Recombinant Expression and Functional Assessment of Uricase from a Pertinent Origin of the Enzyme, Streptomyces sp. Strain 17-1.

Background: Uricase or urate oxidase, as a therapeutic enzyme, is extensively applied to oxidize accumulated uric acid in the body to soluble form to treat related illnesses. Objectives: This study was conducted with the aim of searching for potential sources of uricase-producing Streptomyces from Eshtehard salt desert in Alborz province, Iran and heterologous expression, purification and functional assay of the enzyme. Materials and Methods: Main screening was conducted by cultivation of the strains on a medium enriched with 0.3 percent (w/v) uric acid. The uricase gene from the most potent strain was then recombinantly expressed in E. coli BL21 (DL3). Results: Out of the tested strains, only seven showed uricase activity. The highest level of native uricase activity (11.5735 U.mL-1) belonged to strain 17-1, which had the closest similarity to Streptomyces nigra. A recombinant uricase with a molecular mass of approximately 38 kDa was produced. The purified uricase exhibited a specific activity of about 28.29±0.59 U.mg-1, which is among the highest level of uricase activity reported by other studies. Conclusions: This enzyme is a promising candidate for further applicable investigations and large-scale production in terms of its large volume of soluble expression and selective competitive activity.

Induction of heat shock protein expression in SP2/0 transgenic cells and its effect on the production of monoclonal antibodies.

The objective of the current investigation was to evaluate the induction of heat shock proteins (HSPs) in SP2/0 transgenic cells and the effect of these proteins on the production of monoclonal antibodies (mAbs). The SP2/0 cell line expressing the PSG-026 antibody, a biosimilar candidate of golimumab, the culture parameters, and the target protein expression were not justified for industrial production and were used for the experiments. Paracetamol and heat shock were used as chemical and physical inducers of HSPs, respectively. The results showed that paracetamol and heat shock increased the expression of HSP70 and HSP27 at the mRNA and protein levels. The expression of HSPs was greater in paracetamol-treated cells than in heat shock-treated cells. Paracetamol treatment at concentrations above 0.5 mM significantly reduced cell viability and mAb expression. However, treatment with 0.25 mM paracetamol results in delayed cell death and increased mAb production. Heat shock treatment at 45°C for 30 minutes after enhanced mAb expression was applied after pre-treatment with paracetamol. In bioreactor cultures, pretreatment of cells with paracetamol improved cell viability and shortened the lag phase, resulting in increased cell density. The production of mAbs in paracetamol-treated cultures was markedly greater than that in the control. Analysis of protein quality and charge variants revealed no significant differences between paracetamol-treated and control cultures, indicating that the induction of HSPs did not affect protein aggregation or charge variants. These findings suggest that inducing and manipulating HSP expression can be a valuable strategy for improving recombinant protein production in biopharmaceutical processes.

Enhancing resistance and cell survival in Acipenser ruthenus liver, gill, and kidney cells: The potential of heat shock protein inducers against PAH-benzo[a]pyrene stress.

The Caspian Sea has faced many environmental challenges, such as oil pollution. Heat shock proteins (HSPs) play a critical role in stress conditions and physiological changes caused by disease or injury. By evaluating the effects of various HSP inducers (HSPi), including Pro-Tex® (NOP: 800 mM), amygdalin (AMG: 80 mM), and a novel synthetic compound derived from pirano piranazole (SZ: 80 µm) on isolated cells from Sterlet Sturgeon (Acipenser ruthenus) treated with 75% IC50 PAH-benzo[a]pyrene (BaP; B75). This study examines whether there is a correlation between exposure to the BaP pollutant and HSPs in fish. In vitro, after culturing cells from the liver, kidney, and gills, they were treated with HSPi compounds in the presence and absence of BaP. Western blotting was used to assess HSP27, HSP70, and HSP90 expression patterns. A variety of enzyme activities were measured before (without treatment) and after treatment with HSPis and HSPi + B75, including cytochrome P450 (CYP450) activity, specific enzyme activity for acetylcholinesterase (AChE), antioxidant capacity, liver indicator enzymes, cortisol levels, and immunity parameters. When compared to the control group, cells treated with B75 showed the lowest AChE enzyme activity (p < 0.0001). CYP450 activity was highest in group B75, while HSPi caused the opposite effect (p < 0.0001). HSPi + B75 increased HSP levels and antioxidant parameters while decreasing cortisol and liver indicator enzymes (p < 0.0001). HSPi may be a powerful and reliable method for enhancing the resistance of A. ruthenus to BaP stresses before exposure. Treating cells with HSP-inducing compounds, such as NOP, AMG, and SZ, can assist them in managing stress and increase HSP (27, 70, and 90) protein expression. Furthermore, the study findings suggest that HSPis can also mitigate the adverse effects of stress, ultimately increasing cell survival and resistance.

