RESUMEN

Parasporin-2Aa1 (PS2Aa1) is a toxic protein of 37 KDa (30 kDa, activated form produced by proteolysis) that was shown to be cytotoxic against specific human cancer cells, although its mechanism of action has not been elucidated yet. In order to study the role of some native peptide fragments of proteins on anticancer activity, here we investigated the cytotoxic effect of peptide fragments from domain-1 of PS2Aa1 and one of the loops present in the binding region of the virus spike protein from Alphacoronavirus (HCoV-229E), the latter according to scientific reports, who showed interaction with the human APN (h-APN) receptor, evidence corroborated through computational simulations, and thus being possible active against colon cancer cells. Peptides namely P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa were synthesized using the Fmoc solid-phase synthesis and characterized by mass spectrometry (MS). Additionally, one region from loop 1 of HCoV-229E, Loop1-HCoV-229E, was also synthesized and characterized. The A4W-GGN5 anticancer peptide and 5-fluorouracil (5-FU) were taken as a control in all experiments. Circular dichroism revealed an α-helix structure for the peptides derived from PS2Aa1 (P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa) and ß-laminar structure for the peptide derived from Alphacoronavirus spike protein Loop1-HCoV-229E. Peptides showed a hemolysis percentage of less than 20% at 100 µM concentration. Besides, peptides exhibited stronger anticancer activity against SW480 and SW620 cells after exposure for 48 h. Likewise, these compounds showed significantly lower toxicity against normal cells CHO-K1. The results suggest that native peptide fragments from Ps2Aa1 may be optimized as a novel potential cancer-therapeutic agents.

Asunto(s)

Antineoplásicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Endotoxinas/farmacología , Fragmentos de Péptidos/farmacología , Glicoproteína de la Espiga del Coronavirus/farmacología , Alphacoronavirus , Animales , Antineoplásicos/síntesis química , Antineoplásicos/toxicidad , Antígenos CD13/metabolismo , Células CHO , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Cricetulus , Endotoxinas/toxicidad , Hemólisis/efectos de los fármacos , Humanos , Simulación del Acoplamiento Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/toxicidad , Conformación Proteica en Hélice alfa , Oveja Doméstica , Glicoproteína de la Espiga del Coronavirus/toxicidad , Relación Estructura-Actividad

RESUMEN

The midgut from lepidopteran insects has a particular way to release proteins to the lumen, named microapocrine secretion that could be an adaptation to release secretory contents into the lumen at water absorbing regions. In this process small vesicles (microapocrine vesicles) bud from the midgut microvilli as double membrane vesicles, where the inner membrane comes from the secretion vesicle and the outer one from the microvillar membrane. The molecular machinery associated with this process may be recruited by specific midgut microvilli membrane domains. To address to this, Spodoptera frugiperda midgut microvillar membranes, prepared by magnesium treatment and free from cytoskeleton with the hyperosmotic Tris procedure, were submitted to detergent extraction and fractionated by density gradient ultracentrifugation. Detergent-resistant membrane domains (DRM) were recovered and their proteins identified by proteomics. Microapocrine vesicles were isolated by washing the luminal surface of the midgut epithelium, followed by freezing and thawing plus centrifugation to recover only membranes. Proteins from purified microvillar membranes and microapocrine vesicle membranes were identified by proteomics. Comparison of the two populations suggests that the budding of microapocrine vesicles surrounded by microvillar membrane is not a random process, because only around 50% of the microvillar membrane proteins are in the microapocrine vesicles. From the 16 proteins from DRM, 14 were enriched in the microapocrine membrane vesicles. These results suggest that on budding, the microapocrine vesicle membrane is enclosed by DRM and a surrounding area of the microvillar membrane. It is proposed that the DRMs somehow recruit the proteins composing the secretory machinery.

Asunto(s)

Spodoptera/metabolismo , Animales , Glándulas Apocrinas/metabolismo , Antígenos CD13/metabolismo , Colesterol/metabolismo , Detergentes , Sistema Digestivo/metabolismo , Proteínas Ligadas a GPI/metabolismo , Proteínas de Insectos/metabolismo , Proteínas de la Membrana/metabolismo , Microvellosidades/metabolismo , Octoxinol , Proteómica

