Epigenetic inactivation of the miR-124-1 in haematological malignancies

miR-124-1 is a tumour suppressor microRNA (miR). Epigenetic deregulation of miRs is implicated in carcinogenesis. Promoter DNA methylation and histone modification of miR-124-1 was studied in 5 normal marrow controls, 4 lymphoma, 8 multiple myeloma (MM) cell lines, 230 diagnostic primary samples of acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL), MM, and non-Hodgkin's lymphoma (NHL), and 53 MM samples at stable disease or relapse. Promoter of miR-124-1 was unmethylated in normal controls but homozygously methylated in 4 of 4 lymphoma and 4 of 8 myeloma cell lines. Treatment of 5-Aza-2'-deoxycytidine led to miR-124-1 demethylation and re-expression of mature miR-124, which also associated with emergence of euchromatic trimethyl H3K4 and consequent downregulation of CDK6 in myeloma cells harboring homozygous miR-124-1 methylation. In primary samples at diagnosis, miR-124-1 methylation was absent in CML but detected in 2% each of MM at diagnosis and relapse/progression, 5% ALL, 15% AML, 14% CLL and 58.1% of NHL (p<0.001). Amongst lymphoid malignancies, miR-124-1 was preferentially methylated in NHL than MM, CLL or ALL. In primary lymphoma samples, miR-124-1 was preferentially hypermethylated in B- or NK/T-cell lymphomas and associated with reduced miR-124 expression. In conclusion, miR-124-1 was hypermethylated in a tumour-specific manner, with a heterochromatic histone configuration. Hypomethylation led to partial restoration of euchromatic histone code and miR re-expression. Infrequent miR-124-1 methylation detected in diagnostic and relapse MM samples showed an unimportant role in MM pathogenesis, despite frequent methylation found in cell lines. Amongst haematological cancers, miR-124-1 was more frequently hypermethylated in NHL, and hence warrants further study.     

Mechanisms of the vasorelaxant effect of 1-hydroxy-2, 3, 5-trimethoxy-xanthone, isolated from a Tibetan herb, Halenia elliptica, on rat coronary artery

1-Hydroxy-2, 3, 5-trimethoxyxanthone (HM-1) is a xanthone isolated from Halenia elliptica, a Tibetan medicinal herb. HM-1 (0.33-42.1 microM) produced a concentration-dependent relaxation in rat coronary artery rings pre-contracted with 1 microM 5-hydroxytryptamine (5-HT), with an EC(50) of 1.67+/-0.27 microM. Removal of the endothelium significantly affected the vasodilator potency of HM-1, resulting in 46% decrease in E(max) value. The endothelium-dependent effects of HM-1 was confirmed when its vasorelaxant effect was inhibited after addition of nitric oxide synthase (NOS) inhibitor N(omega)-nitro-l-arginine methyl ester (100 microM) or the soluble guanylate cyclase inhibitor 1H-[1, 2, 4] oxadiazolo [4,3-alpha] quinoxalin-1-one (ODQ, 10 microM). Atropine (100 nM), flurbiprofen (10 microM), propranolol (100 microM), pyrilamine (10 microM), cimetidine (10 microM) and SQ22536 (100 microM) had no effect on the vasorelaxant activity of HM-1 indicated the non-involvement of other receptor/enzyme systems. In endothelium-denuded coronary artery rings, the vasorelaxant effect of HM-1 was unaffected by potassium channel blockers such as tetraethylammonium (10 mM), iberiotoxin (100 nM), barium chloride (100 microM) and 4-aminopyridine (1 mM). The involvement of Ca(2+) channel in 5-HT-primed artery ring preparations incubated with Ca(2+)-free buffer was confirmed when HM-1 (9.93 microM) partially abolished the CaCl(2)-induced vasoconstriction (87% inhibition in intact-endothelium artery rings; 50% inhibition in endothelium-denuded rings). In the KCl-primed preparations incubated with Ca(2+)-free buffer, HM-1 (9.93 microM) produced a 27.3% inhibition in endothelium-denuded rings. HM-1 (3.31-33.1 microM) had minimal relaxant effects (14.4%-20.3%) on the contractile response generated by 10 microM phorbol 12,13-diacetate (PDA) in Ca(2+)-free solutions, suggesting minimal effects on intracellular Ca(2+) mechanisms. These findings suggest the vasodilator action of HM-1 involved both an endothelium-dependent mechanism involving NO and an endothelium-independent mechanism by inhibiting Ca(2+) influx through L-type voltage-operated Ca(2+) channels; a minor contribution to the effects of HM-1 may be related to inhibition of the protein kinase C-mediated release of intracellular Ca(2+) stores.     

