DL-3-n-butylphthalide enhances synaptic plasticity in mouse model of brain impairments

Synaptic impairment results in cognitive dysfunction of Alzheimer’s disease (AD). As a plant extract, it is found that DL-3-n-butylphthalide (L-NBP) rescues abnormal cognitive behaviors in AD animals. However, the regulatory effects of L-NBP on synaptic plasticity remains unclear. APP/PS1 mice at 12 months old received oral L-NBP treatment for 12 weeks. A water maze test assessed cognitive performances. In vitro patch-clamp recordings and in vivo field potential recordings were performed to evaluate synaptic plasticity. The protein expression of AMPA receptor subunits (GluR1 and GluR2) and NMDA receptor subunits (NR1, NR2A, and NR2B) was examined by Western blot. In addition, glutaminase activity and glutamate level in the hippocampus were measured by colorimetry to evaluate presynaptic glutamate release. L-NBP treatment could significantly improve learning and memory ability, upregulate NR2A and NR2B protein expressions, increase glutaminase activity and glutamate level in the hippocampus, and attenuate synaptic impairment transmission in the AD mice. L-NPB plays a beneficial role in AD mice by regulating NMDA receptor subunits’ expression and regulating presynaptic glutamate release.

Highly Active Thermophilic L-Asparaginase from Melioribacter roseus Represents a Novel Large Group of Type II Bacterial L-Asparaginases from Chlorobi-Ignavibacteriae-Bacteroidetes Clade

L-asparaginase (L-ASNase) is a biotechnologically relevant enzyme for the pharmaceutical, biosensor and food industries. Efforts to discover new promising L-ASNases for different fields of biotechnology have turned this group of enzymes into a growing family with amazing diversity. Here, we report that thermophile Melioribacter roseus from Ignavibacteriae of the Bacteroidetes/Chlorobi group possesses two L-ASNases—bacterial type II (MrAII) and plant-type (MrAIII). The current study is focused on a novel L-ASNase MrAII that was expressed in Escherichia coli, purified and characterized. The enzyme is optimally active at 70 °C and pH 9.3, with a high L-asparaginase activity of 1530 U/mg and L-glutaminase activity ~19% of the activity compared with L-asparagine. The kinetic parameters KM and Vmax for the enzyme were 1.4 mM and 5573 µM/min, respectively. The change in MrAII activity was not significant in the presence of 10 mM Ni2+, Mg2+ or EDTA, but increased with the addition of Cu2+ and Ca2+ by 56% and 77%, respectively, and was completely inhibited by Zn2+, Fe3+ or urea solutions 2–8 M. MrAII displays differential cytotoxic activity: cancer cell lines K562, Jurkat, LnCap, and SCOV-3 were more sensitive to MrAII treatment, compared with normal cells. MrAII represents the first described enzyme of a large group of uncharacterized counterparts from the Chlorobi-Ignavibacteriae-Bacteroidetes clade.

Pre-Clinical Evaluation of Novel L-Asparaginase Mutants for the Treatment of Acute Lymphoblastic Leukemia

