Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 20.296
Filtrar
1.
J Cell Sci ; 136(2)2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36655611

RESUMO

Proteins entering the secretory pathway need to attain native disulfide pairings to fold correctly. For proteins with complex disulfides, this process requires the reduction and isomerisation of non-native disulfides. Two key members of the protein disulfide isomerase (PDI) family, ERp57 and ERdj5 (also known as PDIA3 and DNAJC10, respectively), are thought to be required for correct disulfide formation but it is unknown whether they act as a reductase, an isomerase or both. In addition, it is unclear how reducing equivalents are channelled through PDI family members to substrate proteins. Here, we show that neither enzyme is required for disulfide formation, but ERp57 is required for isomerisation of non-native disulfides within glycoproteins. In addition, alternative PDIs compensate for the absence of ERp57 to isomerise glycoprotein disulfides, but only in the presence of a robust reductive pathway. ERdj5 is required for this alternative pathway to function efficiently indicating its role as a reductase. Our results define the essential cellular functions of two PDIs, highlighting a distinction between formation, reduction and isomerisation of disulfide bonds.


Assuntos
Oxirredutases , Isomerases de Dissulfetos de Proteínas , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/metabolismo , Oxirredutases/metabolismo , Dobramento de Proteína , Glicoproteínas/metabolismo , Dissulfetos/metabolismo , Oxirredução
2.
Methods Mol Biol ; 2617: 165-176, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36656523

RESUMO

Cytoplasmic expression of recombinant proteins requiring disulfide bridges in Escherichia coli usually leads to the formation of insoluble inclusion bodies (IBs). The reason for this phenomenon is found in the reducing environment of the cytoplasm, preventing the formation of disulfide bridges and therefore resulting in inactive protein aggregates. However, IBs can be refolded in vitro to obtain the protein in its active conformation. In order to correctly form the required disulfide bridges, cystines are fully reduced during solubilization and, with the help of an oxidizing agent, the native disulfide bridges are formed during the refolding step. Here, a protocol to identify suitable redox conditions for solubilization and refolding is presented. For this purpose, a multivariate approach spanning the unit operations solubilization and refolding is used.


Assuntos
Síndrome do Intestino Irritável , Humanos , Proteínas Recombinantes/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Oxirredução , Dissulfetos/metabolismo , Redobramento de Proteína , Dobramento de Proteína , Solubilidade
3.
J Am Chem Soc ; 145(3): 1964-1972, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36633218

RESUMO

Multicyclic peptides with stable 3D structures are a kind of novel and promising peptide formats for drug design and discovery as they have the potential to combine the best characteristics of small molecules and proteins. However, the development of multicyclic peptides is largely limited to naturally occurring products. It remains a big challenge to develop multicyclic peptides with new structures and functions without recourse to the existing natural scaffolds. Here, we report a general and robust method relying on the utility of new disulfide-directing motifs for designing and discovering diverse multicyclic peptides with potent protein-binding capability. These peptides, referred to as disulfide-directed multicyclic peptides (DDMPs), are tolerant to extensive sequence manipulations and variations of disulfide-pairing frameworks, enabling the development of de novo DDMP libraries useful for ligand and drug discovery. This study opens a new avenue for creating a new generation of multicyclic peptides in sequence and structure space inaccessible by natural scaffolds, thus would greatly benefit the field of peptide drug discovery.


Assuntos
Dissulfetos , Biblioteca de Peptídeos , Ligantes , Peptídeos/química , Desenho de Fármacos
4.
PLoS One ; 18(1): e0280778, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36696377

