Changes in muscle lipid metabolism induced in vitro by 1,25-dihydroxy-vitamin D-3.

1,25-Dihydroxy-vitamin D-3 has been shown to increase phosphatidylcholine and decrease phosphatidylethanolamine levels in skeletal muscle in vitro. To elucidate the metabolic pathway and mechanism involved in the effect of the sterol, chick embryo myoblast and vitamin D-deficient chick soleus muscle cultures were treated with 1,25-dihydroxy-vitamin D-3 (5.0 X 10(-10)-3.6 X 10(-11) M, total concentration) for 12-14 h and 1 h, respectively, in the absence and presence of protein and RNA synthesis inhibitors. Lipids were then labelled with [3H]glycerol and [14C]acetate. A significant increase in phosphatidylcholine and triacylglycerol labelling and a decrease in phosphatidylethanolamine labelling were observed in response to the hormone. Cycloheximide suppressed these changes in both types of preparations. Puromycin and actinomycin D were also effective blockers in cultured muscle cells. In addition, double-labelling of control and 1,25-dihydroxy-vitamin D-3-treated myoblasts with [3H]choline and [14C]ethanolamine were performed. The sterol did not affect [3H]choline labelling of total cell lipid extracts and phosphatidylcholine. However, the total lipid fraction of treated cells was labelled to a greater extent with [14C]ethanolamine. In addition, an increased incorporation of this precursor into phosphatidylcholine accompanied by a proportional decrease in phosphatidylethanolamine could be shown in cells pretreated with the metabolite. These changes were abolished by cycloheximide and actinomycin D. The results suggest that 1,25-dihydroxy-vitamin D-3 stimulates methylation of phosphatidylethanolamine into phosphatidylcholine in myoblasts by a nuclear mechanism. The data are consistent with the presence in the cells of a receptor specific for the hormone.

Modulation of DNA synthesis in cultured muscle cells by 1,25-dihydroxyvitamin D-3.

Biphasic effects of 1,25-dihydroxyvitamin D-3 on DNA synthesis were shown in primary cultured (24 h) chick embryo myoblasts exposed to physiological concentrations of the hormone. The sterol stimulated [3H]thymidine incorporation into DNA in proliferating myoblasts, e.g., at early stages of culture prior to cell fusion or in high serum-treated cells. The opposite effects were observed during the subsequent stage of myoblast differentiation in low-serum media. The mitogenic effect of 1,25-dihydroxyvitamin D-3 was correlated with an increase in c-myc mRNA and a decrease in c-fos mRNA levels, whereas its inhibitory action on DNA synthesis was accompanied by increased myofibrillar and microsomal protein synthesis and an elevation of creatine kinase activity, the latter suggesting a stimulation of muscle cell differentiation by the sterol. These data are in agreement with the results of previous morphological studies. Treatment of myoblasts with the calcium ionophore X-537 A or the phorbol ester TPA caused only a transient stimulation of [3H]thymidine incorporation into DNA, which occurred earlier than the response elicited by 1,25-dihydroxyvitamin D-3, suggesting that changes in intracellular Ca2+ and kinase C activity are not major mediators of the hormone effects. A similar temporal profile of changes in calmodulin mRNA levels as that of [3H]thymidine incorporation into DNA was observed after treatment of myoblasts with the sterol, in accordance with the role of calmodulin in the regulation of cell proliferation. 1,25-dihydroxyvitamin D-3 may play a function in embryonic muscle growth and differentiation.

Effects of 1,25-dihydroxyvitamin D-3 on phospholipid metabolism in chick myoblasts.

1,25-Dihydroxyvitamin D-3 has been shown to increase phosphatidylcholine and decrease phosphatidylethanolamine levels of myoblasts. Recent studies have suggested that the metabolite stimulates the methylation of phosphatidylethanolamine into phosphatidylcholine. In addition, the sterol increases the arachidonate content of phosphatidylcholine. Experiments were carried out to identify the steps of muscle cell lipid metabolism affected by 1,25-dihydroxyvitamin D-3. Primary cultures of chick embryo myoblasts pretreated with physiological concentrations of 1,25-dihydroxyvitamin D-3 were labelled with [14C]ethanolamine. The sterol increased the incorporation of precursor into dimethylphosphatidylethanolamine and phosphatidylcholine, whereas it decreases the labelling of phosphatidylethanolamine. Prior treatment with cycloheximide and actinomycin D blocked these changes. 1,25-Dihydroxyvitamin D-3 also stimulated the incorporation of [14C]ethanolamine into CDP-ethanolamine. In addition, the sterol increased the incorporation of [3H]arachidonic acid into the phosphatidylcholine fraction but did not affect the incorporation of [14C]palmitic acid. The incorporation of labelled fatty acids into diacylglycerol was not changed by the sterol, whereas it stimulated incorporation of both precursors into triacylglycerol. The data indicate that 1,25-dihydroxyvitamin D-3 enhances the synthesis of phosphatidylcholine through a stimulation of de novo synthesis and methylation of phosphatidylethanolamine via a nuclear mechanism. The sterol may also increase the polyunsaturated fatty acid content of phosphatidylcholine by means of an activation of its deacylation-reacylation cycle.

