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Accurately identifying and quantifying toxicants is crucial for medico-legal investigations in forensic toxicology; however, low analyte concentrations and the complex samples matrix make this work difficult. Therefore, a simplified sample preparation procedure is crucial to streamline the analysis to minimize sample handling errors, reduce cost and improve the overall efficiency of analysis of toxicants. To address these challenges, an innovative disposable in-tip cellulose paper (DICP) device has been developed for the extraction of three pesticides viz. Chlorpyrifos, Quinalphos and Carbofuran from postmortem blood samples. The DICP device leverages cellulose paper strips housed within a pipette tip to streamline the extraction process, significantly reducing solvent usage, time, and labor while maintaining high analytical accuracy. The extraction of pesticides from postmortem blood using the DICP device involves a streamlined process characterized by adsorption and desorption. The diluted blood samples were processed through the DICP device via repeated aspirating and dispensing calyces to adsorb the pesticides onto the cellulose paper. The adsorbed pesticides are then eluted using acetone, which is collected for GC-MS analysis. The method was meticulously optimized, achieving a limit of quantification in the range of 0.009-0.01 µg mL-1. The intra-day and inter-day precisions were consistently less than 5 % and 10 %, respectively, with accuracy ranging from 94-106 %. Relative recoveries for the analytes were observed to be between 60 % and 93.3 %, and matrix effects were determined to be less than 10 %. The method's sustainability was validated with a whiteness score of 98.8, an AGREE score of 0.64, a BAGI score of 70 and ComplexMoGAPI score of 77. Applicability was demonstrated through successful analysis of real postmortem blood samples and proficiency testing samples, highlighting its potential utility in forensic toxicology.
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Carnosine is beta-alanyl histidine, a dipeptide, endogenously produced in our body by the carnosine synthase enzyme. It is an antioxidant, thus protecting from the deleterious effect of advanced glycation end products (AGEs). Similarly, aminoguanidine (AG) also prevents AGEs formation by scavenging free radicals such as reactive oxygen species (ROS)/reactive carbonyl species (RCS). This study used experimental and computational techniques to perform a comparative analysis of carnosine and AG and their inhibiting properties against glycated human serum albumin (HSA). Fructose-mediated glycation of albumin produced fluorescent structures, such as pentosidine and malondialdehyde. These AGEs were significantly reduced by carnosine and AG. At 20 mM, carnosine and AG quenches pentosidine fluorescence by 66% and 83%, respectively. A similar inhibitory effect was observed for malondialdehyde. Protein hydrophobicity and tryptophan fluorescence were restored in the presence of carnosine and AG. Aminoguanidine decreased fibrillation in HSA, while carnosine did not significantly affect aggregation/fibrillation. In addition, molecular docking study observed binding scores of -5.90 kcal/mol and -2.59 kcal/mol by HSA-aminoguanidine and HSA-carnosine complex, respectively. Aminoguanidine forms one conventional hydrogen bond with ARG A:10 and a salt bridge with ASP A:13, ASP A:259, ASP A:255, and ASP A:256 from the amine group. Similarly, carnosine forms only hydrogen bonds with GLU A:501 and GLN A:508 from the amine and hydroxy group. The root mean square deviation (RMSD) calculated from simulation studies was 1 nm upto 70 ns for the HSA-aminoguanidine complex and the spectrum of HSA-carnosine was significantly deviated and not stabilized. The superior inhibitory activity of aminoguanidine could be due to additional salt bridge bonding with albumin. Conclusively, aminoguanidine can be the better treatment choice for diabetes-associated neurological diseases.
