Pre- and Posttranslational Regulation of β-Endorphin Biosynthesis in the CNS: Effects of Chronic Naltrexone Treatment

Abstract: There appear to be two anatomically distinct β-endorphin (βE) pathways in the brain, the major one originating in the arcuate nucleus of the hypothalamus and a smaller one in the area of the nucleus tractus solitarius (NTS) of the caudal medulla. Previous studies have shown that these two proopiomelanocortin (POMC) systems may be differentially regulated by chronic morphine treatment, with arcuate cells down-regulated and NTS cells unaffected. In the present experiments, we examined the effects of chronic opiate antagonist treatment on βE biosynthesis across different CNS regions to assess whether the arcuate POMC system would be regulated in the opposite direction to that seen after opiate agonist treatment and to determine whether different βE-containing areas might be differentially regulated. Male adult rats were administered naltrexone (NTX) by various routes for 8 days (subcutaneous pellets, osmotic minipumps, or repeated intraperitoneal injections). Brain and spinal cord regions were assayed for total βE-ir, different molecular weight immunoreactive β-endorphin (βE-ir) peptides, and POMC mRNA. Chronic NTX treatment, regardless of the route of administration, reduced total βE-ir concentrations by 30–40% in diencephalic areas (the arcuate nucleus, the remaining hypothalamus, and the thalamus) and the midbrain, but had no effect on βE-ir in the NTS or any region of the spinal cord. At the same time, NTX pelleting increased POMC mRNA levels in the arcuate to ～ 140% of control values. These data suggest that arcuate POMC neurons are up-regulated after chronic NTX treatment (whereas NTS and spinal cord systems remain unaffected) and that they appear to be under tonic inhibition by endogenous opioids. Chromatographic analyses demonstrated that, after chronic NTX pelleting, the ratio of full length βE_1–31 to more processed βE-ir peptides (i.e., βE_1–27 and βE_1–26) tended to increase in a dose-dependent manner in diencephalic areas. Because βE_1–31 is the only POMC product that possesses opioid agonist properties, and βE_1–27 has been posited to function as an endogenous anatgonist of βE_1–31, the NTX-induced changes in the relative concentrations of βE_1–31 and βE_1–27/βE_1–26 may represent a novel regulatory mechanism of POMC cells to alter the opioid signal in the synapse.     

Accumulation of the β-subunit of polygalacturonase 1 in normal and mutant tomato fruit

Polyclonal antiserum raised against the native PG1 isoform of tomato fruit (Lycopersicon esculentum Mill.) polygalacturonase [poly(1,4--d-galacturonide) glycanohydrolase, EC 3.2.1.15] bound to each of the subunits of the protein and also to a range of other fruit proteins. Affinity purification was used to remove antibody molecules that bound to the native form of the PG2 isoform. The resulting serum bound to native PG1, denatured PG2 and -subunits of PG1 but not to native PG2 or other fruit proteins. This anti-PG1 serum was used to monitor the occurrence of the PG1 -subunit and PG2 in detergent extracts of tomato tissues. The -subunit polypeptide was detected in pericarp but not locule tissue of fruit, including fruit of the rin and nor mutants. It increased in amount in the pericarp tissues from an early stage to the mature green stage, clearly prior to any appreciable accumulation of the PG2 subunit. The -subunit polypeptide was not detected in stem or leaf tissues. A PG2-specific antiserum was used to study the interaction of PG2 with the isolated -subunit. The PG2 isoform was bound to the -subunit over a wide range of salt concentrations and pH; the interaction was independent of the presence of reducing agents. It is concluded that strong non-covalent forces are involved in the interaction. The results are consistent with a model in which the -subunit is positioned in the cell wall structure and provides a specific binding site for the active PG2 subunit when this is synthesised during ripening.Abbreviations B breaker - MG mature green - M_r relative molecular mass - nor non-ripening mutant - PAGE polyacrylamide gel electrophoresis - PG polygalacturonase - rin ripening inhibitor mutant - SDS sodium dodecyl sulphate     

Effects of background adaptation on α-MSH and β-endorphin in secretory granule types of melanotrope cells of Xenopus laevis

Placing the clawed toad Xenopus laevis on a black background stimulates the melanotrope cells in the pars intermedia of the pituitary gland to release proopiomelanocortin (POMC)-derived peptides, including -MSH and N-acetyl-endorphin. In this study three types of secretory granules, electron-dense(130 nm Ø), moderately electron-dense ( 160 nm Ø) and electronlucent ( 180 nm Ø), have been identified in these cells. Apparently, only dark granules are formed by the Golgi apparatus and lucent granules release their contents via exocytosis. Immuno-electron microscopy (immunogold double labelling) of glutaraldehyde-fixed and freeze-substituted material shows that desacetyl--MSH and N-acetyl--endorphin coexist in all three granule types. Quantification of immunostaining revealed that immunoreactivities to these peptides are lowest in the dark granules and highest in the light ones. It is proposed that intragranular processing of POMC to immunoreactive desacetyl--MSH and N-acetyl--endorphin involves an increase in granule size and a decrease in granule electron density. Black background-induced activation of the melanotrope cell is reflected by an increase in immunoreactivity of the secretory granules to each of the antisera. This suggests that cell activation stimulates the formation of peptides by intragranular processing of POMC and/or of intermediate POMC-processing products. In addition, cell activation evoked an increase in the percentage of the granule population that reacts with anti-N-acetyl--endorphin, probably by stimulating intragranular acetylation of -endorphin. Apparently, this acetylation is a regulated event that occurs in the cytoplasm, independently from the acetylation of desacetyl--MSH which takes place near the plasmalemma at the time of granule exocytosis.     

