Escherichia coli tryptophan synthase (TS) contains ${\alpha}_2{\beta}_2$, which catalyzes the final two steps in Trp biosynthesis. A molecular tunnel exists between the two active sites of ${\alpha}$ and ${\beta}$ subunits in TS. Via intersubunit communication, TS increases catalytic efficiency, including substrate channeling. The ${\beta}$ subunit of TS is composed of two domains, one of which, the COMM (communication) domain, plays an important role in intersubunit communication. The ${\alpha}$ subunit has a TIM barrel structure. This protein has functional regions at the C terminal of ${\beta}$ pleated sheets and in its loop regions. Three regions of the ${\alpha}$ subunit (${\alpha}L6$ [${\alpha}-loop$ L6], ${\alpha}L2$, and ${\alpha}L3$) are implicated in intersubunit communication. In the present study, conformational changes in ${\alpha}L6$ were monitored by measuring the sensitivity of mutant proteins in these regions to trypsin. The addition of a ${\alpha}$ subunit-specific ligand, D,L-${\alpha}$-glycerophosphate (GP), partially restored the sensitivity of mutant proteins to trypsin. In contrast, the addition of the ${\beta}$ subunit-specific ligand L-serine (Ser) resulted in varied sensitivity to trypsin, with an increase in PT53 (substitution of Pro with Thr at residue 53) and DG56, decrease in NS104 and wild type, and no change in GD51 and PH53. This finding may be related to several reaction intermediates formed under this condition. The addition of both GP and Ser led to a highly stable state of the complex. The present results are consistent with the current model. The method used herein may be useful for screening residues involved in intersubunit communication.
Poly(2,6-dimethyl-1,4-phenylene ether) (PPE) was synthesized using $Cu(NO_2)_2{\cdot}3H_2O$ or CuCl catalyst with various amounts of ligand and base in several different solvent systems. CuCl/1-methylimidazole/ammonium hydroxide was found to be an effective catalyst system which showed the highest polymer yield and molecular weight. The effects of catalyst/monomer ratio, different amine ligands, and the content of mono-functional reagent 2,4,6-trimethylphenol (TMP) additive on the polymer yield and molecular weight were investigated. Among the co-solvent systems used in this polymerization, chloroform/methanol 9/1(v/v) gave the highest polymer yield and molecular weight ($\overline{M_n}$ 55 K, $\overline{M_w}$ 92 K, PDI 1.7). The catalytic activity between CuCl and CuI was compared by oxygen-uptake experiments and the formation of sideproduct, 5,5'-tetramethyl-4,4'-diphenoquinone (DPQ), was analyzed by ultraviolet spectroscopy.
Objectives: This study aimed to investigate the effects of rhubarb extract on osteoclast differentiation in bone marrow-derived macrophages (BMMs). Osteoclasts are vital for bone resorption and remodeling. Osteoclast dysregulation can contribute to various bone-related disorders that directly affect oral health. Rhubarb, a medicinal plant with anti-inflammatory properties, has been shown to modulate bone metabolism. Methods: BMMs were isolated from the femurs and tibias of 5-week-old C57BL/6 mice and cultured in the presence of mouse macrophage colony-stimulating factor (M-CSF) for 3 days. Subsequently, BMMs were treated with M-CSF and receptor activator of nuclear factor-κB ligand (RANKL) to induce osteoclast differentiation. Results: Rhubarb extract effectively suppressed osteoclast differentiation in BMMs. Furthermore, rhubarb extract inhibited the mRNA expression of tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CTSK), which are essential for osteoclastogenesis. Moreover, it inhibited the RANKL-induced expression of nuclear factor of activated T cell c1 (NFATc1), a crucial transcription factor in osteoclast differentiation. Conclusions: These results suggest that rhubarb extract promotes oral health by inhibiting osteoclastogenesis in BMMs. Thus, rhubarb extract shows promise as a therapeutic agent for bone-related disorders that directly affect oral health, particularly those associated with abnormal osteoclast activity. Further research and exploration of the underlying mechanisms are warranted to fully understand their potential clinical applications.
