Reproductive parameters in Guizhou snub‐nosed monkeys (Rhinopithecus brelichi)

In this study, we present data on reproductive parameters and birth seasonality of Guizhou snub‐nosed monkeys (Rhinopithecus brelichi). Our analyses are based on data from a small captive population collected over 15 years and on 5 years of observations of free‐ranging snub‐nosed monkeys. Captive females (n=4) mature at an age of 70.8±6.7 months and reproduce for the first time at 103.4±7.5 months. The mean interbirth interval was 38.2±4.4 months if the infant survived more than 6 months, which is longer than that in R. roxellana and R. bieti. In the wild and in captivity, births are very seasonal and occur only in a period from the end of March to the end of April. Our data suggest that population growth in Guizhou snub‐nosed monkeys is slow compared with the other two Chinese snub‐nosed monkey species. The risk of extinction is therefore particularly high in this species, given the small overall population size and slow population recovery potential. Am. J. Primatol. 71:266–270, 2009. © 2008 Wiley‐Liss, Inc.     

Determination of ximelagatran, melagatran and two intermediary metabolites in plasma by mixed-mode solid phase extraction and LC-MS/MS

An analytical method was developed for the determination of ximelagatran, an oral direct thrombin inhibitor, its active metabolite melagatran, and the two intermediate metabolites, OH-melagatran and ethyl-melagatran in human plasma. Extraction of plasma was carried out on a mixed mode bonded sorbent material (C8/SO(3)(-)). All four analytes, including their isotope-labelled internal standards, were eluted at high ionic strength with a mixture of 50% methanol and 50% buffer (0.25 M ammonium acetate and 0.05 M formic acid, pH 5.3) with an extraction recovery above 80%. The extracts were demonstrated to be clean in terms of a low concentration of albumin and lysoPC. The sample extraction was fully automated and performed in 96-well plates using a Tecan Genesis pipetting robot. Analysis of the extracts were performed with liquid chromatography followed by positive electrospray ionization mass spectrometry. The low organic content and the low pH of the extracts allowed for, after dilution 1:3 with buffer, direct injection onto the LC-column. The four analytes were separated on a C18 analytical LC-column using gradient elution with the acetonitrile concentration varying from 10 to 30% (v/v) and the ammonium acetate and acetic acid concentration kept constant at 10 and 5 mmol/L, respectively, at a flow rate of 0.75 mL/min. Linearity was achieved over the calibrated range 0.010-4.0 micromol/L with accuracy and relative standard deviation in the range 96.9-101.2% and 6.6-17.1%, respectively at LLOQ, and in the range 94.7-102.6% and 2.7-6.8%, respectively at concentrations above 3 x LLOQ. The method replaces a manual method, and displays the advantages of having a fully automated sample clean-up, no evaporation/reconstitution step, high recovery, and complete LC-separation of all four analytes.     

Evaluation of the prevalence and economic burden of adverse drug reactions presenting to the medical emergency department of a tertiary referral centre: a prospective study

Background  

Adverse drug reactions (ADRs) are now recognized as an important cause of hospital admissions, with a proportion ranging from 0.9–7.9%. They also constitute a significant economic burden. We thus aimed at determining the prevalence and the economic burden of ADRs presenting to Medical Emergency Department (ED) of a tertiary referral center in India     

A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90

P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the Dmd^mdx mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca²⁺ influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of p2rx7 also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome—autophagy—and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.     

Dietary antioxidants prevent age-related retinal pigment epithelium actin damage and blindness in mice lacking αvβ5 integrin

In the aging human eye, oxidative damage and accumulation of pro-oxidant lysosomal lipofuscin cause functional decline of the retinal pigment epithelium (RPE), which contributes to age-related macular degeneration. In mice with an RPE-specific phagocytosis defect due to lack of αvβ5 integrin receptors, RPE accumulation of lipofuscin suggests that the age-related blindness we previously described in this model may also result from oxidative stress. Cellular and molecular targets of oxidative stress in the eye remain poorly understood. Here we identify actin among 4-hydroxynonenal (HNE) adducts formed specifically in β5(-/-) RPE but not in neural retina with age. HNE modification directly correlated with loss of resistance of actin to detergent extraction, suggesting cytoskeletal damage in aging RPE. Dietary enrichment with natural antioxidants, grapes or marigold extract containing macular pigments lutein/zeaxanthin, was sufficient to prevent HNE-adduct formation, actin solubility, lipofuscin accumulation, and age-related cone and rod photoreceptor dysfunction in β5(-/-) mice. Acute generation of HNE adducts directly destabilized actin but not tubulin cytoskeletal elements of RPE cells. These findings identify destabilization of the actin cytoskeleton as a consequence of a physiological, sublethal oxidative burden of RPE cells in vivo that is associated with age-related blindness and that can be prevented by consuming an antioxidant-rich diet.     

