Familial breast/ovarian cancer and BRCA1/2 genetic screening: the role of immunohistochemistry as an additional method in the selection of patients.

Only 20-25% of families screened for BRCA1/2 mutations are found positive. Because only a positive result is informative, we studied the role of BRCA1/2 immunohistochemistry as an additional method for patient selection. From 53 high-risk-affected probands, 18 (34%) had available paraffin blocks of their tumors and were selected for this study. Mutation screening was done by conformation-sensitive gel electrophoresis and multiplex ligation-dependent probe amplification. For immunohistochemistry, 21 neoplastic specimens (15 breast carcinomas, 5 ovary neoplasms, and 1 rectal adenocarcinoma) were analyzed with BRCA1 (monoclonal antibody, Ab-1, oncogene) and BRCA2 (polyclonal antibody, Ab-2, oncogene) antibodies. Absence of the BRCA1 protein was confirmed in negative tumors by Western blotting. Seven patients were positive for BRCA1/2 mutations: 5 for BRCA1 and 2 for BRCA2. Four out of five positive patients had tumors negative for BRCA1 immunostaining, and the remaining 13 BRCA1-negative patients had positive BRCA1 immunostaining in all tumor samples. Sensitivity to predict for BRCA1 mutation carriers was 80%, and specificity was 100%, with a positive predictive value of 100% and a negative predictive value of 93%. This correlation was statistically significant (p=0.001). No correlation was observed for BRCA2. If larger studies confirm these results, high-risk patients with BRCA1-negative tumors should be screened first for this gene.     

Synthetic approaches to 6-substituted 9-(3-azido-3,4-dideoxy-β-d,l-erythro-pentopyranosyl)purines

Methyl 3-azido-2-O-benzoyl-3,4-dideoxy-β-dl-erythro-pentopyranoside (6) was synthesized through two routes in five steps from methyl 2,3-anhydro-4-deoxy-β-dl-erythro-pentopyranoside (1). The first route proceeded via selective azide displacement of the 3-tosyloxy group of methyl 4-deoxy-2,3-di-O-tosyl-α-dl-threo-pentopyranoside, followed by detosylation and benzoylation. The second route consisted, with a better overall yield, in the azide displacement of the mesyloxy group of methyl O-benzoyl-4-deoxy-3-O-methylsulfonyl-α-dl-threo-pentopyranoside (10), obtained by benzylate opening of 1, followed by benzoylation, debenzylation, and mesylation. Compound 6 was transformed into its glycosyl chloride, further treated by 6-chloropurine to give the nucleoside 9-(3-azido-2-O-benzoyl-3,4-dideoxy-β-dl-erythro-pentopyranosyl)-6-chloropurine (13). When treated with propanolic ammonia, 13 yielded 9-(3-azido-3,4-dideoxy-β-dl-erythro-pentopyranosyl)adenine.     

Multilocus and morphological analysis of south-eastern Iberian Wall lizards (Squamata,Podarcis)

The phylogenetic relationships among the wall lizards of the Podarcis hispanicus complex that inhabit the south-east (SE) of the Iberian Peninsula and other lineages of the complex remain unclear. In this study, four mitochondrial and two nuclear markers were used to study genetic relationships within this complex. The phylogenetic analyses based on mtDNA gene trees constructed with ML and BI, and a species tree using *BEAST support three divergent clades in this region: the Valencia, Galera and Albacete/Murcia lineages. These three lineages were also corroborated in species delimitation analyses based on mtDNA using bPTP, mPTP, GMYC, ABGD and BAPS. Bayesian inference species delimitation method (BPP) based on both nuclear data and a combined data set (mtDNA + nuclear) showed high posterior probabilities for these three SE lineages (≥0.94) and another Bayesian analysis (STACEY) based on combined data set recovered the same three groups in this region. Divergence time dating of the species tree provided an estimated divergence of the Galera lineage from the other SE group (Podarcis vaucheri, (Albacete/Murcia, Valencia)) at 12.48 Ma. During this period, the Betic–Rifian arc was isolated, which could have caused the isolation of the Galera form distributed to the south of the Betic Corridor. Although lizards from the Albacete/Murcia and Galera lineage are morphologically similar, they clearly represent distinct genetic lineages. The noteworthy separation of the Galera lineage enables us to conclude that this lineage must be considered as a new full species.     

FLAVONOID EVOLUTION IN DENDROSERIS (COMPOSITAE,LACTUCEAE) FROM THE JUAN FERNANDEZ ISLANDS,CHILE

Leaf flavonoid chemistry was examined from the three subgenera and 11 species of the endemic genus Dendroseris (Compositae, Lactuceae) of the Juan Fernandez Islands, Chile. Eight of the species are restricted to the older island (Masatierra, ca. 4 million years old), which is also closer to the mainland. Three species, one from each subgenus, are restricted to Masafuera, which is younger geologically (1–2 million years old) and 145 km further west of Masatierra. A total of 16 compounds was identified, with the 7-0-glucosides of the flavones apigenin and luteolin accounting for 12 of the constituents. Two glucosides of the flavonol quercetin were detected. Despite considerable interpopulation variation within species, six of the taxa have distinctive flavonoid profiles. Although there are few absolute differences among the subgenera, they can be distinguished chemically. Subgenus Rea contains the greatest number of compounds, and a previous cladistic analysis based on morphological features suggested this subgenus as most primitive. Subgenus Phoenicoseris is considered highly derived morphologically, and it has a reduced flavonoid chemistry. Very little reduction in flavonoid diversity was seen in the morphologically specialized subg. Dendroseris as compared to subg. Rea. A trend in reduction of numbers of compounds was seen for two of the three species on the younger island of Masafuera when compared to their presumed ancestors on Masatierra. Flavonoids of selected species of Hieracium and Hypochaeris, presumptive mainland progenitors of Dendroseris, reveal a close chemical affinity with the former genus.     

