Respuesta a "Fumador, exfumador y COVID-19" / Reply to "Smoker, Former Smoker and COVID-19"

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Sesame water-soluble proteins fraction contains endopeptidases and exopeptidases with high activity: A natural source for plant proteases.

Recently, the interest in the plant proteases has greatly increased. However, only a few of proteases are isolated from the hugely produced oilseeds for the practical utilizations. In this study, the raw sesame milk prepared from peeled sesame seeds was separated into floating, skim, and precipitate fractions by centrifugation. The predominant aspartic endopeptidases and serine carboxypeptidases, which exerted high synergetic activity at pH 4.5-5 and 50-60 °C, were identified in the skim by the liquid chromatography tandem mass spectrometry, Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protease inhibitor assay, trichloroacetic acid-nitrogen soluble index (TCA-NSI), and free amino acid analyses. By incubating the mixture (protein content, 2%) of skim and precipitate at pH 4.5 and 50 °C for 6 h, the TCA-NSI and free amino acids achieved to 38.42% and 3148 mg/L, respectively. Moreover, these proteases efficiently degraded the proteins from soybean, peanut, and bovine milk.

Vacuolar proteases from Candida glabrata: acid aspartic protease PrA, neutral serine protease PrB and serine carboxypeptidase CpY. The nitrogen source influences their level of expression / Proteasas vacuolares de Candida glabrata: aspartil proteasa ácida PrA, proteasa neutra PrB y carboxipeptidasa CpY. La fuente de nitrógeno influye en sus niveles de expresión

Background. The Saccharomyces cerevisiae vacuole is actively involved in the mechanism of autophagy and is important in homeostasis, degradation, turnover, detoxification and protection under stressful conditions. In contrast, vacuolar proteases have not been fully studied in phylogenetically related Candida glabrata. Aims. The present paper is the first report on proteolytic activity in the C. glabrata vacuole. Methods. Biochemical studies in C. glabrata have highlighted the presence of different kinds of intracellular proteolytic activity: acid aspartyl proteinase (PrA) acts on substrates such as albumin and denatured acid hemoglobin, neutral serine protease (PrB) on collagen-type hide powder azure, and serine carboxypeptidase (CpY) on N-benzoyl-tyr-pNA. Results. Our results showed a subcellular fraction with highly specific enzymatic activity for these three proteases, which allowed to confirm its vacuolar location. Expression analyses were performed in the genes CgPEP4 (CgAPR1), CgPRB1 and CgCPY1 (CgPRC), coding for vacuolar aspartic protease A, neutral protease B and carboxypeptidase Y, respectively. The results show a differential regulation of protease expression depending on the nitrogen source. Conclusions. The proteases encoded by genes CgPEP4, CgPRB1 and CgCPY1 from C. glabrata could participate in the process of autophagy and survival of this opportunistic pathogen (AU)

Antecedentes. La vacuola de Saccharomyces cerevisiae está involucrada activamente en el mecanismo de autofagia, desarrollando una labor importante en la homeostasis, degradación, recambio proteico, desintoxicación y protección de la célula en condiciones de estrés. Por el contrario, las proteasas vacuolares de Candida glabrata aún no han sido estudiadas por completo. Objetivos. El presente trabajo describe por primera vez la actividad proteolítica vacuolar en C. glabrata. Métodos. Los estudios bioquímicos realizados en C. glabrata pusieron de manifiesto la presencia de diferentes actividades proteolíticas: aspartil proteinasa ácida, que actúa sobre sustratos como la albúmina y la hemoglobina ácida desnaturalizada; serín proteasa neutra, con actividad sobre el substrato de tipo colágeno hide powder azure, y serín carboxipeptidasa, que actúa sobre N-benzoil-tyr-pNa. Resultados. La obtención de una fracción subcelular mostró una elevada actividad enzimática específica de las tres proteasas, lo que permitió confirmar su localización vacuolar. Se realizaron análisis de la expresión de los genes CgPEP4 (CgAPR1), CgPRB1 y CgCPY1 (CgPRC1), codificantes de las actividades proteolíticas aspartil proteasa A, proteasa neutra B y carboxipeptidasa Y, respectivamente. Los resultados reflejan una regulación diferencial de la expresión de la proteasa, dependiendo de la fuente de nitrógeno. Conclusiones. Las proteasas codificadas por los genes CgPEP4, CgPRB1 y CgCPY1 podrían participar en el proceso de autofagia y supervivencia de este patógeno oportunista (AU)

Responses of proteolytic enzymes in embryonic axes of germinating bean seeds under copper stress.

