Role of the inositol polyphosphate-4-phosphatase type II Inpp4b in the generation of ovarian teratomas

AbstractTeratomas are a unique class of tumors composed of ecto-, meso- and endodermal tissues, all foreign to the site of origin. In humans, the most common teratoma is the ovarian teratoma. Not much is known about the molecular and genetic etiologies of these tumors. Female carriers of the Tgkd transgene are highly susceptible to developing teratomas. Ovaries of Tgkd/+ hemizygous female mice exhibit defects in luteinization, with numerous corpora lutea, some of which contain central trapped, fully-grown oocytes. Genetically, Tgkd teratomas originate from mature oocytes that have completed meiosis I, suggesting that Tgkd teratomas originate from these trapped oocytes. The insertion of Tgkd 3' of the Inpp4b gene is associated with decreased expression of Inpp4b and changes in intracellular PI3 Kinase/AKT signaling in follicular granulosa cells. Because Inpp4b is not expressed in fully-grown wild-type or Tgkd oocytes, these findings suggest that hyperactivation of the PI3K/AKT pathway caused by the decrease in INPP4B in granulosa cells promotes an ovarian environment defective in folliculogenesis and conducive to teratoma formation

Preimplantation expression of the somatic form of Dnmt1 suggests a role in the inheritance of genomic imprints

BACKGROUND: Identical DNA methylation differences between maternal and paternal alleles in gametes and adults suggest that the inheritance of genomic imprints is strictly due to the embryonic maintenance of DNA methylation. Such maintenance would occur in association with every cycle of DNA replication, including those of preimplantation embryos. RESULTS: The expression of the somatic form of the Dnmt1 cytosine methyltransferase (Dnmt1s) was examined in cleavage-stage preimplantation mouse embryos. Low concentrations of Dnmt1s are found in 1-, 2-, 4-, and 8-cell embryos, as well as in morulae and blastocysts. Dnmt1s is present in the cytoplasm at all stages, and in the nuclei of all stages except the 1-cell, pronuclear-stage embryo. The related oocyte-derived Dnmt1o protein is also present in nuclei of 8-cell embryos, along with embryo-synthesized Dnmt1s. Dnmt1s protein expressed in 1-cell and 2-cell embryos is derived from the oocyte, whereas the embryo synthesizes its own Dnmt1s from the 2-cell stage onward. CONCLUSION: These observations suggest that Dnmt1s provides maintenance methyltransferase activity for the inheritance of methylation imprints in the early mouse embryo. Moreover, the ability of Dnmt1o and Dnmt1s proteins synthesized at the same time to substitute for one another's maintenance function, but the lack of functional interchange between oocyte- and embryo-synthesized Dnmt1 proteins, suggests that the developmental source is the critical determinant of Dnmt1 function during preimplantation development

Dissection of Structure and Function of the N-Terminal Domain of Mouse DNMT1 Using Regional Frame-Shift Mutagenesis

Deletion analysis of mouse DNMT1, the primary maintenance methyltransferase in mammals, showed that most of the N-terminal regulatory domain (amino acid residues 412–1112) is required for its enzymatic activity. Although analysis of deletion mutants helps to identify regions of a protein sequence required for a particular activity, amino acid deletions can have drastic effects on protein structure and/or stability. Alternative approaches represented by rational design and directed evolution are resource demanding, and require high-throughput selection or screening systems. We developed Regional Frame-shift Mutagenesis (RFM) as a new approach to identify portions required for the methyltransferase activity of DNMT1 within the N-terminal 89–905 amino acids. In this method, a short stretch of amino acids in the wild-type protein is converted to a different amino acid sequence. The resultant mutant protein retains the same amino acid length as the wild type, thereby reducing physical constrains on normal folding of the mutant protein. Using RFM, we identified three small regions in the amino-terminal one-third of the protein that are essential for DNMT1 function. Two of these regions (amino acids 124–160 and 341–368) border a large disordered region that regulates maintenance methylation activity. This organization of DNMT1's amino terminus suggests that the borders define the position of the disordered region within the DNMT1 protein, which in turn allows for its proper function

Human IL-12 p40 as a reporter gene for high-throughput screening of engineered mouse embryonic stem cells

