• Tuberculosis and Respiratory Diseases
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• v.47 no.3
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• pp.347-355
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• 1999

Background: Phospholipase C(PLC) plays a central role in cellular signal transduction and is important in cellular growth, differentiation and transformation. There are currently ten known mammalian isozymes of PLC reported to this date. Hydrolysis of phosphatidylinositol 4,5-bisphosphate($PIP_2$) by PLC produces two important second messengers, inositol 1,4,5-trisphosphate($IP_3$) and diacylglycerol. PLC-${\gamma}1$, previously, was known to be activated mainly through growth factor receptor tyrosine kinase. Other mechanisms of activating PLC-yl have been reported such as activation through tau protein in the presence of arachidonic acid in bovine brain and activation by $IP_3$, phosphatidic acid, etc. Very recently, another PLC-${\gamma}1$ activator protein such as tau has been found in bovine lung tissue, which now is considered to be AHNAK protein. But there has been no report concerning AHNAK and its associated disease to this date. In this study, we examined the expression of the PLC-${\gamma}1$ activator, AHNAK, in lung cancer specimens and their paired normal. Methods: From surgically resected human lung cancer tissues taken from twenty-eight patients and their paired normal counterparts, we evaluated expression level of AHNAK protein using immunoblot analysis of total tissue extract Immunohistochemical stain was performed with primary antibody against AHNAK protein. Results: Twenty-two among twenty-eight lung cancer tissues showed overexpression of AHNAK protein (eight of fourteen squamous cell lung cancers, all of fourteen adenocarcinomas). The resulting bands were multiple ranging from 70 to 200 kDa in molecular weight and each band was indistinct and formed a smear, reflecting mobility shift mainly due to proteolysis during extraction process. On immunohistochemistry, lung cancer tissues showed a very heavy, dense staining with anti-AHNAK protein antibody as compared to the surrounding normal lung tissue, coresponding well with the results of the western blot Conclusion: The overexpression of PLC-${\gamma}1$ activator protein, AHNAK in lung cancer may provide evidence that the AHNAK protein and PLC-${\gamma}1$ act in concerted manner in carcinogenesis.

• Journal of Applied Biological Chemistry
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• v.57 no.3
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• pp.219-225
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• 2014

Azadirachta indica extract (AIE) has been regarded as a promising source of environment-friendly organic materials owing to their low mammalian toxicity. However, quite a bit of research has been reported that AIE may cause clastogens in human lymphocytes. Therefore, this study was conducted to evaluate the antimutagenic and genotoxicity of two samples of AIE. Antimutagenic test was experimented by using bacterial reverse mutation test. In the bacterial reverse mutation test, five strains Salmonella Typhimurim of two samples of AIE in order to evaluate its mutagenic potential. Bacterial reverse mutation test was also performed on positive control and negative control groups in the presence of the metabolic activation system (S-9 mix) and metabolic non-activation system. In the chromosome aberration test, Chinese hamster lung cells were exposed to AIE for 6 or 24 h with BPS, or for 6 h with S-9 mix. Negative and positive control groups were experimented for chromosome aberration test. As a result, the number of mutated colonies induced by 4-NQO were reduced by AIE treatment in all strains, indicating that AIE may have antimutagenic effects. Bacterial reverse mutation and chromosomal aberration were not shown at all concentration of AIE, regardless of activation of the metabolic system. we concluded that two AIE samples used in this study have no genotoxic effects to human, according to the genotoxicity battery system suggested by ICH (International Conference on Harmonization).

