Olfactory receptor 43 reduces hepatic lipid accumulation and adiposity in mice

Olfactory receptors are primarily expressed in nasal olfactory epithelium, but these receptors are also ectopically expressed in diverse tissues. In this study, we investigated the biological functions of Olfr43, a mouse homolog of human OR1A1, in cultured hepatocytes and mice to assess its functionality in lipid metabolism. Olfr43 was expressed in mouse hepatocytes, and Olfr43 activation by a known ligand, (−)-carvone, stimulated cAMP response element-binding protein (CREB) activity. In ligand-receptor binding studies using site-directed mutagenesis, (−)-carvone binding required two residues, M257 and Y258, in Olfr43. In the mouse study, oral administration of (−)-carvone for 5 weeks in high-fat diet-fed mice improved energy metabolism, including reductions in hepatic steatosis and adiposity, and improved glucose and insulin tolerance. In mouse livers and cultured mouse hepatocytes, Olfr43 activation simulated the CREB-hairy and enhancer of split 1 (HES1)-peroxisome proliferator-activated receptor (PPAR)-γ signaling axis, leading to a reduction in hepatic triglyceride accumulation in the mouse liver. Thus, long-term administration of (−)-carvone reduces hepatic steatosis. The knockdown of Olfr43 gene expression in cultured hepatocytes negated these effects of (−)-carvone. In conclusion, an ectopic olfactory receptor, hepatic Olfr43, regulates energy metabolism via the CREB-HES1-PPARγ signaling axis.     

Chemical sensing of DNT by engineered olfactory yeast strain

With the increasing threat of environmental toxicants including biological and chemical warfare agents, fabricating innovative biomimetic systems to detect these harmful agents is critically important. With the broad objective of developing such a biosensor, here we report the construction of a Saccharomyces cerevisiae strain containing the primary components of the mammalian olfactory signaling pathway. In this engineered yeast strain, WIF-1alpha, olfactory receptor signaling is coupled to green fluorescent protein expression. Using this 'olfactory yeast', we screened for olfactory receptors that could report the presence of the odorant 2,4-dinitrotoluene, an explosive residue mimic. With this approach, we have identified the novel rat olfactory receptor Olfr226, which is closely related to the mouse olfactory receptors Olfr2 and MOR226-1, as a 2,4-dinitrotoluene-responsive receptor.     

Identification and functional characterization of an odorant receptor in pea aphid,Acyrthosiphon pisum

The sensitive olfactory system is necessary for survival of insects.Odorant receptors (ORs)are located on the dendrites of olfactory receptor neurons and play a critical role in odor detection.Insect ORs are functionally analyzed via heterologous expression in a Xenopus oocyte system using a two-electrode voltage-clamp (TEVC)electrophysiological recording.Here,we have identified a novel OR in the pea aphid,Acyrthosiphon pisum,then we cloned and named it ApisOR4.We analyzed the ApisOR4 tissue expression patterns and found expression only in antennae tissues.Further functional analysis using TEVC revealed that ApisOR4 is broadly tuned to eight volatiles,which elicit electrophysiological response in pea aphid antennae.This study provides an initial functional analysis of aphid ORs and identifies candidate volatiles to be used in developing new strategies for aphid control.     

腺苷酸环化酶3缺失下调小鼠嗅觉受体基因表达

主要嗅觉表皮组织（MOE）是哺乳动物感知气味分子的重要器官,气味诱导是嗅觉受体神经元（ORN）活动的起点,嗅觉受体（OR）结合气味分子后通过环腺苷酸（cAMP）信号通路向下游传递信号。腺苷酸环化酶3（AC3）是此通路中的重要分子。为了探讨AC3缺失对小鼠MOE内ORs基因表达的影响,本文以AC3敲除型小鼠(AC3-/-)和野生型小鼠(AC3+/+)为材料,采用荧光定量PCR（qRT-PCR）、荧光原位杂交（FISH）技术分析了部分ORs基因及与其相关因子在MOE中的表达。qRT-PCR表明,3月龄AC3-/-小鼠MOE中嗅觉受体 Olfr15、Olfr16、Olfr533、Olfr536、Olfr1507和Olfr642的表达量均显著下降。出生后PND7、PND30和PND90 三个不同发育时期的AC3-/-小鼠MOE原位杂交显示,嗅觉受体Olfr15、Olfr536和Olfr1507表达的细胞数目均减少。进一步qRT-PCR分析发现,3月龄AC3-/-小鼠嗅觉受体相关因子Rtp1、Rtp2、Reep1、Lhx2、Emx2和Ric-8b的表达也均发生显著下调。由此推测,AC3缺失导致的ORs及其相关因子的表达下调可能是嗅觉行为障碍的原因之一。     

