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PubMed1999

VanX, a bacterial D-alanyl-D-alanine dipeptidase: resistance, immunity, or survival function?

The zinc-containing D-alanyl-D-alanine (D-Ala-D-Ala) dipeptidase VanX has been detected in both Gram-positive and Gram-negative bacteria, where it appears to have adapted to at least three distinct physiological roles. In pathogenic vancomycin-resistant enterococci, vanX is part of a five-gene cluster that is switched on to reprogram cell-wall biosynthesis to produce peptidoglycan chain precursors terminating in D-alanyl-D-lactate (D-Ala-D-lactate) rather than D-Ala-D-Ala. The modified peptidoglycan exhibits a 1, 000-fold decrease in affinity for vancomycin, accounting for the observed phenotypic resistance. In the glycopeptide antibiotic producers Streptomyces toyocaensis and Amylocatopsis orientalis, a vanHAX operon may have coevolved with antibiotic biosynthesis genes to provide immunity by reprogramming cell-wall termini to D-Ala-D-lactate as antibiotic biosynthesis is initiated. In the Gram-negative bacterium Escherichia coli, which is never challenged by the glycopeptide antibiotics because they cannot penetrate the outer membrane permeability barrier, the vanX homologue (ddpX) is cotranscribed with a putative dipeptide transport system (ddpABCDF) in stationary phase by the transcription factor RpoS (sigma(s)). The combined action of DdpX and the permease would permit hydrolysis of D-Ala-D-Ala transported back into the cytoplasm from the periplasm as cell-wall crosslinks are refashioned. The D-Ala product could then be oxidized as an energy source for cell survival under starvation conditions.

Bacteria↗

PubMed2002

Inhibition of expected movements in Tourette's Syndrome.

Tourette's Syndrome (TS) is a basal ganglia (BG) disorder characterized by involuntary motor and vocal tics, impairment of voluntary motor function, and attentional dysfunctions. This experiment aimed to investigate motor reprogramming and inhibitory control in adult TS patients. Ten TS patients and 10 age- and sex-matched controls performed sequences of movements in response to the illumination of LED lights, and were required to interrupt a predictable, reciprocating sequence of leftward and rightward movements in order to move to an unpredictable target that occurred either in the direction opposite to that expected, or in the opposite direction and at twice the expected distance. Results showed that TS patients were no slower than controls in reprogramming the direction and distance of movements, thus showing no overall deficits in inhibitory control. Of interest, however, was a directional asymmetry (right bias) in movement preparation time in TS patients. Patients with TS may have a superiority in allocating attentional resources toward the right side, contralateral to the implicated reduced BG volume in this disorder. These findings extend earlier work reporting anomalous BG lateralization in TS.

Adult↗

PubMed2002

Induction of Oct-3/4 expression in somatic cells by gap junction-mediated cAMP signaling from blastomeres.

We report the induction of embryonic gene expression in epithelial HC-11 cells upon communication with blastomeres in compacting mouse embryos. In contrast to NIH3T3 fibroblasts, HC-11 epithelial cells form gap junctions with blastomeres after injection into cleavage-stage embryos, as shown by targeting of phosphorylated connexin43 (pCx43) to areas of cell-to-blastomere contact and dye coupling. This was accompanied by expression of the embrvo-specific transcription factor, Oct-3/4, in the HC-11 cells. Dye coupling and Oct-3/4 expression were abolished with heptanol and 18beta- glycyrrhetinic acid, two gap junction blockers. Oleamide, which blocks gap junction-mediated communication but not electrical conductance, also inhibited Oct-3/4 expression in HC-11 cells, suggesting that Oct-3/4 induction results from transfer of molecules of < 1 kDa through gap junctions. Inhibition of cAMP signaling in blastomeres abolishes Oct-3/4 expression in somatic cells despite gap junction formation. In addition, reprogramming of NIH3T3 fibroblasts in an extract of HC-11 cells enabled assembly of pCx43 and Oct-3/4 expression after contact of the reprogrammed cells with blastomeres. We propose that gap junction-mediated cAMP signaling between blastomeres and somatic cells results in changes in somatic cell gene expression.

