No abstract

There are three isoforms of the inositol 1,4,5-trisphosphate receptor (InsP 3 R), each of which has a distinct effect on Ca 2؉ signaling. However, it is not known whether each isoform similarly plays a distinct role in the activation of Ca 2؉ -mediated events. To investigate this question, we examined the effects of each InsP 3 R isoform on transmission of Ca 2؉ signals to mitochondria and induction of apoptosis. Each isoform was selectively silenced using isoform-specific small interfering RNA in Chinese hamster ovary cells, which express all three InsP 3 R isoforms. ATP-induced cytosolic Ca 2؉ signaling patterns were altered, regardless of which isoform was silenced, but in a different fashion depending on the isoform. ATP also induced Ca 2؉ signals in mitochondria, which were inhibited more effectively by silencing the type III InsP 3 R than by silencing either the type I or type II isoform. The type III isoform also co-localized most strongly with mitochondria. When apoptosis was induced by activation of either the extrinsic or intrinsic apoptotic pathway, induction was reduced most effectively by silencing the type III InsP 3 R. These findings provide evidence that the type III isoform of the InsP 3 R plays a special role in induction of apoptosis by preferentially transmitting Ca 2؉ signals into mitochondria.Cytosolic Ca 2ϩ (Ca i 2ϩ ) 3 is a versatile second messenger that can simultaneously regulate multiple processes within an individual cell (1). This complex regulatory action of Ca 2ϩ is thought to result in part from the varied spatial and temporal patterns of Ca 2ϩ signals that can occur (1). These signaling patterns in turn are thought to result from special properties of the inositol-1,4,5-trisphosphate (InsP 3 ) receptor (InsP 3 R), which is the principal intracellular Ca 2ϩ release channel in most types of cells (2). The InsP 3 R is gated by InsP 3 and is localized to the endoplasmic reticulum (3) and to a lesser extent the nucleus (4, 5). There are three isoforms of the InsP 3 R, each of which has a different affinity for InsP 3 (6) and distinct functional properties at the single channel level (7,8). Each InsP 3 R isoform also has distinct effects on Ca 2ϩ signaling patterns in intact cells (9, 10). Although some cells and tissues express a single or predominant isoform of the receptor, most cells instead express two or all three InsP 3 R isoforms (9, 11-13). The presence of multiple isoforms within an individual cell suggests that Ca 2ϩ released from each isoform might mediate distinct cellular events. Apoptosis is one Ca 2ϩ -mediated event that may be influenced differently by each InsP 3 R isoform. Morphologically, apoptosis is characterized by membrane blebbing, chromatin condensation, DNA fragmentation, and eventually the formation of apoptotic bodies, which are phagocytosed by neighboring cells (14). Studies at the molecular level suggest that the InsP 3 R plays an important role in the development of apoptosis (15-17). Initial evidence suggested that Ca 2ϩ -dependent apoptotic death was ...

In this study the time course of homing and the body distribution of systemically delivered bone marrow mesenchymal stem cells (BM-MSCs) after myocardial infarction (MI) were evaluated. BM-MSCs were isolated from Wistar rats, expanded in vitro, and their phenotypical characterization was performed by flow cytometer. Rats were randomly divided into three groups: control, sham MI, and MI. BM-MSCs (5 × 10 6 ) were labeled with 99m Tc-HMPAO and injected through the tail vein 7 days after MI. Gamma camera imaging was performed at 5, 15, 30, and 60 min after cell inoculation. Due to the 99m Tc short half-life, cell migration and location were also evaluated in heart sections using DAPI-labeled cells 7 days after transplantation. Phenotypical characterization showed that BM-MSCs were CD90 + , CD73 + , CD54+ , and CD45 − . Five minutes after 99m Tc-HMPAO-labeled cell injection, they were detected in various tissues. The cells migrated mainly to the lungs (approximately 70%) and, in small amounts, to the heart, kidneys, spleen, and bladder. The number of cells in the heart and lungs decreased after 60 min. MI markedly increased the amount of cells in the heart, but not in the lungs, during the period of observation (4.55 ± 0.32 vs. 6.34 ± 0.67% of uptake in infarcted hearts). No significant differences were observed between control and sham groups. Additionally, 7 days after DAPI-labeled cells injection, they were still detected in the heart but only in infarcted areas. These results suggest that the migration of systemically delivered BM-MSCs to the heart is time dependent and MI specifically increases BM-MSCs homing to injured hearts. However, the systemic delivery is limited by cell entrapment in the lungs.Key words: Myocardial infarction; Mesenchymal stem cells; Homing; Body distribution INTRODUCTIONstem cells (BM-MSCs) that can differentiate into different cellular types (5,14,27). Many reports have shown that cellular therapy using Mesenchymal stem cells (MSCs) have been isolated from multiple adult tissue sources, such as cord blood, BM-MSCs is able to improve cardiac function after myocardial ischemia in different species, including huplacenta, adipose and dermal tissues, synovial fluid, deciduous teeth, and amniotic fluid (19). This broad distrimans (2,30). However, the mechanisms by which BMMSCs induce their beneficial effects in the heart are still bution of sources combined with their ability to differentiate into multiple mesenchymal phenotypes, such as controversial. Thus, it has been proposed that BM-MSCs can differentiate into both vascular endothelial cells and bone, cartilage, tendon, and adipose tissue, has led them to be referred as potential therapeutic candidates for sevcardiomyocytes, activate local factors, fuse with resident cells, or even a combination of these mechanisms that eral diseases and degenerative processes, including myocardial infarction (MI) (18,37). In addition to these ultimately lead to a restoration of the cardiac structure and function (6,7,32 based therapy for myocardial repai...

No abstract

No abstract

BackgroundRecombinant proteins expressed in Escherichia coli vectors are generally contaminated with endotoxin. In this study, we evaluated the ability of Polymyxin B to neutralize the effect of LPS present as contaminant on Schistosoma mansoni recombinant proteins produced in E. coli in inducing TNF-α and IL-10. Peripheral blood mononuclear cells from individuals chronically infected with S. mansoni were stimulated in vitro with recombinant Sm22.6, Sm14 and P24 antigens (10 μg/mL) in the presence of Polymyxin B (10 μg/mL).ResultsThe levels of cytokines were measured using ELISA. There was greater than 90 % reduction (p < 0.05) in the levels of TNF-α and IL-10 when Polymyxin B was added to the cultures stimulated with LPS. In cultures stimulated with S. mansoni recombinant proteins in the presence of Polymyxin B, a reduction in the levels of TNF-α and IL-10 was also observed. However, the percentage of reduction was lower when compared to the cultures stimulated with LPS, probably because these proteins are able to induce the production of these cytokines by themselves.ConclusionThis study showed that Polymyxin B was able to neutralize the effect of endotoxin, as contaminant in S. mansoni recombinant antigens produced in E. coli, in inducing TNF-α and IL-10 production.

No abstract

No abstract