RESUMO
Cytosolic thymidine kinase (EC 2.7.1.75), the initial enzyme in the thymidine salvage pathway, was detected in crude homogenates of adult female Brugia pahangi and Dirofilaria immitis, with respective specific activities of 100 and 460 nmol/h/mg protein. Partially purified filarial thymidine kinases were found to have molecular weights of approximately 180 000, to be most active in the presence of Mg2+ and ATP, to have a sharp pH optimum (pH 7.0) and to be heat-labile in the absence of added thymidine. For both, the respective Km values for thymidine and ATP were 60 muM and 1.6 mM, and 5-iodo-2'-deoxyuridine was as good a substrate as thymidine. A distinguishing property was the 3-fold higher sensitivity of the B. pahangi enzyme to feedback inhibition by thymidine 5'-triphosphate. Adult female B. pahangi took up and incorporated [methyl-3 H] thymidine into DNA when they were exposed to this radiolabeled deoxynucleoside in vivo, but the thymidine salvage pathway in these worms was essentially nonfunctional in vitro.
Assuntos
Brugia/enzimologia , Dirofilaria/enzimologia , Filarioidea/enzimologia , Timidina Quinase/metabolismo , Animais , Brugia/metabolismo , DNA/metabolismo , Dirofilaria/metabolismo , Timidina/metabolismoRESUMO
Among various ubiquinone (Q) isoprenologues tested, only Q7 was more efficient than menadione in promoting the oxidation of 5-methyltetrahydrofolate (CH3FH4) by 5,10-methylenetetrahydrofolate reductase isolated from adult Brugia pahangi, whereas Q10 was the best cofactor in the same reaction catalysed by the analogous enzyme from adult Dirofilaria immitis. Menoctone (3-[1-cyclohexyloctyl] -2-hydroxy-1,4-naphthoquinone) was a strong competitive inhibitor of both these ubiquinone isoprenologues in the respective reactions. When incubated in the presence of D,L-[14C]-mevalonate, adult B. pahangi and D. immitis synthesized radiolabelled Q9 only, in addition to other isoprenoid derivatives in the neutral lipid fraction. In view of the inability of Q9 to promote the oxidation of CH3FH4 by 5,10-methylenetetrahydrofolate reductase from B. pahangi, it seems unlikely that this filaria uses Q9 as a cofactor in this reaction. Conceivably, D. immitis could use Q9 as a cofactor in its enzymatic oxidation of CH3FH4, since in this circumstance, it was a better cofactor than menadione.
Assuntos
Brugia/metabolismo , Dirofilaria/metabolismo , Filarioidea/metabolismo , Ubiquinona/biossíntese , Brugia/enzimologia , Dirofilaria/enzimologia , Naftoquinonas/metabolismo , Tetra-Hidrofolatos/metabolismo , Ubiquinona/metabolismo , Vitamina K/metabolismoRESUMO
The diagnosis of canine heartworm infection is based upon the presence of circulating Dirofilaria immitis microfilariae or on techniques for the detection of serum antibodies or antigens. In the first of these, discrimination between D. immitis, D. repens and Acanthocheilonema dracunculoides microfilariae is based upon the acid phosphatase histochemical stain. In this paper, we propose an alternative technique for histochemical staining using a commercial kit test of naphthol-AS-OL (Leucognost-SP). This offers the advantages of speed and simplicity as compared to the standard Barka procedure.
