RESUMO
Nearly all of the known activities required for mitochondrial DNA (mtDNA) replication and expression are nuclear-encoded gene products, necessitating communication between these two physically distinct intracellular compartments. A significant amount of both general and specific biochemical information about mtDNA replication in mammalian cells has been known for almost two decades. Early studies achieved selective incorporation of the thymidine analog 5-Bromo-2-deoxy-Uridine (BrdU) into mtDNA of thymidine kinase-deficient (TK[-]) cells. We have revisited this approach from a cellular perspective to determine whether there exist spatiotemporal constraints on mtDNA replication. Laser-scanning confocal microscopy was used to selectively detect mtDNA synthesis in situ in cultured mammalian cells using an immunocytochemical double-labeling approach to visualize the incorporation of BrdU into mtDNA of dye-labeled mitochondria. In situ detection of BrdU-incorporated mtDNA was feasible after a minimum of 1-2 h treatment with BrdU, consistent with previous biochemical studies that determined the time required for completion of a round of mtDNA replication. Interestingly, the pattern of BrdU incorporation into the mtDNA of cultured mammalian cells consistently radiated outward from a perinuclear position, suggesting that mtDNA replication first occurs in the vicinity of nuclear-provided materials. Newly replicated mtDNA then appears to rapidly distribute throughout the dynamic cellular mitochondrial network.
Assuntos
Replicação do DNA/fisiologia , DNA Mitocondrial/análise , Animais , Anticorpos Antinucleares , Plaquetas/citologia , Plaquetas/fisiologia , Plaquetas/ultraestrutura , Bromodesoxiuridina , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , DNA Mitocondrial/biossíntese , DNA Mitocondrial/imunologia , Imunofluorescência , Genoma , Células HeLa/citologia , Células HeLa/fisiologia , Células HeLa/ultraestrutura , Humanos , Mamíferos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Osteossarcoma , Células PC12/citologia , Células PC12/fisiologia , Células PC12/ultraestrutura , RatosRESUMO
The synaptic vesicle protein synaptotagmin I binds Ca2+ and is required for efficient neurotransmitter release. Here, we measure the response time of the C2 domains of synaptotagmin to determine whether synaptotagmin is fast enough to function as a Ca2+ sensor for rapid exocytosis. We report that synaptotagmin is "tuned" to sense Ca2+ concentrations that trigger neuronal exocytosis. The speed of response is unique to synaptotagmin I and readily satisfies the kinetic constraints of synaptic vesicle membrane fusion. We further demonstrate that Ca2+ triggers penetration of synaptotagmin into membranes and simultaneously drives assembly of synaptotagmin onto the base of the ternary SNARE (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment receptor) complex, near the transmembrane anchor of syntaxin. These data support a molecular model in which synaptotagmin triggers exocytosis through its interactions with membranes and the SNARE complex.
Assuntos
Proteínas de Ligação ao Cálcio , Cálcio/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular , Animais , Cinética , Fusão de Membrana/fisiologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Membranas/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Ratos , Sequências Repetitivas de Ácido Nucleico , Proteínas SNARE , Homologia de Sequência , Sinaptotagmina I , SinaptotagminasRESUMO
GABAA receptors, along with the receptors for acetylcholine, glycine, and serotonin, are members of a ligand-gated ion channel superfamily (Ortells and Lunt, 1995). Because of the paucity of crystallographic information for these ligand-gated channels, little is known about the structure of their binding sites or how agonist binding is transduced into channel gating. We used the substituted cysteine accessibility method to obtain secondary structural information about the GABA binding site and to systematically identify residues that line its surface. Each residue from alpha1 Y59 to K70 was mutated to cysteine and expressed with wild-type beta2 subunits in Xenopus oocytes or HEK 293 cells. The sulfhydryl-specific reagent N-biotinylaminoethyl methanethiosulfonate (MTSEA-Biotin) was used to covalently modify the cysteine-substituted residues. Receptors with cysteines substituted at positions alpha1 T60, D62, F64, R66, and S68 reacted with MTSEA-Biotin, and alpha1 F64C, R66C, and S68C were protected from reaction by agonist. We conclude that alpha1 F64, R66, and S68 line part of the GABA binding site. The alternating pattern of accessibility of consecutive engineered cysteines to reaction with MTSEA-Biotin indicates that the region from alpha1 Y59 to S68 is a beta-strand.
