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p53 gene alterations are among the most common events observed in colorectal cancer,and are accompanied frequently by DNA aneuploidy and high proliferative activity. The prognostic significance of such mutations remains controversial. We prospectively evaluated the prognostic significance of p53 mutations, DNA-ploidy, and S phase fraction (SPF) in a consecutive series of 160 colorectal cancer patients (median follow-up 71 months). Tumor DNA was screened for p53 mutations by PCR/single-strand conformational polymorphism/sequencing. DNA-ploidy and SPF were assessed by DNA flow cytometry. p53 mutations were detected in 68 of 160 (42.5%) cases. In 56% (38 of 68) of these, p53 mutations were found in conserved areas of the gene and in 44% (30 of 68 cases) outside the conserved regions. Eighteen of the 68 cases (26%) had mutations in the L3 loop, 11 of 68 (16%) in the L1 loop-sheet-alpha helix motif, and 39 of 68 (58%) outside L3 and loop-sheet-alpha helix. Seventy-five percent of the cases (120 of 160) showed DNA aneuploidy, whereas 18% of these (22 of 120) were multiclonal. The major independent predictors for both disease relapse and death were advanced Dukes' stage, p53 mutations affecting L3 loop, DNA-aneuploid tumors, and high SPF (18.5%). Our results show that mutations in L3 functional domain, more than any mutations, are important biological indicators to predict the outcome of patients indicating that these mutations have biological relevance in terms of colorectal cancer disease course.

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

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.

Point mutations in codon 12 and 13 of K-ras are frequently found in DNA of colorectal cancer. It has been suggested that particular mutations at these sites may be associated with specific tumour phenotypes. To shed light on the molecular mechanisms on which depends this specificity we set up a system of HT-29 cells stably transfected with a cDNA coding for K-rasG13D under the control of an inducible promoter. Proliferation assay performed on one of the positives clones, showed a decreased growth rate in response to K-rasG13D expression and preliminary gene expression analysis showed an up-regulation of the cell-cycle inhibitor p21 WAF1.

Omaggio a Charles Darwin: 1809-2009: duecento anni di evoluzionismo

Babesia bigemina is a tick-borne haemoparasite affecting cattle in different parts of the world. One of the few antigens characterized in this species codifies for the Apical Membrane Antigen 1 (AMA-1), a transmembrane protein recently identified. In this work, we characterized the ama-1 gene from four Italian B.bigemina strains, two obtained from Ragusa, Sicily (ITA1 and ITA3), one obtained from Benevento, Campania (ITA2) and one coming from Perugia, Umbria (ITA4). Italian sequences were compared to the Australian strain, whose sequence is available in the Sanger Institute web site, and to some strains coming from different parts of the world. This study provides useful information on the features of B.bigemina AMA-1 gene from Italian strains of the parasite and some predictive information of the protein obtained by bioinformatics. A comparison among Italian strains of B.bigemina showed a very high degree of both nucleotide and aminoacidic identity. The gene resulted highly conserved among Italian and foreign strains and the protein was included in the AMA-1 family. By the means of bioinformatics it was possible to compare some features of B.bigemina AMA-1 protein with the already characterized Plasmodium falciparum orthologue. Predictive results show that a structural and thus functional homology between B.bigemina and P.falciparum AMA-1 can be supposed. Moreover several putative antigenic peptides were detected and they could be used for investigations about the recognition by specific antibodies. These data put the basis for a further study on B.bigemina AMA-1 antigen.

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.

Northern blot hybridization is a useful tool for analyzing transcript patterns. To get a picture of what really occurs in vivo, it is necessary to use a protocol allowing full protection of the RNA integrity and recovery and unbiased transfer of the entire transcripts population. Many protocols suffer from severe limitations including only partial protection of the RNA integrity and/or loss of small sized molecules. Moreover, some of them do not allow an efficient and even transfer in the entire sizes range. These difficulties become more prominent in streptomycetes, where an initial quick lysis step is difficult to obtain. We present here an optimized northern hybridization protocol to purify, fractionate, blot, and hybridize Streptomyces RNA. It is based on grinding by a high-performance laboratory ball mill, followed by prompt lysis with acid phenol-guanidinium, alkaline transfer, and hybridization to riboprobes. Use of this protocol resulted in sharp and intense hybridization signals relative to long mRNAs previously difficult to detect.

Babesia bigemina is an endemic parasite 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 transmembrane antigen recently identified. We characterized the ama-1 gene from three Italian B. bigemina strains. Italian sequences were compared to those of the Australian strain. 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.

Southern hybridisation of genomic DNA extracted from a human primary colorectal carcinoma revealed amplification of a fragment containing the wild-type c-myc locus. Two additional rearranged DNA fragments, lying upstream of c-myc, fused to distant non-contiguous sequences from the same chromosome, with an opposite configuration (head to head vs. head to tail), were also found to be amplified. Sequences analysis suggested that these rearrangements resulted from illegitimate recombination at two distinct points within the DNA sequence just upstream of the c-myc ORF and further that these events triggered two different amplification mechanisms, only one of which, involving a strand invasion event following DNA double strand breaks, increased the copy number of the c-myc ORF.

The Ras oncogene is mutated in about 30% of the human tumors and its mutations are always point mutations concerning codon 12, 13 and 61. These mutations cause in the proteins a reduced GTPase activity, so that they become constitutively active. In human cells there are three main isoforms of Ras (H, K, N-ras) which can trigger alternative pathways of signal trasduction. In order to investigate the effects of expression of different oncogenic Ras isoforms in colorectal carcinoma cells (HT-29), we obtained stable clones of HT-29 cells transfected with cDNAs codifying H-RasG12V and K-Ras G12V called respectively H12 and K12 and K-RasG13D called K13, under the control of an hormone-inducible promoter. We had previously observed changes in shape and growth rate when mutated Ras was expressed. We then analyzed the differential expression of genes such as the cell-cycle inhibitor p21 and the EMT associated gene snail. Q-RT PCR assay showed an increase in p21 level only in H12 cells but not in K13 and K12 compared with the respective controls. Instead, snail mRNA level was increased in all thre clones. We investigated also by Semiquantitative RT-PCR H-RasG12V, p21 and snail levels in H12 cells induced for a long time and we noted a decreased level of H-RASG12V associated with no p21 and snail expression. Our results show that specific mutations of different Ras isoforms have different effects on morphology and gene expression in Ht-29. Other studies are of course necessary to shed more light on the role played by Ras mutations in tumor development.

RNA processing and degradation are important steps in the regulation of gene expression. The genes present on the DNA of filamentous phage f1 are expressed at very different levels. A group of genes which are expressed at a very low level (genes III, VI, I and IV is located in the region IF of the phage DNA. The current model proposes that expression of the genes III. VI and I (which form an operon) is regulated by readthrough transcription at a weak terminator located at the end of gene III, while that of gene IV by a terminator located at the end of it. In contrast with this, we found that very long transcripts complementary to the entire f1 DNA are synthesized in the infected cells and that they are quickly processed to shorter, mature transcripts. We discovered that these processing events and differential stability of specific transcripts, are the key elements which regulate the expression of these 4 genes.

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 over-expression 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.