RESUMEN
In this report, we examined the possible functions of the cell death protease, caspase-3, in the axotomy-induced apoptosis of facial motoneurons in newborn rodents. Using in situ hybridization and Western blot, we found higher levels of caspase-3 mRNA and pro-caspase-3 protein expression in motoneurons of neonatal and 2-week-old rats than adult rats. Following facial motoneuron axotomy, caspase-3 mRNA and protein expression increased in motoneurons of both neonatal and adult rats. However, using an antibody directed to the activated form of the caspase-3 protease, we found that catalytically active caspase-3 was present only in axotomized neonatal motoneurons. As motoneurons in neonatal but not adult rodents are susceptible to axotomy-induced apoptosis, we hypothesized that caspase-3 may play a role in their demise. To determine the necessity of caspase-3 activation in axotomy-induced apoptosis, we counted the number of surviving motoneurons at 4 and 7 days following axotomy in wild type mice and caspase-3 gene-deleted mice. There were nearly three times more surviving motoneurons in caspase-3 gene-deleted mice than in wild type mice at both 4 days (mean 1074 vs. 464, P<0.005) and 7 days (mean 469 vs. 190, P<0.005) following injury, indicating a slower rate of death. Examination of the dying motoneurons using TUNEL staining (for fragmented DNA) and bisbenzimide staining (for nuclear morphology) revealed incomplete nuclear condensation in caspase-3-deficient motoneurons. These results demonstrate that caspase-3 activation plays important roles in the rapid demise of axotomized neonatal motoneurons.
Asunto(s)
Apoptosis/genética , Caspasas/metabolismo , Nervio Facial/fisiopatología , Neuronas Motoras/metabolismo , Degeneración Nerviosa/enzimología , Factores de Edad , Animales , Animales Recién Nacidos , Axotomía , Caspasa 3 , Caspasas/genética , Nervio Facial/cirugía , Femenino , Feto , Eliminación de Gen , Masculino , Ratones , Ratones Noqueados , Neuronas Motoras/patología , Degeneración Nerviosa/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Tomato ringspot nepovirus (TomRSV) RNA-1 encodes a putative NTP-binding protein (NTB), a putative viral genome-linked protein (VPg), a putative RNA-dependent RNA polymerase (Pol) and a serine-like protease (Pro), which have been suggested to be involved in viral RNA replication. Proteolytic processing of protease precursors containing these proteins was studied in Escherichia coli and in vitro. The TomRSV protease could cleave the precursor proteins and release the predicted mature proteins or intermediate precursors. Although processing was detected at all three predicted cleavage sites (NTB-VPg, VPg-Pro and Pro-Pol), processing at the VPg-Pro cleavage site was inefficient, resulting in accumulation of the VPg-Pro intermediate precursor in E. coli and in vitro. In addition, the presence of the VPg sequence in the precursor resulted in increased cleavage at the Pro-Pol cleavage site in E. coli and in vitro. Direct N-terminal sequencing of the genomic RNA-linked VPg, of the mature protease purified from E. coli extracts and of radiolabelled mature polymerase purified from in vitro translation products revealed the sequences of the NTB-VPg, VPg-Pro and Pro-Pol dipeptide cleavage sites to be Q/S, Q/G and Q/S, respectively. In vitro processing at the NTB-VPg and Pro-Pol cleavage sites was not detected upon mutation or deletion of the conserved glutamine at the -1 position of the cleavage site. These results are discussed in light of the cleavage site specificity of the TomRSV protease.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Precursores Enzimáticos/metabolismo , Nepovirus/enzimología , Procesamiento Proteico-Postraduccional , ARN Polimerasa Dependiente del ARN/metabolismo , Solanum lycopersicum/virología , Proteínas Virales , Proteasas Virales 3C , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/inmunología , Sitios de Unión , Western Blotting , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Precursores Enzimáticos/biosíntesis , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Escherichia coli/genética , Ratones , Datos de Secuencia Molecular , Peso Molecular , Mutación , Nepovirus/genética , Nepovirus/metabolismo , Nucleótidos/metabolismo , Biosíntesis de Proteínas , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Conejos , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/metabolismoRESUMEN
Large-scale surveys in Africa for blackeye cowpea mosaic (B1CMV) and cowpea aphid-borne mosaic (CABMV) showed that several CABMV isolates from Southern Africa were either not or poorly recognized by monoclonal antibodies prepared to isolates collected in West Africa. Selection of three new monoclonal antibodies prepared against the Maputo (Mozambique) isolate of CABMV, and their incorporation into a revised panel of monoclonal antibodies, resulted in the assignment of four of these new CABMV isolates to existing serotypes (II, IV, and V) and three others to a new serotype (VI). The South African isolate of passiflora mosaic virus was shown to be related to CABMV isolates in serotype IV. It is proposed that CABMV isolates be assembled into a distinct species in the legume-infecting, aphid-transmissible potyviruses.
