Molecular cloning of glycoprotein Ib-binding protein, flavocetin-A, which inhibits platelet aggregation.

Flavocetin-A is a strong platelet aggregation inhibitor isolated from the venom of Trimeresurus flavoviridis. It binds specifically to platelet glycoprotein Ib with high affinity and inhibits von Willebrand factor-dependent platelet aggregation. The apparent molecular weight of flavocetin-A is 149 kDa. It consists of two subunits, alpha (17 kDa) and beta (14 kDa). The amino acid sequences of the alpha and beta subunits were determined from cloned cDNAs. Deduced amino acid sequences showed signal peptide-sequences of 23 amino acids for both alpha and beta subunits, mature peptide sequences of 135 amino acids for the alpha subunit, and 125 amino acids for the beta subunit. The amino acid sequences of alpha and beta subunits show high degrees of homology to those of C-type lectin-like venom proteins such as habu coagulation factors IX/X-binding protein (IX/X-bp), botrocetin, and alboaggregin-B. The cysteinyl residues of flavocetin-A, IX/X-bp, and botrocetin are conserved, except that flavocetin-A contains Cys 135 in the alpha subunit and Cys 3 in the beta subunit. We assumed that the arrangements of disulfide bridges in flavocetin-A are similar to those of IX/X-bp and botrocetin, and the additional Cys 135 of the alpha subunit and Cys 3 of the beta subunit are involved in novel disulfide bridges. These findings suggested that the additional disulfide bridges formed with Cys 135 of the alpha subunit and Cys 3 of the beta subunit cause polymerization of C-type lectin-like heterodimers.

Characterization of leukemic cells in CD2/CD19 double positive acute lymphoblastic leukemia.

In the diagnosis of leukemia, CD2 which is a T-cell associated marker and CD19 which is a B-cell associated marker are widely used to determine the lineage of leukemic cells. It is known that the cells of acute lymphoblastic leukemia (ALL) express both CD2 and CD19 in some cases. The origins of these cells are generally thought to be a common precursor for T- and B-lymphocytes. However, cytoplasmic staining of CD3 which is a more specific marker for T-lineage and cytoplasmic staining of mb-1 (CD79a) which is more specific for B-lineage were not performed in previous reports and the determination of the cell lineages of these cells was unclear. We had two cases of ALL whose blasts were CD2/CD19 double positive. The first case was assessed as B-lineage because the cells expressed cytoplasmic CD79a and lacked cytoplasmic CD3. The immunoglobulin (Ig) heavy chain gene was rearranged. The other cell surface markers including CD22 and HLA-DR also suggested that these cells were B-lineage. The CD2 expression may be a coincidence and should not be taken as a T-cell marker in this case. It was difficult to determine the lineage in the second case because both cytoplasmic CD79a and cytoplasmic CD3 were expressed and neither TCR beta chain nor Ig heavy chain genes were rearranged. The other surface markers were not useful to determine the lineage. We concluded that this case was really an unclassified ALL. Accordingly, cytoplasmic staining of CD3 and CD79a should be carried out in the diagnosis of leukemia when it is difficult to determine the cell lineage.

Characterization of membrane-associated prothrombin activator in normal and injured murine tissues.

Thrombin is a multifunctional enzyme involved in coagulation, cell modulation and inflammation. We recently reported a novel membrane-associated prothrombin activator, abbreviated as MAPA, found in cultured fibroblasts and glial cell lines. In this study, we examined the physiological role of this enzyme. MAPA-like activity was detected in the liver, kidney, lung and heart but not in the spleen or brain in normal mice. To examine whether MAPA participates in biological reactions, hepatic and renal injury were induced by administration of CCl4 and HgCl2, respectively. MAPA-like activity was specifically increased in the injured tissues: the activity was elevated by about 100-fold in 48 h in the liver and increased by about 5-fold in 12 h in the kidney. Their enzymatic properties were the same as those of MAPA in 8C feline kidney fibroblast cells. Phospholipids are required for activation of prothrombin by MAPA obtained from both 8C cells and tissues. These results suggest that MAPA activates prothrombin on the cell surface in injured tissue and participates in inflammation and regeneration associated with tissue injury.

The number of noradrenergic and adrenergic neurons in the brain stem does not change with age in male Sprague-Dawley rats.

We examined whether or not the number of noradrenergic and adrenergic neurons changes with age, using peroxidase antiperoxidase (PAP) immunohistochemistry with specific antisera against tyrosine hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT). TH- and PNMT-immunoreactive neurons were counted in every noradrenergic and adrenergic neuron group of young (3-month-old) and old (24-month-old) rats. The differences in the counted number of TH- and PNMT-immunoreactive neurons did not reach statistical significance between the young and old rats.

Effect of dietary vitamin E on lipofuscin accumulation with age in the rat brain.

We investigated the effect of dietary vitamin E on lipofuscin accumulation with age in the hippocampus, the inferior olive and the cerebellum of young (3-5 months old) middle-aged (12-14 months old) and old (24-26 months old) male Sprague-Dawley rats. The rats were fed either a vitamin E-deficient diet, vitamin E-supplemented diet or a control diet after reaching four weeks old. We employed both quantitative light microscopy using semithin sections and qualitative fluorescence microscopy for the analysis of lipofuscin accumulation with age. The concentrations of alpha-tocopherol were measured simultaneously in both the plasma and the three brain regions investigated. The effect of vitamin E deficiency was statistically significant only in the inferior olive of young rats and in all the three brain regions of middle-aged rats. The effect of vitamin E supplementation was statistically significant in all three brain regions of middle-aged rats. There was no statistically significant effect of vitamin E deficiency or supplementation on lipofuscin accumulation with age as compared with the control rats in all three brain regions of old rats. It was thus revealed that dietary vitamin E clearly had a significant effect on lipofuscin accumulation with age in the rat brain up until middle age, and that the same effect became indistinct in the latter half of their life.

Fine structure of tyrosine hydroxylase-like immunoreactive neurons and terminals of the hypothalamic arcuate nucleus and median eminence in young and aged rats.

Tyrosine hydroxylase (TH)-like immunoreactive neurons, fibers, and terminals in the hypothalamic arcuate nucleus and median eminence of young (8 weeks old) and aged (24 months old) rats were investigated at light and electron microscopic levels by means of the peroxidase anti-peroxidase (PAP) method. In the arcuate nucleus, TH-like immunoreactive neuronal perikarya of young animals contained well-developed cell organelles such as rough-surfaced endoplasmic reticulum (rER), mitochondria, Golgi apparatus, polysomes, as well as dense granules. Immunoreactive neuronal elements appeared as both presynaptic and postsynaptic elements. In aged rats, TH-like immunoreactive neuronal perikarya as well as non-immunoreactive ones were found to include many lysosomes or inclusions, and many neuronal elements with degradative changes were observed. In the external layer of the median eminence of young animals, many immunoreactive nerve endings were found around the pericapillary spaces of portal vessels and often terminated adjacent to the basement membranes of the pericapillary spaces. Immunoreactive fibers having close contact with non-immunoreactive fibers were also observed. In aged animals, degradative changes such as axons with swollen and watery appearance and myelin figures were observed in the neuronal elements, although many immunoreactive nerve endings were seen around the pericapillary spaces.