Analysis of chemotactic molecules in bone marrow-derived mesenchymal stem cells and the skin: Ccl27-Ccr10 axis as a basis for targeting to cutaneous tissues

Background aimsAdult stem cells produce a plethora of extracellular matrix molecules and have a high potential as cell-based therapeutics for connective tissue disorders of the skin. However, the primary challenge of the stem cell-based approach is associated with the inefficient homing of systemically infused stem cells to the skin.MethodsWe examined chemotactic mechanisms that govern directional migration of mesenchymal stem cells (MSCs) into the skin by conducting a comprehensive expression analysis of chemotactic molecules in MSCs and defined cutaneous tissues from normal and hereditary epidermolysis bullosa (EB)-affected skin.ResultsAnalysis of chemokine receptors in short-term and long-term MSC cultures showed tissue culture-dependent expression of several receptors. Assessment of epidermis-derived and dermis-derived chemokines showed that most chemotactic signals that originate from the skin preferentially recruit different sets of leukocytes rather than MSCs. Analysis of the chemotactic molecules derived from EB-affected non-blistered skin showed only minor changes in expression of selected chemokines and receptors. Nevertheless, the data allowed us to define the Ccl27-Ccr10 chemotactic axis as the most potent for the recruitment of MSCs to the skin. Our in vivo analysis demonstrated that uniform expression of Ccr10 on MSCs and alteration of Ccl27 level in the skin enhance extravasation of stem cells from circulation and facilitate their migration within cutaneous tissue.ConclusionsCollectively, our study provides a comprehensive analysis of chemotactic signals in normal and EB-affected skin and proof-of-concept data demonstrating that alteration of the chemotactic pathways can enhance skin homing of the therapeutic stem cells.     

Ectopic expression of olfactory receptors and associated G-protein subunits in the head integument of the amphihaline migratory fish hilsa Tenualosa ilisha

The chemosensory nature of the tissue from the dorsal surface of the head (also termed sensory pad; SP) of the amphihaline diadromous fish hilsa Tenualosa ilisha was investigated for odorant receptor (OR), olfactory marker protein (OMP) and G-protein subunits (Gαs-olf, Gαq, Gαo, Gαi3) through immunolocalization and immunoblotting techniques. The immunolocalization of OR, OMP and G-protein subunits showed clear expression of these proteins in the tissues of the SP. Robust expressions of these proteins in the SP were detected with immunoblot analysis. The strong expression of these proteins in the SP indicates that the tissues from this area in riverine T. ilisha may play significant role in chemosensing and signalling through ectopic expression of olfactory receptor proteins which are otherwise reported in olfactory organs in vertebrates. Being migratory in nature, ectopic expression of these receptors in T. ilisha probably helps them to prevent damage to epidermal tissues of the SP, or they may also utilize them as a chemo and mechanosensory tool to optimize chemo-communications during migration.     

