PI3-kinase and mTOR inhibitors differently modulate the function of the ABCG2 multidrug transporter

The ATP-binding cassette (ABC) transporter ABCG2 plays an important role in tissue detoxification and confers multidrug resistance to cancer cells. Identification of expressional and functional cellular regulators of this multidrug transporter is therefore intensively pursued. The PI3-kinase/Akt signaling axis has been implicated as a key element in regulating various cellular functions, including the expression and plasma membrane localization of ABCG2. Here we demonstrate that besides inhibiting their respective target kinases, the pharmacological PI3-kinase inhibitor LY294002 and the downstream mTOR kinase inhibitor rapamycin also directly inhibit ABCG2 function. In contrast, wortmannin, another commonly used pharmacological inhibitor of PI3-kinase does not interact with the transporter. We suggest that direct functional modulation of ABCG2 should be taken into consideration when pharmacological agents are applied to dissect the specific role of PI3-kinase/Akt/mTOR signaling in cellular functions.     

Up-regulation of myocardial connexin-43 in spontaneously hypertensive rats fed red palm oil is most likely implicated in its anti-arrhythmic effects

The purpose of this study was to test our hypothesis that red palm oil (RPO) intake may affect abnormalities of myocardial connexin-43 (Cx43) and protein kinase Cε (PKCε) signaling, and consequently the propensity of the spontaneously hypertensive rat heart (SHR) heart to arrhythmias. SHR and Wistar-Kyoto (WKY) rats fed a standard rat chow plus red palm oil (200?μL/day) for 5?weeks were compared with untreated rats. Cytosolic but not particulate PKCε expression as well as Cx43-mRNA, total Cx43 proteins, and its phoshorylated forms were increased, and disordered localization of Cx43 was attenuated in the left ventricle of RPO-fed SHR compared with untreated rats. These alterations were associated with suppression of early post-ischemic-reperfusion-related ventricular tachycardia and electrically inducible ventricular fibrillation. However, the treatment dose of RPO caused down-regulation of myocardial Cx43, but did not alter its cell membrane distribution or overall PKCε expression in WKY rats. It was, however, associated with poor arrhythmia protection, suggesting overdosing. Results indicate that SHR benefit from RPO intake, particularly because of its apparent anti-arrhythmic effects. This protection can be, in part, attributed to the preservation of cell-to-cell communication via up-regulation of myocardial Cx43, but not with PKCε activation.     

An Introduction to Proteoglycans and Their Localization

Proteoglycans comprise a core protein to which one or more glycosaminoglycan chains are covalently attached. Although a small number of proteins have the capacity to be glycanated and become proteoglycans, it is now realized that these macromolecules have a range of functions, dependent on type and in vivo location, and have important roles in invertebrate and vertebrate development, maintenance, and tissue repair. Many biologically potent small proteins can bind glycosaminoglycan chains as a key part of their function in the extracellular matrix, at the cell surface, and also in some intracellular locations. Therefore, the participation of proteoglycans in disease is receiving increased attention. In this short review, proteoglycan structure, function, and localizations are summarized, with reference to accompanying reviews in this issue as well as other recent literature. Included are some remarks on proteoglycan and glycosaminoglycan localization techniques, with reference to the special physicochemical properties of these complex molecules.     

Population estimates of Alfred mantas (<Emphasis Type="Italic">Manta alfredi</Emphasis>) in central Maldives atolls: North Male,Ari and Baa

The Alfred manta, Manta alfredi, is economically important in the Maldives due to the large number of tourists attracted to SCUBA dive and snorkel with them. It is important to understand the size of populations around main diving points and throughout the Maldives so that manta based tourism activities can be developed more successfully and the impact of tourism can be monitored. Using natural markings to identify individual manta rays, Petersen and Jolly-Seber probabilistic estimators were used to assess population size for main dive sites and the central atolls of North Male, Ari and Baa. Between 2000 and 2009, 1835 individual mantas were identified from 3373 sightings. There was a significant gender bias with a male: female ratio of 1:1.8. Only 24.7% of manta rays were re-sighted between 1 and 34 times with a maximum gap of 7 years and 11 months between sightings. Estimates for populations around main dive sites were 181–562. The estimated population of M. alfredi for North Male atoll is 537 (SE ± 53.2). Estimated atoll populations were extrapolated to obtain an estimate of Manta alfredi population for the entire Maldives of 9,677 individuals.     

