Expression of myotonic dystrophy protein kinase gene during in vivo and in vitro mouse myogenesis

Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disorder characterized by a great variability in its clinical manifestations. The mutational basis underlying DM consists of an unstable (CTG)n trinucleotide repeat in the 3' untranslated region of the myotonic dystrophy protein kinase gene (DMPK). Conflicting results on DMPK gene expression in congenitally affected infants (CDM) have been published. Moreover, the prominence of satellite cells seen in muscle of CDM infants supports the notion that the congenital form is associated with an arrest in muscle development and suggests a role for the DMPK gene during differentiation and maturation of muscle. In order to clarify these findings, a comparative study of DMPK and myogenic factor mRNA levels was performed in developing mouse muscle tissues and cultured muscle cells at different developmental stages. Results show that DMPK gene expression is upregulated at a late stage of muscular development. This upregulation does not seem to depend on a given muscle specific bHLH factor.     

Fluorescence in situ hybridization (FISH): DNA probe production and hybridization criteria

We describe methods for the production of fluorescence in situ hybridization (FISH) probes and the utilization of these probes for the detection of complementary DNA sequences with accuracy and sensitivity for application in both basic research and clinical diagnosis. Due to the frequent use of FISH in many laboratories, it is important to apply the most convenient and reproducible approach. This review describes some of the most recent techniques, and includes versatile, effective and simple methods of probe production and fluorescence in situ hybridization. We also describe methods for the production of region-specific and chromosome-specific DNA probes and hybridization techniques for the visualization of these probes.     

Iraqi-Jewish kindreds with optic atrophy plus (3-methylglutaconic aciduria type 3) demonstrate linkage disequilibrium with the CTG repeat in the 3' untranslated region of the myotonic dystrophy protein kinase gene

Iraqi-Jewish optic atrophy plus is an autosomal recessive condition characterized by infantile optic atrophy, an early onset movement disorder, and 3-methylglutaconic aciduria. Other features include spastic paraplegia, mild ataxia, mild cognitive deficiency and dysarthria. This disorder was identified in inbred Iraqi-Jewish kindreds in which relationships between most of the affected individuals were unknown. In this study we identify linkage to chromosome 19q13.2-q13.3 by using a DNA pooling strategy to perform a genome wide screen followed by a high density search for shared segments among affected individuals in candidate regions identified in the initial genome wide screen. A significantly high positive lod score of 6.14 at zero recombination was obtained for the CTG repeat in the 3' untranslated region of the myotonic dystrophy protein kinase gene. The existence of multiple recombinant individuals indicates the disease interval can be further narrowed with additional markers. Linkage disequilibrium was seen in six polymorphic markers across a 1 Mb interval. This region is well characterized and contains several candidate genes.     

Normal levels of DM RNA and myotonin protein kinase in skeletal muscle from adult myotonic dystrophy (DM) patients

A major question about the pathogenesis of myotonic dystrophy (DM) is how the (CTG)n repeat mutation alters expression of the DM gene and how that is related to disease causation. Most previous studies have found a decrease in DM RNA and protein in patient tissue. In contrast to these reports we find, unexpectedly, that independent of the size of the CTG repeat: (1) there are equal levels of RNA products of mutant and normal alleles, and (2) levels of Mt-PK in skeletal muscle from DM patients is unaltered from normal. These findings are consistent with the recent hypothesis that mutant DM DNA or RNA may cause disease by disrupting the function of other, yet unidentified, genes.     

Fluorescence in situ hybridization mapping of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to 7q31.3

We have used the fluorescence in situ hybridization (FISH) technique to refine the localization of the cystic fibrosis transmembrane conductance regulator (CFTR) gene on human chromosome 7. The result shows that the gene is most likely located within band q31.3.     

Myotonic dystrophy: antisense oligonucleotide inhibition of DMPK gene expression in vitro

Antisense phosphorothioate oligonucleotides, targeted against the first codon starting region of DMPK mRNA, were successfully used in K562 and HepG2 cells to decrease DMPK expression. The most effective antisense oligo, MIO1, when added to K562 cells, shows a 75% reduction of the DMPK gene expression 6 hours after addition. The same molecule, when encapsulated in liposomes, delays myotonin mRNA decrease at 24 hours after cell treatment. This considerable success with such inhibition in vitro could be utilised to generate a cell model to study myotonic dystrophy (DM) chemio-physiological alterations.     

