68 research outputs found

    Mice deficient in ubiquitous mitochondrial creatine kinase are viable and fertile

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    AbstractCreatine kinase isoenzymes (CK; EC 2.7.3.2) play a pivotal role in high-energy phosphoryl metabolism through subcellular compartmentation of the creatine-phosphate < = > ATP conversion reaction. In mouse, protein subunits constituting the ubiquitous mitochondrial CK (UbCKmit) and cytosolic B-CK isoforms are co-expressed in various cells and tissues with high and fluctuating energy demands such as brain, retina, smooth muscle, uterus, placenta and spermatozoa. Using targeted mutagenesis via homologous recombination in embryonic stem cells, we have generated mice that are deficient in UbCKmit subunits. These mice are viable and show no overt physical or behavioural abnormalities. Matings between UbCKmit-deficient mice produced normal numbers of offspring, showing that both females and males are completely fertile. Motility patterns of isolated spermatozoa were analyzed and found not to be impaired by absence of UbCKmit. From these results we conclude that UbCKmit is not essential for mouse viability, fertility, maintenance of pregnancy, or delivery

    Radiation hazard in soil from Ajaokuta North-central Nigeria

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    Background: Measurement of the radia�on dose distribu�on is important in assessing the health risk a popula�on and serve as reference in documen�ng changes to environmental radioac�vity in soil due to man-made ac�vi�es. Materials and Methods: The ac�vity concentra�on of 238U, 232Th and 40K in soil samples obtained from different loca�ons in Ajaokuta Local Government area was measured using Hyper Pure germanium Detec�on System (HPGe). Results: The calculated average concentra�on of the radionuclides ranged from 12 ± 1 Bqkg-1 to 59 ± 2 Bqkg-1 for 238U, 14 ± 1 Bqkg-1 to 78 ± 5 Bqkg-1 for 232Th and 49 ± 2 Bqkg-1 to 1272 ± 23 Bqkg-1 for 40K. In order to evaluate the radiological hazards due to natural radionuclides within Ajaokuta, the absorbed dose rate, gamma index, radium equivalent and excess life�me cancer risk were es�mated. According to measured data from the top soil (0-10 cm), the es�mated radium equivalent (Raeq) ranges from 55.7 Bqkg-1 at Steel Complex to 253.3 Bqkg-1 obtained from Forest samples. Conclusion: The mean absorbed dose rate, annual effec�ve dose and gamma radia�on index evaluated were 66.2 nGyh-1, 81.2 μSvy-1 and 1.05 respec�vely which are higher than the recommended limit for normal background radia�on. Thus, we conclude that people living in these loca�ons may be exposed to higher radia�on

    Somatic CTG•CAG repeat instability in a mouse model for myotonic dystrophy type 1 is associated with changes in cell nuclearity and DNA ploidy

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    Contains fulltext : 52050.pdf ( ) (Open Access)BACKGROUND: Trinucleotide instability is a hallmark of degenerative neurological diseases like Huntington's disease, some forms of spinocerebellar ataxia and myotonic dystrophy type 1 (DM1). To investigate the effect of cell type and cell state on the behavior of the DM1 CTG*CAG repeat, we studied a knock-in mouse model for DM1 at different time points during ageing and followed how repeat fate in cells from liver and pancreas is associated with polyploidization and changes in nuclearity after the onset of terminal differentiation. RESULTS: After separation of liver hepatocytes and pancreatic acinar cells in pools with 2n, 4n or 8n DNA, we analyzed CTG*CAG repeat length variation by resolving PCR products on an automated PAGE system. We observed that somatic CTG*CAG repeat expansion in our DM1 mouse model occurred almost uniquely in the fraction of cells with high cell nuclearity and DNA ploidy and aggravated with aging. CONCLUSION: Our findings suggest that post-replicative and terminal-differentiation events, coupled to changes in cellular DNA content, form a preconditional state that influences the control of DNA repair or recombination events involved in trinucleotide expansion in liver hepatocytes and pancreatic acinar cells

