Oxygen tension controls the expression of the monocarboxylate transporter MCT4 in cultured mouse cortical astrocytes via a hypoxia-inducible factor-1α-mediated transcriptional regulation.

The monocarboxylate transporter MCT4 is a high capacity carrier important for lactate release from highly glycolytic cells. In the central nervous system, MCT4 is predominantly expressed by astrocytes. Surprisingly, MCT4 expression in cultured astrocytes is low, suggesting that a physiological characteristic, not met in culture conditions, is necessary. Here we demonstrate that reducing oxygen concentration from 21% to either 1 or 0% restored in a concentration-dependent manner the expression of MCT4 at the mRNA and protein levels in cultured astrocytes. This effect was specific for MCT4 since the expression of MCT1, the other astrocytic monocarboxylate transporter present in vitro, was not altered in such conditions. MCT4 expression was shown to be controlled by the transcription factor hypoxia-inducible factor-1α (HIF-1α) since under low oxygen levels, transfecting astrocyte cultures with a siRNA targeting HIF-1α largely prevented MCT4 induction. Moreover, the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG) induced MCT4 expression in astrocytes cultured in presence of 21% oxygen. In parallel, glycolytic activity was enhanced by exposure to 1% oxygen as demonstrated by the increased lactate release, an effect dependent on MCT4 expression. Finally, MCT4 expression was found to be necessary for astrocyte survival when exposed for a prolonged period to 1% oxygen. These data suggest that a major determinant of astrocyte MCT4 expression in vivo is likely the oxygen tension. This could be relevant in areas of high neuronal activity and oxygen consumption, favouring astrocytic lactate supply to neurons. Moreover, it could also play an important role for neuronal recovery after an ischemic episode.

Cardiovascular response to beta-adrenergic blockade or activation in 23 inbred mouse strains.

We report the characterisation of 27 cardiovascular-related traits in 23 inbred mouse strains. Mice were phenotyped either in response to chronic administration of a single dose of the beta-adrenergic receptor blocker atenolol or under a low and a high dose of the beta-agonist isoproterenol and compared to baseline condition. The robustness of our data is supported by high trait heritabilities (typically H(2)>0.7) and significant correlations of trait values measured in baseline condition with independent multistrain datasets of the Mouse Phenome Database. We then focused on the drug-, dose-, and strain-specific responses to beta-stimulation and beta-blockade of a selection of traits including heart rate, systolic blood pressure, cardiac weight indices, ECG parameters and body weight. Because of the wealth of data accumulated, we applied integrative analyses such as comprehensive bi-clustering to investigate the structure of the response across the different phenotypes, strains and experimental conditions. Information extracted from these analyses is discussed in terms of novelty and biological implications. For example, we observe that traits related to ventricular weight in most strains respond only to the high dose of isoproterenol, while heart rate and atrial weight are already affected by the low dose. Finally, we observe little concordance between strain similarity based on the phenotypes and genotypic relatedness computed from genomic SNP profiles. This indicates that cardiovascular phenotypes are unlikely to segregate according to global phylogeny, but rather be governed by smaller, local differences in the genetic architecture of the various strains.

Innate Immune Sensing of Fusarium culmorum by Mouse Dendritic Cells.

Chronic inhalation of grain dust is associated with asthma and chronic bronchitis in grain worker populations. Exposure to fungal particles was postulated to be an important etiologic agent of these pathologies. Fusarium species frequently colonize grain and straw and produce a wide array of mycotoxins that impact human health, necessitating an evaluation of risk exposure by inhalation of Fusarium and its consequences on immune responses. Data showed that Fusarium culmorum is a frequent constituent of aerosols sampled during wheat harvesting in the Vaud region of Switzerland. The aim of this study was to examine cytokine/chemokine responses and innate immune sensing of F. culmorum in bone-marrow-derived dendritic cells and macrophages. Overall, dendritic cells and macrophages responded to F. culmorum spores but not to its secreted components (i.e., mycotoxins) by releasing large amounts of macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, monocyte chemoattractant protein (MCP)-1, RANTES, and interleukin (IL)-12p40, intermediate amounts of tumor necrosis factor (TNF), IL-6, IL-12p70, IL-33, granulocyte colony-stimulating factor (G-CSF), and interferon gamma-induced protein (IP-10), but no detectable amounts of IL-4 and IL-10, a pattern of mediators compatible with generation of Th1 or Th17 antifungal protective immune responses rather than with Th2-dependent allergic responses. The sensing of F. culmorum spores by dendritic cells required dectin-1, the main pattern recognition receptor involved in β-glucans detection, but likely not MyD88 and TRIF-dependent Toll-like receptors. Taken together, our results indicate that F. culmorum stimulates potently innate immune cells in a dectin-1-dependent manner, suggesting that inhalation of F. culmorum from grain dust may promote immune-related airway diseases in exposed worker populations.

