Novel aminobenzyl-acetamidine derivative modulate the differential regulation of NOSs in LPS induced inflammatory response: Role of PI3K/Akt pathway

Background

Previous reports suggest that NO may contribute to the pathophysiology of septic shock. Recently, we have synthesized and characterized a series of benzyl- and dibenzyl derivative of N-(3-aminobenzyl)acetamidine, a potent and selective inhibitor of iNOS, in vitro assay. We evaluated the molecular mechanisms by which these compounds are involved in the regulation of NOSs expression.

Methods

H9c2 cells were stimulated with lipopolysaccharide (LPS) in the presence or absence of acetamidine-derivative. The NOSs mRNA and protein, and activation of signaling pathways (Akt and NF-κB) were assayed.

Results

The induction of endotoxic shock in H9c2 with LPS caused an increase of inducible NOS and a down-regulation of constitutive NOS. The molecular mechanism involved in the modulation of NOSs expression in H9c2 cells upon LPS stimulation resulted in the modification of the redox state responsible for NF-kB nuclear translocation via NIK -IKKα/β-IkBα, simultaneously to the inactivation of the PI3K/Akt pathway. The compounds acted as an anti-inflammatory modulator.

Conclusion

These results suggest that LPS regulates the opposite NOS expression in H9c2 cells by modifying the redox state of these cells responsible for the NF-kB nuclear translocation via NIK–IKKα/β‐IkBα, simultaneous to the inactivation of the PI3K/Akt pathway. The new molecule acts as an anti-inflammatory modulator in LPS-induced inflammation in H9c2 cells by the restoration of eNOS and nNOS expressions, mechanistically involving the PI3K/Akt pathway.

General significance

This study delineates the underlying mechanisms of opposite NOSs expression in H9c2 cells stimulated with LPS.     

Low sclerostin levels: a predictive marker of persistent inflammation in ankylosing spondylitis during anti-tumor necrosis factor therapy?

Introduction

Sclerostin levels have been reported to be low in ankylosing spondylitis (AS), but there is no data regarding the possible role of this Wnt inhibitor during anti-tumor necrosis factor (TNF) therapy. The present study longitudinally evaluated sclerostin levels, inflammatory markers and bone mineral density (BMD) in AS patients under anti-TNF therapy.

Methods

Thirty active AS patients were assessed at baseline, 6 and 12 months after anti-TNF therapy regarding clinical parameters, inflammatory markers, BMD and baseline radiographic damage (mSASSS). Thirty age- and sex-matched healthy individuals comprised the control group. Patients'' sclerostin levels, sclerostin binding low-density lipoprotein receptor-related protein 6 (LRP6) and BMD were evaluated at the same time points and compared to controls.

Results

At baseline, AS patients had lower sclerostin levels (60.5 ± 32.7 vs. 96.7 ± 52.9 pmol/L, P = 0.002) and comparable sclerostin binding to LRP6 (P = 0.387) than controls. Improvement of Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), Bath Ankylosing Spondylitis Metrology Index (BASMI), Ankylosing Spondylitis quality of life (ASQoL) was observed at baseline vs. 6 vs. 12 months (P < 0.01). Concomitantly, a gradual increase in spine BMD (P < 0.001) and a positive correlation between baseline mSASSS and spine BMD was found (r = 0.468, P < 0.01). Inflammatory parameters reduction was observed comparing baseline vs. 6 vs. 12 months (P <0.01). Sclerostin levels progressively increased [baseline (60.5 ± 32.7) vs. 6 months (67.1 ± 31.9) vs. 12 months (72.7 ± 32.3) pmol/L, P <0.001]. At 12 months, the sclerostin levels remained significantly lower in patients compared to controls (72.7 ± 32.3 vs. 96.70 ± 52.85 pmol/L, P = 0.038). Moreover, sclerostin serum levels at 12 months were lower in the 10 patients with high C reactive protein (CRP) (≥ 5 mg/l) compared to the other 20 patients with normal CRP (P = 0.004). Of note, these 10 patients with persistent inflammation also had lower sclerostin serum levels at baseline compared to the other patients (P = 0.023). Univariate logistic regression analysis demonstrated that AS patients with lower sclerostin serum levels had an increased risk to have high CRP at 12 months (odds ratio = 7.43, 95% CI 1.23 to 45.01, P = 0.020) than those with higher sclerostin values.

Conclusions

Persistent low sclerostin levels may underlie continuous inflammation in AS patients under anti-TNF therapy.     

Induction of intracellular carbonic anhydrases during the adaptation to low inorganic carbon concentrations in wild-type and ca-1 mutant cells of Chlamydomonas reinhardtii