The changes in protease activities in embryonic axes during the first days of bean (Phaseolus vulgaris L.) seed germination were investigated in response to copper stress. Synthetic substrates and specific protease inhibitors have been used to define qualitatively and quantitatively different catalytic classes, particularly endoproteases (EP), carboxypeptidases (CP) and aminopeptidases (AP), then identify which ones were affected in the presence of copper. In fact, a failure in storage proteins mobilization and a disorder of nitrogen supply at enzymatic level occurred in Cu. In fact, Cu inhibited azocaseinolytic activity (ACA) and cysteine-, aspartic-, serine-, and metallo-endopeptidases activities (Cys-EP, Asp-EP, Ser-Ep, and Met-EP, respectively). Besides, Cu affected leucine- and proline-aminopeptidases (LAP and PAP, respectively) and glycine-carboxypeptidases (Gly-CP). The proteolytic responses might also be associated with the decrease in defense capacity in the Cu-treated embryos.

Graphene oxide-peptide nanocomplex as a versatile fluorescence probe of protein kinase activity based on phosphorylation protection against carboxypeptidase digestion.

The research on complicated kinomics and kinase-target drug discovery requires the development of simple, cost-effective, and multiplex kinase assays. Herein, we propose a novel and versatile biosensing platform for the detection of protein kinase activity based on graphene oxide (GO)-peptide nanocomplex and phosphorylation-induced suppression of carboxypeptidase Y (CPY) cleavage. Kinase-catalyzed phosphorylation protects the fluorophore-labeled peptide probe against CPY digestion and induces the formation of a GO/peptide nanocomplex resulting in fluorescence quenching, while the nonphosphopeptide is degraded by CPY to release free fluorophore as well as restore fluorescence. This GO-based nanosensor has been successfully applied to sensitively detect two model kinases, casein kinase (CKII) and cAMP-dependent protein kinase (PKA) with low detection limits of 0.0833 mU/µL and 0.134 mU/µL, respectively. The feasibility of this GO-based sensor was further demonstrated by the assessment of kinase inhibition by staurosporine and H-89, in vitro kinase assay in cell lysates, and simultaneous detection of CKII and PKA activity. Moreover, the GO-based fluorescence anisotropy (FA) kinase assay has been also developed using GO as a FA signal amplifier. The proposed sensor is homogeneous, facile, universal, label-free, and applicable for multiplexed kinase assay, presenting a promising method for kinase-related biochemical fundamental research and inhibitor screening.

Quantitative peptidomics of mice lacking peptide-processing enzymes.

Peptidomics is defined as the analysis of peptides present in a tissue extract, usually using mass spectrometry-based approaches. Unlike radioimmunoassay-based detection techniques, peptidomics measures the precise form of each peptide, including post-translational modifications, and can readily distinguish between longer and shorter forms of the same peptide. Also, peptidomics is not limited to known peptides and can detect hundreds of peptides in a single experiment. Quantitative peptidomics enables comparisons between two or more groups of samples and is perfect for studies examining the effect of gene knockouts on tissue levels of peptides. We describe the method for quantitative peptidomics using isotopic labels based on trimethylammonium butyrate, which can be synthesized in five different isotopic forms; this permits multivariate analysis of five different groups of tissue extracts in a single liquid chromatography/mass spectrometry run.

Sensitive luciferin derived probes for selective carboxypeptidase activity.

