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ETHNOPHARMACOLOGICAL RELEVANCE: Abutilon indicum, a shrub of the Malvaceae family, is found abundantly in tropical countries like India. A. indicum is widely used for its high medicinal properties. Traditionally, A. indicum seed powder is consumed to treat piles, constipation, chronic cystitis, gonorrhea, gleet, and pregnancy-related problems. Despite having numerous medicinal properties and widespread traditional use of A. indicum seeds, scientific validation, and toxicity studies have yet to be documented. AIMS OF THE STUDY: The primary objective of this study is to conduct a comprehensive study on phytochemical profiling, in-vitro cytotoxicity, mutagenicity, and in-vivo acute and sub-acute toxicity, and genotoxicity on animal models of methanolic extract of A. indicum seed (MAS). MATERIALS AND METHODS: The qualitative analysis of MAS was explored through FTIR and HR LC-MS. For in-vitro cytotoxicity, the HEK-293 cell line was used, and the TA100 (Staphylococcus typhimurium) bacterial strain was used for the Ames mutagenicity test. A single oral dose of 250, 500, 1000, or 2000 mg/kg body weight of MAS was given to each male and female rat for acute toxicity study and observed for 14 days for any toxicity signs. In the sub-acute toxicity study, 250, 500, or 1000 mg/kg body weight of MAS was administered orally to each rat for 28 days. The experimental animals were weighed weekly, and general behavior was monitored regularly. After 28 days of the experiment, the rats were sacrificed, and different serum biochemical, hematological, and histological analyses were performed. The blood samples of different doses of MAS were used for genotoxicity study through comet assay. RESULTS: FTIR analysis found different functional groups, which indicated the presence of phenolics, flavonoids, and alkaloids. HR LC-MS analysis depicts several components with different biological functions. The cell cytotoxicity and Ames mutagenicity results showed minimal toxicity and mutagenicity up to a certain dose. The acute toxicity study conducted in Wistar albino rats demonstrated zero mortality among the animals, and the LD50 value for seed extract was determined to be 2000 mg/kg body weight. Sub-acute toxicity assessments indicated that the administration of seed extract resulted in no adverse effects at dosages of 250 and 500 mg/kg body weight. However, at higher doses, specifically 1000 mg/kg body weight, the liver of the experimental rats exhibited some toxic effects. In the genotoxicity study, minimal DNA damage was found in 250 and 500 mg/kg doses, respectively, but slightly greater DNA damage was found in 1000 mg/kg doses in both male and female rats. CONCLUSIONS: The consumption of A. indicum seed powder is deemed safe; however, doses exceeding 500 mg/kg body weight may raise concerns regarding use. These findings pave the path for the creation of innovative medicines with improved efficacy and safety profiles.
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Cisplatin, a powerful chemotherapy medication, has long been a cornerstone in the fight against cancer due to chemotherapeutic failure. The mechanism of cisplatin resistance/failure is a multifaceted and complex issue that consists mainly of apoptosis inhibition through autophagy sensitization. Currently, researchers are exploring ways to regulate autophagy in order to tip the balance in favor of effective chemotherapy. Based on this notion, the current study primarily identifies the differentially expressed genes (DEGs) in cisplatin-treated autophagic ACHN cells through the Illumina Hi-seq platform. A protein-protein interaction network was constructed using the STRING database and KEGG. GO classifiers were implicated to identify genes and their participating biological pathways. ClueGO, David, and MCODE detected ontological enrichment and sub-networking. The network topology was further examined using 12 different algorithms to identify top-ranked hub genes through the Cytoscape plugin Cytohubba to identify potential targets, which established profound drug efficacy under an autophagic environment. Considerable upregulation of genes related to autophagy and apoptosis suggests that autophagy boosts cisplatin efficacy in malignant ACHN cells with minimal harm to normal HEK-293 growth. Furthermore, the determination of cellular viability and apoptosis by AnnexinV/FITC-PI assay corroborates with in silico data, indicating the reliability of the bioinformatics method followed by qRT-PCR. Altogether, our data provide a clear molecular insight into drug efficacy under starved conditions to improve chemotherapy and will likely prompt more clinical trials on this aspect.