RESUMO

Integrin αMß2 (Mac-1, CD11b/CD18, CR3) is an important adhesion receptor expressed on monocytes. Mac-1 is responsible for mediating cell migration, phagocytosis, degranulation as well as cell-cell fusion. It is also the most promiscuous integrin in terms of ligand specificity with over 100 ligands, most of which use the αMI-domain as their binding site. Despite the importance of αMI-domain in defining ligand interactions of Mac-1, structural studies of αMI-domain's interactions with ligands are lacking. In particular, solution NMR studies of αMI-domain's interaction with ligands have not been possible because the most commonly used active αMI-domain mutants (I316G and ΔK315) are not sufficiently stable and soluble to be used in solution NMR. The goal of this study is to identify an αMI-domain active mutant that's amenable to NMR characterization. By screening known activating mutations of αMI-domain, we determined that the Q163C/Q309C mutant, which converts the αMI-domain into its active form through the formation of an intramolecular disulfide bond, can be produced with a high yield and is more stable than other active mutants. In addition, the Q163C/Q309C mutant has better NMR spectral quality than other active mutants and its affinity for ligands is comparable to other active mutants. Analysis of the Co2+-induced pseudocontact shifts in the Q163C/Q309C mutant showed the structure of the mutant is consistent with the active conformation. Finally, we show that the minor fraction of the Q163C/Q309C mutant without the disulfide bond can be removed through the use of carboxymethyl sepharose chromatography. We think the availability of this mutant for NMR study will significantly enhance structural characterizations of αMI-domain-ligand interactions.


Assuntos
Dissulfetos , Antígeno de Macrófago 1 , Adesão Celular , Sequência de Aminoácidos , Ligantes , Antígeno de Macrófago 1/metabolismo
5.
Proc Natl Acad Sci U S A ; 120(5): e2215575120, 2023 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-36696445

RESUMO

Chloroplast division involves the coordination of protein complexes from the stroma to the cytosol. The Min system of chloroplasts includes multiple stromal proteins that regulate the positioning of the division site. The outer envelope protein PLASTID DIVISION1 (PDV1) was previously reported to recruit the cytosolic chloroplast division protein ACCUMULATION AND REPLICATION OF CHLOROPLAST5 (ARC5). However, we show here that PDV1 is also important for the stability of the inner envelope chloroplast division protein PARALOG OF ARC6 (PARC6), a component of the Min system. We solved the structure of both the C-terminal domain of PARC6 and its complex with the C terminus of PDV1. The formation of an intramolecular disulfide bond within PARC6 under oxidized conditions prevents its interaction with PDV1. Interestingly, this disulfide bond can be reduced by light in planta, thus promoting PDV1-PARC6 interaction and chloroplast division. Interaction with PDV1 can induce the dimerization of PARC6, which is important for chloroplast division. Magnesium ions, whose concentration in chloroplasts increases upon light exposure, also promote the PARC6 dimerization. This study highlights the multilayer regulation of the PDV1-PARC6 interaction as well as chloroplast division.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Plastídeos/metabolismo , Cloroplastos/metabolismo , Dissulfetos/metabolismo , Dinaminas/metabolismo
6.
Anal Chim Acta ; 1243: 340800, 2023 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-36697173

RESUMO

Protein structure dynamics in solution and from solution to gas phase are important but challenging topics. Great efforts and advances have been made especially since the wide application of ion mobility mass spectrometry (IM-MS), by which protein collision cross section (CCS) in gas phase could be measured. Due to the lack of efficient experimental methods, protein structures in protein databank are typically referred as their structures in solution. Although conventional structural biology techniques provide high-resolution protein structures, complicated and stringent processes also limit their applicability under different solvent conditions, thus preventing the capture of protein dynamics in solution. Enabled by the combination of mobility capillary electrophoresis (MCE) and IM-MS, an efficient experimental protocol was developed to characterize protein conformation dynamics in solution and from solution to gas phase. As a first attempt, key factors that affecting protein conformations were distinguished and evaluated separately, including pH, temperature, softness of ionization process, presence and specific location of disulfide bonds. Although similar extent of unfolding could be observed for different proteins, in-depth analysis reveals that pH decrease from 7.0 to 3.0 dominates the unfolding of proteins without disulfide bonds in conventional ESI-MS experiments; while harshness of the ionization process dominates the unfolding of proteins with disulfide bonds. Second, disulfide bonds show capability of preserving protein conformations in acidic solution environments. However, by monitoring protein conformation dynamics and comparing results from different proteins, it is also found that their capability is position dependent. Surprisingly, disulfide bonds did not show the capability of preserving protein conformations during ionization processes.


