The Arg233Lys AQP0 Mutation Disturbs Aquaporin0-Calmodulin Interaction Causing Polymorphic Congenital Cataract

Calmodulin (CaM) directly interacts with the aquaporin 0 (AQP0) C-terminus in a calcium dependent manner to regulate the water permeability of AQP0. We previously identified a missense mutation (p.R233K) in the putative CaM binding domain of AQP0 C-terminus in a congenital cataract family. This study was aimed at exploring the potential pathogenesis of this mutation causative of cataract and mainly identifying how it influenced the binding of AQP0 to CaM. Wild type and R233K mutant AQP0 with EGFP-tag were transfected separately into Hela cells to determine the expression and subcellular localizations. The co-immunoprecipitation (CoIP) assay was used to detect the interaction between AQP0 and CaM. AQP0 C-terminus peptides were synthesized with and without R233K, and the binding abilities of these peptides to CaM were assessed using a fluorescence binding assay. Localizations of wild type and R233K mutant AQP0 were determined from EGFP fluorescence, and the chimeric proteins were both localized abundantly in the plasma membrane. Protein expression levels of the culture cells showed no significant difference between them. The results from CoIP assay implied that R233K mutant presented more weakly in association with CaM than wild type AQP0. The AQP0 C-terminal mutant peptide was found to have 2.5-fold lower binding affinity to CaM than wild type peptide. These results suggested that R233K mutation did not affect the expression, location and trafficking of the protein but did influence the interaction between AQP0 and CaM. The binding affinity of AQP0 C-terminus to CaM was significantly reduced. Due to lack of the modulation of the Ca2+-calmodulin complex, the water permeability of AQP0 was subsequently augmented, which might lead to the development of this cataract

Indirect Effects of Wnt3a/β-Catenin Signalling Support Mouse Spermatogonial Stem Cells In Vitro

Proper regulation of spermatogonial stem cells (SSCs) is crucial for sustaining steady-state spermatogenesis. Previous work has identified several paracrine factors involved in this regulation, in particular, glial cell line-derived neurotrophic factor and fibroblast growth factor 2, which promote long-term SSC self-renewal. Using a SSC culture system, we have recently reported that Wnt5a promotes SSC self-renewal through a β-catenin-independent Wnt mechanism whereas the β-catenin-dependent Wnt pathway is not active in SSCs. In contrast, another study has reported that Wnt3a promotes SSC self-renewal through the β-catenin-dependent pathway, as it can stimulate the proliferation of a spermatogonia cell line. To reconcile these two contradictory reports, we assessed Wnt3a effects on SSCs and progenitor cells, rather than a cell line, in vitro. We observed that Wnt3a induced β-catenin-dependent signalling in a large subset of germ cells and increased SSC numbers. However, further investigation revealed that cell populations with greater β-catenin-signalling activity contained fewer SSCs. The increased maintenance of SSCs by Wnt3a coincided with more active cell cycling and the formation of germ cell aggregates, or communities, under feeder-free conditions. Therefore, the results of this study suggest that Wnt3a selectively stimulates proliferation of progenitors that are committed to differentiation or are in the process of exiting the SSC state, leading to enhanced formation of germ cell communities, which indirectly support SSCs and act as an in vitro niche

Distribution and New Host Plants of Seed Beetles (Col.: Chrysomelidae: Bruchinae) from Iran

This report is part of a national project for gathering and classifying the arthropod seed feeders in different provinces of Iran between 2008–2014. In this paper, nineteen host species with their areas of distribution are presented for twelve species of seed beetles (Chrysomelidae: Bruchinae). Most of the identified host plants (84%) belong to the family Fabaceae (Leguminosae). In addition, all known hosts for these beetles are discussed. The identified species in this study were confirmed by Dr. Alex Delobel in the Natural history Museum of Paris. The studied material is deposited in the arthropod collection of Research Institute of Forests and Rangelands

A Unique Combination of Male Germ Cell miRNAs Coordinates Gonocyte Differentiation

The last 100 years have seen a concerning decline in male reproductive health associated with decreased sperm production, sperm function and male fertility. Concomitantly, the incidence of defects in reproductive development, such as undescended testes, hypospadias and testicular cancer has increased. Indeed testicular cancer is now recognised as the most common malignancy in young men. Such cancers develop from the pre-invasive lesion Carcinoma in Situ (CIS), a dysfunctional precursor germ cell or gonocyte which has failed to successfully differentiate into a spermatogonium. It is therefore essential to understand the cellular transition from gonocytes to spermatogonia, in order to gain a better understanding of the aetiology of testicular germ cell tumours. MicroRNA (miRNA) are important regulators of gene expression in differentiation and development and thus highly likely to play a role in the differentiation of gonocytes. In this study we have examined the miRNA profiles of highly enriched populations of gonocytes and spermatogonia, using microarray technology. We identified seven differentially expressed miRNAs between gonocytes and spermatogonia (down-regulated: miR-293, 291a-5p, 290-5p and 294*, up-regulated: miR-136, 743a and 463*). Target prediction software identified many potential targets of several differentially expressed miRNA implicated in germ cell development, including members of the PTEN, and Wnt signalling pathways. These targets converge on the key downstream cell cycle regulator Cyclin D1, indicating that a unique combination of male germ cell miRNAs coordinate the differentiation and maintenance of pluripotency in germ cells

MIP/Aquaporin 0 Represents a Direct Transcriptional Target of PITX3 in the Developing Lens