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Recent research has shown that phytocomponents may be useful in the treatment of renal toxicity. This study was conducted to evaluate the renal disease hirsutidin in the paradigm of renal toxicity induced by cisplatin. Male Wistar rats were given cisplatin (3 mg/kg body weight/day, for 25 days, i.p.) to induce renal toxicity. Experimental rats were randomly allocated to four different groups: group I received saline, group II received cisplatin, group III received cisplatin + hirsutidin (10 mg/kg) and group IV (per se) received hirsutidin (10 m/kg) for 25 days. Various biochemical parameters were assessed, oxidative stress (superoxide dismutase (SOD), glutathione transferase (GSH), malonaldehyde (MDA) and catalase (CAT)), blood-chemistry parameters (blood urea nitrogen (BUN) and cholesterol), non-protein-nitrogenous components (uric acid, urea, and creatinine), and anti-inflammatory-tumor necrosis factor-α (TNF-α), interleukin-1ß(IL-1ß). IL-6 and nuclear factor-kB (NFκB) were evaluated and histopathology was conducted. Hirsutidin alleviated renal injury which was manifested by significantly diminished uric acid, urea, urine volume, creatinine, and BUN, compared to the cisplatin group. Hirsutidin restored the activities of several antioxidant enzyme parameters-MDA, CAT, GSH, and SOD. Additionally, there was a decline in the levels of inflammatory markers-TNF-α, IL-1ß, IL-6, and NFκB-compared to the cisplatin group. The current research study shows that hirsutidin may act as a therapeutic agent for the treatment of nephrotoxicity induced by cisplatin.
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We evaluated mechanistic insights into luteolin (LUT)-loaded elastic liposomes (OLEL1) permeated across rat skin. HSPiP software-based parameters, thermal analysis, infrared analysis, and morphological evaluations were employed to understand mechanistic observations of drug permeation and deposition. HSPiP provided HSP values (δd, δp, and δh) of OLEL1 (based on composition), LUT, excipients, and rat skin (literature value and by-default value). Rat skin was studied via Fourier transform infrared (FTIR), differential scanning calorimetry (DSC), fluorescence microscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM) studies. The δd and δh estimation of the skin and phosphatidylcholine showed close relation in terms of δd and δh. Similarly, OLEL1 and the skin might interact with each other mainly through δd and δp forces as evidenced by the predicted values. The untreated skin showed characteristic stretching and vibrations as compared to lower frequencies caused by OLEL1. DSC showed changes in the thermal behavior of the skin after OLEL1 treatment as compared to the untreated skin. Visualization of these changes was evident under fluorescence microscopy and SEM for confirmed substantial reversible surface perturbation of the skin protein layer for improved vesicle permeation and subsequent internalization with the inner skin matrix. The AFM study confirmed the nanoscale surface roughness variation caused substantially by OLEL1 and OLEL1 placebo as compared to the untreated control and drug solution. Thus, the study clearly demonstrated mechanistic insights into LUT-loaded vesicles across rat skin for enhanced permeation and drug deposition.
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Alpha-amylase (α-amylase) not long ago has acquire recognition as a possible drug target for the management of diabetes. Here, we have investigated the binding and enzyme activity of α-amylase by hesperidin; a naturally occurring flavanone having wide therapeutic potential. Hesperidin exerted an inhibitory influence on α-amylase activity with an IC50 value of 16.6 µM. Hesperidin shows a significant binding toward α-amylase with a binding constant (Ka) of the order of 104 M-1. The evaluation of thermodynamic parameters (∆H and ∆S) suggested that van der Waals force and hydrogen bonding drive seemingly specific hesperidin-α-amylase complex formation. Glycation and oxidation studies were performed using human serum albumin (HSA) as ideal protein. Hesperidin inhibited fructosamine content ≈40% at 50 µM and inhibited advanced glycation end products (AGEs) formation by 71.2% at the same concentration. Moreover, significant recovery was evident in free -SH groups and carbonyl content of HSA. Additionally, molecular docking also entrenched in vitro observations and provided an insight into the important residues (Trp58, Gln63, His101, Glu233, Asp300, and His305) at the heart of hesperidin-α-amylase interaction. This study delineates mechanistic insight of hesperidin-α-amylase interaction and provides a platform for use of hesperidin to treat AGEs directed diseases.
