[Altered expression of 15-hydroxyprostaglandin dehydrogenase in chronic rhinosinusitis with nasal polyps].

Objective:To investigate the expression level and regulatory mechanism of 15-hydroxyprostaglandin dehydrogenase（HPGD） in chronic rhinosinusitis with nasal polyps（CRSwNP）. Methods:The expression pattern and level of HPGD in CRSwNP and control was observed using immunofluorescence, and western blot was used for analysis of HPGD expression in nasal polyp tissues. The effect of recombinant human high mobility group box-1（HMGB1） on HPGD expression in primary human nasal epithelial cells was observed, and the potential blocking effect of RAGE neutralizing antibody on HMGB1-induced HPGD expression was investigated. Results:The expression of HPGD was elevated in CRSwNP patients compared to the control, while the protein mainly localized at CD68-positive cells and epithelial cells. Recombinant human HMGB1 stimulated an increase in HPGD expression in primary human nasal mucosal epithelial cells at a time-dependent manner. Additionally, increased phosphorylation levels of MEK and elevated RAGE expression were also observed at 12 hours, but decreased at 24 hours after the incubation of HMGB1. The increase in the expression of HPGD induced by HMGB1 in primary human nasal epithelial cells was partly inhibited with RAGE neutralizing antibody. Conclusion:Elevated HPGD expression is observed in CRSwNP, predominantly in macrophages and epithelial cells. HMGB1 regulates HPGD expression through the RAGE-MEK signaling pathway, potentially providing a new target for future regulation of PGE2levels in CRSwNP.

Inhibition of IL-4/STAT6/IRF4 signaling reduces the epithelial-mesenchymal transition in eosinophilic chronic rhinosinusitis with nasal polyps.

BACKGROUND: Previous studies have shown that epithelial-to-mesenchymal transition (EMT) in nasal epithelial cells is critical for tissue remodeling of chronic rhinosinusitis with nasal polyps (CRSwNP). However, the precise mechanism underlying the EMT remains poorly understood. This study aimed to investigate the role of interleukin-4 (IL-4)/signal transducer and activator of transcription 6 (STAT6)/interferon regulatory factor 4 (IRF4) signaling pathway on EMT in eosinophilic CRSwNP. METHODS: We performed quantitative real-time polymerase chain reaction, immunohistochemistry, immunofluorescent staining, and Western blotting to evaluate the expression of STAT6, IRF4, and EMT markers in sinonasal mucosal samples. Effects of IL-4-induced EMT were determined using primary human nasal epithelial cells (hNECs) from patients with eosinophilic CRSwNP. Wound scratch assay, cell morphology, Western blotting, and immunofluorescence cytochemistry were performed to evaluate EMT, and EMT-related markers. Next, human THP-1 monocytic cells were stimulated by phorbolate-12-myristate-13-acetate to differentiate into M0 and were subsequently polarized into M1 with lipopolysaccharide and interferon-γ, M2 with IL-4. The markers of the macrophage phenotype were assessed by Western blotting. The co-culture system was built to explore the interaction between macrophages (THP-1 cells) and hNECs. After co-culture with M2 macrophages, EMT-related markers of primary hNECs were evaluated by immunofluorescence cytochemistry and Western blotting. Enzymelinked immunosorbent assays were used to detect transforming growth factor beta 1 (TGF-ß1) in THP-1-derived supernatants. RESULTS: STAT6 and IRF4 mRNA and protein expression were significantly upregulated in both eosinophilic and noneosinophilic nasal polyps compared with control tissues. The expression of STAT6 and IRF4 in eosinophilic nasal polyps was higher than those in noneosinophilic nasal polyps. STAT6 and IRF4 were not only expressed in epithelial cells but also in macrophages. The number of STAT6+CD68+ cells and IRF4+CD68+ cells in eosinophilic nasal polyps was higher than those in noneosinophilic nasal polyps and control tissues. EMT was enhanced in eosinophilic CRSwNP compared to the healthy controls and noneosinophilic CRSwNP. IL-4-stimulated human nasal epithelial cells exhibited EMT characteristics. The hNECs co-cultured with M2 macrophages demonstrated high levels of EMT-related markers. The TGF-ß1 level was significantly induced by IL-4 and elevated (M2) rather than control macrophages. The inhibition of STAT6 by AS1517499 reduced the expression of IRF4 in epithelial cells and macrophages and counteracted IL-4-induced EMT in epithelial cells. CONCLUSION: In eosinophilic nasal polyps, IL-4 induces STAT6 signaling to upregulate IRF4 expression in epithelial cells and macrophages. IL-4 promotes EMT of hNECs through the STAT6/IRF4 signaling pathway. IL-4-induced M2 macrophages enhanced EMT of hNECs. Inhibition of STAT6 can downregulate the expression of IRF4 and suppress the EMT process, thus providing a new strategy for the treatment of nasal polyps.

