Shun Sasaki

RNA Interference-Based Silencing Reveals the Regulatory Role of Fatty Acid-Binding Protein 4 in the Production of IL-6 and Vascular Endothelial Growth Factor in 3T3-L1 Adipocytes

The fatty acid-binding protein 4 (FABP4) is believed to play an important role in maintaining glucose and lipid homeostasis. However, the physiological functions of FABP4 in adipocytes have not been fully elucidated because of difficulties associated with the effective transfection of small interfering RNA (siRNA) to differentiated adipocytes. The aim of this study was to clarify the physiological roles of FABP4 in adipocytes by establishing an efficient, universal technique for endogenous gene silencing in fully differentiated 3T3-L1 cells. Confocal-based three-dimensional observations demonstrated that, in traditionally cultured 3T3-L1 cells, multilayers of undifferentiated cells were formed. As a result, small interfering RNA failed to reach many of the differentiated cells. To solve this problem, we developed a reliable method, denoted as density-based separation followed by replating of enriched adipocytes in a monolayer (DREAM) and, using the developed method, succeeded in a significant knockdown of FABP4. Loss-of-function analyses revealed that FABP4 regulates the expression of IL-6 and vascular endothelial growth factor (VEGF) mediated by the protease-activated receptor 1 (PAR1), a thrombin receptor, in adipocytes. In addition, the basal IL-6 production was partially suppressed by PAR1 knockdown. Moreover, we also demonstrated that IL-6 stimulates the proliferation of primary endothelial cells isolated from murine adipose tissue. These findings indicate that FABP4 may have a crucial role in modulating IL-6 and vascular endothelial growth factor as angiogenesis inducers stimulated by the cellular action of thrombin on adipocytes via PAR1. These findings promise to be helpful for developing an understanding of physiological counterparts with respect to the inflammatory and angiogenic properties of adipose tissue.

Pituitary adenylate cyclase-activating polypeptide promotes eccrine gland sweat secretion

Sweat secretion is the major function of eccrine sweat glands; when this process is disturbed (paridrosis), serious skin problems can arise. To elucidate the causes of paridrosis, an improved understanding of the regulation, mechanisms and factors underlying sweat production is required. Pituitary adenylate cyclase‐activating polypeptide (PACAP) exhibits pleiotropic functions that are mediated via its receptors [PACAP‐specific receptor (PAC1R), vasoactive intestinal peptide (VIP) receptor type 1 (VPAC1R) and VPAC2R]. Although some studies have suggested a role for PACAP in the skin and several exocrine glands, the effects of PACAP on the process of eccrine sweat secretion have not been examined.

Toxic epidermal necrolysis caused by acetaminophen featuring almost 100% skin detachment: Acetaminophen is associated with a risk of severe cutaneous adverse reactions

Toxic epidermal necrolysis (TEN) is an adverse reaction that can be induced by various drugs; the associated mortality rate is 20–25%. A previous report showed a weak association between TEN and acetaminophen. Recently, the US Food and Drug Administration declared that acetaminophen is associated with a risk of serious skin reactions, including TEN. Here, we describe the case of a 43‐year‐old Japanese woman with TEN caused by acetaminophen. She had poorly controlled ulcerative colitis and was treated with high doses of prednisolone, infliximab, acetaminophen and lansoprazole. Nine days after administrating acetaminophen, targetoid erythematous and bullous lesions appeared on the patients trunk, palms and the soles of her feet. The skin lesions expanded rapidly; within 3 weeks, skin detachment was detected across nearly 100% of the patients body. However, no mucosal involvement of the eyes, oral cavity or genitalia was found. We performed lymphocyte transformation tests using various drugs; however, a high stimulation index was obtained only with acetaminophen. The patient recovered following treatment with plasmapheresis, i.v. immunoglobulin therapy, topical medication and supportive therapy. Acetaminophen is included in many prescription and over‐the‐counter products; thus, clinicians should monitor their patients for severe drug reactions, including TEN.

Identification of Arabidopsis thaliana upstream open reading frames encoding peptide sequences that cause ribosomal arrest

Abstract Specific sequences of certain nascent peptides cause programmed ribosomal arrest during mRNA translation to control gene expression. In eukaryotes, most known regulatory arrest peptides are encoded by upstream open reading frames (uORFs) present in the 5′-untranslated region of mRNAs. However, to date, a limited number of eukaryotic uORFs encoding arrest peptides have been reported. Here, we searched for arrest peptide-encoding uORFs among Arabidopsis thaliana uORFs with evolutionarily conserved peptide sequences. Analysis of in vitro translation products of 22 conserved uORFs identified three novel uORFs causing ribosomal arrest in a peptide sequence-dependent manner. Stop codon-scanning mutagenesis, in which the effect of changing the uORF stop codon position on the ribosomal arrest was examined, and toeprint analysis revealed that two of the three uORFs cause ribosomal arrest during translation elongation, whereas the other one causes ribosomal arrest during translation termination. Transient expression assays showed that the newly identified arrest-causing uORFs exerted a strong sequence-dependent repressive effect on the expression of the downstream reporter gene in A. thaliana protoplasts. These results suggest that the peptide sequences of the three uORFs identified in this study cause ribosomal arrest in the uORFs, thereby repressing the expression of proteins encoded by the main ORFs.

