Infantile neuronal ceroid-lipofuscinosis is not an allelic form of Batten disease: exclusion of chromosome 16 region with linkage analyses

Infantile neuronal ceroid-lipofuscinosis (CLN1) is the form of neuronal ceroid-lipofuscinoses (NCL) with the earliest onset of symptoms. The locus of the most common form of these disorders, juvenile NCL (CLN3), has been mapped to chromosome 16. We report here linkage data of the same region in Finnish CLN1 families. Our results indicate that CLN1 is not allelic with CLN3 but represents a different locus, which is not located within about 70 cM in chromosome 16.     

Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Several cooperating binding sites mediate the interaction of a lysosomal enzyme with phosphotransferase.

Lysosomal targeting of soluble lysosomal hydrolases is mediated by mannose 6-phosphate receptors, which recognize and bind mannose 6-phosphate residues in the oligosaccharide chains of proteins destined for delivery to lysosomes. This recognition marker is generated by the sequential action of two enzymes, the first of which, UDP-N-acetylglucosamine phosphotransferase, recognizes lysosomal enzymes on the basis of a structural determinant in their polypeptide chains. This recognition event is a key step in lysosomal targeting of soluble proteins, but the exact nature of the recognition determinant is not well understood. In this study we have characterized the phosphotransferase recognition signals of human lysosomal aspartylglucosaminidase (AGA) using transient expression of polypeptides carrying targeted amino acid substitutions. We found that three lysine residues and a tyrosine residing in three spatially distinct regions of the AGA polypeptide are necessary for phosphorylation of the oligosaccharides. Two of the lysines are especially important for the lysosomal targeting efficiency of AGA, which seems to be mostly dictated by the degree of phosphorylation of the alpha subunit oligosaccharide. On the basis of the results of this and previous studies we suggest a general model for recognition of lysosomal enzymes by the phosphotransferase.     

Congenital nephrotic syndrome of the Finnish type maps to the long arm of chromosome 19.

Congenital nephrotic syndrome of the Finnish type (CNF) is an autosomal recessive disease that is characterized by massive proteinuria and nephrotic syndrome at birth. CNF represents a unique, apparently specific dysfunction of the renal basement membranes, and the estimated incidence of CNF in the isolated population of Finland is 1 in 8,000 newborns. The basic defect is unknown, and no specific biochemical defect or chromosomal aberrations have been described. Here we report the assignment of the CNF locus to 19q12-q13.1 on the basis of linkage analyses in 17 Finnish families. Multipoint analyses and observed recombination events place the CNF locus between multiallelic markers D19S416 and D19S224, and the significant linkage disequilibrium observed suggests that the CNF gene lies in the immediate vicinity of the markers D19S224 and D19S220.     

Melanoma cell lines are susceptible to histone deacetylase inhibitor TSA provoked cell cycle arrest and apoptosis

Melanoma is the most aggressive of skin cancers because of its high resistance to currently available therapy. Although melanoma cells often retain wild-type p53 tumour suppressor protein and express it at high levels, the p53 mediated apoptosis pathway is suppressed. Histone deacetylase (HDAC) inhibitors are a promising group of compounds inducing differentiation, growth arrest and apoptosis in tumour cells in preclinical studies. We have studied the cellular effects of trichostatin A (TSA), a HDAC inhibitor, in a panel of melanoma cell lines and its mechanism of action in relation to p53. TSA stabilized wild-type p53, but p53 protein accumulation was overridden by simultaneous downregulation of p53 mRNA leading to a decrease in p53 protein. While growth arrest was induced in all cell lines studied and apoptosis in most (6/7), these cellular effects were independent of the p53 status of the cells. Inhibiting p53 function by a dominant negative p53 (p53(175His)) confirmed that the HDAC inhibitor induced apoptosis was independent of wild-type p53, even though TSA slightly activated p53 in a reporter assay. The results indicate that while the action of TSA is independent of p53, the activation of the apoptosis pathway by the HDAC inhibitors may provide therapeutic approaches for melanoma treatment.     

Marfan syndrome: exclusion of genetic linkage to five genes coding for connective tissue components in the long arm of chromosome 2

Summary Marfan syndrome represents a heterogeneous connective tissue disease, the symptoms arising in several tissues and organs. The defective gene(s) behind this autosomal dominant condition has not been found despite considerable research. The main targets of the research have been the genes coding for connective tissue components. Several of the candidate genes suspected to be defective in Marfan syndrome are located on the long arm of chromosome 2. These genes include a cluster of two genes coding for fibrillar collagens COL3A1 and COL5A2, and a third member of the collagen gene family: COL6A3. Furthermore, genes for elastin (ELN) and fibronectin (FN) are also located in this area of chromosome 2. We studied this chromosomal area using restriction fragment length polymorphism (RFLP) linkage analysis in five Finnish Marfan families with affected members in three generations. In two point linkage analyses, Lod scores of –3.192 ( = 0.1) to COL3A1, –1.683 ( = 0) to COL6A3 and –2.664 ( = 0.01) to FN were obtained, whereas the linkage analysis between elastin and the disease was non-informative (Lod score 0.444, = 0). With the multipoint linkage analysis that permits simultaneous examination of several loci and more efficient use of family data, we obtained an exclusion of all these loci as the site of the mutation leading to Marfan syndrome in these families.