• 제목/요약/키워드: protein structure

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Severe choline deficiency induces alternative splicing aberrance in optimized duck primary hepatocyte cultures

  • Zhao, Lulu;Cai, Hongying;Wu, Yongbao;Tian, Changfu;Wen, Zhiguo;Yang, Peilong
    • Animal Bioscience
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    • 제35권11호
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    • pp.1787-1799
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    • 2022
  • Objective: Choline deficiency, one main trigger for nonalcoholic fatty liver disease (NAFLD), is closely related to lipid metabolism disorder. Previous study in a choline-deficient model has largely focused on gene expression rather than gene structure, especially sparse are studies regarding to alternative splicing (AS). In modern life science research, primary hepatocytes culture technology facilitates such studies, which can accurately imitate liver activity in vitro and show unique superiority. Whereas limitations to traditional hepatocytes culture technology exist in terms of efficiency and operability. This study pursued an optimization culture method for duck primary hepatocytes to explore AS in choline-deficient model. Methods: We performed an optimization culture method for duck primary hepatocytes with multi-step digestion procedure from Pekin duck embryos. Subsequently a NAFLD model was constructed with choline-free medium. RNA-seq and further analysis by rMATS were performed to identify AS events alterations in choline-deficency duck primary hepatocytes. Results: The results showed E13 (embryonic day 13) to E15 is suitable to obtain hepatocytes, and the viability reached over 95% by trypan blue exclusion assay. Primary hepatocyte retained their biological function as well identified by Periodic Acid-Schiff staining method and Glucose-6-phosphate dehydrogenase activity assay, respectively. Meanwhile, genes of alb and afp and specific protein of albumin were detected to verify cultured hepatocytes. Immunofluorescence was used to evaluate purity of hepatocytes, presenting up to 90%. On this base, choline-deficient model was constructed and displayed significantly increase of intracellular triglyceride and cholesterol as reported previously. Intriguingly, our data suggested that AS events in choline-deficient model were implicated in pivotal biological processes as an aberrant transcriptional regulator, of which 16 genes were involved in lipid metabolism and highly enriched in glycerophospholipid metabolism. Conclusion: An effective and rapid protocol for obtaining duck primary hepatocytes was established, by which our findings manifested choline deficiency could induce the accumulation of lipid and result in aberrant AS events in hepatocytes, providing a novel insight into various AS in the metabolism role of choline.

대황고(大黃膏)의 메티실린 내성 황색 포도상구균에 대한 항균활성 (The antimicrobial activity of Daehwanggo against Methicillin-resistant Staphylococcus aureus)

  • 이순애;공룡;강옥화;서윤수;주전;김상아;송옥희;김민철;한형선;최지나;이영섭;권동렬
    • 대한본초학회지
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    • 제32권2호
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    • pp.87-95
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    • 2017
  • Objectives : Infectious diseases by Methicillin-Resistant Staphylococcus aureus (MRSA) are a growing problem worldwide. Characteristic of MRSA is endlessly mutation to resist antibiotics. Daehwanggo (DHG) is one of the oriental medicine prescriptions contained in Principles and Practice of Eastern Medicine. Daehwanggo was mainly used for external preparation from old times. The purpose of this study is to confirm possibility as supplementary drug of DHG about antibiotics through observation of synergy effect between DHG and commercial antibiotics and to observe restriction on growth of MRSA on any pathway through observation of mechanism. Methods : The minimum inhibitory concentration (MIC) of DHG against MRSA is $500{\sim}2000{\mu}g/m{\ell}$ by broth dilution method. In the checkerboard method, the combinations of DHG with antibiotics has partial synergistic effect or synergy effect and DHG markedly reduced the MICs of the antibiotics oxacillin (OX), gentamicin (GT) against MRSA. In the inhibition of resistance mechanism of DHG against MRSA, the expression of resistance gene and protein about ${\beta}-lactam$ antibiotic was reduced. Also, we observed the effect of DHG about cell membrane permeability against MRSA, and confirmed that DHG suppressed growth of strains by increasing cell membrane permeability. Results : Basis on the result, we speculate that DHG increase antibacterial activity of antibiotics against MRSA by changing the structure of cell wall of MRSA. Conclusions : These data suggest that Daehwanggo possesses possibility as supplementary drug about antibiotics against MRSA.

