Molecular evolution of the Sex-Ratio inversion complex in Drosophila pseudoobscura: analysis of the Esterase-5 gene region

The Sex-Ratio chromosome in Drosophila pseudoobscura is subject to meiotic drive. It is associated with a series of three nonoverlapping paracentric inversions on the right arm of the X chromosome. The esterase-5 gene region has been localized to section 23 within the subbasal inversion of the Sex-Ratio inversion complex, making esterase- 5 a convenient locus for molecular evolutionary analyses of the Sex- Ratio inversion complex and the associated drive system. A 504-bp fragment of noncoding, intergenic DNA from the esterase-5 gene region was amplified and sequenced from 14 Sex-Ratio and 14 Standard X chromosomes of D. pseudoobscura, and from 9 X chromosomes of its two sibling species, Drosophila persimilis and Drosophila miranda. There is extensive sequence differentiation between the Sex-Ratio and Standard chromosomal types. The common Standard chromosome is highly polymorphic, while, as expected from either the neutral mutation theory or the selective sweep hypothesis, the rarer Sex-Ratio chromosome has much less within-chromosome nucleotide polymorphism. We estimate that the Standard and Sex-Ratio chromosomes in D. pseudoobscura diverged between 700,000 and 1.3 Mya, or at least 2 million generations ago. The clustering of D. pseudoobscura Sex-Ratio chromosomes in a neighbor- joining phylogeny indicates a fairly old, monophyletic origin in this species. It appears from these data that Sex-Ratio genes were present prior to the divergence of D. pseudoobscura and D. persimilis and that both the Standard and Sex-Ratio chromosomes of D. persimilis were derived from the Standard chromosome of D. pseudoobscura after the inversion events that isolated the D. pseudoobscura Sex-Ratio chromosome.      

Specific CD45 isoforms differentially regulate T cell receptor signaling.

Multiple isoforms of T cell CD45 tyrosine phosphatase are expressed as a result of alternative RNA splicing among extracellular exons. To discern the presence and identity of distinct functions among CD45 isoforms, we compared thymic T cell activation responses by elevating expression of two CD45 isoforms normally found on quiescent T cells. We report that CD45RABC significantly increased CD4+ thymic T cell proliferation in both a mixed lymphocyte reaction and following anti-T cell receptor (TCR) antibody stimulation. Additionally, CD45RABC enhanced Ca2+ mobilization and phosphotyrosine accumulation, and suppressed the inhibitory effect of anti-CD4 antibodies. By contrast, CD45R0 did not enhance TCR signaling or phosphotyrosine levels in CD4+ thymic T cells and required a TCR co-stimulus to augment cellular proliferation. These studies provide genetic evidence that alternative CD45 isoforms are functionally distinct and disclose a unique mechanism by which T cell immunologic responsiveness can be modified.     

Identification and characterization of major flavonoids and caffeoylquinic acids in three Compositae plants by LC/DAD-APCI/MS

In this study, a liquid chromatography/diode array detector-atmospheric pressure chemical ionization/mass spectrometry (LC/DAD-APCI/MS) was successfully developed to identify and characterize the main flavonoids and caffeoylquinic acids (CQAs) of three common Compositae plants (Chrysanthemum morifolium Raman, Artemisia annua, and Chrysanthemum coronarium) which have been used as herbal medicine. Identifications were performed by comparing the retention time, UV and mass spectra of samples with standards or/and earlier publications. The crude methanolic extracts of these plants were assayed directly using LC/MS without any further pretreatment. The proposed method is rapid and reproducible and is useful for characterization and evaluation of different plant flavonoids and CQAs. A total of 41 different flavonoids and 6 CQAs were identified and confirmed by APCI-MS. The main components of three Compositae plants were also compared. Although there exist some similarities in the flavonoidic content of the leaf and flower of C. morifolium, significant variations in their varieties and concentrations were observed. Artemisia annua processes substantial amount of alkylated derivatives of flavones and Chrysanthemum coronarium contains only CQAs. These findings suggest that although all the plants studied are from the same Compositae family, their flavonoids and phenolic compositions are markedly different. The proposed method is useful for further chromatographic fingerprinting of plant flavonoids.     