Construction and analysis of a conjunctive diagnostic model of HNSCC with random forest and artificial neural network.

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous tumor that is highly aggressive and ranks fifth among the most common cancers worldwide. Although, the researches that attempted to construct a diagnostic model were deficient in HNSCC. Currently, the gold standard for diagnosing head and neck tumors is pathology, but this requires a traumatic biopsy. There is still a lack of a noninvasive test for such a high-incidence tumor. In order to screen genetic markers and construct diagnostic model, the methods of random forest (RF) and artificial neural network (ANN) were utilized. The data of HNSCC gene expression was accessed from Gene Expression Omnibus (GEO) database; we selected three datasets totally, and we combined 2 datasets (GSE6631 and GSE55547) for screening differentially expressed genes (DEGs) and chose another dataset (GSE13399) for validation. Firstly, the 6 DEGs (CRISP3, SPINK5, KRT4, MMP1, MAL, SPP1) were screened by RF. Subsequently, ANN was applied to calculate the weights of 6 genes. Besides, we created a diagnostic model and nominated it as neuralHNSCC, and the performance of neuralHNSCC by area under curve (AUC) was verified using another dataset. Our model achieved an AUC of 0.998 in the training cohort, and 0.734 in the validation cohort. Furthermore, we used the Cell-type Identification using Estimating Relative Subsets of RNA Transcripts (CIBERSORT) algorithm to investigate the difference in immune cell infiltration between HNSCC and normal tissues initially. The selected 6 DEGs and the constructed novel diagnostic model of HNSCC would make contributions to the diagnosis.

Prognostic value of O6-methylguanine-DNA methyltransferase hypermethylation and expression in head and neck cancer: A systematic review and meta-analysis.

BACKGROUND: O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that maintains the stability of genetic information. MGMT is a strong prognostic biomarker in patients with glioblastoma. However, the effect of its gene hypermethylation and expression on the survival rate of head and neck cancer (HNC) patients is still disputed. Therefore, we conducted a meta-analysis to evaluate the prognostic value of MGMT hypermethylation and expression in HNC patients. METHODS: This meta-analysis was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses 2020 guidelines and was registered at the International Prospective Register of Systematic Reviews (CRD42021274728). Literature related to the survival rate of HNC patients and MGMT was systematically searched in PubMed, Embase, The Cochrane Library and Web of Science electronic databases (published from inception to February 1, 2023). The association was evaluated by the combined hazard ratio (HR) and related 95% confidence interval (CI). Two authors independently screened all records and extracted the data. The certainty of evidence was assessed using the Grading of Recommendations Assessment, Development and Evaluation system. All of the statistical tests used in this meta-analysis were conducted with Stata 12.0 software. RESULTS: We included 5 studies with 564 HNC patients for the meta-analysis. All of the included patients were primary tumors and underwent surgical resection without prior radiotherapy or chemotherapy therapy. No significant heterogeneity was noted between MGMT and overall survival, MGMT and disease-free survival, and a fixed-effects model was used. HNC patients with MGMT hypermethylation and low expression had a poor prognosis, with pooled HR for overall survival (HR = 1.23, 95% CI: 1.10-1.38, P < .001) and disease-free survival (HR = 2.28, 95% CI: 1.45-3.58, P < .001). Subgroup analysis stratified by molecular abnormalities, such as hypermethylation or low expression, showed similar results. The insufficient number of trials included in our study encountered high risk of bias and may increase the deviation of the final meta-analysis results. CONCLUSION: HNC patients with MGMT hypermethylation and low expression were more likely to exhibit poorer survival. MGMT hypermethylation and low expression can predict survival in patients with HNC.

