Species heterogeneity of hepatic alpha 1-adrenoceptors: alpha 1A-, alpha 1B- and alpha 1C-subtypes.

alpha 1-Adrenergic activation stimulated phosphorylase and phosphoinositide turnover in hepatocytes from guinea pigs, rats and rabbits. Chlorethylclonidine inhibited these effects in rat and rabbit cells but not in guinea pig hepatocytes; low concentrations of 5-methyl urapidil blocked the alpha 1 actions in guinea pig and rabbit liver cells, but not in rat hepatocytes. Binding competition experiments also showed high affinity for 5-methyl urapidil in liver membranes from guinea pigs and rabbits and low affinity in those from rats. The data indicated that guinea pig hepatocytes express alpha 1A-, rat hepatocytes alpha 1B- and rabbit hepatocytes alpha 1C- adrenoceptors. This was confirmed by Northern analysis using receptor subtype-selective probes.     

Platelet alpha2-macroglobulin and alpha1-antitrypsin.

Subcellular membrane and granule fractions derived from human platelets contain immunologically identifiable alpha2-macroglobulin and alpha1-antitrypsin. These platelet-derived inhibitors show a reaction of immunologic identity when compared to alpha2-macroglobulin and alpha1-antitrypsin purified from human plasma. Further, the platelet protease inhibitors possessed a similar subunit polypeptide chain structure to their plasma counterparts as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. Studies of the binding of radiolabeled trypsin to the various solubilized platelet subcellular fractions suggest that the granule-associated alpha2-macroglobulin and alpha1-antitrypsin, as well as membrane-associated alpha2-macroglobulin were functionally active. Quantitatively, circulating platelets contain relatively small concentrations of these inhibitors as compared to platelet-associated fibrinogen and factor VIIIAGN. Platelet protease inhibitors may modulate the protease-mediated events involved in the formation of hemostatic plugs and thrombi.     

Identifications of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 23 beta-tetrol, 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 24 alpha-tetrol, and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 24 beta-tetrol in cerebrotendinous xanthomatosis

The bile alcohols present in the feces of a patient with cerebrotendinous xanthomatosis were studied. Three bile alcohols which are different from any known natural bile alcohol were isolated as minor components of the fecal bile alcohol fraction. The structures of these compounds were established as 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 23 beta-tetrol, 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 24 alpha-tetrol, and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 24 beta-tetrol by comparison with synthetic samples.     

Immunoaffinity purification of GABAA receptor alpha-subunit iso-oligomers. Demonstration of receptor populations containing alpha 1 alpha 2, alpha 1 alpha 3, and alpha 2 alpha 3 subunit pairs.

Novel methods for the isolation of gamma-aminobutyric acidA (GABAA) receptor alpha subunit iso-oligomers have been developed. Thus, populations of GABAA receptors containing the GABAA receptor alpha 1 subunit, the alpha 2 subunit, and the alpha 3 subunit have been purified from sodium deoxycholate extracts of bovine cerebral cortex with the retention of specific [3H]flunitrazepam-binding activity by anti-alpha 1 324-341, anti-Cys alpha 2 414-424, or anti-Cys alpha 3 454-467 antibody affinity chromatography, respectively. The relative abundance of the different specificity alpha subunits in these preparations was compared with benzodiazepine affinity chromatography-purified GABAA receptors by immunoblotting. In each case, it was found that although the immunoreactivity with the specific alpha subunit antibody that was used for purification was enriched in immunoaffinity-purified receptors, reactivity with the other alpha subunit specificity antibodies, together with anti-gamma 2 1-14 Cys immunoreactivity was found. Immunoprecipitation of GABAA receptors purified by anti-alpha 1 324-341 antibody affinity chromatography by all three anti-alpha subunit antibodies employed, together with the use of anti-alpha 1 324-341 and anti-Cys alpha 2 414-424 antibody affinity columns in series, further substantiated the partial co-purification of the different polypeptides. These results demonstrate the copurification of the gamma 2 subunit with each population of alpha 1, alpha 2, alpha 3 subunit-enriched GABAA receptors. They also show the existence of minor populations of GABAA receptors that contain alpha 1 alpha 2, alpha 1 alpha 3, and alpha 2 alpha 3 subunit pairs within single oligomers.     