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Productos Finales de Glicación AvanzadaRESUMEN
BACKGROUND: Sinapic acid (SA) has been shown to have various pharmacological properties such as antioxidant, antifibrotic, anti-inflammatory, and anticancer activities. Its mechanism of action is dependent upon its ability to curb free radical production and protect against oxidative stress-induced tissue injuries. AIM: To study the hepatoprotective effects of SA against lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced acute liver failure (ALF) in rats. METHODS: Experimental ALF was induced with an intraperitoneal (i.p.) administration of 8 µg LPS and 800 mg/kg D-GalN in normal saline. SA was administered orally once daily starting 7 d before LPS/D-GalN treatment. RESULTS: Data showed that SA ameliorates acute liver dysfunction, decreases serum levels of alanine transaminase (ALT), and aspartate aminotransferase (AST), as well as malondialdehyde (MDA) and NO levels in ALF model rats. However, pretreatment with SA (20 mg/kg and 40 mg/kg) reduced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and levels of inflammatory cytokines (tumor necrosis factor-α and interleukin 6). Also, SA increased the activity of the nuclear factor erythroid-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling pathway. CONCLUSION: In conclusion, SA offers significant protection against LPS/D-GalN-induced ALF in rats by upregulating Nrf2/HO-1 and downregulating NF-κB.
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Enfermedad Hepática Inducida por Sustancias y Drogas , Necrosis Hepática Masiva , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Ácidos Cumáricos , Factor de Transcripción GATA1 , Galactosamina/toxicidad , Hemo Oxigenasa (Desciclizante) , Hemo-Oxigenasa 1 , Lipopolisacáridos/toxicidad , Hígado , FN-kappa B , Ratas , Factor de Necrosis Tumoral alfaRESUMEN
Copper compounds are promising candidates for next-generation metal anticancer drugs. Therefore, we synthesized and characterized a formate bridged 1D coordination polymer [Cu(L)(HCOO)2]n, (L = 2-methoxy-6-methyl-3-((quinolin-8-ylimino)methyl)chroman-4-ol), PCU1, wherein the Cu(ii) center adopts a square pyramidal coordination environment with adjacent CuCu distances of 5.28 Å. Primarily, in vitro DNA interaction studies revealed a metallopolymer which possesses high DNA binding propensity and cleaves DNA via the oxidative pathway. We further analysed its potential on cancerous cells MCF-7, HeLa, A549, and two non-tumorigenic cells HEK293 and HBE. The selective cytotoxicity potential of PCU1 against A549 cells driven us to examine the mechanistic pathways comprehensively by carrying out various assays viz, cell cycle arrest, Annexin V-FTIC/PI assay, autophagy, intercellular localization, mitochondrial membrane potential 'MMP', antiproliferative assay, and gene expression of TGF-ß and MMP-2.
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Adenocarcinoma del Pulmón/tratamiento farmacológico , Antineoplásicos , Cromonas , Complejos de Coordinación , Cobre , Neoplasias Pulmonares/tratamiento farmacológico , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cromonas/química , Cromonas/farmacología , Complejos de Coordinación/química , Complejos de Coordinación/farmacología , Cobre/química , Cobre/farmacología , ADN/química , División del ADN , Humanos , Metaloproteinasa 2 de la Matriz/genética , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Stimulation of complex chaperone activity may be a viable means of therapy for neurodegenerative diseases. These chaperons execute reactivation of thermally and chemically aggregated protein substrates by cooperating with their partner co-chaperons. We optimized the expression and purification conditions of Tid1-L chaperone. Expression of Tid1-L in E. coli resulted in the formation of inclusion bodies which was further purified to soluble active form using 8 M urea and Ni-NTA column. Also, we investigated the events of the reactivation and disaggregation using aggregated G6PDH, luciferase and insulin as substrates. Incubation of aggregated/denatured enzymes with mortalin but not with Tid1 and/or Mge1 resulted in the initiation of the disaggregation reaction albeit very insignificantly. Under the same conditions coincubating the samples with chaperon and its assisted partners Tid1-L and nucleotide exchange factor Mge1 led to (40%) increase in enzyme activity of G6PDH. Similarly, luciferase activity was synergistically enhanced in the presence of mortlain/Tid1-L/Mge1 chaperones machinery. Chaperone-dependent disaggregation of thermally aggregated insulin showed that addition of Hsp70 and Hsp40 chaperones resulted in fast-track of renaissance reaction and inhibition of amyloid. The present study results conclude the quality of cell-control involves interaction of chaperon Hsp70 and its co-chaperones leading to complex formation with chemically/thermally aggregated substrate eventually causing its reactivation and disaggregation.
