RESUMEN
Retrotransposons are transcriptionally activated in different tissues and cell types by a variety of genomic and environmental factors. Transcription of LTR retrotransposons is controlled by cis-acting regulatory sequences in the 5' LTR. Mobilization of two LTR retroelements, Idefix and ZAM, occurs in the unstable RevI line of Drosophila melanogaster, in which their copy numbers are high, while they are low in all other stocks tested. Here we show that both a full-length and a subgenomic Idefix transcript that are necessary for its mobilization are present in the Rev1 line, but not in the other lines. Studies on transgenic strains demonstrate that the 5' LTR of Idefix contains sequences that direct the tissue-specific expression of the retroelement in testes and ovaries of adult flies. In ovaries, expression occurs in the early follicle and in other somatic cells of the germarium, and is strictly associated with the unstable genetic context conferred by the RevI line. Control of tissue-specific Idefix expression by interactions between cis-acting sequences of its LTR and trans-acting genomic factors provides an opportunity to use this retroelement as a tool for the study of the early follicle cell lineage in the germarium.
Asunto(s)
Drosophila melanogaster/genética , Retroelementos , Empalme Alternativo , Animales , Animales Modificados Genéticamente , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto , Genoma , Operón Lac , Masculino , Folículo Ovárico/citología , Folículo Ovárico/crecimiento & desarrollo , Folículo Ovárico/metabolismo , Ovario/citología , Ovario/crecimiento & desarrollo , Ovario/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Secuencias Repetidas Terminales , Testículo/citología , Testículo/crecimiento & desarrollo , Testículo/metabolismo , Distribución TisularRESUMEN
A beta-1,4-endoglucanase named MI-ENG1, homologous to the family 5 glycoside hydrolases, was previously isolated from the plant parasitic root-knot nematode Meloidogyne incognita. We describe here the detection of the enzyme in the nematode homogenate and secretion and its complete biochemical characterization. This study is the first comparison of the enzymatic properties of an animal glycoside hydrolase with plant and microbial enzymes. MI-ENG1 shares many enzymatic properties with known endoglucanases from plants, free-living or rumen-associated microorganisms and phytopathogens. In spite of the presence of a cellulose-binding domain at the C-terminus, the ability of MI-ENG1 to bind cellulose could not be demonstrated, whatever the experimental conditions used. The biochemical characterization of the enzyme is a first step towards the understanding of the molecular events taking place during the plant-nematode interaction.
Asunto(s)
Celulasa/química , Proteínas del Helminto/química , Tylenchoidea/enzimología , Secuencia de Aminoácidos , Animales , Carboximetilcelulosa de Sodio/metabolismo , Cationes Bivalentes , Celulasa/aislamiento & purificación , Celulasa/metabolismo , Celulasa/fisiología , Celulosa/metabolismo , Quelantes/farmacología , Fenómenos Químicos , Química Física , Ácido Edético/farmacología , Escherichia coli , Glucanos/metabolismo , Glicósido Hidrolasas/química , Glicósido Hidrolasas/clasificación , Glicósido Hidrolasas/metabolismo , Proteínas del Helminto/aislamiento & purificación , Proteínas del Helminto/metabolismo , Proteínas del Helminto/fisiología , Interacciones Huésped-Parásitos , Datos de Secuencia Molecular , Raíces de Plantas/parasitología , Plantas Tóxicas , Unión Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Nicotiana/citología , Tylenchoidea/fisiología , Xilanos/metabolismoRESUMEN
MGI 114 (HMAF, 6-hydroxymethylacylfulvene) is a cytotoxic drug currently in phase II human clinical trials. As with other anticancer agents, inadvertent drug extravasation may result in perivascular irritation and/or necrosis. In this study the degree of soft tissue injury produced by MGI 114 after intradermal administration to rats was quantified and four potential antidotes for extravasation injuries caused by MGI 114 were evaluated. Intradermal injections of MGI 114 (0.2 ml, concentrations 0.1, 0.5 or 1.0 mg/ml) and a positive control, doxorubicin (0.2 ml, concentration 2 mg/ml) were administered to male Fischer 344 rats in an experiment designed to establish a model for antidote evaluation. Dermal lesions at the injection sites were measured and quantitated as the total area under the lesion area-time curve (AUC). Physiological saline, sodium thiosulfate, dimethylsulfoxide (DMSO) and local cooling, were then compared as potential antidotes in this model. In the initial study, dermal lesions (erythema, ulcerations and eschar formation) occurred at the MGI 114- and doxorubicin-treated sites. The lesion area resulting from MGI 114 was dose-related and was greatest at approximately 5 days, with resolution by day 7-22. Doxorubicin-induced lesions were comparable in area to those induced by the highest dose of MGI 114, but persisted approximately twice as long. In the antidote study, sodium thiosulfate administration resulted in approximately 20% diminution of lesion area and AUC value when compared to untreated controls. Normal saline caused slight reductions in maximum lesion area, but had little effect on AUC values. Local cooling also caused a modest reduction in the maximum lesion area, but actually resulted in higher AUC values by prolonging eschar duration. DMSO provided near complete tissue protection from intradermal exposure to MGI 114. In this model MGI 114 and doxorubicin were found to produce similar soft tissue injuries, but MGI 114-induced lesions tended to show a more rapid resolution. Topical DMSO treatment was found to produce the most effective protection against MGI 114-induced local tissue irritation and necrosis.
