Pdx1 and Ngn3 overexpression enhances pancreatic differentiation of mouse ES cell-derived endoderm population

In order to define the molecular mechanisms regulating the specification and differentiation of pancreatic β-islet cells, we investigated the effect of upregulating Pdx1 and Ngn3 during the differentiation of the β-islet-like cells from murine embryonic stem (ES) cell-derived activin induced-endoderm. Induced overexpression of Pdx1 resulted in a significant upregulation of insulin (Ins1 and Ins2), and other pancreas-related genes. To enhance the developmental progression from the pancreatic bud to the formation of the endocrine lineages, we induced the overexpression express of Ngn3 together with Pdx1. This combination dramatically increased the level and timing of maximal Ins1 mRNA expression to approximately 100% of that found in the βTC6 insulinoma cell line. Insulin protein and C-peptide expression was confirmed by immunohistochemistry staining. These inductive effects were restricted to c-kit(+) endoderm enriched EB-derived populations suggesting that Pdx1/Ngn3 functions after the specification of pancreatic endoderm. Although insulin secretion was stimulated by various insulin secretagogues, these cells had only limited glucose response. Microarray analysis was used to evaluate the expression of a broad spectrum of pancreatic endocrine cell-related genes as well as genes associated with glucose responses. Taken together, these findings demonstrate the utility of manipulating Pdx1 and Ngn3 expression in a stage-specific manner as an important new strategy for the efficient generation of functionally immature insulin-producing β-islet cells from ES cells.     

Breakdown of mucosal immunity in the gut and resultant systemic sensitization by oral antigens in a murine model for systemic lupus erythematosus

Secreted IgA plays a pivotal role in the mucosal immunity to maintain the front line of body defense. We found that the level of fecal IgA was dramatically decreased in aged (NZB x NZW)F(1) (BWF(1)) mice developing lupus nephritis, whereas levels in similarly aged New Zealand Black (NZB) and New Zealand White (NZW) mice remained unchanged compared with young mice. The number of cells obtained from Peyer's patches was markedly decreased in aged BWF(1) mice. Aged BWF(1) mice showed increased susceptibility to pathogenic bacterial infection. Furthermore, oral administration of OVA failed to inhibit secondary IgG response induced by systemic immunization, suggesting defective oral tolerance in aged BWF(1) mice. A significant amount of orally administered OVA was incorporated directly into the intestinal lamina propria in aged BWF(1) mice whereas it was mainly localized in subepithelial domes and interfollicular region in Peyer's patches in young mice. T cells obtained from renal and pulmonary lymph nodes of aged BWF(1) mice that had been orally administered with OVA showed an Ag-specific T cell proliferation, whereas those from young BWF(1), aged NZB, and aged NZW mice did not. Interestingly, aerosol exposure to OVA of aged BWF(1) mice, which had been orally administered with the same Ag, provoked an eosinophil infiltration in the lung. These results demonstrate that mucosal immunity in the gut is impaired and oral Ags induce systemic sensitization instead of oral tolerance in the development of murine lupus.     

Mutual regulation of protein-tyrosine phosphatase 20 and protein-tyrosine kinase Tec activities by tyrosine phosphorylation and dephosphorylation

PTP20, also known as HSCF/protein-tyrosine phosphatase K1/fetal liver phosphatase 1/brain-derived phosphatase 1, is a cytosolic protein-tyrosine phosphatase with currently unknown biological relevance. We have identified that the nonreceptor protein-tyrosine kinase Tec-phosphorylated PTP20 on tyrosines and co-immunoprecipitated with the phosphatase in a phosphotyrosine-dependent manner. The interaction between the two proteins involved the Tec SH2 domain and the C-terminal tyrosine residues Tyr-281, Tyr-303, Tyr-354, and Tyr-381 of PTP20, which were also necessary for tyrosine phosphorylation/dephosphorylation. Association between endogenous PTP20 and Tec was also tyrosine phosphorylation-dependent in the immature B cell line Ramos. Finally, the Tyr-281 residue of PTP20 was shown to be critical for deactivating Tec in Ramos cells upon B cell receptor ligation as well as dephosphorylation and deactivation of Tec and PTP20 itself in transfected COS7 cells. Taken together, PTP20 appears to play a negative role in Tec-mediated signaling, and Tec-PTP20 interaction might represent a negative feedback mechanism.     

