Gamma glutamylcyclotransferase (GGCT) has been proved to be involved in various cancers, but the biological function of GGCT in gastric cancer is still largely unknown.
Methods
The expression level of GGCT was evaluated by informatics analyses based on the Oncomine database. GGCT gene was then effectively knocked down via lentivirus mediated short hairpin RNA (shRNA) system. Then a series of functional assays, including MTT, colony formation and flow cytometry analysis were conducted on gastric cancer cells following GGCT knockdown.
Results
We found GGCT is commonly up-regulated in gastric cancer tissues. Furthermore, MTT analysis showed that GGCT depletion significantly inhibited cell proliferation in MGC80-3 and AGS cells. Colony formation assay revealed that depletion of GGCT reduced the colony formation ability in gastric cancer cells. What’s more, cell cycle analysis showed that depletion of GGCT induced gastric cancer cell cycle arrested G2/M phase. More importantly, cell apoptosis analysis further revealed that GGCT inhibition induced early and late cell apoptosis in gastric cancer.
Conclusion
This study suggests GGCT is essential for gastric cancer proliferation and its downregulation may provide a potential anticancer therapy for gastric cancer.
Luciferases, enzymes that catalyze bioluminescent reactions in different organisms, have been extensively used for bioanalytical purposes. The most well studied bioluminescent system is that of firefly and other beetles, which depends on a luciferase, a benzothiazolic luciferin and ATP, and it is being widely used as a bioanalytical reagent to quantify ATP. Protein kinases are proteins that modify other proteins by transferring phosphate groups from a nucleoside triphosphate, usually ATP.
Methods
Here, we used a red-light emitting luciferase from Phrixotrix hirtus railroad worm to determine the activity of kinases in a coupled assay, based on luminescence that is generated when luciferase is in the presence of its substrate, the luciferin, and ATP.
Results
In this work we used, after several optimization reactions, creatine kinase isoforms as well as ?NEK7 protein kinase in the absence or presence of ATP analogous inhibitors to validate this new luminescence method.
Conclusion
With this new approach we validated a luminescence method to quantify kinase activity, with different substrates and inhibition screening tests, using a novel red-light emitting luciferase as a reporter enzyme.
Triacylglycerols (TAGs) are the major form of energy storage in eukaryotes. Diacylglycerol acyltransferases (DGATs) catalyze the final and rate-limiting step of TAG biosynthesis. Mammalian DGATs are classified into DGAT1 and DGAT2 subfamilies. It was unclear which DGAT was the major isoform expressed in animal cells. The objective was to identify the major DGAT mRNA expressed in cultured mouse adipocytes and macrophages and compared it to that expressed in tung tree seeds.
Methods
qPCR evaluated DGAT mRNA levels in mouse 3?T3-L1 adipocytes and RAW264.7 macrophages and tung tree seeds.
Results
TaqMan qPCR showed that DGAT2 mRNA levels were 10–30 fold higher than DGAT1 in adipocytes and macrophages, and DGAT mRNA levels in adipocytes were 50–100-fold higher than those in macrophages. In contrast, the anti-inflammatory tristetraprolin/zinc finger protein 36 (TTP/ZFP36) mRNA levels were 2–4-fold higher in macrophages than those in adipocytes and similar to DGAT1 in adipocytes but 100-fold higher than DGAT1 in macrophages. SYBR Green qPCR analyses confirmed TaqMan qPCR results. DGAT2 mRNA as the major DGAT mRNA in the mouse cells was similar to that in tung tree seeds where DGAT2 mRNA levels were 10–20-fold higher than DGAT1 or DGAT3.
Conclusion
The results demonstrated that DGAT2 mRNA was the major form of DGAT mRNA expressed in mouse adipocytes and macrophages and tung tree seeds.
Nitroaromatic and chloronitroaromatic compounds have been a subject of great interest in industry and recently in medical-pharmaceutic field. 2-Chloro-4-nitro/2-chloro-5-nitrobenzoic acids and 4-nitrobenzoic acid are promising new agents for the treatment of main infectious killing diseases in the world: immunodeficiency diseases and tuberculosis.
