Kwan-Sik Min

Hypoglycemic Activity of Gymnema sylvestre Extracts on Oxidative Stress and Antioxidant Status in Diabetic Rats

Diabetes mellitus, which is associated with oxidative damage, has a significant impact on health, quality of life, and life expectancy. An ethanol extract of Gymnema sylvestre leaf was examined in vitro and in vivo to investigate the role of antioxidants in diabetic rats. The extract exhibited strong antioxidant activity in the assays, including TBA (56%), SOD-like (92%), and ABTS (54%). Blood glucose levels in the diabetic rats fed G. sylvestre extract decreased to normal levels. The presence of the antihyperglycemic compounds gymnemagenin and gymnemic acids in G. sylvestre extract was detected by LC/MS analysis. Lipid peroxidation levels were decreased by 31.7% in serum, 9.9% in liver, and 9.1% in kidney in the diabetic rats fed the extract. Feeding G. sylvestre extract to the diabetic rats decreased the activity of glutathione peroxidase in cytosolic liver and glutamate pyruvate transaminase in serum to normal levels.

Equine Follicle-Stimulating Hormone: Molecular Cloning of β Subunit and Biological Role of the Asparagine-Linked Oligosaccharide at Asparagine56 of α Subunit

Abstract Equine FSH (eFSH) and eCG are members of the glycoprotein hormone family. These proteins are heterodimeric, composed of noncovalently associated α and β subunits. We have previously reported that recombinant eCG has potent LH- and FSH-like activities and that the oligosaccharide at Asn56 of the α subunit plays an indispensable role in expressing LH- but not FSH-like activity. In the present study, we cloned eFSH β subunit cDNA and expressed wild-type recombinant eFSH and a partially deglycosylated mutant FSH (eFSH α56/β) to investigate the biological role of the oligosaccharide at Asn56 in FSH activity. The wild-type eFSH and eCG stimulated estradiol production in a dose-dependent manner in the primary cultures of rat granulosa cells, indicating that these equine gonadotropins have FSH activity. Partially deglycosylated eCG (eCG α56/β) also stimulated estradiol production, confirming that the FSH-like activity of eCG is resistant to the removal of the N-linked oligosaccharide. Partially deglycosylated eFSH (eFSH α56/β), however, did not show any FSH activity, indicating that the oligosaccharide at Asn56 was necessary for eFSH. Thus, FSH-like activities of two gonadotropins, eCG and eFSH, are evoked through the distinct molecular mechanisms regarding the biological role of oligosaccharide at Asn56 of the α subunit.

Molecular characterization of bovine placental and ovarian 20α-hydroxysteroid dehydrogenase

The enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD) catalyzes the conversion of progesterone to its inactive form, 20α-hydroxyprogesterone. This enzyme plays a critical role in the regulation of luteal function in female mammals. In this study, we conducted the characterization and functional analyses of bovine 20α-HSD from placental and ovarian tissues. The nucleotide sequence of bovine 20α-HSD showed significant homology to that of goats (96%), humans (84%), rabbits (83%), and mice (81%). The mRNA levels increased gradually throughout the estrous cycle, the highest being in the corpus luteum (CL) 1 stage. Northern blot analysis revealed a 1.2 kb mRNA in the bovine placental and ovarian tissues. An antibody specific to bovine 20α-HSD was generated in a rabbit immunized with the purified, recombinant protein. Recombinant 20α-HSD protein produced in mammalian cells had a molecular weight of ∼37 kDa. Bacterially expressed bovine 20α-HSD protein showed enzymatic activity. The expression pattern of the 20α-HSD protein in the pre-parturition placenta and the CL1 stage of the estrous cycle was similar to the level of 20α-HSD mRNA expression. Immunohistochemical analysis also revealed that bovine 20α-HSD protein was intensively localized in the large luteal cells during the late estrous cycle.

