Comparative study of the lethal effects of near-UV light (360 nm) and 8-methoxypsoralen plus near-UV on plasmid DNA

We have studied the lethality produced on pBR322 by near-UV radiation and by 8-Methoxypsoralen plus near-UV (PUV treatment). Samples of pBR322 DNA were irradiated with increasing fluences of 360 nm-light either in the absence or presence of 400 molecules of 8-Methoxypsoralen (8-MOP) per plasmid molecule. We have estimated to what extent the global lethality of PUVA treatment is due to the presence of psoralen adducts in DNA or to radiation itself. In order to analyse the involvement of DNA repair mechanisms in the removal of plasmid lesions, several strains of E. coli (differing in their repair capacities) were used as recipients of the treated plasmids. Results showed that excision and recombination participate in the repair of near-UV-induced plasmid lesions. Repair of PUV-induced lesions showed an even greater requirement of the excision pathway. Besides, a slight increase on plasmid mutation frequencies was observed after near-UV or PUV treatment in wild type and uvrA cells. Estimation of the contribution of 8-MOP to the global lethality of PUV treatment showed that only the excision pathway was involved in removing psoralen adducts from plasmid DNA, suggesting the involvement of the recombinational pathway in the repair of near-UV-derived lesions.     

Developmental competence of prepubertal goat oocytes selected with brilliant cresyl blue and matured with cysteamine supplementation

The aim of this study was to assess the effect of oocyte selection using the brilliant cresyl blue (BCB) test plus the addition of cysteamine to the in vitro maturation (IVM) medium to improve the in vitro embryo development of prepubertal goat oocytes. The oocytes were exposed to 26 microM BCB and classified according to their cytoplasm coloration: BCB+ (oocytes with blue cytoplasm) and BCB- (unstained oocytes). The oocytes were matured in a conventional IVM medium supplemented with cysteamine 100 microM. The control group consisted of oocytes not exposed to BCB and matured without cysteamine. The IVM-oocytes were inseminated and cultured in synthetic oviductal fluid (SOF) for 7 days. The normal fertilisation rate (oocytes showing 2 pronuclei and 1 sperm tail) of BCB+ oocytes (40%) was higher than those of BCB- (21%) and control oocytes (22%). The percentage of morulae plus blastocysts was higher (P < 0.05) in the BCB+ group than in the BCB- group (23.8 vs. 5.1%, respectively). In conclusion, the integration of the BCB test and the addition of cysteamine in the protocol of in vitro embryo production from prepubertal goat oocytes has improved the developmental rates of embryo development.     

A Humanized Mouse Model of HPV-Associated Pathology Driven by E7 Expression

Human papillomavirus (HPV) is the causative agent of human cervical cancer and has been associated with oropharyngeal squamous cell carcinoma development. Although prophylactic vaccines have been developed, there is a need to develop new targeted therapies for individuals affected with malignant infected lesions in these locations, which must be tested in appropriate models. Cutaneous beta HPV types appear to be involved in skin carcinogenesis. Virus oncogenicity is partly achieved by inactivation of retinoblastoma protein family members by the viral E7 gene. Here we show that the E7 protein of cutaneous beta HPV5 binds pRb and promotes its degradation. In addition, we described an in vivo model of HPV-associated disease in which artificial human skin prepared using primary keratinocytes engineered to express the E7 protein is engrafted onto nude mice. Expression of E7 in the transplants was stably maintained for up to 6 months, inducing the appearance of lesions that, in the case of HPV16 E7, histologically resembled human anogenital lesions caused by oncogenic HPVs. Moreover, it was confirmed through biomarker expression analysis via immunodetection and/or quantitative PCR from mRNA and miRNA that the 16E7-modified engrafted skin shares molecular features with human HPV-associated pretumoral and tumoral lesions. Finally, our findings indicate a decrease of the in vitro capacity of HPV5 E7 to reduce pRb levels in vivo, possibly explaining the phenotypical differences when compared with 16E7-grafts. Our model seems to be a valuable platform for basic research into HPV oncogenesis and preclinical testing of HPV-associated antitumor therapies.     

Vitrification of in vitro produced goat blastocysts: effects of oocyte donor age and development stage

This study examines the effectiveness of the cryotop vitrification method for the cryopreservation of goat blastocysts. To determine the effects of embryo development stage and donor age on in vitro survival rates, good-quality blastocysts from adult and prepubertal goats were sorted into non-expanded, expanded, hatching and completely hatched. In vitro produced (IVP) blastocysts were derived from prepubertal goat oocytes by slicing of ovaries from slaughtered animals while adult goat oocytes were collected by the laparoscopic ovum pick up (LOPU) method. Blastocysts were vitrified/warmed using the cryotop technique. Survival rates were determined in terms of blastocoele re-expansion at 3 and 20 h post-warming. For prepubertal goats, survival rates at 3 h post-warming were significantly higher when expanded blastocysts (78.3%) were vitrified/warmed compared to hatched blastocysts (57.4%), whereas non-expanded (62.5%) or hatching blastocysts (71.4%) showed similar rates. For adult goats, survival rates were significantly higher after warming in expanded (36.4%), hatching (75%) or hatched (50%) blastocysts when compared to non-expanded (0%) blastocysts. When survival rates were assessed at 20 h post-warming, no differences were observed when we compared non-expanded (45.8%), expanded (56.5%), hatching (64.3%) and hatched (50.5%) blastocysts from prepubertal goats; and for blastocysts from adult goats, survival rates were only significantly lower for the non-expanded stage (0%) compared to the other stages. For adult versus prepubertal blastocysts at the same developmental stage, our data indicate significantly higher survival rates at 3 h post-warming for non-expanded and expanded blastocysts from prepubertal goats over their counterparts from adult goats. At 20 h post warming, survival rates were only higher for non-expanded blastocysts from prepubertal goats versus adult goats. Collectively, our data reveal that blastocysts produced in vitro from prepubertal goats return similar survival rates regardless of their development stage, whereas blastocysts derived from adult goats are best for vitrification at the expanded, hatching or hatched stage.     

