Diagnostic anoctamin-5 protein defect in patients with ANO5-mutated muscular dystrophy.

AIMS: Previously, detection of ANO5 protein has been complicated by unspecific antibodies, most of which have not identified the correct protein. The aims of the study were to specify ANO5 protein expression in human skeletal muscle, and to investigate if the ANO5 protein levels are affected by different ANO5 mutations in anoctaminopathy patients. METHODS: Four different antibodies were tested for ANO5 specificity. A sample preparation method compatible with membrane proteins, combined with tissue fractionation was used to determine ANO5 expression in cell cultures expressing ANO5, in normal muscles and eight patient biopsies with six different ANO5 mutations in homozygous or compound heterozygous states, and in other dystrophies. RESULTS: Only one specific monoclonal N-terminal ANO5 antibody was efficient in detecting the protein, showing that ANO5 is expressed as a single 107 kD polypeptide in human skeletal muscle. The truncating mutations c.191dupA and c.1261C>T were found to abolish ANO5 expression, whereas the studied point mutations had variable effects; however, all the ANO5 mutations resulted in clearly reduced ANO5 expression in the patient muscle membrane fraction. Attempts to detect ANO5 using immunohistochemistry were not yet successful. CONCLUSIONS: The data presented here indicate that the ANO5 protein expression is decreased in ANO5-mutated muscular dystrophy and that most of the non-truncating pathogenic ANO5 mutations likely destabilize the protein and cause its degradation. The method described here allows direct analysis of human ANO5 protein, which can be used in diagnostics, for evaluating the pathogenicity of the potentially harmful ANO5 variants of uncertain significance.

Serum squalene and noncholesterol sterols before and after delivery in normal and cholestatic pregnancy.

Mechanisms of hyperlipidemia were studied by measurement of serum lipid concentrations and the ratios of cholesterol precursors (squalene, delta 8-cholestenol, desmosterol, and lathosterol), plant sterols (campesterol and sitosterol), and cholestanol (a 5 alpha-derivative of cholesterol) to cholesterol in nonpregnant women, and normal and cholestatic pregnancies near term, and a few days and 6 weeks after delivery. The ratios of the precursors are known to reflect cholesterol synthesis, those of plant sterols and cholestanol the absorption efficiency and biliary sterol secretion of cholesterol. In normal pregnancy, increased serum cholesterol was associated with up to 2-fold increases in squalene, desmosterol, and lathosterol proportions, and the values remained elevated, especially for desmosterol, during the lactation period. These findings suggest that pregnancy and lactation are associated with increased cholesterol synthesis. The proportions of plant sterols were slightly lower, but that of cholestanol was 2-fold that of the nonpregnant women. In contrast to the latter group, the cholestanol proportions were not related to those of plant sterols or the campesterol/sitosterol ratio. The values, especially of cholestanol, became normal during lactation. In cholestatic pregnancy the changes were basically similar, but the serum values of delta 8-cholestenol increased more, and those of squalene, desmosterol and lathosterol less markedly, and the mean cholestanol proportion was 40% higher and the campesterol/sitosterol ratio 15% lower than in the normal pregnancy. Cholestanol was positively related to serum bilirubin and bile acids in cholestatic pregnancy, yet only one-third of the cholestatic pregnant women exhibited cholestanol values higher than in the healthy pregnant women.

Immunolocalization of retinoic acid receptors in rat, mouse and human ovary and uterus.

We raised an antibody against a synthetic peptide corresponding to amino acids 155-174 of human retinoic acid receptor alpha (RAR-alpha). The sequence is highly homologous in all RARs and their isoforms. When mouse and human RARs (alpha, beta and gamma) expressed in Cos cell were analysed with immunoblot, all receptors gave a specific 51 K signal. Mouse RAR-gamma gave an additional signal corresponding to 58 K. In human teratocarcinoma cells (F9) both 51 and 58K molecule sizes were detected. The RAR expression in F9 cells was slightly down-regulated in charcoal-stripped culture medium and returned to normal level after retinoic acid treatment. The 51 K protein was found in all ovarian and uterine samples, but the quantity of the 58 K protein varied in different species and organs, being highest in the mouse uterus and the rat and human ovary. Using immunohistochemistry the RARs were found in the nuclear compartment. In the rat uterus, positive immunoreaction was found mainly in the nuclei of epithelial, uterine glandular and stromal cells. In the rat ovary, positive reaction was found in the nuclei of germinal epithelial, follicular and stromal cells.

Characterization of human 1,25-dihydroxyvitamin D3 receptor anti-peptide antibodies.