Assuntos
Dissulfetos , Proteínas , Espectrometria de Massas/métodos , Conformação Proteica , Proteínas/química , Domínios Proteicos
7.
Food Res Int ; 163: 112220, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36596149

RESUMO

pH-responsive in situ gelling properties of thiolated citrus high-methoxyl pectin (TCHMP) were investigated in this study. The gelation capacity results revealed that the in situ gelation behavior of TCHMP only occurred when the pH value was higher than 6.25. The gel strength increased from 26.63 g to 42.77 g as the pH value increased from 7.4 to 8.9. Rheological measurements confirmed that the apparent viscosity and viscoelasticity of TCHMP were highly dependent on pH value and dialysis time. Compared with the control group, the apparent viscosity of TCHMP dialyzed in phosphate-buffered saline (PBS) of pH 8.9 for 180 min increased 695-fold. During the dialysis process of TCHMP at different pH values (7.4-8.9), the final thiol groups content decreased and the final disulfide bonds content increased with the increase in pH value. This illustrates that the mechanism of in situ gelation is mainly the oxidation of thiol-thiol groups to form disulfide bonds. These results can put forward new insights into the pH-responsive in situ gelling properties of TCHMP and provide a theoretical basis for the application of TCHMP in neutral and alkaline gel systems.


Assuntos
Citrus , Compostos de Sulfidrila , Concentração de Íons de Hidrogênio , Géis/química , Compostos de Sulfidrila/química , Pectinas/química , Dissulfetos/química
8.
Molecules ; 28(1)2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36615624

RESUMO

The interactions of the functional additives SPS (bis-(sodium-sulfopropyl)-disulfide) and polyethylene glycol (PEG) in the presence of chloride ions were studied by time-of-flight secondary-ion mass spectrometry (TOF-SIMS) in combination with cyclic voltammetry measurements (CV). The PEG, thiolate, and chloride surface coverages were estimated and discussed in terms of their electrochemical suppressing/accelerating abilities. The conformational influence of both the gauche/trans thiolate molecules, as well as around C-C and C-O of PEG, on the electrochemical properties were discussed. The contribution of the hydrophobic interaction of -CH2-CH2- of PEG with chloride ions was only slightly reduced after the addition of SPS, while the contribution of Cu-PEG adducts diminished strongly. SPS and PEG demonstrated significant synergy by significant co-adsorption. It was shown that the suppressing abilities of PEG that rely on forming stable Cu-PEG adducts, identified in the form C2H4O2Cu+ and C3H6OCu+, were significantly reduced after the addition of SPS. The major role of thiolate molecules adsorbed on a copper surface in reducing the suppressing abilities of PEG rely on the efficient capture of Cu2+ ions, diminishing the available copper ions for the ethereal oxygen of PEG.


Assuntos
Cobre , Polietilenoglicóis , Polietilenoglicóis/química , Sódio , Cloretos , Dissulfetos , Espectrometria de Massa de Íon Secundário
9.
Ital J Pediatr ; 49(1): 3, 2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36611205

RESUMO

BACKGROUND: Vitamin B12 is an important vitamin for metabolism and affects many mechanisms in the body including neuronal migration, DNA synthesis, neurotransmitter synthesis, brain and cognitive development. Increased oxidative stress in the body leads to the damage of the child development, but also plays a crucial role in the pathogenesis of many diseases encountered in the childhood period. Our aim is to investigate whether or not B12 deficiency is associated with dynamic thiol/disulfide homeostasis in adolescent patients. METHODS: This is a case-controlled observational study consisting of 45 adolescent patients with vitamin b12 deficiency and a control group consisting of 45 healthy adolescent. Patients between 11 and 18 ages who applied to the outpatient clinic for the first time with one of the complaints of headache were selected due to their decreased school performance, dizziness, and fatigue. Hemogram, vitamin B12, homocysteine levels and oxidative stress parameters such as native and total thiol disulfide levels and ratios of disulfide/native thiol, disulfide/total thiol, and native thiol/total thiol were measured from the patients. RESULTS: Vitamin B12 level was found to be significantly lower in vitamin B12 deficiency group (p < 0.001). The serum disulfide level was found to be 27.5 ± 8.38 in the case group and 20.5 ± 8.36 in the control group (p < 0.001). In the multiple linear regression analysis, it was determined that the independent variables of native thiol, homocysteine and disulfide levels effected of vitamin B12 levels (p < 0.001, p < 0.001, p < 0.005 respectively; R2 = 0.62). CONCLUSION: The results obtained in terms of the effect of vitamin B12 deficiency on oxidative stress in adolescents are remarkable. The increase in oxidative stress parameters in the patient group may also suggest that oxidative stress plays a vital role in vitamin B12 deficiency in adolescence.


