RESUMEN
Decarboxylation of phosphatidylserine (PS) to form phosphatidylethanolamine by PS decarboxylases (PSDs) is an essential process in most eukaryotes. Processing of a malarial PSD proenzyme into its active alpha and beta subunits is by an autoendoproteolytic mechanism regulated by anionic phospholipids, with PS serving as an activator and phosphatidylglycerol (PG), phosphatidylinositol, and phosphatidic acid acting as inhibitors. The biophysical mechanism underlying this regulation remains unknown. We used solid phase lipid binding, liposome-binding assays, and surface plasmon resonance to examine the binding specificity of a processing-deficient Plasmodium PSD (PkPSDS308A) mutant enzyme and demonstrated that the PSD proenzyme binds strongly to PS and PG but not to phosphatidylethanolamine and phosphatidylcholine. The equilibrium dissociation constants (Kd) of PkPSD with PS and PG were 80.4 nM and 66.4 nM, respectively. The interaction of PSD with PS is inhibited by calcium, suggesting that the binding mechanism involves ionic interactions. In vitro processing of WT PkPSD proenzyme was also inhibited by calcium, consistent with the conclusion that PS binding to PkPSD through ionic interactions is required for the proenzyme processing. Peptide mapping identified polybasic amino acid motifs in the proenzyme responsible for binding to PS. Altogether, the data demonstrate that malarial PSD maturation is regulated through a strong physical association between PkPSD proenzyme and anionic lipids. Inhibition of the specific interaction between the proenzyme and the lipids can provide a novel mechanism to disrupt PSD enzyme activity, which has been suggested as a target for antimicrobials, and anticancer therapies.
Asunto(s)
Carboxiliasas , Malaria , Fosfolípidos , Plasmodium , Secuencias de Aminoácidos , Calcio/metabolismo , Calcio/farmacología , Carboxiliasas/antagonistas & inhibidores , Carboxiliasas/química , Carboxiliasas/metabolismo , Precursores Enzimáticos/metabolismo , Liposomas , Ácidos Fosfatidicos/metabolismo , Ácidos Fosfatidicos/farmacología , Fosfatidilcolinas/metabolismo , Fosfatidilcolinas/farmacología , Fosfatidiletanolaminas/metabolismo , Fosfatidiletanolaminas/farmacología , Fosfatidilgliceroles/metabolismo , Fosfatidilgliceroles/farmacología , Fosfatidilinositoles/metabolismo , Fosfatidilinositoles/farmacología , Fosfatidilserinas/metabolismo , Fosfatidilserinas/farmacología , Fosfolípidos/química , Fosfolípidos/metabolismo , Fosfolípidos/farmacología , Unión Proteica , Malaria/parasitología , Proteolisis/efectos de los fármacos , Resonancia por Plasmón de Superficie , Plasmodium/enzimologíaRESUMEN
Effective and safe therapies for the treatment of diseases caused by intraerythrocytic parasites are impeded by the rapid emergence of drug resistance and the lack of novel drug targets. One such disease is human babesiosis, which is a rapidly emerging tick-borne illness caused by Babesia parasites. In this study, we identified fosinopril, a phosphonate-containing, FDA-approved angiotensin converting enzyme (ACE) inhibitor commonly used as a prodrug for hypertension and heart failure, as a potent inhibitor of Babesia duncani parasite development within human erythrocytes. Cell biological and mass spectrometry analyses revealed that the conversion of fosinopril to its active diacid molecule, fosinoprilat, is essential for its antiparasitic activity. We show that this conversion is mediated by a parasite-encoded esterase, BdFE1, which is highly conserved among apicomplexan parasites. Parasites carrying the L238H mutation in the active site of BdFE1 failed to convert the prodrug to its active moiety and became resistant to the drug. Our data set the stage for the development of this class of drugs for the therapy of vector-borne parasitic diseases.
