• Bulletin of the Korean Chemical Society
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• v.35 no.5
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• pp.1279-1284
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• 2014

The binding of TATA-binding protein (TBP) to the TATA-box containing promoter region is aided by many other transcriptional factors including TFIIA and TFIIB. The mechanistic insight into the assembly of RNA polymerase II preinitation complex (PIC) has been gained by either directly altering a function of target protein or perturbing molecular interactions using drugs, RNAi, or aptamers. Aptamers have been found particularly useful for studying a role of a subset of PIC on transcription for their ability to inhibit specific molecular interactions. One major hurdle to the wide use of aptamers as specific inhibitors arises from the difficulty with traditional assays to validate and determine specificity, affinity, and binding epitopes for aptamers against targets. Here, using a technique called the bio-layer interferometry (BLI) designed for a label-free, real-time, and multiplexed detection of molecular interactions, we studied the assembly of a subset of PIC, TBP binding to TATA DNA, and two distinct classes of aptamers against TPB in regard to their ability to inhibit TBP binding to TFIIA or TATA DNA. Using BLI, we measured not only equilibrium binding constants ($K_D$), which were overall in close agreement with those obtained by electrophoretic mobility shift assay, but also kinetic constants of binding ($k_{on}$ and $k_{off}$), differentiating aptamers of comparable KDs by their difference in binding kinetics. The assay developed in this study can readily be adopted for high throughput validation of candidate aptamers for specificity, affinity, and epitopes, providing both equilibrium and kinetic information for aptamer interaction with targets.

• BMB Reports
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• v.33 no.4
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• pp.317-320
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• 2000

The succinic semialdehyde dehydrogenase from bovine brain was inactivated by treatment with phenylglyoxal, a reagent that specifically modifies arginine residues. The inhibition at various phenylglyoxal concentrations shows pseudo-first-order kinetics with an apparent secondorder rate constant of 30 $M^{-1}min^{-1}$ for inactivation. Partial protection against inactivation was provided by the coenzyme $NAD^+$, but not by the substrate succinic semialdehyde. Spectrophotometric studies indicated that complete inactivation of the enzyme resulted from the binding of 2 mol phenylglyoxal per mol of enzyme. These results suggest that essential arginine residues, located at or near the coenzyme-binding site, are connected with the catalytic activity of brain succinic semialdehyde dehydrogenase.

• Journal of Pharmacopuncture
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• v.19 no.2
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• pp.114-121
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• 2016

Objectives: Lung cancer remains a deadly disease with unsatisfactory overall survival. Cisplatin, a standard platinum (Pt)-based chemotherapeutic agent, has the potential to inhibit the growth of lung cancer. Its use, however, is occasionally limited by severe organ toxicity. However, until now, no systematic study has been conducted to verify its efficacy with proper experimental support in vivo. Therefore, we examined whether biosynthesized Pt nanoparticles (NPs) inhibited human lung cancer in vitro and in vivo to validate their use in alternative and complementary medicine. Methods: We evaluated the in vitro and the in vivo anticancer efficiencies of biosynthesized Pt NPs in a subcutaneous xenograft model with A549 cells. Severe combined immune deficient mice (SCID) were divided into four groups: group 1 being the vehicle control group and groups 2, 3 and 4 being the experimental groups. Once the tumor volume had reached $70-75mm^3$, the progression profile of the tumor growth kinetics and the body weights of the mice were measured every week for 6 weeks after oral administration of Pt NPs. Doses of Pt NPs of 500, 1,000 and 2,000 mg/kg of body weight were administered to the experimental groups and a dose of honey was administered to the vehicle control group. The efficacy was quantified by using the delay in tumor growth following the administration of Pt NPs of A549 human-lung-cancer xenografts growing in SCID mice. Results: The in vitro cytotoxicity evaluation indicated that Pt NPs, in a dose-dependent manner, inhibited the growth of A549 cells, and the in vivo evaluation showed that Pt NPs at the mid and high doses effectively inhibited and delayed the growth of lung cancer in SCID mice. Conclusion: These findings confirm the antitumor properties of biosynthesized Pt NPs and suggest that they may be a cost-effective alternative for the treatment of patients with lung cancer.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.6
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• pp.649-660
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• 2018