<p>Abstract</p> <p>Background</p> <p>Establishing a suitable level of exogenous gene expression in mammalian cells in general, and embryonic stem (ES) cells in particular, is an important aspect of understanding pathways of cell differentiation, signal transduction and cell physiology. Despite its importance, this process remains challenging because of the poor correlation between the presence of introduced exogenous DNA and its transcription. Consequently, many transfected cells must be screened to identify those with an appropriate level of expression. To improve the screening process, we investigated the utility of the human interleukin 12 (IL-12) p40 cDNA as a reporter gene for studies of mammalian gene expression and for high-throughput screening of engineered mouse embryonic stem cells.</p> <p>Results</p> <p>A series of expression plasmids were used to study the utility of IL-12 p40 as an accurate reporter of gene activity. These studies included a characterization of the IL-12 p40 expression system in terms of: (i) a time course of IL-12 p40 accumulation in the medium of transfected cells; (ii) the dose-response relationship between the input DNA and IL-12 p40 mRNA levels and IL-12 p40 protein secretion; (iii) the utility of IL-12 p40 as a reporter gene for analyzing the activity of <it>cis</it>-acting genetic elements; (iv) expression of the IL-12 p40 reporter protein driven by an IRES element in a bicistronic mRNA; (v) utility of IL-12 p40 as a reporter gene in a high-throughput screening strategy to identify successful transformed mouse embryonic stem cells; (vi) demonstration of pluripotency of IL-12 p40 expressing ES cells <it>in vitro </it>and <it>in vivo</it>; and (vii) germline transmission of the IL-12 p40 reporter gene.</p> <p>Conclusion</p> <p>IL-12 p40 showed several advantages as a reporter gene in terms of sensitivity and ease of the detection procedure. The IL-12 p40 assay was rapid and simple, in as much as the reporter protein secreted from the transfected cells was accurately measured by ELISA using a small aliquot of the culture medium. Remarkably, expression of Il-12 p40 does not affect the pluripotency of mouse ES cells. To our knowledge, human IL-12 p40 is the first secreted reporter protein suitable for high-throughput screening of mouse ES cells. In comparison to other secreted reporters, such as the widely used alkaline phosphatase (SEAP) reporter, the IL-12 p40 reporter system offers other real advantages.</p

Genomic imprinting and assisted reproduction

Imprinted genes exhibit a parent-of-origin specific pattern of expression. Such genes have been shown to be targets of molecular defects in particular genetic syndromes such as Beckwith-Wiedemann and Angelman syndromes. Recent reports have raised concern about the possibility that assisted reproduction techniques, such as in vitro fertilization or intracytoplasmic sperm injection, might cause genomic imprinting disorders. The number of reported cases of those disorders is still too small to draw firm conclusions and the safety of these widely used assisted reproduction techniques needs to be further evaluated

Low pH immobilizes and kills human leukocytes and prevents transmission of cell-associated HIV in a mouse model

BACKGROUND: Both cell-associated and cell-free HIV virions are present in semen and cervical secretions of HIV-infected individuals. Thus, topical microbicides may need to inactivate both cell-associated and cell-free HIV to prevent sexual transmission of HIV/AIDS. To determine if the mild acidity of the healthy vagina and acid buffering microbicides would prevent transmission by HIV-infected leukocytes, we measured the effect of pH on leukocyte motility, viability and intracellular pH and tested the ability of an acidic buffering microbicide (BufferGel(®)) to prevent the transmission of cell-associated HIV in a HuPBL-SCID mouse model. METHODS: Human lymphocyte, monocyte, and macrophage motilities were measured as a function of time and pH using various acidifying agents. Lymphocyte and macrophage motilities were measured using video microscopy. Monocyte motility was measured using video microscopy and chemotactic chambers. Peripheral blood mononuclear cell (PBMC) viability and intracellular pH were determined as a function of time and pH using fluorescent dyes. HuPBL-SCID mice were pretreated with BufferGel, saline, or a control gel and challenged with HIV-1-infected human PBMCs. RESULTS: Progressive motility was completely abolished in all cell types between pH 5.5 and 6.0. Concomitantly, at and below pH 5.5, the intracellular pH of PBMCs dropped precipitously to match the extracellular medium and did not recover. After acidification with hydrochloric acid to pH 4.5 for 60 min, although completely immotile, 58% of PBMCs excluded ethidium homodimer-1 (dead-cell dye). In contrast, when acidified to this pH with BufferGel, a microbicide designed to maintain vaginal acidity in the presence of semen, only 4% excluded dye at 10 min and none excluded dye after 30 min. BufferGel significantly reduced transmission of HIV-1 in HuPBL-SCID mice (1 of 12 infected) compared to saline (12 of 12 infected) and a control gel (5 of 7 infected). CONCLUSION: These results suggest that physiologic or microbicide-induced acid immobilization and killing of infected white blood cells may be effective in preventing sexual transmission of cell-associated HIV

