Malignant glioma is the most common and aggressive form of primary brain tumor in adults. In this study, we describe the efficacy of nonviral gene delivery carriers, histidine- and arginine- or ...histidine- and lysine-grafted polyamidoamine (PAMAM) dendrimers (PAMAM-H-R and PAMAM-H-K), in delivering a therapeutic and a tumor-selective killer gene, apoptin, using human glioma cells (U87-MG) and newborn human dermal fibroblast cells. We analyzed transfection efficiency using luciferase and a plasmid DNA encoding for enhanced green fluorescent protein and assessed cell viability in both cells. The results show that transfection efficiency of PAMAM-H-R and PAMAM-H-K was greatly increased compared with that of native PAMAM. Moreover, among PAMAM derivatives, cytotoxicity of PAMAM-H-K was very low. We treated both cells with complexes of PAMAM-H-R or PAMAM-H-K and apoptin and analyzed their cellular uptake by flow cytometry and localization by confocal microscopy. Furthermore, cell cycle distribution, caspase 3 activity assay, and JC-1 analysis showed cell death induced by apoptin in U87-MG cells. The present study demonstrates that a PAMAM-H-R/apoptin complex is an effective gene carrier system in glioma cell culture.
This study aimed to find the most sensitive brain region of APP-PS1 mice in early-stage Alzheimer's disease (AD) and to compare the findings with wild-type mouse brain using
1H high resolution magic ...angle spectroscopy (HR-MAS). At 18 and 35 weeks of age, the object recognition test was performed with both APP-PS1 and wild-type mice, and the metabolite concentrations were measured in six brain regions at 38–42 weeks using
1H HR-MAS. Compared to that of wild-type mice, the memory index of the APP-PS1 mice at 18 weeks was not significantly different; however, the memory index of the APP-PS1 mice at 35 weeks was significantly lower. Similar to the results of the
1H HR-MAS, the N-acetyl aspartate (NAA)
+
acetate (Acet) level in APP-PS1 mice was decreased in the hippocampus and temporal cortex, and the myo-inositol (mIns) level was increased in the entire brain. In addition, scyllo-inositol (sIns) was also elevated in the frontal, occipital, and parietal cortices, hippocampus and thalamus. These findings demonstrated that the behavioral abnormalities of the APP-PS1 mice started at about 30 weeks of age and that the hippocampus and temporal cortex were the most sensitive regions during early-stage AD. In addition, the results of this study confirmed that an increase of mIns and sIns precedes the reduction of the NAA level. These findings demonstrated that the metabolism of the APP-PS1 mouse was associated with early-stage AD. Furthermore, the regional neurochemical profile of APP-PS1 mouse can be used to investigate the pathophysiological mechanisms associated with AD.
Abstract The aim of this study was to quantitatively investigate the chronic ethanol-induced cerebral metabolic changes in various regions of the rat brain, using the proton high resolution magic ...angle spinning spectroscopy technique. The rats were divided into two groups (control group: N = 11, ethanol-treated group: N = 11) and fed with the liquid diets for 10 weeks. In each week, the mean intake volumes of liquid diet were measured. The brain tissues, including cerebellum (Cere), frontal cortex (FC), hippocampus (Hip), occipital cortex (OC) and thalamus (Thal), were harvested immediately after the end of experiments. The ex vivo proton spectra for the five brain regions were acquired with the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence at 500-MHz NMR spectrometer. All of the spectra were processed using the LCModel software, with simulated basis-set file, and the metabolite levels were referenced to total creatine. In the ethanol liquid diet group, there were significant increases in the metabolites ratio levels, as compared to control (Cere: alanine, glutathione, and N-acetlyaspartate; FC: phosphocholine and taurine; Hip: alanine, glutamine, and N-acetylaspartate; OC: glutamine; Thal: alanine, γ-aminobutyric acid, glutamate, glycerophosphocholine, phosphocholine, taurine, and free choline). However, in the ethanol liquid diet group, the myo-inositol levels of the OC were significantly lower. The present study demonstrates how chronic ethanol consumption affects cerebral metabolites in the chronic ethanol-treated rat. Therefore, this result could be useful to pursue clinical applications for quantitative diagnosis in human alcoholism.
The glutathione S-transferase (GST) system is useful for increasing protein solubility and purifying soluble GST fusion proteins. However, purifying half of the GST fusion proteins is still ...difficult, because they are virtually insoluble under non-denaturing conditions. To optimize a simple and rapid purification condition for GST-pyruvate kinase muscle 2 (GST-PKM2) protein, we used 1% sarkosyl for lysis and a 1：200 ratio of sarkosyl to Triton X-100 (S-T) for purification. We purified the GST-PKM2 protein with a high yield, approximately 5 mg/L culture, which was 33 times higher than that prepared using a conventional method. Notably, the GST-high-temperature requirement A2 (GST-HtrA2) protein, used as a model protein for functional activity, fully maintained its proteolytic activity, even when purified under our S-T condition. This method may be useful to apply to other biologically important proteins that become highly insoluble in the prokaryotic expression system. BMB reports 2011; 44(4): 279-284
The purpose of this study was to develop a cone-shape phantom for multi-voxel magnetic resonance spectroscopy (MRS) and to evaluate MR spectra using the cone-shape phantom we developed in this study.
