Characterization of the human and mouse ETV1/ER81 transcription factor genes: Role of the two alternatively spliced isoforms in the human

The Ets transcription factors of the PEA3 group - E1AF/PEA3, ETV1/ER81 and ERM - are almost identical in the ETS DNA-binding and the transcriptional acidic domains. To accelerate our understanding of the molecular basis of putative diseases linked to ETV1 such as Ewing's sarcoma we characterized the human ETV1 and the mouse ER81 genes. We showed that these genes are both encoded by 13 exons in more than 90 kbp genomic DNA, and that the classical acceptor and donor splicing sites are present in each junction except for the 5' donor site of intron 9 where GT is replaced by TT. The genomic organization of the ETS and acidic domains in the human ETV1 and mouse ER81 (localized to chromosome 12) genes is similar to that observed in human ERM and human E1AF/PEA3 genes. Moreover, as in human ERM and human E1AF/PEA3 genes, a first untranslated exon is upstream from the first methionine, and the mouse ER81 gene transcription is regulated by a 1.8 kbp of genomic DNA upstream from this exon. In human, the alternative splicing of the ETV1 gene leads to the presence (ETV1α) or the absence (ETV1β) of exon 5 encoding the C-terminal part of the transcriptional acidic domain, but without affecting the alpha helix previously described as crucial for transactivation. We demonstrated here that the truncated isoform (human ETV1β) and the full-length isoform (human ETV1α) bind similarly specific DNA Ets binding sites. Moreover, they both activate transcription similarly through the PKA-transduction pathway, so suggesting that this alternative splicing is not crucial for the function of this protein as a transcription factor. The comparison of human ETV1α and human ETV1β expression in the same tissues, such as the adrenal gland or the bladder, showed no clear-cut differences. Altogether, these data open a new avenue of investigation leading to a better understanding of the functional role of this transcription factor.

Ontogeny of mouse B lymphocytes and inactivation by antigen of early B lymphocytes

Taking advantage of recent findings about membrane fluidity, the authors studied and compared the biosynthetic capacities of fetal or neonatal mouse B (bone marrow derived) lymphocytes (until 10 days after birth) and adult B lymphocytes. Although both early and adult lymphocytes can synthesize surface immunoglobulins, they have a different physiological behavior after interaction with a ligand (anti immunoglobulin sera or antigen), either in vivo or in vitro. Fetal and neonatal lymphocytes bearing surface immunoglobulins do not reexpress their membrane receptors after capping and endocytosis promoted by anti immunoglobulin sera. On the other hand, adult lymphocytes resynthesize completely their receptors after the same treatment. Furthermore, intrafetal injections of hemocyanin in pregnant mice lead to a striking decrease in the number of hemocyanin binding cells. It seems plausible that this non reexpression of surface immunoglobulins could be the first step in tolerance establishment.

Genomic organization and expression of the mouse Brca2 gene

info:eu-repo/semantics/published

Ectopic expression of the Ets transcription factor ER81 in transgenic mouse mammary gland enhances both urokinase plasminogen activator and stromelysin-1 transcription

The PEA3 group members PEA3, ER81 and ERM, which are highly conserved transcription factors from the Ets family, are over-expressed in metastatic mammary tumors. In the current study, we present the characterization of a transgenic mouse strain which over-expresses ER81 in the mammary gland via the long terminal repeat of the mouse mammary tumor virus (LTR-MMTV). Although six genotypically positive transgenic lines were identified, only one expressed the ectopic transcript with an exclusive expression in the lactating and late-pregnancy (18th day) mammary glands. No mammary tumor or mammary deregulation appeared after 2 years of ectopic ER81 expression following lactation. We then sought to identify ER81 target genes, and the urokinase plasminogen activator (uPA) and the stromelysin-1, two enzymes involved in extracellular matrix degradation, were found to be transcriptionally upregulated in lactating mammary glands over-expressing ER81. Since these enzymes are involved in metastasis, this murine model could be further used to enhance mammary cancer metastatic process by crossing these animals with mice carrying non-metastatic mammary tumors. We thus created a transgenic mouse model permitting the over-expression of a functionally active Ets transcription factor in the mammary gland without perturbing its development.

