The antigenic components of adult Platynosomum illiciens were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from cats naturally ...infected with P. illiciens, Dipylidium caninum, Toxocara cati and uninfected cat sera. The whole worm extract (WWE) of P. illiciens was fractionated by Sephadex G-200 gel filtration chromatography. The results showed that WWE fraction and F2 were highly antigenic as well as F1 and F3, which were moderately antigenic. For SDS-PAGE and immunoblotting, the antigenic molecules of WWE and all three fractions were mostly at molecular weights (MW) ranging from 11 to 150 kDa. Four antigenic proteins of 11, 18, 27 and 75 kDa detected in WWE and F1-F3 were found to give a reaction with sera from P. illiciens infected cats, and these proteins were also identified using liquid chromatography-mass spectrometry (LC-MS/MS). For immunolocalization observation, it was revealed that the P. illiciens antigen was present in high concentration in the cytoplasm of vitelline cells in the vitelline glands, the shell of the eggs and the eggs within the uterus, but not in other organs, i.e., tegument, muscle, parenchymal cells, testes and oral and ventral suckers of adult fluke. This finding indicates that these proteins may be potential antigen candidates for the immunodiagnosis of feline platynosomosis caused by P. illiciens.
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•WWE of Platynosomum illiciens was purified and 3 peak fractions were observed.•The 11, 18, 27 and 75 kDa were highly antigenic in WWE and F1-F3 of P. illiciens.•The LC/MS-MS was used to discriminate the 11, 18, 27 and 75 kDa peptide sequences.•P. illiciens antigen was present in the vitelline cells, the eggs shells and eggs.
Beef meat was cooked at 373 K for 10 and 30 min to investigate the effect of the cooking conditions generally used during beef stew and curry preparation on protein digestibility. The cooked meats, ...along with a raw control, were digested using an in vitro digestion model to simulate gastric and small-intestinal conditions. Samples taken at different digestion times were analyzed using SDS-PAGE, RP-HPLC, ninhydrin assays for amino N and transmission electron microscopy. Simulated gastric conditions quickly led to the loss of basic sarcomere structure in raw meat myofibrils whereas the sarcomere structure of the compact cooked meat myofibrils remained intact after 30 min of gastric digestion. Prolonged cooking of meat (30 min) resulted in incomplete digestion of small MW (<10 kDa) peptides, as observed from SDS-PAGE. This agreed with the amount of ninhydrin-reactive amino N released during digestion, which decreased with an increase in cooking time. The RP-HPLC peak areas of the major identified amino acids (tryptophan, tyrosine and phenylalanine) also decreased with an increase in cooking time. This suggested the formation of “limit peptides” during prolonged cooking of beef, which were not further broken down into free amino acids by digestive enzymes and therefore might not be bioavailable.
•Cooked and raw meats were digested under simulated gastro-small intestinal conditions.•The compact cooked meat microstructure was not easily broken down by digestive enzymes.•Prolonged cooking resulted in incomplete digestion of peptides with MW < 10 kDa.•Amino N release during digestion decreased with an increase in cooking time.•This suggests an increase in the formation of “limit peptides” with prolonged cooking.
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•Lower SDS concentrations stimulated amyloid fibrillation in Succinyl ConA.•Succinylation affected SDS-induced ConA amyloid fibrillation.•Higher SDS concentrations induced α-helical ...structure in Succinyl ConA.
The anionic surfactant sodium dodecyl sulfate (SDS) is homologous to the cellular membrane lipids, and is known to stimulate amyloid fibrillation in several proteins. However, the mechanism by which SDS influences aggregation and structural changes in succinylated protein has not been determined. In this study, we observed the effects of variable SDS concentrations on succinyl-ConA aggregation at pH 3.5 and proposed a possible mechanism of SDS-induced succinyl-ConA aggregation. We used several biophysical techniques to identify the changes caused by SDS. Our results suggest that SDS stimulates succinyl-ConA aggregation in a concentration-dependent manner. From turbidity measurements, it was evident that a very low concentration (<0.1 mM) of SDS did not induce succinyl-ConA aggregation and proteins remained soluble. However, aggregations were observed at 0.1–2.5 mM SDS, which then dissipated at SDS concentrations above 2.5 mM. Far-UV CD results suggest that the β-sheet secondary structure of succinyl-ConA transformed into the cross-β-sheet structure in the presence of aggregating SDS concentrations. Notably, at SDS concentrations above 2.5 mM, the succinyl-ConA β-sheet transformed into an α-helical structure. The SDS-induced succinyl-ConA amyloid-like aggregates were confirmed by transmission electron microscopy (TEM). We propose that SDS modulates amyloid fibrillation in succinyl-ConA due to electrostatic and hydrophobic interactions and succinylation affects SDS-induced succinyl-ConA aggregation.
