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STUDY QUESTION: Is there any difference at the serum metabolic level between women with recurrent implantation failure (RIF) and women with recurrent implantation success (RIS) when undergoing in vitro fertilization (IVF)? SUMMARY ANSWER: Eight metabolites, including valine, adipic acid, l-lysine, creatine, ornithine, glycerol, d-glucose and urea, were found to be significantly up-regulated in women with RIF when compared with women with RIS. WHAT IS KNOWN ALREADY: Despite transfer of three high-grade embryos per cycle, RIF following three or more consecutive IVF attempts occurs in a group of infertile women. Conversely, there is a group of women who undergo successful implantation each cycle, yet have a poor obstetric history. STUDY DESIGN, SIZE, DURATION: This study was conducted over a period of 10 years (January 2004-October 2014). Groups of 28 women with RIF (age ≤40 years and BMI ≤28) and 24 women with RIS (age and BMI matched) were selected from couples with primary infertility reporting at the Institute of Reproductive Medicine, Kolkata, India. Women recruited in the RIF group had history of implantation failure in at least three consecutive IVF attempts, in which three embryos of high-grade quality were transferred in each cycle. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood samples were collected from both the groups during the implantation window following overnight fasting for at least 10 h (7-10 days post ovulation). Samples were analyzed using a 700 MHz NMR spectrometer and acquired spectra were subjected to chemometric and statistical analysis. Serum levels of endothelial nitric oxide synthase (eNOS) were measured using an enzyme immunoassay technique. MAIN RESULTS AND THE ROLE OF CHANCE: Valine, adipic acid, l-lysine, creatine, ornithine, glycerol, d-glucose and urea were found to be significantly down-regulated in women with RIS when compared with those with RIF, with fold change values of 0.81, 0.82, 0.79, 0.80, 0.78, 0.68, 0.76 and 0.74, respectively. Further, serum eNOS was found to be significantly lower in women with RIF when compared with RIS (P < 0.05), indicating possible impairment in nitric oxide production. Metabolites, mostly related to energy metabolism, lipid metabolism and the arginine metabolic pathway were found to be considerably altered and are likely to be associated with the RIF phenomenon. However, the interplay between these molecules in RIF is complex and holds merit for further exploration. LIMITATIONS, REASONS FOR CAUTION: In-depth studies of the arginine metabolic pathway in endometrial tissues seem necessary to validate our findings. A limitation of the present study is that the metabolic level changes, eNOS and nitric oxide levels have not been investigated in the endometrial tissues of the two groups of women. It would be interesting to investigate whether there exists a direct link between metabolic dysregulation and genetic factors that affects implantation in RIF women. WIDER IMPLICATIONS OF THE FINDINGS: We speculate that tissue metabolomics can provide an improved understanding of the metabolic dysfunction associated with RIF. The identification of serum metabolic marker(s) in women with RIS may help with strategies of early therapeutic intervention, which may improve the chances of implantation significantly in women otherwise susceptible to IVF failure. STUDY FUNDING/COMPETING INTERESTS: One of the authors, S.R.C. acknowledges the Council of Scientific and Industrial Research (CSIR), Government of India [No: 9/81(1228)/14, EMR-I] for financial support.
Assuntos
Implantação do Embrião , Metabolômica , Adulto , Transferência Embrionária , Feminino , Fertilização in vitro , Humanos , Análise Multivariada , Óxido Nítrico Sintase Tipo III/sangue , Resultado do TratamentoRESUMO
In an attempt to find out the association of metabolic dysregulation with poor endometrial receptivity and pregnancy loss, serum metabonomic profiling of women with idiopathic recurrent spontaneous miscarriage (IRSM) is carried out and compared with fertile controls. (1)H nuclear magnetic resonance (NMR)-based metabonomics was used to obtain serum metabolic profiles of 36 women with IRSM and 28 proven fertile women during the window of implantation. The acquired data were analyzed using multivariate principal component analysis, partial least-squares-discriminant analysis, and orthogonal projection to latent structure with discriminant analysis. A clear metabolic differentiation was evident between IRSM and control samples. The distinguishing metabolites, l-lysine, l-arginine, l-glutamine, l-histidine, l-threonine, l-phenylalanine, and l-tyrosine are significantly up-regulated in IRSM as compared to controls. These altered metabolites may be involved in the molecular mechanism of exaggerated inflammatory response and vascular dysfunction associated with poor endometrial receptivity in women with IRSM. The present work proposes a vital association of metabolic dysfunction with the disease pathogenesis.
