• Molecules and Cells
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• 제25권2호
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• pp.265-271
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• 2008

Single nucleotide polymorphisms (SNPs) are the most common form of human genetic variation. Non-synonymous SNPs (nsSNPs) change an amino acid. Organic anion transporters (OATs) play an important role in eliminating or reabsorbing endogenous and exogenous organic anionic compounds. Among OATs, hOAT4 mediates high affinity transport of estrone sulfate and dehydroepiandrosterone sulfate. The rapid bone loss that occurs in post-menopausal women is mainly due to a net decrease of estrogen. In the present study we searched for SNPs within the exon regions of hOAT4 in Korean women osteoporosis patients. Fifty healthy subjects and 50 subjects with osteoporosis were screened for genetic polymorphism in the coding region of SLC22A11 (hOAT4) using GC-clamp PCR and denaturing gradient gel electrophoresis (DGGE). We found three SNPs in the hOAT4 gene. Two were in the osteoporosis group (C483A and G832A) and one in the normal group (C847T). One of the SNPs, G832A, is an nsSNP that changes the $278^{th}$ amino acid from glutamic acid to lysine (E278K). Uptake of [$3^H$] estrone sulfate by oocytes injected with the hOAT4 E278K mutant was reduced compared with wild-type hOAT4. Km values for wild type and E278K were $0.7{\mu}M$ and $1.2{\mu}M$, and Vmax values were 1.8 and 0.47 pmol/oocyte/h, respectively. The present study demonstrates that hOAT4 variants can causing inter-individual variation in anionic drug uptake and, therefore, could be used as markers for certain diseases including osteoporosis.

• 생명과학회지
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• 제30권5호
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• pp.420-427
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• 2020

Repebody는 비면역 글로블린 인공 항체로 저렴하고 빠르게 생산 가능한 맞춤형 항체이다. 그러나 의료용 repebody의 생산은 저수율 및 복잡한 정제 공정으로 인해 여전히 어려움을 겪고 있다. Pseudomonas fluorescens의 ABC transporter를 사용한다면 생산 공정을 간소화하고 비용을 줄일 수는 있지만 repebody는 양전하를 띠어 분비 효율이 낮다. 따라서 등전점(pI)이 높은 repebody의 등전점을 낮추어 음전하를 띄도록 해야 한다. 이것을 위해 repebody의 N 말단과 C 말단에 연속된 아스파탐산을 붙여 보았지만 분비가 증가하지 않았다. 다른 방법으로 ABC transporter를 통한 repebody 분비 효율을 높이기 위해 repebody의 항원 결합 부위의 반대쪽에 존재하는 열다섯 개의 양전하 아미노산을 아스파탐산으로 변환하여 repebody 표면이 강한 음전하를 띠도록 하였다. 그 결과, 기존 repebody의 발현 단백질 당 분비효율은 21.2%였으나 변형한 과음전하 repebody의 분비효율은 58.5%로 향상되었다. 결론적으로 과음전하를 통해 만들어진 repebody는 P. fluorescens에 의해 세포 바깥에 분비 생산할 수 있었다.

• Molecules and Cells
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• 제22권3호
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• pp.343-352
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• 2006

Sialic acid is a sugar typically found at the N-glycan termini of glycoproteins in mammalian cells. Lec3 CHO cell mutants are deficient in epimerase activity, due to a defect in the gene that encodes a bifunctional UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). Sialic acid modification on the cell surface is partially affected in these cells. We have mutagenized Lec3 CHO cells and isolated six mutants (termed C2m) deficient in the cell surface expression of polysialic acid (PSA). Mutant C2m9 was partially defective in expression of cell-surface PSA and wheat germ agglutinin (WGA) binding, while in the other five mutants, both cell-surface PSA and WGA binding were undetectable. PSA expression was restored by complementation with the gene encoding the CMP-sialic acid transporter (CST), indicating that CST mutations were responsible for the phenotypes of the C2m cells. We characterized the CST mutations in these cells by Northern blotting and RT-PCR. C2m9 and C2m45 carried missense mutations resulting in glycine to glutamate substitutions at amino acids 217 (G217E) and 256 (G256E), respectively. C2m13, C2m39 and C2m31 had nonsense mutations that resulted in decreased CST mRNA stability, and C2m34 carried a putative splice site mutation. PSA and CD15s expression in CST-deficient Lec2 cells were partially rescued by G217E CST, but not by G256E CST, although both proteins were expressed at similar levels, and localized to the Golgi. These results indicate that the novel missense mutations isolated in this study affect CST activity.

