Afshari, M.,
Rahimmalek, M.,
Sabzalian, M.R.,
Miroliaei, M.,
Szumny, A. Industrial Crops and Products (09266690)208
Salvia subg. Perovskia is an aromatic medicinal plant from the Lamiaceae family. Essential oil (EO) content and composition, along with enzymatic and non-enzymatic antioxidants, were evaluated in 18 plant populations under three levels of irrigation for two consecutive years. Based on the GC-MS analysis, the main components of EO were borneol (1.18–36.53%), followed by camphor (0.54–32.17%), 1,8-cineole (12.44–29.26%), δ-3-carene (0.39–21.20%), myrcene (0.59–16.28%), and α- pinene (0.79–12.87%) in the studied treatment. Except for malondialdehyde and hydrogen peroxide in leaves and roots, all the examined parameters showed lower values in the first harvest year compared to those of the second year. The activities of antioxidant enzymes, total phenolics, and flavonoids of the extracts were substantially enhanced as stress intensified to reach maximum values in leaves under severe stress and in roots under moderate stress conditions. The maximum root tanshinones content was observed under moderate water deficit conditions, while the highest EO content was obtained in plants exposed to severe stress conditions. Taking into account the evidence provided by this study, it can be inferred that the use of water deficit stress can serve as an effective method to stimulate and improve antioxidant properties, as well as the quantity and quality of secondary metabolites in Salvia subg. Perovskia. © 2023 Elsevier B.V.
Current Research in Structural Biology (2665928X)7
Nowadays, one of the methods to prevent the progress of Alzheimer's disease (AD) is to prescribe compounds that inhibit the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. Researchers are actively pursuing compounds, particularly of natural origin, that exhibit enhanced efficacy and reduced side effects. The inhibition of AChE and BChE using natural flavonoids represents a promising avenue for regulating AD. This study aims to identify alternative flavonoids capable of modulating AD by down-regulating AChE and BChE activity through a molecular docking approach. Molecular docking analysis identified Ginkgetin and Kolaflavanone as potent inhibitors of AChE and BChE, respectively, among the selected flavonoids. Asn87 and Ala127 involved in the interactions of AChE-Ginkgetin complex through conventional hydrogen bonds. While in the BChE-Kolaflavanone complex, Asn83, Ser79, Gln 47, and Ser287 are involved. In vitro analysis further corroborated the inhibitory potential, with Ginkgetin exhibiting an IC50 of 3.2 mM against AChE, and Kolaflavanone displaying an IC50 of 3.6 mM against BChE. These findings underscore the potential of Ginkgetin and Kolaflavanone as candidate inhibitors for the treatment of AD through the inhibition of AChE and BChE enzymes. Nevertheless, additional in vitro and in vivo studies are imperative to validate the efficacy of these compounds. © 2024 The Authors
Applied Biochemistry and Biotechnology (02732289)196(9)pp. 6296-6314
The pathogenesis of diabetes is related to the amount of advanced glycation end products (AGEs) that are naturally generated from the attachment of glucose with tissue and circular proteins. Human serum albumin (HSA) is more susceptible to AGE occurrence than other circular proteins due to its sensitive sites and high abundance. Considering the location of hydroxyl groups in the structure of flavonoids, which play a major role in suppressing of AGEs generating pathways, the present study was conducted to compare the effect of the chemical peculiarities of five flavonoids: apigenin (AP), naringenin (NA), luteolin (LU), Quercetin (QU), and methylquercetin (MQ), in suppressing AGEs generated in the HSA/glucose system. The results showed that all used flavonoids are capable of quenching the fluorescence intensity of AGEs in vitro. Analytical methods including UV–visible spectroscopy, CD spectro-polarimetry, TNBS, DTNB, DNPH, Congo red assay, ThT, and ANS fluorescence were used to deeper analysis of flavonoid performance. The anti-AGE effects of flavonoids followed the order of LU > QU > MQ > AP > NA. Docking results showed that flavonoids are associated with glycation-prone lysines and arginine residues in the “Sudlow pocket” through non-covalent interactions. Hydroxylation at the C4′ and the double bond between C2-C3 increase the antiglycation potential of used flavonoids, while methylation of the OH group at the C3 position decreases this effect. It was also found that hydroxylation at C3 can play a dual role in anti-glycation ability. These findings may introduce a new approach to the structure-inhibition relationship of flavonoids in the design of operative anti-glycemic agents. © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2024.
Iranian Journal Of Chemistry And Chemical Engineering (10219986)43(5)pp. 1905-1918
In an effort to find effective medicines for the treatment of diabetes, the efficient mechanisms and lack of side effects of herbal medicines have made them the main candidates for regulating blood sugar levels and reducing the side effects of the disease. This search will be based on the discovery of digestive enzymes (α-amylase and α-glucosidase) inhibitors from natural sources and the ways to reduce high blood sugar levels. These enzymes are at the forefront of increasing blood glucose levels because they facilitate the digestion of food polysaccharides into smaller monosaccharide in small intestinal wall. Currently, the arsenal of inhibitors approved for this purpose is limited to veglibose, miglitol and acarbose. Despite the ability to reduce glucose absorption their widespread clinical use is limited due to the occurrence of gastrointestinal ailments. Given the efficacy of various natural compounds in alleviating diabetes symptoms, this review aims to assess the inhibitory potential and mode of action of some phytochemicals on intestinal digestive enzymes. Such an exploration seeks to unveil novel and healthful anti-diabetic agents based on the inhibition of digestive enzymes. © 2024, Iranian Institute of Research and Development in Chemical Industries. All rights reserved.
