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1.
Methods Enzymol ; 667: 611-632, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35525556

RESUMEN

Biochemical analyses of membrane receptor kinases have been limited by challenges in obtaining sufficient homogeneous receptor samples for downstream structural and biophysical characterization. Here, we report a suite of methods for the efficient expression, purification, and visualization by cryo-electron microscopy (cryo-EM) of near full-length Human Epidermal Growth Factor Receptor 3 (HER3), a receptor tyrosine pseudokinase, in the unliganded state. Through transient mammalian cell expression, a two-step purification with detergent exchange into lauryl maltose neopentyl glycol (LMNG), and freezing devoid of background detergent micelle, we obtained ~6Å reconstructions of the ~60kDa fully-glycosylated unliganded extracellular domain of HER3 from just 30mL of suspension culture. The reconstructions reveal previously unappreciated extracellular domain dynamics and glycosylation sites.


Asunto(s)
Detergentes , Micelas , Animales , Microscopía por Crioelectrón/métodos , Humanos , Mamíferos
2.
J Mater Sci Mater Med ; 33(5): 40, 2022 May 04.
Artículo en Inglés | MEDLINE | ID: mdl-35507049

RESUMEN

Detergent treatment is the most commonly used method for the decellularization of ligaments and tendon grafts. However, it is well recognized that detergent treatment can also adversely affect the extracellular matrix. This study found that discission into the aponeurosis layer of the patellar tendon (PT) before decellularization is conducive to extracting cells from the PT using a low quantity of detergent in a short time period. The acellular aponeurosis discission ligament (AADL) retains its native collagen fibril structure and mechanical properties. Moreover, the PT retained cell and tissue compatibility in vitro and in vivo. After implantation into a defective allogeneic PT, we found that the AADL healed well in the host, and its collagen structure exhibited gradual improvement 12 months after implantation with satisfactory reconstruction. IMPACT: The aponeurosis of tendons/ligaments is the main barrier to achieving complete decellularization, and it thus prevents complete recellularization for applications in tissue engineering. Aponeurosis can obstruct the removal of cell components. We found that excising the aponeurosis before decellularization allows for the removal of cellular components with a reduced amount of detergent, thus improving the biological properties of the acellular ligament. To the best of our knowledge, no similar studies have been performed. Graphical abstract.


Asunto(s)
Aponeurosis , Detergentes , Colágeno/análisis , Detergentes/análisis , Detergentes/química , Matriz Extracelular/química , Ligamentos , Ingeniería de Tejidos/métodos , Andamios del Tejido/química
3.
An Acad Bras Cienc ; 94(2): e20190996, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35544842

RESUMEN

The present study aimed to evaluate the effect of alkaline treatments with urea, NaOH and Ca(OH)2 on chemical composition and in situ ruminal degradability of dry matter, crud protein and neutral detergent fiber of sugarcane tip hay. Samples were incubated in the rumen of three cannulated cattle for up to 72 hours in a split plot randomized block design. Ammoniation with 6% urea increased (p<0.05) the crude protein content by 13% and reduced the neutral detergent fiber and insoluble nitrogen content of the hay. When treated with the highest doses of the compounds, there was a high potential degradability of dry matter, crude protein and neutral detergent fiber, and a shorter neutral detergent fiber lag time. Ammoniation with urea promotes a reduction in the content of NDF, hemicellulose and insoluble nitrogen, with an increase in the content of CP in the hay, with emphasis for the level of 6% urea. The ruminal degradation of sugarcane tip hay increases with alkaline treatments using 6% urea or 3% NaOH, however, ammoniation with urea is indicated for the treatment of hay, as this is low cost and can be easily adopted by farmers in the semiarid region.


Asunto(s)
Rumen , Saccharum , Alimentación Animal/análisis , Animales , Bovinos , Detergentes/metabolismo , Detergentes/farmacología , Dieta/veterinaria , Fibras de la Dieta/análisis , Digestión , Grano Comestible/química , Fermentación , Nitrógeno/metabolismo , Rumen/metabolismo , Saccharum/metabolismo , Hidróxido de Sodio/metabolismo , Hidróxido de Sodio/farmacología , Urea/farmacología
4.
Anim Sci J ; 93(1): e13734, 2022 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-35537860

RESUMEN

The objective of this study was to investigate the influence of timing of paddock allocation (AM or PM) in tropical grass on nutritive value of the herbage, dry matter intake (DMI), milk yield and composition, ruminal fermentation, nitrogen excretion, and enteric CH4 emissions of dairy cows. Twenty cows were grouped in pairs and randomly distributed within pair to one of two treatments. PM herbage had greater contents of dry matter (DM), soluble carbohydrates, starch, and nonfibrous carbohydrate to protein ratio and lower contents of neutral detergent fiber and acid detergent fiber. There was no treatment effect on DMI, N excretion, milk yield, and CH4 emissions. However, milk protein and casein yields tended to be greater for PM than AM, while milk urea nitrogen concentration was lower for PM than AM. The increase in nutritive value of the afternoon relative to the morning herbage within the framework of this study was not large enough to increase DMI and milk yield or to decrease CH4 emission intensity by the dairy cows as hypothesized. The findings indicate that PM treatment can be a simple and useful grazing strategy that results in an herbage harvest with greater nutritional value and in lower excretion of urea N into milk.


