Production,optimization, purification,characterization, and anti-cancer application of extracellular L-glutaminase produced from the marine bacterial isolate

Abstract

L-glutaminase from bacterial sources has been proven to be effective and economical agents in cancer therapy, food industry and high-value chemicals like threonine. In the present study, a newly isolated bacterial strain was potentially producing extracellular L-glutaminase, it identified as Bacillus subtilis OHEM11 (MK389501) using the 16S rRNA gene. L-glutaminase production optimized and the optimum factors for production under submerged fermentation were at pH 6.5–7.0 and 35?°C after 28?hr using rhamnose and glutamine as carbon and nitrogen sources, respectively, while bagasse was the best inducer for the production under solid-state fermentation. Ethanol precipitation and ion-exchange chromatography using QFF are the purification steps. L-glutaminase was purified to 2-fold with specific activity 89.78?U/mg and its molecular weight about 54.8?kDa with the alkaline property of the enzyme makes it clear having carcinostatic property; maximum enzyme activity at pH 8.2 and 40?°C and retained about 90% activity for 1?hr. The cytotoxicity effect of L-glutaminase indicated a significant safety on Vero cells with high anticancer activity against NFS-60, HepG-2, and MCF-7 cancer cell lines. The outcomes demonstrated that L-glutaminase could be applied in many biotechnological applications such as pharmaceutical and food processing.     

Design,Synthesis and Molecular Docking Studies of Some Tetrahydropyrimidine Derivatives as Possible Fascin Inhibitors

Eight derivatives of tetrahydropyrimidine scaffold were designed and prepared as hybrid compounds possessing the structural features of both monastrol as an anticancer drug and nifedipine as a fascin blocking agent. All of the compounds were evaluated for their cytotoxic potency and the ability to inhibit 4T1 breast cancer cells migration. Then, they were investigated in silico for their ability to inhibit the fascin protein using molecular docking simulation. The most potent compound was 4d and the weakest one was 4a according to the in vitro cytotoxicity assay. The corresponding IC₅₀ values were 193.70 and 248.75 μm , respectively. The least cytotoxic compound ( 4a ) was one of the strongest ones in binding to the fascin binding site according to the molecular docking results. 4a and 4e inhibited the 4T1 cells migration better than other compounds. They were more potent than nifedipine in inhibiting the migration process. In silico studies proved 4h to be the most potent fascin inhibitor in terms of ΔG_bind although it was not inhibiting migration. The controversy between the in vitro and in silico results may cancel the theory of the involvement of the fascin inhibition in the migration inhibition. However, the considerable antimigratory effects of some of the synthesized compounds encourage performing further in vivo experiments to introduce novel tumor metastasis inhibitors.     

The roles of hyperglycaemia and oxidative stress in the rise and collapse of the natural protective mechanism against vascular endothelial cell dysfunction in diabetes

Vascular endothelial cell (VEC) dysfunction in diabetes has been associated with hyperglycaemia-induced intra- and extracellular glycation of proteins and to overproduction of glucose-derived free radicals. VEC protect their intracellular environment against an increased influx of glucose in face of hyperglycaemia by reducing the expression and plasma membrane abundance of their glucose transporter-1 (GLUT-1). We investigated the hypothesis that glucose-derived free radicals induce this down-regulatory mechanism in VEC, but proved the contrary. In fact, pro-oxidants significantly increased the expression and plasma membrane abundance of GLUT-1 and the rate of glucose transport in VEC while abolishing high-glucose-induced down-regulation of the hexose transport system. The resulting uncontrolled influx of glucose followed by overproduction of glucose-derived ROS further up-regulates the rate of glucose transport, and vice versa. This perpetuating glycoxidative stress finally leads to the collapse of the auto-regulatory protective mechanism and accelerates the development of dysfunctional endothelium in blood vessels.     

