RESUMEN
Identification of all the protein components of the large subunit (39 S) of the mammalian mitochondrial ribosome has been achieved by carrying out proteolytic digestions of whole 39 S subunits followed by analysis of the resultant peptides by liquid chromatography and mass spectrometry. Peptide sequence information was used to search the human EST data bases and complete coding sequences were assembled. The human mitochondrial 39 S subunit has 48 distinct proteins. Twenty eight of these are homologs of the Escherichia coli 50 S ribosomal proteins L1, L2, L3, L4, L7/L12, L9, L10, L11, L13, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, L27, L28, L30, L32, L33, L34, L35, and L36. Almost all of these proteins have homologs in Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae mitochondrial ribosomes. No mitochondrial homologs to prokaryotic ribosomal proteins L5, L6, L25, L29, and L31 could be found either in the peptides obtained or by analysis of the available data bases. The remaining 20 proteins present in the 39 S subunits are specific to mitochondrial ribosomes. Proteins in this group have no apparent homologs in bacterial, chloroplast, archaebacterial, or cytosolic ribosomes. All but two of the proteins has a clear homolog in D. melanogaster while all can be found in the genome of C. elegans. Ten of the 20 mitochondrial specific 39 S proteins have homologs in S. cerevisiae. Homologs of 2 of these new classes of ribosomal proteins could be identified in the Arabidopsis thaliana genome.
Asunto(s)
Mitocondrias Hepáticas/química , Proteínas Ribosómicas/química , Ribosomas/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Electroforesis en Gel Bidimensional , Espectrometría de Masas , Datos de Secuencia Molecular , Peso Molecular , Conformación de Ácido Nucleico , Mapeo Peptídico , ARN Ribosómico/química , Homología de Secuencia de AminoácidoRESUMEN
Articular cartilage contains four distinct zones, extending from the surface to the subchondral bone. Freshly isolated chondrocytes from the superficial zone of articular cartilage retain a collagenase-P-resistant cell-associated matrix. In the studies described here, the protein Del1 was identified as a component of the cell-associated matrix of superficial zone chondrocytes from adult bovine articular cartilage. Very little Del1 was associated with freshly isolated deep zone chondrocytes. Western blot analysis of articular cartilage cell and tissue extracts using polyclonal antibodies specific for Del1 showed Del1 was present in an insoluble cell-associated fraction. Extracts of the superficial zone of articular cartilage were found to be enriched in Del1 compared to the deeper layers of the tissue. Immunohistochemical staining of full-thickness articular cartilage with anti-Del1 antibodies also showed an enrichment of Del1 in the superficial zone. These observations are the first to describe the protein Del1 in a nonendothelial, nonfetal tissue.
Asunto(s)
Proteínas Portadoras/análisis , Cartílago Articular/química , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/inmunología , Bovinos , Células Cultivadas , Condrocitos/química , Inmunohistoquímica , Datos de Secuencia Molecular , Péptidos/análisis , Extractos de Tejidos/químicaRESUMEN
In this report we describe the purification of human superficial zone protein (SZP), the generation of cross-species monoclonal antibodies (MAbs) and the detection of this protein in human and animal body fluids. Human SZPs, used as immunizing antigens, were purified either from culture media of human cartilage organ cultures or from human synovial fluids. The immunizing antigens were mixed with RIBI adjuvant in one of three forms: nonmodified SZP, superficial zone protein-keyhole limpet hemocyanin conjugate (SZP-KLH), or a mixture of superficial zone protein and hyaluronic acid (SZP-HA). A panel of MAbs including GW4.23, S6.79, S13.52, S13.233, and S17.109 were generated and characterized. Monoclonal antibody (MAb) S6.79, an IgG2b with K(D) 3.14 x 10(-9) M from SZP-KLH immunization, is of particular interest. It reacts strongly to a large molecular weight form of SZP in both enzyme-linked immunosorbent assay (ELISA) and Western blotting. It stains the most superficial layer of articular cartilage in immunohistochemistry, whereas the middle and deep zones of cartilage are not stained. When MAb S6.79 was applied to Western blots of human body fluids, a strong 345-kDa band was detected in samples of synovial fluid and weaker bands of similar size were detected in samples of plasma and serum. MAb S6.79 also showed cross-species immunoreactivity with SZP in samples of synovial fluids harvested from bovine, dog, guinea pig, and rabbit, as demonstrated by Western blotting and antibody absorption experiments. This cross-species MAb will be a useful tool in human and animal model studies for monitoring SZP levels and tissue distribution. It may help define the roles of SZP in normal articular joints and may be of diagnostic or prognostic value for the measurement of SZP in pathological conditions such as osteoarthritis, rheumatoid arthritis, and camptodactyly-arthropathy-coxa vara-pericarditis.
