RESUMEN
Alphabaculovirus (lepidopteran-specific nucleopolyhedroviruses, NPV) and Betabaculovirus (granuloviruses, GV) are two main genera of the family Baculoviridae. The virion proteomes of Alphabaculovirus have been well studied; however, the Betabaculovirus virion compositions remain unclear. Pieris rapae granulovirus (PrGV) can kill larvae of P. rapae, a worldwide and important pest of mustard family crops. In this study, the occlusion-derived virus (ODV)-associated proteins of PrGV were identified using three mass spectrometry (MS) approaches. The MS analyses demonstrated that 47 proteins were present in PrGV-ODV. Of the 47 PrGV-ODV proteins, 33 have homologues identified previously in other baculovirus ODV/BVs, whereas 14 (P10, Pr21, Pr29, Pr35, Pr42, Pr54, P45/48, Pr83, Pr84, Pr89, Pr92, Pr111, Pr114 and FGF3) were newly identified ODV proteins. Seven of the 14 newly identified ODV proteins are specific to Betabaculovirus, including Pr35, Pr42, Pr54, Pr83, Pr84, Pr111 and Pr114. Furthermore, the data derived from these MS approaches were validated by immunoblotting analysis using antisera prepared from 11 randomly selected recombinant PrGV-ODV proteins (including 5 Betabaculovirus-unique proteins). Comparison analyses revealed the similar and different compositions between Betabaculovirus and Alphabaculovirus virions, which deepen our understanding of the baculovirus virion structure and provide helpful information on Betabaculovirus--host interaction studies.
Asunto(s)
Mariposas Diurnas/virología , Granulovirus/metabolismo , Cuerpos de Inclusión Viral/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Secuencia Conservada , Granulovirus/genética , Granulovirus/ultraestructura , Sueros Inmunes , Cuerpos de Inclusión Viral/ultraestructura , Peso Molecular , Control Biológico de Vectores , Proteoma/genética , Proteoma/metabolismo , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Proteínas Virales/genéticaRESUMEN
γ-Aminobutyric acid (GABA) is a very important inhibitory neurotransmitter in both vertebrate and invertebrate nervous systems. GABA receptors (GABARs) are known to be the molecular targets of a class of insecticides. Members of the GABAR gene family of the silkworm, Bombyx mori, a model insect of Lepidoptera, have been identified and characterized in this study. All putative silkworm GABAR cDNAs were cloned using the reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Bombyx mori appears to have the largest insect GABAR gene family known to date, including three RDL, one LCCH3, and one GRD subunit. The silkworm RDL1 gene has RNA-editing sites, and the RDL1 and RDL3 genes possess alternative splicing. These mRNA modifications enhance the diversity of the silkworm's GABAR gene family. In addition, truncated transcripts were found for the RDL1 and LCCH3 genes. In particular, the three RDL subunits may have arisen from two duplication events.
Asunto(s)
Bombyx/genética , Genes de Insecto , Proteínas de Insectos/genética , Receptores de GABA/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Bombyx/metabolismo , Evolución Molecular , Amplificación de Genes , Regulación de la Expresión Génica , Orden Génico , Genes Duplicados , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Edición de ARN , Receptores de GABA/química , Receptores de GABA/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Repeated hyperbaric oxygen (HBO) exposure prior to ischemia has been reported to provide neuroprotection against ischemic brain injury. The present study examined the time course of neuroprotection of HBO (3.5 atmosphere absolute, 100% oxygen, 1 h for 5 consecutive days) and the changes of gene/protein expression in rats. First, at 6 h, 12 h, 24 h, and 72 h after HBO sessions, rats were subjected to forebrain ischemia (8 min). Histopathological examination of hippocampal CA1 neurons was done 7 days after ischemia. Second, temporal genomic responses and protein expression were examined at the same time points after HBO sessions without subjecting animals to ischemia. HBO significantly reduced loss of hippocampal CA1 neurons that normally follows transient forebrain ischemia when the last HBO session was 6 h, 12 h, or 24 h before ischemia (survived neurons 55%, 75%, and 53%, respectively), whereas if there was a 72-h delay before the ischemic insult, HBO was not protective (survived neurons only 6%). Statistical analysis on microarray data showed significant upregulation in 60 probe sets including 7 annotated genes (p75NTR, C/EBPdelta, CD74, Edg2, Trip10, Nrp1, and Igf2), whose time course expressions corresponded to HBO-induced neuroprotection. The protein levels of p75NTR, C/EBPdelta, and CD74 were significantly increased (maximum fold changes 2.9, 2.0, and 7.9, respectively). The results suggest that HBO-induced neuroprotection against ischemic injury has time window, protective at 6 h, 12 h and 24 h but not protective at 72 h. Although the precise interaction is to be determined, the genes/proteins relevant to neurotrophin and inflammatory-immune system may be involved in HBO-induced neuroprotection.