Assuntos
Aborto Habitual/sangue , Metaboloma , Aminoácidos/sangue , Estudos de Casos e Controles , Implantação do Embrião , Feminino , Humanos , Espectroscopia de Ressonância Magnética , Metabolômica , Gravidez , Análise de Componente PrincipalRESUMO
PURPOSE: The aim of this study was to analyze the seminal plasma of patients with idiopathic/male factor infertility and healthy controls with proven fertility by NMR spectroscopy, with a hope of establishing difference in biomarker profiles, if any, between the groups. METHODS: A total of 103 subjects visiting the infertility clinic of Manipal University with normozoospermic parameters, oligozoospermia, asthenozoospermia, azoospermia and teratozoospermia were included. Semen characteristics were analysed by standard criteria. Seminal plasma was subjected to NMR spectroscopy at a 700 MHz (1)H frequency. The resultant data was analyzed by appropriate software. RESULTS: The analysis revealed significant differences between the fertile control group and other forms of male infertility. Interestingly, seminal plasma profile of the idiopathic infertility group showed distinct segregation from the control population as well as other infertile groups. The difference in biomarker profiles between the idiopathic infertility and the rest of the groups combined could originate from either the up-regulation or down regulation of a several compounds, including lysine, arginine, tyrosine, citrate, proline and fructose. CONCLUSION: Our data suggests the presence of a metabolic reason behind the origin of idiopathic infertility. (1)H NMR based metabonomic profiling based on concentration of biomarker lysine has the potential to aid in the detection and diagnosis of idiopathic infertility in an efficient manner.
Assuntos
Infertilidade Masculina/metabolismo , Adulto , Biomarcadores/metabolismo , Humanos , Masculino , Metabolômica , Análise Multivariada , Ressonância Magnética Nuclear Biomolecular , Análise do Sêmen/métodosRESUMO
The development of novel antifungal agents with high susceptibility and increased potency can be achieved by increasing their overall lipophilicity. To enhance the lipophilicity of voriconazole, a second generation azole antifungal agent, we have synthesized its carboxylic acid ester analogues, namely p-methoxybenzoate (Vpmb), toluate (Vtol), benzoate (Vbz) and p-nitrobenzoate (Vpnb). The intermolecular interactions of these analogues with model membrane have been investigated using nuclear magnetic resonance (NMR) and differential scanning calorimetric (DSC) techniques. The results indicate varying degree of changes in the membrane bilayer's structural architecture and physico-chemical characteristics which possibly can be correlated with the antifungal effects via fungal membrane. Rapid metabolite profiling of chemical entities using cell preparations is one of the most important steps in drug discovery. We have evaluated the effect of synthesized analogues on Candida albicans. The method involves real time (1)H NMR measurement of intact cells monitoring NMR signals from fungal metabolites which gives Metabolic End Point (MEP). This is then compared with Minimum Inhibitory Concentration (MIC) determined using conventional methods. Results indicate that one of the synthesized analogues, Vpmb shows reasonably good activity.