Asunto(s)
Líquidos Corporales/metabolismo , Proteoglicanos/análisis , Animales , Anticuerpos Monoclonales/inmunología , Líquidos Corporales/inmunología , Bovinos , Reacciones Cruzadas , Perros , Humanos , Inmunoensayo , Proteoglicanos/inmunología , Especificidad de la EspecieRESUMEN
Proteins in the small subunit of the mammalian mitochondrial ribosome were separated by two-dimensional polyacrylamide gel electrophoresis. Four individual proteins were subjected to in-gel Endoprotease Lys-C digestion. The sequences of selected proteolytic peptides were obtained by electrospray tandem mass spectrometry. Peptide sequences obtained from in-gel digestion of individual spots were used to screen human, mouse, and rat expressed sequence tag databases, and complete consensus cDNAs for these species were deduced in silico. The corresponding protein sequences were characterized by comparison to known ribosomal proteins in protein databases. Four different classes of mammalian mitochondrial small subunit ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins are homologs to Escherichia coli S9 and S5 proteins. The presence of these newly identified mitochondrial ribosomal proteins are also investigated in the Drosophila melanogaster, Caenorhabditis elegans, and in the genomes of several fungi.
Asunto(s)
ADN Complementario/genética , Mitocondrias/química , Proteoma/química , Proteínas Ribosómicas/química , Proteínas Ribosómicas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans , Bovinos , Drosophila melanogaster , Escherichia coli , Hongos , Humanos , Espectrometría de Masas , Mitocondrias/ultraestructura , Datos de Secuencia Molecular , Subunidades de Proteína , Proteoma/metabolismo , Especificidad de la EspecieRESUMEN
Identification of all the protein components of the small subunit (28 S) of the mammalian mitochondrial ribosome has been achieved by carrying out proteolytic digestions of whole 28 S subunits followed by analysis of the resultant peptides by liquid chromatography and tandem mass spectrometry (LC/MS/MS). Peptide sequence information was used to search the human EST data bases and complete coding sequences of the proteins were assembled. The human mitochondrial ribosome has 29 distinct proteins in the small subunit. Fourteen of this group of proteins are homologs of the Escherichia coli 30 S ribosomal proteins S2, S5, S6, S7, S9, S10, S11, S12, S14, S15, S16, S17, S18, and S21. All of these proteins have homologs in Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae mitochondrial ribosomes. Surprisingly, three variants of ribosomal protein S18 are found in the mammalian and D. melanogaster mitochondrial ribosomes while C. elegans has two S18 homologs. The S18 homologs tend to be more closely related to chloroplast S18s than to prokaryotic S18s. No mitochondrial homologs to prokaryotic ribosomal proteins S1, S3, S4, S8, S13, S19, and S20 could be found in the peptides obtained from the whole 28 S subunit digests or by analysis of the available data bases. The remaining 15 proteins present in mammalian mitochondrial 28 S subunits (MRP-S22 through MRP-S36) are specific to mitochondrial ribosomes. Proteins in this group have no apparent homologs in bacterial, chloroplast, archaebacterial, or cytosolic ribosomes. All but two of these proteins have a clear homolog in D. melanogaster while all but three can be found in the genome of C. elegans. Five of the mitochondrial specific ribosomal proteins have homologs in S. cerevisiae.
