RESUMEN
Antithrombin (AT3) is one of the most important inhibitors of blood coagulation proteases that belong to the serpin family of protease inhibitors. In this study, a novel alternatively spliced isoform of AT3 was identified, both at transcript and protein level. This novel transcript contains an additional region in the continuation of exon 3b that was included in the transcript due to use of an alternate 5' splice site. The existence of the novel transcript was confirmed in human brain and liver through RT-PCR. An analysis of the complete transcript indicated that the native reactive centre loop (RCL) of AT3 is maintained; however the novel amino acid sequence projects out as an additional loop as evident from MD simulation studies. A unique amino acid sequence present in the novel isoform was used for the development of polyclonal antibody. The expression of novel isoform was confirmed in human brain and liver tissue using Western blot analysis. Interestingly an alignment of RCL like domain with other inhibitory serpins showed significant similarity with the neuroserpin RCL. To the best of our knowledge, this is the first evidence of alternatively spliced AT3 sequence containing an additional loop and could have physiological relevance.
Asunto(s)
Empalme Alternativo , Antitrombina III/química , Heparina/química , Neuropéptidos/química , Serpinas/química , Secuencia de Aminoácidos , Animales , Anticuerpos/química , Anticuerpos/aislamiento & purificación , Antitrombina III/genética , Antitrombina III/metabolismo , Secuencia de Bases , Sitios de Unión , Encéfalo/metabolismo , Expresión Génica , Heparina/metabolismo , Humanos , Hígado/metabolismo , Simulación de Dinámica Molecular , Neuropéptidos/genética , Neuropéptidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Conejos , Serpinas/genética , Serpinas/metabolismo , NeuroserpinaRESUMEN
MYD88 is an adaptor protein known to involve in activation of NF-κB through IL-1 receptor and TLR stimulation. It consists of N-terminal death domain and C-terminal Toll/IL-R homology domain that mediates its interaction with IL-1R associated kinase and IL-1R/TLR, respectively. MYD88 contributes to various types of carcinogenesis due to its involvement in oncogene induced inflammation. In the present study, we have recognized two new alternatively spliced variants of MyD88 gene in mouse using bioinformatics tools and molecular biology techniques in combination. The newly identified non-coding exon (NE-1) from 5' upstream region alternatively splices with either exon E-2 or exon E-5 to produce two novel transcript variants MyD88N1 and MyD88N2 respectively. The transcript variant MyD88N1 was expressed in several tissues studied while the variant MyD88N2 was found to be expressed only in the brain. The analysis of the upstream region of novel exon by in silico approach revealed new promoter region PN, which possess potential signature sequences for diverse transcription factors, suggesting complex gene regulation. Studies of post translational modifications of conceptualized amino acid sequences of these isoforms revealed diversity in properties. Western blot analysis further confirmed the expression of protein isoform MYD88N1.
Asunto(s)
Empalme Alternativo , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Neoplasias/genética , Animales , Encéfalo/metabolismo , Simulación por Computador , Dominio de Muerte , Exones , Regulación de la Expresión Génica , Humanos , Ratones , Factor 88 de Diferenciación Mieloide/química , Regiones Promotoras Genéticas , Procesamiento Proteico-Postraduccional , Distribución Tisular , Factores de TranscripciónRESUMEN
Nur77 is a member of nuclear receptor superfamily that acts as a transcription factor and regulates expression of multiple genes. Subcellular localization of Nur77 protein plays an important role in the survival and cell death. In this study, we have predicted and confirmed alternatively spliced two new transcripts of Nur77 gene in mouse. The newly identified transcripts have their alternatively spliced first exon located upstream of published 5'-UTR of the gene. Transcription factor binding sites in the possible promoter regions of these transcripts were also analyzed. Expression of novel transcript variants was found to be significantly lower than the already published transcript. New transcript variants encode for NUR77 protein isoforms which are significantly smaller in size due to lack of transactivation domain and a part of DNA binding domain. Western blot analysis using NUR77 specific antibody confirmed the existence of these smaller variants in mouse. Localization of these new isoforms was predicted to be majorly outside the nucleus. In silico analysis of the conceptually translated proteins was performed using different bioinformatics tools. The results obtained in this study offer further insight into novel area of research on extensively studied Nur77. © 2017 IUBMB Life, 69(2):106-114, 2017.
