RESUMEN
In Germany, the tissue law came into effect on 1 August 2007. The law implemented the requirements of EC directives on quality and safety of human tissues and cells in the German Transplantation Act ("Transplantationsgesetz," TPG) and in the German Medicinal Products Act. Accordingly, tissue establishments are obligated to keep a record of their activities and to submit an annual report to the Paul-Ehrlich-Institut (PEI). The report shall include the types and quantities of tissues procured, conditioned, processed, stored, and distributed, or otherwise disposed of, imported and exported. For this purpose, the PEI published TPG-based notification forms in the Bundesanzeiger and in the Internet. The data provided by tissue establishments have been anonymized and compiled in a general report. The analysis revealed inconclusive data, which can be due to a number of different causes. To achieve better consistency of data provided in the future, the explanations for completing the notification forms will be amended. Thus far, compiled data are not appropriate to draw conclusions on the availability of tissues and tissue preparations in Germany, but the data can serve as reference points.
Asunto(s)
Unión Europea , Programas Nacionales de Salud/legislación & jurisprudencia , Garantía de la Calidad de Atención de Salud/legislación & jurisprudencia , Trasplante de Tejidos/legislación & jurisprudencia , Unión Europea/estadística & datos numéricos , Femenino , Adhesión a Directriz/legislación & jurisprudencia , Adhesión a Directriz/estadística & datos numéricos , Humanos , Masculino , Programas Nacionales de Salud/estadística & datos numéricos , Seguridad del Paciente/legislación & jurisprudencia , Seguridad del Paciente/estadística & datos numéricos , Técnicas Reproductivas Asistidas/legislación & jurisprudencia , Técnicas Reproductivas Asistidas/estadística & datos numéricos , Bancos de Tejidos/legislación & jurisprudencia , Bancos de Tejidos/estadística & datos numéricos , Donantes de Tejidos/legislación & jurisprudencia , Donantes de Tejidos/estadística & datos numéricos , Trasplante de Tejidos/estadística & datos numéricos , Recolección de Tejidos y Órganos/legislación & jurisprudencia , Recolección de Tejidos y Órganos/estadística & datos numéricosRESUMEN
Only 10% of human transmissible spongiform encephalopathies (TSEs) are associated with mutations of the Prnp region encoding the prion protein (PrP). Recently, the murine PrP-like protein doppel (Dpl) was described and was shown to be overexpressed in certain strains of PrP knockout mice and to cause neurological diseases such as ataxia and Purkinje cell loss. To answer the question of whether there are any polymorphisms within the PrP-like protein gene (Prnd) that might cause or be involved in the development of TSEs, we investigated the complete open reading frame of the human Prnd gene from 58 patients who had died of genetic or sporadic Creutzfeldt-Jakob disease (CJD), Alzheimer's disease or other neurological disorders and from 111 controls. We found five new polymorphisms and one frame shift mutation. One silent polymorphism, which does not lead to an altered amino acid sequence, was also observed. Statistical analysis revealed a significant difference in the distribution of the Prnd genotype at codon 174 between sporadic CJD patients and healthy controls.
Asunto(s)
Enfermedad de Alzheimer/genética , Síndrome de Creutzfeldt-Jakob/genética , Enfermedades del Sistema Nervioso/genética , Polimorfismo Genético , Priones/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Estudios de Casos y Controles , ADN/genética , Análisis Mutacional de ADN , Proteínas Ligadas a GPI , Genotipo , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura AbiertaRESUMEN
The improvement of gene transfer efficiency in growth-arrested cells using human immunodeficiency virus type 1 (HIV-1)-derived vectors led to the development of vectors derived from other members of the lentivirus family. Here we report the generation of a lentiviral vector derived from the apathogenic molecular virus clone SIVagm3mc of the simian immunodeficiency virus from African green monkeys (Cercocebus pygerythrus). Upon pseudotyping with the G-protein of vesicular stomatitis virus (VSV-G), the SIVagm-derived vector was shown to transduce proliferating and growth-arrested mammalian cell lines, including human cells. After in vivo inoculation into the striatum of the adult rat brain, the vector was shown to transduce terminally differentiated neurons and oligodendrocytes as well as quiescent and reactive astrocytes. Moreover, SIVagm transfer vector mRNA was efficiently packaged by HIV-1 vector particles. Homologous [SIV(SIV)] vectors generated by using the SIVagm-derived envelope glycoproteins allowed selective gene transfer into human CD4(+)/CCR5(+) cells. Thus, the SIVagm3mc-derived vector is a useful alternative to HIV-1-derived lentiviral vectors in somatic gene therapy.
