A new method for the preservation of fungus stock cultures by deep-freezing

Recovery of 66 fungus stock cultures including Oomycota, Zygomycota, Ascomycota, Basidiomycota, and mitosporic mycetes were examined after cryopreservation. Almost all the stock cultures remained viable when the mycelia that had grown over the sawdust medium containing 10% glycerol as the cryoprotectant (65% moisture content, W/W) were frozen rapidly at −85°C and then allow to thaw naturally at room temperature. Test stock cultures were preserved for more than 10 years by this preservation method without any programmed precooling and rapid thawing for their cryopreservation. Most of the test fungi could survive for 5 years in medium containing 10% glycerol even after alternate freezing and thawing at intervals of 6 months. When a strain of Flammulina velutipes was tested for mycelial growth rate and productivity of fruit-bodies after cryopreservation for 3 years, the fungus reproduced with its initial capability. These results demonstrate that the sawdust-freezing method using a cryoprotectant is expected to be a reliable and easy preservation method for fungus stock cultures. Received: December 7, 2000 / Accepted: December 19, 2001     

Exaggerated Nighttime Sleep and Defective Sleep Homeostasis in a Drosophila Knock-In Model of Human Epilepsy

Despite an established link between epilepsy and sleep behavior, it remains unclear how specific epileptogenic mutations affect sleep and subsequently influence seizure susceptibility. Recently, Sun et al. (2012) created a fly knock-in model of human generalized epilepsy with febrile seizures plus (GEFS+), a wide-spectrum disorder characterized by fever-associated seizing in childhood and lifelong affliction. GEFS+ flies carry a disease-causing mutation in their voltage-gated sodium channel (VGSC) gene and display semidominant heat-induced seizing, likely due to reduced GABAergic inhibitory activity at high temperature. Here, we show that at room temperature the GEFS+ mutation dominantly modifies sleep, with mutants exhibiting rapid sleep onset at dusk and increased nighttime sleep as compared to controls. These characteristics of GEFS+ sleep were observed regardless of sex, mating status, and genetic background. GEFS+ mutant sleep phenotypes were more resistant to pharmacologic reduction of GABA transmission by carbamazepine (CBZ) than controls, and were mitigated by reducing GABA_A receptor expression specifically in wake-promoting pigment dispersing factor (PDF) neurons. These findings are consistent with increased GABAergic transmission to PDF neurons being mainly responsible for the enhanced nighttime sleep of GEFS+ mutants. Additionally, analyses under other light conditions suggested that the GEFS+ mutation led to reduced buffering of behavioral responses to light on and off stimuli, which contributed to characteristic GEFS+ sleep phenotypes. We further found that GEFS+ mutants had normal circadian rhythms in free-running dark conditions. Interestingly, the mutants lacked a homeostatic rebound following mechanical sleep deprivation, and whereas deprivation treatment increased heat-induced seizure susceptibility in control flies, it unexpectedly reduced seizure activity in GEFS+ mutants. Our study has revealed the sleep architecture of a Drosophila VGSC mutant that harbors a human GEFS+ mutation, and provided unique insight into the relationship between sleep and epilepsy.     

SDF-1 alpha regulates mesendodermal cell migration during frog gastrulation

During frog gastrulation, mesendodermal cells become apposed to the blastocoel roof (BCR) by endoderm rotation, and migrate towards the animal pole. The leading edge of the mesendodermal cells (LEM) contributes to the directional migration of involuting marginal zone (IMZ) cells, but the molecular mechanism of this process is not well understood. Here we show that CXCR4/SDF-1 signaling mediates the directional movement of the LEM in Xenopus embryos. Expression of xCXCR4 was detected in the IMZ, and was complemented by xSDF-1alpha expression in the inner surface of the BCR. Over-expression of xCXCR4 and xSDF-1alpha caused gastrulation defects. An xCXCR4 N-terminus deletion construct and xSDF-1alpha-MO also inhibited gastrulation. Furthermore, explants of LEM migrate towards the dorsal BCR in the presence of xSDF-1alpha, and altered xCXCR4 expression in the LEM inhibited LEM migration. These results suggest that CXCR4/SDF-1 signaling is necessary for the migrations of massive numbers of cells during gastrulation.     