Asunto(s)
Metano , Poaceae , Alimentación Animal/análisis , Animales , Bovinos , Detergentes/metabolismo , Dieta/veterinaria , Femenino , Lactancia , Metano/metabolismo , Nitrógeno/metabolismo , Rumen/metabolismo , Urea/metabolismo
5.
Viruses ; 14(4)2022 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-35458445

RESUMEN

The COVID-19 pandemic caused by SARS-CoV-2 is having devastating effects on a global scale. Since common household disinfectants are often used to minimise the risk of infection in the home and work environment, we investigated the ability of some of these products to inactivate the virus. We tested generic brands of vinegar, bleach, and dishwashing detergent, as well as laboratory-grade acetic acid, sodium hypochlorite, and ethanol. Assays were conducted at room temperature (18-20 °C, 40% relative humidity), and two time points were used to reflect a quick wipe (30 s) and a brief soak (5 min). Vinegar, and its active ingredient, acetic acid, were completely ineffective at virus inactivation even when exposed to the virus at 90% v/v (a final concentration equivalent to 3.6% v/v acetic acid). In contrast, ethanol was capable of inactivating the virus at dilutions as low as 40% v/v. Dishwashing detergent effectively rendered SARS-CoV-2 inactive when diluted 100-fold (1% v/v). Bleach was found to be fully effective against SARS-CoV-2 at 0.21 g/L sodium hypochlorite after a 30 s exposure (1/200 dilution of commercial product). Given reports of infectious virus recovered from the surface of frozen packaging, we tested the persistence of infectiousness after multiple freeze-thaw cycles and found no change in infectious SARS-CoV-2 titre after seven freeze-thaw cycles. These results should help inform readers of how to effectively disinfect surfaces and objects that have potentially been contaminated with SARS-CoV-2 using common household chemicals.


Asunto(s)
COVID-19 , Desinfectantes , Ácido Acético/farmacología , COVID-19/prevención & control , Detergentes/farmacología , Desinfectantes/farmacología , Etanol/farmacología , Humanos , Pandemias , SARS-CoV-2 , Hipoclorito de Sodio/farmacología
6.
J Phys Chem B ; 126(15): 2824-2833, 2022 Apr 21.
Artículo en Inglés | MEDLINE | ID: mdl-35384657

RESUMEN

Photosystems I (PSI) and II (PSII) are pigment-protein complexes capable of performing the light-induced charge separation necessary to convert solar energy into a biochemically storable form, an essential step in photosynthesis. Small-angle neutron scattering (SANS) is unique in providing structural information on PSI and PSII in solution under nearly physiological conditions without the need for crystallization or temperature decrease. We show that the reliability of the solution structure critically depends on proper contrast matching of the detergent belt surrounding the protein. Especially, specifically deuterated ("invisible") detergents are shown to be properly matched out in SANS experiments by a direct, quantitative comparison with conventional matching strategies. In contrast, protonated detergents necessarily exhibit incomplete matching so that related SANS results systematically overestimate the size of the membrane protein under study. While the solution structures obtained are close to corresponding high-resolution structures, we show that temperature and solution state lead to individual structural differences compared with high-resolution structures. We attribute these differences to the presence of a manifold of conformational substates accessible by protein dynamics under physiological conditions.


Asunto(s)
Detergentes , Difracción de Neutrones , Detergentes/química , Difracción de Neutrones/métodos , Complejo de Proteína del Fotosistema I , Reproducibilidad de los Resultados , Dispersión del Ángulo Pequeño
7.
Trop Anim Health Prod ; 54(3): 163, 2022 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-35434744

