Translocation of membrane impermeant molecules to the interior of living cells is a necessity for many biochemical investigations. Myristoylation was studied as a means to introduce peptides into living cells. Uptake of a myristoylated, fluorescent peptide was efficient in the B lymphocyte cell line BA/F3. In contrast, this cell line was resistant to peptide uptake using a cell penetrating peptide derived from the TAT protein. In BA/F3 cells, membrane association was shown to be rapid reaching a maximum within 30 minutes. Cellular uptake of the peptide lagged the membrane association, but occurred within a similar time frame. Experiments performed at 37°C vs. 4°C demonstrated profound temperature dependence in the cellular uptake of myristoylated cargo. Myristoylated peptides with either positive or negative charge were shown to load efficiently. In contrast to TAT-conjugated cargo, pyrenebutyrate did not enhance cellular uptake of the myristoylated peptide. The myristoylated peptide did not adversely affect cell viability at concentrations up to 100 μM. This assessment of myristoyl-based transport provides fundamental data needed in understanding the intracellular delivery of myristoylated peptide cargoes for cellbased biochemical studies.
Regulation of sphingosine and sphingosine-1-phosphate concentrations is of growing interest due to their importance in cellular signal transduction. Furthermore, new pharmaceutical agents moderating the intracellular and extracellular levels of sphingosine metabolites are showing promise in preclinical and clinical trials. In the present work, a quantitative assay relying on capillary electrophoresis with laser-induced fluorescence detection was developed to measure the interconversion of sphingosine and sphingosine-1-phosphate. The assay was demonstrated to be capable of determining the in vitro activity of both kinase and phosphatase using purified enzymes. The K M of sphingosine kinase for its fluorescently labeled substrate was 38 ± 18 μM with a V max of 0.4 ± 0.2 μM/min and a k cat of 3900 s −1 . Pharmacologic inhibition of sphingosine kinase in a concentration-dependent manner was also demonstrated. Moreover, the fluorescent substrate was shown to be readily taken up by mammalian cells making it possible to study the endogenous activity of sphingosine kinase activity in living cells. The method was readily adaptable to the use of either bulk cell lysates or very small numbers of intact cells. This new methodology provides enhancements over standard methods in sensitivity, quantification, and manpower for both in vitro and cell-based assays.The sphingolipids sphingosine and sphingosine-1-phosphate (S1P) play crucial roles as signal transduction molecules involved in cell survival and migration. [1][2][3][4][5][6] These second messengers along with the sphingolipid metabolite ceramide are interconvertible, and their dynamic equilibrium is believed to be a determining factor in whether cells will live or die. 7 In addition to its role as an intracellular second messenger, S1P also acts as an extracellular ligand making it a pleiotropic signaling molecule with wide-ranging function from calcium homeostasis to chemotaxis. 4,[7][8][9] S1P is produced by phosphorylation of sphingosine by sphingosine kinase 1 and 2 (SK1 and SK2) and is reversibly dephosphorylated by two known mammalian phosphatases SPP1 and SPP2. 6,[9][10][11] In addition, S1P can be irreversibly degraded by a pyridoxylphosphate-dependent S1P lyase to hexadecenal and phosphoethanolamine. 6,12 The balance and interplay of these metabolic pathways remain to be fully elucidated. SK1 is thought to be oncogenic, and inhibitors of SK1 appear to act as effective chemotherapeutic agents in animal studies. 7,10,13,14 SK2 is involved in the immune response, and compounds directed at extracellular S1P signaling are showing great promise in clinical trials for autoimmune diseases. 10,[15][16][17][18] Thus, sphingosine signaling is proving to be extremely important in clinical medicine. [17][18][19][20][21] *Corresponding authors. Phone: 919-966-2291 (C.E.S. and N.L.A.). Fax: 919-962-2388 (C.E.S. and N.L.A.). cesims@unc.edu (C.E.S.); nlallbri@unc.edu (N.L.A. Although S1P plays a major role as an extracellular signaling molecule, it is predominately syn...
