Biomedical subjects

D Puett

Publications and source records attributed to D Puett.

At least 55 records · Page 3Linked to original sources

PubMed1995

Binding of vanadium (IV) to the phosphatase calcineurin.

X-band electron spin resonance spectroscopy was used to study the binding of vanadium (IV), or vanadyl, to the brain serine/threonine phosphatase-2B, calcineurin. Spectra were determined on frozen solutions of vanadyl and calcineurin at pH 7.4 in the presence of 20% (v/v) glycerol. The binding of vanadyl to the enzyme was established, and the data suggested the presence of two classes of sites, the higher affinity class of which contained two binding sites for vanadyl. The calcium-binding B subunit of the heterodimeric protein was also shown to bind vanadyl. The holoprotein appeared to be stabilized by vanadyl, and vanadyl enhanced enzymatic activity when assayed with or without calmodulin in the absence of calcium.

Animals↗

PubMed1995

Presence of aromatase inhibitors in cycads.

Cycads, the most primitive of the living gymnosperms, have been used and continue to be used for food and medicinal purposes by many cultures, although toxins must be removed before ingestion. In our quest to identify tropical plants that contain inhibitors of the cytochrome P-450 aromatase and thus may be efficacious in treating estrogen-dependent tumors, we have screened extracts from 5 species of cycad folia encompassing 3 genera: Cycas cairnsiana F. Muell., Cycas revoluta Thunb., Cycas rumphii Miq., Dioon spinulosum Dyer and Encephalartos ferox Bertol. All extracts were found to contain inhibitors of the human enzyme.

Aromatase Inhibitors↗

PubMed1995

Trp-21 is important in the processing and secretion of big endothelin-1.

To investigate the intracellular processing of endothelin-1 (ET-1), the synthesis and secretion of preproET-1 (PPET-1) was studied in transiently transfected COS-7 cells. Replacements at the highly conserved C-terminal region of ET-1 were made by site-directed mutagenesis, with codons for residues Ile-20 and Trp-21 being replaced by one for Ala in the PPET-1 cDNA. The mutant and wildtype PPET-1 cDNAs were expressed in COS-7 cells following transient transfection, and cell media and extracts, collected after 48 h, were assayed for ET-1 and big ET-1 by radioimmunoassay. The concentration of immunoreactive ET-1 in the medium obtained from the Ala-21-ET-1 mutant was 10-fold lower than that from PPET-1 wildtype and (Ala-20-ET-1) PPET-1 transfected cells. Moreover, Ala-21-big ET-1 accumulated in the cells transfected with the cDNA for (Ala-21-ET-1) PPET-1, whereas there was no significant accumulation of ET-1-like or big ET-1-like immunoreactivity in the cells transfected with cDNAs for PPET-1 wildtype and (Ala-20-ET-1) PPET-1. These results suggest that Trp-21 is involved in intracellular processing and secretion of big ET-1.

Alanine↗

PubMed1995

Identification of receptor binding and activation sites in endothelin-1 by use of site-directed mutagenesis.

This study addresses the structural requirements for the intracellular processing and receptor binding properties of endothelin-1 (ET-1). Point mutants of preproendothelin-1 cDNA, with replacement of the codons for Lys9 of ET-1 by ones for Ala and Glu and of Ile20 and Trp21 by ones encoding Ala, were expressed in COS-7 cells. Competitive binding experiments on rat vascular smooth muscle cells (A-10), which were shown to be an ETA receptor-rich cell line, between [125I]ET-1 and synthetic ET-1, wild-type recombinant ET-1, and recombinant [Ala9]ET-1, [Glu9]ET-1, [Ala20]ET-1, and [Ala21]ET-1 yielded Ki values of 0.2 +/- 0.02, 0.2 +/- 0.02, 0.04 +/- 0.01, 1.4 +/- 0.2, 1.6 +/- 0.2, and > 50 nmol/L, respectively. In similar experiments with ETB receptor-rich human Girardi heart cells, the corresponding values were 0.2 +/- 0.03, 0.2 +/- 0.03, 0.2 +/- 0.04, 0.2 +/- 0.06, 1.4 +/- 0.4, and > 50 nmol/L. The ETA receptor-mediated contractile responses to [Glu9]ET-1 and [Ala20]ET-1, measured by using canine coronary artery rings, were decreased approximately fourfold to fivefold compared with the response produced by synthetic or wild-type recombinant ET-1, whereas [Ala9]ET-1 was found to be more potent, and [Ala21]ET-1 did not produce any contraction. These results demonstrate that Ile20 and Trp21 are involved in binding to both receptor subtypes. Of considerable interest was the observation that [Glu9]ET-1 also blunts the ETA receptor subtype-mediated contractile response to ET-1 stimulus.

