• 제목/요약/키워드: Light chain gene

검색결과 85건 처리시간 0.038초

Melatonin inhibits the Migration of Colon Cancer RKO cells by Down-regulating Myosin Light Chain Kinase Expression through Cross-talk with p38 MAPK

  • Zou, Duo-Bing;Wei, Xiao;Hu, Ruo-Lei;Yang, Xiao-Ping;Zuo, Li;Zhang, Su-Mei;Zhu, Hua-Qing;Zhou, Qing;Gui, Shu-Yu;Wang, Yuan
    • Asian Pacific Journal of Cancer Prevention
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    • 제16권14호
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    • pp.5835-5842
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    • 2015
  • Background: Melatonin, which is mainly produced by the pineal gland, has a good inhibitory effect on cell growth of multiple cancer types. However, the underlying molecular mechanisms of anti-tumor activity for colon cancer have not been fully elucidated. In this study, we investigated the effects of melatonin on migration in human colon cancer RKO cells and the potential molecular mechanisms. Materials and Methods: The viability of RKO cells was investigated by MTT assay after treatment with melatonin, SB203580 (p38 inhibitor) and phorbol 12-myristate 13-acetate (PMA, MAPK activator) alone or in combination for 48h. The effects of melatonin, and ML-7, a selective inhibitor of myosin light chain kinase (MLCK), and SB203580, and PMA on the migration of RKO cells were analyzed by in vitro scratch-wound assay. The relative mRNA levels of MLCK was assessed by real-time quantitative RT-PCR. Western blotting analysis was performed to examine the expression of MLCK, phosphorylation of myosin light chain (pMLC) and p38 (pp38). Results: The proliferation and migration of human colon cancer RKO cells were inhibited significantly after treatment with melatonin. The expression levels of MLCK and phosphorylation of MLC of RKO cells were reduced, and real-time quantitative RT-PCR showed that melatonin had significant effects on suppressing the expression of MLCK. Furthermore, the phosphorylation level of p38, which showed the same trend, was also reduced when cells were treated by melatonin. In addition, ML-7 (25umol/l) could down-regulate the phosphorylation of p38. Conclusions: Melatonin could inhibit the proliferation and migration of RKO cells, and further experiments confirmed that p38 MAPK plays an important role in regulating melatonin-induced migration inhibition through down-regulating the expression and activity of MLCK.

Relationships of Cocaine and Amphetamine Regulated Transcript with Serotonin in the Brain

  • Park, S. H.;B. S. Kwon;J. R. Chun;J. W. Jahng;Lee, H. T.;K. S. Chung
    • 한국동물번식학회:학술대회논문집
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    • 한국동물번식학회 2001년도 춘계학술발표대회
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    • pp.51-51
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    • 2001
  • Cocaine and amphetamine-regulated transcript (CART) is a satiety factor that is regulated by leptin. It was reported that the mice intracerebroventricularly injected with CART showed behavioral changes resembled with the typical behavioral alterations found in the mice carrying disorders in the brain serotonergic (5-HT) system. Hence, this study was conducted to find out the relationships between CART and 5-HT. We first examined the mRNA levels of CART after the injections of para-chlorophenylalanine (pCPA, 300 mg/kg i.p., single injection or daily for three consecutive days) in the rat brains by in situ hybridization using the mouse CART cDNA probe cloned in our laboratory. Systemic administrations of pCPA, a potent inhibitor of tryptophan hydroxylase, the rate limiting enzyme of 5-HT biosynthesis, acutely depletes the brain 5-HT transporter (5-HTT) in the dorsal raphe nucleus (DRN), which reuptakes terminal 5-HT. Results indicated that the mRNA level of CART significantly decreased in the arcuate nucleus, paraventricular nucleus, and lateral hypothalamic nucleus by three days of daily injection with pCPA with no noticeable change detected 24 hrs after the single injection. The message levels of 5-HTT in DRN decreased in both single and three days of injections. Secondly, to investigate whether CART affect to 5-HT, mouse genomic CART gene, which is consist of 3 exons and 2 introns and mouse neurofilament light (NF-L) chain promoter were cloned. Then, we constructed neuron specific expression vector, which was transfected into HeLa cell using lipid-mediated transfection system. Expression of GFP and CART linked to NF-L-chain promoter in the transfected HeLa cell were detected by using fluorescent microscope and RT-PCR. These results confirmed normal expression of DNA constructs in vitro. Then, to increase brain specific expression of CART in vivo transgenic mice carrying CART gene controlled the deleted NF-L-chain promoter were generated by the DNA microinjection into pronuclei of fertilized embryos. Transgenic mice were detected by Southern blot. Further study is necessary to examine CART expression and 5-HTT in these transgenic mice. Therefore, these results suggest that there maybe a positive molecular correlation between CART and 5-HT in responding to the stimuli.