Do Kyung Han;Jee Won Shon;Eui Suk Sung;Youn Sook Kim;Won G. An
Journal of Life Science
/
v.33
no.11
/
pp.876-886
/
2023
In some cases of head and neck cancers (HNC), surgical interventions may result in the loss of organs and/or changes to their functions, thereby significantly affecting the patient's quality of life. As a result, the surgical treatment of HNC patients is often limited to specific cases, and alternative treatment modalities, such as chemotherapy, are considered. However, serious adverse effects caused by chemotherapy, such as severe nausea and vomiting, necessitate the need for the development of adjunctive methods to minimize patient suffering. Chuanxiong, Ligusticum chuanxiong (L. chuanxiong), is a natural herb used in Eastern medicine to treat cerebrovascular disorders and headaches. This study aimed to predict the effect and potential of L. chuanxiong as an auxiliary anticancer drug through network-based pharmacology and molecular docking analysis. The study results showed that 40 out of 41 genes of L. chuanxiong shared common targets of HNC and their proteins could be used to target HNC cells to prevent cancer progression. The results of the functional enrichment analysis confirmed that L. chuanxiong is associated with the neuroactive-ligand metabolism and neurotransmitter pathways, indicating its potential medicinal value as an adjuvant in HNC treatment. Lastly, our findings demonstrated that the active ingredient of L. chuanxiong, (Z)-Ligustilide, has the ATP binding site of heat shock protein 90, a protein known to promote the activation of cancer cells. These results suggest that L. chuanxiong is a promising candidate for developing auxiliary anticancer drugs, and further research could potentially lead to the discovery of newer and safer anti-cancer agents.
Mifepristone (MIF) and Tamoxifen (TAM) have been used in the treatment of prostate cancer and breast cancer for more than a decade. MIF can induce apoptosis in both AR-positive and negative prostate cancer cells. Because of its pleiotropic ligand-receptor properties, TAM exerts cytotoxic activity in estrogen (ER)-positive and various ER.negative cancer cells. However, the molecular mechanisms of these two substances are not yet clear. In the present work, we report that the cytotoxic effects of MIF and TAM are due to the modulation of intracellular $Ca^{2+}$ level in DU-145, androgen-insensitive cells. When the cells were treated with micromolar concentrations of either MIF or TAM, the growth and viability were significantly decreased in a dose- and time-dependent manner. The apoptosis induced by MIF or TAM was further proved and analyzed by confocal laser scanning microscopy (CLSM) and fluorescence-activated cell sorting (FACS). In the cells cultivated in a normal 1.5 mM $Ca^{2+}$ medium, both MIF and TAM also induced an increase of the intracellular $Ca^{2+}$ level in a dose-dependent fashion. Since a change in calcium level could not be found in cells of the $Ca^{2+}$-free medium, the increase of intracellular $Ca^{2+}$ level might be due to an increase in extracellular calcium uptake. Our results show that the apoptotic effect was more prominent in TAM treatment compared to MIF treatment in DU-145 cells. The above findings might be due to the difference in the uppermost pathways of apoptosis induced by either MIF or TAM. When we checked the level of procaspase-8 activation, TAM showed minor level of activation, as opposed to MIF, which exerted strong activation. In both treatments, the levels of anti-apoptotic protein Bcl-2 decreased, and pro-apoptotic protein Bax level increased more than 2-fold. The activation of caspase-3, a key protease enzyme in the downstream pathway of apoptosis, was much higher in the cells treated with TAM, compared to the MIF treatment. The overall apoptotic activity shown in the present work was closely related to intracellular $Ca^{2+}$ concentration levels. Therefore, the cytotoxic activity induced by MIF and TAM might have been due to intracellular calcium modulation.
Kim, Cheol-Jin;Park, Hyung-Yeon;Kim, Jae-Eun;Park, Hee-Jin;Lee, Bon-Su;Choi, Yu-Sang;Lee, Joon-Hee;Yoon, Je-Yong
Microbiology and Biotechnology Letters
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v.37
no.3
/
pp.248-257
/
2009
The inhibitors against Vibrio harveyi quorum sensing (QS) signaling were developed by modifying the molecular structure of the major signal, N-3-hydroxybutanoyl-L-homoserine lactone (3-OH-$C_4$-HSL). A series of structural derivatives, N-(3-hydroxysulfonyl)-L-homoserine lactones (HSHLs) were synthesized by the solid-phase organic synthesis method. The in vivo QS inhibition by these compounds was measured by a bioassay system using the V. harveyi bioluminescence, and all showed significant inhibitory effects. To analyze the interaction between these compounds and LuxN, a 3-OH-$C_4$-HSL receptor protein of V. harveyi, we tentatively determined the putative signal binding domain of LuxN based on the sequence homology with other acyl-HSL binding proteins, and predicted the partial 3-D structure of the putative signal binding domain of LuxN by using ORCHESTRA program, and further estimated the binding poses and energies (docking scores) of 3-OH-$C_4$-HSL and HSHLs within the domain. In comparison of the result from this modeling study with that of in vivo bioassay, we suggest that the in silica interpretation of the interaction between ligands and their receptor proteins can be a valuable way to develop better competitive inhibitors, especially in the case that the structural information of the protein is limited.