• Development and Reproduction
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• v.6 no.2
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• pp.117-122
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• 2002

Dopamine(DA), norepinephrine(NE), and epinephrine(E) belong to a class of neurotransmitters known as catecholamine (CA) which are synthesized and secreted by mammalian brain and adrenal medulla. CA regulate several behavior patterns connected with breeding, and regulate GnRH-gonadotropin hormone axis' vitality between hypothalamus-pituitary gland linking with reproduction freeze. The present study examined effects of sex steroid hormone on the transcriptional activities of CA biosynthesis enzymes, tyrosine hydroxylase(TH), dopamine $\beta$ -hydroxylase(DBH), and phenylethaolamine-N-methyl transferase(PNMT). Mature female rats were ovariectomized(OVX) and implanted with 17 $\beta$-estradiol(E$_2$: 500 $\mu\textrm{g}$/ml) or sesame oil. Forty-eight hours after implantation all the animals were sacrificed. Total RNAs were extracted immediately and were applied to semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR). The expression level of TH was appeared by hypothalamus > SNc> adrenal medulla orders in OVX＋Oil group, and by SNc > hypothalamus) adrenal medulla orders in OVX+E$_2$ group. Treatment with E$_2$ significantly increased TH expression in SNc and adrenal medulla but in hypothalamus, the reduced TH expression was observed. The expression level of DBH was appeared by adrenal medulla > SNc > hypothalamus orders in OVX＋Oil group and in OVX＋E$_2$ group. Administration of E$_2$ significantly reduced DBH expression in SNc, and increased in adrenal medulla. Two cDNA products, large(PNMT1) and small(PMNTs) species of 110bp difference, were amplified in SNc and hypothalamus, but only PNMTs was observed in adenal medulla. The PNMTs expression level was in the order of adrenal medulla > hypothalamus > SNc in both OVX＋Oil and OVX＋E$_2$ group. The PNMTs expression in SNc and adrenal medulla was significantly increased byE$_2$. The present report demonstrated that estrogen effects on transcriptional activities for CA biosynthethic enzymes were tissue specific in adrenal medulla as well as different region of brain. These results suggest that it might be crucial relationship between the type of estrogen receptor and CA enzyme gene expression.

• Korean Journal of Plant Tissue Culture
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• v.24 no.1
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• pp.1-35
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• 1997

Fertilized egg, by successive cell divisions, differentiates into different tissues and organs with various structures and functions. Different cells and tissues contain different proteins, products of selective gene expression. Not all the genes in any genomes are equally active, temporal and spatial gene expression being the general rule. Present paper attempts to review the tanscriptional mechanisms or the initiations of transcription from several angles. In some of the organisms the genes in the process of transcription or the genes in the inactive state can be seen under the light microscope. Some bands of Drosophila polytene chromosomes may exhibit a swollen or puff appearance under certain conditions. A puff, unfolded or decondensed form of chromomere, represents sets of intense transcriptional activity or RNA synthesis. The heterochromatic X chromosome whose genes remain inactive in the female mammals can be visualized as a dark staining structure called Barr body, Configuration of chromatin differs between transcribed and nontranscribed chromatin. Modification to the chromatin facilitates RNA synthesis. The movement of large polymerase molecule along the DNA would probably be facilitated if some modifications of the chromatin configuration is effected. Methylation of cytosines in CG sequences is associated with inactive genes. Methylation can play a role in determination of mammalian cells during embryogenesis. Demethylation is necessary for the gene to be expressed during development A histone modification that is also known to be correlated with transcriptional capacity of chromatin is acetylation of the lysine residues of the core histones. Chromatin containing a high level of histone acetylation is very sensitive to DNase 1. For the transcription to occur TBP must first bind to the TATA box. Another TF, TF IIB, then binds to the promoter-TBP complex, facilitating the access of RNA polymerase to the transcription initiation site. As recently as eight years ago researchers assumed that histones were irrelevant to the regulation of gene expression. Histones combine with the DNA to form nucleosome of the chromatin. Histones are vital participant in gene regulation. Histone and basal factors compete for access to TATA box. When DNA is exposed to basal factors before histones are introduced, the basal factors assemble on TATA boxes preventing the access of histones, allowing transcription to occur, for transcription to begin, activator protein at the upstream activation sequence or enhancer must interact with the tail of histone H4 at TATA box and cause the histone role particle to dissociate from the TATA box leading to partial breakup of the histone core particle and allowing the basal factors to bind to the TATA box. New concept of genomic flux in contrast to the old concept of static genome has been developed based on the powerful new molecular techniques. Genomic changes such as repetitive DNAs and transposable elements, it is assumed but not yet proved, may affect some of the developmental patterns that characterize particular cells, tissues, organs, and organisms. In the last decade or so remarkable achievement have been made in the researches of the structures and functions of TFs and the specific target sequences located in promoters or enhancers where these TFs bind. TFs have independent domains that bind DNA and that activate transcription. DNA binding domain of TFs serves to bring the protein into the right location. There are many types of DNA binding domains. Common types of motifs can be found that are responsible for binding to DNA. The motifs are usually quite short and comprise only a small part of the protein structure. Steroid receptors have domains for hormone binding, DNA binding, and activating transcription. The zinc finger motif comprises a DNA binding domain. Leucine zipper consist of a stretch of amino acids with a leucine residue in every seventh position Two proteins form a dimer because they interact by means of leucine zippers on similar α-helical domain. This positions their DNA binding basic domains for interaction with the two halves of a DNA sequence with dyad symmetry of TGACTCA, ACTGAGT.