Verticillium wilt of cotton is a vascular disease mainly caused by the soil‐born filamentous fungus Verticillium dahliae. To study the mechanisms associated with defense responses in wilt‐resistant ...sea‐island cotton (Gossypium barbadense) upon V. dahliae infection, a comparative proteomic analysis between infected and mock‐inoculated roots of G. barbadense var. Hai 7124 (a cultivar showing resistance against V. dahliae) was performed by 2‐DE combined with local EST database‐assisted PMF and MS/MS analysis. A total of 51 upregulated and 17 downregulated proteins were identified, and these proteins are mainly involved in defense and stress responses, primary and secondary metabolisms, lipid transport, and cytoskeleton organization. Three novel clues regarding wilt resistance of G. barbadense are gained from this study. First, ethylene signaling was significantly activated in the cotton roots attacked by V. dahliae as shown by the elevated expression of ethylene biosynthesis and signaling components. Second, the Bet v 1 family proteins may play an important role in the defense reaction against Verticillium wilt. Third, wilt resistance may implicate the redirection of carbohydrate flux from glycolysis to pentose phosphate pathway (PPP). To our knowledge, this study is the first root proteomic analysis on cotton wilt resistance and provides important insights for establishing strategies to control this disease.
The ability to routinely identify and quantify the complete proteome from single cells will greatly advance medicine and basic biology research. To meet this challenge of single-cell proteomics, ...single-molecule technologies are being developed and improved. Most approaches, to date, rely on the analysis of polypeptides, resulting from digested proteins, either in solution or immobilized on a surface. Nanopore biosensing is an emerging single-molecule technique that circumvents surface immobilization and is optimally suited for the analysis of long biopolymers, as has already been shown for DNA sequencing. However, proteins, unlike DNA molecules, are not uniformly charged and harbor complex tertiary structures. Consequently, the ability of nanopores to analyze unfolded full-length proteins has remained elusive. Here, we evaluate the use of heat denaturation and the anionic surfactant sodium dodecyl sulfate (SDS) to facilitate electrokinetic nanopore sensing of unfolded proteins. Specifically, we characterize the voltage dependence translocation dynamics of a wide molecular weight range of proteins (from 14 to 130 kDa) through sub-5 nm solid-state nanopores, using a SDS concentration below the critical micelle concentration. Our results suggest that proteins' translocation dynamics are significantly slower than expected, presumably due to the smaller nanopore diameters used in our study and the role of the electroosmotic force opposing the translocation direction. This allows us to distinguish among the proteins of different molecular weights based on their dwell time and electrical charge deficit. Given the simplicity of the protein denaturation assay and circumvention of the tailor-made necessities for sensing protein of different folded sizes, shapes, and charges, this approach can facilitate the development of a whole proteome identification technique.
