Abstract. Equine chorionic gonadotropin (eCG), which consists of highly glycosylated α-and β-subunits, is a unique member of the gonadotropin family because it elicits response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in species other than the horse. In this study, recombinant tethered-eCG as well as its deglycosylated mutants were produced to determine if α-and β-subunits can be synthesized as a single polypeptide chain (tethered-eCG) and display biological activity. We found that tethered-eCG (T-βα) had both LH-and FSH-like activities comparable to dimeric eCG. Luteinizing hormone-like activity of tethered-eCGs deglycosylated at Asn 56 (T-βα56) was decreased. In contrast, LH-like activity of eCG without O-glycosylated carboxylterminal peptide (CTP) (T-βcα) was slightly decreased but still similar to T-βα. Double mutation at Asn 56 and CTP (T-βcα56) caused marked decrease in the activity, indicating that both glycosylations at Asn 56 and CTP are involved in LH-like activity in the tethered form. Interestingly, FSH-like activity remained in all deglycosylated eCG mutants (T-βα56, T-βcα and T-βcα56) as well as T-βα. The biological roles of oligosaccharides at Asn 56 of eCG α-subunit and O-linked peptide of β-subunit appear to be different in LH-and FSH-like activities in tethered-eCG. Key words: Equine chorionic gonadotropin, Luteinizing hormone, Follicular stimulating hormone, Glycosylation, Tethered hormone (J. Reprod. Dev. 50: [297][298][299][300][301][302][303][304] 2004) quine chorionic gonadotropin (eCG) is a member of the glycoprotein hormone family which includes luteinizing hormone (LH), follicles t i m u l a t i n g h o r m o n e ( F S H ) a n d t h y r o i dstimulating hormone (TSH). This hormone family is characterized by a heterodimeric structure composed of a common α-subunit noncovalently linked to a hormone-specific β-subunit [1]. The β-subunits of eCG and eLH, translated from the same gene, have identical primary structures [2,3]. The difference between eCG and eLH lies in the structure of their carbohydrates, which are both sialylated and sulfated in LH and sialylated in CG [4,5]. Equine chorionic gonadotropin shows both LH-and FSH-like activities in many species but not the horse [6][7][8][9][10][11]. The LH-and FSH-like activities of eCG in vitro observed in heterologous species is of fundamental interest to the study of structurefunction relationships of gonadotropins and their receptors.We previously prepared recombinant eCG in CHO-K1 cells, and found that these recombinant

In the present study, the pig CMP-N-acetylneuraminic acid hydroxylase gene (pcmah), a key enzyme for the synthesis of NeuGc (N-glycolylneuraminic acid), was cloned from pig small intestine and characterized. The ORF (open reading frame) of pcmah was 1734 bp, encoding 577 amino acids and consisting of 14 exons. Organ expression pattern analysis reveals that pcmah mRNA is mainly expressed in pig rectum, tongue, spleen and colon tissues, being the most highly expressed in small intestine. In the ectopic expression of pcmah, when pig kidney PK15 cells and human vascular endothelial ECV304 cells were transfected with the cloned pcmah, the NeuGc contents of these transfectants were greater in comparison with vector transfectants used as controls. In addition, in the functional analysis of NeuGc, HSMC (human-serum-mediated cytotoxicity) was elevated in the ectopic NeuGc-expressing pcmah-transfected cells compared with controls. Moreover, binding of human IgM to the pcmah-transfected cells was significantly increased, whereas binding of IgG was slightly increased, indicating that the human IgM type was a major anti-NeuGc antibody. Furthermore, pcmah silencing by shRNA (short hairpin RNA) resulted in a decrease in NeuGc content and xenoantigenicity in PK15. From the results, it was concluded that the pcmah gene was capable of synthesizing the NeuGc acting as a xenoantigen in humans, confirming the NeuGc-mediated rejection response in pig-human xenotransplantation.

Practical application of animal cloning by somatic cell nuclear transfer (SCNT) has been hampered by an extremely low success rate. To address whether placental dysfunction in SCNT causes fetal loss during pregnancy, we have used a global proteomics approach using 2-DE and MS to analyze the differential protein patterns of three placentae from the afterbirth of cases of postnatal death, derived from SCNT of Korean Native cattle, and three normal placentae obtained from the afterbirth of fetuses derived from artificial insemination. Proteins within a pI range of 4.0-7.0 and 6.0-9.0 were analyzed separately by 2-DE in triplicate. A total of approximately 2000 spots were detected in placental 2-DE gels stained with CBB. In the comparison of normal and SCNT samples, 60 spots were identified as differentially expressed proteins, of which 33 spots were up-regulated proteins in SCNT placentae, while 27 spots were down-regulated proteins. Most of the proteins identified in this analysis appeared to be related with protein repair or protection, cytoskeleton, signal transduction, immune system, metabolism, extracellular matrix and remodeling, transcription regulation, cell structure or differentiation and ion transport. One of up-regulated proteins in SCNT was TIMP-2 protein known to be related to extracellular matrix and remodeling during pregnancy. Western blot analysis showed an increased level of TIMP-2 in SCNT placenta compared to normal. Our results revealed composite profiles of key proteins involved in abnormal placenta derived from SCNT, and suggested expression abnormality of these genes in SCNT placenta, resulting in fetal losses following SCNT.

This study was performed to determine the effects of dietary fat sources, i.e., beef tallow, soybean oil, olive oil and coconut oil (each 3% in feed), on the growth performance, meat quality and gene expression in growing-finishing pigs. A total of 72 crossbred pigs (Landrace×Large White×Duroc) were used at 71±1 kg body weight (about 130 d of age) in 24 pens (320×150 cm) in a confined pig house (three pigs per pen) with six replicate pens per treatment. The growing diet was given for periods of 14±3 d and the finishing diet was given for periods of 28±3 d. The fat type had no significant effect either on growth performance or on chemical composition or on meat quality in growing-finishing pigs. Dietary fat type affected fatty acid composition, with higher levels of unsaturated fatty acids (UFAs) and monounsaturated fatty acids (MUFAs) in the olive oil group. Microarray analysis in the Longissimus dorsi identified 6 genes, related to insulin signaling pathway, that were differentially expressed among the different feed groups. Real time-PCR was conducted on the six genes in the longissimus dorsi muscle (LM). In particular, the genes encoding the protein kinase, cAMP-dependent, regulatory, type II, alpha (PRKAR2A) and the catalytic subunit of protein phosphatase 1, beta isoform (PPP1CB) showed the highest expression level in the olive oil group (respectively, p<0.05, p<0.001). The results of this study indicate that the type of dietary fat affects fatty acid composition and insulin signaling-related gene expression in the LM of pigs.