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Carcinoma de Células Renales , Neoplasias Renales , Humanos , Cisplatino , Redes Reguladoras de Genes , Perfilación de la Expresión Génica/métodos , Células HEK293 , Reproducibilidad de los Resultados , AutofagiaRESUMEN
The current research focused on the green synthesis of silver nanoparticles (AgNPs) using Duabanga grandiflora leaf extract. The green synthesis of AgNPs was confirmed by the surface plasmon resonance band at 453 nm in a UV-Visible analysis. The formulated AgNPs had a diameter of around 99.72 nm with a spherical shape. Fourier transform infrared (FTIR) spectrum revealed the bio-reducing potential of phytochemicals present in D. grandiflora, which fundamentally influenced the synthesis of AgNPs. Zeta potential, dynamic light scattering (DLS), scanning electron microscopic (SEM), energy-dispersive X-ray spectroscopic (EDX), X-ray diffraction (XRD), and transmission electron microscopic (TEM) analyses were executed to reveal the physicochemical attributes of the AgNPs. The AgNPs were further investigated for their antioxidant, antidiabetic, anticancer, and antibacterial potential. The DPPH free radical assay revealed the potential radical scavenging capacity (IC50 = 76.73 µg/ml) of green synthesized AgNPs. α-Amylase inhibitory assay displayed significant inhibitory potential (IC50 = 162.11 µg/ml) of this starch-breaking enzyme by AgNPs, revealing the antidiabetic potential of AgNPs. AgNPs exhibited potential cytotoxic activity (IC50 = 244.57 µg/ml) against malignant human kidney cells. In addition, AgNPs showed outstanding antibacterial activity against both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacterial strains. Interestingly, AgNPs showed cytotoxic and antimicrobial activities at much higher concentrations than radical scavenging and α-amylase inhibitory concentrations. Thus, our finding elaborated the scope of green synthesized AgNPs for diverse therapeutic applications (dose-dependent) for further clinical translation.
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Antibacterianos , Nanopartículas del Metal , Extractos Vegetales , Hojas de la Planta , Plata , Nanopartículas del Metal/química , Plata/química , Plata/farmacología , Extractos Vegetales/química , Extractos Vegetales/farmacología , Hojas de la Planta/química , Humanos , Antibacterianos/farmacología , Antibacterianos/química , Antioxidantes/farmacología , Antioxidantes/química , Tecnología Química Verde , Espectroscopía Infrarroja por Transformada de Fourier , Difracción de Rayos XRESUMEN
In quest of developing an efficient and effective drug against the ACHN human renal adenocarcinoma cell line herein, we report the synthesis and characterization of a novel Pyridinium iodide-tagged Schiff base (5) and its Cu (II)/Zn (II)/Cd (II)-complexes (6). The synthesized compounds are well characterized by Elemental analysis, UV-Visible, FTIR, Magnetic Susceptibility, NMR, HRMS, MALDI, and PXRD techniques. They were then subsequently tested on the ACHN cell lines using MTT assays and their IC50 values were determined, followed by their ROS production capacity. Among the tested compounds Zn (II)-complex 6(b) was found to be the most potent one with a minimum IC50 value while the ligand (5) was the least.
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Silver nanoparticles (Ag NPs) via green synthesis using medicinal plants have been widely used in natural product research due to the economical and eco-friendly properties of NPs. The plant-derived Ag NPs biosynthesis comprises the interaction between silver nitrate (precursor) and bioactive components of plant extract (reducing agents). In this work, Ag NPs were biosynthesized using Osbeckia stellata leaves aqueous extract. Characterization of Ag NPs was done by using ultraviolet-visible absorption (UV-Vis) spectroscopy, dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy-dispersive X-ray analysis (EDX). Further, antioxidant, antidiabetic, cytotoxicity, and antimicrobial activities were evaluated to establish the pharmacological properties of Ag NPs. UV-Vis spectroscopy and FTIR showed an absorption peak of Ag NPs due to the surface plasmonic resonance. In contrast, the particle size in the nanometer range was analyzed by XRD and DLS. The size of the particle was confirmed by the SEM, TEM, and EDX in the nanometer range. This study showed the spherical shape and crystalline nature of NPs. Zeta potential was used to determine the stability of Ag NPs. Biosynthesized Ag NPs showed significantly potent antioxidant, antidiabetic, and cytotoxicity activity. Ag NPs also showed effectiveness against gram-positive (Escherichia coli) and gram-negative (Staphylococcus aureus) bacteria in the antimicrobial activity study. The result concluded that these Ag NPs might be used in biomedical and pharmacological fields.