Nicotine binding to native and substituted peptides comprising residues 188-207 of nicotinic acetylcholine receptor alpha1, alpha2, alpha3, alpha4, alpha5, and alpha7 subunits

Structural determinants of L-[(3)H]nicotine binding to synthetic peptides comprising residues 188-207 of nicotinic acetylcholine receptor alpha subunits were invesitigated by equilibrium binding analysis. Two binding components were detected, one of low affinity (K(d) approximately 1.5 microM) that did not differ significantly among peptides and another of high affinity. The high affinity binding component was higher for the neuronal peptides (K(d) = 14-23 nM) than the muscle alpha1 peptides (K(d) = 52 nM). The following nonconservative substitutions in the alpha4 peptide resulted in a significant decrease in nicotine affinity for the peptide: Y190A, Y190D, C192G, E195A, E195-, P199A, P199-, and Y203A. Substitution of alpha4P199 with a leucine which is present in the alpha1 sequence decreased the affinity of the alpha4 peptide for nicotine and substitution of alpha1L199 with a proline (alpha4) or a glutamine (alpha3) increased the affinity of the alpha1 peptide. It is concluded that aromatic residues contribute to the binding site for nicotine on the alpha4 subunit and that the residue present at position 199 partly determines differences in nicotine affinity for different alpha subunits.     

Acid-induced dissociation of alpha A- and alpha B-crystallin homopolymers.

Homopolymers were constructed from the alpha A and alpha B polypeptides isolated from the lens protein alpha-crystallin. As the pH is lowered from 7.0 to 3.4, these homopolymers dissociate to smaller species with molecular masses ranging from 80 to 250 kDa for the alpha A and around 140 kDa for the alpha B dissociation products. The pKa for this dissociation was 3.8 +/- 0.2 for alpha A and 4.1 +/- 0.1 for alpha B homopolymers. Further decreases in pH, to 2.5, resulted in the presence of only denatured alpha B polypeptides, whereas the alpha A dissociation products remained intact. Fractionation of the acid dissociation products from the alpha A homopolymer at pH 2.5 yielded stable species with molecular masses of 220 +/- 30, 160 +/- 20, and 90 +/- 10 kDa. The majority of the population at acid pH consisted of the 160 kDa species. Conformational analysis of these species revealed that most of the secondary structure of the original alpha A homopolymer was retained but that the tertiary structure was perturbed. Fluorescence quenching and energy transfer measurements suggested that the molecule had undergone acid expansion, with the greatest perturbation observed in the smallest particles. The results from this work suggest that alpha A homopolymers are heterogeneous populations of aggregates of a "monomeric" molecule with a molecular mass of 160 kDa. This "monomeric" molecule may be formed from the association of two tetrameric units.     

Hypoxia-inducible factors 1alpha and 2alpha regulate trophoblast differentiation

Placental development initially occurs in a low-oxygen (O2) or hypoxic environment. In this report we show that two hypoxia-inducible factors (HIFs), HIF1alpha and HIF2alpha, are essential for determining murine placental cell fates. HIF is a heterodimer composed of HIFalpha and HIFbeta (ARNT) subunits. Placentas from Arnt-/- and Hif1alpha-/- Hif2alpha-/- embryos exhibit defective placental vascularization and aberrant cell fate adoption. HIF regulation of Mash2 promotes spongiotrophoblast differentiation, a prerequisite for trophoblast giant cell differentiation. In the absence of Arnt or Hifalpha, trophoblast stem cells fail to generate these cell types and become labyrinthine trophoblasts instead. Therefore, HIF mediates placental morphogenesis, angiogenesis, and cell fate decisions, demonstrating that O2 tension is a critical regulator of trophoblast lineage determination. This novel genetic approach provides new insights into the role of O2 tension in the development of life-threatening pregnancy-related diseases such as preeclampsia.     

Immuno-histochemical expression of alpha1, alpha2 and alpha3 integrin subunits during angiogenesis in vitro

Aortic explants were obtained from mouse fetuses and cultured in collagen gels. Immuno-fluorescence microscopy, antibodies (anti alpha1, alpha2 and alpha3 integrin subunits) were used. Fibroblastic cells migrated from the aortic explant after one day of cultivation. The migrating cells located in the peripheral part of the aortic explant were positive for alpha1 and alpha2 integrin subunit antibodies. Immuno-fluorescence-positive staining for the alpha3 integrin subunit antibody was clearly seen in the migrating cells located near the aortic explant and surrounding tube-like structures. In an immuno-electron microscope study performed by pre-embedding immuno labeling, gold particles associated with the alpha3 integrin subunit were found to reside on the membranes of the cells surrounding the capillary-like tubes. Two synthetic peptides, GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) and KDGEA (Lys-Asp-Gly-Glu-Ala), were added to the growth medium to study their effects on cell migration. KDGEA, a compound containing the recognition sequence for alpha2beta1 integrin, decreased cell migration, while GRGDSP exhibited no effect. The migration of fibroblastic cells is an important phenomenon for tube formation. The present study suggested that the alpha1 and alpha2 integrin subunits are both involved in the cell migration, and more specifically, that the alpha2 integrin subunit participates in cell migration through the KDGEA sequence. The alpha3 integrin subunit played a role in tube formation.     

