p53 and DNA Damage-Induced Apoptosis in Ovarian Tumour Cell Lines

The A2780 human ovarian adenocarcinoma cell line was used to establish methodologies for the detection and quantitation of ionising radiation- and cisplatin- induced apoptosis. Detection of apoptosis in A2780 cells by field inversion gel electrophoresis of DNA was found to be reliable and accurate. Apoptosis was quantitated using fluorescent end-labelling of DNA fragments by TUNEL/PI staining followed by FACs analysis. Cells displaying apoptotic morphology (dense chromatin, pyknotic nuclei around the cell margin, apoptotic bodies) when viewed by confocal microscopy were found to be those which also displayed increased green fluorescence due to TUNEL staining. A2780 cells engaged apoptosis in response to DNA damage generated by both ionising radiation and cisplatin. The DNA of apoptotic A2780 cells degraded in a non- random manner to produce DNA fragments of two sizes: one of 50kbp and one of 300- 1000kbp. The DNA did not degrade further to produce internucleosommal fragments. Maximal apoptosis occurred at either 72h or 96h after both cisplatin and radiation. Ionising radiation-induced apoptosis was reduced in the A2780/cp70 cell line compared to A2780 cells. Cisplatin-induced apoptosis was reduced in nine out of ten cisplatin resistant cell lines derived in vitro from A2780 cells. A higher level of Bcl-2 was observed in A2780/cp70 and this may contribute to the lack of apoptosis seen in these cells. The role of p53 in ionising radiation- and cisplatin-induced apoptosis in A2780 cells was investigated using a dominant negative mutant TP53 (codon 143, val to ala)- transfected cell line, A2780/mp53. A2780/mp53 cells underwent less apoptosis than the vector-transfected control cell line after equal doses of cisplatin or ionising radiation. These results showed that DNA damage caused by cisplatin or ionising radiation resulted in cell death by apoptosis in A2780 cells which was reduced in resistant cell lines. In addition they indicate a role for functional p53 in the ionising radiation- and cisplatin-induced apoptotic responses of A2780 cells

Mrgprd Enhances Excitability in Specific Populations of Cutaneous Murine Polymodal Nociceptors

The Mas-related G protein-coupled receptor D (Mrgprd) is selectively expressed in nonpeptidergic nociceptors that innervate the outer layers of mammalian skin. The function of Mrgprd in nociceptive neurons and the physiologically relevant somatosensory stimuli that activate Mrgprd^-expressing (Mrgprd^+) neurons are currently unknown. To address these issues, we studied three Mrgprd knock-in mouse lines using an ex vivo somatosensory preparation to examine the role of the Mrgprd receptor and Mrgprd+ afferents in cutaneous somatosensation. In mouse hairy skin, Mrgprd, as marked by expression of green fluorescent protein reporters, was expressed predominantly in the population of nonpeptidergic, TRPV1-negative, C-polymodal nociceptors. In mice lacking Mrgprd, this population of nociceptors exhibited decreased sensitivity to cold, heat, and mechanical stimuli. Additionally, in vitro patch-clamp studies were performed on cultured dorsal root ganglion neurons from Mrgprd^(–/–) and Mrgprd^(+/–) mice. These studies revealed a higher rheobase in neurons from Mrgprd^(–/–) mice than from Mrgprd^(+/–) mice. Furthermore, the application of the Mrgprd ligand β-alanine significantly reduced the rheobase and increased the firing rate in neurons from Mrgprd^(+/–) mice but was without effect in neurons from Mrgprd^(–/–) mice. Our results demonstrate that Mrgprd influences the excitability of polymodal nonpeptidergic nociceptors to mechanical and thermal stimuli

Cutaneous C-polymodal fibers lacking TRPV1 are sensitized to heat following inflammation, but fail to drive heat hyperalgesia in the absence of TPV1 containing C-heat fibers

