An Ile93Met substitution in the UCH-L1 gene is not a disease-causing mutation for idiopathic Parkinson’s disease

Objective To ascertain whether a coding mutation(Ⅱe93Met) in ubiquitin carboxy-terminal hydrolase(UCH-L1) gene plays a role in idiopathic Parkinson‘s disease(IPD).Methods Polymerase chain reaction-restriction fragment length polymorphism assay(PCR-RFLP) was used to distingusih the wild-type(two DNA fagments of 34 and 126bp)from the variant allele(three fragments of 34,60 and 66bp)because the mutation created a new site for restriction engonuclease Bsm F1.DNA was isolated from various blood samples using a phenolchlorofom extraction.     

ER membrane ubiquitin ligase Hrdl mediates tau degradation in proteosome-dependent and independent manner

Abnormal accumulation of hyperphosphorylated tau protein in intracellular inclusions is a recognized pathological feature in a number of dementias, which is collectively called tauopathies. To explore the molecular mechanism by which tauopthies are regulated, we investigated the effects of human Hrdl, an endoplasmic reticulum （ER） membrane ubiquitin ligase （E3） with RING finger domain,     

Effects of high doses of glucocorticoids on free amino acids,ribosomes and protein turnover in human muscle

BACKGROUND: Treatment with glucocorticosteroids causes a negative nitrogen balance, but the kinetic mechanisms responsible for this catabolic effect are controversial. We investigated the effects of 60 mg day(-1) prednisolone on protein synthesis and degradation in human skeletal muscle. MATERIALS AND METHODS: Healthy adults (n = 9) were studied in the postabsorptive state, before and after 3 days of prednisolone treatment. The L-[ring 2,6(-3)H(5)]-phenylalanine tracer technique, concentration and size distribution of the ribosomes, mRNA content of the ubiquitin-proteasome pathway components in muscle, phenylalanine flux across the leg, and the free amino acid concentrations in skeletal muscle were used to study muscle protein metabolism. RESULTS: The concentrations of most amino acids in arterial blood increased after prednisolone. There were also increased effluxes of phenylalanine, asparagine, arginine, alanine, methionine and isoleucine from the leg. The rate of protein degradation, as measured by the appearance rate (Ra) of phenylalanine, increased by 67% (P = 0.023) which, together with a doubling of the net release of phenylalanine from the leg (P = 0.007), indicated accelerated protein degradation. The pathway was not identified but there was no significant increase in mRNAs' encoding components of the ubiquitin-proteasome pathway. There was a 6% reduction in polyribosomes (P = 0.007), suggesting a decrease in the capacity for protein synthesis, although there was no measured decrease in the rate of protein synthesis. CONCLUSIONS: These findings indicate that high doses of prednisolone lead to a sharp increase in net protein catabolism, which depends more on enhanced protein breakdown, and an uncertain effect on protein synthesis. The mechanisms stimulating proteolysis and the pathway stimulated to increase muscle protein degradation should be explored.     

Cbl Ubiquitin Ligases Control B Cell Exit from the Germinal-Center Reaction

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Autophagy is activated by proteasomal inhibition and involved in aggresome clearance in cultured astrocytes

A common pathway underlying a variety of neurodegenerative disorders is the aggregation and deposition of misfolded proteins. Proteasomal inhibition has been demonstrated to promote the formation of intracellular inclusions. We have shown before that astrocytes respond to the treatment with the proteasome inhibitor MG‐132 by aggresome formation and cytoskeletal disturbances, but unlike oligodendrocytes do not die by apoptotic cell death and have the capability to recover. This study was undertaken to elucidate if the autophagy‐lysosomal pathway participates in the efficient recovery process in astrocytes and is modulated under conditions of proteasomal inhibition. The data show that the autophagic pathway was stimulated during a 24‐h treatment with the proteasome inhibitor MG‐132 in a time and concentration‐dependent manner. It remained at an elevated level throughout a 24‐h recovery period in the absence of MG‐132 and participates in the aggregate clearing process. In the presence of the specific inhibitor of macroautophagy, 3‐methyladenine, cell viability was impaired, aggregates were not as efficiently removed and HSP25, αB‐crystallin and ubiquitinated proteins remained in the insoluble protein fraction. LC3‐II positive puncta, indicative of autophagosomes, were formed abundantly in the cells after proteasome inhibition and were seen in close association with the aggregates. Hence, the ability of astrocytes to upregulate autophagic degradation might contribute to their resistance against proteasomal stress situations and act as a compensatory mechanism when the proteasome is impaired. © 2010 Wiley‐Liss, Inc.     

Amyotrophic lateral sclerosis and neurosarcoidosis: A case report

Amyotrophic lateral sclerosis (ALS) remains a clinical diagnosis without definable biomarkers. The pathomechanism of motor neuron degeneration in ALS has yet to be elucidated. Here we present a case of limb‐onset ALS, with autopsy findings of Bunina bodies and skein‐like inclusions, as well as sarcoid granulomas predominating among motor neurons. The targeting of the motor neurons by the sarcoid inflammation raises questions regarding the role of cellular immunity in the pathomechanisms for ALS. Muscle Nerve, 2009     

Ubiquitin-proteasome system alterations in a striatal cell model of Huntington's disease

Huntington's disease (HD) is a progressive, autosomal dominant neurodegenerative disease caused by an abnormally expanded CAG repeat in the HD gene. Ubiquitylated aggregates containing mutant huntingtin protein in neurons are hallmarks of HD. Misfolded mutant huntingtin monomers, oligomers, or aggregates may be a result of, and cause, ubiquitin- proteasome dysfunction. To investigate the ubiquitin-proteasome system we designed a series of firefly luciferase reporters targeted selectively to different points along this pathway. These reporters were used to monitor ubiquitin-proteasome system function in a striatal cell culture model of HD. Ubiquitylation processes were not reduced in mutant huntingtin cells but recognition or degradation of ubiquitylated substrates was decreased. We also found mutant huntingtin expressing cells had slight but significant decreases in chymotrypsin-like and caspase-like activities, and an unexpected increase in trypsin-like activity of the proteasome core. General proteasome core inhibitors, as well as selective caspase-like activity inhibitors, were less effective in mutant cells. Finally, treatment with 3-nitropropionic acid, a succinate dehydrogenase inhibitor, had opposite effects on the ubiquitin-proteasome system with activation in wild-type and decreased activity in mutant huntingtin expressing cells. The results of these experiments show clearly selective disruption of the ubiquitin-proteasome system in this cell culture model of HD. The high throughput tools that we have designed and optimized will also be useful in identifying compounds that alter ubiquitin-proteasome system function and to investigate other neurodegenerative diseases such Alzheimer's disease and Parkinson's disease.