RESUMO
Colletotrichum lindemuthianum is a hemibiotrophic fungal pathogen that causes bean anthracnose and it is rated among the top 10 important diseases infecting beans. Currently our knowledge on molecular mechanisms underlying C. lindemuthianum pathogenesis is limited. About five pathogenicity genes have been identified in C. lindemuthianum using Restricted Enzyme Mediated Integration and the transformation using Agroinfection has not been optimized. In this study, a series of experiments were conducted to optimize the key parameters affecting the Agrobacterium tumefaciens-mediated transformation for C. lindemuthianum. The transformation efficiency increased with increase in spore concentration and co-cultivation time. However, the optimum conditions that yielded significant number of transformants were 106 ml-1 spore concentration, co-cultivation time of 72 h, incubation at 25°C and using a cellulose membrane filter for the co-cultivation. The optimized protocol resulted in establishment of large mutant library (2400). A few mutants were melanin deficient and a few were unable to produce conidia. To determine the altered pathogenicity, two new approaches such as detached leaf and twig techniques proved reliable and require fewer resources to screen the large mutant libraries in a short time. Among the 1200 transformants tested for virulence, 90% transformants were pathogenically similar to wild type (race 2047), 96 and 24 were reduced and impaired, respectively. The altered avirulent transformants can prove vital for understanding the missing link between growth and developmental stages of pathogen with virulence. This platform will help to develop strategies to determine the potential pathogenicity genes and to decipher molecular mechanisms of host-pathogen interactions in more detail.
Assuntos
Colletotrichum , Fabaceae , Agrobacterium tumefaciens/genética , Colletotrichum/genética , Fabaceae/microbiologia , Doenças das Plantas/microbiologia , Esporos Fúngicos/genética , Virulência/genéticaRESUMO
Bakanae is the emerging disease threating the rice cultivation globally. Yield reduction of 4-70% is recorded in different parts of the world. A total of 119 Fusarium isolates were collected from rice plants at different geographical locations and seeds of different rice cultivars. The isolates were evaluated for morphological, biochemical and pathogenic diversity. The amplification of TEF-1α gene was carried out for exploring the species spectrum associated with the cultivated and pre-released rice varieties. The production of gibberellin varied from 0.53 to 2.26 µg/25 ml, while as that of Indole acetic acid varied from 0.60 to 3.15 µg/25 ml among the Fusarium isolates. The phylogenetic analysis identified 5 different species of the genus Fusarium viz. Fusarium fujikuroi, F. proliferatum, F. equiseti, F.oxysporum and F. persicinum after nucleotide blasting in NCBI. Only two Fusarium spp. F. fujikuroi and F. proliferatum were found to be pathogenic under virulence assays of the isolates. The isolates showed a considerable variation in morphological and pathogenic characters. The isolates were divided into different groups based on morphology and pathogenicity tests. The isolates showed a considerable variation in morphology, phytohormone profile and virulence indicative of population diversity. Three species F. equiseti, F.oxysporum and F. persicinum which have not been reported as pathogens of rice in India were found to be associated with bakanae disease of rice, however their pathogenicity could not be established.
Assuntos
Fusarium , Oryza/microbiologia , Reguladores de Crescimento de Plantas/biossíntese , Fusarium/citologia , Fusarium/genética , Fusarium/metabolismo , Fusarium/patogenicidade , Genes Fúngicos , Giberelinas/metabolismo , Índia , FilogeniaRESUMO
Venturia inaequalis is a notorious fungal pathogen and show classical gene for gene interaction with its apple host. Neutral markers provide clues about history, evolutionary potential, genetic diversity and population structure of V. inaequalis. The genetic diversity and population structure of fungus indicates that the pathogen is highly diverse with the capacity to breach the scab resistance genes. In the present study, we collected 108 V. inaequalis isolates from three apple cultivars differing in Rvi1 resistance gene. Based on the AMOVA, the variation was mostly distributed among the isolates, providing evidence of non-existence of subpopulation in orchards thus founder population is difficult to arise in Kashmir apple orchards. Pair wise genetic differentiation is less due to regular occurrence of gene flow between the populations residing on different orchard as infected material is transported without stringent quarantine measures. Based on principal coordinate analysis and clustering algorithm as implemented in STRUCTURE, we observed admixture between the two subpopulations, which is quite low, suggesting the existence of pre-zygotic and post-zygotic barriers to gene flow and we cannot rule out the existence of other structures shared by accessions belonging to different varieties. Due to the continuous increase in introduction and monoculture of apple varieties, mixed orchard with different host resistance specificities are more suitable for managing the apple scab in Kashmir valley.
