| 1. |
- Jia, X C, et al.
(författare)
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Expression of human luteinizing hormone (LH) receptor : interaction with LH and chorionic gonadotropin from human but not equine, rat, and ovine species.
- 1991
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Ingår i: Molecular Endocrinology. - 0888-8809 .- 1944-9917. ; 5:6, s. 759-68
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Tidskriftsartikel (refereegranskat)abstract
- Studies on human LH receptors are difficult due to the limited availability of clinical samples. Recent cloning of rat and porcine LH receptor cDNAs indicated that these binding sites are single polypeptides of the G-protein-coupled receptor family with seven transmembrane domains. Based on the conserved sequences of rat and porcine receptors, we performed reverse transcription polymerase chain reaction, using human ovarian mRNA as template and obtained partial human LH receptor cDNA clones. Further screening of a human ovary cDNA library and subsequent ligation of individual cDNA clones generated a human LH receptor cDNA containing the entire amino acid-coding region. Sequence analysis indicated that the human receptor cDNA displays 89% and 82% homology at the nucleotide level with its porcine and rat counterparts, respectively. A region spanning the second extracellular and third transmembrane domains is highly conserved among the human LH, FSH, and TSH receptors. The ovarian LH receptor clone is, however, significantly different from an incompletely spliced LH receptor cDNA recently obtained from a human thyroid library. Unlike the thyroid clone, the ovarian LH receptor cDNA could be expressed in the human fetal kidney cell line (293), and radioligand receptor assay identified high affinity (Kd, 1.2 x 10(-10) M) LH/hCG-binding sites on the plasma membrane. Binding specificity of the human LH receptor was studied using recombinant human CG, LH, and FSH secreted by CHO cells transfected with the respective genes. Human CG and LH displaced [125I]hCG binding with an ED50 of 4.3 and 4.8 ng/ml, respectively. In contrast, recombinant FSH was not effective. Treatment of transfected cells with recombinant gonadotropins also induced dose-dependent increases in extracellular cAMP production (hCG = LH much greater than FSH; ED50 25, 10, and greater than 3000 ng/ml). Although equine, rat, and ovine LH as well as equine CG competed effectively for rat testicular LH receptor binding, these hormones were unable to displace [125I]hCG binding to the human receptor, suggesting evolutionary changes in receptor binding specificity and the importance of using human receptors for clinical studies. Thus, the cloning and expression of the human LH receptor cDNA allowed analysis of interactions between human LH receptor and gonadotropins from diverse species. The present work should provide the basis for future design of therapeutic agents capable of interacting with the human receptor and for understanding the structural basis for LH receptor binding to different gonadotropins.
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| 2. |
- Jia, X C, et al.
(författare)
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Luminescence luteinizing hormone/choriogonadotropin (LH/CG) bioassay : measurement of serum bioactive LH/CG during early pregnancy in human and macaque.
- 1993
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Ingår i: Biology of Reproduction. - 0006-3363 .- 1529-7268. ; 49:6, s. 1310-6
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Tidskriftsartikel (refereegranskat)abstract
- Because of the microheterogeneities of gonadotropins, measurement of immunoreactivity of these glycoproteins does not necessarily reflect changes in their bioactivity. In addition, LH bioactivities in human samples analyzed by a rodent LH bioassay have been discordant with findings based on human granulosa-luteal cells. We have isolated a human LH/choriogonadotropin (CG) receptor cDNA and expressed the recombinant protein. Using 293 cells permanently transfected with the human LH receptor cDNA and a luciferase reporter gene driven by a cAMP-dependent promoter, we have developed a luminescence LH/CG bioassay. After cells were treated with human LH or CG for 20 h, luciferase activity was measured through use of a luminometer. Luciferase activity in the cells was increased in a dose-dependent manner. In contrast, treatment with FSH, thyroid-stimulating hormone, prolactin, growth hormone, adrenocorticotropin, insulin, prostaglandins, and several neurotransmitters had no effect. Because treatment with basic fibroblast growth factor (bFGF) caused significant increases in basal luciferase activity, a fixed amount of bFGF was included in all reactions. Incubation with 0.1 to 30 microliters serum from women during different physiological states stimulated the luciferase activity in parallel with the hCG standard curve. In 4 conception cycles, bioactive LH/hCG levels began to increase 2 wk after the midcycle LH surge, followed by a logarithmic increase from 22 days on. Due to the lack of a homologous RIA for measuring CG levels in monkeys, we analyzed serum bioactive monkey CG (mCG) in macaque during early pregnancy. Bioactive mCG was detected about 12 days after the midcycle LH surge and fertile mating and persisted until Days 21-23, followed by a decline.(ABSTRACT TRUNCATED AT 250 WORDS)
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| 3. |
- Jia, X C, et al.