WNT and NOTCH signaling pathways as activators for epidermal growth factor receptor in esophageal squamous cell carcinoma

Background

Esophageal squamous cell carcinoma (ESCC) is the most common histological type of esophageal cancer, with a poor prognosis. Deregulation of WNT and NOTCH signaling pathways is important in ESCC progression, which can be due to either malfunction of their components or crosstalk with other pathways. Therefore, identification of new crosstalk between such pathways may be effective to introduce new strategies for targeted therapy of cancer. A correlation study was performed to assess the probable interaction between growth factor receptors and WNT/NOTCH pathways via the epidermal growth factor receptor (EGFR) and Musashi1 (MSI1), respectively.

Methods

Levels of MSI1/EGFR mRNA expression in tumor tissues from 48 ESCC patients were compared to their corresponding normal tissues using real-time polymerase chain reaction.

Results

There was a significant correlation between EGFR and MSI1 expression (p?=?0.05). Moreover, there was a significant correlation between EGFR/MSI1 expression and grade of tumor differentiation (p?=?0.02).

Conclusion

This study confirms a direct correlation between MSI1 and EGFR and may support the important role of MSI1 in activation of EGFR through NOTCH/WNT pathways in ESCC.

     

Recent changes to the classification of symbiotic,nitrogen-fixing,legume-associating bacteria: a review

The Rhizobia are collectively comprised of gram negative soil bacteria that have the ability to form symbiotic nitrogen-fixing root and/or stem nodules in association with leguminous plants. The taxonomy of these bacteria is continually in a state of flux, in large part due to rapid development of refined molecular biology techniques. The isolation and characterization of new, and often different, legumes-nodulating bacteria on a variety of plant hosts has resulted in the naming of many new rhizobial species. Here we update the taxonomy of the legume-nodulating bacteria and describe newly identified rhizobia capable of nodulating edible legumes and legume trees. In 1990, there was only one bacterial species that was known to nodulate common bean worldwide (Rhizobium leguminosarum sv. phaseoli), one species that nodulated faba bean (Rhizobium leguminosarum sv. viciae), and two species that nodulated soybean (Bradyrhizobium japonicum and Rhizobium fredii). Today, nearly 14, 11, 6, 5, 5, 4, 3 and 2 species have been defined that are capable of nodulating common bean, soybean, cowpea, chickpea, peanut, lentils, faba bean and pea, respectively. The recent use of whole genome based taxonomy (genomotaxonomy) will surely change how we define this important group of bacteria. The identification of several rhizobial species that are able to nodulate and fix nitrogen with edible legumes may enhance the production of these crops and can compensate for worldwide deficiencies in human nutritional needs in the future.     

Evaluation of Pochonia chlamydosporia var. chlamydosporia as a control agent of Meloidogyne javanica on pistachio

The potential of isolates of Pochonia chlamydosporia var. chlamydosporia as biocontrol agents for root-knot nematodes was investigated in vitro and on pistachio plants. On potato dextrose agar, growth of all isolates started at temperatures above 10°C, reached maximum between 25 and 28°C and slowed down at 33°C. On water agar, all isolates parasitized more than 85% of the eggs of Meloidogyne javanica at 18°C after 3 weeks. Filtrates of isolates grown on malt extract broth did not cause more than 5% mortality on second-stage juveniles of M. javanica after 48 h of incubation. A single application of 10×10³ chlamydospores (produced on sand–barley medium) g^–1 soil, was applied to unsterilised soil planted with pistachio cv. Kalehghochi, and plants were inoculated with 3000 nematode eggs. After 120 days in the glasshouse, nematode multiplication and damage were measured. Ability of fungus isolates to survive in the soil and to grow on roots were estimated by counting colony forming units (cfu) on semi-selective medium. Fungal abundance in soil increased nearly 3-fold and 10×10³ and 20×10³ cfu g^–1 root of pistachio were estimated in pots treated with isolates 40 and 50, respectively. Strain 50 was more abundant in soil and on the roots, infected more eggs (40%) on the roots and controlled 56% of total population of M. javanica on pistachio roots, whereas isolate 40 parasitized 15% of the eggs on the roots and controlled ca. 36% of the final nematode population.     