Preliminary study on the effective site and mechanism of action of Meconopsis quintuplinervia Regel in alleviating acute alcoholic liver injury in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Meconopsis quintuplinervia Regel (MQR) belongs to the opium poppy tree plant species, and it has heat purging, detoxification, diuretic, anti-inflammatory, and analgesic effects. AIM OF STUDY: MQR has liver-protective properties and can alleviate liver heat. Therefore, this study aimed to observe the effect of MQR extract on acute alcoholic liver injury in mice and explore the mechanism of action of ethyl acetate extract of MQR (MQR-E) on alcohol-induced liver injury in combination with the network pharmacology. MATERIALS AND METHODS: To induce acute alcoholic liver injury, 52% of edible wine was administered at 12 mL/kg for 14 days. The pharmacodynamic results were used to screen the active site. MQR-E composition was analyzed based on UPLC-Q-TOF-MS, and relevant MQR-E and alcoholic liver disease (ALD) targets were screened using an online database. Then, Venn analysis of drug and disease-related targets was performed to obtain cross-targets. We investigated the protein-protein interaction network (PPI) of overlapping targets, the core targets were screened using the STRING database, and the DAVID database was chosen for GO and KEGG enrichment analysis of the central targets. RESULTS: Each of the four MQR extracts ameliorated alcoholic liver injury to varying degrees; the best results were achieved with MQR-E. MQR-E reduces liver index, serum transaminases, and fat accumulation, and attenuates ethanol-induced histopathological changes. The activities of hepatic superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were increased, the content of malondialdehyde (MDA) was significantly reduced compared to the EtOH group, and MQR-E effectively mitigated the oxidative stress induced by ethanol in the liver. Thirty-six compounds were identified, and flavonoids were the most abundant. PPI network topology analysis was employed to assess 32 core targets: IL-6, TNF, STAT3, PPARA, and other inflammation and lipid metabolism related genes. Pathway analysis of GO and KEGG enrichment showed that the regulation of inflammatory factors and lipid metabolism were primarily involved. CONCLUSION: We concluded that MQR-E had protective effects against acute alcohol-induced liver injury in mice, and the mechanism could be linked to the inhibition of lipid peroxidation and oxidative stress. The mechanism by which MQR-E ameliorated ALD primarily involved regulating inflammatory factors and lipid metabolism based on the prediction of the network pharmacology.

The preparation and relative bioavailability of an artemisin in self-emulsifying drug delivery system.

The aim of this study is to demonstrate a method for improving the solubility and relative bioavailability of artemisinin using a self-emulsifying drug delivery system (SEDDS). The self-emulsifying drug load, solubility, and emulsifying time were used as the evaluation indices, based on a solubility test and a ternary phase diagram. Optimal Mixture Design in Design-Expert software was used to optimize the prescription of the artemisinin SEDDS. By determining the water distribution coefficient in vitro, combined with the drug concentration-time curve in vivo, a comparison was made of the relative oral bioavailability of the artemisinin SEDDS and the crude drug. The optimal prescription ratio of oleic acid polyethylene glycol glyceride, polyoxyethylene hydrogenated castor oil, and diethylene glycol monoethyl ether in the artemisinin SEDDS was 0.5:0.2:0.3 (wt/wt/wt), with a drug loading capacity of 41.556 mg/g, a solubility of 1.997 mg/mL, and a self-emulsification time of 214 s. The optimal prescription was transparent, slightly yellow, and oil-like. The average loading capacity of artemisinin was 41.912 mg/g, the emulsification time was 231 s, the average particle size was 128.0 nm, the average Zeta potential was -4.29 mV, and the solubility of artemisinin SEDDS in water was 1.997 mg mL-1. It is 33.85 times of the solubility of artemisinin in water, which achieves the purpose of increasing the solubility of artemisinin. The comparison of the oil/water distribution coefficient of the artemisinin SEDDS with that of the crude drug in vitro showed that SEDDS could improve the permeability of artemisinin and promote the absorption in vivo, and the relative bioavailability of the SEDDS agent was at least 1.47 times higher than that of the crude drug. The artemisinin SEDDS could significantly improve the solubility and relative bioavailability of artemisinin.

