• BMB Reports
• /
• 제46권12호
• /
• pp.606-610
• /
• 2013

Glucagon like peptide-1 (GLP-1) regulates glucose mediated-insulin secretion, nutrient accumulation, and ${\beta}$-cell growth. Despite the potential therapeutic usage for type 2 diabetes (T2D), GLP-1 has a short half-life in vivo ($t_{1/2}$ <2 min). In an attempt to prolong half-life, GLP-1 fusion proteins were genetically engineered: GLP-1 human serum albumin fusion (GLP-1/HSA), AGLP-1/HSA which has an additional alanine at the N-terminus of GLP-1, and AGLP-1-L/HSA, in which a peptide linker is inserted between AGLP-1 and HSA. Recombinant fusion proteins secreted from the Chinese Hamster Ovary-K1 (CHO-K1) cell line were purified with high purity (>96%). AGLP-1 fusion protein was resistant against the dipeptidyl peptidase-IV (DPP-IV). The fusion proteins activated cAMP-mediated signaling in rat insulinoma INS-1 cells. Furthermore, a C57BL/6N mice pharmacodynamics study exhibited that AGLP-1-L/HSA effectively reduced blood glucose level compared to AGLP-1/HSA.

• Parasites, Hosts and Diseases
• /
• 제40권1호
• /
• pp.17-24
• /
• 2002

We have cloned a cDNA encoding a cysteine proteinase of the Acanthamoeba healui OC-3A strain isolated from the brain of a granulomatous amoebic encephalitis patient. A DNA probe for an A. healui cDNA library screening was amplified by PCR using degenerate oligonucleotide primers designed on the basis of conserved amino acids franking the active sites of cysteine and asparagine residues that are conserved in the eukaryotic cysteine proteinases. Cysteine proteinase gene of A. healui (AhCPI) was composed of 330 amino acids with signal sequence, a proposed pro-domain and a predicted active site made up of the catalytic residues, $Cys^{25},{\;}His^{159},{\;}and{\;}Asn^{175}$. Deduced amino acid sequence analysis indicates that AhCPI belong to ERFNIN subfamily of C 1 peptidases. By Northern blot analysis. no direct correlation was observed between AhCPI mRNA expression and virulence of Acanthamoeba, but the gene was expressed at higher level in amoebae isolated from soil than amoeba from clinical samples. These findings raise the possibility that AhCPI protein may play a role in protein metabolism and digestion of phagocytosed bacteria or host tissue debris rather than in invasion of amoebae into host tissue.

• 대한화학회지
• /
• 제5권1호
• /
• pp.33-37
• /
• 1961

We have shown that carboxy-peptidase destroys the biological activity of angiotensin octa-and deca-peptides. Since Proline occurs as the seventh amino acid from the amino end of the chain and since carboxypeptidase does not cleave proline from a peptid chain, it is evident that the heptapeptid H.asp-arg-val-tyr-ileu-his-pro.OH is formed by this hydrolysis. This peptide must then be biologically inactive. In order to determine whether the phenyl group of the C-terminal amino acid was the necessary requirement for biological activity of the octapeptide, $ala^8$ angiotensin octapeptide(amino acids of peptides numbered from amino end) was synthesized. For this synthesis the four dipeptides were prepared: carbobenzoxy-L-prolyl-L-alanine-P-nitrobenzyl-ester, m.p. $134-135^{\circ}C,$ carbobenzoxy-L-isoleucyl-imidazole benzyl-L-histidine methyl ester, m.p. $114-116^{\circ}C,$ carbobenzoxy-L-valyl-L-tyrosine hydrazide and carbobenzoxy B-benzyl-L-aspartyl-nitro-L-arginine. The first three dipeptides were obtained as crystalline compounds. Imidazole-benzyl-L-histidine was used in the hope that it would block the histidine imidazole against side reactions in steps subsequent to the formation of the C-terminal tetrapeptide. Also, it was through that the imidazole benzylated peptides would be easier to crystallize. This, however, was not the case. The tetrapeptide, carbobenzoxy-L-isoleucyl-L-im, benzyl-histidyl, L-prolyl-L-alanine-nitrobenzyl ester was not obtained in a crystalline form. Neither could the mono-or dihydrobromide of the tetrapeptide free base be induced to crystallize. Carbobenzoxy-L-valyl-L-tyrosine azide was condensed with the tetrapeptide free base to yield the protected hexapeptide; carbobenzoxy-L-valyl-L-tyrosyl-L-isoleucyl-L-im, benzyl, histidyl-L-Prolyl-L-alanine-nitrobenzyl ester. Upon removal of the carbobenzoxy group with hydrogen bromide in acetic acid an amorphous free base hexapeptide ester was obtained. This compound gave the correct C, H, N analysis and contained the six amino acids in the correct ratio. The octapeptide was obtained by condensing this hexapeptide with carbobenzoxy-B-benzyl-L-aspartyl-nitro, L-arginine using the mixed anhydride method of condensation. This amorphous product was proven to be homogenous by chromatography in two solvent systems and upon hydrolysis yielded the eight amino acids in correct ratio. The five protecting groups were removed from the octapeptide by hydrogenolysis over palladium black catalyst. Biological assay of the free peptide indicated that it possessed less than 0.1 per cent of both pressor and oxytocic activity of the phenylalanine8 angiotensin. This suggests that the phenyl group is a point of attachment between angiotensin and its biological receptor site.

