2,3-Dihydroxy-quinoxaline induces ATPase activity of Herpes Simplex Virus thymidine kinase

2,3-Dihydroxy-quinoxaline, a small molecule that promotes ATPase catalytic activity of Herpes Simplex Virus thymidine kinase (HSV-TK), was identified by virtual screening. This compound competitively inhibited HSV-TK catalyzed phosphorylation of acyclovir with K_i = 250 μM (95% CI: 106–405 μM) and dose-dependently increased the rate of the ATP hydrolysis with K_M = 112 μM (95% CI: 28–195 μM). The kinetic scheme consistent with this experimental data is proposed.     

Enzymatic activities of uridine and thymidine phosphorylase in normal and cancerous uterine cervical tissues

In this study, the preliminary analyses were conducted of enzymatic activities of uridine phosphorylase (UP) and thymidine phosphorylase (TP) in normal tissues and cancer tissues of the uterine cervix. The study was performed on 27 patients of cervical cancer, treated first in our hospital. Normal cervical tissues obtained from 15 patients undergoing hysterectomy for benign diseases were used as controls. The supernatant of the homogenated cervical tissues and the stroma (5-FU and ribose-1-P or deoxyribose-1-P) were analyzed by high performance liquid chromatography, and then the UP and TP activities calculated. TP activity was significantly greater than UP activity (P < 0.0001). Both UP and TP showed significantly greater activity in cancer tissues than in normal tissues (P < 0.0001). In the TP activity of the cancer tissues, there was no significant difference among the histological types, while the TP activity tended to be significantly higher in the cases with lymph node metastasis. These results showed that the TP-mediated route seemed important as the 5FU metabolic pathway in the uterine cervical tissues, and TP enzymatic activity might be associated with lymph node metastasis.     

Concentration of human thymidine kinase 1 discover invisible malignant human tumours

Early discover of risk progression of invisible carcinomas is important for a prerequisite successful treatment. Here, we investigated whether concentration of human thymidine kinase 1 (HTK1) discover invisible malignant human tumours. The HTK1 concentration of tumour cellular based on HTK1 IgY-polyclonal-antibody (HTK1-IgY-pAb) was determined by using a novel automatic chemiluminescence analyser with sandwich biotin-streptavidin (SBSA) platform. Minimum number of cells able to be detected by this technology used cells with low and high concentration of HTK1. The limit visibility by tumour imaging is approximately 1 mm in diameter, corresponding to approximately 10⁹ cells with a cell diameter of 1 µm. Based on a HTK1 standard curve and a molecular weight of HTK1 of 96 kD, the HTK1protein (HTK1p) concentration per cell was calculated to be 0.021 pg. Assuming 200 pg in total protein/cell, approximately 50 × 10⁶ growing malignant cells in the body were calculated to releases HTK1 into 5-liter blood. A HTK1 values of 3.914, 0.435 and 0.009 pmol/L corresponds to 10 × 10⁵, 2 × 10⁵ and 1 × 10⁵ growing malignant cells, respectively. The lowest detectable sensitivity of HTK1 is 0.009 pmol/L in 1 × 10⁵ growing malignant cells and 0.01 pmol/L in blood serum, detectable in health screening. Comparing the novel automatic chemiluminescence analyser with the original ECL dot-blot assay using serum HTK1p (health screening, n = 265) showed high correlation (r = 0.8743, P < .000). In conclusion, the novel automatic chemiluminescence analyser with SBSA platform is a reliable method with high accuracy to determine carcinoma invisible.     

