The N-terminal replacement of an olfactory receptor for the development of a yeast-based biomimetic odor sensor

For the development of a biomimetic odor-sensing system, we investigated the effects of replacing the N-terminus of an olfactory receptor (OR) on its functional expression in the budding yeast, Saccharomyces cerevisiae. Using the mouse olfactory receptor OR226 (mOR226), three types of chimeric ORs were constructed by replacing N-terminal regions of mOR226 with the corresponding regions of the rat I7 receptor, which is known to be functionally expressed in yeast. The replacement of the N-terminal region of mOR226 dramatically affected the expression and localization of the receptor and improved the sensing ability of the yeast cells for the odorant. Furthermore, the replacement of the endogenous yeast G-protein α subunit (Gpa1) by the OR-specific G(olf) drastically elevated the odorant-sensing ability of the yeast cells and caused the cells to display a dose-dependent responsiveness to the odorant. Because of the suitability of yeast cells for screening large-scale libraries, the strategy presented here would be useful for the establishment of advanced biomimetic odor-sensing systems.     

Proteinase-activated Receptor 2 Promotes Cancer Cell Migration through RNA Methylation-mediated Repression of miR-125b

Proteinase activated-receptor 2 (PAR₂) participates in cancer metastasis promoted by serine proteinases. The current study aimed to test the molecular mechanism by which PAR₂ promotes cancer cell migration. In different cancer cells, activation of PAR₂ by activating peptide (PAR₂-AP) dramatically increased cell migration, whereas knock down of PAR₂ inhibited cellular motility. The PAR₂ activation also repressed miR-125b expression while miR-125b mimic successfully blocked PAR₂-induced cell migration. Moreover, Grb associated-binding protein 2 (Gab2) was identified as a novel target gene of miR-125b and it mediated PAR₂-induced cell migration. The correlation of PAR₂ with miR-125b and Gab2 was further supported by the findings obtained from human colorectal carcinoma specimens. Remarkably, knock down of NOP2/Sun domain family, member 2 (NSun2), a RNA methyltransferase, blocked the reduction in miR-125b induced by PAR₂. Furthermore, PAR₂ activation increased the level of N⁶-methyladenosine (m⁶A)-containing pre-miR-125b in NSun2-dependent manner. Taken together, our results demonstrated that miR-125b mediates PAR₂-induced cancer cell migration by targeting Gab2 and that NSun2-dependent RNA methylation contributes to the down-regulation of miR-125b by PAR₂ signaling. These findings suggest a novel epigenetic mechanism by which microenvironment regulates cancer cell migration by altering miRNA expression.     

Biased Signaling in Naturally Occurring Mutations in Human Melanocortin-3 Receptor Gene

The melanocortin-3 receptor (MC3R) is primarily expressed in the hypothalamus and plays an important role in the regulation of energy homeostasis. Recently, some studies demonstrated that MC3R also signals through mitogen-activated protein kinases (MAPKs), especially extracellular signal-regulated kinases 1 and 2 (ERK1/2). ERK1/2 signaling is known to alter gene expression, potentially contributing to the prolonged action of melanocortins on energy homeostasis regulation. In the present study, we performed detailed functional studies on 8 novel naturally occurring MC3R mutations recently reported, and the effects of endogenous MC3R agonist, α-melanocyte stimulating hormone (MSH), on ERK1/2 signaling on all 22 naturally occurring MC3R mutations reported to date. We found that mutants D158Y and L299V were potential pathogenic causes to obesity. Four residues, F82, D158, L249 and L299, played critical roles in different aspects of MC3R function. α-MSH exhibited balanced activity in G_s-cAMP and ERK1/2 signaling pathways in 15 of the 22 mutant MC3Rs. The other 7 mutant MC3Rs were biased to either one of the signaling pathways. In summary, we provided novel data about the structure-function relationship of MC3R, identifying residues important for receptor function. We also demonstrated that some mutations exhibited biased signaling, preferentially activating one intracellular signaling pathway, adding a new layer of complexity to MC3R pharmacology.     

