Biomedical subjects

T H Ji

Publications and source records attributed to T H Ji.

At least 19 recordsLinked to original sources

PubMed1992

Electrofractionation: a technique for detecting and recovering biomolecules.

Electrofractionation (EF) is a technique that allows electrophoretic materials to be detected and recovered following electrophoresis. The EF apparatus utilizes the resolving power of electrophoresis and the mobile phase of liquid chromatography to create a continuous elution system. Our data indicate that EF is able to detect and recover oligonucleotides, as DNA fragments, proteins, and presumably other material that can be analyzed by electrophoresis. EF shares functional similarities with high-performance electrophoresis chromatography (HPEC) but operates by a different strategy and at a fraction of the cost. Moreover, EF can be constructed largely from standard laboratory equipment. The simplicity, rapid analysis times, low cost, and high recovery yields of EF make this system a practical alternative to the conventional detection and purification methods used for biomolecules.

Chemical Fractionation↗

PubMed1991

Conversion of lysine 91 to methionine or glutamic acid in human choriogonadotropin alpha results in the loss of cAMP inducibility.

Human choriogonadotropin (hCG) is a heterodimeric glycoprotein hormone. The alpha subunit comprises 92 amino acids, of which 6 are Lys residues (Morgan, F.G., Birken, S., and Canfield, R.E. (1975) J. Biol. Chem. 250, 5247-5258). Our photoaffinity-labeling studies indicate that several of these Lys residues make contact with the lutropin receptor and are covalently cross-linked to the receptor. Lys-91 of the alpha subunit is of interest because deletion of the two alpha C-terminal residues, Lys-91 and Ser-92, results in a significant reduction in the bioactivity of lutropin and thyrotropin (Cheng, K.-W., Glazer, A.N., and Pierce, J.G. (1973) J. Biol. Chem. 248, 7930-7937). To determine the importance of Lys-alpha 91, we substituted it with Arg, Met, or Glu. The resulting mutant alpha cDNA constructs were co-transfected into CHO cells with the wild type hCG beta cDNA construct. Secreted hCG dimers were assayed for binding to receptors on porcine granulosa cells and stimulation of cAMP synthesis in a murine Leydig tumor cell line. The natural hCG, wild type hCG, and all mutant hCGs recognized the receptor, although with somewhat divergent affinities. However, there was a striking difference in the ability of cAMP induction. The natural hCG, wild type hCG, and Lys-91----Arg mutant hCG induced cAMP synthesis, whereas the Lys-91----Met and Lys-91----Glu mutants did not. These results demonstrate that Lys-91 is important for receptor modulation in the stimulation of cAMP synthesis.

Animals↗

PubMed1991

Asp383 in the second transmembrane domain of the lutropin receptor is important for high affinity hormone binding and cAMP production.

The lutropin (LH), follitropin, and thyrotropin receptors belong to the superfamily of G-protein coupled receptors and have some unique structural features. These glycoprotein hormone receptors comprise a C-terminal half and an N-terminal half of similar size. The C-terminal half is equivalent to the entire structure of other G-protein coupled receptors and has seven transmembrane domains, three cytoplasmic loops, three exoplasmic loops, and a C terminus. In contrast, the hydrophilic N-terminal half is exoplasmic and unique to the glycoprotein hormone receptors. This large N-terminal half of the LH receptor has recently been shown to be capable of binding the hormone. Therefore, these glycoprotein hormone receptors are structurally and functionally different from other G-protein coupled receptors. In an attempt to define the role of the membrane-associated C-terminal half of the LH receptor, we have prepared several mutant receptors in which an Asp or Glu in the seven transmembrane domains has been converted to Asn or Gln, respectively. These include Asp383----Asn in the second transmembrane domain, Glu410----Gln in the third transmembrane domain, and Asp556----Asn in the sixth transmembrane domain. All these mutant receptors were successfully expressed in Cos 7A cells. The Glu410----Gln and Asp556----Asn mutants maintained normal affinities for hormone binding and cAMP production, but the Asp383----Asn mutant showed significantly lower affinities. Although Asp383 of the LH receptor is conserved in all G-protein coupled receptors cloned to date except the substance P receptor, which has Glu in the place of the Asp residue, this is the first observation of the critical role of the Asp in hormone binding and subsequent stimulation of cAMP production.

