Nitric oxide and l-arginine cause an accumulation of utrophin at the sarcolemma: a possible compensation for dystrophin loss in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD), a severe X-linked recessive disorder which results in progressive muscle degeneration, is due to a lack of dystrophin, a membrane cytoskeletal protein. An approach to treatment is to compensate for dystrophin loss with utrophin, another cytoskeletal protein with over 80% homology with dystrophin. Utrophin is expressed, at the neuromuscular junction, in normal and DMD muscles and there is evidence that it may perform the same cellular functions as dystrophin. So, the identification of molecules or drugs that could up-regulate utrophin is a very important goal for therapy. We show that in adult normal and mdx mice (an animal model of Duchenne myopathy) treated with l-arginine, the substrate of nitric oxide synthase (NOS), a pool of utrophin localized at the membrane appeared and increased, respectively. In normal and mdx myotubes in culture, l-arginine, nitric oxide (NO), or hydroxyurea increased utrophin levels and enhanced its membrane localization. This effect did not occur with d-arginine, showing the involvement of NOS in this process. The NO-induced increase in utrophin was prevented by oxadiazolo-quinoxalin-1-one, an inhibitor of a soluble guanylate cyclase implicated in NO effects. These results open the way to a potential treatment for Duchenne and Becker dystrophies.     

Hereditary prothrombin deficiency presenting as intracranial haematoma in infancy

Hereditary deficiency of prothrombin is a rare autosomal recessive bleeding disorder, with severe bleeding diathesis in homozygotes, but rarely resulting in intracranial haematoma. We describe two infants of consanguineous parents, presenting with acute subdural haematoma. Because such haematomas in infancy are highly indicative of trauma caused by child battering and because the socio-economic status of the family was unstable, there was a suspicion of child battering. However, further investigations revealed a bleeding diathesis due to a prothrombin deficiency. DNA analysis of the prothrombin gene showed homozygosity for a novel mutation, substituting Lys for Glu at codon 7 and resulting in decreased specific clotting activity. We discuss the probability of bleeding diathesis versus child battering in the aetiology of intracranial haematoma.     

Histological characteristics of sternoclavicular beta 2-microglobulin amyloidosis and clues for its histogenesis

BACKGROUND: The pathogenesis of beta 2-microglobulin amyloidosis (A beta 2m) has yet to be fully elucidated. METHODS: We describe the distribution and extent of A beta 2m deposition and macrophagic infiltration in cartilage, capsule, and synovium of sternoclavicular joints obtained postmortem from 54 patients after 3 to 244 (median 46) months of dialysis. Twenty-four nonuremic patients served as a control group. The diagnosis of amyloidosis (A) rested on a positive Congo Red staining (typical birefringence) and that of A beta 2m on positive immunostaining of the A deposits with a monoclonal anti-beta 2m antibody. The size of A deposits was measured. RESULTS: A beta 2m was detected in 32 (59%), and non-beta 2m amyloid (Anon beta 2m) was detected in an additional 8 (15%) of the 54 dialyzed patients. A beta 2m deposits were present in the cartilage of all A beta 2m (+) patients (100%). They were localized solely in the cartilage in 27% of the cases, either as a thin patchy layer or as a continuous thicker layer (identified as stage I). A beta 2m was additionally present in the capsule and/or synovium without macrophages in 27% of the cases (identified as stage II). The correlation between the size of cartilaginous deposits and dialysis duration (P = 0.02) as well as with the prevalence (P = 0.03) and size of capsular deposits (P = 0.02) suggests that stage II is a later stage of A deposition. Clusters of macrophages were detected around capsular and synovial amyloid deposits in 46% of the cases (identified as stage III). The longer duration of dialysis in those with stage III as well as the relationship between the size of the A beta 2m deposits and the prevalence of macrophagic infiltration suggests that stage III is the last stage of A beta 2m deposition. Marginal bone erosions were observed in 9 out of 12 patients with stage III deposits. Their size was correlated with that of cartilaginous deposits (P = 0.01). Among the 24 control patients, Anon beta 2m was detected in 12 patients (cartilage 100%, capsule 8%, synovium 30%). CONCLUSIONS: The earliest stage of A beta 2m deposition occurs in the cartilage. A beta 2m subsequently extends to capsule and synovium. These two first stages do not require macrophage infiltration. Macrophages are eventually recruited around larger synovial or capsular deposits in the final stage. Marginal bone erosions develop in this late stage.     