Rahimmalek, M.,
Szumny, A.,
Gharibi, S.,
Pachura, N.,
Miroliaei, M.,
Łyczko, J. Molecules (14203049)28(16)
Kelussia odoratissima Mozaff. is a species of Apiaceae endemic to the Zagros Mountains in Iran. In the present investigation, for the first time, the polyphenolic compounds and flavonoids of its leaves were determined by liquid chromatography-mass spectrometry (LC-MS). As a result, p-coumaric acid, ferulic acid, caffeic acid, chlorogenic acid, acetyl phloroglucinol, vanillic acid, m-coumaric acid, and 4-methylsiringol were determined as the main phenolic compounds, while 3-hydroxyflavone, flavone, quercetin, rutin, neohesperidin, polydatin, and diosmin were the main flavonoid components, of which chlorogenic acid (303.08 µL/gDW), neohesperidin (38.37 µL/gDw), and diosmin (28.62 µL/gDW) were the most abundant. Solid-phase microextraction (SPME) was also used to determine the chemical compounds. Based on SPME, (Z)-undec-6-en-2-one (17.48%) and (Z)-butylidenephthalide (4.348%) were the major components. Based on GC-MS analyses, (Z)-ligustilide was the main compound; however, some new compounds were also determined, including 3-ethylisobenzofuran-1 (3H)-one, (E)-ligugustilide, and E-n-butylidene phthalide. Also, for the first time, we have identified EOs ethyl and isobutyl phthalides on the basis of the obtained EI-MS spectra. Finally, the fragmentation of phthalides is also discussed in this research. © 2023 by the authors.
Sadeghi, M.,
Miroliaei, M.,
Rahimmalek, M.,
Taslimi, P.,
Szumny, A.,
Sadeghian, N. Journal of Oleo Science (13473352)72(8)pp. 787-797
Inhibition of α-amylase, α-glucosidase, and advanced glycation end products (AGEs) is considered a prospective method for the prevention of type II diabetes. As two flavonoids obtained from fruits, swertisin (SW) and apigenin (AP) have similar structures and display various pharmacological properties. To examine the effects of flavonoid structure on inhibition of AGEs adducts and carbohydrate hydrolyzing enzymes activity, molecular docking and molecular dynamic simulations (MDs) were used. The molecular docking method was performed by the Autodock program, and the ligand that showed the most negative binding energy was selected for further investigation. SW showed the potential ability to inhibit the AGEs formation and carbohydrate hydrolyzing enzymes activity. The stability of the receptor/SW complex was evaluated by MDs. Based on the findings of the present study, it was found that SW has the potential to reduce glycation and delay the activity of α-amylase and α-glucosidase enzymes. © 2023 by Japan Oil Chemists’ Society.
Sadeghi, M.,
Miroliaei, M.,
Ghanadian, M.,
Szumny, A.,
Rahimmalek, M. International Journal of Biological Macromolecules (01418130)253
Biflavonoids (BFs) are a group of polyphenols that have a unique biochemical structure. One of the key biomedical mechanisms that BFs can have high potential in managing Diabetes mellitus (DM) is α-glucosidase inhibition. Normally, elevated blood glucose levels are caused by high absorption of glucose in the epithelium of the small intestine. Since α-glucosidase helps increase the absorption of glucose in the small intestine in the final stage of glycan catabolism, inhibition of this essential biochemical process in diabetic patients can be considered a suitable approach in the treatment of this disease. The interaction between the BFs and α-glucosidase are still not clear, and need to be deeply investigated. Herein, the aim is to identify BFs with strong α-glucosidase inhibitory activity. Using docking-based virtual screening approach, the potential binding affinity of 18 selected BFs to α-glucosidase was evaluated. The dynamic activity and stability of α-glucosidase-BFs complexes were then measured by molecular dynamics simulation (MDs). “Strychnobiflavone” showed the best score in α-glucosidase inhibition. Arg315 and Phe303 involved in the interactions of α-glucosidase-strychnobiflavone complex through cation-π and π-π stacking, respectively. Based on in vitro kinetic studies, it was determined that the type of inhibition of “strychnobiflavone” corresponds to the pattern of mixed inhibitors. Furthermore, details of the interactions between strychnobiflavone and α-glucosidase were performed by in silico secondary structure content analysis. The findings showed when “strychnobifone” binds to the enzyme, significant alterations occur in the enzyme conformation affecting its catalytic activity. In general, the findings highlighted the potential of “strychnobiflavone” as a promising candidate for the treatment of diabetes mellitus through α-glucosidase inhibition. Further in vitro and in vivo studies have to confirm the therapeutic benefits of “strychnobiflavone” in conformational diseases such as diabetes mellitus. © 2023
Sadeghi, M.,
Miroliaei, M.,
Kamyabiamineh, A.,
Taslimi, P.,
Ghanadian, M. Arabian Journal Of Chemistry (18785352)16(10)
Advanced glycation end products (AGEs) are a heterogeneous group of complex chemical entities resulting from non-enzymatic reactions between reducing sugars with proteins, lipids and/or nucleic acids. AGEs tend to accumulate in cells and stimulate diverse signaling pathways that are closely related to the emergence of several chronic metabolic disorders. This review is based on keywords “medicinal plant”, “AGEs”, “AGEs complication”, “AGEs inhibitor” and their characteristics. The keywords are widely identified and checked in databases such as Science Direct, PubMed Medline, Scopus, and Google Scholar. The complex processes of AGEs formation and their impact on human health are reviewed along with recent developments in AGEs inhibitors derived from natural compounds. In addition, the mechanisms of action of natural inhibitors such as quercetin, lignan, chlorogenic acid, resveratrol and stilbenes are summarized in the protection of glycation-sensitive sites in proteins, removal of active carbonyl compounds, chelating metal ions, and reduction of blood glucose levels. Despite showing glycation-induced-free radicals scavenging activity, these compounds have not yet been widely used in clinical field. Therefore, such natural compounds with specific molecular frameworks might have great potential to pave the way of new drugs discovery. © 2023
Food and Function (20426496)13(19)pp. 10055-10068
α-Glucosidase is among the intestinal epithelial enzymes that produce absorbable glucose in the final stage of glycan catabolism. It leads to an increase in blood glucose levels as a result of high glucose uptake in diabetic patients. However, inhibition of this essential biochemical process can be a useful therapeutic approach to diabetes mellitus (DM). Eriocitrin (ER) is an abundant “flavanone glycoside” in citrus fruits with rich antioxidant properties whose effects on α-Glu inhibition in the small intestine remain to be determined. Herein, pH-sensitive microgels (MGs) were designed based on cross-linked methacrylate with acrylamide (AM) and acrylic acid (AAc) (molar ratio 70 : 30 of AAc : AM) as a controlled release system for sustained delivery of ER into the small intestine. The presence of amide and acrylate in MGs and the mechanical resistance were determined using FT-IR spectroscopy, rheology, and viscoelastometry. In vitro experiments showed that MGs could protect ER against diffusion in the gastric location and adjust its release in the intestinal milieu. The intestinal α-Glu activity was inhibited by ER (IC50 value of 12.50 ± 0.73 μM) in an uncompetitive dose-dependent manner. The presence of ER altered the structure of α-Glu and reduced the hydrophobic pockets of the enzyme. Molecular docking analysis along with molecular dynamics simulation displayed that ER-α-Glu formation is directed by hydrogen binding with Asp69, Asp215, Glu411, Asp307, and Tyr347 residues. Moreover, in vivo assessment showed that rat blood glucose concentration decreased after ER administration compared with the control group. The results highlight that ER-loaded-MGs can be considered as a useful releasing strategy in treating DM via α-Glu inhibition. © 2022 The Royal Society of Chemistry.