The complete plastid genome and nuclear ribosomal transcription unit sequences of Spiraea prunifolia f. simpliciflora (Rosaceae)

  • Jeongjin CHOI;Wonhee KIM;Jee Young PARK;Jong-Soo KANG;Tae-Jin YANG
    • 식물분류학회지
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    • 제53권1호
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    • pp.32-37
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    • 2023
  • Spiraea prunifolia f. simpliciflora Nakai is a perennial shrub widely used for horticultural and medicinal purposes. We simultaneously obtained the complete plastid genome (plastome) and nuclear ribosomal gene transcription units, 45S nuclear ribosomal DNA (nrDNA) and 5S nrDNA of S. prunifolia f. simpliciflora, using Illumina short-read data. The plastome is 155,984 bp in length with a canonical quadripartite structure consisting of 84,417 bp of a large single-copy region, 18,887 bp of a short single-copy region, and 26,340 bp of two inverted repeat regions. Overall, a total of 113 genes (79 protein-coding genes, 30 tRNAs, and four rRNAs) were annotated in the plastome. The 45S nrDNA transcription unit is 5,848 bp in length: 1,809 bp, 161 bp, and 3,397 bp for 18S, 5.8S, and 26S, respectively, and 261 bp and 220 bp for internal transcribed spacer (ITS) 1 and ITS 2 regions, respectively. The 5S nrDNA unit is 512 bp, including 121 bp of 5S rRNA and 391 bp of intergenic spacer regions. Phylogenetic analyses showed that the genus Spiraea was monophyletic and sister to the clade of Sibiraea angustata, Petrophytum caespitosum and Kelseya uniflora. Within the genus Spiraea, the sections Calospira and Spiraea were monophyletic, but the sect. Glomerati was nested within the sect. Chamaedryon. In the sect. Glomerati, S. prunifolia f. simpliciflora formed a subclade with S. media, and the subclade was sister to S. thunbergii and S. mongolica. The close relationship between S. prunifolia f. simpliciflora and S. media was also supported by the nrDNA phylogeny, indicating that the plastome and nrDNA sequences assembled in this study belong to the genus Spiraea. The newly reported complete plastome and nrDNA transcription unit sequences of S. prunifolia f. simpliciflora provide useful information for further phylogenetic and evolutionary studies of the genus Spiraea, as well as the family Rosaceae.

Three sesquiterpene lactones suppress lung adenocarcinoma by blocking TMEM16A-mediated Ca2+-activated Cl- channels

  • Ruilian Xiu;Jie Jia;Qing Zhang;Fengjiao Liu;Yaxin Jia;Yuanyuan Zhang;Beibei Song;Xiaodan Liu;Jingwei Chen;Dongyang Huang;Fan Zhang;Juanjuan Ma;Honglin Li;Xuan Zhang;Yunyun Geng
    • The Korean Journal of Physiology and Pharmacology
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    • 제27권6호
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    • pp.521-531
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    • 2023
  • Transmembrane protein TMEM16A, which encodes calcium-activated chloride channel has been implicated in tumorigenesis. Overexpression of TMEM16A is associated with poor prognosis and low overall survival in multiple cancers including lung adenocarcinoma, making it a promising biomarker and therapeutic target. In this study, three structure-related sesquiterpene lactones (mecheliolide, costunolide and dehydrocostus lactone) were extracted from the traditional Chinese medicine Aucklandiae Radix and identified as novel TMEM16A inhibitors with comparable inhibitory effects. Their effects on the proliferation and migration of lung adenocarcinoma cells were examined. Whole-cell patch clamp experiments showed that these sesquiterpene lactones potently inhibited recombinant TMEM16A currents in a concentration-dependent manner. The half-maximal concentration (IC50) values for three tested sesquiterpene lactones were 29.9 ± 1.1 µM, 19.7 ± 0.4 µM, and 24.5 ± 2.1 µM, while the maximal effect (Emax) values were 100.0% ± 2.8%, 85.8% ± 0.9%, and 88.3% ± 4.6%, respectively. These sesquiterpene lactones also significantly inhibited the endogenous TMEM16A currents and proliferation, and migration of LA795 lung cancer cells. These results demonstrate that mecheliolide, costunolide and dehydrocostus lactone are novel TMEM16A inhibitors and potential candidates for lung adenocarcinoma therapy.