<p>Abstract</p> <p>Background</p> <p>Previous studies have shown that the TRPV1 ion channel plays a critical role in the development of heat hyperalgesia after inflammation, as inflamed TRPV1-/- mice develop mechanical allodynia but fail to develop thermal hyperalgesia. In order to further investigate the role of TRPV1, we have used an ex vivo skin/nerve/DRG preparation to examine the effects of CFA-induced-inflammation on the response properties of TRPV1-positive and TRPV1-negative cutaneous nociceptors.</p> <p>Results</p> <p>In wildtype mice we found that polymodal C-fibers (CPMs) lacking TRPV1 were sensitized to heat within a day after CFA injection. This sensitization included both a drop in average heat threshold and an increase in firing rate to a heat ramp applied to the skin. No changes were observed in the mechanical response properties of these cells. Conversely, TRPV1-positive mechanically insensitive, heat sensitive fibers (CHs) were not sensitized following inflammation. However, results suggested that some of these fibers may have gained mechanical sensitivity and that some previous silent fibers gained heat sensitivity. In mice lacking TRPV1, inflammation only decreased heat threshold of CPMs but did not sensitize their responses to the heat ramp. No CH-fibers could be identified in naïve nor inflamed TRPV1-/- mice.</p> <p>Conclusions</p> <p>Results obtained here suggest that increased heat sensitivity in TRPV1-negative CPM fibers alone following inflammation is insufficient for the induction of heat hyperalgesia. On the other hand, TRPV1-positive CH fibers appear to play an essential role in this process that may include both afferent and efferent functions.</p

The ADP receptor P2Y<inf>1 </inf>is necessary for normal thermal sensitivity in cutaneous polymodal nociceptors

Background: P2Y1 is a member of the P2Y family of G protein-coupled nucleotide receptors expressed in peripheral sensory neurons. Using ratiometric calcium imaging of isolated dorsal root ganglion neurons, we found that the majority of neurons responding to adenosine diphosphate, the preferred endogenous ligand, bound the lectin IB4 and expressed the ATP-gated ion channel P2X3. These neurons represent the majority of epidermal afferents in hairy skin, and are predominantly C-fiber polymodal nociceptors (CPMs), responding to mechanical stimulation, heat and in some cases cold.Results: To characterize the function of P2Y1 in cutaneous afferents, intracellular recordings from sensory neuron somata were made using an ex vivo preparation in which the hindlimb skin, saphenous nerve, DRG and spinal cord were dissected in continuum, and cutaneous receptive fields characterized using digitally-controlled mechanical and thermal stimuli in male wild type mice. In P2Y1-/- mice, CPMs showed a striking increase in mean heat threshold and a decrease in mean peak firing rate during a thermal ramp from 31-52°C. A similar change in mean cold threshold was also observed. Interestingly, mechanical testing of CPMs revealed no significant differences between P2Y1-/- and WT mice.Conclusions: These results strongly suggest that P2Y1 is required for normal thermal signaling in cutaneous sensory afferents. Furthermore, they suggest that nucleotides released from peripheral tissues play a critical role in the transduction of thermal stimuli in some fiber types. © 2011 Molliver et al; licensee BioMed Central Ltd

Lack of neurotrophin-4 causes selective structural and chemical deficits in sympathetic ganglia and their preganglionic innervation

Neurotrophin-4 (NT-4) is perhaps the still most enigmatic member of the neurotrophin family. We show here that NT-4 is expressed in neurons of paravertebral and prevertebral sympathetic ganglia, i.e., the superior cervical (SCG), stellate (SG), and celiac (CG) ganglion. Mice deficient for NT-4 showed a significant reduction (20-30%) of preganglionic sympathetic neurons in the intermediolateral column (IML) of the thoracic spinal cord. In contrast, neuron numbers in the SCG, SG, and CG were unchanged. Numbers of axons in the thoracic sympathetic trunk (TST) connecting the SG with lower paravertebral ganglia were also reduced, whereas axon numbers in the cervical sympathetic trunk (CST) were unaltered. Axon losses in the TST were paralleled by losses of synaptic terminals on SG neurons visualized by electron microscopy. Furthermore, immunoreactivity for the synaptic vesicle antigen SV2 was clearly reduced in the SG and CG. Levels of catecholamines and tyrosine hydroxylase immunoreactivity were dramatically reduced in the SG and the CG but not in the SCG. Despite this severe phenotype in the sympathetic system, blood pressure levels were not reduced and displayed a pattern more typical of deficits in baroreceptor afferents. Numbers of IML neurons were unaltered at postnatal day 4, suggesting a postnatal requirement for their maintenance. In light of these and previous data, we hypothesize that NT-4 provided by postganglionic sympathetic neurons is required for establishing and/or maintaining synapses of IML neurons on postganglionic cells. Impairment of synaptic connectivity may consequently reduce impulse flow, causing a reduction in transmitter synthesis in postganglionic neurons