The phospholipid and fatty acid composition of skeletal muscle cells during culture in the presence of vitamin D-3 metabolites.

The phospholipid and fatty acid composition of primary cultures (24 h) of chick embryo skeletal muscle myoblasts treated for 4-24 h with physiological concentrations of 1,25-dihydroxyvitamin D-3 and 25-hydroxyvitamin D-3 were analyzed. 25-Hydroxyvitamin D-3 did not alter the relative amounts of individual muscle cell phospholipids whereas 1,25-dihydroxyvitamin D-3 significantly increased phosphatidylcholine content, mainly at the expense of a decrease in phosphatidylethanolamine concentration. The increase in phosphatidylcholine occurred at a faster rate during the first 8 h than in the subsequent 8-24 h treatment period. A similar time course in 1,25-dihydroxyvitamin D3-dependent changes in myoblast calcium uptake has been observe. In addition, this metabolite markedly increased (100%) the arachidonate content of myoblast phosphatidylcholine near the fusion stage of the cells (24 h of treatment). The levels of docosahexaenoate, a minor polyunsaturated fatty acid, in phosphatidylcholine and phosphatidylethanolamine were also substantially elevated by 1,25-dihydroxyvitamin D-3. No significant changes in fatty acid composition in response to 25-hydroxyvitamin D-3 were observed. Modifications in phospholipids and polyunsaturated fatty acids may play a role in the effects of 1,25-dihydroxyvitamin D-3 on muscle cell calcium transport and differentiation.

Induction of specific proteins in cultured skeletal muscle cells by 1,25-dihydroxyvitamin D-3.

The presence in myoblasts of an intracellular receptor specific for 1,25-dihydroxyvitamin D-3 [1,25(OH)2D3) and 1,25(OH)2D3-dependent changes in myoblast Ca2+ transport and phospholipid metabolism which are suppressed by RNA and protein synthesis inhibitors have been shown. In agreement with these observations, incubation of chick embryo myoblasts, precultured for 24 h in a medium containing low levels of vitamin D-3 metabolites, with 1,25(OH)2D3 at conditions which induce maximum cell responses (10(-10) M, 24 h) markedly stimulated the incorporation of [3H]leucine into total cell proteins and this effect was abolished when sterol treatment was performed in the presence of cycloheximide or puromycin. To investigate whether 1,25(OH)2D3 selectively stimulates the de novo synthesis of muscle cell proteins, mixtures of myoblast proteins from control and sterol-treated cultures labelled with [14C]leucine and [3H]leucine, respectively, were separated by SDS-polyacrylamide gel electrophoresis and isoelectric focussing. Examination of 3H/14C ratios in gel fractions revealed that 1,25-(OH)2D3 stimulates the production of proteins of molecular masses (isoelectric points) of 9 kDa (4.1 and 8.5), 17 kDa (7.5), 30 kDa (7.2), 40 kDa (5.5), 55 kDa (4.5) and 100 kDa (8.6). Cell fractionation studies showed the following subcellular distribution: 9 kDa (85% cytosol, 15% microsomes); 17 and 100 kDa (100%, 1200 X g pellet); 30 kDa (65% cytosol, 35% mitochondria); 40 kDa (100% microsomes); 55 kDa (65% microsomes, 35% mitochondria). Marker enzyme data indicated that this distribution is not due to cross-contamination between fractions. Affinity chromatography of double-labelled myoblast proteins on an immobilized lectin showed that the 55 kDa protein contains carbohydrate. Labelling of myoblast proteins with 45CaCl2 after their separation on SDS-polyacrylamide gels showed in addition that the 1,25(OH)2D3-dependent proteins of 9, 17, 40 and 100 kDa are major Ca2+-binding components of the cells. Synthesis of these proteins may mediate the effects of the sterol on myoblast calcium metabolism.