• Journal of Food Hygiene and Safety
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• v.23 no.3
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• pp.264-270
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• 2008

Phytic acid(PA) (Inositol hexaphosphate, $IP_6$) is a naturally occurring polyphosphorylated carbohydrate that is present in substantial amounts in almost all plants and mammalian cells. Recently PA has received much attention for its role in anticancer activity. In the present study, the preventive effects of PA on colon carcinogenesis were investigated. Six-week old Fisher 344 male rats were fed a AIN-93G purified diet and PA(0.5% or 2% PA in water) for 8 weeks. The animals received two ($1^{st}\;and\;2^{nd}$ week) injections of azoxymethane(AOM, 15 mg/kg b.w.) to induce colonic aberrant crypt foci(ACF). After sacrifice, the total numbers of aberrant crypts(AC) and ACF in colonic mucosa were examined after staining with methylene blue. Blood and serum were analyzed with a blood cell differential counter and an automatic serum analyzer. AOM induced the total numbers of $142.3{\pm}22.3$ ACF/colon and $336.6{\pm}55.1$ AC/colon. PA at the doses of 0.5 and 2% decreased the numbers of ACF and AC/colon in a dose-dependent manner. The numbers of ACF/colon and AC/colon by PA at the dose of 0.5% were $124.4{\pm}28.5\;and\;302.7{\pm}67.3$, respectively. PA at the dose of 2% significantly decreased the ACF and AC numbers to $109{\pm}18.1\;and\;254.8{\pm}50.6$, respectively(p<0.01). Especially, 2% PA significantly reduced the number of large ACF(${\geq}4$ AC/ACF) from $26.8{\pm}6.2$ ACF/colon to $15{\pm}6.7$ ACF/colon(p<0.01). Although some parameters in blood counts and serum chemistry were changed compared with the control, no specific toxicity was found. These findings suggest that phytic acid can be a chemopreventive agent for colon carcinogenesis resulting from inhibition of the development of ACF in the F344 rat.

• Korean Journal of Veterinary Research
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• v.39 no.5
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• pp.878-895
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• 1999

