Numerous extracellular stimuli trigger trans-autophosphorylation at Tyr402 of Pyk2, inducing its activation. Pyk2 is a key mediator of several signaling pathways and has been implicated in apoptosis induced by specific stress signals. We investigated whether Pyk2 participates in cerebellar granule n
euron (CGN) apoptosis induced by the suppression of membrane depolarization. We demonstrate that shifting CGN cultures from 25 mM to 5 mM KCl-containing medium induces an early, transient 70% increase in phosphorylated Tyr402 and Tyr580 Pyk2 levels that is triggered by Ca(2+) released from intracellular stores and mediated by calmodulin (CaM). Overexpression of Pyk2 increases CGN survival after 24 h by 70% compared to the control, thus suggesting that Pyk2 is involved in an anti-apoptotic response to K+ lowering. Furthermore, we show that CGN grown in K25 medium exhibit detectable CaM-dependent Pyk2 activity. When silencing Pyk2 activity by expressing a dominant-negative form, only 40% of the transfected neurons were alive 24 h after transfection when compared to the control. Overall, the present findings demonstrate for the first time that Pyk2 is a critical mediator of CGN survival.
Si X, etal., Food Funct. 2017 Jan 25;8(1):232-240. doi: 10.1039/c6fo01225f.
The anti-obesity effects of two types of resistant starch (RS) in high-fat-diet-induced obese rats were investigated. The serum triglycerides, total cholesterol and malondialdehyde concentrations were significantly reduced, and the total antioxidant capacity, superoxide dismutase levels and glutathi
one peroxidase activity were increased by RS2 and RS4 consumption compared to the obesity group. A significant reduction in the serum glucose level and elevations in hepatic lipid metabolic enzyme activities were observed only for RS4 administration. Moreover, the expression levels of the fatty acid synthesis associated genes ACC and Fads1, the triglyceride synthesis and metabolism-related gene SREBP-1, the adipocyte differentiation gene PPARγ, the cholesterol synthesis associated gene HMGCR, and the gluconeogenesis associated gene GAPDH were all significantly down-regulated, whilst the lipid oxidation gene Acox1 and the liver function genes Gsta2, Nqo1, and Gclm were up-regulated in both administered groups. Additionally, RS4 performed well in up-regulating the expressions of Gsta2, Gsta3, Nqo1, and Egfr, and down-regulating LXRα, Igfbp1, and Pml. RS4 exhibited great advantages in reducing oxidative stress compared with RS2.
Si X, etal., Carbohydr Polym. 2017 Feb 10;157:834-841. doi: 10.1016/j.carbpol.2016.10.042. Epub 2016 Oct 17.
This study investigated the interventional effect of resistant starch (RS), chitosan (CS) and chitosan-starch complexes (CL) on blood glucose, lipid composition and oxidative stress in high-fat diet fed rats. Compared with RS or CS alone, CL administration performed more efficiently in controlling b
ody weight and adipose tissue mass, together with an increase in HDL-C concentration, oxidative stress suppression by increasing body antioxidant capacity. Gene expression analysis demonstrated the fatty acid and triglyceride synthesis and metabolism gene SREBP-1, adipocyte differentiation gene PPARγ, cholesterol synthesis gene HMGCR, gluconeogenesis gene GAPDH, were significantly down-regulated, whilst lipid oxidation gene Acox1 and liver functional genes Gstm2, Gclc were up-regulated following CL consumption compared with single RS or CS treatment. Hypolipidemic effects were observed by CL administration and oxidative stress suppression by CL appeared to be associated with elevated antioxidant enzyme activity, increased lipid oxidation, as well as improved fatty acid and cholesterol homeostasis.
BACKGROUND: Following spinal cord injury, a highly inhibitory environment for axonal regeneration develops. One of the main sources of this inhibition is the glial scar that is formed after injury by reactive astrocytes. The inhibitory environment is mainly a result of chondroitin sulphate proteogly
cans (CSPGs). Neuroglycan 2 (NG2), one of the main inhibitory CSPGs, is up-regulated following spinal cord injury. METHODS: Small interfering RNA (siRNA) was designed to target NG2 and this short hairpin RNA (shRNA) was cloned into a lentiviral vector (LV). The neurotrophic factor neurotrophin-3 (NT-3) promotes the growth and survival of developing neurites and has also been shown to aid regeneration. NT-3 was also cloned into a LV. In vitro assessment of these vectors using a coculture system of dorsal root ganglia (DRG) neurones and Neu7 astrocytes was carried out. The Neu7 cell line is a rat astrocyte cell line that overexpresses NG2, thereby mimicking the inhibitory environment following spinal cord injury. RESULTS AND DISCUSSION: These experiments show that both the knockdown of NG2 via shRNA and over-expression of NT-3 can significantly increase neurite growth, although a combination of both vectors did not confer any additional benefit over the vectors used individually. These LVs show promising potential for growth and survival of neurites in injured central nervous system tissue (CNS).
