Obesity is a severe disease that causes diabetes, fatty liver, hypertension, and arteriosclerosis. It involves adipocytokines such as tumor necrosis factor-α, leptin, and resistin.The hypertrophic adipocytes induce the secretion of these adipocytokines. Regulating the growth of adipose tissue is an extremely important approach to prevent these diseases. Hypertrophy of adipose tissue is closely associated with angiogenesis, wherein neovascular vessels supply nutrients.Previous studies have shown that angiogenesis inhibitors can prevent the development of obesity in mice. In this study, we report an anti-obesity therapy targeting the neovascular vessels of adipose tissue via endothelial cell (EC) vaccination. EC vaccine was prepared using EC extract antigen, which was pulsed to dendritic cells, and was administered thrice intradermally. The anti-obesity effect was evaluated in high-fat diet-fed obesity model mice. EC vaccination significantly suppressed the increase in body weight and hyperplasia of adipocytes and improved the accumulation of hepatic lipid droplets in these mice. Adipose tissue-induced vascular-targeted vaccine therapy is a potential novel approach for obesity.
Lysosome-associated membrane protein-1 (LAMP-1) is a type I membrane glycoprotein consisting of a large luminal domain, a membrane-anchoring domain, and a short cytoplasmic tail (CT). The tyrosine-based motif (G378Y379QTI382) in its CT exclusively binds to adapter protein complex-3 (AP-3), which may facilitate the incorporation of LAMP-1 into transport vesicles to late endosomes and lysosomes. Of this sequence, Y379 is critical, and hydrophobic I382 is optimal for the AP-3 binding and the efficient delivery to lysosomes. However, it is not clear how important G378 is in the AP-3 binding and lysosome transport. To clarify its importance, four mutants in which G378 was replaced with alanine, aspartic acid, glutamic acid, and asparagine (designated as G378A, G378D, G378E, and G378N, respectively) were prepared and their interaction strengths with AP-3 and lysosomal abundance were compared with those of wild-type (WT)-LAMP-1. A yeast two-hybrid system was applied to measure the interaction strengths of WT-, G378A-, G378D-, G378E-, and G378N-CTs with the medium subunit of AP-3 (μ3A). The G378A-, G378D-, and G378E-CTs as strongly interacted with μ3A as WT-CT, but the G378N-CT exhibited a very weak interaction with it. In the cell fractionation analyses, the lysosomal levels of G378A, G378D, and G378E were almost the same as that of WT whereas a lesser amount of G378N existed in the lysosomes. Taken together, it is considered that Y379 and I382 are essential for AP-3-mediated vesicular transport of LAMP-1 while the Y379-preceding amino acid is not restricted to glycine but asparagine at this position is less suitable for that.
The bacterial endotoxins test (BET) is a method of detection and quantification of bacterial endotoxin in injectable drugs and medical devices using amoebocyte lysate reagents sourced from the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). Three manufacturers have launched three different types of recombinant reagents, and recently the United States, European and Japanese Pharmacopeias have been evaluating the equivalency of these reagents to natural amoebocyte lysate reagents. Several studies suggested that the recombinant reagents are very similar to amoebocyte lysate reagents, however there is potential for improvement. Previous reports indicate that recombinant reagents have two issues: extremely low endotoxin potency determination for Helicobacter pylori GU2 and high levels of interference from Heparin Calcium impacting endotoxin recovery. A new recombinant cascade reagent (rCR), PyroSmart NextGen®, recently introduced to the market has been developed to solve these issues. PyroSmart NextGen® has demonstrated higher reactivity to H. pylori GU2 and a lower level of interference from Heparin Calcium than other existing recombinant reagents. Additionally, the analytical capability and suitability of PyroSmart NextGen® has been demonstrated when applied to the BET as described in the US, European and Japanese pharmacopeias. PyroSmart NextGen® has also shown comparability to amoebocyte lysate reagents by demonstrating its ability to detect autochthonous endotoxin in water and in Escherichia coli culture. Overall, this study has verified that the rCR, PyroSmart NextGen® is a suitable alternative to amoebocyte lysate reagents.
With the increasing development of genetically modified (GM) crops authorized for use in food, a rapid and accurate method of quantifying the weight-based amount of GM crops is needed to ensure consumers’ rights to choose. Conversion factor (Cf) value is the ratio of the copy number of a GM-specific sequence to an endogenous sequence in the GM crop and is used to convert a copy number ratio of the GM-specific sequence to the endogenous sequence of a sample into weight-based amount of GM crops. However, in the current Japanese official method for GM crops, determining Cf values using real-time PCR instruments capable of rapid measurements has not been established. In this study, Cf values for GM maize and soybean authorized for use as food in Japan were experimentally determined using an Applied Biosystems 7500 Fast Real-Time PCR System, which is capable of rapid measurement. The Cf values were almost the same as those of the PCR instruments described in the Japanese official method, and the weight-based amount of GM maize MON810 measured using this Cf value showed similar results. These results suggest that rapid quantification by this PCR instrument has the same performance as the recommended PCR instruments and may contribute to the labeling regulation of GM crops in Japan.