Computational design of a chimeric toxin against Claudin-4-expressing cancer cells: molecular modeling, docking and molecular dynamics simulation analysis.

Cancer is one of the main reasons of mortality all over the world. Over the time, the major ways for cancer-therapy were based on radiotherapy, chemotherapy and surgery. These methods are not specific enough for that purpose, therefore, new ideas for design of new drugs with higher specificity are considered. Chimeric protein toxins are hybrid proteins consisting of a targeting portion and a toxic one which specifically bind and kill the target cancer cells. The main purpose of this study was designing a recombinant chimeric toxin with biding capability to one of the most key receptors namely claudin-4 which is over-expressed in almost all cancer cells. To design it, we utilized the last 30 C-terminal amino acids of Clostridium perfringens enterotoxin (CPE) as a binding module for claudin-4 and the toxic module which is the A-domain of Shiga toxin from Shigella dysenteriae. Using molecular modeling and docking methods, appropriate binding affinity of the recombinant chimeric toxin to its specific receptor was demonstrated. In the next step, the stability of this interaction was investigated by molecular dynamics simulation. Although partial instability was detected at some time points, however, sufficient stable situation of hydrogens bonds and high binding affinity between the chimeric toxin and receptor were observed in the in silico studies which in turn suggested that this complex could be formed successfully.

Behavioral, histopathological, and biochemical evaluations on the effects of cinnamaldehyde, naloxone, and their combination in morphine-induced cerebellar toxicity.

Long-term morphine use for therapeutic approaches may lead to serious side effects. Several studies have suggested opioid antagonist and antioxidant therapy for reducing adverse effects of morphine. Cinnamaldehyde has a potent anti-oxidant property. In this study, separate and combined effects of cinnamaldehyde and naloxone (an opioid receptor antagonist) on behavioral changes and cerebellar histological and biochemical outcomes were investigated after long-term morphine administration. Seventy-eight rats were divided into two major morphine-treated and morphine-untreated groups. Morphine-treated group was subdivided into seven subgroups for receiving vehicle, normal saline, cinnamaldehyde (1.25, 5, and 20 mg/kg), naloxone, and cinnamaldehyde plus naloxone before morphine. Morphine-untreated group was subdivided into six subgroups and treated with vehicle, cinnamaldehyde (1.25, 5, and 20 mg/kg), naloxone, and their combination. Chemical compounds were administered for 28 consecutive days. Behavioral tests including footprint, rotarod, and beam balance tests were employed. Histopathological and biochemical alterations of cerebellum were determined. Body and cerebellum weights, stride width, time spent on the rotarod, Purkinje cell number, thickness of molecular and granular layers, superoxide dismutase (SOD), and total antioxidant capacity (TAC) decreased as a result of administrating morphine. Morphine increased beam transverse time, malondealdehyde (MDA), tumor necrosis factor-α (TNF-α), and caspase-3 levels. Histopathological changes such as cellular vacuolation and loss were also produced as a result of treatment with morphine. Cinnamaldehyde, naloxone, and their combination treatments improved all the above-mentioned alterations induced by morphine. We concluded that cinnamaldehyde produced a neuroprotective effect through anti-oxidant, anti-inflammatory, apoptotic, and probably naloxone-sensitive opioid receptor interaction mechanisms.

The effects of varying concentrations of glutathione and trehalose in improving microscopic and oxidative stress parameters in Turkey semen during liquid storage at 5 °C.

