Many membrane receptors activate phospholipase C (PLC) during signalling, triggering changes in t... more Many membrane receptors activate phospholipase C (PLC) during signalling, triggering changes in the levels of several plasma membrane (PM) lipids including PtdIns, PtdOH and PtdIns(4,5)P2. It is widely believed that exchange of lipids between the PM and endoplasmic reticulum (ER) is required to restore lipid homeostasis during PLC signalling, yet the mechanism remains unresolved. RDGB is a multi-domain protein with a PITP domain (RDGB-PITPd). We find that in vitro, RDGB-PITPd binds and transfers both PtdOH and PtdIns. In Drosophila photoreceptors that experience high rates of PLC activity, RDGB function is essential for phototransduction. We show that binding of PtdIns to RDGB-PITPd is essential for normal phototransduction; yet this property is insufficient to explain in vivo function since another Drosophila PITP (vib) that also binds PtdIns cannot rescue the phenotypes of RDGB deletion. In RDGB mutants, PtdIns(4,5)P2 resynthesis at the PM following PLC activation is delayed and P...
The brain's capacity to analyse and interpret information is limited ultimately by the input ... more The brain's capacity to analyse and interpret information is limited ultimately by the input it receives. This sets a premium on information capacity of sensory receptors, which can be maximized by optimizing sensitivity, speed and reliability of response. Nowhere is selection pressure for information capacity stronger than in the visual system, where speed and sensitivity can mean the difference between life and death. Phototransduction in flies represents the fastest G-protein-signalling cascade known. Analysis in Drosophila has revealed many of the underlying molecular strategies, leading to the discovery and characterization of signalling molecules of widespread importance.
Adult Drosophila have a compound eye composed of approximately 750 repeating units, called ommati... more Adult Drosophila have a compound eye composed of approximately 750 repeating units, called ommatidia, packed together to form the retina. Each ommatidium is a precise arrangement of 19 cells: eight photoreceptors (primary sensory neurons) and 11 accessory cells. Analysis of visual physiology in Drosophila photoreceptors has been central to understanding a number of important areas of modern biology including the G-protein-coupled receptor cycle, phosphoinositide signaling, and calcium signaling. Analysis of photoreceptor performance and synaptic transmission also can be studied using Drosophila photoreceptors as a model system. Electrophysiological analysis of responses to light is a powerful tool for characterizing and understanding visual transduction in Drosophila photoreceptors. The ommatidia can be dissociated to generate healthy cells under a limited set of conditions. The basolateral plasma membrane of the photoreceptors then becomes accessible to a patch pipette and light responses can be recorded in a whole-cell configuration. This approach has principally been used to study the activity of the light-activated channels in voltage-clamped photoreceptors.
Adult Drosophila have a compound eye composed of approximately 750 repeating units, called ommati... more Adult Drosophila have a compound eye composed of approximately 750 repeating units, called ommatidia, packed together to form the retina. Each ommatidium is a precise arrangement of 19 cells: eight photoreceptors (primary sensory neurons) and 11 accessory cells. Analysis of visual physiology in Drosophila photoreceptors has been central to understanding a number of important areas of modern biology, including the G-protein-coupled receptor cycle, phosphoinositide signaling, and calcium signaling. Analysis of photoreceptor performance and synaptic transmission are areas of neurobiology that have been studied using Drosophila photoreceptors as a model system. Electrophysiological analysis of responses to light is a powerful tool for characterizing and understanding visual transduction in Drosophila photoreceptors. This protocol describes the isolation of dissociated ommatidia for use in voltage-clamp recordings of currents from photoreceptors, allowing detailed analysis of ion channels such as the transient receptor potential and potassium channels in photoreceptors. Mechanical dissociation of ommatidia is typically performed in flies that are <4 h post-eclosion. In animals older than this, the ommatidia become progressively more firmly attached to each other and cannot be dissociated mechanically without causing substantial damage to the constituent photoreceptors. Like the rest of the fly, the eye is surrounded by a cuticle. Thus, before the dissociation process, the eye is dissected from the rest of the head and the retina is scooped out of the cuticular covering.
