STUDIES ON THE COSUBSTRATE SITE OF PROTEASE SOLUBILIZED NADPH-CYTOCHROME P450 REDUCTASE

Modification of the protease solubilized NADPH-cytochrome P450 reductase (= NADPH-cytochrome c reductase) at the critical SH group in the cosubstrate binding site affects K^NADPH_m but not V for the cytochrome c reduction. The increase of K^NADPH_m is dependent on the size and the charge of the substituent introduced. Substitution of the cosubstrate site SH by the CN-, S₂ O₃- and the (N-ethyl) succinimido group effects a 3-, 7- and 23-fold increase of K^NADPH_m respectively. The critical SH group in the NADPH binding region can be specifically radiolabeled by N-ethyl (2,3-¹⁴C) maleimide after preincubation of the reductase with unlabeled NEM in the presence of 1 mM NADP+. The selective reaction at the essential cysteine in the cosubstrate site is demonstrated by peptide mapping of the thermolytic digest and urea SDS gel electrophoresis of the cyanogen bromide fragments of the reductase. Protease solubilized NADPH-cytochrome P450 reductase is inactivated by reagents directed to histidine, orginine and lysine residues. NADP (H) (1 mM) and 2′-AMP (1 mM) give effective protection only for the reaction of 1, 2-cyclohexanedione (12 mM). The functional role of the basic amino acid residues for the cosubstrate binding by the NADPH-cytochrome P450 reductase cannot be established therefore by the modification experiments described. The number of NADPH binding sites in the NADPH-cytochrome P450 reductase is determined to one site/mol reductase by titration of the enzyme with NADP+ monitored by CD-spectroscopy.     

REVIEW: Nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a reasonably well-defined clinicopathological entity; it has been reported more commonly in women than in men or children of both sexes and it appears to be most closely associated with obesity, diabetes mellitus and related abnormalities, such as hyperlipidaemia and hyperglycaemia. However, the association with female gender, obesity and diabetes may not be as close as suggested by the literature and an underlying condition cannot be discerned in all cases. The natural history of the disease is poorly understood; the associated biopsy features span a wide spectrum, reaching from uncomplicated, clinically non-progressive fatty liver (not NASH in a strict sense) to a slowly progressive fatty liver with inflammation and fibrosis, to steatohepatitis with submassive hepatic necrosis, which has a subfulminant course and is often fatal. Non-progressive fatty liver appears to be very common but is of little clinical importance. The slowly progressive form of the disease represents NASH as encountered by most clinicians and pathologists. It is a common liver disease in current practice; patients may present with cirrhosis and even HCC arising from steatohepatitic cirrhosis. Subfulminant NASH has become exceedingly rare because many clinicians are now aware of the hazards of sudden weight loss, particularly in morbidly obese patients. Treatment options for NASH are still limited. The promotion of gradual weight loss in obese patients is the most widely recommended therapy but, unfortunately, this is very difficult to achieve. Avoidance of precipitous weight loss and careful control of diabetes mellitus are important and undisputed parts of patient management. Administration of UDCA as a treatment of NASH is still under study; it may be effective in some patients. The treatment of established steatohepatitic cirrhosis does not differ substantially from that of other types of cirrhosis and includes orthotopic liver transplantation.     

LIGHT MICROSCOPE AND IMMUNOHISTOLOGICAL INVESTIGATIONS ON THE BRAIN OF BORNA DISEASE VIRUS-INFECTED RABBITS

We studied the distribution and composition of perivascular cellular infiltrates and the distribution of the virus-specific antigen of Borna disease in the brains of rabbits inoculated either intracerebrally or intraperitoneally with infectious virus suspensions. Using standard histopathological and immunofluorescence techniques, there was, in all animals, a meningeal inflammatory reaction together with perivenous cellular infiltrates which were most numerous in the structures of the limbic system and basal ganglia, but also prominent in the spinal ganglia and area postrema. Studies with fluorescent anti-IgG antibody (direct technique) showed IgG in 20-25% of the cells in any one perivascular cuff, suggesting that most of the infiltrating cells were T-cells and that B-cells were fewer. Studies with virus-specific antibody showed that, in the early stages of the infection, there was evidence of a globular antigen mainly in neurons, while, at later stages, virus antigen was also demonstrated in oligodendrocytes and microgliocytes. The route of inoculation of the virus had no determining effect on the character or distribution of the cellular infiltrates or on the distribution of virus-specific antigen in the nervous system.