Amino Acid Sequence↗

PubMed1991

Human choriogonadotropin binds to a lutropin receptor with essentially no N-terminal extension and stimulates cAMP synthesis.

The lutropin (LH) receptor, which belongs to the family of G-protein coupled receptors, consists of an extracellular hydrophilic N-terminal extension of 341 amino acids and a membrane-embedded C-terminal region of 333 amino acids. This C-terminal region comprises a short N terminus, seven transmembrane domains, three cytoplasmic loops, three exoplasmic loops, and a C terminus. Recently, it was reported that the N-terminal extension of the LH receptor alone or a naturally occurring variant LH receptor similar to the N-terminal extension is capable of binding the hormone with an affinity slightly higher than that of the native receptor. This finding raises a question as to whether the N-terminal extension represents the entire hormone binding site and, if so, how is hormone binding transduced to the activation of a G-protein? In an attempt to answer this important question, we have prepared truncated receptors containing an N-terminal extension as short as 10 amino acids. Surprisingly, the truncated receptors were not only capable of binding the hormone, albeit with low affinities, but also capable of stimulating cAMP synthesis. These results suggest a possibility that the hormone, at least in part, interacts with the membrane-embedded C-terminal region and modulates it to activate adenylate cyclase. The low hormone binding affinities of the truncated receptors taken together with high affinity hormone binding to the N-terminal extension of the LH receptor indicate the existence of two or more contact points between the receptor and the hormone.

Amino Acid Sequence↗

PubMed1991

Analyses of ovine corpora lutea for tumor necrosis factor mRNA and bioactivity during prostaglandin-induced luteolysis.

It has been suggested that tumor necrosis factor (TNF) participates in the mechanism of regression of the corpus luteum. We measured luteal expression of TNF alpha mRNA and biological activity during prostaglandin-induced luteolysis in sheep. Initiation of functional luteolysis was marked by a sharp decline in concentrations of progesterone in luteal tissue beginning 4 h after administration of luteolysin. Structural regression of corpora lutea was manifested by a reduction in glandular weight at 16 h. A luteal cytotoxic factor with TNF alpha-like bioactivity was isolated after the decrease in tissue progesterone had occurred, but before evidence of luteal resorption. We were unable to detect temporal alterations in TNF alpha mRNA in luteal samples by classical Northern blot or in situ hybridization analyses. These results imply that luteal TNF alpha is derived primarily as a preformed entity from an extraovarian source, such as infiltrating leukocytes. These results raise the possibility that this cytokine might not be involved in the early stages of luteal regression in the ewe, yet could play a secondary role, perhaps in the subsequent opsonization and removal of degenerating cells.

Animals↗

PubMed1991

In situ hybridization analysis of ovarian prostaglandin endoperoxide synthase mRNA throughout the periovulatory period of the ewe.

Cellular alterations in level of expression of mRNA encoding for prostaglandin endoperoxide synthase were quantified within ovarian tissues of sheep obtained before, during and after induction of the preovulatory surge of LH and ovulation with LHRH. This was accomplished by isotopic in situ hybridization using a selective cRNA probe to ovine prostaglandin endoperoxide synthase mRNA. A significant elevation in mRNA was detected within the theca interna of the preovulatory follicle at 8, 16 and 24 hr following administration of LHRH. Very close to the time of ovulation (ie., at 24 hr post-LHRH) a marked rise in mRNA was observed in association with epithelial cells covering the apical surface of the follicle. Ovarian cyclooxygenase metabolites of arachidonic acid produced during the ovulatory process in the ewe originate within the thecal layer and germinal epithelium of the follicle destined to ovulate.