Highly selective luminescent probes, QLUC-TYR and LUC-GLU, for detection of carboxypeptidase activity were synthesized. Caged substrates were first cleaved by corresponding carboxypeptidases, and then they were activated by luciferase to emit light. Enzymatic activities of biologically important carboxypeptidases can be determined using this technology.

Relación del telopéptido carboxiterminal del colágeno tipo i sérico con la densidad mineral ósea y el consumo de fármacos en mujeres posmenopáusicas. Datos preliminares del estudio FRODOS / Relationship of serum type I collagen telopeptide carboxy terminal with bone mineral density and drug consumption in postmenopausal women. Preliminary data from the FRODOS study

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Respuesta: Relación del telopéptido carboxiterminal del colágeno tipo i sérico con la densidad mineral ósea y el consumo de fármacos en mujeres posmenopáusicas. Datos preliminares del estudio FRODOS / Reply: Relationship of serum type I collagen telopeptide carboxy terminal with bone mineral density and drug consumption in postmenopausal women. Preliminary data from the FRODOS study

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The subcellular localization of a C-terminal processing protease in Pseudomonas aeruginosa.

Carboxy (C)-terminal processing proteases (CTP) are a relatively new group of serine proteases. Found in a broad range of organisms - bacteria, archaea, algae, plants and animals - these proteases are involved in the C-terminal processing of proteins. In comparison with amino-terminal processing of bacterial proteins, less is known about C-terminal processing and its physiological function. Bacterial CTPs appear to influence different basal cellular processes. Although CTPs of Gram-negative bacteria are generally referred to as being localized in the periplasm, there is little experimental evidence for this. We show for the first time the subcellular localization of a CTP-3 family protein from Pseudomonas aeruginosa, named CtpA, in the periplasm by a carefully designed fractionation study. Our results provide experimental evidence for the generally accepted hypothesis that CTPs are located in the periplasmic space of Gram-negative bacteria.

Application of standard addition for the determination of carboxypeptidase activity in Actinomucor elegans bran koji.

Leucine carboxypeptidase (EC 3.4.16) activity in Actinomucor elegans bran koji was investigated via absorbance at 507 nm after stained by Cd-nihydrin solution, with calibration curve A, which was made by a set of known concentration standard leucine, calibration B, which was made by three sets of known concentration standard leucine solutions with the addition of three concentrations inactive crude enzyme extract, and calibration C, which was made by three sets of known concentration standard leucine solutions with the addition of three concentrations crude enzyme extract. The results indicated that application of pure amino acid standard curve was not a suitable way to determine carboxypeptidase in complicate mixture, and it probably led to overestimated carboxypeptidase activity. It was found that addition of crude exact into pure amino acid standard curve had a significant difference from pure amino acid standard curve method (p < 0.05). There was no significant enzyme activity difference (p > 0.05) between addition of active crude exact and addition of inactive crude kind, when the proper dilute multiple was used. It was concluded that the addition of crude enzyme extract to the calibration was needed to eliminate the interference of free amino acids and related compounds presented in crude enzyme extract.

Optimization of immunoprecipitation-western blot analysis in detecting GW182-associated components of GW/P bodies.

Characterizing the components of GW/processing bodies is key to elucidating RNA interference and messenger RNA processing pathways. This protocol addresses challenges in isolating a low-abundance protein GW182 and GW body (GWB)-associated proteins by building on previous reports that used polyclonal sera containing autoantibodies to GW/P body components. This protocol uses commercially available monoclonal antibodies to GW182 that are covalently coupled to Protein A or G sepharose beads and then used to immunoprecipitate GW182 and associated proteins from cell extracts. Immunoprecipitates are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and probed by western blot with antibodies directed to proteins of interest. This protocol, which is expected to take 4-5 d, provides a biochemical approach for detecting GW182 and associated proteins in biological samples and thus facilitates the elucidation of the diverse functions of GWBs. It is expected that this protocol can be adapted to the detection of other RNA-binding complexes.

All Trypanosoma cruzi developmental forms present lysosome-related organelles.