Asunto(s)
Isquemia Encefálica/metabolismo , Hipocampo/metabolismo , Oxigenoterapia Hiperbárica , Precondicionamiento Isquémico , Proteínas del Tejido Nervioso/biosíntesis , Prosencéfalo/metabolismo , Animales , Isquemia Encefálica/fisiopatología , Análisis por Conglomerados , Perfilación de la Expresión Génica , Hipocampo/fisiopatología , Masculino , Prosencéfalo/fisiopatología , Biosíntesis de Proteínas/fisiología , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
We examined the effects of cyclosporin A (CsA), a drug that inhibits mitochondrial permeability transition pore, and insulin on ischemic spinal cord damage in rabbits. We assigned rabbits to 5 groups (n = 6 in each); sham barrier-opened group (sham BO), barrier-opened group (BO), barrier-opened-CsA group (BO-CsA), barrier-opened-insulin group (BO-I), and barrier-opened-CsA-insulin group (BO-CsA-I). The blood-spinal cord barrier was opened to facilitate drug penetration by a mild injury to the lumber spinal cord on day 1. CsA (10 mg/kg per day IV) was administered on day 3 to day 5 (total 30 mg/kg). Insulin was administered 30 min before ischemia. In all groups, spinal cord ischemia was produced on day 5 by occluding the abdominal aorta for 13 min. Neurological and histopathological evaluations were performed 4 days after ischemia. In group BO-CsA, blood glucose concentrations were significantly larger compared with the other four groups, and no protection was observed. In contrast, hindlimb motor function in groups BO-I and Bo-CsA-I and histopathology in group BO-CsA-I were significantly better than in groups sham BO, BO, and BO-CsA. The results indicate that insulin protects against ischemic spinal cord injury, whereas the effect of CsA is, at best, minimal.
Asunto(s)
Ciclosporina/farmacología , Insulina/farmacología , Fármacos Neuroprotectores/farmacología , Traumatismos de la Médula Espinal/fisiopatología , Isquemia de la Médula Espinal/fisiopatología , Animales , Glucemia/análisis , Encéfalo/patología , Permeabilidad Capilar , Miembro Posterior/fisiología , Vértebras Lumbares , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Proteínas de Transporte de Membrana Mitocondrial/fisiología , Poro de Transición de la Permeabilidad Mitocondrial , Movimiento , Neuronas/patología , Paraplejía/fisiopatología , Conejos , Médula Espinal/irrigación sanguínea , Isquemia de la Médula Espinal/sangre , Isquemia de la Médula Espinal/patologíaRESUMEN
In dairy cows, the extracellular microenvironment varies significantly from the virgin state to lactation. The function of integrin α6ß4 is dependent on cell type and extracellular microenvironment, and the precise expression profile of α6ß4 and its effects on mammary development remain to be determined. In the present study, real-time PCR and immunohistochemistry were used to analyze the expression and localization of integrin α6ß4 in Holstein dairy cow mammary glands. The effects of integrin α6ß4 on the proliferation induced by mammogenic mitogens were identified by blocking integrin function in purified dairy cow mammary epithelial cells (DCMECs). The results showed that the localization of ß4 subunit and its exclusive partner the α6 subunit were not consistent but were co-localized in basal luminal cells and myoepithelial cells, appearing to prefer the basal surface of the plasma membrane. Moreover, α6 and ß4 subunit messenger RNA (mRNA) levels changed throughout the stages of dairy cow mammary development, reflected well by protein levels, and remained higher in the virgin and pregnancy states, with duct/alveolus morphogenesis and active cell proliferation, than during lactation, when growth arrest is essential for mammary epithelial cell differentiation. Finally, the upregulation of integrin expression by both mammogenic growth hormone and insulin-like growth factor-1 and the inhibited growth of DCMECs by function-blocking integrin antibodies confirmed that integrin α6ß4 was indeed involved in dairy cow mammary development.