Animals↗

PubMed1991

Structure of the luteinizing hormone receptor gene and multiple exons of the coding sequence.

The genomic structure of the LH receptor is important to our understanding of its expression mechanisms, functional domains, relationships with other hormone receptors, and evolution. We have isolated four overlapping cosmid clones and six subgenomic clones of the rat LH receptor gene. They span a total of 95.6 kilobases (kb) and extend from 23 kb upstream of the translation start site to 13 kb down-stream of the stop codon. In addition, part of the human LH receptor gene has been isolated. The coding region of the rat hormone receptor gene spans over 60 kb and consists of 11 exons and 10 introns. Southern blots hybridized with exon 1 and exon 11 probes as well as gene dose analyses demonstrate that a single copy gene encodes the rat LH receptor. Sequence comparison suggests that the porcine and human LH receptor genes have similar, if not identical, exon-intron structures. There is no consensus cAMP-responsive element within 600 basepairs up-stream of the translation start site in spite of the cAMP responsiveness of the LH receptor gene. There are, however, unconventional cAMP-responsive elements in the region: one which is identical, several which are homologous to the activating protein-2-binding elements, CCCCAGGC, and several sequences which are similar to the G-rich cAMP-responsive element found in P450c21, a steroid 21-hydroxylase. The first 10 exons encode the N-terminal half of the molecule, while exon 11 encodes the C-terminal half of the molecule. This last exon is the same in the rat and human genes. The DNA and amino acid sequences of the first 10 exons show significant similarities and reveal repetitive sequence motifs. They have similar sizes which occur in the range of 69 and 183 bases; 8 of them are from 69-81 bases. Despite these remarkable similarities, structural predictions of exons 1-10 show a diversity of structures. The N-terminal half of the LH receptor appears to have a folded structure, with frequent turns and an extensive surface area. Part of the surface is predicted to be covered by amphiphilic helices and beta structures, types of secondary structure frequently found at the interfaces between subunits or between 2 interacting molecules. The introns dividing these exons also share many similarities.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Exons 1-10 of the rat LH receptor encode a high affinity hormone binding site and exon 11 encodes G-protein modulation and a potential second hormone binding site.

We have reported that the rat LH receptor is encoded by 11 exons of a single copy gene. Exons 1-10 encode the N-terminal half and exon 11 the C-terminal half. Since exon splice sites often mark structural transitions of multiexon molecules, we have attempted to define the function of the exons by generating mutant receptors with missing exons. As a first step, we have constructed two LH mutant receptors, one containing exons 1-10 (LH receptor (exon)1-10) and the other containing exon 1 and exon 11 (LH receptor(exon)1&11). These mutant receptors were functionally expressed in Cos 7A cells. The LH mutant receptor(exon)1-10, which lacks the membrane associated C-terminal half of the receptor, showed a high affinity for hCG. Surprisingly, the LH mutant receptor(exon)1&11 recognized hCG with a low affinity and stimulated G-proteins and cAMP production. The results demonstrate that exons 1-10 encode a high affinity hCG binding site and proves an important hypothesis that exon 11 encodes the site for receptor-modulation to activate G-proteins. Furthermore, the results raises an intriguing possibility of a second hormone binding site in the C-terminal half and multistep hormone binding.

Animals↗

PubMed1990

Increased production of ovarian thromboxane in gonadotropin-treated immature rats: relationship to the ovulatory process.

Thromboxane (TX) B2, a stable metabolic product of hydrolysis of TXA2, was measured by radioimmunoassay in tissue extracts of ovaries of immature rats pretreated with pregnant mare's serum gonadotropin and human chorionic gonadotropin. Ovarian concentrations of TXB2 increased before, and remained elevated after, the time of ovulation. In a subsequent study, ovulation was inhibited in a dose-dependent fashion by a reported TXA2 receptor antagonist, AH23848. Nevertheless, inhibition of the preovulatory rise in synthesis of TXB2 by furegrelate (a thromboxane synthetase inhibitor) did not prevent ovulation. Nor was the blockade of ovulation caused by indomethacin (a cyclooxygenase inhibitor) reversed by a TXA2 mimetic (U-46619). It does not appear that a preovulatory increase in ovarian thromboxane is an obligatory component of the ovulatory mechanism of gonadotropin-primed immature rats.

