Modulation of cytochrome P450 activity in the kidney of rats following long-term red wine exposure

Cytochrome P450 (CYP)-dependent oxidation of lauric acid, p-nitrophenol and ethanol by microsomal fractions of kidney were studied in control rats and in animals given either ethanol, red wine, or alcohol-free red wine for 10 weeks. Ethanol increased the total CYP content and specifically CYP 2E1, as well as p-nitrophenol and ethanol oxidation. The effects of ethanol treatment on the content and activity of CYP 2E1 were attenuated when red wine was administered, while the alcohol-free red wine values were similar to those of the control group. Although lauric acid hydroxylation was decreased by red wine treatment, the content of CYP 4A1 was not influenced by drinking fluids. We conclude that red wine administration attenuates the ethanol-induced enhancement of microsomal activities dependent on CYP 2E1 of rat kidney. Our results suggest that the non-alcoholic constituents of red wine could account for this modulation.     

Yeast two-hybrid screening using constitutive-active caspase-7 as bait in the identification of PA28gamma as an effector caspase substrate

Caspase-3 and -7 represent executioner/effector caspases that directly cause apoptotic morphological changes by cleaving various death substrates. The substrates for caspases generally interact with active caspases, but not with inactive zymogens of caspase or procaspases. Here, to isolate proteins that interact with caspase-7, we established a yeast two-hybrid screening system using reversed-caspase-7, a constitutive active mutant of caspase-7 as a bait plasmid. Screening of an adult brain cDNA library led to isolation of proteasome activator 28 subunit, PA28gamma. In vitro translates of PA28gamma were cleaved by both recombinant caspase-3 and -7. Mutagenesis of potential cleavage site DGLD80 to EGLE80 completely abolished caspase-mediated cleavage. Moreover, endogenous PA28gamma was cleaved during not only Fas-induced apoptosis of HeLa cells, but also cisplatin-induced cell death of MCF7 cells, which are devoid of caspase-3. These findings indicate that PA28gamma is an endogenous substrate for caspase-3 and -7 and that yeast two-hybrid screening using reversed-caspase is a novel and useful approach to clone substrates for effector caspases.     

Inhibition of proteasome activity is involved in cobalt-induced apoptosis of human alveolar macrophages

Inhalation of particulate cobalt has been known to induce interstitial lung disease. There is growing evidence that apoptosis plays a crucial role in physiological and pathological settings and that the ubiquitin-proteasome system is involved in the regulation of apoptosis. Cadmium, the same transitional heavy metal as cobalt, has been reported to accumulate ubiquitinated proteins in neuronal cells. On the basis of these findings, we hypothesized that cobalt would induce apoptosis in the lung by disturbance of the ubiquitin-proteasome pathway. To evaluate this, we exposed U-937 cells and human alveolar macrophages (AMs) to cobalt chloride (CoCl(2)) and examined their apoptosis by DNA fragmentation assay, 4',6-diamidino-2'-phenylindol dihydrochloride staining, and Western blot analysis. CoCl(2) induced apoptosis and accumulated ubiquitinated proteins. Exposure to CoCl(2) inhibited proteasome activity in U-937 cells. Cobalt-induced apoptosis was mediated via mitochondrial pathway because CoCl(2) released cytochrome c from mitochondria. These results suggest that cobalt-induced apoptosis of AMs may be one of the mechanisms for cobalt-induced lung injury and that the accumulation of ubiquitinated proteins might be involved in this apoptotic process.     

Interleukin-10 reduces natural killer (NK) sensitivity of tumor cells by downregulating NK target structure expression

We examined the effect of exogenous IL-10 on the sensitivity of rat W14 and W31 tumor cells to natural killer (NK) cell-mediated cytotoxicity in relation to previously identified NK target structure (NKTS) expressed on these cells. We also examined the effect of endogenous interleukin-10 (IL-10) on rat IL-10 cDNA-introduced W31 cells (W31T-H, a high-IL-10-producer clone; W31T-L, a low-IL-10-producer clone). Both exogenous and endogenous IL-10 had no effect on the proliferative activity of these cells, but incubation of cells with recombinant human (rh) IL-10 resulted in a dose-dependent decrease in the expression of NKTS recognized by mAb 109. The expression level of NKTS on W31T-H cells was dramatically decreased compared with that on W31T-L cells and parental W31 cells. In addition, treatment of W31 cells with the culture supernatants of W31T-H cells could downregulate the expression of the NKTS. Moreover, NK sensitivity of W31T-H was suppressed down to a level equivalent to that of W31 cells pretreated with exogenous rhIL-10, and cytolysis could no longer be inhibited by mAb 109. We previously demonstrated that IL-10 downregulated MHC class I expression in this model. Nevertheless, NK susceptibility was also decreased. Taken together, these results suggest that the IL-10-mediated decrease in NKTS expression has a larger effect than decreased MHC class I expression on NK sensitivity. Thus, our data reveal a novel mechanism for an IL-10-mediated escape of tumor cells from host immune surveillance by downregulation of NKTS expression.     

Intervención domiciliaria por terapia ocupacional postalta para prevenir la rehospitalización de personas mayores: protocolo de estudio de un ensayo clínico aleatorizado

Background and objective

Re-admission to hospital by the elderly is a frequent event that is associated with complications. The aim of this article is to describe a randomised clinical trial protocol which has the aim of describing and comparing the impact of a home-based intervention by Occupational Therapists (OT) in the likelihood of re-admission at 6 months.

Injury of skeletal muscle and specific cytokines induce the expression of gap junction channels in mouse dendritic cells

Dendritic cells (DCs) in culture express at least connexin43, a protein subunit of gap junctions, and form gap junction channels, which could be important for T-cells activation. Here, we evaluated whether DCs express connexins in vivo and also to identify components of their microenvironment that regulate the functional expression of gap junctions. In vivo studies were performed in lymph nodes of mice under control conditions or after skeletal muscle damage. In double immunolabeling studies, connexin45 was frequently detected in DEC205(+) DCs in lymph nodes of control animals, whereas connexin43 was rarely found in DCs. However, connexin43 was upregulated in DCs after skeletal muscle damage. Upregulation of connexin43 gene expression by tissue damage was also confirmed in mice carrying a beta-galactosidase reporter gene in a connexin43 allele. The effect of several cytokines on the expression of functional gap junctions between cultured DCs was also tested. Under control conditions, cultured DCs did not communicate via gap junctions. However, after treatment with keratinocyte-conditioned medium or cytokine mixtures containing at least TNF-alpha and IL-1beta, they became transiently coupled through a pathway sensitive to octanol, a gap junction blocker. Cellular coupling induced by effective cytokine mixtures was prevented by IL-6. Single cytokines (TNF-alpha, IL-1beta, IFN-gamma, or IL-6) or other mixtures than the described above did not induce coupling via gap junctions. Increased levels of connexin43 and connexin45 protein and mRNA accompanied the appearance of cellular coupling. These studies provide demonstration of connexin expression and regulation by specific danger signals in DCs.