Assuntos
Dissulfetos , Deficiência de Vitamina B 12 , Criança , Humanos , Adolescente , Compostos de Sulfidrila , Vitaminas , Deficiência de Vitamina B 12/diagnóstico , Vitamina B 12 , Estresse Oxidativo/fisiologia
10.
Int J Mol Sci ; 24(2)2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36674720

RESUMO

PAF and related antifungal proteins are promising antimicrobial agents. They have highly stable folds around room temperature due to the presence of 3-4 disulfide bonds. However, unfolded states persist and contribute to the thermal equilibrium in aqueous solution, and low-populated states might influence their biological impact. To explore such equilibria during dimethyl sulfoxide (DMSO)-induced chemical unfolding, we studied PAF and its inactive variant PAFD19S using nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSC). According to the NMR monitoring at 310 K, the folded structures disappear above 80 v/v% DMSO concentration, while the unfolding is completely reversible. Evaluation of a few resolved peaks from viscosity-compensated 15N-1H HSQC spectra of PAF yielded ∆G = 23 ± 7 kJ/M as the average value for NMR unfolding enthalpy. The NMR-based structures of PAF and the mutant in 50 v/v% DMSO/H2O mixtures were more similar in the mixed solvents then they were in water. The 15N NMR relaxation dynamics in the same mixtures verified the rigid backbones of the NMR-visible fractions of the proteins; still, enhanced dynamics around the termini and some loops were observed. DSC monitoring of the Tm melting point showed parabolic dependence on the DMSO molar fraction and suggested that PAF is more stable than the inactive PAFD19S. The DSC experiments were irreversible due to the applied broad temperature range, but still suggestive of the endothermic unfolding of PAF.


Assuntos
Antifúngicos , Dimetil Sulfóxido , Dimetil Sulfóxido/farmacologia , Dimetil Sulfóxido/química , Antifúngicos/farmacologia , Antifúngicos/química , Varredura Diferencial de Calorimetria , Dissulfetos/química , Proteínas , Espectroscopia de Ressonância Magnética , Água , Termodinâmica , Desnaturação Proteica , Desdobramento de Proteína
11.
Sci Rep ; 13(1): 428, 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36624120

RESUMO

Electroplated Cu has been extensively applied in advanced electronic packaging, and its mechanical properties are critical for reliability. In this study, Cu foils fabricated through electroplating with various bis-(3-sulfopropyl) disulfide (SPS) concentrations are examined using tensile tests. The SPS concentration affects the grain size of the electroplated Cu foils, resulting in different mechanical properties. A significant Hall-Petch effect, [Formula: see text], is demonstrated for the electroplated Cu foils. The different concentrations of impurities identified through time-of-flight secondary ion mass spectrometry correspond to the different grain sizes, determining the transgranular and intergranular fracture during the tensile test. The results demonstrate that the SPS concentration controlling the microstructures of the electroplated Cu results in a Hall-Petch effect on the mechanical properties of the electroplated Cu foils.


