A Potent Class of GPR40 Full Agonists Engages the EnteroInsular Axis to Promote Glucose Control in Rodents

Type 2 diabetes is characterized by impaired glucose homeostasis due to defects in insulin secretion, insulin resistance and the incretin response. GPR40 (FFAR1 or FFA1) is a G-protein-coupled receptor (GPCR), primarily expressed in insulin-producing pancreatic β-cells and incretin-producing enteroendocrine cells of the small intestine. Several GPR40 agonists, including AMG 837 and TAK-875, have been disclosed, but no GPR40 synthetic agonists have been reported that engage both the insulinogenic and incretinogenic axes. In this report we provide a molecular explanation and describe the discovery of a unique and potent class of GPR40 full agonists that engages the enteroinsular axis to promote dramatic improvement in glucose control in rodents. GPR40 full agonists AM-1638 and AM-6226 stimulate GLP-1 and GIP secretion from intestinal enteroendocrine cells and increase GSIS from pancreatic islets, leading to enhanced glucose control in the high fat fed, streptozotocin treated and NONcNZO10/LtJ mouse models of type 2 diabetes. The improvement in hyperglycemia by AM-1638 was reduced in the presence of the GLP-1 receptor antagonist Ex(9–39)NH₂.     

LDL coating pVEGF/polyethylenimine complex enhances vascular endothelial growth factor expression

The major limitations to non-viral gene delivery are relatively low efficiency and cytotoxicity, which need to be addressed in the design of new vectors. In this study, negatively charged low density lipoproteins (LDL) were coated onto positively charged pVEGF/PEI complexes to form pVEGF/PEI/LDL terplexes by a two-step procedure. The biocompatible LDL was introduced to reduce the cytotoxicity of the gene delivery system and increase its affinity to cells. The successful formation of pVEGF/PEI/ LDL terplexes was confirmed by their near-neutral and slightly negative surface charges. The pVEGF/PEI/LDL terplexes were well-defined sub-micron spherical particles. On the cell viability assay, both of the PEI/LDL combined vector and pVEGF/PEI/LDL terplexes exhibited much lower cytotoxicity to HeLa cells and HUVE cells than those of PEI and pVEGF/PEI complexes, attributed to the shielding effect of the LDL. pEGFP/PEI/LDL terplexes showed significantly higher transfection efficiency in comparison to pEGFP/PEI complexes in serum-containing medium. pVEGF/PEI/LDL terplexes at their optimal N/P ratio and LDL/PEI weigh ratio induced higher expression levels of VEGF protein in HUVE cells than those of pVEGF/PEI complexes. Therefore, the pVEGF/PEI/LDL terplexes could be used as a promising gene delivery system to enhance VEGF protein expression.     

Modulation of mouse neural crest cell motility by N-cadherin and connexin 43 gap junctions.

Connexin 43 (Cx43alpha1) gap junction has been shown to have an essential role in mediating functional coupling of neural crest cells and in modulating neural crest cell migration. Here, we showed that N-cadherin and wnt1 are required for efficient dye coupling but not for the expression of Cx43alpha1 gap junctions in neural crest cells. Cell motility was found to be altered in the N-cadherin-deficient neural crest cells, but the alterations were different from that elicited by Cx43alpha1 deficiency. In contrast, wnt1-deficient neural crest cells showed no discernible change in cell motility. These observations suggest that dye coupling may not be a good measure of gap junction communication relevant to motility. Alternatively, Cx43alpha1 may serve a novel function in motility. We observed that p120 catenin (p120ctn), an Armadillo protein known to modulate cell motility, is colocalized not only with N-cadherin but also with Cx43alpha1. Moreover, the subcellular distribution of p120ctn was altered with N-cadherin or Cx43alpha1 deficiency. Based on these findings, we propose a model in which Cx43alpha1 and N-cadherin may modulate neural crest cell motility by engaging in a dynamic cross-talk with the cell's locomotory apparatus through p120ctn signaling.     

Structural and functional characterization of human CXCR4 as a chemokine receptor and HIV-1 co-receptor by mutagenesis and molecular modeling studies

The human CXC chemokine receptor 4 (CXCR4) is a receptor for the chemokine stromal cell-derived factor (SDF-1alpha) and a co-receptor for the entry of specific strains of human immunodeficiency virus type I (HIV-1). CXCR4 is also recognized by an antagonistic chemokine, the viral macrophage inflammatory protein II (vMIP-II) encoded by human herpesvirus type VIII. SDF-1alpha or vMIP-II binding to CXCR4 can inhibit HIV-1 entry via this co-receptor. An approach combining protein structural modeling and site-directed mutagenesis was used to probe the structure-function relationship of CXCR4, and interactions with its ligands SDF-1alpha and vMIP-II and HIV-1 envelope protein gp120. Hypothetical three-dimensional structures were proposed by molecular modeling studies of the CXCR4.SDF-1alpha complex, which rationalize extensive biological information on the role of CXCR4 in its interactions with HIV-1 envelope protein gp120. With site-directed mutagenesis, we have identified that the amino acid residues Asp (D20A) and Tyr (Y21A) in the N-terminal domain and the residue Glu (E268A) in extracellular loop 3 (ECL3) are involved in ligand binding, whereas the mutation Y190A in extracellular loop 2 (ECL2) impairs the signaling mediated by SDF-1alpha. As an HIV-1 co-receptor, we found that the N-terminal domain, ECL2, and ECL3 of CXCR4 are involved in HIV-1 entry. These structural and mutational studies provide valuable information regarding the structural basis for CXCR4 activity in chemokine binding and HIV-1 viral entry, and could guide the design of novel targeted inhibitors.     

