Distribution of prepubertal and adult goat oocyte cortical granules during meiotic maturation and fertilisation: ultrastructural and cytochemical study

The aim of this study was evaluate cortical granule (CG) distribution during in vitro maturation (IVM) and fertilisation of prepubertal goat oocytes compared to CG distribution of ovulated and in vitro fertilised oocytes from adult goats. Oocytes from prepubertal goats were recovered from a slaughterhouse and were matured in M199 with hormones and serum for 27 hr. Ovulated oocytes were collected from gonadotrophin treated Murciana goats. Frozen-thawed spermatozoa were selected by centrifugation in percoll gradient and were capacitated in DMH with 20% steer serum for 1 hr. Ovulated and IVM-oocytes were inseminated in DMH medium with steer serum and calcium lactate for 20 hr. Oocytes and presumptive zygotes were stained with FITC-LCA (Lens culinaris agglutinin labelled with fluorescein isothiocyanate) and observed under a confocal laser scanning microscope. Ultrastructure morphology of oocytes and presumptive zygotes were analysed by transmission electron microscopy (TEM). Prepubertal goat oocytes at germinal vesicle stage show a homogeneous CG distribution in the cytoplasm. IVM-oocytes at Metaphase II (MII) and ovulated oocytes presented CGs located in the cortex with the formation of a monolayer beneath to the plasma membrane. At 20 hr postinsemination (hpi), zygotes from IVM-oocytes showed a complete CG exocytosis whereas zygotes from ovulated oocytes presented aggregates of CGs located at the cortical region. Images by TEM detected that CGs were more electrodense and compacts in oocytes from prepubertal than from adult goats.     

Enterocyte proliferation and intestinal hyperplasia induced by simian virus 40 T antigen require a functional J domain

Transgenic mice expressing the simian virus 40 large T antigen (TAg) in enterocytes develop intestinal hyperplasia that progresses to dysplasia with age. This induction requires TAg action on the retinoblastoma (Rb) family of tumor suppressors and is independent of the p53 pathway. In cell culture systems, the inactivation of Rb proteins requires both a J domain in TAg that interacts with hsc70 and an LXCXE motif that directs association with Rb proteins. Together these elements are sufficient to release E2Fs from their association with Rb family members. We have generated transgenic mice that express a J domain mutant (D44N) in villus enterocytes. In contrast to wild-type TAg, the D44N mutant is unable to induce enterocyte proliferation. Histological and morphological examination revealed that mice expressing the J domain mutant have normal intestines without loss of growth control. Unlike mice expressing wild-type TAg, mice expressing D44N do not reduce the protein levels of p130 and are also unable to dissociate p130-E2F DNA binding complexes. Furthermore, mice expressing D44N in a null p130 background are still unable to develop hyperplasia. These studies demonstrate that the ectopic proliferation of enterocytes by TAg requires a functional J domain and suggest that the J domain is necessary to inactivate all three pRb family members.     

DNA damage in peripheral blood lymphocytes in patients during combined chemotherapy for breast cancer

Combined chemotherapy is used for the treatment of a number of malignancies such as breast cancer. The target of these antineoplastic agents is nuclear DNA, although it is not restricted to malignant cells. The aim of the present study was to assess DNA damage in peripheral blood lymphocytes (PBLs) of breast cancer patients subjected to combined adjuvant chemotherapy (5-fluorouracil, epirubicin and cyclophosphamide, FEC), using a modified comet assay to detect DNA single-strand breaks (SSB) and double-strand breaks (DSB).

Forty-one female patients with advanced breast cancer before and after chemotherapy and 60 healthy females participated in the study. Alkaline and neutral comet assays were performed in PBLs according to a standard protocol, and DNA tail moment was measured by a computer-based image analysis system.

Breast cancer patients before treatment had higher increased background levels of SSB and DSB as compared to healthy women. During treatment, a significant increase in DNA damage was observed after the 2nd cycle, which persisted until the end of treatment. Eighty days after the end of treatment the percentage of PBLs with SSB and DSB remained elevated, but the magnitude of DNA damage (tail moment) returned to baseline levels. There was no correlation between PBL DNA damage and response to chemotherapy.

DNA-SSB and DSB in PBLs are present in cancer patients before treatment and increase significantly after combined chemotherapy. No correlation with response to adjuvant chemotherapy was found. Biomonitoring DNA damage in PBLs of cancer patients could help prevent secondary effects and the potential risks of developing secondary cancers.     