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MYLK Polymorphism Associated with Blood Eosinophil Level among Asthmatic Patients in a Korean Population

  • Lee, Soo Ok;Cheong, Hyun Sub;Park, Byung Lae;Bae, Joon Seol;Sim, Won Chul;Chun, Ji-Yong;Isbat, Mohammad;Uh, Soo-Taek;Kim, Yong Hooun;Jang, An-Soo;Park, Choon-Sik;Shin, Hyoung Doo
    • Molecules and Cells
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    • 제27권2호
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    • pp.175-181
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    • 2009
  • The myosin light chain kinase (MYLK) gene encodes both smooth muscle and nonmuscle cell isoforms. Recently, polymorphisms in MYLK have been reported to be associated with several diseases. To examine the genetic effects of polymorphisms on the risk of asthma and related phenotypes, we scrutinized MYLK by re-sequencing/genotyping and statistical analysis in Korean population (n = 1,015). Seventeen common polymorphisms located in or near exons, having pairwise $r^2$ values less than 0.25, were genotyped. Our statistical analysis did not replicate the associations with the risk of asthma and log-transformed total IgE levels observed among African descendant populations. However, two SNPs in intron 16 (+89872C> G and +92263T> C), which were in tight LD (|D'| = 0.99), revealed significant association with log-transformed blood eosinophil level even after correction multiple testing ($P=0.002/P^{corr}=0.01$ and $P=0.002/P^{corr}=0.01$, respectively). The log-transformed blood eosinophil levels were higher in individuals bearing the minor alleles for +89872C> G and +92263T> C than in those bearing other allele. In additional subgroup analysis, the genetic effects of both SNPs were much more apparent among asthmatic patients and atopic asthma patients. Among atopic asthma patients, the log-transformed blood eosinophil levels were proportionally increased by gene-dose dependent manner of in both +89872C> G and +92263T> C(P = 0.0002 and P = 0.00007, respectively). These findings suggest that MYLK polymorphisms might be among the genetic factors underlying differential increases of blood eosinophil levels among asthmatic patients. Further biological and/or functional studies are needed to confirm our results.

A Novel Mutation in the DNA Binding Domain of NFKB is Associated with Speckled Leukoplakia

  • Govindarajan, Giri Valanthan Veda;Bhanumurthy, Lokesh;Balasubramanian, Anandh;Ramanathan, Arvind
    • Asian Pacific Journal of Cancer Prevention
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    • 제17권7호
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    • pp.3627-3629
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    • 2016
  • Background: Activation and inactivation of nuclear factor of kappa light chain gene enhancer in B cells (NFKB) is tightly regulated to ensure effective onset and cessation of defensive inflammatory signaling. However, mutations within NFKB, or change in activation and inactivation molecules have been reported in a few cancers. Although oral squamous cell carcinoma is one of the most prevalent forms of cancer in India, with a development associated with malignant transformation of precancerous lesions, the genetic status of NFKB and relative rates of change in oral precancerous lesions remain unknown. Hence in the present study we investigated all twenty four exons of NFKB gene in two precancerous lesions, namely oral submucous fibrosis (OSMF) and oral leukoplakia (OL) to understand its occurrence, incidence and assess its possible contribution to malignant transformation. Materials and Methods: Chromosomal DNA isolated from twenty five each of OSMF and OL tissue biopsy samples were subjected to PCR amplification with intronic primers flanking twenty four exons of the NFKB gene. The PCR amplicons were subsequently subjected to direct sequencing to elucidate the mutation status. Results: Sequence analysis identified a novel heterozygous mutation, c.419T>A causing substitution of leucine with glutamine at codon 140 (L140Q) in an OL sample. Conclusions: The identification of a substitution mutation L140Q within the DNA binding domain of NFKB in OL suggests that NFKB mutation may be relatively an early event during transformation. To the best of our knowledge, this study is the first to have identified a missense mutation in NFKB in OL.

Genetic Screening of the Dazl-Interacting Protein Genes

  • Lee, Kyung-Ho;Lee, Seong-Ju;Rhee, Kun-Soo
    • Animal cells and systems
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    • 제10권4호
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    • pp.227-231
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    • 2006
  • Micro-deletions at specific loci of the Y chromosome have been observed frequently in male infertility patients, suggesting that genes in these regions are involved in male germ cell development. DAZ is a representative male infertility gene at the AZFc locus of the Y chromosome. Since DAZ contains an RNA binding motif along with so-called a DAZ domain, it was proposed to participate in RNA metabolism during spermatogenesis. A mouse gene homologous to the human DAZ gene has been cloned and named Dazl (DAZlike). Dazl is autosomal and expressed in the testis and also at a low level in the ovary. Male mice homozygous for the Dazl null allele have small testes with a few spermatogonia and almost complete absence of germ cells beyond the spermatogonial stage, suggesting the requirement of Dazl for entry or progression through meiosis. However, its exact cellular functions have not been understood yet. In order to investigate cellular functions of Dazl, we decided to isolate candidate interacting protein genes of the mouse Dazl, using yeast two-hybrid screening. A number of candidate Dazlinteracting proteins have been isolated, such as Bprp, Acf, Hgs, Murr1, Nbak3 and Ranbp9, but dynein light chain 1 (Dlc1) was most predominant. A strong interaction of Dazl with Dlc1 suggests that Dazl might function as an mRNA adaptor to the dynein motor complex.