The role of mitochondria in sepsis-induced cardiomyopathy

Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. Myocardial dysfunction, often termed sepsis-induced cardiomyopathy, is a frequent complication and is associated with worse outcomes. Numerous mechanisms contribute to sepsis-induced cardiomyopathy and a growing body of evidence suggests that bioenergetic and metabolic derangements play a central role in its development; however, there are significant discrepancies in the literature, perhaps reflecting variability in the experimental models employed or in the host response to sepsis. The condition is characterised by lack of significant cell death, normal tissue oxygen levels and, in survivors, reversibility of organ dysfunction. The functional changes observed in cardiac tissue may represent an adaptive response to prolonged stress that limits cell death, improving the potential for recovery. In this review, we describe our current understanding of the pathophysiology underlying myocardial dysfunction in sepsis, with a focus on disrupted mitochondrial processes.     

Renewing olfactory receptor neurons in goldfish do not require contact with the olfactory bulb to develop normal chemical responsiveness

This study investigated whether contact with the olfactory bulb was necessary for developing and renewing olfactory receptor neurons (ORNs) to attain normal odorant responsiveness, and whether the anatomical and functional recoveries of the olfactory epithelium were similar in both bulbectomized (BE) and bilaterally axotomized (AX) preparations. In vivo electrophysiological recordings were obtained in response to amino acids, a bile acid [taurolithocholic acid sulfate(TLCS)] and a pheromonal odorant [17α, 20β,-dihydroxy-4-pregnen-3-one (17,20P)] from sexually immature goldfish. Both transmission and scanning electron microscopy indicated that the olfactory epithelium degenerated in BE and AX goldfish. Within 1–2 weeks subsequent to the respective surgeries, responses to high concentrations (>0.1 mmol · l⁻¹) of the more stimulatory amino acids remained, whereas responses were no longer obtainable to TLCS and 17,20P. At 4 weeks, responses to amino acid stimuli recovered to control levels, while responses to TLCS and 17,20P were minimal. By 7 weeks post bilateral axotomy, the olfactory epithelium recovered to a condition similar to control sensory epithelium; however, the rate of degeneration and proliferation of receptor neurons in BE preparations appeared to remain in balance, thus blocking further recovery of the olfactory epithelium. At 7 weeks post surgery, odorant responses of AX and BE goldfish to TLCS and 17,20P were still recovering. Accepted: 14 June 1997     

Expression in promoter variant of the ERα gene in bos taurus liver

Due to the variant functions that estrogens play in the regulation of reproduction, development of the mammary gland, growth and differentiation of cells, estrogen receptors and their genes are considered as a candidates for the markers of production and functional traits in farm animals, including cattle. In the earliest study, a 2853-bp bovine ER gene 5′-region was PCR amplified and sequenced. Moreover, for the first time, a polymorphism was described within 5′ region of the bovine ERα gene—A/G transition lying upstream at position 2591 from acceptor splice site +85, possibly within its promoter—which could be recognized with RFLP-BglI. In other study we are found second polymorphism—A/G transition at position 1213 from acceptor splice site +85, located in promoter for exon B. We have examined the specific mRNA expression of ERα in various genotypes using real-time RT-PCR. We used four animals from each genotype group—AG, GG for BglI and AA, AG for SnaBI—to analyse liver ERα expression at the level of Real-time PCR. Liver samples were taken from the 16 young Friesian bulls of the different ERα genotypes, slaughtered at the local abattoir. As shown by Real-Time PCR, on the livers of animals with different genotype ERα mRNA for BglI polymorphism we didn’t found variability, but for SnaBI we have found variability between AG and AA genotypes.     

