Threonine 180 Is Required for G-protein-coupled Receptor Kinase 3- and β-Arrestin 2-mediated Desensitization of the µ-Opioid Receptor in Xenopus Oocytes

To determine the sites in the µ-opioid receptor (MOR) critical for agonist-dependent desensitization, we constructed and coexpressed MORs lacking potential phosphorylation sites along with G-protein activated inwardly rectifying potassium channels composed of Kir3.1 and Kir3.4 subunits in Xenopus oocytes. Activation of MOR by the stable enkephalin analogue, [D-Ala2,MePhe4,Glyol5]enkephalin, led to homologous MOR desensitization in oocytes coexpressing both G-protein-coupled receptor kinase 3 (GRK3) and beta -arrestin 2 (arr3). Coexpression with either GRK3 or arr3 individually did not significantly enhance desensitization of responses evoked by wild type MOR activation. Mutation of serine or threonine residues to alanines in the putative third cytoplasmic loop and truncation of the C-terminal tail did not block GRK/arr3-mediated desensitization of MOR. Instead, alanine substitution of a single threonine in the second cytoplasmic loop to produce MOR(T180A) was sufficient to block homologous desensitization. The insensitivity of MOR(T180A) might have resulted either from a block of arrestin activation or arrestin binding to MOR. To distinguish between these alternatives, we expressed a dominant positive arrestin, arr2(R169E), that desensitizes G protein-coupled receptors in an agonist-dependent but phosphorylation-independent manner. arr2(R169E) produced robust desensitization of MOR and MOR(T180A) in the absence of GRK3 coexpression. These results demonstrate that the T180A mutation probably blocks GRK3- and arr3-mediated desensitization of MOR by preventing a critical agonist-dependent receptor phosphorylation and suggest a novel GRK3 site of regulation not yet described for other G-protein-coupled receptors

PRODUCTION OF PLYWOOD WITH VENNERS OF Maquira coriacea (Karsten) C.C. Berg

In the present study, the quality of plywood made with veneers of Maquira coriacea (Karsten) C.C. Berg, was evaluated. Plywoods of five 60 cm x 60 cm veneers, glued with urea formaldehyde resin with 65% solids, were made using adhesive formulations A (Resin:100, Extender: 20, Catalyst:1.5, Water:20), B (Resin:100, Extender:40, Catalyst:1.7, Water:40) and C (Resin:100, Extender:60, Catalyst:1.9, Water:60) and grammages of 220 g/m2 and 260 g/m2. Panels were pressed at specific pressure of 10 kgf/cm2, temperature at 120 °C and pressing time of 10 minutes. MOR and MOE in static bending were evaluated in the parallel and perpendicular to the grain as well as the glue line shear strength. Plywood made with formulation A and 260 g/m2 grammage shows greater resistance to static bending parallel, with a 43.51 MPa MOR and 4576 MPa MOE, and perpendicular to the grain with a 21.88 MPa MOR and 2129 MPa MOE, as well as the glue line shearing strength (dry: 2.42 MPa and wet: 1.90 MPa). Likewise, the formulation positively influences the MOR and MOE perpendicular and parallel to the grain, while the grammage only in the perpendicular. Shear strength in the glue line meets the requirements of the correspondent standard, finding that the interaction between the amount of glue used and the formulation is not significant. It is concluded that the plywood of Maquira coriacea shows satisfactory resistance properties for indoor use, being recommendable to use, to reduce costs, the formulation C and a grammage of 220 g/m2

Identification of a Novel “Almost Neutral” Mu Opioid Receptor Antagonist in CHO Cells Expressing the Cloned Human Mu Opioid Receptor