Effects of feeding different levels of dietary corn silage on growth performance, rumen fermentation and bacterial community of post-weaning dairy calves

  • Lingyan Li;Jiachen Qu;Huan Zhu;Yuqin Liu;Jianhao Wu;Guang Shao;Xianchao Guan;Yongli Qu
    • Animal Bioscience
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    • 제37권2호
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    • pp.261-273
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    • 2024
  • Objective: The objective of this study was to evaluate the growth performance, rumen fermentation parameters and bacterial community of post-weaning dairy calves in response to five diets varying in corn silage (CS) inclusion. Methods: A total of forty Holstein weaned bull calves (80±3 days of age;128.2±5.03 kg at study initiation) were randomized into five groups (8 calves/group) with each receiving one of five dietary treatments offered as total mixed ration in a 123-d feeding study. Dietary treatments were control diet (CON; 0% CS dry matter [DM]); Treatment 1 (T1; 27.2% CS DM); Treatment 2 (T2; 46.5% CS DM); Treatment 3 (T3; 54.8% CS DM); and Treatment 4 (T4; 67.2% CS DM) with all diets balanced for similar protein and energy concentration. Results: Results showed that calves offered CS had greater average daily gain, body length and chest depth growth, meanwhile altered rumen fermentation indicated by decreased rumen acetate concentrations. Principal coordinate analysis showed the rumen bacterial community structure was affected by varying CS inclusion diets. Bacteroidetes and Firmicutes were the predominant bacterial phyla in the calf rumens across all treatments. At the genus level, the abundance of Bacteroidales_RF16_group was increased, whereas Unclassified_Lachnospiraceae was decreased for calves fed CS. Furthermore, Spearman's correlation test between the rumen bacteria and rumen fermentation parameters indicated that Bacteroidales_RF16_group and Unclassified Lachnospiraceae were positively correlated with propionate and acetate, respectively. Conclusion: The results of the current study suggested that diet CS inclusion was beneficial for post-weaning dairy calf growth, with 27.2% to 46.5% CS of diet DM recommended to achieve improved growth performance. Bacteroidales_RF16_group and Unclassified Lachnospiraceae play an important role in the rumen fermentation pattern for post-weaning calves fed CS.

Biological Functions of N- and O-linked Oligosaccharides of Equine Chorionic Gonadotropin and Lutropin/Chorionicgonadotropin Receptor

