Quantitative Measurement of GPCR Endocytosis via Pulse-Chase Covalent Labeling

G protein-coupled receptors (GPCRs) play a critical role in many physiological systems and represent one of the largest families of signal-transducing receptors. The number of GPCRs at the cell surface regulates cellular responsiveness to their cognate ligands, and the number of GPCRs, in turn, is dynamically controlled by receptor endocytosis. Recent studies have demonstrated that GPCR endocytosis, in addition to affecting receptor desensitization and resensitization, contributes to acute G protein-mediated signaling. Thus, endocytic GPCR behavior has a significant impact on various aspects of physiology. In this study, we developed a novel GPCR internalization assay to facilitate characterization of endocytic GPCR behavior. We genetically engineered chimeric GPCRs by fusing HaloTag (a catalytically inactive derivative of a bacterial hydrolase) to the N-terminal end of the receptor (HT-GPCR). HaloTag has the ability to form a stable covalent bond with synthetic HaloTag ligands that contain fluorophores or a high-affinity handle (such as biotin) and the HaloTag reactive linker. We selectively labeled HT-GPCRs at the cell surface with a HaloTag PEG ligand, and this pulse-chase covalent labeling allowed us to directly monitor the relative number of internalized GPCRs after agonist stimulation. Because the endocytic activities of GPCR ligands are not necessarily correlated with their agonistic activities, applying this novel methodology to orphan GPCRs, or even to already characterized GPCRs, will increase the likelihood of identifying currently unknown ligands that have been missed by conventional pharmacological assays.     

Transient Elastography-Based Liver Profiles in a Hospital-Based Pediatric Population in Japan

Background & Aims

The utility of transient elastography (FibroScan) is well studied in adults but not in children. We sought to assess the feasibility of performing FibroScans and the characteristics of FibroScan-based liver profiles in Japanese obese and non-obese children.

Methods

FibroScan examinations were performed in pediatric patients (age, 1–18 yr) who visited Osaka City University Hospital. Liver steatosis measured by controlled attenuation parameter (CAP), and hepatic fibrosis evaluated as the liver stiffness measurement (LSM), were compared among obese subjects (BMI percentile ≥90%), non-obese healthy controls, and non-obese patients with liver disease.

Results

Among 214 children examined, FibroScans were performed successfully in 201 children (93.9%; median, 11.5 yr; range, 1.3–17.6 yr; 115 male). CAP values (mean±SD) were higher in the obese group (n = 52, 285±60 dB/m) compared with the liver disease (n = 40, 202±62, P<0.001) and the control (n = 107, 179±41, P<0.001) group. LSM values were significantly higher in the obese group (5.5±2.3 kPa) than in the control (3.9±0.9, P<0.001), but there were no significant differences in LSM between the liver disease group (5.4±4.2) and either the obese or control group. LSM was highly correlated with CAP in the obese group (ρ = 0.511) but not in the control (ρ = 0.129) or liver disease (ρ = 0.170) groups.

Conclusions

Childhood obesity carries a high risk of hepatic steatosis associated with increased liver stiffness. FibroScan methodology provides simultaneous determination of CAP and LSM, is feasible in children of any age, and is a non-invasive and effective screening method for hepatic steatosis and liver fibrosis in Japanese obese children.     

Prediction of genetic risk for dyslipidemia

The purpose of the present study was to identify genetic variants that confer susceptibility to dyslipidemia. A total of 5213 individuals from two independent populations were examined: Subject panel A comprised 3794 individuals who visited participating hospitals; subject panel B comprised 1419 community-dwelling elderly individuals. The genotypes for 100 polymorphisms of 65 candidate genes were determined. The chi(2) test and multivariable logistic regression analysis revealed that seven polymorphisms of APOA5, APOC3, APOA1, ACAT2, and LPL were significantly associated with hypertriglyceridemia, six polymorphisms of APOA5, LIPC, and CYP3A4 with low HDL-cholesterol, and three polymorphisms of APOE and CCR2 with high LDL-cholesterol in subject panel A. For validation of these associations, the same polymorphisms were examined in subject panel B. Six polymorphisms of APOA5, APOC3, APOA1, and LPL were again significantly associated with hypertriglyceridemia, three polymorphisms of APOA5 with low HDL-cholesterol, and two polymorphisms of APOE with high LDL-cholesterol. Serum triglyceride, HDL-cholesterol, and LDL-cholesterol concentrations differed significantly among genotypes of these corresponding polymorphisms in both subject panels. These results indicate that polymorphisms of APOA5, APOC3, APOA1, and LPL are determinants of hypertriglyceridemia and that those of APOA5 and APOE are determinants of low HDL-cholesterol and high LDL-cholesterol, respectively, in Japanese individuals.     