Assuntos
Cobre , Dissulfetos , Reprodutibilidade dos Testes , Embalagem de Medicamentos , Grão Comestível
12.
Biochem J ; 480(1): 87-104, 2023 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-36594794

RESUMO

Thioredoxins (Trxs) are ubiquitous proteins that play vital roles in several physiological processes. Alr2205, a thioredoxin-like protein from Anabaena PCC 7120, was found to be evolutionarily closer to the Trx-domain of the NADPH-Thioredoxin Reductase C than the other thioredoxins. The Alr2205 protein showed disulfide reductase activity despite the presence a non-canonical active site motif 'CPSC'. Alr2205 not only physically interacted with, but also acted as a physiological reductant of Alr4641 (the typical 2-Cys-Peroxiredoxin from Anabaena), supporting its peroxidase function. Structurally, Alr2205 was a monomeric protein that formed an intramolecular disulfide bond between the two active site cysteines (Cys-38 and Cys-41). However, the Alr2205C41S protein, wherein the resolving cysteine was mutated to serine, was capable of forming intermolecular disulfide bond and exist as a dimer when treated with H2O2. Overproduction of Alr2205 in E. coli protected cells from heavy metals, but not oxidative stress. To delve into its physiological role, Alr2205/Alr2205C41S was overexpressed in Anabaena, and the ability of the corresponding strains (An2205+ or An2205C41S+) to withstand environmental stresses was assessed. An2205+ showed higher resistance to H2O2 than An2205C41S+, indicating that the disulfide reductase function of this protein was critical to protect cells from this peroxide. Although, An2205+ did not show increased capability to withstand cadmium stress, An2205C41S+ was more susceptible to this heavy metal. This is the first study that provides a vital understanding into the function of atypical thioredoxins in countering the toxic effects of heavy metals/H2O2 in prokaryotes.


Assuntos
Anabaena , Cianobactérias , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Peróxido de Hidrogênio/metabolismo , Cisteína/genética , Cisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Oxirredução , Proteínas de Bactérias/metabolismo , Anabaena/genética , Anabaena/metabolismo , Cianobactérias/metabolismo , Tiorredoxinas/química , Dissulfetos/metabolismo , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxina Dissulfeto Redutase/metabolismo
13.
Microb Cell Fact ; 22(1): 9, 2023 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-36635697

RESUMO

BACKGROUND: A number of antimicrobial peptides (AMPs) hold promise as new drugs owing to their potent bactericidal activity and because they are often refractory to the development of drug resistance. Cryptdins (Crps) are a family of antimicrobial peptides found in the small intestine of mice, comprising six isoforms containing three sets of disulfide bonds. Although Crp4 is actively being investigated, there have been few studies to date on the other Crp isoforms. A prerequisite for detailed characterization of the other Crp isoforms is establishment of efficient sample preparation methods. RESULTS: To avoid degradation during recombinant expression of Crps in E. coli, co-expression of Crps with the aggregation-prone protein human α-lactalbumin (HLA) was used to promote the formation of stable inclusion bodies. Using this method, the production of Crp4 and Crp6 by the BL21 strain was effective, but the expression of other Crp isoforms was not as efficient. The results of a cell-free system study suggested that Crps were degraded, even though a substantial amounts of Crps were synthesized. Therefore, using the Origami™ B strain, we were able to significantly increase the expression efficiency of Crps by promoting the formation of erroneous intermolecular disulfide bonds between HLA and Crps, thereby promoting protein aggregation and inclusion body formation, which prevented degradation. The various Crp isoforms were successfully refolded in vitro and purified using reversed-phase HPLC. In addition, the yield was further improved by deformylation of formyl-Crps. We measured the antibacterial activity of Crps against both Gram-positive and Gram-negative bacteria. Each Crp isoform exhibited a completely different trend in antimicrobial activity, although conformational analysis by circular dichroism did not reveal any significant steric differences. CONCLUSION: In this study, we established a novel and efficient method for the production of the cryptdin family of cysteine-containing antimicrobial peptides. Additionally, we found that there were notable differences in the antibacterial activities of the various Crp family members. The expression system established in this study is expected to provide new insights regarding the mechanisms underlying the different antibacterial activities of the Crp family of peptides.