Asunto(s)
Áfidos/virología , Fabaceae/virología , Virus del Mosaico/clasificación , Plantas Medicinales , Potyvirus/clasificación , África , África Austral , Animales , Geografía , Virus del Mosaico/aislamiento & purificación , Virus del Mosaico/fisiología , Enfermedades de las Plantas/virología , Potyvirus/aislamiento & purificación , Potyvirus/fisiologíaRESUMEN
A study of the capsid proteins of different legume-infecting potyviruses using specific monoclonal antibodies on immunoblots of crude extracts from infected plants revealed that cowpea aphid-borne mosaic virus (CAMV) and blackeye cowpea mosaic virus (BICMV) have coat protein M(r) values of 32K and 35K, respectively. Immunoblot comparisons of BICMV, peanut stripe mosaic virus (PStV), bean common mosaic virus (BCMV) and azuki bean mosaic virus (AzMV) revealed equal reactivity of their 35K coat proteins. Similar comparisons between CAMV and the necrotic strain of BCMV (isolate NL3) showed a serological relationship between their 32K coat proteins, results providing the first evidence of a possible similarity between CAMV and BCMV NL3. Peptides from trypsin digests of the coat proteins of several of these legume-infecting potyviruses were analysed by HPLC. Comparison of the peptide profiles confirmed the serological results in distinguishing the two subgroups. Peptide profiles of coat protein from BICMV, PStV, AzMV and BCMV were almost identical, results suggesting that they could be considered as strains of one virus. In contrast, peptide profiles of various CAMV serotypes and BCMV NL3 were distinct from the first group and exhibited limited similarities to each other.
Asunto(s)
Cápside/análisis , Fabaceae/virología , Plantas Medicinales , Potyvirus/clasificación , Animales , Anticuerpos Antivirales , Áfidos/virología , Cápside/química , Cromatografía Líquida de Alta Presión , Peso Molecular , Fragmentos de Péptidos/análisis , Potyvirus/químicaRESUMEN
The immunoreactivity of a panel of polyclonal antibodies and monoclonal antibodies (MAbs) raised against African isolates of potyviruses from cowpea and African yam bean was examined in ELISAs. A serological study including reference isolates followed by further characterization in differential hosts resulted in separation of the potyviruses into two distinct serogroups, one containing blackeye cowpea mosaic virus (BlCMV) and the other containing cowpea aphid-borne mosaic virus (CAMV). Using biotin-labelled MAbs, the BlCMV isolates were further subdivided into two serotypes and the CAMV isolates into five serotypes. Because both BlCMV and CAMV induce a very similar mosaic disease in cowpea, different ELISA procedures using mixed MAbs were evaluated and a single protocol was developed which allowed reliable diagnosis of both viruses.
Asunto(s)
Fabaceae/microbiología , Técnicas Microbiológicas , Virus del Mosaico/clasificación , Plantas Medicinales , Anticuerpos , Anticuerpos Monoclonales , Ensayo de Inmunoadsorción Enzimática , Virus del Mosaico/inmunologíaRESUMEN
The effects of terfenadine and pseudoephedrine, alone and in combination, have been assessed in a nasal provocation test and in perennial rhinitis. In a double-blind, placebo-controlled cross-over nasal provocation test, twelve men allergic to grass pollen were treated with two daily doses of placebo, terfenadine 60 mg, pseudoephedrine 120 mg, or the combination of the two, for 2 days preceding each test. The allergic reaction threshold, based on rhinorrhoea, sneezing and nasal inspiratory peak flow rate, was raised significantly both by terfenadine and pseudoephedrine, and their effects appeared additive (repeated measures analysis of variance). In a double-blind, randomized clinical study of perennial rhinitis two parallel groups, the efficacy and tolerability of terfenadine and terfenadine-pseudoephedrine were compared in 50 patients. Symptoms and signs in both groups were improved after 14 days of treatment. Differences between groups showed a trend in favour of terfenadine-pseudoephedrine, for swelling of the nasal mucosa (rhinoscopy) they were statistically significant. Both medications were well tolerated overall, although adverse events and reactions were more frequent in the terfenadine-pseudoephedrine group. In conclusion, terfenadine-pseudoephedrine and its constituents taken alone were effective. The combination performed better, but adverse events were somewhat more frequent with the combination than with terfenadine alone.
