Activation of Tyrosine Hydroxylase in the Superior Cervical Ganglion by Nicotinic and Muscarinic Agonists

Both dimethylphenylpiperazinium (DMPP), a nicotinic agonist, and bethanechol, a muscarinic agonist, increase 3,4-dihydroxyphenylalanine (DOPA) synthesis in the superior cervical ganglion of the rat. DMPP causes approximately a fivefold increase in DOPA accumulation in intact ganglia whereas bethanechol causes about a two-fold increase in DOPA accumulation. These effects are additive with each other and with the increase in DOPA accumulation produced by 8-bromo cyclic AMP. The action of DMPP is dependent on extracellular Ca2+ while the actions of bethanechol and 8-bromo cyclic AMP are not dependent on extracellular Ca2+. Cholinergic agonists and cyclic nucleotides produce a stable activation of tyrosine hydroxylase (TH) in the ganglion. The activation of TH by nicotinic and muscarinic agonists can be detected after 5 min of incubation of the ganglia with these agents. The nicotinic response disappears after 30 min of incubation, whereas the muscarinic response persists for at least 30 min. The Ca2+ dependence of the TH activation produced by these agents is similar to the Ca2+ dependence of their effects on DOPA accumulation in intact ganglia. These data are consistent with the hypothesis that nicotinic agonists, muscarinic agonists, and cyclic AMP analogues increase TH activity by three distinct mechanisms. The activation of TH presumably underlies the increase in DOPA synthesis produced by these agents.     

Photoperiod and pinealectomy do not affect cold-induced deposition of brown adipose tissue in the long-evans rat

Brown adipose tissue is a major thermogenic effector of cold-induced nonshivering thermogenesis. Previous studies indicate that melatonin and/or short photo-period are involved in the increase in brown fat deposition seen in certain cold-acclimated rodents. The present study was undertaken, in part, to determine whether the pineal is a necessary component in the cold-induced increase in thermogenic capacity characteristic of the cold-acclimated laboratory rat. Under a 12L:12D light cycle, pinealectomized rats did not differ from sham-operated rats in their ability to increase brown fat deposition in the cold (5 °C). Moreover, in a subsequent set of experiments performed at 9 °C, intact rats maintained at a short photoperiod (9L:15D) exhibited the same degree of brown fat hypertrophy/hyperplasia as did those kept at a long photoperiod (15L:9D). These data thus indicate that: (a) the intact pineal is not necessary for the cold-induced increase in brown adipose tissue occurring in the cold-acclimated rat; and (b) photoperiod does not significantly modulate the magnitude of this increase.     

A high-yielding serum-free, suspension cell culture process to manufacture recombinant adenoviral vectors for gene therapy

We have developed an efficient, reproducible, and scaleable cell culture process for a recombinant adenoviral vector expressing therapeutic transgenes for clinical trials. HEK 293 cells – which support the propagation of E1 deficient adenovirus – were first adapted to serum free media and suspension growth. Subsequent studies focused on the infection, virus production and harvest from suspension culture bioreactors. Future studies are planned to address the kinetics of adenovirus production in HEK 293 as well as in other cell lines. This revised version was published online in August 2006 with corrections to the Cover Date.     

Evaluation of the role of phosphatidylserine translocase activity in ABCA1-mediated lipid efflux

The following two theories for the mechanism of ABCA1 in lipid efflux to apolipoprotein acceptors have been proposed: 1) that ABCA1 directly binds the apolipoprotein ligand and then facilitates lipid efflux and 2) that ABCA1 acts as a phosphatidylserine (PS) translocase, increasing PS levels in the plasma membrane exofacial leaflet, and that this is sufficient to facilitate apolipoprotein binding and lipid assembly. Upon induction of ABCA1 in RAW264.7 cells by cAMP analogues there was a moderate increase in cell surface PS as detected by annexin V binding, whereas apoAI binding was increased more robustly. Apoptosis induced large increases in annexin V and apoAI binding; however, apoptotic cells did not efflux lipids to apoAI. Annexin V did not act as a cholesterol acceptor, and it did not compete for the cholesterol acceptor or cell binding activity of apoAI. ApoAI binds to ABCA1-expressing cells, and with incubation at 37 degrees C apoAI is co-localized within the cells in ABCA1-containing endosomes. Fluorescent recovery after photobleaching demonstrated that apoAI bound to ABCA1-expressing cells was relatively immobile, suggesting that it was bound either directly or indirectly to an integral membrane protein. Although ABCA1 induction was associated with a small increase in cell surface PS, these results argue against the notion that this cell surface PS is sufficient to mediate cellular apoAI binding and lipid efflux.     