The PITX3 bicoid-type homeodomain transcription factor plays an important role in lens development in vertebrates. PITX3 deficiency results in a spectrum of phenotypes from isolated cataracts to microphthalmia in humans, and lens degeneration in mice and zebrafish. While identification of downstream targets of PITX3 is vital for understanding the mechanisms of normal ocular development and human disease, these targets remain largely unknown. To isolate genes that are directly regulated by PITX3, we performed a search for genomic sequences that contain evolutionarily conserved bicoid/PITX3 binding sites and are located in the proximity of known genes. Two bicoid sites that are conserved from zebrafish to human were identified within the human promoter of the major intrinsic protein of lens fiber, MIP/AQP0. MIP/AQP0 deficiency was previously shown to be associated with lens defects in humans and mice. We demonstrate by both chromatin immunoprecipitation and electrophoretic mobility shift assay that PITX3 binds to MIP/AQP0 promoter region in vivo and is able to interact with both bicoid sites in vitro. In addition, we show that wild-type PITX3 is able to activate the MIP/AQP0 promoter via interaction with the proximal bicoid site in cotransfection experiments and that the introduction of mutations disrupting binding to this site abolishes this activation. Furthermore, mutant forms of PITX3 fail to produce the same levels of transactivation as wild-type when cotransfected with the MIP/AQP0 reporter. Finally, knockdown of pitx3 in zebrafish affects formation of a DNA-protein complex associated with mip1 promoter sequences; and examination of expression in pitx3 morphant and control zebrafish revealed a delay in and reduction of mip1 expression in pitx3-deficient embryos. Therefore, our data suggest that PITX3 is involved in direct regulation of MIP/AQP0 expression and that the alteration of MIP/AQP0 expression is likely to contribute to the lens phenotype in cataract patients with PITX3 mutations

Systems biology discoveries using non-human primate pluripotent stem and germ cells: novel gene and genomic imprinting interactions as well as unique expression patterns

The study of pluripotent stem cells has generated much interest in both biology and medicine. Understanding the fundamentals of biological decisions, including what permits a cell to maintain pluripotency, that is, its ability to self-renew and thereby remain immortal, or to differentiate into multiple types of cells, is of profound importance. For clinical applications, pluripotent cells, including both embryonic stem cells and adult stem cells, have been proposed for cell replacement therapy for a number of human diseases and disorders, including Alzheimer's, Parkinson's, spinal cord injury and diabetes. One challenge in their usage for such therapies is understanding the mechanisms that allow the maintenance of pluripotency and controlling the specific differentiation into required functional target cells. Because of regulatory restrictions and biological feasibilities, there are many crucial investigations that are just impossible to perform using pluripotent stem cells (PSCs) from humans (for example, direct comparisons among panels of inbred embryonic stem cells from prime embryos obtained from pedigreed and fertile donors; genomic analysis of parent versus progeny PSCs and their identical differentiated tissues; intraspecific chimera analyses for pluripotency testing; and so on). However, PSCs from nonhuman primates are being investigated to bridge these knowledge gaps between discoveries in mice and vital information necessary for appropriate clinical evaluations. In this review, we consider the mRNAs and novel genes with unique expression and imprinting patterns that were discovered using systems biology approaches with primate pluripotent stem and germ cells

Effects of histocompatibility and host immune responses on the tumorigenicity of pluripotent stem cells

Pluripotent stem cells hold great promises for regenerative medicine. They might become useful as a universal source for a battery of new cell replacement therapies. Among the major concerns for the clinical application of stem cell-derived grafts are the risks of immune rejection and tumor formation. Pluripotency and tumorigenicity are closely linked features of pluripotent stem cells. However, the capacity to form teratomas or other tumors is not sufficiently described by inherited features of a stem cell line or a stem cell-derived graft. The tumorigenicity always depends on the inability of the recipient to reject the tumorigenic cells. This review summarizes recent data on the tumorigenicity of pluripotent stem cells in immunodeficient, syngeneic, allogeneic, and xenogeneic hosts. The effects of immunosuppressive treatment and cell differentiation are discussed. Different immune effector mechanisms appear to be involved in the rejection of undifferentiated and differentiated cell populations. Elements of the innate immune system, such as natural killer cells and the complement system, which are active also in syngeneic recipients, appear to preferentially reject undifferentiated cells. This effect could reduce the risk of tumor formation in immunocompetent recipients. Cell differentiation apparently increases susceptibility to rejection by the adaptive immune system in allogeneic hosts. The current data suggest that the immune system of the recipient has a major impact on the outcome of pluripotent stem cell transplantation, whether it is rejection, engraftment, or tumor development. This has to be considered when the results of experimental transplantation models are interpreted and even more when translation into clinics is planned

A Distinct Expression Pattern in Mammalian Testes Indicates a Conserved Role for NANOG in Spermatogenesis

BACKGROUND: NANOG is a key player in pluripotency and its expression is restricted to pluripotent cells of the inner cell mass, the epiblast and to primordial germ cells. Spermatogenesis is closely associated with pluripotency, because through this process highly specialized sperm cells are produced that contribute to the formation of totipotent zygotes. Nevertheless, it is unknown if NANOG plays a role in this process. METHODOLOGY/PRINCIPAL FINDINGS: In the current study, NANOG expression was examined in testes of various mammals, including mouse and human. Nanog mRNA and NANOG protein were detected by RT-PCR, immunohistochemistry, and western blotting. Furthermore, eGFP expression was detected in the testis of a transgenic Nanog eGFP-reporter mouse. Surprisingly, although NANOG expression has previously been associated with undifferentiated cells with stem cell potential, expression in the testis was observed in pachytene spermatocytes and in the first steps of haploid germ cell maturation (spermiogenesis). Weak expression in type A spermatogonia was also observed. CONCLUSIONS: The findings of the current study strongly suggest a conserved role for NANOG in meiotic and post-meiotic stages of male germ cell developmen