Using heat shock protein (HSP) inducers as an approach to increase the viability of sterlet (Pisces; Acipenseridae; Acipenser ruthenus) cells against environmental diazinon toxicity.

Diazinon (DZN) is an organophosphate pesticide frequently used in agriculture and released into aquatic environments. In this study, sterlet sturgeon cells were exposed to DZN to investigate possible defense mechanisms via HSP induction (HSPi). Liver, kidney, and gill cells of Acipenser ruthenus were isolated and cultured and then treated with HSPi (Pro-Tex®, amygdalin, and a novel pirano-piranazole-based synthesized compound: SZ) in the presence and absence of DZN. MTT assays were used to evaluate the effects of different HSPis and their combinations with DZN. Western blotting analysis was conducted to evaluate HSP27, HSP70, and HSP90 expression patterns in each group. The highest rates of caspase-3 and caspase-8 activities were found in the DZN group, whereas HSPi treatment resulted in the lowest rates. The combination of HSPi+DZN resulted in increased HSP levels and antioxidant parameters but decreased cortisol, immune parameters, and metabolic enzymes. Many of the studied parameters (caspases, acetylcholinesterase, antioxidant, immune, and metabolic parameters) showed significant correlations with HSP expression, indicating that HSPs may be associated with markers of sterlet cell health. The results of this study demonstrate that using HSP inducers may be a powerful and reliable way to increase A. ruthenus resistance prior to exposure to DZN.

Protective effects of HSP Inducer on diazinon-exposed stellate sturgeon (Acipenser stellatus) fry: Insights on HSP70 gene expression, immune response, and enzyme indices.

Aquatic environments face frequent exposure to organophosphate pesticides, such as diazinon, which are frequently utilized in agriculture. The goal of this study was to evaluate the effects of diazinon exposure on fish and to investigate the potential of the HSP inducer (HSPi) in developing a defense mechanism. To achieve this, several factors were analyzed, including the HSP70 gene expression, levels of immunity markers (lysozyme, IgM, and C3), antioxidant status, and the activity of acetylcholine esterase (AChE). Stellate sturgeon (Acipenser stellatus) fry, was exposed to diazinon (25, 50, and 75% of 96h-LC50) for 6 days after pre-treatment with an HSP inducer (HSPi), TEX-OE® (a prickly pear cactus extract), for 4 hours. Two HSPi concentrations, 100 and 200 mg.L-1, were used. Pre-treatment with HSPi significantly enhanced HSP70 gene expression in the gill and liver, as well as immune markers in the blood of Acipenser stellatus. Diazinon-treated groups exhibited higher antioxidant activities of SOD, CAT, and T-AOC. Increased activity also observed in control fish pre-treated with HSPi. However, stellate sturgeon receiving both diazinon and HSPi+diazinon experienced a significant decrease in AChE activity in comparison with control group. Cortisol levels were elevated in the fish that were subjected to diazinon. Those subjected to diazinon after receiving HSPi showed a significant decrease in cortisol levels. In conclusion, the study suggests that HSPi-mediated HSP70 induction may have a protective effect. The presence of an HSP inducer offers a potential strategy to mitigate the consequences of diazinon exposure in stellate sturgeon.

Biological responses of stellate sturgeon fingerlings (Acipenser stellatus) immersed in HSP inducer to salinity changes.

Changes in salinity is a stressful and energy-consuming process in fish which give rise to mortalities, especially in fish fingerlings that are more sensitive during the early stages of their life. In the present study, the effects of three salinities, 3‰ (downstream of river), 8‰ (estuarine), and 13‰ (the maximum salinity in the Caspian Sea), on HSP70 gene expression, cortisol level, immune response (lysozyme, complement C3, IgM), and antioxidant enzyme activities (SOD, CAT, T-AOC) of the stellate sturgeon fingerlings in the presence of HSP inducer compound (TEX-OE®) were evaluated. Our results showed that levels of plasma cortisol and heat shock protein (HSP70) in Acipenser stellatus fingerlings increased due to salinity changes. In the presence of the HSP inducer, HSP70 expression in both gill and liver was significantly increased, whereas cortisol level was notably decreased. Exposure to salinity changes resulted in an increase in antioxidant defense activities (SOD, CAT, and T-AOC) and immune response (lysozyme, IgM, and C3) in the presence of an HSP inducer. In conclusion, an HSP-inducing compounds can have a positive effect in strengthening the immunity and antioxidant system of sturgeon fingerlings by increasing the expression of the HSP70 gene against salinity fluctuations and generally increase the body's physiological tolerance.