Relations between genotype and phenotype in German patients with the Machado-Joseph disease mutation.

OBJECTIVE: Machado-Joseph disease (MJD) is an autosomal dominant cerebellar ataxia with extensive phenotypic variability originally described in families of Portuguese ancestry. Recently, the mutation causing the disease has been identified as an expanded CAG trinucleotide repeat. In this study relations between genotype and phenotype were investigated. METHODS: A series of 180 German patients with degenerative forms of ataxia were clinically and genetically examined. Patients bearing the MJD mutation were assigned to three phenotypes: phenotype 1 characterised by early onset and dystonia or pronounced rigidity associated with ataxia and spasticity. Main symptoms in phenotype 2 were ataxia and spasticity. In phenotype 3 onset was relatively late and peripheral neuropathy accompanied ataxia. Clinical and molecular data were correlated. RESULTS: An expanded CAG array was found in 42 patients from 22 families. Repeat length of CAG varied between 67 and 80 CAG motifs and showed an inverse correlation with the age of onset. For the development of phenotype 1 early onset (< 20 years) seemed more decisive than extensive repeat length. Phenotype 2 was present in all patients with more than 73 CAG motifs and onset between 20 and 40. Phenotype 3 developed in most patients with less than 73 CAG motifs and onset was regularly beyond the age of 40. Intrafamilial variability of both repeat length and phenotype was large reflecting meiotic instability of the expanded CAG repeat. CONCLUSIONS: The MJD mutation is the most frequent cause of dominantly inherited ataxia in Germany. Variations in repeat lengths substantially influence age of onset as well as phenotype but cannot explain why MJD characteristics of Portuguese families such as "bulging eyes", dystonia, and rigidity are essentially missing in German families. Despite the genotypic and phenotypic relations found in this study a reliable individual prognosis of the course of the disease is not possible at a presymptomatic stage.     

Adult from of basal cell navevus syndrome: A family study

Summary A 32-year-old patient had marked reduction of visual acuity due to falciform folds of the retina and retinal detachment, and severe neurological abnormality: bilateral pyramidal involvement, fasciculation in all limbs and gait ataxia. Skull radiographs showed internal frontal hyperostosis; CT scan showed calcification of the falx cerebri, and multiple arachnoid cysts were shown by myelography. A naevoid lesion had previously been removed from the left forearm. There was a history of ophthalmological symptoms in the mother and the daughter of the propositus. His son has café au lait spot on the abdomen and dentigerous cysts. The diagnosis of an adult form of basal cell naevus syndrome with an autosomal dominant mode of inheritance is discussed.This work was supported by grants from the F.R.S.M. of Belgium no. 3.4543.77 (Prof. P. Danis) and no. 3.4538.76 (Prof. C. Coërs) and the Free University of Brussels     

Mitotic and meiotic stability of linear plasmids in yeast.

Circular recombinant DNA plasmids that contain autonomously replicating sequences (ARSs) are maintained in extrachromosomal form in transformed yeast cells. However, these plasmids are unstable, being rapidly lost from cells growing without selection. Although the stability of such a plasmid can be increased by the presence of yeast centromere DNA (CEN), even CEN plasmids are lost at a high rate compared to a bona fide yeast chromosome. Natural yeast chromosomes are linear molecules; therefore, we have asked if linearization can improve the stability of recombinant DNA plasmids. Linear plasmids with and without yeast CENs were constructed in vitro by using termini from the extrachromosomal ribosomal DNA (rDNA) of the ciliated protozoan Tetrahymena thermophila as "telomeres." These linear plasmids transformed yeast at high frequency and were maintained as linear extrachromosomal molecules during mitotic growth. Moreover, linear plasmids containing CENs were also transmitted through meiosis: these plasmids segregate predominantly 2+:2- at the first meiotic division, indicating that Tetrahymena rDNA termini can provide telomere function during yeast meiosis. Linear plasmids without CENs were about as stable in mitosis as the comparable circular plasmid. Thus, the Tetrahymena rDNA termini have no marked positive or negative effect on the mitotic stability of ARS plasmids. However, linear plasmids containing CENs are three to four times less stable in mitotic cells than circular CEN plasmids. This decrease in stability is not due to a functional change in the centromere itself; rather, linearization of a CEN plasmid has a direct detrimental effect on its mitotic stability. These results may reflect the existence of spatial constraints on the positions of centromeres and telomeres, constraints which must be satisfied to achieve stable segregation of chromosomes during mitosis.     

