• The Korean Journal of Physiology and Pharmacology
• /
• 제23권5호
• /
• pp.357-366
• /
• 2019

$G{\alpha}_q$-coupled receptor stimulation was implied in the activation process of transient receptor potential canonical (TRPC)1/4 and TRPC1/5 heterotetrameric channels. The inactivation occurs due to phosphatidylinositol 4,5-biphosphate ($PI(4,5)P_2$) depletion. When $PI(4,5)P_2$ depletion was induced by muscarinic stimulation or inositol polyphosphate 5-phosphatase (Inp54p), however, the inactivation by muscarinic stimulation was greater compared to that by Inp54p. The aim of this study was to investigate the complete inactivation mechanism of the heteromeric channels upon $G{\alpha}_q$-phospholipase $C{\beta}$ ($G{\alpha}_q-PLC{\beta}$) activation. We evaluated the activity of heteromeric channels with electrophysiological recording in HEK293 cells expressing TRPC channels. TRPC1/4 and TRPC1/5 heteromers undergo further inhibition in $PLC{\beta}$ activation and calcium/protein kinase C (PKC) signaling. Nevertheless, the key factors differ. For TRPC1/4, the inactivation process was facilitated by $Ca^{2+}$ release from the endoplasmic reticulum, and for TRPC1/5, activation of PKC was concerned mostly. We conclude that the subsequent increase in cytoplasmic $Ca^{2+}$ due to $Ca^{2+}$ release from the endoplasmic reticulum and activation of PKC resulted in a second phase of channel inhibition following $PI(4,5)P_2$ depletion.

• 한국식물병리학회:학술대회논문집
• /
• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
• /
• pp.65.2-65
• /
• 2003

To protect the plant against several soil-borne pathogens, we are currently constructing disease-resistant transgenic root stock for the growth of cucurbitaceae vegetable plants, watermelon and gourd. We made a watermelon cDNA library from Cladosporium cucumerinum-Infected leaves for substractive hybriazation and differential screening. We isolated the several pathogen inducible cDNA clones, such as caffeoyl-CoA-methyltransferase, LAA induced protein, receptor-like kinase homolog, hydroxyproline-rich glycoprotein, catalase, calmodulin binding protein, mitochondrial ATPase beta subunit, methyl tRNA synthetase and WRKY transcription factors. We previously obtained CaMADS in pepper and galactinol synthase ( CsGolS) in cucumber that were confirmed to be related with disease-resistance. CaMADS and CsGolS2 were transformed into the inbred line 'GO701-2' gourd, the inbred line '6-2-2' watermelon and the Kong-dye watermelon by Agrobacterium tumerfaciens LBA4404. Plant growth regulators (zeatin, BAP and IAA) were used for shoot regeneration and root induction for optimal condition. Putative transgenic plants were selected in medium containing 100mg/L kanamycin and integration of the CaMADS and CsGO/S2 into the genomic DNA were demonstrated by the PCR analysis. We isolated major soil-borne pathogens, such as Monosporascus cannonballus, Didymella bryoniae, Cladosporium cuvumerinum from the cultivation area of watermelon or root stock, and successfully established artificial inoculation method for each pathogen. This work was supported by a grant from BioGreen 21 program, Rural Development Administration, Republic of Korea.

• 대한의생명과학회지
• /
• 제15권4호
• /
• pp.319-326
• /
• 2009

Extracellular ATP elicits diverse physiological effects by binding to the G-protein-coupled P2Y receptors on the plasma membrane. In addition to the short-term effects of extracellular nucleotides on cell functions, there is evidence that such purinergic signalling can have long-term effects on cell proliferation, differentiation and death. The 3T3-L1 cell line derived from mouse embryo is a well-established and commonly utilized in vitro model for adipocytes differentiation and function. However, the distributions and roles of P2Y subtypes are still unknown in the preadipocyte. In this study, we identified the distributions and roles of P2Y subtypes in preadipocyte using $Ca^{2+}$ imaging and realtime PCR. ATP increased the $[Ca^{2+}]_i$ in a concentration-dependent manner. ATP increased $Ca^{2+}$ in absence and/or presence of extracellular $Ca^{2+}$. Suramin, non-selective P2Y blocker, largely blocked the ATP-induced $Ca^{2+}$ response. U73122, a PLC inhibitor, completely inhibited $Ca^{2+}$ mobilization in 3T3-L1 cells. The mRNA expression by realtime PCR of P2Y subtypes was $P2Y_2:P2Y_5:P2Y_6=1.0:12.5:0.3$. In conclusion, we showed that $P2Y_5$ receptor is a dominant purinergic receptor in preadipocytes, and multiple P2Y receptors could involve in differentiation and migration via regulating of intracellular calcium concentration.