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Antioxidantes , Nanopartículas del Metal , Antioxidantes/farmacología , Antioxidantes/química , Hipoglucemiantes/farmacología , Nanopartículas del Metal/química , Pruebas de Sensibilidad Microbiana , Plata/farmacología , Antibacterianos/farmacología , Antibacterianos/química , Espectroscopía Infrarroja por Transformada de Fourier , Extractos Vegetales/farmacología , Extractos Vegetales/químicaRESUMEN
The amyloid ß peptide aggregates to form extracellular plaques in the brains of Alzheimer's disease patients. Certain of its fragments have been found to have similar properties to those of the full-length peptide. The best-studied of these is 25-35, which aggregates into fibrils, is toxic to neurons, and forms ion channels in synthetic lipid bilayers. Here, we investigate possible pore-forming structures of oligomers of this peptide in a POPC/POPG membrane. We consider octameric and decameric ß-barrels of different topology, strand orientation, and shear, evaluate their stability in an implicit membrane model, and subject the best models to multimicrosecond all-atom molecular dynamics simulations. We find two decameric structures that are kinetically stable in membranes on this time scale: an imperfectly closed antiparallel ß-barrel with K28 in the pore lumen and a short parallel ß-barrel with K28 toward the membrane interface. Both structures exhibit dehydrated gaps in the pore lumen, which are larger for the antiparallel barrel. Based on these results, the experimental cation selectivity, the dependence of ion channel activity on voltage direction, and certain mutation data, the parallel model seems more compatible with experimental data.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Humanos , Péptidos beta-Amiloides/química , Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Amiloide/químicaRESUMEN
The toxicity of any drug against normal cells is a health hazard for all humans. At present, health and disease researchers from all over the world are trying to synthesize designer drugs with diminished toxicity and side effects. The purpose of the present study is to enhance the bioavailability and biocompatibility of gemcitabine (GEM) by decreasing its toxicity and reducing deamination during drug delivery by incorporating it inside the hydrophobic cavity of ß-cyclodextrin (ß-CD) without affecting the drug ability of the parent compound (GEM). The newly synthesized inclusion complex (IC) was characterized by different physical and spectroscopic techniques, thereby confirming the successful incorporation of the GEM molecule into the nanocage of ß-CD. The molecular docking study revealed the orientation of the GEM molecule into the ß-CD cavity (-5.40 kcal/mol) to be stably posed for ligand binding. Photostability studies confirmed that the inclusion of GEM using ß-CD could lead to better stabilization of GEM (≥96%) for further optical and clinical applications. IC (GEM-ß-CD) and GEM exhibited effective antibacterial and antiproliferative activities without being metabolized in a dose-dependent manner. The CT-DNA analysis showed sufficiently strong IC (GEM-ß-CD) binding (Ka = 8.1575 × 1010), and this interaction suggests that IC (GEM-ß-CD) may possibly exert its biological effects by targeting nucleic acids in the host cell. The newly synthesized biologically active IC (GEM-ß-CD), a derivative of GEM, has pharmaceutical development potentiality.