Since turkey reproduction is mainly through artificial insemination, short-term preservation of turkey semen is one of the most important issues in turkey reproduction management. The present study investigates the effects of glutathione (GSH) and trehalose on lipid peroxidation degree and turkey semen quality while being stored at 5 °C for 72 h. To this end, semen samples were collected from 20 turkeys with a weekly frequency for 12 weeks. A glucose-based extender was used to dilute the pooled semen. It was divided into seven equal parts with varying levels of glutathione [0.5, 1 and 2 mM), trehalose [50, 75 and 100] and control [extender without antioxidant]. Subsequently, the divided semen samples were stored at 5 °C for 72 h. Several sperm parameters such as motility and motion parameters, plasma membrane integrity (PMI), plasma membrane functionality, DNA integrity, and oxidative parameters were assessed following storage for 0, 24, 48, and 72 h. The obtained results indicated an improvement in the plasma membrane functionality and DNA integrity, along with the percentages of PMI in GSH-2 mM group in comparison to the control group following storage at 5 °C for 72 h (P ≤ 0.05). It is also notable that the 2 and 1 mM concentrations of GSH increased the spermatozoa motility and motion parameters in comparison to the control group, respectively (P ≤ 0.05). The study results indicated that GSH-2, 1 mM and trehalose- 100 mM concentrations reduced lipid peroxidase levels and increased total antioxidant activity, catalase, superoxide dismutase, and glutathione peroxidase in comparison to the control group (P ≤ 0.05). Our study's data show that improvement of semen parameters and oxidative stress parameters of turkey semen can be improved by glutathione at 2 and 1 mM and trehalose at 75 mM while storing it 5 °C.

Cytotoxic effect of sea anemone pore-forming toxin on K562 chronic myeloid leukemia cells.

Chronic myelogenous leukemia (CML) is one of prevalent cancer worldwide. In spite of various designed drugs, chemoresistance remains the main obstacle in cancer cure. Therefore, developing novel strategy for treatment of CML is an urgent need. Fragaceatoxin C (FraC) is novel protein toxin from a sea anemone called actinia fragacea with great impacts against cells by pore formation and disturbing cell membrane integrity. The aim of this study was evaluation of FraC toxin toxicity against K562. The bacteria cells harboring expression||||||| vector of FraC were induced by IPTG and purified by Ni2+-NTA sepharose affinity chromatography. Then, purified toxin activity was evaluated using RBC hemolytic test. Eventually, evaluation of FraC cytotoxicity and apoptosis were performed using MTT and flow cytometery assays, respectively. Our results revealed that FraC toxin decreased K562 cells viability in a dose- and time-dependent manner with a whole destroy of cancer cells at 35.00 µg mL-1 after 72 hr. Furthermore, flow cytometery analysis indicated that FraC toxin enhanced necrosis along with apoptosis in K562 cells in a dose dependent manner. We speculated that FraC toxin could be considered as a novel candidate for cancer cell researches and treatments provided that it should be turned into a specific agent by engineering and directing to cancer cell membrane.

The response of PIK3CA/KRAS-mutant colorectal cancer stem-like cells to RGD-peptide FraC produced by the strawberry anemone: A promising water-soluble peptide-based inhibitor of metastasis-driver gene CXCR4, stem cell regulatory genes and self-renewal.

Colorectal cancer (CRC) is a stem cell-based disease. PIK3CA/KRAS-mutant CRC stem cells (CRCSCs) display high self-renewal, metastatic properties, high activity of PI3K and KRAS signaling pathways with chemoresistant phenotypes. Recently, RGD peptide (containing Arg-Gly-Asp motif)-based therapy of solid tumor cells has attracted much attention. However, little is known whether this method can target self-renewal capacity, key effectors of PI3K and KRAS signaling pathways such as metastasis-driver gene CXCR4 and stem cell regulatory genes with caspase-3 reactivation in CRCSCs overexpressing RGD-dependent integrins. The sea anemone Actinia fragacea produces a water-soluble RGD-peptide fragacea toxin C (FraC) suggesting the possible activity of FraC against PIK3CA/KRAS-mutant CRCSCs. Recombinant FraC was expressed via pET-28a(+)-FraC in E. coli and purified through affinity chromatography followed by performing SDS-PAGE and hemolytic activity assay. Next, PIK3CA/KRAS-mutant HCT-116 cells that serve as an attractive model for CRCSCs were treated with FraC. Thereafter, cell numbers, viability, proliferation, LDH activity, cytotoxicity index, CXCR4 and pluripotency network genes expression, self-renewal capacity, caspase-3 activity with apoptosis were evaluated. Caspase-1, -2, -3,…, -9 sequences were analyzed for RGD-binding motifs. FraC sequence and structure were also evaluated by bioinformatics software. FraC altered cellular morphology to round shapes and disrupted cell connections. 48 h post-treatment with 0.056- to 7.2 µM FraC resulted in 12 %-99 % and 8 %-97.6 % decreases in cell numbers and viabilities respectively and increased LDH activity by 0.2 %-66.7 % in a dose-dependent manner. The results of the cytotoxicity index showed that FraC induces significant toxicity on HCT-116 cells compared to PBMCs and Huvec cells. FraC dramatically decreased the expression of CXCR4 and pluripotency network genes Bmi-1, Sox-2, Oct-4 and Nanog followed by remarkable decreases in self-renewal capacity ranged from 91- to 0 colonies per well for 0.056- to 3.6 µM FraC after 2 weeks. Caspase-3 was found to contain an RGD-binding motif and its activity increased with increasing FraC concentrations followed by apoptosis induction. Potential RGD-binding motifs for FraC were also found in caspase-1, -7, -8 and -9. Unique advantages of FraC peptide, such as low molecular weight, water solubility, high sensitivity of CRC stem-like cells with more selective toxicity to this compound, targeting tumor cell membrane and self-renewal capacity along with the modulation of CXCR4 and stem cell regulatory genes as upstream and downstream effectors of undruggable PI3K and KRAS signaling pathways may open up avenues for FraC peptide-based therapy of PIK3CA/KRAS-mutant CRCSCs with lower toxicity on healthy cells.

