Opioid precursor protein isoform is targeted to the cell nuclei in the human brain

Background: Neuropeptide precursors are traditionally viewed as proteins giving rise to small neuropeptide molecules. Prodynorphin (PDYN) is the precursor protein to dynorphins, endogenous ligands for the kappa-opioid receptor. Alternative mRNA splicing of neuropeptide genes may regulate cell- and tissue-specific neuropeptide expression and produce novel protein isoforms. We here searched for novel PDYN mRNA and their protein product in the human brain. Methods: Novel PDYN transcripts were identified using nested PCR amplification of oligo(dT) selected full-length capped mRNA. Gene expression was analyzed by qRT-PCR, PDYN protein by western blotting and confocal imaging, dynorphin peptides by radioimmunoassay. Neuronal nuclei were isolated using fluorescence activated nuclei sorting (FANS) from postmortem human striatal tissue. lmmunofluorescence staining and con focal microscopy was performed for human caudate nucleus. Results: Two novel human PDYN mRNA splicing variants were identified. Expression of one of them was confined to the striatum where its levels constituted up to 30% of total PDYN mRNA. This transcript may be translated into ASP-PDYN protein lacking 13 N-terminal amino acids, a fragment of signal peptide (SP). Delta SP-PDYN was not processed to mature dynorphins and surprisingly, was targeted to the cell nuclei in a model cellular system. The endogenous PDYN protein was identified in the cell nuclei in human striatum by western blotting of isolated neuronal nuclei, and by confocal imaging. Conclusions and general significance: High levels of alternatively spliced Delta SP-PDYN mRNA and nuclear localization of PDYN protein suggests a nuclear function for this isoform of the opioid peptide precursor in human striatum. (C) 2016 Elsevier B.V. All rights reserved

Monoclonal antibodies to inner ear antigens: II Antigens expressed in sensory cell stereocilia

To develop biological reagents for investigating structure-function relationships in the organ of Corti, we have raised monoclonal antibodies, (MAb) to inner ear tissues. Our first series of antibodies prepared after intrasplenic immunization of mice with guinea pig tissues, identified antigens restricted to supporting cell structures, but no hair cell specific antibodies were developed [Zajic et al., Hear. Res. 52, 59-72, 1991]. In this report we describe the isolation, binding specificity and initial characterization of the stereocilia-binding monoclonal antibodies, KHRI-4, and KHRI-5. Mice were immunized with avian, amphibian and mammalian sensory hair cell-containing tissues and antibodies were screened for selective binding to cochlear extracts in ELISA. In the inner ear, KHRI-4 and KHRI-5 bind specifically to stereocilia in both avian and mammalian cochlear and vestibular tissue preparations using immunofluorescence and immunoperoxidase assays. In other tissues only certain cells of mesothelial origin, such as smooth muscle in gut and the arteriolar vasculature, were stained by KHRI-4 indicating that the antigenic structure defined by this antibody has limited distribution. KHRI-5 binding could be detected in other tissues only at high antibody concentrations suggesting that the gene product identified by this antibody is also weakly expressed in other cell lineages. Western blot analysis showed that KHRI-4 and -5 detect different protein complexes. KHRI-4 identifies an antigenic structure common to gut, cochlea, vestibular tissue and cultured fibroblasts consisting of a ~ 195 and a 230 kDa heterodimer designated p195/230. KHRI-5 binds to a prominent ~ 200-210 kDa band in Western blots of cochlear tissues, gut and fibroblasts. In immunoprecipitation experiments, KHRI-5 precipitated three proteins of Mr ~ 200-210, 230 and 260 kDa indicating that the ~ 200-210 kDa protein carrying the epitope for this antibody is a member of a heterotrimer complex. Our results show that these protein complexes are structural components of stereocilia and that the same proteins are arrayed in conjunction with the actin stress fibers of cultured mesothelial cells. Thus, they are likely to be important for maintaining the actin structure of stereocilia essential to transduction in sensory hair cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28991/1/0000019.pd