Asunto(s)
Núcleo Celular/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Isoformas de Proteínas/genética , ARN Mensajero/genética , Empalme Alternativo/genética , Animales , Exones/genética , Ratones , Regiones Promotoras Genéticas , Unión Proteica , Dominios Proteicos/genéticaRESUMEN
Pirenzepine is an anti-ulcer agent which belongs to the anti-cholinergic group of gastrointestinal disorder drugs and functions as an M1 receptor selective antagonist. Drug-DNA interaction studies are of great significance as it helps in the development of new therapeutic drugs. It provides a deeper understanding into the mechanism through which therapeutic drugs control gene expression. Interaction of pirenzepine with calf-thymus DNA (Ct-DNA) was determined via a series of biophysical techniques. UV-visible absorption and fluorescence spectroscopy confirmed the formation of pirenzepine-Ct-DNA complex. The values of binding constant from various experiments were calculated to be in the order of 103 M-1 which is consistent with the groove binding mode. Various spectrofluorimetric experiments like competitive displacement of well known dyes with drug, iodide quenching experiments and the effect of Ct-DNA denaturation in presence of drug confirmed the binding of pirenzepine to the groove of Ct-DNA. The binding mode was further established by viscometric, circular dichroic and molecular modelling studies. Thermodynamic parameters obtained from isothermal titration calorimetric studies suggest that the interaction of pirenzepine with Ct-DNA is enthalpically driven. The value of TΔS and ΔH calculated from calorimetric studies were found to be 4.3 kcal mol-1 and -2.54 kcal mol-1 respectively, indicating that pirenzepine-Ct-DNA complex is mainly stabilized by hydrophobic interaction and hydrogen bonding. The binding energy calculated was -7.5 kcal mol-1 from modelling studies which was approximately similar to that obtained by isothermal titration calorimetric studies. Moreover, the role of electrostatic interaction in the binding of pirenzepine to Ct-DNA cannot be precluded.
Asunto(s)
ADN/metabolismo , Fármacos Gastrointestinales/metabolismo , Pirenzepina/metabolismo , Animales , Calorimetría , Bovinos , ADN/química , Simulación del Acoplamiento Molecular , Conformación de Ácido Nucleico/efectos de los fármacos , Desnaturalización de Ácido Nucleico/efectos de los fármacos , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , TermodinámicaRESUMEN
The aryl hydrocarbon receptor nuclear translocator (ARNT/HIF1-ß) is an obligatory transcriptional partner of the aryl hydrocarbon receptor (AHR) and hypoxia-inducible factor-1α (HIF-1α). It has a basic helix-loop-helix domain that belongs to period-ARNT-single-minded (PAS) protein family. PAS proteins act as heterodimeric transcription factors with ARNT being master dimerization partner. The ARNT-HIF-1α complex is an important transcriptional regulator of the hypoxic response of the tumor cells. Previous studies have reported two transcript variants of the gene produced by alternative splicing in mouse. One transcript variant contains all 22 exons while the other variant lacks exon-E5. In our study, using combinatorial approach comprising bioinformatics tools and molecular biology techniques involving RT-PCR, semi-nested PCR, sequencing and qPCR, we have identified three novel transcript variants of Arnt gene in mouse. All three new transcripts arise as a result of alternative splicing of newly identified exons with exon-E2, replacing reported exon-E1. These transcripts encode for three protein isofoms having different N-termini. The expression of these transcripts was found to be different in different tissues of adult mice. In silico analysis of the upstream region of the new exons revealed three distinct promoter regions designated as PA, PB and PC present upstream of newly identified exons. These promoters possess potential signature sequences for common as well as different transcription factors suggesting complex regulation of Arnt gene. In silico post translational studies of the conceptually translated amino acid sequences of these transcripts show similarity in some of the properties while differ in others. The diversity at N-termini of protein isoforms suggests the possibility of forming different complexes in different tissues and may also be important for unique interactions with partner molecules.
