Glucose-induced alterations of intracellular ionized magnesium in human lymphocytes

The intracellular ionic content of human erythrocytes may be altered by hyperglycaemia. Despite this, very little is known about the cellular mechanisms linking glucose and cellular magnesium homeostasis. We measured intracellular ionized magnesium in human lymphocytes, by means of a fluorimetric technique, total intracellular magnesium by means of atomic absorption spectrophotometry and intracellular ATP by means of HPLC. The incubation of lymphocytes with D-glucose in the absence of insulin was followed by a significant decrease in intracellular ionized magnesium; this effect did not occur when the cells were incubated with L-glucose. The effect of glucose on intracellular ionized magnesium was blocked by amphotericin B and the EC(50) of the effect of glucose on intracellular ionized magnesium was about 5 mmol/l of glucose. The increase of intracellular ionized magnesium in cells incubated in the absence of glucose was followed by a decrease in intracellular ATP. In a Na(+)-free medium the decrease of intracellular ionized magnesium in the presence of glucose was still present and the incubation of lymphocytes with glucose did not modify total intralymphocyte magnesium. By selective permeabilization of cell membranes, we established that glucose could not increase compartmentalized intracellular ionized magnesium. Our data supports the hypothesis that glucose per se induces a substantial decrease in intracellular ionized magnesium, which is probably due to an augmented binding of intracellular ionized magnesium to cellular ATP.     

Island incidence and mainland population density: mammals from Mediterranean islands

Abstract. The reasons why some species are resistant to extinction or are better invaders of islands than others remain unexplained. In this study, we test the hypothesis that mammals living on the mainland at higher density than predicted by the density/body mass relationship have a much greater chance to colonize a small island successfully, and/or that they are less likely to become extinct when living on small islands. For this, we used data compiled on mammals from a number of Mediterranean islands. We show a nested pattern for mammals on western Mediterranean islands, which suggests that the distribution of mammals on these islands is not the result of a random process. Using two comparative methods, we show that mammal density on the mainland, corrected for body mass, is negatively correlated with island size. Mammals with a high density compared to the density/mass relationship are the best invaders and/or probably have less chance of going extinct on small islands when population size is small.     

Arginine side chain assignments in uniformly 15N-labeled proteins using the novel 2D HE(NE)HGHH experiment

A novel 2D NMR experiment, 2D HE(NE)HGHH, is presented for the assignment ofarginine side chain ¹H and ¹⁵N resonances inuniformly ¹⁵N-labeled proteins. Correlations between¹H, ¹Hand ¹H are established on the basis of³J(¹⁵N,¹H) heteronuclear scalarcoupling constants, and sequence-specific assignments are obtained by overlapof these fragments with ¹H chemical shiftsobtained by assignment procedures starting from the polypeptide backbone.Since guanidino protons exchange quite rapidly with the bulk water, the 2DHE(NE)HGHH pulse scheme has been optimized to avoid saturation and dephasingof the water magnetization during the course of the experiment. As anillustration, arginine side chain assignments are presented for two uniformly¹⁵N-labeled proteins of 7 and 23 kDa molecular weight.     

New method for the resolution of the enantiomers of 5,6-Dihydroxy-2-Methyl-Aminotetralin by selective derivatization and HPLC analysis: Application to biological fluids

A new chiral derivatization procedure for the HPLC resolution of chiral catecholamines and structurally related compounds is described. The homochiral reagent, (+)-(R)-1-phenylethyl isocyanate (RPEIC), was added to separate and quantitate the enantiomers of rac-5,6-dihydroxy-2-methyl-aminotetralin, the main metabolite of rac-5,6-diisobutyryl-2-methyl-aminotetralin, a potent dopamine agonist, by reversed-phase HLPC analysis. To avoid catecholamine degradation in the basic reaction medium and to obtain the selective and quantitative derivatization of the amino group of the compound, the reversible complex formation between diphenylborinic acid (DPBA) and the catechol group, in alkaline medium, was performed before homochiral isocyanate addition. The RPEIC derivatization was completed in 30 min and then the DPBA complex was dissociated by adding dilute acid. The structure of intermediates and urea derivatives was confirmed by mass spectrometry. The use of an electrochemical detector, operating in redox mode, allowed HPLC quantitation of enantiomers at the nanogram level in plasma and urine. The derivatization procedure is also suitable for other catecholamine-related compounds. © 1996 Wiley-Liss, Inc.     

Complex Segregation Analysis of Pedigrees from the Gilda Radner Familial Ovarian Cancer Registry Reveals Evidence for Mendelian Dominant Inheritance

Background

Familial component is estimated to account for about 10% of ovarian cancer. However, the mode of inheritance of ovarian cancer remains poorly understood. The goal of this study was to investigate the inheritance model that best fits the observed transmission pattern of ovarian cancer among 7669 members of 1919 pedigrees ascertained through probands from the Gilda Radner Familial Ovarian Cancer Registry at Roswell Park Cancer Institute, Buffalo, New York.