Role of growth factor receptor bound protein 7 in hepatocellular carcinoma

The human growth factor receptor-bound protein 7 (Grb7) is an adaptor molecule and is related to cell invasion. In this present study, we investigated the clinical and biological significance of Grb7 expression in human hepatocellular carcinoma (HCC). We reviewed 64 consecutive patients who had undergone liver resection for HCC, and we investigated the correlation between Grb7 expression and clinical outcome. To analyze the biological behavior of Grb7 in vitro and in vivo, we established Grb7 stable knockdown HCC cells using RNA interference technology. The positive staining of Grb7 protein was correlated with portal venous invasion (P < 0.01), hepatic venous invasion (P < 0.01), and intrahepatic metastasis (P < 0.05). Positive expression of Grb7 was significantly correlated with focal adhesion kinase (FAK) protein levels in HCC (P < 0.01). The Grb7- and FAK-positive group showed a significantly poorer prognosis as compared with the Grb7- and FAK-negative group (P < 0.05). Grb7 knockdown HCC cells exhibited significantly lower levels of invasion potential (P < 0.05) and motility (P < 0.05) than the control cells in vitro; moreover, Grb7 knockdown HCC cells showed delayed onset of the tumors compared with the control cells in vivo. Grb7 expression can modulate the invasive phenotype of HCC. Grb7 plays an important role in HCC progression and is strongly associated with expression of FAK. Grb7 could be a therapeutic target in HCC.     

Down-regulation of cyclin E1 expression by microRNA-195 accounts for interferon-β-induced inhibition of hepatic stellate cell proliferation

Recent studies have suggested that interferons (IFNs) have an antifibrotic effect in the liver independent of their antiviral effect although its detailed mechanism remains largely unknown. Some microRNAs have been reported to regulate pathophysiological activities of hepatic stellate cells (HSCs). We performed analyses of the antiproliferative effects of IFNs in HSCs with special regard to microRNA-195 (miR-195). We found that miR-195 was prominently down-regulated in the proliferative phase of primary-cultured mouse HSCs. Supporting this fact, IFN-β induced miR-195 expression and inhibited the cell proliferation by delaying their G1 to S phase cell cycle progression in human HSC line LX-2. IFN-β down-regulated cyclin E1 and up-regulated p21 mRNA levels in LX-2 cells. Luciferase reporter assay revealed the direct interaction of miR-195 with the cyclin E1 3'UTR. Overexpression of miR-195 lowered cyclin E1 mRNA and protein expression levels, increased p21 mRNA and protein expression levels, and inhibited cell proliferation in LX-2 cells. Moreover miR-195 inhibition restored cyclin E1 levels that were down-regulated by IFN-β. In conclusion, IFN-β inhibited the proliferation of LX-2 cells by delaying cell cycle progression in G1 to S phase, partially through the down-regulation of cyclin E1 and up-regulation of p21. IFN-induced miR-195 was involved in these processes. These observations reveal a new mechanistic aspect of the antifibrotic effect of IFNs in the liver.     

Venestatin from parasitic helminths interferes with receptor for advanced glycation end products (RAGE)-mediated immune responses to promote larval migration