Augmenter of liver regeneration: An important intracellular survival factor for hepatocytes

Background/Aims: Augmenter of liver regeneration (ALR), a protein synthesized and stored in hepatocytes, is associated with mitochondria, and possesses sulfhydryl oxidase and cytochrome c reductase activities. We sought to determine the effects of ALR depletion in hepatocytes by antisense oligonucleotide transfection. Methods: Rat hepatocytes in primary culture were transfected with antisense oligonucleotide for ALR mRNA (ALR-AS) or scrambled oligonucleotide. Various analyses were performed at times up to 24 h after transfection. Results: Treatment with ALR-AS caused a decrease in ALR mRNA, cellular depletion of ALR protein primarily from mitochondria, and decreased viability. Flow cytometric analysis of ALR-AS-transfected hepatocytes stained with annexin-Vcy3 and 7-aminoactinomycin D revealed apoptosis as the predominant cause of death up to 6 h; incubation beyond this time resulted in necrosis in addition to apoptosis. ALR-AS-transfection caused release of mitochondrial cytochrome c, activation of caspase-3, profound reduction in the ATP content, and cellular release of LDH. Inhibition of caspase-3 inhibited the early phase of ALR-AS-induced death but not the late phase that included ALR and LDH release. Conclusions: These results suggest that ALR is critically important for the survival of hepatocytes by its association with mitochondria and regulation of ATP synthesis. © 2008 European Association for the Study of the Liver

Loss of DNMT1o Disrupts Imprinted X Chromosome Inactivation and Accentuates Placental Defects in Females

The maintenance of key germline derived DNA methylation patterns during preimplantation development depends on stores of DNA cytosine methyltransferase-1o (DNMT1o) provided by the oocyte. Dnmt1omat-/- mouse embryos born to Dnmt1Δ1o/Δ1o female mice lack DNMT1o protein and have disrupted genomic imprinting and associated phenotypic abnormalities. Here, we describe additional female-specific morphological abnormalities and DNA hypomethylation defects outside imprinted loci, restricted to extraembryonic tissue. Compared to male offspring, the placentae of female offspring of Dnmt1Δ1o/Δ1o mothers displayed a higher incidence of genic and intergenic hypomethylation and more frequent and extreme placental dysmorphology. The majority of the affected loci were concentrated on the X chromosome and associated with aberrant biallelic expression, indicating that imprinted X-inactivation was perturbed. Hypomethylation of a key regulatory region of Xite within the X-inactivation center was present in female blastocysts shortly after the absence of methylation maintenance by DNMT1o at the 8-cell stage. The female preponderance of placental DNA hypomethylation associated with maternal DNMT1o deficiency provides evidence of additional roles beyond the maintenance of genomic imprints for DNA methylation events in the preimplantation embryo, including a role in imprinted X chromosome inactivation. © 2013 McGraw et al

Shared Role for Differentially Methylated Domains of Imprinted Genes

For most imprinted genes, a difference in expression between the maternal and paternal alleles is associated with a corresponding difference in DNA methylation that is localized to a differentially methylated domain (DMD). Removal of a gene's DMD leads to a loss of imprinting. These observations suggest that DMDs have a determinative role in genomic imprinting. To examine this possibility, we introduced sequences from the DMDs of the imprinted Igf2r, H19, and Snrpn genes into a nonimprinted derivative of the normally imprinted RSVIgmyc transgene, created by excising its own DMD. Hybrid transgenes with sequences from the Igf2r DMD2 were consistently imprinted, with the maternal allele being more methylated than the paternal allele. Only the repeated sequences within DMD2 were required for imprinting these transgenes. Hybrid transgenes containing H19 and Snrpn DMD sequences and ones containing sequences from the long terminal repeat of a murine intracisternal A particle retrotransposon were not imprinted. The Igf2r hybrid transgenes are comprised entirely of mouse genomic DNA and behave as endogenous imprinted genes in inbred wild-type and mutant mouse strains. These types of hybrid transgenes can be used to elucidate the functions of DMD sequences in genomic imprinting