Hormone-Sensitive Lipase Knockouts

All treatments for obesity, including dietary restriction of carbohydrates, have a goal of reducing the storage of fat in adipocytes. The chief enzyme responsible for the mobilization of FFA from adipose tissue, i.e., lipolysis, is thought to be hormone-sensitive lipase (HSL). Studies of HSL knockouts have provided important insights into the functional significance of HSL and into adipose metabolism in general. Studies have provided evidence that HSL, though possessing triacylglycerol lipase activity, appears to be the rate-limiting enzyme for cholesteryl ester and diacylglycerol hydrolysis in adipose tissue and is essential for complete hormone stimulated lipolysis, but other triacylglycerol lipases are important in mediating triacylglycerol hydrolysis in lipolysis. HSL knockouts are resistant to both high fat diet-induced and genetic obesity, displaying reduced quantities of white with increased amounts of brown adipose tissue, increased numbers of adipose macrophages, and have multiple alterations in the expression of genes involved in adipose differentiation, including transcription factors, markers of adipocyte differentiation, and enzymes of fatty acid and triglyceride synthesis. With disruption of lipolysis by removal of HSL, there is a drastic reduction in lipogenesis and alteration in adipose metabolism

Substrate Micropatterning as a New in Vitro Cell Culture System to Study Myelination

Artículo de publicación ISIMyelination is a highly regulated developmental process whereby oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system ensheathe axons with a multilayered concentric membrane. Axonal myelination increases the velocity of nerve impulse propagation. In this work, we present a novel in vitro system for coculturing primary dorsal root ganglia neurons along with myelinating cells on a highly restrictive and micropatterned substrate. In this new coculture system, neurons survive for several weeks, extending long axons on defined Matrigel tracks. On these axons, myelinating cells can achieve robust myelination, as demonstrated by the distribution of compact myelin and nodal markers. Under these conditions, neurites and associated myelinating cells are easily accessible for studies on the mechanisms of myelin formation and on the effects of axonal damage on the myelin sheath.Regenerative Medicine and Nanomedicine Initiative of the Canadian Institutes of Health Research (CIHR) RMF-7028 FONDECYT 1080252 CIHR Ministry of Industry of Canada Rio Tinto Alcan Molson Foundatio

Attainment of Brown Adipocyte Features in White Adipocytes of Hormone-Sensitive Lipase Null Mice

BACKGROUND: Hormone-sensitive lipase (HSL) is expressed predominantly in adipose tissue, where it plays an important role in catecholamine-stimulated hydrolysis of stored tri- and diglycerides, thus mobilizing fatty acids. HSL exhibits broad substrate specificity and besides acylglycerides it hydrolyzes cholesteryl esters, retinyl esters and lipoidal esters. Despite its role in fatty acid mobilization, HSL null mice have been shown to be resistant to diet-induced obesity. METHODOLOGY/PRINCIPAL FINDINGS: Following a high-fat diet (HFD) regimen, energy expenditure, measured using indirect calorimetry, was increased in HSL null mice. White adipose tissue of HSL null mice was characterized by reduced mass and reduced protein expression of PPARgamma, a key transcription factor in adipogenesis, and stearoyl-CoA desaturase 1, the expression of which is known to be positively correlated to the differentiation state of the adipocyte. The protein expression of uncoupling protein-1 (UCP-1), the highly specific marker of brown adipocytes, was increased 7-fold in white adipose tissue of HSL null mice compared to wildtype littermates. Transmission electron microscopy revealed an increase in the size of mitochondria of white adipocytes of HSL null mice. The mRNA expression of pRb and RIP140 was decreased in isolated white adipocytes, while the expression of UCP-1 and CPT1 was increased in HSL null mice compared to wildtype littermates. Basal oxygen consumption was increased almost 3-fold in white adipose tissue of HSL null mice and was accompanied by increased uncoupling activity. CONCLUSIONS: These data suggest that HSL is involved in the determination of white versus brown adipocytes during adipocyte differentiation The exact mechanism(s) underlying this novel role of HSL remains to be elucidated, but it seems clear that HSL is required to sustain normal expression levels of pRb and RIP140, which both promote differentiation into the white, rather than the brown, adipocyte lineage