The 2011 GeFI collaborative exercise. Concordance study, proficiency testing and Italian population data on the new ENFSI/EDNAP loci D1S1656, D2S441, D10S1248, D12S391, D22S1045.

The 2011 collaborative exercise of the ISFG Italian Working Group GeFI was aimed at validating the five ENFSI/EDNAP miniSTR loci D1S1656, D2S441, D10S1248, D12S391 and D22S1045. The protocol required to type at least 50 multilocus profiles from locally resident individuals and two blind bloodstains in duplicate (i.e., using at least two different commercial kits), and to send the electropherograms to the Organizing Committee. Nineteen laboratories distributed across Italy participated, collecting a total of 960 samples. Full concordance was found for the five new miniSTRs as observed from the comparison of 13,150 alleles. The inspection of the electropherograms allowed the identification of a very limited number of mistypings in the miniSTR genotypes thus contributing to the establishment of an high quality Italian database of frequencies.

Direct organogenesis of Mandevilla illustris (Vell) Woodson and effects of its aqueous extract on the enzymatic and toxic activities of Crotalus durissus terrificus snake venom.

In order to produce explants of Mandevilla illustris (Vell) Woodson for the "Cerrado in vitro", the Germplasm Bank of UNAERP, we carried out a micropropagation protocol using MS or MS/3 medium supplemented with different concentrations of 6-benzyladeninepurine (BA), Zeatin or 2-isopentenyladenine for nodal segment growth, and alpha-naphthaleneacetic acid, indole-3-butyric acid (IBA) or 1,4 dithiothreitol for rooting. For nodal segments, all the cytokinins tested yielded similar results. However, 2.22 micro M BA is more economical to use. MS/3 medium supplemented with 0.49 micro M IBA was the most appropriate medium for rooting, resulting in 29% rooted explants. The crude aqueous extract from the subterranean system (SS) of M. illustris was assayed for its inhibitory action on the enzymatic activity of Crotalus durissus terrificus snake venom, isolated basic phospholipase A2 (CB) and crotoxin. It totally inhibited the phospholipase activity of crude Cdt venom and CB toxin and inhibited the phospholipase activity of crotoxin by 49%. The toxic action of both the crude venom and crotoxin was partially inhibited-there was a prolonged survival time and a 40.0% decrease in lethality.

A new design of equilibrator to monitor carbon dioxide in highly dynamic and turbid environments.

A new design of equilibrator for carbon dioxide monitoring in natural waters is described. It consists in a vertical tube filled with marbles through which water is flowing while equilibrating with a closed air circuit. It offers several advantages compared with classical equilibrators, among which is a fast response time (half-life constant approximately 30 s) and the potential to work in very turbid water. The proposed equilibrator is of particular interest to monitor carbon dioxide in coastal ecosystems, such as estuaries, which are known to be turbid and highly dynamic. Two performance tests and some field results are presented to illustrate the efficiency of the proposed system.

Regional Italian allele frequencies at nine short tandem repeat loci.

An Italian population study was performed on the loci D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317, D7S820 and a portion of the X-Y homologous gene Amelogenin for gender determination using the AmpFlSTR Profiler kit (PE Biosystems, Foster city, CA). This study was done on a population of 618 unrelated Italian individuals from 18 regions in Italy (except for Valle d'Aosta and Sardinia) to determine allele frequencies for each STR locus, and to evaluate STR technology for developing an Italian Offender DNA database.

Italian population allele and genotype frequencies for the AmpliType PM and the HLA-DQ-alpha loci.

The distribution of six genetic loci analyzed by PCR using the commercial AmpliType PM (PolyMarker) kit (Perkin Elmer, Norwalk, CT) was evaluated in 200 unrelated Italian individuals. The examined loci included: Group-specific component (Gc) (1), D7S8 (2), hemoglobin G gammaglobin (HBGG) (3), glycophorin A (GYPA) (4), low density lipoprotein receptor (LDLR) (5), and HLA DQ-alpha (6). The AmpliType PM Kit analysis is based on the reverse dot blot format and the results are interpreted by reading the pattern of blue dots which determine the alleles present at each locus. The population data collected allow the implementation of AmpliType PM into routine casework.