• Biomolecules & Therapeutics
• /
• 제29권1호
• /
• pp.90-97
• /
• 2021

Ultraviolet B (UVB) radiation causes DNA base modifications. One of these changes leads to the generation of 8-oxoguanine (8-oxoG) due to oxidative stress. In human skin, this modification may induce sunburn, inflammation, and aging and may ultimately result in cancer. We investigated whether phloroglucinol (1,3,5-trihydroxybenzene), by enhancing the expression and activity of 8-oxoG DNA glycosylase 1 (Ogg1), had an effect on the capacity of UVB-exposed human HaCaT keratinocytes to repair oxidative DNA damage. Here, the effects of phloroglucinol were investigated using a luciferase activity assay, reverse transcription-polymerase chain reactions, western blot analysis, and a chromatin immunoprecipitation assay. Phloroglucinol restored Ogg1 activity and decreased the formation of 8-oxoG in UVB-exposed cells. Moreover, phloroglucinol increased Ogg1 transcription and protein expression, counteracting the UVB-induced reduction in Ogg1 levels. Phloroglucinol also enhanced the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) as well as Nrf2 binding to an antioxidant response element located in the Ogg1 gene promoter. UVB exposure inhibited the phosphorylation of protein kinase B (PKB or Akt) and extracellular signal-regulated kinase (Erk), two major enzymes involved in cell protection against oxidative stress, regulating the activity of Nrf2. Akt and Erk phosphorylation was restored by phloroglucinol in the UVB-exposed keratinocytes. These results indicated that phloroglucinol attenuated UVB-induced 8-oxoG formation in keratinocytes via an Akt/Erk-dependent, Nrf2/Ogg1-mediated signaling pathway.

• Molecules and Cells
• /
• 제27권5호
• /
• pp.563-570
• /
• 2009

We previously isolated the OsCBT gene, which encodes a calmodulin (CaM)-binding protein, from a rice expression library constructed from fungal elicitor-treated rice suspension cells. In order to understand the function of OsCBT in rice, we isolated and characterized a T-DNA insertion mutant allele named oscbt-1. The oscbt-1 mutant exhibits reduced levels of OsCBT transcripts and no significant morphological changes compared to wild-type plant although the growth of the mutant is stunted. However, oscbt-1 mutants showed significant resistance to two major rice pathogens. The growth of the rice blast fungus Magnaporthe grisea, as well as the bacterial pathogen Xanthomonas oryzae pv. oryzae was significantly suppressed in oscbt-1 plants. Histochemical analysis indicated that the hypersensitive-response was induced in the oscbt-1 mutant in response to compatible strains of fungal pathogens. OsCBT expression was induced upon challenge with fungal elicitor. We also observed significant increase in the level of pathogenesis-related genes in the oscbt-1 mutant even under pathogen-free condition. Taken together, the results support an idea that OsCBT might act as a negative regulator on plant defense.

• 한국어병학회지
• /
• 제20권2호
• /
• pp.119-127
• /
• 2007

Phosphoinositide-specific phospholipase Cδ (PLCδ) plays an important role in many cellular responses and is involved in the production of second messenger. The present study was conducted to characterize the catalytic and regulatory properties of the PLCδ of Misgurnus mizolepis (ML-PLCδ). The ML-PLCδ gene was cloned and expressed under according to the method of the previous report (Kim et al., 2004), and its recombinant protein was purified by successive chromatography using Ni2+-NTA affinity column. The recombinant ML-PLCδ showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bisphosphate (PIP2) or phosphatidylinositol (PI). Its activity was absolutely Ca2+-dependence, which was similar to mammalian PLCδ isozymes. The Ca2+ concentration yielding maximal activation of ML-PLCδ was 100 μM. However, the activity was decreased interestingly by a polyamine, such as spermine and spermidine. In vitro assay using cholate-micelle cell, ML-PLCδ activity was inhibited in dose-dependent manner by sphinogosine but increased by phosphocholine . In the lipid-binding assay, ML-PLCδ was strongly bound to LPA, PI(3)P, PI(4)P, PI(5)P, PI(3,5)P2, PI(4,5)P2, PI(3,4,5)P3 and PA, but it showed the low affinity to S1P, PI(3,4)P2 and PS. Taken together our results, it is suggested that the general catalytic and regulatory properties of ML-PLCδ are similar with those of mammalian PLCδ1 isozymes, but the N-terminal extended piscine phospholipase Cδ1 (ML-PLCδ) might reflect some distinctions in regulatory properties and inositol-lipid binding specificity between piscine ML-PLCδ and mammalian PLCδ isozymes.