RESUMEN

The effects of in ovo feeding with threonine (Thr) on intestinal morphology, ileal gene expression and performance of broiler chicken between 1 and 21 d of age (d) were assessed. On day 17.5 of incubation, fertile eggs were randomly allotted to 5 treatments of Thr injection in the amniotic fluid (0; 1.75; 3.5; 5.25; 7%, corresponding to 17.5; 35; 52.5 and 70 mg Thr/mL). After hatch, chicks were given a commercial corn-soybean diet up to 21 d. Daily feed intake (FI), body weight (BW), and food conversion ratio (FCR) were measured from 1 to 7, 14, and 21 d of age. The ileal gene expression of mucin (MUC2), peptide transporter (PepT1), and aminopeptidase enzyme (APN) were evaluated on day of hatch and at 21 d, as well as intestinal morphometric traits. In ovo feeding with threonine significantly increased final weight (FI) and weight gain (WG) and decreased FCR in the period from 1 to 21 d. Threonine levels affected beneficially the villus height, vilo: crypt ratio and villus area on day of hatch and at 21 d. At hatch, all Thr levels increased the expression of MUC2 and PepT1 compared to the control group. APN expression also increased, but for the lowest and the highest threonine levels (1.75 and 7%). At 21 d, there was no effect of threonine on the expression of MUC2, PepT1, and APN. In conclusion, in ovo threonine feeding beneficially affected the morphological and functional development of the intestinal mucosa, which ensured improved performance of chicks at hatch and at 21 d.

Asunto(s)

Pollos/fisiología , Intestino Delgado/efectos de los fármacos , Treonina/farmacología , Amnios , Animales , Antígenos CD13/genética , Antígenos CD13/metabolismo , Embrión de Pollo , Pollos/crecimiento & desarrollo , Expresión Génica , Íleon/metabolismo , Intestino Delgado/crecimiento & desarrollo , Mucinas/genética , Mucinas/metabolismo , Transportador de Péptidos 1/genética , Transportador de Péptidos 1/metabolismo , Treonina/administración & dosificación

RESUMEN

Helicoverpa armigera is a polyphagous pest sensitive to Cry1Ac protein from Bacillus thuringiensis (Bt). The susceptibility of the different larval instars of H. armigera to Cry1Ac protoxin showed a significant 45-fold reduction in late instars compared to early instars. A possible hypothesis is that gut surface proteins that bind to Cry1Ac differ in both instars, although higher Cry toxin degradation in late instars could also explain the observed differences in susceptibility. Here we compared the Cry1Ac-binding proteins from second and fifth instars by pull-down assays and liquid chromatography coupled to mass spectrometry analysis (LC-MS/MS). The data show differential protein interaction patterns of Cry1Ac in the two instars analyzed. Alkaline phosphatase, and other membrane proteins, such as prohibitin and an anion selective channel protein were identified only in the second instar, suggesting that these proteins may be involved in the higher toxicity of Cry1Ac in early instars of H. armigera. Eleven Cry1Ac binindg proteins were identified exclusively in late instar larvae, like different proteases such as trypsin-like protease, azurocidin-like proteinase, and carboxypeptidase. Different aminopeptidase N isofroms were identified in both instar larvae. We compared the Cry1Ac protoxin degradation using midgut juice from late and early instars, showing that the midgut juice from late instars is more efficient to degrade Cry1Ac protoxin than that of early instars, suggesting that increased proteolytic activity on the toxin could also explain the low Cry1Ac toxicity in late instars.

Asunto(s)

Proteínas Bacterianas/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insectos/metabolismo , Mariposas Nocturnas/metabolismo , Receptores de Superficie Celular/metabolismo , Fosfatasa Alcalina/aislamiento & purificación , Fosfatasa Alcalina/metabolismo , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/toxicidad , Antígenos CD13/aislamiento & purificación , Antígenos CD13/metabolismo , Cromatografía Liquida , Sistema Digestivo/metabolismo , Endotoxinas/toxicidad , Proteínas Hemolisinas/toxicidad , Proteínas de Insectos/aislamiento & purificación , Larva/metabolismo , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Mariposas Nocturnas/crecimiento & desarrollo , Mariposas Nocturnas/patogenicidad , Control Biológico de Vectores , Receptores de Superficie Celular/aislamiento & purificación , Espectrometría de Masas en Tándem

