• The Korean Journal of Physiology and Pharmacology
• /
• 제16권1호
• /
• pp.31-36
• /
• 2012

The receptor activator of NF-${\kappa}B$ ligand (RANKL) signal is an activator of tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of NF-${\kappa}B$ and other signal transduction pathways essential for osteoclastogenesis, such as $Ca^{2+}$ signaling. However, the intracellular levels of inositol 1,4,5-trisphosphate ($IP_3$) and $IP_3$-mediated cellular function of RANKL during osteoclastogenesis are not known. In the present study, we determined the levels of $IP_3$ and evaluated $IP_3$-mediated osteoclast differentiation and osteoclast activity by RANKL treatment of mouse leukemic macrophage cells (RAW 264.7) and mouse bone marrow-derived monocyte/macrophage precursor cells (BMMs). During osteoclastogenesis, the expression levels of $Ca^{2+}$ signaling proteins such as $IP_3$ receptors ($IP_3Rs$), plasma membrane $Ca^{2+}$ ATPase, and sarco/endoplasmic reticulum $Ca^{2+}$ ATPase type2 did not change by RANKL treatment for up to 6 days in both cell types. At 24 h after RANKL treatment, a higher steady-state level of $IP_3$ was observed in RAW264.7 cells transfected with green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of phospholipase C (PLC) ${\delta}$, a probe specifically detecting intracellular $IP_3$ levels. In BMMs, the inhibition of PLC with U73122 [a specific inhibitor of phospholipase C (PLC)[ and of $IP_3Rs$ with 2-aminoethoxydiphenyl borate (2APB; a non-specific inhibitor of $IP_3Rs$) inhibited the generation of RANKL-induced multinucleated cells and decreased the bone-resorption rate in dentin slice, respectively. These results suggest that intracellular $IP_3$ levels and the $IP_3$-mediated signaling pathway play an important role in RANKL-induced osteoclastogenesis.

• Molecules and Cells
• /
• 제27권3호
• /
• pp.321-327
• /
• 2009

Voltage-dependent anion channels (VDACs) are reported to be porin-type, ${\beta}$-barrel diffusion pores. They are prominently localized in the outer mitochondrial membrane and are involved in metabolite exchange between the organelle and the cytosol. In this study, we have investigated a family of VDAC isoforms in Arabidopsis thaliana (AtVDAC). We have shown that the heterologous expression of AtVDAC proteins can functionally complement a yeast mutant lacking the endogenous mitochondrial VDAC gene. AtVDACs tagged with GFP were localized to mitochondria in both yeast and plant cells. We also looked at the response of AtVDACs to biotic and abiotic stresses and found that four AtVDAC transcripts were rapidly up-regulated in response to a bacterial pathogen.

• Molecules and Cells
• /
• 제40권12호
• /
• pp.976-985
• /
• 2017

Iron is an essential divalent ion for aerobic life. Life has evolved to maintain iron homeostasis for normal cellular and physiological functions and therefore imbalances in iron levels exert a wide range of consequences. Responses to iron dysregulation in blood development, however, remain elusive. Here, we found that iron homeostasis is critical for differentiation of Drosophila blood cells in the larval hematopoietic organ, called the lymph gland. Supplementation of an iron chelator, bathophenanthroline disulfate (BPS) results in an excessive differentiation of the crystal cell in the lymph gland. This phenotype is recapitulated by loss of Fer1HCH in the intestine, indicating that reduced levels of systemic iron enhances crystal cell differentiation. Detailed analysis of Fer1HCH-tagged-GFP revealed that Fer1HCH is also expressed in the hematopoietic systems. Lastly, blocking Fer1HCH expression in the mature blood cells showed marked increase in the blood differentiation of both crystal cells and plasmatocytes. Thus, our work suggests a relevance of systemic and local iron homeostasis in blood differentiation, prompting further investigation of molecular mechanisms underlying iron regulation and cell fate determination in the hematopoietic system.