Delayed parturition in cloned calves associated with persistently elevated placentomal TGF-β1 expression

The objective of this study was to investigate hormonal and TGF-beta(1) characterizations of delayed parturition in the SCNT recipients (Korean native beef cattle: Hanwoo). The SCNT blastocysts produced by Hanwoo fetal fibroblast cells were transferred into the synchronized Hanwoo recipients. The artificially inseminated Hanwoo recipients (AI-R) were used as control. All AI-R were labored by natural delivery. The SCNT recipients (SCNT-R) with no signs of delivery were operated by Caesarean section. The blood and placentomes were collected during parturition. The weight of placentomes in SCNT-R (n=12, 301+/-41.22 g) was significantly higher than that of AI-R (n=10, 204.8+/-24.89 g) (p<0.05). There were significantly lower E2 (p<0.05) or higher P4 (p<0.01) and TGF-beta(1) (p<0.01) levels in the SCNT-R compared to that of AI-R, respectively. The SCNT-R showed a higher placentomal TGF-beta(1) protein level compared to that of AI-R (p<0.01). Interestingly, the TGF-beta(1) protein level in SCNT-R with normal delivery was dramatically decreased as same as AI-R, but it was highly maintained in C-sec at days 250 of pregnancy in AI-R. These results suggest that delayed parturition in clone calving may be associated with persistence of elevated TGF-beta-1 expression in late pregnancy.

Molecular diversity of rat placental lactogens

Summary A major function of the placenta is the production of placental lactogens (Pls). Progesterone is essential for pregnancy in all mammals and is secreted by the ovary and placenta, depending on the animal species. In the rat, the main source of progesterone throughout pregnancy is the ovary, and 20α-hydroxysteroid dehydrogenase (20α-HSD) is a key enzyme controlling ovarian progesterone secretion. The primary action of prolactin (PRL) in the maintenance of ovarian progesterone secretion is suppression of the activity of ovarian 20α-HSD. In this review, the sequence homologies between cDNAs for PLs and PRL and the intimate functional relationship between the ovary and placenta are discussed in order to speculate how and why the molecular diversity of rat PLs has developed.

Secretion of Human Protein C in Mouse Milk

To determine the production of recombinant human protein C (rec-hPC) in milk, we created two homozygous mice lines for the goat β-casein/hPC transgene. Females and males of both lines (#10 and #11) displayed normal growth, fertility, and lactated normally. The copy number of the transgene was about fivefold higher in #10 line as compared to #11 line. mRNA expression of the transgene was only detected in the mammary glands of both lines. Furthermore, mRNA expression was fourfold higher on day 7 than on day 1 during lactation. Northern blot analysis of mRNA expression in the #10 line of transgenic (Tg) mice indicated a strong expression of the transgene in the mammary glands after seven days of lactation. Comparison of rec-hPC protein level with that of mRNA in the mammary glands showed a very similar pattern. A 52-kDa band corresponding to the hPC protein was strongly detected in mammary glands of the #10 line during lactation. We also detected two bands of heavy chain and one weak band of light chain in the milk of the #10 and #11 lines. One single band at 52 kDa was detected from CHO cells transfected with hPC cDNA. hPC was mainly localized in the alveolar epithelial cell of the mammary glands. The protein is strongly expressed in the cytoplasm of the cultured mammary gland tissue. hPC protein produced in milk ranged from 2 to 28 ng/mL. These experiments indicated that rec-hPC can be produced at high levels in mice mammary glands.

Internalization of Rat FSH and LH/CG Receptors by rec-eCG in CHO-K1 Cells

ABSTRACT Equine chorionic gonadotropin (eCG) is a unique molecule that elicits the response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in other species. Previous studies from this laboratory had demonstrated that recombinant eCG (rec-eCG) from Chinese hamster ovary (CHO-K1) cells exhibited both FSH- and LH-like activity in rat granulosa and Leydig cells. In this study, we analyzed receptor internalization through rec-eCGs, wild type eCG (eCGβ/α) and mutant eCG (eCGβ/αΔ56) with an N-linked oligosaccharide at Asn56 of the α-subunit. Both the rec-eCGs were obtained from CHO-K1 cells. The agonist activation of receptors was analyzed by measuring stimulation time and concentrations of rec-eCGs. Internalization values in the stably selected rat follicle-stimulating hormone receptor (rFSHR) and rat luteinizing/chorionic gonadotropin receptor (rLH/CGR) were highest at 50 min after stimulation with 10 ng of rec-eCGβ/α. The dose-dependent response was highest when 10 ng of rec-eCGβ/α was used. The deglycosylated eCGβ/αΔ56 mutant did not enhance the agonist-stimulated internalization. We concluded that the state of activation of rFSHR and rLH/CGR could be modulated through agonist-stimulated internalization. Our results suggested that the eLH/CGRs are mostly internalized within 60 min by agonist-stimulation by rec-eCG. We also suggested that the lack of responsiveness of the deglycosylated eCGβ/ αΔ56 was likely because the site of glycosylation played a pivotal role in agonist-stimulated internalization in cells expressing rFSHR and rLH/CGR.