Choi, Ki Young;Kim, Dong Won;Kim, Chang Suk;Park, Byung Bin;Choi, Suk Nam;Hong, Choon Pyo;Ryu, Hae Il
Journal of the Korean Chemical Society
/
v.44
no.5
/
pp.403-409
/
2000
The formation and dissociation rates of $Zn^{2+}$ Complexes with l,4,7,10-tetraaza-13,16-diox-acyclooctadecane-N,N',N",N'"-tetraacetic acid (1), 1,4,7,10-tetraaza-13,16- dioxacyclooctadecane-N,N',N",N'"-tetramethylacetic acid (2), and 1,4,7,10-tetraaza-13,16- dioxacyclooctadecane-N,N',N",N'"-tetrapropionic acid(3) have been measured by stopped-flow and conventional spectrophotometry. Observations were made at 25.0$\pm$0.1 $^{\circ}C$ and at an ionic strength of 0.10 M NaClO$_4$. The formation reactions of $Zn^{2+}$ ion with 1 and 2 took place by the rapid formation of an intermediate complex (ZnH$_3L^+$) in which the $Zn^{2+}$ ion is incompletely coor-dinated. This might then lead to be a final product in the rate-determining step.ln the pH range 4.76-5.76, the diprotonated (H2L2-) form is the kinetically active species despite of its low concentration. The stability con-stants (log$K_{(ZnH$_3$3$L^+$)}$) and specific water-assisted rate constants (koH) of intermediate complexes have been deter-mined from the kinetic data. The dissociation reactions of $Zn^{2+}$ complexes of 1,2, and 3 were investigated with $Cu^{2+}$ ions as a scavenger in acetate buffer. All complexes exhibit acid-independent and acid-catalyzed con-tributions. The effect of buffer and $Cu^{2+}$ concentration on the dissociation rate has also been investigated. The ligand effect on t dissociation rate of $Zn^{2+}$ complexes is discussed in terms of the side-pendant armsand the chelate ring sizes of the ligands.
Purpose: Chitosan has been studied as a non-viral gene delivery vector, drug delivery carrier, metal chelator, food additive, and radiopharmaceutical, among other things. Recently, galactose-graft chitosan was studied as a non-viral gene and drug delivery vector to target hepatocytes. The aim of this study was to investigate the usefulness of nuclear imaging for in vivo evaluation of targeting the hepatocyte by galactose grafting. Methods and Materials: Galactosyl methylated chitosan (GMC) was produced by methylation to lactobionic acid coupled chitosan. Cytotoxicity of $^{99m}Tc$-GMC was determined by MTT assay. Rabbits were injected via their auricular vein with $^{99m}Tc$-GMC and $^{99m}Tc$-methylated chitosan (MC), the latter of which does not contain a galactose group, and images were acquired with a gamma camera equipped with a parallel hole collimator. The composition of the galactose group in galactosylated chitosan (GC), as well as the tri-, di-, or mono-methylation of GMC, was confirmed by NMR spectroscopy. Results: The results of MTT assay indicated that $^{99m}Tc$-GMC was non-toxic. $^{99m}Tc$-GMC specifically accumulated in the liver within 10 minutes of injection and maintained high hepatic uptake. In contrast, $^{99m}Tc$-MC showed faint liver uptake. $^{99m}Tc$-GMC scintigraphy of rabbits showed that the galactose ligand principally targeted the liver while the chitosan functionalities led to excretion through the urinary system. Conclusion: Bioconjugation with a specific ligand endows some degree of targetability to an administered molecule or drug, as in the case of galactose for hepatocyte in vivo, and evaluating said targetabililty is a clear example of the great benefit proffered by nuclear imaging.
