Differentiation potential and GFP labeling of sheep bone marrow‐derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are an important cell population in the bone marrow microenvironment. MSCs have the capacity to differentiate in vitro into several mesenchymal tissues including bone, cartilage, fat, tendon, muscle, and marrow stroma. This study was designed to isolate, expand, and characterize the differentiation ability of sheep bone marrow‐derived MSCs and to demonstrate the possibility to permanently express a reporter gene. Bone marrow was collected from the iliac crest and mononuclear cells were separated by density gradient centrifugation. Sheep MSCs cell lines were stable characterized as CD44+ and CD34? and then transfected with a green fluorescent protein (GFP) reporter gene. The GFP expression was maintained in about half (46.6%) of cloned blastocysts produced by nuclear transfer of GFP+ sheep MSCs, suggesting the possibility to establish multipotent embryonic cells' lines carrying the fluorescent tag for comparative studies on the differentiation capacity of adult stem cells (MSCs) versus embryonic stem cells. We found that sheep MSCs under appropriate culture conditions could be induced to differentiate into adipocytes, chondrocytes, and osteoblast lineages. Our results confirm the plasticity of sheep MSCs and establish the foundation for the development of a pre‐clinical sheep model to test the efficiency and safety of cell replacement therapy. J. Cell. Biochem. 114: 134–143, 2012. © 2012 Wiley Periodicals, Inc.     

Application of a YHB1-GFP reporter to detect nitrosative stress in yeast

Abstract

Yeast flavohemoglobin (Yhb1p) plays a pivotal role in NO^? detoxification and has also been implicated in oxidative/reductive stress responses. In this study, we have used a YHB1-GFP reporter expressing a full-length chromosome-tagged Yhb1-GFP fusion protein to monitor changes in flavohemoglobin levels after cell treatment with oxidants, antioxidants and nitric oxide donors. Only nitric oxide donors were found to induce a dose-dependent increase in Yhb1-GFP expression while hydrogen peroxide, menadione and diamide caused a moderate diminution of YHB1-GFP fluorescence. Additionally, the levels of endogenous and hydroperoxide-induced ROS production in the Δyhb1 mutant were comparable to those in the isogenic wild-type strain. Although peroxides increased NO^? generation while nitrite and nitric oxide donors augmented ROS levels in yeast cells, their effects were generally not more pronounced in Δyhb1 than in wild-type cells. Taken together, these data suggest that yeast flavohemoglobin does not contribute to cross-protection against oxidative and nitrosative stress.     

Substance P signaling mediates BMP-dependent heterotopic ossification

Heterotopic ossification (HO) is a disabling condition associated with neurologic injury, inflammation, and overactive bone morphogenetic protein (BMP) signaling. The inductive factors involved in lesion formation are unknown. We found that the expression of the neuro-inflammatory factor Substance P (SP) is dramatically increased in early lesional tissue in patients who have either fibrodysplasia ossificans progressiva (FOP) or acquired HO, and in three independent mouse models of HO. In Nse-BMP4, a mouse model of HO, robust HO forms in response to tissue injury; however, null mutations of the preprotachykinin (PPT) gene encoding SP prevent HO. Importantly, ablation of SP(+) sensory neurons, treatment with an antagonist of SP receptor NK1r, deletion of NK1r gene, or genetic down-regulation of NK1r-expressing mast cells also profoundly inhibit injury-induced HO. These observations establish a potent neuro-inflammatory induction and amplification circuit for BMP-dependent HO lesion formation, and identify novel molecular targets for prevention of HO.     