The role of OAIS representation information in the digital curation of crystallography data

Reusable high quality data are emerging as the raw material of contemporary e-science. Large volumes of scientific data are now “born-digital” and need to be curated to facilitate use and reuse. Representation Information (RI) as defined by the OAIS Reference Model is increasingly recognised as being vital to the long term curation and preservation of meaningful and reliable digital data. This paper is concerned with an investigation of RI for crystallography data and its role in the curation, maintenance and management of such data. We describe how the explicit recording of relevant RI can facilitate long term access and maintain intelligibility of the Crystallographic Information File format (a critical file format in the crystallography domain).<br/

Spectroscopy of BL Lac Objects: new redshifts and mis-identified sources

We are carrying out a program of high signal to noise optical spectroscopy of BL Lacs with unknown or tentative redshift. Here we report some preliminary results. New redshifts are measured for PKS0754+100 (z=0.266) and 1ES0715-259 (z=0.464) . From lineless spectra of PG1553+113 and PKS1722+119 we set a lower limit of z>0.3 for both sources. In two cases (UM493 and 1620+103) stellar spectra indicate a wrong classification.Comment: 4 pages; Conference proceeding "High Energy Blazar Astronomy", Tuorla Observatory, Finland, 17-21 June 2002; to be published in the PASP conference serie

Functional Human \u3cem\u3eGRIN2B\u3c/em\u3e Promoter Polymorphism and Variation of Mental Processing Speed in Older Adults

We investigated the role of a single nucleotide polymorphism rs3764030 (G \u3e A) within the human GRIN2B promoter in mental processing speed in healthy, cognitively intact, older adults. In vitro DNA-binding and reporter gene assays of different allele combinations in transfected cells showed that the A allele was a gain-of-function variant associated with increasing GRIN2B mRNA levels. We tested the hypothesis that individuals with A allele will have better memory performance (i.e. faster reaction times) in older age. Twenty-eight older adults (ages 65-86) from a well-characterized longitudinal cohort were recruited and performed a modified delayed match-to-sample task. The rs3764030 polymorphism was genotyped and participants were grouped based on the presence of the A allele into GG and AA/AG. Carriers of the A allele maintained their speed of memory retrieval over age compared to GG carriers (p = 0.026 slope of the regression line between AA and AG versus GG groups). To validate the results, 12 older adults from the same cohort participated in a different version of the short-term memory task. Reaction times were significantly slower with age in older adults with G allele (p \u3c 0.001). These findings support a role for rs3764030 in maintaining faster mental processing speed over aging

Keratinocytes can modulate and directly initiate nociceptive responses

How thermal, mechanical and chemical stimuli applied to the skin are transduced into signals transmitted by peripheral neurons to the CNS is an area of intense study. Several studies indicate that transduction mechanisms are intrinsic to cutaneous neurons and that epidermal keratinocytes only modulate this transduction. Using mice expressing channelrhodopsin (ChR2) in keratinocytes we show that blue light activation of the epidermis alone can produce action potentials (APs) in multiple types of cutaneous sensory neurons including SA1, A-HTMR, CM, CH, CMC, CMH and CMHC fiber types. In loss of function studies, yellow light stimulation of keratinocytes that express halorhodopsin reduced AP generation in response to naturalistic stimuli. These findings support the idea that intrinsic sensory transduction mechanisms in epidermal keratinocytes can directly elicit AP firing in nociceptive as well as tactile sensory afferents and suggest a significantly expanded role for the epidermis in sensory processing