• Biomedical Science Letters
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• v.12 no.1
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• pp.17-22
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• 2006

The baculovirus expression system is a powerful method for producing large amounts of the human erythrocyte-type glucose transport protein, heterologously. Characterization of the expressed protein is expected to show its ability to transport sugars directly. To achieve this, it is a prerequisite to know the properties of the endogenous sugar transport system in Spodoptera frugiperda Clone 21 (Sf21) cells, which are commonly employed as a host permissive cell line to support the baculovirus replication. The Sf21 cells can grow well on TC-100 medium that contains 0.1% D-glucose as the major carbon source, strongly suggesting the presence of endogenous glucose transport system. However, unlike the human glucose transport protein that has a broad substrate and inhibitor specificity, very little is known about the nature of the endogenous sugar transport system in Sf21 cells. In order to characterize further the inhibitor recognition properties of the Sf21 cell transporter, the ability of phloretin, cytochalasin B and D-fructose to inhibit 2-deoxy-D-glucose (2dGlc) transport was examined by measuring inhibition constants $(K_i)$. The $K_i's$ for reversible inhibitors were determined from plots of uptake versus inhibitor concentration. The 2dGlc transport in the Sf21 cells was very potently inhibited by phloretin, the aglucone of phlorizin with a $K_i$ similar to the value of about $2{\mu}M$ reported for inhibition of glucose transport in human erythrocytes. However, the Sf21 cell transport system was found to differ from the human transport protein in being much less sensitive to inhibition by cytochalasin B (apparent $K_i$ approximately $10\;{\mu}M$). In contrast, It is reported that the inhibitor binds the human erythrocyte counterpart with a $K_d$ of approximately $0.12\;{\mu}M$. Interestingly, the Sf21 glucose transport system also appeared to have high affinity for D-fructose with a $K_i$ of approximately 5mM, contrasting the reported $K_m$ of the human erythrocyte transport protein for the ketose of 1.5M.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.8
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• pp.1062-1068
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• 2004

Leptin has a major role in the regulation of food intake and energy homeostasis. In addition, leptin participates in many physiological functions including regulation of lipid metabolism. Bovine recombinant leptin protein was produced in E. coli cells in order to understand function of leptin in the regulation of lipid metabolism. The leptin expression vector was constructed in pGEX-4T-3 vector and transformed into E. coli BL21 cells. Expression of the GST-leptin fusion protein was induced with IPTG. The fusion protein was purified using glutathione sepharose 4B batch method, and the recombinant leptin was eluted after thrombin protease digestion. The effect of leptin on glucose transport was examined in the differentiated adipocytes of 3T3-L1 cells. Leptin had no effect on basal and insulin-stimulated glucose transport in 3T3-L1 cells (p>0.05). Effect of recombinant leptin on glucose and acetate transport was examined in adipose tissues of Korean cattle (Hanwoo). Insulin stimulated glucose transport in both intramuscular and subcutaneous adipose tissues (p<0.05), but leptin did not affect glucose transport in both adipose tissues (p>0.05). Insulin stimulated acetate transport in bovine adipose tissues (p<0.05), but leptin did not affect acetate transport (p>0.05). Northern and RT-PCR analyses showed that mRNA levels of uncoupling protein-2 were increased by leptin treatment in 3T3-L1 cells without statistical difference (p>0.05). In conclusion, bovine recombinant leptin did not affect glucose and acetate transport in both 3T3-L1 adipocytes and bovine adipose tissues, while it stimulates UCP-2 mRNA expression in 3T3-L1 cells.

• Molecules and Cells
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• v.46 no.12
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• pp.757-763
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• 2023

In this study, we examine whether a change in the protein levels for FOP in Ankyrin repeat and SAM domain-containing protein 1A (ANKS1A)-deficient ependymal cells affects the intraflagellar transport (IFT) protein transport system in the multicilia. Three distinct abnormalities are observed in the multicilia of ANKS1A-deficient ependymal cells. First, there were a greater number of IFT88-positive trains along the cilia from ANKS1A deficiency. The results are similar to each isolated cilium as well. Second, each isolated cilium contains a significant increase in the number of extracellular vesicles (ECVs) due to the lack of ANKS1A. Third, Van Gogh-like 2 (Vangl2), a ciliary membrane protein, is abundantly detected along the cilia and in the ECVs attached to them for ANKS1A-deficient cells. We also use primary ependymal culture systems to obtain the ECVs released from the multicilia. Consequently, we find that ECVs from ANKS1A-deficient cells contain more IFT machinery and Vangl2. These results indicate that ANKS1A deficiency increases the entry of the protein transport machinery into the multicilia and as a result of these abnormal protein transports, excessive ECVs form along the cilia. We conclude that ependymal cells make use of the ECV-based disposal system in order to eliminate excessively transported proteins from basal bodies.