Results
New ethanolamine nitro/chloronitrobenzoates were synthesized and characterized by X-ray crystallography, UV–vis, FT-IR and elementary analysis techniques. The toxicity of the compounds prepared and correspondent components was evaluated using Hydractinia echinata as test system. A significant lower toxicity was observed for nitro-derivative compared with chloronitro-derivatives and individual components. Crystallographic studies, together with the chemical reactivity and stability profiles resulted from density functional theory and ab initio molecular orbital calculations, explain the particular behavior of ethanolamine 4-nitrobenzoate in biological test.
Conclusions
The experimental and theoretical data reveal the potential of these compounds to contribute to the design of new active pharmaceutical ingredients with lower toxicity.
Colony stimulating factors (CSFs) are endogenous cytokines that have key roles in proliferation and differentiation of hematopoietic progenitor cells and in regulation of mature blood cells performance. The CSFs families members are widely used for therapeutic purposes in many field include microbial infections, in cancer chemotherapy, alzheimer disease, hematopoiesis process, and for some neutropenia- related pathologies. Crown ethers are chemical compounds with therapeutic application that can affect the colony formation in vitro. The primary objective of the present study is to evaluate the effect of TDN (novel crown ether) on colony formation of red bone marrow cells in incubation with lung tissues cells.
Method
In this study, bone marrow cells and lung tissue cells of Balb/C were used as a source of hematopoietic stem cells and a source to production colony-stimulating factors, respectively. These cells were incubated with TDN separately and together.
Results
Briefly, the results of this study show that the effects of TDN has excitatory in concentrations lower than 50 µg/ml on colony formation and greater than 50 µg/ml is toxic to cells and it was inhibited the colony formation. Maximum stimulatory and inhibitory effects are shown in 50 and 400 µg/ml of crown ether and no colony was observed in the latter concentration.
Conclusion
The results from this study indicate that TDN significantly able to stimulate the colon formation while increased concentrations of TDN is inhibited colony formation by induction toxic effects due to excessive production of free radicals.
Etravirine (ETV) was approved as the second generation drug for use in individuals infected with HIV-1 in 2008 by the U.S. FDA with its unique antiviral activity, high specificity, and low toxicity. However, there are some shortcomings of the existing synthetic routes, such as the long reaction time and poor yield.
Results
This article describes our efforts to develop an efficient, practical, microwave-promoted synthetic method for one key intermediate of ETV, which is capable of being operated on a scale-up synthesis level. Through this optimized synthetic procedure, the amination reaction time decreased from 12 h to 15 min and the overall yield improved from 30.4 to 38.5%.
Conclusion
Overall, we developed a practical synthesis of ETV via a microwave-promoted method, and the synthetic procedure could be amenable to scale-up, and production costs could be significantly lowered.
The smoothelin-like 1 protein (SMTNL1) can associate with tropomyosin (Tpm) and calmodulin (CaM), two proteins essential to the smooth muscle contractile process. SMTNL1 is phosphorylated at Ser301 by protein kinase A during calcium desensitization in smooth muscle, yet the effect of SMTNL1 phosphorylation on Tpm- and CaM-binding has yet to be investigated.
Results
Using pull down studies with Tpm-Sepharose and CaM-Sepharose, we examined the interplay between Tpm binding, CaM binding, phosphorylation of SMTNL1 and calcium concentration. Phosphorylation greatly enhanced the ability of SMTNL1 to associate with Tpm in vitro; surface plasmon resonance yielded a 10-fold enhancement in KD value with phosphorylation. The effect on CaM binding is more complex and varies with the availability of calcium.
Conclusions
Combining both CaM and Tpm with SMTNL1 shows that the binding to both is mutually exclusive.
Novel six organic donor-π-acceptor molecules (D-π-A) used for Bulk Heterojunction organic solar cells (BHJ), based on thienopyrazine were studied by density functional theory (DFT) and time-dependent DFT (TD-DFT) approaches, to shed light on how the π-conjugation order influence the performance of the solar cells. The electron acceptor group was 2-cyanoacrylic for all compounds, whereas the electron donor unit was varied and the influence was investigated.
