Calcium ions do not influence the Aß(25-35) triggered morphological changes of lipid membranes.

We have studied the effect of calcium ions (Ca2+) at various concentrations on the structure of lipid vesicles in the presence of amyloid-beta peptide Aß(25-35). In particular, we have investigated the influence of calcium ions on the formation of recently documented bicelle-like structures (BLSs) emerged as a result of Aß(25-35) triggered membrane disintegration. First, we have shown by using small-angle X-ray and neutron scattering that peptide molecules rigidify the lipid bilayer of gel phase DPPC unilamellar vesicles (ULVs), while addition of the calcium ions to the system hinders this effect of Aß(25-35). Secondly, the Aß(25-35) demonstrates a critical peptide concentration at which the BLSs reorganize from ULVs due to heating and cooling the samples through the lipid main phase transition temperature (Tm). However, addition of calcium ions does not affect noticeably the Aß-induced formation of BLSs and their structural parameters, though the changes in peptide's secondary structure, e.g. the increased α-helix fraction, has been registered by circular dichroism spectroscopy. Finally, according to 31P nuclear magnetic resonance (NMR) measurements, calcium ions do not affect the lipid-peptide arrangement in BLSs and their ability to align in the magnetic field of NMR spectrometer. The influences of various concentrations of calcium ions on the lipid-peptide interactions may prove biologically important because their local concentrations vary widely in in-vivo conditions. In the present work, calcium ions were investigated as a possible tool aimed at regulating the lipid-peptide interactions that demonstrated the disruptive effect of Aß(25-35) on lipid membranes.

Sulfated Polysaccharides as a Fighter with Protein Non-Physiological Aggregation: The Role of Polysaccharide Flexibility and Charge Density.

Proteins can lose native functionality due to non-physiological aggregation. In this work, we have shown the power of sulfated polysaccharides as a natural assistant to restore damaged protein structures. Protein aggregates enriched by cross-ß structures are a characteristic of amyloid fibrils related to different health disorders. Our recent studies demonstrated that model fibrils of hen egg white lysozyme (HEWL) can be disaggregated and renatured by some negatively charged polysaccharides. In the current work, using the same model protein system and FTIR spectroscopy, we studied the role of conformation and charge distribution along the polysaccharide chain in the protein secondary structure conversion. The effects of three carrageenans (κ, ι, and λ) possessing from one to three sulfate groups per disaccharide unit were shown to be different. κ-Carrageenan was able to fully eliminate cross-ß structures and complete the renaturation process. ι-Carrageenan only initiated the formation of native-like ß-structures in HEWL, retaining most of the cross-ß structures. In contrast, λ-carrageenan even increased the content of amyloid cross-ß structures. Furthermore, κ-carrageenan in rigid helical conformation loses its capability to restore protein native structures, largely increasing the amount of amyloid cross-ß structures. Our findings create a platform for the design of novel natural chaperons to counteract protein unfolding.

Aggregation of Amyloidogenic Peptide Uperin-Molecular Dynamics Simulations.

Uperin 3.5 is a remarkable natural peptide obtained from the skin of toadlets comprised of 17 amino acids which exhibits both antimicrobial and amyloidogenic properties. Molecular dynamics simulations were performed to study the ß-aggregation process of uperin 3.5 as well as two of its mutants, in which the positively charged residues Arg7 and Lys8 have been replaced by alanine. All three peptides rapidly underwent spontaneous aggregation and conformational transition from random coils to beta-rich structures. The simulations reveal that the initial and essential step of the aggregation process involves peptide dimerization and the formation of small beta-sheets. A decrease in positive charge and an increase in the number of hydrophobic residues in the mutant peptides lead to an increase in the rate of their aggregation.

Interaction of Uperin Peptides with Model Membranes: Molecular Dynamics Study.

The interaction of antimicrobial and amyloid peptides with cell membranes is a critical step in their activities. Peptides of the uperin family obtained from the skin secretion of Australian amphibians demonstrate antimicrobial and amyloidogenic properties. All-atomic molecular dynamics and an umbrella sampling approach were used to study the interaction of uperins with model bacterial membrane. Two stable configurations of peptides were found. In the bound state, the peptides in helical form were located right under the head group region in parallel orientation with respect to the bilayer surface. Stable transmembrane configuration was observed for wild-type uperin and its alanine mutant in both alpha-helical and extended unstructured forms. The potential of mean force characterized the process of peptide binding from water to the lipid bilayer and its insertion into the membrane, and revealed that the transition of uperins from the bound state to the transmembrane position was accompanied by the rotation of peptides and passes through the energy barrier of 4-5 kcal/mol. Uperins have a weak effect on membrane properties.

