Actin Dynamics Regulated by the Balance of Neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) and Cofilin Activities Determines the Biphasic Response of Glucose-induced Insulin Secretion

Actin dynamics in pancreatic β-cells is involved in insulin secretion. However, the molecular mechanisms of the regulation of actin dynamics by intracellular signals in pancreatic β-cells and its role in phasic insulin secretion are largely unknown. In this study, we elucidate the regulation of actin dynamics by neuronal Wiskott-Aldrich syndrome protein (N-WASP) and cofilin in pancreatic β-cells and demonstrate its role in glucose-induced insulin secretion (GIIS). N-WASP, which promotes actin polymerization through activation of the actin nucleation factor Arp2/3 complex, was found to be activated by glucose stimulation in insulin-secreting clonal pancreatic β-cells (MIN6-K8 β-cells). Introduction of a dominant-negative mutant of N-WASP, which lacks G-actin and Arp2/3 complex-binding region VCA, into MIN6-K8 β-cells or knockdown of N-WASP suppressed GIIS, especially the second phase. We also found that cofilin, which severs F-actin in its dephosphorylated (active) form, is converted to the phosphorylated (inactive) form by glucose stimulation in MIN6-K8 β-cells, thereby promoting F-actin remodeling. In addition, the dominant-negative mutant of cofilin, which inhibits activation of endogenous cofilin, or knockdown of cofilin reduced the second phase of GIIS. However, the first phase of GIIS occurs in the G-actin predominant state, in which cofilin activity predominates over N-WASP activity. Thus, actin dynamics regulated by the balance of N-WASP and cofilin activities determines the biphasic response of GIIS.     

Mesenchymal stem cells contribute to endogenous FVIII:c production

Besides the liver, it has been difficult to identify which organ(s) and/or cellular component(s) contribute significantly to the production of human FVIII:c (FVIII). Thus far, only endothelial cells have been shown to constitute a robust extrahepatic source of FVIII, possibly explaining both the diverse presence of FVIII mRNA in the body, and the observed increase in FVIII levels during liver failure. Here, we investigate whether human mesenchymal stem cells (MSC), ubiquitously present in different organs, could also contribute to FVIII production. MSC isolated from human lung, liver, brain, and bone marrow expressed FVIII message as determined by quantitative‐RT‐PCR. Using an antibody specific for FVIII, confocal microscopy, and umbilical cord‐derived endothelial cells (HUVEC) as a negative control, we demonstrated that, in MSC, FVIII protein was not stored in granules; rather, it localized to the perinuclear region. Furthermore, functional FVIII was detected in MSC supernatants and cell lysates by aPTT and chromogenic assays. These results demonstrate that MSC can contribute at low levels to the functional FVIII pool, and advance the understanding of the physiology of FVIII production and secretion. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

Identification of N-substituted 8-azatetrahydroquinolone derivatives as selective and orally active M1 and M4 muscarinic acetylcholine receptors agonists

We designed and synthesized N-substituted 8-azatetrahydroquinolone derivatives as selective M₁ and M₄ muscarinic acetylcholine receptors agonists. Optimization of selected derivatives led to the discovery of compound 7 as a highly potent M₁ and M₄ agonist with weak hERG inhibition. Oral administration of compound 7 improved psychosis-like behavior in rats.     

Development of preparative and analytical methods of the hop bitter acid oxide fraction and chemical properties of its components

The bitter acids in hops (Humulus lupulus L.) and beer, such as α-, β-, and iso-α-acids, are known to affect beer quality and display various physiological effects. However, these compounds readily oxidize, and the effect of the oxides on the properties of beer or their potential health benefits are not well understood. In this study, we developed a simple preparative method for the bitter acid oxide fraction derived from hops and designated the constituents as matured hop bitter acids (MHBA). HPLC-PDA-ESI/HRMS and MS² revealed that MHBA are primarily composed of α-acid-derived oxides, which possess a common β-tricarbonyl moiety in their structures similar to α-, β-, and iso-α-acids. We also developed a quantitative analytical method of whole MHBA by HPLC, which showed high precision and reproducibility. Using our newly developed method, the concentration of whole MHBA in several commercial beers was evaluated. Our results will promote the study of bitter acid oxides.     

