Platelets do not express the oxidized or reduced forms of tissue factor

Background

Expression of tissue factor (TF) antigen and activity in platelets is controversial and dependent upon the laboratory and reagents used. Two forms of TF were described: an oxidized functional form and a reduced nonfunctional form that is converted to the active form through the formation of an allosteric disulfide. This study tests the hypothesis that the discrepancies regarding platelet TF expression are due to differential expression of the two forms.

Methods

Specific reagents that recognize both oxidized and reduced TF were used in flow cytometry of unactivated and activated platelets and western blotting of whole platelet lysates. TF-dependent activity measurements were used to confirm the results.

Results

Western blotting analyses of placental TF demonstrated that, in contrast to anti-TF#5, which is directed against the oxidized form of TF, a sheep anti-human TF polyclonal antibody recognizes both the reduced and oxidized forms. Flow cytometric analyses demonstrated that the sheep antibody did not react with the surface of unactivated platelets or platelets activated with thrombin receptor agonist peptide, PAR-1. This observation was confirmed using biotinylated active site-blocked factor (F)VIIa: no binding was observed. Likewise, neither form of TF was detected by western blotting of whole platelet lysates with sheep anti-hTF. Consistent with these observations, no FXa or FIXa generation by FVIIa was detected at the surface of these platelets. Similarly, no TF-related activity was observed in whole blood using thromboelastography.

Conclusion and significance

Platelets from healthy donors do not express either oxidized (functional) or reduced (nonfunctional) forms of TF.     

Weak antioxidant defenses make the heart a target for damage in copper-deficient rats

Copper deficiency causes more salient pathologic changes in the heart than in the liver of rats. Although oxidative stress has been implicated in copper deficiency-induced pathogenesis, little is known about the selective toxicity to the heart. Therefore, we examined the relationship between the severity of copper deficiency-induced oxidative damage and the capacity of antioxidant defense in heart and liver to investigate a possible mechanism for the selective cardiotoxicity. Weanling rats were fed a purified diet deficient in copper (0.4 μg/g diet) or one containing adequate copper (6.0 μg/g diet) for 4 weeks. Copper deficiency induced a 2-fold increase in lipid peroxidation in the heart (thiobarbituric assay) but did not alter peroxidation in the liver. The antioxidant enzymatic activities of superoxide dismutase, catalase, and glutathione peroxidase were, respectively, 3-, 50- and 1.5-fold lower in the heart than in the liver, although these enzymatic activities were depressed in both organs by copper deficiency. In addition, the activity of glutathione reductase was 4 times lower in the heart than in the liver. The data suggest that a weak antioxidant defense system in the heart is responsible for the relatively high degree of oxidative damage in copper-deficient hearts.     

Effect of culture conditions on manganese peroxidase production and activity by some white rot fungi

The ligninolytic system of white rot fungi is primarily composed of lignin peroxidase, manganese peroxidase (MnP) and laccase. The present work was carried out to determine the best culture conditions for production of MnP and its activity in the relatively little-explored cultures of Dichomitus squalens, Irpex flavus and Polyporus sanguineus, as compared with conditions for Phanerochaete chrysosporium and Coriolus versicolor. Studies on enzyme production under different nutritional conditions revealed veratryl alcohol, guaiacol, Reax 80 and Polyfon H to be excellent MnP inducers. Electronic Publication     

秦艽体细胞胚不同发育阶段几种同工酶的特性

以秦艽（ＧｅｎｔｉａｎａｃｒａｓｓｉｃａｕｌｉｓＤｕｔｈｉｅｅｘＢｕｒｋｉｌｌ）无菌苗幼嫩茎叶为外植体,在ＭＳ培养基上诱导出胚性愈伤组织及体细胞胚。本文比较了秦荒体细胞胚发育过程中几种酶的活性及同工酶变化。过氧化物酶与细胞色素氧化酶均在球形胚阶段出现一活性高峰;酯酶同工酶在体细胞胚发育过程中伴有特征带Ｅ＿２、Ｅ＿６的出现;酸性磷酸酯酶的活性与体细胞胚发育程度呈正相关。所以认为只要综合运用这几种同工酶的实验数据,就可以灵敏检测体细胞胚的发育进程。     

The effects of acute ethanol exposure and ageing on rat brain glutathione metabolism

Abstract

Binge alcohol consumption in adolescents is increasing, and it has been proposed that immature brain deals poorly with oxidative stress. The aim of our work was to study the effect of an acute dose of ethanol on glutathione (GSH) metabolism in frontal cortex, hippocampus and striatum of juvenile and adult rats. We have observed no change in levels of glutathione produced by acute alcohol in the three brain areas studied of juvenile and adult rats. Only in the frontal cortex the ratio of GSH/GSSG was increased in the ethanol-treated adult rats. GSH levels in the hippocampus and striatum were significantly higher in adult animals compared to young ones. Higher glutathione peroxidase (GPx) activity in adult rats was observed in frontal cortex and in striatum. Our data show an increased GSH concentration and GPx activity in different cerebral regions of the adult rat, compared to the young ones, suggesting that age-related variations of total antioxidant defences in brain may predispose young brain structures to ethanol-induced, oxidative stress-mediated tissue damage.     