Altered phosphorylation and intracellular distribution of a (CUG)n triplet repeat RNA-binding protein in patients with myotonic dystrophy and in myotonin protein kinase knockout mice

Myotonic dystrophy (DM) is associated with expansion of CTG repeats in the 3'-untranslated region of the myotonin protein kinase (DMPK) gene. The molecular mechanism whereby expansion of the (CUG)n repeats in the 3'-untranslated region of DMPK gene induces DM is unknown. We previously isolated a protein with specific binding to CUG repeat sequences (CUG-BP/hNab50) that possibly plays a role in mRNA processing and/or transport. Here we present evidence that the phosphorylation status and intracellular distribution of the RNA CUG-binding protein, identical to hNab50 protein (CUG-BP/hNab50), are altered in homozygous DM patient and that CUG-BP/hNab50 is a substrate for DMPK both in vivo and in vitro. Data from two biological systems with reduced levels of DMPK, homozygous DM patient and DMPK knockout mice, show that DMPK regulates both phosphorylation and intracellular localization of the CUG-BP/hNab50 protein. Decreased levels of DMPK observed in DM patients and DMPK knockout mice led to the elevation of the hypophosphorylated form of CUG-BP/hNab50. Nuclear concentration of the hypophosphorylated CUG-BP/hNab50 isoform is increased in DMPK knockout mice and in homozygous DM patient. DMPK also interacts with and phosphorylates CUG-BP/hNab50 protein in vitro. DMPK-mediated phosphorylation of CUG-BP/hNab50 results in dramatic reduction of the CUG-BP2, hypophosphorylated isoform, accumulation of which was observed in the nuclei of DMPK knockout mice. These data suggest a feedback mechanism whereby decreased levels of DMPK could alter phosphorylation status of CUG-BP/hNab50, thus facilitating nuclear localization of CUG-BP/hNab50. Our results suggest that DM pathophysiology could be, in part, a result of sequestration of CUG-BP/hNab50 and, in part, of lowered DMPK levels, which, in turn, affect processing and transport of specific subclass of mRNAs.     

MKBP, a novel member of the small heat shock protein family, binds and activates the myotonic dystrophy protein kinase

Muscle cells are frequently subjected to severe conditions caused by heat, oxidative, and mechanical stresses. The small heat shock proteins (sHSPs) such as alphaB-crystallin and HSP27, which are highly expressed in muscle cells, have been suggested to play roles in maintaining myofibrillar integrity against such stresses. Here, we identified a novel member of the sHSP family that associates specifically with myotonic dystrophy protein kinase (DMPK). This DMPK-binding protein, MKBP, shows a unique nature compared with other known sHSPs: (a) In muscle cytosol, MKBP exists as an oligomeric complex separate from the complex formed by alphaB-crystallin and HSP27. (b) The expression of MKBP is not induced by heat shock, although it shows the characteristic early response of redistribution to the insoluble fraction like other sHSPs. Immunohistochemical analysis of skeletal muscle cells shows that MKBP localizes to the cross sections of individual myofibrils at the Z-membrane as well as the neuromuscular junction, where DMPK has been suggested to be concentrated. In vitro, MKBP enhances the kinase activity of DMPK and protects it from heat-induced inactivation. These results suggest that MKBP constitutes a novel stress-responsive system independent of other known sHSPs in muscle cells and that DMPK may be involved in this system by being activated by MKBP. Importantly, since the amount of MKBP protein, but not that of other sHSP family member proteins, is selectively upregulated in skeletal muscle from DM patients, an interaction between DMPK and MKBP may be involved in the pathogenesis of DM.     

Localization of a human double-stranded RNA-binding protein gene (STAU) to band 20q13.1 by fluorescence in situ hybridization

Asymmetric transport of mRNA within the cells is mediated by RNA-binding proteins that form, along with the mRNAs and perhaps other small RNAs, stable ribonucleoprotein complexes. However, the nature of the protein components of these complexes in vertebrates is still unknown. In Drosophila, genetic studies have identified a number of potential genes that are necessary for localization of mRNAs in oocytes; one of the most studied is the staufen gene. The staufen protein has been shown to bind to localized mRNAs in oocytes and to be expressed in somatic cells as well. To understand the mechanism of mRNA transport in mammals and characterize its components, we recently cloned and sequenced the human staufen homolog cDNA (HGMW-approved symbol STAU). In this paper, we show that the gene is unique in the human genome and report its chromosomal localization by fluorescence in situ hybridization. The human staufen gene maps to chromosome 20q13.1, a region that is associated with certain genetic diseases.     