Intrapancreatic injection of human bone marrow-derived mesenchymal stem/stromal cells alleviates hyperglycemia and modulates the macrophage state in streptozotocin-induced type 1 diabetic mice

Type 1 diabetes mellitus is a progressive disease caused by the destruction of pancreatic β-cells, resulting in insulin dependency and hyperglycemia. While transplanted bone marrow-derived mesenchymal stem/stromal cells (BMMSCs) have been explored as an alternative therapeutic approach for diseases, the choice of delivery route may be a critical factor determining their sustainability. This study evaluated the effects of intrapancreatic and intravenous injection of human BMMSCs (hBMMSCs) in streptozotocin (STZ)-induced type 1 diabetic mouse model. C57/BL6 mice were intraperitoneally injected with 115 mg/kg STZ on day 0. hBMMSCs (1 × 106 cells) or vehicle were injected into the pancreas or jugular vein on day 7. Intrapancreatic, but not intravenous, hBMMSC injection significantly reduced blood glucose levels on day 28 compared with vehicle injection by the same route. This glucose-lowering effect was not induced by intrapancreatic injection of human fibroblasts as the xenograft control. Intrapancreatically injected fluorescence-labeled hBMMSCs were observed in the intra- and extra-lobular spaces of the pancreas, and intravenously injected cells were in the lung region, although the number of cells mostly decreased within 2 weeks of injection. For hBMMSCs injected twice into the pancreatic region on days 7 and 28, the injected mice had further reduced blood glucose to borderline diabetic levels on day 56. Animals injected with hBMMSCs twice exhibited increases in the plasma insulin level, number and size of islets, insulin-positive proportion of the total pancreas area, and intensity of insulin staining compared with vehicle-injected animals. We found a decrease of Iba1-positive cells in islets and an increase of CD206-positive cells in both the endocrine and exocrine pancreas. The hBMMSC injection also reduced the number of CD40-positive cells merged with glucagon immunoreactions in the islets. These results suggest that intrapancreatic injection may be a better delivery route of hBMMSCs for the treatment of type 1 diabetes mellitus.

Ghrelin suppresses proliferation of fetal neural progenitor cells, and induces their differentiation into neurons

Although considerable progress has been made in understanding how the temporal and regional control of neural progenitor cells (NPCs) dictates their fate, their key regulators during neural development are still unknown. Ghrelin, which is isolated from porcine stomach extract, is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). The widespread expression of ghrelin and GHS-R in the central nervous system during development suggests that ghrelin may be involved in developmental neural growth. However, its role in regulating fetal NPCs is still unclear. In this study, we investigated the effects of ghrelin on primary cultured NPCs derived from fetal mouse telencephalon. The expressions of both ghrelin and its receptor were observed in NPCs using RT-PCR, immunoblotting and immunocytostaining. Interestingly, the exposure of fetal NPCs to ghrelin at concentrations of 10(-7) and 10(-9)M suppressed their proliferation, and caused them to differentiate into neurons and to extend neurites. These results strongly suggest that ghrelin plays an autocrine modulatory role in fetal neural development.

Inverse Tunnel Magnetocapacitance in Fe/Al-oxide/Fe3O4

Magnetocapacitance (MC) effect, observed in a wide range of materials and devices, such as multiferroic materials and spintronic devices, has received considerable attention due to its interesting physical properties and practical applications. A normal MC effect exhibits a higher capacitance when spins in the electrodes are parallel to each other and a lower capacitance when spins are antiparallel. Here we report an inverse tunnel magnetocapacitance (TMC) effect for the first time in Fe/AlOx/Fe3O4 magnetic tunnel junctions (MTJs). The inverse TMC reaches up to 11.4% at room temperature and the robustness of spin polarization is revealed in the bias dependence of the inverse TMC. Excellent agreement between theory and experiment is achieved for the entire applied frequency range and the wide bipolar bias regions using Debye-Fröhlich model (combined with the Zhang formula and parabolic barrier approximation) and spin-dependent drift-diffusion model. Furthermore, our theoretical calculations predict that the inverse TMC effect could potentially reach 150% in MTJs with a positive and negative spin polarization of 65% and −42%, respectively. These theoretical and experimental findings provide a new insight into both static and dynamic spin-dependent transports. They will open up broader opportunities for device applications, such as magnetic logic circuits and multi-valued memory devices.

Pituitary adenylate cyclase-activating polypeptide (PACAP) contributes to the proliferation of hematopoietic progenitor cells in murine bone marrow via PACAP-specific receptor

Pituitary adenylate cyclase-activating polypeptide (PACAP, encoded by adcyap1) plays an important role in ectodermal development. However, the involvement of PACAP in the development of other germ layers is still unclear. This study assessed the expression of a PACAP-specific receptor (PAC1) gene and protein in mouse bone marrow (BM). Cells strongly expressing PAC1+ were large in size, had oval nuclei, and merged with CD34+ cells, suggesting that the former were hematopoietic progenitor cells (HPCs). Compared with wild-type mice, adcyap1−/− mice exhibited lower multiple potential progenitor cell populations and cell frequency in the S-phase of the cell cycle. Exogenous PACAP38 significantly increased the numbers of colony forming unit-granulocyte/macrophage progenitor cells (CFU-GM) with two peaks in semi-solid culture. PACAP also increased the expression of cyclinD1 and Ki67 mRNAs. These increases were completely and partially inhibited by the PACAP receptor antagonists, PACAP6-38 and VIP6-28, respectively. Little or no adcyap1 was expressed in BM and the number of CFU-GM colonies was similar in adcyap1−/− and wild-type mice. However, PACAP mRNA and protein were expressed in paravertebral sympathetic ganglia, which innervate tibial BM, and in the sympathetic fibers of BM cavity. These results suggested that sympathetic nerve innervation may be responsible for PACAP-regulated hematopoiesis in BM, mainly via PAC1.