The feasibility of PCR-based diagnosis of Prader-Willi and Angelman syndromes using restriction analysis after bisulfite modification of genomic DNA.

We have developed a novel PCR-based method for studying DNA methylation in the proximal region of 15q, using restriction analysis after bisulfite treatment of genomic DNA. This protocol can be used for the diagnosis of Prader-Willi and Angelman syndromes. Unlike the recently reported methylation-specific PCR protocol, our method avoids the use of multiplex amplification, thus overcoming the need to adjust relative primer amounts and the risk of obtaining false-negative results.

Hyperphosphorylated tau in SY5Y cells: similarities and dissimilarities to abnormally hyperphosphorylated tau from Alzheimer disease brain.

Unlike normal tau, abnormally hyperphosphorylated tau (AD P-tau) from Alzheimer disease (AD) does not promote but instead inhibits microtubule assembly and disrupts already formed microtubules. Tau in the human neuroblastoma cell line SH-SY5Y is hyperphosphorylated at several of the same sites as AD P-tau, and accumulates in the cell body without any association to the cellular microtubule network. The aim of the present study was to elucidate why the SY5Y tau does not affect the viability of the cells.

Quantification of sub-femtomole amounts of Alzheimer amyloid beta peptides.

We evaluated methods for the quantitative Western blot analysis of A beta 1-40 and A beta 1-42. Both chromogenic and chemiluminescent detection methods gave similar sensitivities (0.15 fmol of A beta 1-40 and 0.

Expression studies of CLN3 protein (battenin) in fusion with the green fluorescent protein in mammalian cells in vitro.

The gene for Batten disease, the CLN3 gene, encodes a novel, highly hydrophobic, multitransmembrane protein, predicted to consist of 438 amino acid residues. We have expressed a full-length CLN3 protein in fusion with green fluorescent protein in various cell lines to provide its initial biochemical characterization and subcellular localization. By using Western blotting, Percoll density gradient fractionation, and Triton X-114 extraction, we demonstrate that the product of the CLN3 gene, which we call battenin, in mammalian expression system studied is a highly glycosylated protein of lysosomal membrane.

Analysis of intracellular distribution and trafficking of the CLN3 protein in fusion with the green fluorescent protein in vitro.

CLN3 gene, associated with juvenile neuronal ceroid lipofuscinosis, encodes a novel protein of a predicted 438 amino acid residues. We have expressed a full-length CLN3 protein and fragments thereof in fusion with green fluorescent protein in Chinese hamster ovary and human neuroblastoma cell lines to study its subcellular localization and intracellular trafficking pattern. By using laser scanning confocal microscopy, we demonstrate that the full-length CLN3 fusion protein is targeted to lysosomal compartments.

Studies of membrane association of CLN3 protein.

The product of the CLN3 gene is a novel protein of unknown function. Simulations using amphiphacy algorithms have shown that structurally CLN3 may be another candidate for the family of membranous proteins. Signals controlling intracellular targeting of many membrane proteins are present as short sequences within their cytoplasmic domains.

Cerebellar atrophy in Alzheimer's disease-clinicopathological correlations.

Morphometry of the cerebellum of 11 subjects who died in the severe, final stage of Alzheimer's disease (AD) and of five age-matched subjects without dementia revealed significant atrophy in the AD group, with a decrease in the volume of the molecular layer by 24% and of the granular layer by 22% in comparison with controls. The 32% decrease in the total number of Purkinje cells that was observed correlates with the atrophy of the molecular layer, whereas the 30% reduction in the total number of granule cells correlates with the atrophy of the molecular and granular layers. A unique pattern of Alzheimer-type pathology was observed in the cerebellum: (1) there were no neurofibrillary changes in the cerebellum of either the control or the AD subjects, (2) there was almost the same extent of leptomeningeal and cortical amyloid angiopathy in the normal aged subjects and in the AD patients, and (3) the presence of plaques was noted in the AD group, but not in the control group.

Mitotic index and Alzheimer's disease.