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BACKGROUND: Our aim was to assess the association of water, sanitation and hygiene (WASH) and food practices with culture-confirmed enteric fever in children <15 years of age. METHODS: We followed a cohort of 6000 children from an urban low socioeconomic neighbourhood in South Delhi for 2 years to estimate burden of culture-confirmed enteric fever. Risk ratios (RRs) were estimated to study the association between WASH practices and enteric fever. We assessed the microbiological quality of drinking water and conducted geospatial analysis to evaluate the distribution of enteric fever cases around households with contaminated drinking water. RESULTS: A total of 5916 children in 3123 households completed survey. Piped water (82%) was the major source of household drinking water. One-third (32%) of the households treated water before consumption. Almost all households had sanitary toilets (99.9%) and 16% used shared toilets. Consumption of food from street vendors and unnamed ice creams more than once a week was observed in children from 12.7% and 38.4% households, respectively. Eighty culture-confirmed enteric fever cases were reported. The risk of enteric fever was 71% higher in children belonging to households having food from outside once a week or more (RR 1.71, 95% CI 1.00 to 2.94). The RR for enteric fever in children living in households with availability of safe drinking water was 0.75 (95% CI 0.45 to 1.26). We found that 14.8% of the households had presence of coliforms or Escherichia coli in their household drinking water. The odds of having a case of enteric fever within a 5 and 25 m buffer zone around households with contaminated drinking water were 4.07 (95% CI 0.81 to 20.5) and 1.44 (95% CI 0.69 to 3.00), respectively. CONCLUSION: In addition to WASH practices, optimal food hygiene may have a role in urban low socioeconomic population to control enteric fever. TRIAL REGISTRATION NUMBER: CTRI/2017/09/009719.
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Agua Potable , Fiebre Tifoidea , Niño , Agua Potable/análisis , Escherichia coli , Humanos , Higiene , India/epidemiología , SaneamientoRESUMEN
Agro-researchers are endlessly trying to derive a potential biomolecule having antifungal properties in order to replace the application of synthetic fungicides on agricultural fields. Rot disease often caused by Fusarium solani made severe loss of wheat crops every year. Chitosan and its metallic nano-derivatives hold a broad-spectrum antifungal property. Our interdisciplinary study deals with the application of nickel chitosan nanoconjugate (NiCNC) against Fusarium rot of wheat, in comparison with chitosan nanoparticles (CNPs) and commercial fungicide Mancozeb. CNPs and NiCNC were characterized on the basis of UV-Vis spectrophotometry, HR-TEM, FESEM, EDXS and FT-IR. Both CNPs and NiCNC were found effective against the fungal growth, of which NiCNC at 0.04 mg/mL showed complete termination of F. solani grown in suitable medium. Ultrastructural analysis of F. solani conidia treated with NiCNC revealed pronounced damages and disruption of the membrane surface. Fluorescence microscopic study revealed generation of oxidative stress in the fungal system upon NiCNC exposure. Moreover, NiCNC showed reduction in rot disease incidence by 83.33% of wheat seedlings which was further confirmed through the observation of anatomical sections of the stem. NiCNC application helps the seedling to overcome the adverse effect of pathogen, which was evaluated through stress indices attributes.
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Quitosano , Fungicidas Industriales , Fusarium , Antifúngicos/química , Antifúngicos/farmacología , Quitosano/química , Quitosano/farmacología , Fungicidas Industriales/farmacología , Nanoconjugados , Níquel/farmacología , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/prevención & control , Plantones , Espectroscopía Infrarroja por Transformada de Fourier , TriticumRESUMEN
The assembly of an inclusion complex in an aqueous medium using a metabolizer drug (dyphylline) as guest and ß-cyclodextrin as host has been established, which is extremely appropriate for a variety of applications in modern biomedical sciences. The formation of the inclusion complex is established by 1H NMR, and surface tension and conductivity measurements demonstrate that the inclusion complex was produced with 1:1 stoichiometry. The thermodynamic parameters based on density, viscosity, and refractive index measurements were used to determine the nature of the complex. This research also forecasts how dyphylline will release in the presence of CT-DNA without any chemical modifications. The produced insertion complex (IC) has a higher photostability due to the drug dyphylline being protected by ß-CD. The antibacterial activity of dyphylline greatly improved after complexation and exhibited higher toxicity against Gram-negative (highest against Escherichia coli) in comparison to Gram-positive bacteria. The encapsulation mode of the dyphylline molecule into the cavity of the ß-CD was also investigated using DFT to confirm preliminary results.
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[This corrects the article DOI: 10.1021/acsomega.2c00011.].