Bandara N, etal., Stem Cell Res Ther. 2016 Apr 1;7:48. doi: 10.1186/s13287-016-0307-2.
BACKGROUND: Non-viral-based gene modification of adult stem cells with endothelial nitric oxide synthase (eNOS) may enhance production of nitric oxide and promote angiogenesis. Nitric oxide (NO) derived from endothelial cells is a pleiotropic diffusible gas with positive effects on maintaining vascu
lar tone and promoting wound healing and angiogenesis. Adult stem cells may enhance angiogenesis through expression of bioactive molecules, and their genetic modification to express eNOS may promote NO production and subsequent cellular responses. METHODS: Rat bone marrow-derived mesenchymal stem cells (rBMSCs) were transfected with a minicircle DNA vector expressing either green fluorescent protein (GFP) or eNOS. Transfected cells were analysed for eNOS expression and NO production and for their ability to form in vitro capillary tubules and cell migration. Transcriptional activity of angiogenesis-associated genes, CD31, VEGF-A, PDGFRalpha, FGF2, and FGFR2, were analysed by quantitative polymerase chain reaction. RESULTS: Minicircle vectors expressing GFP (MC-GFP) were used to transfect HEK293T cells and rBMSCs, and were compared to a larger parental vector (P-GFP). MC-GFP showed significantly higher transfection in HEK293T cells (55.51 +/- 3.3 %) and in rBMSC (18.65 +/- 1.05 %) compared to P-GFP in HEK293T cells (43.4 +/- 4.9 %) and rBMSC (15.21 +/- 0.22 %). MC-eNOS vectors showed higher transfection efficiency (21 +/- 3 %) compared to P-eNOS (9 +/- 1 %) and also generated higher NO levels. In vitro capillary tubule formation assays showed both MC-eNOS and P-eNOS gene-modified rBMSCs formed longer (14.66 +/- 0.55 mm and 13.58 +/- 0.68 mm, respectively) and a greater number of tubules (56.33 +/- 3.51 and 51 +/- 4, respectively) compared to controls, which was reduced with the NOS inhibitor L-NAME. In an in vitro wound healing assay, MC-eNOS transfected cells showed greater migration which was also reversed by L-NAME treatment. Finally, gene expression analysis in MC-eNOS transfected cells showed significant upregulation of the endothelial-specific marker CD31 and enhanced expression of VEGFA and FGF-2 and their corresponding receptors PDGFRalpha and FGFR2, respectively. CONCLUSIONS: A novel eNOS-expressing minicircle vector can efficiently transfect rBMSCs and produce sufficient NO to enhance in vitro models of capillary formation and cell migration with an accompanying upregulation of CD31, angiogenic growth factor, and receptor gene expression.
OBJECTIVE: To search for genetic alteration in NKX2.5 gene in patients presenting both congenital heart disease (CHD) and TD. SUBJECTS AND METHODS: Individual phenotypes were carefully analyzed in 86 children with thyroid dysgenesis (TD) using thyroid function tests, scintigraphy, ultrasound and ech
ocardiography. DNA was extracted and NKX2.5 gene coding region was amplified by polymerase chain reaction (PCR) and sequenced. RESULTS: CHD were found in 8.1% of patients with TD. The mutation screening revealed two known polymorphisms in patients with isolated TD or TD associated with CHD. None of them are predicted to result in codon change in conserved domain. The c.63A>G polymorphism was detected in 54/86 patients (49 with isolated TD and 5 with TD combined with CHD). There was a significant association of c.63A>G polymorphism with hypoplasia (p < 0.036). The c.541G>A polymorphism was observed in only one patient with isolated thyroid hypoplasia. CONCLUSION: NKX2.5 mutations were not found. The c.63A>G polymorphism might be associated with thyroid hypoplasia.
Slomka M, etal., BMC Genet. 2015 Sep 23;16:114. doi: 10.1186/s12863-015-0271-3.
BACKGROUND: Multidrug resistance-associated protein 1 (MRP1), encoded by the ABCC1 gene, is an ATP-binding cassette transporter mediating efflux of organic anions and xenobiotics; its overexpression leads to multidrug resistance. In this study, 30 exons (from 31 in total) of the ABCC1 gene as well a
s and their flanking intron sequences were screened for genetic variation, using the High Resolution Melting (HRM) method, for 190 healthy volunteers representing the Polish population. Polymorphism screening is an indispensable step in personalized patient therapy. An additional targeted SNP verification study for ten variants was performed to verify sensitivity of the scanning method. RESULTS: During scanning, 46 polymorphisms, including seven novel ones, were found: one in 3' UTR, 21 in exons (11 of them non-synonymous) and 24 in introns, including one deletion variant. These results revealed some ethnic differences in frequency of several polymorphisms when compared to literature data for other populations. Based on linkage disequilibrium analysis, 4 haplotype blocks were determined for 9 detected polymorphisms and 12 haplotypes were defined. To capture the common haplotypes, haplotype-tagging single nucleotide polymorphisms were identified. CONCLUSIONS: Targeted genotyping results correlated well with scanning results; thus, HRM is a suitable method to study genetic variation in this model. HRM is an efficient and sensitive method for scanning and genotyping polymorphic variants. Ethnic differences were found for frequency of some variants in the Polish population compared to others. Thus, this study may be useful for pharmacogenetics of drugs affected by MRP1-mediated efflux.
