Genetic Background Strongly Modifies the Severity of Symptoms of Hirschsprung Disease, but Not Hearing Loss in Rats Carrying Ednrbsl Mutations

Hirschsprung disease (HSCR) is thought to result as a consequence of multiple gene interactions that modulate the ability of enteric neural crest cells to populate the developing gut. However, it remains unknown whether the single complete deletion of important HSCR-associated genes is sufficient to result in HSCR disease. In this study, we found that the null mutation of the Ednrb gene, thought indispensable for enteric neuron development, is insufficient to result in HSCR disease when bred onto a different genetic background in rats carrying Ednrbsl mutations. Moreover, we found that this mutation results in serious congenital sensorineural deafness, and these strains may be used as ideal models of Waardenburg Syndrome Type 4 (WS4). Furthermore, we evaluated how the same changed genetic background modifies three features of WS4 syndrome, aganglionosis, hearing loss, and pigment disorder in these congenic strains. We found that the same genetic background markedly changed the aganglionosis, but resulted in only slight changes to hearing loss and pigment disorder. This provided the important evidence, in support of previous studies, that different lineages of neural crest-derived cells migrating along with various pathways are regulated by different signal molecules. This study will help us to better understand complicated diseases such as HSCR and WS4 syndrome

Endothelial Cells in Co-culture Enhance Embryonic Stem Cell Differentiation to Pancreatic Progenitors and Insulin-Producing Cells through BMP Signaling

Endothelial cells (ECs) represent the major component of the embryonic pancreatic niche and play a key role in the differentiation of insulin-producing β cells in vivo. However, it is unknown if ECs promote such differentiation in vitro. We investigated whether interaction of ECs with mouse embryoid bodies (EBs) in culture promotes differentiation of pancreatic progenitors and insulin-producing cells and the mechanisms involved. We developed a co-culture system of mouse EBs and human microvascular ECs (HMECs). An increase in the expression of the pancreatic markers PDX-1, Ngn3, Nkx6.1, proinsulin, GLUT-2, and Ptf1a was observed at the interface between EBs and ECs (EB-EC). No expression of these markers was found at the periphery of EBs cultured without ECs or those co-cultured with mouse embryonic fibroblasts (MEFs). At EB-EC interface, proinsulin and Nkx6.1 positive cells co-expressed phospho-Smad1/5/8 (pSmad1/5/8). Therefore, EBs were treated with HMEC conditioned media (HMEC-CM) suspecting soluble factors involved in bone morphogenetic protein (BMP) pathway activation. Upregulation of PDX-1, Ngn3, Nkx6.1, insulin-1, insulin-2, amylin, SUR1, GKS, and amylase as well as down-regulation of SST were detected in treated EBs. In addition, higher expression of BMP-2/-4 and their receptor (BMPR1A) were also found in these EBs. Recombinant human BMP-2 (rhBMP-2) mimicked the effects of the HMEC-CM on EBs. Noggin (NOG), a BMP antagonist, partially inhibited these effects. These results indicate that the differentiation of EBs to pancreatic progenitors and insulin-producing cells can be enhanced by ECs in vitro and that BMP pathway activation is central to this process

Bilberry extract administration prevents retinal ganglion cell death in mice via the regulation of chaperone molecules under conditions of endoplasmic reticulum stress