Time Course,Distribution and Cell Types of Induction of Transforming Growth Factor Betas following Middle Cerebral Artery Occlusion in the Rat Brain

Transforming growth factor-βs (TGF-β1–3) are cytokines that regulate the proliferation, differentiation, and survival of various cell types. The present study describes the induction of TGF-β1–3 in the rat after focal ischemia at 3 h, 24 h, 72 h and 1 month after transient (1 h) or permanent (24 h) middle cerebral artery occlusion (MCAO) using in situ hybridization histochemistry and quantitative analysis. Double labeling with different markers was used to identify the localization of TGF-β mRNA relative to the penumbra and glial scar, and the types of cells expressing TGF-βs. TGF-β1 expression increased 3 h after MCAO in the penumbra and was further elevated 24 h after MCAO. TGF-β1 was present mostly in microglial cells but also in some astrocytes. By 72 h and 1 month after the occlusion, TGF-β1 mRNA-expressing cells also appeared in microglia within the ischemic core and in the glial scar. In contrast, TGF-β2 mRNA level was increased in neurons but not in astrocytes or microglial cells in layers II, III, and V of the ipsilateral cerebral cortex 24 h after MCAO. TGF-β3 was not induced in cells around the penumbra. Its expression increased in only a few cells in layer II of the cerebral cortex 24 h after MCAO. The levels of TGF-β2 and -β3 decreased at subsequent time points. Permanent MCAO further elevated the levels of all 3 subtypes of TGF-βs suggesting that reperfusion is not a major factor in their induction. TGF-β1 did not co-localize with either Fos or ATF-3, while the co-localization of TGF-β2 with Fos but not with ATF-3 suggests that cortical spreading depolarization, but not damage to neural processes, might be the mechanism of induction for TGF-β2. The results imply that endogenous TGF-βs are induced by different mechanisms following an ischemic attack in the brain suggesting that they are involved in distinct spatially and temporally regulated inflammatory and neuroprotective processes.     

High Resolution X Chromosome-Specific Array-CGH Detects New CNVs in Infertile Males

Context

The role of CNVs in male infertility is poorly defined, and only those linked to the Y chromosome have been the object of extensive research. Although it has been predicted that the X chromosome is also enriched in spermatogenesis genes, no clinically relevant gene mutations have been identified so far.

Objectives

In order to advance our understanding of the role of X-linked genetic factors in male infertility, we applied high resolution X chromosome specific array-CGH in 199 men with different sperm count followed by the analysis of selected, patient-specific deletions in large groups of cases and normozoospermic controls.

Results

We identified 73 CNVs, among which 55 are novel, providing the largest collection of X-linked CNVs in relation to spermatogenesis. We found 12 patient-specific deletions with potential clinical implication. Cancer Testis Antigen gene family members were the most frequently affected genes, and represent new genetic targets in relationship with altered spermatogenesis. One of the most relevant findings of our study is the significantly higher global burden of deletions in patients compared to controls due to an excessive rate of deletions/person (0.57 versus 0.21, respectively; p = 8.785×10⁻⁶) and to a higher mean sequence loss/person (11.79 Kb and 8.13 Kb, respectively; p = 3.435×10⁻⁴).

Conclusions

By the analysis of the X chromosome at the highest resolution available to date, in a large group of subjects with known sperm count we observed a deletion burden in relation to spermatogenic impairment and the lack of highly recurrent deletions on the X chromosome. We identified a number of potentially important patient-specific CNVs and candidate spermatogenesis genes, which represent novel targets for future investigations.     

Hyperosmotic stress induces Axl activation and cleavage in cerebral endothelial cells

Because of the relative impermeability of the blood‐brain barrier (BBB), many drugs are unable to reach the CNS in therapeutically relevant concentration. One method to deliver drugs to the CNS is the osmotic opening of the BBB using mannitol. Hyperosmotic mannitol induces a strong phosphorylation on tyrosine residues in a broad spectrum of proteins in cerebral endothelial cells, the principal components of the BBB. Previously, we have shown that among targets of tyrosine phosphorylation are β‐catenin, extracellular signal‐regulated kinase 1/2 and the non‐receptor tyrosine kinase Src. The aim of this study was to identify new signalling pathways activated by hypertonicity in cerebral endothelial cells. Using an antibody array and immunoprecipitation we identified the receptor tyrosine kinase Axl to become tyrosine phosphorylated in response to hyperosmotic mannitol. Besides activation, Axl was also cleaved in response to osmotic stress. Degradation of Axl proved to be metalloproteinase‐ and proteasome‐dependent and resulted in 50–55 kDa C‐terminal products which remained phosphorylated even after degradation. Specific knockdown of Axl increased the rate of apoptosis in hyperosmotic mannitol‐treated cells; therefore, we assume that activation of Axl may be a protective mechanism against hypertonicity‐induced apoptosis. Our results identify Axl as an important element of osmotic stress‐induced signalling.     