Trypanosoma cruzi epimastigote forms concentrate their major protease, cruzipain, in the same compartment where these parasites store macromolecules obtained from medium and for this ability these organelles were named as reservosomes. Intracellular digestion occurs mainly inside reservosomes and seems to be modulated by cruzipain and its natural inhibitor chagasin that also concentrates in reservosomes. T. cruzi mammalian forms, trypomastigotes and amastigotes, are unable to capture macromolecules by endocytosis, but also express cruzipain and chagasin, whose role in infectivity has been described. In this paper, we demonstrate that trypomastigotes and amastigotes also concentrate cruzipain, chagasin as well as serine carboxypeptidase in hydrolase-rich compartments of acidic nature. The presence of P-type proton ATPase indicates that this compartment is acidified by the same enzyme as epimastigote endocytic compartments. Electron microscopy analyzes showed that these organelles are placed at the posterior region of the parasite body, are single membrane bound and possess an electron-dense matrix with electronlucent inclusions. Three-dimensional reconstruction showed that these compartments have different size and shape in trypomastigotes and amastigotes. Based on these evidences, we suggest that all T. cruzi developmental stages present lysosome-related organelles that in epimastigotes have the additional and unique ability of storing cargo.

A multifunctional human Argonaute2-specific monoclonal antibody.

Small regulatory RNAs including small interfering RNAs (siRNAs), microRNAs (miRNAs), or Piwi interacting RNAs (piRNAs) guide regulation of gene expression in many different organisms. The Argonaute (Ago) protein family constitutes the cellular binding partners of such small RNAs and regulates gene expression on the levels of transcription, mRNA stability, or translation. Due to the lack of highly specific and potent monoclonal antibodies directed against the different Ago proteins, biochemical analyses such as Ago complex purification and characterization rely on overexpression of tagged Ago proteins. Here, we report the generation and functional characterization of a highly specific monoclonal anti-Ago2 antibody termed anti-Ago2(11A9). We show that anti-Ago2(11A9) is specific for human Ago2 and detects Ago2 in Western blots as well as in immunoprecipitation experiments. We further demonstrate that Ago2 can be efficiently eluted from our antibody by a competing peptide. Finally, we show that anti-Ago2(11A9) recognizes Ago2 in immunofluorescence experiments, and we find that Ago2 not only localizes to cytoplasmic processing bodies (P-bodies) and the diffuse cytoplasm but also to the nucleus. With the anti-Ago2(11A9) antibody we have generated a potent tool that is useful for many biochemical or cell biological applications.

A combined approach of mass spectrometry, molecular modeling, and site-directed mutagenesis highlights key structural features responsible for the thermostability of Sulfolobus solfataricus carboxypeptidase.

Sulfolobus solfataricus carboxypeptidase (CPSso) is a thermostable zinc-metalloenzyme, consisting of four identical subunits with a M(r) of 43,000. In a previous paper (Occhipinti et al., Biophys J 2003; 85:1165-1175), we developed a structure of the enzyme by molecular modeling and validated it by site-directed mutagenesis and small angle X-ray scattering. Here, we report investigations aimed at further validating the model, as well as at identifying molecular determinants responsible for thermostability. To this end, we took advantage of mass spectrometry techniques, notably LC-MS/MS. The structure was confirmed by such approaches, in that they lead to the identification of a disulfide bridge formed by Cys286 and Cys293, whose location in the model is well suited for giving rise to the crosslink. More notably, we also identified a protease-resistant core consisting of the N- and C-terminal antiparallel alpha-helices, which in the model are predicted to interact with each other via hydrophobic quadrants. On the basis of the model, we also tentatively identified the most tightly interacting residues as Leu7, Ala380, and Leu376. Although the replacement of Ala380 by serine did not detectably impair protein stability, a dramatic drop in thermostability was observed when the two leucines were replaced by either aspartate (L7D; L376D) or asparagine (L7N; L376N). We then investigated the kinetic thermal stability of the wild type and the mutants by determining the thermodynamic activation parameters, DeltaG++, DeltaH++, and DeltaS++. Besides highlighting the key role of the hydrophobic core in thermostability, these results suggest clearly different mechanisms of destabilization by the single mutations, depending on whether the leucines are replaced by asparagines or aspartates.