Asunto(s)
Babesia , Parásitos , Profármacos , Animales , Humanos , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Fosinopril/farmacología , Profármacos/farmacología , Esterasas/metabolismoRESUMEN
BACKGROUND: Solid tumors promote tumor malignancy through interaction with the tumor microenvironment, resulting in difficulties in tumor treatment. Therefore, it is necessary to understand the communication between cells in the tumor and the surrounding microenvironment. Our previous study revealed the cancer malignancy mechanism of Bcl-w overexpressed in solid tumors, but no study was conducted on its relationship with immune cells in the tumor microenvironment. In this study, we sought to discover key factors in exosomes secreted from tumors overexpressing Bcl-w and analyze the interaction with the surrounding tumor microenvironment to identify the causes of tumor malignancy. METHODS: To analyze factors affecting the tumor microenvironment, a miRNA array was performed using exosomes derived from cancer cells overexpressing Bcl-w. The discovered miRNA, miR-6794-5p, was overexpressed and the tumorigenicity mechanism was confirmed using qRT-PCR, Western blot, invasion, wound healing, and sphere formation ability analysis. In addition, luciferase activity and Ago2-RNA immunoprecipitation assays were used to study the mechanism between miR-6794-5p and its target gene SOCS1. To confirm the interaction between macrophages and tumor-derived miR-6794-5p, co-culture was performed using conditioned media. Additionally, immunohistochemical (IHC) staining and flow cytometry were performed to analyze macrophages in the tumor tissues of experimental animals. RESULTS: MiR-6794-5p, which is highly expressed in exosomes secreted from Bcl-w-overexpressing cells, was selected, and it was shown that the overexpression of miR-6794-5p increased migratory ability, invasiveness, and stemness maintenance by suppressing the expression of the tumor suppressor SOCS1. Additionally, tumor-derived miR-6794-5p was delivered to THP-1-derived macrophages and induced M2 polarization by activating the JAK1/STAT3 pathway. Moreover, IL-10 secreted from M2 macrophages increased tumorigenicity by creating an immunosuppressive environment. The in vitro results were reconfirmed by confirming an increase in M2 macrophages and a decrease in M1 macrophages and CD8+ T cells when overexpressing miR-6794-5p in an animal model. CONCLUSIONS: In this study, we identified changes in the tumor microenvironment caused by miR-6794-5p. Our study indicates that tumor-derived miR-6794-5p promotes tumor aggressiveness by inducing an immunosuppressive environment through interaction with macrophage.
Asunto(s)
Exosomas , MicroARNs , Neoplasias , Animales , Neoplasias/genética , Bioensayo , Transporte Biológico , Linfocitos T CD8-positivos , MicroARNs/genética , Microambiente TumoralRESUMEN
The apicomplexan pathogen Babesia microti is responsible for most cases of human babesiosis worldwide. The disease, which presents as a malaria-like illness, is potentially fatal in immunocompromised or elderly patients, making the need for its accurate and early diagnosis an urgent public health concern. B. microti is transmitted primarily by Ixodes ticks but can also be transmitted via blood transfusion. The parasite completes its asexual reproduction in the host red blood cell, where each invading merozoite develops and multiplies to produce four daughter parasites. While various techniques, such as microscopy, PCR, and indirect fluorescence, have been used over the years for babesiosis diagnosis, detection of the secreted B. microti immunodominant antigen BmGPI12 using specific polyclonal antibodies was found to be the most effective method for the diagnosis of active infection and for evaluation of clearance following drug treatment. Here, we report the development of a panel of 16 monoclonal antibodies against BmGPI12. These antibodies detected secreted BmGPI12 in the plasma of infected humans. Antigen capture assays identified a combination of two monoclonal antibodies, 4C8 and 1E11, as a basis for a monoclonal antibody-based BmGPI12 capture assay (mGPAC) to detect active B. microti infection. Using a collection of 105 previously characterized human plasma samples, the mGPAC assay showed 97.1% correlation with RNA-based PCR (transcription-mediated amplification [TMA]) for positive and negative samples. The mGPAC assay also detected BmGPI12 in the plasma of six babesiosis patients at the time of diagnosis but not in three matched posttreatment samples. The mGPAC assay could thus be used alone or in combination with other assays for accurate detection of active B. microti infection.