Solitary Fibrous Tumor of the Paranasal Sinuses with Intracranial Extension: A Rare Case Report and Review of the Literature.

Solitary fibrous tumors (SFTs) are rare mesenchymal neoplasms that were initially identified in the pleura. SFTs in the nasal or paranasal sinuses are especially rare. Most SFTs exhibit indolent behavior, with a low local recurrence rate. A 39-year-old man complained of bilateral nasal congestion, hyposmia, and occasional right eye tears six months prior to hospitalization. Based on MRI and CT imaging, a total gross surgical resection was achieved. Subsequently, postsurgical histopathological examinations were conducted. Under the microscope, pathological mitotic bodies were visible (<5 mitoses per 2 mm2). The immunohistochemical staining results revealed that tumor cells were positive for CD34, BCL-2, STAT-6, and Ki-67 (<5%) but negative for EMA, S-100, PR, GFAP, and SMA. Based on these findings, the patient was diagnosed with SFT.

Construction and analysis of a ceRNA network and patterns of immune infiltration in chronic rhinosinusitis with nasal polyps: based on data mining and experimental verification.

Recent studies have revealed the significant role of the competing endogenous RNA (ceRNA) network in human diseases. However, systematic analysis of the ceRNA mechanism in chronic rhinosinusitis with nasal polyps (CRSwNP) is limited. In this study, we constructed a competitive endogenous RNA (ceRNA) network and identified a potential regulatory axis in CRSwNP based on bioinformatics analysis and experimental verification. We obtained lncRNA, miRNA, and mRNA expression profiles from the Gene Expression Omnibus. After analysis of CRSwNP patients and the control groups, we identified 565 DE-lncRNAs, 23 DE-miRNAs, and 1799 DE-mRNAs by the DESeq2 R package or limma R package. Enrichment analysis of 1799 DE-mRNAs showed that CRSwNP was associated with inflammation and immunity. Moreover, we identified 21 lncRNAs, 8 miRNAs and 8 mRNAs to construct the lncRNA-miRNA-mRNA ceRNA network. A potential MIAT/miR-125a/IRF4 axis was determined according to the degree and positive correlation between a lncRNA and its competitive endogenous mRNAs. The GSEA results suggested that IRF4 may be involved in immune cell infiltration. The validation of another dataset confirmed that MIAT and IRF4 were differentially expressed between the CRSwNP and control groups. The area under the ROC curve (AUC) of MIAT and IRF4 was 0.944. The CIBERSORT analysis revealed that eosinophils and M2 macrophages may be involved in the CRSwNP process. MIAT was correlated with dendritic cells and M2 macrophages, and IRF4 was correlated with dendritic cells. Finally, to validate the key genes, we performed in-silico validation using another dataset and experimental validation using immunohistochemistry, immunofluorescence, and Western blot. In summary, the constructed novel MIAT/miR-125a/IRF4 axis may play a critical role in the development and progression of CRSwNP. We believe that the ceRNA network and immune cell infiltration could offer further insight into novel molecular therapeutic targets for CRSwNP.

Oil-water partition coefficient preparation and detection in the dihydroartemisinin self-emulsifying drug delivery system.