IKKγ (NEMO) is involved in the coordination of the AP-1 and NF-κB pathways

Tumor necrosis factor alpha (TNFα) activates the nuclear factor-kappaB (NF-κB) pathway in various cell types, leading to expression of cell survival and inflammatory proteins. One mechanism of cell survival brought about by NF-κB is the inhibition of Activator Protein-1 (AP-1), which when activated, could lead to cell death. However, TNFα can also induce the AP-1 pathway, and the mechanisms by which these two pathways are regulated in response to TNFα are poorly understood. We proposed that Inhibitor of κB Kinase gamma (IKKγ) (which is also known as NF-κB essential modulator, NEMO) plays a key role in integrating and coordinating these two pathways. Our results showed that IKKγ activates the AP-1 pathway, via a mechanism that is dependent on the first leucine zipper (LZ) domain of IKKγ, by interacting with two proteins of the AP-1 complex, c-Jun and c-Fos, and changing the phosphorylation status of c-Jun. Even though IKKγ is required for the activation of NF-κB, we found that it reduced the activity of NF-κB when it was overexpressed. In summary, we demonstrated that transfected IKKγ, while inhibiting the NF-κB pathway, directly interacts with the AP-1 proteins and activates the AP-1 pathway independent of its effects on NF-κB. Our results indicate that IKKγ regulates TNFα signaling by coordinating cell responses mediated by the AP-1 and NF-κB pathways. A. S. Shifera and J. M. Friedman contributed equally to this article. Marshall S. Horwitz—Deceased: This article is dedicated to his loving memory.     

Prolonged suppression of insulin release by insulin-induced hypoglycemia: demonstration by C-peptide assay.

Pancreatic beta cells secrete the proinsulin connecting peptide (C-peptide) and insulin on an equimolar basis. The C-peptide can thus be used as an indicator of endogenous insulin secretion in the presence of exogenously administered insulin. Using this approach, we have shown suppression of endogenous insulin release in healthy subjects during hypoglycemia induced by intravenous infusion of porcine insulin. Moreover, the suppression persists after the plasma glucose returns to fasting levels, suggesting that the recovery of beta cells from the effects of hypoglycemia is not immediate.     

Human progesterone receptor transformation and nuclear down-regulation are independent of phosphorylation

We have studied the phosphorylation of progesterone receptors (PR) in T47Dco human breast cancer cells using a monoclonal antibody directed against human PR called AB-52. This antibody recognizes both the A- (Mr approximately 94,000) and B- (Mr approximately 120,000) hormone binding proteins of PR, and was used to immunoprecipitate phosphorylated receptors isolated from cells incubated in vivo with [32P]orthophosphate. The specific activity, or phosphorylation levels, relative to protein levels was quantified by combined immunoblotting and autoradiography followed by densitometry. We find that immunopurified untransformed hormone-free receptors, which have a characteristic triplet B, singlet A structure, are phosphoproteins with similar levels of phosphate incorporation in all protein bands. If PR are first transformed to the nuclear binding form by treatment of cells with progesterone, and then labeled with [32P]orthophosphate, the receptor proteins are additionally phosphorylated. These chromatin-bound hormone occupied receptors incorporate five to 10 times more labeled phosphate per total receptor protein than do PR from untreated cells during the same [32P]incubation time. The second round of phosphorylation may also account for mobility shifts of transformed A- and B-receptors observed in sodium dodecyl sulfate-polyacrylamide gels. Both untransformed and transformed species of A- and B-receptors are phosphorylated only on serine residues, and neither the extent of phosphorylation, nor the phosphoamino acids, are affected by treatment of the cells with epidermal growth factor or insulin. We previously reported that after hormone binding and transformation of receptors to the tight chromatin binding state, PR undergo processing, or nuclear down-regulation. AB-52 was used to compare PR protein and phosphorylation levels when cells were treated for 0.5-48 h with progesterone or the synthetic progestin R5020. Both agonists lead to hyperphosphorylation of nuclear PR before phosphorylation levels decrease, in parallel with the drop in protein levels as receptors down-regulate. Treatment of cells with RU 486, an antiprogestin, leads to PR transformation as determined by immunoblotting, but subsequent down-regulation does not occur. After transformation, chromatin-bound RU 486-occupied receptors become intensely phosphorylated however, with specific activities 15 times greater than those of untransformed PR. Since these receptors are phosphorylated but not processed, the hormone-induced nuclear phosphorylation of PR is unlikely to be a signal for receptor processing.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nonshivering thermogenesis and brown fat in spontaneously hypertensive rats