RESUMEN

Bacillus thuringiensis Cry1Ca is toxic to different Spodoptera species. The aims of this work were to identify the Cry1Ca-binding proteins in S. frugiperda, to provide evidence on their participation in toxicity, and to identify the Cry1Ca amino acid residues involved in receptor binding. Pulldown assays using Spodoptera frugiperda brush border membrane vesicles (BBMV) identified aminopeptidase N (APN), APN1, and APN2 isoforms as Cry1Ca-binding proteins. Cry1Ca alanine substitutions in all residues of domain III ß16 were characterized. Two ß16 nontoxic mutants (V505A and S506A) showed a correlative defect on binding to the recombinant S. frugiperda APN1 (SfAPN1). Finally, silencing the expression of APN1 transcript, by double-stranded RNA (dsRNA) feeding, showed that silenced larvae are more tolerant of the Cry1Ca toxin, which induced less than 40% mortality in silenced larvae whereas nonsilenced larvae had 100% mortality. Overall, our results show that Cry1Ca relies on APN1 binding through domain III ß16 to impart toxicity to S. frugiperdaIMPORTANCEBacillus thuringiensis Cry toxins rely on receptor binding to exert toxicity. Cry1Ca is toxic to different populations of S. frugiperda, a major corn pest in America. Nevertheless, the S. frugiperda midgut proteins that are involved in Cry1Ca toxicity have not been identified. Here we identified aminopeptidase N1 (APN1) as a functional receptor of Cry1Ca. Moreover, we showed that Cry1Ca domain III ß16 is involved in APN1 binding. These results give insights on potential target sites for improving Cry1Ca toxicity to S. frugiperda.

Asunto(s)

Bacillus thuringiensis/patogenicidad , Proteínas Bacterianas/metabolismo , Antígenos CD13/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Control Biológico de Vectores/métodos , Spodoptera/microbiología , Animales , Toxinas de Bacillus thuringiensis , Antígenos CD13/genética , Unión Proteica/fisiología , Dominios Proteicos/fisiología

RESUMEN

CD13 is a membrane glycoprotein with aminopeptidase activity, expressed on several cell types, including myeloid cells (dendritic cells, monocytes, macrophages, neutrophils, etc.). CD13 participates in several functions such as proteolytic regulation of bioactive peptides, viral receptor, angiogenesis, and tumor metastasis. CD13 has also been proposed to participate in cell adhesion, as crosslinking of CD13 by certain CD13-specific antibodies induces homotypic aggregation of monocytes and heterotypic adhesion of monocytes to endothelial cells. We generated two monoclonal antibodies (mAbs C and E) that block homotypic aggregation of U-937 monocytic cells induced by CD13-specific mAb 452. Moreover, the mAbs cause detachment of cells whose aggregation was induced by CD13 crosslinking. Both mAbs also inhibit heterotypic adhesion of U-937 monocytes to endothelial cells. mAbs C and E recognize membrane CD13 but bind to epitopes different from that recognized by mAb 452. Crosslinking of CD13 by mAb C or E is required to inhibit adhesion, as monovalent Fab fragments are not sufficient. Thus, C and E antibodies recognize a distinct epitope on CD13, and binding to this epitope interferes with both CD13-mediated cell adhesion and enzymatic activity. These antibodies may represent important tools to study cell-cell interactions mediated by CD13 in physiological and pathological conditions.

Asunto(s)

Antígenos CD13/metabolismo , Epítopos/metabolismo , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacología , Adhesión Celular , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/farmacología , Ratones , Células THP-1 , Células U937

RESUMEN

Bacillus thuringiensis Cry toxins are currently used for pest control in transgenic crops but evolution of resistance by the insect pests threatens the use of this technology. The Cry1AbMod toxin was engineered to lack the alpha helix-1 of the parental Cry1Ab toxin and was shown to counter resistance to Cry1Ab and Cry1Ac toxins in different insect species including the fall armyworm Spodoptera frugiperda. In addition, Cry1AbMod showed enhanced toxicity to Cry1Ab-susceptible S. frugiperda populations. To gain insights into the mechanisms of this Cry1AbMod-enhanced toxicity, we isolated the Cry1AbMod toxin binding proteins from S. frugiperda brush border membrane vesicles (BBMV), which were identified by pull-down assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The LC-MS/MS results indicated that Cry1AbMod toxin could bind to four classes of aminopeptidase (N1, N3, N4 y N5) and actin, with the highest amino acid sequence coverage acquired for APN 1 and APN4. In addition to these proteins, we found other proteins not previously described as Cry toxin binding proteins. This is the first report that suggests the interaction between Cry1AbMod and APN in S. frugiperda.

