The Transient Receptor Potential cation channel V1 (TRPV1) is expressed in peripheral nociceptive neurons and is subject to polymodal activation via various agents including capsaicin, noxious heat, ...low extracellular pH, and direct phosphorylation by protein kinase C (PKC). We have cloned and heterologously expressed mouse TRPV1 (mTRPV1) and characterized its function utilizing FLIPR-based calcium imaging to measure functional responses to various small molecule agonists, low pH and direct phosphorylation via PKC. The various TRPV1 agonists activated mTRPV1 with a rank order of agonist potency of (resiniferatoxin (RTX)
=
arvanil
>
capsaicin
=
olvanil
>
OLDA
>
PPAHV) (EC
50 values of 0.15
±
0.04
nM, 0.27
±
0.07
nM, 9.1
±
1.2
nM, 3.7
±
0.3
nM, 258
±
105
nM, and 667
±
151
nM, respectively). Additionally, mTRPV1 was activated by either low pH or with addition of the PKC activator phorbol 12-myristate 13-acetate (PMA). The TRPV1 antagonists iodinated-resiniferatoxin (I-RTX) or BCTC were both able to block capsaicin, pH and PKC-induced responses of mTRPV1 (IC
50 (I-RTX)
=
0.35
±
0.12
nM, 1.9
±
0.7
nM, and 0.80
±
0.68
nM, IC
50 (BCTC)
=
1.3
±
0.36
nM, 0.59
±
0.16
nM, and 0.37
±
0.15
nM, respectively). However, the antagonist capsazepine was only able to inhibit a capsaicin-evoked response of mTRPV1 with an IC
50 of 1426
±
316
nM. Comparable results were achieved with rat TRPV1, while capsazepine blocked all modes of human TRPV1 activation. Thus, the mTRPV1 cation channel has a molecular pharmacological profile more akin to rat TRPV1 than either human or guinea pig TRPV1 and the molecular pharmacology suggests that capsazepine may be an ineffective TRPV1 antagonist for in vivo models of inflammatory pain in the mouse.
The dopamine D2 receptor (D2R) has been used in adenoviral delivery systems and in tumor cell xenografts as an in vivo reporter gene. D2R reporter gene expression has been non-invasively, ...repetitively and quantitatively imaged by positron emission tomography (PET), following systemic injection of a positron-labeled ligand (3-(2'-18F-fluoroethyl)-spiperone; FESP) and subsequent D2R-dependent sequestration. However, dopamine binding to the D2R can modulate cyclic AMP levels. For optimal utilization of D2R as a reporter gene, it is important to uncouple ligand-binding from Gi-protein-mediated inhibition of cAMP production. Mutation of Asp80 or Ser194 produces D2Rs that still bind 3Hspiperone in transfected cells. The D2R80A mutation completely eliminates the ability of the D2R to suppress forskolin-stimulated cAMP accumulation in response to dopamine, in cells transfected with a D2R80A expression plasmid and in cells infected with replication-defective adenovirus expressing D2R80A. The D2R194A mutation substantially reduces, but does not completely eliminate, dopamine modulation of cAMP levels. Cultured cells infected with adenoviruses expressing D2R and D2R80A demonstrated equivalent 3Hspiperone binding activity. Moreover, hepatic FESP sequestration is equivalent, following intravenous injection of adenoviruses expressing D2R and D2R80A. The D2R80A mutant, which can no longer modulate cAMP levels following ligand binding, has full capability as a PET reporter gene.
