Kinetic Analysis of Prodrug Activation and ATP/UTP Substrate Preference of Nine Human Deoxycytidine Kinase Mutants

Deoxynucleoside analogues are prodrugs that canfunction as inhibitors of both viral and cellular DNA replication processes. They are important in anti-cancer therapy because they hinder DNA synthesis and cellular mitosis. Within the cell, deoxyribonucleotides are synthesized using the salvage pathways by converting the unphosphorylated nucleosides to their mono, di- and tri-phosphate forms using a phosphoryl donor: ATP or UTP. Human deoxycytidine kinase (dCK) is the first and rate-limiting enzyme in this process. The dCK protein uses nucleotide triphosphates to phosphorylate several clinically important nucleoside analogue prodrugs in addition to its natural substrates. The preferred physiological phosphoryl donor for dCK is UTP although it is less prevalent in the human body than ATP. Our objective is to improve the understanding of the phosphate-donor binding loop of dCK by kinetic analysis of a series of mutants of Asp241 and Phe242. These mutants were designed in an attempt to improve the activity of dCK with phosphate donors. Results show several mutants with improved kinetics and some with an ATP donor preference over UTP

Method For Obtaining Wood/Bark Adhesion Measurements on Small Samples

Using the Instron Tester, a procedure has been developed for measuring wood/bark adhesion on small samples. Using very small, specially prepared samples, wood/bark adhesion measurements were made on twenty-four hardwoods and eighteen conifers. High dormant season wood/bark adhesion in hardwoods was found to be associated with the presence of fibers in the inner bark. The procedure appears to be useful in identifying difficult-to-debark species and evaluating methods of reducing adhesion

Morphological and Bark Strength Characteristics Important to Wood/Bark Adhesion in Hardwoods

Dormant season wood/bark adhesion was determined for twenty-four hardwood species using a previously described Instron testing machine method. Wood/bark adhesion was compared, using simple and multiple correlation techniques, with bark specific gravity, inner bark strength, bark toughness, wood specific gravity, wood toughness, percent bark fibers, and percent sclereids. Wood/bark adhesion was found to be positively correlated with percent bark fibers, bark toughness, and inner bark strength. Wood/bark adhesion was negatively correlated with percent sclereids in the bark. Morphologically, it appeared that the presence of fibers increased inner bark strength, sclereids decreased inner bark strength, and inner bark strength had a major influence on wood/bark adhesion. A multiple correlation comparison employing wood toughness and inner bark strength accounted for 69% of the wood/bark adhesion encountered

Circular Permutation in Proteins

This is a ‘‘Topic Page’ ’ article for PLoS Computational Biology. Circular permutation describes a type of relationship between proteins, whereby the proteins have a changed order of amino acids in their protein sequence, such that the sequence of the first portion of one protein (adjacent to the N-terminus) is related to that of the second portion of the other protein (near its C-terminus), and vice versa (see Figure 1). This is directly analogous to the mathematical notion of a cyclic permutation over the set of residues in a protein. Circular permutation can be the result of evolutionary events, post-translational modifications, or artificially engineered mutations. The result is a protein structure with different connectivity, but overall similar three-dimensional (3D) shape. The homology between portions of the proteins can be established by observing similar sequences between N- and C-terminal portions of the tw

Alpha-santalol, a chemopreventive agent against skin cancer, causes G2/M cell cycle arrest in both p53-mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells

<p>Abstract</p> <p>Background</p> <p>α-Santalol, an active component of sandalwood oil, has shown chemopreventive effects on skin cancer in different murine models. However, effects of α-santalol on cell cycle have not been studied. Thus, the objective of this study was to investigate effects of α-santalol on cell cycle progression in both p53 mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells to elucidate the mechanism(s) of action.</p> <p>Methods</p> <p>MTT assay was used to determine cell viability in A431 cells and UACC-62; fluorescence-activated cell sorting (FACS) analysis of propidium iodide staining was used for determining cell cycle distribution in A431 cells and UACC-62 cells; immunoblotting was used for determining the expression of various proteins and protein complexes involved in the cell cycle progression; siRNA were used to knockdown of p21 or p53 in A431 and UACC-62 cells and immunofluorescence microscopy was used to investigate microtubules in UACC-62 cells.</p> <p>Results</p> <p>α-Santalol at 50-100 μM decreased cell viability from 24 h treatment and α-santalol at 50 μM-75 μM induced G₂/M phase cell cycle arrest from 6 h treatment in both A431 and UACC-62 cells. α-Santalol altered expressions of cell cycle proteins such as cyclin A, cyclin B1, Cdc2, Cdc25c, p-Cdc25c and Cdk2. All of these proteins are critical for G₂/M transition. α-Santalol treatment up-regulated the expression of p21 and suppressed expressions of mutated p53 in A431 cells; whereas, α-santalol treatment increased expressions of wild-type p53 in UACC-62 cells. Knockdown of p21 in A431 cells, knockdown of p21 and p53 in UACC-62 cells did not affect cell cycle arrest caused by α-santalol. Furthermore, α-santalol caused depolymerization of microtubules similar to vinblastine in UACC-62 cells.</p> <p>Conclusions</p> <p>This study for the first time identifies effects of α-santalol in G₂/M phase arrest and describes detailed mechanisms of G₂/M phase arrest by this agent, which might be contributing to its overall cancer preventive efficacy in various mouse skin cancer models.</p