Asunto(s)
Antídotos/uso terapéutico , Antineoplásicos/toxicidad , Sesquiterpenos/toxicidad , Piel/efectos de los fármacos , Administración Cutánea , Animales , Frío , Dimetilsulfóxido/uso terapéutico , Relación Dosis-Respuesta a Droga , Masculino , Ratas , Ratas Endogámicas F344 , Piel/patología , Tiosulfatos/uso terapéuticoRESUMEN
A beta-1,4-endoglucanase encoding cDNA (EGases, E.C. 3.2.1.4), named Mi-eng-1, was cloned from Meloidogyne incognita second-stage juveniles (J2). The deduced amino acid sequence contains a catalytic domain and a cellulose-binding domain separated by a linker. In M. incognita, the gene is transcribed in the migratory J2, in males, and in the sedentary adult females. In pre-parasitic J2, endoglucanase transcripts are located in the cytoplasm of the subventral esophageal glands. The presence of beta-1,4-endoglucanase transcripts in adult females could be related to the expression of the gene in esophageal glands at this stage. However, cellulase activity within the egg matrix of adult females suggests that the endoglucanase may also be synthesized in the rectal glands and involved in the extrusion of the eggs onto the root surface. The maximum identity of the predicted MI-ENG-1 catalytic domain with the recently cloned cyst nematode beta-1,4-endoglucanases is 52.5%. In contrast to cyst nematodes, M. incognita pre-parasitic J2 were not found to express a beta-1,4-endoglucanase devoid of a cellulose-binding domain.
Asunto(s)
Celulasa/genética , ADN Complementario/genética , ADN de Helmintos/genética , Tylenchoidea/enzimología , Tylenchoidea/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN/genética , ADN Complementario/aislamiento & purificación , ADN de Helmintos/aislamiento & purificación , Femenino , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Genes de Helminto , Masculino , Datos de Secuencia Molecular , Plantas/parasitología , Tylenchoidea/patogenicidadRESUMEN
A gene encoding a protein with strong homology with Caenorhabditis elegans and C. briggsae acetylcholinesterase ACE-1 was cloned from Meloidogyne incognita and M. javanica pre-parasitic juveniles. Both cDNAs have an ORF of 1968 bp for a deduced translation product of 656 amino acid residues. The key residues essential to acetylcholinesterase (AChE) structure and function are conserved in both sequences. M. incognita and M. javanica AChE share a homology of 98.8% at the amino acid level and 97% at the nucleotide level. Phylogenetic analysis showed that Meloidogyne and Caenorhabditis AChE form a cluster among AChE of triploblastic organisms. This Meloidogyne AChE is expressed in eggs, pre-parasitic juveniles and males and AChE activity was detected in situ in amphids of pre-parasitic juveniles. The opportunity of using AChE as a target in new strategies of nematode control is discussed.