Quantitative mRNA Analysis of Eight Bovine 5‐HT Receptor Subtypes in Brain,Abomasum, and Intestine by Real‐Time RT‐PCR

Abstract

Serotoninergic pathways are involved in economically important bovine gastrointestinal (GI) motility disorders such as displaced abomasum and cecal dilatation/dislocation. The existing research tools to investigate the role of serotoninergic pathways in such disorders in ruminants comprise functional pharmacological methods, e.g., in vitro contractility studies in tissue baths, and electromyographical recordings in vivo. However, no tools for quantification of bovine serotonin receptor [5‐hydroxytryptamine receptor (5‐HTR)] expression were available so far. This study aimed to develop real‐time RT‐PCR assays for quantitative mRNA analysis of bovine 5‐HTR subtypes. Because the bovine 5‐HTR coding sequences (CDSs) were completely unknown, multiple species (human, mouse, and rat) alignment of complete CDS was used for primer design in highly homologous regions. LightCycler real‐time RT‐PCR assays (partial CDS) for the following bovine 5‐HTR subtypes were developed and validated: 5‐HTR_1A, 5‐HTR_1B, 5‐HTR_1D, 5‐HTR_1F, 5‐HTR_2A, 5‐HTR_2B, 5‐HTR_2C, and 5‐HTR₄. Intra‐ and inter‐assay coefficients of variation (CV) for the eight established assays were small, ranging from 0.49% to 2.46%. As a first physiological application, 5‐HTR mRNA expression levels were measured in brain, abomasum, and intestine of 10 healthy, lactating dairy cows. The 5‐HTR expression was quantified by normalization to the housekeeping gene glyceraldehyde‐phosphate‐dehydrogenase (GAPDH). The 5‐HTR subtype expression levels ranged from 0.001% (5‐HTR_2C in intestine) to 1% 5‐HTR/GAPDH (5‐HTR_1B and 5‐HTR₄ in intestine). There were high variations of 5‐HTR subtype mRNA expression within tissues across receptor subtypes and within receptor subtypes across tissues. In conclusion, accurate real‐time RT‐PCR assays for quantitative analysis of bovine 5‐HTR subtype gene expression were developed and validated.     

三阴性乳腺癌组织AR、MMP-9、E-cadhenrin表达与临床病理特征及预后的关系研究

摘要 目的：探讨三阴性乳腺癌（TNBC）组织雄激素受体（AR）、基质金属蛋白酶9（MMP-9）、E-钙黏蛋白（E-cadhenrin）表达与临床病理特征及预后的关系。方法：选取2014年1月至2017年1月徐州医科大学附属沭阳医院收集的91例TNBC患者手术切除的癌组织和癌旁组织以及61例乳腺增生组织（对照组）石蜡标本，免疫组化法检测AR、MMP-9、E-cadhenrin表达情况。分析AR、MMP-9、E-cadhenrin表达与TNBC患者临床病理参数之间的关系，Kaplan-Meier生存曲线、COX风险比例回归分析AR、MMP-9、E-cadhenrin表达与TNBC患者预后的关系。结果：TNBC癌组织中MMP-9阳性表达率高于癌旁组织和对照组，AR、E-cadhenrin阳性表达率低于癌旁组织和对照组（P<0.05）。AR表达与分化程度、组织学分级、淋巴结状态有关，MMP-9表达与组织学分级、淋巴结状态、Ki-67表达有关，E-cadhenrin表达与组织学分级、淋巴结状态有关（P<0.05）。Kaplan-Meier生存分析结果显示MMP-9阳性表达患者生存率均低于MMP-9阴性表达患者，AR、E-cadhenrin阴性表达患者生存率均低于AR、E-cadhenrin阳性表达患者（P<0.05）。COX风险比例回归分析结果显示淋巴结状态N1-2、MMP-9阳性表达、AR、E-cadhenrin阴性表达是TNBC患者预后不良的危险因素（P<0.05）。结论：MMP-9过度表达和AR、E-cadhenrin表达缺乏与TNBC患者肿瘤恶性侵袭行为和预后不良有关，评价AR、MMP-9、E-cadhenrin表达状态可为TNBC患者预后预测提供一定的依据。     

A Review of: “Alpha1 – Acid Glycoprotein (Genetics,Biochemistry, Physiological Functions and Pharmacology) P. Baumann,G.B. Eap,W.E. Müller,J.P. Tillement,Eds. Alan R. Liss,New York, 1989; hardbound, 470 pages, $94.00”

Abstract

Increasing complications in incisional hernia surgery call for novel treatments. A gene expression analysis of injured tissues displays important parameters for tissue regeneration. Until today, no reliable method has been described for a quantitative gene expression analysis of hernia tissues. In this work, a protocol is described for the isolation of DNA‐free total RNA of incisional hernias for the first time. Moreover, real‐time RT PCR assays for collagen type I and III and TGF‐β1 are demonstrated for relative gene expression analyses. Both methods enable relative gene expression analyses of hernia tissues for the first time.     