Assuntos
Antibacterianos , alfa-Defensinas , Humanos , Animais , Camundongos , Antibacterianos/farmacologia , Antibacterianos/química , Escherichia coli/metabolismo , alfa-Defensinas/análise , alfa-Defensinas/química , alfa-Defensinas/metabolismo , Bactérias Gram-Positivas/metabolismo , Bactérias Gram-Negativas/metabolismo , Isoformas de Proteínas/genética , Corpos de Inclusão/metabolismo , Dissulfetos/química
14.
Carbohydr Polym ; 304: 120495, 2023 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-36641180

RESUMO

We report the green synthesis of trimethyl chitosan-functionalized poly(2-hydroxyethyl methacrylate) (PHEMA-TMC) nanogels via surfactant-free emulsion photopolymerization. TMC, a quaternized derivative of chitosan, was synthesized through methylation of chitosan, resulting in quaternary and tertiary amine groups as the main substitution products. TMC tertiary amine moiety and riboflavin (RF) acted as a redox photo-initiating system to generate free radicals for the polymerization under light irradiation. The effects of polymerization parameters such as irradiation time, concentrations of TMC and RF were investigated using MBA as crosslinker. Under the optimal condition of 1 % TMC, 4 % HEMA, 0.8 µM RF, 5 % MBA, and 4 h of polymerization time, the cationic PHEMA-TMC nanogel was synthesized with 76 % monomer conversion and an average diameter of about 106 nm. Moreover, the disulfide-crosslinked PHEMA-TMC nanogel was also synthesized using the disulfide dimethacrylate crosslinker, which exhibited a redox-induced degradation and release of encapsulated melatonin, potentially useful as a redox-responsive drug delivery carrier.


Assuntos
Quitosana , Portadores de Fármacos , Nanogéis , Quitosana/farmacologia , Poli-Hidroxietil Metacrilato , Emulsões , Oxirredução , Aminas , Dissulfetos
15.
Pediatr Surg Int ; 39(1): 75, 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36617603

RESUMO

PURPOSE: This study aimed to examine the diagnostic value of IL-6, thiol-disulfide homeostasis, complete blood count and inflammatory biomarkers in the prediction of acute appendicitis in children. METHODS: The study was designed as a prospective and controlled study in children-the study was conducted at a tertiary referential university hospital between May 2020 and April 2021. Patients were divided between study groups and one control group (CG): 1: confirmed acute appendicitis group (AAP); 2: perforated appendicitis group (PAP); and 3: non-specified abdominal pain (NAP). The age and gender of the patients were determined. The following listed laboratory parameters were compared between groups: TOS: total oxidative status, TAS: total antioxidant status, OSI: oxidative stress index, TT: total thiol, NT (µmol/L): native thiol, DIS: disulfide, IL-6: interleukin 6, TNF-a: tumor necrosis factor-alpha, WBC: white blood cell, NEU: neutrophil, NEU%: neutrophil percentage, LY: lymphocyte, LY%: lymphocyte percentage, PLT: platelet, MPV: mean platelet volume NLR: neutrophil lymphocyte ratio, CRP: C-reactive protein, LCR: lymphocyte CRP ratio, and serum lactate. RESULTS: The TOS level of the PAP group was found to be significantly higher than that in the AAP, NAP and control groups (p = 0.006, < 0.001 and p < 0.001). TAS, TT, and NT levels in the PAP group were significantly lower than those in the AAP, NAP and control groups. OSI was significantly higher in the PAP group than in the other groups. The TT and NT levels of the NAP group were both similar to those of the control group. Serum DIS level was similar between the AAP and PAP groups, AAP and NAP groups, and NAP and control groups. Serum IL-6 and TNF-α levels were found to be significantly higher in the PAP group compared to those in all groups. The WBC, NEU, and NEU% values were found to be significantly higher in the PAP group than those in the NAP and control groups, while LY and LY% values were found to be significantly lower. PAP and AAP groups were found to be similar in terms of WBC, NEU, LYM, NEU%, and LYM% values. PLT and MPV values and serum lactate values did not show a significant difference between the groups. NLR was similar in the AAP and PAP groups. A significant increase in CRP versus a decrease in LCR was detected in the PAP group compared to that in the AAP group. Multivariate analysis demonstrated that only IL-6 has significant estimated accuracy rates as 80% for the control group, 78.8% for AAP, 96.9% for PAP, and 81.6% for NAP. CONCLUSION: Rather than AAP, PAP caused significantly higher oxidative stress (increased TOS and OSI), and lower antioxidation capacity (decreased TT and NT). IL-6 levels can provide a significant stratification. Nevertheless, simply detecting WBC or CRP is not enough to distinguish the specific pathology in acute appendicitis and related conditions.