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The development of preferentially selective cancer chemotherapeutics is a new trend in drug research. Thus, we designed and synthesized novel ternary complexes, [Cu(tryp)(Hnor)2(DMSO)]NO3 (1) and [Zn(tryp)(Hnor)2(DMSO)]NO3 (2) (tryp = DL-Tryptophane; Hnor = Norharmane, ß-carboline; DMSO = Dimethyl sulfoxide), characterized with elemental analysis, FTIR, UV-vis, FL, NMR, ESI-MS, and molar conductivity. Furthermore, the TD-DFT studies with UV-vis and FTIR validated the proposed structures of 1 and 2. Moreover, we evaluated the HOMO-LUMO energy gap and found that 1 has a smaller energy gap than 2. Then, 1 and 2 were assessed for anticancer chemotherapeutic potential against cancer cell lines MCF7 (human breast cancer) and HepG2 (human liver hepatocellular carcinoma) as well as the non-tumorigenic HEK293 (human embryonic kidney) cells. The MTT assay illustrated the preferentially cytotoxic behavior of 1 when compared with that of 2 and cisplatin (standard drug) against MCF7 cells. Moreover, 1 was exposed to MCF7 cells, and the results indicated the arrest of the G2/M phases, which followed the apoptotic pathway predominantly. Generation of ROS, GSH depletion, and elevation in LPO validated the redox changes prompted by 1. These studies establish the great potential of 1 as a candidate for anticancer therapeutics.
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Isoniazid is the most commonly used drug for treatment of tuberculosis, and is administered individually or in combination with other drugs as standard first line therapy. Offsetting its efficacy, severe adverse effects, especially peripheral neuropathy and hepatotoxicity, are associated with isoniazid therapy, limiting its use in tuberculosis. Isoniazid is acetylated in vivo producing hydrazine and acetyl hydrazine, which are responsible for hepatotoxicity. Marked pharmacogenetic differences in acetylation have been reported among different population across the globe. This study evaluates isoniazid acetylation patterns in tuberculosis patients receiving DOT therapy under the Revised National Tuberculosis Control Program (RNTCP) in a specialized tuberculosis hospital in north India. Of 351 patients from whom samples were taken for biochemical analysis of adverse events, 36 were assessed for acetylation patterns. Blood samples were taken 1 h after administration of a 600 mg dose of isoniazid, and plasma concentrations of isoniazid were determined using a validated HPLC method. Of these 36 patients, 20 (55.56%) were slow acetylators and 16 (44.44%) were fast acetylators. Our results are consistent with those of an earlier study conducted in a different region of India. Most biochemical changes produced during long-term isoniazid therapy resolve after therapy is terminated.
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Rivaroxaban (RIVA) inhibits factor Xa and exhibits antithrombotic and anti-inflammatory activities by inhibiting several cellular signaling molecules. Sunitinib (SUN) is FDA approved first-line drug for metastatic renal cancers and advanced cancerous states of gastrointestinal tract. Present hypothesis was aimed to examine the nephroprotective potential of RIVA in SUN-induced nephrotoxicity, mediated through the inhibition of oxidative stress-induced apoptosis and inflammation, via the TNF-α/NFk-B signaling pathways. Wistar rats 200-250 g were selected and divided randomely in 5 groups (n = 6): Group 1 kept as normal control; Group 2 as disease control and exposed to SUN 50 mg/kg thrice-weekly upto 21 days; Groups 3 and 4, were treatment groups and administered SUN 50 mg/kg thrice-weekly as of group 2 and treated with RIVA 5 and 10 mg/kg/daily for 21 days, respectively; and Group 5 fed with RIVA alone (10 mg/kg/daily for 21 days). Serum was separated from blood to estimate serum biochemical parameters and kidney tissues were collected to estimate antioxidant enzyme, mRNA and protein expression. SUN exposure significantly elevated levels of creatinine, urea, uric acid, blood urea nitrogen, albumin, and bilirubin, and decreased serum magnesium and iron levels. Malondialdehyde and catalase levels were significantly increased and glutathione and glutathione reductase levels were significantly decreased. Intracellular levels of caspase-3 and TNF-α were significantly increased; RIVA treatment restored the altered levels. In SUN-exposed animals, western blotting revealed significantly elevated NFk-B, IL-17, and MCP-1 expression, and IKBα levels were significantly downregulated; RIVA restored these levels to normal values.RIVA treatment significantly restored the apoptotic and inflammatory parameters in SUN-damaged renal tissues.
