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We report the effect of pH on the supramolecular complexation of two biothiols, viz., homocysteine (Hcy) and cysteine (Cys), with cucurbit[7]uril (CB[7]). Under basic pH conditions, Cys did not complex with CB[7], whereas Hcy efficiently complexed with CB[7], as confirmed by

This study was undertaken to determine the effects of enzyme-treated Zizania latifolia (ETZL) and of its major compound tricin on skin photo-aging and to investigate the mechanisms involved. It was found ETZL and tricin suppressed matrix metalloproteinase (MMP) production and increased type I-procollagen production in UVB-irradiated human dermal fibroblasts (HDFs). Furthermore, ETZL and tricin significantly up-regulated the expressions of the antioxidant enzymes HO-1 and SOD1, reduced UVB-induced reactive oxygen species (ROS) generation and mitogen-activated protein kinase (MAPK) induction by ROS and thereby attenuated activator protein-1 (AP-1) expression. In addition, ETZL and tricin both reduced the phosphorylations of IκBα and IKKα/ß and κB blocked the nuclear translocation of nuclear factor-κB (NF-κB) p65. These results show that ETZL have skin protective effects against UVB and suggest tricin as major efficacious material in ETZL protecting skin photoaging.

This paper introduces three ways to determine host-guest complexation of cucurbit[7]uril (CB[7]) with homocysteine (Hcy). After preincubating Hcy and cysteine (Cys) with CB[7], Ellman's reagent (DTNB) was used to detect Hcy and Cys. Only Cys reacted with DTNB and Hcy gave a retarded color change. This suggests that the -SH group of Hcy is buried inside CB[7]. Human cystathionine γ-lyase (hCGL) decreased the level of Hcy degradation after preincubating Hcy and CB[7]. These results suggest that the amount of free Hcy available was decreased by the formation of a Hcy-CB[7] complex. The immunological signal of anti-Hcy monoclonal antibody was decreased significantly by preincubating CB[7] with Hcy. The ELISA results also show that ethanethiol group (-CH₂CH₂SH) of Hcy, which is an epitope of anti-Hcy monoclonal antibody, was blocked by the cavity in CB[7]. Overall, CB[7] can act as a host by binding selectively with Hcy, but not Cys. The calculated half-complexation formation concentration of CB[7] was 58.2 nmol using Ellman's protocol, 97.9 nmol using hCGL assay and 87.7 nmol using monoclonal antibody. The differing binding abilities of Hcy and Cys towards the CB[7] host may offer a simple and useful method for determining the Hcy concentration in plasma or serum.

We have previously shown that the bactericidal effect of ZnO nanoparticles in the absence of a light source originates from the released Zn2+ ions. The purpose of this study was to explore antibacterial activity arising from photo-induced reactive oxygen species (ROS) of ZnO nanoparticles under UV-A light irradiation. To achieve this, S. aureus and K. pneumoniae bacteria were exposed to three different ZnO nanoparticles under UV-A light. The concentrations of the ZnO nanoparticles were low, such that the antibacterial effect of the dissolved Zn2+ ions was negligible. From various empirical evidence, we found that the oxygen defects of the ZnO crystals enhanced the photogeneration of ROS and consequently, the ZnO nanoplates (NPs) with the polar facets exhibited the most pronounced antibacterial activity under UV-A stimulation. To enhance the antimicrobial activity of the NPs, we successfully synthesized silver-nanoparticle-decorated ZnO NPs and explored their antibacterial activity compared to that of the NPs.

본 연구는 세균에 대한 항균효과와 난연 특성을 갖는 신소재의 개발이 목표이다. 제올라이트의 기공에 은과 아연을 포함한 무기항균제(inorganic bacteriocide, IB)와 친환경 무기계 난연제인 aluminum trihydroxide(ATH)를 함유한 PVC 복합소재인 PVC/IB/ATH를 제조하였다. 두 종류의 세균으로 탁도 분석법, 진탕 배양법, drop-test, 이중 염색법을 사용하여 PVC/IB/ATH 복합소재의 항균효과 실험결과 99%의 항균효과를 확인하였다. 또한 한계산소농도 지수와 열중량 분석으로 PVC/IB/ATH 복합소재의 난연 특성을 확인한 바, ATH와 무기항균제의 알루미늄 성분에 의존적으로 증가하였다. 또한 PVC/IB/ATH 복합소재의 열적 특성은 소폭 증가하였다. 결론적으로, PVC/IB/ATH의 응용은 의료 산업, 항균 시스템 그리고 난연 물질과 같은 다양한 분야에 이용될 것으로 기대한다.

Zizania latifolia exhibits anti-inflammatory and anti-allergic effects; however, the mechanisms behind these effects are unknown. Here the ethanol extract of Z. latifolia was partitioned using hexane, chloroform, ethyl acetate, butanol, and water. Subsequently, the anti-allergic effects of these fractions were evaluated in vitro. The results showed that the chloroform fraction of Z. latifolia inhibited the release of β-hexosaminidase and tumor necrosis factor (TNF-α) from RBL-2H3 cells stimulated with dinitrophenyl-bovine serum albumin (DNP-BSA). In addition, this fraction suppressed the expression of cyclooxygenase-2 (COX-2) and inhibited the activation of mitogen-activated protein kinases (MAPKs). The results obtained suggest that the chloroform fraction of Z. latifolia inhibited mast cell-mediated allergic inflammatory responses.