RESUMEN

We investigated the consequences of fat supplementation (free oil and rumen-protected oil) on the nutrient intake and digestion of beef cattle at pasture. Five rumen-cannulated Nelore bulls, with a median body weight (BW) of 467.8 ± 32.8 kg and an age of 26 months, were distributed in a Latin square design (5 × 5). The treatments were as follows: WF, no additional fat; PA, rumen-protected palm oil; PS, rumen-protected soybean oil; SO, soybean-free oil and CO, free corn oil. Nutrient intake and digestibility, ruminal pH and ammonia (NH3-N), serum urea and nitrogen balance were analysed. The supplements with different oil sources did not alter (P > 0.05) the intake and digestibility of dry matter (DM), forage DM intake (DM), organic matter (OM), crude protein (CP), neutral detergent fibre (NDF), neutral detergent fibre-corrected ash and protein (apNDF), nonfibre carbohydrates (NFC) or total digestible nutrients (TDN) compared to WF. An increase (P < 0.05) in the intake and digestibility of EE was observed with the inclusion of fat, independent of the source. No differences were observed between WF and other supplements with regard to ruminal parameters (pH and NH3-N) (P > 0.05) and serum urea (P > 0.05). The nitrogen balance was not affected by the fat source (P > 0.05). Supplementation of grazing beef cattle (2 g/kg BW) with free oil (130 g/kg DM supplement) or rumen-protected oil (160 g/kg DM supplement) did not interfere with nutrient intake and digestibility.


Asunto(s)
Alimentación Animal , Rumen , Alimentación Animal/análisis , Animales , Bovinos , Detergentes/metabolismo , Dieta/veterinaria , Suplementos Dietéticos/análisis , Digestión , Fermentación , Masculino , Nitrógeno/metabolismo , Rumen/metabolismo , Urea/metabolismo
8.
Nature ; 604(7905): 377-383, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35388220

RESUMEN

PIEZO channels respond to piconewton-scale forces to mediate critical physiological and pathophysiological processes1-5. Detergent-solubilized PIEZO channels form bowl-shaped trimers comprising a central ion-conducting pore with an extracellular cap and three curved and non-planar blades with intracellular beams6-10, which may undergo force-induced deformation within lipid membranes11. However, the structures and mechanisms underlying the gating dynamics of PIEZO channels in lipid membranes remain unresolved. Here we determine the curved and flattened structures of PIEZO1 reconstituted in liposome vesicles, directly visualizing the substantial deformability of the PIEZO1-lipid bilayer system and an in-plane areal expansion of approximately 300 nm2 in the flattened structure. The curved structure of PIEZO1 resembles the structure determined from detergent micelles, but has numerous bound phospholipids. By contrast, the flattened structure exhibits membrane tension-induced flattening of the blade, bending of the beam and detaching and rotating of the cap, which could collectively lead to gating of the ion-conducting pathway. On the basis of the measured in-plane membrane area expansion and stiffness constant of PIEZO1 (ref. 11), we calculate a half maximal activation tension of about 1.9 pN nm-1, matching experimentally measured values. Thus, our studies provide a fundamental understanding of how the notable deformability and structural rearrangement of PIEZO1 achieve exquisite mechanosensitivity and unique curvature-based gating in lipid membranes.


Asunto(s)
Activación del Canal Iónico , Canales Iónicos , Mecanotransducción Celular , Detergentes , Canales Iónicos/metabolismo , Membrana Dobles de Lípidos , Micelas
9.
Int J Biochem Cell Biol ; 146: 106210, 2022 May.
Artículo en Inglés | MEDLINE | ID: mdl-35390493

RESUMEN

Fluorescence correlation spectroscopy (FCS) is a quantitative spectroscopy technique which could potentially increase throughput and sensitivity of screening for ligand, substrate and inhibitor binding to membrane proteins in solution. However, the purification of membrane proteins in their active forms is complex, as the lipid bilayer provides stability and its removal often causes the protein to become conformationally unstable. This has limited the application of biophysical techniques such as FCS to study the function of membrane proteins. The recent application of native extraction techniques such as styrene maleic acid lipid particles (SMALPs) has resolved this issue and FCS has emerged as a powerful option for studying proteins extracted in this way. This review will discuss the application of FCS to study purified membrane proteins in detergent micelles, nanodiscs and SMALPs and its potential to be used routinely in membrane protein drug discovery.


Asunto(s)
Detergentes , Proteínas de la Membrana , Fluorescencia , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Poliestirenos/química
10.
J Extracell Vesicles ; 11(4): e12200, 2022 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-35362259

RESUMEN

Flow cytometry (FCM) is a popular method used in characterisation of extracellular vesicles (EVs). Circulating EVs are often identified by FCM by exploiting the lipid nature of EVs by staining with Annexin V (Anx5) or lactadherin against the membrane phospholipid phosphatidylserine (PS) and evaluating the specificity of the labels by detergent lysis of EVs. Here, we investigate whether PS labelling and detergent lysis approaches are confounded by lipoproteins, another family of lipid-based nanoparticles found in blood, in both frozen and fresh blood plasma. We demonstrated that Anx5 and lactadherin in addition to EVs stained ApoB-containing lipoproteins, identified by the use of fluorophore-labelled polyclonal ApoB-antibody, and that Anx5 had a significantly larger tendency for labelling lipoprotein-bound PS than lactadherin. Furthermore, detergent lysis resulted in a decrease in both EV and lipoprotein events and especially lipoproteins positive for either Anx5 or lactadherin. Taken together, our findings pose concerns to the use of lipid-based strategies in identifying EVs by FCM and support the use of transmembrane proteins such as tetraspannins to distinguish EVs from lipoproteins.


