Role of Divalent Cations in HIV-1 Replication and Pathogenicity.

Divalent cations are essential for life and are fundamentally important coordinators of cellular metabolism, cell growth, host-pathogen interactions, and cell death. Specifically, for human immunodeficiency virus type-1 (HIV-1), divalent cations are required for interactions between viral and host factors that govern HIV-1 replication and pathogenicity. Homeostatic regulation of divalent cations' levels and actions appear to change as HIV-1 infection progresses and as changes occur between HIV-1 and the host. In people living with HIV-1, dietary supplementation with divalent cations may increase HIV-1 replication, whereas cation chelation may suppress HIV-1 replication and decrease disease progression. Here, we review literature on the roles of zinc (Zn2+), iron (Fe2+), manganese (Mn2+), magnesium (Mg2+), selenium (Se2+), and copper (Cu2+) in HIV-1 replication and pathogenicity, as well as evidence that divalent cation levels and actions may be targeted therapeutically in people living with HIV-1.

Tea polyphenols induced apoptosis of breast cancer cells by suppressing the expression of Survivin.

To study the mechanism of tea polyphenols (TP)-induced apoptosis of breast cancer cells. Proliferation of MCF-7 and SK-BR-3 cells was evaluated by MTT assays. Cellular ultrastructure was examined by electron microscopy. Apoptosis was detected by TUNEL. PCNA、 Cyclin D1、 Cyclin E and Survivin expression was measured by Western blot. Cell proliferation was significantly inhibited by TP. Spindle and round cells were loosely distributed with increased particles after TP treatment. Increased cell size, frequent nuclear atypia and a collapse of apoptosis were observed. The nucleus was pushed towards one side, while the cytoplasm was rich in free ribosome. The membrane of mitochondria was thickening, and the cell apoptotic body was observed. TP treated cells experienced significantly enhanced apoptosis compared with 5-Fu treated or control groups. The expression of survivin was downregulated by TP. To conclude, TP can inhibit cell growth and induce apoptosis through downregulating the expression of survivin in breast cancer.

Magnesium lithospermate B protects neurons from N-methyl-D-aspartic acid injury and attenuates kainic acid-induced neurodegeration in FVB mice.

Magnesium lithospermate B (MLB) is one of the major bioactive components of Radix Salviae miltiorrhizae (Dan Shen), which is a Chinese traditional herbal medicine with therapeutic effects on cardiovascular and cerebrovascular diseases. The aim of this study was to investigate the neuroprotective effects of MLB on N-methyl-D-aspartic acid (NMDA)-injured neurons and against kainic acid (KA)-induced neurodegeneration in mice. In cultured mouse primary hippocampal neurons, MLB significantly reduced NMDA-induced cell death and promoted neurite growth in a dose-dependent manner. In FVB mice, MLB attenuated KA-induced neurodegeneration. Additionally, MLB prevented the decrease in phosphorylated Akt and glycogen synthase kinase-3ß (GSK-3ß) levels both in NMDA-injured neurons and KA-injured mouse brain. This effect was blocked by phosphoinositide 3-kinase (PI3K) inhibitor LY294002 and Akt inhibitor triciribine, thus indicating the neuroprotective effects of MLB are most likely mediated by the PI3K/Akt/GSK-3ß pathway. Taken together, these results show the efficacy and underlying mechanism of MLB against neuronal injury and raise its therapeutic potential in neurodegenerative diseases.

[Extraction of praeruptorin A from Radix Peucedani by supercritical fluid extraction-CO2].

OBJECTIVE: To extract praeruptorin A from Radix Peucedani by supercritical fluid extraction (SFE)-CO2. METHODS: After preliminary experiment, three main factors were acquired that could influence the result of SFE-CO2, including the time, pressure and temperature of the extraction. The optimal extraction process was carried out on orthogonal design, and SFE-CO2 was compared with the traditional methods. RESULTS: In the extraction of the praeruptorin A, the best extraction conditions were 60 degrees C, 20 MPa, and duration for three hours. As cosolvent alcohol was added, the amount of extraction of the praeruptorin A increased, and the amount of SFE-CO2 extraction was higher than those of decoction and heating reflux. CONCLUSION: The SFE-CO2 in extracting praeruptorin A from Radix Peucedani is feasible and reliable.