Supramolecular complexation of two bio-thiols, homocysteine (Hcys) and cysteine (Cys), by cucurbit[7]uril (CB[7]) has been fully investigated by 1H NMR spectroscopy, mass spectrometry, isothermal titration calorimetry, and the results were further verified with computational investigations. NMR titration experimental results obviously indicate that the binding stoichiometry of CB[7] to Hcys is 1:1 and to Cys is 1:2 in aqueous solution. The binding constants and thermodynamic parameters associated with the complexation between CB[7] and the bio-thiols were determined by isothermal titration calorimetry. The energy-minimized structures of the supramolecular complexes of CB[7] with Hcys and Cys were determined and provide good agreement with the experimental results. The CB[7] cavity is sufficient to include the two Cys, but is unable to accommodate two Hcys due to steric hindrance. The differing binding abilities of Hcys and Cys in aqueous solution towards CB[7] host may lead to discriminate them.

Bacterial infection is one of the most common and universal cause of disease spreading associated with medical and surgical environment. Antimicrobial function of plasticized polyvinyl chloride (PVC) will be useful for making hygienic environments. Here, we synthesized the PVC composite by the addition of inorganic bacteriocide containing silver, zinc and zeolite. And we investigated the growth inhibition rate for Staphylococcus aureus and Klebsiella pneumoniae which were analyzed in the presence of PVC composite with different doses of inorganic bacteriocide (1~15 wt%). Bacterial proliferation was significantly inhibited by 3 wt% of inorganic bacteriocide containing PVC composite. And we found the inhibition of bacterial biofilm formation by 5 wt% of inorganic bacteriocide containing PVC composite by the observation of scanning electron microscopy (SEM). Our data suggested that the antimicrobial effect of inorganic bacteriocide was caused by disturbing the bacterial biofilm formation.

We investigate the effect of cell culture conditions, using pristine graphene sheets as growth substrate, on the human nerve cell line (SH-SY5Y). In order to evaluate cell viability and morphology, we applied the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, and fluorescence microscopy of cells stained with Hochest 33342 and Calcein AM. Human nerve cells exhibited 84% viability on pristine graphene sheets compared with control (cell culture polystyrene) after 3 days culturing. Fluorescence data showed that the presence of graphene did not influence cell morphology. These results suggest that graphene sheets may be used for biological applications.

Capture and reforming fume gases from atmosphere became an ultimate task recently. Photocatalytic reduction to useful compounds is a key to the future. Photoinduced reduction of carbon dioxide and ammonia aqueous solution into amine compounds at room temperature and atmospheric pressure using transition metal doped titanium dioxide is reported in this paper. UV-visible spectra showed a high absorbance at 200 nm wavelength which is a characteristic of urea. High Performance Liquid Chromatography revealed presence of amine compounds and urea was found a main product of photocatalytic reaction.

Many applications of nanoparticles have been developed since 1970s. Surface plasmon resonance (SPR) effect can be generated at the surface of nanoparticles by illumination. SPR is the resonant oscillation of conduction electrons at the surface material stimulated by incident light. The collisions between excited electrons and metal atoms can cause the production of thermal energy (photothermal effect). Here, we presented the development of thermo-cosmetics using photothermal effect of gold nanoparticles. Gold nanoparticles (GNPs) were chosen for it's low toxicity. We also and investigated the cell biocompatibility and heating effectiveness for photothermal effect of GNPs. Synthesized GNPs were verified by UV-vis spectrophotometer, where GNP has a characteristic absorbance spectrum. Concentration of GNP was measured by atomic absorption analyzer. The cytotoxicity was confirmed by MTT assay and double staining assay. Photothermal effect of GNP was demonstrated by the thermal increasing properties depending on GNP concentration, which was taken by an IR-thermal camera with a xenon lamp as the light source. If the thermal effect of GNP is applied for thermo-cosmetics, it can supply heat to skin by converting solar energy into thermal energy. Thus, cosmetics containing GNPs can provide benefits to people in the cold region or winter season for maintaining skin temperature, which lead to a positive effect on skin health.

We investigate the effect of cell culture conditions, using functionalized graphene sheets with oxygen (–O) or fluorine (–F) as growth surfaces, on the human nerve cell line (SH-SY5Y). We applied the fluorescence microscopy of cells stained with Hochest 33342, Calcein AM, and MTT assay to evaluate cell viability and morphology. The effect of the graphene on the cells in culture was dependent on the atomic state of the graphene sheet. SH-SY5Y cells exhibited approximately 138% viability (MTT assay) on the oxygenated graphene sheets and 50% viability on the fluorinated graphene sheets, as compared to the data from culturing on pristine graphene.