Asunto(s)

Proteínas Bacterianas/metabolismo , Antígenos CD13/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insectos/metabolismo , Spodoptera/enzimología , Actinas/química , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/química , Antígenos CD13/química , Antígenos CD13/aislamiento & purificación , Endotoxinas/química , Proteínas Hemolisinas/química , Proteínas de Insectos/química , Proteínas de Insectos/aislamiento & purificación , Microvellosidades/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética

RESUMEN

Bacillus thuringiensis Cry2Ab toxin has been used in combination with Cry1Ac for resistance management on the Bt-cotton that is widely planted worldwide. However, little is known regarding Cry2Ab mode of action. Particularly, there is a gap of knowledge on the identification of insect midgut proteins that bind Cry2Ab and mediate toxicity. In the case of Cry1Ab toxin, a transmembrane cadherin protein and glycosyl-phosphatidylinositol (GPI) anchored proteins like aminopeptidase-N1 (APN1) or alkaline-phosphatase (ALP) from Manduca sexta, have been shown to be important for oligomer formation and insertion into the membrane. Binding competition experiments showed that Cry2Ab toxin does not share binding sites with Cry1Ab toxin in M. sexta brush border membrane vesicles (BBMV). Also, that Cry2Ab shows reduced binding to the Cry1Ab binding molecules cadherin, APN1 or ALP. Finally, ligand blot experiments and protein sequence by LC-MS/MS identified APN2 isoform as a Cry2Ab binding protein. Cloning and expression of APN2 confirmed that APN2 is a Cry2Ab binding protein.

Asunto(s)

Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Antígenos CD13/química , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insectos/química , Manduca/enzimología , Receptores de Superficie Celular/química , Secuencia de Aminoácidos , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/aislamiento & purificación , Sitios de Unión , Antígenos CD13/aislamiento & purificación , Antígenos CD13/metabolismo , Endotoxinas/química , Proteínas Hemolisinas/química , Proteínas de Insectos/aislamiento & purificación , Proteínas de Insectos/metabolismo , Resistencia a los Insecticidas , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Ligandos , Manduca/genética , Receptores de Superficie Celular/aislamiento & purificación , Receptores de Superficie Celular/metabolismo

RESUMEN

The lack of a complete assembly of the sensitivity of subcellular aminopeptidase (AP) activities to insulin in different pathophysiological conditions has hampered the complete view of the adipocyte metabolic pathways and its implications in these conditions. Here we investigated the influence of insulin on basic AP (APB), neutral puromycin-sensitive AP (PSA), and neutral puromycin-insensitive AP (APM) in high and low density microsomal and plasma membrane fractions from adipocytes of healthy and obese rats. Catalytic activities of these enzymes were fluorometrically monitoring in these fractions with or without insulin stimulus. Canonical traffic such as insulin-regulated AP was not detected for these novel adipocyte APs in healthy and obese rats. However, insulin increased APM in low density microsomal and plasma membrane fractions from healthy rats, APB in high density microsomal fraction from obese rats and PSA in plasma membrane fraction from healthy rats. A new concept of intracellular compartment-dependent upregulation of AP enzyme activities by insulin emerges from these data. This relatively selective regulation has pathophysiological significance, since these enzymes are well known to act as catalysts and receptor of peptides directly related to energy metabolism. Overall, the regulation of each one of these enzyme activities reflects certain dysfunction in obese individuals.

Asunto(s)

Adipocitos/enzimología , Aminopeptidasas/metabolismo , Insulina/farmacología , Obesidad/enzimología , Adipocitos/ultraestructura , Animales , Antígenos CD13/metabolismo , Membrana Celular/enzimología , Metabolismo Energético/fisiología , Femenino , Masculino , Microsomas/enzimología , Ratas , Ratas Wistar

RESUMEN

Strikingly, in spite of its physiological importance, information about occurrence, biochemical characteristics and mechanisms of regulation of aminopeptidase-N (APN) in the hepatopancreas of intertidal euryhaline crabs is still lacking. In this work, we determined the occurrence, biochemical characteristics, response to environmental salinity and dopamine of APN in the hepatopancreas of the euryhaline crab Neohelice granulata (Dana 1851) from the open mudflat of Mar Chiquita coastal lagoon (Buenos Aires province, Argentina). APN activity was maximal at pH and temperature range of 7.6-9.0 and 37-45 °C, respectively. APN activity exhibited Michaelis-Menten kinetics (apparent Km = 0.19 ± 0.10 mM) (pH 7.6, 37 °C) and appeared to be sensitive to bestatin (I 50 = 15 mM) and EDTA (I 50 = 9 mM). In crabs acclimated to 10 psu (hyper-regulation conditions) and 37 psu (hypo-regulation conditions), APN activity was about 45 and 160% higher, respectively, than in 35 psu (osmoconformation). APN activity in the hepatopancreas was stimulated in vitro (about 137%) by 10(-4) M dopamine. Higher dopamine concentrations produced a similar extent of increase. The responses of APN activity to salinity and dopamine in vitro suggest the role of APN in digestive adjustments upon hyper and hypo-regulatory conditions and its modulation via direct mechanisms on hepatopancreas by dopamine.

