Monocyte Chemoattractant Protein-1 (MCP-1) as a Potential Therapeutic Target and a Noninvasive Biomarker of Liver Fibrosis Associated With Transient Myeloproliferative Disorder in Down Syndrome.

Liver fibrosis is one of the common complications of transient myeloproliferative disorder (TMD) in Down syndrome (DS), but the exact molecular pathogenesis is largely unknown. We herein report a neonate of DS with liver fibrosis associated with TMD, in which we performed the serial profibrogenic cytokines analyses. We found the active monocyte chemoattractant protein-1 expression in the affected liver tissue and also found that both serum and urinary monocyte chemoattractant protein-1 concentrations are noninvasive biomarkers of liver fibrosis. We also showed a prospective of the future anticytokine therapy with herbal medicine for the liver fibrosis associated with TMD in DS.

BRAF V600 mutations in Langerhans cell histiocytosis with a simple and unique assay.

BACKGROUND: BRAF (V-raf murine sarcoma viral oncogene homolog B1) is a serine-threonine protein kinase involved in cell survival, proliferation, and differentiation. The most common missense mutation of BRAF (mainly V600E) contributes to the incidence of various cancers, including Langerhans cell histiocytosis (LCH). BRAF inhibitors molecularly targeting the V600E mutation have been developed to counteract the effect of the mutation. To ensure the administration of effective pharmacotherapy, it is therefore imperative to develop an effective assay to screen LCH patients for the V600E mutation. However, tumor tissues of LCH typically contain many inflammatory cells which make a correct judgement of the mutation status difficult in the DNA sequence analysis. RESULTS: In this study, we present a new, highly sensitive analyzing method combining PCR, restriction enzyme digestion, and a sequencing assay using DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue specimens. TspRI is a restriction enzyme that cleaves the sequence encompassing the wild-type BRAF codon 600 into two fragments, which cannot be used as a template for subsequent BRAF PCR amplification. We therefore evaluated the sensitivity of BRAF V600 mutation detection by amplifying the primary PCR product digested with TspRI and sequencing the secondary PCR products. The V600E mutation was detected in FFPE tissue samples from 32 LCH patients; our assay was able to identify mutations in four samples that gave inconclusive results, and ten that were negative, according to standard PCR and sequencing. CONCLUSIONS: We presented a new and highly sensitive method to detect BRAF V600 mutations. This screening method is expected to play an important role to select the most effective therapies.

Thermodynamic analysis of alcohol effect on thermal stability of proteins.

Thermal unfolding of ribonuclease A and α-chymotrypsinogen A was analyzed in various alcohol solutions of methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, tert-butanol, trifluoroethanol, and glycerol. The change in thermal unfolding ratio with temperature was described well by the van't Hoff equation and the melting temperature and the enthalpy of protein unfolding were obtained. The reciprocal form of the Wyman-Tanford equation, which describes the unfolded-to-folded protein ratio as a function of water activity, was applied to obtain a linear plot. From the slope of this plot and water activity, the stabilization free energy (ΔΔG) in a solution was calculated. This shows an important role of water activity in protein stability. ΔΔG was linearly dependent on alcohol concentration and m-values of alcohols for protein unfolding were obtained. This provides a theoretical basis for the linear extrapolation model (LEM). The m-values for alcohols were negative except for glycerol. The negative higher m-value for longer and linear chain alcohols suggested the important role of the disturbance of hydrophobic interactions as well as the hydrogen-bonding in the mechanism of protein destabilization by alcohols. The number of change in bound-alcohol molecules upon protein unfolding was also obtained.

Lipase-catalyzed butanolysis of triolein in ionic liquid and selective extraction of product using supercritical carbon dioxide.

The lipase-catalyzed butanolysis of triolein was carried out in an ionic liquid, methyltrioctylammonium trifluoroacetate (MTOATFA). The addition of 80% MTOATFA to the reaction system gave the best result in terms of alcoholysis rate. The final product (butyloleate) was selectively extracted from the reaction mixture using supercritical carbon dioxide. The present method is expected to be applicable to the lipase-catalyzed alcoholysis of triacylglycerols.

Atrazine binds to F1F0-ATP synthase and inhibits mitochondrial function in sperm.

Atrazine is a widely used triazine herbicide. Although controversy still exists, a number of recent studies have described its adverse effects on various animals including humans. Of particular interest is its effects on reproductive capacity. In this study, we investigated the mechanisms underlying the adverse effects of atrazine, with a focus on its effects on sperm. Here we show evidence that mitochondrial F(1)F(0)-ATP synthase is a molecular target of atrazine. A series of experiments with sperm and isolated mitochondria suggest that atrazine inhibits mitochondrial function through F(1)F(0)-ATP synthase. Moreover, affinity purification using atrazine as a ligand demonstrates that F(1)F(0)-ATP synthase is a major atrazine-binding protein in cells. The inhibitory activity against mitochondria and F(1)F(0)-ATP synthase is not limited to atrazine but is likely to be applicable to other triazine-based compounds. Thus, our findings may have wide relevance to pharmacology and toxicology.

Mechanism of H-8 inhibition of cyclin-dependent kinase 9: study using inhibitor-immobilized matrices.

BACKGROUND: Positive transcription elongation factor b (P-TEFb), which phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII), is comprised of the catalytic subunit cyclin-dependent kinase 9 (CDK9) and the regulatory subunit cyclin T. The kinase activity and transcriptional activation potential of P-TEFb is sensitive to various compounds, including H-8, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB), and flavopiridol. RESULTS: We investigated the molecular mechanism of the H-8 inhibition of CDK9 using matrices to which H-9, an amino derivative of H-8, was immobilized. CDK9 bound specifically to H-9, and this interaction was competitively inhibited by ATP and DRB, but not by flavopiridol. Mutational analyses demonstrated that the central region of CDK9, which encompasses the T-loop region, was important for its binding to H-9. CONCLUSIONS: H-9-immobilized latex beads are useful for trapping CDK9 and a subset of kinases from crude cell extracts. The flavopiridol-binding region of CDK9 is most likely different from its H-9-binding region. These biochemical data support previously reported observations which were based on crystallographic data.