Assuntos
Antifúngicos/síntese química , Bicamadas Lipídicas/química , Pirimidinas/química , Triazóis/química , 1,2-Dipalmitoilfosfatidilcolina/química , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Varredura Diferencial de Calorimetria , Candida albicans/efeitos dos fármacos , Candida albicans/metabolismo , Ácidos Carboxílicos/química , Ésteres , Éteres de Hidroxibenzoatos/síntese química , Éteres de Hidroxibenzoatos/química , Éteres de Hidroxibenzoatos/farmacologia , Bicamadas Lipídicas/metabolismo , Espectroscopia de Ressonância Magnética , Metaboloma , Testes de Sensibilidade Microbiana , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Temperatura de Transição , Triazóis/metabolismo , Triazóis/farmacologia , VoriconazolRESUMO
This work describes miRNA-based electrochemical biosensor for detection of miRNA30e, a pancreatic cancer biomarker. The screen-printed gold electrode was functionalized using cysteine hydrochloride followed by immobilization of synthesized colloidal gold nanorods (10-12 nm diameter and 25-65 nm length). The gold nanorods modified electrode surface was amino functionalized for covalent attachment of single-stranded DNA probe against miRNA30e (miR30e). This platform was utilized for electrochemical measurements and response analysis of target miRNA30e. Electrochemical impedance spectroscopic measurements showed very poor sensitivity (13.51 Ω/µg/mL/cm2) using charge transfer resistance calibration plots. Cyclic voltammetry and differential pulse voltammetry-based miR30e quantification showed decreasing current response with increasing concentration of miR30e with detection range of 0.1 fg/mL-0.1 µg/mL (14.9 aM-14.9 nM). The sensitivity of DPV sensing (104.4 µA/µg/mL/cm2) was found to be 1.3 times higher than that of CV-based quantification (79.6 µA/µg/mL/cm2). miRNA-based biosensors have the potential of replacing current invasive, time consuming and technically difficult diagnostic procedures. Furthermore, the lower limit of detection of 14.9 aM miRNA30e makes it a promising tool for detection of cancer at early stages and hence increasing survival rate.
Assuntos
Técnicas Biossensoriais , MicroRNAs , Neoplasias Pancreáticas , Humanos , DNA de Cadeia Simples , Cisteína , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Coloide de OuroRESUMO
With increase in cancer burden worldwide and poor survival rates due to delayed diagnosis, it is pertinent to develop a device for early diagnosis. We report an electrochemical biosensor for quantification of miRNA-204 (miR-204) biomarker that is dysregulated in most of the cancers. The proposed methodology uses the gold nanoparticles-modified carbon screen-printed electrode for immobilization of single-stranded DNA probe against miR-204. Colloidal gold nanoparticles were synthesized using L-glutamic acid as reducing agent. Nanoparticles were characterized by UV-visible spectroscopy and transmission electron microscopy. Spherical gold nanoparticles were of 7-28 nm in size. Biosensor fabricated using these nanoparticles was characterized by cyclic voltammetry after spiking 0.1 fg/mL-0.1 µg/mL of miR-204 in fetal bovine serum. Response characteristics of the miR-204 biosensor displayed high sensitivity of 8.86 µA/µg/µL/cm2 with wide detection range of 15.5 aM to 15.5 nM. The low detection limit makes it suitable for early diagnosis and screening of cancer.
Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , MicroRNAs , Neoplasias , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas , Ouro , Humanos , Nanopartículas Metálicas/química , Neoplasias/diagnósticoRESUMO
Resistance to currently available antifungal drugs necessitates development of new drugs using rapid, robust and automated methods to test a large number of newly synthesized drugs in less time. We have compared the effect of ketoconazole, fluconazole and its synthesized analogues on Candida albicans ATCC 10231. A metabolic profile of C.albicans ATCC 10231 in presence of drugs has been compared using (1)H NMR. Signals from metabolites have been monitored with time. MIC determined using conventional methods has been compared with Metabolic End Point (MEP) obtained from NMR spectroscopy. Results indicate that the activity of the fluconazole derivatives is in the order fluconazole p-methoxybenzoate>fluconazole=fluconazole benzoate>fluconazole toluate>fluconazole p-nitrobenzoate.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Fluconazol/análogos & derivados , Espectroscopia de Ressonância Magnética/métodos , Candida albicans/metabolismo , Fluconazol/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
In this study, two bacterial isolates designated Alp6 and Alp7 were isolated from soil collected from dye industries and screened for their ability to degrade carbazole. Growing cells of the isolates Alp6 and Alp7 could degrade 99.9% and 98.5% of carbazole, respectively in 216 h. The specific activity for degradation by the resting cells of Alp6 was found to be 7.96 µmol/min/g dry cell weight, while for Alp7 it was 5.82 µmol/min/g dry cell weight. Phylogenetic analysis based on 16S rDNA gene sequences showed that isolates Alp6 and Alp7 belonged to the genus Acinetobacter. To the best of our knowledge, this is the first report on the Acinetobacter spp. showing utilization of carbazole as carbon and nitrogen source.