Ischemic preconditioing (IPC), i.e., a preliminary brief episode of ischemia and reperfusion, has been shown to reduce the cell damage induced by long ischemia and reperfusion. Superoxide radical which is produced during reperfusion after ischemia was recognized as a factor of the ischemic injury and it is dismutated into $H_2O_2$ and $O_2$ by two types of intracellular superoxide dismutase (SOD), Cu,Zn-SOD in cytoplasm and Mn-SOD in mitochondria. Recently oxygen free radicals are suggested to induce the apoptosis, however mechanism of the reduced apoptosis by ischemic preconditioing was unknown, while many studies performed in mammalian heart indicated that ATP-sensitive $K^+$ ($K_{APT}$) channel activation related with the protective effects. The aim of present study is to investigate 1) whether IP upregulate the Cu,Zn-SOD and Mn-SOD activities, and 2) whether ischemic preconditioning decreases apoptosis via $K_{APT}$ channel activation in timely reperfused skeletal muscle after long ishemia. The experimental animals, Sprague-Dawley rats weighing 250~300g, were divided into 8 groups; 1) control group, 2) ischemic preconditioning only groups, 3) pinacidil, a $K_{APT}$ channel opener, treatment only groups, 4) glibenclamide, a $K_{APT}$ channel blocker, treatment only groups, 5) ischemia groups, 6) ischemia after IPC groups, 7) ischemia and pinacidil treatment groups, and 8) IP and ischemia after glibenclamide pretreatment groups. Animals of the control group were administered with the vehicle (DMSO) alone. Pinacidil (1mg/kg) was administered intravenously 5 minutes after initiation of ischemia, and glibenclamide (0.5mg/kg) was injected intravenously 20 minutes before IPC. In rats that were ischemic preconditioned, the left common iliac artery was occluded for 5 minutes followed by 5 minutes of reperfusion by three times using vascular clamp. Ischemia was done by occlusion of the same artery for 4 hours. The specimens of left rectus femoris muscle were obtained immediately (0 hour), 12 hours, 24 hours after drug administrations, IP or ischemia and reperfusion. The immunoreactivities of SOD and its alterations were observed by use of sheep antihuman Cu,Zn-SOD and Mn-SOD antibodies on the $10{\mu}m$ cryosections. The incidencies of apoptosis were observed by TUNEL methods with in situ apoptosis detection kit on $6{\mu}m$ paraffine section. The results obtained were as follows : 1. After IPC, immunoreactivities of Cu,Zn-SOD mainly in the small-sized fibers were increased by 24 hours, that of Mn-SOD at 0 hour and 24 hours. 2. No significant changes in immunoreactivities of SOD was observed in the pinacidil and in the glibenclamide treatment only groups, and in the ischemia only groups. 3. The immunoreactivities of the Cu,Zn-SOD were increased in the ischemia after IPC groups and the ischemia and pinacidil treatment groups. 4. The immunoreactivities of the Cu,Zn-SOD in the IPC and ischemia after glibenclamide pretreatment groups were not increased except for the 12 hours reperfusion group. But, Mn-SOD immunoreactivities were increased in the 0 hours, 12 hours and 24 hours after reperfusion. 5. In the control group, the IPC only groups, and the pinacidil treatment only groups, negative or trace apoptotic reactions were observed, but the positive apoptotic reaction occured in the glibenclamide treatment groups. 6. Moderate or many number of apoptosis were revealed in the ischemia groups, and also the IPC and ischemia after glibenclamide pretreatment group except for 12 hours and 24 hours after reperfusion. However, the incidence of apoptosis was decreased in the ischemia after IPC groups and in the ischemia and pinacidil treatment groups. 7. There is a coincidence between the increase of Cu,Zn-SOD immunoreactivities and the decrease of apoptosis in the presence of ischemia and reperfusion. These results suggest that the protective effects of ishemic preconditioing may related to the SOD activation, and the ischemic preconditioning decreases the apoptosis partially via $K_{APT}$ channel activation in timely reperfused rat skeletal muscle. It is also suggested that inhibition of apoptosis by IPC may related with the SOD activation.