Sadeghi, M.,
Sadeghi, M.,
Miroliaei, M.,
Miroliaei, M.,
Taslimi, P.,
Taslimi, P.,
Moradi, M.,
Moradi, M. Structural Chemistry (15729001)33(4)pp. 1199-1212
Human serum albumin (HSA) is an important carrier protein in plasma with various functions, and it is exposed to glycation. Alkaloids are diverse compounds that have showed different anti-glycation activities. The present study aimed to investigate the anti-glycation activity of thirty alkaloid compounds. First, the compounds were studied via Lipinski’s rule; then, among thirty alkaloids, twenty-three were further designated for docking study, using AutoDock program. Three compounds which provided the minimum docking score were selected for further assays. Jatrorrhizine displayed potential activity to inhibit the HSA glycation. The stability of the jatrorrhizine-HSA complex was confirmed by molecular dynamics simulation (MDs). To approve the efficacy of this compound, future in vitro and in vivo studies are required. © 2022, The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.
Journal Of Molecular Modeling (16102940)28(5)
There is a growing interest in designing a nanocarrier containing an EGFR targeting affibody to direct toward cancer cells. Here, cytolysin A was cloned at the N-terminus of ZEGFR:1907 affibody to guarantee its surface presentation on the OMVs while targeting the epidermal growth factor receptors (EGFRs). A separate construct including a fusogenic peptide (GALA) was also designed for the endosomal escape of the nanocarrier. Binding of the two constructs ClyA-affiEGFR and ClyA-affiEGFR-GALA to domain III of EGFR was investigated using molecular docking and molecular dynamic simulations. The higher stability of the ClyA-affiEGFR-GALA/EGFR as compared to the ClyA-affiEGFR/EGFR complex was evident. The ClyA-affiEGFR-GALA structure showed a higher RMSD during the first half of the simulation time implying a much less stable behavior. Plateau state of the radius of gyration plot of ClyA-affiEGFR-GALA confirmed a well-folded structure in the presence of the GALA sequence. Solvent accessible surface area for both proteins was in the same range. The data obtained from hydrogen bond analysis revealed a more equilibrated and stable form of the ClyA-affiEGFR-GALA structure upon interaction with EGFR. The data provided here was a requisite for our biological evaluation of the synthesized constructs as a component of a novel drug delivery system. Graphical abstract: [Figure not available: see fulltext.] © 2022, The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.
International Journal of Biological Macromolecules (01418130)217pp. 714-730
Flavonoid glycosides (FGs) appear to be good candidates for controlling blood glucose levels, so regular consumption of vegetables/fruits rich in FGs may prevent the consequences of type 2 diabetes (DM). Inhibition of digestive enzymes using natural FGs is a suitable dietary tool to regulate the hydrolysis of polysaccharides and overcome hyperglycemia. The aim of the current research is to find FGs that can effectively inhibit the digestive enzymes α-glucosidase (α-Gl) and α-amylase (α-Am). Accordingly, twenty-three FGs were selected and filtered through docking-based virtual screening. Based on the molecular docking and molecular dynamics (MD) simulation, among the 23 selected FGs, nicotiflorin and swertisin significantly inhibited α-Gl and α-Am, respectively. In vitro analysis revealed the inhibitory capacity of nicotiflorin on α-Gl was equal to IC50 at 0.148 mg/ml and the inhibitory activity of swertisin on α-Am was equal to IC50 at 1.894 mg/ml. It was found that nicotiflorin and swertisin act much like as a competitive inhibitor on α-Gl and α-Am, respectively. Furthermore, the fluorescence intensity of both enzymes decreased after interaction with two FGs. FT-IR and scanning electron microscopy (SEM) measurements suggested that the interactions could alter the conformation and microenvironment of the enzymes. Moreover, in vivo evaluation showed that the administration of nicotiflorin and swertisin can alleviate the blood glucose level of rats compared to the starch group (p < 0.05). The findings highlight that nicotiflorin and swertisin can be considered as possible inhibitors in treating diabetes mellitus via digestive enzymes inhibition. © 2022
Sadeghi, M.,
Sadeghi, M.,
Miroliaei, M.,
Miroliaei, M.,
Fateminasab, F.,
Fateminasab, F.,
Moradi, M.,
Moradi, M. Journal Of Molecular Modeling (16102940)28(1)
Inflammation is a natural protective response toward various simulators, including tissue damage or pathogens. The cyclooxygenase-2 (COX-2) is a very important protein in triggering pain and inflammation. Previous studies have claimed that Allium sativum offers a wide range of anti-inflammatory therapeutics for human consumption. Drug discovery is a complicated process, though in silico methods can make this procedure simpler and more cost-effective. At the current study, we performed the virtual screening of eight Allium sativum–derived compounds via molecular docking with COX-2 enzyme and confirmed the binding energy by docking score estimate followed by ADMET and drug-likeness investigation. The resulting highest-docking scored compound was exposed to molecular dynamics simulation (MDS) for evaluating stability of the docked enzyme-ligand complex and to gauge the oscillation and conformational alterations for the time of enzyme-ligand interaction. The factors of RMSD, RMSF, hydrogen bond interactions, and Rg after 100 ns of MDS proved the stability of alliin in the active site of COX-2 in comparison with celecoxib (CEL) as the control. Moreover, we investigated the binding affinity analysis of all compounds via MM/PBSA method. The results from this study suggest that alliin (a sulfuric compound) exhibits a higher binding affinity for the COX-2 enzyme compared to the other compounds and CEL. Alliin showed to be a possible anti-inflammatory therapeutic candidate for managing the inflammatory conditions. © 2021, The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.