Assuntos
Acinetobacter/metabolismo , Carbazóis/metabolismo , Poluentes do Solo/metabolismo , Acinetobacter/genética , Acinetobacter/isolamento & purificação , Sequência de Bases , Biodegradação Ambiental , Carbazóis/análise , Dados de Sequência Molecular , Filogenia , Microbiologia do Solo , Poluentes do Solo/análiseRESUMO
This study presents nanoparticle-based vaccine development for Hepatitis E virus (HEV). Gold nanoparticles (GNP) of average size 12 nm were synthesized by citrate reduction method followed by functionalization with cysteamine hydrochloride for nano-conjugation. Immune response of nano-conjugates of GNP with 26 kDa protein (368-606 amino acids) and 54 kDa protein (112-606 amino acids) were evaluated. In vitro release kinetics of GNP-conjugated 54 kDa (GNP54) and 26 kDa (GNP26) proteins showed slower rate of release of 54 kDa protein as compared to 26 kDa protein. Humoral immune response of mice immunized intramuscularly with GNP54, GNP26 and GNP alone, exhibited HEV-specific IgG titer of 7.9 ± 2.9, 5.686 ± 4.098 and 0.698 ± 0.089, respectively, after 14 days of booster immunization. In addition to this, HEV-specific cell-mediated immune response was demonstrated by splenocyte proliferation assay. Analysis of results using one-way ANOVA, showed statistically significant (p value < 0.05) increase in splenocyte proliferation for GNP54- and GNP26-immunized mice in comparison to GNP alone immunized mice. Stimulation index of HEV ORF2 proteins in GNP54/GNP26-immunized mice were comparable to Concanavalin A-treated positive control. These results indicate GNP-based vaccine as a promising candidate for efficiently mediating both humoral and cell-mediated immune response against HEV.
RESUMO
Structure-based drug design has led to the introduction of three drugs--oseltamivir (GS-4104), zanamivir (GG-167) and peramivir (RWJ-270201) which target the enzyme neuraminidase, for treatment of influenza infections. Using comparative docking studies we propose that more potent molecules against neuraminidase can be obtained by appending extra positively charged substituents at the C5 position of the oseltamivir skeleton. This provides an additional interaction with the enzyme and may overcome the problem of resistance encountered with these drugs. To get an insight into the transport and absorption of oseltamivir--the ethyl ester prodrug (GS-4104) as well as its mechanism of action, we have carried out 1H, 13C, 31P NMR, DSC and TEM studies on GS-4104 with model membranes prepared from DMPC/DPPC/POPC. These studies reveal that interactions between GS-4104 and the membrane are both electrostatic (involving H-bonding) and hydrophobic (involving the hydrophobic chain and cyclohexene ring of GS-4104) in nature. The prodrug is seen to increase the fluidity as well as stabilize the bilayer phase of the membrane. This property may be responsible for preventing viral entry into the cells by preventing fusion of the virus outer coat with the cell membrane.
Assuntos
Virus da Influenza A Subtipo H5N1/enzimologia , Membranas Artificiais , Sondas Moleculares/análise , Sondas Moleculares/química , Neuraminidase/química , Oseltamivir/análise , Oseltamivir/química , Varredura Diferencial de Calorimetria , Ligantes , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Estrutura Molecular , Neuraminidase/metabolismo , Ressonância Magnética Nuclear Biomolecular , Oseltamivir/metabolismo , Ligação ProteicaRESUMO
As a part of our ongoing program of developing novel influenza virus inhibitors, some new derivatives of oseltamivir were prepared by modifying the amino group with glycyl, acetyl, benzyl and prolyl moieties. The interactions of these derivatives with neuraminidase have been probed by molecular modeling techniques. Further, the interaction of these derivatives with model membranes prepared from DPPC and the effect on the thermotropic behavior and polymorphism of the bilayers have been investigated by multinuclear NMR and DSC methods. Results indicate that the glycyl derivative of oseltamivir has the most profound effects on the membrane, compared to other derivatives and seems to be the most promising derivative for further pharmacological evaluation as a neuraminidase inhibitor.