Efectos de la desnutrición sobre la colagenización de anastomosis intestinales: análisis de procolágeno y telopéptido carboxiterminal mediante radioinmunoanálisis / Role of malnutrition in intestinal anastomosis collagenization: an analysis of procollagen (PINP) andcarboxyterminal telopeptide (ICTP) by radioimmunoassay

Introducción: diversos factores influyen en la cicatrización correcta de las suturas intestinales tras la práctica de una resección intestinal. Uno de los factores más implicados es el estado nutricional del paciente. Objetivos: evaluar la influencia de la desnutrición inducida sobre la viabilidad de una anastomosis intestinal primaria mediante el análisis del procolágeno (PINP) como marcador de la síntesis de colágeno I, y del telopéptido carboxiterminal del colágeno I (ICTP) como marcador de la destrucción del mismo. Métodos: 40 ratas Wistar y material de radioinmunoensayo. Métodos: diseñamos 2 grupos de ratas, 20 animales por cada grupo: grupo control (A) y grupo “desnutrición” (B). Se analiza PINP e ICTP mediante RIA sobre tejido colónico homogeneizado, preanastomótico y anastomótico. Resultados: existen unos niveles menores de PINP en el colon de las ratas del grupo B comparado con el colon del grupo A (0,3620 y 0,4340 μg/g respectivamente) (p = 0,032). Hay un mayor nivel de ICTP analizado en el colon del grupo B (0,9545 en contraposición a 0,8460 μg/g en el grupo A) (p = 0,875). En las anastomosis del grupo B existe una menor síntesis de PINP en comparación con el grupo A (0,376 y 0,468 μg/g respectivamente, p = 0,002). Conclusiones: la anastomosis colónica incrementa los niveles de PINP e ICTP en el tejido cicatricial (p = 0,000); la malnutrición reduce la colagenización de las anastomosis (p = 0,000)

Introduction: some clinical, anatomo-pathological, and technical factors influence the correct healing of intestinal suture following an intestinal resection. One of the most influential factors is patient nutritional status. Objectives: to evaluate the influence of malnutrition on the viability of primary intestinal anastomosis by the analysis of collagen I deposition. Methods: 40 Wistar rats, radioimmunoassay material. We used 2 groups of rats, 20 animals in each group: a control group (A) and a “malnutrition” group (B). Results: there was a decrease in PINP (procollagen) deposition in the colon of group B rats as compared to the colon of group A (0.3620 and 0.4340 μg/g respectively) (p = 0.032). There is an increase in ICTP (carboxyterminal telopeptide) in the colon of group B (0.9545 as against 0.8460 μg/g in group A) (p = 0.875). In anastomoses of group B there was a decrease in PINP synthesis as compared to group A (0.376 and 0.468 mg/g respectively, p = 0.002). As regards ICTP, there was an increase in group B (p = 0.330). In relation to the control group no differences were observed in ICTP increases in group B (p = 1). Conclusions: colonic anastomosis increases the levels of PINP and ICTP in healed tissue (p = 0.000); malnutrition reduces collagenization in anastomoses (p = 0.000)

Biochemical characterization of human glutamate carboxypeptidase III.

Human glutamate carboxypeptidase II (GCPII) is a transmembrane metallopeptidase found mainly in the brain, small intestine, and prostate. In the brain, it cleaves N-acetyl-L-aspartyl-glutamate, liberating free glutamate. Inhibition of GCPII has been shown to be neuroprotective in models of stroke and other neurodegenerations. In prostate, it is known as prostate-specific membrane antigen, a cancer marker. Recently, human glutamate carboxypeptidase III (GCPIII), a GCPII homolog with 67% amino acid identity, was cloned. While GCPII is recognized as an important pharmaceutical target, no biochemical study of human GCPIII is available at present. Here, we report the cloning, expression, and characterization of recombinant human GCPIII. We show that GCPIII lacks dipeptidylpeptidase IV-like activity, its activity is dependent on N-glycosylation, and it is effectively inhibited by several known inhibitors of GCPII. In comparison to GCPII, GCPIII has lower N-acetyl-L-aspartyl-glutamate-hydrolyzing activity, different pH and salt concentration dependence, and distinct substrate specificity, indicating that these homologs might play different biological roles. Based on a molecular model, we provide interpretation of the distinct substrate specificity of both enzymes, and examine the amino acid residues responsible for the differences by site-directed mutagenesis. These results may help to design potent and selective inhibitors of both enzymes.