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Rice plants are known to be more susceptible to arsenic (As) contamination during the cultivation process. Arsenic is genotoxic and can be a big threat to the rice eating people at large. Studies on an effective mitigation mechanism are the need of the hour. This work was an approach using iron (Fe3+) to form Fe-plaque in the plant root that could trap As. The present research was designed with several experimental set ups for rice cultivation in pot culture using different Fe doses with fertilizer in the soil, and finally, the optimum dose was selected considering the translocation ability, plant health, and molecular and stress biomarkers. The study revealed that on an increase in Fe dose, translocation factor (TF) and stress marker (malondialdehyde content) of the plant decreased gradually and encountered minimum (0.12 and 0.03 mg/kg, respectively) at the dose of 4.5gm/kg. In contrast, higher values of chlorophyll (2.5 mg/kg) and carbohydrate (2.2 mg/kg) and intact DNA content were recorded highlighting the rich health condition of the plant. Thus, the experiment supported well the fact that the dose of Fe as fortified fertilizer can be considered the most effective in reducing soil arsenic accumulation in the rice plants. This approach might save the rice eating people from harmful effects of As contamination in this region of India.
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Arsénico , Oryza , Contaminantes del Suelo , Arsénico/farmacología , Fertilizantes , Humanos , Raíces de Plantas/química , SueloRESUMEN
BACKGROUND: Our aim was to estimate the overall and age-specific incidence of lab-confirmed dengue fever using ELISA based assays among children 6 months to 15 years in Delhi. METHODS: We enrolled a cohort of 984 children aged 6 months to <14 years in South Delhi and followed-up weekly for fever for 24 months or till 15 completed years of child-age. Households of the enrolled children were geo-tagged. NS1, IgM and IgG assays were conducted using ELISA method to confirm dengue fever in children with ≥3 consecutive days of fever. Molecular typing was done in a subset of NS1 positive cases to identify the circulating serotypes. PRINCIPAL FINDINGS: We had a total of 1953 person-years (PY) of follow up. Overall, there were 4208 episodes of fever with peaks during June to November. The overall incidence (95%CI) of fever was 215/100 PY (209 to 222). A total of 74/1250 3-day fever episodes were positive for acute dengue fever (NS1 and/or IgM positive). The overall incidence (95%CI) of acute dengue fever was 37.9 (29.8 to 47.6) per 1000 PY; highest among children aged 5 to 10 years (50.4 per 1000 PY, 95% CI 36.5 to 67.8). Spatial autocorrelation analysis suggested a clustering pattern for the dengue fever cases (Moran's Index 0.35, z-score 1.8, p = 0.06). Dengue PCR was positive in 16 of the 24 specimens tested; DEN 3 was the predominant serotype identified in 15/24 specimens. CONCLUSIONS: We found a high incidence of dengue fever among under 15-year children with clustering of cases in the community. DEN 3 was the most commonly circulating strain encountered. The findings underscore the need for development of affordable pre-vaccination screening strategy as well as newer dengue vaccines for young children while continuing efforts in vector control.
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Dengue , Anticuerpos Antivirales , Niño , Preescolar , Estudios de Cohortes , Dengue/diagnóstico , Dengue/epidemiología , Fiebre/epidemiología , Humanos , Inmunoglobulina M , Incidencia , India/epidemiología , SerogrupoRESUMEN
Our present study intended to investigate the encapsulation of DL-AGT within the lipophilic cavity of a ß-CD molecule. The consequential inclusion system was characterized by UV-visible spectroscopy and 1H NMR, PXRD, SEM, and FT-IR studies. Molecular docking was performed for the inclusion complex to discover the most proper orientation, and it was seen that the drug DL-AGT fits into the cavity of ß-CD in a 1:1 ratio, which was also confirmed from the Job plot. Furthermore, a comparison was done on the basis of cell viability between the drug and its inclusion complex.