Effect of population density of compatible neighbours on inbreeding level within a Primula sieboldii population

We investigated the effect of population density of compatible neighbours on inbreeding level of Primula sieboldii, a heterostylous clonal herb. Pollinator availability, seed set, selfing rate, diversity of pollen donors, and fitness of progenies were compared between less and more isolated genets, which differed in the number of compatible opposite-morph genets within 20 m, the range at which most pollen flow occurred. Although pollinator availability did not differ between the two groups, seed set and diversity of pollen donors in more isolated genets were significantly lower than in less isolated genets. Additionally, the mean selfing rates of less and more isolated genets were 1.3 and 36.7%, respectively, and the mean leaf area of the self-fertilized seedlings was 70 to 40% smaller than that of outcrossed seedlings of the same mother genet. Due to this large inbreeding depression, it is unlikely that self-fertilized seedlings could successfully establish in natural habitats and hence the inbreeding level in the next generation around the more isolated genet would not increase rapidly. However, the possibility of mating between full-sibs would increase because the diversity of pollen donors was low and both pollen and seed dispersal were spatially restricted. Thus the inbreeding level of the next generations would gradually increase around the more isolated genets owing to biparental inbreeding. This study suggested that the population density of compatible neighbours has a critical impact on the future inbreeding level within P. sieboldii populations.     

Functional expression of the Aspergillus flavus PKS–NRPS hybrid CpaA involved in the biosynthesis of cyclopiazonic acid

α-Cyclopiazonic acid (CPA) is an indole tetramic acid mycotoxin. Based on our identification of the polyketide synthase–nonribosomal peptide synthase (PKS–NRPS) hybrid gene cpaA involved in cyclopiazonic acid biosynthesis in Aspergillus fungi, we carried out heterologous expression of Aspergillus flavus cpaA under α-amylase promoter in Aspergillus oryzae and identified its sole product to be the CPA biosynthetic intermediate cyclo-acetoacetyl-l-tryptophan (cAATrp). This result rationalized that the PKS–NRPS hybrid enzyme CpaA catalyzes condensation of the diketide acetoacetyl-ACP formed by the PKS module and l-Trp activated by the NRPS module. This CpaA expression system provides us an ideal platform for PKS–NRPS functional analysis, such as adenylation domain selectivity and product releasing mechanism.     

Macroautophagy-mediated degradation of whole nuclei in the filamentous fungus Aspergillus oryzae

Filamentous fungi consist of continuum of multinucleate cells called hyphae, and proliferate by means of hyphal tip growth. Accordingly, research interest has been focusing on hyphal tip cells, but little is known about basal cells in colony interior that do not directly contribute to proliferation. Here, we show that autophagy mediates degradation of basal cell components in the filamentous fungus Aspergillus oryzae. In basal cells, enhanced green fluorescent protein (EGFP)-labeled peroxisomes, mitochondria, and even nuclei were taken up into vacuoles in an autophagy-dependent manner. During this process, crescents of autophagosome precursors matured into ring-like autophagosomes to encircle apparently whole nuclei. The ring-like autophagosomes then disappeared, followed by dispersal of the nuclear material throughout the vacuoles, suggesting the autophagy-mediated degradation of whole nuclei. We also demonstrated that colony growth in a nutrient-depleted medium was significantly inhibited in the absence of functional autophagy. This is a first report describing autophagy-mediated degradation of whole nuclei, as well as suggesting a novel strategy of filamentous fungi to degrade components of existing hyphae for use as nutrients to support mycelial growth in order to counteract starvation.