Dysregulated gene expression resulting from abnormal epigenetic alterations including histone acetylation and deacetylation has been demonstrated to play an important role in driving tumor growth and ...progression. However, the mechanisms by which specific histone deacetylases (HDACs) regulate differentiation in solid tumors remains unclear. Using pediatric rhabdomyosarcoma (RMS) as a paradigm to elucidate the mechanism blocking differentiation in solid tumors, we identified HDAC3 as a major suppressor of myogenic differentiation from a high-efficiency Clustered regularly interspaced short palindromic repeats (CRISPR)-based phenotypic screen of class I and II HDAC genes. Detailed characterization of the HDAC3-knockout phenotype in vitro and in vivo using a tamoxifen-inducible CRISPR targeting strategy demonstrated that HDAC3 deacetylase activity and the formation of a functional complex with nuclear receptor corepressors (NCORs) were critical in restricting differentiation in RMS. The NCOR/HDAC3 complex specifically functions by blocking myoblast determination protein 1 (MYOD1)-mediated activation of myogenic differentiation. Interestingly, there was also a transient up-regulation of growth-promoting genes upon initial HDAC3 targeting, revealing a unique cancer-specific response to the forced transition from a neoplastic state to terminal differentiation. Our study applied modifications of CRISPR/CRISPR-associated endonuclease 9 (Cas9) technology to interrogate the function of essential cancer genes and pathways and has provided insights into cancer cell adaptation in response to altered differentiation status. Because current pan-HDAC inhibitors have shown disappointing results in clinical trials of solid tumors, therapeutic targets specific to HDAC3 function represent a promising option for differentiation therapy in malignant tumors with dysregulated HDAC3 activity.
Pendred syndrome is an autosomal recessive disorder characterized by the association between sensorineural hearing loss and thyroid swelling or goitre and is likely to be the most common form of ...syndromic deafness. Within the thyroid gland of affected individuals, iodide is incompletely organified with variable effects upon thyroid hormone biosynthesis, whilst the molecular basis of the hearing loss is unknown. The PDSgene has been identified by positional cloning of chromosome 7q31, within the Pendred syndrome critical linkage interval and encodes for a putative ion transporter called pendrin. We have investigated a cohort of 56 kindreds, all with features suggestive of a diagnosis of Pendred syndrome. Molecular analysis of the PDSgene identified 47 of the 60 (78%) mutant alleles in 31 families (includes three homozygous consanguineous kindreds and one extended family segregating three mutant alleles). Moreover, four recurrent mutations accounted for 35 (74%) of PDS disease chromosomes detected and haplotype analysis would favour common founders rather than mutational hotspots within the PDS gene. Whilst these findings demonstrate molecular heterogeneity for PDS mutations associated with Pendred syndrome, this study would support the use of molecular analysis of the PDS gene in the assessment of families with congenital hearing loss.
An ultra-low background PMT for liquid xenon detectors Akerib, D.S.; Bai, X.; Bernard, E. ...
Nuclear instruments & methods in physics research. Section A, Accelerators, spectrometers, detectors and associated equipment,
03/2013, Letnik:
703
Journal Article
Recenzirano
Odprti dostop
Results are presented from radioactivity screening of two models of photomultiplier tubes designed for use in current and future liquid xenon experiments. The Hamamatsu 5.6cm diameter R8778 PMT, used ...in the LUX dark matter experiment, has yielded a positive detection of four common radioactive isotopes: 238U, 232Th, 40K, and 60Co. Screening of LUX materials has rendered backgrounds from other detector materials subdominant to the R8778 contribution. A prototype Hamamatsu 7.6cm diameter R11410 MOD PMT has also been screened, with benchmark isotope counts measured at <0.4238U/<0.3232Th/<8.340K/2.0±0.2 60Co mBq/PMT. This represents a large reduction, equal to a change of ×124238U/×19232Th/×1840K per PMT, between R8778 and R11410 MOD, concurrent with a doubling of the photocathode surface area (4.5–6.4cm diameter). 60Co measurements are comparable between the PMTs, but can be significantly reduced in future R11410 MOD units through further material selection. Assuming PMT activity equal to the measured 90% upper limits, Monte Carlo estimates indicate that replacement of R8778 PMTs with R11410 MOD PMTs will change LUX PMT electron recoil background contributions by a factor of ×125 after further material selection for 60Co reduction, and nuclear recoil backgrounds by a factor of ×136. The strong reduction in backgrounds below the measured R8778 levels makes the R11410 MOD a very competitive technology for use in large-scale liquid xenon detectors.
