Abnormal myotonic dystrophy protein kinase levels produce only mild myopathy in mice.

Myotonic dystrophy (DM) is commonly associated with CTG repeat expansions within the gene for DM-protein kinase (DMPK). The effect of altered expression levels of DMPK, which is ubiquitously expressed in all muscle cell lineages during development, was examined by disrupting the endogenous Dmpk gene and overexpressing a normal human DMPK transgene in mice. Nullizygous (-/-) mice showed only inconsistent and minor size changes in head and neck muscle fibres at older age, animals with the highest DMPK transgene expression showed hypertrophic cardiomyopathy and enhanced neonatal mortality. However, both models lack other frequent DM symptoms including the fibre-type dependent atrophy, myotonia, cataract and male-infertility. These results strengthen the contention that simple loss- or gain-of-expression of DMPK is not the only crucial requirement for development of the disease.

Myotonic dystrophy protein kinase is involved in the modulation of the Ca2+ homeostasis in skeletal muscle cells.

Myotonic dystrophy (DM), the most prevalent muscular disorder in adults, is caused by (CTG)n-repeat expansion in a gene encoding a protein kinase (DM protein kinase; DMPK) and involves changes in cytoarchitecture and ion homeostasis. To obtain clues to the normal biological role of DMPK in cellular ion homeostasis, we have compared the resting [Ca2+]i, the amplitude and shape of depolarization-induced Ca2+ transients, and the content of ATP-driven ion pumps in cultured skeletal muscle cells of wild-type and DMPK[-/-] knockout mice. In vitro-differentiated DMPK[-/-] myotubes exhibit a higher resting [Ca2+]i than do wild-type myotubes because of an altered open probability of voltage-dependent l-type Ca2+ and Na+ channels. The mutant myotubes exhibit smaller and slower Ca2+ responses upon triggering by acetylcholine or high external K+. In addition, we observed that these Ca2+ transients partially result from an influx of extracellular Ca2+ through the l-type Ca2+ channel. Neither the content nor the activity of Na+/K+ ATPase and sarcoplasmic reticulum Ca2+-ATPase are affected by DMPK absence. In conclusion, our data suggest that DMPK is involved in modulating the initial events of excitation-contraction coupling in skeletal muscle.

Mice deficient in ubiquitous mitochondrial creatine kinase are viable and fertile.

Creatine 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.

Use of gene targeting for compromising energy homeostasis in neuro-muscular tissues: the role of sarcomeric mitochondrial creatine kinase.

We have introduced a single knock-out mutation in the mitochondrial creatine kinase gene (ScCKmit) in the mouse germ line via targeted mutagenesis in mouse embryonic stem (ES) cells. Surprisingly, ScCKmit -/- muscles, unlike muscles of mice with a deficiency of cytosolic M-type creatine kinase (M-CK -/-; Van Deursen et al. (1993) Cell 74, 621-631), display no altered morphology, performance or oxidative phosphorylation capacity. Also, the levels of high energy phosphate metabolites were essentially unaltered in ScCKmit mutants. Our results challenge some of the present concepts about the strict coupling between CKmit function and aerobic respiration.

Myoadenylate deaminase deficiency: an enzyme defect in search of a disease.

The frequency of MAD deficiency in cases with exercise intolerance compared with the frequency in series of consecutive muscle biopsies suggests a relation between the deficiency and exercise intolerance. Deficiency cases can be presumed by an impaired NH3 production during ischaemic exercise. The ischaemic exercise test also gives information concerning the familial character of the deficiency.

Major and modified nucleosides as markers in ovarian cancer: a pilot study.

A number of major and modified nucleosides were measured in urine samples of patients with ovarian cancer. The patients were divided into three groups based on histological criteria: benign, borderline, malignant. 44% of the measured marker levels of the benign group were in the normal range, whereas 97% of the borderline and malignant groups were outside the normal range. After a total hysterectomy with adnexectomy and omentectomy in a patient with a borderline type malignancy, six of the seven elevated markers decreased to normal values.

Localization of PEPD to the long arm of chromosome 19.

A panel of human-rodent somatic cell hybrids containing different regions of chromosome 19 has been used to obtain a regional localization for peptidase D. The results assign PEPD to the long arm of chromosome 19, in the region cen-q13.2

Determination of uric acid in serum using isotachophoresis.

