RESUMEN
OBJECTIVE: This comparative analysis aimed to investigate the efficacy of Sivelestat Sodium Hydrate (SSH) combined with Ulinastatin (UTI) in the treatment of sepsis with acute respiratory distress syndrome (ARDS). METHODS: A control group and an observation group were formed with eighty-four cases of patients with sepsis with ARDS, with 42 cases in each group. The control group was intravenously injected with UTI based on conventional treatment, and the observation group was injected with SSH based on the control group. Both groups were treated continuously for 7 days, and the treatment outcomes and efficacy of both groups were observed. The Murray Lung Injury Score (MLIS), Sequential Organ Failure Assessment (SOFA), and Acute Physiology and Chronic Health Evaluation II (APACHE II) were compared. Changes in respiratory function, inflammatory factors, and oxidative stress indicators were assessed. The occurrence of adverse drug reactions was recorded. RESULTS: The total effective rate in the observation group (95.24%) was higher than that in the control group (80.95%) (P < 0.05). The mechanical ventilation time, intensive care unit (ICU) hospitalization time, and duration of antimicrobial medication in the observation group were shorter and multiple organ dysfunction syndrome incidence was lower than those in the control group (P < 0.05). The mortality rate of patients in the observation group (35.71%) was lower than that in the control group (52.38%), but there was no statistically significant difference between the two groups (P > 0.05). MLIS, SOFA, and APACHE II scores in the observation group were lower than the control group (P < 0.05). After treatment, respiratory function, inflammation, and oxidative stress were improved in the observation group (P < 0.05). Adverse reactions were not significantly different between the two groups (P > 0.05). CONCLUSION: The combination of SSH plus UTI improves lung injury and pulmonary ventilation function, and reduces inflammation and oxidative stress in patients with sepsis and ARDS.
Asunto(s)
Quimioterapia Combinada , Glicina , Glicoproteínas , Síndrome de Dificultad Respiratoria , Sepsis , Sulfonamidas , Humanos , Masculino , Sepsis/tratamiento farmacológico , Sepsis/complicaciones , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Femenino , Persona de Mediana Edad , Glicoproteínas/administración & dosificación , Glicoproteínas/uso terapéutico , Anciano , Glicina/análogos & derivados , Glicina/uso terapéutico , Glicina/administración & dosificación , Sulfonamidas/administración & dosificación , Sulfonamidas/uso terapéutico , Resultado del Tratamiento , Respiración Artificial , APACHE , Adulto , Insuficiencia Multiorgánica/etiología , Insuficiencia Multiorgánica/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Puntuaciones en la Disfunción de Órganos , Unidades de Cuidados Intensivos , Inhibidores de Tripsina/administración & dosificación , Inhibidores de Tripsina/uso terapéuticoRESUMEN
Tribolium castaneum, also known as the red flour beetle, is a polyphagous pest that seriously damages agricultural products, including stored and processed grains. Researchers have aimed to discover alternative pest control mechanisms that are less harmful to the ecosystem than those currently used. We conduct the purification and characterization of a protease inhibitor from C. plumieri seeds and an in vitro evaluation of its insecticidal potential against the insect pest T. castaneum. The trypsin inhibitor was isolated from C. plumieri seeds in a single-step DEAE-Sepharose column chromatography and had a molecular mass of 50 kDA. When analyzed for interaction with different proteolytic enzymes, the inhibitor exhibited specificity against trypsin and no activity against other serine proteases such as chymotrypsin and elastase-2. The isolated inhibitor was able to inhibit digestive enzymes of T. castaneum from extracts of the intestine of this insect. Therefore, we conclude that the new protease inhibitor, specific in tryptic inhibition, of protein nature from the seeds of C. plumieri was effective in inhibiting the digestive enzymes of T. castaneum and is a promising candidate in the ecological control of pests.