Genetic consequences of tolerance to methylation DNA damage in mammalian cells.

We previously characterized a clone of CHO cells, clone B, that displayed tolerance to the cytotoxic effects of N-methylnitrosourea (MNU) and 6-thioguanine (6-TG). To determine whether this phenotype affected the mutagenic response of the cells, MNU-induced mutation to 8-azaadenine resistance (8-AAr) was measured in the parental and clone B cells. Comparable mutation frequencies were found in the two cell lines up to 0.5 mM MNU, while at higher MNU concentrations mutations could be reproducibly measured only in clone B cells. Similar amounts of DNA methylated bases were found in the two cell lines after a 30 min treatment with different concentrations of [3H]MNU and the same linear relationship was observed when mutation induction by MNU was plotted as a function of the amount of O6-methylguanine (O6-MeGua) in DNA, indicating that mutation induction in both cell lines was related to the presence of this methylated base. Fifteen MNU-induced 8-AAr mutants were isolated from each cell line and the sequences of the adenine phosphoribosyltransferase (aprt) mutations determined. The type (in 90% of the cases, GC to AT transitions), the sequence context and the strand localization of the mutations indicated that all mutations were targeted at O6-MeGua in DNA and no difference was found between the two lines. These results are consistent with a mechanism of tolerance of O6-MeGua that does not alter the processing of this methylated base into a mutation. Growth in 6-TG induced point mutations in clone B but not in the parental cells. A model is proposed in which the alkylation tolerant variant is altered in a mismatch correction pathway responsible for the cytotoxicity of the methylated base.

Expression of the endogenous O6-methylguanine-DNA-methyltransferase protects Chinese hamster ovary cells from spontaneous G:C to A:T transitions.

We have investigated whether the presence of a DNA repair enzyme, O6-methylguanine-DNA-methyltransferase (MGMT), affects the nature of spontaneous mutations in a mammalian cell line. We compared spontaneous mutations in the adenine phosphoribosyl transferase gene of a Chinese hamster ovary (CHO) cell line that expressed 14,000 MGMT molecules/cell with those in the parental CHO cells lacking this DNA repair activity. The mutation rate/cell/generation of the two CHO cell lines did not differ significantly. However, DNA sequence analysis of spontaneous mutations in the MGMT-proficient CHO cell line revealed a complex picture. No significant difference from the parental CHO cells was found in the number or type of deletions, frameshifts, multiple substitutions, or insertions. The frequency of G:C to T:A transversions was elevated in MGMT-proficient CHO cells. Expression of the enzyme considerably reduced G:C to A:T transitions (25% versus 8.3%). This latter result is the first evidence that this protein is active on an endogenous source of O6-methylguanine that is normally responsible for spontaneous G:C to A:T transition mutations.

Generation of highly metastatic tumors in DBA/2 mice. Oncogenicity of a strain tumor cells.

The sequential intraperitoneal injection of five different A strain tumors into DBA/2 mice resulted in the de novo generation of new DBA/2 tumors. These new tumors share many identical characteristics including the expression of surface H2d, FcR, LY6.2 and tumor antigens. All have the same karyotype which differs in number and in the presence of two metacentric chromosomes from the original A strain tumors. Phenotypically, all are vigorously metastatic after intraperitoneal, subcutaneous, intradermal or intravenous injection. None of the original A strain tumors are metastatic in the A strain host. These tumors have thus provided us with a good model for the study of metastasis while raising questions regarding the oncogenicity of tumors.

Snuff dippers keratosis (snuff-induced leukoplakia).

Patients with chronic snuff-induced keratosis associated with mucosal ulcerations were treated with topically applied vitamin A acid. In each case, after a marked, local inflammatory response initially, topical vitamin A acid showed a definite beneficial effect in promoting healing and involution of the precancerous snuff-induced leukoplakic keratosis. In one patient there was distinct involution of the oral lesions with complete healing and restoration of a normal-appearing mucosa within five weeks after beginning of treatment. In four patients there was healing of superficial ulcerations and regression in the size of the luekoplakic lesions.