Asunto(s)
Empalme Alternativo/genética , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Isoformas de Proteínas/genética , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/biosíntesis , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Hipoxia de la Célula/genética , Exones , Regulación de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Regiones Promotoras Genéticas , Isoformas de Proteínas/biosíntesisRESUMEN
Ferulic acid (FA) is a plant polyphenol showing diverse therapeutic effects against cancer, diabetes, cardiovascular and neurodegenerative diseases. FA is a known antioxidant at lower concentrations, however at higher concentrations or in the presence of metal ions such as copper, it may act as a pro-oxidant. It has been reported that copper levels are significantly raised in different malignancies. Cancer cells are under increased oxidative stress as compared to normal cells. Certain therapeutic substances like polyphenols can further increase this oxidative stress and kill cancer cells without affecting the proliferation of normal cells. Through various in vitro experiments we have shown that the pro-oxidant properties of FA are enhanced in the presence of copper. Comet assay demonstrated the ability of FA to cause oxidative DNA breakage in human peripheral lymphocytes which was ameliorated by specific copper-chelating agent such as neocuproine and scavengers of ROS. This suggested the mobilization of endogenous copper in ROS generation and consequent DNA damage. These results were further validated through cytotoxicity experiments involving different cell lines. Thus, we conclude that such a pro-oxidant mechanism involving endogenous copper better explains the anticancer activities of FA. This would be an alternate non-enzymatic, and copper-mediated pathway for the cytotoxic activities of FA where it can selectively target cancer cells with elevated levels of copper and ROS.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Cobre/metabolismo , Ácidos Cumáricos/farmacología , Daño del ADN , Neoplasias/tratamiento farmacológico , Oxidantes/farmacología , Animales , Antineoplásicos Fitogénicos/química , Células CHO , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quelantes/farmacología , Ensayo Cometa , Cobre/química , Ácidos Cumáricos/química , Cricetulus , Relación Dosis-Respuesta a Droga , Depuradores de Radicales Libres/farmacología , Células HEK293 , Células Hep G2 , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Linfocitos/patología , Neoplasias/metabolismo , Neoplasias/patología , Oxidantes/química , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Carbonilación Proteica/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
6-Mercaptopurine (6MP) is a well-known purine antimetabolite used to treat childhood acute lymphoblastic leukemia and other diseases. Cancer cells as compared to normal cells are under increased oxidative stress and show high copper level. These differences between cancer cells and normal cells can be targeted to develop effective cancer therapy. Pro-oxidant property of 6MP in the presence of metal ions is not well documented. Redox cycling of Cu(II) to Cu(I) was found to be efficiently mediated by 6MP. We have performed a series of in vitro experiments to demonstrate the pro-oxidant property of 6MP in the presence of Cu(II). Studies on human lymphocytes confirmed the DNA damaging ability of 6MP in the presence of Cu(II). Since 6MP possesses DNA damaging ability by producing reactive oxygen species (ROS) in the presence of Cu(II), it may also possess apoptosis-inducing activity by involving endogenous copper ions. Essentially, this would be an alternative and copper-dependent pathway for anticancer activity of 6MP.
Asunto(s)
Antineoplásicos/administración & dosificación , Mercaptopurina/administración & dosificación , Neoplasias/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Apoptosis/efectos de los fármacos , Cobre/metabolismo , Daño del ADN/efectos de los fármacos , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Drug-DNA interactions have been extensively studied in the recent past. Various techniques have been employed to decipher these interactions. DNA is a major target for a wide range of drugs that may specifically or non-specifically interact with DNA and affect its functions. Interaction between small molecules and DNA are of two types, covalent interactions and non-covalent interactions. Three major modes of non-covalent interactions are electrostatic interactions, groove binding and intercalative binding. This review primarily focuses on discussing various techniques used to study non-covalent interactions that occur between drugs and DNA. Additionally, we report several techniques that may be employed to analyse the binding mode of a drug with DNA. These techniques provide data that are reliable and simple to interpret.
Asunto(s)
ADN/metabolismo , Sustancias Intercalantes/farmacología , Preparaciones Farmacéuticas/metabolismo , Animales , Sitios de Unión , ADN/química , Humanos , Sustancias Intercalantes/química , Sustancias Intercalantes/metabolismo , Simulación del Acoplamiento Molecular , Conformación de Ácido Nucleico , Preparaciones Farmacéuticas/química , Electricidad EstáticaRESUMEN
Non-covalent interactions of chlorambucil with calf thymus DNA was investigated using multi-spectroscopic techniques and molecular docking study. Binding constant calculated was found to be 1.54×10(4)M(-1) at 290K, significantly lower than various known intercalators. Quenching process was found to be static as evident by biomolecular quenching constant. Thermodynamic parameters revealed the involvement of hydrophobic interactions and hydrogen bonds in the binding. Chlorambucil was found to interact via external binding mode and follow groove binding as it replaces Hoechst (a typical groove binder) from the groove of DNA but does not replace intercalating dyes including ethidium bromide and acridine orange from the DNA helix. These results were further supported by KI quenching experiments, DNA melting studies, CD spectroscopy and molecular docking.