Stimulation of calmodulin synthesis in proliferating myoblasts by 1,25-dihydroxy-vitamin D3.

Myoblasts contain a receptor specific for 1,25-dihydroxy-vitamin D3. Morphological data have indicated that the hormone stimulates both myoblast proliferation and fusion. The synthesis of myoblast proteins in response to the sterol was studied during the proliferating stage of the cells. Chick embryo myoblast primary cultures (precultured for 24 h in the presence of low levels of 1,25-dihydroxy-vitamin D3 after isolation) were used. Labelling (2 h) of cells incubated in the absence and presence of 1,25-dihydroxy-vitamin D3 (10(-10) M) for 1-12 h with [14C]leucine and [3H]leucine, respectively, followed by coelectrophoresis of double-labelled proteins on sodium dodecyl sulfate polyacrylamide gels showed that the sterol initially stimulates the synthesis of proteins of 60 kDa (1.2 h), 70 kDa (2.4 h) and 80 kDa (4 h). These changes were transient and between 6 and 12 h a protein of 19 kDa was induced. This protein was identified as calmodulin on the basis of its isoelectric point (pI 4.1), Ca2(+)-dependent electrophoretic mobility, ability to bind 45Ca and to interact with an immobilized phenothiazine in a Ca2(+)-dependent manner, and by means of immunoblotting with a specific anti-calmodulin antibody and 3',5'-cyclic AMP phosphodiesterase activation assays. In agreement with these results, hybridization analysis with a specific cDNA probe showed increased calmodulin mRNA levels in myoblasts treated for 4-12 h with 1,25-dihydroxy-vitamin D3. These changes were paralleled by a stimulation of [3H]thymidine incorporation into DNA suggesting that they may be involved in the mitogenic action of the hormone.

Involvement of the 3',5'-cyclic AMP pathway in the induction of calmodulin synthesis in myoblasts by 1,25(OH)2-vitamin D3.

The participation of second messenger pathways in 1,25(OH)2D3-induced stimulation of protein synthesis in chick embryo myoblasts undergoing proliferation was studied. Double-labelling experiments with 14C- and 3H-leucine showed the induction by the hormone of proteins with apparent molecular masses (treatment interval) of 60 kDa (1 to 2 h). 70 kDa (2 to 4 h), 80 kDa (4 h) and a 19 kDa protein (6 to 12 h) previously identified as calmodulin. The PKC activator TPA and the Ca2+ ionophore X-537 A did not mimic the effects of the sterol on protein synthesis whereas similar double-labelling patterns were obtained with forskolin, an adenylate cyclase activator. Dot-blot and Northern hybridization analysis revealed increased calmodulin mRNA levels in response to both the hormone and forskolin. These results involve the cAMP messenger system in 1,25(OH)2D3 stimulation of calmodulin synthesis and may be relevant to understand hormone regulation of muscle cell proliferation.

The interactivity between the CFTR gene and cystic fibrosis would be limited to the initial phase of the disease.

PURPOSE: Cystic fibrosis (CF) is a lethal genetic disorder affecting secretory epithelia, caused by mutations on the CFTR gene. In this paper we study the interactivity between the CFTR gene and CF disease over the time course of CF. METHOD: Cross-sectional analysis of CF patient population data from Latin-America, Canada, and The Netherlands, under the assumption that they represent stationary populations, was used to determined and correlates hazard rates, average cores and CF progression rates. RESULTS: Results suggests the existence of two phases throughout the course of CF. CONCLUSION: While the initial phase was related to the CFTR genotype, the kinetics of the second phase seems to be common to all groups considered. The hypothesis that the interactivity between the CFTR gene and CF disease would be limited in time is presented, suggesting that mutant CFTR would trigger a disease that evolves to become independent from the CFTR gene itself.

High throughput production, screening and analysis of adeno-associated viral vectors.

