Differential Requirements of Sodium for Coupling of Cannabinoid Receptors to Adenylyl Cyclase in Rat Brain Membranes

Abstract: Sodium is generally required for optimal inhibition of adenylyl cyclase by G_l/o-coupled receptors. Canna-binoids bind to specific receptors that act like other members of the G_l/o-coupled receptor superfamily to inhibit adenylyl cyclase. However, assay of cannabinoid inhibition of adenylyl cyclase in rat cerebellar membranes revealed that concentrations of NaCI ranging from 0 to 150 mM had no effect on agonist inhibition. This lack of effect of sodium was not unique to cannabinoid receptors, because the same results were observed using baclofen as an agonist for GABA_B receptors in cerebellar membranes. The lack of sodium dependence was region-specific, because assay of cannabinoid and opioid inhibition of adenylyl cyclase in striatum revealed an expected sodium dependence, with 50 mM NaCI providing maximal inhibition levels by both sets of agonists. This difference in sodium requirements between these two regions was maintained at the G protein level, because agonist-stimulated low K_m GTPase activity was maximal at 50 mM NaCI in striatal membranes, but was maximal in the absence of NaCI in cerebellar membranes. Assay of [³H]WIN 55212–2 binding in cerebellar membranes revealed that the binding of this labeled agonist was sensitive to sodium and guanine nucleotides like other G_l/o-coupled receptors, because both NaCI and the nonhydrolyzable GTP analogue Gpp(NH)p significantly inhibited binding. These results suggest that differences in receptor-G protein coupling exist for cannabinoid receptors between these two brain regions.     

Plasmodesmal-mediated cell-to-cell transport in wheat roots is modulated by anaerobic stress

Summary Cell-to-cell transport of small molecules and ions occurs in plants through plasmodesmata. Plant roots are frequently subjected to localized anaerobic stress, with a resultant decrease in ATP. In order to determine the effect of this stress on plasmodesmal transport, fluorescent dyes of increasing molecular weight (0.46 to 10 kDa) were injected into epidermal and cortical cells of 3-day-old wheat roots, and their movement into neighboring cells was determined by fluorescence microscopy. Anaerobiosis was generated by N₂ gas or simulated by the presence of sodium azide, both of which reduced the ATP levels in the tissue by over 80%. In the absence of such stress, the upper limit for movement, or size exclusion limit (SEL), of cortical plasmodesmata was <1 kDa. The ATP analogue TNP-ADP (mw 681) moved across the plasmodesmata of unstressed roots, indicating that plasmodesmata may be conduits for nucleotide (ATP and ADP) exchange between cells. Upon imposition of stress, the SEL rose to between 5 and 10 kDa. This response of plasmodesmata to a decrease in the level of ATP suggests that they are constricted by an ATP-dependent process so as to maintain a restricted SEL. When roots are subjected to anaerobic stress, an increase in SEL may permit enhanced delivery of sugars to the affected cells of the root where anaerobic respiration could regenerate the needed ATP.Abbreviations F-dextran fluorescein-coupled dextran - LYCH Lucifer Yellow CH - SEL size exclusion limit - TNP-ADP 2-O-(trinitrophenyl)adenosine-5-diphosphate     

Characterization of a new and highly distinguishable HLA-A allele in a Spanish family

Correspondence to: J. L. Vicario.     

Molecular characterization of a fourth isoform of the catalytic subunit of protein phosphatase 2A from Arabidopsis thaliana

We have recently reported the existence of multiple isoforms of the catalytic subunit of protein phosphatase 2A (PP2A) in Arabidopsis thaliana and the molecular cloning of cDNAs encoding three of these proteins (PP2A-1, PP2A-2, PP2A-3). The reported cDNA encoding PP2A-3 was truncated at the 5 terminus, lacking a short fragment of the N-terminal coding sequence. We have now isolated a near full-length cDNA encoding the entire PP2A-3 protein (313 residues). The clone includes 188 nucleotides of 5-untranslated region, where a 44 bp long poly(GA) track is found. We also describe the cloning of a cDNA encoding a fourth isoform of PP2A (PP2A-4). The polypeptide contains 313 residues being 98% identical to PP2A-3 and only 80% identical to both PP2A-1 and PP2A-2. The mRNA for PP2A-4 is 1.4 kb in length and, although predominantly expressed in roots, it is also found in other organs. It is concluded that in A. thaliana the isoforms of PP2A can be grouped in two extremely conserved subfamilies.     

