Rat liver mercaptopyruvate sulfurtransferase (MST) was purified to homogeneity. MST is very similar to rhodanese in physicochemical properties. Further, rhodanese cross-reacts with anti-MST antibody. Both purified authentic MST and expressed rhodanese possess MST and rhodanese activities, although the ratio of rhodanese to MST activity is low in MST and high in rhodanese. In order to compare the active site regions of MST and rhodanese, the primary structure of a possible active site region of MST was determined. The sequence showed 66% homology with that of rat liver rhodanese. An active site cysteine residue (Cys246; site of formation of persulfide in catalysis) and an arginine residue (Arg185; substrate binding site) in rhodanese were also conserved in MST. On the other hand, two other active site residues (Arg247 and Lys248) were replaced by Gly and Ser, respectively. Conversion of rhodanese to MST was tried by site-directed mutagenesis. After cloning of rat liver rhodanese, recombinant wild type and three mutants (Arg247-->Gly and/or Lys248-->Ser) were constructed. The enzymes were expressed in Escherichia coli strain BL21 (DE3) with a T7 promoter system. The mutation of these residues decreases rhodanese activity and increases MST activity.

Endothelin 1 (ET-1), mainly produced from vascular endothelial cells, induces vasoconstriction in physiological conditions. The endothelin receptor antagonist is among the most effective agents for pulmonary hypertension. However, little is known about the production source of ET-1 in inflammation and immunity. Here, we studied whether T cell-mediated ET-1 production system exists and operates independent of the production system in vascular endothelial cells. ET-1 production was readily detectable in the culture supernatant of human PBMCs and murine spleen cells stimulated with anti-CD3 antibody. Immunocytostaining showed that ET-1-producing cells emerged only in PBMCs stimulated with anti-CD3 antibody. Using the Transwell system, both murine and human monocytes sorted with magnetic beads in the inner chamber produced ET-1 when T cells were activated with antigen or anti-CD3 antibody in the outer chamber. This ET-1 production was inhibited by anti-IFN-γ and/or TNF-α antibody. Furthermore, monocytes purified from ETflox/flox;Tie2-Cre( + ) mice, which conditionally lack ET-1 in hematopoietic stem cells and vascular endothelial cells, did not produce ET-1 even when stimulated by antigen-specific T cell activation. This study demonstrates the existence of an immune-mediated ET-1 production induced by T cells upon activation through IFN-γ and TNF-α.

Increased glomerular hydraulic pressure has been suggested as a major causative factor in the development of glomerular sclerosis. The elevation of glomerular pressure increases the magnitude of stretch to mesangial cells. The study was, therefore, designed to investigate the effect of mechanical stretch on expression of TGF-beta and extracellular matrix components in cultured rat mesangial cells. The results showed that mechanical stretch stimulated mRNA expression for TGF-beta1 and TGF-beta3 in a time dependent manner, and that mesangial cells secreted substantial amounts of TGF-beta proteins in response to stretch. Stretch was also shown to stimulate mRNA expression for collagen types I and IV, and fibronectin, major components of mesangial extracellular matrix. The stretch-induced mRNA expression for extracellular matrix components was inhibited by neutralizing antibody to TGF-beta. Moreover, stretch-induced mRNA expression of TGF-beta was inhibited by tyrosine kinase inhibitors, genistein or herbimycin A, whereas Ca channel blockers nitrendipine or Gd3+, and inhibitors for protein kinase A or C had no effect. These findings indicate that stretch induced TGF-beta mRNA primarily through tyrosine kinase-dependent mechanisms in cultured rat mesangial cells, and the secreted TGF-beta may play a significant role for the stretch-induced expression of extracellular matrix proteins. Our results suggest that stretch-induced TGF-beta of mesangial cells might be a mediator in the progression of glomerular sclerosis as an autocrine/paracrine factor.