Asunto(s)
Mitocondrias/química , Ribosomas/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans , Bovinos , Secuencia Conservada , Cristalografía por Rayos X , Bases de Datos Factuales , Drosophila melanogaster , Electroforesis en Gel Bidimensional , Escherichia coli/metabolismo , Etiquetas de Secuencia Expresada , Cromatografía de Gases y Espectrometría de Masas , Genoma , Humanos , Lisina/química , Espectrometría de Masas , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Péptidos/química , ARN Mensajero/química , Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido , Programas Informáticos , Thermus thermophilusRESUMEN
Two proteins known to be involved in promoting apoptosis in mammalian cells have been identified as components of the mammalian mitochondrial ribosome. Proteolytic digestion of whole mitochondrial ribosomal subunits followed by analysis of the peptides present using liquid chromatography-tandem mass spectrometry revealed that the proapoptotic proteins, death-associated protein 3 (DAP3) and the programmed cell death protein 9, are both components of the mitochondrial ribosome. DAP3 has motifs characteristic of guanine nucleotide binding proteins and is probably the protein that accounts for the nucleotide binding activity of mammalian mitochondrial ribosomes. The observations reported here implicate mitochondrial protein synthesis as a major component in cellular apoptotic signaling pathways.
Asunto(s)
Apoptosis , Proteínas de Ciclo Celular/metabolismo , Mitocondrias/metabolismo , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Bovinos , Proteínas de Ciclo Celular/química , Humanos , Técnicas In Vitro , Espectrometría de Masas , Mitocondrias/fisiología , Datos de Secuencia Molecular , Prenilación de Proteína , Proteínas/química , Proteínas de Unión al ARN , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Mammalian mitochondrial small subunit ribosomal proteins were separated by two-dimensional polyacrylamide gel electrophoresis. The proteins in six individual spots were subjected to in-gel tryptic digestion. Peptides were separated by capillary liquid chromatography, and the sequences of selected peptides were obtained by electrospray tandem mass spectrometry. The peptide sequences obtained were used to screen human expressed sequence tag data bases, and complete consensus cDNAs were assembled. Mammalian mitochondrial small subunit ribosomal proteins from six different classes of ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins correspond to Escherichia coli S10 and S14. Homologs of two human mitochondrial proteins not found in prokaryotes were observed in the genomes of Drosophila melanogaster and Caenorhabditis elegans. A homolog of one of these proteins was observed in D. melanogaster but not in C. elegans, while a homolog of the other was present in C. elegans but not in D. melanogaster. A homolog of one of the ribosomal proteins not found in prokaryotes was tentatively identified in the yeast genome. This latter protein is the first reported example of a ribosomal protein that is shared by mitochondrial ribosomes from lower and higher eukaryotes that does not have a homolog in prokaryotes.
Asunto(s)
Proteínas de Drosophila , Mitocondrias/metabolismo , Proteoma/química , Proteínas Ribosómicas/química , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans , Cromatografía Liquida , Secuencia de Consenso , ADN Complementario , Drosophila , Escherichia coli/metabolismo , Humanos , Mamíferos , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Proteoma/genética , Proteoma/aislamiento & purificación , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/aislamiento & purificación , Saccharomyces cerevisiae , Alineación de Secuencia , Homología de Secuencia de Aminoácido , TripsinaRESUMEN
Bovine mitochondrial small subunit ribosomal proteins were separated by two-dimensional electrophoresis. The region containing the most basic protein(s) was excised and the protein(s) present subjected to in-gel digestion with trypsin. Electrospray tandem mass spectrometry was used to provide sequence information on some of the peptide products. Searches of the human EST database using the sequence of the longest peptide analyzed indicated that this peptide was from the mammalian mitochondrial homolog of prokaryotic ribosomal protein S7 (MRP S7(human)). MRP S7(human) is a 28-kDa protein with a pI of 10. Significant homology to bacterial S7 is observed especially in the C-terminal half of the protein. Surprisingly, MRP S7(human) shows less homology to the corresponding mitochondrial proteins from plants and fungi than to bacterial S7.