Progesterone reduces erectile dysfunction in sleep-deprived spontaneously hypertensive rats

Background  

Paradoxical sleep deprivation (PSD) associated with cocaine has been shown to enhance genital reflexes (penile erection-PE and ejaculation-EJ) in Wistar rats. Since hypertension predisposes males to erectile dysfunction, the aim of the present study was to investigate the effects of PSD on genital reflexes in the spontaneously hypertensive rat (SHR) compared to the Wistar strain. We also extended our study to examine how PSD affect steroid hormone concentrations involved in genital events in both experimental models.     

Multiplexed genotyping of ABC transporter polymorphisms with the Bioplex suspension array

We have developed and validated a consolidated bead-based genotyping platform, the Bioplex suspension array for simultaneous detection of multiple single nucleotide polymorphisms (SNPs) of the ATP-binding cassette transporters. Genetic polymorphisms have been known to influence therapeutic response and risk of disease pathologies. Genetic screening for therapeutic and diagnostic applications thus holds great promise in clinical management. The allele-specific primer extension (ASPE) reaction was used to assay 22 multiplexed SNPs for eight subjects. Comparison of the microsphere-based ASPE assay results to sequencing results showed complete concordance in genotype assignments. The Bioplex suspension array thus proves to be a reliable, cost-effective and high-throughput technological platform for genotyping. It can be easily adapted to customized SNP panels for specific applications involving large-scale mutation screening of clinically relevant markers.     

Progesterone reduces erectile dysfunction in sleep-deprived spontaneously hypertensive rats

Background

The rate-limiting step in prostaglandin (PG) biosynthesis is catalyzed by phospholipase A2 (PLA2) enzymes which hydrolyze arachidonic acid from membrane phospholipids. Despite their importance in uterine PG production, little is known concerning the specific PLA2 enzymes that regulate arachidonic acid liberation in the uterine endometrium. The objectives of this study were to evaluate the expression and activities of calcium-independent Group VI and Group IVC PLA2 (PLA2G6 and PLA2G4C) and calcium-dependent Group IVA PLA2 (PLA2G4A) enzymes in the regulation of bovine uterine endometrial epithelial cell PG production.

Methods

Bovine endometrial epithelial cells in culture were treated with oxytocin, interferon-tau and the PLA2G6 inhibitor bromoenol lactone, alone and in combination. Concentrations of PGF2alpha and PGE2 released into the medium were analyzed. Western blot analysis was performed on cellular protein to determine the effects of treatments on expression of PLA2G4A, PLA2G6 and PLA2G4C. Group-specific PLA2 activity assays were performed on cell lysates following treatment with oxytocin, interferon-tau or vehicle (control), alone and in combination. To further evaluate the role of specific PLA2 enzymes in uterine cell PG biosynthesis, cells were transfected with cDNAs encoding human PLA2G6 and PLA24C, treated as described above and PG assays performed.

Results

Constitutive cell production of PGF2alpha was about two-fold higher than PGE2. Oxytocin stimulated production of both PGs but the increase of PGF2alpha was significantly greater. Interferon-tau diminished oxytocin stimulation of both PGs. The PLA2G6 inhibitor, bromoenol lactone, abolished oxytocin-stimulated production of PGF2alpha. Treatments had little effect on PLA2G4A protein expression. In contrast, oxytocin enhanced expression of PLA2G6 and this effect was diminished in the presence of interferon-tau. Expression of PLA2G4C was barely detectable in control and oxytocin treated cells but it was enhanced in cells treated with interferon-tau. Oxytocin stimulated PLA2 activity in assays designed to evaluate PLA2G6 activity and interferon-tau inhibited this response. In assays designed to measure PLA2G4C activity, only interferon-tau was stimulatory. Cells overexpressing PLA2G6 produced similar quantities of the two PGs and these values were significantly higher than PG production by non-transfected cells. Oxytocin stimulated production of both PGs and this response was inhibited by interferon-tau. Bromoenol lactone inhibited oxtocin stimulation of PGF2alpha production but stimulated PGE2 production, both in the absence and presence of oxytocin. Cells over-expressing PLA2G4C produced more PGE2 than PGF2alpha and interferon-tau stimulated PGE2 production.

