Typical sphingomyelin, which has a hydrophobic moiety consisting of a sphingosine base with a trans-double bond and saturated fatty acids chain, forms stretched conformations in membrane lipid bilayers. By contrast, most natural glycerophospholipids, which have unsaturated fatty acyl chains with cis-double bonds, cannot form such conformations. These structural features of SM allow it to make more intimate molecular contacts with cholesterol, generating stronger van der Waals interactions, as compared to unsaturated glycerophospholipids. Furthermore, the bulky phosphocholine head group of SM may contribute to shield the hydrophobic part of cholesterol molecules from water molecules under aqueous environments. Consequently, SM and cholesterol tend to be closely packaged and to form membrane mesodomains with a liquid ordered phase. Because both SM and cholesterol are abundant at the plasma membrane in mammalian cells, the SM/cholesterol-enriched meso-domains exist mainly at the plasma membrane. These SM/cholesterol-enriched meso-domains are often considered to be part of ‘lipid rafts’, although the definition of lipid rafts is still under debate. When cells or isolated membranes are treated with several types of non-ionic detergents at low temperatures, various components associated with the SM/ cholesterol-enriched meso-domains or lipid rafts are distributed to the detergent-insoluble fractions. For convenience, we tentatively regard such detergent-insoluble fractions as a biochemical representative of lipid rafts in the present paper. Glycosylphosphatidyl Remdesivir inositolanchored proteins, flotillin, and gangliosides are targeted to lipid rafts. There are specialized raft domains, caveolae, at the cell surface. Caveolae are invaginations of the plasma membrane and contain polymerized caveolin, which is a hairpin-like integral membrane protein. Raft domains play important roles in membrane trafficking, substrate transport, and signal transduction including IgE receptor signaling, T-cell antigen receptor signaling, and epidermal growth factor receptor signaling. ATP-binding cassette G1 and ABCG4 are members of the ABCG subfamily of proteins, which are half-type ABC proteins. They consist of an N-terminal cytosolic nucleotidebinding domain and a C-terminal transmembrane domain, which has 6 transmembrane a-helices. ABCG1 and ABCG4 form a homodimer or a heterodimer. ABCG1 mediates the efflux of cholesterol, 7-ketocholesterol, SM, and phosphatidylcholine to high-density lipoprotein from cells. ABCG4, which shares 69% identity and 84% similarity at the amino acid level with ABCG1, mediates the efflux of cholesterol to HDL, like ABCG1. ABCG1 is ubiquitously expressed, but highly expressed in the brain, lung, and liver.

Deficiency in SI protein results in osmotic diarrhea due to an inability to hydrolyze intestinal disaccharides into component monosaccharides. Therefore, we conclude that the newly established cell line is comprised of small intestine-derived epithelial cells, which in turn suggests that this cell line can be used in future research on disease inducing porcine diarrhea. Typically, differentiated cells were taken for representing the intestinal villus tip cells, while the undifferentiated cells were used to mimic the basilar crypt cells of the intestines. Thus, the prototypical characteristics of differentiated porcine intestinal epithelial cells are the presence of tight junctions and distinct microvilli on their apical surfaces. Here, we demonstrated that porcine intestinal epithelial cells expressed markers typical of differentiated enterocytes, specifically, ZYM-SIEC02 cells were positive for E-cadherin, ZO-1, Occludin, villin, and sucrose isomaltase, which is the most reliable indicator of intestinal cell differentiation in vitro, indicating the presence of differentiated villus cells in the culture. IPEC-J2 cells were cultured for 1 days or 21 days, representing undifferentiated proliferating and highly differentiated IPEC-J2 cells, respectively. However, in our experiment, all the ZYM-SIEC02 cells we used were cultured to have a confluent monolayer within 3 or 6 days according to the density of cell cultures. In this regard, the characteristics of differentiated ZYM-SIEC02 cell were evidently due to the time that isolated and purified the primary porcine intestinal epithelial cells was long enough for the differentiation of pSIECs, which prepared for the lipofection. Thus, ZYM-SIEC02 cells remain the characteristic of differentiated pSIECs, and markers typical of differentiated Cycloheximide enterocytes were detected. Moreover, we used tissue culture method to isolate epithelial cells from small intestine, it was unavoidable that the growth of fibroblasts over cultured primary intestinal epithelial cells, however, on the other side of the coin, the intestinal epithelial cell differentiation need the heterologous cell-cell contacts for denovo synthesis to trigger cell polarity and differentiation. The addition of EGF may be another important inducement for differentiation as it plays a pivotal role in the regulation of intestinal epithelial proliferation and differentiation. Over time in culture, cells maintained their epithelial morphology as well as expression of markers. These characteristics were not affected by cryogenic freezing and retrieval. An early study by Hayflick et al demonstrated that normal human somatic cells have a finite replicative potential in vitro, and will stop dividing after a finite number of population doublings and enter senescense.