Amino Acid Sequence↗

PubMed1995

Functional expression of yoked human chorionic gonadotropin in baculovirus-infected insect cells.

hCG is a glycoprotein hormone composed of an alpha-subunit, common to all gonadotropins and to TSH, and a hormone-specific beta-subunit. The non-covalent association of the two subunits is an obligatory step for the formation of biologically active hormones. The correct assembly of the heterodimer is also important for efficient secretion of the hormone, receptor binding, and signal transduction. Herein, we have demonstrated that expression of the two subunits from independent promoters present in a single recombinant baculovirus resulted in subunit association and secretion of biologically active holoprotein by the insect cells. To determine whether the active conformation of heterodimer could be achieved when the two subunits were synthesized in tandem on a single polypeptide chain, two single chain or yoked hCG1, the C-terminus of the complete beta-subunit (145 amino acid residues) was conjoined to the N-terminus of the alpha-subunit. Yoked hCG2 was similar, except that it contained the N-terminal 123 amino acid residues of the beta-subunit. Both yoked hCG molecules bound LH/CG receptor with high affinity and stimulated adenylate cyclase and progesterone levels in transformed mouse Leydig (MA-10) cells. Therefore, the alpha- and beta-subunits are able to fold into a biologically active conformation when covalently linked. Interestingly, when compared with urinary hCG, the hormone expressed in baculovirus-infected insect cells binds to the LH/CG receptor with higher affinity, but exhibits diminished signaling, thus providing another example of a partial dissociation between receptor binding and activation.

Animals↗

PubMed1995

Vanadium (IV) inhibits calmodulin-stimulated skeletal muscle myosin light chain kinase activity.

Vanadium, believed to be an essential trace metal, exhibits numerous biological effects. Using electron spin resonance spectroscopy, we have demonstrated that vanadyl, vanadium (IV), the predominant intracellular form of vanadate (vanadium V), binds to calmodulin in the presence of physiological concentrations of magnesium, extending earlier work which showed competitive binding of vanadyl and calcium to calmodulin. In the presence of a magnesium-containing buffer, vanadyl does not lead to calmodulin activation of the calmodulin-dependent enzyme, rabbit skeletal muscle myosin light chain kinase; in the presence of calcium, vanadyl is a potent inhibitor of the calmodulin-activated form of the kinase. Thus, vanadyl can potentially interfere with some of the intracellular actions of calcium, presumably via binding to calmodulin. This observation deserves consideration in view of the potential clinical application of vanadium treatment to mimick insulin action and lower blood glucose.

Animals↗

PubMed1994

On the role of the invariant glutamine at position 54 in the human choriogonadotropin beta subunit.

