Insulin-like growth factor-1 effects on bovine retinal endothelial cell glucose transport: role of MAP kinase

In order to maintain normal metabolism, the neuroretina is completely dependent on the constant delivery of glucose across the retinal microvascular endothelial cells comprising the inner blood-retinal barrier. Glucose uptake into these cells is influenced by various stimuli, including hypoxia and growth factors. Recently, insulin-like growth factor-1 (IGF-1) was shown to enhance retinal endothelial glucose transport in a process that is dependent on protein kinase C (PKC) and phosphatidylinositol-3 kinase (PI3 kinase). In the current study, the role of mitogen-activated protein kinase (MAP kinase) in regulating IGF-1 effects on retinal endothelial cell glucose transport was investigated in a bovine retinal endothelial cell (BREC) culture model. IGF-1 (25 ng/mL) caused a rapid increase in MAP-kinase activity and ERK phosphorylation. Inhibition of MAP kinase with PD98059 (100 microm) blocked IGF-1 enhancement of 2-deoxyglucose uptake. In order to clarify the relationship between PKC, PI3 kinase and MAP kinase in IGF-1 signaling in retinal endothelial cells, the effects of selective inhibitors of MAP kinase (PD98059), PKC (GF109203X), and PI3 kinase (wortmannin, LY294002) on signal transduction by IGF-1 were studied. Inhibition of MAP kinase abolished IGF-1 stimulation of PKC but had no effect on PI3 kinase activity, whereas inhibition of either PKC and PI3 kinase had no effect on MAP kinase phosphorylation or activity in IGF-1-treated cells. Taken together, these data demonstrate that IGF-1 stimulation of BREC glucose transport requires activation of MAP kinase and that MAP kinase is upstream from PKC but is independent of PI3 kinase in mediating the actions of IGF-1 on retinal endothelial cells.     

Cysteine-free mutant of aequorin as a photolabel in immunoassay development

The bioluminescent protein aequorin is a sensitive label that has been employed in a number of analytical applications. A mutant of aequorin with enhanced stability produced recombinantly in our laboratory has been employed as a label in the development of an immunoassay for digoxin. Digoxin is a cardiac glycoside used in the treatment of congestive heart failure. This drug has a very narrow therapeutic range of 0.8-2.0 ng/mL (1.0-2.5 nmol/L), thus requiring therapeutic drug monitoring. In this study, a derivative of digoxigenin was chemically conjugated to the mutant aequorin, and the resulting protein-digoxigenin derivative conjugates were characterized in terms of their luminescence properties. A solid-phase immunoassay for digoxin was then developed. The detection limit of the assay for digoxin was 1 x 10(-12) M. To demonstrate the use of this mutant aequorin as a label in biological sample analysis without any need for pretreatment of the samples, the assay was tested in serum spiked with digoxin. Interference from digoxin analogues was also evaluated to determine the specificity of the assay.     

Bioluminescence detection of proteolytic bond cleavage by using recombinant aequorin

Detection of proteolytic bond cleavage was achieved by taking advantage of the bioluminescence emission generated by the photoprotein aequorin. A genetically engineered HIV-1 protease substrate was coupled with a cysteine-free mutant of aequorin by employing the polymerase chain reaction to produce a fusion protein that incorporates an optimum natural protease cleavage site. The fusion protein was immobilized on a solid phase and employed as the substrate for the HIV-1 protease. Proteolytic bond cleavage was detected by a decrease in the bioluminescence generated by the aequorin fusion protein on the solid phase. A dose-response curve for HIV-1 protease was constructed by relating the decrease in bioluminescence signal with varying amounts of the protease. The system was also used to evaluate two competitive and one noncompetitive inhibitor of the HIV-1 protease. Among the advantages of this assay is that by using recombinant methods a complete bioluminescently labeled protease recognition site can be designed and produced. The assay yields very sensitive detection limits, which are inherent to bioluminescence-based methods. An application of this system may be in the high-throughput screening of biopharmaceutical drugs that are potential inhibitors of a target protease.     

Targeting the Ca2+/Calmodulin-dependent protein kinase II by Tetrandrine in human liver cancer cells

Hepatocellular carcinoma (HCC) is the most prevalent malignancy in liver and a leading cause of cancer-related deaths. Despite the pressing need for treatment options, patients with HCC develop significant resistance and adverse side effects to current approved drugs that becomes a major barrier to effective treatment. A natural product Tetrandrine (TET) is a potential alternative treatment option for HCC, with demonstrated effectiveness and low toxicity. However, the mechanisms by which Tetrandrine inhibits HCC are unclear. In the current study, we identify Ca²+/calmodulin-dependent protein kinase II δ (CaMKIIδ) as a potential TET drug target through structural modeling. Screening of a panel of HCC cell lines reveal differential sensitivities toward TET treatment. Interestingly, IC₅₀ of TET inhibition of HCC cell proliferation is positively correlated with CaMKIIδ expression level in these distinct HCC cells. Furthermore, TET treatment resulted in a marked reduction of CaMKIIδ phosphorylation level, and knockdown of CaMKIIδ reduced the sensitivity of HCC cells to TET. Most importantly, CaMKIIδ protein levels in high-grade human HCC samples were significantly elevated as compared to normal liver tissues. Taken together, our studies demonstrate that the natural compound TET targets CaMKIIδ in HCC cells, and that CaMKIIδ level is a potential biomarker to identify HCC patient populations sensitive to Tetrandrine treatment.     