Analysis of visual physiology in Drosophila photoreceptors has been central to understanding a nu... more Analysis of visual physiology in Drosophila photoreceptors has been central to understanding a number of important areas of modern biology, including the G-protein-coupled receptor cycle, phosphoinositide signaling, and calcium signaling. Analysis of photoreceptor performance and synaptic transmission are areas of neurobiology that have been studied using Drosophila photoreceptors as a model system. Electrophysiological analysis of responses to light is a powerful tool for characterizing and understanding visual transduction in Drosophila photoreceptors, and electroretinograms (ERGs) have long been used as a physiological assay in the Drosophila visual system. In these recordings, a microelectrode is placed on the eye and a reference electrode is placed elsewhere on the animal (typically in the thorax). Upon light stimulation, the voltage difference between these two electrodes is measured and displayed in real time. Because recordings are performed in live animals, all photoreceptor cells are intact and therefore surrounded by fluid containing endogenous concentrations of ions. Moreover, synaptic transmission between the photoreceptor and downstream laminar neurons can be detected.
Absolute visual thresholds are limited by &am... more Absolute visual thresholds are limited by "dark noise," which in Drosophila photoreceptors is dominated by brief (∼10 ms), small (∼2 pA) inward current events, occurring at ∼2/s, believed to reflect spontaneous G protein activations. These dark events were increased in rate and amplitude by a point mutation in myosin III (NINAC), which disrupts its interaction with the scaffolding protein, INAD. This phenotype mimics that previously described in null mutants of ninaC (no inactivation no afterpotential; encoding myosin III) and an associated protein, retinophilin (rtp). Dark noise was similarly increased in heterozygote mutants of diacylglycerol kinase (rdgA/+). Dark noise in ninaC, rtp, and rdgA/+ mutants was greatly suppressed by mutations of the Gq α-subunit (Gαq) and the major light-sensitive channel (trp) but not rhodopsin. ninaC, rtp, and rdgA/+ mutations also all facilitated residual light responses in Gαq and PLC hypomorphs. Raising cytosolic Ca(2+) in the submicromolar range increased dark noise, facilitated activation of transient receptor potential (TRP) channels by exogenous agonist, and again facilitated light responses in Gαq hypomorphs. Our results indicate that RTP, NINAC, INAD, and diacylglycerol kinase, together with a Ca(2+)-dependent threshold, share common roles in suppressing dark noise and regulating quantum bump generation; consequently, most spontaneous G protein activations fail to generate dark events under normal conditions. By contrast, quantum bump generation is reliable but delayed until sufficient G proteins and PLC are activated to overcome threshold, thereby ensuring generation of full-size bumps with high quantum efficiency.
The trapping of lipid-laden macrophages in the arterial intima is a critical but reversible step ... more The trapping of lipid-laden macrophages in the arterial intima is a critical but reversible step in atherogenesis. However, information about possible treatments for this condition is lacking. Here, we hypothesized that combining the polyphenol-rich fractions (PHC) of commonly consumed spices (Allium sativum L (Liliaceae), Zingiber officinale R (Zingiberaceae), Curcuma longa L (Zingiberaceae)) and herbs (Terminalia arjuna (R) W & A (Combretaceae) and Cyperus rotundus L (Cyperaceae)) prevents foam cell formation and atherogenesis. Using an in vitro foam cell formation assay, we found that PHC significantly inhibited lipid-laden macrophage foam cell formation compared to the depleted polyphenol fraction of PHC (F-PHC). We further observed that PHC attenuated the LDL and LPS induced CD36, p-FAK and PPAR-γ protein expression in macrophages and increased their migration. NK-κB-DNA interaction, TNF-α, ROS generation, and MMP9 and MMP2 protein expression were suppressed in PHC-treated macr...
