Binding of smoothelin-like 1 to tropomyosin and calmodulin is mutually exclusive and regulated by phosphorylation

Background

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 K _D 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.

     

KCTD20, a relative of BTBD10, is a positive regulator of Akt

Background

BTBD10 binds to Akt and protein phosphatase 2A (PP2A) and inhibits the PP2A-mediated dephosphorylation of Akt, thereby keeping Akt activated. Previous studies have suggested that BTBD10 plays an important role in preventing motor neuronal death and accelerating the growth of pancreatic beta cells. Because levels of BTBD10 expression are much lower in many non-nervous tissues than nervous tissues, there may be a relative of BTBD10 that has BTBD10-like function in non-neuronal cells.

Results

A 419-amino-acid BTBD10-like protein, named KCTD20 (potassium channel tetramerization protein domain containing 20), was to found to bind to all Akt isoforms and PP2A. Overexpression of KCTD20 increased Akt phosphorylation at Thr308, as BTBD10 did, which suggests that KCTD20 as well as BTBD10 positively regulates the function of Akt. KCTD20 was ubiquitously expressed in non-nervous as well as nervous tissues.

Conclusions

KCTD20 is a positive regulator of Akt and may play an important role in regulating the death and growth of some non-nervous and nervous cells.

     

Kohzo Ito,Kazuaki Kato and Koichi Mayumi: Monographs in supramolecular chemistry No 15: Polyrotaxane and Slide-Ring Materials

Objective

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.

     

Phosphatase of regenerating liver-3 directly interacts with integrin β1 and regulates its phosphorylation at tyrosine 783

Background

Phosphatase of regenerating liver-3 (PRL-3 or PTP4A3) has been implicated in controlling cancer cell proliferation, motility, metastasis, and angiogenesis. Deregulated expression of PRL-3 is highly correlated with cancer progression and predicts poor survival. Although PRL-3 was categorized as a tyrosine phosphatase, its cellular substrates remain largely unknown.

Results

We demonstrated that PRL-3 interacts with integrin β1 in cancer cells. Recombinant PRL-3 associates with the intracellular domain of integrin β1 in vitro. Silencing of integrin α1 enhances PRL-3-integrin β1 interaction. Furthermore, PRL-3 diminishes tyrosine phosphorylation of integrin β1 in vitro and in vivo. With site-specific anti-phosphotyrosine antibodies against residues in the intracellular domain of integrin β1, tyrosine-783, but not tyrosine-795, is shown to be dephosphorylated by PRL-3 in a catalytic activity-dependant manner. Phosphorylation of Y783 is potentiated by ablation of PRL-3 or by treatment with a chemical inhibitor of PRL-3. Conversely, depletion of integrin α1 decreases the phosphorylation of this site.

Conclusions

Our results revealed a direct interaction between PRL-3 and integrin β1 and characterized Y783 of integrin β1 as a bona fide substrate of PRL-3, which is negatively regulated by integrin α1.

     

Effect of the expression and knockdown of citrate synthase gene on carbon flux during triacylglycerol biosynthesis by green algae <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Background

The regulation of lipid biosynthesis is essential in photosynthetic eukaryotic cells. This regulation occurs during the direct synthesis of fatty acids and triacylglycerols (TAGs), as well as during other controlling processes in the main carbon metabolic pathway.

Results

In this study, the mRNA levels of Chlamydomonas citrate synthase (CrCIS) were found to decrease under nitrogen-limited conditions, which suggests suppressed gene expression. Gene silencing by RNA interference (RNAi) was conducted to determine whether CrCIS suppression affected the carbon flux in TAG biosynthesis. Results showed that the TAG level increased by 169.5%, whereas the CrCIS activities in the corresponding transgenic algae decreased by 16.7% to 37.7%. Moreover, the decrease in CrCIS expression led to the increased expression of TAG biosynthesis-related genes, such as acyl-CoA:diacylglycerol acyltransferase and phosphatidate phosphatase. Conversely, overexpression of CrCIS gene decreased the TAG level by 45% but increased CrCIS activity by 209% to 266% in transgenic algae.

Conclusions

The regulation of CrCIS gene can indirectly control the lipid content of algal cells. Our findings propose that increasing oil by suppressing CrCIS expression in microalgae is feasible.

     

Up-regulation of DcR3 in microbial toxins-stimulated HUVECs involves NF-κB signalling

Background

Sepsis is a severe condition characterised by the body’s systemic inflammatory response to infection. The specific sepsis-related biomarkers should be used in clinical diagnosis, therapeutic response monitoring, rational use of antibiotics, and prognosis (risk stratification), etc.

Results

In this study, we investigated the expression level of Decoy Receptor 3 (DcR3) and the mechanism of high expression in sepsis patients. Septic cell model experiments were performed by treating human umbilical vein endothelial cells (HUVECs) and Jurkat cells with lipopolysaccharide (LPS), lipoteichoic acid (LTA) and zymosan, respectively. SP600125, SB203580 and ammonium pyrrolidinedithiocarbamate (PDTC) were used to inhibit JNK1/2, p38MAPK and NF-κB signalling pathways in septic cell model, respectively. These results showed that DcR3 levels were higher in sepsis group than control. DcR3 mRNA and protein levels in HUVECs were increased following treatment with LPS, LTA and zymosan, and also increased in Jurkat cells treated by LPS, but not by LTA or zymosan. When HUVECs were treated with the NF-κB inhibitor PDTC, DcR3 expression was decreased compared with controls. However, SP600125 and SB203580 had no effect on DcR3 mRNA or protein levels.

Conclusions

The results indicated that DcR3 secretion proceeded through the NF-κB signalling pathway in HUVECs.

