Biomedical subjects

Mirco Castoldi

Publications and source records attributed to Mirco Castoldi.

4 recordsLinked to original sources

PubMed2006

A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA).

MicroRNAs represent a class of short (approximately 22 nt), noncoding regulatory RNAs involved in development, differentiation, and metabolism. We describe a novel microarray platform for genome-wide profiling of mature miRNAs (miChip) using locked nucleic acid (LNA)-modified capture probes. The biophysical properties of LNA were exploited to design probe sets for uniform, high-affinity hybridizations yielding highly accurate signals able to discriminate between single nucleotide differences and, hence, between closely related miRNA family members. The superior detection sensitivity eliminates the need for RNA size selection and/or amplification. MiChip will greatly simplify miRNA expression profiling of biological and clinical samples.

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PubMed2006

Chromokinesin Xklp1 contributes to the regulation of microtubule density and organization during spindle assembly.

Xklp1 is a chromosome-associated kinesin required for Xenopus early embryonic cell division. Function blocking experiments in Xenopus egg extracts suggested that it is required for spindle assembly. We have reinvestigated Xklp1 function(s) by monitoring spindle assembly and microtubule behavior under a range of Xklp1 concentrations in egg extracts. We found that in the absence of Xklp1, bipolar spindles form with a reduced efficiency and display abnormalities associated with an increased microtubule mass. Likewise, centrosomal asters assembled in Xklp1-depleted extract show an increased microtubule mass. Conversely, addition of recombinant Xklp1 to the extract reduces the microtubule mass associated with spindles and asters. Our data suggest that Xklp1 affects microtubule polymerization during M-phase. We propose that these attributes, combined with Xklp1 plus-end directed motility, contribute to the assembly of a functional bipolar spindle.

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PubMed2003

Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer.

Microtubules can be assembled in vitro from purified alpha/beta tubulin heterodimers in the presence of GTP. Tubulin is routinely obtained from animal brain tissue through repetitive cycles of polymerization-depolymerization, followed by ion-exchange chromatography to remove any contaminating microtubule-associated proteins and motors. Here, we show that only two cycles of polymerization-depolymerization of pig brain tubulin in the presence of a high-molarity PIPES buffer allow the efficient removal of contaminating proteins and production of a high-concentration tubulin solution. The proposed protocol is rapid and yields more active tubulin than the traditional ion-exchange chromatography-based procedures.

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PubMed2002

Structural and functional characterization of the human and mouse fibulin-1 gene promoters: role of Sp1 and Sp3.

Fibulin-1 is a multifunctional extracellular protein involved in diverse biological processes including cardiovascular development, haemostasis and cancer. To investigate the transcriptional regulation of the gene encoding fibulin-1 we cloned and analysed about 4.0 kb of the 5'-flanking regions of both the human and mouse fibulin-1 genes. The human and mouse fibulin-1 promoters share little sequence similarity except for a short region of approx. 150-170 bp immediately upstream of the translation start site. The conserved region contains a TATA-like sequence (ATAATT) and multiple consensus binding sites for Sp1 and activator protein 2 (AP-2). That the short conserved region in each gene confers basal promoter activity is demonstrated by transient transfections of promoter deletion constructs for both the human and mouse genes into cells that express fibulin-1 constitutively. Co-transfections of promoter constructs with expression plasmids for Sp1, Sp3 and Sp4 into Drosophila SL2 cells indicate that Sp1 and Sp3 are essential for transcriptional activation and that these two factors act synergistically. Electrophoretic mobility-shift assays show that Sp1 and Sp3, but not AP-2, bind to the basal promoter of the human fibulin-1 gene. The results demonstrate the functional importance of Sp1 and Sp3 in regulating the expression of the fibulin-1 gene.

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