Asunto(s)
Industria Lechera , Integrina alfa6beta4/genética , Glándulas Mamarias Animales/crecimiento & desarrollo , Mitógenos/farmacología , Regulación hacia Arriba/genética , Animales , Western Blotting , Bovinos , Proliferación Celular/efectos de los fármacos , Separación Celular , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hormona del Crecimiento/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Integrina alfa6beta4/metabolismo , Laminina/metabolismo , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/efectos de los fármacos , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Involvement of adenosine and adenosine triphosphate-sensitive potassium (KATP) channels in the development of ischemic tolerance has been suggested in global ischemia, but has not been studied extensively in focal cerebral ischemia. This study evaluated modulating effects of adenosine A1 receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine) and mitochondrial KATP channel blocker 5HD (5-hydroxydecanoate) on the development of tolerance to focal cerebral ischemia in rats. Preconditioning with 30-minute middle cerebral artery occlusion (MCAO) reduced cortical and subcortical infarct volume following 120-minute MCAO (test ischemia) given 72 hours later. The neuroprotective effect of preconditioning was attenuated by 0.1 mg/kg DPCPX given before conditioning ischemia (30-minute MCAO), but no influence was provoked when it was administered before test ischemia. DPCPX had no effect on infarct volume after conditioning or test ischemia when given alone. The preconditioning-induced neuroprotection disappeared when 30 mg/kg 5HD was administered before test ischemia. These results suggest a possible involvement of adenosine A1 receptors during conditioning ischemia and of mitochondrial KATP channels during subsequent severe ischemia in the development of tolerance to focal cerebral ischemia.
Asunto(s)
Antagonistas del Receptor de Adenosina A1 , Ataque Isquémico Transitorio/fisiopatología , Mitocondrias/química , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/fisiología , Receptor de Adenosina A1/fisiología , Adenosina/fisiología , Adenosina Trifosfato/farmacología , Animales , Ácidos Decanoicos/farmacología , Hidroxiácidos/farmacología , Ataque Isquémico Transitorio/mortalidad , Ataque Isquémico Transitorio/patología , Precondicionamiento Isquémico , Masculino , Neuronas/patología , Canales de Potasio/efectos de los fármacos , Ratas , Ratas Wistar , Xantinas/farmacologíaRESUMEN
Milk fat is the major energy component of milk, and regulation of its production relies on transcription factors sterol regulatory element-binding protein 1 (SREBP1) and peroxisome proliferator-activated receptor gamma (PPARγ). As one of the target genes of SREBP1 and PPARγ, fatty acid-binding protein 3(FABP3) is the main protein allowing for rapid diffusion and selective targeting of long-chain fatty acids toward specific organelles for metabolism. Whether FABP3 plays an important role in milk fat synthesis signaling pathway is largely unknown. In this study, we observed the effects of FABP3 overexpression and gene silencing in dairy cow mammary epithelial cells, as well as the effects of oleic acid, stearic acid, and palmitic acid on the expressions of FABP3 and lipid droplet formation, by using quantitative reverse transcriptase (qRT)-PCR, Western blotting, and fluorescent immunostaining techniques. FABP3 upregulated the expression of SREBP1 and PPARγ to increase lipid droplet accumulation. Oleic acid, stearic acid, and palmitic acid also increased lipid droplet accumulation by affecting expression of FABP3. These findings shed new insights for understanding the mechanism of FABP3 in regulating milk fat synthesis.