Cisplatin nephrotoxicity has been recognized as a complex multifactorial process that includes oxidative stress pathways involved in apoptosis. Increased oxidative stress is one of the earliest features associated with the development of cisplatininduced nephropathy. Several investigators have demonstrated that Paclitaxel interaction of cisplatin with SH-groups leads to glutathione depletion, along with a decline of cellular antioxidant system and accumulation of reactive oxygen species or their products. Mitochondrial injury seems to play an important role in cisplatin-induced nephrotoxicity. Several functional and structural alterations of the mitochondria have been observed in cell cultures and in vivo animal models of cisplatin nephrotoxicity. This was evidenced by decreased mitochondrial mass with reduction of activities of oxidative phosphorylation complexes and manganese superoxide dismutase. This selectivity for mitochondria is probably caused by the accumulation of positively charged aquated complexes of cisplatin in the negatively charged inner space of the mitochondria. Thus, increased oxidative stress in cisplatin nephrotoxicity may be simply a consequence of disrupted respiratory chain and decreased antioxidant activity since mitochondria are a major source and target for damage by ROS. Oxidative stress and mitochondrial damage have been proposed as important factors that are involved in the activation of apoptotic pathway and cisplatin-induced cell death in vitro as well as in vivo. These events, together, result in the loss of renal function during cisplatin nephrotoxicity, triggering acute renal failure and tubular injury. A wide variety of antioxidants have been reported to exhibit protective effects against the deleterious effects of cisplatin-induced nephrotoxicity. As superoxide anions are the major injurious oxidant species generated by mitochondria, earlier studies have focused on the protective role for mitochondrial localized MnSOD in several models of free radicals-mediated cell injury. Tempol is a membrane-permeable radical scavenger which has SOD and catalase activities. Tempol has been reported experimentally to ameliorate oxidative stressmediated renal dysfunction and glomerular injury. Moreover, tempol ameliorated endothelial cell dysfunction in diabetic rats and reduced infarct size in an experimental model of regional myocardial ischemia/reperfusion. A phase I clinical trial in patients receiving whole brain radiotherapy suggested that tempol may be effective at preventing radiation-induced alopecia with only mild toxicity. Tempol, as an antioxidant, has been previously demonstrated to prevent injury induced by cisplatin using established renal epithelial cell line, LLC-PK1. However, no study has investigated the effect of tempol in an in vivo experimental model of cisplatin-induced nephrotoxicity in addition to mitochondrial role in its possible mediated protection. Therefore, the goal of the present study was directed to examine the implication of this membrane-permeable SOD-mimetic agent, tempol, in the prevention of mitochondrial dysfunction in cisplatin-induced nephrotoxicity. Moreover, the effect of tempol on cisplatin antineoplastic efficacy was investigated.

