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- Fluorescent prey traps in carnivorous plants. [Journal Article, Research Support, Non-U.S. Gov't]
- Plant Biol (Stuttg) 2013 May; 15(3):611-5.
Carnivorous plants acquire most of their nutrients by capturing ants, insects and other arthropods through their leaf-evolved biological traps. So far, the best-known attractants in carnivorous prey traps are nectar, colour and olfactory cues. Here, fresh prey traps of 14 Nepenthes, five Sarracenia, five Drosera, two Pinguicula species/hybrids, Dionaea muscipula and Utricularia stellaris were scanned at UV 366 nm. Fluorescence emissions of major isolates of fresh Nepenthes khasiana pitcher peristomes were recorded at an excitation wavelength of 366 nm. N. khasiana field pitcher peristomes were masked by its slippery zone extract, and prey capture rates were compared with control pitchers. We found the existence of distinct blue fluorescence emissions at the capture spots of Nepenthes, Sarracenia and Dionaea prey traps at UV 366 nm. These alluring blue emissions gradually developed with the growth of the prey traps and diminished towards their death. On excitation at 366 nm, N. khasiana peristome 3:1 CHCl3–MeOH extract and its two major blue bands showed strong fluorescence emissions at 430–480 nm. Masking of blue emissions on peristomes drastically reduced prey capture in N. khasiana pitchers. We propose these molecular emissions as a critical factor attracting arthropods and other visitors to these carnivorous traps. Drosera, Pinguicula and Utricularia prey traps showed only red chlorophyll emissions at 366 nm.
- Fluorescence 'turn-on' sensor for F(-) derived from vitamin B6 cofactor. [JOURNAL ARTICLE]
- Analyst 2013 May 22.
A novel vitamin B6 Schiff base analog () was synthesized by combining vitamin B6 cofactor pyridoxal with 2-aminophenol. Receptor displays a color change detectable by the naked-eye from yellow to red in the presence of fluoride and acetate due to the formation of hydrogen bonding host-guest complexes in 1 : 1 stoichiometry. Importantly, receptor showed fluoride-selective 'turn-on' fluorescent response with a detection limit (3σ) of 7.39 × 10(-8) M.
- Acetylcholine Promotes Ca(2+)and NO-Oscillations in Adipocytes Implicating Ca(2+)→NO→cGMP→cADP-ribose→Ca(2+) Positive Feedback Loop - Modulatory Effects of Norepinephrine and Atrial Natriuretic Peptide. [Journal Article]
- PLoS One 2013; 8(5):e63483.
This study investigated possible mechanisms of autoregulation of Ca(2+) signalling pathways in adipocytes responsible for Ca(2+) and NO oscillations and switching phenomena promoted by acetylcholine (ACh), norepinephrine (NE) and atrial natriuretic peptide (ANP).Fluorescent microscopy was used to detect changes in Ca(2+) and NO in cultures of rodent white adipocytes. Agonists and inhibitors were applied to characterize the involvement of various enzymes and Ca(2+)-channels in Ca(2+) signalling pathways.ACh activating M3-muscarinic receptors and Gβγ protein dependent phosphatidylinositol 3 kinase induces Ca(2+) and NO oscillations in adipocytes. At low concentrations of ACh which are insufficient to induce oscillations, NE or α1, α2-adrenergic agonists act by amplifying the effect of ACh to promote Ca(2+) oscillations or switching phenomena. SNAP, 8-Br-cAMP, NAD and ANP may also produce similar set of dynamic regimes. These regimes arise from activation of the ryanodine receptor (RyR) with the implication of a long positive feedback loop (PFL): Ca(2+)→ NO→cGMP→cADPR→Ca(2+), which determines periodic or steady operation of a short PFL based on Ca(2+)-induced Ca(2+) release via RyR by generating cADPR, a coagonist of Ca(2+) at the RyR. Interplay between these two loops may be responsible for the observed effects. Several other PFLs, based on activation of endothelial nitric oxide synthase or of protein kinase B by Ca(2+)-dependent kinases, may reinforce functioning of main PFL and enhance reliability. All observed regimes are independent of operation of the phospholipase C/Ca(2+)-signalling axis, which may be switched off due to negative feedback arising from phosphorylation of the inositol-3-phosphate receptor by protein kinase G.This study presents a kinetic model of Ca(2+)-signalling system operating in adipocytes and integrating signals from various agonists, which describes it as multivariable multi feedback network with a family of nested positive feedback.