• Applied Biological Chemistry
• /
• 제39권6호
• /
• pp.494-500
• /
• 1996

중요한 환경문제로 대두되고 있는 중금속 오염의 대처 방안으로 식물을 이용한 경제적인 정화 방법을 모색하고자 미나리의 중금속 흡수능력을 검정하고 카드늄 결합단백질을 동정하였다. 미나리의 중금속 흡수능력은 미나리를 카드늄 $(Cd^{2+})$, 크롬 $(Cr^{3+})$ 및 납 $(Pb^{2+})$의 농도를 달리한 배양액에서 재배한 후 미나리에 잔류하는 중금속을 정량함으로써 측정되었다. 중금속의 처리 기간은 3일과 7일로 하였고, 생장 저해가 일어나는 농도까지 처리하였다. 카드늄은 16.68 ppm, 크롬은 20 ppm까지 지속적으로 잔류량이 증가하였고 그 농도 이상에서는 생장 저해가 일어나면서 잔류량의 증가율이 둔화되었다. 식물체 부위별 카드늄과 크롬의 잔류량은 뿌리에서 월등하게 높게 나타났고 줄기와 잎의 순으로 낮아졌다. 납의 경우는 카드늄과 크롬에 비하여 잔류량이 가장 높은 뿌리에서 4배 정도 잔류량이 높게 나타났다. 20 ppm의 중금속 용액에서 7일간 재배한 미나리 뿌리에는 건조중량 1 g에 대하여, 카드늄이 6.1 mg, 크롬은 5.2 mg 그리고 납은 23.6 mg이 잔류하고 있었다. 이것은 잔류하고 있는 중금속 중에서 카드늄은 80%, 크롬은 92%그리고 납은 96%이상이 미나리 뿌리에 잔류하고 있는 것이다. 20 ppm카드늄이 함유된 배양액에서 7일간 자란 미나리 뿌리 추출액을 Sephadex G-50과 DEAE-Cellulose 크로마토그래피를 수행하여, polyacrylamide gel 전기영동 상으로 단일 단백질 분리대의 중금속 결합단백질을 정제하였다. 이 단백질의 분자량은 gel filtration 상에서 약 5,000 Da이었고 아미노산의 조성을 보면 산성 아미노산이 27.3%, cystein이 9.9%로서 중금속 결합단백질의 특성을 가지고 있었다. 그러나 그 아미노산 조성은 지금까지 알려진 phytochelatin과는 다른 새로운 중금속 결합단백질로 나타났다.

• BMB Reports
• /
• 제40권5호
• /
• pp.723-730
• /
• 2007

Kinesin is an ATP-driven microtubule motor protein that plays important roles in control of microtubule dynamics, intracellular transport, cell division and signal transduction. The kinesin superfamily is composed of numerous members that are classified into 14 subfamilies. Animal kinesins have been well characterized. In contrast, plant kinesins have not yet to be characterized adequately. Here, a novel plant-specific kinesin gene, GhKCH2, has been cloned from cotton (Gossypium hirsutum) fibers and biochemically identified by prokaryotic expression, affinity purification, ATPase activity assay and microtubule-binding analysis. The putative motor domain of GhKCH2, $M_{396-734}$ corresponding to amino acids Q396-N734 was fused with 6$\times$His-tag, soluble-expressed in E. coli and affinity-purified in a large amount. The biochemical analysis demonstrated that the basal ATPase activity of $M_{396-734}$ is not activated by $Ca^{2+}$, but stimulated 30-fold max by microtubules. The enzymatic activation is microtubule-concentration-dependent, and the concentration of microtubules that corresponds to half-maximum activation was about 11 ${\mu}M$, much higher than that of other kinesins reported. The cosedimentation assay indicated that $M_{396-734}$ could bind to microtubules in vitro whenever the nucleotide AMP-PNP is present or absent. As a plant-specific microtubule-dependent kinesin with a lower microtubule-affinity and a nucleotide-independent microtubule-binding ability, cotton GhKCH2 might be involved in the function of microtubules during the deposition of cellulose microfibrils in fibers or the formation of cell wall.

