Published papers

Background: It is becoming increasingly recognised that opioids are responsible for tumour growth. However, the effects of opioids on tumour growth have been controversial.Methods:The effects of-opioid receptor (KOR) agonist on the growth of non-small cell lung cancer (NSCLC) cells were assessed by a cell proliferation assay. Western blotting was performed to ascertain the mechanism by which treatment with KOR agonist suppresses tumour growth. Results: Addition of the selective KOR agonist U50,488H to gefitinib-sensitive (HCC827) and gefitinib-resistant (H1975) NSCLC cells produced a concentration-dependent decrease in their growth. These effects were abolished by co-treatment with the selective KOR antagonist nor-BNI. Furthermore, the growth-inhibitory effect of gefitinib in HCC827 cells was further enhanced by co-treatment with U50,488H. With regard to the inhibition of tumour growth, the addition of U50, 488H to H1975 cells produced a concentration-dependent decrease in phosphorylated- glycogen synthase kinase 3Β (p-GSK3Β).Conclusion:The present results showed that stimulation of KOR reduces the growth of gefitinib-resistant NSCLC cells through the activation of GSK3Β. © 2012 Cancer Research UK All rights reserved.

Glycosylation of proteins shows two striking features: first, there are a large number of glycan structures that are involved in various biological events (diversity) and, second, certain structures are selectively added to specific sites within proteins (specificity) in a context-dependent manner. However, the mechanisms that regulate diversity and specificity are unknown. Over the last few decades, genetic studies in Drosophila have been used to examine the functions mediated by glycosylation in vivo. Recent improvements in the combined use of biochemical/biophysical, genetic, and cell biology approaches has allowed the rapid and detailed analysis of the structure, function and formation of glycans in Drosophila. This chapter aims to provide an overview of the different types of glycans and their biological function(s) in Drosophila. The regulation of glycan synthesis in Drosophila and mammals is also discussed. © 2012 by Nova Science Publishers, Inc. All rights reserved.

Once the safety issue has been overcome, induced pluripotent stem cells (iPSCs), which do not entail ethical or immunological concerns, may become the preferred cell source for regenerative medicine. Various types of iPSCs have been established by different methods, and each type exhibits different biological properties. Before iPSC-based clinical applications can be initiated, detailed evaluations of the cells, including their differentiation potentials and tumorigenic activities in different contexts, should be investigated to establish their safety and effectiveness for cell transplantation therapies. Recently, we demonstrated the directed neural differentiation of mouse iPSCs and examined their therapeutic potential in a mouse spinal cord injury (SCI) model. Mouse iPSC-derived neural stem/progenitor cells (NS/PCs), which had been pre-evaluated as non-tumorigenic by their transplantation into nonobese diabetic-severe combined immunodeficiency (NOD-scid) mouse brain, were transplanted into the spinal cord 9 days after SCI. Mouse iPSC-derived NS/PCs differentiated into all three neural lineages without forming teratomas or other tumors. They also participated in re-myelination and induced the axonal re-growth, promoting motor functional recovery. Nevertheless, our results constitute only the first step toward clinical application. The safety and effectiveness of human iPSC-derived NS/PCs need to be more intensively investigated in future preclinical studies, for example, using non-human primate SCI models. In particular, human iPSCs established by delivering reprogramming factors using a safer method than retrovirus system, such as an integration-free virus system, virus-free system, or transgene-free system should be evaluated.

Examples of direct differentiation by defined transcription factors have been provided for beta-cells, cardiomyocytes and neurons. In the human visual system, there are four kinds of photoreceptors in the retina. Neural retina and iris-pigmented epithelium (IPE) share a common developmental origin, leading us to test whether human iris cells could differentiate to retinal neurons. We here define the transcription factor combinations that can determine human photoreceptor cell fate. Expression of rhodopsin, blue opsin and green/red opsin in induced photoreceptor cells were dependent on combinations of transcription factors: A combination of CRX and NEUROD induced rhodopsin and blue opsin, but did not induce green opsin; a combination of CRX and RX induced blue opsin and green/red opsin, but did not induce rhodopsin. Phototransduction-related genes as well as opsin genes were up-regulated in those cells. Functional analysis; i.e. patch clamp recordings, clearly revealed that generated photoreceptor cells, induced by CRX, RX and NEUROD, responded to light. The response was an inward current instead of the typical outward current. These data suggest that photosensitive photoreceptor cells can be generated by combinations of transcription factors. The combination of CRX and RX generate immature photoreceptors: and additional NEUROD promotes maturation. These findings contribute substantially to a major advance toward eventual cell-based therapy for retinal degenerative diseases.

The Golgi apparatus is an intracellular organelle playing central roles in post-translational modification and in the secretion of membrane and secretory proteins. These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the cis-, medial- and trans-cisternae of the Golgi. While trafficking through the Golgi, proteins are sequentially modified with glycan moieties by different glycosyltransferases. Therefore, it is important to analyze the glycosylation function of the Golgi at the level of cisternae. Markers widely used for cis-, medial- and trans-cisternae/trans Golgi network (TGN) in Drosophila are GM130, 120 kDa and Syntaxin16 (Syx16); however the anti-120 kDa antibody is no longer available. In the present study, Drosophila Golgi complex-localized glycoprotein-1 (dGLG1) was identified as an antigen recognized by the anti-120 kDa antibody. A monoclonal anti-dGLG1 antibody suitable for immunohistochemistry was raised in rat. Using these markers, the localization of glycosyltransferases and nucleotide-sugar transporters (NSTs) was studied at the cisternal level. Results showed that glycosyltransferases and NSTs involved in the same sugar modification are localized to the same cisternae. Furthermore, valuable functional information was obtained on the localization of novel NSTs with as yet incompletely characterized biochemical properties, © 2012 by Japan Society for Cell Biology.

