• The Korean Journal of Physiology and Pharmacology
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• 제5권1호
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• pp.47-53
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• 2001

Drinking excessive alcohol has been recognized as a risk factor for hypertension. However, the mechanism by which alcohol intake causes hypertension still remains elusive. We tested the hypothesis that ethanol itself acts as a stress factor on vasculature and indirectly modulates vascular contractility. After end of exposure to 1, 2.5 and 5% ethanol for 45 min, rat aortic strips were subjected to contractile responses, immunoblot for Hsp70 and the measurement of levels of myosin light chain phosphorylation. Exposure to 5% ethanol not only augmented contractions to KCl or phenylephrine, but also increased expression of Hsp70 and the levels of myosin light chain phosphorylation. There were no significant differences in contractions produced by $1\;{\mu}mol/L$ phorbol 12,13-dibutyrate, a protein kinase C activator, whether the tissues were exposed to 5% ethanol or not. This is the first report to show that even short exposure to ethanol has a delayed effect to increase vascular smooth muscle contractility through a modulation of thick filament regulation. It may be a mechanism by which ingestion of alcohol induces hypertension.

• Biomolecules & Therapeutics
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• 제16권3호
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• pp.249-254
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• 2008

The present study was undertaken to determine whether hypoxia influences on the agonist-induced vascular smooth muscle contraction and, if so, to investigate the related mechanism. The measurement of isometric contractions using a computerized data acquisition system was combined with molecular experiments. Hypoxia significantly inhibited fluoride-induced contraction regardless of endothelial function, but there was no relaxation on thromboxane $A_2$ mimetic U-46619-induced contraction suggesting that other pathway such as $Ca^{2+}$ entry or thin filament regulation was not affected. In addition, hypoxia significantly decreased fluoride-induced increase of phospho-myosin-targeting subunit of myosin light chain phosphatase (pMYPT1). Interestingly, hypoxia didn't inhibit significantly phenylephrine-induced contraction suggesting that myosin light chain kinase (MLCK) activity or thin filament regulation is less important on the hypoxia-induced vasorelaxation in the denuded muscle than Rho-kinase activity. In conclusion, this study provides the evidence and possible related mechanism concerning the vasodilation effect of hypoxia on the agonist-specific contraction in rat aortic rings regardless of endothelial function.

• Fisheries and Aquatic Sciences
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• 제15권4호
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• pp.325-335
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• 2012

The present study characterized the fast skeletal myosin light chain-2 gene (mlc2f) in the euryhaline Javanese ricefish (Oryzias javanicus: Beloniformes). Coding nucleotide and deduced amino acid sequences of Javanese ricefish mlc2f were well conserved in the vertebrate lineage. Javanese ricefish mlc2f showed a typical seven-exon structure, and its promoter exhibited transcription factor binding motifs common to most muscle-specific genes. However, Javanese ricefish mlc2f also displayed tandem duplications of intronic sequences in both intron 1 and intron 3. Based on quantitative reverse transcription-polymerase chain reaction, the mlc2f transcripts were highly predominant in skeletal muscles of adults and were differentially modulated during embryonic development. Microinjection of the mlc2f promoter-driven red fluorescent protein (RFP) reporter construct successfully exhibited heterologous expression of the fluorescent reporter, primarily in muscular areas of hatchlings, although the distribution pattern of RFP signals was not uniform due to the mosaic nature of the introduced transgene. Data from this study indicate that the Javanese ricefish mlc2f gene has undergone "intra-intronic" duplication events in a species-specific manner and that the mlc2f regulator may also be useful in heterologous expression assays of the skeletal muscles of this species.

• 한국양식학회지
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• 제16권1호
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• pp.8-14
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• 2003

