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Mississippi Academy of Sciences: Seventy-Third Annual Meeting--program corrections.

Cellular, Molecular, and Developmental Biology

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Thursday afternoon:



Melissa Adams, Glen Shearer, University of Southern Mississippi

The dimorphic fungus, Histoplasma capsulatum (Hc) can be found in the soil as a saprophytic multicellular mold. In the lungs of an infected host, a shift to the unicellular parasitic yeast occurs. The mold to yeast shift is required for the progression of the disease. Sulfur metabolism plays a critical role in the dimorphic process. Sulfhydryl groups, especially cysteine, are a necessary nutrient for the transition to the yeast state. Enzymes involved in the cysteine metabolism pathway are being studied in order to evaluate their role in the dimorphism. Cystiene dioxygenase (CDO1), Gamma-glutamyl cysteine synthetase (GSH1), and Glutathione synthetase (GSH2) are involved in the cysteine metabolic pathway. The isolation of the cDNA of these three genes was accomplished by RACE PCR. The expression level of each gene in both the yeast and mold morphotypes of four Hc strains was examined by northern blotting and realtime PCR. CDO1, which was previously isolated in our lab, is expressed in both the mold and yeast phases of the organism while GSH1 and GSH2 appear to be expressed in only the yeast phase. Experiments are underway to see if the enzyme levels correlate with transcription levels.



Davida Crossley, Glen Shearer, The University of Southern Mississippi, Hattiesburg, MS,

Histoplasma capsulatum is the causative agent for the respiratory disease histoplasmosis which infects an estimated 500,000 Americans each year. The dimorphic fungus grows in the soil as a multi-cellular mold. Once the soil is disturbed, spores are released and are inhaled into the lungs. In the lungs, the fungus converts to a unicellular yeast. The mold-to-yeast conversion is a requirement for pathogenesis. Therefore, to understand the molecular basis of dimorphism, we have isolated several mold-specific and yeast-specific genes. The subject of this study is the mold-specific M46 gene. Northern blot analysis has shown that M46 is expressed in G186AS and Downs strains, but is transcriptionally silent in G184AS and G217B strains. The reason for lack of transcription in the latter strains is currently being studied. The putative protein sequences of all four strains is highly conserved. Fusion of the reporter gene, GFP to each of the four Hc promoters has shown that all four strains have a functional M46 promoter. We now hypothesize that the reason for lack of expression of M46 in strains G184AS and G217B may be due to a missing or non-functional trans-regulating factor(s). In order to characterize the presence of the trans-regulating factors, gel shift analysis is underway. Comparison of electrophoretic mobility shifts of the DNA-protein complexes from the expressing M46 stains and non-expressing M46 stains may give insight on the reason for lack of expression from stains G184AS and G217B.


O2.18 IMMUNOISOLATION OF CFTRENRICHED ENDOCYTIC VESICLES, Ghanshyam Heda (1), Christopher Marino (2), (1) Mississippi University for Women, Columbus, MS, (2) V.A. Medical Center, Memphis, TN, (3) The University of Tennessee Health Sciences Center, Memphis, TN

Cystic fibrosis (CF) is caused by mutations that affect the cellular processing of CFTR protein. Half-life of [DELTA]F508, the most common CFTR mutation, at the plasma membrane is shorter (~4 h) than that of wild-type CFTR (>48 h). Effective correction of the CF defect, however, requires stable expression of [DELTA]F508-CFTR at the plasma membrane. We hypothesize that surface instability of [DELTA]F508-CFTR may result from its selective targeting to cellular sites different than that of wild-type CFTR, and is controlled by certain regulatory proteins. OBJECTIVES: To identify regulatory proteins by comparing the proteome of AF508 or wild-type CFTR-enriched endosomal vesicles. METHODS: [LL-CPK.sub.1] cells transfected with AF508 or wild-type CFTR were treated with 5 mM sodium butyrate at 27[degrees]C to upregulate plasma membrane CFTR levels. Endosomal vesicle fraction was obtained by continuous (12-48%) iodixonol density gradient centrifugation. CFTR-enriched endosomes were visualized by immuno-EM, affinity purified by AminoLink[R] gel, and analyzed by 2D-PAGE. RESULTS: Immunoblotting showed that endosomal fraction was enriched with Rab5 (96.23+2.38%, n=6) and Rab11 (95.01+3.96%, n=6), markers of early and recycling endosomes respectively. CFTR enriched endosomes eluted from anti-CFTR linked AminoLink[R] gel retained ~30% of total endosomal proteins in 2-D gels, suggesting their specificity to CFTR-enriched endosomes. Further, CFTR-enriched endosomes contained 3 proteins (~45-50 kDa, pI ~5.5-6.5) that were significantly enriched and remains to be identified. CONCLUSION: Regulatory proteins may be differentially associated with CFTR-enriched endosomes in cells expressing [DELTA] F508 or wild-type CFTR. Their identification may help us understand the mechanisms that regulate endocytic trafficking and plasma membrane CFTR expression.

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Rupesh Patel, Davida Crossley, GLen Shearer The University of Southern Mississippi, Hattiesburg, MS, Histoplasma capsulatum is a dimorphic fungus that causes histoplasmosis, a respiratory disease. The fungus grows as a multicellular saprophytic mold in soils with bird and bat excreta, at 25 o C. Conversion to unicellular parasitic yeast takes place in the lungs, at 37 o C. The yeast causes histoplasmosis; hence, the mold-to-yeast transition is required for the development of the disease. The M46 gene was studied in this project to help determine the mechanism of this conversion. Though the function of M46 is unknown, recent data states that M46 is only expressed in the mold form. Therefore in aid to determine the function of M46, the location of M46 in the cell will be determined. GFP (Green Fluorescent Protein) was fused to the c-terminus of M46 from strains WU24 and G186AS. Calcofluor white was used as a control to stain the cell wall. The distribution of GFP as determined by fluorescence microscopy, throughout the hyphae indicates that M46 localizes in the cytoplasm. We hypothesized that the M46 protein is located in the cytoplasm. Cytoplasmic proteins generally function as signaling proteins, metabolic regulators, etc. To help determine the function of M46, future work includes the n-terminus construct and creation of a knockout.
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Publication:Journal of the Mississippi Academy of Sciences
Date:Apr 1, 2009
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