Inhibition of growth of Leishmania mexicana mexicana by Leishmania mexicana amazonensis during "in vitro" co-cultivation

Inhibition of one Leishmania subspecies by exometabolites of another subspecies, a phenomenon not previously reported, is suggested by our recent observations in cell cloning experiments with Leishmania mexicana mexicana and Leishmania mexicana amazonensis. Clones were identified using the technique of schizodeme analysis. The phenomenon observed is clearly relevant to studies of parasite isolation, leishmanial metabolism, cross-immunity and chemotherapy.     

Implication of lipid peroxidation in triethyltin poisoning in the rat

Triethyltin intoxication induces, in vivo, a significant increase of malondialdehyde concentration in rat brain. After treatment with a Ginkgo biloba extract, an extract known to possess antiedematous and radical scavenging properties, the malondialdehyde level in the brain is significantly decreased. This suggests that a lipid peroxidation process is associated with cerebral oedema induced by triethyltin.     

Genetic and molecular analysis of spontaneous respiratory deficient (res-) mutants of Escherichia coli K-12

Respiratory deficient (res-) mutants of E. coli are slow growing microcolonial, anaerobic, catalase and benzidine negative strains whose broad phenotypic alteration may result from pleiotropic mutations in genes of the hemin biosynthetic pathway. They are easily recovered from platings of sensitive cells on concentrations of gentamicin higher than the minimal inhibitory concentration. These mutants show a dramatic change in their biochemical diagnostic profile resulting primarily from deficiencies in the active transport mechanisms of the cell. Using well-marked F- and Hfr strains, 157 mutants were analyzed from 3 different parent strains; all but 2 resulted from mutations in 3 loci of the hemin biosynthetic pathway. Of these a marked skew to hemB- mutations was seen, with more than 80% mapping there. The possibility that this hot spot resulted from transpositional activity was tested by Southern hybridization of EcoRI digests of the chromosomal DNA, using as a probe, a 2.8-kb fragment containing the hemB gene. The WT and other hemB+ control strains contained a 14.6-kb fragment. Of 18 hemB strains tested, 14 showed deletion and insertion mutations which fell into four classes based on the variation in the size of the fragment or on the absence of hybridization. The latter resulted from complete deletion of the hemB gene. An increase in fragment size from 1.5-kb to 3.4-kb was observed in some of the strains.     

Magnum morphogenesis during the natural development of the quail oviduct: analysis of egg white proteins and progesterone receptor concentration

The histological development of the quail oviduct and the changes in concentrations of progesterone receptor, ovalbumin, conalbumin, ovomucoid and ovoglycocomponents are analyzed during the period spanning 7-35 days of age. The initiation of luminal epithelial cell proliferation is the first event of magnum growth. The epithelial cells begin to evaginate into subepithelial stroma and form tubular glands. Meanwhile, luminal epithelium starts cellular pleomorphism through ciliogenesis. No egg white proteins are detectable in the developing glands; at the same time, the concentration of the progesterone receptor increases from about 5500 sites/cell to 30,300 sites/cell. Tubular gland cells then begin to synthetize and accumulate egg white proteins, mucous cells differentiate in the luminal epithelium, and the cell proliferation decreases and finally stops. Compared with earlier studies dealing with the blood levels of estrogen and progesterone in developing quails during the same period, and the cellular changes induced in the oviducts of ovariectomized and ovariectomized-hypophysectomized quail by exogenous steroids, these results distinguish between the cellular responses that are physiologically controlled by estradiol and other responses that have multihormonal regulation.     

Increase in the immunogenicity of cancer cells exposed to microwaves

A suspension of 10(7) melanoma cells, submitted to microwave hyperthermia (2,450 MHz, 20 minutes, 44 degrees C) leads to a partial protection in mice inoculated 26 days afterwards with a suspension of 10(7) active cells of B16 melanoma (95% vitality). The period of 26 days between the two injections corresponds to the moment where the sera antibodies have an highest level. The kinetics of the primary response of the humoral immunity shows that B16 melanoma proliferation and number of deads can be related to an hypogammaglobulinemia.     

Alteration of membrane phospholipid methylation by adenosine analogs does not affect T lymphocyte activation

Membrane phospholipid methylation has been described during activation of various immune cells. Moreover recent data indicated modulation of immune cells functions by adenosine. As S-Adenosyl-methionine and S-Adenosyl-homocysteine are adenosine analogs and modulators of transmethylation reactions, the effects of SAH and SAM were investigated on membrane phospholipid methylation and lymphocyte activation. SAM (10(-5) M) was shown to induce the membrane phospholipid methylation as assessed by the 3H-methyl-incorporation in membrane extract. This effect was inhibited by SAH. In contrast SAM and SAH did not affect the phytohemagglutinin-induced proliferative response of peripheral blood mononuclear cells. SAH neither modified the early internalization of membrane CD3 antigens nor did it prevent the late expression of HLA-DR antigens on lymphocytes activated by phytohemagglutinin. These results indicate that in vitro alteration of phospholipid methylation does not affect subsequent steps of human T lymphocyte activation and proliferation.