The Effects of pH and Temperature on Cysteine Protease (Cathepsin B) Activity in Miracidia and Eggs of Fasciola hepatica.

BACKGROUND: Fascioliasis is a worldwide zoonotic disease caused by the trematodes Fasciola hepatica in humans and animals. Proteases are essential for the survival of parasites and have important activities such as penetration, tissue migration, and egg hatching. This study was conducted to analyze cysteine protease of the miracidia and eggs of F. hepatica, and to assess the effects of pH and temperature on the proteases activity and stability. METHODS: Adults F. hepatica were isolated from infected livers and were morphologically identified in 2018. Eggs collected from the adults and incubated in distilled water at 28 °C for 16 d to produce miracidia. The extract was collected from miracidia and eggs. A substrate for cathepsin B (Z-Arg-Arg-Pna) was used to assess the enzyme activity at different (2-12) pH levels. After homogenization, protein level was measured with Bradford method. Estimation of optimum temperature and pH was performed in the temperature range of 10-90 ° C and pH values from 2-12. RESULTS: The highest activity of the miracidia and eggs enzyme extracts for Z-Arg-Arg-pNA was at pH 4. The miracidia extract was most stable at neutral pH and the eggs extract was most stable in acidic pH. The optimum temperature activity for both stages was 40 °C. These proteases were stable up to 40 °C. CONCLUSION: Upon the importance of pH and temperature in the life cycle of F. hepatica, the current findings can be used for induction of some modifications in pH and preventing the activity of the enzymes for decrement of the efficacy of miracidia penetration into the intermediate snails and egg hatching of this zoonotic parasite.

In Vitro Inhibitory Effect of Recombinant Human Calprotectin on Nalm6 Leukemia Cell Line.

BACKGROUND & PURPOSE: In evaluating new drugs for the treatment of various types of cancer, investigations have been made to discover a variety of anti-tumor compounds with less side effects on normal cells. Investigations have shown that the heterodimers S100A8 and S100A9 inhibit the enzyme casein kinase 2 and then prevent the activation of the E7 oncoprotein. Therefore, the aim of this study was to evaluate the effect of calprotectin as an antitumor compound on the Nalm6 (B cell precursor leukemia cell line). MATERIALS & METHODS: Transformation of genes encoding S100A8 and S100A9 human, designed in the pQE32 plasmid, was performed by the thermal shock method into E. coli M15 bacteria. After bacterial growth in LB medium, the expression of two S100A8 and S100A9 subunits, the solubility of the protein by SDS-PAGE method was determined. Finally, the S100A8 / A9 complex was equally placed in the microtube. In the next step, the cytotoxic effects of calprotectin produced on the Nalm6 cell line were evaluated using the wst1 test. Then, the apoptosis in these cells was measured using flow cytometry methods with Annexin-V coloration. RESULTS: In the current study, the results showed that the cytotoxic effects of Calprotectin are time and concentration- dependent. Therefore, it can reduce the tumor expression and had a beneficial effect by induced apoptosis in Nalm6 cell line. CONCLUSION: Calprotectin has an anti-tumor effect on the Nalm6 cell line by increasing apoptosis.

