Muscarinic Receptor-Stimulated Phosphoinositide Turnover in Human SK-N-SH Neuroblastoma Cells: Differential Inhibition by Agents that Elevate Cyclic AMP

The possibility that an increased intracellular concentration of cyclic AMP (cAMP) can regulate the extent of muscarinic receptor-stimulated phosphoinositide (PPI) turnover in the human neuroblastoma cell line SK-N-SH was examined. Addition of either forskolin (or its water-soluble analog, L-85,8051), theophylline, isobutylmethylxanthine, or cholera toxin, agents that interact with either the catalytic unit of adenylate cyclase, cAMP phosphodiesterase, or the guanine nucleotide binding protein linked to adenylate cyclase activation, resulted in a 45-181% increase in cAMP concentration and a 27-70% inhibition of carbachol-stimulated inositol phosphate release. Through the use of digitonin-permeabilized cells, the site of inhibition was localized to a step at, or distal to, the guanine nucleotide binding protein that regulates phospholipase C activity. In contrast, when intact SK-N-SH cells were exposed to prostaglandin E1, the ensuing increases in cAMP were not accompanied by an inhibition of stimulated PPI turnover. These differential effects of increased cAMP concentrations on stimulated PPI turnover may reflect the compartmentation of cAMP within SK-N-SH cells.     

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.     

Opiate binding properties of naturally occurring N- and C-terminus modified beta-endorphins

Beta-endorphin is further processed within the pituitary and brain by either N-terminal acetylation, carboxy-terminal proteolysis, or both. These naturally occurring analogues are stored intracellularly and, in some tissues, represent the majority of beta-endorphin immunoreactivity detected by antisera. It is therefore critical to determine their relative potencies at the opiate receptor. This study demonstrates that cleavage of the C-terminus tetrapeptide brings about a 10-fold decrease in opiate binding potency of either camel or human beta-endorphin. N-Acetylation, on the other hand, causes over a thousand fold loss in opiate potency rendering the peptide effectively inactive. Since unmodified beta-endorphin is approximately equipotent at multiple opiate receptors, we tested for possible differential shifts towards mu or delta-type receptors which may result from the modification. Our results show no change in selectivity, but simply an overall loss of potency.     

Dynorphin immunocytochemistry in the rat central nervous system

The distribution of dynorphin in the central nervous system was investigated in rats pretreated with relatively high doses (300–400 μg) of colchicine administered intracerebroventricularly. To circumvent the problems of antibody cross-reactivity, antisera were generated against different portions as well as the full dynorphin molecule (i.e., residues 1–13, 7–17, or 1–17). For comparison, antisera to [Leu]enkephalin (residues 1–5) were also utilized. Dynorphin was found to be widely distributed throughout the neuraxis. Immunoreactive neuronal perikarya exist in hypothalamic magnocellular nuclei, periaqueductal gray, scattered reticular formation sites, and other brain stem nuclei, as well as in spinal cord. Additionally, dynorphin-positive fibers or terminals occur in the cerebral cortex, olfactory bulb, nucleus accumbens, caudate-putamen, globus pallidus, hypothalamus, substantia nigra, periaqueductal gray, many brain stem sties, and the spinal cord. In many areas studied, dynorphin and enkephalin appeared to form parallel but probably separate anatomical systems. The results suggest that dynorphin occurs in neuronal systems that are immunocytochemically distinct from those containing other opioid peptides.     

Detection and identification of phytoplasma associated with witches’ broom and little leaf disease in Arundo donax: first report from India

During the survey of two successive years 2012–2013, in nearby places of Gorakhpur districts, Uttar Pradesh, India, Arundo donax plants were found to be exhibiting witches’ broom, excessive branching accompanied with little leaf symptoms with considerable disease incidence. Nested PCR carried out with universal primers pair R16F2n/R16R2 employing the PCR (P1/P7) product as a template DNA (1:20) resulted in expected size positive amplification ~1.2 kb in all symptom-bearing plants suggested the association of phytoplasma with witches’ broom disease of Narkat plants. BLASTn analysis of the 16S rRNA gene sequence showed the highest (99%) sequence identity with Candidatus phytoplasma asteris (16SrI group). In phylogenetic analysis, the sequence data showed close relationships with the members of 16SrI phytoplasma and clustered within a single clade of 16SrI group and closed to B subgroup representatives. This is a first report of 16Sr I-B group phytoplasma associated with witches’ broom accompanied with little leaf disease of Narkat in India.     

