Tissue distribution of indices of lysine catabolism in growing swine.

The primary pathway of lysine degradation in pigs presumably depends on the bifunctional protein α-aminoadipate δ-semialdehyde synthase (AASS), which contains lysine α-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH) activities. In liver, AASS is restricted to the mitochondrial matrix and lysine is presumptively transported through the plasma membrane by a cationic AA transporter (CAT1/2) and through the inner mitochondrial membrane by 1 or both mitochondrial ornithine transporters (ORC-1/ORC-2). Lysyl oxidase (LO) may represent an alternative pathway of lysine oxidation. The objective of this experiment was to analyze the distribution of indices of lysine catabolism in various pig tissues. We assessed LKR, SDH, and LO activities, lysine oxidation, mRNA abundance of LKR, CAT1/2, and ORC1/2, and AASS protein abundance (via SDH antibody) in liver, heart, kidney medulla and cortex, triceps, longissimus, whole intestine, enterocytes, and intestine stripped of enterocytes in 10 growing pigs, weighing ∼25 kg. The LKR activity differed across tissues (P<0.001) and was greatest in liver, intestine, and kidney samples, and LKR mRNA abundance (P<0.001) was greatest in liver; although, LKR activity and mRNA abundance were detected in all other tissues. Activity of SDH (P<0.001) and SDH mRNA abundance (P<0.001) were affected by tissue and were greatest in liver compared with all other tissues analyzed. The AASS protein abundance (P<0.001) was greatest in whole intestine and liver. Activity of LO (P<0.0001) was greatest in muscle samples. The abundance of ORC-1 (P<0.001) and ORC-2 mRNA (P<0.001) differed among tissues, and ORC-1 was greatest in liver, kidney, and intestinal preparations, and ORC-2 mRNA abundance was greatest in liver and intestine. Interestingly, LKR activity was correlated with ORC-1 (r=0.32, P<0.05) and ORC-2 (r=0.41, P<0.05) expression. The expression of CAT-1 was uniform in all tissues, whereas CAT-2 (P<0.01) was greatest in liver. In conclusion, these data indicate that extra-hepatic tissues contribute to lysine catabolism as do enzymes other than LKR.

Determination and reversal of resistance to epirubicin intravesical chemotherapy. A confocal imaging study.

OBJECTIVE: To evaluate the use of confocal microscopy in the study of resistance to epirubicin and to determine the effect of temperature, viability and a resistance-reversing agent on the intracellular distribution of this drug in sensitive and resistant derivatives of a superficial bladder cancer cell line. MATERIALS AND METHODS: Viable and non-viable adherent cells were incubated in epirubicin solutions under various conditions. After incubation, the distribution of intracellular epirubicin fluorescence was visualized using confocal microscopy and a x50 water-immersion lens. RESULTS: There was a striking and consistent difference between resistant and sensitive cells in the intracellular distribution of the drug. In addition to having greater overall levels of epirubicin fluorescence, sensitive cells accumulated epirubicin predominantly in the nucleus. Epirubicin fluorescence in resistant cells was cytoplasmic and granular in appearance. When incubated at 0 degrees C, both cell lines showed no nuclear uptake and thus resembled resistant cells at 37 degrees C. However, dead cells rapidly acquired brightly fluorescent nuclei. The resistance-reversing agent verapamil appeared to cause reversion of the resistant to the sensitive phenotype. CONCLUSION: Confocal microscopy allows epirubicin-sensitive and resistant cultured tumour cells to be differentiated reliably and provides information about the mechanisms of action of, and resistance to, epirubicin. Applying this technique to clinical specimens should enable patients who have the resistant phenotype to be detected and the efficacy of intravesical resistance-reversing agents to be evaluated in such cases.