Assuntos
Ascomicetos/fisiologia , Especificidade de Hospedeiro , Interações Hospedeiro-Parasita/fisiologia , Malus/microbiologia , Ascomicetos/genética , Evolução Biológica , Análise por Conglomerados , Interações Hospedeiro-Parasita/genética , Índia , Malus/genética , Doenças das Plantas/microbiologiaRESUMO
We report the synthesis and characterization of graphene functionalized with iron (Fe3+) oxide (G-Fe3O4) nanohybrids for radio-frequency magnetic hyperthermia application. We adopted the wet chemical procedure, using various contents of Fe3O4 (magnetite) from 0-100% for making two-dimensional graphene-Fe3O4 nanohybrids. The homogeneous dispersal of Fe3O4 nanoparticles decorated on the graphene surface combined with their biocompatibility and high thermal conductivity make them an excellent material for magnetic hyperthermia. The morphological and magnetic properties of the nanohybrids were studied using scanning electron microscopy (SEM) and a vibrating sample magnetometer (VSM), respectively. The smart magnetic platforms were exposed to an alternating current (AC) magnetic field of 633 kHz and of strength 9.1 mT for studying their hyperthermic performance. The localized antitumor effects were investigated with artificial neural network modeling. A neural net time-series model was developed for the assessment of the best nanohybrid composition to serve the purpose with an accuracy close to 100%. Six Nonlinear Autoregressive with External Input (NARX) models were obtained, one for each of the components. The assessment of the accuracy of the predicted results has been done on the basis of Mean Squared Error (MSE). The highest Mean Squared Error value was obtained for the nanohybrid containing 45% magnetite and 55% graphene (F45G55) in the training phase i.e., 0.44703, which is where the model achieved optimal results after 71 epochs. The F45G55 nanohybrid was found to be the best for hyperthermia applications in low dosage with the highest specific absorption rate (SAR) and mean squared error values.
RESUMO
Plant pathology has been revolutionized by the emergence and intervention of next-generation sequencing technologies (NGS) which provide a fast, cost-effective, and reliable diagnostic for any class of pathogens. NGS has made tremendous advancements in the area of research and diagnostics of plant infecting viromes and has bridged plant virology with other advanced research fields like genome editing technologies. NGS in a broader perspective holds the potential for plant health improvement by diagnosing and mitigating the new or unusual symptoms caused by novel/unidentified viruses. CRISPR-based genome editing technologies can enable rapid engineering of efficient viral/viroid resistance by directly targeting specific nucleotide sites of plant viruses and viroids. Critical genes such as eIf (iso) 4E or eIF4E have been targeted via the CRISPR platform to produce plants resistant to single-stranded RNA (ssRNA) viruses. CRISPR/Cas-based multi-target DNA or RNA tests can be used for rapid and accurate diagnostic assays for plant viruses and viroids. Integrating NGS with CRISPR-based genome editing technologies may lead to a paradigm shift in combating deadly disease-causing plant viruses/viroids at the genomic level. Furthermore, the newly discovered CRISPR/Cas13 system has unprecedented potential in plant viroid diagnostics and interference. In this review, we have highlighted the application and importance of sequencing technologies on covering the viral genomes for precise modulations. This review also provides a snapshot vision of emerging developments in NGS technologies for the characterization of plant viruses and their potential utilities, advantages, and limitations in plant viral diagnostics. Furthermore, some of the notable advances like novel virus-inducible CRISPR/Cas9 system that confers virus resistance with no off-target effects have been discussed.
RESUMO
We have previously reported the involvement of the striatum in acute ethanol-induced motor incoordination and the striatal adenosinergic modulation of ethanol-induced motor incoordination through A1 receptor-mediated mechanism(s). The present study, a continuation of our previous work, was carried out to investigate the possible functional correlation between striatal cyclic AMP and ethanol-induced motor incoordination, and its modulation by striatal adenosine in Sprague-Dawley rats. Forskolin (0.1, 0.5 and 1.0 pmol), a known activator of adenylate cyclase, significantly attenuated ethanol-induced motor incoordination in a dose-dependent manner following its direct intrastriatal microinfusion. Forskolin also antagonized the accentuating effect of intrastriatal N6-cyclohexyladenosine on ethanol-induced motor incoordination. These results suggested that ethanol-induced motor incoordination might be functionally correlated to a decrease in the striatal cyclic AMP levels and that the striatal adenosine A1 receptors might modulate ethanol-induced motor incoordination through cyclic AMP signaling mechanism(s). Further support to this hypothesis was obtained by the actual measurement of the striatal cyclic AMP levels in the same experimental conditions as in motor coordination studies using high-performance liquid chromatography with fluoroscence detection. Regardless of the method (focused microwave irradiation, cervical dislocation or decapitation into a dry ice-ethanol mixture) used to kill the animals, a significant decrease in the striatal cyclic AMP levels was observed due to ethanol. Intrastriatal adenosine A1-selective agonist, N6-cyclohexyladenosine (24 ng), caused a further significant decrease in the striatal cyclic AMP levels in the ethanol- but not in the vehicle-treated animals. The further enhancement in the ethanol-induced decrease in the striatal cyclic AMP levels by intrastriatal N6-cyclohexyladenosine, therefore, functionally correlated with the observed potentiating effect of intrastriatal N6-cyclohexyladenosine on ethanol-induced motor incoordination. The effects of intrastriatal N6-cyclohexyladenosine+ethanol and of ethanol alone on the striatal cyclic AMP levels were blocked by intrastriatal pertussis toxin (500 ng) pretreatment, indicating the involvement of pertussis toxin-sensitive G-proteins (Gi, Go) and possibly of the adenosine A1 receptor coupled to the G-proteins in the striatum. Furthermore, ethanol alone significantly decreased the basal as well as the cyclic AMP-stimulated catalytic activities of the striatal cyclic AMP protein kinase, which were further reduced by intrastriatal N6-cyclohexyladenosine. The results of the present study therefore support an involvement of a cyclic AMP signaling pathway in the striatal adenosinergic modulation of ethanol-induced motor incoordination at the post-adenosine A1 receptor level.