Transient rise in nuclear calcium concentration is implicated in the regulation of events controlling gene expression. Mechanism by which calcium is transported to the nucleus is vehemently debated. Inositol 1,4,5-trisphosphate (InsP3) and inositol-1,3,4,5-tetrakisphosphate (InsP4) receptors have been located to the nucleus and their role in nuclear calcium signaling has been proposed. Outer nuclear membrane was separated from the inner membrane. The two membrane preparations were, as best as possible, devoid of cross contamination as attested by marker enzyme activity, Western blotting with antilamin antibody, and electron microscopy. InsP4 receptor and Ca(2+)-ATPase were located to the outer nuclear membrane. InsP3 receptor was located to the inner nuclear membrane. ATP or InsP4 induced nuclear calcium uptake. External free calcium concentration, in the medium bathing the nuclei, determined the choice for ATP or InsP4-mediated calcium transport. We present a mechanistic model for nuclear calcium transport. According to this model, calcium can reach the nucleus envelope either by the action of ATP or InsP4. However, the calcium release from the nucleus envelope to the nucleoplasm is mediated by InsP3 through the activation of InsP3 receptor, which is located to the inner nuclear membrane. The action of InsP3 in this process was instantaneous and transient and was sensitive to heparin.
Immunocytochemical investigations were performed on Jimpy and control mouse brains using three specific anti-myelin proteolipids antisera: immunoaffinity purified multivalent anti-(PLP + DM-20) proteolipid antibodies, anti-C-terminal hexapeptide 271-276 and anti-tridecapeptide 117-129 antisera. The results show that oligodendrocytes and myelin sheaths in normal mouse brain are labelled to the same extent by the three specific antisera; in contrast, in Jimpy brain these cellular structures are only stained by the multivalent antibodies and the site-specific, anti-tridecapeptide antiserum. The absence of labelling with C-terminal hexapeptide antiserum in mutant brain is interpreted as the result of either a large deletion or a point mutation producing a frameshift in the C-terminal part of the sequences of the proteolipids PLP and DM-20. Furthermore, we show that this mutation prevents the normal transport of proteolipid molecules through the Golgi apparatus. The existence of a minor, extra-Golgi apparatus metabolic route for proteolipids to myelin structures is also discussed.
The passage of immunocompetent cells across the blood-brain barrier (BBB) is regulated at the level of the cerebral capillaries which have specific morphological and biochemical properties. We have developed and characterized an in vitro model of the BBB using immortalized human endothelial cells (ECV 304) induced by rat astrocytes. In this model, endothelial cells are attached together by continuous intercellular junctions with numerous tight junctions, develop a permeability barrier having a significant transcellular electrical resistance, possess high activities of gamma-glutamyl transpeptidase (gamma-GTP) and express the brain-type glucose transporter 1 (GLUT-1). These parameters are also characteristic of brain capillary endothelial cells. Under the culture conditions used, the ECV 304 cells express the intercellular adhesion molecule-1 (ICAM-1) on the external plasma membrane at concentrations which could permit lymphocyte adhesion to be studied.
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