The twelve Cys and eight of the non-Cys residues are invariant in the glycoprotein hormone beta subunits from a variety of mammalian species. beta-Gin-54 of human lutropin (hLH) and choriogonadotropin (hCG) is one of these invariant amino acid residues. A single A-->G mutation in the LH beta gene of a patient presenting with hypogonadism resulted in the replacement of Gin-54 with Arg [1]. The authors also reported that an expressed mutant of hLH beta, with Arg replacing Gin-54, associated with the alpha subunit, but there was no demonstrable binding of the mutant hormone to receptor. We have replaced Gin-54 in hCG beta with Glu and with Lys using site-directed mutagenesis. The expression plasmids pRSV-hCG beta (wild-type and mutants) were transiently transfected into CHO cells containing a stably integrated gene for bovine alpha, and the media were analyzed for holoproteins, which were characterized in vitro using competitive binding and steroidogenic assays with MA-10 cells. hCG beta(Glu-54) bound to alpha almost as well as hCG beta wild-type, and the resulting heterodimer competed with [125l]hCG binding to the LH/CG receptor and stimulated progesterone production to the same extent as the wild-type control. However, the apparent potencies, as judged by ED50s, were less than those of the wild-type control, the effect being more pronounced in binding than in steroidogenesis. In contrast, hCG beta(Lys-54) associated very poorly with alpha. Our results suggest that while Gin-54 in hCG beta participates in receptor binding, its major function appears to involve alpha binding. Such dual functionality leads to interesting models for holoprotein formation and receptor binding.

Animals↗

PubMed1994

A transformed murine Leydig cell line expresses the ETA receptor subtype.

We recently demonstrated that transformed murine Leydig cells (MA-10) responded to endothelin-1 (ET-1) via increased steroidogenesis. This study addresses the endothelin receptor subtype present on this cell line and whether or not the cells produce ET-1. The expression of the preproendothelin-1 (PPET-1) gene was investigated by Northern blot analysis, and PPET-1 mRNA was found to be < 0.2% of that present in pulmonary endothelial cells. The medium from MA-10 cells, maintained under serum-free conditions, was analyzed by radio-immunoassay to determine immunoreactive-ET-1 production and ET-1 levels were found to be below the sensitivity of the assay (< 10 pg/ml). The data from competitive binding experiments with [125I]ET-1 and unlabeled ET-1, ET-3 and receptor subtype selective ligands yielded a single class of high affinity binding sites with ETA receptor subtype characteristics. The results of this study demonstrate that MA-10 cells possess the ETA receptor subtype but do not produce significant quantities of ET-1 under basal conditions.

Animals↗

PubMed1994

Identification of conserved amino acid residues in the beta subunit of human choriogonadotropin important in holoprotein formation.

The beta subunits of mammalian glycoprotein hormones contain 20 invariant amino acid residues, including 12 Cys residues that form six disulfide bonds, as well as other highly conserved residues. Such stringent conservation suggests an important role for those residues in chain folding, holoprotein formation, or receptor binding/activation, but not receptor specificity. Using site-directed mutagenesis, we have prepared and characterized replacements of 14 conserved non-Cys residues in human choriogonadotropin beta for which there is little, if any, information available: Pro7, Thr32, Tyr37, Thr40, Val56, Tyr59, Phe64, Pro70, Gly71, Ser81, Tyr82, Val84, Ala85, and Ser87. The cDNAs were subcloned into a pRSV mammalian expression vector and transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for bovine alpha. Holoprotein formation was assessed by radioimmunoassays, and in vitro competitive binding and steroidogenic assays were used to determine potencies. Our results demonstrated that Tyr37, Thr40, Tyr59, and Ala85 participate directly or indirectly in holoprotein formation, as may Ser81. Replacements of the other conserved amino acid residues resulted in no significant change in subunit assembly or receptor binding/activation as measured, although an Arg at position 81 or 82 reduced secretion. This study has identified several amino acid residues of the beta subunit that are important in alpha binding and has raised interesting questions on the constraints that maintain conservation of amino acid residues during evolution.

Amino Acid Sequence↗

PubMed1994

A label selection approach to assess the role of individual amino groups in human choriogonadotropin receptor binding.