Asunto(s)
Detergentes , Vesículas Extracelulares , Anexina A5/metabolismo , Detergentes/farmacología , Vesículas Extracelulares/metabolismo , Citometría de Flujo/métodos , Lipoproteínas/metabolismo , Fosfatidilserinas/metabolismo
11.
Environ Int ; 162: 107170, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35272140

RESUMEN

Domestic cleaners have an increased risk of asthma-like and other respiratory symptoms and conditions. Uncertainty exists about which products are most hazardous. We aimed to investigate, among professional domestic cleaners, the associations of ocular/respiratory outcomes with using specific types of products at work and with the ability to choose their own products. Among domestic cleaners employed by "service vouchers" companies in Belgium, we administered an online questionnaire on ocular/respiratory symptoms (frequency and time relation to workdays), frequency of use of 40 types of products, and ability to choose one's own products. Work-relatedness was defined as symptoms improving/disappearing on days off-work. We studied associations between frequency of product-use with work-related outcomes (eye irritation, rhinitis symptoms, sore throat, laryngeal symptoms, asthma symptoms, cough) and with chronic bronchitis, using multivariable logistic and elastic net regression. Adjusted odds ratios (OR) with 95%-confidence intervals were obtained per time a product was used per week. Among 1,586 domestic cleaners (99% women), the number of times sprays were used (median 13/week) was significantly associated with all outcomes (ORs between 1.012 and 1.024 per time sprays were used per week). Bleach/disinfectant-containing liquid products were associated with all outcomes, except for laryngeal symptoms (ORs 1.086 to 1.150); ammonia with work-related upper airway symptoms and chronic bronchitis. Cleaners able to choose their own products had fewer work-related eye symptoms (OR 0.728;0.556-0.954), rhinitis (OR 0.735;0.571-0.946) and cough (OR 0.671;0.520-0.865). Using elastic net regression, work-related rhinitis was most strongly associated with mould removal spray (OR 1.108;1.006-1.248), carpet/seat/curtain spray (OR 1.099;1.001-1.304) and ammonia (OR 1.081;1.002-1.372); work-related asthma with carpet/seat/curtain spray (OR 1.103;1.017-1.322), mould removal spray (OR 1.029;0.995-1.199) and drain cleaner (OR 1.023;0.979-1.302). In a large group of domestic cleaners, we documented that cleaning products have a range of adverse respiratory effects. Empowering cleaners to choose their products may reduce the burden of symptoms.


Asunto(s)
Asma , Bronquitis Crónica , Enfermedades Profesionales , Exposición Profesional , Rinitis , Amoníaco , Asma/inducido químicamente , Asma/etiología , Tos/epidemiología , Tos/etiología , Detergentes , Femenino , Humanos , Masculino , Enfermedades Profesionales/inducido químicamente , Enfermedades Profesionales/etiología , Exposición Profesional/efectos adversos , Rinitis/epidemiología , Rinitis/etiología , Recursos Humanos
12.
J Dairy Sci ; 105(5): 4128-4143, 2022 May.
Artículo en Inglés | MEDLINE | ID: mdl-35282921

RESUMEN

Our objective was to evaluate the effects of unprotected choline chloride (Cho) on the ruminal microbiome at 2 dietary neutral detergent fiber (NDF) concentrations. We hypothesized that the effects of Cho on ruminal bacterial populations would depend on NDF. Eight dual-flow continuous-culture fermentors were arranged in a duplicated 4 × 4 Latin square as a 2 × 2 factorial with the following treatments: (1) 30% NDF-control (30% NDF diet, no supplemental choline); (2) 30% NDF-Cho (30% NDF diet plus 1.9 g of choline ion per kg of dry matter); (3) 40% NDF-control (40% NDF diet, no supplemental choline); and (4) 40% NDF-Cho (40% NDF diet plus 1.9 g of choline ion per kg of dry matter). We did 4 fermentation periods of 10 d each and used the last 3 d for collection of samples of solid and liquid digesta effluents for DNA extraction. Overall, 32 solid and 32 liquid samples were analyzed by amplification of the V4 variable region of bacterial 16S rRNA. Data were analyzed with R (R Project for Statistical Computing) and SAS (SAS Institute Inc.) to determine effects of Cho, NDF, and NDF × Cho on taxa relative abundance. The correlation of propionate molar proportion with taxa relative abundance was also analyzed. At the phylum level, relative abundance of Firmicutes in the liquid fraction tended to be greater when Cho was supplemented with a 30% NDF diet. At the order level, Cho increased Coriobacteriales in solid fraction and decreased Fibrobacterales in liquid fraction. Moreover, Cho decreased abundance of Clostridiales and increased Selenomonadales in the solid fraction, only with the 30% NDF diet. For genera, lower abundance of Pseudobutyrivibrio resulted from Cho in solid and liquid fractions. Greater abundance of Succinivibrio in solid and Selenomonas and Selenomonas 1 in liquid resulted from Cho with the 30% NDF diet. Propionate molar proportion was positively correlated with relative abundance of order Selenomonadales in solid and liquid fractions, and with genus Succinivibrio in solid and genera Selenomonas and Selenomonas 1 in liquid. Our results indicate that Cho primarily decreases abundance of bacteria involved in fiber degradation and increases abundance of bacteria mainly involved in nonstructural carbohydrate degradation and synthesis of propionate, particularly when a diet with 30% NDF is provided.