• Journal of fish pathology
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• v.14 no.1
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• pp.54-58
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• 2001

The seasonal trends of plasma protein and glucose concentrations in marbled soles(Limanda yokohamae) and greenlings(Hexagrammos otakii), and the influence of transport stressor on those levels were investigated. Total plasma protein levels of marbled soles and greenlings in late spring and summer were significantly higher than those in winter(January). Plasma glucose levels were consistently increased according to elevation of water temperature both in marbled soles and greenlings. Transport stressor gave rise to decrease of plasma protein levels and increase of blood glucose levels.

• Journal of Plant Biology
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• v.38 no.4
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• pp.343-348
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• 1995

Treatment of Pisum sativum tissue with the protein kinase inhibitor staurosphorine resulted in impairment of 3H-indoleacetic acid transport in etiolated stem segments. The transport inhibitiion was accompanied by an increase in net uptake of labeled auxin in the tissue. The magnitude of auxin accumulation in tissue treated with the phytotropin N-1-naphthylphthalaic acid (NPA) which specifically blocks the efflux of auxin in the plasma membrane was reduced by the protein kinase inhibitor, suggesting that inhibition of protein phosphorylation could lead to hindrance of the auxin-exporting function of NPA receptors. The flavonoid genistein which is also known to inhibit protein kinase likewise reduced NPA-induced auxin accumulation. However, the flavonoid did not bring about auxin accumulation by itself, nor did it inhibit auxin transport. In view of the finding that the flavonoid also competes with NPA for a common binding site, a mechanism for the flavonoid effect on the NPA action will be proposed.

• Biomedical Science Letters
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• v.16 no.3
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• pp.201-206
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• 2010

In order to develop procedures for the rapid isolation of recombinant sugar transporter in functional form from away from the endogenous insect cell transporter, gene fusion techniques were exploited. Briefly, BamH1-digested human HepG2 type glucose transport protein cDNA was first cloned into a transfer vector pBlueBacHis, containing a tract of six histidine residues. Recombinant baculoviruses including the human cDNA were then generated by allelic exchange following transfection of insect cells with wild-type BaculoGold virus DNA and the recombinant transfer vector. Plaque assay was then performed to obtain and purify recombinant viruses expressing the human transport protein. All the cell samples that had been infected with viruses from the several blue plaques exhibited a positive reaction in the immnuassay, demonstrating expression of the glucose transport protein. In contrast, no color development in the immunoassay was observed for cells infected with the wild-type virus or no virus. Immunoblot analysis showed that a major immunoreactive band of apparent Mr 43,000~44,000 was evident in the lysate from cells infected with the recombinant baculovirus. Following expression of the recombinant fusion protein with the metal-binding domain and enterokinase cleavage site, the fusion protein was recovered by competition with imidizole using immobilized metal charged resin. The leader peptide was then removed from the fusion protein by cleavage with porcine enterokinase. Final separation of the recombinant protein of the interest was achieved by passage over $Ni^{2+}$-charged resin under binding conditions. The expressed transport protein bound cytochalasin B and demonstrated a functional similarity to its human counterpart.

• Korea Journal of Cosmetic Science
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• v.1 no.1
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• pp.31-43
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• 2019

Melanosomes are specific melanin-containing intracellular organelles of epidermal melanocytes. In epidermal melanocytes, there are three kinds of key player proteins. Rab27a, melanophilin or Slac2-a and Myosin 5a form a tripartite complex connects the melanosome. Mature melanosomes make movements through the tripartite protein complex along actin filaments.In this study, we found that the haplamine (6-Methoxyflindersine) induced melanosome aggregation around the nucleus in epidermal melanocyte. In an attempt to elucidate the inhibitory effect of haplamine on melanosome transport, effect of haplamineon the expression level of Rab27a, melanophilin and Myosin 5a was measured. The results indicated that haplamine up to 5��M effectively suppressed mRNA and protein expression level of melanophilin.To determine the upstream regulator of melanophilin regulated by haplamine, we checked the level of MITF, c-JUN and USF1. Those are possible transcription factor of melanophilin. Among them,treatment of USF1 siRNA decreased mRNA and protein expression level of USF1 as well as melanophilin. Also, treatment of haplamine decreased mRNA and protein expression level of melanophilin as well as USF1 in a dose-dependent manner. Consequently, we found the inhibitory effect of haplamine on melanosome transport in melan-a melanocyte. Treatment of haplamine reduced melanophilin expression level which is a key protein of melanosome transport. We identified that USF1 could be a major transcription factor of melanophilin regulated by haplamine.