Assuntos
Apendicite , Interleucina-6 , Humanos , Criança , Apendicite/diagnóstico , Dissulfetos , Compostos de Sulfidrila , Estudos Prospectivos , Biomarcadores , Antioxidantes , Homeostase , Lactatos
16.
Molecules ; 28(2)2023 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-36677701

RESUMO

The effects of Steam Flash-Explosion (SFE) on the physicochemical properties and molecular structure of high-temperature denatured defatted rice bran protein isolate (RBPI) were investigated. The mechanism of SFE treatment on high-temperature denatured defatted RBPI was revealed. The analysis of the physical and chemical properties of RBPI showed that the surface hydrophobicity, characteristic viscosity, and thermal stability of rice bran protein isolate were significantly affected by the pressure of saturated steam and pressure holding time. Under the conditions of 2.1 MPa and 210 s, the surface hydrophobicity index decreased significantly from 137.5 to 17.5, and the characteristic viscosity increased significantly. The peak temperature of denaturation decreases from 114.2 to 106.7 °C, and the enthalpy of denaturation decreases from 356.3 to 231.4 J/g. The higher structure (circular dichroic spectrum and endogenous fluorescence spectrum) of rice bran protein isolate was analyzed by volume rejection chromatography (SEC). The results showed that steam flash treatment could depolymerize and aggregate RBPI, and the relative molecular weight distribution changed greatly. The decrease in small molecules with poor solubility was accompanied by the increase in macromolecules (>550 kDa) soluble aggregates, which were the products of a Maillard reaction. The contents of free sulfhydryl and disulfide bonds in high-temperature rice bran meal protein isolate were significantly increased, which resulted in the increase in soluble aggregates containing disulfide bonds. Circular dichroism (CD) analysis showed that the α-helix content of the isolated protein was significantly decreased, the random curl content was increased, and the secondary structure of the isolated protein changed from order to disorder. The results of endogenous fluorescence spectroscopy showed that the high-temperature rice bran meal protein isolate was more extended, tryptophan was in a more hydrophilic microenvironment, the fluorescence intensity was reduced, and the tertiary structure was changed. In addition, the mean particle size and net surface charge of protein isolate increased in the aqueous solution, which was conducive to the development of the functional properties of the protein.


Assuntos
Oryza , Vapor , Oryza/química , Temperatura , Explosões , Dissulfetos
17.
Biomater Adv ; 144: 213235, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36495841

RESUMO

Mucoadhesive thermogels were developed by crosslinking poly(n-isopropylacrylamide) based polymers with chitosan and incorporating disulfide bridges, capable of releasing cysteamine upon interaction with mucin, for the treatment of cystinosis. Through crosslinking with chitosan and incorporating varying concentrations of the disulfide monomer into the polymer backbone, the extent of how mucoadhesive the developed thermogels were could be controlled. Through disulfide bridging with mucin, the thermogels released 6 to 10 µg of the conjugate model 2-mercaptopyridine over five days. Utilizing chitosan as the crosslinker, the developed thermogels were shown to degrade to a statistically higher extent following incubation with lysozyme, the highest concentration tear enzyme, by gravimetric and rheologic analysis. The developed thermogels were extensively tested in vivo utilizing a rat model in which materials were applied directly to the corneal surface and a rabbit model in which thermogels were applied to the inferior fornix. With the developed models, there was no adverse reactions or visual discomfort incurred following application of the thermogels. It has been demonstrated that the thermogels produced can be applied to the inferior fornix and release the stable conjugated payload over several days. The developed thermogel was designed to improve upon the current clinical treatment options for ocular cystinosis which are acidic topical formulations that require reapplication multiple times a day.


