αA-Crystallin-Derived Mini-Chaperone Modulates Stability and Function of Cataract Causing αAG98R-Crystallin

Background

A substitution mutation in human αA-crystallin (αAG98R) is associated with autosomal dominant cataract. The recombinant mutant αAG98R protein exhibits altered structure, substrate-dependent chaperone activity, impaired oligomer stability and aggregation on prolonged incubation at 37°C. Our previous studies have shown that αA-crystallin–derived mini-chaperone (DFVIFLDVKHFSPEDLTVK) functions like a molecular chaperone by suppressing the aggregation of denaturing proteins. The present study was undertaken to determine the effect of αA-crystallin–derived mini-chaperone on the stability and chaperone activity of αAG98R-crystallin.

Methodology/Principal Findings

Recombinant αAG98R was incubated in presence and absence of mini-chaperone and analyzed by chromatographic and spectrometric methods. Transmission electron microscope was used to examine the effect of mini-chaperone on the aggregation propensity of mutant protein. Mini-chaperone containing photoactive benzoylphenylalanine was used to confirm the interaction of mini-chaperone with αAG98R. The rescuing of chaperone activity in mutantα-crystallin (αAG98R) by mini-chaperone was confirmed by chaperone assays. We found that the addition of the mini-chaperone during incubation of αAG98R protected the mutant crystallin from forming larger aggregates that precipitate with time. The mini-chaperone-stabilized αAG98R displayed chaperone activity comparable to that of wild-type αA-crystallin. The complexes formed between mini-αA–αAG98R complex and ADH were more stable than the complexes formed between αAG98R and ADH. Western-blotting and mass spectrometry confirmed the binding of mini-chaperone to mutant crystallin.

Conclusion/Significance

These results demonstrate that mini-chaperone stabilizes the mutant αA-crystallin and modulates the chaperone activity of αAG98R. These findings aid in our understanding of how to design peptide chaperones that can be used to stabilize mutant αA-crystallins and preserve the chaperone function.     

Arsenic hyperaccumulation in the Chinese brake fern (Pteris vittata) deters grasshopper (Schistocerca americana) herbivory

Brake fern, Pteris vittata, not only tolerates arsenic but also hyperaccumulates it in the frond. The hypothesis that arsenic hyperaccumulation in this fern could function as a defense against insect herbivory was tested. Fronds from control and arsenic-treated ferns were presented to nymphs of the grasshopper Schistocerca americana. Feeding damage was recorded by visual observation and quantification of the fresh weight of frond left uneaten and number of fecal pellets produced over a 2-d period. Grasshopper weight was determined before and after 5 d of feeding. Grasshoppers consumed significantly greater amounts of the frond tissue, produced more fecal pellets and had increased body weight on control plants compared with grasshoppers fed arsenic-treated ferns. Very little or none of the arsenic-treated ferns were consumed indicating feeding deterrence. In a feeding deterrent experiment with lettuce, sodium arsenite at 1.0 mm deterred grasshoppers from feeding whereas 0.1 mm did not. In a choice experiment, grasshoppers preferred to feed on lettuce dipped in water compared with lettuce dipped in 1.0 mm sodium arsenite. Our results show that arsenic hyperaccumulation in brake fern is an elemental defense against grasshopper herbivory.     

Development of a bioanalytical liquid chromatography method for quantitation of 9-nitrocamptothecin in human plasma

9-Nitrocamptothecin (9-NC) is an orally administered camptothecin (CPT) that is under evaluation in clinical trials. This compound is not fluorescent, which has hampered development of a sensitive high-performance liquid chromatographic (LC) assay for measurement of drug concentrations in clinical trials. We now report development of an assay that involves reduction of 9-NC to the fluorescent compound 9-aminocamptothecin (9-AC). The method is based on enzymatic reduction of 9-NC using bovine liver S-9 fraction. This method is validated to quantitate 9-NC and 9-AC in patient samples, and yields results comparable to those obtained with an LC/MS method.     

Novel lipid-peroxidation- and cyclooxygenase-inhibitory tannins from Picrorhiza kurroa seeds

From the AcOEt extract of the seeds of Picrorhiza kurroa were isolated picrorhiza acid (1), picrorhizoside A (2), picrorhizoside B (3), picrorhizoside C (4), (-)-shikimic acid (5), gallic acid (6), ellagic acid (7), isocorilagin (8), 1-O-galloyl-beta-D-glucose (9), 1-O,3-O,6-O-trigalloyl-beta-D-glucose (10), and 1-O,2-O,3-O,4-O,6-O-pentagalloyl-beta-D-glucose (11), and their structures were established by extensive NMR and chemical studies. Constituents 1-4 are novel compounds, and the known compounds 5-11 have been isolated for the first time from the seeds of P. kurroa. Compounds 2 and 3 were hydrolyzed and yielded 12, isochebulic acid. Compounds 1-12 showed 89.6, 77.3, 56.1, 50.5, 11.0, 86.4, 50.5, 29.2, 70.9, 50.5, 56.5, and 86.1% inhibition of lipid peroxidation at 5 microg/ml, respectively. The commercial antioxidants BHA (1.8 microg/ml), BHT (2.2 microg/ml), and TBHQ (1.66 microg/ml) inhibited lipid peroxidation at 85.6, 87.1, and 81.1%, respectively. The inhibition of cyclooxygenase-1 (COX-1) by 2-5, 7, 8, and 10-12 at 100 microg/ml was 41.9, 28.4, 32.9, 9.3, 70.7, 34.7, 16.0, 89.6, and 53.4%, respectively. Similarly, compounds 1-8 and 11 and 12, at 100 microg/ml, inhibited COX-2 by 12.6, 15.3, 25.1, 5.3, 13.2, 21.7, 2.0, 42.4, 43.4, and 36.9%, respectively.     