EFFECTS OF VASOACTIVE INTESTINAL PEPTIDE ON ACTIVITIES OF SUCCINIC DEHYDROGENASE AND ALKALINE PHOSPHATASE IN RAT HEPATOMA CELLS

Using cytochemical method,microspectrophotometry and image analysis,effects of va-soactive intestinal peptide(VIP)on activities of succinic dehydrogenase(SDH)and alkalinephosphatase(ALP)in rat hepatoma cells were studied in vitro.The results showed that thehepatoma cell expressed potent positive reactions of SDH and ALP,the positive positionswere located at the cell membranes and/or cytoplasm.Having been treated with VIP,ALPdecreased obviously in activity(P<0. 01,compared with hepatoma cells untreated by VIP).The sites of ALP activty were chiefly located at the cell membranes,particularly at the cell-cell contacts.Cultured rat hepatoma cells had intensive SDH activity in their cytoplasm.Compared with untreated eclls,there was no marked difference in the intensity of SDH activ-ity in VIP-treated hepatoma cells(P>0.05).     

On-off units in the first optic chiasm of the blowfly II. Spatial properties

We recorded from the spiking on-off unit in the first optic chiasm (between lamina and medulla) in the blowfly Calliphora vicina, and investigated its spatial properties. The receptive field extends over (11.4±0.9)° horizontally and (8.7±0.6)° vertically, i.e. about 7 by 5 interommatidial angles. The line spread function of the on-off unit — calculated from its response to moving sinusoidal gratings — has a half-width of (2.3±0.2)°. This half-width is slightly broader than that of the photoreceptor. Lateral inhibition occurs when two different areas of the receptive field are stimulated simultaneously. Fast temporal adaptation (i.e. adaptation to trains of short light pulses) takes place independently in different areas of the receptive field.     

Begomovirus diversity in tomato crops in Costa Rica

Begomoviruses (Geminiviridae family) are characterized by their high recombination rate and a wide range of hosts, making their control difficult. In Costa Rica, various species of bipartite begomoviruses have been reported, which are Pepper golden mosaic virus (PepGMV), Tomato yellow mottle virus (ToYMoV), Tomato leaf curl Sinaloa virus (ToLCSiV) and the monopartite begomovirus Tomato yellow leaf curl virus (TYLCV). Since the TYLCV first report in Costa Rica, neither additional knowledge has been produced on how this begomovirus has spread in the country's territory nor on the distribution of the other bipartite species. A total of 429 tomato samples collected during the years 2015–2016 were used to study these aspects. Each sample was georeferenced and analysed with various techniques such as nucleic acid hybridization, polymerase chain reaction (PCR) and sequencing for the begomoviruses previously reported in Costa Rica. It was found that the presence/absence of the different species can vary, depending on the province. TYLCV is present in the six provinces analysed in this work, with a proportion from 3.7 to 86.6 per cent. Alajuela, Cartago, and Heredia are the provinces most affected by tomato-infecting begomoviruses. Fourteen different haplotypes of TYLCV were detected, but all were identified as TYLCV-IL. The distribution of TYLCV was related to the presence of the whitefly Bemisia tabaci MED, especially in the country's main tomato production areas. This information allows the phytosanitary surveillance services to develop strategies for the integrated management of the disease and to contribute data to the genetic improvement programmes of the crop.     

A novel tomato spotted wilt virus isolate encoding a noncanonical NSm C118F substitution associated with Sw-5 tomato gene resistance breaking

The nonstructural protein NSm of tomato spotted wilt virus (TSWV) has been identified as the avirulence determinant of the tomato single dominant Sw-5 resistance gene. Although Sw-5 effectiveness has been shown for most TSWV isolates, the emergence of resistance-breaking (RB) isolates has been observed. It is strongly associated with two point mutations (C118Y or T120N) in the NSm viral protein. TSWV-like symptoms were observed in tomato crop cultivars (+Sw-5) in the Baja California peninsula, Mexico, and molecular methods confirmed the presence of TSWV. Sequence analysis of the NSm 118–120 motif and three-dimensional protein modelling exhibited a noncanonical C118F substitution in seven isolates, suggesting that this substitution could emulate the C118Y-related RB phenotype. Furthermore, phylogenetic and molecular analysis of the full-length genome (TSWV-MX) revealed its reassortment-related evolution and confirmed that putative RB-related features are restricted to the NSm protein. Biological and mutational NSm 118 residue assays in tomato (+Sw-5) confirmed the RB nature of TSWV-MX isolate, and the F118 residue plays a critical role in the RB phenotype. The discovery of a novel TSWV-RB Mexican isolate with the presence of C118F substitution highlights a not previously described viral adaptation in the genus Orthotospovirus, and hence, the necessity of further crop monitoring to alert the establishment of novel RB isolates in cultivated tomatoes.