RESUMO
OBJECTIVES AND METHODS: Previous studies have shown that CRK3 protein kinase of Leishmania mexicana is a potential drug target. Therefore, the aim of this study was to provide an active protein kinase for chemical inhibitors testing. A system was developed to express and affinity-purify recombinant L. mexicana CRK3 protein from Escherichia coli. RESULTS: Biochemical analysis has confirmed the expression of the pure kinase. The bacterial-expressed kinase was found to be inactive as a monomer. The mutated CRK3-E178 protein kinase was also found to be inactive. CONCLUSION: This study suggests that cyclin binding and phosphorylation status are both important for reconstituting protein kinase activity. Work presented by this paper has confirmed the usefulness of the prokaryotic system for production of pure homogenous recombinant protein kinase of Leishmania parasite, though this system is unable to produce active CRK3 protein kinase
Assuntos
Escherichia coli/genética , Leishmania mexicana/enzimologia , Proteínas Proto-Oncogênicas c-crk/genética , Proteínas Recombinantes/biossíntese , Immunoblotting , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-crk/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-crk/isolamento & purificação , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
We review modelling and experimental work dealing with the mechanisms of generation of electric image. We discuss: (1) the concept of electric image in the context of the reafference principle; (2) how waveform codes an impedance related qualia of the object image, referred to as "electric colour"; (3) that some characteristics of the spatial profiles generated by pre-receptor mechanisms are suitable for edge detection; (4) which parameters of the spatial profiles provide information for distance discrimination; (5) that electric images are distributed representations of the scene.
Assuntos
Peixe Elétrico/fisiologia , Órgão Elétrico/fisiologia , Modelos Biológicos , Animais , Estimulação Elétrica/métodos , EletrofisiologiaRESUMO
The Leishmania mexicana CRK3 gene encodes a cdc2-related protein kinase with activity towards histone H1. Attempts to disrupt both alleles of CRK3 in the promastigote life-cycle stage resulted in changes in cell ploidy, which were avoided only when an extra copy of CRK3 was expressed from an episome. This provides strong evidence that CRK3 is essential to L. mexicana. The cyclin-dependent kinase specific inhibitor flavopiridol inhibited affinity purified histidine tagged CRK3 (CRK3his) with an IC(50) value of 100 nM and inhibited in vitro growth of L. mexicana promastigotes. Incubation of promastigotes with 2.5 microM flavopiridol for 24 h led to cell cycle arrest with an accumulation of 95% of cells in G2 or early mitosis (G2/M). Release from cell cycle arrest resulted in a semi-synchronous re-entry into the cell cycle; samples taken at 2, 4, and 6 h after release from the block were enriched for cells in G1 (68%), S-phase (70%), and G2/M phase (61%), respectively. This method of synchronisation was used to show that the majority of CRK3his activity towards the substrate histone H1 was present at G2/M. These data suggest that CRK3 has an essential role in controlling cell cycle progression at the G2/M-phase transition in L. mexicana promastigotes.
Assuntos
Ciclo Celular/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Leishmania mexicana/citologia , Leishmania mexicana/enzimologia , Animais , Proteína Quinase CDC2 , Ciclo Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Regulação da Expressão Gênica , Leishmania mexicana/genética , Piperidinas/farmacologia , Proteínas de ProtozoáriosRESUMO
Biotinylated dextran amine was injected unilaterally into dorsal regions of the telencephalon of the weakly electric fish Gymnotus carapo in order to study the afferent and efferent connections of specific dorsal regions with ventral regions of the telencephalon and with other regions of the central nervous system. Efferent pathways from the dorsolateral area of the telencephalon project ipsilaterally to the anterior hypothalamic nucleus, the ventral thalamus and magnocellular tegmental nucleus, whose axons reach the spinal cord. Anterograde labeling showed that the central division of the dorsal telencephalon sends efferent projections through the lateral forebrain bundle towards the ipsilateral lateral and medial preglomerular nucleus, the pretectal nucleus, the optic tectum and the dorsal torus semicircularis, regions that are all involved in the processing of electrosensory and/or multisensory information. In addition, when biotinylated dextran amine was injected into the dorsal torus semicircularis, retrogradely labeled neurons were observed in the dorsocentral area of the telencephalon. The dorsocentral area is also a target of the extra-telencephalic afferents originating from rostral, lateral and medial regions of preglomerular complex. Within the telencephalon, neurons of many ventral subdivisions project ipsilaterally to the dorsocentral area. The dorsocentral, dorsolateral and dorsomedial areas are connected ipsilaterally and reciprocally. The dorsocentral area is reciprocally connected with its contralateral homologue through the anterior commissure.