Methodology/Principal Findings

Using the Statistical Analysis for Genetic Epidemiology program, we carried out complex segregation analyses of ovarian cancer affection status by fitting different genetic hypothesis-based regressive multivariate logistic models. We evaluated the likelihood of sporadic, major gene, environmental, general, and six types of Mendelian models. Under each hypothesized model, we also estimated the susceptibility allele frequency, transmission probabilities for the susceptibility allele, baseline susceptibility and estimates of familial association. Comparisons between models were carried out using either maximum likelihood ratio test in the case of hierarchical models, or Akaike information criterion for non-nested models. When assessed against sporadic model without familial association, the model with both parent-offspring and sib-sib residual association could not be rejected. Likewise, the Mendelian dominant model that included familial residual association provided the best-fitting for the inheritance of ovarian cancer. The estimated disease allele frequency in the dominant model was 0.21.

Conclusions/Significance

This report provides support for a genetic role in susceptibility to ovarian cancer with a major autosomal dominant component. This model does not preclude the possibility of polygenic inheritance of combined effects of multiple low penetrance susceptibility alleles segregating dominantly.     

Calcineurin is essential for survival during membrane stress in Candida albicans

The immunosuppressants cyclosporin A (CsA) and FK506 inhibit the protein phosphatase calcineurin and block T-cell activation and transplant rejection. Calcineurin is conserved in microorganisms and plays a general role in stress survival. CsA and FK506 are toxic to several fungi, but the common human fungal pathogen Candida albicans is resistant. However, combination of either CsA or FK506 with the antifungal drug fluconazole that perturbs synthesis of the membrane lipid ergosterol results in potent, synergistic fungicidal activity. Here we show that the C.albicans FK506 binding protein FKBP12 homolog is required for FK506 synergistic action with fluconazole. A mutation in the calcineurin B regulatory subunit that confers dominant FK506 resistance (CNB1-1/CNB1) abolished FK506-fluconazole synergism. Candida albicans mutants lacking calcineurin B (cnb1/cnb1) were found to be viable and markedly hypersensitive to fluconazole or membrane perturbation with SDS. FK506 was synergistic with fluconazole against azole-resistant C.albicans mutants, against other Candida species, or when combined with different azoles. We propose that calcineurin is part of a membrane stress survival pathway that could be targeted for therapy.     

Degradation of trafficking-defective long QT syndrome type II mutant channels by the ubiquitin-proteasome pathway

Mutations in the human ether-a-go-go-related gene (hERG) cause chromosome 7-linked long QT syndrome type II (LQT2). We have shown previously that LQT2 mutations lead to endoplasmic reticulum (ER) retention and rapid degradation of mutant hERG proteins. In this study we examined the role of the ubiquitin-proteasome pathway in the degradation of the LQT2 mutation Y611H. We showed that proteasome inhibitors N-acetyl-L-leucyl-L-leucyl-L-norleucinal and lactacystin but not lysosome inhibitor leupeptin inhibited the degradation of Y611H mutant channels. In addition, ER mannosidase I inhibitor kifunensine and down-regulation of EDEM (ER degradation-enhancing alpha-mannosidase-like protein) also suppressed the degradation of Y611H mutant channels. Proteasome inhibition but not mannosidase inhibition led to the accumulation of full-length hERG protein in the cytosol. The hERG protein accumulated in the cytosol was deglycosylated. Proteasome inhibition also resulted in the accumulation of polyubiquitinated hERG channels. These results suggest that the degradation of LQT2 mutant channels is mediated by the cytosolic proteasome in a process that involves mannose trimming, polyubiquitination, and deglycosylation of mutant channels.     

Poly(ADP-ribose)polymerase activity is reduced in circulating mononuclear cells from type 2 diabetic patients

Poly(ADP-ribose)polymerase (PARP-1), a nuclear enzyme activated by DNA strand breaks, is involved in DNA repair, aging, inflammation, and neoplastic transformation. In diabetes, reactive oxygen and nitrogen species occurring in response to hyperglycemia cause DNA damages and PARP-1 activation. Because circulating mononuclear cells (MNCs) are involved in inflammation mechanisms, these cells were chosen as the experimental model to evaluate PARP-1 levels and activity in patients with type 2 diabetes. MNCs were isolated from 25 diabetic patients (18 M, 7 F, age, 63.5 +/- 10.2 years, disease duration 17.7 +/- 8.2 years) and 11 age and sex matched healthy controls. PARP-1 expression and activity were analyzed by semi-quantitative PCR, Western and activity blot, and immunofluorescence microscopy. PARP-1-mRNA expression was increased in MNCs from all diabetic patients versus controls (P < 0.01), whereas PARP-1 content and activity were significantly lower in diabetic patients (P < 0.0001). To verify whether low PARP-1 levels and activity were due to a proteolytic effect of caspase-3 like, the latter activation was measured by a fluorimetric assay. Caspase-3 activity in MNCs was significantly higher in diabetic patients versus control subjects (P < 0.0001). The different PARP-1 behavior in MNCs from patients with type 2 diabetes could therefore be responsible for the abnormal inflammation and infection responses in diabetes.