Gene cloning and characterization of a novel recombinant 40-kDa heat shock protein from Mesobacillus persicus B48.

The present study reports the recognition and characterization of the gene encoding the co-chaperone DnaJ in the halophilic strain Mesobacillus persicus B48. The new extracted gene was sequenced and cloned in E. coli, followed by protein purification using a C-terminal His-tag. The stability and function of the recombinant DnaJ protein under salt and pH stress conditions were evaluated. SDS-PAGE revealed a band on nearly 40-kDa region. The homology model structure of new DnaJ demonstrated 56% similarity to the same protein from Streptococcus pneumonia. Fluorescence spectra indicated several hydrophobic residues located on the protein surface, which is consistent with the misfolded polypeptide recognition function of DnaJ. Spectroscopic results showed 56% higher carbonic anhydrase activity in the presence of the recombinant DnaJ homolog compared to its absence. In addition, salt resistance experiments showed that the survival of recombinant E. coli+DnaJ was 2.1 times more than control cells in 0.5 M NaCl. Furthermore, the number of recombinant E. coli BL21+DnaJ colonies was 7.7 times that of the control colonies in pH 8.5. Based on the results, DnaJ from the M. persicus can potentially be employed for improving the functional features of enzymes and other proteins in various applications.

A VEGFB-Based Peptidomimetic Inhibits VEGFR2-Mediated PI3K/Akt/mTOR and PLCγ/ERK Signaling and Elicits Apoptotic, Antiangiogenic, and Antitumor Activities.

Vascular endothelial growth factor receptor 2 (VEGFR2) mediates VEGFA signaling mainly through the PI3K/AKT/mTOR and PLCγ/ERK1/2 pathways. Here we unveil a peptidomimetic (VGB3) based on the interaction between VEGFB and VEGFR1 that unexpectedly binds and neutralizes VEGFR2. Investigation of the cyclic and linear structures of VGB3 (named C-VGB3 and L-VGB3, respectively) using receptor binding and cell proliferation assays, molecular docking, and evaluation of antiangiogenic and antitumor activities in the 4T1 mouse mammary carcinoma tumor (MCT) model showed that loop formation is essential for peptide functionality. C-VGB3 inhibited proliferation and tubulogenesis of human umbilical vein endothelial cells (HUVECs), accounting for the abrogation of VEGFR2, p-VEGFR2 and, subsequently, PI3K/AKT/mTOR and PLCγ/ERK1/2 pathways. In 4T1 MCT cells, C-VGB3 inhibited cell proliferation, VEGFR2 expression and phosphorylation, the PI3K/AKT/mTOR pathway, FAK/Paxillin, and the epithelial-to-mesenchymal transition cascade. The apoptotic effects of C-VGB3 on HUVE and 4T1 MCT cells were inferred from annexin-PI and TUNEL staining and activation of P53, caspase-3, caspase-7, and PARP1, which mechanistically occurred through the intrinsic pathway mediated by Bcl2 family members, cytochrome c, Apaf-1 and caspase-9, and extrinsic pathway via death receptors and caspase-8. These data indicate that binding regions shared by VEGF family members may be important in developing novel pan-VEGFR inhibitors that are highly relevant in the pathogenesis of angiogenesis-related diseases.

Polyphyllin D-loaded solid lipid nanoparticles for breast cancer: Synthesis, characterization, in vitro, and in vivo studies.