Asunto(s)
Tribolium , Inhibidores de Tripsina , Animales , Inhibidores de Tripsina/farmacología , Inhibidores de Tripsina/química , Inhibidores de Tripsina/aislamiento & purificación , Tribolium/enzimología , Tribolium/efectos de los fármacos , Proteínas de Insectos/química , Proteínas de Insectos/aislamiento & purificación , Proteínas de Insectos/antagonistas & inhibidores , Semillas/química , Insecticidas/farmacología , Insecticidas/química , Insecticidas/aislamiento & purificación , Proteínas de Plantas/farmacología , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/químicaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. Known as COVID-19, it has affected billions of people worldwide, claiming millions of lives and posing a continuing threat to humanity. This is considered one of the most extensive pandemics ever recorded in human history, causing significant losses to both life and economies globally. However, the available evidence is currently insufficient to establish the effectiveness and safety of antiviral drugs or vaccines. The entry of the virus into host cells involves binding to angiotensin-converting enzyme 2 (ACE2), a cell surface receptor, via its spike protein. Meanwhile, transmembrane protease serine 2 (TMPRSS2), a host surface protease, cleaves and activates the virus's S protein, thus promoting viral infection. Plant protease inhibitors play a crucial role in protecting plants against insects and/or microorganisms. The major storage proteins in sweet potato roots include sweet potato trypsin inhibitor (SWTI), which accounts for approximately 60% of the total water-soluble protein and has been found to possess a variety of health-promoting properties, including antioxidant, anti-inflammatory, ACE-inhibitory, and anticancer functions. Our study found that SWTI caused a significant reduction in the expression of the ACE2 and TMPRSS2 proteins, without any adverse effects on cells. Therefore, our findings suggest that the ACE2 and TMPRSS2 axis can be targeted via SWTI to potentially inhibit SARS-CoV-2 infection.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , Antivirales , Ipomoea batatas , SARS-CoV-2 , Serina Endopeptidasas , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Humanos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/metabolismo , Animales , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Ipomoea batatas/virología , Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , COVID-19/virología , COVID-19/metabolismo , Inhibidores de Tripsina/farmacología , Inhibidores de Tripsina/metabolismo , Internalización del Virus/efectos de los fármacos , Chlorocebus aethiops , Células Vero , Regulación hacia Abajo/efectos de los fármacos , RatonesRESUMEN
Despite the high quality of soybean protein, raw soybeans and soybean meal cannot be directly included in animal feed mixtures due to the presence of Kunitz (KTi) and Bowman-Birk protease inhibitors (BBis), which reduces animal productivity. Heat treatment can substantially inactivate trypsin and chymotrypsin inhibitors (BBis), but such treatment is energy-intensive, adds expense, and negatively impacts the quality of seed proteins. As an alternative approach, we have employed CRISPR/Cas9 gene editing to create mutations in BBi genes to drastically lower the protease inhibitor content in soybean seed. Agrobacterium-mediated transformation was used to generate several stable transgenic soybean events. These independent CRISPR/Cas9 events were examined in comparison to wild-type plants using Sanger sequencing, proteomic analysis, trypsin/chymotrypsin inhibitor activity assays, and qRT-PCR. Collectively, our results demonstrate the creation of an allelic series of loss-of-function mutations affecting the major BBi gene in soybean. Mutations in two of the highly expressed seed-specific BBi genes lead to substantial reductions in both trypsin and chymotrypsin inhibitor activities.
Asunto(s)
Edición Génica , Glycine max , Inhibidor de la Tripsina de Soja de Bowman-Birk , Quimotripsina/metabolismo , Quimotripsina/genética , Sistemas CRISPR-Cas , Edición Génica/métodos , Glycine max/genética , Glycine max/metabolismo , Mutación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Semillas/genética , Semillas/metabolismo , Tripsina/metabolismo , Tripsina/genética , Tripsina/química , Inhibidor de la Tripsina de Soja de Bowman-Birk/metabolismo , Inhibidor de la Tripsina de Soja de Bowman-Birk/genética , Inhibidores de Tripsina/metabolismoRESUMEN
Recovering valuable active substances from the by-products of agricultural processing is a crucial concern for scientific researchers. This paper focuses on the enrichment of soybean trypsin inhibitor (STI) from soybean whey wastewater using either ammonium sulfate salting or ethanol precipitation, and discusses their physicochemical properties. The results show that at a 60% ethanol content, the yield of STI was 3.983 mg/mL, whereas the yield was 3.833 mg/mL at 60% ammonium sulfate saturation. The inhibitory activity of STI obtained by ammonium sulfate salting out (A-STI) was higher than that obtained by ethanol precipitation (E-STI). A-STI exhibited better solubility than E-STI at specific temperatures and pH levels, as confirmed by turbidity and surface hydrophobicity measurements. Thermal characterization revealed that both A-STI and E-STI showed thermal transition temperatures above 90 °C. Scanning electron microscopy demonstrated that A-STI had a smooth surface with fewer pores, while E-STI had a rough surface with more pores. In conclusion, there was no significant difference in the yield of A-STI and E-STI (p < 0.05); however, the physicochemical properties of A-STI were superior to those of E-STI, making it more suitable for further processing and utilization. This study provides a theoretical reference for the enrichment of STI from soybean whey wastewater.
