• Journal of Microbiology and Biotechnology
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• v.18 no.1
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• pp.183-187
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• 2008

Vascular endothelial growth factors (VEGFs) are a family of proteins that mediate angiogenesis. $VEGF_{165}$ is a VEGF-A isoform and has been extensively studied owing to its potential use in therapeutic angiogenesis. This study established Chinese hamster ovary (CHO) cells overexpressing recombinant human $VEGF_{165}$ $(rhVEGF_{165})$ protein. The production rate of the established CHO cells was over 80mg/l of $rhVEGF_{165}$ protein from a 7-day batch culture process using a 7.5-l bioreactor with a 5-l working volume and serum-free medium. The $rhVEGF_{165}$ protein was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure that resulted in a 48% recovery rate. The purified $rhVEGF_{165}$ protein was a glycosylated homodimeric protein with a higher molecular weight (MW) than the protein expressed from insect cells, suggesting that the glycosylation of the $rhVEGF_{165}$ protein in CHO cells differed from that in insect cells. The purified $rhVEGF_{165}$ protein in this study was functionally active with a half-maximal effective concentration of 3.8ng/ml and specific activity of $2.5{\times}10^5U/mg$.

• Journal of Society of Preventive Korean Medicine
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• v.7 no.2
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• pp.65-74
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• 2003

Objective : The aim of present study is to evaluate the inhibitory potential of licorice extract and glycyrrhizin on cytochrome P450(CYP) in human liver microsomes. Methods : Using human liver microsomes, water extract of licorice and glycyrrhizin as an inhibitor were co-incubated with each probe drug representing selective CYP isoform activity. We measured relative metabolic activity in incubation condition compared to that with no extract of licorice using HPLC system. Results : Both water extracts of licorice and glycyrrhizin showed inhibitory effect on CYP-catalyzed reactions. CYP2C19 $(IC_{50}=126.7{\mu}g/ml)$ is most potently inhibited by water extract than other tested CYP isoforms$(IC_{50}>450{\mu}g/ml)$, but glycyrrhizin exhibited potent inhibition on CYP1A2$(IC_{50}=106.9{\mu}g/ml)$ followed by CYP2C9 and CYP2D6. Conclusion: These results indicate that water extract of licorice and glycyrrhizin have inhibitory potential on CYP-catalyzed reaction in human liver microsomes. But the mechanism of inhibition was slightly different between them Water extract of licorice mainly inhibited CYP2C19, and glycyrrhizin primarily inhibited CYP1A2. The inhibition by water extract of licorice and glycyrrhizin on CYP isoforms may cause drug interaction with co-administered drug leading to toxicity or treatment failure.

• Korean Journal of Materials Research
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• v.23 no.5
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• pp.271-275
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• 2013

Cobalt (Co) compounds have been used for centuries to impart rich blue color to glass, glazes and ceramics. Cobalt monoxide (CoO), an oxide of Co, is an inorganic compound that has long been used as a coloring agent in the ceramic industry. Unlike other coloring agents, CoO can be used to develop colors other than blue, and several factors such as its concentration in the glaze and firing condition have been suggested as possible mechanisms. For example, CoO produces a typical blue color called "cobalt blue" at very low concentrations such as 1 wt% in both oxidation and reduction firing conditions; a higher concentration of CoO (5 wt%) develops a darker blue color under the same firing conditions. Interestingly, CoO also develops a purple color at high concentrations above 10 wt%. In this study, we examined the applicability and mechanism of a novel purple glaze containing cobalt(II, III) oxide, one of the well characterized cobalt oxides. Experimental results show that an Augite crystal isoform (Augite-Fe/Co) in which Fe was replaced with Co is the main component contributing to the formation of the purple color. Based on these results, we developed a glaze using chemically synthesized Augite-Fe/Co crystal as a color pigment. Purple color glaze was successfully developed by the addition of 6~15 wt% of $Co_3O_4$ to magnesia lime.

• Animal cells and systems
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• v.12 no.3
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• pp.137-141
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• 2008

Molecular cloning revealed the three isoforms($Ca_v3.1,\;Ca_v3.2,\;and\;Ca_v3.3$) of the T-type calcium channel subfamily. Expression studies exhibited their distinctive electrophysiological and pharmacological properties, accounting for diverse properties of T-type calcium channel currents previously characterized from isolated cells. However, electrophysiological properties of ion channels have shown to be more diversified by their splice variants. We here searched splice variants of rat $Ca_v3.1$ T-type channel by reverse-transcription-polymerase chain reaction(RT-PCR) to further explore diversity of $Ca_v3.1$. Interestingly, analyses of cloned RT-PCR products displayed that there were at least four splicing variants of rat $Ca_v3.1$ in the loop connecting repeats III and IV. Southern blot analyses indicated that the predominantly detected variant in brain was $Ca_v3.1a$(492 bp), which were rarely detected in most of peripheral tissues. Other two variants($Ca_v3.1c$, 546 bp; $Ca_v3.1d$, 525 bp) were detected in most of the tissues examined. The smallest isoform($Ca_v3.1b$, 471 bp) was rarely detected all the tissues. Electrophysiological characterization of the splicing variants indicated that the splice variants differ in inactivation kinetics and the voltage dependence of activation and inactivation as well.

