Mitotic delay in lymphocytes from BRCA1 heterozygotes unable to reduce the radiation-induced chromosomal damage

Double strand breaks (DSB) are critical lesions involved in the formation of chromosomal aberrations. In response to DNA damage, the cell has mechanisms of repair and cell-cycle control to maintain the genome integrity in which BRCA1 gene is implicated. In the present study an evaluation of the radio-induced damage in G(2) phase of the cell cycle in lymphocytes from BRCA1 heterozygotes is presented. For this purpose Calyculin-A-based premature chromosome condensation (PCC) combined with mitotic arrest has been applied to examine with conventional cytogenetics the damage in G(2) and M phase cells, and to evaluate the G(2)-to-M phase transition. Irradiated peripheral blood lymphocytes from seven heterozygote females (BRCA1(+/-)) and seven control females (BRCA1(+/+)) have been analyzed. The mean proportion of G(2) cells in BRCA1(+/-) was significantly higher than in BRCA1(+/+), indicating a higher G(2) delay after IR exposure in cells from BRCA1(+/-) females. On the other hand, whereas the mean frequency of chromatid breaks (chtb) in G(2) cells was not statistically different between both groups, the mean frequency of chtb in M cells of the BRCA1(+/-) group was significantly higher than in the BRCA1(+/+) one. Moreover, the mean proportion of M cells with aberrations was significantly higher in BRCA1(+/-) than in BRCA1(+/+) suggesting that in spite of the higher G(2) delay of BRCA1(+/-) more damaged cells are able to pass the G(2)-to-M transition.     

Colonization of America by Drosophila subobscura: spatial and temporal lethal-gene allelism

About twenty years ago Drosophila subobscura, a western Palearctic species, colonized both North and South America. Lethal genes in the O chromosome has been subject to much research. Lethal gene allelisms between American populations far away have been studied. These allelisms were not negligible, but all cases were due to the lethal gene completely associated to the O5 chromosomal inversion. Here we analyze the lethal genes in a new American population of D. subobscura (Centralia, Washington), located fairly close to a previously studied population (Bellingham, Washington) and separated in space and time with other American populations (Gilroy I and II in California and Santiago de Chile). The frequencies of lethal and semilethal genes of Centralia were 16.9+/-4.6 and 6.2+/-3.0, respectively. The intrapopulational allelism of Centralia was 0.122+/-0.036. Interpopulational allelisms were studied using the lethal genes from the populations separated in space and time from Centralia. The interpopulational allelisms between Centralia and Gilroy I (California) and between Centralia and Bellingham (Washington) were higher than the intrapopulational allelism (0.155+/-0.032 and 0.153+/-0.024, respectively). In all these cases allelism was due to a complete association between a lethal gene and the O5 chromosomal inversion. Accordingly, no other lethal genes are shared in these populations.     

Study in mono and bilayers of the interaction of hepatitis G virus (GBV-C/HGV) synthetic antigen E2(99-118) with cell membrane phospholipids

The interaction of the hepatitis G synthetic peptide E2(99-118) with cell membrane phospholipids of different characteristics such as dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) was studied by Langmuir isotherms. Epifluorescence microscopy and Atomic force microscopy (AFM) was also used to study interactions with DPPC. Compression isotherms of DPPC/E2(99-118) and DPPG/E2(99-118) mixed monolayers showed negative deviation from ideallity consistent with the existence of attractive interactions. The incorporation of the peptide in DPPC monolayer was also confirmed in epifluorescence microscopy and AFM studies. The peptide retarded the formation of DPPC domains and did not let the phospholipid get organized. No important differences in the interactions with DPPC (neutral) or DPPG (anionic) were found, thus suggesting that electrostatics forces do not have a predominant influence in these interactions.     

Structure and dynamics of the potato carboxypeptidase inhibitor by 1H and 15N NMR

The solution structure and backbone dynamics of the recombinant potato carboxypeptidase inhibitor (PCI) have been characterized by NMR spectroscopy. The structure, determined on the basis of 497 NOE-derived distance constraints, is much better defined than the one reported in a previous NMR study, with an average pairwise backbone root-mean-square deviation of 0.5 A for the well-defined region of the protein, residues 7-37. Many of the side-chains show now well-defined conformations, both in the hydrophobic core and on the surface of the protein. Overall, the solution structure of free PCI is similar to the one that it shows in the crystal of the complex with carboxypeptidase A. However, some local differences are observed in regions 15-21 and 27-29. In solution, the six N-terminal and the two C-terminal residues are rather flexible, as shown by 15N backbone relaxation measurements. The flexibility of the latter segment may have implications in the binding of the inhibitor by the enzyme. All the remaining residues in the protein are essentially rigid (S2 > 0.8) with the exception of two of them at the end of a short 3/10 helix. Despite the small size of the protein, a number of amide protons are protected from exchange with solvent deuterons. The slowest exchanging protons are those in a small two-strand beta-sheet. The unfolding free energies, as calculated from the exchange rates of these protons, are around 5 kcal/mol. Other protected amide protons are located in the segment 7-12, adjacent to the beta-sheet. Although these residues are not in an extended conformation in PCI, the equivalent residues in structurally homologous proteins form a third strand of the central beta-sheet. The amide protons in the 3/10 helix are only marginally protected, indicating that they exchange by a local unfolding mechanism, which is consistent with the increase in flexibility shown by some of its residues. Backbone alignment-based programs for folding recognition, as opposite to disulfide-bond alignments, reveal new proteins of unrelated sequence and function with a similar structure.     