Animals↗

PubMed1990

Progesterone induces expression of endometrial messenger RNA encoding for cyclooxygenase (sheep).

Exogenous progesterone given early in the ovine estrous cycle results in precocious luteolysis. It has been suggested that under this circumstance regression of the corpus luteum is caused by an advancement in the timing of uterine secretion of prostaglandin F2 alpha. Uterine tissues were obtained from ewes following administration of progesterone (or injection vehicle), fixed in paraformaldehyde, and embedded in paraffin. Tissue sections were hybridized using an 35S-labeled cRNA probe specific for cyclooxygenase mRNA. There was approximately an eight-fold increase in the level of hybridization signal, localized mainly to uterine glands, consequential to treatment with progesterone. Thus, progesterone appears to control expression of the endometrial gene and(or) the stability of the message encoding for a rate-limiting enzyme involved in metabolism of arachidonic acid to its luteolytic product, thereby resolving the length of the nonpregnant cycle.

Animals↗

PubMed1990

Differential interactions of human choriogonadotropin and its antagonistic aglycosylated analog with their receptor.

Human choriogonadotropin (hCG) is a heterodimeric hormone consisting of an alpha subunit and a beta subunit. hCG and aglycosylated hCG (aghCG) have similar receptor binding affinities but differ in their ability to activate hormone-responsive adenylate cyclase. aghCG is an effective antagonist. The mechanisms of this antagonism and interactions of antagonistic aghCG with the receptor are not understood. To address this critical question, we have examined the interaction of this hormone analog with the receptor. The hormone receptor on porcine granulosa cells is a glycoprotein of 86 kDa and thas three domains of 24 kDa, 28 kDa, and 34 kDa, which are disulfide-linked. They undergo proteolysis, particularly when bound to the hormones, to produce three polypeptide components. These three receptor components can readily be identified through the use of affinity labeling with the hormones. Affinity labeling with an amino-specific homobifunctional reagent and subsequent cleavage indicate that hCG is cross-linked directly to the 24-kDa receptor component. In contrast, aghCG is cross-linked directly to the 34-kDa component. The peptide map of the cross-linked aghCG-34-kDa receptor component produced by papain treatment is different from the peptide map of the cross-linked complex of hCG-24-kDa component. This difference in receptor binding may be a factor determining the success or failure of signal transduction from the receptor to the effector system, guanine nucleotide-binding regulatory protein, and adenylate cyclase.

Affinity Labels↗

PubMed1990

N-linked oligosaccharides are not required for hormone binding of the lutropin receptor in a Leydig tumor cell line and rat granulosa cells.

We have examined roles of carbohydrates of the lutropin receptor in a murine Leydig tumor cell line (MLTC) and primary cultures of rat granulosa cells. We approached this issue by deglycosylating mature receptors with glycosidases and by preventing glycosylation of nascent receptors with tunicamycin B2, an inhibitor of protein glycosylation but not protein synthesis. Deglycosylation of mature receptors with neuraminidase, N-glycanase or both did not affect ligand binding capacity. Regardless of glycosidase treatment, the number of hormone binding sites was similar. The Kas for native receptors, asialoreceptors and aglycoreceptors, are also comparable, being 2.0 x 10(9) M-1, 1.9 x 10(9) M-1 and 1.7 x 10(9) M-1 respectively. In contrast, cells treated with tunicamycin B2 failed to bind the hormone. These results demonstrate that N-oligosaccharides of mature lutropin receptors are not required for ligand binding. In addition, our data suggest, for the first time, that N-glycosylation of the receptor may be necessary for expressing functional receptors on the cell surface and that there exist striking similarities in roles of oligosaccharides of lutropin and its receptor.

Amidohydrolases↗

PubMed1989