Bleomycin Induces Molecular Changes Directly Relevant to Idiopathic Pulmonary Fibrosis: A Model for “Active” Disease

The preclinical model of bleomycin-induced lung fibrosis, used to investigate mechanisms related to idiopathic pulmonary fibrosis (IPF), has incorrectly predicted efficacy for several candidate compounds suggesting that it may be of limited value. As an attempt to improve the predictive nature of this model, integrative bioinformatic approaches were used to compare molecular alterations in the lungs of bleomycin-treated mice and patients with IPF. Using gene set enrichment analysis we show for the first time that genes differentially expressed during the fibrotic phase of the single challenge bleomycin model were significantly enriched in the expression profiles of IPF patients. The genes that contributed most to the enrichment were largely involved in mitosis, growth factor, and matrix signaling. Interestingly, these same mitotic processes were increased in the expression profiles of fibroblasts isolated from rapidly progressing, but not slowly progressing, IPF patients relative to control subjects. The data also indicated that TGFβ was not the sole mediator responsible for the changes observed in this model since the ALK-5 inhibitor SB525334 effectively attenuated some but not all of the fibrosis associated with this model. Although some would suggest that repetitive bleomycin injuries may more effectively model IPF-like changes, our data do not support this conclusion. Together, these data highlight that a single bleomycin instillation effectively replicates several of the specific pathogenic molecular changes associated with IPF, and may be best used as a model for patients with active disease.     

Beyond structure: do intermediate filaments modulate cell signalling?

Intermediate filament (IF) proteins form the largest family of cytoskeletal proteins in mammalian cells. The function of these proteins has long been thought to be only structural. However, this single function does not explain their diverse tissue- and differentiation-specific expression patterns. Evidence is now emerging that IF also act as an important framework for the modulation and control of essential cell processes, in particular, signal transduction events. Here, we review the most recent developments in this growing and exciting new field.     

Impaired Hair Growth and Wound Healing in Mice Lacking Thyroid Hormone Receptors

Both clinical and experimental observations show that the skin is affected by the thyroidal status. In hypothyroid patients the epidermis is thin and alopecia is common, indicating that thyroidal status might influence not only skin proliferation but also hair growth. We demonstrate here that the thyroid hormone receptors (TRs) mediate these effects of the thyroid hormones on the skin. Mice lacking TRα1 and TRβ (the main thyroid hormone binding isoforms) display impaired hair cycling associated to a decrease in follicular hair cell proliferation. This was also observed in hypothyroid mice, indicating the important role of the hormone-bound receptors in hair growth. In contrast, the individual deletion of either TRα1 or TRβ did not impair hair cycling, revealing an overlapping or compensatory role of the receptors in follicular cell proliferation. In support of the role of the receptors in hair growth, TRα1/TRβ-deficient mice developed alopecia after serial depilation. These mice also presented a wound-healing defect, with retarded re-epithelialization and wound gaping, associated to impaired keratinocyte proliferation. These results reinforce the idea that the thyroid hormone nuclear receptors play an important role on skin homeostasis and suggest that they could be targets for the treatment of cutaneous pathologies.     

Further studies on the lethal and mutagenic effects of 8-methoxypsoralen-induced lesions on plasmid DNA

Our previous results on the genotoxic effect of 8-methoxypsoralen-induced lesions on pBR322 suggested an important involvement of an inducible error-free repair pathway in the repair of plasmid lesions. We present herein further results obtained in order to explore that possibility, together with a more general report on the subject. pBR322 treated with increasing concentrations of 8-MOP plus fixed UVA light irradiation was used to transform several E. coli strains differing in their repair capacities, and plasmid survival and mutagenesis were determined. Survival results suggested that crosslinks were completely lethal in pBR322 whereas monoadducts were partially removed from plasmid DNA mainly through an error-free excision pathway. A mutagenic repair pathway did not show a significant contribution to the total repair process. Cell preirradiation stimulated plasmid recovery in recA+ strains, including the umuC strain, thus confirming our previous results indicating that an inducible error-free repair had occurred. Globally, our results showed a strong requirement on the excision pathway for the repair of psoralen-damaged plasmid DNA. In contrast, the recA dependent pathway was needed only for SOS induction. After a theoretical correction of the data for estimating the effect only due to 8-MOP adducts, a different pattern of repair mechanisms appeared to be involved.