Asunto(s)
Mitocondrias/química , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Bovinos , ADN Complementario/genética , Bases de Datos Factuales , Etiquetas de Secuencia Expresada , Humanos , Punto Isoeléctrico , Espectrometría de Masas , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , ARN Ribosómico 16S/química , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , Proteínas Ribosómicas/metabolismo , Alineación de Secuencia , Análisis de Secuencia de Proteína , Homología de Secuencia de AminoácidoRESUMEN
The tumor necrosis factor-alpha-converting enzyme (TACE) is a membrane-anchored zinc metalloprotease involved in precursor tumor necrosis factor-alpha secretion. We designed a series of constructs containing full-length human TACE and several truncate forms for overexpression in insect cells. Here, we demonstrate that full-length TACE is expressed in insect cells inefficiently: only minor amounts of this enzyme are converted from an inactive precursor to the mature, functional form. Removal of the cytoplasmic and transmembrane domains resulted in the efficient secretion of mature, active TACE. Further removal of the cysteine-rich domain located between the catalytic and transmembrane domains resulted in the secretion of mature catalytic domain in association with the precursor (pro) domain. This complex was inactive and function was only restored after dissociation of the complex by dilution or treatment with 4-aminophenylmercuric acetate. Therefore, the pro domain of TACE is an inhibitor of the catalytic domain, and the cysteine-rich domain appears to play a role in the release of the pro domain. Insect cells failed to secrete a deletion mutant encoding the catalytic domain but lacking the inhibitory pro domain. This truncate was inactive and extensively degraded intracellularly, suggesting that the pro domain is required for the secretion of functional TACE.
Asunto(s)
Metaloendopeptidasas/genética , Proteínas ADAM , Proteína ADAM17 , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Línea Celular , Membrana Celular/enzimología , Citoplasma/enzimología , Humanos , Insectos , Cinética , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Transfección , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Two low-molecular-weight proteins have been purified from Brassica napus pollen and a gene corresponding to one of them has been isolated. The gene encodes an 8.6-kD protein with two EF-hand calcium-binding motifs and is a member of a small gene family in B. napus. The protein is part of a family of pollen allergens recently identified in several evolutionarily distant dicot and monocot plants. Homologs have been detected in Arabidopsis, from which one gene has been cloned in this study, and in snapdragon (Antirrhinum majus), but not in tobacco (Nicotiana tabacum). Expression of the gene in B. napus was limited to male tissues and occurred during the pollen-maturation phase of anther development. Both the B. napus and Arabidopsis proteins interact with calcium, and the potential for a calcium-dependent conformational change was demonstrated. Given this affinity for calcium, the cloned genes were termed BPC1 and APC1 (B. napus and Arabidopsis pollen calcium-binding protein 1, respectively). Immunolocalization studies demonstrated that BPC1 is found in the cytosol of mature pollen. However, upon pollen hydration and germination, there is some apparent leakage of the protein to the pollen wall. BPC1 is also concentrated on or near the surface of the elongating pollen tube. The essential nature of calcium in pollen physiology, combined with the properties of BPC1 and its high evolutionary conservation suggests that this protein plays an important role in pollination by functioning as a calcium-sensitive signal molecule.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/metabolismo , Brassica/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Plantas/metabolismo , Polen/metabolismo , Alérgenos/química , Alérgenos/genética , Alérgenos/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Clonación Molecular , Citosol/metabolismo , Genes de Plantas , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Polen/química , Polen/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
A rat cytoplasmic aminopeptidase P was purified from liver cytosol with a procedure including an affinity elution step with 3 microM inositol 1,3,4-trisphosphate. Proteolytic fragments were generated, sequenced and the enzyme was cloned from a rat liver cDNA library. The structure shows high (87.8% and 95.5%, respectively) sequence identity at the nucleotide and amino acid levels with the previously described human putative cytoplasmic aminopeptidase P. The cloned rat enzyme was functionally expressed in Escherichia coli and also in COS-1 cells. Western blot analysis, using an antibody generated against the recombinant protein, and Northern blot hybridization showed ubiquitous expression of the protein in different tissues with the highest expression level in the testis.