• Journal of Animal Science and Technology
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• 제62권2호
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• pp.263-275
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• 2020

Studies on promoting milk protein yield by supplementation of amino acids have been globally conducted. Nevertheless, there is a lack of knowledge of what pathways affected by individual amino acid in mammary epithelial cells that produce milk in practice. Phenylalanine (PHE) and valine (VAL) are essential amino acids for dairy cows, however, researches on mammary cell levels are still lacking. Thus, the aim of this study was conducted to evaluate the effects of PHE and VAL on milk protein synthesis-related and energy-mediated cellular signaling in vitro using immortalized bovine mammary epithelial (MAC-T) cells. To investigate the effects of PHE and VAL, the following concentrations were added to treatment medium: 0, 0.3, 0.6, 0.9, 1.2, and 1.5 mM. The addition of PHE or VAL did not adversely affect cell viability compared to control group. The concentrations of cultured medium reached its maximum at 0.9 mM PHE and 0.6 mM VAL (p < 0.05). Therefore, aforementioned 2 treatments were analyzed for proteomics. Glucose transporter 1 and mammalian target of rapamycin mRNA expression levels were up-regulated by PHE (166% and 138%, respectively) (p < 0.05). Meanwhile, sodium-dependent neutral amino acids transporter type 2 (ASCT2) and β-casein were up-regulated by VAL (173% in ASCT2, 238% in and 218% in β-casein) (p < 0.05). A total of 134, 142, and 133 proteins were detected in control group, PHE treated group, and VAL treated group, respectively. Among significantly fold-changed proteins, proteins involved in translation initiation or energy metabolism were detected, however, expressed differentially between PHE and VAL. Thus, pathway analysis showed different stimulatory effects on energy metabolism and transcriptional pathways. Collectively, these results showed different stimulatory effects of PHE and VAL on protein synthesis-related and energy-mediated cellular signaling in MAC-T cells.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1189-1196
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• 2005

The cyclohexanol dehydrogenase (ChnA), produced by Rhodococcus sp. TK6, which is capable of growth on cyclohexanol as the sole carbon source, has been previously purified and characterized. However, the current study cloned the complete gene (chnA) for ChnA and its flanking regions using a combination of a polymerase chain reaction (PCR) based on the N-terminal amino acid sequence of the purified ChnA and plaque hybridization from a phage library of Rhodococcus sp. TK6. A sequence analysis of the 5,965-bp DNA fragment revealed five potential open reading frames (ORFs) designated as partial pte (phosphotriesterase), acs (acyl-CoA synthetase), scd (short chain dehydrogenase), stp (sugar transporter), and chnA (cyclohexanol dehydrogenase), respectively. The deduced amino acid sequence of the chnA gene exhibited a similarity of up to $53\%$ with members of the short-chain dehydrogenase/reductase (SDR) family. The chnA gene was expressed using the pET21 a(+) system in Escherichia coli. The activity of the expressed ChnA was then confirmed (13.6 U/mg of protein) and its properties investigated.

• Journal of Pharmaceutical Investigation
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• 제25권3호spc1호
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• pp.7-20
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• 1995