BACKGROUND: The aim of the present study is to increase the solubility of dihydroartemisinin (DHA) using the self-emulsifying drug delivery system (SEDDS). METHODS: We first conducted solubility test and ternary phase diagram, then, in order to optimize the formulation of the DHA self-emulsifying agent, the design mixture method was selected in the design expert software. Next, optimal prescription validation and preliminary formulation evaluation were conducted. By comparing the oil-water partition coefficient in vitro, the improvement of the in vivo osmotic absorption of DHA via self-emulsification was evaluated. RESULTS: The optimal prescription ratio of oleic acid polyethylene glycol glyceride, polyoxyethylene hydrogenated castor oil, and diethylene glycol monoethyl ether in the DHA self-emulsifying preparation = 0.511:0.2:0.289 (w/w/w), with a drug-loading capacity of 26.3634 mg/g, solubility of 2.5448 mg/ml, and self-emulsification time of 230 s. The solubility self-emulsification was approximately 20.52 × higher in DHA than in the crude drug. The self-emulsification could improve DHA permeability and promoting in vivo DHA absorption. CONCLUSION: The DHA SEDDS could significantly improve DHA solubility and in vivo absorption.

Survival association of XRCC1 for patients with head and neck squamous cell carcinoma: A systematic review and meta-analysis.

Background: Epidemiologic studies have demonstrated that X-ray repair cross-complementary group 1 (XRCC1) is one of the susceptibility factors in head and neck squamous cell carcinoma (HNSCC) patients. However, its clinical prognostic impact remains controversial. Thus, a meta-analysis was performed to clarify the association between XRCC1 and the survival outcomes in HNSCC patients. Methods: Following the Preferred Reporting Items or Systematic Reviews Meta Analyses (PRISMA) 2020 guidelines, literature searches were systematically performed in PubMed, EMBASE, Web of Science, Wanfang, and Chinese National Knowledge Infrastructure (CNKI) databases with manual retrieval. Hazard ratios (HRs) and 95% confidence intervals (CIs) were collected to estimate the correlation between XRCC1 and the survival outcomes of HNSCC patients. Results: Ten studies including 1995 HNSCC patients who satisfied the inclusion and exclusion criteria were included in this meta-analysis. Pooled analysis indicated that XRCC1 Arg399Gln and XRCC1 high protein expression were significantly correlated with poor overall survival with HR of 1.31 (95% CIs: 1.03-1.66, p = 0.027) and 2.32 (95% CIs: 1.55-3.48 p = 0.000) in HNSCC patients. In addition, our results demonstrated that XRCC1 was significantly associated with poor progression-free survival (HR = 1.42, 95% CIs: 1.15-1.75, p = 0.001) in HNSCC patients. ConclusionThis meta-analysis demonstrated that XRCC1 Arg399Gln and XRCC1 high protein expression increase the risk of poor survival for HNSCC patients. XRCC1 is a potential therapeutic target for HNSCC.

[Research progress of Phyllanthi Fructus and prediction of its Q-markers].

Phyllanthi Fructus, a unique Chinese and Tibetan medicinal plant with both edible and medical values, has high potential of cultivation and development. The resources of Phyllanthi Fructus in China are rich, mainly distributed in Yunnan, Sichuan, Fujian, Guangdong, Guangxi, etc. Phyllanthi Fructus is widely used in the clinical practice of Chinese medicine and plays an important role in Tibetan medicine, Uyghur medicine, Yi medicine, and Mongolian medicine. Phyllanthi Fructus mainly contains phenolic acids,tannins, terpenes, sterols, fatty acids, flavonoids, amino acids and other compounds. Modern pharmacological studies show that Phyllanthi Fructus has antioxidant, anticancer, blood lipid-lowering, liver protective, antimicrobial, anti-inflammatory, and immune regulatory activities. In this paper, the research status of Phyllanthi Fructus was reviewed from the aspects of herbal textual research,chemical composition, and pharmacological action. The quality markers(Q-markers) of Phyllanthi Fructus were predicted and analyzed from the aspects of biogenic pathway, specificity and measurability of chemical components, efficacy, properties, new clinical uses, drug-food homology, and transformation of polyphenols. The results will provide a scientific basis for the quality control, quality evaluation, and standard formulation of Phyllanthi Fructus.

