Histone hyperacetylation induced by histone deacetylase inhibitors is not sufficient to cause growth inhibition in human dermal fibroblasts

Use of specific histone deacetylase inhibitors has revealed critical roles for the histone deacetylases (HDAC) in controlling proliferation. Although many studies have correlated the function of HDAC inhibitors with the hyperacetylation of histones, few studies have specifically addressed whether the accumulation of acetylated histones, caused by HDAC inhibitor treatment, is responsible for growth inhibition. In the present study we show that HDAC inhibitors cause growth inhibition in normal and transformed keratinocytes but not in normal dermal fibroblasts, This was despite the observation that the HDAC inhibitor, suberic bishydroxamate (SBHA), caused a kinetically similar accumulation of hyperacetylated histones, This cell type-specific response to SBHA was not due to the inactivation of SBHA by fibroblasts, nor was it due to differences in the expression of specific HDAC family members. Remarkably, overexpression of HDACs 1, 4, and 6 in normal human fibroblasts resulted in cells that could be growth-inhibited by SBHA. These data suggest that, although histone acetylation is a major target for HDAC inhibitors, the accumulation of hyperacetylated histones is not sufficient to cause growth inhibition in all cell types, This suggests that growth inhibition, caused by HDAC inhibitors, may be the culmination of histone hyperacetylation acting in concert with other growth regulatory pathways.

Lipophilic methotrexate conjugates with glucose-lipoamino acid moieties: Synthesis and in vitro antitumor activity

To obtain methotrexate (MTX) derivatives with a balanced hydrolipophilic character, we synthesized a series of conjugates in which the drug was linked to lipoamino acid (LAA)-glucose residues (LAAG-MTX). These conjugates displayed increased solubility in polar media compared with the corresponding LAA-MTX conjugates previously described. In vitro biological testing of LAAG-MTX indicated that the introduction of the sugar moiety decreased the biological activity of these MTX conjugates. The tetradecyl derivative 6b, however, was effective in inhibiting the dihydrofolate reductase activity in vitro and showed an inhibitory effect on human lymphoblastoid cell growth. (C) 2001 Wiley-Liss, Inc.

Inhibitors of human immunodeficiency virus type 1 reverse transcriptase target distinct phases of early reverse transcription

Early HIV-1 reverse transcription can be separated into initiation and elongation phases. Here we show, using PCR analysis of negative-strand strong-stop DNA [(-)ssDNA] synthesis in intact virus, that different reverse transcriptase (RT) inhibitors affect distinct phases of early natural endogenous reverse transcription (NERT), The effects of nevirapine on NERT were consistent with a mechanism of action including both specific and nonspecific binding events. The nonspecific component of this inhibition targeted the elongation reaction, whereas the specific effect seemed principally to be directed at very early events (initiation or the initiation-elongation switch), In contrast, foscarnet and the nucleoside analog ddATP inhibited both early and late (-)ssDNA synthesis in a similar manner. We also examined compounds that targeted other viral proteins and found that Ro24-7429 (a Tat antagonist) and rosmarinic acid (an integrase inhibitor) also directly inhibited RT, Our results indicate that NERT can be used to identify and evaluate compounds that directly target the reverse transcription complex.

Crystal structure of yeast acetohydroxyacid synthase: A target for herbicidal inhibitors

Acetohydroxyacid synthase (AHAS; EC 4.1.3.18) catalyzes the first step in branched-chain amino acid biosynthesis. The enzyme requires thiamin diphosphate and FAD for activity, but the latter is unexpected, because the reaction involves no oxidation or reduction. Due to its presence in plants, AHAS is a target for sulfonylurea and imidazolinone herbicides. Here, the crystal structure to 2.6 A resolution of the catalytic subunit of yeast AHAS is reported. The active site is located at the dimer interface and is near the proposed herbicide-binding site. The conformation of FAD and its position in the active site are defined. The structure of AHAS provides a starting point for the rational design of new herbicides. (C) 2002 Elsevier Science Ltd.

Back to (non)-Basics: Recent developments in neutral and charge-balanced glycosidase inhibitors

Certain glycosidase inhibitors possess potent antiviral, antitumour and antidiabetic properties. Glyconic acid lactones, the earliest glycosidase inhibitors identified, have planar anomeric carbons that mimic the transition state of glycoside hydrolysis. Other classes include lactams, glycals, epoxides, halides and sulfonium ions, the latter based on the natural product salacinol from an antidiabetic herb.

Study of the retinoic acid regulated genes in the rodent hypothalamus controlling growth and appetite and the potential for the epigenetic control

Dissertação de mestrado em Bioquímica (área de especialização em Biomedicina)

Uric acid is a danger signal activating NALP3 inflammasome in lung injury inflammation and fibrosis.

