奥替普拉在阻断细胞恶性转化时对某些基因表达的影响

奥替普拉（ｏｌｔｉｐｒａｚ）来源于十字花科（Ｃｒｕｃｉｆｅｒａｓｅ）蔬菜，全称５（２吡嗪基）４甲１，２２羟基３硫酮，文献报道它为一种防癌抑癌剂［１］。我们用致癌素香烟凝聚物（ｃｉｇａｒｅｔｅｓｍｏｋｉｎｇｃｏｎｄｅｎｓａｔｅＣＳＣ）诱发...     

Identification of aspartic acid-203 in human thymidine phosphorylase as an important residue for both catalysis and non-competitive inhibition by the small molecule “crystallization chaperone” 5′-O-tritylinosine (KIN59)

Thymidine phosphorylase (TP) is a catabolic enzyme in thymidine metabolism that is frequently upregulated in many solid tumors. Elevated TP levels are associated with tumor angiogenesis, metastasis and poor prognosis. Therefore, the use of TP inhibitors might offer a promising strategy for cancer treatment. The tritylated inosine derivative 5′-O-tritylinosine (previously designated KIN59) is a non-competitive inhibitor of TP which was previously found to be instrumental for the crystallization of human TP. A combination of computational studies including normal mode analysis, automated ligand docking and molecular dynamics simulations were performed to define a plausible binding site for 5′-O-tritylinosine on human TP. A cavity in which 5′-O-tritylinosine could fit was identified in the vicinity of the Gly405-Val419 loop at a distance of about 11 Å from the substrate-binding site. In the X-ray crystal structure, this pocket is characterized by an intricate hydrogen-bonding network in which Asp203 was found to play an important role to afford the loop stabilization that is required for efficient enzyme catalysis. Site-directed mutagenesis of this amino acid residue afforded a mutant enzyme with a severely compromised catalytic efficiency (V_max/K_m of mutant enzyme ∼50-fold lower than for wild-type TP) and pronounced resistance to the inhibitory effect of 5′-O-tritylinosine. In contrast, the D203A mutant enzyme kept full sensitivity to the competitive inhibitors 6-aminothymine and 6-amino-5-bromouracil, which is in line with the kinetic properties of these inhibitors. Our findings reveal the existence of a previously unrecognized site in TP that can be targeted by small molecules to inhibit the catalytic activity of TP.     

Rapid isolation of high-affinity human antibodies against the tumor vascular marker Endosialin/TEM1, using a paired yeast-display/secretory scFv library platform

Endosialin/TEM1 is predominantly expressed on neovasculature, thus ideally suited for diagnostic, targeted imaging and therapy of cancer. To isolate TEM1-specific affinity reagents, we thought to screen a recombinant antibody (scFv) library derived from the repertoire of a patient with thrombotic thrombocytopenic purpura (TTP), as autoimmune disorders may produce self-reactive specificities. The yeast-display scFv library was constructed by homologous recombination of the TTP patient repertoire originally expressed on M13 bacteriophage in the novel vector pAGA2 for yeast-display expression. The TTP yeast-display library (10⁹ members) was screened by magnetic and flow sorting with human TEM1 recombinant protein. A pool of yeast-display scFv able to detect 2 nM of TEM1 was obtained and transformed into yeast-secreted scFv by homologous recombination using the novel p416 BCCP vector for yeast secretion of biotinylated scFv. Anti-TEM1 yeast-secreted scFv were independently validated in vitro by flow cytometry analysis and ELISA assays, then in vivo biotinylated in N-termini to produce biobodies. Biobody-78 bound specifically to Endosialin/TEM1-expressing ovarian tumor in vivo, with functional stability over 48 h. Our results suggest that our novel paired display-secretory yeast libraries can serve as an ideal platform for the rapid isolation of high-affinity reagents, and that anti-TEM1 biobody-78 can be used for in vitro assays including flow cytometry analysis, as well as in vivo for targeted imaging and therapy of cancer.     

Combinational chelation therapy abrogates lead-induced neurodegeneration in rats

Lead, a ubiquitous and potent neurotoxicant causes oxidative stress which leads to numerous neurobehavioral and physiological alterations. The ability of lead to bind sulfhydryl groups or compete with calcium could be one of the reasons for its debilitating effects. In the present study, we addressed: i) if chelation therapy could circumvent the altered oxidative stress and prevent neuronal apoptosis in chronic lead-intoxicated rats, ii) whether chelation therapy could reverse biochemical and behavioral changes, and iii) if mono or combinational therapy with captopril (an antioxidant) and thiol chelating agents (DMSA/MiADMSA) is more effective than individual thiol chelator in lead-exposed rats. Results indicated that lead caused a significant increase in reactive oxygen species, nitric oxide, and intracellular free calcium levels along with altered behavioral abnormalities in locomotor activity, exploratory behavior, learning, and memory that were supported by changes in neurotransmitter levels. A fall in membrane potential, release of cytochrome c, and DNA damage indicated mitochondrial-dependent apoptosis. Most of these alterations showed significant recovery following combined therapy with captopril with MiADMSA and to a smaller extend with captopril + DMSA over monotherapy with these chelators. It could be concluded from our present results that co-administration of a potent antioxidant (like captopril) might be a better treatment protocol than monotherapy to counter lead-induced oxidative stress. The major highlight of the work is an interesting experimental evidence of the efficacy of combinational therapy using an antioxidant with a thiol chelator in reversing neurological dystrophy caused due to chronic lead exposure in rats.     

