Association of SNAREs and Calcium Channels with the Borders of Cytoskeletal Cages Organizes the Secretory Machinery in Chromaffin Cells

In chromaffin cells, SNARE proteins, forming the basic exocytotic machinery are present in membrane clusters of 500–600 nm in diameter. These microdomains containing both SNAP-25 and syntaxin-1 are dynamic and the expression of altered forms of SNAREs modifies not only their motion but also the mobility of the associated granules. It is also clear that SNARE microdomain location defines the place for individual vesicle fusion and that the alteration of cluster dynamics affects the fusion process itself. Interestingly, these SNARE patches colocalize with the borders of F-actin cages forming the cytoskeletal cortical network, and these borders also contain clusters of L- and P/Q type calcium channels. The organization of the secretory machinery in association with the borders of cytoskeletal cages seems to be an effective way to promote fast coupling between calcium entry and catecholamine release as demonstrated with the use of mathematical secretory models.     

The role of the DNA hypermethylating agent Budesonide in the decatenating activity of DNA topoisomerase II

Catenations between sister chromatids result from DNA replication and must be resolved to ensure proper chromatid segregation in mitosis. Functionally active Topoisomerase II (Topo II), through its mechanism of concerted breaking and rejoining of double stranded DNA, is required to carry out this fundamental process. In previous studies we have shown that modifications in DNA sequence by halogenated pyrimidines and by the demethylating agent 5-azacytidine leads to malfunction of Topo II that results in an increased yield of endorreduplicated cells as a result of segregation failure. In the present work we have evaluated the possible influence of the methylating agent Budesonide to modify the frequency of endoreduplicated cells in AA8 Chinese hamster cell population. Our results seem to indicate that when Budesonide was administered for two consecutive cell cycles did induce an increase in the yield of endoreduplicated cells, as previously observed for the hypomethylating agent 5-azaC. We have also examined the possible relationship between extensive hypermethylation induced by Budesonide in DNA and stabilization of cleavable complexes by m-AMSA. Taken as a whole, our results show that the degree of methylation in DNA correlates with the effectiveness of m-AMSA to stabilize the Topo II-DNA complexes and to induce DNA cleavage. These findings evidence for the first time the functional importance of DNA hyper- and hypomethylation changes as epigenetic factors able to modulate Topo II activity for proper chromosome segregation.     

A rational use of glucocorticoids in patients with early arthritis has a minimal impact on bone mass

Introduction  

Glucocorticoid (GC)-induced osteoporosis is a frequent complication in patients with rheumatoid arthritis. However, little information exists about the consequences of GC use in patients with early arthritis. Here we describe the variables underlying the use of GC in early arthritis, as well as its effect on bone-mineral density.     

Metabolic engineering of ketocarotenoids biosynthesis in the unicelullar microalga Chlamydomonas reinhardtii

Most higher plants and microalgae are not able to synthesize ketocarotenoids. In this study the unicellular chlorophyte Chlamydomonas reinhardtii has been genetically engineered with the beta-carotene ketolase cDNA from Haematococcus pluvialis, bkt1 (GeneBank accession no. X86782), involved in the synthesis of astaxanthin, to obtain a transgenic microalga able to synthesize ketocarotenoids. The expression of bkt1 was driven by the Chlamydomonas constitutive promoter of the rubisco small subunit (RbcS2) and the resulting protein was directed to the chloroplast by the Chlamydomonas transit peptide sequences of Rubisco small subunit (RbcS2) or Ferredoxin (Fd). In all transformants containing the bkt1 gene fused to the RbcS2 or the Fd transit peptides a new pigment with the typical ketocarotenoid spectrum was detected. Surprisingly this ketocarotenoid was not astaxanthin nor canthaxanthin. The ketocarotenoid was identified on the basis of its mass spectrum as 3,3'-dihydroxy-beta,varepsilon-carotene-4-one (4-keto-lutein) or its isomer ketozeaxanthin.     

Tight coupling of the t-SNARE and calcium channel microdomains in adrenomedullary slices and not in cultured chromaffin cells

Regulated exocytosis involves calcium-dependent fusion of secretory vesicles with the plasma membrane with three SNARE proteins playing a central role: the vesicular synaptobrevin and the plasma membrane syntaxin1 and SNAP-25. Cultured bovine chromaffin cells possess defined plasma membrane microdomains that are specifically enriched in both syntaxin1 and SNAP-25. We now show that in both isolated cells and adrenal medulla slices these target SNARE (t-SNARE) patches quantitatively coincide with single vesicle secretory spots as detected by exposure of the intravesicular dopamine beta-hydroxylase onto the plasmalemma. During exocytosis, neither area nor density of the syntaxin1/SNAP-25 microdomains changes on the plasma membrane of both preparations confirming that preexisting clusters act as the sites for vesicle fusion. Our analysis reveals a high level of colocalization of L, N and P/Q type calcium channel clusters with SNAREs in adrenal slices; this close association is altered in individual cultured cells. Therefore, microdomains carrying syntaxin1/SNAP-25 and different types of calcium channels act as the sites for physiological granule fusion in "in situ" chromaffin cells. In the case of isolated cells, it is the t-SNAREs microdomains rather than calcium channels that define the sites of exocytosis.     