Selenium is an essential trace element for mammals, but it is very toxic. Therefore, the control of selenium concentrations should be precisely and effectively monitored. Selenium is naturally obtained through foods and seleno-L-methionine (LSeMet) is a major form of selenium. It has been reported that L-SeMet is only converted into Se-adenosyl-L-SeMet. However, a recent study suggested that L-SeMet was directly metabolized into methylselenol () in mouse liver extract by the reaction of cystathionine -lyase (CGL). The canonical reaction of CGL was known to catalyze the cleavage of L-cystathionine to L-cysteine, -ketobutyrate and . In the present study, we found that L-SeMet could be directly converted to using purified homogenous human CGL instead of mouse liver cytosol. Authentic was prepared by reduction of dimethyldiselenide with sodium tetrahydroborate. The gaseous product of the enzymatic reaction with L-SeMet was analyzed by GC/MS spectrometry. The GC/MS data was identical to that of authentic dinitrophenyl selenoether. We also analyzed the kinetic parameters for the formation of from L-SeMet by human and mouse CGL. These results suggest that human CGL is a critical enzyme which is responsible for L-SeMet metabolism.

Zizania latifolia exhibits anti-inflammatory and anti-allergic effects; however, the mechanisms behind these effects are unknown. Here the ethanol extract of Z. latifolia was partitioned using hexane, chloroform, ethyl acetate, butanol, and water. Subsequently, the anti-allergic effects of these fractions were evaluated in vitro. The results showed that the chloroform fraction of Z. latifolia inhibited the release of β-hexosaminidase and tumor necrosis factor (TNF-α) from RBL-2H3 cells stimulated with dinitrophenyl-bovine serum albumin (DNP-BSA). In addition, this fraction suppressed the expression of cyclooxygenase-2 (COX-2) and inhibited the activation of mitogen-activated protein kinases (MAPKs). The results obtained suggest that the chloroform fraction of Z. latifolia inhibited mast cell-mediated allergic inflammatory responses.

Nanocomposite films of poly(ethylene terephthalate)(PET) and nano titania() were prepared by melt compounding with a twin-screw extruder followed by hot pressing for the purpose of investigating antibacterial activity. FE-SEM, EDS and XRD measurements confirmed that nano was successfully dispersed at the level of individual particles in the PET matrix. Silver was introduced through photocatalytic reduction by nano under UV (254 nm) irradiation to the nanocomposite films after being immersed in aqueous solution (100 ppm Ag ion). Even at 1 wt% of content, the nanocomposite with silver photodeposition exhibited strong antimicrobial activity against Klebsiella pneumoniae and Staphylococcus aureus in measurement by shaking flask method.

Medicinal herbs have been shown to have protective functions for skin and hair. We investigated the ef- fects of complex of soluble β-cyclodextrin and phytochemicals on the functions of skin and hair. In previous report, we evaluated the safety of supramolecules and found their anti-microbial effects and anti-fungal effect against Gram (+) and Malassezia furfur which is known to cause dandruff. Here we present that functional supramolecules-con- taining cream promotes the biological skin activity by inducing the collagen formation. And treatment of supra- molecules-containing hair tonic increased the rate of hair growth of mouse. Taken together, supramolecular cosmetic compounds containing water insoluble phytochemicals and water soluble β-cyclodextrin exhibit the potential ability for hair growth promotion and delaying the aging of skin.

We discuss the influence of few-walled carbon nanotubes (FWCNTs) treated with nitric acid and/or sulfuric acid on field emission characteristics. FWCNTs/tetraethyl orthosilicate (TEOS) thin film field emitters were fabricated by a spray method using FWCNTs/TEOS sol one-component solution onto indium tin oxide (ITO) glass. After thermal curing, they were found tightly adhered to the ITO glass, and after an activation process by a taping method, numerous FWCNTs were aligned preferentially in the vertical direction. Pristine FWCNT/TEOS-based field emitters revealed higher current density, lower turn-on field, and a higher field enhancement factor than the oxidized FWCNTs-based field emitters. However, the unstable dispersion of pristine FWCNT in TEOS/N,N-dimethylformamide solution was not applicable to the field emitter fabrication using a spray method. Although the field emitter of nitric acid-treated FWCNT showed slightly lower field emission characteristics, this could be improved by the introduction of metal nanoparticles or resistive layer coating. Thus, we can conclude that our spray method using nitric acid-treated FWCNT could be useful for fabricating a field emitter and offers several advantages compared to previously reported techniques such as chemical vapor deposition and screen printing.