• Animal cells and systems
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• v.1 no.4
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• pp.589-594
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• 1997

The neuronal voltage-sensitive calcium channels (VSCCs) are multisubunit complexes consisting of $\alpha_1,\;\alpha_2-\delta$ and $\beta$ subunits. Heterologous expression and biochemical studies have shown that the activity of VSCCs is regulated by their $\beta$ subunits in a $\beta$ subunit isoform-specific manner. To elucidate the $\beta$ subunit identity of the P/Q-type calcium channel encoded by an $\alpha_{1A}$ subunit, which is exclusively expressed in the Purkinje and granule cell of the cerebellum, we have examined the spatial and temporal expression patterns of $\beta$ subunits and compared them with those of $\alpha_{1A}$ subunit in the developing rat cerebellum. Reverse transcriptase- polymerase chain reaction (RT-PCR) and Northern blot analysis have shown that $\beta_4$ subunit mRNA was prominently expressed in the cerebellum and much more abundant than any other distinct $\beta$ subunits. RNase protection assay has further demonstrated that the expression of $\alpha_{1A}$ and $\beta_4$ subunits increased during cerebellar development, while the amount of $\beta_2$ and $\beta_3$ mRNAs did not significantly change. In addition, a $\beta_4$ transcript was present in cultured cerebellar granule cells, but not in astrocyte cells, and the level of $\beta_4$ mRNA expression increased gradually in vitro seen as in vivo. Based on the spatial and temporal expression patterns of $\beta_4$ subunit, we conclude that $\beta_4$ may predominantly associate, but probably not exclusively, with the $\alpha_{1A}$ subunit in rat cerebellar granule cells.

• Animal cells and systems
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• v.4 no.3
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• pp.273-278
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• 2000

Transforming growth factor-$\beta$ (IGF-$\beta$)s are multifunctional small polypeptides synthesized in most cell types. TGF-$\beta$ exerts pivotal effects on both bone formation and resorption. In addition, increasing lines of evidence implicate TGF-$\beta$ as a potential coupling factor between these two processes during bone remodeling. In the present study, the expression form and the activation mechanism of latent-TGF-$\beta$ were investigated using specific antibodies for each isoform. TGF-$\beta$s were observed to be synthesized and accumulated in a large amount in cultured osteoblastic cells. The estimated molecular weights of intracellular TGF-$\beta$2 and -$\beta$3 were 49 and 55 kDa, respectively. Based on proteolytic digestion study and immunofluorescence observation, these precursor forms seemed to be accumulated in distinct intracellular compartments. To examine whether the internal pool of TGF-$\beta$ was possiblely regulated by external signals, their biological activites were examined in a conditioned media of this cell. Although the intact conditioned media did not contain detectable TGF-$\beta$ activity, heat-treatment or acid-activation of the conditioned media revealed significant TGF-$\beta$ activity. Furthermore, in the presence of estrogen, this activity was dramatically diminished. It is known that activation of latent TGF-$\beta$ can be achieved by different chemical and enzymatic treatments, or by incubation with certain cell types. This extracellular activation was suggested as a key step in the regulation of TGF-$\beta$ activity. In addition to these extracellular activation, this study suggests that the synthesis and intracellular processing are important regulation steps for TGF-$\beta$ action. In addition, this regulation Is specific for TGF-$\beta$ type 2, because the change was not observed in TGF-$\beta$3 in osteoblastic cell line.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.4
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• pp.205-212
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• 2010

Endometritis is one of the primary reasons for reproductive failure. In order to investigate endometritis-associated marker proteins, proteomic analysis was performed on bovine endometrium with endometritis. In bovine endometritis, desmin, $\alpha$-actin-2, heat-shock protein (HSP) 27, peroxiredoxin-6, luteinizing hormone receptor isoform 1, collectin-43 precursor, deoxyribonuclease-I (DNase-I), and MHC class I heavy chain (MHC-Ih) were up-regulated. In contrast, transferrin, interleukin-2 precursor, hemoglobin $\beta$ subunit, and potassium channel tetramerisation domaincontaining 11 (KCTD11) were down-regulated in comparison to normal endometrium. The proteomic results were validated by semiquantitative-PCR and immunoblot analysis. The mRNA levels of desmin, transferrin, $\alpha$-actin-2, HSP27, KCTD11, and MHC-Ih were up-regulated by over 1.5-fold, and showed a pattern similar to their proteomic profiles. Desmin and $\alpha$-actin-2 protein showed positive correlations between proteomic analysis and immunoblot analysis. These results suggest that desmin and $\alpha$-actin-2 may play important roles in endometritis-related function, and could be useful markers for the diagnosis of bovine endometritis.