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Glycine max , Inhibidores de Tripsina , Aguas Residuales , Suero Lácteo , Glycine max/química , Aguas Residuales/química , Suero Lácteo/química , Inhibidores de Tripsina/química , Inhibidores de Tripsina/aislamiento & purificación , Sulfato de Amonio/química , Precipitación Química , Concentración de Iones de Hidrógeno , Solubilidad , Interacciones Hidrofóbicas e Hidrofílicas , TemperaturaRESUMEN
The serine protease trypsin forms a tightly bound inhibitor complex with the bovine pancreatic trypsin inhibitor (BPTI). The complex is stabilized by the P1 residue Lys15, which interacts with negatively charged amino acids at the bottom of the S1 pocket. Truncating the P1 residue of wildtype BPTI to α-aminobutyric acid (Abu) leaves a complex with moderate inhibitor strength, which is held in place by additional hydrogen bonds at the protein-protein interface. Fluorination of the Abu residue partially restores the inhibitor strength. The mechanism with which fluorination can restore the inhibitor strength is unknown, and accurate computational investigation requires knowledge of the binding and unbinding pathways. The preferred unbinding pathway is likely to be complex, as encounter states have been described before, and unrestrained umbrella sampling simulations of these complexes suggest additional energetic minima. Here, we use random acceleration molecular dynamics to find a new metastable state in the unbinding pathway of Abu-BPTI variants and wildtype BPTI from trypsin, which we call the prebound state. The prebound state and the fully bound state differ by a substantial shift in the position, a slight shift in the orientation of the BPTI variants, and changes in the interaction pattern. Particularly important is the breaking of three hydrogen bonds around Arg17. Fluorination of the P1 residue lowers the energy barrier of the transition between the fully bound state and prebound state and also lowers the energy minimum of the prebound state. While the effect of fluorination is in general difficult to quantify, here, it is in part caused by favorable stabilization of a hydrogen bond between Gln194 and Cys14. The interaction pattern of the prebound state offers insights into the inhibitory mechanism of BPTI and might add valuable information for the design of serine protease inhibitors.
Asunto(s)
Aprotinina , Simulación de Dinámica Molecular , Unión Proteica , Tripsina , Tripsina/metabolismo , Tripsina/química , Aprotinina/química , Aprotinina/metabolismo , Animales , Bovinos , Halogenación , Enlace de Hidrógeno , Conformación Proteica , Inhibidores de Tripsina/química , Inhibidores de Tripsina/metabolismo , Inhibidores de Tripsina/farmacologíaRESUMEN
The detection of trypsin and its inhibitors is important for both clinical diagnosis and disease treatment. Abnormal trypsin activity affects pancreatic function and leads to corresponding pathological changes in the body. Therefore, the study presented a riboflavin-induced photo-ATRP electrochemical assay of trypsin activity and its inhibitor, including detection of trypsin activity in real urine samples. Experiments were performed on indium tin oxide (ITO) electrodes modified with sulfhydryl groups of 3-mercaptopropionic acid, and target trypsin-specific cleavage of BSA-Au nanocluster (BSA-Au NCs) was followed by the modification of Au NCs to the electrodes using Au-S. The Au NCs immobilized monodeoxy-monomercapto-ß-cyclodextrin@adamantan-2-amine (SH-ß-CD@2-NH2-Ada) host-guest inclusion complexes to the electrode surfaces via Au-S. In a two-component photo-initiator system consisting of riboflavin as an initiator and ascorbic acid (AA) as a mild reducing agent under mild blue light radiation, a large number of electroactive substances were grafted onto the electrode surface to generate electrochemical signals. In addition, we have successfully realized the detection of clinical drug inhibitors of trypsin. The detection limit of the system is as low as 0.0024 ng/mL, which much littler than the average standard of trypsin in the patient's urine or serum. It's worth noting that this work will provide researchers with a different route to design electrochemical sensors based on non-covalent recognition strategies.
Asunto(s)
Técnicas Electroquímicas , Electrodos , Oro , Riboflavina , Tripsina , Riboflavina/química , Riboflavina/orina , Tripsina/metabolismo , Tripsina/química , Humanos , Oro/química , Biomarcadores/orina , Biomarcadores/sangre , Nanopartículas del Metal/química , Procesos Fotoquímicos , Límite de Detección , Albúmina Sérica Bovina/química , Compuestos de Estaño/química , Inhibidores de Tripsina/química , Inhibidores de Tripsina/farmacología , Inhibidores de Tripsina/orinaRESUMEN
Steroid-induced acute pancreatitis is a rare form of pancreatitis that requires intensive care and has a high morbidity and mortality rate as there is no specific treatment. Management of steroid-induced pancreatitis is generally non-specific and supportive. Here, we are presenting a man in his 40s presented with epigastric pain, fever and vomiting. The patient was diagnosed case of rheumatoid arthritis, for which he was receiving regular 5 mg oral prednisolone therapy. Based on history, and clinical, biochemical and radiological imaging a diagnosis of steroid-induced pancreatitis was made, which was successfully managed with the help of ulinastatin and other supportive treatments. A serine protease inhibitor like ulinastatin may be used early in the clinical management of steroid-induced pancreatitis.