질병과 밀접한 관계가 있는 hCGL 단백질의 경우 대량 배양 시 유도체를 사용하지 않아도 발현이 되는 점과 유전자 측면에서 조작이 쉬운 E.coli를 이용하여도 발현이 된다는 점에 있어서 중요한 이점을 가지고 있다. 본 연구에서는 배양되는 온도와 발현에 관련 있는 유도체의 농도, 600 nm에서의 균 성장 정도에 따른 유도체의 첨가 그리고 배지의 양을 조절하면서 유입되는 aeration의 조건으로 hCGL 단백질 발현의 최적의 조건 확립을 목적으로 하였다. 또 각 발생되는 inclusion body의 양을 측정하면서 보다 많은 가용성 단백질을 발현시키는 조건을 확립하고자 하였다. hCGL 단백질은 저온에서 보다 많은 양의 단백질이 발현되며 inhibitor의 억제를 담당하는 유도체의 농도와는 상관없이 발현이 되었다. 또한 균의 성장 정도에 따라 유도체의 첨가시기를 달리 하였을 때, 발현 비율에 차이는 있었으나 전체적인 단백질 양과 비교해 보면, 이는 hCGL 발현에 큰 영향을 미치지 않는다. 배지의 양을 달리하여 살펴본 aeration에 따른 hCGL 발현 정도는 배지의 부피가 15%일 때 높은 aeration으로 균의 양은 많았으나 목적 단백질인 hCGL의 발현은 aeration이 되지 않는 조건에서 더 잘되는 것을 확인하였다. 그리고 His-TEV-hCGL의 활성은 야생형 hCGL의 활성을 기준으로 하였을 때, L-cystathionine을 기질로 하였을 경우 76%, L-cysteine을 기질로 하였을 경우 88% 수준으로 유사한 활성을 나타내었고, 이는 손쉽게 정제 가능한 His-TEV-hCGL을 야생형을 대신하여 사용할 수 있음을 시사한다. 또한 His-TEV-hCGL이 야생형 hCGL과 같이, 427 nm에서 흡광을 가지는 것으로 보아 보효소PLP를 포함하고 있음을 알 수 있었다. 이로써 homocysteine 대사연구에 필수적인 hCGL 효소를 다량 얻는 방법을 확립하고, 관련 연구에 기여하리라 사료된다. 【There is a growing recognition of the significance of $H_2S$ as a biological signaling molecule involved in vascular and nervous system functions. In mammals, two enzymes in the transsulfuration pathway, cystathionine ${\beta}$ -synthase (CBS) and cystathionine ${\gamma}$ -lyase (CGL), are believed to be chiefly responsible for $H_2S$ biogenesis. Genetic inborn error of CGL leads to human genetic disease, cystathioninuria, by accumulating cystathionine in the body. This disease is secondarily associated with a wide range of diseases including diabetes insipidus and Down's syndrome. Although the human CGL (hCGL) overexpression is essential for the investigation of its function, structure, reaction specificity, substrate specificity, and protein-protein interactions, there is no clear report concerning optimum overexpression conditions. In this study, we report a detailed analysis of the overexpression conditions of the hCGL using a bacterial system. Maximum overexpression was obtained in conditions of low culture temperature after inducer addition, performing low aeration during overexpression, and using a low concentration inducer (0.1 mM, IPTG) for induction. Expressed hCGL was purified by His-tag affinity column chromatography and confirmed by Western blot using hCGL antibody and enzyme activity analysis. We also report that the His tag with TEV site attached protein exhibits 76% activity for ${\alpha}-{\gamma}$ elimination reaction with L-cystathionine and 88% for ${\alpha}-{\beta}$ elimination reaction with L-cysteine compared to those of wild type hCGL, respectively. His tag with TEV site attached protein also exhibits a 420 nm absorption maximum, which is attributed to the binding cofactor, pyridoxal 5'-phosphate (PLP).】

pulegone has been widely used inflavoring agents, in perfumery, and in aromatherapy. Pule-gone is also known as one of the terpenses, which has beenextensively used to enhance the transdermal permeability ofseveral drug molecules such as 5-fluorouracil, propranololhydrochloride, indomethacin, ketoprofen, and tamoxifen.

A diet rich in polyphenols has previously been shown to be associated with a lower risk for cardiovascular dis- ease. Specifically, inhibition of platelet hyperactivation by polyphenols decreases platelet-dependent thrombosis, reducing the risk for cardiovascular disease. However, whether polyphenols in Salvia miltiorrhiza Bunge (SMB) affect platelet functions has not been clearly determined. Here, we investigated the effects of methanol and ethyl acetate (EtOAc) ex- tracts from SMB on free radical scavenging activities, and platelet aggregation and adhesion. We found that the EtOAc fraction was the most effective at scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azinobis(3- ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals (concentration for 50% reduction, RC50 = 2.4 ± 1.5 and 27.9 ± 1.4 μg/mL, respectively), and that, especially for DPPH radicals, the EtOAc fraction showed better scavenging activity than butylated hydroxyanisole (BHA, RC50 = 5.5 ± 0.1 μg/mL). Measurements with a turbidimetric aggregometer revealed that the EtOAc fraction was the most potent inhibitor of ADP- and collagen-induced rat platelet aggregation. In addition, the methanol and EtOAc fraction dose-dependently inhibited thrombin-stimulated platelet adhesion to collagen or fibrino- gen. Collectively, these results suggest that the polyphenol-rich EtOAc fraction from SMB can reduce platelet hyperacti- vation by scavenging free radicals. Thus, the EtOAc fraction of SMB is a potential source of inhibitors of platelet- dependent thrombosis.