TIMAP is a positive regulator of pulmonary endothelial barrier function

TGF-beta-inhibited membrane-associated protein, TIMAP, is expressed at high levels in endothelial cells (EC). It is regarded as a member of the MYPT (myosin phosphatase target subunit) family of protein phosphatase 1 (PP1) regulatory subunits; however, its function in EC is not clear. In our pull-down experiments, recombinant TIMAP binds preferentially the beta-isoform of the catalytic subunit of PP1 (PP1cbeta) from pulmonary artery EC. As PP1cbeta, but not PP1calpha, binds with MYPT1 into functional complex, these results suggest that TIMAP is a novel regulatory subunit of myosin phosphatase in EC. TIMAP depletion by small interfering RNA (siRNA) technique attenuates increases in transendothelial electrical resistance induced by EC barrier-protective agents (sphingosine-1-phosphate, ATP) and enhances the effect of barrier-compromising agents (thrombin, nocodazole) demonstrating a barrier-protective role of TIMAP in EC. Immunofluorescent staining revealed colocalization of TIMAP with membrane/cytoskeletal protein, moesin. Moreover, TIMAP coimmunoprecipitates with moesin suggesting the involvement of TIMAP/moesin interaction in TIMAP-mediated EC barrier enhancement. Activation of cAMP/PKA cascade by forskolin, which has a barrier-protective effect against thrombin-induced EC permeability, attenuates thrombin-induced phosphorylation of moesin at the cell periphery of control siRNA-treated EC. On the contrary, in TIMAP-depleted EC, forskolin failed to affect the level of moesin phosphorylation at the cell edges. These results suggest the involvement of TIMAP in PKA-mediated moesin dephosphorylation and the importance of this dephosphorylation in TIMAP-mediated EC barrier protection.     

Oxidative damage of albumin in advanced liver disease

Albumin has a number of biological functions and the serum albumin level is related to prognosis in advanced liver disease. Oxidative stress is believed to play an important role in the pathogenesis of liver failure. The aim of the present study was to characterize oxidative modification of albumin in patients with various degrees of liver failure and to investigate implications for its binding function. Patients with liver cirrhosis (n=10), acute-on-chronic liver failure (n=8) and healthy controls (n=15) were included in the study. Three fractions of albumin were separated by HPLC according to the redox state of cysteine-34 and detected by fluorescence as well as UV absorption. Carbonyl groups were measured as a marker of oxidative modification in plasma proteins and, by western blotting, on albumin. Progressive oxidative modification of albumin was found with increasing severity of liver failure indicated by an increased content of carbonyl groups and oxidation of cysteine-34. Fluorescence properties of albumin were altered by oxidation and, in patients with acute-on-chronic liver failure, by high plasma levels of bilirubin. This alteration of albumin fluorescence by bilirubin provides evidence for a preferred binding of bilirubin to the fully reduced form of albumin.     

Influence of various biological matrices (plasma, blood microdialysate) on chromatographic performance in the determination of β-blockers using an alkyl-diol silica precolumn for sample clean-up

A HPLC column-switching system with LiChrospher RP-8 ADS precolumn was applied for the determination of beta-blockers (atenolol, pindolol, propranolol) in human plasma. The influence of biological matrices on the changes of the chromatographic parameters such as retention time, peak symmetry, area and selectivity were investigated. After injection of 5 ml plasma a decrease of retention times of the analytes was observed of up to 25% and an increase of asymmetry factors of up to 5%. Peak areas and selectivities were not changed. The observed effect could indicate changes of chromatographic performance caused by contributions of the analytical column or the ADS precolumn. The experiments with microdialysis excluded the contribution of the analytical column. A detailed investigation of experiments have been discussed in this paper.