Asunto(s)

Aclimatación/fisiología , Braquiuros/enzimología , Antígenos CD13/metabolismo , Hepatopáncreas/enzimología , Humedales , Análisis de Varianza , Animales , Argentina , Digestión/efectos de los fármacos , Digestión/fisiología , Dopamina/farmacología , Ácido Edético , Concentración de Iones de Hidrógeno , Cinética , Leucina/análogos & derivados , Salinidad , Temperatura

RESUMEN

Aminopeptidase N (APN or CD13) is a membrane ectopeptidase expressed by many cell types, including myelomonocytic lineage cells: monocytes, macrophages, and dendritic cells. CD13 is known to regulate the biological activity of various peptides by proteolysis, and it has been proposed that CD13 also participates in several functions such as angiogenesis, cell adhesion, metastasis, and tumor invasion. We had previously reported that, in human monocytes and macrophages, CD13 modulates the phagocytosis mediated by receptors for the Fc portion of IgG antibodies (FcγRs). In this work, we analyzed the possible interaction of CD13 with other phagocytic receptors. We found out that the cross-linking of CD13 positively modulates the phagocytosis mediated by receptors of the innate immune system, since a significant increase in the phagocytosis of zymosan particles or heat-killed E. coli was observed when CD13 was cross-linked using anti-CD13 antibodies, in both macrophages and dendritic cells. Also, we observed that, during the phagocytosis of zymosan, CD13 redistributes and is internalized into the phagosome. These findings suggest that, besides its known functions, CD13 participates in phagocytic processes in dendritic cells and macrophages.

Asunto(s)

Antígenos CD13/metabolismo , Células Dendríticas/citología , Células Dendríticas/enzimología , Macrófagos/citología , Macrófagos/enzimología , Fagocitosis , Reactivos de Enlaces Cruzados/farmacología , Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Endocitosis/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Humanos , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Macrófagos/efectos de los fármacos , Masculino , Monocitos/citología , Fagocitos/citología , Fagocitos/efectos de los fármacos , Fagocitos/metabolismo , Fagocitosis/efectos de los fármacos , Fenotipo , Transporte de Proteínas/efectos de los fármacos , Receptor Toll-Like 2/metabolismo , Zimosan/metabolismo

RESUMEN

Aminopeptidase-N (APN1) and alkaline phosphatase (ALP) proteins located in the midgut epithelium of Manduca sexta have been implicated as receptors for Cry1Aa, Cry1Ab, and Cry1Ac insecticidal proteins produced by Bacillus thuringiensis subsp. kurstaki. In this study, we analyzed the roles of ALP and APN1 in the toxicity of these three Cry1A proteins. Ligand blot analysis using brush border membrane vesicles of M. sexta showed that Cry1Aa and Cry1Ab bind preferentially to ALP during early instars while binding to APN was observed after the third instar of larval development. Cry1Ac binds to APN throughout all larval development, with no apparent binding to ALP. ALP was cloned from M. sexta midgut RNA and expressed in Escherichia coli. Surface plasmon resonance binding analysis showed that recombinant ALP binds to Cry1Ac with 16-fold lower affinity than to Cry1Aa or Cry1Ab. Downregulation of APN1 and ALP expression by RNA interference (RNAi) using specific double-stranded RNA correlated with a reduction of transcript and protein levels. Toxicity analysis of the three Cry1A proteins in ALP- or APN1-silenced larvae showed that Cry1Aa relies similarly on both receptor molecules for toxicity. In contrast, RNAi experiments showed that ALP is more important than APN for Cry1Ab toxicity, while Cry1Ac relied principally on APN1. These results indicated that ALP and APN1 have a differential role in the mode of action of Cry1A toxins, suggesting that B. thuringiensis subsp. kurstaki produces different Cry1A toxins that in conjunction target diverse midgut proteins to exert their insecticidal effect.