In this study we evaluated the method to develop red ginseng oil containing high content of phytochemicals by enzymes treatment. To select the optimum extraction process of red ginseng with oils, the antioxidant activities of red ginseng using various enzymes were measured. Red ginseng after 0.5% cellulase treatment for 1 hr at had higher antioxidant activity than the other conditions. We found that red ginseng/soybean oil extracted for 15 days at after 0.5% cellulase treatment increased DPPH radical scavenger activity and decreased the TBA and POV values. However, red ginseng/olive oil had little functional activities compare to the red ginseng/soybean 0il. We also analyzed vitamin A and E by HPLC and found that vitamin E was increased by 0.5% cellulase treatment in the oil. This is the first report that red ginseng oil extracted by enzyme treatment has various beneficial effects.

Our studies of the reaction mechanism of cystathionine beta-synthase from Saccharomyces cerevisiae (yeast) are facilitated by the spectroscopic properties of the pyridoxal phosphate coenzyme that forms a series of intermediates in the reaction of L-serine and L-homocysteine to form L-cystathionine. To characterize these reaction intermediates, we have carried out rapid-scanning stopped-flow and single-wavelength stopped-flow kinetic measurements under pre-steady-state conditions, as well as circular dichroism and fluorescence spectroscopy under steady-state conditions. We find that the gem-diamine and external aldimine of aminoacrylate are the primary intermediates in the forward half-reaction with L-serine and that the external aldimine of aminoacrylate or its complex with L-homocysteine is the primary intermediate in the reverse half-reaction with L-cystathionine. The second forward half-reaction of aminoacrylate with L-homocysteine is rapid. No primary kinetic isotope effect was obtained in the forward half-reaction with L-serine. The results provide evidence (1) that the formation of the external aldimine of L-serine is faster than the formation of the aminoacrylate intermediate, (2) that aminoacrylate is formed by the concerted removal of the alpha-proton and the hydroxyl group of L-serine, and (3) that the rate of the overall reaction is rate-limited by the conversion of aminoacrylate to L-cystathionine. We compare our results with cystathionine beta-synthase with those of related investigations of tryptophan synthase and O-acetylserine sulfhydrylase.

Aminodeoxychorismate lyase is a pyridoxal 5'-phosphate-dependent enzyme that converts 4-aminodeoxychorismate to pyruvate and p-aminobenzoate, a precursor of folic acid in bacteria. The enzyme exhibits significant sequence similarity to two aminotransferases, D-amino acid aminotransferase and branched-chain L-amino acid aminotransferase. In the present study, we have found that aminodeoxychorismate lyase catalyzes the transamination between D-alanine and pyridoxal phosphate to produce pyruvate and pyridoxamine phosphate. L-Alanine and other D- and L-amino acids tested were inert as substrates of transamination. The pro-R hydrogen of C4' of pyridoxamine phosphate was stereospecifically abstracted during the reverse half transamination from pyridoxamine phosphate to pyruvate. Aminodeoxychorismate lyase is identical to D-amino acid aminotransferase and branched-chain L-amino acid aminotransferase in the stereospecificity of the hydrogen abstraction, and differs from all other pyridoxal enzymes that catalyze pro-S hydrogen transfer. Aminodeoxychorismate lyase is the first example of a lyase that catalyzes pro-R-specific hydrogen abstraction. The result is consistent with recent X-ray crystallographic findings showing that the topological relationships between the cofactor and the catalytic residue for hydrogen abstraction are conserved among aminodeoxychorismate lyase, D-amino acid aminotransferase and branched-chain L-amino acid aminotransferase [Nakai, T., Mizutani, H., Miyahara, I., Hirotsu, K., Takeda, S., Jhee, K.-H., Yoshimura, T., and Esaki, N. (2000) J. Biochem. 128, 29-38].

Cystathionine beta-synthase from yeast (Saccharomyces cerevisiae) provides a model system for understanding some of the effects of disease-causing mutations in the human enzyme. The mutations, which lead to accumulation of L-homocysteine, are linked to homocystinuria and cardiovascular diseases. Here we characterize the domain architecture of the heme-independent yeast cystathionine beta-synthase. Our finding that the homogeneous recombinant truncated enzyme (residues 1-353) is catalytically active and binds pyridoxal phosphate stoichiometrically establishes that the N-terminal residues 1-353 compose a catalytic domain. Removal of the C-terminal residues 354-507 increases the specific activity and alters the steady-state kinetic parameters including the K(d) for pyridoxal phosphate, suggesting that the C-terminal residues 354-507 compose a regulatory domain. The yeast enzyme, unlike the human enzyme, is not activated by S-adenosyl-L-methionine. The truncated yeast enzyme is a dimer, whereas the full-length enzyme is a mixture of tetramer and octamer, suggesting that the C-terminal domain plays a role in the interaction of the subunits to form higher oligomeric structures. The N-terminal catalytic domain is more stable and less prone to aggregate than full-length enzyme and is thus potentially more suitable for structure determination by X-ray crystallography. Comparisons of the yeast and human enzymes reveal significant differences in catalytic and regulatory properties.

