Differential G protein activation by fusion proteins between the human delta-opioid receptor and Gi1alpha/Go1alpha proteins

Assessment of the functional activities of agonists acting on the human 6-opioid receptor (hDOR) which couples to G proteins of the Gi/Goalpha class usually involves the measurement of adenylyl cyclase (AC) activity. To assess the relative capacitiy of the hDOR to activate closely related G proteins, fusion proteins were constructed in which the a subunits of either Gi1 or Go1, containing point mutations to render them insensitive to the actions of pertussis toxin (PTx), were linked in frame with the C- terminus of the receptor. Following transient and stable expression in HEK293 cells both constructs bound the antagonist [3H] naltrindole with high affinity. D-ala2, D- leu5 enkephalin [DADLE] effectively inhibited forskolin-stimulated AC activity in intact cells in a concentration-dependent but PTx-insensitive manner. The high affinity GTPase activity of both constructs was also stimulated by DADLE with similar potency. However, enzyme kinetic analysis of agonist stimulation of GTPase activity demonstrated that the GTP turnover number produced in response to DADLE was more than 3 times greater for Gi1alpha than for Go1alpha. As the effect of agonist in both cases was to increase Vmax without increasing the observed Km, for GTP this is consistent with receptor promoting greater guanine nucleotide exchange on, and thus activation of, Gi1alpha compared to Go1alpha. An equivalent fusion protein between the human mu-opioid receptor-1 and Gi1alpha produced a similar DADLE-induced GTP turnover number as the hDOR-Gi1alpha fusion construct, consistent with agonist occupation of these two opioid receptor subtypes being equally efficiently coupled to activation of Gi1alpha. In addition, I have investigated the characteristics of the ternary complex of agonist-GPCR-G protein using high affinity agonist binding studies with the fusion proteins. GDP reduced the binding of the agonist [3H] DADLE but not the antagonist [3H] naltrindole to both the receptor alone and all the hDOR-Gi1alpha (Xaa351) fusion proteins. For the fusion proteins the pEC50 for GDP was strongly correlated with the n-octanol/H2O partition co-efficient of G protein residue351 Fusion proteins in which this residue was either isoleucine or glycine displayed similar association kinetics for [3H] DADLE. However, the rate of dissociation of [3H] DADLE was substantially greater for the glycine-containing fusion protein than that containing isoleucine, indicating the more hydrophobic residue imbued greater stability to the agonist-receptor-G protein ternary complex. This resulted in a higher affinity of binding of [3H] DADLE to the fusion protein containing isoleucine351. In expectation with the binding data, maximal DADLE-stimulated GTP hydrolysis was 2 fold greater with the isoleucine351 containing fusion protein and the potency of DADLE was 7 fold greater with the isoleucine351 fusion protein than for the version containing glycine. These results demonstrate that the stability of the ternary complex between hDOR, Gi1alpha and an agonist (but not antagonist) ligand is dependent upon the nature of residue351 of the G protein and that this determines the effectiveness of information flow from receptor to G protein

Spatial distribution and temporal trends of cyclic and linear siloxanes in sediment from semi-enclosed and industrialized bays of Korea, in 2013 and 2021

Although siloxanes are contaminants of emerging concerns, limited studies have been conducted on contamination and time trends in siloxanes from aquatic environments worldwide. To date, most previous studies have focused on the environmental relevance of cyclic siloxanes in coastal environments. In the present study, cyclic and linear siloxanes were measured in sediment collected from semi-enclosed bays in Korea in 2013 and 2021 to assess occurrence, spatial distribution, temporal trends, and ecological risks. Almost all siloxanes were detected in all sediment samples, indicating continuous contamination for the last decade. The concentrations of cyclic siloxanes in sediment were approximately two times higher than those of linear siloxanes. Decamethylcyclopentasiloxane (D5) and dodecamethylcyclohexasiloxane (D6) were predominantly detected in all sediment samples, possibly due to their high consumption and strong resistance. The overall concentrations of siloxanes in sediment gradually decreased from inner to outer parts of the bays, suggesting that industrial activities largely contributed to siloxane contamination. Moreover, the highest concentrations of siloxanes in both sampling years were observed in sediment near the outfall of wastewater treatment plant, implying a potential source in the coastal environment. Siloxane concentrations in sediment were significantly correlated with sedimentary organic carbon. The concentrations of siloxanes in sediment were not significantly different between 2013 and 2021, indicating ongoing contamination. The concentrations of D5 and D6 in our sediment samples showed a limited potential to pose a threat to benthic organisms