3T3 Cells↗

PubMed2003

Osteogenic potential of rat spleen stromal cells.

Evidence is mounting that an increasing number of cell populations in the adult organism already committed and/or differentiated retain the ability to reprogram themselves and give rise to a different phenotype. Bone marrow stromal cells have long been recognized as early progenitor cells for osteoblasts, chondrocytes, hematopoietic-supportive fibroblasts and adipocytes. Recent reports though have demonstrated a potential of cell populations outside the bone marrow environment to sustain bone formation under specific circumstances. The formation of bone nodules in the spleen of IL-5 transgenic mice has been recently reported (Macias et al. (2001): J. Clin. Invest. 107, 949 - 959). We thus postulated that a cell population exists in the spleen that under particular microenvironmental conditions is able to reprogram itself and pursue a fate other than the tissue-specific one. Therefore we isolated and expanded in vitro spleen-derived stromal cells. After expansion, these cells were challenged with culture conditions designed to induce osteogenic differentiation. We hypothesized that the combination of a proliferating factor (fibroblast growth factor 2) and a differentiating hormone (dexamethasone) would allow us to induce spleen-derived stromal cells to proliferate and at the same time to express osteoblast-specific genes. Thus, spleen-derived stromal cells were isolated from rat spleen and expanded in the presence of fibroblast growth factor 2 and dexamethasone. Once primary cultures reached confluence they were either switched to an osteo-inductive medium or implanted in immunodeficient mice. Although no bone formation was observed in in vivo experiments, in vitro spleen-derived stromal cells were able to deposit a mineralized matrix. Gene expression, as revealed by RT-PCR analysis, evidenced that the deposition of a mineralized matrix was concomitant with the expression of CBFA1 and osteocalcin, along with alkaline phosphatase and bone sialoprotein. Our data suggest that rat spleen-derived stromal cells can undergo osteogenic differentiation in a permissive microenvironment.

Alkaline Phosphatase↗

PubMed1995

Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins.

The reorganization of extracellular matrix (ECM) is an important function in many biological and pathophysiological processes. Culture of fibroblasts in a three-dimensional collagenous environment represents a suitable system to study the underlying mechanisms resulting from cell-ECM interaction, which leads to reprogramming of fibroblast biosynthetic capacity. The aim of this study was to identify receptors that transduce ECM signals into cellular events, resulting in reprogramming of connective tissue metabolism. Our data demonstrate that in human skin fibroblasts alpha 1 beta 1 and alpha 2 beta 1 integrins are the major receptors responsible for regulating ECM remodeling: alpha 1 beta 1 mediates the signals inducing downregulation of collagen gene expression, whereas the alpha 2 beta 1 integrin mediates induction of collagenase (MMP-1). Applying mAb directed against different integrin subunits resulted in triggering the heterodimeric receptors and enhancing the normal biochemical response to receptor ligation. Different signal transduction inhibitors were tested for their influence on gel contraction, expression of alpha 1(I) collagen and MMP-1 in fibroblasts within collagen gels. Ortho-vanadate and herbimycin A displayed no significant effect on any of these three processes. In contrast, genistein reduced lattice contraction, and completely inhibited induction of MMP-1, whereas type I collagen down-regulation was unaltered. Calphostin C inhibited only lattice contraction. Taken together, these data indicate a role of tyrosine-specific protein kinases in mediating gel contraction and induction of MMP-1, as well as an involvement of protein kinase C in the contraction process. The data presented here indicate that different signaling pathways exist leading to the three events discussed here, and that these pathways do not per se depend upon each other.

Antibodies, Monoclonal↗

PubMed2003

Gene expression in cloned bovine fetal liver.