Alzheimer's disease (AD) is the most common form of age-related dementia, characterized by progressive memory loss and cognitive disturbance. According to the amyloid cascade hypothesis, a prevailing theory of AD pathology, accumulation of toxic Aβ42, in the brain is the initiator of AD pathogenesis, subsequently leading to the formation of neurofibrillary tangles, and consequently neuronal loss. Mutations of presenilin 1 (PS1) and presenilin 2(PS2), which are catalytic components of γ-secretase, are causative factors for autosomal dominant early-onset familial AD (FAD). Induced pluripotent stem cell (iPSC) technology provides a new method for elucidating the molecular basis of human diseases, including neurodegenerative diseases. Here we generate iPSCs from fibroblasts of FAD patients with mutations in PS1 (A246E) and PS2 (N141I), and characterize the differentiation of these cells into neurons. We find that FAD-iPSC-derived differentiated neurons have increased toxic Aβ42 secretion, recapitulating the molecular pathogenesis of mutant presenilins. Furthermore, secretion of Aβ42 from these neurons sharply responds to γ secretase inhibitors and modulators, indicating the potential for identification and validation of candidate drugs. Our findings demonstrate that the FAD-iPSC-derived neuron is a valid model of AD and provides an innovative strategy for the study of late-onset neurodegenerative diseases.

Sensory hair cells (HCs) and their associated nonsensory supporting cells (SCs) exhibit a typical mosaic pattern in each of the sensory patches in the inner ear. Notch signaling has been considered to conduct the formation of this mosaic pattern through one of its famous functions, known as 'lateral inhibition'. The two Notch ligands Delta-like1 and Jagged2 are believed to act synergistically at the stage of cell diversification in mammals. In addition, many current studies suggest that Notch signaling has another inductive, but not inhibiting, role in the determination of the prosensory region, which precedes the cell diversification of HCs and SCs and Jagged1 is thought to be an essential ligand in this process. Earlier in ear development, the first cell fate determination begins with the delamination of the neuroblasts from the otic epithelium. The delaminated neuroblasts migrate and coalesce to form cochleovestibular ganglion. Notch signaling pathway is thought to function during the delamination through its lateral inhibitory mechanism. Recently, many experiments examining Notch-related gene expression patterns and direct functional analyses of genes have revealed multiple important functions of Notch in inner ear development. Here, we survey a series of studies and discuss the issues that remain to be elucidated in the future. © 2012 Landes Bioscience and Springer Science+Business Media.

Pluripotent stem cells can provide us with an enormous cell source for in vitro model systems for development. In 2006, new methodology was designed to generate pluripotent stem cells directly from somatic cells, and these cells were named induced pluripotent stem cells (iPSCs). This method consists of technically simple procedures: donor cell preparation, gene transduction, and isolation of embryonic stem cell-like colonies. The iPSC technology enables cell biologists not only to obtain pluripotent stem cells easily but also to study the reprogramming events themselves. Here, we describe the protocols to generate iPSCs from somatic origins by using conventional viral vectors. Specifically, we state the usage of three mammalian species: mouse, common marmoset, and human. As mouse iPSC donors, fibroblasts are easily prepared, while mesenchymal stem cells are expected to give rise to highly reprogrammed iPSCs efficiently. Common marmoset (Callithrix jacchus), a nonhuman primate, represents an alternative model to the usual laboratory animals. Finally, patient-specific human iPSCs give us an opportunity to examine the pathology and mechanisms of dysregulated genomic imprinting. The iPSC technology will serve as a valuable method for studying genomic imprinting, and conversely, the insights from these studies will offer valuable criteria to assess the potential of iPSCs. © 2012 Springer Science+Business Media, LLC.

The renin-angiotensin system (RAS) is a hormone system that has been classically known as a blood pressure regulator but is becoming well recognized as a proinflammatory mediator. In many diverse tissues, RAS pathway elements are also produced intrinsically, making it possible for tissues to respond more dynamically to systemic or local cues. While RAS is important for controlling normal inflammatory responses, hyperactivation of the pathway can cause neural dysfunction by inducing accelerated degradation of some neuronal proteins such as synaptophysin and by activating pathological glial responses. Chronic inflammation and oxidative stress are risk factors for high incidence vision-threatening diseases such as diabetic retinopathy (DR), age-related macular degeneration (AMD), and glaucoma. In fact, increasing evidence suggests that RAS inhibition may actually prevent progression of various ocular diseases including uveitis, DR, AMD, and glaucoma. Therefore, RAS inhibition may be a promising therapeutic approach to fine-tune inflammatory responses and to prevent or treat certain ocular and neurodegenerative diseases. © 2012 Toshihide Kurihara et al.