Role Of MOR Research Articles

Invited Review - Fight on Plankton! or, Phytoplankton Shape and Size as Adaptive Tools to Get Ahead in the Struggle for Life

Abstract A renewed interest in investigating the relationships existing between body size and environmental variables is pervading ecological studies. Phytoplankton has a long tradition as model system in studies of community ecology and several research concepts were developed using these organisms. In this paper we try to review the relevance of analyzing the morphological features of phytoplankton in ecology. Starting with a brief account of allometric relationships existing in phytoplankton, we i) examine the physical context in which phytoplankton grow, and ii) highlight the role of their size in nutrient uptake, and that of their shape in light harvesting. Moreover, the way in which the morphology of phytoplankton organisms cope with the hydrodynamical conditions of a given water-body are considered. In addition, we also included a paragraph on the role of grazing in moulding the size and the shape structure of phytoplankton assemblages. An account on the main research currents about the role of mor...

130 EXPRESSION AND LOCALIZATION OF µ OPIOID RECEPTOR IN PORCINE PRE-IMPLANTATION EMBRYOS

Embryonic stem cells can become any tissue in the body, excluding a placenta. Growth factors, hormones, and neurotransmitters have been implicated in the regulation of their fate. Because various neural precursors express functional neurotransmitter receptors, as G-protein-coupled receptors, it is anticipated that they are involved in cell fate decisions. Moreover, a high level of endogenous opioids linked to G-protein-coupled receptor above all μ opioid receptors (MOR) has been shown to interfere with normal calcium metabolism and with the activity of the mitogen-activated protein kinase (MAPK). Thus it is very important to understand the possible influence of opioid activities in the regulation of stem cell fate. In this study we investigated the presence of MOR on porcine in vitro-produced embryos at one-cell, 4-cell, morula, and blastocyst stages by immunostaining. The COC were collected by aspiration, cultured in NCSU-37 medium supplemented with hormones for 20 to 22 h, and then in maturation medium without hormones for 24 h. After this time, COC were inseminated with frozen-thawed epididymal spermatozoa at the concentration of 10 × 5 sperm cells mL-1 for 3 h. After removal of cumulus cells, putative zygotes were cultured in IVC Pyr-Lac medium for the first 2 days and in IVC Glu medium until Day 6 (the day of IVF was defined as Day 0). Embryos at different stages were collected at 12, 36, 120, and 144 h post fertilization, and kept in 4% (v/v) paraformaldehyde until examination. All samples were washed and incubated for 30 min in PBS-1%BSA. Controls were incubated in PBS-1% BSA for 90 min, whereas embryos were incubated with a 1 : 2500 dilution of the primary rabbit antibody against the third extracellular loop of MOR. Prior to examination, all samples were washed in PBS and incubated with a FITC-conjugated anti rabbit IgG-secondary antibody diluted 1:200 in Evans Blue/PBS1x. Samples were visualized by laser scanning confocal microscope (Nikon). The immunofluorescence localize, by intense brilliant green, the presence of MOR on blastomers of all stage embryos examined, whereas the embryos of negative control did not show any fluorescent region or spotted coloring. Our results support specific implication of the opioid receptors in developmental process of porcine embryos. Their presence suggests a possible role of MOR in embryonic development. Thus it can be speculated that there is a role for MOR in controlling key events of the stem cell life. However, these primary results must be confirmed by the demonstration of protein expression (by Western blot) of MOR in the embryos and deeply studied to understand the exact functional role of MOR in them at this level. JSPS short-term scholarship.

