Ida Albanese

Studies on sea urchin oocytes II. Synthesis of RNA during oogenesis

Abstract Isolated oocytes of the sea urchin Paracentrotus lividus actively incorporate 3H-uridine into RNA. Labeled RNA was analysed by sucrose gradient and acrylamide gel electrophoresis following cell fractionation. Much of the radioactivity is incorporated at the nucleolar level in the form of rRNA precursors. The kinetics of maturation of these latter suggests that this occurs at a slower rate than during embryogenesis. Other non-nucleolar RNA classes are also actively labelled and retained in the nucleus for many hours. These results are confirmed by an autoradiographic investigation.

Studies on sea urchin oocytes: I. Purification and cell fractionation☆

Abstract A procedure is presented which permits a satisfactory purification in bulk of oocytes of Paracentrotus lividus at various stages in the growth period. Methods which allow a quantitative recovery of partially purified germinal vesicles or nucleoli are also reported.

Prognostic significance of proliferative activity, DNA-ploidy, p53 and Ki-ras point mutations in colorectal liver metastases

Abstract. Paired colorectal liver metastases (CLM) and normal tissue samples from a consecutive series of 36 patients were studied prospectively. MIB‐1 expression was studied by immunohistochemistry on paraffin‐embedded sections. DNA ploidy and S‐phase fraction (SPF) measurements were performed by flow cytometry on frozen tissues. Mutations within the p53 (exons 5‐8) and c‐Ki‐ras (codons 12 and 13) genes were detected by PCR single‐strand conformation polymorphism analysis followed by sequencing. A high correlation was observed between the MIB‐1 LI and SPF value (rho=0·81; P<0·01). Moreover, p53 gene mutations were associated with either high MIB‐1 LI and high SPF. In univariate analysis, SPF and MIB‐1 levels were related to risk of death. The association between overall survival and DNA‐ploidy or p53 mutations did not reach statistical significance, but a slightly better survival was observed for patients either with DNA‐diploid tumours or without mutations (P=0·05 and P=0.06, respectively). SPF was shown by multivariate Cox model analysis to be an independent prognostic variable and thus it might be a useful prognostic factor in patients with CLM.

Have p53 gene mutations and protein expression a different biological significance in colorectal cancer

p53 alterations are considered the most common genetic events in many types of neoplasms, including colorectal carcinoma (CRC). These alterations include mutations of the gene and/or overexpression of the protein. The aim of our study was to assess whether in 160 patients undergoing resective surgery for primary operable CRC there was an association between p53 mutations and protein overexpression and between these and other biological variables, such as cell DNA content (DNA‐ploidy) and S‐phase fraction (SPF), and the traditional clinicopathological variables. p53 mutations, identified by PCR‐SSCP‐sequencing analysis, were found in 68/160 patients (43%) and positive staining for p53 protein, detected with the monoclonal antibody DO‐7, was present in 48% (77/160) of the cases, with agreement of 57% (91/160). In particular, a significant association was found between increased p53 expression and genetic alterations localized in the conserved regions of the gene or in the L3 DNA‐binding domain and the specific type of mutation. Furthermore, both overexpression of p53 and mutations in the conserved areas of the gene were found more frequently in distal than in proximal CRCs, suggesting that they might be “biologically different diseases.” Although p53 mutations in conserved areas were associated with flow cytometric variables, overexpression of p53 and mutations in its L3 domain were only related respectively to DNA‐aneuploidy and high SPF. These data may reflect the complex involvement of p53 in the different pathways regulating cell‐cycle progression. In conclusion, the combination of the mutational status and immunohistochemistry of p53, and flow cytometric data may provide an important insight into the biological features of CRCs. J. Cell. Physiol. 191: 237–246, 2002.

Giant RNA is also found in the cytoplasm in sea urchin embryos.

Abstract The kinetics of labelling of nuclear and cytoplasmic RNAs of molecular weights up to 5 · 107 demonstrate that the latter does not originate from artifactual nuclear leakage. Arguments are provided which strongly suggest that the giant cytoplasmic RNA is not an artifact created during the extractive or analytical procedures.