  • Min, K. S.
    • 한국동물번식학회:학술대회논문집
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    • 한국동물번식학회 2000년도 국제심포지움
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    • pp.10-12
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    • 2000
  • Members of the glycoprotein family, which includes CG, LH, FSH and TSH, comprise two noncovalently linked $\alpha$- and $\beta$-subunits. Equine chorionic gonadotropin (eCG), known as PMSG, has a number of interesting and unique characteristics since it appears to be a single molecule that possesses both LH- and FSH-like activities in other species than the horse. This dual activity of eCG in heterologous species is of fundamental interest to the study of the structure-function relationships of gonadotropins and their receptors. CG and LH $\beta$ genes are different in primates. In horse, however, a single gene encodes both eCG and eLH $\beta$-subunits. The subunit mRNA levels seem to be independently regulated and their imbalance may account for differences in the quantities of $\alpha$ - and $\beta$ -subunits in the placenta and pituitary. The dual activities of eCG could be separated by removal of the N-linked oligosaccharide on the $\alpha$-subunit Asn 56 or CTP-associated O-linked oligosaccharides. The tethered-eCG was. efficiently secreted and showed similar LH-like activity to the dimeric eCG. Interestingly, the FSH-like activity of the tethered-eCG was increased markedly in comparison with the native and wild type eCG. These results also suggest that this molecular can implay particular models of FSH-like activity not LH-like activity in the eCG/indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion. A single-chain analog can also be constructed to include additional hormone-specific bioactive generating potentially efficacious compounds that have only FSH-like activity. The LH/CG receptor (LH/CGR), a membrane glycoprotein that is present on testicular Leydig cells and ovarian theca, granulosa, luteal, and interstitial cells, plays a pivotal role in the regulation of gonadal development and function in males as well as in nonpregnant and pregnant females. The LH/CGR is a member of the family of G protein-coupled receptors and its structure is predicted to consist of a large extracellular domain connected to a bundle of seven membrane-spanning a-helices. The LH/CGR phosphorylation can be induced with a phorbol ester, but not with a calcium ionophore. The truncated form of LHR also was down-regulated normally in response to hCG stimulation. In contrast, the cell lines expressing LHR-t63I or LHR-628, the two phosphorylation-negative receptor mutant, showed a delay in the early phase of hCG-induced desensitization, a complete loss of PMA-induced desensitization, and an increase in the rate of hCG-induced receptor down-regulation. These results clearly show that residues 632-653 in the C-terminal tail of the LHR are involved in PMA-induced desensitization, hCG-induced desensitization, and hCG-induced down-regulation. Recently, constitutively activating mutations of the receptor have been identified that are associated with familial male-precocious puberty. Cells expressing LHR-D556Y bind hCG with normal affinity, exhibit a 25-fold increase in basal cAMP and respond to hCG with a normal increase in cAMP accumulation. This mutation enhances the internalization of the free and agonist-occupied receptors ~2- and ~17-fold, respectively. We conclude that the state of activation of the LHR can modulate its basal and/or agonist-stimulated internalization. Since the internalization of hCG is involved in the termination of hCG actions, we suggest that the lack of responsiveness detected in cells expressing LHR-L435R is due to the fast rate of internalization of the bound hCG. This statement is supported by the finding that hCG responsiveness is restored when the cells are lysed and signal transduction is measured in a subcellular fraction (membranes) that cannot internalize the bound hormone.

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한국인삼론(韓國人蔘論) (Current Status of Korean Ginseng Research)