Variaciones termométricas en la planta del pie y piernas valorada en corredores antes y después de correr 30 km

La termometría es una técnica no invasiva que permite cuantificar los cambios en la temperatura cutánea y evaluarla de forma cuantitativa. El aumento significativo de la temperatura puede indicar la existencia de patología. Se ha demostrado que la actividad muscular induce procesos de transferencia de calor entre los músculos y las capas superficiales de tejido. En este estudio queremos cuantificar los cambios de temperatura que se producen en los músculos del pie y miembro inferior tras una carrera de 30 km, para ello hemos utilizado una cámara termográfica de alta resolución. Contamos con la colaboración voluntaria de 32 sujetos sanos a los que procedimos a tomar fotografías de la planta del pie, parte anterior de la pierna, parte posterior de la pierna, parte anterior del muslo y parte posterior del muslo en dos etapas, primero antes de la carrera y segunda toma después de la carrera de 30 km, de esta manera pudimos valorar si había o no variación de temperatura en las zonas seleccionadas. Tras el análisis de los datos obtenidos encontramos significativas variaciones térmicas en Talón, cabeza primer metatarsiano, cabeza segundo metatarsiano, cabeza tercer metatarsiano, cabeza cuarto metatarsiano, cabeza quinto metatarsiano, apófisis estiloides quinto metatarsiano, arco longitudinal interno, maléolo interno, maléolo externo, peroneo lateral largo, vasto interno, vasto externo, recto femoral, tensor de la fascia lata, inserción cuádriceps, gemelo interno, tendón de Aquiles y Biceps femoral.

CYSTATIN RELATED EPIDIDYMAL SPERMATOGENIC PROTEIN RESIDES IN THE OUTER DENSE FIBRES AND LIKELY TRANSIENTLY ASSOCIATES WITH THE SURFACE OF EPIDIDYMAL MOUSE SPERMATOZOA

Cystatin Related Epididymal Spermatogenic protein (CRES) is expressed in both the testis and epididymis and found associated with spermatozoa. It appears as non-glycosylated (14 and 12 kDa) and glycosylated isoforms (19 and 17 kDa). The role of CRES is enigmatic and dependent on localization of its isoforms, which is the objective of this study. The initial approach was to investigate testicular and epididymal origins of these isoforms by immunohistochemistry and immunogold cytochemistry. To further pinpoint CRES localization we then selectively extracted and fractionated epididymal spermatozoa in order to find by immunoblotting which sperm fractions contained CRES isoforms. Immunohistochemical analysis of mouse spermatogenesis showed that CRES was expressed in the tail cytoplasm of elongating spermatids from step 9-16, with a pattern reminiscent of outer dense fibre (ODF) proteins. Ultrastructural immunocytochemistry revealed that the immunogold label was concentrated over growing ODFs and mitochondrial sheath in the testes which persisted in spermatozoa through the epididymis. Sequential extractions of isolated sperm tails with Triton X-100-dithiothreitol (DTT) to remove the mitochondrial sheath, whose extract contained an unrelated 66 kDa immunoreactive band, followed by either sodium dodecyl sulfate (SDS)-DTT or urea-DTT to solubilise accessory fibres of the tail revealed a 14 kDa immunoreactive band associated with the ODF. In addition, Western blots revealed glycosylated and non-glycosylated CRES isoforms in nonyl phenoxylpolyethoxylethanol (NP40) extracts of the caput, but not cauda, sperm. Immunohistochemical analysis of the caput and cauda epithelium showed that CRES is secreted by the Golgi apparatus of the ii initial segment, fills the proximal caput lumen, and disappears by mid caput. Western blots of caput and cauda tissue and luminal fluid revealed 14 and 19 kDa immunoreactive bands in caput tissues and luminal fluid, but not in the cauda. This study concludes that there are two origins of CRES, one arising in the testis and the other in the epididymis. Testicular CRES is ionically and covalently associated with the ODF while epididymal CRES is detergent soluble and is most likely associated temporarily with the surface of caput epididymal sperm.

IMPACT OF RNA VIRUSES ON THE REGULATION OF IL-23 IN MOUSE AND HUMAN MODELS OF INFECTION.

