The molecular basis of spinocerebellar ataxia type 48 caused by a de novo mutation in the ubiquitin ligase CHIP.

The spinocerebellar ataxias (SCAs) are a class of incurable diseases characterized by degeneration of the cerebellum that results in movement disorder. Recently, a new heritable form of SCA, spinocerebellar ataxia type 48 (SCA48), was attributed to dominant mutations in STIP1 homology and U box-containing 1 (STUB1); however, little is known about how these mutations cause SCA48. STUB1 encodes for the protein C terminus of Hsc70 interacting protein (CHIP), an E3 ubiquitin ligase. CHIP is known to regulate proteostasis by recruiting chaperones via a N-terminal tetratricopeptide repeat domain and recruiting E2 ubiquitin-conjugating enzymes via a C-terminal U-box domain. These interactions allow CHIP to mediate the ubiquitination of chaperone-bound, misfolded proteins to promote their degradation via the proteasome. Here we have identified a novel, de novo mutation in STUB1 in a patient with SCA48 encoding for an A52G point mutation in the tetratricopeptide repeat domain of CHIP. Utilizing an array of biophysical, biochemical, and cellular assays, we demonstrate that the CHIPA52G point mutant retains E3-ligase activity but has decreased affinity for chaperones. We further show that this mutant decreases cellular fitness in response to certain cellular stressors and induces neurodegeneration in a transgenic Caenorhabditis elegans model of SCA48. Together, our data identify the A52G mutant as a cause of SCA48 and provide molecular insight into how mutations in STUB1 cause SCA48.

The ubiquitin ligase F-box/G-domain protein 1 promotes the degradation of the disease-linked protein torsinA through the ubiquitin-proteasome pathway and macroautophagy.

DYT1 dystonia is a dominantly inherited, disabling neurological disorder with low penetrance that is caused by the deletion of a glutamic acid (ΔE) in the protein torsinA. We previously showed that torsinA(wt) is degraded through macroautophagy while torsinA(ΔE) is targeted to the ubiquitin-proteasome pathway (UPP). The different catabolism of torsinA(wt) and (ΔE) potentially modulates torsinA(wt):torsinA(ΔE) stoichiometry. Therefore, gaining a mechanistic understanding on how the protein quality control machinery clears torsinA(ΔE) in neurons may uncover important regulatory steps in disease pathogenesis. Here, we asked whether F-box/G-domain protein 1 (FBG1), a ubiquitin ligase known to degrade neuronal glycoproteins, is implicated in the degradation of torsinA(ΔE) by the UPP. In a first set of studies completed in cultured cells, we show that FBG1 interacts with and influences the steady-state levels of torsinA(wt) and (ΔE). Interestingly, FBG1 achieves this effect promoting the degradation of torsinA not only through the UPP, but also by macroautophagy. To determine the potential clinical significance of these findings, we asked if eliminating expression of Fbg1 triggers a motor phenotype in torsinA(ΔE) knock in (KI) mice, a model of non-manifesting DYT1 mutation carriers. We detected differences in spontaneous locomotion between aged torsinA(ΔE) KI-Fbg1 knock out and control mice. Furthermore, neuronal levels of torsinA were unaltered in Fbg1 null mice, indicating that redundant systems likely compensate in vivo for the absence of this ubiquitin ligase. In summary, our studies support a non-essential role for FBG1 on the degradation of torsinA and uncover a novel link of FBG1 to the autophagy pathway.

Lethal toxicity caused by expression of shRNA in the mouse striatum: implications for therapeutic design.