• 생명과학회지
• /
• 제24권7호
• /
• pp.798-803
• /
• 2014

Cathepsin D (CtsD)는 아스파르트산 단백질 분해효소로서 cytochrome C의 방출을 유도하여 apoptosis 기전을 활성화시킨다. 본 연구에서는 3T3-L1 지방전구세포에서 CtsD 발현 조절에 관여하는 microRNA에 대해 조사하였다. 먼저 지방전구세포 사멸시 CtsD 발현 변화를 관찰하기 위하여 DNA damage agent인 doxorubicin을 3T3-L1 세포주에 노출시켜 CtsD 발현이 증가함을 확인하였다. 또한 지방전구세포주에서 CtsD가 과발현되면 세포 생존율이 감소하였다. miRanda program을 이용하여 CtsD 유전자를 표적으로 하는 microRNA를 탐색하여 miR-145를 선발하였다. Luciferase reporter assay에 의해 miR-145가 CtsD 유전자의 3' UTR 부위에 결합하여 luciferase 활성을 감소시킴을 관찰하였다. 3T3-L1 세포주에 miR-145 mimic을 도입한 결과 CtsD mRNA 발현과 단백질 수준이 감소하였다. 또한 세포주에 doxorubicin을 처리한 결과 CtsD 유전자 발현 증가와 상반되게 miR-145 발현이 감소하였다. 이외에도 miR-145 inhibitor을 세포에 도입하면 세포 생존율이 감소하였다. 이러한 결과는 지방전구세포의 세포사멸에 CtsD가 관여할 수 있으며, miR-145에 의해 CtsD 발현이 직접 조절되고 있음을 나타낸다. 따라서, 지방전구세포의 사멸을 유도하기 위해서는 miR-145 발현 제어가 주요한 표적이 될 수 있을 것으로 생각된다. 본 연구결과는 향후 비만 예방 및 치료를 위한 지방세포 사멸기전 규명에 중요한 기초 자료를 제공할 수 있을 것으로 기대한다.

• Biomolecules & Therapeutics
• /
• 제26권2호
• /
• pp.201-209
• /
• 2018

G protein-coupled receptor 119 (GPR119) is expressed in the pancreas and gastrointestinal tract, and its activation promotes insulin secretion in the beta cells of the pancreatic islets as well as the secretion of glucagon-like peptide-1 (GLP-1) in intestinal L cells, consequently improving glucose-stimulated insulin secretion. Due to this dual mechanism of action, the development of small-molecule GPR119 agonists has received significant interest for the treatment of type 2 diabetes. We newly synthesized 1,2,4-triazolone derivatives of GPR119 agonists, which demonstrated excellent outcomes in a cyclic adenosine monophosphate (cAMP) assay. Among the synthesized derivatives, YH18968 showed cAMP=2.8 nM; in GLUTag cell, GLP-1secretion=2.3 fold; in the HIT-T15 cell, and insulin secretion=1.9 fold. Single oral administration of YH18968 improved glucose tolerance and combined treatment with a dipeptidyl peptidase 4 (DPP-4) inhibitor augmented the glucose lowering effect as well as the plasma level of active GLP-1 in normal mice. Single oral administration of YH18968 improved glucose tolerance in a diet induced obese mice model. This effect was maintained after repeated dosing for 4 weeks. The results indicate that YH18968 combined with a DPP-4 inhibitor may be an effective therapeutic candidate for the treatment of type 2 diabetes.