The Ability of Some Polysaccharides to Disaggregate Lysozyme Amyloid Fibrils and Renature the Protein.

The deposition of proteins in the form of insoluble amyloid fibril aggregates is linked to a range of diseases. The supramolecular architecture of such deposits is governed by the propagation of ß-strands in the direction of protofilament growth. In the present study, we analyze the structural changes of hen egg-white lysozyme fibrils upon their interactions with a range of polysaccharides, using AFM and FTIR spectroscopy. Linear anionic polysaccharides, such as κ-carrageenan and sodium alginate, are shown to be capable to disaggregate protofilaments with eventual protein renaturation. The results help to understand the mechanism of amyloid disaggregation and create a platform for both the development of new therapeutic agents for amyloidose treatment, and the design of novel functional protein-polysaccharide complex-based nanomaterials.

Effects of Isoflavone-Rich NADES Extract of Pueraria lobata Roots and Astaxanthin-Rich Phaffia rhodozyma Extract on Prostate Carcinogenesis in Rats.

Prostate cancer (PCa) is one of the most common male malignancies worldwide. In the current study, we evaluated the effects of a natural deep eutectic solvent (NADES) extract of Pueraria lobata roots rich in isoflavones (ISF) and Phaffia rhodozyma extract rich in astaxanthin (ASX) on an N-methyl-N-nitrosourea plus testosterone PCa model in rats. ISF consisted of puerarin, daidzein, genistein, formononetin and other polyphenols, while ASX contained lipids and unsaturated species in addition to astaxanthin. Extracts were administered through a whole promotion period in daily doses shown by our group to successfully inhibit benign prostate hyperplasia (BPH) development - 200 mg/kg for ISF and 25 mg/kg for ASX. Though a similar effect was found for BPH processes accompanying PCa induction, the incidence of PCa in animals treated with placebo, ISF and ASX was 37%, 37% and 41%, respectively, showing no chemopreventive activity of ISF and ASX. PCa development was associated with a decrease in the Ca/Mg ratio in serum and an increase in prostate tissue. Treatment with both extracts produced a normalization effect on Ca balance in serum, which, combined with a decrease in the prostatic index, suggests some positive health effects of ISF and ASX.

Anionic lipids modulate little the reorganization effect of amyloid-beta peptides on membranes.

Amyloid-ß peptide interactions with model lipid membranes have been studied by means of small angle neutron scattering and molecular dynamics simulations. These interactions had been indicated recently as an origin of the membrane structure reorganizations between spherical small unilamellar vesicles and planar bicelle-like structures. In present work, we investigate the influence of charge on the peptide-triggered morphological changes by introducing the anionic lipid DMPS to the underlying DMPC membrane. Changes to the membrane thickness and the overall membrane structure with and without Aß25-35 incorporated have been investigated over a wide range of temperatures. Our results document the previously reported morphological reformations between bicelle-like structures present in gel phase and small unilamellar vesicles present in fluid phase to be independent from the charge existence in the system.

Hepatoprotective Activity of Lignin-Derived Polyphenols Dereplicated Using High-Resolution Mass Spectrometry, In Vivo Experiments, and Deep Learning.

Chronic liver diseases affect more than 1 billion people worldwide and represent one of the main public health issues. Nonalcoholic fatty liver disease (NAFLD) accounts for the majority of mortal cases, while there is no currently approved therapeutics for its treatment. One of the prospective approaches to NAFLD therapy is to use a mixture of natural compounds. They showed effectiveness in alleviating NAFLD-related conditions including steatosis, fibrosis, etc. However, understanding the mechanism of action of such mixtures is important for their rational application. In this work, we propose a new dereplication workflow for deciphering the mechanism of action of the lignin-derived natural compound mixture. The workflow combines the analysis of molecular components with high-resolution mass spectrometry, selective chemical tagging and deuterium labeling, liver tissue penetration examination, assessment of biological activity in vitro, and computational chemistry tools used to generate putative structural candidates. Molecular docking was used to propose the potential mechanism of action of these structures, which was assessed by a proteomic experiment.

κ-Carrageenan Hydrogel as a Matrix for Therapeutic Enzyme Immobilization.