Polyphyllin D (PD), a steroidal saponin in Paris polyphylla, induces apoptosis via the intrinsic apoptotic pathway in different cancer types. However, emerging evidence has shown that the primary issue with PD is its structure's hemolysis and cytotoxicity. This study aimed to develop and optimize PD-loaded SLN formulation and evaluate its efficacy in breast cancer cell lines. Apoptosis, as the mechanism of cell death, was confirmed by flow cytometry following Annexin V/propidium iodide staining and western blot analysis. In in vivo studies, tumor inhibitory efficacy was compared with different doses of PD-loaded SLN on 4T1-implanted BALB/c mice. The half-maximal inhibitory concentration (IC50) of PD- loaded SLN was calculated to be 33.25 and 35.74 µg/mL for MCF7 and MDA-MB-231 cells, respectively. Flow cytometry analysis further confirmed a significant increase in apoptosis after treatment with PD- loaded SLN. When both cell lines were treated with PD-loaded SLN, Bcl2 and HSP70 proteins were down regulated, while Bax, Bad, P53, Apaf-1, p-p53 and Noxa proteins were upregulated. This effect was also confirmed by test performed on BALB/c mice in vivo. Based on results, PD-loaded SLN may be a promising breast cancer treatment, without recognizable side effects.

Synthesis, in vitro biological evaluation and molecular modelling of new 2-chloro-3-hydrazinopyrazine derivatives as potent acetylcholinesterase inhibitors on PC12 cells.

BACKGROUND: The loss of cholinergic neurotransmission in Alzheimer's disease (AD) patients' brain is accompanied by a reduced concentration of Acetylcholine (ACh) within synaptic clefts. Thus, the use of acetylcholinesterase inhibitors (AChEIs) to block the cholinergic degradation of ACh is a promising approach for AD treatment. In the present study, a series of 2-chloro-3-hydrazinopyrazine derivatives (CHP1-5) were designed, synthesized, and biologically evaluated as potential multifunctional anti-AD agents. METHODS: In addition, the chemical structures and purity of the synthesized compounds were elucidated through using IR, 1H and 13C NMR, and elemental analyses. Further, the intended compounds were assessed in vitro for their AChE inhibitory and neuroprotective effects. Furthermore, DPPH, FRAP and ABTS assays were utilized to determine their antioxidant activity. The statistical analysis was performed using one-way ANOVA. RESULTS: Based on the results, CHP4 and CHP5 exhibited strong AChE inhibitory effects with the IC50 values of 3.76 and 4.2 µM compared to the donepezil (0.53 µM), respectively. The study examined the effect and molecular mechanism of CHP4 on the Ab1-42-induced cytotoxicity in differentiated PC12 cells. At concentrations of 0-100 µM, CHP4 was non-toxic in PC12. Additionally, Ab1-42 significantly stimulated tau hyperphosphorylation and induced differentiated PC12 cell death. Further, CHP4 resulted in diminishing the Ab1-42-induced toxicity in PC12 cell significantly. CHP4 at 30 µM concentration significantly increased the Ab1-42-induced HSP70 expression and decreased tau hyperphosphorylation. CONCLUSIONS: According to the results of our studies CHP4 can be considered as safe and efficient AChEI and employed as a potential multifunctional anti-AD agent.

A new and efficient diaminopyrimidine-based colorimetric and fluorescence chemosensor for the highly selective and sensitive detection of Cu2+ in aqueous media and living cells.

In this paper, a new and effective diaminopyrimidine-based chemosensor (DAPCS) was developed for the highly selective and ultra-sensitive detection of Cu2+ ion in aqueous media and living cell. Characterization and structure determining of DAPCS was determined by UV-Vis, FTIR and NMR analyses. It is observed that DAPCS and Cu (II) forms a ligand to metal charge transfer (LMCT) complex which produces distinguishable red color. The results also indicate that the DAPCS easily interacts with Cu2+ ion to form a 1:1 stoichiometry complex (DAPCS -Cu2+), resulting in a bathochromic shift in absorption maximum (429 nm to 449 nm) and remarkable quenching fluorescence intensity at the wavelength of 501 nm in DMSO-H2O solution. Furthermore, the detection limit of DAPCS towards Cu2+ was calculated to be 3.19 µM. Meanwhile, DAPCS was applied as fluorescent probe for detection of Cu2+ ions with the detection limit of 0.014 µM. The optimal pH range of probe DAPCS for quantitative analysis of Cu2+ ions was 9-11, which renders it suitable for detection of Cu2+ under physiological conditions. Additionally, the DAPCS could be applied to detect Cu2+ in real water samples and in HeLa cells, indicating the practical uses of DAPCS in real analyses.

Bio-conjugation of anti-human CD3 monoclonal antibodies to magnetic nanoparticles by using cyanogen bromide: A potential for cell sorting and noninvasive diagnosis.