Synthesis of 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol and 5beta-cholestane-3alpha, 7alpha, 245, 25-pentol.

This paper describes syntheses of 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol and 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol which give higher yields than previously published methods. In addition, 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol was synthesized by a different procedure, namely via performic acid oxidation of the correspinding unsaturated triol, which gave a lower yield but avoided the formation of 5beta-cholestane-3alpha, 7alpha, 12alpha, 25, 26-pentol, which normally tends to contaminate the final product. Structures were confirmed by gas-liquid chromatography, infrared-, proton magnetic resonance- and mass spectrometry, 5beta-Cholestane-3alpha, 7alpha, 12alpha, 25-tetrol and 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol were required for in vivo and in vitro studies of the (hypothetical) 25-hydroxylation pathway of cholic acid biosynthesis.     

Prothymosin alpha is processed to thymosin alpha 1 and thymosin alpha 11 by a lysosomal asparaginyl endopeptidase

Thymosin alpha(1) (T alpha(1)) and thymosin T alpha(11) (T alpha(11)) are polypeptides with immunoregulatory properties first isolated from thymic extracts, corresponding to the first 28 and 35 amino acid residues, respectively, of prothymosin alpha (ProT alpha), a protein involved in chromatin remodeling. It has been widely supposed that these polypeptides are not natural products of the in vivo processing of ProT alpha, since neither was found in extracts in which proteolysis was prevented. Here we show that a lysosomal asparaginyl endopeptidase is able to process ProT alpha to generate T alpha(1) and T alpha(11). In view of its catalytic properties and structural and immunological analyses, this protease was identified as mammalian legumain. It selectively cleaves some of the asparaginyl-glycine residues in the ProT alpha sequence; specifically, Asn(28)-Gly(29) and Asn(35)-Gly(36) residues are cleaved with similar efficiency in vitro to generate T alpha(1) and T alpha(11), respectively. By contrast T alpha(1) is the main product detected in vivo, free in the cytosol, at concentrations similar to that of ProT alpha. The data here reported demonstrate that T alpha(1) is not an artifact but rather is naturally present in diverse mammalian tissues and raise the possibility that it has a functional role.     

Subtypes of alpha 1- and alpha 2-adrenergic receptors.

The adrenergic receptors are members of the superfamily of G protein-coupled receptors. There are three major types of adrenergic receptors: alpha 1, alpha 2, and beta. Each of these three major types can be divided into three subtypes. Within the alpha 1-adrenergic receptors, alpha 1A and alpha 1B subtypes have been defined pharmacologically on the basis of reversible antagonists, such as WB4101 and phentolamine, and the irreversible antagonist chloroethylclonidine. In at least some tissues the mechanism of action of the alpha 1A subtype is related to activation of a calcium channel, whereas the alpha 1B receptor exerts its effect through the second messenger inositol trisphosphate. Both of these receptor subtypes as well as a third, the alpha 1C, have been identified by molecular cloning. Three pharmacological subtypes of the alpha 2-adrenergic receptor have also been identified. Prototypic tissues and cell lines in continuous culture have been developed for each of these subtypes, which facilitated their study. The definition of the alpha 2 subtypes has been based on radioligand binding data and more limited functional data. All three subtypes have been shown to inhibit the activation of adenylate cyclase and thus reduce the levels of cAMP. Three alpha 2-adrenergic receptor subtypes have been identified by molecular cloning in both the human and rat species. There is reasonable agreement between the pharmacological identified subtypes and those identified by molecular cloning.     

Metabolic regulation by alpha 1- and alpha 2-adrenoceptors.

The role of alpha-adrenoceptors in the mediation of autonomic function, particularly in the control of the cardiovascular system, is widely known. However, alpha-adrenoceptors are also important in the regulation of a variety of metabolic processes that occur in the body either through direct action or by stimulation of the release of other mediators that control metabolic function. Thus, alpha 2-adrenoceptor activation by circulating or neuronally released catecholamines inhibits the release of insulin from pancreatic islet beta-cells and, by inhibiting this response, alpha 2-adrenoceptor antagonists have been shown to have an antihyperglycemic effect. The alpha-adrenoceptor-mediated regulation of the release of pituitary hormones is indirect, with alpha-adrenoceptors being located on peptidergic neurons in the hypothalamus that secrete releasing hormones into the hypophysial portal system to regulate the secretion of hormones from the anterior pituitary gland. Thus, the increase in cortisol secretion from the adrenal glands following a meal is produced, at least in part, by an alpha 1-adrenoceptor-mediated increase in vasopressin and CRF-41 secretion from neurons on the hypothalamus that stimulate the release of adrenocorticotrophic hormone secretion from the pituitary gland, which subsequently stimulates the synthesis and release of cortisol from the adrenal medulla. In addition to metabolic regulation by alpha 1- and alpha 2-adrenoceptors within the endocrine system, alpha-adrenoceptors are also a component of the system that regulates certain aspects of metabolism within autonomic effector cells, such as the control of smooth muscle cell division and growth during periods of continued alpha-adrenoceptor activation as a result of activation of second messenger systems.     

