Hepatitis Delta Antigen Mediates the Nuclear Import of Hepatitis Delta Virus RNA

Hepatitis delta virus (HDV) RNA replicates in the nuclei of virus-infected cells. The mechanism of nuclear import of HDV RNA is so far unknown. Using a fluorescein-labeled HDV RNA introduced into partially permeabilized HeLa cells, we found that HDV RNA accumulated only in the cytoplasm. However, in the presence of hepatitis delta antigen (HDAg), which is the only protein encoded by HDV RNA, the HDV RNA was translocated into the nucleus, suggesting that nuclear import of HDV RNA is mediated by HDAg. Deletion of the nuclear localization signal (NLS) or RNA-binding motifs of HDAg resulted in the failure of nuclear import of HDV RNA, indicating that both the NLS and an RNA-binding motif of HDAg are required for the RNA-transporting activity of HDAg. Surprisingly, any one of the three previously identified RNA-binding motifs was sufficient to confer the RNA-transporting activity. We have further shown that HDAg, via its NLS, interacts with karyopherin α2 in vitro, suggesting that nuclear import of the HDAg-HDV RNA complex is mediated by the karyopherin α2β heterodimer. The nuclear import of HDV RNA may be the first biological function of HDAg in the HDV life cycle.     

Detection of polyhydroxyalkanoate synthase activity on a polyacrylamide gel

This study presents a method to detect active polyhydroxyalkanoate (PHA) synthase on a polyacrylamide gel that combines the polyhydroxybutyrate (PHB) polymerization reaction with Sudan Black B staining. After separation of the protein samples on a modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the slab gel was submerged in a buffer containing β-hydroxybutyryl-coenzyme A (3-HBCoA) as substrate and incubated at room temperature for in vitro PHB polymerization. The active PHA synthase catalyzed 3-HBCoA into the PHB polymer and was stained with Sudan Black B. The active PHA synthase appeared as a dark blue band. The activity staining was of high sensitivity, capable of detecting 3.9 ng (0.273 mU) of Cupriavidus necator H16 PHA synthase purified from recombinant Escherichia coli. The detection sensitivity of activity staining was comparable to that of Western blotting analysis. Furthermore, the high sensitivity of activity staining enabled specific detection of the active PHA synthase in the crude extract of wild-type strain C. necator H16. This study provides a rapid, sensitive, and highly specific method for detecting active PHA synthase in gel. The method could be applied to detecting PHA synthase from wild-type bacteria and to the process of enzyme purification.     

Brazilin isolated from Caesalpinia sappan L. acts as a novel collagen receptor agonist in human platelets

Background

Brazilin, isolated from the heartwood of Caesalpinia sappan L., has been shown to possess multiple pharmacological properties.

Methods

In this study, platelet aggregation, flow cytometry, immunoblotting analysis, and electron spin resonance (ESR) spectrometry were used to investigate the effects of brazilin on platelet activation ex vivo. Moreover, fluorescein sodium-induced platelet thrombi of mesenteric microvessels was also used in in vivo study.

Results

We demonstrated that relatively low concentrations of brazilin (1 to 10 μM) potentiated platelet aggregation induced by collagen (0.1 μg/ml) in washed human platelets. Higher concentrations of brazilin (20 to 50 μM) directly triggered platelet aggregation. Brazilin-mediated platelet aggregation was slightly inhibited by ATP (an antagonist of ADP). It was not inhibited by yohimbine (an antagonist of epinephrine), by SCH79797 (an antagonist of thrombin protease-activated receptor [PAR] 1), or by tcY-NH2 (an antagonist of PAR 4). Brazilin did not significantly affect FITC-triflavin binding to the integrin α_IIbβ₃ in platelet suspensions. Pretreatment of the platelets with caffeic acid phenethyl ester (an antagonist of collagen receptors) or JAQ1 and Sam.G4 monoclonal antibodies raised against collagen receptor glycoprotein VI and integrin α₂β₁, respectively, abolished platelet aggregation stimulated by collagen or brazilin. The immunoblotting analysis showed that brazilin stimulated the phosphorylation of phospholipase C (PLC)γ2 and Lyn, which were significantly attenuated in the presence of JAQ1 and Sam.G4. In addition, brazilin did not significantly trigger hydroxyl radical formation in ESR analysis. An in vivo mouse study showed that brazilin treatment (2 and 4 mg/kg) significantly shortened the occlusion time for platelet plug formation in mesenteric venules.

