Rajkumar Rede Patil - Academia.edu (original) (raw)

Papers by Rajkumar Rede Patil

Investigative ophthalmology & visual science, 2001

To identify retinal proteins that are the targets of serum autoantibodies in patients with glauco... more To identify retinal proteins that are the targets of serum autoantibodies in patients with glaucoma. To identify retinal antigens that are recognized by the sera of patients with glaucoma, immunoreactive bands were separated, by using two-dimensional gel electrophoresis of the bovine retinal soluble fraction. A 29-kDa band was then selected for further analysis. Tryptic peptides of the 29-kDa band were analyzed using electrospray mass spectrometry to identify the protein. After protein identification, immunoreactivity against this newly identified protein was studied by Western blot analysis using sera from 65 patients with glaucoma (25 with primary open-angle glaucoma [POAG]; 40 with normal-pressure glaucoma [NPG]) and 25 age-matched healthy subjects. In addition, serum antibody titers were compared in these groups, by using a specific enzyme-linked immunosorbent assay (ELISA). The 29-kDa band was identified as glutathione S-transferase (GST). Western blot analysis revealed that se...

Investigative ophthalmology & visual science, 2001

Recent evidence strongly suggests that activated immunity occurs during the neurodegenerative pro... more Recent evidence strongly suggests that activated immunity occurs during the neurodegenerative process of glaucomatous optic neuropathy. Although activation of lamina cribrosa astrocytes has been identified in glaucomatous optic nerve head, their role on the activated immune responses seen in glaucoma patients is unknown. Here, the authors aimed to study the potential role of lamina cribrosa astrocytes as a component of activated immune responses seen in glaucoma patients. Expression of HLA-DR in optic nerve head astrocytes was studied using immunohistochemistry in postmortem eyes of patients with glaucoma and normal donors. Serum cytokine levels of patients with glaucoma and control subjects were measured using enzyme-linked, immunosorbent assay. In addition, in vitro experiments were performed using astrocyte cultures derived from human optic nerve head or fetal human brain. The cultured astrocytes were incubated under selected stress conditions such as exposure to cytokines, IFN-g...

Investigative ophthalmology & visual science, 1994

To examine the interaction of alpha subunits of guanine nucleotide binding proteins with A1 adeno... more To examine the interaction of alpha subunits of guanine nucleotide binding proteins with A1 adenosine receptors from the SV40-transformed bovine-derived pigmented (PE) and human-derived nonpigmented (NPE) ciliary epithelial cell lines using an immunoprecipitation approach and [3H]DPCPX, a selective radioligand to adenosine receptor. Solubilized preparations of adenosine receptors from PE and NPE cell lines were immunoprecipitated with G protein specific antisera 8730 (anti-Gi alpha), 3646 (anti-Gi alpha 1), 1521 (anti-Gi alpha 2), and 1518 (anti-Gi alpha 3), and of adenosine receptor-G protein complexes were detected by the binding of radioactive [3H]DPCPX. Data indicate that [3H]DPCPX forms high-affinity complex with membrane-bound and solubilized forms of adenosine receptors from PE and NPE cells. Peptide-directed antisera against various G protein alpha subunits indicate that the A1 adenosine receptors from these cells are specifically coupled to Gi alpha complexes. The results f...

Investigative ophthalmology & visual science, 2002

Immunocytochemistry showed strong aquaporin (AQP)-4 water channel expression in Müller cells in m... more Immunocytochemistry showed strong aquaporin (AQP)-4 water channel expression in Müller cells in mouse retina and fibrous astrocytes in optic nerve. This study was designed to test the hypothesis that AQP4 is required for vision by comparing electroretinograms and retinal morphology in wild-type mice and transgenic knockout mice with no AQP4. Electroretinograms were recorded over a 10(5)-fold range of flash intensities in dark-adapted mice and analyzed for a- and b-wave amplitude and latency, a-wave normalized slope, and oscillatory potential amplitude and latency. AQP4 protein was localized in mouse retina by immunocytochemistry, and retinal morphology was studied by light and electron microscopy. Significantly reduced electroretinogram b-wave potentials were recorded in 10-month-old null mice with smaller changes in 1-month-old mice. Immunocytochemistry showed strong AQP4 protein expression in retina of wild-type mice. Morphologic analysis of retina by light and electron microscopy...