- APC(CDH1) Targets MgcRacGAP for Destruction in the Late M Phase. [Journal Article]
- PLoS One 2013; 8(5):e63001.
Male germ cell RacGTPase activating protein (MgcRacGAP) is an important regulator of the Rho family GTPases - RhoA, Rac1, and Cdc42 - and is indispensable in cytokinesis and cell cycle progression. Inactivation of RhoA by phosphorylated MgcRacGAP is an essential step in cytokinesis. MgcRacGAP is also involved in G1-S transition and nuclear transport of signal transducer and activator of transcription 3/5 (STAT3/5). Expression of MgcRacGAP is strictly controlled in a cell cycle-dependent manner. However, the underlying mechanisms have not been elucidated. METHODOLOGYPRINCIPAL FINDINGS: Using MgcRacGAP deletion mutants and the fusion proteins of full-length or partial fragments of MgcRacGAP to mVenus fluorescent protein, we demonstrated that MgcRacGAP is degraded by the ubiquitin-proteasome pathway in the late M to G1 phase via APC(CDH1). We also identified the critical region for destruction located in the C-terminus of MgcRacGAP, AA537-570, which is necessary and sufficient for CDH1-mediated MgcRacGAP destruction. In addition, we identified a PEST domain-like structure with charged residues in MgcRacGAP and implicate it in effective ubiquitination of MgcRacGAP. CONCLUSIONSSIGNIFICANCE: Our findings not only reveal a novel mechanism for controlling the expression level of MgcRacGAP but also identify a new target of APC(CDH1). Moreover our results identify a C-terminal region AA537-570 of MgcRacGAP as its degron.
- Constitutive Somatostatin Receptor Subtype 2 Activity Attenuates GH Synthesis. [JOURNAL ARTICLE]
- Endocrinology 2013 May 21.
Somatostatin signals predominantly through somatostatin receptor (SSTR) subtype 2 to attenuate GH release. However, the independent role of the receptor in regulating GH synthesis is unclear. Because we had previously demonstrated constitutive SSTR2 activity in mouse corticotrophs, we now analyzed GH regulation in rat pituitary somatotroph (GC) tumor cells, which express SSTR2 exclusively and are devoid of endogenous somatostatin ligand. We demonstrate that moderately stable SSTR2 overexpression (GpSSTR2WT cells) was associated with decreased GH promoter activity, GH mRNA, and hormone levels compared with those of control transfectants (GpCon cells). In contrast, levels of GH mRNA and peptide and GH promoter activity were unchanged in GpSSTR2DRY stable transfectants moderately expressing DRY motif mutated SSTR2 (R140A). GpSSTR2DRY did not exhibit an enhanced octreotide response as did GpSSTR2WT cells; however, both SSTR2WT-enhanced yellow fluorescent protein (eYFP) and SSTR2DRY-eYFP internalized on octreotide treatment. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, increased GH synthesis in wild-type GC cells and primary pituitary cultures. GpSSTR2WT cells induced GH synthesis more strongly on SAHA treatment, evident by both higher GH peptide and mRNA levels compared with the moderate but similar GH increase observed in GpCon and GpSSTR2DRY cells. In vivo SAHA also increased GH release from GpSSTR2WT but not from control xenografts. Endogenous rat GH promoter chromatin immunoprecipitation showed decreased baseline acetylation of the GH promoter with exacerbated acetylation after SAHA treatment in GpSSTR2WT compared with that of either GpSSTR2DRY or control cells, the latter 2 transfectants exhibiting similar GH promoter acetylation levels. In conclusion, modestly increased SSTR2 expression constitutively decreases GH synthesis, an effect partially mediated by GH promoter histone deacetylation.
- Axonal Remodeling of the Corticospinal Tract in the Spinal Cord Contributes to Voluntary Motor Recovery After Stroke in Adult Mice. [JOURNAL ARTICLE]
- Stroke 2013 May 21.
BACKGROUND AND PURPOSE:We sought to demonstrate the contribution of axonal remodeling of the corticospinal tract (CST) in the spinal cord to functional outcome after stroke.