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Antiinflamatorios/farmacología , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Glomerulonefritis/tratamiento farmacológico , Mediadores de Inflamación/metabolismo , Riñón/efectos de los fármacos , FN-kappa B/metabolismo , Estrés Oxidativo/efectos de los fármacos , Rivaroxabán/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Modelos Animales de Enfermedad , Glomerulonefritis/inducido químicamente , Glomerulonefritis/metabolismo , Glomerulonefritis/patología , Riñón/metabolismo , Riñón/patología , Masculino , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , SunitinibRESUMEN
Doxorubicin (Dox) is an operational and largely used anticancer drug, used to treat an array of malignancies. Nonetheless, its beneficial use is constrained due to its renal and hepatotoxicity dose dependently. Numerous research findings favor the use of antioxidants may impact Dox-induced liver injury/damage. In the current study, Wistar rats were given naringenin (50 and 100 mg/kg b.wt.) orally for 20 days as prophylactic dose, against the hepatotoxicity induced by single intraperitoneal injection of Dox (20 mg/kg b.wt.). Potency of naringenin against the liver damage caused by Dox was assessed by measuring malonyl aldehyde (MDA) as a by-product of lipid peroxidation, biochemical estimation of antioxidant enzyme system, reactive oxygen species (ROS) level, and inflammatory mediators. Naringenin-attenuated ROS production, ROS-induced lipid peroxidation, and replenished reduced antioxidant armory, namely, catalase (CAT), glutathione reductase (GR), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione (GSH). Naringenin similarly diminished expression of Cox-2 and levels of NF-κB and other inflammatory molecules induced by the Dox treatment. Histology added further evidence to the defensive effects of naringenin on Dox-induced liver damage. The outcomes of the current study reveal that oxidative stress and inflammation are meticulously linked with Dox-triggered damage, and naringenin illustrates the potential effect on Dox-induced hepatotoxicity probably through diminishing the oxidative stress and inflammation.
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Sunitinib (SUN) is an oral tyrosine kinase inhibitor approved in 2006 as a first-line treatment for metastatic renal cell cancer. However, weak selectivity to kinase receptors and cardiotoxicity have limited the use of sunitinib. Rivaroxaban (RIVA) is a Factor Xa inhibitor with cardioprotective action. It inhibits atherosclerosis and numerous inflammatory cascades. The present study was designed to investigate the cardioprotective effects of RIVA in sunitinib-induced cardiotoxicity. Thirty male Wistar rats were divided into five groups. Group 1 was the normal control (control). Group 2 was administered i.p. SUN 25 mg kg-1 thrice weekly for 3 weeks. Groups 3 and 4 received the same treatment as Group 2 followed by the administration of RIVA 5 mg kg-1 day-1 and 10 mg kg-1 day-1, respectively, for 3 weeks. Group 5 received only 10 mg kg-1 day-1 RIVA for 3 weeks. Serum levels of Ca2+, Mg2+, Fe3+/Fe2+, lipid profiles, and cardiac enzymes were measured. Cardiac tissues were isolated for the measurements of oxidant/antioxidant balance gene and protein expressions. Relative to the controls, the administration of SUN significantly altered serum levels of (Ca2+, Mg2+, Fe3+/Fe2+, lipid profiles, and cardiac enzymes), intracellular antioxidant enzymes, and the expression levels of the genes encoding certain proteins. RIVA treatment significantly restored these parameters to near-normal levels. RIVA treatment significantly mitigated SUN-induced cardiac injuries by restoring antioxidant enzyme levels and attenuating the proinflammatory cascades resulting from SUN-induced cardiac injuries.