Orie Nakamura,1 Satoru Moritoh,1,2 Kota Sato,1,3 Shigeto Maekawa,1 Namie Murayama,1 Noriko Himori,1 Kazuko Omodaka,1,3 Tetsuya Sogon,4 Toru Nakazawa1–3,5 1Department of Ophthalmology, Tohoku University Graduate School of Medicine, Miyagi, Japan; 2Department of Retinal Disease Control, Tohoku University Graduate School of Medicine, Miyagi, Japan; 3Department of Ophthalmic Imaging and Information Analytics, Tohoku University Graduate School of Medicine, Miyagi, Japan; 4R&D Department, Wakasa Seikatsu Co., Ltd., Kyoto, Japan; 5Department of Advanced Ophthalmic Medicine, Tohoku University Graduate School of Medicine, Miyagi, Japan Purpose: To investigate the effect of bilberry extract anthocyanins on retinal ganglion cell (RGC) survival after optic nerve crush. Additionally, to determine details of the mechanism of the neuroprotective effect of bilberry extract anthocyanins and the involvement of endoplasmic reticulum stress suppression in the mouse retina.Materials and methods: Anthocyanins in bilberry extract (100 mg/kg/day or 500 mg/kg/day) were administrated orally to C57BL/6J mice. The expression levels of various molecular chaperones were assessed with quantitative reverse-transcription polymerase chain reaction, Western blotting, and immunohistochemistry. RGC survival was evaluated by measuring the gene expression of RGC markers and counting retrogradely labeled RGCs after optic nerve crush.Results: The protein levels of Grp78 and Grp94 increased significantly in mice after bilberry extract administration. Increased Grp78 and Grp94 levels were detected in the inner nuclear layer and ganglion cell layer of the retina, surrounding the RGCs. Gene expression of Chop, Bax, and Atf4 increased in mice after optic nerve crush and decreased significantly after oral bilberry extract administration. RGC survival after nerve crush also increased with bilberry extract administration.Conclusion: These results indicate that oral bilberry extract administration suppresses RGC death. Bilberry extract administration increased Grp78 and Grp94 protein levels, an effect which may underlie the neuroprotective effect of bilberry extract after optic nerve crush. Thus, bilberry extract has a potential role in neuroprotective treatments for retinal injuries, such as those which occur in glaucoma. Keywords: bilberry extract, molecular chaperones, ER stress, retinal ganglion cells, glaucom

Somatostatin release, electrical activity, membrane currents and exocytosis in human pancreatic delta cells.

AIMS/HYPOTHESIS: The aim of this study was to characterise electrical activity, ion channels, exocytosis and somatostatin release in human delta cells/pancreatic islets. METHODS: Glucose-stimulated somatostatin release was measured from intact human islets. Membrane potential, currents and changes in membrane capacitance (reflecting exocytosis) were recorded from individual human delta cells identified by immunocytochemistry. RESULTS: Somatostatin secretion from human islets was stimulated by glucose and tolbutamide and inhibited by diazoxide. Human delta cells generated bursting or sporadic electrical activity, which was enhanced by tolbutamide but unaffected by glucose. Delta cells contained a tolbutamide-insensitive, Ba(2+)-sensitive inwardly rectifying K(+) current and two types of voltage-gated K(+) currents, sensitive to tetraethylammonium/stromatoxin (delayed rectifying, Kv2.1/2.2) and 4-aminopyridine (A current). Voltage-gated tetrodotoxin (TTX)-sensitive Na(+) currents contributed to the action potential upstroke but TTX had no effect on somatostatin release. Delta cells are equipped with Ca(2+) channels blocked by isradipine (L), omega-agatoxin (P/Q) and NNC 55-0396 (T). Blockade of any of these channels interferes with delta cell electrical activity and abolishes glucose-stimulated somatostatin release. Capacitance measurements revealed a slow component of depolarisation-evoked exocytosis sensitive to omega-agatoxin. CONCLUSIONS/INTERPRETATION: Action potential firing in delta cells is modulated by ATP-sensitive K(+)-channel activity. The membrane potential is stabilised by Ba(2+)-sensitive inwardly rectifying K(+) channels. Voltage-gated L- and T-type Ca(2+) channels are required for electrical activity, whereas Na(+) currents and P/Q-type Ca(2+) channels contribute to (but are not necessary for) the upstroke of the action potential. Action potential repolarisation is mediated by A-type and Kv2.1/2.2 K(+) channels. Exocytosis is tightly linked to Ca(2+)-influx via P/Q-type Ca(2+) channels. Glucose stimulation of somatostatin secretion involves both K(ATP) channel-dependent and -independent processes