Asunto(s)
Microbiota , Rumen , Alimentación Animal/análisis , Animales , Bacterias , Colina/metabolismo , Detergentes/metabolismo , Dieta/veterinaria , Fibras de la Dieta/metabolismo , Digestión , Fermentación , Propionatos/metabolismo , ARN Ribosómico 16S/metabolismo , Rumen/metabolismo
13.
J Dairy Sci ; 105(5): 4144-4155, 2022 May.
Artículo en Inglés | MEDLINE | ID: mdl-35307174

RESUMEN

Dairy cattle experience inflammation during the calving transition period, and butyrate and nonsteroidal anti-inflammatory drugs (NSAID) are expected to reduce the inflammation. Our objective was to evaluate the effects of dietary butyrate supplementation and oral NSAID administration on feed intake, serum inflammatory markers, plasma metabolites, and milk production of dairy cows during the calving transition period. Eighty-three Holstein cows were used in the experiment with a 2 × 2 factorial arrangement of treatments. The cows were blocked by parity and calving date, and randomly assigned to a dietary butyrate or control supplement, and NSAID or a placebo oral administration. Experimental diets were iso-energetic containing calcium butyrate at 1.42% of diet dry matter (DM) or the control supplement (1.04% commercial fat supplement and 0.38% calcium carbonate of diet DM). The close-up diets contained 13.3% starch and 42.4% neutral detergent fiber on a DM basis, and were fed from 28 d before expected calving date until calving. The postpartum diets contained 22.1% starch and 34.1% neutral detergent fiber on a DM basis and were fed from calving to 24 d after calving. Oral NSAID (1 mg of meloxicam/kg of body weight) or placebo (food dye) was administered 12 to 24 h after calving. Dietary butyrate supplementation and oral NSAID administration did not affect milk yield or postpartum serum concentrations of amyloid A and haptoglobin. However, butyrate-fed cows increased plasma fatty acid concentration on d -4 relative to calving (501 vs. 340 µEq/L) and tended to increase serum haptoglobin concentration (0.23 vs. 0.10 mg/mL). There was a supplement by drug interaction effect on plasma glucose concentration on d 4; in cows administered the placebo drug, butyrate supplementation decreased plasma glucose concentration compared with control-fed cows (62.8 vs. 70.1 mg/dL). Butyrate-fed cows tended to have lower milk crude protein yield compared with cows fed the control diet (1.21 vs. 1.27 kg/d). Dietary butyrate supplementation and oral NSAID administration did not have overall positive effects on production performance of dairy cows during the calving transition period.


Asunto(s)
Enfermedades de los Bovinos , Lactancia , Administración Oral , Animales , Antiinflamatorios no Esteroideos , Glucemia/metabolismo , Butiratos/metabolismo , Bovinos , Enfermedades de los Bovinos/metabolismo , Detergentes/metabolismo , Dieta/veterinaria , Fibras de la Dieta/metabolismo , Suplementos Dietéticos , Femenino , Haptoglobinas/metabolismo , Inflamación/metabolismo , Inflamación/veterinaria , Leche/metabolismo , Periodo Posparto/metabolismo , Embarazo , Almidón/metabolismo
14.
Tissue Eng Part C Methods ; 28(4): 148-157, 2022 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-35357965