Gintonin, a ginseng-derived novel ingredient, evokes long-term potentiation through N-methyl-D-aspartic acid receptor activation: Involvement of LPA receptors

Ginseng has been shown to have memory-improving effects in human. However, little is known about the active components and the molecular mechanisms underlying its effects. Recently, we isolated novel lysophosphatidic acids (LPAs)-ginseng protein complex derived from ginseng, gintonin. Gintonin activates G protein-coupled LPA receptors with high affinity. Gintonin activated Ca²⁺-activated Clchannels in Xenopus oocytes through the activation of endogenous LPA receptor. In the present study, we investigated whether the activation of LPA receptor by gintonin is coupled to the regulation of N-methyl-d-aspartic acid (NMDA) receptor channel activity in Xenopus oocytes expressing rat NMDA receptors. The NMDA receptor-mediated ion current (I _NMDA ) was measured using the two-electrode voltage-clamp technique. In oocytes injected with cRNAs encoding NMDA receptor subunits, gintonin enhanced I _NMDA in a concentration-dependent manner. Gintonin-mediated I _NMDA enhancement was blocked by Ki16425, an LPA1/3 receptor antagonist. Gintonin action was blocked by a PLC inhibitor, IP₃ receptor antagonist, Ca²⁺ chelator, and a tyrosine kinase inhibitor. The site-directed mutation of Ser1308 of the NMDA receptor, which is phosphorylated by protein kinase C (PKC), to an Ala residue, or co-expression of receptor protein tyrosine phosphatase with the NMDA receptor attenuated gintonin action. Moreover, gintonin treatment elicited a transient elevation of [Ca²⁺]_i in cultured hippocampal neurons and elevated longterm potentiation (LTP) in both concentration-dependent manners in rat hippocampal slices. Gintonin-mediated LTP induction was abolished by Ki16425. These results indicate that gintonin-mediated I _NMDA potentiation and LTP induction in the hippocampus via the activation of LPA receptor might be responsible for ginseng-mediated improvement of memory-related brain functions.     

Pheromone-mediated sexual selection in the mothUtetheisa ornatrix: Olfactory receptor neurons responsive to a male-produced pheromone

Male Utetheisa ornatrixhave a pair of eversible glandular brushes (coremata) which are displayed during precopulatory interactions with the female. Earlier studies have shown that a pheromone associated with the coremata, hydroxydanaidal (HD), is derived by the males from pyrrolizidine alkaloids (PAs) that they sequester as larvae from their foodplants (Crotalariaspp.) The PAs impart a distastefulness upon Utetheisathat protects both larvae and adults against predation. The receptor neurons specialized for detection of HD are housed in sensilla whose morphological features, as revealed by scanning electron microscopy, classify them as sensilla basiconica. The sensitivity and dynamic range of these receptor neurons were largely unaffected by whether the females were raised on an alkaloid-free diet or on a diet supplemented with Crotalariaseeds. Acetylation of the hydroxyl group of HD substantially reduced the activity of the molecule. None of the antennal sensilla examined contained receptor neurons sensitive to a PA (monocrotaline) or its N-oxide.     

A role for acetylcholine receptors in their own aggregation on muscle cells

Both neurotrophic factors and activity regulate synaptogenesis. At neuromuscular synapses, the neural factor agrin released from motor neuron terminals stimulates postsynaptic specialization by way of the muscle specific kinase MuSK. In addition, activity through acetylcholine receptors (AChRs) has been implicated in the stabilization of pre- and postsynaptic contacts on muscle at various stages of development. We show here that activation of AChRs with specific concentrations of nicotine is sufficient to induce AChR aggregation and that this induction requires the function of L-type calcium channels (L-CaChs). Furthermore, AChR function is required for agrin induced AChR aggregation in C2 muscle cells. The same concentrations of nicotine did not induce observable tyrosine phosphorylation on either MuSK or the AChR beta subunit, suggesting significant differences between the mechanisms of agrin and activity induced aggregation. The AChR/L-CaCh pathway provides a mechanism by which neuromuscular signal transmission can act in concert with the agrin-MuSK signaling cascade to regulate NMJ formation.