Asunto(s)
Glicoproteínas , Pancreatitis , Prednisolona , Inhibidores de Tripsina , Humanos , Masculino , Prednisolona/uso terapéutico , Pancreatitis/inducido químicamente , Pancreatitis/tratamiento farmacológico , Adulto , Inhibidores de Tripsina/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Glucocorticoides/uso terapéutico , Glucocorticoides/efectos adversosRESUMEN
Although peach kernels are rich in oil, there is a lack of information about its chemical and biological properties. Therefore, the purpose of this study was to determine the lipid profile, antioxidant capacity, and trypsin inhibitory propriety of peach oil extracted from two varieties (sweet cap and O'Henry) cultivated in Tunisia. The investigated peach kernel oil contains significant amount of unsaponifiable (2.1±0.5-2.8±0.2% of oil) and phenolic compounds (45.8±0.92-74.6±1.3 mg GAE/g of oil). Its n-alkane profile was characterized by the predominance of tetracosane n-C24 (47.24%) followed by tricosane n-C23 (34.43%). An important total tocopherol content (1192.83±3.1 mg/kg oil) has been found in sweet cap cultivar. Although rich in polyphenols and tocopherols, the tested oil did not display an inhibitory effect on trypsin. However, all peach oil samples showed effective antioxidant capacity and the highest values (86.34±1.3% and 603.50±2.6 µmol TE/g oil for DPPH test and ORAC assay, respectively) were observed for sweet cap oil. Peach oil has an excellent potential for application in the food and pharmaceutical industries as source of naturally-occurring bioactive substances.
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Antioxidantes , Fenoles , Aceites de Plantas , Prunus persica , Tocoferoles , Antioxidantes/análisis , Aceites de Plantas/química , Aceites de Plantas/análisis , Fenoles/análisis , Tocoferoles/análisis , Prunus persica/química , Inhibidores de Tripsina/análisis , Polifenoles/análisisRESUMEN
Soybeans are the number one source of plant proteins for food and feed, but the natural presence of protein protease inhibitors (PIs), namely, the Kunitz trypsin inhibitor (KTI) and the Bowman-Birk inhibitor (BBI), exerts antinutritional effects. This communication describes a new methodology for simultaneously quantitating all parameters of PIs in soybeans. It consists of seven steps and featured enzymatically measuring trypsin and chymotrypsin inhibitory activities, respectively, and subsequently determining the contents of reactive KTI and BBI and the contributions of each toward total PI mass and total trypsin or chymotrypsin inhibition by solving a proposed system of linear equations with two variables (C = dB + eK and T = xB + yK). This enzymatic and algebraic (EA) methodology was based on differential inhibitions of KTI and BBI toward trypsin and chymotrypsin and validated by applications to a series of mixtures of purified KTI and BBI, two KTI-null and two conventional soybeans, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The EA methodology allowed calculations of PI composition and the contributions of individual inhibitors toward total inhibition with ease. It was first found that although BBI constituted only about 30% of the total PI mass in conventional raw soybeans, it contributed about 80% toward total chymotrypsin inhibitor activity and about 45% toward trypsin inhibitor activity. Therefore, BBI caused more total protease inhibitions than those of KTI. Furthermore, the so-called KTI-null soybean mutants still contained measurable KTI content and thus should be named KTI-low soybeans.