Asunto(s)

Fosfatasa Alcalina/genética , Proteínas Bacterianas/toxicidad , Antígenos CD13/genética , Endotoxinas/toxicidad , Proteínas Hemolisinas/toxicidad , Manduca/genética , Manduca/microbiología , Fosfatasa Alcalina/metabolismo , Animales , Bacillus thuringiensis/química , Toxinas de Bacillus thuringiensis , Antígenos CD13/metabolismo , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Larva/microbiología , Manduca/crecimiento & desarrollo , Manduca/metabolismo , Interferencia de ARN , ARN Bicatenario/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

An emergent pest is the weevil Asymmathetes vulcanorum, an insect that attacks Colombian potato areas. Here, some Cry proteins from the entomopathogenic bacteria Bacillus thuringiensis were evaluated as biological control strategy. It was found that Cry1B protoxin caused a mortality of 40% with a dose of 8000 ng/cm(2). Also in this research, it was identified a full length cDNA of an aminopeptidase N, a possible Cry protein receptor located in the insect midgut. This is the first report about B. thuringiensis as an alternative method for control of A. vulcanorum in Colombia.

Asunto(s)

Antígenos CD13/genética , Proteínas Hemolisinas/genética , Control Biológico de Vectores/métodos , Gorgojos/genética , Animales , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Antígenos CD13/metabolismo , Proteínas Hemolisinas/metabolismo , Gorgojos/metabolismo

RESUMEN

This study evaluated the hypothesis that neutral (APN) and dipeptidyl-IV (DPPIV) aminopeptidase activity levels would be critical for the susceptibility to arthritis in collagen-induced model (CIA). The macroscopic signs of arthritis in CIA rats were checked and peripheral blood, synovial fluid and synovial tissue from knee joint were withdrawn. Soluble (SF) and solubilized membrane-bound (MF) fractions from the synovial tissue and peripheral blood mononuclear cells (PBMCs) were obtained. APN and DPPIV activities were fluorometrically quantified. Severe swelling in both the entire hind paws was the minimum criterion to select CIA rats with arthritis. These arthritic rats had high APN in plasma, synovial fluid and SF of the synovial tissue, together with low APN and DPPIV in MF of PBMCs and hallmark histological changes in tibio-tarsal joint. CIA rats with no macroscopic signs of arthritis were diagnosed as resistant and they had low APN in MF of the synovial tissue, low DPPIV in SF of PBMCs and high DPPIV in plasma together with histological aspects of tibio-tarsal joint similar to healthy control rats. Data suggested that APN and DPPIV activity levels are related to the development of arthritis, being protective or inducer of the susceptibility. Understanding what is controlling the compartment-specific changes of these peptidases and looking at ways in which to manipulate their activities may lead to a better knowledge of the arthritic processes and novel treatments.

Asunto(s)

Artritis Experimental/enzimología , Artritis Reumatoide/enzimología , Antígenos CD13/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Animales , Articulación del Tobillo/patología , Artritis Experimental/inducido químicamente , Artritis Reumatoide/inducido químicamente , Antígenos CD13/sangre , Recuento de Células , Membrana Celular/enzimología , Colágeno Tipo II , Dipeptidil Peptidasa 4/sangre , Articulación de la Rodilla/enzimología , Articulación de la Rodilla/patología , L-Lactato Deshidrogenasa/metabolismo , Leucocitos Mononucleares , Masculino , Ratas , Ratas Wistar , Fracciones Subcelulares/enzimología , Líquido Sinovial/enzimología

RESUMEN

The climatic variability hypothesis (CVH) states that species are geographically more widespread at higher latitudes because individuals have a broader range of physiological tolerance or phenotypic flexibility as latitude and climatic variability increase. However, it remains unclear to what extent climatic variability or latitude, acting on the phenotype, account for any observed geographical gradient in mean range size. In this study, we analyzed the physiological flexibility within the CVH framework by using an intraspecific population experimental approach. We tested for a positive relationship between digestive-tract flexibility (i.e., morphology and enzyme activities) and latitude and climatic and natural diet variability in populations of rufous-collared sparrows (Zonotrichia capensis) captured in desert (27°S), Mediterranean (33°S), and cold-temperate (41°S) sites in Chile. In accordance with the CVH, we observed a positive relationship between the magnitude of digestive-tract flexibility and environmental variability but not latitude. The greatest digestive flexibility was observed in birds at middle latitudes, which experience the most environmental variability (a Mediterranean climate), whereas individuals from the most stable climates (desert and cold-temperate) exhibited little or no digestive-tract flexibility in response to experimental diets. Our findings support the idea that latitudinal gradients in geographical ranges may be strongly affected by the action of regional features, which makes it difficult to find general patterns in the distribution of species.

