Determination of ammonium and L-Glutamine in hybridoma cell cultures by sequential flow injection analysis

A flow injection anlytical system based on a gas diffusion membrane module for ammonia and an ammonium flow-through potentiometric detector has been set up for measurement of L-glutamine and ammonium ions in hybridoma cell cultures. The main feature of the system is that the same basic analytical concept and equipment is used in both measurements, the only difference being for the determination of L-glutamine, in which the sample flows through an immobilized glutaminase cartridge. The conditions to enable the performance of both analysis consecutively, avoiding potential interferences by unwanted deamination of other compounds in the samples, have been determined. Finally, the proposed system has been compared with reference analytical methods for batch hybridoma cell culture experiments.     

PRODUCTION AND OPTIMIZATION OF L-GLUTAMINASE ENZYME FROM Hypocrea jecorina PURE CULTURE

L-Glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) is the important enzyme that catalyzes the deamination of L-glutamine to L-glutamic acid and ammonium ions. Recently, L-glutaminase has received much attention with respect to its therapeutic and industrial applications. It acts as a potent antileukemic agent and shows flavor-enhancing capacity in the production of fermented foods. Glutaminase activity is widely distributed in plants, animal tissues, and microorganisms, including bacteria, yeasts, and fungi. This study presents microbial production of glutaminase enzyme from Hypocrea jecorina pure culture and determination of optimum conditions and calculation of kinetic parameters of the produced enzyme. The optimum values were determined by using sa Nesslerization reaction for our produced glutaminase enzyme. The optimum pH value was determined as 8.0 and optimum temperature as 50°C for the glutaminase enzyme. The Km and Vmax values, the kinetic parameters, of enzyme produced from Hypocrea jecorina, pure culture were determined as 0.491 mM for Km and 13.86 U/L for Vmax by plotted Lineweaver–Burk graphing, respectively. The glutaminase enzyme from H. jecorina microorganism has very high thermal and storage stability.     

Induction of somatic embryogenesis in gum arabic tree [Acacia senegal (L.) Willd.]

Factors affecting somatic embryogenesis from immature cotyledon of gum arabic tree [Acacia senegal (L.) Willd.] were investigated. Induction of somatic embryogenesis was influenced by plant growth regulator concentrations and addition of amino acids in medium. Best induction of somatic embryogenesis was obtained on MS medium supplemented with 0.45 μM 2, 4-D, 2.32 μM Kin and 15 mM L-glutamine. L-glutamine plays a significant role in the maturation of somatic embryos and most of embryos attained maturity only on L-glutamine (15 mM) containing medium. Maximum percent (75.0 ± 2.5) germination of somatic embryos was recorded on medium containing 0.22 μM BAP.     

Inhibition of UV-induced DNA repair at different steps by quinacrine and anthralin

Inhibition of DNA repair can result in accumulation of unrepaired and partially repaired lesions in DNA. Such lesions are important, not only for their primary disruption of information fidelity, but because they may serve as inducers for repair pathways which may be error prone. Inhibition of UV repair by quinacrine and anthralin (50μM each) was detected in ³H thymidine-labeled mouse L1210 cells by sedimentation of nucleoids on neutral sucrose gradients. Quinacrine delayed strand-nicking (and presumably lesion removal) following uv irradiation and anthralin exerted its strongest effects on some other repair step(s) subsequent to strand-incision with accumulation of strand disruptions. Since anthralin is a potent tumor promoter, it will be interesting to examine other promoters to see if they also cause accumulation of repair ‘intermediates’ which could act as inducers of error prone repair.     

L-谷氨酰胺高产菌的选育和发酵条件

以嗜乙酰乙酸棒杆菌(Corynebacterium acetoacidophilum)LG-3为出发菌株,经紫外线(UV)和硫酸二乙酯(DES)诱变处理,磺胺胍(SG)、高浓度(NH4)2SO4定向筛选,获得1株谷氨酰胺高产菌株LG-65,在未经优化的条件下摇瓶发酵72 h可产谷氨酰胺43.5 g/L,比出发菌株的产量32.4 g/L提高了34.3%.在此基础上,对其发酵条件进行优化,经72 h摇瓶发酵产量可达47～48 g/L,7 L发酵罐补料分批发酵40 h可达55 g/L.     

Effect of anionic micelles on speciation of L-glutamine in presence/absence of metals

Abstract

Protonation equilibria of L-glutamine and speciation of its complexes with Co(II), Ni(II) and Cu(II) have been studied in 0–2.5% w/v SLS–water media using pH-metric method. The protonation constants and binary stability constants have been calculated with the computer program MINIQUAD75. Selection of the best fit chemical models is based on standard deviation in stability constants and residual analysis using crystallographic R-factor and sum of squares of residuals in all mass-balance equations. The trend in the variation of stability constants of the complexes with mole fraction of the medium is attributed to the compartmentalisation of complexation equilibria. Distribution of species and effect of influential parameters on chemical speciation have also been presented.     