The conjugation of monoclonal antibodies with superparamagnetic iron oxide nanoparticles (SPIONs) has appeared as a potential multifunctional clinical tool, which can effectively diagnose cancers and monitor their treatment, specifically. Despite the presence of different methods for conjugating antibodies to iron oxide nanoparticles, novel cost-effective and simpler conjugation techniques should be performed in this regard. In current study, an anti-CD3 monoclonal antibody was conjugated to the Fe3O4 coated by carboxymethyl dextran (CMD) using cyanogen bromide (CNBr). Moreover, EDC/NHS techniques were applied as a positive control. The experimental results showed that the Conjugation was performed and the presence of the antibody conjugated to the MNPs in human xenograft tumors was confirmed using Prussian blue (PB) staining, following magnetic resonance imaging (MRI), 30 min after injection. This conjugation method was shown to be able to separate CD3+ T lymphocytes efficiently from whole blood with high purity. Accordingly, this type of bio-conjugation method can be utilized in the future for cell sorting, and can be applied for adopted cell therapies such as CAR-T cell (Chimeric antigen receptor T cell) therapy, as well as targeted MRI imaging.

A novel sulfamethoxazole derivative as an inhibitory agent against HSP70: A combination of computational with in vitro studies.

In the current study, a novel derivative of sulfamethoxazole (a sulfonamide containing anti-biotic) named ZM-093 (IUPAC name: (E)-4-((4-(bis(2-hydroxyethyl)amino)phenyl)diazenyl)-N-(5-methylisoxazole-3-yl)benzenesulfonamide) was synthesized via common diazotization-coupling reactions from sulfamethoxazole and subsequently characterized through NMR/FT-IR spectroscopy. After evaluation, the compound was geometrically optimized at the DFT level of theory with BL3YP method and 6/31++G (d,p) basis set and from the optimized structure, several molecular descriptors important in the biological reactivity of the compound, such as global reactivity parameters, molecular electrostatic potential, average local ionization energy, and drug-likeness features of the compound were computationally analyzed. The experimental in vitro investigations of the interaction between ZM-093 and heat shock protein 70 (HSP70), a protein that is highly expressed in several types of cancers, exhibited a significant inhibitory effect against the chaperone activity of HSP70 for the titled compound (P-value < 0.01) and the comparison between the experimental studies with the mentioned computational analysis, as well as molecular docking, illustrated that ZM-093 may inhibit HSP70 through binding to its substrate-binding domain. Finally, by taking all the previous results into account, a new method for assessing the inhibitory activity of ligand to HSP70 is introduced based on protonography, a recently developed method that is dependent on the catalytic activity of carbonic anhydrase on polyacrylamide gel electrophoresis.

Effects of heat shock protein inducer on Hsp70 gene expression and immune parameters during Streptococcus iniae infection in a Persian sturgeon fry.

Heat shock proteins (HSPs) as stress-related factors play a fundamental role in innate and adaptive immune responses in fish, which can be considered as strong candidates for the development of new methods for fish disease prevention. It has been proven that Pro-Tex® as a heat shock protein inducer (HSPi) reduces harmful effects of cellular stress by increasing the Hsp70 protein production. We evaluated the effects of Pro-Tex® as an HSPi in a Persian sturgeon, (Acipenser persicus) exposed to a pathogenic bacterium. Therefore, A. persicus fries were pre-treated with 25.00, 50.00 and 100 mg L-1 of Pro-Tex® and then, injected with Streptococcus iniae ATCC29178. The Hsp70 gene expressions were determined in various organs including liver, gill and intestine and lysozyme (LYZ) activities along with supplemental levels of complement component 3 (C3) and immunoglobulin M (IgM) were also determined in sturgeon blood in days 3 and 7 after infection. Expression of Hsp70 gene was increased during the first three days of infection and then, it was found to be down-regulated during the infection process. Also, levels of LYZ activity, C3 and IgM increased in a concentration-dependent manner; but these parameters decreased after 7 days. Our data suggest that induction of Hsp70 is a promising approach in modulation of immune response in A. persicus and it might be employed to confer protection in fish against bacterial infections.

Cloning, purification and biochemical characterization of two glutathione S-transferase isoforms from Rutilus frisii kutum.