The basal (constitutive) activity of G protein-coupled receptors allows for the measurement of inverse agonist activity. Some competitive antagonists turn into inverse agonists under conditions where receptors are constitutively active. In contrast, neutral antagonists have no inverse agonist activity, and they block both agonist and inverse agonist activity. The mu opioid receptor (MOR) demonstrates detectable constitutive activity only after a state of dependence is produced by chronic treatment with a MOR agonist. We therefore sought to identify novel MOR inverse agonists, and novel neutral MOR antagonists in both untreated and agonist-treated MOR cells. CHO cells expressing the cloned human mu receptor (hMOR-CHO cells) were incubated for 20 hr with medium (control) or 10 μM (2S,4aR,6aR,7R,9S,10aS,10bR)-9-(benzoyloxy)-2-(3-furanyl)dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho-[2,1-c]pyran-7-carboxylic acid methyl ester (herkinorin, HERK). HERK-treatment generates a high degree of basal signaling and enhances the ability to detect inverse agonists. [35S]-GTP-γ-S assays were conducted using established methods. We screened 21 MOR “antagonists” using membranes prepared from HERK-treated hMOR-CHO cells. All antagonists, including CTAP and 6β-naltrexol, were inverse agonists. However, LTC-2 7 4 ( (-)-3-cyclopropylmethyl-2,3,4,4aα,5,6,7,7aα-octahydro-1H-benzofuro[3,2-e]isoquinolin-9-ol)) showed the lowest efficacy as an inverse agonist, and, at concentrations less than 5 nM, had minimal effects on basal [35S]-GTP-γ-S binding. Other efforts in this study identified KC-2-009 ((+)-3-((1R,5S)-2-((Z)-3-Phenylallyl)-2-azabicyclo[3.3.1]nonan-5-yl)phenol hydrochloride) as an inverse agonist at untreated MOR cells. In HERK-treated cells, KC-2-009 had the highest efficacy as an inverse agonist. In summary, we identified a novel and selective MOR inverse agonist (KC-2-009), and a novel MOR antagonist (LTC-274) that shows the least inverse agonist activity among 21 MOR antagonists. LTC-274 is a promising lead compound for developing a true MOR neutral antagonist

Mu-Opioid Receptors Transiently Activate the Akt-nNOS Pathway to Produce Sustained Potentiation of PKC-Mediated NMDAR-CaMKII Signaling

BACKGROUND: In periaqueductal grey (PAG) matter, cross-talk between the Mu-opioid receptor (MOR) and the glutamate N-methyl-D-Aspartate receptor (NMDAR)-CaMKII pathway supports the development of analgesic tolerance to morphine. In neurons, histidine triad nucleotide binding protein 1 (HINT1) connects the regulators of G protein signaling RGSZ1 and RGSZ2 to the C terminus of the MOR. In response to morphine, this HINT1-RGSZ complex binds PKCgamma, and afterwards, the interplay between PKCgamma, Src and Gz/Gi proteins leads to sustained potentiation of NMDAR-mediated glutamate responses. METHODOLOGY/PRINCIPAL FINDINGS: Following an intracerebroventricular (icv) injection of 10 nmol morphine, Akt was recruited to the synaptosomal membrane and activated by Thr308 and Ser473 phosphorylation. The Akt activation was immediately transferred to neural Nitric Oxide Synthase (nNOS) Ser1417. Afterwards, nitric oxide (NO)-released zinc ions recruited PKCgamma to the MOR to promote the Src-mediated phosphorylation of the Tyr1325 NMDAR2A subunit. This action increased NMDAR calcium flux and CaMKII was activated in a calcium-calmodulin dependent manner. CaMKII then acted on nNOS Ser847 to produce a sustained reduction in NO levels. The activation of the Akt-nNOS pathway was also reduced by the binding of these proteins to the MOR-HINT1 complex where they remained inactive. Tolerance to acute morphine developed as a result of phosphorylation of MOR cytosolic residues, uncoupling from the regulated G proteins which are transferred to RGSZ2 proteins. The diminished effect of morphine was prevented by LNNA, an inhibitor of nNOS function, and naltrindole, a delta-opioid receptor antagonist that also inhibits Akt. CONCLUSIONS/SIGNIFICANCE: Analysis of the regulatory phosphorylation of the proteins included in the study indicated that morphine produces a transient activation of the Akt/PKB-nNOS pathway. This activation occurs upstream of PKCgamma and Src mediated potentiation of NMDAR activity, ultimately leading to morphine tolerance. In summary, the Akt-nNOS pathway acts as a primer for morphine-triggered events which leads to the sustained potentiation of the NMDAR-CaMKII pathway and MOR inhibition

