A cell-based high-throughput screen to identify synergistic TRAIL sensitizers

We have developed a high-throughput screen (HTS) to search for novel molecules that can synergize with TRAIL, thus promoting apoptosis of ACHN renal tumor cells in a combinatorial fashion. The HTS detects synthetic compounds and pure natural products that can pre-sensitize the cancer cells to TRAIL-mediated apoptosis, yet have limited toxicity on their own. We have taken into account the individual effects of the single agents, versus the combination, and have identified hits that are synergistic, synergistic-toxic, or additive when combined with TRAIL in promoting tumor cell death. Preliminary mechanistic studies indicate that a subset of the synergistic TRAIL sensitizers act very rapidly to promote cleavage and activation of caspase-8 following TRAIL binding. Caspase-8 is an apical enzyme that initiates programmed cell death via the extrinsic apoptotic pathway. Thus, these TRAIL sensitizers may potentially reduce resistance of tumor cells to TRAIL-mediated apoptosis. Two representative sensitizers were found to increase levels of p53 but did not inhibit the proteasome, suggesting that early DNA damage-sensing pathways may be involved in their mechanisms of action.     

First divergence time estimate of spiders,scorpions, mites and ticks (subphylum: Chelicerata) inferred from mitochondrial phylogeny

Spiders, scorpions, mites and ticks (chelicerates) form one of the most diverse groups of arthropods on land, but their origin and times of diversification are not yet established. We estimated, for the first time, the molecular divergence times for these chelicerates using complete mitochondrial sequences from 25 taxa. All mitochondrial genes were evaluated individually or after concatenation. Sequences belonging to three missing genes (ND3, 6, and tRNA-Asp) from three taxa, as well as the faster-evolving ribosomal RNAs (12S and 16S), tRNAs, and the third base of each codon from 11 protein-coding genes (PCGs) (COI-III, CYTB, ATP8, 6, ND1-2, 4L, and 4-5), were identified and removed. The remaining concatenated sequences from 11 PCGs produced a completely resolved phylogenetic tree and confirmed that all chelicerates are monophyletic. Removing the third base from each codon was essential to resolve the phylogeny, which allowed deep divergence times to be calculated using three nodes calibrated with upper and lower priors. Our estimates indicate that the orders and classes of spiders, scorpions, mites, and ticks diversified in the late Paleozoic, much earlier than previously reported from fossil date estimates. The divergence time estimated for ticks suggests that their first land hosts could have been amphibians rather than reptiles. Using molecular data, we separated the spider-scorpion clades and estimated their divergence times at 397 ± 23 million years ago. Algae, fungi, plants, and animals, including insects, were well established on land when these chelicerates diversified. Future analyses, involving mitochondrial sequences from additional chelicerate taxa and the inclusion of nuclear genes (or entire genomes) will provide a more complete picture of the evolution of the Chelicerata, the second most abundant group of animals on earth. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Paleoceanography of the Gulf of Alaska during the past 15,000 years: Results from diatoms, silicoflagellates, and geochemistry

High-resolution records of diatoms, silicoflagellates, and geochemistry covering the past 15,000 years were studied in three cores from the Gulf of Alaska (GOA). Core EW0408-85JC in an oceanic setting on the Kayak Slope displays a paleoceanographic record similar to that at several locations on the California margin during deglaciation. Biologic productivity as reconstructed using geochemical and microfossil proxies increased abruptly during the Bølling–Alleröd (Bø–Al) warm interval (14.7–12.9 cal ka), declined during the Younger Dryas (YD) cold interval (12.9 to 11.7 cal kyr BP), and rose again during the earliest Holocene. At this site, the record after ~ 11 cal kyr BP is dominated by oceanic diatoms and silicoflagellates, with geochemical proxies displaying more subtle variation.Cores EW0408-66JC in the Yakobi Sea Valley near Cross Sound and EW0408-11JC in the Gulf of Esquibel contain an expanded, composite record along the southeast Alaskan margin. Core 66JC contains a detailed record of the Bø–Al and YD. Diatoms and silicoflagellates indicate that coastal upwelling and biosiliceous productivity were strong during the Bø–Al but declined during the YD. Sea ice-related diatoms increased in abundance during the YD, indicating cooler, but less productive waters.The glacial to biogenic marine sediment transition in core 11JC occurs at 1280 cmbsf (centimeters below sea floor), probably representing rising sea level and deglaciation early in the Bø–Al. Freshwater and sea-ice related diatoms are common in the lower part of the core (Bø–Al and YD), but upwelling-related diatoms and silicoflagellates quickly increased in relative abundance up-core, dominating the record of the past 11,000 years. Low oxygen conditions in the bottom water as reconstructed using geochemical proxies (U and Mo concentration) were most intense between ~ 6.5 and 2.8 cal kyr BP, the beginning of which is coincident with increases in abundance of upwelling-related diatoms.The records from these three cores jointly thus made it possible to reconstruct paleoclimatic and paleoceanographic conditions at high northern Pacific latitudes during the last 15 kyr.     

