Wonja Choi

Hydrogen peroxide induces hyphal differentiation in Candida albicans.

ABSTRACT In this study, we demonstrate that hyphal differentiation is induced by the subtoxic concentration of exogenous H2O2 in Candida albicans. This finding is confirmed by the changing intracellular concentration of H2O2. In order to induce the same level of differentiation, low concentrations of exogenous H2O2 are required for the null mutants of the thiol-specific antioxidant and catalase, while higher concentrations are needed for cells treated with ascorbic acid, an antioxidant chemical.

Identification of proteins highly expressed in the hyphae of Candida albicans by two-dimensional electrophoresis

The increase in Candida albicans infections is caused by the increase in therapies resulting in immunocompromised patients. One factor required for C. albicans pathogenicity is the morphological transition from yeast to hypha. The protein profiles of whole extracts from yeasts and hyphae were examined using two‐dimensional electrophoresis to identify the proteins related to the morphological transition. Over 900 protein spots were visualized by silver staining and 11 spots were increased more than three‐fold reproducibly during hyphal differentiation. Six of the 11 spots were identified by peptide mass fingerprints, of which three represented PRA1, two PHR1 and the last TSA1. Vertical streak patterns of Pra1p and Phr1p indicated that post‐translational modifications seem to be caused by variable glycosylation. Comparative proteome analysis between the wild‐type and the deletion mutants, CAMB43 (Δpra1) and CAS10 (Δphr1), further confirmed the identity of PRA1 and PHR1. Interestingly, Pra1p was downregulated in phr1‐deleted mutants. Only PHR1 transcription was increased, indicating that PRA1 and TSA1 are controlled at the post‐translational level. Copyright

Characterization of thiol‐specific antioxidant 1 (TSA1) of Candida albicans

We previously identified several proteins that are differentially expressed in pathogenic hyphae by comparing protein profiles of yeast and hyphae of Candida albicans. One of these, thiol‐specific antioxidant 1 (TSA1), attracted our attention because it may play some roles in surviving an unfavourable oxidative environment created by host cells. Two alleles of the C. albicans TSA1 (CaTSA1) gene are located in opposite orientation on the same chromosome. Using PCR‐directed disruption cassettes and URA‐Blaster, a series of deletion mutants that lack one to four copies were constructed to examine the functions of CaTSA1. Northern and Western analyses showed that both the transcript and protein products of CaTSA1 decreased proportionally to the disrupted copy number and were completely absent in the null mutant, indicating that all four TSA1 copies are equally functional at the transcriptional level. Intracellular H2O2 increased by an order of magnitude in deletion mutants lacking three to four copies, suggesting that CaTsa1p is not a redundant H2O2 scavenger. CaTsa1p was indispensable for yeast‐to‐hyphal transition when C. albicans was cultured under oxidative stress. The level of its oxidized form increased ∼five‐fold in hyphal cells, whereas that of the reduced form increased two‐fold compared to yeast cells. The ratio of oxidized to reduced form was increased three‐fold in hyphal cells. This overall increase was found to be controlled at the post‐transcriptional level. Interestingly, CaTsa1p is translocated to the nucleus of hyphal cells. These findings may be of biological significance for differentiation and pathogenicity. Copyright

Cell cycle-regulated expression and subcellular localization of a kinesin-8 member human KIF18B

The human genome contains genes encoding for over 40 different types of kinesin and kinesin-like proteins. Of these, the functions of 13 kinesins remain uncharacterized. In this study, we constructed a plasmid containing the ORF of KIF18B and revealed that the KIF18B message of approximately 3kb is expressed in a tissue- and cell type-specific manners. A polypeptide of 842 amino acids was deduced from the ORF sequence. We identified another form of 873 amino acids which arises from alternative splicing at the C-terminal end. We also generated an anti-KIF18B antibody which detects a protein band of 120kDa. Western analyses showed that the protein level of KIF18B is elevated at late G(2) through metaphase, very similar to cyclin B1. Immunocytochemical staining revealed that the KIF18B protein is present predominantly in the nucleus and to a lesser extent in the cytoplasm of interphase cells. During mitosis, most KIF18B was found to be closely associated with astral microtubules emanating from the spindle pole during prometaphase and metaphase. Meanwhile, KIF18B was not detected at anaphase and telophase, consistent with the Western blotting data. The nuclear localization signal was roughly determined by using several EGFP-tagged deletion mutants of KIF18B. Together, the expression of KIF18B is regulated in a cell cycle-dependent manner and therefore may play an important role(s) in cell division.