Asunto(s)
Aminopeptidasas/genética , Hígado/enzimología , Secuencia de Aminoácidos , Aminopeptidasas/antagonistas & inhibidores , Aminopeptidasas/biosíntesis , Animales , Secuencia de Bases , Células COS , Clonación Molecular , Citoplasma/enzimología , Escherichia coli/genética , Datos de Secuencia Molecular , Ácido Fítico/farmacología , ARN Mensajero/biosíntesis , RatasRESUMEN
A cell-based, lawn format assay utilizing an in situ photocleavage method has been developed that allows the rapid examination of large bead-based compound libraries as discrete molecules. The format uses frog melanophore cells in a contiguous, adherent, confluent layer in small petri dishes covered with a 0.5-1-mm layer of agarose containing 130 micron diameter TentaGel beads at a density of 2-20 beads/mm2. Employing this technique a 9-mer, 442,368-member peptide library (designed around the 13 amino acid alpha-MSH peptide sequence) made up of 12 separate pools of 36,864 peptides/pool was assayed. Initially, a fraction (approximately 10%) of each pool was scanned (approximately 3700 beads from each pool) in 60-mm petri dishes to identify the most active pools. Upon direct photocleavage of the beads with UV light (365 nm), each petri dish was photographed over a 60-min period with a CCD camera to record changes in light intensity as an index of melanosome dispersion. Active beads were those that were surrounded by a localized decrease in light transmittance indicating melanosome dispersed cells. Upon examination with a dissecting microscope, single beads centrally located to a circular array of dispersed cells were identified and removed from the agarose and sequenced by Edman degradation to determine the peptide sequence. Re-synthesized peptides were re-examined against alpha-MSH receptor to confirm and quantify the activity. Several 9-mer peptides were identified with potencies similar to the natural 13-mer peptide. This method allows for the rapid screening of large bead-based photo-cleavable peptide libraries with the advantage that each compound is screened as a discrete molecule in a well-less format.
Asunto(s)
Técnicas Químicas Combinatorias/métodos , Melanóforos , Biblioteca de Péptidos , Receptores de la Hormona Hipofisaria , Análisis de Secuencia de Proteína/métodos , Animales , Células Cultivadas , XenopusRESUMEN
Diphosphoinositol pentakisphosphate (PP-InsP5 or 'InsP7') and bisdiphosphoinositol tetrakisphosphate ([PP]2-InsP4 or 'InsP8') are the most highly phosphorylated members of the inositol-based cell signaling family. We have purified a rat hepatic diphosphoinositol polyphosphate phosphohydrolase (DIPP) that cleaves a beta-phosphate from the diphosphate groups in PP-InsP5 (Km = 340 nM) and [PP]2-InsP4 (Km = 34 nM). Inositol hexakisphophate (InsP6) was not a substrate, but it inhibited metabolism of both [PP]2-InsP4 and PP-InsP5 (IC50 = 0.2 and 3 microM, respectively). Microsequencing of DIPP revealed a 'MutT' domain, which in other contexts guards cellular integrity by dephosphorylating 8-oxo-dGTP, which causes AT to CG transversion mutations. The MutT domain also metabolizes some nucleoside phosphates that may play roles in signal transduction. The rat DIPP MutT domain is conserved in a novel recombinant human uterine DIPP. The nucleotide sequence of the human DIPP cDNA was aligned to chromosome 6; the candidate gene contains at least four exons. The dependence of DIPP's catalytic activity upon its MutT domain was confirmed by mutagenesis of a conserved glutamate residue. DIPP's low molecular size, Mg2+ dependency and catalytic preference for phosphoanhydride bonds are also features of other MutT-type proteins. Because overlapping substrate specificity is a feature of this class of proteins, our data provide new directions for future studies of higher inositol phosphates.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Monoéster Fosfórico Hidrolasas/metabolismo , Ácido Anhídrido Hidrolasas/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario , Regulación Enzimológica de la Expresión Génica , Humanos , Fosfatos de Inositol/metabolismo , Hígado/enzimología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Pirofosfatasas , Ratas , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