Enhancement of Pharmaceutical Urate Oxidase Thermostability by Rational Design of De Novo Disulfide Bridge.

BACKGROUND AND PURPOSE: As a therapeutic enzyme, urate oxidase is utilized in the reduction of uric acid in various conditions such as gout or tumor syndrome lysis. However, even bearing kinetical advantage over other counterparts, it suffers from structural instability most likely due to its subcellular and fungal origin. OBJECTIVES: In this research, by using rational design and introduction of de novo disulfide bridge in urate oxidase structure, we designed and created a thermostable urate oxidase for the first time. MATERIALS AND METHODS: Utilizing site-directed mutagenesis and only with one point mutation we constructed two separate mutants: Ala6Cys and Ser282Cys which covalently linked subunits of enzyme each other. Single mutation to cysteine created three inter-chain disulfide bridges and one hydrogen bond in Ala6Cys and two disulfide bridges in Ser282Cys. RESULTS: Both mutants showed 10 °C increase in optimum activity compared to wild-type enzyme while the Km values for both increased by 50% and their specific activity compromised. The thermal stability of Ser282Cys increased remarkably by comparing Ala6Cys and wild-type enzymes. Estimation of half life for wild-type enzyme demonstrated 38.5 min, while for Ala6Cys and Ser282Cys were 138 and 115 min, respectively. Interestingly, the optimal pH of both mutants was broaden from 7 to 10, which could make them candidates for industrial applications. CONCLUSION: It seemed that introducing disulfide bridges resulted in local and overall rigidity by bringing two adjacent sites of enzyme together and decreasing the conformational entropy of unfolding state is responsible for the enhancement of thermostability.

The effects of crocin, mesalazine and their combination in the acetic acid-induced colitis in rats.

Crocin, as a carotenoid compound of saffron, exerts a potent antioxidant property. Mesalazine is frequently used in the treatment of ulcerative colitis. This study investigated the effects of separated and combination treatments with crocin and mesalazine in a rat model of ulcerative colitis. Ulcerative colitis was induced by intra-colonic administration of acetic acid (4.00%, 1.00 mL) at 8 cm proximal of the anus. Normal saline, acetic acid, crocin (5.00, 10.00 and 20.00 mg kg-1), mesalazine (100 and 300 mg kg-1) and crocin (5.00 mg kg-1) plus mesalazine (100 mg kg-1) were administered after induction of colitis for eight days. Body weight, organosomatic index (OSI), macroscopic and microscopic evaluations of colon and measurement of malondialdehyde (MDA), superoxide dismutase (SOD), tumor necrosis factor-alpha (TNF-α) contents of colon tissue were determined on day eight after induction of colitis. Crocin (10.00 and 20.00 mg kg-1), mesalazine (300 mg kg-1) and crocin (5.00 mg kg-1) plus mesalazine (100 mg kg-1) significantly (p < 0.05) improved body weight and OSI and reduced macroscopic and microscopic scores. These treatments also significantly (p <0.05) recovered the increased levels of MDA and TNF-α as well as the decreased level of SOD in colon tissue. Crocin and mesalazine did not produce significant effects in intact rats. Based on the results, it is concluded that crocin and mesalazine produced protective effects on colon tissue via antioxidant and anti-inflammatory actions. In addition, a synergistic effect was observed between crocin and mesalazine in attenuating ulcerative colitis.

Safranal, a constituent of saffron, exerts gastro-protective effects against indomethacin-induced gastric ulcer.