Biological Activity of Recombinant Human Thrombopoietin

To investigate the function and secretion of human thrombopoietin (TPO) in mammalian cells, hTPO cDNA was cloned using human liver cDNA, and recombinant hTPO (rec-hTPO) was produced in CHO cell lines. In addition, six N-linked glycosylation sites were substituted for Ala to elucidate the role of each carbohydrate chain. To analyze the biological activity, rec-hTPO protein was injected subcutaneously. Blood was withdrawn for platelet determination. The metabolic clearance rate (MCR) was also analyzed at the 1, 4, 10 and 24 hr after tail vein injection. Wild-type TPO (WT) was efficiently secreted into the medium. However, a hTPO mutant with 116 deleted nucleotides detected by PCR cloning was not secreted. The N-linked glycosylation sites had nearly the same expression quantity as rec-hTPO WT apart from mutants 3 and 4. The glycosylation site of mutant 4 appeared to be an indispensable site for hTPO secretion. Also characterized was the biological activity through an injection with rec-hTPO (10 ng) to ICR mice (7 weeks). The result of the blood analysis showed a considerable increase in the platelet number six days after the injection. To analyze the pharmacokinetics, rec-hTPO was injected into the tail vein (5 ng). The result was 200 pg/ml 1 hr after this injection. Following this, it dramatically decreased and virtually disappeared 10 hours after the injection. Thus, rec-hTPO may be a treatment for thrombopenia by the production of the high active rec-hTPO. In addition, hTPO can permit the development of potent new analogues that stimulate the platelet value.

Signal Transduction of Equine Follicle-Stimulating Hormone Receptor (eFSHR) by rec-eelFSHβ/α, Natural Porcine FSH, and Natural Human FSH

In this study, we analyzed signal transduction by equine follicle-stimulating hormone receptor (eFSHR) on stimulation with recombinant eelFSHβ/α (rec-eelFSHβ/α), natural porcine FSH (pFSH), and natural human FSH (hFSH). cAMP stimulation in CHO-K1 cells expressing eFSHR was determined upon exposure to different doses (0-1450 ng/mL) of these hormones. The EC50 value of rec-eelFSHβ/α was 53.35 ng/mL. The Rmax values of rec-eelFSHβ/α and pFSH were 28.12 and 2.88 ng/mL, respectively. The activity of rec-eelFSHβ/α was much higher than that of natural pFSH. However, signal transduction in CHO PathHunter Parental cells expressing eFSHR was not enhanced by stimulation with natural hFSH. Thus, rec-eelFSHβ/α was completely active in cells expressing eFSHR. However, natural hFSH did not invoke a signal response in cells expressing eFSHR. Particularly, natural pFSH was weakly active in the same cells. These results showed that eelFSHβ/α has potent activity in cells expressing eFSHR. Thus, rec-eelFSHβ/α may efficiently bind to eFSHR, where as natural hFSH does not bind to eFSHR.

Biochemical Characterization of 20α-Hydroxysteroid Dehydrogenase

In this review, we have tried to summarize the evidence and molecular characterization indicating that 20α-hydroxysteroid dehydrogenase (20α-HSD) is a group of the aldo-keto reductase (AKR) family, and it plays roles in the modulation and regulation of steroid hormones. This enzyme plays a critical role in the regulation of luteal function in female mammals. We have studied the molecular expression and regulation of 20α-HSD in cows, pigs, deer, and monkeys. The specific antibody against bovine 20α-HSD was generated in a rabbit immunized with the purified recombinant protein. The mRNA expression levels increased gradually throughout the estrous cycle, the highest being in the corpus luteum (CL) 1 stage. The mRNA was also specifically detected in the placental and ovarian tissues during pregnancy. The 20α-HSD protein was intensively localized in the large luteal cells and placental cytotrophoblast villus, glandular epithelial cells of the endometrium, syncytiotrophoblast of the placenta, the isthmus cells of the oviduct, and the basal part of the primary chorionic villi and chorionic stem villus of the placenta and large luteal cells of the CL in many mammalian species. Further studies are needed to determine the functional significance of the 20αHSD molecule during ovulation, pregnancy, and parturition. This article will review how fundamental information of these enzymes can be exploited for a better understanding of the reproductive organs during ovulation and pregnancy. (