Regulation of MUC1 Expression in Human Mammary Cell Lines by the c-ErbB2 and Ras Signaling Pathways

The MUC1 protein is a highly O-glycosylated transmembrane molecule that is expressed at the luminal surface of most glandular epithelial cells and is upregulated in carcinomas. Here, we report the effect of the activation of the c-ErbB2 --> Ras pathway on the expression of the MUC1 gene in the nontumorigenic mammary cell lines MTSV1-7 and HB2 and in the malignant cell lines T47D and ZR75. Endogenous levels of MUC1 mRNA and protein in HB2 clones permanently overexpressing c-ErbB2 or V12-H-Ras were markedly reduced compared with levels in the parental cell lines. Furthermore, in transient transfection assays, the transcription of a CAT reporter construct driven by the MUC1 promoter was inhibited when cotransfected with a c-ErbB2 or a V12-H-ras expressing vector. Transient transfections using mutant forms of the ras oncogene, and the inhibitor chemical wortmannin, indicated that the pathway activated by c-ErbB2 proceeds via activation of Ras and that the Raf and phosphoinositide 3-kinase pathways are involved. Finally, cotransfection assays using a reporter gene driven by the MUC1 promoter carrying abolishing mutations in some of the cis-acting elements showed that a GC box at -99/-91 is crucial for responsiveness to c-ErbB2 inhibition of transcription.

Characterization of the apical membrane antigen-1 in Italian strains of Babesia bigemina.

Babesia bigemina is a parasite endemic in different parts of the world, including Europe and the Americas. One of the few genes characterized in this species codifies for the Apical Membrane Antigen 1 (AMA-1), a trans-membrane antigen recently identified. In this research, we characterized the ama-1 gene from three Italian B. bigemina strains, two B. bigemina strains obtained from Ragusa, Sicily (ITA1 and ITA3) and a third one obtained from Benevento, Campania (ITA2). Italian sequences were compared with those of the Australian strain obtained from the Sanger Institute web site and to strains from different parts of the world. The results obtained confirmed that this newly described ama-1 gene is highly conserved among Italian and foreign strains which has implications for vaccine development.

Membrane vesicles containing matrix metalloproteinase‐9 and fibroblast growth factor‐2 are released into the extracellular space from mouse mesoangioblast stem cells

Certain proteins, including fibroblast growth factor‐2 (FGF‐2) and matrix metalloproteinase‐9 (MMP‐9), have proved very effective in increasing the efficacy of mesoangioblast stem cell therapy in repairing damaged tissue. We provide the first evidence that mouse mesoangioblast stem cells release FGF‐2 and MMP‐9 in their active form through the production of membrane vesicles. These vesicles are produced and turned over continuously, but are stable for some time in the extracellular milieu. Mesoangioblasts shed membrane vesicles even under oxygen tensions that are lower than those typically used for cell culture and more like those of mouse tissues. These findings suggest that mesoangioblasts may themselves secrete paracrine signals and factors that make damaged tissues more amenable to cell therapy through the release of membrane vesicles. J. Cell. Physiol. 224:144–151, 2010

Cytoplasmic giant RNA in sea urchin embryos. I. Proof that it is not derived from artifactual nuclear leakage

Abstract The analysis of the giant RNA labeling kinetics suggested that the heterogeneous RNA, including classes of very slow electrophoretic mobility, found in the cytoplasm of sea urchin embryos, cannot be attributed to artifactual nuclear leakage. Microsurgical experiments demonstrated that some RNA of heterogeneous and very slow electrophoretic mobility is a genuine component of the cytoplasmic RNA of sea urchin oocytes.

DNA Extraction From Orthoptera Museum Specimens

We describe a procedure for rapid purification of high quality DNA from either fresh or dry Orthoptera, suitable for the PCR amplification of DNA regions more than 800 bp long (even from oldest specimens), which allows genetic analyses on animals from collections without the complete specimen disruption.