Asunto(s)
Proteínas de Unión a Ácidos Grasos/fisiología , Metabolismo de los Lípidos , Glándulas Mamarias Animales/metabolismo , Transducción de Señal , Animales , Bovinos , Células Epiteliales/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Femenino , Regulación de la Expresión Génica , Silenciador del Gen , Gotas Lipídicas/metabolismo , Glándulas Mamarias Animales/citologíaRESUMEN
DSC1 and BSC1 are two founding members of a novel family of invertebrate voltage-gated cation channels with close structural and evolutionary relationships to voltage-gated sodium and calcium channels. In this study, we searched the published genome sequences for DSC1 orthologs. DSC1 orthologs were found in all 48 insect species, and in other invertebrate species belonging to phyla Mollusca, Cnidaria, Hemichordata and Echinodermata. However, DSC1 orthologs were not found in four arachnid species, Ixodes scapularis, Rhipicephalus microplus, Tetranychus urticae and Varroa destructor, two species in Annelida or any vertebrate species. We then cloned and sequenced NlSC1 and BmSC1 full-length cDNAs from the brown planthopper (Nilaparvata lugens) and the silkworm (Bombyx mori), respectively. NlSC1 and BmSC1 share about 50% identity with DSC1, and the expression of NlSC1 and BmSC1 transcripts was most abundant in the head and antenna in adults. All DSC1 orthologs contain a unique and conserved DEEA motif, instead of the EEEE or EEDD motif in classical calcium channels or the DEKA motif in sodium channels. Phylogenetic analyses revealed that DSC1 and its orthologs form a separate group distinct from the classical voltage-gated sodium and calcium channels and constitute a unique family of cation channels. The DSC1/BSC1-family channels could be potential targets of new and safe insecticides for pest control.
Asunto(s)
Bombyx/genética , Genes de Insecto , Hemípteros/genética , Canales Iónicos/genética , Familia de Multigenes , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Bombyx/metabolismo , Expresión Génica , Hemípteros/metabolismo , Canales Iónicos/metabolismo , Datos de Secuencia Molecular , FilogeniaRESUMEN
We previously reported in rats that preconditioning with hyperbaric oxygen (HBO; 100% O(2) 3.5-atomsphere absolute (ATA), 1 h/day for 5 days) provided neuroprotection against transient (8 min) forebrain ischemia possibly through protein synthesis relevant to neurotrophin receptor and inflammatory-immune system. A recent report suggested that HBO-induced neuroprotection is relevant to brain derived neurotrophic factor and its downstream event involving suppression of p38 mitogen activated protein kinase (p38) activation. In the present study, we first performed a dose comparison (1, 2, and 3.5 ATA) of HBO-induced neuroprotection and then investigated pharmacological modification by 10 mg/kg anisomycin (a protein synthesis inhibitor and potent activator for p38) and 200 microg/kg SB203580 (a p38 inhibitor), which were given intraperitoneally 60 and 30 min before every 3.5 ATA-HBO treatment, respectively. Most prominent protective effect on hippocampal CA1 neurons was observed with 3.5 ATA-HBO (survived neurons: 69% [62-73%] vs. untreated: 3.9% [2-8%], 1 ATA: 8.8% [0-26%], 2 ATA-HBO: 46% [22-62%] (median [range]) (7 days after ischemia). Anisomycin abolished a neuroprotective effect (survived neuron: 1.2% [0-7%]). SB203580, when given between administration of anisomycin and HBO treatment, resumed a neuroprotective effect (survived neuron: 52% [37-62%]). The level of phosphorylated p38 at 10-min reperfusion was significantly decreased in 3.5 ATA-HBO group (32% [12-53%] of sham). Single pretreatment with 100 and 200 microg/kg of SB203580 exerted a similar neuroprotective effect (39% [25-51%] and 59% [50-72%]) to 2 and 3.5 ATA-HBO preconditioning, respectively. It is concluded that suppression of p38 phosphorylation plays a key role in HBO-induced neuroprotection and that pretreatment with a p38 inhibitor (SB203580) can provide similar neuroprotection.
