A radiometric assay for glutamine:fructose-6-phosphate amidotransferase

Glutamine:fructose-6-phosphate amidotransferase (GFAT) catalyzes the first step in the biosynthesis of amino sugars by transferring the amino group from l-glutamine to the acceptor substrate, fructose 6-phosphate, generating the products glucosamine 6-phosphate and glutamic acid. We describe a method for the synthesis and purification of the substrate, fructose 6-phosphate, and methods for a radiometric assay of human GFAT1 that can be performed in either of two formats: a small disposable-column format and a high-throughput 96-well-plate format. The method performed in the column format can detect 1 pmol of glucosamine 6-phosphate, much less than that required by previously published assays that measure GlcN 6-phosphate. The column assay demonstrates a broad linear range with low variability. In both formats, the assay is linear with time and enzyme concentration and is highly reproducible. This method greatly improves the sensitivity and speed with which GFAT1 activity can be measured and facilitates direct kinetic measurement of the transferase activity.     

在CHO细胞中表达重组sPDGFRα-Fc及其抑制细胞增殖的研究

目的：筛选在CHO-K1中高表达sPDGFRα-Fc的重组细胞株,并对分泌到培养基的表达产物进行抑制细胞增殖的活性分析。方法：构建带有Fc标签的sPDGFRα基因重组表达载体 pIRES-Neo3-sPDGFRα-Fc;在脂质体介导下,转染CHO-K1细胞,G418筛选2周后获得若干单克隆细胞株,随机挑取单克隆细胞进一步放大培养,RT-PCR筛选阳性单克隆细胞;Real-Time PCR方法鉴定各阳性细胞株中的sPDGFRα-Fc基因的转录水平, Western blot检测进一步验证各细胞中目的蛋白表达水平;筛选出表达最高的细胞株,更换无血清培养基培养,取含有可溶性sPDGFRα的培养基上清冻干浓缩,MTT法检测目的蛋白的抑制细胞增殖能力。结果：成功构建重组表达载体并在CHO-K1中成功表达,各阳性单克隆细胞株的表达量有差异且在转录水平和蛋白表达水平表现一致,从无血清培养基中收集的可溶性sPDGFRα-Fc明显抑制血管内皮细胞的增殖。结论：成功筛选获得CHO-K1中高表达sPDGFRα-Fc的重组细胞株,获得的可溶性sPDGFRα-Fc能抑制细胞增殖,有望成为治疗因PDGF及其受体引起的多种疾病的药物。     

Colorimetric detection of glutamine synthetase-catalyzed transferase activity in glucocorticoid-treated skeletal muscle cells

Induction of the enzyme glutamine synthetase (GS) by corticosteroids correlates with muscle wasting and gluconeogenesis, characteristic side effects of chronic glucocorticoid treatment. This highlights the importance of developing robust high-throughput assays to measure drug-induced GS in whole cells. We have optimized a colorimetric method to measure GS-catalyzed gamma-glutamyltransferase (GT) activity in rat L6 skeletal muscle cells (96-well-plate format) and human skeletal muscle cells (24-well-plate format). We observe a fourfold increase in GT activity in dexamethasone treated L6 cells, as compared to untreated cells, with good reproducibility in the measurements (errors of less than 5%). This assay can distinguish between partial agonists such as halopredone acetate and complete agonists such as prednisolone and measure the potency of known glucocorticoid receptor (GR) antagonists like mifepristone. Importantly, the ability of corticosteroids to induce GS-catalyzed GT activity correlates well with their whole cell GR binding potency, indicating a GR-specific effect. Interestingly, in general, induction of GT activity by commonly administered anti-inflammatory corticosteroid drugs is comparable in rat and human skeletal muscle cells, which emphasizes the potential of a rat model system to study GS induction and muscle wasting by these drugs in humans.     

