Beta-amyloid-induced activation of caspase-3 in primary cultures of rat neurons

It is known that beta-amyloid peptide (Abeta) contributes to the neurodegeneration in Alzheimer's disease (AD) and operates through activation of an apoptotic pathway. Apoptotic signal is driven by a family of cysteine proteases called caspases. The beta-amyloid precursor protein (APP) is directly and efficiently cleaved by caspases during apoptosis, resulting in elevated beta-amyloid peptide formation. Cerebellar neurons from rat pups were treated with the aged Abeta(25-35) at 1 and 5 microM and fluorescence assays of caspase activity performed over 4 days. We observed an increase in caspase activity after 48 h treatment in both 1 and 5 microM treated cells, then (72-96 h) caspase activity decreased to control values. The data presented support the hypothesis that Abeta(25-35)-induced apoptosis is mediated by the activation of Caspase-3 and that this is a transient effect.     

Immunohistochemical classification of non-BRCA1/2 tumors identifies different groups that demonstrate the heterogeneity of BRCAX families.

Around 25% of hereditary breast and ovarian cancer families have mutations in the BRCA1 and BRCA2 genes. The search for other genes has until now failed, probably because there is not one single BRCAX gene, but rather various genes that may each be responsible for a small number of breast cancer families and/or may interact according to a polygenic model. We have studied 50 tumors from probands belonging to non-BRCA1/2 breast cancer families (BRCAX), using 25 immunohistochemical markers. The objective was to classify these tumors and confirm that they are heterogeneous. Unsupervised cluster analysis showed the existence of the following two main groups of tumors: high-grade and estrogen receptor (ER)-negative tumors (50%), and low-grade and ER-positive tumors (50%). In addition we identified five subgroups, three among the high-grade and two among the low-grade groups; one overexpressing HER-2 (18%); one with a basal-like phenotype (14%); one with a normal breast-like phenotype (18%); a luminal A subgroup (36%), and a luminal B subgroup (14%). Hypermethylation of the BRCA1 gene was observed in 42% of the cases, spread across all five subgroups, but only 37% of those had loss of heterozygosity as well. These latter cases were all clustered in the high-grade group and the majority of them in the basal-like subgroup. Our results show that familial non-BRCA1/2 tumors are heterogeneous and suggest a polygenic model for explaining the majority of BRCAX families. In addition we have defined a subset of them that have somatic inactivation of the BRCA1 gene.     

Heat shock proteins (HSPs) 27, 72 and 73 in normal and pre-ulcerative mucosa of the gastric pars oesophagea in swine

Heat shock proteins (HSPs), known to play a key role in cellular homeostasis, may also play a role in the defensive mechanisms of gastric mucosa. By means of appropriate immunohistochemical and immunobiochemical techniques, the expression of HSP27, HSP72 and HSP73 within the epithelium of normal and pre-ulcerative (hyperkeratinized) mucosa of the pars oesophagea of abattoir pigs was assessed. In normal mucosa, HSP72 and HSP73 expression was mainly limited to the basal epithelial cell layer, whereas HSP27 expression was consistently detected within the superficial epithelial cell layers. In hyperkeratinized mucosa, HSP72 and HSP73 immunoreactivity appeared to be more widespread, becoming very intense within epithelial cells affected by hydropic degeneration. Hyperkeratinized mucosa also showed HSP27 immunoreactivity, which was particularly intense in epithelial areas affected by hydropic degeneration. Western blot analysis confirmed HSP27, HSP72 and HSP73 expression in normal and in pre-ulcerative mucosa of the pars oesophagea. Semi-quantitative analysis showed that for all three HSPs the immunoreactivity was more intense in pre-ulcerative mucosa than in normal mucosa. The different expression patterns observed may have functional significance; further studies are needed, however, to define the role of HSPs in swine oesophagogastric lesions, the aetiology and pathogenesis of which are largely unknown.     