Asunto(s)
Babesia microti , Babesiosis , Anciano , Anticuerpos Monoclonales , Antígenos de Protozoos , Babesia microti/genética , Babesiosis/diagnóstico , Humanos , ARNRESUMEN
Human PANK1, PANK2, and PANK3 genes encode several pantothenate kinase isoforms that catalyze the phosphorylation of vitamin B5 (pantothenic acid) to phosphopantothenate, a critical step in the biosynthesis of the major cellular cofactor, Coenzyme A (CoA). Mutations in the PANK2 gene, which encodes the mitochondrial pantothenate kinase (PanK) isoform, have been linked to pantothenate-kinase associated neurodegeneration (PKAN), a debilitating and often fatal progressive neurodegeneration of children and young adults. While the biochemical properties of these enzymes have been well-characterized in vitro, their expression in a model organism such as yeast in order to probe their function under cellular conditions have never been achieved. Here we used three yeast mutants carrying missense mutations in the yeast PanK gene, CAB1, which are associated with defective growth at high temperature and iron, mitochondrial dysfunction, increased iron content, and oxidative stress, to assess the cellular function of human PANK genes and functional conservation of the CoA-controlled processes between humans and yeast. Overexpression of human PANK1 and PANK3 in these mutants restored normal cellular activity whereas complementation with PANK2 was partial and could only be achieved with an isoform, PanK2mtmΔ, lacking the mitochondrial transit peptide. These data, which demonstrate functional conservation of PanK activity between humans and yeast, set the stage for the use of yeast as a model system to investigate the impact of PKAN-associated mutations on the metabolic pathways altered in this disease.
Asunto(s)
Estrés Oxidativo , Neurodegeneración Asociada a Pantotenato Quinasa , Saccharomyces cerevisiae , Humanos , Homeostasis , Hierro/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Estrés Oxidativo/genética , Neurodegeneración Asociada a Pantotenato Quinasa/metabolismo , Ácido Pantoténico , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Phosphatidylserine decarboxylases (PSDs) catalyze the conversion of phosphatidylserine (PS) to phosphatidylethanolamine (PE), a critical step in membrane biogenesis and a potential target for development of antimicrobial and anti-cancer drugs. PSD activity has typically been quantified using radioactive substrates and products. Recently, we described a fluorescence-based assay that measures the PSD reaction using distyrylbenzene-bis-aldehyde (DSB-3), whose reaction with PE produces a fluorescence signal. However, DSB-3 is not widely available and also reacts with PSD's substrate, PS, producing an adduct with lower fluorescence yield than that of PE. Here, we report a new fluorescence-based assay that is specific for PSD and in which the presence of PS causes only negligible background. This new assay uses 1,2-diacetyl benzene/ß-mercaptoethanol, which forms a fluorescent iso-indole-mercaptide conjugate with PE. PE detection with this method is very sensitive and comparable with detection by radiochemical methods. Model reactions examining adduct formation with ethanolamine produced stable products of exact masses (m/z) of 342.119 and 264.105. The assay is robust, with a signal/background ratio of 24, and can readily detect formation of 100 pmol of PE produced from Escherichia coli membranes, Candida albicans mitochondria, or HeLa cell mitochondria. PSD activity can easily be quantified by sequential reagent additions in 96- or 384-well plates, making it readily adaptable to high-throughput screening for PSD inhibitors. This new assay now enables straightforward large-scale screening for PSD inhibitors against pathogenic fungi, antibiotic-resistant bacteria, and neoplastic mammalian cells.
Asunto(s)
Carboxiliasas/análisis , Colorantes Fluorescentes/síntesis química , Espectrometría de Fluorescencia/métodos , Acetofenonas/química , Candida albicans/metabolismo , Carboxiliasas/metabolismo , Membrana Celular/metabolismo , Etanolamina , Fluorescencia , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Mercaptoetanol/química , Mitocondrias , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Estirenos/químicaRESUMEN
Phosphatidylserine decarboxylases (PSDs) catalyze the decarboxylation of phosphatidylserine to generate phosphatidylethanolamine, a critical step in phospholipid metabolism in both prokaryotes and eukaryotes. Most PSDs are membrane-bound, and classical radioisotope-based assays for determining their activity in vitro are not suitable for high-throughput drug screening. The finding that the PkPSD from Plasmodium knowlesi can be purified in a soluble and active form and the recent development of a fluorescence-based distyrylbenzene-bis-aldehyde (DSB-3) assay to measure PSD activity in vitro have laid the groundwork for screening chemical libraries for PSD inhibitors. Using this assay, here we conducted a high-throughput screen of a structurally diverse 130,858-compound library against PkPSD. Further characterization of the hits identified in this screening yielded five PkPSD inhibitors with IC50 values ranging from 3.1 to 42.3 µm Lead compounds were evaluated against the pathogenic yeast Candida albicans in the absence or presence of exogenous ethanolamine, and YU253467 and YU254403 were identified as inhibiting both native C. albicans PSD mitochondrial activity and C. albicans growth, with an MIC50 of 22.5 and 15 µg/ml without ethanolamine and an MIC50 of 75 and 60 µg/ml with ethanolamine, respectively. Together, these results provide the first proof of principle for the application of DSB-3-based fluorescent readouts in high-throughput screening for PSD inhibitors. The data set the stage for future analyses to identify more selective and potent PSD inhibitors with antimicrobial or antitumor activities.