小鼠雌性生殖系统电穿孔与微泡增强超声波转EGFP基因的研究(简报)

非病毒载体转基因法,如注射裸DNA或脂质体转染,不产生细胞毒性,但除了肌肉组织外其他组织的转导效率均不高。电脉冲可使细胞膜产生临时的微孔允许一些分子通过,因此应用此方法可将药物或基因转入动物组织。电穿孔常用于培养的细胞转基因,理论上,低强度、长脉冲,或高强度、短脉冲有利于电转导,选择适合的参数是电转导的关键。本实验比较了不同电压和脉冲时间对小鼠卵巢在体转入绿色荧光蛋白基因的效果,确定了最适的电转导参数,为卵巢疾病的药物、基因治疗和研究卵泡发育中的基因调控提供了实验手段。超声波是临床常用的诊断方法,对人体无害…     

小鼠雌性生殖系统电穿孔与微泡增强超声波转EG即基因的研究（简报）

非病毒载体转基因法。如注射裸DNA或脂质体转染,不产生细胞毒性。但除了肌肉组织外其他组织的转导效率均不高。电脉冲可使细胞膜产生临时的微孔允许一些分子通过。因此应用此方法可将药物或基因转人动物组织。电穿孔常用于培养的细胞转基因,理论上,低强度、长脉冲。或高强度、短脉冲有利于电转导。选择适合的参数是电转导的关键。本实验比较了不同电压和脉冲时间对小鼠卵巢在体转入绿色荧光蛋白基因的效果．确定了最适的电转导参数。为卵巢疾病的药物、基因治疗和研究卵泡发育中的基因调控提供了实验手段。     

Apoptotic cells activate NKT cells through T cell Ig-like mucin-like-1 resulting in airway hyperreactivity

T cell Ig-like mucin-like-1 (TIM-1) is an important asthma susceptibility gene, but the immunological mechanisms by which TIM-1 functions remain uncertain. TIM-1 is also a receptor for phosphatidylserine (PtdSer), an important marker of cells undergoing programmed cell death, or apoptosis. We now demonstrate that NKT cells constitutively express TIM-1 and become activated by apoptotic cells expressing PtdSer. TIM-1 recognition of PtdSer induced NKT cell activation, proliferation, and cytokine production. Moreover, the induction of apoptosis in airway epithelial cells activated pulmonary NKT cells and unexpectedly resulted in airway hyperreactivity, a cardinal feature of asthma, in an NKT cell-dependent and TIM-1-dependent fashion. These results suggest that TIM-1 serves as a pattern recognition receptor on NKT cells that senses PtdSer on apoptotic cells as a damage-associated molecular pattern. Furthermore, these results provide evidence for a novel innate pathway that results in airway hyperreactivity and may help to explain how TIM-1 and NKT cells regulate asthma.     

Proton beam therapy with concurrent chemotherapy is feasible in children with newly diagnosed rhabdomyosarcoma

BackgroundThe optimal treatment for rhabdomyosarcoma (RMS) requires multidisciplinary treatment with chemotherapy, surgery, and radiotherapy. Surgery and radiotherapy are integral to the local control (LC) of RMS. However, postsurgical and radiotherapy-related complications could develop according to the local therapy and tumor location. In this study, we conducted a single-center analysis of the outcomes and toxicity of multidisciplinary treatment using proton beam therapy (PBT) for pediatric RMS.Materials and methodsRMS patients aged younger than 20 years whose RMS was newly diagnosed and who underwent PBT at University of Tsukuba Hospital (UTH) during the period from 2009 to 2019 were enrolled in this study. The patients’ clinical information was collected by retrospective medical record review.ResultsForty-eight patients were included. The 3-year progression-free survival (PFS) and overall survival (OS) rates of all the patients were 68.8% and 94.2%, respectively. The 3-year PFS rates achieved with radical resection, conservative resection, and biopsy only were 65.3%, 83.3%, and 67.6%, respectively (p = 0.721). The 3-year LC rates achieved with radical resection, conservative resection, and biopsy only were 90.9%, 83.3%, and 72.9%, respectively (p = 0.548). Grade 3 or higher mucositis/dermatitis occurred in 14 patients. Although the days of opioid use due to mucositis/dermatitis during the chemotherapy with PBT were longer than those during the chemotherapy without PBT [6.1 and 1.6 (mean), respectively, p = 0.001], the frequencies of fever and elevation of C-reactive protein were equivalent.ConclusionsMultidisciplinary therapy containing PBT was feasible and provided a relatively fair 3-year PFS, even in children with newly diagnosed RMS without severe toxicity.     