Differential susceptibility of alpha A- and alpha B-crystallin to gamma-ray irradiation

Alpha-crystallin, a major protein of all vertebrate lenses, consists of two different subunits, alpha A and alpha B, which form polymeric aggregates with an average molecular mass of 300-800 kDa. Both the alpha A and alpha B subunit have a molecular mass of about 20 kDa. It is not known why alpha crystallin aggregates comprise two different subunits, given that the physicochemical properties of these proteins are very similar. The present study compares the susceptibility of the alpha A and alpha B subunits to gamma-rays. We prepared a recombinant form of human alpha A- and alpha B-crystallin and then irradiated the proteins with gamma-rays. Based on far-UV CD spectra, alpha A-crystallin retained beta-sheet conformation after gamma irradiation up to 3.0 kGy, whereas alpha B-crystallin lost beta-sheet conformation upon exposure to gamma irradiation at >1.0 kGy. Size exclusion chromatography showed that the aggregation and polydispersity of recombinant alpha A-crystallin increased slightly after >1.0 kGy irradiation. In contrast, irradiation of alpha B-crystallin at 1.0 kGy resulted in the formation of huge aggregates and a marked increase in heterogeneity. We have also compared the chaperone activities of gamma-irradiated alpha A- and alpha B-crystallin aggregates. The activity of irradiated alpha A-crystallin was retained while that of the irradiated alpha B-crystallin was became inactive after irradiation of >0.5 kGy. Our results indicate that alpha A-crystallin is more stable to gamma irradiation than alpha B-crystallin.     

Unfolding intermediates in the triple helix to coil transition of bovine type XI collagen and human type V collagens alpha 1(2) alpha 2 and alpha 1 alpha 2 alpha 3

The thermal triple helix to coil transitions of two human type V collagens (alpha 1(2) alpha 2 and alpha 1 alpha 2 alpha 3) and bovine type XI collagen differ from those of the interstitial collagens type I, II, and III by the presence of unfolding intermediates. The total transition enthalpy of these collagens is comparable to the transition enthalpy of the interstitial collagens with values of 17.9 kJ/mol tripeptide units for type XI collagen, 22.9 kJ/mol for type V (alpha 1(2) alpha 2), and 18.5 kJ/mol for type V (alpha 1 alpha 2 alpha 3). It is shown by optical rotatory dispersion and differential scanning calorimetry that complex transition curves with stable intermediates exist. Type XI collagen has two main transitions at 38.5 and 41.5 degrees C and a smaller transition at 40.1 degrees C. Type V (alpha 1(2) alpha 2) shows two main transitions at 38.2 and 42.9 degrees C and two smaller transitions at 40.1 and 41.3 degrees C. Compared to these two collagens type V (alpha 1 alpha 2 alpha 3) unfolds at a lower temperature with two main transitions at 36.4 and 38.1 degrees C and two minor transitions at 40.5 and 42.9 degrees C. The intermediates present at different temperatures are characterized by resistance to trypsin digestion, length measurements of the resistant fragments after rotary shadowing, and amino-terminal sequencing. One of the intermediate peptides has been identified as belonging to the alpha 2 type V chain, starting at position 430 and being about 380 residues long. (The residue numbering begins with the first residue of the first amino-terminal tripeptide unit of the main triple helix. The alpha 2(XI) chain was assumed to be the same length as the alpha 1(XI). One intermediate was identified from the alpha 2(XI) chain and with starting position at residue 495, and three from the alpha 3(XI) with starting positions at residues 519, 585, and 618.     

Metabolism of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 26-tetrol and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 25-tetrol into cholic acid in normal human subjects.

Side chain oxidation and cleavage of precursors in cholic acid synthesis is thought to involve initial hydroxylation at either position 25 or 26 of the side chain. Therefore, the conversion of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 26-tetrol and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 25-tetrol into cholic acid was studied in normal subjects after single intravenous injections of these labeled alcohols. Eighty-six percent and 82% of 5 beta-cholestane, 3 alpha, 7 alpha, 12 alpha, 26-tetrol was converted into cholic acid in two subjects, respectively. However, only 14 and 16% of the injected 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 25-tetrol was converted into cholic acid in two subjects, respectively. Thus, this study indicates that 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 25-tetrol is an inefficient substrate for cholic acid biosynthesis in man and that the major route of cholic acid synthesis probably involves the 26-hydroxylated intermediate.