Magnesium Increases the Protective Effect of Citicoline on Aluminum Chloride-induced Cognitive Impairment

Objective@#Alzheimer’s disease is a popular neurodegenerative disorder which is growing in the elderly people. Exposure to environmental pollutant like aluminum could trigger or accelerate its involved mechanisms like tau phosphorylation. The current study will evaluate the effect of alone or co-administration of Citicoline or/and magnesium on the aluminum chloride induced memory impairment. @*Methods@#Male albino mice were randomly divided into different groups (n = 7). Memory impairment was induced via orally administration of 300 mg/kg Aluminum Chloride for 28 days. Based on respective group, animals received 100, 250, 500 mg/kg of Citicoline or 50, 100, 150 mg/kg of Magnesium sulfate (MgSO4), intraperitoneally. In co-administration, 50 mg/kg of MgSO4 injected concomitantly with 100, 250, or 500 mg/kg of Citicoline. Rivastigmine (2 mg/kg intraperitoneally) was used as a positive control. Memory was evaluated using the Object Recognition Task (ORT) and Passive Avoidance Test (PAT). @*Results@#The studied doses of Citicoline or MgSO4 when administered individually showed significant increase in the discrimination index in ORT and latency time in the PAT compared to the Aluminium chloride (AlCl3) treated group. Concomitant injection of 50 mg/kg MgSO4 with the different doses of Citicoline strongly increased the above indices values in comparison to each alone. @*Conclusion@#The findings show, individual administration of Citicoline or MgSO4 inverted the AlCl3-induced memory impairment in a dose independent manner. The addition of MgSO4 to the Citicoline showed a synergistic effect in the PAT and likely additive effect in the ORT.

Protective Effects of Citicoline and Benfotiamine Each Alone and in Combination on Streptozotocin-induced Memory Impairment in Mice

Objective@#Diabetes mellitus is associated with cognitive disorders such as Alzheimer’s disease. Studies have shown that citicoline and benfotiamine can improve memory and learning through different mechanism of actions. The aim of this study was to compare the individual effects of benfotiamine (100, 200, 300 mg/kg) and citicoline (50, 100, 250, 500 mg/kg, gavage) and their co-administration on memory impairments in diabetic mice. @*Methods@#Diabetes was induced by a single dose of streptozotocin (STZ, 140 mg/kg, intraperitoneal) and benfotiamine and/or citicoline were administered for three weeks. Memory was evaluated using the object recognition task (ORT) and passive avoidance test (PAT). @*Results@#Results from ORT shows that citicoline at 50, 100, 250, and 500 mg/kg and benfotiamine at 100, 200, and 300 mg/kg and their combination (benfotiamine at 100 mg/kg added to citicoline at 50, 100, and 250 mg/kg) are equally effective in reversing the memory loss induced by STZ (p ＜ 0.001). PAT results demonstrate that citicoline at 100, 250, and 500 mg/kg and benfotiamine at above doses did not improve the latency time when administered separately, but benfotiamine at a fixed dose of 100 mg/kg in the presence of citicoline at 50, 100, and 250 mg/kg increased the latency time and improved memory significantly. @*Conclusion@#In conclusion, in PAT, co-administration of benfotiamine and citicoline was more effective than either alone in improving memory. Regarding ORT, although benfotiamine added to citicoline improved memory notably, the difference between combination therapy and single-drug therapy was not considerable.

Molecular gene cloning and sequencing of glutamate decarboxylase gene from Lactobacillus delbrueckii and Lactobacillus reuteri

Glutamate decarboxylase enzyme produces ?-aminobutyric acid [GABA] in a non-reversible decarboxylation reaction of glutamate. GABA is a major inhibitory neurotransmitter of the brain and it is also present at high concentration in other organs such as pancreatic islets. GABA has effects on blood pressure, diabetes, inflammation, sleeplessness and depression. Some bacteria such as Lactobacillus strains are capable of GABA production. Identification of these bacteria is important both for researchers and industry. The aim of this study was molecular gene cloning and sequencing of glutamate decarboxylase [gad] from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 and Lactobacillus reuteri ATCC 23272. These strains were cultured in MRS medium at 37degreeC for 24 hours. For cloning gad gene from these strains, polymerase chain reaction [PCR] was performed using specific primers designed by Oligo7 software. PCR production was extracted from agarose gel and was inserted into PGEM-T vector using T4 DNA ligase enzyme and then it was transformed to E. coli XL1Blue. In the final step, white colonies were selected and after plasmid extraction, the existence of gad gene in recombinants was confirmed by PCR. Gad gene was cloned from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 and Lactobacillus reuteri ATCC 23272. It is for the first time that the gad gene sequences from these bacteria were registered on NCBI with accession numbers KF751355 and KF751352 respectively. The result of this research indicates that the two aforementioned bacteria contain glutamate decarboxylase gene and therefore they possibly can be used for industrial lamda-aminobutyric acid production

Molecular cloning and expression of rat micro -opioid receptor in Escherichia coli [BL21]

The micro [mu] opioid receptors, which mediate the effects of morphine, are widely distributed in brain. The purpose of this study was to design a simple expression system for rat micro -receptor in Escherichia coli [BL21]. In this laboratory study, rat micro -receptor cDNA was isolated from pcDNA3 vector using Xba1 and Hind3 restriction enzymes. pET-15b was digested by Nco1 restriction enzyme. micro -receptor cDNA and pET-15b formed a recombinant DNA that was transformed to Escherichia coli [BL21]. The insert presence was proved by Rsa1 restriction enzyme and the induction of its expression was performed using IPTG. Finally, the presence of desired insert was confirmed using RSA1, and the colonies that had correct orientation in gene containing plasmid were used for further studies. On the SDS-page gel electrophoresis, a 33 kDa band was observed when IPTG was used at 0.5 and 1 mM concentrations, that is equal to calculated molecular weight of rat micro -receptor. At the end of this project, the expression of rat micro -receptor by IPTG induction was successfully performed