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Corpo Estriado/fisiopatologia , AMP Cíclico/metabolismo , Etanol/farmacologia , Transtornos das Habilidades Motoras/induzido quimicamente , Adenosina/análogos & derivados , Adenosina/farmacologia , Animais , Depressores do Sistema Nervoso Central/sangue , Colforsina/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Etanol/sangue , Masculino , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/enzimologia , Transtornos das Habilidades Motoras/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The effect of the methylxanthines caffeine, theophylline and isobutylmethylxanthine (IBMX) on ethanol-induced ataxia and loss of righting reflex was investigated in three strains of mice. A significant potentiation of ethanol-induced ataxia was produced in all strains of mice at 20, 45 and 75 min after ethanol in mice pretreated with 62.5 mg/kg caffeine and 12.5 mg/kg IBMX. In mice pretreated with 40 mg/kg caffeine potentiation of ethanol-induced ataxia was observed only at 20 min after ethanol. Theophylline pretreatment produced no alteration in ethanol-induced ataxia. The results of methylxanthine pretreatment on ethanol-induced ataxia were similar, regardless of a shorter (10 min) or longer (75 min) pretreatment time. The methylxanthines produced no effect on motor coordination or behavior when administered separately. Although ethanol-induced loss of righting reflex was shortened by theophylline, neither caffeine nor IBMX altered the duration of loss of righting reflex. It is possible that inhibition of adenosine uptake, a known effect of the methylxanthines, may be a more likely explanation for the modulation of the behavioral effects of ethanol.
Assuntos
Etanol/administração & dosagem , Desempenho Psicomotor/efeitos dos fármacos , Reflexo/efeitos dos fármacos , Xantinas/administração & dosagem , 1-Metil-3-Isobutilxantina/administração & dosagem , Animais , Cafeína/administração & dosagem , Interações Medicamentosas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Postura , Teofilina/administração & dosagemRESUMO
The effect of acute ethanol on adenosine content in four motor areas of the male Sprague-Dawley rat brain was investigated using HPLC-fluorescence detection. Since basal adenosine levels are difficult to assess due to extremely rapid turnover of adenosine, four different methods of sacrifice were also evaluated for adenosine measurement. The rank order for best results in measuring adenosine content with the various methods of sacrifice was: focused microwave irradiation greater than decapitation into liquid nitrogen greater than immersion into liquid nitrogen greater than decapitation. These differences probably reflect differences in degree of hypoxia and postmortem anoxia, factors well known to elevate adenosine, associated with the sacrifice method. Focused microwave irradiation of appropriate duration was found to be the best method of sacrifice and the results probably most closely reflect true basal adenosine levels. No significant alteration in adenosine content in any brain region examined was observed due to ethanol administration.
Assuntos
Adenosina/metabolismo , Encéfalo/metabolismo , Etanol/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Congelamento , Masculino , Micro-Ondas , Nitrogênio , Ratos , Ratos Endogâmicos , Valores de ReferênciaRESUMO
As an extension of our previous work pertaining to brain adenosinergic modulation of ethanol-induced motor incoordination, the effect of direct intracerebellar administration of the A1-selective adenosine agonist, N6-cyclohexyladenosine (CHA) on ethanol-induced motor incoordination was evaluated. Marked accentuation of ethanol-induced motor impairment by CHA was observed. No change in the normal motor coordination was noted when CHA administration was followed by saline instead of ethanol. Intracerebellar cAMP or its analog, 8-(4-chlorophenylthio)-cAMP, significantly inhibited ethanol's motor impairment in a dose-related manner as well as abolished CHA's accentuating effect on ethanol-induced motor incoordination. These observations suggested a possible involvement of cAMP in the adenosinergic modulation and in the expression of ethanol-induced motor incoordination. Further support was provided by the observation of a marked accentuation and attenuation in a dose-related manner of ethanol-induced motor impairment as well as CHA's accentuation of ethanol's motor impairment by intracerebellar miconazole and forskolin, respectively. However, equimolar intracerebellar doses of miconazole and forskolin (inhibitor and stimulator of adenylyl cyclase, respectively) failed to significantly alter ethanol-induced motor incoordination probably due to their mutual functional antagonism. The expression of adenosinergic modulation and that of ethanol-induced motor impairment most likely involved Gi protein-coupled receptor(s) (such as adenosine receptors). The involvement of receptors linked to pertussis toxin-sensitive G-proteins was suggested because intracerebellar pertussis toxin pretreatment markedly inhibited ethanol-induced motor incoordination as well as CHA's accentuation of ethanol's motor impairment. Finally, cAMP, unlike its antagonism to CHA's accentuation, failed to antagonize the accentuation of ethanol-induced motor impairment by intracerebellar GABA(A) agonist (+)-muscimol. This indicated selectivity of cAMP participation in G protein coupled receptor (such as adenosine)-mediated response and not in ionic channel coupled receptor (such as GABA(A))-mediated mechanism. Overall, the data suggested a possible involvement of cerebellar adenylyl cyclase-cAMP signalling pathway in the adenosinergic modulation of ethanol's ataxia.
