Besides its well-recognized role in hemostasis and thrombosis, thromboxane A 2 synthase (TXAS) is proposed to be involved in thrombopoiesis and lymphocyte differentiation. To evaluate its various physiologic roles, we generated TXAS-deleted mice by gene targeting. TXAS ؊/؊ mice had normal bone marrow megakaryocytes, normal blood platelet counts, and normal CD4 and CD8 lymphocyte counts in thymus and spleen. Platelets from TXAS ؊/؊ mice failed to aggregate or generate thromboxane B 2 in response to arachidonic acid (AA) but produced increased prostaglandin-E 2 (PGE 2 ), PGD 2 , and PGF 2␣ . AA infusion caused a progressive drop of mean arterial pressure (MAP), cardiac arrest, and death in wild-type (WT) mice but did not induce shock in TXAS ؊/؊ mice or in WT and TXAS ؊/؊ mice treated with antagonist to the thromboxane-prostanoid (TP) receptor. The TXAS ؊/؊ mice were able to maintain normal MAP upon AA insult when TP was present but were unable to do so when TP was blocked by an antagonist, suggesting a role of endoperoxide accumulation in influencing MAP. We conclude that TXAS is not essential for thrombopoiesis and lymphocyte differentiation. Its deficiency causes a mild hemostatic defect and protects mice against arachidonate-induced shock and death. The TXAS-deleted mice will be valuable for investigating the roles of arachidonate metabolic shunt in various pathophysiologic processes. IntroductionThromboxane A 2 synthase (TXAS) is a 60-kDa transmembrane protein that is distributed in platelets, monocytes, and several other cell types. [1][2][3][4][5] It belongs to the large cytochrome p450 (CYP) family. 6 It is considered to be an atypical CYP as it does not possess mono-oxygenase activity. It is an isomerase catalyzing the conversion of prostaglandin H 2 (PGH 2 ) to thromboxane A 2 (TXA 2 ), HHT (l2-L-hydroxy-5, 8,10-heptadecatrienoic acid), and malondialdehyde (MDA). TXA 2 is a potent stimulator of platelet activation and aggregation and vascular constriction. 7 These actions of TXA 2 have been demonstrated to be mediated by binding to a G-proteincoupled-specific receptor, the thromboxane-prostanoid (TP) receptor. 8 The cellular and tissue distribution of TP receptors is closely correlated with that of TXAS. 9 Since TXA 2 has an extremely short half-life and acts as an autocoid, the concordant tissue distribution between TXAS and TP facilitates the rapid actions of TXA 2 . TXA 2 plays a major role in hemostasis, thrombosis, vasoconstriction, and vascular cell proliferation. Patients whose platelets are defective in producing TXA 2 have a bleeding disorder, whereas overproduction of TXA 2 is associated with diverse vascular events including acute coronary syndrome, ischemic stroke, pulmonary hypertension, abnormal renal hemodynamics, and preeclampsia. [10][11][12][13][14][15][16][17][18] The important role of TXA 2 in hemostasis and vascular disorders is further supported by the defects observed in TP-deleted mice generated by gene targeting. TP Ϫ/Ϫ mice have normal embryogenesis, postnatal growth, and ferti...

The Chinese medicinal herbs Radix Isatidis and Viola yedoensis Makino have been suggested to possess antiviral activity. This study tests whether these and other Chinese and Western herbal medicinal formulas can modulate the immune functions involving virus-suppression in BALB/c mouse. We first confirmed the extract from Viola yedoensis Makino, but not from Radix Isatidis, the traditional Chinese medicine (TCM) formula Chui-Uren-Chien (CUC), or a Western homeopathic medicinal drink Método Canova, could inhibit the replications of herpes simplex virus-1 and enterovirus 71 in the human neuroblastoma SK-N-SH cell line. Subsequently, the same herbal extracts and drink underwent toxicity and immunomodulatory tests on mice of 5-7 weeks old. After 8 weeks of feeding different herbal medicinal formulas, no hepatic or renal toxicity was noted in any tested animal; whereas among the immune function evaluations, only the mice treated with CUC extract were found to be associated with significant increases (p < 0.05) in both the level of plasma IgG and the percentage of monocyte in blood mononuclear cells as well as the activation of macrophage Raw264.7 cells for nitric oxide production, suggesting its role in modulating the non-specific immune response. Analyses using protein arrays showed CUC was the most potent herbal medicinal formula eliciting fluctuations in plasma cytokine and chemokine concentrations. Taking all experimental data together, we conclude Chui-Uren-Chien possesses immunomodulatory capability in mouse, but none of the herbal medicinal formulas tested here are involved in strengthening antiviral immunity.