An operational system is described for the isotachophoretic determination of uric acid in serum, making use of column coupling. The method has been compared with a standard enzymatic procedure. With the present technique small amounts of serum (ca. 3 microliter) can be applied without any pretreatment. Urate recovery was 99.0-100.5%. Under the non-physiological measuring conditions used, 12-28% of control serum uric acid was bound to macromolecules of molecular weight exceeding 25,000. The day-to-day variations of the isotachophoretic procedure were smaller than those of the enzymatic method, whereas standard deviations were comparable. The isotachophoretic procedure is less influenced by certain metabolites.

Rapid prenatal diagnosis of HG-PRT deficiency using ultra-microchemical methods.

A previously developed simple ultramicromethod has been used for the rapid prenatal diagnosis of hypoxanthine-guanine phosphoribosyl transferase (HG-PRT) deficiency. The method is based on the incubation of small numbers of visually selected, lyophilized fibroblasts (in the present study five cells per incubation) with radioactive substrate in an end volume of 0.3 microliter. Fibroblasts derived from the amniotic fluid of a 15-week male fetus in a heterozygote for the X-linked Lesch-Nyhan syndrome showed a severe degree of HG-PRT deficiency. In total 50 fibroblasts were used. The diagnosis was confirmed upon termination of the pregnancy by the demonstration of HG-PRT deficiency in fetal erythrocytes and cultured skin fibroblasts.

Isotachophoresis of urinary purines and pyrimidines: the use of spacers and enzymes for identification.

An isotachophoretic system is described for the separation and identification of urinary purine and pyrimidine bases and nucleosides. For a better discrimination and interpretation of the UV profiles, well-defined non-UV-absorbing substances were introduced as spacers. Treatment of urine samples with purified enzymes before analysis resulted in specific shifts in the metabolite profiles, providing a sensitive and specific means of identifying a number of metabolites. With an injected volume of 3 microliters (untreated urine diluted 1:5) the present method allows reproducible separations within 20 min of at least twenty different nucleosides and bases.

Modulation of gene activity by consecutive gene targeting of one creatine kinase M allele in mouse embryonic stem cells.

The cytosolic creatine kinases (CK's; EC 2.7.3.2) BB, BM and MM are dimeric isoenzymes which have an important role in energy metabolism and display characteristic tissue- and stage-specific patterns of expression in mammals. To study the functional role of the distribution of the CK isoenzymes we have focussed on the modulation of expression of the genes encoding the individual B and M subunits, starting at the muscle creatine kinase (CKM) gene which is transcriptionally inactive during early embryogenesis. Using repeated rounds of gene targeting in mouse embryonic stem (ES) cells, two types of mutant cell lines were obtained. First, we generated a cell line in which insertion of a neomycin resistance (neor) gene had disrupted one of the CKM alleles. Subsequently, from this cell line, following introduction of an insertion type vector designed for replacement of the muscle specific CKM-enhancer by the constitutively acting polyoma virus enhancer PyF441, several independent doubly targeted clones were isolated which all had insertions in the previously neo-disrupted CKM allele. In some of these ES clones, the targeted enhancer replacement resulted in gene correction and functional activation of the silent CKM gene. Dimerisation between the ectopically expressed CKM subunits and CKB subunits which are normally present at high levels in ES cells, led to the formation of the BM isoform of CK in these clones.

Changes in glycolytic network and mitochondrial design in creatine kinase-deficient muscles.

Skeletal muscles respond with high plasticity to pathobiological conditions or changes in physiological demand by remodeling cytoarchitectural and metabolic characteristics of individual myocytes. We have previously shown that muscles of mice without mitochondrial and/or cytosolic creatine kinases (ScCKmit(-/-) and/or M-CK(-/-)) partly compensate for the defect(s) by redirecting metabolic pathways and ultrastructural characteristics. Here, we show by semiquantitative Western blot analysis that the compensatory changes involve mutation- and fiber-type-specific coordinated regulation of divergent but functionally coupled groups of proteins. Fast-twitch gastrocnemius muscle of CK(--/--) mice display a two- to fourfold upregulation of mitochondrial cytochrome c oxidase, inorganic phosphate carrier, adenine nucleotide translocator, and voltage-dependent anion channel proteins. In parallel, cytosolic myoglobin is upregulated. Slow-twitch soleus muscle responds with changes in the glycolytic enzyme pattern, including a shift in lactate dehydrogenase isoenzyme composition. Adaptations in the network for oxidative adenosine triphosphate (ATP) production are already apparent at 17 days of age.