During the last few decades, polysaccharide hydrogels attract more and more attention as therapeutic protein delivery systems due to their biocompatibility and the simplicity of the biodegradation of natural polymers. The protein retention by and release from the polysaccharide gel network is regulated by geometry and physical interactions of protein with the matrix. In the present work, we studied the molecular details of interactions between κ-carrageenan and three lipases, namely the lipases from Candida rugosa, Mucor javanicus, and Rhizomucor miehei-which differ in their size and net charge-upon protein immobilization in microparticles of polysaccharide gel. The kinetics of protein release revealed the different capability of κ-carrageenan to retain lipases, which are generally negatively charged; that was shown to be in line with the energy of interactions between polysaccharides and positively charged epitopes on the protein surface. These data create a platform for the novel design of nanocarriers for biomedical probes of enzymatic origin.

Spectroscopic study of antimicrobial peptides: Structure and functional activity.

Amphibians are a natural source of a large number of peptides with a wide range of functional activities. Here, a complex of spectroscopic methods including NMR-, FTIR-, CD-, and UV-spectroscopy was applied to characterize the structure and functional activity of megin-1, a peptide isolated from amphibian skin. The three-dimensional structure of two forms of the peptide was determined using solution NMR spectroscopy. Thermodynamic characteristics of the process of peptide transformation from linear to cyclic form were obtained. Antibacterial and antimycotic properties of the peptide, as well as its protease inhibitory activities, were analyzed.

Beta-rich intermediates in denaturation of lysozyme: accelerated molecular dynamics simulations.

Amyloid fibrillar aggregates play a critical role in many neurodegenerative disorders. Conversion of globular proteins into fibrils is associated with global conformational rearrangement and involves the transformation of α-helices to ß-sheets. In the present work, the accelerated molecular dynamics technique was applied to study the unfolding of hen egg-white lysozyme at elevated temperatures, and the transformation of the native structure to a disordered one was analyzed. The influence of the disulfide bonds on the conformational dynamics and the energy landscape of denaturation process was considered. Our results show that formation of the metastable ß-enriched conformers of individual protein molecules may precede the aggregation process. These ß-rich intermediates can play a role of bricks making up fibrils.Communicated by Ramaswamy H. Sarma.

Astaxantin and Isoflavones Inhibit Benign Prostatic Hyperplasia in Rats by Reducing Oxidative Stress and Normalizing Ca/Mg Balance.

Benign prostatic hyperplasia (BPH) is a common pathology among aging men. Despite the broad pharmacological interventions, the available remedies to treat BPH are yet not devoid of side effects. Herbal compounds are suggested to be an alternative option for the BPH treatment. In our study, we evaluated the effect of kudzu isoflavones and astaxanthin on the BPH animal model. The animals were randomly divided into five groups: control; testosterone-induced BPH group; and three BPH-induced groups, which received intragastrically for 28 days finasteride (5 mg/kg) as a positive control, isoflavones (200 mg/kg), and astaxanthin (25 mg/kg). BPH was induced by castration of animals and subsequent subcutaneous injections of prolonged testosterone (25 mg/kg). Prostate index and histology, biochemical parameters, and antioxidant activity were evaluated. A significant decrease in prostate weight, immunohistochemical markers, and normalization of prostate Ca/Mg ratio was found in all treatment groups. Astaxanthin treatment also resulted in decreased epithelial proliferation and normalized superoxide dismutase activity. In conclusion, both isoflavones and astaxanthin inhibited BPH development at a level comparable to finasteride in terms of prostate weight, prostatic epithelium proliferation, and prostate tissue cumulative histology score. These results suggest that isoflavones and especially astaxanthin could serve as a potential alternative therapy to treat BHP.

Amyloid-beta peptide (25-35) triggers a reorganization of lipid membranes driven by temperature changes.

The amyloid-beta peptide (Aß) is considered a key factor in Alzheimer's disease (AD) ever since the discovery of the disease. The understanding of its damaging influence has however shifted recently from large fibrils observed in the inter-cellular environment to the small oligomers interacting with a cell membrane. We studied the effect of temperature on the latter interactions by evaluating the structural characteristics of zwitterionic phosphatidylcholine (PC) membranes with incorporated Aß25-35 peptide. By means of small angle neutron scattering (SANS), we have observed for the first time a spontaneous reformation of extruded unilamellar vesicles (EULVs) to discoidal bicelle-like structures (BLSs) and small unilamellar vesicles (SULVs). These changes in the membrane self-organization happen during the thermodynamic phase transitions of lipids and only in the presence of the peptide. We interpret the dramatic changes in the membrane's overall shape with parallel changes in its thickness as the Aß25-35 triggered membrane damage and a consequent reorganization of its structure. The suggested process is consistent with an action of separate peptides or small size peptide oligomers rather than the result of large Aß fibrils.

Therapeutic effect of iodised serum milk protein, lycopene and their combination on benign prostatic hyperplasia induced in rats.