Aim:The objective of the present study was to propose plasmalogens as a beneficial factor in human plasma by showing a highly positive correlation with high-density lipoprotein (HDL) and a significant reduction with aging. Methods: For 148 elderly subjects suspected of coronary artery disease (CAD), clinical characteristics such as coronary stenosis, hyperlipidemia, abnormal glucose tolerance, and hypertension were investigated, and serum biochemical markers including plasmalogens were determined. Results: Serum plasmalogens levels tended to fall in significant coronary stenosis and abnormal glucose tolerance. Correlative analyses among serum biochemical markers revealed that plasmalogens positively correlate with HDL-related values, particularly apolipoprotein A-(apo A-), and that the molar ratio of choline plasmalogen (ChoPlas) to ethanolamine plasmalogen (EtnPlas) correlates positively with low-density lipoprotein (LDL) particle size, and negatively with apo A-and fasting triglyceride (TG) levels. Comparison of plasmalogens in elderly subjects with those of 119 healthy young subjects showed a marked decrease in serum plasmalogens levels by aging. Conclusion: These results suggest that serum plasmalogens, antioxidant phospholipids, function as a beneficial factor as well as HDL, and that the measurement of serum plasmalogens is useful in clinical diagnosis.

Purpose: To show whether single nucleotide polymorphisms (SNP) of drug metabolic genes were associated with toxicity of 2′,2′-difluoro 2′-deoxycytidine (gemcitabine)-based chemoradiotherapy and overall survival (OS) of patients with pancreatic cancer.Experimental Design: We evaluated 17 SNPs of the CDA, dCK, DCTD, RRM1, hCNT1, hCNT2, hCNT3, and hENT1 genes in 154 patients with potentially resectable pancreatic adenocarcinoma who were enrolled in clinical trials at The University of Texas M.D. Anderson Cancer Center (Houston, TX) from February 1999 to January 2006, with follow-up until April 2009. Patients received neoadjuvant concurrent gemcitabine and radiation therapy with or without gemcitabine-cisplatin induction therapy. The association of genotypes with toxicity or OS was tested, respectively, by logistic regression and Cox regression analysis.Results: None of the 17 SNPs, individually, had a significant association with OS. A combined genotype effect of CDA A-76C, dCK C-1205T, DCTD T-47C, hCNT3 C-69T, hENT1 T-549C, and hENT1 C913T on OS was observed. Patients carrying 0 to 1 (n = 43), 2 to 3 (n = 77), or 4 to 6 (n = 30) variant alleles had median survival time of 31.5, 21.4, and 17.5 months, respectively. The hazard ratio of dying was 1.71 (95% confidence interval, 1.06-2.76) and 3.16 (95% confidence interval, 1.77-5.63) for patients carrying two to three or four to six at-risk genotypes (P = 0.028 and P < 0.001), respectively, after adjusting for clinical predictors. CDA C111T, dCK C-1205T, dCK A9846G, and hCNT3 A25G, individually and jointly, had a significant association with neutropenia toxicity.Conclusions: These observations suggest that polymorphic variations of drug metabolic genes were associated with toxicity of gemcitabine-based therapy and OS of patients with resectable pancreatic cancer. Clin Cancer Res; 16(1); 320-9. ©2010 AACR.2′,2′-Difluoro 2′-deoxycytidine (gemcitabine) is the standard first-line agent for treatment of pancreatic cancer. However, 75% of patients do not benefit from this therapy (1), and other than stage, it is not clear what factors predict clinical response to gemcitabine. A major dose-limiting side effect of gemcitabine is hematologic toxicity such as neutropenia and thrombocytopenia, which often result in dose reduction or longer intervals between gemcitabine administrations. However, there is no available biomarker that predicts the toxicity of gemcitabine.Gemcitabine is a nucleoside analogue and a prodrug that requires cellular uptake and intracellular phosphorylation ( Fig. 1; ref. 2). Five of the nucleotide transporters found in humans-human concentrative nucleotide transporter (hCNT) 1 to 3 (a.k.a. solute carrier family 28 A1-A3) and human equilibrative nucleotide transporter (hENT) 1 and 2 (solute carrier family 29)-seem to be responsible for cellular uptake of gemcitabine (2). Once inside the cell, gemcitabine is phosphorylated by deoxycytidine kinase (dCK) to its monophosphate form. This first stage of phosphorylation is the rate-limiting step for furth...