Halder SK, etal., Cancer Res. 2006 Jun 15;66(12):6156-66.
The development and progression of malignancies is a complex multistage process that involves the contribution of a number of genes giving growth advantage to cells when transformed. The role of transforming growth factor-beta (TGF-beta) in carcinogenesis is complex with tumor-suppressor or prooncog
enic activities depending on the cell type and the stage of the disease. We have previously reported the identification of a novel WD-domain protein, STRAP, that associates with both TGF-beta receptors and that synergizes with the inhibitory Smad, Smad7, in the negative regulation of TGF-beta-induced transcription. Here, we show that STRAP is ubiquitously expressed and is localized in both cytoplasm and nucleus. STRAP is up-regulated in 60% colon and in 78% lung carcinomas. Stable expression of STRAP results in activation of mitogen-activated protein kinase/extracellular signal-regulated kinase pathway and in down-regulation of the cyclin-dependent kinase inhibitor p21(Cip1), which results in retinoblastoma protein hyperphosphorylation. In addition, we have observed that Smad2/3 phosphorylation, TGF-beta-mediated transcription, and growth inhibition are induced in STRAP-knockout mouse embryonic fibroblasts compared with wild-type cells. Ectopic expression of STRAP in A549 lung adenocarcinoma cell line inhibits TGF-beta-induced growth inhibition and enhances anchorage-independent growth of these cells. Moreover, overexpression of STRAP increases tumorigenicity in athymic nude mice. Knockdown of endogenous STRAP by small interfering RNA increases TGF-beta signaling, reduces ERK activity, increases p21(Cip1) expression, and decreases tumorigenicity. Taken together, these results suggest that up-regulation of STRAP in human cancers may provide growth advantage to tumor cells via TGF-beta-dependent and TGF-beta-independent mechanisms, thus demonstrating the oncogenic function of STRAP.
Lisinski I, etal., Biochem Biophys Res Commun. 2006 Jun 16;344(4):1179-85. Epub 2006 Apr 21.
To identify novel regulatory components involved in the recycling of the insulin-responsive glucose transporter GLUT4, we have used the yeast two-hybrid system to isolate GLUT4-binding proteins from a rat adipose cell cDNA library. We found a 49-kDa protein (p49/STRAP
/span>) that specifically interacts with an acidic amino acid motif (Q7IGSEDG) in the N-terminus of GLUT4. Confocal immunofluorescence microscopy of primary rat adipose cells shows co-localization of myc-p49 with GLUT4 and also with the ER-resident protein calnexin. Insulin stimulation had no effect on GLUT4-binding and subcellular distribution of p49 in adipose cells. However, overexpression of the GLUT4-binding domain of p49 in adipose cells reduces protein synthesis and cell-surface expression of GLUT4, but not of GLUT8. Moreover, cell-surface expression of a p49-binding-deficient GLUT4 mutant (ED/QN) is also reduced. Kinetic analysis of HA-epitope-tagged GLUT4 protein synthesis indicates a possible role of p49 in biosynthesis and/or processing of GLUT4 in adipose cells.
Jin L and Datta PK, Cell Cycle. 2014;13(24):3909-20. doi: 10.4161/15384101.2014.973310.
We have previously reported the identification of a novel WD-domain protein, STRAP that plays a role in maintenance of mesenchymal morphology by regulating E-cadherin and that enhances tumorigenicity partly by downregulating CDK inhibitor p21(Cip1). However, the
functional mechanism of regulation of E-cadherin and p21(Cip1) by STRAP is unknown. Here, we have employed STRAP knock out and knockdown cell models (mouse embryonic fibroblast, human cancer cell lines) to show how STRAP downregulates E-cadherin and p21(Cip1) by abrogating the binding of Sp1 to its consensus binding sites. Moreover, ChIP assays suggest that STRAP recruits HDAC1 to Sp1 binding sites in p21(Cip1) promoter. Interestingly, loss of STRAP can stabilize Sp1 by repressing its ubiquitination in G1 phase, resulting in an enhanced expression of p21(Cip1) by >4.5-fold and cell cycle arrest. Using Bioinformatics and Microarray analyses, we have observed that 87% mouse genes downregulated by STRAP have conserved Sp1 binding sites. In NSCLC, the expression levels of STRAP inversely correlated with that of Sp1 (60%). These results suggest a novel mechanism of regulation of E-cadherin and p21(Cip1) by STRAP by modulating Sp1-dependent transcription, and higher expression of STRAP in lung cancer may contribute to downregulation of E-cadherin and p21(Cip1) and to tumor progression.