Asunto(s)
Antineoplásicos Alquilantes/química , Clorambucilo/química , ADN/química , Naranja de Acridina/química , Animales , Bisbenzimidazol/química , Bovinos , Etidio/química , Enlace de Hidrógeno , Cinética , Simulación del Acoplamiento Molecular , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , Análisis Espectral , TermodinámicaRESUMEN
Ibuprofen is an important nonsteroidal anti-inflammatory drug endowed with various pharmacological and biological activities. In the present study, the photochemical properties of ibuprofen were evaluated by assaying the generation of various reactive oxygen species (ROS) such as superoxide, singlet oxygen and the hydroxyl radical. ROS generated by ibuprofen in the presence of white light causes DNA strand scission as observed by plasmid nicking assay. Ibuprofen induced ROS generation is also capable of causing DNA degradation in lymphocytes as observed by photocomet assay. ROS generation properties of ibuprofen were further strengthened by the formation of carbonyl groups in BSA and TBARS in linoleic acid as observed by carbonyl assay and lipid peroxidation assay respectively. We have also investigated the mode of interaction of ibuprofen with calf thymus DNA through a series of in vitro experiments. UV-visible spectroscopy established the formation of a complex between ibuprofen and Ct DNA. The steady state fluorescence experiments at different temperatures revealed a binding constant of â¼10(4) L mol(-1), which is indicative of intercalative binding between ibuprofen and the DNA helix. Analysis of the various thermodynamic parameters ΔG, ΔH and ΔS calculated at different temperatures indicated that the hydrogen bonds played a major role in the interaction. The intercalative binding mode is further confirmed by competitive displacement assays, urea denaturation, iodide quenching, viscosity measurements and CD analysis. In silico molecular docking revealed the binding of ibuprofen within the GC base pairs of DNA, confirming the intercalative binding mode.
Asunto(s)
Antiinflamatorios no Esteroideos/efectos adversos , ADN/metabolismo , Ibuprofeno/efectos adversos , Sustancias Intercalantes/efectos adversos , Especies Reactivas de Oxígeno/metabolismo , Animales , Bovinos , ADN/química , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Humanos , Luz , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Simulación del Acoplamiento Molecular , Fotólisis/efectos de los fármacos , Fotólisis/efectos de la radiación , TermodinámicaRESUMEN
The antioxidant defense mechanisms play a critical role in mitigating the deleterious effects of reactive oxygen species (ROS). Catalase stands out as a paramount enzymatic antioxidant. It efficiently catalyzes the decomposition of hydrogen peroxide (H2O2) into water and oxygen, a potentially harmful byproduct of cellular metabolism. This reaction detoxifies H2O2 and prevents oxidative damage. Catalase has been extensively studied as a therapeutic antioxidant. Its applications range from direct supplementation in conditions characterized by oxidative stress to gene therapy approaches to enhance endogenous catalase activity. The enzyme's stability, bioavailability, and the specificity of its delivery to target tissues are significant hurdles. Furthermore, studies employing conventional catalase formulations often face issues related to enzyme purity, activity, and longevity in the biological milieu. Addressing these challenges necessitates rigorous scientific inquiry and well-designed clinical trials. Such trials must be underpinned by sound experimental designs, incorporating advanced catalase formulations or novel delivery systems that can overcome existing limitations. Enhancing catalase's stability, specificity, and longevity in vivo could unlock its full therapeutic potential. It is necessary to understand the role of catalase in disease-specific contexts, paving the way for precision antioxidant therapy that could significantly impact the treatment of diseases associated with oxidative stress.