Nuclear transfer (NT) is a method of animal reproduction that bypasses fertilization and propagates known combinations of genes. Currently NT is an inefficient process. Attempts have been made to increase the efficiency of this procedure, but most have been deemed unsuccessful. Some problems associated with NT are unusually large birth weights, and physical abnormalities in developing liver, heart, and brain. Despite numerous studies performed on NT animals, the factors behind the anomalies remain unknown. It is possible that nuclear reprogramming is the basis of poor development rates, meaning, when the donor cells are fused with enucleated eggs the nuclei may not regain the full ability to direct cell differentiation in subsequent mitotic divisions. If reprogramming is not carried out precisely, then some genes may not be correctly expressed in NT animals. The purpose of this study was to determine if differential gene expression between the livers of NT fetuses when compared to an embryo transfer (ET) derived fetus could be detected and the genes identified. An Angus fetus at 45 d of gestation was collected and a non-clonal cell line established for use as NT donor cells. Two NT fetuses were propagated and compared to the original. Differential Display Reverse Transcription Polymerase Chain Reaction (ddRT-PCR) was used to identify genes that were differentially expressed. Differentially abundant cDNAs were subcloned, sequenced and their corresponding mRNAs were verified by semi-quantitative RT-PCR. Twenty-three Expressed Sequence Tags (ESTs) were sequenced in Bos taurus and submitted to GenBank. The results of ddRT-PCR identified 39 genes/ESTs that were potentially differentially expressed. Fifteen of the genes were tested by semi-quantitative RT-PCR, but no significant differences were detected.

Animals↗

PubMed1996

Species specificity of ribosomal gene transcription: a factor associated with human RNA polymerase I prevents transcription of mouse rDNA.

An intrinsic property of class I gene transcription by RNA polymerase I (Pol I) is the species specificity of the initiation reaction. Previous studies have demonstrated that species-specific rDNA promoter recognition is brought about by a TBP-TAF complex, termed TIF-IB in mouse and SL1 in man. We have compared the ability of affinity-purified TIF-IB and SL1 to direct transcription from the homologous rDNA template both in a reconstituted transcription system and in nuclear extracts prepared from mouse and human cells. We show that Pol I from both species and the individual transcription factors, with the exception of TIF-IB/SL1, are functionally interchangeable in the reconstituted transcription system containing purified proteins. In nuclear extracts, however, species-specific differences are obvious. Whereas SL1 reprograms a heterologous mouse extract to recognize the human promoter, TIF-IB fails to reprogram a human extract unless it is complemented with mouse Pol I. Crude human, but not mouse, Pol I exhibits species-specific differences that disappear after purification. We propose that in extracts and less purified fractions human Pol I exists as 'holoenzyme' containing associated protein(s) that prevent assembly of TIF-IB-directed initiation complexes at the murine rDNA promoter.

Animals↗

PubMed2001

Developmental potential of mouse embryos reconstructed from metaphase embryonic stem cell nuclei.

Mice have recently been successfully cloned from embryonic stem (ES) cells. However, these fast dividing cells provide a heterogeneous population of donor nuclei, in terms of cell cycle stage. Here we used metaphases as a source of donor nuclei because they offer the advantage of being both unambiguously recognizable and synchronous with the recipient metaphase II oocyte. We showed that metaphases from ES cells can provide a significantly higher development rate to the morula or blastocyst stage (56--70%) than interphasic nuclei (up to 28%) following injection into a recipient oocyte. Selective detachment of mitotic cells after a demecolcin treatment greatly facilitates and accelerates the reconstruction of embryos by providing a nearly pure population of cells in metaphase and did not markedly affect the developmental rate. Most of the blastocysts obtained by this procedure were normal in terms of both morphology and ratio of inner cell mass and total cell number. After transfer into pseudopregnant recipients at the one- or two-cell stage, the ability of metaphase to be fully reprogrammed was demonstrated by the birth of two pups (1.5% of activated oocytes). Although the implantation rate was quite high (up to 32.9% of activated oocytes), the postimplantation development was characterized by a high and rapid mortality. Our data provide a clear situation to explore the long-lasting effects that can be induced by early reprogramming events.

Animals↗

PubMed2002

Limited demethylation leaves mosaic-type methylation states in cloned bovine pre-implantation embryos.