METHODS:Bilateral pyramidotomy (BPT) or sham-BPT was performed in mice with transgenic yellow fluorescent protein labeling in the CST subjected to middle cerebral artery occlusion (MCAo). Foot-fault and single pellet reaching tests were performed 3 days after MCAo and weekly thereafter. Mice were euthanized at day 14 or 28 after stroke. Immunofluorescent staining for growth-associated protein-43 and Synaptophysin was performed on cervical sections.
RESULTS:Functional improvements were evident during the initial 14 days in both MCAo-sham-BPT and MCAo-BPT mice (P<0.01, versus day 3). Progressive recovery was present during the subsequent 14 days in MCAo-sham-BPT mice (P<0.001, versus day 14) but not in MCAo-BPT mice. In the stroke-affected cervical gray matter of MCAo-sham-BPT mice, growth-associated protein-43-Cy3 staining on CST axons were significantly increased at day 14 after stroke compared with normal mice (P<0.001), and CST axonal density and Synaptophysin-Cy3 staining of CST-yellow fluorescent protein axonal terminals were significantly increased at day 28 compared with day 14 after MCAo (P<0.001).
CONCLUSIONS:Our data demonstrate that voluntary motor recovery is associated with CST axonal outgrowth and synaptic formation in the denervated side of the spinal gray matter during the later phase after stroke, suggesting that the CST axonal plasticity in the spinal cord contributes to neurological recovery.
- Epididymal specific, selenium-independent GPX5 protects cells from oxidative stress-induced lipid peroxidation and DNA mutation. [JOURNAL ARTICLE]
- Hum Reprod 2013 May 21.
STUDY QUSTION: Can selenium (Se) independent, epididymal-specific glutathione peroxidase 5 (GPX5) protect CHO-K1 cells from oxidative damage and, more specifically, from lipid peroxidation and DNA mutation? SUMMARY ANSWER: CHO-K1 cells expressing GPX5 have increased resistance to oxidative challenge and, more specifically, decreased levels of lipid peroxidation and decreased levels of the downstream DNA lesion 8-oxo-7,8-dihydroguanine (8-oxodG) compared with control cells. WHAT IS KNOWN ALREADY: GPX5 associates with sperm during transit of the epididymis, and has been postulated to protect sperm from peroxide-mediated attack. However, its function as an active glutathione peroxidase has been questioned due to substitution of the classical selenocysteine residue at its active site. Indirect evidence for a functional role for GPX5 has been provided by in vivo studies, in particular from the GPX5 knockout mouse whereby offspring sired by GPX5(-/-) males have a higher rate of spontaneous abortion and developmental defects, attributed to increased oxidative injury (8-oxodG) to sperm DNA, but only when the GPX5(-/-) males are over 1 year of age. Interestingly, we have previously shown severely reduced levels of GPX5 in humans.
STUDY DESIGN, SIZE, DURATION: To look more directly at its role in protection against oxidative damage, we have used an in vitro system, generating a CHO-K1 mammalian cell line expressing recombinant rat GPX5. PARTICIPANTS/MATERIALS, SETTING,
METHODS:We have used the recombinant CHO-K1 cells to determine whether GPX5 is able to protect these cells from an administered oxidative challenge, using a range of approaches. We compared the viability of GPX5-expressing cells with control cells by both MTT and trypan blue exclusion assays. We next investigated whether GPX5 protects the cells specifically from lipid peroxidation, by using the fluorescent reporter molecule C11-BODIPY((581/591)), and thus from downstream DNA mutation, by comparing levels of the DNA lesion 8-oxodG. We also investigated whether GPX5 can be transferred to rat sperm via epididymosomes. MAIN
RESULTSAND THE ROLE OF CHANCE: GPX5-expressing CHO-K1 cells had increased viability compared with control cells following oxidative challenge (P < 0.005). We also found that GPX5-expressing CHO-K1 cells had significantly lower levels of C11-BODIPY((581/591)) oxidation, and hence lipid peroxidation, compared with control cells. Levels of 8-oxodG DNA damage were also markedly lower in the nuclei of GPX5-expressing cells than in control cells. Finally, we showed that GPX5 can be transferred to rat sperm via epididymosomes. LIMITATIONS, REASONS FOR CAUTION: GPX5 is not active in glutathione peroxidase assays using H2O2 as the substrate. However, the related non-mammalian Se-independent GPXs show preference for electron donors other than glutathione, with a number utilizing thioredoxin as a reducing equivalent. Hence, the in vitro activity of GPX5 needs to be assessed using a range of alternative substrates and electron donors. GPX5 is secreted by the epididymis and associates with the sperm plasma membrane. We showed that this transfer can occur via epididymosomes; however, the mechanism for transfer and the identity of a potential binding partner in the sperm membrane needs to be determined. Finally, our study utilized an in vitro system that needs to be translated to human sperm. WIDER IMPLICATIONS OF THE
FINDINGS:Our study supports an important role for GPX5 as an antioxidant, possibly acting as a phospholipid hydroperoxidase and participating in the maintenance of cell and DNA integrity. STUDY FUNDING/COMPETING INTEREST(S): This project was funded in part by the BBSRC. The authors declare no conflict of interest.