Thermal Stability and Activity Regain of Horseradish Peroxidase in Aqueous Mixtures of Imidazolium-Based Ionic Liquids

Thermal deactivation kinetics of horseradish peroxidase (HRP) were studied from 45 to 90 °C in phosphate buffer and 5–25% (v,w/v) 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF₄] and 1-butyl-3-methylimidazolium chloride [BMIM][Cl]. HRP activity at 25 °C was not affected by the presence of ionic liquids up to 20% (v,w/v). Increasing the ionic liquids concentration up to 25% (v,w/v) changed the biphasic character of deactivation kinetics to an apparent single first-order step. The presence of 5–10% (v/v) [BMIM][BF₄] significantly improved HRP thermal stability with lower activation energies for the deactivation second phase (83–87 kJ mol⁻¹). After deactivation, enhanced activity regain of the enzyme, up to 70–80% of the initial activity, was found in 25% (v/v) [BMIM][BF₄] and 10% (w/v) [BMIM][Cl] and correlated to prevalence of the deactivation first phase.     

Liposome Vector Containing Biosurfactant-Complexed DNA as Herpes Simplex Virus Thymidine Kinase Gene Delivery System

For injectable-sized liposome complexed with DNA (lipoplexes) with high transfection efficiency of genes, we initially prepared small-sized liposomes by addition of biosurfactant. For selectivity of gene expression, the thymidine kinase (MK-tk) gene controlled by midkine was used for herpes simplex virus thymidine kinase (HSV-tk) gene therapy. Liposomes composed of 3([N-(N′,N′–dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), L-dioleoylphosphatidylethanolamine (DOPE), and a biosurfactant, such as β-sitosterol β-D-glucoside (Sit-G) for Sit-G-liposomes and mannosylerythrytol lipid A (MEL) for MEL-liposomes, produced about 300-nm-sized lipoplexes. Sit-G- and MEL-liposomes showed higher transfection efficiency of the luciferase marker gene and thymidine kinase activity in the presence of serum in the cells. The treatment with transfection of MK-tk gene by Sit-G-liposome and injection of ganciclovir significantly reduced tumor growth in a solid tumor model, compared with that by Sit-G-liposome alone. This finding suggested that Sit-G-liposome is a potential vector for HSV-tk gene therapy.     

Role of p53/FAK association and p53 phosphorylation in staurosporine-mediated apoptosis: Wild type versus mutant p53-R175H

A novel survival role of focal adhesion kinase (FAK) that involves its nuclear translocation and direct association with p53 has been demonstrated. Here we examined the relationship between the p53/FAK interaction and Ser46 phosphorylation of p53 (p-p53^Ser46) in the apoptotic regulation of human esophageal squamous cell carcinoma (HOSCC) cell lines, expressing either wild type (wt) p53 or mutant (mt) p53-R175H. In contrast to the wt p53 cell lines, the mt p53-R175H cell line was resistant to staurosporine (STS)-mediated detachment and caspase-3 activation. Furthermore, despite the resistance of mt p53-R175H to Ser46 phosphorylation, both wt and mt HOSCC cells translocate FAK into the nucleus and maintain the p53/FAK interaction post STS treatment. These findings provide unique insight into how tumor cells harboring the R175H mutant may resist chemotherapeutic intervention.

Structured summary

MINT-7294020: FAK (uniprotkb:Q05397) physically interacts (MI:0915) with p53 (uniprotkb:P04637) by anti-bait coimmunoprecipitation (MI:0006)     

Distribution of intraventricularly injected horseradish peroxidase in cerebrospinal fluid compartments of the rat spinal cord

Summary The circulation of the cerebrospinal fluid along the central canal and its access to the parenchyma of the spinal cord of the rat have been analyzed by injection of horseradish peroxidase (HRP) into the lateral ventricle. Peroxidase was found throughout the central canal 13 min after injection, suggesting a rapid circulation of cerebrospinal fluid along the central canal of the rat spinal cord. It was cleared from the central canal within 2 h, in contrast with the situation in the brain tissue, where it remained in the periventricular areas for 4 h. In the central canal, HRP bound to Reissner's fiber and the luminal surface of the ependymal cells; it penetrated through the intercellular space of the ependymal lining, reached the subependymal neuropil, the basement membrane of local capillaries, and appeared in the lumen of endothelial pinocytotic vesicles. Furthermore, it accumulated in the labyrinths of the basement membrane contacting the basolateral aspect of the ependymal cells. In ependymocytes, HRP was found in single pinocytotic vesicles. The blood vessels supplying the spinal cord were classified into two types. Type-A vessels penetrated the spinal cord laterally and dorsally and displayed the tracer along their external wall as far as the gray matter. Type-B vessels intruded into the spinal cord from the medial ventral sulcus and occupied the anterior commissure of the gray matter, approaching the central canal. They represented the only vessels marked by HRP along their course through the gray matter. HRP spread from the wall of type-B vessels, labeling the labyrinths, the intercellular space of the ependymal lining, and the lumen of the central canal. This suggests a communication between the central canal and the outer cerebrospinal fluid space, at the level of the medial ventral sulcus, via the intercellular spaces, the perivascular basement membrane and its labyrinthine extensions.