Asunto(s)
Quimotripsina , Glycine max , Inhibidor de la Tripsina de Soja de Bowman-Birk , Inhibidor de la Tripsina de Soja de Kunitz , Tripsina , Quimotripsina/antagonistas & inhibidores , Quimotripsina/química , Quimotripsina/metabolismo , Glycine max/química , Glycine max/enzimología , Tripsina/química , Tripsina/metabolismo , Inhibidor de la Tripsina de Soja de Bowman-Birk/análisis , Inhibidor de la Tripsina de Soja de Kunitz/análisis , Inhibidores de Tripsina/análisisRESUMEN
Broad-spectrum disease resistance (BSR) is crucial for controlling plant diseases and relies on immune signals that are subject to transcriptional and post-translational regulation. How plants integrate and coordinate these signals remains unclear. We show here that the rice really interesting new gene (RING)-type E3 ubiquitin ligase OsRING113 targets APIP5, a negative regulator of plant immunity and programmed cell death (PCD), for 26S proteasomal degradation. The osring113 mutants in Nipponbare exhibited decreased BSR, while the overexpressing OsRING113 plants showed enhanced BSR against Magnaporthe oryzae (M. oryzae) and Xanthomonas oryzae pv. oryzae (Xoo). Furthermore, APIP5 directly suppressed the transcription of the Bowman-Birk trypsin inhibitor genes OsBBTI5 and AvrPiz-t-interacting protein 4 (APIP4). Overexpression of these two genes, which are partially required for APIP5-mediated PCD and disease resistance, conferred BSR. OsBBTI5 and APIP4 associated with and stabilized the pathogenesis-related protein OsPR1aL, which promotes M. oryzae resistance. Our results identify an immune module with integrated and coordinated hierarchical regulations that confer BSR in plants.
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Resistencia a la Enfermedad , Regulación de la Expresión Génica de las Plantas , Oryza , Enfermedades de las Plantas , Proteínas de Plantas , Inhibidores de Tripsina , Ubiquitina-Proteína Ligasas , Xanthomonas , Oryza/microbiología , Oryza/metabolismo , Oryza/genética , Resistencia a la Enfermedad/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/inmunología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Xanthomonas/patogenicidad , Inhibidores de Tripsina/metabolismo , Inhibidores de Tripsina/farmacología , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Inmunidad de la Planta , Plantas Modificadas Genéticamente , MagnaportheRESUMEN
Typical Kunitz proteins (I2 family of the MEROPS database, Kunitz-A family) are metazoan competitive inhibitors of serine peptidases that form tight complexes of 1:1 stoichiometry, mimicking substrates. The cestode Echinococcus granulosus, the dog tapeworm causing cystic echinococcosis in humans and livestock, encodes an expanded family of monodomain Kunitz proteins, some of which are secreted to the dog host interface. The Kunitz protein EgKU-7 contains, in addition to the Kunitz domain with the anti-peptidase loop comprising a critical arginine, a C-terminal extension of â¼20 amino acids. Kinetic, electrophoretic, and mass spectrometry studies using EgKU-7, a C-terminally truncated variant, and a mutant in which the critical arginine was substituted by alanine, show that EgKU-7 is a tight inhibitor of bovine and canine trypsins with the unusual property of possessing two instead of one site of interaction with the peptidases. One site resides in the anti-peptidase loop and is partially hydrolyzed by bovine but not canine trypsins, suggesting specificity for the target enzymes. The other site is located in the C-terminal extension. This extension can be hydrolyzed in a particular arginine by cationic bovine and canine trypsins but not by anionic canine trypsin. This is the first time to our knowledge that a monodomain Kunitz-A protein is reported to have two interaction sites with its target. Considering that putative orthologs of EgKU-7 are present in other cestodes, our finding unveils a novel piece in the repertoire of peptidase-inhibitor interactions and adds new notes to the evolutionary host-parasite concerto.
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Echinococcus granulosus , Proteínas del Helminto , Echinococcus granulosus/enzimología , Echinococcus granulosus/genética , Echinococcus granulosus/metabolismo , Animales , Perros , Proteínas del Helminto/metabolismo , Proteínas del Helminto/genética , Proteínas del Helminto/química , Inhibidores de Tripsina/metabolismo , Inhibidores de Tripsina/química , Bovinos , Secuencia de Aminoácidos , Tripsina/química , Tripsina/metabolismoRESUMEN
This review aims to provide an updated overview of the effects of protein extraction/recovery on antinutritional factors (ANFs) in plant protein ingredients, such as protein-rich fractions, protein concentrates, and isolates. ANFs mainly include lectins, trypsin inhibitors, phytic acid, phenolic compounds, oxalates, saponins, tannins, and cyanogenic glycosides. The current technologies used to recover proteins (e.g., wet extraction, dry fractionation) and novel technologies (e.g., membrane processing) are included in this review. The mechanisms involved during protein extraction/recovery that may enhance or decrease the ANF content in plant protein ingredients are discussed. However, studies on the effects of protein extraction/recovery on specific ANFs are still scarce, especially for novel technologies such as ultrasound- and microwave-assisted extraction and membrane processing. Although the negative effects of ANFs on protein digestibility and the overall absorption of plant proteins and other nutrients are a health concern, it is also important to highlight the potential positive effects of ANFs. This is particularly relevant given the rise of novel protein ingredients in the market and the potential presence or absence of these factors and their effects on consumers' health.