(författare)
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Synergistic effect of glucocorticoids and androgens on the hormonal induction of tissue plasminogen activator activity and messenger ribonucleic acid levels in granulosa cells.
- 1990
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Ingår i: Molecular and Cellular Endocrinology. - 0303-7207 .- 1872-8057. ; 68:2-3, s. 143-51
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Tidskriftsartikel (refereegranskat)abstract
- Tissue-type plasminogen activator (tPA) is secreted by rat granulosa cells in response to treatment with activators of protein kinase A (follitropin, FSH), protein kinase C (gonadotropin-releasing hormone, GnRH) and tyrosine kinase (epidermal growth factor, EGF). Because steroid hormones have been shown to enhance the gonadotropin stimulation of ovarian differentiation, we investigated the effects of steroid hormones, alone or together with various kinase activators, on tPA activities and mRNA levels in cultured rat granulosa cells. Treatment of cells with dexamethasone (DEX; a glucocorticoid agonist) or R1881 (an androgen agonist) caused an increase in tPA secretion and mRNA levels. In addition, the stimulation of tPA activity and mRNA levels by FSH (50 ng/ml) was synergistically enhanced by cotreatment with DEX or R1881 in a time-dependent manner with 2.8- and 1.6-fold increase at 9 h after incubation as compared to cells treated with FSH alone. In contrast, treatment with diethylstilbestrol had no effect on tPA levels. Furthermore, tPA activity and mRNA levels induced by GnRH and EGF were also increased by cotreatment with DEX or R1881 as compared with cells treated with GnRH or EGF alone. Likewise, the stimulation of tPA mRNA levels by dibutyryl cAMP, a protein kinase A activator, and phorbol myristate acetate (PMA), a protein kinase C activator, was enhanced by cotreatment with DEX or R1881. These results demonstrate that glucocorticoid and androgen enhance tPA secretion and mRNA levels stimulated by FSH, GnRH and EGF in granulosa cells. The rat granulosa cells provide a useful model for studying the mechanism of regulation of tPA gene expression by steroid hormones.
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| 4. |
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| 5. |
- Li, Jia-Yi, et al.