Mesostigmatic mites associated with birds and mammals in Iran. A review

There are diverse relationships between mites and birds or mammals. These mites may play an important role in epizootics and in the perpetuation of some significant diseases. The purpose of this study is to revise the current knowledge of mesostigmatic mites occurring on birds, including their nests and mammals and their substrates, in Iran and to compare the results with other regions of the Palearctic ecozone. This study presents a revised list of 38 species of mesostigmatic mite occuring on birds (17 species) and mammals (24 species) or in their nests/substrates in Iran. Dermanyssus gallinae, Ornithonyssus sylviarum and Parasitus hyalinus were found on both birds and mammals. The species composition of mites reported in Iran was compared with other regions of the Palearctic ecozone. Parasites, specifically those from genera Dermanyssus, Ornithonyssus and Liponyssoides, may be hazardous to human health. Species from these genera were predominant among the reported mites.     

Critical role of Glu175 on stability and folding of bacterial luciferase: stopped-flow fluorescence study

Bacterial luciferase is a heterodimeric enzyme, which catalyzes the light emission reaction, utilizing reduced FMN (FMNH2), a long chain aliphatic aldehyde and O(2), to produce green-blue light. This enzyme can be readily classed as slow or fast decay based on their rate of luminescence decay in a single turnover. Mutation of Glu175 in alpha subunit to Gly converted slow decay Xenorhabdus Luminescence luciferase to fast decay one. The following studies revealed that changing the luciferase flexibility and lake of Glu-flavin interactions are responsible for the unusual kinetic properties of mutant enzyme. Optical and thermodynamics studies have caused a decrease in free energy and anisotropy of mutant enzyme. Moreover, the role of Glu175 in transition state of folding pathway by use of stopped-flow fluorescence technique has been studied which suggesting that Glu175 is not involved in transition state of folding and appears as surface residue of the nucleus or as a member of one of a few alternative folding nuclei. These results suggest that mutation of Glu175 to Gly extended the structure of Xenorhabdus Luminescence luciferase, locally.     

Foam cell‐derived 4‐hydroxynonenal induces endothelial cell senescence in a TXNIP‐dependent manner

Vascular endothelial cell (VEC) senescence is considered an early event in the development of atherosclerotic lesions. Stressful stimuli, in particular oxidative stress, have been linked to premature senescence in the vasculature. Foam cells are a major source of reactive oxygen species and may play a role in the induction of VEC senescence; hence, we investigated their involvement in the induction of VEC senescence in a co‐culture transwell system. Primary bovine aortic endothelial cells, exposed to the secretome of THP‐1 monocyte‐derived foam cells, were analysed for the induction of senescence. Senescence associated β‐galactosidase activity and the expression of p16 and p21 were increased, whereas phosphorylated retinoblastoma protein was reduced. This senescent phenotype was mediated by 4‐hydroxnonenal (4‐HNE), a lipid peroxidation product secreted from foam cells; scavenging of 4‐HNE in the co‐culture medium blunted this effect. Furthermore, both foam cells and 4‐HNE increased the expression of the pro‐oxidant thioredoxin‐interacting protein (TXNIP). Molecular manipulation of TXNIP expression confirmed its involvement in foam cell‐induced senescence. Previous studies showed that peroxisome proliferator‐activated receptor (PPAR)δ was activated by 4‐hydroalkenals, such as 4‐HNE. Pharmacological interventions supported the involvement of the 4‐HNE‐PPARδ axis in the induction of TXNIP and VEC senescence. The association of TXNIP with VEC senescence was further supported by immunofluorescent staining of human carotid plaques in which the expression of both TXNIP and p21 was augmented in endothelial cells. Collectively, these findings suggest that foam cell‐released 4‐HNE activates PPARδ in VEC, leading to increased TXNIP expression and consequently to senescence.