Assuntos
Antivirais/síntese química , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Neuraminidase/antagonistas & inibidores , Oseltamivir/análogos & derivados , 1,2-Dipalmitoilfosfatidilcolina/química , Varredura Diferencial de Calorimetria , Domínio Catalítico , Simulação por Computador , Desenho de Fármacos , Bicamadas Lipídicas/química , Lipossomos/química , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oseltamivir/síntese químicaRESUMO
Glucose-dependent insulinotropic polypeptide (gastric inhibitory polypeptide, or GIP), a 42-amino acid incretin hormone, modulates insulin secretion in a glucose-concentration-dependent manner. Its insulinotropic action is highly dependent on glucose concentration that surmounts the hypoglycemia side effects associated with current therapy. In order to develop a GIP-based anti-diabetic therapy, it is essential to establish the 3D structure of the peptide and study its interaction with the GIP receptor (GIPR) in detail. This will give an insight into the GIP-mediated insulin release process. In this article, we report the solution structure of GIP(1-42, human)NH(2) deduced by NMR and the interaction of the peptide with the N-terminus of GIPR using molecular modelling methods. The structure of GIP(1-42, human)NH(2) in H(2)O has been investigated using 2D-NMR (DQF-COSY, TOCSY, NOESY, (1)H-(13)C HSQC) experiments, and its conformation was built by constrained MD simulations with the NMR data as constraints. The peptide in H(2)O exhibits an alpha-helical structure between residues Ser8 and Asn39 with some discontinuity at residues Gln29 to Asp35; the helix is bent at Gln29. This bent gives the peptide an 'L' shape that becomes more pronounced upon binding to the receptor. The interaction of GIP with the N-terminus of GIPR was modelled by allowing GIP to interact with the N-terminus of GIPR under a series of decreasing constraints in a molecular dynamics simulation, culminating with energy minimization without application of any constraints on the system. The canonical ensemble obtained from the simulation was subjected to a detailed energy analysis to identify the peptide-protein interaction patterns at the individual residue level. These interaction energies shed some light on the binding of GIP with the GIPR N-terminus in a quantitative manner.
Assuntos
Polipeptídeo Inibidor Gástrico/química , Polipeptídeo Inibidor Gástrico/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , Receptores dos Hormônios Gastrointestinais/química , Receptores dos Hormônios Gastrointestinais/metabolismo , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
We report a nanoparticles based electrochemical immunosensor to detect and quantify triiodothyronine (T3) hormone. Immunosensor developed using gold nanoparticles and anti-T3 antibody, was employed for quantification of T3 antigen using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) technique. The electrochemical response of the developed immunosensor correlates well with the amount of antigen present in the sample. With increase in antigen concentration the immunocomplex formation on electrode surface increases and hence redox current decreases. The immunosensor shows a lower limit of detection of 1 pg/mL and dynamic range from 1 to 500 pg/mL. Sensitivity of the immunosensor was found to be 29.81 µA/pg/mL/cm².
Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Técnicas Eletroquímicas , Eletrodos , Ouro , Hormônios , Imunoensaio , Limite de DetecçãoRESUMO
The emergence and spread of multidrug-resistant strains of Klebsiella pneumoniae is a major concern that necessitates the development of unique therapeutics. The essential requirement of serine acetyltransferase (SAT/CysE) for survival of several human pathogens makes it a very promising target for inhibitor designing and drug discovery. In this study, as an initial step to structure-based drug discovery, CysE from K. pneumonia was structurally and biochemically characterized. Subsequently, blind docking of selected natural products into the X-ray crystallography determined 3D structure of the target was carried out. Experimental validation of the inhibitory potential of the top-scorers established quercetin as an uncompetitive inhibitor of Kpn CysE. Molecular dynamics simulations carried out to elucidate the binding mode of quercetin reveal that this small molecule binds at the trimer-trimer interface of hexameric CysE, a site physically distinct from the active site of the enzyme. Detailed analysis of conformational differences incurred in Kpn CysE structure on binding to quercetin provides mechanistic understanding of allosteric modulation. Binding of quercetin to CysE leads to conformation changes in the active site loops and proximal loops that affect its internal dynamics and consequently its affinity for substrate/co-factor binding, justifying the reduced enzyme activity.