Multicompartmental distribution of the tuberous sclerosis gene products,hamartin and tuberin

Mutations of the TSC1 and TSC2 genes give rise to the clinical disorder of tuberous sclerosis characterized by the development of hamartomas predominantly affecting the central nervous system, kidney, skin, lung, and heart. The function of the gene products, hamartin and tuberin, is not well understood but we have previously suggested a role in vesicular transport. To define the subcellular compartment(s) involved with these two proteins, biochemical characterization of hamartin and tuberin was performed in primary tissues and cell lines. Fractionation of cell lysates identified both proteins in the cytosolic, microsomal, and cytoskeletal compartments. In each of these fractions, hamartin and tuberin formed a stable complex in coimmunoprecipitation analyses. Further, they colocalized extensively in discrete, vesicular structures in the cytoplasm. Within the microsomal compartment, hamartin and tuberin behaved as peripheral membrane proteins that associate with the cytosolic leaflet of membranous domains. Immunoisolation of tuberin-bound vesicles using magnetic beads showed an enrichment of rap1, rab5, and caveolin-1, all of which have been found in specialized lipid microdomains, caveolae. Our data suggest that hamartin and tuberin are multicompartmental proteins that partially reside in caveolin-1-enriched structures and potentially affect their signaling.     

Electrical stimulation influences satellite cell proliferation and apoptosis in unloading-induced muscle atrophy in mice

Muscle atrophy caused by disuse is accompanied by adverse physiological and functional consequences. Satellite cells are the primary source of skeletal muscle regeneration. Satellite cell dysfunction, as a result of impaired proliferative potential and/or increased apoptosis, is thought to be one of the causes contributing to the decreased muscle regeneration capacity in atrophy. We have previously shown that electrical stimulation improved satellite cell dysfunction. Here we test whether electrical stimulation can also enhance satellite cell proliferative potential as well as suppress apoptotic cell death in disuse-induced muscle atrophy. Eight-week-old male BALB/c mice were subjected to a 14-day hindlimb unloading procedure. During that period, one limb (HU-ES) received electrical stimulation (frequency: 20 Hz; duration: 3 h, twice daily) while the contralateral limb served as control (HU). Immunohistochemistry and western blotting techniques were used to characterize specific proteins in cell proliferation and apoptosis. The HU-ES soleus muscles showed significant improvement in muscle mass, cross-sectional area, and peak tetanic force relative to the HU limb (p<0.05). The satellite cell proliferative activity as detected within the BrdU+/Pax7+ population was significantly higher (p<0.05). The apoptotic myonuclei (detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) and the apoptotic satellite cells (detected by cleaved Poly [ADP-ribose] polymerase co-labeled with Pax7) were reduced (p<0.05) in the HU-ES limb. Furthermore the apoptosis-inducing factor and cleaved caspase-3 were down-regulated while the anti-apoptotic Bcl-2 protein was up-regulated (p<0.05), in the HU-ES limb. These findings suggest that the electrical stimulation paradigm provides an effective stimulus to rescue the loss of myonuclei and satellite cells in disuse muscle atrophy, thus maintaining a viable satellite cell pool for subsequent muscle regeneration. Optimization of stimulation parameters may enhance the outcome of the intervention.     

Biochemical characterization of CD39L4

Nucleotides are involved in regulating a number of important processes ranging from inflammation to platelet aggregation. Enzymes that can modulate levels of nucleotides in the blood therefore represent important regulatory components in these physiological systems. CD39L4 is a soluble E-nucleoside triphosphate dephosphohydrolase (E-NTPDase) with specificity for nucleotide diphosphates (NDPs). In this study, stable mammalian and insect cell lines were generated expressing CD39L4 protein to purify and characterize the recombinant protein. We demonstrate that recombinant CD39L4 protein expressed in human embryonic carcinoma 293 cells is glycosylated by comparing the molecular masses before and after glycosidase treatment. Activity measurements of CD39L4 isolated from tunicamycin-treated, transiently transfected COS-7 cells indicate that glycosylation is not required for full ADPase activity. Recombinant human CD39L4 protein isolated from stable insect cells was glycosylated differently, but also demonstrated relative activity comparable to that of the mammalian protein. When denatured by SDS under nonreducing conditions, a fraction of the CD39L4 protein migrates as a 110 kDa disulfide-linked dimer. We determined that the monomer is the most active form of CD39L4 by measuring the activity of sucrose density gradient fractions of monomers and partially purified dimers. The physiological significance of the biochemical and enzymatic characterization is discussed.     

