Knockdown of ACAT-1 reduces amyloidogenic processing of APP

Previous studies have shown that acyl-coenzyme A:cholesterol acyl transferase (ACAT), an enzyme that controls cellular equilibrium between free cholesterol and cholesteryl esters, modulates proteolytic processing of APP in cell-based and animal models of Alzheimer's disease. Here we report that ACAT-1 RNAi reduced cellular ACAT-1 protein by approximately 50% and cholesteryl ester levels by 22% while causing a slight increase in the free cholesterol content of ER membranes. This correlated with reduced proteolytic processing of APP and 40% decrease in Abeta secretion. These data show that even a modest decrease in ACAT activity can have robust suppressive effects on Abeta generation.     

Site-directed solid-state NMR measurement of a ligand-induced conformational change in the serine bacterial chemoreceptor

The challenging nature of studies of membrane proteins has made it difficult to determine the molecular mechanism of transmembrane signaling. For the bacterial chemoreceptor family, there are crystal structures of the internal and external domains, structural models of the transmembrane domain, and evidence for subtle ligand-induced conformational changes, but the signaling mechanism remains controversial. We have used a novel site-directed solid-state NMR distance measurement approach, using (13)C(19)F REDOR, to measure a ligand-induced change of 1.0 +/- 0.3 A in the distance between helices alpha 1 and alpha 4 of the ligand-binding domain in the intact, membrane-bound serine receptor. This distance change is shown not to be due to motion of the side chain and thus is due to motion of either the alpha 1 or the alpha 4 helix. Additional distance measurements can be used to determine the type of backbone motion and to follow it to the cytoplasm, to test and refine current proposals for the mechanism of transmembrane signaling. This is a promising general method for high-resolution measurements of local structure in intact, membrane-bound proteins.     

Cytologic features of secretory meningioma

OBJECTIVE: To describe the cytologic features of secretory meningioma on crush preparations. STUDY DESIGN: In five cases, the diagnosis of secretory meningioma was made and crush preparations were available. In each case, crush preparations were made at the time of intraoperative consultation from an open biopsy specimen or stereotactic biopsy. RESULTS: Hematoxylin and eosin-stained crushes showed the presence of clusters of cohesive cells containing variable numbers of inclusions among less cohesive typical meningothelial cells. In two cases, the inclusions were especially prominent. Inclusions varied in size from 3 to 40 microns, had a well-defined rim and contained finely granular or hyaline material and a central core. CONCLUSION: Secretory meningiomas demonstrate distinct cytologic features on crush preparations. Recognition of these inclusions is important since their prominence in some stereotactic smear preparations may lead to diagnostic problems.     

Factor V Leiden in patients with venous thrombosis in Slovak population

Resistance to activated protein C determined by factor V Leiden (FVL) is the most frequent inherited risk factor of venous thrombosis. The purpose of our work was to reveal the frequency of FVL in Slovak patients with venous thromboses, to characterise the nature of venous thromboses in this inherited thrombophilia, and to consider the screening approach to investigation of FVL in patients with venous thromboses. 350 patients with a diagnosis of venous thromboembolic disease from various regions of Slovakia were investigated. FVL, detected by polymerase chain reaction, was found in 128/350 (37%) patients with venous thromboses. 118/128 (92%) patients were heterozygous and 10/128 (8%) were homozygous carriers. In 108/128 (84%) patients with FVL the thromboembolic disease occurred spontaneously. Phlebothrombosis occurred predominantly in the lower limbs--117/128 (91%) patients, atypical localisations were rare. The first thromboembolic event was manifested before 40 years of age in 69% of patients. The family history was positive in 60/128 (47%) FVL carriers with thromboembolic disease. Recurrent thrombosis occurred in 30% of patients with FVL. In agreement with findings in other European countries, the prevalence of FVL was high in Slovak patients with thromboembolic disease. The investigation of FVL seems to be justified in patients before 40 years of age with venous thrombosis of lower limbs, in the absence of triggering factors and with a family history of venous thromboembolic disease.     

MMDB: Entrez's 3D-structure database

Three-dimensional structures are now known within most protein families and it is likely, when searching a sequence database, that one will identify a homolog of known structure. The goal of Entrez's 3D-structure database is to make structure information and the functional annotation it can provide easily accessible to molecular biologists. To this end, Entrez's search engine provides several powerful features: (i) links between databases, for example between a protein's sequence and structure; (ii) pre-computed sequence and structure neighbors; and (iii) structure and sequence/structure alignment visualization. Here, we focus on a new feature of Entrez's Molecular Modeling Database (MMDB): Graphical summaries of the biological annotation available for each 3D structure, based on the results of automated comparative analysis. MMDB is available at: http://www.ncbi.nlm.nih.gov/Entrez/structure.html.     