Oxygen consumption was measured before and during infusion of the catecholamine isoproterenol in age-matched spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) normotensive rats. Mass-independent rates of oxygen consumption of anesthetized 7-week-old rats were similar in the WKY and SHR rats (11.08 +/- 0.74 and 11.33 +/- 0.82 ml O2 min-1 kg-.67, respectively). Catecholamine infusion elicited increased total oxygen consumption in both WKY and SHR animals (15.0 +/- 1.0 and 14.9 +/- 1.2 ml O2 min-1 kg-.67, respectively), and the magnitude of these increases did not significantly differ. To assess whether there were changes in the metabolic state of brown adipose tissue, the major site of catecholamine-induced thermogenesis in rats, enzymes whose activity is proportional to aerobic capacity were assayed in vitro. In both the interscapular and cervical brown fat depots, maximal citrate synthase and maximal HOAD (beta-hydroxyacyl-CoA dehydrogenase) activities were similar in SHR and WKY rats. There were also no significant differences in brown fat protein content, suggesting no differential growth of this tissue in the two rat strains. Our results indicate that the nonshivering thermogenic capacity of the hypertensive SHR rats does not differ from that of the normotensive WKY animals.     

Diverse Lifestyles and Strategies of Plant Pathogenesis Encoded in the Genomes of Eighteen Dothideomycetes Fungi

The class Dothideomycetes is one of the largest groups of fungi with a high level of ecological diversity including many plant pathogens infecting a broad range of hosts. Here, we compare genome features of 18 members of this class, including 6 necrotrophs, 9 (hemi)biotrophs and 3 saprotrophs, to analyze genome structure, evolution, and the diverse strategies of pathogenesis. The Dothideomycetes most likely evolved from a common ancestor more than 280 million years ago. The 18 genome sequences differ dramatically in size due to variation in repetitive content, but show much less variation in number of (core) genes. Gene order appears to have been rearranged mostly within chromosomal boundaries by multiple inversions, in extant genomes frequently demarcated by adjacent simple repeats. Several Dothideomycetes contain one or more gene-poor, transposable element (TE)-rich putatively dispensable chromosomes of unknown function. The 18 Dothideomycetes offer an extensive catalogue of genes involved in cellulose degradation, proteolysis, secondary metabolism, and cysteine-rich small secreted proteins. Ancestors of the two major orders of plant pathogens in the Dothideomycetes, the Capnodiales and Pleosporales, may have had different modes of pathogenesis, with the former having fewer of these genes than the latter. Many of these genes are enriched in proximity to transposable elements, suggesting faster evolution because of the effects of repeat induced point (RIP) mutations. A syntenic block of genes, including oxidoreductases, is conserved in most Dothideomycetes and upregulated during infection in L. maculans, suggesting a possible function in response to oxidative stress.     

Effect of photoperiod on body weight and food intake of obese and lean Zucker rats

Although the rat is usually not considered to be sensitive to photoperiod, under some experimental conditions photoperiod responses are unmasked. In addition, we have observed photoperiod-induced changes in body weight gain in lean and obese Zucker rats. In this experiment, body mass, food intake, body composition, brown adipose tissue (BAT) thermogenic state, and blood concentrations of corticosterone, insulin, and glucose were evaluated under one of two lighting conditions: a short (10 h light: 14 h dark) or a long (14 h light: 10 h dark) photoperiod. Plasma corticosterone and glucose concentrations measured under fasting conditions were unaffected by photoperiod in either genotype. The amount of BAT mitochondrial protein isolated was less in long photoperiod rats. BAT mitochondrial GDP binding was unaffected by photoperiod in the lean rats, but tended to be lower in long photoperiod obese rats than in short photoperiod obese rats. Although, photoperiod had no effect on daily food intake of rats exposed to the short versus long photoperiod, body mass was heaviest in obese rats raised in long photoperiod. Plasma insulin was increased in both lean and obese rats in long photoperiod. In addition, fat storage appeared to shift to internal depots in the lean rats exposed to long photoperiod. Our data demonstrate that photoperiod does have an effect on male Zucker rats with respect to body weight and fat distribution, with the obese rats being more sensitive to changes in photoperiod than the lean rats.