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Proteínas de Plantas , Animales , Fraccionamiento Químico/métodos , Valor Nutritivo , Proteínas de Plantas/química , Inhibidores de Tripsina/aislamiento & purificación , Inhibidores de Tripsina/análisis , Inhibidores de Tripsina/química , HumanosRESUMEN
Trypsin inhibitors derived from plants have various pharmacological activities and promising clinical applications. In our previous study, a Bowman-Birk-type major trypsin inhibitor from foxtail millet bran (FMB-BBTI) was extracted with antiatherosclerotic activity. Currently, we found that FMB-BBTI possesses a prominent anticolorectal cancer (anti-CRC) activity. Further, a recombinant FMB-BBTI (rFMB-BBTI) was successfully expressed in a soluble manner in host strain Escherichia coli. BL21 (DE3) was induced by isopropyl-ß-d-thiogalactoside (0.1 mM) at 37 °C for 3.5 h by the pET28a vector system. Fortunately, a purity greater than 93% of rFMB-BBTI with anti-CRC activity was purified by nickel-nitrilotriacetic acid affinity chromatography. Subsequently, we found that rFMB-BBTI displays a strikingly anti-CRC effect, characterized by the inhibition of cell proliferation and clone formation ability, cell cycle arrest at the G2/M phase, and induction of cell apoptosis. It is interesting that the rFMB-BBTI treatment had no obvious effect on normal colorectal cells in the same concentration range. Importantly, the anti-CRC activity of rFMB-BBTI was further confirmed in the xenografted nude mice model. Taken together, our study highlights the anti-CRC activity of rFMB-BBTI in vitro and in vivo, uncovering the clinical potential of rFMB-BBTI as a targeted agent for CRC in the future.
Asunto(s)
Neoplasias Colorrectales , Extractos Vegetales , Proteínas de Plantas , Setaria (Planta) , Inhibidores de Tripsina , Animales , Humanos , Masculino , Ratones , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Expresión Génica , Ratones Endogámicos BALB C , Ratones Desnudos , Extractos Vegetales/química , Extractos Vegetales/farmacología , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Setaria (Planta)/genética , Setaria (Planta)/química , Inhibidores de Tripsina/farmacología , Inhibidores de Tripsina/aislamiento & purificación , Inhibidores de Tripsina/químicaRESUMEN
The incidence of Candida species resistant to traditional antifungal drugs is increasing globally. This issue significantly impacts patients' lives and increases healthcare expenses, confirming the need to develop novel therapeutic strategies. Recently, a thermostable trypsin inhibitor named ShTI (11.558 kDa), which has antibacterial effects on Staphylococcus aureus, was isolated from Salvia hispanica L. (chia) seeds. This study aimed to assess the antifungal effect of ShTI against Candida species and its synergism with fluconazole and to evaluate its mode of action. Preliminary toxicological studies on mouse fibroblasts were also performed. ShTI exhibited antifungal effects against C. parapsilosis (ATCC® 22,019), C. krusei (ATCC® 6258), and six clinical fluconazole-resistant strains of C. albicans (2), C. parapsilosis (2), and C. tropicalis (2). The minimum inhibitory concentration (MIC) values were 4.1 µM (inhibiting 50% of the isolates) and 8.2 µM (inhibiting 100% of the isolates). Additionally, when combined with fluconazole, ShTI had a synergistic effect on C. albicans, altering the morphological structure of the yeast. The mode of action of ShTI against C. krusei (ATCC® 6258) and C. albicans involves cell membrane permeabilization, the overproduction of reactive oxygen species, the formation of pseudohyphae, pore formation, and consequently, cell death. In addition, ShTI (8.65 and 17.3 µM) had noncytotoxic and nongenotoxic effects on L929 mouse fibroblasts. These findings suggest that ShTI could be a promising antimicrobial candidate, but further research is necessary to advance its application as a novel antifungal agent.
