MicroRNA-199a is induced in dystrophic muscle and affects WNT signaling, cell proliferation, and myogenic differentiation.

In patients with Duchenne muscular dystrophy (DMD), the absence of a functional dystrophin protein results in sarcolemmal instability, abnormal calcium signaling, cardiomyopathy, and skeletal muscle degeneration. Using the dystrophin-deficient sapje zebrafish model, we have identified microRNAs (miRNAs) that, in comparison to our previous findings in human DMD muscle biopsies, are uniquely dysregulated in dystrophic muscle across vertebrate species. MiR-199a-5p is dysregulated in dystrophin-deficient zebrafish, mdx(5cv) mice, and human muscle biopsies. MiR-199a-5p mature miRNA sequences are transcribed from stem loop precursor miRNAs that are found within the introns of the dynamin-2 and dynamin-3 loci. The miR-199a-2 stem loop precursor transcript that gives rise to the miR-199a-5p mature transcript was found to be elevated in human dystrophic muscle. The levels of expression of miR-199a-5p are regulated in a serum response factor (SRF)-dependent manner along with myocardin-related transcription factors. Inhibition of SRF-signaling reduces miR-199a-5p transcript levels during myogenic differentiation. Manipulation of miR-199a-5p expression in human primary myoblasts and myotubes resulted in dramatic changes in cellular size, proliferation, and differentiation. MiR-199a-5p targets several myogenic cell proliferation and differentiation regulatory factors within the WNT signaling pathway, including FZD4, JAG1, and WNT2. Overexpression of miR-199a-5p in the muscles of transgenic zebrafish resulted in abnormal myofiber disruption and sarcolemmal membrane detachment, pericardial edema, and lethality. Together, these studies identify miR-199a-5p as a potential regulator of myogenesis through suppression of WNT-signaling factors that act to balance myogenic cell proliferation and differentiation.

Primary collagen VI deficiency is the second most common congenital muscular dystrophy in Japan.

OBJECTIVES: To determine the frequency of primary collagen VI deficiency in congenital muscular dystrophy (CMD) in Japan and to establish the genotype-phenotype correlation. METHODS: We performed immunohistochemistry for collagen VI in muscles from 362 Japanese patients with CMD, and directly sequenced the three collagen VI genes, COL6A1, COL6A2, and COL6A3, in patients found to have collagen VI deficiency. RESULTS: In Japan, primary collagen VI deficiency accounts for 7.2% of congenital muscular deficiency. Among these patients, five had complete deficiency (CD) and 29 had sarcolemma-specific collagen VI deficiency (SSCD). We found two homozygous and three compound heterozygous mutations in COL6A2 and COL6A3 in all five patients with CD, and identified heterozygous missense mutations or in-frame small deletions in 21 patients with SSCD in the triple helical domain (THD) of COL6A1, COL6A2, and COL6A3. All mutations in SSCD were sporadic dominant. No genotype-phenotype correlation was seen. CONCLUSION: Primary collagen VI deficiency is the second most common CMD after Fukuyama type CMD in Japan. Dominant mutations located in the N-terminal side from the cysteine residue in the THD of COL6A1, COL6A2, and COL6A3 are closely associated with SSCD.

Reduced cell anchorage may cause sarcolemma-specific collagen VI deficiency in Ullrich disease.

BACKGROUND: COL6 gene mutations are associated with Ullrich congenital muscular dystrophy (UCMD), which is clinically characterized by muscle weakness from early infancy, hyperlaxity of distal joints, and multiple proximal joint contractures. We previously reported that the majority of patients with UCMD have sarcolemma-specific collagen VI deficiency (SSCD). More recently, we found heterozygous COL6A1 glycine substitutions in patients with UCMD with SSCD. OBJECTIVE: To elucidate how COL6A1 glycine mutation leads to SSCD. METHODS: We evaluated the synthesis, formation, and binding of collagen VI to the extracellular matrix in fibroblasts with p.G284R mutation in COL6A1. RESULTS: Collagen VI was normally secreted into the cultured medium in fibroblasts harboring p.G284R mutation. When the medium with normal collagen VI was added to collagen VI-deficient fibroblast culture, collagen VI bound surrounding the cells, while collagen VI with p.G284R mutation did not. Cell adhesion of fibroblasts with p.G284R mutation was markedly reduced similarly to that of collagen VI-deficient cells. Interestingly, this reduction in adhesion of the cells with p.G284R mutation was recovered by the addition of the medium with normal collagen VI, which would suggest a therapeutic strategy for a replacement therapy. CONCLUSION: Heterozygous glycine substitution in COL6A1 may cause decreased binding of collagen VI microfibrils to the extracellular matrix resulting in sarcolemma-specific collagen VI deficiency.