Assuntos
Antibacterianos/química , Klebsiella pneumoniae/enzimologia , Serina O-Acetiltransferase/química , Regulação Alostérica/efeitos dos fármacos , Antibacterianos/farmacologia , Clonagem Molecular , Estabilidade Enzimática , Expressão Gênica , Humanos , Cinética , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Desnaturação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Serina O-Acetiltransferase/antagonistas & inibidores , Serina O-Acetiltransferase/genética , Serina O-Acetiltransferase/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
The upsurge of drug resistant tuberculosis is major health threat globally. To counteract, antimicrobial peptides are being explored as possible alternatives. However, certain limitations of peptide-based drugs such as potential toxicity, high cost and relatively low stability need to be addressed to enhance their clinical applicability. Use of computer predicted short active motifs of AMPs along with nanotechnology could not only overcome the limitations of AMPs but also potentiate their antimicrobial activity. Therefore, present study was proposed to in silico identify short antimicrobial motif (Pep-H) of human neutrophil peptide-1 (HNP-1) and explore its antimycobacterial activity in free form and using nanoparticles-based delivery systems. Based on colony forming unit analysis, motif Pep-H led to killing of more than 90% M. tb in vitro at 10 µg/ml, whereas, similar activity against intracellularly growing M. tb was observed at 5 µg/ml only. Thereafter, chitosan (244 nm) and gold nanoparticles (20 nm) were prepared for Pep-H with both the formulations showing minimal effects on the viability of human monocyte derived macrophages (MDMs) and RBC integrity. The antimycobacterial activity of Pep-H against intracellular mycobacteria was enhanced in both the nanoformulations as evident by significant reduction in CFU (>90%) at 5-10 times lower concentrations than that observed for free Pep-H. Thus, Pep-H is an effective antimycobacterial motif of HNP-1 and its activity is further enhanced by chitosan and gold nanoformulations.
Assuntos
Antituberculosos/farmacologia , Quitosana/química , Portadores de Fármacos/química , Ouro/química , Mycobacterium tuberculosis/efeitos dos fármacos , alfa-Defensinas/farmacologia , Antituberculosos/química , Sistemas de Liberação de Medicamentos , Descoberta de Drogas , Humanos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Tuberculose/tratamento farmacológico , alfa-Defensinas/químicaRESUMO
Characterizing the low energy excited states in the energy landscape of a protein is one of the exciting and demanding problems in structural biology at the present time. These describe the adaptability of the protein structure to external perturbations. In this context, we used here non-linear dependence of amide proton chemical shifts on temperature to identify residues accessing alternative conformations in SUMO-1 in the native state as well as in the near-native states created by sub-denaturing concentrations of urea. The number of residues accessing alternative conformations increases and the profiles of curved temperature dependence also change with increasing urea concentration. In every case these alternative conformations lie within 2 kcal/mol from the ground state, and are separated from it by low energy barriers. The residues that access alternative conformations span the length of the protein chain but are located at particular regions on the protein structure. These include many of the loops, beta2 and beta5 strands, and some edges of the helices. We observed that some of the regions of the protein structure that exhibit such fluctuations coincide with the protein's binding surfaces with different substrate like GTPase effector domain (GED) of dynamin, SUMO binding motifs (SBM), E1 (activating enzyme, SAE1/SAE2) and E2 (conjugating enzyme, UBC9) enzymes of sumoylation machinery, reported earlier. We speculate that this would have significant implications for the binding of diversity of targets by SUMO-1 for the variety of functions it is involved in.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Proteína SUMO-1/química , Termodinâmica , Sequência de Aminoácidos , Simulação por Computador , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Desnaturação Proteica , TemperaturaRESUMO
Hepatitis E virus infection is responsible for acute viral hepatitis and associated with high mortality and still birth in pregnant women in developing countries. We report expression of truncated forms of HEV ORF2 as potential vaccine candidates for nanoparticle-based delivery. These two truncated ORF2 proteins (54 kDa and 26 kDa) have been reported to be highly immunogenic and can be used as nanoparticle-based vaccine candidate. The bacterial expressed protein was purified by affinity chromatography and further confirmed by western blot using anti-HEV antibody. The chitosan nanoemulsion was synthesized using ultrasonic waves. The nanoparticle size was found to be 120-160 nm and the entrapment efficiency of purified truncated ORF2 proteins within these nanoparticles was 70% (26 kDa) and 59% (54 kDa). In cell cytotoxicity analysis, 100 µg/mL nanoemulsion was found suitable for cell viability in both HeLa and THP1 cell lines. Release kinetics of encapsulated proteins at physiological pH 7.4 showed 26-59% and 9.7-40% release of 26 kDa and 54 kDa protein within 1 h that gradually increased with time (48 h). Encapsulated proteins were found to be unstable at pH 1.2.