Migraine is a neurological disorder characterized by recurrent and disabling severe headaches. Although several anticonvulsant drugs that block voltagedependent $Na^+$ channels are widely used for migraine, far less is known about the therapeutic actions of carbamazepine on migraine. In the present study, therefore, we characterized the effects of carbamazepine on tetrodotoxin-resistant (TTX-R) $Na^+$ channels in acutely isolated rat dural afferent neurons, which were identified by the fluorescent dye DiI. The TTX-R $Na^+$ currents were measured in medium-sized DiIpositive neurons using the whole-cell patch clamp technique in the voltage-clamp mode. While carbamazepine had little effect on the peak amplitude of transient $Na^+$ currents, it strongly inhibited steady-state currents of transient as well as persistent $Na^+$ currents in a concentration-dependent manner. Carbamazepine had only minor effects on the voltage-activation relationship, the voltage-inactivation relationship, and the use-dependent inhibition of TTX-R $Na^+$ channels. However, carbamazepine changed the inactivation kinetics of TTX-R $Na^+$ channels, significantly accelerating the development of inactivation and delaying the recovery from inactivation. In the current-clamp mode, carbamazepine decreased the number of action potentials without changing the action potential threshold. Given that the sensitization of dural afferent neurons by inflammatory mediators triggers acute migraine headaches and that inflammatory mediators potentiate TTX-R $Na^+$ currents, the present results suggest that carbamazepine may be useful for the treatment of migraine headaches.

• Microbiology and Biotechnology Letters
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• v.34 no.4
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• pp.368-372
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• 2006

Quercetin is a flavonoid molecule that is known to tie in various sources of natural products such as vegetables and fruits. It has been proven that quercetin plays a crucial role in the prevention of colon cancer as well as homeostasis as radical scavenger in human body. It is also well-known that glycosidases, including $\alpha$-glucosidase, are involved in a variety of degenerative metabolic disorders. In the course of screening useful $\alpha$-glucosidase inhibitors, we screened out quercetin as a $\alpha$-glucosidase inhibitor from chemical libraries. Quercetin was shown to be a reversible, slow-binding, and noncompetitive inhibitor of yeast a-glucosidase with a K$_i$ value of $6.3\times10^{-8}$ M when it was included with an enzyme mixture. Together, these results show that quercetin has potential in treating disorders including diabetes, although the further mechanistic study is needed.

• KSBB Journal
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• v.4 no.2
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• pp.94-98
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• 1989

In vitro attachment experiments of bacteria to surface of host plant cell were carried out using C14 labeled cells of A. rhizogenes strain A4 and carrot protoplasts isolated from suspension culture of cells. Protoplasts were cocultivated with A. rhizogenes at various times after their isolation. Attachment kinetics showed that adherence of bacteria to protoplasts attained a maximum level within 120mins of co-cultivation. Maximum attachment occured at pH 6.0 and 24-35$^{\circ}C$. Bacterial attachment was observed at botg carrot cells with and without primary cell wall. The inhibition of transformation on the carrot root discs by A. rhizogenes was observed when non-related strain and heat inactivated bacterial strain cells were pretreated.

• BMB Reports
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• v.42 no.1
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• pp.47-52
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• 2009

Alanine racemase catalyzes the interconversion of L-alanine and D-alanine and plays a crucial role in spore germination and cell wall biosynthesis. In this study, alanine racemase produced by Bacillus anthracis was expressed and purified as a monomer in Escherichia coli and the importance of lysine 41 in the cofactor binding octapeptide and tyrosine 270 in catalysis was evaluated. The native enzyme exhibited an apparent $K_m$ of 3 mM for L-alanine, and a $V_{max}$ of $295\;{\mu}moles/min/mg$, with the optimum activity occurring at $37^{\circ}C$ and a pH of 8-9. The activity observed in the absence of exogenous pyridoxal 5'-phosphate suggested that the cofactor is bound to the enzyme. Additionally, the UV-visible absorption spectra indicated that the activity was pH independece, of VV-visible absorption spectra suggests that the bound PLP exists as a protonated Schiff's base. Furthermore, the loss of activity observed in the apoenzyme suggested that bound PLP is required for catalysis. Finally, the enzyme followed non-competitive and mixed inhibition kinetics for hydroxylamine and propionate with a $K_i$of $160\;{\mu}M$ and 30 mM, respectively.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.11
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• pp.6709-6714
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• 2013