Expression of a putative dioxygenase gene adjacent to an insertion mutation is involved in the short internodes of columnar apples (<Emphasis Type="Italic">Malus</Emphasis> × <Emphasis Type="Italic">domestica</Emphasis>)

Determining the molecular mechanism of fruit tree architecture is important for tree management and fruit production. An apple mutant ‘McIntosh Wijcik’, which was discovered as a bud mutation from ‘McIntosh’, exhibits a columnar growth phenotype that is controlled by a single dominant gene, Co. In this study, the mutation and the Co gene were analyzed. Fine mapping narrowed the Co region to a 101 kb region. Sequence analysis of the Co region and the original wild-type co region identified an insertion mutation of an 8202 bp long terminal repeat (LTR) retroposon in the Co region. Segregation analysis using a DNA marker based on the insertion polymorphism showed that the LTR retroposon was closely associated with the columnar growth phenotype. RNA-seq and RT-PCR analysis identified a promising Co candidate gene (91071-gene) within the Co region that is specifically expressed in ‘McIntosh Wijcik’ but not in ‘McIntosh’. The 91071-gene was located approximately 16 kb downstream of the insertion mutation and is predicted to encode a 2-oxoglutarate-dependent dioxygenase involved in an unknown reaction. Overexpression of the 91071-gene in transgenic tobaccos and apples resulted in phenotypes with short internodes, like columnar apples. These data suggested that the 8202 bp retroposon insertion in ‘McIntosh Wijcik’ is associated with the short internodes of the columnar growth phenotype via upregulated expression of the adjacent 91071-gene. Furthermore, the DNA marker based on the insertion polymorphism could be useful for the marker-assisted selection of columnar apples.     

Influences of two coexisting endosymbionts,CI‐inducing Wolbachia and male‐killing Spiroplasma,on the performance of their host Laodelphax striatellus (Hemiptera: Delphacidae)

The small brown planthopper Laodelphax striatellus (Hemiptera: Delphacidae) is reported to have the endosymbiont Wolbachia, which shows a strong cytoplasmic incompatibility (CI) between infected males and uninfected females. In the 2000s, female‐biased L. striatellus populations were found in Taiwan, and this sex ratio distortion was the result of male‐killing induced by the infection of another endosymbiont, Spiroplasma. Spiroplasma infection is considered to negatively affect both L. striatellus and Wolbachia because the male‐killing halves the offspring of L. striatellus and hinders the spread of Wolbachia infection via CI. Spiroplasma could have traits that increase the fitness of infected L. striatellus and/or coexisting organisms because the coinfection rates of Wolbachia and Spiroplasma were rather high in some areas. In this study, we investigated the influences of the infection of these two endosymbionts on the development, reproduction, and insecticide resistance of L. striatellus in the laboratory. Our results show that the single‐infection state of Spiroplasma had a negative influence on the fertility of L. striatellus, while the double‐infection state had no significant influence. At late nymphal and adult stages, the abundance of Spiroplasma was lower in the double‐infection state than in the single‐infection state. In the double‐infection state, the reduction of Spiroplasma density may be caused by competition between the two endosymbionts, and the negative influence of Spiroplasma on the fertility of host may be relieved. The resistance of L. striatellus to four insecticides was compared among different infection states of endosymbionts, but Spiroplasma infection did not contribute to increase insecticide resistance. Because positive influences of Spiroplasma infection were not found in terms of the development, reproduction, and insecticide resistance of L. striatellus, other factors improving the fitness of Spiroplasma‐infected L. striatellus may be related to the high frequency of double infection in some L. striatellus populations.     

Osteoactivin fragments produced by ectodomain shedding induce MMP-3 expression via ERK pathway in mouse NIH-3T3 fibroblasts

Intact osteoactivin, a novel type I membrane glycoprotein, were shed at a dibasic motif in the juxtamembrane region in C2C12 myoblasts. Extracellular fragments were secreted into the culture media by a putative metalloprotease. Extracellular fragments of osteoactivin, but not control protein, induced matrix metalloprotease-3 (MMP-3) expression in NIH-3T3 fibroblasts. Epidermal growth factor (ERK) kinase inhibitors inhibited the osteoactivin-mediated MMP-3 expression, whereas the extracellular fragment of osteoactivin activated ERK1/2 and p38 in the mitogen-activated protein kinase pathway. Our results suggest that the extracellular fragments of osteoactivin produced by shedding act as a growth factor to induce MMP-3 expression via the ERK pathway in fibroblasts.     

Genetic Diversity of the Cryptococcus gattii Species Complex in Mato Grosso State,Brazil

Mycopathologia - Cryptococcosis is caused by fungi of the genus Cryptococcus. Owing to its importance, this study aimed to analyze the genetic diversity of C. gattii isolates from animals, humans,...