A Sensitive Assay for Monitoring Wild-Type and Mutant Myocilin Secretion

The 54th Annual Medical Student Research Forum at UT Southwestern Medical Center (Monday, January 19, 2016, 2-5 p.m., D1.700)Each year the Medical Student Research Program awards students for the best oral presentation and the best poster presentation as judged by faculty across campus. This author received an award as one of the best poster presentations at this forum.Primary open angle glaucoma (POAG)-associated mutations in myocilin (MYOC) cause protein 'non-secretion', rendering secreted MYOC difficult to quantitatively detect using time-consuming conventional techniques. This study focused on developing an assay which could be used to quickly and easily detect mutant and wild-type (WT) MYOC secretion. We fused Gaussia luciferase (eGLuc2) to MYOC variants and expressed the constructs in HEK-293T cells. The secretion, intracellular soluble and insoluble portions of WT and Y437H MYOC eGLuc2 constructs were evaluated by western blotting and compared to FLAG-tagged constructs. Secreted and soluble MYOC eGLuc2 was measured by a GLuc assay. The secretion of nine additional MYOC mutants was assayed in conditioned media from HEK-293T and NTM-5 cells to test the general applicability of the assay. MYOC eGLuc2 behaved similarly to FLAG MYOC with respect to secretion, soluble intracellular levels, and in response to drug treatment. The GLuc assay could sensitively detect Y437H MYOC secretion 30 min after a media change. eGLuc2 fused variants followed predicted trends; non-pathogenic variants (D208E, G244V) were secreted at WT-like levels, whereas predicted disease-causing variants (C245Y, G246R, E300K, Y437H, I477N) demonstrated substantial secretion defects ranging from 0.20 - 4.0% of WT MYOC levels in HEK-293T cells. These variants were slightly more tolerated in NTM-5 cells, resulting in secretion levels ranging from 1.0 - 12% of WT MYOC levels. Secretion defects caused by the C245Y, G246R, and Y437H mutations were partially rescued by permissive growth temperatures. Interestingly, a combination of growth temperature reduction and cycloheximide treatment of transfected cells indicate that the pool of protein that is rescued at lower temperatures arises from intracellular stores, and is not from newly synthesized MYOC under permissive temperature. Fusion of eGLuc2 to MYOC does not significantly change the behavior of MYOC. The use of the eGLuc2-tagged version of MYOC can be utilized to develop a deeper understanding of MYOC folding and secretion. This newly developed MYOC reporter system has the potential to be used in small molecule and/or genetic high-throughput screens to identify modulators of MYOC secretion.Southwestern Medical Foundatio

New Perspectives in Bougainvillea Breeding

This article does not have an abstract

Effect of certain plant growth regulations on the growth and the metabolism of isolated cotyledons and hypocotyls segments.

The present study aims at studying the effects of Gibberellins (GA3 and GA4+7) and of fusicoccin in addition to those of cytokinins (Kinetin and benzylaminipurine) on expansion growth and on certain aspects of metabolism in cucumber cotyledons. The effects have been studied both without and with 5mm pottassium (supplied as KCl) to find out-how far presence of potassium, which also includes expansion, can modify the effect their growth regulations

Cytogenetical studies on Bougainvillea. I. A case of interchange heterozygosity

A case of interchange heterozygosity has been found inB. peruviana cv "Princess Margaret Rose" in which there is a regular formation of 15 bivalents and an interchange multiple of 4 chromosomes. The multiple is always associated with the nucleolus at diakinesis, indicating that one of the chromosome involved is nucleolar. The nucleolar pair of chromosomes shows a slight heteromorphicity which may be due to an unequal interchange. Although 80% of interchange multiples orientate non-disjunctionally, yet 65% pollen is stainable. The pollen is ineffective in self pollination, but highly effective in crosses with 2x and 3x cultivars ofB. spectabilis. The higher pollen stainability indicates that the deficiencies and duplications caused by non-disjunction do not have serious physiological offects on pollen grains and that its genome can withstand rearrangements

Cytogenetics of cultivated Bougainvilleas. V. Induced tetraploidy and restoration of fertility in sterile cultivars

Induced tetraploidy restores fertility in sterile cultivars of Bougainvillea. However, like the fertile diploid cultivars induced tetraploids are self-incompatible, but set seeds readily on crossing. Diploid progenitors show irregular meiosis, while in tetraploid counterparts there is predominant bivalent pairing. Low frequency of quadrivalents at 4x level implies that pairing at diploid level is between chromosomes heterozygous for cryptic structural changes. This also accounts for lower number of chiasmata per chromosome in diploids in comparison with their tetraploid counterparts. Sterility at 2x level appears to be due to recombination of cryptic structural differences and/or unequal distribution or aberrant behaviour of univalents. Predominant bivalent pairing accompanted by high fertility is a strong pointer towards preferential pairing in induced tetraploids