Feasibility Study of Phragmites karka and Christella dentata Grown in West Bengal as Arsenic Accumulator

A survey was undertaken, in arsenic (As) contaminated area of the Nadia district, West Bengal, India, to find native As accumulator plants. As was determined both in soil and plant parts. The results showed that the mean translocation factor of Pteris vittata L, Phragmites karka (Cav.) Trin. Ex. Steud and Christella dentata Forssk were higher than 1. It thus appeared that these plants can be efficient accumulators of As.

Phytoremediation ability of C. dentata and P. karka was evaluated and compared with known As-hyperaccumulators -P. vittata and Adiantum capillus veneris L. Plants were grown in the As spiked soil (25, 50, 75 and 100 mg kg^?1). As accumulation was found to be highest in P. vittata, 117.18 mg kg^?1 in leaf at 100 mg kg^?1 As treatment, followed by A. capillus veneris, P. karka and C. dentata being 74, 83.87 and 40.36 mg kg^?1, respectively. Lipid peroxidation increased after As exposure in all plants. However, the antioxidant enzyme activity and molecules concentration also increased which helped the plants to overcome As-induced oxidative stress. The study indicates that P. karka and C. dentata could be considered as As-accumulators and find application for As-phytoextraction in field conditions.     

In vivo relevance for photoprotection by the vitamin D rapid response pathway

Vitamin D is produced by exposure of 7-dehydrocholesterol in the skin to UV irradiation (UVR) and further converted in the skin to the biologically active metabolite, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) and other compounds. UVR also results in DNA damage producing cyclobutane pyrimidine dimers (CPD). We previously reported that 1,25(OH)₂D₃ at picomolar concentrations, protects human skin cells from UVR-induced apoptosis, and decreases CPD in surviving cells. 1,25(OH)₂D₃ has been shown to generate biological responses via two pathways—the classical steroid receptor/genomic pathway or a rapid, non-genomic pathway mediated by a putative membrane receptor. Whether the rapid response pathway is physiologically relevant is unclear. A cis-locked, rapid-acting agonist 1,25(OH)₂lumisterol₃ (JN), entirely mimicked the actions of 1,25(OH)₂D₃ to reduce fibroblast and keratinocyte loss and CPD damage after UVR. The effects of 1,25(OH)₂D₃ were abolished by a rapid-acting antagonist, but not by a genomic antagonist. Skh:hr1 mice exposed to three times the minimal erythemal dose of solar-simulated UVR and treated topically with 1,25(OH)₂D₃ or JN immediately after UVR showed reduction in UVR-induced UVR-induced sunburn cells (p < 0.01 and <0.05, respectively), CPD (p < 0.01 for both) and immunosuppression (p < 0.001 for both) compared with vehicle-treated mice. These results show for the first time an in vivo biological response mediated by a rapid-acting analog of the vitamin D system. The data support the hypothesis that 1,25(OH)₂D₃ exerts its photoprotective effects via the rapid pathway and raise the possibility that other D compounds produced in skin may contribute to the photoprotective effects.     

Competitive binding assay using fluorescence resonance energy transfer for the identification of calmodulin antagonists

The ubiquitous calcium regulating protein calmodulin (CaM) has been utilized as a model drug target in the design of a competitive binding fluorescence resonance energy transfer assay for pharmacological screening. The protein was labeled by covalently attaching the thiol-reactive fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) to an engineered C-terminal cysteine residue. Binding of the environmentally sensitive hydrophobic probe 2,6-anilinonaphthalene sulfonate (2,6-ANS) to CaM could be monitored by an increase in the fluorescence emission intensity of the 2,6-ANS. Evidence of fluorescence resonance energy transfer (FRET) from 2,6-ANS (acting as a donor) to MDCC (the acceptor in this system) was also observed; fluorescence emission representative of MDCC could be seen after samples were excited at a wavelength specific for 2,6-ANS. The FRET signal was monitored as a function of the concentration of calmodulin antagonists in solution. Calibration curves for both a selection of small molecules and a series of peptides based upon known CaM-binding domains were obtained using this system. The assay demonstrated dose-dependent antagonism by analytes known to hinder the biological activity of CaM. These data indicate that the presence of molecules known to bind CaM interfere with the ability of FRET to occur, thus leading to a concentration-dependent decrease of the ratio of acceptor:donor fluorescence emission. This assay can serve as a general model for the development of other protein binding assays intended to screen for molecules with preferred binding activity.