Light-stable rhodopsin. II. An opsin mutant (TRP-265→PHE) and a retinal analog with a nonisomerizable 11-cis configuration form a photostable chromophore

In order to prepare a completely light-stable rhodopsin, we have synthesized an analog, II, of 11-cis retinal in which isomerization at the C11-C12 cis-double bond is blocked by formation of a cyclohexene ring from the C10 to C13-methyl. We used this analog to generate a rhodopsin-like pigment from opsin expressed in COS-1 cells and opsin from rod outer segments (Bhattacharya, S., Ridge, K.D., Knox, B.E., and Khorana, H. G. (1992) J. Biol. Chem. 267, 6763-6769). The pigment (lambda max, 512 nm) formed from opsin and analog II (rhodospin-II) showed ground state properties very similar to those of rhodopsin, but was not entirely stable to light. In the present work, 12 opsin mutants (Ala-117→Phe, Glu-122→Gln(Ala, Asp), Trp-126→Phe(Leu, Ala), Trp-265→Ala(Tyr, Phe), Tyr-268→Phe, and Ala-292→Asp), where the mutations were presumed to be in the retinal binding pocket, were reconstituted with analog II. While all mutants formed rhodopsin-like pigments with II, blue-shifted (12-30 nm) chromophores were obtained with Ala-117→Phe, Glu-122→Gln(Ala), Trp-126→Leu(Ala), and Trp-265→Ala(Tyr, Phe) opsins. The extent of chromophore formation was markedly reduced in the mutants Ala-117→Phe and Trp-126→Ala. Upon illumination, the reconstituted pigments showed varying degrees of light sensitivity; the mutants Trp-126→Phe(Leu) showed light sensitivity similar to wild-type. Continuous illumination of the mutants Glu-122→Asp, Trp-265→Ala, Tyr-268→Phe, and Ala-292→Asp resulted in hydrolysis of the retinyl Schiff base. Markedly reduced light sensitivity was observed with the mutant Trp-265→Tyr, while the mutant Trp-265→Phe was light-insensitive. Consistent with this result, the mutant Trp-265→Phe showed no detectable light-dependent activation of transducin or phosphorylation by rhodopsin kinase

Characterization of a novel PTEN mutation in MDA-MB-453 breast carcinoma cell line

<p>Abstract</p> <p>Background</p> <p>Cowden Syndrome (CS) patients with germ line point mutations in the <it>PTEN </it>gene are at high risk for developing breast cancer. It is believed that cells harboring these mutant <it>PTEN </it>alleles are predisposed to malignant conversion. This article will characterize the biochemical and biological properties of a mutant PTEN protein found in a commonly used metastatic breast cancer cell line.</p> <p>Methods</p> <p>The expression of PTEN in human breast carcinoma cell lines was evaluated by Western blotting analysis. Cell line MDA-MB-453 was selected for further analysis. Mutation analysis of the <it>PTEN </it>gene was carried out using DNA isolated from MDA-MB-453. Site-directed mutagenesis was used to generate a PTEN E307K mutant cDNA and ectopic expressed in PC3, U87MG, MCF7 and <it>Pten</it>^-/-mouse embryo fibroblasts (MEFS). Histidine (His)-tagged PTEN fusion protein was generated in <it>Sf9 </it>baculovirus expression system. Lipid phosphatase and ubiquitination assays were carried out to characterize the biochemical properties of PTEN E307K mutant. The intracellular localization of PTEN E307K was determined by subcellular fractionation experiments. The ability of PTEN E307K to alter cell growth, migration and apoptosis was analyzed in multiple PTEN-null cell lines.</p> <p>Results</p> <p>We found a mutation in the <it>PTEN </it>gene at codon 307 in MDA-MB-453 cell line. The glutamate (E) to lysine (K) substitution rendered the mutant protein to migrate with a faster mobility on SDS-PAGE gels. Biochemically, the PTEN E307K mutant displayed similar lipid phosphatase and growth suppressing activities when compared to wild-type (WT) protein. However, the PTEN E307K mutant was present at higher levels in the membrane fraction and suppressed Akt activation to a greater extent than the WT protein. Additionally, the PTEN E307K mutant was polyubiquitinated to a greater extent by NEDD4-1 and displayed reduced nuclear localization. Finally, the PTEN E307K mutant failed to confer chemosensitivity to cisplatinum when re-expressed in <it>Pten</it>^-/-MEFS.</p> <p>Conclusions</p> <p>Mutation at codon 307 in PTEN C2 loop alters its subcellular distribution with greater membrane localization while being excluded from the cell nucleus. This mutation may predispose breast epithelial cells to malignant transformation. Also, tumor cells harboring this mutation may be less susceptible to the cytotoxic effects of chemotherapeutics.</p

Stability And Efficiency Of Some Amorphous Silicon Photovoltaic Modules

Amorphous silicon photovoltaic modules from four US manufacturers were tested for their power generation. The modules were exposed in outdoor sunlight over a period of one year. Current-voltage (I-V) curve measurements were made on each module daily with the automated I-V curve tracer. From these measurements, module electrical performance characteristics were determined. It is shown that the power degradation of the amorphous silicon modules tested was 18 to 33% after the one-year period of sunlight exposure. The degradation in the voltage parameters is generally lower than in the current parameters, being less than 7% for open-circuit voltage. About half of the degradation in the peak power is due to the degradation of the fill factor. The power output of the modules had nearly stabilized in the first eight months of the sunlight exposures and their stabilized efficiences were in the range of 2.6 to 4.4%