OBJECT: To increase knowledge of the safety and efficacy of the use of gamma knife radiosurgery in patients with movement disorders, the authors describe their own experience in this field and include blinded independent assessments of their results. METHODS: Fifty-five patients underwent radiosurgical placement of lesions either in the thalamus (27 patients) or globus pallidus (28 patients) for treatment of movement disorders. Patients were evaluated pre- and postoperatively by a team of observers skilled in the assessment of gait and movement disorders who were blinded to the procedure performed. The observers were not associated with the surgical team and concomitantly and blindly also assessed a group of 11 control patients with Parkinson's disease who did not undergo any surgical procedures. All stereotactic lesions were made with the Leksell gamma unit using the 4-mm secondary collimator helmet and a single isocenter with maximum doses from 120 to 160 Gy. Clinical follow-up evaluation indicated that 88% of patients who underwent thalamotomy became tremor free or nearly tremor free. Statistically significant improvements in performance were noted in the independent assessments of Unified Parkinson's Disease Rating Scale (UPDRS) scores in the patients undergoing thalamotomy. Of patients undergoing pallidotomy who had exhibited levodopainduced dyskinesias, 85.7% had total or near-total relief of that symptom. Clinical assessment indicated improvements in bradykinesia and rigidity in 64.3% of patients who underwent pallidotomy. Independent blinded assessments did not reveal statistically significant improvements in Hoehn and Yahr scores or UPDRS scores. On the other hand, 64.7% of patients showed improvements in subscores of the UPDRS, including activities of daily living (58%), total contralateral score (58%), and contralateral motor scores (47%). Total ipsilateral score and ipsilateral motor scores were both improved in 59% of patients. One (1.8%) of 55 patients experienced a homonymous hemianopsia 9 months after pallidotomy due to an unexpectedly large lesion. No other complications of any kind were seen. Neuropsychological test scores that were obtained for the combined pallidotomy and thalamotomy treatment groups preoperatively and at 6 months postoperatively demonstrated an absence of cognitive morbidity. Follow-up neuroimaging confirmed correct lesion location in all patients, with a mean maximum deviation from the planned target of 1 mm in the vertical axis. Measurements of lesions at regular intervals on postoperative magnetic resonance images demonstrated considerable variability in lesion volumes. The safety and efficacy of functional lesions made with the gamma knife appear to be similar to those made with the assistance of electrophysiological guidance with open functional stereotactic procedures. CONCLUSIONS: Functional lesions may be made safely and accurately using gamma knife radiosurgical techniques. The efficacy is equivalent to that reported for open techniques that use radiofrequency lesioning methods with electrophysiological guidance. Complications are very infrequent with the radiosurgical method. The use of functional radiosurgical lesioning to treat movement disorders is particularly attractive in older patients and in those with major systemic diseases or coagulopathies; its use in the general movement disorder population seems reasonable as well.