Asunto(s)
Vectores Genéticos , Glicoproteínas de Membrana , Virus de la Inmunodeficiencia de los Simios , Animales , Linfocitos T CD4-Positivos , División Celular , Línea Celular Transformada , Femenino , Genes env , Vectores Genéticos/genética , VIH-1/genética , Humanos , Lentivirus/genética , Neuroglía , Neuronas , Ratas , Ratas Endogámicas F344 , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Transducción Genética , Virus de la Estomatitis Vesicular Indiana/genética , Proteínas del Envoltorio Viral/genética , ViriónRESUMEN
Ca2+/calmodulin-dependent protein kinases I and IV (CaMKI and CaMKIV, respectively) require phosphorylation on an equivalent single Thr in the activation loop of subdomain VIII for maximal activity. Two distinct CaMKI/IV kinases, CaMKKalpha and CaMKKbeta, were purified from rat brain and partially sequenced (Edelman, A. M., Mitchelhill, K., Selbert, M. A., Anderson, K. A., Hook, S. S., Stapleton, D., Goldstein, E. G., Means, A. R., and Kemp, B. E. (1996) J. Biol. Chem. 271, 10806-10810). We report here the cloning and sequencing of cDNAs for human and rat CaMKKbeta, tissue and regional brain localization of CaMKKbeta protein, and mRNA and functional characterization of recombinant CaMKKbeta in vitro and in Jurkat T cells. The sequences of human and rat CaMKKbeta demonstrate 65% identity and 80% similarity with CaMKKalpha and 30-40% identity with CaMKI and CaMKIV themselves. CaMKKbeta is broadly distributed among rat tissues with highest levels in CaMKIV-expressing tissues such as brain, thymus, spleen, and testis. In brain, CaMKKbeta tracks more closely with CaMKIV than does CaMKKalpha. Bacterially expressed CaMKKbeta undergoes intramolecular autophosphorylation, is regulated by Ca2+/CaM, and phosphorylates CaMKI and CaMKIV on Thr177 and Thr200, respectively. CaMKKbeta activates both CaMKI and CaMKIV when coexpressed in Jurkat T cells as judged by phosphorylated cAMP response element-binding protein-dependent reporter gene expression. CaMKKbeta activity is enhanced by elevation of intracellular Ca2+, although substantial activity is observed at the resting Ca2+ concentration. The strict Ca2+ requirement of CaMKIV-dependent phosphorylation of cAMP response element-binding protein, is therefore controlled at the level of CaMKIV rather than CaMKK.
Asunto(s)
Encéfalo/enzimología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Clonación Molecular , ADN Complementario , Activación Enzimática , Humanos , Hibridación in Situ , Cinética , Datos de Secuencia Molecular , Especificidad de Órganos , Fosforilación , Proteínas Serina-Treonina Quinasas/química , ARN Mensajero/análisis , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Transcripción GenéticaRESUMEN
It has been observed that the activity of Ca2+-calmodulin (CaM)-dependent protein kinase I is enhanced up to 50-fold by its phosphorylation in vitro by a distinct CaM kinase I kinase (Lee, J. C., and Edelman, A. M. (1994) J. Biol. Chem. 269, 2158-2164). It has, however, been unclear whether this event represents an acute form of cellular regulation. We demonstrate here the phosphorylation and activation of CaM kinase I in PC12 pheochromocytoma cells in response to elevation of intracellular Ca2+. Treatment of PC12 cells with the Ca2+-ionophore, ionomycin, or with a depolarizing concentration of KCl, led to rapid, biphasic phosphorylation of CaM kinase I and to increases in CaM kinase I activity of 5.1- and 7. 3-fold, respectively. Depolarization-induced activation of CaM kinase I was reduced by approximately 80% by blockade of Ca2+ influx through L-type voltage-dependent Ca2+ channels and completely abolished by removal of extracellular Ca2+. The ability of PC12 cell CaM kinase I to be phosphorylated and activated by purified CaM kinase I kinase in vitro was markedly reduced by prior depolarization of the cells, consistent with intracellular phosphorylation and activation of CaM kinase I by CaM kinase I kinase. These results demonstrate the existence in PC12 cells of a CaM kinase I cascade, the function of which may be to sensitize cells to signal-induced elevations of intracellular Ca2+.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Calcio/fisiología , Animales , Canales de Calcio/fisiología , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina , Citoplasma/fisiología , Activación Enzimática , Potenciales de la Membrana , Células PC12 , Fosforilación , RatasRESUMEN
We have purified to near homogeneity from rat brain two Ca(2+)-calmodulin-dependent protein kinase I (CaM kinase I) activating kinases, termed here CaM kinase I kinase-alpha and CaM kinase I kinase-beta (CaMKIK alpha and CaMKIK beta, respectively). Both CaMKIK alpha and CaMKIK beta are also capable of activating CaM kinase IV. Activation of CaM kinase I and CaM kinase IV occurs via phosphorylation of an equivalent Thr residue within the "activation loop" region of both kinases, Thr-177 and Thr-196, respectively. The activities of CaMKIK alpha and CaMKIK beta are themselves strongly stimulated by the presence of Ca(2+)-CaM, and both appear to be capable of Ca(2+)-CaM-dependent autophosphorylation. Automated microsequence analysis of the purified enzymes established that CaMKIK alpha and -beta are the products of distinct genes. In addition to rat, homologous nucleic acids corresponding to these CaM kinase kinases are present in humans and the nematode, Caenorhabditis elegans. CaMKIK alpha and CaMKIK beta are thus representatives of a family of enzymes, which may function as key intermediaries in Ca(2+)-CaM-driven signal transduction cascades in a wide variety of eukaryotic organisms.
