Epigenetic Priming of AML Blasts for All-trans Retinoic Acid-Induced Differentiation by the HDAC Class-I Selective Inhibitor Entinostat

All-trans retinoic acid (ATRA) has only limited single agent activity in AML without the PML-RARα fusion (non-M3 AML). In search of a sensitizing strategy to overcome this relative ATRA resistance, we investigated the potency of the HDAC class-I selective inhibitor entinostat in AML cell lines Kasumi-1 and HL-60 and primary AML blasts. Entinostat alone induced robust differentiation of both cell lines, which was enhanced by the combination with ATRA. This “priming” effect on ATRA-induced differentiation was at least equivalent to that achieved with the DNA hypomethylating agent decitabine, and could overall be recapitulated in primary AML blasts treated ex vivo. Moreover, entinostat treatment established the activating chromatin marks acH3, acH3K9, acH4 and H3K4me3 at the promoter of the RARβ2 gene, an essential mediator of retinoic acid (RA) signaling in different solid tumor models. Similarly, RARβ2 promoter hypermethylation (which in primary blasts from 90 AML/MDS patients was surprisingly infrequent) could be partially reversed by decitabine in the two cell lines. Re-induction of the epigenetically silenced RARβ2 gene was achieved only when entinostat or decitabine were given prior to ATRA treatment. Thus in this model, reactivation of RARβ2 was not necessarily required for the differentiation effect, and pharmacological RARβ2 promoter demethylation may be a bystander phenomenon rather than an essential prerequisite for the cellular effects of decitabine when combined with ATRA. In conclusion, as a “priming” agent for non-M3 AML blasts to the differentiation-inducing effects of ATRA, entinostat is at least as active as decitabine, and both act in part independently from RARβ2. Further investigation of this treatment combination in non-M3 AML patients is therefore warranted, independently of RARβ2 gene silencing by DNA methylation.     

β1 Integrin Signaling Maintains Human Epithelial Progenitor Cell Survival In Situ and Controls Proliferation,Apoptosis and Migration of Their Progeny

β1 integrin regulates multiple epithelial cell functions by connecting cells with the extracellular matrix (ECM). While β1 integrin-mediated signaling in murine epithelial stem cells is well-studied, its role in human adult epithelial progenitor cells (ePCs) in situ remains to be defined. Using microdissected, organ-cultured human scalp hair follicles (HFs) as a clinically relevant model for studying human ePCs within their natural topobiological habitat, β1 integrin-mediated signaling in ePC biology was explored by β1 integrin siRNA silencing, specific β1 integrin-binding antibodies and pharmacological inhibition of integrin-linked kinase (ILK), a key component of the integrin-induced signaling cascade. β1 integrin knock down reduced keratin 15 (K15) expression as well as the proliferation of outer root sheath keratinocytes (ORSKs). Embedding of HF epithelium into an ECM rich in β1 integrin ligands that mimic the HF mesenchyme significantly enhanced proliferation and migration of ORSKs, while K15 and CD200 gene and protein expression were inhibited. Employing ECM-embedded β1 integrin-activating or -inhibiting antibodies allowed to identify functionally distinct human ePC subpopulations in different compartments of the HF epithelium. The β1 integrin-inhibitory antibody reduced β1 integrin expression in situ and selectively enhanced proliferation of bulge ePCs, while the β1 integrin-stimulating antibody decreased hair matrix keratinocyte apoptosis and enhanced transferrin receptor (CD71) immunoreactivity, a marker of transit amplifying cells, but did not affect bulge ePC proliferation. That the putative ILK inhibitor QLT0267 significantly reduced ORSK migration and proliferation and induced massive ORSK apoptosis suggests a key role for ILK in mediating the ß1 integrin effects. Taken together, these findings demonstrate that ePCs in human HFs require β1 integrin-mediated signaling for survival, adhesion, and migration, and that different human HF ePC subpopulations differ in their response to β1 integrin signaling. These insights may be exploited for cell-based regenerative medicine strategies that employ human HF-derived ePCs.     

Improvement of catalytic activity of lipase from Candida rugosa via sol-gel encapsulation in the presence of calix(aza)crown

Lipase from Candida rugosa (CRL) was encapsulated within a chemically inert sol-gel support in the presence of calix(aza)crowns as the new additives. The catalytic activity of the encapsulated lipases was evaluated both in the hydrolysis of p-nitrophenyl palmitate (p-NPP) and the enantioselective hydrolysis of racemic Naproxen methyl ester. It has been observed that the percent activity yields of the calix(aza)crown based encapsulated lipases were higher than that of the free lipase. Improved enantioselectivity was observed with the calix(aza)crown-based encapsulated lipases as compared to encapsulated free lipase. The reaction of Naproxen methyl ester resulted in 48.4% conversion for 24 h and 98% enantiomeric excess for the S-acid, corresponding to an E value of >300 (E = 166 for the encapsulated free enzyme). Moreover, the encapsulated lipases were still retained about 18% of their conversion ratios after the sixth reuse in the enantioselective reaction.     