AIMS: Several natural products have been evaluated for management of gastric ulcer induced by non-steroidal anti-inflammatory drugs. Safranal, a plant-derived chemical, has a potent antioxidant and anti-inflammatory properties. The present study was aimed to evaluate possible gastro-protective effects of safranal against indomethacin-induced gastric ulcer in rats. Lansoprazole (a proton pump inhibitor) was used as a reference drug. MATERIALS AND METHODS: Thirty rats were divided into five groups. Groups 1 and 2 received vehicle. Groups 3, 4 and 5 treated with 0.063, 0.25 and 1 mg/kg safranal. Group 6 received 30 mg/kg lansoprazole. All groups except of group 1 received indomethacin (50 mg/kg) ingestion. Six hours later, animals were euthanized and their stomachs were removed. Gastric contents volume and pH were measured. Gastric ulcer area and protective index were evaluated using image J software. Histological changes were evaluated by light microscope. Malondialdehyde (MDA) level, superoxide dismutase (SOD) activity, total antioxidant capacity (TAC) content, tumor necrosis factor-alpha (TNF-α) and Caspase-3 levels were determined in the gastric tissue. KEY FINDINGS: Safranal and lansoprazole normalized gastric volume and pH, reduced gastric ulcer area and produced gastric protection. Indomethacin-induced histological changes and tissue biochemical alterations were ameliorated by the above-mentioned treatments. SIGNIFICANCE: The results of the present study suggest the involvement of anti-secretory, anti-oxidant, anti-inflammatory and anti-apoptotic mechanisms in gastro-protective effect of safranal. In addition, gastro-protective effect of safranal was comparable to lansoprazole.

Crocin exerts improving effects on indomethacin-induced small intestinal ulcer by antioxidant, anti-inflammatory and anti-apoptotic mechanisms.

Crocin is a plant-derived carotenoid and bears potent antioxidant property. Ranitidine (a histamine H2 receptor blocker) is used for peptic ulcer treatment. The present study was planned to investigate the effects of crocin and ranitidine on indomethacin-induced ulcer in small intestine of rats. Animals were randomized into two major groups including indo-methacin (10.00 mg kg-1, ulcer group, 48 rats) and normal saline (1.00 mL kg-1, intact group, 48 rats) groups. Each of these two major groups was subdivided into eight subgroups for intra-peritoneal (IP) injections of normal saline, crocin (2.50, 10.00 and 40.00 mg kg-1), ranitidine (5.00 and 20.00 mg kg-1), crocin (2.50 and 10.00 mg kg-1) plus ranitidine (5.00 mg kg-1). Indomethacin induced intestinal ulcer was characterized by bleeding, inflammation, epithelial hyperplasia and crypt loss. This non-steroidal anti-inflammatory drug (NSAID), indomethacin decreased goblet cell number and superoxide dismutase (SOD) activity and increased small intestine weight, organo-somatic index (OSI), malodealdehyde (MDA), tumor necrosis factor-α (TNF-α) and caspase-3 contents of intestine. Crocin resolved all the above-mentioned parameter changes induced by indomethacin. These treatments produced no significant effects on the above-mentioned parameters of intact group. The results of the present study showed tissue protective and anti-ulcer effects of crocin on small intestine by antioxidant, anti-inflammatory and anti-apoptotic mechanisms. Ranitidine alone showed no effect; however, in combination with crocin it exerted recovery effects. It is recommended that crocin, be considered as a therapeutic agent for NSAIDs-induced intestinal damage management.

Evaluating cytotoxic effects of recombinant fragaceatoxin C pore forming toxin against AML cell lines.

OBJECTIVES: Current therapeutic strategies for cancer are associated with side effects and lack of specificity in treatments. Biological therapies including monoclonal antibodies and immune effectors have been the subject of multiple research projects. Pore-forming proteins may become the other biological strategy to overcome the problems associated with current treatments. But detailed mechanisms of their action on target membranes remained to be elucidated. We aimed to study the cytotoxic effects of recombinant form of fragaceatoxin C on AML cell lines HL-60 and KG-1. MATERIALS AND METHODS: We cloned the FraC gene in pET-28a (+) bacterial expression vector and the expressed recombinant FraC protein was purified by affinity chromatography. Then, cytotoxic effects of the recombinant protein were examined on two AML cell lines, HL-60 and KG-1. Effects of serum and calcium ion were explored by hemolysis assay in more details. RESULTS: Our results showed that the recombinant C-terminal polyhistidine-tagged FraC protein has potent cytotoxic effects on both AML cell lines, with IC50=5.6, and 4.6 µg.ml-1 for HL-60 and KG-1 cells, respectively. Serum showed dose-dependent and also time-dependent inhibitory effects on the hemolytic and cytotoxic activities of the FraC protein. Pre-incubation of the toxin with different concentrations of calcium ion also inhibited hemolytic activity of FraC toxin. CONCLUSION: Results of the present study showed that FraC has potential anti-tumor effects. By detailed investigation of the inhibition mechanism of serum and calcium effects in the future, it can be possible to design target sites for clinical applications of the toxin.