Asunto(s)
Isquemia Encefálica/prevención & control , Citoprotección/fisiología , Oxigenoterapia Hiperbárica , Precondicionamiento Isquémico , Prosencéfalo/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Análisis de Varianza , Animales , Anisomicina/farmacología , Western Blotting , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Isquemia Encefálica/fisiopatología , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/patología , Región CA1 Hipocampal/fisiopatología , Recuento de Células , Supervivencia Celular/fisiología , Citoprotección/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/administración & dosificación , Conducta Exploratoria/fisiología , Imidazoles/administración & dosificación , Masculino , Memoria a Corto Plazo/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Fosforilación , Prosencéfalo/efectos de los fármacos , Prosencéfalo/fisiopatología , Inhibidores de la Síntesis de la Proteína/farmacología , Piridinas/administración & dosificación , Ratas , Ratas WistarRESUMEN
Bombyx mori nucleopolyhedrovirus (BmNPV) ORF41 (Bm41), homologous to Ac52, is a gene present in most lepidopteran nucleopolyhedroviruses. Bm41 transcripts and encoded protein in BmNPV-infected cells can be detected from 3 and 6 h post-infection, respectively. Immunoassays have shown that Bm41 is not a viral structural protein and is detected in both the nuclei and cytoplasm of infected cells. A Bm41-disrupted virus (vBm(De)) and a repaired virus (vBm(Re)) were generated to investigate the function of Bm41. The results showed that Bm41 was essential for viral replication, and the disruption of Bm41 resulted in a much lower viral titer. Transmission electron microscopy revealed that disruption of Bm41 affected normal nucleocapsid envelopment and polyhedra formation in the nucleus. The disruption of Bm41 might severely affect odv-ec27 and polyhedrin expression. The disrupted virus reduced BmNPV infectivity in an LD(50) bioassay and took 18-23 h longer to kill larvae than wild-type virus in an LT(50) bioassay.
Asunto(s)
Bombyx/virología , Nucleopoliedrovirus/fisiología , Proteínas Virales/genética , Replicación Viral/genética , Animales , Células Cultivadas , Replicación del ADN/genética , ADN Viral/genética , Técnicas de Inactivación de Genes , Larva/virología , Microscopía Electrónica de Transmisión , Mutación , Proteínas de la Nucleocápside/metabolismo , Nucleopoliedrovirus/genética , Nucleopoliedrovirus/patogenicidad , Nucleopoliedrovirus/ultraestructura , Sistemas de Lectura Abierta/genética , Factores de Tiempo , Proteínas Virales/metabolismoRESUMEN
We performed three sets of experiments to investigate the safety of intrathecal magnesium and to determine its optimal dose for protection, if any, against ischemic spinal cord injury in rabbits. First, we examined neurotoxicity of 0.3, 1, 2, or 3 mg/kg of magnesium sulfate (n = 6 each). Significant sensory dysfunction was observed in the 3-mg/kg group 7 days after administration. Motor dysfunction was found in two rabbits in both the 2- and 3-mg/kg groups. The area of destruction in laminae V-VII was observed in one, two, and one rabbit in the 1-, 2-, and 3-mg/kg groups, respectively. Second, we investigated the temporal profile (6 h, 48 h, and 96 h [n = 3 each]) of histopathologic changes after 3 mg/kg of magnesium sulfate and confirmed similar changes in the rabbits with motor dysfunction at 48 and 96 h. Third, we evaluated the effects of 0.3 mg/kg or 1 mg/kg of magnesium sulfate or saline (n = 6 each) administered before ischemia on hindlimb motor function and histopathology after spinal cord ischemia (15 min). Magnesium did not improve neurologic or histopathologic outcome 96 h after reperfusion. The results indicate that intrathecal magnesium has a risk of neurotoxicity and shows no evidence of protective effects against ischemic spinal cord injury.