Alzheimer's disease (AD), a progressive neurodegenerative disorder, is diagnosed definitively by increased numbers of beta-amyloid plaques and neurofibrillary tangles in brain biopsy or autopsy specimens. There are no simple straightforward laboratory tests currently available for clinical diagnosis. We have found consistent reduction in mitotic index levels in skin fibroblast cultures from AD individuals compared with age- and sex-matched controls.

Analysis of the incubation periods, induction of obesity and histopathological changes in senescence-prone and senescence-resistant mice infected with various scrapie strains.

The similarity in histopathological changes seen in scrapie-infected mice and in an uninfected senescence-accelerated mouse strain led to a study in which the mouse strain that is prone to senescence (SAMP8), a strain that is resistant to senescence (SAMR1) and a progenitor strain (AKR) of these two strains were infected with three different scrapie strains, ME7, 139A and 22L. For each scrapie strain, the incubation period was shortest in AKR mice and longest in SAMR1 mice. The induction of obesity was a function of scrapie strain and not mouse strain; ME7 caused obesity in all mouse strains, whereas the average weights of mice injected with 139A and 22L did not differ significantly from mice injected with homogenates of normal mouse brain.

Earlier onset of Alzheimer's disease in men with Down syndrome.

Background: Virtually all individuals with Down syndrome (DS) have neuropathologic changes characteristic of Alzheimer's disease (AD) beginning at 40 years of age. Few studies have examined factors that influence age at onset of AD in DS. We investigated whether sex differences in age at onset and risk of AD among adults with DS are similar to those observed in the general population and whether the effect of sex on risk of AD is modified by apolipoprotein E (APOE) genotype.

Factors secreted by activated microglia and monocytes reduce amyloidogenesis in vascular smooth muscle cells.

Smooth muscle cells cultured from amyloid-beta-affected arteries accumulate amyloid-beta peptide A beta. We now show that accumulation of "A beta" deposits in this model can be significantly reduced by culture in conditioned media from microglia and monocytes. Reduced A beta accumulation was associated with (i) lower secretion of A beta, (ii) increased secretion, but not cellular levels of amyloid-beta-precursor protein (A beta PP), and (iii) increased cell proliferation and metabolic activity.

Early menopause in women with Down's syndrome.

We used the AAMR's Adaptive Behavior Scale to ascertain current menstrual status in a population-based sample of 157 women with Down's syndrome (DS) and 187 women with other intellectual disability, all 40 years of age or older. The age-adjusted likelihood of menopause was twice as high in women with DS syndrome as in women with other intellectual disability (OR = 2.3; 95% CI = 1.

Upregulation of actin gene expression in cells expressing exogenous beta-amyloid precursor protein.

We investigated the effects of beta-amyloid peptide precursor (APP) overexpression upon the levels of other mRNAs. Using quantitative slot-blot hybridization and immunoblot analysis we observed that enhanced levels of APP elevated the levels of beta-actin and beta-actin mRNA. Our results also suggest that the cytoplasmic domain of APP is crucial for the elevation in beta-actin gene expression.

Expression studies of CLN3 protein.

Expression of the gene for Batten disease (CLN3) was studied in Escherichia coli and in a cell-free rabbit reticulocyte expression systems. A full-length recombinant fusion CLN3 protein was not produced in the bacterial systems used. However, both N-terminal fragment encompassing 246 amino acids and short C-terminal fragment containing 428-438 amino acids of the CLN3 protein were successfully overexpressed in bacteria.

Immunodiagnosis of prion disease.

The immunodiagnosis of prion diseases is of critical importance due to the transmissibility of these conditions and their fatal prognosis. A panel of monoclonal and polyclonal antibodies have been generated for use in the study and diagnosis of these diseases. This manuscript describes the generation and characterization of these antibodies as well as their diagnostic application.

Distribution, levels, and activity of glycogen synthase kinase-3 in the Alzheimer disease brain.