Genetic abnormalities in mitochondrial complex assembling factors are associated with leukoencephalopathy. We present a 1‐year‐old girl with consciousness disturbance after a respiratory infection. ...Brain MRI revealed leukoencephalopathy with bilaterally symmetrical hyperintensity in the substantia nigra, medial thalamic nuclei, and basal nuclei, as well as cavities in the cerebral white matter and corpus callosum. Lactate levels in the spinal fluid were high, while magnetic resonance spectroscopy of the cerebral white matter and basal nuclei showed high peak lactate levels, suggesting mitochondrial dysfunction. The respiratory enzyme activity of complex I was reduced to 17% to 21% in skeletal muscle. Whole exome sequencing identified compound heterozygous variations in NDUFAF3, involved in the assembly of mitochondrial complex I (c.342_343insGTG:p.117Valdup, c.505C > A:p.Pro169Thr). Two‐dimensional, blue‐native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate‐PAGE revealed reductions in Q‐module (NDUFS2, NDUFS3, and NDUFA9) and P‐module (NDUFB10 and NDUFB11) subunits, indicating disruption of mitochondrial complex I assembly. Our report expands the spectrum of clinical phenotypes associated with pathogenic variants of NDUFAF3.
Cavitating leukoencephalopathy
The tolerability of single daily gavage doses of 0.5% or 2.0% (wt/vol) sodium lauryl sulfate (SLS) in 11- to 12-week-old male CD-1 mice was evaluated in a study of 3 months in duration. Live-phase, ...gross necropsy, and histopathologic parameters were evaluated. Mortality of 14% occurred in mice administered formulations containing SLS. Clinical observations in mice administered SLS included abnormal respiration (audible, irregular, and/or labored), swollen abdomen, rough haircoat, hunched appearance, and hypoactivity. Necropsy findings in mice administered SLS consisted of enlarged intestines containing abnormal contents with gas. There were no instances of mechanical gavage–related injury. Histologic evaluation of the respiratory tract revealed injury to the nasal passages and nasopharynx, including, but not limited to, inflammation, exudate, apoptosis/necrosis of epithelium, and atrophy of epithelium or olfactory nerves. Collectively, the data indicated that under the experimental conditions of our 3-month study in male CD-1 mice, once-daily gavage administration of vehicle formulations containing SLS at 0.5% or 2.0% resulted in nasal injury and 14% mortality supportive of gastroesophageal reflux. Sponsors utilizing formulations containing SLS in toxicity studies in CD-1 mice should exclude gastroesophageal reflux as a confounding factor in studies with morbidity or mortality associated with respiratory distress or evidence of aerophagia.
An acidic phospholipase A2 enzyme (NnPLA2-I) interacts with three finger toxins (cytotoxin and neurotoxin) from Naja naja venom to form cognate complexes to enhance its cytotoxicity towards rat L6 ...myogenic cells. The cytotoxicity was further enhanced in presence of trace quantity of venom nerve growth factor. The purified rat myoblast cell membrane protein showing interaction with NnPLA2-I was identified as vimentin by LC-MS/MS analysis. The ELISA, immunoblot and spectrofluorometric analyses showed greater binding of NnPLA2-I cognate complex to vimentin as compared to the binding of individual NnPLA2-I. The immunofluorescence and confocal microscopy studies evidenced the internalization of NnPLA2-I to partially differentiated myoblasts post binding with vimentin in a time-dependent manner. Pre-incubation of polyvalent antivenom with NnPLA2-I cognate complex demonstrated better neutralization of cytotoxicity towards L6 cells as compared to exogenous addition of polyvalent antivenom 60–240 min post treatment of L6 cells with cognate complex suggesting clinical advantage of early antivenom treatment to prevent cobra venom-induced cytotoxicity. The in silico analysis showed that 19–22 residues, inclusive of Asp48 residue, of NnPLA2-I preferentially binds with the rod domain (99–189 and 261–335 regions) of vimentin with a predicted free binding energy (ΔG) and dissociation constant (KD) values of −12.86 kcal/mol and 3.67 × 10−10 M, respectively; however, NnPLA2-I cognate complex showed greater binding with the same regions of vimentin indicating the pathophysiological significance of cognate complex in cobra venom-induced cytotoxicity.
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•NnPLA2-I, an acidic PLA2 enzyme forms cognate complex with 3FTxs from N. naja venom•NnPLA2-I binds to membrane-bound vimentin of rat myoblasts leading to its internalization•The cognate complex demonstrates superior binding and cytotoxicity towards myoblasts•A trace quantity of NGF further enhances binding and cytotoxicity of cognate complex•Immediate PAV administration can reduce NnV induced cytotoxicity in myogenic cells
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