Cloning by nuclear transfer (NT) has been riddled with difficulties: most clones die before birth and survivors frequently display growth abnormalities. The cross-species similarity in abnormalities observed in cloned fetuses/animals leads us to suspect the fidelity of epigenetic reprogramming of the donor genome. Here, we found that single-copy sequences, unlike satellite sequences, are demethylated in pre-implantation NT embryos. The differential demethylation pattern between genomic sequences was confirmed by analyzing single blastocysts. It suggests selective demethylation of other developmentally important genes in NT embryos. We also observed a reverse relationship between methylation levels and inner cell mass versus trophectoderm (ICM/TE) ratios, which was found to be a result of another type of differential demethylation occurring in NT blastocysts where unequal methylation was maintained between ICM and TE regions. TE-localized methylation aberrancy suggests a widespread gene dysregulation in an extra-embryonic region, thereby resulting in placental dysfunction familiar to cloned fetuses/animals. These differential demethylations among genomic sequences and between differently allocated cells produce varied overall, but specified, methylation patterns, demonstrating that epigenetic reprogramming occurs in a limited fashion in NT embryos.

Animals↗

PubMed1988

Is the Suppressor-mutator element controlled by a basic developmental regulatory mechanism?

We report the results of genetic studies on derivatives of two different alleles of the maize a locus with an insertion of the Suppressor-mutator (Spm) transposable element in which the element is inactive, but can be reactivated readily. We present evidence that the mechanism that determines whether the element is in an active or inactive phase has two genetically distinguishable components. One determines whether or not the element is genetically active (the phase setting) and the other determines the stability of the setting in development, its heritability, and its phase in the next generation (the phase program). We show that the element's phase can be reset in a reproducible pattern during plant development. We also show that the Spm element can be reprogrammed to undergo a subsequent phase change without a concomitant phase change. The capacity to reset and reprogram the Spm element is differentially expressed within the plant in a pattern that is correlated with the developmental fate of apical and lateral meristems, suggesting the involvement of a basic developmental determination mechanism.

Alleles↗

PubMed2001

Genome-wide methylation patterns in normal and uniparental early mouse embryos.

In the normal diploid mouse embryo, active demethylation of the paternal genome but not of the maternal genome occurs within only a few hours and in a highly coordinated fashion as the zygote proceeds through the first G1 phase. This zygotic demethylation may be necessary to reprogram the sperm genome for somatic development. Immunofluorescence staining with an antibody against 5-methylcytosine shows that the cellular machinery of the fertilized egg cannot demethylate the second maternal genome in parthenogenetic, gynogenetic and triploid digynic embryos or remethylate the additional (already demethylated) paternal genome in androgenetic and triploid diandric embryos. This suggests that differential zygotic demethylation results from differences in the remodeling of paternal and maternal chromatin structures after fertilization, i.e. sperm nuclear decondensation and protamine-histone exchange. A proportion of embryos derived from normal matings display abnormal methylation patterns some of which are indistinguishable from those in androgenetic or gynogenetic embryos. We conclude that methylation reprogramming defects in mammalian zygotes contribute to the high incidence of early pregnancy failure.

Animals↗

PubMed2002

Epigenetic risks related to assisted reproductive technologies: risk analysis and epigenetic inheritance.

A broad spectrum of assisted reproductive technologies has become available for couples with fertility problems. Follow-up studies of children born as a result of assisted reproduction have shown that neonatal outcome and malformation rates are not different from those of the general population, except for a low birthweight and a slight increase in chromosomal abnormalities. The safety aspect of assisted reproduction at the epigenetic level has not been well studied. Epigenetics refers to phenomena where modifications of DNA methylation and/or chromatin structure underlie changes in gene expression and phenotype characteristics. This article intends to analyse epigenetic risks related to assisted reproduction on the basis of an overview of epigenetic reprogramming events in the gamete and early embryo. Two epigenetic modifications, methylation and imprinting, are considered in more detail. The interference of in-vitro embryo culture, immature sperm cells and nuclear transfer with epigenetic reprogramming is discussed, as well as the possibility of epigenetic inheritance.

Animals↗

PubMedDate not supplied

Oocyte maturation and embryonic failure.