- Conditional viral tracing reveals that steroidogenic factor 1-positive neurons of the dorsomedial subdivision of the ventromedial hypothalamus project to autonomic centers of the hypothalamus and hindbrain. [JOURNAL ARTICLE]
- J Comp Neurol 2013 May 21.
Excitation of neurons in the ventromedial hypothalamus (VMH), especially those residing in the dorsomedial part of the nucleus (VMHdm), evokes sympathetic nervous system (SNS) outflow, modulating a number of physiological functions including feeding and blood glucose homeostasis. However, the anatomical basis of VMH-mediated SNS activation has thus far proved elusive. To understand how VMH neurons exercise output functions and describe an anatomical link between these neurons and the SNS, we identified downstream neural targets of the VMHdm by injecting an adenoviral vector encoding Cre recombinase (Cre)-regulated farnesylated green fluorescent protein (GFPf ) into the VMHdm of mice that express Cre in neurons expressing the VMH-specific transcription factor steroidogenic factor 1 (SF1). We confirm previously described projection patterns of the VMHdm and report the existence of a formerly unidentified projection pathway to a number of autonomic centers in the brainstem. These VMH efferents travel caudally through the periaqueductal gray (PAG) and then ventrally through the lateral lemniscus to the ventral surface of the brain, where they eventually reach caudal autonomic centers including the C1 catecholamine cell group of the rostral ventrolateral medulla (RVLM) and the nucleus of the solitary tract (NTS), where VMH efferents make close contacts with catecholaminergic neurons. We also found that VMHdm fibers reach a number of brainstem areas, including the retrotrapezoid nucleus (RTN), which are important in regulating respiration. Thus, the present study indicates that the VMH may modulate sympathetic and autonomic activity via synaptic contacts in the RTN, NTS, and RVLM and provides significant anatomical evidence to support a role of the VMH in respiratory regulation. J. Comp. Neurol. , 2013. © 2013 Wiley Periodicals, Inc.
- [Effects of esophageal cancer on the nerve fiber growth and guidance]. [English Abstract, Journal Article]
- Zhonghua Wei Chang Wai Ke Za Zhi 2013 May; 16(5):474-8.
To establish the co-culture model of cancer cells and nerve, and to study the influence of esophageal cancer on nerve fibers.Mouse dorsal root ganglion (DRG) was cultured in sterile conditions by primary isolation. Co-culture model was established using matrigel matrix-embedded DRG and EC109 (esophageal cancer cell line) transfected with green fluorescent protein. Morphological changes of DRG, number and area of neurites were quantified with microscopy and image analysis. Furthermore, the mRNA expression of nerve growth factor(NGF) and brain derived neurotrophic factor(BDNF) was detected by real-time PCR.In mixture cultivation model of EC109 and DRG cells, directional outgrowth of neurite projecting to EC109 was observed, and the length of neurite was markedly longer in proximal field compared to distal field. The number and area of neurite were 87 and 346 μm(2) in proximal field respectively, and 23 and 141 μm(2) in distal field on the 7th day. The expressions of NGF and BDNF were up-regulated in esophageal cancer cells.The esophageal cancer may play an important role in nerve fiber growth and guidance, which may be associated with the up-regulation of NGF and BDNF expressions.
- Fluorescent DNA Hydrogels Composed of Nucleic Acid-Stabilized Silver Nanoclusters. [JOURNAL ARTICLE]
- Small 2013 May 21.
Y-shaped DNA units functionalized with Ag-nanoclusters are crosslinked by nucleic acids to yield fluorescent hydrogels with controlled luminescence properties.