Complete nucleotide sequence and gene organization of the mitochondrial genome of Paa spinosa (Anura: Ranoidae)

The mt genome of Paa spinosa (Anura: Ranoidae) is a circular molecule of 18,012 bp in length, containing 38 genes (including an extra copy of tRNA-Met gene). This mt genome is characterized by three distinctive features: a cluster of rearranged tRNA genes (LTPF tRNA gene cluster), a tandem duplication of tRNA-Met gene (Met1 and Met2), and distinct repeat regions at both 5′ and 3′-sides in the control region. Comparing the locations and the sequences of all tRNA-Met genes among Ranoidae, and constructing NJ tree of the nucleotide of those tRNA-Met genes, we suggested a tandem duplication of tRNA-Met gene can be regarded as a synapomorphy of Dicroglossinae. To further investigate the phylogenetic relationships of anurans, phylogenetic analyses (BI, ML and MP) based on the nucleotide dataset and the corresponding amino acid dataset of 11 protein-coding genes (except ND5 and ATP8) arrived at the similar topology.     

Glucosamine inhibits IL‐1β‐mediated IL‐8 production in prostate cancer cells by MAPK attenuation

Inflammation is a complex process involving cytokine production to regulate host defense cascades. In contrast to the therapeutic significance of acute inflammation, a pathogenic impact of chronic inflammation on cancer development has been proposed. Upregulation of inflammatory cytokines, such as IL‐1β and IL‐8, has been noted in prostate cancer patients and IL‐8 has been shown to promote prostate cancer cell proliferation and migration; however, it is not clear whether IL‐1β regulates IL‐8 expression in prostate cancer cells. Glucosamine is widely regarded as an anti‐inflammatory agent and thus we hypothesized that if IL‐1β activated IL‐8 production in prostate cancer cells, then glucosamine ought to blunt such an effect. Three prostate cancer cell lines, DU‐145, PC‐3, and LNCaP, were used to evaluate the effects of IL‐1β and glucosamine on IL‐8 expression using ELISA and RT‐PCR analyses. IL‐1β elevated IL‐8 mRNA expression and subsequent IL‐8 secretion. Glucosamine significantly inhibited IL‐1β‐induced IL‐8 secretion. IL‐8 appeared to induce LNCaP cell proliferation by MTT assay; involvement of IL‐8 in IL‐1β‐dependent PC‐3 cell migration was demonstrated by wound‐healing and transwell migration assays. Inhibitors of MAPKs and NFκB were used to pinpoint MAPKs but not NFκB being involved in IL‐1β‐mediated IL‐8 production. IL‐1β‐provoked phosphorylation of all MAPKs was notably suppressed by glucosamine. We suggest that IL‐1β can activate the MAPK pathways resulting in an induction of IL‐8 production, which promotes prostate cancer cell proliferation and migration. In this context, glucosamine appears to inhibit IL‐1β‐mediated activation of MAPKs and therefore reduces IL‐8 production; this, in turn, attenuates cell proliferation/migration. J. Cell. Biochem. 108: 489–498, 2009. © 2009 Wiley‐Liss, Inc.     

Epac1‐induced cellular proliferation in prostate cancer cells is mediated by B‐Raf/ERK and mTOR signaling cascades

cAMP‐dependent, PKA‐independent effects on cell proliferation are mediated by cAMP binding to EPAC and activation of Rap signaling. In this report, we employed the analogue 8‐CPT‐2‐O‐Me‐cAMP to study binding to EPAC and subsequent activation of B‐Raf/ERK and mTOR signaling in human cancer cells. This compound significantly stimulated DNA synthesis, protein synthesis, and cellular proliferation of human 1‐LN prostate cancer cells. By study of phosphorylation‐dependent activation, we demonstrate that EPAC‐mediated cellular effects require activation of the B‐Raf/ERK and mTOR signaling cascades. RNAi directed against EPAC gene expression as well as inhibitors of ERK, PI 3‐kinase, and mTOR were employed to further demonstrate the role of these pathways in regulating prostate cancer cell proliferation. These studies were then extended to several other human prostate cancer cell lines and melanoma cells with comparable results. We conclude that B‐Raf/ERK and mTOR signaling play an essential role in cAMP‐dependent, but PKA‐independent, proliferation of cancer cells. J. Cell. Biochem. 108: 998–1011, 2009. © 2009 Wiley‐Liss, Inc.     