RESUMEN

Collagens, elastin, fibrillin, decorin, and laminin are key constituents of the extracellular matrix and basement membrane of mammalian organs. Thus, changes in their quantities may influence the mechanochemical regulation of resident cells. Since maintenance of a native stromal composition is a requirement for three-dimensional (3D) matrix-based recellularization techniques in tissue engineering, we studied the influence of the decellularization detergents on these proteins in porcine kidney, liver, pancreas, and skin. Using a quick thawing/quick microwave-assisted decellularization protocol and two different detergents, sodium dodecyl sulfate (SDS) vs Triton X-100 (TX100), at identical concentration, variations in matrix conservation of stromal proteins were detected by liquid chromatography-mass spectrometry coupled to light and scanning electron microscopies, in dependence on each detergent. In all organs tested except pancreas, collagens were retained to a statistically significant level using the TX100-based protocol. In contrast fibrillin, elastin (except in kidney), and decorin (only in liver) were better preserved with the SDS-dependent protocol. Irrespective of the detergent used, laminin always remained at an irrelevant level. Our results prompt attention to the type of detergent in organ decellularization, suggesting that its choice may influence morphoregulatory inputs peculiar to the type of 3D bioartificial mammalian organ to be reconstructed. Impact statement Simple change of the protocol's main detergent leads to a very substantial difference in the panel of the stromal proteins detected by qualitative and semiquantitative mass spectrometry in acellular porcine matrices. This remarkable methodological variable promises to yield proteomic reference panels in a number of different species-specific acellular matrices allowing for selective retainment of peculiar mechanochemical inputs, to differently address the development of the seeded cells in relation to the type of organ to be bioartificially reconstructed.


Asunto(s)
Detergentes , Andamios del Tejido , Animales , Colágeno/metabolismo , Decorina/metabolismo , Detergentes/química , Detergentes/metabolismo , Detergentes/farmacología , Elastina/metabolismo , Matriz Extracelular/metabolismo , Fibrilinas/metabolismo , Laminina/metabolismo , Mamíferos , Espectrometría de Masas , Octoxinol/metabolismo , Proteómica , Porcinos , Ingeniería de Tejidos/métodos , Andamios del Tejido/química
15.
Sci Rep ; 12(1): 3532, 2022 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-35241773

RESUMEN

Extraction of integral membrane proteins with poly(styrene-co-maleic acid) provides a promising alternative to detergent extraction. A major advantage of extraction using copolymers rather than detergent is the retention of the lipid bilayer around the proteins. Here we report the first functional investigation of the mammalian insulin receptor which was extracted from cell membranes using poly(styrene-co-maleic acid). We found that the copolymer efficiently extracted the insulin receptor from 3T3L1 fibroblast membranes. Surprisingly, activation of the insulin receptor and proximal downstream signalling was detected upon copolymer extraction even in the absence of insulin stimulation. Insulin receptor and IRS1 phosphorylations were above levels measured in the control extracts made with detergents. However, more distal signalling events in the insulin signalling cascade, such as the phosphorylation of Akt were not observed. Following copolymer extraction, in vitro addition of insulin had no further effect on insulin receptor or IRS1 phosphorylation. Therefore, under our experimental conditions, the insulin receptor is not functionally responsive to insulin. This study is the first to investigate receptor tyrosine kinases extracted from mammalian cells using a styrene-maleic acid copolymer and highlights the importance of thorough functional characterisation when using this method of protein extraction.


Asunto(s)
Detergentes , Receptor de Insulina , Insulina , Ligandos , Maleatos/farmacología , Fosforilación , Polímeros , Poliestirenos
16.
J Bacteriol ; 204(4): e0059921, 2022 Apr 19.
Artículo en Inglés | MEDLINE | ID: mdl-35258322

RESUMEN

Nucleoid-associated proteins (NAPs) silence xenogenes by blocking RNA polymerase binding to promoters and hindering transcript elongation. In Escherichia coli, H-NS and its homolog SptA interact with YmoA proteins Hha and YdgT to assemble nucleoprotein filaments that facilitate transcription termination by Rho, which acts in synergy with NusG. Countersilencing during initiation is facilitated by proteins that exclude NAPs from promoter regions, but auxiliary factors that alleviate silencing during elongation are not known. A specialized NusG paralog, RfaH, activates lipopolysaccharide core biosynthesis operons, enabling survival in the presence of detergents and antibiotics. RfaH strongly inhibits Rho-dependent termination by reducing RNA polymerase pausing, promoting translation, and competing with NusG. We hypothesize that RfaH also acts as a countersilencer of NAP/YmoA filaments. We show that deletions of hns and hha+ydgT suppress the growth defects of ΔrfaH by alleviating Rho-mediated polarity within the waa operon. The absence of YmoA proteins exacerbates cellular defects caused by reduced Rho levels or Rho inhibition by bicyclomycin but has negligible effects at a strong model Rho-dependent terminator. Our findings that the distribution of Hha and RfaH homologs is strongly correlated supports a model in which they comprise a silencing/countersilencing pair that controls expression of chromosomal and plasmid-encoded xenogenes. IMPORTANCE Horizontally acquired DNA drives bacterial evolution, but its unregulated expression may harm the recipient. Xenogeneic silencers recognize foreign genes and inhibit their transcription. However, some xenogenes, such as those encoding lipo- and exopolysaccharides, confer resistance to antibiotics, bile salts, and detergents, necessitating the existence of countersilencing fitness mechanisms. Here, we present evidence that Escherichia coli antiterminator RfaH alleviates silencing of the chromosomal waa operon and propose that plasmid-encoded RfaH homologs promote dissemination of antibiotic resistance genes through conjugation.