• Biomolecules & Therapeutics
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• v.20 no.1
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• pp.43-49
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• 2012

Stimulation of mast cells through the high affinity IgE receptor (Fc${\varepsilon}$RI) induces degranulation, lipid mediator release, and cytokine secretion leading to allergic reactions. Although various signaling pathways have been characterized to be involved in the Fc${\varepsilon}$RI-mediated responses, little is known about the precious mechanism for the expression of tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) in mast cells. Here, we report that rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR), reduces the expression of TNF-${\alpha}$ in rat basophilic leukemia (RBL-2H3) cells. IgE or specific antigen stimulation of RBL-2H3 cells increases the expression of TNF-${\alpha}$ and activates various signaling molecules including S6K1, Akt and p38 MAPK. Rapamycin specifically inhibits antigeninduced TNF-${\alpha}$ mRNA level, while other kinase inhibitors have no effect on TNF-${\alpha}$ mRNA level. These data indicate that mTOR signaling pathway is the main regulation mechanism for antigen-induced TNF-${\alpha}$ expression. TNF-${\alpha}$ mRNA stability analysis using reporter construct containing TNF-${\alpha}$ adenylate/uridylate-rich elements (AREs) shows that rapamycin destabilizes TNF-${\alpha}$ mRNA via regulating the AU-rich element of TNF-${\alpha}$ mRNA. The antigen-induced activation of S6K1 is inhibited by specific kinase inhibitors including mTOR, PI3K, PKC and $Ca^{2+}$chelator inhibitor, while TNF-${\alpha}$ mRNA level is reduced only by rapamycin treatment. These data suggest that the effects of rapamycin on the expression of TNF-${\alpha}$ mRNA are not mediated by S6K1 but regulated by mTOR. Taken together, our results reveal that mTOR signaling pathway is a novel regulation mechanism for antigen-induced TNF-${\alpha}$ expression in RBL-2H3 cells.

• Development and Reproduction
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• v.11 no.3
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• pp.187-193
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• 2007

Ethane 1,2-dimethane sulfonate(EDS), a Leydig cell specific toxicant, has been widely used to create the reversible testosterone withdrawal rat model. Though the maintenance of epididymal structure and function is highly dependent on the testosterone secreted from testis, its derivatives, dihydroxytestosterone(DHT) and estrogen, might have crucial roles. The aim of present study was to monitor the expression patterns of sex steroid receptors, cytochrome P450 aromatase(P450arom) and $5{\alpha}$-reductase in the rat epididymis up to 7 weeks after EDS injection. Adult male rats($350{\sim}400g$) were injected with a single does of EDS(75 mg/kg i.p.) and sacrificed on weeks 0, 1, 2, 3, 4, 5, 6 and 7. The transcriptional activities of the target genes were evaluated by semi-quantitative RT-PCRs. The transcript level of estrogen receptor alpha($ER{\alpha}$) in EDS group was significantly higher than control level on week 1(P<0.01). After week 2, there was no significant difference in $ER{\alpha}$ levels between EDS group and control. The transcript level of estrogen receptor beta($ER{\beta}$) in EDS group was significantly higher than control level on week 1(P<0.05), lowered on weeks 2 and 3(P<0.05 and P<0.01, respectively), fluctuated during weeks 4 and 6, and elevated on week 7(P<0.05). The androgen receptor (AR) message levels increased significantly week 2(P<0.01), then returned to control level on week 3. In contrast, expression of cytochrome P450 aromatase(P450arom) decreased sharply during weeks $1{\sim}3$(P<0.01 on weeks 1 and 2; P<0.05 on week 3), then went back to control level on week 4. The mRNA level of $5{\alpha}$-reductase type 2($5{\alpha}$-RT2) increased significantly on week 4(P<0.01), then returned to control level. The present study indicated that EDS administration could induce reversible alterations in the transcriptional activities of sex steroid hormone receptors and androgenconverting enzymes in rat epididymis. EDS injection model will be useful to clarify the regulation mechanism of mammalian epididymal physiology.