Therapeutic RNA interference (RNAi) has emerged as a promising approach for the treatment of many incurable diseases, including cancer, infectious disease or neurodegenerative disorders. Demonstration of efficacy and safety in animal models is necessary before planning human application. Our group and others have previously shown the potential of this approach for the dominantly inherited neurological disease DYT1 dystonia by achieving potent short-hairpin RNA (shRNA)-mediated silencing of the disease protein, torsinA, in cultured cells. To establish the feasibility of this approach in vivo, we pursued viral delivery of shRNA in two different mouse models. Surprisingly, intrastriatal injections of adeno-associated virus serotype 2/1 (AAV2/1) vectors expressing different shRNAs, whether targeting torsinA expression or mismatched controls, resulted in significant toxicity with progressive weight loss, motor dysfunction and animal demise. Histological analysis showed shRNA-induced neurodegeneration. Toxicity was not observed in animals that received control AAV2/1 encoding no shRNA, and was independent of genotype, occurring in both DYT1 and wild-type animals. Interestingly, the different genetic background of both mouse models influenced toxicity, being earlier and more severe in 129/SvEv than in C57BL/6 mice. In conclusion, our studies demonstrate that expression of shRNA in the mammalian brain can lead to lethal toxicity. Furthermore, the genetic background of rodents modifies their sensitivity to this form of toxicity, a factor that should be taken into consideration in the design of preclinical therapeutic RNAi trials.

Transcriptional and proteomic profiling in a cellular model of DYT1 dystonia.

DYT1, the most common inherited dystonia, is caused by a common dominant mutation in the TOR1A gene that leads to a glutamic acid deletion in the protein torsinA. Wild-type torsinA locates preferentially in the endoplasmic reticulum while the disease-linked mutant accumulates in the nuclear envelope. As a result, it has been proposed that DYT1 pathogenesis could result either from transcriptional dysregulation caused by abnormal interactions of mutant torsinA with nuclear envelope proteins, or from a loss of torsinA function in the endoplasmic reticulum that would impair specific neurobiological pathways. Aiming to determine whether one or both of these potential mechanisms are implicated in DYT1 pathogenesis, we completed unbiased transcriptional and proteomic profiling in well-characterized neural cell lines that inducibly express wild-type or mutant torsinA. These experiments demonstrated that the accumulation of mutant torsinA in the nuclear envelope is not sufficient to cause transcriptional dysregulation. However, we detected expression changes at the protein level that, together with other reports, suggest a potential implication of torsinA on energy metabolism and regulation of the redox state. Furthermore, several proteins identified in this study have been previously linked to other forms of dystonia. In conclusion, our results argue against the hypothesis of transcriptional dysregulation in DYT1 dystonia, suggesting potential alternative pathogenic pathways.

[Therapeutic RNA interference for neurodegenerative diseases]. / ARN de interferencia terapéutico para enfermedades neurodegenerativas.

INTRODUCTION: The discovery of RNA interference (RNAi), a biological way to control gene expression, has revolutionised the field of biomedical research. Of the many applications of RNAi, perhaps its use for therapeutic purposes is the most promising. One group of diseases where the use of therapeutic RNAi is being actively explored is that of neurodegenerative disorders. AIM: To provide both the clinical neurologist and the basic researcher with an updated summary of the development of therapeutic RNAi for neurodegenerative diseases. DEVELOPMENT: The technical progress made in the manipulation of RNAi in neurons and the recent advances in our knowledge of the pathogenesis of neurodegenerative processes have been valuable aids in designing therapeutic RNAi for both hereditary and idiopathic diseases. Several pre-clinical trials have been successfully completed in animal models, thus clearing the way towards the design of clinical trials in humans. Nevertheless, before reaching that point more experimental studies need to be carried out in animals to prove the effectiveness of this mode of therapy and, still more important, to be able to predict the possible side effects of this procedure in the human brain. CONCLUSIONS: Although it is still in the pre-clinical stages, the use of RNAi for therapeutic purposes in neurodegenerative diseases is producing some very promising results. The evolution of this field over the next few years may be critical for beginning its clinical application.