Blood-brain barrier disruption induces in vivo degeneration of nigral dopaminergic neurons

We have evaluated the possibility that changes in the vascular system may constitute a contributing factor for the death of nigral dopaminergic neurons in Parkinson's disease. Thus, we have employed intranigral injections of vascular endothelial growth factor (VEGF), the most potent inducer of blood-brain barrier (BBB) permeability. A single dose of 1 mug of VEGF, chosen from a dose-response study, highly disrupted the BBB in the ventral mesencephalon in a time-dependent manner. A strong regional correlation between BBB disruption and loss of tyrosine hydroxylase-positive neurons was evident. Moreover, Fluoro-Jade B labelling showed the presence of dying neurons in the substantia nigra in response to VEGF injection. High number of TUNEL-positive nuclei was observed in this area along with activation of caspase 3 within nigral dopaminergic neurons. Analysis of the glial population demonstrated a strong inflammatory response and activation of astroglia in response to BBB disruption. We conclude that disruption of the BBB may be a causative factor for degeneration of nigral dopaminergic neurons.     

Ultracentrifugation of serum samples allows detection of hepatitis C virus RNA in patients with occult hepatitis C

Occult hepatitis C virus (HCV) infection of patients with abnormal liver function tests of unknown origin who are anti-HCV and serum HCV RNA negative but who have HCV RNA in the liver has been described. As HCV replicates in the liver cells of these patients, it could be that the amount of circulating viral particles is under the detection limit of the most sensitive techniques. To prove this hypothesis, serum samples from 106 patients with occult HCV infection were analyzed. Two milliliters of serum was ultracentrifuged over a 10% sucrose cushion for 17 h at 100,000 x g(av), where av means average, and HCV RNA detection was performed by strand-specific real-time PCR. Out of the 106 patients, 62 (58.5%) had detectable serum HCV RNA levels after ultracentrifugation, with a median load of 70.5 copies/ml (range, 18 to 192). Iodixanol density gradient studies revealed that HCV RNA was positive at densities of 1.03 to 1.04 and from 1.08 to 1.19 g/ml, which were very similar to those found in the sera of patients with classical chronic HCV infection. Antigenomic HCV RNA was found in the livers of 56 of 62 (90.3%) patients with detectable serum HCV RNA levels after ultracentrifugation, compared to 27 of 44 (61.4%) negative patients (P < 0.001). No differences in the median loads of antigenomic HCV RNA between patients with an those without serum HCV RNA (4.5 x 10(4) [range, 7.9 x 10(2) to 1.0 x 10(6)] versus 2.3 x 10(4) [range, 4.0 x 10(2) to 2.2 x 10(5)]) were found. Alanine aminotransferase and gamma-glutamyl transpeptidase levels, liver necroinflammatory activity, and fibrosis did not differ between both groups. In conclusion, HCV RNA can be detected in the sera of patients with occult HCV infection after circulating viral particles are concentrated by ultracentrifugation.     

Proteomic analysis of phytopathogenic fungus <Emphasis Type="Italic">Botrytis cinerea</Emphasis> as a potential tool for identifying pathogenicity factors,therapeutic targets and for basic research

Botrytis cinerea is a phytopathogenic fungus causing disease in a substantial number of economically important crops. In an attempt to identify putative fungal virulence factors, the two-dimensional gel electrophoresis (2-DE) protein profile from two B. cinerea strains differing in virulence and toxin production were compared. Protein extracts from fungal mycelium obtained by tissue homogenization were analyzed. The mycelial 2-DE protein profile revealed the existence of qualitative and quantitative differences between the analyzed strains. The lack of genomic data from B. cinerea required the use of peptide fragmentation data from MALDI-TOF/TOF and ESI ion trap for protein identification, resulting in the identification of 27 protein spots. A significant number of spots were identified as malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The different expression patterns revealed by some of the identified proteins could be ascribed to differences in virulence between strains. Our results indicate that proteomic analysis are becoming an important tool to be used as a starting point for identifying new pathogenicity factors, therapeutic targets and for basic research on this plant pathogen in the postgenomic era.