Tgfbi/Bigh3 silencing activates ERK in mouse retina.

BIGH3 is a secreted protein, part of the extracellular matrix where it interacts with collagen and integrins on the cell surface. BIGH3 can play opposing roles in cancer, acting as either tumor suppressor or promoter, and its mutations lead to different forms of corneal dystrophy. Although many studies have been carried out, little is known about the physiological role of BIGH3. Using the cre-loxP system, we generated a mouse model with disruption of the Bigh3 genomic locus. Bigh3 silencing did not result in any apparent phenotype modifications, the mice remained viable and fertile. We were able to determine the presence of BIGH3 in the retinal pigment epithelium (RPE). In the absence of BIGH3, a transient decrease in the apoptotic process involved in retina maturation was observed, leading to a transient increase in the INL thickness at P15. This phenomenon was accompanied by an increased activity of the pro-survival ERK pathway.

DNA polymerase activity in trophoblast giant cells in the mouse /

In the developing mouse embryo, the diploid trophectoderm is known to undergo a diploid to giant cell transformation. These cells arise by a process of endoreduplication, characterized by replication of the entire genome without subsequent mitosis or cell division, leading to polyploidy and the formation of giant nuclei. Studies of 13.5 day rat trophoblast derived from the parietal yolk sac have indicated a relatively low rate of DNA polymerase a activity, the noinnal eukaryotic replicase, in comparison to that of DNA polymerase g. These results have suggested that endoreduplication in trophoblast giant cells may not employ the normal replicase enzyme, DNA polymerase a. In order to determine whether a 'switch' from DNA polymerase to DNA polymerase is a necessary concomitant of the diploid to giant cell transformation, two distinct populations of trophoblast giant cells, the primary giant cell derived from the mural trophectoderm and the secondary giant cell derived from the polar trophoectoderm were used. These two populations of trophoblast giant cells can be obtained from the tissue outgrowths of 3.5da blastocysts and the extraembryonic ectoderm (EX) and ectoplacental cone (EPC) of 7.5 day embryos respectively. Tissue outgrowths were treated with aphidicolin, a specific reversible inhibitor of eukaryotic DNA polymerase a, on various days after explantation. The effect of aphidicolin treatment was assessed both qualitatively, using autoradiography and quantitatively by scintillation counting and Feulgen staining. 3 DNA synthesis was measured in control and treated cultures after a Hthymidine pulse. Scintillation counts of the embryo proper revealed that DNA synthesis was consistently inhibited by greater than 907. in the presence of aphidicolin. Inhibition of DNA synthesis in the EX and EPC varied between 81-957. and 82-987. respectively, indicating that most DNA synthesis was mediated by DNA polymerase a, but that a small but significant amount of residual synthesis was indicated. A qualitative approach was then applied to determine whether the apparent residual DNA synthesis was restricted to a subpopulation of giant cells or whether all giant cells displayed a low level of DNA synthesis. Autoradiographs of the ICM of blastocysts and the embryo proper of 7.5da embryos, which acted as diploid control population, was completely inhibited regardless of duration in explant culture. In contrast, primary trophoblast giant cells derived from blastocysts and secondary giant cells derived from the EX and EPC were observed to possess some heavily labelled cells after aphidicolin treatment. These results suggest that although DNA polymerase a is the primary replicating enzyme responsible for endoreduplication in mouse trophoblast giant cells, some nonactivity is also observed. A DNA polymerase assay employing tissue lysates of outgrown 7.5da embryo, EX and EPC tissues was used to attempt to confirm the presence of higher nonactivity in tissues possessing trophoblast giant cells. Employing a series of inhibitors of DNA polymerases, it would appear that DNA polymerase a is the major polymerase active in all tissues of the 7.5da mouse embryo. The nature of the putative residual DNA synthetic activity could not be unequivically determined in this study. Therefore, these results suggest that both primary and secondary trophoblast giant cells possess and use DNA polymerase a in endoreduplicative DNA synthesis. It would appear that the high levels of DNA polymerase g activity reported in trophoblast tissue derived from the 13.5 da rat yolk sac was not a general feature of all endoreduplication.