ARN de interferencia terapéutico para enfermedades neurodegenerativas / Therapeutic RNA interference for neurodegenerative diseases

Introducción. El descubrimiento del ARN de interferencia (ARNi), una vía biológica de control de expresión génica,ha revolucionado el campo de la investigación biomédica. De las muchas aplicaciones del ARNi, quizás su explotación terapéutica sea la más prometedora. Un grupo de enfermedades donde el uso de ARNi terapéutico se está explorando activamente lo constituyen los trastornos neurodegenerativos. Objetivo. Presentar tanto al neurólogo clínico como al investigador básico un sumario actualizado del desarrollo de ARNi terapéutico para enfermedades neurodegenerativas. Desarrollo. El progresotécnico en la manipulación del ARNi en neuronas y los avances recientes en el conocimiento de la patogenia de los procesos neurodegenerativos han facilitado el diseño de ARNi terapéutico tanto para enfermedades hereditarias como idiopáticas. Varios ensayos preclínicos se han completado con éxito en modelos animales, allanando el camino hacia el diseño de ensayos clínicos en humanos. Sin embargo, antes de llegar a ese punto hay que completar más estudios experimentales en animalespara demostrar la eficacia de esta modalidad terapéutica y, más importante aún, para predecir los posibles efectos adversos de esta intervención en el cerebro humano. Conclusión. Aunque todavía en estadios preclínicos, la explotación del ARNi con fines terapéuticos en enfermedades neurodegenerativas está generando resultados muy prometedores. La evoluciónde este campo en los próximos años puede ser crítica para iniciar su aplicación clínica

Introduction. The discovery of RNA interference (RNAi), a biological way to control gene expression, has revolutionised the field of biomedical research. Of the many applications of RNAi, perhaps its use for therapeutic purposes is the most promising. One group of diseases where the use of therapeutic RNAi is being actively explored is that of neurodegenerativedisorders. Aim. To provide both the clinical neurologist and the basic researcher with an updated summary of the development of therapeutic RNAi for neurodegenerative diseases. Development. The technical progress made in the manipulation of RNAi in neurons and the recent advances in our knowledge of the pathogenesis of neurodegenerative processes have been valuableaids in designing therapeutic RNAi for both hereditary and idiopathic diseases. Several pre-clinical trials have been successfully completed in animal models, thus clearing the way towards the design of clinical trials in humans. Nevertheless, before reaching that point more experimental studies need to be carried out in animals to prove the effectiveness of this mode of therapy and, still more important, to be able to predict the possible side effects of this procedure in the human brain.Conclusions. Although it is still in the pre-clinical stages, the use of RNAi for therapeutic purposes in neurodegenerative diseases is producing some very promising results. The evolution of this field over the next few years may be critical for beginning its clinical application

Consequences of the DYT1 mutation on torsinA oligomerization and degradation.

DYT1 is the most common inherited dystonia, a neurological syndrome that causes disabling involuntary muscle contractions. This autosomal dominant disease is caused by a glutamic acid deletion near the carboxy-terminus in the protein torsinA. Cell- and animal-based studies have shown how the DYT1 mutation causes mutant torsinA to redistribute from the endoplasmic reticulum to the nuclear envelope, acting through a dominant negative effect over the wild type protein. As a result, the wild type:mutant torsinA expression ratio would be important for disease pathogenesis, and events that influence it, such as a differential degradation process for each protein, might modulate DYT1 pathobiology. The DYT1 mutation also triggers the formation of abnormal intermolecular disulfide bonds in torsinA, although the significance of this finding is unclear. How the protein quality control machinery handles torsinA, and whether this process is affected by its abnormal oligomerization remain unknown. Here, we first explored how the disease-linked mutation influences the catabolic process of human torsinA, demonstrating that the differences in subcellular localization between both forms of torsinA lead to divergences in their degradation pathways and, whereas torsinA is normally recycled through autophagy, the proteasome is also required for the efficient clearance of the mutated form. Subsequently, we determined that the abnormal disulfide bond-dependent oligomerization of mutant torsinA is not a result of its redistribution to the nuclear envelope, but a direct consequence of the mutation. Finally, we established that the presence of disulfide links in mutant torsinA oligomers interfere with their degradation by the proteasome, thus relying on autophagy as the main pathway for clearance. In conclusion, the abnormal subcellular localization and oligomerization of DYT1-linked torsinA influences its catabolic process, opening the door to the modulation of the wild type:mutant torsinA ratio through pharmacological manipulation of protein degradation pathways.