Asunto(s)
Proteínas de Escherichia coli , Antibacterianos/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Detergentes/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Factores de Elongación de Péptidos/química , Transactivadores/genética , Factores de Transcripción/metabolismo , Transcripción Genética
17.
J Bacteriol ; 204(4): e0006022, 2022 Apr 19.
Artículo en Inglés | MEDLINE | ID: mdl-35315684

RESUMEN

The Mycobacterium tuberculosis type-7 protein secretion system ESX-1 is a major driver of its virulence. While the functions of most ESX-1 components are characterized, many others remain poorly defined. In this study, we examined the role of EspK, an ESX-1-associated protein that is thought to be dispensable for ESX-1 activity in members of the Mycobacterium tuberculosis complex. We show that EspK is needed for the timely and optimal secretion of EsxA and absolutely essential for EspB secretion in M. tuberculosis Erdman. We demonstrate that only the EsxA secretion defect can be alleviated in EspK-deficient M. tuberculosis by culturing it in media containing detergents like Tween 80 or tyloxapol. Subcellular fractionation experiments reveal EspK is exported by M. tuberculosis in an ESX-1-independent manner and localized to its cell wall. We also show a conserved W-X-G motif in EspK is important for its interaction with EspB and enabling its secretion. The same motif, however, is not important for EspK localization in the cell wall. Finally, we show EspB in EspK-deficient M. tuberculosis tends to adopt higher-order oligomeric conformations, more so than EspB in wild-type M. tuberculosis. These results suggest EspK interacts with EspB and prevents it from assembling prematurely into macromolecular complexes that are presumably too large to pass through the membrane-spanning ESX-1 translocon assembly. Collectively, our findings indicate M. tuberculosis EspK has a far more active role in ESX-1-mediated secretion than was previously appreciated and underscores the complex nature of this secretion apparatus. IMPORTANCE Mycobacterium tuberculosis uses its ESX-1 system to secrete EsxA and EspB into a host to cause disease. We show that EspK, a protein whose role in the ESX-1 machinery was thought to be nonessential, is needed by M. tuberculosis for optimal EsxA and EspB secretion. Culturing EspK-deficient M. tuberculosis with detergents alleviates EsxA but not EspB secretion defects. We also show that EspK, which is exported by M. tuberculosis in an ESX-1-independent manner to the cell wall, interacts with and prevents EspB from assembling into large structures inside the M. tuberculosis cell that are nonsecretable. Collectively, our observations demonstrate EspK is an active component of the ESX-1 secretion machinery of the tubercle bacillus.


Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Sistemas de Secreción Tipo VII , Proteínas Bacterianas/metabolismo , Detergentes/metabolismo , Humanos , Mycobacterium tuberculosis/metabolismo , Tuberculosis/microbiología , Sistemas de Secreción Tipo VII/metabolismo
18.
Sci Rep ; 12(1): 3809, 2022 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-35264651

RESUMEN

The experiment objective was to assess the shifting effect from cassava chip (CC) to fresh cassava root (FC) affected feed utilization, rumen metabolism, cyanide-using bacteria, and milk quality in lactating Thai Friesian dairy cows fed diets based on high sulfur-containing pellet (PS). Four lactating Thai Friesian dairy cows of 481.5 ± 31.3 kg BW (about 4 years old were allocated with four treatments in a 4 × 4 Latin square design. The four treatments were: replacement FC for CC at 0%, 60%, 80%, and 100% dry matter (DM), respectively. Feed intakes for four diets in terms of total dry matter intake (kg/day and % BW) was linearly correlated with levels of replacement of FC (p < 0.01). Digestibilities of crude protein (CP), DM, organic matter (OM), amylase-treated neutral detergent fiber (aNDF), and acid detergent fiber (ADF) did not change with increasing levels of FC in the diet (p > 0.05). Moreover, the total bacterial counts and cyanide population utilizing bacteria cubically increased with an increase of FC replacement (p < 0.01). The effect of CC substitution with FC in the PS diet was cubically increased on blood thiocyanate concentrations (p < 0.01). In addition, the propionate (C3) concentration at 0 and 4 h post-feeding changed significantly among treatments (p < 0.01), which were linearly improved with an increasing dose of replacement FC and were highest when FC was replaced at 100%. The yield of 3.5% fat-corrected milk was high in the treatment with the replacement of FC as compared to the control (p < 0.01). The yield of fat and milk fat percentages was high (p < 0.01) in the group with the replacement of FC as compared to feed with no diet replaced. The milk thiocyanate concentration was cubically enhanced when levels of FC replacement increased (p < 0.01) and was the highest when FC was replaced at 100%. As the amount of FC replacement was raised, the somatic cell count in the milk decreased linearly (p < 0.01). In conclusion, the replacement of FC at 100% in PS could enhance the feed intake, microbial populations, total volatile fatty acid (VFA), C3 concentration, milk yield, and milk quality.


