RESUMEN
Little is known about the microbial ecology of the subsurface oil sands in Northern Alberta, Canada. Biodegradation of low molecular weight hydrocarbons by indigenous microbes has enriched high molecular weight hydrocarbons, resulting in highly viscous bitumen. This extreme subsurface environment is further characterized by low nutrient availability and limited access to water, thus resulting in low microbial biomass. Improved DNA isolation protocols and increasingly sensitive sequencing methods have allowed an in-depth investigation of the microbial ecology of this unique subsurface environmental niche. Community analysis was performed on core samples (n = 62) that were retrieved from two adjacent sites located in the Athabasca Oil Sands at depths from 220 to 320 m below the surface. Microbial communities were dominated by aerobic taxa, including Pseudomonas and Acinetobacter. Only one core sample microbial community was dominated by anaerobic taxa, including the methanogen Methanoculleus, as well as Desulfomicrobium and Thauera. Although the temperature of the bitumen-containing subsurface is low (8°C), two core samples had high fractions of the potentially thermophilic taxon, Thermus. Predominance of aerobic taxa in the subsurface suggests the potential for in situ aerobic hydrocarbon degradation; however, more studies are required to determine the functional role of these taxa within this unique environment.
Asunto(s)
Bacterias Aerobias/clasificación , Bacterias Aerobias/aislamiento & purificación , Biodegradación Ambiental , Hidrocarburos/metabolismo , Yacimiento de Petróleo y Gas/microbiología , Alberta , Bacterias Aerobias/genética , Hongos/clasificación , Hongos/genética , Hongos/aislamiento & purificación , Microbiota/genética , ARN Ribosómico 16S/genética , Microbiología del SueloRESUMEN
ß-Glucosidase 2 (GBA2) is an enzyme that cleaves the membrane lipid glucosylceramide into glucose and ceramide. The GBA2 gene is mutated in genetic neurological diseases (hereditary spastic paraplegia and cerebellar ataxia). Pharmacologically, GBA2 is reversibly inhibited by alkylated imino sugars that are in clinical use or are being developed for this purpose. We have addressed the ambiguity surrounding one of the defining characteristics of GBA2, which is its sensitivity to inhibition by conduritol B epoxide (CBE). We found that CBE inhibited GBA2, in vitro and in live cells, in a time-dependent fashion, which is typical for mechanism-based enzyme inactivators. Compared with the well characterized impact of CBE on the lysosomal glucosylceramide-degrading enzyme (glucocerebrosidase, GBA), CBE inactivated GBA2 less efficiently, due to a lower affinity for this enzyme (higher KI) and a lower rate of enzyme inactivation (k(inact)). In contrast to CBE, N-butyldeoxygalactonojirimycin exclusively inhibited GBA2. Accordingly, we propose to redefine GBA2 activity as the ß-glucosidase that is sensitive to inhibition by N-butyldeoxygalactonojirimycin. Revised as such, GBA2 activity 1) was optimal at pH 5.5-6.0; 2) accounted for a much higher proportion of detergent-independent membrane-associated ß-glucosidase activity; 3) was more variable among mouse tissues and neuroblastoma and monocyte cell lines; and 4) was more sensitive to inhibition by N-butyldeoxynojirimycin (miglustat, Zavesca®), in comparison with earlier studies. Our evaluation of GBA2 makes it possible to assess its activity more accurately, which will be helpful in analyzing its physiological roles and involvement in disease and in the pharmacological profiling of monosaccharide mimetics.