Protein kinase C and receptor kinase gene expression in olfactory receptor neurons

Recent biochemical evidence indicates that protein kinase C (PKC) and G-protein-coupled receptor kinases (GRKs) are involved in olfactory signal termination and desensitization. The polymerase chain reaction (PCR) was used to investigate the expression of PKC and GRK genes in olfactory tissue and in isolated olfactory receptor neurons from channel catfish (Ictalurus punctatus). Sequence analysis of cloned PKC PCR products showed that the α, β, δ, ϵ, and τ isotypes were expressed in olfactory tissue. Sequence analysis of PCR products obtained from isolated olfactory receptor neurons showed that PKCβ and PKCδ were expressed in the receptor cells. A 600-bp GRK PCR product was obtained from isolated olfactory neurons that shared 86% and 92% amino acid sequence identity to the mammalian β-adrenergic receptor kinase gene products βARK1 and βARK2, respectively. Go6976, a specific inhibitor of calcium-regulated PKC activity, completely inhibited odorant-stimulated PKC activity in isolated olfactory cilia. This result suggested that odorant-stimulated PKC activity is mediated by the calcium-sensitive PKCβ isotype. Taken together, these results are consistent with the conclusion that PKCβ and βARK mediate odorant receptor phosphorylation and olfactory signal termination. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 387–394, 1997     

Copper exposure and the degeneration of olfactory receptors in rainbow trout (Oncorhynchus mykiss)

Abstract

Sublethal concentrations of copper in water cause the degeneration of olfactory receptors in rainbow trout (Oncorhynchus mykiss). Receptor cell loss has been correlated to the loss of olfaction in fish and may cause difficulties in olfactory mediated behaviors such as migration. This study investigated the effects of three levels of copper (100, 75 and 50 mg L^?1) on the olfactory epithelium of rainbow trout. Twenty fish randomly allocated between three exposure groups and one control were exposed for 24 hours under static renewal conditions. Light and scanning electron microscopic observations of olfactory tissue were taken to determine the extent of degeneration of receptors. In addition, levels of copper and zinc in the brain tissues were analyzed to determine if the olfactory route was a significant route of copper exposure and transfer to fish brain tissue. Results indicate that degeneration of receptors is related to the concentration of copper. Levels of copper in brain were found to be below detection of the instrument. Levels of zinc were extremely variable ranging from 52 to 132 ng zinc g^?1 brain tissue.     

Genomic organization and evolution of olfactory receptors and trace amine-associated receptors in channel catfish,Ictalurus punctatus

BackgroundChannel catfish (Ictalurus punctatus) live in turbid waters with limited visibility to chase prey within a certain distance. This can be compensated through detecting specific water-soluble substances by the olfactory receptors (ORs) and trace amine associated receptors (TAARs) expressed on the olfactory epithelium.MethodsWe identified the OR and TAAR repertoires in channel catfish, and characterized the genomic organizations of these two gene families by data mining available genomic resources.ResultsA total of 47 putative OR genes and 36 putative TAAR genes were identified in the channel catfish genome, including 27 functional OR genes and 28 functional TAAR genes. Phylogenetic and orthogroup analyses were conducted to illustrate the evolutionary dynamics of the vertebrate ORs and TAARs. Collinear analysis revealed the presence of two conserved orthologous blocks that contain OR genes between the catfish genome and zebrafish genome. The complete loss of a conserved motif in fish OR family H may contribute to the divergence of family H from other families. The dN/dS analysis indicated that the highest degree of selection pressure was imposed on TAAR subfamily 14 among all fish ORs and TAARs.ConclusionsThe present study provides understanding of the evolutionary dynamics of the two gene families (OR and TAAR) associated with olfaction in channel catfish.General significanceThis is the first systematic study of ORs and TAARs in catfish, which could provide valuable genomic resources for further investigation of olfactory mechanisms in teleost fish.     