Gliosis in the Mice Hippocampus Without Neuronal Death After Systemic Administration of High Dosage of Tetanus Toxin

Tetanus toxin (TeT), an exotoxin, has been studied to cause tetanus in mammalian brains, and it can block the release of some neurotransmitters and affect seizure propagation. In the present study, we investigated neuronal damage/death and glial changes in the mouse hippocampus after systemic administration (intraperitoneal injection) of TeT 10 and 100 ng/kg. In both the 10 and 100 ng/kg TeT-treated groups, no neuronal death occurred in any subregions of the mouse hippocampus until 24 h post-treatment; however, there were changes in glia in the hippocampus depending on time course and dosage. The morphology of GFAP-immunoreactive astrocytes and Iba-1-immunoreactive microglia was apparently changed in the 100 ng/kg TeT treated-group compared to the 10 ng/kg TeT treated-group. In the 100 ng/kg TeT treated-group, they were increased in size and their immunoreactivity was distinctively increased from 12 h post-treatment. We also found that their protein levels were increased in the hippocampus at 12 h post-treatment of 100 ng/kg TeT. In conclusion, these results indicate that the systemic administration of 100 ng/kg TeT induced a distinctive microglia changes in the mouse hippocampus without any neuronal death/damage.     

Insulin-producing cells from human adipose tissue-derived mesenchymal stem cells detected by atomic force microscope

We successfully differentiated human adipose tissue-derived mesenchymal stem cells (haMSCs) into insulin-producing cells (IPCs) in vitro and did not use any insulin which might be absorbed by cells during in vitro culture. Expression of insulin gene was massively increased by 28,000-fold at day 12 compared with haMSCs (P < 0.05). IPCs could secrete insulin after glucose was stimulated. The higher the concentration of glucose, the more production of insulin was noted. We reported AFM images of IPCs for the first time. AFM images showed that the sizes of cells were similar to each other, and all IPC surface had a porous structure in the cytoplasm area. In sugar-free group, the size of holes was similar (diameter, 1,086.98 ± 156.70 nm; depth, 185.22 ± 52.14 nm). In higher sugar-stimulated group, there were more holes with bigger diameter and smaller depth. (diameter, 3,183.65 ± 2,229.18 nm; depth 109.42 ± 56.26 nm, P < 0.05). We found that the hole diameter and depth could change with the concentration of glucose in media. Concurrently, laser scanning confocal microscopy images indicated that cortical actin network beneath plasma membrane in IPCs was dense and continuous. After glucose stimulation, we found the actin web depolymerized and became discontinuous in IPCs. We speculated that diameter augmentation of holes located in the cytoplasm area in IPCs was one manifestation of excytosis increase.     

文心兰浅绿条纹突变体的生理生化及叶绿素荧光特性研究

以文心兰浅绿条纹突变体为材料,分析叶片光合色素含量和组成、叶绿素合成前体物质含量以及叶绿素荧光参数的变化,观察突变体叶绿体超微结构的改变,以探寻其叶色变异的生理基础。结果表明:(1)突变体叶绿素a(Chl a)、叶绿素b(Chl b)、类胡萝卜素(Car)和总叶绿素(Chl)含量分别比叶色正常植株显著降低了37.1%、34.0%、30.8%和36.3%。(2)突变体叶绿素生物合成受阻于胆色素原(PBG)到尿卟啉原Ⅲ(UrogenⅢ)的反应步骤。(3)突变体叶绿体发育存在明显的缺陷,基粒数目及基粒片层的垛叠层数明显减少,嗜锇颗粒及囊泡较多。(4)突变体初始荧光(Fo)比正常植株高39%,最大荧光(Fm)、最大光化学效率(Fv/Fm)、PSⅡ有效光化学效率(Fv′/Fm′)和PSⅡ实际光化学效率(ΦPSⅡ)均显著低于正常植株,但光化学淬灭系数(qP)和非光化学淬灭系数(NPQ)与正常植株无显著差异。研究结果说明,文心兰叶绿素生物合成受阻和叶绿体结构发育不良,导致叶绿素的含量下降,致使突变体叶片呈现浅绿条纹,光能利用率降低。     

The Role of Physical Activity in Harm Reduction among Betel Quid Chewers from a Prospective Cohort of 419,378 Individuals

ObjectiveTo assess the benefits of regular exercise in reducing harms associated with betel quid (BQ) chewing.MethodsThe study cohort, 419,378 individuals, participated in a medical screening program between 1994 and 2008, with 38,324 male and 1,495 female chewers, who consumed 5–15 quids of BQ a day. Physical activity of each individual, based on “MET-hour/week”, was classified as “inactive” or “active”, where activity started from a daily 15 minutes/day or more of brisk walking (≥3.75 MET-hour/week). Hazard ratios for mortality and remaining years in life expectancy were calculated.ResultsNearly one fifth (18.7%) of men, but only 0.7% of women were chewers. Chewers had a 10-fold increase in oral cancer risk; and a 2-3-fold increase in mortality from lung, esophagus and liver cancer, cardiovascular disease, and diabetes, with doubling of all-cause mortality. More than half of chewers were physically inactive (59%). Physical activity was beneficial for chewers, with a reduction of all-cause mortality by 19%. Inactive chewers had their lifespan shortened by 6.3 years, compared to non-chewers, but being active, chewers improved their health by gaining 2.5 years. The improvement, however, fell short of offsetting the harms from chewing.ConclusionsChewers had serious health consequences, but being physically active, chewers could mitigate some of these adverse effects, and extend life expectancy by 2.5 years and reduce mortality by one fifth. Encouraging exercise, in addition to quitting chewing, remains the best advice for 1.5 million chewers in Taiwan.