Introduction Acute Lymphoblastic Leukemia (ALL) accounts for 20% of all hematological malignancies. L-Asparaginase has been a mainstay of ALL for the last 6 decades and is also included in the WHO list of essential medicines for ALL. Escherichia coli L-asparaginase (EcA) was the first asparaginase to be approved for clinical use. However being isolated from bacteria, EcA has many side-effects which in turn affects the tolerability and efficacy of the drug. EcA administration may cause strong immunogenic and hypersensitive reactions in the patients, necessitating withdrawal of the drug. Sensitive individuals react to repeated EcA administration with formation of anti-drug antibodies (ADAs) that bind to and inactivate the enzyme leading to inadequate plasma levels of EcA. Another serious drawback of EcA is the glutaminase activity which leads to neurotoxicity. Other side effects include hepatotoxicity, thromboembolism and pancreatitis. Although a number of attempts have been made to alleviate these problems by rational protein engineering, the optimization of therapy with EcA for ALL patients still remains a challenge. In an attempt to deal with these problems, we created several EcA mutants. On the basis of their activity, stability and antigenicity we short-listed four EcA mutants (Mutant A, B, C and D) having favourable properties for further development. Methods We identified and mutated several B-cell epitopes and amino acid residues at the EcA interface that are responsible for activity, stability and antigenicity. Enzyme activity was measured at 37 oC (optimum temperature for EcA). Glutaminase activity of the mutants was measured and compared to the wild type EcA. The cytotoxicity of the EcA variants was verified in ALL sensitive REH cell lines by performing MTT assay after 24 h incubation. Further the antigenicity of the mutants was assessed by performing indirect ELISA where the binding of the mutants to the commercially available l-asparaginase antibody was analysed. Further, in vivo immunogenicity was evaluated by immunizing Balc C mice with primary and booster doses of EcA mutants over 66 days followed by the measurement of IgG and IgM titers. In addition, the binding of wild-type EcA and mutants to pre-existing anti-asparaginase antibodies in serum isolated from primary and relapsed ALL patients receiving asparaginase therapy was studied by indirect ELISA. Pharmacokinetics of the mutants was evaluated in female Balb C mice by plotting the asparaginase activity-time curve till 24 h following administration of a single i.v. dose of 50 IU/kg and compared with the wildtype. Finally the safety of the EcA mutants was determined by performing single-dose acute toxicity study at 3 dose levels in Balb C mice. Results At 37 oC, we did not find any significant difference in asparaginase activity of any EcA variant with the wild-type. All four variants showed markedly reduced glutaminase activity as compared to wild-type EcA (P<0.05). In MTT assay Mutant D showed 34.02%, Mutant B (32.4%), Mutant C (31.4%), Mutant A (24.22%), and wild type EcA (24.37%) reduction in REH cell viability in comparison to untreated cells. Binding to commercially available anti-asparaginase antibody was 49.09%, 32.63%, 27.43% less for Mutant D, Mutant B and Mutant C respectively compared to wild type EcA. Mice immunized with Mutant D showed 5-fold lower titres of IgG and 4-fold lower titres of IgM in comparison to wild type. Similarly, when compared to wild type, mice immunized with Mutant C showed 2.5-fold lower titres of IgG and 3.5-fold lower titres of IgM. At the same time Mutants B, C and D showed 2-3 fold less binding to pre-existing anti-asparaginase antibodies in samples collected from primary ALL patients undergoing asparaginase therapy. Similarly mutants B, C and D showed approximately 2-fold less binding to pre-existing anti-asparaginase antibodies in samples collected from relapsed ALL patients. Pharmacokinetic profiling showed that half life of Mutant A (267.28 ± 9.74), Mutant B (213.29 ± 6.53) and Mutant D (273.83 ± 35.45) was significantly longer than the wild type (102.17 ± 7.7). In acute toxicity study, we did not observe any significant toxicity of the mutants over the wildtype EcA. The findings are summarized in the figure. Conclusion Considering the immunogenicity, antigenicity and pharmacokinetics, mutant D emerged as a potent drug candidate for further development in the treatment of ALL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Characterization of a New L-Glutaminase Produced by Achromobacter xylosoxidans RSHG1, Isolated from an Expired Hydrolyzed L-Glutamine Sample

As significant biocatalyst, L-glutaminases find potential applications in various fields, from nourishment to the pharmaceutical industry. Anticancer activity and flavor enhancement are the most promising applications of L-glutaminases. In this study, L-glutaminase was isolated and purified from an old glutamine sample. A selected bacterial isolate was characterized taxonomically by morphological characters, biochemical testing and 16S rDNA sequence homology testing. The taxonomical characterization of the isolate identified it as Achromobacter xylosoxidans strain RSHG1. The isolate showed maximum enzyme production at 30 °C, pH 9, with Sorbitol as a carbon source and L-Glutamine as a nitrogen and inducer source. L-Glutaminsae was purified by using column chromatography on a Sephadex G-75. The enzyme has a molecular weight of 40 KDa, pH optimal 7 and is stable in the pH range of 6–8. The optimum temperature for the catalyst was 40 °C and stable at 35–50 °C. The kinetic studies of the purified L-glutaminase exhibited Km and Vmax of 0.236 mM and 443.8 U/mg, respectively. L-Glutaminase activity was increased when incubated with 20 mM CaCl2, BaCl2, ZnSO4, KCl, MgSO4 and NaCl, whereas EDTA, CoCl2, HgCl, ZnSO4 and FeSO4 decreased the activity of the enzyme. The addition of 8% NaCl enhanced the glutaminase activity. L-Glutaminase immobilized on 3.6% agar was stable for up to 3 weeks.

Process conditions optimization and study of pH and thermal stability of free and immobilized gutaminase-free recombinant L-asparaginase II of Pactobacterium carotovorum MTCC 1428 from Escherichia coli

L-asparaginase (EC 3.5.1.1) is used for the treatment of acute lymphocytic leukemia and food processing of starch rich foods for reducing the acrylamide formation. In our current efforts, we have immobilized the purified glutaminase-free recombinant Lasparaginase II of Pectobacterium carotovorum MTCC 1428 from Escherichia coli BL21 (DE3) on glutaraldehyde activated chitosan beads. The purified recombinant L-asparaginase II has no partial glutaminase activity which is a pre-requisite to reduce the possibility of side effects during the course of anticancer therapy. In order to study the best conditions for the performance of free enzymes and immobilized enzymes, response surface methodology was used to optimize the pH and temperature of the process conditions. It was found that the most favorable pH and temperature for the free enzyme were pH 7.83 and 47.64°C while for the immobilized enzyme, the optimum pH and temperature levels were found to be 7.88 and 48.07 °C. Furthermore, the thermal and pH studies of free and immobilized enzymes were studied under various combinations of pH and temperature and finally the thermodynamic parameters of free and immobilized glutaminase-free recombinant asparaginase were evaluated.

