Identification and Characterization of RBM44 as a Novel Intercellular Bridge Protein

Intercellular bridges are evolutionarily conserved structures that connect differentiating germ cells. We previously reported the identification of TEX14 as the first essential intercellular bridge protein, the demonstration that intercellular bridges are required for male fertility, and the finding that intercellular bridges utilize components of the cytokinesis machinery to form. Herein, we report the identification of RNA binding motif protein 44 (RBM44) as a novel germ cell intercellular bridge protein. RBM44 was identified by proteomic analysis after intercellular bridge enrichment using TEX14 as a marker protein. RBM44 is highly conserved between mouse and human and contains an RNA recognition motif of unknown function. RBM44 mRNA is enriched in testis, and immunofluorescence confirms that RBM44 is an intercellular bridge component. However, RBM44 only partially localizes to TEX14-positive intercellular bridges. RBM44 is expressed most highly in pachytene and secondary spermatocytes, but disappears abruptly in spermatids. We discovered that RBM44 interacts with itself and TEX14 using yeast two-hybrid, mammalian two-hybrid, and immunoprecipitation. To define the in vivo function of RBM44, we generated a targeted deletion of Rbm44 in mice. Rbm44 null male mice produce somewhat increased sperm, and show enhanced fertility of unknown etiology. Thus, although RBM44 localizes to intercellular bridges during meiosis, RBM44 is not required for fertility in contrast to TEX14

Characterization of Spermatogonial Stem Cells Lacking Intercellular Bridges and Genetic Replacement of a Mutation in Spermatogonial Stem Cells

Stem cells have a potential of gene therapy for regenerative medicine. Among various stem cells, spermatogonial stem cells have a unique characteristic in which neighboring cells can be connected by intercellular bridges. However, the roles of intercellular bridges for stem cell self-renewal, differentiation, and proliferation remain to be elucidated. Here, we show not only the characteristics of testis-expressed gene 14 (TEX14) null spermatogonial stem cells lacking intercellular bridges but also a trial application of genetic correction of a mutation in spermatogonial stem cells as a model for future gene therapy. In TEX14 null testes, some genes important for undifferentiated spermatogonia as well as some differentiation-related genes were activated. TEX14 null spermatogonial stem cells, surprisingly, could form chain-like structures even though they do not form stable intercellular bridges. TEX14 null spermatogonial stem cells in culture possessed both characteristics of undifferentiated and differentiated spermatogonia. Long-term culture of TEX14 null spermatogonial stem cells could not be established likely secondary to up-regulation of CDK4 inhibitors and down-regulation of cyclin E. These results suggest that intercellular bridges are essential for both maintenance of spermatogonial stem cells and their proliferation. Lastly, a mutation in Tex14+/− spermatogonial stem cells was successfully replaced by homologous recombination in vitro. Our study provides a therapeutic potential of spermatogonial stem cells for reproductive medicine if they can be cultured long-term

Temporal evolution of proto-Izu–Bonin–Mariana arc volcanism over 10 Myr: Constraints from statistical analysis of melt inclusion compositions