Molecular vision, Jan 3, 2010

Changes in the expression of water channels (aquaporins; AQP) have been reported in several disea... more Changes in the expression of water channels (aquaporins; AQP) have been reported in several diseases. However, such changes and mechanisms remain to be evaluated for retinal injury after optic nerve crush (ONC). This study was designed to analyze changes in the expression of AQP4 (water selective channel) and AQP9 (water and lactate channel) following ONC in the rat. Rat retinal ganglion cells (RGCs) were retrogradely labeled by applying FluoroGold onto the left superior colliculus 1 week before ONC. Retinal injuries were induced by ONC unilaterally. Real-time PCR was used to measure changes in AQP4, AQP9, thy-1, Kir4.1 (K(+) channel), and beta-actin messages. Changes in AQP4, AQP9, Kir4.1, B cell lymphoma-x (bcl-xl), and glial fibrillary acidic protein (GFAP) expression were measured in total retinal extracts using western blotting. The number of RGCs labeled retrogradely from the superior colliculus was 2,090+/-85 cells/mm(2) in rats without any treatment, which decreased to 1,091...

Molecular vision, Jan 10, 2010

The purpose of this study was to determine the effects of down-regulation of Aquaporin 1 (AQP1) a... more The purpose of this study was to determine the effects of down-regulation of Aquaporin 1 (AQP1) and Aquaporin 5 (AQP5) on cell proliferation and migration in human corneal endothelial (HCEC) and human corneal epithelial (CEPI17) cell lines, respectively. AQP1 and AQP5 were down regulated using siRNA following lipofectamine-mediated transfection in corneal endothelial and epithelial cells, respectively. Down-regulation was confirmed using RT-PCR, indirect immunofluorescence, and immunoblot analysis. Total internal reflection fluorescence (TIRF) microscopy was used to detect cell surface aquaporin expression. Cell proliferation was determined by SRB (sulfrodamine B) assay. Cell migration was determined by in vitro wound healing and migration assay. In HCEC cells, AQP1 was localized to the cytosol as well as cell membrane and its down-regulation resulted in decreased cell proliferation and migration with a significant decrease in phosphorylated ERK (pERK). In CEPI17 cells AQP5 protein ...

Molecular vision, 2013

To examine the bradykinin (BK) B₂-receptor system in human and monkey ciliary muscle (CM) using i... more To examine the bradykinin (BK) B₂-receptor system in human and monkey ciliary muscle (CM) using immunohistochemical techniques, and to pharmacologically characterize the associated biochemical signal transduction systems in human CM (h-CM) cells. BK-induced modulation of intraocular pressure (IOP) in pigmented Dutch-Belt rabbits and cynomolgus monkeys was also studied. Previously published procedures were used throughout these studies. The human and monkey ciliary bodies expressed high levels of B₂-receptor protein immunoreactivity. Various kinins differentially stimulated [Ca²⁺](i) mobilization in primary h-CM cells (BK EC₅₀=2.4±0.2 nM > Hyp³,β-(2-thienyl)-Ala⁵,Tyr(Me)⁸-(®)-Arg⁹-BK (RMP-7) > Des-Arg⁹-BK EC₅₀=4.2 µM [n=3-6]), and this was blocked by B₂-selective antagonists, HOE-140 (IC₅₀=1.4±0.1 nM) and WIN-63448 (IC₅₀=174 nM). A phospholipase C inhibitor (U73122; 10-30 µM) and ethylene glycol tetraacetic acid (1-2 mM) abolished the BK-induced [Ca²⁺](i) mobilization. Total pr...

Journal of Power Sources, 2015

h i g h l i g h t s Non-isothermal electrochemical model developed for lithium composite cathode ... more h i g h l i g h t s Non-isothermal electrochemical model developed for lithium composite cathode cell. Experimentally obtained composite cathode property implemented in the model. Model validated for a wide range of temperature and discharge rate. Results analyzed to determine the heat generation pattern in the cell.