PPAR-γ Ligand Inhibits Nasopharyngeal Carcinoma Cell Proliferation and Metastasis by Regulating E2F2.

PURPOSE: Peroxisome proliferator-activated receptor-γ (PPAR-γ) is a nuclear hormone receptor with a key role in lipid metabolism. Previous studies have identified various roles of PPAR-γ in cell cycle progression, cellular proliferation, and tumor progression. However, no report has described a role for PPAR-γ in human nasopharyngeal carcinoma (NPC). Notably, some studies have reported a relationship between PPAR-γ and E2F transcription factor 2 (E2F2), which has been identified as a regulator of cell cycle, apoptosis, and the DNA damage response. Notably, E2F2 has also been reported to correlate with a poor prognosis in patients with various malignancies. METHODS: We used immunohistochemical (IHC) and western blot methods to evaluate PPAR-γ and E2F2 expression and function in nonkeratinizing NPC and nasopharyngitis (NPG) tissue samples, as well as western blotting and CCK8 analyses in the NPC cell lines, CNE1 and CNE2. RESULTS: We observed lower levels of PPAR-γ expression in nonkeratinizing NPC tissues compared with NPG tissues and determined an association between a low level of PPAR-γ expression with a more advanced tumor stage. Furthermore, strong E2F2 expression was detected in nonkeratinizing NPC tissues. We further demonstrated that rosiglitazone, a PPAR-γ agonist, reduced E2F2 expression and proliferation in NPC cell lines. CONCLUSIONS: Our study results revealed a novel role for the PPAR-γ-E2F2 pathway in controlling NPC cell proliferation and metastasis.

[Efficacy of intratympanic steroid injection as supplementary or initial treatment for sudden sensorineural hearing loss].

OBJECTIVE: To observe the efficacy of intratympanic steroid injection as supplementary or initial treatment for sudden sensorineural hearing loss (SSNHL). METHOD: A total of 68 patients diagnosed with SSNHL were randomized into group A (45 cases including systemic steroid for 33 cases and systemic steroid + intratympanic steroid as supplementary treatment for 12 cases) and group B (23 cases, initial intratympanic steroid). Then observe the therapeutic effect in two groups. RESULT: The total effective rate was 55.6% in group A and 56.5% in group B. No statistical difference was detected between these two groups (P > 0.05). There was statistical difference after therapy of intratympanic steroid as supplementary treatment for 12 patients due to poor hearing improvement after systemic steroid in group A (P < 0.05). CONCLUSION: Both systemic and intratympanic steroid injection for SSNHL are effective. The efficiency of intratympanic steroid injection as supplementary or initial treatment for SSNHL is similar to that of systemic steroid. The intratympanic steroid injection for SSNHL as initial protocol or as supplementary treatment when poor hearing improvement after systemic steroid is recommended.

Synthesis and biological evaluation of 7-azaisoindigo derivatives.

A series of novel 7-azaisoindigo derivatives 3-14 were designed, synthesized, and structurally characterized by IR, 1H-NMR, 13C-NMR, mass spectra, and elemental analyses. Their antiproliferative activities were evaluated in a hormone-independent prostate cancer cell line DU145. Among them, compounds 8, 9, 14 showed the highest activities. Our study also showed that compounds 7, 11, 12 exhibited higher inhibitory activities on CDK2/cyclin A than that of the positive control meisoindigo. Western blot analysis on DU145 cells treated with compounds 7 and 9 demonstrated that 7-azaisoindigo derivatives could decrease the level of CDK2 activity (phosphorylation) and the expression of cyclin D1, and increase the expression of endogenous cyclin-dependent inhibitor p27.

R3(BDelta23 27)R/I5 chimeric peptide, a selective antagonist for GPCR135 and GPCR142 over relaxin receptor LGR7: in vitro and in vivo characterization.