BMP2 induces osteoblast apoptosis in a maturation state and noggin-dependent manner

Large doses of bone morphogenetic protein 2 (BMP2) are used clinically to induce bone formation in challenging bone defects. However, complications after treatment include swelling, ectopic bone formation, and adjacent bone resorption. While BMP2 can be effective, it is important to characterize the mechanism of the deleterious effects to optimize its use. The aim of this study was to determine the effect of BMP2 on apoptosis in osteoblast lineage cells and to determine the role of the BMP inhibitor Noggin in this process. Human mesenchymal stem cells (MSCs), immature osteoblast‐like MG63 cells, and mature normal human osteoblasts (NHOst) were treated with BMP2. A model system of increased endogenous BMP signaling was created by silencing Noggin (shNOG‐MG63). Finally, the BMP pathway regulating apoptosis in NHOst was examined using BMP signaling inhibitors (5Z‐7‐oxozeaenol, dorsomorphin, H‐8). Apoptosis was characterized by caspase‐3, BAX/BCL2, p53, and DNA fragmentation. BMP2 induced apoptosis in a cell‐type dependent manner. While the effect was minor in MSCs, MG63 cells had modest increases and NHOst cells had robust increases apoptosis after BMP2 treatment. Apoptosis was significantly higher in shNOG‐MG63 than MG63 cells. 5Z‐7‐oxozeaenol and dorsomorphin eliminated the BMP2‐induced increase in DNA fragmentation in NHOst, suggesting roles for TAB/TAK1 and Smad signaling. These results indicate that the apoptotic effect of BMP2 is dependent on cell maturation state, inducing apoptosis in committed osteoblasts through Smad and TAB/TAK1 signaling, and is regulated by Noggin. Dose and delivery must be optimized in therapeutic applications of BMP2 to minimize complications. J. Cell. Biochem. 113: 3236–3245, 2012. © 2012 Wiley Periodicals, Inc.     

Level of nitrated proteins in the plasma,platelets and liver of patients with liver cirrhosis

Abstract

Over-expression of nitric oxide synthase (NOS) and nitric oxide (NO) formation are associated with the pathogenesis of liver cirrhosis. NO-related stress alters the functions of biomolecules, especially proteins, probably as a result of nitration. The aim of this study was to assess the level of protein nitration and its correlation with the severity of the disease. Liver cirrhosis patients with different grades of severity (grades A, B, and C according to the Child–Pugh classification) were enrolled in this study. Nitroprotein content, arginine, citrulline, NO in terms of total nitrite, nitrosothiol (RSNO) and protein carbonyls were measured in blood. Immunohistochemical detection of nitroprotein was carried out in liver sections of cirrhosis patients. A significant elevation in the levels of serum and platelet arginine, arginase, citrulline, plasma, and platelet nitroproteins, RSNO, total nitrite, protein carbonyls and also a significant amount of nitrated proteins by immunohistochemical detection in tissue were observed in cirrhosis patients. The alterations were highly significant in grade C patients with bleeding complications when compared to those of grade B and A patients. In platelets, both cytosolic and cytoskeletal proteins were found to be nitrated significantly. The level of nitrite seems to have positive correlation with the level of nitroproteins in different grades of cirrhosis. The level of nitroproteins in plasma, platelets and liver tissue can be correlated with the severity of liver cirrhosis.     

Prophylactic role of Enhydra fluctuans against arsenic-induced hepatotoxicity via anti-apoptotic and antioxidant mechanisms

Abstract

Organisms produce reactive species throughout their lives, and this may result in damage to proteins and other biological molecules. Oxidatively damaged proteins are normally selectively degraded and replaced, but this process appears to be less efficient in senescent, long-lived, post-mitotic cells, as is evidenced by their accumulation in the form of lipofuscin inside the lysosomal compartment. A great deal of research has focused on changes to the proteolytic machinery in the ageing cell, in particular the proteasome, although failure of heat shock proteins (HSPs) to bind and deliver oxidised proteins efficiently to the degradation machinery could also contribute to their aggregation and accumulation. Oxidised proteins can be protease-resistant and may even directly inhibit the proteolytic machinery of the cell. The critical role that is played by HSPs in preventing accumulation of oxidised proteins is often overlooked. In this review, we examine the key role played by HSPs in recognising, removing and preventing the formation of oxidised and damaged proteins in cells. We also examine the evidence supporting the view that failure of one of these pathways could underlie ageing and age-related diseases. Finally, we discuss how modulation of HSP-activity could influence the ageing process and the progression of age-related diseases.