Actualización en terapéutica antiparasitaria. I. Parasitosis intestinales / Update on treatment of intestinal parasitic infections

Las infecciones parasitarias presentan una distribución mundial. El incremento en los viajes internacionales, la inmigración, el uso de fármacos inmunosupresores y la difusión del SIDA han provocado que los profesionales sanitarios puedan detectar infecciones causadas por parásitos infrecuentes actualmente. Esta revisión incluye una actualización de los fármacos de primera elección y alternativas terapéuticas frente a la mayoría de las parasitosis intestinales

Parasitic infections are found throughout the world. With increasing travel, immigration, use of immunosuppressive drugs and the spread of AlDS, physicians anywhere may see infections caused by previously unfamiliar parasites. This paper includes an updated summary of the current drug therapies (first-choice and altemative drugs) for most intestinal parasitic disease

Severe interscapular pain and increased creatine kinase activity: the answer was in the ankles.

Severe thoracic back pain with increased creatine kinase activity is a clinical presentation that suggests a variety of life threatening conditions. If initial examination is unrevealing, multiple diagnostic tests are usually performed attempting to identify the origin of the problem, sometimes neglecting apparently unrelated subtle physical findings. A patient is described in whom this was the initial presentation of a sensory demyelinating neuropathy, resulting in a diagnostic challenge. This case expands the differential diagnosis of severe thoracic back pain and increased creatine kinase activity, and illustrates the importance of physical examination in reaching a final diagnosis.

Utilidad de una prueba treponémica rápida para el serodiagnóstico de sífilis / Evaluation of a rapid treponemal test for the serodiagnosis of syphilis

Evaluar una nueva prueba treponémica, Syphilis Fast, para el serodiagnóstico rápido de sífilis en comparación con otras pruebas serológicas. Pacientes y métodos. Un total de 287 sueros de 280 pacientes fueron analizados mediante una prueba no treponémica, RPR (Rapid Plasma Reagin test) y dos pruebas treponémicas (Syphilis Fast y Mercia Syphilis Total EIA). Todos los sueros reactivos fueron confirmados mediante FTA Abs. La población incluida en el estudio comprendía: inmigrantes y viajeros procedentes de África subsahariana, pacientes con prácticas de riesgo para enfermedades de transmisión sexual y VIH (homosexuales o bisexuales, adictos a drogas por vía parenteral, pacientes con múltiples parejas sexuales), inmigrantes que ejercían la prostitución, niños adoptados, mujeres embarazadas y pacientes con síntomas neurológicos o con lesiones cutáneas genitales. Resultados. De los 280 pacientes, 28 (10 por ciento) eran VIH+. La concordancia del FTA Abs, considerada la prueba de referencia o gold standard en serodiagnóstico de sífilis, con Syphilis Fast y Mercia Syphilis Total EIA fue de 98,3 por ciento (282/"287, tres resultados falsos positivos y dos falsos negativos) y de 976 por ciento (280/287, siete resultados falsos negativos), respectivamente. Comparativamente Syphilis Fast fue más sensible que Mercia Syphilis Total EIA para la detección de sífilis (98,4 vs. 94,3 por ciento) y ligeramente menos específico (98,2 por ciento vs. 100 por ciento). Conclusiones Syphilis Fast es una prueba rápida, coste-efectiva, fácil de usar y precisa. Los resultados de este estudio indican que Syphilis Fast puede ser una alternativa a otras pruebas treponémicas para el serodiagnóstico de sífilis en los laboratorios clínicos (AU)

Estudio epidemiológico de las parasitosis intestinales 2000-2001 en población ambulatoria del Área 1 de la Comunidad de Madrid / Epidemiological study of intestinal parisitism 2000-2001 of outpatients in a health area (Madrid, Spain)

