A 368-base-pair cis-acting HWP1 promoter region, HCR, of Candida albicans confers hypha-specific gene regulation and binds architectural transcription factors Nhp6 and Gcf1p

To elucidate the molecular mechanisms controlling the expression of the hypha-specific adhesin gene HWP1 of Candida albicans, its promoter was dissected and analyzed using a green fluorescent protein reporter gene. A 368-bp region, the HWP1 control region (HCR), was critical for activation under hypha-inducing conditions and conferred developmental regulation to a heterologous ENO1 promoter. A more distal region of the promoter served to amplify the level of promoter activation. Using gel mobility shift assays, a 249-bp subregion of HCR, HCRa, was found to bind at least four proteins from crude extracts of yeasts and hyphae with differing binding patterns dependent on cell morphology. Four proteins with DNA binding activities were identified by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis after separation by anion-exchange and heparin-Sepharose chromatography. One protein with high similarity to Nhp6, an HMG1 family member in Saccharomyces cerevisiae, and another with weak similarity to an HMG-like condensation factor from Physarum polycephalum implicated changes in chromatin structure as a critical process in hypha-specific gene regulation. Proteins with strong homology to histones were also found. These studies are the first to identify proteins that bind to a DNA segment that confers developmental gene regulation in C. albicans and suggest a new model for hypha-specific gene regulation.     

Proteomic identification of macrophage migration-inhibitory factor upon exposure to TiO2 particles

Inhalation of particulate matter aggravates respiratory symptoms in patients with chronic airway diseases, but the mechanisms underlying this response remain poorly understood. We used a proteomics approach to examine this phenomenon. Treatment of epithelial cells with BSA-coated titanium dioxide (TiO(2)) particles altered 20 protein spots on the two-dimensional gel, and these were then analyzed by nano-LC-MS/MS. These proteins included defense-related, cell-activating, and cytoskeletal proteins implicated in the response to oxidative stress. The proteins were classified into four groups according to the time course of their expression patterns. For validation, RT-PCR was performed on extracts of in vitro TiO(2)-treated cells, and lung issues from TiO(2)-treated rats were analyzed by immunohistochemical staining and enzyme immunoassay. TiO(2) treatment was found to increase the amount of mRNA for macrophage migration-inhibitory factor (MIF). MIF was expressed primarily in epithelium and was elevated in lung tissues and bronchoalveolar lavage fluids of TiO(2)-treated rats as compared with sham-treated rats. Carbon black and diesel exhaust particles also induced expression of MIF protein in the epithelial cells.     

Effects of tert-butyl acetate on maternal toxicity and embryo-fetal development in Sprague-Dawley rats

This study investigated the potential adverse effects of tert-butyl acetate (TBAc) on maternal toxicity and embryo-fetal development after maternal exposure of pregnant rats from gestational days 6 through 19. TBAc was administered to pregnant rats by gavage at 0, 400, 800, and 1,600 mg/kg/day. All dams were subjected to a Caesarean section on day 20 of gestation, and their fetuses were examined for any morphological abnormalities. At 1,600 mg/kg, maternal toxicity manifested as increases in the incidence of clinical signs and death, lower body weight gain and food intake, increases in the weights of adrenal glands and liver, and a decrease in thymus weight. Developmental toxicity included a decrease in fetal weight, an increase in the incidence of skeletal variation, and a delay in fetal ossification. At 800 mg/kg, only a minimal developmental toxicity, including an increase in the incidence of skeletal variation and a delay in fetal ossification, were observed. In contrast, no adverse maternal or developmental effects were observed at 400 mg/kg. These results show that a 14-day repeated oral dose of TBAc is embryotoxic at a maternally toxic dose (i.e., 1,600 mg/kg/day) and is minimally embryotoxic at a nonmaternally toxic dose (i.e., 800 mg/kg/day) in rats. However, no evidence for the teratogenicity of TBAc was noted in rats. It is concluded that the developmental findings observed in the present study are secondary effects to maternal toxicity. Under these experimental conditions, the no-observed-adverse-effect level of TBAc is considered to be 800 mg/kg/day for dams and 400 mg/kg/day for embryo-fetal development.     

Targeting bladder tumor cells in vivo and in the urine with a peptide identified by phage display

Bladder cancer is one of the most common tumors of the genitourinary tract. Here, we use phage display to identify a peptide that targets bladder tumor cells. A phage library containing random peptides was screened for binding to cells from human bladder tumor xenografts. Phage clones were further selected for binding to a bladder tumor cell line in culture. Six clones displaying the consensus sequence CXNXDXR(X)/(R)C showed selective binding to cells from primary human bladder cancer tissue. Of these, the CSNRDARRC sequence was selected for further study as a synthetic peptide. Fluorescein-conjugated CSNRDARRC peptide selectively bound to frozen sections of human bladder tumor tissue, whereas only negligible binding to normal bladder tissue was observed. When the fluorescent peptide was introduced into the bladder lumen, in a carcinogen-induced rat tumor model, it selectively bound to tumor epithelium. Moreover, when the peptide was intravenously injected into the tail vein, it homed to the bladder tumor but was not detectable in normal bladder and control organs. Next, we examined whether the peptide can detect tumor cells in urine. The fluorescent peptide bound to cultured bladder tumor cells but not to other types of tumor cell lines. Moreover, it bound to urinary cells of patients with bladder cancer, while showing little binding to urinary cells of patients with inflammation or healthy individuals. The CSNRDARRC peptide may be useful as a targeting moiety for selective delivery of therapeutics and as a diagnostic probe for the detection of bladder cancer.     

In situ proteolysis for protein crystallization and structure determination

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.