The goal of the present study was directed to examine the potential role of a membranepermeable SOD-mimetic agent, tempol, in alleviating mitochondrial dysfunction in cisplatin-induced nephrotoxicity and to evaluate the effect of tempol on cisplatin antineoplastic efficacy. Cisplatin induced weight loss might be due to gastrointestinal toxicity and reduction of food ingestion. The present data show that administration of cisplatin to mice caused marked elevation of serum creatinine and urea levels as well as significant glucosuria and proteinuria. Cisplatin causes acute renal failure due to its preferential accumulation within the proximal tubular cells in the outer medulla of the kidney. The alterations in glomerular function in cisplatin-treated mice may be secondary to the oxidative stress status observed in the present study, which induces mesangial cells contraction, alteration of filtration surface area and modification of ultrafiltration coefficient factors that decrease the glomerular filtration rate. Pretreatment with tempol normalized serum creatinine level and caused significant reduction of serum urea, glucosuria and proteinuria compared to cisplatin group, indicating improvement of glomerular and tubular functions. The present results clearly indicate a significant degree of oxidative stress in both mitochondrial and postmitochondrial fractions in renal tissues of cisplatin-treated mice. This was demonstrated by a significant elevation of TBARS content and a significant reduction of GSH content along with inhibition of SOD and catalase activities in both mitochondrial and postmitochondrial fractions. The enhanced state of oxidative stress in cisplatin-treated group was associated with significant reduction of mitochondrial oxidative phosphorylation capacity and complexes I and III activities together with significant elevation of mNOS protein expression and caspase-3 activity. This was correlated with a pronounced reduction of ATP content indicating marked deterioration of mitochondrial respiration and energy production. GSH is one of the essential components for maintaining cell integrity because of its reducing properties and participation in the cell metabolism. The depletion of the renal GSH level has been observed in response to oxidative stress caused by cisplatin treatment. Platinum was shown to preferentially bind to GSH and protein thiol in kidneys after cisplatin treatment. The concentration of platinum-bound proteins was higher in the mitochondrial fraction than in the cytosolic fraction. Formation of these complexes limits the amount of drug available for DNA binding and therefore, a positive correlation has been AB1010 reported between GSH levels and resistance to cisplatin. Cisplatin affects many enzymes that protect the cells from oxidative damage, among which Cu, Zn-SOD, Mn-SOD and catalase. SOD plays an important role in the dismutation of superoxide anions. Decreased SOD activity as observed in this study could lead to incomplete scavenging of superoxide anions that are produced during the normal metabolic process with further initiation and propagation.

Similar to the effect of co-culture with neonatal mouse retinal cells, Taurine and RA promoted upregulation of retinal progenitor markers in human LNS. This suggests that defined culture conditions may replace the use of animal tissue in the future. We did not observe LNS cell migration or integration into the host retina following sub-retinal transplantation into neonatal mice. Cell integration into the retina remains challenging. Despite being derived from the same origin as neural retina, iris or CB derived cells have also shown limited ability for retinal integration. The proportions of cells which integrate into embryonic retinal explants or retina from degenerate animal models are small. Studies using retinal progenitor cells from embryonic retina have also shown little integration into host retina, although mature retinal phenotypes have been observed following sub-retinal transplantation. MacLaren et al. investigated the optimal cell resource for functional integration into adult retina. The cells which migrated and integrated were shown to be post-mitotic rod precursor cells. Therefore, the ontogenetic stage of transplanted cells is important for successful cell integration. Grafted LNS cells in this study were not fully committed post-mitotic cells. This may explain why cell integration was not observed. The host Cycloheximide microenvironment is also essential for inducing cell differentiation and migration. In a study involving transplantation of IPE derived cells, the grafted cells expressed the photoreceptor specific marker rhodopsin when they were transplanted into the SRS of embryonic chicken eyes. On the contrary, they did not express rhodopsin or other neural markers when they were transplanted into the vitreous cavity. This is in accordance with our observation that the LNS cells transplanted into the vitreous do not express photoreceptor markers. LNS display plasticity, the potential to cross the tissue/germ layer boundary and generate cells other than their origin. However, LNS have limited potential to generate photoreceptorlike cells. The highest rhodopsin expression level noted using LNS derived cells was,3% of that observed compared to using neonatal mouse retinal tissue. Reports on other ocular stem-like/ progenitor cells also show limited success in the generation of photoreceptor regardless of cell origin. Recently two independent groups showed CE-derived cells failed to give rise to photoreceptor cells. Retinal neurosphere cells derived from neonatal mice also had a low efficiency in generation of rhodopsin positive cells during spontaneous differentiation. It has been suggested that cell reprogramming is likely to be needed for robust photoreceptor cell production. LNS cells would also be an optimal cell resource for reprogramming and/or transdifferentiation and subsequent retinal repair. They are readily accessible, highly proliferative and multipotent ocular stem cells. iPSCs have been generated from mouse and human somatic cells by ectopic expression of four transcription factors including OCT4, SOX2, c-Myc and KLF4. Due to risks such as insertional mutagenesis or tumour formation, it is desirable to use the minimal number of transcription factors and to eliminate oncogenic factors.