Monosex populations can solve the problem of stunted black crappie populations in small impoundments and can increase the potential of black crappie as an aquaculture species. In this study, we ...investigated the effective mode and duration of exogenous androgen administration to produce monosex male black crappie populations. We conducted two experiments using the synthetic androgens, 17α-methyltestosterone (MT) and trenbolone acetate (TBA). In the first experiment, the same age (45 days old), two different size fry with mean total lengths (±S.D., N=50) of 20.1±1.4 and 26.1±2.0 mm were either fed 60 mg MT/kg diet for 45 days or immersed in a 1 mg MT/l solution on 10 occasions for 5 h/day every 3–5 days between 45 and 86 days post-hatch (dph). In the second experiment, a different cohort of fry was immersed in a 1 mg TBA/l solution for 5 h/day every 3–5 days either on seven occasions between 40 and 66 dph or on 10 occasions between 45 and 86 dph. Mean total lengths (±S.D., N=50) of fry at 40 and 45 dph were 20.3±0.9 and 21.6±1.2 mm, respectively. Both modes of MT administration were ineffective for altering sex ratios in larger black crappie fry. Oral administration of MT to smaller fry resulted in 23% intersex fish. MT administration via short periodic baths of smaller fry produced 96% male populations. TBA was as potent as MT to induce masculinization in black crappie: both immersion treatments produced all male populations. Results of the present study indicated that initial size of fry as well as initial age should be considered when selecting appropriate size black crappie fry for hormone treatments. Monosex male populations in black crappie can effectively be produced through a series of (seven or less) short (5 h) immersions of young fry (40−45 days old and 20−21 mm) in a 1 mg/l aqueous solution of MT or TBA. Besides high effectiveness, immersion method will be the preferred mode of androgen administration to black crappie because it reduces variation in hormone uptake associated with the oral administration of steroids and provides more flexibility in feeding during the period of gonadal differentiation.
The genomes of plant and animal species are influenced by ancestral whole-genome duplication (WGD) events, which have profound impacts on the regulation and function of gene networks. To gain insight ...into the consequences of WGD events, we characterized the sequence conservation and expression patterns of ohnologs in the highly duplicated activin receptor signaling pathway in rainbow trout (RBT). The RBT activin receptor signaling pathway is defined by tissue-specific expression of inhibitors and ligands and broad expression of receptors and Co-Smad signaling molecules. Signaling pathway ligands exhibited shared expression, while inhibitors and Smad signaling molecules primarily express a single dominant ohnolog. Our findings suggest that gene function influences ohnolog evolution following duplication of the activin signaling pathway in RBT.
Using the statistical technique of fuzzy clustering, regimes of inflation and unemployment are explored for the United States, the United Kingdom and Germany between 1871 and 2009. We identify for ...each country three distinct regimes in inflation/unemployment space. Similarities exist across countries in both the regimes and the timings of the transitions between regimes. However, the typical rates of inflation and unemployment experienced in the regimes are substantially different. Further, even within a given regime, the results from the cluster analysis reveal persistent fluctuations in the degree of attachment to that regime of inflation/unemployment observations over time. The economic implications of this are that, first, the inflation/unemployment relationship or Phillips curve experiences from time to time major shifts. Second, that it is also inherently unstable even in the short run. It is likely that the factors which govern the inflation/unemployment trade-off are so multi-dimensional that it is hard to identify periods of short-run Phillips curves which can be assigned to particular historical periods with any degree of accuracy or predictability. The analysis shows that reliance on a trade-off between inflation and unemployment for policy purposes is misplaced even in the short run.
Chimeric cowpea mosaic virus (CPMV) particles displaying foreign peptide antigens on the particle surface are suitable for development of peptide-based vaccines. However, commonly used PEG ...precipitation-based purification methods are not sufficient for production of high quality vaccine candidates because they do not allow for separation of chimeric particles from cleaved contaminating species. Moreover, the purified particles remain infectious to plants. To advance the CPMV technology further, it is necessary to develop efficient and scalable purification strategies and preferably eliminate the infectivity of chimeric viruses. CPMV was engineered to display a 25 amino acid peptide derived from the
Bacillus anthracis protective antigen on the surface loop of the large coat protein subunit. The engineered virus was propagated in cowpea plants and assembled into chimeric virus particles displaying 60 copies of the peptide on the surface. An effective inactivation method was developed to produce non-infectious chimeric CPMV virus-like particles (VLPs). Uncleaved VLPs were separated from the contaminating cleaved forms by anion exchange chromatography. The yield of purified chimeric VLPs was 0.3
g
kg
−1 of leaf tissue. The results demonstrate the ability to generate multi-gram quantities of non-infectious, chimeric CPMV VLPs in plants for use in the development of peptide-based vaccines.
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