy vector and sequenced. By aligning the isolated nucleotide sequences of the two varieties and the sequences from different A. calamus chemotypes present in Genbank, sequence diversities were found in the spacer region. Furthermore, the PCR products were digested by using EcoRI. The restriction profile of the spacer domain resulted different for the two cytotypes. Along with chemical analysis of alcoholic extracts, sequence analysis coupled to restriction mapping was demonstrated to represent a powerful tool to distinguish the A. calamus diploid cytotype from the others. The security and effective usage of the diploid beta-asarone-free A. calamus was also discussed.     

Long-Term Trends in Matrilineal Inbreeding Among the Japanese Macaques of Arashiyama B Troop

In a long-term study of sexual behavior in Japanese macaques, we found that matrilineal inbreeding accounted for 2.9% of the copulations recorded for the Arashiyama B troop during 7 mating seasons between 1968 and 1984. Of the 906 copulatory dyads, 46 (5.1%) occurred among kin. Close matrilineal kin dyads (r = 1/2–1/8, 1.1% of the total of copulatory dyads) strongly avoided matrilineal inbreeding, but for remote kin dyads (r > 1/8, 4.0% of the total) the tendency was weaker in some years. Among the possible determinants of matrilineal inbreeding, we found that it tended to occur among younger and lower-ranking males as an effect of troop demographic changes. There is no significant association between female rank and matrilineal inbreeding. Our results are consistent with the hypothesis that different degrees of kin relatedness are discriminated by individuals with respect to mate choice.     

IL-21 counteracts the regulatory T cell-mediated suppression of human CD4+ T lymphocytes

High expression of IL-21 and/or IL-21R has been described in T cell-mediated inflammatory diseases characterized by defects of counterregulatory mechanisms. CD4(+)CD25(+) regulatory T cells (Treg) are a T cell subset involved in the control of the immune responses. A diminished ability of these cells to inhibit T cell activation has been documented in immune-inflammatory diseases, raising the possibility that inflammatory stimuli can block the regulatory properties of Treg. We therefore examined whether IL-21 controls CD4(+)CD25(+) T cell function. We demonstrate in this study that IL-21 markedly enhances the proliferation of human CD4(+)CD25(-) T cells and counteracts the suppressive activities of CD4(+)CD25(+) T cells on CD4(+)CD25(-) T cells without affecting the percentage of Foxp3(+) cells or survival of Treg. Additionally, CD4(+)CD25(+) T cells induced in the presence of IL-21 maintain the ability to suppress alloresponses. Notably, IL-21 enhances the growth of CD8(+)CD25(-) T cells but does not revert the CD4(+)CD25(+) T cell-mediated suppression of this cell type, indicating that IL-21 makes CD4(+) T cells resistant to suppression rather than inhibiting CD4(+)CD25(+) T cell activity. Finally, we show that IL-2, IL-7, and IL-15, but not IL-21, reverse the anergic phenotype of CD4(+)CD25(+) T cells. Data indicate that IL-21 renders human CD4(+)CD25(-) T cells resistant to Treg-mediated suppression and suggest a novel mechanism by which IL-21 could augment T cell-activated responses in human immune-inflammatory diseases.     

DQ α and β RFLP reveals the composition of the DQ molecule recognized by T-cell clones

Pst I RFLP, revealed with DQ and DQ probes, was compared with Taq I RFLP using a panel of DR-homozygous cell lines and HLA-typed family members. Taq I patterns, characteristic for each DR-associated DQ and allelic forms, were recognized in the homozygous state and then proven to segregate in the heterozygous members of informative families. The presence of both specific and chains was found to be necessary to form the type of DQ molecule specifically recognized by two alloreactive T-cell clones. Particular and associations also seem to be responsible for some Dw splits of the DRw6-positive cells. Taq I RFLP analysis may be more complex than the Pst I analysis, but is certainly more informative and complete, considering the type of information we were seeking by performing these types of experiments.Abbreviations used in this paper BSA bovine serum albumin - GLO glyoxalase - kb kilobase(s) - LCL lymphoblastoid cell line - MHC major histocompatibility complex - PBL peripheral blood lymphocyte - PLT primed lymphocyte test - RFLP restriction fragment length polymorphism - SDS sodium dodecyl sulfate - SSC standard sodium citrate - SSCP sodium, sodium citrate, sodium phosphate - TBE Tris-borate, boric acid, ethylenediaminetetraacetate (EDTA) - TCGF T-cell growth factor     

A nuclear export signal and phosphorylation regulate Dok1 subcellular localization and functions

Dok1 is believed to be a mainly cytoplasmic adaptor protein which down-regulates mitogen-activated protein kinase activation, inhibits cell proliferation and transformation, and promotes cell spreading and cell migration. Here we show that Dok1 shuttles between the nucleus and cytoplasm. Treatment of cells with leptomycin B (LMB), a specific inhibitor of the nuclear export signal (NES)-dependent receptor CRM1, causes nuclear accumulation of Dok1. We have identified a functional NES (348LLKAKLTDPKED359) that plays a major role in the cytoplasmic localization of Dok1. Src-induced tyrosine phosphorylation prevented the LMB-mediated nuclear accumulation of Dok1. Dok1 cytoplasmic localization is also dependent on IKKbeta. Serum starvation or maintaining cells in suspension favor Dok1 nuclear localization, while serum stimulation, exposure to growth factor, or cell adhesion to a substrate induce cytoplasmic localization. Functionally, nuclear NES-mutant Dok1 had impaired ability to inhibit cell proliferation and to promote cell spreading and cell motility. Taken together, our results provide the first evidence that Dok1 transits through the nucleus and is actively exported into the cytoplasm by the CRM1 nuclear export system. Nuclear export modulated by external stimuli and phosphorylation may be a mechanism by which Dok1 is maintained in the cytoplasm and membrane, thus regulating its signaling functions.     

