Nobuo Takasu

Intracellular localization of group II phospholipase A2 in rat vascular smooth muscle cells and its possible relationship to eicosanoid formation

We investigated the localization of group II phospholipase A2 (PLA2-II) in rat vascular smooth muscle cells (VSMCs) by applying immunofluorescence and immunoelectron microscopy with its polyclonal antibody. In unstimulated cells, no immunolabelling was detected in the cells. On the other hand, in the cells stimulated with tumor necrosis factor (TNF) and/or forskolin (FK), intense fluorescence was detected in the cytoplasm. The immunoperoxidase reactions were detected in the cisternae of rough endoplasmic reticulum (rER), trans-cisternae of Golgi apparatus, and small vesicles beneath the plasma membrane. Western blot analysis showed VSMCs secrete PLA2-II after stimulation. Secreted PLA2-II was associated with the plasma membrane and extracellular matrix. Colchicine inhibited PLA2-II synthesis and its secretion to the extracellular space. These observations indicate that in VSMCs PLA2-II is synthesized at rER. transported to Golgi apparatus, discharged into extracellular space via the small vesicles, and microtubules may concern with its process. Furthermore, in VSMCs treated with TNF or TNF + FK, prostaglandin E2 formation was also increased. Actinomycin D and cycloheximide inhibited the potentiation of the prostaglandin E2 formation induced by TNF or TNF + FK, indicating that both RNA and protein synthesis are required for the potentiation. These results suggest an involvement of PLA2-II in the prostaglandin formation.

Monoclonal antibody P-31 recognizes a novel intermediate filament-associated protein (p250) in rat podocytes

The visceral glomerular epithelial cells (GECs) or podocytes of the renal glomerulus constitute a highly specialized epithelium. To study the nature of podocytes, we established mouse monoclonal antibodies against GEC. Clone P-31 reacted exclusively with the cytoplasm of GEC by immunofluorescence. Immunoblot analysis with P-31 showed that a single band of 250 kDa was detectable in a glomerular lysate. The 250-kDa polypeptide (p250) was recovered from Triton X-100-insoluble fractions of isolated glomeruli, suggesting that this molecule is associated with the cytoskeleton. Immunogold staining with P-31 demonstrated that the gold particles were located at the intersections of vimentin-type intermediate filaments of podocytes. In developing kidney, this protein first appeared in immature GECs during the S-shaped body stage. In puromycin aminonucleoside nephrosis, p250 was dramatically increased in glomeruli where enhanced desmin expression was observed in GECs. These results indicate that p250 is a novel intermediate filament-associated protein and plays a role in the organization of the intermediate filament network in both normal and diseased conditions.The visceral glomerular epithelial cells (GECs) or podocytes of the renal glomerulus constitute a highly specialized epithelium. To study the nature of podocytes, we established mouse monoclonal antibodies against GEC. Clone P-31 reacted exclusively with the cytoplasm of GEC by immunofluorescence. Immunoblot analysis with P-31 showed that a single band of 250 kDa was detectable in a glomerular lysate. The 250-kDa polypeptide (p250) was recovered from Triton X-100-insoluble fractions of isolated glomeruli, suggesting that this molecule is associated with the cytoskeleton. Immunogold staining with P-31 demonstrated that the gold particles were located at the intersections of vimentin-type intermediate filaments of podocytes. In developing kidney, this protein first appeared in immature GECs during the S-shaped body stage. In puromycin aminonucleoside nephrosis, p250 was dramatically increased in glomeruli where enhanced desmin expression was observed in GECs. These results indicate that p250 is a novel intermediate filament-associated protein and plays a role in the organization of the intermediate filament network in both normal and diseased conditions.

A unique photoreceptive structure in the arrowworms Sagitta crassa and Spadella schizoptera (Chaetognatha)

SummaryThe ultrastructure of photoreceptors in two species of chaetognaths, Sagitta crassa and Spadella schizoptera, was studied by electron microscopy using a goniometer specimen stage as well as by freeze-fracture techniques. In contrast to earlier descriptions, the photoreceptor is made up of a stack of lamellae with pores. The lamellae, each 30–45 nm in thickness, are piled one on top of one another at intervals of 10–20 nm. The lamellar surface is often perpendicular to the incident light. The pores are 35–55 nm in diameter and arranged in an orderly square lattice with a center-to-center distance of 80–95 nm. These perforated lamellar structures, resembling annulate lamellae, are entirely new as photoreceptive structures.

Group IIA phospholiase A2 induces neuronal cell death via apoptosis

1, Dept. of Cell Science, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine 2, Dept. of Anatomy & Neuroscience, Osaka University Medical School The gene, DP5 was identified as a novel gene specifically induced in cultured sympathetic neurons undergoing apoptosis. The protein encoded by DP5 belongs to the Bcl-2 family but it has only a BH3 domain. So far, four genes (bik/nbk, bid, bim and blk) have been discovered, all of which encode BH3 domain-only proteins (BOPs) with little sequence homology except for their BH3 domains. Although the BOPs have an apoptosis-inducing activity in common when over-expressed, their detailed tissue distribution or the regulatory mechanisms of thier production are not elucidated. In the present study, we analyzed the expression patterns of three BOP genes (DP5. bik and bid) in the developing and adult murine nervous system. We also investigated the relationship between their expression patterns and the excitotoxic cell death in the central nervous system of mice treated with kainic acid. Their differential expression patt,erns during the development of murine nervous system and in response to the epileptic seizure activity suggt’st that the BOPs have distinct roles in the neuronal development and pathology of the excitotoxic cell death.