Estrogen receptor (ER)-positive breast cancer is a distinct subpopulation of breast cancer exhibiting a high response rate to endocrine therapy. However, not all ER-positive patients respond to the therapy, and a subgrouping of ER-positive patients based on the physiology of estrogen signaling is expected to be useful for predicting the prognosis. This study has revealed that selected estrogen-regulated genes (ERGs) are useful in identification of a poor-prognosis population among ER-positive breast cancer patients. First, the expression levels of 11 ERGs, selected based on our earlier microarray study in cultured cells, were analyzed by means of real-time reverse transcription-PCR in 14 ER-positive human breast cancer tissues. The patients were clearly divided into two groups in cluster analysis. Then, we examined the expression levels of two representative ERGs, histone deacetylase 6 (HDAC6) and insulin-like growth factor binding protein 4 (IGFBP-4), in 62 ER-positive patients with immunohistochemistry to assess the impact of ERG expression on prognosis (median follow-up 4409 days). Positive HDAC6 staining was significantly correlated with a lower disease-free survival rate. Moreover, when the expression level of HDAC6 was assessed in combination with IGFBP-4 expression in the nucleus, the poor-prognosis patients were more accurately identified. This study has identified new candidate ERGs for prediction of prognosis, and we suggest that combined assessment of the expression levels of these ERGs will contribute to the clinically useful stratification of ER-positive breast cancer patients. (Cancer Sci 2004; 95: 496-502) reast cancer is the leading cancer in most countries in terms of cumulative incidence rate in females. The female hormone estrogen mediates the proliferation and progression of breast cancer, and endocrine therapy, which inhibits estrogen synthesis or estrogen binding to its receptor, has been a standard therapy for estrogen receptor (ER)-positive breast cancer. Tamoxifen has served as the standard endocrine agent for postmenopausal patients for more than 25 years.1) While the proportional reduction in recurrence is as much as 47% in ER-positive breast cancer patients receiving adjuvant tamoxifen therapy over 5 years,2) a significant number of patients still relapse after the therapy. Therefore, an effective way to stratify ER-positive patients is crucial for improved choice of therapy Many investigators have considered that expression of estrogen-regulated genes (ERGs) can provide predictive markers, 3) because their expression may indicate the presence of a functional estrogen signaling pathway. Progesterone receptor (PgR) is one of the representative ERGs, and ER-negative and PgRpositive tumors occasionally respond to tamoxifen treatment. However, in a recent meta-analysis on adjuvant endocrine treatment, 2) the reduction in recurrence was 23% for ER-negative and PgR-positive patients, 37% for ER-positive and PgR-positive patients, and 32% for ER-positive and PgR-negative patients, and the...

CMCD is a rare congenital disorder characterized by persistent or recurrent skin, nail, and mucosal membrane infections caused by Candida albicans. Heterozygous GOF STAT1 mutations have been shown to confer AD CMCD as a result of impaired dephosphorylation of STAT1. We aimed to identify and characterize STAT1 mutations in CMCD patients and to develop a simple diagnostic assay of CMCD. Genetic analysis of STAT1 was performed in patients and their relatives. The mutations identified were characterized by immunoblot and reporter assay using transient gene expression experiments. Patients' leukocytes are investigated by flow cytometry and immunoblot. Six GOF mutations were identified, three of which are reported for the first time, that affect the CCD and DBD of STAT1 in two sporadic and four multiplex cases in 10 CMCD patients from Japan. Two of the 10 patients presented with clinical symptoms atypical to CMCD, including other fungal and viral infections, and three patients developed bronchiectasis. Immunoblot analyses of patients' leukocytes showed abnormally high levels of pSTAT1 following IFN-γ stimulation. Based on this finding, we performed a flow cytometry-based functional analysis of STAT1 GOF alleles using IFN-γ stimulation and the tyrosine kinase inhibitor, staurosporine. The higher levels of pSTAT1 observed in primary CD14(+) cells from patients compared with control cells persisted and were amplified by the presence of staurosporine. We developed a flow cytometry-based STAT1 functional screening method that would greatly facilitate the diagnosis of CMCD patients with GOF STAT1 mutations.

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