Assuntos
Peixe Elétrico/anatomia & histologia , Células Receptoras Sensoriais/anatomia & histologia , Telencéfalo/anatomia & histologia , Vias Aferentes/anatomia & histologia , Animais , Mapeamento Encefálico , Dominância Cerebral/fisiologia , Vias Eferentes/anatomia & histologia , Neurônios/ultraestrutura , Medula Espinal/anatomia & histologiaRESUMO
The present study describes a measurement-based model of electric image generation in the weakly electric mormyrid fish Gnathonemus petersii. Measurements of skin impedance, internal resistivity and fish body dimensions have been used to generate an electrical-equivalent model of the fish and to calculate electrical images and equivalent dipole sources for elementary resistive objects. These calculations allow us to understand how exafferent and reafferent signals are sensed by electroreceptors. An object's electric image consists of the modulation of the transcutaneous voltage profile generated by the fish's own discharge. The results suggest a set of rules for electrolocation: (1) the side of the fish where modulation is larger indicates the side on which the object is situated; (2) the object's position in the electroreceptive field is indicated by the point of maximum modulation of the transcutaneous voltage; (3) the degree of focus of the image indicates the distance to the object. In addition, center-surround opposition originating at pre-receptor level is proposed. Both experimental measurements and modeling indicate that fish skin impedance is relatively low (400-11 000 
cm2) and mainly resistive. This low skin impedance appears to enhance the local electric organ discharge modulation, the center-surround effect, the signal-to-noise ratio for electrolocation and the active space for electrocommunication.
Assuntos
Peixe Elétrico/fisiologia , Modelos Biológicos , Desempenho Psicomotor/fisiologia , AnimaisRESUMO
A cdc2-related protein kinase gene, crk3, has been isolated from the parasitic protozoan Leishmania mexicana. Data presented here suggests that crk3 is a good candidate to be the leishmanial cdc2 homologue but that the parasite protein has some characteristics which distinguish it from mammalian cdc2. crk3 is predicted to encode a 35.6-kDa protein with 54% sequence identity with the human cyclin-dependent kinase cdc2 and 78% identity with the Trypanosoma brucei CRK3. The trypanosomatid CRK3 proteins have an unusual, poorly conserved 19-amino acid N-terminal extension not present in human cdc2. crk3 is single copy, and there is 5-fold higher mRNA in the replicative promastigote life-cycle stage than in the non-dividing metacyclic form or mammalian amastigote form. A leishmanial suc-binding cdc2-related kinase (SBCRK) histone H1 kinase, has previously been described which binds the yeast protein, p13(suc1), and that has stage-regulated activity (Mottram J. C., Kinnaird, J., Shiels, B. R., Tait, A., and Barry, J. D. (1993) J. Biol. Chem. 268, 21044-21051). CRK3 from cell extracts of the three life-cycle stages was found to bind p13(suc1) and the leishmanial homologue p12(cks1). CRK3 fused with six histidines at the C terminus was expressed in L. mexicana and shown to have SBCRK histone H1 kinase activity. Depletion of histidine-tagged CRK3 from L. mexicana cell extracts, by Ni-nitrilotriacetic acid agarose selection, reduced histone H1 kinase activity binding to p13(suc1). These data imply that crk3 encodes the kinase subunit of SBCRK. SBCRK and histidine-tagged CRK3 activities were inhibited by the purine analogue olomoucine with an IC50 of 28 and 42 microM, respectively, 5-6-fold higher than human p34(cdc2)/cyclinB.
Assuntos
Proteína Quinase CDC2/genética , Leishmania mexicana/genética , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/metabolismo , Inibidores Enzimáticos/farmacologia , Teste de Complementação Genética , Humanos , Cinetina , Dados de Sequência Molecular , Mutação , Ligação Proteica , Purinas/farmacologia , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
The generation of homozygous null mutants for the crk1 Cdc2-Related Kinase of Leishmania mexicana was attempted using targeted gene disruption. Promastigote mutants heterozygous for crk1 were readily isolated with a hyg-targeting fragment, but attempts to create null mutants by second-round transfections with a bie-targeting fragment yielded two classes of mutant, neither of which was null. First, the transfected fragment formed an episome; second, the cloned transfectants were found to contain wild-type crk1 alleles as well as hyg and ble integrations. DNA-content analysis revealed that these mutants were triploid or tetraploid. Plasticity in chromosome number following targeting has been proposed as a means by which Leishmania avoids deletion of essential genes. These data support this theory and implicate crk1 as an essential gene, validating CRK1 as a potential drug target. L mexicana transfected with a Trypanosoma brucel homologue, tbcrk1, was shown to be viable in an immcrk1 null background, thus showing complementation of function between these trypanosomatid genes. The expression of crk1 was further manipulated by engineering a six-histidine tag at the C-terminus of the kinase, allowing purification of the active complex by affinity selection on Nl(2+)-nitriloacetic acid (NTA) agarose.