...A cone-shape MRS phantom was developed with a combination of cone-shape vials. The cylindrical main body was made of acrylic resin and the cone-shape vials were fabricated from poly-ethylene cones. Each cone of the phantom was filled with various metabolite materials. 1.5T GE and 3T Philips systems were used for the single voxel spectroscopy (SVS) as well as for the multi-voxel spectroscopy (MVS). Identification and quantification of the metabolite materials in the cone-shape phantom were done by the SAGE post-program. The MR images and spectra of the cone-shape phantom were obtained from the assigned slice position. The high order shimming control provided enhanced resolution in the SVS and MVS. The area and amplitude were proportional to the metabolite volume in the voxel. The present study demonstrated that the cone-shape phantom was useful for the metabolite quantification. Thus, we propose that the cone-shape phantom can be used for the evaluation of quality control of the MR spectra obtained from SVS and MVS.
The purpose of this study is to investigate the metabolic alterations associated with acute alcohol treatment in zebrafish by 1H nuclear magnetic resonance spectroscopy (NMRS). The brain metabolism ...of zebrafish was investigated after acute alcohol treatment (one-hour long exposure of adult fish to 0.00%, 0.25%, 0.50%, or 1.00% ethyl alcohol) with whole brain extraction. The results of this study showed that glutamate (Glu) was significantly decreased, scyllo-inositol (sIns) showed a small apparent increase only in the highest acute treatment dose group, and myoinositol (mIns) showed a significant decrease. Glu/tCr and mIns/tCr levels were significantly reduced regardless of the alcohol dose, and sIns/tCr was increased in the highest alcohol treatment dose group. The present NMR study revealed that specific metabolites, such as Glu and mIns, were substantially decreased in case of acute alcohol exposed zebrafish brain.
The purpose of this study was to characterize the neurochemical profiles of various parts of the canine brain using proton nuclear magnetic resonance spectroscopy, tissue extraction, and external ...simulated phantom concentration quantification. The occipital, frontal, and temporal lobes, thalami, cerebellar cortices, and spinal cords of five pure bred adult beagles were collected, and heavy water solutions for the nuclear magnetic resonance sample were prepared using the methanol–chloroform–water extraction method. The metabolite concentrations in canine brain tissues were measured and compared with those found in human and rat brain tissues. In addition, the cross peaks of Lac, Glu/Gln, and mIns were identified using two-dimensional correlation spectroscopy in the canine frontal cortex. The present study demonstrated the absolute quantification of canine neuronal parts using in vitro high-resolution magnetic resonance spectroscopy, with tissue extraction used to accurately measure metabolite concentrations, thus providing valuable metabolic information regarding the various canine neuronal regions.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the degeneration of motor neurons. Mutations in Cu/Zn superoxide dismutase (SOD1), including G93A, were reportedly linked ...to familial ALS. SOD1 is a key antioxidant enzyme, and is also one of the major targets for oxidative damage in the brains of patients suffering from Alzheimers disease (AD). Several lines of evidence suggest that intracellular amyloid beta (Abeta) is associated with the pathogenesis of AD. In this report we demonstrate that intracellular Abeta directly interacts with SOD1, and that this interaction decreases the enzymatic activity of the enzyme. We observed Abeta-SOD1 aggregates in the perinuclear region of H4 cells, and mapped the SOD1 binding region to Abeta amino acids 26-42. Interestingly, intracellular Ab binds to the SOD1 G93A mutant with greater affinity than to wild-type SOD1. This resulted in considerably less mutant enzymatic activity. Our study implicates a potential role for Abeta in the development of ALS by interacting with the SOD1 G93A mutant.
The glutathione S-transferase (GST) system is useful for increasing protein solubility and purifying soluble GST fusion proteins. However, purifying half of the GST fusion proteins is still ...difficult, because they are virtually insoluble under non-denaturing conditions. To optimize a simple and rapid purification condition for GST-pyruvate kinase muscle 2 (GST-PKM2) protein, we used 1% sarkosyl for lysis and a 1 : 200 ratio of sarkosyl to Triton X-100 (S-T) for purification. We purified the GST-PKM2 protein with a high yield, approximately 5 mg/L culture, which was 33 times higher than that prepared using a conventional method. Notably, the GST-high-temperature requirement A2 (GST-HtrA2) protein, used as a model protein for functional activity, fully maintained its proteolytic activity, even when purified under our S-T condition. This method may be useful to apply to other biologically important proteins that become highly insoluble in the prokaryotic expression system.
The purpose of this study is to investigate the metabolic alterations associated with acute alcohol treatment in zebrafish by
1
H nuclear magnetic resonance spectroscopy (NMRS). The brain metabolism ...of zebrafish was investigated after acute alcohol treatment (one-hour long exposure of adult fish to 0.00%, 0.25%, 0.50%, or 1.00% ethyl alcohol) with whole brain extraction. The results of this study showed that glutamate (Glu) was significantly decreased, scyllo-inositol (sIns) showed a small apparent increase only in the highest acute treatment dose group, and myoinositol (mIns) showed a significant decrease. Glu/tCr and mIns/tCr levels were significantly reduced regardless of the alcohol dose, and sIns/tCr was increased in the highest alcohol treatment dose group. The present NMR study revealed that specific metabolites, such as Glu and mIns, were substantially decreased in case of acute alcohol exposed zebrafish brain.
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