Discovery of chemerin as the new chemoattractant of human mesenchymal stem cells

Background The homing capacity of human mesenchymal stem cells (hMSCs) to the injured sites enables systemic administration of hMSCs in clinical practice. In reality, only a small proportion of MSCs are detected in the target tissue, which is a major bottleneck for MSC-based therapies. We still dont know the mechanism how MSCs are chemo-attracted to certain target organ and engrafted through trans-endothelial migration. In this study, we aimed to determine the mechanism how the circulating hMSCs home to the injured liver. Methods and results When we compare the cytokine array between normal and injured mouse liver at 1-day thioacetamide (TAA)-treatment, we found that chemerin, CXCL2, and CXCL10 were higher in the injured liver than normal one. Among three, only chemerin was the chemoattractant of hMSCs in 2D- and 3D-migration assay. Analysis of the signal transduction pathways in hMSCs showed that chemerin activated the phosphorylation of JNK1/2, ERK1/2 and p38, and finally upregulated CD44, ITGA4, and MMP-2 that are involved in the transendothelial migration and extravasation of MSCs. Upstream transcription regulators of CD44, ITGA4, and MMP-2 after chemerin treatment were MZF1, GATA3, STAT3, and STAT5A. To develop chemerin as a chemoattractant tool, we cloned gene encoding the active chemerin under the CMV promoter (CMV-aChemerin). We analyzed the migration of hMSCs in the 3D model for space of the Disse, which mimics transmigration of hMSCs in the liver. CMV-aChemerin-transfected hepatocytes were more effective to attract hMSC than control hepatocytes, leading to the enhanced transendothelial migration and homing of hMSCs to liver. The homing efficiency of the intravascularly-delivered hMSCs to liver was evaluated after systemic introduction of the CMV-aChemerin plasmid packed in liposome-vitamin A conjugates which target liver. CMV-aChemerin plasmid targeting liver significantly enhanced homing efficiency of hMSCs to liver compared with control plasmid vector. Conclusions Chemerin is the newly found chemoattractant of hMSCs and may be a useful tool to manipulate the homing of the intravascularly-administered hMSC to the specific target organ.This work was supported by the Korea Health Technology R&D Project Strategic Center of Cell and Bio Therapy [Grant number HI17C2085] and Korea Research-Driven Hospital [Grant number HI14C1277] through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare (MHW), Republic of Korea. The funders had no role in the study design, data collection and analysis, deci‑sion to publish, or preparation of the manuscript

Simultaneous determination of 13 mycotoxins in feedstuffs using QuEChERS extraction

Mycotoxins are secondary metabolites produced by various fungi and are known to have a significant negative impact on human and animal health. When feedstuffs are contaminated with mycotoxins, their toxicities may be caused a variety of diseases. In this study, the residual mycotoxins in feedstuffs were analyzed using LC-MS/MS incorporated with QuEChERS extraction. Analytical method validation was performed for LOD, LOQ, linearity, and recoveries with consideration of matrix effects prior to the residual analysis. They were all reached to the accepted range of validation level. Using 39 feedstuff samples (5 g) for mycotoxin analysis, nine samples were contaminated by four major mycotoxins such as fumonisin B1 (FB1), deoxynivalenol, fumonisin B2, and zearalenone. Among them, FB1 was detected at the highest concentration as 18.0943 mg/kg. The total sum of fumonisins in 39 samples did not exceed the maximum residual level (MRL) criterion set by Korean Food and Drug Administration. Altogether, intensive management of mycotoxins in Korean feedstuffs should be implemented with proper and routine monitoring, even their residual concentrations are not exceeded over the MRL levels because of high frequent detection found in this study

A Role of Canonical Transient Receptor Potential 5 Channel in Neuronal Differentiation from A2B5 Neural Progenitor Cells

Store-operated Ca2+ entry (SOCE) channels are the main pathway of Ca2+ entry in non-excitable cells such as neural progenitor cells (NPCs). However, the role of SOCE channels has not been defined in the neuronal differentiation from NPCs. Here, we show that canonical transient receptor potential channel (TRPC) as SOCE channel influences the induction of the neuronal differentiation of A2B5+ NPCs isolated from postnatal-12-day rat cerebrums. The amplitudes of SOCE were significantly higher in neural cells differentiated from proliferating A2B5+ NPCs and applications of SOCE blockers, 2-aminoethoxy-diphenylborane (2-APB), and ruthenium red (RR), inhibited their rise of SOCE. Among TRPC subtypes (TRPC1-7), marked expression of TRPC5 and TRPC6 with turned-off TRPC1 expression was observed in neuronal cells differentiated from proliferating A2B5+ NPCs. TRPC5 small interfering RNA (siRNA) blocked the neuronal differentiation from A2B5+ NPCs and reduced the rise of SOCE. In contrast, TRPC6 siRNA had no significant effect on the neuronal differentiation from A2B5+ NPCs. These results indicate that calcium regulation by TRPC5 would play a key role as a switch between proliferation and neuronal differentiation from NPCs

Comparison between Kidney and Hemoperfusion for Paraquat Elimination

The mortality rate of acute paraquat (PQ) poisoning depends on the PQ concentration in the blood. It has been shown that the kidneys eliminate PQ effectively. However, early renal function deterioration is frequently observed in acute PQ intoxication. This study is designed to compare the efficacy of PQ elimination with hemoperfusion (HP) and kidneys, taking into account the functional deterioration of the kidneys. The amount of renal and HP excretion of PQ were measured during the procedure of HP in patients with acute PQ intoxication. The PQ clearance and the actual amount of PQ elimination by the HP cartridge during the HP procedure were 111±11 mL/min (range; 13.2-162.2 mL/min) and 251.4±506.3 mg (range; 4.6-1,655.7) each. While, the renal clearance and actual amount of renal elimination of PQ was 79.8±56.0 mL/min (range; 9.7-177.0) and 75.4±73.6 mg (range; 4.9-245.8). As the creatinine clearance decreased, the PQ elimination by HP was as effective as or more effective than the renal elimination. In conclusion, early HP must be provided for life saving treatment in patients with acute PQ intoxication