Parasitic helminths can reside in humans owing to their ability to disrupt host protective immunity. Receptor for advanced glycation end products (RAGE), which is highly expressed in host skin, mediates inflammatory responses by regulating the expression of pro-inflammatory cytokines and endothelial adhesion molecules. In this study, we evaluated the effects of venestatin, an EF-hand Ca²⁺-binding protein secreted by the parasitic helminth Strongyloides venezuelensis, on RAGE activity and immune responses. Our results demonstrated that venestatin bound to RAGE and downregulated the host immune response. Recombinant venestatin predominantly bound to the RAGE C1 domain in a Ca²⁺-dependent manner. Recombinant venestatin effectively alleviated RAGE-mediated inflammation, including footpad edema in mice, and pneumonia induced by an exogenous RAGE ligand. Infection experiments using S. venezuelensis larvae and venestatin silencing via RNA interference revealed that endogenous venestatin promoted larval migration from the skin to the lungs in a RAGE-dependent manner. Moreover, endogenous venestatin suppressed macrophage and neutrophil accumulation around larvae. Although the invasion of larvae upregulated the abundance of RAGE ligands in host skin tissues, mRNA expression levels of tumor necrosis factor-α, cyclooxygenase-2, endothelial adhesion molecules vascular cell adhesion protein-1, intracellular adhesion molecule-1, and E-selectin were suppressed by endogenous venestatin. Taken together, our results indicate that venestatin suppressed RAGE-mediated immune responses in host skin induced by helminthic infection, thereby promoting larval migration. The anti-inflammatory mechanism of venestatin may be targeted for the development of anthelminthics and immunosuppressive agents for the treatment of RAGE-mediated inflammatory diseases.     

Purification and Chemical Properties of an Inhibitor of Plant Virus Infection from Fruiting Bodies of Lentinus edodes

A new inhibitor of plant virus infection from fruiting bodies of Lentinus edodes was purified by a method using fractionation with DEAE-Cellulose, gel filtration on Sephadex G-75, and column chromatography on CM-Toyopearl 650M. The purified inhibitor thus obtained was homogeneous on disc and SDS disc electrophoreses, and the molecular weight of the inhibitor was 23,000. The inhibitor consisted of 17.4% nitrogen, which was found to be a basic simple protein, but contained no neutral sugar, hexosamine nor sialic acid. The inhibitor was estimated to be composed of about 199 amino acid residues. This inhibitor was named “fruiting body protein (FBP).”     

Effect of cyanocobalamin and p-toluic acid on the fatty acid composition of Schizochytrium limacinum (Thraustochytriaceae, Labyrinthulomycota)

Changes in the fatty acid composition of docosahexaenoic acid (DHA)-producing Schizochytrium limacinum SR21 were investigated. The addition of cyanocobalamin, which is an active component of vitamin B₁₂, decreased the content of odd-chain fatty acids such as pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0). Cyanocobalamin may upregulate the cobalamin-dependent methylmalonyl-CoA mutase, which converts propionic acid to succinic acid, thereby decreasing the content of odd-chain fatty acids. The addition of p-toluic acid resulted in a decrease in docosapentaenoic acid (DPA, 22:5n-6) content and an increase in eicosapentaenoic acid (EPA, 20:5n-3) content in a dose-dependent manner. Two additional peaks of fatty acids, characterized as Δ4,7,10,14-eicosatetraenoic acid (20:4n-7) and Δ4,7,10,14-docosatetraenoic acid (22:4n-9), were detected.     

The characteristics of desaturation of Trichoderma sp. AM076 and formation of 9,12,15-hexadecatrienoic acid (16 : 3ω1) through Δ15 desaturation of 9,12-hexadecadienoic acid

We investigated the characteristics of desaturation in Trichoderma sp. AM076. Although 6,9,12-octadecatrienoic acid (18 : 3ω6) was detected when Trichoderma sp. AM076 was cultivated in the presence of 6,9-octadecadienoic acid (18 : 2ω9), the desaturation products of 6,9,12-octadecatrienoic acid (18 : 3ω6) and 6-octadecenoic acid (18 : 1Δ6) were not detected. These results suggest that the double bonds at the Δ6 position of 18 : 3ω6 and 18 : 1Δ6 disturb their Δ15 and Δ9 desaturation, respectively. This fungus also introduced a double bond at the Δ15 position of 9,12-hexadecadienoic acid (16 : 2ω4), thereby yielding a novel C16 polyunsaturated fatty acid (PUFA) identified as 9,12,15-hexadecatrienoic acid (16 : 3ω1). Further investigations revealed that the mutant having enhanced accumulation of linolenic aid (18 : 3ω3) accumulates 16 : 3ω1 as one of the major PUFAs, together with 9,12-octadecadienoic acid (16 : 2ω4), when grown with palmitoleic acid (16 : 1ω7). These results suggest that, in this strain, the reaction that catalyzes the conversion of linoleic acid to linolenic acid, similar to the conversion of 16 : 2ω4 to 16 : 3ω1, is not ω3 desaturation but Δ15 desaturation.