Background: Breast cancer is a leading cause of death in women. The available chemotherapy drugs have been associated with many side effects. Bromelain has novel medicinal qualities including anti-inflammatory, anti-thrombotic, fibrinolytic and anti-cancer functions. Commercially available bromelain is obtained through tedious methods; therefore, recombinant bromelain may provide a cheaper and simpler choice with similar quality. Materials and Methods: This study aimed to assess the effects of commercial and recombinant bromelain on the cytokinetic behavior of MCF-7 breast cancer cells and their potential as therapeutic alternatives in cancer treatment. Cytotoxic activities of commercial and recombinant bromelain were determined using (sulforhodamine) SRB assay. Next, cell viability assays were conducted to determine effects of commercial and recombinant bromelain on MCF-7 cell cytokinetic behavior. Finally, the established growth kinetic data were used to modify a model that predicts the effects of commercial and recombinant bromelain on MCF-7 cells. Results: Commercial and recombinant bromelain exerted strong effects towards decreasing the cell viability of MCF-7 cells with $IC_{50}$ values of 5.13 ${\mu}g/mL$ and 6.25 ${\mu}g/mL$, respectively, compared to taxol with an $IC_{50}$ value of 0.063 ${\mu}g/mL$. The present results indicate that commercial and recombinant bromelain both have anti-proliferative activity, reduced the number of cell generations from 3.92 to 2.81 for commercial bromelain and to 2.86 for recombinant bromelain, while with taxol reduction was to 3.12. Microscopic observation of bromelain-treated MCF-7 cells demonstrated detachment. Inhibition activity was verified with growth rates decreased dynamically from 0.009 $h^{-1}$ to 0.0059 $h^{-1}$ for commercial bromelain and to 0.0063 $h^{-1}$ for recombinant bromelain. Conclusions: Commercial and recombinant bromelain both affect cytokinetics of MCF-7 cells by decreasing cell viability, demonstrating similar strength to taxol.

• BMB Reports
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• v.31 no.1
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• pp.25-30
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• 1998

Kinetic studies of L-Alanine dehydrogenase from Bacillus subtilis-catalyzed reactions in the presence of $Zn^{2+}$ were carried out. The substrate (L-alanine) saturation curve is hyperbolic in the absence of the metal ion but it becomes sigmoidal when $Zn^{2+}$ is added to the reaction mixture indicating the positive cooperative binding of the substrate in the presence of zinc ion. The cooperativity of substrate binding depends on the xinc ion concentration: the Hill coefficients ($n_H$) varied from 1.0 to 1.95 when the zinc ion concentration varied from 0 to $60\;{\mu}m$. The inhibition of AlaDH by $Zn^{2+}$ is reversible and noncompetitive with respect to $NAD^+$ ($K_i\;=\;5.28{\times}10^{-5}\;M$). $Zn^{2+}$ itself binds to AlaDH with positive cooperativity and the cooperativity is independent of substrate concentration. The Hill coefficients of substrate biding in the presence of $Zn^{2+}$ are not affected by the enzyme concentration indicating that $Zn^{2+}$ binding does not change the polymerization-depolymerization equilibria of the enzyme. Among other metal ions, $Zn^{2+}$ appears to be a specific reversible inhibitor inducing conformational change through the intersubunit interaction. These results indicate that $Zn^{2+}$ is an allosteric competitive inhibitor and substrate being a non-cooperative per se, excludes the $Zn^{2+}$ from its binding site and thus exhibits positive cooperativity. The allosteric mechanism of AlaDh from Bacillus subtilis is consistent with both MWC and Koshland's allosteric model.

• BMB Reports
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• v.31 no.1
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• pp.83-88
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• 1998

Recently a B cell surface molecule, CD40, has emerged as a receptor mediating a co-stimulatory signal for B cell proliferation and differentiation. To investigate the mechanism of synergy between interleukin-4 (IL-4) and CD40 ligation in B cell activation, we have examined the effect of CE40 cross-linking on the IL-4 receptor expression in human B cells using anti-CE40 antibody. We observed that IL-4 and anti-CD40 both induce IL-4 receptor gene expression with a rapid kinetics resulting in a noticeable accumulation of IL-4 receptor mRNA within 4 h. While IL-4 caused a dose-dependent induction of surface IL-4 receptor expression, the inclusion of anti-CD40 in the IL-4-treated culture, further up-regulated the IL-4-induced IL-4 receptor expression as analyzed by flow cytometry. Pretreatment of B cells with inhibitors of protein tyrosine kinase (PTK) resulted in a significant inhibition of both the IL-4- and anti-CD40-induced IL-4 receptor mRNA levels, while protein kinase C (PKC) inhibitors had no effects. These results suggest that IL-4 and CD40 ligation generate B cell signals, which via PTK-dependent pathways, lead to the synergistic induction of IL-4 receptor gene expression. The rapid induction of IL-4 receptor gene expression through the tyrosine kinase-mediated signal transduction by B cell activating stimuli, would provide cells capacity for an efficient response to IL-4 in the early phase of IL-4 action, and may in part constitute the molecular basis of the reported anti-CD40 co-stimulatory effect on the IL-4-induced response.