International Ocean Discovery Program (IODP) Expedition 351 ‘Izu–Bonin–Mariana (IBM) Arc Origins’ drilled Site U1438, situated in the northwestern region of the Philippine Sea. Here volcaniclastic sediments and the igneous basement of the proto-IBM volcanic arc were recovered. To gain a better understanding of the magmatic processes and evolution of the proto-IBM arc, we studied melt inclusions hosted in fresh igneous minerals and sampled from 30–40 Myr old deposits, reflecting the maturation of arc volcanism following subduction initiation at 52 Ma. We performed a novel statistical analysis on the major element composition of 237 representative melt inclusions selected from a previously published dataset, covering the full age range between 30 and 40 Ma. In addition, we analysed volatiles (H2O, S, F and Cl) and P2O5 by secondary ion mass spectrometry for a subset of 47 melt inclusions selected from the dataset. Based on statistical analysis of the major element composition of melt inclusions and by considering their trace and volatile element compositions, we distinguished five main clusters of melt inclusions, which can be further separated into a total of eight subclusters. Among the eight subclusters, we identified three major magma types: (1) enriched medium-K magmas, which form a tholeiitic trend (30–38 Ma); (2) enriched medium-K magmas, which form a calc-alkaline trend (30–39 Ma); (3) depleted low-K magmas, which form a calc-alkaline trend (35–40 Ma). We demonstrate the following: (1) the eruption of depleted low-K calc-alkaline magmas occurred prior to 40 Ma and ceased sharply at 35 Ma; (2) the eruption of depleted low-K calc-alkaline magmas, enriched medium-K calc-alkaline magmas and enriched medium-K tholeiitic magmas overlapped between 35 and 38–39 Ma; (3) the eruption of enriched medium-K tholeiitic and enriched medium-K calc-alkaline magmas became predominant thereafter at the proto-IBM arc. Identification of three major magma types is distinct from the previous work, in which enriched medium-K calc-alkaline magmas and depleted low-K calc-alkaline magmas were not identified. This indicates the usefulness of our statistical analysis as a powerful tool to partition a mixture of multivariable geochemical datasets, such as the composition of melt inclusions in this case. Our data suggest that a depleted mantle source had been replaced by an enriched mantle source owing to convection beneath the proto-IBM arc from >40 to 35 Ma. Finally, thermodynamic modelling indicates that the overall geochemical variation of melt inclusions assigned to each cluster can be broadly reproduced either by crystallization differentiation assuming P = 50 MPa (∼2 km deep) and ∼2 wt% H2O (almost saturated H2O content at 50 MPa) or P = 300 MPa (∼15 km deep) and ∼6 wt% H2O (almost saturated H2O content at 300 MPa). Assuming oxygen fugacity (fO2) of log fO2 equal to +1 relative to the nickel–nickel oxide (NNO) buffer best reproduces the overall geochemical variation of melt inclusions, but assuming more oxidizing conditions (log fO2 = +1 to +2 NNO) probably reproduces the geochemical variation of enriched medium-K and calc-alkaline melt inclusions (30–39 Ma)

Cholesterol and oxysterol sulfates:Pathophysiological roles and analytical challenges

Cholesterol and oxysterol sulfates are important regulators of lipid metabolism, inflammation, cell apoptosis, and cell survival. Among the sulfate-based lipids, cholesterol sulfate (CS) is the most studied lipid both quantitatively and functionally. Despite the importance, very few studies have analysed and linked the actions of oxysterol sulfates to their physiological and pathophysiological roles. Overexpression of sulfotransferases confirmed the formation of a range of oxysterol sulfates and their antagonistic effects on liver X receptors (LXRs) prompting further investigations how are the changes to oxysterol/oxysterol sulfate homeostasis can contribute to LXR activity in the physiological milieu. Here, we aim to bring together for novel roles of oxysterol sulfates, the available techniques and the challenges associated with their analysis. Understanding the oxysterol/oxysterol sulfate levels and their pathophysiological mechanisms could lead to new therapeutic targets for metabolic diseases

Analysis of the pattern of suprahyoid muscle activity during pharyngeal swallowing of foods by healthy young subjects

We previously developed the TP technique to discriminate between the activity patterns of skeletal muscles. In this study we aim to identify the TP value(s) that can be used to sensitively evaluate the activity patterns of the suprahyoid (SH) muscles during swallowing. We also analyse the effect of food textural properties on the activity patterns of the SH muscle during oral and pharyngeal swallowing. Three test foods consisting of 3%, 6% and 9% of a thickening agent, Mousse-up (MU) were prepared. Their textural properties differed significantly. Swallowing of 9% MU involved a significantly longer average duration than 3% MU. The average T50 value for 6% MU was significantly larger than that for 3% MU. However, the average T20 and T80 values of the test foods did not differ. Thus, the T50 value is particularly suitable for evaluating SH muscle swallowing patterns. Moreover, test foods that vary in their textural properties elicit different durations and patterns of SH muscle activity

Regulation of Tumor Suppressor p53 and HCT116 Cell Physiology by Histone Demethylase JMJD2D/KDM4D