Asunto(s)
Acetilcolinesterasa/genética , Genes de Helminto , Tylenchoidea/genética , Acetilcolinesterasa/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis/genética , Clonación Molecular , Femenino , Masculino , Datos de Secuencia Molecular , Filogenia , Plantas/parasitología , Alineación de Secuencia , Tylenchoidea/enzimologíaRESUMEN
An extract of the desert plant Yucca shidigera was assessed for its possible benefit in ruminal fermentation. The extract bound ammonia in aqueous solution when concentrations of ammonia were low (up to 0.4 mM) and when the extract was added at a high concentration to the sample (20%, vol/vol). The apparent ammonia-binding capability was retained after autoclaving and was decreased slightly following dialysis. Acid-precipitated extract was inactive. No evidence of substantial ammonia binding was found at higher ammonia concentrations (up to 30 mM). When Y. shidigera extract (1%, vol/vol) was added to strained rumen fluid in vitro, a small (6%) but significant (P < 0.05) decrease in ammonia concentration occurred, apparently because of decreased proteolysis. Inclusion of Y. shidigera extract (1%, vol/vol) in the growth medium of the rumen bacterium Streptococcus bovis ES1 extended its lag phase, while growth of Butyrivibrio fibrisolvens SH13 was abolished. The growth of Prevotella (Bacteroides) ruminicola B(1)4 was stimulated, and that of Selenomonas ruminantium Z108 was unaffected. Protozoal activity, as measured by the breakdown of 14C-leucine-labelled S. ruminantium in rumen fluid incubated in vitro, was abolished by the addition of 1% extract. The antimicrobial activities were unaffected by precipitating tannins with polyvinylpyrrolidone, but a butanol extract, containing the saponin fraction, retained its antibacterial and antiprotozoal effects. Saponins from other sources were less effective against protozoa than Y. shidigera saponins. Y. shidigera extract, therefore, appears unlikely to influence ammonia concentration in the rumen directly, but its saponins have antimicrobial properties, particularly in suppressing ciliate protozoa, which may prove beneficial to ruminal fermentation and may lead indirectly to lower ruminal ammonia concentrations.
Asunto(s)
Amoníaco/metabolismo , Extractos Vegetales/farmacología , Rumen/metabolismo , Ovinos/metabolismo , Animales , Cilióforos/efectos de los fármacos , Extractos Vegetales/química , Extractos Vegetales/metabolismo , Rumen/efectos de los fármacos , Rumen/microbiología , Rumen/parasitología , Ovinos/microbiología , Ovinos/parasitologíaRESUMEN
Alterations in lipid metabolism and cellular morphology in the liver were examined in female rats treated with 100 mg Ethmozine/kg/day for 7 days. The incorporation of either [3H]acetate with [methyl-14C]choline, or [methyl-14C]methionine was used to monitor the effect of the drug on neutral and phospholipid syntheses. Ethmozine (ETH) reduced the incorporation of choline into phosphatidylcholine (PC) by 50%, but the transmethylation of phosphatidylethanolamine to form PC was unaffected. The formation of lyso-PC was reduced by one-half irrespective of the donor radiolabel. An accumulation of both micro- and macrovesicles (fat) was found in the centri- and midlobular zones of the liver, which is likely the result of increased synthesis and decreased secretion of triacylglycerol (TAG). Incorporation of acetate into TAG was increased fivefold by ETH treatment, and to a lesser degree into cholesterol and cholesterylester/squalene.