Assuntos
Adenosina/análogos & derivados , Córtex Cerebelar/fisiologia , AMP Cíclico/farmacologia , Etanol/farmacologia , Atividade Motora/efeitos dos fármacos , Receptores Purinérgicos P1/fisiologia , Adenosina/administração & dosagem , Adenosina/farmacologia , Toxina Adenilato Ciclase , Análise de Variância , Animais , Córtex Cerebelar/efeitos dos fármacos , Colforsina/farmacologia , AMP Cíclico/administração & dosagem , AMP Cíclico/análogos & derivados , Infusões Parenterais , Masculino , Camundongos , Miconazol/administração & dosagem , Miconazol/farmacologia , Toxina Pertussis , Agonistas do Receptor Purinérgico P1 , Tionucleotídeos/farmacologia , Fatores de Tempo , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The effect of intracerebellar microinfusion of antisense oligodeoxynucleotide to Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and other naturally occurring cannabinoid receptor (CB(1)) mRNA on Delta(9)-THC-induced motor impairment was investigated in mice. Delta(9)-THC (15-30 microgram/microliter intracerebellar) resulted in a significant motor impairment in a dose-related manner. The intracerebellar pretreatment with antisense oligodeoxynucleotide (3.0 microgram/100 nl/12 h; six administrations/mouse) virtually abolished Delta(9)-THC (15 and 25 microgram/1 microliter intracerebellar)-induced motor impairment. However, intracerebellar pretreatment with the mismatched oligodeoxynucleotide in exactly the same manner as the antisense was completely ineffective in altering the Delta(9)-THC-induced motor impairment. These results strongly suggest the involvement of CB(1) receptor in the expression of Delta(9)-THC-induced motor impairment. The intracerebellar microinfusion of adenosine A(1)-selective agonist, N(6)-cyclohexyladenosine (CHA) (4 ng/100 nl) significantly enhanced Delta(9)-THC-induced motor impairment, suggesting a cerebellar A(1) adenosinergic modulation of motor impairment. A pretreatment with the antisense and the mismatched oligodeoxynucleotide also markedly attenuated and did not alter, respectively, the cerebellar A(1) adenosinergic modulation (enhancement) of Delta(9)-THC-induced motor impairment. There was no change in the normal motor coordination due to intracerebellar pretreatment with antisense and its mismatch, in the presence as well as absence of intracerebellar CHA indicating the selectivity of interactions with Delta(9)-THC. The Delta(9)-THC-induced motor incoordination was also significantly enhanced dose-dependently by systemic (i.p.) ethanol administration suggesting behavioral synergism between the two psychoactive drugs. Pretreatment (intracerebellar) with pertussis toxin (PTX) markedly attenuated Delta(9)-THC- and Delta(9)-THC+CHA-induced motor incoordination suggesting coupling of CB(1) receptor to PTX-sensitive G-protein (G(i)/G(o)). These data suggested co-modulation by cerebellar cannabinoid and adenosine system of Delta(9)-THC-induced motor impairment. Conversely, the results in the present study also suggested co-modulation by cerebellar adenosine A(1) and CB(1) receptors of ethanol-induced motor impairment, thereby indicating a possible common signal transduction pathway in the expression of motor impairment produced by Delta(9)-THC as well as ethanol.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Ataxia Cerebelar/induzido quimicamente , Cerebelo/química , Dronabinol/análogos & derivados , Etanol/farmacologia , Receptores Adrenérgicos alfa 1/metabolismo , Receptores de Droga/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Relação Dose-Resposta a Droga , Dronabinol/farmacologia , Sinergismo Farmacológico , Masculino , Camundongos , Camundongos Endogâmicos , Movimento/efeitos dos fármacos , Oligonucleotídeos Antissenso , Toxina Pertussis , Receptores Adrenérgicos alfa 1/genética , Receptores de Canabinoides , Receptores de Droga/genética , Fatores de Virulência de Bordetella/farmacologia , Xantinas/farmacologiaRESUMO
Cannabinoids are known to impair motor function in humans and laboratory animals. We have demonstrated an accentuation of cannabinoid (CP55,940)-induced motor incoordination in mice by the adenosine A(1) receptor-selective agonist N(6)-cyclohexyladenosine (CHA) (4 ng) using an intracerebellar (ICB) microinjection method. This effect was mediated by the A(1) receptor because pre-treatment with ICB 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (100 ng), an adenosine A(1) receptor selective antagonist, completely abolished the accentuation. Furthermore, ICB pre-treatment with DPCPX (100 ng) before ICB CP55,940 (15 microg) attenuated the motor incoordination suggesting a modulation by an endogenous adenosine A(1) system. ICB microinjection of CHA or DPCPX prior to ICB vehicle had no effect on normal motor coordination. ICB microinjection of dipyridamole (25 microg), an adenosine transport inhibitor, significantly accentuated the motor incoordination by ICB CP55,940 (15 microg), providing further support for the involvement of endogenous adenosine in the action of CP55,940. Tolerance to the motor incoordinating effect of ICB CP55,940 was demonstrated following 3 days of i.p. CP55,940 (0.1, 1 or 2 mg/kg every 12 or 24 h; total of six or three injections, respectively). Interestingly, animals which exhibited tolerance to ICB CP55,940 also demonstrated tolerance to the accentuating effect of ICB CHA suggesting cross-tolerance between adenosine agonists and cannabinoids. Cross-tolerance was also demonstrated following 3 days of i.p. CHA (0.25 or 1 mg/kg every 24 h; total of three injections) as further evidence of the modulatory role of the cerebellar adenosine system in the acute manifestation of CP55,940-induced motor incoordination. The involvement of cerebellar adenosine and the A(1) receptor in cannabinoid actions is circumstantially supported by previous evidence that CB(1) receptors and A(1) receptors are both localized on cerebellar granule cell parallel fiber terminals and basket cell neurons where they serve to inhibit the release of neurotransmitters.