• Horticultural Science & Technology
• /
• 제30권3호
• /
• pp.243-249
• /
• 2012

Hexokinase is the first enzyme in the hexose assimilation pathway; it acts as a sensor for plant sugar responses, and it is also important in determining the fruit sugar levels. The full-length cDNA of a hexokinase gene was isolated from loquat through reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends, which was designated as EjHXK1. EjHXK1 is 1,839 bp long and contains an entire open reading frame encoding 497 amino acids. The predicted protein of EjHXK1 shares 72%-81% similarity with other plant hexokinases. Phylogeny analysis indicated that EjHXK1 is closely related to maize and rice hexokinases. Transient expression of the 35S: EjHXK1-GFP fusion protein was observed on the cell membrane and cytoplasm. Real-time RT-PCR indicated that EjHXK1 is expressed in loquat leaves, stems, flowers, and fruits. EjHXK1 transcripts were higher during early fruit development, but decreases before maturation, which is consistent with hexokinase enzyme activity during fruit development and conducive for hexose accumulation in mature fruits. These results imply that EjHXK1 may play important roles in the regulation of sugar flux during fruit ripening.

• Korean Journal of Pharmacognosy
• /
• 제47권2호
• /
• pp.179-185
• /
• 2016

Ginger (Zingiber officnale Roscoe, Zingiberaceae), which is a well-known food seasoning, has been used as a traditional medicine for the treatment of gastrointestinal disorder, vomiting and cough in Korea, China and Japan. Ethanol extract from the dried ginger (DG) was successively partitioned as methylene chloride, ethyl acetate, n-butanol and $H_2O$ soluble fractions. Among those fractions the ethyl acetate soluble fraction (EDG) showed the most potent DPPH radical scavenging and superoxide quenching activities. To know the effect of antioxidant activities of EDG, we tested the activities of superoxide dismutase (SOD) and catalase together with oxidative stress tolerance and intracellular ROS level in Caenorhabditis elegans. To investigate whether EDG-mediated increased stress tolerance was due to regulation of stress-response gene, we quantified SOD-3 expression using transgenic strain including CF1553. Consequently, EDG elevated SOD and catalase activities of C. elegans, reduced intracellular ROS accumulation in a dose-dependent manner. Moreover, EDG-treated CF1553 worms exhibited significantly higher SOD-3::GFP intensity.

• Asian-Australasian Journal of Animal Sciences
• /
• 제23권2호
• /
• pp.272-278
• /
• 2010

In this study, we cloned, sequenced and characterized porcine y+L Amino Acid Transporter-1 (y+LAT1). By screening a translated EST database with the protein sequence of the human $y^{+}$LAT1 and by using rapid amplification of cDNA ends (RACE), the full-length cDNA encoding porcine $y^{+}$LAT1 was isolated from porcine intestine RNA. It was 2,111 bp long, encoding a 511 amino acid trans-membrane glycoprotein composed of 12 transmembrane domains. The predicted amino acid sequence was found to be 91%, 90%, 87% and 87% identical to those of cattle, human, mouse and rat $y^{+}$LAT1 respectively. Real-time RT-PCR results indicated that the small intestine had the highest $y^{+}$LAT1 mRNA abundance and the lung had the lowest $y^{+}$LAT1 mRNA abundance. Baby hamster kidney (BHK) cells transfected with green fluorescent protein (GFP) tagged porcine $y^{+}$LAT1 cDNA indicated that the cellular localization of the gene product in BHK was on the plasma membrane.

• Fisheries and Aquatic Sciences
• /
• 제17권3호
• /
• pp.377-380
• /
• 2014

RNA interference (RNAi)-mediated transcriptional knock-down of Crassostrea gigas big defensin 1 and 2 genes (Cg-BigDef1 and Cg-BigDef2) was investigated. The cDNA sequences of Cg-BigDef1 and Cg-BigDef2 were identical, excluding an additional fragment of 20 nucleotides in Cg-BigDef1; thus, a long double-stranded RNA (dsRNA) targeting the mRNA of Cg-BigDef2 effectively downregulated both Cg-BigDef2 and Cg-BigDef1. In addition, long dsRNA targeting green fluorescent protein (GFP) did not affect transcription of the two big defensin genes. These results suggest that the transcriptional downregulation of Cg-BigDef1 and Cg-BigDef2 was mediated by sequence-specific RNA interference (RNAi). Despite injection of long dsRNA targeting Cg-BigDef2 into only the adductor muscle, knock-down of Cg-BigDef1 and Cg-BigDef2 was observed in the adductor muscle, hemocytes, mantle, and gills, suggestive of systemic spread of RNAi in C. gigas. Furthermore, the inhibitory effect of dsRNA persisted until 72 h post-injection, indicative of a long-lasting RNAi-mediated knock-down of target genes.