• Asian Pacific Journal of Cancer Prevention
• /
• 제16권8호
• /
• pp.3339-3344
• /
• 2015

Background: This case-control study aimed to determine if there were any associations between the two single nucleotide polymorphisms (SNPs) in Gc, rs7041 (Asp416Glu) and rs4588 (Thr420Lys) and 3 common cancers (breast, lung and colorectal) in Thai patients. Materials and Methods: Two hundred and eighty two colorectal, 101 breast and 113 lung cancer patients were recruited from one institute during 2011-2013. The controls were age-matched volunteers who had a negative history of index cancers. In addition, vitamin D levels were compared among different genotypes in the 2 SNPs. Results: The minor allele frequencies of rs7041 (G) and rs4588 (A) were 0.32 and 0.24, respectively. Under the dominant model, the study found significant associations between minor-allele genotypes of the SNP rs7041 (TG/GG) and lung cancer (odds ratio [OR] 1.78, 95% CI 1.05-3.03). When subgroup analysis was performed according to sex and age at diagnosis, the study found that the minor-allele genotypes of rs7041 (TG/GG) were significantly associated with colorectal cancer in patients whose age at diagnosis was more than 60 years (OR 1.67, 95%CI 1.06-2.61) and the minor-allele genotypes of rs4588 (CA/AA) were significantly associated with colorectal cancer in males aged 60 years or less (OR 2.34, 95%CI 1.25-4.37). When SNP combinations (rs7041-rs4588) were examined, the TT-CA combination had a significant protective association with lung cancer (OR 0.44, 95% CI 0.22-0.85). On evaluation of serum 25(OH)D levels in 205 individuals without cancer (males 144, females 61), the proportion of subjects with low serum vitamin D (< 20 ng/ml) in those harboring CA or AA genotypes of rs4588 (41.7%) was significantly higher than the CC genotype (15.5%, p-value < 0.01). Conclusions: Genetic polymorphisms in Gc were associated with lung and colorectal cancers in Thai patients. Lower serum 25(OH)D in minor variants of rs4588 may explain this association.

• Journal of Microbiology
• /
• 제41권3호
• /
• pp.239-247
• /
• 2003

The LRV1-4 capsid protein possesses an endoribonuclease activity that is responsible for the single site-specific cleavage in the 5' untranslated region (UTR) of its own viral RNA genome and the formation of a conserved stem-loop structure (stem-loop IV) in the UTR is essential for the accurate RNA cleavage by the capsid protein. To delineate the nucleotide sequences, which are essential for the correct formation of the stem-loop structure for the accurate RNA cleavage by the viral capsid protein, a wildtype minimal RNA transcript (RNA 5' 249-342) and several synthetic RNA transcripts encoding point-mutations in the stem-loop region were generated in an in vitro transcription system, and used as substrates for the RNA cleavage assay and RNase mapping studies. When the RNA 5' 249-342 transcript was subjected to RNase T1 and A mapping studies, the results showed that the predicted RNA secondary structure in the stem-loop region using FOLD analysis only existed in the presence of Mg$\^$2＋/ ions, suggesting that the metal ion stabilizes the stem-loop structure of the substrate RNA in solution. When point-mutated RNA substrates were used in the RNA cleavage assay and RNase T1 mapping study, the specific nucleotide sequences in the stem-loop region were not required for the accurate RNA cleavage by the viral capsid protein, but the formation of a stem-loop like structure in a region (nucleotides from 267 to 287) stabilized by Mg$\^$2＋/ ions was critical for the accurate RNA cleavage. The RNase T1 mapping and EMSA studies revealed that the Ca$\^$2＋/ and Mn$\^$2＋/ ions, among the reagents tested, could change the mobility of the substrate RNA 5' 249-342 on a gel similarly to that of Mg$\^$2＋/ ions, but only Ca$\^$2＋/ ions identically showed the stabilizing effect of Mg$\^$2＋/ ions on the stem-loop structure, suggesting that binding of the metal ions (Mg$\^$2＋/ or Ca$\^$2＋/) onto the RNA substrate in solution causes change and stabilization of the RNA stem-loop structure, and only the substrate RNA with a rigid stem-loop structure in the essential region can be accurately cleaved by the LRV1-4 viral capsid protein.