Significantly reduced expression of the proteoglycan decorin in Alzheimer''s disease fibroblasts

Aims—To investigate whether proteoglycan synthesis is altered in skin fibroblasts in patients with Alzheimer's disease compared with normal subjects.     

Reelin immunoreactivity in the adult sea lamprey brain

The expression of reelin, a large extracellular matrix glycoprotein, was studied in the brain of pre-spawning adult sea lampreys by immunohistochemistry using two monoclonal antibodies against this protein. Reelin immunoreactive (reln-ir) neurons were observed in the olfactory bulb, and pallial and subpallial regions in the telencephalon. In the diencephalon, reln-ir cells were observed in some hypothalamic nuclei, in the nucleus of Bellonci, and in the habenula. In the mesencephalon, this protein was detected in several nuclei related with the centrifugal visual system, although the optic tectum was devoid of immunoreactivity. The hindbrain showed several nuclei with immunopositive neurons, including the branchiomeric nerve motor nuclei and also some groups of non-giant cells of the reticular formation. The rostral spinal cord showed some immunopositive neurons mainly located in lateral and ventral positions. Overall, the pattern of distribution of reelin in the adult sea lamprey correlates with the previously reported in other adult vertebrates. Furthermore, the wide distribution of reelin in the adult lamprey brain is consistent with a possible existence of different roles for this protein not related with development in the central nervous system (CNS) of vertebrates (i.e. neuronal plasticity and/or maintenance).     

GLP-1 signalling and effects on glucose metabolism in myocytes from type 2 diabetic patients

Changes in the activity of glycogen synthase a and related kinases (phosphatidylinositol-3-kinase, protein kinase B, p44/42 MAP kinases and p70s6 kinase) evoked by GLP-1 in human myocytes from normal subjects were recently implied in the effect of this hormone upon D-glucose transport and glycogen synthesis in the same cells. The major aims of the present study were i) to investigate the possible extension of this knowledge to myocytes obtained from type 2 diabetic patients, ii) to compare in these patients the response to GLP-1, insulin or the structurally related GLP-1 peptides, exendin (1-39)amide and exendin(9-39)amide, and iii) to explore possible differences in the responsiveness to these agents between normal and diabetic subjects. Apart from the much higher basal PI3K activity and impaired response to insulin of p44/42 MAP kinases in the diabetic patients, the changes in enzyme activity caused by either hormone or peptide, although not identical, were essentially comparable. Nevertheless, significant differences in glucose transport and metabolism parameters were observed in the diabetic patients vs. normal subjects: in the diabetic patients, basal 2-deoxy-glucose uptake and glycogen synthase a activity were lower, accompanied by a similar increasing effect of GLP-1 or insulin; yet, the basal value for glycogen synthesis was higher, coinciding with a lesser relative increment in response to GLP-1 or insulin.     

High-Throughput Generation of P. falciparum Functional Molecules by Recombinational Cloning

Large-scale functional genomics studies for malaria vaccine and drug development will depend on the generation of molecular tools to study protein expression. We examined the feasibility of a high-throughput cloning approach using the Gateway system to create a large set of expression clones encoding Plasmodium falciparum single-exon genes. Master clones and their ORFs were transferred en masse to multiple expression vectors. Target genes (n = 303) were selected using specific sets of criteria, including stage expression and secondary structure. Upon screening four colonies per capture reaction, we achieved 84% cloning efficiency. The genes were subcloned in parallel into three expression vectors: a DNA vaccine vector and two protein expression vectors. These transfers yielded a 100% success rate without any observed recombination based on single colony screening. The functional expression of 95 genes was evaluated in mice with DNA vaccine constructs to generate antibody against various stages of the parasite. From these, 19 induced antibody titers against the erythrocytic stages and three against sporozoite stages. We have overcome the potential limitation of producing large P. falciparum clone sets in multiple expression vectors. This approach represents a powerful technique for the production of molecular reagents for genome-wide functional analysis of the P. falciparum genome and will provide for a resource for the malaria resource community distributed through public repositories.