A number of studies have implicated a proline-directed protein kinase, glycogen synthase kinase-3 (GSK-3) in the hyperphosphorylation of tau in Alzheimer's disease (AD). Toward understanding the role of GSK-3 in the abnormal hyperphosphorylation of tau in AD we have found that GSK-3 is prominently present in neuronal cell bodies and their processes and co-localizes with neurofibrillary changes in AD brain. Furthermore, the levels of GSK-3 as determined by indirect ELISA are approximately 50% increased in the postsynaptosomal supernatant from AD brains as compared to the controls.

Profilin and gelsolin stimulate phosphatidylinositol 3-kinase activity.

Actin-binding proteins such as profilin and gelsolin bind to phosphatidylinositol (PI) 4,5-bisphosphate (PI 4,5-P2) and regulate the concentration of monomeric actin. We report here that profilin and gelsolin stimulate PI 3-kinase-mediated phosphorylation of PI 4,5-P2 (lipid kinase activity) in a concentration-dependent manner. This effect is specific to profilin and gelsolin because other cytoskeletal proteins such as tau or actin do not affect PI 3-kinase activity.

Osteoglophonic dysplasia: review and further delineation of the syndrome.

We report on a boy with clinical and radiologic findings of osteoglophonic dysplasia. He had craniostenosis, "bizarre," expansile cystic lesions in the diaphyses, delayed tooth eruption, and progressive rib expansion typical of the syndrome. Initially delayed psychomotor development with later normal intelligence, early feeding and breathing difficulty, and speech delay are also characteristic of the disorder.

Onset of dementia is associated with apolipoprotein E epsilon4 in Down's syndrome.

We examined the influence of apolipoprotein E (apoE) genotype on risk of dementia in 82 adults with Down's syndrome (DS). Compared with those with an apoE 3/3 genotype, the group of adults with DS with apoE 2/4, 3/4, and 4/4 genotypes were 5 times more likely to become demented (RR = 4.7; 95% CI = 1.

Tissue differences in fragile X mosaics: mosaicism in blood cells may differ greatly from skin.

The fragile X mutation is diagnosed from the structure of the FMR1 gene in blood cell DNA. An estimated 12 to 41% of affected males are mosaics who carry both a "full mutation" allele from which there is no gene expression and a "premutation" allele which has normal gene expression. We compared the DNA in blood cells and skin fibroblasts from four mosaic fragile X males to see if there was a difference in the relative amounts of premutation and full mutation alleles within the tissues of these individuals.

Suppression of Alzheimer amyloid precursor protein (APP) expression by exogenous APP mRNA.

To establish a cell line expressing enhanced levels of beta-amyloid precursor protein (beta-APP), we constructed plasmid DNAs expressing beta-APP-751 mRNA and transfected them into COS-1 cells. Using a modified version of the reverse transcriptase polymerase chain reaction which is RNA sensitive to study the beta-APP iso-RNAs, we have made the unexpected observation that enhanced expression of beta-APP-751 mRNA resulted in a significant reduction of beta-APP-770 and -695 mRNA levels. Suppression of beta-APP-770 and -695 was also observed in cells expressing truncated and chimeric beta-App-751 mRNAs.

Glutamate agonist activity: implications for antipsychotic drug action and schizophrenia.

Antagonist action at dopamine D2 receptors appears to explain many, but not all of the effects of antipsychotic drugs. Because of the interactions of dopamine with glutamate, and the implication of the latter in the etiology of schizophrenia, possible effects of antipsychotic drugs on glutamate receptors were assessed in the present experiments. These studies showed that, at clinically relevant concentrations, the conventional neuroleptic haloperidol and the atypical antipsychotic clozapine had potent augmenting influences on the NMDA receptor.

Intercellular adhesion molecule-1 (ICAM-1) upregulation in human brain tumors as an expression of increased blood-brain barrier permeability.

The distribution of intercellular adhesion molecule (ICAM-1) binding sites was studied in the microvasculature of several types of human brain tumor biopsies (angioma, glioblastoma multiforme and meningioma). Immunoelectron microscopy was performed with the application of immuno-HRP or -gold probes using a pre-embedding technique. Ultrastructural analysis demonstrated a pronounced ICAM-1 upregulation on the luminal EC and/or perivascular surfaces.