Interluekin-23 (IL-23) is a pro-inflammatory cytokine critical to the regulation of innate and adaptive immune responses. The main role for this cytokine is in the proliferation and differentiation of the IL-17 producing CD4 T helper cell, Th17. Virus infection deregulates IL-23 expression and function, but little is known about the mechanism behind this phenomena. Here, I demonstrate a reduction of Toll like receptor (TLR) ligand-induced IL-23 expression in lymphocytic choriomeningitis virus (LCMV)-infected bone marrow-derived dendritic cells (BMDCs), indicating that a function of these cells is disrupted during virus infection. I propose a mechanism of TLR ligand-induced IL-23 expression inhibition upon LCMV infection via the deactivation of p38, AP-1, and NF-κB. Further analysis revealed a direct relationship between LCMV infection with the IL-10 and SOCS3 expression. To understand IL-23 function, I characterized IL-23-induced JAK/STAT signalling pathway and IL-23 receptor expression on human CD4 T cells. My results demonstrate that IL-23 induces activation of p-JAK2, p-Tyk2, p-STAT1, p-STAT3, and p-STAT4 in CD4 T cells. For the first time I show that IL-23 alone induces the expression of its own receptor components, IL-12Rβ1 and IL-23Rα, in CD4 T cells. Blocking JAK2, STAT1, and STAT3 activation with specific inhibitors detrimentally effected expression of IL-23 receptor demonstrating that activation of JAK/STAT signalling is important for IL-23 receptor expression. I also addressed the effect of viral infection on IL-23 function and receptor expression in CD4 T cells using cells isolated from HIV positive individuals. These studies were based on earlier reports that the expression of IL-23 and the IL-23 receptor are impaired during HIV infection. I demonstrate that the phosphorylation of JAK2, STAT1, and STAT3 induced by IL-23, as well as IL-23 receptor expression are deregulated in CD4 T cells isolated from HIV positive individuals. This study has furthered the understanding of how the expression and function of IL-23 is regulated during viral infections.

Time for treating bone fracture using rhBMP-2: a randomised placebo controlled mouse fracture trial.

Although the mechanisms of osteoinduction by bone morphogenic proteins (BMPs) are increasingly understood, the most appropriate time to administer BMPs exogenously is yet to be clarified.The purpose of this study was to investigate when BMP may be administered to a fracture arena to maximise the enhancement of healing.Forty mice with externally fixed left femoral fractures were randomised into four groups: Group I, the control group was given a placebo of 30 ll saline at day 0; Groups II, III and IV were given 30 ll saline plus 2.5 lg rhBMP-2, at post-operative days 0, 4 or 8, respectively.Sequential radiographs were taken at days 0, 8, 16.On day 22 the mice were sacrificed and both femora were harvested for biomechanical assessment in 3-point bending and histological evaluation.Radiographic analysis indicated that healing of fractures in Groups II and III was significantly greater (p <0.05) than those in Groups I and IV, at both 16 and 22 days post-fracture. The highest median bone mineral content at the fracture site was evidenced in Group III and II.Furthermore, Group III also had the highest relative ultimate load values, followed by Groups II, IV and I.Greater percentage peak loads were observed between Group I and both Groups II and III (p <0.05). Histological examination confirmed that at 22 days post-fracture, only fractures in Groups II and III had united with woven bone, and Groups I and IV still had considerable amounts of fibrous tissue and cartilage at the fracture gap.Data presented herein indicates that there is a time after fracture when rhBMP administration is most effective, and this may be at the time of surgery as well as in the early fracture healing phases.

Mouse Uterine Natural Killer Cell Functions During Early Pregnancy

Early pregnancy is characterized by complex interactions between blood vessels, leukocytes, and conceptus-derived trophoblasts within the gestational uterus. Uterine Natural Killer (uNK) cells become the most abundant leukocyte during decidualization and produce a wide array of angiogenic factors, yet little is known regarding their early pregnancy functions. To characterize the role(s) of uNK cells, whole mount in situ immunohistochemistry of live early implant sites was performed. A timecourse examination of murine early pregnancy (virgin, and gd4.5-9.5) implantation sites was performed. Comparison of Gd6.5, 8.5 and 9.5 implant sites from BALB/c+/+ controls (BALB/c) and BALB/c-Rag2-/-Il2rg-/- (alymphoid) identified anomalies that result from the absence of lymphocytes. In alymphoid decidua basalis, mesometrial angiogenesis was widespread but pruning of nascent vessels within alymphoid decidua basalis was deficient. As early gestation progressed, vessels of alymphoid decidua basalis showed no evidence for remodeling. Alymphoid implantation sites showed ~24h delay in uterine lumen closure and embryonic development. To determine if uNK cells would normalize the anomalies observed in alymphoid implantation sites, adoptive cell transfer of NK+ B- T- marrow to alymphoid mice was performed. All of the above anomalies were reversed by adoptive transfer of NK+B-T- marrow. My results suggest that uNK cells support vascular growth and development which ensures the decidua can support the growing conceptus early in pregnancy prior to formation and function of the placenta. Human decidual NK cells may fill similar roles and be important targets for strategies designed to correct intra-uterine growth restriction.