Asunto(s)
Globo Pálido/cirugía , Trastornos del Movimiento/cirugía , Radiocirugia , Tálamo/cirugía , Actividades Cotidianas , Adulto , Anciano , Anciano de 80 o más Años , Dopaminérgicos/efectos adversos , Discinesia Inducida por Medicamentos/fisiopatología , Discinesia Inducida por Medicamentos/cirugía , Electroencefalografía , Femenino , Estudios de Seguimiento , Marcha/fisiología , Hemianopsia/etiología , Humanos , Levodopa/efectos adversos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Destreza Motora/fisiología , Trastornos del Movimiento/fisiopatología , Rigidez Muscular/fisiopatología , Rigidez Muscular/cirugía , Pruebas Neuropsicológicas , Enfermedad de Parkinson/fisiopatología , Radiocirugia/efectos adversos , Radiocirugia/instrumentación , Radiocirugia/métodos , Dosificación Radioterapéutica , Seguridad , Método Simple Ciego , Temblor/cirugíaRESUMEN
Tumour-necrosis factor-alpha (TNF-alpha) is a cytokine that contributes to a variety of inflammatory disease states. The protein exists as a membrane-bound precursor of relative molecular mass 26K which can be processed by a TNF-alpha-converting enzyme (TACE), to generate secreted 17K mature TNF-alpha. We have purified TACE and cloned its complementary DNA. TACE is a membrane-bound disintegrin metalloproteinase. Structural comparisons with other disintegrin-containing enzymes indicate that TACE is unique, with noteable sequence identity to MADM, an enzyme implicated in myelin degradation, and to KUZ, a Drosophila homologue of MADM important for neuronal development. The expression of recombinant TACE (rTACE) results in the production of functional enzyme that correctly processes precursor TNF-alpha to the mature form. The rTACE provides a readily available source of enzyme to help in the search for new anti-inflammatory agents that target the final processing stage of TNF-alpha production.
Asunto(s)
Desintegrinas/genética , Metaloendopeptidasas/genética , Precursores de Proteínas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas ADAM , Proteína ADAM17 , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Clonación Molecular , Secuencia Conservada , Desintegrinas/aislamiento & purificación , Desintegrinas/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/aislamiento & purificación , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , PorcinosRESUMEN
Tumor necrosis factor-alpha is a potent cytokine, secreted primarily by activated monocytes and macrophages, that possesses a broad range of immunomodulating properties. Involvement of this cytokine has been validated in disease states such as arthritis and Crohn's disease and implicated in diverse neuroimmunological pathologies such as multiple sclerosis, Alzheimers and stroke. TNF-alpha is initially synthesized as a 26 kDa precursor molecule that is subsequently processed to the mature form by cleavage of the Ala76 Val77 bond. The 17 kDa carboxy-terminal protein is then secreted to function in a paracrine manner. The enzyme that processes precursor TNF-alpha has previously been identified as a microsomal metalloprotease called TNF-alpha converting enzyme (TACE). We have now purified and partially cloned the enzyme. TACE represents a novel target for therapeutic intervention in a variety of inflammatory and neuroimmunological diseases.
Asunto(s)
Metaloendopeptidasas/química , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas ADAM , Proteína ADAM17 , Animales , Metaloendopeptidasas/genética , Metaloendopeptidasas/aislamiento & purificaciónRESUMEN
The occurrence of NADH --> NAD transhydrogenation and lipoamide dehydrogenase activities was demonstrated for cysticercoids of the intestinal cestode, Hymenolepis diminuta. In addition, both activities were catalyzed by the mitochondria of 6-, 10-, and 14-day H. diminuta and by the mitochondria from immature, mature, and pregravid/gravid regions of the adult cestode. A developmentally related increase in NADH --> NAD activity was suggested and the levels of both activities in the immature region of the helminth were consistent with it being a region of high metabolic activity. Adult H. diminuta mitochondrial lipoamide dehydrogenase was purified to homogeneity. The native enzyme was a homodimer with a monomeric and dimeric molecular mass of 47 and 93 kDa, respectively. Spectral analyses revealed that the enzyme contained flavin. More importantly, the purified enzyme catalyzed appreciable NADH --> NAD transhydrogenation activity, a premier finding for the phylum Platyhelminthes. The ratio of NADH --> NAD transhydrogenation to lipoamide reduction was 1:5. Both activities were inhibited by Cu2+ and Cd2+ with the NADH --> NAD activity being more resistant to inhibition. Interestingly, aside from NADH diaphorase activity, the cestode enzyme displayed NADH-ferricyanide reductase and, to a lesser degree, NADPH --> NAD transhydrogenation activities. The partial amino acid sequence of H. diminuta lipoamide dehydrogenase indicated that this enzyme was most similar to the corresponding enzymes of other parasitic helminths. Moreover, the phenylalanine for leucine substitution found in the redox-active disulfide site of the lipoamide dehydrogenases of some anaerobic systems was noted for the H. diminuta enzyme.
