Mitophagy is required for mitochondrial biogenesis and myogenic differentiation of C2C12 myoblasts

Myogenesis is a crucial process governing skeletal muscle development and homeostasis. Differentiation of primitive myoblasts into mature myotubes requires a metabolic switch to support the increased energetic demand of contractile muscle. Skeletal myoblasts specifically shift from a highly glycolytic state to relying predominantly on oxidative phosphorylation (OXPHOS) upon differentiation. We have found that this phenomenon requires dramatic remodeling of the mitochondrial network involving both mitochondrial clearance and biogenesis. During early myogenic differentiation, autophagy is robustly upregulated and this coincides with DNM1L/DRP1 (dynamin 1-like)-mediated fragmentation and subsequent removal of mitochondria via SQSTM1 (sequestosome 1)-mediated mitophagy. Mitochondria are then repopulated via PPARGC1A/PGC-1α (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha)-mediated biogenesis. Mitochondrial fusion protein OPA1 (optic atrophy 1 [autosomal dominant]) is then briskly upregulated, resulting in the reformation of mitochondrial networks. The final product is a myotube replete with new mitochondria. Respirometry reveals that the constituents of these newly established mitochondrial networks are better primed for OXPHOS and are more tightly coupled than those in myoblasts. Additionally, we have found that suppressing autophagy with various inhibitors during differentiation interferes with myogenic differentiation. Together these data highlight the integral role of autophagy and mitophagy in myogenic differentiation.     

A petal‐specific InMYB1 promoter from Japanese morning glory: a useful tool for molecular breeding of floricultural crops

Production of novel transgenic floricultural crops with altered petal properties requires transgenes that confer a useful trait and petal‐specific promoters. Several promoters have been shown to control transgenes in petals. However, all suffer from inherent drawbacks such as low petal specificity and restricted activity during the flowering stage. In addition, the promoters were not examined for their ability to confer petal‐specific expression in a wide range of plant species. Here, we report the promoter of InMYB1 from Japanese morning glory as a novel petal‐specific promoter for molecular breeding of floricultural crops. First, we produced stable InMYB1_1kb::GUS transgenic Arabidopsis and Eustoma plants and characterized spatial and temporal expression patterns under the control of the InMYB1 promoter by histochemical β‐glucuronidase (GUS) staining. GUS staining patterns were observed only in petals. This result showed that the InMYB1 promoter functions as a petal‐specific promoter. Second, we transiently introduced the InMYB1_1 kb::GUS construct into Eustoma, chrysanthemum, carnation, Japanese gentian, stock, rose, dendrobium and lily petals by particle bombardment. GUS staining spots were observed in Eustoma, chrysanthemum, carnation, Japanese gentian and stock. These results showed that the InMYB1 promoter functions in most dicots. Third, to show the InMYB1 promoter utility in molecular breeding, a MIXTA‐like gene function was suppressed or enhanced under the control of InMYB1 promoter in Arabidopsis. The transgenic plant showed a conspicuous morphological change only in the form of wrinkled petals. Based on these results, the InMYB1 promoter can be used as a petal‐specific promoter in molecular breeding of floricultural crops.     

Cell wall structure and biogenesis in Aspergillus species

Aspergillus species are among the most important filamentous fungi from the viewpoints of industry, pathogenesis, and mycotoxin production. Fungal cells are exposed to a variety of environmental stimuli, including changes in osmolality, temperature, and pH, which create stresses that primarily act on fungal cell walls. In addition, fungal cell walls are the first interactions with host cells in either human or plants. Thus, understanding cell wall structure and the mechanism of their biogenesis is important for the industrial, medical, and agricultural fields. Here, we provide a systematic review of fungal cell wall structure and recent findings regarding the cell wall integrity signaling pathways in aspergilli. This accumulated knowledge will be useful for understanding and improving the use of industrial aspergilli fermentation processes as well as treatments for some fungal infections.     

Septicemia from Lactobacillus rhamnosus GG,from a Probiotic Enriched Yogurt,in a Patient with Autologous Stem Cell Transplantation

Probiotics and Antimicrobial Proteins - Probiotic-rich foods are consumed without much restriction. We report here, a case of septic shock caused by yogurt derived Lactobacillus species in a...     