Cytoprotective effects of the lipoidic‐liquiform pro‐vitamin C tetra‐isopalmitoyl‐ascorbate (VC‐IP) against ultraviolet‐A ray‐induced injuries in human skin cells together with collagen retention,MMP inhibition and p53 gene repression

Irradiation with ultraviolet‐A (UVA) ray at doses of 20–100 J/cm² diminished the cell viability of human keratinocytes HaCaT and human melanoma cells HMV‐II, both of which were protected by pre‐irradiational administration with the ascorbic acid (Asc) derivative, VC‐IP (2,3,5,6‐O‐tetra‐2′‐hexyldecanoyl‐L‐ascorbic acid; vitamin C‐isopalmityl tetraester), which is the first lipoidic‐liquiform pro‐vitamin C by itself that is materialized by esterization of all four intramolecular hydroxyl groups of an Asc molecule with branched chain fatty groups, resulting in molecular fluidity higher than that of the corresponding straight chains. Irradiation with UVA to HaCaT keratinocytes was shown to cause the formation of 8‐hydroxydeoxyguanosine (8‐OHdG), translocation of phosphatidylserine in the inner layer into the outer layer of cell membrane, and lowering of a mitochondrial membrane potential, all of which were repressed by pre‐irradiational administration with VC‐IP. Expression of p53 gene, another hallmark of UV‐induced DNA damages, was promoted by UVA irradiation to the keratinocytes but also repressed by VC‐IP. Administration with VC‐IP of 10–50 µM to human fibroblasts NHDF achieved the enhancement of collagen synthesis, repression of matrix metalloprotease‐2/9 activity, and increasing of intracellular Asc contents more markedly than that with Asc itself of the same concentrations. Thus UVA‐induced diverse harmful effects could be prevented by VC‐IP, which was suggested to ensue intrinsically from the persistent enrichment of intracellular Asc, through esterolytic conversion of VC‐IP to a free‐form Asc molecule, resulting in relief to UVA‐caused oxidative stress. J. Cell. Biochem. 106: 589–598, 2009. © 2009 Wiley‐Liss, Inc.     

Expression of cGMP signaling elements in the Grueneberg ganglion

The Grueneberg ganglion (GG) is a cluster of neurons localized to the vestibule of the anterior nasal cavity. Based on axonal projections to the olfactory bulb of the brain, as well as expression of olfactory receptors and the olfactory marker protein, it is considered a chemosensory subsystem. Recently, it was observed that in mice, GG neurons respond to cool ambient temperatures. In mammals, coolness-induced responses in highly specialized neuronal cells are supposed to rely on the ion channel TRPM8, whereas in thermosensory neurons of the nematode worm Caenorhabditis elegans, detection of environmental temperature is mainly mediated by cyclic guanosine monophosphate (cGMP) pathways, in which cGMP is generated by transmembrane guanylyl cyclases. To unravel the molecular mechanisms underlying coolness-induced responses in GG neurons, potential expression of TRPM8 in the murine GG was investigated; however, no evidence was found that this ion channel is present in the GG. By contrast, a substantial number of GG neurons was observed to express the transmembrane guanylyl cyclase subtype GC-G. In the nose, GC-G expression appears to be confined to the GG since it was not detectable in other nasal compartments. In the GG, coolness-stimulated responses are only observed in neurons characterized by the expression of the olfactory receptor V2r83. Interestingly, expression of GC-G in the GG was found in this V2r83-positive subpopulation but not in other GG neurons. In addition to GC-G, V2r83-positive GG cells also co-express the phosphodiesterase PDE2A. Thus, in summary, coolness-sensitive V2r83-expressing GG neurons are endowed with a cGMP cascade which might underlie thermosensitivity of these cells, similar to the cGMP pathway mediating thermosensation in neurons of C. elegans. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. J. Fleischer and K. Mamasuew contributed equally to this work.     

Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell membrane, resulting in a voltage across the membrane. This voltage, termed the induced membrane voltage (also induced transmembrane voltage, or induced transmembrane potential difference) and denoted by ΔΦ, exists only as long as the external field is present. If the resting voltage is present on the membrane, the induced voltage superimposes (adds) onto it. By using one of the potentiometric fluorescent dyes, such as di-8-ANEPPS, it is possible to observe the variations of ΔΦ on the cell membrane and to measure its value noninvasively. di-8-ANEPPS becomes strongly fluorescent when bound to the lipid bilayer of the cell membrane, with the change of the fluorescence intensity proportional to the change of ΔΦ. This video shows the protocol for measuring ΔΦ using di-8-ANEPPS and also demonstrates the influence of cell shape on the amplitude and spatial distribution of ΔΦ.