Asunto(s)
Antioxidantes , Catalasa , Estrés Oxidativo , Catalasa/metabolismo , Catalasa/química , Humanos , Estrés Oxidativo/efectos de los fármacos , Animales , Antioxidantes/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Peróxido de Hidrógeno/metabolismo , Terapia Genética/métodosRESUMEN
Patients with advanced prostate cancer (PCa) are more likely to develop bone metastases. Tumor cells thrive in the bone microenvironment, interacting with osteoblasts and osteoclasts. Given the PI3K/AKT pathway's metastatic potential and signal integration's ability to modulate cell fates in PCa development, drugs targeting this system have great therapeutic promise. Hydroxychloroquine (HCQ) is an anti-malarial medication commonly used to treat clinical conditions such as rheumatology and infectious disorders. We explored the anti-neoplastic effect of HCQ on PC3 and C4-2B cell lines in the bone microenvironment. Interestingly, HCQ treatment substantially decreases the viability, proliferation, and migration potential of PCa cells in the bone microenvironment. HCQ induces apoptosis and cell cycle arrest, even in the presence of osteoblast-secreted factors. Mechanistically, HCQ inhibited the activity of the PI3K/AKT signaling pathway, which ultimately regulates the proliferation and migration of PCa cells in the bone. The binding energy for docking HCQ with PI3K was -6.7 kcal/mol, and the complex was stabilized by hydrogen bonds, hydrophobic forces, and van der Waals forces. Molecular simulations further validated the structural integrity of the HCQ-PI3K complex without altering PI3K's secondary structure. Our findings underscore the efficacy of HCQ as a potential therapeutic agent in treating PCa.
Asunto(s)
Proliferación Celular , Hidroxicloroquina , Simulación de Dinámica Molecular , Fosfatidilinositol 3-Quinasas , Neoplasias de la Próstata , Microambiente Tumoral , Humanos , Masculino , Hidroxicloroquina/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Microambiente Tumoral/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasas/metabolismo , Simulación del Acoplamiento Molecular , Movimiento Celular/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Apoptosis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Huesos/efectos de los fármacos , Huesos/metabolismo , Huesos/patología , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patologíaRESUMEN
H2 receptor antagonists are the medication given for treating stomach ulcers, but lately, reports have shown their role in healing several malignant ulcers. The present work entails the interaction of H2 blocker nizatidine with calf thymus (ct)-DNA for determining the binding mode and energetics of the interaction. Multi-spectroscopic, calorimetric, viscometric and bioinformatic analysis revealed that nizatidine interacted with ct-DNA via groove-binding mode and is characterised by exothermic reaction. Moreover, assessment of genotoxic potential of nizatidine in vitro was carried out in peripheral human lymphocytes by alkaline comet assay. DNA damage occurred at high concentrations of nizatidine. Genotoxicity of nizatidine was also evaluated in vivo by assessing cytogenetic biomarkers viz. micronuclei formation and chromosomal aberration test. Nizatidine was able to induce micronuclei formation and chromosomal damage at high dose. Additionally, cytotoxic activity of nizatidine was determined in cancer cell lines, namely HeLa and HCT-116 and compared with the normal human cell line HEK-293 employing MTT assay. It was observed that nizatidine was more toxic towards HeLa and HCT-116 than HEK-293. Cell morphology analysis by compound inverted microscopy further strengthens the finding obtained through MTT assay.
Asunto(s)
Daño del ADN , Nizatidina , Humanos , Células HEK293 , Ensayo Cometa , ADNRESUMEN
Gamma-subunit of nicotinic acetylcholine receptor is encoded by chrng gene of mouse. This gene is located on chromosome 1, spans 6.5 kb, and contains 12 exons and 11 introns. Previous studies have reported three transcript variants (C1-3) produced by alternative splicing; C1 contains all the 12 reported exons, C2 uses an in-frame alternate splice site in exon-2, and C3 produced by exon-5 skipping. These variants differ in their channel kinetics and opening times. In our study, we report the presence of two new transcript variants (T1 and T2) of chrng expressed in mouse postnatal day 3 and adult skeletal muscles. These transcripts contain novel first coding exon either N1 or N2. N1 is located in the 5' UTR, while N2 is an extended exon-2. 5' extension of exon-2 contains an initiation codon which produces a novel transcript variant. Either of the two exons can splice with the internal exons to produce mature transcripts making different 5' ends of the transcripts. Consequently, the proteins encoded by these two transcripts differ at N-termini. The presence of N2 exon containing transcript was further supported by the availability of EST from the database. These new variants display heterogeneous properties. They differ in the presence of signal peptide, phosphorylation, and acetylation of their amino acid residues of the new N-termini of the gamma subunit.