Assuntos
Mapeamento Cromossômico , Receptores de GABA-A , Animais , Sítios de Ligação/fisiologia , Biotina , Cisteína , Relação Dose-Resposta a Droga , Metanossulfonato de Etila/análogos & derivados , Agonistas GABAérgicos/farmacologia , Humanos , Indicadores e Reagentes , Rim/citologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Muscimol/farmacologia , Mutagênese Sítio-Dirigida/fisiologia , Oócitos/fisiologia , Estrutura Terciária de Proteína , Receptores de GABA-A/química , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Homologia de Sequência de Aminoácidos , Trítio , Xenopus , Ácido gama-Aminobutírico/farmacologiaRESUMO
Topical aciclovir cream (ACV, Zovirax Cream) containing 40% propylene glycol (PG), the optimum found for skin penetration, is clinically effective in the treatment of recurrent herpes labialis. One hundred and thirty-nine ACV generic creams were analysed and 80% of these contained less than 20% PG. From this, we hypothesised that these generics might be bioinequivalent to the innovator cream. A pilot in vitro skin permeation study compared the innovator cream with two generics containing about 15% PG. Next, 10 generics containing 0-15% PG were tested in an independent laboratory. Finally, a PG dose-ranging study was conducted in Zovirax cream base. In all studies, human skin was used and ACV analysed by LC-MS-MS. In the pilot study, the innovator cream delivered 7.5-fold more ACV than the two generics. Superiority was confirmed in the second study against all 10 ACV generic creams. By grouping the creams according to PG content, a relationship to ACV skin permeation was suggested. The PG dose effect was confirmed in the third study. These studies suggest that not all marketed ACV creams are bioequivalent to the clinically proven innovator. Given the magnitude of the differences seen, there is concern over therapeutic inequivalence of generic ACV creams to the innovator cream.
Assuntos
Aciclovir/farmacocinética , Medicamentos Genéricos/farmacocinética , Pele/metabolismo , Aciclovir/química , Cromatografia Líquida , Cultura em Câmaras de Difusão , Medicamentos Genéricos/química , Excipientes/química , Humanos , Técnicas In Vitro , Espectrometria de Massas , Pomadas , Projetos Piloto , Polietilenoglicóis/química , Pele/efeitos dos fármacos , Absorção Cutânea/efeitos dos fármacos , Equivalência TerapêuticaRESUMO
RNase MRP is a ribonucleoprotein originally identified on the basis of its ability to cleave RNA endonucleolytically from origins of mitochondrial DNA replication, rendering it a likely candidate for a role in priming leading-strand synthesis of mtDNA. In addition, a nuclear role for RNase MRP has been identified in yeast (Saccharomyces cerevisiae) ribosomal RNA processing. Consistent with a duality of function, RNase MRP has been localized to both mitochondria and nucleoli by in situ techniques. The RNA component of this ribonucleoprotein has been characterized from several different species. We previously cloned the gene for Xenopus laevis MRP RNA and showed that RNase MRP RNA is differentially expressed during amphibian development; in addition, the microinjected X. laevis RNase MRP RNA gene is correctly and efficiently transcribed in vivo. This article presents an analysis of the intracellular movement of in vivo-transcribed RNase MRP RNA in microinjected mature X. laevis oocytes. Although X. laevis MRP RNA is assembled into a ribonucleoprotein form and transported in an expected manner, human and mouse MRP RNAs exhibit markedly different transport patterns even though they are highly conserved in primary sequence. Furthermore, the only currently assigned protein (Th autoantigen) binding site in MRP RNA can be deleted without loss of nuclear export capacity. These results indicate that subtle determinants must exist for nucleocytoplasmic partitioning of this RNP and that the conserved Th autoantigen binding region appears unnecessary for the transit of in vivo-transcribed MRP RNA to the cytoplasm of mature X. laevis oocytes.