Glutathione S-transferases are an important multifunctional family of intracellular enzymes that their detoxification function has been reported in fishes since 1970, but no studies have been conducted on Rutilus frisii kutum GSTs yet. In the present study, RkGSTA and RkGSTM encoding genes were cloned and sequenced and their nucleotide sequences were submitted to NCBI GenBank. In order to reduce the expression challenges of recombinant proteins including low solubility, low yield and insufficient purity issues in E. coli, the pKJE7 chaperone plasmid was used to increase the recovery of expressed proteins in the soluble fractions. Best expression clone was selected for purification by Ni-NTA affinity chromatography. The three-dimensional structural models were constructed by I-TASSER. The optimum temperature of purified RkGSTA and RkGSTM was 35 and 30 °C, with optimum activity at pH 9.0 and 8.5, respectively. The thermostability and pH stability results indicated that RkGSTA is more heat-tolerant than RkGSTM though both of them retained more than 80% of their activities at pH 6.5 to 9.0. Overall, this study represents a comprehensive perspective on the structural and biochemical aspects of this enzyme that would be even used in further researches such as drug design studies in order to eliminate toxicant compounds from the body and environment of fishes to protect them against undesired harmful damages.

Artesunate, as a HSP70 ATPase activity inhibitor, induces apoptosis in breast cancer cells.

The present study aims to evaluate the inhibitory effects of artesunate (a semi-synthetic derivative of artemisinin) on HSP70 and Bcl-2 expression in two breast cancer cell lines, 4T1 and MCF-7. In addition, to determine in vitro inhibitory effect of artesunate against the ATPase activity of purified recombinant HSP70, it was tested in a carbonic anhydrase refolding assay with purified HSP70. Our results demonstrated that the artesunate not only induced apoptosis but also lead to the inhibition of HSP70 ATPase activity the in vitro (P < 0.001). The extent of HSP70 refolding inhibition increased with increasing µM concentrations of artesunate. Incubation of HSP70 with 50 µM artesunate showed significant inhibition of refolding activity by 38%. The IC50 values of artesunate for 4T1 cells, were lower than MCF-7 cells, indicating the higher sensitivity of the triple-negative phenotype. Furthermore, artesunate significantly down-regulated the expression of Bcl-2 and HSP70 while enhancing the expression of cleaved caspase-9 in MCF-7 and 4T1 cells. It also induced caspase-9 activity at 18 h in a dose-dependent manner in two breast cancer cell lines. Generally, our results show that the artesunate induces caspase-dependent apoptosis through the inhibition of HSP70 expression.

Rapid and simple screening of the apoptotic compounds based on Hsp70 inhibition using luciferase as an intracellular reporter.

HSP70 is a powerful antiapoptotic protein that can block the extrinsic and intrinsic pathways of apoptosis. The present study describes a rapid, sensitive, and inexpensive system using luciferase as a reporter for the functional analysis of apoptotic compounds. For this approach, the co-transformation of Escherichia coli cells was performed with two expression vectors containing Hsp70 and firefly luciferase. It was found that the luciferase inactivated by heat treatment (40-46 °C for 10 min) was approximately reactivated at room temperature and regained 70% of its initial activity before heat inactivation after 60 min. The results show that the reactivation of thermally inactivated luciferase was inhibited in living cells by treatment with VER-155008 and pifitrin-µ as Hsp70 inhibitors, with half-maximal inhibitory concentration of 124 and 384 µM, respectively. The sensitivity of this method for detecting VER-155008 and pifitrin-µ was about 8 and 25 µM, respectively. Also, this reporter system showed no response to doxorubicin and dactinomycin, which bind to DNA, and we used these anticancer compounds as control compounds. Therefore, for the first time, a rapid and simple real-time system using luciferase as a reporter is introduced for the screening of apoptosis-inducing compounds based on suppression of Hsp70 in E. coli cells.

Evaluation of novel pyrimidine derivatives as a new class of mushroom tyrosinase inhibitor.