Asunto(s)
Carboxiliasas/antagonistas & inhibidores , Inhibidores Enzimáticos/análisis , Colorantes Fluorescentes/química , Ensayos Analíticos de Alto Rendimiento , Estirenos/química , Candida albicans/efectos de los fármacos , Carboxiliasas/genética , Carboxiliasas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Etanolamina/farmacología , Humanos , Concentración 50 Inhibidora , Fosfatidilserinas/metabolismo , Plasmodium knowlesi/enzimología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The life cycle of malaria parasites in both their mammalian host and mosquito vector consists of multiple developmental stages that ensure proper replication and progeny survival. The transition between these stages is fueled by nutrients scavenged from the host and fed into specialized metabolic pathways of the parasite. One such pathway is used by Plasmodium falciparum, which causes the most severe form of human malaria, to synthesize its major phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. Much is known about the enzymes involved in the synthesis of these phospholipids, and recent advances in genetic engineering, single-cell RNA-Seq analyses, and drug screening have provided new perspectives on the importance of some of these enzymes in parasite development and sexual differentiation and have identified targets for the development of new antimalarial drugs. This Minireview focuses on two phospholipid biosynthesis enzymes of P. falciparum that catalyze phosphoethanolamine transmethylation (PfPMT) and phosphatidylserine decarboxylation (PfPSD) during the blood stages of the parasite. We also discuss our current understanding of the biochemical, structural, and biological functions of these enzymes and highlight efforts to use them as antimalarial drug targets.
Asunto(s)
Estadios del Ciclo de Vida/fisiología , Malaria Falciparum/genética , Malaria Falciparum/metabolismo , Fosfolípidos , Plasmodium falciparum , Animales , Antimaláricos/química , Antimaláricos/uso terapéutico , Desarrollo de Medicamentos , Humanos , Malaria Falciparum/tratamiento farmacológico , Fosfolípidos/biosíntesis , Fosfolípidos/genética , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismoRESUMEN
Phosphatidylserine decarboxylases (PSDs) are central enzymes in phospholipid metabolism that produce phosphatidylethanolamine (PE) in bacteria, protists, plants, and animals. We developed a fluorescence-based assay for selectively monitoring production of PE in reactions using a maltose-binding protein fusion with Plasmodium knowlesi PSD (MBP-His6-Δ34PkPSD) as the enzyme. The PE detection by fluorescence (λex = 403 nm, λem = 508 nm) occurred after the lipid reacted with a water-soluble distyrylbenzene-bis-aldehyde (DSB-3), and provided strong discrimination against the phosphatidylserine substrate. The reaction conditions were optimized for enzyme, substrate, product, and DSB-3 concentrations with the purified enzyme and also tested with crude extracts and membrane fractions from bacteria and yeast. The assay is readily amenable to application in 96- and 384-well microtiter plates and should prove useful for high-throughput screening for inhibitors of PSD enzymes across diverse phyla.