Statistical optimization of kojic acid production by a UV-induced mutant strain of Aspergillus terreus

ABSTRACT The ability of four Aspergillus strains for biosynthesis of kojic acid was evaluated among which Aspergillus terreus represented the highest level (2.21 g/L) of kojic acid production. Improvement kojic acid production ability of A. terreus by random mutagenesis using different exposure time to ultraviolet light (5-40 min) was then performed to obtain a suitable mutant of kojic acid production (designated as C5-10, 7.63 g/L). Thereafter, design of experiment protocol was employed to find medium components (glucose, yeast extract, KH2PO4 (NH4)2SO4, and pH) influences on kojic acid production by the C5-10 mutant. A 25-1 fractional factorial design augmented to central composite design showed that glucose, yeast extract, and KH2PO4 were the most considerable factors within the tested levels (p < 0.05). The optimum medium composition for the kojic acid production by the C5-10 mutant was found to be glucose, 98.4 g/L; yeast extract, 1.0 g/L; and KH2PO4, 10.3 mM which was theoretically able to produce 120.2 g/L of kojic acid based on the obtained response surface model for medium optimization. Using these medium compositions an experimental maximum Kojic acid production (109.0 ± 10 g/L) was acquired which verified the efficiency of the applied method.

Carnosine improves functional recovery and structural regeneration after sciatic nerve crush injury in rats.

AIMS: Peripheral nerve injury represents a substantial clinical problem with insufficient or unsatisfactory treatment options. Current researches have extensively focused on the new approaches for the treatment of peripheral nerve injuries. Carnosine is a naturally occurring pleotropic dipeptide and has many biological functions such as antioxidant property. In the present study, we examined the regenerative ability of carnosine after sciatic nerve crush injury using behavioral, biochemical, histological and ultrastructural evaluations. MATERIALS AND METHODS: Seventy-two rats were divided into six groups including control, sham, crush and carnosine (10, 20 and 40 mg/kg) groups. Crush injury in left sciatic nerve was induced by a small haemostatic forceps. Carnosine was administered for 15 consecutive days after induction of crush injury. Sciatic functional index (SFI) was recorded weekly. Histopathological and ultrastructural evaluations were made using light and electron microscopes, respectively. Sciatic nerve tissue malondialdehyde (MDA), superoxide dismutase (SOD) and tumor necrosis factor-alpha (TNF-α) levels were measured. Gastrocnemius muscle weight was determined. KEY FINDINGS: Carnosine at the doses of 20 and 40 mg/kg accelerated SFI recovery. Wallerian degeneration severity and myelinated fibers density, myelin sheath thickness and diameter as well as ultrastructural changes of myelinated axons were improved. It also recovered nerve tissue biochemical (MDA, SOD and TNF-α) changes induced by crush injury. Muscle weight ratio was reached to near normal values. Our results suggest a regenerative effect of carnosine. Inhibition of oxidative stress and inflammatory pathways, along with provocation of myelination and prevention of muscular atrophy might be involved in this effect of carnosine. SIGNIFICANCE: Carnosine treatment might be considered as a therapeutic agent for peripheral nerve regeneration and its functional recovery.

The effects of safranal, a constitute of saffron, and metformin on spatial learning and memory impairments in type-1 diabetic rats: behavioral and hippocampal histopathological and biochemical evaluations.

