Immunodetection and enzymatic characterization of the α3-isoform of Na,K-ATPase in dog heart

AbstractThe expression of the canine α 2 and 3 subunit isoenzymes of NA,K-ATPase has been investigated in plasma membranes isolated from dog heart, brain and kidney by immunoblotting, employing polyclonal anti rat fusion protein, and enzymological techniques. Western blot analysis revealed with purified dog membrane Na,K-ATPase preparations, one immunoreactive signal with rat specific α3 antisera in cardiac tissues, and two immunoreactive signals with rat α3 and α3 antisera in cerebral tissues. These findings suggested the specific expression of α3 polypeptide in dog heart (99 kDa), whereas dog brain expressed the α 2 and 3 polypeptides. The stained bands were superimposed. The antibody to rat brain α1 fusion protein did not cross-react with dog antigens whatever the three tissues tested. Expression of the α3-subunit isoform in dog heart membranes was consistent with a high affinity digitoxigenin-sensitive class of Na,K-ATPase (IC50 = 7 ± 2 nM). A single component with low affinity to digitoxigenin (IC50 = 110 ± 10 nM) characterized the α1 kidney form. The mixture of α2 and α3 isoforms in dog brain exhibited an apparent affinity for digitoxigenin (IC50 = 17 ± 5 nM) lower than the heart. The sodium dependences of the high affinity digitoxigenin sites were for the cardiac α3 form (K0.5 = 10 ± 1.9 mM) and for the cerebral α2 and α3 mixture (K5.0 19.6 ± 4.9 mM). The sensitivities for Na+ of the low affinity sites (α1) were: 6.7 ± 1.4 mM, 6.3 ± 1.2 mM and 11.6 ± 2.9 mM in heart, brain and kidney respectively. This is the first report of the catalytic characteristics of the α3 subunit isoenzyme in canine cardiac plasma membranes

Neuropeptide AF and FF modulation of adipocyte metabolism. Primary insights from functional genomics and effects on beta-adrenergic responsiveness.

The presence of a neuropeptide AF and FF receptor (NPFF-R2) mRNA in human adipose tissue (Elshourbagy, N. A., Ames, R. S., Fitzgerald, L. R., Foley, J. J., Chambers, J. K., Szekeres, P. G., Evans, N. A., Schmidt, D. B., Buckley, P. T., Dytko, G. M., Murdock, P. R., Tan, K. B., Shabon, U., Nuthulaganti, P., Wang, D. Y., Wilson, S., Bergsma, D. J., and Sarau, H. M. (2000) J. Biol. Chem. 275, 25965-25971) suggested these peptides, principally recognized for their pain modulating effects, may also impact on adipocyte metabolism, an aspect that has not been explored previously. Our aim was thus to obtain more insights into the actions of these peptides on adipocytes, an approach initially undertaken with a functional genomic assay. First we showed that 3T3-L1 adipocytes express both NPFF-R1 and NPFF-R2 transcripts, and that NPAF binds adipocyte membranes with a nanomolar affinity as assessed by surface plasmon resonance technology. Then, and following a 24-h treatment with NPFF or NPAF (1 microm), we have measured using real-time quantitative reverse transcriptase-PCR the mRNA steady state levels of already well characterized genes involved in key pathways of adipose metabolism. Among the 45 genes tested, few were modulated by NPFF ( approximately 10%) and a larger number by NPAF ( approximately 27%). Interestingly, NPAF increased the mRNA levels of beta2- and beta3-adrenergic receptors (AR), and to a lesser extent those of beta1-ARs. These variations in catecholamine receptor mRNAs correlated with a clear induction in the density of beta2- and beta3-AR proteins, and in the potency of beta-AR subtype-selective agonists to stimulate adenylyl cyclase activity. Altogether, these data show that NPFF-R1 and NPFF-R2 are functionally present in adipocytes and suggest that besides their well described pain modulation effects, NPAF and to a lesser extent NPFF, may have a global impact on body energy storage and utilization

Joint movement and sealant selection

The joint width should not be less than the minimum required for the sealant, allowing some flexibility in construction tolerances and counting on cyclical movements of structure. Authors discuss estimating of joint movement and installation temperatures. Examples given.Aussi disponible en fran\ue7ais : Le mouvement des joints et le choix du mat\ue9riau de calfeutrementPeer reviewed: NoNRC publication: Ye

The hexanoyl-CoA precursor for cannabinoid biosynthesis is formed by an acyl-activating enzyme in Cannabis sativa trichomes

The psychoactive and analgesic cannabinoids (e.g. \u3949-tetrahydrocannabinol, THC) in Cannabis sativa are formed from the short-chain fatty acyl-CoA precursor hexanoyl-CoA. Cannabinoids are synthesized in glandular trichomes present mainly on female flowers. We quantified hexanoyl-CoA using LC-MS/MS and found levels of 15.5 pmol g 121 fresh weight in female hemp flowers with lower amounts in leaves, stems and roots. This pattern parallels the accumulation of the end-product cannabinoid, cannabidiolic acid (CBDA). To search for the acyl-activating enzyme (AAE) that synthesizes hexanoyl-CoA from hexanoate, we analyzed the transcriptome of isolated glandular trichomes. We identified 11 unigenes encoding putative AAEs, including CsAAE1 which shows high-transcript abundance in glandular trichomes. In vitro assays show that recombinant CsAAE1 activates hexanoate and other short- and medium-chain fatty acids. This activity and the trichome-specific expression of CsAAE1 suggests that it is the hexanoyl-CoA synthetase that supplies the cannabinoid pathway. CsAAE3 encodes a peroxisomal enzyme that activates a variety of fatty acid substrates including hexanoate. Although phylogenetic analysis shows that CsAAE1 groups with peroxisomal AAEs, it lacks a peroxisome targeting sequence 1 (PTS1) and localizes to the cytoplasm. We suggest that CsAAE1 may have been recruited to the cannabinoid pathway through the loss of its PTS1, thereby redirecting it to the cytoplasm. To probe the origin of hexanoate, we analyzed the trichome EST dataset for enzymes of fatty acid metabolism. The high abundance of transcripts encoding desaturases and a lipoxygenase suggests that hexanoate may be formed through a pathway involving the oxygenation and breakdown of unsaturated fatty acids.Peer reviewed: YesNRC publication: Ye