Multiple PIP2 dependent molecular processes including receptor activated phospholipase C activity... more Multiple PIP2 dependent molecular processes including receptor activated phospholipase C activity occur at the neuronal plasma membranes, yet levels of this lipid at the plasma membrane are remarkably stable. Although the existence of unique pools of PIP2 supporting these events has been proposed, the mechanism by which they are generated is unclear. In Drosophila photoreceptors, the hydrolysis of PIP2 by G-protein coupled phospholipase C activity is essential for sensory transduction of photons. We identify dPIP5K as an enzyme essential for PIP2 re-synthesis in photoreceptors. Loss of dPIP5K causes profound defects in the electrical response to light and light-induced PIP2 dynamics at the photoreceptor membrane. Overexpression of dPIP5K was able to accelerate the rate of PIP2 synthesis following light induced PIP2 depletion. Other PIP2 dependent processes such as endocytosis and cytoskeletal function were unaffected in photoreceptors lacking dPIP5K function. These results provide e...
ABSTRACT TiO2 thin films with 0.2 wt%, 0.4 wt%, 0.6wt%, and 0.8 wt% Fe were prepared on glass and... more ABSTRACT TiO2 thin films with 0.2 wt%, 0.4 wt%, 0.6wt%, and 0.8 wt% Fe were prepared on glass and silicon substrates using sol–gel spin coating technique. The optical cut-off points are increasingly red-shifted and the absorption edge is shifted over the higher wavelength region with Fe content increasing. As Fe content increases, the optical band gap decreases from 3.03 to 2.48 eV whereas the tail width increases from 0.26 to 1.43 eV. The X-ray diffraction (XRD) patterns for doped films at 0.2 wt% and 0.8 wt% Fe reveal no characteristic peaks, indicating that the film is amorphous whereas undoped TiO2 exhibits (101) orientation with anatase phase. Thin films of higher Fe content exhibit a homogeneous, uniform, and nano structured highly porous shell morphology.
Many membrane receptors activate phospholipase C (PLC) during signalling, triggering changes in t... more Many membrane receptors activate phospholipase C (PLC) during signalling, triggering changes in the levels of several plasma membrane (PM) lipids including PtdIns, PtdOH and PtdIns(4,5)P2. It is widely believed that exchange of lipids between the PM and endoplasmic reticulum (ER) is required to restore lipid homeostasis during PLC signalling, yet the mechanism remains unresolved. RDGB is a multi-domain protein with a PITP domain (RDGB-PITPd). We find that in vitro, RDGB-PITPd binds and transfers both PtdOH and PtdIns. In Drosophila photoreceptors that experience high rates of PLC activity, RDGB function is essential for phototransduction. We show that binding of PtdIns to RDGB-PITPd is essential for normal phototransduction; yet this property is insufficient to explain in vivo function since another Drosophila PITP (vib) that also binds PtdIns cannot rescue the phenotypes of RDGB deletion. In RDGB mutants, PtdIns(4,5)P2 resynthesis at the PM following PLC activation is delayed and P...
The brain's capacity to analyse and interpret information is limited ultimately by the input ... more The brain's capacity to analyse and interpret information is limited ultimately by the input it receives. This sets a premium on information capacity of sensory receptors, which can be maximized by optimizing sensitivity, speed and reliability of response. Nowhere is selection pressure for information capacity stronger than in the visual system, where speed and sensitivity can mean the difference between life and death. Phototransduction in flies represents the fastest G-protein-signalling cascade known. Analysis in Drosophila has revealed many of the underlying molecular strategies, leading to the discovery and characterization of signalling molecules of widespread importance.
