Failure of 2 Hydroxyestradiol to interact with dopamine inhibition of human prolactin secretion in vitro and with dopamine receptors of prolactin-secreting adenomas

The ability of 2-Hydroxyestradiol, a catecholestrogen, and 17 beta Estradiol to interact with the dopamine inhibition of prolactin and with dopamine receptors has been tested on dispersed human prolactin-secreting cells obtained from ten pituitary adenomas. There is a 80% inhibition of prolactin secretion obtained by addition of dopamine in a superfusion system. This inhibition is not affected by preexposure to the steroids, or by their introduction into the perifusion medium. Moreover 2 Hydroxyestradiol and 17 beta Estradiol do not interact with the binding of 3H Domperidone to DA receptors.     

Regulation of calcium accumulation and efflux from platelet vesicles: Possible role for cyclic-AMP-dependent phosphorylation and calmodulin

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ activity of about 10 nmol (min·mg)^?1 and an ATP-dependent Ca²⁺ uptake of about 10 nmol (min·mg)^?1. When incubated in the presence of Mg[γ-³²P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [³²P]phosphate-labeled Ca²⁺ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca²⁺ or Ca²⁺ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of ¹²⁵I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol ($\text{M}{}_{\mathrm{r}}\text{= 94 000, 87 000, 60 000 and 43 000}$). Some minor calmodulin-binding proteins were enriched in the membrane fractions ($\text{M}{}_{\mathrm{r}}\text{= 69 000, 57 000, 39 000 and 37 000}$). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca²⁺ uptake is essentially unaltered, while the Ca²⁺ capacity is diminished as a consequence of a doubling in the rate of Ca²⁺ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca²⁺ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 μM calmodulin result in increased levels of vesicle phosphorylation.     

Fluorography--limitations on its use for the quantitative detection of 3H- and 14C-labeled proteins in polyacrylamide gels

The suitability of fluorography for the detection of 3H- and 14C-labeled proteins on polyacrylamide gradient gels has been investigated. It was found that the absorbance of the fluorographic film image produced by a given level of radioactivity decreased as the acrylamide concentration in the gel increased. The use of Coomassie brilliant blue protein dyes to stain the gel prior to fluorography reduced the absorbance of the fluorographic image. It is concluded that quantitative fluorography can only be applied to unstained gels of a uniform acrylamide concentration.     

Ammoniacal silver staining of proteins: mechanism of glutaraldehyde enhancement

When the conditions for detecting proteins by ammoniacal silver staining (B. R. Oakley, D. R. Kirsch, and N. R. Morris (1980) Anal. Biochem. 105, 361-363.) following gel electrophoresis were varied, it was noted that glutaraldehyde pretreatment was necessary for maximal staining, which could not be explained simply as the result of "fixation." Further studies indicated that glutaraldehyde enhancement of protein staining with this silver reagent was probably due to oxidation of the aldehyde groups by silver ions, resulting in metallic silver depositions within the gel which act as nucleation sites for additional metallic silver localization in the protein bands upon the addition of formaldehyde developer. This proposed mechanism is consistent with the Tollen's reaction, as well as some aspects of the photographic process. Consistent with this notion, silver-staining intensities are directly related to mole percentage lysine of various standard proteins.     

Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila,Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria

Ribosomal subunits of Caldariella acidophila (max.growth temp., 90°C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70°C) and Escherichia coli (max. growth temp., 47°C) with respect to (a) bihelical content of rRNA; (b) G·C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. 1. Subunits of C. acidophila ribosomes (T_m = 90–93°C) exhibit considerable thermal tolerance over their B. acidocaldarius (T_m = 77°C) and E. coli counterparts (T_m = 72°C). 2. Based on the ‘melting’ hyperchromicities of the intact ribosomal subunits a 51–55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67–70%, appears to be involved in hydrogen bonding in the naked rRNA species. 3. The G·C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63–64% G·C, compared to 58.5% G·C for B. acidocaldarius and 55% G·C for E. coli. 4. The increment in ribosome T_m values with increasing thermophily is greater than the increase in T_m for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). 5. The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release rRNA from Celite-bound E. coli ribosomes. 6. Compared to E. coli, the C. acidophila 50 and 30 S ribosomal subunits are considerably less susceptible to treatment designed to promote ribosome unfolding through depletion of magnesium ions.     