Rabbit and chicken antibodies were raised against two peptides synthesized according to the structure of human 1,25-dihydroxyvitamin D3 receptor (hVDR): rabbit alpha hVDR-103 against the N-terminal amino acids 5-18 and alpha hVDR-104 against the amino acids 172-186 in the hinge region and chicken alpha hVDR-cab11 against the amino acids 172-186, respectively. The specificity of the antibodies was tested by peptide saturation, SDS-PAGE immunoblotting, gel shift assay and sucrose gradient centrifugation. Immunoblotting of a soluble extract (cytosol) from osteosarcoma cell line MG-63 showed a single band with an M(r) of about 48,000 and human intestine cytosol a broad band (50-63,000) for both antibodies. The antibodies recognized activated (3.2S) hVDR by shifting the centrifugation sedimentation profile to 5-6S. The antibodies showed nuclear immunostaining of unoccupied VDR in human osteosarcoma cells MG-63, U2-Os and SaOs-2. The immunoreaction could be saturated with the corresponding synthetic peptide. In immunoblot alpha hVDR-103 reacted with human and rat VDR, whereas alpha hVDR-104 recognized human VDR only. Similarly in immunohistochemistry, alpha hVDR-103 showed staining with hVDR and rVDR, whereas alpha hVDR-104 reacted only with hVDR. All antibodies recognized the native hVDR as verified with sucrose gradient centrifugation or immunoprecipitation but only alpha hVDR-103 and alpha hVDR-cab11 in gel shift assay of hVDR associated with the vitamin D-responsive element of human osteocalcin gene promoter.

The adrenergic mechanism of the haemodynamic effects of massive doses of hydrocortisone in controlled haemorrhagic shock in the dog.

Haemorrhagic shock was induced in anaesthetized dogs by bleeding them into a blood reservoir system. By adjusting the blood level of the reservoir at a certain distance over the heart level the mean arterial blood pressure was kept at 6.7 kPa (50 mmHg). As has been found earlier, massive doses of hydrocortisone (80-560 mg.kg-1 body weight) caused a dose-dependent decrease in the total peripheral resistance. The degree of vasodilation distinctly increased during concomitant alpha-receptor blockade induced by phenoxybenzamine. The beta-receptor blocking drug propranolol efficiently inhibited the vasodilation caused by hydrocortisone and phenoxybenzamine. The findings fit with the hypothesis that massive doses of hydrocortisone induce an increased stimulation of the adrenergic beta2-receptors of the vascular smooth muscles.

Haemodynamic effects of different corticosteroids in controlled haemorrhagic shock in the dog.

The haemodynamic effects of massive doses of hydrocortisone (80-320 mg.kg-1), methylprednisolone (4-32 mg.kg-1), betamethasone (1.6-12.8 mg.kg-1) and aldosterone (0.1-0.8 mg.kg-1) and the interaction with phenoxybenzamine and propranolol have been studied during controlled haemorrhagic shock in the anaesthetized dog. Hydrocortisone was the only steroid which showed any significant vasodilating ability when given alone. The alpha-receptor blocking agent phenoxybenzamine distinctly decreased the total peripheral resistance. The effect of the phenoxybenzamine was increased in combination with hydrocortisone or methylprednisolone, especially if the steroid was given as the first drug. The vasodilation found was efficiently abolished by the beta-receptor blocking agent propranolol. The ability of hydrocortisone or methylprednisolone to potentiate phenoxybenzamine was not shared by betamethasone or aldosterone. Thus, the haemodynamic effect of the steroid does not seem to be correlated to either a glucocorticoid nor a mineralocorticoid effect. It is suggested that the steroid effect studied is related to the ability of hydrocortisone or methylprednisolone to block the extra neuronal amine uptake which decreases the rate of elimination of the sympathetic transmitter from the vicinity of the adrenergic receptor of the vascular smooth muscle.

Haemodynamic effects of massive doses of hydrocortisone and the interaction with phenoxybenzamine in controlled haemorrhagic shock in the dog.

Haemorrhagic shock was produced in anaesthetized dogs by bleeding into a blood reservoir system. The blood level of the reservoir was adjusted at a level above the heart, corresponding to a mean arterial blood pressure of 6.7 kPa (50 mm Hg). The dogs were treated with hydrocortisone and the adrenergic alpha-receptor blocking agent phenoxybenzamine during the hypotension period. Hydrocortisone (80-160 mg kg-1) was found to induce vasodilation, which, however, was of a very small magnitude and was of short duration. Phenoxybenzamine given after hydrocortisone caused very pronounced vasodilation. Hydrocortisone (80 mg kg-1) given after phenoxybenzamine also showed a vasodilator activity, which seemed to be greater than that of the same dose of hydrocortisone given alone. Thus, the vasodilator action of hydrocortisone does not seem to be due to an alpha-receptor blockade of the drug. The vasodilator action of phenoxybenzamine given after hydrocortisone was greater than that of even higher doses of the drug given alone. From the present findings and the fact that corticosteroids are known to potentiate the sympathomimetic action of catecholamines, it is suggested that the hydrocortisone-induced potentiation of the vasodilation action of phenoxybenzamine found is related to an increased vascular adrenergic beta-receptor tone.