Identification of specific ligands for orphan olfactory receptors. G protein-dependent agonism and antagonism of odorants

Olfactory receptors are the largest group of orphan G protein-coupled receptors with an infinitely small number of agonists identified out of thousands of odorants. The de-orphaning of olfactory receptor (OR) is complicated by its combinatorial odorant coding and thus requires large scale odorant and receptor screening and establishing receptor-specific odorant profiles. Here, we report on the stable reconstitution of OR-specific signaling in HeLa/Olf cells via G protein alphaolf and adenylyl cyclase type-III to the Ca2+ influx-mediating olfactory cyclic nucleotide-gated CNGA2 channel. We demonstrate the central role of Galphaolf in odorant-specific signaling out of OR. The employment of the non-typical G protein alpha15 dramatically altered the odorant specificities of 3 of 7 receptors that had been characterized previously by different groups. We further show for two OR that an odorant may be an agonist or antagonist, depending on the G protein used. HeLa/Olf cells proved suitable for high-throughput screening in fluorescence-imaging plate reader experiments, resulting in the de-orphaning of two new OR for the odorant (-)citronellal from an expression library of 93 receptors. To demonstrate the G protein dependence of its odorant response pattern, we screened the most sensitive (-)citronellal receptor Olfr43 versus 94 odorants simultaneously in the presence of Galpha15 or Galphaolf. We finally established an EC50-ranking odorant profile for Olfr43 in HeLa/Olf cells. In summary, we conclude that, in heterologous systems, odorants may function as agonists or antagonists, depending on the G protein used. HeLa/Olf cells provide an olfactory model system for functional expression and de-orphaning of OR.     

非小细胞肺癌组织PAK4、PAK5蛋白表达与上皮-间质转化、临床病理特征和预后的关系分析

摘要 目的：探讨非小细胞肺癌（NSCLC）组织p21激活激酶（PAK）4、PAK5蛋白表达与上皮-间质转化（EMT）、临床病理特征和预后的关系。方法：选取2018年1月～2019年12月我院收治的100例NSCLC患者,收集手术切除的癌组织和癌旁组织标本,采用免疫组化法检测NSCLC组织和癌旁组织中PAK4、PAK5和EMT相关蛋白[E-钙粘蛋白（E-Cad）、N-钙粘蛋白（N-Cad）和波形蛋白（VIM）]表达。分析PAK4、PAK5蛋白表达与NSCLC患者病理特征的关系和与EMT相关蛋白的相关性。根据NSCLC组织中PAK4、PAK5表达分为阳性/阴性表达组,采用K-M法绘制PAK4、PAK5阳性/阴性表达NSCLC患者的生存曲线,多因素Cox回归分析NSCLC患者死亡的影响因素。结果：与癌旁组织相比,NSCLC组织中PAK4、PAK5、N-Cad、VIM蛋白阳性表达率升高,E-Cad蛋白阳性表达率降低（P＜0.05）。二列相关性分析显示,NSCLC组织PAK4、PAK5与E-Cad蛋白阳性表达率呈负相关,与N-Cad、VIM蛋白阳性表达率呈正相关（P均＜0.001）。不同分化程度、TNM分期、淋巴结转移NSCLC患者PAK4、PAK5蛋白阳性表达率比较,差异有统计学意义（P＜0.05）。100例NSCLC患者3年总生存率为56.00%（56/100）。K-M生存曲线分析显示,PAK4、PAK5阳性表达组总生存率低于阴性表达组（P＜0.05）。多因素Cox回归分析显示,低分化、TNM分期为ⅢA期、淋巴结转移和PAK4、PAK5蛋白阳性表达为NSCLC患者死亡的独立危险因素（P＜0.05）。结论：NSCLC组织PAK4、PAK5蛋白表达升高,与EMT、分化程度、TNM分期、淋巴结转移和预后有关,可能成为NSCLC诊治的新靶点。     

Metabotropic glutamate receptor expression in olfactory receptor neurons from the channel catfish,Ictalurus punctatus

