Synthesis and evaluation of the antioxidative potential of minoxidil–polyamine conjugates

A series of conjugates (MNX–CO–PA) of minoxidil (MNX) with the polyamines (PAs) putrescine (PUT), spermidine (SPD) and spermine (SPM) as well as dopamine were produced through activation of MNX with N,N′-carbonyldiimidazole, followed by reaction with dopamine or selectively protected PAs and acid-mediated deprotection. These conjugates together with conjugates of the general type MNX–PA or PA–MNX–PA, readily produced using literature protocols, were tested as antioxidants. The most potent inhibitors of lipid peroxidation were the conjugates MNX–SPM (2, 94%), SPM–MNX–SPM (4, 94%) and MNX–N⁴-SPD (7, 91%) and MNX (91%). The most powerful lipoxygenase (LOX) inhibitors were MNX (IC₅₀ = 20 μM) and the conjugates MNX–N⁸-SPD (9, IC₅₀ = 22.1 μM), MNX–CO–dopamine (11, IC₅₀ = 28 μM) and MNX–N¹-SPD (8, IC₅₀ = 30 μM). The most interesting conjugates 2, MNX–CO–PUT (5), 8 and 11 as well as MNX were generally found to exhibit weaker (22–36.5%) or no (conjugate 8) anti-inflammatory activity than indomethacin (47%) with the exception of MNX which showed almost equal potency (49%) to indomethacin. The cytocompatibility of conjugates and MNX at the highest concentration of 100 μM showed a survival percentage of 87–107%, with the exception of conjugates with SPM (compound 2) and MNX–CO–SPM (6), which showed considerable cytotoxicity (survival percentage 8–14%). Molecular docking studies were carried on conjugate 9 and the parent compound MNX and were found to be in accordance with our experimental biological results.     

Revolutionizing membrane protein overexpression in bacteria

The bacterium Escherichia coli is the most widely used expression host for overexpression trials of membrane proteins. Usually, different strains, culture conditions and expression regimes are screened for to identify the optimal overexpression strategy. However, yields are often not satisfactory, especially for eukaryotic membrane proteins. This has initiated a revolution of membrane protein overexpression in bacteria. Recent studies have shown that it is feasible to (i) engineer or select for E. coli strains with strongly improved membrane protein overexpression characteristics, (ii) use bacteria other than E. coli for the expression of membrane proteins, (iii) engineer or select for membrane protein variants that retain functionality but express better than the wild-type protein, and (iv) express membrane proteins using E. coli-based cell-free systems.     

Biogeographical patterns of freshwater micro‐ and macroorganisms: a comparison between phytoplankton,zooplankton and fish in the eastern Mediterranean

Aim We investigated the biogeographical patterns of phytoplankton, zooplankton and fish in freshwater ecosystems. We tested whether spatial distance or environmental heterogeneity act as potential factors controlling community composition. Location Northern and central Greece, eastern Mediterranean. Method Data on 310 phytoplankton, 72 zooplankton and 37 fish species were collected from seven freshwater systems. Species occurrence data were used to generate similarity matrices describing community composition. We performed Mantel tests to compare spatial patterns in community composition of phytoplankton, zooplankton and fish. Next, we examined the correlation between geographical distance and the degree of similarity in community composition. The analysis was repeated for different taxonomic, trophic and size‐based groups of the microorganisms studied. We assessed different environmental variables (topographic and limnological) as predictors of community composition. Results Phytoplankton community composition showed a strong positive correlation with environmental heterogeneity but was not correlated with the geographical distance between systems. Zooplankton community composition was unrelated to geographical distance and was only weakly correlated with environmental variables. In contrast, fish community similarity decayed significantly with distance. We found no relationship along all pairwise comparisons of the compositional matrices of the three groups. The pairwise comparisons of the different taxonomic, trophic and size‐based groups of the microorganism communities studied were in accordance with the results for the entire microorganism community. Main conclusions Our results support the proposition that the biogeography of microorganisms does not demonstrate a distance–decay pattern and further suggest that, in reality, the drivers of distribution depend on the specific community examined. In contrast, the biogeography of macroorganisms was affected by geographical distance. These differences reflect the dispersal abilities of the different organisms. The microorganisms exhibit passive dispersal through the air, with local environmental conditions structuring their community composition. On the other hand, for macroorganisms such as fish, the terrestrial environment could pose barriers to their dispersal; with fish structuring distinctive communities over greater distances. Overall, we suggest that the biogeography of freshwater phytoplankton and zooplankton reflects contemporary environmental conditions, while the biogeographical patterns for fish inhabiting the same systems are related to factors affecting their dispersal ability.     