Asunto(s)
Antiarrítmicos/farmacología , Metabolismo de los Lípidos , Hígado/efectos de los fármacos , Fenotiazinas/farmacología , Animales , Colesterol/metabolismo , Colina/metabolismo , Femenino , Hígado/metabolismo , Hígado/patología , Moricizina , Fosfolípidos/metabolismo , Ratas , Triglicéridos/metabolismoRESUMEN
7-Chloro-5-(2-fluorophenyl)-1,3-dihydro-1-(2,2,2-trifluoroethyl)-2H-1,4- benzodiazepine-2-thione (quazepam, Sch 16134, Dormalin) was evaluated for evidence of systemic toxicity, carcinogenicity and reproductive toxicity in several laboratory animal species including the hamster. Mutagenic potential was also assessed in one in vivo and three in vitro assays. In some studies, diazepam was used as a comparative control. Oral LD50 values were greater than 5000 mg/kg in the mouse and rat while i.p. LD50 values were approximately 900 and 2900 mg/kg in the mouse and rat, respectively. Studies in hamsters for 4 weeks at doses up to 500 mg/kg/d and for 51 weeks at doses up to 120 mg/kg/d demonstrated that the liver was the principal target organ in this species with the effects upon the liver related to dose and duration of dosing. Studies in the squirrel monkey for 13 and 52 weeks at doses up to 50 mg/kg/d demonstrated a transient ataxia, hypoactivity and somnolence during the initial two weeks of dosing. No unusual necropsy or microscopic observations were noted in the 13-week study. Male reproductive organs of quazepam-dosed monkeys were reduced in weight after 52 weeks. Moderate to marked impairment of spermatogenesis and higher liver weights with moderate to marked fatty change in both sexes were observed in groups given diazepam. Abrupt withdrawal of quazepam or diazepam after 52 weeks of dosing was associated at all dose levels with excitability, hyperactivity and convulsions. Two quazepam- and all diazepam-dosed monkeys died.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Ansiolíticos/toxicidad , Benzodiazepinas/toxicidad , Animales , Peso Corporal/efectos de los fármacos , Carcinógenos , Cricetinae , Perros , Evaluación de Medicamentos , Femenino , Masculino , Mesocricetus , Ratones , Mutágenos , Embarazo , Ratas , Saimiri , Especificidad de la Especie , TeratógenosRESUMEN
A neurologic deficit characterized by hypokinesia, postural flexion, and to a lesser extent, rigidity, tremor and myoclonus, has been observed in cynomolgus monkeys following administration of 1-methyl-4-(1-methylpyrrol-2-yl)-4-piperidinol (MMPP), a novel 4-substituted piperidine. The syndrome, similar to that described for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), developed within 3-7 days after oral or i.v. dosing, and was accompanied by lesions in the substantia nigra. The behavioral syndrome was seen to a lesser extent in dogs but not in rats. MMPP contains a hydroxyl group on the 4-position of the pyridine ring; the corresponding dehydration product was inactive.
Asunto(s)
Neurotoxinas/toxicidad , Enfermedad de Parkinson Secundaria/inducido químicamente , Peróxidos/toxicidad , Ácidos Ftálicos , Animales , Conducta Animal/efectos de los fármacos , Modelos Animales de Enfermedad , Perros , Relación Dosis-Respuesta a Droga , Femenino , Macaca fascicularis , Masculino , Neurotoxinas/administración & dosificación , Enfermedad de Parkinson Secundaria/patología , Enfermedad de Parkinson Secundaria/fisiopatología , Peróxidos/administración & dosificación , Ratas , Ratas Endogámicas , Sustancia Negra/efectos de los fármacosRESUMEN
SCH 19927 [R,R)-(-)-2-Hydroxy-5-[1-hydroxy-2-[(1-methyl -3-phenylpropyl)amino]ethyl]benzamide hydrochloride) is a beta-adrenergic blocking agent which has vasodilating properties. In a subchronic oral toxicity study in beagle dogs, SCH 19927 was given by gavage at doses of 30, 60, and 90 mg/kg. Lesions were observed at weeks 13 and 19 in the tapetum lucidum, a light reflecting structure of the eye. The lesions consisted of focal to multifocal areas of discoloration of the tapetal portion of the ocular fundus, pigmentation in the tapetal area, and, in one dog, subretinal edema resulting in a focal retinal detachment. Light and electron microscopic examination of the ocular lesions demonstrated tapetal cell degeneration and necrosis with macrophages, lymphocytes, and occasional plasma cells in the tapetum and adjacent choroid. Local cellular infiltrates within the retina internal to the pigmented epithelium were observed in one dog (60 mg/kg) which was demonstrated to have focal retinal edema during the study. In a repeat study the lesion again occurred in tapetal beagle dogs but not in atapetal beagle dogs (90 mg/kg) or cynomolgous monkeys (360 mg/kg). The lesion had not occurred in a previous subchronic study in albino rats. These results demonstrated that the tapetum lucidum was a target organ of toxicity for SCH 19927 and indicated that the finding was without observable toxicological significance in animals, including man, whose eyes do not have this structure.