Benign prostatic hyperplasia (BPH) is a common chronic disease in ageing men. Synthetic inhibitors of 5α-reductase commonly used in BPH treatment have limited effectiveness and may cause side effects. Evaluation of iodised serum milk protein and lycopene therapeutic effect in rat BPH model was the aim of the present study. BPH was induced in male Wistar rats by surgical castration and subsequent testosterone administrations (25 mg/kg, 7 injections). Rats with induced BPH received lycopene (5 mg/kg), iodised serum milk protein (200 µg/kg) or their combination for 1 month daily. The efficacy of the treatment was evaluated by the prostate weight, prostatic index and ventral lobe epithelium thickness. In lycopene and iodised serum milk protein-treated rats, prostate weight and prostatic index were significantly reduced compared to control group; and lycopene and iodised serum milk protein used in combination yielded an additive effect. Thus, further investigation of combined supplementation with micronutrients and plant-derived substances in BPH models may help to find new opportunities or its safe and effective treatment.

Fibril fragments from the amyloid core of lysozyme: An accelerated molecular dynamics study.

Protein aggregation and formation of amyloid fibrils are associated with many diseases and present a ubiquitous problem in protein science. Hen egg white lysozyme (HEWL) can form fibrils both from the full length protein and from its fragments. In the present study, we simulated unfolding of the amyloidogenic fragment of HEWL encompassing residues 49-101 to study the conformational aspects of amyloidogenesis. The accelerated molecular dynamics approach was used to speed up the sampling of the fragment conformers under enhanced temperature. Analysis of conformational transformation and intermediate structures was performed. During the unfolding, the novel short-living and long-living ß-structures are formed along with the unstructured random coils. Such ß-structure enriched monomers can interact with each other and propagate into fibril-like forms. The stability of oligomers assembled from these monomers was evaluated in the course of MD simulations with explicit water. The residues playing a key role in fibril stabilization were determined. The work provides new insights into the processes occurring at the early stages of amyloid fibril assembly.

Rhizosphere Bacterium Rhodococcus sp. P1Y Metabolizes Abscisic Acid to Form Dehydrovomifoliol.

The phytohormone abscisic acid (ABA) plays an important role in plant growth and in response to abiotic stress factors. At the same time, its accumulation in soil can negatively affect seed germination, inhibit root growth and increase plant sensitivity to pathogens. ABA is an inert compound resistant to spontaneous hydrolysis and its biological transformation is scarcely understood. Recently, the strain Rhodococcus sp. P1Y was described as a rhizosphere bacterium assimilating ABA as a sole carbon source in batch culture and affecting ABA concentrations in plant roots. In this work, the intermediate product of ABA decomposition by this bacterium was isolated and purified by preparative HPLC techniques. Proof that this compound belongs to ABA derivatives was carried out by measuring the molar radioactivity of the conversion products of this phytohormone labeled with tritium. The chemical structure of this compound was determined by instrumental techniques including high-resolution mass spectrometry, NMR spectrometry, FTIR and UV spectroscopies. As a result, the metabolite was identified as (4RS)-4-hydroxy-3,5,5-trimethyl-4-[(E)-3-oxobut-1-enyl]cyclohex-2-en-1-one (dehydrovomifoliol). Based on the data obtained, it was concluded that the pathway of bacterial degradation and assimilation of ABA begins with a gradual shortening of the acyl part of the molecule.

Old Rats Are More Susceptible to Induction of Benign Prostatic Hyperplasia (BPH) at Comparative to Young Mature.

AIMS: The aim of the experiments was to find out the factors on which age-related sensitivity to the occurrence of BPH depends. METHODS: 45 Male Wistar rats aged 3 and 24 months were used. In each age group, there were intact rats and animals with induced BPH (by surgical castration + testosterone injections, 25 mg/kg x 7). On the 36th day of the experiment, blood was taken from rats to determine serum testosterone, cholesterol, triglycerides and glucose; then, the animals were autopsied, their prostates were weighed, and their morphology was studied. RESULTS: Young mature intact rats had much higher testosterone levels (6.2±0.93 nmol/l) than old intact (3.8±0.55 nmol/l), while the ratio of prostate weight was inverse. The weight of the prostate and prostatic index in old rats with induced BPH was significantly higher not only in comparison with the old intact rats but also with young animals after BPH induction. Morphologically, the inflammatory foci were determined not only in the prostates of old rats, which induced BPH, but also in intact animals. Besides, in old intact rats, the foci of prostate hyperplasia were often noted. CONCLUSION: Our experimental model indicates the important role of non-bacterial prostatitis in the pathogenesis of BPH. No metabolic disorders in BPH induction were revealed. The sensitivity of the prostate of old rats to BPH development is increasing despite the low concentrations of testosterone in the body. Age sensitivity to BPH is probably determined by a higher expression of androgen receptors in old animals.