Anti-tumor necrosis factor therapy improves insulin resistance, beta cell function and insulin signaling in active rheumatoid arthritis patients with high insulin resistance

Introduction

Prevalence of insulin resistance and the metabolic syndrome has been reported to be high in rheumatoid arthritis (RA) patients. Tumor necrosis factor (TNF), a pro-inflammatory cytokine with a major pathogenetic role in RA, may promote insulin resistance by inducing Ser³¹² phosphorylation (p-Ser³¹²) of insulin receptor substrate (IRS)-1 and downregulating phosphorylated (p-)AKT. We examined whether anti-TNF therapy improves insulin resistance in RA patients and assessed changes in the insulin signaling cascade.

Methods

Prospective study of RA patients receiving anti-TNF agents (infliximab, n = 49, adalimumab, n = 11, or etanercept, n = 1) due to high disease activity score in 28 joints (DAS28 > 5.1). A complete biochemical profile was obtained at weeks 0 and 12 of treatment. Insulin resistance, insulin sensitivity and pancreatic beta cell function were measured by the Homeostasis Model Assessment (HOMA-IR), the Quantitative Insulin Sensitivity Check Index (QUICKI) and the HOMA-B respectively. Protein extracts from peripheral blood mononuclear cells were assayed by western blot for p-Ser³¹² IRS-1 and p-AKT. RA patients treated with abatacept (CTLA4.Ig) were used as a control group for insulin signaling studies.

Results

At study entry, RA patients with high insulin resistance (HOMA-IR above median) had significantly higher mean DAS28 (P = 0.011), serum triglycerides (P = 0.015), and systolic blood pressure levels (P = 0.024) than patients with low insulin resistance. After 12 weeks of anti-TNF therapy, patients with high insulin resistance demonstrated significant reduction in HOMA-IR (P < 0.001), HOMA-B (P = 0.001), serum triglycerides (P = 0.039), and increase in QUICKI (P < 0.001) and serum HDL-C (P = 0.022). Western blot analysis in seven active RA patients with high insulin resistance showed reduction in p-Ser³¹² IRS-1 (P = 0.043) and increase in p-AKT (P = 0.001) over the study period. In contrast, the effect of CTLA4.Ig on p-Ser³¹² IRS-1 and p-AKT levels was variable.

Conclusions

Anti-TNF therapy improved insulin sensitivity and reversed defects in the insulin signaling cascade in RA patients with active disease and high insulin resistance. The impact of these biochemical changes in modifying cardiovascular disease burden in active RA patients remains to be seen.     

Synthesis of hydroxycoumarins and hydroxybenzo[f]- or [h]coumarins as lipid peroxidation inhibitors

Substituted hydroxycoumarins and 7- or 8-hydroxybenzo[f]coumarins were prepared by the treatment of phenols and naphthalenediols, respectively, with malic acid and H₂SO₄ under microwave irradiation. 7- or 8-Hydroxybenzo[f]coumarins and 6-hydroxybenzo[h]coumarin were synthesized by the reaction of naphthalenediols with ethylpropiolate in the presence of ZnCl₂ in refluxing dioxane. The compounds were tested in vitro for their ability: (i) to interact with 1,1-diphenyl-2-picryl-hydrazyl (DPPH) stable free radical, (ii) to inhibit lipid peroxidation, (iii) to scavenge the superoxide anion, (iv) to inhibit the activity of soybean lipoxygenase and (v) to inhibit in vivo the carrageenin-induced rat paw edema. Most of them are potent superoxide anion scavengers and inhibit in vitro lipid peroxidation. The majority of the compounds did not show high lipoxygenase inhibitory activity. No differences were observed between biological responses of hydroxycoumarins and hydroxybenzocoumarins. Compound 3i was found to present a promising antioxidant profile.     

