Methods for Fractionation and Identification of Nonhistone Proteins of Rat Liver Chromatin

Combinations of methods were devised for fractionation and characterization of proteins obtained from rat liver chromatin. The chromosomal proteins were extracted from DNA by treatment with 3M NaCl, 7M urea, or by heating in 1% sodium dodecyl sulfate. The proteins dissociatdd by sodium dodecyl sulfate were fractionated by gel filtration in the presence of the detergent. A number of discrete components, each containing a population of polypeptides of limited heterogeneity with respect to molecular weight, were isolated by this method. Proteins obtained by urea-salt dissociation were initially subfractionated into histone and nonhistone components. The nonhistone proteins were examined by sodium dodecyl polyacrylamide gel electrophoresis and by analytical and preparative isoelectric focusing methods, in an effort to provide reproducible methods for identification of specific families of proteins

A CRAF/glutathione-S-transferase P1 complex sustains autocrine growth of cancers with KRAS and BRAF mutations

The Ras/RAF/MEK/ERK pathway is an essential signaling cascade for various refractory cancers, such as those with mutant KRAS (mKRAS) and BRAF (mBRAF). However, there are unsolved ambiguities underlying mechanisms for this growth signaling thereby creating therapeutic complications. This study shows that a vital component of the pathway CRAF is directly impacted by an end product of the cascade, glutathione transferases (GST) P1 (GSTP1), driving a previously unrecognized autocrine cycle that sustains proliferation of mKRAS and mBRAF cancer cells, independent of oncogenic stimuli. The CRAF interaction with GSTP1 occurs at its N-terminal regulatory domain, CR1 motif, resulting in its stabilization, enhanced dimerization, and augmented catalytic activity. Consistent with the autocrine cycle scheme, silencing GSTP1 brought about significant suppression of proliferation of mKRAS and mBRAF cells in vitro and suppressed tumorigenesis of the xenografted mKRAS tumor in vivo. GSTP1 knockout mice showed significantly impaired carcinogenesis of mKRAS colon cancer. Consequently, hindering the autocrine loop by targeting CRAF/GSTP1 interactions should provide innovative therapeutic modalities for these cancers

Expressions of glutathione S-transferase alpha, mu, and pi in brains of medically intractable epileptic patients

<p>Abstract</p> <p>Background</p> <p>Glutathione S-transferases (GSTs) play an important role in metabolizing anti-epileptic drugs (AEDs) in liver. Expressions of GSTs in brain, which may result in poor efficacy of AEDs, have not been well studied. Using clinical cortex specimen from 32 intractable epileptic subjects and 8 non-epileptic controls, the present study investigated the correlation between GSTs and intractable epilepsy.</p> <p>Results</p> <p>Three different GST isoforms (α, μ, and π) were detected with immunohistochemistry. GST-α expression was not seen in any cortex specimens. Sixty three percent (63%) of control and 53% of intractible epileptic specimens showed GST-μ immunoreactivity. No significant difference in intensity of GST-μ staining was observed between these two groups. GST-π expression was found in endothelial cells and glial cells/astrocytes. Fifty percent (50%) of the control patients and 66% of the epileptic patients were GST-π positive. The grading of epileptic patients was significantly higher than that of control patients (<it>p </it>< 0.01).</p> <p>Conclusion</p> <p>High levels of GST-π in endothelial cells and glial cells/astrocyte correlate to medical intractable epilepsy, suggesting that GST-π contributes to resistance to AED treatment.</p

Association between long-term neuro-toxicities in testicular cancer survivors and polymorphisms in glutathione-s-transferase-P1 and -M1, a retrospective cross sectional study

<p>Abstract</p> <p>Background</p> <p>To assess the impact of polymorphisms in Glutathione S-transferase (GST) -P1, -M1, and -T1 on self-reported chemotherapy-induced long-term toxicities in testicular cancer survivors (TCSs).</p> <p>Methods</p> <p>A total of 238 TCSs, who had received cisplatin-based chemotherapy at median twelve years earlier, had participated in a long-term follow-up survey which assessed the prevalence of self-reported paresthesias in fingers/toes, Raynaud-like phenomena in fingers/toes, tinnitus, and hearing impairment. From all TCSs lymphocyte-derived DNA was analyzed for the functional A→G polymorphism at bp 304 in <it>GSTP1</it>, and deletions in <it>GST-M1 </it>and <it>GST-T1</it>. Evaluation of associations between GST polymorphisms and self-reported toxicities included adjustment for prior treatment.</p> <p>Results</p> <p>All six evaluated toxicities were significantly associated with the cumulative dose of cisplatin and/or bleomycin. Compared to TCSs with either <it>GSTP1-AG </it>or <it>GSTP1</it>-<it>AA</it>, the 37 TCSs with the genotype <it>GSTP1-GG</it>, were significantly less bothered by paresthesias in fingers and toes (p = 0.039, OR 0.46 [0.22–0.96] and p = 0.023, OR 0.42 [0.20–0.88], respectively), and tinnitus (p = 0.008, OR 0.33 [0.14–0.74]). Furthermore, absence of functional GSTM1 protected against hearing impairment (p = 0.025, OR 1.81 [1.08–3.03]).</p> <p>Conclusion</p> <p>In TCSs long-term self-reported chemotherapy-induced toxicities are associated with functional polymorphisms in <it>GSTP1 </it>and <it>GSTM1</it>. Hypothetically, absence of GST-M1 leaves more glutathione as substrate for the co-expressed GST-P1. Also intracellular inactivation of pro-apoptotic mediators represents a possible explanation of our findings. Genotyping of these GSTs might be a welcomed step towards a more individualized treatment of patients with metastatic testicular cancer.</p

Glutathione S-transferases in the organ of Corti of the rat: Enzymatic activity, subunit composition and immunohistochemical localization

Glutathione S-transferases (GSTs), a family of ubiquitous cytosolic isozymes, catalyze the detoxification of electrophilic substrates with reduced glutathione and participate in intracellular binding and transport of lipophilic substances. This study measured GST activity biochemically in the inner ear of the rat; determined the isozyme profile by Western blotting; and identified, immunohistochemically. the distribution of the [mu] and [pi] class GSTs in the organ of Corti. GST enzymatic activity in inner ear tissues ranged from 117 to 348 nmoles glutathione converted/min/mg protein, values somewhat higher than those found in brain (130) and much lower than in liver (1011). Of the GST isoforms, the [pi] class (identified by antibodies against the Yp subunit) was most prominent, the [mu] class (Yb1 subunit) clearly evident while the [alpha] class (Ya subunit) was barely detectable on Western blots. Immunocytochemical analysis showed differential distribution of the Yh1 and Yp subunits. The Yb1 subunit was present in the sensory cells, while supporting cells were not specifically stained. At the subcellular level, the isozyme was localized in the apical zones of inner (IHCs) and outer hair cells (OHCs) close to the cuticular plate. The extent of staining, however, varied between OHCs and IHCs. In the OHCs, staining appeared in discrete spots in the apical areas only, whereas in IHCs staining extended further towards the center of the cells. The Yp subunit was mainly localized to Deiters cell processes and pillar cells. Both Yb1 and Yp colocalized with tubulin-specific antibody.The functional significance of GST in the cochlear receptor cells is speculative. However, a role anologous to that in other tissues (detoxification, prostaglandin synthesis) can be assumed. In addition, an association of GST with the microtubule system is possible based on immunohistochemical colocalization with tubulin.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30402/1/0000022.pd