Fluorescence in situ hybridization analysis of chromosome 1p36 deletions in human MYCN amplified neuroblastoma

Artificial illumination is an important factor in the management of layers. In this study, a new monochromatic light system was developed for egg layers. Prelaying pullets (Lohmann) were marked and housed in nine light and temperature control rooms (15 battery cages, 3 hens per cage; n = 45), divided into three light treatments: 0.1 and 0.01 W/m2 light intensity using light emitting diode (LED) lamps and 0.1 W/m2 using mini-fluorescent bulbs (PL) (control). In each of the LED rooms, three wavelengths were tested: 560 (n = 9), 660 (n = 9), 880 (n = 6), and 660 intermitted lighting (15 min light 45 min dark, 660IN) (n = 9). Birds were exposed to 12 h light and 12 h of darkness using PL lamps. At 21 wk of age, the light period was increased to 12.75 h by using 5.5 h of LED lamps and 7.25 of PL light source for Groups 1 and 2, the third group received 12.75 h of PL light. Until 28 wk of age, light hours increased by 0.5 h/w using LED light for Groups 1 and 2 and PL source for the third group, reaching 16 h of light at 28 wk of age. Egg production and feed consumption were recorded daily; egg components were recorded weekly for 10 mo. A significant reduction in egg production was observed in all 880nm groups; no differences in egg production and quality were found in the other groups. Feed consumption was significantly lower by 7% in all 0.01 W/m2 groups. We suggest that an important reduction in rearing costs of laying hens may be obtained by using this system.     

Detection of the leghemoglobin gene on two chromosomes of Phaseolus vulgaris by in situ PCR linked-fluorescent in situ hybridization (FISH)

OBJECTIVE: To investigate the effect of phentolamine, a sympathetic blocking agent, on the spontaneous electrical activity (SEA) recorded from a locus of a myofascial trigger spot (MTrS), equivalent to a human trigger point, in rabbit skeletal muscle. DESIGN: Randomized control trial. SETTING: A university medical laboratory. PATIENTS OR OTHER PARTICIPANTS: Nine adult New Zealand rabbits. INTERVENTION: In the experimental group phentolamine mesylate (1mg/kg) was injected into the external iliac artery, followed by flushing with normal saline. The control group was treated with normal saline instead of phentolamine using the same procedure. MAIN OUTCOME MEASURES: SEA was recorded from multiple active loci of MTrSs in the biceps femoris muscle: initially SEA in the same locus was recorded before and immediately after phentolamine (or normal saline) injection; then SEA was recorded from 25 different active loci. The mean of the average integrated signal (AIS) of SEA was analyzed, comparing the effects of phentolamine and normal saline on SEA. RESULTS: In the same active locus, the AIS of SEA showed statistically a linear decay with time after phentolamine injection, with a correlation coefficient of .56 at p < .05. However, no statistical relationship could be derived for the control group data with time by using regression analysis, probably because of large variations among the rabbits and movement artifacts during the experiment. In 25 different loci in the phentolamine group, the mean of the AIS of SEA (7.92 microV) was significantly lower than that of the control group (9.89 microV) at p < .05. CONCLUSIONS: The results support the hypothesis that the autonomic nervous system is involved in the pathogenesis of myofascial trigger points. The application of the AIS as an evaluation index seems to be feasible in the quantitative measurement of SEA.     

Myotonic dystrophy protein kinase gene expression in skeletal muscle from congenitally affected infants

Thiothrix spp., sulfide-oxidizing filamentous bacteria, were found to be a principal bacterial component of aquatic biofilms causing biofouling in selected municipal water storage tanks, private wells, and drip irrigation systems in Florida. Treatments of up to 200 ppm chlorine in the affected systems could not prevent return of the biofouling problem. The water originated from the upper Floridan aquifer and associated surficial aquifers in central and north Florida. Samples were examined where visible biofilms had a white, filamentous appearance, indicative of Thiothrix spp. The detection of Thiothrix spp. was confirmed by enzyme-liked immunosorbent assay (ELISA), indirect immunofluorescence (IIF), and microbiological procedures. It was estimated through immunocytochemical procedures that Thiothrix spp. comprised 18% of the biofilm in the municipal water storage tanks. These observations confirm that specific biological and chemical interactions may induce physical changes leading to significant biofouling.