Assuntos
Adenosina/análogos & derivados , Canabinoides/farmacologia , Ataxia Cerebelar/induzido quimicamente , Cerebelo/efeitos dos fármacos , Tolerância a Medicamentos/fisiologia , Abuso de Maconha/metabolismo , Neurônios/efeitos dos fármacos , Receptores Purinérgicos P1/efeitos dos fármacos , Adenosina/farmacologia , Analgésicos/farmacologia , Animais , Ataxia Cerebelar/metabolismo , Ataxia Cerebelar/fisiopatologia , Cerebelo/metabolismo , Cerebelo/fisiopatologia , Cicloexanóis/farmacocinética , Dipiridamol/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas/fisiologia , Masculino , Abuso de Maconha/fisiopatologia , Camundongos , Neurônios/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Desempenho Psicomotor/efeitos dos fármacos , Desempenho Psicomotor/fisiologia , Receptores de Canabinoides , Receptores de Droga/efeitos dos fármacos , Receptores de Droga/metabolismo , Receptores Purinérgicos P1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Trítio/farmacocinética , Xantinas/farmacologiaRESUMO
Many epidemiological studies have suggested a high correlation between the use of tobacco and ethanol, the two most frequently abused psychoactive drugs. Recently, we reported behavioral interactions between (-)-nicotine, (-)-cotinine and ethanol within the CNS. The present report is a confirmation and an extension of that study. Using a 2 g/kg ethanol-induced motor incoordination (EIMI) as the test response, possible behavioral interactions between (-)-nicotine, (-)-cotinine and ethanol and between (-)-nicotine, (-)-cotinine and adenosine agonist + ethanol in the cerebellum were investigated. (-)-Nicotine, 0.625, 1.25 and 5 ng intracerebellarly (ICB) significantly attenuated EIMI in a dose-related manner. Likewise, ICB injection of 1.25, 2.5, and 5 ng (-)-cotinine, a major metabolite of nicotine, significantly attenuated EIMI after the same i.p. dose of ethanol as in case of (-)-nicotine but less markedly compared to (-)-nicotine. No change in normal motor coordination was observed when the highest dose of (-)-nicotine or (-)-cotinine was injected ICB followed by saline control, suggesting selectivity of their behavioral interactions with ethanol. The attenuation of EIMI by (-)-nicotine and (-)-cotinine was blocked by ICB hexamethonium (1 microgram) and trimethaphan (100 ng), the purported nicotinic-cholinergic antagonists. Finally, the ICB injection of adenosine agonists, N6-cyclohexyladenosine (CHA) or 5'-N-ethylcarboxamidoadenosine (NECA), produced marked accentuation of EIMI which was significantly antagonized by ICB (-)-nicotine and (-)-cotinine. The data obtained in the present study suggested, for the first time, a cerebellar adenosinergic-nicotinic cholinergic interaction and modulation of EIMI. The data also suggested participation of cerebellar nicotinic-cholinergic receptors in EIMI.
Assuntos
Adenosina/fisiologia , Cerebelo/fisiologia , Etanol/farmacologia , Movimento/efeitos dos fármacos , Nicotina/farmacologia , Sistema Nervoso Parassimpático/fisiologia , Animais , Cotinina/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Hexametônio , Compostos de Hexametônio/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos , Nicotina/antagonistas & inibidores , Estereoisomerismo , Trimetafano/farmacologiaRESUMO
Previous studies from our laboratory have provided strong evidence that brain adenosine modulates acute ethanol (i.p.)-induced motor incoordination (MI) through receptor mediated mechanism(s). Recently, we have reported the involvement of the striatum in ethanol-induced MI as well as the striatal adenosinergic modulation of the ethanol-induced motor deficit. The present study was thus designed to further characterize the modulatory effect of striatal adenosine on ethanol-induced MI and to look for its functional correlation with chloride flux within the rat striatum. Intrastriatal microinfusion of adenosine A1 receptor agonist N6-cyclohexyladenosine (CHA) and antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), significantly accentuated and attenuated, respectively, the motor incoordinating effect of ethanol while having no effect on the normal motor coordination in saline-treated control animals. These data confirmed the role of striatal adenosine in ethanol-induced MI. The selectivity of interactions between adenosine A1 agonist and antagonist and ethanol was further confirmed by the study in which neither intrastriatal CHA nor DPCPX significantly altered the MI induced by sodium pentobarbital. Previously, we have shown that intrastriatal Ro15-4513 not only significantly attenuated ethanol-induced MI but also blocked its accentuation by intrastriatal CHA. It is well known that Ro15-4513 antagonizes many, but not all, CNS effects of ethanol by blocking the ethanol potentiation of GABA-stimulated uptake of chloride. Therefore, experiments using striatal microsac preparations were carried out to investigate the possible modulation of chloride conductance by CHA and its relationship to ethanol. High concentrations of CHA (10 and 100 nM) increased the total chloride uptake by the striatal microsacs. Corresponding to the ethanol-adenosine interaction observed behaviorally, a much lower concentration (1 nM) of CHA, being ineffective itself, significantly enhanced the stimulatory action of ethanol on chloride uptake. This effect was blocked by either Ro15-4513 (100 nM) or DPCPX (10 nM). The modulatory effect of GABA and/or ethanol on chloride influx was also evaluated, and the results supported the appropriateness to use striatal microsac preparations in the present study. Overall, the data suggested a functional interaction between ethanol and striatal adenosine and further supported the hypothesis that striatal adenosine might, in part, modulate ethanol-induced MI through its effect on chloride conductance through chloride channels coupled to GABA-benzodiazepine receptor complex.