Asunto(s)
Manihot , Leche , Alimentación Animal/análisis , Animales , Bovinos , Cianuros/metabolismo , Detergentes/farmacología , Dieta/veterinaria , Digestión , Femenino , Fermentación , Lactancia , Leche/metabolismo , Rumen/metabolismo , Azufre/metabolismo
19.
J Virol ; 96(8): e0166821, 2022 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-35343783

RESUMEN

Binding to the receptor, CD4, drives the pretriggered, "closed" (state-1) conformation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer into more "open" conformations (states 2 and 3). Broadly neutralizing antibodies, which are elicited inefficiently, mostly recognize the state-1 Env conformation, whereas the more commonly elicited poorly neutralizing antibodies recognize states 2/3. HIV-1 Env metastability has created challenges for defining the state-1 structure and developing immunogens mimicking this labile conformation. The availability of functional state-1 Envs that can be efficiently cross-linked at lysine and/or acidic amino acid residues might assist these endeavors. To that end, we modified HIV-1AD8 Env, which exhibits an intermediate level of triggerability by CD4. We introduced lysine/acidic residues at positions that exhibit such polymorphisms in natural HIV-1 strains. Env changes that were tolerated with respect to gp120-gp41 processing, subunit association, and virus entry were further combined. Two common polymorphisms, Q114E and Q567K, as well as a known variant, A582T, additively rendered pseudoviruses resistant to cold, soluble CD4, and a CD4-mimetic compound, phenotypes indicative of stabilization of the pretriggered state-1 Env conformation. Combining these changes resulted in two lysine-rich HIV-1AD8 Env variants (E.2 and AE.2) with neutralization- and cold-resistant phenotypes comparable to those of natural, less triggerable tier 2/3 HIV-1 isolates. Compared with these and the parental Envs, the E.2 and AE.2 Envs were cleaved more efficiently and exhibited stronger gp120-trimer association in detergent lysates. These highly cross-linkable Envs enriched in a pretriggered conformation should assist characterization of the structure and immunogenicity of this labile state. IMPORTANCE The development of an efficient vaccine is critical for combating HIV-1 infection worldwide. However, the instability of the pretriggered shape (state 1) of the viral envelope glycoprotein (Env) makes it difficult to raise neutralizing antibodies against HIV-1. Here, by introducing multiple changes in Env, we derived two HIV-1 Env variants that are enriched in state 1 and can be efficiently cross-linked to maintain this shape. These Env complexes are more stable in detergent, assisting their purification. Thus, our study provides a path to a better characterization of the native pretriggered Env, which should assist vaccine development.


Asunto(s)
Vacunas contra el SIDA , Infecciones por VIH , VIH-1 , Productos del Gen env del Virus de la Inmunodeficiencia Humana , Vacunas contra el SIDA/genética , Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , Detergentes , Glicoproteínas/química , Glicoproteínas/inmunología , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH/prevención & control , VIH-1/química , VIH-1/genética , VIH-1/inmunología , Humanos , Lisina , Conformación Proteica , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología
20.
Chemistry ; 28(21): e202200116, 2022 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-35238091

RESUMEN

Membrane proteins are of biological and pharmaceutical significance. However, their structural study is extremely challenging mainly due to the fact that only a small number of chemical tools are suitable for stabilizing membrane proteins in solution. Detergents are widely used in membrane protein study, but conventional detergents are generally poor at stabilizing challenging membrane proteins such as G protein-coupled receptors and protein complexes. In the current study, we prepared tandem triazine-based maltosides (TZMs) with two amphiphilic triazine units connected by different diamine linkers, hydrazine (TZM-Hs) and 1,2-ethylenediamine (TZM-Es). These TZMs were consistently superior to a gold standard detergent (DDM) in terms of stabilizing a few membrane proteins. In addition, the TZM-Es containing a long linker showed more general protein stabilization efficacy with multiple membrane proteins than the TZM-Hs containing a short linker. This result indicates that introduction of the flexible1,2-ethylenediamine linker between two rigid triazine rings enables the TZM-Es to fold into favourable conformations in order to promote membrane protein stability. The novel concept of detergent foldability introduced in the current study has potential in rational detergent design and membrane protein applications.


Asunto(s)
Detergentes , Proteínas de la Membrana , Detergentes/química , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de la Membrana/química , Estabilidad Proteica , Triazinas
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