Molecular Biology and Physiology of Water and Solute Transport, 2000

Ciliary epithelium is responsible for the secretion of aqueous humor into posterior chamber of th... more Ciliary epithelium is responsible for the secretion of aqueous humor into posterior chamber of the eye. Most of aqueous humor secretion is driven by the active transport of ions from plasma into the posterior chamber followed by rapid movement of water and water soluble substances (Jacob and Civan, 1996). Both the outer nonpigmented (NPE) and inner pigmented (PE) epithelial layers exhibit properties of transporting epithelia (Helbig et al., 1988; Wolosin et al., 1993). The NPE is thought to provide the direct driving force for aqueous humor formation. Na+/K+-ATPase which is located on the basolateral membranes of NPE drives sodium secretion and provides the main ion motive force for sodium dependent cotransporters (Coca-Prados and Lopez-Briones, 1987; Flugel and Lutjen-Drecoll, 1988; Okami et al., 1989; Ghosh et al., 1990). Recently, it has been proposed that the rate limiting step in active secretion of aqueous humor is a CI- transport via a DIDS sensitive, cAMP dependent chloride channel (Chen et al., 1994; Jacob and Civan, 1996). Although the Na+/K+-ATPase active transport system is a primary transducer of cellular metabolic energy into fluid transport, it does not completely account for a variety of observations and functions that are prerequisites for the effective transport of fluid and electrolytes by the duallayered ciliary epithelium. For example, cell volume regulation which is coupled to ion transport through parallel K+ and, more importantly, Cl- channel activity appear to be an intrinsically important aspects of normal aqueous secretion (Civan et al., 1992; Yantorno el al., 1992; Fischbarg, 1997).

Investigative ophthalmology & visual science, 2001

To determine the expression and localization of tumor necrosis factor (TNF)-alpha and TNF-alpha r... more To determine the expression and localization of tumor necrosis factor (TNF)-alpha and TNF-alpha receptor-1 in the retina of normal and glaucomatous eyes. Using immunohistochemistry and in situ hybridization, retinal expression and localization of TNF-alpha and TNF-alpha receptor-1 were studied in retina sections from 20 eyes of donors with glaucoma, and 20 eyes of age-matched normal donors. According to immunohistochemistry, the intensity of the immunostaining and the number of labeled cells for TNF-alpha or its receptor were greater in retina sections of glaucomatous eyes than in control eyes of age-matched normal donors. In situ hybridization showed that mRNA signals for TNF-alpha or TNF-alpha receptor-1 were similarly more intense in glaucomatous eyes than in age-matched control eyes. Both protein and mRNA of TNF-alpha or TNF-alpha receptor-1 were predominantly localized to the inner retinal layers. Double-immunofluorescence labeling demonstrated that retinal immunostaining for T...

RESULTS. According to immunohistochemistry, the intensity of the immunostaining and the number of... more RESULTS. According to immunohistochemistry, the intensity of the immunostaining and the number of labeled cells for TNF-a or its receptor were greater in retina sections of glaucomatous eyes than in control eyes of age-matched normal donors. In situ hybridization showed that mRNA signals for TNF-a or TNF-a receptor-1 were similarly more intense in glaucomatous eyes than in age-matched control eyes. Both protein and mRNA of TNF-a or TNF-a receptor-1 were predominantly localized to the inner retinal layers. Double-immunofluorescence labeling demonstrated that retinal immunostaining for TNF-a was predominantly positive in the glial cells, whereas immunostaining for TNF-a receptor-1 was mainly positive in the retinal ganglion cells.