Asunto(s)
Envejecimiento/metabolismo , Empalme Alternativo/genética , Feto/metabolismo , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Receptores Nicotínicos/genética , Secuencia de Aminoácidos , Animales , Biología Computacional , Electroforesis en Gel de Agar , Exones/genética , Femenino , Intrones/genética , Masculino , Ratones , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Memantine belongs to the class of cognition enhancers that functions as NMDA receptor antagonist, used to treat Alzheimer's disease. The interaction of memantine with DNA was not investigated. In the present study, the interaction of memantine with ct-DNA, as well as its cytotoxicity on cancer cells, was evaluated. UV-visible spectroscopy, steady-state fluorescence spectroscopic studies revealed the interaction between memantine and ct-DNA. The quenching studies, chemical denaturation, (CD), and DNA melting studies showed the groove binding mode of memantine with ct-DNA. The thermodynamic parameters revealed that the interaction between memantine and ct-DNA is enthalpically driven, and the stabilizing forces involved were hydrogen bonding and van der Waals interaction. The groove-binding was also observed by molecular docking studies, which corroborated the findings of spectroscopic investigations. Density function theory calculations confirmed the existence of electron donor and recipient groups. The stability of memantine and DNA interaction, as well as the critical residues involved in the interaction, was identified by molecular dynamics simulations. Memantine showed cytotoxicity towards the cancer cells as compared to normal cells, as observed by MTT assay. Inverted compound microscopy analysis of memantine treated cancer cell lines further confirmed the results obtained by MTT assay.Communicated by Ramaswamy H. Sarma.
Asunto(s)
ADN , Memantina , Línea Celular , ADN/química , Memantina/farmacología , Simulación del Acoplamiento Molecular , Conformación de Ácido Nucleico , Espectrometría de Fluorescencia , TermodinámicaRESUMEN
Advanced glycation endproducts (AGEs) are the final product of glycation, highly reactive in nature and contribute directly or indirectly to numerous complications related to diabetes. In this study, the antiglycation activity of glyburide was investigated using HSA as model protein, both against glucose and methylglyoxal mediated glycation. The possible mechanism of action was also deciphered using biophysical and computational tools. Approximately 70% inhibition of both early and advanced glycation end products were recorded in the presence of glyburide. Free lysine modification was reduced by glyburide treatment and improvement in biochemical markers such as free thiol groups and carbonyl content was observed. Interaction studies revealed that glyburide showed moderate to strong binding affinity towards HSA with binding constant in the order of 106 M-1. The interaction of glyburide with HSA was entropically favourable and spontaneous in nature. Molecular dynamics simulation deciphered that glyburide-HSA complex was quite stable where RMSD, RMSF, Rg, SASA, and secondary structure of HSA remained approximately same over the entire simulation period. The average binding energy of the MD simulation for glyburide-HSA complex was found to be -15.386 kJ mol-1. The findings demonstrate the antiglycation potential of glyburide and its possible mechanism of action.
Asunto(s)
Gliburida/química , Productos Finales de Glicación Avanzada/química , Productos Finales de Glicación Avanzada/metabolismo , Complicaciones de la Diabetes/metabolismo , Diabetes Mellitus/metabolismo , Glucosa/química , Gliburida/farmacología , Glicosilación , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Secundaria de Proteína , Albúmina Sérica/química , Albúmina Sérica/metabolismoRESUMEN
Serine/threonine kinase 11 (STK11) is a protein kinase that is encoded by Stk11 gene located on chromosome 19 and 10 in humans and mouse respectively. It acts as a master kinase of adenine monophosphate-activated protein kinase (AMPK) pathway that coordinates the regulation of cellular energy metabolism and cell division. STK11 exerts effect by activating more than 14 kinases including AMPK and AMPK-related kinases. It is also known to regulate cell polarity and acts as tumor suppressor. Alternative splicing of pre-mRNA is a mechanism which results in multiple transcript variants of a single gene. In human, two STK11 isoforms have been reported, an alternatively spliced isoform which has variation at its C-terminal and mostly expressed in testis (LKB1S). Another isoform exhibiting oncogenic properties lacks few residues at its N-terminal (ΔN-LKB1). In the present study, we report the identification of a new transcript variant Stk11N which is generated through alternative splicing. The new variant was found to have differential and tissue specific expression at Postnatal-7 and adult stages of mouse. As compared to the known variant Stk11C, the conceptually translated amino acid sequences of the new variant differ from exon-E2 onwards. In silico post translational studies of the new and published variant show similarity in some of the properties while differ in properties like nuclear export signals, phosphorylation, glycosylation, etc. Thus, alternative splicing of Stk11 gene generating new variant with heterogeneous properties suggests for complex regulation of these variants in controlling the AMPK pathway and other functions.