Russian Journal of Plant Physiology (16083407)68(5)pp. 890-900
Abstract: Azospirillum brasilense may regulate MN052803-LTP expression and the activity of defensive enzymes in wheat (Triticum aestivum L.) cultivars to improve salinity tolerance. In a primary experiment, germination indexes of 18 wheat cultivars were measured and Sorkhtokhm and Qods were selected as tolerant and sensitive cultivars to salinity, respectively. Selected cultivars inoculated with A. brasilense (Sp245 produce more ABA and Sp7 as standard strain) and grown-up to five days, then salinity (200 mM NaCl) was applied to seedling via Hoagland’s nutrient solution. The relative expression of MN052803-LTP (authors recorded in the Gen Bank) of roots and shoots was measured at 12, 24, and 48 hours after salinity applied. The results showed that MN052803-LTP expression increased in the order of salinity, inoculation, and inoculation plus salinity. Meanwhile, phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) activity increased in the same order in 12 days-old seedlings. In a similar experiment, 10 mM dithiothreitol (DTT) was used as a reducer and inoculation as a stimulator of MN052803-LTP expression, then the relative expression and phosphatidylcholines (PC) content were measured. Although the MN052803-LTP expression and PC was reduced due to the application of DTT, inoculation eliminates its inhibitory effect. The highest amount of PC was observed in inoculated plants, and the lowest in the plants treated with DTT. Probably, A. brasilense improves salt tolerance of wheat cultivars through MN052803-LTP expression, and PC content via repairing the membrane damages by supplying the membrane phospholipids, such as phosphatidylcholine, and accumulation of antioxidant compounds by activating PAL and TAL via membrane lipid-dependent signaling cascades. © 2021, Pleiades Publishing, Ltd.
Iranian Journal of Diabetes and Metabolism (23454008)20(3)pp. 200-209
Background: Diabetes mellitus is a metabolic syndrome characterized by elevated blood glucose. The α-glucosidase enzymes that are found in the small intestine are responsible for the hydrolysis of carbohydrates. The aim of this study was to Bioinformatics evaluation of T.foenum active compounds in suppression of α-glucosidase enzyme. Methods: This study was a descriptive-analytical method. For this purpose, the compounds separation of Trigonella foenum were first downloaded from PubChem database and then the α-glucosidase enzyme file was obtained from PDB database. The toxicity class of compounds and the Lipinski rules were predicted by Toxtree & Protox II and the Swiss ADME server, respectively. Finally, molecular docking and enzyme interaction with the compounds in Trigonella foenumwere performed by AutoDock Tools 1.5.6 and Molegro Virtual Docker 6.0. Interaction results were also analyzed using Discovery Studio 3.5 & Ligplot 2.1 software. Results: The results indicated that all selected of compounds in Trigonella foenumwere in follow with Lipinski's rules, proper binding energy, and lack of toxicity were appropriate options for α-glucosidase inhibition. But among these compounds, Vitexin had the lowest binding energy and the most inhibitory effect on the α-glucosidase enzyme, with-4.8 kcal/mol. These compounds also had lower binding energy than standard inhibitor (Voglibose). Conclusion: From the results of this study, it can be concluded that among the most important compounds in Trigonella foenum, the Vitexin compound power inhibitor that due to more hydrogen and hydrophobic interactions with the α-glucosidase enzyme active site. © 2021, Tehran University of Medical Sciences. All rights reserved.
Scientific Reports (20452322)11(1)
Non-enzymatic glycation of DNA and the associated effects are among pathogenic factors in diabetes mellitus. Natural polyphenols have anti-diabetic activity. Herein, the protective role of one of the phytochemicals, rosmarinic acid (RA), was evaluated in glycation (with fructose) of human DNA and expression of Akt genes in the hippocampus of diabetic rats. In-vitro studies using fluorescence, agarose gel electrophoresis, fluorescence microscopy, and thermal denaturation analyses revealed that glycation causes DNA damage and that RA inhibits it. In-vivo studies were performed by induction of diabetes in rats using streptozotocin. The diabetic rats were given RA daily through gavage feeding. The expression of Akt genes (inhibitors of apoptosis) in the hippocampus was evaluated using RT-qPCR. In diabetic rats, Akt1 and Akt3 were significantly down-regulated compared to the control group. Treating the diabetic rats with RA returned the expression of Akt1 and Akt3 relatively to the normal condition. Past studies have shown that diabetes induces apoptosis in the hippocampal neurons. Given that glycation changes the genes expression and causes cell death, apoptosis of the hippocampal neurons can be due to the glycation of DNA. The results also suggest that RA has reliable potency against the gross modification of DNA under hyperglycemic conditions. © 2021, The Author(s).
Frontiers in Pharmacology (16639812)12
Bacterial outer membrane vesicles (OMVs) have recently drawn a great deal of attention due to their therapeutic efficiency and ability to target specific cells. In the present study, we sought to probe engineered OMVs as novel and promising carriers to target breast cancer cells. Following the fusion of the affiEGFR-GALA structure to the C-terminal of ClyA as an anchor protein, the ClyA-affiEGFR-GALA construct was successfully expressed on the surface of ∆msbB/∆pagP E. coli W3110-derived OMVs. Morphological features of the engineered and wild-type OMVs were identical. The engineered OMVs induced no endotoxicity, cytotoxicity, or immunogenicity, indicating the safety of their application. These OMVs could specifically bind to EGF receptors of MDA-MB-468 cells expressing high levels of EGFR and not to those with low levels of EGFR (HEK293T cells). Interestingly, despite a lower binding affinity of the engineered OMVs relative to the positive control Cetuximab, it was strong enough to identify these cells. Moreover, confocal microscopy revealed no uptake of the modified OMVs by the EGFR-overexpressing cells in the presence of EGFR competitors. These results suggest that OMVs might internalize into the cells with EGF receptors, as no OMVs entered the cells with any EGFR expression or those pretreated with EGF or Cetuximab. Regarding the EGFR-binding affinity of the engineered OMVs and their cellular uptake, they are presented here as a potential carrier for cell-specific drug delivery to treat a wide variety of cancer cells. Interestingly, the engineered OMVs are capable of reaching the cytoplasm while escaping the endosome due to the incorporation of a fusogenic GALA peptide in the construct. Copyright © 2021 Sepahdar, Miroliaei, Bouzari, Khalaj and Salimi.
