Progressive axonopathy when oligodendrocytes lack the myelin protein CMTM5.

Oligodendrocytes facilitate rapid impulse propagation along the axons they myelinate and support their long-term integrity. However, the functional relevance of many myelin proteins has remained unknown. Here, we find that expression of the tetraspan-transmembrane protein CMTM5 (chemokine-like factor-like MARVEL-transmembrane domain containing protein 5) is highly enriched in oligodendrocytes and central nervous system (CNS) myelin. Genetic disruption of the Cmtm5 gene in oligodendrocytes of mice does not impair the development or ultrastructure of CNS myelin. However, oligodendroglial Cmtm5 deficiency causes an early-onset progressive axonopathy, which we also observe in global and tamoxifen-induced oligodendroglial Cmtm5 mutants. Presence of the WldS mutation ameliorates the axonopathy, implying a Wallerian degeneration-like pathomechanism. These results indicate that CMTM5 is involved in the function of oligodendrocytes to maintain axonal integrity rather than myelin biogenesis.

Cathepsin B inactivation attenuates the apoptotic injury induced by ischemia/reperfusion of mouse liver.

BACKGROUND: A major mechanism underlying warm ischemia/reperfusion (I/R) injury during liver transplantation is the activation of the caspase chain, which leads to apoptosis. Recently, it was demonstrated that the release of cathepsin B, a cysteine protease, from the cytosol in liver injury induces mitochondrial release of cytochrome c and the activation of caspase-3 and -9, thereby leading to apoptosis. The aim of this study was to ascertain if cathepsin B inactivation attenuates the apoptotic injury due to I/R in mouse liver. METHODS: A model of segmental (70%) hepatic ischemia was used. Eighteen mice were anesthetized and randomly divided into three groups: (1) CONTROL GROUP: sham operation (laparotomy); (2) Ischemic group: midline laparotomy followed by occlusion of all structures in the portal triad to the left and median lobes for 60 min (ischemic period); (3) STUDY GROUP: like group 2, but with intraperitoneal administration of a pharmacological inhibitor of cathepsin B (4 mg/100 g) 30 min before induction of ischemia. Serum liver enzyme levels were measured by biochemical analysis, and intrahepatic caspase-3 activity was measured by fluorometric assay; apoptotic cells were identified by morphological criteria, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) fluorometric assay, and immunohistochemistry for caspase-3. RESULTS: Showed that at 6 h of reperfusion, there was a statistically significant reduction in liver enzyme levels in the animals pretreated with cathepsin B inhibitor (p<0.05). On fluorometric assay, caspase-3 activity was significantly decreased in group 3 compared to group 2 (p<0.0001). The reduction in postischemic apoptotic hepatic injury in the cathepsin B inhibitor -treated group was confirmed morphologically, by the significantly fewer apoptotic hepatocyte cells detected (p<0.05); immunohistochemically, by the significantly weaker activation of caspase-3 compared to the ischemic group (p<0.05); and by the TUNEL assay (p<0.05). CONCLUSION: The administration of cathepsin B inhibitor before induction of ischemia can attenuate postischemic hepatocyte apoptosis and thereby minimize liver damage. Apoptotic hepatic injury seems to be mediated through caspase-3 activity. These findings have important implications for the potential use of cathepsin B inhibitors in I/R injury during liver transplantation.

Cosolvent effects on gel-entrapped oxidoreductase: the glucose oxidase model.

An intrinsic problem often involved in biotransformations carried out by immobilized cells is the poor solubility of substrate and product in water. Increase in hydrophobic substrate availability to such gel-entrapped cells may be attained by the replacement of a fraction of the aqueous medium by water-miscible solvents (cosolvents). The introduction of cosolvents results in increased solubility, but may simultaneously affect enzymic activity and stability. Recently, criteria and guidelines for cosolvent selection on the basis of its effect on intracellular enzyme stability were reported (Freeman, A., and Lilly, M.D. (1987) Appl. Microbiol. Biotechnol. 25, 495-501). In order to understand the impact of the preferable or unsuitable cosolvents on enzyme kinetics and stability, the effects of 1-5 M concentrations of a series of cosolvents (e.g., ethylene glycol, dimethylsulfoxide, N,N-dimethylformamide, ethanol) on a well-characterized, highly specific enzyme model (glucose oxidase) were investigated. The presence of 1-5 M of the cosolvents studied imposed 10-50% reduction in Vmax of the enzyme, but Km was only mildly affected (+/- 25%). This inhibition was attributed to cosolvent effect on small, reversible, conformational changes in the enzyme native structure. Determination of the rate constant of thermal inactivation (at 55 degrees C) of glucose oxidase, in the presence of cosolvents, was employed for the quantitative evaluation of cosolvent effect on enzyme stability. A clear pattern of cosolvent preference in respect to its denaturing effect was obtained, which was identical to the pattern previously observed in a study of oxidoreductases operating from within a whole cell. In both cases diols (e.g., ethylene glycol) were found to be the preferable group of cosolvents. Our results indicate that a soluble enzyme and an intracellular enzyme operating from a whole cell are affected by cosolvents via the same mechanism.