Assuntos
Endorribonucleases/genética , Transcrição Gênica , Animais , Autoantígenos , Sequência de Bases , Sítios de Ligação , Primers do DNA , Camundongos , Dados de Sequência Molecular , Oócitos , Regiões Promotoras Genéticas , RNA , Xenopus laevisRESUMO
We hypothesised that the depletion of propylene glycol from topical formulations applied at clinically relevant doses (approximately mg/cm2) would limit its penetration enhancement effect. The in vitro percutaneous permeation of a model drug-loperamide hydrochloride-in formulations containing propylene glycol was therefore investigated under finite dose conditions. The flux of loperamide and propylene glycol across dermatomed human skin was measured. The first study examined the effect of topical loading of a gel containing 12% propylene glycol. The second study investigated the effect of propylene glycol content in creams containing 15 and 40%. Both studies showed a correlation between the amount of propylene glycol dosed on the skin and the amount of drug that had permeated. The substantial permeation of propylene glycol and relatively small permeation of loperamide, strongly suggests, that the time dependent permeation of the drug was due to the depletion of propylene glycol at the skin surface and not to the depletion of the drug itself. As often doses applied in in vitro skin permeation experiments do not match the intended clinical dosage-they are usually much greater-this study suggests that the penetration enhancement effect of propylene glycol can be overestimated in in vitro studies.
Assuntos
Loperamida/administração & dosagem , Propilenoglicol/administração & dosagem , Pele/efeitos dos fármacos , Administração Cutânea , Cultura em Câmaras de Difusão , Sinergismo Farmacológico , Feminino , Humanos , Loperamida/farmacocinética , Pessoa de Meia-Idade , Permeabilidade/efeitos dos fármacos , Propilenoglicol/farmacocinética , Pele/metabolismoRESUMO
The influence of hydroxypropyl methylcellulose (HPMC), methylcellulose (MC), polyvinyl pyrrolidone (PVP) and polyethylene glycol (PEG400) on the crystallization of hydrocortisone acetate (HA) was studied. Supersaturation was created by the cosolvent technique. Spontaneous nucleation was observed when no polymer was used as the additive. In the presence of the polymer, nucleation was delayed. The nucleation time decreased with increasing supersaturation at a particular polymer concentration and increased with increasing polymer concentration at a particular supersaturation. Habit modification from a well-defined polar prismatic morphology to a wing-shaped morphology was observed when HPMC was used as the additive. The effect of PVP and PEG400 on the morphology of HA was less pronounced compared to the cellulose polymers. The mechanism of nucleation retardation by the polymers is explained in terms of association of HA with the polymer through hydrogen bonding. The growth may be inhibited by the hydrodynamic boundary layer, in which the polymers accumulate as well as by the adsorption of the polymer onto the crystal surface. The habit modification of HA by HPMC is due to different extents of adsorption on different faces of the crystal, the extent of which is dependent on the hydrogen bonding functional groups that are exposed at each face of the crystal.
Assuntos
Hidrocortisona/análogos & derivados , Hidrocortisona/química , Lactose/análogos & derivados , Metilcelulose/análogos & derivados , Cristalização , Géis , Ligação de Hidrogênio , Lactose/farmacologia , Metilcelulose/farmacologia , Oxazinas , Polietilenoglicóis/farmacologia , Povidona/farmacologiaRESUMO
A systematic investigation on the influence of two cellulose polymers, methyl cellulose (MC) and hydroxypropyl cellulose (HPMC) on supersaturation and permeation of hydrocortisone acetate (HA) is reported. Diffusion of HA from a 0.5% Carbopol gel across a model silicone membrane was investigated using the Franz-cell technique. At constant polymer concentration, the flux increases proportionally with the degree of saturation up to 4.8x but decreases thereafter. For a particular degree of supersaturation (4.8x), the flux increases with the concentration of polymer up to 1% and decreases at higher concentrations. The behaviour is found to be consistent with crystallisation experiments. The results suggest that optimisation of supersaturation and polymer content is necessary to achieve both high permeation rates and inherent stability.