Phototoxicity of phenothiazine derivatives. II. Photosensitized cross-linking of erythrocyte membrane proteins

Irradiation in the presence of O₂, with near-UV light of five promazine (PZ) derivatives added to erythrocyte ghost membranes, causes covalent cross-linking between proteins as revealed by a progressive decrease in the amounts of proteins separable by electrophoresis after denaturation. The induction of cross-links in the two spectrin subunits is a single-hit process as a function of the irradiation time; relatively the rate constants (in min^?1) of the photoreactions were 0.060 with chlorpromazine (CPZ), 0.039 with methoxypromazine (MTPZ), 0.031 with PZ, 0.029 with triflupromazine (TFPZ) and 0.006 with acepromazine (ACPZ).A main photochemical intermediate implicated in the spectrin aggregation seems to be the cation radical of the PZ derivatives. Indeed, (i) the chemically generated cation radicals can induce the reaction in the dark; (ii) the photoaggregation is regularly reduced upon addition of increasing concentrations of NaN₃; (iii) NaN₃ similarly affects the amount of cross-links induced by the isolated cation radicals. Hydroxyl radicals are also involved in the photocross-linking when the reaction is initiated only by MTPZ and not by the other sensitizers.In the absence of oxygen during irradiation, PZ, MTPZ and ACPZ completely loose their cross-linking activities whereas CPZ and TFPZ remain as efficient as in the presence of oxygen.     

Puromycin aminonucleoside increases the synthesis of ribonuclease inhibitor and other proteins in kidney

The incorporation of labelled amino acids into proteins was measured in vivo in the kidney of control rats and rats that received puromycin aminonucleoside. There was an increase in the synthesis of kidney proteins after the aminonucleoside that was similar to the increased synthesis previously observed in cell-free and slice preparations. The increased synthesis in vivo and in vitro especially involved proteins of the prealbumin fraction of average molecular weights of approx. 50 000, 35 000, 25000, 18 000, and 10 000. The largest of these proteins was identified as kidney ribonuclease inhibitor and additional evidence was obtained for the increased synthesis of the kidney inhibitor after aminonucleoside.     

An improved method for the measurement of total lipid-bound sialic acids after cleavage of α2,8 sialic acid linkage withVibrio cholerae sialidase in the presence of cholic acid,SDS and Ca2+

In the measurement of total lipid-bound sialic acids involving periodic acid oxidation, as in the periodate-resorcinol assay, the inner sialic acids of disialoglycolipids (such as GD₃ and GD₂) are not involved because their 2,8 ketosidic linkages are resistant to periodic acid oxidation, even after acid/enzyme hydrolysis or alkali pretreatment. However, the sialic acids from these glycolipids can be recovered completely after cleavage of 2,8 linkages byV. cholerae sialidase in the presence of cholic acid, sodium dodecyl sulphate and calcium. Interestingly, removal of calcium or detergent(s) or both significantly minimizes the sialidase action on the disialyl residues of these gangliosides. Therefore, we recommend sialidase (Vibrio cholerae) pretreatment of the glycolipids in the presence of cholic acid, SDS and Ca²⁺ for complete recovery of sialic acids from di- and polysialogangliosides and for accurate measurement of total lipid-bound sialic acids by periodate-resorcinol assay.Presented at the Second International Glycobiology Symposium which was held in San Francisco, CA, USA (14 February 1994).     

Effects of detergents on the secondary structures of prion protein peptides as studied by CD spectroscopy.

Pathogenic prion proteins (PrP(Sc)) are thought to be produced by alpha-helical to beta-sheet conformational changes in the normal cellular prion proteins (PrP(C)) located solely in the caveolar compartments. In order to inquire into the possible conformational changes due to the influences of hydrophobic environments within caveolae, the secondary structures of prion protein peptides were studied in various kinds of detergents by CD spectra. The peptides studied were PrP(129-154) and PrP(192-213); the former is supposed to assume beta-sheets and the latter alpha-helices, in PrP(Sc). The secondary structure analyses for the CD spectra revealed that in buffer solutions, both PrP(129-154) and PrP(192-213) mainly adopted random-coils (approximately 60%), followed by beta-sheets (30%-40%). PrP(129-154) showed no changes in the secondary structures even in various kinds of detergents such as octyl-beta-D-glucopyranoside (OG), octy-beta-D-maltopyranoside (OM). sodium dodecyl sulfate (SDS), Zwittergent 3-14 (ZW) and dodecylphosphocholine (DPC). In contrast, PrP(192-213) changed its secondary structure depending on the concentration of the detergents. SDS, ZW, OG and OM increased the alpha-helical content, and decreased the beta-sheet and random-coil contents. DPC also increased the alpha-helical content, but to a lesser extent than did SDS, ZW, OG or OM. These results indicate that PrP(129-154) has a propensity to adopt predominantly beta-sheets. On the other hand, PrP(192-213) has a rather fickle propensity and varies its secondary structure depending on the environmental conditions. It is considered that the hydrophobic environments provided by these detergents may mimic those provided by gangliosides in caveolae, the head groups of which consist of oligosaccharide chains containing sialic acids. It is concluded that PrP(C) could be converted into a nascent PrP(Sc) having a transient PrP(Sc) like structureunder the hydrophobic environments produced by gangliosides.