The cDNA-deduced primary structure of human sex hormone-binding globulin and location of its steroid-binding domain

AbstractWe have sequenced a cDNA for sex hormone-binding globulin (SHBG) isolated from a phage λgt 11 human liver cDNA library. The library was screened with a radiolabeled rat androgen-binding protein (ABP) cDNA, and the abundance of SHBG cDNAs was 1 in 750 000 plaques examined. The largest human SHBG cDNA (1194 base-pairs) contained a reading frame for 381 amino acids. This comprised 8 amino acids of a signal peptide followed by 373 residues starting with the known NH2-terminal sequence of human SHBG, and ending with a termination codon. The predicted polypeptide Mr of SHBG is 40 509, and sites of attachment of one O-linked (residue 7) and two N-linked oligosaccharide (residues 351 and 367) chains were identified. Purified SHBG was photoaffinity-labeled with Δ6-[3H]testosterone and cleaved with trypsin. The labeled tryptic fragment was isolated by reverse-phase HPLC, and its NH2-terminal sequence was determined. The results suggest that a portion of the steroid-binding domain of SHBG is located between residue 296 and the 35 predominantly hydrophilic residues at the C-terminus of the protein

Ionic lanthanum passage across cerebral endothelium exposed to hyperosmotic arabinose

Hyperosmotic media infused into the cerebral circulation open the blood-brain barrier to protein and colloid. The mechanism whereby such substances cross the affected vessels is still disputed. We describe here the transendothelial route taken by ionic lanthanum (La 3+ ), a small electron-dense tracer which, unlike colloidal lanthanum, can be administered to the living animal. In adult rats, 2.9 ml of hyperosmotic (1.4 M) arabinose was infused into the internal carotid artery as a 30-s bolus, followed by 5 mM LaCl 3 . To find the extravasated La 3+ , which is invisible by light microscopy, horseradish peroxidase (HRP) was injected simultaneously into the femoral vein. The hyperosmotic treatment resulted in exudation of both HRP and La 3+ primarily around cerebral arterioles. The La 3+ crossed arterioles through successive tight junctions between endothelial cells. Although the tight junctions were not discernibly opened, they must have become permeable because the extracellular pools between successive tight junctions were penetrated by the La 3+ . These pools are normally inaccessible to La 3+ . Luminal and abluminal pits and cytoplasmic vesicles, some of them containing La 3+ , formed intraendothelial clusters. Their role, if any, in the transfer of ion remains remains uncertain.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47227/1/401_2004_Article_BF00685347.pd

Endogenous Bax Translocation in SH-SY5Y Human Neuroblastoma Cells and Cerebellar Granule Neurons Undergoing Apoptosis

Changes at the mitochondria are an early, required step in apoptosis in various cell types. We used western blot analysis to demonstrate that the proapoptotic protein Bax translocated from the cytosolic to the mitochondrial fraction in SH-SY5Y human neuroblastoma cells undergoing staurosporine- or EGTA-mediated apoptosis. Levels of mitochondrial Bax increased 15 min after staurosporine treatment. In EGTA-treated cells, increased levels of mitochondrial Bax were seen at 4 h, consistent with a slower onset of apoptosis in EGTA versus staurosporine treatments. We also demonstrate the concomitant translocation of cytochrome c from the mitochondrial to the cytosolic fractions. We correlated these translocations with changes in caspase-3-like activity. An increase in caspase-3-like activity was evident 2 h after staurosporine treatment. Inhibition of the mitochondrial permeability transition had no effect on Bax translocation or caspase-3-like activity in staurosporine-treated SH-SY5Y cells. In primary cultures of cerebellar granule neurons undergoing low K + -mediated apoptosis, Bax translocation to the mitochondrial fraction was evident at 3 h. Cytochrome c release into the cytosol was not significant until 8 h after treatment. These data support a model of apoptosis in which Bax acts directly at the mitochondria to allow the release of cytochrome c.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66150/1/j.1471-4159.1999.0721899.x.pd