Triple helix DNA oligomer melting measured by fluorescence polarization anisotropy

A synthetic DNA triple helix sequence was formed by annealing a pyrimidinic 21 mer single strand sequence onto the complementary purinic sequence centred on a 27 mer duplex DNA. Melting of the third strand was monitored by UV spectrophotometry in the temperature range 10-90 degrees C. The T(m) of the triplex, 37 degrees C, was well separated from the onset of duplex melting. When the same triple helix was formed on the duplex bearing one nick in the center of the pyrimidinic sequence the T(m) of the triplex was shifted to approximately 32 degrees C and overlapped the melting of the duplex. We have used fluorescence polarization anisotropy (FPA) measurements of ethidium bromide (EB) intercalated in duplex and triplex samples to determine the hydrodynamic parameters in the temperature range 10-40 degrees C. The fluorescence lifetime of EB in the samples of double and triple stranded DNA is the same (21.3 +/- 0.5 ns) at 20 degrees C, indicating that the geometries of the intercalation sites are similar. The values for the hydration radii of the duplex, normal triplex, and nicked triplex samples were 10.7 +/- 0.2, 12.2 +/- 0.2, and 12.0 +/- 0.2 A. FPA measurements on normal triplex DNA as a function of temperature gave a melting profile very similar to that derived by UV absorption spectroscopy. For the triplex carrying a nick, the melting curve obtained using FPA showed a clear shift compared with that obtained for the normal triplex sample. The torsional rigidity of the triplex forms was found to be higher than that of the duplex form.     

Effect of caffeic acid on tert-butyl hydroperoxide-induced oxidative stress in U937

Nonvitamin phenolic compounds are ubiquitous in food plants and therefore potentially present in human plasma in a diet-dependent concentration. The aim of this study was to evaluate the ability of caffeic acid, a phenolic acid with antioxidant activity, to affect cellular response in U937 human monocytic cells to t-butyl hydroperoxide-induced oxidative stress. In our experimental conditions caffeic acid was incorporated into cells without any cytotoxic effect. Caffeic acid-treated cells showed an increased resistance to oxidative challenge, as revealed by an higher percent of survival and the maintenance of an higher proliferative capacity in respect to control cells. This effect seems to be due to the ability of caffeic acid to reduce glutathione depletion and to inhibit lipid peroxidation during tBOOH treatment. It can be concluded that caffeic acid exerts an antioxidant action inside the cell, responsible for the observed modulation of the cellular response to oxidative challenge. Due to its presence in the diet, therefore, caffeic acid may play a role in the modulation of oxidative processes in vivo.     

Determination of D-amino acids using a D-amino acid oxidase biosensor with spectrophotometric and potentiometric detection

An amperometric and a colorimetric biosensor to detect and quantify D-amino acids selectively has been devised using D-amino acid oxidase from Rhodotorula gracilis. The sensor is characterised by a proportional response between 0.2-3 mM and 0.1-1 mM D-alanine for the amperometric (at a working potential of 1400 mV vs Ag/AgCl) and colorimetric system, respectively.     

Polymorphisms of the prion protein gene in Italian patients with Creutzfeldt-Jakob disease

Creutzfeldt-Jakob disease (CJD) is a transmissible neurodegenerative disorder characterized by the accumulation of the amyloid protein PrP in the CNS. Two coding polymorphisms of the PrP gene (PRNP) are a methionine (Met) to valine (Val) change at codon 129, and a deletion in the octapeptide coding region. In the United Kingdom, homozygosity at codon 129 appears to be associated with a predisposition to develop CJD. However, in Japan, where allelic frequencies and genotype distribution are significantly different, such an association has not been demonstrated. To determine whether such deletion(s) or codon 129 polymorphisms of PRNP predispose to the development of CJD in Italian patients, 31 sporadic CJD patients with no known PRNP mutations, and 186 unrelated control subjects were studied. Genotypic frequencies at codon 129 in these Italian CJD patients revealed a significant excess of methionine alleles, and a different genotype distribution in comparison with the normal Italian population. Deletions of a 24-bp segment located in the PrP octapeptide coding region were found in two control subjects, but in none of the sporadic CJD patients. These data suggest that Met homozygosity at codon 129 may contribute, with other enviromental or endogenous factors, to CJD development.     

Steroid biochemistry and categorization of breast cyst fluid: relation to breast cancer risk

Patients bearing macrocysts of the breast are at higher risk of later developing cancer. The fluid filling the cysts (breast cysts fluid, BCF) contains unusual amounts of steroid conjugates, first androgen and estrogen sulfates. Measuring BCF cations (K⁺, Na⁺) allows categorization of cysts into two major subsets (type I and type II) that are associated with a different degree and/or turnover of apocrine metaplastic cells in the lining epithelium. Type I cysts (high K⁺/ Na⁺ ratio) accumulate huge amounts of dehydroepiandrosterone sulfate, estrone sulfate, androstane-3, 17β-diol glucuronide, androsterone glucuronide and contain more testosterone and dihydrotestosterone than type II. Conversely, type II cysts (low K⁺/Na⁺ ratio) contain more progesterone and pregnenolone. A cohort study was started in 1983 at the Cancer Prevention Center, Ravenna, Italy, with the aim of evaluating the relationships between the biochemistry of BCF and the incidence of breast cancer in women with gross cystic disease (GCD) of the breast. The bimodal distribution of the cationic pattern has been confirmed from data obtained in 798 patients aspirated. The risk of cyst relapse was significantly higher among women with type I cysts or with multiple cysts at presentation. Twelve incident cases of breast cancer have been diagnosed among women whose BCF was categorized. Eleven out of 12 cases had type I or multiple cysts. The cumulative incidence of breast cancer among patients bearing type I cysts was 2.5%. We conclude that women with GCD bearing type I cysts have an increased breast cancer risk when compared with the counterpart bearing type II cysts or the general population.     

