Effects of thioredoxin on established airway remodeling in a chronic antigen exposure asthma model

The development and treatment of asthma remains a subject of considerable interest in the medical community. Previous studies implicate an important role of cytokines in the pathology of asthma. In this current study, we examined whether redox-active protein thioredoxin 1 (TRX1) could prevent airway remodeling in an ovalbumin (OVA)-driven mouse chronic antigen exposure asthma model. Balb/c mice were sensitized and then challenged nine times with OVA (days 19-45). In this protocol, airway remodeling was established by day 34. Administration of recombinant human TRX1 during antigen challenge (days 18-32) significantly inhibited airway remodeling, eosinophilic pulmonary inflammation, airway hyperresponsiveness and resulted in decreased lung expression of eotaxin, macrophage inflammatory protein-1alpha and IL-13. Airway remodeling and eosinophilic pulmonary inflammation was also prevented in chronic OVA-exposed Balb/c human TRX1 transgenic mice. Importantly, TRX1-administration, after the establishment of airway remodeling (days 35-45), resulted in improved airway pathology. Our results suggest TRX1 prevents the development of airway remodeling, and also improves established airway remodeling by inhibiting production of chemokines and Th2 cytokines in the lungs.     

Escape from tolerance of organic nitrate by induction of cytochrome P450.

The mechanism of organic nitrate tolerance is poorly defined. We studied the rat P450-catalyzed conversion of organic nitrate to nitric oxide (NO) by purified P450 isoforms relationship between P450 expression and nitrate tolerance following continuous infusion of organic nitrates in rats. The hypotensive effect of an nitroglycerin (NTG) bolus injection was abolished in rats that had been previously provided a continuous 48 h infusion of NTG. This effect was accompanied by a gradual but marked decrease in plasma and urinary nitrate levels following a peak at 18-24 h. Nitrate tolerance was reversible; the decline in the hypotensive effect and P450 levels observed after 2 d of continuous infusion was followed by restoration to control levels 2 d after cessation of the infusion. Similarly, the hypotensive action disappeared in P450-depleted, and -inhibited rats. At 48 h after infusion, NTG-induced NO generation of the vessels increased in acetone (a P450 inducer) -pretreated rats. The appearance and disappearance of P450 paralleled the conversion of organic nitrates to NO. Our observations indicate that nitrate tolerance is in large part the result of decreased P450 expression and activity. Interventions that maintain or increase P450 activity may be a strategy to provide relief from ischemic conditions in humans.     

Role of phospholipids in reconstituted cytochrome P450 3A form and mechanism of their activation of catalytic activity.

Cytochrome P-450 coded for by the 3A gene family requires specific conditions in a reconstituted system, if its catalytic activity is to be efficient. We investigated the mechanism of activation of the catalytic activity of cytochrome P450 3A by phospholipids. Rat P450 PB-1 (3A2), human P450NF (3A4), and rabbit P450 3c (3A6) were used. They had low activity in a reconstituted system (system I) with dilauroylphosphatidylcholine (DLPC) but had high activity with a mixture of phospholipids (DLPC, dioleoylphosphatidylcholine, and phosphatidylserine) and sodium cholate (system II). P450 3A forms are cationic (having a high content of lysine residues) and needed the anionic phospholipid phosphatidylserine to have sufficient activity. Double-reciprocal plots of the metabolic rate of cytochrome P-450 versus the concentration of NADPH-cytochrome P-450 reductase showed that cytochrome P-450 and the reductase interacted more in system II than in system I. P450 PB-1 did not absorb at 450 nm in the presence of reductase, CO, DLPC, and NADPH, although other cytochrome P-450s absorbed at around 450 nm in such a mixture. However, P450 PB-1 was reduced in the presence of the phospholipid mixture and sodium cholate instead of DLPC. These results suggested that the stimulation of catalytic activity by phospholipids involved increased interaction between cytochrome P-450 and the reductase. Studies of proteolytic digestion and chemical cross-linking in systems I and II showed that a P450 3A form needed disaggregation of cytochrome P-450 and/or the reductase, not the formation of an aggregated complex necessary for the catalytic activity of other cytochrome P-450s.     

MafG controls the hypoxic response of cells by accumulating HIF-1alpha in the nuclei

We identified MafG as a protein that interacts with HIF-1alpha, a key factor in hypoxic response, using the yeast two-hybrid system. Interaction between MafG and HIF-1alpha was confirmed by surface plasmon resonance and by translocation to the nucleolus with the NoLS signal. A knockdown of MafG reduced erythropoietin (EPO) mRNA levels as well as luciferase reporter activity with the hypoxia response element. The knockdown of MafG did not change total HIF-1alpha protein, but reduced the accumulation of HIF-1alpha in the nuclei. These results suggest that MafG regulates the hypoxic response of cells by detaining HIF-1alpha in the nuclei.     