Synergistic Regulation of Cytosolic Ca2+ Concentration by Adenosine and alpha1-Adrenergic Agonists in Mouse Striatal Astrocytes

Adenosine has a broad array of actions on neurons but astrocytes also possess adenosine receptors. We have previously shown that adenosine, by acting on astrocytes in the striatum, can modulate neuronal responses mediated by receptors coupled to phospholipase C through an astrocyto - neuronal interaction. In addition, adenosine was found to potentiate the alpha1-adrenergic production of inositol phosphates in astrocytes. The mechanism involved in this potentiation was further investigated by examining the effects of adenosine and alpha1-adrenergic receptor agonists on cytosolic Ca2+ in cultured striatal astrocytes from the embryonic mouse in primary culture. When used alone, methoxamine, a selective agonist of alpha-adrenergic receptors or 2-chloroadenosine, a stable analogue of adenosine, induced a transitory increase in cytosolic Ca2+, but their combined addition led to a sustained increase in cytosolic Ca2+, which seems to be due to a Ca2+ influx, because it was not observed in the absence of external Ca2+. Voltage independent Ca2+ channels contribute to this process and different blockers of voltage-operated calcium channels, such as dihydropyridines, phenylalkylamines, La3+ or Co2+ were ineffective in suppressing the sustained cytosolic Ca2+ elevation. Three observations suggest the implication of arachidonic acid in the observed potentiation: (i) arachidonic acid induced a sustained elevation of cytosolic Ca2+ similar to that evoked by the coapplication of methoxamine and 2-chloroadenosine; (ii) the addition of arachidonic acid during the calcic plateau produced by the combined application of the agonists did not increase further cytosolic Ca2+ levels; (iii) in the presence of methoxamine, 2-chloroadenosine induced a release of arachidonic acid. The stimulation of phospholipase C and the resulting activation of protein kinase C induced by methoxamine seem to be required for the potentiating effect of 2-chloroadenosine on cytosolic Ca2+. In fact, the direct activation of protein kinase C by an exogenous diacylglycerol analogue mimicked the effect of methoxamine because, in this condition, 2-chloroadenosine alone evoked a sustained elevation of cytosolic Ca2+. Therefore, methoxamine, through the successive activation of phospholipase C and protein kinase C, could allow a lipase, probably phospholipase A2, to be stimulated by 2-chloroadenosine. Arachidonic acid has already been shown to trigger the opening of K+ channels and the formation of inositol phosphates in other cell types. Therefore, in striatal astrocytes, 2-chloroadenosine, through an arachidonic acid-mediated hyperpolarization, could increase the Ca2+ driving force and thus improve Ca2+ influx through inositol phosphate-gated channels. This hypothesis is further supported by the suppressing effect of a 50 mM KCI-induced depolarization on the long lasting elevation of cytosolic Ca2+ seen in the combined presence of 2-chloroadenosine and methoxamine.     

Uptake of the daunorubicin-DNA complex in cultured fibroblasts

Summary The uptake and fate of the daunorubicin-DNA complex have been studied in cultured rat embryo fibroblasts. ¹²⁵I-DNA was digested by the cells and appeared as low molecular weight fragments in the incubation medium. Subcellular fractionation of fibroblasts, previously incubated with the complex, showed that daunorubicin was localized to nuclei and lysosomes. At a high incubation concentration (17.5 M), the accumulation of the free drug exceeded that of the complex. However, at a lower concentration (1.75 M), the accumulation of the complex was as high as that of the free drug.The results are consistent with an uptake of the complex into cultured rat embryo fibroblasts by endocytosis. However, it cannot be excluded that the complex partly dissociates in the incubation medium, and that daunorubicin and DNA thereafter enter the cells separately.     

Duplicated exstrophy in one of identical twins

The characteristic of duplicated exstrophy is the presence of a patch of exstrophic bladder mucosa in the infraumbilical region with a low-set umbilicus, in addition to the normal bladder. Musculoskeletal defects, diastasis pubis, and rectus abdominis can also be found in these patients. We report the first case of a female twin baby with appearance of a low-set umbilicus and diastasis pubis associated with a pubic sinus. The external genitalia and urinary continence were normal. Duplicated exstrophic mucosal remnant was excised, and the histopathologic study of the specimen confirmed urothelium.