Mutations in the pyridoxal phosphate binding site of the tryptophan synthase beta subunit (S377D and S377E) alter cofactor chemistry [Jhee, K.-H., et al. (1998) J. Biol. Chem. 273, 11417-11422]. We now report that the S377D, S377E, and S377A beta2 subunits form alpha2 beta2 complexes with the alpha subunit and activate the alpha subunit-catalyzed cleavage of indole 3-glycerol phosphate. The apparent Kd for dissociation of the alpha and beta subunits is unaffected by the S377A mutation but is increased up to 500-fold by the S377D and S377E mutations. Although the three mutant alpha2 beta2 complexes exhibit very low activities in beta elimination and beta replacement reactions catalyzed at the beta site in the presence of Na+, the activities and spectroscopic properties of the S377A alpha2 beta2 complex are partially repaired by addition of Cs+. The S377D and S377E alpha2 beta2 complexes, unlike the wild-type and S377A alpha2 beta2 complexes and the mutant beta2 subunits, undergo irreversible substrate-induced inactivation by L-serine or by beta-chloro-L-alanine. The rates of inactivation (kinact) are similar to the rates of catalysis (kcat). The partition ratios are very low (kcat/kinact = 0.25-3) and are affected by alpha subunit ligands and monovalent cations. The inactivation product released by alkali was shown by HPLC and by fluorescence, absorption, and mass spectroscopy to be identical to a compound previously synthesized from pyridoxal phosphate and pyruvate. We suggest that alterations in the cofactor chemistry that result from the engineered Asp377 in the active site of the beta subunit may promote the mechanism-based inactivation.

Dropwort (Oenanthe javanica) has been used for many years for the treatment of inflammatory conditions, including hepatitis. We investigated the protective effects of fermented field water-dropwort extract (FDE) on tert-butyl hydroperoxide (t-BHP)-induced hepatotoxicity in HepG2 cells and carbon tetrachloride (CCl4)-induced liver damage in rats. Pretreatment with FDE prior to the t-BHP treatment of HepG2 cells inhibited cell death and lactate dehydrogenase (LDH) leakage in a dose-dependent manner. In addition FDE significantly prevented the increase of hepatic enzyme markers (ALT, AST) in vivo. Moreover, FDE administration for 7 days significantly affected CYP2E1, CYP4A2, and PPARγ gene expressions. CYP2E1 and CYP4A2 gene expression in the liver, increased 2 and 22-fold by CCl4 administration, respectively, was attenuated to normal levels by pretreatment with FDE. PPARγ gene expression, completely blocked by CCl4 treatment, was increased by FDE pretreatment compared to normal control group. Histopathological examination of the livers also revealed that FDE reduced the incidence of liver lesions. Caffeic acid and chlorogenic acid were identified as major constituents of FDE. These results demonstrate the protective effects of FDE against hepatocytotoxicity induced by CCl4 and t-BHP in rats and HepG2 cells, thus indicating the potential of FDE as a therapeutic for acute liver diseases.

The unique properties of graphene have earned much interest in the fields of materials science and condensed-matter physics in recent years. However, the biological applications of graphene remain largely unexplored. In this study, we investigated the conditions and viability of a cell culture exposed to graphene onto glass and SiO2/Si, using a human nerve cell line, SH-SY5Y. Cell viability was 84% when cultured on glass and SiO2/Si coated with graphene as compared with culturing on polystyrene surface. Fluorescence data showed that the presence of graphene did not influence cell morphology. These findings suggest that graphene may be used for biological applications.

The essential oil of silver fir (Abies alba) is known to help respiratory system and have easing and soothing effect for muscle. In the present study, we investigated the chemical composition, cytotoxicity and its biological activities of silver fir (Abies alba) essential oil. The composition of the oil was analyzed by GC-MS and bornyl acetate (30.31%), camphene (19.81%), 3-carene (13.85%), tricyclene (12.90%), dl-limonene (7.50%), alpha-pinene (2.87%), caryophyllene (2.18%), beta-phellandrene (2.13%), borneol (1.74%), bicyclo[2.2.1]hept-2-ene,2,3-dimethyl (1.64%) and alpha-terpinene (1.24%) were the major components in the oil. The results tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay indicated that the oil showed no cytotoxic effect, at concentrations of 1 and 5%, for as long as 24 and 3 h, respectively. The antiradical capacity was evaluated by measuring the scavenging activity of the essential oil on the 2,20-diphenylpicrylhydrazyl (DPPH) and 2,2'-azino-bis 3-ethyl benzothiazoline-6-sulfonic acid (ABTS) radicals. The oil was able to reduce the both radicals dose-dependently, and the concentration required for 50% reduction (RC(50)) against DPPH radicals (2.7 +/- 0.63%) was lower than ABTS radicals (8.5 +/- 0.27%). The antibacterial activity of the oil was also evaluated using disc diffusion method against Staphylococcus aureus, Streptococcus mutans, Listeria monocytogenes, Acinetobacter baumannii, Escherichia coli, and Vibrio parahaemolyticcus. The oil exhibited no antibacterial activity against all the bacterial strains tested except S. aureus of mild activity.

