Potential of biotechnological conversion of lignocellulose hydrolyzates by Pseudomonas putida KT2440 as a model organism for a bio‐based economy

Lignocellulose‐derived hydrolyzates typically display a high degree of variation depending on applied biomass source material as well as process conditions. Consequently, this typically results in variable composition such as different sugar concentrations as well as degree and the presence of inhibitors formed during hydrolysis. These key obstacles commonly limit its efficient use as a carbon source for biotechnological conversion. The gram‐negative soil bacterium Pseudomonas putida KT2440 is a promising candidate for a future lignocellulose‐based biotechnology process due to its robustness and versatile metabolism. Recently, P. putida KT2440_xylAB which was able to metabolize the hemicellulose (HC) sugars, xylose and arabinose, was developed and characterized. Building on this, the intent of the study was to evaluate different lignocellulose hydrolyzates as platform substrates for P. putida KT2440 as a model organism for a bio‐based economy. Firstly, hydrolyzates of different origins were evaluated as potential carbon sources by cultivation experiments and determination of cell growth and sugar consumption. Secondly, the content of major toxic substances in cellulose and HC hydrolyzates was determined and their inhibitory effect on bacterial growth was characterized. Thirdly, fed‐batch bioreactor cultivations with hydrolyzate as the carbon source were characterized and a diauxic‐like growth behavior with regard to different sugars was revealed. In this context, a feeding strategy to overcome the diauxic‐like growth behavior preventing accumulation of sugars is proposed and presented. Results obtained in this study represent a first step and proof‐of‐concept toward establishing lignocellulose hydrolyzates as platform substrates for a bio‐based economy.     

Study of the mechanisms of crocetin-induced differentiation and apoptosis in human acute promyelocytic leukemia cells

Crocetin, the major carotenoid in saffron, exhibits potent anticancer effects. However, the antileukemic effects of crocetin are still unclear, especially in primary acute promyelocytic leukemia (APL) cells. In the current study, the potential antipromyelocytic leukemia activity of crocetin and the underlying molecular mechanisms were investigated. Crocetin (100 µM), like standard anti-APL drugs, all-trans retinoic acid (ATRA, 10 µM) and As₂O ₃ (arsenic trioxide, 50 µM), significantly inhibited proliferation and induced apoptosis in primary APL cells, as well as NB4 and HL60 cells. The effect was associated with the decreased expressions of prosurvival genes Akt and BCL2, the multidrug resistance (MDR) proteins, ABCB1 and ABCC1 and the inhibition of tyrosyl-DNA phosphodiesterase 1 (TDP1), while the expressions of proapoptotic genes CASP3, CASP9, and BAX/BCL2 ratio were significantly increased. In contrast, crocetin at relatively low concentration (10 µM), like ATRA (1 µM) and As ₂O ₃ (0.5 µM), induced differentiation of leukemic cells toward granulocytic pattern, and increased the number of differentiated cells expressing CD11b and CD14, while the number of the immature cells expressing CD34 or CD33 was decreased. Furthermore, crocetin suppressed the expression of clinical marker promyelocytic leukemia/retinoic acid receptor-α ( PML/RARα) in NB4 and primary APL cells, and reduced the expression of histone deacetylase 1 ( HDAC1) in all leukemic cells. The results suggested that crocetin can be considered as a candidate for future preclinical and clinical trials of complementary APL treatment.     

Caseoperoxidase,mixed β-casein–SDS–hemin–imidazole complex: a nano artificial enzyme

A novel peroxidase-like artificial enzyme, named “caseoperoxidase”, was biomimetically designed using a nano artificial amino acid apo-protein hydrophobic pocket. This four-component nano artificial enzyme containing heme–imidazole–β-casein–SDS exhibited high activity growth and k_cat performance toward the native horseradish peroxidase demonstrated by the steady state kinetics using UV–vis spectrophotometry. The hydrophobicity and secondary structure of the caseoperoxidase were studied by ANS fluorescence and circular dichroism spectroscopy. Camel β-casein (Cβ-casein) was selected as an appropriate apo-protein for the heme active site because of its innate flexibility and exalted hydrophobicity. This selection was confirmed by homology modeling method. Heme docking into the newly obtained Cβ-casein structure indicated one heme was mainly incorporated with Cβ-casein. The presence of a main electrostatic site for the active site in the Cβ-casein was also confirmed by experimental methods through Wyman binding potential and isothermal titration calorimetry. The existence of Cβ-casein protein in this biocatalyst lowered the suicide inactivation and provided a suitable protective role for the heme active-site. Additional experiments confirmed the retention of caseoperoxidase structure and function as an artificial enzyme.     

