Mutations in DNAJC5, encoding cysteine-string protein alpha, cause autosomal-dominant adult-onset neuronal ceroid lipofuscinosis

Autosomal-dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is characterized by accumulation of autofluorescent storage material in neural tissues and neurodegeneration and has an age of onset in the third decade of life or later. The genetic and molecular basis of the disease has remained unknown for many years. We carried out linkage mapping, gene-expression analysis, exome sequencing, and candidate-gene sequencing in affected individuals from 20 families and/or individuals with simplex cases; we identified in five individuals one of two disease-causing mutations, c.346_348delCTC and c.344T>G, in DNAJC5 encoding cysteine-string protein alpha (CSPα). These mutations-causing a deletion, p.Leu116del, and an amino acid exchange, p.Leu115Arg, respectively-are located within the cysteine-string domain of the protein and affect both palmitoylation-dependent sorting and the amount of CSPα in neuronal cells. The resulting depletion of functional CSPα might cause in parallel the presynaptic dysfunction and the progressive neurodegeneration observed in affected individuals and lysosomal accumulation of misfolded and proteolysis-resistant proteins in the form of characteristic ceroid deposits in neurons. Our work represents an important step in the genetic dissection of a genetically heterogeneous group of ANCLs. It also confirms a neuroprotective role for CSPα in humans and demonstrates the need for detailed investigation of CSPα in the neuronal ceroid lipofuscinoses and other neurodegenerative diseases presenting with neuronal protein aggregation.     

Involvement of the mitochondrial compartment in human NCL fibroblasts

Neuronal ceroid lipofuscinosis (NCL) are a group of progressive neurodegenerative disorders of childhood, characterized by the endo-lysosomal storage of autofluorescent material. Impaired mitochondrial function is often associated with neurodegeneration, possibly related to the apoptotic cascade. In this study we investigated the possible effects of lysosomal accumulation on the mitochondrial compartment in the fibroblasts of two NCL forms, CLN1 and CLN6. Fragmented mitochondrial reticulum was observed in all cells by using the intravital fluorescent marker Mitotracker, mainly in the perinuclear region. This was also associated with intense signal from the lysosomal markers Lysotracker and LAMP2. Likewise, mitochondria appeared to be reduced in number and shifted to the cell periphery by electron microscopy; moreover the mitochondrial markers VDCA and COX IV were reduced following quantitative Western blot analysis. Whilst there was no evidence of increased cell death under basal condition, we observed a significant increase in apoptotic nuclei following Staurosporine treatment in CLN1 cells only. In conclusion, the mitochondrial compartment is affected in NCL fibroblasts invitro, and CLN1 cells seem to be more vulnerable to the negative effects of stressed mitochondrial membrane than CLN6 cells.     

Facultative predation and scavenging by mammalian carnivores: seasonal,regional and intra‐guild comparisons

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Long-term platinum retention after treatment with cisplatin and oxaliplatin

Background  

The aim of this study was to evaluate long-term platinum retention in patients treated with cisplatin and oxaliplatin.     

Purification and partial sequence of human osteoclast-activating factor: identity with interleukin 1 beta

The lymphokine osteoclast-activating factor (OAF) was purified to homogeneity. OAF was produced by human peripheral blood mononuclear cells stimulated with concanavalin A and phorbol myristate acetate under serum-free culture conditions. OAF was purified by sequential gel filtration, ion-exchange, and reverse-phase HPLC by following bone resorptive activity. Homogeneity was indicated by the criteria of a single 17,800-dalton band on silver-stained polyacrylamide gels, a single pI 6.8 band on isoelectric focusing, and a single aminoterminal sequence. Purified OAF stimulated half-maximal release of calcium from fetal rat long bones at a concentration of approximately 0.66 ng/ml. The amino-terminal sequence of OAF was determined and found to be identical to that of interleukin 1 beta. Homogeneous OAF possessed an activity of 8.2 X 10(6) U/mg in the thymocyte proliferation assay. Because the m.w., isoelectric point, amino-terminal sequence, and specific activity in the thymocyte proliferation assay are the same for homogeneous OAF and interleukin 1 beta, we conclude that they are the same molecule, and that interleukin 1 beta is the major protein with OAF activity produced by lectin-stimulated peripheral blood mononuclear cells.     

Use of simian virus 40 large T-beta-galactosidase fusion proteins in an immunochemical analysis of simian virus 40 large T antigen.

Simian virus 40 large T antigen is a multifunctional protein that is encoded by the early region of the viral genome. We constructed fusion proteins between simian virus 40 large T antigen and beta-galactosidase by cloning HindIII fragments A and D of the virus into the HindIII sites of expression vectors pUR290, pUR291, and pUR292. Large amounts of the fusion protein were synthesized when the DNA fragment encoding part of simian virus 40 large T antigen was in frame with the lacZ gene of the expression vector. Using Western blotting and a competition radioimmunoassay, we assessed the binding of existing anti-T monoclonal and polyclonal antibodies to the two fusion proteins. Several monoclonal antibodies reacted with the protein encoded by the fragment A construction, but none reacted with the protein encoded by the fragment D construction. However, mice immunized with pure beta-galactosidase-HindIII fragment D fusion protein produced good levels of anti-T antibodies, which immunoprecipitated simian virus 40 large T antigen from lytically infected cells, enabling derivation of monoclonal antibodies to this region of large T antigen. Therefore, the fusion proteins allowed novel epitopes to be discovered on large T antigen and permitted the precise localization of epitopes recognized by existing antibodies. The same approach can also be used to produce antibodies against defined regions of any gene.