RATIONALE: Lung injury leads to pulmonary inflammation and fibrosis through myeloid differentiation primary response gene 88 (MyD88) and the IL-1 receptor 1 (IL-1R1) signaling pathway. The molecular mechanisms by which lung injury triggers IL-1beta production, inflammation, and fibrosis remain poorly understood. OBJECTIVES: To determine if lung injury depends on the NALP3 inflammasome and if bleomycin (BLM)-induced lung injury triggers local production of uric acid, thereby activating the NALP3 inflammasome in the lung. Methods: Inflammation upon BLM administration was evaluated in vivo in inflammasome-deficient mice. Pulmonary uric acid accumulation, inflammation, and fibrosis were analyzed in mice treated with the inhibitor of uric acid synthesis or with uricase, which degrades uric acid. MEASUREMENTS AND MAIN RESULTS: Lung injury depends on the NALP3 inflammasome, which is triggered by uric acid locally produced in the lung upon BLM-induced DNA damage and degradation. Reduction of uric acid levels using the inhibitor of uric acid synthesis allopurinol or uricase leads to a decrease in BLM-induced IL-1beta production, lung inflammation, repair, and fibrosis. Local administration of exogenous uric acid crystals recapitulates lung inflammation and repair, which depend on the NALP3 inflammasome, MyD88, and IL-1R1 pathways and Toll-like receptor (TLR)2 and TLR4 for optimal inflammation but are independent of the IL-18 receptor. CONCLUSIONS: Uric acid released from injured cells constitutes a major endogenous danger signal that activates the NALP3 inflammasome, leading to IL-1beta production. Reducing uric acid tissue levels represents a novel therapeutic approach to control IL-1beta production and chronic inflammatory lung pathology.

New inhibitors of Helicobacter pylori urease holoenzyme selected from phage-displayed peptide libraries.

Urease is an important virulence factor for Helicobacter pylori and is critical for bacterial colonization of the human gastric mucosa. Specific inhibition of urease activity has been proposed as a possible strategy to fight this bacteria which infects billions of individual throughout the world and can lead to severe pathological conditions in a limited number of cases. We have selected peptides which specifically bind and inhibit H. pylori urease from libraries of random peptides displayed on filamentous phage in the context of pIII coat protein. Screening of a highly diverse 25-mer combinatorial library and two newly constructed random 6-mer peptide libraries on solid phase H. pylori urease holoenzyme allowed the identification of two peptides, 24-mer TFLPQPRCSALLRYLSEDGVIVPS and 6-mer YDFYWW that can bind and inhibit the activity of urease purified from H. pylori. These two peptides were chemically synthesized and their inhibition constants (Ki) were found to be 47 microM for the 24-mer and 30 microM for the 6-mer peptide. Both peptides specifically inhibited the activity of H. pylori urease but not that of Bacillus pasteurii.

Transport and metabolism of [214-C]abscisic acid in maize root

The tips of intact maize (cv. LG 11) roots, maintained vertically, were pretreated with a droplet of buffer solution or a bead of anion exchange resin, both containing [214-C]abscisic acid (ABA). A significant basipetal ABA movement was observed and two metabolites of ABA (possibly phaseic acid and dihydrophaseic acid) were found. ABA pretreatment enhanced the gravireaction of 10 mm apical root segments kept both in the dark and in the light. The possibility that ABA could be one of the endogenous growth inhibitors produced or released by the cap cells is discussed.

Differential outcome of concurrent radiotherapy plus epidermal growth factor receptor inhibitors versus radiotherapy plus cisplatin in patients with human papillomavirus-related head and neck cancer.

BACKGROUND Human papillomavirus (HPV)-related head and neck cancer has been associated with an improved prognosis in patients treated with radiotherapy (RT) +/- chemotherapy (CT); however, RT combined with epidermal growth factor receptor (EGFR) inhibitors has not been fully studied in this group of patients. METHODS Immunohistochemical expression of p16 and PCR of HPV16 DNA were retrospectively analyzed in tumor blocks from 108 stage III/IV head and neck cancer patients treated with RT+CT (56) or RT+EGFR inhibitors (52). Disease-free survival (DFS) and overall survival (OS) were analyzed by the Kaplan-Meier method. RESULTS DNA of HPV16 was found in 12 of 108 tumors (11%) and p16 positivity in 18 tumors (17%), with similar rates in both arms of treatment. After a median follow-up time of 35 months (range 6-135), p16-positive patients treated with RT+EGFR inhibitors showed improved survival compared with those treated with RT+CT (2-year OS 88% vs. 60%, HR 0.18; 95% CI 0.04 to 0.88; p = 0.01; and 2-year DFS 75% vs. 47%, HR 0.17; 95% CI 0.03 to 0.8; p = 0.01). However, no differences were observed in p16-negative patients (2-year OS 56% vs. 53%, HR 0.97; 95% CI 0.55 to 1.7; p = 0.9; and 2-year DFS 43% vs. 45%, HR 0.99; 95% CI 0.57 to 1.7; p = 0.9). CONCLUSIONS This is the first study to show that p16-positive patients may benefit more from RT+EGFR inhibitors than conventional RT+CT. These results are hypothesis-generating and should be confirmed in prospective trials.