• Journal of the korean academy of Pediatric Dentistry
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• v.25 no.2
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• pp.352-367
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• 1998

Intracellular pH (pHi) plays an important role in the regulation of cellular processes by influencing the acitivity of various enzymes in cells. Therefore, almost every type of mammalian cell possesses an ability to regulate its pHi. One of the most prominent mechanisms in the regulation of pHi is $Na^+/H^+$ exchanger. This exchanger has been known to be activated when cells are stimulated by the binding of agonist to the muscarinic receptors. Therefore, the aims of this study were to compare the rates of $H^+$ extrusion through $Na^+/H^+$ exchanger before and during muscarinic stimulation and to investigate the possible existence of $HCO_3^-$ transporter which is responsible for the continuous supply of $HCO_3^-$ ion to saliva. Acinar cells were isolated from the rat mandibular salivary glands and loaded with pH-sensitive fluoroprobe, 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein(BCECF), for 30min at room temperature. Cells were attached onto the coverglass in the perfusion chamber and the changes in pHi were measured on the iverted microscope using spectrofluorometer. 1. By switching the perfusate from $HCO_3^-$-free to $HCO_3^-$-buffered solution, pHi decreased by $0.39{\pm}0.02$ pH units followed by a slow increase at an initial rate of $0.04{\pm}0.007$ pH units/min. The rate of pHi increase was reduced to $0.01{\pm}0.002$ pH units/min by the simultaneous addition of 1 mM amiloride and $100{\mu}M$ DIDS. 2. An addition and removal of $NH_4^+$ caused a decrease in pHi which was followed by an increase in pHi. The increase of pHi was almost completely blocked by 1mM amiloride in $HCO_3^-$-free perfusate which implied that the pHi increase was entired dependent on the activation of $Na^+/H^+$ exchanger in $HCO_3^-$-free condition. 3. An addition of $10{\mu}M$ carbachol increased the initial rate of pHi recovery from $0.16{\pm}0.01$ pH units/min to $0.28{\pm}0.03pH$ units/min. 4. The initial rate of pHi decrease induced by 1mM amiloride was also increased by the exposure of the acinar cells to $10{\mu}M$ carbachol ($0.06{\pm}0.008pH$ unit/min) compared with that obtained before carbachol stimulation ($0.03{\pm}0.004pH$ unit/min). 5. The intracellular buffering capacity ${\beta}1$ was $14.31{\pm}1.82$ at pHi 7.2-7.4 and ${\beta}1$ increased as pHi decreased. 6. The rate of $H^+$ extrusion through $Na^+/H^+$ exchanger was greatly enhanced by the stimulation of the cells with $10{\mu}M$ carbachol and there was an alkaline shift in the activity of the exchanger. 7. An intrusion mechanism of $HCO_3^-$ was identified in rat mandibular salivary acinar cells. Taken all together, I observed 3-fold increase in $Na^+/H^+$ exchanger by the stimulation of the acinar cells with $10{\mu}M$ carbachol at pH 7.25. In addition, I have found an additional mechanism for the regulation of pHi which transported $HCO_3^-$ into the cells.

• Journal of Life Science
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• v.34 no.5
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• pp.333-338
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• 2024

Kinesin-1 is a heterotetrameric protein composed of two heavy chains (KHCs, also known as KIF5s) with a motor domain and two light chains (KLCs) without a motor domain. KIF5 has three subtypes, namely, KIF5A, KIF5B, and KIF5C, which share high amino acid homology except in their carboxy (C)-terminal region. KIF5A is responsible for transporting cargo within the cell. The adaptor proteins that bind to the C-terminal region of KIF5A mediate between kinesin-1 and cargo. However, the proteins regulating the intracellular cargo transport of kinesin-1 have not yet been fully identified. In this study, we identified ADP-ribosylation factor GTPase-activating protein 1 (ArfGAP1), which is involved in the intracellular trafficking of lysosomes, as a binding partner of KIF5A. KIF5A binds to the C-terminal region of ArfGAP1, and ArfGAP1 binds to the C-terminal region of KIF5A but does not interact with KIF5B, KIF5C, kinesin light chain 1 (KLC1), or KIF3A. When co-expressed in mammalian cells, ArfGAP1 co-localized with KIF5A and co-immunoprecipitated with KIF5A, KIF5B, and KLC1, but not with KIF3B. These results suggest that kinesin-1 may be regulated by ArfGAP1 in the intracellular transport of cargo.