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BACKGROUND: Cancer remains a major world health issue due to its high morbidity and mortality rate. Plant based natural products (NPs) have played vital role in discovery of valuable anti-cancer drugs. Darjeeling Himalayan region has a rich diversity of therapeutic plants that can be utilized for development of novel drugs. AIM: We previously reported cytotoxic potential of rhizome extract of A.rivularis, a Darjeeling himalayan herb. Present study reports isolation and characterization of a phytosteroid from the plant rhizome in a bioassay-guided approach and evaluation of its anti-tumorigenic potential. RESULTS: The phytosteroid was characterized as stigmasta-5(6), 22(23)-dien-3-beta-yl acetate (A11) by various spectrometric techniques (IR, NMR, MS etc.). The catalytic inhibition and structural alteration of human dihydrofolate reductase (hDHFR) by A11 was evaluated using methotrexate (MTX), a DHFR inhibitor anticancer drug as a reference. A11 inhibited hDHFR activity with IC50 values of 1.20 µM A11 caused concentration dependent quenching of tryptophan fluorescence of hDHFR suggesting its effect on alteration of enzyme structure. Molecular docking of A11 on crystal structure of hDHFR revealed significant interaction with free energy of binding and Ki values of -10.86 kcal/mol and 11 nM, respectively. Subsequent in vitro studies at cellular level showed a relatively greater cytotoxic effect of A11 against human kidney (ACHN, IC50 60 µM) and liver (HepG2, IC5070 µM) cancer cells than their respective normal cells (HEK-293, IC50 350 µM and WRL-68, IC50 520 µM). Scanning electron microscopy of A11 treated cells revealed the morphological feature of apoptosis, like cell rounding and surface detachment, membrane blebbing, loss of cilia and increased number of pores of decreased sizes. A11 mediated apoptosis of cancer cells was found to be correlated with induction of intracellular of reactive oxygen species (ROS) level and fragmentation of genomic DNA.
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Antineoplásicos , Fitosteroles , Saxifragaceae , Acetatos/farmacología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Ésteres , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Saxifragaceae/metabolismo , Esteroides , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
The myth of inactivity of inorganic materials in a biological system breaks down by the discovery of nanozymes. From this time, the nanozyme has attracted huge attention for its high durability, cost-effective production, and easy storage over the natural enzyme. Moreover, the multienzyme-mimicking activity of nanozymes can regulate the level of reactive oxygen species (ROS) in an intercellular system. ROS can be generated by peroxidase (POD), oxidase (OD), and Fenton-like catalytic reaction by a nanozyme which kills the cancer cells by oxidative stress; therefore, it is important in CDT (chemo dynamic therapy). Our current study designed to investigate the enzyme mimicking behavior and anticancer ability of cerium-based nanomaterials because the cerium-based materials offer a high redox ability while maintaining nontoxicity and high stability. Our group synthesized CeZrO4 nanoparticles by a green method using ß-cyclodextrin as a stabilizer and neem leaf extract as a reducing agent, exhibiting POD- and OD-like dual enzyme activities. The best enzyme catalytic activity is shown in pH = 4, indicating the high ROS generation in an acidic medium (tumor microenvironment) which is also supported by the Fenton-like behavior of CeZrO4 nanoparticles. Inspired by the high ROS generation in vitro method, we investigated the disruption of human kidney cells by this nanoparticle, successfully verified by the MTT assay. The harmful effect of ROS in a normal cell is also investigated by the in vitro MTT assay. The results suggested that the appreciable anticancer activity with minimal side effects by this synthesized nanomaterial.
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Collective cell migration (CCM), in which cell-cell integrity remains preserved during movement, plays an important role in the progression of cancer. However, studies describing CCM in cancer progression are majorly focused on the effects of extracellular tissue components on moving cell plasticity. The molecular and cellular mechanisms of CCM during cancer progression remain poorly explored. Here, we report that proteolipid protein 2 (PLP2), a colonic epithelium-enriched transmembrane protein, plays a vital role in the CCM of invasive human colorectal cancer (CRC) epithelium by modulating leading-edge cell dynamics in 2D. The extracellular pool of PLP2, secreted via exosomes, was also found to contribute to the event. During CCM, the protein was found to exist in association with ZO-1 (also known as TJP1) and to be involved in the positioning of the latter at the migrating edge. PLP2-mediated positioning of ZO-1 at the leading edge further alters actin cytoskeletal organization that involves Rac1 activation. Taken together, our findings demonstrate that PLP2, via its association with ZO-1, drives CCM in CRC epithelium by modulating the leading-edge actin cytoskeleton, thereby opening up new avenues of cancer research. This article has an associated First Person interview with the first author of the paper.