Cisplatin chemotherapy is used commonly to treat a variety of cancers despite severe side effects such as nausea, vomiting, and anorexia that compromise quality of life and limit treatment adherence. The neural mechanisms mediating these side effects remain elusive despite decades of clinical use. Recent data highlight the dorsal vagal complex (DVC), lateral parabrachial nucleus (lPBN), and central nucleus of the amygdala (CeA) as potential sites of action in mediating the side effects of cisplatin. Here, results from immunohistochemical studies in rats identified a population of cisplatin-activated DVC neurons that project to the lPBN and a population of cisplatinactivated lPBN calcitonin gene-related peptide (CGRP, a marker for glutamatergic neurons in the lPBN) neurons that project to the CeA, outlining a neuroanatomical circuit that is activated by cisplatin. CeA gene expressions of AMPA and NMDA glutamate receptor subunits were markedly increased after cisplatin treatment, suggesting that CeA glutamate receptor signaling plays a role in mediating cisplatin side effects. Consistent with gene expression results, behavioral/pharmacological data showed that CeA AMPA/kainate receptor blockade attenuates cisplatin-induced pica (a proxy for nausea/behavioral malaise in nonvomiting laboratory rodents) and that CeA NMDA receptor blockade attenuates cisplatin-induced anorexia and body weight loss in addition to pica, demonstrating that glutamate receptor signaling in the CeA is critical for the energy balance dysregulation caused by cisplatin treatment. Together, these data highlight a novel circuit and CGRP/glutamatergic mechanism through which cisplatin-induced malaise and energy balance dysregulation are mediated.
The threat of nuclear war is a psychological concern because it began with and dramatically influences the human mind.Problems such as nuclear weapons, pollution, and ecological imbalance stem directly from our own behavior and can May 1985 * American Psychologist
Effective conservation of short-distance migrants requires an understanding of intraspecific variation in migratory patterns across small spatial scales. Until the advent of ultra-light geolocation devices, our knowledge of the migratory connectivity of songbirds was limited. For the Hermit Thrush (Catharus guttatus), subspecies delineations and connectivity patterns have been unclear in the portion of their breeding range in western North America from southeastern Alaska to northwestern Washington, where individuals wintering in the San Francisco Bay Area of California purportedly breed. To determine breeding locations and migratory timing of the Bay Area’s wintering Hermit Thrushes, we deployed geolocators at sites to the north and south of the San Francisco Bay. We compared results from these two regions to one another and to connectivity patterns suggested by subspecies definitions. We collected morphometrics to identify regional differences. Hermit Thrushes that wintered in the North Bay had a wider and more southerly breeding distribution from the British Columbia coast to northwestern Washington, whereas South Bay thrushes migrated to southeastern Alaska and the British Columbia coast. In general, North Bay thrushes departed wintering grounds and arrived on breeding grounds earlier than South Bay thrushes, but we cannot eliminate sex as a factor in these differences. Regional morphology differed only in bill length. Intraspecific isolation in glacial refugia during the Late Pleistocene may explain these fine-scale geographic variations in migration patterns and morphology.
This paper examines the relationship between modern theories of microeconomics and macroeconomics and, more generally, it evaluates the prospects of theoretically reducing macroeconomics to microeconomics. Many economists have shown strong interest in providing “microfoundations” for macroeconomics and much of their work is germane to the issue of theoretical reduction. Especially relevant is the work that has been done on what is called The Problem of Aggregation. On some accounts, The Problem of Aggregation just is the problem of reducing macroeconomics to microeconomics. I show how to separate these problems and then try to determine to what extent particular kinds of solutions to The Problem of Aggregation succeed in reducing macroeconomics to microeconomics as well. I argue that reduction is not possible by this means given the current state of microeconomics. I also describe how reduction may be possible by means of (dis)aggregation if microeconomics is supplemented in a certain way with the results of experimental research on individual economic agents.
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