Asunto(s)
Carboxiliasas/química , Fluorescencia , Proteínas de Unión a Maltosa/química , Fosfatidiletanolaminas/química , Plasmodium knowlesi/enzimología , Proteínas Protozoarias/química , Carboxiliasas/genética , Proteínas de Unión a Maltosa/genética , Fosfatidiletanolaminas/análisis , Plasmodium knowlesi/genética , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Lipid asymmetries between the outer and inner leaflet of the lipid bilayer exist in nearly all biological membranes. Although living cells spend great effort to adjust and maintain these asymmetries, little is known about the biophysical phenomena within asymmetric membranes and their role in cellular function. One reason for this lack of insight into such a fundamental membrane property is the fact that the majority of model-membrane studies have been performed on symmetric membranes. Our aim is to overcome this problem by employing a targeted, enzymatic reaction to prepare asymmetric liposomes with phosphatidylserine (PS) primarily in the inner leaflet. To achieve this goal, we use a recombinant version of a water soluble PS decarboxylase from Plasmodium knowlesi, which selectively decarboxylates PS in the outer leaflet, converting it to phosphatidylethanolamine. The extent of decarboxylation is quantified using high-performance thin-layer chromatography, and the local concentration of anionic PS in the outer leaflet is monitored in terms of the ζ potential. Starting, for example, with 21 mol % 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine sodium salt, the assay leads to liposomes with 21 mol % in the inner and 6 mol % PS in the outer leaflet. This asymmetry persists virtually unchanged for at least 4 days at 20°C and at least 2 days at 40°C. The use of a highly specific enzyme carries the advantage that a minor component such as PS can be adjusted without affecting or being affected by the other lipid species present in the model membrane. The phenomena governing the residual outside PS content are addressed but warrant further study.
Asunto(s)
Proteínas Bacterianas/metabolismo , Carboxiliasas/metabolismo , Membrana Celular/metabolismo , Membrana Dobles de Lípidos/metabolismo , Liposomas/química , Fosfatidilserinas/metabolismo , Plasmodium knowlesi/enzimología , Membrana Celular/química , Liposomas/metabolismo , Fosfatidiletanolaminas/metabolismoRESUMEN
Phosphatidylethanolamine (PE) and phosphatidylserine (PS) are ubiquitously expressed and metabolically interconnected glycerophospholipids in eukaryotes and prokaryotes. In Trypanosoma brucei, PE synthesis has been shown to occur mainly via the Kennedy pathway, one of the three routes leading to PE synthesis in eukaryotes, while PS synthesis has not been studied experimentally. We now reveal the importance of T. brucei PS synthase 2 (TbPSS2) and T. brucei PS decarboxylase (TbPSD), two key enzymes involved in aminophospholipid synthesis, for trypanosome viability. By using tetracycline-inducible down-regulation of gene expression and in vivo and in vitro metabolic labeling, we found that TbPSS2 (i) is necessary for normal growth of procyclic trypanosomes, (ii) localizes to the endoplasmic reticulum and (iii) represents the unique route for PS formation in T. brucei. In addition, we identified TbPSD as type I PS decarboxylase in the mitochondrion and found that it is processed proteolytically at a WGSS cleavage site into a heterodimer. Down-regulation of TbPSD expression affected mitochondrial integrity in both procyclic and bloodstream form trypanosomes, decreased ATP production via oxidative phosphorylation in procyclic form and affected parasite growth.
Asunto(s)
Carboxiliasas/metabolismo , Transferasas de Grupos Nitrogenados/metabolismo , Trypanosoma brucei brucei/enzimología , Descarboxilación , Mitocondrias/metabolismo , Fosforilación Oxidativa , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/genéticaRESUMEN
Cellular senescence, a distinctive type of irreversible growth arrest, develops in response to various stimuli. Bcl-w, an oncogene and member of the Bcl-2 family, has been reported to promote tumorigenicity in various cancer cells. Here, we sought to explore the potential role of Bcl-w in premature senescence, which has received relatively little research attention. Our findings demonstrate that Bcl-w enhances the activity of senescence-associated ß-galactosidase (SA-ß-gal) and promotes histone H3 tri-methylation at lysine 9 (H3K9me3) and expressions of p53, Notch2, p21, and p16-hallmarks of the senescent phenotype-in human U251 glioblastoma and H460 lung carcinoma cells. It is also known that microRNAs (miRNAs) regulate processes related to tumor development, such as cell proliferation, differentiation, survival, metabolism, inflammation, invasion, angiogenesis, and senescence. In this context, we found that miR-93-5p inhibited premature cellular senescence by directly suppressing Bcl-w and p21 expressions. Collectively, these findings suggest that targeting miR-93-5p-regulated Bcl-w may be a useful strategy for preventing premature senescence.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Senescencia Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , MicroARNs/farmacología , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Humanos , MicroARNs/genética , Fenotipo , Células Tumorales CultivadasRESUMEN
Phospholipid biosynthesis is critical for the development, differentiation and pathogenesis of several eukaryotic pathogens. Genetic studies have validated the pathway for phosphatidylethanolamine synthesis from phosphatidylserine catalyzed by phosphatidylserine decarboxylase enzymes (PSD) as a suitable target for development of antimicrobials; however no inhibitors of this class of enzymes have been discovered. We show that the Plasmodium falciparumâ PSD can restore the essential function of the yeast gene in strains requiring PSD for growth. Genetic, biochemical and metabolic analyses demonstrate that amino acids between positions 40 and 70 of the parasite enzyme are critical for proenzyme processing and decarboxylase activity. We used the essential role of Plasmodiumâ PSD in yeast as a tool for screening a library of anti-malarials. One of these compounds is 7-chloro-N-(4-ethoxyphenyl)-4-quinolinamine, an inhibitor with potent activity against P. falciparum, and low toxicity toward mammalian cells. We synthesized an analog of this compound and showed that it inhibits PfPSD activity and eliminates Plasmodium yoelii infection in mice. These results highlight the importance of 4-quinolinamines as a novel class of drugs targeting membrane biogenesis via inhibition of PSD activity.