Adult Drosophila have a compound eye composed of approximately 750 repeating units, called ommati... more Adult Drosophila have a compound eye composed of approximately 750 repeating units, called ommatidia, packed together to form the retina. Each ommatidium is a precise arrangement of 19 cells: eight photoreceptors (primary sensory neurons) and 11 accessory cells. Analysis of visual physiology in Drosophila photoreceptors has been central to understanding a number of important areas of modern biology including the G-protein-coupled receptor cycle, phosphoinositide signaling, and calcium signaling. Analysis of photoreceptor performance and synaptic transmission also can be studied using Drosophila photoreceptors as a model system. Electrophysiological analysis of responses to light is a powerful tool for characterizing and understanding visual transduction in Drosophila photoreceptors. The ommatidia can be dissociated to generate healthy cells under a limited set of conditions. The basolateral plasma membrane of the photoreceptors then becomes accessible to a patch pipette and light responses can be recorded in a whole-cell configuration. This approach has principally been used to study the activity of the light-activated channels in voltage-clamped photoreceptors.
Adult Drosophila have a compound eye composed of approximately 750 repeating units, called ommati... more Adult Drosophila have a compound eye composed of approximately 750 repeating units, called ommatidia, packed together to form the retina. Each ommatidium is a precise arrangement of 19 cells: eight photoreceptors (primary sensory neurons) and 11 accessory cells. Analysis of visual physiology in Drosophila photoreceptors has been central to understanding a number of important areas of modern biology, including the G-protein-coupled receptor cycle, phosphoinositide signaling, and calcium signaling. Analysis of photoreceptor performance and synaptic transmission are areas of neurobiology that have been studied using Drosophila photoreceptors as a model system. Electrophysiological analysis of responses to light is a powerful tool for characterizing and understanding visual transduction in Drosophila photoreceptors. This protocol describes the isolation of dissociated ommatidia for use in voltage-clamp recordings of currents from photoreceptors, allowing detailed analysis of ion channels such as the transient receptor potential and potassium channels in photoreceptors. Mechanical dissociation of ommatidia is typically performed in flies that are <4 h post-eclosion. In animals older than this, the ommatidia become progressively more firmly attached to each other and cannot be dissociated mechanically without causing substantial damage to the constituent photoreceptors. Like the rest of the fly, the eye is surrounded by a cuticle. Thus, before the dissociation process, the eye is dissected from the rest of the head and the retina is scooped out of the cuticular covering.
Analysis of visual physiology in Drosophila photoreceptors has been central to understanding a nu... more Analysis of visual physiology in Drosophila photoreceptors has been central to understanding a number of important areas of modern biology, including the G-protein-coupled receptor cycle, phosphoinositide signaling, and calcium signaling. Analysis of photoreceptor performance and synaptic transmission are areas of neurobiology that have been studied using Drosophila photoreceptors as a model system. Electrophysiological analysis of responses to light is a powerful tool for characterizing and understanding visual transduction in Drosophila photoreceptors, and electroretinograms (ERGs) have long been used as a physiological assay in the Drosophila visual system. In these recordings, a microelectrode is placed on the eye and a reference electrode is placed elsewhere on the animal (typically in the thorax). Upon light stimulation, the voltage difference between these two electrodes is measured and displayed in real time. Because recordings are performed in live animals, all photoreceptor cells are intact and therefore surrounded by fluid containing endogenous concentrations of ions. Moreover, synaptic transmission between the photoreceptor and downstream laminar neurons can be detected.