Taurine, a ${\beta}-amino$ acid, plays an important role as a neuromodulator and is necessary for the normal development of the brain. Since de novo synthesis of taurine in the brain is minimal and in vivo studies suggest that taurine dose not cross the blood-brain barrier, we examined whether the choroid plexus, the blood-cerebrospinal fluid (CSF) barrier, plays a role in taurine transport in the central nervous system. The uptake of $[^3H]-taurine$ into ATP depleted choroid plexus from rabbit was substantially greater in the presence of an inwardly directed $Na^+$ gradient taurine accumulation was negligible. A transient in side-negative potential gradient enhanced the $Na^+-driven$ uptake of taurine into the tissue slices, suggesting that the transport process is electrogenic, $Na^+-driven$ taurine uptake was saturable with an estimated $V_{max}$ of $111\;{\pm}\;20.2\;nmole/g/15\;min$ and a $K_M\;of\;99.8{\pm}29.9\;{\mu}M$. The estimated coupling ratio of $Na^+$ and taurine was $1.80\;{\pm}\;0.122.$ $Na^+-dependent$ taurine uptake was significantly inhibited by ${\beta}-amino$ acids, but not by ${\alpha}-amino$ acids, indicating that the transporter is selective for ${\beta}-amino$ acids. Since it is known that the physiological concentration of taurine in the CSF is lower than that in the plasma, the active transport system we characterized may face the brush border (i.e., CSF facing) side of the choroid plexus and actively transport taurine out of the CSF. Therefore, we examined in vivo elimination of taurine from the CSF in the rat to determine whether elimination kinetics of taurine from the CSF is consistent with the in vitro study. Using a stereotaxic device, cannulaes were placed into the lateral ventricle and the cisterna magna of the rat. Radio-labelled taurine and inulin (a marker of CSF flow) were injected into the lateral ventricle, and the concentrations of the labelled compounds in the CSF were monitored for upto 3 hrs in the cisterna magna. The apparent clearance of taurine from CSF was greater than the estimated CSF flow (p<0.005) indicating that there is a clearance process in addition to the CSF flow. Taurine distribution into the choroid plexus was at least 10 fold higher than that found in other brain areas (e. g., cerebellum, olfactory bulb and cortex). When unlabelled taurine was co-administered with radio-labelled taurine, the apparent clearance of taurine was reduced (p<0.0l), suggesting a saturable disposition of taurine from CSF. Distribution of taurine into the choroid plexus, cerebellum, olfactory bulb and cortex was similarly diminished, indicating that the saturable uptake of taurine into these tissues is responsible for the non-linear disposition. A pharmacokinetic model involving first order elimination and saturable distribution described these data adequately. The Michaelis-Menten rate constant estimated from in vivo elimination study is similar to that obtained in the in vitro uptake experiment. Collectively, our results demonstrate that taurine is transported in the choroid plexus via a $Na^+-dependent,saturable$ and apparently ${\beta}-amino$ acid selective mechanism. This process may be functionally relevant to taurine homeostasis in the brain.

• Biomolecules & Therapeutics
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• 제16권3호
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• pp.219-225
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• 2008

Taurine is the most abundant amino acid in many tissues and is found to be enhancing the bone tissue formation or inhibits the bone loss. Although it is reported that taurine reduces the alveolar bone loss through inhibiting the bone resorption, its functions of taurine and expression of taurine transporter (TauT) in bone have not been identified yet. The purpose of this study is to clarify the uptake mechanism of taurine in osteoblast using mouse osteoblast cell lines. In this study, mouse stromal ST2 cells and mouse osteoblast-like MC3T3-E1 cells as osteoblast cell lines were used. The activity of taurine uptake was assessed by measuring the uptake of [$^3H$]taurine in the presence or absence of inhibitors. TauT mRNA was detected in ST2 and MC3T3-E1 cells. [$^3H$]Taurine uptake by these cells was dependent on the presence of extracellular calcium ion. The [$^3H$]taurine uptake in ST2 cells treated with 4 mM calcium was increased by 1.7-fold of the control which was a significant change. In contrast, in $Ca^{++}$-free condition and L-type calcium channel blockers (CCBs), taurine transport to osteocyte was significantly inhibited. In oxidative stress conditions, [$^3H$]taurine uptake was decreased by TNF-$\alpha$ and $H_2O_2$. Under the hyperosmotic conditions, taurine uptake was increased, but inhibited by CCBs in hyperosmotic condition. These results suggest that, in mouse osteoblast cell lines, taurine uptake by TauT was increased by the presence of extracellular calcium, whereas decreased by CCBs and oxidative stresses, such as TNF-$\alpha$ and $H_2O_2$.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1997년도 춘계학술대회
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• pp.119-119
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• 1997