Analysis of transglutaminase protein substrates by functional proteomics

Transglutaminases are calcium-dependent enzymes that catalyze a post-translational modification of proteins through the formation of epsilon -(gamma-glutamyl)lysine bonds. Although specific roles for transglutaminases have been described, recent findings have provided evidence that dysregulation of transglutaminases may contribute to many pathological processes including celiac disease and neurodegenerative diseases. A crucial step in the elucidation of biological and pathological roles of transglutaminases requires the identification of protein substrates. A strategy based on a functional proteomic analysis was set up using two well-characterized biotinylated transglutaminase substrates as affinity probes: 5-(biotinamido)pentylamine and the synthetic biotinylated peptide TVQQEL, the amino- and acyl-donor probes, respectively. A pool of known tissue type transglutaminase protein substrates was selected in order to test the procedure. Results obtained in this paper indicate that the whole strategy can be successfully applied in order to identify transglutaminases protein substrates as well as the amino acid site sensitive toward enzyme activity.     

Colonization of the Americas by Drosophila Subobscura: Lethal-Gene Allelism and Association with Chromosomal Arrangements

Drosophila subobscura is a Palearctic species that has recently colonized the Americas. It was first found in 1978 in Puerto Montt, Chile, and in 1982 in Port Townsend, WA. The colonization and rapid expansion of the species in western South and North America provides distinctive opportunities for investigating the process of evolution in action. The inversion polymorphism in the O chromosome from populations of central California and northern Washington, separated by 1300 km, corresponds to a previously observed latitudinal cline, also observed in Europe. Recessive lethal genes are not randomly distributed among the chromosomal arrangements. The incidence of lethal allelism is high, yielding unrealistically low estimates of the effective size of these populations (on the order of 1000 individuals). The high incidence of lethal allelism is likely to be a consequence of the low number of the American colonizers (on the order of 10-100 individuals), but the persistence of the allelism over several years suggests that some lethal-carrying chromosomes may be heterotic owing to shared associations between lethal and other genes.     

Significance of SIMS microscopy for the radioiodine detection in animal and human thyroid tissue.

We defined the SIMS conditions for radioiodine detection in animal and man thyroid follicles, in tissue sections (3 microns) chemically fixed and resin embedded. Two radioisotopes were tested: 125I and 129I, of high (14 mCi 125I micrograms-1) and low specific activity (1.07 10(-6) mCi 129I micrograms-1). In animal study, Wistar rats fed a normal iodine diet (10 micrograms 127I day-1) were injected ip 24 h before sacrifice either with 125I (7 10(-3) micrograms) or with 129I at a dose identical to iodine diet (10 micrograms) or 3 times higher (30 micrograms). No SIMS signal of 125I was obtained in vivo due to its too low concentration, while radioiodine distribution was evidenced with both doses of 129I. Local concentration of previously stored 127I in follicular lumen was not modified, when compared to control (4.14 +/- 0.03 micrograms/mg, m +/- SE), by 125I or 129I at a dose of 10 micrograms, but was nearly doubled with 129I at a dose of 30 micrograms, proof of a pharmacological effect on thyroid iodine regulation. In human study 129I was excluded due to its long half-life (1.6 10(7) years), and 125I was tested only in vitro on two surgical specimens of normal perinodular thyroid tissue maintained in mini-organ culture for 48 h in presence of 100 microCi/ml of 125I. The 125I was detectable, its concentration was 1,000-fold higher than that of 127I (1.5 +/- 0.004 micrograms/mg). For both in vivo and in vitro studies, a positive correlation exists between newly organified radioiodine (125I or 129I) and previously stored iodine (127I).(ABSTRACT TRUNCATED AT 250 WORDS)     

Insights into the molecular mechanisms of protein platination from a case study: the reaction of anticancer platinum(II) iminoethers with horse heart cytochrome c

The interactions of anticancer metallodrugs with proteins are attracting a growing interest in the current literature because of their relevant pharmacological and toxicological consequences. To understand in more depth the nature of those interactions, we have investigated the reactions of four anticancer platinum(II) iminoether complexes, namely, trans- and cis-EE (trans- and cis-[PtCl2{(E)-HN=C(OCH3)CH3}2], respectively) and trans- and cis-Z (trans- and cis-[PtCl2(NH3){(Z)-HN=C(OCH3)CH3}], respectively), with horse heart cytochrome c (cyt c). Our investigation was performed using mainly electrospray ionization mass spectrometry (ESI MS) but was also supported by NMR, inductively coupled plasma optical emission spectroscopy (ICP OES), and absorption electronic spectroscopy. ESI MS spectra clearly revealed the formation of a variety of platinum-protein adducts predominantly corresponding to monoplatinated cyt c species. From a careful analysis of the major ESI MS peaks, specific information on the nature of the protein-bound metallic fragments and on the underlying metallodrug-cyt c reactions was gained for the various cases. We found that trans-EE produces a major cyt c adduct (12 667 Da) that is different from that produced by either cis-EE or by trans-Z and cis-Z (12 626 Da). In particular, occurrence of extensive hydrolysis/aminolysis (the latter fostered by ammonium carbonate buffer) of the iminoether ligands and formation of the corresponding amides/amidines has been unambiguously documented. The reactivity of the iminoether ligands is greatly enhanced by the presence of cyt c as inferred from comparative NMR solution studies. Additional ESI MS measurements recorded on enzymatically cleaved samples of platinated cyt c adducts, together with NMR investigation of the cyt c/trans-EE adduct, strongly suggest that protein platination primarily occurs at Met 65. The biological and pharmacological implications of the described protein platination processes are discussed.