Objetivo: Conocer la prevalencia de parasitosis intestinales en población ambulatoria de nuestra Área Sanitaria de Salud. Diseño: Estudio retrospectivo observacional. Marco poblacional Área Sanitaria 1 de la Comunidad de Madrid, España. Material y métodos: Durante el período de estudio (enero 2000-diciembre 2001) han sido procesadas 6.137 muestras fecales (5.783 heces y 354 muestras para diagnóstico de oxiuros, 312 torundas vaselinizadas y 42 celos) procedentes de 3.396 pacientes. Las heces fueron procesadas mediante técnica de concentración en formol-acetato de etilo. La detección de ooquistes de coccidios se llevó a cabo mediante tinción de ácido-alcohol resistencia de Kinyoun modificada, de frotis directos y concentrados. Resultados: La prevalencia de parasitación intestinal ha sido del 13,5 per cent (827 muestras fecales positivas, pertenecientes a 464 pacientes, detectándose un total de 1.029 parásitos). La distribución de los parásitos observados y su prevalencia ha sido: Blastocystis hominis (39,3 per cent), Giardia intestinalis (19,3 per cent), Entamoeba coli y Endolimax nana (12,0 per cent cada uno), Enterobius vermicularis (6,9 per cent), Cryptosporidium parvum (3,6 per cent), Entamoeba histolytica/dispar (3,2 per cent), Trichuris trichiura (1,8 per cent), Hymenolepis nana (0,8 per cent), Taenia saginata (0,5 per cent), Ascaris lumbricoides (0,4 per cent) y Uncinarias (0,1 per cent). Los parásitos intestinales identificados más frecuentemente han sido: Giardia intestinalis (52,8 per cent), Enterobius vermicularis (18,8 per cent), Cryptosporidium parvum (9,8 per cent) y Entamoeba histolytica/dispar (8,7 per cent). Han sido analizados el origen de las muestras, edad, distribución por sexos y procedencia de los pacientes así como la incidencia estacional de los parásitos intestinales detectados. Conclusiones: Nuestros hallazgos sugieren que las infecciones parasitarias todavía constituyen un problema de salud pública. El conocimiento de la situación en cada área facilita su manejo y control (AU)

Increment of the platelet count in temporal arteritis: response to therapy and ischemic complications.

Temporal arteritis (TA) is a vasculitis involving mainly cranial branches of the aorta that can lead to ischemic complications such as amaurosis or ischemic stroke. Increment in the platelet count has been described in the acute period of the disease. We studied retrospectively the platelet count in patients with TA, its association with ischemic complications and quantified its response to therapy. We found thrombocytosis in 44% of 34 patients with TA, with a mean reduction in the platelet count of 25.1% after therapy. We were also able to quantify the increment in the platelet count at the onset of the disease and its response to prednisone in a group of 5 patients. We did not find any relation between platelet count and ischemic complications of the disease or the result of the temporal artery biopsy. In conclusion TA is associated with an increase in the platelet count, with a 25% reduction after prednisone therapy. These two determinations are not related to ischemic complications of the disease.

[Isolation and characterization of Azotobacter sp. for the production of poly-beta-hydroxyalkanoates]. / Aislamiento y caracterización de Azotobacter sp. para la producción de poli-beta-hidroxialcanoatos.

A microorganism (Azotobacter sp.) was isolated from soil samples from the Agronomy Faculty campus and its ability to accumulate poly-beta-hydroxyalkanoate (PHAs) polymers was analyzed. The isolated strain (named No 8) accumulated 8 micrograms PHA/m of culture media as intracytoplasmic granules. The following properties of the strain were analyzed: utilization of different carbon sources, antibiotic resistance, optimal pH and temperature, pigment production, nitrogen fixation, proteolytic activity, acid and hydrogen sulphide production, optimal growth temperature, cyst formation, growth on phenol and sodium fluoride, catalase, pleomorfism and mobility. The synthesized polymer showed valuable properties respect to organic solvents resistance.