Assuntos
Infecções por Dipetalonema/veterinária , Dipetalonema/classificação , Dirofilaria immitis/classificação , Dirofilaria/classificação , Dirofilariose/diagnóstico , Doenças do Cão/diagnóstico , Fosfatase Ácida/análise , Animais , Diagnóstico Diferencial , Dipetalonema/enzimologia , Infecções por Dipetalonema/diagnóstico , Dirofilaria/enzimologia , Dirofilaria immitis/enzimologia , Doenças do Cão/parasitologia , Cães , Histocitoquímica/métodos , Histocitoquímica/veterinária , Microfilárias/classificação , Microfilárias/enzimologia , Kit de Reagentes para Diagnóstico/veterináriaRESUMO
A multilocus genetic analysis based on protein electrophoresis was carried out on 39 adult specimens of Dirofilaria repens and 31 of D. immitis. Seventeen enzymatic loci provided reliable electrophoretic patterns which were compared in the two species. All loci except one were monomorphic. Fixed alternative allozymes were found at 13 loci while only 3 loci shared apparently the same allozyme in the two species. The polymorphic locus, Pgm, also showed alternative allozymes. This remarkably high genetic divergence, which presumably reflects a very old speciation process, allows an easy characterization of D. repens and D. immitis and supports their classification in the subgenera Nochtiella and Dirofilaria, respectively.
Assuntos
Dirofilaria immitis/genética , Dirofilaria/genética , Animais , Dirofilaria/classificação , Dirofilaria/enzimologia , Dirofilaria immitis/enzimologia , Cães , Eletroforese em Gel de Amido , Feminino , Frequência do Gene , Marcadores Genéticos , Isoenzimas/genética , Masculino , Especificidade da EspécieRESUMO
Microfilarial infections could be detected by the Difil Test in 11 (2.2%) of 479 blood samples of clinically asymptomatic dogs from the South of Switzerland. Dirofilaria repens and D. immitis were identified in 3 (0.6%) and 8 dogs (1.6%), respectively, by the acid phosphatase activity of the microfilariae. 10 dogs with microfilaremia had been abroad or a stay outside Switzerland could not be excluded. One dog diagnosed with D. immitis could have had acquired the infection in the canton Tessin according to information given by the owner. Dogs with microfilaremia are a potential source of infection for mosquitoes. An indigenous cycle of infection in the South of Switzerland is possible since the mean average temperature in summer is above 18 degrees C which is necessary for optimal parasite development in the vector. A strict control of imported dogs or animals exposed to the disease in endemic regions as well as the therapy of infected dogs in the South of Switzerland is advisable.
Assuntos
Dirofilariose/epidemiologia , Doenças do Cão/epidemiologia , Fosfatase Ácida/sangue , Animais , Dirofilaria/enzimologia , Dirofilaria/isolamento & purificação , Dirofilaria immitis/enzimologia , Dirofilaria immitis/isolamento & purificação , Cães , Feminino , Masculino , Microfilárias/enzimologia , Microfilárias/isolamento & purificação , Suíça/epidemiologiaRESUMO
Two membrane-bound glutamate dehydrogenases were found in adult Dirofilaria immitis, an NAD-linked enzyme (EC 1.4.1.2) in the cytosol (C-GDH) and an enzyme equally reactive with NAD or NADP (EC 1.4.1.3) in the mitochondria (M-GDH). The cytosolic enzyme had a pH optimum of 7.8-8.0 and exhibited 30% more activity at 25 C than at 37 C (pH 8.0). The mitochondrial enzyme had a pH optimum at 8.4 and exhibited 27% more activity at 37 C than at 25 C (pH 8.4); it was also more sensitive to heat denaturation. Gel filtration of worm subfractions separated four peaks of C-GDH activity with molecular weights of approximately 610, 285, 180, and less than 100 thousand, and a single major peak of M-GDH activity with a molecular weight of about 335,000. When assayed at pH 8, 37 C, and 200 microM NADH, the Km for the substrate, alpha-ketoglutarate, was equivalent for the two enzymes, but the Km for ADP (activator) was five times greater for M-GDH. When the two enzymes were assayed at pH 8.0, 37 C, and 100 microM NADH, 1 mM ADP approximately doubled and 1 mM ATP halved the velocity observed for each enzyme with no effector present. Under these assay conditions AMP, IDP, GDP, and GTP had opposite effects on the reaction velocities for the two enzymes. When the assay conditions were changed, the effects of added purine nucleotides varied, even directionally. Addition of up to 5 mM glutamate (product) had no significant effect on C-GDH kinetics, nor on the substrate Km of M-GDH.(ABSTRACT TRUNCATED AT 250 WORDS)