Human choriogonadotropin (hCG) was trace-labeled with [3H]acetic anhydride and then incubated with transformed murine Leydig cells (MA-10). The bound hormone was recovered, subunits (alpha and beta) were separated and then cleaved, and the peptides were purified by high performance liquid chromatography. Analysis of the labeling patterns of peptides from the initial preparation and the bound hCG fraction enabled us to determine the protection factor of several amino groups, which is a measure of the effects of acetylation on receptor binding. The largest protection factors, indicating lower labeling in the bound fraction, were found on beta and involved the alpha-amino group/Lys2 (analyzed together) and Lys104, which exhibited 6-fold and 5-fold selections against binding, respectively. Thus, acetylation at either of these amino groups does not prevent binding but results in selection against hormone association with receptor. Other amino groups were analyzed (e.g. Lys122 on beta; the alpha-amino group and lysines 44/45 (analyzed as a pair), 51, and 75 on alpha), and the selection factors indicated either no change or only modest changes (2-fold) in selection for or against binding. These results indicate that the alpha-amino group/Lys2 and Lys104 of the hormone-specific beta subunit participate, either directly or indirectly, in receptor binding.

Acetylation↗

PubMed1994

Immunoreactive endothelin-1 concentrations in follicular fluid of women with and without endometriosis undergoing in vitro fertilization-embryo transfer.

OBJECTIVE: To determine the concentrations of immunoreactive (IR) endothelin-1 in human follicular fluid (FF) and whether IR-endothelin-1 levels are different in women with endometriosis-associated infertility. DESIGN: Follicular fluid and plasma samples, obtained from women with and without endometriosis undergoing IVF-ET, were collected at the time of oocyte aspiration and analyzed for IR-endothelin-1 levels. SETTING: Infertility clinic in an academic research environment. RESULTS: Overall, 90% of FF samples and 60% of plasma samples contained IR-endothelin-1 detectable above the threshold of assay sensitivity. Immunoreactive endothelin-1 levels (mean +/- SEM) in FF samples from women with and without endometriosis-associated infertility were 74 +/- 12 and 37 +/- 6 pg/mL, respectively. There was no difference in IR-endothelin-1 levels in FF samples between controlled ovarian hyperstimulation cycles with or without leuprolide acetate. No significant differences were detected in plasma IR-endothelin-1 levels in women with endometriosis-associated infertility when compared with those without. CONCLUSIONS: These results demonstrate the presence of IR-endothelin-1 in human FF obtained at the time of oocyte aspiration for IVF-ET and higher levels of IR-endothelin-1 in FF of women with endometriosis-associated infertility.

Adult↗

PubMed1994

Endothelin-1 promotes mitogenesis in airway smooth muscle cells.

Endothelin exists as three isoforms (ET-1, ET-2, and ET-3) and exhibits vasoconstricting, bronchoconstricting, and growth-promoting properties in vascular smooth muscle. In the airways, ET-1 immunoreactivity and mRNA have been detected and localized to the epithelium, smooth muscle, and endothelium in different species, including humans. It has been suggested that ET-1 may have a role in the airway smooth muscle hyperplasia and hypertrophy seen in patients with bronchial asthma. We studied ovine airway smooth muscle cells (SMC) in vitro and showed saturable binding of [125I]ET-1 with a dissociation constant (Kd) of 0.4 nM and high affinity binding sites (Bmax) for ET-1 (104 fmol/10(6) cells). This binding was functional as ET-1 promoted mitogenesis of these muscle cells as measured by increased cell number in the absence of serum. Twenty-four hours after exposing the cells to graded doses of ET-1 from 1 pM to 1 microM, cell number increased significantly over control in a dose-dependent manner. ET-1 also enhanced the transient expression of c-fos mRNA by 2.5-fold over control, with maximal expression occurring at 30 min. These observations provide evidence that: (1) airway SMC possess high affinity binding sites for ET-1, and (2) ET-1 is mitogenic for airway SMC as determined by increased cell number and amplification of c-fos mRNA expression. ET-1 may have a fundamental role in influencing the growth of smooth muscle in the airways.

Animals↗

PubMed1994

A region in the human glycoprotein hormone alpha-subunit important in holoprotein formation and receptor binding.