We have investigated the infrared spectra for CO adsorbed on silica supported nickel(Ni-Si$O_2$), silica supported copper(Cu-Si$O_2$), silica supported nickel-copper alloys(Ni/Cu-Si$O_2$) of several compositions with varying CO pressures(0.2 $torr{\sim}$50 torr) at room temperature and on pumping to vacumn at room temperature within the frequency range of 1500 $cm^{-1}{\sim}2500\;cm^{-1}$. Four bands(2059.6 $cm^{-1},\;{\sim}$2036.5 $cm^{-1},\;{\sim}$ 1868.7 $cm^{-1},\;{\sim}$ 1697.1 $cm^{-1}$) were observed for Ni-Si$O_2$, two bands($\sim$2115.5 $cm^{-1},\;{\sim}$1743.0 $cm^{-1}$) were observed for Cu-Si$O_2$ and five bands(${\sim}2123.2\;cm^{-1}$, 2059.6 $cm^{-1},\;{\sim}$2036.4 $cm^{-1},\;{\sim}$1899.5 $cm^{-1},\;{\sim}$1697.1 $cm^{-1}$) were observed for Ni/Cu-Si$O_2$. These absorption bands correspond with those of the previous reports approximately. The bands below 1800 $cm^{-1}$ were only observed with Ni metal or Ni/Cu alloy crystal plane containing step at room temperature and the ${\sim}1697.1\;cm^{-1}$ bands observed with Ni-Si$O_2$ and Ni/Cu-Si$O_2$ may be ascribed to CO molecule adsorbed on the adsorption sites near step. The bands below 2000 $cm^{-1}$ were rarely observed with Cu metal crystal plane at room temperature and the 1743.0 $cm^{-1}$ bands may be ascribed to CO molecule adsorbed on the adsorption sites near step. The band shifts of adsorbed CO with varing Cu contents from 0 to 0.5 mole fraction at the same CO pressure or at the same pumping time to vacumn were below 21 $cm^{-1}$. and comparatively small than those with other ⅠB metal addition. It may means ligand effect of Cu d electron is small.
Purpose : To evaluate the NMR relaxation properties of newly developed high performance paramagnetic complexes. Materials and methods : 4-aminomethylcyclohexane carboxylic acid (0.63g, 4 mmol) was mixed with the suspension solution of DMF (15mL) and DTPA-bis-anhydride (0.71g, 2 mmol) to synthesize the ligand. The ligand was then mixed with Gd2O3 (0.18g, 0.5 mmol) to synthesize Gd-chelate. For the measurement of magnetic relaxivity of paramagnetic compounds, the compounds were diluted to 1mM and then the relaxation times were measured at 1.5T(64 MHz). Inversion-recovery pulse sequence was employed for T1 relaxation measurement and CPMG(Carr-Purcell-Meiboon-Gill) pulse sequence was employed for T2 relaxation measurement. Using MATLAB(Version 7.1) program, T1 magnetic relaxation map, R1 map, T2 magnetic relaxation map and R2 map were developed to represent magnetic relaxation time and magnetic relaxivity as image. Results : Compared to $R1=4.9mM^{-1}sec^{-1}$ and $R2=4.8mM^{-1}sec^{-1}$ of Omniscan (Gadodiamide), which is commercially available paramagnetic MR agent, R1 of SUK090(Gd-C32H74N5O24) was $12.46mM^{-1}sec^{-1}$ and R1 of SUK091(Gd-C34H78N5O24) was $12.77mM^{-1}sec^{-1}$. However, R1 of SUK092(Gd-C30H56N5O17) was decreased to $2.09mM^{-1}sec^{-1}$. In case of R2, SUK090(Gd-C32H74N5O24) was $8.76mM^{-1}sec^{-1}$ and SUK091(Gd-C34H78N5O24) was $7.60mM^{-}1sec^{-1}$ whereas SUK092(Gd-C30H56N5O17) was decreased to $1.82mM^{-1}sec^{-1}$. Conclusion : Among three new paramagnetic complexes, SUK090(Gd-C32H74N5O24) and SUK091(Gd-C34H78N5O24) showed higher T1, T2 magnetic relaxation rates than that of commercially available paramagnetic MR agent and thus expected to have more contrast enhancement effect.
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