To generate expressed sequence tags for genomics research involving genetic linkage analysis, to examine gene expression profiles in muscles of channel catfish in a non-normalized muscle cDNA library, to compare gene expression in young and mature channel catfish muscles using the EST reagents and gene filters to demonstrate the feasibility of functional genomics research in small laboratories. 102 randomly picked cDNA clones were analyzed from the catfish muscle cDNA library. Of the sequences generated, 90.2% of ESTs was identified as known genes by identity comparisons. These 92 clones of known gene products represent transcriptional products of 24 genes. The 10 clones of unknown gene products represent 8 genes. The major transcripts (70.1% of the analyzed ESTs) in the catfish muscle are from many major genes involved in muscle contraction, relaxation, energy metabolism and calcium binding such as alpha actin, creatine kinase, parvalbumin, myosin, troponins, and tropomyosins. Gene expression of the unique ESTs was comparatively studied in the young and adult catfish muscles. Significant differences were observed for aldolase, myostatin, myosin light chain, parvalbumin, and an unknown gene. While myosin light chain and an unknown gene (CM 192) are down-regulated in the mature fish muscle, the aldolase, myostatin, and parvalbumin are significantly up-regulated in the mature fish muscle. Although the physiological significance of the changes in expression levels needs to be further addressed, this research demonstrates the feasibility and power of functional genomics in channel catfish. Channel catfish muscle gene expression profiles provide a valuable molecular muscle physiology blueprint for functional comparative genomics.

• Asian Pacific Journal of Cancer Prevention
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• 제15권18호
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• pp.7687-7692
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• 2014

Aim: To observe the effects of a novel all-trans retinoid acid (ATRA) derivative, N-(3-trifluoromethyl-phenyl)-retinamide (ATPR), on lung adenocarcinoma A549 cells and to explore the potential mechanism of ATPR inhibiting of A549 cell migration. Materials and Methods: The cytotoxicity of ATRA and ATPR on A549 cells was assessed using MTT assay. Wound healing assays were used to analyze the influences of ATRA, ATPR, ML-7 (a highly selective inhibitor of myosin light chain kinase (MLCK)), PMA (an activator of MAPKs) and PD98059 (a selective inhibitor of ERK1/2) on the migration of A549 cells. Expression of MLCK and phosphorylation of myosin light chain (MLC) were assessed by Western blotting. Results: ATRA and ATPR inhibited the proliferation of A549 cells in a dose- and time-dependent manner, and the effect of ATPR was much more remarkable compared with ATRA. Relative migration rate and migration distance of A549 cells both decreased significantly after treatment with ATPR or ML-7. The effect on cell migration of PD98059 combining ATPR treatment was more notable than that of ATPR alone. Moreover, compared with control groups, the expression levels of MLCK and phosphorylated MLC in A549 cells were both clearly reduced in ATRA and ATPR groups. Conclusions: ATPR could suppress the migration and invasion of A549 cells, and the mechanism might be concerned with down-regulating the expression of MLCK in the ERK-MAPK signaling pathway, pointing to therapeutic prospects in lung cancer.

• The Korean Journal of Physiology and Pharmacology
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• 제13권3호
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• pp.201-207
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• 2009

Our previous study demonstrated that flavone inhibits vascular contractions by decreasing the phosphorylation levelof the myosin phosphatase target subunit (MYPT1). In the present study, we hypothesized that flavone attenuates vascular contractions through the inhibition of the RhoA/Rho kinase pathway. Rat aortic rings were denuded of endothelium, mounted in organ baths, and contracted with either 30 nM U46619 (a thromboxane A2 analogue) or 8.0 mM NaF 30 min after pretreatment with either flavone (100 or 300 $({\mu}M$) or vehicle. We determined the phosphorylation level of the myosin light chain ($MLC_{20}$), the myosin phophatase targeting subunit 1 (MYPT1) and the protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phophatase of 17-kDa (CPI17) by means of Western blot analysis. Flavone inhibited, not only vascular contractions induced by these contractors, but also the levels of $MLC_{20}$ phosphorylation. Furthermore, flavone inhibited the activation of RhoA which had been induced by either U46619 or NaF. Incubation with flavone attenuated U46619 or NaF-induced phosphorylation of $MYPT1^{Thr855}$ and $CPI17^{Thr38}$, the downstream effectors of Rho-kinase. In regards to the $Ca^{2+}$-free solution, flavone inhibited the phosphorylation of $MYPT1^{Thr855}$ and $CPI17^{Thr38}$, as well as vascular contractions induced by U 46619. These results indicate that flavone attenuates vascular contractions, at least in part, through the inhibition of the RhoA/Rho-kinase pathway.