Synthesis and structure-activity relationships of N-{1-[(6-fluoro-2-naphthyl)methyl]piperidin-4-yl}benzamide derivatives as novel CCR3 antagonists

A novel class of potent CCR3 receptor antagonists were designed and synthesized starting from N-{1-[(6-fluoro-2-naphthyl)methyl]piperidin-4-yl}benzamide (1),which was found by subjecting our chemical library to high throughput screening (HTS). The CCR3 inhibitory activity of the synthesized compounds against eotaxin-induced Ca(2+) influx was evaluated using CCR3-expressing preB cells. Systematic chemical modifications of 1 revealed that the 6-fluoro-2-naphthylmethyl moiety was essential for CCR3 inhibitory activity in this new series of CCR3 antagonists. Further structural modifications of the benzamide and piperidine moieties of 1 led to the identification of exo-N-{8-[(6-fluoro-2-naphthyl)methyl]-8-azabicyclo[3.2.1]oct-3- yl}biphenyl-2-carboxamide [corrected] (31) as a potent CCR3 antagonist with an IC(50) value of 0.020 microM.     

Interaction between bisphenol derivatives and protein disulphide isomerase (PDI) and inhibition of PDI functions: requirement of chemical structure for binding to PDI

Bisphenol A (BPA) is an endocrine disrupting chemical and several biological effects have been reported. Previously, protein disulphide isomerase (PDI) was isolated as a target molecule of bisphenol A. In this study, to clarify the effects of BPA on PDI functions, we investigated the relationship between the chemical structure of BPA derivatives and the effects on PDI-mediated isomerase and chaperone activity. We also investigated the effects of changes in the isomerase domain of PDI on the binding of chemicals, using PDI mutants and oxidized or reduced PDI. Among six chemicals, only chemicals, which have a phenol group, can bind to PDI and these chemicals also have an inhibitory effect on PDI-mediated isomerase activity. Changes in the structure of the PDI isomerase domain did not affect chemical-binding activity. On the other hand, the chemicals used in this study have low effects on chaperone activity of PDI. Substitutions in Cys residues (Cys398 and Cys401) of the isomerase active site changed chaperone activity. The present study indicates that phenolic compounds specifically bind to PDI and inhibit isomerase activity. This study provides useful information to predict the biological effects of chemicals and structural studies of PDI containing the function of chemical binding.     

The decreased in vivo clearance of CYP2D6 substrates by CYP2D6*10 might be caused not only by the low-expression but also by low affinity of CYP2D6

CYP2D6 exhibits genetic polymorphism with interindividual differences in metabolic activity. We have found a significant influence on the pharmacokinetics of venlafaxine by the CYP2D6*10 allele in a Japanese population. CYP2D6.10, which is translated from CYP2D6*10, has two amino acid substitutions: Pro34 --> Ser and Ser486 --> Thr. In this study, CYP2D6.10 was expressed in Saccharomyces cerevisiae and its catalytic activity for CYP2D6 substrates was investigated. The CYP2D6*10B- and *10C-associated cDNA were isolated from human lymphocyte genotyped as CYP2D6*10. In addition, three forms of CYP2D6, Pro34/Thr486 (PT), Ser34/Ser486 (SS), and Pro34/Ser486 (wild type, CYP2D6.1), were constructed by PCR-site mutagenesis to clarify the effects of the two amino-acid substitutions. The expression of CYP2D6 protein was confirmed by immunoblotting using CYP2D antibody. The absorbance at 450 nm was measured by CO-reduced difference spectra from five all microsome preparations. The CYP2D6 forms with Pro34 --> Ser amino acid substitution were at a lower expression than CYP2D6.1 from the findings of immunoblotting and spectral analysis. The apparent K(m) values of CYP2D6.1, CYP2D6.10A, and CYP2D6.10C were 1.7, 8.5, and 49.7 microM, respectively, for bufuralol 1'-hydroxylation, and 9.0, 51.9, and 117.4 microM, respectively, for venlafaxine O-demethylation, respectively. The V(max) values were not significantly different among the three variants. These findings suggest that the decreased in vivo clearance by CYP2D6*10 was caused not only by low expression of but also the increased K(m) value of CYP2D6.     

CYP4B1 is a possible risk factor for bladder cancer in humans

In experimental animals such as rats and rabbits, CYP4B1 has an important role in mutagenic activation of procarcinogens in bladders. In human bladders, it is not clear whether CYP4B1 has such role or not. In the present study, human bladder microsomes activated 2-aminofluorene which is a typical substrate for CYP4B1 and is a bladder carcinogen. CYP4B1 was detected in the human bladder microsomes by immunoblotting. Furthermore, we developed a microassay for CYP4B1 mRNA by performing real-time RT-PCR. Using this method, CYP4B1 mRNA levels were assayed in transurethal resection samples from the bladders of patients with bladder tumors. The bladder-tumor patients had a significantly higher expression of CYP4B1 than the nonbladder tumor patients. These findings suggest that a high expression of CYP4B1 increases the risk of bladder tumor by activation of carcinogenic aromatic amines. This approach could be an important tool in the assessment of human bladder cancer risk.