Safranal is one of saffron constituents and has antioxidant and neuroprotective properties. Metformin is used as an anti-diabetic drug. This study was planned to investigate the separate and combined treatment effects of safranal and metformin on diabetes-induced learning and memory impairments by behavioral and hippocampal histopathological and biochemical evaluations. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ), treatments with safranal (0.025, 0.1 and 0.4 mg/kg), metformin (50 and 200 mg/kg), and a combination of low doses of this chemicals were initiated after confirmation of diabetes and continued for 37 days. Blood glucose concentration was measured before and on days 15, 25 and 35 after injection of streptozotocin. Learning and memory tested using Morris Water Maze (MWM) on days 40-45 and on day 45 hippocampal specimens were collected for determination of malodialdehyde (MDA), tumor necrosis factor-alpha (TNF-α) and Caspase-3 levels and superoxide dismutase (SOD) activity. The hippocampus was also designed for light microscopy evaluation. Hyperglycemia, spatial learning and memory impairments, hippocampal neuron loss, increase of hippocampal MDA, TNF-α and caspase-3 levels and decrease of SOD activity were observed in diabetic rats. Safranal (0.1 and 0.4 mg/kg), metformin (200 mg/kg) and safranal (0.025 mg/kg) with metformin (50 mg/kg) improved the above-mentioned behavioral, histopathological and biochemical changes. Safranal and metformin and their combination improved learning and memory impairments in STZ-induced diabetic rats. Antioxidant, anti-inflammatory and antiapoptotic mechanisms might be involved. It is recommended that safranal be considered for diabetes management.

Recombinant production of Aspergillus Flavus uricase and investigation of its thermal stability in the presence of raffinose and lactose.

Aspergillus flavus uricase (Rasburicase) with a molecular mass of 135 kDa is currently used for the treatment of gout and hyperuricemia occurring in tumor lysis syndrome. To characterize the effects of raffinose and lactose osmolytes on the uricase structure, its coding sequence was cloned, expressed in E. coli BL21, and purified by Ni-NTA agarose affinity chromatography. Thermal inactivation studies at 40 °C showed that nearly 15% of UOX activity was preserved, while the presence of raffinose and lactose reduced its activity to 35 and 45% of its original activity, respectively. Investigation of UOX thermal stability at 40 °C in the course of time showed that the enzyme relatively lost almost 60% of its original activity after 40 min, whereas more than 50% of UOX activity is preserved in the presence of lactose. Estimation of thermal inactivation rate constant, k in, showed that the UOX k in and UOX k in in the presence of raffinose was unchanged (0.018 min-1), whereas for the presence of lactose, it was 0.015 min-1. Half-life and T m analysis showed that UOX half-life is almost 38 min and addition of raffinose did not change the half-life, whereas the presence of lactose had remarkable impact on UOX half-life (46 min). The presence of raffinose increased UOX T m to a lesser extent, whereas lactose notably enhanced the T m from 27 to 37 °C. Overall, our findings show that lactose has protective effects on UOX stability, while for raffinose, it is relatively compromised.

Synergism between NF-kappa B inhibitor, celastrol, and XIAP inhibitor, embelin, in an acute myeloid leukemia cell line, HL-60.

AIM OF STUDY: Embelin and celastrol, inhibitors of XIAP and NF-κB proteins respectively, have been derived from natural sources and shown anti-tumor activities against different cancer cell lines. Some interactions have recently been discovered between XIAP and NF-κB pathways, but the effects of these inhibitors in combination have not been investigated yet. We have studied possible synergistic effects of embelin in combination with celastrol, in an acute myeloid leukemia model, HL-60 cell line. MATERIALS AND METHODS: Cytotoxicity of embelin and celastrol, separately and in combination, was determined by MTT assay and flow cytometry. Chou-Talalay's method was used to assess the synergistic effect of two components. Immunocytochemistry and western blot analysis of the two tumor marker proteins. (survivin and COX-2) was also performed to investigate downstream effects of two components. RESULTS: Analysis of MTT assay and flow cytometry showed that there is a substantial synergistic effect in some affected fractions of drug-treated HL-60. cells, while in other affected fractions a mild synergism or additive effect was observed. Immunocytochemistry and western blot analysis revealed that the expression of survivin and COX-2 proteins was reduced in treated cells. CONCLUSION: Embelin and celastrol showed potent antitumor activity and synergistic effects in combination. Therefore targeting XIAP and NF-κB pathways simultaneously can be investigated in more detail to make use of embelin and celastrol as a combination therapy of cancer.

In solution cation-induced secondary and tertiary structure alterations of human calprotectin.