Assuntos
Celulose/análogos & derivados , Hidrocortisona/análogos & derivados , Metilcelulose/química , Celulose/química , Cromatografia Líquida de Alta Pressão , Difusão , Géis , Hidrocortisona/química , Membranas Artificiais , Permeabilidade , Silicones , Solubilidade , SolventesRESUMO
Permeation of hydrocortisone acetate (HA) from supersaturated solutions was studied across a model silicone membrane. Supersaturated solutions were prepared using the cosolvent technique with propylene glycol and water (or aqueous polymer solutions) as the cosolvents. In the absence of the polymer, the flux of HA was similar at all degrees of saturation and was not significantly different from the value obtained for a saturated solution. Flux enhancement, as a result of supersaturation, was observed with all the polymers. The flux increased with increasing polymer concentration, reached a maximum and decreased at higher polymer percentages. The amount of polymer required for maximum enhancement differed for each polymer. The decrease of flux at high polymer concentrations is attributed to changes in microviscosity and a marginal increase in solubility. The infrared spectroscopic and differential scanning calorimetry data suggest that HA-polymer interactions occurred through hydrogen bonding thus explaining the proposed mechanism of the anti-nucleant properties of the polymers.
Assuntos
Hidrocortisona/análogos & derivados , Hidrocortisona/farmacocinética , Membranas Artificiais , Polímeros , Tecnologia Farmacêutica , Transporte Biológico , Varredura Diferencial de CalorimetriaRESUMO
Isothermal microcalorimetry may be used to determine kinetic and thermodynamic parameters for chemical reactions. This paper reports rate constants, determined as a function of temperature, and the activation enthalpy for the degradation of solid benzoyl peroxide as determined by isothermal microcalorimetry. Studies were conducted on aqueous suspension phase, solid benzoyl peroxide. In addition, supporting evidence is cited from work carried out in this laboratory on the solution phase degradation of benzoyl peroxide using UV-visible spectrophotometry. The activation energy obtained by microcalorimetry was E(a)=137.8+/-6.6 kJ mol(-1) and the activation energy obtained from UV-visible spectrophotometry was E(a)=112.7+/-4.2 kJ mol(-1).
Assuntos
Peróxido de Benzoíla , Calorimetria/métodos , Estabilidade de Medicamentos , CinéticaRESUMO
Recent developments in the analysis of microcalorimetric data output allow the possibility of determining both thermodynamic and kinetic parameters for complex reaction systems. Such experiments routinely take around 50 h, hence qualifying for the description rapid. The methods have earlier been applied to a study of the stability of benzoyl peroxide itself in aqueous suspension. This paper reports the results of isothermal microcalorimetric study of the stability of benzoyl peroxide in the presence of a wide range of excipients and in formulated materials. The results are shown to assist in formulation design, are achieved rapidly and are derived from direct experimental study of the complex systems themselves. That is, no ancillary information is required nor are the studies invasive or destructive.
Assuntos
Peróxido de Benzoíla/química , Calorimetria/métodos , Química Farmacêutica/métodos , Estabilidade de Medicamentos , CinéticaRESUMO
A novel diffusion cell has been constructed which allows study of membrane diffusion processes without the need for sampling of the receiver compartment, that is highly sensitive and, being based around a diode array spectrophotometer also allows for continuous, real-time recording of multi-species concentration changes in the receiving compartment. The system is controlled to operate isothermally (via a Peltier control system) at temperatures between 15 and 85 degrees C. To examine the performance of this novel design, the transfer of tetracaine from a preparation in PEG 400 (20% tetracaine in PEG 400) has been studied. The results have been used to determine flux, lag time and related parameters. The performance of the novel cell is compared with results from traditional Franz cell diffusion studies.
Assuntos
Membrana Celular/metabolismo , Algoritmos , Anestésicos Locais/química , Transporte Biológico , Membrana Celular/química , Difusão , Cultura em Câmaras de Difusão , Cinética , Politetrafluoretileno , Espectrofotometria Ultravioleta , Tetracaína/químicaRESUMO
The solubility of penciclovir (C(10)N(5)O(3)H(17)) in a novel film formulation designed for the treatment of cold sores was determined using X-ray, thermal, microscopic and release rate techniques. Solubilities of 0.15-0.23, 0.44, 0.53 and 0.42% (w/w) resulted for each procedure. Linear calibration lines were achieved for experimentally and theoretically determined differential scanning calorimetry (DSC) and X-ray powder diffractometry (XRPD) data. Intra- and inter-batch data precision values were determined; intra values were more precise. Microscopy was additionally useful for examining crystal shape, size distribution and homogeneity of drug distribution within the film. Whereas DSC also determined melting point, XRPD identified polymorphs and release data provided relevant kinetics.