Suppression of nasopharyngeal carcinoma cell by targeting β-catenin signaling pathway

Aim: To investigate the role of β-catenin in pathogenesis of nasopharyngeal carcinoma (NPC). Methods: Cellular proliferation, apoptosis, matrix penetration assay, and western blotting were employed to determine cell biological changes in NPC cell lines transfected with β-catenin siRNA. Immunohistochemistry staining was used to detect β-catenin and Ki-67 expression in NPC tissue. Results: β-Catenin was upregulated in NPC cell lines and tissues compared with chronic nasopharyngitis tissue. β-Catenin knockdown dramatically inhibited cellular growth, migration and invasion, but induced apoptosis of NPC cells. Further study showed that downstream genes of β-catenin signaling pathway including cyclin D1, c-Myc, MMP2 and MMP9 expression were suppressed in NPC cell lines transfected with β-catenin siRNA. Conclusion: Targeting β-catenin signaling pathway may be a noval strategy for NPC therapy.     

β-Elemene inhibits the metastasis of multidrug-resistant gastric cancer cells through miR-1323/Cbl-b/EGFR pathway

Backgroundβ-Elemene is a natural agent extracted from the traditional Chinese herbal medicine Curcuma wenyujin that is a promising novel plant-derived drug with broad-spectrum anticancer activity. Our previous study identified an enhanced capacity for metastasis in multidrug resistant (MDR) gastric cancer and breast cancer cells. However, the anti-metastatic effects of β-Elemene on MDR cancer cells remain unknown.PurposeIn this study, we posit the hypothesis that β-elemene possesses antimetastatic effects on MDR cancer cells.MethodsCell viability assay was used to assess the resistance of SGC7901/ADR cells and the cytotoxic effects of β-Elemene. Wound healing, transwell assay and lung metastatic mice model were used to the anti-metastasis effects of β-Elemene. MicroRNA microarray analysis was used to explore potential regulated miRNAs. Luciferase reporter assay was used to identify the direct target. Human MMP antibody array, western blot, immunoprecipitation, qRT-PCR analyses and immunohistochemistry were conducted to investigate the underlying anti-metastasis mechanism of β-Elemene.ResultsIn this study, we found that β-Elemene significantly inhibited the metastatic capacity of MDR gastric cells in vivo and in vitro. Mechanistically, we found that β-Elemene regulated MMP-2/9 expression and reversed epithelial-mesenchymal transition. Further studies showed that β-Elemene upregulated Cbl-b expression, resulting in inhibition of the EGFR-ERK/AKT pathways, which regulate MMP-2/9. Additionally, we confirmed that β-Elemene upregulated Cbl-b by inhibiting miR-1323 expression. Finally, we found that numbers of metastatic tumor nodules were significantly decreased in the lungs of nude mice after β-Elemene treatment.ConclusionOur results suggested that β-Elemene inhibits the metastasis of MDR gastric cancer cells by modulating the miR-1323/Cbl-b/EGFR signaling axis.     

Scanning assay of β-galactosidase activity

β-galactosidase, encoded by the lacZ gene in E. coli, can cleave lactose and structurally related compounds to galactose and glucose or structurally related products. Its activity can be measured using an artificial substrate, o-nitrophenyl-β-D-galactopyranoside (ONPG). Miller firstly described the standard quantitative assay of β-galactosidase activity in the cells of bacterial cultures by disrupting the cell membrane with the permeabilization solution instead of preparing cell extracts. Therefore, β-galactosidase became one of the most widely used reporters of gene expression in molecular biology to reflect intracellular gene expression difference. But the Miller assay procedure could not monitor the β-galactosidase reaction in real time and its results were greatly influenced by some operations in the Miller procedure, such as permeabilization time, reaction time and concentration of the cell suspension. A scanning method based on the Miller method to determine the intracellular β-galactosidase activity in E. coli Tuner (DE3) expressing β-galactosidase in real time was developed and the permeabilization time of cells was optimized for that. The comparison of 3 assays of β-galactosidase activity (Miller, colorimetric and scanning) was made. The results proved that scanning method for the determination of enzyme activity with using ONPG as substrate is simple, fast and reproducible.     

