Mechanism of action of serotonin on frog adrenal cortex

The mechanism of action of serotonin (5-HT) on frog adrenal cortex has been investigated in vitro using the perifusion system technique. The direct effect of 5-HT on corticosteroid secreting cells was demonstrated, using enzymatically dispersed adrenocortical cells. Melatonin and 5-HTP appeared to be less potent than 5-HT to enhance corticosteroid secretion. In contrast Trp and 5-HIAA were totally devoid of effect on steroid secretion. To investigate the type of receptor involved in the stimulatory effect of 5-HT on adrenocortical cells, adrenal slices were stimulated with 5-HT in absence or presence of various antagonists. We observed that classical antagonists of 5-HT1, 5-HT2 and 5-HT3 type receptors failed to block 5-HT-induced corticosteroid secretion in our model. These results show that 5-HT exerts a direct effect on corticosteroid-secreting cells. Our data also indicates that the type of receptor involved in the action of 5-HT in frog adrenal cortex differs from mammalian 5-HT receptors.     

Immunohistochemical distribution of the 52-kDa protein in mammary tumors: a marker associated with cell proliferation rather than with hormone responsiveness

We have previously described a secreted glycoprotein of mol. wt 52,000 (52-kDa protein) which is induced by estrogen in some human breast cancer cell lines. This protein has been identified as the proenzyme of a lysosomal cathepsin-D-like protease which is secreted in large proportions in breast cancer cells. To determine which information may be generated by this marker when detected in mammary tumors, in comparison with hormone receptors, we used monoclonal antibodies interacting specifically with the 52-kDa protein and its related cellular processed products (mols. wts 48 and 34 kDa). A high concentration of this protein has been shown in proliferative ductal mastopathies and cysts, suggesting its value in detecting high-risk mastopathies. We now present the immunoperoxidase distribution of this protein in breast carcinoma compared to the cytosolic hormone receptors assayed in parallel. In 232 breast cancers, no correlation was found between the cellular 52-kDa protein content and cytosolic estrogen or progesterone receptor concentrations. This absence of correlation was also shown by the constitutive production of this protein by estrogen-receptor-negative breast cancer cell lines and confirmed by double immunostaining of breast cancer cell aspirates showing a dissociation between the cytoplasmic staining of this 52-kDa lysosomal protease and the nuclear staining of the estrogen receptor. These clinical results, associated with the in vitro mitogenic and proteolytic activities of this protein, strongly suggest that the 52-kDa protein staining in tissue is associated with tumor proliferation and/or invasion, rather than with hormone responsiveness.     

Polar and nonpolar atomic environments in the protein core: Implications for folding and binding

Hydrophobic interactions are believed to play an important role in protein folding and stability. Semi-empirical attempts to estimate these interactions are usually based on a model of solvation, whose contribution to the stability of proteins is assumed to be proportional to the surface area buried upon folding. Here we propose an extension of this idea by defining an environment free energy that characterizes the environment of each atom of the protein, including solvent, polar or nonpolar atoms of the same protein or of another molecule that interacts with the protein. In our model, the difference of this environment free energy between the folded state and the unfolded (extended) state of a protein is shown to be proportional to the area buried by nonpolar atoms upon folding. General properties of this environment free energy are derived from statistical studies on a database of 82 well-refined protein structures. This free energy is shown to be able to discriminate misfolded from correct structural models, to provide an estimate of the stabilization due to oligomerization, and to predict the stability of mutants in which hydrophobic residues have been substituted by site-directed mutagenesis, provided that no large structural modifications occur. © 1994 Wiley-Liss, Inc.     

Effect of calcium on corticosteroid secretion by isolated frog interrenal gland

The direct effect of extracellular calcium concentrations on corticosteroidogenesis has been examined in the frog, using a perifusion system technique. The release of corticosterone and aldosterone in the effluent medium was monitored by specific radioimmunoassays. Increasing concentrations of Ca2+ (from 2 to 15 mM) gave rise to a dose-related stimulation of corticosteroid release, whereas the increment of either Na+ or K+ concentrations did not modify steroid production. Iterative administration of a moderate concentration of calcium (6 mM) led to a reproducible stimulation of steroid secretion whereas the same dose infused during 6 h induced a transient rise in corticosteroid secretion followed by a plateau. The direct effect of Ca2+ on steroidogenesis was confirmed by the dose-dependent stimulation of steroid secretion induced by the calcium ionophore A 23187. Perifusion with a calcium-free medium or blockade of Ca2+ channels by 4 mM Co2+ both resulted in a significant decrease in steroid production. Conversely, the administration of verapamil (up to 10(-4) M) did not affect steroidogenesis. These results provide evidence that extracellular calcium ions are required for basal production of corticosteroids in amphibians and that Ca2+ influx does not occur through voltage-dependent channels. Since, in the frog, blood Ca2+ concentrations vary in a rather large range, these results suggest that circulating Ca2+ levels may regulate corticosteroid production in these animals.     

Three-dimensional structure of phenylalanyl-transfer RNA synthetase from Thermus thermophilus HB8 at 0.6-nm resolution.

The three-dimensional structure of the heterodimeric alpha 2 beta 2 enzyme phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 has been determined by X-ray crystallography, using the multiple-isomorphous-replacement method at 0.6 nm resolution. Trigonal crystals of space group P3(2)21 have cell dimensions a = b = 17.6 nm and c = 14.2 nm. Assuming one heterodimeric molecule/asymmetric unit, the ratio of unit cell volume/molecular mass was V = 0.00244 nm3/Da, which is in the middle of the range normally observed. However, after a rotation-function calculation and measurement of the density of the native crystals, we postulate the existence of only the alpha beta dimer in the asymmetric units. This implies 73% solvent content in the unit cell. Three heavy-atom derivatives [K2PtCl4, KAu(CN)2 and Hg(CH3COO)2] and the solvent-flattening procedure were used for electron-density-map calculations. This map confirmed our hypothesis and revealed a remarkably large space filled by solvent, with alpha beta dimer only in the asymmetric unit. The phenylalanyl-tRNA synthetase from T. thermophilus molecule has a 'quasi-linear' subunit organization. As can be concluded at this level of resolution, there is no contact between small alpha subunits in the functional heterodimer.     

Age-related insulin resistance: a review.

Impaired glucose tolerance occurs with age. This impairment is multifactorial including a decrease in insulin-mediated glucose uptake by peripheral tissues and a delay in insulin-induced suppression of hepatic glucose output. A post-binding defect in insulin action such as a reduced capacity to transcribe more glucose transporter mRNA and/or a reduced translocation of preformed glucose transporters to plasma membrane is incriminated. However, insulin resistance with age is not a constant finding and other mechanism(s) has (have) to be involved in old individuals with impaired glucose tolerance and normal tissue insulin sensitivity.     

Adenylate cyclase and guanylate cyclase activity in the isolated kidney glomerulus of the rat]

Isolated rat renal glomeruli contain an adenylate cyclase system and guanylate cyclase system. Adenylate cyclase was strikingly activated by purified parathyroid hormone, epinephrine, prostaglandin I2 and histamine. The demonstration of PTH activated adenylate cyclase in glomeruli raises the possibility of a role of this hormone in regulation of glomerular filtration rate. Guanylate cyclase was strikingly activated by CA2+, nitrate derivatives such as sodium nitroprusside. Its role remained still unknown.