Calcineurin activity is regulated both by redox compounds and by mutant familial amyotrophic lateral sclerosis-superoxide dismutase

Calcineurin (CN) is a protein phosphatase involved in a wide range of cellular responses to calcium-mobilizing signals, and a role for this enzyme in neuropathology has been postulated. We have investigated the possibility that redox modulation of CN activity is relevant to neuropathological conditions where an imbalance in reactive oxygen species has been described. We have monitored CN activity in cultured human neuroblastoma SH-SY5Y cells and obtained evidence that CN activity is promoted by treatment with ascorbate or dithiothreitol and impaired by oxidative stress. Evidence for the existence of a redox regulation of this enzyme has been also obtained by overexpression of wild-type antioxidant Cu,Zn superoxide dismutase (SOD1) that promotes CN activity and protects it from oxidative inactivation. On the contrary, overexpression of mutant SOD1s associated with familial amyotrophic lateral sclerosis (FALS) impairs CN activity both in transfected human neuroblastoma cell lines and in the motor cortex of brain from FALS-transgenic mice. These data suggest that CN might be a target in the pathogenesis of SOD1-linked FALS.     

细胞色素P450 2D6缺陷型等位基因的家系分析

利用等位基因特民扩增法(ASA)为基础的基因分型法,对细胞色素P4502D6 (CYP2D6)缺陷型等位基因携带者的9个家庭共38个进行了基因分型,并与用右旋美沙芬为 探针的表型分型法进行对比,发现两种方法的结果是一致的,CYP2D6酶缺陷型等位基因呈常染色体隐性遗传。 Abstract:A genotyping method based on the principle of allele-specific amplification and a phenotyping procedure with dextromethorphan as a probe were employed in familial study of nine families with 38 members for the cytochrome P450 2D6(CYP2D6)deficient alleles——CYP2D6A,CYP2D6B,CYP2D6D and CYP2D6T.The results showed that the CYP2D6 deficient alleles were inherited as an autosomal recessive trait.     

Effect of maternal diet rich in omega‐6 and omega‐9 fatty acids on the liver of LDL receptor‐deficient mouse offspring

BACKGROUND: Omega‐6 fatty acids are important to fetal development. However, during gestation/lactation, these fatty acids may contribute toward the development of fat tissue. Omega‐9 fatty acids are associated with a reduction in serum lipids and protection from liver disease. OBJECTIVES: The present study investigated the effect of the maternal intake of omega‐6 and omega‐9 in hypercholesterolemic mothers on the liver of the offspring. METHODS: LDL receptor–deficient mice were fed a diet rich in either omega‐6 (E6D) or omega‐9 (E9D) for 45 days prior to mating and until the birth of the offspring, evaluating the effect on the offspring liver in comparison to a standard diet (STD). RESULTS: Mothers fed with the E6D experienced an increase in total cholesterol (TC) and the offspring exhibited an increase in TC, hepatic triglycerides (TG), and CC‐chemokine ligand (CCL)2/monocyte chemoattractant protein (MCP)‐1 as well as a reduction in HDL. Histological analysis on this group revealed steatosis, leukocyte infiltrate, and increased CCL2/MCP‐1 expression. The ultrastructural analysis revealed hepatocytes with lipid droplets and myofibroblasts. The offspring of mothers fed the standard diet exhibited low serum TC, but microvesicular steatosis was observed. The offspring of mothers fed the E9D exhibited lower serum and hepatic TG as well as higher LDL in comparison to the other diets. The histological analyses revealed lower steatosis and leukocyte infiltrate. CONCLUSIONS: The findings suggest that hypercholesterolemic mothers with a diet rich in omega‐6 fatty acids predispose their offspring to steatohepatitis, whereas a diet rich in omega‐9 has a protective effect. Birth Defects Res (Part B) 89:164–170, 2010. © 2010 Wiley‐Liss, Inc.     