Asunto(s)
1-Desoxinojirimicina/análogos & derivados , Inhibidores Enzimáticos/farmacocinética , Inositol/análogos & derivados , beta-Glucosidasa/antagonistas & inhibidores , 1-Desoxinojirimicina/farmacocinética , 1-Desoxinojirimicina/farmacología , Animales , Células COS , Línea Celular Tumoral , Ataxia Cerebelosa/tratamiento farmacológico , Ataxia Cerebelosa/enzimología , Chlorocebus aethiops , Inhibidores Enzimáticos/farmacología , Glucosilceramidasa , Humanos , Concentración de Iones de Hidrógeno , Inositol/farmacocinética , Inositol/farmacología , Ratones , Paraplejía Espástica Hereditaria/tratamiento farmacológico , Paraplejía Espástica Hereditaria/enzimología , beta-Glucosidasa/metabolismoRESUMEN
Identification of novel pro-survival factors in the brain is paramount for developing neuroprotective therapies. The multifunctional S100 family proteins have important roles in many human diseases and are also upregulated by brain injury. However, S100 functions in the nervous system remain unclear. Here we show that the S100A4 protein, mostly studied in cancer, is overexpressed in the damaged human and rodent brain and released from stressed astrocytes. Genetic deletion of S100A4 exacerbates neuronal loss after brain trauma or excitotoxicity, increasing oxidative cell damage and downregulating the neuroprotective protein metallothionein I+II. We identify two neurotrophic motifs in S100A4 and show that these motifs are neuroprotective in animal models of brain trauma. Finally, we find that S100A4 rescues neurons via the Janus kinase/STAT pathway and, partially, the interleukin-10 receptor. Our data introduce S100A4 as a therapeutic target in neurodegeneration, and raise the entire S100 family as a potentially important factor in central nervous system injury.
Asunto(s)
Citoprotección , Metástasis de la Neoplasia/patología , Neuronas/patología , Proteínas S100/metabolismo , Secuencias de Aminoácidos , Animales , Lesiones Encefálicas/tratamiento farmacológico , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/patología , Muerte Celular/efectos de los fármacos , Citoprotección/efectos de los fármacos , Femenino , Eliminación de Gen , Células HEK293 , Humanos , Quinasas Janus/metabolismo , Ácido Kaínico , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/metabolismo , Neurotoxinas/toxicidad , Estrés Oxidativo/efectos de los fármacos , Péptidos/farmacología , Péptidos/uso terapéutico , Ratas , Receptores de Interleucina-10/metabolismo , Proteína de Unión al Calcio S100A4 , Proteínas S100/química , Factores de Transcripción STAT/metabolismo , Convulsiones/tratamiento farmacológico , Convulsiones/patología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The emergence of multidrug-resistant cancers and the lack of targeted therapies for many cancers underscore an unmet need for new therapeutics with novel modes of action towards cancer cells. Host-defense peptides often exhibit selective cytotoxicity towards cancer cells and show potential as anti-cancer therapeutics. Here, we screen 26 naturally occurring variants of the peptide pleurocidin for cytotoxic and anti-cancer activities, and investigate the underlying mechanism of action. Cytotoxicities were assessed in vitro using cell-based assays and in vivo using zebrafish embryos. Morphological changes were assessed by both transmission and scanning electron microscopy, and functional assays were performed on zebrafish embryos to investigate the mechanism of cell death. A total of 14 peptides were virtually inactive against HL60 human leukemia cells, whereas 12 caused >50% death at ≤32 µg/ml. Morphological changes characteristic of oncosis were evident by electron microscopy after only 1 minute of treatment with 32 µg/ml of variant NRC-03. Only two peptides were hemolytic. Four peptides showed no toxicity towards zebrafish embryos at the highest concentration tested (25 µM; â¼64 µg/ml) and one peptide was highly toxic, killing 4-hour-post-fertilization (hpf) embryos immediately after exposure to 1 µM peptide. Four other peptides killed embryos after 24 hours of exposure at 1 µM. Most peptides caused mortality at one or more developmental stages only after continuous exposure (24 hours) with higher lethal doses (≥5 µM). Pleurocidin NRC-03 bound to embryos and induced the release of superoxide, caused an increase in the number of TUNEL-positive nuclei, and caused membrane damage and the loss of embryonic epithelial integrity, marked by the exclusion of cells from the outer epithelium and the appearance of F-actin within the circumferential cells of the repair site. Our results indicate that specific pleurocidin variants are attractive cancer-selective agents that selectively induce cell death in target cells but leave non-target cells such as erythrocytes and non-transformed cells unaffected.