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Antiinflamatorios/farmacología , Antioxidantes/farmacología , Cardiopatías/prevención & control , Miocitos Cardíacos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Rivaroxabán/farmacología , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Biomarcadores/sangre , Cardiotoxicidad , Modelos Animales de Enfermedad , Cardiopatías/inducido químicamente , Cardiopatías/metabolismo , Cardiopatías/patología , Interleucina-17/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Masculino , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Ratas Wistar , Transducción de Señal , Proteína Smad2/genética , Proteína smad3/genética , Sunitinib , Factor de Crecimiento Transformador beta/genéticaRESUMEN
BACKGROUND: Doxorubicin is an effective, potent and commonly used anthracycline-related anticancer drug; however, cardiotoxicity compromises its therapeutic potential. Apremilast, a novel phosphodiesterase type 4-inhibitor, reported to have anti-inflammatory effects and modulating many inflammatory mediators. METHODS: The present study investigated the influence of apremilast against doxorubicin-induced cardiotoxicity in male Wistar rats. A total, 24 animals were divided into four groups of six animal each. Group 1, served as control and received normal saline. Group 2 animals, received doxorubicin (20mgkg-1, ip). Group 3 and 4, treatment group, received doxorubicin (20mgkg-1, ip) with the same schedule as group-2, plus apremilast (10 and 20mgkg-1day-1, po) respectively. Oxidative stress, caspase-3 enzyme activity, gene expression and protein expression were tested. RESULTS: The results of the present study demonstrated that administration of apremilast reversed doxorubicin-induced cardiotoxicity. CONCLUSION: These findings suggested that apremilast can attenuate doxorubicin-induced cardiotoxicity via inhibition of oxidative stress mediated activation of nuclear factor-kappa B signaling pathways.
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Apoptosis/efectos de los fármacos , Doxorrubicina/efectos adversos , Doxorrubicina/antagonistas & inhibidores , Inflamación/inducido químicamente , FN-kappa B/metabolismo , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Talidomida/análogos & derivados , Animales , Cardiotoxicidad/prevención & control , Caspasa 3/metabolismo , Catalasa/metabolismo , Relación Dosis-Respuesta a Droga , Glutatión/metabolismo , Glutatión Reductasa/metabolismo , Masculino , Malondialdehído/metabolismo , Miocardio/metabolismo , ARN Mensajero/biosíntesis , Ratas , Talidomida/farmacologíaRESUMEN
Amyloid fibrils are playing key role in the pathogenesis of various neurodegenerative diseases. Generally anionic molecules are known to induce amyloid fibril in several proteins. In this work, we have studied the effect of anionic food additive dye i.e., tartrazine (TZ) on the amyloid fibril formation of human serum albumins (HSA) and bovine serum albumin (BSA) at pHs7.4 and 3.5. We have employed various biophysical methods like, turbidity measurements, Rayleigh Light Scattering (RLS), Dynamic Light Scattering (DLS), intrinsic fluorescence, Congo red assay, far-UV CD, transmission electron microscopy (TEM) and atomic force microscopy (AFM) to decipher the mechanism of TZ-induce amyloid fibril formation in both the serum albumins at pHs7.4 and 3.5. The obtained results suggest that both the albumins forms amyloid-like aggregates in the presence of 1.0 to 15.0mM of TZ at pH3.5, but no amyloid fibril were seen at pH7.4. The possible cause of TZ-induced amyloid fibril formation is electrostatic and hydrophobic interaction because sulfate group of TZ may have interacted electrostatically with positively charged amino acids of the albumins at pH3.5 and increased protein-protein and protein-TZ interactions leading to amyloid fibril formation. The TEM, RLS and DLS results are suggesting that BSA forms bigger size amyloids compared to HSA, may be due to high surface hydrophobicity of BSA.
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Albúmina Sérica Bovina/química , Albúmina Sérica Humana/química , Tartrazina/farmacología , Dicroismo Circular , Rojo Congo/química , Dispersión Dinámica de Luz , Humanos , Hidrodinámica , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Microscopía de Fuerza Atómica , Modelos Moleculares , Nefelometría y Turbidimetría , Agregado de Proteínas , Estructura Secundaria de Proteína , Albúmina Sérica Bovina/ultraestructura , Albúmina Sérica Humana/ultraestructura , Tartrazina/químicaRESUMEN
In case of studies associated with human genetics, genomics, and pharmacogenetics the genomic DNA is extracted from the buccal cells, whole blood etc. Several methods are exploited by the researchers to extract DNA from the whole blood. One of these methods, which utilizes cell lysis and proteolytic properties of sodium dodecyl sulfate (SDS) and proteinase K respectively, might also be called SDS-PK method. It does not include any hazardous chemicals such as phenol or chloroform and is inexpensive. However, several researchers report the same method with different formulas and conditions. During our experiments with whole blood DNA extraction we experienced problems such as protein contamination, DNA purity and yield when followed some SDS-PK protocols reported elsewhere. A260/A280 and A260/A230 ratios along with PCR amplification give a clear idea about the procedure that was followed to extract the DNA. In an effort to increase the DNA purity from human whole blood, we pointed out some steps of the protocol that play a crucial role in determining the extraction of high quality DNA.