Embryonic development is readily compromised by imperfections introduced during the process of oocyte maturation. We discuss the nature and causes of these imperfections, particularly in oocytes exposed to inappropriate hormonal regimes in vivo or to culture systems designed to induce the maturation of oocytes in vitro. The acquisition of developmental competence involves the synthesis and storage of a wide range of molecules during oocyte growth followed by the reprogramming and ordered utilization of these stored products during maturation, fertilization and early embryogenesis. The regulatory signals for these molecular changes are produced by the follicle cells in response to circulating levels of gonadotrophins; we report that some ovarian stimulation protocols distort these signals thereby disrupting molecular reprogramming of the oocyte and reducing subsequent developmental competence. The aspiration of immature oocytes from antral follicles followed by their maturation in vitro is a potential alternative to hormonal stimulation of patients in IVF treatment. Although relatively successful in a variety of animals, the production of fully viable human embryos by in-vitro maturation is still unsatisfactory despite the use of a wide variety of culture protocols. Our data suggests that the key to maturation and embryo viability in vitro resides in the follicle cell compartment rather than the oocyte. Because of rapid luteinization changes, follicle cells in culture probably fail to provide the maturing oocyte with the necessary ordered set of instructive signals and nutrients needed for the acquisition of developmental competence. Although much remains to be discovered about the nature, concentration and transmission of signals, nevertheless it is already clear that different steroids, matrix metalloproteinases and growth factors are involved in conferring viability on the maturing oocyte. Major improvements in the yield of viable embryos from in-vitro matured oocytes can be anticipated from a systematic analysis of somatic signals from the pre-ovulatory follicle.

Animals↗

PubMed2002

Reversible CD8 expression induced by common cytokine receptor gamma chain-dependent cytokines in a cloned CD4(+) T(h)1 cell line.

T cells that are intrathymically lineage committed are believed to maintain their CD4 or CD8 co-receptor expression. Here, we investigated whether intrathymic lineage commitment involves irreversible genetic modification or whether co-receptor expression can be reprogrammed depending on external stimuli. The CD4(+) T(h)1 clone 2D6 established from splenic T cells as an IL-12-dependent line survived in culture with IL-2, IL-7 or IL-15 alone. Surprisingly, CD8 expression occurred in 2D6 cells upon replacement of IL-12 with any one of the three cytokines that stimulate the common cytokine receptor gamma chain, yielding CD4(+)CD8(+) 2D6 cells. CD8 expression declined when IL-2 was replaced with IL-12 and CD8 induction was inhibited when IL-12 was included in IL-2 or IL-7 culture. Our observations show that even a lineage-committed mature T cell can be reprogrammed for co-receptor expression in response to particular external stimuli.

Animals↗

PubMed2026

Transient YAP activation uncovers the neurogenic potential of proliferative mammalian Müller glia.

The Hippo pathway effector YAP promotes spontaneous proliferation of Müller glia (MG), suggesting that bypassing Hippo signaling and activating YAP could enhance retinal regeneration. However, whether proliferative adult MGs retain meaningful neurogenic competence remains unclear. Here, using viral delivery of a Hippo-resistant YAP variant to wild-type adult MGs, we achieved transient YAP activation in adult MGs, inducing proliferation followed by cell-cycle withdrawal and differentiation. Intersectional genetic lineage tracing and EdU labeling, combined with transcriptomic analyses, revealed that YAP-activated MGs predominantly regenerate MGs, whereas only a subset gives rise to bipolar cell-like neurons. These results indicate that proliferative MGs acquire a state resembling that of late-stage retinal progenitors, with limited neurogenic lineage potential. We conclude that YAP-activated cell-cycle reentry inefficiently reprograms adult MGs toward photoreceptor or ganglion cell fates. These findings define the limited competence of proliferative adult MGs to contribute to neurogenic fates and provide a rigorous framework for assessing in vivo glial reprogramming strategies.

AAV↗

PubMed2003

Quantification of histone acetyltransferase and histone deacetylase transcripts during early bovine embryo development.