Asunto(s)
Antifúngicos , Candida , Farmacorresistencia Fúngica , Fluconazol , Pruebas de Sensibilidad Microbiana , Salvia , Semillas , Inhibidores de Tripsina , Antifúngicos/farmacología , Antifúngicos/toxicidad , Fluconazol/farmacología , Fluconazol/toxicidad , Candida/efectos de los fármacos , Salvia/química , Semillas/química , Animales , Ratones , Inhibidores de Tripsina/farmacología , Extractos Vegetales/farmacología , Extractos Vegetales/química , Fibroblastos/efectos de los fármacos , Sinergismo Farmacológico , Candidiasis/microbiología , Candidiasis/tratamiento farmacológicoRESUMEN
Lentils have a valuable physicochemical profile, which can be affected by the presence of antinutrients that may impair the benefits arising from their consumption. Different treatments can be used to reduce these undesirable compounds, although they can also affect the general composition and behaviour of the lentils. Thus, the effect of different processing methods on the physicochemical and techno-functional properties, as well as on the antinutritional factors of different lentil varieties was studied. Phytic acid was eliminated during germination, while tannins and trypsin inhibitors are mostly affected by cooking. Functional properties were also altered by processing, these being dependent on the concentration of different nutrients in lentils. All the studied treatments affected the physicochemical profile of lentils and their functional properties. Cooking and germination appear to be the most effective in reducing antinutritional factors and improving the physicochemical profile of the lentils, meeting the current nutritional demands of today's society.
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Culinaria , Germinación , Lens (Planta) , Valor Nutritivo , Semillas , Lens (Planta)/química , Semillas/química , Semillas/crecimiento & desarrollo , Ácido Fítico/análisis , Ácido Fítico/química , Taninos/análisis , Taninos/química , Inhibidores de Tripsina/análisis , Inhibidores de Tripsina/química , Manipulación de AlimentosRESUMEN
This study investigated the function of the MDR49 gene in Aedes aegypti. MDR49 mutants were constructed using CRISPR/Cas9 technology; the mutation led to increased sensitivity to ivermectin (LC50: from 1.3090 mg L-1 to 0.5904 mg L-1), and a reduction in midgut trypsin activity. These findings suggest that the P-gp encoded by MDR49 confers resistance to ivermectin and impacts the reproductive function in Ae. aegypti. RNA interference technology showed that knockdown of MDR49 gene resulted in a significant decrease in the expression of VGA1 after a blood meal, as well as a decrease in the number of eggs laid and their hatching rate. LC-MS revealed that following ivermectin treatment, the MDR493d+2s/3d+2s strain larvae exhibited significantly higher drug concentrations in the head and fat body compared to the wild type. Modeling of inward-facing P-gp and molecular docking found almost no difference in the affinity of P-gp for ivermectin before and after the mutation. However, modeling of the outward-facing conformation demonstrated that the flexible linker loop between TM5 and TM6 of P-gp undergoes changes after the mutation, resulting in a decrease in trypsin activity and an increase in sensitivity to ivermectin. These results provide useful insights into ivermectin resistance and the other roles played by the MDR49 gene.
Asunto(s)
Aedes , Proteínas de Insectos , Ivermectina , Animales , Aedes/efectos de los fármacos , Aedes/genética , Aedes/metabolismo , Ivermectina/farmacología , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Tripsina/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Fertilidad/efectos de los fármacos , Resistencia a los Insecticidas/genética , Inhibidores de Tripsina/metabolismo , Inhibidores de Tripsina/farmacología , Simulación del Acoplamiento Molecular , Insecticidas/farmacologíaRESUMEN
α-Amylase/trypsin inhibitor proteins (ATI) are discussed as possible triggers for non-celiac gluten sensitivity. The potential of high-performance thin-layer chromatography (HPTLC) was studied for the first time to analyse the inhibitory properties of ATIs from flour of wheat, spelt, and einkorn. Inhibition by each flour of the digestive enzymes trypsin or α-amylase was determined by the reduction of released metabolisation products in comparison to non-digested flour, and positive (acarbose) and negative (water) controls. Firstly, amylolysis was carried out in miniaturized form on the HPTLC surface (HPTLC-nanoGIT) after in-vial pre-incubation of the amylase with the inhibitors from flour. α-Amylase inhibition was evident via the reduction of released saccharides, as analysed by normal phase HPTLC. A strong influence of the flour matrix on the assay results (individual saccharides) was evident, caused by an increased amylolysis of further polysaccharides present, making HPTLC analysis more reliable than currently used spectrophotometric sum value assays. The detection and visualization of such matrix influence helps to understand the problems associated with spectrophotometric assays. Only maltotriose was identified as a reliable marker of the amylolysis. The highest α-amylase inhibition and thus the lowest saccharide response was detected for maltotriose in refined spelt, whereas the lowest α-amylase inhibition and thus the highest saccharide response was detected for maltotriose in refined wheat. A comparison of refined and whole grain flours showed no clear trend in the responses. Secondly, trypsin inhibition and proteolysis were performed in-vial, and any inhibition was evident via the reduction of released peptides, analysed by reversed-phase HPTLC. Based on the product pattern of the proteolysis, einkorn and whole wheat showed the highest trypsin inhibition, whereas refined wheat and refined spelt showed the lowest inhibition. Advantageously, HPTLC analysis provided important information on changes in individual saccharides or peptides, which was more reliable and sustainable than spectrophotometric in-vial assays (only sum value) or liquid column chromatography analysis (targeting only the ATI proteins).