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Neoplasias Colorrectales , Exosomas , Movimiento Celular , Neoplasias Colorrectales/genética , Citoesqueleto , Humanos , Proteínas con Dominio MARVEL , Proteínas de la Membrana/genética , ProteolípidosRESUMEN
The binuclear metalloenzyme Helicobacter pylori arginase is important for pathogenesis of the bacterium in the human stomach. Despite conservation of the catalytic residues, this single Trp enzyme has an insertion sequence (-153ESEEKAWQKLCSL165-) that is extremely crucial to function. This sequence contains the critical residues, which are conserved in the homolog of other Helicobacter gastric pathogens. However, the underlying basis for the role of this motif in catalytic function is not completely understood. Here, we used biochemical, biophysical and molecular dynamics simulations studies to determine that Glu155 of this stretch interacts with both Lys57 and Ser152. These interactions are essential for positioning of the motif through Trp159, which is located near Glu155 (His122-Trp159-Tyr125 contact is essential to tertiary structural integrity). The individual or double mutation of Lys57 and Ser152 to Ala considerably reduces catalytic activity with Lys57 to Ala being more significant, indicating they are crucial to function. Our data suggest that the Lys57-Glu155-Ser152 interaction influences the positioning of the loop containing the catalytic His133 so that this His can participate in catalysis, thereby providing a mechanistic understanding into the role of this motif in catalytic function. Lys57 was also found only in the arginases of other Helicobacter gastric pathogens. Based on the non-conserved motif, we found a new molecule, which specifically inhibits this enzyme. Thus, the present study not only provides a molecular basis into the role of this motif in function, but also offers an opportunity for the design of inhibitors with greater efficacy.
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Arginasa/química , Proteínas Bacterianas/química , Helicobacter pylori/enzimología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Aminoácidos/química , Animales , Arginasa/antagonistas & inhibidores , Arginasa/genética , Arginina/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Catálisis , Cobalto/metabolismo , Secuencia Conservada , Polarización de Fluorescencia , Gastritis/microbiología , Gastritis/veterinaria , Helicobacter/enzimología , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/veterinaria , Helicobacter pylori/genética , Humanos , Hidrólisis , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mutación Missense , Mutación Puntual , Estructura Secundaria de Proteína , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
We report two cases of co-infection with Salmonella Typhi and Salmonella Paratyphi A identified by blood culture and confirmed by serotyping from an ongoing fever surveillance cohort in an urban slum in New Delhi. Co-infections such as these have important implications on diagnosis, treatment options including choice of antimicrobial(s), disease outcome and strategy for prevention.
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Arginase is a bimetallic enzyme that utilizes mainly Mn2+ or Co2+ for catalytic function. In human homolog, the substitution of Mn2+ with Co2+ significantly reduces the Km value without affecting the kcat. However, in the Helicobacter pylori counterpart (important for pathogenesis), the kcat increases nearly 4-fold with Co2+ ions both in the recombinant holoenzyme and arginase isolated from H. pylori grown with Co2+ or Mn2+. This suggests that the active site of arginase in the two homologs is modulated differently by these two metal ions. To investigate the underlying mechanism for metal-induced difference in catalytic activity in the H. pylori enzyme, we used biochemical, biophysical and microsecond molecular dynamics simulations studies. The study shows that the difference in binding affinity of Co2+ and Mn2+ ions with the protein is linked to a different positioning of a loop (-122HTAYDSDSKHIHG134-) that contains a conserved catalytic His133. Consequently, the proximity of His133 and conserved Glu281 is varied. We found that the Glu281-His133 interaction is crucial for catalytic function and was previously unexplored in other homologs. We suggest that the proximity difference between these two residues in the Co2+- and Mn2+-proteins alters the proportion of protonated His133 via variation in its pKa. This affects the efficiency of proton transfer - an essential step of l-arginine hydrolysis reaction catalyzed by arginase and thus activity. Unlike in human arginase, the flexibility of the above segment observed in H. pylori homolog suggests that this region in the H. pylori enzyme may be explored to design its specific inhibitors.