High-throughput screen for small molecule inhibitors of Mint1-PDZ domains.

Several hundred PDZ (postsynaptic density-95, Drosophila disks-large, ZO-1) domain-containing proteins have been identified in the human genome. PDZ domains play a critical role in organization and function of cellular signaling pathways. Thus, small molecule inhibitors of PDZ domain association with their targets have wide potential applications as research and therapeutic agents. PDZ domains typically bind to a carboxyl-terminal tail of the target protein. Here we describe a high-throughput screening (HTS) assay for small molecule inhibitors of association between Mint1-PDZ domains and N-type Ca2+ channel carboxyl-terminal peptide (NC peptide). The performance of a homogeneous time-resolved fluorescence resonance energy transfer (HTRF) and an amplified luminescent proximity homogeneous assay (ALPHA) were systematically compared in parallel pilot HTS experiments with glutathione S-transferase-Mint1-PDZ1/2 protein and biotinylated NC peptide. Both of the two assays showed similar sensitivities in our target protein assay. Using HTRF-based assay we screened a library of 100,000 small molecule compounds and identified a number of potential "hits." The activity of isolated "hits" was confirmed by ALPHA assay. However, further evaluation revealed that isolated "hits" most likely act as "promiscuous binders," not as specific Mint-PDZ inhibitors, and that additional screening will be required to identify the true Mint-PDZ inhibitors. The assays described provided an example of HTS for a small molecule inhibitor of Mint-PDZ domain that can be easily adapted to other PDZ domain-mediated interactions.

Diverse effects of Stat1 on the regulation of hsp90alpha gene under heat shock.

Stat1 has been known as a regulator of gene expression and a mediator of IFNgamma signaling in mammalian cells, while its effect in a heat shock response remains unclear. We used RNAi knockdown, point mutations, ChIP and promoter activity assays to study the effect of Stat1 on the heat-shock induction of the hsp90alpha gene under heat shock conditions. We found that Stat1 regulates the heat shock induction of its target genes, the hsp90alpha gene in a heat shock response while the constitutive activity of the gene remains unaffected. The result of Stat1 in complex with Stat3 and HSF1 that bound at the GAS to lead a moderate heat shock induction was designated as an "intrinsic" induction of the hsp90alpha gene. Additionally a reduced or an elevated level of heat shock induction was also controlled by the Stat1 on hsp90alpha. These diverse effects on the hsp90alpha gene were a "reduced" induction with over-expressed Stat1 elicited by transfection of wild-type Stat1 or IFNgamma treatment, bound at the GAS as homodimer; and an "enhanced" heat shock induction with a mutation-mediated prohibition of Stat1/GAS binding. In conclusion, the status and efficacy of Stat1 bound at the GAS of its target gene are pivotal in determining the impact of Stat1 under heat shock. The results provided the first evidence on the tumor suppressor Stat1 that it could play diverse roles on its target genes under heat shock that also shed lights on patients with fever or under thermotherapy.

Cerebrosides of baifuzi, a novel potential blocker of calcium-activated chloride channels in rat pulmonary artery smooth muscle cells.