Filamentous Fungi Producing l-Asparaginase with Low Glutaminase Activity Isolated from Brazilian Savanna Soil

l-asparaginase is an enzyme used as treatment for acute lymphoblastic leukemia (ALL) due to its ability to hydrolyze l-asparagine, an essential amino acid synthesized by normal cells unlike neoplastic cells. The adverse effects of l-asparaginase formulations are associated with its glutaminase activity and bacterial origin; therefore, it is important to find new sources of l-asparaginase-producing eukaryotic microorganisms with low glutaminase activity. This work evaluated the biotechnological potential of filamentous fungi isolated from Brazilian Savanna soil and plants for l-asparaginase production. Thirty-nine isolates were screened for enzyme production using the plate assay, followed by measuring enzymatic activity in cells after submerged fermentation. The variables influencing l-asparaginase production were evaluated using Plackett–Burman design. Cell disruption methods were evaluated for l-asparaginase release. Penicillium sizovae 2DSST1 and Fusarium proliferatum DCFS10 showed the highest l-asparaginase activity levels and the lowest glutaminase activity levels. Penicillium sizovae l-asparaginase was repressed by carbon sources, whereas higher carbon concentrations enhanced l-asparaginase by F. proliferatum. Maximum enzyme productivity, specific enzyme yield and the biomass conversion factor in the enzyme increased after Plackett–Burman design. Freeze-grinding released 5-fold more l-asparaginase from cells than sonication. This study shows two species, which have not yet been reported, as sources of l-asparaginase with possible reduced immunogenicity for ALL therapy.

Rapid Screening of Cultural Parameters for Extracellular Halophilic Glutaminase Production from Tetragenococcus Muriaticus FF5302 using the Plackett-Burman’s Experimental Design

The Plackett-Burman’s experiemental design was used to efficiently select the key cultural parameters for the production of halophilic glutaminase by moderating halophilic bacterium Tetragenococcus muriaticus FF5302. Eleven variables were selected, which involved glutamine, peptone, yeast extract, glucose, fructose, KCl, MgSO4, NaCl, temperature, pH, and inoculum size, to identify the most significant variables affecting halophilic glutaminase production in 12 experimental trials. The results of the statistical analyses demonstrated that glutamine, pH, and temperature had significant effects on halophilic glutaminase production. The maximum halophilic glutaminase activity of 199.27 U mL-1 was observed after 120 h of fermentation. After Plackett-Burman’s design experiments, the glutaminase activity was found to be 2.28 folds increase compared to basal conditions. Thus, the cultivation of T. muriaticus FF5302 under the optimal condition could enhance the production of halophilic glutaminase enzyme effectively.

Anticancer Potential of L-Asparaginase: An Overview

Asparaginase, derived from microbial origin hydrolyses L-asparagine to L-aspartic acid. The enzyme finds principal use in the treatment of Acute Lymphoblastic Leukemia during childhood that primarily occurs between two to ten years of age. L-Asparaginase finds its use in management of haemopoietic disorders especially in pediatrics that is caused due to proliferation and enlargement of lymphoblast in bone marrow and in blood as well as other part of the body. L- Asparaginase from bacterial sources exhibit quaternary and tertiary structural forms. However for using it in therapeutic and clinical application it should not generate any fatal allergic reaction to the patient. Such effects can occur due to the enzyme associated L-Glutaminase activity and also due to the endotoxins from bacteria in enzyme preparations. Therefore, with the recent development in biotechnology with respect to production and purification techniques it is possible to get pure L- asparaginase from microbial origin. The present article provides an insight into the mechanism of action of L-Asparaginase as an anticancer agent and its industrial applications.

Effect of Crop Cover and Stage of Crop Growth on Soil L-Glutaminase Activity

A pot culture experiment was conducted at glass house of Department of Soil Science and Agricultural Chemistry, College of Agriculture, Rajendranagar, Hyderabad. The aim of the present experiment was to study the influence of crop cover and stage of crop growth on soil L- glutaminase activity in an Alfisol and Vertisol. The experiment was under taken with six crops viz., two cereals (Rice, Maize), two legumes (Groundnut, Greengram), one oilseed (Sunflower) and one vegetable (Bhendi) crop. The experiment was conducted in Completely Randomized Block design with three replications along with the uncropped control. The results obtained with regard to the effect of these crops on soil L-glutaminase activity showed that there was an increase in enzyme activity with age of the crop upto 60 DAS and it varied with crops grown. The increased enzyme activity (μg of NH4+ released g-1 soil 4h-1) varied from 5.56 to 12.17 for groundnut, 5.58 to 11.25 for greengram, 5.43 to 10.87 for sunflower, 5.48 to 8.61 for rice, 5.39 to 8.23 maize and 5.31 to 7.92 for bhendi in Vertisol. In Alfisol the L-glutaminase activity (μg of NH4+ released g-1 soil 4h-1) under different crop cover found to vary from 6.72 to 13.59 (groundnut), 6.68 to 12.71 (greengram), 6.63 to 11.96 (sunflower), 6.61 to 10.25 (rice), 6.59 to 9.47 (maize), 6.62 to 9.26 (bhendi). A close perusal of the data indicates that the L-glutaminase activity followed the sequence groundnut > greengram > sunflower > rice > maize > bhendi, in both Alfisol and Vertisol.