Both relaxin-3 and its receptor (GPCR135) are expressed predominantly in brain regions known to play important roles in processing sensory signals. Recent studies have shown that relaxin-3 is involved in the regulation of stress and feeding behaviors. The mechanisms underlying the involvement of relaxin-3/GPCR135 in the regulation of stress, feeding, and other potential functions remain to be studied. Because relaxin-3 also activates the relaxin receptor (LGR7), which is also expressed in the brain, selective GPCR135 agonists and antagonists are crucial to the study of the physiological functions of relaxin-3 and GPCR135 in vivo. Previously, we reported the creation of a selective GPCR135 agonist (a chimeric relaxin-3/INSL5 peptide designated R3/I5). In this report, we describe the creation of a high affinity antagonist for GPCR135 and GPCR142 over LGR7. This GPCR135 antagonist, R3(BDelta23-27)R/I5, consists of the relaxin-3 B-chain with a replacement of Gly23 to Arg, a truncation at the C terminus (Gly24-Trp27 deleted), and the A-chain of INSL5. In vitro pharmacological studies showed that R3(BDelta23-27)R/I5 binds to human GPCR135 (IC50=0.67 nM) and GPCR142 (IC50=2.29 nM) with high affinity and is a potent functional GPCR135 antagonist (pA2=9.15) but is not a human LGR7 ligand. Furthermore, R3(BDelta23-27)R/I5 had a similar binding profile at the rat GPCR135 receptor (IC50=0.25 nM, pA2=9.6) and lacked affinity for the rat LGR7 receptor. When administered to rats intracerebroventricularly, R3(BDelta23-27)R/I5 blocked food intake induced by the GPCR135 selective agonist R3/I5. Thus, R3(BDelta23-27)R/I5 should prove a useful tool for the further delineation of the functions of the relaxin-3/GPCR135 system.

Recent progress in relaxin-3-related research.

Relaxin-3 (R3) is the latest member of the insulin (INSL) superfamily, which is composed of peptides with diverse sequences held together by characteristic disulfide links connecting A and B peptide chains. R3 has nearly exclusive expression in the brainstem and was demonstrated to be an additional ligand for LGR7. We recently identified R3 as a ligand for two orphan G-protein coupled receptors, GPCR135 and GPCR142. The predominant brain expression for both R3 and GPCR135, coupled with their high affinity interaction, strongly suggests that R3 is the endogenous ligand for GPCR135. Both R3 and GPCR135 from different species are highly conserved from genetic sequences to in vitro pharmacology. By contrast, GPCR142 is a pseudogene in the rat, and the mouse gene is less conserved with human GPCR142, suggesting that GPCR142 may have a diminished role as a receptor for R3 in the rodent. In addition, the tissue expression pattern of GPCR142, which is primarily in peripheral tissue, is drastically different from that of R3, suggesting that GPCR142 may have an endogenous ligand other than R3. Sequence analysis among INSL/relaxin family members shows that INSL5 is the closest member of R3. We were able to demonstrate that INSL-5 is an agonist for GPCR142 but not for GPCR135. We also showed that the mRNA expression pattern of INSL-5 overlaps with that of GPCR142. By substituting the A chain of R3 with the A chain of INSL-5, we devised a chimeric peptide (R3/I5) that is about 1000-fold selective for GPCR135 and GPCR142 over LGR7. Autoradiographic distribution of GPCR135 binding sites using [125I]R3/I5 in rat brain shows that GPCR135 receptor is most prominent in areas known for the processing of sensory signals.

Molecular and pharmacological characterization of serotonin 5-HT2A and 5-HT2B receptor subtypes in dog.

