Search

Organoids mimicking the formation of the brain cortex have been demonstrated to be powerful tools for developmental studies as well as pathological investigations of brain malformations. Here, we report an integrated approach for the quantification of temporal neural production (neurogenic rate) in organoids derived from embryonic brains. Spherical tissue fragments with polarized cytoarchitectures were incubated in multiple cavities arranged in a polymethylmethacrylate chip. The time-dependent neurogenic rate in the organoids was monitored by the level of EGFP under the promoter of Tbr2, a transcription factor that is transiently expressed in neural fate-committed progenitors during corticogenesis. Importantly, our monitoring system exhibited a quick response to DAPT, a drug that promotes neural differentiation. Furthermore, we successfully quantified the temporal neurogenic rate in a large number of organoids by applying image processing that semi-automatically recognized the positions of organoids and measured their signal intensities from sequential images. Taken together, we provide a strategy to quantitate the neurogenic rate in brain organoids in a time-dependent manner, which will also be a potent method for monitoring organoid formation and drug activity in other tissue types., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Aurora-A kinase (AurA) is a key regulator of cellular processes involving microtubules. It has also been implicated in actin-dependent events, but the mechanisms that underlie the processes are not fully understood. Here we provide genetic and biochemical evidence suggesting that AurA negatively regulates Drok, the only known Rho-kinase orthologue in Drosophila. AurA directly phosphorylates Drok in vitro, and the overexpression of the nonphosphorylatable forms of Drok in vivo causes similar, but much stronger effects than that of wild-type Drok. The defects induced by the nonphosphorylatable forms of Drok are compensated by reducing the function of myosin downstream. Thus, phosphorylation of Drok by AurA normally suppresses Drok activity. We propose that AurA directly regulates actin-dependent processes by phosphorylating Rho-kinase., (Copyright © 2013 The authors. Published by Elsevier Inc. All rights reserved.)

Drosophila neuroblasts provide an excellent model for asymmetric cell divisions, where cell-fate determinants such as Miranda localize at the basal cortex and segregate to one daughter cell. Mechanisms underlying this process, however, remain elusive. We found that Mo25 and the GC kinase Fray act in this regulation. mo25 and fray mutants show an indistinguishable defect in Miranda localization. On the other hand, Drosophila Mo25 interacts with the tumor suppressor kinase Lkb1 in vivo, as have shown in mammals. Overexpression of Lkb1, which accumulates in the cell cortex, drastically relocalizes both Mo25 and Fray from the cytoplasm to the cortex, causing the same phenotype as mo25-mutant neuroblasts. Recovery from this defect caused by Lkb1 overexpression requires simultaneous overexpression of Mo25 and Fray. We suggest from those results that Mo25 and Fray operate together or in the same pathway in Drosophila asymmetric processes, and that their function counterbalances Lkb1.

We cloned full-length cDNA (PcCYP1f) encoding one of the cytochrome P450s in the lignin-degrading basidiomycete Phanerochaete chrysosporium, which showed high homology to P450s in the CYP53 family. PcCYP1f was expressed as an active microsomal protein using the methylotrophic yeast Pichia pastoris expression system. Using the microsomal fraction containing PcCYP1f, a typical P450 CO-difference spectrum was obtained with absorption maximum at 448nm. Recombinant PcCYP1f catalyzed the hydroxylation of benzoic acid into 4-hydroxybenzoic acid in the presence of NADPH and P. chrysosporium cytochrome P450 oxidoreductase. In contrast to other CYP53 P450s, this enzyme was shown to catalyze the hydroxylation of 3-hydroxybenzoate into 3,4-dihydroxybenzoate. Furthermore, 2- and 3-methylbenzoate were also shown to be substrates of PcCYP1f. This is the first report showing the expression of a functionally active Phanerochaete P450. Finally, real-time quantitative PCR analysis revealed that PcCYP1f is induced at a transcriptional level by exogenous addition of benzoic acid.