Genetic and epigenetic alterations of Ras signalling pathway in colorectal neoplasia: analysis based on tumour clinicopathological features

Activation of RAS signalling induced by K-ras/BRAF mutations is a hallmark of colorectal tumours. In addition, Ras association domain families 1 and 2 (RASSF1 and RASSF2), the negative regulators of K-ras, are often inactivated by methylation of the promoter region in those tumours. However, reports showing differences in the occurrence of these alterations on the basis of tumour characteristics have been scarce. We analysed K-ras/BRAF mutations and the methylation status of RASSF1 and RASSF2 promoter regions in 120 colorectal adenomas with respect to their clinicopathological features. K-ras/BRAF mutations and RASSF2 methylation were observed in 49 (41%) and 30 (25%) of the samples, respectively, while RASSF1 methylation was observed in only 3 (2.5%). Adenomas with RASSF2 methylation often carried K-ras/BRAF mutations simultaneously (22 out of 30, P<0.01). Multivariate analysis revealed that the concomitance of these alterations was frequently observed in serrated adenomas (odds ratio (OR) 11.11; 95% confidence interval (CI) 1.96–63.00), but rarely in adenomas located in the sigmoid or descending colon (OR 0.13; 95% CI 0.03–0.58). A comparison between adenomas and cancers showed a significantly higher prevalence of these alterations in cancers than in adenomas in the proximal colon (58 vs 27%, P=0.02). Frequency and the time point of the occurrence of Ras signalling disorders differ according to colorectal neoplasia's characteristics, particularly the location

Effects of magnolol on UVB-induced skin cancer development in mice and its possible mechanism of action

Background Magnolol, a plant lignan isolated from the bark and seed cones of Magnolia officinalis, has been shown to have chemopreventive effects on chemically-induced skin cancer development. The objectives of this investigation are to study the anticarcinogenic effects of magnolol on UVB-induced skin tumor development in SKH-1 mice, a model relevant to humans, and determine the possible role of apoptosis and cell cycle arrest involved in the skin tumor development. Methods UVB-induced skin carcinogenesis model in SKH-1 mice was used for determining the preventive effects of magnolol on skin cancer development. Western blottings and flow cytometric analysis were used to study the effects of magnolol on apoptosis and cell cycle. Results Magnolol pretreated groups (30, 60 μ g) before UVB treatments (30 mJ/cm2, 5 days/week) resulted in 27-55% reduction in tumor multiplicity as compared to control group in SKH-1 mice. Magnolol pretreatment increased the cleavage of caspase-8 and poly-(-ADP-ribose) polymerase (PARP), increased the expression of p21, a cell cycle inhibitor, and decreased the expression of proteins involved in the G2/M phase of cell cycle in skin samples from SKH-1 mice. Treatment of A431 cells with magnolol decreased cell viability and cell proliferation in a concentration dependent manner. Magnolol induced G2/M phase cell cycle arrest in A431 cells at 12 h with a decreased expression of cell cycle proteins such as cyclin B1, cyclin A, CDK4, Cdc2 and simultaneous increase in the expression of Cip/p21, a cyclin-dependent kinase inhibitor. Magnolol induced apoptosis in vivo and in vitro with an increased cleavage of caspase-8 and PARP. Phospho-signal transducers and activators of transcription 3 (Tyr705), B-Raf, p-MEK, and p-AKT were down-regulated, whereas phosphorylation of ERK was induced by magnolol in A431 cells. Conclusions Magnolol pretreatments prevent UVB-induced skin cancer development by enhancing apoptosis, causing cell cycle arrest at G2/M phase, and affecting various signaling pathways. Magnolol could be a potentially safe and potent anticarcinogenic agent against skin cancer