JMJD2D, also known as KDM4D, is a histone demethylase that removes methyl moieties from lysine 9 on histone 3 and from lysine 26 on histone 1.4. Here, we demonstrate that JMJD2D forms a complex with the p53 tumor suppressor in vivo and interacts with the DNA binding domain of p53 in vitro. A luciferase reporter plasmid driven by the promoter of p21, a cell cycle inhibitor and prominent target gene of p53, was synergistically activated by p53 and JMJD2D, which was dependent on JMJD2D catalytic activity. Likewise, overexpression of JMJD2D induced p21 expression in U2OS osteosarcoma cells in the absence and presence of adriamycin, an agent that induces DNA damage. Furthermore, downregulation of JMJD2D inhibited cell proliferation in wild-type and even more so in p53−/− HCT116 colon cancer cells, suggesting that JMJD2D is a pro-proliferative molecule. JMJD2D depletion also induced more strongly apoptosis in p53−/− compared to wild-type HCT116 cells. Collectively, our results demonstrate that JMJD2D can stimulate cell proliferation and survival, suggesting that its inhibition may be helpful in the fight against cancer. Furthermore, our data imply that activation of p53 may represent a mechanism by which the pro-oncogenic functions of JMJD2D become dampened

Widespread Gene Conversion of Alpha-2-Fucosyltransferase Genes in Mammals

The alpha-2-fucosyltransferases (α2FTs) are enzymes involved in the biosynthesis of α2fucosylated glycan structures. In mammalian genomes, there are three α2FT genes located in tandem—FUT1, FUT2, and Sec1—each contained within a single exon. It has been suggested that these genes originated from two successive duplications, with FUT1 being generated first and FUT2 and Sec1 second. Despite gene conversion being considered the main mechanism of concerted evolution in gene families, previous studies of primates α2FTs failed to detect it, although the occurrence of gene conversion between FUT2 and Sec1 was recently reported in a human allele. The primary aim of our work was to initiate a broader study on the molecular evolution of mammalian α2FTs. Sequence comparison leads us to confirm that the three genes appeared by two rounds of duplication. In addition, we were able to detect multiple gene-conversion events at the base of primates and within several nonprimate species involving FUT2 and Sec1. Gene conversion involving FUT1 and either FUT2 or Sec1 was also detected in rabbit. The extent of gene conversion between the α2FTs genes appears to be species-specific, possibly related to functional differentiation of these genes. With the exception of rabbits, gene conversion was not observed in the region coding the C-terminal part of the catalytic domain. In this region, the number of amino acids that are identical between FUT1 and FUT2, but different in Sec1, is higher than in other parts of the protein. The biologic meaning of this observation may be related to functional constraints

Fluid release from the subducted Cocos plate and partial melting of the crust deduced from magnetotelluric studies in southern Mexico: implications for the generation of volcanism and subduction dynamics

In order to study electrical conductivity phenomena that are associated with subduction related fluid release and melt production, magnetotelluric (MT) measurements were carried out in southern Mexico along two coast to coast profiles. The conductivity-depth distribution was obtained by simultaneous two-dimensional inversion of the transverse magnetic and transverse electric modes of the magnetotelluric transfer functions. The MT models demonstrate that the plate southern profile shows enhanced conductivity in the deep crust. The northern profile is dominated by an elongated conductive zone extending >250 km below the Trans-Mexican Volcanic Belt (TMVB). The isolated conductivity anomalies in the southern profile are interpreted as slab fluids stored in the overlying deep continental crust. These fluids were released by progressive metamorphic dehydration of the basaltic oceanic crust. The conductivity anomalies may be related to the main dehydration reactions at the zeolite → blueschist → eclogite facies transitions and the breakdown of chlorite. This relation allows the estimation of a geothermal gradient of ∼8.5°C/km for the top of the subducting plate. The same dehydration reactions may be recognized along the northern profile at the same position relative to the depth of the plate, but more inland due to a shallower dip, and merge near the volcanic front due to steep downbending of the plate. When the oceanic crust reaches a depth of 80–90 km, ascending fluids produce basaltic melts in the intervening hot subcontinental mantle wedge that give rise to the volcanic belt. Water-rich basalts may intrude into the lower continental crust leading to partial melting. The elongated highly conductive zone below the TMVB may therefore be caused by partial melts and fluids of various origins, ongoing migmatization, ascending basaltic and granitic melts, growing plutons as well as residual metamorphic fluids. Zones of extremely high conductance (>8000 S) in the continental crust on either MT profile might indicate extinct magmatism