Cloning and expression of functional reteplase in escherichia coli TOP10

Production of tissue Plasminogen Activator protein [t-PA] in prokaryotes systems has many problems such as the lack of active protein production, multiple purification steps, and renaturation process which has been shown to be costly and time-consuming. In this study, reteplase which is the nonglycosylated active domain of t-PA was used to transform TOP10 Escherichia coli [E. coli] bacteria to resolve some of the above mentioned problems. Reteplase cDNA was ligated into pBAD/gIII plasmid which allowed secretion of this protein into the periplasmic space and would allow the correct formation of disulfide bonds in protein structure. The presence of reteplase cDNA in pBAD/gIII plasmid was confirmed by restriction digestion and sequencing. After induction of the expression of this protein by adding 0.0002% L-Arabinose to the medium, the proteins in periplasmic space as well as the inclusion bodies formed inside the cell were extracted. Subsequently, these proteins were purified and detected by Western blot method. Our results showed that the amount of reteplase extracted from periplasmic space was much lower than the extracted inclusion bodies and large quantities of the recombinant protein were present as inclusion bodies. Therefore, it was more efficient to use inclusion body extraction method for protein isolation and purification. We produced active reteplase after its expression in E. coli TOP10 and isolation of inclusion bodies produced the best results for purification and extraction of this protein

Constructing a mouse Oct4 promoter/EGFP vector, as a whole-cellular reporter to monitor the pluripotent state of cells

The transcription factor Oct-4, is an important marker of undifferentiating level and a key regulating factor for maintenance of pluripotency in cells. Establishment of an Oct-4 promoter-based reporter ystem is an appropriate tool for monitoring the differentiation of embryonic stem cells both in vivo and in vitro. In the present study, we report construction of a recombinant vector, pDB2 Oct4 promoter/EGFP, in which expression of Enhanced Green Fluorescent Protein [EGFP] was controlled by the mouse Oct-4 promoter. In transfected mouse embryonic stem cells with this vector, EGFP was predicted to be specifically expressed in pluripotency state. After transfection, high-level expression of EGFP under the control of Oct-4 promoter was observed in manipulated embryonic stem cells. Thus, our new cellular reporter showed that both the properties of embryonic cells and expression the EGFP could be of great help in studying the differentiating and reprogramming mechanisms of mESCs

Synthesis and evaluation of the anxiolytic activity of some phthalimide derivatives in mice model of anxiety

The aim of the present study was to synthesis a series of phthalimides based on our previous works and examine their anxiolytic properties. Using a three steps process, phthalimides were prepared from the corresponding di-methyl phthalate derivatives. Phthalic anhydride was nitrated to produce 3-nitrophthalic acid. Ring closer of either 3-nitrophthalic acid or di-methyl phthalate with urea were carried out in reflux condition. Final compounds were prepared by base catalyzed condensation of 4-methylbenzoyl chloride, benzoyl chloride and benzyl chloride with the resulting imides. From the tested compounds, only N-benzoyl 3-nitro-phthalimide was shown to produce anxiolytic activity by increasing the number of entries and time spent in open arms at 10 mg/kg

Molecular detection of lipase A gene in putative bacillus subtilis strains isolated from soil

The present study was undertaken to screen the soil samples collected in Iran for the presence of the Bacillus subtilis lipase A gene. The bacterial colonies obtained from the collected soil samples were examined by physical appearance, biochemical tests and the polymerase chain reaction [PCR]. Only four colonies were identified as putative B. subtilis strains and all contained the lipase A gene. However, the intensities of the DNA bands were different and correlated with the differences obtained from the biochemical tests. Polymorphism of the lipase gene was also determined in samples using SSCP assay. In conclusion, this study demonstrates an easy and reliable method for detection of the lipase gene in B. subtilis strains. Further screening of the soil by this method will enable the detection and identification of industrially more favorable lipases

Effects of Otostegia persica [Burm.] boiss on morphine withdrawal syndrome in mice

Effect of Otostegia persica on naloxone-induced morphine withdrawal syndrome was studied in male mice. Dependence was induced using daily subcutaneous injections of morphine for three days. Morphine was injected to mice at doses of 30 and 45mg/kg on day 1 and 60 and 90mg/kg on day 2 [8:00 am and 6:00 pm]. On day 3, morphine [90mg/kg] was injected 1h before oral administration and 1.5h before intraperitoneal [i.p.] injection of hydroalcoholic and hexane extracts of the plant. Naloxone was injected [5mg/kg, i.p.] 2h after the final dose of morphine and the withdrawal signs including jumping, rearing, diarrhoea, piloerection, tremor and ptosis were recorded during a period of 30 minutes. While oral and i.p. administration of hydroalcoholic extract reduced the number of jumping and rearing, the hexane extract could not exert any significant change. Also the hydroalcoholic extract [1500mg/kg] significantly [p<0.05] reduced diarrhoea, piloerection, tremor and ptosis. The hexane extract only significantly [p<0.05] inhibited diarrhoea. Results of this study indicated that the extract of Otostegia persica contained component[s] that alleviate morphine withdrawal syndrome and the responsible constituent[s] is [are] found in polar fraction since the hexane extract had only a negligible effect