响应面法优化L-谷氨酰胺发酵培养基的研究

目的:采用响应面法对L-谷氨酰胺发酵培养基成分进行优化,以提高L-谷氨酰胺发酵产量。方法:首先利用Plackett-Burman试验设计筛选出影响L-谷氨酰胺产量的主要因素葡萄糖、玉米浆和(NH4)2SO4,在此基础上采用最陡爬坡实验逼近最大响应区域,并利用中心组合设计及响应面分析法进行回归分析。结果:通过求解回归方程得到L-谷氨酰胺发酵培养基最佳浓度为葡萄糖100 g/L、玉米浆4.5 ml/L、(NH4)2SO437.2 g/L,L-谷氨酰胺产量理论最大值达41.0 g/L。结论:经模型验证,预测值与验证试验平均值接近,在优化条件下谷氨酰胺产量提高了37.6%。     

Availability of the key metabolic substrates dictates the respiratory response of cancer cells to the mitochondrial uncoupling

Active glycolysis and glutaminolysis provide bioenergetic stability of cancer cells in physiological conditions. Under hypoxia, metabolic and mitochondrial disorders, or pharmacological treatment, a deficit of key metabolic substrates may become life-threatening to cancer cells. We analysed the effects of mitochondrial uncoupling by FCCP on the respiration of cells fed by different combinations of Glc, Gal, Gln and Pyr. In cancer PC12 and HCT116 cells, a large increase in O₂ consumption rate (OCR) upon uncoupling was only seen when Gln was combined with either Glc or Pyr. Inhibition of glutaminolysis with BPTES abolished this effect. Despite the key role of Gln, addition of FCCP inhibited respiration and induced apoptosis in cells supplied with Gln alone or Gal/Gln. For all substrate combinations, amplitude of respiratory responses to FCCP did not correlate with Akt, Erk and AMPK phosphorylation, cellular ATP, and resting OCR, mitochondrial Ca^2 + or membrane potential. However, we propose that proton motive force could modulate respiratory response to FCCP by regulating mitochondrial transport of Gln and Pyr, which decreases upon mitochondrial depolarisation. As a result, an increase in respiration upon uncoupling is abolished in cells, deprived of Gln or Pyr (Glc). Unlike PC12 or HCT116 cells, mouse embryonic fibroblasts were capable of generating pronounced response to FCCP when deprived of Gln, thus exhibiting lower dependence on glutaminolysis. Overall, the differential regulation of the respiratory response to FCCP by metabolic environment suggests that mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function, and can be explored for selective cancer treatment.     

Ameliorative effects of L-glutamine on haematological parameters in heat-stressed Red Sokoto goats

The aim of the study was to evaluate haematological responses in Red Sokoto goats (RSGs) administered with L-glutamine during the hot-dry season. Experimental animals included 28 clinically healthy RSGs divided into treated group (n = 14); each administered L-glutamine at 0.2 g/kg body weight, dissolved in 10 mL distilled water, and control group (n = 14); each administered 10 mL distilled water, per os once daily for 21 days. The ambient temperature and relative humidity recorded daily for 4 weeks were used to calculate the temperature-humidity index. Three millilitres of blood sample was collected from each goat by jugular venipuncture for haematology, while rectal temperature (RT), heart rate (HR) and respiratory rate (RR) were also measured once weekly at weeks 0 (before), 1, 2, 3 (during) and 4 (after L-glutamine administration). The haematological, RT, HR and RR data obtained weekly were analysed using repeated-measures one-way ANOVA, followed by Tukey's post-hoc test to evaluate differences between periods, and between treated and control groups. The PCV, haemoglobin concentration and RBC count were higher (P < 0.05) in the treated group compared to the control group during the period of L-glutamine administration. These differences were sustained till week 4. Beginning from week 1 of the study, the total leucocyte count in treated group (10.10 ± 0.25 × 10³/μL) was higher (P < 0.05) than the count in control group (7.23 ± 0.41 × 10³/μL), this trend was also maintained throughout the study. The neutrophil:lymphocyte ratio during weeks 3 and 4 of the experiment was lower (P < 0.05) in the treated compared to the control group. RT was lower (P < 0.05) in treated group than the control group. In conclusion, L-glutamine administration ameliorated the adverse effects of heat stress on the haematological parameters in RSGs during the hot-dry season.