Journal of Chemical Technology and Biotechnology (02682575)95(8)pp. 2243-2250
BACKGROUND: Widespread application of enzymes for research and industrial purposes is dependent on enhancing the catalytic capacity. One promising approach to this end is the immobilization of enzymes on novel nanomaterials. In this study, a novel nanocarrier was developed for the immobilization of enzyme on polycaprolactone (PCL)-functionalized magnetic nanoparticles. RESULT: Structural and physicochemical characterization of the PCL-modified magnetic nanocarrier was achieved via Fourier-transform infrared (FTIR) spectra, scanning electron microscopy (SEM), transmission electron microscopy (TEM), thermogravimetric analysis (TGA) and vibration sample magnetometry (VSM). In order to determine the optimal conditions for immobilization, different parameters such as pH, temperature, reaction time and enzyme concentration were evaluated. Based on the results, the optimal biocatalytic activity of immobilized α-amylase was obtained at 65 °C and pH 6.5. Under appropriate conditions, the immobilized α-amylase showed improved storage stability in comparison with free one. Additionally, the reusability of the immobilized system demonstrated >50% enzyme activity, at optimal pH and temperature, after five reaction cycles. CONCLUSION: This finding could be useful in the application of immobilized enzymes for analytical purposes, industrial exploitations, biotechnology, biomedical engineering and other bio-applications. © 2020 Society of Chemical Industry. © 2020 Society of Chemical Industry
International Journal of Biological Macromolecules (01418130)151pp. 797-805
Despite most studied activities of natural biophenols rely on antioxidant properties, little clues explored their key structural components with regard to opposing action on glycation-induced aggregation. Herein, human hemoglobin (hHb)/fructose system used to decipher if structural peculiarities of two biophenols “chlorogenic acid (CGA) and curcumin (CUR)” are effective toward AGEs-bridged aggregate formation. Suppression in amyloid cross-β formation was monitored by CD spectroscopy, fluorescence microscopy, ANS and AGE fluorescence. Reduction in molten globule structure of modified-Hb by CGA was corroborated with helix structure, thiol group and lysine residues content estimation for native, glycated and biophenols treated samples. ThT and Congo red assays showed the cross-β breaking properties of CGA. Molecular docking outcomes revealed the positioning of CGA/CUR is driven by “aromatic interactions” with Trp β1180 and Tyr α2540. These interactions are modulated by the structural constraints such as number of hydroxyl groups and their methylation status directing the biophenols to the amyloidogenic core. The results are applicable to formulation of small-molecule nutraceuticals for treatment of conformational diseases. © 2018
Industrial Crops and Products (09266690)154
The Lamiaceae family is a major source of species among medicinal plants that are highly valued for their antioxidant properties and multiple pharmaceutical uses in folk medicine. Satureja, Origanum, and Dracocephalum are some of the major genera belonging to the Lamiaceae family. The present study used seven Iranian endemic species of the Lamiaceae family, viz. one from each of S. hortensis, S. bachtiarica, S. sahendica, and D. kotschyi species as well as three ecotypes from O. vulgare and compared them in terms of their antioxidant activities. S. hortensis exhibited the highest total phenolic (266.82 mg Tannic acid/g DW) and total flavonoid (22.63 mg Quercetin/g DW) contents with the highest antioxidant activity (IC50 = 95.52 μg/mL), while the least activity was recorded for the aqueous extract of Origanum. High performance liquid chromatography (HPLC) was also performed to determine the major polyphenols. Rosmarinic acid was identified as the predominant phenolic compound in all the studied Lamiaceae species. Three Satureja species richer in phenolics and flavonoids with a substantial antioxidant activity were also selected to investigate their antiglycation properties. For comparison, the antiglycative activity was also assessed in bovine serum albumin/methylglyoxal system. The highest and lowest anti-glycative activities were recorded for S. hortensis and S. sahendica, respectively. Finally, S. hortensis was found to produce the highest amounts of such polyphenols as rosmarinic, syringic, caffeic, and ferulic acids basically considered to be the main phenolic acids responsible for the antioxidant and anti-glycative activities of the plant extracts. © 2020 Elsevier B.V.
Industrial and Engineering Chemistry Research (15205045)58(23)pp. 9844-9854
Both physical and chemical procedures were applied to immobilize yeast alcohol dehydrogenase (yADH) on titania nanoparticles. It was found that chemical immobilization is an authentic method due to providing a strong bond between the enzyme and the support. The immobilization was confirmed by Fourier transform infrared spectroscopy and transmission electron microscopy. The immobilization parameters such as enzyme concentration, time of immobilization, and glutaraldehyde concentration were optimized based on the maximum immobilization yield and the best enzyme catalytic performance. Activity and kinetics of yADH before and after immobilization were studied, and the stability of enzyme at different pHs and temperatures was investigated. The optimum pH for the incubation and activity of yADH was obtained at 7.0, and the activity of immobilized yADH reached more than 80% of its initial activity after 30 days of storage at 4 °C. The reusability of yADH was improved by immobilization as revealed by retaining 84% of its initial activity following 10 cycles. Finally, we suggested that the immobilization of yADH is a promising method for the removal of substrate inhibition in the reaction of formaldehyde to methanol. © 2019 American Chemical Society.
International Journal of Biological Macromolecules (01418130)113pp. 354-360
Enzyme immobilized on magnetic nanoparticles (MNPs) can be used as efficient recoverable biocatalysts under strong magnetic responses. In the present work, α-amylase was immobilized onto naringin functionalized MNPs via ionic interactions. For this purpose, the MNPs were functionalized with naringin, as a biocompatible flavonoid. The morphology, structure, and properties of functionalized MNPs and the immobilization of α-amylase on synthesized nanocomposite were characterized through different analytical tools including TGA, VSM, FTIR, SEM-EDX and TEM. Furthermore, the optimum conditions of temperature, pH, reaction time and enzyme concentration for immobilization process were investigated. The results showed that the optimal conditions for immobilization of α-amylase onto synthesized nanocarrier occurred at pH 6.5 and 55 °C. The reusability experiments revealed high activity maintenance of immobilized α-amylase even after 10 reaction cycles. Moreover, the storage stability of immobilized enzyme improved via immobilization in comparison with free one and it maintained 60% of its initial activity after 6 weeks storage at 4 °C. The improvements in enzyme catalytic properties via immobilization made this nanobiocatalyst as a good candidate in bio-industrial applications. Furthermore, the synthesized nanocomposite would have the potential for practical applications in other and binary enzyme immobilization. © 2018 Elsevier B.V.
