Synemin and vimentin are components of intermediate filaments in avian erythrocytes

Synemin, a high-molecular-weight protein associated with intermediate filaments in muscle, and vimentin, an intermediate-filament subunit found in many different cell types, have been identified by immunologic and electrophoretic criteria as components of intermediate filaments in mature avian erythrocytes. Desmin, the predominant subunit of intermediate filaments in muscle, has not been detected in these cells. Two dimensional immunoautoradiography of proteolytic fragments of synemin and vimentin demonstates that the erythrocyte proteins are highly homologous, if not identical, to their muscle counterparts. Double immunoflurorescence reaveals that erythrocyte synemin and vimentin co-localize in a cytoplasmic network of sinuous filaments that extends from the nucleus to the plasma membrane and resists aggregation by colcemid. Erythrocytes that are attached to glass cover slips can be sonicated to remove nuclei and nonadherent regions of the plasma membrane; this leaves elliptical patches of adherent membrane that retain mats of vimentin- and synemin-containing intermediate filaments, as seen by immunofluorescence and rotary shadowing. Similarly, mechanical enucleation of erythrocyte ghosts in suspension allows isolation of plasma membranes that retain a significant fraction of the synemin and vimentin, as assayed by electrophoresis, and intermediate filaments, as seen in thin sections. Both synemin and vimentin remain insoluble along with spectrin and actin, in solutions containing nonionic detergent and high salt. However, brief exposure of isolated membrane to distilled water releases the synemin and vimentin together in nearly pure form, before the release of significant amounts of spectrin and actin. These data suggest that avian erythrocyte intermeditate filaments are somehow anchored to the plasma membrane; erythrocytes may thus provide a simple system for the study of intermediate filaments and their mode of interaction with membranes. In addition, these data, in conjunction with previous data from muscle, indicate that synemin is capable of associating with either desmin or vimentin and may thus perform a special role in the structure or function of intermediate filaments in erythrocytes as well as muscle.     

Molecular Cloning, Chromosomal Localization, and Cell Cycle-Dependent Subcellular Distribution of the A-Kinase Anchoring Protein, AKAP95

The cyclic AMP-dependent protein kinase (PKA) type II is directed to different subcellular loci through interaction of the RII subunits with A-kinase anchoring proteins (AKAPs). A full-length human clone encoding AKAP95 was identified and sequenced, and revealed a 692-amino acid open reading frame that was 89% homologous to the rat AKAP95 (V. M. Coghlan, L. K. Langeberg, A. Fernandez, N. J. Lamb, and J. D. Scott (1994)J. Biol. Chem.269, 7658–7665). The gene encoding AKAP95 was mapped to human chromosome 19p13.1-q12 using somatic cell hybrids and PCR. A fragment covering amino acids 414–692 of human AKAP95 was expressed inEscherichia coliand shown to bind RIIα. Competition with a peptide covering the RII-binding domain of AKAP Ht31 abolished RIIα binding to AKAP95. Immunofluorescence studies in quiescent human Hs-68 fibroblasts showed a nuclear localization of AKAP95, whereas RIIα was excluded from the nucleus. In contrast, during mitosis AKAP95 staining was markedly changed and appeared to be excluded from the condensed chromatin and localized outside the metaphase plate. Furthermore, the subcellular localizations of AKAP95 and RIIα overlapped in metaphase but started to segregate in anaphase and were again separated as AKAP95 reentered the nucleus in telophase. Finally, RIIα was coimmunoprecipitated with AKAP95 from HeLa cells arrested in mitosis, but not from interphase HeLa cells, demonstrating a physical association between these two molecules during mitosis. The results show a distinct redistribution of AKAP95 during mitosis, suggesting that the interaction between AKAP95 and RIIα may be cell cycle-dependent.     