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OBJECTIVE: Rat bronchoalveolar lavage fluid (BALF) metabolome can be used to obtain valuable, precise, and accurate information about underlying lung conditions in an experiment. The present study focuses on the evaluation of the lung epithelium metabolome in a rat model using techniques including bronchoalveolar lavage, gas chromatography-mass spectroscopy (GC-MS), and Fourier transform infrared spectroscopy (FT-IR). MATERIALS AND METHODS: Untargeted metabolites in BALF were extracted in ethyl acetate and derivatized by standard methods for the analysis by GC-MS. FT-IR spectra of ethyl acetate extract of BALF were obtained and read for the characteristic fingerprint of rats under investigation. Analyses were done in individual animals to obtain consistent data. BALF cells were counted by flow cytometry to monitor any inflammatory condition in rats. RESULTS: FT-IR analysis finds two peaks which are characteristically different from the extract medium, which is ethyl acetate. FT-IR peaks correspond to that of amino acids and carbohydrates, including ß-D-glucose, α-D-glucose, and ß-D-galactose. GC-MS evaluation of the BALF finds several products of the metabolism or its participants. Main compounds in the BALF detected by GC-MS include succinate, fumarate, glycine, alanine, 2-methyl-3-oxovaleric acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octanoic acid, trans-9-octadecanoic acid, octadecanoic acid, and Prostaglandin F1α. CONCLUSION: Several research reports reveal metabolomic parameters in murine model lung tissue or BALF, but they rarely reported a complete metabolomics model profile, particularly in rats. The present data of GC-MS and FT-IR suggest that the set up can be exploited to study metabolomic alterations in several lung conditions including acute lung toxicity, inflammation, asthma, bronchitis, fibrosis, and emphysema.
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Líquido del Lavado Bronquioalveolar/química , Mucosa Respiratoria/metabolismo , Animales , Líquido del Lavado Bronquioalveolar/citología , Cromatografía de Gases y Espectrometría de Masas , Masculino , Metabolómica , Ratas Wistar , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Dyslipidemia is common in patients with diabetes mellitus (DM) and is considered a risk factor for the progression of diabetic nephropathy (DN). Hyperlipidemia and hyperglycemia act synergistically to induce renal injury. The present study was designed to investigate the protective effects of tocotrienols as tocotrienol-rich fraction (TRF) extracted from palm (PO) and rice bran oils (RBO) against lipid induced nephropathy in type-2 diabetic rats and its probable molecular mechanism. Male Wistar rats (175-200 g) were divided into four groups. The first group served as diabetic control, while the second and third groups received PO-TRF and RBO-TRF, respectively by gavage over a period of sixteen weeks post-induction of diabetes. The fourth group comprised of age-matched rats that served as normal control. The effects of TRF on serum lipid profile, oxidative stress markers, expression of TGF-ß, fibronectin and collagen type IV were analyzed in the kidney of diabetic rats. Treatment with PO-TRF and RBO-TRF significantly improved glycemic status, serum lipid profile and renal function in type-2 diabetic rats. In addition, TRF supplementation down-regulated the expression of TGF-ß, fibronectin and collagen type IV in the kidney of diabetic rats. Transforming growth factor-ß (TGF-ß) plays a critical role in progression of DN, but its modulation by tocotrienols in DN remains unexplored. TRF ameliorated lipid induced nephropathy in type-2 diabetes by its hypoglycemic, hypolipidemic and antioxidant activities as well as by modulation of TGF-ß to prevent increased expression of collagen type IV and fibrinogen. We finally propose a mechanism for the expression of molecular markers that are significant in the events leading to diabetic nephropathy and its modulation by tocotrienols/TRF.