Assuntos
Aciclovir/análise , Aciclovir/farmacocinética , Ácidos Polimetacrílicos/análise , Ácidos Polimetacrílicos/farmacocinética , Aciclovir/análogos & derivados , Aciclovir/química , Varredura Diferencial de Calorimetria/métodos , Guanina , Microscopia Confocal/métodos , Ácidos Polimetacrílicos/química , Solubilidade , Difração de Raios X/métodosAssuntos
Terapia Aversiva/métodos , Eletrochoque , Imaginação , Psicoterapia de Grupo , Feminino , Humanos , Masculino , Hábito de Roer Unhas/terapiaRESUMO
Synaptotagmin 1 binds Ca2+ and membranes via its C2A-domain and plays an essential role in excitation-secretion coupling. In this study, we sought to identify Ca2+- and membrane-induced local conformational changes in the C2A-domain of synaptotagmin and to delineate the C2A-lipid binding interface. To address these questions native phenylalanine residues were replaced, at each face of the domain, with tryptophan reporters. Changes in tryptophanyl fluorescence indicated that Ca2+ induced long range conformational changes throughout C2A, including regions distant from an established Ca2+-binding site. Addition of liposomes resulted in Ca2+-dependent increases in the fluorescence of tryptophans 193, 231, and 234. Only the tryptophan residues at positions 234 and 231, which lie within a Ca2+-binding loop of C2A, exhibited liposome-induced blue shifts in their emission spectra. Quenching experiments, using membrane-imbedded doxyl spin labels, revealed that tryptophan residues 231 and 234 penetrated lipid bilayers. These data delineate the lipid binding interface of C2A and provide the first evidence for adjacent Ca2+- and lipid-binding sites within a C2-domain. The penetration of C2A into membranes may function to bring components of the fusion machinery into contact with the lipid bilayer to initiate exocytosis.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Bicamadas Lipídicas , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Fusão de Membrana , Glicoproteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/genética , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sinaptotagmina I , SinaptotagminasRESUMO
RNase MRP is a ribonucleoprotein endoribonuclease found predominantly in nucleoli, but which has been associated with mitochondria and mitochondrial RNA processing. In order to analyze the intracellular localization of specific RNA components of ribonucleoproteins of this type, a whole-mount method for in situ hybridization in Xenopus laevis oocytes was employed. Results with specific probes (for both mitochondrial and nonmitochondrial RNAs) indicate that this procedure is generally effective for the detection of a variety of nucleic acids that reside in different cellular compartments. Probes used to detect the endogenous RNA component of RNase MRP (MRP RNA) during X. laevis oogenesis revealed a continuous nuclear signal as well as a possible dual localization of MRP RNA in nucleoli and mitochondria at developmental stages temporally consistent with both ribosomal and mitochondrial biogenesis. Genomic DNA encoding MRP RNA was injected into the nuclei of stage VI oocytes and correctly transcribed. The in vivo-transcribed RNA was properly assembled with at least some of its cognate proteins as demonstrated by immunoprecipitation with specific autoantiserum. In addition, detectable levels of the RNA were exported to the cytoplasm. This whole-mount procedure has permitted us to identify MRP RNA in situ at different developmental time points as well as during transcription of the injected gene, and suggests differential localization of MRP RNA during oogenesis consistent with its proposed function in both mitochondria and nucleoli.