BACKGROUND AND AIM: Tyrosinase (EC 1.14.18.1) is responsible for enzymatic browning in fruits and vegetables. Its inhibitors may be applied to efficiently treat hyperpigmentation and are widely used in pharmaceutical and cosmetic products, food supplements and insecticides. Previous studies have shown that heterocyclic compounds with an amino group can inhibit tyrosinase activity. The present study aims to evaluate the inhibitory effect of some novel 2,6-diamino-4-chloropyrimidine derivatives (1a-e) and 2,4,6-triaminopyrimidine (2a-e) including bioactive aniline moiety on the activity of the mushroom tyrosinase. METHODS: In practice, the azo salt was initially synthesized from aniline derivatives and combined subsequently with the 2,4,6-triaminopyrimidine and 2,6-diamino-4 chloropyrimidine followed by crystallization. The structures of resulting compounds were confirmed by FT-IR, 13C NMR, and 1H NMR. The derivatives (0-100 µM) were evaluated for their inhibitory effect on tyrosinase activity using l-3,4-dihydroxyphenylalanine (l-DOPA) as substrate. RESULTS: All compounds showed inhibitory effects against the activity of the enzyme. About 23.72-55.08% inhibition was observed in the presence of 30 µM of each compound. The IC50 values of the synthesized compounds were measured, and their inhibition properties were also visualized by zymography. Based on the results, the compounds 1a-e and 2a-e showed moderate inhibitory activities. Notably, pyrimidine derivatives 1a (IC50=24.68) and 1d (IC50=24.45) also exhibited similar inhibitory activities when compared with the positive control, kojic acid (IC50=25.24 µM). Kinetic studies indicated that the type of inhibition was noncompetitive. CONCLUSION: All results suggest that pyrimidine derivatives, especially 1d and 1a, can be considered as safe and efficient tyrosinase inhibitors.

Molecular cloning, expression, and functional characterization of 70-kDa heat shock protein, DnaK, from Bacillus halodurans.

In the present study, we report cloning, sequencing, and functional characterization dnaK gene of B. halodurans that is the central component in cellular network of molecular chaperones. The 3D structures of DnaK obtained by I-TASSER server showed that the overall structures of DnaK from B. halodurans and human HSP70 chaperone BiP are very similar with a homology of 88.8%. The purified recombinant DnaK consists of a His-tag at C-terminus and show a band on approximately 70-kDa region in SDS-PAGE. The resultant refolding assay revealed that the refolding rate was considerably improved by the addition of the novel DnaK chaperone for the refolding of heat-denatured carbonic anhydrase. Also, salt resistance experiments indicated that E. coli + DnaK survival had enhanced by 4.4-fold as compared with control cells in 0.4 M NaCl. The number of E. coli + DnaK colonies was 2.5-fold higher than control colonies in pH 9.5. We showed that DnaK refolding functions were decreased by increasing Cd2+ in nanomolar concentrations. Hg2+ had a biphasic effect on recombinant DnaK refolding function: inhibition at low and stimulation at high concentrations. It was concluded that the DnaK from B. halodurans can potentially be employed for improving functional properties of proteins in various applications.

Evaluation of thiazolidinone derivatives as a new class of mushroom tyrosinase inhibitors.

Tyrosinase (EC 1.14.18.1) is a key copper-containing metalloenzyme widely distributed in nature and plays determinant role in melanin biosynthesis. The enzyme manifests two unusual catalytic properties including oxidase and monooxygenase activities. Its inhibitors may be applied to efficiently treat of hyperpigmentation and widely used in pharmaceutical and cosmetic products, as well as food supplements and insecticides. The present study aims to evaluate the inhibitory effects of some novel azo-hydrazone tautomeric dyes (4a-e) including bioactive thiazolidinone moiety on the activity of the mushroom tyrosinase. When L-3,4-dihydroxyphenylalanine (L-Dopa) was used as the substrate for the enzyme, the compounds 4d, 4a, and 4e showed strong inhibitory effects against the activity of the enzyme (61%, 56%, and 49% inhibition in the presence of 60µM of each compound, respectively). The IC50 values of the synthetized compounds were measured and their inhibition properties were also visualized by zymography. According to tyrosinase inhibitory activity, the compounds 4a, 4c, 4d and 4e exhibited strong inhibitory activities with IC50 values of 45.83, 140.25, 37.59, and 42.31µM, respectively, compared to the positive control kojic acid (29.44µM). Kinetic study of 4d compound (as the most potent inhibitor) revealed that the compound acts as a reversible competitive inhibitor of the enzyme with the Ki value of 31.0µM. We also simulated the molecular docking with the compound 4d and the results confirmed that the compound strongly interacts with the mushroom tyrosinase residues. All results totally suggest that thiazolidine derivatives, especially 4d, 4a, and 4e, can be considered as safe and efficient tyrosinase inhibitors. They also have the potential to be used in the correspond fields.