Methods
The TD-DFT method, combined with a hybrid exchange-correlation functional using the Coulomb-attenuating method (CAM-B3LYP) in conjunction with a polarizable continuum model of salvation (PCM) together with a 6-31G(d,p) basis set, was used to predict the excitation energies, the absorption and the emission spectra of all molecules.
Results
The trend of the calculated HOMO–LUMO gaps nicely compares with the spectral data. In addition, the estimated values of the open-circuit photovoltage (Voc) for these compounds were presented in two cases/PC60BM and/PC71BM.
Conclusion
The study of structural, electronics and optical properties for these compounds could help to design more efficient functional photovoltaic organic materials.
One strategy to increase the stability of proteins is to reduce the area of water-accessible hydrophobic surface.
Results
In order to test it, we replaced 14 solvent-exposed hydrophobic residues of acetylcholinesterase by arginine. The stabilities of the resulting proteins were tested using denaturation by high temperature, organic solvents, urea and by proteolytic digestion.
Conclusion
Altough the mutational effects were rather small, this strategy proved to be successful since half of the mutants showed an increased stability. This stability may originate from the suppression of unfavorable interactions of nonpolar residues with water or from addition of new hydrogen bonds with the solvent. Other mechanisms may also contribute to the increased stability observed with some mutants. For example, introduction of a charge at the surface of the protein may provide a new coulombic interaction on the protein surface.
Chlorogenic acids (CGAs) are a class of phytochemicals that are formed as esters between different derivatives of cinnamic acid and quinic acid molecules. In plants, accumulation of these compounds has been linked to several physiological responses against various stress factors; however, biochemical synthesis differs from one plant to another. Although structurally simple, the analysis of CGA molecules with modern analytical platforms poses an analytical challenge. The objective of the study was to perform a comparison of the CGA profiles and related derivatives from differentiated tobacco leaf tissues and undifferentiated cell suspension cultures.
Results
Using an UHPLC-Q-TOF-MS/MS fingerprinting method based on the in-source collision induced dissociation (ISCID) approach, a total of 19 different metabolites with a cinnamic acid core moiety were identified. These metabolites were either present in both leaf tissue and cell suspension samples or in only one of the two plant systems. Profile differences point to underlying biochemical similarities or differences thereof.
Conclusion
Using this method, the regio- and geometric-isomer profiles of chlorogenic acids of the two tissue types of Nicotiana tabacum were achieved. The method was also shown to be applicable for the detection of other related molecules containing a cinnamic acid core.
Several standard powdered black pigments were characterized by means of thermogravimetry TG-DTG and allied techniques. These pigments were used to make standard plaster frescoes at this purpose prepared. The latter ones were subjected to Raman and reflectance analysis. The results obtained, together with TG data, were chemometrically processed and used to identify an analogous standard fresco fabricated by an unknown commercial black pigment, obtaining excellent results.
Results
The same colorimetric and reflectometric techniques, coupled with suitable chemometric techniques, were then successfully used to identify the type of black pigment present in an ancient roman fresco of the Imperial Age (30 B.C.).
Conclusion
TG-DTG resulted useful techniques to autenticate powdered black pigments.Colorimetry and Raman, but also the only colorimetry, were useful to identify an ancient black pigment in situ.
The defensive capacities of plant protease Inhibitors (PI) rely on inhibition of proteases in insect guts or those secreted by microorganisms; and also prevent uncontrolled proteolysis and offer protection against proteolytic enzymes of pathogens.
Methods
An array of chromatographic techniques were employed for purification, homogeneity was assessed by electrophoresis. Specificity, Ki value, nature of inhibition, complex formation was carried out by standard protocols. Action of SNTI on insect gut proteases was computationally evaluated by modeling the proteins by threading and docking studies by piper using Schrodinger tools.