Translocation of nucleoside analogs across the plasma membrane in hematologic malignancies

Nucleoside analogs are currently used in the treatment of various hematologic malignancies due to their ability to induce apoptosis of lymphoid cells. For nucleoside-derived drugs to exert their action, they must enter cells via nucleoside transporters from two gene families, SLC28 and SLC29 (CNT and ENT, respectively). Once inside the cell, these drugs must be phosphorylated to their active forms. In contrast, some members of the ATP-binding cassette (ABC) protein family have been identified as responsible for the efflux of the phosphorylated forms of these nucleoside-derived drugs. Here, we review the main nucleoside analogs used in hematologic malignancies and focus especially on those that are currently used in chronic lymphocytic leukemia (CLL). Moreover, we discuss the pharmacological profile of the nucleoside transporters, which determines the bioavailability of and cell sensitivity to these nucleoside-derived drugs. We also discuss the expression of nucleoside transporters and their activities in CLL as well as the possibility of modulating these transporter activities as a means of modulating intracellular drug availability and, consequently, responsiveness to therapy.     

Antidepressant-like effect of a Ginkgo biloba extract (EGb761) in the mouse forced swimming test: role of oxidative stress

EGb761 is a well-defined mixture of active compounds extracted from Ginkgo biloba leaves. This extract is used clinically due to its neuroprotective effects, exerted probably via its potent antioxidant or free radical scavenger action. Previous studies suggest that oxidative stress, via free radical production, may play an important role in depression and animal models for depression-like behavior. Preclinical studies have suggested that antioxidants may have antidepressants properties. The aim of this study was to investigate the antidepressant-like of EGb761 due to its antioxidant role against oxidative stress induced in the forced swimming test, the most widely used preclinical model for assessing antidepressant-like behavior. Male BALB/c mice were pretreated with EGb761 (10 mg/kg, ip) daily for 17 days followed by the forced swimming test and spontaneous locomotor activity. Animals were sacrificed to evaluate lipid peroxidation, different antioxidant enzyme activities, serotonin and dopamine content in midbrain, hippocampus and prefrontal cortex. EGb761 significantly decreased the immobility time (39%) in the forced swimming test. This antidepressant-like effect of EGb761 was associated with a reduction in lipid peroxidation and superoxide radical production (indicated by a downregulation of Mn-superoxide dismutase activity), both of which are indicators of oxidative stress. The protective effect of EGb761 is not related to excitatory or inhibitory effects in locomotor activity, and was also associated with the modulation of serotonergic and dopaminergic neurotransmission. It is suggested that EGb761 produces an antidepressant-like effect, and that an antioxidant effect against oxidative stress may be partly responsible for its observed neuroprotective effects.     

Cognitive impairment and the risk of falling in the elderly

Risk of fall is significantly increased in old people with cognitive decline due to specific associations between gait parameters and cognition. This association has recently been demonstrated, there being increasing evidence that cognitive domains such as attention, executive function and types of memory are critical for the correct regulation of gait. Gait disturbances can appear as early predictors of dementia in elderly patients. In the assessment of the fall risk, the use of dual tasks is novel, simple and relevant, especially in cognitive decline. Evidence for interventions in this population is limited, with vitamin D and physical exercise being the most encouraging, for decreasing the risk of fall in dementia.     

Posttranslational modification of gluten shapes TCR usage in celiac disease

Posttranslational modification of Ag is implicated in several autoimmune diseases. In celiac disease, a cereal gluten-induced enteropathy with several autoimmune features, T cell recognition of the gluten Ag is heavily dependent on the posttranslational conversion of Gln to Glu residues. Evidence suggests that the enhanced recognition of deamidated gluten peptides results from improved peptide binding to the MHC and TCR interaction with the peptide-MHC complex. In this study, we report that there is a biased usage of TCR Vβ6.7 chain among TCRs reactive to the immunodominant DQ2-α-II gliadin epitope. We isolated Vβ6.7 and DQ2-αII tetramer-positive CD4(+) T cells from peripheral blood of gluten-challenged celiac patients and sequenced the TCRs of a large number of single T cells. TCR sequence analysis revealed in vivo clonal expansion, convergent recombination, semipublic response, and the notable conservation of a non-germline-encoded Arg residue in the CDR3β loop. Functional testing of a prototype DQ2-α-II-reactive TCR by analysis of TCR transfectants and soluble single-chain TCRs indicate that the deamidated residue in the DQ2-α-II peptide poses constraints on the TCR structure in which the conserved Arg residue is a critical element. The findings have implications for understanding T cell responses to posttranslationally modified Ags.