Pharmacogenetic selection of transplanted human hepatocytes in immunocompetent rats.

OBJECTIVE: To introduce a genetic survival advantage for transplanted human hepatocytes over host cells in rats. METHODS: Green fluorescent protein (GFP) was introduced into Huh-7 human hepatoma cells to create fluorescent GFP-Huh-7 cells. mRNA of CYP2E1, the enzyme that converts acetaminophen (APA) into hepatotoxic intermediates, was quantified by real-time polymerase chain reaction (PCR). The effects of APA on GFP-Huh-7 and control Huh-7 cells were determined in a cell culture. Immunological tolerance was induced by the injection of GFP-Huh-7 cells into fetal rats in utero. The GFP-Huh-7 cells were transplanted after birth of the rats into tolerant rats followed by APA treatment. Serum alanine aminotransferase (ALT) levels and liver histological data were obtained. GFP-Huh-7 cells were detected by quantitive PCR and microscopy. RESULTS: CYP2E1 mRNA levels in the GFP-Huh-7 cells were 2.7% of parental Huh-7 cells. In 1 mmol/L APA, parental Huh-7 cells decreased by 60% while GFP-Huh-7 cells increased to within 95% of untreated controls after 5 days. In rats in which GFP-Huh-7 cells were transplanted and treated with APA, serum ALT increased to a peak of 200 U/L on day 1 and returned to normal levels by day 3. Fluorescence microscopy of liver specimens from rats transplanted with GFP-Huh-7 cells showed substantial increases in GFP-Huh-7, but not Huh-7 cells by day 7 after APA treatment. Real-time PCR confirmed a 10-fold increase of GFP mRNA in APA-treated rats, but not in those without APA treatment. CONCLUSIONS: The difference in CYP2E1 gene expression between GFP-Huh-7 and rat hepatocytes provides a convenient means for the enrichment of transplanted human cells in rat liver.

Granulomatous liver diseases: a review.

Granulomas that consist of focal accumulations of macrophages are commonly found in the liver due to stimulation of the immune system by a number of agents. Manifestations are variable depending on whether the underlying cause is a systemic disease or a primary hepatic granulomatous reaction. This article describes the common causes, presentation, histopathology, and manifestations of granulomatous diseases as well as various diagnostic and management strategies.

Mitochondrial trafficking in neurons: a key variable in neurodegeneration?

Mitochondria are the proximate target of a number of different neurotoxins. Typically, impairing of the key bioenergetic function of mitochondria by toxins is considered as the main mechanism of action. However, the effective maintenance of energy generation in neurons depends on the biogenesis, trafficking, and degradation of mitochondria in addition to the traditional bioenergetic functions. We have recently demonstrated that glutamate alters both the trafficking and morphology of mitochondria in primary neurons. In addition, several other potential neurotoxins, including nitric oxide and zinc, inhibit mitochondrial movement and, in some cases, alter morphology too. This suggests that some part of the action of neurotoxins might include the impairment of mitochondrial trafficking in neurons, with the resultant failure of local ATP delivery.

Nitric oxide-dependent beta2-adrenergic dilatation of rat aorta is mediated through activation of both protein kinase A and Akt.

Vasorelaxation to beta(2)-adrenoceptor stimulation occurs through both endothelium-dependent and endothelium-independent mechanisms, and the former is mediated through Ca(2+)-independent activation of endothelial-type nitric oxide synthase (NOS-3). Since Ca(2+)-independent NOS-3 activation may occur through its serine phosphorylation via protein kinase A (PKA) or Akt, we determined the PKA and Akt dependency of beta(2)-adrenergic relaxation of rat aorta. Rat aortic rings were pre-incubated with the PKA inhibitor H-89 (10(-7) m), the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (5 x 10(-7) m), Akt inhibitor (10(-5) m), or vehicle, in the absence or presence of the NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME, 10(-4) m). Rings were then contracted with phenylephrine (10(-7) m), and concentration-relaxation responses determined to the beta(2)-adrenoceptor agonist albuterol. Rings exhibited a concentration-dependent relaxation to albuterol: pEC(50) 6.9+/-0.2, E(max) 88.2+/-4.0%. l-NAME attenuated E(max) to 60.2+/-3.5% (P<0.001). In the presence of l-NAME, wortmannin or Akt inhibitor did not influence albuterol responses, whereas H-89 reduced E(max) further, to 27.5+/-2.2% (P<0.001). In the absence of l-NAME, E(max) to albuterol was reduced by H-89, wortmannin or Akt inhibitor, to 56.2+/-2.2, 56.0+/-1.6 and 55.4+/-1.8%, respectively (P<0.001 for each); the combinations H-89 plus wortmannin or H-89 plus Akt inhibitor reduced E(max) further still. Western blotting of NOS-3 immunoprecipitates from rat aortas confirmed that albuterol increased serine phosphorylation of NOS-3, and this increase was attenuated by H-89 or Akt inhibitor. Our results indicate that beta(2)-adrenoceptor stimulation relaxes rat aorta through both NO-dependent and independent mechanisms. The latter is predominantly PKA-mediated, whereas the former occurs through both PKA and PI3K/Akt activation.