Ugi Bis-Amide Derivatives as Tyrosinase Inhibitor; Synthesis,Biology Assessment,and in Silico Analysis

Herein, a straightforward synthetic strategy mediated by Ugi reaction was developed to synthesize novel series of compounds as tyrosinase inhibitors. The structures of all compounds were confirmed by FT-IR, ¹H-NMR, ¹³C-NMR, and CHNOS techniques. The tyrosinase inhibitory activities of all synthesized derivatives 5a – m were determined against mushroom tyrosinase and it was found that derivative 5c possesses the best inhibition with an IC₅₀ value of 69.53±0.042 μM compared to the rest of the synthesized derivatives. Structure–activity relationships (SARs) showed that the presence of 4-MeO or 4-NO₂ at the R² position plays a key role in tyrosinase inhibitory activities. The enzyme kinetics studies showed that compound 5c is an noncompetitive inhibitor. For in silico study, the allosteric site detection was first applied to find the appropriate binding site and then molecular docking and molecular dynamic studies were performed to reveal the position and interactions of 5c as the most potent inhibitor within the tyrosinase active site. The results showed that 5c bind well with the proposed binding site and formed a stable complex with the target protein.     

Caspase-dependent apoptosis induced by two synthetic halogenated flavanones, 3′,7-dichloroflavanone and 3′,6-dichloroflavanone,on human breast and prostate cancer cells

The destruction of cancer cells with chemotherapeutic agents is normally achieved through apoptosis. We previously introduced two synthetic halogenated flavanone derivatives, 3^′,7-dichloroflavanone (3′-7 DCF) and 3^′,6-dichloroflavanone (3′-6 DCF), as potential apoptosis-inducing agents. In the current study, we investigated the ability of these compounds in triggering intrinsic or/and extrinsic pathway of apoptosis in breast and prostate cancer cells. Also, the synergistic effect of 3′-7 DCF with TLR3 (Toll-like receptor 3) agonist in apoptosis induction was evaluated on PC3 and LNCaP human prostate cancer cells. The involved pathway of apoptosis in the treated cells was delineated by caspase-3 activity assay, PARP-1 (poly(ADP-ribose)polymerase-1) cleavage, and procaspase-9 cleavage as markers of the intrinsic pathway and procaspase-8 cleavage as the marker of the extrinsic pathway. With the exception of the normal cells, treatment of all cell lines with both 3′-7 DCF and 3′-6 DCF triggered the cleavage of procaspase-8 and procaspase-9. These results indicate that the intrinsic and the extrinsic pathways of apoptosis are the mechanisms of the toxicity of flavanones in these cancer cell lines. However, the cytoxicity of the compound 3′-7 DCF was not synergistic with TLR3 agonist. Interestingly, the activation of caspases-9 preceeded that of caspase-8 suggesting that the intrinsic pathway is the primary reason for apoptosis induction by the flavanones.     

Effect of chronic lithium administration on endothelium-dependent relaxation of rat mesenteric bed: role of nitric oxide

The mechanism of action of lithium, an effective treatment for bipolar disease, is still unknown. In this study, the mesenteric vascular beds of control rats and rats that were chronically treated with lithium were prepared by the McGregor method, and the mesenteric vascular bed vasorelaxation responses were examined. NADPH-diaphorase histochemistry was used to determine the activity of NOS (nitric oxide synthase) in mesenteric vascular beds. We demonstrated that ACh-induced vasorelaxation increased in the mesenteric vascular bed of rats treated with lithium. Acute No-nitro-L-arginine methyl ester (L-NAME) administration in the medium blocked ACh-induced vasorelaxation in the control group more effectively than in lithium-treated rats, while the vasorelaxant response to sodium nitroprusside, a NO donor, was not different between lithium-treated and control groups. Acute aminoguanidine administration blocked ACh-induced vasorelaxation of lithium-treated rats, but had no effect in the control rats. Furthermore, NOS activity, determined by NADPH-diaphorase staining, was significantly greater in the mesenteric vascular beds from chronic lithium-treated rats than in those from control rats. These data suggest that the enhanced ACh-induced endothelium-derived vasorelaxation in rat mesenteric bed from chronic lithium-treated rats might be associated with increased NOS activity, likely via iNOS. Simultaneous acute L-NAME and indomethacin administration suggests the possible upregulation of EDHF (endothelium-derived hyperpolarizing factor) in lithium-treated rats.     

Anticancer activity of N-heteroaryl acetic acid salts against breast cancer; in silico and in vitro investigation

Molecular Biology Reports - The present research was performed to assess N-heteroaryl acetic acid salts' anticancer activity against the breast cancer cell in order to introduce new inhibitory...     

Synthesis and SAR studies of 3-allyl-4-prenyloxyaniline amides as potent 15-lipoxygenase inhibitors

15-Lipoxygenases are one of the nonheme iron-containing proteins with ability of unsaturated lipid peroxidation in animals and plants. The critical role of the enzymes in formation of inflammations, sensitivities and some of cancers has been demonstrated in mammalians. Importance of the 15-lipoxygenases leads to development of mechanistic studies, products analysis and synthesis of their inhibitors. In this work new series of the 3-allyl-4-allyoxyaniline amides and 3-allyl-4-prenyloxyaniline amides were designed, synthesized and their inhibitory potency against soybean 15-lipoxygenase were determined. Among the synthetic amides, 3-allyl-4-(farnesyloxy)-adamantanilide showed the most potent inhibitory activity by IC(50) value of 0.69μM. SAR studies showed that in spite of prenyl length increases, the effects of the amide size and its electronic properties on the inhibitory potency became predominant. The SAR studies was also showed that the orientation of allyl and prenyloxy moieties toward Fe core of the SLO active site pocket is the most suitable location for enzyme inhibition.