• Fisheries and Aquatic Sciences
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• v.15 no.3
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• pp.221-231
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• 2012

The marine medaka Oryzias dancena choriogenin H gene (odChgH) and its mRNA expression during estradiol-$17{\beta}$ (E2) exposure were characterized. At the amino acid level, the choriogenin H protein is predicted to possess the conserved repetitive N-terminal region, as well as zona pellucida (ZP) and Trefoil factor family (TFF) domains. At the genomic level, odChgH has an eight-exon organization with a distribution pattern of transcription factor binding sites in the 5'-upstream region, which is commonly found in other estrogen-responsive genes. The tissue distribution pattern of odChgH mRNA was found to be gender-specific, whereby females showed a higher expression level and wider tissue distribution than did males. During embryonic development, odChgH mRNA was robustly detected from the stage of visceral blood vessel formation. Experimental E2 exposure of males resulted in odChgH mRNA being induced not only in the liver, but also in other several tissues. The E2-mediated induction was fairly dose-dependent. The basal expression levels of hepatic odChgH mRNA were lower in males that were acclimated to 30 ppt salinity than in those acclimated to 0 or 15 ppt salinity. In contrast, the inducibility of odChgH mRNA during E2 exposure was greater in seawater-acclimated fish than in brackish water- or freshwater-acclimated fish.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.44 no.2
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• pp.126-140
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• 2011

Genetic determinants of two metallothionein isoforms (MT-A and MT-B) were isolated and characterized from the perciform species, rockbream (Oplegnathus fasciatus). Rockbream MT-A and MT-B shared a high degree of homology at amino acid levels with representative orthologs from other perciform species, especially with respect to the conserved cysteine residues. At the genomic level, both MT-A and MT-B genes represent a tripartite structure typical of vertebrate MT genes. However, rockbream MT-B showed unusually large introns (1.2 kb and 0.8 kb for intron I and II, respectively), a phenomenon that has rarely been seen in other vertebrate MT genes. MT-A and MT-B transcripts were ubiquitously detected in a wide array of tissues, wherein brain and eye showed the highest basal expression levels, and the fin exhibited the lowest expression of both isoforms. The basal expression of MT-A in most tissues was significantly higher (ranging from 4- to 10-fold) than that of MT-B. Upon heavy metal exposures to Cd, Cu or Zn at 25 ppb for 48 h, MT-A and MT-B transcripts in the liver were significantly activated by Cd and moderately by Zn. On the other hand, exposure to Cu did not result in alterations of MT-A, nor in the significant suppression of MT-B. Following bacterial challenges with Escherichia coli, Edwardsiella tarda or Streptococcus iniae, MT isoforms in the liver, kidney and spleen were highly modulated and exhibited a pattern that was dependent on the bacterial species, tissues and isoforms. These results suggest that the two MT isoforms could be taken into account as potential indicators of metal toxicity and immune perturbations of this aquaculture-relevant species.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.43-43
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• 2003

Large-conductance $Ca^{2+}$-actived $K^{+}$ channels ($BK_{Ca}$ channels) play a key role in setting the pace of contractile activity in muscle and are involved in the regulation of neurotransmitter release in neuron. $BK_{Ca}$ channels are activated by depolarizing membrane potential and the elevated level of intracellular calcium. Using yeast-two hybrid assay, we have identified a novel protein interacting with the cytosolic carboxyl terminus of rSlo, the brain isoform of rat large-conductance $Ca^{2+}$-activated $K^{+}$ channel $\alpha$-subunit. The novel gene encodes 51 kDa protein and is named as SIRK(rSlo-interacting RGS-like protein). SIRK is expressed in various tissues and localized in the cytosolic and the membrane fraction. Biochemical and immunological studies indicated that SIRK physically interacted with the cytosolic region of rSlo. To investigate whether SIRK can modulate the activity of rSlo, GFP-fused SIRK and rSlo were transiently transfected into COS-7 cells and the effects of SIRK was studied using electrophysiological means. We concluded that the overexpression of SIRK alters the surface expression of rSlo channel with only a limited effect on the biophysical characteristics of the channel.the channel.