Absolute visual thresholds are limited by &am... more Absolute visual thresholds are limited by "dark noise," which in Drosophila photoreceptors is dominated by brief (∼10 ms), small (∼2 pA) inward current events, occurring at ∼2/s, believed to reflect spontaneous G protein activations. These dark events were increased in rate and amplitude by a point mutation in myosin III (NINAC), which disrupts its interaction with the scaffolding protein, INAD. This phenotype mimics that previously described in null mutants of ninaC (no inactivation no afterpotential; encoding myosin III) and an associated protein, retinophilin (rtp). Dark noise was similarly increased in heterozygote mutants of diacylglycerol kinase (rdgA/+). Dark noise in ninaC, rtp, and rdgA/+ mutants was greatly suppressed by mutations of the Gq α-subunit (Gαq) and the major light-sensitive channel (trp) but not rhodopsin. ninaC, rtp, and rdgA/+ mutations also all facilitated residual light responses in Gαq and PLC hypomorphs. Raising cytosolic Ca(2+) in the submicromolar range increased dark noise, facilitated activation of transient receptor potential (TRP) channels by exogenous agonist, and again facilitated light responses in Gαq hypomorphs. Our results indicate that RTP, NINAC, INAD, and diacylglycerol kinase, together with a Ca(2+)-dependent threshold, share common roles in suppressing dark noise and regulating quantum bump generation; consequently, most spontaneous G protein activations fail to generate dark events under normal conditions. By contrast, quantum bump generation is reliable but delayed until sufficient G proteins and PLC are activated to overcome threshold, thereby ensuring generation of full-size bumps with high quantum efficiency.
The trapping of lipid-laden macrophages in the arterial intima is a critical but reversible step ... more The trapping of lipid-laden macrophages in the arterial intima is a critical but reversible step in atherogenesis. However, information about possible treatments for this condition is lacking. Here, we hypothesized that combining the polyphenol-rich fractions (PHC) of commonly consumed spices (Allium sativum L (Liliaceae), Zingiber officinale R (Zingiberaceae), Curcuma longa L (Zingiberaceae)) and herbs (Terminalia arjuna (R) W & A (Combretaceae) and Cyperus rotundus L (Cyperaceae)) prevents foam cell formation and atherogenesis. Using an in vitro foam cell formation assay, we found that PHC significantly inhibited lipid-laden macrophage foam cell formation compared to the depleted polyphenol fraction of PHC (F-PHC). We further observed that PHC attenuated the LDL and LPS induced CD36, p-FAK and PPAR-γ protein expression in macrophages and increased their migration. NK-κB-DNA interaction, TNF-α, ROS generation, and MMP9 and MMP2 protein expression were suppressed in PHC-treated macr...
Multiple PIP2 dependent molecular processes including receptor activated phospholipase C activity... more Multiple PIP2 dependent molecular processes including receptor activated phospholipase C activity occur at the neuronal plasma membranes, yet levels of this lipid at the plasma membrane are remarkably stable. Although the existence of unique pools of PIP2 supporting these events has been proposed, the mechanism by which they are generated is unclear. In Drosophila photoreceptors, the hydrolysis of PIP2 by G-protein coupled phospholipase C activity is essential for sensory transduction of photons. We identify dPIP5K as an enzyme essential for PIP2 re-synthesis in photoreceptors. Loss of dPIP5K causes profound defects in the electrical response to light and light-induced PIP2 dynamics at the photoreceptor membrane. Overexpression of dPIP5K was able to accelerate the rate of PIP2 synthesis following light induced PIP2 depletion. Other PIP2 dependent processes such as endocytosis and cytoskeletal function were unaffected in photoreceptors lacking dPIP5K function. These results provide e...
ABSTRACT TiO2 thin films with 0.2 wt%, 0.4 wt%, 0.6wt%, and 0.8 wt% Fe were prepared on glass and... more ABSTRACT TiO2 thin films with 0.2 wt%, 0.4 wt%, 0.6wt%, and 0.8 wt% Fe were prepared on glass and silicon substrates using sol–gel spin coating technique. The optical cut-off points are increasingly red-shifted and the absorption edge is shifted over the higher wavelength region with Fe content increasing. As Fe content increases, the optical band gap decreases from 3.03 to 2.48 eV whereas the tail width increases from 0.26 to 1.43 eV. The X-ray diffraction (XRD) patterns for doped films at 0.2 wt% and 0.8 wt% Fe reveal no characteristic peaks, indicating that the film is amorphous whereas undoped TiO2 exhibits (101) orientation with anatase phase. Thin films of higher Fe content exhibit a homogeneous, uniform, and nano structured highly porous shell morphology.
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Papers by P. Raghu