Conclusion

Results from these studies indicate that oxytocin stimulation of uterine PGF2alpha production is mediated, at least in part, by up-regulation of PLA2G6 expression and activity. In addition to its known inhibitory effect on oxytocin receptor expression, interferon-tau represses oxytocin-stimulated PLA2G6 expression and activity and this contributes to diminished PGF2alpha production. Furthermore, endometrial cell PGE2 biosynthesis was associated with PLA2G4C expression and activity and interferon-tau was stimulatory to this process.     

Establishment of epithelial and fibroblast cell cultures and cell lines from primary renal cancer nephrectomies

Renal cell carcinoma (RCC) is one of the most lethal urogenital cancers and effective treatment of metastatic RCC remains an elusive target. Cell lines enable the in vitro investigation of molecular and genetic changes leading to renal carcinogenesis and are important for evaluating cellular drug response or toxicity. This study details a fast and easy protocol of establishing epithelial and fibroblast cell cultures or cell lines concurrently from renal cancer nephrectomy tissue. The protocol involves mechanical disaggregation, collagenase digestion and cell sieving for establishing epithelial cells while fibroblast cells were grown from explants. This protocol has been modified from previous published reports with additional antibiotics and washing steps added to eliminate microbial contamination from the surgical source. Cell characterisation was carried out using immunofluorescence and quantitative polymerase chain reaction. Eleven stable epithelial renal tumour cell lines of various subtypes, including rare subtypes, were established with a spontaneous immortalisation rate of 21.6% using this protocol. Eight fibroblast cell cultures grew successfully but did not achieve spontaneous immortalisation. Cells of epithelial origin expressed higher expressions of epithelial markers such as pan‐cytokeratin, cytokeratin 8 and E‐cadherin whereas fibroblast cells expressed high α‐smooth muscle actin. Further mutational analysis is needed to evaluate the genetic or molecular characteristics of the cell lines.     

Mapping the consequence of Notch1 proteolysis in vivo with NIP-CRE

The four highly conserved Notch receptors receive short-range signals that control many biological processes during development and in adult vertebrate tissues. The involvement of Notch1 signaling in tissue self-renewal is less clear, however. We developed a novel genetic approach N(1)IP-CRE (Notch1 Intramembrane Proteolysis) to follow, at high resolution, the descendents of cells experiencing Notch1 activation in the mouse. By combining N(1)IP-CRE with loss-of-function analysis, Notch activation patterns were correlated with function during development, self-renewal and malignancy in selected tissues. Identification of many known functions of Notch1 throughout development validated the utility of this approach. Importantly, novel roles for Notch1 signaling were identified in heart, vasculature, retina and in the stem cell compartments of self-renewing epithelia. We find that the probability of Notch1 activation in different tissues does not always indicate a requirement for this receptor and that gradients of Notch1 activation are evident within one organ. These findings highlight an underappreciated layer of complexity of Notch signaling in vivo. Moreover, NIP-CRE represents a general strategy applicable for monitoring proteolysis-dependent signaling in vivo.     

Removal of basic and reactive dyes using ethylenediamine modified rice hull

Wastewaters from textile industries may contain a variety of dyes that have to be removed before their discharge into waterways. Rice hull, an agricultural by-product, was modified using ethylenediamine to introduce active sites on its surface to enable it to function as a sorbent for both basic and reactive dyes. The sorption characteristics of Basic Blue 3 (BB3) and Reactive Orange 16 (RO16) by ethylenediamine modified rice hull (MRH) were studied under various experimental conditions. Sorption was pH and concentration dependent. Simultaneous removal of BB3 and RO16 occurred at pH greater than 4. The kinetics of dye sorption fitted a pseudo-second order rate expression. Increase in agitation rate had no effect on the sorption of BB3 but increased uptake of RO16 on MRH. Decreasing particle size increased the uptake of dyes in binary dye solutions. Equilibrium data could be fitted into both the Langmuir and Freundlich isotherms. Maximum sorption capacities calculated from the Langmuir model are 14.68 and 60.24 mg/g for BB3 and RO16, respectively in binary dye solutions. This corresponds to an enhancement of 4.5 and 2.4 fold, respectively, compared to single dye solutions. MRH therefore has the potential of being used as an efficient sorbent for the removal of both dyes in textile wastewaters.