Asunto(s)
Empalme Alternativo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Quinasas Activadas por AMP , Secuencia de Aminoácidos , Animales , Enfermedad/genética , Variación Genética , Ratones , Señales de Exportación Nuclear , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Isoformas de ARN/metabolismo , Alineación de Secuencia , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
AIM OF THE STUDY: Rutin and Silibinin are active flavonoid compounds, well-known for possessing multiple biological activities. We have studied how Rutin and Silibinin in combination modulate wide range intracellular signaling cascades as evidenced by in-vitro research. Data obtained from preclinical studies provide evidence to be supportive to bridge basic and translational studies. METHODS: In this study, cytotoxic effect of Rutin and Silibinin individually and in combination on the viability of colon cancer cell line (HT-29) was revealed using the MTT assay. Mechanism involved in the cytotoxic effect were then investigated in terms of apoptosis using comet assay, DNA fragmentation and fluorescent microscopy analyses. The apoptosis associated proteins viz; Caspase-3, 8, 9, Bax, Bcl-2, p53, inflammation associated proteins viz; NFκB, IKK-α IKK-ß and MAPK pathway associated proteins viz; p38 and MK-2 were determined by western-blot and Real Time-PCR analysis. RESULTS: Results suggest that Rutin and Silibinin produce anticancer effects via induction of apoptosis as well as regulating the expressions of genes related to apoptosis, inflammation and MAPK pathway proteins more effectively in combination than individually. CONCLUSION: Our study supports the viability of developing Rutin and Silibinin in combination as a novel therapeutic prodrug for colon cancer treatment and may have a promising role in the development of new anticancer drugs in the future.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Rutina/farmacología , Silibina/farmacología , Caspasa 3 , Línea Celular Tumoral , Sinergismo Farmacológico , Células HT29 , Humanos , Proteínas Proto-Oncogénicas c-bcl-2 , SilimarinaRESUMEN
Bax, a pro-apoptotic member of Bcl-2 family regulates apoptosis through homodimerization/heterodimerization with Bcl-2. Bax-α is the only product of the Bax gene that has been extensively studied. Bax-α exists in inactive form and several conformational changes are required during apoptosis to activate it. Here, we have identified a novel transcript variant of Bax gene in mouse which contains alternatively spliced new first exon that is different from the first exon of previously reported transcript. Conceptual translation of new transcript encodes a protein (Bax-α1), having different N-terminus. The existence of the new transcript variant was confirmed by reverse transcriptase-PCR, semi-nested PCR using primers designed for the newly identified transcript variant. The identity of PCR product obtained after semi-nested PCR was confirmed by DNA sequencing. Relative expression of new transcript variant with respect to reported transcript was also studied with the help of real time PCR. The existence of new transcript variant was further supported by the presence of clusters of overlapping ESTs from the database. Bax-α1 possibly displays heterogeneous properties as predicted by post-translational modification analysis tools. The differences in post-translational modifications might play important roles in divergent function of the new isoform. The three dimensional structure was generated by homology modelling to visualize the differences at N termini of known and newly identified variant.
Asunto(s)
Empalme Alternativo , Proteína X Asociada a bcl-2/genética , Animales , Femenino , Masculino , Ratones , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Proteína X Asociada a bcl-2/química , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Ticlopidine is an anti-platelet drug which belongs to the thienopyridine structural family and exerts its effect by functioning as an ADP receptor inhibitor. Ticlopidine inhibits the expression of TarO gene in S. aureus and may provide protection against MRSA. Groove binding agents are known to disrupt the transcription factor DNA complex and consequently inhibit gene expression. Understanding the mechanism of interaction of ticlopidine with DNA can prove useful in the development of a rational drug designing system. At present, there is no such study on the interaction of anti-platelet drugs with nucleic acids. A series of biophysical experiments were performed to ascertain the binding mode between ticlopidine and calf thymus DNA. UV-visible and fluorescence spectroscopic experiments confirmed the formation of a complex between ticlopidine and calf thymus DNA. Moreover, the values of binding constant were found to be in the range of 103M-1, which is indicative of groove binding between ticlopidine and calf thymus DNA. These results were further confirmed by studying the effect of denaturation on double stranded DNA, iodide quenching, viscometric studies, thermal melting profile as well as CD spectral analysis. The thermodynamic profile of the interaction was also determined using isothermal titration calorimetric studies. The reaction was found to be endothermic and the parameters obtained were found to be consistent with those of known groove binders. In silico molecular docking studies further corroborated well with the experimental results.