Results
We have isolated and purified Soap Nut Trypsin Inhibitor (SNTI) by acetone fractionation, ammonium sulphate precipitation, ion exchange and gel permeation chromatography. The purified inhibitor was homogeneous by both gel filtration and polyacrylamide gel electrophoresis (PAGE). SNTI exhibited a molecular weight of 29 kDa on SDS-PAGE, gel filtration and was negative to Periodic Acid Schiff’s stain. SNTI inhibited trypsin and pronase of serine class. SNTI demonstrated non-competitive inhibition with a Ki value of 0.75?±?0.05×10-10 M. The monoheaded inhibitor formed a stable complex in 1:1 molar ratio. Action of SNTI was computationally evaluated on larval gut proteases from Helicoverpa armigera and Spodoptera frugiperda. SNTI and larval gut proteases were modeled and docked using Schrodinger software. Docking studies revealed strong hydrogen bond interactions between Lys10 and Pro71, Lys299 and Met80 and Van Der Waals interactions between Leu11 and Cys76amino acid residues of SNTI and protease from H. Armigera. Strong hydrogen bonds were observed between SNTI and protease of S. frugiperda at positions Thr79 and Arg80, Asp90 and Gly73, Asp2 and Gly160 respectively.
Conclusion
We conclude that SNTI potentially inhibits larval gut proteases of insects and the kinetics exhibited by the protease inhibitor further substantiates its efficacy against serine proteases.
Stilbene cleaving oxygenases (SCOs), also known as lignostilbene-α,β-dioxygenases (LSDs) mediate the oxidative cleavage of the olefinic double bonds of lignin-derived intermediate phenolic stilbenes, yielding small modified benzaldehyde compounds. SCOs represent one branch of the larger carotenoid cleavage oxygenases family. Here, we describe the structural and functional characterization of an SCO-like enzyme from the soil-born, bio-control agent Pseudomonas brassicacearum.
Methods
In vitro and in vivo assays relying on visual inspection, spectrophotometric quantification, as well as liquid-chormatographic and mass spectrometric characterization were applied for functional evaluation of the enzyme. X-ray crystallographic analyses and in silico modeling were applied for structural investigations.
Results
In vitro assays demonstrated preferential cleavage of resveratrol, while in vivo analyses detected putative cleavage of the straight chain carotenoid, lycopene. A high-resolution structure containing the seven-bladed β-propeller fold and conserved 4-His-Fe unit at the catalytic site, was obtained. Comparative structural alignments, as well as in silico modelling and docking, highlight potential molecular factors contributing to both the primary in vitro activity against resveratrol, as well as the putative subsidiary activities against carotenoids in vivo, for future validation.
Conclusions
The findings reported here provide validation of the SCO structure, and highlight enigmatic points with respect to the potential effect of the enzyme’s molecular environment on substrate specificities for future investigation.
The new REACH legislation requires assessment of a large number of chemicals in the European market for several endpoints. Developmental toxicity is one of the most difficult endpoints to assess, on account of the complexity, length and costs of experiments. Following the encouragement of QSAR (in silico) methods provided in the REACH itself, the CAESAR project has developed several models.
Results
Two QSAR models for developmental toxicity have been developed, using different statistical/mathematical methods. Both models performed well. The first makes a classification based on a random forest algorithm, while the second is based on an adaptive fuzzy partition algorithm. The first model has been implemented and inserted into the CAESAR on-line application, which is java-based software that allows everyone to freely use the models.
Conclusions
The CAESAR QSAR models have been developed with the aim to minimize false negatives in order to make them more usable for REACH. The CAESAR on-line application ensures that both industry and regulators can easily access and use the developmental toxicity model (as well as the models for the other four endpoints).
Intermolecular autophosphorylation at Tyr416 is a conserved mechanism of activation among the members of the Src family of nonreceptor tyrosine kinases. Like several other tyrosine kinases, Src can catalyze the thiophosphorylation of peptide and protein substrates using ATPγS as a thiophosphodonor, although the efficiency of the reaction is low.
