A mouse model to study thrombotic complications of thalassemia.

Patients with ß-thalassemia major and mainly intermedia have an increased risk for developing venous and arterial thrombosis which may be related to circulating pathological red blood cells (RBC) and continuous platelet activation. In the present study we used a modified thalassemic mice model in conjunction with a "real-time" carotid thrombus formation procedure to investigate thrombotic complications of thalassemia. Heterozygous Th3/+ mice, which lack one copy of their ß-major and ß-minor globin genes, exhibit anomalies in RBC size and shape, chronic anemia and splenomegaly which recapitulate the phenotype of human ß-thalassemia intermedia. Flow cytometry measurements showed higher reactive oxygen species generation, indicating oxidative stress, in platelets and RBC of the thalassemic mice compared with wild type mice concomitant with an increase in reduced glutathione content which may represent a compensatory response to oxidative stress, and exposed phosphatidylserine which indicates platelet activation. To elucidate the effect of thalassemia on the development of arterial thrombosis, we studied photochemical-induced real-time thrombus formation in the carotid artery of these mice. The results indicated a significantly shorter "time to occlusion" in the thalassemic mice compared to wild type mice, which was prolonged following in vivo aspirin treatment. We suggest that this mouse model may contribute to our understanding of platelet activation and the hypercoagulable state in thalassemia and lay foundations to screening of anti-platelet drugs as well as anti-oxidants as possible therapeutics for prevention of thrombosis in thalassemia patients.

[Oxidation and female reproduction: the good, the bad and what's between].

Reactive oxygen and nitrogen species constitute an inseparable part of aerobic life on earth. They have been known to science for about 90 years, but only during the last 50 years research in this field has expanded. Initially, scientists focused on the free radicals-induced damage to biological systems. Since the eighties, a new concept has emerged, namely, that alongside the oxidative stress-induced deleterious effect and its association with a variety of diseases, a certain threshold level of oxidation is essential to intracellular signaL transduction. Recently, some data has accumulated regarding the involvement of oxidative processes in various aspects of female reproduction, including ovarian function, fertilization, early development of the embryo and implantation. Nevertheless, there is still a long way before comprehensive and thorough understanding of their role, both at the molecular level and the expression in the clinical setup of fertility patients can be achieved. In this article, we shall address some molecular biochemical processes involved in the activity of free radicals, and review the present knowledge regarding their role in female fertility, including ovarian physiology, follicular and oocyte maturation, development of the early embryo and implantation, as well as their association with reproductive pathologies such as endometriosis, polycystic ovary disease and recurrent pregnancy loss.

Oxidative parameters of embryo culture media may predict treatment outcome in in vitro fertilization: a novel applicable tool for improving embryo selection.

OBJECTIVE: To examine whether the oxidative status of an individual embryo before transfer may predict chances of implantation. DESIGN: A prospective laboratory study. SETTING: An IVF unit in a university-affiliated hospital. PATIENT(S): One hundred thirty-three women undergoing IVF-ET treatment cycles. INTERVENTION(S): Before ET, 10 µL of embryo culture medium was retrieved individually from each embryo and the oxidative status assessed by the thermochemiluminescence (TCL) analyzer. MAIN OUTCOME MEASURE(S): The occurrence of pregnancy. Two parameters were recorded: the TCL amplitude after 50 seconds (H1) and the TCL ratio. These were compared with demographic, clinical, and laboratory parameters and treatment outcome. All data underwent statistical analysis. RESULT(S): Altogether 284 embryos were transferred in 133 ET cycles. Forty-one pregnancies occurred (31%). For embryos transferred after 72 hours (77 transfers), the highest H1 levels in each group of transferred embryos correlated with the occurrence of pregnancy. The combination of maximal intracohort H1 level <210 counts per second with a TCL ratio of ≤ 80% had a positive predictive value of 70.6% for the occurrence of pregnancy. CONCLUSION(S): The oxidative status of the early embryo in IVF is associated with the chances of implantation. Assessment of the oxidative status of embryos in culture media before transfer may serve as an applicable tool for improving embryo selection in light of the legal limitations of the number of transferred embryos allowed.

Involvement of DNA curvature in intergenic regions of prokaryotes.

It is known that DNA curvature plays a certain role in gene regulation. The distribution of curved DNA in promoter regions is evolutionarily preserved, and it is mainly determined by temperature of habitat. However, very little is known on the distribution of DNA curvature in termination sites. Our main objective was to comprehensively analyze distribution of curved sequences upstream and downstream to the coding genes in prokaryotic genomes. We applied CURVATURE software to 170 complete prokaryotic genomes in a search for possible typical distribution of DNA curvature around starts and ends of genes. Performing cluster analyses and other statistical tests, we obtained novel results regarding various factors influencing curvature distribution in intergenic regions, such as growth temperature, A+T composition and genome size. We also analyzed intergenic regions between converging genes in 15 selected genomes. The results show that six genomes presented peaks of curvature excess larger than 3 SDs. Insufficient statistics did not allow us to draw further conclusion. Our hypothesis is that DNA curvature could affect transcription termination in many prokaryotes either directly, through contacts with RNA polymerase, or indirectly, via contacts with some regulatory proteins.

Curvature distribution in prokaryotic genomes.

DNA curvature is known to play a biological role in gene regulation, in particular, initiation of transcription. We applied the software CURVATURE based on the wedge model to predict whether promoter regions of certain prokaryotes may be characterized by higher intrinsic DNA curvature located within or upstream to these regions. The main purpose was to verify our earlier hypothesis that the DNA curvature plays a biological role in gene regulation in mesophilic as compared to hyperthermophilic prokaryotes, i.e., DNA curvature presumably has a functional adaptive significance determined by temperature selection. Therefore, we analyzed all available complete prokaryotic genomes. The analysis showed that there is a group of genomes with a relatively high average DNA curvature upstream of start of genes. Remarkably, all organisms of this group appeared to be mesophilic, which is a full confirmation of the former hypothesis. The conservative patterns of genomic curvature distribution across different mesophilic bacterial and archaeal genomes presented in this study provide a new, convincing indication that curved DNA is evolutionarily preserved and determined by temperature selection. Moreover, we found a rather peculiar property of hyperthermophilic prokaryotes: the coding regions are predicted to be significantly more curved than it would be expected from their dinucleotide composition.