Aislamiento y caracterización de Azotobacter sp. para la producción de poli-beta-hidroxialcanoatos / [Isolation and characterization of Azotobacter sp. for the production of poly-beta-hydroxyalkanoates]

A microorganism (Azotobacter sp.) was isolated from soil samples from the Agronomy Faculty campus and its ability to accumulate poly-beta-hydroxyalkanoate (PHAs) polymers was analyzed. The isolated strain (named No 8) accumulated 8 micrograms PHA/m of culture media as intracytoplasmic granules. The following properties of the strain were analyzed: utilization of different carbon sources, antibiotic resistance, optimal pH and temperature, pigment production, nitrogen fixation, proteolytic activity, acid and hydrogen sulphide production, optimal growth temperature, cyst formation, growth on phenol and sodium fluoride, catalase, pleomorfism and mobility. The synthesized polymer showed valuable properties respect to organic solvents resistance.

Aislamiento y caracterización de Azotobacter sp. para la producción de poli-beta-hidroxialcanoatos. / [Isolation and characterization of Azotobacter sp. for the production of poly-beta-hydroxyalkanoates]

A microorganism (Azotobacter sp.) was isolated from soil samples from the Agronomy Faculty campus and its ability to accumulate poly-beta-hydroxyalkanoate (PHAs) polymers was analyzed. The isolated strain (named No 8) accumulated 8 micrograms PHA/m of culture media as intracytoplasmic granules. The following properties of the strain were analyzed: utilization of different carbon sources, antibiotic resistance, optimal pH and temperature, pigment production, nitrogen fixation, proteolytic activity, acid and hydrogen sulphide production, optimal growth temperature, cyst formation, growth on phenol and sodium fluoride, catalase, pleomorfism and mobility. The synthesized polymer showed valuable properties respect to organic solvents resistance.

[Amebic liver abscess in Tarapoto-Peru]. / Absceso hepático amebiano en Tarapoto-Perú.

UNLABELLED: In order to know the incidence and epidemiologic features of the Amebic Hepatic Abscess we realized this study in the medicine service. 86.67% were males, the average age was 41.38 +/- 18.60 years old being more frequent between 30 and 69 years old (74.48%). The more affected were farmers (60%), students (10%) and Housekeepers (6.67%). The average time of the disease was 12.12 +/- 6.35 dias. The most frequent symptoms were abdominal pain in the upper right quadrant (96.66%), Hepatomegaly (83.33%), Fever (82.22%), Diarrhea (37.77%), Nausea (36.66%), Jaundice (33.33%). The initial diagnosis was AHA (45.55%), acute cholecystitis (14.44%), generalized infectious syndrome (7.77%), acute hepatitis (6.66%). The most frequent studies was: echography (98.85%). AHA alone was in the right lobe (84.05%), left lobe (14.49%). The bigger abscess was of 12 cm in diameter. The treatment was metronidazole + antibioticos (37.78%), metronidazole + antibiotico+percutaneous drainage (24.45%), Metronidazole + surgical drainage (3.33%). The complications were right pleural effusion (8.89%), peritonitis (5.56%) and pioneumothorax (1.11%). The hospitalization time was 14 +/- 8.02 days. There was one death (1.11%). CONCLUSION: The AHA was ones of each 76 deliveries in our medicine service.

[An epidemiological study of tuberculous infection]. / Un estudio epidemiológico de la infección tuberculosa.

We have the stage (or condition) of the tuberculous infection in a groups of 1,450 Schoolchildren aged between 6 and 13 years living in Madrid during the school-year 1988/89. For this we used the PPD-RT 23 tuberculin applying 2UT and considering as positive those reactions which were equal or greater 10mm. The prevalence of the tuberculous infection was 1.47% for children aged 6 years and acquire 4.3% for those aged 13 years with greater risk of 3.02 to acquire the infection in the children aged of thirteen in opposition with those of six years. There are no significant statistical significant statistical differences between boys and girls, nor between private and public colleges. We also found that the annual increment of the infection for the vaccinated-children was of 0.14%, while in no vaccinated it was of 0.64%, being of 0.41% for the whole group. The finally we emphasized the existence of a clear increment of the tuberculous infection related to age-group, being the risk of infection greater in no-vaccinated children aged of 13 and from the other part the necessity to amplify the time duration of this type of studies in order to calculate the RAI in Madrid.