Journal of Ocular Pharmacology and Therapeutics, 1994

Journal of Food Science and Technology, 2011

Journal of Biological Chemistry, 1998

The molecular mechanisms for regulating water balance in many tissues are unknown. Like the kidne... more The molecular mechanisms for regulating water balance in many tissues are unknown. Like the kidney, the eye contains multiple water channel proteins (aquaporins) that transport water through membranes, including two (AQP1 and AQP4) in the ciliary body, the site of aqueous humor production. However, because humans with defective AQP1 are phenotypically normal and because the ocular application of phorbol esters reduce intraocular pressure, we postulated that the water channel activity of AQP4 may be regulated by these agents. We now report that protein kinase C activators, phorbol 12,13-dibutyrate, and phorbol 12-myristate 13acetate strongly stimulate the phosphorylation of AQP4 and inhibit its activity in a dose-dependent manner. Phorbol 12,13-dibutyrate (10 M) and phorbol 12-myristate 13-acetate (10 nM) reduced the rate of AQP4-expressing oocyte swelling by 87 and 92%, respectively. Further, phorbol 12,13-dibutyrate significantly increased the amount of phosphorylated AQP4. These results demonstrate that protein kinase C can regulate the activity of AQP4 through a mechanism involving protein phosphorylation. Moreover, they suggest important potential roles for AQP4 in several clinical disorders involving rapid water transport such as glaucoma, brain edema, and swelling of premature infant lungs.

Investigative Opthalmology & Visual Science, 2003

Experimental Eye Research, 1998

Current Eye Research, 2000

Water channel proteins are important pathways for water movements across cell membranes, includin... more Water channel proteins are important pathways for water movements across cell membranes, including those in the ciliary epithelium, which is the major site of aqueous humor secretion. In this study, we aimed to demonstrate the expression of functionally active aquaporin-1 (AQP1) water channels in cultured human ciliary epithelial cells. Poly A(+) RNA was isolated from cell cultures of Simian Virus 40 (SV-40) transformed human nonpigmented ciliary epithelium (NPE) subjected to RT-PCR reaction using primers specific to AQP1. Northern analysis was used to define the expression of AQP1 in NPE cells. Western immunoblotting with polyclonal antibody raised against AQP1 was used to evaluate the AQP1 protein expression in the plasma membranes of human NPE cells. Light scattering method was used to determine the osmotic water permeability in the suspension of NPE cells. RT-PCR using specific primers for AQP1, Northern analysis and Western immunoblot using AQP1 specific antibody demonstrated the expression of AQP1 in the plasma membranes of NPE cells. Osmotic water permeability (P( f)) measurements confirmed that functional AQP1 water channels are expressed in human NPE cells and the P(f) for these cells was 9.8 x 10( -3) cm/s at 10 degrees C. The presence of AQP1 in human NPE cells suggests that it may have a role in the fluid flow across epithelial membranes. In addition, the existence of AQP1 in the human NPE cells provide an excellent in vivo model to study the regulation of aquaporins and their possible role in the aqueous humor secretion.

Current Eye Research, 2000

To count retrogradely labeled retinal ganglion cells by Fluoro-Gold. Retinal ganglion cells were ... more To count retrogradely labeled retinal ganglion cells by Fluoro-Gold. Retinal ganglion cells were retrogradely labeled using bilateral injections of Fluoro-Gold into the superior colliculus. One week after injections, retinas were dissociated and immunolabeled using specific antibody against Fluoro-Gold. The Fluoro-Gold labeled cells were then counted using flow cytometry. Flow cytometry revealed that approximately 7% of the dissociated retinal cells were ganglion cells retrogradely labeled by Fluoro-Gold (Fig. 1B). Based on the total count of retinal cells per eye, the total number of retinal ganglion cells was estimated at approximately 131,250 +/- 2,542 per rat eye. The coefficient of variation of counts was calculated as 1.98%. The use of flow cytometry facilitates simple, reproducible and rapid quantification of virtually all of the retinal ganglion cells labeled by Fluoro-Gold in a single rat eye.

Carbohydrate Research, 1985

Abstract Synergism between (1→4)-β- d -glucan cellobiohydrolase, endo-(1→4)-β- d -glucanases, and... more Abstract Synergism between (1→4)-β- d -glucan cellobiohydrolase, endo-(1→4)-β- d -glucanases, and β- d -glucosidases of Sclerotium rolfsii for solubilization of native and amorphous celluloses is discussed. Besides synergism between cellobiohydrolase and endo-β-glucanases of S. rolfsii , a synergistic effect between endo-β-glucanases and β-glucosidases [which behaved rather as (1→4)-β- d -glucan glucohydrolases] was observed for solubilization of crystalline and amorphous celluloses. It seems that a cellobiohydrolase initiates the attack on crystalline cellulose and an endo-β- d -glucanase the attack on amorphous cellulose.