• Biomedical Science Letters
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• v.15 no.1
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• pp.55-60
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• 2009

Insect cells such as Spodoptera frugiperda Clone 9 (Sf9) cells are widely chosen as the host for heterologous expression of a mammalian sugar transport protein using the baculovirus expression system. Characterization of the expressed protein is expected to include assay of its function, including its ability to transport sugars and to bind inhibitory ligands such as cytochalasin B. It is therefore very important first to establish the transport characteristics and other properties of the endogenous sugar transport proteins of the host insect cells. However, very little is known of the transport characteristics of Sf9 cells, although their ability to grow on TC-100 medium strongly suggested the presence of endogenous glucose transport system. In order to investigate the substrate and inhibitor recognition properties of the Sf9 cell transporter, the ability of pentoses to inhibit 2-deoxy-D-glucose (2dGlc) transport was investigated by measuring inhibition constants $(K_i)$. To determine the time period over which of sugar into the Sf cells was linear, the uptake of 2dGlc 0.1mM extracellular concentration was measured over periods ranging from 30 seconds to 30 minutes. The uptake was linear for at least 2 minutes at the concentration, implying that uptake made over a 1 minute time course would reflect initial rates of the sugar uptake. The data have also revealed the existence of a saturable transport system for pentose uptake by the insect cells. The transport was inhibited by D-xylose and D-ribose, although not as effective as hexoses. However, L-xylose had a little effect on 2dGlc transport in the Sf9 cells, indicating that the transport is stereoselective. Unlike the human erythrocyte-type glucose transport system, D-ribose had a somewhat greater apparent affinity for the Sf9 cell transporter than D-xylose. It is therefore concluded that Sf9 cells contain an endogenous sugar transport activity that in some aspects resembled the human erythrocyte-type counterpart, although the Sf9 and human transport systems do differ in their affinity for cytochalasin B.

• Toxicological Research
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• v.14 no.3
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• pp.371-377
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• 1998

We have cloned a soluble type human folate receptor(hFR type${\gamma}$) from human thymus cDNA library using the PCR amplification technique. To examine whether hFR type${\gamma}$ has a folate transport activity, CHO cells were transfected with the pcDNAhFR${\gamma}$ expression plasmid, and the stable cell line CHO/hFR${\gamma}$ expressing a high level of the hFR type${\gamma}$ was identified by northern and western blot analysis. The CHO/hFR${\gamma}$ cells produced a [$H^3$]folic acid binding protein in the culture medium. However, we couldn't detect any cell surface [$H^3$] folic acid binding and transport activities. The growth of the CHO/hFR${\gamma}$ cells was more rapidly inhibited than the wild type CHO cells in the low concentration folic acid media. These observations indicate that although soluble type human folate receptor can bind [$H^3$]folate, it does not involve in folate transport.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.10
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• pp.1457-1465
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• 2012

Intestinal phosphate (Pi) absorption across the apical membrane of small intestinal epithelial cells is mainly mediated by the type IIb Na-coupled phosphate co-transporter (NaPi-IIb), but its expression and regulation in the chicken remain unclear. In the present study, we investigated the mRNA and protein levels of NaPi-IIb in three regions of chicken small intestine, and related their expression levels to the rate of net phosphate absorption. Our results showed that maximal phosphate absorption occurs in the jejunum, however the highest expression levels of NaPi-IIb mRNA and protein occurs in the duodenum. In response to a low-Pi diet (TP 0.2%), there is an adaptive response restricted to the duodenum, with increased brush border membrane (BBM) Na-Pi transport activity and NaPi-IIb protein and mRNA abundance. However, when switched from a low-(TP 0.2%) to a normal diet (TP 0.6%) for 4 h, there is an increase in BBM NaPi-IIb protein abundance in the jejunum, but no changes in BBM NaPi-IIb mRNA. Therefore, our study indicates that Na-Pi transport activity and NaPi-IIb protein expression are differentially regulated in the duodenum vs the jejunum in the chicken.