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Triticum , Inhibidores de Tripsina , alfa-Amilasas , Triticum/química , Cromatografía en Capa Delgada/métodos , alfa-Amilasas/antagonistas & inhibidores , alfa-Amilasas/análisis , Inhibidores de Tripsina/análisis , Inhibidores de Tripsina/farmacología , Proteínas de Plantas/análisis , Harina/análisisRESUMEN
This experimental study was designed to evaluate the effect of ulinastatin, a urinary trypsin inhibitor, on postoperative cognitive dysfunction (POCD) in rats under general anesthesia with isoflurane, on the aspect of behavior, as evaluated using a Y-maze test and focusing on microglial activity. Ulinastatin (50,000 U/mL) and normal saline (1 mL) were randomly (1:1) administered intraperitoneally to the ulinastatin and control groups, respectively, before general anesthesia. Anesthesia with isoflurane 1.5 volume% was maintained for 2 h. The Y-maze test was used to evaluate cognitive function. Neuronal damage using caspase-1 expression, the degree of inflammation through cytokine detection, and microglial activation with differentiation of the phenotypic expression were evaluated. Twelve rats were enrolled in the study and evenly allocated into the two groups, with no dropouts from the study. The Y-maze test showed similar results in the two groups before general anesthesia (63 ± 12% in the control group vs. 64 ± 12% in the ulinastatin group, p = 0.81). However, a significant difference was observed between the two groups after general anesthesia (17 ± 24% in the control group vs. 60 ± 12% in the ulinastatin group, p = 0.006). The ulinastatin group showed significantly lower expression of caspase-1. Pro-inflammatory cytokine levels were significantly lower in the ulinastatin group than in the control group. The ulinastatin group had a significantly lower microglial activation (41.74 ± 10.56% in the control group vs. 4.77 ± 0.56% in the ulinastatin, p < 0.001), with a significantly lower activation of M1 phenotypes (52.19 ± 7.83% in the control group vs. 5.58 ± 0.76% in the ulinastatin group, p < 0.001). Administering ulinastatin before general anesthesia prevented neuronal damage and cognitive decline after general anesthesia, in terms of the aspect of behavior, as evaluated by the Y-maze test. The protective effect of ulinastatin was associated with the inhibition of microglial activation, especially the M1 phenotype.
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Disfunción Cognitiva , Glicoproteínas , Isoflurano , Complicaciones Cognitivas Postoperatorias , Ratas , Animales , Isoflurano/farmacología , Microglía , Citocinas/farmacología , Caspasa 1 , Aprendizaje por Laberinto , Inhibidores de Tripsina/farmacologíaRESUMEN
Oxidative stress disorders and diseases caused by drug-resistant bacteria have emerged as significant public health concerns. Plant-based medications like protease inhibitors are growing despite adverse effects therapies. Consecutively, in this study, trypsin inhibitors from Dioscorea bulbifera L. (DbGTi trypsin inhibitor) ground tubers were isolated, purified, characterized, and evaluated for their potential cytotoxicity, antibacterial, and antioxidant activities. DbGTi protein was purified by Q-Sepharose matrix, followed by trypsin inhibitory activity. The molecular weight of the DbGTi protein was found to be approximately 31 kDa by SDS-PAGE electrophoresis. The secondary structure analysis by circular dichroism (CD) spectroscopy revealed that the DbGTi protein predominantly comprises ß sheets followed by α helix. DbGTi protein showed competitive type of inhibition with Vmax = 2.1372 × 10-1 µM/min, Km = 1.1805 × 102 µM, & Ki = 8.4 × 10-9 M and was stable up to 70 °C. DbGTi protein exhibited 58 % similarity with Dioscorin protein isolated from Dioscorea alata L. as revealed by LC-MS/MS analysis. DbGTi protein showed a non-toxic effect, analyzed by MTT, Haemolytic assay and in vivo studies on zebrafish model. DbGTi protein significantly inhibited K. pneumoniae and has excellent antioxidant properties, confirmed by various antioxidant assays. The results of anti-microbial, cytotoxicity and antioxidant assays demonstrate its bioactive potential and non-toxic nature.