A microscopic study of kidney tissue in Familial Mediterranean Fever patients

Familial Mediterranean Fever (FMF) is an autosomal recessive hereditary disease leading mostly to renal failure and nephrotic syndrome. The ultrastructure of kidney has not been fully investigated in FMF associated renal disease. The aim of this study is to provide further evidence on the ultrastructure of kidney in patients with FMF who suffer from renal disease. Renal biopsies obtained from two patients who were diagnosed with FMF renal disease complications were examined. Examination of renal tissue by light and electron microscopy identified degenerations both in tubules and the filtration barrier. Foot processes were partly effaced. Amorphous material was found in thickened glomerular basement membranes. Fibrous material deposits in thick Bowman's capsule wall were also seen. Finally, degeneration in the form of folding of plasma membrane and vacuolization as well as fusion in mitochondria cristae, was observed. Accumulation of tissue remnants in the lumen was also found in tubules.     

Tbx3, a transcriptional factor, involves in proliferation and osteogenic differentiation of human adipose stromal cells

Both, diabetes mellitus (DM) and hypercholesterolemia (HCH) are known as risk factors of ischemic heart disease, however, the effects of experimental DM, as well as of HCH alone, on ischemia/reperfusion-induced myocardial injury are not unequivocal. We have previously demonstrated an enhanced resistance to ischemia-induced arrhythmias in rat hearts in the acute phase of DM. Our objectives were thus to extend our knowledge on how DM in combination with HCH, a model that is relevant to diabetic patients with altered lipid metabolism, may affect the size of myocardial infarction and susceptibility to arrhythmias. A combination of streptozotocin (STZ; 80 mg/kg, i.p.) and the fat–cholesterol diet (1% cholesterol, 1% coconut oil; FCHD) was used as a double-disease model mimicking DM and HCH simultaneosly occurring in humans. Following 5 days after STZ injection and FCHD leading to increased blood glucose and cholesterol levels, anesthetized open-chest diabetic, diabetic–hypercholesterolemic (DM–HCH) and age-matched control rats were subjected to 6-min ischemia (occlusion of LAD coronary artery) followed by 10 reperfusion to test susceptibility to ventricular arrhythmias in the in vivo experiments and to 30-min ischemia and subsequent 2-h reperfusion for the evaluation of the infarct size (IS) in the Langendorff-perfused hearts. The incidence of the most life-threatening ventricular arrhythmia, ventricular fibrillation, was significantly increased in the DM–HCH rats as compared with non-diabetic control animals (100% vs. 50%; p<0.05). Likewise, arrhythmia severity score (AS) was significantly higher in the DM–HCH rats than in the controls (4.9±0.2 vs. 3.5±0.5; p<0.05), but was not increased in the diabetic animals (AS 3.7±0.9; p>0.05 vs. controls). Diabetic hearts exhibited a reduced IS (15.1±3.0% of the area at risk vs. 37.6±2.8% in the control hearts; p<0.05), however, a combination of DM and HCH increased the size of myocardial infarction to that observed in the controls. In conclusion, HCH abrogates enhanced resistance to ischemia-reperfusion injury in the diabetic rat heart.     

The cellular trafficking of the secretory proprotein convertase PCSK9 and its dependence on the LDLR

