Screening for candidate genes involved in tolerance to organic solvents in yeast

Saccharomyces cerevisiae mutant strain, KK-211, isolated from serial culture in medium containing isooctane showed an extremely higher tolerance to the hydrophobic organic-solvents, which are toxic to yeast cells compared to the wild-type parent strain, DY-1. To detect genes that are related to this tolerance, a DNA microarray analysis was performed using mRNAs isolated from strains DY-1 and KK-211. Fourteen genes were identified as being related to the tolerance. The expression of 12 genes including ICT1, YNL190W, and PRY3, was induced while the expression of two genes including PHO84 was repressed in strain KK-211. Two genes, ICT1 and YNL190W showed the same profile in the DNA microarray analysis and a differential display-polymerase chain reaction analysis. But, there is no detectable difference in the expression profile of KK-211 cells cultured with or without isooctane. The results suggest that change in expression levels of multiple genes that confer the modification function of the cell surface, not by a single gene, might be required for yeast cell tolerance to organic solvents.     

Analysis of a processing system for proteases using yeast cell surface engineering: conversion of precursor of proteinase A to active proteinase A

The display of a protease, carboxypeptidase Y (CPY) or procarboxypeptidase Y (proCPY), which is the vacuolar protease, on the yeast-cell surface was successfully performed using yeast-cell-surface engineering for the first time. Through that we could confirm the processing of vacuolar proteases containing proteinase A (PrA) and proteinase B (PrB) which are related to the maturation of proCPY, using a novel cell-surface engineering technique. Various protease-knockout strains of Saccharomyces cerevisiae with the CPY-displaying system were constructed to evaluate the operation of the activation process of CPY. The display of CPY (CPY-agg, which is a fusion protein of CPY with C-terminal half of α-agglutinin) on the cell surface was confirmed by immunofluorescence staining. The activity of the CPY-agg was determined after the conversion of proCPY to active CPY by treatment of whole cells with proteinase K. In the proCPY-displaying CPY-knockout strain and PrB-knockout strain, CPY was displayed as an active (mature) form, but in the proCPY-displaying PrA-knockout strain, CPY was present as an inactive form (proCPY). These facts indicate that PrA had been already activated before its transport to the vacuole and that active mature PrA might convert proCPY to CPY before the transport of proCPY to the vacuole. From these results, it was suggested that by using the yeast-cell-surface engineering at the location of the initial step, the autocatalytic activation from proPrA to PrA might occur before the vacuolar branch separates from the main secretory pathway.     

Two-dimensional HPLC on-line analysis of phosphopeptides using titania and monolithic columns

Conventional and comprehensive two-dimensional (2D) HPLC systems using the combination of titania and monolithic columns were established for the on-line analysis of phosphopeptides. Compared with immobilized metal affinity chromatography of a general method for the analysis of phosphopeptides, the use of titania columns in the analysis permits the specific isolation of phosphopeptides in a higher yield. Using the current 2D HPLC systems, phosphopeptides were specifically isolated from nonphosphorylated peptides by the first-dimension titania column, followed by the high-speed separation of the phosphopeptides by the second-dimension monolithic column. Proteolytic digests of beta-casein were analyzed within 30 min using the comprehensive 2D HPLC system; all phosphopeptides from beta-casein could be efficiently isolated and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The comprehensive 2D HPLC system coupled with mass spectrometry will be useful for high-throughput and on-line phosphoproteome analyses.     

Smad3 mediates TGF-beta1-induced collagen gel contraction by human lung fibroblasts

Transforming growth factor-beta1 (TGF-beta1) is a key mediator in tissue repair and fibrosis. Using small interference RNA (siRNA), the role of Smad2 and Smad3 in TGF-beta stimulation of human lung fibroblast contraction of collagenous matrix and induction of alpha-SMA and the role of alpha-SMA in contraction were assessed. HFL-1 cells were transfected with Smad2, Smad3 or control-siRNA, and cultured in floating Type I collagen gels +/- -TGF-beta1. TGF-beta1 augmented gel contraction in Smad2-siRNA- and control-siRNA-treated cells, but had no effect in Smad3-siRNA-treated cells. Similarly, TGF-beta1 upregulated alpha-SMA in Smad2-siRNA- and control-siRNA-treated cells, but had no effect on Smad3-siRNA-treated cells. Alpha-SMA-siRNA-treated cells did not contact the collagen gels with or without TGF-beta1, suggesting alpha-SMA is required for gel contraction. Thus, Smad3 mediates TGF-beta1-induced contraction and alpha-SMA induction in human lung fibroblasts. Smad3, therefore, could be a target for blocking contraction of human fibrotic tissue induced by TGF-beta1.     