  • 한병훈
    • 생약학회지
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    • 제3권3호
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    • pp.151-160
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    • 1972
  • Recent achievements of scientific research on the pharmacologic activities and the chemical problems of dammalene glycosides, which are considered to be effective principles of Korean ginseng, are reviewed and analyzed in view of structure-activity relationship. 1) S. Shibata and his co-workers detected 12 glycoside spots of dammalene series on the two dimensional T.L.C. of total glycoside fraction from Japanese ginseng, and designated them Ginsenoside Rx(x=a, b, c, g, h, etc.) in the order of increasing Rf-value. The aglycones of those glycosides were characterized to be protopanaxadiol for the Ginsenoside $Rx(x=a,\;b_{1},\;b_{2},\;c,\;d,\;e,\;f)$ and protopanaxatriol for the Ginsenoside $Rx(x=g_{1},\;g_{2},\;g_{3},\;h_{1}\;'h_{2})$. Using Korean ginseng as the material for our study, the author and his coworkers isolated a new dammalene glycoside(Panax Saponin C), which comes under the category of protopanaxadiol glycosides based on the classification of S. Shibata et al., and characterized this saponin to be the glycoside of protopanaxatriol series. Furthermore, Panax Saponin C dissociated into $two\;components(C_{1}\;and\;C_{2}-acetate)$ by acetylation, both of which returned to original Panax Saponin C by deacetylation. Based on this result, more than 13 glycoside components of dammalene series will be expected in the Korean ginseng. 2) The structures of protopanaxadiol and protopanaxatriol, the genuine aglycones of dammalene glycosides, are fully established to be structural analogues by S. Shibata and his co-workers, therefore antagonistic and/or analogical activities will be expected for the pharmacologic activities of these glycoside series of structural analogues. K. Takaki and his co-workers found central nervous system (CNS) stimmulant activity from the glycosides of protopanaxatriol series and CNS-depressant activity from the glycosides of protopanaxadiol series. On the other hand, the author and his co-workers found stimmulating activity on the protein synthesis from both the series of dammalene glycosides with delayed and long-lasting characteristics. This delayed and long-lasting characteristics were also observed in the anti-inflammatory activity of glycosides of protopanaxatriol series on their time course tendency. For the convenience's sake of argument, pluralistic pharmacologic activities of dammalene glycosides, which were observed by many workers at various pharmacologic site, may be classified into two main categories; one is pan-cellular activity and the other is organ specific activity to the certain tissue which is a mass of cells differentiated to a certain direction for their special functions in the body. Based on the data of K. Takaki and those of the authors, following assumption will be probable; Pharmacologic activities of both series of glycosides of protopanaxadiol and protopanaxatriol aglycones may be antagonistic on their tissue-specific activities and analogic on their pan-cellular activities. Therefore, the mixture of these two series of glycosides in an appropriate ratio, as the case of total extract of Korean ginseng, will be probably beneficial to the host by increasing the synthesis of some functional proteins, due to the additive action of pan-cellular activity, and with the disappearance of any significant behavioral symptoms due to the antagonism of tissue specific activity. This fact will probably be the main reason why classical trials of pharmacologists failed in re-discovering the efficacy of Korean ginseng with their behavioral test. 3) The author and his co-workers achieved the synthesis of $C^{14}-labelled\;Panax\;Saponin\;A\;on\;C_{25}-C_{27}\;position\;of\;aglycone$ in the interest of tracer studies in vivo. The method will be applicable to other dammalene glycosides regardless of their chemical structure. 4) The author and his co-workers converted chemically betulafolienetriol, a triterpene component of Betula platyphylla, to the protopanaxadiol, one of genuine aglycone of dammalene glycosides.

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Bacillus amyloliquefaciens에서 Puromycin 과 Magnesium에 의한 $\alpha$-Amylase 의 분비저해 (Disturbance of $\alpha$-Amylase Secretion from Bacillus amyloliquefaciens Cells by the Treatment of Puromycin and Magnesium)