Elevated total plasma homocysteine is an independent risk factor in the development of vascular disease in humans. Cystathionine β-synthase (CBS) is an enzyme that condenses homocysteine with serine to form cystathionine. In this article, we describe the effects of modulating CBS activity using a transgenic mouse that contains the human CBS cDNA under control of the zinc-inducible metallothionein promoter (Tg-CBS). In the presence of zinc, Tg-CBS mice have a 2- to 4-fold increase in liver and kidney CBS activity compared with nontransgenic littermates. Transgenic mice on standard mouse chow had a 45% decrease in their serum homocysteine (12.1 to 7.2 μmol/L; P P =NS). Tg-CBS mice maintained on a high-methionine, low-folate diet also had significantly lower serum homocysteine compared with control animals (179 μmol/L versus 242 μmol/L; P P Cbs deletion animals. Our results show that elevating CBS activity is an effective method to lower plasma homocysteine levels. In addition, the creation of an inducible mouse system to modulate plasma homocysteine will also be useful in the study of homocysteine-related vascular disease.

Our studies of the reaction mechanism of cystathionine beta-synthase from yeast (Saccharomyces cerevisiae) are facilitated by the spectroscopic properties of the pyridoxal phosphate coenzyme. The enzyme catalyzes the reaction of L-serine with L-homocysteine to form L-cystathionine through a series of pyridoxal phosphate intermediates. In this work, we explore the substrate specificity of the enzyme by use of substrate analogues combined with kinetic measurements under pre-steady-state conditions and with circular dichroism and fluorescence spectroscopy under steady-state conditions. Our results show that L-allothreonine, but not L-threonine, serves as an effective substrate. L-Allothreonine reacts with the pyridoxal phosphate cofactor to form a stable 3-methyl aminoacrylate intermediate that absorbs maximally at 446 nm. The rapid-scanning stopped-flow results show that the binding of L-allothreonine as the external aldimine is faster than formation of the 3-methyl aminoacrylate intermediate. The 3-methyl aminoacrylate intermediate reacts with L-homocysteine to form a new amino acid, 3-methyl-L-cystathionine, which was characterized by nuclear magnetic resonance spectroscopy. This new amino acid may be a useful analogue of L-cystathionine.

4-Amino-4-deoxychorismate lyase (ADCL) is a member of the fold-type IV of PLP dependent enzymes that converts 4-amino-4-deoxychorismate (ADC) to p-aminobenzoate and pyruvate. The crystal structure of ADCL from Escherichia coli has been solved using MIR phases in combination with density modification. The structure has been refined to an R-factor of 20.6% at 2.2 A resolution. The enzyme is a homo dimer with a crystallographic twofold axis, and the polypeptide chain is folded into small and large domains with an interdomain loop. The coenzyme, pyridoxal 5'-phosphate, resides at the domain interface, its re-face facing toward the protein. Although the main chain folding of the active site is homologous to those of D-amino acid and L-branched-chain amino acid aminotransferases, no residues in the active site are conserved among them except for Arg59, Lys159, and Glu193, which directly interact with the coenzyme and play critical roles in the catalytic functions. ADC was modeled into the active site of the unliganded enzyme on the basis of the X-ray structures of the unliganded and liganded forms in the D-amino acid and L-branched-chain amino acid aminotransferases. According to this model, the carboxylates of ADC are recognized by Asn256, Arg107, and Lys97, and the cyclohexadiene moiety makes van der Waals contact with the side chain of Leu258. ADC forms a Schiff base with PLP to release the catalytic residue Lys159, which forms a hydrogen bond with Thr38. The neutral amino group of Lys159 eliminates the a-proton of ADC to give a quinonoid intermediate to release a pyruvate in accord with the proton transfer from Thr38 to the olefin moiety of ADC.

Our studies of cystathionine β-synthase fromSaccharomyces cerevisiae (yeast) are aimed at (1) clarifying the cofactor dependence and catalytic mechanism and (2) obtaining a system for future investigations of the effects of mutations that cause human disease (homocystinuria or coronary heart disease). We report methods that yielded high expression of the yeast gene in Escherichia coli and of purified yeast cystathionine β-synthase. The absorption and circular dichroism spectra of the homogeneous enzyme were characteristic of a pyridoxal phosphate enzyme and showed the absence of heme, which is found in human and rat cystathionine β-synthase. The absence of heme in the yeast enzyme facilitates spectroscopic studies to probe the catalytic mechanism. The reaction of the enzyme with l-serine in the absence of l-homocysteine produced the aldimine of aminoacrylate, which absorbed at 460 nm and had a strong negative circular dichroism band at 460 nm. The formation of this intermediate from the product, l-cystathionine, demonstrates the partial reversibility of the reaction. Our results establish the overall catalytic mechanism of yeast cystathionine β-synthase and provide a useful system for future studies of structure and function. The absence of heme in the functional yeast enzyme suggests that heme does not play an essential catalytic role in the rat and human enzymes. The results are consistent with the absence of heme in the closely related enzymesO-acetylserine sulfhydrylase, threonine deaminase, and tryptophan synthase.