Mass-spectrometric measurements of ¹⁸O exchange from ¹³C¹⁸O₂ were used to follow changes in the intracellular carbonic anhydrase (CA) activity of cells of Chlamydomonas reinhardtii Dang, wild type and the ca-1 mutant during adaptation to air. With intact cells as well as with crude homogenates total intracellular CA activity in wild-type cells increased six to tenfold within 4 h after transferring cells from 5% CO₂ (high inorganic carbon, C_i) to ambient air (air adapted). After that time the activity slowly declined to a level similar to that observed with cells which had been continuously grown in air (low-C_i grown). In the ca-1 mutant, total CA was induced to a similar extent during 4 h of adaptation; however, absolute activities were two to three times lower in ca-1 than in the wild type regardless of the CO₂ supply. When crude extracts from wild-type cells were separated into soluble and insoluble fractions, each fraction contained about half of the internal CA activity. Within 4 h of adaptation, both forms of CA activity were simultaneously enhanced by nine to tenfold, reaching levels similar to those found in low-C_igrown cells. In contrast, in the ca-1 mutant the soluble CA activity was only enhanced by about eightfold while the level of insoluble CA was very low even in low-C_i cells. After isolation of intact chloroplasts from wild-type cells and further subfractionation, around 70–80% of total chloroplastic CA activity was found to be in the insoluble fraction while 17–20% remained in the soluble fraction. Both chloroplastic CA activities were inducible within the first 4 h of adaptation to air, with each of them being eight to ten times higher than in high-C_i algae. After that time their activities were similar to the corresponding CA values in low-C_i-grown cells. In contrast, plastids from high-C_i cells of the ca-1 mutant showed 40% less insoluble-CA activity compared to the wild type and this insoluble-CA activity was not increased at all by transferring algae to air. In addition, no soluble-CA activity was detected in chloroplasts from high-C_i and air-adapted ca-1 cells. These results indicate the presence of three intracellular CA activities in high-C_i air-adapted and low-C_i cells of the wild type and that two of them are associated with the chloroplasts. All three activities are completely induced within the first 4 h of adaptation to air in wild-type cells. In contrast, it was not possible to induce any of the chloroplastic CA activities in the ca-1 mutant. The possibility that the soluble chloroplastic CA represents a pyrenoid-located CA is discussed.This work is dedicated to Professor A. Wild on the occasion of his 65th birthday     

Analysis of non-TIR NBS-LRR resistance gene analogs in <Emphasis Type="Italic">Musa acuminata</Emphasis> Colla: Isolation,RFLP marker development,and physical mapping

Background  

Many commercial banana varieties lack sources of resistance to pests and diseases, as a consequence of sterility and narrow genetic background. Fertile wild relatives, by contrast, possess greater variability and represent potential sources of disease resistance genes (R-genes). The largest known family of plant R-genes encode proteins with nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. Conserved motifs in such genes in diverse plant species offer a means for isolation of candidate genes in banana which may be involved in plant defence.     

Chromate removal by yeasts isolated from sediments of a tanning factory and a mine site in Argentina

Twenty-one yeast-like microorganisms were isolated from tannery effluents and from a nickel–copper mine in Argentina. They were tested for their Cu(II), Ni(II), Cd(II) and Cr(VI) tolerance in qualitative assays on solid medium. Three isolates were selected for their multiple tolerance to the different heavy metals and highest tolerance to Cr(VI). According to morphological and physiological analysis and 26S rDNA D1/D2 domain sequences the isolates were characterized as: Lecythophora sp. NGV-1, Candida sp. NGV-9 and Aureobasidium pullulans VR-8. Resistance of the three strains to high Cr(VI) concentrations and their ability to remove Cr(VI) were assessed using YNB-glucose medium supplemented with 0.5 and 1 mM Cr(VI). Chromate removal activity was estimated by measuring remaining Cr(VI) concentration in the supernatant using the colorimetric 1,5-diphenylcarbazide method and total chromium was determined by flame atomic absorption spectroscopy. The results indicate that the initial Cr(VI) concentration negatively influenced growth and the specific growth rate but stimulated the metabolic activity of the three strains; resistance to Cr(VI) by these strains was mainly due to reduction of Cr(VI) rather than chromium bioaccumulation. This study showed the potential ability of these strains as tools for bioremediation of Cr(VI) from contaminated sites.     

In situ RT-PCR allows the detection of ornithine decarboxylase mRNA in paraffin embedded archival human hyperplastic breast tissues

Expression of ornithine decarboxylase (ODC) is induced by c-Myc oncoprotein and is required for cell proliferation and tumour growth. We have studied the expression of ODC mRNA by in situ hybridisation and in situ RT-PCR in archival human hyperplastic breast tissues. A very low signal was detected by in situ hybridisation, while the in situ RT-PCR on human breast archival tissues demonstrated an over-expression of ODC mRNA in epithelial cells characterised by some degree of hyperplasia, maintaining the morphology of the archival tissue intact despite the multiple steps of fixation, permeabilization and thermal cycling.     

Puroindoline A-gene expression is involved in association of puroindolines to starch

Puroindolines largely influence cereal grain hardness. In order to understand how they exert this influence, we carried out a molecular analysis of the pina and pinb genes of many Italian wheat cultivars. On the basis of their pin genotypes they could be divided into three groups: Pina-D1a/Pinb-D1a; Pina-D1a/Pinb-D1b; and Pina-D1b/Pinb-D1a. Five cultivars from each group were chosen to be studied to examine the quantity of puroindolines associated with starch (friabilin) and the amount not associated with starch. In addition, the level of pina expression was measured using RT-PCR. Soft cultivars (Pina-D1a/Pinb-D1a) exhibited the highest level of expression of pina; among the hard cultivars, those with the Pina-D1a/Pinb-D1b genotype showed a lower level of expression, while those with the Pina-D1b/Pinb-D1a genotype did not express pina. Total puroindoline and friabilin content was then measured by flow cytometry. Soft Pina-D1a/Pinb-D1a cultivars displayed high puroindoline content that was primarily starch associated. Hard Pina-D1b/Pinb-D1a cultivars had very low puroindoline content with no puroindoline bound to starch. Hard Pina-D1a/Pinb-D1b cultivars were highly heterogeneous with respect to both the content of puroindolines and the level of association with starch. The accurate quantification of puroindolines in starch-bound and not starch-bound forms in association with molecular analysis, indicates that pina expression and presence controls the abundance of total puroindoline and its association with starch.Communicated by H.F. Linskens