Asunto(s)
Antimaláricos/farmacología , Carboxiliasas/antagonistas & inhibidores , Carboxiliasas/metabolismo , Inhibidores Enzimáticos/farmacología , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/enzimología , Animales , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Carboxiliasas/genética , Clonación Molecular , Femenino , Malaria Falciparum/microbiología , Ratones , Pruebas de Sensibilidad Parasitaria , Fosfatidilserinas/metabolismo , Plasmodium falciparum/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genéticaRESUMEN
BACKGROUND: We sought to validate the accuracy and assess the efficacy of a newly developed electronic weight estimation device (ie, the rolling tape) for paediatric weight estimation. METHODS: We enrolled a convenience sample of children aged <17â years presenting to our emergency department who volunteered to participate in the study. The children's heights and weights were measured, and three researchers estimated these values using the rolling tape and Broselow tape at 5 min intervals. The weight estimates of researcher 1, researcher 2 and the Broselow tape were compared with measured values, and mean percentage error (MPE), root mean square error (RMSE) and percentage of estimates within 10% of the actual measured values were calculated. For 30 randomly selected subjects, we compared the time interval from the start of the measurement to the time that orders for epinephrine, defibrillation dose and instrument size could be given in a simulated arrest scenario. RESULTS: We enrolled 906 children (median age 4.0â years). For researcher 1, researcher 2 and the Broselow tape, MPE values were 0.11% (RMSE 2.61â kg), 1.41% (RMSE, 2.61â kg) and 1.72% (RMSE 5.41â kg), respectively, and the percentages of children with predictions within 10% of their actual weight were 75.1%, 75.7% and 60.6%, respectively. In the 30 simulated cases, the mean time for measurement to ordering was significantly shorter (25.8â s vs 35.5â s, p<0.001) for the rolling tape compared with the Broselow tape method. CONCLUSIONS: The rolling tape is a good weight estimation tool for children compared with other methods. The rolling tape method significantly decreased the time from weight estimation to orders for essential drug dose, instrument size and defibrillation dose for resuscitation.
Asunto(s)
Antropometría/métodos , Peso Corporal , Pesos y Medidas/instrumentación , Pesos y Medidas/normas , Adolescente , Antropometría/instrumentación , Niño , Preescolar , Servicio de Urgencia en Hospital/organización & administración , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Pediatría/métodos , República de CoreaRESUMEN
Phosphatidylserine decarboxylase (PSDs) play a central role in the synthesis of phosphatidylethanolamine in numerous species of prokaryotes and eukaryotes. PSDs are unusual decarboxylase containing a pyruvoyl prosthetic group within the active site. The covalently attached pyruvoyl moiety is formed in a concerted reaction when the PSD proenzyme undergoes an endoproteolytic cleavage into a large ß-subunit, and a smaller α-subunit, which harbors the prosthetic group at its N terminus. The mechanism of PSD proenzyme cleavage has long been unclear. Using a coupled in vitro transcription/translation system with the soluble Plasmodium knowlesi enzyme (PkPSD), we demonstrate that the post-translational processing is inhibited by the serine protease inhibitor, phenylmethylsulfonyl fluoride. Comparison of PSD sequences across multiple phyla reveals a uniquely conserved aspartic acid within an FFXRX6RX12PXD motif, two uniquely conserved histidine residues within a PXXYHXXHXP motif, and a uniquely conserved serine residue within a GS(S/T) motif, suggesting that PSDs belong to the D-H-S serine protease family. The function of the conserved D-H-S residues was probed using site-directed mutagenesis of PkPSD. The results from these mutagenesis experiments reveal that Asp-139, His-198, and Ser-308 are all essential for endoproteolytic processing of PkPSD, which occurs in cis. In addition, within the GS(S/T) motif found in all PSDs, the Gly-307 residue is also essential, but the Ser/Thr-309 is non-essential. These results define the mechanism whereby PSDs begin their biochemical existence as proteases that execute one autoendoproteolytic cleavage reaction to give rise to a mature PSD harboring a pyruvoyl prosthetic group.