• Korean Journal of Pharmacognosy
• /
• 제49권1호
• /
• pp.40-46
• /
• 2018

Chaenomeles sinensis (Thouin) Koehne fruit (Rosaceae) has been used as a traditional medicine in Korea, Japan and China to treat sore throat, diarrhea and inflammation. The ethanol extract of C. sinensis fruit was successively partitioned as methylene chloride, ethyl acetate, n-butanol and $H_2O$ soluble fractions. Among those fractions, the n-butanol fraction showed the most potent DPPH radical scavenging and superoxide quenching activities. To verify antioxidant activities, the n-butanol fraction was checked the activities of superoxide dismutase (SOD) and catalase activities, and intracellular ROS levels and oxidative stress tolerance in Caenorhabditis elegans. Furthermore, to see if increased stress tolerance of worms by treating of the n-butanol fraction was due to regulation of stress-response gene, we quantified SOD-3 expression using transgenic strain. Consequently, the n-butanol fraction elevated SOD and catalase activities of C. elegans, and reduced intracellular ROS accumulation in a dose-dependent manner. Moreover, the n-butanol fraction-treated CF1553 worms exhibited significantly higher SOD-3::GFP intensity.

• Molecules and Cells
• /
• 제28권2호
• /
• pp.93-98
• /
• 2009

A plant-specific gene was cloned from melon fruit. This gene was named downward leaf curling (CmDLC) based on the phenotype of transgenic Arabidopsis plants overexpressing the gene. This expression level of this gene was especially upregulated during melon fruit enlargement. Overexpression of CmDLC in Arabidopsis resulted in dwarfism and narrow, epinastically curled leaves. These phenotypes were found to be caused by a reduction in cell number and cell size on the adaxial and abaxial sides of the epidermis, with a greater reduction on the abaxial side of the leaves. These phenotypic characteristics, combined with the more wavy morphology of epidermal cells in overexpression lines, indicate that CmDLC overexpression affects cell elongation and cell morphology. To investigate intracellular protein localization, a CmDLC-GFP fusion protein was made and expressed in onion epidermal cells. This protein was observed to be preferentially localized close to the cell membrane. Thus, we report here a new plant-specific gene that is localized to the cell membrane and that controls leaf cell number, size and morphology.

• Molecules and Cells
• /
• 제28권1호
• /
• pp.43-48
• /
• 2009

By screening C. elegans mutants for severe defects in germline proliferation, we isolated a new loss-of-function allele of cdc-25.1, bn115. bn115 and another previously identified loss-of-function allele nr2036 do not exhibit noticeable cell division defects in the somatic tissues but have reduced numbers of germ cells and are sterile, indicating that cdc-25.1 functions predominantly in the germ line during postembryonic development, and that cdc-25.1 activity is probably not required in somatic lineages during larval development. We analyzed cell division of germ cells and somatic tissues in bn115 homozygotes with germline-specific anti-PGL-1 immunofluorescence and GFP transgenes that express in intestinal cells, in distal tip cells, and in gonadal sheath cells, respectively. We also analyzed the expression pattern of cdc-25.1 with conventional and quantitative RT-PCR. In the presence of three other family members of cdc-25.1 in C. elegans, defects are observed only in the germ line but not in the somatic tissues in cdc-25.1 single mutants, and cdc-25.1 is expressed predominantly, if not exclusively, in the germ line during postembryonic stages. Our findings indicate that the function of cdc-25.1 is unique in the germ line but likely redundant with other members in the soma.