Using site-directed mutagenesis of the human glycoprotein hormone alpha-subunit, we have shown that single replacements of Ala36 and Pro38 with Glu and Asp, respectively, result in mutant subunits that do not bind significantly to hCG beta. In contrast, the replacement of Lys44 with Ala did not interfere with hCG beta binding, but the resulting holoprotein failed to exhibit high affinity binding to the LH/CG receptor. These results in conjunction with other data suggest that the region of human alpha between positions 33-45 contains several amino acid residues that participate in subunit binding and others that function in receptor binding.

Amino Acid Sequence↗

PubMed1994

Holoprotein formation of human chorionic gonadotropin: differential trace labeling with acetic anhydride.

The effects of holoprotein formation in human CG (hCG) on the reactivities of several of the individual amino groups of each subunit were investigated by differential trace labeling with [3H]acetic anhydride. The alpha- and beta-subunits were labeled separately, as was hCG, under conditions chosen to ensure that an average of less than one amino group was modified per molecule. Although the beta-subunit contains fewer amino groups than the alpha-subunit, most of the 3H incorporation occurred in beta at the N-terminal region. Chemical and enzymatic cleavage of the subunits enabled us to identify several individual amino groups and, from measurements of the incorporated radioactivity of the free subunits and intact hormone, determine their protection factor, which is a measure of the reactivity and thus of the local environment and changes thereof upon holoprotein formation. Lys51 and Lys91 of alpha were approximately 2-fold more reactive and less reactive, respectively, in the alpha beta complex than in the free subunit. The alpha-amino group of alpha was characterized by comparable reactivities in the heterodimer and free subunit, as was Lys44/Lys45 when analyzed as a pair; the reactivity of alpha-Lys44 was slightly less in the holoprotein than in the free subunit. The alpha-amino group and Lys2 of beta could not be resolved by available cleavage procedures; consequently they were analyzed as a pair and found to be some 2-fold less reactive in the heterodimer than in the free subunit, as was Lys104 of beta. From these results, we can conclude that subunit assembly produces changes in the microenvironments of several amino groups, attributable to steric effects, specific intermolecular interactions, and localized conformational changes. Analysis of these data with reference to the recently determined crystal structure of hydrogen fluoride-treated hCG enabled a distinction to be made of these possibilities for several of the amino groups.

Acetic Anhydrides↗

PubMed1994

Identification of a point mutation in the thyrotropin receptor of the hyt/hyt hypothyroid mouse.

The hyt/hyt hypothyroid mouse has an autosomal recessive, fetal-onset, severe hypothyroidism related to TSH hyporesponsiveness and associated with elevated TSH. Our previous work has suggested that the hypothyroidism and TSH hyporesponsiveness may result from a mutation in the hyt/hyt TSH receptor (TSHr) of the thyroid gland. Based on DNA sequencing of the entire coding region of the TSHr gene from the wild-type BALB/cBY +/+ mouse, the +/+ TSHr is 92% and 94% identical at the nucleotide and amino acid residue levels, respectively, compared to the rat TSHr gene. The coding region of the hyt/hyt TSHr, compared to that of the +/+ TSHr, has a single base change, CCG to CTG, at nucleotide position 1666, which leads to the replacement of a highly conserved proline at amino acid position 556 with a leucine in transmembrane domain IV. This mutation was introduced by site-directed mutagenesis into the wild-type human TSHr and transiently expressed in COS-7 cells. Although the size and abundance of the mutant TSHr mRNA suggested that there was no effect on the nature of the mRNA, TSH binding and the response to TSH in transfected cells were abolished. Further studies are necessary to clarify how the Pro to Leu replacement interferes with receptor expression on the cell surface or influences TSH binding. These functional consequences of the mutation appear to account for the observed TSH hyporesponsiveness and hypothyroidism in the hyt/hyt mouse.

Amino Acid Sequence↗

PubMed1993

Amino/carboxyl-terminal deletion mutants of human choriogonadotropin beta.