Vimentin and leukocyte common antigen-negative molding cells in pleural effusions of patients with small cell lung carcinoma

OBJECTIVE: To determine whether the presence of vimentin and leukocyte common antigen (LCA)-negative molding cells (VLNMC) could help in identifying rare small cell lung carcinoma (SCLC) cells. STUDY DESIGN: Thirty-four cell blocks of pleural effusions (PEs) from 26 patients with confirmed SCLC were stained immunohistochemically with vimentin and LCA antibodies and compared with hematoxylin and eosin-stained preparations. RESULTS: VLNMC were present in 22/22 PEs originally diagnosed as positive or atypical/suspicious for SCLC. Focal vimentin staining was seen in SCLC in 10/22 cases, and 1 case showed many vimentin-positive SCLC cells. One of 11 PEs originally interpreted as negative showed rare groups of VLNMC. This was supported by a subsequent PE obviously positive for SCLC. CONCLUSION: Immunoperoxidase stains for vimentin and LCA highlight SCLC in PEs as VLNMC; however, morphologic criteria must prevail in making the final diagnosis.     

1-Methyl-4-phenylpyridinium-induced down-regulation of dopamine transporter function correlates with a reduction in dopamine transporter cell surface expression

The mechanisms whereby 1-methyl-4-phenylpyridinium (MPP(+)) mediates cell death and Parkinsonism are still unclear. We have shown that dopamine transporter (DAT) is required for MPP(+)-mediated cytotoxicity in HEK-293 cells stably transfected with human DAT. Furthermore, MPP(+) produced a concentration- and time-dependent reduction in the uptake of [3H]dopamine. We observed a significant decrease in [3H]WIN 35428 binding in the intact cells with MPP(+). The saturation analysis of the [3H]WIN 35428 binding obtained from total membrane fractions revealed a decrease in the transporter density (B(max)) with an increase in the dissociation equilibrium constant (K(d)) after MPP(+) treatment. Furthermore, biotinylation assays confirmed that MPP(+) reduced both plasma membrane and intracellular DAT immunoreactivity. Taken together, these findings suggest that the reduction in cell surface DAT protein expression in response to MPP(+) may be a contributory factor in the down-regulation of DAT function while enhanced lysosomal degradation of DAT may signal events leading to cellular toxicity.     

Phosphoenolpyruvate-dependent tubulin-pyruvate kinase interaction at different organizational levels

Evidence for the direct binding of pyruvate kinase to tubulin/microtubule and for the inhibitory effect of phosphoenolpyruvate on tubulin-enzyme hetero-association were provided by surface plasmon resonance and pelleting experiments. Electron microscopy revealed that pyruvate kinase induces depolymerization of paclitaxel-stabilized microtubules into large oligomeric aggregates and bundles the tubules in a salt concentration-dependent manner. The C-terminal "tail"-free microtubules did not bind pyruvate kinase, suggesting the crucial role of the C-terminal segments in the binding of kinase. Immunoblotting and polymerization experiments with cell-free brain extract revealed that pyruvate kinase specifically binds to microtubules, the binding of pyruvate kinase impedes microtubule assembly, and phosphoenolpyruvate counteracts the destabilization of microtubules induced by pyruvate kinase. We also showed by immunostaining the juxtanuclear localization of pyruvate kinase in intact L929 cells and that this localization was influenced by treatments with paclitaxel or vinblastine. These findings suggest that the distribution of the enzyme may be controlled by the microtubular network in vivo.     

Cloning a calcium channel alpha2delta-3 subunit gene from a putative tumor suppressor gene region at chromosome 3p21.1 in conventional renal cell carcinoma

We have identified loss of heterozygosity (LOH) of approx. 1 cM region around locus D3S1289 at chromosome 3p21.1 in a conventional renal cell carcinoma (RCC). During construction of a YAC/BAC contig for this region and shotgun sequencing of BACs 277p5, 55m24 and 428i24, we detected four new microsatellites. We narrowed down the target region by analysing these new loci to less than 100 kb within the BAC 55m24 and subsequently cloned a human calcium channel alpha2delta-3 subunit gene. This gene is widely expressed in fetal tissues and different types of adult tumors. The exons of the alpha2delta-3 subunit gene are distributed along approx. 500 kb DNA sequences. As the LOH involved exclusively intronic sequences and sequencing the entire coding region did not reveal any mutation, the alpha2delta-3 subunit gene is probably not a tumor suppressor gene.