Asunto(s)
Carboxiliasas/metabolismo , Plasmodium knowlesi/enzimología , Procesamiento Proteico-Postraduccional/fisiología , Proteolisis , Proteínas Protozoarias/metabolismo , Serina Proteasas/metabolismo , Secuencias de Aminoácidos , Carboxiliasas/genética , Plasmodium knowlesi/genética , Proteínas Protozoarias/genética , Serina Proteasas/genética , Relación Estructura-ActividadRESUMEN
Efficient transmission of Plasmodium species between humans and Anopheles mosquitoes is a major contributor to the global burden of malaria. Gametocytogenesis, the process by which parasites switch from asexual replication within human erythrocytes to produce male and female gametocytes, is a critical step in malaria transmission and Plasmodium genetic diversity. Nothing is known about the pathways that regulate gametocytogenesis and only few of the current drugs that inhibit asexual replication are also capable of inhibiting gametocyte development and blocking malaria transmission. Here we provide genetic and pharmacological evidence indicating that the pathway for synthesis of phosphatidylcholine in Plasmodium falciparum membranes from host serine is essential for parasite gametocytogenesis and malaria transmission. Parasites lacking the phosphoethanolamine N-methyltransferase enzyme, which catalyzes the limiting step in this pathway, are severely altered in gametocyte development, are incapable of producing mature-stage gametocytes, and are not transmitted to mosquitoes. Chemical screening identified 11 inhibitors of phosphoethanolamine N-methyltransferase that block parasite intraerythrocytic asexual replication and gametocyte differentiation in the low micromolar range. Kinetic studies in vitro as well as functional complementation assays and lipid metabolic analyses in vivo on the most promising inhibitor NSC-158011 further demonstrated the specificity of inhibition. These studies set the stage for further optimization of NSC-158011 for development of a class of dual activity antimalarials to block both intraerythrocytic asexual replication and gametocytogenesis.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Malaria Falciparum/transmisión , Metiltransferasas/metabolismo , Plasmodium falciparum/enzimología , Reproducción Asexuada/efectos de los fármacos , Antimaláricos/farmacología , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Malaria Falciparum/enzimología , Masculino , Metiltransferasas/antagonistas & inhibidores , Plasmodium falciparum/crecimiento & desarrollo , Radiometría , Serina/metabolismoRESUMEN
In yeast, a protein complex termed the ER-Mitochondria Encounter Structure (ERMES) tethers mitochondria to the endoplasmic reticulum. ERMES proteins are implicated in a variety of cellular functions including phospholipid synthesis, mitochondrial protein import, mitochondrial attachment to actin, polarized mitochondrial movement into daughter cells during division, and maintenance of mitochondrial DNA (mtDNA). The mitochondrial-anchored Gem1 GTPase has been proposed to regulate ERMES functions. Here, we show that ERMES and Gem1 have no direct role in the transport of phosphatidylserine (PS) from the ER to mitochondria during the synthesis of phosphatidylethanolamine (PE), as PS to PE conversion is not affected in ERMES or gem1 mutants. In addition, we report that mitochondrial inheritance defects in ERMES mutants are a secondary consequence of mitochondrial morphology defects, arguing against a primary role for ERMES in mitochondrial association with actin and mitochondrial movement. Finally, we show that ERMES complexes are long-lived, and do not depend on the presence of Gem1. Our findings suggest that the ERMES complex may have primarily a structural role in maintaining mitochondrial morphology.