Investigative ophthalmology & visual science, 2001

To identify retinal proteins that are the targets of serum autoantibodies in patients with glauco... more To identify retinal proteins that are the targets of serum autoantibodies in patients with glaucoma. To identify retinal antigens that are recognized by the sera of patients with glaucoma, immunoreactive bands were separated, by using two-dimensional gel electrophoresis of the bovine retinal soluble fraction. A 29-kDa band was then selected for further analysis. Tryptic peptides of the 29-kDa band were analyzed using electrospray mass spectrometry to identify the protein. After protein identification, immunoreactivity against this newly identified protein was studied by Western blot analysis using sera from 65 patients with glaucoma (25 with primary open-angle glaucoma [POAG]; 40 with normal-pressure glaucoma [NPG]) and 25 age-matched healthy subjects. In addition, serum antibody titers were compared in these groups, by using a specific enzyme-linked immunosorbent assay (ELISA). The 29-kDa band was identified as glutathione S-transferase (GST). Western blot analysis revealed that se...

Investigative ophthalmology & visual science, 2001

Recent evidence strongly suggests that activated immunity occurs during the neurodegenerative pro... more Recent evidence strongly suggests that activated immunity occurs during the neurodegenerative process of glaucomatous optic neuropathy. Although activation of lamina cribrosa astrocytes has been identified in glaucomatous optic nerve head, their role on the activated immune responses seen in glaucoma patients is unknown. Here, the authors aimed to study the potential role of lamina cribrosa astrocytes as a component of activated immune responses seen in glaucoma patients. Expression of HLA-DR in optic nerve head astrocytes was studied using immunohistochemistry in postmortem eyes of patients with glaucoma and normal donors. Serum cytokine levels of patients with glaucoma and control subjects were measured using enzyme-linked, immunosorbent assay. In addition, in vitro experiments were performed using astrocyte cultures derived from human optic nerve head or fetal human brain. The cultured astrocytes were incubated under selected stress conditions such as exposure to cytokines, IFN-g...

Investigative ophthalmology & visual science, 1994

To examine the interaction of alpha subunits of guanine nucleotide binding proteins with A1 adeno... more To examine the interaction of alpha subunits of guanine nucleotide binding proteins with A1 adenosine receptors from the SV40-transformed bovine-derived pigmented (PE) and human-derived nonpigmented (NPE) ciliary epithelial cell lines using an immunoprecipitation approach and [3H]DPCPX, a selective radioligand to adenosine receptor. Solubilized preparations of adenosine receptors from PE and NPE cell lines were immunoprecipitated with G protein specific antisera 8730 (anti-Gi alpha), 3646 (anti-Gi alpha 1), 1521 (anti-Gi alpha 2), and 1518 (anti-Gi alpha 3), and of adenosine receptor-G protein complexes were detected by the binding of radioactive [3H]DPCPX. Data indicate that [3H]DPCPX forms high-affinity complex with membrane-bound and solubilized forms of adenosine receptors from PE and NPE cells. Peptide-directed antisera against various G protein alpha subunits indicate that the A1 adenosine receptors from these cells are specifically coupled to Gi alpha complexes. The results f...

Investigative ophthalmology & visual science, 2002

Immunocytochemistry showed strong aquaporin (AQP)-4 water channel expression in Müller cells in m... more Immunocytochemistry showed strong aquaporin (AQP)-4 water channel expression in Müller cells in mouse retina and fibrous astrocytes in optic nerve. This study was designed to test the hypothesis that AQP4 is required for vision by comparing electroretinograms and retinal morphology in wild-type mice and transgenic knockout mice with no AQP4. Electroretinograms were recorded over a 10(5)-fold range of flash intensities in dark-adapted mice and analyzed for a- and b-wave amplitude and latency, a-wave normalized slope, and oscillatory potential amplitude and latency. AQP4 protein was localized in mouse retina by immunocytochemistry, and retinal morphology was studied by light and electron microscopy. Significantly reduced electroretinogram b-wave potentials were recorded in 10-month-old null mice with smaller changes in 1-month-old mice. Immunocytochemistry showed strong AQP4 protein expression in retina of wild-type mice. Morphologic analysis of retina by light and electron microscopy...