CTCF-dependent chromatin insulator is linked to epigenetic remodeling

Chromatin insulators are boundary elements between distinctly regulated, neighboring chromosomal domains, and they function by blocking the effects of nearby enhancers in a position-dependent manner. Here, we show that the SNF2-like chromodomain helicase protein CHD8 interacts with the insulator binding protein CTCF. Chromatin immunoprecipitation analysis revealed that CHD8 was present at known CTCF target sites, such as the differentially methylated region (DMR) of H19, the locus control region of beta-globin, and the promoter region of BRCA1 and c-myc genes. RNA interference-mediated knockdown of CHD8 significantly abolished the H19 DMR insulator activity that depends highly on CTCF, leading to reactivation of imprinted IGF2 from chromosome of maternal origin. Further, the lack of CHD8 affected CpG methylation and histone acetylation around the CTCF binding sites, adjacent to heterochromatin, of BRCA1 and c-myc genes. These findings provide insight into the role of CTCF-CHD8 complex in insulation and epigenetic regulation at active insulator sites.     

The evaluation of oral health in stroke patients

doi: 10.1111/j.1741‐2358.2011.00505.x The evaluation of oral health in stroke patients Objective: As tooth loss has been suggested as a potential risk factor for stroke, oral examinations were carried out on stroke patients to review the oral condition of those patients. Method: The subjects were patients consecutively discharged from the recovery rehabilitation unit of Hiroshima City General Rehabilitation Center between April 2008 and December 2009. All patients were offered oral examination and 358 of 443 patients accepted. Patients receiving dental examination were divided into two groups: one group comprising stroke patients and the second, patients with other disorders. These two groups were then compared for the number of remaining teeth by age group. Results: Among the examined patients, the number of remaining teeth in stroke patients in their 50s and 60s was significantly lower than for patients in corresponding age groups (18.4 ± 9.4 vs. 24.5 ± 5.4 and 18.3 ± 9.2 vs. 22.2 ± 7.2, respectively, with p < 0.05 for both age groups) who were hospitalised for other conditions. In addition, the number of remaining teeth in stroke patients in their 50s was also significantly lower than the number reported in the Survey of Dental Diseases (24.1 ± 6.1; p < 0.05). Conclusion: The results of this study suggest an association between tooth loss and early occurrence of stroke.     

Chimeric yeast G-protein α subunit harboring a 37-residue C-terminal gustducin-specific sequence is functional in Saccharomyces cerevisiae

Despite many recent studies of G-protein-coupled receptor (GPCR) structures, it is not yet well understood how these receptors activate G proteins. The GPCR assay using baker's yeast, Saccharomyces cerevisiae, is an effective experimental model for the characterization of GPCR-Gα interactions. Here, using the yeast endogenous Gα protein (Gpa1p) as template, we constructed various chimeric Gα proteins with a region that is considered to be necessary for interaction with mammalian receptors. The signaling assay using the yeast pheromone receptor revealed that the chimeric Gα protein harboring 37 gustducin-specific amino acid residues at its C-terminus (GPA1/gust37) maintained functionality in yeast. In contrast, GPA1/gust44, a variant routinely used in mammalian experimental systems, was not functional.     

Construction of cell surface-engineered yeasts displaying antigen to detect antibodies by immunofluorescence and yeast-ELISA

In order to detect monoclonal antibodies (MAbs) from insufficient and unavailable human proteins, yeast cells were engineered to display human antigens on their surface and consequently endowed with the ability to specifically bind antibodies. Thus, a fusion gene for the expression of the human proteasome subunit alpha 6 (hPSA6) and human profilin I (hProI) were assembled, respectively, with a His.tag marker at the C-terminal and displayed on yeast surface. With anti-His.tag MAb as the primary antibody and the fluorescein isothiocyanate-conjugated goat anti-mouse Immunoglobulin G as the second antibody, the surface display of hPSA6 and hProI were verified by immunofluorescence labeling. The antigen-displayed yeast particles were used for MAbs detection from ascites through both immunofluorescence and yeast-enzyme-linked immunosorbent assay (ELISA) methods. The results were verified by Western blotting and indirect ELISA. By improving the sensitivity, the novel MAbs detection can be applied in the generation and screening of positive hybridoma. It is suggested that by combining the DNA immunization, the present study can evolve into a quick and protein-free way of MAbs production for insufficient and unavailable antigen.