Assuntos
Adenosina/fisiologia , Depressores do Sistema Nervoso Central/farmacologia , Cloretos/fisiologia , Corpo Estriado/metabolismo , Etanol/farmacologia , Atividade Motora/efeitos dos fármacos , Adenosina/análogos & derivados , Adenosina/farmacologia , Marcadores de Afinidade/farmacologia , Animais , Azidas/farmacologia , Benzodiazepinas/farmacologia , Líquido Cefalorraquidiano , Cloretos/farmacocinética , Corpo Estriado/fisiopatologia , Moduladores GABAérgicos/farmacologia , Masculino , Microdiálise , Pentobarbital/farmacologia , Radioisótopos , Ratos , Ratos Sprague-Dawley , Xantinas/farmacologiaRESUMO
Dilazep (i.p.), a coronary vasodilator and an uptake inhibitor of adenosine, dose dependently potentiated acute ethanol-induced motor incoordination in mice. In view of peripheral cardiovascular depressive effects of dilazep, the effect of i.c.v. dilazep (25, 50 and 75 micrograms), and its metabolites, 1,4-bis(3-hydroxypropyl)perhydro-1,4-diazepine (BHPD) (15, 31 and 62 micrograms) and 1-[3-(3,4,5-trimethoxybenzoyloxy)propyl]perhydro-1,4-diazepine (TBPD) (62 and 125 micrograms) on ethanol-induced motor incoordination was studied. Dose-related potentiation of ethanol-induced motor incoordination was noted with dilazep and its metabolites. Whereas dilazep (i.p.) produced no apparent central nervous system (CNS) effects, by i.c.v. route, it caused CNS excitation including tonic-clonic seizures. Adenosine uptake inhibition, Ca2+ entry blockade or direct activation of adenosine receptors was ruled out as the possible mechanism of seizures because dipyridamole, verapamil or N6-(2-phenylisopropyl)-adenosine (R-PIA) administered i.c.v., while potentiating ethanol (i.p.)-induced motor incoordination did not produce seizures. The CNS excitation was minimal with BHPD and none with TBPD. Theophylline pretreatment partially blocked potentiation of ethanol-induced motor incoordination by dilazep and BHPD and not by TBPD. The data suggest dilazep-induced potentiation of ethanol-induced motor incoordination is partially due to central adenosine receptor mechanism and partly due to other yet unknown mechanism(s) and further supported our earlier reports about adenosine involvement in the CNS effects of ethanol. The data also suggest that dilazep (i.c.v.)-induced seizures are due to mechanism(s) other than adenosine uptake inhibition, Ca2+ entry blockade or direct adenosine receptor activation.
Assuntos
Azepinas/farmacologia , Comportamento Animal/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Dilazep/farmacologia , Etanol/farmacologia , Animais , Injeções Intraperitoneais , Injeções Intraventriculares , Masculino , Camundongos , Microinjeções , Desempenho Psicomotor/efeitos dos fármacos , Fatores de Tempo , Verapamil/farmacologiaRESUMO
1. On going work in our laboratory has shown that adenosine modulates ethanol-induced motor incoordination (EIMI) when given systemically as well as directly into the cerebral ventricles, cerebellum and corpus striatum of the rat and/or mouse. 2. The objective of this study was to determine what effect adenosine agonists and antagonists would have within the rat motor cortex on EIMI. 3. The participation of the motor cortex in EIMI was suggested when microinfusion of the anti-ethanol compound, Ro15-4513, an inverse agonist of the benzodiazepine binding site, directly into the motor cortex significantly attenuated EIMI. Further, the adenosine agonists N6-cyclohexyladenosine (CHA) and 2-p-(2-carboxyethyl)-phenethylamino-5'-N-carboxaminoadenosine++ + hydrochloride (CGS-21680) significantly accentuated EIMI in a dose-related manner. The adenosine A1 receptor-selective agonist, CHA, appeared most potent in this modulatory effect when compared to the A2-selective agonist, CGS-21680. 4. The extent of diffusion of the adenosine drugs within the cortical tissue after their microinfusion was also checked by measuring the dispersion of microinfused [3H]CHA. The [3H]CHA dispersion study indirectly confirmed that the results of the present investigation were based on the effect of adenosine drugs within the motor cortex only. 5. Accentuation by the A1- and A2-selective adenosine agonists was significantly attenuated by the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) but not by the A2 receptor-selective antagonist 8-(3-chlorostyryl)caffeine (CSC) further suggesting modulation mainly by the A1-subtype. 6. Pretreatment of the motor cortex with pertussis toxin (PT) significantly reduced the capacity of both A1- and A2-selective adenosine agonists to accentuate EIMI suggesting the involvement of a PT-sensitive Gi/Go protein. 7. These data support earlier work which showed that adenosine modulates EIMI within the central nervous system (CNS), most likely via the A1 receptor, and moreover, extend that work by including the motor cortex as a brain area participating in the adenosinergic modulation of ethanol-induced motor impairment.