Relationship between photoperiod, plasma concentration of ionic calcium and the histology of the prolactin-secreting cells of the rostral pars distalis of the pituitary gland, the corpuscles of stannius and the ultimobranchial gland in the goldfish, Carassius auratus L. /

The relationship between photoperiod, plasma concentration of ionic calcium and the histology of the prolactin-secreting cells of the rostral pars distalis of the pituitary gland, the Corpuscles of Stannius and the Ultimobranchial gland were investigated. Neither the plasma concentration of ionic calcium nor histologically apparent prolactin cell activity could be correlated with photoperiod. Some evidence of a photoperiodic effect on both the Corpuscles of Stannius and the Ultimobranchial gland was obtained. The expected reciprocal relationship between the activity of these glands was not obvious at the histological level . Quantitative and qualitative analysis at the light microscope level revealed, however, that the hormone prolactin-secreting eta cells of the rostral pars distalis and the hypocalcin-secreting cells of the Corpuscles of Stannius may be arranged in a lamellar pattern comprized of synchronous bands of cells in like-phase of a secretory cycle consisting of four stages - synthesis, storage, release and reorganization. Such synchronized cell cycles in these glands have not heretofore been described in literature. It is suggested that the maintenance of at least 255? of the cells in any one phase of the cycle ensures a supply of the required hormone at all times.

Force potentiation in the MDX mouse

Large forces are the primary mechanism of injury in muscular dystrophy, and muscular dystrophy is especially damaging to type IIB muscle fibers. It was hypothesized that post-tetanic potentiation (PTP) would be down-regulated to prevent damage in Xlinked muscular dystrophy (mdx) mice since PTP increases force and PTP effects are greatest in IIB fibers. PTP experiments were performed on the extensor digitorum longus (EDL) of 50 day old mdx (YM) and C57BL/10 (YC) mice and 10 month old mdx (OM) and C57B1710 (OC) mice. Twitch and tetanic forces were lower in mdx than controls and lower in younger than older mice. Contrary to the hypothesis, PTP was higher in both mdx groups compared to controls. OM potentiated more than any other condition (OM: 29.8%, OC: 23.2%, YM: 21.9%, YC: 17.2%). In accordance with literature PTP increased in the older groups. To explain PTP changes, fiber typing and Western blots for myosin light chain kinase (MLCK) were performed. YM and YC had similar fiber type profiles (2% I, 58% IIX/D and 40% IIB). In accordance with literature but contrary to expected conditions for elevated PTP, OM had a slower fiber type profile (1.7% I, 69% IIX/D and 29% IIB) than OC (0.4% I, 61% IIX/D and 38% IIB). No differences were found in MLCK expression. It seems that PTP is up-regulated to maintain muscle function rather than being down-regulated to prevent muscle damage. Ca""^ transient and myosin phosphorylation measurements would be beneficial in explaining increased PTP seen in this study.

Assessment of chromatin activity in mouse and human tissues

Pancreatic deoxyribonuclease preferentially digests active genes during all phases of the cell cycle including mitosis. Recently, a DNAse I-directed in ~ nick translation technique has been used to demonstrate differences in the DNAse I sensitivity of euchromatic and heterochromatic regions of mitotic chromosomes. This ill ~ technique has been used in this study to ask whether facultative heterochromatin of the inactive X chromosome can be distinguished from the active X chromosome in mouse and human tissues. In addition to this, in ~ nick translation has been used to distinguish constitutive heterochromatin in mouse and human mitotic chromosomes. Based on relative levels of DNAse I sensitivity, the inactive X chromosome could not be distinguished from the active X chromosome in either mouse or human tissues but regions of constitutive heterochromatin could be distinguished by their relative DNAse I insensitivity. The use of !D situ nick translation was also applied to tissue sections of 7.5 day mouse embryos to ask whether differing levels of DNAse I sensitivity could be detected between different tissue types. Differences in DNAse I sensitivities were detected in three tissues examined; embryonic ectoderm, an embryo-derived tissue, and two extraembryonic tissues, extraembryonic ectoderm and ectoplacental cone. Embryonic ectoderm and extraembryonic ectoderm nuclei possessed comparable levels of DNAse I sensitivity while ectoplacental cone was significantly less DNAse I sensitive. This suggests that tissue-specific mechanisms such as chromatin structure may be involved in the regulation of gene activity in certain tissue types. This may also shed some light on possible tissue specific mechanisms regulating X chromosome activity in the developing mouse embryo.

Interactions of diploid embryonal carcinoma cells and normal cell mass cells with 2.5 day mouse embryos following aggregation

