Puromycin-purified rat brain microvascular endothelial cell cultures exhibit improved barrier properties in response to glucocorticoid induction

In vitro blood-brain barrier (BBB) models using primary rat brain microvessel endothelial cells (BMEC) are often hampered by a lack of culture purity and poor barrier properties. To address these problems, the translation inhibitor puromycin was used to purify rat BMEC cultures. BMEC purities of 99.8% were routinely attained using puromycin treatment, and this technique proved to be far superior to other purification methods of similar difficulty. In contrast to cultures without puromycin treatment, purity of puromycin-treated cultures was unaffected by initial seeding density. Next, rat BMEC monolayer transendothelial electrical resistance (TEER) was increased by glucocorticoid treatment with either corticosterone (CORT) or hydrocortisone (HC), and a corresponding decrease in monolayer permeability to small molecules was observed. Importantly, cultures treated with both puromycin and glucocorticoid attained significantly higher TEER values (CORT 168 +/- 13 Omega x cm2; HC 218 +/- 66 Omega x cm2) than those treated by the glucocorticoid alone (CORT 57 +/- 5 Omega x cm2; HC 70 +/- 2 Omega x cm2). Glucocorticoid induction resulted in BMEC morphological changes that accompanied the increases in TEER, and BMEC tight junctions exhibited improved integrity as visualized by the localization of tight junction proteins zonula occluden-1, occludin and claudin-5. The combined use of puromycin and glucocorticoid therefore provides an in vitro system that is well suited for molecular level BBB investigations.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

The Global Epidemiology and Contribution of Cannabis Use and Dependence to the Global Burden of Disease: Results from the GBD 2010 Study

AimsEstimate the prevalence of cannabis dependence and its contribution to the global burden of disease.MethodsSystematic reviews of epidemiological data on cannabis dependence (1990-2008) were conducted in line with PRISMA and meta-analysis of Observational Studies in Epidemiology (MOOSE) guidelines. Culling and data extraction followed protocols, with cross-checking and consistency checks. DisMod-MR, the latest version of generic disease modelling system, redesigned as a Bayesian meta-regression tool, imputed prevalence by age, year and sex for 187 countries and 21 regions. The disability weight associated with cannabis dependence was estimated through population surveys and multiplied by prevalence data to calculate the years of life lived with disability (YLDs) and disability-adjusted life years (DALYs). YLDs and DALYs attributed to regular cannabis use as a risk factor for schizophrenia were also estimated.ResultsThere were an estimated 13.1 million cannabis dependent people globally in 2010 (point prevalence0.19% (95% uncertainty: 0.17-0.21%)). Prevalence peaked between 20-24 yrs, was higher in males (0.23% (0.2-0.27%)) than females (0.14% (0.12-0.16%)) and in high income regions. Cannabis dependence accounted for 2 million DALYs globally (0.08%; 0.05-0.12%) in 2010; a 22% increase in crude DALYs since 1990 largely due to population growth. Countries with statistically higher age-standardised DALY rates included the United States, Canada, Australia, New Zealand and Western European countries such as the United Kingdom; those with lower DALY rates were from Sub-Saharan Africa-West and Latin America. Regular cannabis use as a risk factor for schizophrenia accounted for an estimated 7,000 DALYs globally.ConclusionCannabis dependence is a disorder primarily experienced by young adults, especially in higher income countries. It has not been shown to increase mortality as opioid and other forms of illicit drug dependence do. Our estimates suggest that cannabis use as a risk factor for schizophrenia is not a major contributor to population-level disease burden.     

Genetic constraints for thermal coadaptation in <Emphasis Type="Italic">Drosophila subobscura</Emphasis>

Background  

Behaviour has been traditionally viewed as a driver of subsequent evolution because behavioural adjustments expose organisms to novel environments, which may result in a correlated evolution on other traits. In Drosophila subobscura, thermal preference and heat tolerance are linked to chromosomal inversion polymorphisms that show parallel latitudinal clines worldwide, such that "cold-climate" ("warm-climate") chromosome arrangements collectively favour a coherent response to colder (warmer) settings as flies carrying them prefer colder (warmer) conditions and have lower (higher) knock out temperatures. Yet, it is not clear whether a genetic correlation between thermal preference and heat tolerance can partially underlie such response.     

Retrospective study of the skin-sparing mastectomy in breast reconstruction

The final appearance of the reconstructed breast is greatly dependent on the relative amounts of skin and breast tissue excised at the time of the mastectomy and on the exact location of the skin incision. A complete mastectomy may be performed using modified skin incisions to avoid the sacrifice of unnecessary breast skin. The type of skin-sparing incision used varies based on the exact location of the tumor and the size of the breast, but it always includes the nipple-areola complex and the biopsy site. The presence of local recurrence, distant disease, or death was determined in 50 consecutive patients who had skin-sparing mastectomies and immediate breast reconstruction between 1985 and 1991 to ascertain the safety of the procedure. The period of follow-up ranged from 23 to 121 months, with a mean of 57 months and a median of 51.5 months. There was no local recurrence, active distant disease was present in five patients, two patients died of distant disease, and there were two unrelated deaths.     

高等植物中的磷酸烯醇式丙酮酸羧激酶

简要介绍了近年来有关高等植物中磷酸烯醇式丙酮酸羧激酶（ＰＥＰＣＫ）的研究进展,并讨论了此酶的结构、功能和调节等方面的问题。