Molecular docking and enzyme kinetic studies of dihydrotanshinone on metabolism of a model CYP2D6 probe substrate in human liver microsomes

The effects of Danshen and its active components (tanshinone I, tanshinone IIA, dihydrotanshinone and cryptotanshinone) on CYP2D6 activity was investigated by measuring the metabolism of a model CYP2D6 probe substrate, dextromethorphan to dextrorphan in human pooled liver microsomes. The ethanolic extract of crude Danshen (6.25-100 μg/ml) decreased dextromethorphan O-demethylation in vitro (IC(50)=23.3 μg/ml) and the water extract of crude Danshen (0.0625-1 mg/ml) showed no inhibition. A commercially available Danshen pill (31.25-500 μg/ml) also decreased CYP2D6 activity (IC(50)=265.8 μg/ml). Among the tanshinones, only dihydrotanshinone significantly inhibited CYP2D6 activity (IC(50)=35.4 μM), compared to quinidine, a specific CYP2D6 inhibitor (IC(50)=0.9 μM). Crytotanshinone, tanshinone I and tanshinone IIA produced weak inhibition, with IC(20) of 40.8 μM, 16.5 μM and 61.4 μM, respectively. Water soluble components such as salvianolic acid B and danshensu did not affect CYP2D6-mediated metabolism. Enzyme kinetics studies showed that inhibition of CYP2D6 activity by the ethanolic extract of crude Danshen and dihydrotanshinone was concentration-dependent, with K(i) values of 4.23 μg/ml and 2.53 μM, respectively, compared to quinidine, K(i)=0.41 μM. Molecular docking study confirmed that dihydrotanshinone and tanshinone I interacted with the Phe120 amino acid residue in the active cavity of CYP2D6 through Pi-Pi interaction, but did not interact with Glu216 and Asp301, the key residues for substrate binding. The logarithm of free binding energy of dihydrotanshinone (-7.6 kcal/mol) to Phe120 was comparable to quinidine (-7.0 kcal/mol) but greater than tanshinone I (-5.4 kcal/mol), indicating dihydrotanshinone has similar affinity to quinidine in binding to the catalytic site on CYP2D6.     

Clinical,Virological and Immunological Features from Patients Infected with Re-Emergent Avian-Origin Human H7N9 Influenza Disease of Varying Severity in Guangdong Province

BackgroundThe second wave of avian influenza H7N9 virus outbreak in humans spread to the Guangdong province of China by August of 2013 and this virus is now endemic in poultry in this region.MethodsFive patients with H7N9 virus infection admitted to our hospital during August 2013 to February 2014 were intensively investigated. Viral load in the respiratory tract was determined by quantitative polymerase chain reaction (Q-PCR) and cytokine levels were measured by bead-based flow cytometery.ResultsFour patients survived and one died. Viral load in different clinical specimens was correlated with cytokine levels in plasma and broncho-alveolar fluid (BALF), therapeutic modalities used and clinical outcome. Intravenous zanamivir appeared to be better than peramivir as salvage therapy in patients who failed to respond to oseltamivir. Higher and more prolonged viral load was found in the sputum or endotracheal aspirates compared to throat swabs. Upregulation of proinflammatory cytokines IP-10, MCP-1, MIG, MIP-1α/β, IL-1β and IL-8 was found in the plasma and BALF samples. The levels of cytokines in the plasma and viral load were correlated with disease severity. Reactivation of herpes simplex virus type 1(HSV-1) was found in three out of five patients (60%).ConclusionExpectorated sputum or endotracheal aspirate specimens are preferable to throat swabs for detecting and monitoring H7N9 virus. Severity of the disease was correlated to the viral load in the respiratory tract as well as the extents of cytokinemia. Reactivation of HSV-1 may contribute to clinical outcome.     

Moderate Alcohol Use and Cardiovascular Disease from Mendelian Randomization

Background

Observational studies show moderate alcohol use negatively associated with ischemic heart disease (IHD) and cardiovascular disease (CVD). However, healthier attributes among moderate users compared to never users may confound the apparent association. A potentially less biased way to examine the association is Mendelian randomization, using alcohol metabolizing genes which influence alcohol use.

Methods

We used instrumental variable analysis with aldehyde dehydrogenase 2 (ALDH2) genotypes (AA/GA/GG) as instrumental variables for alcohol use to examine the association of alcohol use (10 g ethanol/day) with CVD risk factors (blood pressure, lipids and glucose) and morbidity (self-reported IHD and CVD) among men in the Guangzhou Biobank Cohort Study.

Results

ALDH2 genotypes were a credible instrument for alcohol use (F-statistic 74.6). Alcohol was positively associated with HDL-cholesterol (0.05 mmol/L per alcohol unit, 95% confidence interval (CI) 0.02 to 0.08) and diastolic blood pressure (1.15 mmHg, 95% CI 0.23 to 2.07) but not with systolic blood pressure (1.00 mmHg, 95% CI -0.74 to 2.74), LDL-cholesterol (0.03 mmol/L, 95% CI -0.03 to 0.08), log transformed triglycerides (0.03 mmol/L, 95% CI -0.01 to 0.08) or log transformed fasting glucose (0.01 mmol/L, 95% CI -0.006 to 0.03), self-reported CVD (odds ratio (OR) 0.98, 95% CI 0.76 to 1.27) or self-reported IHD (OR 1.10, 95% CI 0.83 to 1.45).

Conclusion

Low to moderate alcohol use among men had the expected effects on most CVD risk factors but not fasting glucose. Larger studies are needed to confirm the null associations with IHD, CVD and fasting glucose.