Phosphorylation and stabilization of topoisomerase IIα protein by p38γ mitogen-activated protein kinase sensitize breast cancer cells to its poisons

Cancer drugs suppress tumor cell growth by inhibiting specific cellular targets. However, most drugs also activate several cellular nonspecific stress pathways, and the implications of these off-target effects are mostly unknown. Here, we report that p38γ, but not p38α, MAPK is specifically activated by treatment of breast cancer cells with topoisomerase II (Topo II) drugs, whereas paclitaxel (Taxol) does not have this effect. The activated p38γ in turn phosphorylates and stabilizes Topo IIα protein, and this enhances the growth inhibition by Topo II drugs. Moreover, p38γ activity was shown to be necessary and sufficient for Topo IIα expression, the drug-p38γ-Topo IIα axis is only detected in intrinsically sensitive but not resistant cells, and p38γ is co-overexpressed with Topo IIα protein in primary breast cancers. These results reveal a new paradigm in which p38γ actively regulates the drug-Topo IIα signal transduction, and this may be exploited to increase the therapeutic activity of Topo II drugs.     

Detergency effects of nanofibrillar amyloid formation on glycation of human serum albumin

The prolonged glycation of human serum albumin (HSA) results in significant changes in its structure. The identity of these structural changes and the influence of carbohydrates on these changes require further study. Here, we evaluated structural changes and amyloid formation of HSA upon incubation with Glc, Fru, or Rib. Fluorescence spectrophotometry, surface tension analysis, and transmission electron microscopy (TEM) were utilized to evaluate the structures of glycated HSA. The physicochemical properties including excess free energy, protein adsorption at the air-water interface, critical aggregation concentration (CAC), and surface activity indicated an increase in hydrophobicity and partial unfolding of HSA structure upon glycation. Thus, it appears that AGE products can act as detergents. Incubation of HSA with these sugars after 20 wks induced significant amyloid nanofibril formation. Together these results indicate that prolonged glycation of HSA is associated with a transition from helical structure to beta-sheet (amyloid formation).     

Fabrication and Characterization of Curcumin-Loaded Complex Coacervates Made of Gum Arabic and Whey Protein Nanofibrils

Food Biophysics - In this research, gum Arabic (GA) and whey protein nanofibrils (WPN) were employed for the encapsulation of curcumin as a bioactive compound with low water solubility through the...     

Oligomeric forms of insulin amyloid aggregation disrupt outgrowth and complexity of neuron-like PC12 cells

Formation of protein amyloid fibrils consists of a series of intermediates including oligomeric aggregates, proto-fibrillar structures, and finally mature fibrils. Recent studies show higher toxicity for oligomeric and proto-fibrillar intermediates of protein relative to their mature fibrils. Here the kinetic of the insulin amyloid fibrillation was evaluated using a variety of techniques including ThT fluorescence, Congo red absorbance, circular dichroism, and atomic force microscopy (AFM). The solution surface tension changes were attributed to hydrophobic changes in insulin structure and were detected by Du Noüy Ring method. Determination of the surface tension of insulin oligomeric, proto-fibrillar and fibrillar forms indicated that the hydrophobicity of solution is enhanced by the formation of the oligomeric forms of insulin compared to other forms. In order to investigate the toxicity of the different forms of insulin we monitored morphological alterations of the differentiated neuron-like PC12 cells following incubation with native, oligomeric aggregates, proto-fibrillar, and fibrillar forms of insulin. The cell body area, average neurite length, neurite width, number of primary neurites, and percent of bipolar cells and node/primary neurite ratios were used to assess the growth and complexity of PC12 cells exposed to different forms of insulin. We observed that the oligomeric form of insulin impaired the growth and complexity of PC12 cells compared to other forms. Together our data suggest that the lower surface tension of oligomers and their perturbation affects the morphology of PC12 cells, mainly due to their enhanced hydrophobicity and detergent-like structures.     

Thymidylate synthase and methionine synthase polymorphisms are not associated with susceptibility to childhood acute lymphoblastic leukemia in Kurdish population from Western Iran

In order to determine the influence of polymorphism in thymidylate synthase (TS 28-bp repeat) and methionine synthase (MS A2756G) genes on the susceptibility to acute lymphoblastic leukemia (ALL), 73 children with ALL and 128 age and sex matched unrelated healthy individuals from the Kermanshah Province of Iran were screened. The genotyping of TS 28-bp repeat and MS A2756G polymorphisms were performed by polymerase chain reaction (PCR) and PCR–RFLP, respectively. The frequency of TS 2R allele in patients and controls were 41.5 and 38%, respectively (Odds ratios (OR) = 1.13, 95%CI 0.73–1.74, P = 0.56). The allelic frequency of G allele of MS was higher (25%) in patients compared with healthy subjects (23%) (OR = 1.09, 95%CI 0.67–1.75, P = 0.71). Considering MS AA and TS 3R3R genotypes as reference indicated that individuals with MS GG + TS 2R2R genotypes have 1.3-fold increase in the risk of ALL (OR = 1.3, 95%CI 0.6–2.7, P = 0.5). Our results showed that neither TS 28-bp repeat nor MS A2756G polymorphisms are risk factors for susceptibility to ALL in Western Iran.     