Effects of new ubiquinone-imidazo[2,1-b]thiazoles on mitochondrial complex I (NADH-ubiquinone reductase) and on mitochondrial permeability transition pore

In this work we describe the synthesis of a series of imidazo[2,1-b]thiazoles and 2,3-dihydroimidazo[2,1-b]thiazoles connected by means of a methylene bridge to CoQ(0). These compounds were tested as specific inhibitors of the NADH:ubiquinone reductase activity in mitochondrial membranes. The imidazothiazole system when bound to the quinone ring in place of the isoprenoid lateral side chain, may increase the inhibitory effect (with an IC(50) for NADH-Q(1) activity ranging between 0.25 and 0.96 microM) whereas the benzoquinone moiety seems to lose the capability to accept electrons from complex I as indicated by very low maximal velocity elicited by the compounds tested. Moreover the low rotenone sensitivity for almost all of these compounds suggests that they are only partially able to interact with the physiological ubiquinone-reduction site. The compounds were investigated for the capability of increasing the permeability transition of the inner mitochondrial membrane in isolated mitochondria. Unlike CoQ(0), which is considered a mitochondrial membrane permeability transition inhibitor, the new compounds were inducers.     

Male-sterile mutation alters Zea m 1 (β-expansin 1) accumulation in a maize mutant

gaMS-2 is a gametophytic male-sterile mutant of maize, with sterile pollen grains developmentally blocked at the binucleate stage. To characterise differentially expressed proteins in gaMS-2 pollen, we compared protein profiles of anthers and mature pollen from heterozygous GaMS-2/gaMS-2 plants and wild type (wt) plants by two-dimensional electrophoresis (2-DE). A basic protein present at a greatly reduced level in GaMS-2/gaMS-2 anthers was subsequently identified by tandem mass spectrometry as Zea m 1 (a glycoprotein of 31 kDa), the major group-1 allergen of maize pollen and a member of the -expansin 1 family. Moreover, we show that Zea m 1 can be deglycosylated by peptide N-glycosidase F. After deglycosylation, four major isoforms—Zea m 1a (more acetic), Zea m 1b, Zea m1c and Zea m 1d (more basic)—can be discriminated in wt anther in 2-DE immunoblots probed with a monoclonal antibody against the group-1 pollen allergen, whereas all the isoforms, especially Zea m 1a, exist at reduced levels in GaMS-2/gaMS-2 anthers. Furthermore, the reduced Zea m 1 accumulation in the mutant appears to occur in immature pollen but not in anther sporophytic tissues. Finally, we separated sterile pollen grains (at the mononucleate stage) from fertile ones using 42% Percoll solution, and found that Zea m 1 is barely detectable in sterile pollen grains. Together, our results indicate that a reduced Zea m 1 level is associated with the sterile phenotype of gaMS-2.W. Wang and M. Scali contributed equally to this study     

Depletion of the 110-kilodalton isoform of poly(ADP-ribose) glycohydrolase increases sensitivity to genotoxic and endotoxic stress in mice

Poly(ADP-ribosylation) is rapidly stimulated in cells following DNA damage. This posttranslational modification is regulated by the synthesizing enzyme poly(ADP-ribose) polymerase 1 (PARP-1) and the degrading enzyme poly(ADP-ribose) glycohydrolase (PARG). Although the role of PARP-1 in response to DNA damage has been studied extensively, the function of PARG and the impact of poly(ADP-ribose) homeostasis in various cellular processes are largely unknown. Here we show that by gene targeting in embryonic stem cells and mice, we specifically deleted the 110-kDa PARG protein (PARG(110)) normally found in the nucleus and that depletion of PARG(110) severely compromised the automodification of PARP-1 in vivo. PARG(110)-deficient mice were viable and fertile, but these mice were hypersensitive to alkylating agents and ionizing radiation. In addition, these mice were susceptible to streptozotocin-induced diabetes and endotoxic shock. These data indicate that PARG(110) plays an important role in DNA damage responses and in pathological processes.     

Early diagnosis of congenital Trypanosoma cruzi infection using PCR, hemoculture, and capillary concentration, as compared with delayed serology

Congenital Trypanosoma cruzi infection is a highly pathogenic and underreported condition. Early recognition is essential for effective treatment. Umbilical chord blood from newborns (n = 302) to infected mothers was analyzed with microhematocrit, hemoculture, and PCR methods. Each subject was then followed serologically. In calibrated suspensions of T. cruzi in blood, the sensitivity of PCR was 27-fold higher than hemoculture. However, this advantage was not reflected during routine testing of samples from maternities, partly because of the uneven distribution of few parasites in small samples. Levels of detection of congenital infection were 2.9% (8/272) for microhematocrit, 6.3% (18/287) for hemoculture, 6.4% (15/235) for PCR, and 8.9% (27/302) for cumulated results. Evaluation against the standard of delayed serology indicates that the regular application of PCR, hemoculture, and microhematocrit to blood samples allows the rapid detection of about 90% of the congenitally infected newborns, in samples that can be obtained before the mother and child leave the maternity ward.