  • 안순자;김순옥;이동희;송방호
    • 한국미생물·생명공학회지
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    • 제17권5호
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    • pp.412-420
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    • 1989
  • Bacillus amyloliquefaciens의 extracellular $\alpha$-amylase는 세포질막에 결합된 ribosome에서 합성되며 이 때 ribosome은 nascent polypeptide쇄에 의해 막에 부착된 것으로 추측된다. $\alpha$-amylase 생산균주인 E. amyioiiquefaciens 야생주의 정상단백(MW, 58kDa)과 이 균주의 $\alpha$-amylase 구조유전자에 변이가 일어나 carboxy 말단이 결실된 변이주의 이상단백 (33kDa)은 동일조건하의 배양외액에서 그 효소활성이 검출됨을 볼 때, $\alpha$-amylase의 carboxy 말단에는 막 투과의 신호가 없는 것으로 생각된다. 그러나 이들 양균주의 대수증식기 세포에 puromycin을 20$\mu\textrm{g}$/$m\ell$까지 가하여 6시간 동안 처리한 결과 균의 사멸에는 영향을 미치지 않았으나 extracellular $\alpha$-amylase 활성은 10$\mu\textrm{g}$/$m\ell$의 농도에서도 동일양상으로 감소하였다. 이 감소요인은 puromycin 처리시 막결합 ribosome에서의 막과 ribosome의 가교로 작용할 것으로 예상되는 nascent poly peptide 합성이 중단되므로 막으로부터 ribosome이 해리되어 $\alpha$-amylase의 분비가 억제된 것으로 추측된다. 이 현상은 50mM의 magnesium의 첨가효과 가 약하게 나타났기 때문에 ribosome은 nascent Polypeptide의 가교로 막에 부착되어 있는 것으로 생각된다. 또 Iysozyme 처리에 의해 세포벽을 분해한 protoplas가 trypsin을 함께 처리하므로서 세포외액의 $\alpha$-amylase 활성이 상실됨은 막의 외부로 protruding되는 nascent polypeptide가 trypsin에 의해 분해되기 때문으로 생각된다. 3차 구조를 형성한 extracellular $\alpha$-amylase의 경우 trypsin 내성임을 감안할 때 이 분자가 세포막 통과 직후, 세포벽을 통과하기 이전에 folding이 이루어져서 그 후 성숙된 단백으로서 세포벽을 통과하는 것으로 믿어진다.

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유색미 품종별 전분 구조 및 이화학적 특성 (Starch Structure and Physicochemical Properties of Colored Rice Varieties)

  • 박지영;오성환;한상익;이유영;이병원;함현미;최용환;오세관;조준현;송유천
    • 한국작물학회지
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    • 제61권3호
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    • pp.153-162
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    • 2016
  • 유색미 가공소재 탐색 및 용도 다양화를 위해 일품을 포함하여 흑미 메벼 6품종, 적미 메벼 3품종, 흑미 찰벼 5품종, 적미 찰벼 1품종으로 일품과 유색미 15개의 품종의 전분 이화학적 구조적 특성을 분석하여 가공용도 다양화를 위한 기초자료로 활용하고자 한다. 1. 유색미의 일반성분을 분석한 결과, 눈큰흑찰이 조단백, 조지방, 식이섬유이 함량이 다른 품종에 비해 높은 경향이었고, 적미의 모든 품종은 조지방이 흑미에 비해 높은 함량을 나타내었다. 2. 유색미 가루를 이용한 찰벼 품종의 아밀로스 함량은 3~5%, 메벼 품종의 아밀로스 함량은 15~18%를 나타내었고, 메벼 품종 중 조은흑미는 14%대로 다소 낮은 함량을 나타내었다. 또한 일품이 80%로 가장 높은 총 전분 함유량을 보였고, 눈큰흑찰과 흑진주가 70, 71%로 가장 낮은 총 전분 함량을 나타내었다. 3. 유색미 가루의 호화특성을 분석한 결과, 모든 유색미의 호화개시온도는 $68^{\circ}C$였으며, 일품의 호화개시온도인 $67.95^{\circ}C$와 비슷하였다. 최고점도는 일품이 가장 높았으며, 적진주, 홍진주, 건강홍미 순으로 적미 메벼 품종이 흑미 품종과 적진주 찰에 비해 높은 것으로 나타났다. 치반점도 값으로 본 노화정도 찰벼 품종에서 느린 것으로 확인되었고, 조은흑미는 메벼품종 중에서 노화정도가 느린 것으로 분석되었다. 4. 유색미의 전분외형은 대부분 $2{\sim}5{\mu}m$ 크기의 다각형의 치밀한 입자로 이루어져있으며, 결정성 분석에서는 모두 A type의 전분구조를 이루고 있었지만 결정성은 품종마다 다소 차이를 보였다. 5. 아밀로펙틴의 체인 길이 분석에서는 16개의 품종에서 12${\leq}24$인 B사슬 함량이 50%이상을 차지하였고, 다음으로 DP<12 함량이 대부분 33~35%함량을 나타내었다. 중합도 36개 이상의 장쇄사슬은 흑미가 적미에 비해 함량이 높은 경향이었다.