Molecular vision, Jan 3, 2010

Changes in the expression of water channels (aquaporins; AQP) have been reported in several disea... more Changes in the expression of water channels (aquaporins; AQP) have been reported in several diseases. However, such changes and mechanisms remain to be evaluated for retinal injury after optic nerve crush (ONC). This study was designed to analyze changes in the expression of AQP4 (water selective channel) and AQP9 (water and lactate channel) following ONC in the rat. Rat retinal ganglion cells (RGCs) were retrogradely labeled by applying FluoroGold onto the left superior colliculus 1 week before ONC. Retinal injuries were induced by ONC unilaterally. Real-time PCR was used to measure changes in AQP4, AQP9, thy-1, Kir4.1 (K(+) channel), and beta-actin messages. Changes in AQP4, AQP9, Kir4.1, B cell lymphoma-x (bcl-xl), and glial fibrillary acidic protein (GFAP) expression were measured in total retinal extracts using western blotting. The number of RGCs labeled retrogradely from the superior colliculus was 2,090+/-85 cells/mm(2) in rats without any treatment, which decreased to 1,091...

Molecular vision, Jan 10, 2010

The purpose of this study was to determine the effects of down-regulation of Aquaporin 1 (AQP1) a... more The purpose of this study was to determine the effects of down-regulation of Aquaporin 1 (AQP1) and Aquaporin 5 (AQP5) on cell proliferation and migration in human corneal endothelial (HCEC) and human corneal epithelial (CEPI17) cell lines, respectively. AQP1 and AQP5 were down regulated using siRNA following lipofectamine-mediated transfection in corneal endothelial and epithelial cells, respectively. Down-regulation was confirmed using RT-PCR, indirect immunofluorescence, and immunoblot analysis. Total internal reflection fluorescence (TIRF) microscopy was used to detect cell surface aquaporin expression. Cell proliferation was determined by SRB (sulfrodamine B) assay. Cell migration was determined by in vitro wound healing and migration assay. In HCEC cells, AQP1 was localized to the cytosol as well as cell membrane and its down-regulation resulted in decreased cell proliferation and migration with a significant decrease in phosphorylated ERK (pERK). In CEPI17 cells AQP5 protein ...

Molecular vision, 2013

To examine the bradykinin (BK) B₂-receptor system in human and monkey ciliary muscle (CM) using i... more To examine the bradykinin (BK) B₂-receptor system in human and monkey ciliary muscle (CM) using immunohistochemical techniques, and to pharmacologically characterize the associated biochemical signal transduction systems in human CM (h-CM) cells. BK-induced modulation of intraocular pressure (IOP) in pigmented Dutch-Belt rabbits and cynomolgus monkeys was also studied. Previously published procedures were used throughout these studies. The human and monkey ciliary bodies expressed high levels of B₂-receptor protein immunoreactivity. Various kinins differentially stimulated [Ca²⁺](i) mobilization in primary h-CM cells (BK EC₅₀=2.4±0.2 nM > Hyp³,β-(2-thienyl)-Ala⁵,Tyr(Me)⁸-(®)-Arg⁹-BK (RMP-7) > Des-Arg⁹-BK EC₅₀=4.2 µM [n=3-6]), and this was blocked by B₂-selective antagonists, HOE-140 (IC₅₀=1.4±0.1 nM) and WIN-63448 (IC₅₀=174 nM). A phospholipase C inhibitor (U73122; 10-30 µM) and ethylene glycol tetraacetic acid (1-2 mM) abolished the BK-induced [Ca²⁺](i) mobilization. Total pr...

Journal of Power Sources, 2015

h i g h l i g h t s Non-isothermal electrochemical model developed for lithium composite cathode ... more h i g h l i g h t s Non-isothermal electrochemical model developed for lithium composite cathode cell. Experimentally obtained composite cathode property implemented in the model. Model validated for a wide range of temperature and discharge rate. Results analyzed to determine the heat generation pattern in the cell.