Mammalian oocytes are very unique cells with an unlimited developmental potential. These totipotent cells are able to remove existing gene-expression patterns and to impose new ones. However, genome reprogramming is still a mystery. Posttranslational modifications by acetylation of the N-termini portion of histones composing the nucleosome are involved in genome reprogramming. These modifications alter the higher-order chromatin structure to render the DNA accessible to the regulatory and transcriptional machinery. In the present study, we have investigated, to our knowledge for the first time, precise expression patterns of seven genes involved in chromatin structure throughout bovine embryo development. Oocytes harvested from bovine ovaries were used for in vitro production of germinal vesicle oocytes, metaphase II oocytes, 2- and 8-cell embryos, and blastocysts. Total RNA was extracted from pools (triplicates) of 20 oocytes or from embryos of each developmental stage. By means of quantitative reverse transcription-polymerase chain reaction using SYBR Green to detect double-stranded DNA, mRNA expression profiles for histone deacetylases (HDAC1, HDAC2, HDAC3, and HDAC7), histone acetyltransferases (GCN5 and HAT1), and histone H2A were established. Transcripts for all genes were detected at all stages from the oocyte to the blastocyst. The HDAC1, HDAC2 (class I HDAC), and HAT1 (type B HAT) revealed similar expression profiles. The HDAC3 (class I HDAC) tends to have an expression profile similar to those of HDAC1, HDAC2, and HAT1, whereas the HDAC7 (class II HDAC) and GCN5 (type A HAT) profiles were different from those three. These results indicate variable levels of histone deacetylases and histone acetyltransferases throughout embryonic development and may indicate the ones that are involved in somatic remodeling.

Acetyltransferases↗

PubMed2003

Somatic cell-like features of cloned mouse embryos prepared with cultured myoblast nuclei.

Cloning by somatic cell nuclear transfer requires silencing of the donor cell gene expression program and the initiation of the embryonic gene expression program (nuclear reprogramming). Failure to silence the donor cell program could lead to altered embryonic phenotypes. Cloned mouse embryos produced using myoblast nuclei fail to thrive in standard embryo culture media but flourish in somatic cell culture media favored by the donor myoblasts themselves, forming blastocysts at a significant rate, with robust morphologies, high total cell number, and a normal allocation of cells to the inner cell mass in most embryos. Myoblast cloned embryos continue expressing the GLUT4 glucose transporter, which is typically expressed in muscle but not in preimplantation stage embryos. Myoblast clones also exhibit precocious enrichment of GLUT1 at the cell surface. Both myoblast and cumulus cell cloned embryos exhibit enhanced rates of glucose uptake. These observations indicate that silencing of the donor cell genome during cloning either is incomplete or occurs progressively over the course of preimplantation development. As a result, cloned embryos initially exhibit many somatic cell-like characteristics. Tetraploid constructs, which possess a transplanted somatic cell genome plus the oocyte-derived chromosomes, exhibit a more embryonic-like pattern of gene expression and culture preference. We conclude that preimplantation stage cloned embryos have profoundly altered characteristics that are donor cell type specific and that exposure of cloned embryos to standard embryo culture conditions may lead to disruptions in basic homeostasis and inhibition of a range of essential processes including further nuclear reprogramming, contributing to cloned embryo demise.

Animals↗

PubMed2003

Disruption of imprinted gene methylation and expression in cloned preimplantation stage mouse embryos.

Cloning by somatic cell nuclear transfer requires that epigenetic information possessed by the donor nucleus be reprogrammed to an embryonic state. Little is known, however, about this remodeling process, including when it occurs, its efficiency, and how well epigenetic markings characteristic of normal development are maintained. Examining the fate of epigenetic information associated with imprinted genes during clonal development offers one means of addressing these questions. We examined transcript abundance, allele specificity of imprinted gene expression, and parental allele-specific DNA methylation in cloned mouse blastocysts. Striking disruptions were seen in total transcript abundance and allele specificity of expression for five imprinted genes. Only 4% of clones recapitulated a blastocyst mode of expression for all five genes. Cloned embryos also exhibited extensive loss of allele-specific DNA methylation at the imprinting control regions of the H19 and Snprn genes. Thus, epigenetic errors arise very early in clonal development in the majority of embryos, indicating that reprogramming is inefficient and that some epigenetic information may be lost.

Allelic Imbalance↗