Asunto(s)

Ecosistema , Tracto Gastrointestinal/anatomía & histología , Tracto Gastrointestinal/enzimología , Pájaros Cantores/anatomía & histología , Pájaros Cantores/fisiología , Animales , Antígenos CD13/metabolismo , Chile , Clima , Dieta , Isótopos de Nitrógeno/química , Estaciones del Año , Sacarasa/metabolismo , alfa-Glucosidasas/metabolismo

RESUMEN

Bacillus thuringiensis (Bt) bacteria are insect pathogens that rely on insecticidal pore forming proteins known as Cry and Cyt toxins to kill their insect larval hosts. At least four different non-structurally related families of proteins form the Cry toxin group of toxins. The expression of certain Cry toxins in transgenic crops has contributed to an efficient control of insect pests resulting in a significant reduction in chemical insecticide use. The mode of action of the three domain Cry toxin family involves sequential interaction of these toxins with several insect midgut proteins facilitating the formation of a pre-pore oligomer structure and subsequent membrane insertion that leads to the killing of midgut insect cells by osmotic shock. In this manuscript we review recent progress in understanding the mode of action of this family of proteins in lepidopteran, dipteran and coleopteran insects. Interestingly, similar Cry-binding proteins have been identified in the three insect orders, as cadherin, aminopeptidase-N and alkaline phosphatase suggesting a conserved mode of action. Also, recent data on insect responses to Cry toxin attack is discussed. Finally, we review the different Bt based products, including transgenic crops, that are currently used in agriculture.

Asunto(s)

Bacillus thuringiensis , Proteínas Bacterianas , Proteínas de Insectos/metabolismo , Insectos/efectos de los fármacos , Insecticidas , Control Biológico de Vectores/métodos , Proteínas Citotóxicas Formadoras de Poros , Fosfatasa Alcalina/metabolismo , Animales , Bacillus thuringiensis/química , Bacillus thuringiensis/metabolismo , Bacillus thuringiensis/patogenicidad , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/toxicidad , Antígenos CD13/metabolismo , Cadherinas/metabolismo , Insectos/genética , Insectos/metabolismo , Modelos Moleculares , Plantas Modificadas Genéticamente , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Proteínas Citotóxicas Formadoras de Poros/toxicidad , Unión Proteica

RESUMEN

Our planet is undergoing fast environmental changes, which are referred as global change. In this new scenario, it is of paramount relevance to understand the mechanistic basis of animal responses to environmental change. Here we analyze to what extent seasonal changes in the digestive function of the lizard Liolaemus moradoensis is under endogenous (i.e., hard wired) or exogenous (i.e., environmentally determined) control. For this purpose we compared animals collected in the field during autumn, winter and summer, against (experimental) specimens collected in the field at the beginning of autumn and reared in the laboratory under simulated summer conditions until winter. We found that different aspects of the digestive function are under different types of control: small intestine length appears to be under endogenous control (i.e., experimental animals were similar to winter animals), small intestine mass appears to be under exogenous control (i.e., experimental animals were similar to summer animals), and specific enzyme activities did not change throughout the year. Thus, we suspect that processes related with gut length, such as cell division, may be under endogenous control, while others related with gut mass, such as enterocyte size and content, may be determined by exogenous factors, such as the presence of food in the intestinal lumen. Faced with accelerated changing conditions, the ability of vertebrates to cope will be closely related with their plasticity in fitness-associated traits. More studies aimed at determining the levels and limits of physiological flexibility will be necessary to understand this phenomenon.

