Localization of cathepsin D in rat liver. Immunocytochemical study using post-embedding immunoenzyme and protein A-gold techniques

Light and electron microscopic localization of cathepsin D in rat liver was investigated by post-embedding immunoenzyme and protein A-gold techniques. By light microscopy, cytoplasmic granules of parenchymal cells and Kupffer cells were stained for cathepsin D. Weak staining was also noted in sinusoidal endothelial cells. In the parenchymal cells many of positive granules located around bile canaliculi. In the Kupffer cells and the endothelial cells, diffuse staining was noted in the cytoplasm in addition to granular staining. By electron microscopy, gold particles representing the antigenic sites for cathepsin D were seen in typical secondary lysosomes and some multivesicular bodies of the parenchymal cells and Kupffer cells. The lysosomes of the endothelial cells and fat-storing cells were weakly labeled. Quantitative analysis of the labeling density in the lysosomes of these three types of cells demonstrated that the lysosomes of parenchymal cells and Kupffer cells are main containers of cathepsin D in rat liver. The results suggest that cathepsin D functions in the intracellular digestive system of parenchymal cells and Kupffer cells but not so much in that of the endothelial cells.     

Effect of benzodiazepines on 5'-nucleotidase activity in rat brain

Experiments on 330 rats were made to study the influence of benzodiazepines (diazepam, dormicum and phenazepam) on 5'-nucleotidase activity in brain homogenates. It was discovered that diazepam and dormicum in doses of 3 and 4 mg, phenazepam in doses of 3.75 and 5 mg per 200 g bw provoked a 16-20% reduction in 5'-nucleotidase activity. The maximal effect of diazepam (3 and 4 mg doses) was attained 1 h after intraperitoneal injection, that of dormicum (3 mg) 30 min and of phenazepam (5 mg) 1 h after intraperitoneal injection. It is assumed that benzodiazepines are involved in AMP metabolism.     

Alterations in cerebrospinal fluid uridine,hypoxanthine, and xanthine in head-injured patients

1. Examination of the cerebrospinal fluid (CSF) of head-injured patients reveals that the concentration of intraventricular xanthine is elevated and that of uridine is decreased relative to those of adult lumbar CSF. 2. No correlations were observed between CSF lactate and CSF hypoxanthine, xanthine, or uridine, suggesting that changes in purine metabolites and the pyrimidine nucleoside do not index similar cellular events as does lactic acid production. 3. Ventricular CSF from hydrocephalic infants had uridine and hypoxanthine concentrations not significantly different from those of normal adult lumbar CSF, but xanthine was significantly elevated. 4. Since uridine has anticonvulsant properties and is a crucial substrate for cerebral metabolism, it may be useful to evaluate this pyrimidine for use in the management of patients with head injury.     

Comparison of statistics for candidate-gene association studies using cases and parents.

Studies of association between candidate genes and disease can be designed to use cases with disease, and in place of nonrelated controls, their parents. The advantage of this design is the elimination of spurious differences due to ethnic differences between cases and nonrelated controls. However, several statistical methods of analysis have been proposed in the literature, and the choice of analysis is not always clear. We review some of the statistical methods currently developed and present two new statistical methods aimed at specific genetic hypotheses of dominance and recessivity of the candidate gene. These new methods can be more powerful than other current methods, as demonstrated by simulations. The basis of these new statistical methods is a likelihood approach. The advantage of the likelihood framework is that regression models can be developed to assess genotype-environment interactions, as well as the relative contribution that alleles at the candidate-gene locus make to the relative risk (RR) of disease. This latter development allows testing of (1) whether interactions between alleles exist, on the scale of log RR, and (2) whether alleles originating from the mother or father of a case impart different risks, i.e., genomic imprinting.     

Catalytic properties of a human cytomegalovirus-induced protein kinase

Human cytomegalovirus, a DNA virus whose genome contains a fragment of transforming DNA, induces a threonine-serine protein kinase having a molecular mass of 68 kDa (p68). p68 was extracted from cells 96-144 h after infection, and immunoprecipitated with a monoclonal antibody (F6b). Antibody-enzyme complexes were immobilized on heat/formaldehyde-inactivated Staphylococcus aureus. The best substrates for p68 were acidic proteins, phosvitin and casein. Glycogen synthase, phosphorylase alpha and histones were phosphorylated at rates not higher than 1-4% that obtained with phosvitin as substrate. ATP and GTP were equally good substrates of p68. p68 is able to autophosphorylate at the same residues (i.e. threonine and serine) as the protein substrates. Autophosphorylation does not seem to represent an intermediate in substrate phosphorylation. The protein kinase activity of p68 was not enhanced by cAMP, calcium ions, or polyamines like spermine or spermidine. Only at low Mg2+ concentration spermine enhanced by 68% the rate of casein phosphorylation. Heparin, a potent inhibitor of casein kinase II, inhibits p68 activity too, but ten-times higher concentrations were required for the same degree of inhibition. Quercetin, a bioflavonoid, acts as a strong inhibitor of p68 protein kinase activity. The inhibitory effect of quercetin was competitive towards the nucleotide substrate (Ki = 2.8 microM), and non-competitive towards the protein substrate (Ki = 15 microM).