• The Korean Journal of Physiology and Pharmacology
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• 제13권6호
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• pp.409-416
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• 2009

Acute pancreatitis is a multifactorial disease associated with the premature activation of digestive enzymes. The genes expressed in pancreatic acinar cells determine the severity of the disease. The present study determined the differentially expressed genes in pancreatic acinar cells treated with cerulein as an in vitro model of acute pancreatitis. Pancreatic acinar AR42J cells were stimulated with $10^{-8}$ M cerulein for 4 h, and genes with altered expression were identified using a cDNA microarray for 4,000 rat genes and validated by real-time PCR. These genes showed a 2.5-fold or higher increase with cerulein: lithostatin, guanylate cyclase, myosin light chain kinase 2, cathepsin C, progestin-induced protein, and pancreatic trypsin 2. Stathin 1 and ribosomal protein S13 showed a 2.5-fold or higher decreases in expression. Real-time PCR analysis showed time-dependent alterations of these genes. Using commercially available antibodies specific for guanylate cyclase, myosin light chain kinase 2, and cathepsin C, a time-dependent increase in these proteins were observed by Western blotting. Thus, disturbances in proliferation, differentiation, cytoskeleton arrangement, enzyme activity, and secretion may be underlying mechanisms of acute pancreatitis.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2001년도 학술 발표회 진행표 및 논문초록
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• pp.24-24
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• 2001

The present study was undertaken to determine whether p38 mitogen-activated protein kinase participates in the regulation of vascular smooth muscle contraction by endothelin-I (ET-1) in rat thoracic aorta. ET-1 induced a sustained contraction. In contrast, both the intracellular Ca$\^$2＋/ and myosin light chain (MLC) phosphorylations were not sustained.(omitted)

• Asian-Australasian Journal of Animal Sciences
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• 제25권8호
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• pp.1083-1088
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• 2012

The breeding value of marbling score in skeletal muscle is an important factor for evaluating beef quality. In the present study, we investigated proteins associated with the breeding value of the marbling score for bovine sirloin to select potential biomarkers to improve meat quality through comparative proteomic analysis. Proteins isolated from muscle were separated by two-dimensional gel electrophoresis. After analyzing images of the stained gel, seven protein spots for the high marbling score group were identified corresponding to changes in expression that were at least two-fold compared to the low marbling score group. Four spots with increased intensities in the high marbling score group were identified as phosphoglycerate kinase 1, triosephophate isomerase, acidic ribosomal phosphoprotein PO, and capping protein (actin filament) Z-line alpha 2. Spots with decreased intensities in the high marbling score group compared to the low score group were identified as 14-3-3 epsilon, carbonic anhydrase II, and myosin light chain 1. Expression of myosin light chain 1 and carbonic anhydrase 2 was confirmed by Western blotting. Taken together, these data could help improve the economic performance of cattle and provide useful information about the underlying the function of bovine skeletal muscle.

• 대한약리학회지
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• 제31권3호
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• pp.291-297
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• 1995

본 연구의 목적은 protein kinase C의 활성물질인 phorbol ester의 수축에 대한 cGMP 및 cAMP의 조절기전을 명확히 하기 위하여 흰쥐의 대동맥을 재료로 실험을 수행하였다. Sodium nitroprusside는 guanylyl cyclase를 활성화시켜 cGMP를, forskolin은 adenylyl cyclase를 활성화시켜서 cAMP를 증가시키는 것으로 보고되어 있으므로 위의 두 약물을 선택하였다. Phorbol ester는 시간경과와 함께 지속적인 수축을 발생하였으며 30분경 안정상태에 도달하였다. 동시에 20-kDa myosin light chain (MLC)의 인산화도 증가하였으며 30분경 최대치를 나타내었다. Sodium nitroprusside와 forskolin은 phorbol ester에 의한 수축을 농도의존적으로 억제하였으나 sodium nitroprusside가 forskolin보다 더욱 민감하게 억제하였다. Phorbol ester는 $^{45}Ca^{2+}$의 유입을 증가시켰고 sodium nitroprusside와 forskolin은 이 증가된 $^{45}Ca^{2+}$을 유의하게 억제하였다. Phorbol ester에 의하여 증가된 MLC의 인산화는 sodium nitroprusside 및 forskolin 각각의 최대농도로 억제되었다. 이상과 같은 결과로 볼때 아마도 cGMP와 cAMP는 phorbol ester에 의한 수축을 $^{45}Ca^{2+}$ 유입억제에 이은 MLC 인산화 억제에 의하여 이완작용을 나타내는 것으로 추측되며 cGMP가 cAMP보다 protein kinase C 매개의 수축조절에 더 중요하게 작용하리라 추측된다.