Assuntos
Leishmania mexicana/genética , Mutagênese Insercional , Proteínas Quinases , Proteínas de Protozoários/genética , Deleção de Sequência , Alelos , Animais , Northern Blotting , Southern Blotting , Western Blotting , Caseínas/metabolismo , Mapeamento Cromossômico , DNA de Protozoário/análise , Regulação da Expressão Gênica , Teste de Complementação Genética , Histidina/genética , Dados de Sequência Molecular , Ácido Nitrilotriacético/análogos & derivados , Ácido Nitrilotriacético/metabolismo , Compostos Organometálicos/metabolismo , Plasmídeos/genética , Ploidias , Proteínas de Protozoários/metabolismo , RNA de Protozoário/genética , Transfecção , Trypanosoma brucei brucei/genéticaRESUMO
We have isolated a Leishmania mexicana homologue of the fission yeast suc1 gene using PCR with oligonucleotides designed to conserved regions of cdc2 kinase subunits (cks). The product of cks1 is a 12 kDa polypeptide, which has 70% identity with human p9cks1 and 44% identity with fission yeast p13suc1.p12cks1 was detected in the three life-cycle stages of L. mexicana by immunoblotting. Recombinant p12cks1 (p12cks1his) bound to agarose beads was used as a matrix to affinity-select histone H1 kinase complexes from Leishmania, yeast and bovine extracts. Immunoblotting showed that yeast and bovine cdc2 kinase bound to p12cks1his, thus demonstrating functional homology between L. mexicana p12cks1 and yeast p13suc1. Histone H1 kinase activity was found at a high level in the proliferative promastigote and amastigote forms of L. mexicana, but at a low level in the non-dividing metacyclic form. These activities are likely to be the same as the leishmanial p13suc1 binding kinase (SBCRK) described previously [Mottram, Kinnaird, Shiels, Tait and Barry (1993) J. Biol. Chem. 268, 21044-21051]. A distinct cdc2-related kinase, L. mexicana CRK1, was also found to associate with p12cks1his but affinity-depletion experiments showed that CRK1 was not responsible for the histone H1 kinase activity associating with p12cks1his in promastigote cell extracts. The finding that p12cks1 associates with at least two cdc2-related kinases, SBCRK and CRK1, is consistent with the presence of a large gene family of cdc2-related kinases in trypanosomatids, a situation thought to be more similar to higher eukaryotes than yeast.
Assuntos
Proteína Quinase CDC2/química , Proteínas de Ciclo Celular , Proteínas Fúngicas/química , Leishmania mexicana/enzimologia , Protamina Quinase/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas de Schizosaccharomyces pombe , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteína Quinase CDC2/biossíntese , Proteína Quinase CDC2/metabolismo , Bovinos , Cromatografia de Afinidade , Sequência Conservada , Primers do DNA , Humanos , Leishmania mexicana/crescimento & desenvolvimento , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Protamina Quinase/química , Proteínas de Protozoários/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/metabolismo , Homologia de Sequência de AminoácidosRESUMO
In over 3500 consecutive open heart procedures using Swan-Ganz catheterization at our institution, we have experienced three major pulmonary artery injuries secondary to this procedure. Pulmonary artery hemorrhage is a rare but frequently fatal complication and a mortality rate as high as fifty percent has been reported. In two of these cases, major retraction of the heart was needed for adequate exposure of the cardiac pathology. The Swan-Ganz catheter inadvertently was advanced into the wedge position for prolonged intervals of time, and periodic overdistention of the balloon occurred. The third case occurred in the cardiac catheterization laboratory. The need for aggressive surgical approach has been demonstrated. The authors have recommended steps to be taken when massive hemoptysis occurs and Swan-Ganz catheter perforation of the pulmonary artery is suspected. Re-evaluation of the "routine" use of the Swan-Ganz catheter may be necessary and overutilization may be a distinct possibility. When the use of this catheter is deemed appropriate, a more exact positioning of the distal portion of the catheter is mandatory if pulmonary artery perforation is to be avoided.