    Creatine kinase B deficient neurons exhibit an increased fraction of motile mitochondria

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    Contains fulltext : 69450.pdf ( ) (Open Access)BACKGROUND: Neurons require an elaborate system of intracellular transport to distribute cargo throughout axonal and dendritic projections. Active anterograde and retrograde transport of mitochondria serves in local energy distribution, but at the same time also requires input of ATP. Here we studied whether brain-type creatine kinase (CK-B), a key enzyme for high-energy phosphoryl transfer between ATP and CrP in brain, has an intermediary role in the reciprocal coordination between mitochondrial motility and energy distribution. Therefore, we analysed the impact of brain-type creatine kinase (CK-B) deficiency on transport activity and velocity of mitochondria in primary murine neurons and made a comparison to the fate of amyloid precursor protein (APP) cargo in these cells, using live cell imaging. RESULTS: Comparison of average and maximum transport velocities and global transport activity showed that CK-B deficiency had no effect on speed of movement of mitochondria or APP cargo, but that the fraction of motile mitochondria was significantly increased by 36% in neurons derived from CK-B knockout mice. The percentage of motile APP vesicles was not altered. CONCLUSION: CK-B activity does not directly couple to motor protein activity but cells without the enzyme increase the number of motile mitochondria, possibly as an adaptational strategy aimed to enhance mitochondrial distribution versatility in order to compensate for loss of efficiency in the cellular network for ATP distribution

    Creatine Kinase–Mediated ATP Supply Fuels Actin-Based Events in Phagocytosis

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    Phagocytosis requires locally coordinated cytoskeletal rearrangements driven by actin polymerization and myosin motor activity. How this actomyosin dynamics is dependent upon systems that provide access to ATP at phagosome microdomains has not been determined. We analyzed the role of brain-type creatine kinase (CK-B), an enzyme involved in high-energy phosphoryl transfer. We demonstrate that endogenous CK-B in macrophages is mobilized from the cytosolic pool and coaccumulates with F-actin at nascent phagosomes. Live cell imaging with XFP-tagged CK-B and β-actin revealed the transient and specific nature of this partitioning process. Overexpression of a catalytic dead CK-B or CK-specific cyclocreatine inhibition caused a significant reduction of actin accumulation in the phagocytic cup area, and reduced complement receptor–mediated, but not Fc-γR–mediated, ingestion capacity of macrophages. Finally, we found that inhibition of CK-B affected phagocytosis already at the stage of particle adhesion, most likely via effects on actin polymerization behavior. We propose that CK-B activity in macrophages contributes to complement-induced F-actin assembly events in early phagocytosis by providing local ATP supply

    CRISPR/Cas9-induced (CTG⋅CAG)n repeat instability in the myotonic dystrophy type 1 locus: implications for therapeutic genome editing

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    Myotonic dystrophy type 1 (DM1) is caused by (CTG⋅CAG)n-repeat expansion within the DMPK gene and thought to be mediated by a toxic RNA gain of function. Current attempts to develop therapy for this disease mainly aim at destroying or blocking abnormal properties of mutant DMPK (CUG)n RNA. Here, we explored a DNA-directed strategy and demonstrate that single clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-cleavage in either its 5′ or 3′ unique flank promotes uncontrollable deletion of large segments from the expanded trinucleotide repeat, rather than formation of short indels usually seen after double-strand break repair. Complete and precise excision of the repeat tract from normal and large expanded DMPK alleles in myoblasts from unaffected individuals, DM1 patients, and a DM1 mouse model could be achieved at high frequency by dual CRISPR/Cas9-cleavage at either side of the (CTG⋅CAG)n sequence. Importantly, removal of the repeat appeared to have no detrimental effects on the expression of genes in the DM1 locus. Moreover, myogenic capacity, nucleocytoplasmic distribution, and abnormal RNP-binding behavior of transcripts from the edited DMPK gene were normalized. Dual sgRNA-guided excision of the (CTG⋅CAG)n tract by CRISPR/Cas9 technology is applicable for developing isogenic cell lines for research and may provide new therapeutic opportunities for patients with DM1