Single-Chain Insulins as Receptor Agonists

Single chain insulins (SCIs) are single polypeptide chains in which the insulin B-chain links contiguously with the insulin A-chain via an uncleaved connecting peptide. While direct linkage of insulin Band A-chains produces SCIs with little insulin receptor binding, biologists have been interested in bioengineering linker peptides that form a flexible reverse turn, allowing SCIs to activate insulin receptors. In this report, we have investigated a series of cDNAs intended to explore the significance of linker length, cleavability, and the impact of certain site-dependent residues for the bioactivity of recombinant SCIs on insulin receptors. SCI concentration is readily measured by radioimmunoassay with a (proinsulin insulin)-specific polyclonal antibody. While dibasic flanking residues may result in potential endoproteolytic susceptibility, a linker with -Gln-Argflanking sequences resisted cleavage even in secretory granules, ensuring single-chain behavior. Effective SCIs exhibit favorable and specific binding with insulin receptors. SCIs with linkers bearing an Arg residue immediately preceding the A-chain were most bioactive, although efficient receptor interaction was inhibited as SCI linker length increased, approaching that observed for proinsulin. SCIs activate downstream metabolic signaling — stimulating glucose uptake into adipocytes and suppressing gluconeogenic enzyme biosynthesis in hepatocytes — with only limited cross-reactivity on IGF1 receptors. SCIs might theoretically have utility either in immunotherapy or gene therapy in insulin-deficient diabetes. The MOR-1 Opioid Receptor Regulates Glucose Homeostasis by Modulating Insulin Secretion Ting Wen, Bonnie Peng, and John E. Pintar (Mol Endocrinol, published February 12, 2009, 10.1210/me.2008-0345) ABSTRACT In addition to producing analgesia, opioids have also been proposed to regulate glucose homeostasis by altering insulin secretion. A considerable controversy exists, however, regarding the contribution of the opioid receptor (MOR-1) to insulin secretion dynamics. We employed congenic C57Bl/6J MOR-1 KO mice to clarify the role of MOR in glucose homeostasis. We first found that both sexes of MOR-1 KO mice weigh more than WT mice throughout postnatal life and that this increase includes preferentially increased fat deposition. We also found that MOR-1 KO mice exhibit enhanced glucose tolerance that results from insulin hypersecretion that reflects increased beta-cell mass and increased secretory dynamics in the MOR-1 mutant mice compared to WT. Analysis of the isolated islets indicated that islet insulin hypersecretion is mediated directly by MOR expressed on islet cells via a mechanism downstream of ATP-sensitive K channel activation by glucose. These findings indicate that MOR-1 regulates body weight by a mechanism that involves insulin secretion and thus may represent a novel target for new diabetes therapies. T R A N S L A T I O N A L H I G H L I G H T S F R O M M O L E C U L A R E N D O C R I N O L O G YIn addition to producing analgesia, opioids have also been proposed to regulate glucose homeostasis by altering insulin secretion. A considerable controversy exists, however, regarding the contribution of the opioid receptor (MOR-1) to insulin secretion dynamics. We employed congenic C57Bl/6J MOR-1 KO mice to clarify the role of MOR in glucose homeostasis. We first found that both sexes of MOR-1 KO mice weigh more than WT mice throughout postnatal life and that this increase includes preferentially increased fat deposition. We also found that MOR-1 KO mice exhibit enhanced glucose tolerance that results from insulin hypersecretion that reflects increased beta-cell mass and increased secretory dynamics in the MOR-1 mutant mice compared to WT. Analysis of the isolated islets indicated that islet insulin hypersecretion is mediated directly by MOR expressed on islet cells via a mechanism downstream of ATP-sensitive K channel activation by glucose. These findings indicate that MOR-1 regulates body weight by a mechanism that involves insulin secretion and thus may represent a novel target for new diabetes therapies. T R A N S L A T I O N A L H I G H L I G H T S F R O M M O L E C U L A R E N D O C R I N O L O G Y

μ-Opioid Receptors Selectively Regulate Basal Inhibitory Transmission in the Central Amygdala: Lack of Ethanol Interactions

Endogenous opioid systems are implicated in the actions of ethanol. For example, mu-opioid receptor (MOR) knockout (KO) mice self-administer less alcohol than the genetically intact counterpart wild-type (WT) mice (Roberts et al., 2000). MOR KO mice also exhibit less anxiety-like behavior than WT mice (Filliol et al., 2000). To investigate the neurobiological mechanisms underlying these behaviors, we examined the effect of ethanol in brain slices from MOR KO and WT mice using sharp-electrode and whole-cell patch recording techniques. We focused our study in the central nucleus of the amygdala (CeA) because it is implicated in alcohol drinking behavior and stress behavior. We found that the amplitudes of evoked inhibitory postsynaptic currents (IPSCs) or inhibitory postsynaptic potentials (IPSPs) were significantly greater in MOR KO mice than WT mice. In addition, the baseline frequencies of spontaneous and miniature GABA(A) receptor-mediated inhibitory postsynaptic currents were significantly greater in CeA neurons from MOR KO than WT mice. However, ethanol enhancements of evoked IPSP and IPSC amplitudes and the frequency of miniature IPSCs were comparable between WT and MOR KO mice. Baseline spontaneous and miniature excitatory postsynaptic currents (EPSCs) and ethanol effects on EPSCs were not significantly different between MOR KO and WT mice. Based on knowledge of CeA circuitry and projections, we hypothesize that the role of MOR- and GABA receptor-mediated mechanisms in CeA underlying reinforcing effects of ethanol operate independently, possibly through pathway-specific responses within CeA.