Asunto(s)

Cambio Climático , Digestión/fisiología , Lagartos/fisiología , Estaciones del Año , Análisis de Varianza , Animales , Antígenos CD13/metabolismo , Intestino Delgado/anatomía & histología , Intestino Delgado/enzimología , Intestino Delgado/fisiología , Masculino , Tamaño de los Órganos , alfa-Glucosidasas/metabolismo

RESUMEN

Bird species exhibit great diversity in digestive tract morphology and enzymatic activity that is partly correlated with the chemical composition of their natural diets. However, no studies have assessed whether the activities of digestive enzymes of the enterocytes correlate with dietary chemical composition data analyzed as a continuous variable at an evolutionary scale. We used a phylogenetically explicit approach to examine the effect of diet on the hydrolytic activity of three digestive enzymes (maltase, sucrase, and aminopeptidase-N) in 16 species of songbirds (Order Passeriformes) from Central Chile. The total activities (µmol/min) of these enzymes were positively associated with body mass using both conventional least squares regressions and phylogenetically independent contrasts. After removing mass effects, we found a significant negative correlation between the ratio of aminopeptidase-N and maltase to the proportion of seeds found in the gizzard, but this relationship was no longer significant after controlling for phylogeny. When we analyzed the specific nutritional content of the diet, we found that the percentage of nitrogen in diet was negatively correlated with residual maltase activity and positively correlated with the ratio aminopeptidase-N/maltase. Given the large interspecific differences in biochemical capacity, we conclude that these differences reflect genetically determined evolutionary changes associated with the nutrient contents of each species' natural diet.

Asunto(s)

Dieta , Intestinos/enzimología , Passeriformes/fisiología , Filogenia , Animales , Peso Corporal , Antígenos CD13/metabolismo , Intestinos/anatomía & histología , Nitrógeno/administración & dosificación , Passeriformes/anatomía & histología , Sacarasa/metabolismo , alfa-Glucosidasas/metabolismo

RESUMEN

Protein (western blotting) and gene (PCR) expressions, catalytic activity of puromycin-insensitive membrane-bound neutral aminopeptidase (APM/CD13) and in situ regional distribution of CD13 and FOS immunoreactivity (ir) were evaluated in the hypothalamus of monosodium glutamate obese (MSG) and/or food deprived (FD) rats in order to investigate their possible interplay with metabolic functions. Variations in protein and gene expressions of CD13 relative to controls coincided in the hypothalamus of MSG and MSG-FD (decreased 2- to 17-fold). Compared with controls, the reduction of hypothalamic CD13 content reflected a negative balance in its regional distribution in the supraoptic, paraventricular, periventricular and arcuate nuclei. CD13-ir increased in the supraoptic nucleus in MSG (2.5-fold) and decreased in the paraventricular nucleus (2-fold) together with FOS-ir (1.5-fold) in FD. In MSG-FD, FOS-ir decreased (7-fold) in the paraventricular nucleus, while CD13-ir decreased in the periventricular (5.6-fold) and the arcuate (3.7-fold) nuclei. It was noteworthy that all these changes of CD13 were not related to catalytic activity of APM. Data suggested that hypothalamic CD13 plays a role in the regulation of energy metabolism not by means of APM enzyme activity.

Asunto(s)

Antígenos CD13/metabolismo , Hipotálamo Anterior/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Animales , Privación de Alimentos/fisiología , Masculino , Obesidad/inducido químicamente , Obesidad/metabolismo , Ratas , Glutamato de Sodio

RESUMEN

The purpose of this work was to isolate and cultivate mesenchymal stem cells (MSC) derived from equine adipose tissue and conduct cellular characterization with the following markers: CD90, CD44 and CD13. Adipose tissue collection was performed at the base of the horses' tails, followed by immediate isolation and cultivation of the MSC and posterior characterization by flow cytometry for the interspecies reaction test using mouse anti-rat CD90 monoclonal antibody (mAb), fluorescein isothiocyanate (FITC), and tests with specific mAb mouse anti-horse CD13 and mouse anti-horse CD44. The technique used for isolation and cell cultivation proved to be safe and viable. The CD90 mAb expressed cross-reaction with MSC derived from equine adipose tissue and CD44 showed greater expression in cells as the number of culture passages increased. Although marker CD13 expresses reaction in other studies involving MSC in different species, it presented no expression in the experiment realized. The results obtained revealed the immunophenotypic characterization of the surface of isolated and cultivated MSC, classifying these cells as a promising type of progenitor cells that can be applied in equine cellular therapy.

Asunto(s)

Tejido Adiposo/citología , Tejido Adiposo/inmunología , Caballos/anatomía & histología , Caballos/inmunología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/inmunología , Animales , Anticuerpos Monoclonales , Antígenos CD13/metabolismo , Separación Celular , Citometría de Flujo , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Receptores de Hialuranos/metabolismo , Inmunofenotipificación , Células Madre Mesenquimatosas/clasificación , Ratones , Ratas , Antígenos Thy-1/metabolismo