Assuntos
Adenosina/farmacologia , Etanol/farmacologia , Atividade Motora/efeitos dos fármacos , Córtex Motor/efeitos dos fármacos , Adenosina/análogos & derivados , Adenosina/antagonistas & inibidores , Animais , Ataxia/induzido quimicamente , Masculino , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos/fisiologiaRESUMO
In a two-year study, from January 1977 to December 1978, 80 human cases of ophthalmomyiasis were recorded at the Benghazi Central Eye Clinic. Infections were due to larvae of the sheep nasal bot, Ostrus ovis. The annual incidence was estimated to be 10 per 100,000 population, with most of the cases occurring amongst males during the months of May, June and July. A typical case history is described to illustrate the clinical features and the treatment used.
Assuntos
Oftalmopatias/epidemiologia , Miíase/epidemiologia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Dípteros/isolamento & purificação , Oftalmopatias/terapia , Feminino , Humanos , Líbia , Masculino , Pessoa de Meia-Idade , Miíase/terapia , Estações do AnoRESUMO
We have demonstrated that ethanol-induced motor incoordination is modulated by cerebellar adenosine A(1) receptor. This study represents an extension into another important brain motor area, the striatum that, unlike cerebellum, has high density of both A(1) and A(2A) receptors. Direct intra-striatal micro-infusion of Ro15-4513 (0.05, 0.5, 1 ng), a partial inverse-agonist of benzodiazepine, significantly and nearly dose-dependently attenuated ethanol-induced motor incoordination indicating mediation of ethanol's motor incoordination by striatum. Intra-striatal A(1)-selective agonist N(6)-cyclohexyladenosine (CHA; 1, 2, 4 ng), A(1) = A(2A) non-selective agonist, 5'-N-ethylcarboxamidoadenosine (NECA; 1.5, 3, 6 ng), and A(1)-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 25, 50, 100 ng) dose-dependently accentuated and attenuated, respectively, ethanol-induced motor incoordination, strongly suggesting modulation by striatal adenosine A(1) receptor. Intra-striatal DPCPX significantly antagonized not only ethanol-induced motor incoordination but also its potentiation by intra-striatal CHA, R-(+)-N(6)-(2-phenylisopropyladenosine) (R-PIA), or NECA. No change in motor coordination occurred after the highest dose of CHA, R-PIA, or NECA followed by saline. Similarly, the highest intra-striatal dose of Ro15-4513 or DPCPX neither altered motor coordination or locomotor activity indicating relative selectivity of interaction with ethanol. Nearly 25-fold higher dose of A(2A)-selective agonist, CGS-21680, compared to CHA was necessary to produce a comparable potentiation of ethanol's motor incoordination perhaps suggesting a lack of or less significant striatal A(2A) involvement. Intra-striatal pertussis toxin (0.5 microg) pre-treatment markedly attenuated ethanol-induced motor incoordination as well as its potentiation by intra-striatal CHA. These results support that striatum is one of the brain motor areas mediating the motor impairing effects of acute ethanol and that the latter's modulation occurs via A(1)-selective receptors coupled to pertussis toxin-sensitive G proteins.
Assuntos
Adenosina/análogos & derivados , Ataxia/induzido quimicamente , Ataxia/metabolismo , Depressores do Sistema Nervoso Central/farmacologia , Corpo Estriado/metabolismo , Etanol/farmacologia , Receptores Purinérgicos P1/metabolismo , Adenosina/farmacologia , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Marcadores de Afinidade/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Ataxia/tratamento farmacológico , Azidas/farmacologia , Benzodiazepinas/farmacologia , Corpo Estriado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Microinjeções , Atividade Motora/efeitos dos fármacos , Toxina Pertussis , Fenetilaminas/farmacologia , Receptor A2A de Adenosina , Vasodilatadores/farmacologia , Fatores de Virulência de Bordetella/farmacologia , Xantinas/farmacologiaRESUMO
The possible modulation and of co-modulation by the cerebellar GABAB and adenosine A1 receptors of ethanol-induced motor impairment were investigated in the mice using rotorod performance as the test response. Direct cerebellar microinfusion of GABAB agonist, baclofen, and antagonist, phaclofen, into the permanently cannulated mice, produced a dose-dependent accentuation and attenuation, respectively of ethanol-induced motor impairment. The baclofen and phaclofen exhibited accentuation and attenuation, respectively, via GABAB receptors linked to pertussis toxin-sensitive G protein. A comodulation by the cerebellar adenosine A1 receptors was also observed because intracerebellar microinfusion of adenosine agonists NB-cyclohexyladenosine (CHA), 5'-N-ethylcarbox-amidoadenosine (NECA), and 2-p-(2-carboxyethyl)-phenyl-ethylamino-5'-N-ethylacarbox- amidoadenosine (CGS-21680), and antagonist, 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX), also accentuated and attenuated, respectively, ethanol-induced motor impairment. The accentuation of ethanol-induced motor impairment by baclofen was further enhanced after the intracerebellar microinfusion of CHA, suggesting a co-modulation by the co-localized adenosine A1 receptors. A similar response was observed after the intracerebellar microinfusion of adenosine A1 = A2 agonist NECA and the several-fold higher dose of adenosine A2-selective agonist CGS-21680. Ethanol-induced motor impairment was markedly blocked by intracerebellar A1-selective antagonist, DPCPX, as well as by the intracerebellar pertussis toxin pretreatment suggesting again a co-modulation by the adenosine A1 receptors and the involvement of pertussis toxsin-sensitive G protein, respectively. The almost 25-fold higher dose of CGS-21680 to accentuate and DPCPX to attenuate, respectively, ethanol-induced motor impairment together with the reported cerebellar localization of adenosine A1 subtype only, suggested A1 receptor activation by NECA and CGS-21680. The functional similarity between GABAB and adenosine A1, receptors associated with their anatomical co-localization on the cerebellar granule cells, mainly axons and axonal terminals, may suggest a possible common adenylate cyclase catalytic unit as the basis of modulation of ethanol's motor impairment by these two receptor mechanisms.