β-Galactosidase activity assay using far-red-shifted fluorescent substrate DDAOG

β-Galactosidase (β-gal) is commonly used as a reporter gene in biological research, and a wide variety of substrates have been developed to assay its activity. One substrate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) β-d-galactopyranoside (DDAOG), can be cleaved by β-gal to produce 7-hydroxy-9H(I,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). On excitation, DDAO generates a far-red-shifted fluorescent signal. Using this substrate, we developed a β-gal activity assay method. The DDAO signal was stable for at least 18 h. The signal intensity was linearly related to both the enzyme amount and substrate concentration. An optimized buffer for the β-gal/DDAOG assay was also formulated. When compared with the colorimetric substrate o-nitrophenyl-β-d-galactopyranoside (ONPG), the signal-to-background ratio of the DDAOG method was approximately 12-fold higher. The β-gal/DDAOG assay method was also tested in transiently transfected cells employing both pharmacologically and genetically inducible gene expression systems. The ability to detect signal induction is comparable to a similar assay using luciferase as the signal generating moiety. The β-gal/DDAOG assay method should provide a fluorescent reporter assay system for the wide variety of β-gal systems currently in use.     

Kinetic study of a substrate cycle involving a chemical step: highly amplified determination of phenolic compounds

A kinetic analysis of a substrate cycle in which one of the two steps was substituted by a chemical reaction has been made. The model is illustrated by the amplified determination, in a continuous assay, of phenolic compounds at low concentrations using the enzyme tyrosinase and β-NADH to reduce the o-quinone product of catalytic activity. Progress curves corresponding to β-NADH disappearance were not linear and followed first-order kinetics. Knowledge of the kinetics of the reaction has allowed us to achieve detection limits as low as 50 nM in a simple 10-min assay. There is no analytical solution to the non-linear differential equation system that describes the kinetics of the reaction, therefore, computer simulations of its dynamic behaviour are also presented, good agreement with the experimental results being obtained. The method is applicable to the measurement of any other metabolite, and its amplification capacity as well as the simplicity of determining kinetic parameters enable it to be implemented in a bioreactor for automation purposes.     

High-performance liquid chromatographic quantification of 4-methylumbelliferyl-β-d-glucuronide as a probe for human β-glucuronidase activity in tissue homogenates

An internally standardized HPLC method to determine the concentration of 4-methylumbelliferone liberated from 4-methylumbelliferyl-β-d-glucuronide by human β-glucuronidase was developed. The assay allows the precise and rapid measurement of specific enzyme activity in human tissue homogenates. Without prior extraction the incubation mixture can be separated using a C₈ column followed by fluorescence detection. The assay showed good accuracy and precision with a detection limit of 20 nM and a limit of quantification of 167 nM. The suitability of the method was shown in enzyme kinetic experiments with human liver homogenates.     

Introduction of silent mutations in a codon-optimized xylanase (xynB) results in enhanced protein expression in HEK293A cells

Xylanase is used extensively to improve feed conversion rates to enhance the performance of poultry and pigs. By expressing xylanase in simple-stomached animals, new breeds of genetically modified animals with enhanced feed conversion rates may be obtained. However, expression of heterologous proteins derived from lower organisms in mammalian cells is usually inefficient. When common codons of a ‘‘one amino acid-one codon”-optimized xylanase from Streptomyces olivaceoviridis were replaced with rare codons, xylanase expression in human embryonic kidney 293A cells increased by 1.4- to 2.3-fold as determined by flow cytometry, western blot and enzymatic activity assay. Quantitative RT-PCR assay indicated that the enhanced expression could not be attributed to altered mRNA levels. This study provides an alternative strategy for improving expression levels of heterologous proteins in mammalian cells, which is potentially helpful for generating genetically modified animals with enhanced feed conversion ability.     

A specific method for the quantitative determination of β-glucose-1-phosphate

A specific spectrophotometric determination of β-glucose-1-phosphate has been devised. It allows β-glucose-1-phosphate to be measured in the presence of α-glucose-1-phosphate and of a one hundred-fold excess of glucose-6-phosphate. Phosphoglucomutase for β-glucose-1-phosphate obtained from cells of Euglena gracilis var. bacillaris must be prepared for the assay.     