Calcium-activated chloride channels (CaCCs) are crucial regulators of vascular tone by promoting a depolarizing influence on the resting membrane potential of vascular smooth muscle cells. However, the lack of a special blocker of CaCCs has limited the investigation of its functions for long time. Here, we report that CB is a novel potential blocker of I(Cl(Ca)) in rat pulmonary artery smooth muscle cells (PASMC). Cerebrosides (CB) were isolated from Baifuzi which is dried root tuber of the herb Typhonium giganteum Engl used for treatment of stroke in traditional medicine. Using the voltage-clamp technique, sustained Ca(2+)-activated Cl(-) current (I(Cl(Ca))) was evoked by a K(+)-free pipette solution containing 500nM Ca(2+) which exhibited typical outwardly rectifying and voltage-/time-dependence characterization. Data showed that CB played a distinct inhibitory role in modulating the CaCCs. Moreover, we investigated the kinetic effect of CB on I(Cl(Ca)) and found that it could slow the activation dynamics of the outward current, accelerate the decay of the inward tail current and change the time-dependence characterization. We conclude that CB is a novel potent blocker of CaCCs. The interaction between CB and CaCCs is discussed.

Role of PGI2 in the formation and maintenance of hyperdynamic circulatory state of portal hypertensive rats.

AIM: To investigate the role of prostacyclin (PGI(2)) and nitric oxide (NO) in the development and maintenance of hyperdynamic circulatory state of chronic portal hypertensive rats. METHODS: Ninety male Sprague-Dawley rats were divided into three groups: intrahepatic portal hypertension (IHPH) group by injection of CCl(4), prehepatic portal hypertension (PHPH) group by partial stenosis of the portal vein and sham-operation control (SO) group. One week after the models were made, animals in each group were subdivided into 4 groups: saline controlled group (n = 23), Nomega-nitro-L-arginine (L-NNA) group (n = 21) group, indomethacin (INDO) group (n = 22) and high-dose heparin group (n = 24). The rats were administrated 1 mL of saline, L-NNA (3.3 mg/kg.d) and INDO (5 mg/kg.d) respectively through gastric tubes for one week, then heparin (200 IU/Kg/min) was given to rats by intravenous injection for an hour. Splanchnic and systemic hemodynamics were measured using radioactive microsphere techniques. The serum nitrate/nitrite (NO(2)(-)/NO(3)(-)) levels as a marker of production of NO were assessed by a colorimetric method, and concentration of 6-keto-PGF1alpha, a stable hydrolytic product of PGI(2), was determined by radioimmunoassay. RESULTS: The concentrations of plasma 6-keto-PGF1alpha (pg/mL) and serum NO(2)(-)/NO(3)(-) (micromol/L) in IHPH rats (1123.85+/-153.64, 73.34+/-4.31) and PHPH rats (891.88+/-83.11, 75.21+/-6.89) were significantly higher than those in SO rats (725.53+/-105.54, 58.79+/-8.47) (P<0.05). Compared with SO rats, total peripheral vascular resistance (TPR) and spanchnic vascular resistance (SVR) decreased but cardiac index (CI) and portal venous inflow (PVI) increased obviously in IHPH and PHPH rats (P<0.05). L-NNA and indomethacin could decrease the concentrations of plasma 6-keto-PGF1alpha and serum NO(2)(-)/NO(3)(-) in IHPH and PHPH rats (P<0.05). Meanwhile, CI, FPP and PVI lowered but MAP, TPR and SVR increased (P<0.05). After deduction of the action of NO, there was no significant correlation between plasma PGI(2) level and hemodynamic parameters such as CI, TPR, PVI and SVR. However, after deduction of the action of PGI(2), NO still correlated highly with the hemodynamic parameters, indicating that there was a close correlation between NO and the hemodynamic parameters. After administration of high-dose heparin, plasma 6-keto-PGF(1alpha) concentrations in IHPH, PHPH and SO rats were significantly higher than those in rats administrated vehicle (P<0.05). On the contrary, levels of serum NO(2)(-)/NO(3)(-) in IHPH, PHPH and SO rats were significantly lower than those in rats administrated Vehicle (P<0.05). Compared with those rats administrated vehicle, the hemodynamic parameters of portal hypertensive rats, such as CI and PVI, declined significantly after administration of high-dose heparin (P<0.05), while TPR and SVR increased significantly (P<0.05). CONCLUSION: It is NO rather than PGI(2) that is a mediator in the formation and maintenance of hyperdynamic circulatory state of chronic portal hypertensive rats.