Assuntos
Quitosana , Cistinose , Ratos , Animais , Coelhos , Polímeros , Géis , Dissulfetos , Mucinas
18.
Mol Pharm ; 20(1): 461-472, 2023 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-36525349

RESUMO

The oral absorption of paclitaxel (PTX) is restricted by poor solubility in the gastrointestinal tract (GIT), low permeability, and high first-pass metabolism. Lipid carriers, such as a self-microemulsifying drug delivery system (SMEDDS), have been deemed as promising vehicles for promoting oral delivery of PTX. Herein, a lipophilic disulfide-bridged linoleic prodrug (PTX-S-S-LA) was synthesized and efficiently incorporated into SMEDDS to facilitate the oral absorption of PTX. This study mainly aims to evaluate the usefulness of the disulfide-bridged linoleic prodrug incorporated with SMEDDS and provides a new strategy for efficient oral delivery of PTX. The prodrug SMEDDS showed a markedly higher drug loading efficiency (3-fold) compared to that of parent PTX. PTX-S-S-LA SMEDDS significantly increased the drug partition (about 1.9-fold) in the intestinal micellar aqueous phase compared to PTX in the in vitro lipolysis study. Additionally, the gastrointestinal (GI) biodistribution study demonstrated that SMEDDS could enhance the GI biological adhesion and go through the lymphatic system to transport. Moreover, it was found that the reduction-sensitive prodrug (PTX-S-S-LA) has good stability in the GIT, leading to an improved antitumor efficiency without significant GI toxicity. Overall, the PTX-linoleic prodrug (PTX-S-S-LA) in combination with SMEDDS provides a promising way to enable effective oral delivery of PTX.


Assuntos
Pró-Fármacos , Paclitaxel , Dissulfetos , Distribuição Tecidual , Emulsões , Sistemas de Liberação de Medicamentos , Solubilidade , Disponibilidade Biológica , Administração Oral
19.
Redox Biol ; 59: 102583, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36567215

RESUMO

Protein disulfide isomerases (PDIs) catalyze redox reactions that reduce, oxidize, or isomerize disulfide bonds and act as chaperones of proteins as they fold. The characteristic features of PDIs are the presence of one or more catalytic thioredoxin (TRX)-like domains harboring typical CXXC catalytic motifs responsible for redox reactions, as well as non-catalytic TRX-like domain. As increasing attention is paid to oxidative post-translational modifications of cysteines (Cys ox-PTMs) with the recognition that they control cellular signaling, strategies to identify sites of Cys ox-PTM by redox proteomics have been optimized. Exploration of an available Cys redoxome dataset supported by modeled structure provided arguments for the existence of an additional non-catalytic thiol-disulfide motif, distinct from those contained in the TRX type patterns, typical of PDIAs. Further structural analysis of PDIA3 and 6 allows us to consider the possibility that this hypothesis could be extended to other members of PDI. These elements invite future studies to decipher the exact role of these non-catalytic thiol-disulfide motifs in the functions of PDIs. Strategies that would allow to validate this hypothesis are discussed.


Assuntos
Isomerases de Dissulfetos de Proteínas , Compostos de Sulfidrila , Isomerases de Dissulfetos de Proteínas/metabolismo , Dissulfetos/química , Tiorredoxinas/metabolismo , Proteômica , Oxirredução , Cisteína/metabolismo
20.
Toxicon ; 222: 106994, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36529153

RESUMO

Sticholysin I (StI) is a water-soluble protein with the ability to bind membranes where it oligomerizes and forms pores leading to cell death. Understanding the assembly property of this protein may be valuable for designing potential biotechnological tools, such as stable or structurally defined nanopores. In order to get insights into the stabilization of StI oligomers by disulfide bonds, we designed and characterized single and double cysteine mutants at the oligomerization interface. The oligomer formation was induced in the presence of lipid membranes and visualized by SDS-PAGE. The contribution of the oligomeric structures to the membrane binding and pore-forming capacities of StI was assessed. Single and double cysteine introduction at the protein-protein oligomerization interface does not considerably affect the conformation and function of the monomeric protein. In the presence of membranes, a cysteine double mutation at positions 15 and 59 favored formation of different size oligomers stabilized by disulfide bonds. The results of this work highlight the relevance of these positions (15 and 59) to be considered for developing biosensors based on nanopores from StI.


Assuntos
Cisteína , Toxinas Biológicas , Cisteína/química , Dissulfetos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...