Bladder cancer is highly recurrent after therapy, which has an enormous impact on the health and financial condition of the patient. It is worth developing diagnostic tools for bladder cancer. In our previous study, we found that the bladder carcinogen BBN increased urothelial global DNA CpG methylation and decreased GSTM1 protein expression in mice. Here, the correlation of BBN-decreased GSTM1 and GSTM gene CpG methylation status was analyzed in mice bladders. BBN treatment decreased the protein and mRNA expression of GSTM1, and the CpG methylation ratio of GSTM1 gene promoter was slightly increased in mice bladders. Unlike mouse GSTM1, the human GSTM1 gene tends to be deleted in bladder cancers. Among 7 human bladder cancer cell lines, GSTM1 gene is really null in 6 cell lines except one, T24 cells. The CpG methylation level of GSTM1 was 9.9% and 5-aza-dC did not significantly increase GSTM1 protein and mRNA expression in T24 cells; however, the GSTM5 gene was CpG hypermethylated (65.4%) and 5-aza-dC also did not affect the methylation ratio and mRNA expression. However, in other cell lines without GSTM1, 5-aza-dC increased GSTM5 expression and decreased its CpG DNA methylation ratio from 84.6% to 61.5% in 5637, and from 97.4% to 75% in J82 cells. In summary, two biomarkers of bladder tumor were provided. One is the GSTM1 gene which is down-regulated in mice bladder carcinogenesis and is usually deleted in human urothelial carcinoma, while the other is the GSTM5 gene, which is inactivated by DNA CpG methylation.

Summary. Epitopes recognized by factor VIII (FVIII) inhibitors of Chinese origin were analysed by immunoblotting with full-length recombinant FVIII (rFVIII), thrombinactivated FVIII (FVIIIa) and 16 FVIII fusion proteins synthesized by bacteria. Twenty-eight patients, 12 with haemophilia A and 16 with autoimmune diseases, were recruited. Antibodies from 22 patients showed reactivity with rFVIII, 20 with FVIIIa, and one reacted only with FVIII fusion proteins. Of these 22 cases, most were reactive with A2-a2 and A3-C1-C2 of FVIII(a). Of the nine cases that depicted binding to the fusion proteins, three were reactive with the A domains, three with only the B domain, and the other three with both the A and B (or C) domains. An epitope for a neutralizing antibody of a haemophilia A patient, designated TWN-112, was localized to residues 323±390, specified by FVIII fusion proteins. The same epitope also appeared on an FVIII-expression phage library screening. Immunoabsorption of antibodies from with the epitope reduced the neutralizing activity of the inhibitor by 33%. The incidence of a1 of FVIII is higher, and that of a3 is lower, than previously reported. Two novel epitopes, reported for the first time in this paper, were localized on the 8B2 (amino acid residues 1022±1204) and 8A2(V) (residues 673±740) fusion proteins. These two epitopes were able to reduce inhibitory antibody activity by 24% and 25% respectively. Changes of FVIII fragment specificity were also observed in one of six patients for whom multiple samples, collected at different times, were available. Our initial finding showed that the FVIII inhibitors in these Chinese patients shared epitopes with those of patients from very different genetic backgrounds, suggesting a common mechanism for the development of FVIII inhibitors.

Despite a plethora of literature has documented that osteoarthritis (OA) is veritably associated with oxidative stress-mediated chondrocyte death and matrix degradation, yet the possible involvement of synoviocyte abnormality as causative factor of OA has not been thoroughly investigated. For this reason, we conduct the current studies to insight into how synoviocytes could respond to an episode of folate-deprived (FD) condition. First, when HIG-82 synoviocytes were cultivated under FD condition, a time-dependent growth impediment was observed and the demise of these cells was demonstrated to be apoptotic in nature mediated through FD-evoked overproduction of reactive oxygen species (ROS) and drastically released of cytosolic calcium (Ca2+) concentrations. Next, we uncovered that FD-evoked ROS overproduction could only be strongly suppressed by either mitochondrial complex II inhibitors (TTFA and carboxin) or NADPH oxidase (NOX) inhibitors (AEBSF and apocynin), but not by mitochondrial complex I inhibitor (rotenone) and mitochondrial complex III inhibitor (antimycin A). Interestingly, this selective inhibition of FD-evoked ROS by mitochondrial complex II and NOX inhibitors was found to correlate excellently with the suppression of cytosolic Ca2+ release and reduced the magnitude of the apoptotic TUNEL-positive cells. Taken together, we present the first evidence here that FD-triggered ROS overproduction in synoviocytes is originated from mitochondrial complex II and NOX. Both elevated ROS in tandem with cytosolic Ca2+ overload serve as final arbitrators for apoptotic lethality of synoviocytes cultivated under FD condition. Thus, folate supplementation may be beneficial to patients with OA.