    A Tail-Anchored Myotonic Dystrophy Protein Kinase Isoform Induces Perinuclear Clustering of Mitochondria, Autophagy, and Apoptosis

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    Contains fulltext : 79678.pdf (publisher's version ) (Open Access)BACKGROUND: Studies on the myotonic dystrophy protein kinase (DMPK) gene and gene products have thus far mainly concentrated on the fate of length mutation in the (CTG)n repeat at the DNA level and consequences of repeat expansion at the RNA level in DM1 patients and disease models. Surprisingly little is known about the function of DMPK protein products. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate here that transient expression of one major protein product of the human gene, the hDMPK A isoform with a long tail anchor, results in mitochondrial fragmentation and clustering in the perinuclear region. Clustering occurred in a variety of cell types and was enhanced by an intact tubulin cytoskeleton. In addition to morphomechanical changes, hDMPK A expression induces physiological changes like loss of mitochondrial membrane potential, increased autophagy activity, and leakage of cytochrome c from the mitochondrial intermembrane space accompanied by apoptosis. Truncation analysis using YFP-hDMPK A fusion constructs revealed that the protein's tail domain was necessary and sufficient to evoke mitochondrial clustering behavior. CONCLUSION/SIGNIFICANCE: Our data suggest that the expression level of the DMPK A isoform needs to be tightly controlled in cells where the hDMPK gene is expressed. We speculate that aberrant splice isoform expression might be a codetermining factor in manifestation of specific DM1 features in patients

    New polymorphic DNA marker close to the fragile site FRAXA

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    Abstract DNA from a human-hamster hybrid cell line, 908-K1B17, containing a small terminal portion of the long arm of the human X chromosome as well as the pericentric region of 19q was used as starting material for the isolation of an X-chromosome-specific DNA segment, RN1 (DXS369), which identifies a XmnI RFLP. Linkage analysis in fragile X families resulted in a maximum lod score of 15.3 at a recombination fraction of 0.05 between RN1 and fra(X). Analysis of recombinations around the fra(X) locus assigned RN1 proximal to fra(X) and distal to DXS105. Analysis of the marker content of hybrid cell line 908K1B17 suggests the localization of RN1 between DXS98 and fra(X). Heterozygosity of DXS369 is approximately 50%, which extends the diagnostic potential of RFLP analysis in fragile X families significantly

    Recovery in the myogenic program of congenital myotonic dystrophy myoblasts after excision of the expanded (CTG)n repeat

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    The congenital form of myotonic dystrophy type 1 (cDM) is caused by the large-scale expansion of a (CTG•CAG)n repeat in DMPK and DM1-AS. The production of toxic transcripts with long trinucleotide tracts from these genes results in impairment of the myogenic differentiation capacity as cDM’s most prominent morpho-phenotypic hallmark. In the current in vitro study, we compared the early differentiation programs of isogenic cDM myoblasts with and without a (CTG)2600 repeat obtained by gene editing. We found that excision of the repeat restored the ability of cDM myoblasts to engage in myogenic fusion, preventing the ensuing myotubes from remaining immature. Although the cDM-typical epigenetic status of the DM1 locus and the expression of genes therein were not altered upon removal of the repeat, analyses at the transcriptome and proteome level revealed that early abnormalities in the temporal expression of differentiation regulators, myogenic progression markers, and alternative splicing patterns before and immediately after the onset of differentiation became normalized. Our observation that molecular and cellular features of cDM are reversible in vitro and can be corrected by repeat-directed genome editing in muscle progenitors, when already committed and poised for myogenic differentiation, is important information for the future development of gene therapy for different forms of myotonic dystrophy type 1 (DM1)
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