     

Sustained activation of mTORC1 in macrophages increases AMPKα-dependent autophagy to maintain cellular homeostasis

Background

The mechanistic target of rapamycin complex 1 (mTORC1) is a well-conserved serine/threonine protein kinase that controls autophagy as well as many other processes such as protein synthesis, cell growth, and metabolism. The activity of mTORC1 is stringently and negatively controlled by the tuberous sclerosis proteins 1 and 2 complex (TSC1/2).

Results

In contrast to the previous studies using Tsc1 knockout mouse embryonic fibroblasts (MEF) cells, we demonstrated evidence that TSC1 deficient macrophages exhibited enhanced basal and mycobacterial infection-induced autophagy via AMPKα-dependent phosphorylation of ULK1 (Ser555). These effects were concomitant with constitutive activation of mTORC1 and can be reversed by addition of amino acids or rapamycin, and by the knockdown of the regulatory-associated protein of mTOR, Raptor. In addition, increased autophagy in TSC1 deficient macrophages resulted in suppression of inflammation during mycobacterial infection, which was reversed upon amino acid treatment of the TSC1 deficient macrophages. We further demonstrated that TSC1 conditional knockout mice infected with Mycobacterium tuberculosis, the causative agent of tuberculosis, resulted in less bacterial burden and a comparable level of inflammation when compared to wild type mice.

Conclusions

Our data revealed that sustained activation of mTORC1 due to defects in TSC1 promotes AMPKα-dependent autophagic flux to maintain cellular homeostasis.

     

Knockdown of GGCT inhibits cell proliferation and induces late apoptosis in human gastric cancer

Background

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.

     

DFT and TD-DFT calculation of new thienopyrazine-based small molecules for organic solar cells

Background

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 (V_oc) 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.

     

Use of a special Brazilian red-light emitting railroad worm Luciferase in bioassays of NEK7 protein Kinase and Creatine Kinase

Background

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.

     

Identification of the major diacylglycerol acyltransferase mRNA in mouse adipocytes and macrophages

Background

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.

     

Mutation of exposed hydrophobic amino acids to arginine to increase protein stability

Background

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.

     

Structure and function of a lignostilbene-α,β-dioxygenase orthologue from <Emphasis Type="Italic">Pseudomonas brassicacearum</Emphasis>

Background

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.

     

Synthesis,structure and toxicity evaluation of ethanolamine nitro/chloronitrobenzoates: a combined experimental and theoretical study

Background

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.

     

CAESAR models for developmental toxicity

Background

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).

     

Characterization of black pigment used in 30 BC fresco wall paint using instrumental methods and chemometry

Background and methods

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.

     

Auto-thiophosphorylation activity of Src tyrosine kinase

Background

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.

     

Development of a practical synthesis of etravirine via a microwave-promoted amination

Background

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.

     

Quick assessment of the economic value of olive mill waste water

Background

Olive biophenols are emerging as a valued class of natural products finding practical application in the food, pharmaceutical, beverage, cosmetic and nutraceutical industries due to their powerful biological activity which includes antioxidant and antimicrobial properties. Olive mill waste water (OMWW), a by-product in olive oil manufacturing, is rich in biophenols such as hydroxytyrosol and tyrosol. The amount of biophenols depends on the cultivar, the geographical area of cultivation, and the seasonal conditions. The goal of this study was to develop a straightforward method to assess the economic value of OMWW via quantification of hydroxytyrosol and tyrosol.

Results

The amount of hydroxytyrosol and tyrosol phenolic compounds in the OMWW from four different cultivars grown in four different regions of Sicily was analyzed using liquid–liquid and solid–liquid analytical protocols developed ad hoc. Results showed significant differences amongst the different cultivars and their geographical origin. In all samples, the concentration of hydroxytyrosol was generally from 2 to 10 times higher than that of tyrosol. In general, the liquid–liquid extraction protocol gave higher amounts of extracted biophenols. The cultivar Cerasuola had the highest amount of both hydroxytyrosol and tyrosol. The cultivar Nocellara Etnea had the lowest content of both biophenols.

Conclusions

A quick method to assess the economic value of olive mill waste water via quantification of hydroxytyrosol and tyrosol in olive phenolic enriched extracts is now available.

     

Effect of IAPP on the proteome of cultured Rin-5F cells

Background

Islet amyloid polypeptide (IAPP) or amylin deposits can be found in the islets of type 2 diabetes patients. The peptide is suggested to be involved in the etiology of the disease through formation of amyloid deposits and destruction of β islet cells, though the underlying molecular events leading from IAPP deposition to β cell death are still largely unknown.

Results

We used OFFGEL? proteomics to study how IAPP exposure affects the proteome of rat pancreatic insulinoma Rin-5F cells. The OFFGEL? methodology is highly effective at generating quantitative data on hundreds of proteins affected by IAPP, with its accuracy confirmed by In Cell Western and Quantitative Real Time PCR results. Combining data on individual proteins identifies pathways and protein complexes affected by IAPP. IAPP disrupts protein synthesis and degradation, and induces oxidative stress. It causes decreases in protein transport and localization. IAPP disrupts the regulation of ubiquitin-dependent protein degradation and increases catabolic processes. IAPP causes decreases in protein transport and localization, and affects the cytoskeleton, DNA repair and oxidative stress.

Conclusions

Results are consistent with a model where IAPP aggregates overwhelm the ability of a cell to degrade proteins via the ubiquitin system. Ultimately this leads to apoptosis. IAPP aggregates may be also toxic to the cell by causing oxidative stress, leading to DNA damage or by decreasing protein transport. The reversal of any of these effects, perhaps by targeting proteins which alter in response to IAPP, may be beneficial for type II diabetes.