Methanol Skin Mucus Extract of Mrigal (Cirrhinus mrigala) Fish Peptide Targeting Viral Particles of Infectious Pancreatic Necrosis Virus (IPNV) and Infectious Salmon Anemia Virus (ISAV): an in silico Approach

International Journal of Peptide Research and Therapeutics - The teleost fish skin mucus acts as an important physical and biological barrier that prevents fish from the surrounding environment....     

Chlorinated benzenes cause concomitantly oxidative stress and induction of apoptotic markers in lung epithelial cells (A549) at nonacute toxic concentrations

In industrialized countries, people spend more time indoors and are therefore increasingly exposed to volatile organic compounds that are emitted at working places and from consumer products, paintings, and furniture, with chlorobenzene (CB) and 1,2-dichlorobenzene (DCB) being representatives of the halogenated arenes. To unravel the molecular effects of low concentrations typical for indoor and occupational exposure, we exposed human lung epithelial cells to CB and DCB and analyzed the effects on the proteome level by 2-D DIGE, where 860 protein spots were detected. A set of 25 and 30 proteins were found to be significantly altered due to exposure to environmentally relevant concentrations of 10(-2) g/m(3) of CB or 10(-3) g/m(3) of DCB (2.2 and 0.17 ppm), respectively. The most enriched pathways were cell death signaling, oxidative stress response, protein quality control, and metabolism. The involvement of oxidative stress was validated by ROS measurement. Among the regulated proteins, 28, for example, voltage-dependent anion-selective channel protein 2, PDCD6IP protein, heat shock protein beta-1, proliferating cell nuclear antigen, nucleophosmin, seryl-tRNA synthetase, prohibitin, and protein arginine N-methyltransferase 1, could be correlated with the molecular pathway of cell death signaling. Caspase 3 activation by cleavage was confirmed for both CB and DCB by immunoblotting. Treatment with CB or DCB also caused differential protein phosphorylation, for example, at the proteins HNRNP C1/C2, serine-threonine receptor associated protein, and transaldolase 1. Compared to previous results, where cells were exposed to styrene, for the chlorinated aromatic substances besides oxidative stress, apoptosis was found as the predominant cellular response mechanism.     

Melanin production in Alternaria helianthi

Abstract

Most of the plant pathogenic fungi produce a dark phenolic polymer called melanin. The high performance liquid chromatography (HPLC) analysis of the mycelial extract of Alternaria helianthi revealed an accumulation of scytalone and a shunt metabolite 2-hydroxyjuglone which confirms the production of dihydroxynapthalene type of melanin. The growth and melanin of A. helianthi increased when grown in host extract broth at 6.5 pH and a temperature beyond 30°C had an inhibitory effect on the pathogen. The production and type of melanin produced in Alternaria helianthi is reported for the first time.     

Anionic surfactant based reverse micellar extraction of l-asparaginase synthesized by Azotobacter vinelandii

Bioprocess and Biosystems Engineering - l-Asparaginase synthesized by Azotobacter vinelandii via submerged fermentation in the presence of sucrose was successfully extracted using Reverse micellar...     

64Cu-labeled alpha-melanocyte-stimulating hormone analog for microPET imaging of melanocortin 1 receptor expression

The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor (melanocortin type 1 receptor, or MC1R) plays an important role in the development and growth of melanoma cells. It was found that MC1R was overexpressed on most murine and human melanoma, making it a promising molecular target for melanoma imaging and therapy. Radiolabeled alpha-MSH peptide and its analogs that can specifically bind with MC1R have been extensively explored for developing novel agents for melanoma detection and radionuclide therapy. The goal of this study was to evaluate a 64Cu-labeled alpha-MSH analog, Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-Lys(DOTA)-NH2 (DOTA-NAPamide), as a potential molecular probe for microPET imaging of melanoma and MC1R expression in melanoma xenografted mouse models. 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated NAPamide was synthesized and radiolabeled with 64Cu (t1/2=12 h) in NH4OAc (0.1 M; pH 5.5) buffered solution for 60 min at 50 degrees C. Cell culture studies reveal rapid and high uptake and internalization of 64Cu-DOTA-NAPamide in B16F10 cells. Over 90% of receptor-bound tracer is internalized at 3 h incubation. A cellular retention study demonstrates that the receptor-bound 64Cu-DOTA-NAPamide is slowly released from the B16F10 cells into the medium; 66% of the radioactivity is still associated with the cells even after 3 h incubation. The biodistribution of 64Cu-DOTA-NAPamide was then investigated in C57BL/6 mice bearing subcutaneous murine B16F10 melanoma tumors with high capacity of MC1R and Fox Chase Scid mice bearing human A375M melanoma with a relatively low number of MC1R receptors. Tumor uptake values of 64Cu-DOTA-NAPamide are found to be 4.63 +/- 0.45% and 2.49 +/- 0.31% ID/g in B16F10 and A375M xenografted melanoma at 2 h postinjection (pi), respectively. The B16F10 tumor uptake at 2 h pi is further inhibited to 2.29 +/- 0.24% ID/g, while A375M tumor uptake at 2 h pi remains 2.20 +/- 0.41% ID/g with a coinjection of excess alpha-MSH peptide. MicroPET imaging of 64Cu-DOTA-NAPamide in B16F10 tumor mice clearly shows good tumor localization. However, low A375M tumor uptake and poor tumor to normal tissue contrast were observed. This study demonstrates that 64Cu-DOTA-NAPamide is a promising molecular probe for alpha-MSH receptor positive melanoma PET imaging as well as MC1R expression imaging in living mice.