A putative catabolite-repressed cell wall protein from the mycoparasitic fungus Trichoderma harzianum

A cDNA clone encoding a putative cell wall protein (Qid3) was isolated from a library prepared from chitin-induced mRNA in cultures of the mycoparasitic fungus Trichoderma harzianum. The predicted 14 kDa protein shows a potential signal peptide, several hydrophobic domains and certain motifs that are structurally similar to proline-rich and glycine-rich plant cell wall proteins. Expression of the qid3 gene is derepressed in the absence of glucose. When introduced in yeast, qid3 expression causes cell division arrest into cytokinesis and cell separation, probably due to its cell wall localization.     

Clonal analysis in hybrids between Drosophila melanogaster and Drosophila simulans

We have analysed the viability of cellular clones induced by mitotic recombination in Drosophila melanogaster/D. simulans hybrid females during larval growth. These clones contain a portion of either melanogaster or simulans genomes in homozygosity. Analysis has been carried out for the X and the second chromosomes, as well as for the 3L chromosome arm. Clones were not found in certain structures, and in others they appeared in a very low frequency. Only in abdominal tergites was a significant number of clones observed, although their frequency was lower than in melanogaster abdomens. The bigger the portion of the genome that is homozygous, the less viable is the recombinant melano-gaster/simulans hybrid clone. The few clones that appeared may represent cases in which mitotic recombination took place in distal chromosome intervals, so that the clones contained a small portion of either melanogaster or simulans chromosomes in homozygosity. Moreover, Lhr, a gene of D. simulans that suppresses the lethality of male and female melanogaster/simulans hybrids, does not suppress the lethality of the recombinant melanogaster/simulans clones. Thus, it appears that there is not just a single gene, but at least one per tested chromosome arm (and maybe more) that cause hybrid lethality. Therefore, the two species, D. melanogaster and D. simulans, have diverged to such a degree that the absence of part of the genome of one species cannot be substituted by the corresponding part of the genome of the other, probably due to the formation of co-adapted gene complexes in both species following their divergent evolution after speciation. The disruption of those coadapted gene complexes would cause the lethality of the recombinant hybrid clones.     

Pigment production from in vitro cultures of Alkanna tinctoria Tausch

Summary An in vitro culture of Alkanna tinctoria Tausch cells was set up in order to investigate the possibility of producing alkannin, a red naphthoquinone naturally present in the root bark of this plant. Furthermore, an in vitro culture of callusderived roots was established and the production of alkannin evaluated. In the different experimental conditions investigated, differences in the production of alkannin derivatives as well as in the type of pigments produced, were observed. The potential use of this technology is discussed.     

Low temperature optical spectroscopy of cobalt-substituted hemocyanin from Carcinus maenas

In this work we report the optical absorption spectra of three cobalt-substituted derivatives of hemocyanin (He) from Carcinus maenas, in the temperature range 300–20 K. The derivatives studied are the mononuclear (Co²⁺)-He with a single cobalt ion in the Cu_A site, the binuclear (Co²⁺)₂-He and the binuclear mixed metal (Co²⁺-Cu¹⁺)-He. At low temperature three main bands are clearly resolved; the temperature dependence of their zeroth, first and second moments sheds light on the stereodynamic properties in the surroundings of the chromophore. Within the limits of the reported analysis, in the binuclear derivatives the motions coupled to the chromophore appear to be essentially harmonic in the whole temperature range investigated; moreover the data are consistent with the presence of an exogenous ligand strongly bound to the two metal ions. For the mononuclear derivative an essentially harmonic behavior is evident only up to 200 K where the data are consistent with the presence of an exogenous ligand much less strongly bound, while at higher temperatures the behavior of the spectra indicates the onset of very large anharmonic contributions to motions, that plausibly involve the above exogenous ligand and, quite likely, the entire active site.Abbreviations He Hemocyanin - M₀ zeroth moment - M₁ first moment - M₂ second moment - (Co^2–)₂-He binuclear bicobalt hemocyanin derivative - (Co²⁺)-He mononuclear monocobalt hemocyanin derivative - (Co²⁺-Cu¹⁺)-He binuclear mixed metals hemocyanin derivative - LFT ligand field theory - CT charge transfer - EPR electronic paramagnetic resonance - XANES X-ray absorption near edge structure Correspondence to: L. Cordone