Assuntos
Ataxia/induzido quimicamente , Cerebelo/química , Etanol/toxicidade , Receptores de GABA-B/efeitos dos fármacos , Receptores Purinérgicos P1/fisiologia , Adenosina/análogos & derivados , Adenosina/farmacologia , Toxina Adenilato Ciclase , Animais , Baclofeno/análogos & derivados , Baclofeno/farmacologia , Comportamento Animal/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Cerebelo/fisiologia , Relação Dose-Resposta a Droga , Agonistas GABAérgicos/farmacologia , Proteínas de Ligação ao GTP/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos , Microinjeções , Atividade Motora/efeitos dos fármacos , Muscimol/farmacologia , Toxina Pertussis , Receptores Purinérgicos P1/efeitos dos fármacos , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The purpose of this study was to investigate the possible in vivo effects of (-)-nicotine, ethanol, and an adenosine agonist N6-cyclohexyladenosine (CHA) when injected individually as well as in various combinations on glucose utilization in the fresh cerebellar slices of mice. Mice received ICV (-)-nicotine or CHA followed 5 min later by a test dose of ethanol (2 g/kg; IP). Animals were killed 20 min postethanol treatment and fresh slices (300 microns) of cerebellum were incubated in a glucose medium in Warburg flasks using 14C-glucose as a tracer. Trapped 14CO2 was counted to estimate glucose utilization. Ethanol treatment markedly accentuated glucose utilization, whereas the pretreatment with (-)-nicotine (125 and 250 ng, ICV) resulted in a significant attenuation in the ethanol-induced increase in glucose utilization. However, ICV (-)-nicotine (125 ng) alone did not produce any change in the cerebellar glucose utilization. The attenuation of ethanol-induced increase in glucose utilization by (-)-nicotine was nearly totally blocked by ICV hexamethonium, a purported nicotinic antagonist, suggesting participation of cholinergic-nicotinic receptors. The (-)-nicotine pretreatment also significantly attenuated both the ICV CHA (25 ng)-induced increase in glucose utilization and the accentuation of ethanol-induced increase in glucose utilization by CHA. The antagonistic effect of (-)-nicotine on CHA- and ethanol-induced increase in glucose utilization indicating an interaction between (-)-nicotine and ethanol and between (-)-nicotine and adenosine may suggest involvement of postreceptor (nicotinic and adenosine) mechanisms including ionic channels.
Assuntos
Adenosina/análise , Cerebelo/efeitos dos fármacos , Etanol/farmacologia , Glucose/metabolismo , Nicotina/farmacologia , Adenosina/farmacologia , Animais , Interações Medicamentosas , Hexametônio/farmacologia , Masculino , CamundongosRESUMO
A high correlation between alcohol use and smoking has long been suggested by epidemiological data. We examined the potential behavioral interactions between ethanol and nicotine using ethanol-induced motor incoordination as the test response in mice. Effect of pretreatment of various doses of (-)-nicotine, (-)-cotinine, a major metabolite of nicotine, and (+)-nicotine administered ICV on ethanol (IP)-induced motor incoordination was investigated. (-)-Nicotine (0.19, 0.38, 0.77 and, 1.54 nmoles ICV) produced significant attenuation of motor incoordination due to ethanol (2 g/kg IP) in a nearly dose-related manner which was blocked by ICV hexamethonium and trimethaphan, both purported nicotinic antagonists. (-)-Cotinine (0.35, 0.70, 1.41 nmole ICV) produced similar attenuation but was les potent than (-)-nicotine. Attenuation by (+)-nicotine (0.19, 0.38, and 0.77 nmoles ICV) was also significant but only at 0.77 nmole dose level. (+)-Nicotine-induced attenuation of motor incoordination by ethanol was antagonized by nicotinic antagonists. Data obtained suggest a central behavioral interaction between ethanol and nicotine at least through the participation of cholinergic nicotinic receptors.