(författare)
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GAP 43-like immunoreactivity in normal adult rat sciatic nerve, spinal cord, and motoneurons: axonal transport and effect of spinal cord transection
- 1993
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Ingår i: Neuroscience. - 0306-4522. ; 57:3, s. 759-76
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Tidskriftsartikel (refereegranskat)abstract
- Using immunofluorescence and cytofluorimetric scanning techniques in the rat, the fast anterograde and retrograde axonal transport of growth-associated protein-43-like immunoreactivity in normal sciatic nerves, and after spinal cord transection in the lower thoracic region, were investigated. Spinal roots and motor endplates in the peroneal muscles were also studied. For comparison, anti-synaptophysin (p38) was used. In intact adult animals, the amounts of immunoreactive growth-associated protein-43 increased linearly, both proximally and distally to the crush site, between 1 and 24 h after crushing the sciatic nerve. The accumulations were present in thick as well as in thin axons. Distal accumulations in the sciatic nerve were about 40-60% of the proximal amounts, indicating a recycling of organelles with growth-associated protein-43-like immunoreactivity. During the week after spinal cord transection, no clear changes were observed; the anterograde transport of growth-associated protein-43-like immunoreactivity showed a tendency to decrease at day 1 and then a tendency to increase, reaching 120% of control at seven days (not significant). Transported p38-like immunoreactivity showed similar but smaller changes. In the lumbar spinal cord gray matter many nerve terminals with growth-associated protein-43-like immunoreactivity were seen in intact animals. After spinal transection, these terminals gradually decreased, suggesting that they belonged to descending pathways. However, p38-positive terminals were not obviously decreased. After crushing ventral and dorsal roots, accumulations of pf growth-associated protein-43-like immunoreactivity were present in thick axons in the ventral roots and in thin to medium-sized axons in the dorsal roots. In peroneal muscles, growth-associated protein-43-like immunoreactivity was present in some (but not all) motor endplates in all groups. These results indicate that: (i) growth-associated protein-43 is normally present in nerve terminals of many descending projections of the spinal cord; (ii) growth-associated protein-43-like immunoreactivity is expressed and bidirectionally transported in neurons (motor as well as sensory) of normal sciatic nerves; (iii) growth-associated protein-43-like immunoreactivity is present in some adult motor endplates; and (iv) inhibited supraspinal input causes minor, if any, alterations--paralleled by p38--in axonal transport of growth-associated protein-43-like immunoreactivity.
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| 6. |
- Li, Jia-Yi, et al.
(författare)
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Influence of spinal cord transection on the presence and axonal transport of CGRP-, chromogranin A-, VIP-, synapsin I-, and synaptophysin-like immunoreactivities in rat motor nerve
- 1992
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Ingår i: J Neurobiol. - 0022-3034. ; 23:8, s. 1094-110
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Tidskriftsartikel (refereegranskat)abstract
- Using immunofluorescence and cytofluorimetric scanning (CFS), we investigated the short-term (1-7 days) influence of lower thoracic spinal cord transection on lumbar motor neurons. The content of calcitonin gene-related peptide- (CGRP) like immunoreactivity (LI), chromogranin A (Chr A)-LI, vasoactive intestinal polypeptide (VIP)-LI, Syn I-LI, and synaptophysin (p38)-LI in motor perikarya, and the anterograde and retrograde axonal transport of these substances in the sciatic nerve, were studied in nerve crush (6 h) experiments. During the week after transection, CGRP-LI in perikarya decreased, whereas Chr A-LI increased. VIP-LI, co-localized with Chr A-LI in motor perikarya, did not change after transection. The antero- and retrograde transport of CGRP-LI in the sciatic nerve, occurring in both motor and sensory axons, appeared unchanged in cytofluorimetric scanning (CFS) graphs, but the microscopical picture clearly showed that large motor axons had a decreased content of CGRP-LI at 3 and 7 days posttransection, whereas thinner axons were unchanged in fluorescence intensity. The anterograde transport of Chr A-LI, present in both motor and postganglionic adrenergic axons, was decreased 1 and 3 days after lesion, but returned to control by day 7. There was a marked decrease in anterograde transport of VIP-LI, present mainly in postganglionic sympathetic axons, at day 3, but at 7 days transport was normal. The amounts of transported p38, the synaptic vesicle marker, were in the normal range during the whole period. Syn I-LI accumulation anterogradely was somewhat decreased at 3 and 7 days posttransection, and at 1 day the retrograde accumulation was significantly increased. The results suggest that removal of supraspinal input to intact lower motor neurons causes alterations in metabolism and axonal transport of organelle-associated substances, partly probably related to the complex pattern of transmitter leakage from degenerating, descending nerve terminals. These alterations appear to take place also in postganglionic sympathetic neurons in the sciatic nerve, that originate in the lumbar sympathetic chain.
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