The effect of the changes in chemical composition due to thermal treatment on the mechanical properties of Pinus densiflora

Wood’s chemical composition has a close relationship to its mechanical properties. Therefore, chemical analysis such as FTIR spectroscopy offers a reasonable non-destructive method to predict wood strength. Pine (Pinus densiflora) specimens were thermal-treated in different conditions (aerobic and anaerobic) and evaluated by 3-point bending test for modulus of rupture (MOR) and by FTIR spectroscopy for chemical composition. Density and moisture content changes were also assessed in this study. The result showed that both density and equilibrium moisture content at 20 ◦C with 65 % humidity change little at low treatment temperatures, but they decrease at high treatment temperatures and when treated in the presence of oxygen. The MOR was improved by the reactions that occurred, including cellulose crystallisation, lignin condensation and cross-linking, whereas it was decreased by degradation reactions. The MOR were well predicted by two FTIR peak at 1318 cm-1 (relatingto CH2 bond and condensation of G-ring of lignin), and at 1730 cm-1 pertaining to changes to carbonyl groups in hemicelluloses) and density. It was concluded that FTIR spectroscopy provides a suitable method for wood non-destructive mechanical testing

Selective targeting of mu opioid receptors to primary cilia

Opioid receptors are therapeutically important G protein-coupled receptors (GPCRs) with diverse neuromodulatory effects. The functional consequences of opioid receptor activation are known to depend on receptor location in the plasma membrane, but mechanisms mediating selective localization of receptors to any particular membrane domain remain elusive. Here, we demonstrate the targeting of the mu opioid receptor (MOR) to the primary cilium, a discrete microdomain of the somatic plasma membrane, both in vivo and in cultured cells. We further show that ciliary targeting is specific to MORs, requires a 17-residue sequence unique to the MOR cytoplasmic tail, and additionally requires the Tubby-like protein 3 (TULP3) ciliary adaptor protein. Our results reveal the potential for opioid receptors to undergo selective localization to the primary cilium. We propose that ciliary targeting is mediated through an elaboration of the recycling pathway, directed by a specific C-terminal recycling sequence in cis and requiring TULP3 in trans

Pengaruh Suhu Kempa Terhadap Kualitas Balok Laminasi Kombinasi Bambu Petung Dengan Bambu Apus Untuk Komponen Kapal

Penggunaan kayu untuk industri terus mengalami peningkatan baik untuk pemakain structural maupun non structural. Permintaan akan kayu tersebut tidak dapat terpenuhi akibat kurangnya kualitas kayu yang baik. Disisi lain pemanfaatan bambu selama ini belum optimal walapun hasil beberapa penelitian menunjukan bahwa bamboo memiliki kekuatan dan keunggulan dibandingkan dengan material bangunan lainya. Maka dilakukan penelitian tentang laminasi bambu. Penelitian ini bertujuan untuk mengetahui nilai kadar air, kerapatan, kuat Tarik, MOR, modulus elastisitas dari laminasi bambu petung kombinasi bambu apus akibat pengaruh suhu kempa (80°C, 100°C, 120°C, 130°C, 140°C). Dalam penelitian ini dibuat balok laminasi bambu petung kombinasi bambu apus untuk uji kuat tarik mengacu pada standar SNI 03-3399-1994 dan uji kuat lentur mengacu pada standar SNI 03- 3960- 1995. Hasil penelitian untuk untuk pengujian Tarik memiliki kadar air rata-rata 11.81 %, berat jenis terbesar 0.7294 g/cm³ untuk suhu kempa 140°C, kekuatan tarik rata-rata sebesar 97.84 Mpa untuk suhu kempa 120°C. untuk laminasi bambu untuk pengujian lentur memiliki nilai kadar air rata – rata sebesar 11.58%, berat jenis sebesar 0.7219 g/cm³ untuk suhu kempa 140°C, modulus of repture sebesar 101.59 Mpa untuk suhu kempa 140°C , modulus elastisitas 9171 Mpa untuk suhu kempa 140°C