Crystal structures of the tryptophan synthase α2β2complex show binding sites in the β subunit for pyridoxal phosphate, for substrates, and for a monovalent cation. Cations, including guanidine at low concentration, activate tryptophan synthase. Molecular modeling indicates that guanidine could bind to the cation site in the β subunit. Our results demonstrate that the conformational states of the tryptophan synthase α2β2 complex are regulated by monovalentcations, chaotropic agents, pH, α subunit ligands, and temperature.

To better understand how an enzyme controls cofactor chemistry, we have changed a tryptophan synthase residue that interacts with the pyridine nitrogen of the pyridoxal phosphate cofactor from a neutral Ser (beta-Ser377) to a negatively charged Asp or Glu. The spectroscopic properties of the mutant enzymes are altered and become similar to those of tryptophanase and aspartate aminotransferase, enzymes in which an Asp residue interacts with the pyridine nitrogen of pyridoxal phosphate. The absorption spectrum of each mutant enzyme undergoes a pH-dependent change (pKa approximately 7.7) from a form with a protonated internal aldimine nitrogen (lambdamax = 416 nm) to a deprotonated form (lambdamax = 336 nm), whereas the absorption spectra of the wild type tryptophan synthase beta2 subunit and alpha2 beta2 complex are pH-independent. The reaction of the S377D alpha2 beta2 complex with L-serine, L-tryptophan, and other substrates results in the accumulation of pronounced absorption bands (lambdamax = 498-510 nm) ascribed to quinonoid intermediates. We propose that the engineered Asp or Glu residue changes the cofactor chemistry by stabilizing the protonated pyridine nitrogen of pyridoxal phosphate, reducing the pKa of the internal aldimine nitrogen and promoting formation of quinonoid intermediates.

We here describe the stereochemical aspects of the reactions of pyridoxal 5'-phosphate (PLP)-dependent enzymes, and the relationship between the stereochemistry of the enzyme reaction and molecular evolution of the enzyme. The reactions of PLP-dependent enzymes proceed through the formation of an anionic Schiff base intermediate between the substrate and the coenzyme. Three stereochemical possibilities exist for the formation and cleavage of bonds in the intermediate: the reaction occurs stereospecifically on either the si- or the re-face of the planar intermediate, or alternatively, non-stereospecifically on both faces. The stereospecificities for hydrogen transfer between C-4' of the cofactor and substrate in the transamination catalyzed by various PLP-dependent enzymes have been studied. The stereospecificities reflect the active-site structures of the enzymes, especially the topographical situation of a coenzyme-substrate Schiff base and a catalytic base for the hydrogen transfer. The aminotransferases and other PLP-enzymes catalyzing the transamination as a side-reaction so far studied catalyze only the si-face specific hydrogen transfer. This suggests that these PLP enzymes have similar active-site structures and are evolved divergently from a common ancestral protein. We recently established a new method for the identification of stereospecificity for the hydrogen transfer, and found that D-amino acid aminotransferase and branched chain L-amino acid aminotransferase, which have significant sequence similarity to each other, catalyze the re-face hydrogen transfer on the intermediate. The X-ray crystallographic studies of D-amino acid aminotransferase showed that the relative arrangement of the catalytic base of the enzyme active center to the C4' of the bound cofactor is opposite to that of other aminotransferases catalyzing the si-face hydrogen transfer. The folding of D-amino acid aminotransferase is also different from those of the other aminotransferase so far studied. Therefore, the classifications of the aminotransferases based on their primary structures, three dimensional structures, and stereochemistry of their hydrogen transfer coincide with one another. We also found that PLP-dependent amino acid racemases, the primary structures of which are similar to none of the other PLP-enzymes, catalyze the non-stereospecific hydrogen transfer on both faces of the planar intermediate. Stereospecificities for the hydrogen transfer suggest convergent evolution of the PLP-dependent enzymes. The stereochemical aspects of the enzyme reactions give a clue to the molecular evolution of the enzymes as well as the primary structures and three-dimensional structures of the enzymes.

Thermostable L-ornithine: alpha-ketoglutarate delta-aminotransferase (L-ornithine: 2-oxo-acid 5-aminotransferase) [EC 2.6.1.13] was purified to homogeneity from Bacillus sp. YM-2. The enzyme has a molecular weight of about 82,000 and consists of two subunits with identical molecular weights. The enzyme catalyzes transamination from L-ornithine to alpha-ketoglutarate, producing L-glutamate and L-glutamate gamma-semialdehyde, which is spontaneously dehydrated to L-delta 1-pyrroline-5-carboxylate, and the enzyme is most active at 70 degrees C. In addition to L-ornithine, the enzyme unexpectedly acts on D-ornithine, the reaction rate being 6% of that for L-ornithine. The enzyme contains 1 mol each of pyridoxal 5'-phosphate and another vitamin B6 compound per mol. The enzyme released the bound pyridoxal 5'-phosphate, as judged from the absorption at 425 nm on incubation with 2.0 M guanidine hydrochloride. The resultant inactive enzyme still gave a 340-nm peak and contained 1 mol of the vitamin B6 compound. The partial amino acid sequence shows high homology with those of mammalian and yeast ornithine delta-aminotransferases.