Biological Functions of N- and O-linked Oligosaccharides of Equine Chorionic Gonadotropin and Lutropin/Chorionic Gonadotropin Receptor

  • Min, K.S.
    • 한국가축번식학회지
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    • 제24권4호
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    • pp.357-364
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    • 2000
  • Members of the glycoprotein family, which includes CG, LH, FSH and TSH, comprise two noncovalently linked $\alpha$- and $\beta$-subunits. Equine chorionic gonadotropin (eCG), known as PMSG, has a number of interesting and unique characteristics since it appears to be a single molecule that possesses both LH- and FSH-like activities in other species than the horse. This dual activity of eCG in heterologous species is of fundamental interest to the study of the structure-function relationships of gonadotropins and their receptors. CG and LH $\beta$ genes are different in primates. In horse, however, a single gene encodes both eCG and eLH $\beta$ -subunits. The subunit mRNA levels seem to be independently regulated and their imbalance may account for differences in the quantities of $\alpha$ - and $\beta$-subunits in the placenta and pituitary. The dual activities of eCG could be separated by removal of the N-linked oligosaccharide on the $\alpha$-subunit Asn 56 or CTP-associated O-linked oligosaccharides. The tethered-eCG was efficiently secreted and showed similar LH-like activity to the dimeric eCG. Interestingly, the FSH-like activity of the tethered-eCG was increased markedly in comparison with the native and wild type eCG. These results also suggest that this molecular can implay particular models of FSH-like activity not LH-like activity in the eCG/indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion. A single-chain analog can also be constructed to include additional hormone-specific bioactive generating potentially efficacious compounds that have only FSH-like activity. The LH/CG receptor (LH/CGR), a membrane glycoprotein that is present on testicular Leydig cells and ovarian theca, granulosa, luteal, and interstitial cells, plays a pivotal role in the regulation of gonadal development and function in males as well as in nonpregnant and pregnant females. The LH/CGR is a member of the family of G protein-coupled receptors and its structure is predicted to of a large extracellular domain connected to a bundle of seven membrane-spanning a-helices. The LH/CGR phosphorylation can be induced with a phorbol ester, but not with a calcium ionophore. The truncated form of LHR also was down-regulated normally in response to hCG stimulation. In contrast, the cell lines expressing LHR-t631 or LHR-628, the two phosphorylation-negative receptor mutant, showed a delay in the early phase of hCG-induced desensitization, a complete loss of PMA-induced desensitization, and an increase in the rate of hCG-induced receptor down-regulation. These results clearly show that residues 632~653 in the C-terminal tail of the LHR are involved in PMA-induced desensitization, hCG-induced desensitization, and hCG-induced down-regulation. Recently, constitutively activating mutations of the receptor have been identified that are associated with familial male-precocious puberty. Cells expressing LHR-D556Y bind hCG with normal affinity, exhibit a 25-fold increase in basal cAMP and respond to hCG with a normal increase in cAMP accumulation. This mutation enhances the internalization of the free and agoinst-occupied receptors ~2- and ~17- fold, respectively. We conclude that the state of activation of the LHR can modulate its basal and/or agonist-stimulated internalization. Since the internalization of hCG is involved in the termination of hCG actions, we suggest that the lack of responsiveness detected in cells expressing LHR-L435R is due to the fast rate of internalization of the bound hCG. This statement is supported by the finding that hCG responsiveness is restored when the cells are lysed and signal transduction is measured in a subcellular fraction (membranes) that cannot internalize the bound hormone.

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