S100 alpha, CAPL, and CACY: molecular cloning and expression analysis of three calcium-binding proteins from human heart.

Elevated levels of intracellular calcium are a major cause of myocardial dysfunction. To find possible mediators of the deregulated calcium we searched for EF-hand calcium-binding proteins of the S100 family. By PCR technology we identified three members of the S100 protein family (S100 alpha, CACY, and CAPL) in the human heart. We cloned the corresponding cDNAs and examined their expression levels in various human tissues by Northern blot analysis. All three proteins are expressed at high levels in the human heart. Whereas CACY and CAPL mRNAs are expressed ubiquitously, S100 alpha mRNA is restricted to heart, skeletal muscle, and brain. Interestingly, the expression pattern of S100 alpha, CACY, and CAPL in human tissues differs significantly from that in rodent tissues.     

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.     

Transcriptional control, translation and function of the products of the five open reading frames of the Escherichia coli nir operon

Five open reading frames designated nirB, nirD, nirE, nirC and cysG have been identified from the DNA sequence of the Escherichia coli nir operon. Complementation experiments established that the NirB, NirD and CysG polypeptides are essential and sufficient for NADH-dependent nitrite reductase activity (EC 1.6.6.4). A series of plasmids has been constructed in which each of the open reading frames has been fused in-phase with the beta-galactosidase gene, lacZ. Rates of beta-galactosidase synthesis during growth in different media revealed that nirB, -D, -E and -C are transcribed from the FNR-dependent promoter, p-nirB, located just upstream of the nirB gene: expression is co-ordinately repressed by oxygen and induced during anaerobic growth. Although the nirB, -D and -C open reading frames are translated into protein, no translation of nirE mRNA was detected. The cysG gene product is expressed from both p-nirB and a second, FNR-independent promoter, p-cysG, located within the nirC gene. No NADH-dependent nitrite reductase activity was detected in extracts from bacteria lacking either NirB or NirD, but a mixture of the two was as active as an extract from wild-type bacteria. Reconstitution of enzyme activity in vitro required stoichiometric quantities of NirB and NirD and was rapid and independent of the temperature during mixing. NirD remained associated with NirB during the initial stages of purification of the active enzyme, suggesting that NirD is a second structural subunit of the enzyme.