RESUMO
Assesment of human epidermal growth factor receptor 2 status is a key factor prompting definitive treatment decisions that help in reducing mortality rates associated with breast cancer. In this article, highly sensitive and low-cost impedimetric immunosensor using single-chain fragment variable antibody fragments was developed for quantitative detection of human epidermal growth factor receptor 2 from serum employing gold nanoparticle-modified disposable screen-printed carbon electrodes. The gold nanoparticles facilitate fast electron transfer and offer a biocompatible surface for immobilization of small antibody fragments in an oriented manner, resulting in improved antigen binding efficiency. The single-chain fragment variable antibody fragment-modified screen printed immunosensor exhibits wide dynamic range of 0.01-100â¯ngâ¯mL-1 and detection limit of 0.01â¯ngâ¯mL-1. The advantages offered by this platform in terms of high sensitivity, broad dynamic range and low-cost demonstrates great potential for improved monitoring of human epidermal growth factor receptor 2 levels for the management of breast and other cancers.
Assuntos
Técnicas Biossensoriais , Neoplasias da Mama/sangue , Imunoensaio , Receptor ErbB-2/isolamento & purificação , Anticorpos Imobilizados/imunologia , Carbono/química , Feminino , Ouro , Humanos , Nanopartículas Metálicas/química , Receptor ErbB-2/sangueRESUMO
SUMO-1 (1-97) is a crucial protein in the machinery of post-translational modifications. We observed by circular dichroism and fluorescence spectroscopy that urea-induced unfolding of this protein is a complex process with the possibility of occurrence of detectable intermediates along the way. The tertiary structure is completely lost around approximately 4.5 M urea with a transition mid-point at 2.53 M urea, while the secondary structure unfolding seems to show two transitions, with mid-points at 2.42 M and 5.69 M urea. We have elucidated by systematic urea titration, the equilibrium residue level structural and dynamics changes along the entire folding/unfolding transition by multidimensional NMR. With urea dilution, the protein is seen to progressively lose most of the broad beta-domain structural preferences present at 8 M urea, acquire some helical propensities at 5 M urea, and lose some of them again on further dilution of urea. Between 3 M and 2 M urea, the protein starts afresh to acquire native structural features. These observations are contrary to the conventional notion that proteins fold with monotonously increasing native-type preferences. For folding below approximately 3 M urea, the region around the alpha1 helix appears to be a potential folding initiation site. The folding seems to start with a collapse into native-like topologies, at least in parts, and is followed by formation of secondary and tertiary structure, perhaps by cooperative rearrangements. The motional characteristics of the protein show sequence-dependent variation as the concentration of urea is progressively reduced. At the sub-nanosecond level, the features are extremely unusual for denatured states, and only certain segments corresponding to the flexible regions in the native protein display these motions at the different concentrations of urea.
Assuntos
Aminoácidos/metabolismo , Dobramento de Proteína , Proteína SUMO-1/química , Proteína SUMO-1/metabolismo , Ureia , Aminoácidos/química , Humanos , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Desnaturação ProteicaRESUMO
Pharmacophore mapping is one of the major elements of drug design in the absence of structural data of the target receptor. The tool initially applied to discovery of lead molecules now extends to lead optimization. Pharmacophores can be used as queries for retrieving potential leads from structural databases (lead discovery), for designing molecules with specific desired attributes (lead optimization), and for assessing similarity and diversity of molecules using pharmacophore fingerprints. It can also be used to align molecules based on the 3D arrangement of chemical features or to develop predictive 3D QSAR models. This review begins with a brief historical overview of the pharmacophore evolution followed by a coverage of the developments in methodologies for pharmacophore identification over the period from inception of the pharmacophore concept to recent developments of the more sophisticated tools such as Catalyst, GASP, and DISCO. In addition, we present some very recent successes of the widely used pharmacophore generation methods in drug discovery.