Methylenetetrahydrofolate reductase C677T polymorphism in patients with gastric and colorectal cancer

This study was designed to investigate, in the Turkish population, an association of methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and the risk of gastric and colorectal cancer. Our study was carried out in 35 patients with gastric cancer (20 men, 15 women) and 144 controls (75 men, 69 women) and 52 colorectal cancer (31 men, 21 women). MTHFR C677T genotypes were determined by polymerase chain reaction, restriction fragment length polymorphism techniques. No differences were observed in the distribution of MTHFR genotypes or allele frequencies in cases versus controls. The homozygous mutation (T/T) in the MTHFR gene was identified in 14.3% of gastric cancer versus 10.4% of controls. MTHFR C677T frequencies of the CC, CT and TT genotypes among colorectal cancer patients were 34.6%, 51.9% and 13.5%, respectively. MTHFR C677T polymorphism may not be important in an individual's susceptibility to gastric and colorectal cancer in Turkey and may not be a useful marker for identifying patients at high risk of developing gastric and colorectal cancer.     

Synthesis and antimicrobial activity of some new thiazolyl thiazolidine-2,4-dione derivatives

In this study, a series of thiazolyl thiazolidine-2,4-dione derivatives (Va-f and VIa-f) were synthesized and evaluated for their antibacterial and antifungal activities against Staphylococcus aureus (ATCC 25923), methicillin resistant S. aureus (MRSA ATCC 43300), methicillin resistant S. aureus (MRSA isolate), and Escherichia coli (ATCC 23556) and C. albicans (ATCC10145). All the compounds were found active against used microorganisms.     

The Effects of Leptin on Rat Brain Development; An Experimental Study

International Journal of Peptide Research and Therapeutics -     

Features of Large Hinge-Bending Conformational Transitions. Prediction of Closed Structure from Open State

We performed a detailed analysis of conformational transition pathways for a set of 10 proteins, which undergo large hinge-bending-type motions with 4–12 Å RMSD (root mean-square distance) between open and closed crystal structures. Anisotropic network model-Monte Carlo (ANM-MC) algorithm generates a targeted pathway between two conformations, where the collective modes from the ANM are used for deformation at each iteration and the conformational energy of the deformed structure is minimized via an MC algorithm. The target structure was approached successfully with an RMSD of 0.9–4.1 Å when a relatively low cutoff radius of 10 Å was used in ANM. Even though one predominant mode (first or second) directed the open-to-closed conformational transition, changes in the dominant mode character were observed for most cases along the transition. By imposing radius of gyration constraint during mode selection, it was possible to predict the closed structure for eight out of 10 proteins (with initial 4.1–7.1 Å and final 1.7–2.9 Å RMSD to target). Deforming along a single mode leads to most successful predictions. Based on the previously reported free energy surface of adenylate kinase, deformations along the first mode produced an energetically favorable path, which was interestingly facilitated by a change in mode shape (resembling second and third modes) at key points. Pathway intermediates are provided in our database of conformational transitions (http://safir.prc.boun.edu.tr/anmmc/method/1).     

Targeting Spare CC Chemokine Receptor 5 (CCR5) as a Principle to Inhibit HIV-1 Entry

CCR5 binds the chemokines CCL3, CCL4, and CCL5 and is the major coreceptor for HIV-1 entry into target cells. Chemokines are supposed to form a natural barrier against human immunodeficiency virus, type 1 (HIV-1) infection. However, we showed that their antiviral activity is limited by CCR5 adopting low-chemokine affinity conformations at the cell surface. Here, we investigated whether a pool of CCR5 that is not stabilized by chemokines could represent a target for inhibiting HIV infection. We exploited the characteristics of the chemokine analog PSC-RANTES (N-α-(n-nonanoyl)-des-Ser(1)-[l-thioprolyl(2), l-cyclohexylglycyl(3)]-RANTES(4-68)), which displays potent anti-HIV-1 activity. We show that native chemokines fail to prevent high-affinity binding of PSC-RANTES, analog-mediated calcium release (in desensitization assays), and analog-mediated CCR5 internalization. These results indicate that a pool of spare CCR5 may bind PSC-RANTES but not native chemokines. Improved recognition of CCR5 by PSC-RANTES may explain why the analog promotes higher amounts of β-arrestin 2·CCR5 complexes, thereby increasing CCR5 down-regulation and HIV-1 inhibition. Together, these results highlight that spare CCR5, which might permit HIV-1 to escape from chemokines, should be targeted for efficient viral blockade.     

HPLC fluorescence method for the determination of nizatidine in human plasma and its application to pharmacokinetic study

A sensitive high‐performance liquid chromatographic (HPLC) method was developed for the determination of nizatidine in human plasma. Nizatidine was derivatized by 4‐fluoro‐7‐nitrobenzofurazan (NBD‐F). Chromatographic separation was performed on a Inertsil C₁₈ column (150 mm × 4.6 mm, 5 µm) using isocratic elution by a mobile phase consisting of methanol/water (55:45) at a flow rate of 1.2 mL/min. Amlodipine was used as the internal standard (IS). Fluorescence detector was used operated at 461 nm (excitation) and 517 nm (emission), respectively. The calibration curve was linear over the range of 50–2000 ng/mL. This method was successfully applied to a pharmacokinetic study after oral administration of a dose (150 mg) of nizatidine. Copyright © 2013 John Wiley & Sons, Ltd.