We report the cloning, molecular characterization, and pharmacological characterization of the canine 5-HT2A and 5-HT2B receptors. The canine and human 5-HT2A receptors share 93% amino acid homology. The canine and human 5-HT2B receptors are also highly conserved (87% homology) with the exception of the carboxyl termini where the canine protein is 62 amino acids shorter. Both the canine 5-HT2A and 5-HT2B receptors have high affinity for [3H]5-HT (KD=4.50+/-0.89 nM and 3.10+/-0.82 nM, respectively) and, in general, the pharmacology of these two receptors matches closely the pharmacology of their human homologs for the 19 serotonergic ligands tested. However, the functional response (Ca2+ mobilization) of the canine 5-HT2B receptor to several agonists was weaker compared to the human 5-HT2B receptor. Using quantitative reverse transcriptase polymerase chain reaction, a high expression level of canine 5-HT2A receptor mRNA was detected in the brain and lower levels in peripheral tissues, whereas the highest levels of canine 5-HT2B receptor mRNA were observed in lungs and smooth muscles. A significant level of canine 5-HT2B receptor mRNA was detected in brain tissue. The availability of the full sequence and pharmacology of the canine 5-HT2A and canine 5-HT2B receptors provides useful information for the interpretation of previous and future pharmacological studies of 5-HT2A/2B ligands in dog.

Identification and pharmacological characterization of prokineticin 2 beta as a selective ligand for prokineticin receptor 1.

Prokineticins 1 and 2 (PK1 and PK2) have been recently identified from humans and other mammals and play multiple functional roles. PK proteins are ligands for two G protein-coupled receptors, PK receptor 1 (PKR1) and PK receptor 2 (PKR2). Here, we report the molecular cloning and pharmacological characterization of an alternatively spliced product of the PK2 gene encoding 21 additional amino acids compared with PK2, designated PK2L (for PK2 long form). PK2L mRNA is broadly expressed, as is PK2. However, PK2L mRNA expression is lower in brain, undetectable in kidney, and much higher in lung and spleen than that of PK2. We expressed PK2L in mammalian cells and characterized the resulting peptide in comparison with PK1 and PK2. Biochemical characterization indicates that secreted PK2L protein is processed into a smaller peptide by proteolytic cleavage. We designate this smaller form of peptide as PK2beta. Coexpression of furin with PK2L significantly increased the PK2beta processing efficiency. Functional studies showed that PK1, PK2, and PK2beta stimulate intracellular Ca(2+) responses in PKR1-expressing cells with similar potencies. However, the PK2beta stimulus of Ca(2+) responses in PKR2-expressing cells is at least 10-fold less potent than that of PK1 or PK2. Differences in receptor selectivity combined with differential tissue expression patterns suggest PK2 and PK2beta might have different functions in vivo. PKRs have been reported to couple to G(q) and G(i) proteins. In this report, we show that PKs not only stimulate Ca(2+) mobilization but also induce cAMP accumulation in PKR-expressing cells.

Pharmacological characterization of relaxin-3/INSL7 receptors GPCR135 and GPCR142 from different mammalian species.

Relaxin-3 has recently been identified as a ligand for two structurally related G-protein-coupled receptors, human GPCR135 and GPCR142. This current study reports the characterization of mouse and rat GPCR135 as well as GPCR142 from mouse, monkey, cow, and pig at the molecular and pharmacological levels. Mouse and rat GPCR135 exhibit high homology (>85%) to the human GPCR135 and have very similar pharmacological properties to that of the human GPCR135. Human and mouse/rat relaxin-3 both bind to and activate mouse, rat, and human GPCR135 at high affinity with IC(50) or EC(50) values close to 0.5 nM. In contrast, the mouse GPCR142 is less well conserved (74% homology) with human GPCR142. The rat GPCR142 gene was found to be a pseudogene. We further cloned GPCR142 genes from monkey, cow, and pig and found that they are highly homologous (>84%) to human GPCR142. Pharmacological characterization of GPCR142 from different species demonstrated that relaxin-3 binds to GPCR142 from different species at high affinity (IC(50) < 5 nM). However, relaxin-3 does not stimulate a Ca(2+) response in cells coexpressing Galpha(16) and mouse GPCR142, whereas it does for cells expressing GPCR142 from other species tested. Our results suggest that GPCR142 may have a diminished role as a receptor for relaxin-3 in rodents, or perhaps GPCR142 functions as a receptor for another ligand in nonrodents. Boels and Schaller recently reported bradykinin as a ligand for GPCR142 (also known as GPR100). In this report, we demonstrate that bradykinin activates neither GPCR135 nor GPCR142, whereas relaxin-3 does.