Asunto(s)
Efedrina/farmacología , Rinitis Alérgica Perenne/tratamiento farmacológico , Terfenadina/farmacología , Adulto , Método Doble Ciego , Sinergismo Farmacológico , Quimioterapia Combinada , Efedrina/administración & dosificación , Efedrina/uso terapéutico , Femenino , Humanos , Masculino , Pruebas de Provocación Nasal , Terfenadina/administración & dosificación , Terfenadina/uso terapéuticoRESUMEN
Twenty tomato spotted wilt virus (TSWV) isolates were serologically compared in ELISA employing five different procedures using a rabbit polyclonal antiserum against nucleocapsid proteins (NuAbR) and mouse monoclonal antibodies (MAbs), two directed to nucleocapsid proteins (N1 and N2) and four directed to glycoproteins G1 to G4. All the antisera were raised against TSWV-CNPH1. The 20 isolates were differentiated into two distinct serogroups. Serogroup I consisting of 16 isolates strongly reacted with NuAbR. The other four isolates were poorly recognized by NuAbR and were placed in another serogroup, designated II. The panel of MAbs differentiated the TSWV isolates into three serotypes. The 16 isolates forming serogroup I reacted strongly with the MAbs generated and were identified as serotype I isolates. The four isolates which made up serogroup II were split into serotypes II and III. The serotype II isolates did not respond or responded poorly with MAbs N1, N2 and G3. The two other isolates placed in serotype III were recognized by N1 but not by N2 and G3. Two isolates became defective after several mechanical passages and failed to respond or responded very poorly with MAbs directed to glycoproteins. Our results show that ELISA employing polyclonal and monoclonal antisera is a useful tool to differentiate TSWV isolates and to detect defective forms. The results also strongly suggest that TSWV nucleocapsid proteins are less conserved than the glycoproteins.
Asunto(s)
Virus de Plantas/clasificación , Anticuerpos Monoclonales , Anticuerpos Antivirales , Cápside/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Virus de Plantas/aislamiento & purificación , Plantas/microbiología , Serotipificación , Proteínas del Núcleo Viral/aislamiento & purificaciónRESUMEN
The immunoreactivity of a panel of monoclonal antibodies raised to tomato spotted wilt virus (TSWV) was examined in enzyme-linked immunosorbent assays (ELISA) and dot immunobinding assays (DIBA) procedures. MAbs 6.12.15 and 2.9 were specific for the nucleocapsid protein of TSWV. The sensitivity of the two immunoassays was compared with that of a dot-blot hybridization technique using riboprobes (RNA transcripts) to TSWV M RNA. Using deproteinized plant extracts or purified virus preparations, as little as 1 pg RNA could be detected. Although an ELISA using MAb 6.12.15, a DIBA procedure using MAb 3.22.6 and the dot-blot hybridization, detected several TSWV isolates in different host species equally well, the ELISA was most precise and most suitable for routine diagnosis in the field.
Asunto(s)
Anticuerpos Monoclonales , Virus de Plantas/inmunología , Sondas ARN , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Especificidad de Anticuerpos , Antígenos Virales/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Estudios de Evaluación como Asunto , Immunoblotting/métodos , Ratones , Ratones Endogámicos BALB C , Hibridación de Ácido Nucleico , ARN/genéticaRESUMEN
A panel of monoclonal antibodies (mAb) produced against peanut clump virus (PCV) was used to characterize five serotypes of the virus. Four different formats of enzyme-linked immunosorbent assays (ELISA) were compared to establish the most suitable one for diagnosis of infected plants and for serotype differentiation. Since most mAb retained their activity when used for coating microtitre plates, a dual mAb-type assay was found to be most suitable. The same mAb could be used in ELISA as coating and as biotinylated antibody. Because of the ability of mAb to recognize subtle conformational alterations in the viral antigen, it is important to carefully select the ELISA format used for comparing different viral isolates.