Asunto(s)
Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Fosfatidilserinas/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , ADN Mitocondrial/metabolismo , GTP Fosfohidrolasas/química , Proteínas Fluorescentes Verdes/metabolismo , Espectrometría de Masas/métodos , Microscopía Fluorescente/métodos , Proteínas Mitocondriales/metabolismo , Modelos Biológicos , Mutación , Fosfatidiletanolaminas/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Polyamines are polycationic molecules that are crucial in a wide array of cellular functions. Their biosynthesis is mediated by aminopropyl transferases (APTs), promising targets in antimicrobial, antineoplastic and antineurodegenerative therapies. A major limitation, however, is the lack of high-throughput assays to measure their activity. We developed the first fluorescence-based assay, DAB-APT, for measurement of APT activity using 1,2-diacetyl benzene, which forms fluorescent conjugates with putrescine, spermidine and spermine with fluorescence intensity increasing with increasing carbon chain length. The assay has been validated using APT enzymes from S. cerevisiae and P. falciparum and is suitable for high-throughput screening of large chemical libraries. Given the importance of APTs in infectious diseases, cancer and neurobiology, our DAB-APT assay has broad applications, holding promise for advancing research and drug discovery efforts.
RESUMEN
Fungal infections, a leading cause of mortality among eukaryotic pathogens, pose a growing global health threat due to the rise of drug-resistant strains. New therapeutic strategies are urgently needed to combat this challenge. The PCA pathway for biosynthesis of Co-enzyme A (CoA) and Acetyl-CoA (AcCoA) from vitamin B5 (pantothenic acid) has been validated as an excellent target for the development of new antimicrobials against fungi and protozoa. The pathway regulates key cellular processes including metabolism of fatty acids, amino acids, sterols, and heme. In this study, we provide genetic evidence that disruption of the PCA pathway in Saccharomyces cerevisiae results in a significant alteration in the susceptibility of fungi to a wide range of xenobiotics, including clinically approved antifungal drugs through alteration of vacuolar morphology and drug detoxification. The drug potentiation mediated by genetic regulation of genes in the PCA pathway could be recapitulated using the pantazine analog PZ-2891 as well as the celecoxib derivative, AR-12 through inhibition of fungal AcCoA synthase activity. Collectively, the data validate the PCA pathway as a suitable target for enhancing the efficacy and safety of current antifungal therapies.
Asunto(s)
Antifúngicos , Mitocondrias , Saccharomyces cerevisiae , Vacuolas , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efectos de los fármacos , Vacuolas/metabolismo , Antifúngicos/farmacología , Antifúngicos/metabolismo , Ácido Pantoténico/metabolismo , Farmacorresistencia Fúngica/genética , Inactivación MetabólicaRESUMEN
High-dose radiation (HDR) is widely used for cancer treatment, but the effectiveness of low-dose radiation (LDR) in the treatment of various diseases is controversial. Therefore, to safely utilize LDR for therapeutic purposes, further research on its numerous biological effects of LDR is required. Interest in the increased use of medical imaging devices or the effects of surrounding living environmental radiation on the human body, particularly on fibrosis, is rapidly increasing. Therefore, this study aimed to verify the relationship between LDR and pulmonary fibrosis by evaluating the changes in fibroblasts after LDR treatment and their associated signaling mechanisms. LDR increased the expression of fibrosis markers COL1A1 and α-SMA, cell proliferation, and migration by activating YAP1 and Twist in fibroblasts. Meanwhile, miRNA was employed as a tool to inhibit LDR-induced fibrosis and it was found that miR-765 simultaneously targeted COL1A1, α-SMA, and YAP1. At the cellular level, miR-765 reduced the proliferation and migration of fibroblasts by suppressing the expression of LDR-induced fibrosis factors COL1A1, α-SMA, and YAP1. The efficacy of miR-765 in vivo was confirmed using bleomycin (BLM)-induced fibrotic mouse model. The characteristics of pulmonary fibrosis were reduced after injection of miR-765-overexpressing cells into BLM-induced fibrotic mice. In addition, the suppression of miR-765 expression in the plasma of patients with pulmonary fibrosis confirmed the negative relationship between pulmonary fibrosis and miR-765 expression. Therefore, this study demonstrates that miR-765 is a potential novel diagnostic biomarker and major target for the development of therapeutic agents to inhibit pulmonary fibrosis.