Asunto(s)
Dihidrolipoamida Deshidrogenasa/metabolismo , Hymenolepis/enzimología , Mitocondrias/enzimología , NAD/metabolismo , Secuencia de Aminoácidos , Animales , Dihidrolipoamida Deshidrogenasa/química , Electroforesis en Gel de Poliacrilamida , Femenino , Hidrogenación , Hymenolepis/crecimiento & desarrollo , Masculino , Datos de Secuencia Molecular , Ratas , Ratas Sprague-DawleyRESUMEN
The characterization of the multiple inositol polyphosphate phosphatase (MIPP) is fundamental to our understanding of how cells control the signalling activities of 'higher' inositol polyphosphates. We now describe our isolation of a 2.3 kb cDNA clone of a rat hepatic form of MIPP. The predicted amino acid sequence of MIPP includes an 18 amino acid region that aligned with approximately 60% identity with the catalytic domain of a fungal inositol hexakisphosphate phosphatase (phytase A); the similarity encompassed conservation of the RHGXRXP signature of the histidine acid phosphatase family. A histidine-tagged, truncated form of MIPP was expressed in Escherichia coli and the enzymic specificity of the recombinant protein was characterized: Ins(1,3,4,5,6)P5 was hydrolysed, first to Ins(1,4,5,6)P4 and then to Ins(1,4,5)P3, by consecutive 3- and 6-phosphatase activities. Inositol hexakisphosphate was catabolized without specificity towards a particular phosphate group, but in contrast, MIPP only removed the beta-phosphate from the 5-diphosphate group of diphosphoinositol pentakisphosphate. These data, which are consistent with the substrate specificities of native (but not homogeneous) MIPP isolated from rat liver, provide the first demonstration that a single enzyme is responsible for this diverse range of specific catalytic activities. A 2.5 kb transcript of MIPP mRNA was present in all rat tissues that were examined, but was most highly expressed in kidney and liver. The predicted C-terminus of MIPP is comprised of the tetrapeptide SDEL, which is considered a signal for retaining soluble proteins in the lumen of the endoplasmic reticulum; the presence of this sequence provides a molecular explanation for our earlier biochemical demonstration that the endoplasmic reticulum contains substantial MIPP activity [Ali, Craxton and Shears (1993) J. Biol. Chem. 268, 6161-6167].
Asunto(s)
Hígado/enzimología , Monoéster Fosfórico Hidrolasas/biosíntesis , Monoéster Fosfórico Hidrolasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/análisis , ADN Complementario/aislamiento & purificación , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/aislamiento & purificación , ARN Mensajero/biosíntesis , Ratas , Proteínas Recombinantes/metabolismoRESUMEN
The bovine liver mitochondrial protein synthesis elongation factor Tu.Ts complex (EF-TU.Tsmt) has been purified and partial peptide sequence information has been obtained for EF-Tumt. A complete cDNA has been obtained encoding bovine EF-Tumt and a nearly complete cDNA has been obtained for human EF-Tumt. The bovine cDNA has a 5' untranslated leader, an open reading frame of 1356 nucleotides and a 3' untranslated region of 189 base pairs. NH2-terminal sequencing of the mature protein indicates that the transit peptide for the mitochondrial localization of this protein is 43 amino acids in length. The human and bovine factors are 95% identical. The deduced protein sequences show considerable identity to bacterial and organellar EF-Tu sequences. At least two genes for EF-Tumt are present in the bovine system. Northern analysis indicates that EF-Tumt is synthesized in all tissues but that the level of expression varies over a wide range. EF-TUmt has been expressed in E. coli as a His-tagged protein and purified to near homogeneity. The expressed form of the factor is active in the poly(U)-directed polymerization of phenylalanine although it is less active than the native EF-Tu.Tsmt complex.