A Na+-phosphate cotransporter homologue (SLC17A4 protein) is an intestinal organic anion exporter

The SLC17 anion transporter family comprises nine members that transport various organic anions in membrane potential (Δψ)- and Cl(-)-dependent manners. Although the transport substrates and physiological relevance of the majority of the members have already been determined, little is known about SLC17A4 proteins known to be Na(+)-phosphate cotransporter homologue (NPT homologue). In the present study, we investigated the expression and transport properties of human SLC17A4 protein. Using specific antibodies, we found that a human NPT homologue is specifically expressed and present in the intestinal brush border membrane. Proteoliposomes containing the purified protein took up radiolabeled p-aminohippuric acid (PAH) in a Cl(-)-dependent manner at the expense of an electrochemical gradient of protons, especially Δψ, across the membrane. The Δψ- and Cl(-)-dependent PAH uptake was inhibited by diisothiocyanostilbene-2,2'-disulfonic acid and Evans blue, common inhibitors of SLC17 family members. cis-Inhibition studies revealed that various anionic compounds, such as hydrophilic nonsteroidal anti-inflammatory drugs, pravastatin, and urate inhibited the PAH uptake. Proteoliposomes took up radiolabeled urate, with the uptake having properties similar to those of PAH uptake. These results strongly suggested that the human NPT homologue acts as a polyspecific organic anion exporter in the intestines. Since SLC17A1 protein (NPT1) and SLC17A3 protein (NPT4) are responsible for renal urate extrusion, our results reveal the possible involvement of a NPT homologue in urate extrusion from the intestinal duct.     

The Autophagy-related Protein Kinase Atg1 Interacts with the Ubiquitin-like Protein Atg8 via the Atg8 Family Interacting Motif to Facilitate Autophagosome Formation

In autophagy, a cup-shaped membrane called the isolation membrane is formed, expanded, and sealed to complete a double membrane-bound vesicle called the autophagosome that encapsulates cellular constituents to be transported to and degraded in the lysosome/vacuole. The formation of the autophagosome requires autophagy-related (Atg) proteins. Atg8 is a ubiquitin-like protein that localizes to the isolation membrane; a subpopulation of this protein remains inside the autophagosome and is transported to the lysosome/vacuole. In the budding yeast Saccharomyces cerevisiae, Atg1 is a serine/threonine kinase that functions in the initial step of autophagosome formation and is also efficiently transported to the vacuole via autophagy. Here, we explore the mechanism and significance of this autophagic transport of Atg1. In selective types of autophagy, receptor proteins recognize degradation targets and also interact with Atg8, via the Atg8 family interacting motif (AIM), to link the targets to the isolation membrane. We find that Atg1 contains an AIM and directly interacts with Atg8. Mutations in the AIM disrupt this interaction and abolish vacuolar transport of Atg1. These results suggest that Atg1 associates with the isolation membrane by binding to Atg8, resulting in its incorporation into the autophagosome. We also show that mutations in the Atg1 AIM cause a significant defect in autophagy, without affecting the functions of Atg1 implicated in triggering autophagosome formation. We propose that in addition to its essential function in the initial stage, Atg1 also associates with the isolation membrane to promote its maturation into the autophagosome.     

TRIM50 Protein Regulates Vesicular Trafficking for Acid Secretion in Gastric Parietal Cells

Of the TRIM/RBCC family proteins taking part in a variety of cellular processes, TRIM50 is a stomach-specific member with no defined biological function. Our biochemical data demonstrated that TRIM50 is specifically expressed in gastric parietal cells and is predominantly localized in the tubulovesicular and canalicular membranes. In cultured cells ectopically expressing GFP-TRIM50, confocal microscopic imaging revealed dynamic movement of TRIM50-associated vesicles in a phosphoinositide 3-kinase-dependent manner. A protein overlay assay detected preferential binding of the PRY-SPRY domain from the TRIM50 C-terminal region to phosphatidylinositol species, suggesting that TRIM50 is involved in vesicular dynamics by sensing the phosphorylated state of phosphoinositol lipids. Trim50 knock-out mice retained normal histology in the gastric mucosa but exhibited impaired secretion of gastric acid. In response to histamine, Trim50 knock-out parietal cells generated deranged canaliculi, swollen microvilli lacking actin filaments, and excess multilamellar membrane complexes. Therefore, TRIM50 seems to play an essential role in tubulovesicular dynamics, promoting the formation of sophisticated canaliculi and microvilli during acid secretion in parietal cells.