An unexpected localization of basonuclin in the centrosome, mitochondria, and acrosome of developing spermatids.

Basonuclin is a zinc finger protein that was thought to be restricted to keratinocytes of stratified squamous epithelia. In epidermis, basonuclin is associated with the nuclei of mitotically active basal cells but not in terminally differentiating keratinocytes. We report here the isolation of a novel form of basonuclin, which we show is also expressed in stratified epithelia. Most unexpectedly, we find both forms in testis, where a surprising localization pattern was uncovered. While basonuclin RNA expression occurs in mitotically active germ cells, protein was not detected until the meiotic stage, where basonuclin localized to the appendage of the distal centriole of spermatocytes and spermatids. Near the end of spermiogenesis, basonuclin also accumulated in the acrosome and mitochondrial sheath surrounding the flagellum. Intriguingly, a perfect six-amino acid residue mitochondrial targeting sequence (Komiya, T., N. Hachiya, M. Sakaguchi, T. Omura, and K. Mihara. 1994. J. Biol. Chem. 269:30893-30897; Shore, G.C., H.M. McBride, D.G. Millar, N.A. Steenaart, and M. Nguyen. 1995. Eur. J. Biochem. 227: 9-18; McBride, H.M., I.S. Goping, and G.C. Shore. 1996. J. Cell. Biol. 134:307-313) is present in basonuclin 1a but not in the 1b form. Moreover, three distinct affinity-purified peptide antibodies gave this unusual pattern of basonuclin antibody staining, which was confirmed by cell fractionation studies. Our findings suggest a unique role for basonuclin in centrosomes within the developing spermatid, and a role for one of the protein forms in germ cell mitochondrial function. Its localization with the acrosome suggests that it may also perform a special function during or shortly after fertilization.

Ganglioside GT1b induces keratinocyte differentiation without activating protein kinase C.

The altered patterns of expression of gangliosides during density-dependent growth inhibition, oncogenic transformation, and embryogenesis suggest that gangliosides, sialylated membrane glycolipids, may affect cellular proliferation and differentiation. Gangliosides of the "b" pathway of ganglioside synthesis, including GM3, GD3, and GD1b, inhibit the proliferation of cultured keratinocytes without increasing differentiation. We have examined the effect on keratinocyte proliferation and differentiation of supplemental ganglioside GT1b, a more highly sialylated ganglioside of the "b" synthetic pathway that is also present in cultured keratinocytes. In contrast to the lack of effect on differentiation of these other gangliosides, we noted significant induction of keratinocyte differentiation by GT1b, as evidenced by early desmosome formation, and increased cornified envelope formation and expression of involucrin and of the differentiation-specific keratin K1. The addition of GT1b did not cause a shift in intracellular free calcium or alter protein kinase C activity. Alterations in the membrane concentration of ganglioside GT1b, a minor ganglioside component of the keratinocyte membrane, may participate in regulating keratinocyte differentiation.

Postmortem viability and early changes in organ culture of human and rabbit aortic endothelial cells.

In order to understand the role of the postmortem interval (PMI) on endothelial cell changes both in human and rabbit aortas, we have examined the ultrastructural cytomorphologic alterations of these cells. Human aorta (HA) and rabbit aorta (RA) were maintained in calcium-free, glucose-supplemented Hank's balanced salt solution (HBSS). Rabbit endothelial cells (REC) on the aorta (organ culture) assayed morphologically survive for at least 12 h in culture solution. The predominant morphological change in the RA was the formation of multiple subendothelial vacuoles (SEV). These vacuoles may form as the results of increased permeability of endothelial cells to ions and fluid or cell contraction. Cell to cell connection remained intact. Individual and dispersed endothelial cells were observed 8 h after removal from the animal when incubated in calcium-free HBSS. These necrotic endothelial cells were scattered among viable endothelial cells. Human aortic endothelial cells were also well preserved in the same media for periods of 6-8 h postmortem. Increased extracellular calcium (1.3 mM) in the incubation media caused accelerated cell death. These findings suggest that aortic endothelial cells can be preserved for longer periods of postmortem time than would be expected and that the use of calcium-free HBSS media supplemented with glucose improves endothelial cell viability in vitro.