Metabotropic glutamate receptors (mGluRs) were identified in olfactory receptor neurons of the channel catfish, Ictalurus punctatus, by polymerase chain reaction. DNA sequence analysis confirmed the presence of two subtypes, mGluR1 and mGluR3, that were coexpressed with each other and with the putative odorant receptors within single olfactory receptor neurons. Immunocytochemical data showed that both mGluR subtypes were expressed in the apical dendrites and some cilia of olfactory neurons. Pharmacological analysis showed that antagonists to each mGluR subtype significantly decreased the electrophysiological response to odorant amino acids. α-Methyl-L -CCG1/(2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG), a known antagonist to mGluR3, and (S)-4-carboxyphenylglycine (S-4CPG), a specific antagonist to mGluR1, each significantly reduced olfactory receptor responses to L -glutamate. S-4CPG and MCCG reduced the glutamate response to 54% and 56% of control, respectively, which was significantly greater than their effect on a neutral amino acid odorant, methionine. These significant reductions of odorant response by the antagonists, taken with the expression of these receptors throughout the dendritic and ciliated portions of some olfactory receptor neurons, suggest that these mGluRs may be involved in olfactory reception and signal transduction. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 94–104, 1998     

受体酪氨酸激酶Axl对胶质母细胞瘤细胞增殖、凋亡和侵袭的影响

摘要 目的：研究受体酪氨酸激酶Axl在胶质母细胞瘤组织和细胞系U-118MG细胞中的表达情况及其对U-118MG细胞增殖、凋亡、侵袭的影响。方法：收集2015年3月至2018年5月在本院进行手术切除并经病理分型证实的胶质母细胞瘤组织标本（n=30）,另取脑外伤手术中因作内减压而切除的正常脑组织作为对照（n=28）。采用荧光实时定量 (qRT-PCR)检测正常脑组织和胶质母细胞瘤肿瘤组织中Axl mRNA表达水平;采用Western blot检测人小神经胶质HM细胞、U-118MG细胞以及Axl-shRNA转染后U-118MG细胞中Axl蛋白表达水平;采用CCK-8检测Axl-shRNA转染后U-118MG细胞增殖能力;采用流式细胞术检测Axl-shRNA转染后U-118MG细胞凋亡水平;采用Transwell小室实验检测Axl-shRNA转染后U-118MG细胞的侵袭能力。结果：在胶质母细胞瘤组织中Axl mRNA表达水平显著高于正常脑组织（P<0.05）;U-118MG细胞Axl蛋白表达水平显著高于人小神经胶质细胞系HM细胞,差异有统计学意义（P<0.05）;转染Axl-shRNA后,U-118MG细胞中Axl蛋白表达水平显著降低（P<0.05）。与U-118MG细胞和转染control-shRNA细胞相比, 转染Axl-shRNA的U-118MG细胞增殖能力降低（P<0.05）,凋亡水平升高（P<0.05）,侵袭能力降低（P<0.05）。结论：在胶质母细胞瘤组织和U-118MG细胞中,Axl表达水平显著增高,并且Axl表达水平与U-118MG细胞增殖、凋亡及侵袭密切关联。     

Immunolocalisation of P2X and P2Y nucleotide receptors in the rat nasal mucosa

Purinoceptor subtypes were localised to various tissue types present within the nasal cavity of the rat, using immunohistochemical methods. P2X₃ receptor immunoreactivity was localised in the primary olfactory neurones located both in the olfactory epithelium and vomeronasal organs (VNO) and also on subepithelial nerve fibres in the respiratory region. P2X₅ receptor immunoreactivity was found in the squamous, respiratory and olfactory epithelial cells of the rat nasal mucosa. P2X₇ receptor immunoreactivity was also expressed in epithelial cells and colocalised with caspase 9 (an apoptotic marker), suggesting an association with apoptosis and epithelial turnover. P2Y₁ receptor immunoreactivity was found within the respiratory epithelium and submucosal glandular tissue. P2Y₂ receptor immunoreactivity was localised to the mucus-secreting cells within the VNO. The possible functional roles of these receptors are discussed.