Expression of the cystine-glutamate exchanger (xc −) in retinal ganglion cells and regulation by nitric oxide and oxidative stress

The cystine-glutamate exchanger, system x_c ⁻, mediates the Na⁺-independent exchange of cystine into cells, coupled to the efflux of intracellular glutamate. System x_c ⁻ plays a critical role in glutathione homeostasis. Early studies of brain suggested that system x_c ⁻ was present primarily in astrocytes but not neurons. More recent work indicates that certain brain neurons have an active system x_c ⁻. In the retina, system x_c ⁻ has been demonstrated in Müller and retinal pigment epithelial cells. We have recently suggested that two protein components of system x_c ⁻, xCT and 4F2hc, are present in ganglion cells of the intact retina. Here, we have used (1) molecular and immunohistochemical assays to determine whether system x_c ⁻ is present in primary ganglion cells isolated from neonatal mouse retinas and (2) functional assays to determine whether its activity is regulated by oxidative stress in a retinal ganglion cell line (RGC–5). Primary mouse ganglion cells and RGC–5 cells express xCT and 4F2hc. RGC–5 cells take up [³H]glutamate in the absence of Na⁺, and this uptake is blocked by known substrates of system x_c ⁻ (glutamate, cysteine, cystine, quisqualic acid). Treatment of RGC–5 cells with NO and reactive oxygen species donors leads to increased activity of system x_c ⁻ associated with an increase in the maximal velocity of the transporter with no significant change in the substrate affinity. This is the first report of system x_c ⁻ in primary retinal ganglion cells and RGC–5 cells. Oxidative stress upregulates this transport system in RGC–5 cells, and the process is associated with an increase in xCT mRNA and protein but no change in 4F2hc mRNA or protein. This work was supported by National Institutes of Health grants EY014560 and EY012830.     

Mono-(2-ethylhexyl) phthalate (MEHP) regulates glucocorticoid metabolism through 11β-hydroxysteroid dehydrogenase 2 in murine gonadotrope cells

Di-(2-ethylhexyl) phthalate (DEHP) and its metabolite mono-(2-ethylhexyl) phthalate (MEHP) have been classified as toxicants to the reproductive system at the testis level and DEHP may also impair reproductive axis function at the pituitary levels. However, MEHP is 10-fold more potent than DEHP in toxicity and little is known about the toxicological effect of MEHP on pituitary. In this study, we demonstrated that 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), not 11β-HSD1, is strongly expressed in murine gonadotrope LβT2 cells. Interestingly, MEHP inhibited Hsd11b2 mRNA level and 11β-HSD2 enzyme activity in LβT2 cells at as low as 10⁻⁷ M. Corticosterone (CORT) at a concentration of 10⁻⁶ M significantly inhibited LβT2 cell proliferation after 2-day culture, and 10⁻⁶ M RU486, an antagonist of glucocorticoid receptor (GR), reversed this inhibition. However, in the presence of 10⁻⁵ or 10⁻⁴ M MEHP, the minimal concentration of CORT to inhibit the proliferation of LβT2 cells was lowered to 10⁻⁷ M, and 10⁻⁶ M RU486 was not able to completely reverse the CORT effect. In conclusion, along with the regulation of GR, 11β-HSD2 may have a key role in glucocorticoid metabolism in LβT2 cells. MEHP may participate in the glucocorticoid metabolism in LβT2 cells through inhibition of 11β-HSD2 enzyme activity. Such perturbation may be of pathological significance as MEHP may interfere with the reproductive system at pituitary level through regulation of glucocorticoid metabolism, especially in neonates with higher risk of phthalates exposure.     

Computational Modeling and Molecular Physiology Experiments Reveal New Insights into Shoot Branching in Pea

Bud outgrowth is regulated by the interplay of multiple hormones, including auxin, cytokinin, strigolactones, and an unidentified long-distance feedback signal that moves from shoot to root. The model of bud outgrowth regulation in pea (Pisum sativum) includes these signals and a network of five RAMOSUS (RMS) genes that operate in a shoot-root-shoot loop to regulate the synthesis of, and response to, strigolactones. The number of components in this network renders the integration of new and existing hypotheses both complex and cumbersome. A hypothesis-driven computational model was therefore developed to help understand regulation of shoot branching. The model evolved in parallel with stepwise laboratory research, helping to define and test key hypotheses. The computational model was used to verify new mechanisms involved in the regulation of shoot branching by confirming that the new hypotheses captured all relevant biological data sets. Based on cytokinin and RMS1 expression analyses, this model is extended to include subtle but important differences in the function of RMS3 and RMS4 genes in the shoot and rootstock. Additionally, this research indicates that a branch-derived signal upregulates RMS1 expression independent of the other feedback signal. Furthermore, we propose xylem-sap cytokinin promotes sustained bud outgrowth, rather than acting at the earlier stage of bud release.     

转Bt cry1Ab基因水稻对稻田弹尾虫种群数量的影响

以转Bt cry1Ab基因水稻克螟稻1号(KMD1)和克螟稻2号(KMD2)及其亲本非转基因水稻秀水11(XS11)为材料,于2003年9月和翌春4月,调查了KMD1和KMD2对水稻灌浆期和收割后休田期稻田土表落叶层中弹尾虫种群数量的影响.结果表明,KMD植株表达的cry1Ab杀虫蛋白可在稻田环境中残留160 d以上;在水稻灌浆期采用吸虫器法在稻田落叶层中采集到灰橄榄长角跳虫(Entomobrya griseoolivata)和钩圆跳虫(Bourletiella christianseni)等2种弹尾虫,其中灰橄榄长角跳虫在KMD1和KMD2稻田中的种群密度显著高于XS11稻田;在水稻收割后休田期采用网袋法采集到灰橄榄长角跳虫、钩圆跳虫、球角跳虫(Hypogastrura matura)和等节跳虫(Isotoma monochaeta)等4种弹尾虫,其中转Bt基因水稻稻田中的灰橄榄长角跳虫和球角跳虫的种群密度显著高于XS11稻田,且其植株组织残体生物量损失率显著高于XS11.