Insulin-like growth factor-1 increases endothelin receptor A levels and action in cultured rat aortic smooth muscle cells

Insulin is known to cause an increase in endothelin-1 (ET-1) receptors in vascular smooth muscle cells (SMCs), but the effect of insulin-like growth factor 1 (IGF-1) on ET-1 receptor expression is not known. We therefore carried out the present study to determine the effect of IGF-1 on the binding of ET-1 to, and ET type A receptor (ETAR) expression and ET-1-induced 3H-thymidine incorporation in, vascular SMCs. In serum-free medium, IGF-1 treatment increased the binding of 125I-ET-1 to SMC cell surface ET receptors from a specific binding of 20.1%+/-3.1% per mg of protein in control cells to 45.1%+/-8.6% per mg of protein in cells treated with IGF-1 (10 nM). The effect of IGF-1 was dose-related, with a significant effect (1.4-fold) being seen at 1 nM. The minimal time for IGF-1 treatment to be effective was 30 min and the maximal effect was reached at 6 h. Immunoblotting analysis showed that ETAR expression in IGF-1-treated cells was increased by 1.7-fold compared to controls. Levels of ETAR mRNA measured by the RT-PCR method and Northern blotting were also increased by 2-fold in IGF-1-treated SMCs. These effects of IGF-1 were abolished by cycloheximide or genistein. Finally, ET-1-stimulated thymidine uptake and cell proliferation were enhanced by IGF-1 treatment, with a maximal increase of 3.2-fold compared to controls. In conclusion, in vascular SMCs, IGF-1 increases the expression of the ET-1 receptor in a dose- and time-related manner. This effect is associated with increased thymidine uptake and involves tyrosine kinase activation and new protein synthesis. These findings support the role of IGF-1 in the development of atherosclerotic, hypertensive, and diabetic vascular complications.     

Variation in cell-substratum adhesion in relation to cell cycle phases

The quantification of focal adhesion sites offers an assessable method of measuring cell-substrate adhesion. Such measurement can be hindered by intra-sample variation that may be cell cycle derived. A combination of autoradiography and immunolabelling techniques, for scanning electron microscopy (SEM), were utilised simultaneously to identify both S-phase cells and their focal adhesion sites. Electron-energy 'sectioning' of the sample, by varying the accelerating voltage of the electron beam, combined with backscattered electron (BSE) imaging, allowed for S-phase cell identification in one energy 'plane' image and quantitation of immunogold label in another. As a result, it was possible simultaneously to identify S-phase cells and their immunogold-labelled focal adhesions sites on the same cell. The focal adhesion densities were calculated both for identified S-phase cells and the remaining non-S-phase cells present. The results indicated that the cell cycle phase was a significant factor in determining the density of focal adhesions, with non-S-phase cells showing a larger adhesion density than S-phase cells. Focal adhesion morphology was also seen to correspond to cell cycle phase; with 'dot' adhesions being more prevalent on smaller non-S-phase and the mature 'dash' type on larger S-phase cells. This study demonstrated that when quantitation of focal adhesion sites is required, it is necessary to consider the influence of cell cycle phases on any data collected.     

Use of 4-Thiouridine and 4-Thiothymidine in Studies on Pyrimidine Nucleoside Phosphorylases

The new substrates 4-thiouridine and 4-thiothymidine were proposed for spectrophotometric measurement of the activity of uridine (UP) and thymidine (TP) phosphorylases. At pH 7.5, 4-thiouridine has an absorbance maximum at 330 nm, and the difference in extinction coefficient () between 4-thiouridine and 4-thiouracil is 3000 ^–1cm^–1. 4-Thiouridine proved to be a good substrate for UP: the Michaelis ( ) and catalytic (k _cat) constants were estimated respectively at 130 M and 49 s^–1 at 25°C. Even a greater (5000 M^–1cm^–1 at 336 nm) was observed for the 4-thiothymidine/4-thiothymine pair.     

In vivo tracing of canonical Wnt signaling in Xenopus tadpoles by means of an inducible transgenic reporter tool

The canonical Wnt pathway is recurrently used during embryogenesis and adult life. To track the cellular output of Wnt signaling in a living organism, we designed a hormone-inducible Wnt responsive system, capable to dynamically and specifically report Wnt pathway activities through eGFP expression. In contrast to previous in vivo reporters, our system essentially avoids interference of consecutive signals by remaining dormant until addition of hormone, which makes it a valuable tool to map canonical Wnt signaling in post-embryonic stages. Transgenic Xenopus laevis embryos were analyzed revealing at tadpole stage in specific tissues and organs cell populations with high Wnt pathway activity.