Human choriogonadotropin (hCG) contains an alpha subunit common to other members of the glycoprotein hormone family, lutropin (LH), follitropin, and thyrotropin, and a hormone-specific beta subunit. hCG beta contains a carboxyl-terminal extension of 25-30 amino acid residues not present in the other beta subunits; also, CG beta and lutropin beta have an additional 6 or 7 amino-terminal residues that are not present in follitropin beta and thyrotropin beta. To delineate the contribution of these extensions in hCG beta, site-directed mutagenesis was used to prepare several deletion fragments. Plasmids containing cDNAs for wild-type and mutant hCG beta were transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for bovine alpha. Medium from the transfected cells was used in two in vitro assays with a transformed murine Leydig cell line, MA-10. The deletion fragments, des(1-7), des(111-145), and des(1-7, 111-145), associated with alpha as well as hCG beta wild-type; moreover, the potencies of the three mutant hormones were comparable to that of control. In contrast, des(1-7, 101-145)hCG beta yielded very little heterodimer, although that which formed was partially active. These results define the shortest known core fragment of hCG beta, amino acid residues 8-110, that retains significant functionality in vitro.

Animals↗

PubMed1993

Inhibition of growth and appearance of estrogen-dependent rat mammary tumors by 10-propargylestr-4-ene-3,17-dione, an aromatase inhibitor.

The aromatase inhibitor 10-propargylestr-4-ene-3,17-dione (PED) has been evaluated in vivo as an anticancer agent. Prolonged administration of PED to rats bearing dimethylbenzanthracene-induced mammary tumors resulted in significant regression of hormone-responsive tumors within several days. Greater than 50% regression was generally observed after 14 days of treatment, irrespective of dose (1, 5, or 50 mg/kg body weight/day). In addition to tumor regression, a significantly increased incidence in tumor stasis was observed over the course of PED treatment. While all doses of PED examined were equipotent for both tumor regression and stasis, a dose-dependent inhibition of new tumor formation was observed in PED-treated rats. In control animals an average of 1.2 new tumors was observed during the experimental period; in contrast, averages of 0.5 tumors appeared in animals receiving 1 mg PED/kg body weight/day, 0.1 tumors at 5 mg/kg, and at 50 mg of PED/kg body weight/day, no new tumors occurred during the time PED was administered. The effects of PED on both regression of existing tumors and appearance of new tumors were reversed by co-administration of estradiol. Thus, PED impairs estrogen-dependent mammary tumor growth, resulting in cessation of new growth and regression of responsive tumors.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1993

Replacement of the invariant tyrosine in the CAGY region of the human chorionic gonadotropin beta subunit.

The mammalian glycoprotein hormone beta subunits contain a highly conserved amino acid sequence, Cys-Ala-Gly-Tyr-Cys (residues 34-38 of human chorionic gonadotropin beta), that is denoted as the 'CAGY region'. Using site-directed mutagenesis we have replaced Tyr-37 in hCG beta, i.e., the invariant Tyr in all known mammalian CG, LH, FSH, and TSH beta subunits, with two hydrophobic amino acid residues, Phe and Leu. The resultant mutant forms were characterized for alpha subunit binding and the resulting heterodimers were analyzed for biological activity using two in vitro assays with transformed murine Leydig cells (MA-10). Chinese hamster ovary cells containing a stably integrated gene for bovine alpha were transiently transfected with a eukaryotic expression vector containing a Rous sarcoma viral promoter and the wild-type and mutant cDNAs. The hCG beta(Phe-37) mutant bound to alpha essentially to the same extent as hCG beta wild-type, while the hCG beta(Leu-37) mutant formed somewhat less heterodimer. The heterologous heterodimeric mutant and wild-type gonadotropins were equipotent in a competitive binding assay with [125I]hCG. In a steroidogenic assay, the mutant hormones were active, but they appeared slightly less potent than the wild-type form. Thus, this invariant Tyr can be replaced with another aromatic amino acid residue or with a hydrophobic, but not aromatic amino acid residue in hCG beta without any dramatic effect on function. These results indicate that Tyr-37 in hCG beta, while not obligatory, may participate, either directly or indirectly, in subunit assembly and that the hydroxyl group may function in a modulatory role in signaling.

Amino Acid Sequence↗