The effect of ischemia and reperfusion on purine nucleoside phosphorylase was studied in an isolated perfused rat liver model. This enzyme is localized primarily in the cytoplasm of the endothelial and Kupffer cells; some activity is associated with the parenchymal cells. Levels of this enzyme accurately predicted the extent of ischemia and reperfusion damage to the microvascular endothelial cell of the liver. Livers from Lewis rats were subjected to 30, 45 and 60 min of warm (37° C) no flow ischemia that was followed by a standard reperfusion period lasting 45 min. Purine nucleoside phosphorylase was measured at the end of the no flow ischemia and reperfusion periods as was superoxide generation (O₂–). Bile production was monitored throughout the no flow ischemia and reperfusion periods. Control perfusions were carried out for 120 min. A significant rise in purine nucleoside phosphorylase levels as compared with controls was observed at the end of ischemia in all the three groups. The highest level, 203.5 ± 29.2 mU/ml, was observed after 60 min of ischemia. After the reperfusion period, levels of purine nucleoside phosphorylase decreased in the 30– and 45–min groups 58.17 ± 9.66 mU/ml and 67.5 ± 17.1 mU/ml, respectively. These levels were equal to control perfusions. In contrast, after 60 min of ischemia, levels of purine nucleoside phosphorylase decreased early in the reperfusion period and then rose to 127.8 ± 14.8 mU/ml by the end of reperfusion (p < 0.0001). Superoxide generation at the beginning of reperfusion was higher than in controls with similar values observed at the end of 30, 45 and 60 min of ischemia. During reperfusion, production of superoxide continued. Bile production was significantly lower at the end of 30 min (0.044 ± 0.026 μl/min/gm), 45 min (0.029 ± 0.0022 μ/min/gm) and 60 min of ischemia (0.022 ± 0.008 μ/min/gm) when compared with bile production by control livers during the corresponding time (0.680 ± 0.195, 0.562 ± 0.133 and 0.480 ± 0.100 μ/min/gm respectively; p < 0.001). During reperfusion, rates of bile production were normal after 30 and 45 min of ischemia. In contrast, significantly lower rates of bile production, 0.046 ± 0.36 μ/min/gm (p < 0.001) occurred during reperfusion after 60 min of ischemia. Control livers during the same period produced 0.330 ± 0.056 μl/min/gm of bile. The results indicate that purine nucleoside phosphorylase levels may be a good index of oxidative injury to the liver in ischemia reperfusion and reliably predict the functional state of the organ after reperfusion.

Traditional chemotherapy and radiotherapy for cancer treatment face serious challenges such as drug resistance and toxic side effects. Complementary / Alternative medicine is increasingly being practiced worldwide due to its safety beneficial therapeutic effects. We hypothesized that a super combination (SC) of known phytochemicals used at bioavailable levels could induce 100% killing of breast cancer (BC) cells without toxic effects on normal cells and that microarray analysis would identify potential genes for targeted therapy of BC. Mesenchymal Stems cells (MSC, control) and two BC cell lines were treated with six well established pro-apoptotic phytochemicals individually and in combination (super cocktail), at bioavailable levels. The compounds were ineffective individually. In combination, they significantly suppressed BC cell proliferation (>80%), inhibited migration and invasion, caused cell cycle arrest and induced apoptosis resulting in 100% cell death. However, there were no deleterious effects on MSC cells used as control. Furthermore, the SC down-regulated the expression of PCNA, Rb, CDK4, BcL-2, SVV, and CD44 (metastasis inducing stem cell factor) in the BC cell lines. Microarray analysis revealed several differentially expressed key genes (PCNA, Rb, CDK4, Bcl-2, SVV, P53 and CD44) underpinning SC-promoted BC cell death and motility. Four unique genes were highly up-regulated (ARC, GADD45B, MYLIP and CDKN1C). This investigation indicates the potential for development of a highly effective phytochemical combination for breast cancer chemoprevention / chemotherapy. The novel over-expressed genes hold the potential for development as markers to follow efficacy of therapy.

The main site of injury induced during small bowel preservation is perceived to be the basement membrane and the endothelium of the highly vascularized mucosa, an aspect evaluated here in further detail. The effects of preservation were studied using a specific basement membrane stain (laminin antibody), an endothelial cell stain (factor 8 antibody) and standard histology. In addition, mucosal glutaminase activity reflecting entero-cyte integrity was measured as monitor of the extent of preservation injury. Using a rat model, small bowel grafts were harvested, the vascular bed and bowel lumen were flushed, and the grafts were stored (4°C) for 1, 6, 9, and 12 hr and transplanted into syngeneic hosts. After cold storage prior to transplantation, full-thickness small bowel biopsies were obtained for the various tissue preparations. Histologic evaluation at the end of the preservation period revealed separation of the vil-lous epithelium from the lamina propria that increased with extending preservation time. Tissue staining with the laminin antibody disclosed progressive changes with increasing preservation intervals. Staining with the factor 8 antibody demonstrated also progressive changes, but failed to reflect in a gradual fashion increasing endothelial cell injury. Histologic injury became more pronounced after transplantation and reperfusion, then showing destruction of epithelial cells; the extent of injury correlated with the duration of preservation. Glutaminase activity was maintained after cold storage, indicating that the enterocytes remained intact during this period, but when assayed after reperfusion, glutaminase decreased with increasing preservation intervals and increasing histologic mucosal damage. We conclude that cold ischemic injury involves primarily the endothelium and the basement membrane, which progresses to global mucosal impairment with reperfusion.