Effect of two pyrimidine analogs on accumulation of tubulin in NHIK 3025 cells

Summary Accumulation of tubulin as compared with the accumulation of total cellular protein in human NHIK 3025 cells treated with the sulfone 2-(2-thenyl)sulfonyl-5-bromopyrimidine (NY 4137) and the sulfoxide 2-(2thenyl)sulfinyl-5-bromopyrimidine (NY 4138), two mitotic inhibitors, were investigated by two-parametric flow cytometry. Following a 4 h treatment with NY 4137 tubulin accumulation is inhibited while total protein continues to accumulate. After treatment for 4h with NY 4138 the accumulation of total protein is approximately constant, while the accumulation of tubulin is reduced although not to the same degree as that found for NY 4137-treated cells. In addition, the percentage tubulin SH-groups (6.89 ± 0.14) remaining after treatment of purified rat brain tubulin with NY 4137 or NY 4138 was determined. Treatment with 0.0125 mM NY 4137 reduced the number of tubulin SH-groups detectable with dithiobis benzoate or from 6.89 ± 0.14 before treatment to about 4 after treatment. However, practically all SH-groups of tubulin remain detectable following treatment with the same concentration of NY 4138. From the results described in this report we infer that NY 4137 binds to tubulin SH-groups and that inhibition of tubulin accumulation follows as a secondary effect.     

Stage-related chondrogenic potential of avian mandibular ectomesenchymal cells

We have examined the in vitro stage-related chondrogenic potential of avian mandibular ectomesenchymal cells using micromass cultures. Our results indicate that mandibular ectomesenchymal cells as early as stage 16, soon after the formation of the mandibular arches and well before the initiation of in vivo chondrogenesis, have chondrogenic potential which is expressed in micromass culture. There is an increase in the total area of the cultures occupied by cartilage when cells from increasing stages of development are used. The nodular pattern of chondrogenesis in these cultures indicates that mandibular ectomesenchymal cells are a heterogenous population from the time of mandibular arch formation. In addition, we studied the temporal expression of the genes for extracellular matrix proteins during in vitro chondrogenesis and correlated the morphological changes with the pattern of gene expression. Low levels of type II collagen mRNA are present in the cultures prior to detection of any stainable cartilage matrix and increase 5 fold just before the onset of chondrogenesis in vitro. On the other hand mRNA for cartilage proteoglycan core protein was not detected until the second day of culture when stainable cartilage matrix was present and progressively increased thereafter. Messenger RNA for type I collagen was present at the time of initiation of cultures and continuously increased during the culture period. Our experiments also indicated that embryonic epithelia can inhibit the in vitro chondrogenesis of mandibular ectomesenchymal cells and that the inhibitory effect of embryonic epithelia is independent of its age and site of origin.     

Network Analysis of Breast Cancer Progression and Reversal Using a Tree-Evolving Network Algorithm

The HMT3522 progression series of human breast cells have been used to discover how tissue architecture, microenvironment and signaling molecules affect breast cell growth and behaviors. However, much remains to be elucidated about malignant and phenotypic reversion behaviors of the HMT3522-T4-2 cells of this series. We employed a “pan-cell-state” strategy, and analyzed jointly microarray profiles obtained from different state-specific cell populations from this progression and reversion model of the breast cells using a tree-lineage multi-network inference algorithm, Treegl. We found that different breast cell states contain distinct gene networks. The network specific to non-malignant HMT3522-S1 cells is dominated by genes involved in normal processes, whereas the T4-2-specific network is enriched with cancer-related genes. The networks specific to various conditions of the reverted T4-2 cells are enriched with pathways suggestive of compensatory effects, consistent with clinical data showing patient resistance to anticancer drugs. We validated the findings using an external dataset, and showed that aberrant expression values of certain hubs in the identified networks are associated with poor clinical outcomes. Thus, analysis of various reversion conditions (including non-reverted) of HMT3522 cells using Treegl can be a good model system to study drug effects on breast cancer.     

TSE clearance during plasma products separation process by Gradiflow(TM).

BACKGROUND AND OBJECTIVES: Recent experimental evidence from rodent models suggests a potential risk for transmissible spongiform encephalopathy (TSE) transmission by blood. The emergence of a new variant Creutzfeldt-Jakob disease (vCJD) has raised increased concerns about the safety of blood components and plasma products derived from vCJD-infected donors. Recent risk-minimisation strategies have included a ban on the use of UK-sourced plasma for the preparation of licensed blood products and leukodepletion of blood donations for fear of possible transmission of the human TSE via blood or blood components. The aim of this study was to investigate the capability and efficacy of a preparative electrophoresis system (Gradiflow) in the removal of TSE contaminants during the separation of plasma products. MATERIALS AND METHODS: Using hamster adapted scrapie 263 K as a model for TSE agent, albumin and IgG separation from human plasma by Gradiflow were performed separately by spiking a 263 K scrapie microsomal fraction to the feed material at each process step. Samples from pre- and post-Gradiflow separation process were titrated to the end-point for the detection of the disease-associated, proteinase K resistant form of the pathogenic prion protein (PrP(Sc)) by Western blot. RESULTS: Under all conditions tested, a greater than 3 log(10) reduction was achieved with no PrP(Sc) detected in any of the pooled products for either of the IgG or albumin separations. These data show that Gradiflow processing has clear advantages for concurrent purification of plasma products and in-process TSE removal. CONCLUSIONS: Our findings suggest that Gradiflow process is a viable alternative to remove causative TSE agents during plasma products separation, potentially eliminating the risk of TSE agents transmission.     

Effects of sodium butyrate on oxidative stress and behavioral changes induced by administration of d-AMPH

Several evidences have demonstrated that oxidative stress has a central role in bipolar disorder (BD). Recently, studies have been suggested histone deacetylases (HDAC) as a possible target for new medications in treatment of mood disorders. In this study, we investigated the effects of sodium butyrate (SB, a histone deacetilase inhibitor) on oxidative stress in rats submitted to an animal model of mania induced by d-amphetamine (d-AMPH). Wistar rats were first given d-AMPH or saline (Sal) for 14 days, and then, between days 8 and 14, rats were treated with SB or Sal. Locomotor activity and risk-taking behavior were assessed by open-field test and oxidative stress was measured in prefrontal cortex, amygdala, hippocampus and striatum. The results showed that SB reversed and prevented d-AMPH-induced behavioral effects. The d-AMPH administration induced oxidative damage in all brain structures analyzed. Depending on the cerebral area and technique, SB was able to reverse this impairment. The present study reinforces the need for more studies of HDAC inhibitors as possible target for new medications in treatment for BD.