Epigallocatechin-3-gallate up-regulates tumor suppressor gene expression via a reactive oxygen species-dependent down-regulation of UHRF1

Ubiquitin-like containing PHD and Ring finger 1 (UHRF1) contributes to silencing of tumor suppressor genes by recruiting DNA methyltransferase 1 (DNMT1) to their hemi-methylated promoters. Conversely, demethylation of these promoters has been ascribed to the natural anti-cancer drug, epigallocatechin-3-gallate (EGCG). The aim of the present study was to investigate whether the UHRF1/DNMT1 pair is an important target of EGCG action. Here, we show that EGCG down-regulates UHRF1 and DNMT1 expression in Jurkat cells, with subsequent up-regulation of p73 and p16^INK4A genes. The down-regulation of UHRF1 is dependent upon the generation of reactive oxygen species by EGCG. Up-regulation of p16^INK4A is strongly correlated with decreased promoter binding by UHRF1. UHRF1 over-expression counteracted EGCG-induced G1-arrested cells, apoptosis, and up-regulation of p16^INK4A and p73. Mutants of the Set and Ring Associated (SRA) domain of UHRF1 were unable to down-regulate p16^INK4A and p73, either in the presence or absence of EGCG. Our results show that down-regulation of UHRF1 is upstream to many cellular events, including G1 cell arrest, up-regulation of tumor suppressor genes and apoptosis.     

Distribution of Mahogany/Attractin mRNA in the rat central nervous system

The Mahogany/Attractin gene (Atrn) has been proposed as a downstream mediator of Agouti signaling because yellow hair color and obesity in lethal yellow (A(y)) mice are suppressed by the mahogany (Atrn(mg)) mutation. The present study examined the distribution of Atrn mRNA in the brain and spinal cord by in situ hybridization. Atrn mRNA was found widely distributed throughout the central nervous system, with high levels in regions of the olfactory system, some limbic structures, regions of the brainstem, cerebellum and spinal cord. In the hypothalamus, Atrn mRNA was found in specific nuclei including the suprachiasmatic nucleus, the supraoptic nucleus, the medial preoptic nucleus, the paraventricular hypothalamic nucleus, the ventromedial hypothalamic nucleus, and the arcuate nucleus. These results suggest a broad spectrum of physiological functions for the Atrn gene product.     

Molecular signatures of proliferation and quiescence in hematopoietic stem cells

Stem cells resident in adult tissues are principally quiescent, yet harbor enormous capacity for proliferation to achieve self renewal and to replenish their tissue constituents. Although a single hematopoietic stem cell (HSC) can generate sufficient primitive progeny to repopulate many recipients, little is known about the molecular mechanisms that maintain their potency or regulate their self renewal. Here we have examined the gene expression changes that occur over a time course when HSCs are induced to proliferate and return to quiescence in vivo. These data were compared to data representing differences between naturally proliferating fetal HSCs and their quiescent adult counterparts. Bioinformatic strategies were used to group time-ordered gene expression profiles generated from microarrays into signatures of quiescent and dividing stem cells. A novel method for calculating statistically significant enrichments in Gene Ontology groupings for our gene lists revealed elemental subgroups within the signatures that underlie HSC behavior, and allowed us to build a molecular model of the HSC activation cycle. Initially, quiescent HSCs evince a state of readiness. The proliferative signal induces a preparative state, which is followed by active proliferation divisible into early and late phases. Re-induction of quiescence involves changes in migratory molecule expression, prior to reestablishment of homeostasis. We also identified two genes that increase in both gene and protein expression during activation, and potentially represent new markers for proliferating stem cells. These data will be of use in attempts to recapitulate the HSC self renewal process for therapeutic expansion of stem cells, and our model may correlate with acquisition of self renewal characteristics by cancer stem cells.     

Differential distribution and regulation of OX1 and OX2 orexin/hypocretin receptor messenger RNA in the brain upon fasting

To further understand the functions of the orexin/hypocretin system, we examined the expression and regulation of the orexin/hypocretin receptor (OX1R and OX2R) mRNA in the brain by using quantitative in situ hybridization. Expression of OX1R and OX2R mRNA exhibited distinct distribution patterns. Within the hypothalamus, expression for the OX1R mRNA was largely restricted in the ventromedial (VMH) and dorsomedial hypothalamic nuclei, while high levels of OX2R mRNA were contained in the paraventricular nucleus, VMH, and arcuate nucleus as well as in mammilary nuclei. In the amygdala, OX1R mRNA was expressed throughout the amygdaloid complex with robust labeling in the medial nucleus, while OX2R mRNA was only present in the posterior cortical nucleus of amygdala. High levels of OX2R mRNA were also observed in the ventral tegmental area. Moreover, both OX1R and OX2R mRNA were observed in the hippocampus, some thalamic nuclei, and subthalamic nuclei. Furthermore, we analyzed the effect of fasting on levels of OX1R and OX2R mRNA in the hypothalamic and amygdaloid subregions. After 20 h of fasting, levels of OX1R mRNA were significantly increased in the VMH and the medial division of amygdala. An initial decrease (14 h) and a subsequent increase (20 h) in OX1R mRNA levels after fasting were observed in the dorsomedial hypothalamic nucleus and lateral division of amygdala. Levels of OX2R mRNA were augmented in the arcuate nucleus, but remained unchanged in the dorsomedial hypothalamic nucleus, paraventricular hypothalamic nucleus, and amygdala following fasting. The time-dependent and region-specific regulatory patterns of OX1R and OX2R suggest that they may participate in distinct neural circuits under the condition of food deprivation.