Hypotonic swelling-induced Ca2+ release by an IP3-insensitive Ca2+ store

Hypotonicswelling increases the intracellular Ca²⁺ concentration([Ca²⁺]_i) in vascular smooth muscle cells(VSMC). The source of this Ca²⁺ is not clear. To study thesource of increase in [Ca²⁺]_i in response tohypotonic swelling, we measured [Ca²⁺]_i infura 2-loaded cultured VSMC (A7r5 cells). Hypotonic swelling produced a40.7-nM increase in [Ca²⁺]_i that was notinhibited by EGTA but was inhibited by 1 µM thapsigargin. Priordepletion of inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ stores with vasopressin did not inhibit the increasein [Ca²⁺]_i in response to hypotonic swelling.Exposure of ⁴⁵Ca²⁺-loaded intracellular storesto hypotonic swelling in permeabilized VSMC produced an increase in⁴⁵Ca²⁺ efflux, which was inhibited by 1 µMthapsigargin but not by 50 µg/ml heparin, 50 µM ruthenium red, or25 µM thio-NADP. Thus hypotonic swelling of VSMC causes a release ofCa²⁺ from the intracellular stores from a novel sitedistinct from the IP₃-, ryanodine-, and nicotinic acidadenine dinucleotide phosphate-sensitive stores.

     

Nitrogen fixation and indole acetic acid production by Azospi-rillum sp. as influenced by an insecticide, carbofuran

Both indole acetic acid (IAA) accumulation and nitrogen fixation were increased in Azospirillum cultures isolated from rice roots and soils by carbofuran (2, 3-dihydro-2, 2-dimethyl-7-benzofuranyl-N-methyl carbamate), an insecticide widely used in rice cultivation. Addition of carbofuran at 5 and 10 parts/10₆ significantly stimulated nitrogen fixation in Azospirillum. Indole acetic acid accumulation by Azospirillum cultures was more pronounced at a lower level (250 g/50 ml) of carbofuran. Evidence is provided for carbofuran degradation by Azospirillum cultures. The 7-benzofuranol (2, 3-dihydro-2–2-dimethyl-7 benzofuranol), a major degradation product of carbofuran, however, did not enhance the IAA accumulation. The higher accumulation of IAA in Azospirillum in the presence of carbofuran is probably related to the increased growth due to fixed N present in the insecticide. Results indicate the involvement of parent compound carbofuran and/or compounds other than the 7-benzofuranol in the higher accumulation of IAA by Azospirillum sp.     

Analysis of gamma c-family cytokine target genes. Identification of dual-specificity phosphatase 5 (DUSP5) as a regulator of mitogen-activated protein kinase activity in interleukin-2 signaling

Interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21 form a family of cytokines based on their sharing the common cytokine receptor gamma chain, gamma(c), which is mutated in X-linked severe combined immunodeficiency (SCID). As a step toward further elucidating the mechanism of action of these cytokines in T-cell biology, we compared the gene expression profiles of IL-2, IL-4, IL-7, and IL-15 in T cells using cDNA microarrays. IL-2, IL-7, and IL-15 each induced a highly similar set of genes, whereas IL-4 induced distinct genes correlating with differential STAT protein activation by this cytokine. One gene induced by IL-2, IL-7, and IL-15 but not IL-4 was dual-specificity phosphatase 5 (DUSP5). In IL-2-dependent CTLL-2 cells, we show that IL-2-induced ERK-1/2 activity was inhibited by wild type DUSP5 but markedly increased by an inactive form of DUSP5, suggesting a negative feedback role for DUSP5 in IL-2 signaling. Our findings provide insights into the shared versus distinctive actions by different members of the gamma(c) family of cytokines. Moreover, we have identified a DUSP5-dependent negative regulatory pathway for MAPK activity in T cells.     

Dimethyl fumarate protects thioacetamide‐induced liver damage in rats: Studies on Nrf2, NLRP3, and NF‐κB

The present study was designed to investigate the hepatoprotective potential of dimethyl fumarate (DMF) against thioacetamide (TAA)‐induced liver damage. Wistar rats were treated with DMF (12.5, 25, and 50 mg/kg/day, orally) and TAA (200 mg/kg intraperitoneally, every third day) for 6 consecutive weeks. TAA exposure significantly reduced body weight, increased liver weight and index, and intervention with DMF did not ameliorate these parameters. DMF treatment significantly restored TAA‐induced increase in the levels of aspartate aminotransferase, alanine aminotransferase, γ‐glutamyl transferase, total bilirubin, uric acid, malondialdehyde, reduced glutathione, and histopathological findings such as inflammatory cell infiltration, deposition of collagen, necrosis, and bridging fibrosis. DMF treatment significantly ameliorated TAA‐induced hepatic stellate cell activation, increase in inflammatory cascade markers (NACHT, LRR, and PYD domains‐containing protein 3; NLRP3, apoptosis‐associated speck like protein containing a caspase recruitment domain; ASC, caspase‐1, nuclear factor‐kappa B; NF‐κB, interleukin‐6), fibrogenic makers (α‐smooth muscle actin; ɑ‐SMA, transforming growth factor; TGF‐β1, fibronectin, collagen 1) and antioxidant markers (nuclear factor (erythroid‐derived 2)‐like factor 2; Nrf2, superoxide dismutase‐1; SOD‐1, catalase). The present findings concluded that DMF protects against TAA‐induced hepatic damage mediated through the downregulation of inflammatory cascades and upregulation of antioxidant status.     

Interferon and Ribavirin Combination Treatment Synergistically Inhibit HCV Internal Ribosome Entry Site Mediated Translation at the Level of Polyribosome Formation

Purpose

Although chronic hepatitis C virus (HCV) infection has been treated with the combination of interferon alpha (IFN-α) and ribavirin (RBV) for over a decade, the mechanism of antiviral synergy is not well understood. We aimed to determine the synergistic antiviral mechanisms of IFN-α and RBV combination treatment using HCV cell culture.

Methods

The antiviral efficacy of IFN-α, RBV alone and in combination was quantitatively measured using HCV infected and replicon cell culture. Direct antiviral activity of these two drugs at the level of HCV internal ribosome entry site (IRES) mediated translation in Huh-7 cell culture was investigated. The synergistic antiviral effect of IFN-α and RBV combination treatment was verified using both the CalcuSyn Software and MacSynergy Software.

Results

RBV combination with IFN-α efficiently inhibits HCV replication cell culture. Our results demonstrate that IFN-α, interferon lambda (IFN-λ) and RBV each inhibit the expression of HCV IRES-GFP and that they have a minimal effect on the expression of GFP in which the translation is not IRES dependent. The combination treatments of RBV along with IFN-α or IFN-λ were highly synergistic with combination indexes <1. We show that IFN-α treatment induce levels of PKR and eIF2α phosphorylation that prevented ribosome loading of the HCV IRES-GFP mRNA. Silencing of PKR expression in Huh-7 cells prevented the inhibitory effect of IFN-α on HCV IRES-GFP expression. RBV also blocked polyribosome loading of HCV-IRES mRNA through the inhibition of cellular IMPDH activity, and induced PKR and eIF2α phosphorylation. Knockdown of PKR or IMPDH prevented RBV induced HCV IRES-GFP translation.

Conclusions

We demonstrated both IFN-α and RBV inhibit HCV IRES through prevention of polyribosome formation. The combination of IFN-α and RBV treatment synergistically inhibits HCV IRES translation via using two different mechanisms involving PKR activation and depletion of intracellular guanosine pool through inhibition of IMPDH.