Molecular Biology and Physiology of Water and Solute Transport, 2000

Ciliary epithelium is responsible for the secretion of aqueous humor into posterior chamber of th... more Ciliary epithelium is responsible for the secretion of aqueous humor into posterior chamber of the eye. Most of aqueous humor secretion is driven by the active transport of ions from plasma into the posterior chamber followed by rapid movement of water and water soluble substances (Jacob and Civan, 1996). Both the outer nonpigmented (NPE) and inner pigmented (PE) epithelial layers exhibit properties of transporting epithelia (Helbig et al., 1988; Wolosin et al., 1993). The NPE is thought to provide the direct driving force for aqueous humor formation. Na+/K+-ATPase which is located on the basolateral membranes of NPE drives sodium secretion and provides the main ion motive force for sodium dependent cotransporters (Coca-Prados and Lopez-Briones, 1987; Flugel and Lutjen-Drecoll, 1988; Okami et al., 1989; Ghosh et al., 1990). Recently, it has been proposed that the rate limiting step in active secretion of aqueous humor is a CI- transport via a DIDS sensitive, cAMP dependent chloride channel (Chen et al., 1994; Jacob and Civan, 1996). Although the Na+/K+-ATPase active transport system is a primary transducer of cellular metabolic energy into fluid transport, it does not completely account for a variety of observations and functions that are prerequisites for the effective transport of fluid and electrolytes by the duallayered ciliary epithelium. For example, cell volume regulation which is coupled to ion transport through parallel K+ and, more importantly, Cl- channel activity appear to be an intrinsically important aspects of normal aqueous secretion (Civan et al., 1992; Yantorno el al., 1992; Fischbarg, 1997).

Investigative ophthalmology & visual science, 2001

To determine the expression and localization of tumor necrosis factor (TNF)-alpha and TNF-alpha r... more To determine the expression and localization of tumor necrosis factor (TNF)-alpha and TNF-alpha receptor-1 in the retina of normal and glaucomatous eyes. Using immunohistochemistry and in situ hybridization, retinal expression and localization of TNF-alpha and TNF-alpha receptor-1 were studied in retina sections from 20 eyes of donors with glaucoma, and 20 eyes of age-matched normal donors. According to immunohistochemistry, the intensity of the immunostaining and the number of labeled cells for TNF-alpha or its receptor were greater in retina sections of glaucomatous eyes than in control eyes of age-matched normal donors. In situ hybridization showed that mRNA signals for TNF-alpha or TNF-alpha receptor-1 were similarly more intense in glaucomatous eyes than in age-matched control eyes. Both protein and mRNA of TNF-alpha or TNF-alpha receptor-1 were predominantly localized to the inner retinal layers. Double-immunofluorescence labeling demonstrated that retinal immunostaining for T...

RESULTS. According to immunohistochemistry, the intensity of the immunostaining and the number of... more RESULTS. According to immunohistochemistry, the intensity of the immunostaining and the number of labeled cells for TNF-a or its receptor were greater in retina sections of glaucomatous eyes than in control eyes of age-matched normal donors. In situ hybridization showed that mRNA signals for TNF-a or TNF-a receptor-1 were similarly more intense in glaucomatous eyes than in age-matched control eyes. Both protein and mRNA of TNF-a or TNF-a receptor-1 were predominantly localized to the inner retinal layers. Double-immunofluorescence labeling demonstrated that retinal immunostaining for TNF-a was predominantly positive in the glial cells, whereas immunostaining for TNF-a receptor-1 was mainly positive in the retinal ganglion cells.