Effect of Adjuvants on Antibody Titer of Synthetic Recombinant Light Chain of Botulinum Neurotoxin Type B and its Diagnostic Potential for Botulism

  • Jain, Swati;Ponmariappan, S.;Kumar, Om;Singh, Lokendra
    • Journal of Microbiology and Biotechnology
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    • 제21권7호
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    • pp.719-727
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    • 2011
  • Botulism is a neuroparalytic disease caused by Clostridium botulinum, which produces seven (A-G) antigenically diverse neurotoxins (BoNTs). BoNTs are the most poisonous substances known to humans, with a median lethal dose ($LD_{50}$) of approximately 1 ng/kg of body weight. Owing to their extreme potency and lethality, they have the potential to be used as a bioterrorism agent. The mouse bioassay is the gold standard for the detection of botulinum neurotoxins; however, it requires at least 3-4 days for completion. Attempts have been made to develop an ELISA-based detection system, which is potentially an easier and more rapid method of botulinum neurotoxin detection. The present study was designed using a synthetic gene approach. The synthetic gene encoding the catalytic domain of BoNT serotype B from amino acids 1-450 was constructed with PCR overlapping primers (BoNT/B LC), cloned in a pQE30 UA vector, and expressed in an E. coli M15 host system. Recombinant protein production was optimized at 0.5 mM IPTG final concentration, 4 h post induction, resulting in a maximum yield of recombinant proteins. The immunogenic nature of the recombinant BoNT/B LC protein was evaluated by ELISA. Antibodies were raised in BALB/c mice using various adjuvants. A significant rise in antibody titer (p<0.05) was observed in the Alum group, followed by the Titermax Classic group, Freund's adjuvant, and the Titermax Gold group. These developed high-titer antibodies may prove useful for the detection of botulinum neurotoxins in food and clinical samples.

Disentangling Evolutionary Pattern and Haplotype Distribution of Starch Synthase III-1 (SSIIIb) in Korean Rice Collection

  • Bhagwat Nawade ;Yong-Jin Park
    • 한국작물학회:학술대회논문집
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    • 한국작물학회 2022년도 추계학술대회
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    • pp.214-214
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    • 2022
  • Soluble starch synthases (SSs) elongate α-glucans from ADP-Glc to the glucan nonreducing ends and play a critical role in synthesizing resistant starch in the rice. A total of 10 SSs isoforms were reported in rice, including granules-bound starch synthase I (GBSSI), GBSSII, starch synthase I (SSI), SSIIa (SSII-3), SSIIb (SSII-2), SSIIc (SSII-1), SSIIIa (SSIII-2), SSIIIb (SSIII-1), SSIVa (SSIV-1), and SSIVb (SSIV-2). SSIII proteins are involved in forming the B chain and elongating cluster filling chains in amylopectin metabolism. The functions of SSIIIb (SSIII-1) are less clear as compared to SSs. Here, we sought to shed light on the genetic diversity profiling of the SSIII-1 gene in 374 rice accessions composed of 54 wild-type accessions and 320 bred cultivars (temperate japonica, indica, tropical japonica, aus, aromatic, and admixture). In total, 17 haplotypes were identified in the SSIII-1 coding region of 320 bred cultivars, while 44 haplotypes were detected from 54 wild-type accessions. The genetic diversity indices revealed the most negative Tajima's D value in the temperate-japonica, followed by the wild type, while Tajima's D values in other ecotypes were positive, indicating balancing selection. Nucleotide diversity in the SSIII-1 region was highest in the wild group (0.0047) while lowest in temperate-japonica. Lower nucleotide diversity in the temperate-japonica is evidenced by the negative Tajima's D and suggested purifying selection. The fixation index (FST) revealed a very high level of gene flow (low FST) between the tropical-japonica and admixture groups (FST=-0.21) followed by admixture and wild groups (-0.04), indica and admixture groups (0.02), while low gene flow with higher FST estimates between the temperate-japonica and aus groups (0.72), tropical-japonica and aromatic groups (0.71), and temperate-japonica and admixture groups (0.52). Taken together, our study offers insights into haplotype diversity and evolutionary fingerprints of SSIII-1. It provides genomic information to increase the resistant starch content of cooked rice.

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