Chemistry and Biodiversity (16121872)15(8)
A comparative study was carried out on the methanolic extracts from six Achillea species and the examined polyphenols from these plants on the formation of advanced glycation end-products (AGE) in vitro. A. pachycephala which was richer in flavonoids (15 mg quercetin/g W) and phenolics (111.10 mg tannic acid/g DW) with substantial antioxidant activity (IC50 = 365.5 μg/ml) presented strong anti-AGE properties. Chlorogenic acid, luteolin, quercetin and caffeic acid were identified as the major polyphenols in the extracts by HPLC. In general, polyphenolic content follows the order of A. pachycephalla > A. nobilis > A. filipendulina > A. santolina > A. aucheri > A. millefolium. Most extracts exhibited marked anti-AGE ability in the bovine serum albumin (BSA)/methylglyoxal (MG) system, though A. pachycephala showed the highest potential. The formation of AGEs was assessed by monitoring the production of fluorescent products and circular dichroism (CD) spectroscopy. Diminution in free radical production (assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays) is discussed as potential mechanism for delay or reduced AGE. The results demonstrate the antiglycative, antioxidant and antimicrobial potential of Achillea species which can be attributed to polyphenols content and the effectiveness on generation of AGEs, thus Achillea species can be considered as natural sources for slowing down glycation related diseases. © 2018 Wiley-VHCA AG, Zurich, Switzerland
Miroliaei, M.,
Aminjafari, A.,
Ślusarczyk, S.,
Nawrot-hadzik, I.,
Rahimmalek, M.,
Matkowski, A. Pharmacognosy Magazine (09731296)13(51)
Background: Protein glycation and glycotoxicity belong to the main oxidative-stress related complications in diabetes. Perovskia species are used in Asian folk medicine as antidiabetic herbs. Objective: The aim of this study was to verify the ability of the methanolic extract from Perovskia atriplicifolia Benth. roots to diminish glycation of albumin and to prevent cell damage in vitro. Furthermore, we tested the extract for in vitro antioxidant activity and inhibition of elastase and collagenase. Material and Methods: The aqueous methanol extract was analyzed by UHPLC-MS for the content of polyphenols and terpenoids. The prevention of glycated albumin-induced cell damage was tested in four mammalian cell lines (peripheral blood mononuclear cells, human embryonic kidney cells - HEK293, normal human fibroblasts, and Chinese hamster ovary cells) with the 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazoly)-3-(4-sulfophenyl) tetrazolium assay. Results: Glycated albumin is significantly more toxic than native human serum albumin (LC50from 35.00 to 48.34 μ g/mL vs. 5.47-9.10 μ g/mL, respectively). The extract, rich in rosmarinic acid (344.27 mg/g dry mass), mitigated the glycated albumin toxicity, and increased glycated albumin-treated cell survival by more than 50%. The inhibition of advanced glycation endproduct formation was confirmed by monitoring conformational changes. The free radical scavenging activity was higher than Trolox and metal reducing power was one-third to half that of ascorbic acid. The activity of elastase and collagenase was inhibited by 54.75% ± 6.87% and 60.03% ± 7.22%, respectively. Conclusions: The results confirm antiglycative and antiglycotoxic potential of Perovskia root and its traditional antidiabetic use. The high activity can be attributed to rosmarinic acid abundance. © 2017 Pharmacognosy Magazine | Published by Wolters Kluwer - Medknow.
Aminjafari, A.,
Miroliaei, M.,
Angelova, V.T.,
Emamzadeh, R.,
Djukic, M.M.,
Djuric, A.,
Saso, L. Electronic Journal Of Biotechnology (07173458)24pp. 43-48
Background: Synthesized aminocoumarins are heterocyclic compounds possessing potential for the treatment of insulin-dependent diabetes mellitus with unexplored anti-glycative action. Results: In this study 4-aminocoumarin derivatives (4-ACDs) were evaluated in vitro for antiglycation (AG) activities by using the human serum albumin (HSA)/glucose system, for 8 weeks of incubation. The glycation and conformational alteration of HSA in the presence of the tested compounds were evaluated by Congo red assay, fluorescence and circular dichroism spectroscopy. The antioxidant (AO) capacity were also tested by four different assays including: DPPH (2,2′-diphenyl-1-picrylhydrazyl radical), ABTS (2,2-azinobis (3-ethylbenzothiazoline-6-sulphonate) diammonium salt), FRAP (ferric reducing antioxidant power) and β-carotene-linoleic acid assay. The tested compounds showed AG and AO effects. The intensity of the accomplished AO potential is related to the type of the used assay. Significant alterations in the secondary (monitored by CD spectropolarimetry) and tertiary structure (assessed by spectrofluorimetry) of HSA upon glycation were mitigated by the 4-ACDs, suggesting their suppressive role in the late stage (post-Amadori) of the HSA glycation. Conclusions: By the analogues, in vitro ascertained AO and AG properties of 4-ACDmay be recognized as rationale for their protective role against oxidative changes of proteins, thereby precluding diabetic complications in humans. © 2016 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved.
Computational Biology and Chemistry (14769271)58pp. 9-18
Development of a protein-based drug delivery system has major impact on the efficacy and bioavailability of unstable and water insoluble drugs. In the present study, the binding modes of a nonspecific lipid transfer protein (nsLTP2) from Oryza sativa with various nucleosides and analogous molecules were identified. The 3-D structure of the protein was designed and validated using modeler 9.13, Molegro virtual docker and procheck tool, respectively. The binding affinity and strength of interactions, key contributing residues and specificity toward the substrates were accomplished by computational docking and model prediction. The protein presented high affinity to acyclovir and vidarabine as purine-analogous drugs. Binding affinity is influenced by the core template and functional groups of the ligands which are structurally different cause the variation of interaction energies with nsLTP2. Nonetheless, all the evaluated analogous drugs occupy the proximity space at the nsLTP active site with high similarity in their binding modes. Our findings hold great promise for the future applications of nsLTPs in various aspects of pharmaceutical science and molecular biology. © 2015 Published by Elsevier Ltd.