Asunto(s)
Encéfalo/enzimología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , Isoenzimas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/aislamiento & purificación , Cromatografía Liquida , Activación Enzimática , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de AminoácidoRESUMEN
AMP-activated protein kinase (AMPK) and Ca2+/calmodulin (CaM)-dependent protein kinase I (CaMKI) are protein kinases that are regulated both by allosteric activation (AMP and Ca2+/CaM, respectively) and by phosphorylation by upstream protein kinases (AMPK kinase (AMPKK) and CaMKI kinase (CaMKIK), respectively). We now report that AMPKK can activate CaMKI and that, conversely, CaMKIK can activate AMPK. CaMKIK is 68-fold more effective at activating CaMKI than AMPK, while AMPKK is 17-fold more effective at activating AMPK than CaMKI. Our results suggest that CaMKIK and AMPKK are distinct enzymes dedicated to their respective kinase targets but with some overlap in their substrate specificities. The availability of alternative substrates for AMPKK and CaMKIK allowed the unequivocal demonstration that AMP and Ca2+/calmodulin promote the activation of AMPK and Ca2+/calmodulin promote the activation of AMPK and CaMKI, respectively, via three independent mechanisms: 1) direct activation of AMPK and CaMKI, 2) activation of AMPKK and CaMKIK, and 3) by binding to AMPK and CaMKI, inducing exposure of their phosphorylation sites. Since AMP and Ca2+/calmodulin each has a triple effect in its respective system, in vivo, the two systems would be expected to be exquisitely sensitive to changes in concentration of their respective activating ligands.
Asunto(s)
Adenosina Monofosfato/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Calcio/farmacología , Calmodulina/farmacología , Complejos Multienzimáticos/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Activación Enzimática/efectos de los fármacos , Técnicas In Vitro , Ligandos , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Péptidos/química , Fosforilación , Proteínas Quinasas/química , Proteínas Quinasas/genética , Especificidad por SustratoRESUMEN
Human Ca(2+)-calmodulin (CaM) dependent protein kinase I (CaMKI) encodes a 370 amino acid protein with a calculated M(r) of 41,337. The 1.5 kb CaMKI mRNA is expressed in many different human tissues and is the product of a single gene located on human chromosome 3. CaMKI 1-306, was unable to bind Ca(2+)-CaM and was completely inactive thereby defining an essential component of the CaM-binding domain to residues C-terminal to 306. CaMKI 1-294 did not bind CaM but was fully active in the absence of Ca(2+)-CaM, indicating that residues 295-306 are sufficient to maintain CaMKI in an auto-inhibited state. CaMKI was phosphorylated on Thr177 and its activity enhanced approximately 25-fold by CaMKI kinase in a Ca(2+)-CaM dependent manner. Replacement of Thr177 with Ala or Asp prevented both phosphorylation and activation by CaMKI kinase and the latter replacement also led to partial activation in the absence of CaMKI kinase. Whereas CaMKI 1-306 was unresponsive to CaMKI kinase, the 1-294 mutant was phosphorylated and activated by CaMKI kinase in both the presence and absence of Ca(2+)-CaM although at a faster rate in its presence. These results indicate that the auto-inhibitory domain in CaMKI gates, in a Ca(2+)-CaM dependent fashion, accessibility of both substrates to the substrate binding cleft and CaMKI kinase to Thr177. Additionally, CaMKI kinase responds directly to Ca(2+)-CaM with increased activity.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , ADN Complementario/genética , Treonina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calcio/metabolismo , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Calmodulina/metabolismo , Línea Celular , Mapeo Cromosómico , Cromosomas Humanos Par 3 , Clonación Molecular , Activación Enzimática , Humanos , Datos de Secuencia Molecular , Mutación , Fosforilación , Proteínas Recombinantes de FusiónRESUMEN
Purified pig brain Ca(2+)-calmodulin (CaM)-dependent protein kinase Ia kinase (Lee, J. C., and Edelman, A. M. (1994) J. Biol. Chem. 269, 2158-2164) enhances, by up to 24-fold, the activity of recombinant CaM kinase IV in a reaction also requiring Ca(2+)-CaM and MgATP. The addition of brain extract, although capable of activating CaM kinase IV by itself, provides no further activation beyond that induced by purified CaM kinase Ia kinase, consistent with the lack of a requirement of additional components for activation. Activation is accompanied by the development of significant (38%) Ca(2+)-CaM-independent CaM kinase IV activity. In parallel fashion to its activation, CaM kinase IV is phosphorylated in a CaM kinase Ia kinase-, Ca(2+)-CaM-, and MgATP-dependent manner. Phosphorylation occurs on multiple serine and threonine residues with a Ser-P:Thr-P ratio of approximately 3:1. The identical requirements for phosphorylation and activation and a linear relationship between extent of phosphorylation of CaM kinase IV and its activation state indicate that CaM kinase IV activation is induced by its phosphorylation. Replacement of Thr-196 of CaM kinase IV with a nonphosphorylatable alanine by site-directed mutagenesis abolishes both the phosphorylation and activation of CaM kinase IV, demonstrating that Thr-196 phosphorylation is essential for activation.