A common variant in the telomerase RNA component is associated with short telomere length

Background

Telomeres shorten as cells divide. This shortening is compensated by the enzyme telomerase. We evaluated the effect of common variants in the telomerase RNA component (TERC) gene on telomere length (TL) in the population-based Health Aging and Body Composition (Health ABC) Study and in two replication samples (the TwinsUK Study and the Amish Family Osteoporosis Study, AFOS).

Methodology

Five variants were identified in the TERC region by sequence analysis and only one SNP was common (rs2293607, G/A). The frequency of the G allele was 0.26 and 0.07 in white and black, respectively. Testing for association between TL and rs2293607 was performed using linear regression models or variance component analysis conditioning on relatedness among subjects.

Results

The adjusted mean TL was significantly shorter in 665 white carriers of the G allele compared to 887 non-carriers from the Health ABC Study (4.69±0.05 kbp vs. 4.86±0.04 kbp, measured by quantitative PCR, p = 0.005). This association was replicated in another white sample from the TwinsUK Study (6.90±0.03 kbp in 301 carriers compared to 7.06±0.03 kbp in 395 non-carriers, measured by Southern blots, p = 0.009). A similar pattern of association was observed in whites from the family-based AFOS and blacks from the Health ABC cohort, although not statistically significant, possibly due to the lower allele frequency in these populations. Combined analysis using 2,953 white subjects from 3 studies showed a significant association between TL and rs2293607 (β = −0.19±0.04 kbp, p = 0.001).

Conclusion

Our study shows a significant association between a common variant in TERC and TL in humans, suggesting that TERC may play a role in telomere homeostasis.     

Plasma Membrane-Associated Glycohydrolases Along Differentiation of Murine Neural Stem Cells

The activities of plasma membrane associated sialidase Neu3, total β-glucosidase, CBE-sensitive β-glucosidase, non-lysosomal β-glucosyl ceramidase GBA2, β-galactosidase, β-hexosaminidase and sphingomyelinase were determined at three different stages of differentiation of murine neural stem cell cultures, corresponding to precursors, commited progenitors, and differentiated cells. Cell immunostaining for specific markers of the differentiation process, performed after 7 days in culture in presence of differentiating agents, clearly showed the presence of oligodendrocytes, astrocytes and neurons. Glial cells were the most abundant. Sialidase Neu3 after a decrease from progenitors to precursors, showed an increase parallel to the differentiation process. All the other glycosidases increased their activity along differentiation. The activity of CBE-sensitive β-glucosidase and GBA2 were very similar at the precursor stage, but CBE-sensitive β-glucosidase increased 7 times while GBA2 only two in the differentiated cells. In addition, we analysed also sphingomyelinase as enzyme specifically associated to sphingolipids. The activity of this enzyme increased from precursors to differentiated cells.     

Potential role of the methylation of VEGF gene promoter in response to hypoxia in oxygen‐induced retinopathy: beneficial effect of the absence of AQP4

Hypoxia‐dependent accumulation of vascular endothelial growth factor (VEGF) plays a major role in retinal diseases characterized by neovessel formation. In this study, we investigated whether the glial water channel Aquaporin‐4 (AQP4) is involved in the hypoxia‐dependent VEGF upregulation in the retina of a mouse model of oxygen‐induced retinopathy (OIR). The expression levels of VEGF, the hypoxia‐inducible factor‐1α (HIF‐1α) and the inducible form of nitric oxide synthase (iNOS), the production of nitric oxide (NO), the methylation status of the HIF‐1 binding site (HBS) in the VEGF gene promoter, the binding of HIF‐1α to the HBS, the retinal vascularization and function have been determined in the retina of wild‐type (WT) and AQP4 knock out (KO) mice under hypoxic (OIR) or normoxic conditions. In response to 5 days of hypoxia, WT mice were characterized by (i) AQP4 upregulation, (ii) increased levels of VEGF, HIF‐1α, iNOS and NO, (iii) pathological angiogenesis as determined by engorged retinal tufts and (iv) dysfunctional electroretinogram (ERG). AQP4 deletion prevents VEGF, iNOS and NO upregulation in response to hypoxia thus leading to reduced retinal damage although in the presence of high levels of HIF‐1α. In AQP4 KO mice, HBS demethylation in response to the beginning of hypoxia is lower than in WT mice reducing the binding of HIF‐1α to the VEGF gene promoter. We conclude that in the absence of AQP4, an impaired HBS demethylation prevents HIF‐1 binding to the VEGF gene promoter and the relative VEGF transactivation, reducing the VEGF‐induced retinal damage in response to hypoxia.