Journal of Ocular Pharmacology and Therapeutics, 1994

Journal of Food Science and Technology, 2011

Journal of Biological Chemistry, 1998

The molecular mechanisms for regulating water balance in many tissues are unknown. Like the kidne... more The molecular mechanisms for regulating water balance in many tissues are unknown. Like the kidney, the eye contains multiple water channel proteins (aquaporins) that transport water through membranes, including two (AQP1 and AQP4) in the ciliary body, the site of aqueous humor production. However, because humans with defective AQP1 are phenotypically normal and because the ocular application of phorbol esters reduce intraocular pressure, we postulated that the water channel activity of AQP4 may be regulated by these agents. We now report that protein kinase C activators, phorbol 12,13-dibutyrate, and phorbol 12-myristate 13acetate strongly stimulate the phosphorylation of AQP4 and inhibit its activity in a dose-dependent manner. Phorbol 12,13-dibutyrate (10 M) and phorbol 12-myristate 13-acetate (10 nM) reduced the rate of AQP4-expressing oocyte swelling by 87 and 92%, respectively. Further, phorbol 12,13-dibutyrate significantly increased the amount of phosphorylated AQP4. These results demonstrate that protein kinase C can regulate the activity of AQP4 through a mechanism involving protein phosphorylation. Moreover, they suggest important potential roles for AQP4 in several clinical disorders involving rapid water transport such as glaucoma, brain edema, and swelling of premature infant lungs.

Investigative Opthalmology & Visual Science, 2003

Experimental Eye Research, 1998

Current Eye Research, 2000

Water channel proteins are important pathways for water movements across cell membranes, includin... more Water channel proteins are important pathways for water movements across cell membranes, including those in the ciliary epithelium, which is the major site of aqueous humor secretion. In this study, we aimed to demonstrate the expression of functionally active aquaporin-1 (AQP1) water channels in cultured human ciliary epithelial cells. Poly A(+) RNA was isolated from cell cultures of Simian Virus 40 (SV-40) transformed human nonpigmented ciliary epithelium (NPE) subjected to RT-PCR reaction using primers specific to AQP1. Northern analysis was used to define the expression of AQP1 in NPE cells. Western immunoblotting with polyclonal antibody raised against AQP1 was used to evaluate the AQP1 protein expression in the plasma membranes of human NPE cells. Light scattering method was used to determine the osmotic water permeability in the suspension of NPE cells. RT-PCR using specific primers for AQP1, Northern analysis and Western immunoblot using AQP1 specific antibody demonstrated the expression of AQP1 in the plasma membranes of NPE cells. Osmotic water permeability (P( f)) measurements confirmed that functional AQP1 water channels are expressed in human NPE cells and the P(f) for these cells was 9.8 x 10( -3) cm/s at 10 degrees C. The presence of AQP1 in human NPE cells suggests that it may have a role in the fluid flow across epithelial membranes. In addition, the existence of AQP1 in the human NPE cells provide an excellent in vivo model to study the regulation of aquaporins and their possible role in the aqueous humor secretion.

Current Eye Research, 2000

To count retrogradely labeled retinal ganglion cells by Fluoro-Gold. Retinal ganglion cells were ... more To count retrogradely labeled retinal ganglion cells by Fluoro-Gold. Retinal ganglion cells were retrogradely labeled using bilateral injections of Fluoro-Gold into the superior colliculus. One week after injections, retinas were dissociated and immunolabeled using specific antibody against Fluoro-Gold. The Fluoro-Gold labeled cells were then counted using flow cytometry. Flow cytometry revealed that approximately 7% of the dissociated retinal cells were ganglion cells retrogradely labeled by Fluoro-Gold (Fig. 1B). Based on the total count of retinal cells per eye, the total number of retinal ganglion cells was estimated at approximately 131,250 +/- 2,542 per rat eye. The coefficient of variation of counts was calculated as 1.98%. The use of flow cytometry facilitates simple, reproducible and rapid quantification of virtually all of the retinal ganglion cells labeled by Fluoro-Gold in a single rat eye.

Carbohydrate Research, 1985

Abstract Synergism between (1→4)-β- d -glucan cellobiohydrolase, endo-(1→4)-β- d -glucanases, and... more Abstract Synergism between (1→4)-β- d -glucan cellobiohydrolase, endo-(1→4)-β- d -glucanases, and β- d -glucosidases of Sclerotium rolfsii for solubilization of native and amorphous celluloses is discussed. Besides synergism between cellobiohydrolase and endo-β-glucanases of S. rolfsii , a synergistic effect between endo-β-glucanases and β-glucosidases [which behaved rather as (1→4)-β- d -glucan glucohydrolases] was observed for solubilization of crystalline and amorphous celluloses. It seems that a cellobiohydrolase initiates the attack on crystalline cellulose and an endo-β- d -glucanase the attack on amorphous cellulose.