2D-QSAR and 3D-QSAR/CoMFA analyses of the N-terminal substituted anthranilic acid based CCK1 receptor antagonists: ‘Hic Rhodus,hic saltus’

A research is presented on quantitative structure–activity relationship (QSAR) studies on the more recent class of non-peptidic CCK₁ receptor antagonists. Our results suggest that the balance of hydrophobicity and volume dependent polarizability term plays a key role in the antagonism of CCK₁ receptor. The size of the substitution of ligands at particular position which induce steric fit is crucial as well as their hydrophobic contribution. Indicator variables were used after the best model was found to account for the usual structural features.The CoMFA results show a good variance explanation and the best self-predictivity is slightly lower than 60% with both leave-one-out and random-group methods. The CoMFA molecular fields showed the importance of steric hindrance of the substituent. From the GRIND models it can be deduced that the shape differences of the molecules are secondary in the regulation of the activity, or better, that their polar substituents are capable of occupying the same zones of the space in the most of the cases.     

Structural model and functional characterization of the Bemisia tabaci CYP6CM1vQ, a cytochrome P450 associated with high levels of imidacloprid resistance

The neonicotinoid imidacloprid is one of the most important insecticides worldwide. It is used extensively against the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae), an insect pest of eminent importance globally, which was also the first pest to develop high levels of resistance against imidacloprid and other neonicotinoids in the field. Recent reports indicated that in both the B and Q biotypes of B. tabaci, the resistant phenotype is associated with over-expression of the cytochrome P450 gene CYP6CM1. In this study, molecular docking and dynamic simulations were used to analyze interactions of imidacloprid with the biotype Q variant of the CYP6CM1 enzyme (CYP6CM1vQ). The binding mode with the lowest energy in the enzyme active site, the key amino acids involved (i.e. Phe-130 and Phe-226), and the putative hydroxylation site (lowest distance to carbon 5 of the imidazolidine ring system of imidacloprid) were predicted. Heterologous expression of the CYP6CM1vQ confirmed the accuracy of our predictions and demonstrated that the enzyme catalyses the hydroxylation of imidacloprid to its less toxic 5-hydroxy form (K_cat = 3.2 pmol/min/pmol P450, K_m = 36 μM). The data identify CYP6CM1vQ as a principle target for inhibitor design, aimed at inactivating insecticide-metabolizing P450s in natural insect pest populations.     

Regulation of the Ras‐GTPase activating protein neurofibromin by C‐tail phosphorylation: implications for protein kinase C/Ras/extracellular signal‐regulated kinase 1/2 pathway signaling and neuronal differentiation

PKC, Ras, and ERK1/2 signaling is pivotal to differentiation along the neuronal cell lineage. One crucial protein that may play a central role in this signaling pathway is the Ras GTPase‐activating protein, neurofibromin, a PKC substrate that may exert a positive role in neuronal differentiation. In this report, we studied the dynamics of PKC/Ras/ERK pathway signaling, during differentiation of SH‐SY5Y neuroblastoma cells upon treatment with the PKC agonist, phorbol ester 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA). Surprisingly, we observed that, among other PKC‐dependent signaling events, TPA induced a rapid and sustained decrease of neurofibromin immunoreactivity which was not due to proteolysis. Instead, we identified a specific phosphorylation event at the C‐tail of neurofibromin. This phosphorylation was acute and correlated perfectly with the signaling dynamics of the Ras/ERK pathway. Moreover, it persisted throughout prolonged treatment and TPA‐induced differentiation of SH‐SY5Y cells, concurrently with sustained activation of ERK1/2. Most importantly, C‐tail phosphorylation of neurofibromin correlated with a shift of neurofibromin localization from the nucleus to the cytosol. We propose that PKC‐dependent, sustained C‐tail phosphorylation is a requirement for prolonged recruitment of neurofibromin from the nucleus to the cytosol in order for a fine regulation of Ras/ERK pathway activity to be achieved during differentiation.     

Synthesis and characterization of new aromatic aldehyde/ketone 4-(β-d-glucopyranosyl)thiosemicarbazones

A series of 22 aromatic aldehyde/ketone 4-(β-d-glucopyranosyl)thiosemicarbazones have been synthesized by condensation of 4-(per-O-acetylated-β-d-glucopyranosyl)thiosemicarbazide with an aldehyde or a ketone, and then, deacetylation of the resulting product. The compounds were fully characterized by spectroscopic techniques, elemental analysis, and for two derivatives by X-ray analysis. The data indicate the β configuration, and the E configuration pertaining to the stereochemistry of the CN bond. However, a partial conversion of the E-form into the Z-form is possible in solution after several hours.