Spontaneous teratocarcinomas are ovarian or testicular tumors which have their origins in germ cells. The tumors contain a disorganized array of benign differentiated cells as well as an undifferentiated population of malignant stem cells, the embryonal carcinoma or EC cells. These pluripotent stem cells in tissue culture share many properties with the transient pluripotent cells of the early embryo, and might therefore serve as models for the investigation of developmental events ill vitro. The property of EC cells of prime interest in this study is an in vivo phenomenon. Certain EC cell lines are known to be regulated ill vivo and to differentiate normally in association with normal embryonic cells, resulting in chimeric mice. These mice have two genetically distinct cell populations, one of which is derived from the originally malignant EC cells. This has usually been accomplished by injection of the EC cells into the Day 3 blastocyst. In this study, the interactions between earlier stage embryos and EC cells have been tested by aggregating clumps of EC cells with Day 2 embryos. The few previous aggregation studies produced a high degree of abnormality in chimeric embryos, but the EC cells employed had known chromosomal abnormalities. In this study, two diploid EC cell lines (P19 and Pi0) were aggregated with 2.5 day mouse embryos, and were found to behave quite differently in the embryonic environment. P19 containing aggregates generally resorbed early, and the few embryos recovered at midgestation were normal and non-chimeric. Pi0 containing aggregates survived in high numbers to midgestation, and the Pi0 cells were very successful in colonizing the embryo. All these embryos were chimeric, and the contribution by the EC cells to each chimera was very high. However, these heavily chimeric embryos were all abnormal. Blastocyst injection had previously produced some abnormal embryos with high Pl0 contributions in addition to the live born mice, which had lower EC contributions. This study now adds more support to the hypothesis that high EC contributions may be incompatible with normal development. The possibility that the abnormalities were due to the mixing of temporally asynchronous embryonic cell types in the aggregates was tested by aggregating normal pluripotent cells taken from 3.5 day embryos with 2.5 day embryos. Early embryo loss was very high, and histological studies showed that the majority of these embryos died by 6.5 days development. Some embryos escaped this early death such that some healthy chimeras were recovered, in contrast to recovery of abnormal chimeric embryos following Pl0-morula aggregations, and non-chimeric embryos following P19-morula aggregations. This somewhat surprising adverse effect on development following aggregation of normal cell types suggests that there are developmental difficulties associated with the mixing of asynchronous cell types in aggregates. However, the greater magnitude of the adverse effects when the aggregates contained tumor derived cells suggests that EC cells should not be considered the complete equivalent of the pluripotent cells of the early embryo.

THE INFLUENCE OF LENGTH CHANGE SPEED AND DIRECTION ON DYNAMIC FUNCTION POTENTIATION IN FAST MOUSE MUSCLE

The purpose of this study was to test the hypothesis that the potentiation of dynamic function was dependent upon both length change speed and direction. Mouse EDL was cycled in vitro (25º C) about optimal length (Lo) with constant peak strain (± 2.5% Lo) at 1.5, 3.3 and 6.9 Hz before and after a conditioning stimulus. A single pulse was applied during shortening or lengthening and peak dynamic (concentric or eccentric) forces were assessed at Lo. Stimulation increased peak concentric force at all frequencies (range: 19 ± 1 to 30 ± 2%) but this increase was proportional to shortening speed, as were the related changes to concentric work/power (range: -15 ± 1 to 39 ± 1 %). In contrast, stimulation did not increase eccentric force, work or power at any frequency. Thus, results reveal a unique hysteresis like effect for the potentiation of dynamic output wherein concentric and eccentric forces increase and decrease, respectively, with work cycle frequency.

THE INFLUENCE OF LENGTH CHANGE SPEED AND DIRECTION ON DYNAMIC FUNCTION POTENTIATION IN FAST MOUSE MUSCLE

The purpose of this study was to test the hypothesis that the potentiation of dynamic function was dependent upon both length change speed and direction. Mouse EDL was cycled in vitro (250 C) about optimal length (Lo) with constant peak strain (± 2.5% Lo) at 1.5,3.3 and 6.9 Hz before and after a conditioning stimulus. A single pulse was applied during shortening or lengthening and peak dynamic (concentric or eccentric) forces were assessed at Lo. Stimulation increased peak concentric force at all frequencies (range: 19±1 to 30 ± 2%) but this increase was proportional to shortening speed, as were the related changes to concentric work/power (range: -15 ± 1 to 39 ± 1 %). In contrast, stimulation did not increase eccentric force, work or power at any frequency. Thus, results reveal a unique hysteresis like effect for the potentiation of dynamic output wherein concentric and eccentric forces increase and decrease, respectively, with work cycle frequency.

Identification of renin progenitors in the mouse bone marrow that give rise to B-cell leukaemia

cell of origin and triggering events for leukaemia are mostly unknown. Here we show that the bone marrow contains a progenitor that expresses renin throughout development and possesses a B-lymphocyte pedigree. This cell requires RBP-J to differentiate. Deletion of RBP-J in these renin-expressing progenitors enriches the precursor B-cell gene programme and constrains lymphocyte differentiation, facilitated by H3K4me3 activating marks in genes that control the pre-B stage. Mutant cells undergo neoplastic transformation, and mice develop a highly penetrant B-cell leukaemia with multi-organ infiltration and early death. These reninexpressing cells appear uniquely vulnerable as other conditional models of RBP-J deletion do not result in leukaemia. The discovery of these unique renin progenitors in the bone marrow and the model of leukaemia described herein may enhance our understanding of normal and neoplastic haematopoiesis.

Place Card - Mouse

A handmade place card with an illustration of a mouse in a orange and black polka dot dress, sipping from a small glass.