Calprotectin (CP) is widely considered to have diverse roles including growth inhibitory and apoptosis induction in a number of tumor cell lines and antimicrobial activities. As CP has been proposed to bind metal ions with high affinity, we have studied its functional and primarily its structural behavior upon Zn(2+) and Mn(2+) chelation solely and along with Ca(2+). We employed fluorescence spectroscopy and circular dichroism to determine the resulting modifications. Based upon our findings it is clear that treating CP with ions effectively weakened its natural growth inhibitory activity. Moreover, structural analysis of Zn(2+) and Mn(2+)-treated CPs indicated remarkable alterations in the regular secondary structures in favor of irregular structures while Zn(2+) and Mn(2+) treatment of CP after incubation with Ca(2+) displayed no remarkable shifts. Tertiary structure investigation using fluorescence spectroscopy showed that CP undergoes conformational changes upon Zn(2+) and Mn(2+) treatment whereby Trp residues of protein is slightly exposed to the hydrophilic environment, compactness of CP is compromised, whereas in Ca(2+)-treated CP, the tertiary structure integrity is intact upon Zn(2+) and Mn(2+) chelation. Interestingly, CP structural modifications upon Zn(2+) and Mn(2+) treatment was significantly comparable, probably due to similar radii and charges of ions. Taken all together, we have concluded that CP maintains its normal nature in Ca(2+)-loaded state when treated with Zn(2+) and Mn(2+) ions. It can be suggested that Ca(2+) not only stabilize CP structure but also helps CP to keep its structure upon metal ions chelation which is involved in host organism defense system.

Calprotectin induces cell death in human prostate cancer cell (LNCaP) through survivin protein alteration.

Calprotectin (CP), an abundant heterodimeric cytosolic protein of neutrophils, conveys a variety of functions such as tumor cell growth arrest and antimicrobial activity. We investigated CP activity and its possible apoptosis-inducing mechanism of action against an antiandrogen therapy-resistance prostate cancer cell line LNCaP. Cell viability and Annexin V FITC assays were performed in order to investigate its cell death activity and apoptosis, respectively. In order to address cell death inducing mechanism(s), immunocytochemistry and immunobloting analysis, reactive oxygen species (ROS) and nitric oxide (NO) measurements were performed. The effective concentration of CP against LNCaP promoting LNCaP cell death was 200 µg/mL. ROS and NO levels of cells remarkably were enhanced following treatment with 50 and 100 µg/mL of CP, respectively. Protein expression of anti-apoptotic protein survivin was significantly decreased after administration of tumor cells with CP. Our data indicate that CP regulates the LNCaP cells viability via survivin-mediated pathway and ROS and NO enhancement. Thus, inhibition of survivin expression, enhancement of ROS and NO level by CP or other similar pharmaceutical agents might be effective in lowering the malignant proliferation of human prostate cancer cells.

Human calprotectin: effect of calcium and zinc on its secondary and tertiary structures, and role of pH in its thermal stability.

Calprotectin, a heterodimeric complex belonging to the S100 protein family, has been found predominantly in the cytosolic fraction of neutrophils. In the present study, human calprotectin was purified from neutrophils using two-step ion exchange chromatography. The purified protein was used for circular dichroism study and fluorescence analysis in the presence of calcium and zinc at physiological concentrations, as well as for assessment of its inhibitory activity on the K562 leukemia cell line. The thermal stability of the protein at pH 7.0 (physiological pH) and 8.0 (similar to intestinal pH) was also compared. The results of cell proliferation analysis revealed that human calprotectin initiated growth inhibition of the tumor cells in a dose-dependent manner. The intrinsic fluorescence emission spectra of human calprotectin (50 microg/ml) in the presence of calcium and zinc ions show a reduction in fluorescence intensity, reflecting a conformational change within the protein with exposure of aromatic residues to the protein surface that is important for the biological function of calprotectin. The far ultraviolet-circular dichroism spectra of human calprotectin in the presence of calcium and zinc ions at physiological concentrations show a decrease in the alpha-helical content of the protein and an increase in beta- and other structures. Our results also show that increasing the pH level from 7.0 to 8.0 leads to a marked elevation in the thermal stability of human calprotectin, indicating a significant role for pH in the stability of calprotectin in the gut.