Conclusion

To the best of our knowledge, this study provides the first evidence that brazilin acts a novel collagen receptor agonist. Brazilin is a plant-based natural product, may offer therapeutic potential as intended anti-thrombotic agents for targeting of collagen receptors or to be used a useful tool for the study of detailed mechanisms in collagen receptors-mediated platelet activation.     

Salvianolic acid B attenuates cyclooxygenase-2 expression in vitro in LPS-treated human aortic smooth muscle cells and in vivo in the apolipoprotein-E-deficient mouse aorta

Inflammation plays an essential role in atherosclerosis and post-angioplasty restenosis and the synthesis and release of inflammatory cytokines from vascular smooth muscle cells is an important contributor to these pathologies. It is assumed that drugs that prevent the overproduction of inflammatory cytokines may inhibit cardiovascular disorders. In the present study, the effects of a water-soluble antioxidant, salvianolic acid B (Sal B), derived from a Chinese herb, on the expression of cyclooxygenase (COX) in lipopolysaccharide (LPS)-treated human aortic smooth muscle cells (HASMCs) and in the aortas of cholesterol-fed apoE deficient mice were investigated. In unstimulated HASMCs, COX-2 mRNA and protein were almost undetectable, but were strongly upregulated in response to LPS. In contrast, HASMCs with or without LPS treatment showed constitutive expression of COX-1 mRNA and protein. The activation of COX-2 protein synthesis in LPS-stimulated HASMCs was shown to involve the activation of the extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun NH(2)-terminal kinase (JNK), and p38 mitogen-activated protein kinase pathway. Incubation of HASMCs with Sal B before LPS stimulation resulted in pronounced downregulation of COX-2 expression. Sal B treatment suppressed ERK1/2 and JNK phosphorylation and attenuated the increase in prostaglandin E(2) production and NADPH oxidase activity in LPS-treated HASMCs. When apoE-deficient mice were fed a 0.15% cholesterol diet with or without supplementation with 0.3% Sal B for 12 weeks, the intima/media area ratio in the thoracic aortas was significantly reduced in the Sal B group (0.010 +/- 0.009%) compared to the apoE-deficient group (0.114 +/- 0.043%) and there was a significant reduction in COX-2 protein expression in the thickened intima. These results demonstrate that Sal B has anti-inflammatory properties and may explain its anti-atherosclerotic properties. This new mechanism of action of Sal B, in addition to its previously reported inhibition of LDL oxidation, may help explain its efficacy in the treatment of atherosclerosis.     

Fish Oil and Oxidative Stress by Inflammatory Leukocytes

Several papers have claimed that mitochondria contain nitric oxide synthase (NOS) and make nitric oxide (NO^?) in amounts sufficient to affect mitochondrial respiration. However, we found that the addition of l-arginine or the NOS inhibitor l-NMMA to intact rat liver mitochondria did not have any effect on the respiratory rate in both State 3 and State 4. We did not detect mitochondrial NO^? production by the oxymyoglobin oxidation assay, or electrochemically using an NO^? electrode. An apparent NO^? production detected by the Griess assay was identified as an artifact. NO^? generated by eNOS added to the mitochondria could easily be detected, although succinate-supplemented mitochondria appeared to consume NO^?.

Our data show that NO^? production by normal rat liver mitochondria cannot be detected in our laboratory, even though the levels of production claimed in the literature should easily have been measured by the techniques used. The implications for the putative mitochondrial NOS are discussed.     

Increased Healthcare Service Utilizations for Patients with Dementia: A Population-Based Study

Background

The majority of previous studies investigating the health care utilization of people with dementia were conducted in Western societies. There is little information on the economic burden on the healthcare system attributable to dementia in Asian countries. This study thus investigated differences in utilization of healthcare services between subjects with and those without a diagnosis of dementia using Taiwan’s National Health Insurance population-based database.