Cisplatin effect on digital cytomorphometric and bioinformatic tumor cell characteristics in rat ovarian cancer model-a preliminary study.

BACKGROUND: Ovarian cancer is one of the most common diseases of the female reproductive system. The aim of this study was the investigation of the antitumor cisplatin effect on ascitic fluid tumor cells in the ovarian cancer experimental model by digital cytomorphometry and cell bioinformatic analysis. METHODS: Female Wistar rats were inoculated by ovarian cancer strain. After ovarian cancer transplantation rats were divided into two groups: control group-without cisplatin treatment, the experimental group-under cisplatin treatment. The ascitic fluid was taken on the 9-th day after tumor cell inoculation. Digital cytomorphometric and cytobioinformatic analysis were used to study ascitic fluid cancer cell morphofunctional changes under cisplatin treatment. RESULTS: Digital cytomorphometric characteristics of rat ovarian cancer ascitic cells were obtained. Tumor cells were classified into four groups according to their geometric size: small, medium, large, "gigantic". The algorithm and the computer program based on tumor cell morphometric characteristics were created to calculate such cell bioinformatic characteristic as information redundancy coefficient R. Three parameters were determined as the criteria of cisplatin effect on cancer cells: cell number, nuclear/cytoplasmic ratio, R-value. CONCLUSIONS: Data obtained suggest that cisplatin reduces the total cell number, the nuclear/cytoplasmic ratio and R-value thus indicates a decrease in cellular resistance and adaptive potential. The digital cytomorphometry and bioinformatics could be recommended as a testing system in the experimental or clinical study.

Cation-Zwitterionic Lipid Interactions Are Affected by the Lateral Area per Lipid.

Interactions of the divalent cations Ca2+ and Mg2+ with the zwitterionic lipid bilayers prepared of a fully saturated dipalmitoylphosphatidylcholine (DPPC) or a di-monounsaturated dioleoylphosphatidylcholine (DOPC) were studied by using the neutron scattering methods and molecular dynamics simulations. The effect on the bilayer structural properties confirms the direct interactions in all cases studied. The changes are observed in the bilayer thickness and lateral area. The extent of these structural changes, moreover, suggests various mechanisms of the cation-lipid interactions. First, we have observed a small difference when studying DPPC bilayers in the gel and fluid phases, with somewhat larger effects in the former case. Second, the hydration proved to be a factor in the case of DOPC bilayers, with the larger effects in the case of less hydrated systems. Most importantly, however, there was a qualitative difference between the results of the fully hydrated DOPC bilayers and the others examined. These observations then prompt us to suggest an interaction model that is plausibly governed by the lateral area of lipid, though affected indirectly also by the hydration level. Namely, when the interlipid distance is small enough to allow for the multiple lipid-ion interactions, the lipid-ion-lipid bridges are formed. The bridges impose strong attractions that increase the order of lipid hydrocarbon chains, resulting in the bilayer thickening. In the other case, when the interlipid distance extends beyond a limiting length corresponding to the area per lipid of ∼65 Å2, Mg2+ and Ca2+ continue to interact with the lipid groups by forming the separate ion-lipid pairs. As the interactions proposed affect the lipid membrane structure in the lateral direction, they may prove to play their role in other mechanisms lying within the membrane multicomponent systems and regulating for example the lipid-peptide-ion interactions.

Structural changes introduced by cholesterol and melatonin to the model membranes mimicking preclinical conformational diseases.

The structure and dynamics of membranes depend on many external and internal factors that in turn determine their biological functions. One of the widely accepted and studied characteristics of biomembranes is their fluidity. We research a simple system with variable fluidity tweakable via its composition. The addition of cholesterol is employed to increase the order of lipid chains, thus decreasing the membrane fluidity, while melatonin is shown to elevate the chain disorder, thus also the membrane fluidity. We utilize the densitometric measurements to show a shift of studied systems closer or further from the gel-to-fluid phase transition. The structural changes represented by changes to membrane thickness are evaluated from small angle neutron scattering. Finally, we look at the ability of the two additives to control the interactions between membrane and amyloid-beta peptides. Our results suggest that fluidizing effect of melatonin can promote an insertion of peptide within the membrane interior. Intriguingly, the latter structure relates possibly to an Alzheimer's disease preventing mechanism postulated in the case of melatonin.