Search

A system for the production of soluble interferon (IFN)-lambda2 was developed by fusing the IFN-lambda2, NusA protein, polyhistidine and S peptide genes and then expressing the fused product (Nus-His-S-tagged IFN-lambda2) in Escherichia coli. The expressed fusion protein was purified by Ni-NTA affinity chromatography. The fusion tag was removed from recombinant IFN-lambda2 by cleavage with enterokinase. N-Terminal sequencing confirmed the identity of the purified protein. When compared with commercial IFN-alpha2b, IFN-lambda2 had similar antiviral activity. The production and characterization of biologically active IFN-lambda2 will be beneficial for its potential role in clinical applications.

T-type Ca 2+ channels are important regulators of peripheral sensory neuron excitability. Accordingly, T-type Ca 2+ currents are often increased in various pathological pain conditions, such as inflammation or nerve injury. Here we investigated effects of inflammation on functional expression of T-type Ca 2+ channels in small-diameter cultured dorsal root ganglion (DRG) neurons. We found that overnight treatment of DRG cultures with a cocktail of inflammatory mediators bradykinin (BK), adenosine triphosphate (ATP), norepinephrine (NE) and prostaglandin E 2 (PGE2) strongly increased the population size of the small-diameter neurons displaying low-voltage activated (LVA, T-type) Ca 2+ currents while having no effect on the peak LVA current amplitude. When applied individually, BK and ATP also increased the population size of LVA-positive neurons while NE and PGE2 had no effect. The PLC inhibitor U-73122 and B 2 receptor antagonist, Hoe-140, both abolished the increase of the population of LVA-positive DRG neurons. Inflammatory treatment did not affect Ca V 3.2 mRNA or protein levels in DRG cultures. Furthermore, an ubiquitination inhibitor, MG132, did not increase the population of LVA-positive neurons. Our data suggest that inflammatory mediators BK and ATP increase the abundance of LVA-positive DRG neurons in total neuronal population by stimulating the recruitment of a ‘reserve pool’ of Ca V 3.2 channels, particularly in neurons that do not display measurable LVA currents under control conditions. [ABSTRACT FROM AUTHOR]

Background: Esophageal squamous cell carcinoma (ESCC) is a highly aggressive cancer whose underlying molecular mechanisms are poorly understood. The natural antisense transcript (NAT) WRAP53 regulates p53 expression and WRAP53 protein is a component of telomerase. NATs play key roles in carcinogenesis, and although WRAP53 is known to increase cancer cell survival, its role in ESCC clinicopathology is unknown. The aim of this study was to investigate WRAP53 expression in ESCC and to correlate it with clinicopathological characteristics. Methods: WRAP53 mRNA and protein expression was measured by quantitative PCR (qRT-PCR) and western blotting, respectively, in 4 ESSC cells lines and in 45 paired ESCC and non-neoplastic esophageal mucosa tissues. To correlate WRAP53 protein expression with clinicopathological characteristics, immunohistochemistry (IHC) was performed on 134 ESCC and 85 non-neoplastic esophageal mucosa tissues. Results: Expression of WRAP53 was detected in all ESCC cell lines and was upregulated in the ESCC tissues compared with the corresponding non-neoplastic tissues (P<0.01). More cells expressed WRAP53 protein in the ESCC tissues than in the non-neoplastic tissues (P<0.01). Overexpression of WRAP53 was significantly correlated with tumor infiltration depth (P = 0.000), clinical stage (P = 0.001), and lymph node metastasis (P = 0.025). Wrap53 expression was not correlated with age, gender, or tumor differentiation. Conclusion: This report indicates increased expression of WRAP53 in ESCC and that WRAP53 overexpression is correlated with tumor progression. WRAP53 may play a significant role in ESCC; accordingly, WRAP53 could be a useful biomarker for ESCC. [ABSTRACT FROM AUTHOR]

Non-alcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation unrelated to excess alcohol intake, in which the hepatic expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is inhibited. Long non-coding RNA HOTAIR suppresses PTEN expression in hepatic stellate cells during liver fibrosis, and it is involved in liver lipid dysregulation. In this study, we evaluated whether the PTEN down-regulation and lipid accumulation in hepatic cells might be mediated by HOTAIR during the development of NAFLD. Free fatty acids (FFAs) treatment promoted triglyceride accumulation in HepG2 cells, significantly increasing HOTAIR expression and inhibiting PTEN expression (both at mRNA and protein levels).The effects on HOTAIR and PTEN expressions disappeared after withdrawal of the FFAs treatment. SiRNA-mediated HOTAIR knockdown prevented PTEN down-regulation and triglyceride accumulation in HepG2 cells treated with FFAs; and CAPE (an NF-κBp65 inhibitor) treatment prevented the HOTAIR up-regulation and PTEN down-regulation. FFAs could induce the up-regulation of HOTAIR in HepG2 cells probably dependent on the NF-κB signaling, and consequently suppress PTEN expression and promote triglyceride accumulation. Aberrant up-regulation of HOTAIR mediated by excessive circulating FFAs levels may be a crucial mechanism associated with liver steatosis. [ABSTRACT FROM PUBLISHER]

H2S is emerging as an important regulator of ion channels [1], effects which may account for many of this gasotransmitter’s biological actions. Here, we have explored its ability to modulate T-type Ca2+ channels. H2S (applied as NaHS) caused a concentration-dependent inhibition of currents recorded in Cav3.2-expressing HEK293 cells using the whole-cell patch-clamp technique. Inhibition was voltage-independent and maximal inhibition (ca. 35%) was observed at 1mM NaHS. In contrast, Cav3.1 channel activity was unaffected, and Cav3.3 channel activity only modestly inhibited (ca.13%), over this concentration range. The redox-sensitive extracellular residue H191 [2] was essential for H2S sensitivity: currents evoked in mutant Cav3.2 (H191Q)-expressing cells were insensitive to NaHS, and the analogous reverse mutation in Cav3.1 (Q172H [3]) conferred sensitivity to NaHS on Cav3.1. As previously reported [4], the zinc chelator TPEN (10μM) augmented Cav3.2 activity, but augmented currents were also inhibited by NaHS, suggesting H2S inhibition occurred independently of zinc binding by this channel. We also explored the ability of H2S to inhibit native T-type currents: in rat aortic smooth muscle (A7r5) cells currents were modestly (<5%), but consistently, inhibited by 100μM NaHS. Inhibition was greater in HL-1 (murine atrial myocytes) and NG 108-15 (rodent neuroblastoma × glioma hybrid) cells, and in primary cultures of rat dorsal root ganglia, where inhibitions of ca. 20% were observed. We are currently exploring whether differential effects are attributable to differential channel isoform expression. Our data clearly indicate that H2S selectively inhibits Cav3.2 T-type Ca2+ channels and requires the presence of H191. [ABSTRACT FROM AUTHOR]