Occurrence of prohormone convertase-like substances in the neural complex cells of the ascidian Halocynthia roretzi.

Our previous study on the distribution of adrenocorticotropin (ACTH)-like substances in the neural complex (cerebral ganglion, dorsal strand, and neural gland) of an ascidian Halocynthia roretzi revealed that some of the cells in the cerebral ganglion and the cells scattered along the dorsal strand were immunopositive with antiserum against ACTH. In order to ascertain whether these cells are equipped with prohormone convertases, we performed immunohistochemical studies on the neural complex by using antisera against PC1 and PC2. A considerable number of cells around the dorsal strand and a few cells in the neural ganglion were immunopositive with PC1 and/or PC2 antibodies. Immunoelectron microscopic study demonstrated that some granulated cells situated in the cerebral ganglion and along the dorsal strand contained PC1- or PC2-like substances within their secretory granules. Western blot analysis revealed the presence of 66-kDa PC1-like and 70-kDa PC2-like substances in the neural complex. Moreover, immunostaining of consecutive sections showed that the majority of the cells containing PC1- and/or PC2-like substances corresponded to the cells immunoreactive with antisera against ACTH and CLIP but not to those immunoreactive with an antiserum against PRL. Cells belonging to the neural gland neither contained electron-dense granules nor showed immunoreactivity with any antisera employed in this experiment. The possibility that some of the cells situated in the cerebral ganglion and along the dorsal strand are progenitors of vertebrate adenohypophyseal cells is discussed.

Hypoglycemia associated with the production of insulin-like growth factor II in adrenocortical carcinoma.

A 78-year-old woman was hospitalized for congestive heart failure and repeated hypoglycemic attacks. The laboratory data showed a serum insulin level within the normal range and an increased level of serum insulin-like growth factor (IGF) II. Abdominal ultrasonogram and computed tomography scan revealed a huge mass lying above the left kidney. She was diagnosed as having an adrenocortical carcinoma. After the removal of the tumor, the plasma glucose level and the serum level of IGF-II were normalized. The tumor cells stained positively for IGF-II immunohistochemically. These findings suggested that the hypoglycemia was due to IGF-II produced by the adrenocortical carcinoma.

Effect of prolactin and androgen on the expression of the female-attracting pheromone silefrin in the abdominal gland of the newt, Cynops ensicauda.

Silefrin is a sodefrin-like, female-attracting pheromone comprising 10 amino acids that was isolated from the abdominal gland of the sword-tailed newt, Cynops ensicauda. Hormonal effects on the silefrin precursor mRNA expression and silefrin content in the abdominal gland were investigated in the present study by using Northern blot analysis and radioimmunoassay, respectively. In the abdominal gland of newts treated with prolactin (PRL) plus testosterone propionate (TP), silefrin precursor mRNA expression was markedly enhanced as compared with that in the newts injected with saline, PRL, or TP. Values for radioimmunoassayable silefrin content in the abdominal gland paralleled those for the silefrin precursor mRNA levels. Moreover, silefrin precursor mRNA signals, as revealed by in situ hybridization, as well as stainability of immunoreactive silefrin were much more intense in the epithelial cells of the abdominal gland of the PRL-plus-TP-treated animals than in those of controls. We thus conclude that PRL and androgen are important factors for enhancing silefrin synthesis.

Silefrin, a sodefrin-like pheromone in the abdominal gland of the sword-tailed newt, Cynops ensicauda.

Sodefrin-like female-attracting pheromone was purified from the abdominal glands of male sword-tailed newts, Cynops ensicauda, by gel-filtration chromatography and reversed-phase high-performance liquid chromatography. The final product comprises 10 amino acid residues with the sequence SILSKDAQLK which coincided with the sequence deduced from its precursor cDNA. This peptide was designated silefrin. The sequence of silefrin was different from that of sodefrin by two amino acid residues, with substitutions Leu for Pro and Gln for Leu at positions 3 and 8, respectively. Both native and synthetic silefrin exerted an equipotent activity in attracting conspecific females.