Dual role of imidazole as activator/inhibitor of sweet almond (Prunus dulcis) β-glucosidase

The activity of Prunus dulcis (sweet almond) β-glucosidase at the expense of p-nitrophenyl-β-d-glucopyranoside at pH 6 was determined, both under steady-state and pre-steady-state conditions. Using crude enzyme preparations, competitive inhibition by 1–5 mM imidazole was observed under both kinetic conditions tested. However, when imidazole was added to reaction mixtures at 0.125–0.250 mM, we detected a significant enzyme activation. To further inspect this effect exerted by imidazole, β-glucosidase was purified to homogeneity. Two enzyme isoforms were isolated, i.e. a full-length monomer, and a dimer containing a full-length and a truncated subunit. Dimeric β-glucosidase was found to perform much better than the monomeric enzyme, independently of the kinetic conditions used to assay enzyme activity. In addition, the sensitivity towards imidazole was found to differ between the two isoforms. While monomeric enzyme was indeed found to be relatively insensitive to imidazole, dimeric β-glucosidase was observed to be significantly activated by 0.125–0.250 mM imidazole under pre-steady-state conditions. Further, steady-state assays revealed that the addition of 0.125 mM imidazole to reaction mixtures increases the K_m of dimeric enzyme from 2.3 to 6.7 mM. The activation of β-glucosidase dimer by imidazole is proposed to be exerted via a conformational transition poising the enzyme towards proficient catalysis.     

IFN-γ Induces Gastric Cancer Cell Proliferation and Metastasis Through Upregulation of Integrin β3-Mediated NF-κB Signaling

Interferon γ (IFN-γ), a multifunctional cytokine, was upregulated in the resected gastric cancer tissue. However, whether IFN-γ is involved in the regulation of gastric cancer has not been well elucidated. Herein, we aimed to investigate the effects and mechanism of IFN-γ on gastric cancer. In this study, we found a vital role of IFN-γ in enhancing proliferation, inhibiting apoptosis, and promoting cell migration and invasion in gastric cancer cells SGC-7901 and MGC-803. Additionally, IFN-γ activated nuclear factor κB (NF-κB) signaling pathway by upregulating the phosphorylation expression of p65 and IκBα, and induced the expression of integrin β3 in vitro. Therefore, to further investigate the relationship between IFN-γ and integrin β3, SGC-7901 cells were transfected with integrin β3 siRNA. And then cells expressed lower cell viability, migration, and invasion rates, while cell apoptosis was significantly enhanced. Meanwhile, expression of integrin β3, MMP-2, MMP-9, and NF-κB, including p65 and IκBα, and the nuclear translocation of NF-κB/p65 were dramatically repressed, whereas IFN-γ significantly improved the effects. Moreover, in vivo, the experiment of xenograft model and pulmonary metastasis model also retarded in integrin β3 siRNA group. And the expression of integrin β3, MMP-2, MMP-9, and NF-κB was repressed. However, the treatment with IFN-γ improved tumor volume, lung/total weight, tumor nodules, and the protein expression described above compared with integrin β3 siRNA group. Overall, the results indicated that IFN-γ induces gastric cancer cell proliferation and metastasis partially through the upregulation of integrin β3-mediated NF-κB signaling. Hence, the inhibition of IFN-γ or integrin β3 may be the key for the treatment of gastric cancer.     

XLMR protein related to neurite extension (Xpn/KIAA2022) regulates cell–cell and cell–matrix adhesion and migration

X-linked mental retardation (XLMR) is a common cause of moderate to severe intellectual disability in males. XLMR protein related to neurite extension (Xpn, also known as KIAA2022) has been implicated as a gene responsible for XLMR in humans. Although Xpn is highly expressed in the developing brain and is involved in neurite outgrowth in PC12 cells and neurons, little is known about the functional role of Xpn. Here, we show that Xpn regulates cell–cell and cell–matrix adhesion and migration in PC12 cells. Xpn knockdown enhanced cell–cell and cell–matrix adhesion mediated by N-cadherin and β1-integrin, respectively. N-Cadherin and β1-integrin expression at the mRNA and protein levels was significantly increased in Xpn knockdown PC12 cells. Furthermore, overexpressed Xpn protein was strongly expressed in the nuclei of PC12 and 293T cells. Finally, depletion of Xpn perturbed cellular migration by enhancing N-cadherin and β1-integrin expression in a PC12 cell wound healing assay. We conclude that Xpn regulates cell–cell and cell–matrix adhesion and cellular migration by regulating the expression of adhesion molecules.     