The Role of MOR and the CI Operator Sites on the Genetic Switch of the Temperate Bacteriophage TP901-1

A genetic switch controls whether the temperate bacteriophage TP901-1 will enter a lytic or a lysogenic life cycle after infection of its host, Lactococcus lactis. We studied this bistable switch encoded in a small DNA fragment of 979 bp by fusing it to a reporter gene on a low-copy-number plasmid. The cloned DNA fragment contained the two divergently oriented promoters, PR and PL, transcribing the lysogenic and lytic gene clusters; the two promoter-proximal genes, cI and mor; and the three CI operator sites, OR, OL and OD. We show that mor encodes a protein and that this protein in concert with CI is required for the bistability. Furthermore, interaction of CI at OR represses transcription from the lysogenic promoter, PR. Thus, CI regulates its own transcription. Interaction of CI at OL represses transcription from the lytic promoter, PL. The presence of only OL (absence of OR and OD) is enough to maintain a bistable system. The distantly located operator site, OD, functions as a helper site by increasing binding of CI at OR and OL. In the immune state, OD increases repression of the lytic promoter, PL. Our results strongly support the model that a hexameric form of CI binds cooperatively to the three operator sites in the immune state forming a CI–DNA loop structure. Finally, we show that in the anti-immune state, repression of the lysogenic promoter is independent of the known CI operator sites but requires the presence of both CI and MOR.

185 EXPRESSION AND LOCALIZATION OF µ-OPIOID RECEPTOR IN CANINE OOCYTES

Endogenous opioid peptides (EOP), through G-protein-coupled receptors, control metabolism and many physiological and pathological conditions. Once EOP are linked to their receptors, above all µ-opioid receptor (MOR), a block of the Ca2+ channel occurs (Sciorsci et al. 2000 Immunopharm. Immunotox. 22, 575–626). The disruption of Ca2+ homeostasis interferes with many Ca2+-mediated/dependent actions. Our previous studies demonstrated the presence of MOR in human, bovine, and equine oocytes, in sperm cells of several species (equine, canine, etc.), in mare's tube, in ovine, bovine and mouse embryos. The presence of MOR on the male canine gamete lets us hypothesize its presence on the female gamete, too. In this study we demonstrated the presence of MOR on canine oocytes by immunofluorescence (IF) and western blot (WB) analysis, and we speculate on its possible functional role. Canine ovaries were obtained from healthy bitches randomly chosen among those arriving at our veterinary hospital for surgical ovariectomy without considering the period of their reproductive cycle. Oocytes were collected by ovary slicing and tested to check for the presence of MOR. For IF, oocytes were washed in 100 mm glycine in PBS and incubated for 30 min in PBS-1% BSA. Control oocytes were incubated with primary rabbit polyclonal antibody against the rat 3rd extracellular loop of MOR (Chemicon, Temecula, CA, USA). All oocytes were incubated for 2 h at room temperature with a FITC-conjugated anti-rabbit IgG-secondary antibody diluted 1:200 in Evans blue/PBS, washed, and visualized by laser scanning confocal microscope. For the WB, crude plasma membranes were obtained from pools of oocytes. They were lysed in Laemli buffer and loaded on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels. After electrophoresis, proteins were electrotransferred (semi-dry apparatus, BioRad, Milano, IT) to Immobilon-P membranes (Millipore, Bedford, MA, USA). Filters were blocked for 1 h and blotted overnight at 4�C against the same primary antibody used for IF, diluted 1:7500 in blocking buffer. After washing, membranes were incubated with a 1:10 000 dilution of peroxidase-conjugated goat anti-rabbit IgG secondary antibody for 2 h at room temperature. Reactive bands were visualized by Supersignal West Pico Chemiluminescent substrate (Pierce, Milano, IT). A negative control was performed. The IF highlighted, by clear brilliant green, the MOR's localization on canine oocytes. The negative control did not present any fluorescent region or spotted coloring. The WB revealed the presence of one immunoreactive band of approximately 65 kDa, thus confirming the results obtained by IF. No reactivity was evident when the primary antibody was adsorbed with an excess of immunizing peptide. The presence of MOR on canine oocytes indicates its possible role in the modulation of oocyte metabolism. These data strongly confirm previous evidence from our research unit on the involvement of the opioidergic system during gamete development and interaction, thus allowing us to speculate on a primary role of MOR in controlling key events of the reproductive activity.