Results
Here, we have characterized the ability of Src to auto-thiophosphorylate. Auto-thiophosphorylation of Src at Tyr416 in the activation loop proceeds efficiently in the presence of Ni2+, resulting in kinase activation. Other tyrosine kinases (Ack1, Hck, and IGF1 receptor) also auto-thiophosphorylate in the presence of Ni2+. Tyr416-thiophosphorylated Src is resistant to dephosphorylation by PTP1B phosphatase.
Conclusions
Src and other tyrosine kinases catalyze auto-thiophosphorylation in the presence of Ni2+. Thiophosphorylation of Src occurs at Tyr416 in the activation loop, and results in enhanced kinase activity. Tyr416-thiophosphorylated Src could serve as a stable, persistently-activated mimic of Src.
The extracts from the aerial parts of Portulaca quadrifida have been reported to show the total flavonoid content, antioxidant and antibacterial activities.
Results
Our results revealed that the total flavonoid content of methanol and chloroform extracts is 2.335?±?0.0097 and 1.7312?±?0.0082 mgQE/100 g respectively. The two extracts also showed good antioxidant activity and total phenolic content as well as weak to moderate antibacterial activity against some bacteria.
Conclusions
The extracts the aerial parts of the P. quadrifida showed good total flavonoid content, DPPH radical scavenging activity and antibacterial activity. In addition to this, the extracts also showed the presence of some important compounds by phytochemical analysis.
The potential for a compound to cause hepatotoxicity and nephrotoxicity is a matter of extreme interest for human health risk assessment. To assess liver and kidney toxicity, repeated-dose toxicity (RDT) studies are conducted mainly on rodents. However, these tests are expensive, time-consuming and require large numbers of animals. For early toxicity screening, in silico models can be applied, reducing the costs, time and animals used. Among in silico approaches, structure–activity relationship (SAR) methods, based on the identification of chemical substructures (structural alerts, SAs) related to a particular activity (toxicity), are widely employed.
Results
We identified and evaluated some SAs related to liver and kidney toxicity, using RDT data on rats taken from the hazard evaluation support system (HESS) database. We considered only SAs that gave the best percentages of true positives (TP).
Conclusions
It was not possible to assign an unambiguous mode of action for all the SAs, but a mechanistic explanation is provided for some of them. Such achievements may help in the early identification of liver and renal toxicity of substances.
The analysis of mineral elements composition was determined in three wild edible herbs (Cichorium intybus L., Sonchus asper L. and Borago officinalis) collected in seven different sampling sites which were characterized by different pollution grade. The detection of mineral elements (Ca, K, Mg and Na), micronutrients (Cu, Fe, Li, Mn and Zn) and heavy metals (As, Cd, Hg, Ni and Pb) was performed.
Results
The results obtained show that in most cases a direct relationship appeared between the amount of elements and the sampling sites. The highest concentrations of heavy metals were found in samples grown in polluted soils. These evaluations showed that contaminants in plants may reflect the environmental state in which they develop.
Conclusion
The examined species are a good source of mineral elements and micronutrients, making them particularly adapt to integrate a well-balanced diet. The accumulation of heavy metals showed that contaminants in plants may reflect the environmental state in which they develop. Results showed high concentrations of heavy metals in samples taken in locations characterized by high human activity and in some samples from the local market, of which no one knows the collection area.
Construction of electrochemical impedance sensors by the self-assembly technique has become a promising strategy for the `label-free' detection of protein-ligand interactions. However, previous impedance sensors are devoid of an inherent electrochemical signal, which limits the standardization of the sensors for protein recognition in a reproducible manner.
Results
We designed and synthesized an anthraquinonyl glycoside (AG) where the anthraquinone (AQ) moiety can bind to the surface of a graphene-based working electrode while the glycoside serving as a ligand for lectin. By measuring the inherent voltammetric signal of AQ, the glycosides decorated on the working electrode could be simply quantified to obtain electrodes with a unified signal window. Subsequently, impedance analysis showed that the `standardized' electrodes gave a reproducible electrochemical response to a selective lectin with no signal variation in the presence of unselective proteins.
Conclusion
Anthraquinone-modified ligands could be used to facilitate the standardization of electrochemical impedance sensors for the reproducible, selective analysis of ligand-protein interactions.