Biosensor analysis of beta-lactams in milk using the carboxypeptidase activity of a bacterial penicillin binding protein.

The applicability of a beta-lactam receptor protein for detection of beta-lactam antibiotics in milk using surface plasmon resonance (SPR) biosensor technology was investigated. The advantage of using a receptor protein instead of antibodies for detection of beta-lactams is that a generic assay, specific for the active form of the beta-lactam structure, is obtained. Two assays based on the enzymatic activity of the DD-carboxypeptidase from Actinomadura R39 were developed, using a Biacore SPR biosensor. The carboxypeptidase converts a tri-peptide into a di-peptide, a reaction which is inhibited in the presence of beta-lactams. Polyclonal antibodies against the 2 peptides were developed and used to measure the amount of enzymatic product formed (di-peptide assay) or the amount of remaining enzymatic substrate (tri-peptide assay), respectively. The 2 assays showed similar performances with respect to detection limits (1.2 and 1.5 microg/kg, respectively) and precision (coefficient of variation <5%) for penicillin G in milk. Several other beta-lactams were detected at or near their respective maximum residue limit. Furthermore, the 2 peptide assays were evaluated against 5 commercial kit tests in the screening of 195 producer milk samples. The biosensor assays showed 0% false-negative and 27% false-positive results, whereas the figures were 0% false-negative and 27-53% false-positive results for other screening tests investigated.

Determination of enzymatic activity by capillary electrophoresis.

Enzymes are biological catalysts that play an important role in biochemical reactions necessary for normal growth, maturation and reproduction through whole live world. Their accurate quantitation in biological samples is important in many fields of biochemistry, not only in routine biochemistry and in fundamental research, but also in clinical and pharmacological research and diagnosis. Since the direct measurement of enzymes by masses is impossible, they must be quantified by their catalytic activities. Many different methods have been applied for this purpose so far. Although photometric methods are undoubtedly the most frequently used, separation methods will further gain their position in this field. The article reviews different possibilities for the assay of enzymatic activity by means of capillary electrophoresis (CE). Both the off-line and on-line enzyme assays based on CE are discussed.

A vitellogenic-like carboxypeptidase expressed by human macrophages is localized in endoplasmic reticulum and membrane ruffles.

Carboxypeptidase, vitellogenic-like (CPVL) is a serine carboxypeptidase of unknown function that was first characterized in human macrophages. Initial studies suggested that CPVL is largely restricted to the monocytic lineage, although it may also be expressed by cells outside the immune system. Here, we use a new monoclonal antibody to characterize the properties and localization of CPVL in human macrophages to elucidate a possible function for the protease. CPVL is up-regulated during the maturation of monocytes (MO) to macrophages, although the protein can be seen in both. In primary macrophages, CPVL is glycosylated with high mannose residues and colocalizes with markers for endoplasmic reticulum, while in MO it is more disperse and less clearly associated with endoplasmic reticulum. CPVL is highly expressed in lamellipodia and membrane ruffles, which also concentrate markers of the secretory pathway (MIP-1alpha and tumour necrosis factor-alpha) and major histocompatibility complex (MHC) class I and II molecules. CPVL can be seen on early latex bead and Candida albicans phagosomes, but it is not retained in the maturing phagosome, unlike MHC class I/II. CPVL has a mixed cytosolic and membrane-associated localization but is not detectable on the outer plasma membrane. We propose that CPVL may be involved in antigen processing, the secretory pathway and/or in actin remodelling and lamellipodium formation.