The functional diversity of cytochrome P450s (P450s) of the white-rot basidiomycete, Phanerochaete chrysosporium, was studied. A series of compounds known to be P450 substrates of other organisms were utilized for metabolic studies of P. chrysosporium. Metabolic conversions of benzoic acid, camphor, 1,8-cineol, cinnamic acid, p-coumaric acid, coumarin, cumene, 1,12-dodecanediol, 1-dodecanol, 4-ethoxybenzoic acid, and 7-ethoxycoumarin were observed with P. chrysosporium for the first time. 1-Dodecanol was hydroxylated at seven different positions to form 1,12-, 1,11-, 1,10-, 1,9-, 1,8-, 1,7-, and 1,6-dodecandiols. The effect of piperonyl butoxide, a P450 inhibitor, on the fungal conversion of 1-dodecanol was also investigated, indicating that hydroxylation reactions of 1-dodecanol were inhibited by piperonyl butoxide in a concentration-dependent manner. With 11 substrates, 23 hydroxylation reactions and 2 deethylation reactions were determined and 6 products were new with the position of hydroxyl group incorporated. In conclusion, fungal P450s were shown to have diverse and unique functions.

A homeobox gene, defective proventriculus (dve), is expressed in various tissues including the ventral ectoderm and midgut. Here, we show the expression pattern of dve in the ventral ectoderm, in which dve expression is induced by Spitz, a ligand for Drosophila epidermal growth factor receptor (EGFR). In spitz mutants, dve expression is only lost in the ventral ectoderm and overexpression of Spitz induces ectopic dve activation in the ventral ectoderm. Dve expression in the middle midgut depends on Decapentaplegic (Dpp) signaling, while expression of a dominant-negative form of Drosophila EGFR (DER(DN)) also causes a marked decrease in dve expression in the middle midgut. Furthermore, heterozygous mutation of thick veins (tkv), a Dpp receptor, strongly enhances the effect of DER(DN). These results indicate that EGFR signaling is crucial for dve expression in the ventral ectoderm and is required in the middle midgut where it cooperates with Dpp signaling.

Prostaglandins (PGs), hepatocyte growth factor (HGF), and induction of cyclooxygenase (PG synthetase, COX) play important roles in the repair process of gastric mucosa. We hypothesized that nonsteroidal anti-inflammatory drugs (NSAIDs), including indomethacin (IND), retard the healing of ulcers by suppressing these factors. In this study, we investigated the effects of cytokines, growth factors, and IND on production of PG and HGF, and induction of COX using cultured human gastric fibroblasts. Exogenous PGs significantly increased HGF production in a dose-dependent manner. Among various potential stimulants tested, interleukin-1 beta (IL-1 beta) dramatically increased PGE2 production and significantly stimulated HGF production. IL-1 beta induced COX-2 but not COX-1 protein. IND significantly reduced both basal and IL-1 beta-induced PGE2 release and HGF production. These results suggest that the IL-1 beta-PG-HGF pathway plays a role in the repair process of gastric mucosa. Further, NSAIDs may delay the healing of gastric mucosal ulcer, in part through suppression of HGF expression via inhibition of endogenous PG production.

The regulation of cyclin D-dependent kinase activity in tissue regeneration in vivo has not been fully described. In young adult rat liver after 70% partial hepatectomy, the association of cyclin D1 with cdk4 was significantly promoted during G1 phase and was maximal at 18 hr, corresponding mainly to late G1. Cyclin D1-dependent kinase activity also strongly increased during G1 phase. The timing of the induction of cyclin D1 / cdk4 complex assembly correlated well with that of cyclin D1-dependent kinase activity. At 18 hr after partial hepatectomy, the amounts of CDK inhibitors p21(CIP1) and p27(KIP1) were also maximal, while only one-tenth of p21(CIP1) and of p27(KIP1) was associated with cyclin D1. These findings suggest that cyclin D1, cdk4 and their association act as promoting factors, and that both p21(CIP1) and p27(KIP1) may have physiological functions as adaptor proteins in additions to their roles as CDK inhibitors in rat liver regeneration., (Copyright 1998 Academic Press.)

The Drosophila central nervous system comprises an enormous diversity of neurons that are originated from neuronal stem cells, neuroblasts. They generate a specific series of ganglion mother cells, each of which is once cleaved into a pair of neurons. Among genes known to control neurogenesis, prospero (pros) was recently identified as a gene required for gene expression specifying properties of some identified neurons. Here we report that pros encodes a nuclear protein containing a homeodomain-like sequence. In neuronal lineages of the central nervous system, pros protein is specifically detected in ganglion mother cells, although their parental neuroblasts have begun expressing a significant level of pros transcripts, suggesting a post-transcriptional control of pros expression. Our results provoke that in neuronal cell differentiation ganglion mother cells might play a pivotal role associating with the pros function.