Valacyclovir is a L-valyl ester prodrug of acyclovir which is a highly effective and selective antiviral agent in the treatment of herpes virus diseases. Valacyclovir is rapidly and almost completely converted to acyclovir and increases the oral bioavailability of acyclovir three to five fold. However, the intestinal absorption mechanism of valacyclovir is not clear. If the improved absorption mechanism of valacyclovir is fully understood, it will provide a rationale of designing the amino acid ester prodrugs of polar drugs containing hydroxyl group. The main objective of our present study is to characterize the membrane transport mechanism of valacyclovir. Methods : Intestinal absorption of valacyclovir was investigated by using in-situ rat perfusion study and its wall permeability was estimated by modified boundary layer model. The membrane transport mechanism was also investigated through the uptake study in Caco-2 cells and in CHO-hPepTl cells. Results : In the rat perfusion study, the wall permeability of valacyclovir was ten times higher than acyclovir and showed concentration dependency, Valacyclovir also demonstrated a D,L stereo-selectivity with L-isomer having an approximately five-fold higher permeability than D-isomer. Mixed dipeptides and cephalexin, which are transported by dipeptide carriers, strongly competed with valacyclovir for the intestinal absorption, while L-valine did not show any competition with valacyclovir. This indicated that the intestinal absorption of valacyclovir could be dipeptide carrier-mediated. In addition, the competitive uptake study in Caco-2 cells presented that dipeptides reduced the valacyclovir uptake but valine did not. Also, in IC$\sub$50/ study, valacyclovir showed strong inhibition on the $^3$H-gly-sar uptake in CHO-hPepTl cells over-expressing a human intestinal peptide transporter. Taken together, the result from our present study indicated that valacyclovir utilized the peptide transporter for the intestinal absorption.

• Journal of Microbiology and Biotechnology
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• 제18권2호
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• pp.242-247
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• 2008

In the fungal pathogen Candida albicans, the yeast-to-hyphal transition occurs in response to a broad range of environmental stimuli and is considered to be a major virulence factor. To address whether the zinc homeostasis affects the growth or pathogenicity of C. albicans, we functionally characterized the zinc-finger protein Csr1 during filamentation. The deduced amino acid sequence of Csr1 showed a 49% similarity to the zinc-specific transcription factor, Zap1 of Saccharomyces cerevisiae. Sequential disruptions of CSR1 were carried out in diploid C. albicans. The csr1/csr1 mutant strain showed severe growth defects under zinc-limited growth conditions and the filamentation defect under hypha-inducing media. The colony morphology and the germ-tube formation were significantly affected by the csr1 mutation. The expression of the hyphae-specific gene HWP1 was also impaired in csr1/csr1 cells. The C. albicans homologs of ZRTl and ZRT2, which are zinc-transporter genes in S. cerevisiae, were isolated. High-copy number plasmids of these genes suppressed the filamentation defect of the csr1/csr1 mutant strain. We propose that the filamentation phenotype of C. albicans is closely associated with the zinc homeostasis in the cells and that Csr1 plays a critical role in this regulation.

• Asian-Australasian Journal of Animal Sciences
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• 제23권1호
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• pp.98-105
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• 2010

A total of 16 crossbred post-weaning pigs (10.64${\pm}$0.27 kg BW) were individually penned and assigned to one of two treatments to investigate the influences of diquat-induced oxidative stress on performance and arginine metabolism. Pigs in the oxidative stress group were injected intra-peritoneally with 10 mg/kg BW of diquat, while the control group were injected with isotonic saline. All pigs were fed ad libitum. The experiment lasted for 7 days. The results indicated that compared with control treatment, oxidative stress induced by diquat significantly decreased average daily gain, intake and feed conversion. The treatment decreased activities of antioxidant enzymes, increased concentration of malondialdehyde in plasma, increased cationic amino acid transporter-1 mRNA level and activity of ornithine aminotransferase and concentrations of arginine and citrulline in the jejunum, decreased the concentrations of arginine in plasma and kidney, and decreased induced nitric oxide synthase mRNA level. It is concluded that oxidative stress induced by diquat can influence absorption and metabolism of arginine and consequently modify the requirement of arginine for post-weaning pigs.