Assuntos
Endorribonucleases/genética , Oogênese/fisiologia , RNA/metabolismo , Ribonucleoproteínas , Animais , Endorribonucleases/metabolismo , Feminino , Hibridização In Situ , Ribonucleoproteínas/metabolismo , Transcrição Gênica , Xenopus laevisRESUMO
PO-B was originally characterized as a transcriptional regulatory factor of the pro-opiomelanocortin (POMC) gene; however, it has become increasingly clear that this protein may be active in tissues outside the pituitary, since it is present in diverse cell types, including differentiated HL-60 promyelocytic leukemia cells. We previously showed that PO-B DNA-binding is progressively induced during differentiation of promyelomonocytic leukemic HL-60 cells to the macrophage-like lineage (with phorbol esters). We now report that PO-B DNA-binding in HL-60 cells is similarly induced during differentiation to the granulocytic lineage (with either retinoic acid or dimethylsulfoxide). Either a genetic or pharmacologic blockade of HL-60 differentiation prohibited these inductive effects. These studies have prompted our interest in the dynamics of other transcription factor changes during HL-60 differentiation. Of these, we observed that another transcription factor (AP-1) is also robustly induced at the DNA-binding level during macrophage-like HL-60 differentiation, but not during granulocytic differentiation. Conversely, the DNA-binding of the transcription factor AP-2 was slightly reduced by TPA-induced HL-60 differentiation but unchanged during granulocyte differentiation. From these data, we conclude that the induction of PO-B DNA binding is a general marker of HL-60 myelomonocytic differentiation, but that qualitative aspects of the induction of additional distinct transcription factors, such as AP-1, may contribute to lineage-specific determinants of cell fate.
Assuntos
Diferenciação Celular , DNA/metabolismo , Lactonas/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Transcrição/metabolismo , Tretinoína/farmacologia , Antineoplásicos/farmacologia , Briostatinas , Diferenciação Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Dimetil Sulfóxido/farmacologia , Granulócitos , Células HL-60 , Humanos , Macrolídeos , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição AP-2RESUMO
Biochemical and genetic studies indicate that synaptotagmin I functions as a Ca2+ sensor during synaptic vesicle exocytosis and as a membrane receptor for the clathrin adaptor complex, AP-2, during endocytosis. These functions involve the interaction of two conserved domains, C2A and C2B, with effector proteins. The C2B domain mediates Ca2+-triggered synaptotagmin oligomerization, binds AP-2 and is important for the interaction of synaptotagmin with Ca2+ channels. Here, we report that these are conserved biochemical properties: Ca2+ promoted the hetero-oligomerization of synaptotagmin I with synaptotagmins III and IV, and all three synaptotagmin isoforms bound the synprint region of the alpha1B subunit of N-type Ca2+ channels. Using chimeric and truncated C2 domains, we defined a common region of C2B that mediates oligomerization and AP-2 binding. Within this region, two adjacent lysine residues were identified that were critical for synaptotagmin oligomerization, AP-2, and synprint binding. Competition experiments demonstrated that the synprint fragment was an effective inhibitor of synaptotagmin oligomerization and also blocked binding of synaptotagmin to AP-2. In a model for the structure of C2B, the common effector binding site localized to a putative Ca2+-binding loop and a concave region formed by two beta-strands. These studies provide the first structural information regarding C2B target protein recognition and provide the means to selectively disrupt synaptotagmin-effector interactions for functional studies.
Assuntos
Proteínas de Ligação ao Cálcio , Proteínas de Ligação a DNA/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Biopolímeros , Glicoproteínas de Membrana/química , Modelos Moleculares , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Ligação Proteica , Ratos , Homologia de Sequência de Aminoácidos , Sinaptotagmina I , Sinaptotagminas , Fator de Transcrição AP-2RESUMO
Mitochondria are essential organelles in all eukaryotic cells where cellular ATP is generated through the process of oxidative phosphorylation. Protein components of the respiratory assembly are gene products of both mitochondrial and nuclear genes. The mitochondrial genome itself encodes several protein and nucleic acid components required for such oxidative phosphorylative processes, but the vast majority of genes encoding respiratory chain components are nuclear. Similarly, the processes of replication and transcription of mitochondrial DNA rely exclusively upon RNA and protein species encoded by nuclear genes. We have analyzed two key nuclear-encoded proteins involved in mitochondrial DNA replication and transcription as a function of the presence or absence of mitochondrial DNA. Mitochondrial DNA polymerase (DNA polymerase gamma), the nuclear-encoded enzyme which synthesizes mtDNA, is expressed and translated in cells devoid of mitochondrial DNA itself. In contrast, mitochondrial transcription factor A protein levels are tightly linked to the mtDNA status of the cell. These results demonstrate that the DNA polymerase gamma protein is stable in the absence of mitochondrial DNA, and that there appears to be no regulatory mechanism present in these cells to alter levels of this protein in the complete absence of mitochondrial DNA. Alternatively, it is possible that this enzyme plays an additional, as yet undefined, role in the cell, thereby mandating its continued production.