Altered tubulin assembly dynamics with N-homocysteinylated human 4R/1N tau in vitro

Tau isoforms promote neuronal integrity through binding and stabilization of microtubule proteins (MTP). It has been shown that hyperphosphorylation of tau contributes to Alzheimer’s disease (AD) pathology and related tauopathies. However, other pathogenic modifications of tau have not been well characterized. It is well accepted that elevated level of homocysteine (Hcy) is associated with neurodegenerative diseases such as AD. As a result of N-homocysteinylation of lysine residues, Hcy becomes a component of proteins, as a protein–homocystamide adduct, which affects protein structure and function. Here we demonstrate that N-homocysteinylation of human tau (4R/1N isoform) inhibits its function via impaired tau–tubulin specific binding and MTP assembly dynamics in vitro.     

Design, synthesis, and anticancer activity of phosphonic acid diphosphate derivative of adenine-containing butenolide and its water-soluble derivatives of paclitaxel with high antitumor activity

Synthesis of adenine derivative of triphosphono-gamma-(Z)-ethylidene-2,3-dimethoxybutenolide 4 was accomplished by treatment of phosphonate 3 with 5-phosphoribosyl 1-pyrophosphate in the presence of 5-phosphoribosyl 1-pyrophosphate synthetase. It was found that triphosphonate 4 functions as an irreversible stoichiometric inactivator of the Escherichia coli ribonucleoside diphosphate reductase (RDPR). Triphosphonate 4 exhibited potent inhibitory activity against murine leukemias (L1210 and P388), breast carcinoma (MCF7), and human T-lymphoblasts (Molt4/C8 and CEM/0) cell lines. Paclitaxel ester derivatives of adenine-containing triphosphono-gamma-(Z)-ethylidene-2,3-dimethoxybutenolide 8-10 were also synthesized. Like triphosphonate 4, compound 8 exhibited inhibitory property toward RDPR. It also induced microtubule assembly similar to paclitaxel (5). The structure of the chlorodiester linker in 8 was found to account for this dual property. After treatment of MCF7 cells with compounds 4, 5, and 8, fluorescence microscope examination demonstrated the presence of nucleus shrinkage or segmentation. Bifunctional prodrug 8 exhibited higher lipophilicity than 4 and higher water-solubility than 5. Pro-dual-drug 8 exhibited more pronounced anticancer activity relative to that of the triphosphonate 4 and paclitaxel (5). In contrast, compound 9, resulting from the linkage of triphosphonate 4 and paclitaxel (5) through a diester unit, was only found to function as a highly water-soluble prodrug for paclitaxel (5). It induced microtubule assembly in vitro, but did not show inhibitory property toward RDPR. On the other hand, compound 10, an aggregate of triphosphonate 4 and paclitaxel (5), neither functioned as an inhibitor of RDPR nor exhibited microtubule assembly stimulating activity in vitro.     

Comparative analysis of refolding of chemically denatured beta-lactoglobulin types A and B using the dilution additive mode

The kinetic refolding of beta-lactoglobulin (BLG), types A and B, by beta-cyclodextrin, glucose and sorbitol has been investigated in aqueous solution using fluorescence, far UV-CD and UV-spectrophotometric techniques. A new Pd-complex has been used to denature the protein. CD and fluorescence studies indicated that when incubated with sugar, the denatured BLG is refolded into the native-like structure through the dilution additive mode resulting in a higher yield of active protein than without sugar. CD studies show that these sugars can induce a non-native alpha-helical structure in denatured BLG-A and -B, then aid in the refolding of the protein. Based on the present study, these sugars have a different effect on BLG-A than BLG-B because of their differences in protein thermal stability. BLG-A has a higher thermal stability than BLG-B due to differences in the amino acid sequences.     

The interaction of beta-lactoglobulin with ciprofloxacin and kanamycin; a spectroscopic and molecular modeling approach

A vast research has been conducted to find suitable and safe carriers for vital and pH-sensitive drugs including antibiotics. This article reports the use of easily accessible and abundant purified beta-lactoglobulin (β-LG) protein as the potential carrier of widely used Kanamycin (Kana) and Ciprofloxacin (Cip) antibiotics. Spectroscopic techniques (Fluorescence, UV–vis, Circular Dichroism) combined with molecular docking were used to determine the binding mechanism of these drugs. Fluorescence studies showed moderate binding affinity with the calculated binding constants K_Cip = 60.1 (±0.2)?×?10³ M^?1 and K_kana = 2.5 (±0.6)?×?10³ M^?1 with the order of Cip > Kana. Results of UV–vis were consistent with fluorescence measurements and demonstrated a stronger complexation for Cip rather than Kana. The secondary structure of β-LG was preserved upon interaction with Kana; however, a reduction in β-sheet content from 39.1 to 31.9% was convoyed with an increase in α-helix from 12.8 to 20.5% due to complexation of Cip. Molecular docking studies demonstrated that preferred binding sites of these drugs are not the same and several amino acids are involved in stabilizing the interaction. Based on the achieved results, Kana and Cip can spontaneously bind to β-LG and this protein may serve as their transport vehicle.