Mutations in the proprotein convertase PCSK9 gene are associated with autosomal dominant familial hyper- or hypocholesterolemia. These phenotypes are caused by a gain or loss of function of proprotein convertase subtilisin kexin 9 (PCSK9) to elicit the degradation of the low-density lipoprotein receptor (LDLR) protein. Herein, we asked whether the subcellular localization of wild-type PCSK9 or mutants of PCSK9 and the LDLR would provide insight into the mechanism of PCSK9-dependent LDLR degradation. We show that the LDLR is the dominant partner in regulating the cellular trafficking of PCSK9. In cells lacking the LDLR, PCSK9 localized in the endoplasmic reticulum (ER). In cells expressing the LDLR, PCSK9 sorted to post-ER compartments (i.e. endosomes in cell lines and Golgi apparatus in primary hepatocytes), where it colocalized with the LDLR. In cell lines, PCSK9 also colocalized with the LDLR at the cell surface, requiring the presence of the C-terminal Cys/His-rich domain of PCSK9. We provide evidence that PCSK9 promotes the degradation of the LDLR by an endocytic mechanism, as small interfering RNA-mediated knockdown of the clathrin heavy chain reduced the functional activity of PCSK9. We also compared the subcellular localization of natural mutants of PCSK9 with that of the wild-type enzyme in human hepatic (HuH7) cells. Whereas the mutants associated with hypercholesterolemia (S127R, F216L and R218S) localized to endosomes/lysosomes, those associated with hypocholesterolemia did not reach this compartment. We conclude that the sorting of PCSK9 to the cell surface and endosomes is required for PCSK9 to fully promote LDLR degradation and that retention in the ER prevents this activity. Mutations that affect this transport can lead to hyper- or hypocholesterolemia.     

Parkinson's disease: from causes to mechanisms

Parkinson's disease (PD) is a common age-related, progressive neurodegenerative disease of unknown etiology. Environmental factors have long been suspected to participate in the pathogenesis of PD due to the existence of neurotoxins that preferentially damage the dopaminergic nigrostriatal pathway. In the past few years, novel insights into the degenerative process have been provided by the discovery of genes responsible for rare monogenic parkinsonian syndromes. Compelling evidence is accumulating, suggesting that the products of several of these genes can interact with environmental toxins and intervene in molecular pathways controlling the functional integrity of mitochondria.     

Selective silencing of a mutant transthyretin allele by small interfering RNAs

Familial amyloidotic polyneuropathy (FAP) is a hereditary systemic amyloidosis caused by dominantly acting missense mutations in the gene encoding transthyretin (TTR). The most common mutant TTR is of the Val30Met type, which results from a point mutation. Because the major constituent of amyloid fibrils is mutant TTR, agents that selectively suppress mutant TTR expression could be powerful therapeutic tools. This study has been performed to evaluate the use of small interfering RNAs (siRNAs) for the selective silencing of mutant Val30Met TTR in cell culture systems. We have identified an siRNA that specifically inhibits mutant, but not wild-type, TTR expression even in cells expressing both alleles. Thus, this siRNA-based approach may have potential for the gene therapy of FAP.     

Inhibition of cholesterol absorption by the combination of dietary plant sterols and ezetimibe: effects on plasma lipid levels

Consumption of plant sterols and treatment with ezetimibe both reduce cholesterol absorption in the intestine. However, the mechanism of action differs between the two treatments, and the consequences of combination treatment are unknown. Therefore, we performed a double-blind, placebo-controlled, crossover study for the plant sterol component with open-label ezetimibe treatment. Forty mildly hypercholesterolemic subjects were randomized to the following treatments for 4 weeks each: 10 mg/day ezetimibe combined with 25 g/day control spread; 10 mg/day ezetimibe combined with 25 g/day spread containing 2.0 g of plant sterols; 25 g/day spread containing 2.0 g of plant sterols; and placebo treatment consisting of 25 g/day control spread. Combination treatment of plant sterols and ezetimibe reduced low density lipoprotein cholesterol (LDL-C) by 1.06 mmol/l (25.2%; P < 0.001) compared with 0.23 mmol/l (4.7%; P = 0.006) with plant sterols and 0.94 mmol/l (22.2%; P < 0.001) with ezetimibe monotherapy. LDL-C reduction conferred by the combination treatment did not differ significantly from ezetimibe monotherapy (-0.12 mmol/l or -3.5%; P = 0.13). Additionally, the plasma lathosterol-to-cholesterol ratio increased with all treatments. Sitosterol and campesterol ratios increased after plant sterol treatment and decreased upon ezetimibe and combination therapy. Our results indicate that the combination of plant sterols and ezetimibe has no therapeutic benefit over ezetimibe monotherapy in subjects with mild hypercholesterolemia.