Rahgozar, S.,
Amirian, T.,
Qi m., M.,
Shahshahan, Z.,
Entezar-e-ghaem, M.,
Tehrani, H.G.,
Miroliaei, M.,
Krilis s.a., S.A.,
Giannakopoulos b., B. PLoS ONE (19326203)10(8)
Objective: Angiotensinogen exists in two distinct redox forms in plasma, the oxidized sulfhydryl-bridge form and the reduced, unbridged, free thiol form. The oxidized form of angiotensinogen compared to the free thiol form preferentially interacts with renin resulting in increased generation of angiotensin. The predictive potential of the ratio of free-thiol to oxidized angiotensinogen in the plasma for pre-eclampsia was first suggested by the Read group in ref 10. We propose an improved method for determining the ratio and validate the method in a larger cohort of pregnant women. Methods: Plasma samples from 115 individuals with pre-eclampsia and from 55 healthy pregnant control subjects were collected sequentially over a 2 year period. Using two distinct enzyme-linked immunosorbent assays (ELISAs) the plasma levels of total and free thiol angiotensinogen were quantified. The oxidized angiotensinogen plasma level is derived by subtracting the level of free thiol, reduced angiotensinogen from the total angiotensinogen levels in the plasma. Results: The relative proportion of free thiol angiotensinogen, expressed as a percentage of that observed with an in-house standard, is significantly decreased in pre-eclamptic patients (70.85% ± 29.49%) (mean ± SD) as compared to healthy pregnant controls (92.98 ± 24.93%) (mean ± SD) p ≤ 0.0001. The levels of total angiotensinogen did not differ between the two groups. © 2015 Rahgozar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Tousheh, M.,
Miroliaei, M.,
Asghar rastegari, A.,
Ghaedi, K.,
Esmaeili, A.,
Matkowski, A. Computers in Biology and Medicine (00104825)43(11)pp. 1732-1738
A computational study was carried out to identify the structural determinant controlling the affinity, specificity and binding strength of several saturated and unsaturated fatty acids with Oryza sativa (Indica group) nonspecific lipid transfer protein (nsLTP2). Association between the number, position and conformation of hydrophobic patches and lipid binding properties of the protein was evidenced by docking analysis. Binding affinity is influenced by the number of carbon atoms, location of double bonds and hydroxyl group in the acyl chain. The results may direct at developing applications in LTP-mediated transport and controlled release of low molecular weight drugs. © 2013 Elsevier Ltd.
Journal of Spinal Cord Medicine (20457723)36(3)pp. 231-236
Background: Induction of p75 neurotrophin receptor (p75NTR) could be one of the first steps that initiate apoptotic cascade after injury, or it may indicate regeneration responses undertaken by the injured system, possibly in collaboration with resident tropomyosin-receptor-kinase (Trk). Objective: To measure quantitative changes in messenger RNA (mRNA) expression levels of p75NTR, Trk A, and caspase-9 in rat's injured spinal cord (SCI). The reciprocal interaction between Trk and p75NTR signaling pathways can dictate cellular responses to neurotrophins. p75NTR can regulate Trk-dependent responses, but the role of Trk in regulating p75NTR-dependent signaling is not well documented. Design: Using real-time polymerase chain reaction, this study analyzed changes in the mRNA abundance of the mentioned genes at 6, 24, and 72 hours and 7 and 10 days after SCI in adult male rats. SCI was induced at T9 level by transsection. Results: Results show a complicated temporal and spatial pattern of alteration with different degrees and direction (up- or down-regulation) in p75NTR, Trk A, and caspase-9 mRNA expression levels after SCI. The greatest variation was seen in center regions following SCI. This study shows that alteration in p75NTR, Trk A, and caspase-9 expression starts as early as 6 hours after SCI. Alterations in p75NTR, Trk A, and caspase-9 expression within the spinal cord may play a key role in the apoptotic cell death. Conclusion: Results suggest that the role of p75NTR is to eliminate damaged cells by activating the apoptotic machinery, especially at the center of damage and during first week after injury. © The Academy of Spinal Cord Injury Professionals, Inc. 2013.
Food Chemistry (18737072)129(2)pp. 267-271
Lemon balm (Melissa officinalis) is a medicinal herb possessing functional compounds with unexplored anti-glycative action. The anti-glycative activity of Lemon balm extract was evaluated in the bovine serum albumin (BSA)/glucose system. The level of glycation, conformational alterations and protein binding to RAGE receptors were assessed by specific fluorescence, Congo red binding assay, circular dichroism, ligand and Western blotting. Ethanol fractions of Melissa leaf exhibited the highest inhibitory effects on the formation of advanced glycation end products (AGEs) and the late stage of glycation process. Significant alteration in the secondary structure of albumin was observed upon glycation, which was mitigated by applying the herb extract. Moreover, upon treatment with balm extract, glycated albumin adopts a secondary structure impeding its detection by RAGE receptors of microglial cells. Our results represent the anti-glycative properties of Melissa extract and its application for possible treatment of AGE-associated diseases. © 2011 Elsevier Ltd. All rights reserved.
Barzegar, A.,
Moosavi-movahedi, A.,
Pedersen, J.Z.,
Miroliaei, M. Enzyme and Microbial Technology (18790909)45(2)pp. 73-79
The present study demonstrates the comparative thermal, conformational and kinetic stabilities of the three closely related enzymes; the mesophilic yeast alcohol dehydrogenase (YADH), horse liver alcohol dehydrogenase (HLADH), and the extreme-thermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH). The mid-point unfolding temperatures for TBADH and HLADH were at least 10 °C and 6 °C higher, respectively, than that of YADH. When YADH was completely inactivated by thermal stress, the residual activities of HLADH and TBADH were 70% and 100%, respectively. The optimum temperature (Topt) activities of HLADH and TBADH were at least 40 °C and 55 °C higher, respectively, than that of YADH. Due to the higher rigidity of HLADH and TBADH, the enzymatic activation energies of HLADH and TBADH were higher than that of YADH. Geometric X-ray analysis indicated a comparatively higher coil (turn and loop) percentage in TBADH and HLADH than in YADH. Pairwise alignment for TBADH/HLADH exhibited a similarity score approximately 2.5-fold greater than that of the TBADH/YADH pair. Multiple alignments made with ClustalW revealed a higher number of conserved proline residues in the two most stable enzymes (HLADH/TBADH). These extra prolines tend to occur in surface loops and are likely to be responsible for the increased stability of TBADH and HLADH, by loop rigidification. © 2009 Elsevier Inc. All rights reserved.