Methods

This study comprised 5,666 subjects with a dementia diagnosis and 5,666 age- and gender-matched comparison subjects without a dementia diagnosis. We individually followed each subject for a 1-year period starting from their index date to evaluate their healthcare resource utilization. Healthcare resource utilization included the number of outpatient visits and inpatient days, and the mean costs of outpatient and inpatient treatments. In addition, we divided healthcare resource utilization into psychiatric and non-psychiatric services.

Results

As for utilization of psychiatric services, subjects with a dementia diagnosis had significantly more outpatient visits (2.2 vs. 0.3, p<0.001) and significantly higher outpatient costs (US$124 vs. US$16, p<0.001) than comparison subjects. For non-psychiatric services, subjects with a dementia diagnosis also had significantly more outpatient visits (34.4 vs. 31.6, p<0.001) and significantly higher outpatient costs (US$1754 vs. US$1322, p<0.001) than comparison subjects. For all healthcare services, subjects with a dementia diagnosis had significantly more outpatient visits (36.7 vs. 32.0, p<0.001) and significantly higher outpatient costs (US$1878 vs. US$1338, p<0.001) than comparison subjects. Furthermore, the total cost was about 2-fold greater for subjects with a dementia diagnosis than for comparison subjects (US$3997 vs. US$2409, p<0.001).

Conclusions

We concluded that subjects who had received a clinical dementia diagnosis had significantly higher utilization of all healthcare services than comparison subjects.     

Differential expression of circulating vascular cell adhesion molecule-1 in subjects with coronary artery disease and cardiac syndrome X without known diabetes mellitus

Context: Inflammation is one of the mechanisms underlying cardiac syndrome X (CSX).

Objectives: Few studies have compared the expression of inflammatory or adhesion molecules between coronary artery disease (CAD) versus CSX.

Materials and methods: Ninety-two CSX and 145 CAD subjects without known diabetes mellitus underwent coronary angiogram for angina.

Results: Vascular cell adhesion molecule (VCAM)-1 (median, 507 versus 431?ng/ml, p?=?0.001) was significantly higher in the CAD group. In the binary regression, VCAM-1 was a significant differential factor for CAD versus CSX.

Discussion and conclusion: Adhesion molecules might be implicated in the differential expression of macro versus microvascular coronary disease.

Trial registration number: NCT01198730 at https://clinicaltrials.gov     

Immunomodulatory Protein from Ganoderma microsporum Induces Pro-Death Autophagy through Akt-mTOR-p70S6K Pathway Inhibition in Multidrug Resistant Lung Cancer Cells

Chemoresistance in cancer therapy is an unfavorable prognostic factor in non-small cell lung cancer (NSCLC). Elevation of intracellular calcium level in multidrug resistant (MDR) sublines leads to sensitization of MDR sublines to cell death. We demonstrated that a fungal protein from Ganoderma microsporum, GMI, elevates the intracellular calcium level and reduces the growth of MDR subline via autophagy and apoptosis, regardless of p-glycoprotein (P-gp) overexpression, in mice xenograft tumors. In addition, we examined the roles of autophagy in the death of MDR A549 lung cancer sublines by GMI, thapsigargin (TG) and tunicamycin (TM) in vitro. Cytotoxicity of TG was inhibited by overexpressed P-gp. However, TM-induced death of MDR sublines was independent of P-gp level. Combinations of TG and TM with either docetaxel or vincristine showed no additional cytotoxic effects on MDR sublines. TG- and TM-mediated apoptosis of MDR sublines was demonstrated on Annexin-V assay and Western blot and repressed by pan-caspase inhibitor (Z-VAD-FMK). Treatment of MDR sublines with TG and TM also augmented autophagy with accumulation of LC3-II proteins, breakdown of p62 and formation of acidic vesicular organelles (AVOs). Inhibition of ATG5 by shRNA silencing significantly reduced autophagy and cell death but not apoptosis following TG or TM treatment. GMI treatment inhibited the phosphorylation of Akt/S473 and p70S6K/T389. Interestingly, the phosphorylation of ERK was not associated with GMI-induced autophagy. We conclude that autophagy plays a pro-death role in acquired MDR and upregulation of autophagy by GMI via Akt/mTOR inhibition provides a potential strategy for overcoming MDR in the treatment of lung cancers.