Alpha-inhibin gene expression occurs in the ovine adrenal cortex, and is regulated by adrenocorticotropin

Inhibin is a glycoprotein hormone composed of two nonidentical subunits. It is produced by the ovary and testis and plays a vital role in gonadal function by inhibiting the secretion of FSH. More recently, additional activities associated with inhibin peptides have been identified. Inhibin heterodimers (alpha-beta) are reported to act directly on ovarian granulosa cells and inhibit estrogen production induced by FSH. Furthermore, homodimers of beta-inhibin subunits stimulate the secretion of FSH, an activity that is directly opposite to that of inhibin. Each of these inhibin-related activities are concerned with the hypothalamic-pituitary-gonadal axis. We have investigated further the complexity of inhibin activity by determining whether inhibin genes are expressed in nongonadal tissue. RNA hybridization experiments demonstrate that the alpha-inhibin gene is expressed in the sheep adrenal cortex and hybridization histochemistry shows that this gene is expressed in each of the functional zones within the cortex. Dot blot analysis showed that the level of alpha mRNA within the adrenal is influenced by ACTH, one of the major regulators of adrenal cortex function. These observations imply that there are inhibin-related peptides not directly associated with the gonads. beta-inhibin gene expression was not clearly detected in the adrenal and we conclude that if expression occurs then it does so at extremely low levels.     

Distribution of glomerular peripolar cells in different mammalian species

Summary Peripolar cells are granulated glomerular epithelial cells that form a cuff around the vascular pole of the glomerulus. Quantitation of these cells in 17 species of mammals (including man, several laboratory animals and a variety of other species) indicated that they were detectable by light microscopy in all but one of the mammals that were examined (the Australian hopping mouse). In adult mammals with detectable peripolar cells, the peripolar cell index (the percentage of randomly sectioned glomeruli that displayed peripolar cells in histological sections of kidney) ranged from 0.15 (for echidna) to 11.86 (for sheep). Newborn lambs and rats showed strikingly high values (23.30 and 10.76, respectively) compared with their adult counterparts. Using electron microscopy, peripolar cells were observed in all species that were examined, including the Australian hopping mouse. Morphologically, peripolar cells were similar in all species although their size and granule population varied. They showed a predominantly outer cortical glomerular distribution and a close anatomical relationship with the renin-containing myoepithelioid cells. These findings indicate that peripolar cells are present in a wide variety of species and support the view that such cells may play a significant role in the regulation of normal renal function.     

Tissue and species distribution of the secreted carbonic anhydrase isoenzyme.

The secreted carbonic anhydrases, CA VI, are high molecular mass, oligomeric enzymes originally found in the sheep parotid gland and saliva. The enzymes have been purified from the saliva or parotid glands of several different species. All the CA VI enzymes studied have an apparent subunit Mr of about 45,000 as previously reported for the sheep enzyme. By Western analysis, CA VI from human, cow and dog cross-reacted with antibody raised against the purified sheep enzyme whereas that of the mouse did not. The N-terminal sequences of the sheep, human, cow and mouse enzymes are reported. The sheep, cow and human N-terminal sequences are similar to one another while the mouse sequence is substantially different. Nevertheless, the amino acids in the aromatic cluster I (Trp-5, Tyr-7, Trp-16 and Tyr/Phe-20) have all been conserved, as is the case with the cytoplasmic carbonic anhydrases. Eighteen tissues from the sheep have been examined for the presence of CA VI by Western analysis but it has been found only in the salivary glands. Northern analysis and hybridization histochemistry show that the mRNA for CA VI in sheep is expressed specifically in the acinar cells of the parotid and submandibular glands.     

The gene for human carbonic anhydrase VI(CA6) is on the tip of the short arm of chromosome 1

The gene encoding the human secreted carbonic anhydrase isozyme CAVI(CA6) maps to chromosome 1 by Southern analysis of a somatic cell hybrid panel and to 1p36.22----p36.33 by in situ hybridization. CA6 is therefore not linked to the cytoplasmic carbonic anhydrase genes on chromosome 8 or to CA7 on chromosome 16.