Scientia Iranica (23453605)14(4)pp. 303-307
The aim of this work was to evaluate the possibilities of immobilization of lactoperoxidase (LPO) on concanavalin A-Sepharose 4B support. Significant biospecific interaction of this heme-containing glycoenzyme with Con A was established by using α-D-mannopyranoside and α-D-glucopyranoside, which are involved in the linking of the carbohydrate moieties of the enzyme with lectin. The preparation obtained indicated improved kinetic parameters (Km and Vmax), compared with the soluble form. No significant differences were observed between the optimal pH and temperature of the anchored and free enzymes. The thermal stability of the biospecifically immobilized preparation was substantially higher than that of the unbound enzyme. In addition, seven cycles of enzymatic conversion and washing of the column showed the remarkable operational stability of immobilized LPO. © Sharif University of Technology, August 2007.
Scientia Iranica (23453605)14(2)pp. 112-117
A thermophilic alcohol dehydrogenase from Thermoanaerobacter brockii (TBADH) was immobilized by adsorption on Fractosil, methyl-, octyl-, and hexadecyl-Fractosil. As compared to its free form, the thermal stability of the enzyme was enhanced upon immobilization and its pH and temperature optima were altered. The immobilized preparations were used in continuous catalytic operations using a packed-bed reactor. It was demonstrated that the inorganic supports are suitable candidates for operating reactors, especially at high temperatures. Furthermore, larger volumes of the substrates could be converted to a product using enzyme preparations immobilized on the derivatized (hydrophobic) Fractosil, as compared with the unsubstituted form. © Sharif University of Technology, April 2007.
Enzyme and Microbial Technology (18790909)40(4)pp. 896-901
The present communication reports on changes in the secondary and tertiary structures of native and apo-yeast alcohol dehydrogenase upon heating at 50 °C, as evident from circular dichroism (CD) studies. The presence of sugars provided significant protection with trehalose being the most effective. Exposure of hydrophobic clusters in the protein molecule upon heat denaturation was confirmed by fluorescence studies using 1-anilinonaphthalene-8-sulfonate (ANS) as a hydrophobic reporter probe. All sugars, and especially trehalose, reduced the affinity of both forms of the enzyme for this probe. The effectiveness of sugars in diminishing ANS fluorescence enhancement is in accordance with their ability to protect aggregation of the proteins, reported earlier [Miroliaei M, Nemat-Gorgani M. Sugars protect native and apo yeast alcohol dehydrogenase against irreversible thermoinactivation. Enzyme Microb Technol 2001;29:554-9]. It is concluded that prevention of the mechanisms of irreversible thermoinactivation may occur with retention of the secondary and tertiary structural properties of the proteins. © 2006 Elsevier Inc. All rights reserved.
International Journal of Biological Macromolecules (01418130)39(4-5)pp. 192-196
Structural properties of modified forms of glucose oxidase made by stepwise specific modification have been investigated. By a single step modification, one of the modified forms resulted in the conversion of native structure of glucose oxidase to molten globule like form [S. Hosseinkhani, B. Ranjbar, H. Naderi-Manesh, M. Nemat-Gorgani, FEBS Lett. 561 (2004) 213-216]. Chemical modification of lysine residues in glucose oxidase was carried out using different concentration of citraconic anhydride. Modification brought about changes in the tertiary structure with some degree of alteration in secondary structure. FTIR, far and near-UV CD spectropolarimetry, intrinsic and extrinsic fluorescence spectroscopy showed structural changes of glucose oxidase in a concentration dependent manner. This was supported by comparative study of secondary and tertiary structure. © 2006 Elsevier B.V. All rights reserved.
International Journal of Biochemistry and Cell Biology (13572725)34(2)pp. 169-175
A comparative study was performed regarding the catalytic activity and stability of two related enzymes (thermophilic alcohol dehydrogenase from Thermoanaerobacter brockii and its mesophilic counterpart from yeast) in the presence of a number of miscible and immiscible organic solvents. The study was performed in view of the practical usefulness of organic solvents for alcohol dehydrogenases which have been shown to catalyse a variety of industrially-important dehydrogenation reactions. A number of organic solvents of different physicochemical characteristics were used and substantial stabilization was achieved. The non-polar solvents utilized showed the ability to enhance thermal stability of both proteins. Protection against thermal denaturation was especially pronounced by n-dodecane, the solvent with the highest log P used in the present study. Dimethylformamide and dioxane, employed as two miscible organic solvents, showed the ability to cause substrate inhibition and changes in protein conformation as indicated by kinetic and fluorescence studies. A higher resistance of the thermophilic protein to the deleterious effect of pyridine and thermostabilization of the mesophilic enzyme by non-polar solvents are especially emphasized. Combined differences in protein structure and nature of organic solvents are suggested to explain the differences in stability and catalytic activity observed in the present investigation. © 2002 Published by Elsevier Science Ltd.
Enzyme and Microbial Technology (18790909)29(8-9)pp. 554-559
In the present study, irreversible thermoinactivation of holo- and apo-yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) and protection by sugars (mannitol, sorbitol, sucrose and trehalose) were investigated at 50°C and pH 7.8. The apo-protein was obtained by removing the structural zinc with the catalytic zinc remaining on the enzyme. Thiol group oxidation, aggregation and deamidation were examined. Hypochlorous acid and cupric chloride were used in relation to thiol group oxidation. Zn2+ mobilization was measured spectrophotometrically using the metallochromic indicator 4-(2-pyridylazo)resorcinol (PAR). The presence of sugars provided significant protection against all the three deleterious processes. It is concluded that use of sugars may provide an effective approach for stabilization of holo- and apo-YADH via alteration of protein microenvironment. © 2001 Elsevier Science Inc. All rights reserved.
