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.     

Construction and Characterization of a Magnaporthe grisea Bacterial Artificial Chromosome Library

Diaz-Perez, S. V., Crouch, V. W., and Orbach, M. J. 1996. Construction and characterization of a Magnaporthe grisea bacterial artificial chromosome library. Fungal Genet. Biol. 20, 280-288. A bacterial artificial chromosome (BAC) library of Magnaporthe grisea containing 4128 clones with an average insert size of 66-kb has been constructed. This library represents seven genome equivalents of M. grisea and has been demonstrated to be representative of the genome by screening for the presence of several single-copy genes and DNA markers. The utility of the library for use in map-based cloning projects was shown by the spanning of a nine-cosmid, 207-kb DNA contig with only 3 BAC clones. In addition, using a lys1-3 auxotroph, we have shown that BAC clones at least 113 kb can be transformed into M. grisea to screen for complementation of mutations. Thus, BACs isolated in chromosome walks can be rapidly screened for the presence of the sought after gene. The ease of construction of BAC libraries and of isolation and manipulation of BAC clones makes the BAC system an ideal one for physical analyses of fungal genomes.     

Purification of the enhancing factor from mouse intestines

A unique polypeptide, called enhancing factor (EF), which enhances the binding of labeled epidermal growth factor (EGF) to cells, has been isolated. It has been purified to homogeneity from the acid-soluble proteins of mouse intestines. Earlier, EF was partially purified by two cycles of gel-permeation chromatography on Bio-Gel columns. We now report the final purification of EF on high-performance liquid chromatography (HPLC), using a reverse-phase column (mu Bondapak C18). The purity of the protein was confirmed when a single peak was obtained in HPLC. Also, a single protein band was obtained in SDS-PAGE. Purified EF has the same properties in vitro as those reported earlier for partially purified EF.     

Interleukin-6 Is a Risk Factor for Atrial Fibrillation in Chronic Kidney Disease: Findings from the CRIC Study

Atrial fibrillation (AF) is the most common sustained arrhythmia in patients with chronic kidney disease (CKD). In this study, we examined the association between inflammation and AF in 3,762 adults with CKD, enrolled in the Chronic Renal Insufficiency Cohort (CRIC) study. AF was determined at baseline by self-report and electrocardiogram (ECG). Plasma concentrations of interleukin(IL)-1, IL-1 Receptor antagonist, IL-6, tumor necrosis factor (TNF)-α, transforming growth factor-β, high sensitivity C-Reactive protein, and fibrinogen, measured at baseline. At baseline, 642 subjects had history of AF, but only 44 had AF in ECG recording. During a mean follow-up of 3.7 years, 108 subjects developed new-onset AF. There was no significant association between inflammatory biomarkers and past history of AF. After adjustment for demographic characteristics, comorbid conditions, laboratory values, echocardiographic variables, and medication use, plasma IL-6 level was significantly associated with presence of AF at baseline (Odds ratio [OR], 1.61; 95% confidence interval [CI], 1.21 to 2.14; P = 0.001) and new-onset AF (OR, 1.25; 95% CI, 1.02 to 1.53; P = 0.03). To summarize, plasma IL-6 level is an independent and consistent predictor of AF in patients with CKD.     

Changes in Vegetation Characteristics Downwind of an Aluminium Factory in India

Vegetation characteristics were assessed at different distancesdownwind of an aluminium factory to elucidate changes in communityattributes. The importance value index (IVI) of sensitive speciesdecreased and those of tolerant species increased with the increasingpollution load around the factory. Plant community compositionchanged with distance from this source. Species richness andthe Shannon-Weiner index increased with the increasing distancefrom the source, while concentration of dominance declined from1 to 11 km. The similarity coefficient indicated that the thirdand fifth sites are closer to each other, and first and fourthsites are farthest. The shape of the species sequence vs. theIVI distribution curve slipped from the shape of log normaldistribution at sites receiving higher pollution load. The woodylayer is found to be more affected compared to the herbaceouslayer. The aluminium factory impact was most pronounced up to4 km downwind of the source. Data on ambient air quality werefound to be directly related to the unfavourable changes incommunity attributes.Copyright 1994, 1999 Academic Press Importance value index, aluminium factory, community attributes, herbaceous layer, woody layer, sensitive and tolerant species     

Implementation and analysis of temperature control strategies for outdoor photobiological hydrogen production

For outdoor photobiological hydrogen production, the effective control of temperature in photobioreactors is a challenge. In this work, an internal cooling system for outdoor tubular photobioreactors was designed, built, and tested. The temperatures in the reactors with bacteria were consistently higher than those without bacteria, and were also strongly influenced by solar irradiation and ambient air temperature. The cooling protocol applied successfully kept the reactor temperatures below the threshold limit (38 °C) required for the bioprocess and provided a uniform distribution of temperature along the reactor tube length. The biomass growth and hydrogen production were similar in the reactors cooled co-currently and counter-currently. The biomass growth rate was 0.1 l/h, the maximum hydrogen production rate was 1.28 mol/m³/h, and the overall hydrogen yield obtained was 20 %. The change in the biomass was fitted using the logistic model while cumulative hydrogen production was fitted using the modified Gompertz equation.