• The Korean Journal of Pain
• /
• 제37권1호
• /
• pp.26-33
• /
• 2024

Background: Sitagliptin is an antidiabetic drug that inhibits dipeptidyl peptidase-4 enzyme. This study aimed to investigate the antinociceptive and anti-inflammatory effects of sitagliptin in formalin and carrageenan tests and determine the possible mechanism(s) of its antinociceptive activity. Methods: Male Swiss mice (25-30 g) and male Wistar rats (180-220 g) were used for formalin and carrageenan tests, respectively. In the formalin test, paw licking time and in the carrageenan test, paw thickness were considered as indexes of pain behavior and inflammation respectively. Three doses of sitagliptin (2.5, 5, and 10 mg/kg) were used in these tests. Also, several antagonists and enzyme inhibitors were used to evaluate the role of adrenergic, serotonergic, dopaminergic, and opioid receptors as well as the NO/cGMP/K_ATP pathway in the antinociceptive effect of sitagliptin (5 mg/kg). Results: Sitagliptin showed significant antinociceptive and anti-inflammatory effects in the formalin and carrageenan tests respectively. In the carrageenan test, all three doses of sitagliptin significantly (P < 0.001) reduced paw thickness. Pretreatment with yohimbine, prazosin, propranolol, naloxone, and cyproheptadine could not reverse the antinociceptive effect of sitagliptin (5 mg/Kg), which indicates that adrenergic, opioid, and serotonin receptors (5HT₂) are not involved in the antinociceptive effects. L-NAME, methylene blue, glibenclamide, ondansetron, and sulpiride were able to reverse this effect. Conclusions: NO/cGMP/K_ATP, 5HT₃ and D₂ pathways play an important role in the antinociceptive effect of sitagliptin. Additionally significant anti-inflammatory effects observed in the carrageenan test might contribute in reduction of pain response in the second phase of the formalin test.

• 생명과학회지
• /
• 제24권7호
• /
• pp.777-783
• /
• 2014

항암제로 알려진 cisplatin과 t-BHP를 토끼에 투여하여 유도된 급성 신부전 시 산조인 추출액을 처리하였을 때 신장 세포의 보호에 미치는 항산화 효과를 조사하였다. 신장을 분리한 후 신피질 절편 실험에서 세포의 손상을 유발하는 지질과산화 및 LDH 실험에서 t-BHP 단독 처리 시 대조군에 비하여 각각 3배, 5배 이상 증가하였으나 산조인 추출액 0.5%를 동시 처리하였을 때는 대조군 수준으로 감소하였다. Creatinine 측정과 지질과산화 실험에서 cisplatin $5mg{\cdot}kg^{-1}$을 복강 투여한 군의 creatinine 농도가 $2.13{\pm}0.1mg{\cdot}dl^{-1}$로 나타났으나 산조인 추출액 $50mg{\cdot}kg^{-1}{\cdot}day^{-1}$을 7일간 전처리 후 cisplatin 투여 48시간 경과한 군은 $0.84{\pm}0.1mg{\cdot}dl^{-1}$로 creatinine의 농도가 약 60% 감소되는 신장보호 효과를 나타내었고, 지질과산화 검사는 cisplatin 단독 투여 시 대조군에 비하여 1.6배 높게 나타났으나 산조인 추출액 전처리 시 1.1배로 대조군과 유사하였다. 병리조직 검사는 cisplatin 단독 처리군에서 근위곱슬세관이 대조군에 대하여 더 붉게 염색되었으며 근위곱슬세관은 내강의 융모세포가 탈락하여 공포를 형성하였다. 그러나 산조인 추출액을 7일간 전처리한 군에서는 근위곱슬세관이 대조군과 유사한 염색소견을 보였고 근위곱습세관도 내강의 융모세포 탈락이 거의 나타나지 않았다. 따라서 cisplatin과 t-BHP에 의해 유발된 신장세포 손상에 대하여 산조인 추출액이 항산화 효과를 보였다.