Hirudisins. Hirudin-derived thrombin inhibitors with disintegrin activity.

Recombinant hirudin variants have been designed which inhibit alpha-thrombin by the hirudin mechanism and which in addition exhibit disintegrin activity. These proteins, called "hirudisins," have been engineered by replacing the Ser-Asp-Gly-Glu sequence at the tip of hirudin's finger-like structure (residues 32-35) by Arg-Gly-Asp-Ser (RGDS) to yield hirudisin and Lys-Gly-Asp-Ser (KGDS) to obtain hirudisin-1. Comparison of thrombin inhibition activities showed that hirudisin is 2-fold more potent (K(i) = 160 +/- 70 fM) than hirudisin-1 (K(i) = 370 +/- 44 fM) and recombinant (r)-hirudin (K(i) = 270 +/- 50 fM). alpha-Thrombin-stimulated platelet aggregation was effectively inhibited by r-hirudin, hirudisin, and hirudisin-1 with IC50 of 5.7 to 6.8 nM. Unlike r-hirudin, hirudisin inhibits ADP-induced platelet aggregation (IC50 = 65 microM) 3- to 5-fold stronger than the linear GRGDS- and RGDS-peptide. Direct interaction of hirudisin with purified glycoprotein IIb-IIIa demonstrated that antiplatelet aggregation activity is due to the integrin-directed RGD motif. Disintegrin activity of hirudisin relative to that of reduced and carboxymethylated hirudisin suggests that the conformational strain favors binding to integrins. On the basis of these results, hirudisins appear to be interesting molecules for the design of potential antithrombotic agents with antithrombin as well as antiplatelet aggregation activities.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

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.     

Global Epidemiology of Mental Disorders: What Are We Missing?

Background

Population-based studies provide the understanding of health-need required for effective public health policy and service-planning. Mental disorders are an important but, until recently, neglected agenda in global health. This paper reviews the coverage and limitations in global epidemiological data for mental disorders and suggests strategies to strengthen the data.

Methods

Systematic reviews were conducted for population-based epidemiological studies in mental disorders to inform new estimates for the global burden of disease study. Estimates of population coverage were calculated, adjusted for study parameters (age, gender and sampling frames) to quantify regional coverage.

Results

Of the 77,000 data sources identified, fewer than 1% could be used for deriving national estimates of prevalence, incidence, remission, and mortality in mental disorders. The two major limitations were (1) highly variable regional coverage, and (2) important methodological issues that prevented synthesis across studies, including the use of varying case definitions, the selection of samples not allowing generalization, lack of standardized indicators, and incomplete reporting. North America and Australasia had the most complete prevalence data for mental disorders while coverage was highly variable across Europe, Latin America, and Asia Pacific, and poor in other regions of Asia and Africa. Nationally-representative data for incidence, remission, and mortality were sparse across most of the world.

Discussion

Recent calls to action for global mental health were predicated on the high prevalence and disability of mental disorders. However, the global picture of disorders is inadequate for planning. Global data coverage is not commensurate with other important health problems, and for most of the world''s population, mental disorders are invisible and remain a low priority.     

Nutlin-3 protects kidney cells during cisplatin therapy by suppressing Bax/Bak activation

Nutlins, the newly developed small molecule antagonists of MDM2, activate p53 and induce apoptosis in cancer cells, offering a novel strategy of chemotherapy. Recent studies have further suggested synergistic effects of nutlins with other chemotherapeutic drugs. However, it is unclear whether nutlins increase or decrease the side effects of these drugs in normal non-malignant cells or tissues. Cisplatin is a widely used chemotherapy drug, which has a major side effect of kidney injury. Here we show that Nutlin-3 protected kidney cells against cisplatin-induced apoptosis. The cytoprotective effects of Nutlin-3 were not related to its regulation of p53 or consequent gene expression during cisplatin treatment. Moreover, the protective effects were shown in MDM2-, MDM4-, or p53-deficient cells. On the other hand, Nutlin-3 suppressed mitochondrial events of apoptosis during cisplatin incubation, including Bax activation and cytochrome c release. Nutlin-3 attenuated cisplatin-induced oligomerization of Bax and Bak but not their interactions with Bcl-XL. In isolated mitochondria, Nutlin-3 inhibited cytochrome c release induced by Ca2+, Bim peptide, and recombinant tBid. Importantly, it blocked both Bax and Bak oligomerization under these conditions. Together, the results have uncovered a new pharmacological function of nutlins, i.e. suppression of Bax and Bak, two critical mediators of apoptosis.     

A novel Arabidopsis thaliana protein is a functional peripheral-type benzodiazepine receptor

A key element in the regulation of mammalian steroid biosynthesis is the 18 kDa peripheral-type benzodiazepine receptor (PBR), which mediates mitochondrial cholesterol import. PBR also possess an affinity to the tetrapyrrole metabolite protoporphyrin. The bacterial homolog to the mammalian PBR, the Rhodobacter TspO (CrtK) protein, was shown to be involved in the bacterial tetrapyrrole metabolism. Looking for a similar mitochondrial import mechanism in plants, protein sequences from Arabidopsis and several other plants were found with significant similarities to the mammalian PBR and to the Rhodobacter TspO protein. A PBR-homologous Arabidopsis sequence was cloned and expressed in E. coli. The recombinant gene product showed specific high affinity benzodiazepine ligand binding. Moreover, the protein applied to E. coli protoplasts caused an equal benzodiazepine-stimulated uptake of cholesterol and protoporphyrin IX. These results suggest that the PBR like protein is involved in steroid import and is directing protoporphyrinogen IX to the mitochondrial site of protoheme formation.     

On the mechanism of drug-induced acceleration of phospholipid translocation in the human erythrocyte membrane

Small amphiphilic compounds (M(r)<200 Da) such as anaesthetics and hexane derivatives with different polar groups produced a concentration-dependent acceleration of the slow passive transbilayer movement of NBD-labelled phosphatidylcholine in the human erythrocyte membrane. Above a threshold concentration characteristic for each compound, the flip rate gradually increased at increasing concentrations in the medium. For compound concentrations required to produce a defined flip acceleration, corresponding membrane concentrations were estimated using reported octanol/water partition coefficients. The effective threshold membrane concentrations (50-150 mmol l(-1)) varied in the order: hexylamine>isoflurane=hexanoic acid>hexanol=chloroform>hexanethiol=1,1,2,2-tetrachloroethane>chlorohexane. Apolar hexane, which mainly distributes in the apolar membrane core, was much less effective and supersaturating concentrations were required to enhance flip. Localization of the drug at the lipid-water interface seems to be required for flip acceleration. Such a localization may increase the lateral pressure in this region and the bilayer curvature stress with concomitant decrease of order and rigidity at the interface. This unspecific bilayer perturbation is proposed to enhance the probability of formation of hydrophobic defects in the bilayer, facilitating penetration of the polar head group of the phospholipid into the apolar membrane core.