Recombinant adeno-associated viruses (rAAV) are promising candidates as gene vectors, as they transduce non-dividing cells and permit lasting transgene expression in a wide spectrum of tissues. In this paper, we describe a robust procedure for the high throughput production, screening and characterization of rAAV vectors. The technology includes the production of rAAV from rapid small scale plasmid preparations and the analysis of virus productivity (physical and infectious particles) and activity (transgene expression, replication). rAAV are produced by triple transfection (rAAV plasmid and AAV- and adenovirus (Ad)-helper plasmids) on 293 human embryo kidney (HEK) cells. The titers of physical and infectious particles are obtained by dot blot hybridization and by a serial dilution assay, followed by either dot blot hybridization or real-time PCR, respectively. rAAV can be produced and characterized from plasmid mixtures containing as little as 1/100 productive molecules. Experiments on rAAV replication kinetics and Ad helper functions are discussed. All steps are performed in 96-well microtiter plates. The process is reproducible, high throughput, linear and ready for automation.

Optimised helper virus-free production of high-quality adeno-associated virus vectors.

BACKGROUND: Clinical development of adeno-associated virus (AAV) requires standardised, safe, efficient and scalable procedures for the manufacture of the rAAV vector, including production, purification and testing. Several strategies have been reported for the approach to the manufacturing problem. We report a helper virus-free process that produces high quality rAAV stocks. METHODS: rAAV were produced by triple transfection, a helper virus-free process. After lysis of the cells in the presence of nuclease, the rAAV produced were purified by HPLC through two ion-exchange columns in tandem followed by dialysis. rAAV stocks were thoroughly characterised for biological activity and for the presence of residual contaminants. The titer of infectious particles and of rep + particles was determined by dRA assay. Contaminating DNA and RNA were determined by fluorescent dye binding and real-time PCR. The protein content of the rAAV stocks was characterised by SDS-PAGE, ELISA test, Western blot and specific enzymatic assays for putative residual contaminating protein. The in vivo biological activity of the stocks was evaluated in mouse muscle. RESULTS: rAAV stocks obtained following this procedure elicit: 2-5 x 10(12) pp/ml; 3-6 x 10(10) ip/ml; < 10(3) rep + particles/ml; <0.3 mUeq/ml of residual benzonase activity; non-detectable Ad or beta-galactosidase proteins; <35 pg/ml of cellular genomic DNA; in vivo expression in mouse muscle without any immune reaction detected. CONCLUSIONS: This work demonstrates the possibility of producing purified high-quality rAAV free of helper virus. The procedure described in this paper is easily adaptable for large-scale production of clinical rAAV vectors.

Paraplejia espástica e insuficiencia suprarrenal primaria: un caso de adrenomieloneuropatia / Spastic paraplegia and primary adrenal insuficiency: a case of adrenomyeloneuropathy

Un varón de 40 años se internó por dificultad creciente para la marcha que había comenzado 2 años antes y que lo había reducido a una silla o a la cama. Hacía 7 años se le había diagnosticado enfermedad de Addison y tomaba regularmente 20 mg/día de hidrocortisona oral. Su padre, un tío paterno y 2 hermanas están sanos; un hermano afectado por oligofrenia, disartria y dificultad para caminar falleció a los 9 años de edad. A su ingreso, el paciente estaba lúcido y orientado,; tenía hiperpigmentaciín leve de piel y mucosas, alopecía en cuero cabeludo y cola de cejas, testículos de 3,5 ml y paraplejía espástica; los miembros inferiores conservaban la sensibilidad táctil y dolorosa. La radiografía de tórax y la reacción de Mantoux eran normales. Las pruebas hormonales de laboratorio confirmaron la insuficiencia suprarrenal primaria (con conservación de la función de la zona glomerulosa) y un hipogonadismo primario; la función tiroidea era normal. La velocidad de conducción motora en miembros inferiores era baja (30-32 m/seg), con aumento de las latencias proximal y distal; esto y el EMG eran compatibles con polineuropatía de tipo mielinopático. Se comprobó leve atrofia cortical difusa (TAC) de cráneo); los potenciales evocados auditivos de tronco cerebral mostraron baja amplitud del complejo IV-V y tiempo de conducción central prolongado (5,3 mseg). Los ácidos grasos séricos eran cuantitativa y cualitativamente normales por cromatografía gaseosa. En la biopsia de nervio safeno se observó desmielinización segmentaria y degeneración axonal, sin infiltrados inflamatorios; el estudio ultraestructural demostró, en el citoplasma de algunas células de Schwann, inclusiones bilaminares, en su mayoría curvalíneas, que confirmaron el diagnósticos clínico de adrenomieloneuropatía. Esta rara enfermedad familiar de transmisión recesiva ligada al cromosoma X se origina en un trastorno del metabolismo de los ácidos grasos saturados de cadena muy larga, con compromiso funcional del sistema nervioso y de células productoras de hormonas esteroides