GST pulldown技术检测HEK293T细胞Daam1的活性

目的:运用GST pulldown技术建立真核细胞中Daam1活性检测的可靠方法?方法:构建GST-RhoA的原核表达载体,并使其在大肠杆菌BL21菌株中大量表达?用GST pulldown和Western blot技术分别检测HEK293T细胞中活化的Daam1及Daam1蛋白的表达?结果:在成功构建了GST-RhoA的原核表达载体,并使其在原核细胞中大量表达融合蛋白GST-RhoA后,用GST pulldown和Western blot技术证实HEK293T细胞中有活化的Daam1和Daam1总蛋白的表达?结论:本工作所建立的GST pulldown技术可以检测HEK293T细胞中Daam1的活性,从而为进一步深入研究Daam1在真核细胞中的功能提供了技术保障?     

Oxidative stress and glutathione S-transferase genetic polymorphisms in medical staff professionally exposed to ionizing radiation

Purpose: Ionizing radiation (IR) is considered as a diagnostic and therapeutic tool in medicine. However, chronic occupational exposure of medical staff to IR may affect the antioxidant status and, as a result, DNA damage and cancers as well. The objective of our study was to evaluate the oxidative stress profile caused by IR in 29 Tunisian medical staff from radiology and radiotherapy departments, and to find an association between the GSTM1 null, GSTT1 null, and GSTP1 Ile105Val polymorphisms and oxidative stress biomarkers.

Materials and methods: The oxidant biomarkers malondialdehyde (MDA) and advanced oxidation protein product (AOPP) and the activities of the antioxidant superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) enzymes were spectrophotometrically determined in erythrocytes hemolysates. The analysis of GSTT1 null, GSTM1 null, and GSTP1 Ile105Val polymorphisms was determined for each participant using PCR methods.

Results: A significant increase of white blood cell (WBC) numbers (p?<?.05) and a significant decrease by 11% of hemoglobin (Hb) (p?<?.01) were noted in the exposed subjects in our study. Moreover, we report a significant increase of MDA level and the activities of SOD and CAT enzymes of the IR-exposed group compared to controls (p?<?.001). Interestingly, a close association was noted between the genotypes GSTP1 low active, GSTT1 null, GSTM1 null, and both GSTT1/GSTM1 null and oxidative stress biomarkers, especially with MDA level, SOD, and CAT activities.

Conclusions: Our findings indicate that the medical staff exposed to low IR levels were under risk of significant oxidative stress that was enhanced by their glutathione S-transferase (GST) polymorphisms.     

Alpha glutathione S-transferase: a potential marker of ischemia-reperfusion injury of the intestine after cardiac surgery?

Background

The aim of the study was to assess the utility of α glutathione S-transferase (αGST) as a potential marker of intestinal ischemia-reperfusion injury in children after cardiac surgery.

Methods

Twenty-six patients undergoing cardiac surgery were enrolled in this longitudinal experimental study. Blood samples were drawn for analysis at specified time points during surgery and analyzed for αGST levels. Clinical indices of splanchnic morbidity were assessed up to discharge from hospital. Results were analyzed using Mann-Whitney tests and linear mixed effects models.

Results

Two groups were identified. Group 1 (n = 16) showed no intestinal morbidity and group 2 (n = 10) had signs of intestinal morbidity. Statistical differences were shown between the 2 groups with respect to time with aortic cross-clamp (ACC) in situ, time on cardiac bypass, duration of operation, time to enteral feeding and full feeding, time on mechanical ventilation, and time in the intensive care unit postoperatively. The serum concentration of αGST was significantly higher for group 2 and this rise was greatest after removal of the ACC.

Conclusions

αGST showed significant elevation in patients with prolonged bypass times and ACC times. These patients also displayed signs of intestinal morbidity, suggesting that this marker may be useful in screening patients at risk for intestinal pathology. This rise in αGST was associated with a prolonged ischemia time, and was greatest after the cross-clamp was released, suggesting that it is a postischemic reperfusion phenomenon leading to its elevation. A low αGST level appears to exclude significant intestinal ischemia.