Histone deacetylase1 promotes TGF-β1-mediated early chondrogenesis through down-regulating canonical Wnt signaling

Cartilage formation during both embryonic development and bone repairing processes involves mesenchymal stem cells (MSCs) differentiation. Wnt/β-catenin signaling pathway inhibits early chondrogenesis and is down-regulated during Transforming growth factor-β1 (TGF-β1)-induced chondrogenesis. However, the regulatory molecules that participate in the process is unknown. This study was designed to investigate the underlying mechanisms that down-regulate Wnt/β-catenin pathway during chondrogenesis. TGF-β1-induced micromass cultures of C3H10T1/2 were used as chondrocyte differentiation model. Gene expression profile was detected by realtime-PCR. Regulatory role of HDAC1 on β-catenin was investigated by luciferase assay, chromatin immunoprecipitation (ChIP) assay, co-immunoprecipitation (Co-IP) assay and in vitro ubiquitination assay. In this study, we showed that HDAC1 was induced and suppressed β-catenin gene expression through direct binding to its promoter. Besides, HDAC1 could also interact with deacetylate β-catenin protein through its deacetylase domain, which causes degradation of β-catenin. Our results indicate that HDAC1 plays an important role in chondrogenesis and may represent a therapeutic target for modulation of cartilage development.     

Combinatorial strategy of sorbitol feeding and low-temperature induction leads to high-level production of alkaline β-mannanase in Pichia pastoris

A process for efficient production of an alkaline β-mannanases from Bacillus sp. N16-5 was established by heterologous expression using Pichia pastoris. A high producing strain was generated by removing the native β-mannanases signal peptide and increasing the copy number of the mature β-mannanases gene. High cell density fermentation of this strain in 1-L bioreactor led to a production level of 4164 U/mL after 96 h of induction. Sorbitol co-feeding and temperature-lowering strategies both increased the β-mannanase production levels. Combined usage of these two strategies achieved the most effective result—the enzyme level reached 6336 U/mL within 84 h, which to our best knowledge is the highest production level reported for the expression of extreme β-mannanase thus far. The strategy described in this work can also be adapted to express other important industrial enzymes with extreme properties.     

High lysosomal activities in cystic fibrosis tracheal gland cells corrected by adenovirus-mediated CFTR gene transfer

Human tracheal gland serous (HTGS) cells are now believed to be a major target of cystic fibrosis (CF) gene therapy. To evaluate the efficiency of adenovirus-mediated gene transfer in these cells we tested the adenovirus construction containing β-galactosidase cDNA. We observed that the endogenous β-galactosidase activity in cultured CF-HTGS cells was too strong to allow us to detect any exogenous β-galactosidase activity. Immunohistological study on sections of human tracheal tissue confirmed the presence of β-galactosidase in the serous component of the submucosal glands. We then looked for other lysosomal activities in normal and CF-HTGS cells. We showed that normal cells already have elevated enzyme values and that CF-HTGS cells contained 2–4-fold more β-galactosidase, α-fucosidase, α-mannosidase and β-glucuronidase activities than normal cells. An analysis of their kinetic constants has shown that this difference could be attributed to a lower K_m of CF lysosomal enzymes. More importantly, these differences are eliminated after adenovirus-mediated CFTR gene transfer and not after β-galactosidase gene transfer.     

A high-capacity screen for adipogenic differentiation

Glycerol-3-phosphate dehydrogenase (GPDH) is highly expressed in mature adipocytes. Activity of this enzyme is therefore routinely measured to assess adipogenic differentiation in cell cultures. Existing protocols for GPDH assays require relatively large amounts of cells, and throughput is limited due to multiple steps needed for cell harvest and enzyme extraction. We present here a new protocol allowing GPDH determinations to be performed in a 96-well-plate format. From the start of cell culture to the final readout all steps are carried out using the same multiwell plate, with a minimum of handling required. Our method is suitable for setting up high-throughput assays of adipogenic differentiation.