The extremely conserved C-terminal region of Reelin is not necessary for secretion but is required for efficient activation of downstream signaling

Reelin is a very large secreted glycoprotein essential for correct development of the mammalian brain. It is also implicated in higher functions and diseases of human brain. However, whether or not secretion of Reelin is regulated and how Reelin transmits signals remain largely unknown. Reelin protein is composed of an N-terminal F-spondin-like domain, Reelin repeats, and a short and highly basic C-terminal region (CTR). The primary sequence of CTR is almost completely conserved among vertebrates except fishes, indicating its importance. A prevailing idea regarding the function of CTR is that it is required for the secretion of Reelin, although this remains unproven. Here we aimed to clarify the function of Reelin CTR. Neither deleting most of CTR nor replacing CTR with unrelated amino acids affected secretion efficiency, indicating that CTR is not absolutely required for the secretion of Reelin. We also found that Reelin mutants without CTR were less potent in activating the downstream signaling in cortical neurons. Although these mutants were able to bind to the Reelin receptor ectodomain as efficiently as wild-type Reelin, quite interestingly, their ability to bind to the isolated cell membrane bearing Reelin receptors or receptor-expressing cells (including cortical neurons) was much weaker than that of wild-type Reelin. Therefore, it is concluded that the CTR of Reelin is not essential for its secretion but is required for efficient activation of downstream signaling events, presumably via binding to an unidentified "co-receptor" molecule(s) on the cell membrane.     

The telomerase activities in several organs and strains of rats with ageing

Telomerase activity is known to be implicated both in cell immortalization and carcinogenesis. Telomerase activity has not been detected in most human somatic tissues. However, we previously confirmed that the activity is present both in methylazoxymethanol acetate-induced rat colonic adenocarcinoma and non-treated colonic mucosa, presumably indicating the tissue-specific activity of the enzyme in rats. To determine the standard activity of rat telomerase in various organs in relation to differences in sex, age and strain, we examined the activity by using the telomeric repeat amplification protocol (TRAP) assay. The testis, liver, and colon mucosa showed the activity. The brain had very low or negative activity in 5-week-old male rats of the F344, SD, Wistar, Donryu or ACI strains. Age (5-week-old and 9-month-old) or sex difference for the activity was not apparent in rats of these strains. In general, telomerase activity in the fetal brain, liver and kidney was stronger than in the adult organ. The telomerase activity of each organ was different from that of human. This difference may indicate that the rat has a specific mechanism for maintaining the telomeric repeats of the chromosome even in somatic tissues. The basic information resulting from this study may be useful for the study of the role of telomerase in tumorigenesis in animal experiment models.     

The pro-peptide of Streptomyces mobaraensis transglutaminase functions in cis and in trans to mediate efficient secretion of active enzyme from methylotrophic yeasts

Transglutaminase (TGase) from the actinomycete Streptomyces mobaraensis is a useful enzyme in the food industry, and development of an efficient production system for it would be desirable. Herein we report secretion of TGase in an enzymatically active form by methylotrophic yeasts as expression hosts. Secretory production of active TGase required a pro-peptide from TGase. When an artificial Kex2-endopeptidase recognition site was placed between the pro-peptide and mature TGase, secretion and in vitro maturation of TGase depended on Kex2-dependent cleavage. Unexpectedly, coexpression of unlinked pro-peptide with mature TGase yielded efficient secretion of the active enzyme. These results indicate that the pro-peptide from TGase functions not only in an intramolecular but also in an intermolecular manner. Site-directed mutagenesis of putative N-glycosylation sites increased the productivity of the active TGase further. A recombinant Candida boidinii strain was found to secrete active TGase up to 1.83 U/ml (about 90 mg/l) after 119 h of cultivation.     

Fungicide activity through activation of a fungal signalling pathway

Fungicides generally inhibit enzymatic reactions involved in fungal cellular biosynthesis. Here we report, for the first time, an example of fungicidal effects through hyperactivation of a fungal signal transduction pathway. The OSC1 gene, encoding a MAP kinase (MAPK) related to yeast Hog1, was isolated from the fungal pathogen Colletotrichum lagenarium that causes cucumber anthracnose. The osc1 knockout mutants were sensitive to high osmotic stress and showed increased resistance to the fungicide fludioxonil, indicating that Osc1 is involved in responses to hyperosmotic stress and sensitivity to fludioxonil. The Osc1 MAPK is phosphorylated under high osmotic conditions, indicating activation of Osc1 by high osmotic stress. Importantly, fludioxonil treatment also activates phosphorylation of Osc1, suggesting that improper activation of Osc1 by fludioxonil has negative effects on fungal growth. In the presence of fludioxonil, the wild-type fungus was not able to infect the host plant because of a failure of appressorium-mediated penetration, whereas osc1 mutants successfully infected plants. Analysis using a OSC1-GFP fusion gene indicated that Osc1 is rapidly translocated to the nucleus in appressorial cells after the addition of fludioxonil, suggesting that fludioxonil impairs the function of infection structures by activation of Osc1. Furthermore, fludioxonil activates Hog1-type MAPKs in the plant pathogenic fungi Cochliobolus heterostrophus and Botrytis cinerea. These results strongly suggest that fludioxonil acts as a fungicide, in part, through activation of the MAPK cascade in fungal pathogens.