Inhibition of rotavirus ECwt infection in ICR suckling mice by N-acetylcysteine, peroxisome proliferator-activated receptor gamma agonists and cyclooxygenase-2 inhibitors

Live attenuated vaccines have recently been introduced for preventing rotavirus disease in children. However, alternative strategies for prevention and treatment of rotavirus infection are needed mainly in developing countries where low vaccine coverage occurs. In the present work, N-acetylcysteine (NAC), ascorbic acid (AA), some nonsteroidal anti-inflammatory drugs (NSAIDs) and peroxisome proliferator-activated receptor gamma (PPARγ) agonists were tested for their ability to interfere with rotavirus ECwt infectivity as detected by the percentage of viral antigen-positive cells of small intestinal villi isolated from ECwt-infected ICR mice. Administration of 6 mg NAC/kg every 8 h for three days following the first diarrhoeal episode reduced viral infectivity by about 90%. Administration of AA, ibuprofen, diclofenac, pioglitazone or rosiglitazone decreased viral infectivity by about 55%, 90%, 35%, 32% and 25%, respectively. ECwt infection of mice increased expression of cyclooxygenase-2, ERp57, Hsc70, NF-κB, Hsp70, protein disulphide isomerase (PDI) and PPARγ in intestinal villus cells. NAC treatment of ECwt-infected mice reduced Hsc70 and PDI expression to levels similar to those observed in villi from uninfected control mice. The present results suggest that the drugs tested in the present work could be assayed in preventing or treating rotaviral diarrhoea in children and young animals.

Maladies peptiques [Progress in 2008 for acid related diseases]

The treatment of reflux disease did not change in the review period. PPI therapy remains the first line treatment and surgery the second line approach. Endoscopic anti-reflux procedures should be only performed in controlled studies. Beside the classic triple therapy, sequential treatment of Helicobacter pylori infection can today be considered as a first line therapy. PPI are effective in the prevention of gastroduodenal lesions and in the treatment of dyspeptic symptoms induced by NSAIDs treatment. Only patients younger then 65 years and without any risk factors do not need a preventive PPI prescription during classic NSAIDS treatment.

Design of potent inhibitors of human RAD51 recombinase based on BRC motifs of BRCA2 protein: modeling and experimental validation of a chimera peptide.

We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochemical testing to construct a highly efficient chimera peptide from eight existing human BRC motifs. We built a molecular model of all BRC motifs complexed with HsRad51 based on the crystal structure of the BRC4 motif-HsRad51 complex, computed the interaction energy of each residue in each BRC motif, and selected the best amino acid residue at each binding position. This analysis enabled us to propose four amino acid substitutions in the BRC4 motif. Three of these increased the inhibitory effect in vitro, and this effect was found to be additive. We thus obtained a peptide that is about 10 times more efficient in inhibiting HsRad51-ssDNA complex formation than the original peptide.

New inhibitors of Helicobacter pylori urease holoenzyme selected from phage-displayed peptide libraries.

Urease is an important virulence factor for Helicobacter pylori and is critical for bacterial colonization of the human gastric mucosa. Specific inhibition of urease activity has been proposed as a possible strategy to fight this bacteria which infects billions of individual throughout the world and can lead to severe pathological conditions in a limited number of cases. We have selected peptides which specifically bind and inhibit H. pylori urease from libraries of random peptides displayed on filamentous phage in the context of pIII coat protein. Screening of a highly diverse 25-mer combinatorial library and two newly constructed random 6-mer peptide libraries on solid phase H. pylori urease holoenzyme allowed the identification of two peptides, 24-mer TFLPQPRCSALLRYLSEDGVIVPS and 6-mer YDFYWW that can bind and inhibit the activity of urease purified from H. pylori. These two peptides were chemically synthesized and their inhibition constants (Ki) were found to be 47 microM for the 24-mer and 30 microM for the 6-mer peptide. Both peptides specifically inhibited the activity of H. pylori urease but not that of Bacillus pasteurii.